For intratracheal infections, C. burnetii Nine Mile phase I (NMI) clone 7 (RSA493) was grown in embryonated yolk sacs, then purified using gradient centrifugation as described previously (22). To produce WCV, cultures of C. burnetii NMI RSA493 were grown in ACCM-2 media as described in Omsland et al. (23), then inactivated in 2% formalin for 48 hours (14, 23). WCV was administered as 2 µg, 10 µg, 30 µg, or 50 µg doses by dry weight (1 mg WCV = 3.7x10¹⁰ cells) (24). Experiments involving live C. burnetii NMI RSA493 were performed in biosafety level 3 (BSL3) facilities at Texas A&M Health Science Center.

Female C57Bl/6NHsd (C57) and BALB/c mice (BAL), 6-8 weeks old, were purchased from Envigo (Huntingdon, UK) and female SKH1-Elite (SK) mice, 6-8 weeks old, were purchased from Charles River Laboratories (Wilmington, MA). Mice were housed in microisolator cages under pathogen-free conditions and given free access to food and water. Animals were housed in approved animal biosafety level 3 or level 2 facilities and all experiments were performed under an animal use protocol approved by the Institutional Animal Care and Use Committee at Texas A&M University.

For infection-sensitization (NMI), mice were intratracheally inoculated with 10⁵ or 10⁶ genomic equivalents (GE) of live C. burnetii as previously described with some modifications (22). Briefly, mice were anesthetized by intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine. Mice were then placed on a Mouse Intubation Platform (Penn-Century; Wyndmoor, USA) at a 45° angle and a 20-gauge catheter, needle removed, was inserted into the trachea. Live bacteria were administered through the catheter in 30 µL of sterile PBS. Infection-sensitized mice were monitored for clinical signs and weighed three times per week for two weeks post-inoculation. For vaccine-sensitization (WCV), mice were anesthetized as above and 50 µg of WCV in 50 µL of sterile PBS was administered subcutaneously (SC) in the middle of the back. Unsensitized control mice (PBS) were given a subcutaneous vaccination with 50 µL PBS alone. Mice were rested for 5-6 weeks post-sensitization prior to elicitation.

For elicitation of vaccine reactions mice were anesthetized as above. In haired mice, vaccination sites were first shaved using electric clippers followed by the application of a depilatory cream for 30 seconds. Mice were vaccinated SC with 2 µg, 10 µg, or 30 µg of WCV in 50 µL of sterile PBS or with 50 µL sterile PBS alone into the right and left flanks. Vaccine sites were visually monitored daily for two weeks and local induration was measured using calipers, then mice were euthanized and tissues were collected for histopathology or flow cytometry.

Serum samples were collected from either the submandibular vein or by intra-cardiac stick at pre-sensitization, post-sensitization, and post-elicitation time points for measurement of anti-C. burnetii IgG titers. Briefly, flat-bottomed 96-well plates were coated in 5 µg/mL C. burnetii antigen overnight then blocked in 3% powdered milk for 2 hours. Serum was pooled from 5 mice per group, diluted in PBS with 1% powdered milk, and serial dilutions were applied to the plate and incubated for 2 hours at 37°C. After washing three times with PBS with 0.05% tween 20, plates were then incubated with HRP-conjugated goat anti-mouse IgG antibody (1:10,000) for 2 hours at 37°C. 3,3′,5,5′-Tetramethylbenzidine (TMB) was used as substrate and OD was measured at 490 nm by a Biotek 800 TS Absorbance Reader.

Vaccination sites, lungs, and spleens were collected into 10% neutral buffered formalin and fixed for a minimum of 48 hours. Tissues were serially trimmed (2-4 sections per tissue) and placed in cassettes before submission to AML Laboratories (Jacksonville, FL, USA) for processing, embedding, and sectioning at 5 µm. Slides were stained with hematoxylin and eosin (HE). Histopathology of vaccine sites was assessed on de-identified, HE-stained slides using semi-quantitative scoring by a board-certified veterinary pathologist. Vaccine sites were scored from 0-5 based on lesion size, immune cell infiltrate, and areas of suppurative necrosis. Briefly, 0: no lesions, 1: minimal immune cell infiltrate, 2: mild, focal immune cell infiltrate, 3: moderate multifocal immune cell infiltrate, 4: moderate to severe, diffuse immune cell infiltrate, 5: severe, diffuse immune cell infiltrate with areas of suppurative necrosis.

For immunohistochemistry (IHC), unstained slides were deparaffinized and rehydrated by incubating in three washes of xylene, three washes of 100% ethanol, and one wash in 95% ethanol, 70% ethanol, 50% ethanol, and deionized water for 3 minutes each. Slides were then incubated in 3% hydrogen peroxide at room temperature for 10 minutes. Antigen retrieval was performed by incubating slides in Tris-EDTA buffer (10mM Tris Base, 1 mM EDTA, 0.05% Tween 20, pH 9.0) at 100°C for 20 min followed by washing in tap water for 10 min. Slides were blocked in TBS with 1% powdered milk for 2 hours. Primary antibodies were diluted in TBS with 0.5% bovine serum albumin (BSA) and applied to slides for 2 hours at room temp or overnight at 4°C. Slides were washed in TBS with 0.025% Triton-X 100 then incubated with HRP-conjugated goat anti-rabbit IgG diluted in TBS with 0.5% BSA for 2 hours at room temp or overnight. For fluorescence, Tyramide Reagents (Thermofisher) were applied to the slide per manufacturer directions for 10 min at room temp, then antigen retrieval was repeated for 2 min to strip antibodies before repeating the antibodies for the next antigen. Finally, TrueVIEW Autofluorescence Quenching Kit with DAPI (Vector, cat. SP-8500) was used to decrease autofluorescence, stain nuclei with DAPI and coverslip the slides. Slides were allowed to cure overnight at room temp.

HE-stained and IHC slides were scanned at 20X magnification using an Olympus VS120 Slide Scanner (Integrated Microscopy and Imaging Laboratory, Texas A&M University). For HE slides, brightfield images were collected. For fluorescent slides, DAPI, FITC, and TxRed channels were used to acquire images. Lesion measurements and cell counts on slide images were performed using QuPath v0.2.0-m8 (25). Neutrophils, macrophages, and lymphocytes were evaluated by morphology and quantified by counting cells within ten representative 100 µm² fields on H&E. For CD3+ T cells and CD19+ B cells, all cells within the lesions area were quantified using positive cell analysis on IHC slides.

Primary antibodies were purchased from Cell Signaling Technology: anti-CD3ϵ (D7A6E) and anti-CD19 (D4V4B). HRP-conjugated Goat anti-rabbit IgG (Novus Biologicals) diluted 1:1000 was used as the secondary antibody. Fluorescent Tyramide Regents (Thermofisher) used were AlexaFluor 488 and AlexaFluor 555 or AlexaFluor 594.

For spleens, single cell suspensions were produced by pressing spleens through a 70 µm cell strainer in cold 3 mL FACs buffer (PBS with 2% fetal bovine serum, 0.1% NaN3, pH 7.2) then washing with FACs buffer. Splenocytes were centrifuged then resuspended in ACK Lysing Buffer (Thermofisher) for 1 min to remove red blood cells. Cells were resuspended in 1 mL RPMI complete and an aliquot was mixed with 10 µL trypan blue to quantify cells using a Countess II (Thermofisher). Splenocytes were then diluted to 10⁷ cells/mL.

For vaccination sites, a 10 mm punch biopsy was used to collect the skin and subcutis at the elicitation site. Using a razor blade the subcutis and dermis were scraped from the overlying epidermis and placed in a gentleMACS C-tube for processing using the gentleMACS mouse adipose tissue dissociation kit (Miltenyi Biotec). Vaccine sites were dissociated using the Miltenyi Biotec gentleMACS Octo Dissociator with heaters. Cell suspensions were centrifuged and resuspended in 1 mL RPMI complete and cells were quantified as above then diluted to 10⁷ cells/mL.

Cells were centrifuged and resuspended in 1:1000 anti-mouse CD16/CD32 in FACs buffer and incubated on ice for 10 min. Next cells were stained with 1 µL/mL Zombie Violet or Zombie Aqua live/dead dye (Biolegend) and incubated on ice for 5 min. Cells were then incubated in fluorochrome-conjugated cell surface antibodies on ice for 30 min. For intracellular staining, cells were fixed and permeabilized using FoxP3/Transcription Factor Staining Buffer Set (eBioscience) followed by incubation with intracellular antibodies for 30 min on ice. Cells were then resuspended in FACs buffer and kept at 4°C until analysis. For intracellular cytokine and FoxP3 expression, cell suspensions from spleens and vaccine sites were aliquoted into a round-bottomed 96-well plate with or without 20 µg/mL WCV and cultured for 18 hours at 37°C with 5% CO₂ followed by the addition of GolgiPlug (BD Biosciences) for 6 hours prior to antibody staining. Stimulation of cells by 50ng/mL of phorbol 12-myristate 13-acetate (PMA) and 1mM ionomycin for 6 hours was used as a positive control (Data not shown).

Flow cytometric antibodies included CD3ϵ-PE/Cy7, CD8-FITC, CD4-APC/Cy7, IL17a-BV711, IFNγ-APC, IL4-PE, FoxP3-BV421, CD69-BV711, CD44-PE, and CD62L-APC (BioLegend). Cells were counted using the BD LSRFortessa X-20 Flow Cytometer and analyzed using FlowJo v10.6.2 (FlowJo LLC.).

Statistical analyses were calculated using Prism v7.0 (GraphPad Software Inc.). Results were compared using one-way ANOVA with Dunnett’s correction for multiple comparisons. Differences were considered significant if p-value ≤ 0.05 (*), ≤ 0.01 (**), ≤ 0.001 (***), or ≤ 0.0001 (****).

Guinea pigs are the current animal model of choice for evaluation of hyper-reactive lesions to C. burnetii vaccines, however, antibodies targeting guinea pig cell markers are limited which inhibits immunologic investigation of these responses. To address this, we developed a novel mouse model of the C. burnetii whole cell vaccine reactogenic response. To develop our model, we tested the ability to reproduce C. burnetii reactogenic responses in SKH1(SK), C57Bl/6 (C57), and BALBc (BAL) mice. SK mice are outbred, immune-competent, and hairless due to a mutation in the Hr gene (26). C57 and BAL mice were chosen as Th1- and Th2-biased strains, respectively, to compare responses by immunophenotype (27). C57, BAL, and SK mice (n=4-5/group) were sensitized to C. burnetii by intratracheal inoculation with 10⁵ or 10⁶ GE C. burnetii RSA493 (NMI), by SC vaccination with 50 µg WCV in 50 µL sterile PBS (WCV), or by SC vaccination with 50 µL sterile PBS (PBS) (22). Mice were monitored for 14 days post-sensitization by measuring weight change. Infection-sensitized SK and C57 mice showed transient weight loss followed by recovery by day 14 while infection-sensitized BAL, all vaccination-sensitized, and all control groups showed no overt weight loss over the observation period ( Figure 1A ). For elicitation, mice were given a SC vaccination of 10 µg WCV in 50 µL sterile PBS and PBS alone into the right and left flanks, respectively, and vaccination sites were monitored for swelling and erythema daily for 14 days until euthanasia. Day 14 post-elicitation was chosen as the endpoint based on previous reports and our own work in guinea pigs showing induration is most severe at approximately 11-15 days post-vaccination (11). Mice in all experimental groups showed no evidence of weight loss during elicitation ( Supplementary Figure 1 ).

Blood samples were collected prior to sensitization (pre-sensitization), one day before elicitation (post-sensitization), and at necropsy (post-elicitation) to evaluate antigen-specific antibody formation in serum as a measure of sensitization. Anti-C. burnetii IgG titers showed seroconversion at post-sensitization and post-elicitation time points in both infection- and vaccine-sensitized groups in all mouse strains. SK and BAL mice as well as infection-sensitized C57 mice produced IgG titers of ≥1:1600. Vaccine-sensitized C57 mice produced modest IgG titers of 1:200. No anti-C. burnetii IgG titers were detected in unsensitized controls at any time point ( Figure 1B ).

Focal swellings at the WCV site were observed beginning at day 8 in infection-sensitized and day 10 in vaccine-sensitized SK mice, but only occurred in 3 of 5 mice in each of these groups ( Figure 2A and Supplementary Figure 1 ). The vaccine site swellings measured 2 mm thick using skin calipers compared to 1 mm in normal mouse skin. Swellings were not observed grossly in the control SK mice or any of the C57 and BAL mouse groups, however, hair regrowth in both strains and skin pigmentation in C57 mice were common during the elicitation period.

Vaccination sites were collected in formalin at 14 days post-elicitation for histopathology. HE-stained slides of vaccination sites showed more severe lesions in infection- and vaccine-sensitized C57 and SK mice compared to unsensitized controls, but vaccine lesions in BAL mouse groups did not significantly differ ( Figure 2B ). Local reactive lesions consisted of infiltrates of macrophages, neutrophils, and lymphocytes with multifocal areas of suppurative necrosis and degeneration ( Figure 2C ). Infection- and vaccine-sensitized lesions in C57 and SK mice did not significantly differ by the type of cellular infiltrate or overall severity of lesions.

Although C. burnetii WCV reactogenicity is mainly associated with previous sensitization, higher doses of WCV can produce significant local inflammation through innate responses that may obscure the hypersensitivity response (16). To target the hypersensitivity response, we continued by assessing multiple elicitation doses to determine which dose maximized the cellular influx produced by adaptive immunity without obscuring the lesions with excessive innate responses. C57 mice were sensitized with WCV or PBS as described above then administered a SC elicitation dose of 2 µg, 10 µg, 30 µg, and sterile PBS in a volume of 50 µL into either the right or left flank. At 14 days post-elicitation, vaccine sites were collected in formalin for histopathology and immunohistochemistry. Both sensitized and unsensitized groups showed dose-dependent responses to vaccination, however sensitized mice consistently showed significantly more severe responses than unsensitized mice at the same dose based on semi-quantitative scoring of HE-stained slides ( Figures 3A, B ). However, the mean size of the lesions only significantly differed between sensitization groups at the 30 µg dose ( Figure 3C ). Interestingly, suppurative necrosis was only evident in sensitized mice at the 10 µg and 30 µg doses ( Figure 3C ). Individual cell counts of neutrophils, lymphocytes, and macrophages showed variable differences within dose groups. Neutrophils were increased in sensitized mice compared to unsensitized mice at all doses, while macrophages were only increased at the 2 µg dose and lymphocytes were only increased at the 10 µg dose when comparing sensitization ( Figure 4A ).

Immunohistochemistry was used to further differentiate lymphocytes into CD3+ T cells and CD19+ B cells. At the 10 µg dose, both B and T cells were significantly increased in sensitized mice compared to unsensitized, but there was no difference in these populations at the 30 µg dose ( Figure 4B ). In all sections, T cells were distributed evenly throughout the lesion except in deep, often perivascular, clusters of lymphocytes forming ectopic lymphoid follicles (ELF) where they formed dense aggregates with CD19+ B cells. There were significantly more ELFs in sensitized mice at the 10 µg dose but not the 30 µg dose ( Figure 4C ) compared to unsensitized mice. In contrast, B cells were only present within ELFs ( Figure 4D ).

Because delayed-type hypersensitivities are mediated by T cells and experiments showed a marked influx of CD3+ T cells on immunohistochemistry of vaccine site lesions, we used flow cytometry to further characterize T cell subsets that are increased during WCV reactogenic responses both locally and systemically. To do so, we extracted cells from the vaccine sites and spleens of mice at 14 days post-elicitation. Vaccine-sensitized and unsensitized mice as described above were vaccinated SC at four separate sites in the right and left flank, then spleens and vaccine sites were collected at 14 days post-elicitation. The four vaccine sites from each mouse were pooled prior to cell separation for flow cytometric evaluation. The 10 µg elicitation dose was chosen for these experiments since this dose produced maximal differences in lymphocyte responses between sensitization groups. Extracted cells were stimulated with WCV and stained for surface markers and expression of IFNγ, IL4, IL17a, and FoxP3 as markers for Th1, Th2, Th17, and Treg cells, respectively. Unsensitized mice elicited with injections of PBS only were used to compare responses to normal cell populations in the skin and spleen (Sham).

Local vaccine sites showed marked influx of total cells and T cells compared to unsensitized and sham mice ( Figures 5A, B ). CD4+ and CD8+ T cells in sensitized mice were similarly increased at vaccine sites compared to unsensitized and sham groups ( Figures 5A, B ). Evaluation of cytokine production by CD4 T cells in sensitized mice showed a significant increase in IFNγ production compared to the sham group but not unsensitized mice. Although the mean of IFNγ+ CD4+ T cells in unsensitized mice was increased compared to sham mice, this result was not significant (p=0.0818) ( Figures 6A, B ). Similarly, CD4+ IL17a-secreting cells were elevated in sensitized mice compared to unsensitized and sham mice ( Figures 6A, B ). However, unsensitized mice did not have an increase in IL17a-secretion by CD4+ T cells compared to sham mice. In contrast, IL4+ CD4+ T cells were significantly decreased in sensitized mice compared to sham ( Figures 6A, B ). FoxP3+ CD4+ T cells and production of IFNγ, IL4, and IL17a by CD8+ T cells did not significantly differ between experimental groups ( Figures 6A, B and Supplementary Figure 2 ).

We next evaluated the local effector, central, and resident memory T cells populations within vaccine sites using the surface markers CD44, CD62L, and CD69. CD44 is upregulated in activated T cells and is important for recruitment to sites of inflammation while CD62L is a lymph node homing receptor (28). CD44+CD62L+ central memory T cells (T_CM) normally circulate within the blood and lymphoid tissues and rapidly expand in response to antigen re-stimulation while CD44+CD62L- effector memory T cells (T_EM) home to peripheral tissues in response to chemoattractants and produce cytokines in response to re-stimulation (29, 30). Similar to T_EM, resident memory T cells (T_RM) downregulate CD62L but also upregulate CD69 which sequesters sphingosine 1-phosphate receptor 1 (S1PR1) preventing tissue egress. This causes T_RM to remain within the peripheral tissues after sensitization to provide tissue-specific immune memory (29, 31). Evaluation of memory T cell populations from vaccine sites showed significant increases in CD4 and CD8 T_EM and T_CM populations in sensitized mice compared to unsensitized and sham controls ( Figures 7A, B ). CD4+ T_RM from sensitized mice were also significantly increased compared to unsensitized and sham controls, while CD8+ T_RM from sensitized mice were significantly increased compared to sham mice but not unsensitized mice. There were no significant differences in memory T cell populations between unsensitized and sham mice.

WCV reactogenic responses reported in humans are not confined to the vaccine site and may manifest as fever, fatigue, malaise, and joint pain (9, 15). To determine if these systemic responses are reflected in systemic T cell expansion and activation, we evaluated T cell subpopulations extracted from spleens of sensitized, unsensitized, and sham mice at 14 days post-elicitation. The total numbers of cells extracted from the spleens of mice did not significantly differ between experimental groups ( Figure 8B ). Evaluation of splenocytes showed a mild, but significant decrease in the proportion of CD4+ T cells in sensitized compared to unsensitized and sham controls in response to elicitation ( Figures 8A, B ). Both CD4+ and CD8+ T cells showed significant increases in the proportions of T_CM compared to unsensitized and sham groups ( Figures 8A, B ). CD8+ T_EM from sensitized mice were decreased compared to sham but not unsensitized mice, while CD4+ T_EM did not differ among groups ( Figures 8A, B ).

Similar to local vaccine sites, splenic CD4 T cells from sensitized mice produced significantly more IFNγ+ and IL17a+ cells compared to unsensitized and sham controls. CD4 T cells from sensitized mice also showed significantly less IL4+ cells than sham mice ( Figures 9A, B ). FoxP3+ CD4 T cells did not differ across experimental groups ( Figures 9A, B ). Among CD8 T cells in the spleen, IFNγ+ cells were significantly increased in unsensitized mice compared to sensitized mice, but not sham mice. There were no significant differences in IL4+ and IL17a+ CD8 T cells among experimental groups ( Supplementary Figure 3 ).