BACKGROUND: The use of IgYs in a variety of methods in different areas of research, diagnostics, medical application and biotechnology should be considered widely. OBJECTIVES: Development of antibodies against extra cellular domain of influenza M2 (M2e) protein in egg yolk of laying hens. METHODS: A Fusion construct harboring C-terminal of bovine heat shock protein 70 (Hsp70) and influenza M2e coding genes was injected to laying hens. Serum and egg yolk antibodies were screened for the presence of anti-M2e antibodies by indirect enzyme-linked immunosorbent assay (ELISA).RESULTS: Anti-M2e antibodies were detected in egg yolks and sera of injected hens from 13 and 7 days post injection (PI), with the peak titer detected on 41 and 35 days PI, respectively. CONCLUSIONS: Anti-M2e IgY titers could be an index for expression potential of pcDNA3.1-M2e-HspCterminal construct in laying hens. This construct could be considered as a promising tool in production of anti-M2e polyclonal, monospecific IgY antibodies. Such anti-M2e antibodies could be exploited for influenza diagnostic and therapeutic measures.

Aim and background: The extra domain of the M2 protein of influenza is a weak immunogen, Hence this is not a suitable candidate for the vaccine development. The B subunit of the Cholera enterotoxin is nontoxic causing attachment of the holotoxin to the OMI at the surface of the epithelial intestine cells, while, CTB is highly immunogenic. So fusion of these two genes would be very applicable in influenza vaccine development.Material and methods: using PCR with specific primers including restriction sites ctxB was amplified. Standard strain of influenza type A (AJPUERTO RICO/8/34) was prepared and M2e was amplified using RT-PCR with specific primers. The second PCR reaction was performed for the fusion of the M2e and ctxB using the F primer of M2e and R primer of ctxB. PCR product was digested with BamHI and EcoRI and cloned into the pET28a. Verification of the cloning was performed using sequence analyzing. After analysis, the recombinant pET28a1M2e-ctxB was transferred into E. coli Top10. The final confirmation was performed using colony PCR, double digesting and sequence analysis.Results: Sequence analysis showed that M2e has been fused to ctxB in an exact frame. Moreover the fused gene was cloned into the restriction site of the BamHI and EcoR1 in pET28a.Conclusion: Despite stability of the sequence of M2e it is not immunogen but fusion to strong immunogens such as CTB would be a new suggestion for Influenza recombinant vaccine production.

Background and Aims: The ectodomain of matrix protein of influenza virus is a weak immunogen that is highly conserved among all subtypes of influenza A virus. Tandem repeats of these genes along with linker were used to enhance immunogenicity of M2e protein and so it can be served as a universal vaccine in both humans and livestock.Materials and Methods: In this study, the sequences of extra-domain of matrix protein of influenza A registered in NCBI was converted into codons compatible for Bacillus subtilis using JAVA codon adaptation tool software.Results: A cassette consist of four repeats of this codon optimized sequence, spaced by appropriate linkers and flanked by BamHI and HindIII restriction sites was designed and thoroughly used for the synthesis. The cassette then was cloned into pMR12 shuttle expression vector.Conclusion: Two kinds of prokaryotic host, E. coli BL21and Bacillus subtilis WB600 were transformed by pMR12+4M2e. The fidelity of the construct in both transformants was confirmed by enzymatic analysis and PCR.

BACKGROUND: Influenza outbreak has become a great lifethreatening disease in the world. Nasal vaccines can induce systemic IgG and mucosal IgA antibody responses, which establish two layers of immune defense against the infectious pathogens like influenza. Mucosal vaccines must overcome several limitations, including the mucociliary clearance and inefficient uptake of soluble antigens. Therefore, nasal vaccines require potent adjuvants and delivery systems.OBJECTIVES: In this study we evaluated the effect of N-trimethyl chitosan (TMC) as a potent vehicle for DNA encoding M2e/HSP70c in order for intranasal administration in mice.METHODS: Ectodomain of the conserved influenza matrix protein 2 (M2e), which has been found to induce heterosubtypic immunity, was fused to HSP70359-610 or C-terminus of Mycobacterium tuberculosis HSP70 (HSP70c) in pcDNA3.1 vector (pcDNA/M2e-HSP70c) and then encapsulated into a derivative of chitosan, N-trimethyl chitosan (TMC). After encapsulation of the plasmid, physical properties of the particles were investigated using ZetasizerÒ 3000 the particles were then administered through the intranasal delivery in BALB/c mice.RESULTS: It was found that the particles had a size ranging between 90-120nm and positive surface charge. The intranasal immunization with M2e- HSP70c+TMC in BALB/c mice significantly induced higher M2e specific IgG than those induced in control groups (pcDNA/M2e-HSP70c without TMC, pcDNA/M2e, bearing M2e alone, and PBS).CONCLUSIONS: The present study showed that the encapsulation of M2e/ HSP70c into N-trimethyl chitosan (TMC) could strongly induce the humoral immune response against the M2e-HSP70c plasmid without lowering the adjuvant efficacy of HSP70c.

Background: Sequence variations in glycoproteins of influenza virus surface impel us to design new candidate vaccines yearly. Ectodomain of influenza M2 protein is a surface and highly conserved protein. M2e in influenza vaccines may eliminate the need for changing vaccine formulation every year.Objectives: In this study, a recombinant baculovirus containing M2e and cholera toxin subunit B fusion gene was generated with transposition process to express in large amounts in insect cell lines.Materials and Methods: M2e-ctxB fusion gene was created and cloned into pFastBac HT. The recombinant vector was transformed into DH10Bac cells to introduce the fusion gene into the bacmid DNA via a site-specific transposition process. The recombinant bacmid was then extracted from white colonies and further analyzed using PCR, DNA sequence analyzing, and indirect immunofluorescence assay.Results: PCR and DNA sequence analyzing results showed that the fusion gene was constructed as a single open reading frame and was successfully inserted into bacmid DNA. Moreover, indirect immunofluorescence results showed that the fusion gene was successfully expressed.Conclusions: Baculovirus expression vector system is valuable to produce M2e based influenza vaccines due to its simple utilization and ease of target gene manipulation. The expressed protein in such systems can improve the evaluating process of new vaccination strategies.

The present study was aimed to construct a fusion plasmid harboring the extracellular domain of the influenza A M2-protein (M2e), which was fused to the N-terminus of the truncated HSP70 (HSP70359–610) molecule as a new approach for future vaccine research against influenza A. The amplified fragments, M2e and HSP70359-610 genes, were gel-purified. The products were then single digested with BamHI restriction enzyme separately. The digested products were again gel-purified and ligated by T4 DNA ligase to form M2e- HSP359-610 gene. The PCR product containing both M2e and HSP359-610 genes as a single open reading frame (ORF) was gel-purified and double digested with EcoRI and XbaI restriction enzymes and then ligated into the EcoRI / XbaI double digested pPICZαA expression vector to form recombinant expression vector. Finally, the fused gene was sequenced, and then confirmed according to the related deposited gene in Genbank. The extracellular domain of the M2 protein, M2e, which consists of N-terminal 24 residues, showed to be remarkably conserved, and the N-terminal epitope SLLTEVET (residues 2-9) was conserved among all subtypes of influenza A viruses. Because of M2e limited potency; hence, low immunogenicity, it seems by linking this M2e-peptide to an appropriate carrier such as mycobacterium tuberculosis C-terminal 28-kDa domain of HSP70 (hsp70 359–610) we can render it very immunogenic, but further study needs to express it in both prokaryotic and eukaryotic systems and then evaluate this fusion protein in animal model.