Immunization of macaques with single-cycle simian immunodeficiency virus (SIV) stimulates diverse virus-specific immune responses and reduces viral loads after challenge with SIVmac239

Abstract

Genetically engineered simian immunodeficiency viruses (SIV) that is limited to a single cycle of infection was evaluated as a nonreplicating AIDS vaccine approach for rhesus macaques. Four Mamu-A*01(+) macaques were inoculated intravenously with three concentrated doses of single-cycle SIV (scSIV). Each dose consisted of a mixture of approximately equivalent amounts of scSIV strains expressing the SIV(mac)239 and SIV(mac)316 envelope glycoproteins with mutations in nef that prevent major histocompatibility complex (MHC) class I downregulation. Viral loads in plasma peaked between 10(4) and 10(5) RNA copies/ml on day 4 after the first inoculation and then steadily declined to undetectable levels over the next 4 weeks. SIV Gag-specific T-cell responses were detected in peripheral blood by MHC class I tetramer staining (peak, 0.07 to 0.2% CD8(+) T cells at week 2) and gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assays (peak, 50 to 250 spot forming cells/10(6) peripheral blood mononuclear cell at week 3). Following the second and third inoculations at weeks 8 and 33, respectively, viral loads in plasma peaked between 10(2) and 10(4) RNA copies/ml on day 2 and were cleared over a 1-week period. T-cell-proliferative responses and antibodies to SIV were also observed after the second inoculation. Six weeks after the third dose, each animal was challenged intravenously with SIV(mac)239. All four animals became infected. However, three of the four scSIV-immunized animals exhibited 1 to 3 log reductions in acute-phase plasma viral loads relative to two Mamu-A*01(+) control animals. Additionally, two of these animals were able to contain their viral loads below 2,000 RNA copies/ml as late as 35 weeks into the chronic phase of infection. Given the extraordinary difficulty in protecting against SIV(mac)239, these results are encouraging and support further evaluation of lentiviruses that are limited to a single cycle of infection as a preclinical AIDS vaccine approach.

FIG. 1.

Comparison of the relative infectivity of six different strains of scSIV on an immortalized human CD4⁺ T-cell line. (A) Six different strains of scSIV were generated with differences in the env and nef genes. These included strains expressing the SIV_mac239 (239) and the SIV_mac316 (316) envelope glycoproteins together with wild-type nef (Nef), a nef mutant deficient for MHC class I downregulation (Nef^sff), or the gene for EGFP at the nef locus (3). (B) T2-SEAP cells (1 × 10⁶) were infected with 250 ng p27 equivalents of each virus. Four days after infection, the infectivity of each strain was determined by flow cytometry and by SEAP production in the culture supernatant. Fixed cells were analyzed for both Gag and EGFP expression. Intracellular Gag staining was performed on permeabilized cells using the SIV Gag-specific monoclonal antibody 2F12 (23) and a phycoerythrin-conjugated donkey anti-mouse F(ab′)₂ fragment. SEAP activity was measured by chemiluminescent detection using a 96-well format lumaplate reader.

FIG. 2.

Viral RNA loads in plasma after intravenous inoculation of four macaques with single-cycle SIV. Each animal was inoculated intravenously with three doses of concentrated scSIV at weeks 0, 8, and 33. The first two doses used identical amounts of the same cryopreserved stocks and contained a total of 7.8 μg p27 equivalents of scSIV (a mixture of 4.4 μg p27 of scSIV_mac239Nef^sff and 3.4 μg p27 of scSIV_mac316Nef^sff). The third dose contained a total of 11.6 μg p27 equivalents of scSIV (5.2 μg p27 of scSIV_mac239Nef^sff and 6.4 μg p27 of scSIV_mac316Nef^sff) from separate stocks. Viral RNA loads in plasma were determined using a quantitative reverse transcription-PCR assay (35) with a nominal sensitivity of 25 copy equivalents (eq.)/ml (dotted line) and an interassay coefficient of variation of <25%.

FIG. 3.

SIV Gag-specific CD8⁺ T-cell responses in four Mamu-A*01⁺ rhesus macaques immunized with scSIV. Whole blood was stained with monoclonal antibodies to CD3 and CD8 together with Mamu-A*01-Gag_181-189 tetramers and analyzed by flow cytometry. After gating on the CD3⁺ CD8⁺ lymphocyte population, the percentage of tetramer-positive cells was determined at each time point. The arrows at weeks 0, 8, and 33 indicate inoculations with scSIV. Values greater than 0.05% (dotted line) are considered positive.

FIG. 4.

SIV-specific IFN-γ T-cell responses in four scSIV-immunized macaques. PBMC were stimulated overnight with pools of overlapping Gag (A) and Env (B) peptides. IFN-γ-producing cells were enumerated in ELISPOT assays. The average numbers of SFC per million PBMC and the standard deviations (error bars) were determined from duplicate wells plated at 3 × 10⁵ PBMC/well. Responses greater than 50 SFC/10⁶ PBMC (dotted line) are considered positive. Arrows indicate scSIV inoculations at weeks 0, 8, and 33.

FIG. 5.

SIV-specific T-cell proliferative responses in four scSIV-immunized macaques. PBMC were stimulated in quadruplicate wells at 1 × 10⁵ cells/well with AT-2-inactivated SIV_mac239 (7) and matched microvesicle control supernatants prepared from uninfected cells. Five days after stimulation, virus-specific proliferation was determined in an 18-hour [³H]thymidine incorporation assay. Stimulation indices were calculated by dividing the average counts per minute for virus-stimulated wells by the average counts per minute for control wells. Stimulation indices greater than 3 were considered positive (dotted line). Arrows indicate scSIV inoculations at weeks 8 and 33.

FIG. 6.

Virus-specific antibody responses in scSIV-immunized animals. SIV Western blot strips were probed with plasma samples collected at weeks 0, 8, 11, 33, and 35 at a 1/100 dilution according to the manufacturer's instructions. The positions of major viral proteins are indicated on the left side of the blot. The serum control band (ctrl) represents an internal positive control for the reactivity of each strip. +, positive; −, negative.

FIG. 7.

Neutralizing antibody responses at the time of challenge. Serial twofold dilutions of plasma were tested for the ability to block the infection of CEMx174SIV-SEAP cells (38) with SIV_mac251_LA (A), SIV_mac316 (B), and SIV_mac239 (C). Serial twofold dilutions of plasma were incubated with SIV_mac251_LA (0.25 ng p27), SIV_mac239 (1.0 ng p27), and SIV_mac316 (5.0 ng p27) for 1 hour before the addition of 4 × 10⁴ target cells. SEAP activity was measured in culture supernatants collected from duplicate wells 3 days after infection with SIV_mac251_LA and SIV_mac239 and from quadruplicate wells 5 days after infection with SIV_mac316. The dashed lines indicate 50% neutralization.

FIG. 8.

Viral loads in plasma and CD4⁺ T-cell counts after an intravenous SIV_mac239 challenge. Four scSIV-immunized animals and two unvaccinated control animals were challenged intravenously with 10 animal infectious doses (1.5 pg p27) of SIV_mac239. (A) Viral RNA loads in plasma were determined using a quantitative reverse transcription-PCR assay (35) with a nominal sensitivity of 25 copy equivalents (eq.)/ml and an interassay coefficient of variation of <25%. (B) CD4⁺ T-cell counts in peripheral blood were monitored by flow cytometry and complete blood count analysis. All six macaques were Mamu-A*01⁺. The solid symbols represent the scSIV-immunized animals, and the open symbols represent the unvaccinated control animals.

FIG. 9.

SIV-specific T-cell responses postchallenge. (A) SIV Gag_181-189-specific CD8⁺ T-cell responses were monitored by staining whole blood with monoclonal antibodies to CD3 and CD8 together with Mamu-A*01-Gag_181-189 tetramers. The percentage of tetramer-positive CD8⁺ T cells was determined by flow cytometry after gating on the CD3⁺ CD8⁺ lymphocyte population. (B) SIV-specific T-cell proliferative responses were monitored by stimulating PBMC with AT-2-inactivated SIV_mac239 (7) and matched microvesicle control supernatants. Five days after stimulation, virus-specific proliferation was determined in an 18-hour [³H]thymidine incorporation assay. Stimulation indices greater than 3 were considered positive (dotted line). (C) IFN-γ-producing T-cell responses to the SIV Gag, Env, Tat/Rev, and Nef proteins. PBMC were stimulated with pools of overlapping peptides, and cells expressing IFN-γ were enumerated in ELISPOT assays. The average numbers of SFC per million PBMC and standard deviations (error bars) were determined from duplicate wells plated at 3 × 10⁴, 1 × 10⁵, or 3 × 10⁵ PBMC/well.