Formalin-Inactivated Coxiella burnetii Phase I Vaccine-Induced Protection Depends on B Cells To Produce Protective IgM and IgG

Abstract

To further understand the mechanisms of formalin-inactivated Coxiella burnetii phase I (PI) vaccine (PIV)-induced protection, we examined if B cell, T cell, CD4⁺ T cell, or CD8⁺ T cell deficiency in mice significantly affects the ability of PIV to confer protection against a C. burnetii infection. Interestingly, compared to wild-type (WT) mice, PIV conferred comparable levels of protection in CD4⁺ T cell- or CD8⁺ T cell-deficient mice and partial protection in T cell-deficient mice but did not provide measurable protection in B cell-deficient mice. These results suggest that PIV-induced protection depends on B cells. In addition, anti-PI-specific IgM was the major detectable antibody (Ab) in immune sera from PIV-vaccinated CD4⁺ T cell-deficient mice, and passive transfer of immune sera from PIV-vaccinated CD4⁺ T cell-deficient mice conferred significant protection. These results suggest that T cell-independent anti-PI-specific IgM may contribute to PIV-induced protection. Our results also suggested that PIV-induced protection may not depend on complement activation and Fc receptor-mediated effector functions. Furthermore, our results demonstrated that both IgM and IgG from PIV-vaccinated WT mouse sera were able to inhibit C. burnetii infection in vivo, but only IgM from PIV-vaccinated CD4⁺ T cell-deficient mouse sera inhibited C. burnetii infection. Collectively, these findings suggest that PIV-induced protection depends on B cells to produce protective IgM and IgG and that T cell-independent anti-PI-specific IgM may play a critical role in PIV-induced protection against C. burnetii infection.

INTRODUCTION

Coxiella burnetii is an obligate intracellular Gram-negative bacterium that causes acute and chronic Q fever in humans. It undergoes lipopolysaccharide (LPS) phase variation in which its virulent smooth LPS phase I (PI) converts to an avirulent rough LPS phase II (PII) upon serial passages in eggs and tissue cultures (1). Although formalin-inactivated C. burnetii phase I vaccine (PIV) was able to provide near-complete protection in animal models as well as in human vaccinees (2–4), the mechanism of PIV-induced protective immunity against C. burnetii infection is not well understood. In addition, it is unique among intracellular bacterial pathogens in that killed C. burnetii whole-cell vaccine can induce long-lasting protective immunity against challenge with virulent C. burnetii (5, 6). Therefore, elucidation of the mechanism of protective immunity elicited by PIV may provide critical information for an understanding of the mechanisms of vaccine-induced immunity against intracellular bacterial pathogens.

Both humoral and cell-mediated immune responses are considered to be important for host defense against C. burnetii infection, while cell-mediated immunity probably plays a critical role in eliminating the organisms. Abinanti and Marmion (7) first reported that mixtures of antibody (Ab) and C. burnetii were not infectious in experimental animals, suggesting that Ab may play a role in the control of C. burnetii infection. Several in vitro studies indicated that treatment of C. burnetii with immune sera made the organisms more susceptible to phagocytosis and to destruction by normal polymorphonuclear leukocytes or macrophages (8–10). These studies provided strong support for the notion that humoral immunity is important in the development of the acquired resistance to C. burnetii infection. However, the observation that treatment of athymic mice with immune sera 24 h before challenge with C. burnetii had no effect on bacterial multiplication within the spleens of the T cell-deficient animals (11) suggests that T cell-mediated immunity plays a critical role for elimination of C. burnetii. Despite C. burnetii being an obligate intracellular pathogen, two recent studies (12, 13) demonstrated that passive transfer of immune sera from PIV-vaccinated mice was able to confer significant protection against C. burnetii infection, suggesting that Ab-mediated immunity is critical for PIV-induced protective immunity. Therefore, an understanding of the mechanisms of Ab-mediated immunity will provide critical information for developing novel vaccines against Q fever.

In this study, to further understand the role of humoral and cellular immunity in PIV-induced protection and to determine whether T cell-dependent or -independent antigens are critical for PIV-induced protection, we examined if B cell, T cell, CD4⁺ T cell, or CD8⁺ T cell deficiency in mice significantly affects the ability of PIV to confer protection against a C. burnetii infection. Our results suggest that PIV-induced protection depends on B cells to produce protective IgM and IgG and that T cell-independent anti-PI-specific IgM may play a critical role in PIV-induced protection against C. burnetii infection.

MATERIALS AND METHODS

Animals.

Specific-pathogen-free (SPF) 6-week-old female BALB/c, C57BL/6, CD4⁺ T cell-deficient (B6.129S2-Cd4tm1Mak/J), CD8⁺ T cell-deficient (B6.129S2-Cd8atm1Mak/J), B cell-deficient (B6.129S2-Igh-6tm1Cgn/J), and T cell-deficient (nude) (NU/J) mice were purchased from Jackson Laboratories (Bar Harbor, ME). Fc receptor (FcR) (FcγRI/FcγRIII/FcϵRI)-deficient mice (B6.129P2-Fcer1gtm1Rav N12) were obtained from Taconic Laboratories (Germantown, NY). All mice were housed in sterile microisolator cages under SPF conditions at the University of Missouri laboratory animal facilities according to the Guide for the Care and Use of Laboratory Animals (14). The research protocols described in this report were approved by the Institutional Biosafety Committee and the Animal Care and Use Committee of the University of Missouri. All C. burnetii infection experiments were conducted in animal biohazard safety level 3 (ABL3) facilities at the University of Missouri Laboratory of Infectious Disease Research (LIDR).

C. burnetii strain.

Nine Mile phase I (NMI) clone 7 (RSA493) organisms were grown in L929 cells and purified as described previously (15). Purified C. burnetii NMI was inactivated by the use of a 1% formaldehyde solution as described previously (16) and used as killed C. burnetii phase I (PI) antigen in vaccination and enzyme-linked immunosorbent assays (ELISAs). The protein concentration of inactivated PI antigens was measured by using a Micro BCA protein assay kit (Pierce, Rockford, IL).

PIV vaccination and challenge with virulent C. burnetii.

Wild-type (WT) mice (C57BL/6) and B cell-, T cell-, CD4⁺ T cell-, or CD8⁺ T cell-deficient mice were vaccinated with 4 μg of PIV plus adjuvant. At each vaccination, one mouse was subcutaneously injected with a mixture of 50 μl of antigen in phosphate-buffered saline (PBS) and 50 μl of aluminum hydroxide (Sigma). C57BL/6 mice were vaccinated with adjuvant only and used as unvaccinated controls. Vaccinated and unvaccinated control mice were challenged at 28 days postvaccination by intraperitoneal (i.p.) injection with 1 × 10⁷ organisms of NMI, as described previously (17). Mice were sacrificed at 14 days postchallenge. Serum samples were collected from each vaccination group of mice at prechallenge and stored at −80°C until use. The protective efficacy of PIV was evaluated by comparing splenomegaly as well as bacterial burden and histopathological changes in the spleen with those of controls.

Depletion of complement.

Cobra venom factor (CVF) has been shown to degrade complement and results in near-complete depletion of complement activity (18, 19). Depletion of the complement system in unvaccinated BALB/c mice and PIV-vaccinated BALB/c and FcR-deficient mice was performed as described previously (20). Twenty-four hours prior to challenge, mice were injected i.p. twice with 10 μg of CVF (Complement Technology, Tyler, TX) at 4-h intervals. After challenge for 1 week, mice were treated with CVF as described above. To confirm the efficiency of the in vivo complement depletion by CVF, the concentration of complement factor 3 (C3) in mouse sera from CVF-treated mice was measured by using a mouse C3 ELISA kit (Immunology Consultants Laboratory, Newberg, OR).

Quantitative ELISA.

The concentrations of anti-PI-specific IgM and IgG in mouse sera from PIV-vaccinated mice at prechallenge were measured by quantitative ELISA. One hundred microliters of serially diluted mouse IgM (Bethyl, Montgomery, TX) or IgG (Equitech-Bio, Kerrville, TX) in 0.05 M carbonate-bicarbonate coating buffer (pH 9.6) was added to each well of a 96-well microtiter plate and used as the standard. One hundred microliters of formalin-inactivated NMI antigen at a concentration of 0.5 μg/ml in the above-described coating buffer was added to each well of the same 96-well microtiter plate for measuring anti-PI-specific IgM and IgG. After the antigens were coated at 4°C overnight, the 96-well microtiter plates were blocked with 1% bovine serum albumin (BSA) in PBST buffer (0.05% Tween 20 in PBS) and then incubated with 100 μl of 0.5% BSA for standard wells or 1:400-diluted mouse sera in 0.5% BSA for sample wells at 37°C for 2 h. After washing 5 times with PBST buffer, the plates were incubated with 100 μl of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM or IgG (1:1,000 dilution) at 37°C for 2 h. Sigma Fast O-phenylenediamine dihydrochloride tablet sets (Sigma, St. Louis, MO) were used as substrates, and the IgM and IgG concentrations were measured by the Spectra Max M2 system using the SoftMax program (Molecular Devices Corporation, Sunnyvale, CA).

Passive transfer of immune sera from PIV-vaccinated WT or CD4 T cell-deficient mice.

Immune serum was collected from PIV-vaccinated (4 μg/mouse, once) C57BL/6 or CD4 T cell-deficient mice at 28 days postvaccination, pooled as equal amounts from each mouse, and inactivated at 56°C for 30 min. Each recipient naive BALB/c mouse received 300 μl of pooled sera by i.p. injection 72 h prior to infection with C. burnetii. Mice receiving 300 μl of normal mouse sera were used as negative controls. All mice were infected with 1 × 10⁷ NMI organisms by i.p. injection at 72 h after passive transfer. Mice were sacrificed at 14 days postinfection. Mouse spleen weight was measured, and a portion of spleen from each mouse was taken for real-time PCR analysis. The ability of immune sera to confer protection in naive recipient mice was evaluated by comparing splenomegaly, bacterial burden, and histopathological changes in the spleen with those in controls.

Purification of IgG and IgM from immune sera.

Immune sera from PIV-vaccinated WT or CD4 T cell-deficient mice were collected as described above. IgG was purified from pooled immune sera by protein G-Sepharose affinity chromatography (GE Healthcare). The flowthrough fraction from the protein G column was collected for purification of IgM from the same pooled immune sera. IgM was purified from the flowthrough fraction of the protein G column by precipitation with 21% polyethylene glycol 6000 (PEG 6000). IgM was precipitated by the addition of an equal volume of 21% (wt/vol) PEG 6000 to the above-described flowthrough fraction, and the mixture was slowly stirred at room temperature for 30 min and then centrifuged at 4,000 × g at 4°C for 30 min. The pellet was dissolved in PBS and dialyzed against PBS to remove the residual PEG. The concentrations of purified IgM and IgG were assessed by measurement of absorption in a UV spectrophotometer at 280 nm. The purities and specificities of the purified IgM and IgG were analyzed by SDS-PAGE and Western blotting with HRP-conjugated goat anti-mouse IgM or IgG. The concentrations of anti-PI-specific IgM and IgG in purified IgM and IgG were measured by a quantitative ELISA, as described above.

C. burnetii inhibition assay in vivo.

We examined whether treatment of C. burnetii with immune sera or purified IgM and IgG from immune sera can inhibit C. burnetii infection in vivo. Inhibition of C. burnetii with immune sera was performed by incubating 1 × 10⁷ virulent C. burnetii NMI organisms with 30 μl of normal mouse sera or immune sera from PIV-vaccinated C57BL/6 or CD4 T cell-deficient mice at 4°C overnight. Inhibition of C. burnetii with purified IgM and IgG was performed by incubating 1 × 10⁷ virulent C. burnetii NMI organisms with 300 μg of purified IgM or IgG from PIV-vaccinated C57BL/6 or CD4 T cell-deficient mouse sera at 4°C overnight. In addition, C. burnetii organisms treated with purified IgM or IgG from naive C57BL/6 mice, as described above, were used as negative controls. Six-week-old BALB/c mice were infected by i.p. injection with 1 × 10⁷ normal mouse serum-, immune serum-, or purified IgM- or IgG-treated C. burnetii organisms. The ability of immune sera, IgM, and IgG to inhibit C. burnetii infection in BALB/c mice was evaluated by comparing splenomegaly, bacterial burden, and histopathological changes in the spleen at 14 days postinfection with those in controls.

C. burnetii inhibition assay in vitro.

To determine whether immune sera can directly neutralize or kill C. burnetii, mouse bone marrow-derived macrophages were used to examine if treatment of C. burnetii with immune sera can inhibit C. burnetii infection in vitro. Bone marrow-derived macrophages were prepared as described previously (21). C. burnetii NMI organisms were incubated with PBS, normal mouse sera, or immune sera at a concentration of 1:10 or 1:100 at 4°C overnight. PBS-, normal mouse serum-, or immune serum-treated NMI organisms were inoculated into macrophages at a multiplicity of infection (MOI) of 100 and incubated for various times. The C. burnetii infection rate was determined at 4 h and at 5 days postinfection by an indirect immunofluorescence assay (IFA), as described previously (22). For cells growing for more than 24 h, infected medium was removed and replaced with fresh medium 24 h following infection. Cells were fixed with 2% paraformaldehyde for 15 min and permeabilized with cold methanol for 10 min. Rabbit anti-NMI polyclonal antibodies (1:500) were used to stain intracellular C. burnetii, followed by incubation with goat anti-rabbit IgG (Invitrogen) (1:500). Host nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), and slides were examined by fluorescence microscopy.

Quantitative PCR assay.

A High Pure PCR Template Preparation kit (Roche Molecular Biochemicals, Indianapolis, IN) with modifications was used for extraction of DNA templates from spleens and C. burnetii-infected macrophages, as described previously (12, 23). Real-time PCR was performed as described previously (23), with modifications, by using an Applied Biosystems 7300/7500 real-time PCR system. The recombinant plasmid DNA (com1 gene ligated into the pET23a vector) (17) was used as a standard DNA to quantify com1 gene copy numbers in spleen samples.

Histopathology.

Spleens were collected from mice at 14 days after challenge with C. burnetii, fixed in 10% formalin–PBS for at least 48 h, prepared as 5-μm paraffin-embedded sections by standard methods, and sliced. Slides were stained with hematoxylin and eosin and examined in a blinded fashion for evaluation of histopathology.

Statistical analysis.

A two-tailed Student t test was used to compare the significance between different immunization groups. P values of <0.05 were considered significant.

RESULTS

PIV-induced protection depends on B cells.

Although C. burnetii infection in mice does not cause death and clear clinical signs, infection can induce significant splenomegaly. Splenomegaly has been used as an indicator to monitor the severity of C. burnetii infection in mice (17). In addition, because C. burnetii is difficult to grow on a plate and does not form clear plaques in cell culture, it is difficult to use traditional methods to measure the C. burnetii burden in animal tissues. Recently, a quantitative real-time PCR procedure was developed and used to measure the number of C. burnetii organisms in the spleen (24). One recent study (12) demonstrated that splenomegaly was correlated with infection dose and also with bacterial load in the spleen, as measured by real-time PCR, suggesting that splenomegaly can be a useful indicator to evaluate the protective efficacy of vaccines. In this study, splenomegaly, bacterial burden, and histopathological changes in the spleen were examined at 14 days postchallenge and used as indicators to evaluate the protective efficacy of PIV in B cell, T cell, CD4⁺ T cell, or CD8⁺ T cell deficiency in mice. As shown in Fig. 1A, splenomegaly in PIV-vaccinated CD4⁺ or CD8⁺ T cell-deficient mice was similar to that in PIV-vaccinated WT mice, while splenomegaly in PIV-vaccinated B cell-deficient mice was significantly greater than that in PIV-vaccinated WT mice (P < 0.001). In addition, compared to unvaccinated WT mice, splenomegaly was significantly reduced in PIV-vaccinated T cell-deficient mice, but it was significantly greater than that in PIV-vaccinated WT mice (P < 0.05). In support of the splenomegaly results, C. burnetii genome copy numbers were comparable in the spleens from PIV-vaccinated WT, CD4⁺ T cell-deficient, and CD8⁺ T cell-deficient mice, but significantly higher C. burnetii genome copy numbers were detected in the spleen from PIV-vaccinated B cell-deficient (P < 0.01) and T cell-deficient (P < 0.05) mice (Fig. 1B). Histopathological differences were observed in the spleens from different groups of mice. As shown in Fig. 1C, moderate to large multifocal accumulations of macrophages (encircled by a black border) were present in red pulp of spleens from unvaccinated WT and PIV-vaccinated B cell-deficient and T cell-deficient mice. However, few or no small accumulations of macrophages (Fig. 1C, encircled by a black border) were observed in red pulp of spleens from PIV-vaccinated WT, CD4⁺ T cell-deficient, and CD8⁺ T cell-deficient mice. These observations indicate that PIV vaccination provided similar levels of protection against the C. burnetii challenge-induced inflammatory response in WT, CD4⁺ T cell-deficient, and CD8⁺ T cell-deficient mice but did not protect B cell-deficient and T cell-deficient mice against the inflammatory response. Interestingly, PIV-vaccinated T cell-deficient mice were partially protected from development of splenomegaly against C. burnetii challenge, but vaccination did not prevent C. burnetii replication and pathological changes in the spleen. Thus, PIV conferred a comparable level of protection against C. burnetii challenge in WT, CD4⁺ T cell-deficient, and CD8⁺ T cell-deficient mice and partial protection in T cell-deficient mice but did not provide measurable protection in B cell-deficient mice. These results suggest that PIV-induced protection depends on B cells. In addition, the observation that CD4⁺ T cell deficiency in mice did not affect the ability of PIV to confer protection suggests that T cell-independent antigens may be the key protective antigens responsible for PIV-induced protection.

Fig 1.

Evaluation of protective efficacy of PIV in WT or B cell-, CD4⁺ T cell-, CD8⁺ T cell-, or T cell-deficient mice by comparing splenomegaly, bacterial burden, and pathological changes in the spleen with those of controls at 14 days postchallenge. Each mouse was subcutaneously injected with a mixture of 50 μl of PI antigen in PBS and 50 μl of aluminum hydroxide. WT mice were vaccinated with adjuvant only and used as unvaccinated controls. Vaccinated and unvaccinated control mice were challenged at 28 days postvaccination by i.p. injection with 1 × 10⁷ C. burnetii organisms. (A) Splenomegaly was measured by spleen weight as a percentage of body weight. (B) Bacterial burden in the spleen was determined by real-time PCR and is reported as log₁₀ C. burnetii com1 gene copy numbers. (C) Pathological changes in the spleen at 14 days postchallenge. The data presented in each group are the averages with standard deviations for three mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001. WTV, WT mice vaccinated with PIV; KO, knockout.

Anti-PI antigen-specific IgM may contribute to PIV-induced protection.

To understand the role of Abs in PIV-induced protection, immune sera were collected from PIV-vaccinated WT and B cell-, CD4⁺ T cell-, CD8⁺ T cell-, or T cell-deficient mice at 28 days postvaccination in the above-described experiment for measuring the concentrations of anti-PI-specific IgM and IgG. As shown in Fig. 2A, compared to PIV-vaccinated WT mice, a significantly low concentration of IgM was detected in PIV-vaccinated CD4⁺ T-cell deficient (P < 0.05) and T cell-deficient (P < 0.001) mice, but a comparable concentration of IgM was detected in CD8⁺ T cell-deficient mice. These results indicate that mice vaccinated with PIV can induce both T cell-dependent and -independent IgM responses to PI antigens. Interestingly, the IgM concentration in T cell-deficient mice was significantly lower than that in CD4⁺ T cell-deficient mice (P < 0.05). This result suggests that other subsets of T cells, such as CD4⁻ CD8⁻ T cells or γδ T cells, may be involved in inducing IgM responses to PI antigens. In addition, anti-PI-specific IgM was undetectable in unvaccinated WT and B cell-deficient mice. Figure 2B shows the concentrations of anti-PI-specific IgG in immune sera. Compared to PIV-vaccinated CD4⁺ T cell-deficient and T cell-deficient mice, a significantly high concentration of IgG was detected in PIV-vaccinated WT and CD8⁺ T cell-deficient mice, but there was no significant difference in IgG concentrations between PIV-vaccinated WT and CD8⁺ T cell-deficient mice. These results suggest that CD4⁺ T cells are required for mice to generate an antigen-specific IgG response to PI antigens and that IgM may be responsible for PIV-induced protection in CD4⁺ T cell-deficient mice.

Fig 2.

Concentrations of anti-PI-specific IgM and IgG in immune sera from PIV-vaccinated WT and B cell-, CD4⁺ T cell-, CD8⁺ T cell-, or T cell-deficient mice by ELISA with PI antigen at 28 days postvaccination. (A) Concentrations of anti-PI-specific IgM. (B) Concentrations of anti-PI-specific IgG. The data presented in each group are the averages with standard deviations for three mice. *, P < 0.05; ***, P < 0.001.

Immune sera from PIV-vaccinated CD4⁺ T cell-deficient mice conferred significant protection.

The observations that vaccination with PIV protected CD4⁺ T cell-deficient mice against C. burnetii challenge and that IgM was the major anti-PI antigen-specific Ab in immune sera from PIV-vaccinated CD4⁺ T cell-deficient mice suggest that anti-PI-specific IgM may be responsible for PIV-induced protection in CD4⁺ T cell-deficient mice. To test this hypothesis, we examined if passive transfer of immune sera from CD4⁺ T cell-deficient mice vaccinated with PIV would protect naive recipient mice against a C. burnetii challenge. The protective efficacy of immune sera was evaluated by comparing splenomegaly, bacterial burden, and histopathological changes in the spleen with those for mice receiving normal mouse sera. As shown in Fig. 3A, compared to mice receiving normal mouse sera, splenomegaly was significantly reduced in mice receiving immune sera from PIV-vaccinated WT or CD4⁺ T cell-deficient mice. In support of the splenomegaly results, compared to mice receiving normal mouse sera, significantly low C. burnetii genome copy numbers were detected in spleens from mice receiving immune sera from PIV-vaccinated WT or CD4⁺ T cell-deficient mice (Fig. 3B). In addition, histopathological differences in the spleens of mice receiving normal mouse sera and those receiving immune sera were observed. Compared to mice receiving normal mouse sera, multifocal accumulations of macrophages (encircled by a black border) were few and small in red pulp of spleens from mice receiving immune sera from PIV-vaccinated WT or CD4⁺ T cell-deficient mice (Fig. 3C). This indicates that immune sera from PIV-vaccinated WT or CD4⁺ T cell-deficient mice provided similar levels of protection against the C. burnetii infection-induced inflammatory response. These results demonstrated that passive transfer of immune sera from PIV-vaccinated CD4⁺ T cell-deficient mice was able to confer significant protection against C. burnetii challenge in naive recipient mice. These results support the hypothesis that anti-PI-specific IgM may be the key protective component responsible for PIV-induced protection against C. burnetii infection in CD4⁺ T cell-deficient mice.

Fig 3.

Evaluation of the efficacy of immune sera from PIV-vaccinated CD4⁺ T cell-deficient mice to confer protection in naive recipient mice against C. burnetii infection by comparing splenomegaly, bacterial burden, and pathological changes in the spleen with those with control sera at 14 days postchallenge. (A) Splenomegaly was measured by spleen weight as a percentage of body weight. (B) Bacterial burden in the spleen was determined by real-time PCR and is reported as log₁₀ C. burnetii com1 gene copy numbers. (C) Pathological changes in the spleen at 14 days postinfection. Wt-NS, normal mouse sera from C57BL/6 mice; Wt-IS, immune sera from PIV-vaccinated C57BL/6 mice; CD4 KO-IS, immune sera from PIV-vaccinated CD4⁺ T cell-deficient mice (C57BL/6 background). The data presented in each group are the averages with standard deviations for three mice. *, P < 0.05.

Complement and Fc receptor deficiencies did not affect the ability of PIV to confer protection.

To determine whether complement and FcR-mediated effector functions are involved in PIV-induced protection, we examined if complement and/or FcR deficiency in mice would significantly affect the ability of PIV to confer protection against C. burnetii infection. Depletion of the complement system was performed by treating mice with CVF. CVF is a C3b analog and can form a stable C3-convertase upon binding to serum components, which can rapidly cleave all circulating C3, resulting in depletion of C3. The efficiency of complement depletion by CVF was confirmed by comparing the concentration of C3 in mouse sera from CVF-treated mice with that in control mouse sera at 7 days posttreatment. As shown in Fig. S1 in the supplemental material, compared to PBS-treated mice, a significantly low concentration of C3 was detected in mouse sera from CVF-treated mice. This result suggests that two doses of CVF treatment can deplete C3 in mice for at least 7 days. The protective efficacy of PIV in complement-depleted and/or FcR-deficient mice against C. burnetii challenge was evaluated by comparing splenomegaly, bacterial burden, and histopathological changes in the spleens at 14 days postchallenge with the spleens of controls. As shown in Fig. 4A, compared to unvaccinated control mice, splenomegaly was significantly reduced in PIV-vaccinated complement-depleted FcR-deficient and complement-depleted FcR-deficient mice to levels similar to those of PIV-vaccinated WT mice (P < 0.001 compared with unvaccinated control mice). In support of the splenomegaly results, compared to unvaccinated control mice, significantly low C. burnetii genome copy numbers were detected in spleens from PIV-vaccinated complement-depleted FcR-deficient and complement-depleted FcR-deficient mice (P < 0.01 compared with unvaccinated control mice) (Fig. 4B). As shown in Fig. 4C, compared to unvaccinated control mice (WT and CVF groups), multifocal accumulations of macrophages (encircled by a black border) were few and small in red pulp of spleens from PIV-vaccinated complement-depleted FcR-deficient mice and complement-depleted FcR-deficient mice. These results indicate that (i) depletion of complement in PIV-vaccinated mice did not affect the ability of PIV to confer protection, (ii) FcR deficiency in mice did not affect the ability of PIV to confer protection, and (iii) both complement and FcR deficiency in mice did not affect the ability of PIV to confer protection against C. burnetii infection. These findings suggest that complement activation and FcR-mediated effector functions may not be required for PIV-induced protection.

Fig 4.

Evaluation of the protective efficacy of PIV in complement- and/or FcR-deficient mice by comparing splenomegaly, bacterial burden, and pathological changes in the spleen with those of control mice at 14 days postchallenge. (A) Splenomegaly was measured by spleen weight as a percentage of body weight. (B) Bacterial burden in the spleen was determined by real-time PCR and is reported as log₁₀ C. burnetii com1 gene copy numbers. (C) Pathological changes in the spleen at 14 days postchallenge. WT, wild-type BALB/c mice; WT-V, BALB/c mice vaccinated with PIV; CVF, BALB/c mice treated with CVF; FcR KO-V, FcR-deficient mice vaccinated with PIV; FcR KO-V-CVF, FcR-deficient mice vaccinated with PIV and then treated with CVF. The data presented in each group are the averages with standard deviations for four mice. **, P < 0.01; ***, P < 0.001.

Immune sera from PIV-vaccinated CD4⁺ T cell-deficient mice can inhibit C. burnetii infection in vivo.

The observation that complement and FcR deficiencies did not affect the ability of PIV to confer protection against C. burnetii infection suggests that complement activation and FcR-mediated effector functions may not be the essential mechanisms for Ab-mediated immunity in PIV-vaccinated mice. To determine whether Ab can directly neutralize or kill C. burnetii, we examined if treatment of virulent C. burnetii with immune sera from PIV-vaccinated mice could inhibit C. burnetii infection in BALB/c mice. The ability of immune sera to inhibit C. burnetii infection in BALB/c mice was evaluated by comparing splenomegaly, bacterial burden, and histopathological changes in the spleen at 14 days postinfection with those in controls. As shown in Fig. 5A, compared to mice infected with normal mouse serum-treated C. burnetii, splenomegaly was significantly reduced in mice infected with immune serum-treated C. burnetii. Interestingly, compared to mice infected with C. burnetii treated by immune sera from PIV-vaccinated CD4⁺ T cell-deficient mice, splenomegaly was significantly low in mice infected with C. burnetii treated by immune sera from PIV-vaccinated WT mice. In support of the splenomegaly results, compared to mice infected with normal mouse serum-treated C. burnetii, significantly low C. burnetii genome copy numbers were detected in spleens from mice infected with immune serum-treated C. burnetii (Fig. 5B). In addition, compared to mice infected with C. burnetii treated by immune sera from PIV-vaccinated CD4⁺ T cell-deficient mice, significantly low C. burnetii genome copy numbers were detected in spleens from mice infected with C. burnetii treated by immune sera from PIV-vaccinated WT mice. As shown in Fig. 4C, histopathological differences in the spleens of mice infected with normal mouse serum-treated C. burnetii and the spleens of mice infected with immune serum-treated C. burnetii were observed. Compared to mice infected with normal mouse serum-treated C. burnetii, multifocal accumulations of macrophages (Fig. 4C, encircled by a black border) were few and small in red pulp of the spleens from mice infected with C. burnetii treated by immune sera from PIV-vaccinated WT or CD4⁺ T cell-deficient mice, but there was no observable pathological difference in the spleens of mice infected with C. burnetii treated by immune sera from PIV-vaccinated WT and CD4⁺ T cell-deficient mice. These results demonstrated that immune sera from both PIV-vaccinated WT and CD4⁺ T cell-deficient mice were able to inhibit C. burnetii infection in vivo, while immune sera from PIV-vaccinated WT mice had stronger inhibiting activity than did immune sera from PIV-vaccinated CD4⁺ T cell-deficient mice.

Fig 5.

Evaluation of the ability of immune sera from PIV-vaccinated WT and CD4⁺ T cell-deficient mice to inhibit C. burnetii infection in vivo by comparing splenomegaly, bacterial burden, and pathological changes in the spleen with those with normal mouse sera at 14 days postinfection. (A) Splenomegaly was measured by spleen weight as a percentage of body weight. (B) Bacterial burden in the spleen was determined by real-time PCR and is reported as log₁₀ C. burnetii com1 gene copy numbers. (C) Pathological changes in the spleen at 14 days postinfection. Wt-NS, BALB/c mice infected with 1 × 10⁷ C. burnetii organisms treated by normal mouse sera; Wt-IS, BALB/c mice infected with 1 × 10⁷ C. burnetii organisms treated with immune sera from PIV-vaccinated WT mice; CD4 KO-IS, BALB/c mice infected with 1 × 10⁷ C. burnetii organisms treated with immune sera from PIV-vaccinated CD4⁺ T cell-deficient mice. The data presented in each group are the averages with standard deviations for three mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Immune sera were unable to inhibit C. burnetii infection in vitro.

To determine whether immune sera from PIV-vaccinated mice can directly neutralize or kill C. burnetii, we examined if treatment of C. burnetii with immune sera could inhibit C. burnetii infection in macrophages. As shown in Fig. 6A, compared to macrophages infected with PBS- or normal mouse serum-treated C. burnetii, the C. burnetii infection rate was significantly (P < 0.001) increased in macrophages infected with immune serum-treated C. burnetii at both 4 h and 5 days postinfection. In addition, the infection rate in macrophages infected with immune serum-treated C. burnetii at 5 days postinfection was significantly higher than the infection rate at 4 h postinfection. These results indicate that treatment of C. burnetii with immune sera did not inhibit the ability of C. burnetii to infect macrophages. To determine whether immune serum-treated C. burnetii can replicate in macrophages, C. burnetii genome copy numbers in C. burnetii-infected macrophages were measured by real-time PCR at 5 days postinfection. As shown in Fig. 6B, compared to macrophages infected with PBS- or normal mouse serum-treated C. burnetii, significantly high C. burnetii genome copy numbers were detected in macrophages infected with immune serum-treated C. burnetii. This observation indicates that immune serum-treated C. burnetii can replicate in macrophages, suggesting that treatment of C. burnetii with immune sera from PIV-vaccinated mice cannot inhibit C. burnetii infection of macrophages in vitro.

Fig 6.

Evaluation of the ability of immune sera from PIV-vaccinated WT mice to inhibit C. burnetii infection in mouse bone marrow-derived macrophages by comparing infection rates and C. burnetii genomic copy numbers with those with PBS and normal mouse serum controls at different time points postinfection. (A) C. burnetii infection rate determined by IFA. A total of 200 cells were counted per sample to determine the infection rate. (B) C. burnetii genomic copy number determined by real-time PCR. ***, P < 0.001.

Purified IgM from PIV-vaccinated CD4⁺ T cell-deficient mouse sera can inhibit C. burnetii infection in vivo.

To determine whether IgM and/or IgG is responsible for immune serum-induced inhibition of C. burnetii infection, we examined if treatment of C. burnetii with purified IgM or IgG from immune sera would inhibit C. burnetii infection in BALB/c mice. The purities and specificities of the purified IgM and IgG from immune sera were confirmed by SDS-PAGE and Western blotting (see Fig. S2A and S2B in the supplemental material). In addition, anti-PI-specific IgM was detected in purified IgM from both PIV-vaccinated WT and CD4⁺ T cell-deficient mouse sera, but anti-PI-specific IgG was detected only in the purified IgG from PIV-vaccinated WT mouse sera (see Fig. S2C in the supplemental material). The ability of IgM and IgG from immune sera to inhibit C. burnetii infection in BALB/c mice was evaluated by comparing splenomegaly, bacterial burden, and histopathological changes in the spleen at 14 days postinfection with those of controls. As shown in Fig. 7A, compared to mice infected with C. burnetii treated by IgM or IgG from normal mouse sera, splenomegaly was significantly (P < 0.001) reduced in mice infected with C. burnetii treated by IgM from PIV-vaccinated WT or CD4⁺ T cell-deficient mouse sera or IgG from PIV-vaccinated WT mouse sera, but it was similar to that in mice infected with C. burnetii treated by IgG from PIV-vaccinated CD4⁺ T cell-deficient mouse sera. In addition, splenomegaly in mice infected with C. burnetii treated by IgM from PIV-vaccinated WT or CD4⁺ T cell-deficient mouse sera was similar to that in mice infected with C. burnetii treated by IgG from PIV-vaccinated WT mouse sera. In support of the splenomegaly results, compared to mice infected with C. burnetii treated by IgM or IgG from normal mouse sera, significantly low (P < 0.001) C. burnetii genome copy numbers were detected in the spleens from mice infected with C. burnetii treated by IgM from PIV-vaccinated WT or CD4⁺ T cell-deficient mouse sera or IgG from PIV-vaccinated WT mouse sera, but a similar number of C. burnetii genome copies was detected in spleens from mice infected with C. burnetii treated by IgG from PIV-vaccinated CD4⁺ T cell-deficient mouse sera (Fig. 7B). As shown in Fig. 7C, histopathological differences were observed in the spleens from different groups of mice. Large numbers of moderate to large accumulations of macrophages (Fig. 7C, encircled by a black border) were present in red pulp of spleens from mice infected with C. burnetii treated by IgM or IgG from normal mouse sera or IgG from PIV-vaccinated CD4⁺ T cell-deficient mouse sera. In contrast, only few small to moderate accumulations of macrophages (Fig. 7C, encircled by a black border) appeared in red pulp of spleens from mice infected with C. burnetii treated by IgM from PIV-vaccinated WT or CD4⁺ T cell-deficient mouse sera or IgG from PIV-vaccinated WT mouse sera. These results indicate that both IgM and IgG from PIV-vaccinated WT mouse sera were able to inhibit C. burnetii infection in vivo, but only IgM from PIV-vaccinated CD4⁺ T cell-deficient mouse sera inhibited C. burnetii infection. These findings suggest that both anti-PI-specific IgM and IgG contributed to PIV-induced protection in WT mice, while IgM is the key protective component and is responsible for PIV-induced protection in CD4⁺ T cell-deficient mice. Thus, T cell-independent anti-PI-specific IgM may play a critical role in PIV-induced protection against C. burnetii infection.

Fig 7.

Evaluation of the ability of purified IgM and IgG from immune sera to inhibit C. burnetii infection in vivo by comparing splenomegaly as well as bacterial burden and pathological changes in the spleen with those of normal mouse IgM and IgG controls at 14 days postinfection. (A) Splenomegaly was measured by spleen weight as a percentage of body weight. (B) Bacterial burden in the spleen was determined by real-time PCR and is reported as log₁₀ C. burnetii com1 gene copy numbers. (C) Pathological changes in the spleen at 14 days postinfection. NIgM, BALB/c mice infected with 1 × 10⁷ C. burnetii organisms treated with IgM from normal mouse sera; NIgG, BALB/c mice infected with 1 × 10⁷ C. burnetii organisms treated with IgG from normal mouse sera; WTIgM, BALB/c mice infected with 1 × 10⁷ C. burnetii organisms treated with purified IgM from PIV-vaccinated C57BL/6 mouse sera; WTIgG, BALB/c mice infected with 1 × 10⁷ C. burnetii organisms treated with purified IgG from PIV-vaccinated C57BL/6 mouse sera; CD4 KO-IgM, BALB/c mice infected with 1 × 10⁷ C. burnetii organisms treated with purified IgM from PIV-vaccinated CD4⁺ T cell-deficient mouse sera; CD4 KO-IgG, BALB/c mice infected with 1 × 10⁷ C. burnetii organisms treated with purified IgG from PIV-vaccinated CD4⁺ T cell-deficient mouse sera. The data presented in each group are the averages with standard deviations for four mice. ***, P < 0.001.

DISCUSSION

Early studies suggested that both humoral and cell-mediated immune responses are important for host defense against C. burnetii infection. A recent study (24) demonstrated that T cells and gamma interferon (IFN-γ) are essential for clearance of primary C. burnetii infection. In addition, Read et al. (25) recently demonstrated that either CD4⁺ T cells alone or CD8⁺ T cells alone were able to control a primary pulmonary C. burnetii infection, suggesting that CD4⁺ or CD8⁺ T cells are required for host defense against a primary C. burnetii infection. These studies suggested that T cell-mediated immunity may be the essential mechanism for host defense against primary C. burnetii infection. However, the role of CD4⁺ or CD8⁺ T cells in PIV-induced protective immunity remains unclear. Despite the obligate intracellular life-style of C. burnetii, two recent studies (12, 13) demonstrated that passive transfer of immune sera from PIV-vaccinated mice was able to provide significant protection against C. burnetii infection in naive mice, suggesting that Abs play an important role in PIV-induced protective immunity. Our results demonstrated that PIV conferred comparable levels of protection in CD4⁺ T cell- or CD8⁺ T cell-deficient mice and partial protection in T cell-deficient mice but did not provide measurable protection in B cell-deficient mice, suggesting that B cells are critical for PIV-induced protection against C. burnetii infection, but CD4⁺ or CD8⁺ T cells may be necessary to confer full protection. Thus, the mechanisms of vaccine-induced protection may be different with host defense against a primary C. burnetii infection. This study together with previous studies suggest that Ab-mediated immunity may be the essential mechanism for vaccine-induced protective immunity, while T cell-mediated immunity may be more critical for host defense against a primary C. burnetii infection.

The result that anti-PI-specific IgM concentrations in PIV-vaccinated CD4⁺ T cell-deficient and T cell-deficient mice were significantly lower than those in PIV-vaccinated WT and CD8⁺ T cell-deficient mice suggests that CD4⁺ T cells are required for generation of IgM in response to PIV vaccination and that PIV can induce a T cell-dependent IgM response. The observation that the IgM concentration in T cell-deficient mice was significantly lower than that in CD4⁺ T-cell deficient mice suggests that other subsets of T cells, such as CD4⁻CD8⁻ T cells or γδ T cells, may also be involved in inducing an IgM response to PI antigens. Interestingly, PIV-vaccinated T cell-deficient mice were partially protected from development of splenomegaly against C. burnetii challenge but did not prevent C. burnetii replication and pathological changes in the spleen, while CD4⁺ T cell or CD8⁺ T cell deficiency in mice did not affect the ability of PIV to confer protection. These results suggest that although Ab-mediated immunity is critical for PIV-induced protection, CD4⁺ T cells or CD8⁺ T cells are required for controlling C. burnetii replication and the inflammatory response in the spleen. This hypothesis was supported by a previous study (12) that demonstrated that passive transfer of immune sera from PIV-vaccinated mice protected SCID mice against body weight loss but did not significantly reduce the splenomegaly and bacterial burdens in the spleen. These data together suggest that anti-PI-specific Abs play a critical role in protection from the development of clinical disease at an early stage against C. burnetii challenge, while T cell-mediated immunity is required for clearance and complete elimination of the organisms at the late stage of infection.

Recent evidence demonstrated that Ab can mediate protection against intracellular pathogens through various mechanisms, including direct bactericidal activity, complement activation, opsonization, cellular activation via Fc or complement receptors, and Ab-dependent cellular cytotoxicity (26, 27). Although it has been shown that immune serum opsonization was able to increase the ability of guinea pig macrophages to take up and kill C. burnetii (28), Hinrichs and Jerrells (29) observed that immune serum had no negative effect on the intracellular replication of the organism. Kazar et al. (9) indicated that macrophages were unable to control the growth of Ab-opsonized C. burnetii. Most recently, Shannon and Heinzen (30) demonstrated previously that human monocyte-derived macrophages do not control growth of Ab-opsonized C. burnetii in vitro. Those studies suggested that Abs were able to increase the ability of phagocytes to take up Ab-opsonized C. burnetii but did not affect the ability of phagocytes to control the organism's replication. Since those studies were conducted with in vitro systems, the results may not accurately reflect what is occurring in vivo. In addition, it is unknown if complement and FcR-mediated effector functions are contributing to PIV-induced protection against C. burnetii infection. The result that depletion of complement in PIV-vaccinated BALB/c mice did not affect the ability of PIV to confer protection against C. burnetii challenge suggests that Ab-mediated complement-dependent immunity may not be contributing to PIV-induced protection. This hypothesis was supported by a recent study that demonstrated that Abs can provide protection in mice deficient in all three complement pathways by passive transfer of immune sera from PIV-vaccinated mice (13). These data suggest that complement activation may not be the essential mechanism for Abs to confer protection in PIV-vaccinated mice. The observation that PIV was able to confer complete protection against C. burnetii challenge in FcR-deficient mice indicated that FcR-mediated effector functions are not involved in Ab-mediated immunity in PIV-vaccinated mice. Shannon et al. (13) recently demonstrated that although FcR-dependent, Ab-opsonized C. burnetii was able to stimulate activation of mouse bone marrow-derived dendritic cells in vitro, FcR deficiency in mice did not affect the ability of immune sera from PIV-vaccinated mice to confer protection against a C. burnetii challenge in vivo. These data suggest that Ab-mediated immunity against C. burnetii infection may not be dependent on FcR-mediated effector functions. However, it remains unknown whether both complement and FcR deficiency in mice significantly affects the ability of Abs to confer protection against C. burnetii infection. Interestingly, our results demonstrated that PIV conferred significant protection against C. burnetii challenge in complement-depleted, PIV-vaccinated, FcR-deficient mice, suggesting that both complement and FcR deficiency in mice did not affect the ability of PIV to confer protection. Our results together with previous evidence suggest that complement activation and FcR-mediated effector functions may not be the essential mechanisms in PIV-induced protection against C. burnetii infection.

Until now, there has been no clear evidence to demonstrate that anti-C. burnetii-specific Abs have the ability to directly neutralize or kill C. burnetii. One early study (31) demonstrated that purified human anti-PI IgM was able to suppress C. burnetii replication in the mouse spleen when mixed with a suspension of organisms prior to inoculation of mice. This study suggests that anti-PI IgM may be able to inhibit C. burnetii replication via its ability to neutralize or kill the organisms. However, further detailed studies using both in vitro and in vivo systems are required to clearly determine whether anti-PI IgM can neutralize or kill C. burnetii. To address this question, we evaluated the ability of immune sera from PIV-vaccinated WT mice to inhibit C. burnetii infection in both in vivo and in vitro systems. The in vivo C. burnetii inhibition experiment demonstrated that treatment of C. burnetii with immune sera from PIV-vaccinated mice significantly inhibited C. burnetii infection in BALB/c mice. This result supports the hypothesis that anti-C. burnetii-specific Abs can neutralize or kill C. burnetii. In contrast, the in vitro C. burnetii inhibition assay indicated that treatment of C. burnetii with immune sera did not inhibit its ability to infect macrophages and that immune serum-treated C. burnetii can replicate in macrophages, suggesting that treatment of C. burnetii with immune sera from PIV-vaccinated mice cannot inhibit C. burnetii infection of macrophages in vitro. This result correlated with several previous in vitro studies (9, 29, 30) suggesting that anti-C. burnetii-specific Abs can increase the ability of phagocytes to take up Ab-opsonized C. burnetii but may not be able to affect the ability of phagocytes to control the organism's replication. However, the results from the in vitro studies do not support the hypothesis that anti-C. burnetii-specific Abs have the ability to neutralize or kill C. burnetii. C. burnetii is difficult to grow in non-cell-culture systems and does not form clear plaques in cell cultures, which limits our ability to directly test whether immune sera or Abs can neutralize or kill C. burnetii. In addition, the conflicting results between in vivo and in vitro model systems may be due to the inability of the in vitro C. burnetii inhibition assay to mimic the in vivo situation, and it is difficult to use a cell culture system to determine what happens after macrophages take up Ab-opsonized C. burnetii in mice. Although the mechanisms of immune serum-mediated inhibition of C. burnetii infection in mice remain undefined, this study provided clear evidence to demonstrate that treatment of C. burnetii with immune sera from PIV-vaccinated mice can inhibit C. burnetii infection and that the in vivo C. burnetii inhibition model can be used to evaluate the ability of immune sera or Abs to inhibit C. burnetii infection in vivo. Future studies to separate the F(ab) fragment from the Fc portion of the protective Ab and test its ability to neutralize or kill C. burnetii in both in vitro and in vivo systems would be helpful to determine whether anti-C. burnetii-specific Abs can neutralize or kill C. burnetii.

The observations that immune sera from PIV-vaccinated CD4⁺ T cell-deficient mice were able to inhibit C. burnetii in mice and that anti-PI-specific IgM was the major detectable Ab in PIV-vaccinated CD4⁺ T cell-deficient mice suggest that anti-PI-specific IgM may be the protective component in immune sera and responsible for inhibition of C. burnetii infection. To test this hypothesis, we examined if treatment of C. burnetii with purified IgM or IgG from PIV-vaccinated CD4⁺ T cell-deficient mouse sera would inhibit C. burnetii infection in BALB/c mice. Interestingly, treatment of C. burnetii with purified IgM was able to significantly inhibit C. burnetii infection, but IgG treatment did not affect the ability of C. burnetii to cause infection in mice. These results suggest that anti-PI-specific IgM is the protective component in immune sera from PIV-vaccinated CD4⁺ T cell-deficient mice and that T cell-independent anti-PI-specific IgM may play a critical role in PIV-induced protection.

It was noticed that immune sera from PIV-vaccinated WT mice showed stronger inhibiting activity than immune sera from PIV-vaccinated CD4⁺ T cell-deficient mice. This observation suggests the following two possibilities: (i) anti-PI specific IgG may play a role in inhibiting C. burnetii infection, and (ii) PIV-vaccinated WT mice generated T cell-dependent anti-PI-specific IgM, which contributed to inhibiting C. burnetii infection, but PIV-vaccinated CD4⁺ T cell-deficient mice did not produce T cell-dependent anti-PI-specific IgM. Interestingly, anti-PI-specific IgM was detected in purified IgM from both PIV-vaccinated WT and CD4⁺ T cell-deficient mouse sera, but anti-PI-specific IgG was detected only in the purified IgG from PIV-vaccinated WT mouse sera. This result supports the hypothesis that anti-PI-specific IgG may play a role in the ability of immune sera from PIV-vaccinated WT mice to inhibit C. burnetii infection. The observation that treatment of C. burnetii with purified IgG from PIV-vaccinated WT mouse sera was able to inhibit C. burnetii infection in mice demonstrates that anti-PI-specific IgG is a protective component and contributes to PIV-induced protection. These data suggest that anti-PI-specific IgG and T cell-dependent anti-PI-specific IgM may be involved in the ability of immune sera from PIV-vaccinated WT mice to induce stronger inhibiting activity. Thus, anti-PI-specific IgG and T cell-dependent and T cell-independent anti-PI-specific IgM may be required for the PIV-vaccinated host to confer full protection against C. burnetii infection.

In summary, our results suggest that PIV-induced protection depends on B cells to produce protective IgM and IgG and that T cell-independent anti-PI-specific IgM may play a critical role in PIV-induced protection. Therefore, to develop new generation vaccines against Q fever, future studies should be focused on identifying the antigens that can activate B cells to produce anti-PI-specific IgG and T cell-dependent and T cell-independent anti-PI-specific IgM.