Immunogenicity Studies Of Rabies Vaccines Based On The G Protein Recombinant Lentiviral Vectors

Rabies is a kind of infectious zoonotic disease caused by Rabies virus(RABV),which is still a serious public health problem in many countries.The production of traditional inactivated rabies vaccine is risky and costly.For post-exposure vaccination,more vaccination times and immunization doses are still required,and the protection effect of traditional rabies vaccine after high exposure is also limited,so it is still necessary to develop a new generation of rabies vaccine.Rabies virus G protein,as the only structural protein that can induce the production of protective neutralizing antibodies,is the focus at new rabies vaccine development.The lentiviral vector system has good potential in the development of new rabies vaccine due to its efficient gene delivery mechanism,biological safety and immune stimulation ability.In this study,several recombinant lentivirus vector vaccines based on G protein were designed and prepared,and the immunogenicity and application potential of these vaccines were explored to lay a foundation for the development of a new rabies vaccine with lower cost,fewer injections and stronger immunogenicity.In the first chapter of this study,three kinds of recombinant lentiviral vector vaccines based on the rabies virus G protein were designed and prepared based on the lentiviral vector system.The G protein gene was cloned into a lentiviral expression plasmid or a lentiviral envelope plasmid and co-transfected into HEK293T cells to package recombinant lentivirus.The specificity of the recombinant lentiviral particle antigen were detected by ELISA and Western Blot.The recombinant lentivirusmediated G protein expression in HEK293T cells were detected by Western Blot.All recombinant lentivirus were purified by sucrose density gradient centrifugation and their titers were determined by RT-PCR.The neutralization test of recombinant lentivirus was used to establish the detection method of rabies virus neutralization antibody test.The results showed that the recombinant lentiviral expression plasmid and envelope plasmid that correctly carried the G protein gene and could express the G protein in HEK 293T cells were successfully constructed.Three kinds of recombinant lentiviruses including VSV-G/LV-RABV-G,RABV-G/LV-RABV-G and RABV-G/LVeGFP were successfully prepared.ELISA and Western Blot results showed that the recombinant lentivirus RABV-G/LV-eGFP and RABV-G/LV-RABV-G can react specifically with rabies virus neutralizing antibody with good antigen specificity.All recombinant lentiviruses can successfully infect HEK293T cells.VSV-G/LV-RABV-G and RABV-G/LV-RABV-G recombinant lentivirus can mediate the expression of G protein in HEK293T cells.All recombinant lentiviral vectors were successfully purified by sucrose density gradient centrifugation.And the correlation analysis of recombinant lentivirus neutralization test showed that the titer of serum rabies virus neutralization antibody could be determined effectively.In the second chapter of this study,an experimental immunization study of recombinant lentiviral vector vaccines was conducted.Each recombinant lentiviral vector vaccine was given single intramuscular injection,single intraperitoneal injection,or booster immunization injection with a dose of 1×107 TU in mice,and serum samples were collected from the orbital venous plexus using capillary glass tubes before and after immunization.Rabies virus neutralizing antibody titers of all serum samples were detected by recombinant lentivirus neutralization test.The results showed that the VSVG/LV-RABV-G vaccine immunized by single intramuscular injection or intraperitoneal injection,and the RABV-G/LV-RABV-G vaccine immunized by single intramuscular injection could induce neutralizing antibody level higher than 0.5 IU/ml in mice 3 days after injection,and the neutralizing antibody titers remained at a relatively high level at 10 weeks after injection.At the same time,it can induce IgM antibody production in 3 and 7 days after immunization and early and persistent IgG antibody levels.RABVG/LV-eGFP vaccine can induce neutralizing antibody levels higher than 0.5 IU/ml in mice 7 days after single intramuscular immunization and last for 4 weeks,but there is no significant increase in IgM and IgG antibody levels.The RABV-G/LV-eGFP vaccine assisted by CPG-ODN adjuvant can induce neutralizing antibody levels higher than 0.5 IU/ml 7 days after single intramuscular injection in mice,and the neutralizing antibody titer still maintains a high level at 10 weeks.It could also induce IgM antibody production in 3 and 7 days after immunization and early and persistent IgG antibody levels.In addition,IgG antibody levels and neutralizing antibody titers were significantly increased in 1 week and 2 weeks after booster immunization.In summary,three kinds of recombinant lentivirus vector vaccines with different immune stimulation principles were successfully designed and prepared based on the rabies virus G protein and lentivirus vector system.The antigenicity and infectivity of each vaccine were good.High virus titer can be obtained after purification,and at the same time,a recombinant lentiviral neutralization test that can effectively determine the titer of rabies virus neutralizing antibody was established.All the recombinant lentiviral vector vaccines have great application value in single-dose vaccination and emergency exposure immunization,and were excellent candidate vaccine to replace the traditional rabies vaccine.