Inhibition of influenza virus haemolytic and haemagglutination activities by monoclonal antibodies to haemagglutinin glycopolypeptides HA1 and HA2.

Acid treatment of influenza virus enhanced haemagglutination inhibiting (HI) activity of some anti-HA1 monoclonal antibodies (MoAbs). These changes in the HI-activity could be either due to alteration in the mutual orientation of MoAb (e.g. IC8, IB8) binding epitope to receptor site or to an increase in the number of epitopes accessible to the corresponding MoAbs (e.g. IVA1). HI test with pH 5-virus revealed similar (although not identical) antigenic differences among related virus strains as the HI test with pH 7-virus. Anti-HA2 MoAbs were negative in the HI test with both pH 5- and pH 7-virus. Anti-HA1 MoAbs showed a HI activity with pH 5-treated BHA similar to that with pH 5-treated virus. Surprisingly one out of eight anti-HA2 MoAbs (IIF4) exhibited a relatively high HI activity to pH 5-BHA-mediated haemagglutination. Virus-induced red blood cell haemolysis was efficiently inhibited with several anti-HA1 MoAbs (e.g. IC8, IB8, and IIB4) while other anti-HA1 antibodies, including IVA1 and IVG6 with preferential reactivity with pH 5-treated antigens in RIA, gave no inhibition. As a rule, anti-HA2 MoAbs were poor haemolysis inhibitors.

Changes in the influenza virus haemagglutinin at acid pH detected by monoclonal antibodies to glycopolypeptides HA1 and HA2.

Monoclonal antibodies (Mabs) specific to the HA1 and HA2 subunits of the influenza virus haemagglutinin (HA) were used to show that changes in the antigenicity of the HA molecule at acid pH involve both HA subunits. In solid phase RIA (intact virus adsorbed) the acid-induced change was detected in the form of greatly increased binding of anti-HA 1 Mabs (IVA 1 and IVG 6) and anti-HA2 Mab (IIF 4). This increased binding could be most probably explained by alterations in accessibility of epitopes to the corresponding Mabs. Other Mabs examined (including 7 anti-HA2 Mabs specific to 3 independent antigenic sites) had either similar reactivities with both untreated and pH 5-treated virus or slightly but significantly increased binding to pH 5-treated virus. No effect of pH 5 treatment on antibody binding was observed with purified BHA in solid phase RIA. Nevertheless a similar pH 5-induced conformational change in the isolated BHA (like in intact viral HA in solid phase RIA) was detected in competitive binding assay carried out in liquid phase.

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.

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. IV. Immunogenic properties of separated haemagglutinin glycopolypeptides.

Highly purified haemagglutinin glycopolypeptides HA1 and HA2 were effective in eliciting an antibody response. HA2 had a markedly greater immunogenic potential than HA1. In gel double immunodiffusion, sera from rabbits immunized with HA2 produced more distinct precipitin lines than sera obtained by immunization with HA1. Both kinds of rabbit sera gave precipitation with homologous antigen and with bromelain-released and purified haemagglutinin (B-HA). In radioimmunoassay, sera from rabbits immunized with HA2 revealed considerable titres for 125I-labelled HA2 binding and reacted preferentially with 125I-labelled HA2. In general, sera from rabbits immunized with HA1 exhibited low titres for 125I-labelled HA1 binding: usually they reacted also with 125I-labelled B-HA and 125I-labelled HA2. Only rabbits injected with a few doses of HA1 at short intervals revealed preferential binding for 125I-labelled HA1. Glycopolypeptides HA1 and HA2 failed to induce haemagglutination-inhibiting and virus neutralizing antibodies in rabbits.

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.

Radioimmunoassay of influenza A virus haemagglutinin. I. Preparation and properties of radioactive 125I-labelled bromelain-released haemagglutinin.

Haemagglutinin released from influenza A virus recombinant MRC11 [antigenically identical to the strain A/Port Chalmers/1/73 (H3N2)] by bromelain treatment and purified by rate zonal centrifugation (further on B-HA) was examined for eventual contamination by neuraminidase. According to specific enzymatic activities corresponding to MRC11 virus and B-HA alone respectively, B-HA contained less than 0.1% of enzymatically active neuraminidase orginally present in the virus. Gel double diffusion tests, specifities of rabbit antisera induced by B-HA, as well as radioimmunoprecipitation experiments demonstrated that B-HA was devoid of any antigenically active neuraminidase. Precipitation of 125I-labelled B-HA with antisera to influenza virus recombinants with N2 neuraminidase has been evidently caused by antibodies to host antigenic determinaant(s) present in these sera. With respect to purity as well as radioimmunoprecipitation properties, B-HA is quite suitable for radioimmunoassay experiments.

[Preparation of porcine IgA and its detection using the fluorescent antibody technic]. / Príprava prasacieho IgA a jeho detekcia metódou fluorescencných protilatok

The preparation of immunoglobulin A (IgA) from porcine colostrum, intestinal content and serum is described. The best results were achieved with colostrum, from which an antigen of satisfactory purity was prepared by purification on Sephadex G-200, on DEAE cellulose and subsequent filtration on Sephadex G-200. The serum to this antigen raised in rabbits was adsorbed to an immunoadsorbent from porcine serum (PS) or porcine IgG. The adsorbtion of the serum against secretory IgA (SIgA) to PS removed its undesirable heterologous and nonspecific reactivity. The anti-SIgA serum adsorbed in this way still reacted with IgA from porcine serum. In the direct and indirect immunofluorescent staining we detected the main antigenic determinants of the SIgA molecule, i. e. the heavy chains and the secretory component.