Scientific Publications

A randomized, double-blind, placebo-controlled phase I trial was conducted in 32 HIV-uninfected healthy volunteers to assess the safety and immunogenicity of 3 doses of DNA vaccine (Advax) plus 1 dose of recombinant modified vaccinia virus Ankara (MVA) (TBC-M4) or 3 doses of TBC-M4 alone (groups A and B, respectively). Both vaccine regimens were found to be safe and well tolerated. Gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) assay responses were detected in 1/10 (10%) individuals in group A after three Advax primes and in 9/9 individuals (100%) after the MVA boost. In group B, IFN-γ ELISPOT responses were detected in 6/12 (50%) and 7/11 (64%) individuals after the second and third MVA vaccinations, respectively. Responses to all vaccine components, but predominantly to Env, were seen. The breadth and magnitude of the T cell response and viral inhibition were greater in group A than in group B, indicating that the quality of the T-cell response was enhanced by the DNA prime. Intracellular cytokine staining indicated that the T-cell responses were polyfunctional but were skewed toward Env with a CD4(+) phenotype. At 2 weeks after the last vaccination, HIV-specific antibody responses were detected in all (100%) group B and 1/11 (9.1%) group A vaccinees. Vaccinia virus-specific responses were detected in all (100%) group B and 2/11 (18.2%) group A vaccinees. In conclusion, HIV-specific T-cell responses were seen in the majority of volunteers in groups A and B but with a trend toward greater quality of the T-cell response in group A. Antibody responses were better in group B than in group A.

We report the molecular identification, cloning and initial biological characterization of 12 full-length HIV-1 subtype A, D and A/D recombinant transmitted/founder (T/F) genomes. T/F genomes contained intact canonical open reading frames and all T/F viruses were replication competent in primary human T-cells, although subtype D virus replication was more efficient (p<0.05). All 12 viruses utilized CCR5 but not CXCR4 as a co-receptor for entry and exhibited a neutralization profile typical of tier 2 primary virus strains, with significant differences observed between subtype A and D viruses with respect to sensitivity to monoclonal antibodies VRC01, PG9 and PG16 and polyclonal subtype C anti-HIV IgG (p<0.05 for each). The present report doubles the number of T/F HIV-1 clones available for pathogenesis and vaccine research and extends their representation to include subtypes A, B, C and D.

The envelope glycoproteins (Env) represent a critical component of a successful antibody-mediated human immunodeficiency virus type 1 (HIV-1) vaccine. However, immunization with soluble Env was reported to induce short-lived antibody responses, suggesting that Env has unusual immunogenic properties. Here, we directly compared the magnitude and durability of B-cell responses induced by HIV-1 Env and an unrelated soluble viral protein, influenza virus hemagglutinin (HA), in simultaneously inoculated macaques. We demonstrate robust peak responses followed by rapid contraction of circulating antibody and memory B cells for both antigens, suggesting that short-lived responses are not unique to HIV-1 Env but may be a common feature of soluble protein vaccines.

HIV-1 incidence estimates and correlates of HIV-1 acquisition in African MSM are largely unknown.

The need for an effective vaccine to prevent the global spread of human immunodeficiency virus type 1 (HIV-1) is well recognized. Passive immunization and challenge studies in non-human primates testify that broadly neutralizing antibodies (BrNAbs) can accomplish protection against infection. In recent years, the introduction of new techniques has facilitated the discovery of an unprecedented number of new human BrNAbs that target and delineate diverse conserved epitopes on the envelope glycoprotein spike (Env). The epitopes of these BrNAbs can serve as templates for immunogen design aimed to induce similar antibodies. Here we will review the characteristics of the different classes of BrNAbs and their target epitopes, as well as factors associated with their development and implications for vaccine design.

In HIV-1 infection, the early set-point viral load strongly predicts both viral transmission and disease progression. The factors responsible for the wide spectrum of set-point viral loads are complex and likely reflect an interplay between the transmitted virus and genetically defined factors in both the transmitting source partner and the seroconverter. Indeed, analysis of 195 transmission pairs from Lusaka, Zambia, revealed that the viral loads in transmitting source partners contributed only ∼2% of the variance in early set-point viral loads of seroconverters (P = 0.046 by univariable analysis). In multivariable models, early set-point viral loads in seroconverting partners were a complex function of (i) the viral load in the source partner, (ii) the gender of the seroconverter, (iii) specific HLA class I alleles in the newly infected partner, and (iv) sharing of HLA-I alleles between partners in a transmission pair. Each of these factors significantly and independently contributed to the set-point viral load in the newly infected partner, accounting for up to 37% of the variance observed and suggesting that many factors operate in concert to define the early virological phenotype in HIV-1 infection.

A new generation of extremely broad and potent neutralizing antibodies (bNAbs) has been isolated from HIV-infected subjects. This has refocused interest in the sites of vulnerability targeted by these bNAbs and in the potential for designing Envelope (Env) immunogens that display these sites. Standard methods for evaluating HIV-1 vaccine candidates do not enable epitope mapping on the HIV Env spike, the target for NAbs. To meet the need for rapid analysis of Ab specificity, we designed a multiplexed, quantitative mapping assay that can test for serum Ab competition for the binding of an HIV-1 Env gp120 to a panel of bNAbs directed to different sites of vulnerability on the Env that do not compete for one another in the assay. Using serum samples from rabbits immunized with various DNA prime/gp120 protein boost vaccines we were able to detect serum Ab competition for multiple classes of bNAbs in the postimmune samples that were significantly higher than background competition detected in samples obtained prior to vaccination. Importantly, application of this novel assay to our ongoing HIV-1 Env viral vector studies in mice has allowed us to distinguish qualitative differences in the Ab elicited by various regimens that ELISA cannot. Furthermore, pooled immunoglobulin from HIV-infected donors (HIVIg) competes for binding to the bNAb panel whereas a control pool from HIV-negative donors does not, highlighting the utility of this assay for human studies. This novel assay will add value in rational immunogen design and in the detailed, qualitative evaluation of binding and, potentially, neutralizing Abs elicited by natural infections and HIV-1 vaccine candidates.

A randomized, double-blind, placebo controlled phase I trial.

An effective vaccine remains the best solution to stop the spread of human immunodeficiency virus (HIV). Cellular immune responses have been repeatedly associated with control of viral replication and thus may be an important element of the immune response that must be evoked by an efficacious vaccine. Recombinant viral vectors can induce potent T-cell responses. Although several viral vectors have been developed to deliver HIV genes, only a few have been advanced for clinical trials. The live-attenuated yellow fever vaccine virus 17D (YF17D) has many properties that make it an attractive vector for AIDS vaccine regimens. YF17D is well tolerated in humans and vaccination induces robust T-cell responses that persist for years. Additionally, methods to manipulate the YF17D genome have been established, enabling the generation of recombinant (r)YF17D vectors carrying genes from unrelated pathogens. Here, we report the generation of seven new rYF17D viruses expressing fragments of simian immunodeficiency virus (SIV)mac239 Gag, Nef, and Vif. Studies in Indian rhesus macaques demonstrated that these live-attenuated vectors replicated in vivo, but only elicited low levels of SIV-specific cellular responses. Boosting with recombinant Adenovirus type-5 (rAd5) vectors resulted in robust expansion of SIV-specific CD8(+) T-cell responses, particularly those targeting Vif. Priming with rYF17D also increased the frequency of CD4(+) cellular responses in rYF17D/rAd5-immunized macaques compared to animals that received rAd5 only. The effect of the rYF17D prime on the breadth of SIV-specific T-cell responses was limited and we also found evidence that some rYF17D vectors were more effective than others at priming SIV-specific T-cell responses. Together, our data suggest that YF17D - a clinically relevant vaccine vector - can be used to prime AIDS virus-specific T-cell responses in heterologous prime boost regimens. However, it will be important to optimize rYF17D-based vaccine regimens to ensure maximum delivery of all immunogens in a multivalent vaccine.

Low levels of HIV-1 transmitted drug resistance (TDR) have previously been reported from many parts of sub-Saharan Africa (sSA). However, recent data, mostly from urban settings, suggest an increase in the prevalence of HIV-1 TDR. Our objective was to determine the prevalence of TDR mutations among HIV-1-infected, antiretroviral (ARV)-naive adults enrolling for care in a rural HIV clinic in Kenya. Two cross-sectional studies were carried out between July 2008 and June 2010. Plasma samples from ARV-naive adults (>15 years old) at the time of registering for care after HIV diagnosis and before starting ARVs were used. A portion of the pol subgenomic region of the virus containing the protease and part of the reverse transcriptase genes was amplified and sequenced. TDR mutations were identified and interpreted using the Stanford HIV drug resistance database and the WHO list for surveillance of drug resistance strains. Overall, samples from 182 ARV-naive adults [mean age (95% CI): 34.9 (33.3-36.4) years] were successfully amplified and sequenced. Two TDR mutations to nucleoside reverse transcriptase inhibitors [n=1 (T215D)] and protease inhibitors [n=1 (M46L)] were identified, giving an overall TDR prevalence of 1.1% (95% CI: 0.1-3.9). Despite reports of an increase in the prevalence of HIV-1 TDR in some urban settings in sSA, we report a prevalence of HIV-1 TDR of less than 5% at a rural HIV clinic in coastal Kenya. Continued broader surveillance is needed to monitor the extent of TDR in sSA.

Broadly neutralizing HIV antibodies (bnAbs) are typically highly somatically mutated, raising doubts as to whether they can be elicited by vaccination. We used 454 sequencing and designed a novel phylogenetic method to model lineage evolution of the bnAbs PGT121-134 and found a positive correlation between the level of somatic hypermutation (SHM) and the development of neutralization breadth and potency. Strikingly, putative intermediates were characterized that show approximately half the mutation level of PGT121-134 but were still capable of neutralizing roughly 40-80% of PGT121-134 sensitive viruses in a 74-virus panel at median titers between 15- and 3-fold higher than PGT121-134. Such antibodies with lower levels of SHM may be more amenable to elicitation through vaccination while still providing noteworthy coverage. Binding characterization indicated a preference of inferred intermediates for native Env binding over monomeric gp120, suggesting that the PGT121-134 lineage may have been selected for binding to native Env at some point during maturation. Analysis of glycan-dependent neutralization for inferred intermediates identified additional adjacent glycans that comprise the epitope and suggests changes in glycan dependency or recognition over the course of affinity maturation for this lineage. Finally, patterns of neutralization of inferred bnAb intermediates suggest hypotheses as to how SHM may lead to potent and broad HIV neutralization and provide important clues for immunogen design.

Vaccination and SIV challenge of macaque species is the best animal model for evaluating candidate HIV vaccines in pre-clinical studies. As such, robust assays optimized for use in nonhuman primates are necessary for reliable ex vivo measurement of immune responses and identification of potential immune correlates of protection. We optimized and qualified an 8-color intracellular cytokine staining assay for the measurement of IFNγ, IL-2, and TNF from viable CD4 and CD8 T cells from cryopreserved rhesus macaque PBMC stimulated with peptides. After optimization, five laboratories tested assay performance using the same reagents and PBMC samples; similar results were obtained despite the use of flow cytometers with different configurations. The 8-color assay was then subjected to a pre-qualification study to quantify specificity and precision. These data were used to set positivity thresholds and to design the qualification protocol. Upon completion of the qualification study, the assay was shown to be highly reproducible with low inter-aliquot, inter-day, and inter-operator variability according to the qualification criteria with an overall variability of 20-40% for each outcome measurement. Thus, the 8-color ICS assay was formally qualified according to the ICH guidelines Q2 (R1) for specificity and precision indicating that it is considered a standardized/robust assay acceptable for use in pre-clinical trial immunogenicity testing.

Broadly neutralizing HIV antibodies (bNAbs) can recognize carbohydrate-dependent epitopes on gp120. In contrast to previously characterized glycan-dependent bNAbs that recognize high-mannose N-glycans, PGT121 binds complex-type N-glycans in glycan microarrays. We isolated the B-cell clone encoding PGT121, which segregates into PGT121-like and 10-1074-like groups distinguished by sequence, binding affinity, carbohydrate recognition, and neutralizing activity. Group 10-1074 exhibits remarkable potency and breadth but no detectable binding to protein-free glycans. Crystal structures of unliganded PGT121, 10-1074, and their likely germ-line precursor reveal that differential carbohydrate recognition maps to a cleft between complementarity determining region (CDR)H2 and CDRH3. This cleft was occupied by a complex-type N-glycan in a 'liganded' PGT121 structure. Swapping glycan contact residues between PGT121 and 10-1074 confirmed their importance for neutralization. Although PGT121 binds complex-type N-glycans, PGT121 recognized high-mannose-only HIV envelopes in isolation and on virions. As HIV envelopes exhibit varying proportions of high-mannose- and complex-type N-glycans, these results suggest promiscuous carbohydrate interactions, an advantageous adaptation ensuring neutralization of all viruses within a given strain.