RESUMO
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.
RESUMO
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.
Assuntos
Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/tratamento farmacológico , Amplificação de Genes , Imunogenicidade da Vacina , RNA Mensageiro/genética , Vacinas Atenuadas/uso terapêutico , Vacinas Virais/uso terapêutico , Células A549 , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Modelos Animais de Doenças , Emulsões/química , Encefalomielite Equina Venezuelana/virologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transfecção , Vacinas Virais/farmacologia , Replicação ViralRESUMO
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.
Assuntos
Adjuvantes Imunológicos , Encefalomielite Equina Venezuelana/prevenção & controle , Imunogenicidade da Vacina , Interleucina-12/imunologia , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Encefalite Equina Venezuelana , Encefalomielite Equina Venezuelana/imunologia , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Injeções Intramusculares , Interleucina-12/genética , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Licensure of medical countermeasure vaccines to protect against aerosolized Venezuelan equine encephalitis virus (VEEV) requires the use of the Animal Rule to assess vaccine efficacy, because human studies are not feasible or ethical. We therefore performed a retrospective study of VEE cases that occurred in at-risk laboratory workers and support personnel during the United States Biowarfare Program (1943-1969) to better define percutaneous- and aerosol-acquired VEE in humans and to compare these results with those described for the NHP model (in which high-dose aerosol VEEV challenge led to more severe encephalitis than parenteral challenge). Record review and analysis of 17 aerosol- and 23 percutaneous-acquired human cases of VEE included incubation period, symptoms, physical examination findings, and markers of infection. Human VEE disease by both exposure routes presented as acute febrile illness, typically with fever, chills, headache, back pain, malaise, myalgia, anorexia, and nausea. Aerosol exposure more commonly led to upper respiratory tract-associated findings of sore throat (59% compared with 26%), pharyngeal erythema (76% compared with 52%), neck pain (29% compared with 4%), and cervical lymphadenopathy (29% compared with 4%). Other disease manifestations, including encephalitis, were similar between the 2 exposure groups. The increase in upper respiratory tract findings in aerosol-acquired VEE in humans has not previously been reported but is supported by the mouse model, which showed nasal mucosal necrosis, necrotizing rhinitis, and an increase in upper respiratory tract viral burden associated with aerosol VEEV challenge. Fever, viremia, and lymphopenia were common markers of VEE disease in both humans and NHP, regardless of the exposure route. Taken collectively, our findings provide support for use of the nonlethal NHP model for advanced development of medical countermeasures against aerosol- or percutaneous-acquired VEE.
Assuntos
Encefalomielite Equina Venezuelana/prevenção & controle , Primatas/virologia , Vacinas Virais/uso terapêutico , Aerossóis , Animais , Anticorpos Antivirais/sangue , Armas Biológicas , Modelos Animais de Doenças , Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/transmissão , Humanos , Período de Incubação de Doenças Infecciosas , Testes de Neutralização , Primatas/imunologia , Estudos Retrospectivos , Resultado do TratamentoRESUMO
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.
Assuntos
Alphavirus , Vírus da Encefalite Equina do Leste/imunologia , Encefalomielite Equina/imunologia , Encefalomielite Equina/prevenção & controle , Vetores Genéticos , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Aerossóis , Alphavirus/genética , Alphavirus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/imunologia , Modelos Animais de Doenças , Eletroporação , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Imunidade Celular/imunologia , Imunização , Camundongos , Coelhos , Vacinas de DNA/administração & dosagem , Vacinas Virais/administração & dosagemRESUMO
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.
Assuntos
Ebolavirus/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Ebolavirus/genética , Vírus da Encefalite Equina Venezuelana/genética , ELISPOT , Epitopos de Linfócito T/genética , Feminino , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Interferon gama/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ligação Proteica , Linfócitos T/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
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.
Assuntos
Encefalomielite Equina Venezuelana/prevenção & controle , Vacinas de DNA/administração & dosagem , Vacinas Virais/administração & dosagem , Adolescente , Adulto , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Método Duplo-Cego , Eletroporação , Vírus da Encefalite Equina Venezuelana , Feminino , Humanos , Imunogenicidade da Vacina , Injeções Intradérmicas , Injeções Intramusculares , Masculino , Pessoa de Meia-Idade , Vacinas de DNA/uso terapêutico , Vacinas Virais/uso terapêutico , Adulto JovemRESUMO
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.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/prevenção & controle , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Modelos Animais de Doenças , Eletroporação , Vírus da Encefalite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana/patologia , Feminino , Febre/prevenção & controle , Glicoproteínas/genética , Glicoproteínas/imunologia , Linfopenia/prevenção & controle , Macaca , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Fatores de Tempo , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Viremia/prevenção & controleRESUMO
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.
Assuntos
Anticorpos Neutralizantes/sangue , Encefalomielite Equina Venezuelana/prevenção & controle , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Vírus da Encefalite Equina Venezuelana/imunologia , Feminino , Macaca fascicularis , Masculino , Projetos Piloto , Proteínas Estruturais Virais/imunologia , Viremia/imunologiaRESUMO
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.