Onset and duration of protective immunity to IA/IB and IE strains of Venezuelan equine encephalitis virus in vaccinated mice.

Three vaccines developed for protection against IA/IB subtypes of Venezuelan equine encephalitis (VEE) virus were evaluated in mice for the ability to protect against systemic and mucosal challenges with a virulent virus of the IE subtype. The vaccines were the formaldehyde-inactivated C-84 and live attenuated TC-83 vaccines currently administered to people under investigational new drug (IND) status, and a new live attenuated vaccine candidate, V3526. V3526 was superior for inducing protection to VEE IA/IB within a week of vaccination, and protection persisted for at least a year. All three vaccines induced long-term clinical protection against peripheral or mucosal challenge with IE virus, with the mucosal immunity induced by attenuated vaccines lasting longer than that induced by the inactivated vaccine. These data show that the molecularly cloned V3526 vaccine induces equivalent or improved immunity to homologous and heterologous VEE viruses than the existing vaccines.     

Improved mucosal protection against Venezuelan equine encephalitis virus is induced by the molecularly defined, live-attenuated V3526 vaccine candidate

The genetically engineered, live-attenuated Venezuelan equine encephalitis (VEE) virus vaccine candidate, V3526, was evaluated as a replacement for the TC-83 virus vaccine. Protection from lethal subcutaneous or aerosol challenge was evaluated in vaccinated mice clinically and immunohistochemically. Subcutaneous administration of V3526 induced systemic and mucosal protection more efficiently than did the TC-83 vaccine. The bronchial IgA responses induced in mice by subcutaneous administration of vaccines significantly corresponded to the ability to survive aerosol challenge with virulent virus. Furthermore, V3526 delivered by aerosol induced more complete mucosal protection than either vaccine administered subcutaneously. The ability of V3526 to induce protection in mice warrants its consideration for further testing as a potential vaccine candidate for human use.     

Toxicity assessment of Venezuelan Equine Encephalitis virus vaccine candidate strain V3526

DynPort Vaccine Company (DVC) LLC, a CSC Company, is under a contract with the United States Department of Defense Joint Vaccine Acquisition Program (JVAP) to develop, test and license safe and efficacious vaccines against biowarfare agents. As part of this program DVC is conducting a comprehensive toxicological assessment of the safety of V3526, a live attenuated Venezuelan Equine Encephalitis (VEE) vaccine candidate. Our review of the published pre-clinical toxicology literature, together with the data collected from DVC-sponsored investigations indicated V3526 was comparatively safer and more efficacious than TC-83, the current VEE Investigational New Drug (IND) status vaccine. Non-clinical toxicity studies on experimental systems ranging from mouse, guinea pig, equine and non-human primates (NHP) consistently revealed the V3526 vaccine candidate superior in terms of safety and related toxicological parameters such as neurovirulence when compared with the TC-83. Our experimental investigations indicated that V3526 may conform to the key requirements of a VEE virus vaccine, in that, (a) it elicits a high level of immunogenic response in mice and hamsters, both sensitive to VEE-induced pathologies, and (b) it induces a protective response in the NHP model when challenged with either virulent IA/B or IE viruses. Additional studies are underway to further confirm these findings.     

Venezuelan equine encephalitis virus vaccine candidate (V3526) safety, immunogenicity and efficacy in horses

A new vaccine, V3526, is a live-attenuated virus derived by site-directed mutagenesis from a virulent clone of the Venezuelan equine encephalitis virus (VEEV) IA/B Trinidad donkey (TrD) strain, intended for human use in protection against Venezuelan equine encephalitis (VEE). Two studies were conducted in horses to evaluate the safety, immunogenicity, ability to boost and protective efficacy of V3526 against challenges of TrD and VEEV IE 64A99. Horses were vaccinated subcutaneously (SC) with 10(7), 10(5), 10(3) or 10(2) plaque-forming units (pfu) of V3526. Control horses were sham immunized. In the first study, challenge viruses (TrD or 64A99) were administered SC 28 days post-vaccination (PV). No viremia and only mild fluctuation in white blood cell counts were observed PV. None of the V3526 vaccinated horses showed clinical signs of disease or pathology of VEE post-challenge (PC). In contrast, control horses challenged SC with 10(4)pfu TrD became viremic and showed classical signs of VEE beginning on Day 3 PC, including elevated body temperature, anorexia, leukopenia and malaise. Moderate to severe encephalitis was found in three of five control horses challenged with TrD. Control horses challenged with 64A99 failed to develop detectable viremia, but did exhibit a brief febrile episode at 1-3 days PC. None of the 10 immunized horses challenged with 64A99 became pyrexic. Twenty four of 25 horses immunized with V3526 in the first study developed serum neutralizing antibody to TrD and 64A99 within 14 days PV. Vaccinations with V3526, at doses as low as 10(2)pfu, were safe and efficacious in protecting horses against a virulent TrD virus challenge. The second study supported that repeat dosing resulted in an increase in serum neutralizing antibody to TrD.     

1,5-Iodonaphthyl azide-inactivated V3526 protects against aerosol challenge with virulent venezuelan equine encephalitis virus

Venezuelan equine encephalitis virus (VEEV) is a New World alphavirus. VEEV is highly infectious in aerosolized form and has been identified as a bio-terrorism agent. There is no licensed vaccine for prophylaxis against VEEV. The current IND vaccine is poorly immunogenic and does not protect against an aerosol challenge with virulent VEEV. We have previously shown that VEEV inactivated by 1,5-iodonaphthyl azide (INA) protects against footpad challenge with virulent VEEV. In this study, we inactivated an attenuated strain of VEEV, V3526, with INA and evaluated its protective efficacy against aerosol challenge with wild type VEEV. We demonstrated that among three routes of immunization, intramuscular immunization with INA-inactivate V3526 (INA-iV3526) provided complete protection against aerosol challenge with virulent VEEV. Our data suggests that INA-iV3526 can be explored further for development as an effective vaccine candidate against aerosol challenge of virulent VEEV.     

Safety and protective efficacy of INA-inactivated Venezuelan equine encephalitis virus: implication in vaccine development

We have previously shown that a hydrophobic alkylating compound, 1,5-iodonaphthyl-azide (INA) can efficiently inactivate the virulent strain of Venezuelan equine encephalitis virus (VEEV), V3000 in vitro. In this study, we have evaluated the safety of INA-inactivated V3000 and V3526 and the protective efficacy of INA-inactivated V3000. INA-inactivated V3000 and V3526 did not cause disease in suckling mice. RNA isolated from the INA-inactivated V3000 and V3526 was also not infectious. Immunization of adult mice with INA-inactivated V3000 induced an anti-VEEV antibody response and protected mice from virulent VEEV challenge. The protective efficacy of INA-inactivated V3000 increased with the use of adjuvants. Results suggest that inactivation of enveloped viruses by INA may occur by two independent mechanisms and the INA-inactivated VEEV elicit a protective antibody response in mice.     

Stability of RNA virus attenuation approaches

The greatest risk from live-attenuated vaccines is reversion to virulence. Particular concerns arise for RNA viruses, which exhibit high mutation frequencies. We examined the stability of 3 attenuation strategies for the alphavirus, Venezuelan equine encephalitis virus (VEEV): a traditional, point mutation-dependent attenuation approach exemplified by TC-83; a rationally designed, targeted-mutation approach represented by V3526; and a chimeric vaccine, SIN/TC/ZPC. Our findings suggest that the chimeric strain combines the initial attenuation of TC-83 with the greater phenotypic stability of V3526, highlighting the importance of the both initial attenuation and stability for live-attenuated vaccines.     

Neurovirulence evaluation of Venezuelan equine encephalitis (VEE) vaccine candidate V3526 in nonhuman primates

Assessment of neurovirulence is a standard test for vaccines derived from virulent neurotropic viruses. This study evaluated the potential neurovirulence of V3526, a live attenuated vaccine derived from a full-length infectious clone of Venezuelan equine encephalitis virus (VEEV) Trinidad donkey strain (TrD), a comparator VEEV vaccine (TC-83), TrD, and process control material (PCM) in juvenile rhesus macaques. Following intrathalamic/intraspinal (i.t./i.s. ) or subcutaneous (s.c.) inoculations, animals were observed for periods of 18, 91 or 181 days for paresis, paralysis, neurological disorders and other signs of clinical illness. Blood was collected for measurement of viremia, VEEV neutralizing antibodies, hematologic parameters, and liver enzymes. Gross necropsies and histopathological examinations were conducted with emphasis on detecting lesions in the brain and spinal cord. Elevated temperatures (1-2 degrees C) were noted in several of the TrD and vaccine inoculated animals on Day 6 following inoculation and mean temperatures for the V3526 i.t./i.s. and TC-83 groups were higher than PCM group throughout the study Day 18. No significant differences were seen for weight or clinical chemistry results between vaccine and PCM inoculated groups. Clinically significant signs (Grades 3 or 4) were noted in three of 21 V3526 i.t./i.s. and three of 12 TC-83 inoculated animals, however, these signs resolved within 3 weeks for all V3526 i.t./i.s. and for two of three TC-83 inoculated animals. At Day 18 extensive lesions indicative of a viral infection were seen in brain sections of all four TrD inoculated animals and one of seven V3526 i.t./i.s. inoculated animals. Only scattered lesions, characterized by foci of gliosis and vessels with perivascular inflammation, were found in the sections from four TC-83 and six V3526 i.t./i.s. inoculated animals. The minimal histological changes observed at Day 18 resolved to baseline levels by Day 181 comparable to the PCM group. V3526 was immunogenic and essentially nonneurovirulent when administered via the clinically relevant subcutaneous route.     

Venezuelan equine encephalitis vaccine with rearranged genome resists reversion and protects non-human primates from viremia after aerosol challenge

Live-attenuated V4020 vaccine for Venezuelan equine encephalitis virus (VEEV) containing attenuating rearrangement of the virus structural genes was evaluated in a non-human primate model for immunogenicity and protective efficacy against aerosol challenge with wild-type VEEV. The genomic RNA of V4020 vaccine virus was encoded in the pMG4020 plasmid under control of the CMV promoter and contained the capsid gene downstream from the glycoprotein genes. It also included attenuating mutations from the VEE TC83 vaccine, with E2-120Arg substitution genetically engineered to prevent reversion mutations. The population of V4020 vaccine virus derived from pMG4020-transfected Vero cells was characterized by next generation sequencing (NGS) and indicated no detectable genetic reversions. Cynomolgus macaques were vaccinated with V4020 vaccine virus. After one or two vaccinations including by intramuscular route, high levels of virus-neutralizing antibodies were confirmed with no viremia or apparent adverse reactions to vaccinations. The protective effect of vaccination was evaluated using an aerosol challenge with VEEV. After challenge, macaques had no detectable viremia, demonstrating a protective effect of vaccination with live V4020 VEEV vaccine.     

Environmental hazard assessment of Venezuelan equine encephalitis virus vaccine candidate strain V3526

A hazard assessment of Venezuelan equine encephalitis (VEE) virus sub-types and vaccine candidates was performed according to standard risk assessment procedures. Data from published literature demonstrates a considerable degree of safety of V3526 when compared to TC-83 vaccine, the protective measure that has been used to protect laboratory workers for over four decades. V3526 is a new recombinant vaccine candidate that is a vastly different product with a diminished hazard to public health and the general environment. A weight-of-evidence (WOE)-based scheme was employed to assign weights for relevance, quality, and adequacy of evidence in published literature on medical pathology, epidemiology, pre-clinical investigational studies, and environmental studies. The results of this assessment indicated that V3526 has a low adverse impact on public health and the general environment. Although there are currently no human infectivity or pathogenicity data for V3526, existing evidence from published experimental animal studies reveals a diminished hazard for environmental transmission and distribution. Recently, the US Centers for Disease Control and Prevention (CDC) excluded V3526 from select agent requirements set forth under the Health and Human Services (HHS) regulations in Title 42 C.F.R. Part 73 and the US Department Agriculture (USDA) regulations set forth in Title 7 C.F.R. Part 331 and Title 9 C.F.R. Part 121. This paper summarizes the background, rationale, and hazard analysis used for assessing the environmental hazard of the VEE vaccine candidate strain V3526.     

Evaluation of formalin inactivated V3526 virus with adjuvant as a next generation vaccine candidate for Venezuelan equine encephalitis virus

V3526, a genetically modified strain of Venezuelan equine encephalitis virus (VEEV), was formalin inactivated for evaluation as a next generation vaccine candidate for VEEV. In this study, we tested formalin-inactivated V3526 (fV3526) with and without adjuvant for immunogenicity and efficacy in BALB/c mice and results were compared to the existing inactivated VEEV vaccine, C84. Mice were vaccinated intramuscularly (IM) or subcutaneously (SC) with fV3526 formulations and challenged with VEEV IAB Trinidad donkey (VEEV TrD) strain by SC or aerosol exposure. Efficacy following SC or aerosol challenge was not significantly different between the fV3526 formulations or compared to C84 despite C84 being administered in more doses and higher concentration of viral protein per dose. These data support further evaluation of fV3526 formulations as a next generation VEEV vaccine.     

Potential for Central American mosquitoes to transmit epizootic and enzootic strains of Venezuelan equine encephalitis virus.

Experimental studies were undertaken to compare the vector competence of Culex (Melanoconion) taeniopus Dyar and Knab, Culex (Melanoconion) ocossa Dyar and Knab, and Psorophora confinnis (Lynch Arribalzalga) from Central America for epizootic (IAB) and enzootic (IE) strains of Venezuelan equine encephalitis (VEE) virus. Virus infection and dissemination rates were significantly higher in Cx. taeniopus orally exposed to IE as compared to those orally exposed to IAB virus. In contrast, both infection and dissemination rates were similar in Cx. ocossa exposed to either IAB or IE strains of VEE virus. Thus, susceptibility to epizootic and enzootic strains of VEE virus seems to be species specific within the subgenus Culex (Melanoconion). Both species transmitted each strain of VEE virus after intrathoracic inoculation, indicating that a midgut barrier affected vector competence in these species. Psorophora confinnis was equally susceptible to both IAB and IE viruses, but apparently had a salivary gland barrier, as only 1 of 16 mosquitoes with a disseminated infection transmitted VEE virus by bite.     

Venezuelan equine encephalitis virus infection of cotton rats

Venezuelan equine encephalitis virus (VEEV) is an emerging pathogen of equids and humans, but infection of its rodent reservoir hosts has received little study. To determine whether responses to infection vary among geographic populations, we inoculated 3 populations of cotton rats with 2 enzootic VEEV strains (Co97-0054 [enzootic ID subtype] and 68U201 [enzootic IE subtype]). The 3 populations were offspring from wild-caught cotton rats collected in a VEE-enzootic area of south Florida, USA; wild-caught cotton rats from a non-VEE-enzootic area of Texas, USA; and commercially available (Harlan) colony-reared cotton rats from a non-VEE-enzootic region. Although each population had similar early viremia titers, no detectable disease developed in the VEE-sympatric Florida animals, but severe disease and death affected the Texas and Harlan animals. Our findings suggest that the geographic origins of cotton rats are important determinants of the outcome of VEE infection and reservoir potential of these rodents.     

委内瑞拉马脑炎的研究进展

委内瑞拉马脑炎（Venezuelan equine encephalitis，VEE）是由委内瑞拉马脑炎病毒复合物（VEEV复合物）引起的自然疫源性疾病，历史上引起了多次暴发流行。VEEV还是一种潜在的、可以用于战争和恐怖活动的生物武器。本文就VEE的病原学、流行病学、临床表现、实验室检测、治疗和预防等方面予以综述。     

Recombinant RNA replicons derived from attenuated Venezuelan equine encephalitis virus protect guinea pigs and mice from Ebola hemorrhagic fever virus

RNA replicons derived from an attenuated strain of Venezuelan equine encephalitis virus (VEE), an alphavirus, were configured as candidate vaccines for Ebola hemorrhagic fever. The Ebola nucleoprotein (NP) or glycoprotein (GP) genes were introduced into the VEE RNA downstream from the VEE 26S promoter in place of the VEE structural protein genes. The resulting recombinant replicons, expressing the NP or GP genes, were packaged into VEE replicon particles (NP-VRP and GP-VRP, respectively) using a bipartite helper system that provided the VEE structural proteins in trans and prevented the regeneration of replication-competent VEE during packaging. The immunogenicity of NP-VRP and GP-VRP and their ability to protect against lethal Ebola infection were evaluated in BALB/c mice and in two strains of guinea pigs. The GP-VRP alone, or in combination with NP-VRP, protected both strains of guinea pigs and BALB/c mice, while immunization with NP-VRP alone protected BALB/c mice, but neither strain of guinea pig. Passive transfer of sera from VRP-immunized animals did not confer protection against lethal challenge. However, the complete protection achieved with active immunization with VRP, as well as the unique characteristics of the VEE replicon vector, warrant further testing of the safety and efficacy of NP-VRP and GP-VRP in primates as candidate vaccines against Ebola hemorrhagic fever.     

Directed molecular evolution improves the immunogenicity and protective efficacy of a Venezuelan equine encephalitis virus DNA vaccine

We employed directed molecular evolution to improve the cross-reactivity and immunogenicity of the Venezuelan equine encephalitis virus (VEEV) envelope glycoproteins. The DNA encoding the E1 and E2 proteins from VEEV subtypes IA/B and IE, Mucambo virus (MUCV), and eastern and western equine encephalitis viruses (EEEV and WEEV) were recombined in vitro to create libraries of chimeric genes expressing variant envelope proteins. ELISAs specific for all five parent viruses were used in high-throughput screening to identify those recombinant DNAs that demonstrated cross-reactivity to VEEV, MUCV, EEEV, and WEEV after administration as plasmid vaccines in mice. Selected variants were then used to vaccinate larger cohorts of mice and their sera were assayed by both ELISA and by plaque reduction neutralization test (PRNT). Representative variants from a library in which the E1 gene from VEEV IA/B was held constant and only the E2 genes of the five parent viruses were recombined elicited significantly increased neutralizing antibody titers to VEEV IA/B compared to the parent DNA vaccine and provided improved protection against aerosol VEEV IA/B challenge. Our results indicate that it is possible to improve the immunogenicity and protective efficacy of alphavirus DNA vaccines using directed molecular evolution.     

Vaccination of macaques with SIV immunogens delivered by Venezuelan equine encephalitis virus replicon particle vectors followed by a mucosal challenge with SIVsmE660

VEE replicon particles (VRP), non-propagating vaccine vectors derived from Venezuelan equine encephalitis virus (VEE), were engineered to express immunogens from the cloned isolate SIVsmH-4, combined in a vaccine cocktail and inoculated subcutaneously to immunize rhesus macaques. The virulent, uncloned challenge stock, SIVsmE660, represented a type of heterologous challenge and the intrarectal challenge modeled infection across a mucosal surface. Prechallenge neutralizing antibodies against SIVsmH-4 were induced in all vaccinates, and a prechallenge cellular immune response could be detected in one of six. Post-challenge, virus loads were reduced at the peak, at set point and at termination (41 weeks post-challenge), although these differences did not reach statistical significance. Significantly elevated levels of CD4+ T cells were observed post-challenge. A strong correlation was noted between a net increase in CD4+ T cell count and lowered virus load at set point.     

Immune interference after sequential alphavirus vaccine vaccinations

We compared the effect of order of administration of investigational alphavirus vaccines on neutralizing antibody response. Volunteers who received the inactivated eastern and western equine encephalitis (EEE and WEE) vaccines before live attenuated Venezuelan (VEE) vaccine had significantly lower rates of antibody response than those receiving VEE vaccine before EEE and WEE vaccines (66.7% vs. 80.6%; p = 0.026). The odds of having a VEE antibody non-response among those initially receiving EEE and WEE vaccines, adjusted for gender, were significant (odds ratio [OR] = 2.20; 95% CI = 1.2–4.1 [p = 0.0145]) as were the odds of non-response among females adjusted for group (OR = 1.81; 95% CI = 1.2–2.7 [p = 0.0037]). Antibody interference and gender effect have major implications for vaccine strategy among those receiving multiple alphavirus vaccines and those developing next generation vaccines for these threats.     

Equine amplification and virulence of subtype IE Venezuelan equine encephalitis viruses isolated during the 1993 and 1996 Mexican epizootics

To assess the role of horses as amplification hosts during the 1993 and 1996 Mexican Venezuelan equine encephalitis (VEE) epizootics, we subcutaneously infected 10 horses by using four different equine isolates. Most horses showed little or no disease and low or nonexistent viremia. Neurologic disease developed in only 1 horse, and brain histopathologic examination showed meningeal lymphocytic infiltration, perivascular cuffing, and focal encephalitis. Three animals showed mild meningoencephalitis without clinical disease. Viral RNA was detected in the brain of several animals 12-14 days after infection. These data suggest that the duration and scope of the recent Mexican epizootics were limited by lack of equine amplification characteristic of previous, more extensive VEE outbreaks. The Mexican epizootics may have resulted from the circulation of a more equine-neurotropic, subtype IE virus strain or from increased transmission to horses due to amplification by other vertebrate hosts or transmission by more competent mosquito vectors.