Expression of influenza A virus-derived peptides on a rotavirus VP6-based delivery platform

Abstract Recombinant protein technology enables the engineering of modern vaccines composed of a carrier protein displaying poorly immunogenic heterologous antigens. One promising carrier is based on the rotavirus inner-capsid VP6 protein. We explored different VP6 insertion sites for the presentation of two peptides (23 and 140 amino acids) derived from the M2 and HA genes of influenza A virus. Both termini and three surface loops of VP6 were successfully exploited as genetic fusion sites, as demonstrated by the expression of the fusion proteins. However, further studies are needed to assess the morphology and immunogenicity of these constructs.

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Design, synthesis and primary immunologic evaluation of M2e-CRM197 conjugate as a universal influenza vaccine candidate

Current Pharmaceutical Biotechnology ◽

10.2174/1389201021666201104145006 ◽

2020 ◽

Vol 21 ◽

Author(s):

Lu Xu ◽

Chun Zhang ◽

Jing Zhang ◽

Rong Yu ◽

Zhiguo Su

Keyword(s):

Immune Responses ◽

Influenza Vaccine ◽

Influenza A Virus ◽

Influenza A ◽

Vaccine Development ◽

Acute Infection ◽

Acid Hydrazide ◽

Influenza Viruses ◽

Carrier Protein ◽

Universal Influenza Vaccine

Background: Influenza is a contagious respiratory illness caused by acute infection of influenza viruses, among which influenza A virus causes epidemic seasonal infection nearly every year. Along with unpredictability of evolving influenza A virus and time-consuming vaccine development cycles, novel universal influenza vaccine designed to induce broadly cross-reactive immune responses against frequently mutant influenza A virus strains are greatly urgent. Objective: The aim of this study was to synthesize a novel vaccine through the dual-site specific conjugation of the constant epitope of 23 amino acids (M2e) of influenza A virus with highly immunogenic carrier protein of cross-reacting material (CRM197) under denaturation, and evaluate its primary immunogenicity in mice. Methods: The antigen (M2e) and the carrier protein (CRM197) were linked with different type of hetero-functionalized linkers, α-maleimide-ε-hydrazide polyethylene glycol 2k (MAL-PEG-HZ) and N-β-maleimidopropionic acid hydrazide (BMPH) separately. The immunogenicity of the M2e-CRM197 conjugates with different type of linkers was evaluated in mice, and the M2e-specific total IgG and IgG-isotypes were determined by ELSIA. Results: Immunogenicity study revealed that anti-M2e antibody could be induced by the conjugate products, M2e-PEGCRM197 and M2e-BMPH-CRM197, were approximately 30 and 90-fold higher than that of M2e group. In addition, the antiM2e antibody level induced by M2e-PEG-CRM197 conjugate was three times higher than that of M2e-BMPH-CRM197 conjugate, and the former could simultaneously activate both cellar and humoral immune responses. Conclusions: The M2e-CRM197 conjugated vaccines we synthesized in this study are highly immunogenic compared with M2e alone. Besides, evidences were presented here indicated that the hydrophilic, non-immunogenic and biocompatible chain of the cross-linker might be a better choice for development of conjugate vaccine.

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Highly Immunogenic Nanoparticles Based on a Fusion Protein Comprising the M2e of Influenza A Virus and a Lipopeptide

Viruses ◽

10.3390/v12101133 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1133

Author(s):

Anna A. Zykova ◽

Elena A. Blokhina ◽

Roman Y. Kotlyarov ◽

Liudmila A. Stepanova ◽

Liudmila M. Tsybalova ◽

...

Keyword(s):

Escherichia Coli ◽

Influenza A Virus ◽

Fusion Proteins ◽

Influenza A ◽

Transmembrane Protein ◽

Antibody Responses ◽

Nanosized Particles ◽

Vaccine Candidates ◽

Promising Target ◽

Study Results

The highly conserved extracellular domain of the transmembrane protein M2 (M2e) of the influenza A virus is a promising target for the development of broad-spectrum vaccines. However, M2e is a poor immunogen by itself and must be linked to an appropriate carrier to induce an efficient immune response. In this study, we obtained recombinant mosaic proteins containing tandem copies of M2e fused to a lipopeptide from Neisseria meningitidis surface lipoprotein Ag473 and alpha-helical linkers and analyzed their immunogenicity. Six fusion proteins, comprising four or eight tandem copies of M2e flanked by alpha-helical linkers, lipopeptides, or a combination of both of these elements, were produced in Escherichia coli. The proteins, containing both alpha-helical linkers and lipopeptides at each side of M2e repeats, formed nanosized particles, but no particulate structures were observed in the absence of lipopeptides. Animal study results showed that proteins with lipopeptides induced strong M2e-specific antibody responses in the absence of external adjuvants compared to similar proteins without lipopeptides. Thus, the recombinant M2e-based proteins containing alpha-helical linkers and N. meningitidis lipopeptide sequences at the N- and C-termini of four or eight tandem copies of M2e peptide are promising vaccine candidates.

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