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

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.


Introduction
Rocky Mountain spotted fever (RMSF) is a serious and potentially life-threatening infectious disease, which is caused by Rickettsia rickettsii, an obligate intracellular Gram-negative bacterium naturally transmitted by tick bites [1]. Initial signs and symptoms of RMSF include sudden potential mechanism of the efficient immunoprotections conferred by the combination of rOmpB-4 and CMR was also investigated.

Bacterial strains
Rickettsia rickettsii (Sheila Smith strain) were cultured in Vero cells and isolated by isopycnic density gradient centrifugation as per conventional methods [29]. The number of R. rickettsii or viable rickettsial organisms in suspension was detected by quantitative polymerase chain reaction (qPCR) specific for R. rickettsii [30] or plaque assay [31]. Coxiella burnetii (Xinqiao strain, phase I) was grown in the acidified citrate cysteine medium (ACCM) as described previously [18]. The purified C. burnetii organisms were inactivated with formalin and extracted 2 times with chloroform-methanol (4:1) to obtain CMR fraction according to the procedures described previously [23]. The purified organisms were inactivated with formalin as whole cell antigens (WCA).

Mice
Male C3H/HeN mice at 6-7 weeks old were purchased from Vital River Laboratories (Beijing, China). All animal experiments were carried out according to the guidelines of authors' institution. The protocol was approved by the Institute of Animal Care and Use Committee (IACUC No: AMMS-2014-020) at Academy of Military Medical Sciences (AMMS) and all efforts were made to minimize mice suffering.
doi:10.1371/journal.pone.0124664.g001 cognate primer pairs (S1 Table), respectively. Each of 5 ompB fragments was inserted into pET32a (+) plasmid (Novagen, Madison, WI) to construct a recombinant plasmid that was used to transform Escherichia coli BL21 cells (Novagen, Madison, WI) according to conventional procedures [32]. The expressed recombinant OmpB fragments (rOmpB-1 to 5, rOmpBs) were respectively purified from the cognate gene-transformed E. coli cells using Ni-NTA affinity resin (Qiagen GmbH, Hilden, Germany) as per the manufacture's instruction and the purified rOmpBs were subjected to 10% SDS-PAGE and immunoblotted with sera from mice infected with R. rickettsii following the methods described previously [33]. The endotoxin of the purified recombinant proteins were removed with Toxin Eraser (GenScript, Piscataway, NJ) [34].
Evaluation of protective efficacy of rOmpBs C3H/HeN mice (n = 5) were immunized with the 5 rOmpBs, respectively. Briefly, each mouse was injected subcutaneously (i.s.) with 30 μg of each rOmpB in 200 μl PBS mixed with complete Freund's adjuvant (CFA, Sigma-Aldrich, MO). Then, 20 μg of cognate rOmpB in 200 μl PBS mixed with incomplete FA (IFA, Sigma-Aldrich, MO) were injected intraperitoneally (i. p.) on day 28 and 42 after primary immunization. In parallel, WCA of R. rickettsii and PBS alone were used to immune mice at same doses and procedures described above as positive and negative controls, respectively. Fourteen days after last immunization, each mouse was challenged i.p. with a sublethal dose of viable R. rickettsii (6 × 10 6 PFU). On day 5 after the challenge, each mouse was sacrificed to determine rickettsial loads in spleen, liver, and lung by qPCR described previously [35].
In addition, other 2 groups of mice (n = 5) were immunized and boosted with rOmpB-4 mixed with CMR (rOmpB-4-CMR group) and mixed with PBS (rOmpB-4 group) at the same doses and procedures described above, respectively. Fourteen days after final immunization, each mouse was challenged i.p. with sublethal dose of C. burnetii (1 × 10 7 PFU). Five days later, mice were sacrificed and their livers, spleens, and lungs were harvested for determination of C. burnetii by qPCR [36] Additionally, the spleen weight of mouse in R. rickettsii or C. burnetii infection groups was also determined.

Histopathological analysis
A part of spleen, liver, or lung from each sacrificed mouse per group was collected for histopathological examination. The tissue samples were fixed in 4% (vol/vol) formaldehyde overnight, embedded in paraffin, sectioned at 5-μm thickness, and stained by hematoxylin and eosin for evaluation of histopathology under an Olympus DP71 microscope.

Determination of specific antibodies in mouse sera
Blood samples were collected from the tail veins of mice per immunized group and pooled together on day 7, 14, 21, and 28 after primary immunization, respectively. Anti-rOmpB-4 IgGs were detected by enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well plate (Nunc, Shanghai, China) was coated with 1.5 μg/ml rOmpB-4 overnight and incubated with mouse sera at the dilution of 1:1000. Then, the IgG, IgG1, or IgG2a to rOmpB-4 was determined with goat anti-mouse IgG, IgG1, or IgG2a HRP-conjugated antibodies (1:5000) and a TMB substrate kit (eBioscience, San Diego, CA) according to previous methods [32]. Absorbance at 450nm was analysed with a UVM 340 microplate reader (Asys Hitech GmbH, Eugendorf, Austria). Anti-C. burnetii phase I/II IgGs were detected by indirect immunofluorescence assay (IFA) as per methods described previously [37]. The phase I or II C. burnetii-coated slide was incubated with sera from mice immunized with rOmpB-4 mixed with CMR at two-fold dilution (initial at the dilution of 1:100) in PBS for 45 min at 37°C. After three washes with PBS, the C. burnetii cells on the slides were incubated with a 1:100 dilution of FITC-conjugated goat anti-mouse IgGs (eBioscience, San Diego, CA) for 45 min at 37°C. After another three washes, the coxiella cells on the slides were observed under a fluorescence microscope (Olympus BX60).

Serum neutralization assay of R. rickettsii
The human endothelial hybrid cell line (EA.hy 926, ATCC), the host cells of R. rickettsii, were cultured in DMEM containing 15% heat-inactivated FBS. The pooled sera collected from rOmpB-4-CMR group, rOmpB-4 group, or CMR group mice on day 28 after primary immunization were inactivated at 56°C for 30 min and filter sterilized [38]. And then 150 μl of each serum sample was mixed with R. rickettsii cells in 150 μl of DMEM (3 ×10 7 PFU/ml) at room temperature for 60 min. After which the serum-rickettsial mixture was added to 3 × 10 5 host cells in 2.7 ml of DMEM containing 1% heat-inactivated FBS. This mixture was divided into 3 replicate wells in a 24-well plate (Corning, Corning, NY) and cultured at 37°C for 4 h [38]. After 3 times washing, the remaining cells in each well were collected for DNA extraction with DNeasy Blood & Tissue Kit (Qiagen GmbH, Hilden, Germany). The DNA samples were evaluated by qPCR with primers specific for R. rickettsii [35].

Cytokine determination
The blood samples were collected from the tail vein of rOmpB-4-CMR group, rOmpB-4 group, or CMR group mice and pooled together to obtain a serum sample on days 7, 14, or 21 after primary immunization. And IFN-γ and TNF-α in the serum sample were determined using a Luminex Bio-Plex 200 IS 100 instrument (BIO-RAD, Hercules, CA) with multiplex kits and related reagents produced by Affymetrix (Santa Clara, CA).

Statistical analysis
All statistics were computed using SAS statistical software (version 9.1, SAS Institute Inc., Cary, NC). The statistical significances of the differences in protective efficacy among rOmpBs were assayed using 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. The differences in protective efficacy among groups after challenge with R. rickettsii or C. burnetii and the differences in serum neutralization and ELISA or cytokines were assayed using Student's t-test or Wilcoxon Two-Sample test, and P<0.05 was considered significantly different.
The 5 rOmpBs were used to immunize C3H/HeN mice, respectively. After challenge with R. rickettsii, the rickettsial load in livers or spleens of mice immunized with rOmpB-1, rOmpB-2, rOmpB-4, rOmpB-5, or WCA of R. rickettsii and that in lungs of mice immunized with any of the 5 rOmpBs or WCA of R. rickettsii were significantly lower compared with mice mockimmunized with PBS. And mice immunized with rOmpB-4 consistently harbored lower levels of R. rickettsii in these organs compared with mice immunized with any other rOmpB (Fig 2A-2C).

Immunoprotection induced by rOmpB-4 and/or CMR
To evaluate the potentiation of C. burnetii CMR, mice immunized with rOmpB-4 combined with CMR, or rOmpB-4/CMR alone were challenged with R. rickettsii. As a result, the rickettsial load in livers (P < 0.05, Fig 3A), spleens (P < 0.01, Fig 3B), or lungs (P < 0.01, Fig 3C) of mice immunized with rOmpB-4 combined with CMR was significantly lower than that with rOmpB-4 or CMR alone. The rickettsial load in livers or spleens of mice immunized with CMR alone were lower than that in cognate organs of mice immunized with rOmpB-4 alone, but which was not significantly different (P > 0.05, Fig 3A and 3B). The spleen weight of mice immunized with rOmpB-4 combined with CMR was significantly lighter than that of mice immunized with rOmpB-4 alone (P < 0.05, Fig 3D).
Additionally, mice immunized with rOmpB-4 combined with C. burnetii CMR or rOmpB-4 alone were challenged with C. burnetii, after which the coxiella load in livers, spleens or lungs of mice immunized with rOmpB-4 combined with CMR was significantly lower than that of mice immunized with rOmpB-4 alone (P<0.05, Fig 4A-4C), and the spleen weight of mice immunized with rOmpB-4 combined with CMR was significantly lighter than that of mice immunized with rOmpB-4 alone (P < 0.05, Fig 4D).

Histopathological examination
As shown in Fig 5A, lobular hepatitis, inflammatory cell infiltration, macrophagocytes, and interstitial pneumonia were observed in liver, spleen, and lung from mice infected by R. rickettsii or C. burnetii, respectively. After challenge with R. rickettsii, the focal zone of inflammatory infiltrates in livers (Fig 5B), the number of macrophage number in spleens (Fig 5C), and the mean thickness of alveolar wall in lungs (Fig 5D) of mice immunized by rOmpB-4 combined with CMR-C were significantly less or thinner compared with those of mice immunized by either rOmpB-4 or CMR-C. Meanwhile, after challenge with C. burnetii, these pathological alters in livers, spleens, and in lungs of mice immunized by rOmpB-4 combined with CMR-C were also significantly slighter than those of mice immunized by rOmpB-4 alone (Fig 5A-5D). 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 rOmpB-4 combined with CMR produced a significantly higher level of IgG (Fig 6A, P<0.001), IgG1 (Fig 6B, P<0.001) or IgG2a (Fig 6C, P<0.001) compared mice immunized with rOmpB-4 or CMR alone on days 7, 14, 21, and 28 after primary immunization. Additionally, the ratio of IgG2a/IgG1 to rOmpB-4 of mice immunized with rOmpB-4 combined with CMR was 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. burnetiispecific 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).  markedly higher than that of mice immunized with rOmpB-4 or CMR alone on days 7, 14, and 21 after primary immunization ( Fig 6D).
The IgGs to C. burnetii phase I or Phase II antigen were detected by IFA. The results displayed that both anti-phase I and Phase II IgGs were dramatically rising till day 21 after primary immunization and then keeping at the high level on day 28 (Fig 7).
Neutralization of rickettsiae with sera from mice immunized with rOmpB-4 and/or CMR As Fig 8 showed, the total amount of rickettsiae treated with sera from mice immunized with rOmpB-4 combined with C. burnetii CMR was significantly lower than that treated with sera from mice with rOmpB-4 alone (P<0.05) or CMR alone (P<0.001), while that treated with sera from mice immunized with rOmpB-4 alone was lower, but not significantly, than that treated with sera from mice immunized with CMR alone (P>0.05). Cytokines in sera from mice immunized with rOmpB-4 and/or CMR As shown in Fig 9, the content of TNF-α or IFN-γ in sera from mice immunized with rOmpB-4 combined with C. burnetii CMR was significantly higher than those in sera from mice immunized with rOmpB-4 (P<0.05) or CMR (P<0.001) alone through the immunization process, especially day 14 after primary immunization.  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).
doi:10.1371/journal.pone.0124664.g008 Discussion R. rickettsii and C. burnetii are both the obligate intracellular pathogens parasiting respectively in vascular endothelial cells and monocytes/macrophages. OmpB, an important virulence factor of rickettsiae [28,39], is the most abundant and high-molecular-mass surface protein of rickettsiae [40]. OmpB has been proved to be a good protective antigen capable of eliciting specific protective immunoresponses in animal models by eliciting the production of specific antibodies to inhibit rickettsial adhesion and invasion of host cells as well as opsonize macrophages or activate complements to kill rickettsiae in hosts [40], making it good candidate for subunit vaccine against rickettsial infection [39]. Here ompB gene of R. rickettsii was divided into 5 fragments to result in 5 recombinant proteins and our results showed that any of the 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. doi:10.1371/journal.pone.0124664.g009 rOmpBs except rOmpB-3 could offer a significant protection against R. rickettsii infection and rOmpB-4 was the best one among the protective rOmpBs.
C. burnetii has been proved to be a potent immunomodulator which can modulate host immune responses positively [23]. Mice injected with phase I C. burnetii has been showed to increase resistance to tumors and bacteria [19], virus [20] or protozoans [21]. The non-specific immunopreotection by phase I C. burnetii has been recognized as involving the enhancement of humoral immunoresponses [41] and cellular immunoresponses including the increase of IFN-γ/β level in sera and the percentage of T-cells and la-positive T-cells in spleens [20] as well as macrophage functions [42] which may involve the oxygen radical microbicidal system [23]. High levels of GM-CSF and IL-1 detected after in vivo or in vitro exposure to phase I C. burnetii can enhance dendritic cell activity [43]. Furthermore, phase I C. burnetii can increase expression of la MHC class II antigen to induce production of IFN-γ and other lymphokines in hosts [23]. All these strongly suggest that phase I C. burnetii is a good immunopotentiator which can stimulate antigen processing cells, leading to enhanced antigen processing and subsequent humoral and cellular immunoresponses.
Meanwhile, phase I C. burnetii also can negatively modulate host immune responses, inducing immune suppression [44] and adverse reactions giving rise to severe local inflammatory responses such as persistent indurated masses or sterile abscesses at the injection site in immunization [45]. Phase I C. burnetii lipopolysaccharide is important in the development of immunity [46], but it's also a major virulence factor containing determinants of immune suppressive and adverse components. Fortunately, the components that can induce the immune suppression and adverse reaction are efficiently dissociated from phase I C. burnetii by chloroformmethanol extraction [47]. The obtained phase I C. burnetii CMR has been shown to be nontoxic, immunogenic, and protective in animals and humans [48].
In the present study, rOmpB-4 was chosen to combine with C. burnetii CMR in immunization of mice so as to explore whether CMR could promote rOmpB-4 to induce more efficient protection against R. rickettsii infection. Our results showed that mice immunized with rOmpB-4 combined CMR had a significantly lower of rickettsial load or slighter histopathologic lesions in livers, spleens, or lungs compared with mice immunized with rOmpB-4 or CMR alone. The result demonstrated that C. burnetii CMR could potentiate rOmpB-4 to induce much more efficient protection against R. rickettsii infection, especially in lungs of mice. Previous studies found that R. rickettsii organisms primarily infect the microvascular endothelium, leading to systemic spread of the organisms and the major pathophysiological effects, such as increased microvascular permeability and edema in vital organs, the lung and brain [49]. Thus the effective inhibition of rickettsial infection in lung is critical for protection against R. rickettsii infection.
CMR of C. burnetii has been well demonstrated to be a safer Q fever vaccine compared with WCV, and vaccination with the CMR can effectively prime the immune system to mount significant anamnestic responses after infection [50]. In the present study, mice immunized with rOmpB-4 combined with C. burnetii CMR showed that the coxiella load in livers, spleens or lungs was significantly lower than that of mice immunized without CMR. This result suggested that in the combination immunization, C. burnetii CMR could keep intrinsic immunoprotective efficacy against C. burnetii infection in addition to be a good immunopotentiator capable to enhance the specific protection conferred by rOmpB-4.
Mice immunized with rOmpB-4 combined with CMR produced a significantly higher level of IgGs to rOmpB-4 and the total amount of rickettsiae in host cells was markedly reduced by sera from the mice in neutralization assay. These results suggested that CMR could efficiently promote humoral responses to rOmpB-4, producing greater amount of specific antibodies to block rickettsia invasion of vascular endothelial cells. Moreover, both IgG1 and IgG2a to rOmpB-4 were markedly in mice immunized with rOmpB-4 combined with CMR, indicating that this combined immunization induced both T helper cell type 2 (Th2) and T helper cell type 1 (Th1) specific immunoresponses [51]. Whereas, the ratio of IgG2a/IgG1 to rOmpB-4 in these mice was markedly higher than that of mice immunized with rOmpB-4 alone during the early phase of vaccination, indicating that CMR could more efficiently increase production of specific IgG2a and the specific immunoresponse was skewing toward Th1 pathway. Previous studies have proved that IgG2a may promote specific immunoprotections and facilitate the removal of intracellular pathogens from hosts. Specifically, the Fc portion of IgG2a can interact with complement components [52] and Fc receptors of macrophages [53] to elicit antibodydependent cell-mediated cytotoxicity [54] and opsonophagocytosis by macrophages [55], which contributes to clearance of rickettsiae from infected hosts.
In addition, both IFN-γ and TNF-α in mice immunized with rOmpB-4 combined with C. burnetii CMR were significantly higher than those in mice immunized with rOmpB-4 or CMR alone, indicating that C. burnetii CMR could effectively facilitate production of both Th1-specific IFN-γ and important inflammatory cytokine TNF-α. IFN-γ and TNF-α produced in Th1-oriented immunoresponses are critical for eradication of rickettsial infection in hosts since they can act synergistically to activate endothelial cells and other cells infected by rickettsiae to kill intracellular rickettsiae via a nitric oxide synthesis-dependent mechanism [56]. Moreover, IFN-γ produced by activated T cells, NK cells, macrophages and/or dendritic cells [57] is a potent activator of macrophages and Th1 immunoresponses [58]. TNF-α is also a potent activator of macrophages and the TNF-α-activated macrophages in turn produce greater amounts of IFN-γ to synergize with TNF-α, mediating more potent killing of intracellular bacteria [59,60].

Conclusion
In the present study, C. burnetii CMR could effectively potentiate a protein fragment rOmpB-4 derived from OmpB of R. rickettsii to elicit markedly enhanced protection against R. rickettsii infection through increasing production of specific antibodies, particular IgG2a, and cytokines IFN-γ and TNF-α, which acted synergistically to resist R. rickettsii infection in mice. In addition, C. burnetii CMR could keep intrinsic efficacy to induce immunoprotection against C. burnetii infection in the combined immunization in addition to act as a potent immunopotentiator. Therefore, the combination of protective rOmpBs of R. rickettsii and CMR of C. burnetii may provide candidates for development of vaccines against both R. rickettsii and C. burnetii infection.
Supporting Information S1

Author Contributions
Conceived and designed the experiments: BHW WPG. Performed the experiments: WPG PCW XLX. Analyzed the data: JJ. Contributed reagents/materials/analysis tools: XMY. Wrote the paper: WPG BHW.