Universal anti-influenza vaccines based on viral HA2 and M2e antigens.

Aquatic birds are the main reservoir of influenza A viruses (IAVs). These viruses can infect humans repeatedly and cause acute respiratory disease with potential of spread in the form of epidemics. In addition, avian influenza viruses that overcome the interspecies barrier and adapt to humans can cause a world-wide pandemic with severe consequences to human health. Therefore, scientists are focused on the development of a "universal" vaccine with a broad protective efficacy, i.e. against different subtypes of influenza A viruses and not only against the currently co-circulating human epidemic strains. Nowadays, several new vaccine design strategies have been described. Most of them utilize the conserved stem part of influenza surface glycoprotein hemagglutinin (HA) or the ectodomain of M2 (M2e) protein with proton-channel activity. A comparison of the efficacy of novel vaccines and their protective mechanisms against influenza infection is discussed in this review and should be considered for the construction of the most effective broadly protective vaccine with minimal side effects. This is the essential goal in influenza virus research today, especially when the infection with new human coronavirus SARS-CoV-2 can interfere with the course of influenza virus infection. Keywords: influenza A virus; HA2 gp; M2 ectodomain; universal vaccine.

Biological properties of influenza A virus mutants with amino acid substitutions in the HA2 glycoprotein of the HA1/HA2 interaction region.

Influenza A viruses (IAVs) enter into cells by receptor-dependent endocytosis. Subsequently, conformational changes of haemagglutinin are triggered by low environmental pH and the N terminus of HA2 glycoprotein (gp) is inserted into the endosomal membrane, resulting in fusion pore formation and genomic vRNA release into the cytoplasm. However, the pH optimum of membrane fusion is host- and virus-specific and can have an impact on virus pathogenicity. We prepared mutants of neurotropic IAV A/WSN/33 (H1N1) with aa substitutions in HA2 gp at the site of HA1/HA2 interaction, namely T642H (HA2 numbering position 64, H1 numbering position HA407; referred to as mutant '64'), V662H ('66') (HA409); and a double mutant ('D') with two aa substitutions (T642H, V662H). These substitutions were hypothesized to influence the pH optimum of fusion. The pH optimum of fusion activity was measured by a luciferase assay and biological properties of viruses were monitored. The in vitro and in vivo replication ability and pathogenicity of mutants were comparable (64) or lower (66, D) than those of the wild-type virus. However, the HA2 mutation V662H and double mutation T642H, V662H shifted the fusion pH maximum to lower values (ranging from 5.1 to 5.3) compared to pH from 5.4 to 5.6 for the wild-type and 64 mutant. The decreased replication ability and pathogenicity of 66 and D mutants was accompanied by higher titres in late intervals post-infection in lungs, and viral RNA in brains compared to wild-type virus-infected mice. These results have implications for understanding the pathogenicity of influenza viruses.

Different mechanisms of the protection against influenza A infection mediated by broadly reactive HA2-specific antibodies.

Influenza A viruses (IAVs) cause yearly repeating infections in humans. The current vaccination approach is based on the production of virus-neutralizing antibodies. Virus-neutralizing antibodies, however, are closely strain-specific due to the IAV variability. Therefore, antibodies produced during the previous influenza season do not provide sufficient protection against new infection, and, hence, annual revaccination is needed. The utilization of the influenza conserved stem domain of hemagglutinin (HA), the HA2 gp, led to a new vaccine design based on cross-reactive cellular and especially humoral immune responses represented by HA2-specific antibodies. The HA2-specific antibodies exhibit cross-reactivity with HA2 gp within one subtype or even among subtypes and play a role in protective immunity against influenza infection. There are several elimination mechanisms of viral replication mediated by HA2-specific antibodies. After recognition of the epitope, they prevent the conformational rearrangement of HA or the insertion of the fusion protein into the endosomal membrane and, consequently, the fusion pore formation. In this case, no release of viral genetic information into the target cell is enabled and virus cannot replicate. The HA2-specific antibodies are involved in the elimination of pathogen via the Fc fragment by activation of the cytotoxic mechanisms of innate immunity as are the antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), or complement-dependent cytotoxicity (CDC), resulting in virus elimination and earlier recovery of the host from the infection. Though the protective effect of HA2-specific antibodies on the course of IAV infection was shown, few cases of worsening of IAV infection mediated by HA2-specific antibodies have been described. The identification of antigenic epitopes on HA2 gp that induce antibodies with such deteriorating effect on influenza infection can help to eliminate the unsuitable epitopes of HA2 gp as immunogens during the design of heteroprotective vaccine against influenza and can remove the side effects linked with the observations mentioned above. Keywords: influenza A virus; HA2 stem domain of hemagglutinin; immunization strategies; HA2-specific antibodies.

Avian influenza A virus adaptation to the equine host and identification of host-specific markers.

Avian influenza A viruses (IAVs) are able to overcome the interspecies barrier and adapt to the new non-avian host. The process of adaptation requires the adaptive changes of IAV genome resulting in amino acid substitutions. The aim of this work was the description of amino acid substitutions in avian influenza A viruses (IAVs) occurring during their adaptation to equine host. Today, viruses of the equine influenza H3N8 subtype, first isolated in 1963, represent a single genetic lineage of IAV causing a respiratory disease in horses. We compared the amino acid sequences of the conserved proteins PB2, PB1, PA, NP, M1, M2, NS1 and NEP of equine influenza H3N8 subtype IAV with sequences of avian viruses, both available in the NCBI's Influenza Virus Resource Database. The amino acid substitutions persisting in equine IAV isolates and occurring in avian IAV at f both hosts.

The role of fusion activity of influenza A viruses in their biological properties.

Influenza A viruses (IAVs) cause acute respiratory infections of humans, which are repeated yearly. Human IAV infections are associated with significant morbidity and mortality and therefore they represent a serious health problem. All human IAV strains are originally derived from avian IAVs, which, after their adaptation to humans, can spread in the human population and cause pandemics with more or less severe course of the disease. Presently, however, the potential of avian IAV to infect humans and to cause the disease cannot be predicted. Many studies are therefore focused on factors influencing the virulence and pathogenicity of IAV viruses in a given host. The virus-host interaction starts by virus attachment via the envelope glycoprotein hemagglutinin (HA) to the receptors on the cell surface. In addition to receptor binding, HA mediates also the fusion of viral and endosomal membranes, which follows the virus endocytosis. The fusion potential of HA trimer, primed by proteolytic cleavage, is activated by low pH in endosomes, resulting in HA refolding into the fusion-active form. The HA conformation change is predetermined by its 3-D structure, is pH-dependent, irreversible and strain-specific. The process of fusion activation of IAV hemagglutinin is crucial for virus entry into the cell and for the ability of the virus to replicate in the host. Here we discuss the known data about the characteristics of fusion activation of HA in relation to IAV virulence and pathogenicity.

The influence of dual infection with herpes and influenza viruses on the differential blood cell count of mice.

Based on our previous results, which confirmed the role of latent gammaherpesvirus infection in alteration of immune homeostasis, we studied the influence of simultaneous infection with gammaherpes and influenza viruses on selected parameters of innate immunity, particularly on the subpopulations of peripheral blood cell leukocytes. The aim was to analyze changes of differential blood cell count of BALB/c mice persistently infected with murine gammaherpesvirus 68 (MHV-68) and subsequently co-infected with influenza A virus (IAV), in comparison to mice infected with MHV-68 or with IAV only. Our results showed that ongoing gammaherpesvirus latency in mice caused a decreased number of leukocytes after acute infection with IAV in comparison to a single acute IAV infection. However, increased proportion of neutrophils was measured in peripheral blood of IAV- infected and co-infected mice. Dual infection had no effect on the proportion of monocytes or basophilic and eosinophilic granulocytes. The number of atypical lymphocytes, usually accompanying the persistent infection with MHV-68, decreased in co-infected mice as a consequence of the acute infection with IAV. Persistent infection with gammaherpesvirus may thus modulate the host immune response to influenza A virus and the acute IAV infection can influence the immune homeostasis established by latent MHV-68 infection.

Immune response of mice to non-adapted avian influenza A virus.

Human infections with avian influenza A viruses (IAVs) without or with clinical symptoms of disease were recently reported from several continents, mainly in high risk groups of people, who came into the contact with infected domestic birds or poultry. It was shown that avian IAVs are able to infect humans directly without previous adaptation, however, their ability to replicate and to cause a disease in this new host can differ. No spread of these avian IAVs among humans has been documented until now, except for one case described in Netherlands in the February of 2003 in people directly involved in handling IAV (H7N7)-infected poultry. The aim of our work was to examine whether a low pathogenic avian IAV can induce a virus-specific immune response of biological relevancy, in spite of its restricted replication in mammals. As a model we used a low pathogenic virus A/Duck/Czechoslovakia/1956 (H4N6) (A/Duck), which replicated well in MDCK cells and produced plaques on cell monolayers, but was unable to replicate productively in mouse lungs. We examined how the immune system of mice responds to the intranasal application of this non-adapted avian virus. Though we did not prove the infectious virus in lungs of mice following A/Duck application even after its multiple passaging in mice, we detected virus-specific vRNA till day 8 post infection. Moreover, we detected virus-specific mRNA and de novo synthesized viral nucleoprotein (NP) and membrane protein (M1) in lungs of mice on day 2 and 4 after exposure to A/Duck. Virus-specific antibodies in sera of these mice were detectable by ELISA already after a single intranasal dose of A/Duck virus. Not only antibodies specific to the surface glycoprotein hemagglutinin (HA) were induced, but also antibodies specific to the NP and M1 of IAV were detected by Western blot and their titers increased after the second exposure of mice to this virus. Importantly, antibodies neutralizing virus A/Duck were proved in mouse immune sera after the second dose of virus and a slight increase of mRNA expression of immune mediators tumor necrosis factor alpha (TNF-α) and IP10 has been observed in lungs of these mice 48 hr after the infection. These observations correspond to the limited replication ability of the virus in mice and provided an important information about its ability to induce virus-specific antibodies, including those neutralizing virus, even without the previous virus adaptation to the new mammalian host. Such antibodies could consequently influence the immune potential of exposed individuals and their defensive capability against the newly emerged, even more virulent IAV.

Simultaneous infection with gammaherpes and influenza viruses enhances the host immune defense.

We have studied the impact of simultaneous infection of mice with murine gammaherpesvirus (MHV) and influenza A virus (IAV) on the immune response and pathogenesis of both infections. After a persistent MHV-68 herpesviral infection had been established, the same mice were super-infected with IAV. Individual parameters of MHV infection (viral DNA detection in organs and blood) and numbers of leukocytes in lungs and spleens were determined. With regard to the assumed reactivation of MHV-68 (mainly in lungs, spleen, thymus and peritoneal exudate cells) we focused our attention on the detection of transcripts, typical either for lytic infection (ORF50) and/or for latency (ORF73). Herpesviral DNA was detected in above mentioned organs in several intervals during the acute phase of IAV co-infection, but the expression of monitored transcripts was lower, i.e. it has decreased. Though the reason for such limited expression during acute influenza superinfection remains unclear, it is unambiguous that lower MHV-68 expression was detected in lungs and peritoneal exudate cells (PECs) from 3rd to 10th day after co-infection with IAV. Furthermore, our study showed that the ongoing gammaherpesvirus latency in co-infected mice affected the number of cytotoxic T-lymphocytes and neutrophils during the acute IAV infection and lowered their deviations from that of non-infected mice. Therefore, we suppose that co-infection with herpes and influenza viruses could be mutually beneficial for the host by promoting its defense against both viruses.

Virus-neutralizing antibody response of mice to consecutive infection with human and avian influenza A viruses.

In this work we simulated in a mouse model a naturally occurring situation of humans, who overcame an infection with epidemic strains of influenza A, and were subsequently exposed to avian influenza A viruses (IAV). The antibody response to avian IAV in mice previously infected with human IAV was analyzed. We used two avian IAV (A/Duck/Czechoslovakia/1956 (H4N6) and the attenuated virus rA/Viet Nam/1203-2004 (H5N1)) as well as two human IAV isolates (virus A/Mississippi/1/1985 (H3N2) of medium virulence and A/Puerto Rico/8/1934 (H1N1) of high virulence). Two repeated doses of IAV of H4 or of H5 virus elicited virus-specific neutralizing antibodies in mice. Exposure of animals previously infected with human IAV (of H3 or H1 subtype) to IAV of H4 subtype led to the production of antibodies neutralizing H4 virus in a level comparable with the level of antibodies against the human IAV used for primary infection. In contrast, no measurable levels of virus-neutralizing (VN) antibodies specific to H5 virus were detected in mice infected with H5 virus following a previous infection with human IAV. In both cases the secondary infection with avian IAV led to a significant increase of the titer of VN antibodies specific to the corresponding human virus used for primary infection. Moreover, cross-reactive HA2-specific antibodies were also induced by sequential infection. By virtue of these results we suggest that the differences in the ability of avian IAV to induce specific antibodies inhibiting virus replication after previous infection of mice with human viruses can have an impact on the interspecies transmission and spread of avian IAV in the human population.

HA2 glycopolypeptide of influenza A virus and antiviral immunity.

Influenza A viruses (IAVs) cause acute respiratory infections in humans against which an effective prevention has not yet been developed due to their high variability and broad host specificity. The permanent threat of arising new influenza pandemic is represented by avian viruses which after their interspecies transmission can cause a disease with a devastating impact on humans lacking the specific immunity. Since the current vaccines inducing virus-neutralizing (VN) antibodies are targeted at a variable globular part of hemagglutinin (HA), their efficacy is limited and they need permanent updating. On the other hand, conserved IAV antigens such as proton channel M2, membrane protein M1 or nucleoprotein (NP) do not induce VN antibodies, but they do induce heterosubtypic protection resulting in the reduction of virus replication and an improved recovery from the disease. From this point of view recent attention has also been focused on the conserved part of HA, its HA2 glycoprotein (HA2). The main aspects revealing a contribution of HA2 gp to protective immunity are discussed in this review.

Two distinct regions of HA2 glycopolypeptide of influenza virus hemagglutinin elicit cross-protective immunity against influenza.

UNLABELLED: Currently, a new trend in development of vaccines against influenza with broader spectrum of efficacy is focused on conserved antigens of influenza virus. The HA2 glycopolypeptide (HA2 gp) is one of conserved antigens, potentially suitable as immunogens inducing cross-protection against influenza. We selected two distinct domains of HA2 gp originating from influenza A virus (IAV) of H3 subtype for induction of antiviral immune response: the ectodomain (EHA2) comprising aa 23-185 and the fusion peptide (FP) comprising N-terminal aa 1-38. BALB/c mice were immunized with three doses of EHA2 and FP, respectively, and subsequently challenged with 2 LD50 of IAV of homologous (H3) or heterologous (H7) HA subtype. Both peptides induced significant antibody response and protected mice against the lethal infection. The most efficient protection was achieved with EHA2 against homologous virus. KEYWORDS: influenza A virus; cross-protection; HA2 glycopolypeptide; HA2 ectodomain; fusion peptide; mice; vaccine.

Evaluation of anti-influenza efficiency of polyclonal IgG antibodies specific to the ectodomain of M2 protein of influenza A virus by passive immunization of mice.

We attempted to quantify the protective potential of polyclonal IgG antibodies specific to the ectodomain of M2 protein (eM2) of influenza A virus (IAV) against lethal influenza infection of mice. For this purpose, eM2 conjugated with keyhole limpet hemocyanin (KLH) or KLH alone were administered with Freund's adjuvant intraperitoneally (i.p.) to BALB/c mice. IgG antibodies specific to the KLH-eM2 conjugate (anti-KLH-eM2 IgGs) and KLH (anti-KLH IgGs), respectively, were purified from ascitic fluids. Analysis of the preparation of anti-KLH-eM2 IgGs by ELISA revealed that it contained about 25% of anti-eM2 IgGs and 75% of anti-KLH IgGs. Taking into account this finding mice were passively immunized by intravenous route with 320, 160, 80, and 40 µg of anti-eM2 IgGs per mouse, respectively, while 320 µg of anti-KLH IgGs were used in control. Following subsequent infection with 3 LD50 IAV the survival of mice was determined. An absolute protection (100% survival) was obtained with 320 µg of anti-eM2 IgGs, and a relatively strong significant protection (~80% survival, p = 0.024) with 160 µg. The amount 160 µg of IgGs represents approx. 100 µg IgGs per 1 ml of blood.

Heterosubtypic protective immunity against influenza A virus induced by fusion peptide of the hemagglutinin in comparison to ectodomain of M2 protein.

Several types of influenza vaccines are available, but due to the highly unpredictable variability of influenza virus surface antigens (hemagglutinin (HA) and neuraminidase) current vaccines are not sufficiently effective against broad spectrum of the influenza viruses. An innovative approach to extend the vaccine efficacy is based on the selection of conserved influenza proteins with a potential to induce inter-subtype protection against the influenza A viruses. A promising new candidate for the preparation of broadly protective vaccine may be a highly conserved N-terminal part of HA2 glycopolypeptide (HA2 gp) called fusion peptide. To study its capacity to induce a protective immune response, we immunized mice with the fusion peptide (aa 1-38 of HA2 gp). The protective ability of fusion peptide was compared with the ectodomain aa 2-23 of M2 protein (eM2) that is antigenically conserved and its immunogenic properties have already been well documented. Corresponding peptides (both derived from A/Mississippi/1/85 (H3N2) virus) were synthesized and conjugated to the keyhole limpet hemocyanin (KLH) and used for the immunization of mice. Both antigens induced a significant level of specific antibodies. Immunized mice were challenged with the lethal dose of homologous (H3N2) or heterologous A/PR/8/34 (H1N1) influenza A viruses. Immunization with the fusion peptide led to the 100% survival of mice infected with 1 LD50 of homologous as well as heterologous virus. Survival rate decreased when infectious dose was raised to 2 LD50. The immunization with eM2 induced effective cross-protection of mice infected even with 3 LD50 of both challenge viruses. The lower, but still effective protection induced by the fusion peptide of HA2 gp suggested that besides ectodomain of M2, fusion peptide could also be considered as a part of cross-protective influenza vaccine. To our knowledge, this is the first report demonstrating that active immunization with the conjugated fusion peptide of HA2 gp provided the effective production of antibodies, what contributed to the cross-protection against influenza infection.

Trends in development of the influenza vaccine with broader cross-protection.

Influenza A viruses cause in humans acute respiratory infections, which spread yearly in the form of epidemics or pandemics. A high variability and broad host specificity of influenza A viruses are the main reasons of repeated influenza infections. Therefore, no effective prevention against influenza is available today. The main problem of insufficient protection efficacy is that virus-neutralizing antibodies induced by current vaccines are closely strain-specific and the vaccines need to be updated each year. Therefore, various novel approaches to vaccine preparation have been developed with the aim to widen the spectrum of their efficacy. These approaches comprise using new adjuvants as components of the inactivated vaccines, new techniques of live attenuated vaccine preparation (reverse genetics), and new vaccine design focused on the conserved antigens of influenza A viruses inducing protective immunity not only against the influenza viruses antigenically similar (homologous) to vaccine strains, but also against heterologous viruses, even of different subtypes. In this review examples of new approaches to the induction of intersubtype immunity against influenza and their utilization in vaccine preparation are described.

Multiorgan distribution of human influenza A virus strains observed in a mouse model.

Multiorgan spread and pathogenesis of influenza infection with three human influenza A viruses was studied in mice. Mouse-adapted viruses A/Dunedin/4/73(H3N2), A/Mississippi/1/85(H3N2), and A/PR/8/34(H1N1) differed considerably in virulence (p.f.u./LD(50)): 79,000 p.f.u. for Dunedin, 5,000 p.f.u. for Mississippi, and 65 p.f.u. for PR/8, which qualified Dunedin as low virulent, Mississippi as intermediate, and PR/8 as highly virulent. All three viruses were detected in lungs, heart, and thymus by cultivation and RT-PCR. Moreover, vRNA of all viruses was found in liver and spleen, of Dunedin and PR/8 also in kidneys and that of Dunedin and Mississippi in blood. Only vRNA of Dunedin was demonstrated in brain. Lung damage accompanied by histopathological changes and thymus reduction were most extensive after infection with the highly virulent virus PR/8. We assume that the ability to spread to multiple organs may be a more common property of influenza viruses in mammalian hosts than previously believed.

Broadly cross-reactive monoclonal antibodies against HA2 glycopeptide of Influenza A virus hemagglutinin of H3 subtype reduce replication of influenza A viruses of human and avian origin.

The reactivity of monoclonal antibodies (MAbs) prepared to the HA2 glycopeptide (gp) of A/Dunedin/4/73 (H3N2) hemagglutinin was tested against influenza A viruses of H3, H4, and H7 subtypes. Only one (CF2) out of six MAbs reacted with influenza A viruses of all three subtypes (H3, H4 and H7). The inter-subtype reactivity of this MAb (CF2) is in accord with the highly conservative sequence in the previously defined MAb-binding site I, i.e. the aa 1-38 of N-terminus of HA2 gp. MAb CF2 as well as inter-subtype cross-reactive MAb IIF4, recognizing the binding site II of HA2 gp, were tested for their effect on replication of influenza A viruses. Both these MAbs reduced the number of plaques of viruses of homologous (H3) as well as heterologous (H4) virus subtypes, the latter less efficiently. The potential of these MAbs to influence in vivo replication of influenza A viruses of various subtypes is discussed.

Evaluation of clinical specimens for influenza A virus positivity using various diagnostic methods.

The diagnostic method for Influenza A virus, utilizing the SERION ELISA Antigen kit (SERION EIA), if results were evaluated according to the manufacturer's instructions, has repeatedly failed to detect a great number of clinical samples positive by virus isolation and RT-PCR. Therefore we compared the SERION EIA with the one-step 44/107L-Px immunocapture enzyme immunoassay (44/107L-Px EIA), developed in our laboratory (Tkácová and Varecková, J. Virol. Methods 60, 65-71, 1996). Seventy-three clinical specimens, of which 65 were positive by virus isolation (used as reference method), were tested by both EIAs. By the SERION EIA, out of the 65 reference-positive samples only 8 (12%) were positive, 5 (8%) were ambiguous, and 52 (80%) were negative, which corresponded to the sensitivity of 12%. On the contrary, the sensitivity of the 44/107L-Px EIA was 74%. However, the calculation of cut-off values for the evaluation of positivity of clinical specimens in these two assays were not the same. If the evaluation procedure used for the 44/107L-Px EIA was applied to the SERION EIA, the sensitivity and the specificity of both EIAs became comparable, namely 71% and 100% for the SERION EIA and 74% and 100% for the 44/107L-Px EIA, respectively. From these results it follows that not the detection ability of the SERION EIA, but the evaluation procedure recommended by its manufacturer led to a loss of large number of positive specimens.

Differences in antibody responses of mice to intranasal or intraperitoneal immunization with influenza A virus and vaccination with subunit influenza vaccine.

Two antigenically related but different influenza A virus strains of H3N2 subtype, A/Dunedin/ 4/73 (H3N2) (Dunedin) and A/Mississippi/1/85 (H3N2) (Mississippi), were used for intranasal (i.n.) and intraperitoneal (i.p.) immunization of mice and respective antibody responses were compared. In ELISA, using purified influenza A virus as antigen, the highest titer of antiviral antibodies was observed after a repeated i.n. infection, in which the Dunedin strain was followed by the Mississippi strain and vice versa. Similarly, in virus neutralization (VN) test, the highest titer of VN antibodies was found after a repeated i.n. infection. The subunit vaccine INFLUVAC, when administered intramuscularly (i.m.), induced only a poor antibody response as assayed by ELISA. Moreover, the INFLUVAC vaccination elicited a 100-fold lower titer of VN antibodies than the i.n. infection and an approx a 10-fold lower titer than the i.p. immunization. A repeated INFLUVAC vaccination did not lead to a significant increase of VN antibody titer. Also the antibody response to HA2gp--a conserved part of influenza hemagglutinin (HA) that might contribute to the induction of specific antiviral antibodies--was followed. Similarly to the VN antibody response, the highest HA2 antibody titer was induced after a repeated i.n. infection, whereas the lowest HA2 antibody titer was observed after a single or repeated INFLUVAC vaccination. Overall, the HA2 antibody titers remarkably well corresponded to the VN potential of the examined sera.

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.