Hemagglutination Inhibition Activity Research Articles

Two distinct serum mannose-binding lectins function as beta inhibitors of influenza virus: identification of bovine serum beta inhibitor as conglutinin

Normal bovine and mouse sera contain a component, termed beta inhibitor, that inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H1 and H3 subtypes. We have previously shown these beta inhibitors to be mannose-binding lectins that apparently act by binding to carbohydrate on the viral hemagglutinin, blocking access of the receptor-binding site to receptors on host cells (E. M. Anders, C. A. Hartley, and D. C. Jackson, Proc. Natl. Acad. Sci. USA 87:4485-4489, 1990). For the H3-subtype virus A/Memphis/1/71 x A/Bel/42 (H3N1), sensitivity to beta inhibitors is determined by the oligosaccharide at residue 165 of the hemagglutinin, this glycosylation site being lost in a resistant mutant selected by growth in the presence of bovine serum. In the present study, we sequenced the hemagglutinin genes of additional bovine serum-resistant mutants derived from influenza viruses A/Philippines/2/82 (H3N2) and A/Brazil/11/78 (H1N1). The results confirm the importance of carbohydrate at residue 165 for inhibitor sensitivity of H3 viruses and implicate carbohydrate at residue 87 (94a in the H3 numbering system) as an important determinant in the sensitivity of H1-subtype viruses to the bovine inhibitor. Unlike the two H3 mutants, which had also gained resistance to hemagglutination inhibition by mouse serum, the H1 bovine serum-resistant mutant remained sensitive to the mouse beta inhibitor, suggesting that inhibition by the two types of sera is mediated by distinct mannose-binding lectins. In support of this hypothesis, the beta inhibitors in bovine and mouse sera were shown to differ in their pattern of inhibition by monosaccharides and in their sensitivity to 2-mercaptoethanol. In these and other properties, the bovine inhibitor closely resembled conglutinin, a Ca(2+)-dependent N-acetylglucosamine- and mannose-binding lectin present in bovine serum but absent from the serum of other species. Furthermore, polyclonal and monoclonal anticonglutinin antibodies abrogated the hemagglutination-inhibiting activity of bovine serum. Direct binding of conglutinin to the parent viruses and reduced binding to their respective mutants were confirmed by radioimmunoassay.

Serological study using α- N-acetylgalactosaminidase from Acremonium sp.

α- N-Acetylgalactosaminidase, produced by Acremonium sp. destroyed blood group A active substance. This enzyme converted human erythrocytes from group A to O(H). The enzyme was ascertained to liberate N-acetylgalactosamine from group A erythrocytes membranes accompanying a decrease in hemagglutination inhibition activity of A erythrocyte-anti-A serum by the erythrocyte membranes. Glycoproteins and glycolipids extracted from A erythrocytes reduced their blood group A activity after the enzyme treatment. The enzyme also worked on Forssman hapten glycolipid.

Protection from pseudorabies virus challenge in mice by a combination of purified gII, gIII and gVI antigens.

The antigens gII, gIII and gVI were purified from the lysates of the pseudorabies virus (PRV) -infected HmLu-1 cells using Sepharose 4 B coupled with MAbs against these antigens. Mice immunized with either gII, gIII, gVI antigen or a mixture of them were challenged intraperitoneally with 8.5 x 10(3) plaque forming units of PRV. All the mice immunized with 1.5 and 4.5 micrograms of the mixture and 4.5 micrograms of the gIII antigen survived. The sera of mice immunized with the mixture had virus neutralizing activity which was independent of complement, hemagglutination inhibition activity, and recognized major (93 kilodaltons) and minor (129, 74, 68 and 50 kilodaltons) PRV proteins under reducing conditions in western blotting. The serological activity levels in the lower survival group were not different from those in the complete survival. These results indicate that the mixture of each glycoprotein is more effective for eliciting of protective immunities in mice and that serological activity do not always correlate with protection.

Monoclonal antibody characterization of Jamestown Canyon (California serogroup) virus topotypes isolated in Canada.

Jamestown Canyon (JC) virus of the California (CAL) serogroup has been isolated in 12 American states and 6 Canadian provinces. A study was undertaken to produce monoclonal antibodies (MAbs) to JC virus and to use these MAbs to assay for possible heterogeneity among naturally occurring JC topotypes in Canada. MAbs were produced to the prototype strain of JC virus using BALB/c mice. Twenty-seven secreting MAbs were obtained and three of these MAbs were propagated and studied. All three MAbs, M1 (IgG1), M2 (IgG2b), and M3 (IgG2a), were reactive by immunofluorescent antibody assay against JC-infected vero cells and by ELISA against JC antigen. MAb M2 reacted with all members of the Melao complex, MAb M1 reacted only with Keystone virus, while MAb M3 exhibited no reactivity with other CAL serogroup viruses. Only MAb M3 possessed neutralization and hemagglutination inhibition activities against JC virus. The MAbs were also tested by ELISA and for neutralizing activity against 13 JC topotypes isolated in 5 provinces from Newfoundland to Saskatchewan. ELISA confirmed closer identity of the Canadian topotypes to JC as opposed to the closely related South River virus. The MAbs verified all Canadian topotypes to be JC virus but revealed different patterns of reactivity between these topotypes and prototype JC virus.

Natural infection of dogs by influenza C virus

To date, only one seroepidemiological survey, carried out in Japan, gave a strong indication that dogs may be naturally infected by the influenza C virus, long considered to be exclusively human. In the present work, 134 serum samples were collected during the winter of 1988/89 from dogs aged 6 months to 16 years in northern France. Samples were tested for the presence of antibodies to influenza C virus by both haemagglutination inhibition (HI) and ELISA. Using antibody absorption by staphylococcal protein A, we demonstrated the specificity of the results. In 62% of cases, the results were identical using the two methods. Significant HI activity was found in 32% of the 134 tested sera and titres ranged from 20 to 320. Of the sera tested, 42% were positive by ELISA and titres ranged from 500 to 8,000. The discordant results are discussed. The population tested was divided into five age groups: less than 4 years, 4 to 6 years, 7 to 9 years, 10 to 11 years and greater than 12 years. The distribution of antibodies in the tested canine population, in contrast to that of humans, did not show a significant degree of association with age.

Production and characterization of monoclonal antibodies to psittacine beak and feather disease virus.

Monoclonal antibodies specific for the virus that causes psittacine beak and feather disease (PBFD) were produced by fusing spleen cells from mice immunized with purified concentrated PBFD virus with mouse myeloma cell line Sp2/0. The resulting hybridomas were tested for reactivity against whole purified virus by an enzyme-linked immunosorbent assay (ELISA) system. Four clones, designated 15H8, 8E3, 11G12, and 2C3, were subcloned by limiting dilution. Isotyping indicated that clone 15H8 was secreting IgG, whereas the remaining clones secreted IgM. The secreted immunoglobulins were characterized by reactivity against purified PBFD virus using immunoblotting procedures, by immunohistochemical staining of virus-induced lesions in infected tissues, and by inhibition of PBFD virus agglutination of cockatoo erythrocytes. Antibodies secreted by clones 15H8 and 8E3 had the strongest activity against purified whole virus. Only immunoglobulin secreted by the clone 15H8 could be used to detect viral antigen in infected tissues. None of the monoclonal antibodies had hemagglutination-inhibition activity.

Influenza C virus infection in France

Little is known of the epidemiology of influenza C virus infections in western Europe and of the exact role of this agent in acute viral respiratory infections. Several tests may be used for detecting antibodies against this agent but the significance of their respective results is not clear. A total of 301 samples of serum was collected from persons aged from 4 months to 88 years living in France in 1988. The samples were tested for the presence of antibodies to influenza C virus by haemagglutination-inhibition (HI) tests and ELISA. The specificity of the results was checked by immunoblotting and by antibody absorption with staphylococcal protein A. Significant HI activity was found in 61% of the 301 samples tested, titres ranging from 20-320; 70% were positive by ELISA with titres ranging from 500 to 32,000. The population tested was divided into four age groups: 0-15 years; 16-30 years; 31-50 years and 51-88 years. The highest rates for positive samples were found in the 16-30 year group (76 and 79% by HI tests and ELISA respectively) as well as significant HI and ELISA geometric mean titres. Positive samples were less common in young children (46 and 50% by HI tests and ELISA respectively) and in the oldest group (44 and 54% respectively). The 31-50 years age group formed an intermediate class. The high prevalence of antibody as well as the significant titres indicate intense circulation of influenza C virus, especially among young adults.

Isolation of cDNA clones coding for humoral lectin of silkworm, Bombyx mori, larvae

In the hemolymph of the silkworm, Bombyx mori, lectin with hemagglutinating activity against sheep red blood cells increases at larval-larval ecdysis and at spinning stage ( Suzuki and Natori, 1983) and is induced by infection with cytoplasmic polyhedrosis virus. A Bombyx lectin polypeptide with molecular weight approx 280K is responsible for hemagglutinating activity, since antiserum raised against this polypeptide inhibited hemagglutinating activity. The site of synthesis of Bombyx lectin was determined by primary tissue cultures of fat body and hemocytes. A hemagglutinating activity assay demonstrated that hemocyte is responsible for the release of hemagglutinin into the culture medium. Isolation of cDNA clones coding for Bombyx lectin was carried out on the cDNA library prepared in an expression vector α gt11 starting with poly(A) + RNA from spinning larval hemocytes. As a result of immunoscreening, several positive clones were obtained, and the cDNA clones were characterized.

Immunogenicity of experimental live attenuated Japanese encephalitis vaccine viruses and comparison with wild-type strains using monoclonal and polyclonal antibodies

A total of 105 hybridomas secreting anti-Japanese encephalitis (JE) virus monoclonal antibodies (mAbs) were generated from six fusions against four strains of JE virus: wild-type strains SA14 and G8924 and live attenuated vaccines SA14-5-3 and SA14-14-2 (PDK-9). Most of the mAbs (87%) elicited haemagglutination inhibition activity while only a minority (24%) elicited neutralization. None of the mAbs prepared against SA14-5-3, parent of SA14-14-2, elicited neutralization while the only mAbs prepared against SA14-14-2 that elicited neutralization recognized flavivirus cross-reactive epitopes. In comparison, mAbs raised against wild-type strains showed that a spectrum of epitopes with different specificities, including JE type-specific epitopes, elicited neutralizing activity. Two mAbs, prepared against SA14-5-3 virus, were found to be vaccine-specific and five, prepared against strains SA14 and G8924, were wild-type-specific.

Antigenic and Functional Characterization Of Rinderpest Virus Envelope Proteins Using Monoclonal Antibodies

A total of 24 monoclonal antibodies (MAbs) against the haemagglutinin (H) and the fusion protein (F) of rinderpest virus (RPV) were used to characterize their antigenic structure and biological properties, and to analyse natural variation in the envelope proteins of morbilliviruses. The anti-H and anti-F MAbs defined seven and three distinct antigenic sites, respectively. The MAbs to six sites on H were able to neutralize the infectivity of RPV. The addition of guinea-pig complement or anti-mouse immunoglobulin increased the virus-neutralizing antibody titre of most of the anti-H MAbs, including those lacking neutralizing activity. One of the antigenic sites on H was conserved among morbilliviruses and the MAbs to this site had haemagglutination inhibition activity against measles virus (MV). The remaining sites were specific for RPV and varied antigenically between strains of RPV. The anti-F MAbs lacked neutralizing activity, but two of the five MAbs did show activity in the presence of complement or anti-mouse immunoglobulin. On the whole, the antigenic sites on F were conserved in some strains of MV, but not in canine distemper virus. All of the sites on the surface proteins were sensitive to SDS and, although those on F were not affected by 2-mercaptoethanol, five of the seven sites on H were destroyed by it. These results suggest that the epitopes on the envelope proteins are conformation-dependent.

Production, characterization and protective effect of monoclonal antibodies to Haemophilus paragallinarum serotype A

Monoclonal antibodies (mAbs) to Haemophilus paragallinarum serotype A were obtained by fusion of murine myeloma cells (P3-X63-Ag8-U1) and spleen cells from BALB/c mice immunized with whole cells of strain 221. Enzyme linked immunosorbent assay with whole cells was used to show that the monoclonal antibodies are specific for serotype A of H. paragallinarum. Four monoclonal antibodies indicated hemagglutination-inhibition (HAI) activity against serotype A; their titers were 10 4–10 5. By western blotting, two of these monoclonal antibodies reacted with a protein of molecular weight 39 000. Chickens treated with mAbs possessing HAI activity survived without clinical signs of infection. No challenge strain was isolated from these chickens, indicating that four mAbs with HAI activity suppressed growth of the challenge strain in the nasal cavity, whereas mAbs without HAI activity showed no passive protective effect. These results demonstrated that HI antibodies contributed to protection, and strongly suggest that hemagglutinin (HA) antigen, especially the epitopes which were recognized by these mAbs are important for protective immunity in chickens.

Prevalence of antibody to influenza C virus among pigs in Hyogo Prefecture, Japan

The prevalence of influenza C virus among pigs in Hyogo Prefecture, Japan, was investigated by serological techniques. Out of 240 sera tested, 45 (19%) showed haemagglutination inhibition (HI) to influenza C virus. Pig sera with high HI titres also scored high in neutralization tests and ELISAs. When fractionated by sucrose density gradient ultracentrifugation, the HI/ELISA reactivities corresponded to antibodies of the IgM and IgG classes. Radioimmunoprecipitation tests revealed that some, but not all, of the pig sera with high HI activities precipitated HEF glycoprotein of influenza C virus. These results suggested that the HI activities of pig sera in Hyogo Prefecture were due to the presence of antibody to influenza C virus. Sera with IgM class antibody to influenza C virus were found throughout the year. However, the question of whether or not pigs serve as a natural reservoir for human influenza C virus still remains to be solved.

VP4 monotype specificities among porcine rotavirus strains of the same VP4 serotype

The porcine rotavirus OSU strain was used to produce monoclonal antibodies (MAbs) directed against the outer capsid protein VP4. From two separate fusions, eight MAbs that inhibited hemagglutination activity of the OSU strain were selected. All MAbs immunoprecipitated both the OSU VP4 protein derived from a lysate of infected MA104 cells and the OSU VP4 protein expressed in Sf9 cells by a recombinant baculovirus. By immunoprecipitation of in vitro-translated OSU gene 4 transcripts of different length, the eight MAbs were found to be specific for the VP8 subunit of VP4. All MAbs neutralized the OSU strain but failed to neutralize human, bovine, and simian rotavirus strains. Antiserum to the expressed OSU VP4 protein was used to study the distribution of VP4 antigenicity among porcine rotaviruses. At least two distinct specificities were identified among 14 rotavirus strains that had been previously assigned to four distinct VP7 serotypes. Five groups of monotype specificities of the VP4 protein were identified by the eight anti-VP4 MAbs among 11 porcine strains that share the same VP4 serotype.

Measurement of IgM responses to a subunit influenza a vaccine by sucrose-gradient centrifugation and membrane-filtration enzyme immunoassays

IgM responses to a deoxycholate-split influenza vaccine containing the surface antigens of the H3N2 virus A/Philippines/2/82 were studied in five volunteers, three of whom were seronegative by haemagglutination inhibition (HI) tests. Responses were measured by a sucrose-gradient centrifugation technique, in which IgM-specific HI activity was computed as a proportion of total IgM and IgG-specific HI activity, and by a membrane filtration-enzyme immunoassay (MF-EIA). Responses weeks, and the IgM induced was 5–40% of total HI-specific activity. The response was also observed in the presence of low levels of pre-existing antibody. Levels of IgM antibody, when measured by MF-EIA, could be easily detected in two of the volunteers, while those of two others were very low and there was no response in a fifth. No biphasic virus-specific response to vaccination, involving first IgM and then IgG, could be measured by either technique. From these studies, the sucrose-gradient fractionation technique appears to be the more sensitive procedure.

The localization and function of the neutralizing protein, VP4, in human rotavirus.

The binding sites of a monoclonal antibody (mAb) to human rotavirus VP4 (K-1532) on the virion surface were examined with electron microscopy and the Markham photorotation method. The mAb, which had both hemagglutination inhibiting and neutralizing activities, appeared to bind to the outer margin of double-shelled particles of human rotavirus, especially to the parts overlaying the tubelike channels, which seemed to be the "cover lid" of the tubelike channels that extruded radially from the core region. This binding point may be a putative fusion site of the virus (Mackow et al. 1988) as well as a viral nucleus component ejection gate (Suzuki et al. 1986). Rotavirus particles treated with the mAb were shown to be capable of binding to MA104 cells by ultra-thin section examination. These observations suggest that neutralization via VP4 is related to virus uncoating but not to attachment of rotavirus.

Infection enhancement of influenza A H1 subtype viruses in macrophage‐like P388D1 cells by cross‐reactive antibodies

The contribution of cross-reactive hemagglutination inhibition (HI) antibodies to infection enhancement of influenza A H1 subtype NWS virus and two antigenic drift strains was investigated in a macrophage-like cell line P388D1. When P388D1 cells, previously treated with neuraminidase (NA) to remove the viral receptors, were infected with NWS virus exposed to rabbit antiviral immunoglobulin (IgG) showing various levels of cross-HI titers, virus yields were enhanced in the presence of a subneutralizing antibody, depending on their cross-HI titers. By flow cytometric analysis using a fluorescein isothiocyanate (FITC)-labeled NWS virus, the efficiency of attachment of virus-rabbit IgG complexes to Fc receptors on NA-treated cells showed close correlation with its cross-HI titer. These data suggest that cross-reactive HI antibodies could contribute to infection enhancement through the formation of potent infectious immune complexes with drift strains to mediate virus infection via Fc receptor uptake. Two monoclonal antibodies (mAB) in mouse IgG subclasses IgG1 and IgG2a showing strain-specific or cross-reactive HI activity were tested for their infection enhancement characteristics. A strain-specific mAB enhanced infection of homologous NWS virus, but not that of two other drift strains in either antibody dilution. In contrast, a cross-reactive mAB caused infection enhancement of all three virus strains in the presence of the subneutralizing antibody. This indicates that cross-reactivity, but not the IgG subclass, acts as an enhancing factor to this phenomenon. The antibody, with the same specificity as cross-reactive mAB, was detected semiquantitatively by competitive enzyme-linked immunosorbent assay (ELISA) with results almost consistent with cross-HI titers of polyclonal rabbit antiviral IgGs. These data suggest that the antibody detected by this assay might be one of the potent antibodies governing cross-HI activity as a whole antibody and causing infection enhancement of drift strains.

Feline herpesvirus type 1 glycoproteins eliciting virus neutralizing and hemagglutination-inhibiting antibodies

Monoclonal antibodies (MoAbs) to feline herpesvirus type 1 (FHV-1) were produced to identify the virus-specified immunogenic proteins. When antigens recognized by the MoAbs were investigated by immunoblot and immunoprecipitation assays using FHV-1-infected cell lysate, four groups of immunogenic proteins were identified. MoAbs directed against 60 kDa, 113 kDa, and 143 kDa/108 kDa glycoproteins had virus neutralizing activities, but those against 170 kDa protein did not. Furthermore, MoAbs to the 60 kDa glycoprotein also showed hemagglutination-inhibition activity, indicating that this glycoprotein might be the hemagglutinin.

Analysis of Properties of Monoclonal Antibodies to Transmissible Gastroenteritis Virus using TO‐163 Strain

Nineteen monoclonal antibodies (MAbs), reactive in enzyme-linked immunosorbent assay (ELISA), with porcine transmissible gastroenteritis (TGE) virus TO-163 were obtained. Of these MAbs, 5 showed neutralizing (NT) activity (x 3,200 to 25,600) against TO-163. One of the MAbs which had NT activity showed hemagglutination inhibition activity (x 5,120) too. 14 hybridomas of polypeptide specificity against TO-163 strain were developed from which 11, 2, and 1 were specific for protein E2, N, and E1, respectively. Immunofluorescence staining patterns in TGE virus-infected cells reacted with MAbs were divided into three groups (types I, II and III). The fluorescence staining of E2 specific MAbs having NT activity were limited to the perinuclear area. All MAbs having NT activity showed the same fluorescence staining pattern.

Canine herpesvirus 47kD and mutated 41kD glycoproteins are responsible for hemagglutination.

Monoclonal antibodies (MoAbs) were used to identify the hemagglutinin of canine herpesvirus (CHV). The inhibition of viral hemagglutination (HA) activity was observed with MoAbs against 41 kD glycoprotein, while no hemagglutination-inhibition (HI) activity was observed with those against 145/112 kD and 80 kD glycoproteins, suggesting that the 41 kD glycoprotein is the hemagglutinin of plaque-selected virus of CHV YP11 strain used as immunogen for MoAb production. All of the HI MoAbs also showed HI activities against HA antigens which were prepared from cells infected with other CHV strains, namely, F-205 V and Glasgow CHV2 reference strains, eight Japanese isolates, and the original YP11 strain. However, on immunoblotting analysis, a 47 kD protein band was detected in these strains by the HI MoAbs. These data suggest that the 47 kD glycoprotein is the common molecule of the hemagglutinin among CHV strains and the plaque-selected virus of YP11 strain appears to be a mutant whose molecular weight of the hemagglutinin changed into 41 kD.

Protection of mice from lethal influenza by adoptive transfer of non-neutralizing haemagglutination-inhibiting IgG obtained from the lungs of infected animals treated with defective interfering virus.

The lungs of C3H/HeMg mice infected with a lethal dose of the influenza virus A/WSN (H1N1) contained antibody to the viral neuraminidase glycoprotein but not to the haemagglutinin (HA). When coinoculated with a life-saving amount of defective interfering (DI) WSN, lungs were found to contain both anti-neuraminidase and haemagglutination-inhibiting (HI) antibodies which peaked at 3 and 5 days post-infection (p.i.), respectively. Mice coinoculated with WSN and beta-propiolactone-inactivated DI WSN had only anti-neuraminidase antibody in the lungs. The HI activity was unusual in that there was no detectable neutralizing activity. The HI activity has been affinity-purified by adsorption to and elution from WSN HA and consisted entirely of IgG, comprising isotypes IgG1, IgG2a and IgG2b. The antibody was specific for WSN HA, of low activity and had disappeared from the lungs by 20 days p.i. Transfer of affinity-purified HA-specific, non-neutralizing antibody to mice 24 h before infection with WSN alone protected 60% from death. We conclude that the antibody contributes significantly to the life-saving activity of DI WSN virus and that the ability to stimulate such antibody represents a novel property of this DI virus.