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1.  A Multisystem Approach for Development and Evaluation of Inactivated Vaccines for Venezuelan Equine Encephalitis Virus (VEEV) 
A multisystem approach was used to assess the efficiency of several methods for inactivation of Venezuelan equine encephalitis virus (VEEV) vaccine candidates. A combination of diverse assays (plaque, in vitro cytopathology and mouse neurovirulence) was used to verify virus inactivation, along with the use of a specific ELISA to measure retention of VEEV envelope glycoprotein epitopes in the development of several inactivated VEEV candidate vaccines derived from an attenuated strain of VEEV (V3526). Incubation of V3526 aliquots at temperatures in excess of 64°C for periods >30 minutes inactivated the virus, but substantially reduced VEEV specific monoclonal antibody binding of the inactivated material. In contrast, V3526 treated either with formalin at concentrations of 0.1% or 0.5% v/v for 4 or 24 hours, or irradiated with 50 kilogray gamma radiation rendered the virus non-infectious while retaining significant levels of monoclonal antibody binding. Loss of infectivity of both the formalin inactivated (fV3526) and gamma irradiated (gV3526) preparations was confirmed via five successive blind passages on BHK-21 cells. Similarly, loss of neurovirulence for fV3526 and gV3526 was demonstrated via intracerebral inoculation of suckling BALB/c mice. Excellent protection against subcutaneous challenge with VEEV IA/B Trinidad donkey strain was demonstrated using a two dose immunization regimen with either fV3526 or gV3526. The combination of in vitro and in vivo assays provides a practical approach to optimize manufacturing process parameters for development of other inactivated viral vaccines.
doi:10.1016/j.jviromet.2009.11.006
PMCID: PMC2815040  PMID: 19903494
Venezuelan equine encephalitis virus (VEEV); Formalin inactivated vaccines; Gamma irradiated vaccines; Neurovirulence; Alphavirus
2.  Evaluation in Nonhuman Primates of Vaccines against Ebola Virus 
Emerging Infectious Diseases  2002;8(5):503-507.
Ebola virus (EBOV) causes acute hemorrhagic fever that is fatal in up to 90% of cases in both humans and nonhuman primates. No vaccines or treatments are available for human use. We evaluated the effects in nonhuman primates of vaccine strategies that had protected mice or guinea pigs from lethal EBOV infection. The following immunogens were used: RNA replicon particles derived from an attenuated strain of Venezuelan equine encephalitis virus (VEEV) expressing EBOV glycoprotein and nucleoprotein; recombinant Vaccinia virus expressing EBOV glycoprotein; liposomes containing lipid A and inactivated EBOV; and a concentrated, inactivated whole-virion preparation. None of these strategies successfully protected nonhuman primates from robust challenge with EBOV. The disease observed in primates differed from that in rodents, suggesting that rodent models of EBOV may not predict the efficacy of candidate vaccines in primates and that protection of primates may require different mechanisms.
doi:10.3201/eid0805.010284
PMCID: PMC3369765  PMID: 11996686
Keywords: Ebola; macaque; vaccine; Vaccinia virus; replicon
3.  Replication and Clearance of Venezuelan Equine Encephalitis Virus from the Brains of Animals Vaccinated with Chimeric SIN/VEE Viruses 
Journal of Virology  2006;80(6):2784-2796.
Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic pathogen. Recent outbreaks in Venezuela and Colombia in 1995, involving an estimated 100,000 human cases, indicate that VEEV still poses a serious public health threat. To develop a safe, efficient vaccine that protects against disease resulting from VEEV infection, we generated chimeric Sindbis (SIN) viruses expressing structural proteins of different strains of VEEV and analyzed their replication in vitro and in vivo, as well as the characteristics of the induced immune responses. None of the chimeric SIN/VEE viruses caused any detectable disease in adult mice after either intracerebral (i.c.) or subcutaneous (s.c.) inoculation, and all chimeras were more attenuated than the vaccine strain, VEEV TC83, in 6-day-old mice after i.c. infection. All vaccinated mice were protected against lethal encephalitis following i.c., s.c., or intranasal (i.n.) challenge with the virulent VEEV ZPC738 strain (ZPC738). In spite of the absence of clinical encephalitis in vaccinated mice challenged with ZPC738 via i.n. or i.c. route, we regularly detected high levels of infectious challenge virus in the central nervous system (CNS). However, infectious virus was undetectable in the brains of all immunized animals at 28 days after challenge. Hamsters vaccinated with chimeric SIN/VEE viruses were also protected against s.c. challenge with ZPC738. Taken together, our findings suggest that these chimeric SIN/VEE viruses are safe and efficacious in adult mice and hamsters and are potentially useful as VEEV vaccines. In addition, immunized animals provide a useful model for studying the mechanisms of the anti-VEEV neuroinflammatory response, leading to the reduction of viral titers in the CNS and survival of animals.
doi:10.1128/JVI.80.6.2784-2796.2006
PMCID: PMC1395430  PMID: 16501087
4.  A DNA Vaccine for Venezuelan Equine Encephalitis Virus Delivered by Intramuscular Electroporation Elicits High Levels of Neutralizing Antibodies in Multiple Animal Models and Provides Protective Immunity to Mice and Nonhuman Primates ▿ 
We evaluated the immunogenicity and protective efficacy of a DNA vaccine expressing codon-optimized envelope glycoprotein genes of Venezuelan equine encephalitis virus (VEEV) when delivered by intramuscular electroporation. Mice vaccinated with the DNA vaccine developed robust VEEV-neutralizing antibody responses that were comparable to those observed after administration of the live-attenuated VEEV vaccine TC-83 and were completely protected from a lethal aerosol VEEV challenge. The DNA vaccine also elicited strong neutralizing antibody responses in rabbits that persisted at high levels for at least 6 months and could be boosted by a single additional electroporation administration of the DNA performed approximately 6 months after the initial vaccinations. Cynomolgus macaques that received the vaccine by intramuscular electroporation developed substantial neutralizing antibody responses and after an aerosol challenge had no detectable serum viremia and had reduced febrile reactions, lymphopenia, and clinical signs of disease compared to those of negative-control macaques. Taken together, our results demonstrate that this DNA vaccine provides a potent means of protecting against VEEV infections and represents an attractive candidate for further development.
doi:10.1128/CVI.00030-11
PMCID: PMC3122536  PMID: 21450977
5.  IRES-driven Expression of the Capsid Protein of the Venezuelan Equine Encephalitis Virus TC-83 Vaccine Strain Increases Its Attenuation and Safety 
The live-attenuated TC-83 strain is the only licensed veterinary vaccine available to protect equids against Venezuelan equine encephalitis virus (VEEV) and to protect humans indirectly by preventing equine amplification. However, TC-83 is reactogenic due to its reliance on only two attenuating point mutations and has infected mosquitoes following equine vaccination. To increase its stability and safety, a recombinant TC-83 was previously engineered by placing the expression of the viral structural proteins under the control of the Internal Ribosome Entry Site (IRES) of encephalomyocarditis virus (EMCV), which drives translation inefficiently in insect cells. However, this vaccine candidate was poorly immunogenic. Here we describe a second generation of the recombinant TC-83 in which the subgenomic promoter is maintained and only the capsid protein gene is translated from the IRES. This VEEV/IRES/C vaccine candidate did not infect mosquitoes, was stable in its attenuation phenotype after serial murine passages, and was more attenuated in newborn mice but still as protective as TC-83 against VEEV challenge. Thus, by using the IRES to modulate TC-83 capsid protein expression, we generated a vaccine candidate that combines efficient immunogenicity and efficacy with lower virulence and a reduced potential for spread in nature.
Author Summary
Venezuelan equine encephalitis virus (VEEV) is transmitted by mosquitoes and widely distributed in Central and South America, causing regular outbreaks in horses and humans. Often misdiagnosed as dengue, VEEV infection in humans can lead to lifelong neurological sequelae and is fatal in up to >80% of equine cases, representing a significant socio-economic burden and constant public health threats for developing countries of Latin America. The only available vaccine, the live-attenuated TC-83 strain, is restricted to veterinary use due to its high reactogenicity in humans and risk for reversion to virulence, which could initiate an epidemic. By using an attenuation approach that allows the modulation of the virus capsid protein expression, we generated a new version of TC-83 that is more attenuated but still induces a protective immune response in mice. Additionally, this new vaccine cannot infect mosquitoes, which prevents the risk of spreading in nature. The attenuation approach we describe can be applied to a lot of other alphaviruses to develop vaccines against diseases regularly emerging and threatening developing countries.
doi:10.1371/journal.pntd.0002197
PMCID: PMC3649961  PMID: 23675542
6.  Recombinant Sindbis/Venezuelan Equine Encephalitis Virus Is Highly Attenuated and Immunogenic 
Journal of Virology  2003;77(17):9278-9286.
Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic virus. VEEV was a significant human and equine pathogen for much of the past century, and recent outbreaks in Venezuela and Colombia (1995), with about 100,000 human cases, indicate that this virus still poses a serious public health threat. The live attenuated TC-83 vaccine strain of VEEV was developed in the 1960s using a traditional approach of serial passaging in tissue culture of the virulent Trinidad donkey (TrD) strain. This vaccine presents several problems, including adverse, sometimes severe reactions in many human vaccinees. The TC-83 strain also retains residual murine virulence and is lethal for suckling mice after intracerebral (i.c.) or subcutaneous (s.c.) inoculation. To overcome these negative effects, we developed a recombinant, chimeric Sindbis/VEE virus (SIN-83) that is more highly attenuated. The genome of this virus encoded the replicative enzymes and the cis-acting RNA elements derived from Sindbis virus (SINV), one of the least human-pathogenic alphaviruses. The structural proteins were derived from VEEV TC-83. The SIN-83 virus, which contained an additional adaptive mutation in the nsP2 gene, replicated efficiently in common cell lines and did not cause detectable disease in adult or suckling mice after either i.c. or s.c. inoculation. However, SIN-83-vaccinated mice were efficiently protected against challenge with pathogenic strains of VEEV. Our findings suggest that the use of the SINV genome as a vector for expression of structural proteins derived from more pathogenic, encephalitic alphaviruses is a promising strategy for alphavirus vaccine development.
doi:10.1128/JVI.77.17.9278-9286.2003
PMCID: PMC187387  PMID: 12915543
7.  Stability of RNA Virus Attenuation Approaches 
Vaccine  2011;29(12):2230-2234.
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.
doi:10.1016/j.vaccine.2011.01.055
PMCID: PMC3060670  PMID: 21288800
vaccine stability; RNA viruses; alphavirus; Venezuelan equine encephalitis virus
8.  Host responses to live-attenuated Venezuelan equine encephalitis virus (TC-83) 
Human Vaccines & Immunotherapeutics  2012;8(8):1053-1065.
Venezuelan equine encephalitis virus (VEEV) is a positive-strand RNA Alphavirus endemic in Central and South America, and the causative agent of fatal encephalitis in humans. In an effort to better understand the mechanisms of infection, including differences between people who produce a neutralizing antibody response to the vaccine and those who do not, we performed whole genome transcriptional analysis in human PBMCs exposed in vitro to the live-attenuated vaccine strain of VEEV, TC-83. We compared the molecular responses in cells from three groups of individuals: naïve; previously vaccinated individuals who developed a neutralizing antibody response to the vaccine (responders); and those who did not develop a neutralizing antibody response to the vaccine (nonresponders). Overall, the changes in gene expression were more intense for the naïve group after TC-83 challenge and least potent in the nonresponder group. The main canonical pathways revealed the involvement of interferon and interferon-induced pathways, as well as toll-like receptors TLR- and interleukin (IL)-12-related pathways. HLA class II genotype and suppression of transcript expression for TLR2, TLR4 and TLR8 in the nonresponder group may help explain the lack of vaccine response in this study group. Because TL3 and TLR7 transcripts were elevated in all study groups, these factors may be indicators of the infection and not the immunological state of the individuals. Biomarkers were identified that differentiate between the vaccine responder and the vaccine nonresponder groups. The identified biomarkers were contrasted against transcripts that were unique to the naïve population alone upon induction with TC-83. Biomarker analysis allowed for the discernment between the naïve (innate) responses; the responder (recall) responses; and the nonresponder (alternative) changes to gene transcription that were caused by infection with TC-83. The study also points to the existence of HLA haplotypes that may discriminate between vaccine low- and high-responder phenotypes.
doi:10.4161/hv.20300
PMCID: PMC3551876  PMID: 22617845
biomarkers; gene expression; microarray; neutralizing antibody; vaccination; vaccine responder; Venezuelan equine encephalitis virus
9.  Development of a novel monoclonal antibody with reactivity to a wide range of Venezuelan equine encephalitis virus strains 
Virology Journal  2009;6:206.
Background
There is currently a requirement for antiviral therapies capable of protecting against infection with Venezuelan equine encephalitis virus (VEEV), as a licensed vaccine is not available for general human use. Monoclonal antibodies are increasingly being developed as therapeutics and are potential treatments for VEEV as they have been shown to be protective in the mouse model of disease. However, to be truly effective, the antibody should recognise multiple strains of VEEV and broadly reactive monoclonal antibodies are rarely and only coincidentally isolated using classical hybridoma technology.
Results
In this work, methods were developed to reliably derive broadly reactive murine antibodies. A phage library was created that expressed single chain variable fragments (scFv) isolated from mice immunised with multiple strains of VEEV. A broadly reactive scFv was identified and incorporated into a murine IgG2a framework. This novel antibody retained the broad reactivity exhibited by the scFv but did not possess virus neutralising activity. However, the antibody was still able to protect mice against VEEV disease induced by strain TrD when administered 24 h prior to challenge.
Conclusion
A monoclonal antibody possessing reactivity to a wide range of VEEV strains may be of benefit as a generic antiviral therapy. However, humanisation of the murine antibody will be required before it can be tested in humans.
Crown Copyright © 2009
doi:10.1186/1743-422X-6-206
PMCID: PMC2783036  PMID: 19925641
10.  IRES-Based Venezuelan Equine Encephalitis Vaccine Candidate Elicits Protective Immunity in Mice 
Virology  2013;437(2):81-88.
Venezuelan equine encephalitis virus (VEEV) is an arbovirus that causes periodic outbreaks that impact equine and human populations in the Americas. One of the VEEV subtypes located in Mexico and Central America (IE) has recently been recognized as an important cause of equine disease and death, and human exposure also appears to be widespread. Here, we describe the use of an Internal Ribosome Entry Site (IRES) from encephalomyocarditis virus to stably attenuate VEEV, creating a vaccine candidate independent of unstable point mutations. Mice infected with this virus produced antibodies and were protected against lethal VEEV challenge. This IRES-based vaccine was unable to establish productive infection in mosquito cell cultures or in intrathoracically injected Aedes taeniorhynchus, demonstrating that it cannot be transmitted from a vaccinee. These attenuation, efficacy and safety results justify further development for humans or equids of this new VEEV vaccine candidate.
doi:10.1016/j.virol.2012.11.013
PMCID: PMC3767167  PMID: 23351391
Venezuelan equine encephalitis virus; vaccine; alphavirus; internal ribosome entry site
11.  Novel Vaccine against Venezuelan Equine Encephalitis Combines Advantages of DNA Immunization and a Live Attenuated Vaccine 
Vaccine  2012;31(7):1019-1025.
DNA vaccines combine remarkable genetic and chemical stability with proven safety and efficacy in animal models, while remaining less immunogenic in humans. In contrast, live-attenuated vaccines have the advantage of inducing rapid, robust, long-term immunity after a single-dose vaccination. Here we describe novel iDNA vaccine technology that is based on an infectious DNA platform and combines advantages of DNA and live attenuated vaccines. We applied this technology for vaccination against infection with Venezuelan equine encephalitis virus (VEEV), an alphavirus from the Togaviridae family. The iDNA vaccine is based on transcription of the full-length genomic RNA of the TC-83 live-attenuated virus from plasmid DNA in vivo. The in vivo-generated viral RNA initiates limited replication of the vaccine virus, which in turn leads to efficient immunization. This technology allows the plasmid DNA to launch a live-attenuated vaccine in vitro or in vivo. Less than 10 ng of pTC83 iDNA encoding the full-length genomic RNA of the TC-83 vaccine strain initiated replication of the vaccine virus in vitro. In order to evaluate this approach in vivo, BALB/c mice were vaccinated with a single dose of pTC83 iDNA. After vaccination, all mice seroconverted with no adverse reactions. Four weeks after immunization, animals were challenged with the lethal epidemic strain of VEEV. All iDNA-vaccinated mice were protected from fatal disease, while all unvaccinated controls succumbed to infection and died. To our knowledge, this is the first example of launching a clinical live-attenuated vaccine from recombinant plasmid DNA in vivo.
doi:10.1016/j.vaccine.2012.12.050
PMCID: PMC3556218  PMID: 23287629
DNA vaccine; live attenuated virus; infectious DNA; Venezuelan equine encephalitis; VEE; TC-83
12.  4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus 
The EMBO Journal  2011;30(18):3854-3863.
4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus
This study uses high-resolution cryo-electron microscopy to provide a complete structural model of the VEEV alphavirus, bridging the gap between incomplete crystal structures and lower resolution electron microscopy analyses.
Venezuelan equine encephalitis virus (VEEV), a member of the membrane-containing Alphavirus genus, is a human and equine pathogen, and has been developed as a biological weapon. Using electron cryo-microscopy (cryo-EM), we determined the structure of an attenuated vaccine strain, TC-83, of VEEV to 4.4 Å resolution. Our density map clearly resolves regions (including E1, E2 transmembrane helices and cytoplasmic tails) that were missing in the crystal structures of domains of alphavirus subunits. These new features are implicated in the fusion, assembly and budding processes of alphaviruses. Furthermore, our map reveals the unexpected E3 protein, which is cleaved and generally thought to be absent in the mature VEEV. Our structural results suggest a mechanism for the initial stage of nucleocapsid core formation, and shed light on the virulence attenuation, host recognition and neutralizing activities of VEEV and other alphavirus pathogens.
doi:10.1038/emboj.2011.261
PMCID: PMC3173789  PMID: 21829169
alphavirus; bioweapon; cryo-EM; modelling; VEEV
13.  Immune Parameters Correlate with Protection Against Ebola Virus Infection in Rodents and Nonhuman Primates 
Science translational medicine  2012;4(158):158ra146.
Ebola virus causes severe hemorrhagic fever in susceptible hosts. Currently, no licensed vaccines or treatments are available; however, several experimental vaccines have been successful in protecting rodents and nonhuman primates (NHPs) from the lethal Zaire ebolavirus (ZEBOV) infection. The objective of this study was to evaluate immune responses correlating with survival in these animals after lethal challenge with ZEBOV. Knockout mice with impaired ability to generate normal T and/or B cell responses were vaccinated and challenged with ZEBOV. Vaccine-induced protection in mice was mainly mediated by B cells and CD4+ T cells. Vaccinated, outbred guinea pigs and NHPs demonstrated the highest correlation between survival and levels of total immunoglobulin G (IgG) specific to the ZEBOV glycoprotein (ZGP). These results highlight the relevance of total ZGP-specific IgG levels as a meaningful correlate of protection against ZEBOV exposure.
doi:10.1126/scitranslmed.3004582
PMCID: PMC3789651  PMID: 23115355
14.  A mouse-based assay for the pre-clinical neurovirulence assessment of vaccinia virus-based smallpox vaccines 
Post-vaccinal encephalitis, although relatively uncommon, is a known adverse event associated with many live, attenuated smallpox vaccines. Although smallpox vaccination ceased globally in 1980, vaccine manufacture has resumed in response to concerns over the possible use of smallpox virus as an agent of bioterrorism. To better support the production of safer smallpox vaccines, we previously reported the development of a mouse model in which a relatively attenuated vaccine strain (Dryvax®) could be discerned from a more virulent laboratory strain (WR). Here we have further tested the performance of this assay by evaluating the neurovirulence of several vaccinia virus-based smallpox vaccines spanning a known range in neurovirulence for humans. Our data indicate that testing of 10 to 100 pfu of virus in mice following intracranial inoculation reliably assesses the virus’s neurovirulence potential for humans.
doi:10.1016/j.biologicals.2009.09.007
PMCID: PMC2849879  PMID: 19896867
15.  Salmonella typhimurium virulence in a burned-mouse model. 
Infection and Immunity  1989;57(9):2842-2846.
Various features of salmonellosis were examined in a burned-mouse model. In this model, which uses an outbred mouse strain, a challenge dose of ca. 100 CFU with any of several strains of Salmonella typhimurium caused a fatal infection. A variety of mutated strains attenuated for virulence in Salmonella-susceptible parenterally infected mice were also attenuated in the burned-mouse model. When administered as live vaccines injected intraperitoneally the same attenuated strains provided between slight and complete protection against subsequent lethal challenge subcutaneously at the site of a burn. The correspondence of results obtained in the burned-mouse model with those seen in other mouse models coupled with the unique advantages of the burned-mouse model argue for the usefulness of the model in studies of salmonellosis and in testing of strains constructed for use as live vaccines.
PMCID: PMC313536  PMID: 2668190
16.  Immunization with SARS Coronavirus Vaccines Leads to Pulmonary Immunopathology on Challenge with the SARS Virus 
PLoS ONE  2012;7(4):e35421.
Background
Severe acute respiratory syndrome (SARS) emerged in China in 2002 and spread to other countries before brought under control. Because of a concern for reemergence or a deliberate release of the SARS coronavirus, vaccine development was initiated. Evaluations of an inactivated whole virus vaccine in ferrets and nonhuman primates and a virus-like-particle vaccine in mice induced protection against infection but challenged animals exhibited an immunopathologic-type lung disease.
Design
Four candidate vaccines for humans with or without alum adjuvant were evaluated in a mouse model of SARS, a VLP vaccine, the vaccine given to ferrets and NHP, another whole virus vaccine and an rDNA-produced S protein. Balb/c or C57BL/6 mice were vaccinated IM on day 0 and 28 and sacrificed for serum antibody measurements or challenged with live virus on day 56. On day 58, challenged mice were sacrificed and lungs obtained for virus and histopathology.
Results
All vaccines induced serum neutralizing antibody with increasing dosages and/or alum significantly increasing responses. Significant reductions of SARS-CoV two days after challenge was seen for all vaccines and prior live SARS-CoV. All mice exhibited histopathologic changes in lungs two days after challenge including all animals vaccinated (Balb/C and C57BL/6) or given live virus, influenza vaccine, or PBS suggesting infection occurred in all. Histopathology seen in animals given one of the SARS-CoV vaccines was uniformly a Th2-type immunopathology with prominent eosinophil infiltration, confirmed with special eosinophil stains. The pathologic changes seen in all control groups lacked the eosinophil prominence.
Conclusions
These SARS-CoV vaccines all induced antibody and protection against infection with SARS-CoV. However, challenge of mice given any of the vaccines led to occurrence of Th2-type immunopathology suggesting hypersensitivity to SARS-CoV components was induced. Caution in proceeding to application of a SARS-CoV vaccine in humans is indicated.
doi:10.1371/journal.pone.0035421
PMCID: PMC3335060  PMID: 22536382
17.  Antibody to the E3 Glycoprotein Protects Mice against Lethal Venezuelan Equine Encephalitis Virus Infection▿  
Journal of Virology  2010;84(24):12683-12690.
Six monoclonal antibodies were isolated that exhibited specificity for a furin cleavage site deletion mutant (V3526) of Venezuelan equine encephalitis virus (VEEV). These antibodies comprise a single competition group and bound the E3 glycoprotein of VEEV subtype I viruses but failed to bind the E3 glycoprotein of other alphaviruses. These antibodies neutralized V3526 virus infectivity but did not neutralize the parental strain of Trinidad donkey (TrD) VEEV. However, the E3-specific antibodies did inhibit the production of virus from VEEV TrD-infected cells. In addition, passive immunization of mice demonstrated that antibody to the E3 glycoprotein provided protection against lethal VEEV TrD challenge. This is the first recognition of a protective epitope in the E3 glycoprotein. Furthermore, these results indicate that E3 plays a critical role late in the morphogenesis of progeny virus after E3 appears on the surfaces of infected cells.
doi:10.1128/JVI.01345-10
PMCID: PMC3004303  PMID: 20926570
18.  Improved Efficacy of a Gene Optimised Adenovirus-based Vaccine for Venezuelan Equine Encephalitis Virus 
Virology Journal  2009;6:118.
Background
Optimisation of genes has been shown to be beneficial for expression of proteins in a range of applications. Optimisation has increased protein expression levels through improved codon usage of the genes and an increase in levels of messenger RNA. We have applied this to an adenovirus (ad)-based vaccine encoding structural proteins (E3-E2-6K) of Venezuelan equine encephalitis virus (VEEV).
Results
Following administration of this vaccine to Balb/c mice, an approximately ten-fold increase in antibody response was elicited and increased protective efficacy compared to an ad-based vaccine containing non-optimised genes was observed after challenge.
Conclusion
This study, in which the utility of optimising genes encoding the structural proteins of VEEV is demonstrated for the first time, informs us that including optimised genes in gene-based vaccines for VEEV is essential to obtain maximum immunogenicity and protective efficacy.
doi:10.1186/1743-422X-6-118
PMCID: PMC2732613  PMID: 19646224
19.  Pegylated Alpha Interferon Is an Effective Treatment for Virulent Venezuelan Equine Encephalitis Virus and Has Profound Effects on the Host Immune Response to Infection 
Journal of Virology  2000;74(11):5006-5015.
Venezuelan equine encephalitis virus (VEEV) is a highly infectious alphavirus endemic in parts of Central and South America. The disease is transmitted by mosquitoes, and the natural reservoir is the small rodent population, with epidemics occurring in horses and occasionally humans. Following infection, VEEV replicates in lymphoid tissues prior to invasion of the central nervous system. Treatment of VEEV-infected BALB/c mice with polyethylene glycol-conjugated alpha interferon (PEG IFN-α) results in a greatly enhanced survival from either a subcutaneous or an aerosol infection. Virus is undetectable within PEG IFN-α-treated individuals by day 30 postinfection (p.i.). Treatment results in a number of changes to the immune response characteristics normally associated with VEEV infection. Increased macrophage activation occurs in PEG IFN-α-treated BALB/c mice infected with VEEV. The rapid activation of splenic CD4, CD8, and B cells by day 2 p.i. normally associated with VEEV infection is absent in PEG IFN-α-treated mice. The high tumor necrosis factor alpha production by macrophages from untreated mice is greatly diminished in PEG IFN-α-treated mice. These results suggest key immunological mechanisms targeted by this lethal alphavirus that can be modulated by prolonged exposure to IFN-α.
PMCID: PMC110852  PMID: 10799574
20.  Transmission Potential of Two Chimeric Chikungunya Vaccine Candidates in the Urban Mosquito Vectors, Aedes aegypti and Ae. albopictus 
Chikungunya virus (CHIKV) is an emerging, mosquito-borne alphavirus that has caused major epidemics in Africa and Asia. We developed chimeric vaccine candidates using the non-structural protein genes of either Venezuelan equine encephalitis virus (VEEV) attenuated vaccine strain TC-83 or a naturally attenuated strain of eastern equine encephalitis virus (EEEV) and the structural genes of CHIKV. Because the transmission of genetically modified live vaccine strains is undesirable because of the potentially unpredictable evolution of these viruses as well as the potential for reversion, we evaluated the ability of these vaccines to infect the urban CHIKV vectors, Aedes aegypti and Ae. albopictus. Both vaccine candidates exhibited significantly lower infection and dissemination rates compared with the parent alphaviruses. Intrathoracic inoculations indicated that reduced infectivity was mediated by midgut infection barriers in both species. These results indicate a low potential for transmission of these vaccine strains in the event that a vaccinee became viremic.
doi:10.4269/ajtmh.2011.11-0049
PMCID: PMC3110354  PMID: 21633043
21.  Alpha-beta T cells provide protection against lethal encephalitis in the murine model of VEEV infection 
Virology  2007;367(2):307-323.
We evaluated the safety and immunogenicity of a chimeric alphavirus vaccine candidate in mice with selective immunodeficiencies. This vaccine candidate was highly attenuated in mice with deficiencies in the B and T cell compartments, as well as in mice with deficient gamma-interferon responsiveness. However, the level of protection varied among the strains tested. Wild type mice were protected against lethal VEEV challenge. In contrast, alpha/beta (αβ) TCR-deficient mice developed lethal encephalitis following VEEV challenge, while mice deficient in gamma/delta ( γδ) T cells were protected. Surprisingly, the vaccine potency was diminished by 50% in animals lacking interferon-gamma receptor alpha chain (R1)-chain and a minority of vaccinated immunoglobulin heavy chain-deficient (μMT) mice survived challenge, which suggests that neutralizing antibody may not be absolutely required for protection. Prolonged replication of encephalitic VEEV in the brain of pre-immunized mice is not lethal and adoptive transfer experiments indicate that CD3+ T cells are required for protection.
doi:10.1016/j.virol.2007.05.041
PMCID: PMC2067255  PMID: 17610927
alphavirus immunity; recombinant vaccines; Venezuelan equine encephalitis virus; pathogenesis
22.  The 5′UTR-specific mutation in VEEV TC-83 genome has a strong effect on RNA replication and subgenomic RNA synthesis, but not on translation of the encoded proteins 
Virology  2009;387(1):211-221.
Venezuelan equine encephalitis virus (VEEV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. Viruses in the VEEV serocomplex continuously circulate in the Central and South Americas. The only currently available attenuated strain VEEV TC-83 is being used only for vaccination of at-risk laboratory workers and military personnel. Its attenuated phenotype was shown to rely only on two point mutations, one of which, G3A, was found in the 5′ untranslated region (5′UTR) of the viral genome. Our data demonstrate that the G3A mutation strongly affects the secondary structure of VEEV 5′UTR, but has only a minor effect on translation. The indicated mutation increases replication of the viral genome, downregulates transcription of the subgenomic RNA, and, thus, affects the ratio of genomic and subgenomic RNA synthesis. These findings and the previously reported G3A-induced, higher sensitivity of VEEV TC-83 to IFN-α/β suggest a plausible explanation for its attenuated phenotype.
doi:10.1016/j.virol.2009.02.027
PMCID: PMC2675632  PMID: 19278709
23.  C3H/HeN mouse model for the evaluation of antiviral agents for the treatment of Venezuelan equine encephalitis virus infection 
Antiviral research  2008;78(3):230-241.
The TC-83 vaccine strain of Venezuelan equine encephalitis virus (VEEV) causes encephalitis and death in C3H/HeN mice infected by intranasal instillation. Since TC-83 is exempt as a Select Agent, this mouse model was used in the evaluation of antiviral therapies. Virus titers in the brains of infected mice peaked on 4 dpi and persisted at high levels until death at 9.4 ± 0.5 dpi. Mouse brains appeared histologically normal on 2 dpi, but developed meningoencephalitis, neuropil vacuolation, and gliosis by 8 dpi. Results from a protein cytokine array showed significant elevations over time in IL-1α, IL-1β, IL-6, IL-12, MCP-1, IFNγ, TNFα, MIP-1α, and RANTES in homogenized brain samples of infected mice. Immunohistochemical staining showed a colocalization of viral antigen with neuron markers. Treatment with interferon-α B/D or ampligen significantly improved survival, brain virus titer and cytokine levels, mean day-to-death, and weight change in infected mice. The time-course of infection and disease parameters of mice infected with TC-83 VEEV were similar in many ways to disease parameters in mice infected with other VEEV strains. Thus, infection of C3H/HeN mice with TC-83 VEEV may serve as a suitable model for the evaluation of antiviral compounds for the treatment of this viral disease.
doi:10.1016/j.antiviral.2008.01.007
PMCID: PMC2396595  PMID: 18313150
24.  Immune Responses of Mice with Different Genetic Backgrounds to Improved Multiepitope, Multitarget Malaria Vaccine Candidate Antigen FALVAC-1A ▿  
Clinical and Vaccine Immunology : CVI  2008;15(11):1674-1683.
FALVAC-1A is a second-generation multitarget, multiepitope synthetic candidate vaccine against Plasmodium falciparum, incorporating elements designed to yield a stable and immunogenic molecule. Characteristics of the immunogenicity of FALVAC-1A were evaluated in congenic (H-2b, H-2k, and H-2d) and outbred strains of mice. The influences of four adjuvants (aluminum phosphate, QS-21, Montanide ISA-720, and copolymer CRL-1005) on different aspects of the immune response were also assessed. FALVAC-1A generated strong antibody responses in all mouse strains. The highest mean enzyme-linked immunosorbent assay (ELISA) antibody concentrations against FALVAC-1A were observed in the outbred ICR mice, followed by B10.BR, B10.D2, and C57BL/6 mice, though this order varied for the different adjuvants, with no statistical differences between mouse strains. In all mouse strains, the highest anti-FALVAC-1A antibody titers in ELISAs were induced by FALVAC-1A in copolymer and ISA-720 formulations, followed by QS-21 and AlPO4. These antibodies were of all four subclasses, though immunoglobulin G1 (IgG1) predominated, with the exception of FALVAC-1A with the QS-21 adjuvant, which induced predominantly IgG2c responses. Both sporozoites and blood stages of P. falciparum were recognized by anti-FALVAC-1A sera in the immunofluorescence assay. In addition to antibody, cellular immune responses were detected; these responses were studied by examining spleen cells producing gamma interferon and interleukin-4 in enzyme-linked immunospot assays. In summary, FALVAC-1A was found to be highly immunogenic and elicited functionally relevant antibodies that can recognize sporozoites and blood-stage parasites in diverse genetic backgrounds.
doi:10.1128/CVI.00164-08
PMCID: PMC2583527  PMID: 18784343
25.  A DNA Vaccine-Encoded Nucleoprotein of Influenza Virus Fails To Induce Cellular Immune Responses in a Diabetic Mouse Model▿  
Influenza virus infections cause yearly epidemics and are a major cause of lower respiratory tract illnesses in humans worldwide. Influenza virus has long been recognized to be associated with higher morbidity and mortality in diabetic patients. Vaccination is an effective tool to prevent influenza virus infection in this group of patients. Vaccines employing recombinant-DNA technologies are an alternative to inactivated virus and live attenuated virus vaccines. Internal highly conserved viral nucleoprotein (NP) can be delivered as a DNA vaccine to provide heterosubtypic immunity, offering resistance against various influenza virus strains. In this study, we investigated the efficacy of an NP DNA vaccine for induction of cell-mediated immune responses and protection against influenza virus infection in a mouse model of diabetes. Healthy and diabetic BALB/c mice were immunized on days 0, 14, and 28 by injection of NP DNA vaccine. Two weeks after the last immunization, the cellular immune response was evaluated by gamma interferon (IFN-γ), lymphocyte proliferation, and cytotoxicity assays. The mice were challenged with influenza virus, and the viral titers in the lungs were measured on day 4. Diabetic mice showed significantly smaller amounts of IFN-γ production, lymphocyte proliferation, and cytotoxicity responses than nondiabetic mice. Furthermore, higher titers of the influenza virus were detected after challenge in the lungs of the diabetic mice. The present data suggest that the NP DNA vaccine with the protocol of immunization described here is not able to induce efficient cellular immune responses against influenza virus infection in diabetic mice.
doi:10.1128/CVI.00445-09
PMCID: PMC2849332  PMID: 20164252

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