Collectively, DNA prime, rAd5 boost vaccine-induced gp41-reactive mAbs were more polyreactive than gp120-reactive mAbs (p <0.003, Cochran-Mantel-Haenszel Test) (Table 2). == Physique 2. (2,3). However, it is unknown if a similar gp41-reactive Ab response would occur in the setting of HIV-1 Env vaccination. A DNA primary, recombinant adenovirus serotype 5 (rAd5) boost vaccine that included HIVgag, polandnefgenes, as well as a trivalent mixture of clade A, B and Cenvgp140 genes made up of both gp120 and gp41 components was studied in the HIV Vaccine Trials Network (HVTN) [phase Ib (HVTN 082), phase II (HVTN 204), phase IIb (HVTN 505) efficacy trial] and other clinical trials [phase I/II (RV172), phase I (V001)] (47). This vaccine was the first vaccine made up of the ectodomain of the Env gp41 component, NR2B3 covalently linked to gp120, to be tested in an efficacy trial, and was designed to primarily generate CD8 T cell responses, although this vaccine generated Env Ab responses as well (810). However, the phase IIb HVTN 505 efficacy trial showed no vaccine efficacy (11). Thus, these vaccine trials made up of Env gp41 provided an opportunity to determine if the Env Ab response in the setting of Env vaccination was dominated by gp41-reactive Abs derived from Env-IM cross-reactive B cells. == Isolation of Env-reactive Memory B Cells and Vaccinee Plasma Serologies == We found that the DNA primary, rAd5 boost antibody response to HIV-1 Env was dominantly focused on gp41 compared to gp120. This specificity was exhibited by both serologic analysis Nomegestrol acetate and by vaccine-Env circulation cytometry-sorted memory B cells. Plasma IgG binding assays were performed Nomegestrol acetate on plasma of a random sample of 40 Phase IIb efficacy trial vaccine recipients who were HIV-1 unfavorable at the final month 24 visit (11) (Physique 1A), and plasma of 8 HIV-1 uninfected Phase Ib and II DNA primary, rAd5 boost trial participants with high titers of plasma binding Abs to recombinant (r)gp140 vaccine-Envs and/or neutralization of clade C MW965 HIV-1 isolate (Physique 1B). Plasma binding gp41-reactive Ab titers were 10 fold higher than gp120-reactive Ab titers, including Ab reactivity with vaccine-gp120s (p<0.0001(Physique 1A),p<0.01(Physique 1B); Wilcoxon signed rank test). Thus, the non-protective DNA primary, rAd5 boost gp140 vaccine induced a dominant HIV-1 Env gp41 plasma Ab response. == Physique 1. Characteristics of HIV-1-reactive antibodies (Abs) induced by DNA primary, rAd5 boost vaccine. == A) Plasma Ab titers (AUC, area under the curve) to gp120 and gp41 proteins in a subset of 40 HVTN 505 vaccine recipients; reddish circles represent vaccine responders (percents) to the antigens tested, and blue represent non-responders. (B) Plasma binding Abdominal muscles from 8 HVTN 082 and 204 trial participants (vaccine responders) were screened for gp120 and gp41 reactivity via binding Ab multiplex assay (BAMA). (C) HIV-1-reactive Abs were isolated from single memory B cells of the 8 HVTN 082 and 204 trial participants and screened for binding gp120 and gp41 proteins via ELISA. Statistics: (A) ***p < 0.0001, Wilcoxon signed rank test, B.MN gp41 vs. gp120 (maximum; which is the highest value of all gp120 Envs for each participant), *p < 0.05McNemars test, B.MN gp41 vs. each gp120 (not shown); (B) *p < 0.05**p < 0.01, Wilcoxon signed rank test [Graphpad Prism]. Next, we performed single memory B cell sorting by circulation cytometry using peripheral blood B cells from phase Ib and phase II DNA primary, rAd5-boost trial participants. Vaccine-Env gp140 and V1V2 subunits, as well as a consensus group M gp140 Env (termed CON-S) (12) were used as fluorophore-labeled recombinant proteins to identify Env-specific memory B cells present in peripheral blood mononuclear cells (PBMCs) of vaccinees 4 weeks after final vaccination (Physique S1) (Table S1). We analyzed 8 phase Ib and phase II DNA primary, rAd5-boost trial participants; from these 8 vaccinees we isolated 221 HIV-1 Env-reactive Abdominal muscles (Physique 1C,Table S2). Of the 221 HIV-1 Env-reactive Abs, there were 131 unique VHDJHrearrangements (Table S3). Amazingly, 205/221 (93%) of the HIV-1 Env-reactive Abs Nomegestrol acetate and 115/131 (88%) of the unique.