RV144 HIV Vaccine Trial Research Articles

A Quantitative Approach to Unravel the Role of Host Genetics in IgG-FcγR Complex Formation After Vaccination.

Fc-mediated immune functions have been correlated with protection in the RV144 HIV vaccine trial and are important for immunity to a range of pathogens. IgG antibodies (Abs) that form complexes with Fc receptors (FcRs) on innate immune cells can activate Fc-mediated immune functions. Genetic variation in both IgGs and FcRs have the capacity to alter IgG-FcR complex formation via changes in binding affinity and concentration. A growing challenge lies in unraveling the importance of multiple variations, especially in the context of vaccine trials that are conducted in homogenous genetic populations. Here we use an ordinary differential equation model to quantitatively assess how IgG1 allotypes and FcγR polymorphisms influence IgG-FcγRIIIa complex formation in vaccine-relevant settings. Using data from the RV144 HIV vaccine trial, we map the landscape of IgG-FcγRIIIa complex formation predicted post-vaccination for three different IgG1 allotypes and two different FcγRIIIa polymorphisms. Overall, the model illustrates how specific vaccine interventions could be applied to maximize IgG-FcγRIIIa complex formation in different genetic backgrounds. Individuals with the G1m1,17 and G1m1,3 allotypes were predicted to be more responsive to vaccine adjuvant strategies that increase antibody FcγRIIIa affinity (e.g. glycosylation modifications), compared to the G1m-1,3 allotype which was predicted to be more responsive to vaccine boosting regimens that increase IgG1 antibody titers (concentration). Finally, simulations in mixed-allotype populations suggest that the benefit of boosting IgG1 concentration versus IgG1 affinity may be dependent upon the presence of the G1m-1,3 allotype. Overall this work provides a quantitative tool for rationally improving Fc-mediated functions after vaccination that may be important for assessing vaccine trial results in the context of under-represented genetic populations.

Non-neutralizing antibodies targeting the immunogenic regions of HIV-1 envelope reduce mucosal infection and virus burden in humanized mice.

Antibodies are principal immune components elicited by vaccines to induce protection from microbial pathogens. In the Thai RV144 HIV-1 vaccine trial, vaccine efficacy was 31% and the sole primary correlate of reduced risk was shown to be vigorous antibody response targeting the V1V2 region of HIV-1 envelope. Antibodies against V3 also were inversely correlated with infection risk in subsets of vaccinees. Antibodies recognizing these regions, however, do not exhibit potent neutralizing activity. Therefore, we examined the antiviral potential of poorly neutralizing monoclonal antibodies (mAbs) against immunodominant V1V2 and V3 sites by passive administration of human mAbs to humanized mice engrafted with CD34+ hematopoietic stem cells, followed by mucosal challenge with an HIV-1 infectious molecular clone expressing the envelope of a tier 2 resistant HIV-1 strain. Treatment with anti-V1V2 mAb 2158 or anti-V3 mAb 2219 did not prevent infection, but V3 mAb 2219 displayed a superior potency compared to V1V2 mAb 2158 in reducing virus burden. While these mAbs had no or weak neutralizing activity and elicited undetectable levels of antibody-dependent cellular cytotoxicity (ADCC), V3 mAb 2219 displayed a greater capacity to bind virus- and cell-associated HIV-1 envelope and to mediate antibody-dependent cellular phagocytosis (ADCP) and C1q complement binding as compared to V1V2 mAb 2158. Mutations in the Fc region of 2219 diminished these effector activities in vitro and lessened virus control in humanized mice. These results demonstrate the importance of Fc functions other than ADCC for antibodies without potent neutralizing activity.

An HIV Vaccine Protective Allele in FCGR2C Associates With Increased Odds of Perinatal HIV Acquisition.

In the Thai RV144 HIV-1 vaccine trial, a three-variant haplotype within the Fc gamma receptor 2C gene (FCGR2C) reduced the risk of HIV-1 acquisition. A follow-on trial, HVTN702, of a similar vaccine candidate found no efficacy in South Africa, where the predominant population is polymorphic for only a single variant in the haplotype, c.134-96C>T (rs114945036). To investigate a role for this variant in HIV-1 acquisition in South Africans, we used the model of maternal-infant HIV-1 transmission. A nested case-control study was conducted of infants born to mothers living with HIV-1, comparing children with perinatally-acquired HIV-1 (cases, n = 176) to HIV-1-exposed uninfected children (controls, n = 349). All had received nevirapine for prevention of mother-to-child transmission. The FCGR2C copy number and expression variants (c.−386G>C, c.−120A>T c.169T>C, and c.798+1A>G) were determined using a multiplex ligation-dependent probe amplification assay and the c.134-96C>T genotype with Sanger sequencing. The copy number, genotype and allele carriage were compared between groups using univariate and multivariate logistic regression. The FCGR2C c.134-96C>T genotype distribution and copy number differed significantly between HIV-1 cases and exposed-uninfected controls (P = 0.002, P Bonf = 0.032 and P = 0.010, P Bonf = > 0.05, respectively). The FCGR2C c.134-96T allele was overrepresented in the cases compared to the controls (58% vs 42%; P = 0.001, P Bonf = 0.016). Adjusting for birthweight and FCGR2C copy number, perinatal HIV-1 acquisition was associated with the c.134-96C>T (AOR = 1.89; 95% CI 1.25-2.87; P = 0.003, P Bonf = 0.048) and c.169C>T (AOR = 2.39; 95% CI 1.45-3.95; P = 0.001, P Bonf = 0.016) minor alleles but not the promoter variant at position c.−386G>C. The c.134-96C>T variant was in strong linkage disequilibrium with the c.169C>T variant, but remained significantly associated with perinatal acquisition when adjusted for c.169C>T in multivariate analysis. In contrast to the protective effect observed in the Thai RV144 trial, we found the FCGR2C variant c.134-96T-allele associated with increased odds of perinatal HIV-1 acquisition in South African children. These findings, taken together with a similar deleterious association found with HIV-1 disease progression in South African adults, highlight the importance of elucidating the functional relevance of this variant in different populations and vaccination/disease contexts.

Assessment of an LSDV-Vectored Vaccine for Heterologous Prime-Boost Immunizations against HIV.

The modest protective effects of the RV144 HIV-1 vaccine trial have prompted the further exploration of improved poxvirus vector systems that can yield better immune responses and protection. In this study, a recombinant lumpy skin disease virus (LSDV) expressing HIV-1 CAP256.SU gp150 (Env) and a subtype C mosaic Gag was constructed (LSDVGC5) and compared to the equivalent recombinant modified vaccinia Ankara (MVAGC5). In vitro characterization confirmed that cells infected with recombinant LSDV produced Gag virus-like particles containing Env, and that Env expressed on the surface of the cells infected with LSDV was in a native-like conformation. This candidate HIV-1 vaccine (L) was tested in a rabbit model using different heterologous vaccination regimens, in combination with DNA (D) and MVA (M) vectors expressing the equivalent HIV-1 antigens. The four different vaccination regimens (DDMMLL, DDMLML, DDLMLM, and DDLLMM) all elicited high titers of binding and Tier 1A neutralizing antibodies (NAbs), and some regimens induced Tier 1B NAbs. Furthermore, two rabbits in the DDLMLM group developed low levels of autologous Tier 2 NAbs. The humoral immune responses elicited against HIV-1 Env by the recombinant LSDVGC5 were comparable to those induced by MVAGC5.

Recombinant MVA-prime elicits neutralizing antibody responses by inducing antigen-specific B cells in the germinal center

The RV144 HIV-1 vaccine trial has been the only clinical trial to date that has shown any degree of efficacy and associated with the presence of vaccine-elicited HIV-1 envelope-specific binding antibody and CD4+ T-cell responses. This trial also showed that a vector-prime protein boost combined vaccine strategy was better than when used alone. Here we have studied three different priming vectors—plasmid DNA, recombinant MVA, and recombinant VSV, all encoding clade C transmitted/founder Env 1086 C gp140, for priming three groups of six non-human primates each, followed by a protein boost with adjuvanted 1086 C gp120 protein. Our data showed that MVA-priming favors the development of higher antibody binding titers and neutralizing activity compared with other vectors. Analyses of the draining lymph nodes revealed that MVA-prime induced increased germinal center reactivity characterized by higher frequencies of germinal center (PNAhi) B cells, higher frequencies of antigen-specific B-cell responses as well as an increased frequency of the highly differentiated (ICOShiCD150lo) Tfh-cell subset.

IgG3 collaborates with IgG1 and IgA to recruit effector function in RV144 vaccinees.

While the RV144 HIV vaccine trial led to moderately reduced risk of HIV acquisition, emerging data from the HVTN702 trial point to the critical need to reexamine RV144-based correlates of reduced risk of protection. While in RV144, the induction of V2-binding, non-IgA, IgG3 antibody responses with nonneutralizing functions were linked to reduced risk of infection, the interactions between these signatures remain unclear. Thus, here we comprehensively profile the humoral immune response in 300 RV144 vaccinees to decipher the relationships between humoral biomarkers of protection. We found that vaccine-specific IgG1, IgG3, and IgA were highly correlated. However, ratios of IgG1:IgG3:IgA provided insights into subclass/isotype polyclonal functional regulation. For instance, in the absence of high IgG1 levels, IgG3 antibodies exhibited limited functional activity, pointing to IgG3 as a critical contributor, but not sole driver, of effective antiviral humoral immunity. Higher IgA levels were linked to enhanced antibody effector function, including neutrophil phagocytosis (ADNP), complement deposition (ADCD), and antibody-dependent NK degranulation (CD107a), some of which were increased in infected vaccinees in a case/control data set, suggesting that IgA-driven functions compromised immunity. These data highlight the interplay between IgG1, IgG3, and IgA, pointing to the need to profile the relationships between subclass/isotype selection.

Polyfunctional Fc Dependent Activity of Antibodies to Native Trimeric Envelope in HIV Elite Controllers.

Antibody dependent (AD) functions such as AD cellular cytotoxicity (ADCC) were associated with lower viral load (VL) in untreated HIV progressors and protection from HIV infection in the modestly protective RV144 HIV vaccine trial. Target cells used to measure ADCC, AD complement deposition (ADCD), and AD cellular trogocytosis (ADCT) have been either HIV envelope (Env) gp120-coated CEM.NKr.CCR5 cells or HIV infected cell cultures. In HIV infected cell cultures, uninfected bystander cells take up gp120 shed from infected cells. Both gp120-coated and gp120+ bystander cells expose CD4 induced (CD4i) epitopes, which are normally hidden in native trimeric Env expressed by genuinely HIV infected cells since Nef and Vpu downmodulate cell surface CD4. Antibody dependent assays using either of these target cells probe for CD4i Abs that are abundant in HIV+ plasma but that do not recognize HIV-infected cells. Here, we examined ADCC, ADCD, and ADCT functions using a target cell line, sorted HIV-infected cell line cells, whose HIV infection frequency nears 100% and that expresses HIV Env in a native trimeric closed conformation. Using sorted HIV-infected cells (siCEM) as targets, we probed the binding and AD functions of anti-gp120/Env Abs in plasma from HIV-infected untreated progressor (UTP, n = 18) and treated (TP, n = 24) subjects, compared to that in Elite controllers (EC, n = 37) and Viral Controllers (VC, n = 16), which are rare subsets of HIV-infected individuals who maintain undetectable or low VL, respectively, without treatment. Gp120-coated beads were used to measure AD cellular phagocytosis. Equivalent concentrations of input IgG in plasma from UTPs, ECs, and VCs supported higher levels of all AD functions tested than plasma from TPs. When AD activities were normalized to the concentration of anti-gp120/Env-specific Abs, between-group differences largely disappeared. This finding suggests that the anti-gp120/Env Abs concentrations and not their potency determined AD functional levels in these assays. Elite controllers did differ from the other groups by having AD functions that were highly polyfunctional and highly correlated with each other. PCR measurement of HIV reservoir size showed that ADCC activity was higher in ECs and VCs with a reservoir size below the limit of detection compared to those having a measurable HIV reservoir size.

Topical Tenofovir Pre-exposure Prophylaxis and Mucosal HIV-Specific Fc-Mediated Antibody Activities in Women

The RV144 HIV-vaccine trial highlighted the importance of envelope-specific non-neutralizing antibody (nNAb) Fc-mediated functions as immune correlates of reduced risk of infection. Since pre-exposure prophylaxis (PrEP) and HIV-vaccines are being used as a combination prevention strategy in at risk populations, the effects of PrEP on nNAb functions both mucosally and systemically remain undefined. Previous animal and human studies demonstrated reduced HIV-specific antibody binding avidity post-HIV seroconversion with PrEP, which in turn may affect antibody functionality. In seroconverters from the CAPRISA 004 tenofovir gel trial, we previously reported significantly higher detection and titres of HIV-specific binding antibodies in the plasma and genital tract (GT) that distinguished the tenofovir from the placebo arm. We hypothesized that higher HIV-specific antibody titres and detection reflected corresponding increased antibody-dependent neutrophil-mediated phagocytosis (ADNP) and NK-cell-activated antibody-dependent cellular cytotoxic (ADCC) activities. HIV-specific V1V2-gp70, gp120, gp41, p66, and p24 antibodies in GT and plasma samples of 48 seroconverters from the CAPRISA 004 tenofovir gel trial were tested for ADCP and ADCC at 3, 6- and 12-months post-HIV-infection. GT gp41- and p24-specific ADNP were significantly higher in the tenofovir than the placebo arm at 6 and 12 months respectively (p < 0.05). Plasma gp120-, gp41-, and p66-specific ADNP, and GT gp41-specific ADCC increased significantly over time (p < 0.05) in the tenofovir arm. In the tenofovir arm only, significant inverse correlations were observed between gp120-specific ADCC and gp120-antibody titres (r = −0.54; p = 0.009), and gp41-specific ADNP and gp41-specific antibody titres at 6 months post-infection (r = −0.50; p = 0.015). In addition, in the tenofovir arm, gp41-specific ADCC showed significant direct correlations between the compartments (r = 0.53; p = 0.045). Certain HIV-specific nNAb activities not only dominate specific immunological compartments but can also exhibit diverse functions within the same compartment. Our previous findings of increased HIV specific antibody detection and titres in women who used tenofovir gel, and the limited differences in nNAb activities between the arms, suggest that prior PrEP did not modulate these nNAb functions post-HIV seroconversion. Together these data provide insight into envelope-specific-nNAb Fc-mediated functions at the site of exposure which may inform on ensuing immunity during combination HIV prevention strategies including PrEP and HIV vaccines.

Protein-based, but not viral vector alone, HIV vaccine boosting drives an IgG1-biased polyfunctional humoral immune response.

The RV144 HIV-1 vaccine trial results showed moderate reduction in viral infections among vaccinees as well as induction of antibody-dependent cellular cytotoxicity and vaccine-specific IgG and IgG3 responses directed at variable loop regions 1 and 2 of the HIV envelope protein. However, with the recent failure of the HVTN 702 clinical trial, comprehensive profiling of humoral immune responses may provide insight for these disappointing results. One of the changes included in the HVTN 702 study was the addition of a late boost, aimed at augmenting peak immunity and durability. The companion vaccine trial RV305 was designed to permit the evaluation of the immunologic impact of late boosting with either the boosting protein antigen alone, the canarypox viral vector ALVAC alone, or a combination of both. Although previous data showed elevated levels of IgG antibodies in both boosting arms, regardless of ALVAC-HIV vector incorporation, the effect on shaping antibody effector function remains unclear. Thus, here we analyzed the antibody and functional profile induced by RV305 boosting regimens and found that although IgG1 levels increased in both arms that included protein boosting, IgG3 levels were reduced compared with the original RV144 vaccine strategy. Most functional responses increased upon protein boosting, regardless of the viral vector-priming agent incorporation. These data suggest that the addition of a late protein boost alone is sufficient to increase functionally potent vaccine-specific antibodies previously associated with reduced risk of infection with HIV.

A Systematic Approach to HIV-1 Vaccine Immunogen Selection.

A tremendous loss of financial and human resources from seven large-scale HIV vaccine efficacy trials suggest a need for a systematic approach to vaccine selection. We conducted a systematic analysis of three important envelope glycoprotein (Env) vaccine candidates: BG505 SOSIP.664, 1086.C gp140, and 1086.C gp120 to determine the most promising by comparing their structure and antigenicity. We found that the BG505 SOSIP trimer and 1086.C gp140 clearly outperformed the 1086.C gp120 monomer. BG505 SOSIP.664 bound the strongest to the most potent and broadest broadly neutralizing antibodies (bnAbs) PG9, PGT145, VRC01, and PGT121. Of interest, although BG505 SOSIP.664 did not bind to the CH58 mAb, 1086.C gp140 bound strongly to this mAb, which belongs to a class of non-neutralizing antibodies that may be protective based on correlates of protection studies of the RV144 HIV vaccine trial. The 1086.C gp120 monomer was the least antigenic of the three vaccine immunogens, binding the weakest to bnAbs and CH58 mAb. Taken together, the evidence provided here combined with previous preclinical immunogenicity and efficacy data strongly argue that the BG505 SOSIP.664 trimer and 1086.C gp140 are likely to be better vaccine immunogens than the monomeric 1086.C gp120, which was just recently tested and shown to be nonefficacious in a phase IIb/III trial. Thus, to best utilize our financial and valuable human resources, we propose a systematic approach by not only comparing structure and antigenicity, but also immunogenicity and efficacy of Env vaccine candidates in the preclinical phase to the selection of only the most promising vaccine candidates for clinical testing.

Update on Fc-Mediated Antibody Functions Against HIV-1 Beyond Neutralization.

Antibodies (Abs) are the major component of the humoral immune response and a key player in vaccination. The precise Ab-mediated inhibitory mechanisms leading to in vivo protection against HIV have not been elucidated. In addition to the desired viral capture and neutralizing Ab functions, complex Ab-dependent mechanisms that involve engaging immune effector cells to clear infected host cells, immune complexes, and opsonized virus have been proposed as being relevant. These inhibitory mechanisms involve Fc-mediated effector functions leading to Ab-dependent cellular cytotoxicity, phagocytosis, cell-mediated virus inhibition, aggregation, and complement inhibition. Indeed, the decreased risk of infection observed in the RV144 HIV-1 vaccine trial was correlated with the production of non-neutralizing inhibitory Abs, highlighting the role of Ab inhibitory functions besides neutralization. Moreover, Ab isotypes and subclasses recognizing specific HIV envelope epitopes as well as pecular Fc-receptor polymorphisms have been associated with disease progression. These findings further support the need to define which Fc-mediated Ab inhibitory functions leading to protection are critical for HIV vaccine design. Herein, based on our previous review Su & Moog Front Immunol 2014, we update the different inhibitory properties of HIV-specific Abs that may potentially contribute to HIV protection.

Human Immunodeficiency Virus C.1086 Envelope gp140 Protein Boosts following DNA/Modified Vaccinia Virus Ankara Vaccination Fail To Enhance Heterologous Anti-V1V2 Antibody Response and Protection against Clade C Simian-Human Immunodeficiency Virus Challenge.

The RV144 human immunodeficiency virus type 1 (HIV-1) vaccine trial showed a strong association between anti-gp70 V1V2 scaffold (V1V2) and anti-V2 hot spot peptide (V2 HS) antibody responses and reduced risk of HIV infection. Accordingly, a primary goal for HIV vaccines is to enhance the magnitude and breadth of V1V2 and V2 HS antibody responses in addition to neutralizing antibodies. Here, we tested the immunogenicity and efficacy of HIV-1 C.1086 gp140 boosts administered sequentially after priming with CD40L-adjuvanted DNA/simian-human immunodeficiency virus (SHIV) and boosting with modified vaccinia virus Ankara (MVA)-SHIV vaccines in rhesus macaques. The DNA/MVA vaccination induced robust vaccine-specific CD4 and CD8 T cell responses with a polyfunctional profile. Two gp140 booster immunizations induced very high levels (∼2 mg/ml) of gp140 binding antibodies in serum, with strong reactivity directed against the homologous (C.1086) V1V2, V2 HS, V3, and gp41 immunodominant (ID) proteins. However, the vaccine-induced antibody showed 10-fold (peak) and 32-fold (prechallenge) weaker binding to the challenge virus (SHIV1157ipd3N4) V1V2 and failed to bind to the challenge virus V2 HS due to a single amino acid change. Point mutations in the immunogen V2 HS to match the V2 HS in the challenge virus significantly diminished the binding of vaccine-elicited antibodies to membrane-anchored gp160. Both vaccines failed to protect from infection following repeated SHIV1157ipd3N4 intrarectal challenges. However, only the protein-boosted animals showed enhanced viral control. These results demonstrate that C.1086 gp140 protein immunizations administered following DNA/MVA vaccination do not significantly boost heterologous V1V2 and V2 HS responses and fail to enhance protection against heterologous SHIV challenge.IMPORTANCE HIV, the virus that causes AIDS, is responsible for millions of infections and deaths annually. Despite intense research for the past 25 years, there remains no safe and effective vaccine available. The significance of this work is in identifying the pros and cons of adding a protein boost to an already well-established DNA/MVA HIV vaccine that is currently being tested in the clinic. Characterizing the effects of the protein boost can allow researchers going forward to design vaccines that generate responses that will be more effective against HIV. Our results in rhesus macaques show that boosting with a specific HIV envelope protein does not significantly boost antibody responses that were identified as immune correlates of protection in a moderately successful RV144 HIV vaccine trial in humans and highlight the need for the development of improved HIV envelope immunogens.

AAV-Mediated Expression of Broadly Neutralizing and Vaccine-like Antibodies Targeting the HIV-1 Envelope V2 Region

HIV-1 infection continues to be a global health challenge and a vaccine is urgently needed. Broadly neutralizing antibodies (bNAbs) are considered essential as they inhibit multiple HIV-1 strains, but they are difficult to elicit by conventional immunization. In contrast, non-neutralizing antibodies that correlated with reduced risk of infection in the RV144 HIV vaccine trial are relatively easy to induce, but responses are not durable. To overcome these obstacles, adeno-associated virus (AAV) vectors were used to provide long-term expression of antibodies targeting the V2 region of the HIV-1 envelope protein, including the potent CAP256-VRC26.25 bNAb, as well as non-neutralizing CAP228 antibodies that resemble those elicited by vaccination. AAVs mediated effective antibody expression in cell culture and immunocompetent mice. Mean concentrations of human immunoglobulin G (IgG) in mouse sera increased rapidly following a single AAV injection, reaching 8–60 μg/mL for CAP256 antibodies and 44–220 μg/mL for CAP228 antibodies over 24 weeks, but antibody concentrations varied for individual mice. Secreted antibodies collected from serum retained the expected binding and neutralizing activity. The vectors generated here are, therefore, suitable for the delivery of V2-targeting HIV antibodies, and they could be used in a vectored immunoprophylaxis (VIP) approach to sustain the level of antibody expression required to prevent HIV infection.

Common helical V1V2 conformations of HIV-1 Envelope expose the \u03b14\u03b27 binding site on intact virions

The α4β7 integrin is a non-essential HIV-1 adhesion receptor, bound by the gp120 V1V2 domain, facilitating rapid viral dissemination into gut-associated lymphoid tissues. Antibodies blocking this interaction early in infection can improve disease outcome, and V1V2-targeted antibodies were correlated with moderate efficacy reported from the RV144 HIV-1 vaccine trial. Monoclonal α4β7-blocking antibodies recognise two slightly different helical V2 conformations, and current structural data suggests their binding sites are occluded in prefusion envelope trimers. Here, we report cocrystal structures of two α4β7-blocking antibodies from an infected donor complexed with scaffolded V1V2 or V2 peptides. Both antibodies recognised the same helix-coil V2 conformation as RV144 antibody CH58, identifying a frequently sampled alternative conformation of full-length V1V2. In the context of Envelope, this α-helical form of V1V2 displays highly exposed α4β7-binding sites, potentially providing a functional role for non-native Envelope on virion or infected cell surfaces in HIV-1 dissemination, pathogenesis, and vaccine design.

Glycan modifications to the gp120 immunogens used in the RV144 vaccine trial improve binding to broadly neutralizing antibodies.

To date, the RV144 HIV vaccine trial has been the only study to show that immunization can confer protection from HIV infection. While encouraging, the modest 31.2% (P = 0.04) efficacy achieved in this study left significant room for improvement, and created an incentive to optimize the AIDSVAX B/E vaccine immunogens to increase the level of vaccine efficacy. Since the completion of the RV144 trial, our understanding of the antigenic structure of the HIV envelope protein, gp120, and of the specificity of broadly neutralizing monoclonal antibodies (bN-mAbs) that bind to it, has significantly improved. In particular, we have learned that multiple families of bN-mAbs require specific oligomannose glycans for binding. Both of the monomeric gp120 immunogens (MN- and A244-rgp120) in the AIDSVAX B/E vaccine used in the RV144 trial were enriched for glycans containing high levels of sialic acid, and lacked critical N-linked glycosylation sites required for binding by several families of bN-mAbs. The absence of these epitopes may have contributed to the low level of efficacy achieved in this study. In this report, we describe our efforts to improve the antigenic structure of the rgp120 immunogens used in the vaccine by optimizing glycan-dependent epitopes recognized by multiple bN-mAbs. Our results demonstrated that by shifting the location of one PNGS in A244-rgp120, and by adding two PNGS to MN-rgp120, in conjunction with the production of both proteins in a cell line that favors the incorporation of oligomannose glycans, we could significantly improve the binding by three major families of bN-mAbs. The immunogens described here represent a second generation of gp120-based vaccine immunogens that exhibit potential for use in RV144 follow-up studies.

Predominant envelope variable loop 2-specific and gp120-specific antibody-dependent cellular cytotoxicity antibody responses in acutely SIV-infected African green monkeys

BackgroundThe initial envelope (Env)-specific antibody response in acutely HIV-1-infected individuals and simian immunodeficiency virus (SIV)-infected rhesus monkeys (RMs) is dominated by non-neutralizing antibodies targeting Env gp41. In contrast, natural primate SIV hosts, such as African green monkeys (AGMs), develop a predominant Env gp120-specific antibody response to SIV infection. However, the fine-epitope specificity and function of SIV Env-specific plasma IgG, and their potential role on autologous virus co-evolution in SIV-infected AGMs and RMs remain unclear.ResultsUnlike the dominant linear gp41-specific IgG responses in RMs, SIV-infected AGMs demonstrated a unique linear variable loop 2 (V2)-specific plasma IgG response that arose concurrently with high gp120-directed antibody-dependent cellular cytotoxicity (ADCC) activity, and SIVsab-infected cell binding responses during acute infection. Moreover, SIV variants isolated from SIV-infected AGMs exhibited high amino acid mutation frequencies within the Env V1V2 loop compared to those of RMs. Notably, the linear V2-specific IgG epitope in AGMs overlaps with an analogous region of the HIV V2 loop containing the K169 mutation epitope identified in breakthrough viruses from RV144 vaccinees.ConclusionVaccine-elicited Env V2-specific IgG responses have been proposed as an immune correlate of reduced risk in HIV-1/SIV acquisition in humans and RMs. Yet the pathways to elicit these potentially-protective V2-specific IgG responses remain unclear. In this study, we demonstrate that SIV-infected AGMs, which are the natural hosts of SIV, exhibited high plasma linear V2-specific IgG binding responses that arose concurrently with SIV Env gp120-directed ADCC-mediating, and SIV-infected cell plasma IgG binding responses during acute SIV infection, which were not present in acutely SIV-infected RMs. The linear V2-specific antibody response in AGMs targets an overlapping epitope of the proposed site of vaccine-induced immune pressure defined in the moderately protective RV144 HIV-1 vaccine trial. Identifying host factors that control the early elicitation of Env V2-specific IgG and ADCC antibody responses in these natural SIV hosts could inform vaccination strategies aimed at rapidly inducing potentially-protective HIV-1 Env-specific responses in humans.

Monoclonal Antibodies, Derived from Humans Vaccinated with the RV144 HIV Vaccine Containing the HVEM Binding Domain of Herpes Simplex Virus (HSV) Glycoprotein D, Neutralize HSV Infection, Mediate Antibody-Dependent Cellular Cytotoxicity, and Protect Mice from Ocular Challenge with HSV-1

The RV144 HIV vaccine trial included a recombinant HIV glycoprotein 120 (gp120) construct fused to a small portion of herpes simplex virus 1 (HSV-1) glycoprotein D (gD) so that the first 40 amino acids of gp120 were replaced by the signal sequence and the first 27 amino acids of the mature form of gD. This region of gD contains most of the binding site for HVEM, an HSV receptor important for virus infection of epithelial cells and lymphocytes. RV144 induced antibodies to HIV that were partially protective against infection, as well as antibodies to HSV. We derived monoclonal antibodies (MAbs) from peripheral blood B cells of recipients of the RV144 HIV vaccine and showed that these antibodies neutralized HSV-1 infection in cells expressing HVEM, but not the other major virus receptor, nectin-1. The MAbs mediated antibody-dependent cellular cytotoxicity (ADCC), and mice that received the MAbs and were then challenged by corneal inoculation with HSV-1 had reduced eye disease, shedding, and latent infection. To our knowledge, this is the first description of MAbs derived from human recipients of a vaccine that specifically target the HVEM binding site of gD. In summary, we found that monoclonal antibodies derived from humans vaccinated with the HVEM binding domain of HSV-1 gD (i) neutralized HSV-1 infection in a cell receptor-specific manner, (ii) mediated ADCC, and (iii) reduced ocular disease in virus-infected mice.IMPORTANCE Herpes simplex virus 1 (HSV-1) causes cold sores and neonatal herpes and is a leading cause of blindness. Despite many trials, no HSV vaccine has been approved. Nectin-1 and HVEM are the two major cellular receptors for HSV. These receptors are expressed at different levels in various tissues, and the role of each receptor in HSV pathogenesis is not well understood. We derived human monoclonal antibodies from persons who received the HIV RV144 vaccine that contained the HVEM binding domain of HSV-1 gD fused to HIV gp120. These antibodies were able to specifically neutralize HSV-1 infection in vitro via HVEM. Furthermore, we showed for the first time that HVEM-specific HSV-1 neutralizing antibodies protect mice from HSV-1 eye disease, indicating the critical role of HVEM in HSV-1 ocular infection.

Plasticity and Epitope Exposure of the HIV-1 Envelope Trimer.

We recently showed that mutations in the HIV-1 envelope (Env) destabilize the V3 loop, rendering neutralization-resistant viruses sensitive to V3-directed monoclonal antibodies (MAbs). Here, we investigated the propagation of this effect on other Env epitopes, with special emphasis on V2 loop exposure. Wild-type JR-FL and 19 mutant JR-FL pseudoviruses were tested for neutralization sensitivity to 21 MAbs specific for epitopes in V2, the CD4 binding site (CD4bs), and the CD4-induced (CD4i) region. Certain glycan mutants, mutations in the gp120 hydrophobic core, and mutations in residues involved in intraprotomer interactions exposed epitopes in the V2i region (which overlies the α4β7 integrin binding site) and the V3 crown, suggesting general destabilization of the distal region of the trimer apex. In contrast, other glycan mutants, mutations affecting interprotomer interactions, and mutations affecting the CD4bs exposed V3 but not V2i epitopes. These data indicate for the first time that V3 can move independently of V2, with V3 pivoting out from its "tucked" position in the trimer while apparently leaving the V2 apex intact. Notably, none of the mutations exposed V2 epitopes without also exposing V3, suggesting that movement of V2 releases V3. Most mutations increased sensitivity to CD4bs-directed MAbs without exposure of the CD4i epitope, implying these mutations facilitate the trimers' maintenance of an intermediate energy state between open and closed conformations. Taken together, these data indicate that several transient Env epitopes can be rendered more accessible to antibodies (Abs) via specific mutations, and this may facilitate the design of V1V2-targeting immunogens.IMPORTANCE Many epitopes of the HIV envelope (Env) spike are relatively inaccessible to antibodies (Abs) compared to their exposure in the open Env conformation induced by receptor binding. However, the reduced infection rate that resulted from the vaccine used in the RV144 HIV-1 vaccine trial was correlated with the elicitation of V2- and V3-directed antibodies. Previously, we identified various mechanisms responsible for destabilizing the V3 loop; here, we determined, via mutation of numerous Env residues, which of these elements maintain the V1V2 loop in an inaccessible state and which expose V1V2 and/or V3 epitopes. Notably, our data indicate that V3 can move independently of V2, but none of the mutations studied expose V2 epitopes without also exposing V3. Additionally, V1V2 can be rendered more accessible to Abs via specific mutations, facilitating the development of engineered V2 immunogens.

Boosting of HIV envelope CD4 binding site antibodies with long variable heavy third complementarity determining region in the randomized double blind RV305 HIV-1 vaccine trial.

The canary pox vector and gp120 vaccine (ALVAC-HIV and AIDSVAX B/E gp120) in the RV144 HIV-1 vaccine trial conferred an estimated 31% vaccine efficacy. Although the vaccine Env AE.A244 gp120 is antigenic for the unmutated common ancestor of V1V2 broadly neutralizing antibody (bnAbs), no plasma bnAb activity was induced. The RV305 (NCT01435135) HIV-1 clinical trial was a placebo-controlled randomized double-blinded study that assessed the safety and efficacy of vaccine boosting on B cell repertoires. HIV-1-uninfected RV144 vaccine recipients were reimmunized 6–8 years later with AIDSVAX B/E gp120 alone, ALVAC-HIV alone, or a combination of ALVAC-HIV and AIDSVAX B/E gp120 in the RV305 trial. Env-specific post-RV144 and RV305 boost memory B cell VH mutation frequencies increased from 2.9% post-RV144 to 6.7% post-RV305. The vaccine was well tolerated with no adverse events reports. While post-boost plasma did not have bnAb activity, the vaccine boosts expanded a pool of envelope CD4 binding site (bs)-reactive memory B cells with long third heavy chain complementarity determining regions (HCDR3) whose germline precursors and affinity matured B cell clonal lineage members neutralized the HIV-1 CRF01 AE tier 2 (difficult to neutralize) primary isolate, CNE8. Electron microscopy of two of these antibodies bound with near-native gp140 trimers showed that they recognized an open conformation of the Env trimer. Although late boosting of RV144 vaccinees expanded a novel pool of neutralizing B cell clonal lineages, we hypothesize that boosts with stably closed trimers would be necessary to elicit antibodies with greater breadth of tier 2 HIV-1 strains.Trial Registration: ClinicalTrials.gov NCT01435135

Adjuvanting a Simian Immunodeficiency Virus Vaccine with Toll-Like Receptor Ligands Encapsulated in Nanoparticles Induces Persistent Antibody Responses and Enhanced Protection in TRIM5α Restrictive Macaques.

The results of the RV144 HIV vaccine trial, which demonstrated a rapid waning of protective immunity with time, have underscored the need to develop strategies to enhance the durability of protective immune responses. Our recent work in mice has highlighted the capacity of nanoparticle-encapsulated TLR ligands (NP) to induce potent and durable antibody responses that last a lifetime in mice. In the present study, we evaluated the ability of these NP adjuvants to promote robust and durable protective immune responses against SIV in nonhuman primates. Our results demonstrate that immunization of rhesus macaques with NP adjuvants mixed with soluble SIV Env or a virus-like particle form of Env (VLP) induces potent and durable Env-specific antibody responses in the serum and in vaginal secretions. These responses were superior to those induced by alum adjuvant, and they resulted in enhanced protection against a low-dose intravaginal challenge with a heterologous strain of SIV in animals with TRIM5a restrictive alleles. These results highlight the potential for such NP TLR L adjuvants in promoting robust and durable antibody responses against HIV in the next generation of HIV immunogens currently being developed.