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

Abstract

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.

Keywords: (6 max); Complete protection; IgA; IgG; Passive immunization; Rhesus monkey; SHIV-C mucosal challenge.

Fig. 1

Antiviral T-cell responses after previous SHIV-1157ipEL-p challenge (unpublished data and [13]). PBMC were stimulated with overlapping peptides representing SIVmac239 Gag and proliferation of CD4⁺ and CD8⁺ cells was measured using the CFSE dilution method as described in Materials and Methods. The y-axis indicates % proliferating cells. PBMC isolated from two naïve macaques (RCy-5 and RSf-12) were used as a negative control and PBMC from a previously vaccinated, aviremic animal RAt-9 [47] served as a positive control, respectively. (A) Positive (RAt-9) [47] and negative (RCy-5 and RSf-12) controls. (B) T-cell responses of RMs that had received wild-type (IgG1_wt ) or afucosylated (IgG1_kif ) versions of HGN194 IgG1 systemically (i.v.) (unpublished data). (C) T-cell responses of animals that had previously received HGN194 IgG1, dimeric IgA1 or dimeric IgA2 topically (i.r.) (unpublished data and [13]).

Fig. 2

Antibody responses in RMs previously given passive immunization with different forms of HGN194 (unpublished data and [13]). (A) and (B) Only animals that had received HGN194 systemically were analyzed. Mucosally treated RMs had been tested earlier and HGN194 had not been detected in the plasma (data not shown). (A) Residual concentration of HGN194 IgG1 at different time points after administration. HGN194 IgG1 was used as a standard. Secondary goat anti-monkey HRP-conjugated Ab was RM IgG adsorbed. (B) RM anti-human IgG responses at different time points after HGN194 IgG1 i.v. administration. +C, positive control (goat anti-human Ab HRP-conjugated). (C) and (D) HIV Env binding ELISA analysis of RM plasma samples collected at different time points after virus challenge. (C) Anti-Env plasma responses of Group A RMs. Black bar, pooled naïve RM plasma was used as a negative control; open bar, plasma of RRi-11 [48] was used as positive control. SHIV-1157ip gp120 served as antigen. The secondary Ab was mouse anti-monkey HRP-conjugated secondary Ab with minimal cross-reactivity to human IgG. (D) Anti-Env plasma responses of Group B RMs. Experimental conditions are same as for (C).

Fig. 3

Study timeline and design. Three groups of RMs were enrolled. Group A (n = 6) received the combination of i.v. HGN194 IgG1 (1.45 mg/kg); and i.r. HGN194 dIgA2 (1.25 mg). Group B RMs (n = 6) received i.v. HGN194 IgG1 (1.45 mg/kg) only. Group C (n = 2) RMs served as virus-only controls. Small arrow, mAb administrations; big open arrow, 24 h after IgG1 administration and 30 min after dIgA2 topical application (Group A only) animals were challenged i.r. with 31.5 AID₅₀ of SHIV-1157ipEL-p.

Fig. 4

The combination of HGN194 IgG1 + dIgA2 completely protected RMs from high-dose mucosal virus challenge. (A) Gray open symbols, viral RNA loads for individual RMs for Group A (IgG1 + dIgA2); black filled symbols, vRNA loads for Group B (IgG1) RMs; dashed black lines, vRNA loads for Group C (controls) RMs. (B) Kaplan–Meier analysis of time until vRNA load exceeded 50 copies/ml. Log rank test significance P value is indicated. Gray dashed line, Group A; black solid line, Group B.

Fig. 5

Analysis of HGN194 IgG1 levels in plasma. (A) HGN194 IgG1 pharmacokinetics in RM groups. Black arrow indicates SHIV-1157ipEL-p challenge; black filled symbols, RM of Group A; gray open symbols, RMs of Group B. (B) Analysis of HGN194 IgG1 half-life in RMs. Circles, RMs of Group A; squares, RMs of Group B. Statistical analysis was performed by Mann–Whitney test (P < 0.05).

Fig. 6

The combination of HGN194 IgG1 + dIgA2 neutralized virus equally well as the individual mAbs. The concentration of IgG1 + dIgA2 combination is the sum of concentrations of individual mAbs. MAbs VRC01 and Fm-6 were used as positive and negative controls, respectively (not shown). (A) Human PBMC-based assay; (B) TZM-bl cell assay; and (C) A3R5 cell assay.

Fig. 7

Blocking of IgG1-mediated transcytosis enhanced by low pH. Virus was incubated with the IgG1 mAbs HGN194 or Fm-6 (isotype control) ranging from 1 to 100 μg/ml. The IgG1 mAbs were used alone or in combinations with 200 μg/ml of dIgA2. Experiments involving dIgA1 served as positive control. Next, virus or virus/mAb mixtures were added to the top well of a transwell/transcytosis system (Methods). Percent of transcytosis inhibition was calculated in comparison with the number of vRNA copies determined for wells with virus alone. Negative values on the y-axis represent enhanced transcytosis. All experiments were repeated at least twice.