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P01.14

Epitope competition could influence the kinetics of specific CD8+ T cell reponses during repeated DNA vaccination


Epitope competition could influence the kinetics of specific CD8+ T cell responses during repeated DNA vaccinationYanqin Ren1#, Yanmin Wan1#, Jing Wang1,2, Jianqing Xu1,2* 1Scientific Research Unit, Shanghai Public Health Clinical Center, Fudan University, Shanghai, China;   # authors contribute equally2Key Laboratory of Medical Molecular Virology, Shanghai Medical College & Institute of Biomedical Sciences, Fudan University, Shanghai, China;  * corresponding author
Abstract:
How to improve the immunogenicity of DNA vaccine is a topic discussed for a long time. During repeated DNA vaccine immunization, the T cell responses revealed to be not match with the expectations. Thus, some strategies trying to induce more width T cell response, in addition to pursuing high level of frequency of T cell responses. To understand the mechanisms lie in the repeated DNA vaccine immunization may help us to design more efficient HIV-1 vaccine, and one of the most important mechanisms among these is why the same immunogen would induce different frequency of T cell responses in different vaccine components.  One of the answer is competition! Here we designed, synthesized and tested two CD8+ T cell epitope, to elucidate the influences of epitope competition on specific T cell responses and learn the kinetics of epitope competition during repeated DNA vaccination. We immunized mice with single epitope vaccine or mixed epitope vaccine or fusion epitope vaccine, and we collected peripheral blood  after every vaccination and detected the frequency of IFN-γ+CD8+ T cells. Our results showed that epitope competition became anabatic during repeated DNA vaccination in fusion epitope vaccine group,  and epitope competition could influence the kinetics of epitope specific CD8+ T cell responses. In addition, separate expression of different epitopes may help to balance the magnitudes of T cell responses among different epitopes.
Introduction:
Previous study suggested that specific T cell responses against HIV-1 Gag were suppressed when being co-expressed with HIV-1 Env, while Gag itself has strong immunogenicity, which suggests that there may exist competition between HIV-1 Gag and Env when co-expressing. Epitope competition was postulated to be one of the underlying mechanisms. In our previous study, we identified several dominant and/or non-dominant CD8+ T cell epitopes, so we constructed a dominant epitope vaccine and a non-dominant epitope vaccine. The objective of our current study is to elucidate the influences of epitope competition on specific T cell responses and learn the kinetics of epitope competition during repeated DNA vaccination.
Method:
DNA vaccines: vaccines expressing Gag92, Env203 or Gag92/Env203 fusion epitope (Gag92 and Env203 were mouse T cell epitopes identified in our previous work) were synthesized and cloned to pSV1.0.
Mice: C57BL/6 mice were immunized with 100ug single epitope DNA vaccines(pSV-gag92 or pSV-env203), or 100ug fusion epitope DNA vaccine(pSV-gag92/env203) or the mixed single epitope DNA vaccines(50ug pSV-gag92+50ug pSV-env203) by intramuscular immunization every two weeks.
Peripheral blood collection: eye venous blood were collected into sodium citrate anticoagulation. Then, cracked the red blood cells with RBC lysis buffer, and washed white blood cells with R10, and do cell counting after that.
Spleen: after all the vaccination on the schedule, vaccinated mice were sacrificed with leading strength dislocated. spleen were gained and grinded with gauze, and then cracked the red blood cells with RBC lysis buffer, washed with R10, and then count the white blood cells. 1×106 cells per well were placed into 96-well round bottom plate and stimulated with Gag92 peptide or Env203 peptide at 37 ℃,5% CO2 for 7h.
Intracellular cytokine staining: Epitope specific CD8 T cell responses were analyzed by the method of intracellular cytokines staining. After stimulation with peptides, cells were stained with surface marker---rat anti-mouse CD3(PerCP/Cy5.5), rat anti-mouse CD8(Pacific Blue), and intracellular marker---rat anti-mouse IFN-γ(PE). After staining, cells were detected with BD FACSria II.
Statistics analysis: flow cytometry data was analyzed with FlowJo VX.0.6. column statistics were calculated by mean ±SD. Comparisons between two groups were done by the method of unpaired t test and comparisons among three or more groups were done by using the method of one way ANOVA(GraphPad Software, Inc.). Significant difference was defined as P≤0.05.
Results:
To enhance the immunogenicity of single epitopes, we designed 6×gag92 and 6×env203 derived from RL42 subtype of HIV-1, and fusion gene gag92/env203 were synthesized with the same strategy. In order to understand the dynamic of epitope competition elicited by fusion gene, four groups of female C56BL/6 mice were vaccinated with single epitope vaccine (pSV-gag92 or pSV-env203), or mixed epitope vaccine (pSV-gag92+pSV-env203), or fusion epitope vaccine (pSV-gag92/env203). A fifth group of mice were inoculated with empty plasmid vector as mock control. Vaccination Schedule was illustrated in Table 1.
1. Competition existed between two epitopes, and Env203 is a dominant epitope, while Gag92 is a non-dominant epitope.  After 4-time immunization with epitope vaccines, we sacrificed the mice, gained the splenocytes and detected the frequency of epitope specific T cell response(IFN-γ) with intracellular cytokine staining.  We found that despite the mock control, all group of mice show that the frequency of Env203 specific T cell response is significantly higher than that of Gag92, which indicate that Env203 is a dominant epitope, while Gag92 is a sub-dominant epitope. Moreover, data from fusion epitope vaccine group(blue dots) suggested that there were competition between the two epitopes(Fig. 1).
2. Epitope competition became anabatic during repeated DNA vaccination in fusion epitope vaccine group. We collected the peripheral blood of vaccinated mice and assayed the epitope specific T cell response on 9th or 10th day after every intramuscular immunization. The frequency of Env203-specific IFN-γ+CD8+ T cell[(1.001±0.2676)%] was significantly higher than Gag92-specific IFN-γ+CD8+ T cell[(0.1563±0.09369)%] in pSV-gag92/env203 immunizing group. While, no significant difference was observed in the group immunized with mixed DNA vaccines (pSV-gag92+pSV-env203). Moreover, the mixed single epitope DNA vaccines could induce higher rate of Gag92-specific IFN-γ+CD8+ T cell response[(0.4220±0.4497)%]than pSV-gag92/env203.
3. Epitope competition could influence the kinetics of epitope specific CD8+ T cell responses during repeated DNA vaccination. Dynamic observation showed the specific IFN-γ+CD8+ T cell responses against non-dominant epitope(Gag92) could not be efficiently boosted during repeated vaccination of pSV-gag92/env203 fusion gene vaccine and only dominant epitope(Env203) specific IFN-γ+CD8+ T cell responses could be improved. While, the mixed single epitope DNA vaccines could induce much more balanced CD8+ T cell responses against both epitopes.
Conclusion:
Epitope competition could significantly decrease the frequency of specific T cell response against non-dominant epitope. This is true not only at the end but also during the process of repeated DNA vaccination. And separate expression of different epitopes may help to balance the magnitudes of T cell responses among different epitopes.