Flaviviruses such as West Nile (WN), Japanese encephalitis (JE), St. Louis encephalitis, and tick-borne encephalitis (TBEV) viruses are important neurotropic human pathogens, causing outbreaks of diseases in humans and domestic animals in many regions of the world. The emergence of WN in North America has resulted in a significant increase in the disease being observed in birds, horses, and humans. During the 1999-2010 outbreaks of WN in the USA, there were at least 29,982 reported human cases of WN illness that included 1,303 deaths. Since there is no antiviral drug and no vaccine effective against WN infection, it is considered a significant public health threat in the USA. Tick-borne encephalitis is a severe disease affecting thousands of people throughout Eurasia. Despite the use of formalin-inactivated vaccines in endemic areas, an increasing incidence of TBEV during the past 2 decades emphasizes the need for an alternative vaccine that will induce a more durable immunity and protection against TBEV. In an effort to develop the efficacious live attenuated vaccines against neurotropic flaviviruses, we explored several strategies, and progress toward this goal in the past year is reviewed below. In the first approach, the live attenuated TBEV or WN vaccine candidates are being developed in the Neurotropic Flaviviruses Section of the LID using a strategy based on chimerization of a neurovirulent TBEV or WN with a non-neuroinvasive, mosquito-borne dengue-4 virus (DEN4d30) that contains an attenuating mutation, a 30 nucleotide deletion in the 3'-noncoding region (3'NCR). Chimeric TBEV/DEN4d30 virus that contains the structural protein genes of a highly virulent TBEV demonstrates moderate levels of immunogenicity and protective efficacy in mice and monkeys, but retains an unacceptably high level of neurovirulence based on the results of clinical observations and analysis of virus-induced histopathology in the CNS of monkeys. Further attenuation of TBEV/DEN4d30 neurovirulence was achieved by introducing amino acid substitutions that had previously been shown to reduce replication of tick-borne Langat virus (LGT) or DEN4 in suckling mouse brain. When attenuating mutations such as amino acid substitutions in the envelope E (Lys315 >Glu) and NS5 (Asp654Arg655 >AlaAla) proteins were introduced into the TBEV/DEN4d30 genome, the resulting virus (TBEV/DEN4d30-E315-NS5-654,655) demonstrated the desired properties of an acceptable live attenuated vaccine. In FY 2011, we demonstrated that this virus was genetically stable in the brain of suckling mice and safe for the CNS as shown by a reduced level of cellular inflammatory response and neuronal degeneration. TBEV/DEN4 and its d30 mutant induced severe neuroinflammation, whereas virus-induced inflammatory changes were not observed in the brains of mice with TBEV/DEN4d30-E315-NS5-654,655 or mock-inoculated controls. The vaccine candidate exhibits a limited potential for transmission in nature since it was unable to infect or replicate in mosquitoes and ticks. In addition, we demonstrated that the immunogenicity and protective efficacies of our vaccine candidate and commercial inactivated TBEV vaccine were similar in mice challenged with wild-type European or Far Eastern strains of TBEV. Thus, TBEV/DEN4d30-E315-NS5-654,655 virus is a promising TBEV vaccine candidate, but its ability to induce a long-lasting protective immunity against TBEV, the effect of boost immunizations, and its level of neurovirulence in the CNS of non-human primates need to be evaluated prior to testing in humans. A live attenuated WN/DEN4d30 virus vaccine developed in the LID to protect humans against WN disease was well-tolerated, safe, and induced a potent and durable WN antibody response in healthy adult volunteers. Further studies of neurovirulence in monkeys are necessary to provide the additional evidence of safety of this vaccine for the CNS of non-human primates prior to its use in the risk group of volunteers >50 years of age. In FY 2011, we performed a comparative analysis of neuropathogenesis in rhesus monkeys following intrathalamic inoculation with the wild type WN virus or the WN/DEN4d30, DEN4d30, or yellow fever (YF) 17D vaccine. The clinical and virology data indicated that the WN/DEN4d30 vaccine is a most attenuated virus in the CNS compared to either comparator virus as demonstrated by a lack of virus replication in the brain and spinal cord and the absence of clinical signs of neurological disease. In the coming year, we plan to further evaluate the spatiotemporal distributions of viral antigens, virus-induced histopathology, cellular inflammatory response, and neuronal degeneration in the CNS of monkeys. In the second approach for the design of safe and effective live flavivirus vaccines, we explored the ability of the CNS-expressed cellular microRNAs to modulate the neurotropism and pathogenesis of flaviviruses. The inclusion of a single target copy for a brain tissue-expressed miRNA (let-7c, mir-9, mir-124a, mir-128a, or mir-218) into the TBEV/DEN4 genome was sufficient to prevent the development of otherwise lethal encephalitis in adult mice. However, in FY2011, we demonstrated that the efficacy of miRNA-mediated inhibition of virus replication in the immature CNS of suckling mice depends on the genetic stability of the miRNA-targeted virus and it can revert to virulent phenotype by accumulating mutations within the miRNA target sequence. Following intracerebral inoculation of suckling mice with TBEV/DEN4 viruses carrying a single miRNA-target copy, the brain-isolated viruses from moribund mice contained partially or completely deleted miRNA-target sequence. The risk of virus escape from miRNA-mediated inhibition in the CNS was significantly minimized by increasing the number of target sites in the viral genome for broadly CNS-expressed miRNAs (mir-9 and mir-124a). We found that a location of miRNA-targets in the 3'NCR of the TBEV/DEN4 genome as well as the distance between the inserted miRNA target sites affect the level of miRNA-mediated inhibition of virus replication. Multiple miRNA-target insertions (2, 3, or 4 copies) into the 3'NCR of viral genome altered virus neuroinvasiveness for immunodeficient mice and significantly attenuated its neurovirulence for suckling mice, but did not impaired its immunogenicity and protective efficacy. Importantly, the miRNA-targeting of a large portion of the 3'NCR with the multiple miRNA-binding sites greatly reduced the probability of virus to escape from miRNA-mediated suppression even in the immature CNS of newborn mice. These findings suggest that a microRNA-targeting approach to control the virus tissue tropism could provide a new basis for future design of safe and effective live virus vaccines against neurotropic flaviviruses.
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