INTRODUCTION
Influenza virus has two antigenic proteins that are called NA and HA.[ 1 ] The type A is the acute type that mutates and transfers between different species and causes pandemics.[ 2 ] Induction of neutralizing antibodies against the viral surface hemagglutinin (HA), stimulating the humoral immunity which provides higher response but not as stable as cellular immunity.[ 3 ] Using two antigenic proteins causing both humoral and cellular immunity seems to be more efficient. Hence, designing a construct based on the conserved proteins of influenza virus causing both cellular and humoral immunity together seems to be more reliable for providing a stable immunity. One of the most conserved proteins of influenza A virus is the nucleoprotein (NP) that provides cross-protection against various subtypes of the virus.[ 4 ] NP is an internal conserved protein, stimulating the host cellular immunity. The cellular immunity is stable but not effective enough to create resistance. NP is a protein that shows more than 95% similarity in sequence among different serotypes isolated during the last two decades.[ 5 ] Moreover, influenza-specific cytotoxic T lymphocyte (CTL) is able to lyse influenza-infected cells by recognition of NP, the major target in virus for CTL responses. Thus, the specific immunity induced against NP contributes to the clearance of the virus from the infected tissue and prohibits the spread of the viral infection. However, one of the concerns in using NP as a target for immunization is its limited potency to produce efficient immunity because of its small size. HA is the main surface glycoprotein and the indicator antigen. After penetrating the host body, HA cleaves into HA1 and HA2. The HA2 part of the protein is the most conserved part causing the hosts humoral immunity.[ 6 ] In this study, we designed and built a construct made of both HA2 and NP together in order to stimulate both cellular and humoral immunities after injection as a vaccine .
MATERIALS AND METHODS
Bioinformatics analysisPrimary structural analysis. Bioinformatics systems such as Expasy’s ProtParam and ProtScale were used to study the physiochemical characteristics of the designed fusion construct. The GRAVY value definition is based on the sum of hydropathy values of all amino acids divided by the protein length. The instability index estimates the in vitro stability of a protein.[ 7 ] The secondary structure of HA2-NP fusion protein was determined by using PHD SECONDARY STRUCTURE PREDICTION METHOD (https:// npsa-prabi.ibcp.fr). The PHD site can predict the percentage of α-helix, extended strand, random coil, and so on available in the secondary structure.[ 8 ] The antigenic sites were predicted by inserting the protein data bank (PDB) format of HA2-NP fusion protein in the ElliPro tool (Switzerland) (http://tools. iedb.org/ ellipro/). ElliPro gives an area under the curve (AUC) value of 0.732 for the prediction of antibody epitopes, which shows a correlation between antigenicity, solvent accessibility, and flexibility in proteins. It gives us a reliable prediction of antibody, or B-cell, epitopes.
Determining the availability of the antigenic area
Antigenic areas of the chimeric protein HA2-NP obtained by using Imtech, Raghava, and Bcepred was identified in pYMOL program in purple (London, United Kingdom). The three-dimensional (3D) model was predicted by Phyre2, I-TASSER, and PyMOL (London, United Kingdom). (Data not shown). Structural templates having the highest sequence with the target protein were used in comparative homology modeling methods. Homologous proteins were identified by uploading the PDB or the FASTA format of the fusion protein against proteins 3D structures deposited in PDB, using Position-Specific Iterative Basic Local Alignment Search Tool (PSI-BLAST) (Cambridgeshire, United Kingdom. The PROCHECK analysis, root-mean-square deviation calculation, and structure superimposition were performed by the Protein Structure Validation Software (PSVS) server (USA), by uploading the PDB format received by the I-TASSER server. PSVS suits for the consistent and rapid evaluation of the quality of protein structures with a focus on nuclear magnetic resonance structures and homology models. Ramachandran Plot estimates statistical approximation about phi- psi angles of residues. It can show if the angles of residues are in the right location or not. The MolProbity set of programs is used to regenerate the hydrogen atoms (using the reduce program), and calculate atomic clashes (using the probe program). It is also used to visualize any steric overlaps in the structure of the molecule (using the Prekin program (Prekin Arcata, United states of America) and Mage (Mag Chicago, USA) modeler). Two sets of primers were designed by Gene Runner 3.05 (http://www.generunner.net/) (Hastings Software) software to amplify the genes of interest, HA2, and NP from influenza A virus H1N1. The construct containing the HA2-NP fusion protein was designed to be expressed in pET28a in BL21 host cell. According to the cloning scheme, the proper restriction cut sites were created at 5’ end of each primer, and checked by NEB cutter. Thus, there were no more cutting sites. To amplify the 300-bp specific region of NP, polymerase chain reaction (PCR) was performed on extracted DNA using high-fidelity Taq polymerase (Fermentas, Lithuania) and specific primers were as follows: 5’GAAGCTT /GATTATGAGGGACGG3’ as forward primer and 3’GCTCGAG /CCTCTGATAAGTTG5’ as reverse primer. Amplification reaction was carried out under the following profile: 5 min at 94°C followed by 30 cycles at 94°C for 30 s, 46°C for 45 s, and 72°C for 2 min, with a final extension step at 72°C for 10 min. PCR products were analyzed by 1.5% (w/v) agarose gel electrophoresis. Sticky ends were designed to be added to NP to stick to pGEM vector.
Cloning Amplicons of NP embedded to pGEM vector, transformed into E. coli top10F’ for proliferation and purified by fermentas kit, and then digested with Xho1 and HindIII enzymes. The isolated band in 1.5% agarose gel was extracted by gel cutting and cleaned up by QIAGENE kit (Germany). The NP was ligated to the digested pET28a/HA2 in the presence of T4 ligase then transformed to E. coli top10F’competent cells and incubate in the presence of kanamycin and tetracycline antibiotics( 18 h , 37°C). Then colony PCR was performed to indicate the presence of NP in the cells with absorbed plasmids, using the same primers. The PCR product was confirmed using electrophoresed in 1.5% agarose gel. At the last step, the restriction analyzed recombinant vector pET28a/HA2-NP was sent for sequencing and results were confirmed.
Expression of recombinant fusion protein in prokaryotic systemThe recombinant vector was transformed to competent E. coli BL21 then cultured on LB agar plates containing 1 μg/mL kanamycin antibiotic, incubated at 37°C overnight. One colony from each type was inoculated in 15 mL of LB broth containing 1 μg/mL kanamycin and incubated until reaching the OD600 of 0.5-1. For expression of HA2-NP fusion protein , cultures were induced by adding isopropyl β- d-1- thiogalactopyranoside (IPTG) in final concentration of 1 mM. Time course fractionating from cultures started after inducing and continued for 4 h at 37°C temperature. The expression of fusion protein (HA2- NP) was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using the method of Laemmli.[ 9 ] The new fusion protein was confirmed by western blotting using anti –HA2 which prepared before in Pasteur Institute of Iran.[ 10 ] The Ni-NTA purification system was used to purify the 6xHis-tagged HA2-NP recombinant fusion protein . Results were checked by SDS-PAGE.
RESULTS
Bioinformatics analysisPrimary structure analysis
By using the ProtParam program, the HA2-NP fusion protein using the whole NP segment was predicted to be unstable. Choosing a specific fragment of NP with the most efficient antigenic part by its physicochemical properties showed more stability and higher instability index (proteins with the instability index less than 40 are known as stable proteins). The grand average hydrophobicity decreased (became more hydrophilic), and the molecular weight also decreased [Table 1 ]. The isoelectric pH was found to be 6.60 and the half-life increased from 4.4 to 30 h [Table 2 ]. The results by Scratch, aligning two HA2 and NP protein sequences together as a fusion protein , showed an increasing effective score in the antigenicity than using NP and HA2 proteins separately and the solubility was apparent [Table 1 ].
Table 1: Antigenicity and physicochemical property necessary comparisons
Table 2: Half-life comparison of HA2 with complete NP vs. 300-bp fragment of NPs
Secondary Structure Prediction
PHD server results showed 44.74% predicted α-helix, 14.02% extended strand, and 41.24% random coil. No 310-helix, Pi-helix, β-bridge, β-turn, bend region, ambiguous states, or any other states were predicted. Residues with a scale reliability index of prediction of 5 and over were predicted at more than 82%. PSVS server also showed inter-chain break(s) between 371 and 382 amino acids.
Prediction of Linear B-cell Epitopes of HA2-NP
As the instability index and average hydrophobicity of the HA2-NP fusion protein was inappropriate, we decided to pick out a specific part of the NP peptide sequence. The raw amino-acid sequence of the whole NP was uploaded in FASTA format in Imtech and Raghava. The overlap of the most hydrophilic, flexible, accessible, turns, exposed surfaces, polar, and antigenic segments was observed. The part of NP earning the most overlap of them, based on the NPs most antigenic parts,[ 11 ] was NP150-450 as the efficient antigenic part for the HA2-NP fusion protein . JSmol and Ellipro showed an exposed location of antigenic parts of the HA2-NP fusion protein [Figure 1 ]. The PSVS server indicated a dimer oligomerization of the 3D structure of the HA2-NP fusion protein .
Figure 1: Antibody, or B-cell, epitopes predicted for HA2-NP
Structure Validation
Ramachandran plot summary for selected residues from PROCHECK
Most of the residues were in the allowed and favored regions (favored=85.8% and allowed= 95.7). [Figure 2 ]. The MolProbity offers the quality validation for 3D structures of proteins. It provides detailed all-atom contact analysis of any steric problems within the molecules and can calculate and display the H-bond and Van Der Waals contacts in the interfaces between components. An integral step in the process is the addition and full optimization of all hydrogen atoms, both polar and nonpolar. The Verify3D of Global quality scores showed the maximum deviation for residues, which did not match the standard 3D structure MolProbity, (Duke University of Medical Science Duke, Germany).
Figure 2: Residue plot Of Ramachandran analysis (based on data from Richardson Lab’s MolProbity). (a) General Case, (b) Glycine, (c) Proline, (d)Pre-Proline
Amplification to purification
The NP 300-bp fragment was amplified by PCR and the results were confirmed by agarose gel electrophoresis [Figure 3a ]. As a result of enzymatic digestion pGEM/ NP vector, 2 mL of the sample was electrophoresised to ensure the accuracy of the cut [Figure 3b ]. The positive PCR colonies’ plasmids were sequenced by CinnaGene (Tehran, Iran) company and the results were analyzed using alignment in the GenBank (Maryland, United States). (Date not shown) The confirmed recombinant plasmid was transformed into BL21 expression cells, followed by electrophoresis in polyacrylamide gel that showed protein bands of HA2-NP in the predicted weight in the range of 35-48 kDa appeared [Figure 3c ]. HA2-NP proteins were induced to express themselves and gradually after induction, the expression levels increased. Three hours after induction, the HA2-NP protein was collected and reached the highest level. Then the presence of the specific HA2-NP fusion protein was confirmed by the Western blotting method using anti-6X His tag antibody [Figure 3e ]. The extraction of Proteins from Prokaryotic cells was performed using denaturation with 8M urea and sonication. The results of polyacrylamide gel electrophoresis indicated that, in the soup HA2-NP proteins were extracted and purified to an acceptable level [Figure 3d ]. This purified protein was confirmed using homemade specific polyclonal antibody against HA2 [Figure 3f ].
Figure 3: (a) sample dilution of primer and testing solutions. (b) pGEM/NP restriction analysis: (1) 100-bp marker, (2) sample digested sample with enzymes, (3) undigested, (4) empty well, (5) digested sample with enzymes and separating piece, and (6) undigested sample. (c) Electrophoresis samples of expression pET28a/HA2-NP in E. coli strain BL21 (DE3) at 28 °C after induction with 1-mM IPTG concentration: (1) marker proteins, (2) the bacteria before induction with IPTG, (3-5) hours of sample bacteria in the first, second, and third, and (6) 18 h after induction with IPTG. (d) purified protein electrophoresis of HA2-NP, 1) Marker protein, 2 to 6) washing samples at 50 mM imidazole (E) (e)Western blotting with a polyclonal antibody anti-HA2 and anti-rabbit examples of the expression pET28a/HA2-NP in E. coli strain BL21 (DE3) at 28 °C for 4 h after induction with 1-mM IPTG: (1) protein markers, (2) sample before induction of expression with IPTG, and (3) HA2-NP samples after 4-h speech (4)HA2-NP-Histag samples after expression (f)
DISCUSSION
Influenza virus genome causes changes in its antigenic features, resulting as the major reason of the virus resistance against prophylactic proceedings. To prevent seasonal flu, some vaccines have been used so far. Inactivated vaccines are safe (except some allergic reactions related to egg production system), but because of poor response in high-risk populations and also the need to change each year, researchers are working on a vaccine formulation that is stable and causes acceptable cross immunity against multiple viral subtypes. As the subunit vaccines have the same high quality and adequate performance and acceptable cost and features as compared with other methods of vaccine production, they are very effective, and developing countries are also capable of producing them.[ 11 ] Using conserved antigenic proteins that do not affect the influenza antigenic changes, with the ability to stimulate the immune system on a permanent basis, seems to be a perfect solution for universal vaccines. Studies on the effects of influenza viral proteins led to choose the NP and HA2 proteins as chimeric protein for the vaccine composition. The stalk-like structure of the HA molecule (HA2) is highly protected and is capable of stimulating humoral immunity.[ 6 ] NP can provide a stable cellular immunity in human body. Other advantages of the NP protein in vaccines are the creation of cross immunity against multiple subtypes of influenza virus .[ 13 ] These two proteins are protected and annual changes of the virus will not affect them. The combination of humoral and cellular immunity will stay highly permanent against the virus. In previous studies, NP and HA2 were both expressed in prokaryotic system individually and created immune protection.[ 6 , 13 ] In this study, we managed to create a chimeric protein that has both antigenic properties of HA2 and NP at the same time in order to be able to provide an acceptable protection in mice and also human. The reason why other conserved proteins of influenza were not used instead is that NP and M1 are internal conserved proteins of influenzas. Cellular immunity generated against both NP and M1 can infect the cells through MHC-I route. CTL can stimulate cross immunity, resulting in protection between influenza subtypes. Humoral immunity created by NP can also inhibit germination of infected cells. As a result, they stimulate cellular and humoral immunities as well as a cross between several subtypes. The conservation of the NP amino-acid sequence of the H1N1 strain of influenza is 96%, and as mentioned above, it can induce strong T-helper cell and humoral immunity, which dedicates long-term immunity response against the virus.
HA2 and M2 proteins are surface antigens of influenza virus . The experimental reasons for choosing the NP and HA2 instead of other proteins are the prior knowledge that the created immune protection was stronger than M2. The most CD8+ T cell and positive IFN-γ specific responses are detected against HA and the most CD4+ T cell and positive IFN-γ responses are generated against NP.[ 14 ]
There are reasons for using the 300-bp NP segment instead of the full NP in HA2-NP fusion protein . The NP418-426 of NP is the most important epitope to stimulate CTL.[ 11 ] In addition, the studies in primary structural analysis and prediction of linear B-cell epitopes of HA2-NP showed that the HA2-NP fusion protein using NP150-450 instead of full NP was chosen as the most overlapping of antigenic factors such as flexibility, hydrophobicity, accessibility, turns, exposed surface, polarity, and antigenic showed better physicochemical properties such as higher stability, lower grand average hydrophobicity, higher half-life, lower molecular weight, and number of amino acids. In addition, it showed a suitable antigenicity and solubility in high levels of express terms and reduction as compared to the full NP.
The ElliPro structure- based method performs gives a prediction of AUC value of 0.732, when the most significant prediction is considered for each protein. The advantage of subunit vaccines as compared with live-attenuated vaccines is that these vaccines contain only immunogenetic parts of the pathogen and their manipulation removes the risk of recurrence.[ 15 ] The advantage of subunit vaccines against vaccines that are made in eggs is that they are safe and do not make allergic reactions.[ 16 ]
Subunit NP-based vaccine showed that the vaccine may also produce immune protection against influenza virus as MVA vaccines do.[ 12 ] The difference is that the manufacturing method is much easier and cheaper. The recombinant protein can be created in E. coli expression system. The advantage of this method compared to the inactivated vaccine is its safety in high-risk populations such as infants and the elderly, and the lack of need to change every year which prompted researchers, to look for a vaccine more efficient, requiring no change and making an acceptable cross-protection.[ 17 ] The benefits of recombinant protein production in prokaryotic system instead of eukaryotic system are the cost, the production of protein in large quantities, the possibility of creating proteins with high stability, and easy purification . The product stability and high- molecular-weight expression of cassette and purification of protein is easier in bacteria. As stated above, the bioinformatics analysis on the physicochemical properties of the chimeric protein showed acceptable results. The foreign gene sequence that may mark the end of an E. coli transcription and early termination of transcription and protein expression was checked with bioinformatic program (NEB cutter). Previous records showed HA2 gene expression in E. coli by IPTG induction at 37°C.[ 18 ] The NP protein was cloned in plasmid pET28a and expressed in E. coli expression system[ 13 ] and purified with Ni-NTA chromatography column.[ 19 ]
These methods seemed to be an acceptable way for producing HA2-NP chimeric protein. The HA2-NP is being produced for the first time in this study. The reasons for the use of prokaryotic system to produce HA2 as a glycoprotein are as follows. Using two antigenic proteins of HA2 and NP causing humoral and cellular immunities, respectively, seems to be a good option for stable subunit vaccine production. The viral HA is the major target recognized by neutralizing antibodies. However, glycans act as antigenic sites shield on HA. Glycans can also interfere with the receptor binding properties of HA.[ 20 ] In the absence of glycans on HA, the antibody titer of cross- reactivity increases.[ 21 ] As the E. coli (BL21) host shows lack of glycosylation on HA2 expressed in this system, it can create a greater safety as compared with the live- attenuated vaccines.
Financial support and sponsorship
Nil
Conflicts of interest
There are no conflicts of interest.
Acknowledgments
I am using this opportunity to express my gratitude Hadis Shokouhi who supported me throughout the course of this MBA project.
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