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Transmitted/founder (TF) simian-human immunodeficiency viruses (SHIVs) express HIV-1 envelopes modified at position 375 to efficiently infect rhesus macaques while preserving authentic HIV-1 Env biology. TF SHIV.C.CH505 is an extensively characterized virus shown to recapitulate key features of HIV-1 immunobiology, including CCR5-tropism, a tier 2 neutralization profile, reproducible early viral kinetics, and authentic immune responses. SHIV.C.CH505 is used frequently in nonhuman primate studies of HIV, but viral loads after months of infection are variable and typically lower than those in people living with HIV. We hypothesized that additional mutations besides Δ375 might further enhance virus fitness without compromising essential components of CH505 Env biology. From sequence analysis of SHIV.C.CH505-infected macaques across multiple experiments, we identified a signature of envelope mutations associated with higher viremia. We then used short-termin vivomutational selection and competition to identify a minimally adapted SHIV.C.CH505 with just five amino acid changes that substantially improve virus replication fitness in macaques. Next, we validated the performance of the adapted SHIVin vitroandin vivoand identified the mechanistic contributions of selected mutations.In vitro, the adapted SHIV shows improved virus entry, enhanced replication on primary rhesus cells, and preserved neutralization profiles.In vivo, the minimally adapted virus rapidly outcompetes the parental SHIV with an estimated growth advantage of 0.14 days-1and persists through suppressive antiretroviral therapy to rebound at treatment interruption. Here, we report the successful generation of a well-characterized, minimally adapted virus, termed SHIV.C.CH505.v2, with enhanced replication fitness and preserved native Env properties that can serve as a new reagent for NHP studies of HIV-1 transmission, pathogenesis, and cure.Author SummaryThe power of the nonhuman primate model of HIV to predict outcomes in people living with HIV (PLWH) depends on authentic virus-host interactions. In pursuit of viruses that generate infection that mirrors the effects of HIV-1 in PLWH, we developed a minimally adapted version of a commonly used virus, SHIV.C.CH505, which has better fitness than the parental virus while retaining important biological properties. First, we studied virus sequences from SHIV.C.CH505-infected rhesus macaques to identify a signature of mutations common to animals with higher viral loads. We then tested viruses containing the various mutations in the lab and in animals to determine the most fit version and to identify the contribution of each mutation. Ultimately, we identified a minimally adapted version of SHIV.C.CH505 with just 5 amino acid substitutions that enhances virus replication and preserves CH505 envelope properties, including sensitivity to clinically relevant broadly neutralizing antibodies. This new virus, called SHIV.C.CH505.v2 replicates well in macaques over time and persists through antiretroviral therapy. SHIV.C.CH505.v2 could be an important component of nonhuman primate studies of HIV prevention, therapy, and cure.

The systemic nature of SARS-CoV-2 infection is highly recognized, but poorly characterized. A non-invasive and unbiased method is needed to clarify whole body spatiotemporal dynamics of SARS-CoV-2 infection after transmission. We recently developed a probe based on the anti-SARS-CoV-2 spike antibody CR3022 to study SARS-CoV-2 pathogenesis in vivo. Herein, we describe its use in immunoPET to investigate SARS-CoV-2 infection of three rhesus macaques. Using PET/CT imaging of macaques at different times post-SARS-CoV-2 inoculation, we track the 64Cu-labelled CR3022-F(ab’)2 probe targeting the spike protein of SARS-CoV-2 to study the dynamics of infection within the respiratory tract and uncover novel sites of infection. Using this method, we uncovered differences in lung pathology between infection with the WA1 isolate and the delta variant, which were readily corroborated through computed tomography scans. The 64Cu-CR3022-probe also demonstrated dynamic changes occurring between 1- and 2-weeks post-infection. Remarkably, a robust signal was seen in the male genital tract (MGT) of all three animals studied. Infection of the MGT was validated by immunofluorescence imaging of infected cells in the testicular and penile tissue and severe pathology was observed in the testes of one animal at 2-weeks post-infection. The results presented here underscore the utility of using immunoPET to study the dynamics of SARS-CoV-2 infection to understand its pathogenicity and discover new anatomical sites of viral replication. We provide direct evidence for SARS-CoV-2 infection of the MGT in rhesus macaques revealing the possible pathologic outcomes of viral replication at these sites.Graphic AbstractPET/CT detected SARS-CoV-2 infection of 4 different tissues in the male genital tract illuminates the cause of COVID-19 clinical sequalae of male sexual health and fertility Figure 1.Diagram shows schematic illustration of the male genital tract of the rhesus macaque. Virus icon shows sites of SARS-CoV-2 PET signal. Text highlighting the clinical sequalae associated with each sight of infection is shown in text adjacent to each infection site.

CD4 T cell depletion in the mucosa has been well documented during acute HIV and SIV infections. The demonstration the HIV/SIVcan use the alpha4beta7 receptor for viral entry suggests that these viruses selectively target CD4 T cells in the mucosa that express high levels of alpha4beta7 receptor.Mucosal samples obtained from SIV infected rhesus macaques during the early phase of infection were used for immunophenotypic analysis. CD4 T cell subsets were sorted based on the expression of beta7 and CD95 to quantify the level of SIV infection in different subsets of CD4 T cells. Changes in IL-17, IL-21, IL-23 and TGFbeta mRNA expression was determined using Taqman PCR.CD4 T cells in the mucosa were found to harbor two major population of cells; -25% of CD4 T cells expressed the alpha4(+)beta7(hi) phenotype, whereas the rest of the 75% expressed an alpha4(+)beta7(int) phenotype. Both the subsets were predominantly CD28(+)Ki-67(-)HLA-DR(-) but CD69(+), and expressed detectable levels of CCR5 on their surface. Interestingly, however, alpha4(+)beta7(hi)CD4 T cells were found to harbor more SIV than the alpha4(+)beta7(int) subsets at day 10 pi. Early infection was associated with a dramatic increase in the expression of IL-17, and IL-17 promoting cytokines IL-21, IL-23, and TGFbeta that stayed high even after the loss of mucosal CD4 T cells.Our results suggest that the differential expression of the alpha4beta7 receptor contributes to the differences in the extent of infection in CD4 T cell subsets in the mucosa. Early infection is associated dysregulation of the IL-17 network in mucosal tissues involves other non-Th-17 cells that likely contributes to the pro-inflammatory environment in the mucosa during acute stages of SIV infection.

Recent progress in HIV-1 and SIV pathogenesis has revealed that mucosal tissues, primarily the gastrointestinal tract, are major sites for early viral replication and CD4+ T-cell destruction, and may be the major viral reservoir, even in patients receiving HAART. This is likely attributable to the fact that the majority of mucosal CD4+ T-cells co-expressing chemokine receptors requited for HIV-1 entry, reside in mucosal tissues. Furthermore, the intestinal mucosal immune system is continuously bombarded by dietary antigens, resulting in continual lymphocyte activation, dissemination, and homing of these activated lymphocytes (including CCR5+CD4+ T-cells) throughout mucosal tissues. Thus, the intestinal immune system represents a very large target for HIV-infection, which is continually generating newly activated CD4+ T-cells that are the preferred target of infection. Thus, HIV-1 appears uniquely adapted to persist and thrive in the mucosal-tissue environment. The selective loss of intestinal CD4+ T-cells from immune-effector sites is also likely to explain, at least in part, the preponderance of opportunistic infections at mucosal sites. It is increasingly evident that effective therapies and vaccines must be directed towards eliminating HIV-1 in mucosal tissue reservoirs, protecting mucosal CD4+ T-cells and stimulating effective mucosal immune responses.

There is increasing evidence suggesting an unexpected potential for non-neutralizing antibodies to prevent HIV infection. Consequently, identification of functional B cell epitopes for HIV is also important for developing preventative and therapeutic strategies. We therefore explored the antigen-specific immune responses following SIV or SHIV infection in rhesus macaques to determine functional B cell epitopes and identify factors that may influence control of plasma viremia. Functional B cell epitopes are a minimum of 10 amino acids in length for SIV and occur in similar regions despite differences in the route of mucosal SIV inoculation. Peptide-specific IgG responses generate primarily against gp41, V1-V3 regions of gp120, and the NC and p6 regions of Gag. Controller macaques showed much greater IgG responses to Nef, Tat, and p15 and p27 regions of Gag protein compared to absent or minimal responses in non-controller macaques. Antigen-specific IgM and IgA responses share similar epitopes to that of IgG, however the responses are much more limited. Moreover, antigen-specific IgA responses did not correlate with plasma viremia. Our study has shown that Env-specific IgG responses are not reliable prognostic indicators, unlike Gag, Nef, and Tat-specific IgG responses for SIV/SHIV controllers. Therefore, generation of early functional Gag, Nef and Tat-specific IgG responses may be important therapeutic strategies for the control or prevention of HIV/AIDS.

Attenuated vaccines in primates have provided better protection than most approaches to date. We hypothesized that SHIVsf162P3 infection might “immunize” macaques against intravenous challenge with a highly pathogenic virus. Twelve rhesus macaques of Chinese (Ch) and 2 of Indian (In) origin were intravaginally infected with SHIVsf162P3 and followed until their level of virus had subsided to undetectable or very low levels in plasma. SHIV vaccinated macaques were challenged intravenously with either 100, 10TCID50 SIVmac251 or plasma with high viral loads. Four “naïve” Ch macaques were similarly challenged with SIVmac251 as controls. Remarkably, Ch macaques “immunized” with SHIVsf162P3 were able to control viral replication of pathogenic SIVmac compared to naive (non-immunized) Ch controls. Viral loads after challenge in immunized Ch macaques ranged from undetectable to 104 in plasma compared to105 to 107 in naïve Ch controls. Indian macaques had higher levels of virus than Ch-macaques. but had better levels of protection compared to naïve challenged macaques. Control (naïve) macaques had rapid depletion of intestinal CD4+ T cells but levels remained fairly stable in immunized macaques. Control of viral load in “immunized” macaques did not correlate with the presence of neutralizing antibody and/or cell-mediated immune responses. Combined, these data suggest that effective control of SIV replication through immunization may be possible, but correlates of resistance have yet to be determined.