Speaker at Global conference on Pharmaceutics and Drug Delivery Systems 2019 - Geraldine Mayeux
University Grenoble Alpes, France
Title : Cell-free expression of a membrane antigen for vaccine uses

Abstract:

Pseudomonas aeruginosa is an opportunistic bacterium and is the second-leading cause of nosocomial infections and pneumonia in hospitals, which are mainly acquired in intensive care units. The pathogen is also responsible for the chronic colonization of the respiratory tract that is a major cause of morbidity and mortality in patients with cystic fibrosis. 
     Different vaccine strategies such as multivalent DNA vaccines, recombinant fusion proteins, viral vector expressing bacterial proteins or bioengineered bacterial pathogens have been proposed to induce a protection against P. aeruginosa infections. However, even though some of these vaccines are still in clinical phases, most of them failed to achieve some specific primary goals and are no longer in trials. 
        Here, we overexpressed a porin, a well conserved membrane antigen from clinical isolates, in a cell-free protein synthesis system in the presence of liposomes. We used different biochemical and biophysical approaches to demonstrate that this membrane protein is mainly inserted in a correct orientation into the lipid bilayer and harbors a porin activity and native conformation. This expression system produced monomer and oligomer of the protein resulting in the formation of pores of large size and containing a large proportion of the porin into an open channel conformer. Using these recombinant proteoliposomes, we demonstrated that immunization of mice resulted in total protection of these mice against a lethal dose of P. aeruginosa. Administration of sera from immunized animals into infected mice was also capable of fully protecting them against lethal infection. These results demonstrate that by using the bacterial cell-free expression system, a production of a fully functional recombinant vaccine against P. aeruginosa is feasible.
Audience take away:
• Cell-free expression systems represent an attractive alternative to the classical overexpression systems for producing membrane proteins. Limitations in the production of folded membrane proteins for functional and structural studies can be easily overcome by such approaches.
• The production of a bacterial membrane antigen in a cell-free system in the presence of liposomes results in the insertion into the liposome of a fully active protein harboring conformational epitopes which are not present in the composition of the other recombinant vaccines developed so far.
• Immunizations of mice with this recombinant proteoliposome result in a total protection of these mice against a lethal dose of P. aeruginosa.
• The vaccine response after administration of the proteoliposome is higher than vaccine platforms developed by other groups or in clinical trials.
• Producing recombinant proteoliposomes in a cell-free system for vaccine use is feasible and easy.

Biography:

Pseudomonas aeruginosa is an opportunistic bacterium and is the second-leading cause of nosocomial infections and pneumonia in hospitals, which are mainly acquired in intensive care units. The pathogen is also responsible for the chronic colonization of the respiratory tract that is a major cause of morbidity and mortality in patients with cystic fibrosis. 
     Different vaccine strategies such as multivalent DNA vaccines, recombinant fusion proteins, viral vector expressing bacterial proteins or bioengineered bacterial pathogens have been proposed to induce a protection against P. aeruginosa infections. However, even though some of these vaccines are still in clinical phases, most of them failed to achieve some specific primary goals and are no longer in trials. 
        Here, we overexpressed a porin, a well conserved membrane antigen from clinical isolates, in a cell-free protein synthesis system in the presence of liposomes. We used different biochemical and biophysical approaches to demonstrate that this membrane protein is mainly inserted in a correct orientation into the lipid bilayer and harbors a porin activity and native conformation. This expression system produced monomer and oligomer of the protein resulting in the formation of pores of large size and containing a large proportion of the porin into an open channel conformer. Using these recombinant proteoliposomes, we demonstrated that immunization of mice resulted in total protection of these mice against a lethal dose of P. aeruginosa. Administration of sera from immunized animals into infected mice was also capable of fully protecting them against lethal infection. These results demonstrate that by using the bacterial cell-free expression system, a production of a fully functional recombinant vaccine against P. aeruginosa is feasible.

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