Study On Multiepitope Vaccine For WNV

West Nile virus (WNV ), a member of the family Haviviridae, genus Havivirus, causes a febrile illness that can lead to fatal meningitis or encephalitis in humans, horses, and birds. WNV was first isolated from a febrile woman in the West Nile district of Uganda in 1937. Afterwards WNV infections were reported in Africa, Middle East, Western Europe, Central Asia and other areas. The ability of West Nile virus to infect birds, and to be carried in mosquitoes, has allowed it to spread at an alarming pace. From 1999, WNV epidemic appeared in New York and spread to different areas of United States. According to the reports of Center for Disease Control (CDC) of United States, human WNV cases reached to 9175 and 230 dead in 2004. So far, there are no effective treatments or vaccines approved for clinical use against West Nile virus and only the inactivated vaccines were used in equines.Epitopes are the antigenic determinants, which are recognized by the T-cell receptors and B-cell receptors. T-cell epitopes, as presented in the context of the MHC molecule and recognized by the T-cell receptor, are the minimal essential units that stimulate cellular (T cell) immune responses. B-cell epitopes, as presented on the external surface of antigen and recognized by the B-cell receptor, stimulate humoral (B cell) immune responses. The ideal antigens include T-cell epitopes and B-cell epitopes, which can stimulate complete immune responses, and educe its effects of prevention and treatments. Epitope vaccine became the emphasis of noval virus vaccines in recent years. However, there are no research on WNV epitopes vaccines reported so far.In view of above consideration, a recombinant WNV plasmid was constructed in this study to investigate its potential as a candidate multiepitope DNA vaccine to prevent WNV infection. The potential multiepitopes were determined through analysis of WNV complete cDNA sequence. Multiepitopes genes were constructed by seriesly connecting the preference epitopes. The codon-optimized multiepitopes genes were cloned into prokaryotic plasmids and expressed in Escherichia coli. The immunogenicity of recombinant proteins were observed in rabbits. Based on this, the eukaryotic recombinant plasmids including the codon-optimized multiepitopes genes were also constructed. The immunogenicity and immunoprotection of the multiepitope genes were observed in BALB/C mice. The results would be helpful to develop WNV multiepitopes DNA vaccine with high immnoprotection.Firstly, the candidate epitopes were determined through analysis of WNV complete cDNA sequence from China 01 strain by Bio-Sun Software. Combined together with the reported epitopes, the multiepitopes (210bp) we chosen were consisted of four sequences (encoding protein C 98-106 AA residues, protein PrM 73-91 AA residues , protein E 307-332 AA residues, protein NS1 252-260 AA residues, respectively) and three connecting peptides. The codon usage of frequency of WNV was analyzed and the result showed that codon usage is different between human genome and the WNV genes. To increase the expression level of the multiepitopes protein in mammalian and prokaryotic cells, we replaced all codons by those more frequently used in human genome as well as those in Escherichia coli without modifying the proteins sequence.Secondly, the codon-optimized cDNA sequences obtained by over-lapping PCR amplification were cloned into pET-43a(+) vector. The recombinant plasmids pET-43-M was transfected into Escherichia coli and expressed. Analyzed by SDS-PAGE and Western blotting methods, the expression products Nus-M protein reacted strongly with the positive serum against WNV. To observe the immunogenicity of recombinant proteins, the Nus-M were purified and hypodermically injected into rabbits with Freund's adjuvant. The rabbits were vaccinated three times with two weeks interval at a dose of 500μg in a volume of 100μl. Specific antibodies against Nus-M could be detected by IFA and ELISA after the second injection. The antibody titer reached to peak after the third injection and decreased later. The results showed that the recombinant Nus-M protein could induce specific antibodies with high titer.Thirdly, the recombinant eukaryotic plasmids pcDNA—M were also constructed and transiently transfected into BHK21 cells. The transcription of the recombinant plasmids in eukaryotic cells was detected by RT-PCR. Specific proteins of WNV were observed in cells and specific reactivity to the WNV antiserum was confirmed by IFA. This indicated that the recombinant plasmids in eukaryotic cells could expressed recombinant multiepitope protein with WNV antigenicity.Fourthly, to observe the immunogenicity of recombinant plasmids pcDNA—M, five-week-old female BALB/C mice were immunized three times by intramuscular injection in the hind leg muscles at a dose of 100μg in a volume of 100μl. At the same time, the levels of inducing antidodies among the three groups vaccinated by naked recombinant DNA, recombinant DNA plus recombinant protein boost and recombinant DNA plus adjuvant were also evaluated respectively. The results showed that the three groups could all induced specific neutralizing antibodies against WNV with titers of 1:52, 1:85 and 1:90 respectively. To observe the cellular immune responses of vaccinated mice, spleen cells were isolated from mice and stimulated with CoA in vitro. Four days later, the proliferation of cells and CTL activity were determined. The results showed that naked recombinant DNA, recombinant DNA plus recombinant protein boost and DNA plus adjuvant could all induce specific cellular immune responses in mice. The simulation indexes were 4.10+1.34, 3.80+0.12 and 7.81+0.13. The CTL activities were 18.9% (E/T=20 / 1) , 32.1% (E/T=20 / 1) and 47.7% (E/T=-20 / 1) . All of the parameters were obviously higher than those of the controls.Fifthly, the immunoprotection of multiepitope recombinant plasmids against WNV challenge was observed in mice. Different groups of vaccinated mice were intracalvarium injected with WNV at a dose of 100 LD50 in a volume of 30μl. The cases of death and the protection ratios were calculated at the end of the second week. The ratios of recombinant DNA plus recombinant protein boost and DNA plus adjuvant groups (62.5%) were higher than that of naked DNA group (50%). The ratio of recombinant proteins was the lowest (25%). All the data indicated that multiepitope gene vaccine could provide protection against WNV challenge in mice.This study exhibited that the WNV multiepitope gene vaccine could induce both humoral and cellular immune responses and provide protection againstWNVchallenge in mice. These lay a solid foundation for further developing high-titer multiepitope gene vaccine for WNV.