WO2010110737A1

WO2010110737A1 - Monoclonal antibody against a conserved domain of m2e polypeptide in influenza viruses

Info

Publication number: WO2010110737A1
Application number: PCT/SG2009/000102
Authority: WO
Inventors: Prabakaran Mookkan; Nayana Prabhu Padubidhri; Sumathy Velumani; Hwei-Sing Jimmy Kwang
Original assignee: Temasek Life Sciences Laboratory Limited
Priority date: 2009-03-23
Filing date: 2009-03-23
Publication date: 2010-09-30

Abstract

The invention provides monoclonal antibodies and related binding proteins that bind specifically to the ectodomain of the M2 membrane protein of influenza A virus. The monoclonal antibodies and related binding proteins are useful for the detection of influenza A virus, including H5 subtypes such as the pathogenic H5N1 avian influenza virus. Accordingly, the invention provides means for the diagnosis, surveillance, treatment and prevention of dangerous viral infections.

Description

Monoclonal Antibody Against a Conserved Domain of M2e Polypeptide in Influenza Viruses

FIELD OF THE INVENTION

This invention relates to antibodies and related binding proteins for the detection of influenza A virus. More particularly, the invention relates to monoclonal antibodies and related binding proteins useful for the detection of avian influenza virus (AIV), particularly the highly pathogenic H5 subtypes of AIV and to methods and products for the diagnosis and surveillance of such influenza A virus infections in animals and humans.

BACKGROUND OF THE INVENTION

Avian influenza is a common disease in birds. Subtype H5N1 AIV has caused an outbreak of avian influenza that is spreading incessantly to many regions of the world (1 ).¹ The affected areas include Europe, the Middle East and, particularly, Asia. According to the World Health Organization ("WHO"), as of April 2006, about one hundred human deaths had occurred as a result of H5N1 avian influenza, and the situation seems to be deteriorating. See WHO website (2). While AIV infection in humans is rare, there have been times in the past in which the occurrence of new AIV subtypes that are able to cross species barriers have caused deadly influenza pandemics (3-5). Influenza viruses are classified according to their nucleoprotein and matrix protein antigenic specificity. These viruses are categorized mainly into A, B and C serotypes, with type A having eight RNA segments that encode ten viral proteins. All known type A influenza viruses originated in birds. This category of virus can infect other species, such as horses, pigs, owls and seals, and poses a threat to humans as well (18). Influenza A virus is further divided into subtypes according to the antigenic nature of the envelope glycoproteins, hemagglutinins ("HAs"), H1 through H16, and neuraminidases ("NAs"), N1 through N9 (5-7). An integral membrane protein of influenza A virus is called M2. This 97 amino acid protein forms an ion channel crossing the membrane of a virus particle or infected cell with an ectodomain protruding from the surface. The 23 amino acids that make up the ectodomain of M2 (known as M2e) scarcely vary from one influenza strain to the next, even back to the 1918 Spanish flu.

Because of the risk that influenza infection poses to wildlife, domesticated animals and humans, there is a pressing need for a fast, specific and reliable method for detecting the virus in tissue specimens. In particular, the ability to detect the virus in preserved specimens, such as formalin fixed specimens embedded in paraffin and in frozen sections, is important to the ability to diagnose the disease and monitor its progress.

There also is a need for a method of treating avian and mammalian species infected with avian influenza A virus, particularly H5 subtype virus strains, and most particularly H5N1 virus strains. In particular, there is a need for a safe and convenient method of treating humans infected with such a virus. SUMMARY OF THE INVENTION

In accordance with the present invention, monoclonal antibodies and related binding proteins that are specific for the M2e epitope of the M2 membrane protein of influenza A virus are provided. The mAbs to M2e epitopes are able to recognize influenza A viruses, including all of the H1-H16 subtypes, preferably H5 subtype virus strains, and most preferably H5N1 virus, with good specificity and sensitivity.

In particular, mAb designated 3H5 targets antigenic sites on the M2 ectodomain of Influenza A virus. Accordingly, the invention comprises a binding protein having substantially the immunological binding characteristics for antigenic sites on M2e as mAb 3H5.

In a further aspect, the invention comprises a method for detecting influenza A virus in a specimen which comprises detecting the binding of influenza A virus with a mAb or binding protein having substantially the immunological binding characteristics of mAb 3H5. In particular, the invention relates to immunofluorescence assays, immunohistochemical assays and ELISA methods that utilize such binding proteins to detect influenza A viruses such as H subtypes, in a preferred embodiment subtypes H1-H16, yet more preferably H5 subtype virus strains, and most preferably H5N1 virus.

In another aspect, the invention relates to kits for the detection of influenza A virus which comprise binding proteins having substantially the immunological binding characteristics of mAb 3H5.

The invention further relates to methods of treating subjects infected with influenza A virus strain, including any of the H1 -H 16 subtypes, preferably H5 subtype virus strains, and most preferably H5N1 virus, which comprise administering to such subjects effective amounts of a monoclonal antibody or binding protein having substantially the immunological binding characteristics of mAb 3H5. In particular, the invention relates to methods of treating avian and mammalian subjects, particularly human subjects.

The invention further relates to methods of providing cross-clade protection against infection against influenza A virus which comprise administering to such subjects effective amounts of one or more recombinant monoclonal antibodies or binding proteins or fragments thereof having substantially the immunological binding characteristics of mAb 3H5.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a photomicrograph of rhodamine conjugated anti-M2e mAb 3H5 bound to H5N1 infected MDCK cells.

Figures 2(a) and 2(b) illustrate the epitope mapping of 3H5. Figure 2(a) represents six fragments of the M2e polypeptide tagged with GST. The GST-tagged polypeptide sequence labeled M2e corresponds to SEQ ID NO: 1. The sub-sequences labeled 1-5 correspond to SEQ ID NOs: 2-6, respectively. Figure 2(b) is a Western blot analysis of the reactivities of mAb 3H5 with M2e polypeptide fragments protein of H5N1 virus expressed in E. coli's total cell lysate.

Figure 3 graphically represents the conservation of residues within the motif recognized by 3H5 across all available H5N1 sequences.

Figure 4 is a graphical representation of body weight changes in mice intraperitoneal^ treated with 10 mg/kg, 5 mg/kg, or 0 mg/kg (PBS) of anti-M2e mAb 3H5 after 1 day post viral challenge with mouse-adapted Indonesian HPAI H5N1 (A/TLL013/0β-Clade 2.1) virus. Mice were monitored for body weight loss throughout a 14 day observation period. The results are expressed in terms of percent body weight (at the beginning of the trial) respectively (* represents no survival of any animals in the group).

Figure 5 is a graphical representation of the percent survival of mice intraperitoneal^ treated with 10 mg/kg, 5 mg/kg, or 0 mg/kg (PBS) of anti-M2e mAb 3H5 after 1 day post viral challenge with mouse-adapted Indonesian HPAI H5N1 (A/TLL013/06-Clade 2.1) virus. Mice were monitored for percent survival throughout a 14 day observation period. The results are expressed in terms of percent survival.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to monoclonal antibodies and related binding proteins that bind specifically to influenza A virus. Monoclonal antibodies ("mAbs") are a substantially homogeneous population of antibodies derived from a single antibody-producing cell. Thus, all antibodies in the population are identical and of the same specificity for a given epitope. The specificity of the mAb responses provides a basis for an effective diagnostic reagent. Monoclonal antibodies and binding proteins derived therefrom also have utility as therapeutic agents.

The present invention is directed to mAbs and related antigen-binding proteins that bind specifically to the M2 ectodomain epitope of influenza A viruses. In particular, the mAb or related antigen-binding protein possesses the immunological binding characteristics of mAb 3H5 as produced by hybridoma 3H5, deposited with the American Type Culture Collection (ATCC) on June 25, 2008, in accordance with the provisions of the Budapest Treaty, and assigned Accession Number PTA-9300. The hybridoma comprises a further embodiment of the invention and provides a continuous source of the mAbs and binding proteins of the invention. The invention further relates to methods for the detection and diagnosis of influenza A virus infection and assay kits that comprise the mAbs or binding proteins of the invention.

The invention additionally relates to methods of treating a subject infected with influenza A virus strain through the administration of effective amounts of an antibody or related binding protein of the invention. In particular, in this embodiment the subject is infected with influenza A virus, particularly an H5 subtype such as an H5N1 subtype. The antibodies of this invention also can be administered to subjects on the advent of a possible influenza pandemic as a precautionary measure. In this instance, effective amounts of antibodies to be administered may be about half of the amounts used to treat influenza A virus infections.

Various terms are used herein, which have the following meanings:

The term "immunological binding characteristics" of a mAb or related binding protein, in all of its grammatical forms, refers to the specificity, affinity and cross-reactivity of the mAb or binding protein for its antigen.

The term "ectodomain epitope" refers to a consecutive sequence of from about 6 to about 13 amino acids which form an antibody binding site. The ectodomain epitope of the mAb of this invention preferably is in the region from about amino acid 14 to about amino acid 19 of the M2 membrane protein encoded by the H5N1 viral gene. The ectodomain epitope, in the form that binds to the mAb or binding protein, may be in a denatured protein that is substantially devoid of tertiary structure.

The term "binding protein" refers to a protein, including those described below, that includes the antigen binding site of a mAb of the present invention or a mAb having the immunological binding characteristics of a mAb of the present invention.

The present invention advantageously provides methods for preparing monoclonal antibodies having the binding characteristics of mAb 3H5 by immunizing an animal with AIV subtype H5N1 , preparing monoclonal antibodies having the binding characteristics of 3H5 by immunizing an animal with M2e antigen. Any such antigen can be used as an immunogen to generate antibodies with the desired immunological binding characteristics. Such antibodies include, but are not limited to, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and proteins comprising the antigen binding sequence of mAb 3H5.

The mAb of the present invention can be produced by any technique that provides for the production of antibody molecules by continuous cell lines in culture. Such methods include, but are not limited to, the hybridoma technique (8), as well as the trioma technique, the human B-cell hybridoma technique (9), and the EBV-hybridoma technique to produce human monoclonal antibodies (10). Human antibodies can be used and can be obtained by using human hybridomas (11) or by transforming human B cells with EBV virus in vitro (10). Moreover, techniques developed for the production of "chimeric antibodies" or "humanized antibodies" (12-14) by introducing sequences from a murine antibody molecule of the present invention, e.g., mAb 3H5, together with genes from a human antibody molecule of appropriate biological activity can be used. Chimeric antibodies are those that contain a human Fc portion and a murine (or other non-human) Fv portion. Humanized antibodies are those in which the murine (or other non-human) complementarity determining regions (CDR) are incorporated into a human antibody. Both chimeric and humanized antibodies are monoclonal. Such human or humanized chimeric antibodies are preferred for use in in vivo diagnosis or therapy of human diseases or disorders.

According to the invention, techniques described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to provide single chain antibodies of the present invention. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (15) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for the antibody of the present invention, or its derivatives or analogs.

Antibody fragments that contain the idiotype of the antibody molecule can be generated by known techniques. For examples, such fragments include but are not limited to: the F(ab')₂ fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent. Such antibody fragments can be generated from any of the polyclonal or monoclonal antibodies of the invention.

In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), immunofluorescence assays and Immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or other reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Methods and means for detecting binding in an immunoassay are known in the art and are within the scope of the present invention.

The foregoing antibodies can be used in methods known in the art relating to the detection or localization of influenza A virus, e.g., Western blotting, ELISA, radioimmunoassay, immunofluorescence assay, immunohistochemical assay, and the like. The techniques disclosed herein may be applied to the qualitative and quantitative determination of the influenza A virus and to the diagnosis and surveillance of animals or humans infected with the virus.

The present invention also includes assay and test kits for the qualitative and/or quantitative determination of the influenza A virus. Such assay systems and test kits may comprise a labeled component prepared, e.g., by labeling with a radioactive atom, a fluorescent group or an enzyme, coupling a label to the mAb or related binding protein of the present invention, or to a binding partner thereof. Such assay or test kits further may comprise reagents, diluents and instructions for use, as is well known to those skilled in immunoassay techniques.

In certain embodiments of the invention, such kits will contain at least the mAb or related binding protein of the invention, means for detecting immunospecific binding of said mAb or related binding protein to influenza A virus in a biological sample, and instructions for use, depending upon the method selected, e.g., "competitive," "sandwich," "DASP" and the like. The kits may also contain positive and negative controls. They can be configured to be used with automated analyzers or automated immunohistochemical slide staining instruments.

An assay kit of the invention can further comprise a second antibody or binding protein that may be labeled or may be provided for attachment to a solid support (or attached to a solid support or surface). Such an antibody or binding protein can be, for example, one that binds to influenza A virus. Such second antibodies or binding proteins can be polyclonal or monoclonal antibodies.

Monoclonal antibodies to influenza A protein can be prepared by immunizing animals with influenza A virus, M2 protein, or fragments thereof. A preferred method involves amplification of the H5-subtype M2e gene followed by expression of the gene, recovery and purification of M2e recombinant proteins and use of the purified proteins as immunogens. For example, H5N1 AIV is propagated by inoculation of chicken embryos with available strains of the virus, followed by isolation of the viral RNA. The M2e gene is amplified by reverse transcriptase polymerase chain reaction (RT-PCR) and then may be cloned into a baculovirus vector that is used to express H5 proteins in insect cells. The proteins so produced then can be used to immunize mice or other suitable species for production of hybridomas.

Hybridomas are screened for their ability to produce high affinity mAbs that are capable of specifically binding to H5 proteins and distinguishing them from other AIV subtypes. The hybridoma clones secreting mAbs to M2e antigen were screened by Immunofluorescence (IFA) assay against Indonesian H5N1 Influenza strains and other non-H5 subtypes. MDCK cells were infected with H5N1 strains or non H5-subtypes. The cells were then incubated with hybridoma culture fluid or test sample and followed by incubation with a 1 :40 dilution of fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse Immunoglobulin (DAKO, Denmark). The FITC visualization was recorded using epifluorescence microscope. Screening can also be done by indirect ELISA using influenza antigen as a coating antigen.

M2 contains 97 amino acids and its ectodomain, M2e, contains 23 amino acids. To study the distribution of epitopes on the M2 protein, truncated and mutated fragments are advantageously tested for binding with mAbs, e.g., by Western blot or a similar technique. Epitopes can be identified that are binding targets for mAbs that give a good performance in detecting denatured M2 protein, such as that occurring in formalin-fixed tissue, using immunohistochemical staining methods. Mapping of the M2 mAb in this manner provides a platform for further study and a more effective clinical diagnosis of the infectious virus.

Antibody 3H5 exhibits positive results in immunofluorescence assay, and in Western blot analysis strong bands that correspond to the recombinant H5N1-M2e protein (MW ~29 kDa including GST tag) are observed.

This invention provides convenient, highly specific and sensitive means for detecting influenza A virus. One such means is the immunofluorescence microscopy format. In a preferred embodiment mAb 3H5 is conjugated with rhodamine to detect virus-infected MDCK cells. While not bound by any particular theory, a possible explanation of these results is that the antibody reacts with an epitope on the M2e protein.

The preferred ELISA test of this invention is able to detect M2 antigen from influenza A virus infections globally, including, but not limited to, avian and human infections.

In a further embodiment of the invention, the antibodies and related binding proteins of the invention can be administered to treat subjects suffering from an influenza A virus infection, particularly an infection from an H5 AlV, in particular H5N1 subtype. The antibodies and related binding proteins of the invention also can be administered to subjects as a preventive measure in the event of an influenza pandemic or threatened pandemic. The antibodies and related binding proteins can be administered in a single dose or in repeated administrations, optionally in a slow release form. Administration can be made by any means that enables the antibody to reach its site of action in the body of the subject being treated, e.g., intravenously, intramuscularly, intradermal^, orally or nasally. Typically, the antibody is administered in a pharmaceutically acceptable diluent or carrier, such as a sterile aqueous solution, and the composition can further comprise one or more stabilizers, adjuvants, solubilizers, buffers, etc. The exact method of administration, composition and particular dosage will be determined and adjusted at the time of therapy, depending upon the individual needs of the subject, taking into account such factors as the subject's age, weight, general health, and the nature and extent of his or her symptoms, as well as the frequency of treatment to be given. Generally, the dosage of antibody administered is within the range of about 0.1 mg/kg to about 10 mg/kg body weight when the antibody is administered to treat patients suffering from an H5 AIV infection. Typically, the dosage is reduced by about half, i.e. to within the range of about 0.05 mg/kg to about 5 mg/kg body weight, when administered as a preventive measure. It is within the skill of those in the art to determine the appropriate dose based upon the severity of the infection.

In a further embodiment, the antibodies and related binding proteins of the invention can be administered to treat a fixed tissue specimen infected with influenza A virus with an effective amount of a binding protein that binds specifically to an ectodomain epitope of the M2 membrane protein of an influenza A virus that has substantially the immunological binding characteristics of monoclonal antibody 3H5.

In another embodiment, the highly immunogenic 3H5 mAb can be used to treat or prevent influenza A virus strains from different clades, including lethal influenza H5N1 strains.

A neutralization escape mutant can evade neutralization by certain monoclonal antibodies (e.g. antibodies against neutralizing epitope of HA1 protein) that are effective in neutralizing its parent virus.

A single antibody or binding protein of the invention can be administered for therapeutic purposes or administered sequentially or simultaneously with other antibodies. If antibodies to one or more generations of neutralization escape mutants have been produced, such antibodies and the 3H5 antibody described above can be administered as therapeutic antibody "cocktails."

The following examples are provided to illustrate a preferred mode of practicing the invention. The invention is not limited to the details of the examples, but is commensurate with the full scope of the appended claims.

EXAMPLE 1

Production of Hybridomas

Viruses designated A/lndonesia/CDC669/06 and A/lndonesia/TLL013/06 were obtained from the Ministry of Health (Indonesia). The virus was propagated in the allantoic cavity of 11 day old chicken embryos and the allantoic fluid was harvested from the eggs after 48 hours. Virus titers were determined using hemagglutination assays (16). The virus was then clarified and stored at -80°C. Monoclonal antibody (IgM) was purified from clarified fluids using protein A affinity column (SIGMA ALDRICH; St. Louis, Missouri, USA) and Immunopure^® IgM purification kit (PIERCE BIOTECHNOLOGY; Rockford, Illinois, USA) in accordance with manufacturer's instructions. The concentrations of IgM were measured by using an ND-1000 spectrophotometer (NANODROP TECHNOLOGIES; Wilmington, Delaware, USA).

H5N1 (A/CDC/669/lndonesia/06) viral RNA was extracted using Trizol (INVITROGEN, Carlsbad, CA, USA).The M2e gene was amplified from the cDNA and cloned into pET28a vector (INVITROGEN, Carlsbad, CA, USA) followed by transformation into Escherichia coli BL21 (DE3) competent cells for protein expression. The fusion protein expression was induced by adding 1 mmol/L IPTG for 3 hours and was purified on Ni-NTA column (QIAGEN, Germany).

BALB/c mice were immunized two times subcutaneously at regular intervals of 2 weeks with 25 μg of rM2e antigen with adjuvant (SEPPIC, France). Mice were boosted with 25 μg of recombinant antigen 3 days before the fusion of splenocytes with SP2/0 cells (17).

EXAMPLE 2

Epitope mapping of 3H5

The monoclonal antibodies were confirmed to bind to the recombinant M2e protein by Western blotting analysis. A series of six fragments of the M2e polypeptide were designed and tagged with glutathione S-transferase (GST), so as to detect the exact epitope region of the monoclonal antibodies. The following fragments were cloned:

Table 1 : Epitope mapping of M2e fragments

A total of 1280 human and avian influenza A H5N1 viruses with full-length sequences available on the NCBI influenza-database (ncbi.nlm.nih.gov/genomes/FLU/-atabase/multiple.cgi) were analyzed for the presence of the epitope region recognized by mAb 3H5.

Immunofluorescence Assay: Madin Darby Canine Kidney cells (MDCK) were infected with H5N1 Indonesian strains and non-H5 subtypes. At 24-48h post-infection, the cells were fixed with 4% para-formaldehyde for 30 min at room temperature and washed three times with PBS. The fixed cells were incubated with hybridoma culture fluid at 37°C for 1 h and followed by incubation with 1 :40 dilution of fluorescein isothiocyanate (FITC) or rhodamine-conjugated rabbit anti-mouse Ig (DAKO, Denmark) and observed by epi-fluorescence microscopy (OLYMPUS 1X71 microscope).

The mAb (3H5) recognizing distinct antigenic sites on the M2 ectodomain were generated by immunizing mice with rM2e protein from A/lndonesia/CDC669 (H5N1 ). The epitope mapping results revealed that 3H5 mAbs strongly interacted with C-terminus peptide (CM2, aa13-24) and further mapping confirmed that the epitope was ¹EWECKC¹ (SEQ ID NO: 6) (aa 14-19) (Fig. 2). The isotype of the mAb was found to be IgM. Without being bound to any particular theory, antibodies specific to the antigenically conserved M2 ectodomain appear to block the ion channel and inhibit virus replication.

Further analyses of the mAb 3H5 recognizing the epitope ΕWECKC (SEQ ID NO: 6) (aa 14-19) were made, lmmunofluorescent assay among the available strains showed that the 3H5 mAb recognizes both EWECKC (SEQ ID NO: 6) and EWECRC (SEQ ID NO: 7) sequences (Table 2). Thus, the antibody is able to recognize epitopes with conservative mutations such as Lys -> Arg, herein referred to as conservative variants of the M2 ectodomain epitope.

Table 2: sequences of M2e epitopes recognized by 3H5

As used herein, the term "conservative variant" or grammatical variations thereof denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the replacement of a hydrophobic residue such as isoleucine, valine, leucine or methionine for another, the replacement of a polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.

EXAMPLE 3

Characterization of H5-Subtype Monoclonal Antibodies

BALB/c mice were immunized two times subcutaneously at regular intervals of 2 weeks with 25 μg of rM2e antigen with adjuvant (SEPPIC, France). Mice were boosted with 25 μg of recombinant antigen 3 days before the fusion of splenocytes with SP2/0 cells (17). The fused cells were seeded in 96-well plates, and their supernatants were screened by immunofluorescence assays. Selected mAbs were screened for isotype using a one-minute isotyping kit (AMERSHAM BIOSCIENCE, England).

3H5 mAb strongly reacted with MDCK cells infected with A/CDC594/H5N1 and yielded positive cytoplasmic immunofluorescence patterns. Rhodamine conjugated anti-M2e mAb 3H5 recognized H5N1 (epitope 'EWECRC SEQ ID NO: 6) infected MDCK cells (Fig. 1). Other non-H5 subtypes infected MDCK cells also gave a fluorescence signal (data not shown). Based on the sensitivity and specificity of the monoclonal antibody by IFA, 3H5 mAb was selected for mapping of the linear epitope and further studies. The isotype of 3H5 mAb was determined as IgM class. Analysis of the variation of the epitope EWECKC (SEQ ID NO: 6) or EWECRC

(SEQ ID NO: 7) (recognized by 3H5 mAb) in 1280 H5N1 M2e sequences in the NCBI database revealed that the composition of the epitope region was EWECKC (SEQ ID NO: 6) in 437 sequences (34.14%) and EWECRC (SEQ ID NO: 6) in 767 sequences (64.53%) and present in all the currently identified H5N1 viruses, indicating that the mAb can recognize any of the existing H5N1 strains (Fig. 3). A small number of other variations were found in 17 H5N1 strains (1.33% of all available sequences) wherein the sequence of H5N1 is mutated to DWECKC (SEQ ID NO: 8) or GWGCRC (SEQ ID NO: 9). As disclosed above, mAb 3H5 will recognize conservative variants of the M2e sequence in all influenza A virus subtypes.

EXAMPLE 4

Therapeutic efficacy studies of the anti-M2e mAb against H5N1 infection:

To determine the therapeutic efficacy, inbred SPF BALB/c mice aged 4-6 weeks were used. Five mice (n=5) per group were anesthetized with Ketamine/Xylazine and intranasally infected with 5MLD50 (Mouse lethal dose 50%) of highly pathogenic avian influenza H5N1 strain (A/lndonesia/TLL013/06 from clade 2.1). Fifty percent mouse lethal dose (MLD50) was determined as previously described by the Reed and Muench method.

Therapeutic efficacy: To determine the therapeutic efficacy of the mAbs, each group of mice was treated via intra-peritoneal route with 5 mg/kg, 10 mg/kg or 0 mg/kg (PBS) of anti-M2e mAb 3H5 one day after viral challenge. Mice were observed daily to monitor body weight and mortality. Monitoring continued until all animals died or until day 14 after challenge.

The progress of infection was indicated by varying trends of decrease in body weight in the different groups. In mice challenged with the H5N1 virus, untreated mice showed the most rapid decline in bodyweight (FIG. 4), culminating in 100% mortality within 7 days after viral challenge (FIG. 5). The body weight of succumbed mice from this group, on day 5, was less than 77 % of the original body weight.

Groups of mice treated with 10 mg/kg 3H5 mAb lost up to 14% of their original body weight by day 5, but they steadily regained their body weight after the next 1 -2 days (FIG. 4). 10 mg/kg 3H5 mAb provided 100% protection against H5N1 virus infection (FIG. 5). The groups of mice treated with 5 mg/kg of 3H5 showed a loss of body weight of up to 20% on day 5 (FIG. 4). The mortality studies showed that 5 mg/kg 3H5 mAb provided 60% protection against 5 MLD₅₀ of H5N1 viral infections (FIG. 5).

REFERENCES

1. Mase, M., K. Tsukamoto, T. Imada, K. Imai, N. Tanimura, K. Nakamura, Y. Yamamoto, T. Hitomi, T. Kira, T. Nakai, M. Kiso, T. Horimoto, Y. Kawaoka, and S. Yamaguchi. 2005. Characterization of H5N1 influenza A viruses isolated during the 2003-2004 influenza outbreaks in Japan. Virology, 332:167-76.

2. http://www.who.int/csr/disease/avian influenza/country/cases table 2006 04 27/en/index.html.

3. Cauthen, A. N., D. E. Swayne, S. Schultz-Cherry, M. L. Perdue, and D. L. Suarez 2000. Continued circulation in China of highly pathogenic avian influenza viruses encoding the hemagglutinin gene associated with the 1997 H5NI outbreak in poultry and humans. J. Virol., 74:6592-9.

4. Fouchier, R. A., T. M. Bestebroer, S. Herfst, L. Van Der Kemp, G. F. Rimmelzwaan, and A.D. Osterhaus. 2000. Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene. J. Clin. Microbiol., 38:4096-5101.

5. Horimoto, T., N. Fukuda, K. Iwatsuki-Horimoto, Y. Guan, W. Lim, M. Peiris, S. Sugii, T. Odagiri, M. Tashiro, and Y. Kawaoka. 2004. Antigenic differences between H5N1 human influenza viruses isolated in 1997 and 2003. J. Vet. Med. ScL, 66:303-5.

6. Iwasaki, T., S. Itamura, H. Nishimura, Y. Sato, M. Tashiro, T. Hashikawa, and T. Kurata. 2004. Productive infection in the murine central nervous system with avian influenza virus A (H5N1) after intranasal inoculation. Acta Neuropathol. (Berl), 108:485-92.

7. Stevens, J., O. Blixt, T. M. Tumpey, J. K. Taubenberger, J. C. Paulson, and I. A. Wilson. 2006. Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science, 312:404-10.

8. Kohler and Milstein. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256:495-497.

9. Kozbor D, Lagarde AE, Roder JC. Human hybridomas constructed with antigen-specific Epstein-Barr virus-transformed cell lines. Proc Natl Acad Sci U S A. 1982 79(21 ):6651 -5.

10. Cole et al., 1985. The EBV-Hybridoma Technique and its Application to Human Lung Cancer. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96.

11. Cote et al., 1983. Generation of human monoclonal antibodies reactive with cellular antigens. Proc. Natl. Acad. Sci. U.S.A., 80:2026-2030. 12. Morrison et al., 1984. Isolation of transformation-deficient Streptococcus pneumoniae ... resistance determinant of pAM beta 1. J. Bacteriol. 159-870. 13. Neuberger et al., 1984. Recombinant Antibodies Possessing Novel Effector

Functions, Nature 312:604-608. 14.Takeda et al., 1985. Construction of chimaeric processed immunoglobulin genes containing mouse variable and human constant region sequences,

Nature 314:452-454. 15. Huse et al., 1989. Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda, Science 246: 1275-1281. 16. Anonymous. 1995. Laboratory biosafety manual. World Health Organization.

Ann 1st Super Sanita, 31 , 1-121.) 17.Yokoyama W.M, C. J. E., A.M Kruisbeek, D.H Margulies, E.M.Shevach,

W.Strober (eds). 2001. Production of monoclonal antibodies. Currents protocols in immunology:2.5.1- 2.6.9.

18.Zhou, N. N., D. A. Senne, J. S. Landgraf, S. L. Swenson, G. Erickson, K. Rossow, L. Liu, K. Yoon, S. Krauss, and R. G. Webster. 1999. Genetic reassortment of avian, swine, and human influenza A viruses in American pig: Virol., 73:8851-6.

Claims

1. A binding protein that binds specifically to an ectodomain epitope of the M2 membrane protein of an influenza A virus and has substantially the immunological binding characteristics of monoclonal antibody 3H5.

2. The binding protein of claim 1 which is a monoclonal antibody, a single chain antibody, an antibody fragment, a chimeric antibody or a humanized antibody.

3. The binding protein of claim 1 which is a monoclonal antibody.

4. Monoclonal antibody 3H5 as produced by hybridoma 3H5 which is deposited with the American Type Culture Collection with Accession Number PTA-9300.

5. A method for detecting influenza A virus in a biological specimen which comprises contacting the specimen with a first binding protein that binds specifically to an ectodomain epitope of the M2 membrane protein of an influenza A virus, said binding protein having substantially the immunological binding characteristics of monoclonal antibody 3H5.

6. The method of claim 5 wherein the first binding protein is a monoclonal antibody, a single chain antibody, an antibody fragment, a chimeric antibody or a humanized antibody.

7. The method of claim 5 wherein the first binding protein is a monoclonal antibody.

8. The method of claim 7 wherein the monoclonal antibody is antibody 3H5 as produced by hybridoma 3H5 which is deposited with the American Type Culture Collection with Accession Number PTA-9300.

9. The method of claim 5 which further comprises contacting the specimen with a second binding protein that specifically binds to an ectodomain epitope of the M2 membrane protein of an influenza A virus, wherein the first binding protein is a capture binding protein and the second binding protein is a detector binding protein that contains or is conjugated to a detectable element.

10. The method of claim 9 wherein at least one of the first and second binding proteins is a monoclonal antibody.

11.The method of claim 9 wherein the first and second binding proteins are monoclonal antibodies.

12. The method of claim 9 wherein the first binding protein is immobilized onto a solid surface.

13. The method of claim 9 wherein the second binding protein contains a radioactive atom, is conjugated to a fluorescent molecule, or is conjugated to an enzyme.

14. A kit for detecting influenza A virus in a biological specimen which comprises a first binding protein that binds specifically to an ectodomain epitope of the M2 membrane protein of an influenza A virus, said first binding protein having substantially the immunological binding characteristics of monoclonal antibody 3H5 together with reagents for the detection of binding of said binding protein to said envelope glycoprotein.

15. The kit of claim 14 which comprises a second binding protein that specifically binds to an ectodomain epitope of the M2 membrane protein of an influenza A virus, wherein the first binding protein is a capture binding protein and the second binding protein is a detector binding protein that contains or is conjugated to a detectable element.

16. The kit of claim 15 wherein at least one of the first and second binding proteins is a monoclonal antibody.

17. The kit of claim 15 wherein the first and second binding proteins are monoclonal antibodies.

18. The kit of claim 15 wherein the first binding protein is immobilized onto a solid surface.

19. The kit of claim 15 wherein the second binding protein contains a radioactive atom, is conjugated to a fluorescent molecule, or is conjugated to an enzyme.

20. A method of treating a fixed tissue specimen infected with influenza A virus with an effective amount of a binding protein that binds specifically to an ectodomain epitope of the M2 membrane protein of an influenza A virus and has substantially the immunological binding characteristics of monoclonal antibody 3H5.

21.A method of treating a subject infected with influenza A virus which comprises administering to said subject an effective amount of a binding protein that binds specifically to an ectodomain epitope of the M2 membrane protein of an influenza A virus and has substantially the immunological binding characteristics of monoclonal antibody 3H5.

22. The method of claim 21 , wherein the binding protein is a recombinant monoclonal antibody, single chain antibody, antibody fragment, chimeric antibody, or humanized antibody.

23. The method of claim 21 , wherein the binding protein is a recombinant monoclonal antibody.

24. The method of claim 23, wherein the binding protein is monoclonal antibody 3H5.

25. The method of claim 21 , further comprising administration of at least one other antibody.

26. The method of claim 25, further comprising administration of at least one neutralization escape mutant antibody.

27. The method of claim 21 , wherein the influenza A virus is an H5 subtype.

28. The method of claim 27, wherein the influenza A virus is an H5N1 subtype.

29. A method of protecting a subject from influenza A virus infection comprising administering to said subject a binding protein that binds specifically to an epitope of a glycoprotein of an influenza A virus and has substantially the immunological binding characteristics of monoclonal antibody 3H5 in an amount effective to protect the subject from influenza A virus infection.

30. The method of claim 29, wherein the binding protein is a recombinant monoclonal antibody, single chain antibody, antibody fragment, chimeric antibody, or humanized antibody.

31.The method of claim 30, wherein the binding protein is a recombinant monoclonal antibody comprising the variable region of mAb 3H5.

32. The method of claim 29, wherein the influenza A virus is an H5 subtype.

33. The method of claim 32, wherein the influenza A virus is an H5N1 subtype.