Background: Design and construction of a universal vaccine based on conserved influenza antigens is the best way to protect populations from unforeseen influenza outbreaks. The ectodomain of matrix protein 2 (M2e) and hemagglutinin stalk domain (HA2) are considered as conserved influenza antigens. Genetic linkage of adjuvant HSP70 to these antigens can improve the efficacy of the vaccine. Objectives: The aim of this study was to produce a chimer protein to confer cross-protection against different subtypes of influenza A virus. Methods: The chimer formwas subjected to in silico modeling and Immunoinformatics prediction analysis. The heat shock protein 70 (HSP70c) gene was cloned into the pET28a vector downstream of 3M2e-HA2 and expressed in Escherichia coli host. The desired chimer protein was purified with the Ni-TED column. Results: Validation analysis of the tertiary structure model showed that the model is in the range of native conformations. High score epitopes by Immunoinformatics tools were predicted. The expression of purified 3M2e-HA2-HSP70c was confirmed by the western-blotting assay. Conclusions: The genetic fusion of adjuvant HSP70 to the target antigen may improve the stimulation of immune responses. In silico analysis revealed the appropriate epitopes characterization of conserved antigens. Hence, the constructed chimer protein could be considered as a potential universal vaccine candidate to protect against influenza infection.

Introduction: Influenza virus has several conserved peptides which have the capacity to be used as suitable candidates for appropriate and stable vaccine production against different types of influenza viruses. One of these peptides is HA2, the hemagglutinin stalk domain which mediates the membrane fusion and is conserved amongst different sub-types of influenza virus. This peptide is a good candidate for participation in vaccine structure to induce immunity and antibody production. The ensued antibody may hamper the membrane fusion and subsequently the virus propagation. Methods: The peptide sequence of HA2 from influenza virus A/Tehran/18/2010 (H1N1) was analyzed using in silico tools in order to evaluate its physicochemical properties and identification of its best immunogenic sites. The confirmed sequence was amplified and cloned into a pET28a vector and the recombinant protein was over-expressed prokaryotically and confirmed by Western blotting. Results: The bioinformatics data showed that HA2 peptide stability and immunogenicity. The generated HA2 construct was confirmed by PCR, endonuclease restriction enzyme analysis and sequencing. The expression of HA2 was confirmed by SDS-PAGE and Western blot analysis. The results on the cell lysate demonstrated the high expression of HA2 subunit of the influenza virus hemagglutinin. Conclusion: One of the disadvantages of the current flu vaccines is that they cannot produce efficient broad-spectrum cellular and humoral immune responses against all subtypes of the virus due to the genetic variation of the virus. Therefore, such a conserved protein is potentially a good candidate for production of a broad-spectrum vaccine to prevent influenza epidemics and pandemics.

The H5N1 subtype of Influenza virus, have a very high pathogenicity and lethality in humans and animals, and the need to develop new vaccines with a wide range of effects against this pathogen seems to be very crucial. Today, a highly regarded solution to this problem is to design and produce recombinant vaccines using the conserved peptide of influenza viruses. By searching in international databases, the peptide sequence of M2e fragment of H5N1 viruses isolated from Iran and a variety of conserved peptide sequences of fragments of HA1, HA2, NA and NP of other H5N1 virus was obtained for both MHC receptors in mice. Then these fragments along with a PADRE sequence were connected by bioinformatics programs to design a fusion epitopic construct construct. Afterwards the designed construct was optimized for expression in E.coli BL21. After expression, and purifications of fusion epitopic construct, it was injected subcutaneously (SC) into the hindlimb muscles of 6 – 8 old week female BALB/c mice. Three weeks after the end of the second immunization, both groups of immunized and control mice were weighed and checked for any side effects at the injection sites. Eventually the mice were euthanized and blood was collected from their heart to determine the total IgG antibody before and after immunization by ELISA. No local inflammation or complications were observed at the SC injection sites until the end of the experiment and autopsy of mice showed no bleeding or lesions in organs especially liver and spleen. The weight of the mice did not change significantly during the immunization period. The total IgG level measured by average OD value in the serum of immunized mice showed five times more (5.881 ng/ml) compared to the control group (1.143 ng/ml). Our results demonstrated a highly significant IgG antibody response following SC administration of immunogenic recombinant peptide in mice.

The H5N1 subtype of Influenza virus, have a very high pathogenicity and lethality in humans and animals, and the need to develop new vaccines with a wide range of effects against this pathogen seems to be very crucial. Today, a highly regarded solution to this problem is to design and produce recombinant vaccines using the conserved peptide of influenza viruses. By searching in international databases, the peptide sequence of M2e fragment of H5N1 viruses isolated from Iran and a variety of conserved peptide sequences of fragments of HA1, HA2, NA and NP of other H5N1 virus was obtained for both MHC receptors in mice. Then these fragments along with a PADRE sequence were connected by bioinformatics programs to design a fusion epitopic construct construct. Afterwards the designed construct was optimized for expression in E.coli BL21. After expression, and purifications of fusion epitopic construct, it was injected subcutaneously (SC) into the hindlimb muscles of 6 – 8 old week female BALB/c mice. Three weeks after the end of the second immunization, both groups of immunized and control mice were weighed and checked for any side effects at the injection sites. Eventually the mice were euthanized and blood was collected from their heart to determine the total IgG antibody before and after immunization by ELISA. No local inflammation or complications were observed at the SC injection sites until the end of the experiment and autopsy of mice showed no bleeding or lesions in organs especially liver and spleen. The weight of the mice did not change significantly during the immunization period. The total IgG level measured by average OD value in the serum of immunized mice showed five times more (5.881 ng/ml) compared to the control group (1.143 ng/ml). Our results demonstrated a highly significant IgG antibody response following SC administration of immunogenic recombinant peptide in mice.

Introduction: The genus Exiguobacterium includes psychrotrophic, mesophilic, and moderate thermophilic strains and species that have been isolated from extreme environments, including very cold or hot environments. The genus Exiguobacterium has received much attention in biotechnology, and related industries due to secretory enzymes with high enzymatic activity and consequently stable enzymes capable of tolerating extreme conditions. The aim of the present study was to introduce and describe the phenotypic and the genotypic characterization of a novel species of genus Exiguobacterium isolated from the Ilam mountains, Iran. Materials and Methods: Genotypic features were analyzed using universal genes (gyrB, hsp70, rpoB, and citC) belonging to the Exiguobacterium genus. Also, cspC1, nusA and dnaJ genes were used to confirm the profile of new psychrotrophic strains. The distinctive phenotypic characteristics of the new strain with the most famous strains of the genus Exiguobacterium were investigated. All statistical analyses were conducted using R Packages for data visualization and exploratory data. Results: A motile, Gram-stain-positive, rod-shaped, non-sporing, tolerant up to 5% NaCl, grew at 0-25  ° C and pH 6 and 9, designated Exiguobacterium sp. HA2 was isolated from the soil. The major isoprenoid quinone is MK-7 and in the smaller amount are MK-6 and MK-8. The major cellular fatty acids (>10 %) were isoC13: 0, isoC15: 0 and C16: 0. To adapt to low temperatures, Exiguobacterium sp. HA2 upregulated expresses cold shock response including cold shock protein (CSP) and transcription elongation protein NusA. Also, downregulated expression of heat shock protein DnaJ domain protein. Conclusions: In the current study we investigated the difference in the genotype, phenotypic, and functional characteristics of Exiguobacterium sp. HA2. It can be regarded as representing a novel psychrotrophic species within the genus Exiguobacterium.

Background & Objective: DNA vaccines as a new generation of vaccines require adjuvant to improve vaccine immunogenicity; adjuvants can also increase the DNA vaccine efficacy. In this study, the effects of the host’ s interferon-inducible Mx protein as bio adjuvant and conventional alum adjuvant were evaluated. Materials & Methods: The BALB/c mice were immunized by different prime-boost strategies of the alum and Mx adjuvanted-HA2 DNA vaccine; they were challenged with the specific influenza virus. The potency and safety were evaluated. Humoral immune response was assayed by haemagglutination and virus neutralization tests. The induction of cell-mediated immune responses was determined using an MTT assay. The safety of vaccine regarding side effects occurrence was assessed by observation and histopathologic evaluation. Results: Mx as a host defense peptide was able to increase the immune response against influenza better than alum adjuvant (p<0. 01). By HA2 and Mx in prime and boost strategy, the highest level of specific antibodies developed; they are capable of inducing cell-mediated immune responses. Results indicated that Mx in the DNA vaccine could induce stronger immune responses without any side effects; but alum had some local and general reactions. Conclusion: The Mx could effectively enhance immune responses; it has the potential to enhance the vaccine immunogenicity.