Scientific Publications

The discovery of novel conditions for highly beta-stereoselective (>9 : 1) mannosylation of OH-2 of mannosides using a straightforward perbenzylthioglycoside donor has allowed ready assembly of beta-mannosyl oligosaccharides including the repeating trisaccharide motif of the O5 antigen of pathogen Klebsiella pneumoniae.

The small molecule CCR5 inhibitors are a new class of drugs for treating infection by human immunodeficiency virus type 1 (HIV-1). They act by binding to the CCR5 co-receptor and preventing its use during HIV-1-cell fusion. Escape mutants can be raised against CCR5 inhibitors in vitro and will arise when these drugs are used clinically. Here, we have assessed the responses of CCR5 inhibitor-resistant viruses to other anti-retroviral drugs that act by different mechanisms, and their sensitivities to neutralizing antibodies (NAbs). The rationale for the latter study is that the resistance pathway for CCR5 inhibitors involves changes in the HIV-1 envelope glycoproteins (Env), which are also targets for NAbs. The escape mutants CC101.19 and D1/85.16 were selected for resistance to AD101 and vicriviroc (VVC), respectively, from the primary R5 HIV-1 isolate CC1/85. Each escape mutant was cross-resistant to other small molecule CCR5 inhibitors (aplaviroc, maraviroc, VVC, AD101 and CMPD 167), but sensitive to protein ligands of CCR5: the modified chemokine PSC-RANTES and the humanized MAb PRO-140. The resistant viruses also retained wild-type sensitivity to the nucleoside reverse transcriptase inhibitor (RTI) zidovudine, the non-nucleoside RTI nevirapine, the protease inhibitor atazanavir and other attachment and fusion inhibitors that act independently of CCR5 (BMS-806, PRO-542 and enfuvirtide). Of note is that the escape mutants were more sensitive than the parental CC1/85 isolate to a subset of neutralizing monoclonal antibodies and to some sera from HIV-1-infected people, implying that sequence changes in Env that confer resistance to CCR5 inhibitors can increase the accessibility of some NAb epitopes. The need to preserve NAb resistance may therefore be a constraint upon how escape from CCR5 inhibitors occurs in vivo.

Antibodies, through passive or active immunization, play a central role in prophylaxis against many infectious agents. While neutralization is a primary function of antibodies in protection against most viruses, the relative contribution of Fc-dependent and complement-dependent anti-viral activities of antibodies was found to vary between different viruses in recent studies. The multiple hit model explains how antibodies neutralize viruses, and recent data on the stoichiometry of antibody neutralization suggest that the organization of viral surface proteins on viruses, in addition to virus size, influences the level of antibody occupancy required for neutralization. These new findings will improve our strategies in therapeutic antibody engineering and rational vaccine design.

Allyl sulfides undergo efficient cross-metathesis in aqueous media with Hoveyda-Grubbs second generation catalyst 1. The high reactivity of allyl sulfides in cross-metathesis was exploited in the first examples of cross-metathesis on a protein surface. S-Allylcysteine was incorporated chemically into the protein, providing the requisite allyl sulfide handle. Preliminary efforts to genetically incorporate S-allylcysteine into proteins are also reported.

The need to broaden research directed at answering fundamental questions in HIV vaccine discovery through laboratory, nonhuman primate (NHP), and clinical research has recently been emphasized. In addition, the importance of attracting and retaining young researchers, developing better NHP models, and more closely linking NHP and clinical research is being stressed. In an era of a level budget for biomedical research at the U.S. National Institutes of Health (NIH), HIV/AIDS vaccine research efforts will need to be carefully prioritized such that resources to energize HIV vaccine discovery can be identified. This article summarizes progress and challenges in HIV vaccine research, the priorities arising from a recent summit at NIAID, and the actions needed, some already under way, to address those priorities.

Following CD4 receptor binding to the HIV-1 envelope spike (Env), the conserved N-heptad repeat (NHR) region of gp41 forms a coiled-coil that is a precursor to the fusion reaction. Although it has been a target of drug and vaccine design, there are few monoclonal antibody (mAb) tools with which to probe the antigenicity and immunogenicity specifically of the NHR coiled-coil. Here, we have rescued HIV-1-neutralizing anti-NHR mAbs from immune phage display libraries that were prepared (i) from b9 rabbits immunized with a previously described mimetic of the NHR coiled-coil, N35(CCG)-N13, and (ii) from an HIV-1 infected individual. We describe a rabbit single-chain Fv fragment (scFv), 8K8, and a human Fab, DN9, which specifically recognize NHR coiled-coils that are unoccupied by peptide corresponding to the C-heptad repeat or CHR region of gp41 (e.g. C34). The epitopes of 8K8 and DN9 were found to partially overlap with that of a previously described anti-NHR mAb, IgG D5; however, 8K8 and DN9 were much more specific than D5 for unoccupied NHR trimers. The mAbs, including a whole IgG 8K8 molecule, neutralized primary HIV-1 of clades B and C in a pseudotyped virus assay with comparable, albeit relatively modest potency. Finally, a human Fab T3 and a rabbit serum (both non-neutralizing) were able to block binding of D5 and 8K8 to a gp41 NHR mimetic, respectively, but not the neutralizing activity of these mAbs. We conclude from these results that NHR coiled-coil analogs of HIV-1 gp41 elicit many Abs during natural infection and through immunization, but that due to limited accessibility to the corresponding region on fusogenic gp41 few can neutralize. Caution is therefore required in targeting the NHR for vaccine design. Nevertheless, the mAb panel may be useful as tools for elucidating access restrictions to the NHR of gp41 and in designing potential improvements to mimetics of receptor-activated Env.

The objective of this study was to assess the impact of temporary closure of an HIV research clinic on the health of study participants. Primary data were collected quarterly from couples enrolled in research studies at an established HIV study site. There were 632 participating couples enrolled when the project closed, 475 of whom returned when it re-opened six months later. HIV sero-incidence, mortality rates and risk-taking behaviours were compared before and during the closure. Perceived impact of the closure was measured in returning participants. Demographic data collected at the last pre-closure study visit were used to look at the differences between returning and non-returning study participants. Serologic data from those who returned were compared pre- and post-closure to examine changes in HIV incidence. Mortality rates were estimated from reported deaths, and were compared pre- and during project closure. Perceptions of the impact of the closure among returning participants were examined through an interviewer administered questionnaire. It was found that couples who returned were not demographically different from couples who did not return. Most participants reported no problems with finding alternate sources of condoms and the incidence of HIV did not change significantly during the closure. Eighty-four percent respondents reported that the closure had a negative impact on them, 87% of whom rated loss of medical care as the main impact. The mortality rate among HIV-positive participants doubled from 6.7/100 person years to 12.4/100 person years during the closure (p=0.01). Results indicate that couples voluntary counselling and testing (CVCT) established durable risk-reduction behaviours that persisted during project closure. ThIn ae loss of healthcare was perceived as the most negative impact on participants, reflected in increased mortality rates. Research projects should make transition plans and budget for mechanisms to reduce the negative impact on participants of project closures.

We assessed the utility of the modified Kigali combined (MKC) staging system for predicting survival in HIV-infected Zambian adults in a prospective, longitudinal, open cohort. From 1995 to 2004, HIV-discordant couples (one HIV-infected partner and one HIV-negative partner) were recruited from couples' voluntary counseling and testing centers in Lusaka, Zambia and followed at 3-month intervals. MKC stage, which incorporates clinical stage with erythrocyte sedimentation rate (ESR), hematocrit, and body mass index (BMI), was determined at enrollment. Kaplan-Meier survival and Cox proportional hazard methods were used to calculate median survival and relative hazards. We enrolled 1479 HIV-discordant couples with a combined 7305 person-years of follow-up. Among HIV-infected participants over the 9-year study period, there were 333 confirmed deaths. The time to 50% mortality was 8.5 years with MKC stage 1 and 2 disease compared to 3.7 years with MKC stage 4 disease at enrollment. Survival rates at 3 years were 85% with MKC stage 1 and 2 disease, 74% with MKC stage 3 disease, and 51% with MKC stage 4 disease. A total of 275 HIV-negative partners seroconverted during follow-up. In comparison, survival rates at 3 years were 94% for HIV-negative participants and 92% for participants who seroconverted during follow-up. In multivariate analysis, MKC stage 4 disease (HR = 3.7, 95% CI = 2.7-5.0) remained a strong predictor of mortality. Incorporating ESR, hematocrit, and BMI with clinical staging is a powerful, low-cost tool to identify HIV-infected adults at high risk for mortality.

Although antibodies against the third variable loop (V3) of the HIV-1 viral envelope glycoprotein are among the first neutralizing antibodies to be detected in infected individuals, they are normally restricted in their specificity. X-ray crystallographic studies of V3-specific antibodies have contributed to a more thorough understanding of recognition of this epitope and of conserved features in the V3 loop that could potentially aid in the design of a multi-component vaccine. The human antibody 447-52D exhibits relatively broad neutralization of primary viral isolates compared with other V3-loop antibodies. A crystal structure of Fab 447-52D in complex with a V3 peptide (UG1033) was determined at 2.1 angstroms resolution. The structure was determined using an epitaxially twinned data set and in-house programs to detect and remove overlapping reflections. Although the processed data have lower than desired completeness and slightly higher than normal R values for the resolution, good-quality electron-density maps were obtained that enabled structure determination. The structure revealed an extended CDR H3 loop that forms a beta-sheet with the peptide, with the predominant contacts being main-chain hydrogen bonds. The V3 peptide and Fab show high structural homology with the previously reported structures of other Fab 447-52D complexes, reinforcing the idea that the V3 loop may adopt a small set of conserved structures, particularly around the crown of the beta-hairpin.

Although alcohol abuse is highly prevalent in many countries in sub-Saharan Africa, little is known about the relationship between alcohol consumption and risky sexual behavior in these settings. An understanding of this relationship is particularly important given the high prevalence of HIV that exists in many of these countries. This study analyzes data collected from members of cohabiting HIV-discordant couples regarding alcohol consumption and self-reported condom use. After controlling for demographic and socioeconomic co-factors, alcohol use by male partners of HIV-discordant couples was associated with self-reported unprotected sex at follow-up. Counseling about alcohol use should be part of HIV testing and counseling programs, particularly among those found to be HIV-positive.

The glycan shield of human immunodeficiency virus type 1 (HIV-1) gp120 contributes to viral evasion from humoral immune responses. However, the shield is recognized by the HIV-1 broadly neutralizing antibody (Ab), 2G12, at a relatively conserved cluster of oligomannose glycans. The discovery of 2G12 raises the possibility that a carbohydrate immunogen may be developed that could elicit 2G12-like neutralizing Abs and contribute to an AIDS vaccine. We have previously dissected the fine specificity of 2G12 and reported that the synthetic tetramannoside (Man(4)) that corresponds to the D1 arm of Man(9)GlcNAc(2) inhibits 2G12 binding to gp120 as efficiently as Man(9)GlcNAc(2) itself, indicating the potential use of Man(4) as a building block for creating immunogens. Here, we describe the development of neoglycoconjugates displaying variable copy numbers of Man(4) on bovine serum albumin (BSA) molecules by conjugation to Lys residues. The increased valency enhances the apparent affinity of 2G12 for Man(4) up to a limit which is achieved at approximately 10 copies per BSA molecule, beyond which no further enhancement is observed. Immunization of rabbits with BSA-(Man(4))(14) elicits significant serum Ab titers to Man(4). However, these Abs are unable to bind gp120. Further analysis reveals that the elicited Abs bind a variety of unbranched and, to a lesser extent, branched Man(9) derivatives but not natural N-linked oligomannose containing the chitobiose core. These results suggest that Abs can be readily elicited against the D1 arm; however, potential differences in the presentation of Man(4) on neoglycoconjugates, compared to glycoproteins, poses challenges for eliciting anti-mannose Abs capable of cross-reacting with gp120 and HIV-1.

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.

Sub-Saharan Africa has a high rate of HIV infection, most of which is attributable to heterosexual transmission. Few attempts have been made to assess the extent of HIV transmission within marriages, and HIV-prevention efforts remain focused on abstinence and non-marital sex. We aimed to estimate the proportion of heterosexual transmission of HIV which occurs within married or cohabiting couples in urban Zambia and Rwanda each year.