Studies On Rabies Virus MRNA Vaccine And Its Delivery System

Rabies(Rabies)is a serious and acute zoonotic infectious disease,there were about 59,000 people die of rabies each year,especially in Asia and Africa.Rabies virus(RABV)is the causative agent of rabies,which consists of structural proteins and negative-strand RNA.Glycoprotein(G)is an important component of RABV and the primary antigen of RABV,it can stimulate the body to produce a specific immune response.Rabies is extremely bad,once it develops,the prognosis is very poor.Vaccination is still the most effective strategy to prevent and control the spread of rabies virus.The production of traditional rabies inactivated vaccines is expensive and the availability is also low in remote areas;The new rabies DNA vaccines and virus vector vaccines have potential safety risks;m RNA vaccines are a new generation of nucleic acid vaccines,which are rapid,cheap,safe and effective,They can also stimulate the body to produce a strong immune response with effective delivery vectors.Therefore,the development of rabies m RNA vaccine has important application value.Hepatitis B virus core protein virus-like particles(HBc VLPs)are 21-hedral hollow nanoparticle composed of hepatitis B virus core protein(HBc),which can be controlled to assemble,disassemble and display the epitope.The development of HBc VLPs vectors has broad research prospects.Tuftsin is a physiologically active tetrapeptide produced by the body’s spleen tissue with a strong ability to strengthen the immune system.The single Tuftsin peptide is short,and it is easy to degrade in vivo.The stability of Tuftsin complex is higher,it also has important clinical application value.Tuftsin chimeric HBc VLPs gave functionalized Tuftsin-HBc VLP nanocarriers.c KGM is a cationized mannose polymer that can specifically bind to nucleic acids to form nano-scale dense complexes.It can also bind to mannose receptors on cell membranes and has mannose receptor targeting capabilities.As a delivery vector targeting dendritic cells,c KGM has broad application prospects.In this study,we designed and prepared rabies m RNA vaccine and Tuftsin-HBc VLP,c KGM vector,used c KGM vector to deliver the m RNA vaccine,and analyzed the immunogenicity of the vaccine.1.Design,preparation and in vitro expression detection of rabies m RNA vaccineFour kinds of rabies virus G protein nucleic acid sequences were designed: RABV-A,RABV-B,RABV-C,RABV-D,and cloning vectors were constructed.RABV G m RNA was prepared in vitro for qualitative and quantitative analysis.Western blot results showed that RABV-C m RNA could be translated and expressed in vitro,laying a foundation for the study of immunogenicity of later m RNA vaccine.2.Preparation and detection of Tuftsin-HBc VLP and c KGMThe Tuftsin-HBc VLP nucleic acid sequence was designed,and the PET43.1a-Tuftsin-HBc VLP plasmid was constructed.E.coli BL21(DE3)was used as the host strain,Isopropyl β-D-Thiogalactoside(IPTG)was used for the expression of Tuftsin-HBc VLP.Tuftsin-HBc VLP was purified and characterized.Gel blocking results showed bright bands in the gel pores of the VLP / EGFP m RNA complex,suggesting that VLP could contain m RNA.No generation of green fluorescent protein was observed in 293 T cells in vitro,it suggested that the vector may not be suitable for delivery of m RNA.3.Preparation and detection of c KGM carrier CKGM-EGFP m RNA nanoparticles were prepared.Gel blocking results showed that c KGM / EGFP m RNA was retained in the wells.Potential and structural characterization showed that c KGM / EGFP m RNA was positively charged and was round or oval.The expression of green fluorescent protein was observed in 293 T cells after the transfection of ckgm-egfp m RNA nanoparticles..4.Study on the immunogenicity of rabies m RNA vaccineUsing c KGM vector,c KGM/m RNA vaccine was inoculated intradermally into female BALB/c mice 3 times,and the serum was tested by ELISA and FAVNT.The ELISA results displayed that there was specific Ig G antibody in the serum of the m RNA vaccine group of mice.The FAVNT results showed that specific virus neutralizing antibodies were detected in the serum of the mice of the c KGM/m RNA vaccine group;the ELISpot results of IFN-γ showed that splenocytes of the mice in the c KGM/m RNA vaccine group produced more spot forming cells(SFCs),after surface vaccination,the mouse spleen cells were activated,suggesting that the mice produced a strong immune response,especially cellular immune response.