Mucosal transmission and induction of simian AIDS by CCR5-specific simian/human immunodeficiency virus SHIV(SF162P3)

Abstract

Nonhuman primate models are increasingly used in the screening of candidate AIDS vaccine and immunization strategies for advancement to large-scale human trials. The predictive value of such macaque studies is largely dependent upon the fidelity of the model system in mimicking human immunodeficiency virus (HIV) type 1 infection in terms of viral transmission, replication, and pathogenesis. Herein, we describe the efficient mucosal transmission of a CCR5-specific chimeric simian/human immunodeficiency virus, SHIV(SF162P3). Female rhesus macaques were infected with SHIV(SF162P3) after a single atraumatic application to the cervicovaginal mucosa. The disease course of SHIV(SF162P3)-infected monkeys is similar and as varied as natural HIV infection in terms of viral replication, gradual loss of CD4(+) peripheral blood mononuclear cells, and the development of simian AIDS-defining opportunistic infections. The SHIV(SF162P3)/macaque model should facilitate direct preclinical assessment of HIV vaccine strategies in addition to antiviral compounds directed towards envelope target cell interactions. Furthermore, this controlled model provides the setting to investigate immunologic responses and putative host-specific susceptibility factors that alter viral transmission and subsequent disease progression.

FIG. 1

Infection of macaque T353 with serially passaged SHIV_SF162. Shown are values for plasma viremia (left y axis) and absolute number of CD3⁺ CD4⁺ PBMCs per μl of blood (right y axis) at various weeks postinfection (x axis). Mononuclear lymphocytes (LNMC) from this passage 3 macaque (T353) were purified by mechanical disruption followed by gradient centrifugation (Lymphocyte Separation Media; Bio-Whittaker, Walkersville, Md.), washed in Hanks balanced buffer solution, and cultured with an equal number of phytohemagglutinin- or recombinant interleukin 2-stimulated human PBMCs. Cultures were maintained for 10 to 14 days and monitored for the production of viral p27^gag antigen using a commercially available antigen enzyme-linked immunosorbent assay as described below. Viral antigen-positive cultures were collected, subjected to light centrifugation to remove cellular debris, and passed through a 0.45-μm-pore-size filter. Cell viral supernatants designated SHIV_SF162P3 were stored in 1-ml aliquots at −80°C. Stock virus was tested for TCID on stimulated human PBMC using the method of Reed and Munch (16).

FIG. 2

Coreceptor usage of SHIV_SF162P3 was compared to those of molecular clones SHIV_SF162 (CCR5 specific) and SHIV_SF33A.2 (CXCR4 specific) for replication in PBMCs isolated from a CCR5 wild-type (wt) donor (CCR5^+/+ [wt/wt PBMC]) and a donor homozygous for Δ32 (CCR5^−/− [Δ32/Δ32]). Culture supernatants were collected 10 days after infection and analyzed for p27^gag content by antigen enzyme-linked immunosorbent assay (Cellular Products, Buffalo N.Y.). These values represent peak p27^gag production. Activated T-cell cultures were exposed to cell-free SHIV_SF162P3 (multiplicity of infection of 0.1) for 4 h, washed twice in phosphate-buffered saline to remove residual inoculum, and maintained in RPMI complete medium supplemented with recombinant interleukin 2. Culture supernatants were collected at regular intervals and analyzed for p27^gag production by antigen enzyme-linked immunosorbent assay.

FIG. 3

IVAG infection with cell-free SHIV_SF162P3. Whole blood was collected at defined intervals from animals R061 (A), T290 (B), R513 (C), and T637 (D), and samples were monitored for plasma viremia (■) (copies of viral RNA per milliliter of plasma), CD4⁺ cell number per microliter of whole blood (□), and CD8⁺ cell number per microliter of whole blood (○). To detect plasma viremia, we used the branched-DNA signal amplification assay (Bayer Diagnostics, Emeryville, Calif.) with a sensitivity limit of 1,500 copies of viral RNA/ml of plasma. The following human antibodies were used: Leu-3a-phycoerythrin and Leu-2a-peridinin chlorophyll protein (Becton Dickinson, Mountainview, Calif.), which recognize the CD4 and CD8 proteins, respectively. The monoclonal antibody FN-18–fluorescein isothiocyanate (Biosource International, Camarillo, Calif.) that recognizes the monkey CD3 molecule was also used. T-cell subsets were enumerated using TruCount absolute count tubes (Becton Dickinson) according to the manufacturer. Flow cytometry analysis was performed using the FACS Caliber device. PI, postinfection.

FIG. 4

Humoral immune responses were detected in the plasma of macaques infected with SHIV_SF162P3 using a strip immunoblot assay, RIBA HIV-1/2 (Chiron Corp., Emeryville, Calif.), as per the manufacturer's instructions Lanes: 1, animal R061; 2, animal T290; 3, animal R513; 4, animal T637 −, negative control; +, positive control. Plasma was obtained 23 weeks postinoculation.

FIG. 5

Histologic section of lung obtained at necropsy form macaque R061. Alveolar septae are thickened, and hypercellular white alveolar spaces are filled with eosinic, foamy material. These changes are typical of interstitial PCP. Hematoxylin and eosin stain was used. The presence of P. carinii was confirmed by Gomorri methenamine silver staining.