Pseudomonas aeruginosa PAO-1 Lipopolysaccharide-Diphtheria Toxoid Conjugate Vaccine: Preparation, Characterization and Immunogenicity

Abstract

Background: Treatment of Pseudomonas aeruginosa PAO-1 infections through immunological means has been proved to be efficient and protective.

Objectives: The purpose of this study was to produce a conjugate vaccine composed of detoxified lipopolysaccharide (D-LPS) P. aeruginosa and diphtheria toxoid (DT).

Materials and methods: Firstly, LPS was purified and characterized from P. aeruginosa PAO1 and then detoxified. D-LPS was covalently coupled to DT as a carrier protein via amidation method with adipic acid dihydrazide (ADH) as a spacer molecule and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAC) as a linker. The molar ratio of LPS to DT in the prepared conjugate was 3:1. The immunogenicity of D-LPS-DT conjugate vaccine in mice model was evaluated as well.

Results: The conjugate was devoid of endotoxin activity and 0.125 U/mL of D-LPS was acceptable for immunization. D-LPS-DT conjugate was nonpyrogenic for rabbits and nontoxic for mice. Mice immunization with D-LPS-DT conjugate vaccine elicited the fourfold higher IgG antibody compared to D-LPS. Anti-LPS IgG antibody was predominantly IgG1 subclass and then IgG3, IgG2a and IgG2b, respectively.

Conclusions: Vaccine based on the conjugation of P. aeruginosa PAO-1 LPS with DT increased anti-LPS antibodies and had a significant potential to protect against Pseudomonas infections.

Keywords: Diphtheria Toxoid; Lipopolysaccharides; Pseudomonas aeruginosa PAO-1; Vaccines, Conjugate.

Figure 1.. Silver Nitrate-Stained SDS-PAGE of Extracted Pseudomonas aeruginosa LPS

Lane 1: 10 μg of LPS, lane 2: 5 μg of LPS.

Figure 2.. Gel Filtration Profile of D-LPS-DT Conjugate Through a CL-2B Sepharose Column

Figure 3.. Induction of Anti-LPS Antibodies in BALB/C Mice for Days 28 (A) and 42 (B)