Generation and evaluation of clade C simian-human immunodeficiency virus challenge stocks

Abstract

The development of a panel of mucosally transmissible simian-human immunodeficiency virus (SHIV) challenge stocks from multiple virus clades would facilitate preclinical evaluation of candidate HIV-1 vaccines and therapeutics. The majority of SHIV stocks that have been generated to date have been derived from clade B HIV-1 env sequences from viruses isolated during chronic infection and typically required serial animal-to-animal adaptation for establishing mucosal transmissibility and pathogenicity. To capture essential features of mucosal transmission of clade C viruses, we produced a series of SHIVs with early clade C HIV-1 env sequences from acutely HIV-1-infected individuals from South Africa. SHIV-327c and SHIV-327cRM expressed env sequences that were 99.7 to 100% identical to the original HIV-1 isolate and did not require in vivo passaging for mucosal infectivity. These challenge stocks infected rhesus monkeys efficiently by both intrarectal and intravaginal routes, replicated to high levels during acute infection, and established chronic setpoint viremia in 13 of 17 (76%) infected animals. The SHIV-327cRM challenge stock was also titrated for both single, high-dose intrarectal challenges and repetitive, low-dose intrarectal challenges in rhesus monkeys. These SHIV challenge stocks should facilitate the preclinical evaluation of vaccines and other interventions aimed at preventing clade C HIV-1 infection.

Importance: We describe the development of two related clade C SHIV challenge stocks. These challenge stocks should prove useful for preclinical testing of vaccines and other interventions aimed at preventing clade C HIV-1 infection.

FIG 1

Cloning strategy for the generation of SHIVs expressing early HIV-1 env sequences. Two unique restriction sites, ClaI and AgeI, in the SHIV-KB9 infectious clone were utilized. Fragments containing KB9 sequence from the ClaI site to the env ATG start codons and early clade C env sequences with AgeI site designed at the corresponding 3′ end were synthesized and replaced the corresponding regions in the SHIV-KB9-AC.

FIG 2

Highlighter amino acid sequence alignment of env derived from the SHIV-327c and SHIV-327cRM stocks and the parental CA327 HIV-1 env. Amino acid substitutions that differed from the parental HIV-1 CA327 env sequence (GenBank accession number JN681220) are indicated in color. Dashes indicate amino acid sequences identical to the parental sequence. The two relevant mutations in SHIV-327cRM (L124F, G408R) are indicated. A total of 36 sequences from SHIV-327c and 35 sequences from SHIV-327cRM were generated by SGA.

FIG 3

Coreceptor tropism of early SHIV stocks. (A) TZM-bl cells were incubated for 1 h with different concentrations of the CXCR4 inhibitor AMD-3100 or the CCR5 inhibitor TAK-779 and subsequently were infected with 100 TCID₅₀ of the indicated SHIV stocks. The luciferase activity was quantified after 48 h. (B) GHOST cell lines expressing CXCR4 (X4) and/or CCR5 (R5) coreceptors were used, and inoculated with 100-TCID₅₀ SHIV stocks. Cell culture supernatant was collected for SIV p27 determination after 4 days of infection. All assays were done in triplicate. The means with the standard deviations are shown.

FIG 4

i.r. challenge with the clade C SHIV-405c and SHIV-459c stocks in rhesus monkeys. Six animals were challenged once with 1 ml of undiluted SHIV-405c (n = 3) (A) or SHIV-459c (n = 3) (B) stocks by the i.r. route. The upper panel shows plasma viral loads, and the lower panel shows the percentage of CD4⁺ T cells in peripheral blood. The dotted line reflects the limit of detection of the assay (50 RNA copies/ml).

FIG 5

i.r. challenge with the clade C SHIV-327c and SHIV-327cRM stocks in rhesus monkeys. Six animals were challenged once with 1 ml of undiluted SHIV-327c (n = 3) (A) or SHIV-327cRM (n = 3) (B) stocks by the i.r. route. The upper panel shows plasma viral loads, and the lower panel shows the percentage of CD4⁺ T cells in peripheral blood. The dotted line reflects the limit of detection of the assay (50 RNA copies/ml).

FIG 6

i.r. titration of the SHIV-327cRM stock in rhesus monkeys (1:10 dilution). Three animals were challenged once with 1 ml of 1:10-diluted SHIV-327cRM stock by the i.r. route. The upper panel shows plasma viral loads, and the lower panel shows the percentage of CD4⁺ T cells in peripheral blood. The dotted line reflects the limit of detection of the assay (50 RNA copies/ml).

FIG 7

i.r. titration of the SHIV-327cRM stock in rhesus monkeys (1:100 dilution). Two animals were challenged six times (red arrows) with 1 ml of 1:100-diluted SHIV-327cRM stock by the i.r. route. The upper panel shows plasma viral loads, and the lower panel shows the percentage of CD4⁺ T cells in peripheral blood. The dotted line reflects the limit of detection of the assay (50 RNA copies/ml).

FIG 8

i.vag. challenge with the clade C SHIV-327c and SHIV-327cRM stocks in rhesus monkeys. Six animals were pretreated 4 weeks prior to challenge with Depo-Provera and were then challenged once with 1 ml of undiluted (A) SHIV-327c (n = 3) or (B) SHIV-327cRM (n = 3) stocks by the i.vag. route. The upper panel shows plasma viral loads, and the lower panel shows the percentage of CD4⁺ T cells in peripheral blood. The dotted line reflects the limit of detection of the assay (50 RNA copies/ml).