Article Publication: Nature Biotechnology

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Article Publication: PLOS ONE

Article Summary: BACKGROUND: An understanding of the health of potential volunteers in Africa is essential for the safe and efficient conduct of clinical trials, particularly for trials of preventive technologies such as vaccines that enroll healthy individuals. Clinical safety laboratory values used for screening, enrolment and follow-up of African clinical trial volunteers have largely been based on values derived from industrialized countries in Europe and North America. This report describes baseline morbidity during recruitment for a multi-center, African laboratory reference intervals study. METHODS: Asymptomatic persons, aged 18-60 years, were invited to participate in a cross-sectional study at seven sites (Kigali, Rwanda; Masaka and Entebbe, Uganda; Kangemi, Kenyatta National Hospital and Kilifi, Kenya; and Lusaka, Zambia). Gender equivalency was by design. Individuals who were acutely ill, pregnant, menstruating, or had significant clinical findings were not enrolled. Each volunteer provided blood for hematology, immunology, and biochemistry parameters and urine for urinalysis. Enrolled volunteers were excluded if found to be positive for HIV, syphilis or Hepatitis B and C. Laboratory assays were conducted under Good Clinical Laboratory Practices (GCLP). RESULTS AND CONCLUSIONS: Of the 2990 volunteers who were screened, 2387 (80%) were enrolled, and 2107 (71%) were included in the analysis (52% men, 48% women). Major reasons for screening out volunteers included abnormal findings on physical examination (228/603, 38%), significant medical history (76, 13%) and inability to complete the informed consent process (73, 13%). Once enrolled, principle reasons for exclusion from analysis included detection of Hepatitis B surface antigen (106/280, 38%) and antibodies against Hepatitis C (95, 34%). This is the first large scale, multi-site study conducted to the standards of GCLP to describe African laboratory reference intervals applicable to potential volunteers in clinical trials. Approximately one-third of all potential volunteers screened were not eligible for analysis; the majority were excluded for medical reasons.

Article Publication: Nature medicine

Article Summary: The adenovirus type 5 (Ad5)-based vaccine developed by Merck failed to either prevent HIV-1 infection or suppress viral load in subsequently infected subjects in the STEP human Phase 2b efficacy trial. Analogous vaccines had previously also failed in the simian immunodeficiency virus (SIV) challenge–rhesus macaque model. In contrast, vaccine protection studies that used challenge with a chimeric simian-human immunodeficiency virus (SHIV89.6P) in macaques did not predict the human trial results. Ad5 vector–based vaccines did not protect macaques from infection after SHIV89.6P challenge but did cause a substantial reduction in viral load and a preservation of CD4+ T cell counts after infection, findings that were not reproduced in the human trials. Although the SIV challenge model is incompletely validated, we propose that its expanded use can help facilitate the prioritization of candidate HIV-1 vaccines, ensuring that resources are focused on the most promising candidates. Vaccine designers must now develop T cell vaccine strategies that reduce viral load after heterologous challenge.

Article Publication: Indian journal of medical research

Article Summary: India bears a heavy disease burden of HIV/AIDS infected and affected people. A safe, effective and accessible preventive AIDS vaccine, used along with other preventive interventions, is urgently needed to stem the epidemic. This review highlights the extensive preparedness activities undertaken from 2002 by the International AIDS Vaccine Initiative (IAVI), its Indian government and non government partners with the Indian scientific, political, media and community stakeholders and the capacity building process, before the conduct of the first ever AIDS vaccine trials in India in early 2005. Issues addressed included mistrust of clinical research due to past history of some unethical trials, transparency, community involvement, stigma and discrimination, provision for care and treatment of participants, informed consent, gender considerations, approval process, and operational aspects. The strong political support along with preparedness activities led to the successful conduct of AIDS vaccine trials enrolling equitably healthy women and men from all sections of society. This has paved the way for future vaccine trials in the country.

Article Publication: Vaccine

Article Summary: Lessons from IAVI-006, a Phase I clinical trial to evaluate the safety and immunogenicity of the pTHr.HIVA DNA and MVA.HIVA vaccines in a prime-boost strategy to induce HIV-1 specific T-cell responses in healthy volunteers

Article Publication: Journal of experimental medicine

Article Summary: An effective AIDS vaccine will need to protect against globally diverse isolates of HIV. To address this issue in macaques, we administered a live-attenuated simian immunodeficiency virus (SIV) vaccine and challenged with a highly pathogenic heterologous isolate. Vaccinees reduced viral replication by approximately 2 logs between weeks 2-32 (P ]]>Fri, 29 Jan 2010 19:20:34 GMThttp://www.iavireport.org/pages/IAVIAuthors.aspx?AbsId=1046Article Author: Burton DR, Desrosiers RC, Doms RW, Koff WC (IAVI), Kwong PD, Moore JP, Nabel GJ, Sodroski J, Wilson IA, Wyatt RT.

Article Publication: Nature immunology

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Article Publication: Clinical and vaccine immunology

Article Summary: The interferon-gamma (IFN-) ELISPOT assay is used routinely to evaluate the potency of HIV and other vaccine candidates. In order to compare candidates and pool data across multiple trial laboratories, validated standardized methods must be applied across laboratories. Proficiency panels are a key part of a comprehensive quality assurance program to monitor inter- and intra-laboratory as well as assay performance over time. Seven IAVI-sponsored trial sites participated in the proficiency panels. At each laboratory two operators independently processed identical panels containing frozen peripheral blood mononuclear cell (PBMC) samples from different donors using four blinded stimuli. PBMC recovery and viability after overnight rest and IFN- ELISPOT assay performance were assessed. All sites demonstrated good performance in PBMC thawing and resting: median recovery (78%) and viability (95%). The laboratories were able to detect similar antigen-specific T cell responses ranging from 50 to >3000 spot forming cells per million PBMC. An approximate range of a half log across operators within or across sites was seen when comparing antigen specific responses. Consistently low background responses were seen in all laboratories. This proficiency panel demonstrates the ability of seven laboratories, located across three continents, to process PBMC samples and to rank volunteers with differential magnitudes of IFN- ELISPOT responses. This panel also illustrates the ability to standardize the IFN- ELISPOT assay across multiple laboratories when common training, reagents such as FCS and standard operating procedures are adopted. These results are encouraging for laboratories that are using cell-based immunology assays to test HIV and other vaccines.

Article Publication: Journal of virology

Article Summary: An effective AIDS vaccine must control highly diverse circulating strains of HIV-1. Among HIV-1 gene products, the envelope (Env) protein contains variable as well as conserved regions. In this report, an informatic approach to the design of T-cell vaccines directed to HIV-1 Env M group global sequences was tested. Synthetic Env antigens were designed to express mosaics that maximize the inclusion of common potential T-cell epitope (PTE) 9-mers and minimize the inclusion of rare epitopes likely to elicit strain-specific responses. DNA vaccines were evaluated using intracellular cytokine staining (ICS) in inbred mice with a standardized panel of highly conserved 15-mer PTE peptides. 1, 2 and 3 mosaic sets were developed that increased theoretical epitope coverage. The breadth and magnitude of T-cell immunity stimulated by these vaccines were compared to natural strain Envs; additional comparisons were performed on mutant Envs, including gp160 or gp145 with or without V regions and gp41 deletions. Among them, the 2 or 3 mosaic Env sets elicited the optimal CD4 and CD8 responses. These responses were most evident in CD8 T cells; the 3 mosaic set elicited responses to an average of 8 peptide pools compared to 2 pools for a set of 3 natural Envs. Synthetic mosaic HIV-1 antigens can therefore induce T-cell responses with expanded breadth and may facilitate the development of effective T-cell-based HIV-1 vaccines.

Article Publication: Expert reviews in molecular medicine

Article Summary: Antigens administered via the oral and, to a lesser extent, the nasal route are potentially able to invoke tolerance, resulting in a nonreactive immune response. This has been a hurdle for mucosal vaccine development and yet the desire to induce protective local and systemic responses, with pain-free and more convenient products, has been the impetus driving mucosal vaccine R&D. Nevertheless, few mucosal vaccines have reached the marketplace and products are still treated with caution, particularly where live organisms are utilized. In this review, we examine the use of delivery systems with adjuvant properties as key components in a vaccine strategy that does not require the use of live vectors to overcome tolerance and have exemplified their success in mucosal vaccines, concentrating on the nasal and oral routes of administration.

Article Publication: AIDS research and human retroviruses

Article Summary: Vaccines inducing pathogen-specific cell-mediated immunity are being developed using attenuated adenoviral (Ad) vectors. We report the results of two independent Phase I trials of similar replication-deficient Ad5 vaccines containing a near-consensus HIV-1 clade B gag transgene. Healthy HIV-uninfected adults were enrolled in two separate, multicenter, dose-escalating, blinded, placebo-controlled studies to assess the safety and immunogenicity of a three-dose homologous regimen of Ad5 and MRKAd5 HIV-1 gag vaccines given on day 1, week 4, and week 26. Adverse events were collected for 29 days following each intradeltoid injection. The primary immunogenicity endpoint was the proportion of subjects with a positive unfractionated Gag-specific IFN-gamma ELISPOT response measured 4 weeks after the last dose (week 30). Analyses were performed after combining data for each dose group from both protocols, stratifying by baseline Ad5 titers. Overall, 252 subjects were randomized to receive either vaccine or placebo, including 229 subjects (91%) who completed the study through week 30. Tolerability and immunogenicity did not appear to differ between the Ad5 and MRKAd5 vaccines. The frequency of injection-site reactions was dose dependent. Systemic adverse events were also dose dependent and more frequent in subjects with baseline Ad5 titers <200 versus >/=200, especially after the first dose. The percent of ELISPOT responders and the ELISPOT geometric means overall were significantly higher for all four vaccine doses studied compared to placebo, and were generally higher in vaccine recipients with baseline Ad5 titers <200 versus >/=200. Ad5 titers increased after vaccination in a dose-dependent fashion. Both Ad5-vectored HIV-1 vaccines were generally well tolerated and induced cell-mediated immune responses against HIV Gag-peptides in the majority of healthy adults with baseline Ad5 titers <200. Preexistent and/or vaccine-induced immunity to the Ad5 vector may dampen the CMI response to HIV Gag.

Article Publication: Blood

Article Summary: There exists a unique group of individuals who are able to durably control HIV in the absence of therapy. The mechanisms of control in these individuals remain poorly defined. In this study we examined CD8+ T-cell responses in blood and rectal mucosa from 17 "elite controllers" (viral load < 75 copies/ml), 11 "viremic controllers" (75-2,000 copies/mL), 14 non-controllers (>10,000 copies/mL), and 10 antiretroviral-treated individuals (<75 copies/mL). Production of IFN-, IL-2, TNF-, MIP-1, and CD107a by CD8+ T-cells in response to HIV-1 Gag stimulation was measured using flow cytometry. Our hypothesis was that 'polyfunctional' T-cells producing multiple antiviral factors would be most abundant in mucosal tissues of HIV controllers. Mucosal CD8+ T-cell responses were significantly stronger and more complex in controllers than in antiretroviral-suppressed individuals (P=0.0004). The frequency of 4-function responses in rectal mucosa was higher in controllers than in non-controllers and patients on therapy (P<0.0001). Mucosal responses in controllers were frequently stronger and more complex than blood responses. These findings demonstrate that many controllers mount strong, complex HIV-specific T-cell responses in rectal mucosa. These responses may play an important role in mucosal immune surveillance, as suggested by their relative enrichment among individuals who control HIV in the absence of therapy.

Article Publication: Journal of virology

Article Summary: HIV-1 elite controllers (EC) maintain viremia below the limit of commercial assay detection (<50 RNA copies/ml) in the absence of antiviral therapy, but the mechanisms of control remain unclear. HLA-B57 and the closely related allele B*5801 are particularly associated with enhanced control, and recognize the same Gag240-249 TW10 epitope. The typical escape mutation (T242N) within this epitope diminishes viral replication capacity in chronically infected persons; however, little is known about TW10 epitope sequences in residual replicating viruses in B57/B*5801 EC, and the extent to which mutations within this epitope may influence steady state viremia. Here we analyzed TW10 in a total of 50 B57/B*5801 positive subjects (23 EC and 27 viremic subjects). Autologous plasma viral sequences from both EC and viremic subjects frequently harbored the typical CTL selected mutation T242N [15/23 (65.2%) vs. 23/27 (85.1%), respectively, p=0.18]. However, other unique mutants were identified in HIV controllers both within and flanking TW10 that were associated with an even greater reduction in in vitro viral replication capacity. In addition, strong CTL responses to many of these unique TW10 variants were detected by IFN-gamma ELISPOT assay. These data suggest a dual mechanism for durable control of HIV replication, consisting of viral fitness loss resulting from CTL escape mutations together with strong CD8 T cell immune responses to the arising variant epitopes.

Article Publication: PLoS medicine

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Article Publication: Virology

Article Summary: Human immunodeficiency virus type 1 (HIV-1) entry into host cells is mediated by the trimeric envelope glycoprotein complex (Env). Accordingly, the Env proteins are the targets for neutralizing antibodies (NAbs) and are the focus of vaccines intended to induce NAbs. Because the Env complex is labile, soluble recombinant Env (gp140) trimers require engineering to stabilize them sufficiently for use as immunogens. Trimeric forms of gp140 trimers can be created that are either cleavage-competent or cleavage-defective at the junction between the gp120 and gp41 subunits. As functional trimers are cleaved at this site, the question arises as to whether cleavage affects the antigenic structure of the Env complex in a way that is relevant to vaccine design. Here, we present a comparative analysis of the antigenicity profiles of cleaved and uncleaved gp140 trimers derived from the KNH1144 (subtype A) virus that are otherwise closely sequence-matched. While cleavage did not affect the exposure of NAb epitopes on the gp140 trimers, non-neutralizing antibodies to gp41 epitopes bound much more strongly to uncleaved trimers. Hence cleavage does alter the structure of the HIV-1 Env complex.

Article Publication: Vaccine

Article Summary: The safety and immunogenicity of plasmid pTHr DNA, modified vaccinia virus Ankara (MVA) human immunodeficiency virus type 1 (HIV-1) vaccine candidates were evaluated in four Phase I clinical trials in Kenya and Uganda. Both vaccines, expressing HIV-1 subtype A gag p24/p17 and a string of CD8 T-cell epitopes (HIVA), were generally safe and well-tolerated. At the dosage levels and intervals tested, the percentage of vaccine recipients with HIV-1-specific cell-mediated immune responses, assessed by a validated ex vivo interferon gamma (IFN-gamma) ELISPOT assay and Cytokine Flow Cytometry (CFC), did not significantly differ from placebo recipients. These trials demonstrated the feasibility of conducting high-quality Phase 1 trials in Africa.

Article Publication: Vaccine

Article Summary: In an earlier study, our group vaccinated rhesus macaques with vesicular stomatitis virus (VSV) vectors expressing Gag, Pol, and Env proteins from a hybrid simian/human immunodeficiency virus (SHIV). This was followed by a single boost with modified vaccinia virus Ankara (MVA) vectors expressing the same proteins. Following challenge with SHIV89.6P, vaccinated animals cleared challenge virus RNA from the blood by day 150 and maintained normal CD4 T cell counts for 8 months. Here we report on the long-term (>5-year post-challenge) status of these animals and the immunological correlates of long-term protection. Using real-time PCR, we found that viral DNA in peripheral blood mononuclear cells (PBMCs) of the vaccinees declined continuously and fell to below detection (<5copies/10(5)cells) by approximately 3 years post-challenge. SHIV DNA was also below the limit of detection in the lymph nodes of two of the four animals at 5 years post-challenge. We detected long-term persistence of multi-functional Gag-specific CD8(+) T cells in both PBMCs and lymph nodes of the two protected animals with the Mamu A*01(+) MHC I allele. All animals also maintained SHIV89.6P neutralizing antibody titers for 5 years. Our results show that this vaccine approach generates solid, long-term control of SHIV infection, and suggest that it is mediated by both cytotoxic T lymphocytes and neutralizing antibody.

Article Publication: Vaccine

Article Summary: We developed the method to efficiently construct recombinant vaccinia viruses based on LC16m8Delta strain that can replicate in mammalian cells but is still safe in human. Immunization in a prime-boost strategy using DNA and LC16m8Delta expressing SIV Gag elicited 7-30-fold more IFN-gamma-producing T cells in mice than that using DNA and non-replicating vaccinia DIs recombinant strain. As the previous study on the DNA-prime and recombinant DIs-boost anti-SIV vaccine showed protective efficacy in the macaque model [Someya K, Ami Y, Nakasone T, Izumi Y, Matsuo K, Horibata S, et al. Induction of positive cellular and humoral responses by a prime-boost vaccine encoded with simian immunodeficiency virus gag/pol.

Article Publication: Journal of virology

Article Summary: Although there is increasing evidence that individuals already infected with HIV-1 can be infected with a heterologous strain of the virus, the extent of protection against superinfection conferred by the first infection and the biologic consequences of superinfection are not well understood. We explored these questions in the simian immunodeficiency virus (SIV)/rhesus monkey model of HIV-1/AIDS. We infected cohorts of rhesus monkeys with either SIVmac251 or SIVsmE660, and then exposed animals to the reciprocal virus through intrarectal inoculations. Employing a quantitative real-time polymerase chain reaction (qRT-PCR) assay, we determined the replication kinetics of the two strains of virus for 20 weeks. We found that primary infection with a replication-competent virus did not protect against acquisition of infection by a heterologous virus, but did confer relative control of the superinfecting virus. In animals that became superinfected, there was a reduction in peak replication and rapid control of the second virus. The relative susceptibility to superinfection was not correlated with CD4(+) T cell count, CD4(+) memory T cell subsets, cytokine production by virus-specific CD8(+) or CD4(+) cells, or neutralizing antibodies at the time of exposure to the second virus. Although there were transient increases in viral load of the primary virus and a modest decline in CD4(+) T cell counts after superinfection, there was no evidence of disease acceleration. These findings indicate that an immunodeficiency virus infection confers partial protection against a second immunodeficiency virus infection, but this protection may be mediated by mechanisms other than classical adaptive immune responses.

Article Publication: Journal of immunology

Article Summary: An efficient pathway of cross-presentation common to a range of dendritic cell (DC) populations was identified by targeting Ag to MHC class II molecules. This finding was achieved by conjugating Ag to M1, which is a modified version of the superantigen streptococcal mitogenic exotoxin Z-2 that binds to MHC class II molecules but cannot directly stimulate T cells. M1 conjugates were efficiently presented to CD4(+) and CD8(+) T cells by bone marrow-derived DC and Langerhans cells in vitro. Whereas nonconjugated Ag was preferentially cross-presented by splenic CD8alpha(+) DC in vivo, M1-conjugated Ag was cross-presented by all dendritic subtypes assessed. Potent effector T cell responses with antitumor activity were elicited when M1 conjugates were injected together with an adjuvant. This method of Ag delivery has significant potential in therapeutic applications.

Article Publication: Vaccine

Article Summary: HIV vaccines of the first generation are, in contrast to most currently licensed vaccines, unlikely to provide sterilizing immunity against infection with HIV. However, they are expected to exert a beneficial effect on the maintenance of CD4 T-cell counts by reducing viral load. There is a recognized need for clarifying the path to licensure of such novel vaccines and defining the clinical trial endpoints. This was the focus of discussions of a 2-day workshop organized by the WHO, UNAIDS and the ANRS, in support of the Global HIV Vaccine Enterprise, which took place in Paris, France (5-6 September 2007). This expert consultation, the proceedings of which are presented here, was intended to review the fundamental principles and approaches to validate surrogate markers in clinical research, as well as the significance of viral load for the individual course of disease and for secondary transmission. Recommendations were also made for additional research to inform decision-making regarding potential licensure and delivery of vaccines which do not prevent HIV acquisition but do reduce viral load.

Article Publication: Journal of general virology

Article Summary: Candidate human immunodeficiency virus (HIV) vaccine regimens based on DNA boosted with recombinant modified vaccinia Ankara (MVA) have been in development for some time, and there is evidence for improved immunogenicity of newly developed constructs. This study describes immune responses to candidate DNA and MVA vaccines expressing multiple genes (gag, RT, tat, nef and env) from HIV-1 subtype C in chacma baboons (Papio ursinus). The vaccine regimen induced (i) strong T-cell responses, with a median of 4103 spot forming units per 10(6) peripheral blood mononuclear cells by gamma interferon (IFN-gamma) ELISPOT, (ii) broad T-cell responses targeting all five vaccine-expressed genes, with a median of 12 peptides targeted per animal and without any single protein dominating the response, (iii) balanced CD4(+) and CD8(+) responses, which produced both IFN-gamma and interleukin (IL)-2, including IL-2-only responses not detected by the ELISPOT assay, (iv) vaccine memory, which persisted 1 year after immunization and could be boosted further, despite strong anti-vector responses, and (v) mucosal T-cell responses in iliac and mesenteric lymph nodes in two animals tested. The majority of peptide responses mapped contained epitopes previously identified in human HIV infection, and two high-avidity HIV epitope responses were confirmed, indicating the utility of the baboon model for immunogenicity testing. Together, our data show that a combination of DNA and MVA immunization induced robust, durable, multifunctional CD4(+) and CD8(+) responses in baboons targeting multiple HIV epitopes that may home to mucosal sites. These candidate vaccines, which are immunogenic in this pre-clinical model, represent an alternative to adenoviral-based vaccines and have been approved for clinical trials.

Article Publication: Vaccine

Article Summary: Modified Vaccinia Ankara (MVA) is a replication-defective strain of vaccinia virus (VV) that is being investigated in humans as an alternative vaccine against smallpox. Understanding the parameters of a MVA vaccine regimen that can effectively enhance protective immunity will be important for clinical development. The present studies utilize cohorts of rhesus monkeys immunized with recombinant MVA (rMVA) or recombinant VV (rVV) vaccine vectors to investigate the magnitude, breadth, and durability of anti-VV immunity elicited by a single or multi-dose vaccine regimen. These data demonstrate that a single immunization with rMVA elicits weaker cellular and humoral immunity compared to a single inoculation with rVV. However, vaccine-elicited antibody responses, but not T cell responses, are significantly enhanced with repeated immunizations of rMVA. Importantly, only monkeys receiving up to four inoculations with rMVA generated neutralizing antibody (NAb) responses that were comparable in magnitude and durability to those elicited in monkeys receiving two inoculations with rVV. These data also show that the breadth of antibody responses against protein antigens associated with two antigenically distinct forms of infectious VV are similar in rMVA and rVV immunized monkeys. Together, these studies suggest that a multi-dose vaccine regimen utilizing up to four inoculations of MVA generates robust and durable antibody-mediated immunity comparable to that elicited by replication-competent VV.

Article Publication: Virology

Article Summary: ittle is known about the neutralization properties of HIV-1 in India to optimally design and test vaccines. For this reason, a functional Env clone was obtained from each of ten newly acquired, heterosexually transmitted HIV-1 infections in Pune, Maharashtra. These clones formed a phylogenetically distinct genetic lineage within subtype C. As Env-pseudotyped viruses the clones were mostly resistant to IgG1b12, 2G12 and 2F5 but all were sensitive to 4E10. When compared to a large multi-subtype panel of Env-pseudotyped viruses (subtypes B, C and CRF02_AG) in neutralization assays with a multi-subtype panel of HIV-1-positive plasma samples, the Indian Envs were remarkably complex. With the exception of the Indian Envs, results of a hierarchical clustering analysis showed a strong subtype association with the patterns of neutralization susceptibility. From these patterns we were able to identify 19 neutralization cluster-associated amino acid signatures in gp120 and 14 signatures in the ectodomain and cytoplasmic tail of gp41. We conclude that newly transmitted Indian Envs are antigenically complex in spite of close genetic similarity. Delineation of neutralization-associated amino acid signatures provides a deeper understanding of the antigenic structure of HIV-1 Env.

Article Publication: Journal of virology

Article Summary: Defining the antiviral efficacy of CD8 T cells is important for immunogen design, and yet most current assays do not measure the ability of responses to neutralize infectious virus. Here we show that HIV-specific CTL clones and cell lines derived from infected persons and targeting diverse epitopes differ by over 1000 fold in their ability to retard infectious virus replication in autologous CD4 T cells during a 7 day period in vitro, despite comparable activity as assessed by IFN gamma Elispot assay. Cell lines derived from peripheral blood mononuclear cells (PBMC) stimulated in vitro with peptides representing targeted Gag epitopes consistently neutralized HIV better than Env-specific lines from the same person although ineffective inhibition of virus replication is not a universal characteristic of Env-specific responses at the clonal level. Gag-specific cell lines were of higher avidity than Env-specific lines although avidity did not correlate with the ability of Gag- or Env-specific lines to contain HIV replication. The greatest inhibition was observed with cell lines restricted by the protective HLA alleles B*27 and B*57, but stimulation with targeted Gag epitopes resulted in greater inhibition than did stimulation with targeted Env epitopes even in non-B*27/B*57 subjects. These results assessing functional virus neutralization by HIV-specific CD8 T cells indicate that there are marked epitope and allele-specific differences in virus neutralization by in vitro expanded CD8 T cells, a finding not revealed by standard IFN gamma Elispot assay currently in use in vaccine trials, which may be of critical importance in immunogen design and testing of candidate AIDS vaccines.

Article Publication: Journal of virology

Article Summary: Currently there is limited information about the quality of immune responses elicited by candidate human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env)-based immunogens in primates. Here we describe a comprehensive analysis of neutralizing antibody and T-cell responses obtained in cynomolgus macaques by three selected immunization regimens. We used the previously described YU2-based gp140 protein trimers administered in an adjuvant, preceded by two distinct priming strategies: either alphavirus replicon particles expressing matched gp140 trimers or gp120 core proteins stabilized in the CD4-bound conformation. The rationale for priming with replicon particles was to evaluate the impact of the expression platform on trimer immunogenicity. The stable core proteins were chosen in an attempt to expand selectively lymphocytes recognizing common determinants between the core and trimers to broaden the immune response. The results presented here demonstrate that the platform by which Env trimers were delivered in the priming (either protein or replicon vector) had little impact on the overall immune response. In contrast, priming with stable core proteins followed by a trimer boost strikingly focused the T-cell response on the core sequences of HIV-1 Env. The specificity of the T-cell response was distinctly different from that of the responses obtained in animals immunized with trimers alone and was shown to be mediated by CD4(+) T cells. However, this regimen showed limited or no improvement in the neutralizing antibody responses, suggesting that further immunogen design efforts are required to successfully focus the B-cell response on conserved neutralizing determinants of HIV-1 Env.

Article Publication: PLoS pathogens

Article Summary: Strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection were evaluated for the ability to elicit protective immunity against wild-type SIV(mac)239 infection of rhesus macaques by two different vaccine regimens. Six animals were inoculated at 8-week intervals with 6 identical doses consisting of a mixture of three different envelope variants of single-cycle SIV (scSIV). Six additional animals were primed with a mixture of cytoplasmic domain-truncated envelope variants of scSIV and boosted with two doses of vesicular stomatitis virus glycoprotein (VSV G) trans-complemented scSIV. While both regimens elicited detectable virus-specific T cell responses, SIV-specific T cell frequencies were more than 10-fold higher after boosting with VSV G trans-complemented scSIV (VSV G scSIV). Broad T cell recognition of multiple viral antigens and Gag-specific CD4(+) T cell responses were also observed after boosting with VSV G scSIV. With the exception of a single animal in the repeated immunization group, all of the animals became infected following an intravenous challenge with SIV(mac)239. However, significantly lower viral loads and higher memory CD4(+) T cell counts were observed in both immunized groups relative to an unvaccinated control group. Indeed, both scSIV immunization regimens resulted in containment of SIV(mac)239 replication after challenge that was as good as, if not better than, what has been achieved by other non-persisting vaccine vectors that have been evaluated in this challenge model. Nevertheless, the extent of protection afforded by scSIV was not as good as typically conferred by persistent infection with live, attenuated SIV. These observations have potentially important implications to the design of an effective AIDS vaccine, since they suggest that ongoing stimulation of virus-specific immune responses may be essential to achieving the degree of protection afforded by live, attenuated SIV.

Article Publication: Nature immunology

Article Summary: The International AIDS Vaccine Initiative has established a consortium to elucidate mechanisms of protection conferred by live attenuated simian immunodeficiency virus vaccines in monkeys. Here, the strategies defining key components of the protective immune response elicited by these vaccines are discussed.

Article Publication: Journal of virology

Article Summary: The broadly neutralizing human monoclonal antibodies (mAbs) 2F5 and 4E10, both targeting the highly conserved HIV-1 envelope membrane proximal external region (MPER), are among the mAbs with the broadest heterologous neutralizing activity and are of considerable interest for HIV-1 vaccine development. We have identified serum antibodies from an HIV- infected subject that were both broadly neutralizing and specifically targeted MPER epitopes that overlap the 2F5 epitope. These MPER-specific antibodies were made 15-20 months following transmission and concomitant with the development of autoantibodies. Our findings suggest that multiple events (i.e. genetic predisposition and HIV-1 immune dysregulation) may be required for induction of broadly reactive gp41 MPER antibodies in natural infection

Article Publication: Vaccine

Article Summary: Correlates of immune protection from HIV vaccines remain undefined. The first HIV vaccine efficacy trial in the US and Europe VAX004, was designed to assess whether rgp120 envelope subunits (AIDSVAX B/B, VaxGen) can induce partial or complete protection from HIV-1 infection. No effectiveness in the reduction of either the acquisition of infection or levels of plasma viremia after HIV infection was noted. We found evidence of vaccine-specific CD8+ T cells in volunteers who received the vaccine, regardless of behavioral risk. Surprisingly, the CD8-response is significantly higher in participants who would go on to contract HIV infection. These results suggest that AIDSVAX immunization may boost preexisting immune responses-due to pre-infection exposure, and a vaccine-induced immune profile may serve as a biological marker for HIV susceptibility.

Article Publication: PLoS ONE

Article Summary: Clinical laboratory reference intervals have not been established in many African countries, and non-local intervals are commonly used in clinical trials to screen and monitor adverse events (AEs) among African participants. Using laboratory reference intervals derived from other populations excludes potential trial volunteers in Africa and makes AE assessment challenging. The objective of this study was to establish clinical laboratory reference intervals for 25 hematology, immunology and biochemistry values among healthy African adults typical of those who might join a clinical trial.

Equal proportions of men and women were invited to participate in a cross sectional study at seven clinical centers (Kigali, Rwanda; Masaka and Entebbe, Uganda; two in Nairobi and one in Kilifi, Kenya; and Lusaka, Zambia). All laboratories used hematology, immunology and biochemistry analyzers validated by an independent clinical laboratory. Clinical and Laboratory Standards Institute guidelines were followed to create study consensus intervals. For comparison, AE grading criteria published by the U.S. National Institute of Allergy and Infectious Diseases Division of AIDS (DAIDS) and other U.S. reference intervals were used. 2,990 potential volunteers were screened, and 2,105 (1,083 men and 1,022 women) were included in the analysis. While some significant gender and regional differences were observed, creating consensus African study intervals from the complete data was possible for 18 of the 25 analytes. Compared to reference intervals from the U.S., we found lower hematocrit and hemoglobin levels, particularly among women, lower white blood cell and neutrophil counts, and lower amylase. Both genders had elevated eosinophil counts, immunoglobulin G, total and direct bilirubin, lactate dehydrogenase and creatine phosphokinase, the latter being more pronounced among women. When graded against U.S.-derived DAIDS AE grading criteria, we observed 774 (35.3%) volunteers with grade one or higher results; 314 (14.9%) had elevated total bilirubin, and 201 (9.6%) had low neutrophil counts. These otherwise healthy volunteers would be excluded or would require special exemption to participate in many clinical trials.

To accelerate clinical trials in Africa, and to improve their scientific validity, locally appropriate reference ranges should be used. This study provides ranges that will inform inclusion criteria and evaluation of adverse events for studies in these regions of Africa.

Article Publication: Tropical medicine and international health

Article Summary: Summary Objectives To assess willingness to participate in HIV vaccine trials and possible barriers to participation. Methods Questionnaire survey of participants completing a 2-year community-based HIV Vaccine Preparedness Study, followed by cross sectional analysis of data. Results 95% of participants were willing to participate in a trial with similar attributes to the Vaccine Preparedness Study. Certain hypothetical trial attributes significantly reduced willingness to participate: The requirement to delay pregnancy (for females) had the largest effect, reducing willingness to participate from 97% to 23% (P < 0.0001). Larger blood draws had the second largest effect: 95-55% (P < 0.0001). The possibility of receiving either candidate vaccine or placebo had the third largest effect: 95-73% (P < 0.0001). Monthly study visits had the fourth largest effect: 95-92% (P < 0.0001). Trial duration longer than 2 years had the least effect: 95-93% (P = 0.0025). Combined attributes reduced willingness to participate from 95% to 43% (McNemar's chi(2) = 521.00; P < 0.0001) overall and 97-11% (McNemar's chi(2) = 531.00; P < 0.0001) for female participants. Physical harm concerns (adjusted OR = 34.9; 95% CI, 10.4-118) and a low risk behaviour index (adjusted OR = 0.09; 95% CI, 0.01-0.73) were associated with unwillingness to participate. Conclusions We found a high level of willingness to participate in HIV vaccine trials in this population. However, certain HIV vaccine trial requirements were associated with reduced willingness to participate. Community as well as individual concerns will have to be carefully addressed in planned HIV vaccine trials

Article Publication: Southeast Asian journal of tropical medicine and public health

Article Summary: According to the Joint UN Program on AIDS (UNAIDS), an estimated 4.9 million adults and children are living with HIV in Asia and the Pacific. Refinement and development of existing and new prevention and treatment technologies--including safe, effective, and accessible AIDS vaccines--are urgent public health priorities. The Asian region faces several challenges for AIDS vaccine development. There are multiple genetic variants of HIV-1 driving the epidemic in the region and too few vaccine candidates in the pipeline targeting those subtypes. Low HIV incidence throughout the region means that trial sites must recruit larger numbers of volunteers and shift their focus to higher-risk populations where incidence is higher. Also, the cultural, economic, and political diversity of the region may render collaboration very complex, but also beneficial at a regional level. Recognizing that collaborating as a region could foster and accelerate AIDS vaccine development, participants at the Sapporo International Consultation recommended that an AIDS Vaccine Asian Network (AVAN) be created to facilitate interactions between donors and funding opportunities, increase regional clinical trial and production capacity, support region-specific advocacy and communication strategies, contribute to the Global HIV Vaccine Enterprise Scientific Plan, prepare a regional approach for future vaccine deployment, and develop a regional platform for clinical trials including harmonized legal, regulatory, and ethical frameworks.

Article Publication: Biologicals

Article Summary: At the International AIDS Society Conference on Pathogenesis, Treatment and Prevention held in Sydney, Australia, in July 2007, the International AIDS Vaccine Initiative (IAVI) convened a satellite symposium entitled 'Accelerating the Development of Replicating Viral Vectors for AIDS Vaccines.' Its purpose was to highlight the rationale for accelerating the development of replicating viral vectors for use as vaccines against HIV-1, and to bring together vaccine scientists, regulatory officials, and public health specialists from industrialized and developing nations to discuss the major issues facing the development and testing of replicating viral vector-based vaccines.

Article Publication: AIDS research and human retroviruses

Article Summary: A novel prophylactic AIDS vaccine candidate, consisting of single-stranded DNA for HIV-1 subtype C gag, protease, and part of reverse transcriptase genes, enclosed within a recombinant adeno-associated virus serotype-2 protein capsid (tgAAC09) induced T cell responses and antibodies in nonhuman primates. In this randomized, dose escalation phase I trial, HIV-uninfected healthy volunteers (50 in Europe, 30 in India) received a single intramuscular injection of tgAAC09 at 3 x 10(9) DNase resistant particles (DRP) (n = 16), 3 x 10(10) DRP (n = 23), 3 x 10(11) DRP (n = 25), or placebo (n = 16). Twenty-one participants in Europe received a second (boost) dose of 3 x 10(11) DRP tgAAC09 or placebo at least 24 weeks after the first injection. The vaccine was safe and well-tolerated after initial and boost vaccination. Local and systemic reactogenicity was experienced by 13-25% of participants and was not dose related. No vaccine-related serious adverse events were reported. Modest HIV-specific T cell responses were detected in 7/64 vaccinees (40-385 SFC/10(6) PBMC), with 16% (4/25) responders in the highest dose group. All responses were to Gag epitopes. tgAAC09 appears to be safe, well-tolerated, and modestly immunogenic. Further evaluation of higher doses of tgAAC09 and boost injections is ongoing in Africa.

Article Publication: AIDS

Article Summary: This report summarizes the discussions and recommendations from a consultation held in New York City, USA (31 January-2 February 2006) organized by the joint World Health Organization-United Nations Programme on HIV/AIDS HIV Vaccine Initiative and the International AIDS Vaccine Initiative. The consultation discussed issues related to the design and implementation of phase IIB 'test of concept' trials (phase IIB-TOC), also referred to as 'proof of concept' trials, in evaluating candidate HIV vaccines and their implications for future approval and licensure. The results of a single phase IIB-TOC trial would not be expected to provide sufficient evidence of safety or efficacy required for licensure. In many instances, phase IIB-TOC trials may be undertaken relatively early in development, before manufacturing processes and capacity are developed sufficiently to distribute the vaccine on a large scale. However, experts at this meeting considered the pressure that could arise, particularly in regions hardest hit by AIDS, if a phase IIB-TOC trial showed high levels of efficacy. The group largely agreed that full-scale phase III trials would still be necessary to demonstrate that the vaccine candidate was safe and effective, but emphasized that governments and organizations conducting trials should consider these issues in advance. The recommendations from this meeting should be helpful for all organizations involved in HIV vaccine trials, in particular for the national regulatory authorities in assessing the utility of phase IIB-TOC trials in the overall HIV vaccine research and development process.

Article Publication: Lancet

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Article Publication: AIDS

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Article Publication: Journal of infectious diseases

Article Summary: BACKGROUND: We conducted a prospective study to evaluate methods of detecting clients with sexually transmitted diseases (STDs) who were acutely coinfected with human immunodeficiency virus (HIV) in Lilongwe, Malawi. METHODS: After informed consent was obtained, all clients with acute STDs were offered voluntary HIV counseling and testing by 2 rapid antibody tests. Samples from rapid test-negative or -discordant subjects were pooled (50 : 5 : 1) and tested for HIV RNA. Western blots were performed on all rapid test-discordant specimens with detectable HIV RNA. A subset of specimens received p24 antigen testing with standard and/or ultrasensitive methods. Patients with possible acute HIV infection were followed to confirm seroconversion. RESULTS: A total of 1450 clients (34% female and 66% male) agreed to testing, of whom 588 (40.55%) had established HIV infection and 21 (1.45%) had acute infection. Discordant rapid antibody tests identified 7 of 21 (33.3% sensitivity), standard p24 antigen identified 12 of 16 (75% sensitivity), and ultrasensitive p24 antigen identified 15 of 17 (88% sensitivity) acute cases. By definition, the sensitivity of the RNA assay was 100%. CONCLUSIONS: Real-time pooled RNA testing for the detection of acute HIV infection is feasible in resource-limited settings. However, parallel rapid testing and p24 antigen testing are technologically simpler and together may detect approximately 90% of acute cases.

Article Publication: Journal of acquired immune deficiency syndromes

Article Summary: A successful HIV vaccine would have a substantial impact on acquisition of infection, progression of disease among the infected, or infectiousness of the infected. Current vaccine candidates are anticipated to have their major effect on viremia, however, with the expectation that this would induce or be concordant with a reduced rate of AIDS, death, or infectiousness. Although direct assessment of disease progression or infectiousness may be impractical, available potential surrogates for these endpoints may be misleading. This article summarizes the proceedings of a National Institute of Allergy and Infectious Disease-sponsored workshop to explore the use of surrogate endpoints for licensure of an HIV vaccine. Early, medium, and late endpoints were discussed, along with challenges such as surrogate validity, the confounding effect of antiretroviral therapy initiation, and potential selection bias in the vaccine and placebo recipients who become infected. Results from 5 hypothetic HIV vaccine clinical trials with ambiguously successful results were presented to an expert panel for interpretation and discussion of next steps. Key recommendations included assessing magnitude and durability of surrogate effects, generalization across populations, and directed improvement of vaccines. Use of acquisition and a postinfection surrogate as coprimary endpoints was supported, along with use of composite endpoints and exploration of heterogeneity in vaccine efficacy by characteristics of the host and virus.

Article Publication: East African medical journal

Article Summary: Background: HIV rapid tests (RT) are a quick and non-technically demanding means to perform HIV voluntary counselling and testing (VCT) but understanding their limitations is vital to delivering quality VCT. Objective: To determine the sensitivity and specificity of HIV rapid tests used for research and voluntary counselling and testing at four sites in East Africa. Design: Cross-sectional study. Setting: Masaka District, Uganda; a sugar plantation in Kakira, Uganda; Coastal Villages in the Kilifi District of Kenya; and the Urban slum of Kangemi located West of Nairobi, Kenya. Subjects: Six thousands two hundred and fifty five consenting volunteers were enrolled into the study, and 675 prevalent HIV infections were identified. Results: The RT sensitivity tended to be high for all assays at all sites (97.63-100%) with the exception of the Uni-Gold assay (90.24% in Kangemi, 96.58% in Kilifi). Twenty four RT results were recorded as ‘weak positives', 22 (92%) of which were negative by ELISA. There was a high rate of RT false positives in Uganda (positive predictive values ranging from 45.70% to 86.62%).Conclusions: The sensitivity and specificity of the RT varied significantly across sites. The rate of RT misclassification in Uganda suggests that a multiple test algorithm may be preferable to a single test as screener for HIV VCT .

Article Publication: European journal of immunology

Article Summary: Induction of a long-term immunological memory, which can expand and defend the host upon pathogen encounter, is the "holy grail" of vaccinology. Here, using a sensitive cultured IFN-gamma ELISPOT assay, we show that 50% (15 out of 30) of healthy, HIV-1/2-uninfected volunteers who received pTHr.HIVA DNA prime-modified vaccinia virus Ankara. HIVA boost vaccine regimen 1 to 3 1/2 years ago had detectable HIV-1-specific T-cell responses. These T cells, predominantly of the CD4(+) subtype, could proliferate and produce multiple cytokines in response to in vitro peptide stimulation. Peptide mapping studies showed that the vaccine-induced CD4(+) T cells were mostly directed toward epitopes targeted in HIV-1-infected individuals. In addition, we used the same assay to re-evaluate 51 volunteers from past vaccine trial IAVI-006 and corrected the previously reported 10% of vaccine responders to 50%. Thus, we confirmed that cultured assays are a valuable tool for studying T-cell memory. These results are discussed in the context of the current state-of-affairs of the HIV-1 vaccine field.

Article Publication: Virology

Article Summary: Identifying the earliest neutralizing antibody specificities that are elicited following infection or vaccination by HIV-1 is an important objective of current HIV/AIDS vaccine research. We have shown previously that transplantation of HIV-1 V3 epitopes into an HIV-2 envelope (Env) scaffold provides a sensitive and specific means to detect and quantify HIV-1 V3 epitope specific neutralizing antibodies (Nabs) in human sera. Here, we employ this HIV-2/HIV-1 V3 scaffolding strategy to study the kinetics of development and breadth of V3-specific Nabs in longitudinal sera from individuals acutely infected with clade C or clade B HIV-1 and in human subjects immunized with clade B HIV-1 immunogens. HIV-2/HIV-1 chimeras containing V3 sequences matched to virus type (HIV-2 or HIV-1), subtype (clade B or C), or strain (autologous or heterologous) were used as test reagents. We found that by 3-8 weeks post infection, 12 of 14 clade C subjects had a median IC(50) V3-specific Nab titer of 1:700 against chimeric viruses containing a heterologous clade C V3. By 5 months post-infection, all 14 subjects were positive for V3-specific Nabs with median titers of 1:8000 against heterologous clade C V3 and 1:1300 against clade B V3. Two acutely infected clade B patients developed heterologous clade B V3-specific Nabs at titers of 1:300 and 1:1800 by 13 weeks of infection and 1:5000 and 1:11000 by 7 months of infection. Titers were not different against chimeras containing autologous clade B V3 sequences. Each of 10 uninfected normal human volunteers who were immunized with clade B HIV-1 Env immunogens, but none of five sham immunized control subjects, developed V3-specific Nabs titers as high as 1:3000 (median 1:1300; range 1:700-1:3000). None of the HIV-1 infected or vaccinated subjects had antibodies that neutralized primary HIV-1 virus strains. These results indicate that high-titer, broadly reactive V3-specific antibodies are among the first to be elicited during acute and early HIV-1 infection and following vaccination but these antibodies lack neutralizing potency against primary HIV-1 viruses, which effectively shield V3 from antibody binding to the functional Env trimer.

Article Publication: Journal of virology

Article Summary: Previous studies demonstrated that immunization of macaques with simian immunodeficiency virus (SIV) Gag-pol and Env recombinants of the attenuated poxvirus, modified vaccinia virus Ankara (MVA) provided protection from high viremia and AIDS following challenge with a pathogenic strain of SIV. Although all animals became infected, plasma viremia was significantly reduced in animals that received the MVA-SIV recombinant vaccines as compared with animals that received non-recombinant MVA. Most importantly, the reduction in viremia resulted in a significant increase in median and cumulative survival. In continued analysis of these animals over the subsequent 9 years, they maintain a survival advantage, although all but two of the macaques have progressed to AIDS. Importantly, improved survival correlated with preservation of memory CD4(+) T cells in the peripheral blood. The greatest survival advantage was observed in macaques immunized with regimens containing SIV Env and the titer of neutralizing antibodies to the challenge virus prior to or shortly following challenge correlated with preservation of CD4(+) T cells. These data are consistent with a role for neutralizing antibodies in non-sterilizing protection from high viremia and associated memory CD4(+) T cell loss.

Article Publication: Journal of virology

Article Summary: While mycobacteria have been proposed as vaccine vectors because of their persistence and safety, little has been done systematically to optimize their immunogenicity in nonhuman primates. We successfully generated rBCG expressing SIV Gag and Pol as multigenic, non-integrating vectors, but rBCG expressing SIV Env was unstable. A dose and route determination study in rhesus monkeys revealed that intramuscular administration of rBCG was associated with local reactogenicity; whereas intravenous and intradermal administration of 10(6) to 10(8) cfu rBCG was well-tolerated. Following single or repeat rBCG inoculations, monkeys developed high frequency BCG PPD IFNgamma ELISpot responses. However, those same animals developed only modest SIV-specific CD8(+) T cell responses. Nevertheless, high frequency SIV-specific cellular responses were observed in the rBCG-primed monkeys following boosting with rAd5 expressing the SIV antigens. These cellular responses were of greater magnitude and more persistent than those generated after vaccination with rAd5 alone. The vaccine-elicited cellular responses were predominantly polyfunctional CD8(+) T cells. These findings support the further exploration of mycobacteria as priming vaccine vectors.

Article Publication: AIDS

Article Summary: OBJECTIVES:: The p1 region of HIV-1 gag contains the frameshift stem-loop, gag-pol transframe and a protease cleavage site that are crucial for viral assembly, replication and infectivity. Identifying and characterizing CD8 epitopes that are under host immune selection in this region will help in designing effective vaccines for HIV-1. DESIGN:: An approach combining bioinformatical analysis and interferon gamma enzyme-linked immunosorbent spot (ELISPOT) assays is used to identify and characterize the epitopes. Potential human leukocyte antigen (HLA)-restricted epitopes were identified by correlating the positively-selected mutations with host HLA alleles. METHODS:: ELISPOT analysis with overlapping peptides was used to confirm and characterize the epitopes. RESULTS:: Four positively-selected residues were significantly associated with HLA class I alleles, including HLA B*1302 (K4R, P = 0.0008 and I5L, P = 0.0108), A*7401 (S9N, P = 0.0002) and A*30 genotypes (P7S, P = 0.009), suggesting epitopes restricted by these alleles are present in this region. ELISPOT analysis with patient peripheral blood mononuclear cells (PBMCs) identified seven novel epitopes restricted by the 3 alleles. Two types of epitopes were observed in this region based on the ELISPOT responses, Type I: the positively-selected variation does not affect CD8 T-cell responses; and Type II: the CD8 T-cell responses are determined by the epitope variants. CONCLUSION:: We identified and characterized seven novel CD8 epitopes in the p1 spacer protein region. Classifying the effects of positively-selected variants on CD8 T-cell responses will help in designing effective vaccines for HIV-1.

Article Publication: Biochemistry

Article Summary: The third variable region (V3) of the HIV-1 envelope glycoprotein gp120 is a target for virus neutralizing antibodies. The V3 sequence determines whether the virus will manifest R5 or X4 phenotypes and use the CCR5 or CXCR4 chemokine co-receptor, respectively. Previous NMR studies revealed that both R5- and X4-V3 peptides bound to antibodies 0.5beta and 447-52D form beta-hairpin conformations with the GPGR segment at the turn. In contrast, in their free form, linear V3 peptides and a cyclic peptide consisting of the entire 35-residue V3 loop were highly unstructured in aqueous solution. Herein we evaluated a series of synthetic disulfide constrained V3-peptides in which the position of the disulfide bonds, and therefore the ring size, was systematically varied. NMR structures determined for singly and doubly disulfide constrained V3-peptides in aqueous solution were compared with those found for unconstrained V3JRFL and V3IIIB peptides bound to 447-52D and to 0.5beta, respectively. Our study indicated that cyclic V3 peptides manifested significantly reduced conformational space compared to their linear homologues and that in all cases cyclic peptides exhibited cross strand interactions suggestive of beta-hairpin like structures. Nevertheless, the singly constrained V3-peptides retained significant flexibility and did not form an idealized beta-hairpin. Incorporation of a second disulfide bond results in significant overall rigidity and in one case, a structure close to that of V3MN peptide bound to 447-52D Fab was assumed and in another case a structure close to that formed by the linear V3IIIB peptide bound to antibody 0.5beta was assumed.

Article Publication: Chembiochem

Article Summary: Two HIV-1 recognition domains for the human monoclonal antibodies (MAb) 2F5, which recognises the core sequence ELDKWA, and 4E10, which recognises the core sequence NWFNIT, serve as promising models for immunogens in vaccine development against HIV-1. However, the failure of these recognition domains to generate broadly reactive neutralizing antibodies, and the putative membrane-binding properties of the antibodies raised to these recognition domains, suggest that additional features or recognition motifs are required to form an efficient immunogen, which could possibly include the membrane components. In this study we used an extended peptide epitope sequence derived from the gp41 native sequence (H-NEQELLELDKWASLWNWFNITNWLWYIK-NH), which contains the two recognition domains for 2F5 and 4E10, to examine the role of model cell (POPC) and viral (POPC/cholesterol/sphingomyelin) membranes in the recognition of these two antibodies. By using a surface plasmon resonance biosensor, the binding of 2F5 and 4E10 to membranes was compared and contrasted in the presence and absence of prebound peptide epitope. The recognition of the peptide epitope by each MAb was found to be distinct; 2F5 exhibited strong and almost irreversible binding to both membranes in the presence of the peptide, but bound weakly in the absence of the peptide epitope. In contrast, 4E10 exhibited strong membrane binding in the presence or absence of the peptide epitope, and the binding was essentially irreversible in the presence of the peptide epitope. Overall, these results demonstrate that both 2F5 and 4E10 can bind to membranes prior to epitope recognition, but that high-affinity recognition of gp41-derived epitope sequences by 2F5 and 4E10 occurs in a membrane context. Moreover, 4E10 might utilise the membrane to access and bind to gp41; such membrane properties of 2F5 and 4E10 could be exploited in immunogen design.

Article Publication: AIDS research and human retroviruses

Article Summary: Abstract Epitopes, also known as antigenic determinants, are small clusters of specific atoms within macromolecules that are recognized by the immune system. Such epitopes can be targeted with vaccines designed to protect against specific pathogens. The third variable loop (V3 loop) of the HIV-1 pathogen's gp120 surface envelope glycoprotein can be a highly sensitive neutralization target. We derived sequence motifs for the V3 loop epitopes recognized by the human monoclonal antibodies (mAbs) 447-52D and 2219. Searching the HIV database for the occurrence of each epitope motif in worldwide viruses and correcting the results based on published WHO epidemiology reveal that the 447-52D epitope we defined occurs in 13% of viruses infecting patients worldwide: 79% of subtype B viruses, 1% of subtype C viruses, and 7% of subtype A/AG sequences. In contrast, the epitope we characterized for human anti-V3 mAb 2219 is present in 30% of worldwide isolates but is evenly distributed across the known HIV-1 subtypes: 48% of subtype B strains, 40% of subtype C, and 18% of subtype A/AG. Various assays confirmed that the epitopes corresponding to these motifs, when expressed in the SF162 Env backbone, were sensitively and specifically neutralized by the respective mAbs. The method described here is capable of accurately determining the worldwide occurrence and subtype distribution of any crystallographically resolved HIV-1 epitope recognized by a neutralizing antibody, which could be useful for multivalent vaccine design. More importantly, these calculations demonstrate that globally relevant, structurally conserved epitopes are present in the sequence variable V3 loop.

Article Publication: Nature

Article Summary: Antibodies to conserved epitopes on the human immunodeficiency virus (HIV) surface protein gp140 can protect against infection in non-human primates, and some infected individuals show high titres of broadly neutralizing immunoglobulin (Ig)G antibodies in their serum. However, little is known about the specificity and activity of these antibodies. To characterize the memory antibody responses to HIV, we cloned 502 antibodies from HIV envelope-binding memory B cells from six HIV-infected patients with broadly neutralizing antibodies and low to intermediate viral loads. We show that in these patients, the B-cell memory response to gp140 is composed of up to 50 independent clones expressing high affinity neutralizing antibodies to the gp120 variable loops, the CD4-binding site, the co-receptor-binding site, and to a new neutralizing epitope that is in the same region of gp120 as the CD4-binding site. Thus, the IgG memory B-cell compartment in the selected group of patients with broad serum neutralizing activity to HIV is comprised of multiple clonal responses with neutralizing activity directed against several epitopes on gp120

Article Publication: Journal of virology

Article Summary: The magnitude and character of adenovirus type 5 (Ad5) -specific T cells was determined in volunteers with and without pre-existing neutralizing antibodies (NA) to Ad5 who received rAd5-based HIV vaccines. There was no correlation between T cell responses and NA to Ad5. There was no increase in magnitude or activation state of Ad5-specific CD4(+) T cells at time points where antibodies to Ad5 and T cell responses to the recombinant gene products could be measured. These data indicate that rAd5-based vaccines containing deletions in the E1, E3 and E4 region do not induce appreciable expansion of vector-specific CD4(+) T cells.

Article Publication: Nature

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Article Publication: Science

Article Summary: The spread of HIV between immune cells is greatly enhanced by cell-cell adhesions called virological synapses, although the underlying mechanisms have been unclear. With use of an infectious, fluorescent clone of HIV, we tracked the movement of Gag in live CD4 T cells and captured the direct translocation of HIV across the virological synapse. Quantitative, high-speed three-dimensional (3D) video microscopy revealed the rapid formation of micrometer-sized "buttons" containing oligomerized viral Gag protein. Electron microscopy showed that these buttons were packed with budding viral crescents. Viral transfer events were observed to form virus-laden internal compartments within target cells. Continuous time-lapse monitoring showed preferential infection through synapses. Thus, HIV dissemination may be enhanced by virological synapse-mediated cell adhesion coupled to viral endocytosis.

Article Publication: Proceedings of the national academy of sciences of the United States of America

Article Summary: HIV-1 latency in resting CD4(+) T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART). Eliminating the latent HIV-1 reservoir may require the reactivation of viral gene expression in latently infected cells. Most approaches for reactivating latent HIV-1 require nonspecific T cell activation, which has potential toxicity. To identify factors for reactivating latent HIV-1 without inducing global T cell activation, we performed a previously undescribed unbiased screen for genes that could activate transcription from the HIV-1 LTR in an NF-kappaB-independent manner, and isolated an alternatively spliced form of the transcription factor Ets-1, DeltaVII-Ets-1. DeltaVII-Ets-1 activated HIV-1 transcription through 2 conserved regions in the LTR, and reactivated latent HIV-1 in cells from patients on HAART without causing significant T cell activation. Our results highlight the therapeutic potential of cellular factors for the reactivation of latent HIV-1 and provide an efficient approach for their identification.

Article Publication: Journal of infectious diseases

Article Summary: Effective strategies for preventing human immunodeficiency virus infection are urgently needed, but recent failures in key clinical trials of vaccines and microbicides highlight the need for new approaches validated in relevant animal models. Here, we show that 2 new chemokine (C-C motif) receptor 5 inhibitors, 5P12-RANTES (regulated on activation, normal T cell expressed and secreted) and 6P4-RANTES, fully protect against infection in the rhesus vaginal challenge model. These highly potent molecules, which are amenable to low-cost production, represent promising new additions to the microbicides pipeline.

Article Publication: Pharmacoeconomics

Article Summary: BACKGROUND: An AIDS vaccine could play a very significant role in reversing the HIV pandemic, saving millions of lives. For a vaccine to have such an impact, it must be widely available and adopted and taken up rapidly in the countries most affected. A demand-forecasting model provides a valuable tool that can guide R&D spending decisions and identify policy actions to help achieve these goals. OBJECTIVE: To identify the key determinants of vaccine demand, model global adoption and uptake dynamics, estimate potential demand and revenues associated with future preventive AIDS vaccines, and to conduct sensitivity analyses to assess the impact of each parameter on demand. METHODS: A discrete, deterministic, linear, predictive mathematical model based on stratified population averages with a 30-year time horizon was developed to assess scenarios of future demand. This forecasting model was used to explore the effects of vaccine characteristics and a variety of regulatory, political, financial and health service factors on future demand and revenues. The intervention modelled was a preventive AIDS vaccine (efficacy: 30-90%; duration of protection: 3-5 years; in a two-dose prime-boost combination). The main outcome measure was the number of complete courses of vaccine administered. RESULTS: The model suggests that demand for a preventive AIDS vaccine with a medium efficacy (50%) and duration of protection (3 years) would average 68 million courses annually over a 30-year period. Under different scenarios, demand would peak at 38-152 million courses annually. On the basis of tiered pricing across public and private markets ($US2-100 per dose), these levels of peak demand would translate into $US2.5-5.5 billion in peak annual sales revenues. Private markets and high-income countries account for small volumes but large shares of projected revenues, while low-income countries account for large volumes and more modest, but still significant, sales revenues. Vaccinations to 'catch-up' those who are missed or not eligible for routine annual programmes (whether adolescent or high-risk populations) would account for 20-35% of cumulative vaccination courses across all scenarios. Demand was found to be sensitive to vaccine efficacy, duration of protection and price. Efforts to expedite regulatory review processes, improve immunization infrastructure and reduce political constraints could increase demand for an AIDS vaccine by 40 million additional courses a year compared with the medium efficacy (baseline) vaccine forecast. CONCLUSIONS: Our model can provide vaccine developers with credible estimates of market potential for an AIDS vaccine, and with a tool that can be used to improve forecasts over time as AIDS vaccine science progresses. It can also help governments to identify and pursue those policies that could best strengthen demand and uptake of a safe and effective preventive AIDS vaccine.

Article Publication: Virology

Article Summary: Although a live attenuated HIV vaccine is not currently considered for safety reasons, a strategy inducing both T cells and neutralizing antibodies to native assembled HIV-1 particles expressed by a replicating virus might mimic the advantageous characteristics of live attenuated vaccine. To this aim, we generated a live attenuated recombinant measles vaccine expressing HIV-1 Gag virus-like particles (VLPs) covered with gp160DeltaV1V2 Env protein. The measles-HIV virus replicated efficiently in cell culture and induced the intense budding of HIV particles covered with Env. In mice sensitive to MV infection, this recombinant vaccine stimulated high levels of cellular and humoral immunity to both MV and HIV with neutralizing activity. The measles-HIV virus infected human professional antigen-presenting cells, such as dendritic cells and B cells, and induced efficient presentation of HIV-1 epitopes and subsequent activation of human HIV-1 Gag-specific T cell clones. This candidate vaccine will be next tested in non-human primates. As a pediatric vaccine, it might protect children and adolescents simultaneously from measles and HIV.

Article Publication: Expert opinion on biological therapy

Article Summary: Background: Vaccines capable of eliciting cellular and humoral immune responses in tandem could provide prophylactic and therapeutic responses against infectious diseases and cancer. These responses can be induced systemically and at mucosal surfaces by activating the mucosal immune system, but rarely successfully due to challenges associated with mucosal delivery. Objective: To investigate delivery strategies to improve the effectiveness of mucosal vaccines. Methods: Different challenges are associated with different types of vaccines. We consider administration routes, schedules, carrier systems and adjuvants that can be used to overcome these challenges to enhance mucosal vaccination. Results/conclusions: The use of particle-mediated delivery systems is an effective strategy to enhance mucosal vaccination by protecting immunogenic material during delivery, providing targeted delivery systems, and allowing incorporation of adjuvant material

Article Publication: Journal of acquired immune deficiency syndromes

Article Summary: Despite the pressing need for an AIDS vaccine, the determinants of protective immunity to HIV remain concealed within the complexity of adaptive immune responses. We dissected immunodominant virus-specific CD8(+) T cell populations in Mamu-A*01(+) rhesus macaques with primary SIV infection to elucidate the hallmarks of effective immunity at the level of individual constituent clonotypes, which were identified according to the expression of distinct T cell receptors (TCRs). The number of public clonotypes, defined as those that expressed identical TCR beta-chain amino acid sequences and recurred in multiple individuals, contained within the acute phase CD8(+) T cell population specific for the biologically constrained Gag CM9 (CTPYDINQM; residues 181-189) epitope correlated negatively with the virus load set point. This independent molecular signature of protection was confirmed in a prospective vaccine trial, in which clonotype engagement was governed by the nature of the antigen rather than the context of exposure and public clonotype usage was associated with enhanced recognition of epitope variants. Thus, the pattern of antigen-specific clonotype recruitment within a protective CD8(+) T cell population is a prognostic indicator of vaccine efficacy and biological outcome in an AIDS virus infection.

Article Publication: Nature reviews. Microbiology

Article Summary: Ebola virus infection is a highly lethal disease for which there are no effective therapeutic or preventive treatments. Several vaccines have provided immune protection in laboratory animals, but because outbreaks occur unpredictably and sporadically, vaccine efficacy cannot be proven in human trials, which is required for traditional regulatory approval. The Food and Drug Administration has introduced the 'animal rule', to allow laboratory animal data to be used to show efficacy when human trials are not logistically feasible. In this Review, we describe immune correlates of vaccine protection against Ebola virus in animals. This research provides a basis for bridging the gap from basic research to human vaccine responses in support of the licensing of vaccines through the animal rule.

Article Publication: PLoS medicine

Article Summary: In June 2003, an international group of scientists proposed the creation of a Global HIV Vaccine Enterprise [1]. The authors invited discussion of this proposal, and challenged scientists to identify new strategies and mechanisms to accelerate the global effort to develop a safe and effective HIV vaccine. This paper describes the processes that led to agreement on the major roadblocks in HIV vaccine development, summarizes current scientific priorities, and describes an initial strategic approach to address those priorities. Specific research is not prescribed. Rather, the intent is to stimulate both researchers and funders to explore new, more collaborative, cooperative, and transparent approaches to address the major obstacles in HIV vaccine development identified in the plan, in addition to continuing the productive, high-quality programs already underway.

Article Publication: Journal of molecular biology

Article Summary: Yeast display is a powerful technology for the isolation of monoclonal antibodies against a target antigen. Antibody libraries have been displayed on the surface of yeast as both single chain variable (scFv) and antigen binding (Fab) fragments. Here, we combine these two formats to display well-characterized monoclonal antibodies as single chain Fabs (scFabs) on the surface of yeast and construct the first scFab yeast display antibody library. When expressed on the surface of yeast, two out of three anti-HIV-1 monoclonal antibodies bound with higher affinity as scFabs than scFvs. Also, the soluble scFab preparations exhibited binding and neutralization profiles comparable to that of the corresponding Fab fragments. Display of an immune HIV-1 scFab library on the surface of yeast, followed by rounds of sorting against HIV-1 gp120, allowed for the selection of thirteen antigen-specific clones. When the same cDNA was used to construct the library in an scFv format, a similar number, but a lower affinity set of clones were selected. Based on these results, yeast displayed scFab libraries can be constructed and selected with high efficiency, characterized without the need for a re-formatting step, and used to isolate higher affinity antibodies than scFv libraries.

Article Publication: Current opinion in HIV and AIDS

Article Summary: PURPOSE OF REVIEW: We will highlight recent advances in defining the attributes of immune responses that control AIDS virus replication using animal models, and also point out key gaps in our understanding that should be addressed in future research. RECENT FINDINGS: Many different vaccine approaches are currently being evaluated in animal models. Almost all of them elicit strong cellular or humoral immune responses in macaques. Commonly used prime-and-boost strategies have had varying degrees of success in diminishing chronic phase virus loads in vaccinated animals, but few have shown durable reduction in replication of the CCR5-tropic simian immunodeficiency viruses (SIVs) that most closely resemble field isolates of HIV. Investigators are therefore turning to other systems, including live attenuated vaccines and cohorts of spontaneous SIV controllers, to help identify the properties of successful host responses to pathogenic SIV. These responses likely incorporate multiple coordinated effector mechanisms. SUMMARY: There is no effective AIDS vaccine on the horizon. CD4+ and CD8+ T cell responses and antibodies have all been implicated in control of SIV replication seen in various experimental systems, but the correlates of protection against AIDS virus replication have not yet been definitively identified. Animal models will remain a necessary component of this research.

Article Publication: Current opinion in HIV and AIDS

Article Summary: PURPOSE OF REVIEW: Here we summarize evaluations of T cell immune responses in HIV vaccine clinical trials, highlighting investigations addressing optimization and validation of assays, reagents for determining multiclade HIV-specific responses, and current findings in phase I and II clinical testing. RECENT FINDINGS: Maintaining peripheral blood mononuclear cell quality and function is critical for optimal detection of T cell responses. Two recent reports emphasize the necessity for processing and freezing peripheral blood mononuclear cell within 8-12 h of venipuncture at local processing sites. Analytical designs of HIV peptide panels for use in T cell assays permit standardization of measurements between vaccines and laboratories, and evaluation of responses representing global HIV-1 strains. Phase I trials indicate superior induction of T cell response frequencies and magnitude using recombinant DNA and adenovirus serotype 5 (Ad5) vaccine with HIV-1 gene inserts. Additionally, use of a unique adjuvant with a recombinant Nef/Tat/gp120 vaccine induced high titer anti-HIV neutralizing antibodies, T cell proliferation, but not CD8 T cell responses. SUMMARY: T cell functional assays have achieved the standardization and validation to support licensure of vaccine candidates. Studies in progress will determine if these measurements are critical in defining correlates of immune protection.

Article Publication: PLoS medicine

Article Summary: In 2004, about 3 million people died of AIDS [1]. Because sexual intercourse accounts for the vast majority of HIV transmissions worldwide, it is important to understand the events that occur in the genital or rectal mucosa during transmission. Here we dissect a number of factors in the transmitter and recipient that are relevant before, during, and after transmission. We weigh the evidence suggesting that the transmitted virus differs from the virus that predominates in the transmitter. We discuss the prospect for protection by innate and adaptive immune mechanisms at mucosal surfaces as well as by locally applied inhibitors of viral replication, microbicides. A better understanding of sexual transmission will enable more rational designs of vaccines and microbicides and potential combinations of the two.

Article Publication: Current opinion in HIV and AIDS

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Article Publication: Topics in HIV medicine

Article Summary: Several interesting new vaccine-related studies were presented at the 16th Conference on Retroviruses and Opportunistic Infections this year. Transmitted viruses appear to be derived from cell-free virus rather than cell-associated virus, at least in men who have sex with men. Follow-up studies from the Step (HIV Vaccine Trials Network 502) trial indicated that if individuals mounted certain vaccine-induced responses, they may control viral replication after infection. Finally, adeno-associated-virus-derived neutralizing antibodies completely protected macaques from infection, suggesting novel mechanisms of viral control.

Article Publication: Proceedings of the National Academy of Sciences of the United States of America

Article Summary: Monoclonal antibodies b12 and 4E10 are broadly neutralizing against a variety of strains of the human immunodeficiency virus type 1 (HIV-1). The epitope for b12 maps to the CD4-binding site in the gp120 subunit of HIV-1's trimeric gp120-gp41 envelope spike, whereas 4E10 recognizes the membrane-proximal external region (MPER) of gp41. Here, we constructed and compared a series of architectures for the b12 and 4E10 combining sites that differed in size, valency, and flexibility. In a comparative analysis of the ability of the b12 and 4E10 constructs to neutralize a panel of clade B HIV-1 strains, we observed that the ability of bivalent constructs to cross-link envelope spikes on the virion surface made a greater contribution to neutralization by b12 than by 4E10. Increased distance and flexibility between antibody combining sites correlated with enhanced neutralization for both antibodies, suggesting restricted mobility for the trimeric spikes embedded in the virion surface. The size of a construct did not appear to be correlated with neutralization potency for b12, but larger 4E10 constructs exhibited a steric occlusion effect, which we interpret as evidence for restricted access to its gp41 epitope. The combination of limited avidity and steric occlusion suggests a mechanism for evading neutralization by antibodies that target epitopes in the highly conserved MPER of gp41.

Article Publication: Journal of virology

Article Summary: All HIV vaccine efficacy trials to date have ended in failure. Structural features of the Env glycoprotein and its enormous variability have frustrated efforts to induce broadly-reactive neutralizing antibodies. To explore the extent to which vaccine-induced cellular immune responses, in the absence of neutralizing antibodies, can control replication of a heterologous, mucosal viral challenge, we vaccinated eight macaques with a DNA/Ad5 regimen expressing all of the proteins of SIVmac239 except Env. Vaccinees mounted high-frequency T cell responses against 11-34 epitopes. We challenged the vaccinees and eight naïve animals with the heterologous biological isolate SIVsmE660, using a regimen intended to mimic typical human HIV exposures resulting in infection. Viral loads in the vaccinees were significantly less at both peak (1.9 log reduction p<0.03) and at set point (3.3 log reduction p<0.003) than those of control naïve animals. Five of eight vaccinated macaques controlled acute peak viral replication to less than 80,000 vRNA copy Eq/ml and to less than 100 vRNA copy Eq/ml in the chronic phase. Our results demonstrate that broad vaccine-induced cellular immune responses can effectively control replication of a pathogenic, heterologous AIDS virus, suggesting that T cell based vaccines may have greater potential than previously appreciated.

Article Publication: Journal of virology

Article Summary: Proteasomes are the major source of proteases responsible for the generation of peptides bound to MHC class I molecules. Antigens, adjuvants, and cytokines can modulate the composition and enzymatic activity of proteasomes and thus alter the epitopes generated. In the present study, we examined the effect of HIV-1 p24 on proteasomes from a dendritic cell line (JAWS II), a macrophage cell line (C2.3), and from murine primary bone marrow-derived macrophages and dendritic cells. HIV-1 p24 down-regulated PA28beta and the beta2i subunit of the immunoproteasome complex in JAWS II cells but did not decrease the immunoproteasomes subunits in macrophages, whereas in primary dendritic cells, PA28alpha, beta2i, and beta5i were down regulated. Exposure of JAWS II cells and primary dendritic cells to HIV-1 p24 for 90 min significantly decreased the presentation of ovalbumin to a SIINFEKL-specific CD8(+) T cell hybridoma. The decrease in antigen presentation and the down modulation of the immunoproteasome subunits in JAWS II cells and primary dendritic cells could be overcome by pre-treating the cells with IFN-gamma for 6 h or by exposing the cells to HIV-1 p24 encapsulated in liposomes containing lipid A [L(p24 + LA)]. These results suggest that early antigen processing kinetics could influence the immunogenicity of CD8(+) T cell epitopes generated.

Article Publication: PLOS One

Article Summary: BACKGROUND: Generation of robust cell-mediated immune responses at mucosal surfaces while reducing overall inflammation is a primary goal for vaccination. Here we report the use of a recombinant nanoparticle as a vaccine delivery platform against mucosal infections requiring T cell-mediated immunity for eradication. METHODOLOGY/PRINCIPAL FINDINGS: We encapsulated an immunogenic protein, the major outer membrane protein (MOMP) of Chlamydia muridarum, within hollow, vault nanocapsules (MOMP-vaults) that were engineered to bind IgG for enhanced immunity. Intranasal immunization (i.n) with MOMP-vaults induced anti-chlamydial immunity plus significantly attenuated bacterial burden following challenge infection. Vault immunization induced anti-chlamydial immune responses and inflammasome formation but did not activate toll-like receptors. Moreover, MOMP-vault immunization enhanced microbial eradication without the inflammation usually associated with adjuvants. CONCLUSIONS/SIGNIFICANCE: Vault nanoparticles containing immunogenic proteins delivered to the respiratory tract by the i.n. route can act as "smart adjuvants" for inducing protective immunity at distant mucosal surfaces while avoiding destructive inflammation

Article Publication: Clinical pharmacology and therapeutics

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Article Publication: PLoS medicine

Article Summary: Global clinical laboratory work performed under harmonized operations is a central component for the successful conduct of phase I–III clinical trials in multiple fields of science and medicine. However, global harmonization of clinical laboratories for the analysis of specimens from clinical trials operations (i.e., for safety, diagnostic, endpoint laboratory assays) faces international challenges (e.g., laboratory logistical and technical factors), and it is subject to different interpretations of regulations and guidance materials published by the federal government, accrediting, and non-accrediting organizations (e.g., Good Laboratory Practice [GLP] [1], Clinical Laboratory Improvement Amendments [CLIA] [2], College of American Pathologists [3], International Organization for Standardization [ISO] 15189 [4], and International Conference on Harmonization [ICH] Good Clinical Practice [GCP] [5]). In an effort to harmonize and gain consensus on international clinical laboratory operations, Good Clinical Laboratory Practice (GCLP) guidelines were originated by merging GLP and ICH-GCP principles, and were first published and copyrighted by the British Association of Research Quality Assurance (BARQA) (BARQA-GCLP) [6]. Subsequently, the Division of AIDS (DAIDS), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health expanded the existing knowledge on GCLP standards by publishing guidelines on GCLP (NIAID-GCLP) [7], with increased implementation guidance based on applicable portions of GLP, CLIA, the College of American Pathologists, and the International Organization for Standardization (ISO 15189). Both of these GCLP approaches were created to ensure that clinical laboratory results are reliable, repeatable, auditable, and comparable between multiple clinical laboratories. Nevertheless, differences in the implementation of GCLP by clinical laboratories have created critical inconsistencies for routine management of operations in support of clinical trials and have caused an urgent need to clarify and harmonize four central GCLP elements for optimal management and clinical laboratory operations. These GCLP elements—discussed in this paper—are training, auditing, assay validation, and proficiency testing. The differences regarding the implementation of universal standards of GCLP for clinical laboratory operations (i.e., clinical laboratories performing safety, diagnostic, and endpoint assays) in the conduct of clinical trials have been experienced in the HIV study field. However, it is expected that this problem will have broader implications in clinical trials, involving multiple fields. This paper addresses for the first time an attempt to harmonize these GCLP approaches into a single set of recommendations for optimal operations and management that can be followed by clinical laboratories, not only in the HIV field, but also possibly in other science and medical fields.

Article Publication: Journal of virology

Article Summary: Effective vaccines for human immunodeficiency virus-1 (HIV-1) will likely need to stimulate protective immunity in the intestinal mucosa, where HIV-1 infection causes severe CD4(+) T cell depletion. While replication-competent adenoviral vectors (rAd) can stimulate Ad-specific mucosal immunity after replication, oral delivery of replication-defective rAd vectors encoding specific immunogens has proven challenging. In this study, we have systematically identified barriers to effective gut delivery of rAd vectors and identify sites and strategies to induce potent cellular and humoral immunity. Vector-mediated gene transfer by rAd5 was susceptible to low pH buffer, gastric and pancreatic proteases, and extracellular mucins. Using ex vivo organ explants, we found that transduction with rAd5 was highest in the ileum and colon compared to other intestinal segments. Transgene expression was 100-fold higher after direct surgical introduction into the ileum than observed with oral gavage, with rAd5 showing greater potency than rAd35 or rAd41 vectors. A single immunization of rAd5 encoding HIV-1 gp140B to the ileum stimulated potent CD8(+) T cell responses in the intestinal and systemic compartments, and these responses were further enhanced by intramuscular rAd5 boosting. These studies suggest that induction of primary immune responses by rAd5 gut immunization elicits potent antigen-specific mucosal responses after subsequent systemic boosting.

Article Publication: Journal of immunology

Article Summary: In the phase IIb STEP trial an HIV-1 vaccine based on adenovirus (Ad) vectors of the human serotype 5 (AdHu5) not only failed to induce protection but also increased susceptibility to HIV-1 infection in individuals with preexisting neutralizing Abs against AdHu5. The mechanisms underlying the increased HIV-1 acquisition rates have not yet been elucidated. Furthermore, it remains unclear if the lack of the vaccine's efficacy reflects a failure of the concept of T cell-mediated protection against HIV-1 or a product failure of the vaccine. Here, we compared two vaccine regimens based on sequential use of AdHu5 vectors or two different chimpanzee-derived Ad vectors in rhesus macaques that were AdHu5 seropositive or seronegative at the onset of vaccination. Our results show that heterologous booster immunizations with the chimpanzee-derived Ad vectors induced higher T and B cell responses than did repeated immunizations with the AdHu5 vector, especially in AdHu5-preexposed macaques.

Article Publication: Mucosal immunology

Article Summary: HIV prevention mandates an understanding of the mechanisms of mucosal immunity with attention to some unique features of the epidemic and mucosal environment in the developing world. An effective vaccine will have to induce mucosal protection against a highly diverse virus, which is equipped with a number of immune evasion strategies. Its development will require assessment of mucosal immune responses, and it will have to protect a mucosal environment where inflammation and altered immune responses are common because of the presence of other mucosal infections, such as sexually transmitted infections and parasites, and where nutritional status may also be compromised. Ideally, not only prevention methods would protect adults but also provide cover against gastrointestinal transmission through maternal milk. Prevention might also be complemented by microbicides and circumcision, two alternative approaches to mucosal protection. It seems unlikely that a single solution will work in all instances and intervention might have to act at multiple levels and be tailored to local circumstances. We review here some of the mucosal events associated with HIV infection that are most relevant in an African setting.

Article Publication: AIDS

Article Summary: OBJECTIVE: To determine the spectrum of antiviral antibodies in HIV-1-infected individuals in whom viral replication is spontaneously undetectable, termed HIV controllers (HICs). DESIGN: Multicenter French trial ANRS EP36 studying the viral control in HICs. METHODS: Neutralizing Antibody (nAb) activities (neutralization assay, competition with broadly reactive monoclonal antibodies, and reactivity against the viral MPER gp41 region), FcgammaR-mediated antiviral activities, antibody-dependent cell cytotoxicity (ADCC), as well as autoantibody levels, were quantified in plasma from 22 controllers and from viremic individuals. The levels of these different antibody responses and HIV-specific CD8 T cell responses quantified by enzyme-linked immunosorbent spot (ELISPOT) IFNgamma assay were compared in each controller. RESULTS: The levels of antibody against the gp120 CD4 binding site, gp41, as well as Env epitopes near to the sites bound by broadly nAbs 2F5 and 1b12 were not different between HICs and viremic individuals. We did not find significant autoantibody levels in HICs. The magnitude and breadth of nAbs were heterogeneous in HICs but lower than in viremic individuals. The levels of nAbs using FcgammaR-mediated assay inhibition were similar in both groups. Regardless of the type of antibody tested, there was no correlation with HIV-specific CD8 T cell responses. ADCC was detectable in all controllers tested and was significantly higher than in viremic individuals (P < 0.0002). CONCLUSION: There was no single anti-HIV-1 antibody specificity that was a clear correlate of immunity in controllers. Rather, for most antibody types, controllers had the same or lower levels of nAbs than viremic individuals, with the possible exception of ADCC antibodies.

Article Publication: AIDS

Article Summary: OBJECTIVE: To determine the mechanism of interaction between the HIV-1 gp41-specific broadly neutralizing monoclonal antibody (mAb) 2F5, its epitope in the membrane proximal external region and a domain located in the fusion peptide proximal region in the N-terminal region of gp41. Knowledge of these interactions would be useful for the design of antigens used to induce 2F5-like antibodies. METHODS: The binding and avidity of the mAb 2F5 were analyzed using enzyme-linked immunosorbent assays, epitope mapping and surface plasmon resonance analysis. Inhibition of virus neutralization by 2F5 was analyzed using peptides corresponding to the gp41 sequence. RESULTS: Using transmembrane envelope proteins of gammaretroviruses, we had previously induced neutralizing antibodies that recognize two epitopes, one located in the N-terminal part of the transmembrane protein (designated E1) and the other in the C-terminal membrane proximal external region (E2). The E2 epitope corresponds to the mAb 2F5/4E10 epitope in the gp41 of HIV and we have now identified a corresponding E1 domain in gp41. Although 2F5 did not bind directly to E1, the presence of E1 peptides increased the binding of 2F5 to peptides carrying its epitope. Neutralization of HIV-1 by 2F5 was inhibited more effectively by both gp41-derived peptides E1 and E2 together than with the peptide E2 alone. CONCLUSION: The interaction between the E1 and E2 domains of gp41 increased the efficacy of mAb 2F5 binding to its E2 epitope. Such an interaction may occur after gp41 folding into a six-helix bundle. Antigens containing both domains might be used to induce broadly neutralizing 2F5-like antibodies.

Article Publication: Journal of virology

Article Summary: The development of a rapid and efficient system to identify HIV-1 infected individuals with broad and potent HIV-1 specific neutralizing antibody responses is an important step toward the discovery of critical neutralization targets for rational AIDS vaccine design. In this study, samples from HIV-1 infected volunteers from diverse epidemiological regions were screened for neutralization responses using pseudovirus panels composed of clades A, B, C, D and circulating recombinant forms (CRFs). Initially, 463 sera and plasma samples from Australia, Rwanda, Uganda, the United Kingdom, and Zambia were screened to explore neutralization patterns and selection ranking algorithms. Samples were identified that neutralized representative isolates from at least 4 clade/CRF groups with titers above pre-specified thresholds, and ranked based on a weighted average of their log-transformed neutralization titers. Linear regression methods selected a 5 pseudovirus subset, representing clades A, B, C, and one CRF01_AE, that could identify samples with IC50 neutralization titers >/=100 to at least 4 clade groups. This reduced panel was then used to screen 1,234 new samples from the Ivory Coast, Kenya, South Africa, Thailand, and the United States and 1% were identified as elite neutralizers. These elite neutralizers provide promising starting material for the isolation of broadly neutralizing monoclonal antibodies to assist in HIV-1 vaccine design.

Article Publication: PLoS pathogens

Article Summary: Developing an immunogen that elicits broadly neutralizing antibodies (bNAbs) is an elusive but important goal of HIV vaccine research, especially after the recent failure of the leading T cell based HIV vaccine in human efficacy trials. Even if such an immunogen can be developed, most animal model studies indicate that high serum neutralizing concentrations of bNAbs are required to provide significant benefit in typical protection experiments. One possible exception is provided by the anti-glycan bNAb 2G12, which has been reported to protect macaques against CXCR4-using SHIV challenge at relatively low serum neutralizing titers. Here, we investigated the ability of 2G12 administered intravenously (i.v.) to protect against vaginal challenge of rhesus macaques with the CCR5-using SHIV(SF162P3). The results show that, at 2G12 serum neutralizing titers of the order of 1ratio1 (IC(90)), 3/5 antibody-treated animals were protected with sterilizing immunity, i.e. no detectable virus replication following challenge; one animal showed a delayed and lowered primary viremia and the other animal showed a course of infection similar to 4 control animals. This result contrasts strongly with the typically high titers observed for protection by other neutralizing antibodies, including the bNAb b12. We compared b12 and 2G12 for characteristics that might explain the differences in protective ability relative to neutralizing activity. We found no evidence to suggest that 2G12 transudation to the vaginal surface was significantly superior to b12. We also observed that the ability of 2G12 to inhibit virus replication in target cells through antibody-mediated effector cell activity in vitro was equivalent or inferior to b12. The results raise the possibility that some epitopes on HIV may be better vaccine targets than others and support targeting the glycan shield of the envelope.

Article Publication: Journal of virology

Article Summary: The global spread of HIV and the human suffering left in its wake have made AIDS a global heath priority. ...

Article Publication: Vaccine

Article Summary: Recombinant vesicular stomatitis viruses (rVSVs) are being developed as potential HIV-1 vaccine candidates. To characterize the in vivo replication and dissemination of rVSV vectors in mice, high doses of a highly attenuated vector expressing HIV-1 Gag, rVSV(IN)-N4CT9-Gag1, and a prototypic reference virus, rVSV(IN)-HIVGag5, were delivered intramuscularly (IM), intranasally (IN), or intravenously (IV). We used quantitative, real-time RT-PCR (Q-PCR) and standard plaque assays to measure the temporal dissemination of these viruses to various tissues. Following IM inoculation, both viruses were detected primarily at the injection site as well as in draining lymph nodes; neither virus induced significant weight loss, pathologic signs, or evidence of neuroinvasion. In contrast, following IN inoculation, the prototypic virus was detected in all tissues tested and caused significant weight loss leading to death. IN administration of rVSV(IN)-N4CT9-Gag1 resulted in detection in numerous tissues (brain, lung, nasal turbinates, and lymph nodes) albeit in significantly reduced levels, which caused little or no weight loss nor any mortality. Following IV inoculation, both prototypic and attenuated viruses were detected by Q-PCR in all tissues tested. In contrast to the prototype, rVSV(IN)-N4CT9-Gag1 viral loads were significantly lower in all organs tested, and no infectious virus was detected in the brain following IV inoculation, despite the presence of viral RNA. These studies demonstrated significant differences in the biodistribution patterns of and the associated pathogenicity engendered by the prototypic and attenuated vectors in a highly susceptible host.

Article Publication: Immunology and cell biology

Article Summary: There is a logarithmic increase in the cost and complexity of the research and development process when transitioning a promising candidate vaccine from the laboratory into the clinic. Managing complex development programs involving people from diverse technical, cultural and geographical backgrounds is a specialised skill. It is essential that the group is clear on their objectives and how their activities affect others, that communication is open, inclusive and effective, and that the most rigorous, scientific approach based on statistical principles in compliance with regulatory requirements is used. Applying these standards to all vaccine development programs will filter out inappropriate candidates more readily and enhance the efficiency of vaccine development. The challenges of developing a new vaccine are illustrated in human immunodeficiency virus (HIV) vaccinology. Selecting vaccine candidates for HIV requires the ability to evaluate the large number of potential antigens in imperfect and non-standardised animal models. Further, using these models to evaluate questions such as dose scaling to humans, optimal route of administration, the use of adjuvants and potential formulation improvements adds variable to variable, making the interpretation of results particularly challenging. This may lead to the promotion of a poor candidate or the elimination of a good one. The absence of precise immunological correlates of protection and the prohibitive cost of confirmatory clinical trials are further significant barriers. However, there are practical steps that can be taken to standardise early vaccine evaluation, which would result in more efficient development of new vaccines for HIV and other disease areas with similarly challenging development issues (such as hepatitis C virus, influenza, Mycobacterium tuberculosis and malaria).Immunology and Cell Biology advance online publication, 12 May 2009; doi:10.1038/icb.2009.30.

Article Publication: Proceedings of the National Academy of Sciences of the United States of America

Article Summary: A vaccine capable of stimulating protective antiviral antibody responses is needed to curtail the global AIDS epidemic caused by HIV-1. Although rarely elicited during the course of natural infection or upon conventional vaccination, the membrane-proximal ectodomain region (MPER) of the HIV-1 glycoprotein of M(r) 41,000 (gp41) envelope protein subunit is the target of 3 such human broadly neutralizing antibodies (BNAbs): 4E10, 2F5, and Z13e1. How these BNAbs bind to their lipid-embedded epitopes and mediate antiviral activity is unclear, but such information might offer important insight into a worldwide health imperative. Here, EPR and NMR techniques were used to define the manner in which these BNAbs differentially recognize viral membrane-encrypted residues configured within the L-shaped helix-hinge-helix MPER segment. Two distinct modes of antibody-mediated interference of viral infection were identified. 2F5, like 4E10, induces large conformational changes in the MPER relative to the membrane. However, although 4E10 straddles the hinge and extracts residues W672 and F673, 2F5 lifts up residues N-terminal to the hinge region, exposing L669 and W670. In contrast, Z13e1 effects little change in membrane orientation or conformation, but rather immobilizes the MPER hinge through extensive rigidifying surface contacts. Thus, BNAbs disrupt HIV-1 MPER fusogenic functions critical for virus entry into human CD4 T cells and macrophages either by preventing hinge motion or by perturbing MPER orientation. HIV-1 MPER features important for targeted vaccine design have been revealed, the implications of which extend to BNAb targets on other viral fusion proteins.

Article Publication: Nature immunology

Article Summary: New modes of humoral recognition have been identified by studies of antibodies that neutralize human immunodeficiency virus type 1 and influenza A viruses. Understanding how such modes of antibody-antigen recognition can occur in the context of sophisticated mechanisms of humoral evasion has implications for the development of effective vaccines. Here we describe eight modes of antibody recognition first observed with human immunodeficiency virus type 1. Similarities to four of these modes have been identified with antibodies to a conserved 'stem' epitope on influenza A viruses. We outline how each of these different modes of antibody recognition is particularly suited to overcoming a specific viral evasion tactic and assess potential routes of re-elicitation in vaccine settings

Article Publication: Journal of virology

Article Summary: There is an urgent need for HIV vaccines that induce robust mucosal immunity. Influenza A viruses (both H1N1 and H3N2) were engineered to express SIV CD8 T cell epitopes and evaluated following administration to the respiratory tract of 11 pigtail macaques. Influenza virus was readily detected from respiratory tract secretions although the infections were asymptomatic. Animals seroconverted to influenza virus and generated CD8 and CD4 T cell responses to influenza virus proteins. SIV-specific CD8 T cell responses bearing the mucosal homing marker beta7 integrin were induced by vaccination of naive animals. Further, SIV-specific CD8 T cell responses could be boosted by recombinant influenza-SIV vaccination of animals with already established SIV infection. Sequential vaccination with influenza-SIV recombinants of different subtypes (H1N1 followed by H3N2 or vice versa) produced only a limited boost in immunity, probably reflecting T cell immunity to conserved internal proteins of influenza A virus. SIV challenge of macaques vaccinated with an influenza virus expressing a single SIV CD8 T cell resulted in a large anamnestic recall CD8 T cell response but immune escape rapidly ensued and there was no impact on chronic SIV viremia. Although our results suggest influenza-HIV vaccines hold promise for the induction of mucosal immunity to HIV, broader antigen cover will be needed to limit CTL escape.

Article Publication: Vaccine

Article Summary: The attenuated poxvirus vectors MVA and NYVAC are now in clinical trials against HIV/AIDS. Due to the vectors restricted replication capacity in human cells, approaches to enhance their immunogenicity are highly desirable. Here, we have analyzed the ability of a soluble form of hexameric CD40L (sCD40L) to stimulate specific immune responses to HIV antigens when inoculated in mice during priming with DNA and in the booster with MVA or NYVAC, expressing the vectors HIV-1 Env, Gag, Pol and Nef antigens from clade B. Our findings revealed that sCD40L in DNA/poxvirus combination enhanced the magnitude about 2-fold (DNA-B/MVA-B) and 4-fold (DNA-B/NYVAC-B), as well as the breath of the HIV antigen specific cellular immune responses. sCD40L was necessary in both prime and boost inoculations triggering a potent polarization of the Th response towards a Th1 type. In DNA-B/NYVAC-B regime the addition of sCD40L significantly enhanced the humoral immune response against HIV gp160, but not in DNA-B/MVA-B combination. These findings provided evidence for the immunostimulatory benefit of sCD40L when DNA and the poxvirus vectors MVA and NYVAC are used as immunogens.

Article Publication: PLoS pathogens ‎

Article Summary: The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein, gp120, possesses conserved binding sites for interaction with the primary virus receptor, CD4, and also for the co-receptor, generally CCR5. Although gp120 is a major target for virus-specific neutralizing antibodies, the gp120 variable elements and its malleable nature contribute to evasion of effective host-neutralizing antibodies. To understand the conformational character and immunogenicity of the gp120 receptor binding sites as potential vaccine targets, we introduced structure-based modifications to stabilize gp120 core proteins (deleted of the gp120 major variable regions) into the conformation recognized by both receptors. Thermodynamic analysis of the re-engineered core with selected ligands revealed significant stabilization of the receptor-binding regions. Stabilization of the co-receptor-binding region was associated with a marked increase in on-rate of ligand binding to this site as determined by surface plasmon resonance. Rabbit immunization studies showed that the conformational stabilization of core proteins, along with increased ligand affinity, was associated with strikingly enhanced humoral immune responses against the co-receptor-binding site. These results demonstrate that structure-based approaches can be exploited to stabilize a conformational site in a large functional protein to enhance immunogenic responses specific for that region.

Article Publication: Vaccine

Article Summary: The presence of vector-specific immune responses may hamper the induction of responses to a foreign antigen encoded by the vector. We evaluated the impact of pre-existing immunity to vaccinia virus on the induction of HIV-specific responses after immunization of healthy volunteers with a HIV-1 DNA prime-MVA boost vaccine. Following three priming immunizations with HIV-1 DNA plasmids, the volunteers were boosted with a single injection of recombinant MVA encoding HIV-1 proteins. Pre-existing immunity to vaccinia virus did not reduce the proportion of individuals who responded to HIV-1, but did lower the magnitude of responses. Our results suggest that vaccinia-based vectors can be used to efficiently induce immune responses to vectored HIV-1 antigens, even in individuals with pre-existing immunity to vaccinia virus.

Article Publication: Journal of virology

Article Summary: The HIV-1 variants that are transmitted to newly infected individuals are the primary targets of interventions such as vaccines and microbicides aimed at preventing new infections. Newly acquired subtype A, B, and C variants have been the focus of neutralization studies, although many of these viruses, particularly from subtypes A and B, represent viruses circulating more than a decade ago. In order to better represent the global diversity of transmitted HIV-1 variants, an additional thirty-one sexually transmitted Kenyan HIV-1 envs representing several recent infections with subtype A, as well as subtypes A/D, C, and D were cloned and their neutralization profile was characterized. Most envs were resistant to neutralization by the monoclonal antibodies (MAbs) b12, 4E10, 2F5, and 2G12, suggesting that targeting the epitopes of these MAbs may not be effective against variants that are spreading in endemic areas. However, significant cross-subtype neutralization by plasma was observed, indicating that there may be other epitopes, not yet defined by the limited available MAbs, which could be recognized more broadly.

Article Publication: Journal of virology

Article Summary: Identification of the determinants of sensitivity and resistance to broadly neutralizing antibodies is a high priority for HIV research. Analysis of the swarm of closely related envelope protein variants in HIV infected individuals revealed a mutation that markedly affected sensitivity to neutralization by antibodies and antiviral entry inhibitors targeting both gp41 and gp120. This mutation mapped to the C34 helix of gp41 and disrupted an unexplored structural feature consisting of a ring of hydrogen bonds in the gp41 trimer. This mutation appeared to affect the assembly of the 6 helix bundle required for virus fusion and to alter the conformational equilibria so as to favor the pre-hairpin intermediate conformation required for the binding of the MPER specific neutralizing antibodies, 2F5 and 4E10, and the antiviral drug, Fuzeon. The "swarm analysis" method we describe furthers our understanding of the relationship between the structure, function and antigenicity of the HIV envelope protein and represents a new approach to the identification of vaccine antigens

Article Publication: Expert review of vaccines

Article Summary: The first clinical evaluations of adenovirus (Ad)-based vectors for gene therapy were initiated in the mid-1990s and led to great anticipation for future utility. However, excitement surrounding gene therapy, particularly Ad-based therapy, was diminished upon the death of Jesse Gelsinger, and recent discouraging results from the HIV vaccine STEP trial have brought efficacy and safety issues to the forefront again. Even so, Ad vectors are still considered among the safest and most effective vaccine vectors. Innate and pre-existing immunity to Ad mediate much of the acute toxicities and reduced therapeutic efficacies observed following vaccination with this vector. Thus, innovative strategies must continue to be developed to reduce Ad-specific antigenicity and immune recognition. This review provides an overview and critique of the most promising strategies, including results from preclinical trials in mice and nonhuman primates, which aim to revive the future of Ad-based vaccines.

Article Publication: Science

Article Summary:

Article Publication: Journal of virology

Article Summary: The membrane proximal external region (MPER) of the HIV envelope glycoprotein (gp41) is critical for viral fusion and infectivity and is the target of three of the five known broadly neutralizing HIV-1 antibodies, 2F5, Z13, and 4E10. Here we report the crystal structure of the Fab fragment of Z13e1, an affinity-enhanced variant of monoclonal antibody Z13, in complex with a 12-residue peptide corresponding to the core epitope (W(670)NWFDITN(677)) at 1.8 A resolution. The bound peptide adopts an S-shaped conformation composed of two tandem, perpendicular helical turns. This conformation differs strikingly from the alpha-helical structure adopted by an overlapping MPER peptide bound to 4E10. Z13e1 binds to an 'elbow' in the MPER at the membrane interface making relatively few interactions with conserved aromatics (Trp672 and Phe673) that are critical for 4E10 recognition. Comparison of the Z13e1 and 4E10 epitope structures reveals a conformational switch such that neutralization can occur by recognition of different conformations and faces of the largely amphipathic MPER. The Z13e1 structure provides significant new insights into the dynamic nature of the MPER, which is likely critical for membrane fusion, and has significant implications for mechanisms of HIV-1 neutralization by MPER antibodies and for design of HIV-1 immunogens.

Article Publication: Journal of virology

Article Summary: In this study we examined whether HIV-1 is equally susceptible to neutralization by a given antibody when the epitope of this antibody is introduced at different positions within the viral Envelope glycoprotein (Env). To this end we introduced two exogenous 'epitope tags' in different locations within three major Env regions in two distinct HIV-1 isolates. We examined how the introduction of the exogenous epitopes affects Env-expression, Env-incorporation into virions, Env fusogenic potential and viral neutralization susceptibility. Our data indicate that even within the same Env region, the exact positioning of the epitope impacts the neutralization susceptibility of the virus by the antibody that binds to that epitope. Our data also indicate that even if the same epitope is introduced in the exact same position on two different Envs, its exposure, and as a result the neutralization susceptibility of the virus, can be very different. In contrary to previous studies conducted with HIV-1 isolates, other than the ones used here, but similarly to results obtained with SIV, we observed that tagging the fourth variable region of Env (V4) did not result in neutralization by the anti-tag antibodies. Our data indicate that epitopes in V4 are not properly exposed within the functional HIV-1 trimeric Env spike and suggest that V4 may not be a good target for vaccine-elicited NAbs.

Article Publication: Nature medicine

Article Summary: Most existing viral vaccines generate antibodies that either block initial infection or help eradicate the virus before it can cause disease. For HIV-1, obstacles to eliciting protective neutralizing antibodies (NAbs) have often seemed insurmountable. The target of HIV-specific NAbs, the viral envelope glycoprotein (Env), is highly variable in amino acid sequence and glycosylation pattern. Conserved elements of HIV-1 Env seem to be poorly immunogenic, and previous attempts to generate broadly reactive NAbs by vaccination have proven ineffective. However, recent studies show that antibodies in the sera of some HIV-1–infected individuals can neutralize diverse HIV-1 isolates. Detailed analyses of these sera provide new insights into the viral epitopes targeted by broadly reactive NAbs. The findings discussed here suggest that the natural NAb response to HIV-1 can inform future vaccine design. A concerted effort of structure-based vaccine design will help guide the development of improved antibody-based vaccines for HIV-1

Article Publication: Nature medicine

Article Summary: Neutralizing antibodies are thought to be crucial for HIV vaccine protection, but studies in animal models suggest that high antibody concentrations are required. This is a major potential hurdle for vaccine design. However, these studies typically apply a large virus inoculum to ensure infection in control animals in single-challenge experiments. In contrast, most human infection via sexual encounter probably involves repeated exposures to much lower doses of virus. Therefore, animal studies may have provided an overestimate of the levels of antibodies required for protection in humans. We investigated whether plasma concentrations of antibody corresponding to relatively modest neutralization titers in vitro could protect macaques from repeated intravaginal exposure to low doses of a simian immunodeficiency virus-HIV chimera (SHIV) that uses the CC chemokine receptor 5 (CCR5) co-receptor. An effector function-deficient variant of the neutralizing antibody was also included. The results show that a substantially larger number of challenges is required to infect macaques treated with neutralizing antibody than control antibody-treated macaques, and support the notion that effector function may contribute to antibody protection. Overall, the results imply that lower amounts of antibody than previously considered protective may provide benefit in the context of typical human exposure to HIV-1.

Article Publication: Vaccine

Article Summary: Mycobacterium bovis BCG is considered an attractive live bacterial vaccine vector. In this study, we investigated the immune response of baboons to a primary vaccination with recombinant BCG (rBCG) constructs expressing the gag gene from a South African HIV-1 subtype C isolate, and a boost with HIV-1 subtype C Pr55(gag) virus-like particles (Gag VLPs). Using an interferon enzyme-linked immunospot assay, we show that although these rBCG induced only a weak or an undetectable HIV-1 Gag-specific response on their own, they efficiently primed for a Gag VLP boost, which strengthened and broadened the immune responses. These responses were predominantly CD8+ T cell-mediated and recognised similar epitopes as those targeted by humans with early HIV-1 subtype C infection. In addition, a Gag-specific humoral response was elicited. These data support the development of HIV-1 vaccines based on rBCG and Pr55(gag) VLPs.

Article Publication: Vaccine

Article Summary: Integrase (IN) defective lentiviral vectors have a high safety profile and might prove useful as immunizing agents especially against HIV-1. However, IN defective SIV-based vectors must be developed in order to test their potential in the non-human primate models (NHP) of AIDS. To this aim we tested a novel SIV-based IN defective lentiviral vector for its ability to induce sustained immune responses in mice. BALB/c mice were immunized once intramuscularly with a SIV-based IN defective lentiviral vector expressing the model antigen enhanced green fluorescence protein (eGFP). Immune responses were evaluated 90 days after the injection and compared with those elicited with the IN competent counterpart. The IN defective vector was able to efficiently elicit specific and long-lasting polyfunctional immune responses as evaluated by enzyme-linked immunospot (ELISPOT) assays for interferon-gamma (IFN-gamma) in spleens, bone marrow (BM) and draining lymph nodes, and by intracellular staining (ICS) for IFN-gamma, Interleukin-2 (IL-2) and tumor necrosis factor (TNF-alpha) in both splenocytes and BM cells without integration of the vector into the host genome. This is the first demonstration that an IN defective SIV-based lentiviral vector provides effective immunization, thus paving the way for the construction of IN defective vectors expressing SIV antigen(s) and test their efficacy against a SIV virus challenge in the NHP model of AIDS.

Article Publication: Journal of virology

Article Summary: Defining the specificities of the anti-HIV-1 envelope antibodies able to mediate broad heterologous neutralization will assist in identifying targets for an HIV-1 vaccine. We screened 70 plasmas from HIV-1 chronically infected individuals for neutralization breadth. Of these, 16 (23%) were found to neutralize 80% or more of the viruses tested. Anti-CD4bs antibodies were found in almost all plasmas independent of their neutralization breadth, but they mainly mediated neutralization of the laboratory strain HxB2 with little effect over the primary virus Du151. Adsorption with Du151 monomeric gp120 reduced neutralizing activity to some extent in most plasma samples when tested against the matched virus, although these antibodies did not always confer cross-neutralization. For one plasma this activity was mapped to a site overlapping the CD4i epitope and CD4bs. Anti-MPER (r=0.69, P<0.001) and anti-CD4i (r=0.49, P<0.001) antibody titers were found to correlate with neutralization breadth. These anti-MPER antibodies were not 4E10 or 2F5-like but spanned the 4E10 epitope. Furthermore, we found anti-cardiolipin antibodies correlated with neutralization breadth (r=0.67, P<0.001) and anti-MPER antibodies (r=0.6, P<0.001). Our study suggests that more than one epitope on the envelope glycoprotein is involved in the cross-reactive neutralization elicited during HIV-1 natural infection, many of which are yet to be determined, and the possible involvement of polyreactive antibodies in this phenomenon.

Article Publication: Journal of virology

Article Summary: Rare serotype and chimeric recombinant adenovirus (rAd) vectors that evade anti-Ad5 immunity are currently being evaluated as potential vaccine vectors for both HIV-1 and other pathogens. We have recently reported that a heterologous rAd prime-boost regimen expressing SIV Gag afforded durable partial immune control of an SIV challenge in rhesus monkeys. However, a single-shot immunization may ultimately be preferable for global vaccine delivery. We therefore evaluated the immunogenicity and protective efficacy of a single immunization of hexon-chimeric rAd5HVR48 vectors expressing SIV Gag, Pol, Nef, and Env against a homologous SIV challenge in rhesus monkeys. Inclusion of Env resulted in improved control of peak and setpoint SIV RNA levels following challenge. In contrast, DNA vaccine priming did not further improve the protective efficacy of rAd5HVR48 vectors in this system.

Article Publication: Journal of virology

Article Summary: The function of lentiviral Vif proteins is to neutralize the host antiviral cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F). Vif bridges a Cullin5-based E3 ubiquitin ligase with A3G and A3F, and mediates their degradation by proteasomes. Recent studies have found that Vif uses different domains to bind to A3G and A3F. A (14)DRMR(17) domain binds to A3F, (40)YRHHY(44) binds to A3G, and (69)YxxL(72) binds to both A3G and A3F. Here, we report another functional domain of Vif. Previously, we demonstrated that HIV-1 Vif failed to mediate A3G proteasomal degradation when all 16 lysines were mutated to arginines. Here we show that K26, and to a lesser extend K22, are critical for A3G neutralization. K22 and K26 are part of a conserved (21)WxSLVK(26) (x: N, K, or H) motif, which is found in most primate lentiviruses, and which shows species-specific variation. Both K22 and K26 in this motif only regulated Vif specificity to A3G, whereas the SLV residues regulated Vif specificity to both A3F and A3G. Interestingly, SLV and K26 in HIV-1 Vif did not directly mediate Vif interaction with either A3G or A3F. Previously, other groups have reported an important role for W21 in A3F and A3G neutralization. Thus, (21)WxSLVK(26) is a novel functional domain that regulates Vif activity to both A3F and A3G, and is a potential drug target to inhibit Vif activity and block HIV-1 replication.

Article Publication: Biochemical and biophysical research communications

Article Summary: HIV-1 gp120 is an alleged B cell superantigen, binding certain VH3+ human antibodies. We reasoned that a CD4-VH3 fusion protein could possess higher affinity for gp120 and improved HIV-1 inhibitory capacity. To test this we produced several human IgG1 immunoligands harboring VH3. Unlike VH3-IgG1 or VH3-CD4-IgG1, CD4-VH3-IgG1 bound gp120 considerably stronger than CD4-IgG1. CD4-VH3-IgG1 exhibited approximately 1.5-2.5-fold increase in neutralization of two T-cell laboratory-adapted strains when compared to CD4-IgG1. CD4-VH3-IgG1 improved neutralization of 7/10 clade B primary isolates or pseudoviruses, exceeding 20-fold for JR-FL and 13-fold for Ba-L. It enhanced neutralization of 4/8 clade C viruses, and had negligible effect on 1/4 clade A pseudoviruses. We attribute this improvement to possible pairing of VH3 with CD4 D1 and stabilization of an Ig Fv-like structure, rather than to superantigen interactions. These novel findings support the current notion that CD4 fusion proteins can act as better HIV-1 entry inhibitors with potential clinical implications.

Article Publication: Virology

Article Summary: The membrane-proximal external region (MPER) of gp41 is considered as a prime target for the induction of neutralizing antibodies, since it contains the epitopes for three broadly neutralizing antibodies (2F5, 4E10 and Z13). Here we present a novel gp41 construct (HA-gp41) comprising gp41 HR2 and MPER fused to two triple-stranded coiled-coil domains at both ends. HA-gp41 is trimeric, has a high helical content in solution and forms rod-like structures as revealed by negative staining electron microscopy. Immunization of rabbits with HA-gp41 induced antibodies directed against MPER, which failed to exert significant neutralization capacity against envelopes from primary isolates. Thus trimerisation of MPER regions does not suffice to induce a potent neutralizing antibody response specific for conserved regions within gp41.

Article Publication: Virology

Article Summary: The conserved nature of the epitopes of the four broadly neutralizing antibodies (BNAbs), b12, 2G12, 2F5, and 4E10, may imply that the sensitivity of HIV-1 for these BNAbs remains fairly constant over the course of infection. Here, we demonstrate that viruses isolated early during the course of infection were mostly sensitive to HIVIg and antibody neutralization, although variation was observed in neutralization sensitivity of coexisting viruses to the different antibodies as well as between viruses from different patients. HIV-1 resistance to HIVIg developed relatively early during follow-up in three out of five patients, while early, b12 sensitive viruses in three out of five patients were replaced by b12 resistant variants relatively late in infection. In contrast, viruses generally remained sensitive to 2F5 and 4E10 neutralization over the course of infection, although 2F5 and/or 4E10 resistant variants did emerge later in infection in four out of five patients. In most patients, HIV-1 resistance to 2F5 or 4E10 did not correlate with mutations at critical amino acid positions in their defined epitopes. Viruses resistant to 2G12-mediated neutralization were present throughout the course of infection. As viral resistance against BNAb-mediated neutralization generally developed when autologous serum neutralizing activity had faded, it seems unlikely that these changes are driven by escape from autologous humoral immunity.

Article Publication: PLoS medicine

Article Summary: BACKGROUND: The antibody response to HIV-1 does not appear in the plasma until approximately 2-5 weeks after transmission, and neutralizing antibodies to autologous HIV-1 generally do not become detectable until 12 weeks or more after transmission. Moreover, levels of HIV-1-specific antibodies decline on antiretroviral treatment. The mechanisms of this delay in the appearance of anti-HIV-1 antibodies and of their subsequent rapid decline are not known. While the effect of HIV-1 on depletion of gut CD4(+) T cells in acute HIV-1 infection is well described, we studied blood and tissue B cells soon after infection to determine the effect of early HIV-1 on these cells. METHODS AND FINDINGS: In human participants, we analyzed B cells in blood as early as 17 days after HIV-1 infection, and in terminal ileum inductive and effector microenvironments beginning at 47 days after infection. We found that HIV-1 infection rapidly induced polyclonal activation and terminal differentiation of B cells in blood and in gut-associated lymphoid tissue (GALT) B cells. The specificities of antibodies produced by GALT memory B cells in acute HIV-1 infection (AHI) included not only HIV-1-specific antibodies, but also influenza-specific and autoreactive antibodies, indicating very early onset of HIV-1-induced polyclonal B cell activation. Follicular damage or germinal center loss in terminal ileum Peyer's patches was seen with 88% of follicles exhibiting B or T cell apoptosis and follicular lysis. CONCLUSIONS: Early induction of polyclonal B cell differentiation, coupled with follicular damage and germinal center loss soon after HIV-1 infection, may explain both the high rate of decline in HIV-1-induced antibody responses and the delay in plasma antibody responses to HIV-1. Please see later in the article for Editors' Summary.

Article Publication: Vaccine

Article Summary: Recombinant Adenovirus serotype 5 (Ad5) vectors have been used as vaccine platforms in numerous animal and human clinical studies. The immune response induced by Ad5 vaccines can be mitigated due to pre-existing Ad5 immunity. We previously reported the use of a novel Ad5 platform to induce cellular immune responses (CMI) against HIV-1 Gag in Ad5 hyper immune mice. Here, the effectiveness of the Ad5 [E1-, E2b-] vaccine platform was evaluated using a triad mixture of HIV-1 Gag, Pol, and Nef as antigenic transgenes. Broad CMI was induced following vaccination with the HIV-1 expressing vectors in Ad5 naive and Ad5 immunized mice. A mixture of the three vaccines induced CMI against each transgene product even in the presence of hyper Ad5 immunity. These studies revealed that CMI responses to immunization with Ad5 [E1-, E2b-]-gag, Ad5 [E1-, E2b-]-pol or Ad5 [E1-, E2b-]-nef vectors were transgene specific and did not induce CMI responses against irrelevant antigens such as carcinoembryonic antigen (CEA), herpes simplex virus glycoprotein B (HSV), cytomegalovirus (CMV) or influenza virus antigens. We are evaluating this recombinant triad viral vector as an HIV-1 vaccine in a non-human primate model and the data indicate that the vaccine is worthy of clinical evaluation.

Article Publication: Virology journal

Article Summary: ABSTRACT: BACKGROUND: Recombinant Salmonella vaccine vectors may potentially be used to induce specific CD4+ T cell responses against foreign viral antigens. Such immune responses are required features of vaccines against pathogens such as human immunodeficiency virus type 1 (HIV-1). The aim of this study was to investigate the induction of systemic HIV-1-specific CD4+ T helper (Th) responses in mice after oral immunization with a live attenuated Salmonella vaccine vector that expressed HIV-1 subtype C Gag. Groups of BALB/c mice were vaccinated orally three times (4 weeks apart) with this recombinant Salmonella. At sacrifice, 28 days after the last immunization, systemic CD4+ Th1 and Th2 cytokine responses were evaluated by enzyme-linked immunospot assay and cytometric bead array. HIV-1 Gag-specific IgG1 and IgG2a humoral responses in the serum were determined by enzyme-linked immunosorbent assay. RESULTS: Mice vaccinated with the recombinant Salmonella elicited both HIV-1-specific Th1 (interferon-gamma (IFN-g) and tumour necrosis factor-alpha (TNF-a)) and Th2 (interleukin-4 (IL-4) and interleukin-5 (IL-5)) cytokine responses. The vaccine induced 70 (IFN-g) spot-forming units (SFUs)/10e6 splenocytes and 238 IL-4 SFUs/10e6 splenocytes. Splenocytes from vaccinated mice also produced high levels of Th1 and Th2 cytokines upon stimulation with a Gag CD4 peptide. The levels of IFN-g, TNF-a, IL-4 and IL-5 were 7.5-, 29.1-, 26.2- and 89.3-fold above the background, respectively. Both HIV-1 Gag-specific IgG1 and IgG2a antibodies were detected in the sera of vaccinated mice. CONCLUSIONS: The study highlights the potential of orally-delivered attenuated Salmonella as mucosal vaccine vectors for HIV-1 Subtype C Gag to induce Gag-specific CD4+ Th1 and Th2 cellular immune responses and antibodies which may be important characteristics required for protection against HIV-1 infection.

Article Publication: Vaccine

Article Summary: SOSIP gp140 trimers represent a soluble, stabilized, proteolytically cleaved form of the HIV-1 envelope (Env) glycoproteins. SOSIP gp140 derived from a subtype A HIV-1 isolate, KNH1144, forms exceptionally stable trimers that resemble virion-associated Env in antigenicity and topology. Here, we used electron microscopy to demonstrate that KNH1144 SOSIP gp140 trimers bound three soluble CD4 molecules in a symmetrical orientation similar to that seen for native Env. We compared the immunogenicities of KNH1144 SOSIP gp140 trimers and gp120 monomers in rabbits and found that the trimers were superior at eliciting neutralizing antibodies (NAbs) to homologous virus as well as neutralization-sensitive subtype B and C viruses. The NAb specificities for SOSIP antisera mapped in part to the CD4 binding site on gp120. We also observed adjuvant-dependent induction of antibodies to the residual levels of host cell proteins (HCPs) contained in the purified Env preparations. When present, HCP antibodies enhanced pseudovirus infection. Our findings are relevant for the further development of Env-based vaccines for HIV-1.

Article Publication: Retrovirology

Article Summary: ABSTRACT: BACKGROUND: Studies have shown that in the genome of human immunodeficiency virus (HIV-1) regions responsible for interactions with the host's immune system, namely, cytotoxic T-lymphocyte (CTL) epitopes tend to cluster together in relatively conserved regions. On the other hand, "epitope-less" regions or regions with relatively low density of epitopes tend to be more variable. However, very little is known about relationships among epitopes from different genes, in other words, whether particular epitopes from different genes would occur together in the same viral genome. To identify CTL epitopes in different genes that co-occur in HIV genomes, association rule mining was used. RESULTS: Using a set of 189 best-defined HIV-1 CTL/CD8+ epitopes from 9 different protein-coding genes, as described by Frahm, Linde & Brander (2007), we examined the complete genomic sequences of 62 reference HIV sequences (including 13 subtypes and sub-subtypes with approximately 4 representative sequences for each subtype or sub-subtype, and 18 circulating recombinant forms). The results showed that despite inclusion of recombinant sequences that would be expected to break-up associations of epitopes in different genes when two different genomes are recombined, there exist particular combinations of epitopes (epitope associations) that occur repeatedly across the world-wide population of HIV-1. For example, Pol epitope LFLDGIDKA is found to be significantly associated with epitopes GHQAAMQML and FLKEKGGL from Gag and Nef, respectively, and this association rule is observed even among circulating recombinant forms. CONCLUSIONS: We have identified CTL epitope combinations co-occurring in HIV-1 genomes including different subtypes and recombinant forms. Such co-occurrence has important implications for design of complex vaccines (multi-epitope vaccines) and/or drugs that would target multiple HIV-1 regions at once and, thus, may be expected to overcome challenges associated with viral escape.

Article Publication: European journal of immunology

Article Summary: All HIV-1 'systemic vaccine trials' in humans have yielded poor outcomes. Thus, it is important to understand whether the route of delivery influences the quality of protective CTL immunity. Using heterologous poxvirus immunisation we have shown that systemically (i.m./i.m.) immunised CD8(+) T cells generated higher levels of IL-4/IL-13 compared to mucosal delivery and expression also correlated with i.m./i.m. immunised mice eliciting CTL of lower avidity. Studies using IL-4(-/-) and IL-13(-/-) KO mice have shown that the capacity to express IFN-gamma, IL-4 and/or IL-13 by K(d)Gag(197-205)-specific CTL differed between these groups and was inversely correlated with CTL avidity (IL-13(-/-)>IL-4(-/-)>BALB/c), although no significant differences in the magnitude of CTL responses were observed between IL-13(-/-) and wild type mice. When IL-13 was reconstituted in IL-13(-/-) splenocytes in vitro, their ability to bind tetramers also decreased significantly. Our data reveal that total absence of IL-13 can greatly enhance CTL avidity. In contrast, extracellular IL-4 appears to be important in maintaining long-term Th1/Th2 balance in CTL, even though expression of IL-4 by CTL markedly reduced avidity. STAT6(-/-) mice also showed memory CTL of higher avidity. Furthermore, CCL5 expression in K(d)Gag(197-205)-specific CTL was also regulated by IL-4/IL-13.

Article Publication: Journal of acquired immune deficiency syndromes

Article Summary: OBJECTIVES:: To evaluate a social network approach to develop an adolescent cohort for HIV vaccine preparedness and investigate characteristics that influence recruitment. METHODS:: We summarize baseline data from a prospective cohort study that included 4 sessions over 6 months. Fifty-nine HIV-infected adolescent and adult patients of a family-based HIV clinic named significant others and indicated willingness to involve them in this study. Sixty-two adolescent and adult significant others not known to be HIV infected were enrolled. Logistic regression was used to estimate factors associated with willingness. RESULTS:: Participants identified 624 social network members including 276 adolescents (44%). Network member's awareness of the index's HIV positivity (P < 0.01) and older age (P = 0.05) affected willingness. Respondents were less willing to invite drug-risk alters (P = 0.006). Adolescents were willing to invite more adolescents than were adults (P < 0.0001). Adolescents younger than 18 years old reported fewer sexual and drug-using risk behaviors than expected. CONCLUSIONS:: HIV-infected patients are willing to recruit their social networks, provided concerns about disclosure of HIV status are addressed. Using social networks to identify and recruit adolescent populations is appropriate and feasible for vaccine preparedness activities, future vaccine trials, and other prevention programs, but procedures are needed to selectively identify and retain high-risk youth.

Article Publication: Aids

Article Summary: OBJECTIVE:: Using clinical isolates from a recent passive immunization trial with antibody 2G12, we probed the capacity of frequently used neutralization formats - the primary peripheral blood mononuclear cell (PBMC)-based and the TZM-bl cell-based assay systems - to predict in-vivo activity of 2G12. DESIGN:: Antibodies and entry inhibitors of established efficacy were used to neutralize HIV-1 isolates in different in-vitro assay setups. METHODS:: Single round infection with Env-pseudotyped and multiple round infection with replication-competent virus was studied on PBMCs and a variety of engineered cell lines. RESULTS:: Six out of 12 isolates with high sensitivity to 2G12 in the replication-competent PBMC assay lacked sensitivity to the monoclonal antibody in the env-pseudotype TZM-bl assay. Outcome of passive immunization with 2G12 corroborated the PBMC-assay in-vitro data, as escape mutations to 2G12 emerged, proving the monoclonal antibody's impact on HIV in vivo. Failure to inhibit pseudotype infection of TZM-bl was not due to sequence differences or the pseudotype infection per se, as infection of PBMCs with the identical pseudotyped viruses was sensitive to 2G12 inhibition. Similar shifts in efficacy, though less extreme, were noted for other neutralizing antibodies and inhibitors. Exploration of causes for the observed differences between assay systems revealed that both target cell and virus producer properties influence sensitivity of virus entry to inhibition. CONCLUSION:: Our observation that neutralization assay systems employing engineered reporter cell lines can miss in-vivo relevant neutralizing activities strongly argues that preclinical assessment should not be restricted to a single assay type

Article Publication: Idrugs

Article Summary: In September 2007, a clinical trial (STEP trial) evaluating the candidate HIV vaccine MRK Ad5 HIV-1 gag/pol/nef from Merck & Co Inc was halted at the first interim analysis because the vaccine demonstrated no positive impact on virus acquisition or virus load following infection. Additionally, there was an increased rate of HIV infection in vaccinees who had prior immunity to adenovirus type 5 (Ad5) and/or were circumcised. This failure of the vaccine, as well as the apparent harm caused to some study participants, generated a massive pessimism regarding HIV vaccine development. Concerns regarding the future of HIV vaccine research led to a summit convened by the NIAID in March 2008 to provide new directions in HIV vaccine research. A shift in emphasis focused on three areas: discovery research, animal models and clinical research. In each of these areas, notable new activities have occurred: a wealth of information has emerged from the STEP trial, promising results have been reported on assay development for markers of vaccine-induced immune function, and evaluations of promising new experimental vaccines have occurred in nonhuman primates. Overall, the progress in the field of HIV vaccine research since September 2007 has reinforced the need for a balanced approach between basic vaccine discovery and development, as well as the importance of addressing questions in nonhuman primate studies and clinical trials. This article discusses how past product failures have invigorated the field of HIV vaccine research by addressing critical questions and suggesting additional possible approaches to follow. As a result of the insight gained, a new sense of optimism exists in the field of HIV vaccine research.

Article Publication: Mucosal immunology

Article Summary: AIDS is mainly a sexually transmitted disease, and accordingly, mucosal tissues are the primary sites of natural human immunodeficiency virus type-1 (HIV-1) transmission. Mucosal immunoglobulin A (IgA) antibody specific for HIV-1 envelope gp41 subunit is one correlate of protection in individuals who are highly sexually exposed to HIV-1 but remain persistently IgG seronegative (HEPS). Understanding these peculiar IgAs at the gene and functional level is possible only with monoclonal IgAs. We have constructed a mucosal Fab IgA library from HEPS and have characterized a series of HIV-1 IgAs specific for gp41 that, in vitro, are transcytosis-blocking and infection-neutralizing. Characterization of their IgA genes shows that Fab specific for the gp41 membrane-proximal region harbors a long heavy-chain CDR3 loop (CDRH3) similar to the two broadly neutralizing IgG monoclonal antibodies, 2F5 and 4E10. Furthermore, the selected Fab IgA shows extensive somatic mutations that cluster in the CDR regions, indicating that affinity maturation due to an antigen-driven process had occurred in HEPS individuals, presumably upon multiple exposures to HIV. This analysis of HEPS monoclonal IgA gives a unique opportunity to correlate an antibody function (resistance to a pathogen in vivo) with an antibody gene. Such neutralizing monoclonal IgAs could be used in microbicide formulation.Mucosal Immunology advance online publication 8 July 2009. doi:10.1038/mi.2009.89.

Article Publication: Vaccine

Article Summary: The laboratory strain of murine cytomegalovirus (MCMV), K181, has been successfully engineered as a vaccine expressing murine zona pellucida 3 (mZP3) for viral vectored immunocontraception (VVIC) in mice. However, certain laboratory strains of mice are resistant to infection with K181 and therefore demonstrate resistance to VVIC. Cmv1 is the best characterised innate resistance mechanism to MCMV and was first described in C57BL/6 mice. Resistance in C57BL/6 mice is due to early and strong activation of natural killer (NK) cells by an MCMV gene product, m157, that binds directly to the NK cell activating receptor Ly49H. In this study a wild strain of MCMV, G4, which expresses a variant m157 incapable of activating Ly49H, was engineered to express murine zona pellucida 3 (mZP3) and assessed for its ability to sterilise female C57BL/6 mice. When infected with K181-mZP3 female C57BL/6 mice remained fully fertile. In contrast, female C57BL/6 mice were sterilised by a single intraperitoneal inoculation of G4-mZP3. Infertility was induced by G4-mZP3 in three strains of mice that express Ly49H, on two different histocompatibility-2 (H-2) backgrounds. Finally, enhanced immunocontraception was observed in mice expressing H-2(k) mediated resistance to MCMV when infected with G4-mZP3 compared to K181-mZP3. These data indicate that when using viral vaccine vectors, variant vector strains may be used to circumvent powerful innate immune responses against the vector and promote effective vaccination. This study highlights the importance of vaccine vector genetics in vaccination strategies.

Article Publication: Cell host microbe

Article Summary: Over 800 million people worldwide are infected with hepatitis viruses, human immunodeficiency virus (HIV), and malaria, resulting in more than 5 million deaths annually. Here we discuss the potential and challenges of humanized mouse models for developing effective and affordable therapies and vaccines, which are desperately needed to combat these diseases.

Article Publication: Nature medicine

Article Summary: The mechanisms underlying possible increased HIV-1 acquisition in adenovirus 5 (Ad5)-seropositive subjects vaccinated with Ad5-HIV-1 vectors in the Merck STEP trial remain unclear. We find that Ad5 serostatus does not predict Ad5-specific CD4(+) T cell frequency, and we did not observe durable significant differences in Ad5-specific CD4(+) T cells between Ad5-seropositive and Ad5-seronegative subjects after vaccination. These findings indicate no causative role for Ad5-specific CD4(+) T cells in increasing HIV-1 susceptibility in the STEP trial.

Article Publication: Nature medicine

Article Summary: The immunologic basis for the potential enhanced HIV-1 acquisition in adenovirus serotype 5 (Ad5)-seropositive individuals who received the Merck recombinant Ad5 HIV-1 vaccine in the STEP study remains unclear. Here we show that baseline Ad5-specific neutralizing antibodies are not correlated with Ad5-specific T lymphocyte responses and that Ad5-seropositive subjects do not develop higher vector-specific cellular immune responses as compared with Ad5-seronegative subjects after vaccination. These findings challenge the hypothesis that activated Ad5-specific T lymphocytes were the cause of the potential enhanced HIV-1 susceptibility in the STEP study.

Article Publication: Journal of infectious diseases

Article Summary: Simulation studies were conducted to estimate the statistical power of repeated low-dose challenge experiments performed in nonhuman primates to detect the effect of a candidate human immunodeficiency virus vaccine. The effect of various design parameters on power was explored. Results of simulation studies indicate that repeated low-dose challenge studies with a total sample of size 50 (25 animals/arm) typically provide adequate power to detect a 50% reduction in the per-exposure probability of infection resulting from vaccination. Power generally increases with the maximum number of allowable challenges per animal, the per-exposure risk of infection in control animals, and the proportion of animals susceptible to infection

Article Publication: Journal immunology

Article Summary: Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8(+) cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8(+) T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2D(d) better than the native sequences, and clones with distinct specificities were elicited by alternative vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8(+) T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.

Article Publication: Nature

Article Summary: African primates are naturally infected with over 40 different simian immunodeficiency viruses (SIVs), two of which have crossed the species barrier and generated human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2)1, 2. Unlike the human viruses, however, SIVs do not generally cause acquired immunodeficiency syndrome (AIDS) in their natural hosts3. Here we show that SIVcpz, the immediate precursor of HIV-1, is pathogenic in free-ranging chimpanzees. By following 94 members of two habituated chimpanzee communities in Gombe National Park, Tanzania, for over 9 years, we found a 10- to 16-fold higher age-corrected death hazard for SIVcpz-infected (n = 17) compared to uninfected (n = 77) chimpanzees. We also found that SIVcpz-infected females were less likely to give birth and had a higher infant mortality rate than uninfected females. Immunohistochemistry and in situ hybridization of post-mortem spleen and lymph node samples from three infected and two uninfected chimpanzees revealed significant CD4+ T-cell depletion in all infected individuals, with evidence of high viral replication and extensive follicular dendritic cell virus trapping in one of them. One female, who died within 3 years of acquiring SIVcpz, had histopathological findings consistent with end-stage AIDS. These results indicate that SIVcpz, like HIV-1, is associated with progressive CD4+ T-cell loss, lymphatic tissue destruction and premature death. These findings challenge the prevailing view that all natural SIV infections are non-pathogenic and suggest that SIVcpz has a substantial negative impact on the health, reproduction and lifespan of chimpanzees in the wild.

Article Publication: Clinical and vaccine immunology

Article Summary: Background: Preexisting immunity to adenovirus (Ad) type 5 diminishes immune responses to vaccines using Ad5 as a vector. Alternate Ad serotypes as vaccine vectors might overcome Ad5-specific neutralizing antibodies and enhance immune responses in populations with a high prevalence of Ad5 immunity. Methods: Healthy HIV-seronegative adults were enrolled in a blinded, randomized, dose-escalating, placebo-controlled study. In Part A, subjects with baseline Ad6 titers 200) and Ad6 titers (18) received MRKAd5+6 HIV-1 trigene vaccine at Weeks 0, 4, and 26. Immunogenicity was assessed by ELISPOT at Week 30. Results: No serious adverse events occurred. MRKAd6 trigene vaccine recipients responded more often to Nef than Gag or Pol. In Part A, ELISPOT response rates to >/=2 vaccine antigens were 14%, 63%, and 71% at the 10(9), 10(10), and 10(11) vg/dose, respectively. All responders had a positive Nef-specific ELISPOT. In Part B, Nef-ELISPOT response rates at 10(10) vg/dose of the MRKAd5+6 trigene vaccine were 50% in low Ad5/low Ad6 stratum (n=8), 78% in low Ad5/high Ad6 stratum (n=9), 75% in the high Ad5/low Ad6 stratum (n=8), and 44% in the high Ad5/high Ad6 stratum (n=9). Conclusions: The MRKAd6 and MRKAd5+6 trigene vaccines elicited dose-dependent responses predominantly to Nef and were generally well tolerated, indicating that Ad6 should be considered a candidate vector for future vaccines. Although small sample sizes limit conclusions drawn from this exploratory study, combining two Ad vectors may be a useful vaccine strategy to circumvent isolated immunity to a single Ad type.

Article Publication: Clinical experimental immunology

Article Summary: Many pathogens use mucosal surfaces to enter and propagate within the host, making particularly desirable vaccines that target immune responses specifically to mucosal compartments. The majority of mucosal vaccine design strategies to date have been empirical in nature. However, an emerging body of basic immunological knowledge is providing new insights into the regulation of tissue-specific lymphocyte trafficking and differentiation. These insights afford the opportunity for the rational design of vaccines that focus immune responses at mucosal surfaces. Mucosal cellular immunity may prove critical for protection in the context of HIV infection, and thus there has been considerable interest in developing vaccines that target HIV-specific cellular immune responses to the gastrointestinal and vaginal mucosa. However, the optimal strategies for eliciting mucosal cellular immune responses through vaccination remain to be determined. Here, we review both recent vaccine studies and emerging paradigms from the basic immunological literature that are relevant to the elicitation of potent and protective mucosal cellular immune memory. Increasing the synergy between these avenues of research may afford new opportunities for mucosal vaccine design.

Article Publication: Journal of immunology

Article Summary: Successful vaccines (i.e., tetanus and diphtheria) can induce long-lived Ab levels that are maintained by bone marrow plasma cells and plasma Ab levels do not correlate with numbers of blood memory B cells. Destruction of CD4(+) T cells early in HIV-1 acute infection may result in insufficient induction of neutralizing Ab responses; thus, an HIV-1 vaccine should elicit high levels of durable Abs by long-lived plasma cells to be protective. We asked if HIV-1 envelope-specific memory responses were sustained by memory B cells in the settings of HIV-1 gp120 envelope vaccination and chronic HIV-1 infection. Levels of anti-HIV-1 envelope plasma Abs and memory B cells were found to correlate in both settings. Moreover, whereas the expected half-life of plasma Ab levels to protein vaccines was >10 years when maintained by long-lived plasma cells, anti-envelope Ab level half-lives were approximately 33-81 wk in plasma from antiretroviral drug-treated HIV-1(+) subjects. In contrast, anti-p55 Gag Ab level half-life was 648 wk, and Ab titers against influenza did not decay in-between yearly or biennial influenza vaccine boosts in the same patients. These data demonstrated that HIV-1 envelope induces predominantly short-lived memory B cell-dependent plasma Abs in the settings of envelope vaccination and HIV-1 infection. The inability to generate high titers of long-lived anti-envelope Abs is a major hurdle to overcome for the development of a successful HIV-1 vaccine.

Article Publication: Human gene therapy

Article Summary: Strategies to improve vaccine efficacy are still required, especially in case of chronic infections, including the human immunodeficiency virus (HIV). DNA vaccines have potential advantages over conventional vaccines; however, low immunological efficacy has been demonstrated in many experiments involving large animals and in clinical trials. To improve the immunogenicity of DNA vaccine, we have designed a plasmid vector exploiting the binding capacity of the bovine papillomavirus E2 protein and we have used the electroporation (EP) to increase the DNA uptake after intra-dermal (i.d.) inoculation. We demonstrated in nonhuman primates (NHP), efficient induction of anti-HIV immunity to an improved DNA vaccine vector encoding an artificial fusion protein, consisting of several proteins and selected epitopes from HIV-1. We show that a DNA vaccine delivery method combining i.d. injection and non-invasive EP dramatically increased the expression of the vaccine antigen selectively in the epidermis and our observations strongly suggest the involvement of Langerhans cells in the strength and quality of the anti-HIV immune response. Although the humoral responses to the vaccine were transient, the cellular responses were exceptionally robust and persisted, at high levels, more than two years after the last vaccine boost. The immune responses were characterized by the induction of significant proportions of T cells producing both interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) cytokines, in both subpopulations, CD4+ and CD8+. This strategy is an attractive approach for vaccination in humans because of its high efficacy and the possible use of newly developed devices for EP.

Article Publication: Virology

Article Summary: Studies were performed to induce cross-clade neutralizing antibodies (Abs) by testing various combinations of prime and boost constructs that focus the immune response on structurally-conserved epitopes in the V3 loop of HIV-1 gp120. Rabbits were immunized with gp120 DNA containing a V3 loop characterized by the GPGR motif at its tip, and/or with gp120 DNA with a V3 loop carrying the GPGQ motif. Priming was followed by boosts with V3-fusion proteins (V3-FPs) carrying the V3 sequence from a subtype B virus (GPGR motif), and/or with V3 sequences from subtypes A and C (GPGQ motif). The broadest and most consistent neutralizing responses were generated when using a clade C gp120 DNA prime and with the V3(B)-FP boost. Immune sera displayed neutralizing activity in three assays against pseudoviruses and primary isolates from subtypes A, AG, B, C, and D. Polyclonal Abs in the immune rabbit sera neutralized viruses that were not neutralized by pools of human anti-V3 monoclonal Abs. Greater than 80% of the neutralizing Abs were specific for V3, showing that the immune response could be focused on a neutralizing epitope and that vaccine-induced anti-V3 Abs have cross-clade neutralizing activity.

Article Publication: Journal of virology

Article Summary: The binding of neutralizing antibodies 2F5 and 4E10 to HIV-1 gp41 involves both the viral membrane and gp41 membrane proximal external region (MPER) epitopes. In this study we have used several biophysical tools to examine the secondary structure, orientation and depth of immersion of gp41 MPER peptides in liposomes and to determine how the orientation of the MPER with lipids affects binding kinetics of mAbs 2F5 and 4E10. The binding of 2F5 and 4E10 to both their respective nominal and a bi-epitope (includes 2F5 and 4E10 epitopes) MPER peptide-liposome conjugates was best described by a two-step encounter-docking model. Analysis of the binding kinetics and the effect of temperature on binding stability of 2F5 and 4E10 to MPER peptide-liposome conjugates revealed that the docking of 4E10 was relatively slower and thermodynamically less favorable. Fluorescence quenching and FRET experiments showed that the 2F5 epitope was more solvent exposed, whereas the 4E10 epitope was immersed into the polar-apolar interfacial region of the lipid bilayer. CD spectroscopic study demonstrated that the nominal and bi-epitope MPER peptides when anchored to liposomes adopted ordered structures with varying helical content. Furthermore, anchorage of MPER peptides to membrane via a hydrophobic anchor sequence was required for efficient mAb docking. These results support the model that the ability of 2F5 and 4E10 to bind to membrane lipid is required for stable docking to membrane embedded MPER residues. These data have important implications in the design and use of peptide-liposome conjugates as immunogens for the induction of MPER neutralizing antibodies.

Article Publication: Journal of virology

Article Summary: An ideal HIV-1 vaccine would elicit potent cellular and humoral immune responses that recognize diverse strains of virus. In the present study, combined methodologies (flow cytometry, Vbeta repertoire analysis, CDR3 sequencing) were used to determine the clonality of CD8(+) T lymphocytes taking part in the recognition of variant epitope peptides elicited in Mamu-A*01(+) rhesus monkeys immunized with vaccines encoding diverse HIV-1 envelopes. Monkeys immunized with clade B Envs generated CD8(+) T cell lymphocytes that cross-recognized both clade B- and clade C-p41A epitope peptides using a large degree of diversity in Vbeta gene usage. However, in two monkeys immunized with clade C Env, one monkey exhibited p41A-specific CTL with the capacity for cross-recognition of variant epitopes while the other did not. These studies demonstrate that the cross-reactive potential of variant p41A-epitope peptide-specific CTL populations can differ between monkeys that share the same restricting MHC class I molecule and receive the same vaccine immunogens.

Article Publication: Journal of virology

Article Summary: Intramuscular (IM) inoculation of rhesus macaques with one or more doses of recombinant vesicular stomatitis virus (rVSV) expressing HIV-1 gag (rVSVgag) typically elicits peak cellular immune responses of 500-1000 interferon gamma (IFN-gamma) ELISPOTS/10(6) PBMCs. Here, we describe the generation of a novel recombinant mumps virus (rMuV) expressing HIV-1 gag (rMuVgag) and measure the gag-specific cellular immune responses detected in rhesus macaques following vaccination with a highly attenuated form of rVSV expressing HIV-1 gag (rVSVN4CT1gag1) and rMuVgag in various prime-boost combinations. Notably, peak gag-specific cellular immune responses of 3000-3500 ELISPOTS/10(6) PBMCs were detected in macaques that were primed with rMuVgag and boosted with rVSVN4CT1gag1. Lower peak cellular immune responses were detected in macaques that were primed with rVSVN4CT1gag1 and boosted with rMuVgag, although longer term gag-specific responses appeared to remain higher in this group of macaques. These findings indicate that rMuVgag may significantly enhance gag-specific cellular immune responses when administered with rVSVN4CT1gag1 in heterologous prime-boost regimens.

Article Publication: Journal of virology

Article Summary: Recent findings suggest that most sexual transmission of HIV-1 occurs during the acute phase of infection when viral replication is most intense. Here we show that vaccine-elicited cellular immune responses can significantly reduce SIV levels in the semen during the period of primary infection in monkeys. A vaccine that decreases the quantity of HIV-1 in the semen of males during primary infection might decrease HIV-1 transmission in human populations and therefore affect the spread of AIDS.

Article Publication: Journal of virology

Article Summary: Emerging data suggest that a cytotoxic T lymphocyte response against a diversity of epitopes confers greater protection against an HIV/SIV infection than does a more focused response. To facilitate the creation of vaccine strategies that will generate cellular immune responses with the greatest breadth, it will be important to understand the mechanisms employed by the immune response to regulate the relative magnitudes of dominant and nondominant epitope-specific cellular immune responses. In this study, we generated dominant Gag p11C- and subdominant Env p41A-specific CD8(+) T lymphocyte responses in Mamu-A*01(+) rhesus monkeys through vaccination with plasmid DNA and recombinant adenovirus (rAd) encoding simian-human immunodeficiency virus (SHIV) proteins. Infection of vaccinated Mamu-A*01(+) rhesus monkeys with a SHIV Gag Deltap11C mutant virus generated a significantly increased expansion of Env p41A-specific CD8(+) T lymphocyte responses in the absence of secondary Gag p11C-specific CD8(+) T lymphocyte responses. These results indicate that the presence of the Gag p11C-specific CD8(+) T lymphocyte response following virus challenge may exert suppressive effects on primed Env p41A-specific CD8(+) T lymphocyte responses. These findings suggest that immunodomination exerted by dominant responses during SHIV infection may diminish the breadth of recall responses primed during vaccination.

Article Publication: Nature medicine

Article Summary: We have identified a new human immunodeficiency virus in a Cameroonian woman. It is closely related to gorilla simian immunodeficiency virus (SIVgor) and shows no evidence of recombination with other HIV-1 lineages. This new virus seems to be the prototype of a new HIV-1 lineage that is distinct from HIV-1 groups M, N and O. We propose to designate it HIV-1 group P.

Article Publication: Nature

Article Summary: Single-stranded RNA viruses encompass broad classes of infectious agents and cause the common cold, cancer, AIDS and other serious health threats. Viral replication is regulated at many levels, including the use of conserved genomic RNA structures. Most potential regulatory elements in viral RNA genomes are uncharacterized. Here we report the structure of an entire HIV-1 genome at single nucleotide resolution using SHAPE, a high-throughput RNA analysis technology. The genome encodes protein structure at two levels. In addition to the correspondence between RNA and protein primary sequences, a correlation exists between high levels of RNA structure and sequences that encode inter-domain loops in HIV proteins. This correlation suggests that RNA structure modulates ribosome elongation to promote native protein folding. Some simple genome elements previously shown to be important, including the ribosomal gag-pol frameshift stem-loop, are components of larger RNA motifs. We also identify organizational principles for unstructured RNA regions, including splice site acceptors and hypervariable regions. These results emphasize that the HIV-1 genome and, potentially, many coding RNAs are punctuated by previously unrecognized regulatory motifs and that extensive RNA structure constitutes an important component of the genetic code.

Article Publication: Expert opinon on biological therapy

Article Summary: Adenovirus (Ad)-based gene transfer has been successfully utilised in gene therapy and vaccine applications. To date, an increasing number of human clinical trials utilise recombinant Ad-based vectors as a gene transfer platform. In particular, progress has been made recently in utilising Ad-based vectors as a vaccine platform in HIV, cancer immunotherapy approaches and in vaccination for other infections. Despite these successes, the scientific and bio-industrial communities have recently recognised that innate and pre-existing immunity against Ad vectors can constitute a serious obstacle to the development and application of this technology. It is essential to overcome vector-mediated immune responses, such as production of inflammatory cytokines and pre-existing immunity to Ad, because the induction of these responses not only shortens the period of gene expression but also leads to serious side effects. This review focuses on the biology of Ad infection and the approaches that are being adopted to overcome immunity against the Ad-based vectors

Article Publication: Reviews in medical virology

Article Summary: Although the development of an effective HIV-1 vaccine has proved very challenging for more than two decades, it remains the best hope to control the HIV pandemic. Since Brazil has particular epidemiological features, as well as adequate policies and infrastructure, the country has been an interesting site for HIV vaccine trials. Since 1995, eight trials were performed in Brazil enrolling over 2000 subjects. Peptide vaccine candidates were initially designed to elicit neutralising antibodies as an attempt to provide sterilising immunity against HIV-1. This strategy, however, has proved extremely difficult, and candidates were poorly immunogenic. Therefore, the next vaccine candidates focused mainly on the induction of cell mediated immune responses that would limit AIDS progression and transmission by suppressing viremia. Such candidates were naked DNA or viral vectors in either prophylactic or therapeutic approaches. Even though several candidates were immunogenic, protective immune responses against HIV-1 remain to be achieved. However, several studies with non-human primates and human elite controllers demonstrate that effective immune responses against HIV-1 may be elicited, supporting the belief that an HIV-1 vaccine is possible. Much has been learned, and now the development of an effective HIV-1 vaccine requires resetting priorities with focus on basic research, considering the merits of neutralising antibodies and CMI, as well as the role of innate immunity on HIV-1 protection. In this new perspective, large-scale trials should be replaced by smaller preliminary efficacy studies. Copyright (c) 2009 John Wiley & Sons, Ltd.

Article Publication: Virology

Article Summary: In non-human primate models of AIDS, attenuated lentiviruses provide the most reliable protection from challenge with pathogenic virus but the extent to which the vaccine virus replicates after challenge is unclear. At 7 and 14 days after vaginal challenge with pathogenic SIVmac239, plasma SIVenv RNA levels were significantly lower in female macaques immunized 6 months earlier with live, attenuated SHIV89.6 compared to unimmunized control animals. In 2 SHIV-immunized, unprotected macaques SIV replication produced moderate-level plasma viremia with dissemination of challenge virus to all tissues on day 14 after challenge. In protected, SHIV-immunized monkeys, SIV replication was controlled in all tissues, from the day of challenge through 14 days post-challenge. Further, in CD8(+) T cell-depleted SHIV-immunized animals, SIV replication and dissemination were more rapid than in control animals. These findings suggest that replication of a pathogenic AIDS virus can be controlled at the site of mucosal inoculation by live-attenuated lentivirus immunization.

Article Publication: Nature immunology

Article Summary: Contact-dependent communication between immune cells generates protection but also facilitates viral spread. Here we found that macrophages formed long-range actin-propelled conduits in response to negative factor (Nef), a human immunodeficiency virus type 1 (HIV-1) protein with immunosuppressive functions. Conduits attenuated immunoglobulin G2 (IgG2) and IgA class switching in systemic and intestinal lymphoid follicles by shuttling Nef from infected macrophages to B cells through a guanine-exchange factor-dependent pathway involving the amino-terminal anchor, central core and carboxy-terminal flexible loop of Nef. By showing stronger virus-specific IgG2 and IgA responses in patients with Nef-deficient virions, our data suggest that HIV-1 exploits intercellular 'highways' as a 'Trojan horse' to deliver Nef to B cells and evade humoral immunity systemically and at mucosal sites of entry.

Article Publication: Journal of infectious diseases

Article Summary: Background. Human immunodeficiency virus type 1 (HIV-1) elite controllers are able to control virus replication to levels below the limits of detection by commercial assays, but the actual level of viremia in these individuals is not well defined. Here, we quantify plasma HIV-1 RNA in elite controllers and correlate this with specific immunologic parameters. Methods. Plasma HIV-1 RNA levels were quantified in 90 elite controllers with use of a real time reverse-transcriptase polymerase chain reaction assay with a sensitivity of 0.2 copies/mL. HIV-1-specific immune responses and longitudinal CD4(+) T cell counts were examined. Results. The median plasma HIV-1 RNA level was 2 copies/mL (interquartile range, 0.2-14 copies/mL). A longitudinal analysis of 31 elite controllers demonstrated 2-5-fold fluctuations in viremia in the majority of individuals; 6 had persistent levels below 1 copy/mL. Viremia correlated directly with HIV-1-specific neutralizing antibodies and Western blot reactivity but not with CD8(+) T cell responses. Absolute CD4(+) T cell decrease was more common among individuals with detectable viremia ([Formula: see text]). Conclusions. Low-level viremia is present in the majority of elite controllers and is associated with higher HIV-1-specific antibody responses. Absolute CD4(+) T cell loss is more common among viremic individuals, suggesting that even very low-level viremia has negative consequences over time.

Article Publication: Vaccine

Article Summary: Recombinant modified vaccinia virus Ankara (rMVA) expressing HIV-1 genes are promising vaccine candidates. Toward the goal of conducting clinical trials with one or a cocktail of recombinant viruses, four rMVAs expressing env and gag-pol genes from primary HIV-1 isolates representing predominant subtypes from Kenya, Tanzania, Uganda, and Thailand (A, C, D, and CRF01_AE, respectively) were constructed. Efficient expression, processing, and function of Env and Gag were demonstrated. All inserted genes were shown to be genetically stable after repeated passage in cell culture. Strong HIV-specific cellular and humoral immune responses were elicited in mice immunized with each individual vaccine candidate. The MVA/CMDR vaccine candidate expressing CRF01_AE genes has elicited HIV-specific T-cell responses in two independent Phase I clinical trials. Further testing of the other rMVA is warranted.

Article Publication: FEBS letters

Article Summary: The global transcriptional profile of PBMCs stimulated with HIV candidate vaccine (Virus-Like Particles, VLPs) has been evaluated in HIV-infected patients with low/high viral load compared to healthy volunteers. Baseline activation of chemokine production was observed in PBMC from HIV infected patients and innate immune stimulation with HIV-VLPs was not blunted. The immune profile among HIV-infected patients was found to be qualitatively similar but quantitatively extremely variable. This diversity was independent of viral load and it might be dependent on individual immunogenetic traits or concurrent immunological status. This ex-vivo screening strategy represents an efficient tool for guiding modifications/optimizations of vaccination strategies and understanding failures in individuals enrolled in clinical trials.

Article Publication: Vaccine

Article Summary: Pre-existing immunity to human adenovirus serotype 5 (AdHu5) has been shown to suppress the immunogenicity of recombinant Ad5 (rAdHu5) vector-based vaccines for human immunodeficiency virus type 1 (HIV-1) in both preclinical studies and clinical trials. As a potential solution to this problem we developed adenovirus vaccine vectors of chimpanzee origin. In the present study we assessed the immunogenicity of various chimpanzee adenovirus vectors in a prime/boost regimen to HIV-1 envelope and SIV Gag-Pol in rhesus monkeys and their ability to protect against pathogenic viral challenge. Although rAdHu5-primed monkeys had higher magnitude T cell responses than rAdC7 or rAdC68 prior to challenge, the rAdC7-rAdC1/C5 and rAdHu5-rAdC1/C5 immunizations resulted in comparable magnitude recall cellular immune responses and comparable level of control of viremia post-challenge

Article Publication: Nature medicine

Article Summary: The design of an effective AIDS vaccine has eluded the efforts of the scientific community to the point that alternative approaches to classic vaccine formulations have to be considered. We propose here that HIV vaccine research could greatly benefit from the study of natural simian immunodeficiency virus (SIV) infections of African nonhuman primates. Natural SIV hosts (for example, sooty mangabeys, African green monkeys and mandrills) share many features of HIV infection of humans; however, they usually do not develop immunodeficiency. These natural, nonprogressive SIV infections represent an evolutionary adaptation that allows a peaceful coexistence of primate lentiviruses and the host immune system. This adaptation does not result in reduced viral replication but, rather, involves phenotypic changes to CD4(+) T cell subsets, limited immune activation and preserved mucosal immunity, all of which contribute to the avoidance of disease progression and, possibly, to the reduction of vertical SIV transmission. Here we summarize the current understanding of SIV infection of African nonhuman primates and discuss how unraveling these evolutionary adaptations may provide clues for new vaccine designs that might induce effective immune responses without the harmful consequences of excessive immune activation.

Article Publication: European journal of immunology

Article Summary: Heterologous adenovirus-based vectors hold promise as preventative HIV vaccines but their capacity to induce effective T-cell immunity in established infection has not been explored. We vaccinated rhesus macaques chronically infected with SIVmac251 and undergoing antiretroviral therapy (ART) with human adenovirus serotype 5-based vectors expressing SIV Gag, Env, and Nef with and without IL-15 and evaluated vaccine immunogenicity. Vaccination increased Ag-specific T cells 20-fold but did not expand the breadth of epitopes recognized or the quality of response, as the majority of CD8(+) and CD4(+) T cells produced only one cytokine irrespective of vaccination. Immunization transiently restored blood CD4(+) central memory T cells (Tcm) and boosted CD4(+) and CD8(+) Tcm and effector cell responses but did not prevent virus rebound upon cessation of ART. Boosting with human adenovirus serotype 35-based vectors during a second ART cycle increased Ag-specific T cells to 50-fold above pre-vaccination levels and boosted CD4(+) Tcm numbers but did not expand the breadth or quality of immunity or control virus levels following drug discontinuation. The number of blood CD4(+) Tcm correlated positively with complexity of T-cell responses and negatively with virus load, suggesting that more complete restoration of this subset through vaccination would be beneficial.

Article Publication: Journal of virology

Article Summary: The region of the HIV-1 envelope glycoprotein gp120 that engages its primary cellular receptor CD4, forms a site of vulnerability to neutralizing antibodies. The monoclonal antibody b12 exploits the conservation and accessibility of the CD4-binding site to neutralize many, though not all HIV-1 isolates. To understand the basis of viral resistance to b12, we used the atomic-level definition of b12-gp120 contact sites to study a panel of diverse circulating viruses. A combination of sequence analysis, computational modeling, and site-directed mutagenesis were used to determine the influence of amino-acid variants on binding and neutralization by b12. We found that several substitutions within the dominant b12-contact surface, called the CD4-binding loop, mediated b12 resistance and that these substitutions resided just proximal to the known CD4-contact surface. Hence, viruses varied key b12-contact residues which are proximal to, but not part of, the CD4-contact surface. This explained how viral isolates were able to evade b12 neutralization while maintain functional binding to CD4. In addition, some viruses were resistant to b12 despite minimal sequence variation at b12-contact sites. Such neutralization resistance could usually be reversed by alterations at residues thought to influence the quaternary configuration of the viral envelope spike. To design immunogens that elicit neutralizing antibodies directed to the CD4-binding site, researchers will need to address the antigenic variation within this region of gp120, and the restricted access to the CD4-binding site imposed by the native configuration of the trimeric viral envelope spike.

Article Publication: Journal of virology

Article Summary: The administration of vectors designed to elicited cell-mediated immune responses may have other consequences that are clinically significant. To explore this possibility, we evaluated T cell activation during the first two months following rAd5 prime or boost immunizations in rhesus monkeys. We also evaluated the kinetics of T lymphocyte activation in both the systemic and mucosal compartments following rAd5 administration in monkeys with pre-existing immunity to Ad5. The rAd5 immunization induced lower frequency Gag epitope-specific CD8(+) T cells in the colonic mucosa than in the peripheral blood. There was evidence of an expansion of the SIV Gag-specific CD8(+) T cell responses, but not the Ad5 hexon-specific T cell responses, following homologous rAd5 boost. A striking but transient T lymphocyte activation in both the systemic and mucosal compartments of rhesus monkeys was observed following rAd5 immunization. These findings indicate that the administration of a vaccine vector such as Ad5 can induce a global activation of T cells.

Article Publication: Journal of virology

Article Summary: We identified three cross-neutralizing plasma samples with high titer anti-MPER peptide binding antibodies from among 156 HIV-1 chronically infected individuals. In order to establish if these antibodies were directly responsible for the observed neutralization breadth, we used MPER-coated magnetic beads to deplete plasmas of these specific antibodies. Depletion of anti-MPER antibodies from BB34, CAP206 and SAC21 resulted in a 77%, 68% and 46% decrease respectively in the number of viruses neutralized. Antibodies eluted from the beads showed similar neutralization profiles as the original plasmas, with potencies comparable to the known anti-MPER MAbs, 4E10, 2F5 and Z13e1. The anti-MPER neutralizing antibodies in BB34 were present in the IgG3 subclass-enriched fraction. Alanine scanning of the MPER showed that the antibodies from these three plasmas had specificities distinct from the known MAbs, requiring one to three crucial residues at positions 670, 673 and 674. These data demonstrate the existence of MPER-specific cross-neutralizing antibodies in plasma although the ability to elicit such potent anti-viral antibodies during natural infection appears to be rare. Nevertheless, the identification of three novel antibody specificities within the MPER supports its further study as a promising target for vaccine design.

Article Publication: Journal of virology

Article Summary: AIDS vaccination has a pressing need for more potent vaccination vectors capable of eliciting strong, diversified and long-lasting cellular immune responses against HIV. Lentiviral vectors have a proven efficiency not only as gene delivery vehicles for gene therapy applications but also as vaccination tools. This is likely due to their ability to transduce non-dividing cells, including dendritic cells, enabling sustained endogenous antigen presentation and thus the induction of high proportions of specific cytotoxic T cells and long-lasting memory T cells. We show in a first proof-of-concept pilot study that a prime/boost vaccination strategy using lentiviral vectors pseudotyped with a glycoprotein G from two non-cross-reactive VSV serotypes elicited robust and broad cellular immune responses against the vector-encoded antigen, SIV GAG, in cynomolgus macaques. Vaccination conferred strong protection against a massive intra-rectal challenge with SIVmac251 as evidenced both by the reduction of viremia at the peak of acute infection (a mean of over 2 log10 fold reduction) and the full preservation of the CD28+ CD95+ memory CD4+ T cells during the acute phase, a strong correlate of protection against pathogenesis. Although vaccinees continued to display lower viremia than control macaques during the early chronic phase, these differences were not statistically significant by day 50 post-challenge. A non-optimized SIV GAG antigen was voluntary chosen to underline the strong potential of the lentiviral vector system for vaccination. Given that a stronger protection can be anticipated from a modern HIV-1 antigen design, gene transfer vectors derived from HIV-1 appear as promising candidates for vaccination against HIV-1 infection

Article Publication: Journal of virology

Article Summary: Understanding the mechanisms underlying potential altered susceptibility to HIV-1 infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1 infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific CTL responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTL, which have been associated with disease control, were detected after but not before seroconversion in LSC63. Furthermore, for the majority of the protein coding regions of HIV-1 variants in LSC63 (except gp41, nef and 3' half of pol), the genetic distances between the infecting and exposed (P63) viruses were comparable to the distances between random subtype B HIV-1 sequences and the exposed (P63) viruses. These results suggest that broad pre-infection immune responses were not able to prevent acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.

Article Publication: Clinical pharmacology & therapeutics

Article Summary:

Article Publication: Science

Article Summary: Broadly neutralizing antibodies (bNAbs), which develop over time in some HIV-1–infected individuals, define critical epitopes for HIV vaccine design. Using a systematic approach, we have examined neutralization breadth in the sera of about 1800 HIV-1–infected individuals, primarily infected with non–clade B viruses, and have selected donors for monoclonal antibody (mAb) generation. We then used a high-throughput neutralization screen of antibody-containing culture supernatants from approximately 30,000 activated memory B cells from a clade A–infected African donor to isolate two potent mAbs that target a broadly neutralizing epitope. This epitope is preferentially expressed on trimeric Envelope protein and spans conserved regions of variable loops of the gp120 subunit. The results provide a framework for the design of new vaccine candidates for the elicitation of bNAb responses.

Article Publication: Human vaccines

Article Summary: The U.S. vaccine and immunization enterprise has been successful in introducing new vaccines to the U.S. and global markets. The vaccine sector of the pharmaceutical industry is experiencing growth projected to increase through the next decade. Despite the successes and future promise, challenges exist for funding the research and development and delivery of new vaccines. Adult and adolescent vaccination coverage of routinely recommended vaccines remains low and financial barriers to vaccination have emerged due to the increased cost of vaccination including the increased price of newer vaccines. Because the development and manufacture of new vaccines rely on the sales of established vaccines and other products, high coverage rates are important to the vaccine industry. Moreover, government and foundation support can assist in the research and development and introduction of new vaccines, especially for those vaccines that are necessary for use in the developing world. Novel approaches are needed to encourage and expand partnerships to advance vaccine research and development. Manufacturers in emerging economies have an increasing role in providing innovative vaccines to the world

Article Publication: Aids

Article Summary: OBJECTIVES:: There is a need to develop HIV-1 vaccine formulations that incorporate inexpensive antigens and clinically acceptable potent adjuvants for inducing neutralizing antibodies. The purpose of this initial vaccine study was to produce peptide- and lipid-induced murine mAbs that replicate the characteristics of the 2F5 and/or 4E10 human antibodies in binding both to the membrane proximal external region (MPER) of glycoprotein 41 and the adjacent lipid bilayer for neutralizing HIV-1 infection of CD4 lymphocytes. RESEARCH DESIGNS AND METHODS:: Liposomes containing a synthetic MPER peptide as a peptide antigen, phosphatidylinositol-4-phosphate (PIP) as a lipid antigen, and monophosphoryl lipid A as a potent adjuvant were used as a formulation to immunize mice. mAbs were then produced and tested for binding to MPER, glycoprotein 41, and PIP and for the ability to neutralize HIV-1 infection of CD4 cells in a human peripheral blood mononuclear cell assay. RESULTS:: Polyclonal antisera contained antibodies that bound both to MPER and PIP. Immunoglobulin M mAbs were produced that bound both to the core MPER site of 2F5, or that overlapped with the 4E10 site, and that simultaneously bound PIP. High concentrations of these mAbs neutralized infection of peripheral blood lymphocytes by a primary infectious molecular clone of HIV-1. CONCLUSION:: Liposomes containing MPER peptide as an antigen, PIP as a lipid antigen, and lipid A as an adjuvant induce anti-MPER-specific multispecific antibodies that simultaneously bind glycoprotein 41 MPER and adjacent lipid and neutralize HIV-1 infection in a human peripheral blood mononuclear cell assay.

Article Publication: Vaccine

Article Summary: We developed highly expressing clade B and AE DNA and envelope protein (Env) vaccines for evaluation in mice and macaques as DNA prime/protein boost regimens. High levels of Env-specific antibodies were induced in mice, albeit with limited neutralizing activity in vitro. A combined clade B and AE regimen induced high titer Env-specific antibody in two pigtail macaques that neutralized several strains of HIV-1. However, upon mucosal challenge with SHIV(SF162P3) no protection from infection was observed. Although the vaccines tested provide a platform for inducing robust humoral immunity, further refinements to broaden coverage against divergent strains and induce mucosal immunity are needed.

Article Publication: Vaccine

Article Summary: DNA- and modified virus Ankara (MVA)-vectored candidate vaccines expressing human immunodeficiency virus type 1 (HIV-1) clade A-derived p24/p17 gag fused to a string of HLA class I epitopes, called HIVA, were tested in phase I trials in healthy, HIV-1/2-uninfected adults in Oxford, United Kingdom. Eighteen volunteers were vaccinated with pTHr.HIVA DNA (IAVI-001) alone, 8 volunteers received MVA.HIVA (IAVI-003) alone and 9 volunteers from study IAVI-001 were boosted with MVA.HIVA 9-14 months after DNA priming (IAVI-005). Immunogenicity results observed in these trials was published previously [Mwau M, Cebere I, Sutton J, Chikoti P, Winstone N, Wee EG-T, et al. An HIV-1 clade A vaccine in clinical trials: stimulation of HIV-specific T cell responses by DNA and recombinant modified vaccinia virus Ankara (MVA) vaccines in humans. J Gen Virol 2004;85:911-9]. Here, we report on the safety of the two vaccines and the vaccine regimes. Overall, both candidate vaccines were safe and well tolerated. There were no reported vaccine-related adverse events over the 6-month period of the study and up to 2 years after the last vaccination. There were no moderate or severe local symptoms recorded after the pTHr.HIVA DNA intramuscular administration. Almost all participants experienced local reactogenicity events such as redness and induration after MVA.HIVA intradermal injection. Thus, the results from these initial small phase I trials administering the pTHr.HIVA DNA and MVA.HIVA vaccines either alone or in a prime-boost regime to healthy HIV-1/2-negative adults indicated that the vaccines were safe and warranted further testing of this approach in larger phase I/II studies.

Article Publication: Lancet

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Article Publication: Lancet

Article Summary: Mother-to-child transmission (MTCT) of HIV-1 is the major mode of paediatric infection. The rapidly increasing incidence of MTCT worldwide has resulted in an urgent need for preventive strategies. Antiretroviral regimens can prevent intrapartum HIV transmission; however, these regimens do not prevent HIV transmission through breastfeeding. Furthermore, children who escape MTCT are again at risk of infection when they become sexually active as adolescents. An infant vaccine regimen, begun at birth, would hence be a more attractive strategy and might also provide the basis for lifetime protection. Unique features of MTCT and paediatric HIV disease could be helpful in understanding correlates of immune protection and could facilitate rapid assessment of vaccine efficacy. Thus, there is compelling rationale to develop safe, effective HIV vaccines for use in infants and children. Here, we discuss the scientific and logistical challenges for the development of paediatric HIV vaccines; available vaccines and completed or planned paediatric vaccine trials are also discussed.

Article Publication: Lancet

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Article Publication: Vaccine

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Article Publication: Lancet

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Article Publication: Reviews in medical virology

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Article Publication: Expert reviews of vaccine

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Article Publication: Journal of acquired immune deficiency syndromes

Article Summary: Because increasing numbers of HIV vaccine candidates are being tested globally, it is essential to differentiate vaccine- from virus-induced antibodies. Most of the currently tested vaccines contain multiple viral components. As a result, many vaccine recipients give positive results in FDA-licensed HIV serodetection tests. We have identified conserved sequences in Env-gp41 and Gag-p6, which are recognized soon after infection but are not included in most HIV vaccine candidates. A new HIV serodetection assay, the HIV-SELECTEST, was established that distinguishes between vaccine-induced antibodies and seroconversion due to true HIV infections. It is important to make this assay globally relevant, because many clinical trials are conducted around the world where most HIV infections are due to non-B subtype HIV-1. Therefore, the current study examined the reactivity of plasma samples from >3,000 infections with diverse HIV subtypes worldwide. The HIV-SELECTEST performed at >99% specificity and sensitivity. Both recent and established infections with clades A, B, C, D, E, F, G, J, and CRFs were detected. Antibodies elicited by other vaccinations or infections endemic to the clinical trial sites did not react in this assay. Therefore, HIV-SELECTEST could be an important differential diagnostic tool for HIV vaccine trials, blood banks, and population screening worldwide.

Article Publication: Journal of Acquired Immune Deficiency Syndromes

Article Summary: OBJECTIVE: Allocation of funds to program areas where they may have an impact is critical to the success of any HIV control program. We examined the cost-effectiveness of providing first-line treatment for male trichomoniasis in Malawi, a condition not commonly considered in syndromic management throughout sub-Saharan Africa. METHODS: We used decision tree analysis to assess program costs and outcomes among a 1-year population of male sexually transmitted disease (STD) clinic attendees estimated at 10,000 in Lilongwe. Our main outcomes were program costs from the government perspective and HIV infections averted. We conducted univariate and multivariate sensitivity analyses on selected parameters. RESULTS: In our study population of male STD clinic attendees with an HIV prevalence of 44% and a Trichomonas vaginalis prevalence of 20%, including universal metronidazole as a first-line treatment for trichomoniasis at $0.05 per dose would increase program costs by $277 (year 2000 US dollars) and avert 23 cases of HIV. The incremental cost-effectiveness ratio (ICER) over the current STD management guidelines was $15.42 per case of HIV averted. The number of HIV infections averted under sensitivity analysis ranged from 2 to 52, with attendant ICERs varying from cost savings to $162.92. Consideration of wider social benefits, such as the costs of HIV infections to the individual or the government, would further enhance the cost-effectiveness of this program. CONCLUSIONS: As part of a larger program to control STDs, incorporating metronidazole to treat male trichomoniasis could represent a cost-effective means to reduce HIV transmission in this high-risk group

Article Publication: Immunology letters

Article Summary: Reliable and effective methods for induction of cytotoxic T-lymphocytes (CTL) are constantly persued. Central to this search is work in animal models, which allow to test novel vaccine strategies and ultimately lead to a more efficient planning of clinical trials. Here, human immunodeficiency virus (HIV) vaccine candidates were constructed as a string of partially overlapping CTL epitopes (20 human, 3 macaque and 1 mouse) delivered and expressed using plasmid DNA and modified virus Ankara (MVA; an attenuated vaccinia virus), which are both vaccine vehicles acceptable for use in humans. In mice, these vaccines were shown to induce virus-specific interferon-gamma-producing and cytolytic CD8+ T-cells after a single intramuscular needle injection. When immunization protocols were sought which would improve the level of induced HIV-specific T-cells, DNA priming-MVA boosting was found to be the most potent protocol. The multi-epitope DNA also elicited CTL when delivered intradermally using the Accell gene delivery device (gene gun). Finally, a combined intradermal gene gun DNA-MVA vaccination regimen induced in macaques high frequencies of circulating CTL, which were comparable to those observed in simian immunodeficiency virus (SIV)-infected monkeys. Further optimization of this method in non-human primates is under way. Thus, a vaccination regimen for an effective elicitation of CTL has been developed which might facilitate evaluation of the role(s) that these lymphocytes play in the control of SIV and HIV infections.

Article Publication: Journal of virology

Article Summary: The quest to create an HIV-1 vaccine capable of eliciting broadly neutralizing antibodies (bnAbs) against Env has been challenging. Amongst others, one difficulty in creating a potent immunogen resides in the substantial overall sequence variability of the HIV envelope protein. The membrane-proximal region (MPER) of gp41 is a particularly conserved tryptophan-rich region spanning residues 659 to 683, which is recognized by three bnAbs (2F5, Z13 and 4E10). In this study, we first describe the variability of residues in the gp41 MPER and report on the invariant nature of 15 out of 25 amino acids comprising this region. Subsequently, we evaluate the ability of the bnAb 2F5 to recognize 31 varying sequences of the gp41 MPER at a molecular level. In 19 cases, resulting crystal structures show the various MPER peptides bound to the 2F5 Fab'. A variety of amino acid substitutions outside the (664)DKW(666) core epitope are tolerated. However, changes at the (664)DKW(666) motif itself are restricted to those residues that preserve the aspartate's negative charge, the hydrophobic alkyl-pi stacking arrangement between the beta-turn lysine and tryptophan, and the positive charge of the former. We also characterize a possible molecular mechanism of 2F5 escape by sequence variability at position 667, which is often observed in HIV-1 clade C isolates. Based on our results, we propose a somewhat more flexible molecular model of epitope recognition by bnAb 2F5, which could guide future attempts at designing small molecule MPER-like vaccines capable of eliciting 2F5-like bnAbs.

Article Publication: Gene therapy

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Article Publication: Virology

Article Summary: The extraordinarily high level of genetic variation of HIV-1 env genes poses a challenge to obtain antibodies that cross-react with multiple subtype Env glycoproteins. To determine if cross-reactive monoclonal antibodies (mAbs) to highly conserved epitopes in HIV-1 envelope glycoproteins can be induced, we immunized mice with wild-type or consensus HIV-1 Env proteins and characterized a panel of ten mAbs that reacted with varying breadth to subtypes A, B, C, D, F, G, CRF01_AE, and a highly divergent SIVcpzUS Env proteins by ELISA and Western blot analysis. Two mAbs (3B3 and 16H3) cross-reacted with all tested Env proteins, including SIVcpzUS Env. Surface plasmon resonance analyses showed both 3B3 and 16H3 bound Env proteins with high affinity. However, neither neutralized primary HIV-1 pseudoviruses. These data indicate that broadly reactive non-neutralizing monoclonal antibodies can be elicited, but that the conserved epitopes that they recognize are not present on functional virion trimers. Nonetheless, such mAbs represent valuable reagents to study the biochemistry and structural biology of Env protein oligomers.

Article Publication: Human vaccines

Article Summary: As yet, the only human infectious disease eradicated from our planet is smallpox, caused by variola virus a member of the poxvirus family. The vaccination success, with the declaration by WHO in 1980 of a worldwide free of smallpox, was largely due to the availability of a quite effective and stable live vaccine, as well as the restricted human host for virus infection. Variola was considered one of the most devastating diseases of human mankind. With the sudden appearance of the HIV/AIDS in 1981, an infection which spread rapidly to become a pandemic in a short time, causing up to date more than 22 million deaths, about 40 million people infected and a current incidence of about 3 million deaths per year, this dreadful pandemic has become one of the most severe diseases in the World, specially in poor countries. While different antiviral drugs have been developed that block virus replication at various stages of infection, however the rapid virus escape that follows during the drug therapy due to mutations, makes the development of vaccines the most secure option to control and eradicate the disease. Numerous vaccines have been developed, but to date the clinical trials have failed to show any efficacy against HIV infection. Due to the proven success of vaccinia virus in the control of smallpox as well as of poxvirus recombinants against veterinary diseases, a major effort has been directed to document the advantages of poxvirus vectors as vaccines against multiple diseases. Two of the most promising poxvirus vectors are the highly attenuated modified vaccinia virus Ankara (MVA) and the modified Copenhagen strain NYVAC. In this commentary I describe the biological characteristics of the attenuated poxvirus vectors, MVA and NYVAC, with emphasis in their application in HIV preclinical and clinical trials, and considerations as future HIV vaccines.

Article Publication: Vaccine

Article Summary: A Phase I human vaccine trial of a novel polypeptide vaccine of HIV T helper epitopes (EP-1043) and a DNA vaccine of HIV CTL epitopes was conducted in 84 healthy adult volunteers. The vaccine immunogenicity was assessed by an intracellular cytokine staining assay for IL-2, IL-4, TNF-alpha and IFN-gamma. Sixty eight percent (32/47) of subjects had a positive CD4+ T response after receiving two vaccinations of the polypeptide vaccine. The responding CD4+ T cells made various combinations of IL-2, IL-4, IFN-gamma, and TNF-alpha. The study demonstrated that the EP-1043 vaccine is safe, well-tolerated, and immunogenic.

Article Publication: Vaccine

Article Summary: Coordinated interactions between helper and cytotoxic T-lymphocytes (HTL and CTL) are needed for optimal effector cell functions and the establishment of immunological memory. We, therefore, designed a mixed format vaccine based on the use of highly conserved HIV-derived T-lymphocyte epitopes wherein the HTL epitopes were delivered as a recombinant protein and the CTL epitopes which were encoded in a DNA vaccine plasmid. Immunogenicity testing in HLA transgenic mice and GLP preclinical safety testing in rabbits and guinea pigs were used to document the utility of this approach and to support Phase 1 trial clinical testing. Both vaccine components were immunogenic and safely co-administered.

Article Publication: Vaccine

Article Summary: Reducing mother to child transmission (MTCT) of HIV in resource poor countries continues to be a major challenge. Here, we construct a hazard model to assess the effectiveness of combinations of HIV vaccine, Nevirapine (NVP), and HIV-specific monoclonal antibody (HIVAB) in reducing MTCT of HIV during the intrapartum and breastfeeding periods. The model shows that an intervention that uses three doses of vaccine with 30% initial immunity and 30% boost effect with subsequent doses (giving rise to maximum immunity approximately 66% with 3 doses) could reduce MTCT to 7.7% when used with NVP and to 5.9% when used with NVP and HIVAB. Using a vaccine with 50% initial immunity and 50% boost can reduce the rate to 4.3%. These results indicate that even an imperfect vaccine, when used in combination with other therapies, can be of considerable benefit in preventing MTCT in resource poor countries.

Article Publication: Lancet

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Article Publication: International reviews of immunology

Article Summary: The HIV epidemic continues to represent one of the major problems worldwide, particularly in the Asia and Sub-Saharan regions of the world, with social and economical devastating effects. Although antiretroviral drugs have had a dramatically beneficial impact on HIV-infected individuals that have access to treatment, it has had a negligible impact on the global epidemic. Hence, the inexorable spreading of the HIV pandemic and the increasing deaths from AIDS, especially in developing countries, underscore the urgency for an effective vaccine against HIV/AIDS. However, the generation of such a vaccine has turned out to be extremely challenging. Here we provide an overview on the rationale for the use of non-structural HIV proteins, such as the Tat protein, alone or in combination with other HIV early and late structural HIV antigens, as novel, promising preventative and therapeutic HIV/AIDS vaccine strategies.

Article Publication: Annual review of medicine

Article Summary: Developing an HIV-1 vaccine that can elicit antibodies to prevent infection has been a formidable challenge. Although no single immunogen has generated antibodies that can neutralize diverse isolates, progress has been made in understanding (a) the structure of the HIV-1 envelope glycoprotein, which is targeted by neutralizing antibodies, (b) how HIV-1 evades antibodies made by an infected host, and (c) how rare monoclonal antibodies can exhibit broadly neutralizing activity. Advances in structural and molecular biology coupled with new approaches to isolate neutralizing antibodies from HIV-1-infected individuals are enhancing our understanding of what humoral immune responses will be required for a vaccine. This review summarizes progress in understanding the host antibody response to HIV-1 and current strategies for applying this information to develop an effective vaccine.

Article Publication: Aids

Article Summary: Neutralizing activity of secretory immunoglobulin A (S-IgA) directed against the V1/V2 domain of HIV-1 was studied in parotid saliva of HIV-1- infected patients in Colombian and French cohorts. Purified V1/V2-specific S-IgA antibodies were found to neutralize clades A, B and C primary isolates in five out 76 and 82 patients from each cohort, respectively. These results suggest that neutralizing S-IgA antibodies targeting the V1/V2 domain may provide protection against HIV-1 infection in vivo and may be beneficial in mucosal vaccines.

Article Publication: Journal of virology

Article Summary: A recent clinical trial of a T-cell-based AIDS vaccine delivered with recombinant adenovirus 5 (rAd5) vectors showed no efficacy in lowering viral load and was associated with increased risk of HIV-1 infection. Preexisting immunity to adenovirus serotype 5 (Ad5) in humans could therefore affect both immunogenicity and vaccine efficacy. Here, we hypothesize that vaccine-induced immunity is differentially affected, depending on whether subjects are exposed to Ad5 by natural infection or by vaccination. Sera from vaccine subjects receiving a DNA/rAd5 AIDS vaccine with or without prior immunity to Ad5 were examined for the specificity of their Ad5 neutralizing antibodies and their effect on HIV-1 immune responses. We report that rAd5 neutralizing antibodies were directed to different components of the virion depending on whether they were elicited by natural infection or vaccination in HIV vaccine trial subjects. Neutralizing antibodies elicited by natural infection were directed largely to the Ad5 fiber, while exposure to rAd5 through vaccination elicited antibodies primarily to capsid proteins other than fiber. Notably, pre-existing immunity to Ad5 fiber from natural infection significantly reduced the CD4 and CD8 cell responses to HIV Gag after DNA/rAd5 vaccination. The specificity of Ad5 neutralizing antibodies therefore differs depending on the route of exposure, and natural Ad5 infection compromises Ad5 vaccine-induced immunity to weak immunogens such as HIV-1 Gag. These results have implications for future AIDS vaccine trials and the design of next generation gene-based vaccine vectors.

Article Publication: Cell host and microbes

Article Summary: Monoclonal antibodies that effectively neutralize HIV-1 have been widely sought, yet few have been isolated. Now, technological advances in sera evaluation, B cell stimulation, microneutralization, and antibody cloning have allowed Burton and colleagues to identify two broadly neutralizing monoclonal antibodies, PG9 and PG16, which provide insights for HIV vaccine design.

Article Publication: Nature reviews immunology

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Article Publication: Vaccine

Article Summary: The native HIV-1 Tat protein was chosen as vaccine candidate for phase I clinical trials based on its role in the natural infection and AIDS pathogenesis, on the association of Tat-specific immune response with the asymptomatic stage as well as on its sequence conservation among HIV clades. A randomized, double blind, placebo-controlled phase I study (ISS P-001) was conducted in healthy adult volunteers without identifiable risk of HIV infection. Tat was administered 5 times monthly, subcute in alum or intradermic alone at 7.5mug, 15mug or 30mug, respectively (ClinicalTrials.gov identifier: NCT00529698). Vaccination with Tat resulted to be safe and well tolerated (primary endpoint) both locally and systemically. In addition, Tat induced both Th1 and Th2 type specific immune responses in all subjects (secondary endpoint) with a wide spectrum of functional antibodies that are rarely seen in natural infection, providing key information for further clinical development of the Tat vaccine candidate.

Article Publication: Vaccine

Article Summary: Broad CTL response against HIV-1 is one factor that helps to control the viral replication. We have constructed a DNA vaccine that encodes a large artificial fusion protein (MultiHIV) and shown it to be immunogenic in mice, swine and macaques. Inbred mice revealed CTL response only against two epitopes due to limited MHC class I variability. To assess the quality of the CTL response we addressed this question in domestic swine. Number of presented epitopes varied between 7 and 14 among the five selected animals. Epitopes detected in swine are localised in the same antigenic regions recognised in humans. This can be explained by the fact that swine MHC-I (SLA-I) complex is remarkably similar to human HLA-I. These results also indicate that immunogenicity profile of vaccines in domestic swine may predict the outcome of human immunisation.

Article Publication: Vaccine

Article Summary: The capacity of immune complexes to augment antibody (Ab) responses is well established. The enhancing effects of immune complexes have been attributed mainly to Fc-mediated adjuvant activity, while the ability of Abs to induce antigenic alterations of specific epitopes as a result of immune complex formation has been less well studied. Previously we have shown that the interaction of anti-CD4-binding site (CD4bs) Abs with HIV-1 gp120 induces conformation changes that lead to enhanced antigenicity and immunogenicity of neutralizing epitopes in the V3 loop. The present study shows that significant increases in the antigenicity of the V3 and C1 regions of gp120 were attained for several subtype B gp120s and a subtype C gp120 upon immune complex formation with the anti-CD4bs monoclonal Ab (mAb) 654-D. Such enhancement was observed with immune complexes made with other anti-CD4bs mAbs and anti-V2 mAbs, but not with anti-C2 mAbs, indicating this activity is determined by antigen specificity of the mAb that formed the immune complex. When immune complexes of gp120(LAI)/654-D and gp120(JRFL)/654-D were tested as immunogens in mice, serum Abs to gp120 and V3 were generated at significantly higher titers than those induced by the respective uncomplexed gp120s. Notably, the anti-V3 Ab responses had distinct fine specificities; gp120(JRFL)/654-D stimulated more cross-reactive anti-V3 Abs than gp120(LAI/)654-D. Neutralizing activities against viruses with heterologous envelope were also detected in sera of mice immunized with gp120(JRFL)/654-D, although the neutralization breadth was still limited. Overall this study shows the potential use of gp120/Ab complexes to augment the immunogenicity of HIV-1 envelope gp120, but further improvements are needed to elicit virus-neutralizing Ab responses with higher potency and breadth.

Article Publication: Journal of virology

Article Summary: The membrane-proximal external region (MPER) of HIV-1, located at the C-terminus of the gp41 ectodomain, is conserved and crucial for viral fusion. Three broadly neutralizing monoclonal antibodies (bnMAbs), 2F5, 4E10 and Z13e1, are directed against linear epitopes mapped to the MPER, making this conserved region an important potential vaccine target. However, no MPER antibodies have been definitively shown to provide protection against HIV challenge. Here, we show that both MAbs 2F5 and 4E10 can provide complete protection against mucosal SHIV challenge in macaques. MAb 2F5 or 4E10 was administered intravenously at 50 mg/kg to groups of 6 male Indian rhesus macaques one day prior to and again one day following intrarectal challenge with SHIVBaL. In both groups, 5 out of 6 animals showed complete protection and sterilizing immunity, while for 1 animal in each group a low level of viral replication following challenge could not be ruled out. The study confirms the protective potential of 2F5 and 4E10 and supports emphasis on HIV immunogen design based on the MPER region of gp41.

Article Publication: Journal of medical primatology

Article Summary: BACKGROUND: Pathogenic HIV and SIV infections characteristically deplete central memory CD4(+) T cells and induce chronic immune activation, but it is controversial whether this also occurs after vaccination with attenuated SIVs and whether depletion or activation of CD4(+) T-cell play roles in protection against wild-type virus challenge. METHODS: Rhesus macaques were vaccinated with SIVmac239Deltanef and quantitative and phenotypic polychromatic flow cytometry analyses were performed on mononuclear cells from blood, lymph nodes and rectal biopsies. RESULTS: Animals vaccinated with SIVmac239Deltanef demonstrated no loss of CD4(+) T cells in any tissue, and in fact CCR5(+) and CD28(+)CD95(+) central memory CD4(+) T cells were significantly increased. In contrast, CD4(+) T-cell numbers and CCR5 expression significantly declined in unvaccinated controls challenged with SIVmac239. Also, intracellular Ki67 increased acutely as much as 3-fold over baseline in all tissues after SIVmac239Deltanef vaccination then declined following primary infection. CONCLUSION: We demonstrated in this study that SIVmac239Deltanef vaccination did not deplete CD4(+) T cells but transiently activated and expanded the memory cell population. However, increases in numbers and activation of memory CD4(+) T cells did not appear to influence protective immunity.

Article Publication: Proceedings of the National Academy of Sciences of the United States of America

Article Summary: In the recently halted HIV type 1 (HIV-1) vaccine STEP trial, individuals that were seropositive for adenovirus serotype 5 (Ad5) showed increased rates of HIV-1 infection on vaccination with an Ad5 vaccine. We propose that this was due to activation and expansion of Ad5-specific mucosal-homing memory CD4 T cells. To test this hypothesis, Ad5 and Ad11 antibody titers were measured in 20 healthy volunteers. Dendritic cells (DCs) from these individuals were pulsed with replication defective Ad5 or Ad11 and co-cultured with autologous lymphocytes. Cytokine profiles, proliferative capacity, mucosal migration potential, and susceptibility to HIV infection of the adenovirus-stimulated memory CD4 T cells were measured. Stimulation of T cells from healthy Ad5-seropositive but Ad11-seronegative individuals with Ad5, or serologically distinct Ad11 vectors induced preferential expansion of adenovirus memory CD4 T cells expressing α4β7 integrins and CCR9, indicating a mucosal-homing phenotype. CD4 T-cell proliferation and IFN-γ production in response to Ad stimulation correlated with Ad5 antibody titers. However, Ad5 serostatus did not correlate with total cytokine production upon challenge with Ad5 or Ad11. Expanded Ad5 and Ad11 memory CD4 T cells showed an increase in CCR5 expression and higher susceptibility to infection by R5 tropic HIV-1. This suggests that adenoviral-based vaccination against HIV-1 in individuals with preexisting immunity against Ad5 results in preferential expansion of HIV-susceptible activated CD4 T cells that home to mucosal tissues, increases the number of virus targets, and leads to a higher susceptibility to HIV acquisition.

Article Publication: Science

Article Summary: The site on HIV-1 gp120 that binds to the CD4 receptor is vulnerable to antibodies. However, most antibodies that interact with this site cannot neutralize HIV-1. To understand the basis of this resistance, we determined co-crystal structures for two poorly neutralizing, CD4–binding site (CD4BS) antibodies, F105 and b13, in complexes with gp120. Both antibodies exhibited approach angles to gp120 similar to those of CD4 and a rare, broadly neutralizing CD4BS antibody, b12. Slight differences in recognition, however, resulted in substantial differences in F105- and b13-bound conformations relative to b12-bound gp120. Modeling and binding experiments revealed these conformations to be poorly compatible with the viral spike. This incompatibility, the consequence of slight differences in CD4BS recognition, renders HIV-1 resistant to all but the most accurately targeted antibodies.

Article Publication: Tropical medicine & international health

Article Summary: Objectives To assess the degree of haematological and biochemistry abnormalities associated with splenomegaly in asymptomatic adults in order to determine whether they may be eligible for inclusion in HIV biomedical prevention trials. Methods Asymptomatic adults (50% women) aged 18–60 with splenomegaly (≥grade II by Hackett's classification) who agreed to provide blood and urine specimens for laboratory testing were invited to participate in a cross-sectional study. Volunteers who were menstruating, pregnant, infected with HIV, syphilis or Hepatitis B and C, or had significant clinical findings were excluded. Haematological and biochemistry laboratory evaluations were performed for enroled volunteers, and the results were compared to local reference ranges. The proportion of volunteers with out-of-range (OOR) values was estimated for each parameter. Linear regression models were fitted to investigate the association between grade of splenomegaly and laboratory values. Results The proportion of volunteers with OOR haematology values ranged from 4.5% (mean corpuscular volume) and 15% (CD4 cells) to 31% (basophils). Increasing spleen size was significantly associated with anaemia, thrombocytopenia and low CD4 count. OOR biochemistry values were found in about 10% of volunteers. Increasing spleen size was associated with reduced creatinine phosphokinase and creatinine (in men) and raised lactate dehydrogenase. Conclusions In areas with a high prevalence of splenomegaly, most asymptomatic individuals with this condition have haematology and biochemistry values that fall within the local reference ranges, and they could therefore be eligible for inclusion in HIV biomedical prevention trials. However, the effect of splenomegaly on certain parameters should be taken into account during interpretation of laboratory-based adverse events.

Article Publication: AIDS research and human retroviruses

Article Summary: Abstract A recombinant modified vaccinia Ankara virus vaccine candidate (TBC-M4) expressing HIV-1 subtype C env, gag, tat-rev, and nef-RT genes was tested in a randomized, double-blind, dose escalation Phase I trial in 32 HIV-uninfected healthy volunteers who received three intramuscular injections of TBC-M4 at 0, 1, and 6 months of 5 x 10(7) plaque-forming units (pfu) (low dosage, LD) (n = 12) or 2.5 x 10(8) pfu (high dosage, HD) (n = 12) or placebo (n = 8). Local and systemic reactogenicity was experienced by approximately 67% and 83% of vaccine recipients, respectively. The reactogenicity events were mostly mild in severity. Severe but transient systemic reactogenicity was seen in one volunteer of the HD group. No vaccine-related serious adverse events or events suggesting perimyocarditis were seen. A higher frequency of local reactogenicity events was observed in the HD group. Cumulative HIV-specific IFN-gamma ELISPOT responses were detected in frozen PBMCs from 9/11 (82%), 12/12 (100%), and 1/8 (13%) volunteers after the third injection of the LD, HD, and placebo groups, respectively. Most of the responses were to gag and env proteins (maximum of 430 SFU/10(6) PBMCs) persisting across multiple time points. HIV-specific ELISA antibody responses were detected in 10/11, 12/12, and 0/8 volunteers post-third vaccination, in the LD, HD, and placebo groups, respectively. No neutralizing activity against HIV-1 subtype C isolates was detected. TBC-M4 appears to be generally safe and well-tolerated. The immune response detected was dose dependent, modest in magnitude, and directed mostly to env and gag proteins, suggesting further evaluation of this vaccine in a prime-boost regimen.

Article Publication: Journal of immunology

Article Summary: Rational vaccines designed to engender T cell responses require intimate knowledge of how epitopes are generated and presented. Recently, we vaccinated 8 Mamu-A *02(+) rhesus macaques with every SIV protein except Envelope (Env). Surprisingly, one of the strongest T cell responses engendered was against the Env protein, the Mamu-A *02-restricted epitope, Env(788-795)RY8. In this paper, we show that translation from an alternate reading frame of both the Rev-encoding DNA plasmid and the rAd5 vector engendered Env(788-795)RY8-specific CD8(+) T cells of greater magnitude than "normal" SIV infection. Our data demonstrate both that the pathway from vaccination to immune response is not well understood and that products of alternate reading frames may be rich and untapped sources of T cell epitopes.

Article Publication: Current HIV Research

Article Summary: A current debate in the HIV-1 vaccine field concerns the ability of an immunodeficiency virus to elicit a protective response. One argument is that HIV-1 superinfections are frequent in healthy individuals, because virus evades conventional immune surveillance, a serious obstacle to vaccine design. The opposing argument is that protection from superinfection is significant, reflecting a robust immune response that might be harnessed by vaccination to prevent disease. In an experiment designed to address the debate, two macaques received an I.V. inoculation with SHIV KU-1-d (a derivative of SHIV KU-1) and were rested for >10 months. Infection elicited diverse neutralizing antibody activities in both animals. Animals were then exposed to SHIV 89.6P (I.V.), a virus carrying a heterologous envelope protein relative to the vaccine strain. Infection was monitored by viral load and CD4+ T-cell measurements. All control animals were infected and most succumbed to disease. In contrast, protection from superinfection was statistically significant in test monkeys; one animal showed no evidence of superinfection at any time point and the second showed evidence of virus at only one time point over a 6-month observation period. Neither animal showed signs of disease. Perhaps this protective state may serve as a 'gold-standard' for HIV-1 vaccine development, as a similar degree of protection against immunodeficiency virus infections in humans would be much desired.

Article Publication: Journal of immunological methods

Article Summary: Methods to prime human CD4(+) T cells in vitro would be of significant value for the pre-clinical evaluation of vaccine candidates and other immunotherapeutics. However, to date, there is no reliable method for the induction of primary human T cell responses in the laboratory. Here, we optimized a culture strategy incorporating highly purified lymphocytes and dendritic cells, in the absence of any exogenous growth factors, for the in vitro sensitization of naïve CD4(+) T cells against a variety of protein antigens. This fully autologous approach, which was superior to the more traditional PBMC assay for supporting the induction of primary human T helper cell responses in culture, elicited effector cells capable of producing a variety of Th cytokines, including IFNgamma, TNFalpha, IL-2, IL-5, IL-17 and IL-21, and memory cells that could be restimulated multiple times with a specific antigen. Through simple modifications to this culture method, we evaluated the role of dendritic cell maturation state and regulatory T cells on the sensitization of naïve T helper cells, which highlights its utility for addressing basic questions of human immunobiology. Finally, using the formulated yellow fever vaccine, YF-VAX (R), we provide a proof-of-concept demonstration of the utility of the system for evaluating the T cell immunogenicity of vaccine candidates in a pre-clinical setting .

Article Publication: AIDS care

Article Summary: Quality improvement (QI) has been widely implemented in health services but has not been widely applied in HIV prevention research. Most prevention research centers have commonly employed traditional approaches (e.g., checklists) to quality control that document what has been done but not the quality of what has been done. Unlike other health settings, prevention research settings have unique characteristics and ethical requirements that require the development or adaptation of specific quality indicators. A QI model for health services was adapted for use in prevention research settings and was piloted between August 2006 and July 2007 at three research centers in East Africa. Four hundred and twenty-six volunteers exit interviews were administered in two cycles. Quantitative and qualitative data were analyzed using Excel worksheets. QI meeting reports and QI plans were used to complement data from exit interviews.

Article Publication: Journal of virology

Article Summary: The prophylactic efficacies of several multi-valent replication-incompetent adenovirus serotype 5 (Ad5) vaccines were examined in rhesus macaques using an intrarectal high-dose SIVmac239 challenge model. Cohorts of Mamu-A*01(+)/B*17(-) Indian rhesus macaques were immunized with one of several combinations of Ad5 vectors expressing Gag, Pol, Nef and Env gp140; for comparison, a Mamu-A*01(+) cohort was immunized using the Ad5 vector alone. There was no sign of immunological interference between antigens in the immunized animals. In general, expansion of the antigen breadth resulted in more favorable virological outcomes. In particular, the order of efficacy trended as follows: Gag/Pol/Nef/Env approximately Gag/Pol > Gag approximately Gag/Pol/Nef > Nef. However, the precision in ranking the vaccines based on the study results may be limited by the cohort size and as such, warrant additional testing. Implications of these results in light of the recent discouraging results of Phase IIb study of the trivalent Ad5 HIV-1 vaccine will be discussed .

Article Publication: Journal of virology

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Article Publication: AIDS

Article Summary: BACKGROUND: The identification of populations at risk of HIV infection is a priority for trials of preventive technologies, including HIV vaccines. To quantify incidence traditionally requires laborious and expensive prospective studies. METHODS: The BED IgG-Capture enzyme immunoassay (EIA) was developed to estimate HIV-1 incidence using cross-sectional data by measuring increasing levels of HIV-specific IgG as a proportion of total IgG. To evaluate this assay, we tested 189 seroconversion samples taken at 3-monthly intervals from 15 Rwandan and 26 Zambian volunteers with known time of infection and cross-sectional specimens from 617 Kenyan and Ugandan volunteers with prevalent infection. RESULTS: The BED-EIA-estimated incidence in Uganda was unexpectedly high, at 6.1%/year [95% confidence interval (CI) 4.2-8.0] in Masaka and 6.0%/year (95% CI 4.3-7.7) in Kakira. Prospective incidence data in Masaka from the same population was 1.7%/year before and 1.4%/year after the study. Kenyan estimates were 3.5%/year in Kilifi (95% CI 2.1-4.9) and 3.4%/year in Nairobi (95% CI 1.5-5.3). From the Rwandan and Zambian data, the sensitivity of the assay was 81.2% and the specificity was 67.8%. After approximately one year, subjects misclassified as recently infected tended to have lower plasma viral loads compared with those not misclassified as recent (median copies/ml 14 773 versus 93 560; P = 0.02). Clinical presentation, sex and HIV subtype were not significantly associated with BED-EIA misclassification in seroconverter samples. CONCLUSION: These data suggest that this assay does not perform reliably in all populations. Further research is warranted before using this assay to estimate incidence from prevalent HIV samples.

Article Publication: South African medical journal

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Article Publication: AIDS

Article Summary: BACKGROUND: The role of homosexuality and anal sex practices in the African HIV -1 epidemic is not well described. We aimed to assess the risk factors for prevalent HIV-1 infection among men who have sex with men (MSM) to guide HIV-1 prevention efforts. METHODS: Socio-behavioural characteristics, signs and symptoms of sexually transmitted diseases (STD), and serological evidence of HIV-1 were determined for 285 MSM at enrolment into a vaccine preparedness cohort study. We used multivariate logistic regression to assess risk factors for prevalent HIV-1 infection. RESULTS: HIV-1 prevalence was 43.0% [49/114, 95% confidence interval (CI), 34-52%] for men who reported sex with men exclusively (MSME), and 12.3% (21/171, 95% CI, 7-17%) for men who reported sex with both men and women (MSMW). Eighty-six (75%) MSME and 69 (40%) MSMW reported recent receptive anal sex. Among 174 MSM sexually active in the last week, 44% reported no use of condoms with casual partners. In the previous 3 months, 210 MSM (74%) reported payment for sex, and most clients (93%) were local residents. Prevalent HIV-1 infection was associated with recent receptive anal sex [odds ratio (OR), 6.1; 95% CI, 2.4-16], exclusive sex with men (OR, 6.3; 95% CI, 2.3-17), and increasing age (OR, 1.1 per year; 95% CI, 1.04-1.12). Only four MSM reported injecting drug use. CONCLUSIONS: The high prevalence of HIV-1 in Kenyan MSM is probably attributable to unprotected receptive anal sex. There is an urgent need for HIV-1 prevention programmes to deliver targeted risk-reduction interventions and STD services to MSM in Kenya.

Article Publication: Journal of virology

Article Summary: Here we describe a novel vaccine vector for expressing HIV antigens. We show that recombinant attenuated yellow fever vaccine virus 17D expressing SIVmac239 Gag sequences can be used as a vector to generate SIV-specific CD8+ T cell responses in the rhesus macaque. Priming with recombinant BCG expressing SIV antigens increased the frequency of these SIV-specific CD8+ T cell responses after recombinant YF17D boosting. These recombinant YF17D-induced SIV-specific CD8+ T cells secreted several cytokines, were largely effector memory and suppressed viral replication in CD4+ T cells .

Article Publication: Journal of virology

Article Summary: Human immunodeficiency virus (HIV-1) entry into cells is mediated by a trimeric complex consisting of noncovalently associated gp120 (exterior) and gp41 (transmembrane) envelope glycoproteins. The binding of gp120 to receptors on the target cell alters the gp120-gp41 relationship and activates the membrane-fusing capacity of gp41. Interaction of gp120 with the primary receptor, CD4, results in the exposure of the gp120 third variable (V3) loop, which contributes to binding the CCR5 or CXCR4 chemokine receptors. Here we show that insertions in the V3 stem or polar substitutions in a conserved hydrophobic patch near the V3 tip result in decreased gp120-gp41 association (in the unliganded state) and decreased chemokine receptor binding (in the CD4-bound state). Subunit association and syncytium-forming ability of the envelope glycoproteins from primary HIV-1 isolates were disrupted more by V3 changes than those of laboratory-adapted HIV-1 envelope glycoproteins. Changes in the gp120 beta2, beta19, beta20 and beta21 strands, which evidence suggests are proximal to the V3 loop in unliganded gp120, also resulted in decreased gp120-gp41 association. Thus, a gp120 element composed of the V3 loop and adjacent beta strands contributes to quaternary interactions that stabilize the unliganded trimer. CD4 binding dismantles this element, altering the gp120-gp41 relationship and rendering the hydrophobic patch in the V3 tip available for chemokine receptor binding.

Article Publication: Journal of virology

Article Summary: HIV-1 envelope glycoprotein (Env) on whole virions is heterogeneous, so molecular analysis of Env with monoclonal antibodies (mAbs) is challenging. Virus capture assays (VCAs) involving immobilized mAbs are typically used, but these assays suffer from immobilization artifacts and do not provide binding constants. Furthermore, we show here that certain HIV-1 neutralizing mAbs, including 2G12, 4E10, 2F5, Z13e1 and D5, will capture virion particles completely devoid of Env. We modified the VCA such that mAbs and virions are incubated in solution and unbound mAbs removed prior to the capture step. This modification nearly eliminated evidence of Env-independent binding by mAbs to virions and allowed determination of apparent affinity constants (IC50s) in solution. Three important qualitative observations were further revealed. First, neutralizing mAbs 2F5, 4E10 and Z13e1 against the membrane-proximal external region (MPER) of HIV-1 gp41 were found to capture virions efficiently only if a significant amount of uncleaved gp160 or synthetic MPER peptide was present. Second, we show how non-native forms of Env vary by Env genotype, and that Env from HIV-1JR-FL is more homogeneously trimeric than that from HIV-1JR-CSF. Third, we determined that Env containing all or parts of gp41, including uncleaved gp160, binds spontaneously to free virions. This exogenous Env is an indiscriminate molecular 'bridge' between Env-specific Ab and virions and can affect VCA analyses, particularly using pseudotyped virions. Heterogeneity in Env from endogenous and exogenous sources might also subvert humoral immunity to HIV-1, so in-solution VCAs may help to dissect this heterogeneity for vaccine design purposes.

Article Publication: Trends in immunology

Article Summary: CD8(+) cytotoxic T lymphocyte (CTL) responses are crucial in establishing the control of persistent virus infections. Population studies of HIV-1-infected individuals suggest that CD8(+) CTL responses targeting epitopes that take the greatest toll on virus replication are instrumental in immune control. A major question for vaccine design is whether incorporating epitopes responsible for controlling a persistent virus will translate into protection from natural infection or serve solely as a fail-safe mechanism to prevent overt disease in infected individuals. Here, we discuss qualitative parameters of the CD8(+) CTL response and mechanisms operative in the control of persistent virus infections and suggest new strategies for design and delivery of HIV vaccines. Published by Elsevier Ltd.

Article Publication: Journal of infectious diseases

Article Summary: Makerere University Walter Reed Project, Kampala, Uganda; 2Walter Reed Project, US Army Medical Research Unit-Kenya, Kericho, Kenya; 3Mbeya Medical Research Programme, Mbeya, Tanzania; 4Klinikum of the Ludwigs Maximilians University, Munich, Germany; 5US Military HIV Research Program, 6The Emmes Corporation, Rockville, and 7Vaccine Research Centre, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland; 8International AIDS Vaccine Initiative, New York, New York; 9Centers for Disease Control and Prevention, Atlanta, Georgia; and 10Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand. Background. Human immunodeficiency virus (HIV) vaccine development remains a global priority. We describe the safety and immunogenicity of a multiclade DNA vaccine prime with a replication-defective recombinant adenovirus serotype 5 (rAd5) boost. Methods. The vaccine is a 6-plasmid mixture encoding HIV envelope (env) subtypes A, B, and C and subtype B gag, pol, and nef, and an rAd5 expressing identical genes, with the exception of nef. Three hundred and twenty-four participants were randomized to receive placebo ([Formula: see text]), a single dose of rAd5 at 10(10) ([Formula: see text]) or 10(11) particle units ([Formula: see text]), or DNA at 0, 1, and 2 months, followed by rAd5 at either 10(10) ([Formula: see text]) or 10(11) particle units ([Formula: see text]) boosting at 6 months. Participants were followed up for 24 weeks after the final vaccination. Results. The vaccine was safe and well tolerated. HIV-specific T cell responses were detected in 63% of vaccinees. Titers of preexisting Ad5 neutralizing antibody did not affect the frequency and magnitude of T cell responses in prime-boost recipients but did affect the response rates in participants that received rAd5 alone ([Formula: see text]). Conclusion. The DNA/rAd5 vaccination regimen was safe and induced HIV type 1 multi-clade T cell responses, which were not significantly affected by titers of preexisting rAd5 neutralizing antibody. Trial Registration. ClinicalTrials.gov identifier: NCT00123968.

Article Publication: Vaccine

Article Summary: BACKGROUND: Two parallel studies evaluated safety and immunogenicity of a prophylactic HIV-1 vaccine in 192 HIV-seronegative, low-risk volunteers. Modified vaccinia virus Ankara (MVA) and plasmid DNA (pTHr) expressed HIV-1 clade A gag p24 and p17 fused to a string of 25 overlapping CD8+ T cell epitopes (HIVA). METHODS: These studies compared intramuscular, subcutaneous, and intradermal MVA at dosage levels ranging from 5x10(6)-2.5x10(8) pfu. In Study IAVI-010, DNA vaccine was given as a prime at months 0 and 1, followed by MVA as a boost at months 5 and 8. In Study IAVI-011, MVA alone was given at months 0 and 2. Regular safety monitoring was performed. Immunogenicity was measured by the interferon (IFN)-gamma ELISPOT assay on peripheral blood mononuclear cells (PBMC). RESULTS: No serious adverse events were attributed to either vaccine; most adverse events were mild or moderate, although MVA resulted in some severe local reactions. Five vaccine recipients had at least one positive IFN-gamma ELISPOT response, but none were sustained. CONCLUSION: This HIV-1 vaccine candidate was in general safe and well-tolerated. Local reactions were common, but tolerable. Detectable immune responses were infrequent.

Article Publication: Journal of virology

Article Summary: Monoclonal antibodies (MAbs) that neutralize the human immunodeficiency virus type one (HIV-1) have been isolated from HIV-1 infected individuals or animals immunized with recombinant HIV-1 Envelope glycoprotein constructs (Env). The epitopes of these neutralizing antibodies (NAbs) were shown to be located on either the variable or conserved regions of the HIV-1 Env and to be linear or conformational. However, one neutralizing MAb, 2909, which was isolated from an HIV-1-infected subject, recognizes a more complex, quaternary epitope that is present on the virion-associated functional trimeric Env spike of the SF162 HIV-1 isolate. Here, we discuss the isolation of eleven anti-HIV NAbs isolated from three rhesus macaques infected with the simian/human immunodeficiency virus SHIVSF162P4, and which also recognize quaternary epitopes. A detailed epitope mapping analysis of three of these rhesus antibodies revealed that their epitopes overlap that of the human MAb 2909. Despite this overall similarity in binding, however, differences in specific amino acid and glycosylation pattern requirements for MAb 2909 and the rhesus MAbs were identified. These results highlight similarities in the B cell responses of humans and macaques to structurally complex neutralization epitopes on related viruses, HIV-1 and SHIV.

Article Publication: PLoS one

Article Summary: Corti D, Langedijk JP, Hinz A, Seaman MS, Vanzetta F, Fernandez-Rodriguez BM, Silacci C, Pinna D, Jarrossay D, Balla-Jhagjhoorsingh S, Willems B, Zekveld MJ, Dreja H, O'Sullivan E, Pade C, Orkin C, Jeffs SA, Montefiori DC, Davis D, Weissenhorn W, McKnight A, Heeney JL, Sallusto F, Sattentau QJ, Weiss RA, Lanzavecchia A.

Article Publication: PLoS one

Article Summary: We conducted a Phase I dose escalation trial of ADVAX, a DNA-based candidate HIV-1 vaccine expressing Clade C/B' env, gag, pol, nef, and tat genes. Sequences were derived from a prevalent circulating recombinant form in Yunnan, China, an area of high HIV-1 incidence. The objective was to evaluate the safety and immunogenicity of ADVAX in human volunteers.

Article Publication: PLoS One

Article Summary: We conducted a Phase I dose-escalation trial of ADMVA, a Clade-B'/C-based HIV-1 candidate vaccine expressing env, gag, pol, nef, and tat in a modified vaccinia Ankara viral vector. Sequences were derived from a prevalent circulating HIV-1 recombinant form in Yunnan, China, an area of high HIV incidence. The objective was to evaluate the safety and immunogenicity of ADMVA in human volunteers.

Article Publication: PLoS medicine

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Article Publication: Health affairs

Article Summary: A new model was developed to examine the potential impacts of an AIDS vaccine in developing countries. The findings suggest that even a modestly efficacious first-generation vaccine could have a profound effect on the AIDS pandemic. A vaccine with 50 percent efficacy provided to 30 percent of the population would reduce new annual infections by 34 percent (seventeen million infections avoided) over fifteen years and result in substantial financial savings. A more efficacious vaccine, combined with expanded delivery, would do even more to control the pandemic. It therefore makes sense to continue investing in AIDS vaccine research and development and the eventual manufacture and widespread distribution of a vaccine.

Article Publication: Journal of infectious diseases

Article Summary: We have characterized an assay measuring CD8 T cell-mediated inhibition of human immunodeficiency virus (HIV) type 1 replication, demonstrating specificity and reproducibility and employing a panel of primary HIV-1 isolates. The assay uses relatively simple autologous cell culture and enzyme-linked immunosorbent assay, avoids generation of T cell clones, and can be performed with <2 million peripheral blood mononuclear cells. Efficient CD8 T cell-mediated cross-clade inhibition of HIV-1 replication in vitro was demonstrated in antiretroviral therapy-naive HIV-1-infected subjects with controlled viral replication in vivo but not in viremic subjects. An HIV-1 vaccine candidate, consisting of DNA and recombinant adenovirus 5 vectors tested in a phase I clinical trial, induced CD8 T cells that efficiently inhibited HIV-1 in a HLA-I-dependent manner. Assessment of direct antiviral T cell function by this assay provides additional information to guide vaccine design and the prioritizing of candidates for further clinical trials

Article Publication: Nature protocols

Article Summary: The development of an HIV vaccine will require a more precise understanding of the immunological and virological underpinnings of HIV infection. Magnetofection, the process of magnetizing HIV by coupling it to ferrous nanoparticles and coordinating infection using a magnetic field, synchronizes the viral replication cycle at attachment while recapitulating the events of natural infection. Although spinoculation also concentrates virus onto target cells to increase infection, it does not synchronize infection. The synchronization of HIV infection in vitro facilitates the study of events in the viral replication cycle and the antiviral immune response on timelines previously impossible. Furthermore, by infecting a high percentage of cells in a short time frame, magnetofection increases the throughput of in vitro assays. Once a virus stock is generated, magnetofection of target cells is rapid, requiring only 1-2 h. Here we present a detailed protocol for this assay and review its applications for studying the immune response to HIV.

Article Publication: PLoS medicine

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Article Publication: AIDS research and human retroviruses

Article Summary: We compared HIV-1 strains in incident and prevalent infections in a cohort of men having sex with men (MSM) and female sex workers (FSW) near Mombasa, Kenya and conducted a cross-sectional study of viral isolates from a sample of HIV-1-infected MSM and FSW in Kilifi, Coast Province, Kenya. RNA extracted from plasma of 13 MSM, 9 FSW, and one heterosexual male was amplified by nested RT-PCR and the products were directly sequenced. HIV-1 strains from 21 individuals were characterized with one or more complete genome sequences, and two were sequenced in the Nef gene. The envelope quasispecies was also studied in one individual. Among MSM, eight strains were subtype A and five were recombinant. There were two epidemiologically linked pairs of sequences; one pair was subtype A and the other pair was a complex AA2CD recombinant of identical structure. Another MSM was dually infected with DG recombinant strains of related, but nonidentical, structure. MSM also harbored AC and AD recombinant strains. The FSW harbored seven subtype A strains, an AD recombinant, and an AA2D strain related to CRF16_A2D. The one heterosexual male studied had a subtype A infection. This MSM epidemic in Kenya appears to be of local origin, harboring many strains typical of the broader Kenyan epidemic. Characteristics of a close social network were identified, with extended chains of transmission, novel recombinant strains possibly generated within the network, and a relatively high proportion of recombinant and dual infections.

Article Publication: Nature medicine

Article Summary: The worldwide diversity of HIV-1 presents an unprecedented challenge for vaccine development. Antigens derived from natural HIV-1 sequences have elicited only a limited breadth of cellular immune responses in nonhuman primate studies and clinical trials to date. Polyvalent 'mosaic' antigens, in contrast, are designed to optimize cellular immunologic coverage of global HIV-1 sequence diversity. Here we show that mosaic HIV-1 Gag, Pol and Env antigens expressed by recombinant, replication-incompetent adenovirus serotype 26 vectors markedly augmented both the breadth and depth without compromising the magnitude of antigen-specific T lymphocyte responses as compared with consensus or natural sequence HIV-1 antigens in rhesus monkeys. Polyvalent mosaic antigens therefore represent a promising strategy to expand cellular immunologic vaccine coverage for genetically diverse pathogens such as HIV-1.

Article Publication: Nature medicine

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Article Publication: Glycobiology

Article Summary: Immunologically "self" carbohydrates protect the HIV-1 surface glycoprotein, gp120, from antibody recognition. However, one broadly neutralising antibody, 2G12, neutralises primary viral isolates by direct recognition of Manalpha1-->2Man motifs formed by the host-derived oligomannose glycans of the viral envelope. Immunogens, capable of eliciting antibodies of similar specificity to 2G12, are therefore candidates for HIV/AIDS vaccine development. In this context, it is known that the yeast mannan polysaccharides exhibit significant antigenic mimicry with the glycans of HIV-1. Here we report that modulation of yeast polysaccharide biosynthesis directly controls the molecular specificity of cross-reactive antibodies to self oligomannose glycans. Saccharomyces cerevisae mannans are typically terminated by alpha1-->3-linked mannoses that cap a Manalpha1-->2Man motif that otherwise closely resembles the part of the oligomannose epitope recognised by 2G12. Immunisation with S. cerevisiae deficient for the alpha1-->3 mannosyltransferase gene (DeltaMnn1), but not with wild-type S. cerevisiae, reproducibly elicited antibodies to the self oligomannose glycans. Carbohydrate microarray analysis of DeltaMnn1 immune sera revealed fine carbohydrate specificity to Manalpha1-->2Man units, closely matching that of 2G12. These specificities were further corroborated by ELISA with chemically defined glycoforms of gp120. These antibodies exhibited remarkable similarity in the carbohydrate specificity to 2G12 and displayed statistically significant, albeit extremely weak, neutralisation of HIV-1 compared to control immune sera. These data confirm the Manalpha1-->2Man motif as the primary carbohydrate neutralisation determinant of HIV-1 and show that the genetic modulation of microbial polysaccharides is a route towards immunogens capable of eliciting antibody responses to the glycans of HIV-1.

Article Publication: Molecular cell

Article Summary: The entry of human immunodeficiency virus (HIV-1) into cells is initiated by binding of the gp120 exterior envelope glycoprotein to the receptor, CD4. How does CD4 binding trigger conformational changes in gp120 that allow the gp41 transmembrane envelope glycoprotein to mediate viral-cell membrane fusion? The transition from the unliganded to the CD4-bound state is regulated by two potentially flexible topological layers (layers 1 and 2) in the gp120 inner domain. Both layers apparently contribute to the noncovalent association of unliganded gp120 with gp41. After CD4 makes initial contact with the gp120 outer domain, layer 1-layer 2 interactions strengthen gp120-CD4 binding by reducing the off rate. Layer 1-layer 2 interactions also destabilize the activated state induced on HIV-1 by treatment with soluble CD4. Thus, despite lack of contact with CD4, the gp120 inner-domain layers govern CD4 triggering by participating in conformational transitions within gp120 and regulating the interaction with gp41. Copyright © 2010 Elsevier Inc. All rights reserved.

Article Publication: Nature

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Article Publication: Vaccine

Article Summary: Hidajat, R., S. Kuate, D. Venzon, V. Kalyanaraman, I. Kalisz, J. Treece, Y. Lian, S. W. Barnett and M. Robert-Guroff

Article Publication: Journal of biomedicine and biotechnology

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Article Publication: Nature

Article Summary: Without therapy, most people infected with human immunodeficiency virus (HIV) ultimately progress to AIDS. Rare individuals (‘elite controllers’) maintain very low levels of HIV RNA without therapy, thereby making disease progression and transmission unlikely. Certain HLA class I alleles are markedly enriched in elite controllers, with the highest association observed for HLA-B57 (ref. 1). Because HLA molecules present viral peptides that activate CD8+ T cells, an immune-mediated mechanism is probably responsible for superior control of HIV. Here we describe how the peptide-binding characteristics of HLA-B57 molecules affect thymic development such that, compared to other HLA-restricted T cells, a larger fraction of the naive repertoire of B57-restricted clones recognizes a viral epitope, and these T cells are more cross-reactive to mutants of targeted epitopes. Our calculations predict that such a T-cell repertoire imposes strong immune pressure on immunodominant HIV epitopes and emergent mutants, thereby promoting efficient control of the virus. Supporting these predictions, in a large cohort of HLA-typed individuals, our experiments show that the relative ability of HLA-B alleles to control HIV correlates with their peptide-binding characteristics that affect thymic development. Our results provide a conceptual framework that unifies diverse empirical observations, and have implications for vaccination strategies.

Article Publication: Current opinion in immnology

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Article Publication: Journal of virological methods

Article Summary: The introduction of antiretroviral (ARV) therapy in resource-poor settings is effective in suppressing HIV-1 replication and prolonging life of infected individuals. This has led to a demand for affordable HIV-1 drug resistance assays, since treatment failure due to development of drug resistance is common. This study developed and evaluated an affordable "in-house" genotyping assay to monitor HIV-1 drug resistance in Africa, particularly South Africa. An "in-house" assay using automated RNA extraction, and subtype C specific PCR and sequencing primers was developed and successfully evaluated 396 patient samples (viral load ranges 1000-1.6 million RNA copies/ml). The "in-house" assay was validated by comparing sequence data and drug resistance profiles from 90 patient and 10 external quality control samples to data from the ViroSeq HIV-1 Genotyping kit. The "in-house" assay was more efficient, amplifying all 100 samples, compared to 91 samples using Viroseq. The "in house" sequences were 99.2% homologous to the ViroSeq sequences, and identical drug resistance mutation profiles were observed in 96 samples. Furthermore, the "in-house" assay genotyped 260 of 295 samples from seven African sites, where 47% were non-subtype C. Overall, the newly validated "in-house" drug resistance assay is suited for use in Africa as it overcomes the obstacle of subtype diversity.

Article Publication: Proceedings of the National Academy of Sciences

Article Summary: Development of an effective vaccine against HIV-1 will likely require elicitation of broad and potent neutralizing antibodies against the trimeric surface envelope glycoprotein (Env). Monoclonal antibodies (mAbs) PG9 and PG16 neutralize ∼80% of HIV-1 isolates across all clades with extraordinary potency and target novel epitopes preferentially expressed on Env trimers. As these neutralization properties are ideal for a vaccine-elicited antibody response to HIV-1, their structural basis was investigated. The crystal structure of the antigen-binding fragment (Fab) of PG16 at 2.5 Å resolution revealed its unusually long, 28-residue, CDR H3 forms a unique, stable subdomain that towers above the antibody surface. A 7-residue “specificity loop” on the “hammerhead” subdomain was identified that, when transplanted from PG16 to PG9 and vice versa, accounted for differences in the fine specificity and neutralization of these two mAbs. The PG16 electron density maps also revealed that a CDR H3 tyrosine was sulfated, which was confirmed for both PG9 (doubly) and PG16 (singly) by mass spectral analysis. We further showed that tyrosine sulfation plays a role in binding and neutralization. An N-linked glycan modification is observed in the variable light chain, but not required for antigen recognition. Further, the crystal structure of the PG9 light chain at 3.0 Å facilitated homology modeling to support the presence of these unusual features in PG9. Thus, PG9 and PG16 use unique structural features to mediate potent neutralization of HIV-1 that may be of utility in antibody engineering and for high-affinity recognition of a variety of therapeutic targets.

Article Publication: Journal of virology

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Article Publication: Biologicals

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Article Publication: Journal of virology

Article Summary: An effective HIV vaccine will likely need to reduce mucosal transmission and, if infection occurs, control virus replication. To determine whether our best SIV vaccine can achieve these lofty goals, we vaccinated eight Indian rhesus macaques with SIVmac239Deltanef and challenged them intrarectally (i.r.) with repeated, low doses of the pathogenic heterologous swarm isolate SIVsmE660. We detected a significant reduction in acquisition of SIVsmE660 in comparison to naïve controls (Log-rank test; p=0.023). After ten mucosal challenges we detected replication of the challenge strain in only five of the eight vaccinated animals. In contrast, seven of the eight control animals became infected with SIVsmE660 after these ten challenges. Additionally, the SIVsmE660-infected vaccinated animals controlled peak acute virus replication significantly better than the naive controls (Mann-Whitney test; p=0.038). Four of the five SIVsmE660 vaccinees rapidly brought virus replication under control by week 4 post-infection. Unfortunately, two of these four vaccinated animals lost control of virus replication during the chronic phase of infection. Bulk sequencing analysis of the circulating virus in these animals indicated that recombination had occurred between the vaccine and challenge strains and likely contributed to the increased virus replication in these animals. Overall, our results suggest that a well-designed HIV vaccine might both reduce the rate of acquisition and control viral replication.

Article Publication: Proceedings of the National Academy of Sciences

Article Summary: The envelope spike of HIV is one of the most highly N-glycosylated structures found in nature. However, despite extensive research revealing essential functional roles in infection and immune evasion, the chemical structures of the glycans on the native viral envelope glycoprotein gp120-as opposed to recombinantly generated gp120-have not been described. Here, we report on the identity of the N-linked glycans from primary isolates of HIV-1 (clades A, B, and C) and from the simian immunodeficiency virus. MS analysis reveals a remarkably simple and highly conserved virus-specific glycan profile almost entirely devoid of medial Golgi-mediated processing. In stark contrast to recombinant gp120, which shows extensive exposure to cellular glycosylation enzymes (>70% complex type glycans), the native envelope shows barely detectable processing beyond the biosynthetic intermediate Man(5)GlcNAc(2) (<2% complex type glycans). This oligomannose (Man(5-9)GlcNAc(2)) profile is conserved across primary isolates and geographically divergent clades but is not reflected in the current generation of gp120 antigens used for vaccine trials. In the context of vaccine design, we also note that Manalpha1-->2Man-terminating glycans (Man(6-9)GlcNAc(2)) of the type recognized by the broadly neutralizing anti-HIV antibody 2G12 are 3-fold more abundant on the native envelope than on the recombinant monomer and are also found on isolates not neutralized by 2G12. The Manalpha1-->2Man residues of gp120 therefore provide a vaccine target that is physically larger and antigenically more conserved than the 2G12 epitope itself. This study revises and extends our understanding of the glycan shield of HIV with implications for AIDS vaccine design.

Article Publication: New england journal of medicine

Article Summary: From July 18 through July 23, 2010, delegates from around the globe will convene for the biennial International AIDS Conference in Vienna. They will discuss our current risk of losing the war against the human immunodeficiency virus (HIV). Despite an unprecedented outpouring of resources and proliferation of programs, today, for every two patients who begin receiving treatment for HIV, five people are newly infected. Furthermore, new guidelines from the World Health Organization recommending that infected persons begin receiving treatment earlier will significantly increase the number of patients targeted for therapy. If we are to control this pandemic, we must recognize the urgent need to develop and deploy better prevention tools and, most important, a safe and effective HIV vaccine

Article Publication: Sexually transmitted infections

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