Postinoculation PMPA treatment, but not preinoculation immunomodulatory therapy, protects against development of acute disease induced by the unique simian immunodeficiency virus SIVsmmPBj

Abstract

The fatal disease induced by SIVsmmPBj4 clinically resembles endotoxic shock, with the development of severe gastrointestinal disease. While the exact mechanism of disease induction has not been fully elucidated, aspects of virus biology suggest that immune activation contributes to pathogenesis. These biological characteristics include induction of peripheral blood mononuclear cell (PBMC) proliferation, upregulation of activation markers and Fas ligand expression, and increased levels of apoptosis. To investigate the role of immune activation and viral replication on disease induction, animals infected with SIVsmmPBj14 were treated with one of two drugs: FK-506, a potent immunosuppressive agent, or PMPA, a potent antiretroviral agent. While PBMC proliferation was blocked in vitro with FK-506, pig-tailed macaques treated preinoculation with FK-506 were not protected from acutely lethal disease. However, these animals did show some evidence of modulation of immune activation, including reduced levels of CD25 antigen and FasL expression, as well as lower tissue viral loads. In contrast, macaques treated postinoculation with PMPA were completely protected from the development of acutely lethal disease. Treatment with PMPA beginning as late as 5 days postinfection was able to prevent the PBj syndrome. Plasma and cellular viral loads in PMPA-treated animals were significantly lower than those in untreated controls. Although PMPA-treated animals showed acute lymphopenia due to SIVsmmPBj14 infection, cell subset levels subsequently recovered and returned to normal. Based upon subsequent CD4(+) cell counts, the results suggest that very early treatment following retroviral infection can have a significant effect on modifying the subsequent course of disease. These results also suggest that viral replication is an important factor involved in PBJ-induced disease. These studies reinforce the idea that the SIVsmmPBj model system is useful for therapy and vaccine testing.

FIG. 1

Replication of SIVsmmPBj in the presence of FK-506. Pig-tailed macaque PBMC were stimulated with ConA (A) or used unstimulated (B) for virus replication studies. Cells (1 × 10⁷ to 2 × 10⁷) were inoculated with virus (in amounts equivalent to 10 ng of p27) derived from the molecular clone PBj6.6 either in the presence or in the absence of the indicated concentrations of FK-506. Cell-free supernatants harvested at the indicated times postinfection were tested for the presence of RT activity. The results are representative of two separate experiments.

FIG. 2

Immunocytochemical localization of virus, apoptosis, and activation markers in intestinal tissue from PBj6.6-infected macaques. (A through H) Ileum sections from high-dose PBj6.6-infected pig-tailed macaques that were either left untreated (POd) (A, C, E, and G) or treated with FK-506 (PKf) (B, D, F, and H) were used to investigate the local effects of immunosuppressive therapy on SIVsmmPBj14 infection. Apoptotic nuclei were visualized by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling method (A and B). SIV antigen-positive cells (C and D), CD25-positive cells (E and F), and Fas ligand (CD95L)-positive cells (G and H) were identified by immunohistochemistry using specific antisera (see Materials and Methods). (I) Quantitation of the mean number of positive cells for each item pictured in panels A through H for all animals in the FK-506 cohort. Nine random fields of ileum tissue were examined to determine the mean number of positive cells for each evaluation. In the high-virus-dose group, statistically significant differences were observed between treated and untreated animals in the numbers of CD25⁺ cells (P < 0.005), SIV antigen levels (P < 0.01), and FasL expression (P < 0.001).

FIG. 2

Immunocytochemical localization of virus, apoptosis, and activation markers in intestinal tissue from PBj6.6-infected macaques. (A through H) Ileum sections from high-dose PBj6.6-infected pig-tailed macaques that were either left untreated (POd) (A, C, E, and G) or treated with FK-506 (PKf) (B, D, F, and H) were used to investigate the local effects of immunosuppressive therapy on SIVsmmPBj14 infection. Apoptotic nuclei were visualized by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling method (A and B). SIV antigen-positive cells (C and D), CD25-positive cells (E and F), and Fas ligand (CD95L)-positive cells (G and H) were identified by immunohistochemistry using specific antisera (see Materials and Methods). (I) Quantitation of the mean number of positive cells for each item pictured in panels A through H for all animals in the FK-506 cohort. Nine random fields of ileum tissue were examined to determine the mean number of positive cells for each evaluation. In the high-virus-dose group, statistically significant differences were observed between treated and untreated animals in the numbers of CD25⁺ cells (P < 0.005), SIV antigen levels (P < 0.01), and FasL expression (P < 0.001).

FIG. 3

Replication of SIVsmmPBj in the presence of PMPA. Pig-tailed macaque PBMC were stimulated with ConA (A) or were used unstimulated (B) for virus replication studies. Cells (1 × 10⁷ to 2 × 10⁷) were inoculated with virus (equivalent to 10 ng of p27) derived from the molecular clone PBj6.6 either in the presence or in the absence of the indicated concentrations of PMPA. Cell-free supernatants harvested at the indicated times postinfection were tested for the presence of RT activity. The results are representative of two separate experiments.

FIG. 4

Longitudinal analysis of circulating CD4⁺ cells in SIVsmmPBj14-infected macaques treated with PMPA. Blood samples from pig-tailed macaques infected with the PBj6.6 virus and treated with PMPA beginning at either 3 days (PAi and PNi) or 5 days (PEe and PZh) postinfection were used for the enumeration of absolute circulating CD4⁺ cells at the indicated time points by fluorescence-activated cell sorter analysis.