IgM,IgG, and IgA Antibody Responses to Influenza A(H1N1)pdm09 Hemagglutinin in Infected Persons during the First Wave of the 2009 Pandemic in the United States

The novel influenza A(H1N1)pdm09 virus caused an influenza pandemic in 2009. IgM, IgG, and IgA antibody responses to A(H1N1)pdm09 hemagglutinin (HA) following A(H1N1)pdm09 virus infection were analyzed to understand antibody isotype responses. Age-matched control sera collected from U.S. residents in 2007 and 2008 were used to establish baseline levels of cross-reactive antibodies. IgM responses often used as indicators of primary virus infection were mainly detected in young patient groups (≤5 years and 6 to 15 years old), not in older age groups, despite the genetic and antigenic differences between the HA of A(H1N1)pdm09 virus and pre-2009 seasonal H1N1 viruses. IgG and IgA responses to A(H1N1)pdm09 HA were detected in all age groups of infected persons. In persons 17 to 80 years old, paired acute- and convalescent-phase serum samples demonstrated ≥4-fold increases in the IgG and IgA responses to A(H1N1)pdm09 HA in 80% and 67% of A(H1N1)pdm09 virus-infected persons, respectively. The IgG antibody response to A(H1N1)pdm09 HA was cross-reactive with HAs from H1, H3, H5, and H13 subtypes, suggesting that infections with subtypes other than A(H1N1)pdm09 might result in false positives by enzyme-linked immunosorbent assay (ELISA). Lower sensitivity compared to hemagglutination inhibition and microneutralization assays and the detection of cross-reactive antibodies against homologous and heterologous subtype are major drawbacks for the application of ELISA in influenza serologic studies.     

Detection of Nonhemagglutinating Influenza A(H3) Viruses by Enzyme-Linked Immunosorbent Assay in Quantitative Influenza Virus Culture

To assess the efficacy of novel antiviral drugs against influenza virus in clinical trials, it is necessary to quantify infectious virus titers in respiratory tract samples from patients. Typically, this is achieved by inoculating virus-susceptible cells with serial dilutions of clinical specimens and detecting the production of progeny virus by hemagglutination, since influenza viruses generally have the capacity to bind and agglutinate erythrocytes of various species through their hemagglutinin (HA). This readout method is no longer adequate, since an increasing number of currently circulating influenza A virus H3 subtype (A[H3]) viruses display a reduced capacity to agglutinate erythrocytes. Here, we report the magnitude of this problem by analyzing the frequency of HA-deficient A(H3) viruses detected in The Netherlands from 1999 to 2012. Furthermore, we report the development and validation of an alternative method for monitoring the production of progeny influenza virus in quantitative virus cultures, which is independent of the capacity to agglutinate erythrocytes. This method is based on the detection of viral nucleoprotein (NP) in virus culture plates by enzyme-linked immunosorbent assay (ELISA), and it produced results similar to those of the hemagglutination assay using strains with good HA activity, including A/Brisbane/059/07 (H1N1), A/Victoria/210/09 (H3N2), other seasonal A(H1N1), A(H1N1)pdm09, and the majority of A(H3) virus strains isolated in 2009. In contrast, many A(H3) viruses that have circulated since 2010 failed to display HA activity, and infectious virus titers were determined only by detecting NP. The virus culture ELISA described here will enable efficacy testing of new antiviral compounds in clinical trials during seasons in which nonhemagglutinating influenza A viruses circulate.     

Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays

From May to December 1997, 18 cases of mild to severe respiratory illness caused by avian influenza A (H5N1) viruses were identified in Hong Kong. The emergence of an avian virus in the human population prompted an epidemiological investigation to determine the extent of human-to-human transmission of the virus and risk factors associated with infection. The hemagglutination inhibition (HI) assay, the standard method for serologic detection of influenza virus infection in humans, has been shown to be less sensitive for the detection of antibodies induced by avian influenza viruses. Therefore, we developed a more sensitive microneutralization assay to detect antibodies to avian influenza in humans. Direct comparison of an HI assay and the microneutralization assay demonstrated that the latter was substantially more sensitive in detecting human antibodies to H5N1 virus in infected individuals. An H5-specific indirect enzyme-linked immunosorbent assay (ELISA) was also established to test children's sera. The sensitivity and specificity of the microneutralization assay were compared with those of an H5-specific indirect ELISA. When combined with a confirmatory H5-specific Western blot test, the specificities of both assays were improved. Maximum sensitivity (80%) and specificity (96%) for the detection of anti-H5 antibody in adults aged 18 to 59 years were achieved by using the microneutralization assay combined with Western blotting. Maximum sensitivity (100%) and specificity (100%) in detecting anti-H5 antibody in sera obtained from children less than 15 years of age were achieved by using ELISA combined with Western blotting. This new test algorithm is being used for the seroepidemiologic investigations of the avian H5N1 influenza outbreak.     

Serum antibody responses in naturally occurring influenza A virus infection determined by enzyme-linked immunosorbent assay, hemagglutination inhibition, and complement fixation

Serum antibody responses to influenza A virus infection were examined in 388 normal subjects during a trial of chemoprophylaxis in an outbreak of influenza A in 1980-1981 in which both A/H1N1 and A/H3N2 viruses circulated. Paired serum specimens obtained over a 6-week period were tested for antibodies to both A/H1N1 and A/H3N2 viruses by conventional hemagglutination inhibition, complement fixation, and an enzyme-linked immunosorbent assay (ELISA). Antibody responses detected by ELISA were determined by calculation of the area generated between titration curves of paired sera (area method), as well as by a conventional endpoint dilution method (endpoint method). Forty-two significant antibody rises were detected; 42 by ELISA (area method), 33 by ELISA (endpoint method), 32 by hemagglutination inhibition, and 13 by complement fixation. ELISA (area method) detected rises more frequently than either ELISA (endpoint method) (P less than 0.01), hemagglutination inhibition (P less than 0.005), or complement fixation (P less than 0.001). Another sensitive assay, the microneutralization test, detected significantly fewer rises (33, P less than 0.025) than the ELISA (area method). In the 42 subjects with ELISA (area method) rises, corroborating evidence of influenza A infection by other techniques (virus isolation, microneutralization, hemagglutination inhibition, or complement fixation tests) were available for 39 (93%). ELISA (area method) rises were subtype specific in all serum pairs in which other documentation of subtype-specific infection was available (38 of 38). Thus, ELISA (area method) was the single most sensitive assay for detection of serum antibody rises in this setting and possessed a high degree of subtype specificity.     

Safety of and serum antibody response to cold-recombinant influenza A and inactivated trivalent influenza virus vaccines in older adults with chronic diseases.

Forty older adults with chronic diseases were vaccinated intranasally with either influenza A/California/10/78 (H1N1) (CR37) or influenza A/Washington/897/80 (H3N2) (CR48) virus. No clinically significant morbidity or decrement in pulmonary function occurred postvaccination. Two (15%) recipients of CR37 virus and twelve (44%) recipients of CR48 virus became infected with vaccine virus, as indicated by a fourfold rise in serum hemagglutination inhibition antibody titer; a fourfold rise in serum immunoglobulin G (IgG) or IgA antibody titer, indicated by enzyme-linked immunosorbent assay; isolation of vaccine virus from nasal washings; or all of these. Within 1 year after cold-recombinant vaccine virus vaccination, 18 vaccines received inactivated trivalent influenza virus vaccine parenterally. Of the vaccinees, 13 (72%) developed a fourfold rise in serum antibody titer to H1N1 antigen and 16 (89%) developed a fourfold rise in serum antibody titer to H3N2 antigen. We conclude that administration of these cold-recombinant vaccine viruses to older adults with chronic diseases was safe, but that serum antibody response rates were lower than those achieved with subsequently administered inactivated influenza virus vaccine given parenterally. However, the higher seroconversion rates attained by using the inactivated trivalent influenza virus vaccine do not necessarily mean that it is more efficacious in preventing infection or severe illness or both due to natural wild-type influenza A virus.     

Use of the enzyme-linked immunosorbent assay to detect serum antibody responses of volunteers who received attenuated influenza A virus vaccines.

Sera from volunteers who received live influenza A wild-type or ts recombinant virus were tested by hemagglutination inhibition (HI) assay, neuraminidase inhibition (NI) assay, and the enzyme-linked immunosorbent assay (ELISA) to determine which assay system was the most sensitive in detecting an immunological response to infection. The ELISA was performed with inactivated whole virus antigen, and the optical density at each of five serial twofold dilutions of pre- and postimmunization sera was measured. The difference in the amount of ELISA antibody in pre- and postinoculation serum specimens was taken to be proportional to the area between the respective titration curves. The ELISA was more sensitive than the HI or NI test in detecting a seroresponse in volunteers infected with A/Hong Kong/123/77 (H1N1), A/New Jersey/8/76 (Hswine N1), or A/Alaska/6/77 (H3N2) ts recombinant virus. These results suggest that the ELISA should be used to determine the frequency of infection with attenuated viruses as well as the 50% human infectious dose of candidate live influenza A vaccine viruses.     

Rapid detection of type A influenza viruses with monoclonal antibodies to the M protein (M1) by enzyme-linked immunosorbent assay and time-resolved fluoroimmunoassay.

Monoclonal antibodies (MAbs) to the M protein (M1) were used in the development of direct detection systems for type A influenza viruses in clinical specimens. Optimal detection by an enzyme-linked immunosorbent assay was achieved when MAbs were used as capture antibodies and rabbit polyclonal antibodies were used as sandwich antibodies. Detection by the enzyme-linked immunosorbent assay required amplification of the virus. direct detection in clinical specimens (nasopharyngeal aspirates) was accomplished when MAbs recognizing two distinct antigenic sites of M1 were used in a time-resolved fluoroimmunoassay. Type A influenza viruses could be detected equally well in specimens obtained during epidemics of both H3N2 and H1N1 influenza viruses.     

Generation and characterisation of monoclonal antibodies specific to avian influenza H7N9 haemagglutinin protein

Introduction: Emerging virulent strains of influenza virus pose a serious public health threat with potential pandemic consequences. A novel avian influenza virus, H7N9, breached the species barrier from infected domestic poultry to humans in 2013 in China. Since then, it has caused numerous infections in humans with a close contact to poultry. Materials and Methods: In this study, we describe the preliminary characterisation of five murine monoclonal antibodies (MAbs) developed against recombinant haemagglutinin (rHA) protein of avian H7N9 A/Anhui/1/2013 virus by their Western blot and enzyme-linked immunosorbent assay (ELISA) reactivity and binding affinity. Results: Of the five MAbs, four were highly specific to H7N9 HA and did not show any cross-reactivity in ELISA with rHA protein from pandemic as well as seasonal H1N1, H2N2, H3N2, H5N1 and influenza virus B (B/Brisbane/60/2008). However, one of the MAbs, MA-24, in addition to HA protein of H7N9 also reacted strongly with HA protein of H3N2 and weakly with HA of pandemic and seasonal H1N1 and H2N2. All the five MAbs also reacted with H7N9 rHA in Western blot. The MAbs bound H7N9 rHA with an equilibrium dissociation constant (K_D) ranging between 0.14 and 25.20 nM, indicating their high affinity to HA. Conclusions: These antibodies may be useful in developing diagnostic tools for the detection of influenza H7N9 virus infections.     

Molecular and phylogenetic analysis of human influenza virus among Iranian patients in Shiraz, Iran

Influenza is a viral respiratory pathogen responsible for frequent seasonal epidemics. There are currently three major human influenza viruses in global circulation namely A/H1N1, A/H3N2, and B. The objective of this study was to determine the human influenza virus genotypes in Shiraz, the capital of the Fars province of Iran. Three hundred patients suspected with human influenza virus infection were enrolled in this survey (2004-2005). The throat samples were cultured and titrated by hemagglutination (HA) assay. Typing and subtyping were performed by an in-house developed multiplex RT-PCR. Moreover, the phylogenetic analysis was carried out for HA gene. A total of 24 samples were found to be positive for human influenza virus infection, 17 H1N1 and 7 H3N2. These results were in agreement with the HI assay. The phylogenetic analysis results revealed that the Iranian H1N1 isolated were close to the A/New Caledonia/20/99 vaccine strain genetically and the Iranian H3N2 isolates were also related closely to the Fujian/411/021 and California/7/2004 vaccine strains. However, a slight genetic drift was found in these isolates. In conclusion, it was demonstrated that both influenza A subtypes A/H1N1 and A/H3N2 were dominant among Iranian patients in Shiraz during the 2004/5 winter season.     

Type- and subtype-specific detection of influenza viruses in clinical specimens by rapid culture assay.

A rapid culture assay which allows for the simultaneous typing and subtyping of currently circulating influenza A(H1N1), A(H3N2), and B viruses in clinical specimens was developed. Pools of monoclonal antibodies (MAbs) against influenza A and B viruses and MAbs HA1-71 and HA2-76, obtained by immunizing mice with the denatured hemagglutinin subfragments HA1 and HA2 of influenza virus A/Victoria/3/75, were used for immunoperoxidase staining of antigens in infected MDCK cells. MAb HA1-71 reacted exclusively with influenza A viruses of the H3 subtype, while MAb HA2-76 reacted with subtypes H1, H3, H4, H6, H8, H9, H10, H11, and H12, as determined with 78 human, 4 swine, and 10 avian influenza virus reference strains subtyped by the hemagglutination inhibition test. To determine if the technique can be used as a rapid diagnostic test, 263 known influenza virus-positive frozen nasal or throat swabs were inoculated into MDCK cells. After an overnight incubation, the cells were fixed and viral antigens were detected by immunoperoxidase staining. Influenza A viruses of the H1 and H3 subtypes were detected in 31 and 113 specimens, respectively. The subtypes of 10 influenza A virus-positive specimens could not be determined because they contained too little virus. Influenza B viruses were detected in 84 specimens, and 25 specimens were negative. We conclude that this assay is a rapid, convenient, non-labor-intensive, and relatively inexpensive test for detecting, typing, and subtyping influenza viruses in clinical specimens.     

Detection of influenza A virus homo‐ and heterosubtype‐specific memory B‐cells using a novel protein microarray‐based analysis tool

The emergence of the A(H1N1) 2009 pandemic influenza virus was initially seen as a major world‐wide health concern since a low degree of immunity to this virus strain was anticipated. However, age‐specific infection attack rates and age‐specific differences in seroresponse indicate that pre‐existing immunity may have played a significant role in protection especially in older age groups. This study describes the use of a protein microarray as a multiplex analysis tool for detection of influenza virus H1 strain‐specific memory B‐cells before and after infection with A(H1N1)pdm09. The discrimination was based on detection of specific antibodies in culture supernatants from polyclonally stimulated B‐cells against recombinant influenza virus HA1 proteins representing influenza virus subtypes H1 through H9. The protein microarray proved sensitive and specific for antibody detection in culture supernatants of B‐cells, and with the potential to deduce a person's history of infection with particular influenza virus variants, including A(H1N1)pdm09. Blood samples obtained from different age groups prior to the pandemic in 2009 partly showed the presence of B‐cells producing antibodies binding to the closely related A(H1N1) 1918 pandemic influenza virus, and of which the magnitude increased with age. These cross‐reactive antibodies were produced by single memory B‐cells present in these donors, and either bind to epitopes on HA1 which are shared within different H1 strains (homosubtypic response) or shared between different subtypes (heterosubtypic response). J. Med. Virol. 85:899–909, 2013. © 2013 Wiley Periodicals, Inc.     

In elderly persons live attenuated influenza A virus vaccines do not offer an advantage over inactivated virus vaccine in inducing serum or secretory antibodies or local immunologic memory.

In a double-blind, randomized trial, 102 healthy elderly subjects were inoculated with one of four preparations: (i) intranasal bivalent live attenuated influenza vaccine containing cold-adapted A/Kawasaki/86 (H1N1) and cold-adapted A/Bethesda/85 (H3N2) viruses; (ii) parenteral trivalent inactivated subvirion vaccine containing A/Taiwan/86 (H1N1), A/Leningrad/86 (H3N2), and B/Ann Arbor/86 antigens; (iii) both vaccines; or (iv) placebo. To determine whether local or systemic immunization augmented mucosal immunologic memory, all volunteers were challenged intranasally 12 weeks later with the inactivated virus vaccine. We used a hemagglutination inhibition assay to measure antibodies in sera and a kinetic enzyme-linked immunosorbent assay to measure immunoglobulin G (IgG) and IgA antibodies in sera and nasal washes, respectively. In comparison with the live virus vaccine, the inactivated virus vaccine elicited higher and more frequent rises of serum antibodies, while nasal wash antibody responses were similar. The vaccine combination induced serum and local antibodies slightly more often than the inactivated vaccine alone did. Coadministration of live influenza A virus vaccine did not alter the serum antibody response to the influenza B virus component of the inactivated vaccine. The anamnestic nasal antibody response elicited by intranasal inactivated virus challenge did not differ in the live, inactivated, or combined vaccine groups from that observed in the placebo group not previously immunized. These results suggest that in elderly persons cold-adapted influenza A virus vaccines offer little advantage over inactivated virus vaccines in terms of inducing serum or secretory antibody or local immunological memory. Studies are needed to determine whether both vaccines in combination are more efficacious than inactivated vaccine alone in people in this age group.     

Comprehensive serological analysis of two successive heterologous vaccines against H5N1 avian influenza virus in exotic birds in zoos

In 2005, European Commission directive 2005/744/EC allowed controlled vaccination against avian influenza (AI) virus of valuable avian species housed in zoos. In 2006, 15 Spanish zoos and wildlife centers began a vaccination program with a commercial inactivated H5N9 vaccine. Between November 2007 and May 2008, birds from 10 of these centers were vaccinated again with a commercial inactivated H5N3 vaccine. During these campaigns, pre- and postvaccination samples from different bird orders were taken to study the response against AI virus H5 vaccines. Sera prior to vaccinations with both vaccines were examined for the presence of total antibodies against influenza A nucleoprotein (NP) by a commercial competitive enzyme-linked immunosorbent assay (cELISA). Humoral responses to vaccination were evaluated using a hemagglutination inhibition (HI) assay. In some taxonomic orders, both vaccines elicited comparatively high titers of HI antibodies against H5. Interestingly, some orders, such as Psittaciformes, which did not develop HI antibodies to either vaccine formulation when used alone, triggered notable HI antibody production, albeit in low HI titers, when primed with H5N9 and during subsequent boosting with the H5N3 vaccine. Vaccination with successive heterologous vaccines may represent the best alternative to widely protect valuable and/or endangered bird species against highly pathogenic AI virus infection.     

Development of two types of rapid diagnostic test kits to detect the hemagglutinin or nucleoprotein of the swine-origin pandemic influenza A virus H1N1

Since its emergence in April 2009, pandemic influenza A virus H1N1 (H1N1 pdm), a new type of influenza A virus with a triple-reassortant genome, has spread throughout the world. Initial attempts to diagnose the infection in patients using immunochromatography (IC) relied on test kits developed for seasonal influenza A and B viruses, many of which proved significantly less sensitive to H1N1 pdm. Here, we prepared monoclonal antibodies that react with H1N1 pdm but not seasonal influenza A (H1N1 and H3N2) or B viruses. Using two of these antibodies, one recognizing viral hemagglutinin (HA) and the other recognizing nucleoprotein (NP), we developed kits for the specific detection of H1N1 pdm and tested them using clinical specimens of nasal wash fluid or nasopharyngeal fluid from patients with influenza-like illnesses. The specificities of both IC test kits were very high (93% for the HA kit, 100% for the NP kit). The test sensitivities for detection of H1N1 pdm were 85.5% with the anti-NP antibody, 49.4% with the anti-HA antibody, and 79.5% with a commercially available influenza A virus detection assay. Use of the anti-NP antibody could allow the rapid and accurate diagnosis of H1N1 pdm infections.     

Development of an antigen-capture enzyme-linked immunosorbent assay using monoclonal antibodies for detecting H6 avian influenza viruses

The H6 subtype of avian influenza virus (AIV) infection occurs frequently in wild and domestic birds. AIV antigen detection is preferred for controlling AIV as birds are infected before they produce antibodies. The purpose of this study was to develop an early diagnostic method for AIV detection. Six monoclonal antibodies (mAbs) developed from a field H6N1 AIV strain were tested for their ability to bind to viruses. The two that showed the greatest binding ability to AIVs were used for antigen detection. An antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect H6 AIVs was developed using these mAbs. One mAb was coated onto an ELISA plate as the capture antibody. The other mAb was used as the detector antibody after labeling with horseradish peroxidase. The antigen-capture ELISA detected H6N1 AIVs but not H5 AIVs, human H1N1, H3N2 influenza or other viruses. This antigen-capture ELISA could be used to specifically detect H6N1 AIV.     

Expression and purification of recombinant H5HA1 protein of H5N1 avian influenza virus in E. coli and its application in indirect ELISA

The PCR amplified HA1 fragment of H5N1 (H5HA1) avian influenza virus (AIV) hemagglutinin gene was cloned into pET28a (+) expression vector and expressed in Rosetta Blue (DE3) pLysS cells. The recombinant H5HA1 (rH5HA1) protein purified by passive gel elution after SDS-PAGE of the inclusion bodies reacted specifically with H5N1 serum in Western blot analysis. A subtype specific indirect enzyme linked immunosorbent assay (iELISA) using the rH5HA1 protein as the coating antigen was developed for detecting antibodies to H5 subtype of AIV. The assay had 89.04% sensitivity and 95.95% specificity when compared with haemagglutination inhibition test. The Kappa value of 0.842 indicated a perfect agreement between the tests. The iELISA developed can be used for serosurveillance of avian influenza in chickens.     

Recombinant protein comprising multi-neutralizing epitopes induced high titer of antibodies against influenza A virus

In previous studies, we suggested that epitope-vaccine might be a new strategy against virus infection. Based on this hypothesis, we designed and expressed a recombinant immunogen (multi-epitope-peptide) comprising repeats of three neutralizing-epitopes (neutralizing epitopes: aa92-105, 127-133 and 183-195) of hemagglutininin (HA) of influenza virus (H3N2) in E. coli. After vaccination, the recombinant multi-epitope protein could induce a high level of antibodies with predefined multi-epitope-specificity in mice and rabbits. The epitope-specific antibodies in sera were tested using three different epitope-peptides (synthetic peptides) in ELISA assay, and the serum dilutions from 1 : 6400 to 1 : 25600 were confirmed. In western blot analysis, both the antiserum and the antibodies purified by synthetic epitope-peptide coupled sepharose columns could recognize natural HA from influenza virus particles (strain A/Wuhan/359/95 H3N2). In hemagglutination inhibition (HI) tests, these three antisera at the dilutions from 1 : 20 to 1 : 80 showed inhibitory activity. Interestingly, antisera and purified antibodies induced by the epitope-vaccine could partially inhibit plaque-formation of influenza virus (strain A/Wuhan/359/95) on MDCK cell monolayers. These results suggest that the recombinant multi-epitope vaccine can simultaneously induce multi-antiviral activities against influenza virus, which may provide a new way to develop effective vaccines against influenza virus.     

Natural influenza A virus infection of mice elicits strong antibody response to HA2 glycopolypeptide

Two influenza viruses, A/Dunedin/4/73 (H3N2) and A/Mississippi/1/85 (H3N2) were adapted to BALB/c mice. Groups of BALB/c mice were intranasally (i.n.) infected with either single dose of particular virus strain or successively with both virus strains and titers of serum antibodies against influenza virus antigens ("influenza virus antibodies") and those just against the HA2 part of hemagglutinin (HA) ("HA2 antibodies") were determined. Successive infection with virus strains Dunedin and Mississippi in interval of 21 days led to the strong increase of the proportion of anti-HA2 antibodies in sera, though whole antiviral titres remained in general unchanged. These observations confirmed that the HA2 glycopolypeptide (gp) part of influenza virus HA is very strong immunogen in natural infection.