Interactions between human immunodeficiency virus type 1 and vaccinia virus in human lymphoid tissue ex vivo

Abstract

Vaccinia virus (VACV) has been attracting attention recently not only as a vector for various vaccines but also as an immunization tool against smallpox because of its potential use as a bioterrorism agent. It has become evident that in spite of a long history of studies of VACV, its tissue pathogenesis remains to be fully understood. Here, we investigated the pathogenesis of VACV and its interactions with human immunodeficiency virus type 1 (HIV-1) in the context of human lymphoid tissues. We found that ex vivo-cultured tonsillar tissue supports productive infection by the New York City Board of Health strain, the VACV strain of the Dryvax vaccine. VACV readily infected both T and non-T (B) lymphocytes and depleted cells of both of these subsets equally over a 12-day period postinfection. Among T lymphocytes, CD8(+) cells are preferentially depleted in accordance with their preferential infection: the probability that a CD8(+) T cell will be productively infected is almost six times higher than for a CD4(+) T cell. T cells expressing CCR5 and the activation markers CD25, CD38, and HLA-DR are other major targets for infection by VACV in lymphoid tissue. As a consequence, VACV predominantly inhibits the replication of the R5(SF162) phenotype of HIV-1 in coinfected tissues, as R5-tropic HIV-1 requires activated CCR5(+) CD4(+) cells for productive infection. Human lymphoid tissue infected ex vivo by VACV can be used to investigate interactions of VACV with other viruses, in particular HIV-1, and to evaluate various VACV vectors for the purpose of recombinant vaccine development.

FIG. 1.

Replication of VACV in human lymphoid tissue ex vivo. Tissue blocks (27 from each human tonsil) were infected with either NYCBH or MVA. Each of the 27 tissue blocks from a single donor was inoculated with 5 μl of viral stock containing approximately 1 × 10⁶ genome equivalents. The culture medium was changed every 3 days, and VACV replication was monitored in these medium samples by means of real-time PCR (A) or flow cytometry (B and C). A. Typical replication kinetics of NYCBH and MVA are presented. The inset represents the average kinetics of replication of NYCBH (n = 13); results are shown as means ± SEM. Each point represents the measurement of medium pooled from three wells, each of which contained nine tissue blocks. To merge the data from different experiments, we normalized the amount of VACV DNA for each experiment as a percentage of the maximum production of VACV over the 12-day period. B. Expression of VACV antigen by lymphocytes as detected with flow cytometry. Density plots of lymphocytes, isolated from VACV-infected (right panel) and matched uninfected tissue (left panel), indicate preferential staining of CD3⁻ cells for cell-associated vaccinia virus antigen on day 12 postinfection. One representative experiment (out of 11) is shown. C. Frequencies of VACV-infected CD3⁻, CD3⁺, CD3/CD4⁺, and CD3/CD8⁺ cells detected at day 12 postinfection. Cells were stained at day 12 for CD3, CD4, CD8, and cell-associated vaccinia virus antigens. Data are presented as means ± SEM (error bars) of VACV-infected cells from 11 donors.

FIG. 2.

Preferential depletion of the CD4⁺ CCR5⁺ cell subset in VACV-inoculated ex vivo-cultured human lymphoid tissues. Tissue blocks (27 from each individual human tonsil donor) were infected with NYCBH. At day 12 postinfection, cells were stained for CD3, CD4, or CD8. Data are means ± SEM (error bars) of the numbers of lymphocyte subsets normalized by the weight of the tissue blocks and by the numbers of corresponding cells in matched uninfected control tissues. A. Depletion of CD3⁻, CD3⁺, CD3⁺/CD4⁺, and CD3⁺/CD8⁺ cells by VACV in human lymphoid tissue ex vivo (n = 12). B. Depletion of CCR5⁺ versus CCR5⁻ cells within the CD3⁺ versus the CD3⁻ cell subsets (n = 9). C. Depletion of CCR5⁺ versus CCR5⁻ cells within the CD3⁺/CD4⁺ versus the CD3⁺/CD8⁺ cell subsets (n = 9).

FIG. 3.

Preferential depletion of activated T cells in human lymphoid tissues inoculated ex vivo with VACV. Tissue blocks (27 from each individual human tonsil donor) were infected with NYCBH. At day 12 postinfection, cells were stained for the following activation markers: CD3, CD25, CD38, CD69, and HLA-DR. Data are means ± SEM (error bars) of the numbers of lymphocytes positive or negative for any of the activation markers listed above normalized by the weight of the tissue blocks and by the numbers of corresponding cells in matched uninfected control tissues. A. Depletion of activated CD3⁺ cells by VACV in human lymphoid tissue ex vivo (n = 12). B. Fraction of VACV-infected activated T cells detected at day 12 postinfection (n = 7).

FIG. 4.

Interactions of VACV and HIV-1 in human lymphoid tissues coinfected ex vivo. Tissue blocks were coinfected with VACV and either R5_SF-162 or X4_LAI.04 or were infected singly with one of these isolates. HIV-1 replication was evaluated based on measurement of the p24 core antigen. VACV replication was evaluated based on measurement of VACV genome equivalents in the culture medium by using real-time PCR. A. Typical kinetics of HIV-1 replication in tissue blocks infected singly with HIV-1 or coinfected with VACV. Each point represents a measurement of the pooled medium bathing 27 blocks. B. Average HIV-1 replication in tissues coinfected with VACV and HIV-1, relative to that in matched tissues infected singly with HIV-1. Data are means ± SEM (error bars) of experiments with tissues from 13 donors infected ex vivo with R5_SF-162 and from 6 donors infected ex vivo with X4_LAI.04. C. Average VACV replication in tissues coinfected with VACV and HIV-1, relative to that in matched tissues infected singly with VACV. Data are means ± SEM (error bars) of experiments with tissues from 13 donors infected ex vivo with R5_SF-162 and from 6 donors infected ex vivo with X4_LAI.04.