This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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 completed and data is being analyzed and prepared for submission for two manuscripts.Selection of animals for alloimmunization: Eight adult male Rh were used in this study. Genotypic analysis was performed to provide haplotype information which could indicate a basis for host-contribution to SIV pathogenicity. Initially, 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. Initial stimulation indices of all possible pair combinations demonstrated that the 6 Ind Rh responded more strongly to PBMC from Ch Rh than other Ind Rh, and Ch Rh responded more strongly to other Ch Rh than to Ind Rh. Vaccination: PBMC from 3 Ch Rh origin were pooled, adjuvanted with liposome DNA adjuvant, and used to alloimmunize each other as well as 3 Ind Rh. The two remaining Ind Rh were vaccinated with self PBMC. 6 animals were alloimmunized with 5 X106 mixed Ch Rh 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. Post-immunization immune responses: 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. Killed SIV grown in ChRh vs. CEM cells elicited a greater stimulation index in InRh vs ChRh regardless of immunization group. Further, PBMC exposed to killed ChRhSIVmac239 produced more IFNg than PBMC exposed to SIVmac239 grown in CEM cells. Serum neutralization titers vs SIVmac239 propagated in ChRh PBMC increased in all animals immunized with ChRh cells, but did not increase in the two animals that were self-immunized.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; however, a positive correlation between CD69 (r=~.75) and MHCII (r~.93) expression (as measured by MFI) was noted.Challenge: SIVmac239 grown and characterized on CEMX174 cells was used to infect ConA and IL2 stimulated PBMC from all 8 Rh. Virus was quantitated by SIV p28 ELISA. Virus from the three Ch Rh 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 after the second boost, animals in both the alloimmunization and the autologous control group were challenged. Outcome: All 8 animals became infected with SIVmac239. Initial VLs were identical among the three groups; however, by day 43 PI, ChRh macaque plasma viremia was approximately two logs lower than what was observed in InRh, and InRh developed clinical signs leading to euthanasia starting at d. 60 PI. Among CD4, CD8, CD4/8, circulating memory T cell phenotype, CD20, and coreceptor-bearing cells (CCR5, CXCR4), only CD20 cell levels were preserved in Ch vs In Rh despite striking differences in viral control and clinical disease.Conclusions:1. Liposomal-CpG adjuvant appeared to enhance immune activation parameters associated with immunization to a greater degree than immunization with allo- vs self PBMC.2. Anti-ChRh PBMC immune responses, as indicated by MLR, IFNg production, and increase in SN titers was detected prior to challenge with SIVmac239 grown in ChRh PBMC.3. Despite development of non-self IR, all animals challenged with 400TCID50 SIVmac239 grown in ChRh PBMC developed high plasma viremia levels within 10-14 days of challenge. Pre-challenge immune activation by exposure to adjuvant may have hightened susceptibility to infection. The challenge dose administered by IV route may have exceeded any reasonable level of protection that vaccination would have afforded.4. ChRh were able to down-modulate viral infection so that by day 43 PI animals had significantly lower viral loads than InRh. While InRh succumbed to immunodeficiency disease by day 120 PI, none of the ChRh showed clinical signs by this time.5. This study is the first demonstration that the previously noted inherent resistance of ChRh to SIV infection is maintained during challenge with SIVmac239 grown in ChRh PBMC. Further, though many PBMC phenotypes were monitored during infection, only B cell numbers showed a slight correlation with the lower VL and blunted disease course of SIV disease in ChRh.
Showing the most recent 10 out of 352 publications
