Vojtech Mucha

Antibodies induced by the HA2 glycopolypeptide of influenza virus haemagglutinin improve recovery from influenza A virus infection.

The haemagglutinin (HA) of influenza A virus consists of two glycopolypeptides designated HA1 and HA2. Antibodies recognizing HA1 inhibit virus haemagglutination, neutralize virus infectivity and provide good protection against infection, but do not cross-react with the HA of other subtypes. Little is known regarding the biological activities of antibodies against HA2. To study the role of antibodies directed against HA2 during influenza virus infection, two vaccinia virus recombinants (rVVs) were used expressing chimeric molecules of HA, in which HA1 and HA2 were derived from different HA subtypes. The KG-11 recombinant expressed HA1 from A/PR/8/34 (H1N1) virus and HA2 from A/NT/60 (H3N2) virus, whilst KG-12 recombinant expressed HA1 from A/NT/60 virus and HA2 from A/PR/8/34 virus. Immunization of BALB/c mice with rVV expressing HA2 of the HA subtype homologous to the challenge virus [A/PR/8/34 (H1N1) or A/Mississippi/1/85 (H3N2)] did not prevent virus infection, but nevertheless resulted in an increase in mice survival and faster elimination of virus from the lungs. Passive immunization with antibodies purified from mice immunized with rVVs confirmed that antibodies against HA2 were responsible for the described effect on virus infection. Based on the facts that HA2 is a rather conserved part of the HA and that antibodies against HA2, as shown here, may moderate virus infection, future vaccine design should deal with the problem of how to increase the HA2 antibody response.

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.

Preparation of monoclonal antibodies for the diagnosis of influenza A infection using different immunization protocols

Four immunization protocols were used to obtain cross-reactive influenza type A-specific monoclonal antibodies: (1) repeated administration of purified influenza virus, (2) immunization with bromelain-treated viral particles free of HA and NA, (3) sequential immunization with two strains of different subtypes, and (4) immunization with bromelain-treated particles following tolerization of mice to surface glycoproteins by cyclophosphamide. The fourth approach was shown to be the most effective since a high proportion of hybridomas producing cross-reactive influenza virus type A-specific MAbs were obtained. MAbs of type A specificity were immunochemically characterized and examined for their ability to detect virus in clinical specimens. It was demonstrated that two pairs of the newly prepared MAbs provided excellent reagents for viral detection in clinical specimens using time-resolved fluoroimmunoassay.

Monoclonal antibodies demonstrate accessible HA2 epitopes in minor subpopulation of native influenza virus haemagglutinin molecules

SummaryHaemagglutinin (HA) was detected on the surface of influenza virus infected cell with monoclonal antibodies (MAbs) against both HA glycopolypeptides, HA1 and HA2, however, the reactivity of HA2-specific MAbs was remarkably lower as compared with HA1-specific MAbs. Quantitative analysis with two MAbs, IB8 (anti-HA1) and IIF4 (anti-HA2) respectively, revealed that HA2 epitope was reachable for antibody only in minor subpopulation of the HA representing approximately 7% of all molecules (spikes). The basis of the HA heterogeneity is discussed.

Epitope specificity of anti-HA2 antibodies induced in humans during influenza infection.

Please cite this paper as: Stanekováet al. (2012) Epitope specificity of anti‐HA2 antibodies induced in humans during influenza infection. Influenza and Other Respiratory Viruses 6(6), 389–395.

The Carboxyterminal Domains of Human IFN-α2 and IFN-α8 Are Antigenically Homologous

The antigenic properties of human hybrid IFN-α8(60)/α1(92)/α 8 were compared with those of human IFN alpha 1 and IFN-α 2 using monoclonal antibodies (mAb). Hybrid IFN demonstrated a significantly closer antigenic relationship to the subtype α2 than to the subtype α1. In particular, high homology was observed between antigenic structures located in the C-terminal domains (93–166) of IFN-α8 and IFN-α2, whereas the corresponding N-terminal receptor-binding domains (30–53) showed distinct antigenic characteristics. The 100% homology between IFN-α8 and IFN-α2 in the region 114–131 (helix D) indicated the role of this region in formation of the common antigenic structure. In IFN-α 8/1/8, this shared antigenic structure was important for antiviral activity and exhibited immunodominant properties, consistent with functional and antigenic properties of the corresponding structure in IFN-α2. Based on this antigenic homology, we suggest that IFN-α8 and IFN-α2 are evolutionarily more closely related to each other tha...

Radioiodination of human interferon-α2 interferes with binding of C-terminal specific antibodies

Radioimmunoassays based on reactivity between a monoclonal antibody (mAb) and human 125I-interferon (IFN)-alpha2 are frequently exploited in interferon research. In general, epitopes of antibodies specific for human IFN-alpha2 are located on the two immunodominant structures formed in the N- and C-terminal domains, respectively. We found that labelling of IFN-alpha2 with Na(125)I by the chloramine-T method did not affect the binding of antibodies recognising the N-terminal region 30-53. In contrast, radioiodination of IFN was associated with a dramatic decrease in IFN reactivity with mAbs specific for the C-terminus (residues approximately 120-145 approximately ). We suggest that steric hindrance araising from the incorporation of 125I into the tyrosine residues at positions 123, 130 and 136 may be responsible for the change in immunoreactivity. The adverse effect of radioiodination of IFN-alpha2 on the binding potency of C-terminal specific mAbs must be taken into consideration in experiments based on the interaction of such antibodies (i.e. NK2) with the labelled antigen.

Comparison of 44/107L one-step immunocapture enzyme-immunoassay and time-resolved fluoroimmunoassay for influenza A diagnosis

One-step immunocapture enzyme-immunoassay (EIA) was compared with time-resolved fluoroimmunoassay (TR-FIA) for rapid diagnosis of influenza A infection by antigen detection. The high-affinity monoclonal antibodies (MAbs) recognising two independent epitopes on the conservative nucleoprotein were used for capture (MAb 44) and detection (MAb 107L) of antigen by both assays. The detection limit for purified recombinant influenza A virus nucleoprotein was approximately 10 pg by EIA and 5 pg by TR-FIA. The performance of the methods was evaluated by testing 43 known positive and 50 negative clinical specimens (nasopharyngeal washes and aspirates). The sensitivity and specificity was 93% and 92% for EIA and 100% and 98% for TR-FIA, respectively, in comparison to the reference A3/A1 TR-FIA. The relationship of 44/107L immunoassays has been evaluated: in comparison to 44/107L TR-FIA (100%), EIA confirmed 93% of positive and 94% of negative samples. In conclusion, the capture-detector pair of MAbs 44 and 107L can be used for the sensitive detection of influenza A viral antigen in clinical samples by both immunocapture methods. Despite the slightly lower accuracy of the EIA, widespread availability and economy of the EIA methodology makes it an advantageous alternative for the laboratory diagnosis of influenza A virus infections.