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

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 reco...

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Autores principales:	A. A. Kaloshin, E. M. Zimina, E. O. Kalinichenko, N. A. Mihailova
Formato:	article
Lenguaje:	RU
Publicado:	Sankt-Peterburg : NIIÈM imeni Pastera 2021
Materias:	pseudomonas aeruginosa outer membrane protein f (oprf) toxoid recombinant protein recombinant vaccine Infectious and parasitic diseases RC109-216
Acceso en línea:	https://doaj.org/article/6c0485af93414a16a637224f05e158d9
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spelling	oai:doaj.org-article:6c0485af93414a16a637224f05e158d92021-11-22T07:09:54ZGeneration and evaluation of specific activity and authenticity of recombinant vaccine designed preventing against Pseudomonas aeruginosa2220-76192313-739810.15789/2220-7619-OES-1369https://doaj.org/article/6c0485af93414a16a637224f05e158d92021-09-01T00:00:00Zhttps://www.iimmun.ru/iimm/article/view/1369https://doaj.org/toc/2220-7619https://doaj.org/toc/2313-7398The 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.A. A. KaloshinE. M. ZiminaE. O. KalinichenkoN. A. MihailovaSankt-Peterburg : NIIÈM imeni Pasteraarticlepseudomonas aeruginosaouter membrane protein f (oprf)toxoidrecombinant proteinrecombinant vaccineInfectious and parasitic diseasesRC109-216RUInfekciâ i Immunitet, Vol 11, Iss 4, Pp 763-770 (2021)
institution	DOAJ
collection	DOAJ
language	RU
topic	pseudomonas aeruginosa outer membrane protein f (oprf) toxoid recombinant protein recombinant vaccine Infectious and parasitic diseases RC109-216
spellingShingle	pseudomonas aeruginosa outer membrane protein f (oprf) toxoid recombinant protein recombinant vaccine Infectious and parasitic diseases RC109-216 A. A. Kaloshin E. M. Zimina E. O. Kalinichenko N. A. Mihailova Generation and evaluation of specific activity and authenticity of recombinant vaccine designed preventing against Pseudomonas aeruginosa
description	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.
format	article
author	A. A. Kaloshin E. M. Zimina E. O. Kalinichenko N. A. Mihailova
author_facet	A. A. Kaloshin E. M. Zimina E. O. Kalinichenko N. A. Mihailova
author_sort	A. A. Kaloshin
title	Generation and evaluation of specific activity and authenticity of recombinant vaccine designed preventing against Pseudomonas aeruginosa
title_short	Generation and evaluation of specific activity and authenticity of recombinant vaccine designed preventing against Pseudomonas aeruginosa
title_full	Generation and evaluation of specific activity and authenticity of recombinant vaccine designed preventing against Pseudomonas aeruginosa
title_fullStr	Generation and evaluation of specific activity and authenticity of recombinant vaccine designed preventing against Pseudomonas aeruginosa
title_full_unstemmed	Generation and evaluation of specific activity and authenticity of recombinant vaccine designed preventing against Pseudomonas aeruginosa
title_sort	generation and evaluation of specific activity and authenticity of recombinant vaccine designed preventing against pseudomonas aeruginosa
publisher	Sankt-Peterburg : NIIÈM imeni Pastera
publishDate	2021
url	https://doaj.org/article/6c0485af93414a16a637224f05e158d9
work_keys_str_mv	AT aakaloshin generationandevaluationofspecificactivityandauthenticityofrecombinantvaccinedesignedpreventingagainstpseudomonasaeruginosa AT emzimina generationandevaluationofspecificactivityandauthenticityofrecombinantvaccinedesignedpreventingagainstpseudomonasaeruginosa AT eokalinichenko generationandevaluationofspecificactivityandauthenticityofrecombinantvaccinedesignedpreventingagainstpseudomonasaeruginosa AT namihailova generationandevaluationofspecificactivityandauthenticityofrecombinantvaccinedesignedpreventingagainstpseudomonasaeruginosa
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Generation and evaluation of specific activity and authenticity of recombinant vaccine designed preventing against Pseudomonas aeruginosa

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