The Polymorphic Membrane Protein G Has a Neutral Effect and the Plasmid Glycoprotein 3 an Antagonistic Effect on the Ability of the Major Outer Membrane Protein to Elicit Protective Immune Responses against a Chlamydia muridarum Respiratory Challenge

Abstract

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen. The number of chlamydial infections continuous to increase and there is an urgent need for a safe and efficacious vaccine. To assess the ability of the Chlamydia muridarum polymorphic membrane protein G (PmpG) and the plasmid glycoprotein 3 (Pgp3) as single antigens, and in combination with the major outer-membrane protein (MOMP) to induce protection, BALB/c mice were immunized utilizing CpG-1826 and Montanide ISA 720 VG as adjuvants. Following vaccination with MOMP, significant humoral and cell-mediated immune responses were observed, while immunization with PmpG, or Pgp3, elicited weaker immune responses. Weaker immune responses were induced with MOMP+Pgp3 compared with MOMP alone. Following the intranasal challenge with C. muridarum, mice vaccinated with MOMP showed robust protection against body-weight loss, inflammatory responses in the lungs and number of Chlamydia recovered from the lungs. PmpG and Pgp3 elicited weaker protective responses. Mice immunized with MOMP+PmpG, were no better protected than animals vaccinated with MOMP only, while Pgp3 antagonized the protection elicited by MOMP. In conclusion, PmpG and Pgp3 elicited limited protective immune responses in mice against a respiratory challenge with C. muridarum and failed to enhance the protection induced by MOMP alone. The virulence of Pgp3 may result from its antagonistic effect on the immune protection induced by MOMP.

Keywords: Chlamydia muridarum; adjuvants; major outer membrane protein; mouse; plasmid glycoprotein 3; polymorphic membrane protein G; vaccine.

Figure 1

Characterization of the recombinant C. muridarum proteins used as antigens. (A): Silver-stained 10% SDS-PAGE of the C. muridarum recombinant proteins used as antigens. Lane 1: MOMP; Lane 2: PmpG (passenger domain); Lane 3: Pgp3 and Lane 4: MW protein standards. (B): Silver-stained 10% SDS-PAGE of the C. muridarum heated and non-heated Pgp3. Lane 1: Pgp3 was denatured by heat (10 min at 100 °C) before loading. Lane 2: Pgp3 was loaded on the gel without boiling. The gel was run overnight at 4 °C. (C): Detection by Western blot of MOMP, PmpG and Pgp3 in C. muridarum EB using serum from vaccinated mice. C. muridarum EBs were probed with sera from mice immunized with: Lane 1: PmpG; Lane 2: MOMP+PmpG; Lane 3: Pgp3; Lane 4: MOMP+Pgp3; Lane 5: MOMP; Lane 6: PBS, and Lane 7: MW protein standards.

Figure 2

Vaccine-induced serum IgG1 and IgG2a antibody responses (GMT±SE) against C. muridarum EB the day before the i.n. challenge. Serum samples from immunized mice were collected the day before the i.n. challenge with C. muridarum and the titers of IgG2a and IgG1 determined to evaluate the Th1 versus Th2 humoral immune responses using EB as the antigen in an ELISA. BLD < 100. * p < 0.05 by the Mann–Whitney’s U-test. ** p < 0.1 by the Mann–Whitney’s U-test.

Figure 3

Vaccine-induced serum IgG antibody responses (GMT±SE) against MOMP, PmpG, or Pgp3 the day before the C. muridarum i.n. challenge. Blood was collected the day before the i.n. challenge and probed with MOMP, PmpG or Pgp3, individually or in combination, using an ELISA. BLD < 100. * p < 0.05 by the Mann–Whitney’s U-test.

Figure 4

Detection of serum antibodies specific for C. muridarum MOMP peptides (OD₄₀₅ ± SD). Serum samples from immunized mice were collected the day before the i.n. challenge and their reactivity to 25-mer overlapping peptides corresponding to the whole amino acid sequence of mature C. muridarum MOMP were analyzed by ELISA. MOMP and C. muridarum EB were used as positive controls.

Figure 5

Vaccine elicited levels of neutralizing antibody (GMT±SE) in sera the day before the C. muridarum i. n. challenge. Serum samples were incubated with C. muridarum live EBs and the neutralization titer was determined by the dilution of the sera that decreased the number of C. muridarum IFU by 50% compared with the PBS immunized group. BLD (horizontal broken line) < 50.

Figure 6

C. muridarum-specific IgA and IgG (GMT) in vaginal washes from vaccinated mice the day before the i.n. challenge. Vaginal washes were collected and pooled from each group of mice and the C. muridarum specific IgA and IgG titer determined using EB as the antigen in an ELISA. BLD (horizontal broken line) < 10.

Figure 7

Evaluation of the cytokine responses from T-cells isolated from the spleens of vaccinated mice the day before the i.n. challenge. Spleens from four mice from each immunized group were collected the day before the i.n. challenge, T-cells separated with nylon wool and stimulated with C. muridarum EB, and ConA as a positive control. Levels of IFN-γ (pg/mL ± SE) and IL-4 (pg/mL ± SE) were determined as indicators of Th1 and Th2 responses, respectively. * p < 0.05 by the Mann–Whitney’s U-test. ** p < 0.1 by the Mann–Whitney’s U-test. BLD (horizontal broken line) IFN-γ < 15 pg/mL; IL-4 < 4 pg/mL.

Figure 8

Body weight loss following the i.n. challenge with C. muridarum. Mice were immunized with several vaccine formulations and challenged i.n. with 10⁴ C. muridarum IFU at four weeks after the last immunization. Daily percentage changes in mean body weight following the i.n. challenge. ^a p < 0.05 by the repeated-measures ANOVA compared with the PBS immunized group. ^b p < 0.05 by the repeated-measures ANOVA compared with the MOMP immunized group. ^c p < 0.05 by the repeated-measures ANOVA compared with the MOMP+PmpG immunized group. ^d p < 0.05 by the repeated-measures ANOVA compared with the MOMP+Pgp3 immunized group. ^e p < 0.05 by the repeated-measures ANOVA compared with the PmpG immunized group.

Figure 9

Systemic and local disease burden and local immune responses in the lungs in vaccinated mice following the i.n. challenge with C. muridarum. (A) Percentage change in mean body weight at D10 following the i.n. challenge. The mean is shown as a horizontal line. Each symbol represents a single animal. ^a p < 0.05 by the Student’s t-test compared with the PBS immunized group. ^b p < 0.05 by the Student’s t-test compared with the MOMP immunized group. ^c p < 0.1 by the Student’s t-test compared with the MOMP immunized group. ^d p < 0.05 by the Student’s t-test compared with the MOMP+PmpG immunized group. ^e p < 0.05 by the Student’s t-test compared with the MOMP+Pgp3 immunized group. (B) Lungs weight (g) at D10 after the i.n. challenge. The mean is shown as a horizontal line. Each symbol represents a single animal. ^a p < 0.05 by the Student’s t-test compared with the PBS immunized group. ^b p < 0.05 by the Student’s t-test compared with the MOMP immunized group. ^d p < 0.05 by the Student’s t-test compared with the MOMP+PmpG immunized group. (C) Number of C. muridarum IFU recovered from the lungs at D10 after the i.n. challenge. The median is shown as a horizontal line. Each symbol represents a single animal. ^f p < 0.05 by the Mann–Whitney’s U-test compared with the PBS immunized group. ^g p < 0.05 by the Mann–Whitney’s U-test compared with the MOMP immunized group. ^h p < 0.05 by the Mann–Whitney’s U-test compared with the MOMP+PmpG immunized group. ⁱ p < 0.05 by the Mann–Whitney’s U-test compared with the MOMP+Pgp3 immunized group. BLD (horizontal broken line) < 50 C. muridarum IFU. (D) Levels of IFN-γ (pg/mL) detected in the lungs at D10 after the i.n. challenge. The mean is shown as a horizontal line. Each symbol represents a single animal. ^a p < 0.05 by the Student’s t-test compared with the PBS immunized group. ^b p < 0.05 by the Student’s t-test compared with the MOMP immunized group. ^d p < 0.05 by the Student’s t-test compared with the MOMP+PmpG immunized group. BLD (horizontal broken line) < 15 pg/mL.