Heterologous Immunity Between HBV And MCMV

Objective1. To develop HBV infection mouse models and explore the mechanisms involved in HBV clearence2. To investigate the possible interference between a resolved HBV infection and a sequential acute MCMV infection and the related immunological mechanisms3. To investigate the possible interplay between a persistent HBV infection and a sequential acute HBV infection and the related immunological mechanismsMethods1. Ten ug pAAV/HBV1.2was hydrodynamically injected into6-8wks old male C57BL/6mice from different providers (Harlan、DEREG、ZTL、Charles River) via the tail vein. Serum HBsAg levels were monitored at different time points.2. By hydrodynamic infection,2ug,5ug, and lOug pAAV/HBV1.2were injected into three groups of Harlan mice through the tail vein, respectively. Serum HBsAg levels were monitored at different time points.3. Three groups of Harlan mice were transfected10ug of pAAV/HBV1.2, its variants with mutantions in the HBV core region (HBc150, HBc175), and another plasmid pSM2with different plasmid backbone to pAAV/HBV1.2and two tandem whole HBV genomes, respectively. Serum HBsAg levels were monitored at different time points. Intracellular IFNy staining combined with cell surface marker staining was used to detect frequencies of HBV specific T cells in PBMC of pAAV/HBV1.2or pSM2transfected mice at different time points.4. Harlan mice were hydrodynamically injected with10ug of pAAV/HBV1.2, and6weeks later were sequentially injected i.p. with5×104PFU MCMV in100ul PBS. Serum HBsAg levels were monitored at different time points. One week post MCMV injection splenocytes were harvested to detected MCMV and HBV specific T cell frequencies and the supernatant of the homogenized livers was used to determine MCMV titers by plaque assay.5. Intracellular cytokine staining in combination with H-2b dimer or pentamer staining after coated with MCMV or HBV specific peptide were performed to determine if there existed cross-reactivities between HBV and MCMV.6. Harlan mice were injected i.p. with1×105PFU MCMV in100ul PBS, and6weeks later were sequentially hydrodynamically injected with10ug of pSM2. Serum HBsAg levels were monitored at different time points. Sixteen days post pSM2transfection splenocytes were harvested to detected frequencies of NK and Treg cells as well as MCMV and HBV specific T cell; the supernatant of the homogenized livers was used to determine MCMV titers by plaque assay.7. NK cells, CD8cells and INF-γ were blocked separately in pSM2transfected mice using their specific monoclonal antibodies. Serum HBsAg levels and HBV specific T cell responses were monitored at different time points.Results1. Serum HBsAg of the C57BL/6from different providers could be detected one day post pAAV/HBV1.2transfection, peaked at4d.p.i. or1w.p.i., and then declined. Six and seven weeks later serum HBsAg were not detectable any more from Harlan and DEREG mice. HBsAg antigenemia persisted until8w.p.i. in approximately50%ZTL and Charles River mice.2. All the Harlan mice separately transfected with2ug,5ug, and lOug pAAV/HBV1.2were eliminated of the serum HBsAg5weeks after the plasmid transfection and there were no significant differences in the clearance peed of serum HBsAg.3. Around75%of the pAAV/HBV1.2transfected Harlan mice clear serum HBsAg4weeks after transfection, and it happened for66%of HBc150and HBc175transfected mice3w.p.i and5w.p.i, respecitively. However, all of the pSM2transfected mice cleared their serum HBsAg within2weeks.4. HBcAg and HBsAg specific T cell responses could be detected in PBMC of pSM2transfected Harlan mice at2w.p.i., and the responses to HBcAg were significantly higher that those to HBsAg. In mice which were injected with pAAV/HBV1.2and its mutants such immune responses can not be detected. 5. There were not significant differences in the frequency of MCMV specific T cells and MCMV titers in the liver between mice with resolved HBV infection and naive mice following acute MCMV infection. Seven weeks after HBV transfection and2weeks after clearance of serum HBsAg, HBV specific T cell responses were detected in the spleen of some MCMV infected mice, but not in only HBV transfected mice. Splenocytes from some MCMV-infected mice showed cross-reactivity between the HBV specific peptide c1and the MCMV specific peptide c1, but this phenomemon was not observed in HBV-immune splenocytes by using peptide coated H-2b dimer or pentamer staining.6. Serum HBsAg of pSM2tranfected naive and MCMV persistent Harlan mice increased rapidly after pAAV/HBV1.2transfection, peaked at4d.p.i., and then declined gradually. MCMV persistent mice cleared serum HBsAg slower and their serum HBsAg levels at9,11d.p.i. were higher than those of the naive mice. The difference in serum HBsAg at9d.p.i. was significantly different. There was no significant difference in MHC expression on macrophages (F4/80+). However, the frequencies of IFNy-producing HBV c1specific T cells and NK cells were significantly lower in pSM2transfected MCMV persistent mice than in only pSM2transfected mice. Blockade of NK or IFNysignificantly enhanced srum HBsAg levels, and blocking IFNy also reduced HBV specific T cell responses. Additionally, Tregs were significantly higher in pSM2transfected MCMV persistent mice than in only pSM2transfected mice.7. There were no significant differences in MCMV specific T cell number and MCMV titers in the spleen between HBV-MCMV co-infected mice and mice infected with MCMV alone.Conclusions1. Mouse genetic background and plasmid backbone or the length of HBV insert determines HBV persistence. Because of private specificity of TCR repertoires, even the same strain mice with different providers show different capacity of clearing HBV infection. HBV specific T cell responses are main effectors for HBV clearance and determine the duration of HBV persistence.2. HBV infection has no effect on the immunity to and outcome of MCMV infection. However persistent MCMV can influence the immunity and outcome of sequentially acute HBV infection in two ways:one is that MCMV-immune splenocytes can protect the host against HBV infection through cross-reactive T cells, but it is not the case in the other way around. The other is that after infection with HBV, MCMV persistent mice can decrease the numbers of NK and HBV specific T cells, thereby reducing the secretion of IFNγin the serum and slowering HBV clearance ultimately. This phenomenon may be involved with increased Tregs in MCMV persistent mice, which depress the function of HBV specific T cell responses.