Modulation of Immunogenicity and Conformation of HA1 Subunit of Influenza A Virus H1/N1 Hemagglutinin in Tubular Immunostimulating Complexes

The HA1 subunit of the influenza virus hemagglutinin (HA) is a valuable antigen for the development of vaccines against flu due to the availability of most antigenic sites which are conformational. Therefore, a novel adjuvanted antigen delivery system, tubular immunostimulating complexes (TI-complexes) comprising monogalactosyldiacylglycerol (MGDG) from different marine macrophytes as a lipid matrix for an antigen, was applied to enhance the immunogenicity of recombinant HA1 of influenza A H1N1 and to study the relation between its immunogenicity and conformation. The content of anti-HA1 antibodies and cytokines was estimated by ELISA after the immunization of mice with HA1 alone, and HA1 was incorporated in TI-complexes based on different MGDGs isolated from green algae Ulva lactuca, brown algae Sargassum pallidum, and seagrass Zostera marina. Conformational changes of HA1 were estimated by differential scanning calorimetry and intrinsic fluorescence. It was shown that the adjuvant activity of TI-complexes depends on the microviscosity of MGDGs, which differently influence the conformation of HA1. The highest production of anti-HA1 antibodies (compared with the control) was induced by HA1 incorporated in a TI-complex based on MGDG from S. pallidum, which provided the relaxation of the spatial structure and, likely, the proper presentation of the antigen to immunocompetent cells

Immunogenicity and Protective Activity of a Chimeric Protein Based on the Domain III of the Tick-Borne Encephalitis Virus E Protein and the OmpF Porin of Yersinia pseudotuberculosis Incorporated into the TI-Complex

Tick-borne encephalitis (TBE) is a widespread, dangerous infection. Unfortunately, all attempts to create safe anti-TBE subunit vaccines are still unsuccessful due to their low immunogenicity. The goal of the present work was to investigate the immunogenicity of a recombinant chimeric protein created by the fusion of the EIII protein, comprising domain III and a stem region of the tick-borne encephalitis virus (TBEV) E protein, and the OmpF porin of Yersinia pseudotuberculosis (OmpF-EIII). Adjuvanted antigen delivery systems, the tubular immunostimulating complexes (TI-complexes) based on the monogalactosyldiacylglycerol from different marine macrophytes, were used to enhance the immunogenicity of OmpF-EIII. Also, the chimeric protein incorporated into the most effective TI-complex was used to study its protective activity. The content of anti-OmpF-EIII antibodies was estimated in mice blood serum by enzyme-linked immunosorbent assay (ELISA). To study protective activity, previously immunized mice were infected with TBEV strain Dal’negorsk (GenBank ID: FJ402886). The animal survival was monitored daily for 21 days. OmpF-EIII incorporated into the TI-complexes induced about a 30–60- and 5–10-fold increase in the production of anti-OmpF-EIII and anti-EIII antibodies, respectively, in comparison with the effect of an individual OmpF-EIII. The most effective vaccine construction provided 60% protection. Despite the dramatic effect on the specific antibody titer, the studied TI-complex did not provide a statistically significant increase in the protection of OmpF-EIII protein. However, our results provide the basis of the future search for approaches to design and optimize the anti-TBEV vaccine based on the OmpF-EIII protein

Recombinant Fusion Protein Joining E Protein Domain III of Tick-Borne Encephalitis Virus and HSP70 of Yersinia pseudotuberculosis as an Antigen for the TI-Complexes

Domain III (DIII) of the tick-borne encephalitis virus (TBEV) protein E contains epitopes, which induce antibodies capable of neutralizing the virus. To enhance the immunogenicity of this protein, which has a low molecular weight, the aim of the present work was to express, isolate, and characterize a chimeric protein based on the fusion of the bacterial chaperone HSP70 of Yersinia pseudotuberculosis and EIII (DIII + stem) as a prospective antigen for an adjuvanted delivery system, the tubular immunostimulating complex (TI-complex). The chimeric construction was obtained using pET-40b(+) vector by ligating the respective genes. The resulting plasmid was transformed into DE3 cells for the heterologous expression of the chimeric protein, which was purified by immobilized metal affinity chromatography (IMAC). ELISA, differential scanning calorimetry, intrinsic fluorescence, and computational analysis were applied for the characterization of the immunogenicity and conformation of the chimeric protein. Mice immunization showed that the chimeric protein induced twice the number of anti-EIII antibodies in comparison with EIII alone. In turn, the incorporation of the HSP70/EIII chimeric protein in the TI-complex resulted in a twofold increase in its immunogenicity. The formation of this vaccine construction was accompanied by significant conformational changes in the chimeric protein. Using HSP70 in the content of the chimeric protein represents an efficient means for presenting the main antigenic domain of the TBEV envelope protein to the immune system, whereas the incorporation of this chimeric protein into the TI-complex further contributes to the development of a stronger immune response against the TBEV infection