Evaluation of Alginate-Exotoxin A Conjugated Vaccine Immunogenicity Against Pseudomonas aeruginosa Infection in Mouse Model

Background and Objective: Pseudomonas aeruginosa is an opportunistic pathogen, and alginate is its most important factor in pathogenicity. The objective of the study is the immunogenicity evaluation of P. Pseudomonas aeruginosa alginate conjugated to exotoxin A as a vaccine candidate in mice. Materials and Methods: The mucoid strain of Pseudomonas aeruginosa 6494 was used to prepare alginate, and the separation of exotoxin A was done with the standard strain of PAO1. Alginate was extracted by means of sedimentation with cold ethanol, dialysis, enzymatic digestion and chromatography. To improve immunogenicity, purified antigen was coupled to exotoxin A with ADH as a spacer and EDAC as a linker. Based on confirmation tests, the resulting conjugate was devoid of specific toxicity and pyrogenic effect. Four groups of BALB/c female mice (each group was included 15 mice) were selected in the next step. The first group with the ALG, the second group with the D-ALG-ETA, and the third one with the ETA were vaccinated. The fourth group (control group) was vaccinated with normal saline. Vaccination was performed in three injectable doses with two-week intervals. Subsequently, serum samples were collected, and antibody responses were measured by the ELISA method for total IgG, IgG1, IgG2a, IgG2b, IgG3. Results: After the second and third doses with ALG-ETA showed a significant increase in antibody titer against ALG-ETA in comparison with pure ALG. The titers of IgG2a, IgG2b, IgG, IgG1, IgG3 antibodies that produced against alginate increased in the third injection compared to the first conjugate injection, which was 2.9, 3, 5.2, 10, and 9.2, respectively. Conclusion: These results show that ALG from Pseudomonas aeruginosa increases anti-alginate antibodies in conjugate form with exotoxin A and can be considered an appropriate, effective adjuvant.