Production of Mucosally Transmissible SHIV Challenge Stocks from HIV-1 Circulating Recombinant Form 01_AE env Sequences

Abstract

Simian-human immunodeficiency virus (SHIV) challenge stocks are critical for preclinical testing of vaccines, antibodies, and other interventions aimed to prevent HIV-1. A major unmet need for the field has been the lack of a SHIV challenge stock expressing circulating recombinant form 01_AE (CRF01_AE) env sequences. We therefore sought to develop mucosally transmissible SHIV challenge stocks containing HIV-1 CRF01_AE env derived from acutely HIV-1 infected individuals from Thailand. SHIV-AE6, SHIV-AE6RM, and SHIV-AE16 contained env sequences that were >99% identical to the original HIV-1 isolate and did not require in vivo passaging. These viruses exhibited CCR5 tropism and displayed a tier 2 neutralization phenotype. These challenge stocks efficiently infected rhesus monkeys by the intrarectal route, replicated to high levels during acute infection, and established chronic viremia in a subset of animals. SHIV-AE16 was titrated for use in single, high dose as well as repetitive, low dose intrarectal challenge studies. These SHIV challenge stocks should facilitate the preclinical evaluation of vaccines, monoclonal antibodies, and other interventions targeted at preventing HIV-1 CRF01_AE infection.

Fig 1. Highlighter amino acid sequence alignment of env derived SHIV-AE6, SHIV-AE6RM, and SHIV-AE16 stocks compared to the parental strains.

(A) SHIV-AE6, (B) SHIV-AE16, and (C) SHIV-AE6RM amino acid sequences are shown. Amino acids that differ from the parental sequence (GenBank accession numbers KP109513 and KT581086) are indicated in color, while dashes indicate identical amino acid positions. The D168H mutation in SHIV-AE6RM is depicted by the red box. A total of 31 sequences for each SHIV challenge stock was generated by SGA.

Fig 2. Coreceptor tropism of early SHIV stocks.

(A-B) TZM-bl cells were incubated for 1 h with different concentrations of the CCR5 inhibitor TAK-779 or the CXCR4 inhibitor AMD-3100 and subsequently were infected with 100 TCID₅₀ of the indicated SHIV stocks. The luciferase activity was quantified after 48 h. (C) 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 standard deviation are shown.

Fig 3. i.r. challenge with SHIV-AE6, SHIV-AE6RM, SHIV-AE16 stocks in rhesus monkeys.

Twelve animals were challenged once with 1 ml of undiluted (A) SHIV-AE6 (n = 4), (B) SHIV-AE6RM (n = 4), and (C) SHIV-AE16 (n = 4) 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 reflected the limit of detection of the assay (50 RNA copies/ml).

Fig 4. Intrarectal titration of the SHIV-AE16 stock in rhesus monkeys (1:1 and 1:10 dilution).

Twelve animals were challenged six times (red arrows) with two week intervals through week 10 with 1ml of (A) 1:1 diluted or (B) 1:10 diluted SHIV-AE16 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 reflected the limit of detection of the assay (50 RNA copies/ml).

Fig 5. Log viral DNA copies/10⁶ cells from week 12 PBMC, LMNC, and colorectal samples obtained from rhesus monkeys infected with SHIV-AE16 stock at (A) 1:1 and (B) 1:10 dilution.

The dotted line depicts the limit of detection of the assay (3 copies/10⁶ cells).