Generation and evaluation of specific activity and authenticity of recombinant vaccine designed preventing against Pseudomonas aeruginosa

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Abstract

The aim of the study was to obtain and characterize a recombinant vaccine for the immunoprophylaxis of infections caused by Pseudomonas aeruginosa. A set of two highly immunogenic P. aeruginosa recombinant proteins was used to create related vaccine. The first vaccine component was presented by recombinant outer membrane protein F (OprF), whereas the second one — by recombinant atoxic exotoxin A (toxoid). These antigens allowed to develop vaccine inducing immune response against the surface bacterial cues and promote production of neutralizing antibodies against exotoxin A, one of the most dangerous P. aeruginosa pathogenicity factors. Recombinant proteins were synthesized in Escherichia coli cells and isolated by two-step purification. In case of recombinant OprF protein, it was initially (stage 1) isolated as a precipitate containing hydrophobic fraction of producer cell-derived proteins, whereas for recombinant toxoid we purified inclusion bodies. At stage 2, a Ni Sepharose column affinity chromatography was performed. Next, purified recombinant proteins were dialyzed against buffer solution containing 50 mМ Tris-HCl (pH 9.0) and 0.01% Tween 20 followed by filtration sterilization. Three lots of the Pseudomonas Recombinant Vaccine (PRV) were obtained for the study, wherein the recombinant antigens were absorbed with aluminum hydroxide. Recombinant OprF and toxoid protein in vaccine formula were used at a dose of 25 μg and 50 μg, respectively. Absorption completeness of the recombinant antigens within the vaccine was evaluated by polyacrylamide gel electrophoresis with PRV preparations concentrated by ultrafiltration in spin columns. Authenticity of recombinant vaccine was assessed by using customized method by desorbing antigens followed by ultrafiltration concentration in spin columns. Final concentrated desorbed vaccine preparations were analyzed by polyacrylamide gel electrophoresis and immunoblotting, which allowed to confirm presence of specific recombinant antigens in the vaccine. The experimental PRV series demonstrated specific activity (protective properties) after inoculation in animals (mice) by using a dual vaccination protocol, wherein mice were immunized intraperitonially twice with a two-week interval. Next, two weeks later mice were infected by toxigenic P. aeruginosa strain PA-103 culture cells. The index of protective efficiency (IE) for experimental vaccine series was at least a value of three (IE: 3.0–3.3) that was by 1.5-fold higher than that for using single vaccine components (IE: 2.0–2.3). Thus, we confirmed an additive effect of using a set preparation to protect against infection caused by toxigenic Pseudomonas aeruginosa strains.

About the authors

A. A. Kaloshin

Mechnikov Research Institute of Vaccines and Sera

Author for correspondence.
Email: alex-k-1973@yandex.ru
ORCID iD: 0000-0001-8679-2421

Alexei A. Kaloshin,  PhD (Biology), Leading Researcher, Laboratory of Protective Antigens 

105064, Moscow, Malyi Kazennyi lane, 5А

Phone: +7 (965) 136-51-45 (mobile), 8 (495) 916-25-87 (office)

Fax: +7 (495) 917-54-60 

Russian Federation

E. M. Zimina

Mechnikov Research Institute of Vaccines and Sera

Email: ziminka@ya.ru
ORCID iD: 0000-0003-2167-8905

Junior Researcher, Laboratory of Protective Antigens

Moscow 

 

Russian Federation

E. O. Kalinichenko

Mechnikov Research Institute of Vaccines and Sera

Email: gladius.domini@gmail.com
ORCID iD: 0000-0002-0048-3968

PhD Student, Junior Researcher, Laboratory of Mechanisms of Immunity Regulation

Moscow 

Russian Federation

N. A. Mihailova

Mechnikov Research Institute of Vaccines and Sera

Email: n_michailova@inbox.ru
ORCID iD: 0000-0002-6652-2093

PhD, MD (Medicine), Head of the Laboratory of Protective Antigens

Moscow 

Russian Federation

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