Many of arthropod-borne viruses are emerging and re-emerging human pathogens that have caused outbreaks of the devastating and often fatal diseases and represent a serious public health problem in many regions of the world. During the past decades, neurotropic arboviruses mainly belonging to the Flaviviridae and Togaviridae families (CHIKV, JE, TBEV, WN, SLE, and ZIKV viruses) unexpectedly spread in many new geographic areas of the world which resulted in severe impact on wildlife and increase in the number of flavivirus-associated illnesses in humans. Currently, WN is the major source of viral encephalitis in the USA. During the 1999-2015 epidemics, nearly 3 million people were infected with WN in the US, with about 20,170 CDC-reported cases of neuroinvasive disease and 1884 deaths. Over the last decade, CHIKV and ZIKV viruses had emerged explosively in the Latin America affecting an estimated 1 to 2 million people. Of main public health concern with ZIKV epidemics is the increase of the severity disease that has never been observed in other flavivirus infections and associated with catastrophic fetal abnormalities including microcephaly and spontaneous abortion. Compared to the natural transmission cycle of other RNA viruses, arboviruses need to replicate in two hosts: vertebrate and invertebrate vector (mosquito, tick or other arthropod). Surprisingly, unlike other members of the Flaviviridae family, ZIKV was able to establish long-term persistence in vertebrate hosts and utilize a sexual mode of virus transmission, posing new challenges for controlling virus outbreaks. The spread of tick- and mosquito-borne viruses in new areas and the increase in incidence of arbovirus-associated illness highlight the need for efficacious vaccines, antiviral drugs, and novel approaches for vector control. Live attenuated vaccine candidates against neurotropic flaviviruses (JE, WN, SLE, TBEV) are being developed in the LID using the strategies based on (1) chimerization of a neurovirulent virus with a non-neuroinvasive dengue type 4 flavivirus (DEN4), (2) introduction of attenuating mutations, (3) targeting of viral genome for cellular microRNAs (miRNAs), and (4) genetic order gene repositioning within the virus genome. The pathogenesis of neurotropic infections involves two distinct properties of the viruses: neuroinvasiveness and neurovirulence. Thus, the research in our lab aims first to prevent the virus entry into the central nervous system (CNS) and second to restrict its replication in the neurons. The most attenuated vaccine candidates were then evaluated for safety, immunogenicity, their ability to protect mice and monkeys against challenge with wild-type virus. JE, SLE and WN vaccines: A WN vaccine (WN/DEN4d30 virus) is a chimeric virus in which the prM and E genes of the DEN4 virus have been replaced by those of WN. In previous clinical trials, a WN/DEN4d30 has been shown to be safe and effective in healthy adults aged 18-50 years. However, older adults are more susceptible to the neuroinvasive WN disease that can be accompanied by a high mortality rate. Based on our previous data, a dose of 10,000 PFU was selected for 2015-16 clinical studies to further evaluate safety and immunogenicity of the WN vaccine in flavivirus-naive elderly volunteers, adults 50-65 years of age, and have found it to be safe, well-tolerated, and immunogenic. Immunization with a single vaccine dose produces high levels of neutralizing antibody against wild-type WN strains, inducing a 95% rate of seroconversion in the volunteers. These results suggest that a single dose of vaccine may be sufficient to induce a protective antibody response. As outbreaks of WN tend to be unpredictable, a safe and effective vaccine in the vulnerable elderly population will be an important public health tool against severe neuroinvasive WN disease. During recent years, we developed a general approach to control flavivirus tissue tropism and pathogenesis by targeting virus genome (TBEV/DEN4 chimera) with cellular brain-expressed microRNAs (Teterina et al., 2014). This work demonstrated that miRNA targeting is a rational method for flavivirus attenuation and was utilized in FY2016 for WN, JE, and SLE vaccine development with an end goal of generating the trivalent vaccine. Next generation of vaccine candidates was engineered by introducing multiple targets for the CNS-specific miRNAs into WN/DEN4 and SLE/DEN4 genomes. Each monovalent vaccine candidate (WN/DEN4 or SLE/DEN4 miRNA-targeted viruses) demonstrated reduced neuroinvasiveness and neurovirulence in suckling mice and provided complete protection against parental WN or SLE challenge. In addition, a single dose of bivalent vaccine formulations (1000 or 1000 PFU of WN/DEN4 and SLE/DEN4 miRNA-targeted viruses) induced the strong neutralizing WN- and SLE-specific antibody responses and protected mice against both wild-type viruses. In addition, JE/DEN4 viruses were generated recently in which the targets for brain-specific mir-124 miRNA in the E protein gene were combined with additional miRNA-targets located in the 3'NCR. Currently, we are working to identify the most attenuated JE vaccine candidate for neuropathogenesis in mice that will be used as a third component of the combined trivalent vaccine against WN, SLE and JE. TBEV vaccine: The TBEV vaccine candidates are chimeric viruses carrying the prM and E structural protein genes of Far Eastern TBEV and remaining sequences derived from either (1) a non-neuroinvasive DEN4 (TBEV/DEN4), (2) a naturally attenuated, tick-borne Langat virus (LGT) strain E5 (TBEV/E5) or (3) a most immunogenic LGT strain T1674 (TBEV/LGT). The large set of modified TBEV/DEN4 and TBEV/LGT was generated by introducing the cassettes of brain-specific miRNA-targets into three distant regions of chimeric DEN4- or LGT-based genome, etc., in the duplicated capsid gene region, the duplicated E gene region, and the 3NCR. In FY2016, we identified 11 most promising vaccine candidates that were restricted for replication in the brain of newborn mice and had significantly limited neuroinvasiveness in immunodeficient mice. Currently, comparative studies for evaluation of protective efficacy against TBEV challenge in mice and immunogenicity in rhesus monkeys of these selected miRNA-targeted viruses (DEN4- and LGT-based viruses) are underway. Flaviviruses are transmitted in nature by their specific arthropod vector, and the environmental safety of live attenuated vaccines is a significant concern. Hypothetically, the DEN4- or LGT-based vaccine viruses have a potential for reversion back to wild-type that could be associated with possible dissemination of these mutated viruses by mosquitoes or ticks after feeding on vaccinees. Using a DEN4 virus as a model system, we demonstrated that targeting of mosquito-borne DEN4 genome for mosquito-specific mir-184 and mir-275 miRNAs abolishes virus transmission by its natural vectors: Aedes aegypti and Aedes. albopictus mosquitoes, which are the competent vectors for many arboviruses. Unlike mosquito-borne viruses, tick-borne flaviviruses can be efficiently transmitted between co-feeding ticks at each life cycle stage of tick development (larvae, nymphs and adults). In FY2016, we showed that insertion of target sequences for tick-specific miRNAs (mir-1, mir-275 and mir-279) individually or in combination into tick-borne LGT genome caused selective attenuation of virus replication in tick-derived cells. In addition, simultaneous dual miRNA targeting for tick- and vertebrate CNS-specific miRNAs led to silencing of virus replication in live Ixodes ticks and abolished virus neurotropism in highly permissive newborn mice. We believe that the developed miRNA co-targeting approach can be adapted to support the design of environmentally safe vaccines.
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