Development of novel SHIVs from HIV-1 clades for preclinical evaluation
Pastores, Kevin Clyde Gasmena
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The lack of an animal model that recapitulates the prominent features of HIV-1 infection in humans limits the search for preventative and curative strategies against HIV-1. As stated by the National Institutes of Health (NIH), developing highly pathogenic simian-human immunodeficiency viruses (SHIVs) that can establish persistent infections and AIDS progression in rhesus macaques (RMs) remains vital for advancing the field. The HIV-1 envelope (Env) serves as a major target in vaccine studies. SHIVs - which are chimeras of the simian immunodeficiency virus (SIV) backbone and a humanized Env, are utilized for preclinical evaluation of vaccines and therapeutics aimed at targeting the Env glycoprotein. However, SHIVs presently available poorly infect RM due to weak binding interactions between Env and rhesus CD4 (rhCD4). Position 375 (Env375) lies within the rhCD4 binding pocket of HIV-1 Env. Accordingly, substituting the wild-type (WT) amino acid in Env375 for bulky hydrophobic and/or basic amino acids may strengthen Env-rhCD4 interactions. These mutations should increase the pathogenicity of our original SHIV challenge stocks (SHIV162p3 and SHIVAE16) and allow for the development of an animal model that closely mirrors HIV-1 acquisition and chronic AIDS infection in humans. OBJECTIVES: To develop an animal model that recapitulates HIV-1 infection and AIDS progression in humans. MATERIALS AND METHODS: SHIV design involved insertion of human env sequences into a modified SIV backbone (provided by Dr. George Shaw, University of Pennsylvania). Site-directed mutagenesis was employed to introduce amino acid substitutions at Env375 for the following residues: serine, histidine, methionine, tryptophan, tyrosine, and phenylalanine. These constructs were then utilized for transfection of human embryonic kidney cells (293T) with viral supernatants collected 72 hours post-transfection. 293T viral supernatants were then used to infect human and rhesus PBMCs with the resulting supernatant harvested every three days and subjected to ELISA to monitor viral growth. Viruses were further characterized by RT-PCR for quantification and TCID50 assays to determine infectious dose. Resulting SHIVs were then used for challenge studies in rhesus macaques. Sixteen rhesus macaques were divided into groups of four that received the following SHIV challenge stocks: (1) original SHIV162p3, (2) modified SHIV162p3, (3) original SHIVAE16, and (4) modified SHIVAE16. Following infection, animal plasma and sera were collected and subjected to post-challenge analyses such as cellular assays and measurements of plasma viral RNA levels. RESULTS: Several analyses were conducted to study the pathogenicity of the modified SHIV162p3 and SHIVAE16 relative to the original stocks. Regarding SHIV infectivity, several versions of the modified SHIVs exhibited greater in vitro infectivity titers than the original stocks. Additionally, preliminary viral load analyses conducted in vivo indicate that all sixteen RMs challenged with original and modified SHIVs were infected at comparable levels. Furthermore, CD4+ T cell counts were measured and all sixteen animals exhibited declines in CD4+ T cell percentages. CONCLUSION: In sum, the modified SHIVs may improve animal models by closely recapitulating the events of HIV-1 infection in humans and serve better for future studies of preventative and curative treatments against HIV-1.
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