Development Of New Approaches To Vaccines Against TBEV and West Nile Virus

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Abstract

Vaccines against tick-borne encephalitis virus (TBEV).[unreadable] [unreadable] The viruses of the TBEV complex frequently cause human neurological disease of varying severity with up to 30% mortality. A live attenuated TBEV vaccine is expected to induce a more durable protective immunity than that induced by the inactivated TBEV vaccines that are currently available in Europe. To protect humans against TBEV disease, two promising live attenuated vaccines are being developed in the LID using a strategy based on chimerization of a non-neuroinvasive, mosquito-borne dengue-4 virus (DEN4) with a neurotropic, tick-borne Langat virus (LGT) or TBEV. Chimerization of LGT with DEN4 resulted in: decreased neurovirulence and neuroinvasiveness in mice; highly restricted viremia in monkeys; highly restricted replication in human cells of neural origin and in mosquitoes. A second chimeric virus (TBEV/DEN4d30) was generated by replacing the membrane and envelope protein genes of DEN4 with the corresponding genes of a Far Eastern TBEV and introducing a 30 nucleotide deletion (d30) in the 3 UTR of the genome. Unlike the LGT/DEN4 virus, TBEV/DEN4d30 was neither attenuated for neurovirulence in suckling mice nor restricted in replication in the human neuroblastoma cells. However, it was attenuated for neuroinvasiveness in immunodeficient mice. In addition, both LGT/DEN4 and TBEV/DEN4d30 were found to be greatly attenuated, immunogenic, and efficacious in rhesus monkeys. A single dose of either LGT/DEN4 or TBEV/DEN4d30 protected monkeys against LGT challenge. [unreadable] The clinical phase I evaluation of LGT/DEN4 vaccine in humans was conducted at the Vanderbilt University Medical Center. A single 1000 PFU dose of LGT/DEN4 vaccine was safe and induced a moderate neutralizing antibody response against LGT in 80% of volunteers; however the antibody response against TBEV was less frequent and lower in magnitude, indicating that the improvement in immunogenicity against TBEV is desirable. We anticipate that the antigenically closely related TBEV/DEN4d30 chimera will induce a more satisfactory immune response against highly neurovirulent TBEV in humans than did LGT/DEN4. [unreadable] Prior to clinical evaluation of the TBEV/DEN4d30 vaccine candidate in humans, it was necessary to further examine its neurovirulence in mice and monkeys. Since a LGT virus is the only member of the TBEV complex that was previously tested in humans as a live vaccine, TBEV/DEN4d30 and LGT were compared in adult mice and rhesus monkeys for their abilities to replicate and induce central nervous system (CNS) pathology following intracerebral (IC) inoculation. LGT replicates efficiently in the brain of mice, attains a peak virus titer of 9.6log10 PFU/g, and causes fatal encephalitis. However, TBEV/DEN4d30 attained a peak virus titer of only 1.9log10 PFU/g, indicating that TBEV/DEN4d30 was remarkably attenuated compared to LGT. In monkeys, although both viruses induced a moderate to severe cellular inflammatory response in the CNS, the level of virus replication was low. These two viruses demonstrated different patterns of replication and spatiotemporal distributions of viral antigen and histopathological lesions in the CNS. TBEV/DEN4d30 replicated poorly in the spinal cord and induced less severe neuronal changes compared to LGT. The topographical correlation between histopathological lesions and localization of viral antigen was poor. Dissociation between the kinetics of viral replication and development of inflammation in the CNS suggests involvement of immunopathological mechanisms in the neuropathogenesis of TBEV/DEN4d30 infection. Further studies are needed to investigate the cellular inflammatory responses induced by TBEV/DEN4d30 in the CNS and evaluate their contributions to neuronal cell death. Also, neurovirulence of TBEV/DEN4d30 needs to be compared with that of yellow fever 17D vaccine, which is safe and efficacious in humans. Since TBEV/DEN4d30 exhibits high neurovirulence in young mice and was able to induce severe inflammatory lesions in the CNS of monkeys, its additional attenuation is desirable, and this might be achieved by introducing one or more attenuation mutations identified previously for the LGT or DEN4 virus.[unreadable] [unreadable] Vaccines against West Nile virus (WN) and St. Louis encephalitis virus (SLE).[unreadable] [unreadable] A live attenuated virus vaccine is being developed in the LID to protect humans against WN disease. Previously, we have reported the generation of a clinical lot of WN/DEN4d30 virus and its preclinical evaluation for safety and immunogenicity in mice, geese, and monkeys. Unlike its WN parent, the vaccine candidate was not neuroinvasive in SCID mice and had greatly reduced replication in brains of suckling mice. WN/DEN4d30 failed to infect geese, indicating that the vaccine virus would be restricted in its ability to be transmitted from vaccinees to domestic or wild birds. Furthermore, the WN/DEN4d30 vaccine was safe and readily induced WN neutralizing antibodies even in monkeys immune to each of the four serotypes of DEN. These studies indicate that the vaccine should be efficacious for the recipient and safe for the environment. In a clinical trial of the WN/DEN4d30 vaccine in healthy adult volunteers, the vaccine was well tolerated, safe, and induced a potent and durable antibody response against WN. These results are encouraging and deserved further evaluation of this vaccine in humans. [unreadable] Before the 1999 outbreak of WN, SLE had been the dominant flavivirus causing encephalitis in the USA. SLE vaccine development was initiated using the chimeric approach found successful for WN and TBEV. Chimeric SLE/DEN4 and SLE/DEN4d30 viruses that contained the structural protein genes of SLE lacked detectable neurovasiveness in Swiss mice and were less neuroinvasive than SLE in SCID mice. However, a SLE/DEN4 virus was neither attenuated for neurovirulence in suckling mice nor restricted in replication in monkey, contrary to the observation for WN/DEN4. Chimerization of SLE with DEN4d30 resulted in a virus that did not reduce neurovirulence in young mice and was over-attenuated and not immunogenic in monkeys. Efforts are underway to further attenuate a SLE/DEN4 virus to achieve the optimal balance between attenuation and immunogenicity of this virus in mice and monkeys.