The tick-borne encephalitis virus complex of flavivirus family includes tick-borne encephalitis virus (formerly called Russian spring-summer encephalitis virus), Langat, Louping ill, Powassan, Kyassanur forest disease, Negishi, and Omsk hemorrhagic fever viruses. Tick-borne encephalitis viruses (TBEV) causes serious disease with high mortality throughout Russia, India, North China and European countries. In addition, a virulent member of the TBEV complex, Powassan virus, is present in Canada and the Eastern and Western US. Previously, despite the considerable evolutionary distance between the mosquito-borne dengue viruses of flavivirus family and tick-borne encephalitis viruses, we succeeded in constructing viable chimeric flaviviruses that contained C-preM-E or preM-E structural protein genes of a virulent Far Eastern Russian TBEV with the remaining nonstructural protein genes and 5'- and 3'-noncoding sequences derived from dengue type 4 virus (DEN4). The preM-E chimera (designated TBEV(ME)/DEN4) retained the neurovirulence for mice of its TBEV parent from which its preM and E genes were derived, but it lacked the peripheral neuroinvasiveness of TBEV, i.e., the ability to spread from a peripheral site in the body to the central nervous system and cause fatal encephalitis. However, mice previously inoculated with the chimeric virus by a peripheral route were completely resistant to subsequent intraperitoneal challenge with a lethal dose of the highly virulent TBEV. Later, neurovirulence demonstrable by intracerebral inoculation of the TBEV(ME)/DEN4 chimera was significantly reduced by a single mutation introduced into its preM, E or NS1 viral protein. These amino acid substitutions also caused a restriction in viral replication in both simian and mosquito cells. Nonetheless, parenteral inoculation of these further attenuated chimeric mutants induced complete resistance in mice to fatal encephalitis caused by intracerebral inoculation of the neurovirulent TBEV(ME)/DEN4 chimera. These encouraging results provide the foundation for a new strategy for the development of a live attenuated TBEV vaccine. Unfortunately, this strategy could not be pursued because the P3/P4 biosafety facility at NIH required for study of TBEV and its chimeras was no longer available to us. As a consequence, our strategy was modified by substituting the Langat (LGT) virus for the highly virulent TBEV. LGT is the least virulent of all TBEV-complex flaviviruses but it has a very close antigenic relationship to the highly virulent Far Eastern TBEV as well as a high level of sequence homology. Langat virus has not been associated with disease in humans in the laboratory or under natural circumstances. One of the goals of the current project was to evaluate the feasibility of developing a live attenuated TBEV vaccine using LGT virus antigens. For this reason, we determined the complete nucleotide sequence of the wild type LGT virus (TP21 strain) and a more attenuated virus derived from it (strain E5) by multiple passages in chick embryo tissue. Later, full-length DEN4 cDNA was employed to engineer chimeric LGT/DEN4 constructs by substituting the structural or nonstructural protein genes of LGT TP21 or LGT E5 for the corresponding DEN4 genes. Only two viable chimeric viruses were recovered and each contained the preM and E genes of LGT virus strain TP21 or strain E5 and all other sequences from DEN4. Chimerization of LGT TP21 or LGT E5 with DEN4 significantly reduced the replicative capacity of these chimeras in simian cells compared to either parental virus. This is in contrast to our previous observation that chimera TBEV(ME)/DEN4 replicated 1000 times more efficiently in simian cells than did DEN4. Also, chimeric TP21/DEN4 or E5/DEN4 exhibited a significant reduction in neurovirulence compared to its LGT parent when tested by direct inoculation into the brain of suckling mice. Significantly, chimerization of LGT TP21 with DEN4 also reduced or eliminated neuroinvasiveness of this tick-borne flavivirus for normal mice. Diminished neuroinvasiveness of the TP21/DEN4 chimera was demonstrated more dramatically in test performed in SCID mice which were 10/7 times more sensitive than normal mice for detection of this phenotype. SCID mice inoculated IP with 10/7 PFU of chimeric virus remained healthy during the 6 week post-inoculation observation interval. Overall, the current observations are consistent with earlier findings that chimerization of TBEV with DEN4 significantly reduced or eliminated its neuroinvasiveness for mice. Significantly, all of the mice that had been immunized previously IP with TP21/DEN4 or E5/DEN4 developed a high titer of neutralizing antibodies and were completely protected against intraperitoneal challenge of TP21. Both LGT/DEN4 chimeras appear to be promising live virus vaccine candidates because these viruses induced an asymptomatic infection in mice that provided effective protective immunity against challenge with 1000 intraperitoneal LD50 of wild type LGT TP21.
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