Introduction

Influenza infection in humans is one of the most importantrespiratory diseases in all over the world. The infection could befetal specially in children [1]. Influenza virus A, is responsible forprevalence of pandemics and the most of the yearly flu epidemics[2]. This type is further categorized into the subtypes according tothe properties of Hemagglutinin (HA) and neuraminidase (NA)antigens on the surface of viral particle [3]. The unexpected variabilityof the Influenza A virus makes annual epidemics in population andis the principal reason of the lack of impressive prevention againstInfluenza infection to date [4].

In April of 2009, a swine-origin influenza H1N1 virus (S-OIV)has appeared. 2009 pandemic H1N1 influenza A virus is a reassortantstrain of the influenza virus containing gene segments from swine, avian and human [5]. 2009 pandemic H1N1 influenza A virus isantigenically similar to the 1918 pandemic H1N1 Influenza A virus[6]. Until 2013, pandemic H1N1 has led to more than 1.4 millioninfections with about 25,000 deaths worldwide [7]. The most of theserious consequences caused by influenza infection occurred inindividuals with underlying medical conditions. The highest mortalityrates were found in immunocompromised individuals, patients withchronic neurological disease and respiratory disease [8]. Pregnantwomen were also under threat [9]. Due to Ser31Asn substitutionin the transmembrane Matrix 2 protein, the 2009 pandemic H1N1Influenza A virus is resistant to Amantadine antiviral drug. 2009pandemic H1N1 Influenza A virus is sensitive to Oseltamivir andZanamivir antiviral drugs [10,11].

Hemagglutinin is the main antigen responsible for theattachment of virus to the specific receptors on the surface of host cells, providing entrance into the host cell via endocytosis. It definesthe virulence, host ranges and transitivity of influenza viruses. Theprecursor of hemagglutinin (HA0) is synthesized in the infected hostcells and undergoes a enzymatic cleavage to obtain two subunits,HA1 and HA2 [12]. HA exhibit conserved and variable domain[13]. Hu et al [14] reported seven conserved epitopes of HA1 of 2009pandemic H1N1 Influenza A virus containing 5-23, 63-81, 142-158,178-195, 201-216, 246-269, and 277-287, which are responsible forattachment of the virus to the swine cells. According to the previousresearches conducted by Kubota-Koketsu et al [15] and Yamashita[16], two highly conserved epitopes 173-181 and 227-239 werereported which antibodies against these epitopes could neutralizethe different subtypes of influenza virus. This glycoprotein also hasa principal role in the emergence of human pandemic influenzaviruses [17]. The HA protein is the main antigenic stimulator of theacquired immunity [18] and is the major target for virus-neutralizingantibodies [19]. The prominent immune response against influenzaHA is presumed to be conducted to the head of the glycoprotein(amino acids 52-277) particularly against determined antigenic zonesthat enclose the receptor binding site [20]. It is well documented thatantibodies against HA in the circulation protect against intensiveviral pneumonia following the secretion of antibodies from the bloodinto the lungs of mice [21,22].

Because of the production of vaccines and new anti-viraldrugs are time-consuming, passive immunization by neutralizingantibodies provides urgent treatment for influenza pandemicemergency, particularly, for acutely exposed individuals [23].Neutralizing monoclonal antibodies successfully applied for therapyand prophylaxis of viral infection [7,24]. Due to HAMA (human antimouseimmunoglobulin antibody) responses induced by monoclonalantibodies, limited protections are reported [25,26]. Single chainfragment variable are small fragments (~25-30 kDa) comprised ofvariable heavy (VH) and light chain (VL) of antibody, which arejoined with a flexible peptide linker contains 15-20 amino acids [27].Human scFv antibodies have advantageous properties including:human origin, low molecular weight, higher tissue penetration,lower retention times in non desired tissues, better clearanceof theimmunocomplexes, preservation the binding specificity of the intactmonoclonal antibody and minimally immunogenic. Large quantitiesand cost-effective production of scFv antibodies in bacteria is possible[28-30]. Many scFvs are used successfully in clinical trial and treatment[30,31]. Highly specific scFv antibodies already produced to severalimportant pathogens, for example, against H5N1 Influenza viruses[32,33], Urease and lysate of H.pylori bacteria [34], p24 of HIV-1[35], thermolabile hemolysin (TLH) of Vibrio parahemolyticus [36],Varicella-zoster virus [37] and Human Papillomavirus type 16 [38].

In the present study, a phage antibody display library of singlechain variable fragments (scFv) was utilized for isolation scFvantibodies against the conserved sequence of the hemagglutinin of2009 pandemic H1N1 influenza virus. The antigenic specificities ofthese antibodies were evaluated by enzyme-linked immunosorbentassay (phage ELISA).

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Materials and Methods

scFvs selection

A phage antibody display library of scFv was developed as described previously [39]. Phage clones displaying scFv were selectedfrom the library after four rounds of panning. Briefly, immunotubes(Nunc, Roskilde, Denmark) were coated with peptides as epitopes(amino acids 173-181 and 227-239 of hemagglutinin of influenzavirus at 4°C overnight. The phage-rescued supernatant (1010 PFU/mL) diluted with blocking solution was added to the tubes andincubated for 1h at room temperature. The recombinant phagemidwas introduced into competent E. coli TG1 cells, and rescued withhelper phage M13KO7 (Amersham, Biosciences). Four rounds ofpanning were performed. To confirm the existence of VH-Linker-VLinserts, PCR was performed on the clones obtained after panning.DNA fingerprinting of the scfv from individual E. coli clones weredetermined by using MvaI restriction enzyme (Roche DiagnosticGmbH, Mannheim, Germany). The common patterns were revealedby electrophoresis. One clone with the most frequent patternwas selected against each epitope and phage-rescued for furtherevaluations.

Determination of phage-antibody concentration

For determination of phage-antibody concentration of eachrescued phage, phage antibody supernatant was added to 1ml oflog phase TG1 E. coli and incubated with shaking at 37°C for 1 h.Serial dilution of bacteria was prepared and cultured on to 2TY Agar/Ampicillin medium at 30°C overnight. Number of clones per dilutionwas determined and phage concentration titer per milliliter wascalculated.

Evaluation of reactivity of scFvs by phage ELISA

Specificity of individual scFv was assessed by phage ELISA. The96 well ELISA plate was coated with peptides (dilution: 100 μg/ml inPBS) at 4°C overnight. An unrelated peptide was used as a negativecontrol. The wells were blocked with 3% skimmed milk for 2 h at 37°C.Following washing with PBS/Tween 20 and PBS, the phage-rescuedsupernatant of each clone containing the selected scFvs was added tothe wells. M13KO7 helper phage was added to peptide coated wellsas a negative antibody control. After incubation and washing, antifdbacteriophage antibody (Sigma, UK) was added and incubated for1hr. Following washing, HRP-conjocated anti-Rabbit IgG (Sigma,UK) antibody was added and left at room temperature for 1 h. 100μlof TMB solution was added to each well and the reaction was stoppedwith 100 μl of 1 M Sulfuric acid. The optical density of each well wasdetected at 450 nm by ELISA reader (BP-800, Biohit, USA).

Statistical analysis

Mann- Whitney test was used to compare the mean ratio of thephage ELISA results of phage display scFvs and of the control (nopeptide).

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Results

Anti- hemagglutinin of 2009 pandemic H1N1 influenza Avirus scFvs

Figure 1 shows DNA-Fingerprinting of 20 panned clones againstpeptide 1(A) and peptide 2(B). Two common patterns obtainedagainst each peptide, scFv-I and svFv-II with the frequencies of 25%(5/20) and 55% (11/20) against peptide 1 and scFv -I’ and scFv II’with the frequencies of 20% (4/20) and 75% (15/20) against peptide 2. The common patterns are shown by common symbol in Figure 1.Lanes (1, 5, 8, 9, 17) and lanes (2, 3, 6, 7, 10, 11, 12, 13, 16, 19, 20)show the common patterns of scFv-I and scFv-II against peptide 1,respectively. Lanes (3, 8, 9, 17) and lanes (1, 2, 4, 5, 6, 7, 10, 12, 13, 14,15, 16, 18, 19, 20) represent common patterns of scFv-I’ and scFv-II’ which were selected against peptide 2, respectively.

Figure 1: DNA fingerprinting of 20 panned clones (lanes 1-20) of peptide 1 (A) and peptide 2 (B). M: Marker.

Phage ELISA

The binding specificity of the selected scFvs to the related peptideswas measured by phage ELISA assay. All of four isolated scFvs (scFvI, scFvII, scFvI’, scFvII’) were found to bind with their related peptides(Figure 2). The absorbance obtained for reaction of scFv antibodieswith related peptide were significantly higher than the wells withno peptide (P value< 0.05). The M13KO7 helper phage showed noreactivity to the peptides.

Figure 2: Phage ELISA results of scFv-I (A), scFv-II (B) against peptide 1 and scFv-I′ (C) and scFv-II′ antibody (D) against peptide 2. All of the four antibodiesbound to the related peptides significantly higher than the wells with no peptide (P value< 0.05). No reactivity was detected for unrelated peptide, unrelated scFv(scFv against IL25R) and M13KO7 helper phage with the peptides.

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Discussion

One of the major causes of the global mortality is Influenzainfection and the infection remains a consistent health problem. Noimpressive prevention against Influenza infection has been identifiedtill now. Immunotherapy with antibodies is a helpful tool in thetreatment of infectious disease. Immunothreapy with neutralizingantibodies has been utilized against various viral infections includingprotection against Respiratory syncytial virus in cotton rats [40],prophylactic effect in experimentally infected chimpanzees againstHepatitis C virus [41], protection in mice and Guinea pigs inoculatedwith the rabies virus [42] and immunity against influenza viruses[43,44]. A strategy for defense against various viral infections is theproduction of antibodies to protective epitopes. Detection of theseepitopes is the main step in the development of effective antibodies.Between the 11 proteins that are encoded by gene segments ofinfluenza A virus, hemagglutinin is the major target of neutralizingantibodies as it is responsible for the attachment of the virus to thehost cells and determines the virulence of influenza virus. Conservedregions of hemagglutinin of influenza virus are principal targets forimmunotherapy against the virus. Highly conserved amino acids 173-181 and 227-239 were recognized in hemagglutinin of 2009 pandemicH1N1 influenza A virus [15,16,45]. These epitopes have beenidentified via B-1 and D-1 human monoclonal antibodies, respectively[16]. Considering the advantages of the scFv antibodies over intactantibodies, in the present study this format of antibody was used fortargeting of these epitopes. We chose peptide EGRMNYYWTLVEP(amino acids resides 227-239) of hemagglutinin fragmentsGKEVLVLWG (amino acides residues 173-181) and H1N1 InfluenzaA virus as immunodominant epitopes. Hemagglutinin fragmentsGKEVLVLWG (amino acids residues 173-181) and H1N1 Influenza A virus as immunodominant epitopes. Maneewatch et al. [33]isolated HuscFv antibodies against hemagglutinin of H5N1 influenzavirus. They reported that two of the selected HuscFvs had the highestneutralization effect against different strain of the H5N1 virus inMDCK culture and one HuscFv could rescue C57BL/6 mice thatwere infected with H5N1 influenza virus intranasally. In the previousstudies, the selection of scFv antibodies against the related peptidewas done by panning procedure. A number of specific scFvs have beenselected against key antigens using panning process [36,46,47]. In thisstudy we isolated four scFv antibodies (scFv-I, scFv-II, scFv-I’, scFv-II’) against peptide 1 and peptide 2 of hemagglutinin of influenza virus.The binding specificity of scFv antibodies to their related peptideswere measured by phage ELISA Assay. Results demonstrated thatthe selected scFvs against peptide 1 and 2 specifically bound to thecorresponding peptides compared to controls (Figure 2). The meanabsorbance of scFv-I, scFv-II and scFv-I’, scFv-II’ in reaction with thecorresponding peptides were significantly higher than the wells withno peptide, unrelated scFv, unrelated peptide and M13KO7 (P.value< 0.05). This represented the specificities of the four selected scFvs for the related peptides.

Immunotherapy via neutralizing antibodies against all speciesof influenza virus might be an ideal alternative for individuals whoare at high risk. This group includes: newborns, the old people andthe Immunodeficient patients who active vaccination probably doesnot help them. Neutralizing scFvs against hemagglutinin of influenzavirus would yield a new tool for therapy of influenza infection sinceHA has a principal role in the advent of human pandemic influenzaviruses. The well-known properties of scFv antibodies, speciallyhuman origin and wide penetration to the targets have made theseantibodies more applicable in immunotherapy. The high affinityspecific scFv antibodies against highly conserved neutralizingsequences of hemagglutinin of influenza were isolated in thisstudy suggest further evaluation of these antibodies to select highneutralizing scFvs to prevent against influenza infection in high riskindividuals.

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Acknowledgment

The authors would like to acknowledge Shiraz University ofMedical Sciences for financial support. The present paper wasextracted from the Msc thesis written by Samaneh Alizadeh grant No:6713.

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