| Rabies is an acute zoonotic infectious disease caused by rabies virus with a fatality rate of 100 %.According to statistics,about 59,000 people die of rabies every year,most of them in Asia and Africa.China is a region with a high incidence of rabies.Rabies virus consists of an RNA genome and five structural proteins.Rabies virus attaches to the target cell by G protein and enters the peripheral cell rapidly.Rabies virus finally reaches the brain after retrograde axon transport and trans-synaptic diffusion.G protein is the main antigen to induce the generation of neutralizing antibodies,which can stimulate the immune response of the body.Therefore,rabies virus G protein(RABV-G)has been widely used as the target antigen in the development of rabies vaccine.Compared to traditional vaccines,DNA vaccines and viral carrier vaccines,mRNA vaccine preparation methods are relatively simple,relatively low cost,short production cycle,high theoretical security,and under the safe and efficient delivery carrier delivery,mRNA vaccine can induce the body to produce strong adaptive immune response.Therefore,the development of rabies mRNA vaccines is important.This research was designed to synthesize rabies mRNA vaccines,and the mRNA vaccine was delivered by a safe and effective carrier,and the immunogenicity of the vaccine was studied.This paper includes the following sections:Preparation and screening of rabies mRNA vaccines Three types of rabies virus G protein(RABV-G)nucleic acid sequence templates were designed,then RABV-G mRNA molecules were prepared by in vitro transcription,and quality of the mRNA was analyzed by using Agilent 2100 analyzer.Using commercial transfection reagent to transfect HEK-293T cells,verify the in vitro expression effects of mRNA through Western Blot.The results showed that the synthesized RABV-G mRNA was consistent with the expected results,and the purity was good.The western blot results showed that all the three RABV-G mRNA achieved in vitro antigen expression,among which the best expression of was achieved by RABV-G mRNA-1902P.So,it was selected for the subsequent immunogenicity analysis.Study on LPX-delivered rabies mRNA vaccine.Evaluation of the delivery ability of lipid nanopolymer LPX Firstly,LPX/mRNA lipid polymeric complex were prepared,and particle size,polymer dispersion index and surface potential were measured.HEK-293T cells were transfected with LPX/eGFP mRNA,and the expression of the green fluorescent protein was detected by fluorescence microscope.LPX/Fluc mRNA were intramuscularly injected into mice,and the expression of luciflucase in mice was detected by the IVIS Spectrum instrument to verify the expression ability of LPX/Fluc mRNA in vivo.HEK-293T cells were transfected with mRNA-1902P synthesized fromLPX envelope vectors,and the expression of the rabies G protein was detected by Western blot.The results showed that the average particle size of LPX/mRNA was 140.3±4.317 nm,and the Zeta potential was13.63±0.55 mV.The expression of the green fluorescent protein could be observed under fluorescence microscope,indicating that LPX/eGFP mRNA were well expressed in vitro.The bioluminescence signal of LPX/Fluc mRNA was detected by the IVIS Spectrum instrument,which indicated that the expression effect of LPX/Fluc mRNA was good in vivo.Western blot results showed that both LPX/ mRNA-1902P could be translated into the target rabies G proteins in vitro.Evaluation of immunoefficacy Effects of Rabies LPX/RABV-G mRNA vaccines Mice were immunized with LPX/ RABV-G mRNA vaccine by intramuscular injection.The immunoefficacy of the rabies mRNA vaccine was evaluated by fluorescence antibody virus neutralization test(FAVN)and ELIspot test.The antibody titers of 10 μg group and 30 μg group were45-135 IU/mL and 135-307 IU/ml respectively,which were 90-614 times of the effective protective antibody threshold(0.5 IU/ml)recognized by WHO.ELIspot assay showed that the secretion of the antigen-specific IFN-γ(Th1)and IL-2(Th1)in the splenocytes of the LPX/mRNA-1902P vaccine immunized mice significantly increased,while the secretion of IL-4(Th2)and IL-6(Th2)did not significantly change.The results showed that LPX/mRNA-1902P vaccine induced a Th1-biased antigen-specific cellular immune response in mice.Evaluation of Immunoprotective Effects of Rabies LPX/RABV-G mRNA vaccinesRabies virus was injected into the brain of mice 45 days post the first immunization,and challenge protection test was performed.During the experiment,the health condition was observed,and the weight was recorded;after the challenge experiment was completed,the number of rabies virus RNA copies in the mouse brain tissue was measured.The experimental results showed that all the mice in the vaccine group survived well and no rabies virus was detected in the mouse brain tissue.Study on LNP-delivered rabies mRNA vaccine Evaluation of the delivery ability of lipid nanopolymer LPX Firstly,LNP/mRNA lipid nanoparticles were prepared,and particle size,polymer dispersion index and surface potential were measured.HEK-293T cells were transfected with LNP/eGFP mRNA,and the expression of the green fluorescent protein was detected by fluorescence microscope.LNP/Fluc mRNA were intramuscularly injected into mice,and the expression of luciflucase in mice was detected by the IVIS Spectrum instrument to verify the expression ability of LNP/Fluc mRNA in vivo.HEK-293T cells were transfected with mRNA-1902P synthesized fromLNP envelope vectors,and the expression of the rabies G protein was detected by Western blot.The results showed the average particle size of LNP/mRNA was 89.8 ± 0.4 nm,and the Zeta potential was 10.3 ± 0.6 mV.The expression of the green fluorescent protein could be observed under fluorescence microscope,indicating that LNP/eGFP mRNA were well expressed in vitro.The bioluminescence signal of LNP/Fluc mRNA was detected by the IVIS Spectrum instrument,which indicated that the expression effect of LNP/Fluc mRNA were good in vivo.Western blot results showed that both LNP/mRNA-1902P could be translated into the target rabies G protein antigen in vitro.Evaluation of immunoefficacy of Rabies LNP/RABV-G mRNA vaccines Mice were immunized with LNP/ RABV-G mRNA(5 μg)vaccine by intramuscular injection.The immunoefficacy of the rabies mRNA vaccine was evaluated by fluorescence antibody virus neutralization test(FAVN).Fluorescence antibody virus neutralization showed that the neutralizing antibody titers were higher than 0.5 IU/mL at 5 days post first immunization and the neutralizing antibody titers were 45-177.7 IU/mL.Evaluation of Immunoprotective Effects of Rabies LNP/RABV-G mRNA vaccinesRabies virus was injected into the brain of mice 45 days post the first immunization,and challenge protection test was performed.During the experiment,the mouse health condition was observed,and the weight was recorded;after the challenge experiment was completed,the number of rabies virus RNA copies in the mouse brain tissue was measured.The experimental results showed that all the mice in the vaccine group survived well and no rabies virus was detected in the mouse brain tissue.Conclusion: This study successfully prepared rabies mRNA vaccine,which can induce specific humoral and cellular immune response in mice. |
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