Influenza Virus M2 Protein Research Articles

Anti-influenza A (H1N1) virus effect of gallic acid through inhibition of virulent protein production and association with autophagy.

Influenza remains one of the most serious infectious diseases. Gallic acid is one of the most common and representative phenolic acids found in various plants. This is an interesting subject to explore how gallic acid could inhibit H1N1 influenza virus infection by reducing the production of virulent proteins and interrupting autophagy machinery for influenza virus replication on the host cell. Cellular viability was assessed by XTT assay. The inhibitory effects on the H1N1 influenza virus were assessed by hemagglutination assay, plaque assay, and qRT-PCR. Western blot analysis was used for detecting protein levels of M1, M2, NP, LC3B, and beclin-1. Autophagy activity was demonstrated by acridine orange staining assay. The result demonstrated that there was no cytotoxic effect of gallic acid on A549 cells, and gallic acid could restore the cellular viability of H1N1 influenza virus-infected A549 cells within the experimental concentration treatment. Moreover, gallic acid could effectively restrain viral activity of the H1N1 influenza virus. After the treatment of gallic acid, the production of virulent H1N1 influenza virus proteins, that is, M1, M2, and NP protein were reduced. As for autophagic mechanism, both of the LC3B II conversion and the level ratio of LC3B II to LC3B I were notably decreased. The acridine orange staining assay also revealed decreased accumulation of autophagosomes in H1N1 influenza virus-infected cells. In conclusion, gallic acid suppresses H1N1 influenza viral infectivity through restoration of autophagy pathway and inhibition of virulent M1, M2, and NP protein production.

A Thermal-Stable Protein Nanoparticle That Stimulates Long Lasting Humoral Immune Response.

A thermally stable vaccine platform is considered the missing piece of vaccine technology. In this article, we reported the creation of a novel protein nanoparticle and assessed its ability to withstand extended high temperature incubation while stimulating a long-lasting humoral immune response. This protein nanoparticle was assembled from a fusion protein composed of an amphipathic helical peptide derived from the M2 protein of the H5N1 influenza virus (AH3) and a superfolder green fluorescent protein (sfGFP). Its proposed structure was modeled according to transmission electronic microscope (TEM) images of protein nanoparticles. From this proposed protein model, we created a mutant with two gain-of-function mutations that work synergistically on particle stability. A protein nanoparticle assembled from this gain-of-function mutant is able to remove a hydrophobic patch from its surface. This gain-of-function mutant also contributes to the higher thermostability of protein nanoparticles and stimulates a long lasting humoral immune response after a single immunization. This assembled nanoparticle showed increasing particle stability at higher temperatures and salt concentrations. This novel protein nanoparticle may serve as a thermally-stable platform for vaccine development.

Establishment of Recombinant Eimeria acervulina Expressing Multi-Copies M2e Derived from Avian Influenza Virus H9N2.

The potential of Eimeria parasites as live vaccine vectors has been reported with successful genetic manipulation on several species like E. tenella, E. mitis and E. necatrix. Among seven Eimeria species infecting chickens, E. acervulina is a highly prevalent, moderately pathogenic species. Thus, it is valuable for the study of transfection and for use as a potential as vaccine vector. In this study, a plasmid containing expression cassette with enhanced yellow fluorescent protein (EYFP), red fluorescent protein (RFP) and 12 copies of extracellular domain of H9N2 avian influenza virus M2 (M2e) protein was used for the transfection. Nucleofected sporozoites were inoculated into birds through wing vein. Recombinant E. acervulina oocysts with 0.1% EYFP+ and RFP+ populations were collected from the feces of the inoculated birds. The fluorescent rate of transgenic parasites reached over 95% after nine successive propagations with a pyrimethamine selection in vivo and fluorescent-activated cell sorting (FACS) of progeny oocysts. The expression of M2e in the transgenic parasites (EaM2e) was confirmed by Western blot and its cytoplasm localization in sporozoites was displayed by an indirect immunofluorescent assay (IFA). Meanwhile, we found that the fecundity of EaM2e was equivalent to that of wild type E. acervulina (EaWT). Taken together, the stable transfection of E. acervulina was successfully established. Future studies will focus on whether transgenic E. acervulina can serve as a live vaccine vector.

Dysregulation of ion transport in the lung epithelium infected with SARS-CoV-2.

Dysregulation of ion transport in the lung epithelium infected with SARS-CoV-2.

Immunogenicity and Protective Efficacy of Influenza A DNA Vaccines Encoding Artificial Antigens Based on Conservative Hemagglutinin Stem Region and M2 Protein in Mice.

Background: Development of a universal vaccine capable to induce antibody responses against a broad range of influenza virus strains attracts growing attention. Hemagglutinin stem and the exposed fragment of influenza virus M2 protein are promising targets for induction of cross-protective humoral and cell-mediated response, since they contain conservative epitopes capable to induce antibodies and cytotoxic T lymphocytes (CTLs) to a wide range of influenza virus subtypes. Methods: In this study, we generated DNA vaccine constructs encoding artificial antigens AgH1, AgH3, and AgM2 designed on the basis of conservative hemagglutinin stem fragments of two influenza A virus subtypes, H1N1 and H3N2, and conservative M2 protein, and evaluate their immunogenicity and protective efficacy. To obtain DNA vaccine constructs, genes encoding the designed antigens were cloned into a pcDNA3.1 vector. Expression of the target genes in 293T cells transfected with DNA vaccine constructs has been confirmed by synthesis of specific mRNA. Results: Immunization of BALB/c mice with DNA vaccines encoding these antigens was shown to evoke humoral and T-cell immune responses as well as a moderated statistically significant cross-protective effect against two heterologous viruses A/California/4/2009 (H1N1pdm09) and A/Aichi/2/68 (H3N2). Conclusions: The results demonstrate a potential approach to creating a universal influenza vaccine based on artificial antigens.

Influenza A virus M2 protein triggers mitochondrial DNA-mediated antiviral immune responses

Cytosolic mitochondrial DNA (mtDNA) activates cGAS-mediated antiviral immune responses, but the mechanism by which RNA viruses stimulate mtDNA release remains unknown. Here we show that viroporin activity of influenza virus M2 or encephalomyocarditis virus (EMCV) 2B protein triggers translocation of mtDNA into the cytosol in a MAVS-dependent manner. Although influenza virus-induced cytosolic mtDNA stimulates cGAS- and DDX41-dependent innate immune responses, the nonstructural protein 1 (NS1) of influenza virus associates with mtDNA to evade the STING-dependent antiviral immunity. The STING-dependent antiviral signaling is amplified in neighboring cells through gap junctions. In addition, we find that STING-dependent recognition of influenza virus is essential for limiting virus replication in vivo. Our results show a mechanism by which influenza virus stimulates mtDNA release and highlight the importance of DNA sensing pathway in limiting influenza virus replication.

Envelope-deforming antiviral peptide derived from influenza virus M2 protein

Molecules interfering with lipid bilayer function exhibit strong antiviral activity against a broad range of enveloped viruses, with a lower risk of resistance development than that for viral protein-targeting drugs. Amphipathic peptides are rich sources of such membrane-interacting antivirals. Here, we report that influenza viruses were effectively inactivated by M2 AH, an amphipathic peptide derived from the M2 protein of the influenza virus. Although overall hydrophobicity (<H>) of M2 AH was not related to antiviral activity, modification of the hydrophobic moment (<μH>) of M2 AH dramatically altered the antiviral activity of this peptide. M2 MH, a derivative of M2 AH with a <μH> of 0.874, showed a half maximal inhibitory concentration (IC50) of 53.3 nM against the A/PR/8/34 strain (H1N1), which is 16-times lower than that of M2 AH. The selectivity index (IC50/CC50), where CC50 is the half maximal cytotoxic concentration, was 360 for M2 MH and 81 for M2 AH. Dynamic light scattering spectroscopy and electron microscopy revealed that M2 AH-derived peptides did not disrupt liposomes but altered the shape of viruses. This result suggests that the shape of virus envelope was closely related to its activity. Thus, we propose that deforming without rupturing the membranes may achieve a high selectivity index for peptide antivirals.

Protection against homo and hetero-subtypic influenza A virus by optimized M2e DNA vaccine

ABSTRACT Current influenza vaccines provide hemagglutinin strain-specific protection, but rarely provide cross-protection against divergent strains. It is, therefore, particularly important to develop a universal vaccine against conserved proteins or conserved regions of the virus. In this study, we used N-terminal extracellular region of the influenza virus M2 protein (M2e) as the target antigen and constructed two optimized M2e DNA vaccines (p-tPA-p3M2e and p-p3M2e) with increased antigenic epitope density and enhanced antigen secretion. Both vaccines induced high M2e-specific humoral and cellular immune responses in the vaccinated mice. These two vaccines also conferred protection against a lethal infection of homo-subtypic H1N1 virus, with p-tPA-p3M2e being the most effective. In addition, p-tPA-p3M2e also showed cross-protection against different subtypes of the influenza virus (H9N2, H6N6, and H10N8) at varying rates (80%, 40%, and 20%, respectively). After passive immunization, M2e DNA vaccine-induced antibodies in the sera provided complete protection against homologous virus challenge. An analysis of the mechanism underlying this immunization-mediated protection indicates that M2e-specific IgG and T-cell immune responses may play critical roles in the prevention of infection and viral clearance. Taken together, our results indicate that this optimized M2e DNA vaccine is a promising candidate for the development of a universal, broad-spectrum influenza virus vaccine.

Chimeric protein consisting of 3M2e and HSP as a universal influenza vaccine candidate: from in silico analysis to preliminary evaluation.

The 23-amino acid ectodomain of influenza virus M2 protein (M2e) is highly conserved among human influenza virus variants and represents an attractive target for developing a universal vaccine. Although this peptide has limited potency and low immunogenicity, the degree of M2e density has been shown to be a critical factor influencing the magnitude of epitope-specific responses. The aim of this study was to design a chimer protein consisting of three tandem repeats of M2e peptide sequence fused to the Leishmania major HSP70 gene and evaluate its characteristics and immunogenicity. The structure of the deduced protein and its stability, aliphatic index, biocomputed half-life and the anticipated immunogenicity were analyzed by bioinformatics software. The oligonucleotides encoding 3M2e and chimer 3M2e-HSP70 were expressed in Escherichia coli and affinity purified. The immunogenicity of the purified recombinant proteins was preliminary examined in mouse model. It was predicted that fusion of HSP70 to the C-terminal of 3M2e peptide led to increased stability, hydropathicity, continuous B cell epitopes and antigenic propensity score of chimer protein. Also, the predominant 3M2e epitopes were not hidden in the chimer protein. The initial in vivo experiment showed that 3M2e-HSP chimer protein stimulates specific immune responses. In conclusion, the results of the current study suggest that 3M2e-HSP chimer protein would be an effective universal subunit vaccine candidate against influenza infection.

Virus-Like Particles Are a Superior Platform for Presenting M2e Epitopes to Prime Humoral and Cellular Immunity against Influenza Virus.

Influenza virus M2 protein has a highly conserved ectodomain (M2e) as a cross-protective antigenic target. We investigated the antigenic and immunogenic properties of tandem repeat M2e (5xM2e) proteins and virus-like particles (5xM2e VLP) to better understand how VLP and protein platform vaccines induce innate and protective adaptive immune responses. Despite the high antigenic properties of 5xM2e proteins, the 5xM2e VLP was superior to 5xM2e proteins in inducing IgG2a isotype antibodies, T cell responses, plasma cells and germinal center B cells as well as in conferring cross protection. Mice primed with 5xM2e VLP were found to be highly responsive to 5xM2e protein boost, overcoming the low immunogenicity and protective efficacy of 5xM2e proteins. Immunogenic differences between VLPs and proteins in priming immune responses might be due to an intrinsic ability of 5xM2e VLP to stimulate dendritic cells secreting T helper type 1 (Th1) cytokines. We also found that 5xM2e VLP was effective in inducing inflammatory cytokines and chemokines, and in recruiting macrophages, monocytes, neutrophils, and CD11b+ dendritic cells at the injection site. Therefore, this study provides evidence that 5xM2e VLP is an effective vaccine platform, inducing cross-protection by stimulating innate and adaptive immune responses.

Expression in Plants of a Recombinant Protein Based on Flagellin Linked to Conservative Fragments of M2 Protein and Hemagglutintin of Influenza Virus

The composition of traditional influenza vaccines require nearly annual updates due to the high variability of the influenza virus. The use of conservative viral antigens, the extracellular domain of the transmembrane protein M2, and fragments of the second subunit of hemagglutinin provides the opportunity to create recombinant broad-spectrum vaccines. Bacterial flagellin was used as a mucosal adjuvant to increase the immunogenicity of these conservative antigens. Recombinant proteins based on flagellin simultaneously containing M2e and a fragment of the hemagglutinin stem region were expressed in Nicotiana benthamiana plants using a self-replicating vector based on the potato virus X genome. Methods for their isolation from plants and purification have been developed. The developed expression system can be used to produce a new candidate influenza vaccine in plants.

Symmetric dimeric adamantanes for exploring the structure of two viroporins: influenza virus M2 and hepatitis C virus p7.

BackgroundAdamantane-based compounds have been identified to interfere with the ion-channel activity of viroporins and thereby inhibit viral infection. To better understand the difference in the inhibition mechanism of viroporins, we synthesized symmetric dimeric adamantane analogs of various alkyl-spacer lengths.MethodsSymmetric dimeric adamantane derivatives were synthesized where two amantadine or rimantadine molecules were linked by various alkyl-spacers. The inhibitory activity of the compounds was studied on two viroporins: the influenza virus M2 protein, expressed in Xenopus oocytes, using the two-electrode voltage-clamp technique, and the hepatitis C virus (HCV) p7 channels for five different genotypes (1a, 1b, 2a, 3a, and 4a) expressed in HEK293 cells using whole-cell patch-clamp recording techniques.ResultsUpon testing on M2 protein, dimeric compounds showed significantly lower inhibitory activity relative to the monomeric amantadine. The lack of channel blockage of the dimeric amantadine and rimantadine analogs against M2 wild type and M2-S31N mutant was consistent with previously proposed drug-binding mechanisms and further confirmed that the pore-binding model is the pharmacologically relevant drug-binding model. On the other hand, these dimers showed similar potency to their respective monomeric analogs when tested on p7 protein in HCV genotypes 1a, 1b, and 4a while being 700-fold and 150-fold more potent than amantadine in genotypes 2a and 3a, respectively. An amino group appears to be important for inhibiting the ion-channel activity of p7 protein in genotype 2a, while its importance was minimal in all other genotypes.ConclusionSymmetric dimeric adamantanes can be considered a prospective class of p7 inhibitors that are able to address the differences in adamantane sensitivity among the various genotypes of HCV.

Efficient Transient Expression of Recombinant Proteins in Plants by the Novel pEff Vector Based on the Genome of Potato Virus X.

Agroinfiltration of plant leaves with binary vectors carrying a gene of interest within a plant viral vector is a rapid and efficient method for protein production in plants. Previously, we constructed a self-replicating vector, pA7248AMV, based on the genetic elements of potato virus X (PVX), and have shown that this vector can be used for the expression of recombinant proteins in Nicotiana benthamiana. However, this vector is almost 18 kb long and therefore not convenient for genetic manipulation. Furthermore, for efficient expression of the target protein it should be co-agroinfiltrated with an additional binary vector expressing a suppressor of post-transcriptional gene silencing. Here, we improved this expression system by creating the novel pEff vector. Its backbone is about 5 kb shorter than the original vector and it contains an expression cassette for the silencing suppressor, P24, from grapevine leafroll-associated virus-2 alongside PVX genetic elements, thus eliminating the need of co-agroinfiltration. The pEff vector provides green fluorescent protein expression levels of up to 30% of total soluble protein. The novel vector was used for expression of the influenza vaccine candidate, M2eHBc, consisting of an extracellular domain of influenza virus M2 protein (M2e) fused to hepatitis B core antigen. Using the pEff system, M2eHBc was expressed to 5–10% of total soluble protein, several times higher than with original pA7248AMV vector. Plant-produced M2eHBc formed virus-like particles in vivo, as required for its use as a vaccine. The new self-replicating pEff vector could be used for fast and efficient production of various recombinant proteins in plants.

Chemical Synthesis of Monomeric, Dimeric and Tetrameric Forms of the Ectodomain of Influenza Matrix 2 Protein

AbstractThe highly conserved ectodomain of M2 protein (M2e) of influenza virus A is one of the most studied vaccine targets for induction of cross‐strain protection. It has been reported that the native form of M2 is a homo‐tetramer linked by disulfide bonds and that antibodies reactive to this form correlate with M2‐mediated protection. Although vaccine constructs mimicking the quaternary structure of M2 have been described, the chemical syntheses of disulfide‐linked dimeric and tetrameric forms of M2e have not yet been reported. By exploiting orthogonal chemistry during the syntheses, we successfully assembled dimeric and tetrameric forms of M2e but found that the antibodies that they elicited had protection properties similar to those of the monomeric form. There was, therefore no advantage mimicking the native conformation to improve vaccine efficacy. Nevertheless, the synthetic strategies have the potential for use in preparation of other dimeric or tetrameric constructs.

Characterization of the M2e antibody response following highly pathogenic H5N1 avian influenza virus infection and reliability of M2e ELISA for identifying infected among vaccinated chickens

A surveillance method able to differentiate between vaccinated and infected poultry is required for those countries that practice vaccination against highly pathogenic avian influenza H5N1. The external domain of the M2 protein (M2e) of influenza virus is a potentially useful differentiating-infected from vaccinated animals (DIVA) antigen but little is known about the M2e antibody response and factors influencing its detection. In this study, the M2e antibody response was characterized in layer birds vaccinated and challenged with an Indonesian H5N1 virus isolate, using a single M2e peptide or four-branched multiple antigenic peptide form of M2e (MAP-M2e) as antigens in two separate ELISAs. Anti-M2e antibodies were absent in chicks with high level of maternal haemagglutination inhibition antibodies and also in all layers vaccinated once, twice or three times with an inactivated commercial H5N1 vaccine. In contrast, anti-M2e antibodies were detected in vaccinated layers challenged with H5N1 virus and their presence was associated with virus isolation and an increase in haemagglutination inhibition titres. The number of birds that developed M2e antibodies, as well as the strength and duration of the M2e antibody response were strongly influenced by the length of the interval between vaccination and challenge. Birds challenged at six weeks after vaccination all developed M2e antibodies by 14 days that lasted until at least 56 days after infection. In birds challenged at two weeks after vaccination, only a proportion of birds developed M2e antibodies by 14 days that lasted only until 28 days post-infection. Both single M2e peptide and MAP-M2e ELISAs had high diagnostic specificity but the diagnostic sensitivity of MAP-M2e ELISA was significantly higher and more effective in detecting M2e antibody in immune and infected birds. The results show that MAP-M2e ELISA would be useful for surveillance in countries using vaccination to control highly pathogenic avian influenza H5N1.

Investigation of the curvature induction and membrane localization of the influenza virus M2 protein using static and off-magic-angle spinning solid-state nuclear magnetic resonance of oriented bicelles.

A wide variety of membrane proteins induce membrane curvature for function; thus, it is important to develop new methods to simultaneously determine membrane curvature and protein binding sites in membranes with multiple curvatures. We introduce solid-state nuclear magnetic resonance (NMR) methods based on magnetically oriented bicelles and off-magic-angle spinning (OMAS) to measure membrane curvature and the binding site of proteins in mixed-curvature membranes. We demonstrate these methods on the influenza virus M2 protein, which not only acts as a proton channel but also mediates virus assembly and membrane scission. An M2 peptide encompassing the transmembrane (TM) domain and an amphipathic helix, M2(21-61), was studied and compared with the TM peptide (M2TM). Static (31)P NMR spectra of magnetically oriented 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles exhibit a temperature-independent isotropic chemical shift in the presence of M2(21-61) but not M2TM, indicating that the amphipathic helix confers the ability to generate a high-curvature phase. Two-dimensional (2D) (31)P spectra indicate that this high-curvature phase is associated with the DHPC bicelle edges, suggestive of the structure of budding viruses from the host cell. (31)P- and (13)C-detected (1)H relaxation times of the lipids indicate that the majority of M2(21-61) is bound to the high-curvature phase. Using OMAS experiments, we resolved the (31)P signals of lipids with identical headgroups based on their distinct chemical shift anisotropies. On the basis of this resolution, 2D (1)H-(31)P correlation spectra show that the amide protons in M2(21-61) correlate with the DMPC but not DHPC (31)P signal of the bicelle, indicating that a small percentage of M2(21-61) partitions into the planar region of the bicelles. These results show that the amphipathic helix induces high membrane curvature and localizes the protein to this phase, in good agreement with the membrane scission function of the protein. These bicelle-based relaxation and OMAS solid-state NMR techniques are generally applicable to curvature-inducing membrane proteins such as those involved in membrane trafficking, membrane fusion, and cell division.

Influenza virus M2 protein ion channel activity helps to maintain pandemic 2009 H1N1 virus hemagglutinin fusion competence during transport to the cell surface.

The influenza virus hemagglutinin (HA) envelope protein mediates virus entry by first binding to cell surface receptors and then fusing viral and endosomal membranes during endocytosis. Cleavage of the HA precursor (HA0) into a surface receptor-binding subunit (HA1) and a fusion-inducing transmembrane subunit (HA2) by host cell enzymes primes HA for fusion competence by repositioning the fusion peptide to the newly created N terminus of HA2. We previously reported that the influenza virus M2 protein enhances pandemic 2009 influenza A virus [(H1N1)pdm09] HA-pseudovirus infectivity, but the mechanism was unclear. In this study, using cell-cell fusion and HA-pseudovirus infectivity assays, we found that the ion channel function of M2 was required for enhancement of HA fusion and HA-pseudovirus infectivity. The M2 activity was needed only during HA biosynthesis, and proteolysis experiments indicated that M2 proton channel activity helped to protect (H1N1)pdm09 HA from premature conformational changes as it traversed low-pH compartments during transport to the cell surface. While M2 has previously been shown to protect avian influenza virus HA proteins of the H5 and H7 subtypes that have polybasic cleavage motifs, this study demonstrates that M2 can protect HA proteins from human H1N1 strains that lack a polybasic cleavage motif. This finding suggests that M2 proton channel activity may play a wider role in preserving HA fusion competence among a variety of HA subtypes, including HA proteins from emerging strains that may have reduced HA stability. Influenza virus infects cells when the hemagglutinin (HA) surface protein undergoes irreversible pH-induced conformational changes after the virus is taken into the cell by endocytosis. HA fusion competence is primed when host cell enzymes cleave the HA precursor. The proton channel function of influenza virus M2 protein has previously been shown to protect avian influenza virus HA proteins that contain a polybasic cleavage site from pH-induced conformational changes during biosynthesis, but this effect is less well understood for human influenza virus HA proteins that lack polybasic cleavage sites. Using assays that focus on HA entry and fusion, we found that the M2 protein also protects (H1N1)pdm09 influenza A virus HA from premature conformational changes as it transits low-pH compartments during biosynthesis. This work suggests that M2 may play a wider role in preserving HA function in a variety of influenza virus subtypes that infect humans and may be especially important for HA proteins that are less stable.

Development of a heat-stable and orally delivered recombinant M2e-expressing B. subtilis spore-based influenza vaccine

Highly conserved ectodomain of influenza virus M2 protein (M2e) is an important target for the development of universal influenza vaccines. Today, the use of chemical or genetic fusion constructs have been undertaken to overcome the low immunogenicity of M2e in vaccine formulation. However, current M2e vaccines are neither orally delivered nor heat-stable. In this study, we evaluated the immune efficacy of an orally delivered recombinant M2e vaccine containing 3 molcules of M2e consensus sequence of influenza A viruses, termed RSM2e3. To accomplish this, CotB, a spore coat of Bacillus subtilis (B. subtilis), was used as a fusion partner, and heat-stable nonpathogenic B. subtilis spores were used as the carrier. Our results showed that CotB-M2e3 fusion had no effect on spore structure or function in the resultant recombinant RSM2e3 strain and that heterologous influenza virus M2e protein was successfully displayed on the surface of the recombinant RSM2e3 spore. Importantly, recombinant RSM2e3 spores elicited strong and long-term M2e-specific systemic and mucosal immune responses, completely protecting immunized mice from lethal challenge of A/PR/8/34(H1N1) influenza virus. Taken together, our study forms a solid basis for the development of a novel orally delivered and heat-stable influenza vaccine based on B. subtilis spore surface display.

In Vitro Evaluation of Influenza M2 and Leishmania major HSP70 (221-604) Chimer Protein.

Background:Permanent antigenic variation of influenza viruses causes a major concern to develop an effective human influenza vaccine. Conserved antigens are new vaccine candidates because it is not necessary to match the prepared vaccine with circulating strains. Ion channel M2 protein is conserved among all influenza A viruses, allowing the virus to enter host cells.Objectives:To prepare an effective vaccine against influenza A viruses, a chimerical DNA plasmid encoding Influenza virus M2 protein and Leishmania major HSP70 was constructed.Materials and Methods:Influenza A/New Caledonia/20/99 (H1N1) was inoculated into MDCK cell line and total RNA was extracted. The full length M2 gene was amplified by RT-PCR using designed specific primers, cloned into pGEM-T Easy cloning vector and completely sequenced. The M2 gene was then subcloned into the pcDNA upstream of HSP70 gene. Recombinant plasmids were transfected into COS-7 cells to evaluate protein expression.Results:The recombinant plasmids were confirmed by PCR, restriction enzyme analysis and sequencing. Three dimensional structure of chimer protein was assessed using specific software. Transient protein expression in eukaryotic cells was confirmed by specific mRNA detection, indirect Immunofluorescence test and western blotting.Conclusions:M2-HSP70 chimer protein was successfully expressed in eukaryotic cells. Computational studies of chimer peptide sequence revealed that fusing HSP to the C-terminal of M2 protein does not mask the predominant epitope of M2. HSP70 is a molecular chaperon and immunostimulatory component. Genetically fusing antigens to HSPs leads to the enrichment of DNA vaccine potency. The immunogenicity of this construct with different formulation would be evaluated in further investigations.

Docking and Molecular Dynamics (MD) Simulations in Potential Drugs Discovery: An Application to Influenza Virus M2 Protein

Biological Faculty, Lomonosov Moscow State University (MSU), Leninskie gory 1, Moscow 119234, Russia Department of Physics, University of Osnabruck, Barbarastr 7, Osnabruck 49076, Germany Department of Health Sciences (DISSAL), University of Genoa, Via Antonio Pastore 1, Genoa 16132, Italy Laboratories of Biophysics and Nanotechnologies (LBN), Department of Experimental Medicine (DIMES), University of Genoa, Via Antonio Pastore 3, Genoa 16132, Italy Nanoworld Institute Fondazione EL.B.A.Nicolini (NWI-FEN), Largo Redaelli 7, Pradalunga, Bergamo 24100, Italy