Previously, we showed that substitution of HIV-1 Env residue 375-Ser by bulky aromatic residues enhances binding to rhesus CD4 and enables primary HIV-1 Envs to support efficient replication as simian-human immunodeficiency virus (SHIV) chimeras in rhesus macaques (RMs). Here, we test this design strategy more broadly by constructing SHIVs containing ten primary Envs corresponding to HIV-1 subtypes A, B, C, AE and AG. All ten SHIVs bearing wildtype Env375 residues replicated efficiently in human CD4⁺ T cells, but only one replicated efficiently in primary rhesus cells. This was a subtype AE SHIV that naturally contained His at Env375. Replacement of wildtype Env375 residues by Trp, Tyr, Phe or His in the other nine SHIVs led to efficient replication in rhesus CD4+ T cells in vitro and in vivo Nine SHIVs containing optimized Env375 alleles were grown large-scale in primary rhesus CD4⁺ T cells to serve as challenge stocks in preclinical prevention trials. These virus stocks were genetically homogeneous, native-like in Env antigenicity and tier-2 neutralization sensitivity, and transmissible by rectal, vaginal, penile, oral or intravenous routes. To facilitate future SHIV constructions, we engineered a simplified second-generation design scheme and validated it in RMs. Overall, our findings demonstrate that SHIVs bearing primary Envs with bulky aromatic substitutions at Env375 consistently replicate in RMs, recapitulating many features of HIV-1 infection in humans. Such SHIVs are efficiently transmitted by mucosal routes common to HIV-1 infection and can be used to test vaccine efficacy in preclinical monkey trials.ImportanceSHIV infection of Indian rhesus macaques is an important animal model for studying HIV-1 transmission, prevention, immunopathogenesis and cure. Such research is timely, given recent progress with active and passive immunization and novel approaches to HIV-1 cure. Given the multifaceted roles of HIV-1 Env in cell tropism and virus entry, and as a target for neutralizing and non-neutralizing antibodies, Envs selected for SHIV construction are of paramount importance. Until recently, it has been impossible to strategically design SHIVs bearing clinically relevant Envs that replicate consistently in monkeys. This changed with the discovery that bulky aromatic substitutions at residue Env375 confer enhanced affinity to rhesus CD4. Here, we show that 10 new SHIVs bearing primary HIV-1 Envs with residue 375 substitutions replicated efficiently in RMs and could be transmitted efficiently across rectal, vaginal, penile and oral mucosa. These findings suggest an expanded role for SHIVs as a model of HIV-1 infection.

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