RESUMO
Parasitic nematodes produce an unusual class of fatty acid and retinol (FAR)-binding proteins that may scavenge host fatty acids and retinoids. Two FARs from Brugia malayi (Bm-FAR-1 and Bm-FAR-2) were expressed as recombinant proteins, and their ligand binding, structural characteristics, and immunogenicities examined. Circular dichroism showed that rBm-FAR-1 and rBm-FAR-2 are similarly rich in α-helix structure. Unexpectedly, however, their lipid binding activities were found to be readily differentiated. Both FARs bound retinol and cis-parinaric acid similarly, but, while rBm-FAR-1 induced a dramatic increase in fluorescence emission and blue shift in peak emission by the fluorophore-tagged fatty acid (dansyl-undecanoic acid), rBm-FAR-2 did not. Recombinant forms of the related proteins from Onchocerca volvulus, rOv-FAR-1 and rOv-FAR-2, were found to be similarly distinguishable. This is the first FAR-2 protein from parasitic nematodes that is being characterized. The relative protein abundance of Bm-FAR-1 was higher than Bm-FAR-2 in the lysates of different developmental stages of B. malayi. Both FAR proteins were targets of strong IgG1, IgG3 and IgE antibody in infected individuals and individuals who were classified as endemic normal or putatively immune. In a B. malayi infection model in gerbils, immunization with rBm-FAR-1 and rBm-FAR-2 formulated in a water-in-oil-emulsion (®Montanide-720) or alum elicited high titers of antigen-specific IgG, but only gerbils immunized with rBm-FAR-1 formulated with the former produced a statistically significant reduction in adult worms (68%) following challenge with B. malayi infective larvae. These results suggest that FAR proteins may play important roles in the survival of filarial nematodes in the host, and represent potential candidates for vaccine development against lymphatic filariasis and related filarial infections.
Assuntos
Antígenos de Helmintos/imunologia , Brugia Malayi/imunologia , Proteínas de Ligação a Ácido Graxo/imunologia , Filariose/prevenção & controle , Proteínas de Ligação ao Retinol/imunologia , Vacinas Sintéticas/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/química , Dicroísmo Circular , Modelos Animais de Doenças , Proteínas de Ligação a Ácido Graxo/química , Feminino , Gerbillinae , Humanos , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Masculino , Carga Parasitária , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas de Ligação ao Retinol/química , Resultado do Tratamento , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/isolamento & purificação , Vitamina A/metabolismoRESUMO
The devastating Ebola virus (EBOV) epidemic in West Africa in 2013-2016 accelerated the progress of several vaccines and antivirals through clinical trials, including the replication-competent vesicular stomatitis virus-based vaccine expressing the EBOV glycoprotein (VSV-EBOV). Extensive preclinical testing in animal models demonstrated the prophylactic and post-exposure efficacy of this vaccine, identified the mechanism of protection, and suggested it was safe for human use. Based on these data, VSV-EBOV was extensively tested in phase 1-3 clinical trials in North America, Europe and Africa. Although some side effects of vaccination were observed, these clinical trials showed that the VSV-EBOV was safe and immunogenic in humans. Moreover, the data supported the use of VSV-EBOV as an emergency vaccine in individuals at risk for Ebola virus disease. In this review, we summarize the results of the extensive preclinical and clinical testing of the VSV-EBOV vaccine.
Assuntos
Portadores de Fármacos , Vacinas contra Ebola/imunologia , Vacinas contra Ebola/isolamento & purificação , Doença pelo Vírus Ebola/prevenção & controle , Vesiculovirus/genética , Animais , Ensaios Clínicos como Assunto , Avaliação Pré-Clínica de Medicamentos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Vacinas contra Ebola/administração & dosagem , Vacinas contra Ebola/genética , Humanos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/isolamento & purificação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
INTRODUCTION: GMZ2 is a recombinant protein consisting of conserved domains of GLURP and MSP3, two asexual blood-stage antigens of Plasmodium falciparum, and is designed with the aim of mimicking naturally acquired anti-malarial immunity. The rationale for combining these two antigens is based on a series of immune epidemiological studies from geographically diverse malaria endemic regions; functional in vitro studies; and pre-clinical studies in rodents and New World monkeys. GMZ2 adjuvanted with alhydrogel® (alum) was well tolerated and immunogenic in three phase 1 studies. The recently concluded phase 2 trial of GMZ2/alum, involving 1849 participants 12 to 60 month of age in four countries in West, Central and Eastern Africa, showed that GMZ2 is well tolerated and has some, albeit modest, efficacy in the target population. Areas covered: PubMed ( www.ncbi.nlm.nih.gov/pubmed ) was searched to review the progress and future prospects for clinical development of GMZ2 sub-unit vaccine. We will focus on discovery, naturally acquired immunity, functional activity of specific antibodies, sequence diversity, production, pre-clinical and clinical studies. Expert commentary: GMZ2 is well tolerated and has some, albeit modest, efficacy in the target population. More immunogenic formulations should be developed.
Assuntos
Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Vacinas Antimaláricas/isolamento & purificação , Plasmodium falciparum/imunologia , Animais , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Avaliação Pré-Clínica de Medicamentos , Humanos , Vacinas Antimaláricas/efeitos adversos , Roedores , Vacinas de Subunidades Antigênicas/efeitos adversos , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/isolamento & purificação , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
The epidemic emergence of Zika virus (ZIKV) in 2015-2016 has been associated with congenital malformations and neurological sequela. Current efforts to develop a ZIKV vaccine build on technologies that successfully reduced infection or disease burden against closely related flaviviruses or other RNA viruses. Subunit-based (DNA plasmid and modified mRNA), viral vectored (adeno- and measles viruses) and inactivated viral vaccines are already advancing to clinical trials in humans after successful mouse and non-human primate studies. Among the greatest challenges for the rapid implementation of immunogenic and protective ZIKV vaccines will be addressing the potential for exacerbating Dengue virus infection or causing Guillain-Barré syndrome through production of cross-reactive immunity targeting related viral or host proteins. Here, we review vaccine strategies under development for ZIKV and the issues surrounding their usage.
Assuntos
Vacinas de DNA/imunologia , Vacinas de DNA/isolamento & purificação , Vacinas Virais/imunologia , Vacinas Virais/isolamento & purificação , Infecção por Zika virus/prevenção & controle , Zika virus/imunologia , Animais , Ensaios Clínicos como Assunto , Dengue/epidemiologia , Avaliação Pré-Clínica de Medicamentos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Síndrome de Guillain-Barré/epidemiologia , Humanos , Camundongos , Vacinas de DNA/efeitos adversos , Vacinas de Produtos Inativados/efeitos adversos , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/isolamento & purificação , Vacinas de Subunidades Antigênicas/efeitos adversos , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/isolamento & purificação , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação , Vacinas Virais/efeitos adversosRESUMO
INTRODUCTION: Respiratory infections have an enormous, worldwide epidemiologic impact on humans and animals. Among the prophylactic measures, vaccination has the potential to neutralize this impact. New technologies for vaccine production and delivery are of importance in this field since they offer the potential to develop new immunization approaches overriding the current limitations that comprise high cost, safety issues, and limited efficacy. Areas covered: In the present review, the state of the art in developing plant-based vaccines against respiratory diseases is presented. The review was based on the analysis of current biomedical literature. Expert commentary: Preclinical and clinical evaluations of several vaccine candidates against influenza, tuberculosis, respiratory syncytial virus, pneumonia, anthrax and asthma are discussed and placed in perspective.
Assuntos
Vacinas Bacterianas/imunologia , Plantas Geneticamente Modificadas/metabolismo , Infecções Respiratórias/prevenção & controle , Vacinas Virais/imunologia , Animais , Vacinas Bacterianas/isolamento & purificação , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Descoberta de Drogas/tendências , Avaliação Pré-Clínica de Medicamentos , Humanos , Plantas Geneticamente Modificadas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação , Vacinas Virais/isolamento & purificaçãoRESUMO
Mycobacterium bovis BCG is the only available vaccine for protection against tuberculosis (TB). While BCG protects children from severe disease, it has little impact on pulmonary disease in adults. A recombinant BCG vaccine BCG ΔureC::hly (strain VPM1002) is in advanced clinical trials and shows promise for improved vaccine safety but little change in efficacy in animal models. A second-generation recombinant BCG vaccine with an additional deletion of the nuoG gene (BCG ΔureC::hly ΔnuoG) shows improved efficacy in a mouse model compared to that of VPM1002. BCG was first used in humans in 1921 and, like Sleeping Beauty pricked by the spinning wheel, we have slept for 100 years, showing a reluctance to invest in clinical development or in biomanufacturing capacity for TB vaccines. The advance of recombinant BCGs should awaken us from our sleep and call us to invest in new-generation TB vaccines and to protect the biomanufacture of our current BCG vaccine.
Assuntos
Vacina BCG/imunologia , Vacina BCG/isolamento & purificação , Descoberta de Drogas/tendências , Animais , Vacina BCG/administração & dosagem , Vacina BCG/genética , Ensaios Clínicos como Assunto , Avaliação Pré-Clínica de Medicamentos , Humanos , Camundongos , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
INTRODUCTION: Lassa virus (LASV), the most prominent human pathogen of the Arenaviridae, is transmitted to humans from infected rodents and can cause Lassa Fever (LF). The sizeable disease burden in West Africa, numerous imported LF cases worldwide, and the possibility that LASV can be used as an agent of biological warfare make a strong case for vaccine development. There are no licensed LASV vaccines and the antiviral treatment is limited to an off-label use of ribavirin that is only partially effective. AREAS COVERED: LASV vaccine development is hampered by high cost of biocontainment requirement, the absence of appropriate small animal models, genetic diversity of LASV species, and by high HIV-1 prevalence in LASV endemic areas. Over the past 15 years several vaccine platforms have been developed. Natural history of LASV and pathogenesis of the disease provide strong justification for replication-competent (RC) vaccine as one of the most feasible approaches to control LF. Development of LASV vaccine candidates based on reassortant, recombinant, and alphavirus replicon technologies is covered in this review. Expert commentary: Two lead RC vaccine candidates, reassortant ML29 and recombinant VSV/LASV, have been successfully tested in non-human primates and have been recommended by international vaccine experts for rapid clinical development. Both platforms have powerful molecular tools to further secure safety, improve immunogenicity, and cross-protection. These platforms are well positioned to design multivalent vaccines to protect against all LASV strains citculatrd in West Africa. The regulatory pathway of Candid #1, the first live-attenuated arenaviral vaccine against Argentine hemorrhagic, will be a reasonable guideline for LASV vaccine efficacy trials.
Assuntos
Febre Lassa/prevenção & controle , Vírus Lassa/imunologia , Vacinas Virais/imunologia , Vacinas Virais/isolamento & purificação , Animais , Descoberta de Drogas/tendências , Avaliação Pré-Clínica de Medicamentos , Humanos , Febre Lassa/imunologia , Primatas , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/isolamento & purificação , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
BACKGROUND: Human rotaviruses are the main cause of severe gastroenteritis in children and are responsible for over 500 000 deaths annually. There are two live rotavirus vaccines currently available, one based on human rotavirus serotype G1P[8], and the other a G1-G4 P[8] pentavalent vaccine. However, the recent emergence of the G9 and other novel rotavirus serotypes in Africa and Asia has prompted fears that current vaccines might not be fully effective against these new varieties. RESULTS: We report an effort to develop an affordable candidate rotavirus vaccine against the new emerging G9P[6] (RVA/Human-wt/ZAF/GR10924/1999/G9P[6]) strain. The vaccine is based on virus-like particles which are both highly immunogenic and safe. The vaccine candidate was produced in Nicotiana benthamiana by transient expression, as plants allow rapid production of antigens at lower costs, without the risk of contamination by animal pathogens. Western blot analysis of plant extracts confirmed the successful expression of two rotavirus capsid proteins, VP2 and VP6. These proteins assembled into VLPs resembling native rotavirus particles when analysed by transmission electron microscopy (TEM). Expression of the rotavirus glycoprotein VP7 and the spike protein VP4 was also tried. However, VP7 expression caused plant wilting during the course of the time trial and expression could never be detected for either protein. We therefore created three fusion proteins adding the antigenic part of VP4 (VP8*) to VP6 in an attempt to produce more appropriately immunogenic particles. Fusion protein expression in tobacco plants was detected by western blot using anti-VP6 and anti-VP4 antibodies, but no regular particles were observed by TEM, even when co-expressed with VP2. CONCLUSION: Our results suggest that the rotavirus proteins produced in N. benthamiana are candidates for a subunit vaccine specifically for the G9P[6] rotavirus strain. This could be more effective in developing countries, thereby possibly providing a higher overall efficacy for the existing vaccines. The production of rotavirus proteins in plants would probably result in lower manufacturing costs, making it more affordable for developing countries. Further investigation is required to evaluate the immunogenic potential of the VLPs and fusion proteins created in this study.
Assuntos
Genótipo , Infecções por Rotavirus/prevenção & controle , Vacinas contra Rotavirus/imunologia , Rotavirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Gastroenterite/prevenção & controle , Gastroenterite/virologia , Humanos , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Rotavirus/classificação , Rotavirus/genética , Infecções por Rotavirus/virologia , Vacinas contra Rotavirus/genética , Vacinas contra Rotavirus/isolamento & purificação , Análise de Sequência de DNA , Nicotiana/genética , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificaçãoRESUMO
Dengue is a significant threat to public health worldwide. Currently, there are no licensed vaccines available for dengue. Takeda Vaccines Inc. is developing a live, attenuated tetravalent dengue vaccine candidate (TDV) that consists of an attenuated DENV-2 strain (TDV-2) and three chimeric viruses containing the prM and E protein genes of DENV-1, -3 and -4 expressed in the context of the attenuated TDV-2 genome backbone (TDV-1, TDV-3, and TDV-4, respectively). TDV has been shown to be immunogenic and efficacious in nonclinical animal models. In interferon-receptor deficient mice, the vaccine induces humoral neutralizing antibody responses and cellular immune responses that are sufficient to protect from lethal challenge with DENV-1, DENV-2 or DENV-4. In non-human primates, administration of TDV induces innate immune responses as well as long lasting antibody and cellular immunity. In Phase 1 clinical trials, the safety and immunogenicity of two different formulations were assessed after intradermal or subcutaneous administration to healthy, flavivirus-naïve adults. TDV administration was generally well-tolerated independent of dose and route. The vaccine induced neutralizing antibody responses to all four DENV serotypes: after a single administration of the higher formulation, 24-67%% of the subjects seroconverted to all four DENV and >80% seroconverted to three or more viruses. In addition, TDV induced CD8(+) T cell responses to the non-structural NS1, NS3 and NS5 proteins of DENV. TDV has been also shown to be generally well tolerated and immunogenic in a Phase 2 clinical trial in dengue endemic countries in adults and children as young as 18 months. Additional clinical studies are ongoing in preparation for a Phase 3 safety and efficacy study.
Assuntos
Vacinas contra Dengue/imunologia , Vacinas contra Dengue/isolamento & purificação , Dengue/prevenção & controle , Imunidade Adaptativa , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linfócitos T CD8-Positivos/imunologia , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Dengue/epidemiologia , Vacinas contra Dengue/efeitos adversos , Vacinas contra Dengue/genética , Avaliação Pré-Clínica de Medicamentos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Imunidade Celular , Imunidade Inata , Injeções Intradérmicas , Camundongos , Primatas , Análise de Sobrevida , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/isolamento & purificação , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
This review focuses on a dengue virus (DENV) vaccine candidate based on a recombinant subunit approach which targets the DENV envelope glycoprotein (E). Truncated versions of E consisting of the N-terminal portion of E (DEN-80E) have been expressed recombinantly in the Drosophila S2 expression system and shown to have native-like conformation. Preclinical studies demonstrate that formulations containing tetravalent DEN-80E adjuvanted with ISCOMATRIX™ adjuvant induce high titer virus neutralizing antibodies and IFN-γ producing T cells in flavivirus-naïve non-human primates. The preclinical data further suggest that administration of such formulations on a 0, 1, 6 month schedule may result in higher maximum virus neutralizing antibody titers and better durability of those titers compared to administration on a 0, 1, 2 month schedule. In addition, the virus neutralizing antibody titers induced by adjuvanted tetravalent DEN-80E compare favorably to the titers induced by a tetravalent live virus comparator. Furthermore, DEN-80E was demonstrated to be able to boost virus neutralizing antibody titers in macaques that have had a prior DENV exposure. A monovalent version of the vaccine candidate, DEN1-80E, was formulated with Alhydrogel™ and studied in a proof-of-principle Phase I clinical trial by Hawaii Biotech, Inc. (NCT00936429). The clinical trial results demonstrate that both the 10 µg and 50 µg formulations of DEN1-80E with 1.25 mg of elemental aluminum were immunogenic when administered in a 3-injection series (0, 1, 2 months) to healthy, flavivirus-naïve adults. The vaccine formulations induced DENV-1 neutralizing antibodies in the majority of subjects, although the titers in most subjects were modest and waned over time. Both the 10 µg DEN1-80E and the 50 µg DEN1-80E formulations with Alhydrogel™ were generally well tolerated.
Assuntos
Ensaios Clínicos Fase I como Assunto , Vacinas contra Dengue/administração & dosagem , Vacinas contra Dengue/imunologia , Dengue/prevenção & controle , Avaliação Pré-Clínica de Medicamentos , Adjuvantes Imunológicos/administração & dosagem , Hidróxido de Alumínio/administração & dosagem , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Colesterol/administração & dosagem , Dengue/epidemiologia , Vacinas contra Dengue/genética , Vacinas contra Dengue/isolamento & purificação , Combinação de Medicamentos , Humanos , Esquemas de Imunização , Interferon gama/metabolismo , Macaca , Fosfolipídeos/administração & dosagem , Saponinas/administração & dosagem , Linfócitos T/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/isolamento & purificação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
Since the early 1990s, DNA immunization has been used as a platform for developing a tetravalent dengue vaccine in response to the high priority need for protecting military personnel deployed to dengue endemic regions of the world. Several approaches have been explored ranging from naked DNA immunization to the use of live virus vectors to deliver the targeted genes for expression. Pre-clinical animal studies were largely successful in generating anti-dengue cellular and humoral immune responses that were protective either completely or partially against challenge with live dengue virus. However, Phase 1 clinical evaluation of a prototype monovalent dengue 1 DNA vaccine expressing prM and E genes revealed anti-dengue T cell IFNγ responses, but poor neutralizing antibody responses. These less than optimal results are thought to be due to poor uptake and expression of the DNA vaccine plasmids. Because DNA immunization as a vaccine platform has the advantages of ease of manufacture, flexible genetic manipulation and enhanced stability, efforts continue to improve the immunogenicity of these vaccines using a variety of methods.
Assuntos
Vacinas contra Dengue/administração & dosagem , Vacinas contra Dengue/imunologia , Dengue/prevenção & controle , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Ensaios Clínicos Fase I como Assunto , Dengue/epidemiologia , Vacinas contra Dengue/genética , Vacinas contra Dengue/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos , Humanos , Primatas , Vacinas de DNA/genética , Vacinas de DNA/isolamento & purificação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
In 2014, an outbreak of Ebola virus spread rapidly in West Africa. The epidemic killed more than 10,000 people and resulted in transmissions outside the endemic countries. WHO hopes for effective vaccines by the end of 2015. Numerous vaccine candidates have been proposed, and several are currently being evaluated in humans. Among the vaccine candidates are vectors derived from adenovirus (Ad). Despite previous encouraging preclinical and Phase I/II trials, Ad vectors used in three Phase II trials targeting HIV were prematurely interrupted because of the lack of demonstrated efficacy. The vaccine was not only ineffective but also led to a higher rate of HIV acquisition. In this context, the authors discuss the potential benefits, risks and impact of using Ad-derived vaccines to control Ebola virus disease.
Assuntos
Vacinas contra a AIDS/efeitos adversos , Adenoviridae/genética , Portadores de Fármacos , Descoberta de Drogas/métodos , Vacinas contra Ebola/imunologia , Vacinas contra Ebola/isolamento & purificação , Vetores Genéticos , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , África Ocidental/epidemiologia , Ensaios Clínicos como Assunto , Avaliação Pré-Clínica de Medicamentos , Vacinas contra Ebola/genética , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Risco , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
BACKGROUND: Bovine viral diarrhea virus (BVDV) infections are endemic in cattle populations worldwide and cause major economic losses. Thus, an effective vaccine is needed against the transmission of BVDV. The glycoprotein E(rns) is one of the envelope proteins of this virus and shows BVDV-related immunogenicity. Here, we report the use of Panax ginseng as an alternative production platform for the expression of glycoprotein E(rns) via Agrobacterium-mediated transformation. RESULT: Polymerase chain reaction (PCR) and reverse transcription (RT)-PCR analyses showed that pBI121-E(rns) was stably integrated into the chromosome of transformants. ELISA assay and Western blot analysis confirmed the antigenicity of plant-derived E(rns) glycoprotein. Immunogenicity was evaluated subcutaneously in deer using a soluble protein extract of dried transgenic ginseng hairy roots. Specific humoral and cell-mediated immune responses against BVDV were detected following immunization. CONCLUSION: These results demonstrated that the E(rns) glycoprotein could be expressed in ginseng hairy roots and that plant-derived glycoprotein E(rns) retained its antigenicity and immunogenicity.
Assuntos
Diarreia/veterinária , Transmissão de Doença Infecciosa/prevenção & controle , Panax/metabolismo , Infecções por Pestivirus/veterinária , Plantas Geneticamente Modificadas/metabolismo , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Cervos , Diarreia/prevenção & controle , Leucócitos Mononucleares/imunologia , Masculino , Panax/genética , Infecções por Pestivirus/prevenção & controle , Plantas Geneticamente Modificadas/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação , Vacinas Virais/administração & dosagem , Vacinas Virais/isolamento & purificaçãoRESUMO
Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious and devastating disease that affects the pig industry worldwide. The glycoprotein E2 of CSFV is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. Several CSFV genotypes, including 1.1, 2.1, 2.2, and 2.3, have been identified in Mainland China. The glycoprotein E2 of genotypes 1.1 and 2.1 was expressed by using a baculovirus system and tested for its protective immunity in rabbits to develop novel CSF vaccines that elicit a broad immune response. Twenty CSFV seronegative rabbits were randomly divided into five groups. Each rabbit was intramuscularly immunized with E2 of genotypes 1.1 (CSFV-1.1E2), 2.1 (CSFV-2.1E2), or their combination (CSFV-1.1 + 2.1E2). A commercial CSF vaccine (C-strain) and phosphate-buffered saline (PBS) were used as positive or negative controls, respectively. All animals were challenged with CSFV C-strain at 4 weeks and then boosted with the same dose. All rabbits inoculated with CSFV-1.1E2, CSFV-2.1E2, and CSFV-1.1 + 2.1E2 elicited high levels of ELISA antibody, neutralizing antibody, and lymphocyte proliferative responses to CSFV. The rabbits inoculated with CSFV-1.1E2 and CSFV-1.1 + 2.1E2 received complete protection against CSFV C-strain. Two of the four rabbits vaccinated with CSFV-2.1E2 were completely protected. These results demonstrate that CSFV-1.1E2 and CSFV-1.1 + 2.1E2 not only elicit humoral and cell-mediated immune responses but also confer complete protection against CSFV C-strain in rabbits. Therefore, CSFV-1.1E2 and CSFV-1.1 + 2.1E2 are promising candidate subunit vaccines against CSF.
Assuntos
Vírus da Febre Suína Clássica/imunologia , Peste Suína Clássica/prevenção & controle , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Baculoviridae/genética , Proliferação de Células , China , Peste Suína Clássica/imunologia , Peste Suína Clássica/patologia , Vírus da Febre Suína Clássica/genética , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Vetores Genéticos , Injeções Intramusculares , Linfócitos/imunologia , Testes de Neutralização , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Suínos , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/isolamento & purificação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação , Proteínas do Envelope Viral/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/isolamento & purificaçãoRESUMO
Recombinant viruses based on the cDNA copy of the tobacco mosaic virus (TMV) genome carrying different versions of the conserved M2e epitope from influenza virus A cloned into the coat protein (CP) gene were obtained and partially characterized by our group previously; cysteines in the human consensus M2e sequence were changed to serine residues. This work intends to show some biological properties of these viruses following plant infections. Agroinfiltration experiments on Nicotiana benthamiana confirmed the efficient systemic expression of M2e peptides, and two point amino acid substitutions in recombinant CPs significantly influenced the symptoms and development of viral infections. Joint expression of RNA interference suppressor protein p19 from tomato bushy stunt virus (TBSV) did not affect the accumulation of CP-M2e-ser recombinant protein in non-inoculated leaves. RT-PCR analysis of RNA isolated from either infected leaves or purified TMV-M2e particles proved the genetic stability of TMVbased viral vectors. Immunoelectron microscopy of crude plant extracts demonstrated that foreign epitopes are located on the surface of chimeric virions. The rodshaped geometry of plant-produced M2e epitopes is different from the icosahedral or helical filamentous arrangement of M2e antigens on the carrier virus-like particles (VLP) described earlier. Thereby, we created a simple and efficient system that employs agrobacteria and plant viral vectors in order to produce a candidate broad-spectrum flu vaccine.
Assuntos
Epitopos/biossíntese , Vírus da Influenza A/genética , Nanotubos , Nicotiana/genética , Plantas Geneticamente Modificadas , Tobamovirus/genética , Proteínas da Matriz Viral/biossíntese , Proteínas do Capsídeo/genética , Epitopos/genética , Perfilação da Expressão Gênica , Vetores Genéticos , Instabilidade Genômica , Vacinas contra Influenza/isolamento & purificação , Microscopia Imunoeletrônica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tombusvirus , Vacinas Sintéticas/isolamento & purificação , Proteínas da Matriz Viral/genéticaRESUMO
A highly purified TUL4-CBD chimeric protein was obtained by one stage purification method. TUL4-CBD protein consists of TUL4 Francisella tularensis mature peptide sequence, Gly-Ser spacer and cellulose binding domain (CBD) of Anaerocellum thermophilum. The TUL4-CBD protein was shown to induce production of specific antibodies to TUL4 protein in laboratory animals.
Assuntos
Adjuvantes Imunológicos , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/imunologia , Celulose/imunologia , Francisella tularensis/imunologia , Esquemas de Imunização , Lipoproteínas/imunologia , Tularemia/imunologia , Adjuvantes Imunológicos/metabolismo , Animais , Azidas , Bactérias Anaeróbias/enzimologia , Celulase/metabolismo , Celulose/metabolismo , Escherichia coli/metabolismo , Adjuvante de Freund , Injeções Subcutâneas , Netuno , Ligação Proteica , Estrutura Terciária de Proteína/fisiologia , Coelhos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Tularemia/sangue , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic peptide or lipid tags to improve their capacity to be incorporated into an adjuvant formulation. In the present study, we have explored the strong interaction between biotin and SA (streptavidin) (K(D) approximately 10(-15) M) to couple recombinant immunogens to iscoms (immunostimulating complexes). Two different concepts were evaluated. In the first concept, a His(6)-tagged SA fusion protein (His(6)-SA) was bound to Ni(2+)-loaded iscom matrix (iscom without associated protein), and biotinylated immunogens were thereafter associated with the SA-coated iscoms. The immunogens were either biotinylated in vivo on E. coli expression or double biotinylated in vivo and in vitro. In the second concept, the recombinant immunogens were expressed as SA fusion proteins, which were directly bound to a biotinylated iscom matrix. A 53-amino-acid malaria peptide (M5), derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, and a 232-amino-acid segment (SRS2') from the central region (from Pro-97 to Lys-328) of the major surface antigen NcSRS2 of the protozoan parasite Neospora caninum, served as model immunogens in the present study. All fusion proteins generated were found to be efficiently expressed and could be recovered to high purity using affinity chromatography. The association between the different immunogen-containing fusion proteins and the corresponding iscom matrix was demonstrated by analytical ultracentrifugation in a sucrose density gradient. However, some fusion proteins were, to a certain extent, also found to associate unspecifically with a regular iscom matrix. Furthermore, selected iscom fractions were demonstrated to induce high-titre antigen-specific antibody responses on immunization of mice. For the particular target immunogen SRS2', the induced antibodies demonstrated reactivity to the native antigen NcSRS2. We believe that the presented concepts offer convenient methods to achieve efficient adjuvant association of recombinant immunogens, and the advantages and disadvantages of the two concepts are discussed.
Assuntos
Proteínas de Bactérias/metabolismo , Biotina/análogos & derivados , Vacinas Sintéticas/biossíntese , Animais , Anticorpos/metabolismo , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Antígenos de Superfície/imunologia , Antígenos de Superfície/metabolismo , Proteínas de Bactérias/química , Sequência de Bases , Biotina/química , Biotina/metabolismo , Feminino , Engenharia Genética , Imunização , Camundongos , Dados de Sequência Molecular , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Vacinas Sintéticas/química , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
Cervical cancer is linked to infection with human papillomaviruses (HPV) and is the third most common cancer among women worldwide. There is a strong demand for the development of an HPV preventive vaccine. Transgenic plants expressing the HPV major capsid protein L1 could be a system to produce virus-like particles for prophylactic vaccination or could even be used as edible vaccines to induce an L1-specific prophylactic immune response. Here, we describe the generation of transgenic tobacco and potato plants carrying the HPV type 16 major structural gene L1 under the control of the cauliflower mosaic virus 35S promoter. All attempts to express either the original, unmodified L1 gene or an L1 gene with a codon usage optimized for expression in plants failed. Surprisingly, small amounts of the protein were detected using an L1 gene optimized for expression in human cells. However, Northern blot analysis revealed that most of the L1 transcripts were degraded. Introduction of the translational enhancer Omega derived from the tobacco mosaic virus strongly increased transcript stability and resulted in accumulation of L1 protein to approximately 0.5 to 0.2% of total soluble protein in transgenic tobacco and potato plants, respectively. The plant-derived L1 protein displayed conformation-specific epitopes and assembled into virus-like particles. Furthermore, we did not find any indications of protein modification of the L1 protein produced in plants. Plant-derived L1 was as immunogenic as L1 expressed in baculovirus-infected insect cells. Feeding of tubers from transgenic potatoes to mice induced an anti-L1 antibody response in 3 out of 24 mice, although this response was only transient in two of the mice. Our data, however, indicate that an anti-L1 response was primed in about half of the 24 animals.
Assuntos
Proteínas do Capsídeo , Papillomaviridae/genética , Animais , Elementos Facilitadores Genéticos , Feminino , Expressão Gênica , Genes Virais , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Oncogênicas Virais/biossíntese , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Papillomaviridae/imunologia , Papillomaviridae/patogenicidade , Papillomaviridae/fisiologia , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/prevenção & controle , Plantas Geneticamente Modificadas , Solanum tuberosum/genética , Nicotiana/genética , Vírus do Mosaico do Tabaco/genética , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/prevenção & controle , Vacinas de Plantas Comestíveis/genética , Vacinas Sintéticas/genética , Vacinas Sintéticas/isolamento & purificação , Vacinas Virais/genética , Vacinas Virais/isolamento & purificaçãoRESUMO
The need for the development of cheap and effective vaccines against pneumococcal disease has necessitated the evaluation of common virulence-associated proteins of Streptococcus pneumoniae as potential vaccine antigens. In this study, we examined the capacity of active immunization with a genetic toxoid derivative of pneumolysin (PdB) and/or a fragment of choline binding protein A (CbpA; also known as PspC, Hic, and SpsA) to protect mice from intraperitoneal challenge with medium to very high doses of a highly virulent capsular type 2 pneumococcal strain, D39. The median survival times for mice immunized with the individual protein antigens in different adjuvant combinations were significantly longer than those for mice that received the respective adjuvants alone. Mice immunized with CbpA alone were significantly better protected than mice immunized with PdB alone. Correspondingly, the median survival times for mice that were immunized with a combination of PdB and CbpA were significantly longer than those for mice that received PdB alone but not significantly different from those that received CbpA alone. Mice immunized with the protein antigens in a mixture of monophospholipid A (MPL) and aluminium phosphate (AlPO4) adjuvants had higher antibody titers than mice that received the antigens in AlPO4 alone. Mice immunized with PdB in MPL plus AlPO4 were also significantly better protected than mice that received PdB in AlPO4 alone.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Infecções Pneumocócicas/prevenção & controle , Estreptolisinas/imunologia , Vacinas Sintéticas/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Histidina , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Vacinação , Vacinas Sintéticas/genética , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
To investigate the role of tegumental glycoprotein Sm25 in protective immunity against schistosomiasis, codons 43-182 of its gene (GP22) were amplified by PCR and cloned in the pET 15b bacterial expression system. Recombinant protein r140 was inducibly expressed in the presence of rifampicin and purified by Ni-affinity chromatography. In different vaccination trials, Balb/c mice and Fischer rats repeatedly immunized with r140 in combination with one of several adjuvants (alum, cholera toxin or complexed into proteosomes) produced high titre anti-r140 responses. These antibodies detected an N-glycanase sensitive. 25 kDa antigen in a detergent solubilized worm fraction using Western immunoblotting. The choice of adjuvant affected the isotype distribution of the specific anti-r140 antibodies. Despite the presence of high antibody titres and isotypes which have been shown to correlate with protective immunity, protection against subsequent cercarial challenge was not observed. In addition, no appreciable effects on worm sex ratios or liver egg yields were detected in mice. Studies involving biotin labelling of membrane proteins in live worms showed that the majority of anti-r140 reactive molecules present in adult schistosomes are biotinylated after permeabilization of the parasite surface. Several possibilities to account for the lack of protective immunity are analysed.