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.     

Antigenic glycopolypeptides HA1 and HA2 of influenza virus haemagglutinin. II. Reactivity with rabbit sera against intact virus and purified undissociated haemagglutinin.

Rabbit sera produced against either intact virus or purified undissociated haemagglutinin were examined for reactivity with highly purified haemagglutinin glycopolypeptides. Sensitive radioimmunoassay for 125I-labelled glycopolypeptides revealed antibody reactive with either glycopolypeptide HA1, or glycopolypeptide HA2. Antibodies against the carbohydrate moiety were responsible only for a part of the binding activity. Under the conditions employed, the binding activity for glycopolypeptide HA2 was much stronger than for glycopolypeptide HA1. Competition assays suggested that immune reactivities were due to distinct antibody populations (i.e. with a specifity for glycopolypeptide HA1 and glycopolypeptide HA2, respectively). The immune reactivity to both haemagglutinin constituents, glycopolypeptides HA1 and HA2, was also shown by gel double diffusion. The precipitin line(s) corresponding to glycopolypeptide HA1 was (were) usually more distinct than precipitin line(s) corresponding to glycopolypeptide HA2. The glycopolypeptides HA1 and HA2 showed the reaction of nonidentity in immunodiffusion analysis.     

Monoclonal antibodies to glycopolypeptides HA1 and HA2 of influenza virus haemagglutinin

Anti-haemagglutinin monoclonal antibodies were prepared and their HA1 or HA2 specificity was determined by solid phase radioimmunoassay (RIA) using purified viral haemagglutinin (HA) and haemagglutinin glycopolypeptides HA1 and HA2, by radioimmunoprecipitation followed with SDS-PAGE, by immunoblotting and by inhibition of virus-induced haemagglutination. The capacity of these methods to estimate HA1 or HA2 specificity of anti-HA monoclonal antibodies (MoAb) was compared. HA1 specificity was demonstrated for all hybridomas originating from lymphocytes of mice immunized with complete influenza virus, except IIF4 hybridoma which was HA2-specific. All hybridomas obtained with lymphocytes from mice immunized with HA glycopolypeptide HA2 were HA2-specific. Anti-HA2 MoAb neither inhibit haemagglutination induced by the virus or by HA subunits nor neutralized viral infectivity, either alone or in mixture. As expected, all anti-HA1 MoAb were H3 subtype-specific, showing usually good reactivity only with viruses close to the virus strain used for immunization. Two anti-HA1 MoAb (IVA1 and IVG6) showed unusual cross-reactivity within the H3 subtype. All anti-HA2 MoAb were broadly cross-reactive within the H3 subtype. Moreover, a half of them showed high cross-reactivity with influenza viruses of the H7 HA subtype. But the same antibodies did not react with HA of H1, H2 and H8 subtypes.     

Antigenic analysis of recent H1N1 influenza viruses with monoclonal antibodies.

Antigenic analysis of recently isolated H1 influenza viruses was performed using haemagglutination inhibition (HI) assay with monoclonal antibodies to the haemagglutinin (HA) subunit. Tests using monoclonal antibodies against the HA of the A/England/333/80 (H1N1) and A/Yamagata/120/86 (H1N1) viruses revealed that the major antigenic drift occurred in 1985 or 1986 and A/Dunedin/6/83-like virus became a major strain after 1986.     

H-2 and viral haemagglutinin expression by influenza-infected cells; the proteins are close but do not cocap.

The proximity of H-2K and D antigens and influenza virus haemagglutinin (HA) molecules on the surface of infected target cells was assessed by a topographical study using monoclonal antibodies to H-2 and to HA. The effect of pretreatment of fixed, infected cells with excess of one monoclonal antibody on the subsequent binding of a second radiolabelled antibody was measured. Using CBA mouse B lymphoblasts which were paraformaldehyde fixed 5 hr postinfection with influenza virus (A/USSR/90/77), pretreatment with monoclonal antibody 30/3 to H-2Kk and Dk partially blocked (Approximately equal to 37%) the binding of one radiolabelled monoclonal anti-HA antibody (264/2). A different monoclonal IgG (W18/1) directed to the same HA molecule was not blocked by similar pretreatment of cells with the anti-H-2 antibody. Interaction of monoclonal antibodies with their sites is highly specific, and mutual blocking of two antibodies requires very closely located sites even if the antibodies are directed to the same molecule. We therefore have evidence for proximity of H-2 and HA molecules; however, we were unable to demonstrate cocapping of H-2K and D antigens with influenza HA.     

Radioimmunoassay of influenza A virus haemagglutinin. II. Antigenic cross-reactions of influenza A (H3 subtype) viruses as determined by radioimmunoassay and haemagglutination inhibition tests

Individual rabbits differed greatly in their antibody response to the "strain-specific" and "cross-reactive" antigenic determinants on the haemagglutinin (HA) subunit of influenza virus recombinant MRC11 (H3N2) and influenza virus Dunedin (H3N2), after immunization with whole virus or bromelain-released haemagglutinin (B-HA). Consequently, diverse cross-reactions between htese viruses and A/Hong Kong/68 virus were found in the haemagglutination inhibition (HI) test as well as in homologous radioimmunoassay (125I-B-HA from MRC11:anti MRC11 serum, and 125I-B-HA from Dunedin: anti Dunedin serum) when sera from different animals were employed. Radioimmunoassay (RIA), over and above to the HI test, was able to differentiate clearly the respective HAs also with antisera reacting to the same HI titre with both corresponding influenza virus strains. Thus it appeared that antigenic differences could be identified with higher sensitivity by homologous RIA than by the HI test and that multiple antigenic determinants were reactive on the 125I-B-HA in the RIA procedure employed. MRC11 and A/HK/68 viruses were also compared by heterologous RIA (125I-B-HA from MRC11: anti A/HK/68 serum). It was found that preferentially antigenic determinants with a high degree of cross-reactivity could be studied in the heterologous system.     

Identification of a linear epitope on the haemagglutinin protein of pandemic A/H1N1 2009 influenza virus using monoclonal antibodies

A novel influenza A/H1N1 virus, emerging from Mexico and the United States in the spring of 2009, caused the pandemic human infection of 2009-2010. The haemagglutinin (HA) glycoprotein is the major surface antigen of influenza A virus and plays an important role in viral infection. In this study, three hybridoma cell lines secreting specific monoclonal antibodies (Mabs) against the HA protein of pandemic influenza A/H1N1 2009 virus were generated with the recombinant plasmid pCAGGS-HA as an immunogen. Using Pepscan analysis, the binding sites of these Mabs were identified in a linear region of the HA protein. Further, refined mapping was conducted using truncated peptides expressed as GST-fusion proteins in E. coli. We found that the ²⁵⁰VPRYA²⁵⁴ motif was the minimal determinant of the linear epitope that could be recognized by the Mabs. Alignment with sequences from the databases showed that the amino acid residues of this epitope were highly conserved among all pandemic A/H1N1 2009 viruses as well as the classical swine H1N1 viruses isolated to date. These results provide additional insights into the antigenic structure of the HA protein and virus-antibody interactions at the amino acid level, which may assist in the development of specific diagnostic methods for influenza viruses.     

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.     

Membrane fusion by surrogate receptor-bound influenza haemagglutinin.

In influenza infections, haemagglutinin (HA) mediates the fusion of virus and cellular membranes at endosomal pH, between pH 5 and 6. In vitro, when reconstituted into virosomes, efficient fusion requires target membranes to contain sialic acid receptors or receptor analogues. In the experiments reported, lipid-associated anti-HA monoclonal Fab' fragments were used as surrogate receptors to investigate the fusion capacity of receptor-bound HA compared with unbound HA. The conclusions are drawn, in contrast to those from previous studies, that bound HA can mediate fusion and that fusion mainly involves bound HA when the liposome targets are densely packed with surrogate receptors.     

Analysis of antigenic determinants on internal and external proteins of influenza virus and identification of antigenic subpopulations of virions in recent field isolates using monoclonal antibodies and immunogold labelling

Summary An electron microscopic immunogold labelling technique employing monoclonal antibodies has been applied to the antigenic analysis of influenza A and B viruses. Reassortant influenza A H3N2 viruses containing haemagglutinin molecules from viruses isolated between 1968 and 1982 were analysed with a panel of monoclonal antibodies raised against viruses which appeared over the same period. The immunogold labelling technique clearly demonstrated the antigenic drift in the haemagglutinin molecule that occurred between 1968 and 1982. When the technique was applied to the examination of viruses from a more geographically restricted influenza epidemic in a semi-closed community, antigenic variants were found. Furthermore the technique enabled the identification of distinct antigenic variant subpopulations within a single clinical isolate. Analysis of the HA of MDCK cell or egg grown virus by this procedure provided data to support the hypothesis that the host cell exerts selective pressure on subpopulations of virus resulting in the emergence of antigenic variants.With 4 Figures     

In vitro synthesis, glycosylation, and membrane insertion of influenza virus haemagglutinin.

The synthesis of influenza virus haemagglutinin (HA) has been studied, using cell-free systems primed with cellular RNA from chick embryo fibroblast (CEF) cells infected with influenza virus (A/Japan/305/57-Bellamy/42(H₂N₁)). In L-cell and wheat germ cell-free systems, a precursor to the HA protein of apparent molecular weight 63,000 was identified by immunoprecipitation and by comparative peptide mapping of this product and the 75,000-dalton glycopolypeptide HA precursor synthesised during infection of CEF cells. The 63,000-dalton precursor was converted to a product of apparent molecular weight 75,000 by addition of dog pancreas membrane vesicles during in vitro translation. The 75,000-dalton protein made in the presence of membranes is protected from trypsin digestion and binds to lectin-Sepharose. These, and further results, suggest that HA is extruded into membrane vesicles and glycosylated during synthesis.     

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     

Quantitative relationships between an influenza virus and neutralizing antibody

In this quantitative study of the interaction of influenza virus with neutralizing antibody we have determined the maximum number of antibody molecules which can bind to the haemagglutinin (HA) of native influenza A/FPV/Rostock/34 (H7N1) particles in aqueous suspension and the minimum number which is required to cause neutralization. Using radiolabelled immunoglobulins approximately one IgG molecule, whether of monoclonal or polyclonal origin, binds per HA spike under conditions of antibody saturation. In the same manner, we have determined that when infectivity is neutralized by 63% (1/e) about 70 molecules of monoclonal IgGs HC2 and HC10 were bound per virus particle and this is supported by independent evidence from electron microscopy. However, the kinetics of neutralization were single-hit or at most, under critical conditions of low temperature (4 degrees) and minimal neutralizing concentrations of antibody, two-hit. This apparent conflict is reconciled by a hypothesis which proposes that neutralization occurs only when antibody binds to certain "neutralization relevant" HA spikes which are in the minority. It is suggested that these only differ from the majority of "neutralization irrelevant" HA spikes by their transmembrane interaction with the core of the virion.     

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.     

Action of rimantadine on the structure of influenza A virus haemagglutinin

Rimantadine prevents the conformational changes of influenza virus haemagglutinin (HA) caused by acid pH and the acquisition of sensitivity to trypsin, protects the haemolytic activity from inactivation and prevents the morphological changes of HA spikes on the virus surface.     

Regulation of antibody production by an antigen-specific EBV-transformed B-lymphoblastoid cell line: effect of high-dose antigen and antigen-pulsed T cells.

A B-cell line (C1B2) secreting monoclonal IgG antibody to influenza virus haemagglutinin (HA3) was obtained by Epstein-Barr virus (EBV) transformation of human tonsillar B cells activated in vitro to influenza A/X31. Antibody secretion by C1B2 was completely inhibited by purified HA3 at concentrations above 100 ng/ml. By contrast, high doses of HA3 had no effect on EBV-transformed B-cell lines making antibody of unrelated specificity. Inhibition of specific antibody secretion by HA3 continued for at least 3 days after the removal of soluble antigen, but this could be partially reversed by treatment with pronase, suggesting that inhibition was due to 'effector cell' blockade by binding of antigen to surface Ig receptors. T cells pulsed with high doses of antigen also suppressed antibody secretion by C1B2, but this effect was probably due to a tolerogenic signal delivered to the B cell by HA3 complexed to the T-cell membrane rather than suppression by antigen-induced Ts, or carryover of free antigen. These experiments demonstrate two independent mechanisms of high-dose tolerance in vitro, and show that monoclonal B-lymphoblastoid lines of known specificity can be used to study regulation of specific antibody production at the level of the B cell.     

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.     

Exploration of the emergence of the Victoria lineage of influenza B virus

Summary. The Victoria lineage represented by B/Victoria/2/87 is one of the two major distinctive haemagglutinin (HA) lineages of influenza B virus, and its recent re-emergence has aroused great concerns. However, it remains unknown when, where, and how this HA lineage emerged in the world. In this study, the HA1 domain of the HA gene of fourteen influenza B viruses isolated in China in 1972–1984 was sequenced. The sequences were phylogenetically analyzed with the HA1 sequences of 41 other important influenza B isolates. The results unveiled some earlier footprints of the Victoria lineage in China, and the epidemic history of the Victoria lineage could be traced back from the year 1985 to 1975. Moreover, phylogenetic analysis, the history of China, and the epidemiology of influenza B virus indicated that the Victoria lineage possibly emerged in China in the 1970s through gradual evolution from a minor lineage.     

Antigenic glycopolypeptides HA1 and HA2 of influenza virus haemagglutinin. I. Gel filtration in 6 M guanidine hydrochloride.

Highly purified glycopolypeptides HA1 and HA2 were separated from bromelain-released haemagglutinin of influenza virus A/Dunedin/4/73 (H3N2) by gel filtration in 6 M guanidine hydrochloride under reducing conditions. The purity of both glycopolypeptides was proved by extensive studies. Despite the lack of C-terminal end, the isolated HA2 glycopolypeptide displayed some hydrophobic properties.     

Antigenic structure of the hemagglutinin of influenza virus B/Hong Kong/8/73 as determined from gene sequence analysis of variants selected with monoclonal antibodies

Antigenic variation among influenza B viruses is different from that of influenza A in several ways. Antigenic shift has not been observed, distinct antigenic variants of influenza B cocirculate, and antigenically similar viruses have been isolated many years apart. To study the mechanism of antigenic drift in influenza B viruses, monoclonal antibodies were used to select antigenic variants of B/Hong Kong/8/73 virus hemagglutinin (HA). Analyses of the nucleotide sequences of the HA gene of B/Hong Kong/8/73 and the eight variants identified specific regions of the influenza B HA molecule involved in antigenicity, and enabled antigenic mapping data to be correlated with the structure of the protein. The altered amino acids in the variants, when compared to the HA of A/Aichi/2/68, were found in two of the four antigenic regions previously identified for type A viruses. In addition, four of the eight variants showed multiple nucleotide changes some of which gave rise to double amino acid changes. In addition, in the present study monoclonal antibodies which belong to the same antigenic group recognize amino acid changes in regions corresponding to antigenic sites A and B of the H3 HA. These results are in contrast to those obtained with HA variants of A/Memphis/1/71 virus. In the influenza A studies only single amino acid changes were found and these correlated well with the three-dimensional structure as determined by D. C. Wiley, I. A. Wilson, and J. J. Skehel, (1981, Nature (London) 289, 366-373); monoclonal antibodies which recognized one region did not recognize any of the other antigenic sites. Our results suggest that although the basic three-dimensional structure of the influenza B HA may be similar to that of A viruses, the B HA molecule may be folded in a more compact manner so that antigenic sites A and B are in closer proximity to each other than in the H3 structure.