Protective multi-epitope candidate vaccine for urinary tract infection

Abstract

Urinary tract infections (UTIs) are induced by exogenous organisms including extraintestinal pathogenic such as Escherichia coli (ExPEC), Proteus mirabilis and Klebsiella pneumonia, which are closely related. These organisms can colonize in the urinary tract and cause UTIs. In this study, a cross-reactive multi-epitope vaccine was designed by two constructs to stimulate the immune system (CD8+ and CD4 + T cells) against ExPEC, Proteus mirabilis and Klebsiella pneumonia strains. Uropathogenic Escherichia coli (UPEC), Proteus mirabilis and Klebsiella pneumoniae are the main bacterial cause of UTI. They were used for designing experimental candidate vaccine, and their immunogenicity and protectivity were assessed. In this study, conserved antigens from their bacterial genomes were considered, and informatics-based immunological vaccine with cross-protective T and B-cells epitopes was designed and evaluated. The vaccine candidate was used as a broad immune system inducer, and its cross-protective immunity and protectivity were confirmed in in vivo experiments.

Keywords: Escherichia coli (ExPEC); Klebsiella pneumonia; Multi-epitope vaccine; Proteus mirabilis; Urinary tract infections (UTIs).

Fig. 1

a) T helper (Th) construct with flagellin (FljB) and PADRE sequence as carrier. FljB: phase 2 flagellin, strain: Salmonella enterica subsp. Enteric aserovar Typhimurium str. LT2, TLR5 agonist, gi|16766083|ref|NP_461698.1| PADRE: Universal T helper pan DR Epitope Linkers: EAAAK, GPGPG, AAA b) CTL construct with cholera toxin B subunit carrier. Cholera toxin B subunit, strain: Vibrio cholerae O1 biovar El Tor str. N16961, gi|15641467|ref|NP_231099.1| HBHA: heparin binding hemagglutinin, strain: Mycobacterium tuberculosis H37Rv, gi|15607616|ref|NP_214989.1|, TLR4 agonist

Fig. 2

Expression evaluation of the recombinant E. coli cell lysate and purified proteins by SDS-PAGE and western blotting analysis. The recombinant proteins from Th and CTL constructs were purified using the nickel resins and evaluated by Western blot using the His-specific monoclonal antibody. a) Th construct expression (55 KD); lane 1: Negative control, lane 2−7: protein expression b) Western blotting of Th construct protein; lane 1–3 protein expression confirmation c) CTL construct expression (30 KD); lane 1: MW, lane 2–5 protein expression d) Western blotting of CTL construct protein; lane 1: MW, lane 2–5 protein expression confirmation

Fig. 3

Evaluation of IgG responses in mice. Mice were immunized with Th and CTL purified proteins. The control group was injected with PBS. P values were calculated between groups received expressed proteins and a P value below 0.05 were considered as significant. a) Total IgG antibody response in different groups. b) Serum IgG1 and IgG2a antibody responses in different groups. The results are the average of three independent experiments. Bars represent mean ± S.D. from 10 mice per groups at serum dilution1:800.

Fig. 4

Evaluation of IgA responses in mice. Serum IgA antibody response after vaccination in different groups was shown. Mice were immunized with Th and CTL purified proteins. The control group was injected with PBS. P values were calculated between groups received expressed proteins and a P value below 0.05 were considered as significant. The results are the average of three independent experiments. Bars represent mean ± S.D. from 10 mice per groups at serum dilution1:800.

Fig. 5

a) IFN-γ, b) IL-4 and c) IL-17 assessment. The cytokine response was evaluated in the immunized mice. Splenocytes of each mice group were stimulated and cultured in the presence of expressed proteins. The supernatant of splenocytes cells were collected. Then splenocytes secretion was analyzed with ELISA for a) IFN-γ, b) IL-4 and c) IL-17. The control group was injected with PBS. P values were calculated between groups received expressed proteins and a P value below 0.05 were considered as significant. Results are the mean stimulation index ± S.D. of mice per group from three independent experiments.

Fig. 6

Evaluation the efficacy of immune responses against UTI. Immune responses in bladder and kidney infection were evaluated by challenge of the immunized mice by standard strains of UPEC (A, B), Proteus mirabilis (C, D) and Klebsiella pneumonia (E, F). Two weeks after the last vaccine dose, the bladders of mice (n = 6) were infected with standard strains. One week after challenge, the levels of standard strains in the kidneys (A), (C), (E) and bladders (B), (D), (F) of mice were determined. Solid lines indicate median of the colonization levels. Statistical significance of the differences between mice groups were determined by kruskal-wallis analysis (Dunn’s multiple comparison test) and are shown by P value levels. P < 0.05 was considered significant.