Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21
Filtrar
1.
J Virol ; 91(15)2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28490598

RESUMO

We have developed pandemic live attenuated influenza vaccines (pLAIVs) against clade 1 H5N1 viruses on an Ann Arbor cold-adapted (ca) backbone that induced long-term immune memory. In 2015, many human infections caused by a new clade (clade 2.2.1.1) of goose/Guangdong (gs/GD) lineage H5N1 viruses were reported in Egypt, which prompted updating of the H5N1 pLAIV. We explored two strategies to generate suitable pLAIVs. The first approach was to modify the hemagglutinin gene of a highly pathogenic wild-type (wt) clade 2.2.1.1 virus, A/Egypt/N03434/2009 (Egy/09) (H5N1), with its unmodified neuraminidase (NA) gene; this virus was designated Egy/09 ca The second approach was to select a low-pathogenicity avian influenza H5 virus that elicited antibodies that cross-reacted with a broad range of H5 viruses, including the Egypt H5N1 viruses, and contained a novel NA subtype for humans. We selected the low-pathogenicity A/duck/Hokkaido/69/2000 (H5N3) (dk/Hok/00) virus for this purpose. Both candidate vaccines were attenuated and immunogenic in ferrets, inducing antibodies that neutralized homologous and heterologous H5 viruses with different degrees of cross-reactivity; Egy/09 ca vaccine antisera were more specific for the gs/GD lineage viruses but did not neutralize recent North American isolates (clade 2.3.4.4), whereas antisera from dk/Hok/69 ca-vaccinated ferrets cross-reacted with clade 2.3.4.4 and 2.2.1 viruses but not clade 1 or 2.1 viruses. When vaccinated ferrets were challenged with homologous and heterologous H5 viruses, challenge virus replication was reduced in the respiratory tract. Thus, the two H5 pLAIV candidates are suitable for clinical development to protect humans from infection with different clades of H5 viruses.IMPORTANCE In response to the continuing evolution of H5N1 avian influenza viruses and human infections, new candidate H5 live attenuated vaccines were developed by using two different approaches: one targeted a specific circulating strain in Egypt, and the other was based on a virus that elicits broadly cross-reactive antibodies against a wide range of H5 viruses. Both candidate vaccines were immunogenic and exhibited protective efficacy in ferrets. Our study permits a comparison of the two approaches, and the data support the further development of both vaccine viruses to optimally prepare for the further spread of clade 2.2.1 or 2.3.4.4 viruses.


Assuntos
Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Reações Cruzadas , Modelos Animais de Doenças , Furões , Genótipo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/genética , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Vacinas contra Influenza/isolamento & purificação , Testes de Neutralização , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Vírus Reordenados/genética , Vírus Reordenados/imunologia , Sistema Respiratório/virologia , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/isolamento & purificação , Carga Viral
2.
Proc Natl Acad Sci U S A ; 112(30): 9430-5, 2015 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-26170284

RESUMO

H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer's formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion.


Assuntos
Anticorpos Monoclonais/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Hemaglutininas/química , Virus da Influenza A Subtipo H5N1/imunologia , Animais , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Sítios de Ligação , Cristalografia por Raios X , Epitopos/química , Humanos , Concentração de Íons de Hidrogênio , Fragmentos de Imunoglobulinas/química , Imunoglobulina G/química , Vacinas contra Influenza/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Ligação Proteica , Conformação Proteica , Solventes/química
3.
Influenza Other Respir Viruses ; 8(2): 169-76, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24734293

RESUMO

OBJECTIVE: The objective was to study passively acquired influenza H1N1 pandemic (H1N1pdm) maternal antibody kinetics and its impact on subsequent influenza infection and vaccination in ferrets during an outbreak of the H1N1pdm. DESIGN AND MAIN OUTCOME MEASURES: Infectivity of the H1N1pdm in the respiratory tract of ferrets was compared with the previous seasonal A/South Dakota/6/2007 (SD07, H1N1). Influenza-specific antibodies were quantitated and antibody-mediated protection against the homologous and heterologous H1N1 virus challenge infection was determined. RESULTS: H1N1pdm virus was approximately 10 times more infectious than SD07 in ferrets, replicated to higher viral titers in the upper respiratory tract and shed for a longer duration. Influenza-specific antibodies after natural infection persisted much longer in the circulation than passively acquired maternal antibodies. The protection conferred by the maternal antibodies was limited to the homologous virus strain and was ineffective against SD07 and H3N2 virus. Serum antibodies from maternal transmission or passive transfer interfered with homologous vaccine strain-mediated antibody responses in the ferret. A booster immunization was required to elicit a high level of antibody. CONCLUSIONS: The findings support the rationale for a prime and boost immunization strategy in young children in whom maternal antibodies are present.


Assuntos
Anticorpos Antivirais/sangue , Imunidade Materno-Adquirida , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/veterinária , Vacinação/métodos , Replicação Viral , Animais , Anticorpos Antivirais/imunologia , Furões , Vacinas contra Influenza/administração & dosagem , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Carga Viral , Eliminação de Partículas Virais
4.
J Infect Dis ; 208(4): 594-602, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23656978

RESUMO

The humoral and cellular immune responses elicited by the trivalent live attenuated influenza vaccine (LAIV) and the trivalent inactivated influenza vaccine (TIV) were evaluated in the ferret model, using newly developed ferret immunological reagents and assays. In contrast to the TIV, which only induced immune responses in primed animals, LAIV induced strong influenza virus-specific serum antibody and T-cell responses in both naive and influenza-seropositive animals. The LAIV offered significant protection against a heterologous H1N1 virus challenge infection in the upper respiratory tract. Influenza virus-specific immunoglobulin A (IgA) and immunoglobulin G (IgG) antibody-secreting cells (ASCs) and influenza virus-specific CD4(+) and CD8(+) T cells were detected in the circulation and local paratracheal draining lymph nodes. The frequency of the influenza-specific ASCs in the local lymph nodes appeared to correlate with the degree of protection in the upper respiratory tract. The protection conferred by the LAIV could be attributed not only to the antibody response but also to the cell-mediated and local mucosal immune responses, particularly in naive ferrets. These findings may explain why the LAIV is immunologically superior and offers immediate protection after a single dose in children.


Assuntos
Anticorpos Antivirais/sangue , Células Produtoras de Anticorpos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Animais , Modelos Animais de Doenças , Furões , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Vírus da Influenza A Subtipo H1N1/imunologia , Linfonodos/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia
5.
Vaccine ; 30(5): 872-8, 2012 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-22172510

RESUMO

The proteolytic enzyme bromelain has been traditionally used to cleave the hemagglutinin (HA) protein at the C-terminus of the HA2 region to release the HA proteins from influenza virions. The bromelain cleaved HA (BHA) has been routinely used as an antigen to generate antiserum that is essential for influenza vaccine product release. The HA of the 2009 pandemic H1N1 influenza A/California/7/2009 (CA09) virus could not be cleaved efficiently by bromelain. To ensure timely delivery of BHA for antiserum production, we generated a chimeric virus that contained the HA1 region from CA09 and the HA2 region from the seasonal H1N1 A/South Dakota/6/2007 (SD07) virus that is cleavable by bromelain. The BHA from this chimeric virus was antigenically identical to CA09 and induced high levels of HA-specific antibodies and protected ferrets from wild-type H1N1 CA09 virus challenge. To determine the molecular basis of inefficient cleavage of CA09 HA by bromelain, the amino acids that differed between the HA2 of CA09 and SD07 were introduced into recombinant CA09 virus to assess their effect on bromelain cleavage. The D373N or E374G substitution in the HA2 stalk region of CA09 HA enabled efficient cleavage of CA09 HA by bromelain. Sequence analysis of the pandemic H1N1-like viruses isolated from 2010 revealed emergence of the E374K change. We found that K374 enabled the HA to be cleaved by bromelain and confirmed that the 374 residue is critical for HA bromelain cleavage.


Assuntos
Bromelaínas/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H1N1/genética , Vacinas contra Influenza/imunologia , Substituição de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Feminino , Furões , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Hidrólise , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Masculino , Mutagênese Sítio-Dirigida , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
6.
J Virol ; 86(5): 2706-14, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22205751

RESUMO

Highly pathogenic avian influenza (HPAI) viruses of the H5 and H7 subtypes typically possess multiple basic amino acids around the cleavage site (MBS) of their hemagglutinin (HA) protein, a recognized virulence motif in poultry. To determine the importance of the H5 HA MBS as a virulence factor in mammals, recombinant wild-type HPAI A/Vietnam/1203/2004 (H5N1) viruses that possessed (H5N1) or lacked (ΔH5N1) the H5 HA MBS were generated and evaluated for their virulence in BALB/c mice, ferrets, and African green monkeys (AGMs) (Chlorocebus aethiops). The presence of the H5 HA MBS was associated with lethality, significantly higher virus titers in the respiratory tract, virus dissemination to extrapulmonary organs, lymphopenia, significantly elevated levels of proinflammatory cytokines and chemokines, and inflammation in the lungs of mice and ferrets. In AGMs, neither H5N1 nor ΔH5N1 virus was lethal and neither caused clinical symptoms. The H5 HA MBS was associated with mild enhancement of replication and delayed virus clearance. Thus, the contribution of H5 HA MBS to the virulence of the HPAI H5N1 virus varies among mammalian hosts and is most significant in mice and ferrets and less remarkable in nonhuman primates.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Especificidade de Hospedeiro , Virus da Influenza A Subtipo H5N1/fisiologia , Mamíferos/virologia , Infecções por Orthomyxoviridae/veterinária , Infecções por Orthomyxoviridae/virologia , Fatores de Virulência/metabolismo , Motivos de Aminoácidos , Animais , Chlorocebus aethiops , Feminino , Furões , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Processamento de Proteína Pós-Traducional , Fatores de Virulência/química , Fatores de Virulência/genética
7.
PLoS One ; 6(7): e21942, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21760928

RESUMO

Priming immunization plays a key role in protecting individuals or populations to influenza viruses that are novel to humans. To identify the most promising vaccine priming strategy, we have evaluated different prime-boost regimens using inactivated, DNA and live attenuated vaccines in ferrets. Live attenuated influenza A/Vietnam/1203/2004 (H5N1) candidate vaccine (LAIV, VN04 ca) primed ferrets efficiently while inactivated H5N1 vaccine could not prime the immune response in seronegative ferrets unless an adjuvant was used. However, the H5 HA DNA vaccine alone was as successful as an adjuvanted inactivated VN04 vaccine in priming the immune response to VN04 ca virus. The serum antibody titers of ferrets primed with H5 HA DNA followed by intranasal vaccination of VN04 ca virus were comparable to that induced by two doses of VN04 ca virus. Both LAIV-LAIV and DNA-LAIV vaccine regimens could induce antibody responses that cross-neutralized antigenically distinct H5N1 virus isolates including A/HongKong/213/2003 (HK03) and prevented nasal infection of HK03 vaccine virus. Thus, H5 HA DNA vaccination may offer an alternative option for pandemic preparedness.


Assuntos
Formação de Anticorpos/imunologia , DNA Viral/imunologia , Furões/imunologia , Furões/virologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Animais , Anticorpos Neutralizantes/sangue , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Virus da Influenza A Subtipo H5N1/imunologia , Cinética , Infecções por Orthomyxoviridae/sangue , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Vacinas de Produtos Inativados/imunologia
8.
PLoS Pathog ; 7(12): e1002443, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22241979

RESUMO

The epidemiological success of pandemic and epidemic influenza A viruses relies on the ability to transmit efficiently from person-to-person via respiratory droplets. Respiratory droplet (RD) transmission of influenza viruses requires efficient replication and release of infectious influenza particles into the air. The 2009 pandemic H1N1 (pH1N1) virus originated by reassortment of a North American triple reassortant swine (TRS) virus with a Eurasian swine virus that contributed the neuraminidase (NA) and M gene segments. Both the TRS and Eurasian swine viruses caused sporadic infections in humans, but failed to spread from person-to-person, unlike the pH1N1 virus. We evaluated the pH1N1 and its precursor viruses in a ferret model to determine the contribution of different viral gene segments on the release of influenza virus particles into the air and on the transmissibility of the pH1N1 virus. We found that the Eurasian-origin gene segments contributed to efficient RD transmission of the pH1N1 virus likely by modulating the release of influenza viral RNA-containing particles into the air. All viruses replicated well in the upper respiratory tract of infected ferrets, suggesting that factors other than viral replication are important for the release of influenza virus particles and transmission. Our studies demonstrate that the release of influenza viral RNA-containing particles into the air correlates with increased NA activity. Additionally, the pleomorphic phenotype of the pH1N1 virus is dependent upon the Eurasian-origin gene segments, suggesting a link between transmission and virus morphology. We have demonstrated that the viruses are released into exhaled air to varying degrees and a constellation of genes influences the transmissibility of the pH1N1 virus.


Assuntos
Vírus da Influenza A Subtipo H1N1 , Influenza Humana/transmissão , Modelos Biológicos , Pandemias , Aerossóis , Animais , Linhagem Celular , Cães , Furões , Genes Virais/genética , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H1N1/ultraestrutura , Influenza Humana/epidemiologia , Influenza Humana/genética , Influenza Humana/metabolismo , Neuraminidase/genética , Neuraminidase/metabolismo , América do Norte , Proteínas Virais/genética , Proteínas Virais/metabolismo
9.
J Clin Invest ; 120(5): 1663-73, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20389023

RESUMO

The target of neutralizing antibodies that protect against influenza virus infection is the viral protein HA. Genetic and antigenic variation in HA has been used to classify influenza viruses into subtypes (H1-H16). The neutralizing antibody response to influenza virus is thought to be specific for a few antigenically related isolates within a given subtype. However, while heterosubtypic antibodies capable of neutralizing multiple influenza virus subtypes have been recently isolated from phage display libraries, it is not known whether such antibodies are produced in the course of an immune response to influenza virus infection or vaccine. Here we report that, following vaccination with seasonal influenza vaccine containing H1 and H3 influenza virus subtypes, some individuals produce antibodies that cross-react with H5 HA. By immortalizing IgG-expressing B cells from 4 individuals, we isolated 20 heterosubtypic mAbs that bound and neutralized viruses belonging to several HA subtypes (H1, H2, H5, H6, and H9), including the pandemic A/California/07/09 H1N1 isolate. The mAbs used different VH genes and carried a high frequency of somatic mutations. With the exception of a mAb that bound to the HA globular head, all heterosubtypic mAbs bound to acid-sensitive epitopes in the HA stem region. Four mAbs were evaluated in vivo and protected mice from challenge with influenza viruses representative of different subtypes. These findings reveal that seasonal influenza vaccination can induce polyclonal heterosubtypic neutralizing antibodies that cross-react with the swine-origin pandemic H1N1 influenza virus and with the highly pathogenic H5N1 virus.


Assuntos
Anticorpos Neutralizantes/química , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Animais , Anticorpos Monoclonais/química , Sítios de Ligação , Cães , Mapeamento de Epitopos/métodos , Feminino , Humanos , Imunoglobulina G/metabolismo , Leucócitos Mononucleares/citologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Orthomyxoviridae/metabolismo , Ligação Proteica
10.
J Virol ; 84(13): 6570-7, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20427525

RESUMO

A live attenuated influenza A/Vietnam/1203/2004 (H5N1) vaccine virus (VN04 ca) has receptor binding specificity to alpha2,3-linked sialosides (alpha2,3SAL), and a single dose induces a minimal serum antibody response in mice and ferrets. In contrast, A/Hong Kong/213/2003 (H5N1) vaccine virus (HK03 ca) binds to both alpha2,6SAL and alpha2,3SAL and generates a stronger serum antibody response in animals. Among the 9 amino acids that differed between the two H5 HA1 proteins, several HK03-specific residues enabled the VN04 ca virus to bind to both alpha2,3SAL and alpha2,6SAL receptors, but only the removal of the 158N glycosylation, together with an S227N change, resulted in more-efficient viral replication in the upper respiratory tract of ferrets and an increased serum antibody response. However, the antibody response was HK03 strain specific and did not significantly cross-neutralize VN04 virus. A second approach was taken to adapt the H5N1 VN04 ca virus in MDCK cells to select HA variants with larger plaque morphology. Although a number of large-plaque-size HA variants with amino acid changes in the HA receptor binding region were identified, none of these mutations affected virus receptor binding preference and immunogenicity. In addition, the known receptor binding site changes, Q226L and G228S, were introduced into the HA protein of the VN04 ca virus. Only in conjunction with the removal of the 158N glycosylation did the virus replicate efficiently in the upper respiratory tract of ferrets and became more immunogenic, yet the response was also HK03 specific. Thus, the mask of the antigenic epitopes by 158N glycosylation at the HA globular head and its alpha2,3SAL binding preference of VN04 ca virus affect virus antigenicity and replication in the host, resulting in a lower antibody response.


Assuntos
Hemaglutininas Virais/imunologia , Hemaglutininas Virais/metabolismo , Virus da Influenza A Subtipo H5N1/imunologia , Virus da Influenza A Subtipo H5N1/fisiologia , Vacinas contra Influenza/imunologia , Receptores Virais/metabolismo , Ligação Viral , Adaptação Biológica , Substituição de Aminoácidos , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linhagem Celular , Cães , Epitopos/genética , Epitopos/imunologia , Furões , Glicosilação , Humanos , Camundongos , Mutação de Sentido Incorreto , Processamento de Proteína Pós-Traducional , Inoculações Seriadas , Vacinas Atenuadas/imunologia , Ensaio de Placa Viral , Tropismo Viral
11.
J Virol ; 84(9): 4611-8, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20181720

RESUMO

The emergence in 1997 and continuance today of a highly lethal H5N1 avian influenza virus (AIV) causing human disease has raised concern about an impending pandemic and the need for a vaccine to prepare for such an occurrence. We previously generated an efficacious vesicular stomatitis virus (VSV)-based AIV vaccine expressing H5 hemagglutinin (HA) from the fifth genomic position of VSV (J. A. Schwartz et al., Virology 366:166-173, 2007). Here we have generated and characterized VSV-based vaccines that express the A/Hong Kong/156/1997 (clade 0) H5 HA from the first position of the VSV genome. These vectors induce broadly cross-neutralizing antibodies against homologous and heterologous H5N1 viruses of different clades in mice. The vaccines provide complete protection against morbidity and mortality after heterologous challenge with clade 0 and clade 1 strains in animals even 1 year after vaccination. Postchallenge pulmonary virus loads show that these vectors provide sterilizing immunity. Therefore, VSV-based AIV vaccines are potent, broadly cross-protective pandemic vaccine candidates.


Assuntos
Proteção Cruzada , Vetores Genéticos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Vesiculovirus/genética , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Peso Corporal , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/genética , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/virologia , Análise de Sobrevida , Fatores de Tempo , Ensaio de Placa Viral
12.
J Virol ; 84(1): 44-51, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19864389

RESUMO

Several live attenuated influenza virus A/California/7/09 (H1N1) (CA09) candidate vaccine variants that possess the hemagglutinin (HA) and neuraminidase (NA) gene segments from the CA09 virus and six internal protein gene segments from the cold-adapted influenza virus A/Ann Arbor/6/60 (H2N2) virus were generated by reverse genetics. The reassortant viruses replicated relatively poorly in embryonated chicken eggs. To improve virus growth in eggs, reassortants expressing the HA and NA of CA09 were passaged in MDCK cells and variants exhibiting large-plaque morphology were isolated. These variants replicated at levels approximately 10-fold higher than the rate of replication of the parental strains in embryonated chicken eggs. Sequence analysis indicated that single amino acid changes at positions 119, 153, 154, and 186 were responsible for the improved growth properties in MDCK cells and eggs. In addition, the introduction of a mutation at residue 155 that was previously shown to enhance the replication of a 1976 swine influenza virus also significantly improved the replication of the CA09 virus in eggs. Each variant was further evaluated for receptor binding preference, antigenicity, attenuation phenotype, and immunogenicity. Mutations at residues 153, 154, and 155 drastically reduced viral antigenicity, which made these mutants unsuitable as vaccine candidates. However, changes at residues 119 and 186 did not affect virus antigenicity or immunogenicity, justifying their inclusion in live attenuated vaccine candidates to protect against the currently circulating 2009 swine origin H1N1 viruses.


Assuntos
Embrião de Galinha/virologia , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vacinas Atenuadas , Substituição de Aminoácidos , Animais , Linhagem Celular , Cães , Hemaglutininas/genética , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Mutagênese Sítio-Dirigida , Neuraminidase/genética
13.
Virology ; 395(2): 280-8, 2009 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-19833372

RESUMO

A recombinant live attenuated influenza virus DeltaH5N1 vaccine with a modified hemagglutinin (HA) and intact neuraminidase genes from A/Vietnam/1203/04 (H5N1) and six remaining genome segments from A/Ann Arbor/6/60 (H2N2) cold-adapted (AA ca) virus was previously shown to be attenuated in chickens, mice and ferrets. Evaluation of the recombinant H5N1 viruses in mice indicated that three independent factors contributed to the attenuation of the DeltaH5N1 vaccine: the attenuating mutations specified by the AA ca loci had the greatest influence, followed by the deletion of the H5 HA multi-basic cleavage site (MBS), and the constellation effects of the AA genes acting in concert with the H5N1 glycoproteins. Restoring the MBS in the H5 HA of the vaccine virus improved its immunogenicity and efficacy, likely as a consequence of increased virus replication, indicating that removal of the MBS had a deleterious effect on the immunogenicity and efficacy of the DeltaH5N1 vaccine in mice.


Assuntos
Adaptação Fisiológica , Temperatura Baixa , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Virus da Influenza A Subtipo H5N1/fisiologia , Vacinas contra Influenza/imunologia , Vacinas Atenuadas/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Galinhas , DNA Viral , Esquema de Medicação , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas Recombinantes , Proteínas Virais
14.
PLoS Med ; 6(4): e1000049, 2009 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-19381279

RESUMO

BACKGROUND: Transmission of highly pathogenic avian H5N1 viruses from poultry to humans have raised fears of an impending influenza pandemic. Concerted efforts are underway to prepare effective vaccines and therapies including polyclonal or monoclonal antibodies against H5N1. Current efforts are hampered by the paucity of information on protective immune responses against avian influenza. Characterizing the B cell responses in convalescent individuals could help in the design of future vaccines and therapeutics. METHODS AND FINDINGS: To address this need, we generated whole-genome-fragment phage display libraries (GFPDL) expressing fragments of 15-350 amino acids covering all the proteins of A/Vietnam/1203/2004 (H5N1). These GFPDL were used to analyze neutralizing human monoclonal antibodies and sera of five individuals who had recovered from H5N1 infection. This approach led to the mapping of two broadly neutralizing human monoclonal antibodies with conformation-dependent epitopes. In H5N1 convalescent sera, we have identified several potentially protective H5N1-specific human antibody epitopes in H5 HA[(-10)-223], neuraminidase catalytic site, and M2 ectodomain. In addition, for the first time to our knowledge in humans, we identified strong reactivity against PB1-F2, a putative virulence factor, following H5N1 infection. Importantly, novel epitopes were identified, which were recognized by H5N1-convalescent sera but did not react with sera from control individuals (H5N1 naïve, H1N1 or H3N2 seropositive). CONCLUSION: This is the first study, to our knowledge, describing the complete antibody repertoire following H5N1 infection. Collectively, these data will contribute to rational vaccine design and new H5N1-specific serodiagnostic surveillance tools.


Assuntos
Antígenos Virais/imunologia , Epitopos , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Aviária/imunologia , Proteínas Virais/imunologia , Animais , Anticorpos Monoclonais , Aves , Convalescença , Mapeamento de Epitopos , Epitopos/sangue , Biblioteca Genômica , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/virologia , Neuraminidase/imunologia , Vietnã , Proteínas da Matriz Viral/imunologia , Proteínas Virais/antagonistas & inibidores , Fatores de Virulência
15.
Infect Immun ; 77(4): 1483-91, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19139195

RESUMO

During pregnancy, Plasmodium falciparum-infected erythrocytes (IE) sequester in the placenta where they induce pathology and increase the risk of low-birth-weight (LBW) babies. The innate immune mediator, mannose-binding lectin (MBL), enhances phagocytosis of pathogens. Since MBL is reported to bind to IE, we hypothesized that it might aid in clearance of IE from the placenta, thereby reducing the risk of LBW babies. To test this hypothesis, molecular genotyping was used to detect polymorphisms at codon 57 (A/C) in exon 1 of MBL2 in 401 pregnant Cameroonian women, with or without placental malaria, who had LBW and normal-weight babies. Polymorphisms in the promoter region at positions -550 (H/L), -221 (X/Y), and +4 (P/Q) were also determined, and plasma MBL levels were measured during pregnancy and at delivery. The expected correlation between genotype and plasma MBL levels was confirmed. However, asymptomatic infections were not associated with an increase in MBL levels in the peripheral blood, and MBL levels were similar in the placental and cord blood of women with or without placental malaria at delivery. There was no evidence that MBL levels at delivery were associated with malaria-related poor pregnancy outcomes. Women with the LXPA haplotype, however, were more likely to have LBW babies, but the risk was not related to malaria. These results do not support the hypothesis that MBL aids in the clearance of parasites from the placenta but suggest that Cameroonian women with LXPA are at risk of having LBW babies due to other causes.


Assuntos
Recém-Nascido de Baixo Peso , Recém-Nascido Prematuro , Malária Falciparum/imunologia , Lectina de Ligação a Manose/genética , Polimorfismo de Nucleotídeo Único , Complicações Parasitárias na Gravidez/imunologia , Animais , Camarões , Eritrócitos/parasitologia , Feminino , Humanos , Recém-Nascido , Malária Falciparum/genética , Malária Falciparum/parasitologia , Lectina de Ligação a Manose/sangue , Parasitemia/genética , Parasitemia/parasitologia , Placenta/imunologia , Placenta/parasitologia , Placenta/patologia , Doenças Placentárias/genética , Doenças Placentárias/imunologia , Doenças Placentárias/parasitologia , Plasmodium falciparum , Gravidez , Complicações Parasitárias na Gravidez/genética , Complicações Parasitárias na Gravidez/parasitologia
16.
PLoS Med ; 4(5): e178, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17535101

RESUMO

BACKGROUND: New prophylactic and therapeutic strategies to combat human infections with highly pathogenic avian influenza (HPAI) H5N1 viruses are needed. We generated neutralizing anti-H5N1 human monoclonal antibodies (mAbs) and tested their efficacy for prophylaxis and therapy in a murine model of infection. METHODS AND FINDINGS: Using Epstein-Barr virus we immortalized memory B cells from Vietnamese adults who had recovered from infections with HPAI H5N1 viruses. Supernatants from B cell lines were screened in a virus neutralization assay. B cell lines secreting neutralizing antibodies were cloned and the mAbs purified. The cross-reactivity of these antibodies for different strains of H5N1 was tested in vitro by neutralization assays, and their prophylactic and therapeutic efficacy in vivo was tested in mice. In vitro, mAbs FLA3.14 and FLD20.19 neutralized both Clade I and Clade II H5N1 viruses, whilst FLA5.10 and FLD21.140 neutralized Clade I viruses only. In vivo, FLA3.14 and FLA5.10 conferred protection from lethality in mice challenged with A/Vietnam/1203/04 (H5N1) in a dose-dependent manner. mAb prophylaxis provided a statistically significant reduction in pulmonary virus titer, reduced associated inflammation in the lungs, and restricted extrapulmonary dissemination of the virus. Therapeutic doses of FLA3.14, FLA5.10, FLD20.19, and FLD21.140 provided robust protection from lethality at least up to 72 h postinfection with A/Vietnam/1203/04 (H5N1). mAbs FLA3.14, FLD21.140 and FLD20.19, but not FLA5.10, were also therapeutically active in vivo against the Clade II virus A/Indonesia/5/2005 (H5N1). CONCLUSIONS: These studies provide proof of concept that fully human mAbs with neutralizing activity can be rapidly generated from the peripheral blood of convalescent patients and that these mAbs are effective for the prevention and treatment of H5N1 infection in a mouse model. A panel of neutralizing, cross-reactive mAbs might be useful for prophylaxis or adjunctive treatment of human cases of H5N1 influenza.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Virus da Influenza A Subtipo H5N1/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/terapia , Adulto , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/virologia , Linhagem Celular Transformada , Modelos Animais de Doenças , Feminino , Genoma Viral , Humanos , Imunização Passiva , Memória Imunológica/imunologia , Virus da Influenza A Subtipo H5N1/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Testes de Neutralização , Infecções por Orthomyxoviridae/imunologia , Vietnã
17.
J Immunol ; 178(5): 2770-7, 2007 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-17312120

RESUMO

Plasmodium falciparum infection during pregnancy can lead to the transplacental passage of malarial Ags that are capable of inducing acquired immune responses in the fetus. Studies have identified cytokines produced by malaria-specific cord blood (CB) T cells, but information on fetal B cells is limited. Thus, CB mononuclear cells from 120 Cameroonian newborns were cultured for 7 days in vitro and supernatants were assessed by ELISA for Abs to an extract of malarial schizonts (MA), recombinant apical merozoite Ag 1 (AMA-1), the 42-kDa C-terminal region of merozoite surface protein 1 (MSP-1(42)), a B epitope of ring-infected erythrocyte surface Ag (RESA), and the dominant B epitope of the circumsporozoite protein (CSP). Only 12% of supernatants contained IgM to MA but 78% had IgG to one or more malarial Ags, with 53% having IgG to AMA-1, 38% to MSP-1(42), 3% to RESA, and 0% to CSP. The Abs to AMA-1 and MSP-1(42) were predominantly IgG1 and IgG3. CB mononuclear cells were also tested for the ability to secrete cytokines in response to MA and a pool of conserved MSP-1 T cell epitopes. Among the Ag-reactive samples, 39.3% produced only Th2-type cytokines, whereas 60.6% produced a combination of Th1- and Th2-type cytokines. Although a Th2 bias was observed, the in utero cytokine environment was adequate to support isotype switching to cytophilic IgGs, the isotypes that are protective in adults. Because many infants living in a low transmission area are born with malaria-specific B and T cells, the influence of in utero priming on neonatal immunity merits further investigation.


Assuntos
Antígenos de Protozoários/imunologia , Feto/imunologia , Malária Falciparum/imunologia , Troca Materno-Fetal/imunologia , Plasmodium falciparum/imunologia , Complicações Parasitárias na Gravidez/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Camarões , Citocinas/imunologia , Doenças Endêmicas , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Sangue Fetal/imunologia , Humanos , Switching de Imunoglobulina/imunologia , Imunoglobulina G/imunologia , Masculino , Gravidez , Células Th1/imunologia , Células Th2/imunologia
18.
PLoS Med ; 3(9): e360, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16968127

RESUMO

BACKGROUND: Recent outbreaks of highly pathogenic influenza A H5N1 viruses in humans and avian species that began in Asia and have spread to other continents underscore an urgent need to develop vaccines that would protect the human population in the event of a pandemic. METHODS AND FINDINGS: Live, attenuated candidate vaccines possessing genes encoding a modified H5 hemagglutinin (HA) and a wild-type (wt) N1 neuraminidase from influenza A H5N1 viruses isolated in Hong Kong and Vietnam in 1997, 2003, and 2004, and remaining gene segments derived from the cold-adapted (ca) influenza A vaccine donor strain, influenza A/Ann Arbor/6/60 ca (H2N2), were generated by reverse genetics. The H5N1 ca vaccine viruses required trypsin for efficient growth in vitro, as predicted by the modification engineered in the gene encoding the HA, and possessed the temperature-sensitive and attenuation phenotypes specified by the internal protein genes of the ca vaccine donor strain. More importantly, the candidate vaccines were immunogenic in mice. Four weeks after receiving a single dose of 10(6) 50% tissue culture infectious doses of intranasally administered vaccines, mice were fully protected from lethality following challenge with homologous and antigenically distinct heterologous wt H5N1 viruses from different genetic sublineages (clades 1, 2, and 3) that were isolated in Asia between 1997 and 2005. Four weeks after receiving two doses of the vaccines, mice and ferrets were fully protected against pulmonary replication of homologous and heterologous wt H5N1 viruses. CONCLUSIONS: The promising findings in these preclinical studies of safety, immunogenicity, and efficacy of the H5N1 ca vaccines against antigenically diverse H5N1 vaccines provide support for their careful evaluation in Phase 1 clinical trials in humans.


Assuntos
Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/uso terapêutico , Influenza Aviária/prevenção & controle , Animais , Galinhas , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Furões , Esquemas de Imunização , Virus da Influenza A Subtipo H5N1/genética , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Influenza Aviária/imunologia , Influenza Aviária/virologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/uso terapêutico
19.
Infect Immun ; 72(9): 5267-73, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15322022

RESUMO

Sequestration of Plasmodium falciparum parasites within the placenta often leads to an accumulation of macrophages within the intervillous space and increased production of tumor necrosis factor alpha (TNF-alpha), a cytokine associated with placental pathology and poor pregnancy outcomes. P. falciparum glycosylphosphatidylinositol (GPI) anchors have been shown to be the major parasite component that induces TNF-alpha production by monocytes and macrophages. Antibodies against P. falciparum GPI (anti-PfGPI), however, can inhibit the induction of TNF-alpha and inflammation. Thus, the study was undertaken to determine whether anti-PfGPI antibodies down-regulate inflammatory-type changes in the placentas of women with malaria. Anti-PfGPI immunoglobulin M (IgM) and IgG levels were measured in 380 pregnant women with or without placental malaria, including those who delivered prematurely and at term. Results showed that anti-PfGPI antibody levels increased with gravidity and age and that malaria infection boosted anti-PfGPI antibodies in pregnant women. However, no association was found between anti-PfGPI antibodies and placental TNF-alpha levels or the presence of acute or chronic placental malaria. Furthermore, anti-PfGPI antibody levels were similar in women with preterm and full-term deliveries and were not associated with an increase in infant birth weight. Thus, these results fail to support a strong role for anti-PfGPI antibodies in the prevention of chronic placental malaria infections and malaria-associated poor birth outcomes.


Assuntos
Anticorpos Antiprotozoários/sangue , Glicosilfosfatidilinositóis/imunologia , Placenta/parasitologia , Plasmodium falciparum/imunologia , Complicações Parasitárias na Gravidez/imunologia , Resultado da Gravidez , Adulto , Fatores Etários , Animais , Camarões , Parto Obstétrico , Feminino , Número de Gestações , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Recém-Nascido , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Placenta/imunologia , Doenças Placentárias/parasitologia , Plasmodium falciparum/química , Gravidez , Fator de Necrose Tumoral alfa/metabolismo
20.
J Infect Dis ; 188(7): 1074-82, 2003 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-14513430

RESUMO

Plasmodium falciparum-infected erythrocytes often are sequestered in the placenta and stimulate the accumulation of maternal mononuclear cells. In this study, the role that chemokines and cytokines play in mediating the inflammatory response was investigated. Placental parasites elicited a statistically significant increase in the levels of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-10, in plasma collected from the intervillous space. Explants of fetal tissue from malaria-positive placentas also secreted significantly enhanced amounts of IFN-gamma. Culture supernatant of maternal intervillous leukocytes obtained from infected placentas contained significantly higher levels of TNF-alpha, IL-10, monocyte chemotactic protein-1, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and IFN-gamma inducible protein-10 than did cultures of white blood cells obtained from uninfected placentas. Taken together, these results show that both fetal and maternal cells secrete inflammatory and immunoregulatory cytokines in response to P. falciparum and suggest that beta-chemokines produced by maternal cells contribute to the accumulation of macrophages in the intervillous space.


Assuntos
Quimiocinas/metabolismo , Citocinas/metabolismo , Malária Falciparum/imunologia , Placenta/imunologia , Plasmodium falciparum/imunologia , Complicações Parasitárias na Gravidez/imunologia , Adulto , Animais , Camarões , Ensaio de Imunoadsorção Enzimática , Feminino , Feto/parasitologia , Hematócrito , Humanos , Imuno-Histoquímica , Contagem de Leucócitos , Malária Falciparum/sangue , Malária Falciparum/parasitologia , Parasitemia/sangue , Parasitemia/imunologia , Parasitemia/parasitologia , Placenta/parasitologia , Plasmodium falciparum/metabolismo , Gravidez , Complicações Parasitárias na Gravidez/sangue , Complicações Parasitárias na Gravidez/parasitologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...