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Insect Cell Surface Expression of Hemagglutinin (HA) of Egyptian H5N1 Avian Influenza Virus Under Transcriptional Control of Whispovirus Immediate Early-1 Promoter
Insect Cell Surface Expression of Hemagglutinin (HA) of Egyptian H5N1 Avian Influenza Virus Under Transcriptional Control of Whispovirus Immediate Early-1 Promoter
Journal of Microbiology and Biotechnology. 2014. Dec, 24(12): 1719-1727
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : May 12, 2014
  • Accepted : August 06, 2014
  • Published : December 28, 2014
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About the Authors
M. R. Gadalla
Department of Virology, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt
A. H. El-Deeb
Department of Virology, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt
M. M. Emara
Qatar Biomedical Research Institute, Qatar Foundation, Education City, 5825, Doha, Qatar
H. A. Hussein
Department of Virology, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt
husvirol@link.net

Abstract
In the present study, whispovirus immediate early 1 promoter (ie-1) was used to initiate surface expression of the hemagglutinin (HA) protein of Egyptian H5N1 avian influenza virus (AIV) by using the baculovirus expression vector system. The HA gene and whispovirus ie-1 promoter sequence were synthesized as a fused expression cassette (ie1-HA) and successfully cloned into the pFastBac-1 transfer vector. The recombinant vector was transformed into DH10Bac competent cells, and the recombinant bacmid was generated via site-specific transposition. The recombinant bacmid was used for transfection of Spodoptera frugiperda ( Sf-9 ) insect cells to construct the recombinant baculovirus and to induce expression of the HA protein of H5N1 AIV. The recombinant glycoprotein expressed in Sf-9 cells showed hemadsorption activity. Hemagglutination activity was also detected in both extra- and intracellular recombinant HAs. Both the HA and hemadsorption activities were inhibited by reference polyclonal anti-H5 sera. Significant expression of the recombinant protein was observed on the surface of infected insect cells by using immunofluorescence. SDS-PAGE analysis of the expressed protein revealed the presence of a visually distinguishable band of ~63 kDa in size, which was absent in the non-infected cell control. Western blot analysis confirmed that the distinct 63 kDa band corresponded to the recombinant HA glycoprotein of H5N1 AIV. This study reports the successful expression of the HA protein of H5N1 AIV. The expressed protein was displayed on the plasma membrane of infected insect cells under the control of whispovirus ie-1 promoter by using the baculovirus expression vector system.
Keywords
Introduction
Baculoviruses are members of the family Baculoviridae, which infect insects and use them as natural hosts. These large rod-shaped double-stranded DNA viruses are widely used as expression vectors for the production of recombinant proteins [9] . Autographa californica multiple nucleopolyhedro virus (AcMNPV) is the most well-known baculovirus expression vector system (BEVS) that has been extensively used to express heterologous proteins in insect cells [4 , 28] . The BEVS possesses many advantages, including the production of high-quality proteins, improvement in protein solubility, and proper post-translational modifications such as glycosylation, acetylation, and phosphorylation [11] . Moreover, the BEVS has the capacity to produce high protein yields that ease scaling-up procedures for manufacturing purposes. Altogether, these advantages qualify the BEVS to be one of the most efficient and reliable systems for recombinant protein production.
The initial attempts to express recombinant proteins using a BEVS depended on cloning the gene of interest downstream of the very late baculovirus promoters (polyhedrin or p10). Although these are very strong promoters that have the ability to produce high yields of the expressed protein(s), they only reach their maximum activity at the late phase of infection, when cells start to enter the death phase. This leads to the improper folding and/or processing of recombinant proteins, especially during the expression of secretory or membrane-associated proteins [8] . To avoid this limitation, various alternative early promoters such as the baculovirus Eco RI restriction fragment T promoter, which is also known as “early to late” promoter (ETL) [5] , and the white spot syndrome virus (WSSV)-ie1 promoter have been utilized in BEVSs [20] Although, both promoters can act as shuttle promoters that drive protein expression in both insect and mammalian cells [6 , 19] , the WSSV-ie1 promoter has demonstrated stronger activity than the baculovirus ETL promoter [6] . Moreover, WSSV-ie1 promoter activation acts independently from other viral proteins or host-specific transcription factors, which enables the initiation of target protein expression a few hours after infection [8] .
Hemagglutinin (HA) is a major envelope glycoprotein that is exposed on the surface of influenza viruses. This protein retains antigenic properties, which elicit an immune response against influenza virus infection. This immune response is primarily mediated by the induction of proper neutralizing antibodies, which are directed specifically towards HA. Therefore, HA represents the main building block of the influenza recombinant subunit vaccine [3] . The ectopic expression of avian influenza HA recombinant protein in insect cells was first reported by Kuroda et al. [14] , who demonstrated that the recombinant HA protein was able to maintain its biological activity and antigenic properties after its expression by using this system. Subsequently, several studies showed successful production of HA recombinant protein by using the BEVS [2 , 17] . This expression ranges from the production of purified recombinant HA protein under the transcriptional control of the very late baculovirus polyhedrin promoter [22] , to its display on the baculovirus surface by genetic fusion with the baculovirus envelope protein gp64 or by using early promoters [1 , 27] . Lu et al. [20] have expressed the HA protein of H5N1 AIV using the WSSV-ie1 promoter on the baculovirus surface. The expression of recombinant HA protein under these conditions has maintained authentic HA cleavage ability and hemaggl tination activity as well as immunogenicity without affecting baculovirus growth. Moreover, the potential of the WSSV-ie1 promoter to produce high yields of HA protein was reflected in its superior ability to induce high levels of anti-HA antibodies in chickens immunized with baculovirus-expressing HA under the WSSV-ie1 promoter, as indicated by hemagglutination inhibition (HI), virus neutralization, and ELISA assays [7] . Importantly, the expression of recombinant HA by using the BEVS in insect cells has been extensively evaluated as a safe and effective subunit vaccine against HPAI H5N1 in both chickens [16] and humans [29] .
In the present study, we have constructed a recombinant baculovirus, expressing HA of Egyptian H5N1 avian influenza virus under transcriptional control of the novel WSSV-ie1 promoter in insect cells. The biological activity of the recombinant Egyptian H5N1 HA protein was detected by hemadsorption and hemagglutination assays, and the antigenicity of the expressed protein was characterized using hemagglutination inhibition, immune-blotting, and indirect immunofluorescence assays. The successful expression of the Egyptian H5N1 HA protein will pave the way for the development of a novel avian influenza subunit vaccine that aims to control the continuously evolving avian influenza virus in Egypt.
Materials and Methods
- Construction of the Expression Vector
The construction of the expression vector was done through two major steps. First, the synthetic expression cassette was generated from the HA gene sequence of the Egyptian highly pathogenic avian influenza H5N1 virus (from clade 2.2.1.1; A/chicken/Egypt/121/2012(H5N1)) and the WSSV-ie1 promoter sequence. The promoter sequence was retrieved from the WSSV complete genome (GenBank Accession No. AF369029 by using the primer sets ie1-F (5’-TCCCTACGTATCAATTTTATGTGGCTAATGGAGA-3’) and ie1-R (5’-TTATAACTAGCTCTCTCTCTCCACTCAAGGTCGACGCGT-3’) [20] . Then this fused synthetic cassette was cloned in the pUC57 plasmid. Second, the original polyhedrin promoter sequence in the transfer plasmid (pFastBac1) was deleted using Sna BI and Hin dIII digestion, and then the transfer vector backbone was ligated to the synthetic expression cassette (ie1-HA) by using T4 DNA ligase (Invitrogen, Carlsbad, CA, USA) to generate the recombinant transfer vector (pFastBac-ie1-HA). This recombinant plasmid was transformed into chemically competent Escherichia coli cells (MAX Efficiency DH5α; Invitrogen, USA). The recombinant plasmid was isolated using the QIAprep Spin Miniprep Kit (Qiagen, Germany), and subjected to PCR analysis by using both the gene cassette (ie1-F) and plasmid specific primers (pFastBac-R) (Bac-To-Bac kit manual; Invitrogen, USA) to verify the correct orientation of the gene within the backbone. The correct orientation and the presence of the cloned cassette were confirmed by restriction digestion using Sna BI and Hin dIII enzymes. Finally, the recombinant bacmids were generated according to the Bac-To-Bac expression system protocol (Invitrogen, USA). The pFastBac-ie1-HA recombinant plasmid was transformed into DH10Bac (Invitrogen, USA) competent cells containing the bacmid DNA and a helper plasmid encoding transposase enzyme for the generation of recombinant bacmids through site-specific transposition, according to protocol of the Bac-To-Bac expression system (Invitrogen, USA). The recombinant bacmid was isolated using the PureLink HiPure Plasmid Miniprep Kit (Invitrogen, USA) and verified for successful transposition of the gene cassette by PCR using pUC/M13 and gene-specific primers:
  • pUC/M13 Forward: 5’-CCCAGTCACGACGTTGTAAAACG-3’
  • pUC/M13 Reverse: 5’-AGCGGATAACAATTTCACACAGG-3’
- Cells and Transfection
Spodoptera frugiperda ( Sf-9 ) insect cells (Invitrogen, USA) were maintained at 27℃ in Grace’s insect medium (Gibco, UK) supplemented with 10% (v/v) fetal bovine serum (Gibco, UK) and 1% penicillin/streptomycin (Sigma-Aldrich, USA). Sf-9 monolayer cells were transfected with the recombinant bacmid by using Cellfectin II (Invitrogen, USA) according to the manufacturer’s protocol. The transfected cells were monitored daily for development of cytopathic effect (CPE), and the baculoviruses expressing recombinant HA (Bac-ie1-HA) were harvested from the cell culture supernatant at 96 h post-transfection. The harvested supernatant was then centrifuged at 3,000 rpm for 5 min, collected, and stored at 4℃.
- Hemadsorption and Hemadsorption Inhibition Assays
Sf-9 insect cells were cultured in p-25 cm 2 sterile tissue culture flasks. Cells were infected with Bac-ie1-HA recombinant virus according to the manufacturer‘s instructions. At 48 h post-infection, the medium was removed, and cells were washed three times via gentle rocking with sterile phosphate-buffered saline (PBS) (pH 7.2). Both infected and non-infected cells were then overlaid with 5 ml of 0.5% chicken red blood cells (RBCs) for 1 h at 37℃, after which the RBCs were washed off several times with PBS and examined with an inverted light microscope (Hund, Germany). For the hemadsorption inhibition assay, the recombinant baculoviruses were incubated with polyclonal anti-H5 antibody (Harbin, China) for 60 min at 37℃. The incubated mixture was used to inoculate Sf-9 cells and was examined microscopically for hemadorption activity by using chicken RBCs at 48 h post-inoculation.
- Hemagglutination and Hemagglutination Inhibition Assays
The hemagglutination assay was performed as previously described [12] . Briefly, 50 µl of the infected cell lysate was serially diluted in an equal volume of PBS and then mixed with an equal volume of 0.5% chicken RBCs in 96-well HA plates. The plates were then incubated for 30 min at room temperature and the degree of RBC agglutination was recorded. The hemagglutination activity is expressed using the hemagglutination activity unit (HAU), which is defined as the highest dilution that causes visible hemagglutination/well. In the hemagglutination inhibition assay, 8 HAU/50 µl of recombinant protein was prepared and incubated with 2-fold serially diluted anti-H5 antisera (Harbin, China) for 30 min at room temperature. This was followed by the addition of 0.5% chicken RBCs. This mixture was incubated for 30 min at room temperature and the hemagglutination inhibition unit (HI) was determined by the reciprocal of the last dilution that contains nonagglutinated RBCs.
- Immunofluorescence Assay
Sf-9 cells were cultured in sterile 6-well plates and infected with the recombinant baculovirus according to the manufacturer’s instructions. At 48 h post-infection, the cells were fixed with 4% paraformaldehyde for 30 min at 4℃. After fixation, the cells were rinsed with cold PBS, blocked for 1 h at 37℃ with 1% bovine serum albumin in PBS, and then incubated with polyclonal antiH5 primary antibody (1:100 dilution in blocking buffer; Harbin, China) for 1 h at 37℃. The cells were then washed three times with PBS and incubated with the secondary antibodies (fluorescein isothiocyanate conjugated rabbit anti-chicken, 1:200 dilution; MP Biomedicals, USA) for 1 h at 37℃. The cells were then washed three times with cold PBS. After the final washes, the cells were mounted with glycerol/PBS (1:1 (v/v)), and the cells expressing the recombinant HA protein were detected and imaged using an inverted fluorescence microscope (Olympus CX41, Japan) equipped with a digital imaging system (Olympus, Japan).
- SDS-PAGE and Western Blot Analysis
The expression of recombinant HA under the transcriptional control of the WSSV-ie1 promoter was detected using SDS-polyacrylamide gel electrophoresis followed by western blot analysis. Recombinant baculovirus-infected or -non-infected Sf-9 monolayer cells were harvested by centrifugation at 3,000 rpm for 5 min and washed three times with cold PBS, and the cell pellet was resuspended in lysis buffer (300 mM NaCl, 20 mM Tris–HCl (pH 8), 1% Triton X-100, and 1× protease inhibitor cocktail (Sigma-Aldrich)) [10] . The resuspended pellet was then incubated for 30 min on ice before another round of centrifugation at 12,000 rpm for 5 min. After centrifugation, cell lysate proteins (10-20 µg) from both infected and non-infected Sf-9 cells were separated on 10% SDS-PAGE and then transferred to nitrocellulose membranes (Carlroth, Belguim). The membranes were then blocked using 10% non-fat dry milk overnight at 4℃. The polyclonal antiH5 primary antibody (Harbin, China) diluted in blocking buffer (1:100) was then added to the membranes and incubated for 1 h at 37℃. The membranes were washed three times with washing buffer (0.05% Tween 20 in PBS) and then incubated with the secondary antibody (anti-chicken IgG alkaline phosphatase antibody; Sigma-Aldrich, USA), diluted 1:6,000 in blocking buffer. After washing with PBS/Tween 20 three successive times, the reaction was developed using BCIP/NBT reagent (Sigma-Aldrich, USA) and photographed using a Nikon digital camera (Nikon, USA).
Results
- Construction of Recombinant Baculovirus Expression Vector Containing the HA Gene of Egyptian H5N1 AIV
Previous studies have shown that avian influenza HA can be expressed in insect cells by using a BEVS without affecting its biological activity or antigenic properties [14] . Such expression has been demonstrated in different forms and by using HA genes of different influenza virus strains and subtypes [1 , 22 , 27] . Therefore, we sought to determine whether the HA gene of Egyptian H5N1 AIV can be expressed in insect cells by using a BEVS. This process is based on two major steps; the first step is to generate an HA transfer vector, and the second is to transfer the HA gene to the bacmid expression vector. To create the HA transfer vector, the full-length HA gene sequence of Egyptian H5N1 AIV along with the WSSV-ie1 promoter sequence was first cloned as a fusion cassette into the baculovirus transfer vector (pFastBac-1) after deletion of the polyhedrin promoter, as described in the Materials and Methods section ( Fig. 1 A). The correct cloning and orientation of this construct were detected using restriction endonuclease and PCR analyses. The DNA restriction endonuclease digestion of the transfer vector with Sna BI and Hin dIII yielded two distinct DNA fragments having sizes of 4,520 bp and 2,236 bp ( Fig. 1 B). The larger fragment precisely corresponded to the pFast-Bac-1 backbone and the smaller fragment was the combined length of the Egyptian HA H5N1 AIV-WSSV-ie1 promoter fused DNA. In line with the restriction analysis data, PCR amplification of the ie1-HA expression cassette sequence yielded a 2,330 bp PCR product that represents the bonded ie1-HA fragment ( Fig. 1 C). These results confirm the successful production of the recombinant transfer vector (pFastBac-ie-HA).
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Confirmation of construction of the recombinant baculovirus expression vector. (A) Expression cassette arrangement in the recombinant transfer vector (pFastBac-ie1-HA) (The figure was constructed using SnapGene software, GSL Biotech). (B) Restriction digestion analysis of plasmid pFastBac-ie1-HA using SnaBI and HindIII (lane 1: double-digested recombinant transfer vector; M: 1 kb gene ruler, 100-10,000 bp; Thermoscientific, USA. (C) PCR amplification of the ie1-HA cassette using gene cassette forward and plasmid reverse primers (M: Vivantis 100 bp plus DNA ladder 100-3,000 bp (Malaysia); lane 2: Amplified ie1-HA gene cassette). (D) PCR amplification products of both recombinant and non-recombinant bacmids using pUC/M13 and gene-specific primers. M: 1 Kb Gene ruler DNA Ladder 250-10,000 bp (Thermoscientific, USA). Lane 1: Amplified PCR product using pUC/M13 forward and reverse primers (4,300 bp). Lane 2: Amplified PCR product using pUC/M13 forward and expression cassette reverse primers (3,700 bp). Lane 3: Amplified PCR product of non-recombinant bacmid (300 bp).
To generate the recombinant HA bacmid expression vector, the HA gene was transferred from the pFastBac-ie-HA vector to the bacmid DNA by genetic transposition. The successfully transposed colonies containing recombinant bacmids appeared on agar as large white colonies in a background of blue colonies, which contain the nonrecombinant bacmid DNA (data not shown). The presence of recombinant HA bacmids was confirmed by PCR using pUC/M13 forward and reverse primers. DNA extracted from positive colonies generated a PCR product of 4,300 bp ( Fig. 1 D, lane 1), and a PCR product of 300 bp ( Fig. 1 D, lane 3) was observed for the non-recombinant bacmid DNA. The pUC/M13 forward and the expression cassettespecific reverse primers were used to confirm the correct orientation and transposition of the ie1-HA gene cassette, which is demonstrated in Fig. 1 D lane 2 as a 3,700 bp PCR product. This recombinant bacmid vector could then be used for the ectopic expression of the Egyptian H5N1 AIV HA through transfection of insect cells.
- Generation of Recombinant Baculoviruses Expressing HA Gene of Egyptian H5N1 AIV
The recombinant as well as the non-recombinant bacmids were employed to transfect Sf -9 monolayer cells by using Cellfectin II reagent, and the transfected cultures were analyzed for CPE at various times post-transfection. Time-course analysis showed that the signs of virus production started to appear only in recombinant bacmidtransfected cells 72-96 h post-transfection ( Fig. 2 ). A slow growth rate, low cell density, giant cell, and granule formation as well as cell degeneration and detachment, which are typical CPE characteristics of baculovirus-infected cells, were clearly observed in recombinant bacmidtransfected cells ( Fig. 2 A). In contrast, non-transfected Sf -9 cells grew normally and kept their cell integrity with a normal size and no granule formation ( Fig. 2 B). These visual observations demonstrate that the virus vector incorporating the HA gene of Egyptian H5N1 AIV is able to replicate in insect cells. To produce higher virus titers, the supernatant containing recombinant baculoviruses from the transfected cultures was collected and used to infect new Sf -9 cells. The produced viruses were aliquoted, and stored for subsequent expression experiments.
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Expression of the recombinant bacmid in Sf-9 cells. (A) Transfected Sf-9 cells at 96 h post-infection showing cellular swelling, granulation, and degeneration (arrows). (B) Control noninfected Sf-9 cells with a healthy appearance and continuous division forming a monolayer sheet. (C) SDS-PAGE gel stained with Coomassie blue: M: Prestained protein ladder (Gendirex). Lane 1: Transfected cell lysate with recombinant bacmid showing a faint band of approximately 63 kDa. Lane 2: Lysate of cells infected with P2 recombinant baculovirus stock showing a strong recombinant HA-specific 63 KDa band (arrow). Lane 3: Non-infected cell lysate. Lane 4: Positive control of inactivated purified H5N1 virus antigen with pattern of different viral proteins, with the arrow showing HA. Lane 5: Non-infected cell pellet.
To determine whether the recombinant HA was successfully expressed in the BEVS, the cell lysates of both infected and non-infected Sf-9 cells were analyzed by SDSPAGE at 96 h post-infection. A clear band of approximately 63 kDa was detected from cells infected with recombinant viruses ( Fig. 2 C, lanes 2), whereas no bands were observed in control non-infected Sf-9 cells ( Fig. 2 C, lanes 3 and 5). These results confirm the successful expression of HA protein of H5N1 AIV under transcriptional control of the WSSV-ie1 promoter.
- Validation of the Biological Activities of the Expressed HA Gene of Egyptian H5N1 AIV
Because cells expressing surface HA are known to adsorb erythrocytes, the expression of recombinant HA on the surface of Sf-9 cells was detected by hemadsorption assay [26] . Sf-9 monolayer cells were left uninfected or infected with recombinant baculovirus containing the HA gene of Egyptian H5N1 AIV, and the ability of these cells to bind chicken RBCs was assessed. Infected Sf-9 cells were able to adsorb chicken erythrocytes and form cellular clumps at 48 h post-infection ( Fig. 3 A), whereas no adsorption was observed in non-infected cells ( Fig. 3 B). These results suggest that the recombinant HA had fully functional and authentic hemadsorption activity, and was successfully synthesized and expressed on the surface of the infected insect cells under the control of the WSSV-ie1 promoter. To confirm these results, we treated the infected cells expressing HA with a specific antiserum directed toward H5N1 AIV HA and tested their ability to adsorb chicken erythrocytes. As shown in Fig. 3 C, no cellular clumps were observed in these cells, indicating the complete inhibition of HA hemadsorption activity after the antibody treatment.
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Hemadsorption and hemadsorption inhibition assays. (A) Sf-9 infected with recombinant baculovirus adsorbing chicken erythrocytes (arrows). (B) Mock-infected Sf-9 cells with PBS. (C) Hemadsorption inhibition assay on Sf-9 insect cells showing no hemadsorption 48 h post-inoculation of the serum virus mixture. Both infected and non-infected cells were incubated with 0.5% chicken RBCs for 1 h, washed three times with PBS to remove unbounded RBCs, and examined under an inverted light microscope (Hund, Germany).
The hemadsorption experiment revealed that the recombinant HA protein of Egyptian H5N1 AIV has a strong capacity to retain its biological activity. To further analyze this biological activity relative to authentic influenza HA, infected or non-infected Sf-9 cell lysates were assayed for the hemagglutination activity of the expressed recombinant HA. Cell lysates were incubated with 0.5% chicken RBCs and the hemagglutination titers of the expressed HA were determined, as described in the Materials and Methods section. For cells infected with the recombinant virus, chicken RBC agglutination was observed in wells up to 1:32 dilutions ( Fig. 4 , row A) demonstrating a hemagglutination titer of 32 HAU/0.05 ml. In contrast, no hemagglutination was observed in cell lysates from noninfected cells ( Fig. 4 , row C). These results indicate that the recombinant HA expressed using the BEVS is a functional protein that exhibits the influenza virus hemagglutininspecific biological activities and thus can be used in subsequent experiments.
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Hemagglutination and hemagglutination inhibition. Row A: hemagglutination activity of the 2-fold serially diluted infected cell lysates mixed with 0.5% chicken RBCs showing 25 HAU/50 µl. The HA titer was determined as the reciprocal of the highest dilution with HA activity. Row B: HI assay using the 8 HAU of the recombinant HA protein against 2-fold serially diluted reference anti-H5 antisera and 0.5% chicken RBCs showing 29 HIU/50 µl. Row C: negative control (0.5% chicken RBCs + non-infected insect cell lysate).
- Validation of the Antigenic Property of the Expressed HA Gene of Egyptian H5N1 AIV
It is well known that specific antibodies bind selectively to the major antigenic sites of the HA protein of influenza viruses to neutralize virus infectivity [25] . To evaluate the antigenicity of Egyptian H5N1 AIV recombinant HA, the HI assay was performed to determine HI titers. As shown in Fig. 4 , row B, the reference standard anti-H5 antisera could efficiently inhibit the hemagglutination activity of the recombinant HA protein and yield a high HI titer (512 HIU) against the recombinant HA. Importantly, the HI activity of the expressed recombinant HA protein was similar to that observed with the reference influenza virus strain (data not shown). These results indicate that the expressed recombinant HA protein is antigenically similar to the HA of native influenza viruses. The antigenicity of recombinant HA was confirmed using two additional assays, immunofluorescence and western blot analyses. For the immunofluorescence analysis, recombinant baculovirusinfected Sf-9 cells as well as non-infected cells were fixed, stained with anti-H5 antisera, and analyzed using immunofluorescence microscopy to detect the presence of HA antigen in these cells. A very bright green HA protein staining was observed in 95% of the cells infected with recombinant baculovirus at 48 h post-infection ( Fig. 5 A). In contrast, minimal or no green fluorescence was observed in non-infected cells ( Fig. 5 B). In agreement with the immunofluorescence data, western blot analysis using the reference anti-H5 antisera detected significantly high expression levels of the recombinant HA in lysates prepared from the baculovirus-infected cells ( Fig. 5 C, lane 1), which gave a distinct band of ~63 kDa, along with that of inactivated purified H5N1 antigen, which was used as a positive control ( Fig. 5 C, lane 2). These data indicate that recombinant HA expressed in the BEVS is antigenic and could possibly be used alone to stimulate immune protection against Egyptian H5N1 AIV.
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Indirect immunofluorescence assay of insect Sf-9 cells. (A) Cells infected with recombinant baculovirus expressing HA of H5N1 avian influenza virus. (B) Cells mock-infected with PBS. Both infected and non-infected cells were incubated for 48 h at 27℃, fixed with 4% paraformaldehyde, and then polyclonal anti-H5 antisera and rabbit anti-chicken fluorescein conjugate were used to detect the expressed HA protein. (C) Western blotting of infected cell lysate using H5N1 polyclonal antisera. M: Prestained protein ladder (Gendirex). Lane 1: P2-infected cell lysate showing specific recombinant HA band at 63 kDa. Lane 2: Inactivated purified H5N1 antigen with pattern of different viral proteins.
Discussion
In addition to its ability to differentiate between infected and vaccinated birds, HA subunit vaccines have the benefit of inducing immunogenicity without using highly pathogenic H5N1 influenza viruses [15] . Remarkably, the baculovirusbased technology for expression of influenza virus proteins has proven to be an extremely powerful platform for the development of novel and customized influenza subunit vaccines, owing to its handling feasibility, relative safety, immunogenicity, high yield, and ability to be scaled-up easily [8] . In addition to the previously mentioned advantages, the proper protein post-translational modifications within this system, which have been proven to retain the conformation and biological activity of expressed proteins, stands out as one of the most important advantages [23] . In this study, we successfully expressed the HA protein of H5N1 AIV under the transcriptional control of the WSSV-ie1 promoter in insect cells by using the Bac-to-Bac baculovirus expression system, aiming to use it as basis for the development of a subunit vaccine against circulating Egyptian H5N1 AIV.
The WSSV- ie1 promoter was identified during the mapping of immediate early genes of white spot syndrome virus isolated from shrimp [18] . This promoter was shown to possess most eukaryotic promoter elements, and hence its high activity in both insect and vertebrate cells [6] Thus, the WSSV-ie1 promoter is an important and interesting research tool in the development of baculovirus-based expression vectors [21] . This has been confirmed by previous studies that demonstrated the surface expression of HA under the control of the WSSV-ie1 promoter in insect cells using the baculovirus expression system [7 , 20] . Consistent with these data, in our present study, the HA protein of Egyptian H5N1 AIV was efficiently expressed under the control of the WSSV-ie1 promoter. A highintensity 63 kDa band corresponding to the expressed HA protein was detected by SDS-PAGE ( Fig. 2 C, lane 2) and confirmed by western blot analysis ( Fig. 5 C, lane 1). These findings raise the question of whether other non-baculovirusbased promoters could be highly active and able to promote the expression of viral proteins in insect cells.
It has been well documented that the expression of some recombinant proteins affects the expressed protein(s) functional activities [24] . The authentic hemagglutinin protein of the influenza virus possesses biological activities that are very important for its functions [13] . Here, we show that the recombinant HA protein expressed in Sf-9 cells infected by the recombinant baculovirus exhibits biological activities and properties similar, if not identical, to authentic HA in influenza viruses. The baculovirusinfected cells expressing recombinant HA demonstrated hemadsorption ( Fig. 3 A) and hemagglutination ( Fig. 4 A) activities. Both biological activities were inhibited by the addition of specific anti-H5 antisera ( Figs. 3 C and 4 B). Therefore, it is reasonable to conclude that the WSSV-ie1 promoter induces an efficient expression of a biologically active HA protein that can be utilized in future research activities. It would be interesting to determine whether this is the case for other baculovirus recombinants.
Maintaining the antigenic properties of any recombinant expressed protein is a cornerstone for its successful use in future applications. In the present study, we were able to confirm the antigenicity of the H5N1 avian influenza recombinant HA protein by hemagglutination inhibition, immunofluorescence, and western blot analyses. Although the reference anti-H5 antibody is specific for the HA of H5 influenza viruses, two nonspecific bands were observed in western blot analysis ( Fig. 5 C). These nonspecific bands may be attributed to the cross-reactivity of the utilized antiH5 antiserum with other cellular or baculoviral proteins. However, these results do not interfere with the high antigenic property observed with the recombinant HA expressed in this study. These observations raised the possibility that a subunit vaccine that contains only the recombinant HA of Egyptian H5N1 could be used to induce high immunogenic activities against this devastating virus. To confirm this notion, experiments to examine this possibility are in progress.
In conclusion, the present study reports the successful expression of the HA protein of H5N1 avian influenza virus under the control of the WSSV-ie1 promoter in insect cells. This recombinant protein retains its authentic biological activity and antigenicity in a manner similar to that of natural HA and thus establishes a strong basis for the development of a customized avian influenza subunit vaccine.
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