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.

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Comparative Neurovirulence and Tissue Tropism of Wild-type and Attenuated Strains of Venezuelan Equine Encephalitis Virus Administered by Aerosol in C3H/HeN and BALB/c Mice

Veterinary Pathology ◽

10.1177/030098589803500508 ◽

1998 ◽

Vol 35 (5) ◽

pp. 386-397 ◽

Cited By ~ 64

Author(s):

K. E. Steele ◽

K. J. Davis ◽

K. Stephan ◽

W. Kell ◽

P. Vogel ◽

...

Keyword(s):

Geometric Mean ◽

Venezuelan Equine Encephalitis Virus ◽

Encephalitis Virus ◽

Wild Type Virus ◽

Tissue Tropism ◽

Venezuelan Equine Encephalitis ◽

Wild Type ◽

Equine Encephalitis Virus ◽

Olfactory Neuroepithelium ◽

The Brain

To assess the potential for aerosol administration of vaccines for Venezuelan equine encephalitis virus (VEE), we compared the neurovirulence and tissue tropism of the wild-type Trinidad donkey (TrD) strain to those of the attenuated TC83 and V3526 strains of VEE in mice. Six to 8-week-old female C3H/HeN and BALB/c mice were aerosol exposed to one of the three VEE strains. Three mice of each strain were euthanatized at different times and their tissues were processed and stained using hematoxylin and eosin, immunohistochemistry, and in situ hybridization. All three viral strains infected the brains of mice and induced encephalitis. TrD spread caudally from the olfactory bulbs to all regions of the brain, caused widespread necrotizing panencephalitis by day 5, and resulted in 100% mortality (geometric mean = 7 days) in both mouse strains. By comparison, TC83 relatively spared the caudal regions of the brain but still caused 100% mortality in the C3H/HeN mice (geometric mean = 12 days), yet it did not kill any BALB/c mice. V3526 infectivity of the brain was the most limited, mainly affecting the neocortex and diencephalon. This virus was not lethal in either mouse strain. The TrD strain also infected the olfactory neuroepithelium, local lymphoid tissues, teeth, and vomeronasal organs, whereas the affinity of TC83 and V3526 outside the brain was essentially limited to the olfactory neuroepithelium. Attenuated VEE strains administered to mice by aerosol have restricted tissue tropism as compared with wild-type virus; however, even attenuated strains can infect the brain and induce encephalitis.

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Approach to Strain Selection and the Propagation of Viral Stocks for Venezuelan Equine Encephalitis Virus Vaccine Efficacy Testing under the Animal Rule

Viruses ◽

10.3390/v11090807 ◽

2019 ◽

Vol 11 (9) ◽

pp. 807 ◽

Cited By ~ 4

Author(s):

Janice M. Rusnak ◽

Pamela J. Glass ◽

Scott C. Weaver ◽

Carol L. Sabourin ◽

Andrew M. Glenn ◽

...

Keyword(s):

Vaccine Efficacy ◽

Model Development ◽

Venezuelan Equine Encephalitis Virus ◽

Lessons Learned ◽

Encephalitis Virus ◽

Strain Selection ◽

Venezuelan Equine Encephalitis ◽

Equine Encephalitis Virus ◽

Viral Vaccine ◽

Animal Rule

Licensure of a vaccine to protect against aerosolized Venezuelan equine encephalitis virus (VEEV) requires use of the U.S. Food and Drug Administration (FDA) Animal Rule to assess vaccine efficacy as human studies are not feasible or ethical. An approach to selecting VEEV challenge strains for use under the Animal Rule was developed, taking into account Department of Defense (DOD) vaccine requirements, FDA Animal Rule guidelines, strain availability, and lessons learned from the generation of filovirus challenge agents within the Filovirus Animal Nonclinical Group (FANG). Initial down-selection to VEEV IAB and IC epizootic varieties was based on the DOD objective for vaccine protection in a bioterrorism event. The subsequent down-selection of VEEV IAB and IC isolates was based on isolate availability, origin, virulence, culture and animal passage history, known disease progression in animal models, relevancy to human disease, and ability to generate sufficient challenge material. Methods for the propagation of viral stocks (use of uncloned (wild-type), plaque-cloned, versus cDNA-cloned virus) to minimize variability in the potency of the resulting challenge materials were also reviewed. The presented processes for VEEV strain selection and the propagation of viral stocks may serve as a template for animal model development product testing under the Animal Rule to other viral vaccine programs. This manuscript is based on the culmination of work presented at the “Alphavirus Workshop” organized and hosted by the Joint Vaccine Acquisition Program (JVAP) on 15 December 2014 at Fort Detrick, Maryland, USA.

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Vaccination of Macaques against Pathogenic Simian Immunodeficiency Virus with Venezuelan Equine Encephalitis Virus Replicon Particles

Journal of Virology ◽

10.1128/jvi.74.1.371-378.2000 ◽

2000 ◽

Vol 74 (1) ◽

pp. 371-378 ◽

Cited By ~ 155

Author(s):

Nancy L. Davis ◽

Ian J. Caley ◽

Kevin W. Brown ◽

Michael R. Betts ◽

David M. Irlbeck ◽

...

Keyword(s):

Viral Load ◽

Simian Immunodeficiency Virus ◽

Limit Of Detection ◽

Rhesus Macaques ◽

Venezuelan Equine Encephalitis Virus ◽

Encephalitis Virus ◽

Effective Vaccine ◽

Venezuelan Equine Encephalitis ◽

Equine Encephalitis Virus ◽

Immunodeficiency Virus

ABSTRACT Vaccine vectors derived from Venezuelan equine encephalitis virus (VEE) that expressed simian immunodeficiency virus (SIV) immunogens were tested in rhesus macaques as part of the effort to design a safe and effective vaccine for human immunodeficiency virus. Immunization with VEE replicon particles induced both humoral and cellular immune responses. Four of four vaccinated animals were protected against disease for at least 16 months following intravenous challenge with a pathogenic SIV swarm, while two of four controls required euthanasia at 10 and 11 weeks. Vaccination reduced the mean peak viral load 100-fold. The plasma viral load was reduced to below the limit of detection (1,500 genome copies/ml) in one vaccinated animal between 6 and 16 weeks postchallenge and in another from week 6 through the last sampling time (40 weeks postchallenge). The extent of reduction in challenge virus replication was directly correlated with the strength of the immune response induced by the vectors, which suggests that vaccination was effective.

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Venezuelan equine encephalitis virus vectors expressing HIV-1 proteins: vector design strategies for improved vaccine efficacy

Vaccine ◽

10.1016/s0264-410x(99)00142-5 ◽

1999 ◽

Vol 17 (23-24) ◽

pp. 3124-3135 ◽

Cited By ~ 53

Author(s):

Ian J Caley ◽

Michael R Betts ◽

Nancy L Davis ◽

Ronald Swanstrom ◽

Jeffrey A Frelinger ◽

...

Keyword(s):

Vaccine Efficacy ◽

Venezuelan Equine Encephalitis Virus ◽

Encephalitis Virus ◽

Venezuelan Equine Encephalitis ◽

Design Strategies ◽

Virus Vectors ◽

Equine Encephalitis Virus ◽

Hiv 1

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Resveratrol Inhibits Venezuelan Equine Encephalitis Virus Infection by Interfering with the AKT/GSK Pathway

Plants ◽

10.3390/plants10020346 ◽

2021 ◽

Vol 10 (2) ◽

pp. 346

Author(s):

Caitlin W. Lehman ◽

Kylene Kehn-Hall ◽

Megha Aggarwal ◽

Nicole R. Bracci ◽

Han-Chi Pan ◽

...

Keyword(s):

Antiviral Activity ◽

Fluorescent Protein ◽

Glycogen Synthase ◽

Venezuelan Equine Encephalitis Virus ◽

Encephalitis Virus ◽

Host Cells ◽

Dynamics Simulation ◽

Luciferase Reporter ◽

Venezuelan Equine Encephalitis ◽

Equine Encephalitis Virus

The host proteins Protein Kinase B (AKT) and glycogen synthase kinase-3 (GSK-3) are associated with multiple neurodegenerative disorders. They are also important for the replication of Venezuelan equine encephalitis virus (VEEV), thereby making the AKT/GSK-3 pathway an attractive target for developing anti-VEEV therapeutics. Resveratrol, a natural phytochemical, has been shown to substantially inhibit the AKT pathway. Therefore, we attempted to explore whether it exerts any antiviral activity against VEEV. In this study, we utilized green fluorescent protein (GFP)- and luciferase-encoding recombinant VEEV to determine the cytotoxicity and antiviral efficacy via luciferase reporter assays, flow cytometry, and immunofluorescent assays. Our results indicate that resveratrol treatment is capable of inhibiting VEEV replication, resulting in increased viability of Vero and U87MG cells as well as reduced virion production and viral RNA contents within host cells for at least 48 h with a single treatment. Furthermore, the suppression of apoptotic signaling adaptors, caspase-3, caspase-7, and annexin V may also be implicated in resveratrol-mediated antiviral activity. We found that decreased phosphorylation of the AKT/GSK-3 pathway, mediated by resveratrol, can be triggered during the early stages of VEEV infection, suggesting that resveratrol disrupts the viral replication cycle and consequently promotes cell survival. Finally, molecular docking and dynamics simulation studies revealed that resveratrol can directly bind to VEEV glycoproteins, which may interfere with virus attachment and entry. In conclusion, our results suggest that resveratrol exerts inhibitory activity against VEEV infection and upon further modification could be a useful compound to study in neuroprotective research and veterinary sciences.

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