The simian immunodeficiency virus deltaNef vaccine, after application to the tonsils of Rhesus macaques, replicates primarily within CD4(+) T cells and elicits a local perforin-positive CD8(+) T-cell response

Abstract

Deletion of the nef gene from simian immunodeficiency virus (SIV) strain SIVmac239 yields a virus that undergoes attenuated growth in rhesus macaques and offers substantial protection against a subsequent challenge with some SIV wild-type viruses. We used a recently described model to identify sites in which the SIVDeltanef vaccine strain replicates and elicits immunity in vivo. A high dose of SIVDeltanef was applied to the palatine and lingual tonsils, where it replicated vigorously in this portal of entry at 7 days. Within 2 weeks, the virus had spread and was replicating actively in axillary lymph nodes, primarily in extrafollicular T-cell-rich regions but also in germinal centers. At this time, large numbers of perforin-positive cells, both CD8(+) T cells and CD3-negative presumptive natural killer cells, were found in the tonsil and axillary lymph nodes. The number of infected cells and perforin-positive cells then fell. When autopsy studies were carried out at 26 weeks, only 1 to 3 cells hybridized for viral RNA per section of lymphoid tissue. Nevertheless, infected cells were detected chronically in most lymphoid organs, where the titers of infectious virus could exceed by a log or more the titers in blood. Immunocytochemical labeling at the early active stages of infection showed that cells expressing SIVDeltanef RNA were CD4(+) T lymphocytes. A majority of infected cells were not in the active cell cycle, since 60 to 70% of the RNA-positive cells in tissue sections lacked the Ki-67 cell cycle antigen, and both Ki-67-positive and -negative cells had similar grain counts for viral RNA. Macrophages and dendritic cells, identified with a panel of monoclonal antibodies to these cells, were rarely infected. We conclude that the attenuated growth and protection observed with the SIVDeltanef vaccine strain does not require that the virus shift its characteristic site of replication, the CD4(+) T lymphocyte. In fact, this immunodeficiency virus can replicate actively in CD4(+) T cells prior to being contained by the host, at least in part by a strong killer cell response that is generated acutely in the infected lymph nodes.

FIG. 1.

Virus load over time in the blood of rhesus macaques after tonsillar infection with SIVΔnef. (A) Cell-associated virus in PBMCs was determined by a limiting-dilution coculture technique, and the endpoint was calculated. Virus loads are expressed as the number of infectious cells per 10⁶ PBMCs. (B) Levels of viral RNA in plasma measured by quantitative reverse transcription-PCR and expressed as RNA equivalents per ml. The detection limit of this assay is 40 RNA equivalents per ml. Individual monkeys are indicated by four-digit numbers.

FIG. 2.

Cell-associated virus load in lymphoid organs and blood of rhesus macaques during acute infection (4 and 7 days postinfection, upper two panels) with attenuated SIVΔnef and in the chronic stage (26 weeks, lower three panels). Mononuclear cell suspensions from each organ and the blood were prepared, and infectious units were determined as described for Fig. 1. Missing bars mean that no virus could be isolated; nd means the organ was not obtained; ne means the culture could not be evaluated because of contamination; the asterisks indicate that the endpoint was not reached. dpi, days postinfection; wpi, weeks postinfection; Ln, lymph node; tons, tonsil; cervic, cervical; retrophar, retropharyngeal; submand, submandibular; axill, axillary; mesent, mesenteric; Peyer’s, Peyer’s patches. Individual monkeys are indicated on the right of each panel by Mm (for Macaca mulatta) and four-digit numbers.

FIG. 3.

Active replication and spread of SIVΔnef following infection via the tonsil. Lymphoid tissues were examined by in situ hybridization with ³⁵S-labeled antisense probes (labeled cells were not found with the control sense probe; not shown). Cells expressing viral RNA appear black (A, B, and D) or green (C). (A) The tonsil during acute infection, 1 week after infection with SIVΔnef. Many RNA -positive cells (black) are in the extrafollicular T-cell-rich regions but are also found in germinal centers (GC). (B) Axillary lymph nodes, to which infection has spread at 2 weeks, contain many infected cells, including in the germinal centers. (C and D) Mesenteric node and auxiliary node 26 weeks after infection contain a few but clear-cut (arrows) RNA-positive cells, typically in the germinal centers.

FIG. 4.

SIVΔnef primarily replicates in CD4⁺ T cells in lymphoid nodes. Tissue sections from lymph node (LN) and tonsil were labeled with the indicated monoclonal antibodies using an immunoperoxidase method (red color) and by in situ hybridization (black) to visualize RNA-positive cells. (A) Two infected CD4⁺ cells (arrows) in the lymphoepithelium of the acutely infected tonsil at 1 week. (B and C) Low- and high-power views of peripheral lymph nodes at 2 weeks to show labeling of infected cells for the CD4 T-cell marker (arrows). (D to F) Double labeling for CD1a (D), DC-LAMP/CD208 (E), and CD68 (F). Most infected cells are negative. Infected T cells can be found close to noninfected dendritic cells (E, arrows). An occasional CD68-positive cell is infected (F, arrow).

FIG. 5.

SIV wild type (A and B) and SIVΔnef (C and D) often replicate in cells that do not immunolabel for the cell cycle antigen Ki-67 (red). Black arrows indicate Ki-67-negative RNA-infected cells, while white arrows indicate Ki-67-positive infected cells.

FIG. 6.

Detection of perforin-positive lymphocytes in tissue sections following tonsillar infection of rhesus macaques with SIVΔnef. Single-color immunolabeling (red) at 1 week in the heavily infected tonsil (A) and weakly infected retropharyngeal lymph node (B) and at 2 weeks (C) and 26 weeks (D) in strongly infected and weakly infected nodes. Two-color immunolabeling, with perforin in blue, shows the cells to be CD4 negative (red color in E and F), either CD3⁺ (arrows, G) or CD3⁻ (arrowheads, G), or CD8⁺ (arrows, H).