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1.
J Infect Dis ; 2018 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-30304515

RESUMO

Background: There remains an important need for prophylactic anti-Ebola virus vaccine candidates that elicit long-lasting immune responses and can be delivered to vulnerable populations that are unable to receive live-attenuated or viral vector vaccines. Methods: We designed novel synthetic anti-Ebola virus glycoprotein (EBOV-GP) DNA vaccines as a strategy to expand protective breadth against diverse EBOV strains and evaluated the impact of vaccine dosing and route of administration on protection against lethal EBOV-Makona challenge in cynomolgus macaques. Long-term immunogenicity was monitored in nonhuman primates for >1 year, followed by a 12-month boost. Results: Multiple-injection regimens of the EBOV-GP DNA vaccine, delivered by intramuscular administration followed by electroporation, were 100% protective against lethal EBOV-Makona challenge. Impressively, 2 injections of a simple, more tolerable, and dose-sparing intradermal administration followed by electroporation generated strong immunogenicity and was 100% protective against lethal challenge. In parallel, we observed that EBOV-GP DNA vaccination induced long-term immune responses in macaques that were detectable for at least 1 year after final vaccination and generated a strong recall response after the final boost. Conclusions: These data support that this simple intradermal-administered, serology-independent approach is likely important for additional study towards the goal of induction of anti-EBOV immunity in multiple at-risk populations.

2.
Vaccine ; 33(35): 4313-20, 2015 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-25887087

RESUMO

Identifying new molecular adjuvants that elicit effective vaccine-induced CD8(+) T cell immunity may be critical for the elimination of many challenging diseases including Tuberculosis, HIV and cancer. Here, we report that co-administration of molecular adjuvant IL-33 during vaccination enhanced the magnitude and function of antigen (Ag)-specific CD8(+) T cells against a model Ag, LCMV NP target protein. These enhanced responses were characterized by higher frequencies of Ag-specific, polyfunctional CD8(+) T cells exhibiting cytotoxic characteristics. Importantly, these cells were capable of robust expansion upon Ag-specific restimulation in vivo and conferred remarkable protection against a high dose lethal LCMV challenge. In addition, we demonstrate the ability of IL-33 to amplifying the frequency of Ag-specific KLRG1(+) effector CD8(+) T cells. These data show that IL-33 is a promising immunoadjuvant at improving T cell immunity in a vaccine setting and suggest further development and understanding of this molecular adjuvant for strategies against many obstinate infectious diseases and cancer.


Assuntos
Adjuvantes Imunológicos , Interleucina-33/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Vacinas de DNA/imunologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Animais , Infecções por Arenaviridae/prevenção & controle , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Imunidade Celular , Memória Imunológica , Interleucina-33/genética , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/patogenicidade , Camundongos Endogâmicos C57BL
3.
Adv Exp Med Biol ; 848: 131-48, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25757619

RESUMO

There is no licensed vaccine or cure for human cytomegalovirus (CMV), a ubiquitous ß-herpes virus that infects 60-95 % of adults worldwide. Infection is a major cause of congenital abnormalities in newborns, contributes to development of childhood cerebral palsy and medulloblastoma, can result in severe disease in immunocompromised patients, and is a major impediment during successful organ transplantation. While CMV has been increasingly associated with numerous inflammatory diseases and cancers, only recently has it been correlated with increased risk of heart disease in adults, the number-one killer in the USA. These data, among others, suggest that subclinical CMV infection, or microinfection, in healthy individuals may play more of a causative role than an epiphenomenon in development of CMV-associated pathologies. Due to the myriad of diseases and complications associated with CMV, an efficacious vaccine would be highly valuable in reducing human morbidity and mortality as well as saving billions of dollars in annual health-care costs and disability adjusted life years (DALY) in the developing world. Therefore, the development of a safe efficacious CMV vaccine or immune therapy is paramount to the public health. This review aims to provide a brief overview on aspects of CMV infection and disease and focuses on current vaccine strategies. The use of new synthetic DNA vaccines might offer one such approach to this difficult problem.


Assuntos
Clonagem Molecular/métodos , Infecções por Citomegalovirus/terapia , Vacinas contra Citomegalovirus/uso terapêutico , Imunoterapia Ativa/métodos , Vacinas de DNA/uso terapêutico , Adulto , Animais , Citomegalovirus/genética , Citomegalovirus/imunologia , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/imunologia , Vacinas contra Citomegalovirus/genética , DNA Recombinante/genética , DNA Recombinante/uso terapêutico , Humanos , Vacinas de DNA/genética
4.
Mol Pharm ; 12(8): 2712-31, 2015 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-25363619

RESUMO

As the Ebola outbreak in West Africa continues and cases appear in the United States and other countries, the need for long-lasting vaccines to preserve global health is imminent. Here, we evaluate the long-term efficacy of a respiratory and sublingual (SL) adenovirus-based vaccine in non-human primates in two phases. In the first, a single respiratory dose of 1.4×10(9) infectious virus particles (ivp)/kg of Ad-CAGoptZGP induced strong Ebola glycoprotein (GP) specific CD8+ and CD4+ T cell responses and Ebola GP-specific antibodies in systemic and mucosal compartments and was partially (67%) protective from challenge 62 days after immunization. The same dose given by the SL route induced Ebola GP-specific CD8+ T cell responses similar to that of intramuscular (IM) injection, however, the Ebola GP-specific antibody response was low. All primates succumbed to infection. Three primates were then given the vaccine in a formulation that improved the immune response to Ebola in rodents. Three primates were immunized with 2.0×10(10) ivp/kg of vaccine by the SL route. Diverse populations of polyfunctional Ebola GP-specific CD4+ and CD8+ T cells and significant anti-Ebola GP antibodies were present in samples collected 150 days after respiratory immunization. The formulated vaccine was fully protective against challenge 21 weeks after immunization. While diverse populations of Ebola GP-specific CD4+ T cells were produced after SL immunization, antibodies were not neutralizing and the vaccine was unprotective. To our knowledge, this is the first time that durable protection from a single dose respiratory adenovirus-based Ebola vaccine has been demonstrated in primates.


Assuntos
Adenoviridae/imunologia , Vacinas contra Ebola/administração & dosagem , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Vacinas Sintéticas/administração & dosagem , Adenoviridae/genética , Animais , Células Cultivadas , Cercopithecus aethiops , Células HEK293 , Doença pelo Vírus Ebola/imunologia , Humanos , Macaca fascicularis , Masculino , Vacinação/métodos , Vacinas Sintéticas/genética , Células Vero
5.
Vaccines (Basel) ; 2(2): 196-215, 2014 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-26344618

RESUMO

DNA vaccine-induced immunity can be enhanced by the co-delivery of synthetic gene-encoding molecular adjuvants. Many of these adjuvants have included cytokines, chemokines or co-stimulatory molecules that have been demonstrated to enhance vaccine-induced immunity by increasing the magnitude or type of immune responses and/or protective efficacy. In this way, through the use of adjuvants, immune responses can be highly customizable and functionally tailored for optimal efficacy against pathogen specific (i.e., infectious agent) or non-pathogen (i.e., cancer) antigens. In the novel study presented here, we examined the use of cellular transcription factors as molecular adjuvants. Specifically the co-delivery of (a) RelA, a subunit of the NF-κB transcription complex or (b) T-bet, a Th1-specific T box transcription factor, along with a prototypical DNA vaccine expressing HIV-1 proteins was evaluated. As well, all of the vaccines and adjuvants were administered to mice using in vivo electroporation (EP), a technology demonstrated to dramatically increase plasmid DNA transfection and subsequent transgene expression with concomitant enhancement of vaccine induced immune responses. As such, this study demonstrated that co-delivery of either adjuvant resulted in enhanced T and B cell responses, specifically characterized by increased T cell numbers, IFN-γ production, as well as enhanced antibody responses. This study demonstrates the use of cellular transcription factors as adjuvants for enhancing DNA vaccine-induced immunity.

6.
Mol Ther ; 21(7): 1432-44, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23670573

RESUMO

Marburg and Ebola hemorrhagic fevers have been described as the most virulent viral diseases known to man due to associative lethality rates of up to 90%. Death can occur within days to weeks of exposure and there is currently no licensed vaccine or therapeutic. Recent evidence suggests an important role for antiviral T cells in conferring protection, but little detailed analysis of this response as driven by a protective vaccine has been reported. We developed a synthetic polyvalent-filovirus DNA vaccine against Marburg marburgvirus (MARV), Zaire ebolavirus (ZEBOV), and Sudan ebolavirus (SUDV). Preclinical efficacy studies were performed in guinea pigs and mice using rodent-adapted viruses, whereas murine T-cell responses were extensively analyzed using a novel modified assay described herein. Vaccination was highly potent, elicited robust neutralizing antibodies, and completely protected against MARV and ZEBOV challenge. Comprehensive T-cell analysis revealed cytotoxic T lymphocytes (CTLs) of great magnitude, epitopic breadth, and Th1-type marker expression. This model provides an important preclinical tool for studying protective immune correlates that could be applied to existing platforms. Data herein support further evaluation of this enhanced gene-based approach in nonhuman primate studies for in depth analyses of T-cell epitopes in understanding protective efficacy.


Assuntos
Doença do Vírus de Marburg/imunologia , Doença do Vírus de Marburg/prevenção & controle , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/imunologia , Animais , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Immunoblotting , Marburgvirus/imunologia , Marburgvirus/patogenicidade , Camundongos Endogâmicos C57BL , Vacinas de DNA/uso terapêutico , Vacinas Virais/imunologia , Vacinas Virais/uso terapêutico
7.
Expert Rev Vaccines ; 12(5): 537-54, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23659301

RESUMO

The human body has developed an elaborate defense system against microbial pathogens and foreign antigens. However, particular microbes have evolved sophisticated mechanisms to evade immune surveillance, allowing persistence within the human host. In an effort to combat such infections, intensive research has focused on the development of effective prophylactic and therapeutic countermeasures to suppress or clear persistent viral infections. To date, popular therapeutic strategies have included the use of live-attenuated microbes, viral vectors and dendritic-cell vaccines aiming to help suppress or clear infection. In recent years, improved DNA vaccines have now re-emerged as a promising candidate for therapeutic intervention due to the development of advanced optimization and delivery technologies. For instance, genetic optimization of synthetic plasmid constructs and their encoded antigens, in vivo electroporation-mediated vaccine delivery, as well as codelivery with molecular adjuvants have collectively enhanced both transgene expression and the elicitation of vaccine-induced immunity. In addition, the development of potent heterologous prime-boost regimens has also provided significant contributions to DNA vaccine immunogenicity. Herein, the authors will focus on these recent improvements to this synthetic platform in relation to their application in combating persistent virus infection.


Assuntos
DNA/imunologia , Vacinas Virais/imunologia , Viroses/imunologia , Viroses/prevenção & controle , Adjuvantes Imunológicos/administração & dosagem , Antígenos Virais/genética , Antígenos Virais/imunologia , Doença Crônica , DNA/administração & dosagem , DNA/genética , Descoberta de Drogas/tendências , Eletroporação , Humanos , Plasmídeos , Vacinação/métodos , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
8.
Hum Vaccin Immunother ; 8(11): 1668-81, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23151448

RESUMO

There is no licensed vaccine or cure for human cytomegalovirus (CMV), a ubiquitous ß-herpesvirus infecting 60-95% of adults worldwide. Infection can cause congenital abnormalities, result in severe disease in immunocompromised patients, and is a major impediment during successful organ transplantation. In addition, it has been associated with numerous inflammatory diseases and cancers, as well as being implicated in the development of essential hypertension, a major risk factor for heart disease. To date, limited data regarding the identification of immunogenic viral targets has frustrated CMV vaccine development. Based upon promising clinical data suggesting an important role for T cells in protecting against disease in the transplantation setting, we designed a novel panel of highly-optimized synthetic vaccines encoding major CMV proteins and evaluated their immune potential in murine studies. Vaccination induced robust CD8+ and CD4+ T cells of great epitopic breadth as extensively analyzed using a novel modified T cell assay described herein. Together with improved levels of CMV-specific T cells as driven by a vaccine, further immune evaluation of each target is warranted. The present model provides an important tool for guiding future immunization strategies against CMV.


Assuntos
Vacinas contra Citomegalovirus/imunologia , Citomegalovirus/imunologia , Linfócitos T/imunologia , Vacinas Sintéticas/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Eletroporação , Feminino , Citometria de Fluxo , Terapia Genética , Camundongos , Camundongos Endogâmicos C57BL
9.
PLoS One ; 7(12): e52165, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23284919

RESUMO

Numerous studies have suggested that an effective Hepatitis C Virus (HCV) vaccine must induce strong cytotoxic and IFN-γ+ T cell responses targeting the non-structural region of the virus. Most importantly, these responses must be able to migrate into and remain functional within the liver, an organ known to cause T cell tolerance. Using three novel HCV DNA vaccines encoding non-structural proteins NS4B, NS5A and NS5B, we assessed the ability of peripheral immunization to induce functional intrahepatic immunity both in the presence and absence of cognate HCV antigen expression within the liver. We have shown that these constructs induced potent HCV-specific CD4+ and CD8+ T cell responses in the spleen of C57BL/6 mice and that these responses were detected within the liver following peripheral immunization. Additionally, using a transfection method to express HCV antigen within the liver, we showed that intrahepatic HCV-specific T cells remained highly functional within the liver and retained the ability to become highly activated as evidenced by upregulation of IFN-γ and clearance of HCV protein expressing hepatocytes. Taken together, these findings suggest that peripheral immunization can induce potent HCV-specific T cell responses able to traffic to and function within the tolerant environment of the liver.


Assuntos
Imunidade Celular/imunologia , Fígado/imunologia , Proteínas não Estruturais Virais/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Feminino , Citometria de Fluxo , Imunofluorescência , Hepacivirus/imunologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal
10.
Hum Vaccin ; 7(12): 1326-35, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22108033

RESUMO

It is believed that an effective HCV vaccine must induce strong HCV-specific cytotoxic IFN-γ⁺ CD8⁺ T cells able to migrate into and become fully activated within the liver, an organ known to suppress T cell responses and induce tolerance. Given the importance of intrahepatic HCV-specific T cells in the clearance of acute infection, the goal of this present study was to determine if peripheral immunization was able to induce functional intrahepatic HCV-specific T cell based immunity both in the presence and absence of HCV antigen expression within the liver. Using a novel HCV NS3/NS4A DNA vaccine, we show that peripheral immunization of C57BL/6 mice results in the formation of a large pool of fully functional HCV-specific cytotoxic IFN-γ⁺ CD8⁺ T cells within the liver and that these cells were highly enriched within the liver as compared to the spleen. Following hepatic expression of cognate HCV antigen using a previously described liver transfection method, we show that this pool of vaccine-induced HCV-specific CD8⁺ T cells retained its ability to become highly activated as shown by the upregulation of IFN-γ and CCR5 expression, as well as by the clearance of HCV NS3 expressing hepatocytes. Taken together, these findings suggest that T cell effector function is preserved within the liver and that selective recruitment of antigen-specific T cells to the liver may play a previously unappreciated role in the process of immune surveillance, which may be exploited for future T cell based HCV vaccines.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Hepacivirus/imunologia , Hepatite C/imunologia , Fígado/imunologia , Vacinas de DNA/administração & dosagem , Animais , Proteínas de Transporte/administração & dosagem , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Feminino , Hepatite C/prevenção & controle , Hepatite C/virologia , Humanos , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Citotóxicos/imunologia , Transfecção , Vacinação , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Proteínas não Estruturais Virais/administração & dosagem , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia
11.
J Immunol ; 187(6): 2932-43, 2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21856939

RESUMO

Recent evidence demonstrates that HIV-1 infection leads to the attenuation of cellular immune responses, which has been correlated with the increased expression of programmed death (PD)-1 on virus-specific CD8(+) T cells. PD-1 is induced upon T cell activation, and its prolonged expression facilitates CD8(+) T cell inhibitory signals when bound to its B7 family ligands, PD-ligand (L)1/2, which are expressed on APCs. Importantly, early reports demonstrated that blockade of the PD-1/PD-L interaction by Abs may help to counter the development of immune exhaustion driven by HIV viral persistence. To better understand the regulation of the PD-1 pathway during HIV infection, we examined the ability of the virus to induce PD-L expression on macrophages and dendritic cells. We found a direct relationship between the infection of APCs and the expression of PD-L1 in which virus-mediated upregulation induced a state of nonresponsiveness in uninfected HIV-specific T cells. Furthermore, this exhaustion phenotype was revitalized by the blockade of PD-L1, after which T cells regained their capacity for proliferation and the secretion of proinflammatory cytokines IFN-γ, IL-2, and IL-12 upon restimulation. In addition, we identify a critical role for the PI3K/serine-threonine kinase signaling pathway in PD-L1 upregulation of APCs by HIV, because inhibition of these intracellular signal transducer enzymes significantly reduced PD-L1 induction by infection. These data identify a novel mechanism by which HIV exploits the immunosuppressive PD-1 pathway and suggest a new role for virus-infected cells in the local corruption of immune responses required for viral suppression.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , Ativação Linfocitária/imunologia , Transdução de Sinais/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos CD/biossíntese , Antígenos CD/imunologia , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/imunologia , Western Blotting , Linfócitos T CD8-Positivos/metabolismo , Separação Celular , Ativação Enzimática/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Infecções por HIV/metabolismo , HIV-1/imunologia , Humanos , Ligantes , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Receptor de Morte Celular Programada 1 , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima
12.
PLoS One ; 6(6): e19681, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21701683

RESUMO

While HIV-1-specific cellular immunity is thought to be critical for the suppression of viral replication, the correlates of protection have not yet been determined. Rhesus macaques (RM) are an important animal model for the study and development of vaccines against HIV/AIDS. Our laboratory has helped to develop and study DNA-based vaccines in which recent technological advances, including genetic optimization and in vivo electroporation (EP), have helped to dramatically boost their immunogenicity. In this study, RMs were immunized with a DNA vaccine including individual plasmids encoding SIV gag, env, and pol alone, or in combination with a molecular adjuvant, plasmid DNA expressing the chemokine ligand 5 (RANTES), followed by EP. Along with standard immunological assays, flow-based activation analysis without ex vivo restimulation and high-throughput gene expression analysis was performed. Strong cellular immunity was induced by vaccination which was supported by all assays including PBMC microarray analysis that identified the up-regulation of 563 gene sequences including those involved in interferon signaling. Furthermore, 699 gene sequences were differentially regulated in these groups at peak viremia following SIVmac251 challenge. We observed that the RANTES-adjuvanted animals were significantly better at suppressing viral replication during chronic infection and exhibited a distinct pattern of gene expression which included immune cell-trafficking and cell cycle genes. Furthermore, a greater percentage of vaccine-induced central memory CD8+ T-cells capable of an activated phenotype were detected in these animals as measured by activation analysis. Thus, co-immunization with the RANTES molecular adjuvant followed by EP led to the generation of cellular immunity that was transcriptionally distinct and had a greater protective efficacy than its DNA alone counterpart. Furthermore, activation analysis and high-throughput gene expression data may provide better insight into mechanisms of viral control than may be observed using standard immunological assays.


Assuntos
Leucócitos Mononucleares/metabolismo , Vacinas contra a SAIDS/imunologia , Vacinas de DNA/imunologia , Animais , Citometria de Fluxo , Perfilação da Expressão Gênica , Interferon gama/metabolismo , Macaca mulatta , Análise de Sequência com Séries de Oligonucleotídeos
13.
PLoS Negl Trop Dis ; 5(1): e928, 2011 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-21264351

RESUMO

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus indigenous to tropical Africa and Asia. Acute illness is characterized by fever, arthralgias, conjunctivitis, rash, and sometimes arthritis. Relatively little is known about the antigenic targets for immunity, and no licensed vaccines or therapeutics are currently available for the pathogen. While the Aedes aegypti mosquito is its primary vector, recent evidence suggests that other carriers can transmit CHIKV thus raising concerns about its spread outside of natural endemic areas to new countries including the U.S. and Europe. Considering the potential for pandemic spread, understanding the development of immunity is paramount to the development of effective counter measures against CHIKV. In this study, we isolated a new CHIKV virus from an acutely infected human patient and developed a defined viral challenge stock in mice that allowed us to study viral pathogenesis and develop a viral neutralization assay. We then constructed a synthetic DNA vaccine delivered by in vivo electroporation (EP) that expresses a component of the CHIKV envelope glycoprotein and used this model to evaluate its efficacy. Vaccination induced robust antigen-specific cellular and humoral immune responses, which individually were capable of providing protection against CHIKV challenge in mice. Furthermore, vaccine studies in rhesus macaques demonstrated induction of nAb responses, which mimicked those induced in convalescent human patient sera. These data suggest a protective role for nAb against CHIKV disease and support further study of envelope-based CHIKV DNA vaccines.


Assuntos
Infecções por Alphavirus/prevenção & controle , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vírus Chikungunya/imunologia , Vacinas de DNA/imunologia , Infecções por Alphavirus/virologia , Animais , Vírus Chikungunya/genética , Vírus Chikungunya/isolamento & purificação , Modelos Animais de Doenças , Eletroporação , Feminino , Humanos , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização/métodos , Vacinação/métodos , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Virologia/métodos
14.
Vaccine ; 29(39): 6755-62, 2011 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-21238574

RESUMO

Protection against infection is the hallmark of immunity and the basis of effective vaccination. For a variety of reasons there is a great demand to develop new, safer and more effective vaccine platforms. In this regard, while 'first-generation' DNA vaccines were poorly immunogenic, new genetic 'optimization' strategies and the application of in vivo electroporation (EP) have dramatically boosted their potency. We developed a highly optimized plasmid DNA vaccine that expresses the lymphocytic choriomeningitis virus (LCMV) nucleocapsid protein (NP) and evaluated it using the LCMV challenge model, a gold standard for studying infection and immunity. When administered intramuscularly with EP, robust NP-specific cellular and humoral immune responses were elicited, the magnitudes of which approached those following acute LCMV infection. Furthermore, these responses were capable of providing 100% protection against a high-dose, normally lethal virus challenge. This is the first non-infectious vaccine conferring complete protective immunity up to 8 weeks after vaccination and demonstrates the potential of 'next-generation' DNA vaccines.


Assuntos
Imunidade Celular , Imunidade Humoral , Coriomeningite Linfocítica/prevenção & controle , Proteínas do Nucleocapsídeo/imunologia , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Formação de Anticorpos , ELISPOT , Feminino , Vetores Genéticos , Células HEK293 , Humanos , Dose Letal Mediana , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/patogenicidade , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/genética , Plasmídeos/metabolismo , Transfecção , Vacinação/métodos , Vacinas de DNA/administração & dosagem , Vacinas Virais/administração & dosagem
15.
Expert Rev Vaccines ; 9(7): 747-63, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20624048

RESUMO

DNA vaccination has been of great interest since its discovery in the 1990s due to its ability to elicit both humoral and cellular immune responses. DNA vaccines consist of a DNA plasmid containing a transgene that encodes the sequence of a target protein from a pathogen under the control of a eukaryotic promoter. This revolutionary technology has proven to be effective in animal models and four DNA vaccine products have recently been approved for veterinary use. Although few DNA vaccines against bacterial infections have been tested, the results are encouraging. Because of their versatility, safety and simplicity a wider range of organisms can be targeted by these vaccines, which shows their potential advantages to public health. This article describes the mechanism of action of DNA vaccines and their potential use for targeting bacterial infections. In addition, it provides an updated summary of the methods used to enhance immunogenicity from codon optimization and adjuvants to delivery techniques including electroporation and use of nanoparticles.


Assuntos
Infecções Bacterianas/prevenção & controle , Vacinas Bacterianas/imunologia , Vacinas de DNA/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Antígenos de Bactérias/genética , Códon , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Expressão Gênica , Vetores Genéticos , Humanos , Plasmídeos , Regiões Promotoras Genéticas
16.
Mol Ther ; 18(9): 1714-23, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20571540

RESUMO

Type III/lambda interferons (IFNs) were discovered less than a decade ago and are still in the process of being characterized. Although previous studies have focused on the function of IFN-lambda 3 (also known as interleukin (IL)-28B) in a small animal model, it is unknown whether these functions would translate to a larger, more relevant model. Thus in the present study, we have used DNA vaccination as a method of studying the influence of IFN-lambda 3 on adaptive immune responses in rhesus macaques. Results of our study show for the first time that IFN-lambda 3 has significant influence on antigen-specific CD8(+) T-cell function, especially in regards to cytotoxicity. Peripheral CD8(+) T cells from animals that were administered IFN-lambda 3 showed substantially increased cytotoxic responses as gauged by CD107a and granzyme B coexpression as well as perforin release. Moreover, CD8(+) T cells isolated from the mesenteric lymph nodes (MLN) of animals receiving IFN-lambda 3 loaded significant amounts of granzyme B upon extended antigenic stimulation and induced significantly more granzyme B-mediated cell death of peptide pulsed targets. These data suggest that IFN-lambda 3 is a potent effector of the immune system with special emphasis on CD8(+) T-cell killing functions which warrants further study as a possible immunoadjuvant.


Assuntos
Granzimas/metabolismo , Interferons/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/metabolismo , Animais , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , ELISPOT , Citometria de Fluxo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Macaca
17.
PLoS Negl Trop Dis ; 4(4): e623, 2010 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-20436958

RESUMO

Chikungunya virus is a mosquito-borne emerging pathogen that has a major health impact in humans and causes fever disease, headache, rash, nausea, vomiting, myalgia, and arthralgia. Indigenous to tropical Africa, recent large outbreaks have been reported in parts of South East Asia and several of its neighboring islands in 2005-07 and in Europe in 2007. Furthermore, positive cases have been confirmed in the United States in travelers returning from known outbreak areas. Currently, there is no vaccine or antiviral treatment. With the threat of an emerging global pandemic, the peculiar problems associated with the more immediate and seasonal epidemics warrant the development of an effective vaccine. In this review, we summarize the evidence supporting these concepts.


Assuntos
Infecções por Alphavirus/epidemiologia , Infecções por Alphavirus/virologia , Vírus Chikungunya/isolamento & purificação , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/virologia , África/epidemiologia , Infecções por Alphavirus/prevenção & controle , Ásia Sudeste/epidemiologia , Vírus Chikungunya/imunologia , Doenças Transmissíveis Emergentes/prevenção & controle , Europa (Continente)/epidemiologia , Humanos , Viagem , Estados Unidos/epidemiologia , Vacinas Virais/imunologia
18.
Virology ; 401(2): 228-35, 2010 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-20304456

RESUMO

Human Ebola virus causes severe hemorrhagic fever disease with high mortality and there is no vaccine or treatment. Antibodies in survivors occur early, are sustained, and can delay infection when transferred into nonhuman primates. Monoclonal antibodies (mAbs) from survivors exhibit potent neutralizing activity in vitro and are protective in rodents. To better understand targets and mechanisms of neutralization, we investigated a panel of mAbs shown previously to react with the envelope glycoprotein (GP). While one non-neutralizing mAb recognized a GP epitope in the nonessential mucin-like domain, the rest were specific for GP1, were neutralizing, and could be further distinguished by reactivity with secreted GP. We show that survivor antibodies, human KZ52 and monkey JP3K11, were specific for conformation-dependent epitopes comprising residues in GP1 and GP2 and that neutralization occurred by two distinct mechanisms; KZ52 inhibited cathepsin cleavage of GP whereas JP3K11 recognized the cleaved, fusion-active form of GP.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Animais , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Haplorrinos , Humanos , Testes de Neutralização , Ligação Proteica , Proteínas do Envelope Viral/imunologia
19.
Curr Opin Investig Drugs ; 11(2): 192-202, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20112169

RESUMO

There is currently no cure for HIV infection, and the possibility of developing a vaccine in the near future appears unlikely. With more than 33 million individuals living with HIV/AIDS worldwide, there is a distinct need for a prophylactic vaccine against HIV infection. However, conventional vaccine strategies aimed at eliciting antibody and T-cell responses have failed to protect against the virus. Current research has been directed toward the more realistic goal of controlling viral replication during the early stages of infection, thus reducing the viral setpoint, through the use of novel vaccine delivery systems and techniques. In this review, several of the key milestones achieved as a result of research efforts aimed at developing an effective HIV vaccine are identified, and future prospects are examined.


Assuntos
Vacinas contra a AIDS/uso terapêutico , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Vacinas contra a AIDS/imunologia , Animais , Ensaios Clínicos como Assunto , Desenho de Drogas , Infecções por HIV/imunologia , Humanos , Replicação Viral/efeitos dos fármacos
20.
Cytometry A ; 77(3): 275-84, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20104580

RESUMO

The capacity for robust proliferation upon re-infection is a hallmark of adaptive immunity and the basis of vaccination. A widely used animal model for the study of human disease is the rhesus macaque (RM), where capacity for proliferation can be assessed ex vivo using carboxyfluorescein succinimidyl ester (CFSE)-based dilution assays. However, we show over the course of the standard ex vivo proliferation assay that CFSE-labeling at commonly used dye concentrations induces significant cell death, but that this phenomenon is dose-dependent. Here, we describe an alternative semiquantitative method for estimating T cell proliferative responses that avoids the putative biases associated with chemical modification. RM peripheral blood mononuclear cells were stimulated ex vivo with cognate peptides for 5 days, immunostained for intracellular Ki-67, and then analyzed by flow cytometry. We describe a gating strategy using Ki-67 and side light scatter, also a marker of blastogenesis, which correlates strongly with data from CFSE dilution. We show that this method is a valid tool for measuring RM antigen-specific cellular proliferation ex vivo and can be used as an alternative to CFSE dilution assays.


Assuntos
Citometria de Fluxo/métodos , Antígeno Ki-67/biossíntese , Linfócitos T/citologia , Animais , Antígenos/química , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Corantes/farmacologia , Relação Dose-Resposta a Droga , Fluoresceínas/química , Deleção de Genes , Leucócitos Mononucleares/citologia , Macaca mulatta , Succinimidas/química , Linfócitos T/microbiologia
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