Development Of A Subunit Vaccine And Monoclonal Antibodies Based On Nipah Virus F Protein Domain Ⅲ

Nipah virus(Ni V)is a single negative-strand RNA virus belonging to the genus Henipavirus of the Paramyxoviridae family.The fatality rate of Ni V infection is as high as 40%-75%.It belongs to the fourth-level biosafety pathogen,but there is still no vaccines and therapeutic antibodies available.The most vaccines under research are live vector vaccines or eukaryotic subunit vaccines expressed in mammalian cells which are both costly.Given that the areas where Ni V frequently occurs are mainly in economically underdeveloped countries,low cost and low price have become important aspects of Ni V vaccine research and development.The development of highly effective therapeutic antibodies is also an urgent need for rapid and efficient control of potential Ni V outbreaks and epidemics.In this study,the most critical third domain(Domain Ⅲ,DⅢ)of Ni V F protein was selected as the target for the preparation of subunit vaccines by using prokaryotic expression system and for the screening of monoclonal antibodies with neutralizing activity.First,by introducing four point mutations of L110 N,I114N,V118 N,and I120 N into the fusion peptide and optimizing the expression conditions,a large amount of soluble DⅢ protein expressed in the prokaryotic system was achieved.The purified four-point mutant protein DⅢM4 was used to immunize BALB/c mice and then was found that it can only induce binding antibodies with no neutralizing activity.This result suggested that the pre-fusion conformation of DⅢM4 was imperfect and its stability was poor.In view of this,the 5B3 monoclonal antibody which can recognize the pre-fusion conformation of F protein DⅢ,was used as a tool to establish an ELISA detection system to identify the pre-fusion conformation and stability of DⅢ.In addition,the fusion protein DⅢM4-3L-Foldon was constructed by connecting the Foldon motif to the C-terminus of DⅢM4 with a flexible connecting peptide.Through this way,DⅢM4 was successfully trimerized to mimic the DⅢ in the F protein trimer which can be formed under natural conditions.The experimental results showed that the pre-fusion conformation and stability of the trimer have been significantly improved compared to the monomer.After immunizing the mice,it was found that although the trimer can induce the production of neutralizing antibodies,only a small part of the mice can produce highly effective neutralizing antiserum,and the remaining mice had no neutralizing antibody responses.Therefore,the interchain disulfide bond mutations were further designed and introduced in the Foldon to stabilize the conformation of the trimer.An immunogen superior to DⅢM4-3L-Foldon called DⅢM4-3L-Foldon C1 with A14C-K18 C mutation was screened out.By contrast,DⅢM4-3L-Foldon C1 had significantly improved the quality and quantity of neutralizing antiserum of immunized mice,but it still had the problem that a part of mice cannot produce effective neutralizing antiserum.Finally,we tested a combination strategy of priming with full-length F DNA and boosting with DⅢM4-3L-Foldon C1 protein twice.And the results showed that 100% mice can produce efficiently neutralizing antibody responses.At the same time,we selected a mouse with efficient neutralizing antiserum after immunized with DⅢM4-3L-Foldon,then screened out 3 monoclonal neutralizing antibodies(1C7,2B12,8A2)and 2 monoclonal binding antibodies(9G5,10H7)by hybridoma technology.The variable region sequences of these monoclonal antibodies were obtained through gene cloning and amplification,and then human-mouse chimeric antibodies were constructed.The experimental results showed that the 8A2 monoclonal antibody had a neutralizing activity equivalent to that of the 5B3 monoclonal antibody,but had a different specific recognition epitope.In conclusion,this study constructed a prokaryotically expressed DⅢM4-3L-Foldon C1 candidate Ni V vaccine targeting F protein DⅢ,which can induce high-efficiency neutralizing antibody responses by boosting immunization twice on the basis of F DNA primary immunization.At the same time,a monoclonal antibody 8A2 with significant neutralizing activity was screened out by hybridoma technology.