Influence of amantadine resistance mutations on the pH regulatory function of the M2 protein of influenza A viruses.

Mutations in the influenza M2 membrane protein which confer resistance to the antiviral drug amantadine are exclusively located within the transmembrane region of the molecule. The influence of specific amino acid substitutions on the activity of the M2 protein in influenza A virus-infected cells is assessed in this report by their effects upon haemagglutinin (HA) stability and virus growth. A number of amino acid substitutions, e.g., L26H, A30T, S31N and G34E reduced the activity of the M2 protein of A/chicken/Germany/34 (Rostock) and caused a substantial increase in expression of the low-pH form of HA. The adverse effects of the mutations on virus replication were evident from changes selected during subsequent passage of the mutant viruses in the presence or absence of amantadine: reversion to wt, the acquisition of a second suppressor mutation in M2, or the appearance of a complementary mutation in HA which increased its pH stability. In contrast, 127T and 127S, mutations which were most readily selected following passage of the wt virus in the presence of drug, caused an increase in M2 activity. Furthermore, in double mutants the 127T mutation suppressed the attenuating effects of the A30T and S31N mutations on M2 activity. The influence of primary structure on the consequences of particular amino acid changes was further emphasized by the contrasting effects of the G34E mutation on the activities of two closely related proteins, causing an increase in the activity of the M2 of A/chicken/Germany/27 (Weybridge) as opposed to the decrease in activity of the Rostock protein. Estimates of differences in trans Golgi pH based on the degree of conversion of HA to the low-pH form, or complementation of differences in pH stability of mutant HAs, indicate that changes in M2 may influence pH within the transport pathway by as much as 0.6. The results thus provide further evidence that M2 regulates transmembrane pH gradients in the trans Golgi. Incompatibility between particular HA and M2 components and the selection of M2 mutants with suboptimal activity stresses the essential relationship between the structures and functions of these two virus proteins.     

Heterosubtypic protective immunity against influenza A virus induced by fusion peptide of the hemagglutinin in comparison to ectodomain of M2 protein

Several types of influenza vaccines are available, but due to the highly unpredictable variability of influenza virus surface antigens (hemagglutinin (HA) and neuraminidase) current vaccines are not sufficiently effective against broad spectrum of the influenza viruses. An innovative approach to extend the vaccine efficacy is based on the selection of conserved influenza proteins with a potential to induce inter-subtype protection against the influenza A viruses. A promising new candidate for the preparation of broadly protective vaccine may be a highly conserved N-terminal part of HA2 glycopolypeptide (HA2 gp) called fusion peptide. To study its capacity to induce a protective immune response, we immunized mice with the fusion peptide (aa 1-38 of HA2 gp). The protective ability of fusion peptide was compared with the ectodomain aa 2-23 of M2 protein (eM2) that is antigenically conserved and its immunogenic properties have already been well documented. Corresponding peptides (both derived from A/Mississippi/1/85 (H3N2) virus) were synthesized and conjugated to the keyhole limpet hemocyanin (KLH) and used for the immunization of mice. Both antigens induced a significant level of specific antibodies. Immunized mice were challenged with the lethal dose of homologous (H3N2) or heterologous A/PR/8/34 (H1N1) influenza A viruses. Immunization with the fusion peptide led to the 100% survival of mice infected with 1 LD50 of homologous as well as heterologous virus. Survival rate decreased when infectious dose was raised to 2 LD50. The immunization with eM2 induced effective cross-protection of mice infected even with 3 LD50 of both challenge viruses. The lower, but still effective protection induced by the fusion peptide of HA2 gp suggested that besides ectodomain of M2, fusion peptide could also be considered as a part of cross-protective influenza vaccine. To our knowledge, this is the first report demonstrating that active immunization with the conjugated fusion peptide of HA2 gp provided the effective production of antibodies, what contributed to the cross-protection against influenza infection.     

Prevention and treatment of bronchopneumonia in mice caused by mouse-adapted variant of avian H5N2 influenza A virus using monoclonal antibody against conserved epitope in the HA stem region

Summary.  The effects of monoclonal antibody (MAb) C179 recognizing a conformational epitope in the middle of the hemagglutinine (HA) stem region were examined in a mouse model in the experiments of prevention and treatment of lethal bronchopneumonia caused by influenza A virus of H5 subtype. To model the lethal infection, avian nonpathogenic strain A/mallard duck/Pennsylvania/ 10218/84 (H5N2) was adapted to mice. This resulted in highly pathogenic pneumovirulent mouse-adapted (MA) variant, which was characterized. Three amino acid changes were found in the HA1 subunit of HA of MA virus. One of these was located inside the region of the conformational epitope recognized by MAb C179. However, this substitution was not significant for the recognition of HA and virus neutralization by MAb C179 in vitro and in vivo. Intraperitoneal administration of two different concentrations of MAb C179 one day before or two days after the virus challenge significantly decreased mortality rate. These results suggest that MAb C179 is efficient not only in the prevention and treatment of H1 and H2 influenza virus bronchopneumonia, as was reported previously, but also of H5-induced bronchopneumonia as well, and demonstrate in vivo the existence of a common neutralizing epitope in the HAs of these three subtypes. Received November 24, 1999 Accepted February 22, 2000     

An epitope shared by the hemagglutinins of H1, H2, H5, and H6 subtypes of influenza A virus

The membrane-inserted hemagglutinin (HA) is the most variable protein of influenza viruses. Here we describe the characterization of a shared epitope in the HA of influenza A virus H1, H2, and H5 subtypes which were completely neutralized by a monoclonal antibody (MAb), directed against this epitope. This MAb (C179) also efficiently precipitated the HAs of these viruses. In addition, MAb C179 did not neutralize H6 subtype strains despite complete amino acid homology of the epitope regions. Furthermore, only the non-glycosylated form of the HA of one of the H6 subtype strains could be precipitated by the MAb. The conformational epitope may be masked by glycosylation, although it could not be excluded that differences in the primary amino acid sequence may cause the decreased accessibility of the epitope in H6 subtype strains.     

Influenza A virus hemagglutinin containing basolateral localization signal does not alter the apical budding of a recombinant influenza A virus in polarized MDCK cells

Morphogenesis of influenza virus is a complex multistep process involving transport of all viral components as either individual or subviral components to the specified assembly site and interaction among the viral components in an ordered fashion to initiate the budding process. Envelope glycoprotein(s) is believed to be the major determinant in selecting the viral budding site since the majority of the viral glycoproteins are directed to the budding site independent of other viral components. Influenza viruses bud from the apical surface of polarized epithelial cells and all three envelope proteins, hemagglutinin (HA), neuraminidase (NA), and M2, are also targeted independently to the apical surface. Since HA is the major viral envelope protein, we decided to test whether basolaterally expressed HA can make the virus bud from the basolateral surface. Accordingly, we introduced the tyrosine-based basolateral-sorting signal to the cytoplasmic tail of HA by changing Cys561 --> Tyr561 and generated a transfectant virus by reverse genetics. Compared to the parent WSN virus, the mutant virus (HAtyr virus) contained less HA on its envelope. While the wild-type (wt) HA was >95% apical, the mutated HA (HAtyr) was approximately 60% basolateral in both transfected and virus-infected polarized MDCK cells. Also, HAtyr protein exhibited a much higher rate of endocytosis than the wt HA, in both apical and basolateral surface of transfected as well as virus-infected cells. However, the HAtyr virus, similar to wt WSN virus, was seen to bud almost exclusively (>99%) from the apical side of polarized MDCK cells. This finding was confirmed by using neuraminidase to facilitate virus release, by treating the collected virus particles with trypsin to cleave HA0 --> HA1 and HA2, by protein analysis of released virus particles, and finally, by electron microscopy. Therefore HA, the major glycoprotein alone, does not determine the budding site, and other factor(s), possibly both viral and host, is responsible for selecting the budding site of influenza virus.     

Design of a recombinant strain of the vaccinia virus containing an expressible gene for influenza A virus hemagglutinin

A recombinant vaccinia virus (VV) strain containing a cloned gene of influenza A/Udorn/307/72 (H3N2) hemagglutinin (HA) gene has been produced. HA expression in CV-1 cells infected with the recombinant virus was determined by enzyme immunoassay. The influenza virus HA titer was 1:64-1:128. When rabbits were inoculated intravenously with the recombinant VaV, antibody titres were 1:5120. The recombinant VaV preparation may be used for generation of monospecific antibody to influenza virus.     

A candidate vaccine against influenza virus intensively improved the immunogenicity of a neutralizing epitope

BACKGROUND: The hemagglutinin (HA) of influenza viruses is one of the major targets of the humoral response. The role of serum antibody to HA in the protection against infection has been demonstrated by long-standing observation. In previous studies, we suggested that an epitope vaccine might be a new strategy against the virus. METHODS: HA sequences of 491 H3 subtype strains from the influenza sequence database were compared and analyzed. To acquire information on the immunogenicity of the F3 epitope, F3-epitope-specific antibody levels in 81 patient sera infected with influenza virus were tested by ELISA. Based on the theory of the epitope vaccine, we designed an epitope peptide F3 (C-KAYSNCYPYDVPDY-G-KAYSNCYPYDVPDY), which contains the repeated F3 epitope KAYSNCYPYDVPDY (aa92-105) on HA (H3N2). The specificity and the titer of the antibodies induced by the epitope vaccine were determined by ELISA. The neutralizing activities of these anti-F3 antibodies were shown by inhibiting influenza virus infection of MDCK cells. RESULTS AND CONCLUSION: Comparison of HA sequences of 491 H3 subtype strains indicates that this epitope is highly conservative. Analysis of the sera from influenza virus-infected patients revealed a very low level of F3 epitope-specific antibodies, suggesting the poor immunogenicity of the F3 epitope on influenza virus. The epitope vaccine based on the F3 epitope induced high levels of F3 epitope-specific antibodies recognizing the epitope peptide F3 (antibody titer in antisera up to 1:25,600). Besides, the antisera could also recognize the natural HA in Western blotting. Interestingly, these antisera induced by the epitope vaccine could inhibit infection of MDCK cells by influenza virus (strain A/Wuhan/359/95) in the neutralization assay. These results suggest that the epitope vaccine can intensively increase the immunogenicity of neutralizing epitopes and may provide a new way to develop an effective vaccine against influenza virus.     

Inhibition of fusion activity of influenza A haemagglutinin mediated by HA2-specific monoclonal antibodies

Summary.  The effect of seven monoclonal antibodies (MAbs) specific to the light chain (HA2) of influenza A haemagglutinin (HA) on its fusion activity was investigated. These MAbs, which are non-virus neutralizing, defined four distinct antigenic sites on HA2 glycopolypeptide and the corresponding epitopes were attributed to the sequence stretches on HA2. The accessibility of all seven HA2 epitopes significantly increased after trypsin cleavage and pH 5 treatment of the HA (X-31). The influence of anti-HA2 MAbs on the fusion process was followed by cell–cell fusion of CHO cells expressing precursor HA, virus-liposome fusion assay, and haemolysis mediated by virus. MAb CF2, which bound directly to the fusion peptide 1–35 of HA2, was positive in all three fusion-inhibition assays and was the only one inhibiting the polykaryon formation of CHO-X-31 cells. Two other MAbs belonging to the same antigenic site but not binding directly to the fusion peptide inhibited virus to liposome fusion (EB12) or inhibited haemolysis (BB8). Moreover, MAb IIF4 binding to distinct antigenic site within 125–175 HA2 inhibited haemolysis, too. Thus, fusion activity of HA may be inhibited by anti-HA2 MAbs, mainly those binding to or near the fusion peptide. These antibodies represent useful probes for studies of influenza virus to cell membrane fusion. Received June 24, 2002; accepted October 2, 2002     

Development of a novel rapid immunochromatographic test specific for the H5 influenza virus

Three anti-H5 influenza virus monoclonal antibody (mAb) clones, IFH5-26, IFH5-115 and IFH5-136, were obtained by immunising a BALB/C mouse with inactivated A/duck/Hokkaido/Vac-1/04 (H5N1). These mAbs were found to recognise specifically the haemagglutinin (HA) epitope of the influenza H5 subtypes by western blotting with recombinant HAs; however, these mAbs have no neutralising activity for A/duck/Hokkaido/84/02 (H5N3) or A/Puerto Ric/8/34 (H1N1). Each epitope of these mAbs was a conformational epitope that was formed from the regions located between 46 to 60 amino acids (aa) and 312 to 322 aa for IFH5-115, from 101 to 113 aa and 268 to 273 aa for IFH5-136 and from 61 to 80 aa and 290 to 300 aa for IFH5-26. The epitopes were located in the loop regions between the receptor region and alpha-helix structure in haemagglutinin 1 (HA1). Influenza A virus H5-specific rapid immunochromatographic test kits were tested as solid phase antibody/alkaline phosphate-conjugated mAb in the following three combinations: IFH5-26/IFH5-115, IFH5-136/IFH5-26 and IFH5-136/IFH5-115. In every combination, only influenza A H5 subtypes were detected. For effective clinical application, rapid dual discrimination immunochromatographic test kits in combination with H5 HA-specific mAb, IFA5-26 and IFA5-115 and the influenza A NP NP-specific mAb, FVA2-11, were developed. The dual discrimination immunochromatographic tests kits detected influenza A virus H5 subtypes as H5 line-positive and all influenza A subtypes as A line-positive simultaneously. The dual discrimination immunochromatographic test kits may be useful for discriminating highly pathogenic avian influenza A H5N1 viruses from seasonal influenza A virus, as well as for confirming influenza infection status in human, avian and mammalian hosts.     

Relation between drug resistance and antigenicity among norakin-resistant mutants of influenza A (fowl plague) virus

Summary Norakin-resistant (NR) mutants of fowl plague virus (A/FPV/Wey-bridge, H7N7) have 1 to 2 (in one instance 3) amino acid substitutions in different positions of the heavy (HA 1) and/or light (HA 2) subunits of the haemagglutinin (HA) molecule. Investigation of NR mutants using the haemagglutination inhibition test with monoclonal antibodies (MAb) to the HA of A/seal/Massachusetts/80 (H7N7) virus revealed that one of the mutants (NR 1) differs antigenically from the wild-type fowl plague virus: its haemagglutination was not inhibited by MAb 55/2 and 58/6. By contrast, MAb-resistant (escape) mutants, selected from the wild-type fowl plague virus under pressure from MAb 55/2 or 58/6, showed reduced drug sensitivity. These findings suggest a possibility of correlation between alteration of influenza virus antigenicity and change of its sensitivity to drugs whose target is the haemagglutinin. This potential effect should be taken into account when antiviral substances directed to surface influenza virus antigens are being developed for use as antiviral drugs.     

Enzyme immunoassay, complement fixation and hemagglutination inhibition tests in the diagnosis of influenza A and B virus infections. Purified hemagglutinin in subtype-specific diagnosis

The efficacy of enzyme immunoassay (EIA) in detecting diagnostic antibody rises to influenza A and B viruses was compared with complement fixation (CF) and hemagglutination inhibition (HI) tests in 455 patients with an acute respiratory infection. EIA and HI detected significantly more diagnostic antibody rises against influenza A than the CF method (96 and 87 vs. 47, respectively). In the case of influenza B significantly more diagnostic influenza B antibody rises were observed by EIA than by CF or HI (59 vs. 37 and 40, respectively). In most of the cases antibody rises in EIA were found in both IgG and IgA isotypes whereas increases in IgM antibodies were seen less frequently. Purified hemagglutinins (HA) were prepared from influenza A HI- and H3-subtypes and from influenza B viruses and used as antigens in EIA and the results were compared with those of HI. Infections caused by influenza A HI-subtype showed good homologous antibody responses in EIA but heterologous antibody responses to H3-subtype and influenza B HAs were frequently observed. Heterologous responses were clearly less frequent in patients with infections caused by the H3-subtype. Influenza B infections occasionally raised HA antibodies against influenza A H1-subtype but not to the H3-subtype. Interestingly, HI detected these heterologous responses at least as frequently as EIA. When whole viruses were used as antigens in EIA, subtype specificity was not observed and cross-reactions between influenza A and B virus antibodies were found. These observations suggest that, although EIA can show greater diagnostic efficacy over HI and CF methods, HI is still the serological method of choice in determining the causative subtype of influenza A virus infection.     

A common antigenic epitope in influenza A virus (H1, H2, H5, H6) hemagglutinin]

Avian influenza A viruses belonging to hemagglutinin (HA) subtypes H5 and H6 were studied in the infectivity neutralization test and radioimmunoprecipitation assay (RIPA) with monoclonal antibody MAb C179. This MAb recognizes a conformational antigenic epitope in the stem region of HA formed by two regions (amino acid positions 318-322 in HA1 subunit and 47-58 in HA2), conserved in all H1 and H2 influenza viruses. MAb C179 reacts with HA of H5 viruses in RIPA and neutralizes these strains as efficiently as H2 viruses. C179 precipitates H6 subtype HA but does not neutralize the infectivity of these viruses. Comparison of amino acid sequences of H2, H5, and H6 strains showed identical epitope recognized by MAb C179 in H5 and H6 HAs, which differs from epitopes of H1 and H2 by two amino acids in the HA2 subunit. Causes of disagreement between immunoprecipitation of H6 HA by MAb C179 and neutralization of this serosubtype by this MAb are discussed.     

Preparation of influenza virus subviral particles lacking the HA1 subunit of hemagglutinin: Unmasking of cross-reactive HA2 determinants

Acid treatment of influenza A and B virus preparations followed by addition of dithiothreitol (DTT) and centrifugation through a sucrose cushion removes the HA1 subunit of hemagglutinin from virus. Rabbit sera made against these subviral particles and untreated virus were tested in a radioimmune precipitation assay using [³⁵S]cysteine-labeled virus. Conditions of the assay permitted discrimination of discrete HA1- and HA2-specific antibody populations. It was found that (a) sera raised to intact influenza A virus preparations contained both HA1- and HA-2 specific antibodies, (b) sera made to subviral particles of influenza A virus contained HA2-specific antibody but had little or no detectable HA1-specific antibody. (c) the HA2-specific antibodies were partially cross-reactive with the HA2 of an influenza A virus of a different subtype, and (d) sera raised against two strains of untreated influenza B viruses contained antibodies which were cross-reactive with the HA2 as well as the NP of influenza A viruses.     

A mouse hepatotropic variant of influenza virus

A hepatotropic variant of avian influenza virus A/Turkey/England 63 (Hav 1, Nav 3) was selected by serial passages in mouse liver. Adaptation to this organ was established after 13 in vivo passages and was found to improve during further passages as shown by increasing rates of replication in livers of ICR mice. The mutant virus finally selected was stable and differed from the original virus mainly in lethality upon intraperitoneal injection in mice, in its ability to grow to high titers in livers of susceptible animals and in plaque morphology in chick embryo fibroblasts. No differences were detected in hemagglutination inhibition and neutralization by standard mouse antisera. Pathogenicity for the liver was independent of the route of inoculation, included other laboratory animals sensitive to influenza virus and could be inhibited by amantadine. Fatal hepatitis in 50 per cent of susceptible mice by the intraperitoneal route required from 10 to 20 EID50-. Pathological changes consisted of severe necrosis of liver parenchyma accompanied by release of F antigen into the serum and were apparently due to virus replication in hepatic cells as evidenced by immunofluorescence. The main implications of this animal model for studies on experimental hepatitis and on myxovirus-host interactions in an organ not usually associated with influenza are discussed.     

HA2-specific monoclonal antibodies as tools for differential recognition of influenza A virus antigenic subtypes

Antigenic reactivity of a set of monoclonal antibodies (MAb) raised against the HA2 subunit of hemagglutinin of H3 subtype was characterized in a rapid culture assay. MAbs FC12 and FE1, known to recognize the same antigenic site (IV), cross-reacted with influenza viruses of H3 and H4 subtypes, regardless of their host origin. No cross-reactivity was detected with other antigenic subtypes tested (H1-H13). The involvement of conserved residues D160, N168, and F171 in the differential recognition of H3 and H4 subtypes is proposed. In contrast, MAb IIF4 that recognizes antigenic site II exhibited a broader inter-subtype reactivity including subtypes H3, H4, H5, H8 and some viruses of H2, H6 and H13 subtypes. The ability of HA2-specific antibodies to differentially react with distinct antigenic subtypes can be utilized in development of diagnostics and in the influenza virus surveillance.     

Characterization of host cell binding variants of influenza virus by monoclonal antibodies

We have previously shown that a plaque-type mutant of influenza virus A/WSN has a growth advantage in MDBK cells because its hemagglutinin (HA) has a greater affinity for host cell receptors than does the HA of the parent virus. We show here that the mutant is also less sensitive than the parent to neutralization by antibodies to epitopes in at least two regions on the HA. WSN-specific monoclonal antibodies which had higher radioimmunoassay (RIA) titers against the parent than the mutant virus also had higher plaque inhibition (PI) and hemagglutination inhibition (HI) titers. In contrast, cross-reacting antibodies bound equally well to the parent and mutant viruses as judged by RIA but those which bound to the Cb region of the HA exhibited higher PI and HI titers against the parent virus. The results suggest that preferential neutralization of the parental virus by antibodies can contribute to the selective advantage of mutants which have increased affinity for cellular receptors.     

A 36 nucleotide deletion mutation in the coding region of the NS1 gene of an influenza A virus RNA segment 8 specifies a temperature-dependent host range phenotype

Previously a spontaneous 36 nucleotide deletion in the coding region of NS1 was detected in the NS gene of a reassortant virus (CR43-3) recovered from a dual infection by the influenza A/Ann Arbor/6/60 cold-adapted (ca) mutant and wild-type (wt) influenza A/Alaska/6/77 (H3N2). The hemagglutinin, neuraminidase and NS genes were derived from the wild type virus parent while the other 5 genes were derived from the ca parent. The CR43-3 reassortant virus exhibited: (i) a host range (hr) phenotype, i.e. the reassortant replicated efficiently in avian cells in tissue culture but failed to grow in mammalian (MDCK) cell culture and (ii) an attenuation (att) phenotype, i.e., the reassortant was restricted in replication in the upper and lower respiratory tract of ferrets and hamsters. Since the CR43-3 reassortant possessed 5 genes from the ca parent which are each known to contain one or more mutations, it was not possible to assign the hr and att phenotypes solely to the NS deletion mutant gene. In order to determine the phenotype(s) specified solely by the mutant NS gene, it was transferred into a reassortant virus (143-1) which derived its seven other genes from the homologous wild type A/Alaska/6/77 virus. The deletion mutant NS gene specified only a partial hr phenotype manifested by a reduction in plaque size in MDCK tissue, but not a reduction in plaque number. Thus, the complete hr manifested by the CR43-3 parent virus is specified by the mutant NS1 gene acting in concert with one or more genes derived from the ca virus. The clone 143-1 virus exhibited the ts phenotype and was restricted in plaque formation at 37 degrees C in MDCK cells, a level of temperature sensitivity previously shown with other ts mutants to correlate with significant restriction of viral replication in the lower respiratory tract of hamsters. However, the clone 143-1 virus grew almost as well as the wt virus in the upper and lower respiratory tracts of hamsters and chimpanzees and thus did not possess the att phenotype. The finding that the ts phenotype was not manifest in vivo in animals with a 37 degrees C core temperature indicates that the mutated NS1 gene specifies a host dependent ts phenotype with replication restricted in vitro (MDCK tissue culture) at 37 degrees C but not in vivo in the lungs of hamsters and chimpanzees. ts+ virus was readily recovered from infected hamsters and chimpanzees indicating that the ts phenotype specified by the 36-base deletion was not stable following replication in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antigenic glycopolypeptides HA1 and HA2 of influenza virus haemagglutinin. III. Reactivity with human convalescent sera.

The immune reactivity to both haemagglutinin glycopolypeptides HA1 and HA2 [prepared from bromelain-released haemagglutinin of influenza virus A/Dunedin/4/73 (H3N2)], was demonstrated by both gel double immundiffusion and radioimmunoassay in human convalescent sera obtained after natural infection during influenza epidemics in 1974/75 and 1976/77. In gel double immunodiffusion, the precipitin line(s) corresponding to glycopolypeptide HA1 were always more distinct than precipin line(s) corresponding to glycopolypeptide HA2. In radioimmunoassay, human convalescent sera revealed higher titres for binding of 125-I-labelled HA2 than for 125-I-labelled HA1. Characterization of human convalescent sera was completed by haemagglutination-inhibition test.     

Avian glycan-specific IgM monoclonal antibodies for the detection and quantitation of type A and B haemagglutinins in egg-derived influenza vaccines

Two IgM monoclonal antibodies (MAbs), Y6F5 and Y13F9, were selected during a screening of clones obtained immunising BALB/c mice with purified envelop proteins of the A/Sydney/5/97 (H3N2) IVR108 influenza strain. These MAbs recognised avian glycans on the haemagglutinin (HA) of the virus. This broad recognition allowed these MAbs to be used as enzyme-labelled secondary antibody reagents in a strain specific enzyme-linked immunosorbent assay (ELISA) in combination with a capture MAb that recognised and allowed the quantitation of the strain specific HA protein present in an egg-produced influenza vaccine. Advantage was taken of these MAbs to develop a universal ELISA in which the MAbs were used both as capture antibody and as enzyme-labelled secondary antibody to detect and quantify the HA protein of any egg-derived influenza vaccine. These avian-glycan specific IgM MAbs may prove to be particularly useful for determining the HA concentration in monovalent egg-derived pandemic influenza vaccines, in which the HA concentration may be lower than 5μg/ml. The HA detection limit in the ELISA assays developed in this study was 1.9μg/ml, as opposed to the 5μg/ml quantitation limit generally accepted for the standard single-radial-immunodiffusion (SRID) assay, the approved technique for quantifying HA content in influenza vaccines. These ELISAs can also be used to quantify influenza HA formulated with emulsion-based or mineral salt adjuvants that could interfere with HA measurement by the SRID assay.