Jennifer D. Watkins

Anti-HIV IgA isotypes: differential virion capture and inhibition of transcytosis are linked to prevention of mucosal R5 SHIV transmission.

Objective:Although passive immunization with anti-HIV-1 Env IgG1 neutralizing monoclonal antibodies (nmAbs) prevented simian–human immunodeficiency virus (SHIV) infection in rhesus monkeys, IgA nmAbs have not been tested. Here, we sought to determine whether human anti-HIV-1 dimeric (d)IgA1, dIgA2, and IgG1 differ in their ability to prevent mucosal R5 SHIV acquisition in rhesus monkeys. Design:DIgA1, dIgA2, and IgG1 versions of nmAb HGN194 were applied intrarectally in three rhesus monkey groups 30 min before intrarectal SHIV challenge. Methods:After a control pharmacokinetic study confirmed that nmAb concentrations in rectal fluids over time were similar for all HGN194 isotypes, control and nmAb-treated animals were challenged intrarectally with an R5 SHIV, and viral loads were monitored. Results:Unexpectedly, dIgA1 provided the best protection in vivo – although all nmAbs showed similar neutralizing activity in vitro. Five out of the six dIgA1-treated rhesus monkeys remained virus-free compared to only one out of six animals given dIgA2 (P = 0.045 by log-rank test) and two out of six rhesus monkeys treated with IgG1 forms of the nmAb (P = 0.12). Protection correlated significantly with virion capture activity by a given nmAb form, as well as inhibition of transcytosis of cell-free virus across an epithelial cell layer in vitro. Conclusions:Our data imply that dIgA1-mediated capturing of virions in mucosal secretions and inhibition of transcytosis can provide significant prevention of lentiviral acquisition – over and above direct virus neutralization. Vaccine strategies that induce mucosal IgA, especially IgA1, should be developed as a first line of defense against HIV-1, a virus predominantly transmitted mucosally.

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.

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.

Identification and Characterization of a Broadly Cross-Reactive HIV-1 Human Monoclonal Antibody That Binds to Both gp120 and gp41

Identification of broadly cross-reactive HIV-1-neutralizing antibodies (bnAbs) may assist vaccine immunogen design. Here we report a novel human monoclonal antibody (mAb), designated m43, which co-targets the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein (Env). M43 bound to recombinant gp140 s from various primary isolates, to membrane-associated Envs on transfected cells and HIV-1 infected cells, as well as to recombinant gp120 s and gp41 fusion intermediate structures containing N-trimer structure, but did not bind to denatured recombinant gp140 s and the CD4 binding site (CD4bs) mutant, gp120 D368R, suggesting that the m43 epitope is conformational and overlaps the CD4bs on gp120 and the N-trimer structure on gp41. M43 neutralized 34% of the HIV-1 primary isolates from different clades and all the SHIVs tested in assays based on infection of peripheral blood mononuclear cells (PBMCs) by replication-competent virus, but was less potent in cell line-based pseudovirus assays. In contrast to CD4, m43 did not induce Env conformational changes upon binding leading to exposure of the coreceptor binding site, enhanced binding of mAbs 2F5 and 4E10 specific for the membrane proximal external region (MPER) of gp41 Envs, or increased gp120 shedding. The overall modest neutralization activity of m43 is likely due to the limited binding of m43 to functional Envs which could be increased by antibody engineering if needed. M43 may represent a new class of bnAbs targeting conformational epitopes overlapping structures on both gp120 and gp41. Its novel epitope and possibly new mechanism(s) of neutralization could helpdesign improved vaccine immunogens and candidate therapeutics.

Development of a tier 1 R5 clade C simian–human immunodeficiency virus as a tool to test neutralizing antibody-based immunoprophylaxis

Background  While some recently transmitted HIV clade C (HIV‐C) strains exhibited tier 1 neutralization phenotypes, most were tier 2 strains (J Virol 2010; 84:1439). Because induction of neutralizing antibodies (nAbs) through vaccination against tier 2 viruses has proven difficult, we have generated a tier 1, clade C simian–human immunodeficiency virus (SHIV‐C) to permit efficacy testing of candidate AIDS vaccines against tier 1 viruses.

Efficiency of Neutralizing Antibodies Targeting the CD4-Binding Site: Influence of Conformational Masking by the V2 Loop in R5-Tropic Clade C Simian-Human Immunodeficiency Virus

ABSTRACT In R5-tropic clade C simian-human immunodeficiency viruses (SHIV-Cs), we identified a 3-asparagine (3N) deletion mutation in the V2 loop stem of gp120 as the major determinant of neutralization escape of the anti-CD4-binding site (anti-CD4-bs) neutralizing monoclonal antibody (nMAb) b12. However, the more potent anti-CD4-bs nMAbs VRC01 and VRC03 were not sensitive to this mutation. Using isogenic tier 1 or tier 2 proviruses differing only in the 3N mutation, we showed that this mutation might result in selective conformational b12 epitope masking. Therefore, human immunodeficiency virus (HIV) Env immunogens targeting the CD4-bs and designed to neutralize tier 2 viruses should take conformational masking by the V2 loop into account.

Isolation of Monoclonal Antibodies with Predetermined Conformational Epitope Specificity

Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs) from B cells. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes) that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs) that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs). To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. We established proof-of-concept of our strategy by isolating mAbs targeting the conformational V3 loop crown of HIV Env; the new mAbs cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents.

High-level, lasting antiviral immunity induced by a bimodal AIDS vaccine and boosted by live-virus exposure: Prevention of viremia

Objective:To characterize the correlates of protection from systemic infection in a vaccinated rhesus macaque, RAt-9, which had been challenged sequentially with two related clade C simian/human immunodeficiency viruses (SHIV-Cs) yet remained aviremic for more than 5 years despite indirect evidence of cryptic infection. Design:To measure long-term anti-SHIV-C immunity, host genetics and gene-expression patterns for protective correlates. Methods:Long-term immune reactivity was evaluated and identification of virus in RAt-9 was attempted by RT-PCR analysis of concentrated plasma and blood transfer to CD8+ cell-depleted infant macaques. Full MHC genotyping of RAt-9, TRIM5&agr; and KIR3DL allelic expression analysis of PBMC, and microarray gene expression analysis were performed. Results:All attempts to detect/isolate virus, including blood transfer to CD8+ cell-depleted infant rhesus macaques, were negative, and the animal maintained normal levels of memory CD4+ T cells in both peripheral blood and gut tissues. However, RAt-9 maintained high levels of anti-SHIV-C humoral and cellular immunity, including reactivity to nonvaccine neoantigens (Nef and Rev), up to 63 months postinitial challenge, suggesting chronic sub-threshold infection. RAt-9 expressed the Mamu A*001 allele negative for B*008 and B*017, had a B13 serotype, and had increased expression of killer-cell immunoglobulin-like receptors (KIRs) previously linked to favorable outcomes of lentiviral infection. Elements of the gene expression profiling coincided with genotyping results. RAt-9 also displayed CD8+ cell noncytotoxic antiviral response (CNAR) activity. Conclusion:Monkey RAt-9 is the first example of a virus-exposed, persistently aviremic animal that has maintained long-term, high-level cellular and humoral antiviral immunity in the absence of an identifiable cryptic reservoir.

Passive immunization with polyclonal anti-SHIV IgG: partial protection or increased acquisition of heterologous tier 2 SHIV – depending on IgG dose

Author(s): Sholukh, AM; Siddappa, NB; Shanmuganathan, V; Lakhashe, SK; Rasmussen, RA; Watkins, JD; Vyas, HK; Mukhtar, MM; Hemashettar, G; Thorat, S; Yoon, JK; Villinger, F; Novembre, FJ; Landucci, G; Forthal, DN; Ratcliffe, S; Robert-Guroff, M; Polonis, V; Montefiori, DC; Ertl, HC; Ruprecht, RM