MONOCLONAL ANTIBODIES AGAINST HEMAGGLUTININ OF H5-SEROTYPE INFLUENZA VIRUSES AND THEIR USES, HYBRIDOMAS PRODUCING SAID ANTIBODIES, COMPOSITIONS AND DIAGNOSTIC KITS
20190352375 ยท 2019-11-21
Inventors
- Violetta SACZYNSKA (Warszawa, PL)
- Violetta CECUDA-ADAMCZEWSKA (Warszawa, PL)
- Anna POREBSKA (Warszawa, PL)
- Katarzyna FLORYS (Warszawa, PL)
- Anna BIERCZYNSKA-KRZYSIK (Warszawa, PL)
- Piotr BARAN (Warszawa, PL)
- Agnieszka ROMANIK-CHRUSCIELEWSKA (Jozefow, PL)
- Grazyna PLUCIENNICZAK (Warszawa, PL)
- Andrzej PLUCIENNICZAK (Warszawa, PL)
- Piotr BOROWICZ (Warszawa, PL)
- Krzysztof KUCHARCZYK (Warszawa, PL)
- Boguslaw Szewczyk (Gdansk, PL)
Cpc classification
C07K2317/76
CHEMISTRY; METALLURGY
C12N2760/16111
CHEMISTRY; METALLURGY
C12N2760/16134
CHEMISTRY; METALLURGY
C12N2760/16122
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
International classification
Abstract
The object of the invention are monoclonal antibodies against hemagglutinin of H5-serotype influenza viruses selected from the group comprising G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-7-24-17 and G-7-27-18, having a broad application in immunoprophyiaxis and immunotherapy of infections evoked by H5-serotype influenza viruses in humans and animals. The invention also provides hybridomas producing said antibodies, as well as compositions and diagnostic kits containing said antibodies for the detection and typing of H5-serotype influenza viruses and antibodies against H5-serotype influenza viruses in biological samples.
Claims
1. A monoclonal antibody against hemagglutinin of H5 serotype influenza viruses, which binds to epitopes of H5 antigens, as well as its fragments and functional variants, selected from the group comprising: (i) G-1-31-22 produced by mouse hybridoma cell line G-1-31-22 deposited with the DSMZ under the number DSM ACC3292, (ii) G-2-14-10 produced by mouse hybridoma cell line G-2-14-10 deposited with the DSMZ under the number DSM ACC3293, (iii) G-5-32-5 produced by mouse hybridoma cell line G-5-32-5 deposited with the DSMZ under the number DSM ACC3294, (iv) G-6-42-42 produced by mouse hybridoma cell line G-6-42-42 deposited with the DSMZ under the number DSM ACC3295, (v) G-7-24-17 produced by mouse hybridoma cell line G-7-24-17 deposited with the DSMZ under the number DSM ACC3296, and (vi) G-7-27-18 produced by mouse hybridoma cell line G-7-27-18 deposited with the DSMZ under the number DSM ACC3297.
2. The monoclonal antibody according to claim 1, characterized in that fragments of monoclonal antibodies include Fab, Fab, F(ab)2, Fv, V.sub.H, V.sub.L, single-chain antibody molecules consisting of V.sub.L and V.sub.H domains bound to each other by peptide linker.
3. The monoclonal antibody according to claim 1, characterized in that hemagglutinin is from influenza virus of H5 serotype, including H5N1, H5N2, H5N3 and H5N9.
4. The monoclonal antibody according to claim 1, characterized in that monoclonal antibody is conjugated with analytically detectable label, prodrug, drug, therapeutic agent, peptide, protein, enzyme, virus, lipid, PEG.
5. A mouse hybridoma cell line selected from the group consisting of: (i) G-1-31-22 deposited with the DSMZ under the number DSM ACC3292, (ii) G-2-14-10 deposited with the DSMZ under the number DSM ACC3293, (iii) G-5-32-5 deposited with the DSMZ under the number DSM ACC3294, (iv) G-6-42-42 deposited with the DSMZ under the number DSM ACC3295, (v) G-7-24-17 deposited with the DSMZ under the number DSM ACC3296, and (vi) G-7-27-18 deposited with the DSMZ under the number DSM ACC3297.
6. A pharmaceutical or diagnostic composition comprising an antibody according to claim 1 and suitable carrier or label.
7. (canceled)
8. The method for in vitro diagnosis of an influenza virus H5 serotype infection, comprising contacting a biological sample with monoclonal antibody according to claim 1 and detecting binding of said antibody with H5 serotype influenza virus or H5 hemagglutinin protein.
9. A diagnostic kit for detection of an influenza virus H5 serotype infection, comprising a monoclonal antibody according to claim 1 or its fragments, variants and immunoconjugates, and reagents for detection of antibodies or their functional equivalents bound with H5 serotype influenza virus or H5 hemagglutinin protein.
10. A diagnostic kit for detection, quantification or semi-quantification of an influenza virus H5 serotype infection in a biological sample, comprising a monoclonal antibody according to claim 1.
11. A diagnostic kit for detection, quantification or semi-quantification of antibodies against H5 serotype influenza viruses in a biological sample, comprising a monoclonal antibody as defined in claim 1.
12-14. (canceled)
15. The diagnostic kit according to claim 9, in which isolated and purified antibodies or their functional equivalents are detected.
16. The diagnostic kit according to claim 11, in which isolated and purified antibodies or their functional equivalents are detected, quantified or semi-quantified.
17. A method of performing a test selected from the group consisting of an immunoenzymatic test, an immunofluorescent test, an immunochemiluminescent test, a radioimmunological test, an immunochromatographic test, an immunodiffusion test, and an immunoprecipitation test, comprising using a kit according to claim 9.
18. A method of performing a test selected from the group consisting of an immunoenzymatic test, an immunofluorescent test, an immunochemiluminescent test, a radioimmunological test, an immunochromatographic test, an immunodiffusion test, and an immunoprecipitation test, comprising using a kit according to claim 10.
19. A method of performing a test selected from the group consisting of an immunoenzymatic test, an immunofluorescent test, an immunochemiluminescent test, a radioimmunological test, an immunochromatographic test, an immunodiffusion test, and an immunoprecipitation test, comprising using a kit according to claim 11.
20. A method of performing a DIVA test to distinguish between infected animals and vaccinated animals, comprising providing a sample and contacting the sample with a kit according to claim 9.
21. A method of performing a DIVA test to distinguish between infected animals and vaccinated animals, comprising providing a sample and contacting the sample with a kit according to claim 10.
22. A method of performing a DIVA test to distinguish between infected animals and vaccinated animals, comprising providing a sample and contacting the sample with a kit according to claim 11.
Description
DESCRIPTION OF THE FIGURES
[0136]
[0137]
[0138]
[0139]
[0140]
[0141]
[0142]
[0143]
[0144]
[0145]
[0146]
[0147]
[0148]
[0149]
[0150]
[0151]
[0152]
[0153]
[0154]
[0155]
[0156] The embodiment of the invention is presented below. The embodiments presented do not limit protection of the subject patent application.
EXAMPLE 1
Procedure for Manufacturing of mAb Against Hemagglutinin of H5 Serotype Influenza Viruses.
[0157] Monoclonal antibodies were obtained using classic hybridoma method developed in mouse model by in vivo immunization. In this method, animals are vaccinated several times with the antigen. Splenocytes isolated from spleens of vaccinated animals are fused with myeloma cells in the presence of 50% PEG and 5% DMSO. Cell culturing in HAT differentiating medium causes splenocytes and non-fused myeloma cells to die, and only hybridomas cells survive. During cloning, individual hybridomas are isolated and supernatant from hybridoma cultures established from a single hybridoma is tested for the presence of antigen-specific antibodies of a given class (isotyping). Hybridoma clones can be cultured in vitro or in an exudative cancer.
[0158] In the first stage of the procedure for mAb manufacturing, mice immunisation was conducted. As the immunogen, purified H5 HA protein (17-530 aa, RRRKKR (SEQ ID NO: 13), 6His (SEQ ID NO: 14)) was used produced in mammalian expression system based on the HA sequence from A/Bar-headed Goose/Qinghai/12/05 (H5N1) strain of AIV by Immune Technology Corporation (ITC). rHA-A/H5N1/Qinghai was previously analysed using mass spectrometry, ELISA assays for antigenicity analysis and HA protein oligomerization, and hemagglutination test. Method for carrying out the tests and their results are shown in Example 2. In Example 2, properties of rHA-A/H5N1/Qinghai immunogen are also described that indicate its usefulness in the procedure of manufacturing mAb against H5 HA. According to the standard method, production of antigen-specific mAbs comprises: mice immunisation, fusion of splenocytes with mouse myeloma cells and hybridoma culture, cloning and selection.
Mice Immunization
[0159] Five, 6-weeks old female Balb/c mice were vaccinated subcutaneously with rHA (10 g/mouse) at two sites in the neck with Complete Freund Adjuvant (CFA). Emulsion of antigen with CFA in a 1:1 ratio (v/v) was prepared using two 1-mL syringes connected with needle for emulsification (Popper & Sons). Immunologically preferable consequences of using 2 syringes method for the preparation of the vaccine in a form of emulsion and the advantage of this method over other emulsification techniques is described in the literature (Koh Y. T. et al., 2006). Vaccination was conducted under anaesthesia. Another two doses of antigen, 10 g each, were given intraperitoneally in a form of PBS. The second vaccination was conducted 2 weeks after the I dose, and the third vaccination was performed 3 weeks after the II dose. The fourth vaccination was performed 17 days after the III dose by intravenous administration of rHA (10 g/mouse) in PBS.
Control of Titer of Antibodies Against H5 HA in Serum
[0160] Eleven days after the administration of the III dose and nine days before the fusion, blood was collected from the retro-orbital sinus from each of the immunized mice using heparinized capillary tubes. Serum obtained after blood centrifugation was aliquoted and frozen at 20 C. In order to titrate antibodies against H5 HA, serum samples were analysed by ELISA using HA protein used for animal vaccination (rHA-A/H5N1/Qinghai). rHA-A/H5N1/Poland of HA sequence from A/swan/Poland/305-135V08/2006 (H5N1) strain of AIV, produced by Oxford Expression Technologies (OET) in baculovirus expression system, was also used in the tests. The protein characterized by high homology to immunogen was described in detail in Example 3. To develop the plates, anti-mouse IgG antibodies specific to were used. Using highly specific secondary antibodies in the tests enabled identification of H5-specific antibodies of IgG class-only, and thus an appropriate evaluation of animals' response to the vaccination.
[0161] ELISA tests were performed with the use of microtitration plates (NUNC). rHA-A/H5N1/Qinghai (ITC), diluted in PBS do 5 g/mL, was applied to MaxiSorp plates, whereas rHA-A/H5N1/Poland (OET, series 1), diluted in PBS to 7 g/mL, to MediSorp plates. Plates with the antigens were incubated overnight at 2-8 C. Non-specific binding sites on the plates were blocked with the use of 10% FBS/PBS. Serum samples from immunised mice were tested as series of 2-fold dilutions in PBS. Negative control was serum samples from non-immunized mice. Dilution buffer was the control for non-specific binding of secondary antibodies (BLK sample). Tested and control samples were incubated on the coated plates for 2 hours at room temperature. Plates were developed by 1-hour incubation at 37 C. with HRP-labelled anti-mouse IgG antibodies, specific for chain (Sigma-Aldrich). Secondary antibodies were diluted 1:1000 in 2% BSA/PBS. TMB (Sigma-Aldrich) was used as a substrate for horseradish peroxidase. Reaction was inhibited using 0.5 M solution of H.sub.2SO.sub.4. Absorption of samples was read at =450 nm. Titer of IgG-class antibodies against H5 HA was defined as serum dilution, at which absorption (A.sub.450) reaches value 1.0.
Hybridomas Preparation, Culture and Cloning
[0162] Immunized mice were sacrificed by cervical dislocation 3 days after intravenous administration of the last boosting dose. Spleens were collected in sterile manner and splenocytes were isolated. Following cells collection and centrifugation (10 min, 1000 rpm, 5 C.), the pellet was resuspended in complete RPMI-1640, the cells were counted and their viability was evaluated in Thoma camera in the presence on trypan blue. Cells of the mouse myeloma cell line SP2/0 (ATCC) were cultured in HT medium do logarithmic phase, centrifugated at 1000 rpm, resuspended in RPMI-1640 and live cells were counted.
[0163] For the fusion, splenocytes isolated from the spleen of mouse with the highest titer of IgG class of antibodies against H5 HA in serum were chosen. Spleen and myeloma cells in RPMI-1640 were mixed in 2:1 ratio. Following centrifugation and precise removing of the supernatant, about 1 mL of the following solution was added for 1 min: 50% PEG 1500, 5% DMSO in PBS without calcium and magnesium ions, at 37 C., with gentle stirring of the pellet. The fusion was stopped by adding heated RPMI-1640 medium with the addition of antibiotics (streptomycin50 g/mL, penicillin50 U/mL)first 4 mL slowly, dropwise, and the next 16 mLrapidly. Cell suspension was centrifugated for 10 min at 1000 rpm, 5 C. The pellet was resuspended in HAT medium and transferred into eight 96 well plates containing a layer of mouse peritoneal macrophages. After a week of incubation at 37 C. in atmosphere of 5% CO.sub.2, cell cultures were feed with HAT medium, and in the following weekwith HT.
[0164] Cloning of hybridomas was performed by further dilutions. Cell suspensions were prepared containing 10 and 5 cells/mL, respectively. Each of the suspensions was aliquoted (100 L/well) in 96 well plate with a layer of macrophages. Wells were observed and those with clones originating from 1 cell were marked.
[0165] After the fusion, hybridomas were cultured in RPMI-1640 medium supplemented with 20% FBS, containing 2 mM glutamine, 2 mM sodium pyruvate, antibiotics (streptomycin50 g/mL, penicillin50 U/mL) and 100 M hypoxanthine, 2.5 M aminopterin and 16 M thymidine (HAT differentiating medium) or 100 M hypoxanthine and 16 M thymidine (HT medium), as required. During cloning, RPMI-1640 medium with the addition of 20% FBS, containing 2 mM glutamine, 2 mM sodium pyruvate and antibiotics (streptomycin50 g/mL, penicillin50 U/mL) was used. Further hybridoma culture was carried out in medium having the same composition as in cloning medium, with the exception of FBS concentration (10%). For freezing obtained clones in liquid nitrogen, complete RPMI-1640 medium containing 30% FBS and 10% DMSO was used. Cell number and viability were determined using trypan blue solution in saline. Hank's salt solution without calcium and magnesium was used for the isolation of peritoneal macrophages. All of the reagents used were company tested regarding to the lack of toxicity for cell cultures and were from Sigma-Aldrich or Gibco.
Hybridoma Selection
[0166] Hybridoma culture supernatant was analysed for the presence of H5 HA-specific antibodies. The analysis was performed by ELISA. Initial hybridoma selection was performed using rHAA/H5N1/Qinghai (ITC) and rHAA/H5N1/Poland (OET) with sequence very similar to the immunogen sequence. Further hybridoma selection was carried out by determining specificity of the generated antibodies against recombinant H5 HA proteins based on hemagglutinin ectodomain (rHA) or HA1 subunit (rHA1) with varied homology for the immunogen, which were produced in mammalian expression system (ITC). In ELISA carried out using supernatants from cultures of non-cloned hybridomas, protein with HA sequence of six H5-serotype AIV strains was used. After cloning, selection of mAbs was performed using rHA or rHA1 based on the HA sequence of nine H5-serotype AIV strains. For positive selection of cloned hybridomas H5N1, H5N2, H5N3 and H5N9 IVs (x-OvO) were also used. At each step of the procedure, antibody isotyping was performed.
[0167] Clones of mAbs with the highest diagnostic value chose as a result of selection were purified from hybridoma supernatants using affinity chromatography. Antibodies were characterized using mass spectrometry, ELISA with recombinant H5 HA proteins (rHA, rHA1) and IVs of H5 serotype. Tests using IVs of serotypes other than H5, i.e. H1-H4 and H6-H16 were also used. In order to evaluate the ability of generated mAbs to inhibit hemagglutination, HI testes were carried out.
[0168] Antigens and tests used for the selection of hybridomas generating antibodies against H5 HA, as well as the method for carrying out antibody isotyping are described in great detail in Example 3. Antigens and tests used for further selection of serotype-specific antibodies, as well as hybridomas generating those antibodies are described in Example 4. Results of mAbs selection at particular stages of the procedure are described in Example 5. Properties of selected mAbs are described in Examples 6-10.
EXAMPLE 2
Immunogen Properties
[0169] Commercially available mAbs against H5 HA are usually obtained using hybridoma method with IVs for animal vaccination (ABR/Thermo Scientific, Acris Antibodies, USBiological). In the production of mAbs, recombinant HA proteins are also used (USBiological). In order to generate antibodies with high application value in prevention, therapy and diagnostics of IV infections, antigen having viral HA-like properties has to be used. In case of production of mAbs for use in influenza diagnostics, it is desirable for the antigen to contain primarily well preserved epitopes specific for the serotype. Therefore, in the procedure of production of mAbs specific for H5 HA serotype by hybridoma method, it was of key importance to use an immunogen fulfilling above requirement.
[0170] Before starting manufacturing procedure for mAbs against H5 HA, information on commercially available H5 HA antigens was collected. Products from Immune Technology Corporation were selected, wherein the company specialises in the production of viral antigens and anti-viral antibodies for diagnostics, monitoring and therapy of viral infectious diseases. At ITC, recombinant H5 HA proteins from several strains of H5N1 virus, as well as HA of serotypes other than H5, are available. Mammalian expression system is used for antigen production. Usefulness of purified, recombinant H5 HA proteins for selection of antibodies and hybridomas producing them, was tested using ELISA for antigenicity and oligomerization studies, which is described in Example 4. In the present Example, results of testing the properties of recombinant H5 HA protein used as an immunogen are shown.
[0171] For the production of mAbs against H5 HA with the use of hybridoma method, recombinant protein was selected with HA sequence of A/Bar-headed Goose/Qinghai/12/05 (H5N1) strain of AIVs (ITC). The protein, which was expressed in mammalian cells without signal sequence, transmembrane and cytoplasmic domain, as well as cleavage region (RRRKKR (SEQ ID NO: 13)), was the fragment of HA (17-530 aa) containing HA1 subunit, part of HA2 subunit and histidine tail6His (SEQ ID NO: 14) at the C-terminus. Due to the eukaryotic origin, produced antigen was a glycoprotein, just as native HA. According to the specification, HA protein of molecular weight 75 kDA (SDS-PAGE) and at least 95% purity, is present mainly in a form of trimers/oligomers. To evaluate its usefulness in the manufacturing procedure for mAbs against H5 HA, the protein, hereinafter described as rHA-A/H5N1/Qinghai, was subjected to analysis using mass spectrometry, ELISA (for studies of HA proteins antigenicity and oligomerization) and hemagglutination test.
Mass Spectrum of rHA-A/H5N1/Qinghai
[0172] The mass spectrum of rHA-A/H5N1/Qinghai was obtained using MALDI TOF/TOF (4800 Plus, AB SCIEX) mass spectrometer. Before performing mass spectrometry, the samples were purified using ZipTip.sub.C4 (Millipore) according to the procedures included in the manufacturer's instruction: User Guide for Reversed-Phase ZipTip. The matrix was -cyano-4-hydroxycinnamic acid (Fluka) in 5 mg/mL concentration dissolved in 0.1% trifluoroacetic acid containing 50% of acetonitrile. Mass spectra were measured in the linear mode (MS Linear Positive Ion), in the range of 20-100 kDa. External calibration was achieved with ProMix3 mixture (LaserBio Labs). Method for data acquisition was established in MALDI TOF/TOF4000 Series Explorer software. Mass spectra of rHA-A/H5N1/Qinghai were processed using Gaussian filter and the procedure for signal detection with the use of Data Explorer Software (V4.9).
[0173]
Antigenicity of rHA-A/H5N1/Qinghai
[0174] rHA-A/H5N1/Qinghai antigenicity studies were conducted using commercially available mAbs and pAbs against H5 HA of influenza virus. Based on the specifications, a list of used antibodies was prepared and is presented below.
TABLE-US-00001 List A The list of monoclonal and polyclonal antibodies used in the studies of HA protein antigenicity. Origin Type Name Cat. no. Immunogen Isotype Application Monoclonal antibodies (mAbs) against H5 HA Recognise H5 HA in HI test, react with H5N1, H5N2 and H5N9 influenza viruses, do not cross-react with IVs of other HA serotypes (H1, H2, H3, H4, H6, H7, H8, H9, H10, H11, H12, H13, H14 and H15). mAb Influenza A USBiological, Purified avian influenza virus mAb HI, iELISA 1 Hemagglutinin H5 17649-41B type A (H5N1) IgG2a* DB (Avian H5N1) Specific for H5 HA protein of influenza A virus [A/Vietnam/1203/04 (H5N1) strain], do not cross-react with IVs of other HA serotypes, react with H5N1 influenza viruses, representatives of different clades and subclades. mAb Influenza A, Avian, USBiological, HA of influenza virus mAb HI, VN, 2 H5N1A/Vietnam/1203/04 17649-42C A/Vietnam/1203/04(H5N1) IgG2a* ELISA, IP, Hemagglutinin (Bird Flu) IHC, WB Recognize H5 HA in HI test. mAb Influenza A USBiological Purified avian influenza virus mAb HI 3 Hemagglutinin H5 17649-42D type A (H5N1) IgG2a* ELISA (Avian H5N1) Detect influenza A H5 antigen in viral samples. mAb Anti-Influenza A H5 ABR/Thermo Purified avian influenza virus mAb ELISA 4 Antigen Monoclonal Scientific type A (H5N1) IgG2a* Antibody MA1-81927 React with H5 HA in HI test, react with H5N1, H5N2 and H5N9 influenza viruses, do not cross-react with IVs of other HA serotypes (H1, H2, H3, H4, H6, H7, H8, H9, H10, H11, H12, H13, H14 and H15). mAb Monoclonal Antibody Acris Purified avian influenza virus mAb HI, iELISA, 5 to Influenza A Antibodies type A (H5N1) IgG2a* DB (Hemagglutinin H5) AM00942PU- H5N1 - Purified N mAb Monoclonal Antibody Acris Purified avian influenza virus mAb HI, iELISA, 6 to Influenza A Antibodies type A (H5N1) IgG2a* DB (Hemagglutinin H5) AM00944PU- H5N1 - Purified N mAb Monoclonal Antibody Acris Purified avian influenza virus mAb HI, iELISA, 7 to Influenza A Antibodies type A (H5N1) IgG2a* DB (Hemagglutinin H5) AM00945PU- H5N1 - Purified N mAb Monoclonal Antibody Acris Purified avian influenza virus mAb HI, iELISA, 8 to Influenza A Antibodies type A (H5N1) IgG2a* DB (Hemagglutinin H5) AM00941PU- H5N1 - Purified N Policlonal antibodies (pAbs) against H5 HA React with H5 HA of human and avian H5N1 influenza viruses. Cross-reactivity to other HA not tested. pAb Anti-HA (H5N1) ITC Hemagglutinin of influenza A pAb WB itd. 1 (Bar headed IT-003-005G virus (H5N1) (1-345 aa) rabbit goose/Qinghai/ (A/Bar-headed IgG* 1A/05) Goose/Qinghai/ 12/05(H5N1)) React with HA and HA2 subunit (H5N1). Cross-reactivity against other serotypes not tested. pAb Anti-HA2 (H5N1) ITC HA2 protein (H5N1) (347-523 pAb WB itd. 2 IT-003-010 aa) rabbit (A/VietNam/1203/2004(H5N1)) IgG* *purified, HIhemagglutination inhibition test, iELISAindirect ELISA (enzyme-linked immunosorbent assay), DBdot blot, VNvirus neutralization test, IPimmunoprecipitation, IHCimmunohistochemistry, WBWestern blot, ITCImmune Technology Corporation
[0175] Monoclonal antibodies were from USBiological (3 clones), ABR/Thermo Scientific (1 clone) and Acris Antibodies (4 clones). mAbs were obtained using purified H5N1 AIVs (7 clones) or recombinant protein with the HA sequence from A/Vietnam/1203/04 (H5N1) strain (1 clone) as immunogens. According to specifications, antibodies described as mAb 1 and mAb 5-8 recognize H5 HA in HI test, bind to H5N1, H5N2 and H5N9 IVs and do not cross-react with HA of H1-H4 and H6-H15 serotypes. Specificity of other mAb clones is described as the ability to recognize H5 HA in viral samples (mAb 4) or HI test (mAb 3). According to the manufacturer's information, antibodies designated as mAb 2, are specific against HA of A/Vietnam/1203/04 (H5N1) strain, react with H5N1 IVs from various clades and subclades and do not cross-react with HA of serotypes other than H5. Specifications for all mAbs used indicate, that antibodies recognize conformational epitopes.
[0176] In the immunoreactivity assays, polyclonal antibodies against HA1 and HA2 subunit of H5 hemagglutinin were also used. Antibodies, described hereafter as pAb 1 and pAb 2, were obtained using HA1 (1-345 aa) from A/Bar-headed Goose/Qinghai/12/05 (H5N1) strain and fragment of HA2 subunit (347-523 aa) of A/VietNam/1203/2004 (H5N1) strain (ITC) as immunogens, respectively. According to specifications, pAb 1 and pAb 2 may be used in Western blot-type assays. It can be expected, that pAbs against HA will recognize both conformational epitopes and linear proteins.
[0177] The assays for the reactivity of rHA-A/H5N1/Qinghai immunogen with commercial antibodies were carried out by ELISA using polystyrene PolySorp, MaxiSorp, MediSorp and MultiSorp (NUNC), as well as Ni-NTA plates (Qiagen). Polystyrene plates were coated with rHA in PBS at 5 g/mL overnight at 2-8 C. Non-specific binding sites on the plates were blocked with the use of 10% FBS/PBS. HA protein diluted in 1% BSA/PBS to 1 g/mL concentration was applied on Ni-NTA plate. In order to control the level of unspecific binding of antibodies, some wells were filled in with 1% BSA/PBS and incubated overnight at 2-8 C., in parallel to the antigen-containing wells. Because Ni-NTA plates from Qiagen are pre-blocked, those tests were carried out without blocking stage. Plate-bound antigen was tested using mAbs and pAbs against H5 HA, enumerated on the List A. Antibodies diluted to 1 g/mL in 2% BSA/PBS, were incubated in coated plates overnight at 2-8 C. Whole-molecule specific HRP-labelled pAbs against mouse IgG (Sigma-Aldrich) were used for the detection of antigen-bound mAb. -chain specific, HRP-labelled mAbs against rabbit IgG (Sigma-Aldrich) were used for the detection of antigen-bound pAb. Secondary antibodies were diluted 1:1000 with 1% BSA/PBST and incubated with the plates for 30 min (polystyrene plates) or for 45 min (Ni-NTA plates) at room temperature. TMB (Sigma-Aldrich) was used as a substrate for HRP. Reaction was stopped using 0.5 M (polystyrene plates) or 1.25 M (Ni-NTA plates) solution of H.sub.2SO.sub.4. Absorption of samples was read at =450 nm.
[0178] The results of rHA-A/H5N1/Qinghai antigenicity studies performed on MediSorp, MaxiSorp and Ni-NTA plates using commercial anti-H5 HA antibodies are shown in
rHA-A/H5N1/Qinghai Oligomerization
[0179] Presence of oligomeric forms in rHA-A/H5N1/Qinghai preparation was tested by sandwich ELISA method, using the same clone of mAb (B513M), designated as mAb 8 (Acris Antibodies), for coating and developing microtiter plates. Detecting antibodies were HRP-labelled by service company (Acris Antibodies).
[0180] To perform the test, MaxiSorp plates (NUNC) were coated with mAb 8 (Acris Antibodies, Cat. No. AM00941PU-N) in PBS (1 g/mL), overnight, at 2-8 C. Non-specific binding sites on the plate were blocked with the use of 2% BSA/PBS. Next, different samples of rHA-A/H5N1/Qinghai protein, prepared as a series of 2-fold dilutions ranging from 10 g/mL to 0.009 ng/mL in 2% BSA/PBS, and BLK sample (2% BSA/PBS), were loaded on the plate to control the level of unspecific binding. Plates were incubated overnight at 2-8 C. HRP-labelled mAb 8 (Acris Antibodies, Cat. No. AM00941HR-Cus) was used to detect oligomeric forms of HA protein. Antibodies were diluted 1:500 in 2% BSA/PBS and incubated on the plate for 1 h at 37 C. TMB (Sigma-Aldrich) was used as a substrate for HRP. Reaction was inhibited using 0.5 M solution of H.sub.2SO.sub.4. Absorption of samples was read at =450 nm.
[0181]
Hemagglutination Activity of rHA-A/H5N1/Qinghai
[0182] rHA-A/H5N1/Qinghai protein, for which oligomeric forms were found using ELISA, was subjected to an additional analysis for the presence of functional oligomers (using hemagglutination test). In this test, the ability of the viral protein to cause erythrocyte agglutination by binding with their surface receptors is used. Evaluation of erythrocyte agglutination ability is commonly used in the analyses of vaccine antigens quality.
[0183] Preparation of fresh erythrocytes collected from the blood of SPF chickens, obtained from sterile culture from Department of Poultry Diseases, National Veterinary Research Institute in Pulawy (Poland), was used in HA test. LPAIV A/turk/It/80 (H5N2) (x-OvO) certified by Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe) in Legnaro (Italy) was used as a positive control. Erythrocytes control without viral antigen (internal control for the environment) was also included in the test. The test was performed using 96 well conical bottom (V) plates (CellStar/Greiner bio-one).
[0184] In order to evaluate hemagglutination activity of rHA-A/H5N1/Qinghai, 50 l PBS-Dulbecco (Sigma-Aldrich), following by suitable amount of antigen and buffer (up to 100 L of the final volume) were added into each well of the plate. Samples containing rHA in amounts from 0.1 g to 10 g were thus prepared. Positive control was performed by serial dilutions of the suspension containing 4 hemagglutination units (HAU) H5N2 LPAIV in PBS-Dulbecco. Buffer only (PBS-Dulbecco) was added into the wells intended for erythrocytes control. Next, 50 L of 1% erythrocyte suspension in PBS-Dulbecco was added into each well and the contents of the wells were gently mixed by pipetting. The plate was incubated for 45 min at room temperature and then the results were read. According to the principle of the test, hemagglutination effect was evaluated visually by comparison of the samples with erythrocytes control. In contrary to the control erythrocytes, agglutinated erythrocytes do not sediment but form unitary suspension.
[0185] Hemagglutination test performed using chicken erythrocytes showed, that rHA-A/H5N1/Qinghai has the ability to evoke hemagglutination, similarly to H5N2 LPAIV. Hemagglutination effect was observed within whole tested range of antigen concentrations, including samples containing 0.1 g of the protein. The results indicate, that rHA-A/H5N1/Qinghai forms functional oligomers, which is the desired feature of vaccine HA. It also applies to HA proteins used as immunogens in the mAbs manufacturing procedure with the use of hybridoma method.
SUMMARY
[0186] The results of the tests indicate, that rHA-A/H5N1/Qinghai protein shares native antigen characteristics: it maintains conformational epitopes recognized by anti-H5 HA antibodies, including HI antibodies, forms oligomeric structures and has the ability to bind to cell surface receptors and agglutinate erythrocytes. This leads to the conclusion, that rHA-A/H5N1/Qinghai protein is valuable immunogen, thus rationalizing its use in the procedure of manufacturing anti-H5 HA mAbs by hybridoma method.
EXAMPLE 3
Specificity and Isotype Assays
[0187] Besides using valuable immunogen, another condition for generating mAbs of high diagnostic value is the use of adequate procedure for selection of serotype-specific antibodies. For this purpose, tests using HA proteins maintaining conformation of native antigen were developed. Two antigens were chosen for an initial selection of hybridomas producing anti-H5 HA antibodies. One of them was rHA-A/H5N1/Qinghai protein for animal vaccination. Properties of rHA-A/H5N1/Qinghai, indicting its usefulness as an immunogen, but also as an antigen for the mAbs selection, were described in detail in Example 2. Another antigen was 17-530 aa (RRRKKR (SEQ ID NO: 13), 6His (SEQ ID NO: 14)) protein with the HA sequence of A/swan/Poland/305-135V08/2006 (H5N1) strain of AIV, produced in baculovirus expression system (OET). Similarly to rHA-A/H5N1/Qinghai, recombinant HA protein from baculovirus expression system, described hereinafter as rHA-A/H5N1/Poland, was glycosylated protein.
[0188] Amino acid sequence of rHA-A/H5N1/Poland was very similar to the immunogen sequence. Using BLAST software, it was shown, that homology between hemagglutinins of A/swan/Poland/305-135V08/2006 (H5N1) and A/Bar-headed Goose/Qinghai/12/05 (H5N1) strains, measured as a percentage of amino acid sequence identity, is 99%, wherein one conservative change of amino acids is found in the signal sequence, and the other three changes are semiconservative and are found in HA1 and HA2 subunit of the protein. Usefulness of rHA-A/H5N1/Poland for verification of specificity of antibodies against H5 HA was evaluated by performing protein antigenicity and oligomerization analysis and protein's ability for hemagglutination.
Properties of rHA-A/H5N1/Poland
[0189] rHA-A/H5N1/Poland (OET) was analyzed using MALDI TOF/TOF (4800 Plus, AB SCIEX) mass spectrometer described in Example 2 for rHA-A/H5N1/Qinghai. Determined molecular weight of the antigen (OET, series 2-8) was 64 kDa (
[0190] The panel of commercially available mAbs and pAbs against H5 HA (List A), described in Example 2, was used in rHA-A/H5N1/Poland antigenicity studies. Analysis was performed by ELISA method using polystyrene plates of various hydrophilicity (NUNC) and Ni-NTA plates (Qiagen), in the same way as antigenicity studies for rHA-A/H5N1/Qinghai (Example 2).
[0191] The oligomerization status for rHA-A/H5N1/Poland (OET, series: 5, 8) was examined using a sandwich ELISA with non-labeled and HRP-labelled commercial antibodies against H5 HA, described on the List A as mAb 8 (Acris Antibodies, Cat. No. AM00941PU-N, AM00941HR-Cus). Recombinant HA was tested as a series of 2-fold dilution in the range of 10 g/mL-0.009 ng/mL. Test method is described in Example 2. The results shown in
[0192] rHA-A/H5N1/Poland (OET, series 5) was subjected to various tests in order to evaluate protein's ability for erythrocyte agglutination. HA protein produced in baculovirus expression system was tested simultaneously with rHA from mammalian expression system (rHA-A/H5N1/Qinghai). Hemagglutination test method is described in Example 2. The test was performed using chicken erythrocytes and showed, that rHA-A/H5N1/Poland has the ability to hemagglutinate. Full hemagglutination effect was observed in the samples containing from 10 g to 2.5 g of the antigen. Partial hemagglutination was observed in the samples containing 1 g of the antigen. For erythrocytes agglutination significantly higher concentration of rHA-A/H5N1/Poland than rHA-A/H5N1/Qinghai was needed (Example 2), which is in line with the results of ELISA for testing the oligomerization state of HA proteins (
[0193] In summary, rHA-A/H5N1/Poland from baculovirus expression system preserves conformational epitopes recognized by anti-H5 HA antibodies, including HI antibodies. In comparison to rHA-A/H5N1/Qinghai, this protein is characterized by the lower level of glycosylation and low degree of oligomerization, which is an important feature of viral HA. Normal conformation of HA1 subunit of the antigen derived from baculovirus expression system rationalizes its use for testing for the specificity of generated anti-H5 HA antibodies simultaneously with the mammalian expression system-derived antigen. Different way of presenting epitopes of HA1 subunit in rHA-A/H5N1/Poland than in rHA-A/H5N1/Qinghai was found preferable for the effectiveness of selection of anti-HA mAbs.
ELISA for Determining Specificity of the Obtained mAbs
[0194] ELISA was used to determine the specificity of the produced antibodies. For this purpose tests were developed using previously characterized proteins: rHA-A/H5N1/Qinghai and rHA-A/H5N1/Poland. Test optimization was performed mainly by the selection of plates for binding particular antigens. The primary criterion for test plate selection was the availability of important conformational epitopes in the plate-bound protein, assessed using commercially available anti-H5 HA mAbs and pAbs (List A). At the stage of test optimization, all types of protein immobilization surfaces offered by NUNC (PolySorp, MediSorp, MaxiSorp, MultiSorp), as well as Ni-NTA plates (Qiagen) were used. Antigens are adsorbed on the polystyrene plates by different fragments of the protein, mainly in a random manner, whereas antigen immobilization on Ni-NTA plates is directional. Tests for optimization of antibody specificity assays were performed in the same conditions as used in the studies of rHA-A/H5N1/Qinghai (Example 2) and rHA-A/H5N1/Poland (Example 3) antigenicity.
[0195] With the use of polystyrene plates of different hydrophilicity it was shown, that using MediSorp and MaxiSorp plates for antigen binding, particularly rHA-A/H5N1/Poland, provides better sensitivity of antibody assays than using other types of polystyrene plates (data not shown). It was also shown, that reactivity of commercial antibodies with rHA-A/H5N1/Qinghai is higher after binding to Ni-NTA plate (
[0196] Eventually, specificity of antibodies was determined by ELISA using Ni-NTA plates (Qiagen) for coating with rHA H5 derived from mammalian expression system and MediSorp plates (NUNC) for coating with rHA H5 derived from baculovirus expression system. rHA-A/H5N1/Qinghai protein diluted in 1% BSA/PBS to 1 g/mL was applied on Ni-NTA plates. MediSorp plates were coated with rHA-A/H5N1/Poland in PBS in 6.2 g/mL or 5 g/mL concentration. In order to control the level of unspecific binding of antibodies, some wells were filled in with 1% BSA/PBS (Ni-NTA plates) or PBS (MediSorp plates) and incubated overnight at 2-8 C., in parallel to the antigen-containing wells. Because Ni-NTA strips from Qiagen are pre-blocked, testing was carried out without blocking stage. Non-specific binding sites on the MediSorp plates were blocked with the use of 10% FBS/PBS. Supernatant from non-cloned hybridomas was analysed in 1:20 dilution (Ni-NTA plates coated with rHA-A/H5N1/Qinghai) or 1:10 (MediSorp plates coated with rHA-A/H5N1/Poland). After cloning of hybridomas, culture supernatants were analysed without dilution. Commercial anti-H5 HA antibodies diluted to 0.01 g/mL in PBS, were used as positive controls (mAb 8, List A). RPMI buffer was the control for non-specific binding of secondary antibodies (BLK sample). Plates with the tested and control samples were incubated overnight at 2-8 C. HRP-labelled anti-mouse IgG antibodies (-chain specific, Sigma-Aldrich) were used for the detection of antigen-bound mAbs. Secondary antibodies were diluted 1:1000 using 2% BSA/PBS and were incubated on the plates for 1 h at 37 C. TMB (Sigma-Aldrich) was used as a substrate for HRP. Reaction was stopped using 1.25 M (Ni-NTA strips) or 0.5 M (MediSorp plates) solution of H.sub.2SO.sub.4. Absorption of samples was read at =450 nm.
Isotyping of the Obtained Antibodies
[0197] Classes and subclasses of the obtained mAbs were determined using Mouse Monoclonal Antibody Isotyping ReagentsISO-2 isotyping kit (Sigma-Aldrich). MaxiSorp plates (NUNC) were coated with antibodies against mouse antibodies of various classes and subclasses (IgG1, IgG2a, IgG2b, IgG3, IgA, IgM) in 1:1000 dilution in PBS, wherein the incubation was carried out for 1 h at 37 C. or overnight at 2-8 C. Non-diluted or diluted samples from hybridoma culture supernatants were incubated in the coated plates (1 h, 37 C.). Control sample was PBS or RPMI (BLK sample). Whole molecule-specific (1:1000) or Fab fragment-specific (1:50 000), diluted in 1% BSA/PBST, HRP-labelled antibodies against mouse IgG antibodies (Sigma-Aldrich), were used for the detection of antibodies bound by the isotype-specific antibodies. Plates with the diluted antibodies were incubated for 30 min at room temperature. TMB (Sigma-Aldrich) was used as a substrate for HRP. Reaction was inhibited using 0.5 M solution of H.sub.2SO.sub.4. Absorption of samples was read at =450 nm. Described procedure is a modified version on manufacturer's instruction for ISO-2 kit. Implemented changes were related to the conditions of sample incubation on the plate, as well as to the antibodies and substrate used to develop the plates.
EXAMPLE 4 ANALYSIS OF SEROTYPE SPECIFICITY
[0198] Development of immunological tests, which may be used to detect infection with IVs of H5 serotype (including H5N1 AIVs) in animals and humans, requires the use of mAbs having broad range of specificities against H5 HA. Therefore, antibodies specific against HA of amino acid sequence identical or very similar to the sequence of immunogen, were further selected for serotype specificity. For this purpose, tests were developed using different forms of H5 HA antigens (positive selection) and HA of serotypes other than H5 (negative selection). For positive selection, recombinant H5 HA proteins based on the ectodomain (rHA) or HA1 subunit (rHA1) of hemagglutinin, having varied homology to the immunogen and AIVs of H5 serotype (H5N1, H5N2, H5N3 and H5N9) were used. Negative selection was performed using AIVs of fifteen serotypes other than H5 (H1-H4 and H6-H16).
Recombinant H5 HA Proteins
[0199] Antigens used in the serotype specificity studies, described as ectodomain-based HA proteins, corresponded to H5 HA from which signal sequence, transmembrane domain and cytoplasmic domain were removed. Antigen fragments corresponding to HA1 subunit, which is characterized by the greater sequence variability than HA2 subunit and contains serotype-specific epitopes, were also present in the panel of HA proteins. The use of antigens described as HA1 subunit-based HA proteins, was increasing probability of finding clones specific against HA of H5 serotype among selected antibodies. Recombinant HA proteins based on the ectodomain (rHA) and HA1 subunit (rHA1) of the antigen, were produced in mammalian expression system (ITC). For the analysis of serotype specificity of produced antibodies, proteins were selected based on the variation of the homology to the immunogen (rHA-A/H5N1/Qinghai). Studies on the specificity of anti-H5 HA antibodies with varied homology were carried out simultaneously to studies on the specificity towards antigens having amino acid sequence identical or significantly similar to the sequence of the immunogen, which were generated using mammalian (rHA-A/H5N1/Qinghai) and baculovirus (rHA-A/H5N1/Poland) expression system. In total, in the analysis of serotype specificity, recombinant proteins with HA sequences from nine IVs of H5 serotype, including H5N1 (8 strains) and H5N2 (1 strain) were used. Information regarding antigens used in the analysis of specificity of generated antibodies, including serotype specificity, is shown below, on the List.
TABLE-US-00002 List B Recombinant hemagglutinin proteins used in the analysis of serotype specificity of generated antibodies. Influenza virus strain of H5 serotype Hemagglutinin Genbank Accession No. HA fragment Antigen (EpiFluDatabase Accession No.) expression system Origin Hemagglutinin ectodomain-based recombinant proteins (rHA) rHA - A/H5N1/Qinghai A/Bar-headed 17-530 aa, RRRKKR ITC Goose/Qinghai/12/05 (H5N1) (SEQ ID NO: 13), 6x His ABE68927 (SEQ ID NO: 14) mammalian rHA - A/H5N1/India A/chicken/India/NIV33487/ 17-530 aa, RRRKKR ITC 2006(H5N1) (SEQ ID NO: 13), 6x His ABQ45850 (SEQ ID NO: 14) mammalian rHA - A/H5N1/Vietnam A/Vietnam/1203/2004(H5N1) 18-530 aa, RRRKKR ITC AAW80717 (SEQ ID NO: 13), 6x His (SEQ ID NO: 14) mammalian rHA - A/H5N1/Guiyang A/goose/Guiyang/337/2006(H5N1) 17-530 aa, RRRKKR ITC ABJ96698 (SEQ ID NO: 13), 6x His (SEQ ID NO: 14) mammalian rHA - A/chicken/Vietnam/NCVD- 18-534 aa, 6x His (SEQ ID ITC A/H5N1/Ck/Vietnam 016/08(H5N1) NO: 14) ACO07033 mammalian rHA - A/American green-winged teal/ 19-506 aa, 6x His ITC A/H5N2/California California/HKWF609/2007(H5N2) mammalian ACF47563 rHA - A/H5N1/Poland A/swan/Poland/305- 17-530 aa, RRRKKR OET 135V08/2006(H5N1) (SEQ ID NO: 13), 6x His (EPI156789) (SEQ ID NO: 14) baculovirus Hemagglutinin HA1 subunit-based recombinant proteins (rHA1) rHA1 - A/Vietnam/1203/2004(H5N1) 1-345 aa, 6x His (SEQ ID ITC A/H5N1/Vietnam AAW80717 NO: 14) mammalian rHA1 - A/H5N1/HK/156 A/Hong Kong/156/97(H5N1) 18-346 aa, 6x His (SEQ ID ITC AAC32088 NO: 14) mammalian rHA1 - A/H5N1/HK/483 A/Hong Kong/483/97(H5N1) 17-346 aa, 6x His(SEQ ID ITC AAC32099 NO: 14) mammalian ITCImmune Technology Corporation (USA) OETOxford Expression Technologies (Great Britain)
[0200] Ectodomain-based HA proteins (rHA) from mammalian expression system were the fragments of antigen containing HA1 subunit and part of the HA2 subunit, expressed without signal sequence (17-530 aa, 18-530 aa, 18-534 aa, 19-506 aa). Cleavage regions (RRRKKR (SEQ ID NO: 13)) were removed in four out of six HA antigens and, according to the specification, those proteins were mainly in the form of trimers/oligomers. HA1 subunit-based recombinant HA proteins (rHA1) were expressed in mammalian cells with (1-345 aa) or without (18-346 aa, 17-346 aa) signal sequence. Conformation of rHA1 proteins was not specified. All recombinant HA proteins contained 6His (SEQ ID NO: 14) tag. Antigens were of at least 95% purity. On the electrophoregrams of purified antigens, rHA and rHA1 from mammalian expression system were present as single bands having molecular weight of 75 kDa and 50 kDa, respectively.
[0201] Usefulness of rHA and rHA1 antigens, as shown on List B, for testing serotype specificity of the obtained antibodies was evaluated by performing tests for antigenicity and oligomerization state. Method for testing antigenicity of rHA-A/H5N1/Qinghai and rHA-A/H5N1/Poland is shown in Examples 2 and 3, respectively. As in the case of rHA-A/H5N1/Qinghai and rHA-A/H5N1/Poland, tests for the antigenicity of rHA and rHA1 were performed using commercially available anti-H5 HA antibodies. mAbs recognising native HA, mAbs showing properties of serotype-specific antibodies and mAbs active in HI test were incorporated into the panel of antibodies. Commercial mAbs and pAbs used for the characterization of HA proteins are shown in the List A and described in detail in Example 2.
[0202] Antigenicity tests for rHA proteins having sequences of A/H5N1/India, A/H5N1/Vietnam, A/H5N1/Guiyang, A/H5N1/Ck/Vietnam, A/H5N2/California strains, as well as rHA1 proteins having sequences of A/H5N1/Vietnam, A/H5N1/HK/156 and A/H5N1/HK/483 strains were performed by ELISA using Ni-NTA plates (Qiagen). HA proteins diluted in 1% BSA/PBS to 1 g/mL concentration were applied on the plates. In order to control the level of unspecific binding of antibodies, some wells were filled in with 1% BSA/PBS and incubated overnight at 2-8 C., in parallel to the antigen-containing wells. In the antigenicity tests for seven out of eight recombinant H5 HA proteins mentioned above, all anti-H5 HA mAbs and pAbs from List A were used. Antibodies diluted to 1 g/mL in 2% BSA/PBS, were incubated in coated plates overnight at 2-8 C. To develop the plates, whole molecule-specific HRP-labelled pAbs against mouse IgG (Sigma-Aldrich) or HRP-labelled monoclonal antibodies against rabbit IgG, -chain specific (Sigma-Aldrich) were used. The secondary antibodies were diluted 1:1000 in 1% BSA/PBST and incubated in the plates for 45 min at room temperature. rHA1-A/H5N1/HK/156 antigenicity tests were performed using antibodies described as mAb 8, diluted to 0.1 g/mL in 2% BSA/PBS. Antigen-bound mAbs were detected using whole molecule-specific, HRP-labelled, anti-mouse IgG pAbs (Sigma-Aldrich), diluted 1:1000 in 2% BSA/PBS and incubated with the plate for 60 min at 37 C. TMB (Sigma-Aldrich) was used as a substrate for horseradish peroxidase. Reaction was inhibited using 1.25 M solution of H.sub.2SO.sub.4. Absorption of samples was read at =450 nm. ELISA was not optimized for particular antigens and antibodies.
[0203]
[0204] Studies of rHA1-A/H5N1/HK/156 antigenicity with the use of mAb 8 having a broad range of specificities against H5 HA (
[0205] The results of antigenicity analysis for recombinant HA proteins, showed in
[0206] Tests for the presence of oligomeric forms in rHA and rHA1 antigen were performed by sandwich ELISA, identically as in the oligomerization studies for rHA-A/H5N1/Qinghai and rHA-A/H5N1/Poland. Test conditions are described in Example 2. The results for ten recombinant H4 HA proteins from List B are shown in
H1-H16 Serotype Influenza Viruses
[0207] AIVs were used in the analysis of serotype specificity of generated antibodies. The viruses (x-OvO) had IZSVe certificates. The list of used AIVs is given below, in the List.
TABLE-US-00003 List C Avian influenza viruses used in the analysis of serotype specificity of generated antibodies. Hemagglutinin Avian influenza virus serotype serotype Strain Origin H1 H1N1 A/duck/It/1447/05(H1N1) x-OvO H2 H2N3 A/duck/Germ/1215/73(H2N3) x-OvO H3 H3N8 A/pass/It/6000/V00(H3N8) x-OvO A/psitt/It/2873/00(H3N8) x-OvO H4 H4N8 A/cockatoo/Eng/72(H4N8) x-OvO H5 H5N1 A/mallard/It/3401/05(H5N1) x-OvO H5N2 A/turk/It/80(H5N2) x-OvO H5N3 A/duck/It/775/04(H5N3) x-OvO H5N9 A/ck/It/22A/98(H5N9) x-OvO H6 H6N2 A/turkey/Canada/65 (H6N2) x-OvO H7 H7N1 A/ck/It/1067/V99(H7N1) x-OvO H7N3 A/ty/It/9289/V02(H7N3) x-OvO H7N4 A/mallard/It/4810-79/04(H7N4) x-OvO H7N7 A/macaw/626/80(H7N7) x-OvO H8 H8N4 A/turk/Ont/6118/68(H8N4) x-OvO H9 H9N2 A/ty/Wis/66(H9N2) x-OvO H9N7 A/turk/Scotland/1/70(H9N7) x-OvO H10 H10N1 A/ostrich/SA/01(H10N1) x-OvO H11 H11N6 A/duck/Eng/56(H11N6) x-OvO H11N9 A/duck/Memphis/546/174(H11N9) x-OvO H12 H12N5 A/duck/Alberta/60/76(H12N5) x-OvO H13 H13N6 A/gull/Maryland/704/77(H13N6) x-OvO H14 H14N5 A/mallard/Gurjev/263/82(H14N5) x-OvO H15 H15N9 A/shearwater/2576/79(H15N9) x-OvO H16 H16N3 A/gull/Denmark/68110/02(H16N3) x-OvO x-OvO Limited (Great Britain), Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe, Italy)
[0208] Twenty-five AIV strains were used in the analysis of serotype specificity of the antibodies, including four H5 serotype AIVs, and the rest of H1, H2, H3, H4, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16 serotype.
Homology of H5 HA Antigens
[0209] In the analysis of serotype specificity of generated antibodies, hemagglutinins or their fragments having sequences of thirteen IV strains of H5 serotype (List B and C) were used. This number includes H5N3 (1 strain), H5N9 (1 strain), H5N2 (2 strains) and, above all, H5N1 viruses (9 strains), classified into six different clades, mentioned in the list below.
TABLE-US-00004 List D Classification of H5N1 influenza viruses, from which sequences of H5 HA antigen were derived. Hemagglutinin Genbank Accession No. (EpiFluDatabase Clade Influenza virus strain of H5 serotype Accession No.) IV H5N1 A/Hong Kong/156/97(H5N1) AAC32088 0 A/Hong Kong/483/97(H5N1) AAC32099 0 A/Vietnam/1203/2004(H5N1) AAW80717 1 A/Bar-headed Goose/Qinghai/12/ ABE68927 2.2 05(H5N1) A/chicken/India/NIV33487/2006(H5N1) ABQ45850 2.2 A/swan/Poland/305-135V08/2006(H5N1) (EPI156789) 2.2 A/goose/Guiyang/337/2006(H5N1) ABJ96698 4 A/chicken/Vietnam/NCVD-016/ ACO07033 7 08(H5N1) A/mallard/Italy/3401/2005(H5N1) ABG57086.1 EA- nonGsGD EA-nonGsGDH5 serotype virus, more similar to low pathogenic viruses of Eurasian origin
[0210] Homology of hemagglutinins, from which sequences of H5 HA antigens were derived, was determined against 1-567 aa (full-length protein) and 17-338 aa (HA1 subunit) fragments of the HA of A/Bar-headed Goose/Qinghai/60/05 (H5N1) strain, which is the source of immunogen sequence (rHA-A/H5N1/Qinghai). Sequences were analysed using BLAST software. Relative homologies of full-length hemagglutinins are shown in the List E, while HA1 subunits of the proteins are shown in the List F. Hemagglutinins of different IV strains are shown in decreasing Max Score order.
TABLE-US-00005 List E Homology of full-length hemagglutinins, from which sequences of H5 HA antigens were derived, against hemagglutinin of A/H5N1/Qinghai strain, determined using BLAST software. Sequences producing significant alignments: Max Total Query E Description score score cover value Ident Accession.sup.x hemagglutinin [Influenza A virus 1186 1186 100% 0.0 100% ABE68927.1 (A/Bar-headed Goose/Qinghai/60/05(H5N1))] hemagglutinin [Influenza A virus 1181 1181 99% 0.0 99% ABQ45850.1 (A/chicken/India/NIV33487/06(H5N1))] hemagglutinin [Influenza A virus 1177 1177 99% 0.0 99% (EPI156789) (A/swan/Poland/305-135V08/2006(H5N1))] hemagglutinin HA [Influenza A virus 1150 1150 99% 0.0 97% AAW80717.1 (A/Viet Nam/1203/2004(H5N1))] hemagglutinin subtype H5 [Influenza A virus 1142 1142 99% 0.0 96% AAC32088.1 (A/Hong Kong/156/97(H5N1))] hemagglutinin subtype H5 [Influenza A virus 1135 1135 99% 0.0 95% AAC32099.1 (A/Hong Kong/483/1997(H5N1))] hemagglutinin [Influenza A virus 1112 1112 99% 0.0 93% ABS89310.1 (A/duck/Italy/775/2004(H5N3))] hemagglutinin [Influenza A virus 1100 1100 98% 0.0 94% ABJ96698.1 (A/goose/Guiyang/337/2006(H5N1))] hemagglutinin [Influenza A virus 1089 1089 99% 0.0 91% ABR37720.1 (A/chicken/Italy/22A/1998(H5N9))] hemagglutinin [Influenza A virus 1082 1082 99% 0.0 89% ACF47563.1 (A/American green-winged teal/California/ HKWF609/2007(H5N2))] hemagglutinin [Influenza A virus 1067 1067 98% 0.0 91% ACS93985.1 (A/turkey/Italy/1980(H5N2))] hemagglutinin [Influenza A virus 1066 1066 99% 0.0 91% ACO07033.1 (A/chicken/Vietnam/NCVD-016/2008(H5N1))] hemagglutinin [Influenza A virus 1058 1073 96% 0.0 93% ABG57086.1 (A/mallard/Italy/3401/2005(H5N1))] .sup.xGenbank (EpiFluDatabase) Accession No.
TABLE-US-00006 List F Homology of sequences forming HA1 subunit of H5 HA antigens, against HA1 subunit of hemagglutinin from A/H5N1/Qinghai strain, determined using BLAST software. Sequences producing significant alignments: Max Total Query E Description score score cover value Ident Accession.sup.x hemagglutinin [Influenza A virus 679 696 100% 0.0 100% ABE68927.1 (A/Bar-headed Goose/Qinghai/60/05(H5N1))] hemagglutinin [Influenza A virus 677 694 100% 0.0 99% ABQ45850.1 (A/chicken/India/NIV33487/06(H5N1))] hemagglutinin [Influenza A virus 677 693 100% 0.0 99% (EPI156789) (A/swan/Poland/305-135V08/2006(H5N1))] hemagglutinin HA [Influenza A virus 654 685 100% 0.0 95% AAW80717.1 (A/Viet Nam/1203/2004(H5N1))] hemagglutinin subtype H5 [Influenza A virus 652 668 100% 0.0 95% AAC32088.1 (A/Hong Kong/156/97(H5N1))] hemagglutinin subtype H5 [Influenza A virus 646 664 100% 0.0 94% AAC32099.1 (A/Hong Kong/483/1997(H5N1))] hemagglutinin [Influenza A virus 636 652 100% 0.0 93% ABS89310.1 (A/duck/Italy/775/2004(H5N3))] hemagglutinin [Influenza A virus 635 650 100% 0.0 93% ABJ96698.1 (A/goose/Guiyang/337/2006(H5N1))] hemagglutinin [Influenza A virus 633 649 100% 0.0 93% ABG57086.1 (A/mallard/Italy/3401/2005(H5N1))] hemagglutinin [Influenza A virus 619 636 100% 0.0 90% ABR37720.1 (A/chicken/Italy/22A/1998(H5N9))] hemagglutinin [Influenza A virus 616 633 100% 0.0 88% ACF47563.1 (A/American green-winged teal/California/ HKWF609/2007(H5N2))] hemagglutinin [Influenza A virus 615 631 100% 0.0 90% ACS93985.1 (A/turkey/Italy/1980(H5N2))] hemagglutinin [Influenza A virus 605 623 100% 0.0 89% ACO07033.1 (A/chicken/Vietnam/NCVD-016/2008(H5N1))] .sup.xGenbank (EpiFluDatabase) Accession No.
[0211] Analysis of full-length hemagglutinin homology for twelve IV strains of H5 serotype against HA from A/H5N1/Qinghai strain showed significant variation of proteins (List E), from which HA antigen sequences were derived, which were used in the analysis of serotype specificity of the generated antibodies. Because serotype-specific epitopes are localised within HA1 HA subunit, high variability of sequences forming this subunit in HA antigens, as shown in homology analysis (List F), is especially important for the effective selection of the desired diagnostic monoclonal antibodies. Antigens having HA1 subunit of very high and relatively low homology against HA1 subunit of HA of A/H5N1/Qinghai strain are included in the HA protein panel. For the proteins which are the most similar to the immunogen, i.e. rHA-A/H5N1/India and rHA-A/H5N1/Poland, homology factors for HA1 subunit: Max Score, Total Score, Identities, reached values of 677, 694 and 693, and 99%, respectively. The same factors for HA1 subunit of A/H5N9/Ck/Italy AIV, rHA-A/H5N2/California, A/H5N2/Tk/Italy AIV and rHA-A/H5N1/Ck/Vietnam antigens reached values of 619-605, 636-623 and 88-90%, respectively.
ELISA for Determining Serotype Specificity of the Obtained mAbs
[0212] Clones specific for the HA having amino acid sequence identical or very similar to the immunogen sequence, i.e. to rHA-A/H5N1/Qinghai and rHA-A/H5N1/Poland, were analysed using previously characterized rHA and rHA1 proteins of various homology against the immunogen, which were obtained using mammalian expression system (ITC). Because of the presence of histidine tags in the recombinant antigens, ELISA was performed using Ni-NTA plates. Preferably for antibody detection, targeted binding of the antigen to Ni-NTA plates provides availability of different epitopes of the antigen. ELISA using MaxiSorp plates (NUNC) was used for the determination of antibody specificity against AIVs. The first supernatant analyses for the presence of anti-AIV of H5 serotype (x-OvO) were performed only after hybridoma cloning (Example 5). Full characterization of antibody specificity against AIVs of H1, H2, H3, H4, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16 serotype (x-OvO), was performed only for selected and purified clones of mAbs (Example 7). Similarly to tests for the analysis of antibody specificity against H5 HA (Example 3), highly specific anti-mouse antibodies were used as detecting antibodies in the tests for analysis of serotype-specificity of antibodies using rHA, rHA1 and various AIV strains. Thus, antibody selection was limited to the serotype-specific antibodies of the desired class, i.e. IgG.
ELISAs Using Recombinant HA Proteins
[0213] In order to perform ELISA using recombinant HA proteins, rHA and rHA1, including rHA-A/H5N1/Qinghai (ITC), were diluted in 1% BSA/PBS and then loaded into Ni-NTA plates (Qiagen). Some wells of the plate were filled in with 1% BSA/PBS and incubated overnight at 2-8 C., in parallel to the antigen-containing wells. Supernatant of non-cloned hybridomas was analyzed without dilution using plates coated with rHA and rHA1 in 1 g/mL. After hybridoma cloning, culture supernatant was analysed without dilution and/or after dilution in PBS on the plates coated with rHA and rHA1 in 1 and 5 g/mL concentration, respectively. Samples for reactivity tests with rHA having sequences of A/H5N1/Qinghai, A/H5N1/India, A/H5N1/Vietnam and A/H5N1/Guiyang strains were diluted 1:3000 or 1:3000 and 1:5000. For the tests on rHA-A/H5N2/California-coated plates, supernatants were analysed without dilution or, additionally, in 1:1500 and 1:3000 dilutions. Samples of supernatants for reactivity analysis with the remaining HA antigens were not diluted. In order to control the level of non-specific binding of antibodies, samples prepared from hybridoma culture supernatants were additionally analysed in non-coated wells of the plate. Assay was carried out in the presence of control samples. Positive control was commercial anti-H5 HA antibodies (mAb 8, List A), whereas negative control was RPMI buffer (BLK sample). Plates with the tested and control samples were incubated overnight at 2-8 C. HRP-labelled anti-mouse IgG antibodies (-chain specific, Sigma-Aldrich) were used for the detection of antigen-bound mAbs. Secondary antibodies were diluted 1:1000 in 1% BSA/PBST or 2% BSA/PBS and incubated in the plates for 45 min at room temperature or for 60 min at 37 C. TMB (Sigma-Aldrich) was used as a substrate for HRP. Reaction was inhibited using 1.25 M solution of H.sub.2SO.sub.4. Absorption of samples was read at =450 nm.
[0214] The results of reactivity studies for commercial mAbs and pAbs on Ni-NTA plates coated with rHA and rHA1 with the HA sequence of A/H5N1/Vietnam strain in 1 g/mL concentration (
ELISAs Using Influenza Viruses of H1-16 Serotypes
[0215] To perform ELISAs, AIV preparations (x-OvO) were diluted in PBS to 4000 HAU/mL based on the value specified by the manufacturer, and then loaded on the MaxiSorp plates (NUNC). Plates with wells containing viral antigen were incubated overnight at 2-8 C., in parallel to PBS-filled wells. Efficiency of coating with particular viruses could be surprisingly varied due to the observed differences in the composition of preparations. After incubation, plates were blocked with 2% BSA/PBS. Hybridoma culture supernatants were analysed without dilution in the plates coated with AlVs of H5 serotype (positive selection). Purified mAb clones were diluted to 20 g/mL in 2% BSA/PBS and analysed in the plates coated with AIVs of H5 serotype (positive selection) and of H1-H4 and H5-H16 serotypes (negative selection). A part of each negative selection plate was coated with H5N3 and H5N9 viruses to confirm correct testing for IVs of serotypes other than H5. Samples containing mAbs and BLK samples (RPMI buffer and/or 2% BSA/PBS) loaded into antigen-coated and non-coated wells of the plate, respectively, provide controls for non-specific binding of antibodies. The control was commercial anti-H5 HA antibodies (mAb 8, List A) diluted to 0.05, 0.01, 0.005 and 0.001 g/mL concentration in 2% BSA/PBS during testing of culture supernatants and to 20 g/mL during testing of purified mAbs. Plates with the obtained mAbs and control samples were incubated overnight at 2-8 C. HRP-labelled anti-mouse IgG antibodies (-chain specific, Sigma-Aldrich) were used for the detection of antigen-bound mAbs. Secondary antibodies were diluted 1:1000 in 2% BSA/PBS and incubated on the plates for 1 h at 37 C. TMB (Sigma-Aldrich) was used as a substrate for HRP. Reaction was inhibited using 0.5 M solution of H.sub.2SO.sub.4. Absorption of samples was read at =450 nm.
SUMMARY
[0216] Screening of monoclonal antibodies for serotype-specificity was performed with ELISA method using previously characterized H5 HA antigens (positive selection) and IVs of serotypes other than H5 (negative selection). For positive selection, recombinant H5 HA proteins were used based on the ectodomain (rHA) or HA1 subunit (rHA1) of hemagglutinin, described in the List B. Recombinant antigens differed in the amino acid sequence and properties. Individual rHA and rHA1 proteins showed characteristic profiles of recognition by commercial anti-HA antibodies (
[0217] In the analysis of serotype specificity of generated antibodies, hemagglutinins were used or their fragments showing characteristics of native antigen, which contained HA sequences of twelve IV strains of H5 serotype (List B and C). This number includes H5N3 (1 strain), H5N9 (1 strain), H5N2 (2 strains) and H5N1 viruses (8 strains), classified into five clades: 0, 1, 2.2, 4 and EA-nonGsGD (List D). Homology of HA1 subunit of conformational HA antigens to HA1 subunit of the immunogen (rHA-A/H5N1/Qinghai), measured as the percentage of identical amino acids in the sequence, was from 99% to 88% (List F). It could be assumed, that the use of different forms of H5 HA antigens with varied homology will enable to evaluate serotype-specificity of novel mAb clones, as the tests for the reactivity of commercial antibodies with recombinant H5 HA proteins revealed the range of specificities of individual clones against H5 hemagglutinins (
[0218] In summary, strategy used for the selection of mAbs provided high chance of identification of antibody clones recognizing different epitopes specific for H5 HA, which would react with IVs of H5 serotype, which belong to different clades and subclades, and at the same time would not cross-react with viruses of serotypes other than H5.
EXAMPLE 5 THE RESULTS OF ANTIBODY SELECTION
[0219] As a result of the use of procedure for obtaining mAbs using rHA-A/H5N1/Qinghai for mice immunization, described in Example 1, 440 hybridomas were obtained, including 58, which produced anti-H5 HA antibodies. Specificities were determined by ELISA using two antigens: rHA-A/H5N1/Qinghai from mammalian expression system (ITC) and rHA-A/H5N1/Poland from baculovirus expression system (OET). Test conditions are described in Example 3. Supernatants from hybridoma cultures containing rHA-A/H5N1/Qinghai-recognizing antibodies were subjected to further analysis using recombinant HA proteins based on the ectodomain (rHA) and HA1 subunit (rHA1) of hemagglutinin, which were produced in mammalian cells (ITC). Assay was performed as described in Example 4. The results indicating correct conformation of HA proteins used are shown in Examples 2, 3 and 4. Antigens contained HA sequences of five H5N1 virus strains and one H5N2 virus strain. Antigen sequences were analysed in BLAST by comparison with HA sequence of A/Bar-headed Goose/Qinghai/60/05 (H5N1) strain. Homology of full-length, source sequences for HA antigens, expressed as percentage of identical amino acids, was from 99% to 89%, and from 99% to 88% for HA1 subunit-forming sequences. Table 1, below, shows the result of serotype specificity assays for the obtained antibodies.
[0220] In particular, Table 1 shows the test results for immunoreactivity of antibodies produced by non-cloned hybridomas. Samples from hybridoma supernatants were tested by ELISA using recombinant proteins based on ectodomain (rHA) and HA1 subunit (rHA1) of hemagglutinin. rHA-A/H5N1/Poland protein was produced in baculovirus expression system (OET, series 1). The remaining rHA and rHA1 proteins were obtained from mammalian expression system (ITC). MediSorp (rHA-A/H5N1/Poland) plates from NUNC and Ni-NTA plates (rHA, rHA1 from mammalian expression system) from Qiagen were used for coating with HA antigens. Homology of hemagglutinins, from which sequences of H5 HA antigens were derived, was determined against 1-567 aa (full-length protein) and 17-338 aa (HA1 subunit) fragments of the HA of A/Bar-headed Goose/Qinghai/60/05 (H5N1) strain, which is the source of immunogen sequence (rHA-A/H5N1/Qinghai). Sequences were compared using BLAST software and expressed as percentage of identical amino acids.
TABLE-US-00007 TABLE 1 HA antigens Homology HA Ident [%] oryginal sequence HA1- HA1 58 hybridomas before cloning (IV strain, H5N1 AIV clade) 567 aa 17-338 aa 25 3 3 9 4 7 3 2 1 1 Hemagglutinin ectodomain-based proteins from mammalian expression system (rHA) rHA - A/H5N1/Qinghai 100 100 + + + + + + + + + + A/Bar-headed Goose/Qinghai/12/05 clade 2.2 rHA - A/H5N1/India 99 99 + + + + + + + + + + A/chicken/India/NIV33487/2006 clade 2.2 rHA - A/H5N1/Vietnam 97 95 + + + + + + + + + + A/Vietnam/1203/2004 clade 1 rHA - A/H5N1/Guiyang 94 93 + + + + + + A/goose/Guiyang/337/2006 clade 4 rHA - A/H5N2/California 89 88 + + + + + A/American green-winged teal/ California/HKWF609/2007 Hemagglutinin ectodomain-based protein from baculovirus expression system (rHA) rHA - A/H5N1/Poland 99 99 + + + + + + + A/swan/Poland/305-135V08/2006 clade 2.2 Hemagglutinin HA1 subunit-based protein from mammalian expression system (rHA1) rHA1 - A/H5N1/Vietnam 95 + + + A/Vietnam/1203/2004 clade 1 6 hybridomas were selected for cloning
[0221] Antibodies produced by the most of non-cloned hybridomas (31/58) recognized recombinant proteins from mammalian and baculovirus expression system, including both rHA and rHA1-A/H5N1/Vietnam. Few hybridomas (6/31) were found to produce antibodies which do not detect proteins with hemagglutinin sequences of the lowest homology to the immunogen, i.e. rHA-A/H5N2/California or rHA-A/H5N1/Guiyang. Tests of the supernatants from the remaining cultures of primary hybridomas (27/58) did not show the presence of rHA1-A/H5N1/Vietnam-recognizing antibodies. Positive signal from rHA-A/H5N1/Vietnam and no signal from rHA1-A/H5N1/Vietnam, observed in the tests of all supernatants from cultures of this group of hybridomas, does not have to indicate, that mAbs are directed against HA2 subunit of the antigen. Rationale behind this interpretation is shown in Example 4. Among 27 hybridomas for which no production of antibodies against rHA1-A/H5N1/Vietnam was shown, 13 produced antibodies recognizing all used rHA proteins from mammalian expression system, while antibodies produced by the remaining 14 hybridomas did not show reactivity against rHA-A/H5N2/California and/or rHA-A/H5N1/Guiyang with the lowest homology to the immunogen. No antibodies recognising baculovirus expression system-derived rHA antigen having HA sequence of the HA from A/H5N1/Poland strain were found in the culture supernatants of some hybridomas (8/27), despite high homology to the immunogen.
[0222] Taking into account, that serotype-specific epitopes are localised in HA1 subunit of HA, and reactivity with antigens of varied homology indicates the range of specificities of the obtained antibodies, the most promising material for further screening was the group of 25 primary hybridomas, which produced antibodies recognizing all antigens used in the serotype-specificity test. From this group, six hybridomas were chosen for cloning: G-1-31, G-2-14, G-5-32, G-6-42, G-7-24 and G-7-27. Immunoreactivity profiles obtained in the tests of supernatants from cultures of selected hybridomas are shown below, in Table 2. The results are shown as signal values (A.sub.450) obtained in ELISAs using recombinant HA proteins.
[0223] In particular, Table 2 shows the results of immunoreactivity tests of antibodies produced by hybridomas selected for cloning. Tests were performed by ELISA using recombinant proteins based on ectodomain (rHA) and HA1 subunit (rHA1) of hemagglutinin. rHA-A/H5N1/Poland protein was produced in baculovirus expression system (OET, series 1). The remaining rHA and rHA1 proteins were obtained from mammalian expression system (ITC). MediSorp (rHA-A/H5N1/Poland) plates from NUNC and Ni-NTA plates (rHA, rHA1 from mammalian expression system) from Qiagen were used for coating with HA antigens. The results are shown as signal values (A.sub.450) obtained from the analyses of hybridoma culture supernatants. Homology of hemagglutinins, from which sequences of H5 HA antigens were derived, was determined against 1-567 aa (full-length protein) and 17-338 aa (HA1 subunit) fragments of the HA of A/Bar-headed Goose/Qinghai/60/05 (H5N1) strain, which is the source of immunogen sequence (rHA-A/H5N1/Qinghai). Sequences were compared using BLAST software and expressed as percentage of identical amino acids.
TABLE-US-00008 TABLE 2 HA antigens Homology HA Ident [%] oryginal sequence HA1- HA1 Hybridomas selected for cloning (no.) (IV strain, H5N1 AIV clade) 567 aa 17-338 aa G-1-31 G-2-14 G-5-32 G-6-42 G-7-24 G-7-27 Hemagglutinin ectodomain-based proteins from mammalian expression system (rHA) rHA - A/H5N1/Qinghai 100 100 >4.0 >4.0 >4.0 >4.0 >4.0 >4.0 A/Bar-headed Goose/Qinghai/12/05 clade 2.2 rHA - A/H5N1/India 99 99 >4.0 >4.0 >4.0 >4.0 >4.0 >4.0 A/chicken/India/NIV33487/2006 clade 2.2 rHA - A/H5N1/Vietnam 97 95 >4.0 >4.0 >4.0 >4.0 >4.0 >4.0 A/Vietnam/1203/2004 clade 1 rHA - A/H5N1/Guiyang 94 93 >4.0 >4.0 >4.0 >4.0 >4.0 >4.0 A/goose/Guiyang/337/2006 clade 4 rHA - A/H5N2/California 89 88 >4.0 >4.0 >4.0 >4.0 >4.0 3.784 A/American green-winged teal/ California/HKWF609/2007 Hemagglutinin ectodomain-based protein from baculovirus expression system (rHA) rHA - A/H5N1/Poland 99 99 3.143 2.594 3.220 3.343 3.104 3.335 A/swan/Poland/305-135V08/2006 clade 2.2 Hemagglutinin HA1 subunit-based protein from mammalian expression system (rHA1) rHA1 - A/H5N1/Vietnam 95 1.532 1.244 1.861 3.638 1.670 1.975 A/Vietnam/1203/2004 clade 1
[0224] In the tests for serotype-specificity of antibodies produced by the selected hybridoma using rHA antigen from mammalian expression system and rHA-A/H5N1/Poland from baculovirus expression system, signals (A.sub.450) of very high (A.sub.450>4) and high (2.6-3.3) values were obtained. Using rHA1-A/H5N1/Vietnam for tests, lower A.sub.450 values were read than in case of rHA1-A/H5N1/Vietnam (1.2-3.6 vs >4), which confirms earlier observations regarding lower detection sensitivity for antibodies against HA1 subunit of HA with the use of short fragments of the protein (1-345 aa) under applied test conditions.
[0225] Cloning of selected hybridomas: G-1-31, G-2-14, G-5-32, G-6-42, G-7-24 and G-7-27 provided 64 clones producing anti-H5 HA antibodies. Specificity of the obtained mAbs against rHA-A/H5N1/Qinghai and rHA-A/H5N1/Poland and their isotyping were determined by ELISA, as described in Example 3. Hybridoma culture supernatants were subjected to further analysis for the presence of serotype-specific antibodies. Comparing to the tests performed with the use of supernatants from uncloned hybridoma cultures, tests for serotype-specificity of antibodies produced by cloned hybridomas was performed using panel of HA antigens, which was broadened by two recombinant, HA1 subunit-based proteins from mammalian expression system: rHA1-A/H5N1/HK/156 and rHA1-A/H5N1/HK/483 (ITC) and AlVs: H5N1, H5N2, H5N3 and H5N9 (x-OvO). The results of antigenicity analysis indicating correct conformation of rHA1 antigens used are shown in
[0226] In particular, Table 3 shows the test results for immunoreactivity of antibodies produced by cloned hybridomas. Tests were performed by ELISA using recombinant proteins based on ectodomain (rHA) and HA1 subunit (rHA1) of hemagglutinin, as well as AIV of H5 serotype. rHA-A/H5N1/Poland protein was produced in baculovirus expression system (OET). The remaining rHA and rHA1 proteins were produced in mammalian expression system (ITC). Influenza viruses of H5 serotype (x-OvO) were derived from IZSVe. MediSorp (rHA-A/H5N1/Poland, series 3), MaxiSorp (AIV) plates from NUNC and Ni-NTA plates (rHA, rHA1 from mammalian expression system) from Qiagen were used for coating with HA antigens. Homology of hemagglutinins, from which sequences of H5 HA antigens were derived, was determined against 1-567 aa (full-length protein) and 17-338 aa (HA1 subunit) fragments of the HA of A/Bar-headed Goose/Qinghai/60/05 (H5N1) strain, which is the source of immunogen sequence (rHA-A/H5N1/Qinghai). Sequences were compared using BLAST software and expressed as percentage of identical amino acids.
TABLE-US-00009 TABLE 3 HA antigens Recombinant HA protein original sequence Hybridomas before cloning (no.) (IV strains, H5N1 AIV clade) Homology HA Ident [%] G-1-31 G-2-14 G-5-32 G-6-42 G-7-24 G-7-27 Influenza virus HA1- HA1 64 hybridomas after cloning (serotype, IV strain, H5N1 AIV clade) 567 aa 17-338 aa 22 1 10 6 14 11 Hemagglutinin ectodomain-based proteins from mammalian expression system (rHA) rHA - A/H5N1/Qinghai 100 100 + + + + + + A/Bar-headed Goose/Qinghai/12/05, clade 2.2 rHA - A/H5N1/India 99 99 + + + + + + A/chicken/India/NIV33487/2006, clade 2.2 rHA - A/H5N1/Vietnam 97 95 + + + + + + A/Vietnam/1203/2004, clade 1 rHA - A/H5N1/Guiyang 94 93 + + + + + + A/goose/Guiyang/337/2006, clade 4 rHA - A/H5N1/Ck/Vietnam .sup.I 91 89 A/chicken/Vietnam/NCVD-016/08, clade 7 rHA - A/H5N2/California 89 88 + + + + + + A/American green-winged teal/ California/HKWF609/2007 Hemagglutinin ectodomain-based protein from baculovirus expression system (rHA) rHA - A/H5N1/Poland 99 99 + + + + + + A/swan/Poland/305-135V08/2006, clade 2.2 Hemagglutinin HA1 subunit-based proteins from mammalian expression system (rHA1) rHA1 - A/H5N1/Vietnam 95 + + + + + + A/Vietnam/1203/2004, clade 1 rHA1 - A/H5N1/HK/156 95 + + + + + + A/Hong Kong/156/97, clade 0 rHA1 - A/H5N1/HK/483 94 + + + + + + A/Hong Kong/483/1997, clade 0 H5 serotype influenza viruses IV - H5N3 93 93 + + + + + + A/duck/Italy/775/2004 IV - H5N1 93 93 + + + + + + A/mallard/Italy/3401/2005, clade EA-nonGsGD IV - H5N9 91 90 + + + + + + A/chicken/Italy/22A/1998 IV - H5N2 91 90 + + + + + + A/turkey/Italy/1980 mAb isotype IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 Selected hybridomas: G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, 1 1 1 2 1 1 clon clon clon clon clon clon .sup.I Antygen showing non-native hemagglutinin conformation
[0227] Obtained mAb clones, all of IgG1 isotype, recognized conformational HA antigens: rHA (5/5) and rHA1 (3/3) from mammalian expression system, rHA-A/H5N1/Poland from baculovirus expression system, and H5 HA in viral particles (4/4). None of the obtained mAb clones was binding to rHA-A/H5N1/Ck/Vietnam, which showed no characteristics of native HA. In a situation, where all obtained mAb clones showed desired, broad range of specificities against H5 HA (Table 3), few clones of mAbs derived from cloning of each of the six primary hybridomas, were selected for further analysis. By this approach, probability of finding clones recognizing different epitopes specific for H5 serotype of hemagglutinin, was increased. One mAb clone was selected from each of hybridomas: G-1-31, G-5-32, G-7-24 and G-7-27, and 2 mAb clones were selected from G-6-42 hybridoma. One G-2-14-10 clone obtained from cloned G-2-14 hybridoma was also selected for further characterization.
[0228] Selected clones of G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 antibodies were purified from hybridoma culture supernatants using HiTrap Protein G HP (GE Healthcare). Preparation was according to manufacturer's instructions. 0.1 M glycine-HCl buffer, pH 2.7, was used for the elution of mAbs. Protein fractions were neutralized during elution using 1 M Tris-HCl, pH 9.0. Buffer exchange and antibody concentrations were performed by centrifugation in Vivaspin 6 Centrifugal Concentrator, 10 000 MWCO (Sartorius Stedim Biotech). Purified mAbs: G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 were tested using mass spectrometry and ELISAs for the analysis of serotype-specificity. Differentiation of the above mentioned antibody clones was performed based on the immunoreactivity profiles and peptide maps of Fab fragments. In order to evaluate the ability of generated mAbs to inhibit hemagglutination, HI testes were carried out. Test results are shown in subsequent Examples 6-10. Possible applications of the obtained antibodies are described in Examples 11 and 12. The object of the present patent application are monoclonal antibodies specific to HA of H5 serotype IVs: G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 and their use.
EXAMPLE 6 MASS SPECTRA OF THE SELECTED ANTIBODY CLONES
[0229] Purified clones of antibodies: G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18, chosen as a result of the selection for serotype-specificity (Table 3) and described in Example 5, were analysed using MALDI TOF/TOF mass spectrometer (4800 Plus, AB SCIEX). Before performing mass spectrometry, the samples were purified using ZipTip.sub.C4 (Millipore) according to the procedures included in the manufacturer's instruction: User Guide for Reversed-Phase ZipTip. The matrix was sinapinic acid (Fluka) in 5 mg/mL concentration, dissolved in 0.1% trifluoroacetic acid containing 50% of acetonitrile. Mass spectra were measured in the linear mode (MS Linear Positive Ion), in the range of 20-170 kDa. External calibration was achieved with IgG standard (AB SCIEX). All mass spectra were processed using Gaussian filter and the procedures for signal detection with the use of Data Explorer Software (V4.9).
[0230]
[0231] Relationship between an increase in molecular weights of antibodies and an increased level of non-enzymatic glycosylation was well documented in the studies of immunoglobulin fractions from diabetic patients (Lapolla A. et al., 1997, 2000a, 2000b). For example, molecular weights of IgG antibodies from healthy individuals and antibody standard, determined with the use of mass spectrometer, were 149 kDa and 148 kDa, respectively, and 152 kDa for antibodies in protein fraction from patients with poorly-controlled diabetes (Lapolla A. et al., 1997, 2000a). The effect of an increased non-enzymatic glycosylation was also obtained in vitro, by incubating protein fraction from plasma of healthy individual or IgG antibody standard in the presence of high glucose concentrations, when an increase of antibody molecular weight was observed, from 149 kDa and 148 kDa to 153 kDa, respectively (Lapolla A. et al., 2000a).
EXAMPLE 7 REACTIVITY OF SELECTED ANTIBODY CLONES WITH HEMAGGLUTININ ANTIGENS
[0232] Monoclonal antibodies: G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18, chosen as a result of the selection for serotype-specificity (Table 3), were purified by affinity chromatography, as described in Example 5. In further studies, reactivity of selected mAb clones with HA of H1-H16 serotypes was determined in order to evaluate specificity and affinity mAbs for H5 HA of different IV strains, and their ability to cross-react with HA of serotypes other than H5. The studies were carried out using H5 HA antigens with different properties. Among those antigens, there were recombinant proteins based on the ectodomain (rHA) or HA1 subunit (rHA1) of hemagglutinin and AIV of H5 serotype (List B and C). Recombinant HA proteins from mammalian and, exceptionally, baculovirus expression system, contained HA1 subunit of, usually, correct conformation, were in monomeric forms (rHA1) or, mostly or partially, formed oligomeric forms (rHA). Four certified AIV strains of H5 serotype: H5N1, H5N2, H5N3 and H5N9 were used in the studies of the obtained mAbs. Conformational antigens contained HA sequences from twelve IV strains of H5 serotype, with varied homology to the immunogen (rHA-A/H5N1/Qinghai)percentage of identical amino acids in the sequences of HA1 subunit of proteins used in the studies was from 99% to 88% (List F). Antigens: rHA-A/H5N1/Qinghai from mammalian expression system and rHA-A/H5N1/Poland from baculovirus expression system, are described in detail in Examples 2 and 3, respectively, whereas the remaining H5 HA antigens, produced in mammalian cells, are described in Example 4.
[0233] Comparing to the tests performed for the supernatants from G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 hybridoma cultures (Example 5), the assays for serotype-specificity of antibodies purified from the supernatants of those hybridomas were performed using panel of antigens, which additionally contained HA ectodomain-based recombinant protein from bacterial expression system and AlVs of H1-H4 and H6-H16 serotypes. The protein (17-522 aa, RRRKKR (SEQ ID NO: 13)) with HA sequence of A/swan/Poland/305-135V08/2006 (H5N1) strain of AIV, was over-expressed in Escherichia coli in The Institute of Biotechnology and Antibiotics (Instytut Biotechnologii i Antybiotykw, IBA) in Warsaw (Poland). Following purification and renaturation, the H5 HA protein was subjected to the analyses with the use of mass spectrometry, ELISAs for HA proteins antigenicity and oligomerization studies, similarly to other recombinant H5 HA antigens. In the immunoreactivity tests, antigen preparation of 80% purity was used. Molecular weight of rHA-A/H5N1/Poland protein from bacterial system, determined using MALDI TOF/TOF mass spectrometer (4800 Plus, AB SCIEX), was 57 kDa. Contrary to the results obtained for rHA-A/H5N1/Qinghai and rHA-A/H5N1/Poland proteins from mammalian and baculovirus expression system (Examples 2, 3), respectively, experimentally determined molecular weight of bacterial HA was in line with the weight calculated on the basis of amino acid composition in GPMAW 8.2 programme (Lighthouse). This is due to the fact, that proteins produced in bacteria do not undergo glycosylation. Conducted analyses revealed, that produced protein has features of native antigen: it retains conformational epitopes recognized by commercial anti-H5 HA antibodies (List A), including also HI antibodies, and it forms oligomeric structures. Two antigenic variants of the protein were prepared in the IBA. In the tests described in the present Example and in Examples 8 and 11, the second antigenic variant was used. In the tests of purified mAbs, twenty-one certified AIV strains representing H1-H4 and H6-H16 serotypes (List C) were used.
Specificity for H5 HA
[0234] Specificity of purified mAbs: G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 against H5 HA was tested by titrating them against rHA-A/H5N1/Qinghai protein from mammalian expression system (ITC), which was used as the immunogen in antibody producing procedure (Examples 1, 2). Also, titration against rHA-A/H5N1/Poland from baculovirus (OET) and bacterial expression system (IBA) with high homology to the immunogen (Lists: E, F), was performed.
[0235] To perform the assay, rHA-A/H5N1/Qinghai (ITC) and rHA-A/H5N1/Poland (OET, series 8), diluted in PBS to 1 g/mL concentration, were loaded into MediSorp plates (NUNC), and rHA-A/H5N1/Poland preparation (IBA) of 80% purity, containing renatured hemagglutinin in 1 g/mL PBS, was loaded into PolySorp, MediSorp, MaxiSorp and MultiSorp plates (NUNC). Some wells of the plate were filled in with PBS instead of the antigen solution. Following the overnight incubation at 2-8 C., the plates were blocked with 10% FBS/PBS. Purified mAb clones were analysed as series of 2-fold dilutions in 2% BSA/PBS. Antibodies diluted in a range of 8 g/mL-0.015 ng/mL were loaded into the plates coated with antigens from mammalian and baculovirus expression system, whereas antibodies diluted in a range of 8 g/mL-7.8 (3.9) ng/mL were loaded into the plates coated with the protein from bacterial expression system. The level of non-specific antibody binding was controlled by samples containing antibodies in 4 g/mL and/or 8 g/mL concentration and 2% BSA/PBS (BLK sample) loaded into non antigen-coated and coated wells, respectively. Plates with the obtained mAbs and control samples were incubated overnight at 2-8 C. HRP-labelled anti-mouse IgG antibodies (-chain specific, Sigma-Aldrich) were used for the detection of antigen-bound mAbs. Secondary antibodies were diluted 1:1000 in 2% BSA/PBS and incubated on the plates for 1 h at 37 C. TMB (Sigma-Aldrich) was used as a substrate for HRP. Reaction was inhibited using 0.5 M solution of H.sub.2SO.sub.4. Absorption of samples was read at =450 nm.
[0236] Titration curves of mAbs: G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 against rHA-A/H5N1/Qinghai from mammalian expression system is shown in
[0237] Concentrations, at which the levels of (A.sub.450) signal were 1.5 and/or 1.0, were determined using Gene5 software (Bio-Tek), by interpolation from linear segments of 4-parametric titration curves. Table 4, below, shows interpolated values obtained for the individual clones of mAbs.
[0238] In particular, Table 4 shows concentration of mAbs: G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17, G-7-27-18, determined by interpolation from linear ranges of 4-parametric titration curves against recombinant HA proteins with high homology to the immunogen. rHA*-A/H5N1/Qinghai protein (17-530 aa, RRRKKR (SEQ ID NO: 13), 6His (SEQ ID NO: 14)) with HA sequence derived from A/Bar-headed Goose/Qinghai/12/05 (H5N1) strain of AIV was produced in (*) mammalian expression system (ITC). rHA**-A/H5N1/Poland (17-530 aa, RRRKKR (SEQ ID NO: 13), 6His (SEQ ID NO: 14)) and rHA***-A/H5N1/Poland (17-522 aa, RRRKKR (SEQ ID NO: 13)) were produced in (**) baculovirus (OET, series 8) and (***) bacterial expression system (IBA), respectively, based on the HA sequence derived from A/swan/Poland/305-135V08/2006 (H5N1) strain of AIV. The analysis was performed by ELISA. Titration of HA protein from mammalian and baculovirus expression system was performed using MediSorp plates (NUNC) coated with the antigens in 1 g/mL concentration, and the results are shown as concentration values interpolated for the level of (A.sub.450) signal, i.e. 1.5. Titration against HA protein from bacterial expression system was performed on PolySorp, MediSorp, MaxiSorp and MultiSorp plates (NUNC) coated with rHA preparation with purity 80%, which contained renatured hemagglutinin in 1 g/mL concentration. The results are shown as means of concentration values (SD) interpolated for A.sub.450 levels of 1.0 and/or 1.5, obtained by assays performed with the use of particular type of polystyrene plates. Titration curves and interpolated values were determined using Gene5 software (Bio-Tek).
TABLE-US-00010 TABLE 4 mAb concentration mAb concentration for for A.sub.450 = 1.0 A.sub.450 = 1.5 [ng/mL] [ng/mL] rHA* rHA** rHA*** rHA*** mAb A/H5N1/ A/H5N1/ A/H5N1/ A/H5N1/ clones Qinghai Poland Poland Poland G-1-31-22 39 38 G-2-14-10 11 15 ~6368 157 G-5-32-5 13 14 G-6-42-42 9 10 ~10 1 ~5 1 G-6-42-71 14 14 ~12 2 ~6 1 G-7-24-17 39 43 G-7-27-18 7 9 ~322 28 ~161 12 *mammalian, **baculovirus, ***bacterial expression system
[0239] Values interpolated from titration curves of mAbs: G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-27-18 against rHA-A/H5N1/Qinghai from mammalian expression system and rHA-A/H5N1/Poland from baculovirus expression system for A.sub.450=1.5 reached values in the range of 7-14 ng/mL and 9-15 ng/mL, respectively, and in the case of G-1-31-22 and G-7-24-17 mAbs, they were 39 ng/mL and 39 ng/mL, and 38 ng/mL, respectively. Values interpolated from titration curves of G-6-42-42, G-6-42-71 mAbs against rHA-A/H5N1/Poland from bacterial expression system for A.sub.450=1.5 reached values of 10 ng/mL1 and 12 ng/mL2, respectively, and which were comparable to the values obtained by titration of those antibodies against antigens from eukaryotic expression system. In case of G-7-27-18 mAb titration, concentration interpolated for A.sub.450=1.5 from the titration curve against bacterial HA protein was 322 ng/mL28 and was, in average, 41-fold higher than against antigens derived from mammalian and baculovirus expression system. Within the range of used G-2-14-10 mAb dilutions, concentration interpolated for A.sub.450=1.0 from the titration curve against bacterial HA protein was 6368 ng/mL157, and was over 1000-fold higher than the concentration interpolated for the same signal level obtained for G-6-42-42 and G-6-42-71 mAbs, and 40-fold higher than the one obtained for G-7-27-18 mAb.
[0240] Data shown in Table 4 indicate similarity of the results obtained for particular mAb clones in the studies using rHA proteins (17-530 aa, RRRKKR (SEQ ID NO: 13), 6His (SEQ ID NO: 14)) from mammalian and baculovirus expression system with high homology, differing in the level of glycosylation (Examples 2, 3) and oligomerization (
Reactivity with Recombinant H5 HA Proteins
[0241] Before performing reactivity studies of the obtained mAbs with recombinant H5 HA proteins, analysis of serially diluted antibodies on Ni-NTA plates coated with rHA-A/H5N1/Vietnam from mammalian expression system (ITC) (List B) was conducted. The assay was performed under conditions described in Example 4. Table 5, below, shows concentrations of mAbs from linear range of titration curves, resulting in A.sub.4502.5.
[0242] In particular, Table 5 shows concentrations of mAbs: G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17, G-7-27-18 from linear range of titration curves against rHA-A/H5N1/Vietnam, for which signal level (A.sub.450) was 2.5. The protein (18-530 aa, RRRKKR (SEQ ID NO: 13), 6His (SEQ ID NO: 14)) with HA sequence derived from A/Vietnam/1203/2004 (H5N1) was produced in (*) mammalian expression system (ITC). The assay was performed by ELISA on Ni-NTA plates (Qiagen) coated with 1 g/mL antigen.
TABLE-US-00011 TABLE 5 mAb concentration for A.sub.450 ~2.5 [ng/mL] mAb clones rHA* A/H5N1/Vietnam G-1-31-22 50.0 G-2-14-10 22.5 G-5-32-5 20.0 G-6-42-42 8.75 G-6-42-71 12.5 G-7-24-17 50.0 G-7-27-18 12.5 *mammalian expression system
[0243] For immunoreactivity studies, Ni-NTA plates (Qiagen) were coated with H5 HA proteins: rHA and rHA1 from mammalian (ITC) and rHA protein from baculovirus (OET, series 8) expression system. Antigens were diluted to 1 g/mL in 1% BSA/PBS. Some wells of the plate were filled in with 1% BSA/PBS and incubated overnight at 2-8 C., in parallel to the antigen-containing wells. Tested mAb clones were diluted in 2% BSA/PBS to concentrations given in Table 5, and then loaded into coated and non-coated wells of the plate. Assay was carried out in the presence of control samples. Positive control was commercial anti-H5 HA antibodies (mAb 8, List A), whereas negative control was 2% BSA/PBS (BLK sample). Plates with the tested and control samples were incubated overnight at 2-8 C. HRP-labelled anti-mouse IgG antibodies (-chain specific, Sigma-Aldrich) were used for the detection of antigen-bound mAbs. Secondary antibodies were diluted 1:1000 in 2% BSA/PBS and incubated on the plates for 1 h at 37 C. TMB (Sigma-Aldrich) was used as a substrate for HRP. Reaction was inhibited using 1.25 M solution of H.sub.2SO.sub.4. Absorption of samples was read at =450 nm.
[0244] The results of reactivity studies for mAbs: G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 with recombinant proteins based on the ectodomain (rHA) and HA1 subunit (rHA1) of hemagglutinin, are shown in
[0245] To provide better data comparability, signal values (A.sub.450) obtained in the studies of reactivity of generated antibodies with recombinant H5 HA proteins having sequences from different viral strains, were expressed as % of A.sub.450 values read in the tests, in which the immunogen (rHA-A/H5N1/Qinghai) was used. The results thus obtained, further described as relative reactivity of the obtained antibodies with recombinant HA proteins, is shown in
[0246] For most of the obtained clones (5/7), decrease in relative reactivities with rHA-A/H5N2/California (19-506 aa, 6His (SEQ ID NO: 14)) from mammalian expression system was found, to values in a range of 58-85%. HA1 subunit of this antigen contained 88% of amino acids identical to the sequence of HA1 subunit of the immunogen (List F). Reduced relative reactivities were also observed in the studies using rHA-A/H5N1/Poland (17-530 aa, RRRKKR (SEQ ID NO: 13), 6His (SEQ ID NO: 14)) from baculovirus expression system, with high homology to the immunogen (99% of sequence identity for HA1 subunit, List F). This was the case for all of the obtained mAbs (7/7), and relative reactivities were from 46% to 87%.
[0247] In the tests using HA1 subunit-based proteins from mammalian expression system: rHA1-A/H5N1/Vietnam (1-345 aa, 6His (SEQ ID NO: 14)), rHA1-A/H5N1/HK/156 (18-346 aa, 6His (SEQ ID NO: 14)) and rHA1-A/H5N1/HK/483 (17-346 aa, 6His (SEQ ID NO: 14)), lower reactivity was found in comparison to the reactivity with rHA proteins from mammalian expression system, which were based on the HA sequences from A/H5N1/Qinghai, A/H5N1/India, A/H5N1/Vietnam and A/H5N1/Guiyang influenza viruses. Relative reactivities with individual rHA1 antigens were 60-89%, 43-81% and 26-55%, respectively. The differences in relative reactivities of the obtained antibody clones with rHA-A/H5N1/Vietnam and rHA1-A/H5N1/Vietnam, were from 21% to 42%. This is in line with previous observations, that sensitivity of detection of anti-HA1 subunit antibodies is lower when rHA1 proteins, and not rHA proteins are used for coating of Ni-NTA plates (Examples 4, 5). Identity of amino acids in rHA1 proteins with sequences of HA of A/H5N1/Vietnam, A/H5N1/HK/156, A/H5N1/HK/483 influenza viruses to the sequence of HA1 subunit of the immunogen, was 95%, 95% and 94%, respectively (List F).
[0248] Reduced relative reactivity with rHA-A/H5N2/California antigen with relatively high oligomerization (
Reactivity with H1-H16 Serotype Influenza Viruses
[0249] The tests for the ability of mAbs: G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 to recognize HA in viral particles were performed by ELISA using twenty five AIV strains, representing H1-H16 serotypes (List C). The assay was performed under conditions described in Example 4. Table 6, below, shows the results of reactivity analysis for the obtained clones of mAbs.
[0250] In particular, Table 6 shows the results of the reactivity tests for mAbs: G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 against LPAIV of the H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16 serotypes (List C). The assay was performed by ELISA by coating MaxiSorp plates (NUNC) with viruses diluted do 4000 HAU/mL based on the value specified by the manufacturer. Influenza viruses (x-OvO) were derived from IZSVe. Purified antibodies were tested in 20 g/mL concentration.
TABLE-US-00012 TABLE 6 HA mAb clones serotype Avian influenza virus (AIV) G-1-31-22 G-2-14-10 G-5-32-5 G-6-42-42 G-6-42-71 G-7-24-17 G-7-27-18 H1 A/duck/It/1447/05(H1N1) H2 A/duck/Germ/1215/73(H2N3) H3 A/pass/It/6000/V00(H3N8) A/psitt/It/2873/00(H3N8) H4 A/cockatoo/Eng/72(H4N8) H5 A/mallard/It/3401/05(H5N1) + + + + + + + A/turk/It/80(H5N2) + + + + + + + A/duck/It/775/04(H5N3) + + + + + + + A/ck/It/22A/98(H5N9) + + + + + + + H6 A/turkey/Canada/65 (H6N2) H7 A/ck/It/1067/V99(H7N1) A/ty/It/9289/V02(H7N3) A/mallard/It/4810-79/04(H7N4) A/macaw/626/80(H7N7) H8 A/turk/Ont/6118/68(H8N4) H9 A/ty/Wis/66(H9N2) A/turk/Scotland/1/70(H9N7) H10 A/ostrich/SA/01(H10N1) H11 A/duck/Eng/56(H11N6) A/duck/Memphis/546/174(H11N9) H12 A/duck/Alberta/60/76(H12N5) H13 A/gull/Maryland/704/77(H13N6) H14 A/mallard/Gurjev/263/82(H14N5) H15 A/shearwater/2576/79(H15N9) H16 A/gull/Denmark/68110/02(H16N3)
[0251] All obtained mAb clones, i.e. G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18, recognized HA of H5N1, H5N2, H5N3 and H5N9 AIVs. The results obtained for the purified antibodies are in line with the results of the tests for the presence of antibodies specific against AIV of H5 serotype in supernatants from cultures of cloned hybridomas, which are shown in Table 3 and described in Example 5. Tests with the use of AIVs of H1-H4 and H6-H16 serotypes showed, that generated antibodies do not cross-react with HA of serotypes other than H5.
[0252]
[0253] To provide better data comparability, signal values (A.sub.450) obtained in the studies of reactivity of generated antibodies against H5N1, H5N9 and H5N2 viruses, were expressed as % of A.sub.450 values read in the tests, in which H5N3 AIVs were used. The results thus obtained, further described as relative reactivity of the obtained antibodies against AIVs of H5 serotype, is shown in
[0254] Analysis of the results leads to the conclusion, that observed variation of reactivities of particular mAb clones with H5N1, H5N2, H5N3 and H5N9 AIVs may be associated with variation of sequences forming HA1 subunit of HA viruses used in the tests and/or with the effectiveness of coating ELISA plates with viral antigens. Information regarding differences in the composition of viral preparations, which could influence conditions of assays for the immunoreactivity of antibodies, is included in the description of ELISA method with the use of influenza viruses (Example 4).
SUMMARY
[0255] Further studies of mAbs: G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18, after their purification from hybridoma culture supernatants, were performed using H5 HA antigens of varied properties. Among the antigens, there were HA ectodomain-based recombinant proteins (rHA) from mammalian, baculovirus and bacterial expression systems, proteins based on the HA1 subunit (rHA1), obtained in mammalian cells, as well as twenty five AIV strains of H1-H16 serotypes (Lists B, C). The vast majority of H5 HA antigens (13/14) showed characteristics of native hemagglutinin (
[0256] Obtained mAb clones recognized all conformational, glycosylated H5 HA antigens: rHA (6/6) and rHA1 (3/3) from eukaryotic expression system (
[0257] The obtained results lead to the conclusion, that mAbs: G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 are directed against conformational epitopes of Hemagglutinin HA1 subunit, show broad range of specificities to the HA of H5 serotype and at the same time, do not cross-react with the HA of H1-H4 and H6-H16 serotypes.
EXAMPLE 8 CLONES DIFFERENTIATION BASED ON IMMUNOREACTIVITY PROFILES
[0258] Based on the results of the reactivity tests for mAbs: G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 with H5 HA antigens (Example 7), reactivity profiles were created for use in comparative analysis of the obtained clones.
[0259] G-6-42-42 and G-6-42-71 antibodies differ clearly from the other obtained clones by characteristic reactivity profiles (
[0260] An important criterion differentiating remaining obtained mAbs: G-1-31-22, G-2-14-10, G-5-32-5, G-7-24-17 and G-7-27-18 are the levels of signals (A.sub.450) for particular antibody concentrations, treated as an indicative measure of the affinity for antigens and shown in
[0261] G-1-31-22 and G-7-24-17 antibodies differ from G-2-14-10, G-5-32-5 and G-7-27-18 antibodies, inter alia, because they show lower binding affinity with rHA-A/H5N1/Qinghai from mammalian expression system and rHA-A/H5N1/Poland from baculovirus expression system adsorbed on MediSorp plates (
[0262] The immunoreactivity profiles, shown in
[0263] G-2-14-10 and G-7-27-18 clones were in a group of antibodies with high binding affinity for rHA-A/H5N1/Qinghai from mammalian expression system and rHA-A/H5N1/Poland from baculovirus expression system adsorbed on MediSorp plates (
SUMMARY
[0264] As a result of applying procedure of mAb generation by hybridoma method (Example 1), 7 antibody clones, denoted G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18, were obtained. Analysis of immunoreactivity profiles revealed, that G-1-31-22, G-2-14-10, G-5-32-5, G-7-24-17 and G-7-27-18 mAbs are different clones, whereas G-6-42-42 and G-6-42-71 mAbs represent one antibody clone, different from the other five clones. Thus, six mAb clones were obtained, which recognized different epitopes specific for H5 hemagglutinin.
EXAMPLE 9
Differentiation of Clones Based on the Peptide Maps of Fab Fragments
[0265] The aim of the studies described below was to obtain Fc and Fab fragments of G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 antibodies by ficin digestion and comparison of their peptide maps generated as a result of trypsinolysis. Based on the mass spectra recorded, peptides characteristic only for one particular or only for some of the obtained antibody clones (discriminatory peptides), as well as those present in all of the analysed mAbs (common peptides) were determined.
[0266] Purified mAbs were concentrated with the use of centrifugal concentrators VivaSpin6, MWCO 10 000 (Sartorius Stedim Biotech) with simultaneous buffer exchange to PBS, pH 7.4. Ficin digestion of antibodies was performed using commercial Pierce Mouse IgG.sub.1 Fab and F(ab).sub.2 Micro Preparation Kit (Pierce/Thermo Scientific). Sample preparation, digestion and purification were conducted strictly according to manufacturer's instructions. Maximally concentrated protein, i.e. 250 g in 125 L solution, was loaded into the ficin-containing column. The digestion time was 5 h. After separation by affinity chromatography, fractions containing Fab and Fc fragments, as well as undigested antibodies, were concentrated using VivaSpin 6 centrifugal concentrators, MWCO 5 000 (Sartorius Stedim Biotech) with simultaneous buffer exchange to PBS, pH 7.4.
[0267] Concentrated fractions were separated using non-reducing electrophoresis (SDS-PAGE) in 2 polyacrylamide gels system (5% stacking gel, pH 6.8 and 12.5% resolving gel, pH 8.8). The protein bands corresponding to the Fab, Fc and undigested IgG were excised from the gel, reduced using 10 mM dithiothreitol (Sigma-Aldrich), alkylated with 50 mM iodoacetamide (Sigma-Aldrich) and digested with 10 ng/mL trypsin solution (Promega). Simultaneously, digestion of the above mentioned fractions with trypsin in solution was performed. Each time, peptide-containing samples were concentrated using concentrator (Koncentrator 5301, Eppendorf) and analysed by MALDI TOF/TOF mass spectrometry.
[0268] Molecular weights and fragmentation ions of the peptides were determined using MALDI TOF/TOF mass spectrometer (4800 Plus, AB SCIEX). The matrix was alpha-cyano-4-hydroxycinnamic acid dissolved in 0.1% trifluoroacetic acid containing 50% of acetonitrile. Peptide masses were recorded in reflector-positive mode (MS Reflector Positive Ion) in the range of 900-8000 Da. External calibration was achieved with a 4700 proteomics analyzer calibration mixture (AB SCIEX). Masses of molecules, included into calculations and shown in the description, are monoisotopic masses of ionized single molecule with hydrogen atom attached. Peptides were identified based on the obtained peptide maps and fragmentation spectra using databases available in Mascot system. Theoretical masses of peptides, whose sequence was confirmed using Mascot system, were calculated with the use of generally available internet service on ExPASy server. Peptides found in protein databases were further referred to, in the text and tables, as identified. In tables, they are designated with * symbol. For a distinction, non-identified peptides were those not assigned to any known protein. In case of those peptides, monoisotopic masses given are means from all obtained mass spectra. Data in the tables below are specific result of both trypsinolysis method used. Symbols + and in the tables referred to the presence and absence, respectively, of a given peptide in peptide maps of particular mAbs.
Peptide Maps of Fc Fragments
[0269] Peptide maps of Fc fragments of G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 mAbs and amino acid sequences of peptides identified in those maps are shown in Table 7(a, b), below.
[0270] More specifically, Table 7 shows (a) peptide maps of Fc fragments of G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 mAbs and (b) amino acid sequences of peptides identified in those maps. Peptide maps, obtained as a result of trypsinolysis, were analysed using MALDI TOF/TOF mass spectrometry. Peptides were identified based on the obtained peptide maps and fragmentation spectra using databases available in Mascot system.
TABLE-US-00013 TABLE 7a Peptide maps of Fc fragments of monoclonal antibodies (mAbs) mAb Mass [Da] clones 1169.6* 1243.7* 1301.7* 1826.9* 2112.0* 2753.3* 2782.3* 1580.9 1771.9 1854.9* 2258.1* 1965.8 2764.1 G-1-31-22 + + + + + + + + + G-2-14-10 + + + + + + + + + + G-5-32-5 + + + + + + + + + + G-6-42-42 + + + + + + + + + + + G-6-42-71 + + + + + + + + + + + G-7-24-17 + + + + + + + + + + + G-7-27-18 + + + + + + + + + + + *.sub.identified peptides
TABLE-US-00014 TABLE7b Aminoacidsequencesofpeptidesidentifiedinpeptidemapsof Fcfragments Mass[Da] AminoAcidSequence 1169.6 IQHQDWTGGK(SEQIDNO:1) 1243.7 VNSAAFPAPIEK(SEQIDNO:2) 1301.7 VHNEGLPAPIVR(SEQIDNO:3) 1826.9 EPQVYVLAPPQEELSK(SEQIDNO:4) 1854.9 SVSELPIMHQDWLNGK(SEQIDNO:5) 2112.0 FSWFVDDVEVNTATTKPR(SEQIDNO:6)(1MC).sup.a 2258.1 SVSELPIMHQDWLNGKEFK(SEQIDNO:7)(1MC).sup.a 2753.3 STVSLTCMVTSFYPDYIAVEWQR(SEQIDNO:8)(CYS_CAM).sup.b 2782.3 DTLTISGTPEVTCVVVDVGHDDPEVK(SEQIDNO:9)(CYS_CAM).sup.b .sup.aMC is 1 omitted restriction site .sup.bCYS_CAM is iodoacetamide-modified cysteine (modification was introduced intentionally before trypsin digestion of protein, in order to avoid reorganisation of disulphide bridges during hydrolysis)
[0271] Analysis of peptide maps of Fc fragments of the obtained mAbs allowed to identify thirteen peptides (Table 7a). Amino acid sequences identified as fragments of IgG antibodies, could be assign to nine of those peptides (Table 7b). The majority of the peptides (9/13) shown in Table 7a, are common peptides, among which seven are identified and two non-identified peptides. Another four peptides are discriminatory fragments (Table 7a). Amino acid sequences of two of them are known (Table 7b). Identical discriminatory peptide profile in Fc fragments was found in two groups of clones, one of which included G-6-42-42, G-6-42-71 and G-7-24-17 mAbs, and the other G-2-14-10 and G-5-32-5 mAbs. Compared to mAbs from the second group, G-7-27-18 mAbs contained additional discriminatory peptide. No discriminatory fragments were found in G-1-31-22 clone.
Peptide Maps of Fab Fragments
[0272] As in case of Fc fragments, analysis of peptide maps of Fab fragments of G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 clones allowed to identify common and discriminatory peptides. Common peptides from Fab fragments of antibodies and amino acid sequences of identified common peptides are shown in Table 8(a, b), below.
[0273] More specifically, Table 8 shows (a) common peptides in peptide maps of Fab fragments of G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 mAbs and (b) amino acid sequences of identified common peptides. Peptide maps, obtained as a result of trypsinolysis, were analysed using MALDI TOF/TOF mass spectrometry. Peptides were identified based on the obtained peptide maps and fragmentation spectra using databases available in Mascot system.
TABLE-US-00015 TABLE 8a Peptide maps of Fab fragments of monoclonal antibodies (mAbs) - common peptides mAb Mass [Da] clones 1696.9* 1666.8 1678.8 2015.0 2028.0 2037.0 2255.2 2421.0 2439.1 G-1-31-22 + + + + + + + + + G-2-14-10 + + + + + + + + + G-5-32-5 + + + + + + + + + G-6-42-42 + + + + + + + + + G-6-42-71 + + + + + + + + + G-7-24-17 + + + + + + + + + G-7-27-18 + + + + + + + + + *.sub.identified peptides
TABLE-US-00016 TABLE8b Aminoacidsequencesofcommon peptides identifiedinpeptidemapsofFabfragments Mass[Da] AminoAcidSequence 1696.9 PAVLNQPSSVSGSLGQR(SEQIDNO:10)
[0274] Analysis of peptide maps of Fab fragments of the obtained antibodies allowed to identify nine common peptides (Table 8a). Among them, only one (1696.9 Da) was identified using Mascot search engine. It was assign amino acid sequence shown in Table 9b. Although for most of the other peptides good quality fragmentation spectra were obtained, they could not be identified based on protein databases.
[0275] Discriminatory peptides in peptide maps of Fab fragments of G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 antibodies, as well as amino acid sequences of identified discriminatory peptides are shown in Table 9 (a, b), below.
[0276] More specifically, Table 9 shows (a) discriminatory peptides in peptide maps of Fab fragments of G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 antibodies and (b) amino acid sequences of identified discriminatory peptides. Peptide maps, obtained as a result of trypsinolysis, were analysed using MALDI TOF/TOF mass spectrometry. Peptides were identified based on the obtained peptide maps and fragmentation spectra using databases available in Mascot system.
TABLE-US-00017 TABLE 9a Peptide maps of Fab fragments of monoclonal antibodies (mAbs) - differentiating peptides mAb Mass [Da] clones 1944.1 1445.7 1515.8 1705.9* 1111.5 1800.8 1580.9 1430.7 1884.0 1647.8 1819.8 1110.5 1872.1* 3300.5* G-1-31-22 + + + + + G-2-14-10 + + + G-5-32-5 + + + + + G-6-42-42 + + + G-6-42-71 + + + G-7-24-17 + + + + + + G-7-27-18 + + + + + + *.sub.identified peptides
TABLE-US-00018 TABLE9b Aminoacidsequencesofdiscriminatory peptidesidentified inpeptidemapsofFabfragments Mass[Da] AminoAcidSequence 1705.9 LWIYGTSDLASGVPAR(SEQIDNO:11) 3300.5 ATLTVDASSSTAYIQLSSLSSEDSAVYFCAR(SEQIDNO:12)
[0277] Analysis of peptide maps of Fab fragments from the obtained antibodies determined fourteen differentiating peptides, among which three of 1705.9 Da, 1872.1 Da and 3300.5 Da molecular weight were identified using protein databases (Table 9a). Sequences shown in Table 9b were assigned to peptides 1705.9 Da and 3300.5 Da. Three differentiating peptides were recognised in Fab fragments of clones G-2-14-10, G-6-42-42 and G-6-42-71, five of clones G-1-31-22 and G-5-32-5, and six of clones G-7-24-17 and G-7-27-18.
Profiles of Differentiating Peptides in Peptide Maps of Fab Fragments
[0278] Peptide maps of Fab fragments of antibodies G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 were subjected to detailed comparative analysis. Table 10 (a-e) shows profiles of discriminatory peptides from Fab fragments of individual mAbs and their comparison with profiles of the other antibody clones. Peptides present in peptide map of only one, particular antibody clone were identified among discriminatory peptides and are further referred to as peptides characteristic for that clone.
[0279] More specifically, Table 10 shows discriminatory peptides in peptide maps of Fab fragments of clones: (a) G-6-42-42 and G-6-42-71, (b) G-2-14-10 and G-5-32-5, (c) G-1-31-22, (d) G-7-24-17, (e) G-7-27-18, obtained as a result of trypsinolysis nad analysed using MALDI TOF/TOF mass spectrometry.
TABLE-US-00019 TABLE 10a Differentiating peptides in peptide maps of Fab fragments of G-6-42-42 and G-6-42-71 clones Mass [Da] mAb clones 1580.9 1515.8 1944.1 G-6-42-42 + + + G-6-42-71 + + + G-7-24-17 + + G-1-31-22 + G-2-14-10 + G-5-32-5 + G-7-27-18
[0280] For G-6-42-42 and G-6-42-71 antibodies, the same discriminatory fragments were observed, derived both from Fab part (Table 10a), and Fc part (Table 7a). Those are the only clones among generated mAbs, for which identical peptide maps were obtained. It was assumed, that they represent one antibody clone. In Fab part of G-6-42-42 (G-6-42-71) antibodies, 1580.9 Da peptide characteristic for this clone was recognized. It was also found, that G-6-42-42 (G-6-42-71) antibodies differ from four out of five other clones in that they contain, at the same time, 1515.8 Da and 1944.1 Da fragments.
TABLE-US-00020 TABLE 10b Differentiating peptides in peptide maps of Fab fragments of G-2-14-10 and G-5-32-5 clones mAb Mass [Da] clones 1110.5 1647.8 1819.8 1705.9* 1445.7 1944.1 G-2-14-10 + + + G-5-32-5 + + + + + G-1-31-22 + + G-7-27-18 + + G-7-24-17 + + G-6-42-42 + (G-6-42-71) *.sub.identified peptides
[0281] As a result of digestion of Fab part of G-2-14-10 clone, three discriminatory peptide were obtained (Table 10b), including one identified, with a mass of 1705.9 Da (Table 9b). Among peptides derived from Fab fragment of G-2-14-10 antibodies, no peptide characteristic for this clone was found. In peptide map of Fab fragment of G-5-32-5 antibodies, five non-identified discriminatory peptides were found, including three characteristic for this clone 1110.5 Da, 1647.8 Da and 1819.8 Da (Table 10b). Both G-2-14-10 mAb, and G-5-32-5 mAb differ from four other clones in that at the same time they contain 1445.7 Da and 1944.1 Da peptides. Profiles of discriminatory peptides, shown in Table 10b, indicate that G-2-14-10 and G-5-32-5 antibodies are different clones, and at the same time each of them is different than other antibody clones.
TABLE-US-00021 TABLE 10c Discriminatory peptides in peptide map of Fab fragment of G-1-31-22 clone Mass [Da] mAb clones 1884.0 1705.9* 1111.5 1445.7 1515.8 G-1-31-22 + + + + + G-7-27-18 + + + G-7-24-17 + + + G-2-14-10 + + G-5-32-5 + G-6-42-42 + (G-6-42-71) *.sub.identified peptides
[0282] Analysis of peptide maps of Fab fragment of G-1-31-22 clone, enabled identification of five discriminatory peptide (Table 10c), including one identified, with a mass of 1705.9 Da (Table 9b). In the peptide map of G-1-32-22 mAb, 1884.0 Da peptide characteristic for this clone was recognized. G-1-31-22 antibodies differ from other clones also by simultaneous presence of non-identified 1111.5 Da, 1445.7 Da and 1515.8 Da peptides.
TABLE-US-00022 TABLE 10d Discriminatory peptides in peptide map of Fab fragment of G-7-24-17 clone mAb Mass [Da] clones 1430.7 1705.9* 1111.5 1515.8 1800.8 1944.1 G-7-24-17 + + + + + + G-1-31-22 + + + G-7-27-18 + + + G-6-42-42 + + (G-6-42-71) G-2-14-10 + + G-5-32-5 + *.sub.identified peptides
[0283] In the peptide map of Fab fragment of G-7-24-17 clone, six discriminatory peptides were found (Table 10d), including one with a mass of 1705.9 Da, which was identified (Table 9b). In Fab part of G-7-24-17 antibodies, 1430.7 Da peptide characteristic for this clone was recognized. G-7-24-17 clone differs also by simultaneous presence of 1111.5 Da, 1515.8 Da, 1800.8 Da and 1944.1 Da peptides.
TABLE-US-00023 TABLE 10e Discriminatory peptides in peptide map of Fab fragments of G-7-27-18 clone mAb Mass [Da] clones 1872.1* 3300.5* 1705.9* 1111.5 1445.7 1800.8 G-7-27-18 + + + + + + G-1-31-22 + + + G-7-24-17 + + + G-2-14-10 + + G-5-32-5 + G-6-42-42 (G-6-42-71) *.sub.identified peptides
[0284] Analysis of peptide map of Fab fragment of G-7-27-18 clone enabled identification of six discriminatory peptides (Table 10e). Among them, there are three peptides with a mass of 1705.9 Da, 1872.1 Da and 3300.5 Da, which were identified (Table 9b). Peptides: 1872.1 Da and 3300.5 Da were found only in peptide map of G-7-27-18 mAb. Moreover, G-7-27-18 clone differs from other antibodies by simultaneous presence of 1111.5 Da, 1445.7 Da and 1800.8 Da peptides.
SUMMARY
[0285] As a result of ficin digestion, Fc and Fab fragments of G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 antibodies were obtained and then their peptide maps were generated as a result of trypsinolysis. Based on the obtained peptide maps and fragmentation spectra, an attempt was made to identify peptides using databases available in Mascot system.
[0286] Most peptides in the maps of Fc fragments of analysed mAbs (Table 7a) were common peptides (9/13; 69%). Amino acid sequences of most of the peptides derived from Fc fragments (9/13; 69%) were found in protein databases (Table 7b). The obtained results are consistent with the current state of knowledge, that within the species and isotype, Fc fragments are conserved part of immunoglobulin, without relevance to the specificity of antibodies. Unlike in the case of Fc fragments, in peptide maps of Fab fragments of tested antibodies (Tables 8a, 9a) the majority were discriminatory peptides (14/23; 61%). Additionally, only small number of peptides (4/23; 17%) was identified in Fab fragments (Tables 8b, 9b), significantly less than in Fc fragments (17% vs 69%). Fab fragments are characterised by a great sequence variability determining their specificity, and therefore databases can be incomplete in those areas.
[0287] Of major importance for the identification of the obtained antibodies was comparative analysis of peptide maps of antibody variable fragments responsible for antigen binding, i.e. Fab fragments. Clones discrimination was based mainly on the presence of peptides characteristic for individual clones or particular groups of clones (discriminatory peptides). The most informative was the presence of peptides having unique amino acid sequences, which were not deposited in protein databases so far (non-identified discriminatory peptides).
[0288] Detailed analysis of peptide maps of Fab fragments showed, that the obtained antibodies differ in the number and the profile of discriminatory peptides (Table 10a-e). Among seven obtained mAbs, six different antibody clones were recognized and it was shown, that G-6-42-42 and G-6-42-71 antibodies are the same clone. This corresponds to the results of immunoreactivity profile-based mAbs discrimination, which are shown in Example 8.
EXAMPLE 10 DETERMINATION OF ACTIVITY OF THE OBTAINED MABS IN HEMAGGLUTINATION INHIBITION TEST
[0289] Monoclonal antibodies generated as a result of mice vaccination with rHA-A/H5N1/Qinghai and selected for the specificity against HA of H5 serotype, were subjected to HI test. The test uses the ability of antibodies binding specifically to particular antigenic sites on HA molecule to block binding between the protein and erythrocyte surface receptors, what is manifested by the lost ability of the antigen to erythrocyte agglutination.
[0290] The studies included purified G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 antibodies. As reference antibodies, commercial anti-H5 HA mAbs (Pierce/Thermo Scientific, Acris Antibodies), denoted mAb 7, mAb 8, mAb 9, were used. According to specifications, antibodies recognize H5 HA in HI test (List A). HI test was performed using H5N3 LPAIVs as an antigen. Hemagglutinin of this AIV strain is characterized by the highest homology with immunogen's source sequence among hemagglutinins of H5 serotype IVs used in the mAb manufacturing procedure (List E, F). It is the case for both full-length protein, and HA1 subunit, where epitopes for HI antibodies are localised. HI test was also performed using H5N2 LPAIV as an antigen. Comparative analyses of H5N3, H5N1, H5N9 and H5N2 AIV hemagglutinin showed, that HA HA1 subunit of H5N2 virus has the lowest homology to HA1 subunit of the immunogen, if all homology factors are taken into account: Max Score, Total Score, Identities (List F). According to the results of ELISA using four LPAIV strains of H5 serotype (Example 7), reactivity of all obtained mAb clones with H5N3 AIV was the highest, whereas with H5N2 AIV was one of the lowest (
TABLE-US-00024 List G Influenza viruses and anti-sera used in the studies of hemagglutination inhibition activity of the obtained mAbs. HI test with low pathogenicity H5N3 avian influenza virus Origin Antigen LPAIV A/duck/It/775/04(H5N3) x-OvO Positive Control Antiserum A/duck/It/775/04(H5N3) x-OvO Negative control Antiserum A/macaw/626/80(H7N7) x-OvO HI test with low pathogenicity H5N2 avian influenza virus Origin Antigen LPAIV A/turk/It/80(H5N2) x-OvO Positive Control Antiserum A/turk/It/80(H5N2) x-OvO Negative control Antiserum A/macaw/626/80(H7N7) x-OvO LPAIVlow-pathogenic (LP) avian influenza virus (AIV) x-OvO Limited (Great Britain), Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe, Italy)
[0291] Fresh preparation of erythrocytes collected from the blood of SPF chickens from sterile culture in DPD, NVR was used in the test. Each performed HI test included positive control with anti-H5N3 AIV or H5N2 AIV antisera incubated with H5N3 or H5N2 viral antigens, respectively. Regardless the antigen used, negative controls were samples containing anti-H7N7 AIV antiserum. For all tested antibodies and antisera, blood cells controls without viral antigen were performed (internal assay control). For each HI test, hemagglutination unit was determined (HAU).
[0292] To evaluate HI activities of the obtained mAbs, series of 2-fold dilutions of selected antibody clones and their mixture in PBS-Dulbecco (Sigma-Aldrich) were prepared in 96 well conical bottom (V) plates (CellStar/Greiner bio-one). Thus, antibody dilutions in the range from 2 to 4096(8192)were prepared. Reference anti-H5 HA mAbs were diluted analogically. On each test plate, serial dilutions of control antisera against H5N3 AIV or H5N2 AIV (positive control) and H7N7 AIV (negative control) were prepared, resulting in dilutions from 2 to 4096(8192). Next, into each of the wells containing mAbs and antisera diluted 4 and more in the final volume of 25 L, 25 L of H5N3 AIV or H5N2 AIV suspension containing 4 HAU was added. 25 L PBS-Dulbecco was added into each of the wells for blood cells control containing 2-fold diluted mAbs and antisera. After 25 min of initial incubation at room temperature, 25 L 1% erythrocyte suspension was added into each well. The results were observed after minimum 30 min incubation with blood cells.
[0293] According to the principle of the test, hemagglutination inhibition was evaluated visually by comparing samples with blood cell controls. In HI-positive samples, virus antigens do not agglutinate erythrocytes and therefore they fall freely on the bottom of the wells, as in the blood cells control samples. HI titer of monoclonal antibodies was defined as the lowest concentration of antibodies causing hemagglutination inhibition. HI titer of antisera was generally defined as the inverse of the highest dilution, which inhibits erythrocyte agglutination by viral antigens. The results of HI tests for G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17, G-7-27-18 mAbs using H5N3 and H5N2 AlVs as antigens are shown in Table 11, below, together with the results for reference antibodies and control antisera.
[0294] More specifically, Table 11 shows HI tests results for G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17, G-7-27-18 mAbs using H5N3 and H5N2 LPAIV (x-OvO) as antigens. Each test performed included positive control (anti-H5N3 AIV or anti-H5N2 AIV antisera) and negative (anti-H7N7 AIV antiserum), and blood cells controls. In HI test using H5N3 AIV, commercial anti-H5 HA mAbs (Pierce/Thermo Scientific, Acris Antibodies) were used as reference antibodies.
TABLE-US-00025 TABLE 11 mAb concentrations [g/mL] Result Antiserum HI HI mAbs and control antisera dilution activity titer HI test with low pathogenicity H5N3 avian influenza virus G-1-31-22 0.3-526 G-2-14-10 0.8-1011 G-5-32-5 0.9-1008 G-6-42-42 0.7-1034 G-6-42-71 0.9-973 G-7-24-17 0.5-877 G-7-27-18 0.9-1023 G-1-31-22 + G-2-14-10 + 0.7-726 G-5-32-5 + G-6-42-42 + G-6-42-71 + G-7-24-17 + G-7-27-18 mAb 9 (Pierce/Thermo 0.1-125 + 7.8 g/mL Sci., Cat. No. MA1- 81928) mAb 7 (Acris Antibodies, 0.1-125 + 2.0 g/mL Cat. No. AM00945PU- N) mAb 8 (Acris Antibodies, 0.1-25 + 1.0 g/mL Cat. No. AM00941PU- N) Antiserum against 4x-2048x + 1:512, 1:1024 H5N3 LPAIV (4096x) Antiserum against 4x-2048x H5N7 LPAIV (4096x) HI test with low pathogenicity H5N2 avian influenza virus G-1-31-22 0.5-526 G-2-14-10 1.0-1011 G-5-32-5 1.0-1008 G-6-42-42 1.0-1034 G-6-42-71 0.9-973 G-7-24-17 0.9-877 G-7-27-18 1.0-1023 Antiserum against 4x-4096x + 1:512 H5N2 LPAIV Antiserum against 4x-4096x H5N7 LPAIV
[0295] HI tests showed, that non of the mAb clones produced as a result of mice vaccination with rHA-A/H5N1/Qinghai and selected for serotype specificity, has the ability to inhibit hemagglutination by H5N3 and H5N2 AIVs. No HI activity was showed for G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17, G-7-27-18 mAbs, despite using a wide variety of antibody concentrations: from submicrogram to couple of hundreds of grams, wherein some clones were tested in concentration of up to 1 mg. Under the same conditions, HI titers of commercial mAbs, denoted mAb 9, mAb 7 and mAb 8, determined using H5N3 AIV, were 7.8 g/mL, 2.0 g/mL and 1.0 g/mL, respectively. Correct assay performance was confirmed by high titers of HI-positive control antisera (anti-H5N3 AIV or anti-H5N2 AIV antisera) and no HI activity of negative control antisera (anti-H7N7 antiserum). Additionally, observation of blood cells control samples did not showed, that tested samples of antibodies and antisera influenced the quality of erythrocytes.
[0296] G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18 antibodies differ from mAb 7 and mAb 8 of narrow and broad range of specificities against H5 HA (
EXAMPLE 11
[0297] The Use of Produced mAbs for the Evaluation of Quality of H5 Hemagglutinin Antigens
[0298] The main challenge for works on subunit vaccines against influenza, based on the use of hemagglutinin produced using genetic engineering methods, is to obtain viral HA-like antigens. Particularly important for vaccine HA quality is the correctness of structure of HA1 subunit, where conformational epitopes for neutralizing antibodies are localized. One of the indicators of HA proteins usefulness for vaccine manufacturing is the reactivity of antibodies of known properties with produced antigens. Antigenicity studies found broad application in the analysis of HA proteins produced in a form of inclusive bodies in bacterial cells, where suitable protein renaturation and purification methods must be used in order to produce valuable vaccine antigen (Saczyska V., 2014). Because newly obtained antibody clones recognize only conformational epitopes of HA1 subunit and show broad range of H5 HA specificities, they can be successfully used in the studies of properties of antigens for production of vaccines against HPIVs of H5 serotype, regardless of the original vaccine antigen sequence. Envisioned is also the use of generated antibodies for controlling stability of antigens and vaccines against influenza. Another possible application for the obtained mAbs is their use to monitor quality of H5 HA proteins intended for use or already used as reagents in diagnostic tests, i.e. for verification whether conformation of serotype-specific epitopes in antigens is correct.
[0299] To determine applicability of the obtained antibodies for the evaluation of H5 HA antigens quality, antigenicity tests for bacterial expression system-produced rHA-A/H5N1/Poland using G-6-42-42, G-6-42-71 and G-7-27-18, were performed. Following purification and renaturation, H5 HA protein (17-522 aa, RRRKKR (SEQ ID NO: 13)), produced on the basis of HA sequence of A/swan/Poland/305-135V08/2006 (H5N1) strain of AIVs (IBA), was subjected to analyses, which showed its correct conformation. Test methods and protein properties are described in detail in Example 7. rHA-A/H5N1/Poland was analysed under reducing conditions before and after denaturation. Denaturation was conducted by diluting protein preparation in 2 concentrated denaturation buffer (4% SDS, 625 mM -mercaptoethanol, 120 mM Tris-HCl, pH 8.0) in 1:1 ratio (v./v.) and by heating the obtained solution in 99 C. for 10 minutes. The antigenicity analysis was performed by ELISA on PolySorp, MediSorp, MaxiSorp and MultiSorp plates (NUNC) coated with conformational and denatured rHA preparation with purity 80%, which contained hemagglutinin in 1 g/mL concentration. Serially 2-fold diluted G-6-42-42, G-6-42-71 and G-7-27-18 mAbs were loaded into plates blocked with 10% FBS/PBS. The assay was performed under conditions described in Example 7. Concentrations interpolated from 4-parameter titration curves with bacterial HA protein were determined using Gene5 software (Bio-Tek).
[0300]
[0301] The results described in the present example confirm earlier findings, that generated G-6-42-42, G-6-42-71 and G-7-27-18 antibodies recognize conformational epitopes of hemagglutinin (Examples 5, 7). Some reactivity level of those antibodies with denatured rHA protein, observed in tests using some types of plates, was probably resulting from partial protein renaturation after dilution in PBS under plate-coating conditions.
[0302] The results confirm usefulness of the produced antibodies for studying properties of newly obtained H5 HA proteins and for the detection of changes of those proteins during their storage. This is the case for both antigens for the production of vaccines against IVs of H5 serotype, e.g. H5N1 HPAIV, and antigens used in diagnostic tests.
EXAMPLE 12
[0303] The Use of Generated mAbs in Diagnostic Tests
[0304] In order to show possible applications of generated mAbs in diagnostic tests, prototype blocking ELISA was developed for detection of anti-H5 HA antibodies (BELISA H5). G-7-27-18 clone was used in BELISA H5. As the antigen, 17-530 aa protein (RRRKKR (SEQ ID NO: 13), 6His (SEQ ID NO: 14)) having sequence of HA of A/swan/Poland/305-135V08/2006 strain of H5N1 virus, produced in baculovirus expression system (OET), was used. The protein properties are described in detail in Example 2. BELISA H5 was optimized. Assays using polystyrene plates with various polarity enabled selection of the best plate for antigen binding and epitop presentation for G-7-27-18 mAb. Preferable conditions for plates incubation at particular stages of purification procedure were determined in additional experiments. Optimum concentration/dilution of reagents were determined by titration.
[0305] In optimal variant of BELISA H5, MediSorp plates (NUNC) were coated with antigen in 0.5 g/mL (50 L/well) overnight, at 2-8 C. Unspecific binding sites on the plates were blocked for 1 h at room temperature using Protein-Free T20 (PBS) Blocking Buffer (200 L/well) from Pierce. Into each well for control mAbs, 100 L of incubation buffer (1% BSA/PBS) was added, and into each of the other wells50 l of incubation buffer was added. Control and tested serum samples were diluted in the wells of the plate by adding 50 L of serum to 50 L of 1% BSA/PBS. The samples were incubated in the plates for 1 h at 37 C., with shaking (150 rpm). Next, 7-27-18 mAb (50 L/well) diluted to 1 g/mL in Antibody Stabilizer PBS (Candor Bioscience) was added into the wells, and the plate was incubated again for 1 h at 37 C., with 150 rpm. HRP-labelled anti-mouse IgG antibodies (-chain specific, Sigma-Aldrich) were used for the detection of antigen-bound mAbs. Secondary antibodies were diluted 1:3500 in HRP-Protector (Candor Bioscience) and incubated on the plates (50 L/well) for 1 h at 37 C., at 150 rpm. TMB (Sigma-Aldrich) was used as a substrate for HRP (50 l/well). After 15 min incubation at room temperature, reaction was inhibited by 0.5 M H.sub.2SO.sub.4 solution (50 L/well). Absorption of samples was read at =450 nm.
[0306] Assays were carried out in the presence of control samples. The control of maximum binding of mAb 7-27-18 with H5 HA antigen (mAb control) was obtained in the wells of the plate, into which serum was not added. Normal chicken serum (Abcam) was used as negative control, whereas antisera obtained by vaccination of SPF chickens with inactivated H5N3 and H5N2 AIVs (x-OvO) were used as positive controls. Strong positive control was anti-H5N3 AIV antiserum, and weak positive control was anti-H5N2 AIV antiserum. 13 BELISA H5s were performed according to the above protocol. mAb control was assayed in 8 repeats on each plate. Remaining control samples and serum test samples were analysed in duplicates. The level of mAb 7-27-18-antigen binding inhibition by sera, expressed as percent, was calculated according to the formula: % inhibition=100[(A.sub.450 sample/A.sub.450 control mAb)100]. Mean A.sub.450 values for control mAb and tested samples were included into calculations.
[0307] Initial evaluation of diagnostic value of BELISA H5 was conducted using commercially available chicken sera (Abcam, x-OvO), sera from DPD, NVRI, as well as serum samples prepared during chicken immunization studies conducted by IBA. Sera for analysis were previously tested for the presence of antibodies against HA of influenza viruses using HI test. HI test allows to classify animals as anti-HA-positive or anti-HA-negative, and to determine serotype specificity of anti-sera. In the validation procedures of test for diagnostics of IV infections, HI test is treated as a so-called gold standard. HI titer for normal chicken sera (Abcam) and sera from immunization studies (IBA) were determined in IBA using H5N2 AIV (x-OvO) as antigen. Antisera (x-OvO), certified by IZSVe, were characterised using specified HI titer values.
[0308] Anti-H5-negative sera were from a group of non-vaccinated chickens differentiated in terms of type, breed, age and conditions under which chickens were raised. Anti-H5-negative sera, as well as sera positive against HA of H1-H4 and H6-H16 serotypes (x-OvO) were obtained by immunization of SPF chicken with inactivated AIVs, shown in List C. Anti-H5-positive sera were obtained by animal vaccination using H5 HA antigens from: SPF chickens with inactivated H5N1, H5N2, H5N3, H5N9 (x-OvO) AIVs shown in List C and free range chickens with recombinant H5 HA protein (rH5-E. coli). rH5-E. coli protein (the first antigen variant) was produced in IBA based on HA sequence from A/swan/Poland/305-135V08/2006 strain of H5N1 virus. The protein properties are described in Example 7. H5 HA antigens with varied sequences were used to obtain antisera. Indicators for relative homology of those antigens are shown in List E and F. Samples used for the initial evaluation of diagnostic values of BELISA H5, are described in detail in the List, below.
TABLE-US-00026 List H Samples of chicken sera analysed by BELISA H5 (IBA) using G-7-27-18 mAb. Number of chickens/ Number Sera series of samples HI titer Origin Controls for bELISA Negative control Series 1 1 <1:8.sup.6 Abcam (Cat. Normal chicken serum No. ab7477) Positive control, strong Series 1 1 1:512.sup.7 x-OvO* anti-H5N3 AIV (1).sup.1 Positive control, weak Series 1 1 1:512.sup.7 x-OvO* anti-H5N2 AIV (3).sup.1 Anti-H5-negative (1) non-laying type SPF 10 18 n.d..sup.8 DPD, NVRI ** chickens.sup.2 laying-type chickens.sup.3 30 130 <1:8.sup.6 IBA*** meat-type chickens.sup.4 42 61 <1:8.sup.6 controls in immunization tests Anti-H5-negative (2) anti AIV: H1-H4, H6-H12, after 20 positive.sup.7 x-OvO* H14-H16.sup.1 1 series anti H13 AIV.sup.1 2 series 2 positive.sup.7 x-OvO* Anti-H5-positive (1) anti H5N1 AIV.sup.1 1 series 1 1:512.sup.7 x-OvO* anti H5N2 AIV.sup.1 3 series 3 1:256; 1:512.sup.7 x-OvO* anti H5N3 AIV.sup.1 3 series 3 1:512.sup.7 x-OvO* anti H5N9 AIV.sup.1 3 series 3 1:256; 1:512.sup.7 x-OvO* Anti-H5-positive (2) anti H5 69 115 1:8 1:512.sup.6 IBA*** laying-type chickens.sup.5 immunization tests .sup.1SPF chickens were vaccinated with inactivated AIVs of serotype H1-H16 (List C) .sup.2Blood was collected on day 63 and/or 77 of life of laying SPF chickens (White Leghorn) (1 or 2 collections from 1 hen). .sup.3Blood was collected from free range laying chickens (Rossa 1) on 49, 56, 63, 70 and 77 day of life of animals (3-5 collections from 1 hen). .sup.4Blood was collected from free-range meat-type chickens (Rossa 308) on 21, 35, 38, 42 and 49 day of life of animals (1-4 collections from 1 hen). .sup.5Free range laying chickens (Rossa 1) were vaccinated with rH5- E. coli protein with HA sequence from A/swan/Poland/305-135V08/2006 strain of H5N1 AIV (EpiFluDatabase Accession No. EPI156789). The blood was collected from the animals 1 and/or 2 weeks following administration of the 2nd dose of antigen, on 56 and/or 63 day of life, or on 70 and/or 77 day of life (1 or 2 collections from 1 hen). .sup.6determined using H5N2 AIV (x-OvO) as antigen and HIU = 1:8 (IBA) .sup.7according to specification (x-OvO) .sup.8not determined *x-OvO Limited (Great Britain), Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe, Italy)) ** Department of Poultry Diseases, National Veterinary Research Institute (Pulawy, Poland) ***Institute of Biotechnology and Antibiotics (Warsaw, Poland)
[0309] Analysis of H5-negative sera with BELISA H5 allowed for determination of cut-off values for the developed test (mean value % inhibition+2SD), and therefore interpretation of the results for the tested samples. Values of % inhibition higher than cut-off values mean, that samples are seropositive, and lowerthat they are seronegative. The results of samples analysis using BELISA H5 H5 relative to cut-off values are presented in
[0310] Mean values of % inhibition of negative control, anti-H5-negative sera from non-vaccinated chickens and vaccinated AIVs of serotypes other that H5 (H1-H4, H6-H16) are shown in
[0311] Characterisation of prototype BELISA H5 was expanded to include an initial identification of the key validation parameters of the diagnostic test. Reproducibility of the assays was determined by performing independent assays for mAbs controls, negative controls (normal chicken serum) and positive controls (antisera against H5N3 and H5N2 AIVs). Analytical (Asp) and diagnostic (Dsp) specificity of the test was determined by calculating the ratio of samples denoted as true negative (TN) among sera from chickens vaccinated with AIV of H1-H4, H6-H16 serotype (Asp) and anti-H5-negative sera from non-vaccinated group of chickens (Dsp). Diagnostic sensitivity (Dse) of BELISA H5 test was determined by calculating the ratio of samples denoted as true positive (TP) among sera from chickens vaccinated with AIVs of H5 serotype (H5N1, H5N2, H5N3 and H5N9) or rH5-E. Coli protein. The results for the validation parameters of BELISA H5 are shown in Table 12, below.
[0312] More specifically, Table 12 shows the results of initially determined validation parameters of blocking ELISA for the detection of antibodies against H5 HA (BELISA H5) in chicken sera, which was developed and optimized in IBA. G-7-27-18 mAb was used in BELISA H5 assay. Analysed samples were previously classified as anti-H5-positive or -negative based on the results of HI test. Reproducibility of assays was determined by performing independent assays for control samples. Analytical (Asp) and diagnostic (Dsp) specificity of the test was evaluated by calculating the ratio of samples denoted as true negative (TN) among anti-H5-negative sera. Diagnostic sensitivity (Dse) was determined by calculating the ratio of samples denoted as true positive (TP) among anti-H5-positive sera.
TABLE-US-00027 TABLE 12 Number Number Sera of of Result Reproducibility of assays [A.sub.450] samples assays Mean SD RSD Control G-7-27-18 mAb 1 13 1.566 0.111 7.1% Negative control 1 13 1.460 0.114 7.8% Normal chicken serum Positive control, strong 1 12 0.439 0.044 10.0% anti-H5N3 AIV (1) Positive control, weak 1 12 0.944 0.083 8.8% anti-H5N2 AIV (3) Asp Analytical specificity (Asp) TN FP TN/(TN + FP) anti-H1(H2-H4, H6-H16) AIV 22 43 43 0 100% Dsp Diagnostic specificity (Dsp) TN FP TN/(TN + FP) anti-H5-negative 209 209 204 5 97.6% Dse TP FN TP/(TP + FN) Diagnostic sensitivity 1 (Dse 1) anti-H5 AIV 10 99 97 2 98.0% Diagnostic sensitivity 2 (Dse 2) anti-H5 115 115 114 1 99.1% TNtrue negative FPfalse positive TPtrue positive FNfalse negative
[0313] The results of assays for control samples, anti-H5 negative and positive samples indicate, that prototype BELISA H5 test is characterized by satisfying reproducibility (RSD: 7.1%-10.0%) and high analytical specificity factors (Asp: 100%), as well as diagnostic specificity and sensitivity (Dsp: 97.6%; Dse 1: 98.0%; Dse 2: 99.1%).
LITERATURE
[0314] Cao Z i wsp. The epitope and neutralization mechanism of AVFluIgG01, a broad-reactive human monoclonal antibody against H5N1 influenza virus. PLoS One. 2012; 7:e38126. [0315] Chiu F F i wsp. Immunological study of HA domain of hemagglutinin of influenza H5N1 virus. Biochem Biophys Res Commun. 2009; 383:27-31. [0316] Corti D i wsp. A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins. Science. 2011; 333:850-6. [0317] Dlugolenski D i wsp. Production of H5-specific monoclonal antibodies and the development of a competitive enzyme-linked immunosorbent assay for detection of H5 antibodies in multiple species. Avian Dis. 2010; 54(1 Suppl):644-9. [0318] Du L i wsp. Identification and structural characterization of a broadly neutralizing antibody targeting a novel conserved epitope on the influenza virus H5N1 hemagglutinin. J Virol. 2013; 87:2215-25. [0319] Ekiert D C i wsp. Antibody recognition of a highly conserved influenza virus epitope. Science. 2009; 324:246-51. [0320] Grabowska I i wsp. Electrochemical biosensors for detection of avian influenza viruscurrent status and future trends. Acta Biochim Pol. 2014; 61:471-8. [0321] Ha Y i wsp. H5 avian and H9 swine influenza virus haemagglutinin structures: possible origin of influenza subtypes. EMBO J. 2002; 21:865-75. [0322] Hvistendahl M. Avian influenza. Enigmatic bird flu strain races across the U.S. Midwest. Science. 2015; 348:741-2. [0323] ICTV, International Committee On Taxonomy Of Viruses, Taxonomy History, ICTV, 2014. [world wide web address: ictvonline.org/virusTaxonomy.asp?taxnode_id=20142704]. [0324] Ip H S i wsp. Novel Eurasian highly pathogenic avian influenza A H5 viruses in wild birds, Washington, USA, 2014. Emerg Infect Dis. 2015; 21:886-90. [0325] Jarocka U i wsp. An immunosensor based on antibody binding fragments attached to gold nanoparticles for the detection of peptides derived from avian influenza hemagglutinin H5. Sensors (Basel). 2014; 14:15714-28. [0326] Koh Y T i wsp. Immunological consequences of using three different clinical/laboratory techniques of emulsifying peptide-based vaccines in incomplete Freund's adjuvant. J Transl Med. 2006; 4:42. [0327] Khler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975; 256:495-7. [0328] Lamb R A, Choppin P W. The gene structure and replication of influenza virus. Annu Rev Biochem. 1983; 52:467-506. [0329] Lapolla A i wsp. Evaluation of IgG glycation levels by matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom. 1997; 11:1342-6. [0330] Lapolla A i wsp. (a) Matrix-assisted laser desorption/ionization mass spectrometry, enzymatic digestion, and molecular modeling in the study of nonenzymatic glycation of IgG. J Am Soc Mass Spectrom. 2000; 11:153-9. [0331] Lapolla A i wsp. (b) The role of mass spectrometry in the study of non-enzymatic protein glycation in diabetes. Mass Spectrom Rev. 2000; 19:279-304. [0332] Lebarbenchon C i wsp. Evaluation of a commercial enzyme-linked immunosorbent assay for detection of antibodies against the H5 subtype of Influenza A virus in waterfowl. Influenza Other Respir Viruses. 2013; 7:1237-40. [0333] Miyagawa E i wsp. Development of a novel rapid immunochromatographic test specific for the H5 influenza virus. J Virol Methods. 2011; 173:213-9. [0334] Oh H L i wsp. An antibody against a novel and conserved epitope in the hemagglutinin 1 subunit neutralizes numerous H5N1 influenza viruses. J Virol. 2010; 84: 8275-8286. [0335] Okuno Y i wsp. A common neutralizing epitope conserved between the hemagglutinins of influenza A virus H1 and H2 strains. J Virol. 1993; 67:2552-8. [0336] Petric M i wsp. Role of the laboratory in diagnosis of influenza during seasonal epidemics and potential pandemics. J Infect Dis. 2006; 194 Suppl 2:S98-110. [0337] Postel A i wsp. Broad spectrum reactivity versus subtype specificity-trade-offs in serodiagnosis of influenza A virus infections by competitive ELISA. J Virol Methods. 2011; 173:49-59. [0338] Prabakaran M i wsp. Development of epitope-blocking ELISA for universal detection of antibodies to human H5N1 influenza viruses. PLoS One. 2009; 4:e4566. [0339] Rowe T i wsp. Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays. J Clin Microbiol. 1999; 37:937-43. [0340] Saczyska V. Influenza virus hemagglutinin as a vaccine antigen produced in bacteria. Acta Biochim Pol. 2014; 61:561-72. [0341] Skehel J J, Wiley D C. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem. 2000; 69:531-69. [0342] Steinhauer D A. Role of hemagglutinin cleavage for the pathogenicity of influenza virus. Virology. 1999; 258:1-20. [0343] Stelzer-Braid S i wsp. A commercial ELISA detects high levels of human H5 antibody but cross-reacts with influenza A antibodies. J Clin Virol. 2008; 43:241-3. [0344] Suarez D L. Overview of avian influenza DIVA test strategies. Biologicals. 2005; 33:221-6. [0345] Sui J i wsp. Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat Struct Mol Biol. 2009; 16:265-73. [0346] Szewczyk B i wsp. Introduction to molecular biology of influenza a viruses. Acta Biochim Pol. 2014; 61:397-401. [0347] Verhagen J H i wsp. Infectious disease. How a virus travels the world. Science. 2015 6; 347:616-7. [0348] Wang S F i wsp. Generating and characterizing monoclonal and polyclonal antibodies against avian H5N1 hemagglutinin protein. Biochem Biophys Res Commun. 2009; 382:691-6. [0349] Wilson I A i wsp. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature. 1981; 289:366-73. [0350] Wu R i wsp. A novel neutralizing antibody against diverse clades of H5N1 influenza virus and its mutants capable of airborne transmission. Antiviral Res. 2014; 106:13-23. [0351] Xu X i wsp. Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96 (H5N1) virus: similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong. Virology. 1999; 261:15-9. [0352] Yang M i wsp. Production and diagnostic application of monoclonal antibodies against influenza virus H5. J Virol Methods. 2009; 162:194-202.