Ectodomain Of M2 Protein Research Articles

A single-shot vaccine approach for the universal influenza A vaccine candidate M2e.

SignificanceAlthough the need for a universal influenza vaccine has long been recognized, only a handful of candidates have been identified so far, with even fewer advancing in the clinical pipeline. The 24-amino acid ectodomain of M2 protein (M2e) has been developed over the past two decades. However, M2e-based vaccine candidates have shortcomings, including the need for several administrations and the lack of sustained antibody titers over time. We report here a vaccine targeting strategy that has the potential to confer sustained and strong protection upon a single shot of a small amount of M2e antigen. The current COVID-19 pandemic has highlighted the importance of developing versatile, powerful platforms for the rapid deployment of vaccines against any incoming threat.

DNA vaccine targeting the ectodomain of influenza M2 protein to endolysosome pathway enhances anti-M2e protective antibody response in mice.

A promising candidate for developing the universal influenza vaccine is the ectodomain of the M2 protein (M2e). We designed and prepared an experimental DNA vaccine with an improved potential to induce anti-M2e immune response. The sequence for truncated NS1 protein followed by 4xM2e was inserted into the expression vector pTriEx-4 (pEx). M2e repeats were fused to the transmembrane domain and cytoplasmic tail of lysosome-associated membrane glycoprotein 2 isoform A (LAMP-2a) to target the M2e to the endo-lysosome pathway, facilitating increased antigen presentation by MHC II. Using confocal microscope immunofluorescence analysis, we confirmed a strong colocalization of pEx 4M2e-LAMP-2a with early endosomes and a weaker colocalization with late endosomes. BALB/c mice immunized with three doses of pEx 4M2e-LAMP-2a DNA vaccine and challenged with 2LD50 mouse-adapted influenza virus developed significantly (up to 16 times) higher anti-M2e antibody response in comparison to mice immunized with pEx 4M2e vaccine using the same immunization protocol. This was in correlation with the increased survival rate (near to 67% vs 50%) observed in animals immunized with pEx 4M2e-LAMP-2a DNA in comparison to mice immunized with pEx 4M2e. Keywords: influenza A; matrix protein 2 ectodomain; NS1; LAMP-2a; DNA vaccine.

CROSS-PROTECTIVE PROPERTIES OF AN INFLUENZA VACCINE BASED ON HBC4M2E RECOMBINANT PROTEIN.

One of the main problems in the area of influenza prophylaxis and pandemic prevention is the development of cross-reactive vaccines, i.e. vaccines directed against all subtypes of human influenza viruses. Such vaccines are being developed in many countries for more than 10 years. A number of vaccines are presently undergoing clinical trials. We created Uniflu candidate vaccine based on recombinant HBc4M2e protein consisting of 4 tandem-connected copies of the highly conserved ectodomain of M2 protein of the influenza A virus. These 4 copies were genetically fused to the carrier protein, namely hepatitis B core antigen. Commercially available Derinat was used as adjuvant in the candidate vaccine. Preclinical studies on laboratory animals (mice, ferrets) demonstrated that immunization with Uniflu leads to significantly higher level of specific immunoglobulins in the blood and bronchoalveolar lavages. Moreover, it produces immunoglobulins belonging to subtype IgG2a that is the most important mediator of antibody-dependent cytotoxicity. The vaccine under review stimulates the proliferation of T-lymphocytes, as well as the formation of CD4+ and CD8+ T-cells synthesizing ɣ-IFN. When infected with the lethal doses (5 LD50) of influenza A viruses of the subtypes H1N1, H2N2, H3N2, and H1N1pdm09, immunized animals typically developed mild form of illness. This kept them alive in 90-100% of cases, which demonstrated almost complete protection from death. Replication of the virus in the lungs of immunized mice was reduced by 1.8-4.8 log10. High immunogenicity of the vaccine, and reduced clinical symptoms following experimental infection, were demonstrated in ferrets as well. The developed recombinant vaccine Uniflu has high specific activity and cross-protection. Uniflu can be proposed as pre-pandemic vaccine, provided that it passes clinical trials.

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.

Sintesis Tiga Peptida Bergugus Pelindung sebagai Prekursor Komponen Vaksin Influenza Universal

Current highly effective conventional vaccine to halt the spread of bird flu has not been invented yet because of susceptiblemutation of influenza virus. In spite of undergoing mutation which causes the amino acid sequence change, influenzaviruses maintain conservation at ectodomain of M2 protein, especially M2e(2-16) (SLLTEVETPIRNEW). The use ofconserved epitope M2e(2-16) in epitope-based vaccine potentially produces universal influenza vaccine. In designingepitope-based vaccine, the M2e(2-16) needs to be coupled with T helper epitope, P25, which is subsequently mentioned asM2e(2-16)-K-P25 (SLLTEVETPIRNEWGKKKL IPNASLIENCTKAEL). The M2e(2-16)-K-P25 was synthesized usingconvergent solid phase peptide synthesis strategy because of the size of the sequence. In this strategy, four peptideprecursors of M2e(2-16)-K-P25; SLLTEVETP (F1), IRNEWGK (F2), KLIPNASLI (F3), and ENCTKAEL (F4); were synthesizedin advance. After the precursors ready, coupling reaction was performed to obtain M2e(2-16)-K-P25. In the previousresearch, F3 has been obtained in high purity through Fmoc/tBu solid phase peptide synthesis method. In this conductedresearch, the three remaining precursors; F1, F2, and F4; were synthesized by the same method. Each peptide was analysedby thin layer chromatography, HPLC, and mass spectroscopy methods. F1, F2 and F4 were successfully synthesized andeach of them was detected at 1490.0, 1874.8 and 1881.9 amu, respectively. However, F1 was not possible to purify becauseof its insolubility in various solvents.

Rapid high-yield expression of a candidate influenza vaccine based on the ectodomain of M2 protein linked to flagellin in plants using viral vectors.

BackgroundThe extracellular domain of matrix protein 2 (M2e) of influenza A virus is a promising target for the development of a universal vaccine against influenza because M2e sequences are highly conserved among human influenza A strains. However, native M2e is poorly immunogenic, but its immunogenicity can be increased by delivery in combination with adjuvants or carrier particles. It was previously shown that fusion of M2e to bacterial flagellin, the ligand for Toll-like receptor (TLR) 5 and powerful mucosal adjuvant, significantly increases the immunogenicity and protective capacity of M2e.ResultsIn this study, we report for the first time the transient expression in plants of a recombinant protein Flg-4M comprising flagellin of Salmonella typhimurium fused to four tandem copies of the M2e peptide. The chimeric construct was expressed in Nicotiana benthamiana plants using either the self-replicating potato virus X (PVX) based vector, pA7248AMV-GFP, or the cowpea mosaic virus (CPMV)-derived expression vector, pEAQ-HT. The highest expression level up to 30 % of total soluble protein (about 1 mg/g of fresh leaf tissue) was achieved with the PVX-based expression system. Intranasal immunization of mice with purified Flg-4M protein induced high levels of M2e-specific serum antibodies and provided protection against lethal challenge with influenza virus.ConclusionsThis study confirms the usefulness of flagellin as a carrier of M2e and its relevance for the production of M2e-based candidate influenza vaccines in plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0164-6) contains supplementary material, which is available to authorized users.

Recombinant M2e Protein-Based ELISA: A Novel and Inexpensive Approach for Differentiating Avian Influenza Infected Chickens from Vaccinated Ones

Available avian influenza (AIV) serological diagnostic tests cannot distinguish vaccinated from naturally infected birds. Differentiation of vaccinated from infected animals (DIVA) is currently advocated as a means of achieving the full control of H5N1. In this study, for the first time, recombinant ectodomain of M2 protein (M2e) of avian influenza virus (H5N1 strain) was used for the DIVA serology test. M2e was cloned into pMAL-P4X vector and expressed in E. coli cells. We used Western blot to recognize the expressed M2e-MBP protein by chicken antisera produced against live H5N1 virus. Also, the specificity of M2e-MBP protein was compared to the M2e synthetic peptide via ELISA. In M2e-MBP ELISA, all sera raised against the live avian influenza viruses were positive for M2e antibodies, whereas sera from killed virus vaccination were negative. Furthermore, M2e-MBP ELISA of the field sera obtained from vaccinated and non-vaccinated chickens showed negative results, while challenged vaccinated chickens demonstrated strong positive reactions. H5N1-originated recombinant M2e protein induced broad-spectrum response and successfully reacted with antibodies against other AIV strains such as H5N2, H9N2, H7N7, and H11N6. The application of the recombinant protein instead of synthetic peptide has the advantages of continues access to an inexpensive reagent for performing a large scale screening. Moreover, recombinant proteins provide the possibility of testing the DIVA results with an additional technique such a Western blotting which is not possible in the case of synthetic proteins. All together, the results of the present investigation show that recombinant M2e-MBP can be used as a robust and inexpensive solution for DIVA test.

Evaluation of anti-influenza efficiency of polyclonal IgG antibodies specific to the ectodomain of M2 protein of influenza A virus by passive immunization of mice

We attempted to quantify the protective potential of polyclonal IgG antibodies specific to the ectodomain of M2 protein (eM2) of influenza A virus (IAV) against lethal influenza infection of mice. For this purpose, eM2 conjugated with keyhole limpet hemocyanin (KLH) or KLH alone were administered with Freund's adjuvant intraperitoneally (i.p.) to BALB/c mice. IgG antibodies specific to the KLH-eM2 conjugate (anti-KLH-eM2 IgGs) and KLH (anti-KLH IgGs), respectively, were purified from ascitic fluids. Analysis of the preparation of anti-KLH-eM2 IgGs by ELISA revealed that it contained about 25% of anti-eM2 IgGs and 75% of anti-KLH IgGs. Taking into account this finding mice were passively immunized by intravenous route with 320, 160, 80, and 40 µg of anti-eM2 IgGs per mouse, respectively, while 320 µg of anti-KLH IgGs were used in control. Following subsequent infection with 3 LD50 IAV the survival of mice was determined. An absolute protection (100% survival) was obtained with 320 µg of anti-eM2 IgGs, and a relatively strong significant protection (~80% survival, p = 0.024) with 160 µg. The amount 160 µg of IgGs represents approx. 100 µg IgGs per 1 ml of blood.

Heterosubtypic protective immunity against influenza A virus induced by fusion peptide of the hemagglutinin in comparison to ectodomain of M2 protein

Several types of influenza vaccines are available, but due to the highly unpredictable variability of influenza virus surface antigens (hemagglutinin (HA) and neuraminidase) current vaccines are not sufficiently effective against broad spectrum of the influenza viruses. An innovative approach to extend the vaccine efficacy is based on the selection of conserved influenza proteins with a potential to induce inter-subtype protection against the influenza A viruses. A promising new candidate for the preparation of broadly protective vaccine may be a highly conserved N-terminal part of HA2 glycopolypeptide (HA2 gp) called fusion peptide. To study its capacity to induce a protective immune response, we immunized mice with the fusion peptide (aa 1-38 of HA2 gp). The protective ability of fusion peptide was compared with the ectodomain aa 2-23 of M2 protein (eM2) that is antigenically conserved and its immunogenic properties have already been well documented. Corresponding peptides (both derived from A/Mississippi/1/85 (H3N2) virus) were synthesized and conjugated to the keyhole limpet hemocyanin (KLH) and used for the immunization of mice. Both antigens induced a significant level of specific antibodies. Immunized mice were challenged with the lethal dose of homologous (H3N2) or heterologous A/PR/8/34 (H1N1) influenza A viruses. Immunization with the fusion peptide led to the 100% survival of mice infected with 1 LD50 of homologous as well as heterologous virus. Survival rate decreased when infectious dose was raised to 2 LD50. The immunization with eM2 induced effective cross-protection of mice infected even with 3 LD50 of both challenge viruses. The lower, but still effective protection induced by the fusion peptide of HA2 gp suggested that besides ectodomain of M2, fusion peptide could also be considered as a part of cross-protective influenza vaccine. To our knowledge, this is the first report demonstrating that active immunization with the conjugated fusion peptide of HA2 gp provided the effective production of antibodies, what contributed to the cross-protection against influenza infection.