Specific subtyping of influenza A virus using a recombinant hemagglutinin protein expressed in baculovirus
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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.
KeywordsInfluenza 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 . 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 .
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 .
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.
The HI test using four HAU of standard H9 antigen was done . 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.
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
Expression of the recombinant HAF and HA1 proteins
Immunogenicity of recombinant HA proteins in chickens
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 , preparation of antiserum of good quality is essential. The BEVS is widely used for recombinant biologics production , 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 .
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 .
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 . 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 . 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 . 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 . 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.
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