Chloroform-Methanol Residue of Coxiella burnetii Markedly Potentiated the Specific Immunoprotection Elicited by a Recombinant Protein Fragment rOmpB-4 Derived from Outer Membrane Protein B of Rickettsia rickettsii in C3H/HeN Mice

Abstract

The obligate intracellular bacteria, Rickettsia rickettsii and Coxiella burnetii, are the potential agents of bio-warfare/bio-terrorism. Here C3H/HeN mice were immunized with a recombinant protein fragment rOmp-4 derived from outer membrane protein B, a major protective antigen of R. rickettsii, combined with chloroform-methanol residue (CMR) extracted from phase I C. burnetii organisms, a safer Q fever vaccine. These immunized mice had significantly higher levels of IgG1 and IgG2a to rOmpB-4 and interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), two crucial cytokines in resisting intracellular bacterial infection, as well as significantly lower rickettsial loads and slighter pathological lesions in organs after challenge with R. rickettsii, compared with mice immunized with rOmpB-4 or CMR alone. Additionally, after challenge with C. burnetii, the coxiella loads in the organs of these mice were significantly lower than those of mice immunized with rOmpB-4 alone. Our results prove that CMR could markedly potentiate enhance the rOmpB-4-specific immunoprotection by promoting specific and non-specific immunoresponses and the immunization with the protective antigen of R. rickettsii combined with CMR of C. burnetii could confer effective protection against infection of R. rickettsii or C. burnetii.

Fig 1. Diagram of preparing recombinant OmpB fragments (rOmpBs).

The full-length sequence of ompB was divided into 5 fragments (named as ompB-1 to -5) according to hydrophilicity, antigenic index, and surface probability (A). Five recombinant OmpB fragments (named as rOmpB-1 to -5) purified from E. coli cell lysate were separated by 10% SDS-PAGE and stained by G-250 Coomassie Brilliant Blue (B) and immunoblotted with sera from mice infected with R. rickettsii (C): lane M, protein molecular mass markers; lanes 1 to 5, rOmpB-1 to -5 (C).

Fig 2. Evaluation of immunoprotective efficacy of rOmpBs.

C3H/HeN mice were immunized with each of the rOmpBs, WCA, or PBS, followed by a sublethal challenge with R. rickettsii. Five days after challenge, the mice were sacrificed and the rickettsial load in their livers (A), spleens (B), or lungs (C) was determined using R. rickettsii-specific qPCR. The rickettsial load is expressed as the ratio of R. rickettsii ompB to murine actin gene copies (S2 Table), and the results were analyzed by variance (ANOVA) procedure or Kruskal-Wallis test (NPAR1WAY Procedure) according to their normality and homogeneity of variances, followed by between-group comparison with Student-Newman-Keuls Test. Data were presented as mean + SEM. (n = 5). Means with different letters are significantly different (P< 0.05).

Fig 3. Comparison of immunoprotective efficacy between combination group and individual group.

C3H/HeN mice were immunized with rOmpB-4 combined with C. burnetii CMR (O+CMR-C), C. burnetii CMR alone (CMR-C), or rOmpB-4 alone (rOmpB-4). Day 14 after the last immunization, mice were challenged with R. rickettsii, and mice were sacrificed and their livers (A), spleens (B) and lungs (C) were collected day 5 post challenge. In R. rickettsii-specific qPCR assay, the data was expressed as the ratio of R. rickettsii ompB to murine actin gene copies (S2 Table). The spleen weight of mice in each group was also been determined (D). The significant difference between two groups was compared with the Student’s t-test or Wilcoxon two-sample test according to their normality and homogeneity of variance. All data were presented as mean + SEM. (n = 5). P<0.05 was considered significantly different. *, P<0.05; **, P<0.01; ns, no significance.

Fig 4. Comparison of protective efficacy between rOmpB-4 combined with C. burnetii CMR and rOmpB-4 alone.

C3H/HeN mice were immunized with rOmpB-4 combined with C. burnetii CMR (O+CMR-C) or PBS (O+PBS). Day 14 after the last immunization, mice were challenged with C. burnetii, and mice were sacrificed and their livers (A), spleens (B) and lungs (C) were collected day 5 post challenge. In C. burnetii-specific qPCR assay, the data was expressed as the ratio of C. burnetii 23S rRNA to murine actin gene copies (S2 Table). The spleen weight of mice in each group was also been determined (D). The significant difference between two groups was compared with the Student’s t-test or Wilcoxon two-sample test according to their normality and homogeneity of variance. Data were presented as mean + SEM. (n = 5). P<0.05 was considered significantly different. *, P<0.05.

Fig 5. Pathological lesions after R. rickettsii or C. burnetii challenge.

Liver, spleen, and lung tissues were collected from mice infected with R. rickettsii or C. burnetii for pathological examination (A, original magnifications 400, bar = 200μm), respectively. The focal zone of inflammatory infiltrates in livers (B), the number of macrophage number in spleens (C), and the mean thickness of alveolar wall in lungs (D) of mice were observed. The lesions in liver, spleen, or lung were quantified (n = 10 lesion high powered fields), and the differences between the groups were compared using Student’s t-test or Wilcoxon two-sample test according to their normality and homogeneity of variance. *, P<0.05; ***, P<0.001; ns, no significance.

Fig 6. Specific antibodies determined by ELISA.

Sera samples were collected from mice immunized with rOmpB-4 combined with C. burnetii CMR (O+CMR-C), C. burnetii CMR alone (CMR-C), or rOmpB-4 alone (rOmpB-4) on days 7, 14, 21, and 28 days after first immunization, respectively. IgG (A), IgG1 (B), or IgG2a (C) to rOmpB-4 in sera was determined by ELISA and the ratio of IgG2a/IgG1 in each serum sample was also compared (D). The statistically significant differences of OD₄₅₀ on 28 days after first immunization among groups were analyzed using the Student’s t-test or Wilcoxon two-sample test according to their normality and homogeneity of variance. Results were expressed as mean ± SD (n = 3). P<0.05 was considered significantly different. ***, P<0.001.

Fig 7. Specific antibodies determined by IFA.

Serum samples were collected from mice immunized with rOmpB-4 combined C. burnetii CMR on days 7, 14, 21, and 28 after first immunization, respectively. Anti-C. burnetii phase I/II IgG titers was evaluated by IFA.

Fig 8. Neutralization of R. rickettsii by sera.

Viable R. rickettsii were incubated with sera from mice immunized with rOmpB-4 combined with C. burnetii CMR (O+CMR-C), rOmpB-4 alone (rOmpB-4), or C. burnetii CMR alone (CMR-C). Sixty minutes later, the mixture of rickettsiae and each serum sample was added into EA.hy 926 cells for a 4-hour incubation. After which the number of R. rickettsii in host cells was determined by R. rickettsii-specific qPCR. Values are presented as the mean with standard deviations. The statistically significant differences among groups were analyzed using the Student’s t-test or Wilcoxon Two-Sample Test based on their normality and equality of variances and are indicated as follows: *, P<0.05; ***, P<0.001; ns, no significance. All data were presented as mean + SD (n = 3).

Fig 9. Detection of TNF-α and IFN-γ in sera.

The pooled sera collected from mice immunized with rOmpB-4 combined with C. burnetii CMR (O+CMR-C), C. burnetii CMR alone (CMR-C), or rOmpB-4 alone (rOmpB-4) on days 7, 14, and 21 after first immunization, respectively. TNF-α (A) or IFN-γ (B) in the sera was detected with a Multiplex MouseTh1/Th2 cytokine kit. The statistically significant differences in TNF-α or IFN-γ levels among groups on 14 days after primary immunization were analyzed using the Student’s t-test or Wilcoxon two-sample test according to their normality and homogeneity of variance. Results were expressed as mean ± SD (n = 3). P<0.05 was considered significantly different. *, P<0.05; ***, P<0.001.