Scientific Publications

Local HIV epidemiology data are critical in determining the suitability of a population for HIV vaccine efficacy trials. The objective of this study was to estimate the prevalence and incidence of, and determine risk factors for HIV transmission in a rural community-based HIV vaccine preparedness cohort in Masaka, Uganda.

To characterize WHO-defined transmitted HIV drug resistance mutation (TDRM) data from recently HIV-infected African volunteers, we sequenced HIV (pol) and evaluated for TDRM the earliest available specimens from ARV-naive volunteers diagnosed within 1 year of their estimated date of infection at eight research centers in sub-Saharan Africa. TDRMs were detected in 19/408 (5%) volunteers. The prevalence of TDRMs varied by research center, from 5/26 (19%) in Entebbe, 6/78 (8%) in Kigali, 2/49 (4%) in Kilifi, to 3/106 (3%) in Lusaka. One of five volunteers from Cape Town (20%) had TDRMs. Despite small numbers, our data suggest an increase in DRMs by year of infection in Zambia (p = 0.004). The prevalence observed in Entebbe was high across the entire study. ARV history data from 12 (63%) HIV-infected sexual partners were available; 3 reported ARV use prior to transmission. Among four partners with sequence data available, transmission linkage was confirmed and two had the same TDRMs as the newly infected volunteer (both K103N). As ARV therapy continues to increase in availability throughout Africa, monitoring incident virus strains for the presence of TDRMs should be a priority. Early HIV infection cohorts provide an excellent and important platform to monitor the development of TDRMs to inform treatment guidelines, drug choices, and strategies for secondary prevention of TDRM transmission.

High-titer autologous neutralizing antibody responses have been demonstrated during early subtype C human immunodeficiency virus type 1 (HIV-1) infection. However, characterization of this response against autologous virus at the monoclonal antibody (MAb) level has only recently begun to be elucidated. Here we describe five monoclonal antibodies derived from a subtype C-infected seroconverter and their neutralizing activities against pseudoviruses that carry envelope glycoproteins from 48 days (0 month), 2 months, and 8 months after the estimated time of infection. Sequence analysis indicated that the MAbs arose from three distinct B cell clones, and their pattern of neutralization compared to that in patient plasma suggested that they circulated between 2 and 8 months after infection. Neutralization by MAbs representative of each B cell clone was mapped to two residues: position 134 in V1 and position 189 in V2. Mutational analysis revealed cooperative effects between glycans and residues at these two positions, arguing that they contribute to a single epitope. Analysis of the cognate gp120 sequence through homology modeling places this potential epitope near the interface between the V1 and V2 loops. Additionally, the escape mutation R189S in V2, which conferred resistance against all three MAbs, had no detrimental effect on virus replication in vitro. Taken together, our data demonstrate that independent B cells repeatedly targeted a single structure in V1V2 during early infection. Despite this assault, a single amino acid change was sufficient to confer complete escape with minimal impact on replication fitness.

The HIV envelope (Env) protein uses a dense coat of glycans to mask conserved domains and evade host humoral immune responses. The broadly neutralizing antibody 2G12, which binds a specific cluster of high-mannose glycans on HIV Env, shows that the glycan shield can also serve as a target for neutralizing antibodies. We have described a triple mutant Saccharomyces cerevisiae strain that expresses high-mannose glycoproteins that bind to 2G12. When used to immunize rabbits, this yeast elicits antibodies that bind to gp120-associated glycans but fail to neutralize virus. Here we sought to determine the reason for these discordant results. Affinity purification of sera over columns conjugated with three 2G12-reactive yeast glycoproteins showed that these proteins could adsorb 80% of the antibodies that bind to gp120 glycans. Despite binding to monomeric gp120, these mannose-specific antibodies failed to bind cell surface-expressed trimeric Env. However, when Env was expressed in the presence of the mannosidase inhibitor kifunensine to force retention of high-mannose glycans at all sites, the purified antibodies gained the abilities to bind trimeric Env and to strongly and broadly neutralize viruses produced under these conditions. Combined, these data show that the triple mutant yeast strain elicits antibodies that bind to high-mannose glycans presented on the HIV envelope, but only when they are displayed in a manner not found on native Env trimers. This implies that the underlying structure of the protein scaffold used to present the high-mannose glycans may be critical to allow elicitation of antibodies that recognize trimeric Env and neutralize virus.

The HIV-1 envelope glycoprotein (Env) spike is challenging to study at the molecular level, due in part to its genetic variability, structural heterogeneity and lability. However, the extent of lability in Env function, particularly for primary isolates across clades, has not been explored. Here, we probe stability of function for variant Envs of a range of isolates from chronic and acute infection, and from clades A, B and C, all on a constant virus backbone. Stability is elucidated in terms of the sensitivity of isolate infectivity to destabilizing conditions. A heat-gradient assay was used to determine T(90) values, the temperature at which HIV-1 infectivity is decreased by 90% in 1 h, which ranged between ∼40 to 49°C (n = 34). For select Envs (n = 10), the half-lives of infectivity decay at 37°C were also determined and these correlated significantly with the T(90) (p = 0.029), though two 'outliers' were identified. Specificity in functional Env stability was also evident. For example, Env variant HIV-1(ADA) was found to be labile to heat, 37°C decay, and guanidinium hydrochloride but not to urea or extremes of pH, when compared to its thermostable counterpart, HIV-1(JR-CSF). Blue native PAGE analyses revealed that Env-dependent viral inactivation preceded complete dissociation of Env trimers. The viral membrane and membrane-proximal external region (MPER) of gp41 were also shown to be important for maintaining trimer stability at physiological temperature. Overall, our results indicate that primary HIV-1 Envs can have diverse sensitivities to functional inactivation in vitro, including at physiological temperature, and suggest that parameters of functional Env stability may be helpful in the study and optimization of native Env mimetics and vaccines.

The genetic diversity of HIV-1 across the globe is a major challenge for developing an HIV vaccine. To facilitate immunogen design, it is important to characterize clusters of commonly targeted T-cell epitopes across different HIV clades. To address this, we examined 39 HIV-1 clade C infected individuals for IFN-γ Gag-specific T-cell responses using five sets of overlapping peptides, two sets matching clade C vaccine candidates derived from strains from South Africa and China, and three peptide sets corresponding to consensus clades A, B, and D sequences. The magnitude and breadth of T-cell responses against the two clade C peptide sets did not differ, however clade C peptides were preferentially recognized compared to the other peptide sets. A total of 84 peptides were recognized, of which 19 were exclusively from clade C, 8 exclusively from clade B, one peptide each from A and D and 17 were commonly recognized by clade A, B, C and D. The entropy of the exclusively recognized peptides was significantly higher than that of commonly recognized peptides (p = 0.0128) and the median peptide processing scores were significantly higher for the peptide variants recognized versus those not recognized (p = 0.0001). Consistent with these results, the predicted Major Histocompatibility Complex Class I IC(50) values were significantly lower for the recognized peptide variants compared to those not recognized in the ELISPOT assay (p0.0001), suggesting that peptide variation between clades, resulting in lack of cross-clade recognition, has been shaped by host immune selection pressure. Overall, our study shows that clade C infected individuals recognize clade C peptides with greater frequency and higher magnitude than other clades, and that a selection of highly conserved epitope regions within Gag are commonly recognized and give rise to cross-clade reactivities.

Acquisition of human immunodeficiency virus type 1 (HIV-1) infection is mediated by a combination of characteristics of the infectious and the susceptible member of a transmission pair, including human behavioral and genetic factors, as well as viral fitness and tropism. Here we report on the impact of established and potential new HLA class I determinants of heterosexual HIV-1 acquisition in the HIV-1-exposed seronegative (HESN) partners of serodiscordant Zambian couples.

To determine the incidence of and risk factors for HIV acquisition in a cohort of HIV-uninfected partners from HIV discordant couples in Masaka, Uganda, and to establish its suitability for HIV vaccine trials.

Antiretroviral chemoprophylaxis before exposure is a promising approach for the prevention of human immunodeficiency virus (HIV) acquisition.

The Comprehensive T Cell Vaccine Immune Monitoring Consortium (CTC-VIMC) was created to provide standardized immunogenicity monitoring services for HIV vaccine trials. The ex vivo interferon-gamma (IFN-γ) ELISpot is used extensively as a primary immunogenicity assay to assess T cell-based vaccine candidates in trials for infectious diseases and cancer. Two independent, GCLP-accredited central laboratories of CTC-VIMC routinely use their own standard operating procedures (SOPs) for ELISpot within two major networks of HIV vaccine trials. Studies are imperatively needed to assess the comparability of ELISpot measurements across laboratories to benefit optimal advancement of vaccine candidates.

The membrane proximal external region (MPER) of gp41 abuts the viral membrane at the base of HIV-1 envelope glycoprotein spikes. The MPER is highly conserved and is rich in Trp and other lipophilic residues. The MPER is also required for the infection of host cells by HIV-1 and is the target of the broadly neutralizing antibodies, 4E10, 2F5, and Z13e1. These neutralizing antibodies are valuable tools for understanding relevant conformations of the MPER and for studying HIV-1 neutralization, but multiple approaches used to elicit MPER binding antibodies with similar neutralization properties have failed. Here we report our efforts to mimic the MPER using linear as well as constrained peptides. Unnatural amino acids were also introduced into the core epitope of 4E10 to probe requirements of antibody binding. Peptide analogs with C-terminal Api or Aib residues designed to be helical transmembrane (TM) domain surrogates exhibit enhanced binding to the 4E10 and Z13e1 antibodies. However, we find that placement of constrained amino acids at nonbinding sites within the core epitope significantly reduce binding. These results are relevant to an understanding of native MPER structure on HIV-1, and form a basis for a chemical synthesis approach to mimic MPER stricture and to construct an MPER-based vaccine.

To assess the burden of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) in high-risk HIV-1 negative men who have sex with men (MSM) in Africa.