RESUMO
Recombinant adenoviral (rAd) vectors elicit potent cellular and humoral immune responses and show promise as vaccines for HIV-1, Ebola virus, tuberculosis, malaria, and other infections. These vectors are now widely used and have been generally well tolerated in vaccine and gene therapy clinical trials, with many thousands of people exposed. At the same time, dose-limiting adverse responses have been observed, including transient low-grade fevers and a prior human gene therapy fatality, after systemic high-dose recombinant adenovirus serotype 5 (rAd5) vector administration in a human gene therapy trial. The mechanism responsible for these effects is poorly understood. Here, we define the mechanism by which Ad5 targets immune cells that stimulate adaptive immunity. rAd5 tropism for dendritic cells (DCs) was independent of the coxsackievirus and adenovirus receptor (CAR), its primary receptor or the secondary integrin RGD receptor, and was mediated instead by a heparin-sensitive receptor recognized by a distinct segment of the Ad5 fiber, the shaft. rAd vectors with CAR and RGD mutations did not infect a variety of epithelial and fibroblast cell types but retained their ability to transfect several DC types and stimulated adaptive immune responses in mice. Notably, the pyrogenic response to the administration of rAd5 also localized to the shaft region, suggesting that this interaction elicits both protective immunity and vector-induced fevers. The ability of replication-defective rAd5 viruses to elicit potent immune responses is mediated by a heparin-sensitive receptor that interacts with the Ad5 fiber shaft. Mutant CAR and RGD rAd vectors target several DC and mononuclear subsets and induce both adaptive immunity and toxicity. Understanding of these interactions facilitates the development of vectors that target DCs through alternative receptors that can improve safety while retaining the immunogenicity of rAd vaccines.
Assuntos
Infecções por Adenoviridae/prevenção & controle , Adenoviridae/imunologia , Células Dendríticas/virologia , Vacinas Virais/efeitos adversos , Vacinas Virais/imunologia , Adenoviridae/patogenicidade , Infecções por Adenoviridae/imunologia , Animais , Células Dendríticas/patologia , Relação Dose-Resposta a Droga , Feminino , Vetores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Coelhos , Receptores Imunológicos/genética , Receptores Imunológicos/fisiologia , Receptores de Peptídeos/genética , Receptores de Peptídeos/fisiologia , Receptores Virais/genética , Receptores Virais/fisiologia , Transdução Genética , Proteínas Virais/efeitos adversos , Proteínas Virais/imunologia , Proteínas Virais/uso terapêutico , Vacinas Virais/uso terapêutico , Replicação Viral/fisiologiaRESUMO
The Vaccine Research Center has developed a number of vaccine candidates for different diseases/infectious agents (HIV-1, Severe Acute Respiratory Syndrome virus, West Nile virus, and Ebola virus, plus a plasmid cytokine adjuvant-IL-2/Ig) based on a DNA plasmid vaccine platform. To support the clinical development of each of these vaccine candidates, preclinical studies were performed to screen for potential toxicities (intrinsic and immunotoxicities). All treatment-related toxicities identified in these repeated-dose toxicology studies have been confined primarily to the sites of injection and seem to be the result of both the delivery method (as they are seen in both control and treated animals) and the intended immune response to the vaccine (as they occur with greater frequency and severity in treated animals). Reactogenicity at the site of injection is generally seen to be reversible as the frequency and severity diminished between doses and between the immediate and recovery termination time points. This observation also correlated with the biodistribution data reported in the companion article (Sheets et al., 2006), in which DNA plasmid vaccine was shown to remain at the site of injection, rather than biodistributing widely, and to clear over time. The results of these safety studies have been submitted to the Food and Drug Administration to support the safety of initiating clinical studies with these and related DNA plasmid vaccines. Thus far, standard repeated-dose toxicology studies have not identified any target organs for toxicity (other than the injection site) for our DNA plasmid vaccines at doses up to 8 mg per immunization, regardless of disease indication (i.e., expressed gene-insert) and despite differences (strengths) in the promoters used to drive this expression. As clinical data accumulate with these products, it will be possible to retrospectively compare the safety profiles of the products in the clinic to the results of the repeated-dose toxicology studies, in order to determine the utility of such toxicology studies for signaling potential immunotoxicities or intrinsic toxicities from DNA vaccines. These data build on the biodistribution studies performed (see companion article, Sheets et al., 2006) to demonstrate the safety and suitability for investigational human use of DNA plasmid vaccine candidates for a variety of infectious disease prevention indications.
Assuntos
Vacinas de DNA/toxicidade , Vacinas Virais/toxicidade , Síndrome da Imunodeficiência Adquirida/genética , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/prevenção & controle , Animais , Ebolavirus/genética , Ebolavirus/imunologia , Feminino , Genes Virais , HIV-1/genética , HIV-1/imunologia , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Injeções Intramusculares , Masculino , Plasmídeos , Regiões Promotoras Genéticas , Coelhos , Síndrome Respiratória Aguda Grave/genética , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/prevenção & controle , Distribuição Tecidual , Vacinas de DNA/administração & dosagem , Vacinas Virais/administração & dosagem , Febre do Nilo Ocidental/genética , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/prevenção & controle , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/imunologiaRESUMO
The Vaccine Research Center has developed a number of vaccine candidates for different diseases/infectious agents (HIV-1, Severe Acute Respiratory Syndrome virus, West Nile virus, and Ebola virus, plus a plasmid cytokine adjuvant-IL-2/Ig) based on a DNA plasmid vaccine platform. To support the clinical development of each of these vaccine candidates, preclinical studies have been performed in mice or rabbits to determine where in the body these plasmid vaccines would biodistribute and how rapidly they would clear. In the course of these studies, it has been observed that regardless of the gene insert (expressing the vaccine immunogen or cytokine adjuvant) and regardless of the promoter used to drive expression of the gene insert in the plasmid backbone, the plasmid vaccines do not biodistribute widely and remain essentially in the site of injection, in the muscle and overlying subcutis. Even though approximately 10(14) molecules are inoculated in the studies in rabbits, by day 8 or 9 ( approximately 1 week postinoculation), already all but on the order of 10(4)-10(6) molecules per microgram of DNA extracted from tissue have been cleared at the injection site. Over the course of 2 months, the plasmid clears from the site of injection with only a small percentage of animals (generally 10-20%) retaining a small number of copies (generally around 100 copies) in the muscle at the injection site. This pattern of biodistribution (confined to the injection site) and clearance (within 2 months) is consistent regardless of differences in the promoter in the plasmid backbone or differences in the gene insert being expressed by the plasmid vaccine. In addition, integration has not been observed with plasmid vaccine candidates inoculated i.m. by Biojector 2000 or by needle and syringe. These data build on the repeated-dose toxicology studies performed (see companion article, Sheets et al., 2006) to demonstrate the safety and suitability for investigational human use of DNA plasmid vaccine candidates for a variety of infectious disease prevention indications.
Assuntos
Vacinas de DNA/farmacocinética , Vacinas Virais/farmacocinética , Síndrome da Imunodeficiência Adquirida/genética , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/prevenção & controle , Animais , Ebolavirus/genética , Ebolavirus/imunologia , Feminino , Genes Virais , HIV-1/genética , HIV-1/imunologia , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Injeções Intramusculares , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos , Plasmídeos , Regiões Promotoras Genéticas , Síndrome Respiratória Aguda Grave/genética , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/prevenção & controle , Distribuição Tecidual , Vacinas de DNA/administração & dosagem , Vacinas Virais/administração & dosagem , Febre do Nilo Ocidental/genética , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/prevenção & controle , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/imunologiaRESUMO
BACKGROUND: The severe acute respiratory syndrome (SARS) virus is a member of the Coronaviridae (CoV) family that first appeared in the Guangdong Province of China in 2002 and was recognized as an emerging infectious disease in March 2003. Over 8000 cases and 900 deaths occurred during the epidemic. We report the safety and immunogenicity of a SARS DNA vaccine in a Phase I human study. METHODS: A single-plasmid DNA vaccine encoding the Spike (S) glycoprotein was evaluated in 10 healthy adults. Nine subjects completed the 3 dose vaccination schedule and were evaluated for vaccine safety and immune responses. Immune response was assessed by intracellular cytokine staining (ICS), ELISpot, ELISA, and neutralization assays. RESULTS: The vaccine was well tolerated. SARS-CoV-specific antibody was detected by ELISA in 8 of 10 subjects and neutralizing antibody was detected in all subjects who received 3 doses of vaccine. SARS-CoV-specific CD4+ T-cell responses were detected in all vaccinees, and CD8+ T-cell responses in approximately 20% of individuals. CONCLUSIONS: The VRC SARS DNA vaccine was well tolerated and produced cellular immune responses and neutralizing antibody in healthy adults.
Assuntos
Anticorpos Antivirais/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Síndrome Respiratória Aguda Grave/prevenção & controle , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Vacinas de DNA , Vacinas Virais , Adolescente , Adulto , Feminino , Humanos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Pessoa de Meia-Idade , Testes de Neutralização , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Síndrome Respiratória Aguda Grave/imunologia , Glicoproteína da Espícula de Coronavírus , Resultado do Tratamento , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/imunologia , Adulto JovemRESUMO
Needle-free delivery of a six-plasmid HIV-1 DNA vaccine encoding EnvA, EnvB, EnvC, and subtype B Gag, Pol, and Nef underwent open-label evaluation in 15 subjects; 14 completed the 0, 1, 2 month vaccination schedule. T cell responses to HIV-specific peptide pools were detected by intracellular cytokine staining of CD4(+) [13/14 (93%)] and CD8(+) [5/14 (36%)], and by ELISpot in 11/14 (79%). Ten of 14 (71%) had ELISA antibody responses to Env proteins. Compared to a four-plasmid product, Gag- and Nef-specific T cell responses were improved, while Env-specific responses were maintained. This candidate vaccine has now advanced to Phase II evaluation.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Vacinas de DNA/administração & dosagem , Vacinas contra a AIDS/efeitos adversos , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Adolescente , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Produtos do Gene env/imunologia , Produtos do Gene gag/imunologia , Produtos do Gene nef/imunologia , Anticorpos Anti-HIV/biossíntese , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , Humanos , Imunidade Celular/imunologia , Masculino , Plasmídeos/genética , Plasmídeos/imunologia , Vacinas Combinadas/administração & dosagem , Vacinas Combinadas/efeitos adversos , Vacinas Combinadas/genética , Vacinas Combinadas/imunologia , Vacinas de DNA/efeitos adversos , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
OBJECTIVE: To evaluate the safety and immunogenicity of a candidate HIV DNA vaccine administered using a needle-free device. DESIGN: In this phase 1, dose escalation, double-blind, placebo-controlled clinical trial, 21 healthy adults were randomized to receive placebo or 0.5, 1.5, or 4 mg of a single plasmid expressing a Gag/Pol fusion protein. Each participant received repeat immunizations at days 28 and 56 after the first inoculation. Safety and immunogenicity data were collected. RESULTS: The vaccine was well tolerated, with most adverse events being mild injection site reactions, including pain, tenderness, and erythema. No dose-limiting toxicities occurred. HIV-specific antibody response was not detected in any vaccinee by enzyme-linked immunosorbent assay. HIV-specific T-cell responses to Gag or Pol as measured by enzyme-linked immunospot assay and intracellular cytokine staining were of low frequency and magnitude. CONCLUSIONS: This candidate HIV DNA vaccine was safe and well tolerated. No HIV-specific antibody responses were detected, and only low-magnitude HIV-specific T-cell responses were detected in 8 (53%) of 15 vaccinees. This initial product led to the development of a 4-plasmid multiclade HIV DNA Vaccine Research Center vaccine candidate in which envelope genes expressing Env from clades A, B, and C and a Nef gene from clade B have been added.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Soronegatividade para HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS/efeitos adversos , Vacinas contra a AIDS/imunologia , Adulto , Método Duplo-Cego , Feminino , Genes gag , Genes pol , Anticorpos Anti-HIV/biossíntese , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/genética , Humanos , Imunidade Celular , Masculino , Pessoa de Meia-Idade , Segurança , Linfócitos T/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/efeitos adversos , Vacinas de DNA/imunologiaRESUMO
BACKGROUND: West Nile virus (WNV) is a mosquito-borne flavivirus that can cause severe meningitis and encephalitis in infected individuals. We report the safety and immunogenicity of a WNV DNA vaccine in its first phase 1 human study. METHODS: A single-plasmid DNA vaccine encoding the premembrane and the envelope glycoproteins of the NY99 strain of WNV was evaluated in an open-label study in 15 healthy adults. Twelve subjects completed the 3-dose vaccination schedule, and all subjects completed 32 weeks of evaluation for safety and immunogenicity. The development of a vaccine-induced immune response was assessed by enzyme-linked immunosorbant assay, neutralization assays, intracelluar cytokine staining, and enzyme-linked immunospot assay. RESULTS: The vaccine was safe and well tolerated, with no significant adverse events. Vaccine-induced T cell and antibody responses were detected in the majority of subjects. Neutralizing antibody to WNV was detected in all subjects who completed the 3-dose vaccination schedule, at levels shown to be protective in studies of horses, an incidental natural host for WNV. CONCLUSIONS: Further assessment of this DNA platform for human immunization against WNV is warranted. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00106769 .
Assuntos
Anticorpos Antivirais/biossíntese , Vacinas de DNA/uso terapêutico , Febre do Nilo Ocidental/prevenção & controle , Vacinas contra o Vírus do Nilo Ocidental/uso terapêutico , Vírus do Nilo Ocidental/imunologia , Adolescente , Adulto , Anticorpos Antivirais/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linfócitos T/imunologia , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vacinas de DNA/virologia , Febre do Nilo Ocidental/virologia , Vacinas contra o Vírus do Nilo Ocidental/genética , Vacinas contra o Vírus do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/genéticaRESUMO
BACKGROUND: Gene-based vaccine delivery is an important strategy in the development of a preventive vaccine for acquired immunodeficiency syndrome (AIDS). Vaccine Research Center (VRC) 004 is the first phase 1 dose-escalation study of a multiclade HIV-1 DNA vaccine. METHODS: VRC-HIVDNA009-00-VP is a 4-plasmid mixture encoding subtype B Gag-Pol-Nef fusion protein and modified envelope (Env) constructs from subtypes A, B, and C. Fifty healthy, uninfected adults were randomized to receive either placebo (n=10) or study vaccine at 2 mg (n=5), 4 mg (n=20), or 8 mg (n=15) by needle-free intramuscular injection. Humoral responses (measured by enzyme-linked immunosorbant assay, Western blotting, and neutralization assay) and T cell responses (measured by enzyme-linked immunospot assay and intracellular cytokine staining after stimulation with antigen-specific peptide pools) were measured. RESULTS: The vaccine was well tolerated and induced cellular and humoral responses. The maximal CD4(+) and CD8(+) T cell responses occurred after 3 injections and were in response to Env peptide pools. The pattern of cytokine expression by vaccine-induced HIV-specific T cells evolved over time, with a diminished frequency of interferon- gamma -producing T cells and an increased frequency of interleukin-2-producing T cells at 1 year. CONCLUSIONS: DNA vaccination induced antibody to and T cell responses against 3 major HIV-1 subtypes and will be further evaluated as a potential component of a preventive AIDS vaccine regimen.
Assuntos
Vacinas contra a AIDS/efeitos adversos , Vacinas contra a AIDS/imunologia , Vetores Genéticos , Infecções por HIV/imunologia , HIV-1/imunologia , Esquemas de Imunização , Testes de Neutralização , Plasmídeos , Vacinação , Vacinas contra a AIDS/administração & dosagem , Adolescente , Adulto , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , Western Blotting , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/análise , Citocinas/biossíntese , Método Duplo-Cego , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas de Fusão gag-pol/genética , Proteínas de Fusão gag-pol/imunologia , Produtos do Gene nef/genética , Produtos do Gene nef/imunologia , Infecções por HIV/sangue , Humanos , Injeções Intramusculares , Masculino , Vacinas de DNA/administração & dosagem , Vacinas de DNA/efeitos adversos , Vacinas de DNA/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
BACKGROUND: The development of an effective human immunodeficiency virus (HIV) vaccine is a high global priority. Here, we report the safety, tolerability, and immunogenicity of a replication-defective recombinant adenovirus serotype 5 (rAd5) vector HIV-1 candidate vaccine. METHODS: The vaccine is a mixture of 4 rAd5 vectors that express HIV-1 subtype B Gag-Pol fusion protein and envelope (Env) from subtypes A, B, and C. Healthy, uninfected adults were randomized to receive 1 intramuscular injection of placebo (n=6) or vaccine at dose levels of 10(9) (n=10), 10(10) (n=10), or 10(11) (n=10) particle units and were followed for 24 weeks to assess immunogenicity and safety. RESULTS: The vaccine was well tolerated but was associated with more reactogenicity at the highest dose. At week 4, vaccine antigen-specific T cell responses were detected in 28 (93.3%) and 18 (60%) of 30 vaccine recipients for CD4(+) and CD8(+) T cells, respectively, by intracellular cytokine staining assay and in 22 (73%) of 30 vaccine recipients by enzyme-linked immunospot assay. Env-specific antibody responses were detected in 15 (50%) of 30 vaccine recipients by enzyme-linked immunosorbant assay and in 28 (93.3%) of 30 vaccine recipients by immunoprecipitation followed by Western blotting. No neutralizing antibody was detected. CONCLUSIONS: A single injection induced HIV-1 antigen-specific CD4(+) T cell, CD8(+) T cell, and antibody responses in the majority of vaccine recipients. This multiclade rAd5 HIV-1 vaccine is now being evaluated in combination with a multiclade HIV-1 DNA plasmid vaccine.
Assuntos
Vacinas contra a AIDS/efeitos adversos , Vacinas contra a AIDS/imunologia , Anticorpos Antivirais/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Náusea/etiologia , Vacinação , Vacinas contra a AIDS/administração & dosagem , Adenovírus Humanos/genética , Adolescente , Adulto , Especificidade de Anticorpos , Western Blotting , Citocinas/análise , Citocinas/biossíntese , Relação Dose-Resposta Imunológica , Método Duplo-Cego , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Proteínas de Fusão gag-pol/imunologia , Produtos do Gene env/imunologia , Vetores Genéticos , Humanos , Injeções Intramusculares , Masculino , Recombinação Genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Produtos do Gene env do Vírus da Imunodeficiência HumanaRESUMO
Ebola viruses represent a class of filoviruses that causes severe hemorrhagic fever with high mortality. Recognized first in 1976 in the Democratic Republic of Congo, outbreaks continue to occur in equatorial Africa. A safe and effective Ebola virus vaccine is needed because of its continued emergence and its potential for use for biodefense. We report the safety and immunogenicity of an Ebola virus vaccine in its first phase I human study. A three-plasmid DNA vaccine encoding the envelope glycoproteins (GP) from the Zaire and Sudan/Gulu species as well as the nucleoprotein was evaluated in a randomized, placebo-controlled, double-blinded, dose escalation study. Healthy adults, ages 18 to 44 years, were randomized to receive three injections of vaccine at 2 mg (n = 5), 4 mg (n = 8), or 8 mg (n = 8) or placebo (n = 6). Immunogenicity was assessed by enzyme-linked immunosorbent assay (ELISA), immunoprecipitation-Western blotting, intracellular cytokine staining (ICS), and enzyme-linked immunospot assay. The vaccine was well-tolerated, with no significant adverse events or coagulation abnormalities. Specific antibody responses to at least one of the three antigens encoded by the vaccine as assessed by ELISA and CD4(+) T-cell GP-specific responses as assessed by ICS were detected in 20/20 vaccinees. CD8(+) T-cell GP-specific responses were detected by ICS assay in 6/20 vaccinees. This Ebola virus DNA vaccine was safe and immunogenic in humans. Further assessment of the DNA platform alone and in combination with replication-defective adenoviral vector vaccines, in concert with challenge and immune data from nonhuman primates, will facilitate evaluation and potential licensure of an Ebola virus vaccine under the Animal Rule.