Expression and testing of Pseudomonas aeruginosa vaccine candidate proteins prepared with the Caulobacter crescentus S-layer protein expression system

• Published in: Vaccine 2001-01, Vol.19 (11), p.1406-1415
• Main Authors: Umelo-Njaka, Elizabeth, Nomellini, John F., Bingle, Wade H., Glasier, Linda G.M., Irvin, Randall T., Smit, John
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Bacterial - biosynthesis; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - immunology; Bacterial Proteins - genetics; Bacterial Proteins - immunology; Bacterial Proteins - isolation & purification; Bacterial Vaccines - genetics; Bacterial Vaccines - isolation & purification; Bacterial Vaccines - pharmacology; Bacteriology; Base Sequence; Biological and medical sciences; Biotechnology; Caulobacter - genetics; Caulobacter crescentus; Caulobactercrescentus; DNA Primers - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Health. Pharmaceutical industry; Industrial applications and implications. Economical aspects; Membrane Glycoproteins; Mice; Mice, Congenic; Mice, Inbred A; Microbiology; Production of active biomolecules; Protein expression; Pseudomonas aeruginosa; Pseudomonas aeruginosa - immunology; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - isolation & purification; RsaA protein; Vaccine; Vaccines, antisera, therapeutical immunoglobulins and monoclonal antibodies; Vaccins
• ISSN: 0264-410X; 1873-2518
• DOI: 10.1016/S0264-410X(00)00362-5

A novel bacterial protein secretion system was used to produce vaccine candidates against Pseudomonas aeruginosa. The surface protein (RsaA) of Caulobacter crescentus was adapted to produce recombinant vaccine proteins based on the pilus tip epitope (‘adhesintope’) of P. aeruginosa. Two versions of the adhesintope, with (PCK) or without (PE) the cysteine residues that flank the epitope were investigated, fused to the C-terminus or inserted into full-length RsaA. When expressed in caulobacter the fusion proteins were secreted as aggregates. Full length RsaA-containing adhesintopes assembled on the cell surface as an S-layer; these were recovered by low pH extraction. Vaccine candidates were evaluated in a mouse challenge model. PCK-containing proteins produced at least 1000-fold higher antibody titers against the adhesintope, compared to the PE version, exceeding titers achievable with any other vaccine preparation method. Immunoglobulin isotyping indicated a balanced IgG1/IgG2 response, though when challenged with P. aeruginosa, the PE- and PCK-containing proteins did not afford mice a significant level of protection. Overall, we describe a new system for vaccine production that shows promise as a fast, economical way to construct, evaluate and produce vaccine proteins.