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1.
J Virol ; 90(24): 11122-11131, 2016 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-27707930

RESUMEN

Half of the world's population is exposed to the risk of dengue virus infection. Although a vaccine for dengue virus is now available in a few countries, its reported overall efficacy of about 60% is not ideal. Protective immune correlates following natural dengue virus infection remain undefined, which makes it difficult to predict the efficacy of new vaccines. In this study, we address the protective capacity of dengue virus-specific antibodies that are produced by plasmablasts a few days after natural secondary infection. Among a panel of 18 dengue virus-reactive human monoclonal antibodies, four groups of antibodies were identified based on their binding properties. While antibodies targeting the fusion loop of the glycoprotein of dengue virus dominated the antibody response, two smaller groups of antibodies bound to previously undescribed epitopes in domain II of the E protein. The latter, largely serotype-cross-reactive antibodies, demonstrated increased stability of binding at pH 5. These antibodies possessed weak to moderate neutralization capacity in vitro but were the most efficacious in promoting the survival of infected mice. Our data suggest that the cross-reactive anamnestic antibody response has a protective capacity despite moderate neutralization in vitro and a moderate decrease of viremia in vivo IMPORTANCE: Antibodies can protect from symptomatic dengue virus infection. However, it is not easy to assess which classes of antibodies provide protection because in vitro assays are not always predictive of in vivo protection. During a repeat infection, dengue virus-specific immune memory cells are reactivated and large amounts of antibodies are produced. By studying antibodies cloned from patients with heterologous secondary infection, we tested the protective value of the serotype-cross-reactive "recall" or "anamnestic" response. We found that results from in vitro neutralization assays did not always correlate with the ability of the antibodies to reduce viremia in a mouse model. In addition, a decrease of viremia in mice did not necessarily improve survival. The most protective antibodies were stable at pH 5, suggesting that antibody binding in the endosomes, after the antibody-virus complex is internalized, might be important to block virus spread in the organism.


Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Virus del Dengue/inmunología , Dengue/prevención & control , Proteínas del Envoltorio Viral/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/química , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/administración & dosificación , Anticuerpos Antivirales/química , Reacciones Cruzadas , Dengue/inmunología , Dengue/virología , Virus del Dengue/clasificación , Virus del Dengue/genética , Modelos Animales de Enfermedad , Mapeo Epitopo , Epítopos/química , Epítopos/inmunología , Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Inmunidad Humoral/efectos de los fármacos , Memoria Inmunológica , Ratones , Pruebas de Neutralización , Unión Proteica , Estabilidad Proteica , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología
3.
J Virol ; 83(6): 2553-62, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19109379

RESUMEN

The HA2 glycopolypeptide (gp) is highly conserved in all influenza A virus strains, and it is known to play a major role in the fusion of the virus with the endosomal membrane in host cells during the course of viral infection. Vaccines and therapeutics targeting this HA2 gp could induce efficient broad-spectrum immunity against influenza A virus infections. So far, there have been no studies on the possible therapeutic effects of monoclonal antibodies (MAbs), specifically against the fusion peptide of hemagglutinin (HA), upon lethal infections with highly pathogenic avian influenza (HPAI) H5N1 virus. We have identified MAb 1C9, which binds to GLFGAIAGF, a part of the fusion peptide of the HA2 gp. We evaluated the efficacy of MAb 1C9 as a therapy for influenza A virus infections. This MAb, which inhibited cell fusion in vitro when administered passively, protected 100% of mice from challenge with five 50% mouse lethal doses of HPAI H5N1 influenza A viruses from two different clades. Furthermore, it caused earlier clearance of the virus from the lung. The influenza virus load was assessed in lung samples from mice challenged after pretreatment with MAb 1C9 (24 h prior to challenge) and from mice receiving early treatment (24 h after challenge). The study shows that MAb 1C9, which is specific to the antigenically conserved fusion peptide of HA2, can contribute to the cross-clade protection of mice infected with H5N1 virus and mediate more effective recovery from infection.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Antivirales/uso terapéutico , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antivirales/aislamiento & purificación , Células CHO , Fusión Celular , Cricetinae , Cricetulus , Mapeo Epitopo , Pulmón/patología , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/prevención & control , Unión Proteica , Análisis de Supervivencia
4.
J Virol Methods ; 147(2): 219-25, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17950911

RESUMEN

Emergence of highly pathogenic avian influenza H7N1 was due to mutation of low pathogenic avian influenza H7N1 strain, which caused outbreaks in Italy between 1999 and 2000, and resulted in complete mortality of infected poultry. This outbreak places increased importance on the early detection of H7N1 AIV. Here we describe the development of a detection method for H7N1 virus from infected chickens using a specific antigen-capture-ELISA (AC-ELISA). A panel of mAbs was developed against the surface antigen HA of H7N1 AIV strain A/chicken/Singapore/94. The mAbs were screened by immunofluorescence assays, ELISA and immunoblotting. Selected mAbs 5E5 and 8F10 were of isotypes IgM and IgG and were conformation- or linear epitope-specific, respectively. These mAbs were used as capture antibodies for AC-ELISA development. The detection limit was as little as 10(2)-10(3) TCID(50) units of virus derived from tissue culture supernatants. Virus from the tracheal swab samples of experimentally infected chickens was detected from days 3 to 7 post-infection using the AC-ELISA, with results being confirmed by RT-PCR. AIV subtypes H4N1, H5N3 H9N2 and H10N5 did not react in the AC-ELISA but were RT-PCR positive, indicating that this AC-ELISA is specific for H7N1 strains.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/virología , Animales , Anticuerpos Antivirales/inmunología , Pollos , Virus de la Influenza A/inmunología , Gripe Aviar/inmunología , Sensibilidad y Especificidad
6.
PLoS One ; 13(1): e0189262, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29298302

RESUMEN

Dengue virus (DENV) is one of the most widespread arboviruses. The four DENV serotypes infect about 400 million people every year, causing 96 million clinical dengue cases, of which approximately 500'000 are severe and potentially life-threatening. The only licensed vaccine has a limited efficacy and is only recommended in regions with high endemicity. We previously reported that 2'-O-methyltransferase mutations in DENV-1 and DENV-2 block their capacity to inhibit type I IFNs and render the viruses attenuated in vivo, making them amenable as vaccine strains; here we apply this strategy to all four DENV serotypes to generate a tetravalent, non-chimeric live-attenuated dengue vaccine. 2'-O-methyltransferase mutants of all four serotypes are highly sensitive to type I IFN inhibition in human cells. The tetravalent formulation is attenuated and immunogenic in mice and cynomolgus macaques and elicits a response that protects from virus challenge. These results show the potential of 2'-O-methyltransferase mutant viruses as a safe, tetravalent, non-chimeric dengue vaccine.


Asunto(s)
Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Metiltransferasas/genética , Mutación , Animales , Anticuerpos Neutralizantes/análisis , Anticuerpos Neutralizantes/inmunología , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Macaca fascicularis , Masculino , Ratones
7.
NPJ Vaccines ; 2: 2, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29263863

RESUMEN

A therapy for dengue is still elusive. We describe the neutralizing and protective capacity of a dengue serotype-cross-reactive antibody isolated from the plasmablasts of a patient. Antibody SIgN-3C neutralized all four dengue virus serotypes at nano to picomolar concentrations and significantly decreased viremia of all serotypes in adult mice when given 2 days after infection. Moreover, mice were protected from pathology and death from a lethal dengue virus-2 infection. To avoid potential Fc-mediated uptake of immune complexes and ensuing enhanced infection, we introduced a LALA mutation in the Fc part. SIgN-3C-LALA was as efficient as the non-modified antibody in neutralizing dengue virus and in protecting mice while antibody-dependent enhancement was completely abrogated. The epitope of the antibody includes conserved amino acids in all three domains of the glycoprotein, which can explain its cross-reactivity. SIgN-3C-LALA neutralizes dengue virus both pre and post-attachment to host cells. These attributes likely contribute to the remarkable protective capacity of SIgN-3C.

8.
Sci Rep ; 7(1): 14224, 2017 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-29079750

RESUMEN

The pathogenesis of severe dengue remains unclear, particularly the mechanisms underlying the plasma leakage that results in hypovolaemic shock in a small proportion of individuals. Maximal leakage occurs several days after peak viraemia implicating immunological pathways. Skin is a highly vascular organ and also an important site of immune reactions with a high density of dendritic cells (DCs), macrophages and T cells. We obtained skin biopsies and contemporaneous blood samples from patients within 24 hours of onset of dengue shock syndrome (DSS), and from healthy controls. We analyzed cell subsets by flow cytometry, and soluble mediators and antibodies by ELISA; the percentage of migratory CD1a+ dermal DCs was significantly decreased in the DSS patients, and skin CD8+ T cells were activated, but there was no accumulation of dengue-specific antibodies. Inflammatory monocytic cells were not observed infiltrating the skin of DSS cases on whole-mount histology, although CD14dim cells disappeared from blood.


Asunto(s)
Células Dendríticas/inmunología , Activación de Linfocitos , Dengue Grave/inmunología , Piel/inmunología , Linfocitos T/inmunología , Adolescente , Adulto , Animales , Anticuerpos Antivirales/inmunología , Antígenos CD1/metabolismo , Estudios de Casos y Controles , Células Dendríticas/metabolismo , Femenino , Humanos , Macaca fascicularis , Masculino , Monocitos/inmunología , Piel/virología , Adulto Joven
9.
Hum Vaccin Immunother ; 12(5): 1265-73, 2016 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-26889737

RESUMEN

Globally, dengue virus (DENV) is one of the most widespread vector-borne viruses. Dengue disease affects populations in endemic areas and, increasingly, tourists who travel to these countries, but there is currently no approved vaccine for dengue. A phase 3 efficacy trial with Sanofi-Pasteur's recombinant, live-attenuated, tetravalent dengue vaccine (CYD-TDV) conducted in South East Asia showed an overall efficacy of 56% against virologically confirmed dengue infections of any severity and any of the 4 serotypes, but the long-term protection of the vaccine has yet to be demonstrated. To address longevity of antibody titers and B cell memory, we recalled study participants from an earlier CYD immunogenicity study (Phase 2) conducted in Singapore that enrolled healthy volunteers in the year 2009. Depending on the age group, 57-84% of the participants initially generated a neutralizing antibody titer ≥ 10 to all 4 DENV serotypes 28 d after the third and final dose. We observed very low antibody titers in blood samples collected from 23 vaccinees 5 y after the first dose, particularly titers of antibodies binding to virus particles compared with those binding to recombinant E protein. The in vivo efficacy of plasma antibodies against DENV-2 challenge was also tested in a mouse model, which found that only 2 out of 23 samples were able to reduce viremia. Although the sample size is too small for general conclusions, dengue immune memory after vaccination with CYD-TDV appears relatively low.


Asunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Memoria Inmunológica , Adulto , Animales , Anticuerpos Neutralizantes/inmunología , Afinidad de Anticuerpos , Asia , Linfocitos B/inmunología , Ensayos Clínicos Fase II como Asunto , Ensayos Clínicos Fase III como Asunto , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Singapur , Factores de Tiempo , Vacunación
10.
EBioMedicine ; 12: 178-188, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-27628668

RESUMEN

Dengue is endemic in tropical countries worldwide and the four dengue virus serotypes often co-circulate. Infection with one serotype results in high titers of cross-reactive antibodies produced by plasmablasts, protecting temporarily against all serotypes, but impairing protective immunity in subsequent infections. To understand the development of these plasmablasts, we analyzed virus-specific B cell properties in patients during acute disease and at convalescence. Plasmablasts were unrelated to classical memory cells expanding in the blood during early recovery. We propose that only a small subset of memory B cells is activated as plasmablasts during repeat infection and that plasmablast responses are not representative of the memory B cell repertoire after dengue infection.


Asunto(s)
Subgrupos de Linfocitos B/inmunología , Virus del Dengue/inmunología , Dengue/inmunología , Dengue/virología , Memoria Inmunológica , Células Plasmáticas/inmunología , Enfermedad Aguda , Animales , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos/inmunología , Antígenos Virales/inmunología , Subgrupos de Linfocitos B/metabolismo , Línea Celular , Evolución Clonal , Reacciones Cruzadas/inmunología , Dengue/genética , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/genética , Inmunofenotipificación , Pruebas de Neutralización , Fenotipo , Células Plasmáticas/metabolismo
11.
Antiviral Res ; 110: 20-30, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25046486

RESUMEN

Dengue is a global emerging infectious disease, with no specific treatment available. To identify novel human host cell targets important for dengue virus infection and replication, an image-based high-throughput siRNA assay screening of a human kinome siRNA library was conducted using human hepatocyte cell line Huh7 infected with a recent dengue serotype 2 virus isolate BR DEN2 01-01. In the primary siRNA screening of 779 kinase-related genes, knockdown of 22 genes showed a reduction in DENV-2 infection. Conversely, knockdown of 8 genes enhanced viral infection. To assess host cell specificity, the confirmed hits were tested in the DENV-infected monocytic cell line U937. While the expression of EIF2AK3, ETNK2 and SMAD7 was regulated in both cell lines after infection, most kinases were hepatocyte-specific. Monocytic cells represent initial targets of infection and an antiviral treatment targeting these cells is probably most effective to reduce initial viral load. In turn, infection of the liver could contribute to pathogenesis, and the novel hepatocyte-specific human targets identified here could be important for dengue infection and pathogenesis.


Asunto(s)
Antivirales/farmacología , Virus del Dengue/crecimiento & desarrollo , Proteínas Quinasas/genética , ARN Interferente Pequeño/farmacología , Replicación Viral/genética , Línea Celular , Dengue/terapia , Hepatocitos/virología , Ensayos Analíticos de Alto Rendimiento , Interacciones Huésped-Patógeno , Humanos , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Interferencia de ARN , Proteína smad7/genética , eIF-2 Quinasa/genética
12.
Immunol Res ; 53(1-3): 229-34, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22407576

RESUMEN

The immune system has multiple, complex, and sometimes opposing roles during cancer progression. While immune-compromised individuals have a higher incidence of cancers, inflammation is also associated with increased risk of disease progression. It is becoming apparent that simple measures of immune responses in the blood are of limited use in cancer. Instead, the importance of the exact identity and functional characteristics of tumor-infiltrating immune cells is increasingly recognized. This realization has led to recent studies that have revealed a critical role for chemokine expression in the tumor microenvironment and suggested a therapeutic potential of manipulating intratumoral expression of chemokines to alter the local immune milieu.


Asunto(s)
Biomarcadores de Tumor/inmunología , Quimiocinas/inmunología , Neoplasias/diagnóstico , Neoplasias/inmunología , Animales , Movimiento Celular/inmunología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunidad , Vigilancia Inmunológica , Ratones , Ratones Transgénicos , Neoplasias/genética , Pronóstico , Microambiente Tumoral
13.
PLoS One ; 6(6): e20737, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21695200

RESUMEN

BACKGROUND: Active serologic surveillance of H5N1 highly pathogenic avian influenza (HPAI) virus in humans and poultry is critical to control this disease. However, the need for a robust, sensitive and specific serologic test for the rapid detection of antibodies to H5N1 viruses has not been met. METHODOLOGY/PRINCIPAL FINDINGS: Previously, we reported a universal epitope (CNTKCQTP) in H5 hemagglutinin (HA) that is 100% conserved in H5N1 human isolates and 96.9% in avian isolates. Here, we describe a peptide ELISA to detect antibodies to H5N1 virus by using synthetic peptide that comprises the amino acid sequence of this highly conserved and antigenic epitope as the capture antigen. The sensitivity and specificity of the peptide ELISA were evaluated using experimental chicken antisera to H5N1 viruses from divergent clades and other subtype influenza viruses, as well as human serum samples from patients infected with H5N1 or seasonal influenza viruses. The peptide ELISA results were compared with hemagglutinin inhibition (HI), and immunofluorescence assay and immunodot blot that utilize recombinant HA1 as the capture antigen. The peptide ELISA detected antibodies to H5N1 in immunized animals or convalescent human sera whereas some degree of cross-reactivity was observed in HI, immunofluorescence assay and immunodot blot. Antibodies to other influenza subtypes tested negative in the peptide-ELISA. CONCLUSION/SIGNIFICANCE: The peptide-ELISA based on the highly conserved and antigenic H5 epitope (CNTKCQTP) provides sensitive and highly specific detection of antibodies to H5N1 influenza viruses. This study highlighted the use of synthetic peptide as a capture antigen in rapid detection of antibodies to H5N1 in human and animal sera that is robust, simple and cost effective and is particularly beneficial for developing countries and rural areas.


Asunto(s)
Anticuerpos Antivirales/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Subtipo H5N1 del Virus de la Influenza A/inmunología , Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Pollos/inmunología , Reacciones Cruzadas/inmunología , Epítopos/química , Epítopos/inmunología , Técnica del Anticuerpo Fluorescente , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Sueros Inmunes/inmunología , Inmunización , Immunoblotting , Subtipo H5N1 del Virus de la Influenza A/clasificación , Datos de Secuencia Molecular , Péptidos/química , Proteínas Recombinantes/aislamiento & purificación , Sensibilidad y Especificidad
14.
PLoS One ; 4(2): e4566, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19238211

RESUMEN

BACKGROUND: Human infections with highly pathogenic H5N1 avian influenza viruses have generally been confirmed by molecular amplification or culture-based methods. Serologic surveillance has potential advantages which have not been realized because rapid and specific serologic tests to detect H5N1 infection are not widely available. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe an epitope-blocking ELISA to detect specific antibodies to H5N1 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (5F8) that binds to an epitope comprising amino acid residues 274-281 (CNTKCQTP) in the HA1 region of H5 hemagglutinin. Database search analysis of publicly available sequences revealed that this epitope is conserved in 100% of the 163 H5N1 viruses isolated from humans. The sensitivity and specificity of the epitope-blocking ELISA for H5N1 were evaluated using chicken antisera to multiple virus clades and other influenza subtypes as well as serum samples from individuals naturally infected with H5N1 or seasonal influenza viruses. The epitope-blocking ELISA results were compared to those of hemagglutinin inhibition (HI) and microneutralization assays. Antibodies to H5N1 were readily detected in immunized animals or convalescent human sera by the epitope-blocking ELISA whereas specimens with antibodies to other influenza subtypes yielded negative results. The assay showed higher sensitivity and specificity as compared to HI and microneutralization. CONCLUSIONS/SIGNIFICANCE: The epitope-blocking ELISA based on a unique 5F8 mAb provided highly sensitive and 100% specific detection of antibodies to H5N1 influenza viruses in human sera.


Asunto(s)
Anticuerpos Antivirales/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Subtipo H5N1 del Virus de la Influenza A/inmunología , Animales , Anticuerpos Monoclonales , Aves , Secuencia Conservada , Epítopos , Hemaglutininas/inmunología , Humanos , Gripe Aviar/diagnóstico , Gripe Humana/diagnóstico , Infecciones por Orthomyxoviridae/diagnóstico , Sensibilidad y Especificidad , Pruebas Serológicas/métodos
15.
Virology ; 380(2): 412-20, 2008 Oct 25.
Artículo en Inglés | MEDLINE | ID: mdl-18786689

RESUMEN

The increasing number of recent outbreaks of HPAI H5N1 in birds and humans brings out an urgent need to develop potent H5N1 vaccine regimens. Here we present a study on the intranasal vaccination of recombinant baculovirus surface-displayed hemagglutinin (BacHA) or inactivated whole H5N1 viral (IWV) vaccine with a recombinant cholera toxin B subunit (rCTB) as a mucosal adjuvant in a BALB/c mouse model. Two groups of mice were vaccinated with different doses (HA titer of log 2(4) or log 2(8)) of either HA surface-displayed baculovirus or inactivated whole viral vaccine virus adjuvanted with different doses (2 mug or 10 mug) of rCTB. The vaccinations were repeated after 28 days. HA specific serum IgG and mucosal IgA antibodies were quantified by indirect ELISA, and serum neutralizing antibody titer were estimated by hemagglutination inhibition (HI) assay and virus neutralization titer assay. Functional protective efficacy of the vaccine was assessed by host challenge against HPAI H5N1 strains. The results revealed that mice co-administered with log 2(8) HA titer of BacHA vaccine and adjuvanted with 10 mug of rCTB had a significantly enhanced serum IgG and mucosal IgA immune response and serum microneutralization titer compared with mice administered with unadjuvanted log 2(4) or log 2(8) HA titer of BacHA alone. Also vaccination with 10 mug of rCTB and log 2(8) HA titer of BacHA elicited higher HA specific serum and mucosal antibody levels and serum HI titer than vaccination with log 2(8) HA titer of inactivated H5N1 virus adjuvanted with the same dose of rCTB. The host challenge study also showed that 10 mug rCTB combined with log 2(8) HA titer of BacHA provided 100% protection against 10MLD(50) of homologous and heterologous H5N1 strains. The study shows that the combination of rH5 HA expressed on baculovirus surface and rCTB mucosal adjuvant form an effective mucosal vaccine against H5N1 infection. This baculovirus surface-displayed vaccine is more efficacious than inactivated H5N1 influenza vaccine when administered by intranasal route and has no biosafety concerns associated with isolation, purification and production of the latter vaccine.


Asunto(s)
Adyuvantes Inmunológicos/farmacología , Baculoviridae/genética , Toxina del Cólera/farmacología , Hemaglutininas Virales/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Adyuvantes Inmunológicos/genética , Administración Intranasal , Animales , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/sangre , Peso Corporal , Toxina del Cólera/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Pruebas de Inhibición de Hemaglutinación , Hemaglutininas Virales/genética , Inmunización Secundaria , Inmunoglobulina A/análisis , Inmunoglobulina G/sangre , Vacunas contra la Influenza/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Membrana Mucosa/inmunología , Pruebas de Neutralización , Análisis de Supervivencia , Vacunas de Productos Inactivados/inmunología , Vacunas Sintéticas/inmunología
16.
Clin Vaccine Immunol ; 14(5): 617-23, 2007 May.
Artículo en Inglés | MEDLINE | ID: mdl-17344345

RESUMEN

The unprecedented spread of highly pathogenic avian influenza virus subtype H5N1 in Asia and Europe is threatening animals and public health systems. Effective diagnosis and control management are needed to control the disease. To this end, we developed a panel of monoclonal antibodies (MAbs) against the H5N1 avian influenza virus (AIV) and implemented an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) to detect the H5 viral antigen. Mice immunized with denatured hemagglutinin (HA) from A/goose/Guangdong/97 (H5N1) expressed in bacteria or immunized with concentrated H5N2 virus yielded a panel of hybridomas secreting MAbs specific for influenza virus HA. The reactivity of each MAb with several subtypes of influenza virus revealed that hybridomas 3D4 and 8B6 specifically recognized H5 HA. Therefore, purified antibodies from hybridomas 3D4 and 8B6, which secrete immunoglobulin G (IgG) and IgM, respectively, were used as the capture antibodies and pooled hyperimmune guinea pig serum IgG served as the detector antibody. The specificity of the optimized AC-ELISA was evaluated by using AIV subtypes H5 H3, H4, H7, H9, and H10. Specimens containing AIV subtype H5 subtype yielded a specific and strong signal above the background, whereas specimens containing all other subtypes yielded background signals. The detection limits of the AC-ELISA were 62.5 ng of bacterium-expressed H5N1 HA1 protein and 124, 62, and 31 50% tissue culture infective doses of influenza virus subtypes H5N1/PR8, H5N2, and H5N3, respectively. Reconstituted clinical samples consisting of H5 AIVs mixed with pharyngeal-tracheal mucus from healthy chickens also yielded positive signals in the AC-ELISA, and the results were confirmed by reverse transcription-PCR. The tracheal swab samples from H9N2-infected chickens did not give positive signals. Taken together, the newly developed MAb-based AC-ELISA offers an attractive alternative to other diagnostic approaches for the specific detection of H5 AIV.


Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Antivirales , Subtipo H5N1 del Virus de la Influenza A/inmunología , Gripe Aviar/virología , Animales , Antígenos Virales/inmunología , Pollos , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina G , Inmunoglobulina M , Gripe Aviar/inmunología , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Proteínas Virales/biosíntesis
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