Development of Epitope-Blocking ELISA for Universal Detection of Antibodies to Human H5N1 Influenza Viruses

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.     

Use of immunoenzyme analysis for the detection and differentiation of influenza viruses A, B and C

Test systems for ELISA, containing antibodies to internal or supercapsid proteins and intended for the detection and differentiation of influenza A, B and C viruses in small amounts, have been developed. The possibility of using these systems for the determination of viruses both in material obtained directly from humans and in chick embryo isolates has been demonstrated.     

应用跨膜区置换的血凝素蛋白建立H7N9禽流感病毒抗体间接ELISA检测方法

【目的】由于H7N9禽流感病毒能够感染鸡,并且已经变异成了高致病性毒株,因此,鸡群中H7N9禽流感疫苗的免疫是一个趋势,而鸡群免疫后抗体检测方法的建立也十分必要。本研究旨在建立一种灵敏、高效、高通量的鸡群H7N9亚型禽流感病毒抗体间接酶联免疫吸附试验(ELISA)检测方法。【方法】通过昆虫杆状病毒表达系统分别表达属于W1、W2-A和W2-B分支H7N9流感病毒的3种野生型血凝素(HA)蛋白,以及跨膜区(TM)置换为H3 HA TM的W2-B分支HA蛋白(H7-53TM)。4种HA蛋白经过离子交换层析纯化后作为抗原,通过ELISA检测H7N9禽流感病毒抗体。【结果】ELISA特异性、敏感性和重复性试验结果显示,跨膜区置换主要影响HA蛋白ELISA检测的重复性,以H7-53TM为抗原的ELISA方法具有较好的重复性,其批内和批间变异系数小于10%,然而3种野生型HA蛋白与部分血清反应批内和批间变异系数大于10%,重复性较差,因此选择H7-53TM蛋白作为ELISA包被抗原。通过受试者工作特征曲线(ROC曲线)分析,以H7-53TM为抗原的ELISA能够精准地区分H7N9亚型流感病毒抗体阳性和阴性血清。通过相关性分析,该ELISA方法与134份鸡血清HI试验结果具有显著强相关性(r=0.854 6,P0.000 1),并且与3个分支疫苗株免疫血清的HI试验结果也具有显著相关性(r0.5,P0.05)。【结论】跨膜区置换能够提高HA蛋白抗原检测H7N9禽流感病毒抗体的重复性,并应用跨膜区置换的HA蛋白建立了一种能够检测不同分支疫苗株免疫的H7N9亚型禽流感病毒抗体间接ELISA检测方法。     

Profiles of Acute Cytokine and Antibody Responses in Patients Infected with Avian Influenza A H7N9

The influenza A H7N9 virus outbreak in Eastern China in the spring of 2013 represented a novel, emerging avian influenza transmission to humans. While clinical and microbiological features of H7N9 infection have been reported in the literature, the current study investigated acute cytokine and antibody responses in acute H7N9 infection. Between March 27, 2013 and April 23, 2013, six patients with confirmed H7N9 influenza infection were admitted to Drum Tower Hospital, Nanjing, China. Acute phase serum cytokine profiles were determined using a high-throughput multiplex assay. Daily H7 hemagglutinin (HA)-specific IgG, IgM, and IgA responses were monitored by ELISA. Neutralizing antibodies specific for H7N9 viruses were determined against a pseudotyped virus expressing the novel H7 subtype HA antigen. Five cytokines (IL-6, IP-10, IL-10, IFNγ, and TNFα) were significantly elevated in H7N9-infected patients when compared to healthy volunteers. Serum H7 HA-specific IgG, as well as IgM and IgA responses, were detected within 8 days of disease onset and increased in a similar pattern during acute infection. Neutralizing antibodies developed shortly after the appearance of binding antibody responses and showed similar kinetics as a fraction of the total H7 HA-specific IgG responses. H7N9 infection resulted in hallmark serum cytokine increases, which correlated with fever and disease persistence. The novel finding of simultaneous development of IgG, IgM, and IgA responses in acute H7N9 infection points to the potential for live influenza viruses to elicit fast and potent protective antibodies to limit the infection.     

Expression of recombinant HA1 protein for specific detection of influenza A/H1N1/2009 antibodies in human serum

The hemagglutinin genes (HA1 subunit) from human and animal 2009 pandemic H1N1 virus isolates were expressed with a baculovirus vector. Recombinant HA1 (rHA1) protein‐based ELISA was evaluated for detection of specific influenza A(H1N1)pdm09 antibodies in serum samples from vaccinated humans. It was found that rHA1 ELISA consistently differentiated between antibodies recognizing the seasonal influenza H1N1 and pdm09 viruses, with a concordance of 94% as compared to the hemagglutination inhibition test. This study suggests the utility of rHA1 ELISA in serosurveillance.     

A novel peptide ELISA for universal detection of antibodies to human H5N1 influenza viruses

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.     

Purification of antibodies to influenza A virus structural proteins by affinity chromatography, and their participation in haemagglutination-inhibition, neutralization and enzyme-linked immunosorbent assay.

Affinity chromatography was used to purify rabbit antibodies to common and strain-specific antigeneic determinants of haemagglutinin, to neuraminidase, and to a combination of the internal proteins of influenza A viruses. The purity of the antibodies was assessed by haemagglutination-inhibition, enzyme-linked immunosorbent assay (ELISA) and competition ELISA. The antibodies were examined for their participation in neutralization and haemagglutination-inhibition assays, and in ELISA. ELISA was found to be the most sensitive technique for detecting antibodies. Antibodies to the common and strain-specific determinants of haemagglutinin were shown to participate in neutralization assay and ELISA, but only those antibodies to the common determinants were detected by haemagglutination-inhibition.     

三株交叉反应性的流感病毒HA抗体与H1N1流感病毒的致病机制分析

目的:结合临床甲型流感病例分析流感病毒可能的致病机理。方法:收集87份陕西省2009年甲型H1N1流感重症,危重症及死亡病例的血常规参数,对其淋巴细胞、红细胞和血小板三个指标分析。制备针对甲型H1N1的单克隆抗体,采用抗体亚类鉴定试剂盒分析其抗体轻链和重链的亚型,通过血凝活性实验检测三株抗体的血凝抑制活性,通过ELISA检测三株抗体与人和小鼠的血红蛋白、红细胞、白细胞膜和血小板膜的反应,通过免疫组化分析三株流感病毒抗体与正常小鼠肺组织的结合。结果:流感病毒感染后的死亡病例中淋巴细胞、红细胞和血小板均明显降低。三株抗体与人和小鼠的淋巴细胞、红细胞和血小板均有不同程度的交叉反应;免疫组化结果同时也证实三株HA抗体与小鼠的肺组织有不同的结合力。结论:流感病毒致病的原因可能与流感病毒感染机体后产生的抗体可与血液和组织中的成分结合有关。     

Specific determination of influenza H7N2 virus based on biotinylated single-domain antibody from a phage-displayed library

The unpredicted spread of avian influenza virus subtype H7N2 in the world is threatening animals and humans. Specific and effective diagnosis and supervision are required to control the influenza. However, the existing detecting methods are laborious, are time-consuming, and require appropriate laboratory facilities. To tackle this problem, we isolated VHH antibodies against the H7N2 avian influenza virus (AIV) and performed an enzyme-linked immunosorbent assay (ELISA) to detect the H7N2 virus. To obtain VHH antibodies with high affinity and specificity, a camel was immunized. A VHH antibody library was constructed in a phage display vector pMECS with diversity of 2.8 × 10⁹. Based on phage display technology and periplasmic extraction ELISA, H7N2-specific VHH antibodies were successfully isolated. According to a pairing test, two VHH antibodies (Nb79 and Nb95) with good thermal stability and specificity can recognize different epitopes of H7N2 virus. The capture antibody (Nb79) was biotinylated in vivo, and the detection antibody (Nb95) was coupled with horseradish peroxidase (HRP). Based on biotin–streptavidin interaction, a novel sandwich immune ELISA was performed to detect H7N2. The immunoassay exhibited a linear range from 5 to 100 ng/ml. Given the above, the newly developed VHH antibody-based double sandwich ELISA (DAS–ELISA) offers an attractive alternative to other diagnostic approaches for the specific detection of H7N2 virus.     

Importance of secretory antibodies in nonsusceptibility to influenza B disease and the prevention of the spread of this virus

Under the conditions of the natural outbreak of influenza B a rise in the level of antibodies in the blood serum and respiratory tract secretions has been found to occur in nonimmunized persons during this disease. Secretory antibodies, along with serum antibodies and in interaction with them, prevent the development of clinically manifest influenza. The asymptomatic form of influenza infection has been revealed: during this form of the disease the response of the body is limited to the local immunity system.     

Multi-Dimensional Measurement of Antibody-Mediated Heterosubtypic Immunity to Influenza

The human immune response to influenza vaccination depends in part on preexisting cross-reactive (heterosubtypic) immunity from previous infection by, and/or vaccination with, influenza strains that share antigenic determinants with the vaccine strains. However, current methods for assessing heterosubtypic antibody responses against influenza, including the hemagglutination-inhibition (HAI) assay and ELISA, are time and labor intensive, and require moderate amounts of serum and reagents. To address these issues we have developed a fluorescent multiplex assay, mPlex-Flu, that rapidly and simultaneously measures strain specific IgG, IgA, and IgM antibodies against influenza hemagglutinin (HA) from multiple viral strains. We cloned, expressed and purified HA proteins from 12 influenza strains, and coupled them to multiplex beads. Assay validation showed that minimal sample volumes (<5 μl of serum) were needed, and the assay had a linear response over a four Log₁₀ range. The assay detected nanogram levels of anti-influenza specific antibodies, had high accuracy and reproducibility, with an average percentage coefficient of variation (%CV) of 9.06 for intra-assay and 12.94 for inter-assay variability. Pre- and post-intramuscular trivalent influenza vaccination levels of virus specific Ig were consistent with HAI titer and ELISA measurements. A significant advantage of the mPLEX-Flu assay over the HAI assay is the ability to perform antigenic cartography, determining the antigenic distances between influenza HA’s, without mathematical correction for HAI data issues. For validation we performed antigenic cartography on 14 different post-influenza infection ferret sera assayed against 12 different influenza HA’s. Results were in good agreement with a phylogenetic tree generated from hierarchical clustering of the genomic HA sequences. This is the first report of the use of a multiplex method for antigenic cartography using ferret sera. Overall, the mPlex-Flu assay provides a powerful tool to rapidly assess the influenza antibody repertoire in large populations and to study heterosubtypic immunity induced by influenza vaccination.     

Antigenic Characterization of Recombinant Hemagglutinin Proteins Derived from Different Avian Influenza Virus Subtypes

Since the advent of highly pathogenic variants of avian influenza virus (HPAIV), the main focus of avian influenza research has been the characterization and detection of HPAIV hemagglutinin (HA) from H5 and H7 subtypes. However, due to the high mutation and reassortation rate of influenza viruses, in theory any influenza strain may acquire increased pathogenicity irrespective of its subtype. A comprehensive antigenic characterization of influenza viruses encompassing all 16 HA and 9 neuraminidase subtypes will provide information useful for the design of differential diagnostic tools, and possibly, vaccines. We have expressed recombinant HA proteins from 3 different influenza virus HA subtypes in the baculovirus system. These proteins were used to generate polyclonal rabbit antisera, which were subsequently employed in epitope scanning analysis using peptide libraries spanning the entire HA. Here, we report the identification and characterization of linear, HA subtype-specific as well as inter subtype-conserved epitopes along the HA proteins. Selected subtype-specific epitopes were shown to be suitable for the differentiation of anti-HA antibodies in an ELISA.     

基于血凝素(HA)的新型流感疫苗研究进展

新型广谱流感疫苗是预防和控制不断变异的流感病毒的重要手段。血凝素(HA)是流感病毒表面的糖蛋白,具有免疫原性,但其变异性强,是A型流感病毒发生抗原变异的主要原因。近年来研究发现,HA存在保守的恒定区,可诱导机体产生流感病毒特异性广谱中和抗体,拮抗多种流感病毒的感染。因此,如何采取不同策略和方法,研发基于HA的新型疫苗成为流感防治研究的重点。就基于HA的新型流感疫苗研究进展作一综述。     

Viral replication and innate host responses in primary human alveolar epithelial cells and alveolar macrophages infected with influenza H5N1 and H1N1 viruses

Highly pathogenic influenza H5N1 virus continues to pose a threat to public health. Although the mechanisms underlying the pathogenesis of the H5N1 virus have not been fully defined, it has been suggested that cytokine dysregulation plays an important role. As the human respiratory epithelium is the primary target cell for influenza viruses, elucidating the viral tropism and innate immune responses of influenza H5N1 virus in the alveolar epithelium may help us to understand the pathogenesis of the severe pneumonia associated with H5N1 disease. Here we used primary cultures of differentiated human alveolar type II cells, alveolar type I-like cells, and alveolar macrophages isolated from the same individual to investigate viral replication competence and host innate immune responses to influenza H5N1 (A/HK/483/97) and H1N1 (A/HK/54/98) virus infection. The viral replication kinetics and cytokine and chemokine responses were compared by quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). We demonstrated that influenza H1N1 and H5N1 viruses replicated productively in type II cells and type I-like cells although with different kinetics. The H5N1 virus replicated productively in alveolar macrophages, whereas the H1N1 virus led to an abortive infection. The H5N1 virus was a more potent inducer of proinflammatory cytokines and chemokines than the H1N1 virus in all cell types. However, higher levels of cytokine expression were observed for peripheral blood monocyte-derived macrophages than for alveolar macrophages in response to H5N1 virus infection. Our findings provide important insights into the viral tropisms and host responses of different cell types found in the lung and are relevant to an understanding of the pathogenesis of severe human influenza disease.     

Diagnostic value of dengue virus-specific IgA and IgM serum antibody detection

The diagnostic value of dengue virus (DV)-specific immunoglobulin A (IgA) serum antibody detection, by an indirect immunofluorescence assay (IFA) was evaluated. For this study, the kinetics of DV-specific IgA serum antibodies was analysed in two experimentally immunised macaques, paired samples from 35 patients suspected of a primary or secondary DV infection, paired sera from patients with high levels of IgA specific antibodies against influenza virus (n = 15), sera from patients with other viral infections (n = 40) and healthy blood donors (n = 10), which served as controls. The presence of DV-specific IgA serum antibodies in humans and in monkeys was compared with that of DV-specific IgM demonstrated in a capture enzyme-linked immunosorbent assay (ELISA). The development of DV-specific IgA and IgM antibodies in macaques proved to be similar to that observed in humans with a DV infection. In sera obtained from suspected primary DV patients during the acute phase and convalescent phase, DV-specific IgA was detected in 1/6 (17%) and 6/6 (100%), whereas IgM was detected in 4/6 (67%) and 5/6 (83%), respectively. In sera from suspected secondary DV patients during the acute phase and convalescent phase, DV-specific IgA was detected in 18/29 (62%) and 28/29 (97%), whereas IgM was detected in 20/29 (69%) and 28/29 (97%), respectively. The control group consisted of five paired serum samples from yellow fever vaccinated individuals and a patient with acute tick-borne encephalitis, 15 paired serum samples from patients with high levels of IgA antibodies specific for influenza virus and 40 serum samples from patients with specific IgM antibodies against other viruses. Ten serum samples from healthy blood donors were included. Among the control serum samples, in one patient, both DV-specific IgA and IgM antibodies were present, and in three sera DV-specific IgM antibodies could be demonstrated. These data suggest that detection of DV-specific IgA serum antibodies by IFA may have additional value for the diagnosis of DV infection.     

Production and characterization of a conserved M2e peptide-based specific IgY antibody: evaluation of the diagnostic potential via conjugation with latex nanoparticles

Abstract

Antibodies play an important role in combating and controlling viral diseases such as influenza. Immunoglobulin Y (IgY) antibodies have several advantages such as a less invasive manufacturing process, ease of isolation, higher affinity compared with IgG antibodies, and cost-effectiveness. To date, although specific IgY production has been performed for different strains of influenza A, to the best of our knowledge, an IgY against the M2e peptide has not been produced. In the current study, IgY antibodies are produced, purified, and characterized using the M2e peptide sequence for the first time with the intent to apply them for the diagnosis of influenza A virus. Anti-M2e IgY antibodies are obtained from eggs using a two-step purification method. The activity and characterization of the antibodies are determined using an enzyme-linked immunosorbent assay, a nano-spectrophotometer, an SDS-Page assay, and a Western Blot analysis. Finally, anti-M2e IgY antibodies are conjugated to the latex nanoparticles, and the reaction between the influenza A virus and the nanoparticles is demonstrated using light microscopy, transmission electron microscopy, and energy dispersive X-ray spectroscopy. In conclusion, this study shows that anti-M2e IgY antibodies can contribute to the diagnosis, treatment, and prevention of the influenza A virus.     

Analysis of the antigenic and immunogenic properties of protein M of the influenza virus by an immunoenzyme method

A test system permitting the detection of influenza virus protein M at a concentration of 0.1-0.5 ng/ml in ELISA has been developed. The use of this system made it possible to detect influenza viruses A and B directly in crude virus-containing material and clinical samples obtained from influenza patients. During the outbreak of influenza in the spring of 1983 ELISA was successfully used for the rapid diagnosis of influenza, and some of its advantages in comparison with the conventional immunofluorescence test were thus demonstrated. To overcome difficulties arising from the low immunogenic potency of protein M, in the process of obtaining diagnostic sera and ascitic fluids the animals were immunized with the conjugate of protein M and polyelectrolite, which ensured considerable activation of humoral immune response.     

Properties of monoclonal antibodies interacting with determinants of hepatitis B virus surface antigen

Fourteen hybridoma clones have been isolated producing the monoclonal antibodies to the surface antigen of the hepatitis B virus (HBsAg). Monoclonal antibodies have been shown to react in high titres with HBsAg in the reactions of PHA, PH and ELISA. The specificity of monoclonal antibodies to two antigenic determinants has been found by the competitive solid phase ELISA technique. Monoclonal antibodies from nine clones react with one determinant while monoclonal antibodies from the rest five clones react with the other nonoverlapping determinant.     

Radioimmunological analysis of the antigenic variability of influenza virus nucleoprotein

Influenza A virus ability to bind anti-NP monoclonal antibodies to two viral strains has been studied by radioimmunoassay on polyethylene film with the subsequent autoradiographic registration of results. Monoclonal antibodies were obtained to the viral strains differing in antigenic formula of outer glycoproteids and isolated at different time. The studied influenza viruses were divided into seven groups due to their ability to bind monoclonal antibodies. The absence of correlation between the antigenic properties of nucleoprotein and glycoproteids has been registered. Variability of some antigenic sites has been analyzed. The human epidemic strains of influenza virus are different in ability to bind monoclonal antibodies from the viral strains that are connected with animals in nature or laboratory practice.     

Improvement of immunofluorescence for diagnosis of AIDS using laser microscopy

One of the most commonly used methods for demonstration of HIV antibodies is indirect immunofluorescence employing HIV-infected, CD4-positive lymphoid cell lines as antigenic substrate. Immunofluorescence with conventional optic equipment is reported to be slightly less sensitive than enzyme-linked immunoassay (ELISA). We have developed an immunofluorescence microscope which is equipped with an argon laser that has the advantages of much brighter fluorescence than conventional techniques, the prevention of fluorescence bleaching, and the possibility of distinguishing specific from nonspecific staining by comparative analysis of the kinetics of the bleaching curves. This microscope has now been used for demonstration of HIV antibodies in indirect immunofluorescence tests on the H9 lymphoid cell line, which is highly efficient in expressing HIV after infection. Titers of ELISA and Western blot-verified HIV-positive patients and appropriate normal controls were compared using four types of microscopic equipment, including the laser immunofluorescence microscope. The latter afforded significantly higher titers than those obtained with conventional immunofluorescence microscopes, and also made possible the distinction between specific and nonspecific staining.