Journal of Medical Virology 86:995–1002 (2014)
Identification of a Highly Conserved and Surface Exposed B-cell Epitope on the Nucleoprotein of Influenza A Virus Xun Gui,1 Pinghui Ge,1 Xuliang Wang,1 Kunyu Yang,2,3 Hai Yu,2 Qinjian Zhao,2 Yixin Chen,1,2* and Ningshao Xia1,2** 1
National Institute of Diagnostics and Vaccine Development in Infectious Disease, State Key Laboratory of Cellular Stress Biology, School of Life Science, Xiamen University, Xiamen, China 2 School of Public Health, Xiamen University, Xiamen, China 3 Xiamen International Travel Health Care Centre, Xiamen, China
Influenza virus still poses a major threat to human health worldwide. The nucleoprotein (NP) of influenza A virus plays an essential role in the viral replication and transcription and hence becomes a promising therapeutic target. NP forms a complicated conformation under native conditions and might denature when performing immunoassays such as western blot in the study of NP function. Therefore, it is useful to make an NP specific monoclonal antibody (mAb) that recognizes linear epitope instead of conformational epitope. In this study, a recombinant NP (rNP) of influenza A virus was over-expressed and used to generate a panel of anti-NP mAbs. These anti-NP mAbs were grouped into three classes based on their reactivity in Western blots. Only Class I mAb can react with linear rNP fragments. One of Class I mAb, 4D2, was characterized further by epitope mapping with a series of overlapping synthetic peptides, indicating that the 4D2 epitope is a surface exposed, linear epitope between amino acid residues 243 and 251. This epitope is highly conserved among different influenza A viruses with an identity of 98.4% (17,922/18,210). Western blot, co-immunoprecipitation, immunofluorescence, and immunohistochemistry experiments all indicated 4D2 is highly specific to NP of influenza A virus. The results demonstrated that 4D2 can be used as a research tool for functional study of NP in the replication cycle of influenza A virus. Further work is needed to understand the function and importance of this epitope. J. Med. Virol. 86: 995–1002, 2014. # 2013 Wiley Periodicals, Inc. KEY WORDS:
influenza A virus; nucleoprotein; monoclonal antibody; conserved epitope
C 2013 WILEY PERIODICALS, INC.
INTRODUCTION Influenza A virus can cause a contagious respiratory disease with potentially fatal outcomes in both humans and animals. Every year about 250,000– 500,000 deaths in the world might result from seasonal outbreak of human H1N1 or H3N2 influenza A virus [Digard and Portela, 2002]. Also, human influenza infection can be attributed to exposure to poultry, such as avian influenza A H5N1 virus occurred since 1997 and avian influenza A H7N9 virus emerged recently in China [Uyeki, 2009; Amendola et al., 2011; Chen et al., 2013; Tang and Chen, 2013]. Because 16 H subtypes and 9 N subtypes of influenza A virus have been detected in avian species, there is concern that more lethal pandemic influenza strains might emerge through genetic reassortment or adaptation in the future and could pose greater threat to humans, like the 1918–1919 Spanish influenza which had killed about 50 million people worldwide [Johnson and Mueller, 2002]. Influenza viruses are members of the Orthomyxoviridae, which is a family of enveloped viruses with Grant sponsor: National Natural Science Foundation of China; Grant number: 81202255.; Grant sponsor: Scientific and Technological Major Special Project of China; Grant number: 2011ZX09102-009-12.; Grant sponsor: Hong Kong, Macao, and Taiwan Science and Technology Cooperation Special Project of China; Grant number: 2012DFH30020. Correspondence to: Yixin Chen, National Institute of Diagnostics and Vaccine Development in Infectious Disease, Xiamen University, Xiamen 361102, China. E-mail: [email protected] Correspondence to: Prof. Ningshao Xia, National Institute of Diagnostics and Vaccine Development in Infectious Disease, Xiamen University, Xiamen 361102, China. E-mail: [email protected] Accepted 20 September 2013 DOI 10.1002/jmv.23812 Published online 17 October 2013 in Wiley Online Library (wileyonlinelibrary.com).
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segmented, single-stranded, negative-sense RNA genomes. On the basis of the antigenic properties of the internal nucleoprotein (NP) and matrix proteins (M1), influenza viruses are divided further into three distinct serotypes (A, B, and C). Generally, NP is used as a major target in diagnosis of influenza and differentiating influenza A, B, and C due to its high degree of sequence conservation. In addition, NP is a promising therapeutic target as part of the major component of the vRNPs complex of influenza virus, which is critical for the viral replication and transcription [Digard and Portela, 2002; Noda et al., 2006]. NP of influenza A virus consists of 498 amino acids and forms a complicated conformation [Fu et al., 1997; Rimmelzwaan et al., 2000; Kreijtz et al., 2008]. While performing immunoassays such as western blot, immunofluorescence assay (IFA), immunohistochemistry assay (IHC), and co-immunoprecipitation (Co-IP) experiment, the NP proteins might be denatured and their native conformations might not be recovered. Therefore, antibodies that recognize linear epitopes instead of conformational epitopes should have some advantages in the study of NP function. However, neither linear B-cell epitope on NP protein nor linear mAb specific to NP was reported. In this study, a panel of anti-NP monoclonal antibodies (mAbs) was generated by using the rNP antigen and characterized by using a series of synthetic peptides. Several linear mAbs against NP of influenza A virus were identified and a highly conserved linear B-cell epitope on NP was identified for the first time by using mAb 4D2. Through the structural analysis and molecular docking, this linear 4D2 epitope was shown to be surface exposed. The high reactivity of this specific mAb 4D2 with influenza A virus, demonstrated with several different methods, makes 4D2 a promising tool for further study of NP function in the viral life cycle. MATERIALS AND METHODS Cells and Viruses The SP2/0 and Madin–Darby canine kidney (MDCK) cells were separately maintained in RPMI1640 (Gibco, Grand Island, NY) and Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillinstreptomycin in a humidified 5% CO2 atmosphere at 37˚C. Influenza A virus strains used in this study include A/WSN/1933 (WSN, seasonal H1N1), A/Brisbane/59/2007 (Bris/59, seasonal H1N1), A/Moscow/10/ 1999 (Mos/10, seasonal H3N2), A/Brisbane/10/2007 (Bris/10, seasonal H3N2), A/California/04/2009 (CA/ 04, 2009 pandemic H1N1), Bar-headed/Qinghai/15C/ 2005 (QH/15C, avian H5N1), Hong Kong/213/2003 (HK/213, avian H5N1), DK/HK/Y280/1997 (Y280, avian H9N2). One influenza B virus strain B/Wisconsin/01/2010 (B/Wis/01) was used as control in this study. J. Med. Virol. DOI 10.1002/jmv
Cloning and Expression of NP Gene The 1,494-bp DNA fragment encoding the NP of CA/04 was amplified with the specific primers. PCR was performed by pre-denaturing at 94˚C for 5 min, denaturing at 94˚C for 45 sec, annealing at 56˚C for 45 sec, and extending at 72˚C for 90 sec for 30 cycles, with a final elongation step for 10 min at 72˚C. PCR products were cloned into the pMD18-T vector (TaKaRa Biotechnology, Dalian, China). The resulting expression plasmid, designated as pMD18-T-NP, was then verified by restriction digestion and sequencing. Subsequently, the NP gene was subcloned into the prokaryotic expression vector pET-22b. The resulting recombinant expression plasmid, designated as pET22b-NP, was transformed into the E. coli BL21 (DE3) competent cells and propagated at 37˚C for 4 hr under induction with 1 mM isopropyl-d-thiogalactopyranoside (IPTG) for the expression of the recombinant NP (rNP). The rNP was purified by Ni-NTA affinity chromatography column (Novagen, Madison, WI) according to the manufacturer’s protocol and detected by Western blot using mouse anti-influenza A immune sera described previously [Chen et al., 2010]. Monoclonal Antibody (mAb) To produce mAb against NP of influenza A virus, 8-week-old SPF BALB/c mice were immunized subcutaneously with 100 mg rNP mixed with an equal volume of Freund’s complete adjuvant (Sigma, St. Louis, MO). Two booster immunizations using the same antigen were carried out with 50 mg rNP emulsified in incomplete Freund’s adjuvant (Sigma) on days 14 and 28. Three days after final boost, spleens of mouse were removed and the spleen cells were fused with Sp2/0-Ag-14. The hybridoma cells were cultured in RPMI-1640 containing HAT for 10– 14 days and culture supernatant from individual hybridoma clones was then screened against rNP by direct binding ELISA on rNP-coated microplates. After three times of limiting dilution, the stable hybridoma cells were propagated and inoculated into the abdominal cavity of the pristine-primed mice to produce ascitic fluid. All animal procedures were approved officially by Xiamen University Institutional Committee for the Use and Care of Laboratory Animal. Reactivity of the anti-NP mAbs with rNP was determined in Western blot and ELISA. The isotyping of the mAbs was performed using a SBA Clonotyping System/horseradishperoxidase (HRP) (Southern Biotechnology Associates, Birmingham, AL). Western Blot Purified rNPs or lysates of influenza virus were subjected to 12% SDS-PAGE gels and then transferred to 0.22 mm nitrocellulose membranes (GE Healthcare, Madison, WI). The blotted membrane was blocked with 5% skim milk in Tris-buffered
Conserved B-cell Epitope on Nucleoprotein of Flu A
saline, and then incubated with mAb at 37˚C for 60 min. After washing three times with PBS containing 0.5% Tween-20 (PBST), the membranes reacted further with alkaline phosphatase (AKP)-conjugated goat anti-mouse antibody (Abcam, Cambridge, UK) at 37˚C for 60 min and visualized with bromochlorindolephosphate/nitro blue tetrazalium (BCIP/NBT) substrate (Amresco, Solon, OH). Indirect ELISA The reactivity of the mAb with rNPs was determined by ELISA. Briefly, 100 ng/well of purified rNP protein in 20 mM bicarbonate buffer (pH 9.6) was coated on 96-well microplates at 4˚C for 12 hr and then blocked with 0.5% BSA at 37˚C for 2 hr. After washing three times with PBST, 100 ml of hybridoma culture supernatants or purified mAbs were added to the wells and incubated at 37˚C for 30 min. The plates were washed three times and incubated with HRP-conjugated goat anti-mouse secondary antibody (Abcam, Cambridge, UK) at 37˚C for 30 min. The color was developed with 100 ml of tetramethylbenzidine (TMB) substrate (Beijing Wantai, Beijing, China) and 50 ml of 2 M H2SO4 for terminating the reaction. The absorbance was measured at 450 nm. Immunofluorescence Assay (IFA) MDCK cells grown on coverslips in 24-well culture plates were infected with 200 TCID50 of influenza A virus strain CA/04. After 24 hr, the cells were washed twice with PBS and fixed with 4.0% paraformaldehyde in PBS for 20 min at room temperature. Then, the cells were washed twice again and permeabilized with 0.5% Triton X-100 for 10 min. After being blocked with 10% BSA for 20 min at room temperature, cells were treated with anti-NP mAb for 30 min at room temperature, washed with PBS, and then stained with fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin G antibody (Roche Diagnostics, Indianapolis, IN) for 30 min at room temperature. Finally, the cells were washed one more time and the plates were observed under a fluorescence microscope.
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expression of NP in lung histological sections was examined by immunohistochemical staining of histological sections. In brief, sections were blocked with 1% bovine serum albumin in PBS, stained with mAb 4D2 at a dilution of 1:2,000 overnight at 4˚C, and then incubated with goat anti-mouse IgG H- and Lchain-specific biotin conjugate (Calbiochem, Darmstadt, Germany) at a dilution of 1:2,000 for 30 min at room temperature. Tissue sections were then incubated with streptavidin/peroxidase complex reagent (Vector Laboratories, Burlingame, CA) for 30 min at room temperature, and color was developed using 3,30 -diaminobenzidine (Vector Laboratories) according to the manufacturer’s instructions [Zhu et al., 2010]. All animal procedures were approved officially by Xiamen University Institutional Committee for the Use and Care of Laboratory Animal. Competition ELISA Competition ELISA was used to detect the reactivity of the peptides with 4D2. Briefly, microplates (Nunc-Immunoplate, Roskilde, Denmark) were coated with 100 ng/well of rNP in carbonate buffer (pH 9.6) at 4˚C overnight. Equal volumes (50 ml) of diluted test peptides (10 mg/ml) and 4D2 (2 mg/ml) were mixed together at 37˚C for 2 hr, and then added to the plates and incubated at 37˚C for 30 min. Then HRP conjugated anti-mouse IgG was added and incubated at 37˚C for 30 min with subsequent washing. Color was developed with TMB substrates and stopped with 2 M H2SO4. The OD was determined at 450 nm on an automated plate reader. If the peptides had reactivity with 4D2, the OD would be low. If the peptides had no reactivity with 4D2, the OD would be high. Epitope Alignment To assess the degree of homology of the epitope recognized by 4D2, all 18,210 influenza A virus strains of different HA subtypes available from GenBank as of April 18, 2013 were selected and their amino acid sequences of NP were aligned and analyzed using Clustal W within the DNASTAR software (version 7.0, Madison, WI).
Co-Immunoprecipitation Assay (Co-IP) Influenza virus grown in MDCK cells was lysed and incubated with mAb 4D2 at 37˚C for 30 min. After the addition of 50 ml of protein A-sepharose beads, the lysates were incubated at 37˚C for 30 min. The NP protein bound to the beads were then eluted into sodium dodecyl sulfate (SDS) running buffer by heating at 95˚C for 10 min. The samples were then analyzed by SDS-PAGE and Western blot. Immunohistochemistry Analysis (IHC) By infection of mouse with influenza A virus strain CA/04 and influenza B virus strain B/Wis/01, the
RESULTS Production of the Recombinant NP Protein The open reading frame of the NP gene of influenza A virus contained 1,494 bp, which encoded a protein with 498 amino acids. In this study, the NP gene derived from 2009 pandemic H1N1 influenza A virus strain A/California/04/2009 (CA/04) was expressed in E. coli strain BL21. The rNP was purified by Ni-NTA affinity chromatography, with the MW of 58 kDa. The Western blot showed that rNP has good reactivity with mouse anti-CA/04 immune sera, indicating that the rNP has similar antigenicity to native NP (Fig. 1). As a control, the mouse anti-CA/04 immune J. Med. Virol. DOI 10.1002/jmv
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Fig. 1. Western blot analysis of the recombinant nucleoprotein derived from the influenza A virus strain CA/04. Samples loaded include purified recombinant NP, lysates of influenza virus, and molecular weight marker. One influenza A virus strain, A/California/04/2009 (CA/04), was used as a positive control, and one influenza B virus strain, B/Wisconsin/01/2010 (B/Wis/01), as a negative control. The mouse anti-CA/04 immune sera were used as the primary antibody at a dilution of 1:500 (with the stock mAb solution with a concentration of 1.0 mg/ml), anti-mouse HRP conjugated IgG was used as a secondary antibody at a dilution of 1:1,000.
sera showed positive to CA/04 of influenza A virus but not with B/Wisconsin/01/2010 (B/Wis/01) of influenza B virus. To characterize the anti-NP mAbs, three rNP fragments derived from CA/04 were also expressed in E. coli BL21, including F1 (1–120 aa), F2 (110–301 aa), and F3 (293–498 aa). Generation and Characterization of mAbs Against NP The rNP was used as immunogen in mice to produce mAbs with hybridoma technology. A panel of
43 mAbs against NP of influenza A virus was generated and was characterized further by ELISA. All mAbs were characterized with rNP and three rNP fragments (F1, F2, and F3) in Western blot and divided further into three classes based on their reactivity to the four different rNP antigens (Table I). Four of the Class I mAbs reacted not only with fulllength rNP but also with F2 fragment, implying that Class I mAbs might recognize linear epitope on NP. Twenty-one of the Class II mAbs reacted only with full-length rNP but not with any other rNP fragment. Eighteen of Class III mAbs didn’t react with any rNP
TABLE I. Representatives From Three Classes of Anti-NP mAbs Were Tabulated Based on Their Reactivity Pattern in Western Blots mAbs specific to NP of CA/04 I (n ¼ 4)
II (n ¼ 21)
III (n ¼ 18)
Recombinant NP antigens
4D2
5B11
17G7
19C10
1F2
14F10
Mouse anti-CA/04 sera
rNP (1–498 aa) F1 (1–120 aa) F2 (110–301 aa) F3 (293–498 aa)
þþ þþ
þþ þþ
þþ
þþ
þþ þ
þþ, strongly positive; þ, positive; , negative. rNP, recombinant NP with full-length NP gene of influenza A virus strain California/04/ 2009 (CA/04). F1, F2, and F3 are recombinant NP fragments derived from influenza A virus strain CA/04. Anti-CA/04 sera are a mouse serum sample immunized with virus strain CA/04.
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antigen, implying that they might be specific to conformational epitopes on NP. Interestingly, the mouse anti-CA/04 sera reacted with F2 but not with F1 and F3, indicating that linear epitope on NP might be located between 110 and 301 aa. ELISA result showed that all three classes of NP mAbs have a good reactivity to rNP as well as polyclonal antisera (Fig. 2). Reactivity of mAb 4D2 With Influenza A Virus CA/04
Fig. 2. ELISA results of three classes of NP mAb representative binding to recombinant NP (rNP). The rNP was coated at 100 ng/well, antibodies were diluted from 104 to 103 mg/ml. Mouse anti-CA/04 sera were used as positive control, anti-NP of influenza B virus mAb 10B6 was used as a negative control.
To know the reactivity of Class I mAb, representative mAb 4D2 was characterized further by using Western blot, IFA, Co-IP, and IHC experiments against influenza A virus strain CA/04 with influenza B virus strain B/Wis/01 as a negative control. Western blot showed that 4D2 had good reactivity with lysates of CA/04, but no reactivity to B/Wis/01 (Fig. 3A), implying that 4D2 may recognize a linear B-cell epitope on NP. On the whole cell level, IFA was used to test the reactivity of 4D2 with CA/04 infected MDCK cells. Consistently, the results showed that 4D2 had good reactivity with CA/04 infected cells, but not with B/Wis/01 infected cells
Fig. 3. Reactivity of anti-NP mAb 4D2 with influenza A virus strain CA/04 with influenza B virus strain B/Wis/01 as a negative control. A: Western blot analysis of CA/04 and B/Wis/01 whole viruses with 4D2. B: Indirect immunofluorescence assay of CA/04 and B/Wis/01 infected MDCK cells with 4D2. C: Co-immunoprecipitation assay of CA/04 and B/Wis/01 whole virus lysates with 4D2. D: Immunohistochemistry analysis of CA/04 and B/Wis/01 infected mouse lung tissues with 4D2.
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(Fig. 3B). In addition, the mAb 4D2 also showed positive reactivity with CA/04, but negative with B/ Wis/01 in Co-IP experiment (Fig. 3C). Then, lung tissues collected from CA/04 infected mice or B/Wis/ 01 infected mice were used to test the reactivity of NP with 4D2 in the IHC experiment. The mAb 4D2 showed good reactivity with NP of CA/04 infected tissues, but no reactivity with B/Wis/01 infected tissues (Fig. 3D). These results indicated that NP specific mAb 4D2 can be used as a specific immunochemical tool to study the function of NP in the replication of influenza A virus. Epitope Mapping of mAb 4D2 Western blot revealed that 4D2 could recognize rNP fragment F2 (110–301 aa), but not F1 or F3 (Fig. 4A). To further characterize the epitope, 15 overlapping
24-mer peptides were synthesized (F2-1 to F2-15), covering the amino acid residues between amino acid residues 111 and 305 of the NP. ELISA showed comparable binding activity of 4D2 to F2-11 (230–253 aa) and F2-12 (242–265 aa), but no activity to other peptides (Fig. 4B). Thus, the sequence 242–253 aa (242VRESRNPGNAEI253) was identified as the the epitope recognized by mAb 4D2. To further identify the core sequence of the epitope, six short peptides (P1–P6) were synthesized for competition ELISA, and the results showed that the core sequence recognized by 4D2 was P4 (243RESRNPGNA251) peptide and located between amino acid residues 243 and 251 because any deletion of 243R (P6 peptide) or 251A (P5 peptide) would abrogate the binding activity of the peptides with mAb 4D2 (Fig. 4C). This linear epitope consists of nine amino acids on NP of influenza A virus was designated as NP9. Conservation of 4D2-Defined Sequences in Influenza A Viruses In order to determine whether the epitope recognized by mAb 4D2 was conserved among different influenza A viruses, all 18,210 influenza A virus strains with different HA subtypes in GenBank were aligned and analyzed. The results showed that the epitope recognized by 4D2 is highly conserved among influenza A viruses. The identity of the epitope in influenza A viruses was 98.4% (17,922/18,210). Western blot analysis showed further that 4D2 has good reactivity with eight influenza A virus strains, including two seasonal H1N1, two seasonal H3N2, one 2009 pandemic H1N1, one avian H9N2, and two avian H5N1 but not with one influenza B virus strain (Fig. 5), demonstrating that this linear epitope is conserved and the linear mAb 4D2 would be suitable for the functional study of different subtype of influenza A virus. Since mAb 4D2 showed binding activity to the NP of H9N2, it is speculated that mAb 4D2 might react with the emerging avian influenza A H7N9 virus in China due to the NP genes of this H7N9 virus was sourced from H9N2 virus [Gao et al., 2013]. 4D2 Epitope in the 3D Structure of NP
Fig. 4. Epitope mapping of the mAb 4D2. A: Western blot result of mAb 4D2 with full-length rNP and overlapping rNP fragments. F2 in red (þ) means positive reactivity, F1 and F3 in black () means negative. B: ELISA result of mAb 4D2 with 15 overlapping 24-mer peptides. The OD at 450 nm value showed on the right, positive reactivity showed in red (F2-11, F2-12), negative reactivity in black. C: The competition ELISA results of mAb 4D2 with six truncated peptides. In competition ELISA, 2 mg/ml of mAb 4D2 was mixed with the peptides, and then the mixture was used for indirect ELISA. The peptide which can block the binding of mAb 4D2 with rNP in competition ELISA showed in red (OD value is low). The results suggest that the epitope recognized by mAb 4D2 is 243RESRNPGNA251(P4 peptide) on NP.
J. Med. Virol. DOI 10.1002/jmv
After identification of the 4D2 epitope on NP of CA/ 04 by using synthetic peptides, we mapped the nine amino acids in 3D model based on the X-ray crystal structure of NP in influenza A virus (PDB accession number is 2Q06). The image showed the nine amino acids are adjacent in protein sequence and located in the surface of NP in 3D structure (Fig. 6), showing this epitope is readily accessible under native conditions. DISCUSSION In this study, rNP derived from the influenza A virus strain CA/04 was expressed in E. coli and used
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Fig. 5. Western blot analysis of mAb 4D2 with different subtypes of influenza A viruses. The influenza A virus strains include two human seasonal H1N1 virus, A/WSN/1933 (WSN) and A/ Brisbane/59/2007 (Bris/59), two human seasonal H3N2 virus, A/Moscow/10/1999 (Mos/10) and A/ Brisbane/10/2007 (Bris/10), one 2009 pandemic H1N1 virus, A/California/04/2009 (CA/04), one avian H9N2 virus, DK/HK/Y280/1997 (Y280), two avian H5N1 virus, Bar-headed/Qinghai/15C/ 2005 (QH15C), and Hong Kong/213/2003 (HK/213). One influenza B virus strain, B/Wisconsin/ 01/2010 (B/Wis/01), was used as a negative control. Samples were loaded with virus lysates, and the anti-CA/04 mouse sera were used as controls. 1.0 mg/ml of 4D2 and anti-CA/04 mouse sera were used at a dilution of 1:500, anti-mouse HRP conjugated IgG was used as a secondary antibody at a dilution of 1:1,000.
to generate a panel of mAbs to NP protein of influenza A virus. After characterizing with four rNP antigens, these NP specific mAbs were grouped into three classes. One of the Class I mAb, 4D2, was identified and mapped further to a conserved linear epitope within nine amino acids (between amino acid residues 243 and 251 in NP of influenza A virus). This linear epitope on NP identified for the first time will be helpful to further understanding of the NP protein structure and function in the influenza virus. The structure has revealed that NP contains the head domain, the body domain and the tail loop region. Deletion mutants also showed that the head
domain plays an important role in NP–NP homooligomerization and PB2 binding [Ye et al., 2006; Ng et al., 2008]. Interestingly, the 4D2-defined epitope is located on the head domain of the X-ray crystal structure. Since this NP B-cell antigenic epitope has not been identified so far, additional investigation is needed to determine whether this epitope has function in viral replication and transcription and whether the epitope is a new antiviral target for drug design. Also, this epitope might be suitable to develop diagnostic kit due to its high degree of homology among different subtypes of influenza A virus isolates.
Fig. 6. Position and conformation of the 4D2 epitope on the 3D structure of the NP molecule. Images were created with the PyMOL Molecular Graphics System, and NP structure was obtained from the Protein Data Bank (PDB accession number is 2Q06). The amino acid numbers are indicated in one monomer. The region recognized by 4D2 is enlarged in the image on the right.
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At present, the propensity of influenza A virus to develop resistance to Tamiflu and adamantanes requires development of new drugs [Regoes and Bonhoeffer, 2006; Saito et al., 2007; Reece, 2007; Dharan et al., 2009; Moscona, 2009; McKimmBreschkin, 2013]. NP is relatively well conserved compared with viral surface spike proteins and plays an important role in the viral replication of influenza [Digard and Portela, 2002]. Recent studies have also demonstrated that NP is a novel target for the development of new antiviral drugs against the influenza virus [Hagiwara et al., 2010; Kao et al., 2010]. Further study in the roles of NP in pathogenicity and host interaction might be helpful to find some measures to control influenza [Sakabe et al., 2011]. It is said that substrates that blocked the nuclear accumulation and transportation of NP can be used to design drugs against influenza A virus because NP is accumulated in the nucleus in the early stage of infection and then distributed in cytoplasm during viral assembly and maturation [Davey et al., 1985; Boulo et al., 2007; Ozawa et al., 2007]. Since the mAb 4D2 showed good reactivity with native NP in cultured virus in Co-IP, IFA, and IHC assay and can be used to detect influenza virus in cells or tissues. It is conceivable that 4D2 (or antibodies targeting the same epitope) could function as a molecular probe for NP conformation, NP accessibility, or NP function in research as well as for some clinical testing applications. ACKNOWLEDGMENTS We thank Dr. Honglin Chen (Department of Microbiology in Hong Kong University) for providing some strains of influenza A and B virus. REFERENCES Amendola A, Ranghiero A, Zanetti A, Pariani E. 2011. Is avian influenza virus A(H5N1) a real threat to human health? J Prev Med Hyg 52:107–110. Boulo S, Akarsu H, Ruigrok RW, Baudin F. 2007. Nuclear traffic of influenza virus proteins and ribonucleoprotein complexes. Virus Res 124:12–21. Chen Y, Liang W, Yang S, Wu N, Gao H, Sheng J, Yao H, Wo J, Fang Q, Cui D, Li Y, Yao X, Zhang Y, Wu H, Zheng S, Diao H, Xia S, Chan KH, Tsoi HW, Teng JL, Song W, Wang P, Lau SY, Zheng M, Chan JF, To KK, Chen H, Li L, Yuen KY. 2013. Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: Clinical analysis and characterisation of viral genome. Lancet 381:1916–1925. Chen Y, Xu F, Gui X, Yang K, Wu X, Zheng Q, Ge S, Yuan Q, Yeo AE, Zhang J, Guan Y, Chen H, Xia N. 2010. A rapid test for the detection of influenza A virus including pandemic influenza A/ H1N1 2009. J Virol Methods 167:100–102. Davey J, Dimmock NJ, Colman A. 1985. Identification of the sequence responsible for the nuclear accumulation of the influenza virus nucleoprotein in Xenopus oocytes. Cell 40:667–675. Dharan NJ, Gubareva LV, Meyer JJ, Okomo-Adhiambo M, McClinton RC, Marshall SA, St George K, Epperson S, Brammer L, Klimov AI, Bresee JS, Fry AM. 2009. Infections with oseltamivir-resistant influenza A(H1N1) virus in the United States. JAMA 301:1034–1041. Digard P, Portela A. 2002. The influenza virus nucleoprotein: A multifunctional RNA-binding protein pivotal to virus replication. J Gen Virol 83:723–734.
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Fu TM, Friedman A, Ulmer JB, Liu MA, Donnelly JJ. 1997. Protective cellular immunity: Cytotoxic T-lymphocyte responses against dominant and recessive epitopes of influenza virus nucleoprotein induced by DNA immunization. J Virol 71:2715–2721. Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, Chen J, Jie Z, Qiu H, Xu K, Xu X, Lu H, Zhu W, Gao Z, Xiang N, Shen Y, He Z, Gu Y, Zhang Z, Yang Y, Zhao X, Zhou L, Li X, Zou S, Zhang Y, Yang L, Guo J, Dong J, Li Q, Dong L, Zhu Y, Bai T, Wang S, Hao P, Yang W, Han J, Yu H, Li D, Gao GF, Wu G, Wang Y, Yuan Z, Shu Y. 2013. Human infection with a novel avian-origin influenza A (H7N9) virus. N Engl J Med 368:1888–1897. Hagiwara K, Kondoh Y, Ueda A, Yamada K, Goto H, Watanabe T, Nakata T, Osada H, Aida Y. 2010. Discovery of novel antiviral agents directed against the influenza A virus nucleoprotein using photo-cross-linked chemical arrays. Biochem Biophys Res Commun 394:721–727. Johnson NP, Mueller J. 2002. Updating the accounts: Global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 76:105–115. Kao RY, Yang D, Lau LS, Tsui WHW, Hu LH, Dai J, Chan MP, Chan CM, Wang P, Zheng BJ, Sun JA, Huang JD, Madar J, Chen GH, Chen HL, Guan Y, Yuen KY. 2010. Identification of influenza A nucleoprotein as an antiviral target. Nat Biotechnol 28:600–605. Kreijtz JH, de Mutsert G, van Baalen CA, Fouchier RA, Osterhaus AD, Rimmelzwaan GF. 2008. Cross-recognition of avian H5N1 influenza virus by human cytotoxic T-lymphocyte populations directed to human influenza A virus. J Virol 82:5161–5166. McKimm-Breschkin JL. 2013. Influenza neuraminidase inhibitors: Antiviral action and mechanisms of resistance. Influenza Other Respi Viruses 7:25–36. Moscona A. 2009. Global transmission of oseltamivir-resistant influenza. N Engl J Med 360:953–956. Ng AK, Zhang H, Tan K, Li Z, Liu JH, Chan PK, Li SM, Chan WY, Au SW, Joachimiak A, Walz T, Wang JH, Shaw PC. 2008. Structure of the influenza virus A H5N1 nucleoprotein: Implications for RNA binding, oligomerization, and vaccine design. Faseb J 22:3638–3647. Noda T, Sagara H, Yen A, Takada A, Kida H, Cheng RH, Kawaoka Y. 2006. Architecture of ribonucleoprotein complexes in influenza A virus particles. Nature 439:490–492. Ozawa M, Fujii K, Muramoto Y, Yamada S, Yamayoshi S, Takada A, Goto H, Horimoto T, Kawaoka Y. 2007. Contributions of two nuclear localization signals of influenza A virus nucleoprotein to viral replication. J Virol 81:30–41. Reece PA. 2007. Neuraminidase inhibitor resistance in influenza viruses. J Med Virol 79:1577–1586. Regoes RR, Bonhoeffer S. 2006. Emergence of drug-resistant influenza virus: Population dynamical considerations. Science 312:389–391. Rimmelzwaan GF, Voeten JTM, Bestebroer TM, Nieuwkoop NJ, Fouchier RAM, Osterhaus ADME. 2000. Antigenic drift in the influenza A virus (H3N2) nucleoprotein and escape from recognition by cytotoxic T lymphocytes. J Virol 74:6800–6807. Saito R, Li D, Suzuki Y, Sato I, Masaki H, Nishimura H, Kawashima T, Shirahige Y, Shimomura C, Asoh N, Degawa S, Ishikawa H, Sato M, Shobugawa Y, Suzuki H. 2007. High prevalence of amantadine-resistance influenza a (H3N2) in six prefectures, Japan, in the 2005–2006 season. J Med Virol 79:1569–1576. Sakabe S, Ozawa M, Takano R, Iwastuki-Horimoto K, Kawaoka Y. 2011. Mutations in PA, NP, and HA of a pandemic (H1N1) 2009 influenza virus contribute to its adaptation to mice. Virus Res 158:124–129. Tang RB, Chen HL. 2013. An overview of the recent outbreaks of the avian-origin influenza A (H7N9) virus in the human. J Chin Med Assoc 76:245–248. Uyeki TM. 2009. Human infection with highly pathogenic avian influenza A (H5N1) virus: Review of clinical issues. Clin Infect Dis 49:279–290. Ye Q, Krug RM, Tao YJ. 2006. The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA. Nature 444:1078–1082. Zhu HC, Wang J, Wang P, Song WJ, Zheng ZY, Chen RR, Guo KY, Zhang TX, Peiris JSM, Chen HL, Guan Y. 2010. Substitution of lysine at 627 position in PB2 protein does not change virulence of the 2009 pandemic H1N1 virus in mice. Virology 401:1–5.
Identification of a Highly Conserved and Surface Exposed B-cell Epitope on the Nucleoprotein of Influenza A Virus Xun Gui,1 Pinghui Ge,1 Xuliang Wang,1 Kunyu Yang,2,3 Hai Yu,2 Qinjian Zhao,2 Yixin Chen,1,2* and Ningshao Xia1,2** 1
National Institute of Diagnostics and Vaccine Development in Infectious Disease, State Key Laboratory of Cellular Stress Biology, School of Life Science, Xiamen University, Xiamen, China 2 School of Public Health, Xiamen University, Xiamen, China 3 Xiamen International Travel Health Care Centre, Xiamen, China
Influenza virus still poses a major threat to human health worldwide. The nucleoprotein (NP) of influenza A virus plays an essential role in the viral replication and transcription and hence becomes a promising therapeutic target. NP forms a complicated conformation under native conditions and might denature when performing immunoassays such as western blot in the study of NP function. Therefore, it is useful to make an NP specific monoclonal antibody (mAb) that recognizes linear epitope instead of conformational epitope. In this study, a recombinant NP (rNP) of influenza A virus was over-expressed and used to generate a panel of anti-NP mAbs. These anti-NP mAbs were grouped into three classes based on their reactivity in Western blots. Only Class I mAb can react with linear rNP fragments. One of Class I mAb, 4D2, was characterized further by epitope mapping with a series of overlapping synthetic peptides, indicating that the 4D2 epitope is a surface exposed, linear epitope between amino acid residues 243 and 251. This epitope is highly conserved among different influenza A viruses with an identity of 98.4% (17,922/18,210). Western blot, co-immunoprecipitation, immunofluorescence, and immunohistochemistry experiments all indicated 4D2 is highly specific to NP of influenza A virus. The results demonstrated that 4D2 can be used as a research tool for functional study of NP in the replication cycle of influenza A virus. Further work is needed to understand the function and importance of this epitope. J. Med. Virol. 86: 995–1002, 2014. # 2013 Wiley Periodicals, Inc. KEY WORDS:
influenza A virus; nucleoprotein; monoclonal antibody; conserved epitope
C 2013 WILEY PERIODICALS, INC.
INTRODUCTION Influenza A virus can cause a contagious respiratory disease with potentially fatal outcomes in both humans and animals. Every year about 250,000– 500,000 deaths in the world might result from seasonal outbreak of human H1N1 or H3N2 influenza A virus [Digard and Portela, 2002]. Also, human influenza infection can be attributed to exposure to poultry, such as avian influenza A H5N1 virus occurred since 1997 and avian influenza A H7N9 virus emerged recently in China [Uyeki, 2009; Amendola et al., 2011; Chen et al., 2013; Tang and Chen, 2013]. Because 16 H subtypes and 9 N subtypes of influenza A virus have been detected in avian species, there is concern that more lethal pandemic influenza strains might emerge through genetic reassortment or adaptation in the future and could pose greater threat to humans, like the 1918–1919 Spanish influenza which had killed about 50 million people worldwide [Johnson and Mueller, 2002]. Influenza viruses are members of the Orthomyxoviridae, which is a family of enveloped viruses with Grant sponsor: National Natural Science Foundation of China; Grant number: 81202255.; Grant sponsor: Scientific and Technological Major Special Project of China; Grant number: 2011ZX09102-009-12.; Grant sponsor: Hong Kong, Macao, and Taiwan Science and Technology Cooperation Special Project of China; Grant number: 2012DFH30020. Correspondence to: Yixin Chen, National Institute of Diagnostics and Vaccine Development in Infectious Disease, Xiamen University, Xiamen 361102, China. E-mail: [email protected] Correspondence to: Prof. Ningshao Xia, National Institute of Diagnostics and Vaccine Development in Infectious Disease, Xiamen University, Xiamen 361102, China. E-mail: [email protected] Accepted 20 September 2013 DOI 10.1002/jmv.23812 Published online 17 October 2013 in Wiley Online Library (wileyonlinelibrary.com).
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segmented, single-stranded, negative-sense RNA genomes. On the basis of the antigenic properties of the internal nucleoprotein (NP) and matrix proteins (M1), influenza viruses are divided further into three distinct serotypes (A, B, and C). Generally, NP is used as a major target in diagnosis of influenza and differentiating influenza A, B, and C due to its high degree of sequence conservation. In addition, NP is a promising therapeutic target as part of the major component of the vRNPs complex of influenza virus, which is critical for the viral replication and transcription [Digard and Portela, 2002; Noda et al., 2006]. NP of influenza A virus consists of 498 amino acids and forms a complicated conformation [Fu et al., 1997; Rimmelzwaan et al., 2000; Kreijtz et al., 2008]. While performing immunoassays such as western blot, immunofluorescence assay (IFA), immunohistochemistry assay (IHC), and co-immunoprecipitation (Co-IP) experiment, the NP proteins might be denatured and their native conformations might not be recovered. Therefore, antibodies that recognize linear epitopes instead of conformational epitopes should have some advantages in the study of NP function. However, neither linear B-cell epitope on NP protein nor linear mAb specific to NP was reported. In this study, a panel of anti-NP monoclonal antibodies (mAbs) was generated by using the rNP antigen and characterized by using a series of synthetic peptides. Several linear mAbs against NP of influenza A virus were identified and a highly conserved linear B-cell epitope on NP was identified for the first time by using mAb 4D2. Through the structural analysis and molecular docking, this linear 4D2 epitope was shown to be surface exposed. The high reactivity of this specific mAb 4D2 with influenza A virus, demonstrated with several different methods, makes 4D2 a promising tool for further study of NP function in the viral life cycle. MATERIALS AND METHODS Cells and Viruses The SP2/0 and Madin–Darby canine kidney (MDCK) cells were separately maintained in RPMI1640 (Gibco, Grand Island, NY) and Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillinstreptomycin in a humidified 5% CO2 atmosphere at 37˚C. Influenza A virus strains used in this study include A/WSN/1933 (WSN, seasonal H1N1), A/Brisbane/59/2007 (Bris/59, seasonal H1N1), A/Moscow/10/ 1999 (Mos/10, seasonal H3N2), A/Brisbane/10/2007 (Bris/10, seasonal H3N2), A/California/04/2009 (CA/ 04, 2009 pandemic H1N1), Bar-headed/Qinghai/15C/ 2005 (QH/15C, avian H5N1), Hong Kong/213/2003 (HK/213, avian H5N1), DK/HK/Y280/1997 (Y280, avian H9N2). One influenza B virus strain B/Wisconsin/01/2010 (B/Wis/01) was used as control in this study. J. Med. Virol. DOI 10.1002/jmv
Cloning and Expression of NP Gene The 1,494-bp DNA fragment encoding the NP of CA/04 was amplified with the specific primers. PCR was performed by pre-denaturing at 94˚C for 5 min, denaturing at 94˚C for 45 sec, annealing at 56˚C for 45 sec, and extending at 72˚C for 90 sec for 30 cycles, with a final elongation step for 10 min at 72˚C. PCR products were cloned into the pMD18-T vector (TaKaRa Biotechnology, Dalian, China). The resulting expression plasmid, designated as pMD18-T-NP, was then verified by restriction digestion and sequencing. Subsequently, the NP gene was subcloned into the prokaryotic expression vector pET-22b. The resulting recombinant expression plasmid, designated as pET22b-NP, was transformed into the E. coli BL21 (DE3) competent cells and propagated at 37˚C for 4 hr under induction with 1 mM isopropyl-d-thiogalactopyranoside (IPTG) for the expression of the recombinant NP (rNP). The rNP was purified by Ni-NTA affinity chromatography column (Novagen, Madison, WI) according to the manufacturer’s protocol and detected by Western blot using mouse anti-influenza A immune sera described previously [Chen et al., 2010]. Monoclonal Antibody (mAb) To produce mAb against NP of influenza A virus, 8-week-old SPF BALB/c mice were immunized subcutaneously with 100 mg rNP mixed with an equal volume of Freund’s complete adjuvant (Sigma, St. Louis, MO). Two booster immunizations using the same antigen were carried out with 50 mg rNP emulsified in incomplete Freund’s adjuvant (Sigma) on days 14 and 28. Three days after final boost, spleens of mouse were removed and the spleen cells were fused with Sp2/0-Ag-14. The hybridoma cells were cultured in RPMI-1640 containing HAT for 10– 14 days and culture supernatant from individual hybridoma clones was then screened against rNP by direct binding ELISA on rNP-coated microplates. After three times of limiting dilution, the stable hybridoma cells were propagated and inoculated into the abdominal cavity of the pristine-primed mice to produce ascitic fluid. All animal procedures were approved officially by Xiamen University Institutional Committee for the Use and Care of Laboratory Animal. Reactivity of the anti-NP mAbs with rNP was determined in Western blot and ELISA. The isotyping of the mAbs was performed using a SBA Clonotyping System/horseradishperoxidase (HRP) (Southern Biotechnology Associates, Birmingham, AL). Western Blot Purified rNPs or lysates of influenza virus were subjected to 12% SDS-PAGE gels and then transferred to 0.22 mm nitrocellulose membranes (GE Healthcare, Madison, WI). The blotted membrane was blocked with 5% skim milk in Tris-buffered
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saline, and then incubated with mAb at 37˚C for 60 min. After washing three times with PBS containing 0.5% Tween-20 (PBST), the membranes reacted further with alkaline phosphatase (AKP)-conjugated goat anti-mouse antibody (Abcam, Cambridge, UK) at 37˚C for 60 min and visualized with bromochlorindolephosphate/nitro blue tetrazalium (BCIP/NBT) substrate (Amresco, Solon, OH). Indirect ELISA The reactivity of the mAb with rNPs was determined by ELISA. Briefly, 100 ng/well of purified rNP protein in 20 mM bicarbonate buffer (pH 9.6) was coated on 96-well microplates at 4˚C for 12 hr and then blocked with 0.5% BSA at 37˚C for 2 hr. After washing three times with PBST, 100 ml of hybridoma culture supernatants or purified mAbs were added to the wells and incubated at 37˚C for 30 min. The plates were washed three times and incubated with HRP-conjugated goat anti-mouse secondary antibody (Abcam, Cambridge, UK) at 37˚C for 30 min. The color was developed with 100 ml of tetramethylbenzidine (TMB) substrate (Beijing Wantai, Beijing, China) and 50 ml of 2 M H2SO4 for terminating the reaction. The absorbance was measured at 450 nm. Immunofluorescence Assay (IFA) MDCK cells grown on coverslips in 24-well culture plates were infected with 200 TCID50 of influenza A virus strain CA/04. After 24 hr, the cells were washed twice with PBS and fixed with 4.0% paraformaldehyde in PBS for 20 min at room temperature. Then, the cells were washed twice again and permeabilized with 0.5% Triton X-100 for 10 min. After being blocked with 10% BSA for 20 min at room temperature, cells were treated with anti-NP mAb for 30 min at room temperature, washed with PBS, and then stained with fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin G antibody (Roche Diagnostics, Indianapolis, IN) for 30 min at room temperature. Finally, the cells were washed one more time and the plates were observed under a fluorescence microscope.
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expression of NP in lung histological sections was examined by immunohistochemical staining of histological sections. In brief, sections were blocked with 1% bovine serum albumin in PBS, stained with mAb 4D2 at a dilution of 1:2,000 overnight at 4˚C, and then incubated with goat anti-mouse IgG H- and Lchain-specific biotin conjugate (Calbiochem, Darmstadt, Germany) at a dilution of 1:2,000 for 30 min at room temperature. Tissue sections were then incubated with streptavidin/peroxidase complex reagent (Vector Laboratories, Burlingame, CA) for 30 min at room temperature, and color was developed using 3,30 -diaminobenzidine (Vector Laboratories) according to the manufacturer’s instructions [Zhu et al., 2010]. All animal procedures were approved officially by Xiamen University Institutional Committee for the Use and Care of Laboratory Animal. Competition ELISA Competition ELISA was used to detect the reactivity of the peptides with 4D2. Briefly, microplates (Nunc-Immunoplate, Roskilde, Denmark) were coated with 100 ng/well of rNP in carbonate buffer (pH 9.6) at 4˚C overnight. Equal volumes (50 ml) of diluted test peptides (10 mg/ml) and 4D2 (2 mg/ml) were mixed together at 37˚C for 2 hr, and then added to the plates and incubated at 37˚C for 30 min. Then HRP conjugated anti-mouse IgG was added and incubated at 37˚C for 30 min with subsequent washing. Color was developed with TMB substrates and stopped with 2 M H2SO4. The OD was determined at 450 nm on an automated plate reader. If the peptides had reactivity with 4D2, the OD would be low. If the peptides had no reactivity with 4D2, the OD would be high. Epitope Alignment To assess the degree of homology of the epitope recognized by 4D2, all 18,210 influenza A virus strains of different HA subtypes available from GenBank as of April 18, 2013 were selected and their amino acid sequences of NP were aligned and analyzed using Clustal W within the DNASTAR software (version 7.0, Madison, WI).
Co-Immunoprecipitation Assay (Co-IP) Influenza virus grown in MDCK cells was lysed and incubated with mAb 4D2 at 37˚C for 30 min. After the addition of 50 ml of protein A-sepharose beads, the lysates were incubated at 37˚C for 30 min. The NP protein bound to the beads were then eluted into sodium dodecyl sulfate (SDS) running buffer by heating at 95˚C for 10 min. The samples were then analyzed by SDS-PAGE and Western blot. Immunohistochemistry Analysis (IHC) By infection of mouse with influenza A virus strain CA/04 and influenza B virus strain B/Wis/01, the
RESULTS Production of the Recombinant NP Protein The open reading frame of the NP gene of influenza A virus contained 1,494 bp, which encoded a protein with 498 amino acids. In this study, the NP gene derived from 2009 pandemic H1N1 influenza A virus strain A/California/04/2009 (CA/04) was expressed in E. coli strain BL21. The rNP was purified by Ni-NTA affinity chromatography, with the MW of 58 kDa. The Western blot showed that rNP has good reactivity with mouse anti-CA/04 immune sera, indicating that the rNP has similar antigenicity to native NP (Fig. 1). As a control, the mouse anti-CA/04 immune J. Med. Virol. DOI 10.1002/jmv
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Fig. 1. Western blot analysis of the recombinant nucleoprotein derived from the influenza A virus strain CA/04. Samples loaded include purified recombinant NP, lysates of influenza virus, and molecular weight marker. One influenza A virus strain, A/California/04/2009 (CA/04), was used as a positive control, and one influenza B virus strain, B/Wisconsin/01/2010 (B/Wis/01), as a negative control. The mouse anti-CA/04 immune sera were used as the primary antibody at a dilution of 1:500 (with the stock mAb solution with a concentration of 1.0 mg/ml), anti-mouse HRP conjugated IgG was used as a secondary antibody at a dilution of 1:1,000.
sera showed positive to CA/04 of influenza A virus but not with B/Wisconsin/01/2010 (B/Wis/01) of influenza B virus. To characterize the anti-NP mAbs, three rNP fragments derived from CA/04 were also expressed in E. coli BL21, including F1 (1–120 aa), F2 (110–301 aa), and F3 (293–498 aa). Generation and Characterization of mAbs Against NP The rNP was used as immunogen in mice to produce mAbs with hybridoma technology. A panel of
43 mAbs against NP of influenza A virus was generated and was characterized further by ELISA. All mAbs were characterized with rNP and three rNP fragments (F1, F2, and F3) in Western blot and divided further into three classes based on their reactivity to the four different rNP antigens (Table I). Four of the Class I mAbs reacted not only with fulllength rNP but also with F2 fragment, implying that Class I mAbs might recognize linear epitope on NP. Twenty-one of the Class II mAbs reacted only with full-length rNP but not with any other rNP fragment. Eighteen of Class III mAbs didn’t react with any rNP
TABLE I. Representatives From Three Classes of Anti-NP mAbs Were Tabulated Based on Their Reactivity Pattern in Western Blots mAbs specific to NP of CA/04 I (n ¼ 4)
II (n ¼ 21)
III (n ¼ 18)
Recombinant NP antigens
4D2
5B11
17G7
19C10
1F2
14F10
Mouse anti-CA/04 sera
rNP (1–498 aa) F1 (1–120 aa) F2 (110–301 aa) F3 (293–498 aa)
þþ þþ
þþ þþ
þþ
þþ
þþ þ
þþ, strongly positive; þ, positive; , negative. rNP, recombinant NP with full-length NP gene of influenza A virus strain California/04/ 2009 (CA/04). F1, F2, and F3 are recombinant NP fragments derived from influenza A virus strain CA/04. Anti-CA/04 sera are a mouse serum sample immunized with virus strain CA/04.
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antigen, implying that they might be specific to conformational epitopes on NP. Interestingly, the mouse anti-CA/04 sera reacted with F2 but not with F1 and F3, indicating that linear epitope on NP might be located between 110 and 301 aa. ELISA result showed that all three classes of NP mAbs have a good reactivity to rNP as well as polyclonal antisera (Fig. 2). Reactivity of mAb 4D2 With Influenza A Virus CA/04
Fig. 2. ELISA results of three classes of NP mAb representative binding to recombinant NP (rNP). The rNP was coated at 100 ng/well, antibodies were diluted from 104 to 103 mg/ml. Mouse anti-CA/04 sera were used as positive control, anti-NP of influenza B virus mAb 10B6 was used as a negative control.
To know the reactivity of Class I mAb, representative mAb 4D2 was characterized further by using Western blot, IFA, Co-IP, and IHC experiments against influenza A virus strain CA/04 with influenza B virus strain B/Wis/01 as a negative control. Western blot showed that 4D2 had good reactivity with lysates of CA/04, but no reactivity to B/Wis/01 (Fig. 3A), implying that 4D2 may recognize a linear B-cell epitope on NP. On the whole cell level, IFA was used to test the reactivity of 4D2 with CA/04 infected MDCK cells. Consistently, the results showed that 4D2 had good reactivity with CA/04 infected cells, but not with B/Wis/01 infected cells
Fig. 3. Reactivity of anti-NP mAb 4D2 with influenza A virus strain CA/04 with influenza B virus strain B/Wis/01 as a negative control. A: Western blot analysis of CA/04 and B/Wis/01 whole viruses with 4D2. B: Indirect immunofluorescence assay of CA/04 and B/Wis/01 infected MDCK cells with 4D2. C: Co-immunoprecipitation assay of CA/04 and B/Wis/01 whole virus lysates with 4D2. D: Immunohistochemistry analysis of CA/04 and B/Wis/01 infected mouse lung tissues with 4D2.
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(Fig. 3B). In addition, the mAb 4D2 also showed positive reactivity with CA/04, but negative with B/ Wis/01 in Co-IP experiment (Fig. 3C). Then, lung tissues collected from CA/04 infected mice or B/Wis/ 01 infected mice were used to test the reactivity of NP with 4D2 in the IHC experiment. The mAb 4D2 showed good reactivity with NP of CA/04 infected tissues, but no reactivity with B/Wis/01 infected tissues (Fig. 3D). These results indicated that NP specific mAb 4D2 can be used as a specific immunochemical tool to study the function of NP in the replication of influenza A virus. Epitope Mapping of mAb 4D2 Western blot revealed that 4D2 could recognize rNP fragment F2 (110–301 aa), but not F1 or F3 (Fig. 4A). To further characterize the epitope, 15 overlapping
24-mer peptides were synthesized (F2-1 to F2-15), covering the amino acid residues between amino acid residues 111 and 305 of the NP. ELISA showed comparable binding activity of 4D2 to F2-11 (230–253 aa) and F2-12 (242–265 aa), but no activity to other peptides (Fig. 4B). Thus, the sequence 242–253 aa (242VRESRNPGNAEI253) was identified as the the epitope recognized by mAb 4D2. To further identify the core sequence of the epitope, six short peptides (P1–P6) were synthesized for competition ELISA, and the results showed that the core sequence recognized by 4D2 was P4 (243RESRNPGNA251) peptide and located between amino acid residues 243 and 251 because any deletion of 243R (P6 peptide) or 251A (P5 peptide) would abrogate the binding activity of the peptides with mAb 4D2 (Fig. 4C). This linear epitope consists of nine amino acids on NP of influenza A virus was designated as NP9. Conservation of 4D2-Defined Sequences in Influenza A Viruses In order to determine whether the epitope recognized by mAb 4D2 was conserved among different influenza A viruses, all 18,210 influenza A virus strains with different HA subtypes in GenBank were aligned and analyzed. The results showed that the epitope recognized by 4D2 is highly conserved among influenza A viruses. The identity of the epitope in influenza A viruses was 98.4% (17,922/18,210). Western blot analysis showed further that 4D2 has good reactivity with eight influenza A virus strains, including two seasonal H1N1, two seasonal H3N2, one 2009 pandemic H1N1, one avian H9N2, and two avian H5N1 but not with one influenza B virus strain (Fig. 5), demonstrating that this linear epitope is conserved and the linear mAb 4D2 would be suitable for the functional study of different subtype of influenza A virus. Since mAb 4D2 showed binding activity to the NP of H9N2, it is speculated that mAb 4D2 might react with the emerging avian influenza A H7N9 virus in China due to the NP genes of this H7N9 virus was sourced from H9N2 virus [Gao et al., 2013]. 4D2 Epitope in the 3D Structure of NP
Fig. 4. Epitope mapping of the mAb 4D2. A: Western blot result of mAb 4D2 with full-length rNP and overlapping rNP fragments. F2 in red (þ) means positive reactivity, F1 and F3 in black () means negative. B: ELISA result of mAb 4D2 with 15 overlapping 24-mer peptides. The OD at 450 nm value showed on the right, positive reactivity showed in red (F2-11, F2-12), negative reactivity in black. C: The competition ELISA results of mAb 4D2 with six truncated peptides. In competition ELISA, 2 mg/ml of mAb 4D2 was mixed with the peptides, and then the mixture was used for indirect ELISA. The peptide which can block the binding of mAb 4D2 with rNP in competition ELISA showed in red (OD value is low). The results suggest that the epitope recognized by mAb 4D2 is 243RESRNPGNA251(P4 peptide) on NP.
J. Med. Virol. DOI 10.1002/jmv
After identification of the 4D2 epitope on NP of CA/ 04 by using synthetic peptides, we mapped the nine amino acids in 3D model based on the X-ray crystal structure of NP in influenza A virus (PDB accession number is 2Q06). The image showed the nine amino acids are adjacent in protein sequence and located in the surface of NP in 3D structure (Fig. 6), showing this epitope is readily accessible under native conditions. DISCUSSION In this study, rNP derived from the influenza A virus strain CA/04 was expressed in E. coli and used
Conserved B-cell Epitope on Nucleoprotein of Flu A
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Fig. 5. Western blot analysis of mAb 4D2 with different subtypes of influenza A viruses. The influenza A virus strains include two human seasonal H1N1 virus, A/WSN/1933 (WSN) and A/ Brisbane/59/2007 (Bris/59), two human seasonal H3N2 virus, A/Moscow/10/1999 (Mos/10) and A/ Brisbane/10/2007 (Bris/10), one 2009 pandemic H1N1 virus, A/California/04/2009 (CA/04), one avian H9N2 virus, DK/HK/Y280/1997 (Y280), two avian H5N1 virus, Bar-headed/Qinghai/15C/ 2005 (QH15C), and Hong Kong/213/2003 (HK/213). One influenza B virus strain, B/Wisconsin/ 01/2010 (B/Wis/01), was used as a negative control. Samples were loaded with virus lysates, and the anti-CA/04 mouse sera were used as controls. 1.0 mg/ml of 4D2 and anti-CA/04 mouse sera were used at a dilution of 1:500, anti-mouse HRP conjugated IgG was used as a secondary antibody at a dilution of 1:1,000.
to generate a panel of mAbs to NP protein of influenza A virus. After characterizing with four rNP antigens, these NP specific mAbs were grouped into three classes. One of the Class I mAb, 4D2, was identified and mapped further to a conserved linear epitope within nine amino acids (between amino acid residues 243 and 251 in NP of influenza A virus). This linear epitope on NP identified for the first time will be helpful to further understanding of the NP protein structure and function in the influenza virus. The structure has revealed that NP contains the head domain, the body domain and the tail loop region. Deletion mutants also showed that the head
domain plays an important role in NP–NP homooligomerization and PB2 binding [Ye et al., 2006; Ng et al., 2008]. Interestingly, the 4D2-defined epitope is located on the head domain of the X-ray crystal structure. Since this NP B-cell antigenic epitope has not been identified so far, additional investigation is needed to determine whether this epitope has function in viral replication and transcription and whether the epitope is a new antiviral target for drug design. Also, this epitope might be suitable to develop diagnostic kit due to its high degree of homology among different subtypes of influenza A virus isolates.
Fig. 6. Position and conformation of the 4D2 epitope on the 3D structure of the NP molecule. Images were created with the PyMOL Molecular Graphics System, and NP structure was obtained from the Protein Data Bank (PDB accession number is 2Q06). The amino acid numbers are indicated in one monomer. The region recognized by 4D2 is enlarged in the image on the right.
J. Med. Virol. DOI 10.1002/jmv
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Gui et al.
At present, the propensity of influenza A virus to develop resistance to Tamiflu and adamantanes requires development of new drugs [Regoes and Bonhoeffer, 2006; Saito et al., 2007; Reece, 2007; Dharan et al., 2009; Moscona, 2009; McKimmBreschkin, 2013]. NP is relatively well conserved compared with viral surface spike proteins and plays an important role in the viral replication of influenza [Digard and Portela, 2002]. Recent studies have also demonstrated that NP is a novel target for the development of new antiviral drugs against the influenza virus [Hagiwara et al., 2010; Kao et al., 2010]. Further study in the roles of NP in pathogenicity and host interaction might be helpful to find some measures to control influenza [Sakabe et al., 2011]. It is said that substrates that blocked the nuclear accumulation and transportation of NP can be used to design drugs against influenza A virus because NP is accumulated in the nucleus in the early stage of infection and then distributed in cytoplasm during viral assembly and maturation [Davey et al., 1985; Boulo et al., 2007; Ozawa et al., 2007]. Since the mAb 4D2 showed good reactivity with native NP in cultured virus in Co-IP, IFA, and IHC assay and can be used to detect influenza virus in cells or tissues. It is conceivable that 4D2 (or antibodies targeting the same epitope) could function as a molecular probe for NP conformation, NP accessibility, or NP function in research as well as for some clinical testing applications. ACKNOWLEDGMENTS We thank Dr. Honglin Chen (Department of Microbiology in Hong Kong University) for providing some strains of influenza A and B virus. REFERENCES Amendola A, Ranghiero A, Zanetti A, Pariani E. 2011. Is avian influenza virus A(H5N1) a real threat to human health? J Prev Med Hyg 52:107–110. Boulo S, Akarsu H, Ruigrok RW, Baudin F. 2007. Nuclear traffic of influenza virus proteins and ribonucleoprotein complexes. Virus Res 124:12–21. Chen Y, Liang W, Yang S, Wu N, Gao H, Sheng J, Yao H, Wo J, Fang Q, Cui D, Li Y, Yao X, Zhang Y, Wu H, Zheng S, Diao H, Xia S, Chan KH, Tsoi HW, Teng JL, Song W, Wang P, Lau SY, Zheng M, Chan JF, To KK, Chen H, Li L, Yuen KY. 2013. Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: Clinical analysis and characterisation of viral genome. Lancet 381:1916–1925. Chen Y, Xu F, Gui X, Yang K, Wu X, Zheng Q, Ge S, Yuan Q, Yeo AE, Zhang J, Guan Y, Chen H, Xia N. 2010. A rapid test for the detection of influenza A virus including pandemic influenza A/ H1N1 2009. J Virol Methods 167:100–102. Davey J, Dimmock NJ, Colman A. 1985. Identification of the sequence responsible for the nuclear accumulation of the influenza virus nucleoprotein in Xenopus oocytes. Cell 40:667–675. Dharan NJ, Gubareva LV, Meyer JJ, Okomo-Adhiambo M, McClinton RC, Marshall SA, St George K, Epperson S, Brammer L, Klimov AI, Bresee JS, Fry AM. 2009. Infections with oseltamivir-resistant influenza A(H1N1) virus in the United States. JAMA 301:1034–1041. Digard P, Portela A. 2002. The influenza virus nucleoprotein: A multifunctional RNA-binding protein pivotal to virus replication. J Gen Virol 83:723–734.
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