RESUMO
We describe an adaptation of conventional ELISA methods to an ELISA-Array format using non-contact Piezo printing of up to 30 spots of purified recombinant viral fusion proteins and vaccine on 96 well high-protein binding plates. Antigens were printed in 1 nanoliter volumes of protein stabilizing buffer using as little as 0.25 nanograms of protein, 2000-fold less than conventional ELISA. The performance of the ELISA-Array was demonstrated by serially diluting n = 9 human post-flu vaccination plasma samples starting at a 1/1000 dilution and measuring binding to the array of Influenza antigens. Plasma polyclonal antibody levels were detected using a cocktail of biotinylated anti-human kappa and lambda light chain antibodies, followed by a Streptavidin-horseradish peroxidase conjugate and the dose-dependent signal was developed with a precipitable TMB substrate. Intra- and inter-assay precision of absorbance units among the eight donor samples showed mean CVs of 4.8% and 10.8%, respectively. The plasma could be differentiated by donor and antigen with titer sensitivities ranging from 1 × 103 to 4 × 106, IC50 values from 1 × 104 to 9 × 106, and monoclonal antibody sensitivities in the ng/mL range. Equivalent sensitivities of ELISA versus ELISA-Array, compared using plasma and an H1N1 HA trimer, were achieved on the ELISA-Array printed at 0.25 ng per 200um spot and 1000 ng per ELISA 96-well. Vacuum-sealed array plates were shown to be stable when stored for at least 2 days at ambient temperature and up to 1 month at 4-8 °C. By the use of any set of printed antigens and analyte matrices the methods of this multiplexed ELISA-Array format can be broadly applied in translational research.
Assuntos
Anticorpos Antivirais/imunologia , Ensaio de Imunoadsorção Enzimática , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Anticorpos Antivirais/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/sangue , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/sangueRESUMO
Early diagnosis of the highly pathogenic H5N1 avian influenza virus (AIV) is significant for preventing and controlling a global pandemic. However, there is no existing electrical biosensor for detecting biomarkers for AIV in clinically relevant samples such as chicken serum. Herein, we report the first use of an aptamer-functionalized field-effect transistor (FET) as a label-free sensor for AIV detection in chicken serum. A DNA aptamer is employed as a sensitive and selective receptor for hemagglutinin (HA) protein, which is a biomarker for AIVs. This aptamer is immobilized on a gold microelectrode that is connected to the gate of a reusable FET transducer. The specific binding of the target protein results in a change in the surface potential, which generates a signal response of the FET transducer. We hypothesize that a conformational change in the DNA aptamer upon specific binding of HA protein may alter the surface potential. The signal of the aptamer-based FET biosensor increased linearly with the increase in the logarithm of HA protein concentration in a dynamic range of 10 pM to 10 nM with a detection limit of 5.9 pM. The selectivity of the biosensor for HA protein was confirmed by employing relevant interfering proteins. The proposed biosensor was successfully applied to the selective detection of HA protein in a chicken serum sample. Owing to its simple and low-cost architecture, portability, and sensitivity, the aptamer-based FET biosensor has potential as a point-of-care diagnosis of H5N1 AIVs in clinical samples.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/instrumentação , Orthomyxoviridae/isolamento & purificação , Soro/virologia , Transistores Eletrônicos , Animais , Galinhas , Glicoproteínas de Hemaglutininação de Vírus da Influenza/sangue , Orthomyxoviridae/metabolismoRESUMO
BACKGROUND: The development of serologic assays that can rapidly assess human exposure to novel influenza viruses remains a public health need. Previously, we developed an 11-plex magnetic fluorescence microsphere immunoassay (MAGPIX) by using globular head domain recombinant hemagglutinins (rHAs) with serum adsorption using two ectodomain rHAs. METHODS: We compared sera collected from two cohorts with novel influenza exposures: animal shelter staff during an A(H7N2) outbreak in New York City in 2016-2017 (n = 119 single sera) and poultry workers from a live bird market in Bangladesh in 2012-2014 (n = 29 pairs). Sera were analyzed by microneutralization (MN) assay and a 20-plex MAGPIX assay with rHAs from 19 influenza strains (11 subtypes) combined with serum adsorption using 8 rHAs from A(H1N1) and A(H3N2) viruses. Antibody responses were analyzed to determine the novel influenza virus exposure. RESULTS: Among persons with novel influenza virus exposures, the median fluorescence intensity (MFI) against the novel rHA from exposed influenza virus had the highest correlation with MN titers to the same viruses and could be confirmed by removal of cross-reactivity from seasonal H1/H3 rHAs following serum adsorption. Interestingly, in persons with exposures to novel influenza viruses, age and MFIs against exposed novel HA were negatively correlated, whereas in persons without exposure to novel influenza viruses, age and MFI against novel HAs were positively correlated. CONCLUSIONS: This 20-plex high-throughput assay with serum adsorption will be a useful tool to detect novel influenza virus infections during influenza outbreak investigations and surveillance, especially when well-paired serum samples are not available.
Assuntos
Anticorpos Antivirais/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A/imunologia , Testes Sorológicos/métodos , Adsorção , Animais , Bangladesh , Estudos de Coortes , Glicoproteínas de Hemaglutininação de Vírus da Influenza/sangue , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Vírus da Influenza A Subtipo H7N2/imunologia , Vírus da Influenza A Subtipo H7N2/isolamento & purificação , Vírus da Influenza A/isolamento & purificação , Influenza Humana/virologia , Cidade de Nova Iorque , Soro/virologiaRESUMO
The H7 subtype avian influenza threatens public health with respect to poultry and humans. Thus, a specific and sensitive diagnostic test is essential for the management of H7 subtype influenza infections. In this study, five mouse monoclonal antibodies (mAbs) against hemagglutinin (HA) of influenza A/Anhui/1/2013 (H7N9) were produced and characterized by the Western blot, immunofluorescence, and hemagglutination inhibition assays. All five specific mAbs reacted with the HA protein of H7N9 but not with that of H1N1, H3N2, or H5N1. With the combination arrays of capture and detection antibodies, the matched pair mAbs (1C4-coated and 2D7-labeled) were selected and employed in a double-antibody sandwich ELISA (DAS-ELISA). Detection limits of the sandwich ELISA were 0.45 ng mL-1 for the HA protein derived from A/Anhui/1/2013 (H7N9); or 1 and 2 HA units/50 µL for A/Anhui/1/2013 (H7N9) and A/GD/17SF003/2016 (H7N9), respectively. These anti-HA mAbs against subtype H7 and the novel DAS-ELISA provide a valuable approach for specific detection of the H7 subtype influenza virus and quantification of its HA protein, especially for the novel epidemic H7N9.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/sangue , Subtipo H7N9 do Vírus da Influenza A/imunologia , Influenza Humana/diagnóstico , Animais , Técnicas Biossensoriais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A Subtipo H3N2 , Virus da Influenza A Subtipo H5N1 , Limite de Detecção , Camundongos , Camundongos Endogâmicos BALB C , Sensibilidade e EspecificidadeRESUMO
In the present study, we fabricated a label-free avian influenza (AIV H5N1) detection biosensor composed of a multi-functional DNA 3 way-Junction (3 W J) on a hollow Au spike-like nanoparticle (hAuSN) using a localized surface plasmon resonance (LSPR) method. To construct the multi-functional DNA (MF-DNA) as a bioprobe, the 3 W J was introduced. The proposed AIV detection bioprobe should contain three functionalities: target recognition, signal amplification, and connection to substrate. To achieve this goal, each piece of the DNA 3 W J was tailored to a hemagglutinin (HA) binding aptamer, FAM dye and thiol group, respectively. The assembly of each DNA 3 W J functional fragment was then confirmed by TBM-Native PAGE. Moreover, the hAuSN was immobilized on the indium-tin-oxide (ITO) substrate for LSPR measurement. The DNA 3 W J was immobilized onto the hAuSN electrode through the thiol-group of DNA 3 W J. The fabricated DNA 3 W J/hAuSN heterolayer on the ITO substrate was investigated by field emission scanning electron microscopy (FE-SEM) and atomic force microscopy (AFM). LSPR experiments were conducted to confirm HA protein binding to the DNA 3 W J/ hAuSN -modified electrode. The proposed biosensor can detected the HA protein in PBS buffer (LOD: 1 pM) as well as in the diluted chicken serum (LOD: 1 pM). The present study details a label-free, simple fabrication method consisted of DNA 3 W J/ hAuSN heterolayer that uses easy-to-tailor elements to detect not only AIV but also various viruses detection platform easily.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , DNA/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/sangue , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/diagnóstico , Nanopartículas Metálicas/química , Animais , Galinhas , Eletrodos , Ouro/química , Influenza Aviária/sangue , Limite de Detecção , Nanopartículas Metálicas/ultraestrutura , Conformação de Ácido Nucleico , Ressonância de Plasmônio de Superfície , Compostos de Estanho/químicaRESUMO
H9N2 avian influenza virus is threatening animals and public health systems. Effective diagnosis is imperative to control the disease. Thus, we developed a panel of monoclonal antibodies (Mabs) against the H9N2 avian inï¬uenza virus (AIV) and implemented a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) to detect the H9 viral antigen. Hybridomas 4D10 and 5G2 were screened to secrete immunoglobulin G (IgG) and IgA, respectively. Antibody 4D10 was used as the capture antibodies and HRP labeled 5G2 as the detector antibody. The speciï¬city of the optimized DAS-ELISA was evaluated by using AIV subtypes H1, H3, H5, H9 and H10. Specimens containing AIV H9 subtype yielded a speciï¬c and strong signal above the background, whereas specimens containing all other subtypes yielded background signals. The detection limit of the DAS-ELISA is 10-2.3 TCID50 (50% Tissue culture infective doses). Negative-positive threshold was 0.211 (OD630). In comparison with virus isolation the sensitivity and speciï¬city of DAS-ELISA were found to be 98.9% and 98.1% respectively. Taken together, the newly developed Mab-based DAS-ELISA offers an attractive alternative to other diagnostic approaches for the speciï¬c detection of H9 subtype AIV.
Assuntos
Ensaio de Imunoadsorção Enzimática , Vírus da Influenza A Subtipo H9N2/imunologia , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Influenza Aviária/diagnóstico , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Embrião de Galinha , Galinhas , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Influenza Aviária/virologia , Células Madin Darby de Rim Canino , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
Many broadly neutralizing antibodies (bnAbs) bind to conserved areas of the hemagglutinin (HA) stalk region and can inhibit the low pH induced HA conformational changes necessary for viral membrane fusion activity. We developed and evaluated a high-throughput virus-free and cell-free ELISA based low pH induced HA Conformational Change Inhibition Antibody Detection Assay (HCCIA) and a complementary proteinase susceptibility assay. Human serum samples (n = 150) were tested by HCCIA using H3 recombinant HA. Optical density (OD) ratios of mAb HC31 at pH 4.8 to pH 7.0 ranged from 0.87 to 0.09. Our results demonstrated that low pH induced HA conformational change inhibition antibodies (CCI) neutralized multiple H3 strains after removal of head-binding antibodies. The results suggest that HCCIA can be utilized to detect and characterize CCI in sera, that are potentially broadly neutralizing, and serves as a useful tool for evaluating universal vaccine candidates targeting the HA stalk.
Assuntos
Anticorpos Neutralizantes/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/química , Ensaio de Imunoadsorção Enzimática/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Ensaios de Triagem em Larga Escala/métodos , Humanos , Concentração de Íons de Hidrogênio , Vírus da Influenza A Subtipo H3N2/química , Influenza Humana/sangue , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologiaRESUMO
Vaccination is becoming a more acceptable option in the effort to eradicate avian influenza viruses (AIV) from commercial poultry, especially in countries where AIV is endemic. The main concern surrounding this option has been the inability of the conventional serological tests to differentiate antibodies produced due to vaccination from antibodies produced in response to virus infection. In attempts to address this issue, at least six strategies have been formulated, aiming to differentiate infected from vaccinated animals (DIVA), namely (i) sentinel birds, (ii) subunit vaccine, (iii) heterologous neuraminidase (NA), (iv) nonstructural 1 (NS1) protein, (v) matrix 2 ectodomain (M2e) protein, and (vi) haemagglutinin subunit 2 (HA2) glycoprotein. This short review briefly discusses the strengths and limitations of these DIVA strategies, together with the feasibility and practicality of the options as a part of the surveillance program directed toward the eventual eradication of AIV from poultry in countries where highly pathogenic avian influenza is endemic.
Assuntos
Anticorpos Antivirais/sangue , Galinhas/sangue , Doenças Endêmicas/prevenção & controle , Monitoramento Epidemiológico/veterinária , Vírus da Influenza A/imunologia , Influenza Aviária/prevenção & controle , Animais , Galinhas/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/sangue , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/patogenicidade , Vacinas contra Influenza/uso terapêutico , Influenza Aviária/epidemiologia , Influenza Aviária/imunologia , Influenza Aviária/virologia , Neuraminidase/sangue , Espécies Sentinelas/sangue , Espécies Sentinelas/imunologia , Testes Sorológicos , Vacinação/métodos , Proteínas não Estruturais Virais/sangueRESUMO
A fluorescent aptasensor system has been designed for the sensitive detection of recombinant hemagglutinin (rHA) protein of the H5N1 influenza virus in human serum. Guanine-richen anti-rHA aptamers by SELEX were immobilized on the surface of the Ag@SiO2 nanoparticles which performed as a metal-enhanced fluorescence (MEF) sensing platform. Thiazole orange (TO) was used as fluorescent tag which reported to the G-quadruplex secondary structural induced by aptamer-rHA binding event. In the absence of rHA protein, TO was free in the solution with almost no fluorescence emission. When rHA protein was added to the solution, the aptamer strand bound rHA protein to form a stable G-quadruplex complex, which can bind TO and excite the fluorescence emission of TO. Moreover, the excited-state TO captured by the G-quadruplex complex was forced to the surface of the Ag@SiO2 nanoparticles and could experience a surface plasmon resonance enhancement which can be transformed into more efficient fluorescence emission signals, therefore, the fluorescence signal of TO can be amplified largely. This system does not require covalent labeling with fluorophores to the aptamer and the background noise is very low. The detection of rHA protein of the H5N1 influenza virus could be operated both in aqueous buffer and human serum with the detection limit of 2 and 3.5ng/mL respectively. More important, the whole detection process can be finished in a PE tube within 30min, which makes it suitable as a self-contained diagnostic kit for H5N1 influenza virus point-of-care (POC) diagnostic.
Assuntos
Aptâmeros de Nucleotídeos/genética , Técnicas Biossensoriais/métodos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Aptâmeros de Nucleotídeos/química , Benzotiazóis , Corantes Fluorescentes , Quadruplex G , Glicoproteínas de Hemaglutininação de Vírus da Influenza/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/isolamento & purificação , Humanos , Nanopartículas Metálicas/ultraestrutura , Nanopartículas , Potássio , Quinolinas , Proteínas Recombinantes/sangue , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Técnica de Seleção de Aptâmeros , Dióxido de Silício , PrataRESUMO
RATIONALE: The biology of fatal pandemic influenza infection remains undefined. OBJECTIVES: To characterize the virologic and immune parameters associated with severity or death in patients who required mechanical ventilation for A(H1N1) 2009 pneumonia of various degrees of severity during the two waves of the 2009-2011 pandemic in Paris, France. METHODS: This multicenter study included 34 unvaccinated patients with very severe or fatal confirmed influenza A(H1N1) infections. It analyzed plasma A(H1N1) 2009 reverse-transcriptase polymerase chain reaction, hemagglutinin 222G viral mutation, and humoral and cellular immune responses to the virus, assessed in hemagglutination inhibition (HI), microneutralization, ELISA, lymphoproliferative, ELISpot IFN-γ, and cytokine and chemokine assays. MEASUREMENTS AND MAIN RESULTS: The patients' median age was 35 years. Influenza A(H1N1) 2009 viremia was detected in 4 of 34 cases, and a 222G hemagglutinin mutation in 7 of 17 cases, all of them with sequential organ failure assessment greater than or equal to 8. HI antibodies were detectable in 19 of 26 survivors and undetectable in all six fatal fulminant cases. ELISA and microneutralization titers were concordant. B-cell immunophenotyping and plasma levels of immunoglobulin classes did not differ between patients who survived and died. After immune complex dissociation, influenza ELISA serology became strongly positive in the bronchoalveolar lavage of the two fatal cases tested. H1N1-specific T-cell responses in lymphoproliferative and IFN-γ assays were detectable in survivors' peripheral blood, and lymphoproliferative assays were negative in the three fatal cases tested. Plasma levels of IL-6 and IL-10 were high in fatal cases and correlated with severity. Finally, a negative HI serology 4 days after the onset of influenza symptoms predicted death from fulminant influenza (P = 0.04). CONCLUSIONS: Early negative A(H1N1) 2009 HI serology can predict death from influenza. This negative serology in fatal cases in young adults reflects the trapping of anti-H1N1 antibodies in immune complexes in the lungs, associated with poor specific helper T-cell response. Clinical trial registered with www.clinicaltrials.gov (NCT 01089400).
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/imunologia , Pneumonia Viral/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Adolescente , Adulto , Anticorpos Neutralizantes/sangue , Biomarcadores/sangue , Feminino , França , Glicoproteínas de Hemaglutininação de Vírus da Influenza/sangue , Humanos , Influenza Humana/sangue , Influenza Humana/complicações , Influenza Humana/diagnóstico , Influenza Humana/mortalidade , Interleucina-10/imunologia , Interleucina-6/imunologia , Masculino , Pessoa de Meia-Idade , Pneumonia Viral/diagnóstico , Pneumonia Viral/mortalidade , Pneumonia Viral/virologia , Valor Preditivo dos Testes , Estudos Prospectivos , Unidades de Cuidados Respiratórios , Fatores de Risco , Sensibilidade e Especificidade , Índice de Gravidade de DoençaRESUMO
BACKGROUND: The influence of prior seasonal influenza vaccination on the antibody response produced by natural infection or vaccination is not well understood. METHODS: We compared the profiles of antibody responses of 32 naturally infected subjects and 98 subjects vaccinated with a 2009 influenza A(H1N1) monovalent MF59-adjuvanted vaccine (Focetria, Novartis), with and without a history of seasonal influenza vaccination. Antibodies were measured by hemagglutination inhibition (HI) assay for influenza A(H1N1)pdm09 and by protein microarray (PA) using the HA1 subunit for seven recent and historic H1, H2 and H3 influenza viruses, and three avian influenza viruses. Serum samples for the infection group were taken at the moment of collection of the diagnostic sample, 10 days and 30 days after onset of influenza symptoms. For the vaccination group, samples were drawn at baseline, 3 weeks after the first vaccination and 5 weeks after the second vaccination. RESULTS: We showed that subjects with a history of seasonal vaccination generally exhibited higher baseline titers for the various HA1 antigens than subjects without a seasonal vaccination history. Infection and pandemic influenza vaccination responses in persons with a history of seasonal vaccination were skewed towards historic antigens. CONCLUSIONS: Seasonal vaccination is of significant influence on the antibody response to subsequent infection and vaccination, and further research is needed to understand the effect of annual vaccination on protective immunity.
Assuntos
Anticorpos Antivirais/sangue , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Vacinação/estatística & dados numéricos , Adolescente , Adulto , Idoso , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Aves , Feminino , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vacinas contra Influenza/administração & dosagem , Influenza Aviária/imunologia , Influenza Aviária/virologia , Influenza Humana/sangue , Influenza Humana/imunologia , Influenza Humana/virologia , Masculino , Pessoa de Meia-Idade , Análise Serial de Proteínas , Vacinação/métodosRESUMO
The domestic poultry population in Vietnam has been vaccinated against highly pathogenic avian influenza (HPAI) H5N1 since 2005. Since then, outbreaks have continued to occur without a clear understanding of the mechanisms involved. The general objective of this study was to understand the epidemiology of the disease in the context of vaccination and to draw some conclusions about vaccination efficacy in the domestic poultry population of the Red River Delta area. Five cross-sectional surveys to measure the serological and virological prevalence in vaccinated and unvaccinated poultry were performed from the end of 2008 to June 2010. The global seroprevalence was 24% (95% confidence interval 19·9-28·2). Determinants of vaccine immunogenicity were identified separately in chickens and ducks as well as determinants of the seroconversion in unvaccinated birds. The results highlight the difficulties in maintaining good flock immunity in poultry populations using inactivated vaccine in the field with two vaccination rounds per year, and in preventing circulation of virus in co-existing unvaccinated poultry.
Assuntos
Galinhas/virologia , Patos/virologia , Gansos/virologia , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/uso terapêutico , Influenza Aviária/prevenção & controle , Animais , Estudos Transversais , Surtos de Doenças/veterinária , Glicoproteínas de Hemaglutininação de Vírus da Influenza/sangue , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/epidemiologia , Reação em Cadeia da Polimerase , Estudos Soroepidemiológicos , Vacinação/veterinária , Vietnã/epidemiologiaRESUMO
Serological studies are the gold standard method to estimate influenza infection attack rates (ARs) in human populations. In a common protocol, blood samples are collected before and after the epidemic in a cohort of individuals; and a rise in haemagglutination-inhibition (HI) antibody titers during the epidemic is considered as a marker of infection. Because of inherent measurement errors, a 2-fold rise is usually considered as insufficient evidence for infection and seroconversion is therefore typically defined as a 4-fold rise or more. Here, we revisit this widely accepted 70-year old criterion. We develop a Markov chain Monte Carlo data augmentation model to quantify measurement errors and reconstruct the distribution of latent true serological status in a Vietnamese 3-year serological cohort, in which replicate measurements were available. We estimate that the 1-sided probability of a 2-fold error is 9.3% (95% Credible Interval, CI: 3.3%, 17.6%) when antibody titer is below 10 but is 20.2% (95% CI: 15.9%, 24.0%) otherwise. After correction for measurement errors, we find that the proportion of individuals with 2-fold rises in antibody titers was too large to be explained by measurement errors alone. Estimates of ARs vary greatly depending on whether those individuals are included in the definition of the infected population. A simulation study shows that our method is unbiased. The 4-fold rise case definition is relevant when aiming at a specific diagnostic for individual cases, but the justification is less obvious when the objective is to estimate ARs. In particular, it may lead to large underestimates of ARs. Determining which biological phenomenon contributes most to 2-fold rises in antibody titers is essential to assess bias with the traditional case definition and offer improved estimates of influenza ARs.
Assuntos
Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Influenza Humana/epidemiologia , Influenza Humana/imunologia , Adolescente , Adulto , Idoso , Anticorpos Antivirais/sangue , Estudos de Coortes , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/sangue , Humanos , Influenza Humana/sangue , Masculino , Pessoa de Meia-Idade , Variações Dependentes do Observador , Estudos Soroepidemiológicos , Testes Sorológicos , Vietnã/epidemiologiaRESUMO
Detection by microneutralization of low-titre antibodies (anti-H5 micro-NT titre ≤ 1:80) against avian influenza virus (H5N1) is usually taken to be a false-positive result. In this prospective study of 242 intensive-care unit patients admitted for severe community-acquired pneumonia, the prevalence of low-titre anti-H5 micro-NT was 2.4%. Prior exposure to poultry was the sole independent risk factor for these low-titre antibodies (adjusted OR 42.41; 95% CI 22.45-64.51; p <0.001). We suggest that low anti-H5 micro-NT titres be interpreted in conjunction with plausible poultry, environmental and human exposure to H5N1.
Assuntos
Infecções Comunitárias Adquiridas/terapia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/sangue , Influenza Humana/diagnóstico , Pneumonia/terapia , Virologia/métodos , Adulto , Animais , Infecções Comunitárias Adquiridas/diagnóstico , Estado Terminal , Feminino , Humanos , Influenza Humana/epidemiologia , Unidades de Terapia Intensiva , Masculino , Testes de Neutralização/métodos , Pneumonia/diagnóstico , PrevalênciaRESUMO
The memory T cell response is polyclonal, with the magnitude and specificity of the response controlled in part by the burst size of T cells expanded from effector/memory precursors. Sensitive assays using HLA class II multimers were used to detect low-frequency Ag-specific T cells directed against influenza viral Ags in subjects immunized with the influenza vaccine. Direct ex vivo tetramer staining of PBMC from five individuals identified frequencies of hemagglutinin (HA) 306-318 tetramer binding CD4(+) T cells in the peripheral blood ranging from 1 in 600 to 1 in 30,000 CD4(+) T cells. These frequencies were validated by counting CFSE(low), tetramer-positive T cells after in vitro expansion. Low frequency of T cells directed to other influenza epitopes, including DRA1*0101/DRB1*0401-restricted matrix protein 60-73, DRA1*0101/DRB1*0101-restricted matrix protein 18-29, DRA1*0101/DRB1*0701-restricted HA 232-244 and DRA1*0101/DRB1*0101-restricted nucleoprotein 206-217 were also determined. T cells which occurred at a frequency as low as 1 in 350,000 could be ascertained by in vitro expansion of precursors. Peripheral HA(306-318)-responsive T cells expanded 2- to 5-fold following influenza vaccination. Examination of phenotypic markers of the HA(306-318)-responsive T cells in the peripheral blood indicated that the majority were CD45RA(-), CD27(+), CD25(-), CD28(+), and CD62L(-), while T cell clones derived from this population were CD45RA(-), CD27(-), CD25(+), CD28(+), and CD62L(-).
Assuntos
Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Antígenos HLA-DR/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Sequência de Aminoácidos , Antígenos Virais/sangue , Linfócitos T CD4-Positivos/química , Células Clonais , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito T/sangue , Epitopos de Linfócito T/imunologia , Fluoresceínas/análise , Glicoproteínas de Hemaglutininação de Vírus da Influenza/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Esquemas de Imunização , Imunofenotipagem , Vírus da Influenza A/imunologia , Vírus da Influenza B/imunologia , Vacinas contra Influenza/análise , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Contagem de Linfócitos , Dados de Sequência Molecular , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/imunologia , Coloração e Rotulagem , Succinimidas/análiseRESUMO
The success of influenza vaccination depends largely on the antigenic match between the influenza vaccine strains and the virus strains actually circulating during the season. In the past, this match has proved to be satisfactory in most seasons. In the 1997/1998 season, however, hemagglutination inhibition (HI) assays with ferret antisera indicated a considerable mismatch between the H3N2 vaccine component and the most prevalent epidemic influenza A(H3N2) virus. The results from antigenic analyses using pre- and postvaccination serum samples from volunteers of various ages, including residents of nursing homes who were more than 60 years of age, were in good agreement with the results obtained with ferret antisera. Homologous serum antibody responses to the H3N2 vaccine component as well as the cross-reactivity of the induced antibodies to the epidemic H3N2 strain, declined with increasing age of the vaccinees. As a consequence of these two effects, 84% of the vaccinees over 75 years of age did not develop HI antibody titers >/= 40 against the major H3N2 virus variant of 1997/1998, suggesting that they were not protected against infection with this virus variant. These findings support the current policy of the World Health Organization (WHO), which is to base worldwide influenza virus surveillance on results predominantly obtained by antigenic analyses of influenza virus isolates with ferret antisera in HI tests. If an antigenic mismatch is observed, the protective efficacy of the vaccine, especially for the elderly, may be insufficient. The observations also support the current policy to include the elderly in serologic efficacy trials.