Venezuelan Equine Encephalitis Virus V3526 Vaccine RNA-Dependent RNA Polymerase Mutants Increase Vaccine Safety Through Restricted Tissue Tropism in a Mouse Model

Background: Venezuelan equine encephalitis virus (VEEV) is an arbovirus endemic to the Americas, for which no vaccines or antiviral agents have been approved. TC-83 and V3526 are the best-characterized vaccine candidates for VEEV. Both are live-attenuated vaccines and have been associated with safety concerns, although fewer concerns exist for V3526. A previous attempt to improve the TC-83 vaccine focused on further attenuating the vaccine by adding mutations that alter the error-incorporation rate of the RNA-dependent RNA polymerase (RdRp). Methods: The research herein examined the effects of these RdRp mutations in V3526 by cloning the 3X and 4X strains, assessing vaccine efficacy against challenge in adult female CD-1 mice, examining neutralizing-antibody titers, investigating vaccine tissue tropism, and testing the stability of the mutant strains. Results: The V3526 RdRp mutants exhibited less tissue tropism in the spleen and kidney than the wild-type V3526, while maintaining vaccine efficacy. Illumina sequencing indicated that the RdRp mutations reverted to wild-type V3526 after five passages in murine pup brains. Conclusions: The observed genotypic reversion is likely to be of limited concern, because wild-type V3526 remains an effective vaccine capable of providing protection. Our results indicate that the V3526 RdRp mutants may be a safer vaccine design than the original V3526.

Bivalent single domain antibody constructs for effective neutralization of Venezuelan equine encephalitis

Venezuelan equine encephalitis virus (VEEV) is a mosquito borne alphavirus which leads to high viremia in equines followed by lethal encephalitis and lateral spread to humans. In addition to naturally occurring outbreaks, VEEV is a potential biothreat agent with no approved human vaccine or therapeutic currently available. Single domain antibodies (sdAb), also known as nanobodies, have the potential to be effective therapeutic agents. Using an immune phage display library derived from a llama immunized with an equine vaccine that included inactivated VEEV, five sdAb sequence families were identified that showed varying ability to neutralize VEEV. One of the sequence families had been identified previously in selections against chikungunya virus, a related alphavirus of public health concern. A key advantage of sdAb is the ability to optimize properties such as neutralization capacity through protein engineering. Neutralization of VEEV was improved by two orders of magnitude by genetically linking sdAb. One of the bivalent constructs showed effective neutralization of both VEEV and chikungunya virus. Several of the bivalent constructs neutralized VEEV in cell-based assays with reductions in the number of plaques by 50% at protein concentrations of 1 ng/mL or lower, making future evaluation of their therapeutic potential compelling.

Examination of Antiviral Resistance in Venezuelan Equine Encephalitis Virus

Venezuelan equine encephalitis virus (VEEV) is a New World Alphavirus that causes Venezuelan equine encephalitis (VEE), which is characterized by a febrile illness that can progress to neurological disease and death. While no major outbreaks of VEE have occurred since 1995, VEEV is a virus of concern as, in addition to its spread through mosquitos, it can be aerosolized and used as a bioweapon. Unfortunately, there are currently no FDA-approved vaccines or antivirals against VEEV. Efforts have been made to discover small molecules with an inhibitory effect on VEEV, but the potential for emergence of antiviral resistance to these compounds will remain a concern because VEEV is an RNA virus with a high mutation rate and grows to high titers. To examine the evolutionary trajectory of antiviral resistance in VEEV, we developed a next-generation sequencing pipeline to examine single-nucleotide polymorphisms that emerged after repeated passaging of the virus with increasing concentrations of antiviral compounds. In addition, we examined the effect of the microenvironment on the evolution of antiviral resistance, both in cell culture and mouse models. We found that VEEV evolves resistance to the compound ML336 and its derivatives through mutations in the nsP2 and nsP4 genes, but the number, timing of emergence, and the extent of penetrance of these SNPs depend on the compound. These mutations emerged more slowly when infecting an astrocyte cell line. We also found that neurons in the mouse brain did not impose a selective pressure on VEEV during an infection. These results demonstrate how the population dynamics of RNA viruses can be tracked over time and the extent to which they are affected by selective pressures, as well as opening questions about how viruses can mutate and adapt at the molecular level.

An alphavirus replicon-based vaccine expressing a stabilized Spike antigen induces protective immunity and prevents transmission of SARS-CoV-2 between cats

Early in the SARS-CoV-2 pandemic concerns were raised regarding infection of new animal hosts and the effect on viral epidemiology. Infection of other animals could be detrimental by causing clinical disease, allowing further mutations, and bares the risk for the establishment of a non-human reservoir. Cats were the first reported animals susceptible to natural and experimental infection with SARS-CoV-2. Given the concerns these findings raised, and the close contact between humans and cats, we aimed to develop a vaccine candidate that could reduce SARS-CoV-2 infection and in addition to prevent spread among cats. Here we report that a Replicon Particle (RP) vaccine based on Venezuelan equine encephalitis virus, known to be safe and efficacious in a variety of animal species, could induce neutralizing antibody responses in guinea pigs and cats. The design of the SARS-CoV-2 spike immunogen was critical in developing a strong neutralizing antibody response. Vaccination of cats was able to induce high neutralizing antibody responses, effective also against the SARS-CoV-2 B.1.1.7 variant. Interestingly, in contrast to control animals, the infectious virus could not be detected in oropharyngeal or nasal swabs of vaccinated cats after SARS-CoV-2 challenge. Correspondingly, the challenged control cats spread the virus to in-contact cats whereas the vaccinated cats did not transmit the virus. The results show that the RP vaccine induces protective immunity preventing SARS-CoV-2 infection and transmission. These data suggest that this RP vaccine could be a multi-species vaccine useful to prevent infection and spread to and between animals should that approach be required.

Structure of Venezuelan equine encephalitis virus in complex with the LDLRAD3 receptor

LDLRAD3 is a recently defined attachment and entry receptor for Venezuelan equine encephalitis virus (VEEV)1, a New World alphavirus that causes severe neurological disease in humans. Here we present near-atomic-resolution cryo-electron microscopy reconstructions of VEEV virus-like particles alone and in a complex with the ectodomains of LDLRAD3. Domain 1 of LDLRAD3 is a low-density lipoprotein receptor type-A module that binds to VEEV by wedging into a cleft created by two adjacent E2–E1 heterodimers in one trimeric spike, and engages domains A and B of E2 and the fusion loop in E1. Atomic modelling of this interface is supported by mutagenesis and anti-VEEV antibody binding competition assays. Notably, VEEV engages LDLRAD3 in a manner that is similar to the way that arthritogenic alphaviruses bind to the structurally unrelated MXRA8 receptor, but with a much smaller interface. These studies further elucidate the structural basis of alphavirus–receptor interactions, which could inform the development of therapies to mitigate infection and disease against multiple members of this family.

Long-term persistence of viral RNA and inflammation in the CNS of macaques exposed to aerosolized Venezuelan equine encephalitis virus

ABSTRACTVenezuelan equine encephalitis virus (VEEV) is a positively-stranded RNA arbovirus of the genus Alphavirus that causes encephalitis in humans. Cynomolgus macaques are a relevant model of the human disease caused by VEEV and are useful in exploring pathogenic mechanisms and the host response to VEEV infection. Macaques were exposed to small-particle aerosols containing virus derived from an infectious clone of VEEV strain INH-9813, a subtype IC strain isolated from a human infection. VEEV-exposed macaques developed a biphasic fever after infection similar to that seen in humans. Maximum temperature deviation correlated with the inhaled dose, but fever duration did not. Neurological signs, suggestive of virus penetration into the CNS, were predominantly seen in the second febrile period. Electroencephalography data indicated a statistically significant decrease in all power bands and circadian index during the second febrile period that returned to normal after fever resolved. Intracranial pressure increased late in the second febrile period. On day 6 post-infection macaques had high levels of MCP-1 and IP-10 chemokines in the CNS, as well as a marked increase of T lymphocytes and activated microglia. More than four weeks after infection, viral genomic RNA was found in the brain, cerebrospinal fluid and cervical lymph nodes. Pro-inflammatory cytokines & chemokines, infiltrating leukocytes and pathological changes were seen in the CNS tissues of macaques euthanized at these times. These data are consistent with persistence of virus replication and/or genomic RNA and potentially, inflammatory sequelae in the central nervous system after resolution of acute VEEV disease.

Inhibitors of Venezuelan Equine Encephalitis Virus Identified Based on Host Interaction Partners of Viral Non-Structural Protein 3

Venezuelan equine encephalitis virus (VEEV) is a new world alphavirus and a category B select agent. Currently, no FDA-approved vaccines or therapeutics are available to treat VEEV exposure and resultant disease manifestations. The C-terminus of the VEEV non-structural protein 3 (nsP3) facilitates cell-specific and virus-specific host factor binding preferences among alphaviruses, thereby providing targets of interest when designing novel antiviral therapeutics. In this study, we utilized an overexpression construct encoding HA-tagged nsP3 to identify host proteins that interact with VEEV nsP3 by mass spectrometry. Bioinformatic analyses of the putative interactors identified 42 small molecules with the potential to inhibit the host interaction targets, and thus potentially inhibit VEEV. Three inhibitors, tomatidine, citalopram HBr, and Z-VEID-FMK, reduced replication of both the TC-83 strain and the Trinidad donkey (TrD) strain of VEEV by at least 10-fold in astrocytoma, astroglial, and microglial cells. Further, these inhibitors reduced replication of the related New World (NW) alphavirus Eastern equine encephalitis virus (EEEV) in multiple cell types, thus demonstrating broad-spectrum antiviral activity. Time-course assays revealed all three inhibitors reduced both infectious particle production and positive-sense RNA levels post-infection. Further evaluation of the putative host targets for the three inhibitors revealed an interaction of VEEV nsP3 with TFAP2A, but not eIF2S2. Mechanistic studies utilizing siRNA knockdowns demonstrated that eIF2S2, but not TFAP2A, supports both efficient TC-83 replication and genomic RNA synthesis, but not subgenomic RNA translation. Overall, this work reveals the composition of the VEEV nsP3 proteome and the potential to identify host-based, broad spectrum therapeutic approaches to treat new world alphavirus infections.