Scientific Publications

Live, attenuated viral vectors that express HIV-1 antigens are being investigated as an approach to generating durable immune responses against HIV-1 in humans. We recently developed a replication-competent, highly attenuated Ad26 vector that expresses mosaic HIV-1 Env (rcAd26.MOS1.HIV-Env, 'rcAd26'). Here we present the results of a first-in-human, placebo-controlled clinical trial to test the safety, immunogenicity and mucosal shedding of rcAd26 given orally.

Killer cell immunoglobulin-like receptors (KIRs) are expressed predominantly on natural killer cells, where they play a key role in the regulation of innate immune responses. Recent studies show that inhibitory KIRs can also affect adaptive T cell-mediated immunity. In mice and in human T cells in vitro, inhibitory KIR ligation enhanced CD8 T cell survival. To investigate the clinical relevance of these observations, we conducted an extensive immunogenetic analysis of multiple independent cohorts of HIV-1-, hepatitis C virus (HCV)-, and human T cell leukemia virus type 1 (HTLV-1)-infected individuals in conjunction with in vitro assays of T cell survival, analysis of ex vivo KIR expression, and mathematical modeling of host-virus dynamics. Our data suggest that functional engagement of inhibitory KIRs enhances the CD8 T cell response against HIV-1, HCV, and HTLV-1 and is a significant determinant of clinical outcome in all three viral infections.

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.

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.

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.

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.

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.

How precursor frequencies and antigen affinities impact interclonal B cell competition is a particularly relevant issue for candidate germline-targeting HIV vaccine designs because of the in vivo rarity of naive B cells that recognize broadly neutralizing epitopes. Knowing the frequencies and affinities of HIV-specific VRC01-class naive human B cells, we transferred B cells with germline VRC01 B cell receptors into congenic recipients to elucidate the roles of precursor frequency, antigen affinity, and avidity on B cell responses following immunization. All three factors were interdependently limiting for competitive success of VRC01-class B cells. In physiological high-affinity conditions using a multivalent immunogen, rare VRC01-class B cells successfully competed in germinal centers (GC), underwent extensive somatic hypermutation, and differentiated into memory B cells. The data reveal dominant influences of precursor frequency, affinity, and avidity for interclonal GC competition and indicate that germline-targeting immunogens can overcome these challenges with high-affinity multimeric designs.

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.