Construction Of A Novel Genetic Engineering Vaccine Against Nipah Virus And Evaluation Of Immune Effect In Mice

Background Nipah virus(Ni V)is a zoonotic virus.Through the analysis of Nipah virus genome,Nipah virus belongs to single strand negative strand RNA virus order and Paramyxoviridae.Compared with other members of Paramyxoviridae,NIV genome and Hendra virus(He V)genome have high homology,so they are classified as genus henipavirus.In recent years,three new types of hennipa virus have been found,namely Ghana virus found in Kumasi,Ghana,cedar virus found in Cedar Grove Town,Queensland,Australia,and Mojiang virus found in Mojiang Hani Autonomous County,Yunnan Province,China.From 1998 to 1999,Nipah virus broke out for the first time in the world.When the epidemic caused one million local pigs in Malaysia to be slaughtered,more than 100 people died of infection,and the case fatality rate was 48%.Since it was discovered,Nipah virus and Hendra virus have broken out locally in the Asia Pacific region for many times.In 2018,the virus was listed as one of the key diseases that can cause serious international outbreaks by the "World Health Organization(WHO)R & D blueprint",and it belongs to the fourth most dangerous level in terms of biosafety.Vaccine is a powerful means to prevent infectious diseases caused by virus.At present,there are a variety of Nipah virus vaccines in the research stage,including recombinant protein vaccine,vesicular stomatitis virus vector vaccine,measles virus vector vaccine,poxvirus vector vaccine,etc.,but no human vaccine has been approved for marketing.Objective In this study,non replicating vaccinia vector vaccine and subunit vaccine expressing Nipah virus fusion protein(F)and attachment protein(G)were constructed.Different immune strategies were used to evaluate the specific humoral and cellular immune responses induced by Nipah virus fusion protein(F)and attachment protein(G)in BALB/c mouse model,so as to provide scientific reference and technical support for the development and application of Nipah virus genetic engineering vaccine in China.Methods and Results 1.Construction and expression identification of non replicating vaccinia virus vector vaccine and subunit vaccine of Nipah virus Plasmid pc DNA3.1-F and pc DNA3.1-G containing target antigen,which expresses the fusion protein(F)and attachment protein(G)of Nipah virus Malaysia strain,and the sequence is optimized by mammalian codon preference.By designing primers,the target antigen was amplified by PCR to obtain the target gene fragment.The target gene fragment was inserted into the homologous recombinant plasmid p JSC1175-lac Z,and homologous recombination was carried out with NTV in chicken embryo fibroblasts.The recombinant non replicating vaccinia virus vaccine(NTV-F,NTV-G)was screened and purified by blue and white spots.The successful expression of F protein and G protein was verified by Western blotting.In order to obtain soluble Ni V F and G protein,we first delete the membrane anchoring region of F protein and G protein to effectively secrete them outside the cell.The 1-488 amino acid sequence of Ni V F protein was retained,and in order to stabilize its pre fusion conformation,we mutated the amino acid site.In order to increase its immunogenicity,trimer domain(TD)was added after residue 488 aa to form trimer.NIV G protein is type II transmembrane protein,and its N-end is located in the membrane,so its 172-602 amino acids were retained,and 6 amino acids were added at the C-end After his labeling,the eukaryotic expression plasmid was inserted to purify it by nickel column affinity chromatography.Through SDS-PAGE and Western blot identification,soluble F protein and G protein with purity ≥ 90%,namely s F and s G,were obtained,which are intended to be used as subunit vaccine.2.Specific immune response induced by DNA vaccine and NTV vaccine BABL/c mice aged 6-8 weeks were immunized to evaluate the specific humoral immune response induced by the vaccine.ELISA was used to detect specific Ig G antibody in mouse serum,construct HIV based Nipah pseudovirus,and establish Ni V neutralizing antibody detection that can be carried out in biosafety secondary Laboratory(BSL-2).The results showed that pc DNA3 1-F、pc DNA3.1-G,NTV-F and NTV-G induced specific Ig G antibodies with high titers when immunized with homologous and heterologous vaccines and combined immunization with two antigens.Among them,the Ig G antibody induced by DNA NTV immunization scheme was the highest,and the antibody induced by each immune group could neutralize HIV-Ni V pseudovirus,including the neutralizing antibody titer induced by pc DNA3.1-G+NTV-G immunization group was the highest,and the neutralizing antibody titer induced by combined immunization of F + G antigen was higher than that of single antigen immunization group.At the same time,we evaluated the specific cellular immune response induced by the vaccine.Two weeks after booster immunization,mice were killed to isolate splenocytes,and their IFN-γ was detected by ELISPOT.The results showed that the vaccine induced a strong CD8 + T cell immune response in mice,and the T cell immune response induced by dna-ntv immunization group was stronger than that of other groups,with significant difference.3.Screening Ni V-F,Ni V-G specific H-2d restricted T cell epitopes 119 peptides covering the full length of Ni V-G and 108 peptides covering the full length of Ni V-F were designed and synthesized(the front and rear peptides overlap 10 amino acids,step forward with 5 amino acids,and each peptide segment contains 15 amino acid sequences).The spleen cells of mice immunized by each immune group were taken,and the Ni V-G and Ni V-F peptide pools designed by matrix were used as stimulants.ELISPOT was used to detect the specific dominant peptides of G protein and F protein,The CTL epitope peptides with strong stimulating activity were determined by matrix analysis.F protein screened 12 dominant peptides,of which F56(IIVRVYFPILTEIQQ)had the strongest stimulating activity,and G screened 12 dominant peptides,of which G72(IKQGDTLYFPAVGFL)and G73(TLYFPAVGFLVRTEF)had the strongest stimulating activity.4.Immunogenicity of recombinant subunit vaccine BABL/c mice aged 6-8 weeks were immunized to evaluate the immune response induced by recombinant proteins s F and s G combined with different adjuvants.The specific Ig G antibody in mouse serum was detected by ELISA,the neutralization antibody level induced by immunization was detected by micro neutralization test,and the specific cellular immune response induced by vaccine was evaluated by ELISPOT test.The results showed that the effect of aluminum adjuvant combined with Cp G was better than that of aluminum adjuvant or Cp G alone.The mice immunized with recombinant protein SF induced better humoral immune response and cellular immune response,and the mice immunized with recombinant protein s G induced better humoral immune response,but the efficiency in stimulating cellular immune response was low.The neutralizing antibody titer induced by s F+s G combined with aluminum adjuvant combined with CPG is the highest,which proves again that the neutralizing protective effect induced by simultaneous immunization of F and G antigens is better than that of single antigen.Conclusion We successfully constructed the non replicating vaccinia virus vector vaccine by using the DNA vaccine expressing the target antigens of Nipah virus F and G,purified and prepared the recombinant subunit vaccines of Ni V s F and Ni V s G,evaluated the immune response of the three vaccines in BALB/c mice with the strategy of homologous,heterologous and combined immunization of two antigens,and selected the specific H-2d epitope peptides of F protein and G protein.The specific Ig G antibody level and T cell immune response induced by DNA NTV immunization group were higher than those in other immunization groups,the neutralizing antibody titer induced by pc DNA3.1-G+NTV-G immunization group was the highest,and the neutralizing antibody titer induced by combined immunization of F and G antigen was higher than that of single antigen immunization group.The effect of recombinant protein combined with aluminum adjuvant and Cp G was better than that of aluminum adjuvant or Cp G alone.Both recombinant proteins SF and SG induced better humoral immune response,and SG protein was inferior to SF protein in stimulating T cell immune response.The neutralizing antibody titer induced by s F+s G combined with aluminum adjuvant combined with Cp G is the highest,which proves again that the neutralizing protective effect induced by simultaneous immunization of F and G antigens is better than that of single antigen.