RESUMO
DNA methylation is an important epigenetic modification in eukaryotes, which plays a significant role in regulating gene expression. When the host is invaded by the influenza virus, gene expression is regulated via changes in DNA methylation levels or patterns, leading to the activation or suppression of relevant signaling pathways or networks, triggering a series of immune responses against viral invasion. Here, we investigated the changes in genomic DNA methylation in the immune organs of chicken infected with H5N1 influenza virus. Genome-wide DNA methylation levels in the spleen, thymus, and bursa of Fabricius of specific pathogen-free (SPF) chicken infected with the Guangdong (G-H5N1) and Anhui (A-H5N1) H5N1 strains, and water (control) were analyzed by fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP). The results indicated that total DNA methylation levels did not differ between spleen genomic DNA in chicken treated with different viral strains and the control (P > 0.05). However, the total DNA methylation levels were significantly upregulated in the thymus (P < 0.01) and bursa (P < 0.05) of chicken in the A-H5N1 group compared to those in the G-H5N1 and control groups. These results provide a basis for the screening of avian influenza-resistance genes or methylation markers, analyzing the epigenetic regulation mechanisms of avian influenza, and performing selective breeding for disease resistance.
Assuntos
Metilação de DNA/genética , Resistência à Doença/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/genética , Animais , Bolsa de Fabricius/imunologia , Bolsa de Fabricius/virologia , Galinhas , Metilação de DNA/imunologia , Resistência à Doença/imunologia , Epigênese Genética , Genoma/genética , Virus da Influenza A Subtipo H5N1/imunologia , Influenza Aviária/imunologia , Influenza Aviária/virologia , Transdução de Sinais , Baço/imunologia , Baço/virologia , Timo/imunologia , Timo/virologiaRESUMO
The unpredictable, evolutionary nature of the influenza A virus (IAV) is the primary problem when generating a vaccine and when designing diagnostic strategies; thus, it is necessary to determine the constant regions in viral proteins. In this study, we completed an in silico analysis of the reported epitopes of the 4 IAV proteins that are antigenically most significant (HA, NA, NP, and M2) in the 3 strains with the greatest world circulation in the last century (H1N1, H2N2, and H3N2) and in one of the main aviary subtypes responsible for zoonosis (H5N1). For this purpose, the HMMER program was used to align 3,016 epitopes reported in the Immune Epitope Database and Analysis Resource (IEDB) and distributed in 34,294 stored sequences in the Pfam database. Eighteen epitopes were identified: 8 in HA, 5 in NA, 3 in NP, and 2 in M2. These epitopes have remained constant since they were first identified (~91 years) and are present in strains that have circulated on 5 continents. These sites could be targets for vaccination design strategies based on epitopes and/or as markers in the implementation of diagnostic techniques.
Assuntos
Epitopos , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A Subtipo H2N2 , Vírus da Influenza A Subtipo H3N2 , Virus da Influenza A Subtipo H5N1 , Vacinas contra Influenza , Simulação por Computador , Epitopos/genética , Epitopos/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H2N2/genética , Vírus da Influenza A Subtipo H2N2/imunologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologiaRESUMO
Avian influenza (AI) vaccines should be used as part of a whole comprehensive AI control programme. Vectored vaccines based on Newcastle disease virus (NDV) are very promising, but are so far licensed in only a few countries. In the present study, the immunogenicity and protection against a highly pathogenic H5N1 influenza challenge were evaluated after vaccination with an enterotropic NDV vector expressing an H5 haemagglutinin (rNDV-H5) in 1-day-old specific pathogen free chickens inoculated once, twice or once followed by a heterologous boost with an inactivated H5N9 vaccine (iH5N9). The heterologous prime/boost rNDV-H5/iH5N9 combination afforded the best level of protection against the H5N1 challenge performed at 6 weeks of age. Two rNDV-H5 administrations conferred a good level of protection after challenge, although only a cellular H5-specific response could be detected. Interestingly, a single administration of rNDV-H5 gave the same level of protection as the double administration but without any detectable H5-specific immune response. In contrast to AI immunity, a high humoral, mucosal and cellular NDV-specific immunity could be detected up to 6 weeks post vaccination after using the three different vaccination schedules. NDV-specific mucosal and cellular immune responses were slightly higher after double rNDV-H5 vaccination when compared with single inoculation. Finally, the heterologous prime/boost rNDV-H5/iH5N9 combination induced a broader detectable immunity including systemic, mucosal and cellular AI and NDV-specific responses.
Assuntos
Animais Recém-Nascidos , Galinhas , Imunidade Humoral/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Influenza Aviária/prevenção & controle , Doenças das Aves Domésticas/prevenção & controle , Vacinas Virais/administração & dosagem , Animais , Vetores Genéticos/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Vírus da Doença de Newcastle/genética , Vacinas Virais/uso terapêutico , Eliminação de Partículas ViraisRESUMO
Having previously demonstrated the feasibility of administering A/H5N1 and seasonal influenza vaccine antigens in an MF59-adjuvanted tetravalent formulation, we now report on long-term antibody persistence and responses to a booster dose of a combined seasonal-pandemic, tetravalent influenza vaccine in adults. The primary objective was the evaluation of responses to a booster dose of tetravalent influenza vaccine containing seasonal (A/H1N1, A/H3N2, and B) and avian (A/H5N1, clade 2) influenza virus strains administered to 265 healthy 18- to 40-year-old volunteers 1 year after priming with one or two clade 1 A/H5N1 doses. Secondary objectives were assessment of reactogenicity, safety, and antibody persistence 1 year after priming with a combined seasonal-pandemic, tetravalent vaccine. Responses to seasonal strains met all European licensure criteria; seroprotection rates were 94 to 100%, 100%, and 61 to 90% for A/H1N1, A/H3N2, and B strains, respectively. Anamnestic responses were observed against homologous and heterologous A/H5N1 strains whether priming with one or two A/H5N1 doses, with a monovalent A/H5N1 vaccine, or with a tetravalent vaccine. A single dose of MF59-adjuvanted A/H5N1 vaccine given alone or as part of a fixed combination with a seasonal influenza vaccine was sufficient to prime adult subjects, resulting in robust antigen-specific and cross-reactive antibody responses to heterologous booster immunization 1 year later. These data support the feasibility of incorporating prepandemic priming into seasonal influenza vaccination programs. (This study has been registered at clinicaltrials.gov under registration no. NCT00481065.).
Assuntos
Anticorpos Antivirais/sangue , Memória Imunológica , Vírus da Influenza A Subtipo H1N1/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/administração & dosagem , Influenza Humana/prevenção & controle , Adjuvantes Imunológicos , Adolescente , Adulto , Anticorpos Antivirais/imunologia , Feminino , Humanos , Imunização Secundária , Vacinas contra Influenza/efeitos adversos , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/virologia , Masculino , Polissorbatos/administração & dosagem , Esqualeno/administração & dosagem , Esqualeno/imunologia , Vacinação , Vacinas Combinadas/administração & dosagem , Vacinas Combinadas/efeitos adversos , Vacinas Combinadas/imunologia , Adulto JovemRESUMO
To determine whether guinea pigs are infected with influenza virus in nature, we conducted a serologic study in domestic guinea pigs in Ecuador. Detection of antibodies against influenza A and B raises the question about the role of guinea pigs in the ecology and epidemiology of influenza virus in the region.
Assuntos
Anticorpos Antivirais/sangue , Cobaias , Gado , Infecções por Orthomyxoviridae/veterinária , Orthomyxoviridae/imunologia , Animais , Equador/epidemiologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Influenza B/imunologia , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologiaRESUMO
H5N1 influenza candidate vaccine viruses were developed using the "6+2" approach. The hemagglutinin (HA) and neuraminidase (NA) genes were derived from the popular H5N1 virus and the remaining six internal segments were derived from the A/Puerto Rico/8/34 strain (H1N1, PR8). However, some of these candidate strains have been reported to produce relatively low yields in vaccine manufacture. In this study, we found that the NA vRNA of the A/Vietnam/1194/2004 strain (H5N1, VN1194) was poorly packaged into recombinant viruses with a backbone of PR8 genes, which resulted in the formation of defective virions that did not include the NA vRNA in the genome. Using recombinant DNA techniques, we constructed a chimeric NA gene with the coding region of VN1194 NA flanked by the packaging signal sequence of the PR8 NA gene (41 bp form the 3' end of the vRNA and 67 bp from the 5' end). The packaging of the NA vRNA was restored to normal levels in the recombinant viruses containing the chimeric NA gene. Recombinant viruses containing the chimeric NA replicated much better in chicken embryonated eggs than viruses with the wild-type NA from VN1194. These findings suggest a novel strategy to improve in ovo growth of vaccine strains and to increase the number of vaccine doses available to save people if a pandemic were to occur.
Assuntos
Virus da Influenza A Subtipo H5N1/crescimento & desenvolvimento , Virus da Influenza A Subtipo H5N1/genética , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Neuraminidase/genética , Proteínas Virais/genética , Animais , Linhagem Celular , Embrião de Galinha , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Virus da Influenza A Subtipo H5N1/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Neuraminidase/imunologia , RNA Viral/genética , Vírus Reordenados/genética , Vírus Reordenados/imunologia , Proteínas Recombinantes de Fusão , Vacinas Sintéticas/imunologia , Proteínas Virais/imunologia , Montagem de Vírus , Cultura de Vírus , Replicação ViralRESUMO
World Health Organization has a great concern about the spreading of avian influenza virus H5N1. To counteract its massive spread, poultry vaccination is highly recommended together with biosecurity measures. In our study, a recombinant vaccine candidate based on the fusion of extracellular segments of hemagglutinin (HA) H5 of avian influenza virus and chicken CD154 (HACD) is tested with the aim of enhancing humoral and cellular immune responses in chickens. Protein expression was carried out by transducing several mammalian cell lines with recombinant adenoviral vectors. HACD purification was assessed by three distinct purification protocols: immunoaffinity chromatography by elution at acidic pH or with a chaotropic agent and size exclusion chromatography. Humoral and cellular immune responses were measured using the hemagglutination inhibition assay and the semiquantitative real time PCR, respectively. The results showed that humoral response against HACD was significantly higher than the obtained with HA alone after booster (P<0.01, P<0.05). From HACD molecules purified by distinct protocols, only the obtained by size exclusion chromatography generated hemagglutinationin-inhibition activity. IFN-γ levels indicated that cellular immune response was significantly higher with HACD, in its pure or impure form, compared to its counterpart HA (P<0.01). These data demonstrate that HACD is able to significantly enhance humoral and cellular immune responses against HA antigen, which make this fusion protein a promising subunit vaccine candidate against H5N1 virus outbreaks.
Assuntos
Ligante de CD40/metabolismo , Galinhas , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Aviária/imunologia , Influenza Aviária/prevenção & controle , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Testes de Inibição da Hemaglutinação , Hemaglutininas/genética , Hemaglutininas/imunologia , Humanos , Imunidade Celular , Vacinas contra Influenza/genética , Influenza Aviária/virologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/imunologiaRESUMO
Since 2006 Egypt has been facing an extensive epidemic of H5N1 highly pathogenic avian influenza (HPAI) with a huge number of outbreaks both in rural and intensively reared poultry areas. The use of efficacious vaccines in this country has been, and still remains, essential for the control and possible eradication of HPAI. The present study was performed to establish whether the administration of inactivated vaccines containing an H5 virus belonging to a different lineage to the Eurasian H5N1 HPAI viruses guarantees protection from clinical signs, provides significant immune response and is able to achieve a reduction of viral shedding in the face of a challenge with a contemporary H5N1 virus isolated in Egypt. Despite the genetic and antigenic differences between the vaccine strain (H5N2/Mexico) and the challenge strain (H5N1/Egypt), confirmed by molecular and serological (haemagglutination inhibition) tests, it was established that the immune response induced by these conventional vaccines is sufficient to prevent infection in the majority of birds challenged with a contemporary H5N1 Egyptian strain. The data reported in this study also indicate that there may be a low degree of correlation between haemagglutination inhibition titres, clinical protection and reduction of shedding.
Assuntos
Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Influenza A Subtipo H5N2/imunologia , Influenza Aviária/epidemiologia , Influenza Aviária/imunologia , Animais , Formação de Anticorpos , Galinhas/imunologia , Galinhas/virologia , Egito/epidemiologia , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Vírus da Influenza A Subtipo H5N2/genética , Vírus da Influenza A Subtipo H5N2/isolamento & purificação , Aves Domésticas/imunologia , Aves Domésticas/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Organismos Livres de Patógenos Específicos , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/uso terapêutico , Vacinas Virais/administração & dosagem , Vacinas Virais/uso terapêutico , Eliminação de Partículas Virais/imunologiaRESUMO
Consecutive lots of H5N1 (A/Vietnam/1194/2004 - NIBRG-14) split virion and whole virus vaccines were produced in a pilot-scale laboratory. The average yields of vaccine doses (15 microg HA) per egg were 0.57 doses for H5N1 split virion vaccine and 1.12 for H5N1 whole virus vaccine, compared to 2.09 doses for the seasonal H3N2 split virion vaccine. H5N1 split virion vaccine lots complied with WHO protein content criteria, while some lots of the H5N1 whole virus vaccine showed protein content per dose higher than the limit established. All lots of both vaccines showed ovalbumin (OVA) concentration below the recommended limit. Dose sparing strategies using adjuvant formulations using aluminum hydroxide (Al(OH)(3)) and monophosphoryl lipid A (MPLA) from Bordetella pertussis were tested in mice. Both 3.75 microg HA and 7.5 microg HA of H5N1 split virion vaccine with Al(OH)(3) or Al(OH)(3) plus MPLA in aqueous suspension showed higher hemagglutination-inhibition (HAI) titers when compared to the same vaccine dose without any adjuvant. Immunization with the H5N1 inactivated whole virus vaccine was also performed using 3.75 microg HA and HAI titers were higher than those induced by the split virion vaccine. Moreover, the use of Al(OH)(3) with MPLA as an emulsion induced a further increase in HAI titers.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Hidróxido de Alumínio/administração & dosagem , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/análise , Bordetella pertussis/química , Feminino , Testes de Inibição da Hemaglutinação , Vacinas contra Influenza/química , Lipídeo A/administração & dosagem , Lipídeo A/análogos & derivados , Lipídeo A/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/análise , Vacinas de Produtos Inativados/química , Vacinas de Produtos Inativados/imunologia , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
In the last years our country has been affected by several outbreaks of infectious diseases such as Cholera and Hanta virus and recently, by pathogens associated to red tide. Chile was able to manage those emergencies using the local health system. The new threat that may emerge and could eventually overcome that capacity, is the possible H5N1 influenza virus outbreak. Influenza is responsible for the most destructive pandemic, the Spanish influenza, that killed over 40 million individuals in 1918. The new influenza strain (H5N1) is at present endemic in poultry in Asia and has been associated to human fatal cases in Hong Kong and Vietnam. Even though this strain is not able yet to be transmitted among humans, evidence has accumulated that such ability could be reached by the new strain, since it was already detected in pigs. That particular evidence may indicate that the virus could adapt to infect humans, since a similar situation was observed in several of the influenza pandemics. The World Health Organization set a "task force" to develop a strategy that may help to control the virus spread. Several countries are already stocking anti-flu drugs and others are developing new vaccine that are currently been assayed in human volunteers. It is possible that we may have a vaccine before the outbreak; this development is even faster than for SARS. The mayor question to be addressed for developing countries is: what will be done if we do not have the vaccine on time?