Biological activity of monoclonal antibodies to operationally defined antigenic regions on the hemagglutinin molecule of A/Seal/Massachusetts/1 /80 (H7N7) influenza virus

Monoclonal antibodies to the hemagglutinin (HA) molecule of A/Seal/Mass/1/80 (H7N7) have been prepared and used to establish an operational antigenic map. Four nonoverlapping antigenic areas on the HA of seal influenza viruses were defined. Monoclonal antibodies belonging to two of the groups (III and IV) failed to inhibit hemagglutination of intact virus yet effectively neutralized viral infectivity. These findings could not be explained by differences in affinities and were interpreted in terms of functional differences between the assays. Antibodies that failed to inhibit hemagglutination may bind nearer to the hydrophobic end of the HA molecule and might not block the receptor binding site for erythrocytes. Antibodies to these areas may inhibit infectivity by interference with cell fusion or viral replication. The monoclonal antibodies that failed to inhibit hemagglutination of intact virus nevertheless did inhibit HA activity of purified isolated rosettes of hemagglutinin. The mechanism of inhibition is not resolved but may involve the access of a larger number of antibody molecules to the HA when it is in the form of rosettes compared to when it is located on the viral membrane. The reactivity of seal influenza virus and other H7 viruses from birds with the monoclonal antibodies show that some avian strains possess HA molecules that are closely related to those of seal virus and may therefore be related to viruses that were important in the evolution of this strain. Since HI tests do not necessarily detect all antibodies that neutralize viral infectivity, the question is raised as to whether HI assays alone can be used for determining the efficacy of all influenza virus vaccines.     

Identification of the binding sites to monoclonal antibodies on A/USSR/90/77 (H1N1) hemagglutinin and their involvement in antigenic drift in H1N1 influenza viruses

We have determined nucleotide sequences of the HA1 portion of the hemagglutinin (HA) gene of the parental A/USSR/90/70 (H1N1) virus and its eight variants selected in vitro with six monoclonal antibodies to study antigenic determinants. The HA1 gene of one of the variants (B-1-23) was cloned in bacteria and its nucleotide sequence was determined by the Maxam-Gilbert method. The nucleotide sequence of the variant was confirmed by the dideoxy chain termination method. The gene sequences of the other viruses were determined by the latter method. Three variants with reduced reactivity in HI test only with the selecting antibodies possessed one amino acid substitution. On the other hand, most other variants which had the changed reactivity to multiple antibodies in HI test possessed more than one substitution. Comparison of the amino acid sequences of the HA1 molecule, deduced from the nucleotide sequences, suggested that the monoclonal antibodies W18 and 264 reacted with epitopes located on the area involving amino acid residues 125C and 189-190, respectively, whereas, the antibodies 22 and 70 reacted with epitopes involving amino acid residues 129, 132, and 157. The epitope recognized by antibody 110 overlapped with that of W18, and the epitope recognized by antibody 385 was located on the area involving at least amino acid residues 129, 159, and 189, which overlapped with some of the above epitopes. The sequence analysis with B-1-23 variant selected with antibody 264 clearly showed that in A/USSR/77 viruses, a single substitution at amino acid residue 190 effectively changes the epitope and caused a significant antigenic variation detectable by postinfection ferret sera.     

A comparative study of monoclonal and monospecific antibodies in determining the immunodominant antigenic sites of influenza virus A (H3N2) hemagglutinin]

Comparative studies of monospecific (MSA) and monoclonal (MCA) antibodies showed MSA to detect three non-overlapping immunodominant sites on the surface of hemagglutinin (HA) molecule whereas MCA established more subtle differences in HA antigenic structure on the level of epitopes with different immunological significance. The activity of MSA and MCA differed in various tests. While MCA were more active in HI and EIA tests, MSA had a higher neutralizing activity, reducing the infectious virus titre by 5.0-7.5 Ig. Similar reduction of the virus biologic activity was observed only with two MCA whereas the other 20 MCA had a poor neutralizing effect (the virus titre reduction not more than by 2.5 Ig). The employment of MSA and MCA gives most complete information on antigenic restructuring of influenza virus HA at the site level and more subtle structures, epitopes, in the process of evolutionary variability.     

Immunological study of antigenic determinants in the isolated hemagglutinin of the influenza A virus (H1N1)

A preparation of isolated subunits of influenza A (H1N1) virus hemagglutinin was examined for antigenic properties by four serological tests: radioimmunoassay, radial immunodiffusion test, complement fixation test, and hemagglutination inhibition test with hyperimmune polyclonal serum to intact virion as well as with monospecific antibodies to individual antigenic determinants obtained by adsorption technique. In isolated hemagglutinin subunits, radioimmunoassay identified three main virus-specific antigenic sites identical to antigenic determinants in the hemagglutinin of the intact virion. No neuraminidase was found in the hemagglutinin preparation but increased serological activity of host cell antigens was observed.     

抗禽流感病毒H7亚型血凝素特异性单克隆抗体的研制

目的:研制禽流感病毒H7亚型血凝素特异性单克隆抗体(mAb)。方法:以H7亚型禽流感诊断抗原为免疫原免疫6～8周雌性BALB/c小鼠,末次加强免疫后取其脾细胞与骨髓瘤细胞Sp2/0-Ag-14进行融合。通过HA和HI试验筛选阳性克隆。应用HI试验和Western blot试验测定mAb的反应性和特异性。结果:共获得4株分泌抗AIVH7亚型HAmAbs的杂交瘤细胞株,分别命名为2E2、2A4、5F5、7G5。这些mAb的腹水HI效价在5×27～5×211之间,其中2E2属于IgM亚类,2A4属于IgG1亚类,5F5、7G5属于IgG2a亚类。Western blot分析结果显示,4株AIVH7亚型HAmAb能与AIVH7蛋白在Mr75000处反应,但不与新城疫病毒(NDV)蛋白发生反应,表明这些mAb能特异性识别AIVH7亚型HA。mAbHI反应性测定结果表明:4株mAb中,2E2、5F5、7G5只与H7亚型AIV发生特异性HI反应,而不与其他亚型AIV以及NDV、传染性支气管炎病毒(IBV)反应,显示出良好的特异性;而2A4除了与H7亚型AIV反应外,还与H15N8标准株发生低水平交叉反应。结论:这些mAb不仅为H7亚型AIV的HA结构分析提供了工具,而且为建立快速廉价的H7亚型禽流感诊断方法提供了核心试剂。     

Is bivalent binding of monoclonal antibodies to different antigenic areas on the hemagglutinin of influenza virus required for neutralization of viral infectivity?

Summary Biological activities of Fab fragments of monoclonal IgG antibodies to each of four nonoverlapping antigenic areas on the hemagglutinin molecule of A/seal/Massachusetts/1/80 (H7N7) influenza virus were examined. Fab fragments of the antibodies belonging to groups I and II neutralized viral infectivity. These Fab fragments inhibited hemagglutination of the virus and virus-induced hemolysis at pH 5.9. On the other hand, Fab fragments of groups III and IV antibodies showed neither neutralization nor hemolysis-inhibition activities, while intact IgG molecules of groups III and IV effectively neutralized viral infectivity and inhibited virus-induced hemolysis, as previously found. These IgG molecules scarcely or did not inhibit hemagglutination of the virus. Neutralization of viral infectivity, however, was observed when the virus was coated with Fab fragments of groups III and IV antibodies and then incubated with anti-Fab fragment antibodies. These findings suggest that bivalent binding of the IgG antibodies of groups III and IV is required for neutralization of viral infectivity through a proposed mechanism by which these antibodies interfere with a low pH-induced conformational change resulting in inhibition of the fusion step of the viral replication process (6).With 2 Figures     

Study of influenza virus hemagglutinin H3 avidity using monoclonal antibodies

The results of a comparative study of the features of antigenic determinants of avid and non-avid variants of influenza virus hemagglutinin H3 and of the influence of the mode of antigen presentation on the degree of its avidity are presented. The avidity of influenza virus hemagglutinin was shown to be determined by the capacity of individual antigenic determinants to interaction with antibodies. The antigenic determinants of avid hemagglutinin possessing a high functional activity in interaction with antibodies may have the spatial configuration which does not change in different modes of the antigen presentation. Isolation of hemagglutinin from virions of non-avid virus variants may lead to increased functional activity of individual antigenic determinants (and the antigen molecule as a whole) probably due to an increased degree of exposure and/or complementarity of the determinants for active centres of antibody.     

Topological mapping antigenic sites on the influenza A/PR/8/34 virus hemagglutinin using monoclonal antibodies

We have previously constructed an antigenic map of the PR8 virus hemagglutinin (HA) using monoclonal antibodies and PR8 virus antigenic variants. Four different antigenic sites were operationally defined: two predominantly “strain-specific” antigenic sites (designated Sa and Sb) and two predominantly “cross-reactive” antigenic sites (designated Ca and Cb). In the present study, we have conducted competitive binding assays using monoclonal antibodies to investigate the topologic relationship among these antigenic sites. It was found that several antibodies directed against the site Sa did not compete with the binding of a radiolabeled antibody to the site Cb and, vice versa, antibodies directed against site Cb did not compete with the binding of a radiolabeled antibody to site Sa. This proved unequivocally that at least part of the “strain-specific” site Sa and of the “cross-reactive” site Cb are topologically distinct. Various degrees of competition were observed, however, between other antibodies directed against operationally defined antigenic sites. This may indicate that parts of these sites are either structurally overlapping or in close proximity. Alternatively, it is possible that the binding of antibody to any of these sites allosterically affects the binding of antibody at topologically distant sites.     

Detection of a heterogeneous population of influenza virus type A/H1N1 with monoreceptor sera to hemagglutinin antigenic determinants

Studies of the antigenic composition of hemagglutinins of influenza H1N1 virus variants isolated in 1981 using monoreceptor antibody to individual antigenic determinants obtained by the selective adsorption method showed some variants to represent heterogeneous populations manifesting antigenic properties of different H1N1 viruses. Passages of the heterogeneous virus in the presence of antiinfluenza serum resulted in cloning a subpopulation with the antigenic properties of the hemagglutinin similar to that of H1N1 viruses which had circulated in the end of the first period of epidemic activity of H1N1 viruses. The method "from-plaque-to-plaque" passages in MDCK cells yielded from the heterogeneous population a homogeneous variant which by the hemagglutinin antigenic properties was similar to H1N1 viruses isolated in 1956-1957.     

Molecular and biological properties of a variant of avian influenza A/Seal/Massachusetts/1/80 (H7N7) virus that is pathogenic for mice

A/Seal/Mass/80 influenza virus has been shown to be closely related antigenically and genetically to avian influenza H7N7 viruses, however, the virus does not replicate efficiently in avian species but does replicate in most mammals, except mice (Hinshaw et al., Infect. Immun., 34, 351-361, 1981). In order to develop a model defining the molecular changes that occur during acquisition of virulence, the A/Seal/Mass/80 virus was adapted to growth in mouse lungs. The adaptation was accompanied by changes in a number of properties of the haemagglutinin as well as by changes in other genes of the virus as determined by RNA: RNA hybridization.     

Antigenic analysis of H4 influenza virus isolates using monoclonal antibodies to defined antigenic sites on the hemagglutinin of A/budgerigar/Hokkaido/1/77 strain

Summary Three non-overlapping antigenic sites were defined on the hemagglutinin of avian influenza virus A/budgerigar/Hokkaido/1/77 (H4N6) by competitive binding assay of monoclonal antibodies to the virus and comparative antigenic analysis of variants selected with monoclonal antibodies. Antigenic relationship among 25 H4 influenza viruses of different bird origin was examined by ELISA with the monoclonal antibodies to each of defined antigenic sites. Two of the three antigenic sites contained epitopes specific to the H4 influenza viruses of budgerigar and mynah origin, and the remaining site contained an epitope which was cross-reactive with almost all of the H4 influenza viruses.     

Determination of the number of nonoverlapping antigenic areas on Hong Kong (H3N2) influenza virus hemagglutinin with monoclonal antibodies and the selection of variants with potential epidemiological significance

Monoclonal antibodies provided evidence for at least three nonoverlapping antigenic areas on the hemagglutinin molecule of A/Mem/V71 (H3N2) influenza virus. This was established by determining the reactivity patterns of 30 different monoclonal antibodies in hemagglutination-inhibition tests and by the failure to select antigenic variants of influenza virus when monoclonal antibodies from two nonoverlapping areas were used in combination. Antigenic analysis showed that most of the variants selected with monoclonal antibodies could not be distinguished from the parental virus with heterogeneous sera, suggesting that they are probably epidemiologically irrelevant. One variant, however, could be distinguished from the parental virus with heterogeneous sera and this variant had a change in sequence at residue 144 of the HAl polypeptide, from glycine in the parent to aspartic acid in the variant. A similar amino acid change has been found in naturally occurring variants at this residue. These studies suggest that some amino acid substitutions are more important than others for producing viruses with epidemiological potential. Antigenic analysis of naturally occurring H3N2 strains with monoclonal antibodies showed that antigenic variation occurs in each nonoverlapping antigenic area of the HA molecule and established that two distinct variants cocirculated in 1968, Hong Kong/1/68 being distinguishable from Aichi/2/68 in at least two antigenic areas. It appears that there may have been two separate lineages of H3N2 viruses, Hong Kong/1/68 giving rise to variants in England and Aichi/2/68 to variants in the USA and Australia.     

Preparation and properties of monoclonal antibodies to influenza virus A/Krasnodar/101/59 (H2N2)

Somatic hybridization produced a set of 6 mouse hybridomas producing monoclonal antibodies of G isotype to influenza A/Krasnodar/101/59 (H2N2) virus. MCA were characterized by solid phase enzyme-immunoassay, hemagglutination-inhibition test, and indirect immunofluorescence technique. According to the results of radioimmunoprecipitation, all 6 hybridomas produced MCA to hemagglutinin of influenza A/Krasnodar/101/59 (H2N2) virus.     

Reassortants with equine 1 (H7N7) influenza virus hemagglutinin in an avian influenza virus genetic background are pathogenic in chickens

Reassortants possessing the hemagglutinin (HA) gene from A/Equine/London/1416/73 (H7N7) [Eq/Lond] and five or more genes from A/Chicken/Pennsylvania/1370/83 (H5N2) [Ck/Penn] were lethal in chickens. This result demonstrates that horses can maintain influenza viruses whose HAs are capable of promoting virulence. Thus, reassortment of equine and avian influenza virus genes could generate viruses that might be lethal in domestic poultry.     

Selection of antigenic variants of H1N1 influenza viruses by means of monoclonal antibodies

Six different monoclonal antibodies to influenza A/Brazil/11/78 virus hemagglutinin were used for selection of antigenic variants of H1N1 viruses: A/USSR/090/77 and A/black-headed gull/ Kaz . SSR/470/79. The group-specific monoclonal antibody completely neutralized the infective activity of the parental viruses (dilutions 1:5 to 1:640). Two antigenic variants of wild type viruses were obtained using cross-reactive antibody. A comparative study of the antigenic structure, biological properties, and peptide maps of the heavy chain of the original viruses, antigenic variants, and some epidemic H1N1 strains was carried out. The selected variants of A/black-headed gull/ Kaz . SSR/470/77 and A/USSR/090/79 viruses were shown to be similar to epidemic H1N1 strains isolated in 1953 and 1978.     

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.     

High-yield reassortant virus containing hemagglutinin and neuraminidase genes of pandemic influenza A/Moscowl/01/2009 (H1N1) virus

The crossing of influenza A/Moscow/01/2009 (H1N1) virus and reassortant strain X31 (H3N2) containing the genes of internal and non-structural proteins of A/Puerto Rico/8/34 (H1N1) strain gave rise to reassortant virus ReM8. The reassortant contained hemagglutinin (HA) and neuraminidase (NA) genes of pandemic 2009 influenza virus and 6 genes of high-yield A/Puerto Rico/8/34 (H1N1) strain. The reassortant ReM8 produced higher yields in the embryonated chicken eggs than the parent pandemic virus, as suggested by infectivity and HA activity titration as well as by ELISA and the measurement of HA protein content by scanning electrophoresis in polyacrylamide gel slabs. High immunogenicity of ReM8 reassortant was demonstrated by immune protection studies in mice. The reassortant virus ReM8 is suitable as a candidate strain for the production of inactivated and subunit influenza vaccines.