This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This pilot project was approved in September 2004. In vivo and in vitro experiments are in progress. Selection of animals for alloimmunization: Eight adult male rhesus macaques are being used in this study. Genotypic analysis was performed to provide haplotype information which could indicate a basis for host-contribution to SIV pathogenicity. Prior to manipulations, blood was collected from each animal, PBMC were purified, and standard MLR assays were set up between each pair of animals. Cells were also cultured alone as negative controls. After five days of culture, 3H thymidine was added to each culture, and stimulation indices (SI) were calculated for each animal vs all other animals and compared to baseline proliferation. Initial stimulation indices of all possible pair combinations demonstrated that the 6 Indian macaques responded more strongly to PBMC from Chinese macaques than other Indian macaques, and Chinese macaques responded more strongly to other Chinese macaques than to Indian macaques. Vaccination: PBMC from 3 macaques of Chinese origin were pooled, adjuvanted with liposome DNA adjuvant, and used to alloimmunize each other as well as 3 macaques of Indian origin. The two remaining Indian origin rhesus were vaccinated with self PBMC. 6 animals were alloimmunized with 5 X106 mixed Chinese PBMC IV and 5X106 Ch PBMC at 3 ID sites. 2 animals were vaccinated with 5X106 self PBMC IV and 5X106 self PBMC at 3 ID sites to serve as autologous controls. Six weeks post vaccination, an intrarectal boost of 1X107 Mixed Ch PBMC was given to the alloimmunized group and the autologous controls were given an intrarectal boost of 1X107 self PBMC. At 12 weeks post vaccination, the alloimmunized group received 5 X106 mixed Chinese PBMC IV and 5X106 Ch PBMC at 3 ID sites and the autologous controls received 5X106 self PBMC IV and 5X106 self PBMC at 3 ID sites. Cells and sera were collected at each boost timepoint and used to assay MLR and proliferation vs killed virus grown in self or autologous cells (Fig. 2). Similar results were obtained following stimulation of cells with killed SIV grown in ChRh vs. CEM cells. Challenge: The stock of SIVmac239 Rh511 grown and characterized on CEMX174 cells at CSU was used to infect ConA and IL2 stimulated PBMC from all 8 macaques and this virus was stored separately at -80oC. Virus was quantitated by SIV p28 ELISA. Virus grown from the three Chinese macaques used in alloimmunization was combined in equal parts according to capsid concentration and stock titer determined by growth on CEMX174 cells. A challenge stock of 400TCID50 /ml was made by a 1/200 dilution. 6 weeks following the second boost, animals in both the alloimmunization and the autologous control group were challenged. Plasma viral loads, lymphocyte counts, and flow cytometry parameters of post-challenge dates are pending. Flow Cytometry: Isolated PBMC were stained for CD4, CD8, and the activation markers MHC II, CD69, and CD25 at 2 pre-immunization dates, 3 days post-immunization (pi), 4 weeks pi, 3 days post-boosts and 4 weeks post-boosts. No significant differences between groups were noted. Monkey IFN? ELISA of Con A stimulated culture supernatants: Post vaccination/boost IFN? levels of Con A stimulated PBMC after 2 days in culture were measured using ELISA. Values were highest in alloimmunized Indian macaques, with alloimmunized Chinese macaques being lowest, mirroring the trend seen with the MLR SIs. Pending In vitro Assays: Pre- and post-immunization western blots of EDTA Plasma from all study animals against whole cell lysate from pooled Chinese PBMC are pending, as are neutralization titers vs SIV grown in ChRhPBMC or CEM cells.
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