Scientific Publications

To evaluate initial reported willingness to participate in a hypothetical HIV vaccine clinical trial and actual participation of volunteers in a longitudinal observational study.

In this Review, we highlight some recent developments in the discovery and application of broadly neutralizing antibodies (bnAbs) to human immunodeficiency virus (HIV); i.e., antibodies able to neutralize diverse isolates of HIV. We consider the characterization of novel bnAbs, recent data on the effects of bnAbs in vivo in humans and animal models, and the importance of both kinds of data for the application of Abs to prophylaxis and therapy and to guide vaccine design. We seek to place newly discovered bnAbs in the context of existing bnAbs, and we explore the various characteristics of the antibodies that are most desirable for different applications.

Background While advances have been made in HIV prevention and treatment, new HIV infections continue to occur. The introduction of pre-exposure prophylaxis (PrEP) as an additional HIV prevention option for those at high risk of HIV may change the landscape of the HIV epidemic, especially in sub-Saharan Africa, which bears the greatest HIV burden.

The broadly neutralizing antibody against HIV-1, b12, binds to the CD4 binding site (CD4bs) on the outer domain (OD) of the gp120 subunit of HIV-1 Env. We have previously reported the design of an E. coli expressed fragment of HIV-1 gp120, b122a, containing about 70% of the b12 epitope with the idea of focusing the immune response to this structure. Since the b122a structure was found to be only partially folded, as assessed by circular dichroism and protease resistance, we attempted to stabilize it by the introduction of additional disulfide bonds. One such mutant, b122a1-b showed increased stability and bound b12 with 30-fold greater affinity as compared to b122a. Various b122a and OD fragment proteins were displayed on the surface of Qβ virus-like particles. Sera raised against these particles in six-month long rabbit immunization studies could neutralize Tier1 viruses across different subtypes with the best results observed with b122a1-b displayed particles. Significantly higher amounts of antibodies directed towards the CD4bs were also elicited by particles displaying b122a1-b. This study highlights the ability of fragment immunogens to focus the antibody response to the conserved CD4bs of HIV-1.

Human immunodeficiency virus 1 (HIV-1) diversity is a significant challenge in developing a vaccine against the virus. B/C recombinants have been found in India and other places but are the predominant clade prevalent in China. HIV-1 envelopes (Envs) are the target of broadly neutralizing antibodies (bNAbs) which develop spontaneously in some HIV-1 infected patients. It has been previously reported with efficiently cleaved clade A, B and C Envs that preferential binding of Envs to bNAbs as opposed to non-NAbs, a desirable property for immunogens, is correlated with efficient cleavage of the Env precursor polypeptide into constituent subunits. These Envs are suitable for designing immunogens as soluble proteins, virus-like particles or for delivery by viral vectors/plasmid DNA. However, a B/C recombinant Env with similar properties has not been reported. Here we show that the chimeric, recombinant B/C clade Env LT5.J4b12C is efficiently cleaved on the plasma membrane and selectively binds to bNAbs.

Protein-minimization is an attractive approach for designing vaccines against rapidly evolving pathogens such as HIV-1 since it can help in focussing the immune response towards conserved conformational epitopes present on complex targets. The outer domain (OD) of HIV-1 gp120 contains epitopes for a large number of neutralizing antibodies and therefore is a primary target for structure-based vaccine design. We have previously designed a bacterially expressed outer domain immunogen (OD) that bound CD4 binding site (CD4bs) ligands with 3-12µM affinity and elicited a modest neutralizing antibody response in rabbits. In this study, we have optimized OD using consensus sequence design, cyclic permutation and structure-guided mutations to generate a number of variants with improved yields, biophysical properties, stabilities and affinities (KD of 10-50 nM) for various CD4bs targeting, broadly neutralizing antibodies, including the germline reverted version of the broadly neutralizing antibody, VRC01. In contrast to OD, the optimized immunogens elicited high anti-gp120 titers in rabbits as early as 6 weeks post-immunization, before any gp120 boost was given. Following two gp120 boosts, sera collected at week 22 showed cross-clade neutralization of Tier 1 HIV-1 viruses. Using a number of different prime: boost combinations, we have identified a cyclically permuted OD fragment as the best priming immunogen, and a trimeric, cyclically permuted gp120 as the most suitable boosting molecule amongst the tested immunogens. This study also provides insights into some of the biophysical correlates of improved immunogenicity.

As the sole target of broadly neutralizing antibodies (bnAbs) to HIV, the envelope glycoprotein (Env) trimer is the focus of vaccination strategies designed to elicit protective bnAbs in humans. Because HIV Env is densely glycosylated with 75-90 N-glycans per trimer, most bnAbs use or accommodate them in their binding epitope, making the glycosylation of recombinant Env a key aspect of HIV vaccine design. Upon analysis of three HIV strains, we here find that site-specific glycosylation of Env from infectious virus closely matches Envs from corresponding recombinant membrane-bound trimers. However, viral Envs differ significantly from recombinant soluble, cleaved (SOSIP) Env trimers, strongly impacting antigenicity. These results provide a benchmark for virus Env glycosylation needed for the design of soluble Env trimers as part of an overall HIV vaccine strategy.

While prior studies have demonstrated that CD8 T cell responses to cryptic epitopes (CE) are readily detectable during HIV-1 infection, their ability to drive escape mutations following acute infection is unknown. We predicted 66 CE in a Zambian acute infection cohort based on escape mutations occurring within or near the putatively predicted HLA-I restricted epitope. The CE were evaluated for CD8 T cell responses in patients with chronic and acute HIV infection. Of the 66 predicted CE, 10 were recognized in 8/32 and 4/11 patients with chronic, and acute infection respectively. The immunogenic CE were all derived from a single antisense reading frame within However, when these CE were tested using longitudinal study samples, CE specific T cell responses were detected but did not consistently select for viral escapes. Thus, while we demonstrated that CE are immunogenic in acute infection, the immune responses to CE are not major drivers of viral escape in the initial stages of HIV infection. This latter finding may be due to either the subdominant nature of CE-specific responses, the low antigen sensitivity, and magnitude of CE responses during acute infections. Although prior studies demonstrated that cryptic epitopes of HIV-1 induce CD8 T cell responses, evidence supporting that targeting these epitopes to drive HIV escape mutations have been substantially limited and none have addressed this question following acute infection. In this comprehensive study, we utilized longitudinal viral sequencing data obtained from three separate acute infection cohorts to predict potential cryptic epitopes based on HLA-I associated viral escape. Our data shows that cryptic epitopes are immunogenic during acute infection and many of these responses are elicited towards translation products of HIV-1 antisense reading frames. However, despite cryptic epitope targeting, our study did not find any evidence of early CD8 mediated immune escape. Nevertheless, improving cryptic epitope specific CD8 T cell responses may still be beneficial in both preventative and therapeutic HIV-1 vaccines.

Broadly neutralizing antibodies (bNAbs), able to prevent viral entry by diverse global viruses, are a major focus of HIV vaccine design, with data from animal studies confirming their ability to prevent HIV infection. However, traditional vaccine approaches have failed to elicit these types of antibodies. During chronic HIV infection, a subset of individuals develops bNAbs, some of which are extremely broad and potent. This review describes the immunological and virological factors leading to the development of bNAbs in such 'elite neutralizers'. The features, targets and developmental pathways of bNAbs from their precursors have been defined through extraordinarily detailed within-donor studies. These have enabled the identification of epitope-specific commonalities in bNAb precursors, their intermediates and Env escape patterns, providing a template for vaccine discovery. The unusual features of bNAbs, such as high levels of somatic hypermutation, and precursors with unusually short or long antigen-binding loops, present significant challenges in vaccine design. However, the use of new technologies has led to the isolation of more than 200 bNAbs, including some with genetic profiles more representative of the normal immunoglobulin repertoire, suggesting alternate and shorter pathways to breadth. The insights from these studies have been harnessed for the development of optimized immunogens, novel vaccine regimens and improved delivery schedules, which are providing encouraging data that an HIV vaccine may soon be a realistic possibility.

A Phase I HIV-1 vaccine trial sponsored by the International AIDS Vaccine Initiative (IAVI) was conducted in India in 2009 to test a subtype C prophylactic vaccine in a prime-boost regimen comprising of a DNA prime (ADVAX) and MVA (TBC-M4) boost. The trial demonstrated that the regimen was safe and well tolerated and resulted in enhancement of HIV-specific immune responses. Preliminary observations on vaccine-induced immune responses were limited to analysis of neutralizing antibodies and IFN-γ ELISPOT response. The present study involves a more detailed analysis of the nature of the vaccine-induced humoral immune response using specimens that were archived from the volunteers at the time of the trial. Interestingly, we found vaccine induced production of V1/V2 and V3 region-specific antibodies in a significant proportion of vaccinees. Variable region antibody levels correlated directly with the frequency of circulating T follicular helper cells (Tfh) and regulatory T cells (Treg). Our findings provide encouraging evidence to demonstrate the immunogenicity of the tested vaccine. Better insights into vaccine-induced immune responses can aid in informing future design of a successfulHIV-1 vaccine.

Protection against acquiring HIV infection may not require a vaccine in the conventional sense, because broadly neutralizing antibodies (bNAbs) alone prevent HIV infection in relevant animal challenge models. Additionally, bNAbs as therapeutics can effectively suppress HIV replication in infected humans and in animal models. Combinations of bNAbs are generally even more effective and bNAb-derived multivalent antibody-like molecules also inhibit HIV replication both in vitro and in vivo. To expand the available array of multi-specific HIV inhibitors, we designed single-component molecules that incorporate two (bispecific) or three (trispecific) bNAbs that recognize HIV Env exclusively, a bispecific CrossMab targeting two epitopes on the major HIV co-receptor, CCR5, and bi- and trispecifics that cross-target both Env and CCR5. These newly designed molecules displayed exceptional breadth, neutralizing 98-100% of a 109-virus panel, as well as additivity and potency compared to the individual parental control IgGs. The bispecific molecules designed as tandem single chain variable fragments (scFvs; 10E8fv-N6fv and m36.4-PRO 140fv) displayed a median IC of 0.0685 and 0.0131 μg/ml, respectively. A trispecific containing 10E8-PGT121-PGDM1400 Env-specific binding sites was equally potent (median IC of 0.0135 μg/ml), while the trispecific molecule targeting Env and CCR5 simultaneously (10E8Fab-PGDM1400fv-PRO 140fv), demonstrated even greater potency with a median IC of 0.007 μg/ml. By design, some of these molecules lacked Fc-mediated effector function; therefore, we also constructed a trispecific prototype possessing reconstituted CH2-CH3 domains to restore Fc receptor binding capacity. The molecules developed here, along with those described previously, possess promise as prophylactic and therapeutic agents against HIV. Broadly neutralizing antibodies (bNAbs) prevent HIV infection in monkey challenge models and suppress HIV replication in infected humans. Combinations of bNAbs are more effective at suppression and antibody-like molecules engineered to have 2 or 3 bNAb combining sites also inhibit HIV replication in monkeys and other animal models. To expand the available array of multi-specific HIV inhibitors, we designed single-component molecules that incorporate two (bispecific) or three (trispecific) bNAb binding sites that recognize the HIV envelope glycoprotein (Env), the HIV co-receptor (CCR5), or that cross-target both Env and CCR5. Several of the bi- and trispecific molecules neutralized most viruses in a diverse cross-clade panel with greater breadth and potency than the individual parental bNAbs. The molecules described here provide additional options to prevent or suppress HIV infection.

An important class of HIV-1 broadly neutralizing antibodies, termed the VRC01 class, targets the conserved CD4-binding site (CD4bs) of the envelope glycoprotein (Env). An engineered Env outer domain (OD) eOD-GT8 60-mer nanoparticle has been developed as a priming immunogen for eliciting VRC01-class precursors and is planned for clinical trials. However, a substantial portion of eOD-GT8-elicited antibodies target non-CD4bs epitopes, potentially limiting its efficacy. We introduced N-linked glycans into non-CD4bs surfaces of eOD-GT8 to mask irrelevant epitopes and evaluated these mutants in a mouse model that expressed diverse immunoglobulin heavy chains containing human IGHV1-202, the germline VRC01 V segment. Compared to the parental eOD-GT8, a mutant with five added glycans stimulated significantly higher proportions of CD4bs-specific serum responses and CD4bs-specific immunoglobulin G B cells including VRC01-class precursors. These results demonstrate that glycan masking can limit elicitation of off-target antibodies and focus immune responses to the CD4bs, a major target of HIV-1 vaccine design.

With the expansion of couples' voluntary HIV counseling and testing (CVCT) in urban Zambia, there is a growing need to evaluate CVCT provider trainings to ensure that couples are receiving quality counseling and care. We evaluated provider knowledge scores, pre- and post-training and predictors of pre- and post-training test scores. Providers operating in 67 government clinics in four Copperbelt Province cities were trained from 2008 to 2013 in three domains: counseling, rapid HIV laboratory testing and data management. Trainees received pre- and post-training tests on domain-specific topics. Pre- and post-training test scores were tabulated by provider demographics and training type, and paired t-tests evaluated differences in pre- and post-training test scores. Multivariable ANCOVA determined predictors of pre- and post-training test scores. We trained 1226 providers, and average test scores increased from 68.8% pre-training to 83.8% post-training (p < 0.001). Test scores increased significantly for every demographic group and training type (p < 0.001) with one exception-test scores did not significantly increase for those receiving counseling or data management training who had less than a high school education. In multivariable analysis, higher educational level and having a medical background were predictive of a higher pre-test score; higher pre-test scores and having a medical background were predictive of higher post-test scores. Pre- and post-test assessments are critical to ensure quality services, particularly as task-shifting from medical to lay staff becomes more common. Assessments showed that our CVCT trainings are successful at increasing knowledge, and that those with lower education may benefit from repeat trainings.