Schedule of immunizations and SIV challenge in six rhesus macaques. The DNA immunization consisted of five plasmid constructs; MCP3p39 gag, MCP3p39 env, CATEDX gag, CATE239 env, and CATEpolNTV. One mg of each plasmid DNA was administered via the intramuscular route. The HSV immunization included three recombinant HSV d106 constructs, d106env, d106gag and d106rev-tat-nef, each administered at a dose of 1.25 to 3x10⁹ pfu per dose. Two-thirds of the HSV vaccine dose was inoculated via the intramuscular route while one-third of the dose was administered via the subcutaneous route. 10 animal infectious doses of cloned homologous pathogenic SIV239 inoculated via the intravenous route was used for challenge.

Antibody responses to a) HSV and b) SIV in immunized monkeys by ELISA. Closed symbols denote the DNA-DNA-HSV-HSV vaccinated macaques while open symbols denote the HSV-HSV-HSV-HSV vaccinated macaques. Arrows show the time of vaccination. Anti-HSV antibody responses in plasma were tested at 1 in 200 dilution while anti-SIV antibody responses are shown at 1 in 20 plasma dilutions.

Neutralizing antibody responses to laboratory passaged SIV251 in six vaccinated rhesus macaques. a) At week 26, on day of challenge. b) At week 28, two weeks post SIV challenge. Serial two-fold dilutions of plasma were incubated with SIV251-lab for 1 hour before addition of 3x10⁴ CEMx174SIV-SEAP reporter cells. One pool of negative rhesus monkey plasma was used as a negative control and two separate pools of SIV+ rhesus monkey plasma were used as positive controls at the time of each assay. In the assays shown 50% neutralizing antibody titers in the positive pool plasma ranged between 1:3200 and 1:5120. Negative control plasma was negative.

Anti-SIV cellular responses during the vaccine phase in immunized monkeys. a) IFN-γ ELISPOT responses to pools of overlapping peptides spanning the SIV239 Gag, Env, Nef, Tat and Rev proteins. Closed symbols denote the DNA-DNA-HSV-HSV vaccinated rhesus macaques. Open symbols denote the HSV-HSV-HSV-HSV vaccinated monkeys. Arrows show the time of vaccinations. Spot forming cell (SFC) frequencies shown after subtraction of background with unstimulated PBMC in medium alone wells. b) Frequency of Mamu-A*01 / Gag CM9 tetramer-positive CD8+ T lymphocytes in immunized monkeys. Closed symbols (left panel) show DNA-DNA-HSV-HV vaccinated macaques. Open symbols (right panel) show HSV-HSV-HSV-HSV vaccinated macaques. Data on Mamu-A*01 positive rhesus macaques shown. Arrows depict time of vaccination. Fresh PBMC were incubated with Mamu-A*01/GagCM9 tetramers at 37°C for 15 min before surface staining with anti-CD3 and anti-CD8 mAb. Data on lymphocytes gated on CD3+CD8+ T lymphocytes shown. c) Peptide titration of IFN-γ ELISPOT responses to the Gag CM9 CTL epitope at week 14 and 22 during the vaccination phase in two Mamu-A*01-positive immunized monkeys. PBMC were incubated with 10-fold dilutions of SIV Gag CM9 peptide for 16–20 hours before measuring IFN-γ ELISPOT responses. Spot forming cell (SFC) frequency per million CD8+ T lymphocytes is shown. The sensitizing peptide dose required to achieve half maximized lysis (SD₅₀) was determined from the titration curves.

Phenotype of vaccine-induced Mamu-A*01 / Gag CM9 tetramer-positive CD8+ T lymphocytes in the peripheral blood of one immunized monkey (Mm 205-87). Representative dot plots of four-color flow cytometry gated on CD3+CD8+ T lymphocytes shown. Percentages in upper right quadrant shows the frequency of Gag CM9 tetramer positive CD8+ T lymphocytes that express the molecule shown on the horizontal axis.

Correlation between IFN-γ ELISPOT responses to SIV Gag peptide pool and a) frequency of Mamu-A*01 / Gag CM9 tetramer-positive CD8+ T lymphocytes in Mamu-A*01+ rhesus monkeys and b) IFN-γ ELISPOT responses to whole SIV Gag p55 antigen in immunized monkeys.

Detection of CD4+ and CD8+ T lymphocyte responses to SIV in immunized monkeys. Representative intracellular cytokine staining data in one monkey shown. a) Gating strategy for analyzing SIV-specific CD4+ and CD8+ T lymphocytes. b) Responses to SIV Env and Gag in one vaccinated monkey. PBMC were stimulated for six hours in the presence of Brefeldin A and evaluated for intracellular cytokine and CD69 expression by four-color flow cytometry. Percentages in upper right quadrant show the frequency of CD3+8+ or CD4+ T lymphocytes co-expressing CD69 and IFN-γ.

Plasma SIV viral loads post intravenous SIV239 challenge in vaccinated macaques.

Comparison of SIV viral loads in vaccinated rhesus monkeys and unvaccinated historical control rhesus macaques infected with the same stock of SIV239. Plasma SIV RNA compared in vaccinated and control macaques using the unpaired nonparametric Mann-Whitney U test.

IFN-γ ELISPOT responses post SIV challenge in vaccinated rhesus macaques. a) IFN-γ ELISPOT responses to SIV Gag, Env, Nef, Tat and Rev from day of challenge to week 12 post challenge. Closed symbols show DNA-DNA-HSV-HSV vaccinated macaques, open symbols show HSV-HSV-HSV-HSV vaccinated macaques. Mm136-90 died three weeks post challenge. b) Regression plots showing relationship between area under curve (AUC) plasma SIV RNA and cellular and humoral immune responses on the day of challenge. c) Regression plots showing relationship between AUC plasma SIV RNA and cellular and humoral immune responses two weeks post challenge. AUC was calculated for plasma SIV RNA values until 24 weeks post challenge. Correlation co-efficient (Rho) and P-Values shown in each plot were calculated by the nonparametric Spearman Rank Correlation test. Plots with an asterisk (*) denote statistically significant correlations. ‘SFC’ – Spot forming cells

Gross, morphological, and immunohistochemical findings in the brain of a rhesus macaque (136-90) that died 3 weeks after SIV challenge. (A) Gross photograph of the formalin-fixed brain showing tan to brown discoloration of affecting the white matter of the left occipital lobe. Compare to normal white matter on the left (arrows). Ruler, 1 cm. (B, C and D) Hematoxylin- and eosin- stained section of the left occipital lobe from the area outlined by the box in (A). (B) Diffuse pallor of the white matter due to necrosis with multifocal hemorrhage and basophilic foci composed of inflammatory cell infiltrates. Small foci of hemorrhage and inflammatory infiltrates extend into the inner layer of the gray matter. Arrow marks interface of gray and white matter. (C) Higher magnification of white matter showing foci of hemorrhage and inflammatory cell infiltrates. Rarefaction of white matter is evident. (D) Higher magnification illustrating necrosis of a blood vessel surrounded by infiltrates of degenerate neutrophils. Magnifications, x20 (B), x100 (C) and x400 (D). (E) Immunohistochemical staining for myelin basic protein (MBP) of occipital cortex. Pallor or lack of staining for MBP corresponds to destruction of myelin in areas of perivascular neutrophilic infiltrates and vascular necrosis as depicted in (B,C and D). Magnification, x20 (E). (F) Hematoxylin- and eosin- stained section of cerebellar white matter showing a glial nodule. Magnification x200 (F).