Functional Analysis Of Critical Sites In Japanese Encephalitis Live Attenuated Vaccine SA14-14-2 Genome And Its Application As Viral Vector

Japanese encephalitis virus(JEV) is the cause of Japanese encephalitis in Southeast Asia and the Pacific region, resulting in about 68,000 cases and 10,000 deaths worldwide annually. JEV is transmitted by Culex mosquitos between vertebrate hosts. JEV infection causes damage to central nervous system, and about half of survivors have severe neurological sequelae. The geographical range of JE is expanding to larger areas. To date, JE has become one of the most important causes of viral encephalitis in the world and has posed great threats to human and public health.Vaccination has been proven to be the most effective method to prevent and control JE. The live attenuated JE vaccine SA14-14-2 was first licensed in 1989 in China. Since then, more than 300 million children have been immunized with SA14-14-2, and the satisfactory attenuation and safety profile with low cost have been demonstrated. In addition, immunization with one dose of the live JE vaccine has been proven to induce protective antibody levels efficiently and provide long-term protection. SA14-14-2 has acquired precertification from World Health Organization in 2013 and has been recommended to be widely used throughout the world.Until now, the attenuation mechanism of the live JE vaccine has not been completely understood. JEV is a member of the family Flaviviridae, genus Flavivirus, with an approximately 11 kb positive-sense, single-stranded RNA genome containing a single open reading frame(ORF) flanked by the 5′ and 3′ untranslated regions(UTRs). The ORF encodes three structural proteins(capsid, premembrane, and envelope) and seven nonstructural proteins(NS1, NS2 A, NS2 B, NS3, NS4 A, NS4 B and NS5). The structural proteins are associated with virus attachment and entry as well as virion assembly, whereas nonstructural proteins take part in virus genome replication. Flavivirus nonstructural protein NS1 is expressed on the surface of cells, within cells, and in the extracellular space. NS1 is associated with virus replication, assembly and the regulation of host immune response. A larger NS1-related protein, called NS1′, has been detected in JEV serogroup flavivirus infected cells as a product of a programmed-1 ribosomal frameshift event in NS2 A. The expression of NS1′ in JEV infected cells and its impact on virulence are still unclear.Besides, the epidemiology and distribution of JEV have changed in recent years. GI virus has gradually replaced GIII as the dominant circulating strains in many areas of Asia in the past two decades, while all JE vaccines in use are derived from GIII viruses. The inactivated vaccines have been reported to induce decreased neutralization antibody titers against heterologous GI viruses. To evaluate the protective efficacy of live JE vaccine against circulating JE viruses and to analyze the influential genotype-dependent factors have important implications to the disease control and prevention.Besides, to construct an expression system based on attenuated JEV can provide a new method for the study about genetically engineering vaccines.In this study, we have found the determinants for NS1′ formation and investigated its impact on JEV virulence. Then, we analyzed and confirmed the genotype-specific amino acids affecting the protective efficacy induced by JE live vaccine. Finally, we evaluated the feasibility of using an attenuated JEV as a viral vector to express exogenetic antigens.The study has been divided into three parts. 1. Analysis of nonstructural protein related virulence determinants of JEV.To analyze the determinant factors affecting NS1′ expression and the virulence of JEV, both BHK-21 and C6/36 cells were infected with JEV virulent strain SA14 and vaccine strain SA14-14-2, respectively. The infected cells were then lysed and subjected to Western blotting assay. The results indicated that in SA14 infected cells, both NS1 and NS1′ can be detected, while in SA14-14-2 infected cells, only NS1 can be detected.Sequence alignment of the NS1-NS2 A genes indicated that the vaccine strain SA14-14-2 has a silent mutation at the 66 th nucleotide in NS2 A coding sequence(G→A) compared to other JEV serogroup isolates. RNA structure prediction revealed that the G66 A mutation in SA14-14-2 destabilized the pseudoknot structure which was necessary for NS1′ production.To elucidate the influence of the silent mutation on NS1′ expression and the virulence of JEV, G66 A mutation was introduced into the infectious c DNA clone of SA14 to generate the mutant virus G66 A. Western blotting assay showed that only NS1 was expressed in the G66 A infected cells, while both NS1 and NS1′ were detected in SA14 infected cells. Moreover, G66 A mutants showed reduced neurovirulence and neuroinvasiveness in mice with decreased replication efficiency in vivo.Our results demonstrated that the G66 A mutation in NS2 A abolished NS1′ formation by destabilizing the pseudoknot structure-dependent frameshift event, and it is a virulence determinant of JEV. 2. Analysis of genotype-specific amino acids affecting the neutralizing antibody titers induced by SA14-14-2.To analyze the protective efficacy of SA14-14-2 against different JEV genotypes, we assessed the SA14-14-2-induced neutralization antibody titers against the vaccine strain and the circulating JEV isolates FJ03, SX06, SC04 and SH53. The results indicated that the live JE vaccine provided reduced antibody titers against circulating strains.To find out the genotype-related determinants of antibody neutralization, bioinformatic analysis revealed two distinct amino acids within envelope protein(E222 and E327) between GI and GIII JEV strains. By using reverse genetic approaches, A222 S and S327 T mutations were introduced into the infectious c DNA clone of a GIII virus. The results of plaque reduction neutralization test(PRNT) revealed that the mutant viruses were less neutralized by SA14-14-2 induced antibody, indicating that A222 S and S327 T mutations may decrease the live attenuated vaccine induced neutralizing antibody levels in human, without altering viral replication, protein expression, plaque morphology and virulence.Next, A222 S or S327 T mutations were engineered into the infectious clone of SA14-14-2, respectively. Immunization of mice with mutant vaccines elicited increased neutralizing antibodies against GI strains SX06 and SH53. Moreover, the mutant strains remained the same genetic stability and attenuation characteristics as their parent strain. Together, these results demonstrated that E222 and E327 are potential genotype-related neutralization determinants, and A222 S and S327 T mutations in SA14-14-2 may help to enhance the protective efficacy of the live JE vaccine against circulating GI strains. 3. Establishment of a viral expression system based on attenuated JEV.Based on the full-length c DNA clone of attenuated JEV, by using reverse genetic techniques, a DNA cassette containing the E3-E2 Domain A coding sequence of Chikungunya virus(CHIKV) followed by IRES sequence from encephalomyocarditis virus was inserted into the downstream of JEV C gene to generate a recombinant virus. Transfection of RNA transcripts into BHK-21 cells caused typical CPE, and RT-PCR indicated that the CHIKV gene sequence was successfully constructed into JEV genome. The recombinant virus exhibited smaller plaque morphology and reduced replication efficiency compared with the attenuated JEV. Indirect immunofluorescence assay indicated that the recombinant virus expressed the CHIKV protein and JEV protein within infected cells. These results indicated that a recombinant JE virus carrying CHIKV antigen gene was successfully constructed, demonstrating the feasibility of using attenuated JEV as a viral vector and exhibiting important implications for the design of next generation vaccines.In conclusion, based on reverse genetic approaches of JEV and bioinformatics techniques, a nucleotide mutation G66 A in NS2 A of SA14-14-2 was demonstrated to destabilize the pseudoknot structure-dependent ribosomal frameshifting and abolish the expression of NS1′, which contributes to the reduced neurovirulence and neuroinvasiveness of SA14-14-2. Meanwhile, considering the influence of JEV genotype shift on the live JE vaccine induced protection, two genotype-dependent amino acids in E protein were found to affect the neutralization efficacy of live JE vaccine induced antibody. In addition, a recombinant attenuated virus carrying CHIKV antigen gene was successfully generated and was shown to be able to express the heterogenous antigen in infected cells, suggesting the feasibility of novel viral vectors based on attenuated JEV strains. The results have significant implications for the understanding of JEV attenuation mechanism, the quality control of JE vaccines, and the improvement of JE vaccine to provide better protection efficacy, as well as the application of JEV based viral vectors.