Neutralization-sensitive R5-tropic simian-human immunodeficiency virus SHIV-2873Nip, which carries env isolated from an infant with a recent HIV clade C infection

Abstract

Human immunodeficiency virus clade C (HIV-C) accounts for >56% of all HIV infections worldwide. To investigate vaccine safety and efficacy in nonhuman primates, a pathogenic, R5-tropic, neutralization-sensitive simian-human immunodeficiency virus (SHIV) carrying HIV-C env would be desirable. We have constructed SHIV-2873Ni, an R5-tropic SHIV carrying a primary pediatric HIV-C env gene isolated from a 2-month-old Zambian infant, who died within 1 year of birth. SHIV-2873Ni was constructed using SHIV-1157ipd3N4 (R. J. Song, A. L. Chenine, R. A. Rasmussen, C. R. Ruprecht, S. Mirshahidi, R. D. Grisson, W. Xu, J. B. Whitney, L. M. Goins, H. Ong, P. L. Li, E. Shai-Kobiler, T. Wang, C. M. McCann, H. Zhang, C. Wood, C. Kankasa, W. E. Secor, H. M. McClure, E. Strobert, J. G. Else, and R. M. Ruprecht. J. Virol. 80:8729-8738, 2006) as the backbone, since the latter contains additional NF-kappaB sites in the long terminal repeats to enhance viral replicative capacity. The parental virus, SHIV-2873Ni, was serially passaged through five rhesus monkeys (RMs); SHIV-2873Nip, the resulting passaged virus, was reisolated from the fourth recipient about 1 year postinoculation. SHIV-2873Nip was replication competent in RM peripheral blood mononuclear cells of all random donors tested and was exclusively R5 tropic, and its env gene clustered with HIV-C by phylogenetic analysis; its moderate [corrected] sensitivity to neutralization led to classification as a tier 2 [corrected] virus. Indian-origin RMs were inoculated by different mucosal routes, resulting in high peak viral RNA loads. Signs of virus-induced disease include depletion of gut CD4(+) T lymphocytes, loss of memory T cells in blood, and thrombocytopenia that resulted in fatal cerebral hemorrhage. SHIV-2873Nip is a highly replication-competent, mucosally transmissible, pathogenic R5-tropic virus that will be useful to study viral pathogenesis and to assess the efficacy of immunogens targeting HIV-C Env.

FIG. 1.

Construction of SHIV-2873Ni. (A) Identification of an infectious env clone. Primary full-length env genes were PCR amplified from genomic DNA of normal human donor PBMC cocultured with infected PBMC of Zambian infant 2873i collected at 2 months of age and cloned into the expression vector pcDNA6/B. Infant 2873i was a rapid progressor who died of AIDS-related disease within 1 year of birth to an HIV-C-positive mother. To identify an infectious envelope, the resultant constructs were cotransfected into 293T cells with HIV-1 ΔEN, an infectious HIV provirus with deletions of env and nef and encoding GFP in lieu of nef. The resulting pseudovirus released into cell supernatants was used to infect CEM.NKR.CCR5 cells, which were screened for GFP expression. HIV-1 ΔEN cotransfected with ADA env was used as positive control. (B) Construction of SHIV-2873Nip. SHIV-1157ipd3N4 (51) was used as the backbone. The 2.2-kb KpnI (K)-BamHI (B) fragment of HIV2873i (spanning most of gp120, the entire gp41 extracellular domain, the transmembrane region [TM], and part of the cytoplasmic domain) was amplified to replace the corresponding region of the proviral backbone. The modified 3′ half was ligated with the 5′ half of SHIV-vpu⁺ proviral DNA to form full-length SHIV-2873Ni. NN, two NF-κB sites are present in the 3′ LTR; during viral replication, this duplication is copied into the 5′ LTR also.

FIG. 2.

Coreceptor usage of SHIV-2873i, SHIV-2873Ni, and SHIV-2873Nip. U87.CD4.CXCR4 cells (A) and U87.CD4.CCR5 cells (B) were exposed to SHIV-2873Ni, SHIV-2873Nip, SHIV_SF162 (HIV clade B env, R5), and SHIV-vpu⁺ (HIV clade B env, X4). The levels of p27 Gag were measured in the supernatants as indicated. SHIV-2873i contains the standard SIVmac239 LTRs with only a single NF-κB site/LTR; it was built using SHIV-vpu⁺ as the backbone.

FIG. 3.

Serial passage of SHIV-2873Ni in RMs. (A) SHIV-2873Ni was passaged in five Indian-origin RMs through serial blood transfer. Animal RWa-9 had been exposed previously to SHIV-2873Ni but had remained uninfected. After receiving blood from donor RAi-8, RM RWa-9 and all monkeys shown in Fig. 2A became infected. (B) Viral loads were measured after serial passage at the time points indicated. †, monkey RNt-9 developed fatal cerebral hemorrhages due to severe thrombocytopenia at week 92 postinoculation. (C and D) Absolute CD4⁺ T-cell numbers, platelet counts, and CD4⁺CD29⁺ memory T cells were assessed during the course of the infection. The dashed line denotes the lowest normal value (10%) for the CD4⁺CD29⁺ T cells.

FIG. 4.

(A) Phylogenetic tree showing the relationship between SHIV-2873Ni and SHIV-2873Nip Env sequences and those of other primary strains of HIV. Phylogenetic trees were constructed from full-length Env sequences by using the neighbor-joining method. Major subtypes of HIV group M were used as reference sequences; sequences from SHIV-1157i, SHIV-1157ip, SHIV-1157ipd3N4 (51), and HIV1084i (14) were also included, since HIV1084i and the env genes in these SHIVs had been derived from the same cohort of infected mothers and their infants in Lusaka, Zambia. The scale bar indicates the genetic distance along the horizontal branches, and the numbers at the nodes are bootstrap values. (B) Evolution of SHIV-2873Nip Env during passage and replication in monkey RNt-9. The deletions of 5 and 3 aa at the end and beginning of the V4 and V5 domains of gp120, respectively, in the adapted SHIV-2873Nip are shown. The Env consensus sequence for SHIV-2873Nip was derived by sequencing eight individual clones carrying infectious env genes.

FIG. 5.

Oral and i.r. inoculation of SHIV-2873Nip. Two monkeys (RBg-10 and RUf-10) were inoculated orally or i.r., respectively, with SHIV-2873Nip stock. (B) Six monkeys were used in a repeated low-dose i.r. titration; the aim was to find a virus dose leading to systemic infection (defined as a viral RNA level of ≥10⁴ copies/ml) after a maximum of five weekly i.r. inoculations. Monkeys remaining uninfected at week 2 after the fifth weekly low-dose virus challenge were given a single high dose of SHIV-2873Nip (30,000 TCID₅₀). Viral loads were measured at the time points indicated. The horizontal dotted line indicates the lower limit of detection (<50 viral RNA copies/ml). (C and D) Estimation of CD4⁺ T-cell loss in gut (C) and blood (D), showing levels of CD4⁺ T cells in rectal biopsy specimens (collected between weeks 6 and 12 after the last, successful inoculation) and blood of RMs inoculated with SHIV-2873Nip compared with uninfected controls. The asterisk in panel C designates the percent CD4⁺ T cells in rectal mucosa of monkey RNt-9 collected at week 84 postinoculation.