While simian immunodeficiency viruses (SIVs) are generally nonpathogenic in their natural hosts, dramatic increases in pathogenicity may occur upon cross-species transmission to new hosts. Deciphering the drivers of these increases in virulence is of major interest for understanding the emergence of new human immunodeficiency viruses (HIVs). We transmitted SIVsab from the sabaeus species of African green monkeys (AGMs) to pigtailed macaques (PTMs). High acute viral replication occurred in all SIVsab-infected PTMs, yet the outcome of chronic infection was highly variable, ranging from rapid progression to controlled infection, which was independent of the dynamics of acute viral replication, CD4؉ T cell depletion, or preinfection levels of microbial translocation. Infection of seven PTMs with plasma collected at necropsy from a rapid-progressor PTM was consistently highly pathogenic, with high acute and chronic viral replication, massive depletion of memory CD4 ؉ T cells, and disease progression in all PTMs. The plasma inoculum used for the serial passage did not contain adventitious bacterial or viral contaminants. Single-genome amplification showed that this inoculum was significantly more homogenous than the inoculum directly derived from AGMs, pointing to a strain selection in PTMs. In spite of similar peak plasma viral loads between the monkeys in the two passages, immune activation/inflammation levels dramatically increased in PTMs infected with the passaged virus. These results suggest that strain selection and a massive cytokine storm are major factors behind increased pathogenicity of SIV upon serial passage and adaptation of SIVs to new hosts following cross-species transmission. IMPORTANCEWe report here that upon cross-species transmission and serial passage of SIVsab from its natural host, the sabaeus African green monkey (AGM), to a new host, the pigtailed macaque (PTM), viral adaptation and increased pathogenicity involve strain selection and a massive cytokine storm. These results permit the design of strategies aimed at preventing cross-species transmission from natural hosts of SIVs to humans in areas of endemicity. Furthermore, our study describes a new animal model for SIV infection. As the outcomes of SIVsab infection in PTMs, African green monkeys, and rhesus macaques are different, the use of these systems enables comparative studies between pathogenic, nonpathogenic, and elite-controlled infections, to gain insight into the mechanisms of SIV immunodeficiency and comorbidities.

We assessed the role of myeloid dendritic cells (mDCs) in the outcome of SIV infection by comparing and contrasting their frequency, mobilization, phenotype, cytokine production and apoptosis in pathogenic (pigtailed macaques, PTMs), nonpathogenic (African green monkeys, AGMs) and controlled (rhesus macaques, RMs) SIVagmSab infection. Through the identification of recently replicating cells, we demonstrated that mDC mobilization from the bone marrow occurred in all species postinfection, being most prominent in RMs. Circulating mDCs were depleted with disease progression in PTMs, recovered to baseline values after the viral peak in AGMs, and significantly increased at the time of virus control in RMs. Rapid disease progression in PTMs was associated with low baseline levels and incomplete recovery of circulating mDCs during chronic infection. mDC recruitment to the intestine occurred in all pathogenic scenarios, but loss of mucosal mDCs was associated only with progressive infection. Sustained mDC immune activation occurred throughout infection in PTMs and was associated with increased bystander apoptosis in blood and intestine. Conversely, mDC activation occurred only during acute infection in nonprogressive and controlled infections. Postinfection, circulating mDCs rapidly became unresponsive to TLR7/8 stimulation in all species. Yet, stimulation with LPS, a bacterial product translocated in circulation only in SIV-infected PTMs, induced mDC hyperactivation, apoptosis and excessive production of proinflammatory cytokines. After infection, spontaneous production of proinflammatory cytokines by mucosal mDCs increased only in progressor PTMs. We thus propose that mDCs promote tolerance to SIV in the biological systems that lack intestinal dysfunction. In progressive infections, mDC loss and excessive activation of residual mDCs by SIV and additional stimuli, such as translocated microbial products, enhance generalized immune activation and inflammation. Our results thus provide a mechanistic basis for the role of mDCs in the pathogenesis of AIDS and elucidate the causes of mDC loss during progressive HIV/SIV infections.

During antiretroviral therapy (ART) that suppresses HIV replication to below the limit-ofquantification, virions produced during ART can be detected at low frequencies in the plasma, termed residual viremia (RV). We hypothesized that a reservoir of HIV-infected cells actively produce and release virions during ART that are potentially infectious, and that following ART-interruption, these virions can complete full-cycles of replication and contribute to rebound viremia. Therefore, we studied the dynamics of RV sequence variants in 3 participants who initiated ART after~3 years of infection and were ART-suppressed for >6 years prior to self-initiated ART-interruptions. Longitudinal RV C2V5env sequences were compared to sequences from pre-ART plasma, supernatants of quantitative viral outgrowth assays (QVOA) of cells collected during ART, post-ART-interruption plasma, and ART-resuppression plasma. Identical, "putatively clonal," RV sequences comprised 8-84% of sequences from each timepoint. The majority of RV sequences were genetically similar to those from plasma collected just prior to ART-initiation, but as the duration of ART-suppression increased, an increasing proportion of RV variants were similar to sequences from earlier in infection. Identical sequences were detected in RV over a median of 3 years (range: 0.3-8.2) of ART-suppression. RV sequences were identical to pre-ART plasma viruses (5%), infectious viruses induced in QVOA (4%) and rebound viruses (5%) (total n = 21/154 (14%) across the 3 participants). RV sequences identical to ART-interruption "rebound" sequences were detected 0.1-7.4 years prior to ART-interruption. RV variant prevalence and persistence were not associated with detection of the variant among rebound sequences. Shortly after ART-re-suppression, variants that had been replicating during ART-interruptions were detected as RV (n = 5). These studies show a dynamic, virion-producing HIV reservoir that contributes to rekindling infection upon ART-interruption. The

Free energy perturbation (FEP) theory coupled to molecular dynamics (MD) or Monte Carlo (MC) statistical mechanics offers a theoretically precise method for determining the free energy differences of related biological inhibitors. Traditionally requiring extensive computational resources and expertise, it is only recently that its impact is being felt in drug discovery. A review of computer-aided anti-HIV efforts employing FEP calculations is provided here that describes early and recent successes in the design of human immunodeficiency virus type 1 (HIV-1) protease and non-nucleoside reverse transcriptase inhibitors. In addition, our ongoing work developing and optimizing leads for small molecule inhibitors of cyclophilin A (CypA) is highlighted as an update on the current capabilities of the field. CypA has been shown to aid HIV-1 replication by catalyzing the cis/trans isomerization of a conserved Gly-Pro motif in the N-terminal domain of HIV-1 capsid (CA) protein. In the absence of a functional CypA, e.g., by the addition of an inhibitor such as cyclosporine A (CsA), HIV-1 has reduced infectivity. Our simulations of acylurea-based and 1-indanylketone-based CypA inhibitors have determined that their nanomolar and micromolar binding affinities, respectively, are tied to their ability to stabilize Arg55 and Asn102. A structurally novel 1-(2,6-dichlorobenzamido) indole core was proposed to maximize these interactions. FEP-guided optimization, experimental synthesis, and biological testing of lead compounds for toxicity and inhibition of wild-type HIV-1 and CA mutants have demonstrated a dose-dependent inhibition of HIV-1 infection in two cell lines. While the inhibition is modest compared to CsA, the results are encouraging.