Live-virus exposure of vaccine-protected macaques alters the anti-HIV-1 antibody repertoire in the absence of viremia

Background: We addressed the question whether live-virus challenges could alter vaccine-induced antibody (Ab) responses in vaccinated rhesus macaques (RMs) that completely resisted repeated exposures to R5-tropic simian-human immunodeficiency viruses encoding heterologous HIV clade C envelopes (SHIV-Cs). Results: We examined the Ab responses in aviremic RMs that had been immunized with a multi-component protein vaccine (multimeric HIV-1 gp160, HIV-1 Tat and SIV Gag-Pol particles) and compared anti-Env plasma Ab titers before and after repeated live-virus exposures. Although no viremia was ever detected in these animals, they showed significant increases in anti-gp140 Ab titers after they had encountered live SHIVs. When we investigated the dynamics of anti-Env Ab titers during the immunization and challenge phases further, we detected the expected, vaccine-induced increases of Ab responses about two weeks after the last protein immunization. Remarkably, these titers kept rising during the repeated virus challenges, although no viremia resulted. In contrast, in vaccinated RMs that were not exposed to virus, anti-gp140 Ab titers declined after the peak seen two weeks after the last immunization. These data suggest boosting of pre-existing, vaccine-induced Ab responses as a consequence of repeated live-virus exposures. Next, we screened polyclonal plasma samples from two of the completely protected vaccinees by peptide phage display and designed a strategy that selects for recombinant phages recognized only by Abs present after – but not before – any SHIV challenge. With this “subtractive biopanning” approach, we isolated V3 mimotopes that were only recognized after the animals had been exposed to live virus. By detailed epitope mapping of such anti-V3 Ab responses, we showed that the challenges not only boosted pre-existing binding and neutralizing Ab titers, but also induced Abs targeting neo-antigens presented by the heterologous challenge virus. Conclusions: Anti-Env Ab responses induced by recombinant protein vaccination were altered by the multiple, live SHIV challenges in vaccinees that had no detectable viral loads. These data may have implications for the interpretation of “vaccine only” responses in clinical vaccine trials

Cooperation Between Systemic and Mucosal Antibodies Induced by Virosomal Vaccines Targeting HIV-1 Env: Protection of Indian Rhesus Macaques Against Low-Dose Intravaginal SHIV Challenges.

A virosomal vaccine inducing systemic/mucosal anti-HIV-1 gp41 IgG/IgA had previously protected Chinese-origin rhesus macaques (RMs) against vaginal SHIVSF162P3 challenges. Here, we assessed its efficacy in Indian-origin RMs by intramuscular priming/intranasal boosting (n=12/group). Group K received virosome-P1-peptide alone (harboring the Membrane Proximal External Region), Group L combined virosome-rgp41 plus virosome-P1, and Group M placebo virosomes. Vaccination induced plasma binding but no neutralizing antibodies. Five weeks after boosting, all RMs were challenged intravaginally with low-dose SHIVSF162P3 until persistent systemic infection developed. After SHIV challenge #7, six controls were persistently infected versus only one Group L animal (vaccine efficacy 87%; P=0.0319); Group K was not protected. After a 50% SHIV dose increase starting with challenge #8, protection in Group L was lost. Plasmas/sera were analyzed for IgG phenotypes and effector functions; the former revealed that protection in Group L was significantly associated with increased binding to FcγR2/3(A/B) across several time-points, as were some IgG measurements. Vaginal washes contained low-level anti-gp41 IgGs and IgAs, representing a 1-to-5-fold excess over the SHIV inoculum's gp41 content, possibly explaining loss of protection after the increase in challenge-virus dose. Virosomal gp41-vaccine efficacy was confirmed during the initial seven SHIV challenges in Indian-origin RMs when the SHIV inoculum had at least 100-fold more HIV RNA than acutely infected men's semen. Vaccine protection by virosome-induced IgG and IgA parallels the cooperation between systemically administered IgG1 and mucosally applied dimeric IgA2 monoclonal antibodies that as single-agents provided no/low protection - but when combined, prevented mucosal SHIV transmission in all passively immunized RMs

Defense-in-depth by mucosally administered anti-HIV dimeric IgA2 and systemic IgG1 mAbs: complete protection of rhesus monkeys from mucosal SHIV challenge.

Although IgA is the most abundantly produced immunoglobulin in humans, its role in preventing HIV-1 acquisition, which occurs mostly via mucosal routes, remains unclear. In our passive mucosal immunizations of rhesus macaques (RMs), the anti-HIV-1 neutralizing monoclonal antibody (nmAb) HGN194, given either as dimeric IgA1 (dIgA1) or dIgA2 intrarectally (i.r.), protected 83% or 17% of the RMs against i.r. simian-human immunodeficiency virus (SHIV) challenge, respectively. Data from the RV144 trial implied that vaccine-induced plasma IgA counteracted the protective effector mechanisms of IgG1 with the same epitope specificity. We thus hypothesized that mucosal dIgA2 might diminish the protection provided by IgG1 mAbs targeting the same epitope. To test our hypothesis, we administered HGN194 IgG1 intravenously (i.v.) either alone or combined with i.r. HGN194 dIgA2. We enrolled SHIV-exposed, persistently aviremic RMs protected by previously administered nmAbs; RM anti-human IgG responses were undetectable. However, low-level SIV Gag-specific proliferative T-cell responses were found. These animals resemble HIV-exposed, uninfected humans, in which local and systemic cellular immune responses have been observed. HGN194 IgG1 and dIgA2 used alone and the combination of the two neutralized the challenge virus equally well in vitro. All RMs given only i.v. HGN194 IgG1 became infected. In contrast, all RMs given HGN194 IgG1+dIgA2 were completely protected against high-dose i.r. SHIV-1157ipEL-p challenge. These data imply that combining suboptimal defenses at the mucosal and systemic levels can completely prevent virus acquisition. Consequently, active vaccination should focus on defense-in-depth, a strategy that seeks to build up defensive fall-back positions well behind the fortified frontline.We thank Dr. J. Mascola for providing mAb VRC01, Dr. S.-L. Hu for providing SHIV-1157ip Env proteins, and Dr. W. Marasco for providing mAb Fm-6. We thank Dr. K. Rogers and K. Kinsley for TRIM5α genotype analysis, Dr. S. Lee for assistance in statistical analysis, V. Shanmuganathan for technical assistance, and Juan Esquivel for assistance with the preparation of the manuscript. This was work supported by the Bill and Melinda Gates Foundation Collaboration for AIDS Vaccine Discovery (CAVD) UCL-VDC Grant 38637 (R.A.W.). This project was also funded in part by NIH grants P01 AI048240, R01 AI100703 and R37 AI034266 to RMR. Base grant P51 OD011132 provided support to the Yerkes National Primate Research Center. The Southwest National Primate Research Center is supported by an NIH primate center base grant (previously NCRR grant P51 RR013986; currently Office of Research Infrastructure Programs/OD P51 OD011133).This is the accepted manuscript of a paper published in Vaccine (Sholukh AM, et al., Vaccine, 2015, 33, 2086-2095, doi:10.1016/j.vaccine.2015.02.020). The final version is available at http://dx.doi.org/10.1016/j.vaccine.2015.02.02

An Anti-HIV-1 V3 Loop Antibody Fully Protects Cross-Clade and Elicits T-Cell Immunity in Macaques Mucosally Challenged with an R5 Clade C SHIV

Neutralizing antibodies have been shown to protect macaques against SHIV challenge. However, genetically diverse HIV-1 clades have evolved, and a key question left unanswered is whether neutralizing antibodies can confer cross-clade protection in vivo. The novel human monoclonal antibody HGN194 was isolated from an individual infected with an HIV-1 clade AG recombinant circulating recombinant form (CRF). HGN194 targets an epitope in the third hypervariable loop (V3) of HIV-1 gp120 and neutralizes a range of relatively neutralization-sensitive and resistant viruses. We evaluated the potential of HGN194 to protect infant rhesus monkeys against a SHIV encoding a primary CCR5-tropic HIV-1 clade C envelope. After high-dose mucosal challenge, all untreated controls became highly viremic while all HGN194-treated animals (50 mg/kg) were completely protected. When HGN194 was given at 1 mg/kg, one out of two monkeys remained aviremic, whereas the other had delayed, lower peak viremia. Interestingly, all protected monkeys given high-dose HGN194 developed Gag-specific proliferative responses of both CD4+ and CD8+ T cells. To test whether generation of the latter involved cryptic infection, we ablated CD8+ cells after HGN194 clearance. No viremia was detected in any protected monkeys, thus ruling out virus reservoirs. Thus, induction of CD8 T-cell immunity may have resulted from transient “Hit and Run” infection or cross priming via Ag-Ab-mediated cross-presentation. Together, our data identified the HGN194 epitope as protective and provide proof-of-concept that this anti-V3 loop mAb can prevent infection with sterilizing immunity after challenge with virus of a different clade, implying that V3 is a potential vaccine target

Passive immunization of macaques with polyclonal anti-SHIV IgG against a heterologous tier 2 SHIV: outcome depends on IgG dose

Background: A key goal for HIV-1 envelope immunogen design is the induction of cross-reactive neutralizing antibodies (nAbs). As AIDS vaccine recipients will not be exposed to strains exactly matching any immunogens due to multiple HIV-1 quasispecies circulating in the human population worldwide, heterologous SHIV challenges are essential for realistic vaccine efficacy testing in primates. We assessed whether polyclonal IgG, isolated from rhesus monkeys (RMs) with high-titer nAbs (termed SHIVIG), could protect RMs against the R5-tropic tier-2 SHIV-2873Nip, which was heterologous to the viruses or HIV-1 envelopes that had elicited SHIVIG. Results: SHIVIG demonstrated binding to HIV Gag, Tat, and Env of different clades and competed with the broadly neutralizing antibodies b12, VRC01, 4E10, and 17b. SHIVIG neutralized tier 1 and tier 2 viruses, including SHIV-2873Nip. NK-cell depletion decreased the neutralizing activity of SHIVIG 20-fold in PBMC assays. Although SHIVIG neutralized SHIV-2873Nip in vitro, this polyclonal IgG preparation failed to prevent acquisition after repeated intrarectal low-dose virus challenges, but at a dose of 400 mg/kg, it significantly lowered peak viremia (P = 0.001). Unexpectedly, single-genome analysis revealed a higher number of transmitted variants at the low dose of 25 mg/kg, implying increased acquisition at low SHIVIG levels. In vitro, SHIVIG demonstrated complement-mediated Ab-dependent enhancement of infection (C’-ADE) at concentrations similar to those observed in plasmas of RMs treated with 25 mg/kg of SHIVIG. Conclusion: Our primate model data suggest a dual role for polyclonal anti-HIV-1 Abs depending on plasma levels upon virus encounter

Molecular Epidemiology of HIV-1 Subtypes in India: Origin and Evolutionary History of the Predominant Subtype C

This thesis describes the translational genomics of HIV-1subtype C in India from its origin to therapeutic response with the aim to improve our knowledge for better therapeutic and preventive strategies to combat HIV/AIDS. In a systemic approach, we identified the molecular phylogeny of HIV-1 subtypes circulating in India and the time to most recent common ancestors (tMRCA) of predominant HIV-1 subtype C strains. Additionally, this thesis also studied drug resistance mutations in children, adolescents and adults, the role of host factors in evolution of drug resistance, and population dynamics of viremia and viral co-receptor tropism in perinatal transmission. Finally, the long term therapeutic responses on Indian national first-line antiretroviral therapy were also studied. In Paper I, we reported an increase in the HIV-1 recombinant forms in the HIV-1 epidemiology using a robust subtyping methodology. While the study confirmed HIV- 1 subtype C as a dominant subtype, its origin was dated back to the early 1970s from a single or few genetically related strains from South Africa, whereafter, it has evolved independently. In Paper II, the lethal hypermutations due to the activity of human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (hA3G) was significantly associated with antiretroviral therapy (ART) failure in Indian HIV-1 subtype C patients. The presence of M184I and M230I mutations were observed due to the editing of hA3G in the proviral compartment but stop codons were also found in the open reading frames and the same drug resistance mutations were absent in plasma virus. Therefore, it is unlikely that the viral variants which exhibit hypermutated sequences and M184I and/or M230I will mature and expand in vivo and hence are unlikely to have any clinical significance. The high concordance of drug resistance genotyping in the plasma and proviral compartments in therapy-naïve patients, gives weight to the idea of using whole blood for surveillance of drug resistance mutations which precludes logistic challenges of cold chain transport. In Papers III and IV, we identified a substantial proportion of HIV-1 subtype C perinatally-infected older children who had a high burden of plasma viremia but also had high CD4+ T-cell counts. In addition, older children with HIV-1 subtype C infection presented a high prevalence of predicted X4 and R5/X4 tropic strains which indicates that HIV-1 subtype C strains required longer duration of infection and greater disease progression to co-receptor transition from R5- to X4-tropic strains (IV). Our studies also indicate that transmitted drug resistance is low among Indian HIV-1 infected children, adolescents (III) and adults (II). In Paper V, in a longitudinal cohort study, a good long-term response to the Indian national first-line therapy for a median of nearly four years with 2.8% viral failure, indicating the overall success of the Indian ART program. Our study also showed that three immunologically well patients with virological rebound and major viral drug resistance mutations (M184V, K103N and Y181C) during one study visit had undetectable viral load at their next visit. These findings suggest that use of multiple parameters like patients’ immunological (CD4+ T-cell count), virological (viral load) and drug resistance data should all be used to optimize the treatment switch to second line therapy. In conclusion, this translational genomics study enhances our knowledge about the HIV-1 subtype C strains circulating in India which are genetically distinct from prototype African subtype C strains. Considerably more research using appropriate models need to be performed to understand the phenotypic and biological characteristics of these strains to guide efficient disease intervention and management strategies

Vaccination against Heterologous R5 Clade C SHIV: Prevention of Infection and Correlates of Protection

A safe, efficacious vaccine is required to stop the AIDS pandemic. Disappointing results from the STEP trial implied a need to include humoral anti-HIV-1 responses, a notion supported by RV144 trial data even though correlates of protection are unknown. We vaccinated rhesus macaques with recombinant simian immunodeficiency virus (SIV) Gag-Pol particles, HIV-1 Tat and trimeric clade C (HIV-C) gp160, which induced cross-neutralizing antibodies (nAbs) and robust cellular immune responses. After five low-dose mucosal challenges with a simian-human immunodeficiency virus (SHIV) that encoded a heterologous R5 HIV-C envelope (22.1% divergence from the gp160 immunogen), 94% of controls became viremic, whereas one third of vaccinees remained virus-free. Upon high-dose SHIV rechallenge, all controls became infected, whereas some vaccinees remained aviremic. Peak viremia was inversely correlated with both cellular immunity (p<0.001) and cross-nAb titers (p<0.001). These data simultaneously linked cellular as well as humoral immune responses with the degree of protection for the first time

An anti-HIV-1 V3 loop antibody fully protects cross-clade and elicits T-cell immunity in macaques mucosally challenged with an R5 clade C SHIV

Neutralizing antibodies have been shown to protect macaques against SHIV challenge. However, genetically diverse HIV-1 clades have evolved, and a key question left unanswered is whether neutralizing antibodies can confer cross-clade protection in vivo. The novel human monoclonal antibody HGN194 was isolated from an individual infected with an HIV-1 clade AG recombinant circulating recombinant form (CRF). HGN194 targets an epitope in the third hypervariable loop (V3) of HIV-1 gp120 and neutralizes a range of relatively neutralization- sensitive and resistant viruses. We evaluated the potential of HGN194 to protect infant rhesus monkeys against a SHIV encoding a primary CCR5-tropic HIV-1 clade C envelope. After high- dose mucosal challenge, all untreated controls became highly viremic while all HGN194-treated animals (50 mg/kg) were completely protected. When HGN194 was given at 1 mg/kg, one out of two monkeys remained aviremic, whereas the other had delayed, lower peak viremia. Interestingly, all protected monkeys given high-dose HGN194 developed Gag-specific proliferative responses of both CD4+ and CD8+ T cells. To test whether generation of the latter involved cryptic infection, we ablated CD8+ cells after HGN194 clearance. No viremia was detected in any protected monkeys, thus ruling out virus reservoirs. Thus, induction of CD8 T-cell immunity may have resulted from transient “Hit and Run” infection or cross priming via Ag-Ab- mediated cross-presentation. Together, our data identified the HGN194 epitope as protective and provide proof-of-concept that this anti-V3 loop mAb can prevent infection with sterilizing immunity after challenge with virus of a different clade, implying that V3 is a potential vaccine target

R5 Clade C SHIV Strains with Tier 1 or 2 Neutralization Sensitivity: Tools to Dissect Env Evolution and to Develop AIDS Vaccines in Primate Models

Background: HIV-1 clade C (HIV-C) predominates worldwide, and anti-HIV-C vaccines are urgently needed. Neutralizing antibody (nAb) responses are considered important but have proved difficult to elicit. Although some current immunogens elicit antibodies that neutralize highly neutralization-sensitive (tier 1) HIV strains, most circulating HIVs exhibiting a less sensitive (tier 2) phenotype are not neutralized. Thus, both tier 1 and 2 viruses are needed for vaccine discovery in nonhuman primate models. Methodology/Principal Findings: We constructed a tier 1 simian-human immunodeficiency virus, SHIV-1157ipEL, by inserting an “early,” recently transmitted HIV-C env into the SHIV-1157ipd3N4 backbone [1] encoding a “late” form of the same env, which had evolved in a SHIV-infected rhesus monkey (RM) with AIDS. SHIV-1157ipEL was rapidly passaged to yield SHIV-1157ipEL-p, which remained exclusively R5-tropic and had a tier 1 phenotype, in contrast to “late” SHIV-1157ipd3N4 (tier 2). After 5 weekly low-dose intrarectal exposures, SHIV-1157ipEL-p systemically infected 16 out of 17 RM with high peak viral RNA loads and depleted gut CD4$^+$ T cells. SHIV-1157ipEL-p and SHIV-1157ipd3N4 env genes diverge mostly in V1/V2. Molecular modeling revealed a possible mechanism for the increased neutralization resistance of SHIV-1157ipd3N4 Env: V2 loops hindering access to the CD4 binding site, shown experimentally with nAb b12. Similar mutations have been linked to decreased neutralization sensitivity in HIV-C strains isolated from humans over time, indicating parallel HIV-C Env evolution in humans and RM. Conclusions/Significance: SHIV-1157ipEL-p, the first tier 1 R5 clade C SHIV, and SHIV-1157ipd3N4, its tier 2 counterpart, represent biologically relevant tools for anti-HIV-C vaccine development in primates