RESUMEN
Viruses in the family Filoviridae, including the commonly known Ebola (EBOV) and Marburg (MARV) viruses, can cause severe hemorrhagic fever in humans and nonhuman primates. Sporadic outbreaks of filovirus disease occur in sub-Saharan Africa with reported case fatality rates ranging from 25% to 90%. The high mortality and increasing frequency and magnitude of recent outbreaks along with the increased potential for spread from rural to urban areas highlight the importance of pandemic preparedness for these viruses. Despite their designation as high-priority pathogens, numerous scientific gaps exist in critical areas. In this review, these gaps and an assessment of potential prototype pathogen candidates are presented for this important virus family.
Asunto(s)
Ebolavirus , Filoviridae , Fiebre Hemorrágica Ebola , Marburgvirus , Animales , Humanos , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/prevención & control , Brotes de EnfermedadesRESUMEN
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
There is a pressing need for sustainable and sensitive immunodiagnostics for use in public health efforts to understand and combat the threat of endemic and emerging infectious diseases. In this proof-of-concept work, we describe an immunodiagnostic approach based on the utilization of virus-like particles (VLPs) in a magnetic bead-based platform for multiplexed detection of antiviral humoral response. A retroviral-based VLP, that presents Venezuelan equine encephalitis virus E1/E2 glycoprotein antigen on its surface, was synthesized and coupled to magnetic beads to create VLP-conjugated microspheres (VCMs). Using these VCMs, IgM and IgG antibodies were detectable in nonhuman primate (NHP) and human clinical serum samples at dilutions of 1 × 10 Basile et al. [4] and greater. We also extended the VCM methodology to an Old World alphavirus, chikungunya virus, demonstrating the flexibility of this approach toward different VLP architectures. When multiplexed on the MAGPIX® platform, this method provided differential detection between Old World and New World alphaviral IgM. This flexible, immunodiagnostic method, based on the MAGPIX® platform, demonstrates compatibility of particulate antigens with bead-based assays, improves sensitivity by up to 2-logs, and has faster sample-to-answer time over traditional methods.
Asunto(s)
Infecciones por Alphavirus/diagnóstico , Inmunidad Humoral , Inmunoensayo/métodos , Proteínas del Envoltorio Viral/inmunología , Alphavirus/genética , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Humanos , Inmunización , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Pruebas Inmunológicas , Cinética , Microesferas , Modelos Animales , Prueba de Estudio Conceptual , Proteínas del Envoltorio Viral/genéticaRESUMEN
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 ViralRESUMEN
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.
Asunto(s)
Encefalomielitis Equina Venezolana/prevención & control , Primates/virología , Vacunas Virales/uso terapéutico , Aerosoles , Animales , Anticuerpos Antivirales/sangre , Armas Biológicas , Modelos Animales de Enfermedad , Virus de la Encefalitis Equina Venezolana/inmunología , Encefalomielitis Equina Venezolana/inmunología , Encefalomielitis Equina Venezolana/transmisión , Humanos , Periodo de Incubación de Enfermedades Infecciosas , Pruebas de Neutralización , Primates/inmunología , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
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 CRESUMEN
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 & dosificaciónRESUMEN
In previous studies, we showed that deoxyribonucleic acid (DNA) vaccines expressing codon-optimized filovirus envelope glycoprotein genes protect mice and nonhuman primates from viral challenge when delivered by intramuscular (IM) electroporation (EP). To determine whether we could achieve equivalent immunogenicity and protective efficacy by a simplified delivery method, we generated DNA vaccine plasmids expressing genetic adjuvants to potentiate immune responses. We tested the Th1-inducing cytokine interleukin-12 and the granulocyte growth factor granulocyte-macrophage colony stimulating factor, both of which have demonstrated significant adjuvant effect when included in clinical DNA vaccine formulations. In addition, because interferon (IFN)-αß is required for DNA vaccine-induced immunity, we tested inclusion of a potent stimulator of the IFN-αß pathway. Our data suggest that IM vaccination of mice with plasmid DNA encoding genetic adjuvants enhances vaccine immunogenicity, resulting in increased anti-Ebola virus (EBOV) immunoglobulin G and T-cell responses. Codelivery of genetic adjuvants also improved EBOV neutralizing capability compared with vaccine alone. Finally, IM vaccination with plasmid EBOV and genetic adjuvants provided complete protection against EBOV challenge. Overall, our data suggest that codelivery of genetic adjuvants with filovirus DNA vaccines using IM delivery can provide comparable efficacy to the same DNA vaccines when delivered using IM-EP devices.
Asunto(s)
Vacunas contra el Virus del Ébola/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Interleucina-12/farmacología , Vacunas de ADN/inmunología , Animales , Células COS , Chlorocebus aethiops , Vacunas contra el Virus del Ébola/administración & dosificación , Electroporación , Femenino , Glicoproteínas/genética , Inmunogenicidad Vacunal , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Plásmidos , Vacunas de ADN/administración & dosificaciónRESUMEN
Ebola virus (EBOV) infection is highly lethal and results in severe febrile bleeding disorders that affect humans and non-human primates. One of the therapeutic approaches for treating EBOV infection focus largely on cocktails of monoclonal antibodies (mAbs) that bind to specific regions of the EBOV glycoprotein (GP) and neutralize the virus. Recent structural studies using cryo-electron microscopy have identified key epitopes for several EBOV mAbs. While such information has yielded deep insights into antibody binding, limitations on resolution of these structures often preclude a residue-level analysis of EBOV epitopes. In this study, we performed combinatorial peptide-based epitope mapping of EBOV GP against a broad panel of mAbs and polyclonal sera derived from several animal species vaccinated with EBOV DNA and replicon vaccines and/or exposed to EBOV infection to identify residue-level determinants of antibody binding. The peptide-based epitope mapping obtained from a wide range of serum and mAb samples, combined with available cryo-EM structure reconstructions revealed fine details of antibody-virus interactions, allowing for a more precise and comprehensive mapping of antibody epitopes on EBOV GP. We show how these residue-level epitope definitions can be used to characterize antigenic variation across different filoviruses, and provide a theoretical basis for predicting immunity and cross-neutralization in potential future outbreaks.
Asunto(s)
Anticuerpos Antivirales/inmunología , Vacunas contra el Virus del Ébola/inmunología , Ebolavirus/inmunología , Mapeo Epitopo , Vacunas de ADN/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Unión ProteicaRESUMEN
We performed epitope mapping studies on the major surface glycoprotein (GP) of Ebola virus (EBOV) using Chemically Linked Peptides on Scaffolds (CLIPS), which form linear and potential conformational epitopes. This method identified monoclonal antibody epitopes and predicted additional epitopes recognized by antibodies in polyclonal sera from animals experimentally vaccinated against or infected with EBOV. Using the information obtained along with structural modeling to predict epitope accessibility, we then constructed 2 DNA vaccines encoding immunodominant and subdominant epitopes predicted to be accessible on EBOV GP. Although a construct designed to produce a membrane-bound oligopeptide was poorly immunogenic, a construct generating a secreted oligopeptide elicited strong antibody responses in mice. When this construct was administered as a boost to a DNA vaccine expressing the complete EBOV GP gene, the resultant antibody response was focused largely toward the less immunodominant epitopes in the oligopeptide. Taken together, the results of this work suggest a utility for this method for immune focusing of antibody responses elicited by vaccination.
Asunto(s)
Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Vacunas contra el Virus del Ébola/inmunología , Ebolavirus/inmunología , Mapeo Epitopo , Glicoproteínas/inmunología , Vacunas de ADN/inmunología , Animales , Formación de Anticuerpos , Antígenos Virales/genética , ADN Viral , Vacunas contra el Virus del Ébola/administración & dosificación , Vacunas contra el Virus del Ébola/genética , Ebolavirus/genética , Epítopos/genética , Epítopos/inmunología , Glicoproteínas/genética , Esquemas de Inmunización , Ratones Endogámicos BALB C , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
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/genéticaRESUMEN
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 JovenRESUMEN
DNA vaccination of transchromosomal bovines (TcBs) with DNA vaccines expressing the codon-optimized (co) glycoprotein (GP) genes of Ebola virus (EBOV) and Sudan virus (SUDV) produce fully human polyclonal antibodies (pAbs) that recognize both viruses and demonstrate robust neutralizing activity. Each TcB was vaccinated by intramuscular electroporation (IM-EP) a total of four times and at each administration received 10 mg of the EBOV-GPco DNA vaccine and 10 mg of the SUDV-GPco DNA vaccine at two sites on the left and right sides, respectively. After two vaccinations, robust antibody responses (titers > 1000) were detected by ELISA against whole irradiated EBOV or SUDV and recombinant EBOV-GP or SUDV-GP (rGP) antigens, with higher titers observed for the rGP antigens. Strong, virus neutralizing antibody responses (titers >1000) were detected after three vaccinations when measured by vesicular stomatitis virus-based pseudovirion neutralization assay (PsVNA). Maximal neutralizing antibody responses were identified by traditional plaque reduction neutralization tests (PRNT) after four vaccinations. Neutralizing activity of human immunoglobulins (IgG) purified from TcB plasma collected after three vaccinations and injected intraperitoneally (IP) into mice at a 100 mg/kg dose was detected in the serum by PsVNA up to 14 days after administration. Passive transfer by IP injection of the purified IgG (100 mg/kg) to groups of BALB/c mice one day after IP challenge with mouse adapted (ma) EBOV resulted in 80% protection while all mice treated with non-specific pAbs succumbed. Similarly, interferon receptor 1 knockout (IFNAR(-/-)) mice receiving the purified IgG (100 mg/kg) by IP injection one day after IP challenge with wild type SUDV resulted in 89% survival. These results are the first to demonstrate that filovirus GP DNA vaccines administered to TcBs by IM-EP can elicit neutralizing antibodies that provide post-exposure protection. Additionally, these data describe production of fully human IgG in a large animal system, a system which is capable of producing large quantities of a clinical grade therapeutic product.
Asunto(s)
Anticuerpos Antivirales/metabolismo , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Profilaxis Posexposición , Vacunas de ADN/inmunología , Animales , Animales Modificados Genéticamente , Anticuerpos Neutralizantes/inmunología , Bovinos/genética , Bovinos/inmunología , Cromosomas Artificiales Humanos/genética , República Democrática del Congo , Vacunas contra el Virus del Ébola/inmunología , Femenino , Fiebre Hemorrágica Ebola/virología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Profilaxis Posexposición/métodos , Receptor de Interferón alfa y beta/genética , Sudán , Vacunación/métodosRESUMEN
Cynomolgus macaques were vaccinated by intramuscular electroporation with DNA plasmids expressing codon-optimized glycoprotein (GP) genes of Ebola virus (EBOV) or Marburg virus (MARV) or a combination of codon-optimized GP DNA vaccines for EBOV, MARV, Sudan virus and Ravn virus. When measured by ELISA, the individual vaccines elicited slightly higher IgG responses to EBOV or MARV than did the combination vaccines. No significant differences in immune responses of macaques given the individual or combination vaccines were measured by pseudovirion neutralization or IFN-γ ELISpot assays. Both the MARV and mixed vaccines were able to protect macaques from lethal MARV challenge (5/6 vs. 6/6). In contrast, a greater proportion of macaques vaccinated with the EBOV vaccine survived lethal EBOV challenge in comparison to those that received the mixed vaccine (5/6 vs. 1/6). EBOV challenge survivors had significantly higher pre-challenge neutralizing antibody titers than those that succumbed.
Asunto(s)
Electroporación , Filoviridae/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Enfermedad del Virus de Marburg/prevención & control , Vacunas de ADN/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Codón , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Ensayo de Immunospot Ligado a Enzimas , Femenino , Filoviridae/genética , Glicoproteínas/genética , Glicoproteínas/inmunología , Inmunoglobulina G/sangre , Inyecciones Intramusculares , Interferón gamma/metabolismo , Leucocitos Mononucleares/inmunología , Macaca fascicularis , Masculino , Pruebas de Neutralización , Plásmidos , Análisis de Supervivencia , Resultado del Tratamiento , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genéticaRESUMEN
Vaccinations against the encephalitic alphaviruses (western, eastern, and Venezuelan equine encephalitis virus) are of significant interest to biological defense, public health, and agricultural communities alike. Although vaccines licensed for veterinary applications are used in the Western Hemisphere and attenuated or inactivated viruses have been used under Investigational New Drug status to protect at-risk personnel, there are currently no licensed vaccines for use in humans. Here, we will discuss the need for a trivalent vaccine that can protect humans against all three viruses, recent progress to such a vaccine, and a strategy to continue development to Food and Drug Administration licensure.
Asunto(s)
Infecciones por Alphavirus/prevención & control , Alphavirus/inmunología , Encefalitis Viral/prevención & control , Vacunas Virales , Alphavirus/genética , Infecciones por Alphavirus/virología , Animales , Virus de la Encefalitis Equina del Este/genética , Virus de la Encefalitis Equina del Este/inmunología , Virus de la Encefalitis Equina Venezolana/genética , Virus de la Encefalitis Equina Venezolana/inmunología , Virus de la Encefalitis Equina del Oeste/genética , Virus de la Encefalitis Equina del Oeste/inmunología , Encefalitis Viral/virología , Humanos , Alineación de Secuencia , VacunaciónRESUMEN
We evaluated the immunogenicity and protective efficacy of DNA vaccines expressing the codon-optimized envelope glycoprotein genes of Zaire ebolavirus, Sudan ebolavirus, and Marburg marburgvirus (Musoke and Ravn). Intramuscular or intradermal delivery of the vaccines in BALB/c mice was performed using the TriGrid™ electroporation device. Mice that received DNA vaccines against the individual viruses developed robust glycoprotein-specific antibody titers as determined by ELISA and survived lethal viral challenge with no display of clinical signs of infection. Survival curve analysis revealed there was a statistically significant increase in survival compared to the control groups for both the Ebola and Ravn virus challenges. These data suggest that further analysis of the immune responses generated in the mice and additional protection studies in nonhuman primates are warranted.
Asunto(s)
Electroporación/métodos , Fiebre Hemorrágica Ebola/prevención & control , Enfermedad del Virus de Marburg/prevención & control , Músculos/metabolismo , Vacunas de ADN/administración & dosificación , Vacunas de ADN/uso terapéutico , Animales , Ebolavirus/inmunología , Ebolavirus/patogenicidad , Femenino , Fiebre Hemorrágica Ebola/inmunología , Enfermedad del Virus de Marburg/inmunología , Marburgvirus/inmunología , Marburgvirus/patogenicidad , Ratones , Ratones Endogámicos BALB CRESUMEN
Venezuelan, western, and eastern equine encephalitis viruses are New World alphaviruses that are recognized as potential agents of biowarfare and bioterrorism owing to their morbidity and mortality in humans, ease of production, considerable stability, and high infectivity in aerosols. As a result, these encephalitic alphaviruses are defined as category B select agents. Studies involving infection of nonhuman primates have been instrumental in gaining an understanding of the in vivo pathogenesis of these viruses and have provided relevant models to evaluate the efficacy of candidate human vaccines. Recent advances have led to refinement and further characterization of these models toward the goal of utility in the licensure of next-generation alphavirus vaccines and therapeutics for use in humans by the Animal Rule.
Asunto(s)
Infecciones por Alphavirus/patología , Infecciones por Alphavirus/virología , Modelos Animales de Enfermedad , Encefalitis Viral/patología , Encefalitis Viral/virología , Primates , Alphavirus/patogenicidad , Infecciones por Alphavirus/tratamiento farmacológico , Infecciones por Alphavirus/prevención & control , Animales , Antivirales/administración & dosificación , Antivirales/efectos adversos , Aprobación de Drogas , Encefalitis Viral/tratamiento farmacológico , Encefalitis Viral/prevención & control , Humanos , Vacunas Virales/administración & dosificación , Vacunas Virales/efectos adversosRESUMEN
Type I interferons (IFNs) are potent mediators of the innate immune response to viral infection. IFNs released from infected cells bind to a receptor (IFNAR) on neighboring cells, triggering signaling cascades that limit further infection. Subtle variations in amino acids can alter IFNAR binding and signaling outcomes. We used a new gene crossbreeding method to generate hybrid, type I human IFNs with enhanced antiviral activity against four dissimilar, highly pathogenic viruses. Approximately 1400 novel IFN genes were expressed in plants, and the resultant IFN proteins were screened for antiviral activity. Comparing the gene sequences of a final set of 12 potent IFNs to those of parent genes revealed strong selection pressures at numerous amino acids. Using three-dimensional models based on a recently solved experimental structure of IFN bound to IFNAR, we show that many but not all of the amino acids that were highly selected for are predicted to improve receptor binding.
Asunto(s)
Antivirales/farmacología , Interferón Tipo I/farmacología , Virus/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Chlorocebus aethiops , Humanos , Interferón Tipo I/química , Interferón Tipo I/genética , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Alineación de Secuencia , Nicotiana/genética , Células VeroRESUMEN
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 & controlRESUMEN
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.