Vaccination of mice with hybrid protein containing Exotoxin S and PcrV with adjuvants alum and MPL protects Pseudomonas aeruginosa infections

Abstract

Pseudomonas aeruginosa as a common pathogen causing urinary tract infections (UTIs) has been resistant to different antibiotics and developing an effective vaccine can be an alternative strategy. In the present study, the immunogenicity and protection efficacy of formulations composed of a hybrid protein composed of P. aeruginosa V-antigen (PcrV) and exoenzyme S (ExoS) with alum and MPL were evaluated. The hybrid protein could increase the specific systemic and mucosal immune responses, as well as cellular responses as compared with control groups. Combining of alum or MPL adjuvant with the hybrid protein significantly improved the levels of IgG1, serum IgA, mucosal IgG, and IL-17 as compared to the ExoS.PcrV alone. After bladder challenge with a P. aeruginosa strain, the bacterial loads of bladder and kidneys were significantly decreased in mice received ExoS.PcrV admixed with alum and ExoS.PcrV admixed with MPL than controls. The present study indicated that immunization of mice with a hybrid protein composed of ExoS and PcrV could induce multifactorial immune responses and opsonize the bacteria and decrease the viable bacterial cells. Because P. aeruginosa have caused therapeutic challenges worldwide, our study proposed ExoS.PcrV + alum and ExoS.PcrV + MPL as promising candidates for the prevention of infections caused by P. aeruginosa.

Figure 1

Measurement of serum IgG responses. Mice were vaccinated subcutaneously three times with PcrV alone, hybrid protein alone (protein), hybrid protein admixed with alum (Protein + Alum), hybrid protein admixed with MPL (Protein + MPL). The control groups were given Alum, MPL, and PBS. Blood samples were collected and anti-hybrid protein IgG levels were measured by ELISA. Significant values are shown with P value. Data are shown as mean ± SD of three independent experiments at dilution 1:800.

Figure 2

Analysis of isotypes antibodies responses in serum. The levels of IgG1 and IgG2a isotypes, as well as IgA responses were detected after the last immunization. (A) IgG1 and IgG2a, and (B) IgA responses. The significant levels between different groups are presented with P value. In addition, asterisks show the statstical difference in IgG2a level between the vaccinated groups with the control groups (p < 0.05). The experiments were repeated three times. The results are mean ± S.D. from 10 mice per groups at dilution 1:800.

Figure 3

Detection of mucosal responses in the urine. After the last immunization, (A) mucosal IgA and (B) mucosal IgG levels were detected by ELISA in the urine samples. The significant difference in IgA levels between the immunized mice groups with control mice is shown with single asterisks. Also, the significant differences in IgG levels are shown with P value. The results are the average of three independent experiments. Data were shown as the mean ± SD (16 mice/ group) at dilution 1:2 of the urine.

Figure 4

The opsonic killing activity of anti-ExoS.PcrV against P. aeruginosa strain PAO1. The P. aeruginosa strain PAO1 was incubated with four dilutions of anti-ExoS.PcrV, mice macrophage and rabbit complement. A significant opsonic killing activity was observed when anti-ExoS.PcrV was used as compared to the control group (49% vs. 2.33%). Data are the mean ± S.D. from three independent experiments.

Figure 5

Production of cytokines from the immunized mice. Two weeks after the last administration, splenocytes of mice were collected and cultured with recombinant ExoS.PcrV for 72 h to determine the levels of (A) IFN-γ, (B) IL-4, and (C) IL-17. The significant differences between the immunized groups and control groups are shown with single asterisks. Furthermore, some significant differences are shown with P value. Data are the mean stimulation index ± S.D. of six mice per group from three independent experiments.

Figure 6

Protective efficacy of the vaccine combinations in a challenge model. Mice were immunized with vaccine formulations and challenged with P. aeruginosa strain by transurethral route. Thereafter, the bacterial burdens in the (A) bladder and (B) kidneys of the mice were counted after 2 days. Symbols show the bacterial count of each mice group with median of the colonization number. Statistical significance of the differences between mice groups is shown by brackets with P value.