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Influence of the Linking Order of Fragments of HA2 and M2e of the influenza A Virus to Flagellin on the Properties of Recombinant Proteins.

ABSTRACT: The ectodomain of the M2 protein (M2e) and the conserved fragment of the second subunit of hemagglutinin (HA2) are promising candidates for broadly protective vaccines. In this paper, we report on the design of chimeric constructs with differing orders of linkage of four tandem copies of M2e and the conserved fragment of HA2 (76-130) from phylogenetic group II influenza A viruses to the C-terminus of flagellin. The 3D-structure of two chimeric proteins showed that interior location of the M2e tandem copies (Flg-4M2e-HA2) provides partial ?-helix formation nontypical of native M2e on the virion surface. The C-terminal position of the M2e tandem copies (Flg-HA2-4M2e) largely retained its native M2e conformation. These conformational differences in the structure of the two chimeric proteins were shown to affect their immunogenic properties. Different antibody levels induced by the chimeric proteins were detected. The protein Flg-HA2-4M2e was more immunogenic as compared to Flg-4M2e-HA2, with the former offering full protection to mice against a lethal challenge. We obtained evidence suggesting that the order of linkage of target antigens in a fusion protein may influence the 3D conformation of the chimeric construct, which leads to changes in immunogenicity and protective potency.

SUBMITTER: Stepanova LA

PROVIDER: S-EPMC5916737 | biostudies-literature | 2018 Jan-Mar

REPOSITORIES: biostudies-literature

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Influence of the Linking Order of Fragments of HA2 and M2e of the influenza A Virus to Flagellin on the Properties of Recombinant Proteins.

Stepanova L A LA Kotlyarov R Y RY Shuklina M A MA Blochina E A EA Sergeeva M V MV Potapchuk M V MV Kovaleva A A AA Ravin N V NV Tsybalova L M LM

Acta naturae 20180101 1

The ectodomain of the M2 protein (M2e) and the conserved fragment of the second subunit of hemagglutinin (HA2) are promising candidates for broadly protective vaccines. In this paper, we report on the design of chimeric constructs with differing orders of linkage of four tandem copies of M2e and the conserved fragment of HA2 (76-130) from phylogenetic group II influenza A viruses to the C-terminus of flagellin. The 3D-structure of two chimeric proteins showed that interior location of the M2e ta ...[more]

PMID: 29713522

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Project description:The study was aimed to evaluate the elicitation of highly pathogenic avian influenza (HPAI) virus (AIV) M2e and HA2-specific immunity in chicken to develop broad protective influenza vaccine against HPAI H5N1. Based on the analysis of Indian AIV H5N1 sequences, the conserved regions of extracellular domain of M2 protein (M2e) and HA2 were identified. Synthetic gene construct coding for M2e and two immunodominant HA2 conserved regions was designed and synthesized after codon optimization. The fusion recombinant protein (~38 kDa) was expressed in a prokaryotic system and characterized by Western blotting with anti-His antibody and anti-AIV polyclonal chicken serum. The M2e-HA2 fusion protein was found to be highly reactive with known AIV-positive and -negative chicken sera by ELISA. Two groups of specific pathogen-free (SPF) chickens were immunized (i/m) with M2e synthetic peptide and M2e-HA2 recombinant protein along with one control group with booster on the 14th day and 28th day with the same dose and route. Pre-immunization sera and whole blood were collected on day 0 followed by 3, 7, 14, 21, and 28 days and 2 weeks after the second booster (42 day). Lymphocyte proliferation assay by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) method revealed that the stimulation index (SI) was increased gradually from days 0 to 14 in the immunized group (p < 0.05) than that in control chicken. Toll-like receptor (TLR) mRNA analysis by RT-qPCR showed maximum upregulation in the M2e-HA2-vaccinated group compared to M2e- and sham-vaccinated groups. M2e-HA2 recombinant protein-based indirect ELISA revealed that M2e-HA2 recombinant fusion protein has induced strong M2e and HA2-specific antibody responses from 7 days post-primary immunization, and then the titer gradually increased after booster dose. Similarly, M2e peptide ELISA revealed that M2e-HA2 recombinant fusion protein elicited M2e-specific antibody from day 14 onward. In contrast, no antibody response was detected in the chicken immunized with synthetic peptide M2e alone or control group. Findings of this study will be very useful in future development of broad protective H5N1 influenza vaccine targeting M2e and HA2.

Project description:Background: Type A influenza viruses are contagious and even life-threatening if left untreated. So far, no broadly protective vaccine is available due to rapid antigenic changes and emergence of new subtypes of influenza virus. In this study, we exploited bioinformatics tools in order to design a subunit chimeric vaccine from the antigenic and highly conserved regions of HA and M2 proteins of H7N9 subtype of influenza virus. We used mucosal adjuvant candidates, including CTxB, STxB, ASP-1, and LTB to stimulate mucosal immunity and analyzed the combination of HA2, M2e, and the adjuvant. Furthermore, to improve the antigen function and to maintain their three-dimensional structure, 12 different linkers including six rigid linkers and six flexible linkers were used. The 3D structure model was generated using a combination of homology and ab initio modeling methods and the molecular dynamics of the model were analyzed, either.Results: Analysis of different adjuvants showed that using CtxB as an adjuvant, results in higher overall vaccine stability and higher half-life among four adjuvant candidates. Fusion of antigens and the CTxB in the form of M2e-linker-CTxB-linker-HA2 has the most stability and half life compared to other combination forms. Furthermore, the KPKPKP rigid linker showed the best result for this candidate vaccine among 12 analyzed linkers. The changes in the vaccine 3D structure made by linker insertion found to be negligible, however, although small, the linker insertion between the antigens causes the structure to change slightly. Eventually, using predictive tools such as Ellipro, NetMHCpan I and II, CD4episcore, CTLpred, BepiPred and other epitope analyzing tools, we analyzed the conformational and linear epitopes of the vaccine. The solubility, proteasome cleavage sites, peptidase and potential chemical cutters, codon optimization, post translational modification were also carried out on the final vaccine.Conclusions: It is concluded that M2e-Linker-CTxB-Linker-HA2 combination of chimeric vaccine retains its 3D structure and antigenicity when KPKPKP used as linker and CTxB used as adjuvant.

Project description:BACKGROUND:Influenza infection could be more effectively controlled if a multi-purpose vaccine with the ability to induce responses against most, or all, influenza A subtypes could be generated. Conserved viral proteins are a promising basis for the creation of a broadly protective vaccine. In the present study, the immunogenicity and protective properties of three recombinant proteins (vaccine candidates), comprising conserved viral proteins fused with bacterial flagellin, were compared. METHODS:Balb/c mice were immunized intranasally with recombinant proteins comprising either one viral protein (the ectodomain of the M2 protein, 'M2e') or two viral proteins (M2e and the hemagglutinin second subunit 'HA2' epitope) genetically fused with flagellin. Further, two different consensus variants of HA2 were used. Therefore, three experimental positives were used in addition to the negative control (Flg-his). The mucosal, humoral, and T-cell immune responses to these constructs were evaluated. RESULT:We have demonstrated that insertion of the HA2 consensus polypeptide (aa 76-130), derived from either the first (HA2-1) or second (HA2-2) virus phylogenetic group, into the recombinant Flg4M2e protein significantly enhanced its immunogenicity and protective properties. Intranasal administration of the vaccine candidates (Flg-HA2-2-4M2e or Flg-HA2-1-4M2e) induced considerable mucosal and systemic responses directed at both the M2e-protein and, in general, the influenza A virus. However, the immune response elicited by the Flg-HA2-1-4M2e protein was weaker than the one generated by Flg-HA2-2-4M2e. These recombinant proteins containing both viral peptides provide complete protection from lethal challenge with various influenza viruses: A/H3N2; A/H2N2; and A/H5N1. CONCLUSION:This study demonstrates that the intranasal administration of Flg-HA2-2-4M2e recombinant protein induces a strong immune response which provides broad protection against various influenza viruses. This construct is therefore a strong candidate for development as a universal vaccine.

Dataset Information

Influence of the Linking Order of Fragments of HA2 and M2e of the influenza A Virus to Flagellin on the Properties of Recombinant Proteins.

Publications

Influence of the Linking Order of Fragments of HA2 and M2e of the influenza A Virus to Flagellin on the Properties of Recombinant Proteins.

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