These plates were washed four times and designed with tetramethylbenzidine substrate (20 l per well) (SureBlue Reserve) for 15 min. host antigens, including autologous intestinal microbiota. Thus, gp41-made up of DNA/rAd5 vaccine induced dominant gp41-microbiota cross-reactive antibodies derived from blood memory B cells in RMs as observed in the HVTN 505 vaccine efficacy D13-9001 trial. These data exhibited that RMs can be used to investigate gp41 immunodominance in candidate HIV-1 vaccines. Moreover, colonization of neonatal RMs occurred within the first week of life, and immunization of neonatal RMs during this time also induced a dominant gp41-reactive antibody response. IMPORTANCE Our results are crucial to current work in the HIV-1 vaccine field evaluating the phenomenon of gp41 immunodominance induced by HIV-1 Env gp140 in RMs and humans. Our data demonstrate that RMs are an appropriate animal model to study this phenomenon and to determine the immunogenicity in new HIV-1 Env trimer vaccine designs. The demonstration of gp41 immunodominance in memory B cells of both adult and neonatal RMs indicated that early vaccination could not overcome gp41 dominant responses. KEYWORDS: HIV-1 envelope, gp41, rhesus macaques, HIV-1 vaccine, microbiome INTRODUCTION Recombinant monoclonal antibodies (MAbs) isolated from blood plasmablasts of individuals with acute HIV-1 infection predominantly targeted Env gp41 and were polyreactive with both host and environmental antigens, including users of the intestinal microbiota (1,C3). Polyreactive gp41-binding MAbs can also be isolated from blood of uninfected individuals, suggesting that D13-9001 this HIV-1 Env gp41-reactive Abs induced during acute HIV-1 infection originated from a subset of gp41 cross-reactive memory B cells previously activated by non-HIV-1 antigens (2, 3). Similarly, in the terminal ileum in HIV-1 infected and uninfected individuals, mutated Env gp41-reactive MAbs were found to cross-react with users of the intestinal microbiota (3). In the setting of vaccination, the multiclade (A, B, and C) DNA/recombinant adenovirus computer virus type 5 (rAd5) vaccine in phase 2a and 2b trials (4, 5) induced Env-reactive Ab responses that were dominated by gp41-reactive Abdominal muscles (6). Vaccine-induced gp41-reactive Abs were nonneutralizing and cross-reacted with host antigens as well as with bacterial proteins RNA polymerase and pyruvate-flavodoxin oxidoreductase, which shared sequence similarities with the heptad repeat 1 region of HIV-1 gp41 (6). Interestingly, pre- and postvaccination clonally related Abs from DNA/rAd5-vaccinated individuals were reactive with both gp41 and users of the intestinal microbiota, but the postvaccination Ab was more affinity matured than the prevaccination Ab, suggesting that this vaccine-induced Ab response originated from a preexisting pool of gp41-microbiota cross-reactive B cells (6). In a second human clinical D13-9001 trial, HIV-1 Vaccine Trials Network (HVTN) trial 205, a DNA primary and MVA boost with gp140 also induced gp41-dominant plasma responses in humans, with titers of gp41-specific IgG higher than those of gp120-specific IgG (7). These studies indicated that two gp41-made up of immunogens induced a dominant gp41-reactive Ab response in humans. Rhesus macaques (RMs) are widely used to investigate human HIV-1 pathogenesis and have utility for screening preclinical efficacy of prevention strategies, including candidate vaccines (8,C11). Thus, determining if gp41-made up of immunogens in HIV-1 vaccines induce a dominant gp41 response in RMs is key to defining if RMs are an appropriate animal model for evaluation of Env-containing vaccines. Studies have suggested that microbial antigens can stimulate immune cells and expand CD4+ T cells and B cell repertoires (12,C19). For example, the intestines of germfree mice have low numbers of lamina propria CD4+ T cells compared to bacterially colonized mice (12). Microbial colonization also influences early B-lineage development TRA1 and prospects to increased receptor editing, suggesting that this gut microbes may shape the preimmune repertoire in gut lamina propria (16)..