RESUMEN

Venezuelan equine encephalitis virus (VEEV) is an encephalitic alphavirus that can cause debilitating, acute febrile illness and potentially result in encephalitis. Currently, there are no FDA-licensed vaccines or specific therapeutics for VEEV. Previous studies have demonstrated that VEEV infection results in increased blood-brain barrier (BBB) permeability that is mediated by matrix metalloproteinases (MMPs). Furthermore, after subarachnoid hemorrhage in mice, MMP-9 is upregulated in the brain and mediates BBB permeability in a toll-like receptor 4 (TLR4)-dependent manner. Here, we demonstrate that disease in C3H mice during VEEV TC-83 infection is dependent on TLR4 because intranasal infection of C3H/HeN (TLR4 WT ) mice with VEEV TC-83 resulted in mortality as opposed to survival of TLR4-defective C3H/HeJ (TLR4 mut ) mice. In addition, BBB permeability was induced to a lesser extent in TLR4 mut mice compared with TLR4 WT mice during VEEV TC-83 infection as determined by sodium fluorescein and fluorescently-conjugated dextran extravasation. Moreover, MMP-9, MMP-2, ICAM-1, CCL2 and IFN-γ were all induced to significantly lower levels in the brains of infected TLR4 mut mice compared with infected TLR4 WT mice despite the absence of significantly different viral titers or immune cell populations in the brains of infected TLR4 WT and TLR4 mut mice. These data demonstrate the critical role of TLR4 in mediating BBB permeability and disease in C3H mice during VEEV TC-83 infection, which suggests that TLR4 is a potential target for the development of therapeutics for VEEV.

Asunto(s)

Barrera Hematoencefálica/metabolismo , Virus de la Encefalitis Equina Venezolana/patogenicidad , Receptor Toll-Like 4/genética , Animales , Encéfalo/virología , Modelos Animales de Enfermedad , Virus de la Encefalitis Equina Venezolana/inmunología , Encefalomielitis Equina Venezolana/virología , Femenino , Ratones , Ratones Endogámicos C3H , Permeabilidad , Receptor Toll-Like 4/metabolismo , Replicación Viral

RESUMEN

Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV) are mosquito-borne viruses in the Americas that cause central nervous system (CNS) disease in humans and equids. In this study, we directly characterized the pathogenesis of VEEV, EEEV, and WEEV in cynomolgus macaques following subcutaneous exposure because this route more closely mimics natural infection via mosquito transmission or by an accidental needle stick. Our results highlight how EEEV is significantly more pathogenic compared to VEEV similarly to what is observed in humans. Interestingly, EEEV appears to be just as neuropathogenic by subcutaneous exposure as it was in previously completed aerosol exposure studies. In contrast, subcutaneous exposure of cynomolgus macaques with WEEV caused limited disease and is contradictory to what has been reported for aerosol exposure. Several differences in viremia, hematology, or tissue tropism were noted when animals were exposed subcutaneously compared to prior aerosol exposure studies. This study provides a more complete picture of the pathogenesis of the encephalitic alphaviruses and highlights how further defining the neuropathology of these viruses could have important implications for the development of medical countermeasures for the neurovirulent alphaviruses.

Asunto(s)

Virus de la Encefalitis Equina del Este/patogenicidad , Virus de la Encefalitis Equina Venezolana/patogenicidad , Virus de la Encefalitis Equina del Oeste/patogenicidad , Encefalomielitis Equina/patología , Encefalomielitis Equina Venezolana/patología , Macaca fascicularis/virología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Femenino , Masculino , Replicación Viral

RESUMEN

DNA vaccines expressing codon-optimized Venezuelan equine encephalitis virus (VEEV) and Ebola virus (EBOV) glycoprotein genes provide protective immunity to mice and nonhuman primates when delivered by intramuscular (IM) electroporation (EP). To achieve equivalent protective efficacy in the absence of EP, we evaluated VEEV and EBOV DNA vaccines constructed using minimalized Nanoplasmid expression vectors that are smaller than conventional plasmids used for DNA vaccination. These vectors may also be designed to co-express type I interferon inducing innate immune agonist genes that have an adjuvant effect. Nanoplasmid vaccinated mice had increased antibody responses as compared to those receiving our conventional pWRG7077-based vaccines when delivered by IM injection, and these responses were further enhanced by the inclusion of the innate immune agonist genes. The Nanoplasmid VEEV DNA vaccines also significantly increased protection against aerosol VEEV challenge as compared to the pWRG7077 VEEV DNA vaccine. Although all mice receiving the pWRG7077 and Nanoplasmid EBOV DNA vaccines at the dose tested survived EBOV challenge, only mice receiving the Nanoplasmid EBOV DNA vaccine that co-expresses the innate immune agonist genes failed to lose weight after challenge. Our results suggest that Nanoplasmid vectors can improve the immunogenicity and protective efficacy of alphavirus and filovirus DNA vaccines.

RESUMEN

Venezuelan equine encephalitis virus (VEEV) is a known biological defense threat. A live-attenuated investigational vaccine, TC-83, is available, but it has a high non-response rate and can also cause severe reactogenicity. We generated two novel VEE vaccine candidates using self-amplifying mRNA (SAM). LAV-CNE is a live-attenuated VEE SAM vaccine formulated with synthetic cationic nanoemulsion (CNE) and carrying the RNA genome of TC-83. IAV-CNE is an irreversibly-attenuated VEE SAM vaccine formulated with CNE, delivering a TC-83 genome lacking the capsid gene. LAV-CNE launches a TC-83 infection cycle in vaccinated subjects but eliminates the need for live-attenuated vaccine production and potentially reduces manufacturing time and complexity. IAV-CNE produces a single cycle of RNA amplification and antigen expression without generating infectious viruses in subjects, thereby creating a potentially safer alternative to live-attenuated vaccine. Here, we demonstrated that mice vaccinated with LAV-CNE elicited immune responses similar to those of TC-83, providing 100% protection against aerosol VEEV challenge. IAV-CNE was also immunogenic, resulting in significant protection against VEEV challenge. These studies demonstrate the proof of concept for using the SAM platform to streamline the development of effective attenuated vaccines against VEEV and closely related alphavirus pathogens such as western and eastern equine encephalitis and Chikungunya viruses.

Asunto(s)

Virus de la Encefalitis Equina Venezolana/inmunología , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Amplificación de Genes , Inmunogenicidad Vacunal , ARN Mensajero/genética , Vacunas Atenuadas/uso terapéutico , Vacunas Virales/uso terapéutico , Células A549 , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Modelos Animales de Enfermedad , Emulsiones/química , Encefalomielitis Equina Venezolana/virología , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Transfección , Vacunas Virales/farmacología , Replicación Viral

RESUMEN

We have previously shown that DNA vaccines expressing codon-optimized alphavirus envelope glycoprotein genes protect both mice and non-human primates from viral challenge when delivered by intramuscular electroporation (IM-EP). To determine if we could achieve equivalent immunogenicity and protective efficacy in the absence of electroporation, we co-delivered our Venezuelan equine encephalitis virus (VEEV) DNA vaccine with DNA plasmids expressing genetic adjuvants designed to augment immune responses. We tested the Th1-inducing cytokine IL-12 as well as the granulocyte growth factor GM-CSF, both of which have demonstrated significant adjuvant effect when included in clinical DNA vaccine formulations. Additionally, as multiple reports have described the necessity of IFN-αß in DNA vaccine immunogenicity, we tested vaccine plasmids encoding a potent stimulator of the IFN-αß pathway. Our data suggest that IM vaccination of mice with plasmid DNA encoding genetic adjuvants enhances VEEV vaccine immunogenicity, resulting in improved T cell responses, as well as skewing of the anti-VEEV IgG antibody isotype. Additionally, IM vaccination of VEEV DNA vaccine and IL-12 provided complete protection against aerosol VEEV challenge. Overall, our data suggest that co-delivery of genetic adjuvants with alphavirus DNA vaccines using IM delivery can influence the type of immune response obtained and provide comparable protective immunity to that achieved by IM-EP delivery of the vaccine without adjuvants.

Asunto(s)

Adyuvantes Inmunológicos , Encefalomielitis Equina Venezolana/prevención & control , Inmunogenicidad Vacunal , Interleucina-12/inmunología , Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Virus de la Encefalitis Equina Venezolana , Encefalomielitis Equina Venezolana/inmunología , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Inyecciones Intramusculares , Interleucina-12/genética , Ratones , Ratones Endogámicos BALB C

RESUMEN

There remains a need for vaccines that can safely and effectively protect against the biological threat agents Venezuelan (VEEV), western (WEEV), and eastern (EEEV) equine encephalitis virus. Previously, we demonstrated that a VEEV DNA vaccine that was optimized for increased antigen expression and delivered by intramuscular (IM) electroporation (EP) elicited robust and durable virus-specific antibody responses in multiple animal species and provided complete protection against VEEV aerosol challenge in mice and nonhuman primates. Here, we performed a comparative evaluation of the immunogenicity and protective efficacy of individual optimized VEEV, WEEV, and EEEV DNA vaccines with that of a 1 : 1 : 1 mixture of these vaccines, which we have termed the 3-EEV DNA vaccine, when delivered by IM EP. The individual DNA vaccines and the 3-EEV DNA vaccine elicited robust and durable virus-specific antibody responses in mice and rabbits and completely protected mice from homologous VEEV, WEEV, and EEEV aerosol challenges. Taken together, the results from these studies demonstrate that the individual VEEV, WEEV, and EEEV DNA vaccines and the 3-EEV DNA vaccine delivered by IM EP provide an effective means of eliciting protection against lethal encephalitic alphavirus infections in a murine model and represent viable next-generation vaccine candidates that warrant further development.

Asunto(s)

Alphavirus , Virus de la Encefalitis Equina del Este/inmunología , Encefalomielitis Equina/inmunología , Encefalomielitis Equina/prevención & control , Vectores Genéticos , Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Aerosoles , Alphavirus/genética , Alphavirus/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos/inmunología , Modelos Animales de Enfermedad , Electroporación , Femenino , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Inmunidad Celular/inmunología , Inmunización , Ratones , Conejos , Vacunas de ADN/administración & dosificación , Vacunas Virales/administración & dosificación

RESUMEN

Immunoinformatics tools were used to predict human leukocyte antigen (HLA) class II-restricted T cell epitopes within the envelope glycoproteins and nucleocapsid proteins of Ebola virus (EBOV) and Sudan virus (SUDV) and the structural proteins of Venezuelan equine encephalitis virus (VEEV). Selected epitopes were tested for binding to soluble HLA molecules representing 5 class II alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, and DRB1*1501). All but one of the 25 tested peptides bound to at least one of the DRB1 alleles, and 4 of the peptides bound at least moderately or weakly to all 5 DRB1 alleles. Additional algorithms were used to design a single "string-of-beads" expression construct with 44 selected epitopes arranged to avoid creation of spurious junctional epitopes. Seventeen of these 44 predicted epitopes were conserved between the major histocompatibility complex (MHC) of humans and mice, allowing initial testing in mice. BALB/c mice vaccinated with the multi-epitope construct developed statistically significant cellular immune responses to EBOV, SUDV, and VEEV peptides as measured by interferon (IFN)-γ ELISpot assays. Significant levels of antibodies to VEEV, but not EBOV, were also detected in vaccinated BALB/c mice. To assess immunogenicity in the context of a human MHC, HLA-DR3 transgenic mice were vaccinated with the multi-epitope construct and boosted with a mixture of the 25 peptides used in the binding assays. The vaccinated HLA-DR3 mice developed significant cellular immune responses to 4 of the 25 (16%) tested individual class II peptides as measured by IFN-γ ELISpot assays. In addition, these mice developed antibodies against EBOV and VEEV as measured by ELISA. While a low but significant level of protection was observed in vaccinated transgenic mice after aerosol exposure to VEEV, no protection was observed after intraperitoneal challenge with mouse-adapted EBOV. These studies provide proof of concept for the use of an informatics approach to design a multi-agent, multi-epitope immunogen and provide a basis for further testing aimed at focusing immune responses toward desired protective T cell epitopes.

Asunto(s)

Ebolavirus/inmunología , Virus de la Encefalitis Equina Venezolana/inmunología , Epítopos de Linfocito T/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Animales , Ebolavirus/genética , Virus de la Encefalitis Equina Venezolana/genética , Ensayo de Immunospot Ligado a Enzimas , Epítopos de Linfocito T/genética , Femenino , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Interferón gamma/metabolismo , Ratones Endogámicos BALB C , Ratones Transgénicos , Unión Proteica , Linfocitos T/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética , Vacunas Virales/administración & dosificación , Vacunas Virales/genética

RESUMEN

Venezuelan equine encephalitis virus (VEEV), a mosquito-borne alphavirus, causes periodic epizootics in equines and is a recognized biological defense threat for humans. There are currently no FDA-licensed vaccines against VEEV. We developed a candidate DNA vaccine expressing the E3-E2-6K-E1 genes of VEEV (pWRG/VEE) and performed a Phase 1 clinical study to assess the vaccine's safety, reactogenicity, tolerability, and immunogenicity when administered by intramuscular (IM) or intradermal (ID) electroporation (EP) using the Ichor Medical Systems TriGrid™ Delivery System. Subjects in IM-EP groups received 0.5mg (N=8) or 2.0mg (N=9) of pWRG/VEE or a saline placebo (N=4) in a 1.0ml injection. Subjects in ID-EP groups received 0.08mg (N=8) or 0.3mg (N=8) of DNA or a saline placebo (N=4) in a 0.15ml injection. Subjects were monitored for a total period of 360 days. No vaccine- or device-related serious adverse events were reported. Based on the results of a subject questionnaire, the IM- and ID-EP procedures were both considered to be generally acceptable for prophylactic vaccine administration, with the acute tolerability of ID EP delivery judged to be greater than that of IM-EP delivery. All subjects (100%) in the high and low dose IM-EP groups developed detectable VEEV-neutralizing antibodies after two or three administrations of pWRG/VEE, respectively. VEEV-neutralizing antibody responses were detected in seven of eight subjects (87.5%) in the high dose and five of eight subjects (62.5%) in the low dose ID-EP groups after three vaccine administrations. There was a correlation between the DNA dose and the magnitude of the resulting VEEV-neutralizing antibody responses for both IM and ID EP delivery. These results indicate that pWRG/VEE delivered by either IM- or ID-EP is safe, tolerable, and immunogenic in humans at the evaluated dose levels. Clinicaltrials.gov registry number NCT01984983.

Asunto(s)

Encefalomielitis Equina Venezolana/prevención & control , Vacunas de ADN/administración & dosificación , Vacunas Virales/administración & dosificación , Adolescente , Adulto , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Método Doble Ciego , Electroporación , Virus de la Encefalitis Equina Venezolana , Femenino , Humanos , Inmunogenicidad Vacunal , Inyecciones Intradérmicas , Inyecciones Intramusculares , Masculino , Persona de Mediana Edad , Vacunas de ADN/uso terapéutico , Vacunas Virales/uso terapéutico , Adulto Joven

RESUMEN

Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50 = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.

Asunto(s)

Antivirales/farmacología , Virus de la Encefalitis Equina Venezolana/efectos de los fármacos , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Quinazolinonas/farmacología , Animales , Línea Celular , Chlorocebus aethiops , Cricetinae , Modelos Animales de Enfermedad , Farmacorresistencia Viral/genética , Virus de la Encefalitis Equina Venezolana/genética , Encefalomielitis Equina Venezolana/virología , Ensayos Analíticos de Alto Rendimiento , Ratones , Ratones Endogámicos C3H , Especificidad de la Especie , Relación Estructura-Actividad , Células Vero , Ensayo de Placa Viral , Replicación Viral/efectos de los fármacos

RESUMEN

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.

Asunto(s)

Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Virus de la Encefalitis Equina Venezolana/inmunología , Encefalomielitis Equina Venezolana/prevención & control , Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Animales , Modelos Animales de Enfermedad , Electroporación , Virus de la Encefalitis Equina Venezolana/genética , Encefalomielitis Equina Venezolana/patología , Femenino , Fiebre/prevención & control , Glicoproteínas/genética , Glicoproteínas/inmunología , Linfopenia/prevención & control , Macaca , Masculino , Ratones , Ratones Endogámicos BALB C , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Factores de Tiempo , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/genética , Viremia/prevención & control

RESUMEN

We have developed a high-resolution genomic mapping technique that combines transposon-mediated insertional mutagenesis with either capillary electrophoresis or massively parallel sequencing to identify functionally important regions of the Venezuelan equine encephalitis virus (VEEV) genome. We initially used a capillary electrophoresis method to gain insight into the role of the VEEV nonstructural protein 3 (nsP3) in viral replication. We identified several regions in nsP3 that are intolerant to small (15 bp) insertions, and thus are presumably functionally important. We also identified nine separate regions in nsP3 that will tolerate small insertions at low temperatures (30°C), but not at higher temperatures (37°C, and 40°C). Because we found this method to be extremely effective at identifying temperature sensitive (ts) mutations, but limited by capillary electrophoresis capacity, we replaced the capillary electrophoresis with massively parallel sequencing and used the improved method to generate a functional map of the entire VEEV genome. We identified several hundred potential ts mutations throughout the genome and we validated several of the mutations in nsP2, nsP3, E3, E2, E1 and capsid using single-cycle growth curve experiments with virus generated through reverse genetics. We further demonstrated that two of the nsP3 ts mutants were attenuated for virulence in mice but could elicit protective immunity against challenge with wild-type VEEV. The recombinant ts mutants will be valuable tools for further studies of VEEV replication and virulence. Moreover, the method that we developed is applicable for generating such tools for any virus with a robust reverse genetics system.

Asunto(s)

Mapeo Cromosómico/métodos , Virus de la Encefalitis Equina Venezolana/genética , Genoma Viral/genética , Mutagénesis Insercional/métodos , Análisis de Secuencia de ADN/métodos , Animales , Células Cultivadas , Chlorocebus aethiops , Virus de la Encefalitis Equina Venezolana/fisiología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Ratones , Ratones Endogámicos BALB C , Homología de Secuencia de Ácido Nucleico , Células Vero , Virulencia/genética , Replicación Viral/genética

RESUMEN

A study to evaluate the immunogenicity and protective efficacy of a Venezuelan equine encephalitis virus (VEEV) DNA vaccine in an aerosol model of nonhuman primate infection was performed. Cynomolgus macaques vaccinated with a plasmid expressing the 26S structural genes of VEEV subtype IAB by particle-mediated epidermal delivery (PMED) developed virus-neutralizing antibodies. No serum viremia was detected in two out of three macaques vaccinated with the VEEV DNA after aerosol challenge with homologous virus, while one displayed a low viremia on a single day postchallenge. In contrast, all three macaques vaccinated with empty vector DNA developed a high viremia that persisted for at least 3 days after challenge. In addition, macaques vaccinated with the VEEV DNA had reduced febrile reactions, lymphopenia, and clinical signs of disease postchallenge as compared to negative control macaques. Therefore, although the sample size was small in this pilot study, these results indicate that a VEEV DNA vaccine administered by PMED can at least partially protect nonhuman primates against an aerosol VEEV challenge.

Asunto(s)

Anticuerpos Neutralizantes/sangre , Encefalomielitis Equina Venezolana/prevención & control , Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/sangre , Virus de la Encefalitis Equina Venezolana/inmunología , Femenino , Macaca fascicularis , Masculino , Proyectos Piloto , Proteínas Estructurales Virales/inmunología , Viremia/inmunología

RESUMEN

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.

Asunto(s)

Evolución Molecular Dirigida , Virus de la Encefalitis Equina Venezolana/inmunología , Encefalomielitis Equina Venezolana/prevención & control , Vacunas de ADN/inmunología , Animales , Anticuerpos Antivirales/sangre , Reacciones Cruzadas , Virus de la Encefalitis Equina Venezolana/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Análisis de Supervivencia , Vacunas de ADN/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Ensayo de Placa Viral