Molecular Biology Reports

, Volume 38, Issue 5, pp 3293–3298 | Cite as

Specific subtyping of influenza A virus using a recombinant hemagglutinin protein expressed in baculovirus

  • Shahla Shahsavandi
  • Ali-Hatef Salmanian
  • Seyed Ali Ghorashi
  • Shahin Masoudi
  • Fatemeh Fotouhi
  • Mohammad Majid Ebrahimi


Influenza A viruses are subtyped according to antigen characterization of hemagglutinin (HA) and neuraminidase surface glycoproteins. The hemagglutination inhibition (HI) assay using reference antiserum is currently applied to serologic screening of subtype-specific antibodies in sera. The reference antiserum is made by injecting chickens with live or inactivated whole virus preparations. Nonspecific inhibitors of antisera prepared by the conventional method may affect the specificity of HI assay. In this study, highly pure recombinant proteins generated using baculovirus expression vector system based on full-length of HA (HAF) and antigenic region of HA1 genes of H9 subtype, and also inactivated whole virus were used to immunization of chickens. Measurable antibody titers were present for treated birds after 3 weeks and generally increased after each boost. The performance of the prepared antisera was evaluated by testing a panel of known standard strains of influenza virus representing five HA subtypes. Relative to the conventional method using whole virus immunization and recombinant HAF protein, the antiserum prepared by recombinant HA1 had a specificity of 100% for all tested subtypes. The antiserum prepared by expression of HA1 protein in baculovirus has the potential for rapid and specific HA subtyping of influenza viruses without producing antibodies specific to other viral proteins.


Influenza A virus Recombinant hemagglutinin Baculovirus Hemagglutination inhibition 


Influenza A viruses belong to Orthomyxoviridae family infect a wide range of host including birds and mammals. The viruses have genomes comprising eight segments of RNA encoding eleven identified polypeptides and classify into subgroups based on antigenic differences in both hemagglutinin (HA), H1 to H16, and neuraminidase (NA), N1 to N9, surface glycoproteins [1]. Of the 16 HA subtypes that have been identified, only H5 and H9 subtypes having crossed-directly to infect humans [2, 3, 4] and close relationship between them [5, 6] present a much greater concern. The co-circulation of the two subtypes and emergence of new strains emphasize the need for improved diagnostic tests to characterize of newly isolated influenza viruses. Virus characterization involves subtype determination of the two surface proteins [7, 8].

Molecular methods such as RT-PCR, real-time RT-PCR and loop-mediated isothermal amplification, are considered for detecting and subtyping of influenza viruses [9, 10, 11, 12] but the most widely used serologic diagnostic technique for subtyping of influenza viruses is hemagglutination inhibition (HI) test using reference antisera [8, 13].

Antibodies to the HA are subtype-specific, so that antibodies against one subtype will not typically react with another subtype. The basis of the HI assay is that antibodies to influenza virus will prevent attachment of the virus to red blood cells. The interaction of erythrocyte sialic acid and sialic acid receptors on the HA protein resulted in cross-linking or clumping of erythrocytes by influenza virus. This hemagglutination can be inhibited by antibodies directed against the HA protein. The common means of producing reference antisera for influenza viruses is by injecting chickens with live or inactivated whole virus preparations, which may stimulate the production of antibodies to other influenza viral proteins [8, 13]. Antibodies against NA can interfere nonspecifically with HA, leading to nonspecific inhibition and possible misidentification of an isolate [14].

In this study, preparing HA antiserum using recombinant proteins generated in baculovirus expression vector system (BEVS) and the potential applications of the antiserum in subtyping of influenza viruses are discussed.

Materials and methods

Production of recombinant HAF and HA1 proteins in baculovirus

Baculovirus expression vectors expressing HA proteins of influenza virus H9N2 strain were prepared. Briefly, the total RNA was extracted from influenza virus H9N2 strain A/Chicken/Iran/SS2/2008 (Acc. No. FJ794818) by using QIAamp Viral RNA Mini Kit (Qiagen, Germany). The coding sequence of HAF and HA1 subunit genes were amplified from the cDNA by RT-PCR using one forward (5′-AACTCGAGATGGAAACAGTATCACAAATAAC-3′), and two reverse HAFR (5′-AAGCTTTTATATACAAATGTTGCACCT-3′) and HA1R (5′-AAGCTTGTAGGCACGTTTCTCAGACC-3′) primers. Underlining indicates XhoI and HindIII restriction enzyme sites at 5′ end of each primer. The purified PCR products with approximately 1.7 and 1.0 kb in length were cloned into the XhoI and HindIII sites of pFastBacTMHTB transfer vector (Invitrogen, USA) according to the manufacturers’ protocol. The integrity of the plasmid was confirmed by sequencing the each DNA in both directions. The resulting recombinant plasmids were transformed into E. coli DH10BacTM competent cell (Invitrogen) to generation recombinant bacmids. The bacmid was analyzed by electrophoresis on 0.5% agarose gel using λ DNA/HindIII marker, and also PCR reaction using gene-specific and pUC/M13 standard primers [15].

Expression of recombinant HAF and HA1 in insect suspension cultures

The Sf9 insect cells were grown as monolayers into 25 cm2 flasks at 27°C in Grace’s medium (Invitrogen) supplemented with 10% (v/v) FBS. The cells seeded at 5 × 105 cells/ml (multiplicity of infection = 0.5) were transfected with the recombinant bacmids. Supernatants containing the recombinant baculoviruses were collected at 72 h post-transfection and examined for hemadsorption assay. Briefly, 0.1 ml of 5% chicken red blood cells in PBS was added to each 0.5 ml insect cells uninfected and infected with recombinant baculoviruses and shaken gently for 10 min at room temperature. Then, 10 μl of the suspensions was placed on a glass plate and observed under a microscope. Plaque purification, amplification and end-point titration procedures were done according to Bac-to-Bac instructions (Invitrogen).

For insect suspension culture an initial cell density of 2.0 × 105 cells/ml were grown in 200 ml volumes in two 1,000 ml baffled glass flasks and incubated at 27°C in a rotary shaker with 80 rpm. Cells were inoculated with each recombinant virus and harvested by centrifugation at 3,000×g for 10 min at 72 h postinfection. The hemadsorption assay was used to monitor the expression of HA during suspension culture. The resulting cell pellets were fractionated by 12% SDS-PAGE. The separated proteins were electrotransferred and immobilized on a nitrocellulose membrane for Western blotting analysis. Hemagglutinin yields were estimated by Bradford assay after purification by Ni-NTA purification system (Invitrogen) according to the manufacturers’ instruction.

H9 reference antiserum preparation in chickens

H9 reference antiserum was prepared in chickens by recombinant protein vaccination and conventional methods. Twenty 3 week-old chickens originated from specific pathogen free eggs (Cuxhaven, Lohman, Germany) were randomly divided into four groups and housed into individual rooms with a negative-pressure ventilation system. Room temperature was maintained at 20–22°C, and water and feed ration were provided ad libitum. The H9N2 influenza virus (SS2 isolate) was inactivated by treatment with β-propiolactone (Sigma-Aldrich, USA) at a final concentration of 0.1% for 90 min at 37°C mixed with ISA-70 adjuvant (Seppic Co. France) at 30:70 (v/v) ratios. 100 μg of each recombinant protein was diluted into phosphate-buffered saline (PBS) and mixed with adjuvant at the same ratio. The chickens were divided into normal control (n = 5) and three treatments groups (each n = 5) included recombinant HAF (rHAF) protein, recombinant HA1 (rHA1) protein and inactivated H9N2 virus. Treatment birds received two separate injections of 0.2 ml intramuscularly into each breast muscle. The control group injected with PBS only. Birds were boostered with the same regime twice at 3 week intervals and were bled weekly to determine HA antibody by HI assay. Sera was harvested and inactivated at 56°C for 30 min before being used.

HI assay

The HI test using four HAU of standard H9 antigen was done [13]. 0.025 ml of PBS was dispensed into each well of a plastic V-bottomed microtitre plate. The same amount of sera from vaccinated chickens was placed into the first well and two-fold dilutions of sera were made across the plate. Four HAU of standard H9 antigen in 0.025 ml was added to each well and incubated for 30 min at room temperature. An equal volume of 1% chicken red blood cells was added to the each well and incubation continued for 40 min at room temperature. Wells containing 0.025 ml red blood cells and 0.05 ml PBS were considered as control. The HI end-point was the highest serum dilution in which agglutination was not observed.

Cross-HI test

To determine the subtype-specificity of the antibodies produced by rHAF, rHA1 proteins and inactivated virus vaccinations, the HI test was applied with standard antigens of H9N2 (Razi Institute, Iran), and H5N1, H3N8, H2N3 and H1N1 (IZS Venezie, Italy) subtypes of influenza virus.


Construction of the recombinant bacmid HAs

The full coding region of the HA and HA1 genes from H9N2 influenza virus were cloned into pFastBac HTB plasmid. The recombinant bacmid HAs were constructed by transforming the pFastBac HTB plasmids into DH10Bac E.coli and identified by electrophoresis on 0.5% agarose gel and PCR with the universal M13 primers. A 300 bp band of negative clone (size of PCR product) and target bands of positive clones were observed (Fig. 1).
Fig. 1

Analyzing of recombinant bacmids. a Agarose gel electrophoresis of bacmid DNA; M λ DNA/EcoRI + HindIII, lane 1 control (blue colony), lane 2 HA1 recombinant bacmid, lane 3 HAF recombinant bacmid. b Gel electrophoresis of bacmid DNA; M 1 kb DNA marker, lane 1 bacmid alone, lane 2 pFastBacHTB, lane 3 HA1 PCR product by specific primers, lane 4 pFastBacHTBHA1 by M13 primers, lane 5 HAF PCR product by specific primers, lane 6 pFastBacHTBHAF by M13 primers

Expression of the recombinant HAF and HA1 proteins

Sf9 cells were transfected with the recombinant bacmids. Supernatants were harvested when the hemadsorption activity assay showed in infected cells containing the HA proteins. Infected cells were larger than uninfected cells and adsorbed by red blood cells, revealed that the recombinant HA protein expressed on the surface of the insect cells. By monitoring of signs of infection the proteins were collected from Sf9 cell cultures 72 h after transfection. Subsequently, the cell lysates were analyzed by SDS-PAGE and Western blot analysis (Fig. 2). The HA and HA1 proteins showed specific bands at ~68 and ~43 kDa position, respectively. These recombinant proteins were purified and protein concentrations were measured as 42 mg/l for HAF and 48 mg/l for HA1.
Fig. 2

Detection of protein expression in insect cells. a SDS-PAGE analysis of HA recombinant proteins stained by Coomassie blue; M Molecular marker, lane 1 recombinant HAF protein, lane 2 recombinant HA1 protein. b Western blot analysis of expressed HA proteins; M Molecular marker, lane 1 recombinant HAF protein, lane 2 recombinant HA1 protein

Immunogenicity of recombinant HA proteins in chickens

HA-specific humoral immunity was evaluated by HI test. During the first 3 weeks following a single immunization, antibody titers were present for treated birds and generally increased after each boost. A 16-fold increase in HI titer of the antibody produced by inactivated virus was observed after first boosting (4.8–9.6 log2 HI titer), which increase to 10.2 log2 HI titer after second boosting (Fig. 3). The HI titer with sera prepared by rHAF immunization produced similar result. The HA antibody raised by rHA1 showed low titer after second boosting, which remained steady after 2 weeks.
Fig. 3

Mean log2 HI titers of 3 week-old SPF chicken groups immunized with inactivated whole H9N2 virus, recombinant HAF protein and recombinant HA1 protein, and boostered at 3 and 6 weeks after primary immunization. Sera were collected from individual birds every week and the HI titers were determined

Cross-HI test

H5, H3, H2 and H1 antigens exhibited cross-reactivity (1:8–1:16) with H9 subtype antisera prepared by rHAF and inactivated virus vaccinations. This was somewhat expected because of the high HA2 subunit sequence similarities between these strains. No cross-reactivity was found in rHA1 vaccine antiserum. The specificity of the rHAF antiserum was equal to conventional antiserum but the antiserum prepared by recombinant HA1 had a specificity of 100% for all tested subtypes.


By using recombinant protein production strategy and stimulate the generation of anti-HA neutralizing antibodies, we were able to introduce an alternative to the conventional method of preparing subtype-specific influenza antiserum. HA glycoprotein is the major antigen of the virus and the target for neutralizing antibodies. It is responsible for pathogenicity [16, 17, 18] and viral binding to host receptors containing sialic acid groups. HA subtyping is crucial for the control and epidemiological study of the disease. In recent years molecular techniques have been used to differentiate subtypes [9, 10, 11, 12], but the standard method for subtyping of the HA of influenza viruses is the HI test with reference antisera to the known HA subtypes [12, 13]. To avoid problems occur with HI, safety and steric hindrance [14], preparation of antiserum of good quality is essential. The BEVS is widely used for recombinant biologics production [19], provides several advantages over currently licensed technology. In order to produce HA subtype-specific antiserum, we used the recombinant baculovirus expression method for production of HA proteins. Recombinant baculoviruses carrying the HAF and HA1 genes of a G1 sublineage H9N2 avian influenza virus (data not shown), A/Chicken/Iran/SS2/2008 were constructed and propagated in Sf9 cells under suspension culture. The molecular weight and binding properties of HA proteins were determined by SDS-PAGE and Western immunoblotting. The presence of the N-terminal 6× His tag and the TEV recognition site increase the size of recombinant protein by at least 3 kDa [16].

We were interested in determining if the expressed HAF and HA1 proteins would be suitable as antigen in HA antisera production for using in HI test. In order to study the immunogenicity of the recombinant HAs, groups of chickens were immunized with these proteins and inactivated whole virus, and the immune response measured by HI assay. The antibody response to these proteins after three booster injections is shown in Fig. 3. A 10.2 log2 HI titer was obtained after three vaccinations with inactivated whole virus and lower HI titers were observed in sera derived from chickens immunized with 100 μg of rHAF and rHA1 proteins. It has been suggested that higher doses of subunit vaccine were needed to induce responses comparable to those elicited by current licensed egg-derived influenza antigens [20].

In cross-HI tests with antiserum prepared by rHAF vaccination and antiserum prepared by a conventional method, minor cross-reactivity was observed between the G1 H9N2 and other influenza virus subtypes used in this study. In contrast, when HI test was applied with antiserum prepared by rHA1 vaccination, no cross-reactivity was observed. Regarding to the less specificity, sequence similarities between these strains were studied. The six internal genes of G1 sublineage H9N2 viruses are similar to those of the highly pathogenic H5N1 viruses isolated from humans in 1997 [6]. Although the degree of sequence diversity between subtypes is great, particularly in the HA1 subtype, more conserved regions are found in HA2 (>90% homology between subtypes). The HA2 subunit, the more conserved part among all influenza A viruses [9, 20] is responsible for mediating the fusion of viral and cell membranes during the entry into the cell [21]. While HA2 does not contribute significantly to the antigenic activity of influenza virus HA, the anti-HA2 antibodies are induced during natural and experimental infections [22, 23]. Vaccination studies have been performed with the HA2 subunit but have not demonstrated robust protective responses in experimental models [24, 25]. Polyclonal anti-HA2 antibodies revealed very low reactivity with purified undissociated HA suggesting that in intact HA the majority of epitopes on HA2 subunits are not accessible for interaction with antibodies [26]. Despite anti-HA1 antibodies, these antibodies do not inhibit hemagglutination or neutralize the infectivity of the virus [22, 27]. Because of HA glycoprotein is the principal target of protective humoral immune responses to influenza virus infections, both rHAF and inactivated whole virus are similarly immunogenic in chickens can provide efficient protection against HA antigenic variants and induce titers of cross-reactive HI antibody. It is shown that when HI tests were conducted with antisera prepared by a conventional method, cross-reactivities were observed among some isolates that shared high HA sequence homology as well as among isolates that shared the same NA subtype [14]. Our study shows that the reference antiserum prepared by rHA1 vaccination could be applied in HI test for influenza subtyping purposes. Biologically, rHA1 protein is as immunogenic as the whole virus and exact match to the natural virus. It is highly purified and does not contain egg proteins, making the antigen safe. Unlike the conventional method, no live influenza viruses and inactivation step are used during antiserum production. In conclusion, the reference antiserum production based on recombinant HA1 developed in this study could be a viable alternative to the conventional method of preparing reference antiserum.


  1. 1.
    Fouchier RA, Munster V, Wallensten A (2005) Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol 79:2814–2822PubMedCrossRefGoogle Scholar
  2. 2.
    Butt KM, Smith GJD, Chen H, Zhang LJ, Connie Leung YH, Xu KM, Lim W, Webster RG, Yuen KY, Malik Peiris JS, Guan Y (2005) Human infection with an avian H9N2 influenza A virus in Hong Kong in 2003. J Clin Microbiol 43:5760–5767PubMedCrossRefGoogle Scholar
  3. 3.
    Guan Y, Poon LLM, Cheung CY, Ellis TM, Lim W, Lipatov AS, Chan KH, Peiris JSM (2004) H5N1 influenza: a protean pandemic threat. PNAS 101:8156–8161PubMedCrossRefGoogle Scholar
  4. 4.
    Shaw M, Cooper L, Xu X, Thompson W, Krauss S, Guan Y, Zhou N, Subbarao K (2002) Molecular changes associated with the transmission of avian influenza A H5N1 and H9N2 viruses to humans. J Med Virol 66:107–114PubMedCrossRefGoogle Scholar
  5. 5.
    Guan Y, Shortridge KF, Krauss S, Chin PS, Dyrting KC, Ellis TM, Webster RG, Peiris M (2000) H9N2 influenza viruses possessing H5N1-like internal genomes continue to circulate in Southeastern China. J Virol 74:9372–9380PubMedCrossRefGoogle Scholar
  6. 6.
    Guan Y, Shortridge KF, Krauss S, Webster RG (1999) Molecular characterization of H9N2 influenza viruses: were they the donors of the internal genes of H5N1 viruses in Hong Kong? PNAS 96:9363–9367PubMedCrossRefGoogle Scholar
  7. 7.
    Peiris M, Yam WC, Chan KH, Ghose P, Shortridge KF (1999) Influenza A H9N2: aspects of laboratory diagnosis. J Clin Microbiol 37:3426–3427PubMedGoogle Scholar
  8. 8.
    Swayne DE, Senne DA, Beard CW (1998) Influenza. In: Isolation and identification of avian pathogens, 4th edn. American association of avian Pathologists, University of Pennsylvania, USA, pp 235–240Google Scholar
  9. 9.
    Phipps LP, Essen SC, Brown IH (2004) Genetic subtyping of influenza A viruses using RT-PCR with a single set of primers based on conserved sequences within the HA2 coding region. J Virol Methods 122:119–122PubMedCrossRefGoogle Scholar
  10. 10.
    Lee CW, Suarez DL (2004) Application of real-time RT-PCR for the quantitation and competitive replication study of H5 and H7 subtype avian influenza virus. J Virol Methods 119:151–158PubMedCrossRefGoogle Scholar
  11. 11.
    Mori Y, Notomi T (2009) Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chem 15:62–69CrossRefGoogle Scholar
  12. 12.
    Ong WT, Omar AR, Ideris A, Seyed Hassan S (2007) Development of a multiplex real-time PCR assay using SYBR Green 1 chemistry for simultaneous detection and subtyping of H9N2 influenza virus type A. J Virol Methods 144:57–64PubMedCrossRefGoogle Scholar
  13. 13.
    Office International des Epizooties (2005) Avian influenza. In: Manual of diagnostic tests and vaccines for terrestrial animals, 5th edn. Office International des Epizooties, ParisGoogle Scholar
  14. 14.
    Lee C-W, Senne DA, Suarez DL (2006) Development and application of reference antisera against 15 hemagglutinin subtypes of influenza virus by DNA vaccination of chickens. Clin Vaccine Immunol 13:395–402PubMedCrossRefGoogle Scholar
  15. 15.
    Bac-to-Bac Baculovirus Expression System 2002. version C. Invitrogen life technologiesGoogle Scholar
  16. 16.
    Okamatsu M, Sakoda Y, Kishida N, Isoda N, Kida H (2008) Antigenic structure of the hemagglutinin of H9N2 influenza viruses. Arch Virol 153:2189–2195PubMedCrossRefGoogle Scholar
  17. 17.
    Gambaryan A, Yamnikova S, Lvov D, Tuzikov A, Chinarev A et al (2005) Receptor specificity of influenza viruses from birds and mammals: new data on involvement of the inner fragments of the carbohydrate chain. Virology 334:276–283PubMedCrossRefGoogle Scholar
  18. 18.
    Skehel JJ, Wiley DC (2000) Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 69:531–569PubMedCrossRefGoogle Scholar
  19. 19.
    Kost T, Condreay PJ, Jarvis DL (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23:567–575PubMedCrossRefGoogle Scholar
  20. 20.
    Jones T, Allard F, Cyr SL, Tran SP, Plante M, Gauthier J, Bellerose N, Lowell GH, Burt DS (2003) A nasal proteosome influenza vaccine containing baculovirus-derived hemagglutinin induces protective mucosal and systemic immunity. Vaccine 21:3706–3712PubMedCrossRefGoogle Scholar
  21. 21.
    Spackman E, Senne DA, Myers TJ, Bulaga LL, Garber LP, Perdue ML, Lohman K, Suarez DL (2002) Development of a real-time RT-PCR assay type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J Clin Microbiol 40:3250–3256CrossRefGoogle Scholar
  22. 22.
    Bosch FX, Garten W, Klenk HD, Rott R (1981) Proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between HA1 and HA2 determines proteolytic cleavability and pathogenicity of avian influenza virus. Virology 113:725–735PubMedCrossRefGoogle Scholar
  23. 23.
    Graves PN, Schulman JL, Young JF, Palese P (1983) Preparation of influenza virus subviral particles lacking the HA1 subunit of hemagglutinin: unmasking of cross-reactive HA2 determinants. Virology 126:106–116PubMedCrossRefGoogle Scholar
  24. 24.
    Gerhard W (2001) The role of the antibody response in influenza virus infection. Curr Top Microbiol Immunol 260:171–190PubMedGoogle Scholar
  25. 25.
    Cox RJ, Brokstad KA (1999) The postvaccination antibody response to influenza virus proteins. APMIS 107:289–296PubMedCrossRefGoogle Scholar
  26. 26.
    Gocník M, Fislová T, Mucha V, Sládková T, Russ G, Kostolanský F, Varečková E (2008) Antibodies induced by the HA2 glycopolypeptide of influenza virus hemagglutinin improve recovery from influenza A virus infection. J Gen Virol 89:958–967PubMedCrossRefGoogle Scholar
  27. 27.
    Vare-kovfi E, Mueha V, Betfikovfi T, Russ G (1993) Monoclonal antibodies demonstrate accessible HA2 epitopes in minor subpopulation of native influenza virus hemagglutinin molecules. Arch Virol 130:45–56CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Shahla Shahsavandi
    • 1
    • 2
  • Ali-Hatef Salmanian
    • 1
  • Seyed Ali Ghorashi
    • 1
  • Shahin Masoudi
    • 2
  • Fatemeh Fotouhi
    • 3
  • Mohammad Majid Ebrahimi
    • 2
  1. 1.National Institute of Genetic Engineering and BiotechnologyTehranIran
  2. 2.Razi Vaccine and Serum Research InstituteKarajIran
  3. 3.Pasteur Institute of IranTehranIran

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