C. burnetii Nine Mile phase I (NMI) clone 7 (RSA 493), Nine Mile phase II (NMII) clone 4 (RSA 439), Scurry phase I (Q177), and Priscilla phase I (Q217) were propagated in acidified citrate cysteine medium-D (ACCM-D), as previously described (32). Bacteria were purified by centrifugation at 15,000 × g for 30 min, followed by two washes with sterile 1× phosphate-buffered saline (PBS), and stored at -80°C until use. All phase I virulent strains used in this study undergo four passages and handled under biosafety level-3 conditions at the UTSA.

Briefly, 200 μl of lysis buffer (1 M Tris, 0.5 M EDTA, 7 mg/ml glucose, 28 mg/ml lysozyme) and 10 μl of proteinase K (20 mg/ml) were added to 10 µl of bacterial stock and incubated at 60°C for 18 h. Next, 21 μl of 10% SDS was added to the samples and incubated at room temperature for 1 h. Finally, DNA was extracted using a High Pure PCR Template Preparation Kit (Roche, Indianapolis, IN) as directed by the manufacturer. Bacterial load was determined using TaqMan assay by quantifying C. burnetii com1 (FAM) (Custom Plus TaqMan™ RNA Assay, Invitrogen). The standard curve was generated using recombinant plasmid DNA (com1 gene ligated into pBluescript vector), and the results are represented genome equivalents (GE). Further, the bacterial viability was verified by serially diluting 10 µl of bacterial stock and plating in ACCM-D agarose plates and observing the colony-forming units by C. burnetii after 7–10 days as previously described (32).

Purified C. burnetii NMI and NMII were inactivated for 48 h in 10% formalin, followed by dialysis in deionized water. Antigen concentrations were then measured using a BCA protein assay kit (Pierce, Rockford, IL) per the manufacturer’s instructions, and the concentration was represented as micrograms (µg). Ten micrograms of formalin-killed PIV and PIIV was dissolved in 50 µl PBS and mixed with 50 µl of alum adjuvant. The formulation was mixed well for 15 min.

Eight-week-old female BALB/c mice, B cell KO (Ighm^tm1Cgn ) mice, T cell KO (Foxn1^nu ) mice, CD4 KO (Cd4^tm1Mak ), and CD8 KO (Cd8a^tm1Mak ) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Since female mice are more resistant than males to C. burnetii infection, female mice were exclusively used in this study. All mice were housed in sterile microisolator cages containing four mice per cage under pathogen-free conditions at the UTSA animal biosafety level 3 laboratory facility. Animals were fed normally as per University regulations. All research protocols used in this study were approved by the Institutional Biosafety Committee and the Animal Care and Use Committee of the UTSA (MU-CP001).

Mice were immunized with viable NMII via intraperitoneal, intranasal, intramuscular, or subcutaneous routes. Mice were anesthetized with isoflurane (5% induction, 1% to 2% maintenance) delivered in oxygen by using an anesthetic vaporizer before each immunization and challenge experiment. Since ≥10⁷ of bacteria were used as the optimized infectious dose for all virulent NMI infections in mice (33) and to avoid unnecessary immune activation, we used 10⁵ GE of NMII as immunization dose in all immunization experiments if not specifically mentioned.

Serum and splenocytes were collected from viable NMII-immunized mice and transferred to naive mice, respectively. Blood was collected through cardiac puncture from euthanized animal, incubated for 30 min at room temperature, and centrifuged at 1,500 × g for 10 min at room temperature, and the serum was collected. Splenocytes were isolated from BALB/c mice at 14 days post vaccination. Spleens were removed and homogenized. The cell suspension was then filtered through a 100-μm-pore-size nylon mesh to remove any connective tissue. Splenocytes were pelleted by centrifugation at 500 × g for 8 min and suspended in 5 ml of ammonium chloride–potassium (ACK) lysis buffer for 5 min at room temperature to lyse red blood cells. Remaining cells were then pelleted by centrifugation at 500 × g for 8 min and resuspended in 2 ml of fluorescence-activated cell sorting (FACS) buffer (PBS supplemented with 0.5% bovine serum albumin [BSA], 2 mM EDTA, and 0.1% sodium azide) for counting. Splenocytes (1 × 10⁷) suspended in 100 μl of PBS were intraperitoneally (IP) injected into each mouse, while 500 μl of pooled serum was injected into each mouse. Three days after adoptive transfer, immune serum- or splenocyte-receiving and control mice were IP challenged with 1 × 10⁷ GE of NMI and observed for 14 days.

For C. burnetii challenge, virulent NMI bacteria grown in ACCM-D were used. Mice were anesthetized and 10⁷ GE of NMI bacteria were dissolved in 400 µl PBS and was IP injected. Body weight was measured at 0, 3, 7, 10, and 14 days post-challenge. Mice were euthanized by CO₂ exposure at 14 days post NMI challenge. Spleen was dissected and weighed, and 40 mg of spleen was used for genomic quantification. Blood was collected by cardiac puncture technique, and serum was separated by spinning blood at 1,500 × g for 10 min at room temperature. If not processed immediately, spleen and serum were stored at -20°C for further use.

Spleen pieces were homogenized in 200 μl of lysis buffer (1 M Tris, 0.5 M EDTA, 7 mg/ml glucose, 28 mg/ml lysozyme) and filtered through a 100-μm-pore-size nylon mesh to remove any connective tissue. Ten microliters of proteinase K (20 mg/ml) was added to each sample prior to incubation at 60°C for 18 h. Next, 21 μl of 10% SDS was added to samples and incubated at room temperature for 1 h. Finally, DNA was extracted using a High Pure PCR Template Preparation Kit (Roche, Indianapolis, IN) as directed by the manufacturer. Bacterial burden was determined using TaqMan assay by quantifying the C. burnetii com1 or CBU_0680 gene and normalized using the mouse tfrc gene. Custom Plus TaqMan™ RNA Assay, FAM for com1, or CBU_0680 was designed and procured from Invitrogen. TaqMan™ Copy Number Reference Assay, mouse, tfrc (VIC) was used to quantify the tfrc genes in the mouse genome. The experiment was conducted using an Applied Biosystems QuantStudio3 real-time PCR system. The standard curve was generated using recombinant plasmid DNA (com1, CBU_0680, mouse tfrc genes ligated into the pBluescript vector), and the results are represented as log₁₀ com1 gene copy number. All the experiments are performed at least in triplicates including both technical and biological replicates.

Sera from vaccinated and unvaccinated control mice were used for quantification of total IgM, IgG, IgG1, IgG2a, and IgG3 subclass antibodies. Microtiter plates (96-well) were coated with 100 μl of inactivated NMI antigen (0.5 μg/ml) or unlabeled anti-IgM or -IgG antibody (0.5 μg/ml, for the standard curve) (Southern Biotech, Birmingham, AL) in 0.05 M carbonate/bicarbonate coating buffer (pH 9.6) for 24 h at 4°C. Plates were blocked with 1% BSA in PBS-T buffer (0.05% Tween 20 in 1× PBS) and then incubated for 2 h with 200 μl of diluted sample serum (1:200 to 1:1,200) or serially diluted pure IgM, IgG, IgG1, IgG2a, or IgG3 (Southern Biotech) at room temperature. Plates were washed four times with PBS-T buffer and then incubated with 100 μl of diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM, IgG, IgG1, or IgG2a (1:4,000 to 1:8,000) (Southern Biotech) at room temperature for 1 h. Plates were washed again four times with PBS-T, followed by the addition of 100 μl of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific). Reactions were stopped using 1 M H₃PO₄, and absorbance was measured at 450 nm using an Infinite F50 (Tecan, Switzerland) microplate reader.

Statistical analysis of body weight loss, splenomegaly, and C. burnetii load in spleen was determined by Welch’s unpaired t-test using GraphPad Prism 9.00 software (GraphPad). For all analyses, p < 0.05 was deemed significant.

To explore whether viable avirulent NMII can interfere the infection caused by the virulent NMI, we examined i) if coinfection of mice with NMI and NMII would significantly affect the ability of NMI to cause disease and ii) if priming of mice with alive NMII before infection with NMI would alter the infection caused by the virulent NMI. Eight-week-old BALB/c mice were IP infected with 1) 1 × 10⁷ virulent NMI, 2) 1 × 10⁷ avirulent NMII, 3) 1 × 10⁷ NMII 3 days before infection with 1 × 10⁷ NMI, 4) 1 × 10⁷ NM I +1 × 10⁷ NMII, or 5) 1 × 10⁷ NMI 3 days before infection with 1 × 10⁷ NMII. Splenomegaly and bacterial burden in the spleen were measured at 14 days post C. burnetii infection. As shown in Figure 1A , compared to NMI-infected mice, splenomegaly was significantly reduced in NMI and NMII-coinfected mice and mice infected with avirulent NMII 3 days before infection with virulent NMI. In addition, splenomegaly in mice infected with NMI 3 days before infection with NMII was similar to that in NMI-infected mice. However, the bacterial burden was similar in the spleens among NMI-infected mice regardless of infection with or without NMII bacteria ( Figure 1B ). These results indicated that NMII bacteria interfered with the severity of NMI infection-induced disease but did not affect NMI bacterial replication in mice. Interestingly, the findings suggest that the viable non-pathogenic NMII bacteria may be capable of inducing protective immunity against virulent C. burnetii infection.

To confirm whether viable non-pathogenic NMII can induce protective immunity against virulent NMI infection, we examined if mouse immunization with viable avirulent NMII bacteria would provide significant protection against virulent NMI challenge. Three groups of BALB/c mice were IP immunized with 1 × 10⁷ viable NMII bacteria and challenged with virulent NMI bacteria at 7, 14, and 28 days post-immunization with NMII, respectively. In addition, mice were infected with 1 × 10⁷ NMI or NMII bacteria and served as controls. Splenomegaly and bacterial burden in the spleen were measured at 14 days post virulent NMI challenge. As shown in Figure 2A , compared to NMI-infected control mice, NMII-immunized mouse-protected NMI infection induced body weight loss at all the time points post-immunization. In addition, splenomegaly and bacterial burden (com1 gene copy number) were significantly reduced in NMII-immunized mice at different times post challenge with virulent NMI ( Figures 2B, C ). Further, to determine the NMI-specific genomic copy number, the CBU_0680 gene, which is not present in NMII, was quantified using RT-PCR. The results showed that the CBU_0680 genomic copy number is similar to the com1 genomic copy number ( Figure 2D ), further supporting that virulent NMI bacterial burden in the spleens is significantly reduced in all NMII-immunized mice regardless of the challenge time with virulent NMI. These results indicated that mice immunized with viable NMII bacteria were able to provide significant protection against virulent C. burnetii NMI challenge as early as 7 days post immunization, suggesting that viable non-pathogenic NMII bacteria may be useful as a live attenuated vaccine against human Q fever.

To determine if immunization dose would affect the ability of non-pathogenic NMII bacteria to confer protection against virulent NMI infection, we tested whether mouse immunization with different doses of viable NMI bacteria would affect their ability to confer protection against virulent NMI challenge. Eight-week-old BALB/c mice were IP immunized with 1) 1 × 10¹, 2) 1 × 10³, 3) 1 × 10⁵, or 4) 1 × 10⁷ of viable NMII bacteria and IP challenged with 1 × 10⁷ virulent NMI bacteria at 28 days post immunization with avirulent NMII. In addition, mice were infected with 1 × 10⁷ NMI and served as unimmunized controls. Splenomegaly and bacterial burden in the spleen were measured at 14 days post virulent NMI challenge. As shown in Figure 3A , compared to unimmunized control mice, all doses of NMII-immunized mice protected NMI infection-induced transitional body weight loss at 3, 7, and 10 days post NMI infection. In addition, splenomegaly and bacterial burden were significantly reduced in NMII-immunized mice regardless of the dose of immunization ( Figures 3B–D ). The lowest dose of 1 × 10¹ viable NMI bacteria-immunized mice also provided significant protection against virulent NMI challenge. These results further demonstrated that mice immunized with viable NMII bacteria could confer significant protection against virulent C. burnetii challenge.

To determine if viable NMII bacteria would induce NMI-specific antibody response, immune sera were collected from mice immunized with 1) 1 × 10¹, 2) 1 × 10³, 3) 1 × 10⁵, or 4) 1 × 10⁷ of viable NMII bacteria at 14 days post-challenge with NMI in the above experiment for measuring the concentrations of anti-NMI-specific IgM and IgG by ELISA. As shown in Figure 4A , NMI-specific IgM titer in mice immunized with live NMII bacteria was significantly lower than that in unimmunized mice, but there was no significant difference among different doses of NMII-immunized mice. In contrast, as shown in Figure 4B , NMI-specific IgG titer was significantly higher in mice immunized with NMII except the lowest dose (10¹) of NMII and the pattern was similar for subclasses IgG1 and IgG2a ( Figures 4C, D ). Interestingly, mice immunized with the lowest dose (10¹) of NMII exhibited lower levels of IgG3 than all other groups including unimmunized group; however, there was no difference in protection, suggesting that IgG3 may not play a major role in NMII-mediated protection ( Figure 4E ). Collectively, these results indicated that viable NMII bacteria induced a significant NMI-specific antibody response. Notably, although the lowest dose of 1 × 10¹ NMII-immunized mice induced a lower level of NMI-specific IgG response, they conferred a similar level of protection against NMI infection as other higher doses of NMII-immunized mice ( Figures 3B, C ), suggesting that NMI-specific IgG response may not correlate with viable NMII bacteria-induced protection.

To identify the optimized route of immunization for viable NMII bacteria vaccine, we examined if the route of immunization would affect the ability of viable NMII bacteria to confer protection against virulent NMI infection. BALB/c mice were IP, IN, IM, or SQ immunized with 1 × 10⁵ of viable NMII bacteria. PBS-vaccinated mice were maintained as unimmunized control. Twenty-eight days post vaccination, mice were IP challenged with 1 × 10⁷ of virulent NMI bacteria. Splenomegaly and bacterial burden in the spleen were measured at 14 days post NMI challenge. As shown in Figure 5A , compared to unimmunized controls, all immunized mice protected the NMI infection-induced transitional body weight loss. Splenomegaly and bacterial burden were also significantly reduced in NMII-immunized mice regardless of the route of immunization ( Figures 5B, C ). In addition, compared to IP-immunized mice, splenomegaly and bacterial burden were significantly lower in IN- and SQ-immunized mice. Although there was no significant difference in splenomegaly between IP- and IM-immunized mice, bacterial burden in IM-immunized mice was significantly lower than IP-immunized mice. Collectively, the results indicated that IN immunization with viable NMII induced a stronger protective immunity against NMI infection than the other immunization routes. Since the IN immunization route is less invasive than IP, IM, and SQ immunization, it would be the optimized immunization route for human vaccination with live NMII vaccine.

To determine if avirulent NMII live vaccine can confer a similar level of protection against virulent C. burnetii infection as the current licensed vaccine, Q-Vax, we compared the protective efficacy between viable NMII bacteria and PIV in BALB/c mice via SQ immunization. As shown in Figure 6A , compared to adjuvant and formalin-killed NMII vaccine-immunized mice, both viable NMII-immunized and PIV-immunized mice were able to protect NMI infection-induced transitional body weight loss at 7 days post challenge and significantly reduced the splenomegaly ( Figure 6B ) and bacterial burden in the spleens ( Figure 6C ) at 14 days post challenge at a comparable level. These results indicated that viable NMII bacteria induced a similar level of protection against virulent NMI infection as the PIV. In addition, the observation that viable NMII bacteria conferred significant protection against virulent NMI infection but killed NMII vaccine did not confer a measurable protection, suggesting that bacterial replication in mice may be required for viable NMII-induced protective immunity.

According to our previous studies, 10 µg formalin-killed PIV provided the highest levels of protection. To determine whether viable NMII bacteria can confer a similar level of long-term protection as the PIV, we compared the protective efficacy against virulent NMI infection between 10⁵ GE of viable NMII bacteria and 10 µg of PIV in BALB/c mice at 120 days post SQ immunization. As shown in Figure 7 , compared to unvaccinated and adjuvant-immunized mice, both viable NMII-immunized and PIV-immunized mice were able to protect NMI infection-induced significant body weight loss at different times post challenge ( Figure 7A ) and significantly reduced the splenomegaly ( Figure 7B ) and bacterial burden in the spleens ( Figure 7C ) at 14 days post challenge at a comparable level. In addition, there was no significant difference in splenomegaly and bacterial burden in the spleens between viable NMII-immunized and PIV-immunized mice. These results demonstrated that viable NMII bacteria induced a similar level of long-term protection against virulent NMI challenge as the PIV. Notably, a nodule-like inflammatory response was observed at the site of injection in PIV-immunized mice but the abnormal response at the site of injection did not appear in viable NMII-immunized mice. This observation indicates that immunization with viable NMII bacteria unlikely will induce adverse reactions as the PIV, suggesting that viable NMII bacteria might be useful as a live attenuated vaccine to replace the existing Q-Vax for prevention of human Q fever.

The results from the above experiments demonstrate that viable NMII bacteria can elicit long-lasting significant protection against challenge with virulent NMI strains. However, it remains unknown if viable NMII bacteria can confer cross-protection against challenge with other virulent C. burnetii isolates. To address this question, we evaluated the cross-protective efficacy of viable NMII bacteria against virulent C. burnetii Priscilla and Scurry strains, which are chronic Q fever-associated isolates. Three groups of BALB/c mice were IN immunized with 1 × 10⁵ of viable NMII bacteria and IP challenge with 1 × 10⁷ of virulent NMI, Priscilla, or Scurry bacteria at 28 days post-immunization. In addition, three groups of PBS-immunized mice were infected with 1 × 10⁷ of NMI, Priscilla, or Scurry bacteria and used as infection control for each respective strain. Splenomegaly and bacterial burden in the spleen were measured at 14 days after virulent C. burnetii infection. As shown in Figure 8 , compared to unimmunized and C. burnetii-infected control mice, viable NMII-immunized mice were able to protect NMI, Priscilla, and Scurry infection-induced transitional body weight loss at 3 days post challenge ( Figure 8A ) and significantly reduced the splenomegaly ( Figure 8B ) and bacterial burden in the spleens ( Figure 8C ) at 14 days post challenge. These results demonstrated that IN immunization of mice with viable NMII bacteria conferred significant protection against various virulent C. burnetii isolate infection, suggesting that viable NMII bacteria can confer broad protection against challenge with heterogeneous isolates of virulent C. burnetii.

To determine the roles of humoral and cellular immunity in viable NMII-induced protection, we tested if adoptive transfer of immune sera or splenocytes from NMII-vaccinated mice can confer protection in naive mice against NMI infection. Immune sera and splenocytes were isolated from PBS or 1 × 10⁵ of viable NMII bacteria-immunized BALB/c mice at 14 days post immunization and transferred to naive BALB/c mice by IP injection, respectively. Immune sera, splenocytes, or PBS receiving mice were challenged with 1 × 10⁷ of NMI bacteria at 3 days after adoptive transfer. Splenomegaly and bacterial burden in the spleen were measured at 14 days post NMI challenge. Compared to mice that received PBS, sera, or splenocytes from PBS-immunized mice, mice receiving immune sera or splenocytes from NMII-immunized mice were able to protect NMI infection-induced significant body weight loss at 3 and 7 days post challenge ( Figure 9A ) and significantly reduced the splenomegaly ( Figure 9B ). In addition, compared to mice that received PBS, sera, or splenocytes from PBS-immunized mice, the bacterial burden in the spleens was significantly reduced in mice receiving splenocytes from NMII-immunized mice but was similar in mice receiving immune sera from NMII-immunized mice ( Figure 9C ). These results suggest that both humoral and cellular immunities are involved in the viable NMII-induced protection but cellular immunity may play a critical role in controlling bacterial replication in mice.

To further understand the mechanisms of viable NMII bacteria-induced protective immunity, we examined if B cell, T cell, CD4⁺ T cell, or CD8⁺ T cell deficiency in mice will significantly affect the ability of viable NMII bacteria to confer protection against virulent NMI infection. C57BL/6J wild-type (WT) and B cell-, T cell-, CD4⁺ T cell-, or CD8⁺ T cell-deficient mice were IP immunized with viable NMII bacteria and challenged with NMI at 28 days post-immunization. Additionally, naive WT mice were infected with NMI and served as unimmunized controls. Splenomegaly and bacterial burden in the spleen were examined at 14 days post challenge. As shown in Figure 10A , NMI infection induced a significant body weight loss in unimmunized WT and NMII-immunized B cell-deficient and T cell-deficient mice at different times post challenge, but there was no body weight loss in NMII-immunized WT, CD4⁺ T cell-deficient, and CD8⁺ T cell-deficient mice. In addition, compared to unimmunized WT mice, NMI infection-induced splenomegaly was significantly reduced in all NMII-immunized mice ( Figure 10B ). However, although splenomegaly was similar between NMII-immunized B cell-deficient and T cell-deficient mice, it was significantly higher than NMII-immunized WT, CD4⁺ T cell-deficient, and CD8⁺ T cell-deficient mice. As shown in Figure 10C , the bacterial burden was comparable in the spleens from NMII-immunized WT, B cell-deficient, CD4⁺ T cell-deficient, and CD8⁺ T cell-deficient mice, but it was significantly higher in the spleen from NMII-immunized T cell-deficient mice. Notably, the bacterial burden in the spleen from NMII-immunized T cell-deficient mice was even significantly higher than that in NMI-infected unimmunized WT mice. Collectively, these results demonstrated that viable NMII bacteria conferred a comparable level of protection against virulent C. burnetii challenge in WT, CD4⁺ T cell-deficient, and CD8⁺ T cell-deficient mice, and a partial protection in B cell-deficient and T cell-deficient mice. The observation that the bacterial burden in the spleens from NMII-immunized T cell-deficient mice was significantly higher than that in NMI-infected unimmunized WT mice suggests that T cells may play a critical role in controlling virulent C. burnetii replication in mice. Thus, viable NMII bacteria-induced protective immunity depends on both B cells and T cells, but T cells may play a critical role in controlling bacterial replication.