Immune responses and viral persistence in SHIV.C.CH848-infected rhesus macaques

Chimeric simian/human immunodeficiency viruses (SHIVs) are widely used in nonhuman primate models to recapitulate HIV infection in humans, yet most SHIVs fail to establish persistent viral infection. We investigated immunological and virological events in rhesus macaques infected with the newly developed SHIV.C.CH848, combined with antiretroviral therapy (cART). Similar to HIV/SIV infection, SHIV.C.CH848 infection established viral reservoirs in CD4+ T cells and myeloid cells, accompanied by productive infection and depletion of CD4+ T cells in systemic and lymphoid tissues throughout SHIV infection. Despite 6-months of cART suppressed viral replication, integrated proviral DNA levels remained stable, especially in CD4+ T cells, and the viral rebound was also observed after ART interruption. Autologous neutralizing antibodies to the parental HIV-1 strain CH848 were detected, with limited viral evolution at 5 months post infection. In comparison, heterogenous neutralizing antibodies in SHIV.C.CH848-infected macaques were not detected except for one (1 of 10) animal at 2 years post infection. These findings suggest that the SHIV.C.CH848, a novel class of transmitted/founder SHIVs, can establish sustained viremia and viral reservoirs in rhesus macaques with clinical immunodeficiency consequences, providing a valuable SHIV model for HIV research.Importance SHIVs have been extensively used in a nonhuman primate (NHP) model for HIV research. Here, we investigate viral reservoir in tissues and immune responses in an NHP model inoculated with newly generated transmitted/founder HIV-1 clade C-based SHIV.C.CH848. The data show T/F SHIVC infection of macaques more closely recapitulates the virologic and clinical features of HIV infection including persistent viremia, and viral rebound once antiretroviral therapy is discontinued. These results suggest this CCR5-tropic, SHIVC virus is valuable for testing responses to HIV vaccines and therapeutics.

experiments. All antibodies and reagents were purchased from BD Biosciences Pharmingen (San Diego, CA) 116 unless otherwise noted. Samples were resuspended in BD Stabilizing Fixative (BD Biosciences) and acquired 117 on a FACS FORTESSA (Becton Dickinson). Data were analyzed with FlowJo software (Tree Star, Ashland, 118 OR).

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(Qiagen) according to the manufacturer's instructions. Viral RNA in plasma was directly isolated using the 7 Technologies). The reaction conditions were performed as following: 25L of the reaction mix, containing 1X 171 PCR buffer, 0.2mM dNTPs, 2mM MgCl 2 , 0.8M of each primer, and 0.5U Taq DNA polymerase (Invitrogen Life 172 Technologies), was programmed to perform a 5 minutes hot start at 95C, followed by 20 cycles of 173 denaturation at 95C for 30 seconds, annealing at 63°C for 30 seconds and extension at 72°C for 3 minutes.

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2.5μL of these amplicons were further amplified in triplicate with each primer/probe pairs by real-time PCR 175 reaction using 40 cycles at 95°C for 15 seconds and 63°C for 1 minute. The highly reproducible calibration 176 curves were generated by plotting Cq values against log-transformed concentrations of serial standard.

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Internal standard curves were also generated using the known copy number of target plasmids (1-500 copies) 178 diluted in cellular DNA from SIV naïve RMs. The calibration curves and the internal regression curves were 179 used for interpolating initial copies of each target in unknown samples. A non-template control (NTC) and 180 extracted cellular DNA from the HUT78/SIVmac239 cell line (positive control) were included in the qPCR 181 reactions. As described above, quantification of SHIV RNA/DNA was expressed as copies per 1 million cells, in 182 which cell numbers were determined by copies of genomic CCR5 DNA per cell.

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Antibody neutralization assays 184 TZM-bl neutralization assays were performed using single-round infection of TZM-bl cells with Env 185 pseudoviruses as described (16,(34)(35)(36). For each of the 10 SHIV.C.CH848 infected rhesus macaques, plasma 186 neutralization was tested at 3 time points: 5 months, 15 months, and 2 years post infection. Briefly, TZM-bl 187 cells were seeded at 10,000 cells per well. After 24 hours, plasma was serially diluted 5-fold, starting at a 188 dilution of 1:20 and incubated with 4,000 IU of virus stock as measured via titration on TZM-bl cells. The sham 189 medium was used in place of plasma in specified control wells. The autologous infectious molecular SHIV 190 clone, SHIV.C.CH848, and the pseudotyped HIV-1 CH848, were used to assess the neutralizing antibody 191 titers. Pseudotyped MLV was used as a negative control. Antibody-virus mixtures were co-incubated for 1 h 192 and then added in triplicate to pre-seeded TZM-bl cells.

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We next performed TZM-bl neutralization assays to characterize neutralizing antibody responses in 274 these macaques at 5, 15, and 24 months post infection ( Table 1). These animals developed autologous 275 neutralizing antibody against SHIV.C.CH848 with variable potency (ID50 titers ranged from less than 1:50 to 276 more than 1:300) at 5 months post infection, consistent with previous reports that autologous neutralizing 277 antibody responses are common and arise relatively early in viremic macaques in the first few weeks to against SHIV.C.CH848 increased in 6 of 9 macaques (animal #9 died before this time point), exhibiting 280 neutralization ID50 titers of ≥1:300 dilution.

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In contrast, none of the animals developed heterogenous neutralizing antibodies against the seven

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Recrudescence of SHIVC is observed within 4 months of treatment interruption, consistent with the viral 323 rebound that occurs in most HIV+ patients after ART interruption, ranging from ~5 days to 48 days (49, 50).

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However, viral rebound was not still observed in one animal at 6 months after ART cessation, compared with 325 stable viremic set point in untreated animals. Unlike SIV-infected macaques that show rapidly viral rebound 326 after analytic treatment interruption, some SHIV.C.CH848-infected animals showed a delayed viral 327 recrudescence, which might be attribute to limited chronic activation and latency reactivation, as indicated by 328 lower levels of plasma inflammatory cytokines and chemokines (e.g. IL-8 and MIP-1) at 3 weeks after 329 treatment discontinuation, compared with those at pre-treatment (data not shown). CCR5-tropic 330 SHIV.C.CH848 efficiently infects rhesus macaques, resulting in persistent high levels of viremia, viral reservoir 331 seeding and depletion of CD4+ T cells, consistent with SHIV AD8 infected macaques (8, 51, 52). However, 332 circulating CD4+ T cells were rapidly restored in SHIV.C.CH848-infected macaques after ART was initiated.

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The funders had no role in study design, data collection and analysis, the decision to publish, or preparation of