Dam-reared neonatal macaques were selected because it is most natural, and because it was previously shown that nursery-reared neonatal macaques have differences in microbiome and immune function.65 Despite potential differences in the microbiomes, V035 generated 6-TAMRA equally or more potent autologous NAbs as the other RMs, but spontaneously controlled viremia by 8 months of infection and did not generate heterologous HIV-1 NAbs. (bnAbs). Thus, having a neonatal model for the induction of heterologous HIV-1 nAbs may provide insights into the mechanisms of neonatal bnAb development. Here, we describe a neonatal model for heterologous HIV-1 nAb induction in pathogenic simian-HIV (SHIV)-infected rhesus macaques (RMs). Viral envelope (alleles (negative [?] and positive [+]), and viral inoculum used to establish infection at the indicated time point post-birth. (C) Viral load dynamics. SIV gag RNA was measured via qPCR to assess plasma viral loads and was reported as RNA copies/mL (log10). Each symbol represents an individual animal; animals with persistent high viral loads of ~103 RNA copies/mL are shown in colored circles, whereas animals with lower viral loads below 103 RNA copies/mL over time are shown in black symbols. The horizontal dash line represents the limit of detection of the assay (1.79 RNA copies/mL). HIV-1 Env evolution Viral-Env evolution in chronic human HIV-1 and macaque SHIV infections of adults have identified mutation patterns associated with selection pressures from NAbs.16,22,23 To explore SHIV Env evolution in RMs, we sequenced 1,212 viral genomes via singlegenome sequencing (SGS) from longitudinal plasma following SHIV infection in RMs with viral loads of 103 RNA copies/mL. Sequences were aligned to the infecting virus, 6-TAMRA and divergence of sequences was observed in all RMs as early as month 6, the first time point tested (Figures 2 and ?and3).3). We found multiple mutations across HIV-1 Env that reached >75% fixation, including mutations in the 133/138 glycan hole (cyan), the 230/289 glycan hole (dark blue), and the 6-TAMRA amino acid 335C347 region (green) (Figure 2), suggesting that these were convergent mutations selected by HIV-1 Env rather than being random mutations. Convergent evolution in the 133/138 glycan hole (V1), the 230/289 glycan hole (C2), and the amino acid 335C347 region (C3) occurred uniformly in all RMs, indicating elicitation of similar autologous HIV-1 nAb responses in these RMs. The potential glycosylation sites at 133/138 and 230/289 were restored by month 6 by mutations at amino acid positions 133, 138, 230, and 289/291, which implicated virus escape from nAbs targeting these glycan holes. In addition, viruses in most RMs developed identical, undefined mutations in V4 or V5 loops, and similar amino acid deletions emerged in the V4 loop in 4 of 11 RMs (gray) (Figure 2). Open in a separate window Figure 2. Longitudinal HIV-1 sequencing in neonatal SHIV infectionEnv sequence evolution visualized using a pixel plot of amino acid alignments of sequences obtained from eleven RMs at different time points. Env sequences were amplified via single-genome sequencing (SGS) of plasma vRNA in samples where vRNA copies were 103/mL. For each animal, 30C60 sequences were generated from each time point (M, months after SHIV infection). The transmitted/founder (T/F) CH848 1017.DT.E169K sequence is depicted schematically at the top. Amino acid substitutions and indels (insertions or deletions) relative to the T/F strain are colored red and black, respectively. Tags indicate 6-TAMRA amino positions (HXB2 numbering) of the mutations that reached 75% frequency of T/F loss in at least two animals. Open in a separate window Figure 3. Mutational patterns in HIV-1 Env nAb epitopes following SHIV infection of neonatal RMs(A) SGS (N, number of sequences; M, months after SHIV infection) in SHIV-infected RMs showed selection and fixation of mutations in strands B and C of the V2 region. Mutations at positions 166, 167, 168, 169, 170, 171, and 172 are colored cyan, dark blue, purple, red, orange, green, and pink, respectively. (B) Sequential V3 loop sequences showed selection and fixation of mutations in the GDIR motif. Lysine (K)-to-arginine (R) reversion at position 327 is colored in blue. Mutations at positions 325 and 326 are highlighted in dark red. Additionally, we found mutations suggestive of nAb selection pressure in the Env V2 and V3 regions, including bnAb epitopes in multiple RMs (Figures 2 and ?and3).3). By month Rabbit Polyclonal to B3GALT4 6 post-infection, a fraction of circulating viruses in 8/11 (73%) RMs (V055, V058, V093, V095, V331, V249, V096, and.