Evaluation Of The Adjuvant Effect Of Toll-like Receptor 7/8 And 9 Agonists In Chimeric DNA Vaccine Against Japanese Encephalitis

Objective:Japanese encephalitis is an acute infectious disease caused by Japanese encephalitis virus,which is a serious challenge to public health.There are no specific therapeutic medications available,so the vaccination remains an important measure to prevent Japanese encephalitis.In our previous study,we constructed the pJME using the genes encoding pr M and E proteins,the major structural antigenic proteins of Japanese encephalitis virus.On the basis of this,we embedded a gene fragment of LC3B and constructed a pJME-LC3 chimeric DNA vaccine which induced the autophagic pathway.In this study,we investigated the effect of TLR7/8 agonist imiquimod and TLR9 agonist Cp G ODN 1826 as vaccine adjuvants on the immune efficacy of Japanese encephalitis DNA chimeric vaccine,which provides a new idea for the development of DNA vaccine.Methods:Fifty clean healthy female Balb/c mice(4-6 weeks old)were selected and housed in a standard SPF grade pathogen-free animal laboratory.After one week of acclimatization,they were randomly and equally divided into 5 groups.The groups were divided as follows:pc DNA3.1(+)as a blank control group,pJME-LC3 as a control group,pJME-LC3+Imiquimod as an experimental group,pJME-LC3+Cp G as an experimental group,pJME-LC3+Imiquimod+Cp G as the experimental group.The immunization doses of pc DNA3.1(+)and pJME-LC3 were 100μg/each,the imiquimod was 50μg/each and the Cp G was 10μg/each.The vaccine and adjuvant mentioned above were injected intramuscularly into the quadriceps muscles of the hind limbs of mice bilaterally and immunized three times at two weeks intervals.One week after the first immunization,three mice in each group were anesthetized and executed,and the inguinal lymphocytes were aseptically isolated.The inguinal lymphocytes were isolated aseptically,and the ability of vaccine adjuvant to induce recruitment/activation of DCs and B cells in secondary lymphoid organs of mice was measured by flow cytometry.One week after the final immunization,the serum and the splenocytes were aseptically isolated,and the proportion of spleen regulatory T cells in immunized mice was detected by flow cytometry.After the antigen stimulation of splenic lymphocytes,cell culture supernatants were collected and the levels of cytokines(IFN-γ,IL-2,IL-4 and IL-10)were measured by ELISA and the secretion levels of nitrite in the supernatant were detected by Griess method.The lymphocytes were collected,the activation of CD4~+/CD8~+T lymphocytes and the ratio of CD4+/CD8+central memory T cells in immunized mice wer detected by flow cytometry,also the CCK-8 assay was used to detect the proliferative capacity of splenocytes.Results:1.pJME-LC3 combined with the vaccine adjuvant promoted the activation of dendritic cells and B cells in secondary lymphoid organs during early immunization:Compared with the pJME-LC3 control group,the proportion of CD11c~+MHC II~+and CD19~+CD40~+double-positive cells in the inguinal lymphocytes of pJME-LC3+IMQ+Cp G group was significantly increased(P<0.05).2.pJME-LC3 combined with the vaccine adjuvant induced high levels of CD4~+/CD8~+T cell activation:For the CD4~+T lymphocyte activation levels,compared to the pJME-LC3 group(3.22±0.20)%,the percentage of CD4~+CD69~+double positive cells was significantly higher in the pJME-LC3+IMQ group(4.49±0.27)%,pJME-LC3+IMQ+Cp G group(5.60±0.56)%,and the difference between groups was statistically significant(P<0.05).For the CD8~+T lymphocyte activation levels,the percentage of CD8~+CD69~+double positive cells in the pJME-LC3+IMQ+Cp G group was(4.54±0.48)%,which was significantly higher than that in the pJME-LC3 group(2.07±0.19)%,the pJME-LC3+IMQ group(2.62±0.17)%and the pJME-LC3+Cp G group(2.97±0.30)%(P<0.05).3.pJME-LC3 combined with the vaccine adjuvant promoted the secretion of Th1-type cytokines from splenic lymphocytes:The use of IMQ and Cp G as pJME-LC3 vaccine adjuvants alone or in combination significantly promoted the secretion of Th1-type cytokines(IFN-γand IL-2)from the spleen lymphocytes,also the combination of IMQ and Cp G as adjuvants showed a synergistic effect.However,for the Th2-type cytokines,the differences in IL-4 expression between groups were not statistically significant(P>0.05),and the combination of IMQ and Cp G significantly inhibited the IL-10 expression in mouse spleen lymphocytes(P<0.05).4.pJME-LC3 combined with the vaccine adjuvant promoted the proliferation of spleen lymphocytes:The use of IMQ alone or in combination with Cp G as vaccine adjuvant significantly increased the proliferation index of splenocytes compared to the value-added index of pJME-LC3 control group(1.74±0.15)%(P<0.05).5.pJME-LC3 combined with the vaccine adjuvant stimulated the innate immune system:Compared with the pJME-LC3 group(10.01±1.47)μM,the nitrite in spleen cell culture supernatants of the pJME-LC3+IMQ group(13.57±0.59)μM,pJME-LC3+Cp G group(14.00±1.38)μM and pJME-LC3+IMQ+Cp G group(17.41±0.67)μM concentrations were significantly increased(P<0.05).6.pJME-LC3 combined with the vaccine adjuvant reduced the percentage of spleen regulatory T cells:Compared with the pJME-LC3 group(11.03±0.72)%,the proportion of Tregs in the pJME-LC3+IMQ group(9.06±0.45)%and the pJME-LC3+IMQ+Cp G group(8.63±0.36)%was significantly decreased(P<0.05).7.pJME-LC3 combined with the vaccine adjuvant promoted the percentage of CD4~+/CD8~+central memory T cells:For CD4~+TCM levels,the proportion of CD4~+TCM in pJME-LC3+Cp G group(20.17±1.92)%and the pJME-LC3+IMQ+Cp G group(26.10±3.30)%were significantly higher compared to the pJME-LC3(14.10±1.11)%group(P<0.05).For CD8~+TCM,the percentage of CD8~+TCM in the pJME-LC3+IMQ+Cp G group was(11.63±1.33)%,which was significantly higher than that in the pJME-LC3 group,pJME-LC3+IMQ and pJME-LC3+Cp G(P<0.05).8.pJME-LC3 combined with the vaccine adjuvant promoted the production of serum JEV-specific Ig G2a antibodies in immunized mice:For serum JEV-specific Ig G2a antibodies,IMQ in combination with Cp G was able to induce a statistically significant increase in Ig G2a antibody levels compared to the pJME-LC3 DNA vaccine group alone(P<0.05).Conclusion:1.The pJME-LC3 DNA vaccine combined with IMQ and Cp G ODN 1826vaccine adjuvant contributed to the activation of the innate immune system,promoting the maturation and activation of antigen-presenting cells in the early stages of immunity.The combination immunization strategy modulates and optimizes the subsequent antigen-specific immune response,generating an effective cellular and humoral immune response and providing a long-lasting immune protection.2.The TLR7/8 agonist imiquimod and TLR9 agonist Cp G ODN 1826 showed a certain adjuvant synergy as adjuvants for the recombinant DNA vaccine against Japanese encephalitis.