Derivation and Characterization of Pathogenic Transmitted/Founder Molecular Clones from Simian Immunodeficiency Virus SIVsmE660 and SIVmac251 following Mucosal Infection

Abstract

Currently available simian immunodeficiency virus (SIV) infectious molecular clones (IMCs) and isolates used in nonhuman primate (NHP) models of AIDS were originally derived from infected macaques during chronic infection or end stage disease and may not authentically recapitulate features of transmitted/founder (T/F) genomes that are of particular interest in transmission, pathogenesis, prevention, and treatment studies. We therefore generated and characterized T/F IMCs from genetically and biologically heterogeneous challenge stocks of SIVmac251 and SIVsmE660. Single-genome amplification (SGA) was used to identify full-length T/F genomes present in plasma during acute infection resulting from atraumatic rectal inoculation of Indian rhesus macaques with low doses of SIVmac251 or SIVsmE660. All 8 T/F clones yielded viruses that were infectious and replication competent in vitro, with replication kinetics similar to those of the widely used chronic-infection-derived IMCs SIVmac239 and SIVsmE543. Phenotypically, the new T/F virus strains exhibited a range of neutralization sensitivity profiles. Four T/F virus strains were inoculated into rhesus macaques, and each exhibited typical SIV replication kinetics. The SIVsm T/F viruses were sensitive to TRIM5␣ restriction. All T/F viruses were pathogenic in rhesus macaques, resulting in progressive CD4؉ T cell loss in gastrointestinal tissues, peripheral blood, and lymphatic tissues. The animals developed pathological immune activation; lymphoid tissue damage, including fibrosis; and clinically significant immunodeficiency leading to AIDS-defining clinical endpoints. These T/F clones represent a new molecular platform for the analysis of virus transmission and immunopathogenesis and for the generation of novel "bar-coded" challenge viruses and next-generation simianhuman immunodeficiency viruses that may advance the HIV/AIDS vaccine agenda. IMPORTANCENonhuman primate research has relied on only a few infectious molecular clones for a myriad of diverse research projects, including pathogenesis, preclinical vaccine evaluations, transmission, and host-versus-pathogen interactions. With new data suggesting a selected phenotype of the virus that causes infection (i.e., the transmitted/founder virus), we sought to generate and characterize infectious molecular clones from two widely used simian immunodeficiency virus lineages (SIVmac251 and SIVsmE660). Although the exact requirements necessary to be a T/F virus are not yet fully understood, we generated cloned viruses with all the necessary characteristic of a successful T/F virus. The cloned viruses revealed typical acute and set point viral-load dynamics with pathological immune activation, lymphoid tissue damage progressing to significant immunodeficiency, and AIDS-defining clinical endpoints in some animals. These T/F clones represent a new molecular platform for studies requiring authentic T/F viruses.

Envelope residue 375 substitutions in simian–human immunodeficiency viruses enhance CD4 binding and replication in rhesus macaques

“…We cloned a replication-competent, pathogenic T/F provirus designated SIVmac766 (Fig. 1A) from a RM infected by atraumatic rectal inoculation with an early passage isolate of SIVmac251 (41,42). Complete tat-rev-vpu-env(gp160) cassettes from a prototypic primary subtype B strain YU2 (51) and from a genetically divergent T/F subtype D HIV-1 strain 191859 (52) were exchanged for the corresponding region of SIVmac766 ( Fig.…”

“…We further surmised that optimization of the SIVmac backbone and simplification of the HIV-1 insert strategy would enhance the chances of success. Our strategy was thus to: (i) molecularly clone a new SIVmac251-derived T/F virus, pSIVmac766 (41,42), that lacked any of four mutations found in SIVmac239 known to be suboptimal for replication (39,43); (ii) substitute into this vector complete tat-rev-vpu-env (gp160) cassettes of T/F or primary HIV-1 strains; and (iii) modify Env residue 375, which is part of the CD4-binding pocket and facilitates CD4 engagement (44), to encode genotypic variants (Met, M; Tyr, Y; His, H; Trp, W; and Phe, F) found naturally at this position across the broad evolutionary spectrum of primate lentiviruses (25) (www.hiv.lanl.gov/) (Fig. S1A).…”

Collapse of Cytolytic Potential in SIV-Specific CD8+ T Cells Following Acute SIV Infection in Rhesus Macaques

“…vRNA was subject to reverse transcription using MuLV (SuperScript III) reverse transcriptase using the following primers; Tat-RT3: 5’–TGGGGATAATTTTACACAAGGC-3’ [41] and SMgag.R1: 5’-CTACTGGTCTTCTCCAAAGAGAGAATTGA-3’ for analysis of TL8 and CM9 epitopes, respectively. Resampling of PCR products was avoided by obtained PCR end point dilutions of each cDNA sample as previously described [42]. Equal quantities of cDNA from each sample were PCR amplified to achieve the desirable sampling depth.…”

Nonhuman primate models for the evaluation of HIV-1 preventive vaccine strategies

“…To date, the majority of NHP/AIDS research, including vaccine studies, has utilized experimental challenge with SIV isolates and clones derived from chronically infected RMs, though very recent efforts have resulted in the generation of several new pathogenic transmitted/founder SIV clones that may represent more relevant vaccine targets [5] (Fig. 1).…”

“…Example SIV challenge viruses shown include those of the SIVmac and SIVsm lineages, including recently described transmitted/founder clones (SIVmac746, 766, SIVsmCG7G, CG7V) derived from these lineages [5]. Representative SHIV challenge viruses are also shown; additional SHIVs have also been recently described [1,2*–4*].…”