The influence of the ratio of protein and aluminum hydroxide on the manifestation of the protective properties of the recombinant fusion protein OprF-aTox-OprI Pseudomonas aeruginosa and the expression of genes of molecules of innate immunity

• Ekaterina M. Zimina
• Aleksey A. Kaloshin
• Natal’ya A. Mikhailova
• Ekaterina D. Merkushova
• Lyudmila V. Gankovskaya

Abstract

Introduction. The main indicator of the quality of a preparation in the creation vaccines based on recombinant proteins is specific activity or immunogenicity. The severity of the protective effect is influenced by the content of antigen and adjuvant in the preparation. An important step in the development of production technology is the selection of the optimal ratio of components. The effect of the ratio of protein and aluminum hydroxide is assessed by the protective properties and by the level of expression of genes for innate immunity sensors and antimicrobial peptide.

Aim of the study - to investigate the effect of the ratio of protein and aluminum hydroxide on the manifestation of protective properties and the level of activity of innate immunity, assessed by the expression of genes for sensory receptors TLR4, NLRC4 and antimicrobial peptide BD2.

Material and methods. The evaluation of immunogenicity was carried out on a mouse model with two-fold immunization with a two-week interval and subsequent infection with a live virulent P. aeruginosa culture. Gene expression was assessed by real-time PCR.

Results. The assessment of the protective properties of the recombinant fusion protein OprF-aTox-OprI in ratios with the sorbent 1 : 1, 1 : 2 and 1 : 3 was carried out, the effect of immunization on the expression of Defb2, Tlr4, Nlrc4, Asc and Caspl genes by murine macrophages was revealed. During the research the protective properties of a recombinant protein with different protein-sorbent ratios (1 : 1, 1 : 2, and 1 : 3), the maximum protective effect was observed when mice were immunized with a preparation with a 1 : 1 ratio. The injecting of the vaccine affected the expression of proteins TLR4, BD2, NLRC4, ASC and CASP1 by macrophages and differed in the severity of the response of the innate immune system depending on the formulation of the preparation used.

Conclusion. As a result of the work performed, the optimal ratio of the fusion recombinant protein OprF-aTox-OprI with the sorbent was determined, corresponding to a value of 1 : 1 and confirmed by the results of immunological studies.

Keywords:Pseudomonas aeruginosa; fusion recombinant protein OprF-aTox-OprI; TLR4; defensins; inflammasome

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