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Short Communication |
kováInstitute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic
Correspondence
E. Vareková
viruevar{at}savba.sk
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ABSTRACT |
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Conserved domains of influenza A viruses that could stimulate a protective immune response have, therefore, been searched for (Mitchell et al., 2003; Mozdzanowska et al., 2003; Spier, 2005; Tamura et al., 2005).
The HA2 glycopolypeptide (HA2 gp), the light chain of influenza haemagglutinin (HA), represents the conserved part of HA (Nobusawa et al., 1991). HA2 gp of influenza virus is responsible for the fusion of the virus and the endosomal membrane during the entry of the virus into the cell (Skehel & Wiley, 2000). Although the epitopes on the HA2 gp of the native virus are less accessible for interaction with antibodies than those on HA1 gp, anti-HA2 antibodies are induced during natural infection in humans (Styk et al., 1979), as well as in immune-naive individuals in an experimental mouse model (Kostolansk et al., 2002; Fislová et al., 2005). These antibodies do not prevent the haemagglutination mediated by the virus and do not neutralize the infectivity of the virus (Becht et al., 1984; Russ et al., 1987; Sánchez-Fauquier et al., 1987, 1991). However, some HA2-specific monoclonal antibodies (mAbs) are able to inhibit the fusion activity of influenza virus in various experimental systems (cell–cell fusion mediated by HA expressed on the cell surface, haemolysis mediated by infuenza virus, and virus–liposome fusion; Vareková et al., 2003a) and can reduce virus replication in vitro (Vareková et al., 2003b). These mAbs are highly cross-reactive between strains of influenza virus of the same subtype and, some of them, even within various subtypes (Russ et al., 1987; Tkáová et al., 1997; Vareková et al., 2002). Therefore, we studied the effect of the above HA2-specific mAbs, delineating four distinct antigenic sites on the HA2 gp of influenza virus A/Dunedin/4/73 (H3N2) (Russ et al., 1987; Vareková et al., 2003a), on the course of in vivo infection of mice. Two main parameters were evaluated: survival of mice for 18 days after infection and the level of infectious virus in the lungs.
Six-week-old BALB/c mice were infected under light ether narcosis 2 h after intravenous (i.v.) application of purified mAbs (200 µg in 200 µl per mouse) using an infectious dose of 1 LD50 influenza A virus (40 µl). Mice were infected with the homologous virus A/Dunedin/4/73 (H3N2), which was used for the immunization of mice during the preparation of hybridomas (Russ et al., 1987), or with the heterologous virus A/Mississippi/1/85 (H3N2), of the same subtype (H3), but evolutionarily distant from A/Dunedin/4/73 (H3N2) (viruses were obtained from the collection of the National Institute for Medical Research, London, UK). Both strains were adapted to mice by six to nine serial lung passages as described previously (Kostolansk et al., 2002). Hybridoma cells producing mAbs specific to the HA2 gp of influenza virus A/Dunedin/4/73 (H3N2) were prepared by standard procedures (Russ et al., 1987) and antibodies were purified as described by Ey et al. (1978).
For passive immunization, the particular anti-HA2 mAb (CF2-IgG1, IIF4-IgG1, CB8-IgG1 or FE1-IgG2a) was used. PBS and an irrelevant mAb (499-IgG1), specific to glycoprotein gB-2 of herpes simplex virus 2 (HSV-2) (Bystrická et al., 1997), were used as controls. For positive control of inhibition of infection, a polyclonal hyperimmune mouse serum specific to influenza A/Dunedin/4/73 (H3N2) (Kostolansk et al., 2002) was applied i.v. (diluted 1 : 4 or 1 : 64). All animal experiments were carried out according to EU standards. The significance of survival of mice was evaluated by using Fisher's exact test. The titre of infectious virus in mouse lungs was determined by a rapid-culture assay on Madin–Darby canine kidney cells (Tkáová et al., 1997). Mouse-lung homogenates made in PBS (1 ml) were sedimented and supernatants were used for titration in twofold dilutions (100 µl). Infectious virus was detected after 18 h incubation as described previously (Vareková et al., 2002). Infected cells were scored microscopically as distinct red staining. The titre of infectious virus was evaluated as the reciprocal value of the highest dilution of virus sample where the cells were still infected (log2 units).
Viral RNA (vRNA) in mouse lungs was detected by RT-PCR. Total RNA from lung-cell homogenate (100 µl) was extracted by using the RNA Insta-Pure system (Eurogentec). Reverse transcription and cDNA amplification were carried out as described previously (Vareková et al., 2006), using oligonucleotide primers specific for the influenza A nucleoprotein (forward, 5'-GTGAGGATGCAACAGCTGGTCTAAC-3'; reverse, 5'-TACCCCTCTTTTTCGAAGTCGTAC-3'; expected size of product, 509 bp). The detection limit of this RT-PCR was 1.12 pg specific cDNA.
Each experimental group of mice infected with the homologous influenza virus A/Dunedin/4/73 after passive immunization with anti-HA2 mAbs included eight individuals; the control group pretreated with PBS included 12 mice. In the control group, 50 % of mice died between days 7 and 13 post-infection (p.i.) (Fig. 1a), whereas in the group of mice passively immunized with anti-Dunedin polyclonal immune serum at two dilutions, all animals (four in each group) survived the infection (results not shown). mAbs CF2, specific to the N-terminal aa 1–38 of HA2 gp, and FE1, binding to aa 125–175 of HA2 gp (Vareková et al., 2003a), improved the survival of mice infected with 1 LD50 homologous virus to 100 % (statistically significant; P=0.0419). mAb IIF4 increased survival to 87 % (P=0.1577). However, no protection was observed after the application of mAb CB8, when all mice died before day 14 p.i. (Fig. 1a). This increased mortality was significant (P=0.0419) and reproducible.
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). Virus was extracted from lungs of two mice in each group at 2 day intervals up to day 10 p.i. It was detectable until day 8 p.i. in the control group receiving non-specific mAb 499. The application of mAb CF2 caused clearance of infectious virus from lungs 2 days earlier than in the control group; moreover, the last detectable titre on the day 6 p.i. (6.64±0 log2 units) was significantly lower than that in the control group (Fig. 2a). Similar to the control group, virus persisted until day 8 p.i. in mice that received mAb CB8, but the last detectable titre was higher by 2 log2 units than in the control. However, the maximal titre (13.14±0.71 log2 units) in this group was reached repeatedly on day 4 p.i. This was different from all other groups, in which the viral titre peaked on day 2 p.i. The i.v. application of polyclonal immune serum caused rapid reduction of virus titre and the most rapid clearance of virus from lungs on day 6 p.i. The strong effect of antiviral polyclonal serum was undoubtedly due to the presence of antibodies with haemagglutination-inhibition and virus-neutralization activities, which prevent binding of virus to the cell receptor (Knossow et al., 2002).
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). Simultaneously, the proportion of macroscopically damaged lung tissue (haemorrhagic and necrotic lesions) was scored as a percentage of the total lung tissue. The highest damage, observed on day 6 p.i. in all groups, was 70 % in the control group and in that after the application of mAb CB8, whilst in the group of mice that received mAb CF2, the maximal damage was only 30 %. The differences were also observed on day 10 p.i. Lesions were still visible in control mice and in the group treated with mAb CB8, but not in the lungs of mice treated with mAb CF2, which were without impairment (results not shown).
To study whether anti-HA2 mAbs have the potential to cross-protect mice against infection within a subtype, influenza virus A/Mississippi/1/85 (H3N2) was used for infection. mAb CF2, specific to the N terminus of HA2 gp, which inhibited the fusion mediated by HA most effectively and inhibited the replication of virus in vitro (Vareková et al., 2003a, b), was chosen. The same methods of infection and immunization were used as described above. The survival of mice, followed for 18 days p.i. in each group (14 individuals), increased after the application of mAb CF2 to 93 % (statistically significant; P=0.0329) from 50 % in the control group, which received irrelevant mAb 499. The improved survival of mice passively immunized with mAb CF2 was, similarly to the infection with A/Dunedin/4/73 virus (Fig. 2a), accompanied by clearance of infectious virus from lungs 2 days earlier than in the control group (Fig. 2b), as well as by earlier diminishing of vRNA: day 8 p.i. in mice immunized with mAb CF2 versus day 10 p.i. in the control group, not immunized with a specific mAb (Fig. 2b). The protection ability of antibody CF2 was also indicated by the significantly lower maximal damage of lungs visible on days 6–8 p.i. in mice infected with A/Mississippi/1/85 following mAb CF2 application: 35 versus 90 % in the control (results not shown).
From the above experiments, we conclude that HA2 gp is able to induce a specific antibody response with protective potential. It is known that HA2 gp is an antigen that elicits a specific, protective immune response via HA2-specific T-cell clones, which are cross-reactive with various strains of influenza A virus within subtype (Katz et al., 1985; Wabuke-Bunoti et al., 1984; Gould et al., 1987; Kuwano et al., 1988; Jackson et al., 1994; Mbawuike et al., 1994; Saikh et al., 1995; Gerhard, 2001). However, no protection mediated by antibodies specific exclusively to the HA2 gp has been described until now.
The three anti-HA2 antibodies (mAbs CF2, IIF4 and FE1) that conferred protection to infected mice were able to inhibit the in vitro fusion activity of influenza virus HA in previous experiments (Vareková et al., 2003a). On the other hand, mAb CB8 (without fusion-inhibition activity) did not protect mice against infection. The protective effect also correlated with the more rapid clearance of infectious virus from lungs, whereas mAb CB8 enhanced infection. It was proposed previously that the enhancement of infection may be mediated by binding of the Fc fragment of antibody, immunocomplexed with the virus, to the cell-surface Fc receptor (Tamura et al., 1994). We excluded this possibility in the case of antibody CB8, as mAbs CF2 and IIF4, of the same isotype as CB8, did not enhance infection. mAb CB8 recognizes the antigenic site between aa 38 and 112 of HA2 gp (Vareková et al., 2003a), which overlaps the epitope comprising aa 93–102 of HA2 gp. This epitope is recognized by cytotoxic T lymphocytes (Jackson et al., 1994; Saikh et al., 1995). We believe that the antibody recognition and binding of this peptide might interfere with the antiviral action of T lymphocytes. This interpretation is also supported by the fact that the viral titre in lungs peaked on day 4 p.i. in CB8-treated mice. To confirm our hypothesis, further studies are required.
mAb CF2, which inhibited the fusion activity of HA most effectively (Vareková et al., 2003a, b), improved the survival of mice even after infection with the heterologous virus A/Mississippi/1/85 (H3N2). We describe here, for the first time, intrasubtype cross-protection of mice against lethal infection mediated by a mAb specific to HA2 gp, particularly to its N terminus (aa 1–38), which is responsible for the insertion of HA2 gp into the target membrane in the process of fusion. The proposed mechanism of inhibition of fusion activity of HA mediated by mAb CF2 and, consequently, the inhibition of in vitro or in vivo virus replication is different from that described by Okuno et al. (1993, 1994) and Smirnov et al. (2000) for mAb C179, specific to the epitope comprising HA1 and HA2 gp, because we showed in a cleavage-protection assay that the binding of mAb CF2 to HA does not prevent pH 5-induced conformational change of the HA trimer (Vareková et al., 2003b).
As HA2-specific antibodies do not prevent attachment of virus to the cell surface, we suppose that they can influence the second stage of infection, after they are endocytosed into the cell together with the virus. Their binding to corresponding epitopes on HA2 gp blocks the fusion of the virus with the endosomal membrane and thus prevents the release of the viral genetic material into the cytoplasm. Such an inhibition effect would be revealed only when the binding of antibodies to the virus is sufficiently strong. This is dependent on their affinities and on the accessibility of corresponding epitopes for antibodies. The higher flexibility of the HA molecule at higher temperatures could lead to the higher binding capacity of anti-HA2 antibodies. Thus, we suppose that, under physiological conditions, where the temperature of the organism is about 37 °C, the inhibition potential of HA2-specific antibodies can be exerted. From that point of view, HA2-specific antibodies induced during natural infection could contribute to earlier recovery from influenza infection with new epidemic strains of a given subtype.
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ACKNOWLEDGEMENTS |
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ná and Jana Mesáro
ová for their excellent technical assistance. The authors are indebted to Dr G. Russ for providing hybridomas producing HA2-specific mAbs, to Dr M. Bystrická for providing mAb 499, specific to gB-2 of HSV-2, and to Dr K. Policová for reading the manuscript. This work was supported by the Scientific Grant Agency of the Ministry of Education of the Slovak Republic and the Slovak Academy of Sciences, grants no. 2/6077/6 and 2/4051/06, and by the Slovak Research and Development Agency under contract no. APVV-51-021605. |
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Bystrická, M., Petríková, M., Zat'oviová, M., Soláriková, L., Kostolansk, F., Mucha, V. & Russ, G. (1997). Monoclonal antibodies to the distinct antigenic sites on glycoproteins C and B and their protective abilities in herpes simplex virus infection. Acta Virol 41, 5–12.[Medline]
Cox, N. J., Tamblyn, S. E. & Tam, T. (2003). Influenza pandemic planning. Vaccine 21, 1801–1803.[CrossRef][Medline]
Ey, P. L., Prowse, S. J. & Jenkin, C. R. (1978). Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using Protein A-Sepharose. Immunochemistry 15, 429–436.[CrossRef][Medline]
Fislová, T., Sládková, T., Gocník, M., Mucha, V., Vareková, E. & Kostolansk, F. (2005). Differences in antibody responses of mice to intranasal or intraperitoneal immunization with influenza A virus and vaccination with subunit influenza vaccine. Acta Virol 49, 243–250.[Medline]
Gerhard, W. (2001). The role of the antibody response in influenza virus infection. Curr Top Microbiol Immunol 260, 171–190.[Medline]
Gould, K. G., Scotney, H., Townsend, A. R., Bastin, J. & Brownlee, G. G. (1987). Mouse H-2k-restricted cytotoxic T cells recognize antigenic determinants in both the HA1 and HA2 subunits of the influenza A/PR/8/34 hemagglutinin. J Exp Med 166, 693–701.
Jackson, D. C., Drummer, H. E. & Brown, L. E. (1994). Conserved determinants for CD4+ T cells within the light chain of the H3 hemagglutinin molecule of influenza virus. Virology 198, 613–623.[CrossRef][Medline]
Katz, J. M., Brown, L. E., Ffrench, R. A. & White, D. O. (1985). Murine helper T lymphocyte response to influenza virus: recognition of haemagglutinin by subtype-specific and cross-reactive T cell clones. Vaccine 3, 257–262.[CrossRef][Medline]
Knossow, M., Gaudier, M., Douglas, A., Barrere, B., Bizebard, T., Barbey, C., Gigant, B. & Skehel, J. J. (2002). Mechanism of neutralization of influenza virus infectivity by antibodies. Virology 302, 294–298.[CrossRef][Medline]
Kostolansk, F., Mucha, V., Slováková, R. & Vareková, E. (2002). Natural influenza A virus infection of mice elicits strong antibody response to HA2 glycopolypeptide. Acta Virol 46, 229–236.[Medline]
Kuwano, K., Scott, M., Young, J. F. & Ennis, F. A. (1988). HA2 subunit of influenza A H1 and H2 subtype induces a protective cross-reactive cytotoxic T lymphocyte response. J Immunol 140, 1264–1268.[Abstract]
Mbawuike, I. N., Dillion, S. B., Demuth, S. G., Jones, C. S., Cate, T. R. & Couch, R. B. (1994). Influenza A subtype cross-protection after immunization of outbred mice with a purified chimeric NS1/HA2 influenza virus protein. Vaccine 12, 1340–1348.[CrossRef][Medline]
Mitchell, J. A., Green, T. D., Bright, R. A. & Ross, T. M. (2003). Induction of heterosubtypic immunity to influenza A virus using a DNA vaccine expressing hemagglutinin-C3d fusion proteins. Vaccine 21, 902–914.[CrossRef][Medline]
Mozdzanowska, K., Feng, J., Eid, M., Kragol, G., Cudic, M., Otvos, L., Jr & Gerhard, W. (2003). Induction of influenza type A virus-specific resistance by immunization of mice with a synthetic multiple antigenic peptide vaccine that contains ectodomains of matrix protein 2. Vaccine 21, 2616–2626.[CrossRef][Medline]
Nobusawa, E., Aoyama, T., Kato, H., Suzuki, Y., Tateno, Y. & Nakajima, K. (1991). Comparison of complete amino acid sequences and receptor-binding properties among 13 serotypes of hemagglutinins of influenza A viruses. Virology 182, 475–485.[CrossRef][Medline]
Okuno, Y., Isegawa, Y., Sasao, F. & Ueda, S. (1993). A common neutralizing epitope conserved between the hemagglutinins of influenza A virus H1 and H2 strains. J Virol 67, 2552–2558.
Okuno, Y., Matsumoto, K., Isegawa, Y. & Ueda, S. (1994). Protection against the mouse-adapted A/FM/1/47 strain of influenza A virus in mice by a monoclonal antibody with cross-neutralizing activity among H1 and H2 strains. J Virol 68, 517–520.
Russ, G., Poláková, K., Kostolansk, F., Styk, B. & Vaníková, M. (1987). Monoclonal antibodies to glycopeptides HA1 and HA2 of influenza virus hemagglutinin. Acta Virol 31, 374–386.[Medline]
Saikh, K. U., Martin, J. D., Nishikawa, A. H. & Dillon, S. B. (1995). Influenza A virus-specific H2d restricted cross-reactive cytotoxic T lymphocyte epitope(s) detected in the hemagglutinin HA2 subunit of A/Udorn/72. Virology 214, 445–452.[CrossRef][Medline]
Sánchez-Fauquier, A., Villanueva, N. & Melero, J. A. (1987). Isolation of cross-reactive, subtype-specific monoclonal antibodies against influenza virus HA1 and HA2 hemagglutinin subunits. Arch Virol 97, 251–265.[CrossRef][Medline]
Sánchez-Fauquier, A., Guillen, M., Martin, J., Kendal, A. P. & Melero, J. A. (1991). Conservation of epitopes recognized by monoclonal antibodies against the separated subunits of influenza hemagglutinin among type A viruses of the same and different subtypes. Arch Virol 116, 285–292.[CrossRef][Medline]
Skehel, J. J. & Wiley, D. C. (2000). Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 69, 531–569.[CrossRef][Medline]
Smirnov, Y. A., Lipatov, A. S., Gitelman, A. K., Claas, E. C. & Osterhaus, A. D. (2000). Prevention and treatment of bronchopneumonia in mice caused by mouse-adapted variant of avian H5N2 influenza A virus using monoclonal antibody against conserved epitope in the HA stem. Arch Virol 145, 1733–1741.[CrossRef][Medline]
Spier, R. E. (2005). On the need for, and the delivery of, cross-protective vaccines. Vaccine 23, 2027–2029.[CrossRef][Medline]
Styk, B., Russ, G. & Poláková, K. (1979). Antigenic glycopolypeptides HA1 and HA2 of influenza virus haemagglutinin. III. Reactivity with human convalescent sera. Acta Virol 23, 1–8.[Medline]
Tamura, M., Webster, R. G. & Ennis, F. A. (1994). Subtype cross-reactive, infection-enhancing antibody responses to influenza A viruses. J Virol 68, 3499–3504.
Tamura, S., Tanimoto, T. & Kurat, T. (2005). Mechanism of broad cross-protection provided by influenza virus infection and their application to vaccines. Jpn J Infect Dis 58, 195–207.[Medline]
Tkáová, M., Vareková, E., Baker, I. C., Love, J. M. & Ziegler, T. (1997). Evaluation of monoclonal antibodies for subtyping of currently circulating human type A influenza viruses. J Clin Microbiol 35, 1196–1198.[Abstract]
Vareková, E., Cox, N. & Klimov, A. (2002). Evaluation of the subtype specificity of monoclonal antibodies raised against H1 and H3 subtypes of human influenza A virus hemagglutinins. J Clin Microbiol 40, 2220–2223.
Vareková, E., Mucha, V., Wharton, S. A. & Kostolansk, F. (2003a). Inhibition of fusion activity of influenza A haemagglutinin mediated by HA2-specific monoclonal antibodies. Arch Virol 148, 469–486.[Medline]
Vareková, E., Wharton, S. A., Mucha, V., Gocník, M. & Kostolansk, F. (2003b). A monoclonal antibody specific to the HA2 glycoprotein of influenza A virus hemagglutinin that inhibits its fusion activity reduces replication of the virus. Acta Virol 47, 229–236.[Medline]
Vareková, E., Blakoviová, H., Gocník, M., Mikas, J., Adamáková, J., Fislová, T., Kostolansk, F. & Mucha, V. (2006). Evaluation of clinical specimens for influenza A virus positivity using various diagnostic methods. Acta Virol 50, 181–186.[Medline]
Wabuke-Bunoti, M. A., Taku, A., Fan, D. P., Kent, S. & Webster, R. G. (1984). Cytolytic T lymphocyte and antibody responses to synthetic peptides of influenza virus hemagglutinin. J Immunol 133, 2194–2201.[Abstract]
Received 11 September 2006;
accepted 22 November 2006.
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