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 TRIM5α 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 simian-human immunodeficiency viruses that may advance the HIV/AIDS vaccine agenda.

Importance: Nonhuman 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.

FIG 1

(A and B) Phylogenetic trees and highlighter alignments of Env sequences from SIvmac251-infected rhesus macaques M766 (A) and M746 (B). Single nucleotide polymorphisms to the consensus sequences are denoted by colored tick marks; gaps are shown in gray and G-to-A mutations in purple. (C) Phylogenetic tree of the 33 T/F env variants (color coded by animal identifier) and sequences from the infection stock used in this study (gray circles). One large cluster (boxed in red) contains 20% of the T/F viruses and 32% of the stock sequences with identical or nearly identical envelopes. The green boxes indicate inoculum-enriched clusters, and the blue boxes indicate transmission-enriched clusters. Nucleotide differences in phylogenetic trees are shown by the scale bar.

FIG 2

Highlighter alignments of 5′ and 3′ half-genomes from animals M766 (A) and M746 (B). Single nucleotide polymorphisms to the consensus sequences are denoted by colored tick marks; gaps are shown in gray and G-to-A mutations in purple. The two overlapping amplicons are shown in black, and the previously determined T/F env sequence is shown in red (Env T/F). The full-genome consensus sequence was identified using the overlapping half-genomes.

FIG 3

In vitro replication curves of 8 T/F IMCs and the control clones SIVmac239 and SIVsmE543. CD8-depleted PBMCs from 2 naive, TRIM5α-permissive (Q/Q) rhesus macaques were inoculated with equivalent numbers of infectious units of each virus. Viral replication was monitored by measuring RT activity in cell-free culture supernatants at days 3, 7, 10, and 14 postinfection. (A) SIVmac lineage clones. (B) SIVsm lineage clones.

FIG 4

Summary of neutralization profiles of the T/F clones against 12 SIV-specific MAbs. The antibodies were specific for CD4 binding site; CD4 induced; variable loops 1, 2, and 3; and sites outside the variable loops (ΔV1V2V3). Color coding indicates the ranges of IC₅₀s, as indicated. For MAbs that did not completely neutralize, a half-maximum IC₅₀ (Half Max) was determined based on the concentration that caused a 50% reduction of the maximal neutralization (V_max) achieved for that antibody. Values of >50 indicate that no neutralization was detected.

FIG 5

(A to D) Neutralization profiles of the T/F clones SIVmac746, SIVmac766, SIVsmCG7G, and SIVsmCG7V following exposure to lineage-homologous or lineage-heterologous sera from chronically infected animals. Viruses were incubated with serially diluted sera from rhesus macaques chronically infected with SIVmac251 or SIVsmE660. Residual infectivity was measured on TZM-bl cells following exposure to the sera. (E and F) Percent inhibition of virus entry was also assessed using monoclonal antibodies specific for V3-induced (E) or CD4-induced (F) epitopes.

FIG 6

In vivo replication kinetics and peripheral CD4 depletion. (A and B) Plasma viral loads from animals infected with SIVmac clones (A) or SIVsm clones (B) highlight the consistency of SIVmac infections and the variable viral loads following SIVsm infection. The legend (right) indicates the infecting virus and TRIM5α genotype for each animal. †, euthanasia due to simian-AIDS-defining conditions. (C and D) Longitudinal absolute peripheral blood CD4⁺ T cell counts for SIVmac-infected animals (C) and SIVsm-infected animals (D). (E) Early gut CD4⁺ T cell depletion shown by immunohistochemistry in SIVmac766-infected animals (p106 and p097) and SIVsmCG7G-infected animals (p114 and p116) from preinfection and acute infection (day 28 postinfection). The brown stain is for CD4⁺ cells, and pink staining for myeloperoxidase identifies myeloid cells. Scale bars, 100 μm.

FIG 7

In vitro replication curves of 4 T/F IMCs (SIVsmCG7G, SIVsmCG7V, SIVmac746, and SIVmac766) and the control clones SIVmac239 and SIVsmE543. Viruses from these clones were used to infect CD8-depleted PBMCs collected from 2 naive rhesus macaques. One macaque (A) possessed a permissive TRIM5α genotype (Q/Q), while the other (B) possessed a restrictive TRIM5α genotype (TFP/TFP). RT activity was measured at days 3, 7, and 10 postinfection, showing significant TRIM5α-specific restriction of SIVsm, but not SIVmac.

FIG 8

Highlighter alignments of 5′ and 3′ half-genomes from all 8 infected animals. Plasma samples were obtained at peak viremia and used to identify the consensus genome for each infected animal. Each alignment represents two animals infected with each clone, SIVmac746 (A), SIVmac766 (B), SIVsmCG7V (C), and SIVsmCG7G (D). Single nucleotide polymorphisms to the T/F IMC sequences are denoted by colored tick marks; gaps are shown in gray and G-to-A mutations in purple. The full-genome consensus sequence (CONSENSUS) for both animals is an exact nucleotide match with that of the IMC clone used to infect each animal.

FIG 9

Unrooted phylogenetic trees of sequences from 6 of the 8 infected animals from plasma taken at necropsy. (A and B) SIVmac-infected animals, with trees representing the 5′ half-genome (A) and the 3′ half-genome (B). (C and D) SIVsm-infected animals, with trees representing the 5′ half-genome (C) and the 3′ half-genome (D). The scale bars represent 0.01-nucleotide changes. Sequences from each animal are color coded. Each TF IMC sequence is labeled in black.

FIG 10

Immunopathology in the lymph nodes for 2 representative animals, p096 (SIVmac746) and p116 (SIVsmCG7G). Biopsy specimens obtained preinfection (Pre.) are compared to samples obtained postinfection, with the animal manifesting an AIDS-defining condition (AIDS). (A) Decrease in CD4⁺ T cells (brown) with unchanged myeloid lineage cells (pink) at AIDS. (B and C) Increased immune activation at AIDS as measured by Ki67 staining (brown), largely restricted to B cell follicles prior to infection but generalized throughout the lymph node when simian AIDS was diagnosed (B), and increased collagen deposition (brown) (C).