Intravenous inoculation of replication-deficient recombinant vaccinia virus DIs expressing simian immunodeficiency virus gag controls highly pathogenic simian-human immunodeficiency virus in monkeys

Abstract

To be effective, a vaccine against human immunodeficiency virus type 1 (HIV-1) must induce virus-specific T-cell responses and it must be safe for use in humans. To address these issues, we developed a recombinant vaccinia virus DIs vaccine (rDIsSIVGag), which is nonreplicative in mammalian cells and expresses the full-length gag gene of simian immunodeficiency virus (SIV). Intravenous inoculation of 10(6) PFU of rDIsSIVGag in cynomologus macaques induced significant levels of gamma interferon (IFN-gamma) spot-forming cells (SFC) specific for SIV Gag. Antigen-specific lymphocyte proliferative responses were also induced and were temporally associated with the peak of IFN-gamma SFC activity in each macaque. In contrast, macaques immunized with a vector control (rDIsLacZ) showed no significant induction of antigen-specific immune responses. After challenge with a highly pathogenic simian-human immunodeficiency virus (SHIV), CD4(+) T lymphocytes were maintained in the peripheral blood and lymphoid tissues of the immunized macaques. The viral set point in plasma was also reduced in these animals, which may be related to the enhancement of virus-specific intracellular IFN-gamma(+) CD8(+) cell numbers and increased antibody titers after SHIV challenge. These results demonstrate that recombinant DIs has potential for use as an HIV/AIDS vaccine.

FIG. 1.

Vector construction and expression of rDIsSIVGag. (a) Construction of rDIsSIVGag. Full-length DNA of SIVmac239 Gag was inserted into the deleted region of vaccinia strain DIs. (b) Detection of SIV Gag protein by Western blot with anti-p27 Gag MAb IB6.

FIG. 2.

SIV Gag-specific IFN-γ SFC in PBMC from rDIsSIVGag- and rDIsLacZ-inoculated and naive macaques. Freshly isolated PBMC were assessed for their ability to produce IFN-γ in response to overlapping peptides covering the full-length SIV Gag protein. The experimental schedule of each grouped animal is presented in Table 2.

FIG. 3.

Proliferative responses to SIV p27 Gag in the macaques inoculated with either rDIsSIVGag or rDIsLacZ. Responses were measured 2 weeks after the second inoculation of recombinant vaccinia virus antigen. Naive macaques did not show any proliferative response to SIV p27 (data not shown). Bars represent the mean values of all animals.

FIG. 4.

Flow cytometric analysis of IFN-γ-producing CD8⁺ T cells specific for SIV Gag. PBMC from macaques were cultured in vitro with overlapping peptides and stained for intracellular IFN-γ. The percentage of IFN-γ-producing CD8⁺ T cells in each macaque PBMC was determined by flow cytometry, and data obtained before (at 26 week p.i. [a]) and after(at 31 week p.i. [b]) the SHIV challenge were compared.

FIG. 5.

Enhancement of anti-SIV Gag antibody titers in rDIsSIVGag-inoculated macaques by SHIV challenge. Titers of binding antibody in plasma to SIV Gag were measured by SIV Gag-specific enzyme-linked immunosorbent assay. The results represent the mean of three independent experiments. Error bars represent the mean ± three SD.

FIG. 6.

Comparison of CD4⁺ T cells and viral load in plasma among rDIsSIVGag-inoculated macaques and vector controls. (a and b) CD4⁺-T-cell counts in peripheral blood of rDIsSIVGag-inoculated animals (a) and vector controls (b). (c and d) Viral set-point levels in plasma in vaccinated macaques (c) and vector controls (d).

FIG. 7.

CD4⁺ T lymphocytes remaining in spleen and mesenteric lymph nodes of macaques inoculated with rDIsSIVGag. Morphological analysis of spleen and mesenteric lymph node from vaccinated macaques. Tissue sections of spleen (a and b) and lymph nodes (c and d) were obtained at autopsy from macaques inoculated with rDIsSIVGag (a and c) or rDIsLacZ (b and d), followed by SHIV challenge. Sections were obtained from samples taken 12 weeks postchallenge and were stained with hematoxylin and eosin. Bar, 100 μm. (e) Quantitative flow cytometric analysis of CD4⁺ T lymphocytes in the spleen, inguinal lymph nodes, and mesenteric lymph nodes. The data represent mean values from the spleen and three different tissues of each inguinal and mesenteric lymph node.