Vaccines against tick-borne encephalitis virus.? ? A live attenuated TBEV vaccine is expected to induce a more durable protective immunity than that induced by the inactivated TBEV vaccine that is not licensed in the USA. Two live attenuated TBEV vaccine candidates 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. Phase I clinical studies in humans with the LGT-based chimeric vaccine indicated that this vaccine was safe, induced a moderate neutralizing antibody response against LGT, but it was weakly immunogenic against the prevalent TBEV strains, indicating that the improvement in immunogenicity against TBEV is required. In FY 2008, we have focused on the identification of a chimeric TBEV/DEN4 vaccine candidate that is suitable for evaluation in humans and that exhibits the following important properties: (1) reduced neurovirulence and restricted replication in the brains of mice; (2) restricted or ablated neuroinvasiveness in immunodeficient mice; (3) decreased replication in rhesus monkeys; (4) ability to induce a protective TBEV-specific immune response in monkeys; and (5) a low level of neurovirulence in the CNS of non-human primates. We considered the second chimeric virus, TBEV/DEN4d30, as a possible vaccine candidate for evaluation in humans since it exhibits an evidence of attenuation for mice and monkeys and has satisfactory immunogenicity and protective efficacy in monkeys. However, it remained possible that this new chimeric virus, containing the structural protein genes derived from the highly neurovirulent Far Eastern strain of TBEV, might gain access to the CNS of vaccine recipients and cause neurological disease. Therefore, prior to clinical evaluation of this vaccine candidate in humans, it was necessary to further examine its neurovirulence in monkeys. We compared the neuropathogenesis of TBEV/DEN4d30, LGT, a former live TBEV vaccine, and yellow fever 17D vaccine (YF 17D) in rhesus monkeys inoculated intracerebrally. The results of histopathological analysis of the CNS together with clinical observations and data on virus replication indicated that chimerization of a neurovirulent TBEV strain with non-neuroinvasive DEN4d30 had an attenuating effect on the neurovirulence of TBEV in monkeys. However, this effect was insufficient to justify bringing this vaccine candidate to trials in humans since TBEV/DEN4d30 exhibited higher neurovirulence in monkeys compared to either LGT or YF 17D, suggesting that it needs to be further attenuated.? Since LGT and TBEV/DEN4d30 evoked a strong cellular inflammatory response in the CNS of monkeys, we have developed a new computerized method for quantitative evaluation of the CNS infiltration with peripheral immune cells (CD3, CD4, CD8, and CD20 lymphocytes) and the response of CNS resident cells (microglial activation and neuronal degradation). We found that immunoreactivity for CD3 and CD4 T cells and CD20 B cells correlated remarkably well with semi-quantitative histopathological scores for cellular inflammatory infiltration in the brains of monkeys inoculated with either YF 17D, LGT or TBEV/DEN4d30 attenuated viruses. Preliminary data from the correlation analyses indicate that a high CD4:CD8 T cell ratio in the CNS infiltrates induced in response to YF 17D infection is a major factor that differentiates this vaccine virus from a more neurovirulent LGT or TBEV/DEN4d30 virus. It is likely that the balanced response of T and B cells within the CNS of monkeys induced with YF 17D vaccine virus plays an important role in the recovery from CNS infection and might serve as a reference to evaluate the safety of new live flavivirus vaccine candidates.? Further attenuation of TBEV/DEN4d30 neurovirulence was undertaken by introducing amino acid substitutions that had previously been shown to reduce replication of LGT or DEN4 in suckling mouse brain. When two attenuating amino acid substitutions in the E and NS5 proteins were introduced into TBEV/DEN4d30 genome, the resulting virus (TBEV/DEN4d30-E-NS5) demonstrated the desired properties of an acceptable live attenuated vaccine candidate as it displayed (1) a 490-fold decrease in neurovirulence in suckling mice, (2) a non-neuroinvasive phenotype in immunodeficient mice, and (3) was still safe and immunogenic in monkeys. However, its neurovirulence and protective efficacy in non-human primates should be evaluated before clinical trials can be initiated. ? ? Vaccines against West Nile virus and St. Louis encephalitis virus.? ? A live attenuated WN/DEN4d30 virus vaccine is being developed in the LID to protect humans against WN disease. In a clinical trial in healthy adult volunteers, the WN/DEN4d30 vaccine was well tolerated, safe, and induced a potent and durable antibody response against WN. Trials to define the optimal dose of WN/DEN4d30 (10,000 or 100,000) and the optimal number of immunizations (one versus two) are in progress.? ? Before the 1999 outbreak of WN, SLE had been the dominant flavivirus causing encephalitis in the USA. Antigenic chimeric viruses (SLE/DEN4 and SLE/DEN4d30) were generated using the chimeric approach proven successful for WN and TBEV. Both chimeras had greatly reduced neuroinvasiveness in immunodeficient mice but retained neurovirulence in suckling mice. Chimerization of SLE with DEN4 resulted in only moderate restriction in replication in rhesus monkeys, whereas the presence of the d30 mutation led to over-attenuation. Introduction of previously described attenuating paired charge-to-alanine mutations in the DEN4 NS5 protein of SLE/DEN4 reduced neurovirulence in mice and replication in rhesus monkeys. Two modified SLE/DEN4 viruses have significantly reduced neurovirulence in mice and conferred protective immunity in monkeys against SLE challenge. These viruses may be considered for use as SLE vaccine candidates and for use as diagnostic reagents with reduced virulence. Efforts are underway to identify a SLE/DEN4 virus that will exhibit a satisfactory balance between attenuation and immunogenicity in monkeys.
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