Summary The RV144 HIV-1 trial of the canary pox vector (ALVAC-HIV) plus the gp120 AIDSVAX B/E vaccine demonstrated an estimated efficacy of 31%, that correlated directly with antibodies to HIV-1 envelope variable regions 1 and 2 (V1–V2). Genetic analysis of trial viruses revealed increased vaccine efficacy against viruses matching the vaccine strain at V2 residue 169. Here, we isolated four V2 monoclonal antibodies from RV144 vaccinees that recognize residue 169, neutralize laboratory-adapted HIV-1, and mediate killing of field isolate HIV-1-infected CD4+ T cells. Crystal structures of two of the V2 antibodies demonstrated residue 169 can exist within divergent helical and loop conformations, which contrasted dramatically with the beta strand conformation previously observed with a broadly neutralizing antibody PG9. Thus, RV144 vaccine-induced immune pressure appears to target a region that may be both sequence variable and structurally polymorphic. Variation may signal sites of HIV-1 envelope vulnerability, providing vaccine designers with new options.

iThe ALVAC-HIV/AIDSVAX-B/E RV144 vaccine trial showed an estimated efficacy of 31%. RV144 secondary immune correlate analysis demonstrated that the combination of low plasma anti-HIV-1 Env IgA antibodies and high levels of antibody-dependent cellular cytotoxicity (ADCC) inversely correlate with infection risk. One hypothesis is that the observed protection in RV144 is partially due to ADCC-mediating antibodies. We found that the majority (73 to 90%) of a representative group of vaccinees displayed plasma ADCC activity, usually (96.2%) blocked by competition with the C1 region-specific A32 Fab fragment. Using memory B-cell cultures and antigen-specific B-cell sorting, we isolated 23 ADCC-mediating nonclonally related antibodies from 6 vaccine recipients. These antibodies targeted A32-blockable conformational epitopes (n ‫؍‬ 19), a non-A32-blockable conformational epitope (n ‫؍‬ 1), and the gp120 Env variable loops (n ‫؍‬ 3). Fourteen antibodies mediated cross-clade target cell killing. ADCC-mediating antibodies displayed modest levels of V-heavy (VH) chain somatic mutation (0.5 to 1.5%) and also displayed a disproportionate usage of VH1 family genes (74%), a phenomenon recently described for CD4-binding site broadly neutralizing antibodies (bNAbs). Maximal ADCC activity of VH1 antibodies correlated with mutation frequency. The polyclonality and low mutation frequency of these VH1 antibodies reveal fundamental differences in the regulation and maturation of these ADCC-mediating responses compared to VH1 bNAbs.

؉ T cells earlier than the CD4-inducible (CD4i) epitope bound by MAb 17b and the gp120 carbohydrate epitope bound by MAb 2G12. Importantly, MAb A32 was a potent mediator of ADCC activity. Finally, an A32 Fab fragment blocked the majority of ADCC-mediating Ab activity in plasma of subjects chronically infected with HIV-1. These data demonstrate that the epitope defined by MAb A32 is a major target on gp120 for plasma ADCC activity.

The RV144 ALVAC/AIDSVax HIV-1 vaccine clinical trial showed an estimated vaccine efficacy of 31.2%. Viral genetic analysis identified a vaccine-induced site of immune pressure in the HIV-1 envelope (Env) variable region 2 (V2) focused on residue 169, which is included in the epitope recognized by vaccinee-derived V2 monoclonal antibodies. The ALVAC/AIDSVax vaccine induced antibody-dependent cellular cytotoxicity (ADCC) against the Env V2 and constant 1 (C1) regions. In the presence of low IgA Env antibody levels, plasma levels of ADCC activity correlated with lower risk of infection. In this study, we demonstrate that C1 and V2 monoclonal antibodies isolated from RV144 vaccinees synergized for neutralization, infectious virus capture, and ADCC. Importantly, synergy increased the HIV-1 ADCC activity of V2 monoclonal antibody CH58 at concentrations similar to that observed in plasma of RV144 vaccinees. These findings raise the hypothesis that synergy among vaccine-induced antibodies with different epitope specificities contributes to HIV-1 antiviral antibody responses and is important to induce for reduction in the risk of HIV-1 transmission. IMPORTANCEThe Thai RV144 ALVAC/AIDSVax prime-boost vaccine efficacy trial represents the only example of HIV-1 vaccine efficacy in humans to date. Studies aimed at identifying immune correlates involved in the modest vaccine-mediated protection identified HIV-1 envelope (Env) variable region 2-binding antibodies as inversely correlated with infection risk, and genetic analysis identified a site of immune pressure within the region recognized by these antibodies. Despite this evidence, the antiviral mechanisms by which variable region 2-specific antibodies may have contributed to lower rates of infection remain unclear. In this study, we demonstrate that vaccine-induced HIV-1 envelope variable region 2 and constant region 1 antibodies synergize for recognition of virus-infected cells, infectious virion capture, virus neutralization, and antibody-dependent cellular cytotoxicity. This is a major step in understanding how these types of antibodies may have cooperatively contributed to reducing infection risk and should be considered in the context of prospective vaccine design.

BackgroundDuring the recent H1N1 influenza pandemic, excess morbidity and mortality was seen in young but not older adults suggesting that prior infection with influenza strains may have protected older subjects. In contrast, a history of recent seasonal trivalent vaccine in younger adults was not associated with protection.Methods and FindingsTo study hemagglutinin (HA) antibody responses in influenza immunization and infection, we have studied the day 7 plasma cell repertoires of subjects immunized with seasonal trivalent inactivated influenza vaccine (TIV) and compared them to the plasma cell repertoires of subjects experimentally infected (EI) with influenza H3N2 A/Wisconsin/67/2005. The majority of circulating plasma cells after TIV produced influenza-specific antibodies, while most plasma cells after EI produced antibodies that did not react with influenza HA. While anti-HA antibodies from TIV subjects were primarily reactive with single or few HA strains, anti-HA antibodies from EI subjects were isolated that reacted with multiple HA strains. Plasma cell-derived anti-HA antibodies from TIV subjects showed more evidence of clonal expansion compared with antibodies from EI subjects. From an H3N2-infected subject, we isolated a 4-member clonal lineage of broadly cross-reactive antibodies that bound to multiple HA subtypes and neutralized both H1N1 and H3N2 viruses. This broad reactivity was not detected in post-infection plasma suggesting this broadly reactive clonal lineage was not immunodominant in this subject.ConclusionThe presence of broadly reactive subdominant antibody responses in some EI subjects suggests that improved vaccine designs that make broadly reactive antibody responses immunodominant could protect against novel influenza strains.

iPlasma from a small subset of subjects chronically infected with HIV-1 shows remarkable magnitude and breadth of neutralizing activity. From one of these individuals (CH0219), we isolated two broadly neutralizing antibodies (bnAbs), CH01 and VRC-CH31, from two clonal lineages of memory B cells with distinct specificities (variable loop 1 and 2 [V1V2] conformational specificity and CD4-binding site specificity, respectively) that recapitulate 95% of CH0219 serum neutralization breadth. These data provide proof of concept for an HIV-1 vaccine that aims to elicit bnAbs of multiple specificities.

Glycogen storage disease type Ia (GSD-Ia) profoundly impairs glucose release by the liver due to glucose-6-phosphatase (G6Pase) deficiency. An adeno-associated virus (AAV) containing a small human G6Pase transgene was pseudotyped with AAV8 (AAV2/8) to optimize liver tropism. Survival was prolonged in 2-week-old G6Pase (-/-) mice by 600-fold fewer AAV2/8 vector particles (vp), in comparison to previous experiments involving this model (2 x 10(9) vp; 3 x 10(11) vp/kg). When the vector was pseudotyped with AAV1, survival was prolonged only at a higher dose (3 x 10(13) vp/kg). The AAV2/8 vector uniquely prevented hypoglycemia during fasting and fully corrected liver G6Pase deficiency in GSD-Ia mice and dogs. The AAV2/8 vector has prolonged survival in three GSD-Ia dogs to >11 months, which validated this strategy in the large animal model for GSD-Ia. Urinary biomarkers, including lactate and 3-hydroxybutyrate, were corrected by G6Pase expression solely in the liver. Glycogen accumulation in the liver was reduced almost to the normal level in vector-treated GSD-Ia mice and dogs, as was the hepatocyte growth factor (HGF) in GSD-Ia mice. These preclinical data demonstrated the efficacy of correcting hepatic G6Pase deficiency, and support the further preclinical development of AAV vector-mediated gene therapy for GSD-Ia.

An immune correlates analysis of the RV144 HIV-1 vaccine trial revealed that antibody responses to the gp120 V1/V2 region correlated inversely with infection risk. The RV144 protein immunogens (A244-rp120 and MN-rgp120) were modified by an N-terminal 11-amino-acid deletion (Δ11) and addition of a herpes simplex virus (HSV) gD protein-derived tag (gD). We investigated the effects of these modifications on gp120 expression, antigenicity, and immunogenicity by comparing unmodified A244 gp120 with both Δ11 deletion and gD tag and with Δ11 only. Analysis of A244 gp120, with or without Δ11 or gD, demonstrated that the Δ11 deletion, without the addition of gD, was sufficient for enhanced antigenicity to gp120 C1 region, conformational V2, and V1/V2 gp120 conformational epitopes. RV144 vaccinee serum IgGs bound more avidly to A244 gp120 Δ11 than to the unmodified gp120, and their binding was blocked by C1, V2, and V1/V2 antibodies. Rhesus macaques immunized with the three different forms of A244 gp120 proteins gave similar levels of gp120 antibody titers, although higher antibody titers developed earlier in A244 Δ11 gp120-immunized animals. Conformational V1/V2 monoclonal antibodies (MAbs) gave significantly higher levels of blocking of plasma IgG from A244 Δ11 gp120-immunized animals than IgG from animals immunized with unmodified A244 gp120, thus indicating a qualitative difference in the V1/V2 antibodies induced by A244 Δ11 gp120. These results demonstrate that deletion of N-terminal residues in the RV144 A244 gp120 immunogen improves both envelope antigenicity and immunogenicity.