Development Of New Approaches To Vaccines Against Neurotropic Flaviviruses

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

The pathogenesis of flaviviral neuroinfection is complex and depends on a series of events involving (1) virus replication in the periphery to produce and sustain the level of viremia sufficient for neuroinvasion, (2) virus entry into the CNS (neuroinvasiveness), (3) virus replication, spread, and introduction of neuropathology once in the CNS (neurovirulence), and (4) virus-mediated stimulation of innate and adaptive immune responses of the host both in the periphery and within the CNS. To prevent the virus entry into the CNS (neuroinvasiveness) and to restrict its replication in the neurons (neurovirulence) or in peripheral organs, we have explored several different approaches and developed the vaccine candidates using the strategies based on (1) chimerization of a neurovirulent virus (JEV, WNV, SLEV or TBEV) with a non-neuroinvasive mosquito-borne dengue type 4 flavivirus (DEN4) or naturally attenuated tick-borne Langat virus (LGT) and (2) targeting of viral genome for cellular microRNAs (miRNAs) expressed in the tissue of interest (etc., brain, reproductive organs, placenta) for selective control of virus tropism. The promising and most attenuated vaccine candidates were then evaluated for safety, immunogenicity, and their ability to protect mice and monkeys against challenge with wild-type virulent parental virus. In addition, a miRNA targeting approach was adapted to design environmentally safe vaccine viruses restricted in their ability to infect and be transmitted by permissive arthropod vectors (mosquitoes or ticks). Pathogenesis and development of vaccines against diseases that caused by neurotropic flaviviruses: In FY2020-21, we identified an optimal adjusted dose of each component in the combined trivalent vaccine based on miRNA-targeted viruses (JEV/DEN4mirT, WNV/DEN4mirT and SLEV/DEN4mirT) and achieved the balanced antibody response in mice against virulent parental viruses. We demonstrated that a single dose of trivalent vaccine formulation (admixture of 100,000 PFU of JEV/DEN4mirT + 10,000 PFU of WNV/DEN4mirT + 100,000 PFU of SLEV/DEN4mirT) induced JEV-, SLEV- and WNV-specific strong neutralizing antibody responses in immunocompromised mice and protected them against lethal challenge (100 lethal dose) with wild-type JEV, WNV or SLEV virus. In our studies on the regulation of processes between the immune and nervous system in a nonhuman primate model of WNV neurological disease, we showed that virus infection disrupts the homeostasis of the immune-neural-synaptic axis via induction of pleiotropic genes; this suggests an unintended off-target negative impact of virus-induced immune responses on neurotransmission in the CNS (Maximova et al., eLife 2021).These data may serve as a resource in the search for new therapeutic approaches to restore homeostasis in interactions between the nervous and immune systems during virus clearance from the CNS. In FY2021, we continued and broadened our research on mechanism of ZIKV pathogenesis in the male and female reproductive organs (Tsetsarkin et al., PLoS Path 2020). We applied our accumulated knowledge to evaluate the relative contributions of the components of male reproductive system (MRS) during male-to-female sexual transmission of ZIKV in mice with altered antiviral responses. Using microRNA targeted ZIKV clones that were engineered to abolish viral infectivity to different parts of the MRS, we pinpointed epithelial cells of the epididymis (rather than cells of the testis, vas deferens, prostate, or seminal vesicles) as a most likely source of the sexually transmitted ZIKV genomes during the early (most productive) phase of ZIKV shedding into the semen. To verify these findings, we developed a novel approach for tracking ZIKV genomes during virus transit through the MRS. This approach relies on analysis of the genetic bottlenecks imposed on ZIKV populations passing through anatomical barriers in the MRS. This approach required generation of a library of viable ZIKV genomes containing unique molecular identifiers (barcodes). Similar to results obtained using microRNA targeted ZIKV clones, the analysis of barcoded ZIKV library showed that the epididymis (specifically, the epididymal epithelial cells) is the main contributor of ZIKV output in the ejaculate of mice. Incorporation of this mechanistic knowledge into the development of a live-attenuated ZIKV vaccine candidate at NFS abolished sexual transmission of the vaccine candidate virus (Pletnev et al., Nature Commun 2021). In collaboration with University of California, Davis (Dr. L. Clark), the M1404I mutation (ns2B protein) was identified in genome of SPH2015 strain of ZIKV, which was recovered from a dead macaque fetus. To analyze the role of this mutation in congenital Zika syndrome, we developed a full-length infectious clone of SPH2015 strain of ZIKV carrying a M1404I mutation and found that this mutation increased the ability of the virus to infect mouse fetuses but decreased its capacity to produce high levels of virus in the blood of nonpregnant macaques and to be transmitted by mosquitoes (Tsetsarkin et al., PLoS Path 2021). These findings underscore the complexity of the arbovirus mutation-fitness dynamics and suggest that intrahost ZIKV mutations capable of augmenting fitness in pregnant vertebrates may not necessarily spread efficiently via mosquitoes during epidemics (Lemos et al., J Virol. 2020). In FY2021, we completed the evaluation of neurovirulence of the second generation of micro-RNA targeted ZIKV vaccine candidates (ZIKV viruses carrying multiple targets for cellular microRNA) in the CNS of newborn mice. This study demonstrated safety and attenuation of the developed viruses for CNS, which propelled these vaccine candidates to enter the Phase I clinical trials in human volunteers.