QuilA® adjuvanted Coxevac® sustains Th1-CD8+-type immunity and increases protection in Coxiella burnetii-challenged goats

Tomaiuolo, Sara; Jansen, Wiebke; Soares Martins, Susana; Devriendt, Bert; Cox, Eric; Mori, Marcella

doi:10.1038/s41541-023-00607-z

QuilA® adjuvanted Coxevac® sustains Th1-CD8⁺-type immunity and increases protection in Coxiella burnetii-challenged goats

Article
Open access
Published: 14 February 2023

Volume 8, article number 17, (2023)
Cite this article

Download PDF

You have full access to this open access article

npj Vaccines

QuilA® adjuvanted Coxevac® sustains Th1-CD8⁺-type immunity and increases protection in Coxiella burnetii-challenged goats

Download PDF

1845 Accesses
6 Citations
3 Altmetric
Explore all metrics

Abstract

Coxevac® is the EMA-approved veterinary vaccine for the protection of cattle and goats against Q fever, a zoonotic bacterial disease due to Coxiella burnetii. Since Coxevac® reduces bacterial shedding and clinical symptoms but does not prevent infection, novel, ready-to-use vaccine formulations are needed to increase its immunogenicity. Here, a goat vaccination-challenge model was used to evaluate the impact of the commercially available saponin-based QuilA® adjuvant on Coxevac® immunity. Upon challenge, the QuilA®-Coxevac® group showed a stronger immune response reflected in a higher magnitude of total IgG and an increase in circulating and splenic CD8⁺ T-cells compared to the Coxevac® and challenged-control groups. The QuilA®-Coxevac® group was characterized by a targeted Th1-type response (IFNγ, IP10) associated with increased transcripts of CD8⁺ and NK cells in spleens and γδ T cells in bronchial lymph nodes. Coxevac® vaccinated animals presented an intermediate expression of Th1-related genes, while the challenged-control group showed an immune response characterized by pro-inflammatory (IL1β, TNFα, IL12), Th2 (IL4 and IL13), Th17 (IL17A) and other immunoregulatory cytokines (IL6, IL10). An intriguing role was observed for γδ T cells, which were of TBX21- and SOX4-types in the QuilA®-Coxevac® and challenged control group, respectively. Overall, the addition of QuilA® resulted in a sustained Th1-type activation associated with an increased vaccine-induced bacterial clearance of 33.3% as compared to Coxevac® only. QuilA® could be proposed as a readily-applied veterinary solution to improve Coxevac® efficacy against C. burnetii infection in field settings.

Contributions of lipopolysaccharide and the type IVB secretion system to Coxiella burnetii vaccine efficacy and reactogenicity

Article Open access 19 March 2021

The adjuvant G3 promotes a Th1 polarizing innate immune response in equine PBMC

Article Open access 22 October 2018

A proof-of-concept study to investigate the efficacy of heat-inactivated autovaccines in Mycobacterium caprae experimentally challenged goats

Article Open access 22 December 2022

Introduction

Q fever is a zoonotic bacterial infection leading to significant public health and economic issues worldwide. The causative agent, Coxiella burnetii, is a highly infectious intracellular Gram-negative bacterium, which is extremely resistant in the environment¹. In ruminants, C. burnetii infection causes abortion, stillbirth and infertility, but distinct clinical outcomes are observed between ruminant species: abortion storms in goats and sheep, and sporadic abortion cases in cattle^2,3. The parturition or immediate postpartum periods are those at the highest risk in terms of bacterial shedding, however, the excretion might occur also beyond these periods by clinically affected or asymptomatic animals^4,5.

Knowledge about the pathogenesis of C. burnetii and the associated immune responses in domestic ruminants is scarce. In pregnant goats, bacterial replication starts in the trophoblasts of the allantochorion^6,7,8. Bacterial DNA can be detected in the placenta 4 weeks post-infection (wpi), suggesting that C. burnetii needs time to multiply until reaching detectable levels⁸. Massive bacterial multiplication occurs however in the last weeks of gestation, before abortion⁷. From 6 wpi, C. burnetii DNA can also be present in other organs than the placenta⁸. Whether the function of these infected organs is affected by the presence of C. burnetii is yet unknown.

Infection of goats with C. burnetii induces considerable antibody responses, indicating that humoral immunity contributes to the defense mechanisms against Q fever. A similar magnitude of antibody responses has been observed in pregnant and non-pregnant goats^9,10. In both, IgM and IgG antibodies against C. burnetii phase II (phII, avirulent) antigens appear from 3 wpi onwards. In non-pregnant goats, phase I (phI, virulent) IgM and IgG titers increase after 3 and 4 wpi, respectively. This anti-phI antibody response is delayed in infected pregnant goats.

Although cell-mediated immune responses are important for protection against infection with intracellular pathogens, both the nature of the induced cell-mediated immune responses and their role in protecting goats from infection with C. burnetii remain poorly described. Roest et al.⁹ showed that cytokine mRNA levels in peripheral blood of pregnant goats increased before (IL10) or after (TNFα, IL1β) parturition. This coincided with an up-regulation of the total IFNγ production in peripheral blood mononuclear cells (PBMCs) at one-week post parturition⁹. Ammerdorffer et al.¹¹ also observed increased IFNγ and TNFα transcript levels in antigen-stimulated PBMCs from infected pregnant goats. Thus, although some studies reported changes in cytokine production by PBMCs in pregnant goats, the identity of the involved cells remains unknown. In addition, no information is available regarding non-pregnant goats.

A deeper understanding of the pathogenesis of C. burnetii and the immune responses raised upon infection of domestic ruminants may contribute to the redefinement and the improvement of ruminant vaccines, deployed as a preventive measure to limit Q fever in animals and halt transmission to humans. In Europe, the Coxevac® non-adjuvanted whole-cell formalin-inactivated ph I vaccine (Ceva Santé Animale, Libourne, France) is used to protect cattle and goats against Q fever¹². Although several studies demonstrated its efficacy in reducing clinical signs (abortions) and bacterial shedding^13,14,15,16, this vaccine does not prevent infection^13,14,15 nor clears the infection in infected animals^17,18. In addition, field data indicated that antibody levels decrease after 9 months below protective levels, hampering targeted annual vaccination programs on herd level¹⁹. If the critical level of protective immunity is not maintained long enough at the individual or herd level, recrudescence will likely take place^19,20. In this case, consistent control of C. burnetii infection in ruminants is only reached through the use of costly and multiannual compliant vaccination protocols^14,19.

Many vaccines require the addition of adjuvants to induce an appropriate immune response for protection upon challenge. Adjuvants are able to increase the magnitude of the vaccine induced-immune responses, and, importantly, to tailor immune responses to each specific pathogen. Indeed, certain adjuvants can drive specific immune responses (i.e., CD4⁺ vs CD8⁺ T cells, Th1 vs Th2 response, distinct antibody isotypes), encourage immune memory responses or activate a quicker initial response²¹. QuilA®, a saponin adjuvant extracted from Quillaja saponaria, is widely used in veterinary vaccines due to its capacity to stimulate both CD4⁺ helper and CD8⁺ cytotoxic cells as well as to induce long-lasting antibody responses^{22,23,24,25,26,27}. It is an EMA-approved veterinary medicinal product and therefore immediately applicable in field settings. Considering both the non-adjuvanted nature of Coxevac® and the QuilA®-driven induction of T cell immunity, we selected this adjuvant as a candidate for improving the efficacy and immunogenicity of Coxevac®.

In this study, we characterized the immune response induced by Coxevac® vaccination and evaluated the influence of QuilA® on vaccine immunogenicity and efficacy in goats. In order to mimic natural infections, goats were challenged for the first time with an aerosolized axenic culture of C. burnetii, isolated from an infected goat in 2010. Considering the intracellular nature of the bacterium, obtaining an axenic culture from field samples is extremely rare and challenging. Our data demonstrated that Coxevac® did not generate an immune response able to confer substantial protection upon challenge. However, the addition of the QuilA® adjuvant enhanced the humoral response and induced a sustained Th1-type cellular response resulting in increased vaccine efficacy.

Results

Effective action of Coxevac® is usually achieved following compliant multiannual vaccination protocols, but field data indicated that protective antibody levels are not maintained long enough to establish standardized yearly vaccination programs¹⁹. Given that Coxevac® does not contain an adjuvant and the need to increase its efficacy, we designed an improved formulation of Coxevac® by including the ready-to-use QuilA® adjuvant in the emulsion. This new formulation was assessed in a vaccination-challenge experiment using Saanen goats and inocula prepared from axenic cultures of the CbBEC2 strain (P6) (Fig. 1a).

**Fig. 1: Overview of the experimental design, daily temperature and bacterial burden of the vaccination-challenge experiment in goats.**

In vitro and in vivo quality controls of the bacterial inoculum

The CbBEC2 strain was previously isolated in axenic medium at the National Reference Centre (Sciensano, Belgium). As the infective capacity of C. burnetii strains is determined by the expression of a phI lipopolysaccharide (LPS)²⁸, we verified the presence of a full-length LPS in this strain. The existence of genes involved in the synthesis of a phI LPS was investigated by Whole Genome Sequencing (WGS). The alignment of the CbBEC2 assembly and the Nine Mile (NM) phI DNA sequence (encoding for the operon involved in the biosynthesis of the complete LPS) resulted in 99.92% similarity, indicating an operon genetic integrity in the CbBEC2 strain. The complete LPS sequence was localized in contig 11 as illustrated in Supplementary Fig. 1a. To further examine the phase properties of the LPS, CbBEC2 at passage 6 (P6) was cultivated for 14 days in Acidified Citrate Cysteine Medium-2 (ACCM-2) and LPS was isolated and profiled on a silver-stained gel (Supplementary Fig. 1b). The phI pattern of CbBEC2 LPS was unique and differed from that of NM phI LPS (bands above 10 kDa). As LPS was isolated from the CbBEC2 strain at P6, we observed co-appearance of intermediate (around 10 kDa) and minor presence of phII (far below 10 kDa) LPS forms. These LPS features could be attributed to the serial passages²⁹ or the intrinsic nature of the strain³⁰. To verify that the infectious potential of the CbBEC2 axenic cultures (P6) was retained after passaging, we compared the proliferative capacity of CbBEC2 from freshly collected splenic harvest at P2 (estimated m.o.i. of 5 × 10³G.E.) to that from the axenic culture at P6 (estimated m.o.i. of 4 × 10³G.E.) in a mouse model. The axenic culture at P6 triggered significantly higher bacterial loads than the P2 in the spleen (Supplementary Fig. 1c, mean week1–week4 of 7.7 × 10⁵G.E./g vs 5.3 × 10⁴G.E./g). Also, the axenic strain triggered significantly higher organ weights in the spleen and liver and significantly higher IgM and IgG levels (Supplementary Fig. 2). Overall, these results validated the infectivity, the genetic features and the quality of the inoculum prepared from CbBEC2 cultures P6, which will be used in the next experiment for the challenge infection of goats.

QuilA®-Coxevac® prime vaccination induces a transient increase of the rectal temperature

None of the control and the Coxevac® goats experienced severe hyperthermia (>40 °C), as indicated by the rectal temperature, at any time point after prime vaccination or boost (Fig. 1b1). One day after prime vaccination, all animals from the QuilA®-Coxevac® group presented a rise in the rectal temperature (average of 39.9 °C ± 0.5), with three goats experiencing severe hyperthermia on days 1 to 3 (Fig. 1b2). After challenge, no major changes were observed in the groups, although a slight increase of the average temperature was observed in the control animals a few days post-challenge. One goat (V2) of the Coxevac® group experienced severe hyperthermia from day 32 post-challenge towards the end of the experiment (Fig. 1b2) and presented clinical signs attributable to mastitis (the left mammary gland was hard, swollen, reddish and sensitive to touch).

QuilA®-Coxevac® vaccination induced a robust and sustained anti-C. burnetii IgG response and an increased protection efficacy against Q fever

After vaccination, IgG antibodies against C. burnetii were significantly more protracted in the QuilA®-Coxevac® than in the Coxevac® group (4 to 12 weeks post vaccination (wpv) vs 4 to 9 wpv) as opposed to the control goats (Fig. 2a). The first goat that reached levels above the threshold defined by the ELISA assay was detected at 2 wpv in goats vaccinated with QuilA®-Coxevac®, whilst only at 4 wpv in the Coxevac® goats (Fig. 2b). All goats from the Coxevac® and QuilA-Coxevac® groups exceeded the threshold after 5 and 6 wpv, respectively.

**Fig. 2: Kinetics of antigen specific-IgG and IFNɣ production upon vaccination and challenge infection.**

Upon challenge, the first goat of the control group that crossed the threshold level was detected at 16.5 wpv (3.5 weeks post-challenge, wpc) and an increase in the total IgG production was reached at 18 wpv (5 wpc) (Fig. 2b). Throughout the experiment, two control goats remained below the threshold defined by the ELISA assay. In the vaccinated groups, IgG levels rose again at 17.5 and 18 wpv in the Coxevac® and QuilA®-Coxevac® goats, respectively. After the challenge, the magnitude of the induced IgG antibodies was significantly higher in the QuilA®-Coxevac® group compared to control and Coxevac® animals (73% S/P vs 18% S/P and 43% S/P, respectively), while the control and Coxevac® group did not differ significantly in IgG levels (Fig. 2a).

As humoral immunity plays a role in the protection against Q fever^31,32,33, we assessed whether vaccination with Coxevac® and QuilA®-Coxevac® was associated with increased protection. The presence of C. burnetii was investigated in several organs (n = 28) and blood after sacrifice (Fig. 1c and Supplementary Tables 1 and 2). The detection limit of the PCR assay was reached when results on DNA extracted from tissues were negative. Thus, splenocytes and bronchial lymph node cells were injected in embryonated eggs for bacterial amplification. An animal was considered positive if at least one of the two results was positive. C. burnetii was detected in 100% of the control and Coxevac® goats and in 66.7% of the QuilA®-Coxevac® goats (Fig. 1c) resulting in an increased vaccine-induced bacterial clearance of 33.3% as compared to Coxevac® only. Also, bacteremia was evaluated in all challenged goats at selected time points post-infection by PCR (Supplementary Table 2). In vaccinated goats, bacteremia was principally detected 1-day post-challenge. Taken together, these results confirmed that all goats were successfully infected with C. burnetii CbBEC2 strain and that QuilA® increased the Coxevac®-induced IgG response and protection.

Differential IFNγ secretion profiles upon antigen-specific stimulation of PBMCs

To further explore cell-mediated immunity upon vaccination and challenge, we assessed the antigen-specific IFNγ response. PBMCs were ex vivo stimulated with C. burnetii antigens and the IFNγ production was measured at different time points. Upon vaccination, the IFNγ secretion profile in the Coxevac® group was detected as a unimodal response with a peak at 5 wpv (after the boost vaccine dose, 61% S/P). In contrast, the IFNγ kinetics of the QuilA®-Coxevac® group resulted in a bimodal response with the first peak of IFNγ production after the prime (2 wpv, 45% S/P) and the second peak after the booster (5 wpv, 25% S/P) vaccination (Fig. 2c). Upon challenge, completely different IFNγ patterns were observed for the three groups. In the control group, moderate levels of IFNγ production were detected immediately after the challenge (13.5 wpv), followed by a sudden increase at 17–17.5 wpv (4–4.5 wpc) and maintained until the end of the experiment. The IFNγ response of the Coxevac® group remained moderate and stable until the end of the experiment. In contrast, a bimodal trend characterized again the response of the QuilA®-Coxevac® group with the highest peak detected promptly after the challenge (13.5–14 wpv) and a second peak at 17.5–18 wpv. The mixed-effects model corroborated statistically significant differences between the three patterns post-challenge. This is illustrated by the interaction between time and group variables of control and Coxevac® (F(13, 102) = 5.02; p < 0.0001), control and QuilA-Coxevac® (F(13, 102) = 4.5; p < 0.0001), Coxevac® and QuilA®-Coxevac® (F(13, 129) = 1.96; p < 0.02) groups.

The two Coxevac® formulations activate different immune cell subsets upon challenge

Considering the differential IFNγ recall response observed in the three groups, we next assessed whether these differences could be attributed to distinctive activation of lymphocyte subpopulations. Therefore, we investigated the kinetics of T (CD4⁺ and CD8⁺) and B (CD21⁺) cells in PBMCs upon vaccination and challenge (Fig. 3a). The frequency of CD4⁺ and CD21⁺ cells did not change between groups after vaccination nor challenge (Fig. 3b). Likewise, vaccination did not influence the percentage of CD8⁺ cells detected before challenge, however, vaccinated groups showed significantly different patterns as compared to the control group after challenge. The results were validated with the mixed-effects model analysis that revealed a significant interaction between time and group variables of the control and Coxevac® (F(8, 64) = 2.48; p < 0.02) and control and QuilA®-Coxevac® (F(8, 64) = 2.68; p < 0.01) groups. At 19 wpv, the CD8⁺ frequency in PBMCs was higher in animals vaccinated with QuilA®-Coxevac® compared to the Coxevac® group (Fig. 3b). This CD8⁺ population was also strongly present in splenocytes of the QuilA®-Coxevac® group at sacrifice (28.0% in QuilA-Coxevac® vs 15.9% in control and 18.3% in Coxevac®) (Fig. 4a). Interestingly, the increased frequency of CD8⁺ cells in spleens was associated with an increased frequency of a granulocytic population (3.4% in QuilA®-Coxevac® vs 1.1% in control and 1.8% in Coxevac®) (Fig. 4b). The frequency of the other sub-populations was comparable between groups in all investigated organs (Fig. 4a and Supplementary Fig. 3).

**Fig. 3: The two Coxevac® formulations activate cell subsets of different nature upon challenge.**

**Fig. 4: Higher frequencies of CD8⁺ cells and granulocytes detected in the spleen of the QuilA®-Coxevac® group at sacrifice.**

For the CD4⁻CD8⁻ cell population in PBMCs, the mixed-effects model did not identify different patterns between groups (F(16, 104) = 0.7; p = 0.7), but revealed significant differences in the average CD4⁻CD8⁻ frequency between groups post-challenge (F(2, 13) = 4.803; p < 0.02). At several time points (13, 13.5, 15, 16, 18, 19 wpv), significant pairwise differences between the Coxevac® and QuilA®-Coxevac® group were present. Additionally, at 19 wpv, the CD4⁻CD8⁻ frequency in the Coxevac® group was also significantly higher compared to control goats (Fig. 3b). To provide a deeper characterization of this CD4⁻CD8⁻ cell subpopulation, we looked at gamma-delta (γδ) T cells and WC1⁺ γδ T cells at 13.5 and 19 wpv. However, no significant differences in the relative percentage were observed between groups in these cell populations (Supplementary Fig. 4).

Distinctive transcriptional patterns are induced in spleens and bronchial lymph nodes of vaccinated and control animals upon challenge

We next sought to further investigate the immune responses activated by the different conditions. As such, we studied the expression profiles of 23 selected genes (comprising cytokines, CDs, receptors and transcription factors) in splenocytes and bronchial lymph node cells upon infection (Figs. 5 and 6). Vaccination with QuilA®-Coxevac® skewed the transcriptional gene expression induced by infection towards an increased expression of CD8, NRC1 and the Th1 cytokine IFNγ in spleen, corroborating the previous results of cell phenotyping and IFNγ production. In this organ, the expression of IL1β, IL17A, CD11b, and TRGC2 was instead more expressed upon challenge in the control group than in the other two groups (Fig. 5a). Gene expression in the respiratory lymph nodes highlighted the differences among groups. Here, IL12p40, IL6, and CD11b were more expressed upon challenge in the control group than in vaccinated groups. TLR6 was also significantly more expressed in the Coxevac® compared to the QuilA®-Coxevac® group (Fig. 6a).

**Fig. 5: Distinctive transcriptional patterns are induced in spleens of vaccinated and control animals upon challenge.**

**Fig. 6: Distinctive transcriptional patterns are induced in bronchial lymph nodes of vaccinated and control animals upon challenge.**

To highlight specific transcriptional patterns, the relative gene expression was further used for high-level comparative analyses. In both splenocytes and bronchial lymph node cells, three different clusters were identified (Figs. 5b and 6b). The first cluster regrouped genes highly and intermediately expressed in the QuilA®-Coxevac® and Coxevac® groups, respectively. These genes included IFNγ, CD8, NRC1, and IP10 in splenocytes and TBX21, TRGC2, and IP10 in bronchial lymph nodes. The second cluster represented genes more expressed in challenged-control goats than the challenged-vaccinated groups. In splenocytes, it regrouped pro-inflammatory cytokines (IL1β, TNFα), Th2 cytokines (IL4 and IL13), IL17A and IL10, as well as CD11b and TRGC2 cellular markers and the SOX4 transcription factor. In bronchial lymph nodes, it consisted again of IL4, IL13, IL17A, and CD11b, but included IL6 and IL12 as well. The third cluster comprised genes that were homogeneously expressed in all groups or for which a specific pattern was unclear.

Specific immune responses distinguish Coxevac®, QuilA®-Coxevac® and control goats following C. burnetii challenge

In an attempt to reduce noise and extract representative information from the entire dataset to highlight immune patterns activated distinctly by vaccinated and control goats upon C. burnetii infection, we used the pattern recognition approach through the principal component analysis (PCA). When we used the complete dataset (n = 68), which includes data from serology (13.5, 17.5 and 19 wpv), IFNγ secretion upon antigen-specific stimulated PBMCs (13.5, 17.5 and 19 wpv), organ and blood (19 wpv) phenotyping and gene expression profiles, we observed that vaccinated animals from the two groups, Coxevac® and QuilA®-Coxevac®, were separated in two clusters (Fig. 7a). In these groups, even if crossing areas were present, the intra-group variance (σ²) was reduced compared to the control group (σ²_Coxevac = 0.71, σ²_{QuilA-Coxevac} = 0.99 vs σ²_Control = 1.35). Conversely, within the control group, the higher intra-group variance highlighted the different responses among animals present in this group, in particular for the C1 goat, who was the least responsive upon challenge. Overall, when considering a high level of complexity of variables, the three groups were quite close in both 2D and 3D plots (Fig. 7a). In contrast, selecting specific variables (n = 22) to reach distinctive clusters among conditions, characteristic features that can accurately describe each group were identified (Fig. 7b). The control group was distinguished by cytokines (i.e., IL1β, IL12, IL6, IL17A), CD11b⁺ cells in the secondary lymphoid organs, γδ T cells in spleen and IFNγ secretion upon antigen-specific stimulation of PBMCs. The Coxevac® group had a distinctive feature, the CD4⁻CD8⁻ lymphocyte population present in both systemic and secondary lymphoid organs. Finally, the QuilA®-Coxevac® group was distinguished by its increased IgG response, the higher frequency of CD8⁺ T cells in blood and secondary lymphoid organs, and by a greater expression of CD8, NRC1, and IFNγ in the spleen.

Discussion

In the present study, we characterized the immune responses elicited by Coxevac® vaccination upon challenge of goats with C. burnetii and evaluated whether adjuvanting this vaccine with QuilA, an EMA-approved veterinary medicinal product, could increase its protective efficacy against Q fever. Adjuvanting vaccines with QuilA® triggers more robust humoral and cellular immune responses against a wide range of pathogens than the non-adjuvanted forms^34,35,36,37. For the first time, an axenic culture of C. burnetii, isolated from a field sample, was used to produce the challenge inoculum. Before intranasal infection of goats with the CbBEC2 strain, we assessed the presence of a full-length LPS and the infectious potential of the bacterial strain in a mouse model, demonstrating the suitability of an axenic culture for the preparation of the challenge dose. Although small rodents and non-human primates have been used to study vaccine-induced protective immune responses, we decided to work with a goat model as they are natural hosts of C. burnetii and the target species of the Coxevax® vaccine.

In our experimental study, the humoral immunity was evaluated after vaccination and challenge in the caprine model by determining C. burnetii-specific IgG levels. Both Coxevac® and QuilA®-adjuvanted Coxevac® induced antigen-specific IgG, whilst only the adjuvanted form elicited a more protracted IgG response after vaccination. Upon challenge, the QuilA®-Coxevac® group mounted a stronger humoral response compared to both the challenged-control and the Coxevac® group, highlighting the enhanced effect promoted by the QuilA® in the formulation. The role of the humoral response for protection against C. burnetii is still undetermined in goats. In mouse models, it has been shown that the humoral response is critical for the prevention of clinical disease, but not for infection control³³. Surprisingly, the antibody response generated by challenged control and Coxevac® goats was comparable. This suggests that the reduction of clinical symptoms (abortions) and bacterial shedding in goats as read-out of vaccine efficacy does not rely exclusively on antibodies^13,14,15,16.

At sacrifice, live C. burnetii were detected in spleens and/or bronchial lymph nodes of all challenged control and Coxevac® goats, confirming that vaccination with Coxevac® was insufficient to prevent the infection^13,14,15. However, C. burnetii DNA was under the detection limit of the PCR assay requiring bacterial sample pre-amplification in embryonated eggs. Globally, the infection was associated with a low bacterial burden in all groups. Reasons for the low infection rate could be explained by the absence of breeding in our animals¹⁰ or the different kinetic of infection of our strain in relation to a previous study⁸. Interestingly, vaccination with QuilA®-adjuvanted Coxevac® induced a moderate but increased protection efficacy.

Considering that production of specific antibodies was not sufficient to clear C. burnetii infection, we explored the vaccine-induced cell-mediated immunity. In mice, T cell-mediated immunity is critical for protection against Q fever^33,38 and IFNγ is important for controlling bacterial replication and elimination^{13,39,40,41,42,43}. To assess cellular C. burnetii-specific responses, PBMCs were ex vivo stimulated in an antigen-specific manner and IFNγ was measured upon vaccination and challenge of goats. In the Coxevac® group, a peak of IFNγ production was reached only one week after the booster dose (5 wpv). In contrast, in the QuilA®-Coxevac® group, a first peak was detected two weeks after the prime vaccination and a second peak one week after the boost (5 wpv). These results suggest that (1) the reaction time for IFNγ production was reduced to one week between the prime and the booster dose in the QuilA®-Coxevac® group and (2) the antigen-specific IFNγ response was accelerated by QuilA®-Coxevac® vaccination compared to Coxevac® only. The mechanism of action of QuilA® remains poorly understood, but the current model entails that the lipophilic moiety of Quillaja saponins promotes direct delivery of exogenous antigens to the intracellular cell compartment of antigen-presenting cells (APCs) promoting histocompatibility complex (MHC) class I presentation with consequent stimulation of CD8⁺ T cells⁴⁴. It is possible that QuilA® favored the process of antigen uptake and presentation to promote immunomodulation towards accelerated IFNγ responses.

Upon challenge, a bimodal pattern characterized again the IFNγ response generated by antigen-stimulated PBMCs of the QuilA®-Coxevac® group, with the highest peak detected immediately after challenge (13.5 wpv) and a second peak at 17.5 and 18 wpv. Considering that goats were exposed to C. burnetii antigens during vaccination, the prompt response of this group as compared to control goats could be explained by a direct consequence of the vaccine prior exposure. By contrast, the IFNγ response of the Coxevac® group upon challenge was less pronounced. One hypothesis is that Coxevac® vaccination could activate different pathways or other lymphocyte subsets, promoting IFNγ-independent responses, as supported by the higher frequency of CD4⁻CD8⁻ cells detected in PBMCs of this group after challenge. The CD4⁻CD8⁻ population is involved in the control of intracellular bacteria, such as Mycobacterium tuberculosis or Francisella tularensis Live Vaccine Strain (LVS)⁴⁵, but further investigations are needed to better characterize this population and its role in protective immunity against C. burnetii.

In challenged control goats, an antigen-specific IFNγ response was detected quickly upon challenge until the end of the experiment. This response was characterized by two phases: an initial moderate production promptly after challenge (13.5 wpv) followed by a sudden increase at 17–17.5 wpv (4–4.5 wpc). This second wave corresponded to the second IFNγ peak detected in the QuilA®-Coxevac® group. Four weeks could be the time needed to mount the adaptive response against C. burnetii, or relates to the kinetic of bacterial multiplication in vivo. Indeed, in pregnant goats, C. burnetii DNA has been detected in the placenta of challenged animals only 4 wpi onwards⁸. The strong IFNγ response induced in the challenged control goats by stimulated PBMCs could be the consequence of a pronounced inflammatory response. In this group, the transcriptional pattern of spleens and respiratory lymph nodes showed indeed an immune response characterized by cytokines, such as pro-inflammatory (IL1β, TNFα, IL12), Th2 (IL4 and IL13), Th17 (IL17A), and other immunoregulatory cytokines (IL6, IL10). Increased levels of IL10, TNFα and IL1β were previously described in peripheral blood of infected pregnant goats before or after parturition⁹. IL1β, IL6 and TNFα production were higher in naturally infected individuals compared to vaccinated ones⁴⁶. Increased levels of inflammatory cytokines (i.e. TNF, IL6, IL12, IL10, IL1b) characterize patients with both acute and chronic Q fever^47,48,49,50. In particular, the chronic course is related to intense inflammation, characterized by upregulation of TNF, IL1β, IL6 and excessive IL10 expression^47,48,49. In addition, IL10 and IL4 support the intracellular replication of C. burnetii in monocytes^51,52 and macrophages^53,54 and the polarization of macrophages toward an M2 program⁵⁵, proposed to be permissive for bacterial replication⁵⁶. Trials of new candidate vaccines inducing a Th2-based response were not able to confer protection similar to Q-Vax, the licensed human vaccine against Q fever⁵⁷, which elicits a Th1-dominant response^33,58. Overall, IL10 and Th2 cytokines seem to interfere negatively with the control of Q fever. Thus, the overexpression of these cytokines in secondary lymphoid organs of challenged control goats may have hampered the establishment of an effective immune response able to clear C. burnetii in organs and to control the infection.

The immune response initiated by QuilA®-Coxevac® goats in secondary lymphoid organs was unambiguous compared to the other groups. An increased frequency of a granulocytic population was detected in spleens. In mouse models, eosinophils and neutrophils are involved in vaccine-induced protection against Q fever^59,60. Whether a similar mechanism exists also in goats deserves further attention, but the increased frequency of splenic granulocytes in QuilA®-Coxevac® vaccinated goats seems to support this hypothesis. Interestingly, the expression level of CD11b, used as a marker for several cell types such as granulocytes, macrophages, NK cells, B lymphocytes and dendritic cells, was significantly increased in challenged control goats compared to the vaccinated-challenged groups. The specificity of this marker should be better investigated in goats, together with its role during C. burnetii infection.

The QuilA®-Coxevac® group was also characterized by an increased CD8⁺ T cell frequency in spleens and PBMCs (at 19 wpv) compared to the other groups as well as a more pronounced mRNA expression of CD8, NRC1 (NK cell receptor), IP10 and IFNγ, than the challenged control goats. Coxevac® vaccinated goats on the other hand presented an intermediate expression of these genes. Thus, the QuilA® adjuvant triggered a splenic Th1-type response probably driven by CD8⁺ and NK cells. This confirms observations from a previous study in mice showing that QuilA skews a Th1-type response and improves antibody levels upon immunization with C. burnetii phII soluble antigens⁶¹. Also, the protection conferred by CD8⁺ T cells results in less lung inflammation and decreased bacterial burden in spleens as compared to the protection elicited by CD4⁺ T cells, suggesting that efficacious vaccines against Q fever should elicit CD8⁺ T-cell immunity⁶². NK cells do not intervene in bacterial clearance but participate in mounting the inflammatory response against Q fever⁴¹. C. burnetii can infect NK cells, which secrete IFNγ and release lytic granules containing bacteria via degranulation⁶³. Whether this also occurs in goats remains to be clarified.

The role of γδ T cells during C. burnertii infection or protective immunity is not characterized and some interesting data are presented here in goats for the first time. γδ T cells are distinguished by their production of IFNγ or IL17A. While the transcription factor T-bet promotes the production of IFNγ by γδ T cells, SOX4 and SOX13 promote IL17A production⁶⁴. In bronchial lymph nodes of the QuilA®-Coxevac® group, IP10, a chemokine produced in response to IFNγ, was grouped with TBX21 (or T-bet), and TRGC2, the receptor of γδ T cells. During C. burnetii infection, IP10 is strongly induced by IFNγ and its expression is increased in infected mice, as a model of acute Q fever^65,66,67. By contrast, in patients with chronic Q fever, IP10 expression is downregulated^68,69. The co-clustering of IP10, TBX21 and TRGC2 genes suggests the involvement of IFNγ-producing γδ T cells after challenge in this group. T-bet has been described to play a key role during C. burnetii primary and protective immunity. Tbet^−/− mice experience more severe infection and a decreased ability to control bacterial replication not only after primary infection but also after vaccination-challenge^60,70. By contrast, primary infection of goats was identified with a rise of SOX4/IL-17A and γδ T cell receptor transcripts, highlighting a different role for γδ T cell subpopulations during vaccination and infection. During acute Q fever infection, circulating γδ T cells increase in patients⁷¹. In vitro studies show that C. burnetii downregulates the IL-17A signaling pathway in murine alveolar macrophages and that the activation of this pathway results in bacterial elimination⁷². On the other hand, Elliott et al.⁵⁹ demonstrated that IL17A is not essential during Q fever infection. To date, the exact role of IL-17A producing γδ T cells in the immune response activated after C. burnetii infection remains to be elucidated. While in our study different γδ T cell transcript profiles were present in vaccinated vs control animals, no changes in the relative frequency of γδ T cells and WC1⁺ γδ T were observed. This suggests that possible effects on γδ T cells due to vaccination or challenge could relate to changes in their functional role rather than their relative abundance.

To have a global view of the immune responses activated by the different conditions, we used a principal component analysis. This analysis corroborated the existence of three distinct clusters and suggests that the activation of a Th1-based response by QuilA®-Coxevac® vaccination helps the clearance of infection. Down-regulation of these pathways would be used as survival strategy by the bacterium, as observed in challenged control goats, which presented a different immune response. The transient increase of the rectal temperature observed after QuilA®-Coxevac® prime vaccination might reduce the acceptance of the vaccine. This could be overcome by reducing the antigen level or using other adjuvants promoting, as QuilA, Th1 responses.

In conclusion, this study described for the first time the immune response generated by Coxevac® vaccination in a goat model of aerosol challenge with an axenic culture of C. burnetii isolated from a field sample. Coxevac® did not induce a significant humoral or cellular response able to confer substantial protection upon challenge. It confirmed previous field studies and presented insights associated with the low immunogenicity of this vaccine and the need to improve its formulation, including reconsidering several aspects such as the antigen, the dose or the inactivation method. Seeing that the addition of QuilA® to the Coxevac® formulation increased the humoral response and elicited a sustained Th1-type cellular response, which improved vaccine efficacy, we propose to test the use of QuilA® to increase Coxevac® efficacy against C. burnetii infection in field settings.

Methods

C. burnetii strain and culture

CbBEC2 is a Belgian strain representative of the SNP2 genomic group, isolated from infected caprine milk. The strain, initially isolated in vivo in BALB/c mice⁷³, was either (1) propagated again in BALB/c mice (Charles Rivers, Wilmington, MA, USA) (Passage P2), from now on indicated as freshly collected splenic harvest, or (2) propagated in African green monkey kidney (Vero) cells (2 passages) and then in ACCM-2^74,75 (3 passages) (P6). The CbBEC2 strain was isolated and maintained at the National Reference Laboratory for Coxiella burnetii and Bartonella (Sciensano, Belgium).

Analysis of C. burnetii LPS

The presence of a full-length LPS, distinctive of virulent phI strains, was assessed in the CbBEC2 strain by WGS and SDS-PAGE silver staining. Genomic DNA extracted from a 14-day ACCM-2 culture (P6) was sequenced on the Illumina MiSeq platform and assembled as described⁷⁶. To ascertain the presence of genes involved in the synthesis of a full length LPS, CbBEC2 assembly was aligned in NCBI BLASTN (Galaxy version 2.10.1) with the NM phI sequence of the operon involved in the biosynthesis of the complete LPS (Genbank accession number: AF387640). Illustration of the alignment was obtained using the CLC sequence viewer software (Version 8.0).

C. burnetii LPS from confluent ACCM-2 cultures was extracted as previously reported²⁹. LPS (6 µl, diluted 1:3) was then electrophoresed (120 V, ±1 h) on 4–20% precast protein gels (Biorad, Hercules, CA, USA) together with the PageRuler prestained protein ladder (ThermoFisher Scientific, Waltham, MA, USA). The gel was fixed for 1 h in a solution composed of 25% (vol/vol) ethanol, 8% (vol/vol) acetic acid, 25% (vol/vol) formaldehyde solution (37 wt. % in H₂O) and washed trice, 20 min each, with 50% (vol/vol) ethanol solution.LPS was visualized using silver staining of the gel by consecutive baths of 0.8 mM sodium thiosulfate pentahydrate (1 min), 10 nM silver nitrate (20 min in the dark), 250 nM sodium carbonate (2.5 min in the dark) and 25 nM Na₂EDTA.2H₂O (10 min). Gels were imaged using a digital camera.

Analysis of the C. burnetii infectious potential in a mouse infection model

Six-week-old female BALB/c mice were injected intraperitoneally with 4–5 × 10³ CbBEC2 C. burnetii cells (200 µl) derived from freshly collected splenic harvest (P2) or a 4-day old axenic ACCM-2 culture (P6). The axenic culture was arrested at the exponential phase of bacterial growth. Dilutions of C. burnetii from stock suspensions were obtained in 0.9% saline solution (Biorad, Hercules, CA, USA). Control mice were injected with 0.9% saline solution only. Inocula were quantified with the monocopy com1 real-time PCR⁷⁷. The cycle threshold (Ct) was used to calculate the genomic equivalents (G.E.) of the suspension pondered against a calibration curve obtained with an external reference (ThermoFisher Scientific, Waltham, MA, USA). A group of six mice was used for each strain type and for the four time-points (48 mice in total). At sacrifice, serum and organs were collected from each animal to evaluate the serological responses (IgM and IgG), the organ weight (spleen and liver) and the bacterial load (G.E. in spleen) as described previously⁷⁷.

Vaccination-challenge experimental protocol in goats

Sixteen 2–3 years old female Saanen goats from the same Q fever free herd (defined as free of disease in the past 8 years by the bulk tank milk surveillance program) were tested for Q fever, brucellosis, Chlamydia abortus, paratuberculosis, Schmallenberg virus, and caprine arthritis encephalitis virus. Assuming a medium level of difference between conditions (effect size = 0.25 using the F test⁷⁸), a power analysis was used to calculate the required number of animals in each group. Goats were randomly distributed into three experimental groups: control (n = 4), vaccine only (Coxevac® group, n = 6) and vaccine plus adjuvant (QuilA®-Coxevac® group, n = 6). Animals were allocated to three independent, separated, BSL-2 housing boxes of 19 m² floor area (3.2 m²/animal), complying with the minimum requirements established by the Belgian law for housing goats of any weight. Prior to challenge, goats were transferred to the BSL-3 area and the animals were housed in three separated boxes of 18 m² floor area (3 m²/animal), under a common filtered-ventilated area. Each housing box was separated by one empty box. Both in the BSL-2 and BSL-3 facilities, circadian rhythm was maintained with artificial light. Feeding was ad libitum and cleaning of the boxes occurred daily. Figure 1a shows the timeline of the vaccination-challenge experiment. After 2 weeks of acclimatization, goats were vaccinated subcutaneously 2 cm above the Spina scapula with 2 ml of Coxevac® (72 QF Units/ml, CEVA Sante Animale, Libourne, France) or with 150 μg of QuilA® (Invivogen, San Diego, CA, USA) diluted in 2 ml of Coxevac®. As controls, goats were injected with 2 ml of PBS (pH 7.4, Gibco). At 4 wpv, goats received a booster dose following the manufacturer’s recommendations.

At 13 wpv, all groups received an intranasal challenge with 10⁶ CbBEC2 C. burnetii derived from 4-day old axenic ACCM-2 culture (P6). Inocula were diluted in 1 ml PBS and quantified with the monocopy com1 real-time PCR. Inoculation (0.5 ml/nostril) was performed using a small nebulizer with 1-mm spray opening (Boehringer Ingelheim, Ingelheim, Germany) fixed on a syringe. Nostrils were held alternately closed during inhalation.

The general health and the rectal temperature of goats were monitored daily by clinical inspections until the end of the experiment (19 wpv). Blood was collected for serological and immunological analyses at the indicated time points (Fig. 1a). At the end of the study, goats were sacrificed by stunning with a penetrating captive bolt followed by immediate exsanguination. Blood and organs were collected for bacterial detection, cell phenotyping and gene expression profiling.

C. burnetii-specific antibody response

Total IgG antibody levels directed against C. burnetii (antigen phI + phII) in goats were measured using the commercially available PrioCHECK^TM Ruminant Q fever Ab Plate Kit (Applied Biosystems, Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions. Briefly, serum samples (diluted 1:400) were added to the plate coated with C. burnetii whole-cell antigens and detected with the HRP-conjugated G protein at 1:100 dilution. The optical densities (OD) were measured at dual wavelengths of 450–620 nm on a microplate reader (Sunrise, Tecan Trading AG, Switzerland). S/P% >40 was considered as positive.

IFNγ ELISA

Blood was sampled from goat jugular vein on BD Vacutainer® Heparin Tubes (BD Bioscience, San Jose, CA, USA) and PBMCs were isolated using Ficoll®-Paque PREMIUM 1.073 (GE Healthcare, Chicago, IL, USA) density gradient centrifugation. After washing, PBMCs were resuspended in RPMI 1640 Medium with GlutaMAX™ (Gibco) and 10% fetal bovine serum (FBS) (Gibco) as supplement. Cells were seeded in 180 uL at 3.0 × 10⁵ cells/well in 96 well-plates (VWR, Radnor, PA, USA) and incubated either with 20 µl 1× PBS, C. burnetii CbBEC2 inactivated antigen (2 × 10⁷/ml) or pokeweed mitogen (PWM) at 5 μg/ml (positive control) for 48 h. After centrifugation, the supernatant was harvested and stored at −20 °C. IFNγ secretion was measured in the supernatant with a sandwich ELISA using the ID screen® Ruminant IFNγ kit (IDvet, Grabels, France) according to the manufacturer’s instructions. Antigen-specific stimulation was validated for each time point by the positive (PWM) and negative (PBS) controls of stimulation. Recombinant goat IFNγ (Cusabio, Houston, TX, USA) was used to validate the recognition of goat IFNγ by the antibodies provided in the kit.

Isolation of splenocytes and lymph node cells

At sacrifice, caprine spleens, bronchial and inguinal lymph nodes were aseptically removed and transported to the laboratory in RPMI 1640 medium with GlutaMAX™ (Gibco) and 10% FBS (Gibco). One cm³ of tissue was disrupted and homogenized in the transport medium for cell isolation. After passage through 70 µM cell strainers (Corning Life Science, Corning, NY, USA), cells were centrifuged (1200 rpm, 10 min) and resuspended in RPMI 1640 medium with GlutaMAX™ (Gibco) and 10% FBS (Gibco) as supplement. Splenocytes were incubated with the BD Pharm Lyse™ lysing solution (BD Bioscience, San Jose, CA, USA), according to the manufacturer’s protocol, for red blood cell lysis. Successively, splenocytes, bronchial and inguinal lymph node cells were immediately stained for cell phenotyping or cryopreserved in 90% FBS (Gibco) with 10% DMSO (Merck, Darmstadt, Germany) for further testing (RNA extraction or C. burnetii detection).

Immunophenotyping with flow cytometry

PBMCs, splenocytes, bronchial and inguinal lymph node cells isolated from goats were immediately (ex vivo) stained for phenotyping in 50 µl of PBS containing a mix of monoclonal antibodies (mAb) against CD4 (Alexa Fluor®647, clone 44.38, MCA2213A647, 1:320 dilution, Bio-rad), CD8 (FITC, clone 38.65, MCA2216F, 1:320 dilution, Bio-rad) and CD21 (RPE, clone CC21, MCA1424PE, 1:10 dilution, Bio-rad). Mouse IgG1-RPE (MCA928PE, 1:10 dilution, Bio-rad) was used as isotype control. The LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (Invitrogen, Waltham, MA, USA), diluted 1:1000 with PBS, was used to stain dead cells. Several vials of PBMCs cryopreserved in 90% FBS (Gibco) with 10% DMSO (Merck, Darmstadt, Germany) were thawed following standard procedures and stained in 50 µl of PBS containing mAb anti-WC1 (Clone CC15, MCA838GA, 1:200 dilution, Bio-rad) or anti-γδ TCR1-N24 δ chain specific (G-GB21A, G-BOV2058, 1:200 dilution, Monoclonal Antibody Centre, Washington State University, Pullman, WA, USA) and the secondary antibody rabbit anti-mouse IgG-RPE (STAR12A, 1:20 dilution, Biorad). The LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogen), diluted 1:1000 in PBS, determined the viability of cells. The staining procedure required a 30 min incubation at 4 °C in the dark. After washing in PBS, cells were preserved in 4% PFA diluted in PBS (Santa Cruz Biotechnology, Dallas, TX, USA) (max. overnight) until flow cytometry was performed with BD FACSVerse™ Cytometer (BD Bioscience, San Jose, CA, USA). Data acquisition and analysis were accomplished using BD FACSuite (BD Bioscience) and FlowJo software (FlowJo, LLC, Ashland, OR, USA), respectively.

Gene expression analysis

RNA was isolated from 5 × 10⁶ cryopreserved splenocytes and bronchial lymph node cells of goats using the RNeasy Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s guidelines. DNA contamination was removed by treatment with the RNase-Free DNase Set (Qiagen). The concentration and purity of RNA samples was assessed using Nanodrop 1000 (Isogen Life Science, De Meern, The Netherlands) and the reverse transcription was performed on equal RNA amounts for each sample with the QuantiTect Reverse Transcription Kit (Qiagen). Gene expression was quantified by real-time PCR using the LightCycler^® 480 SYBR Green I Master (Roche, Basel, Switzerland) following the manufacturer’s recommendations in a Light cycler® 480 Instrument II (Roche) with cycling conditions of pre-heat (10 min at 95 °C), denaturation (40 times 15 s at 95 °C), annealing (40 times 1 min at 60 °C). Primers (Supplementary Table 3) were designed on the NCBI Primer-Blast tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and PCR efficiency was defined for all genes using the standard curve method. Six genes (18S, GAPDH, HMBS, HSP90, SDHA, YWHAZ,) were selected as possible reference genes according to the literature^{9,11,79,80,81,82,83}. Three genes (GAPDH, HMBS, YWHAZ for splenocytes and GAPDH, HMBS, HSP90 for bronchial lymph node cells) were selected as the most stable reference genes using geNorm NormFinder⁸⁴ and Bestkeeper⁸⁵ software packages. The relative gene expression was calculated using the model described in Hellemans et al.⁸⁶, which takes into account primer efficiency and three (or more) reference genes.

C. burnetii detection

The presence of C. burnetii in goats was investigated in blood (post-challenge) and organs listed in Supplementary Table 1. Briefly, DNA was extracted from 200 µl of blood or homogenized tissues (max. 1 g in 1 ml MilliQ water) with the MagMax™ Isolation Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. C. burnetii DNA was detected on a 7500 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific Inc.) by real-time PCR targeting the IS1111 repetitive element as previously described⁷⁶.

Considering that the detection limit of the PCR assay was reached, a volume of 200 µl containing approximately 5 × 10⁵ frozen splenocytes and bronchial lymph nodes cells, suspended in 1× PBS (pH 7.4, Gibco), were injected into embryonated eggs (triplicates for each sample) for bacterial amplification as described⁷³. The yolk sac was harvested and homogenized in 9 ml 0.9% saline solution. The samples (200 µl) were inactivated for 40 min at 80 °C and incubated for 30 min at 37 °C with 180 µl of lysis buffer (20 mM Tris pH8, 2 mM EDTA, 1.2% Triton x-100) containing 20 mg/ml lysozyme (Roche) and 4 µl of RNase A (17,500 U) (Qiagen). DNA was extracted using the QIAGEN kit DNeasy (Qiagen) according to the manufacturer’s guidelines. The real-time PCR was run as described before in a Light cycler® 480 Instrument II (Roche). The cut-off for positivity was set at a Ct-value <40 (accredited validation file). Goats presenting at least one positive result were considered to be positive.

Data analysis

General statistical analyses were performed as indicated in figure legends using GraphPad Prism Software Version 9 (San Diego, CA, USA). A p value ≤0.05 was considered significant.

The clustering analysis was performed in R using data issued from the gene expression profiling. Briefly, the distance matrix was calculated applying the maximum distance, the hierarchical clustering was run with the hclust function using the ward.D2 method and the dendrogram was plotted employing the ggdendro package. The heatmap was created via the ggplot2 package after data transformation with tidyverse package. Finally, the multi plot figure was constructed using the grid package.

The PCA was computed in R using the prcomp function on selected data. PCA results were extracted and visualized using the factoextra package, while the 3D visualization was performed with the scatterplot3D package.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

All data generated or analyzed during this study are included in this published article and its Supplementary Information files.

References

Oyston, P. C. F. & Davies, C. Q fever: the neglected biothreat agent. J. Med. Microbiol. 60, 9–21 (2011).
Article CAS PubMed Google Scholar
Sanford, S. E., Josephson, G. K. & MacDonald, A. Coxiella burnetii (Q fever) abortion storms in goat herds after attendance at an annual fair. Can. Vet. J. 35, 376–378 (1994).
CAS PubMed PubMed Central Google Scholar
Emery, M. P. et al. Coxiella burnetii serology assays in goat abortion storm. J. Vet. Diagn. Invest 26, 141–145 (2014).
Article PubMed Google Scholar
Guatteo, R., Beaudeau, F., Joly, A. & Seegers, H. Coxiella burnetii shedding by dairy cows. Vet. Res. 38, 849–860 (2007).
Article PubMed Google Scholar
Rodolakis, A. et al. Comparison of Coxiella burnetii shedding in milk of dairy bovine, caprine, and ovine herds. J. Dairy Sci. 90, 5352–5360 (2007).
Article CAS PubMed Google Scholar
Maurin, M. & Raoult, D. Q fever. Clin. Microbiol. Rev. 12, 518–553 (1999).
Article CAS PubMed PubMed Central Google Scholar
Sánchez, J. et al. Experimental Coxiella burnetii infection in pregnant goats: a histopathological and immunohistochemical study. J. Comp. Pathol. 135, 108–115 (2006).
Article PubMed Google Scholar
Roest, H.-J. et al. Q fever in pregnant goats: pathogenesis and excretion of Coxiella burnetii. PLoS ONE 7, e48949 (2012).
Article CAS PubMed PubMed Central Google Scholar
Roest, H. I. J., Post, J., van Gelderen, B., van Zijderveld, F. G. & Rebel, J. M. J. Q fever in pregnant goats: humoral and cellular immune responses. Vet. Res. 44, 67 (2013).
Article PubMed PubMed Central Google Scholar
Roest, H. I. J., Dinkla, A., Koets, A. P., Post, J. & van Keulen, L. Experimental Coxiella burnetii infection in non-pregnant goats and the effect of breeding. Vet. Res. 51, 74 (2020).
Article PubMed PubMed Central Google Scholar
Ammerdorffer, A. et al. The effect of C. burnetii infection on the cytokine response of PBMCs from pregnant goats. PLoS ONE 9, e109283 (2014).
Article PubMed PubMed Central Google Scholar
Achard, D. & Rodolakis, A. in The Principles and Practice of Q Fever: The One Health Paradigm (eds Simoes, J. C. C., Anastácio, S. A. & Gabriela Jorge da Silva G. J.) 367–389 (Nova Science Publishers Inc., 2017).
Arricau-Bouvery, N. et al. Effect of vaccination with phase I and phase II Coxiella burnetii vaccines in pregnant goats. Vaccine 23, 4392–4402 (2005).
Article CAS PubMed Google Scholar
Hogerwerf, L. et al. Reduction of Coxiella burnetii prevalence by vaccination of goats and sheep, The Netherlands. Emerg. Infect. Dis. 17, 379–386 (2011).
Article PubMed PubMed Central Google Scholar
de Cremoux, R. et al. Assessment of vaccination by a phase I Coxiella burnetii-inactivated vaccine in goat herds in clinical Q fever situation. FEMS Immunol. Med. Microbiol. 64, 104–106 (2012).
Article PubMed Google Scholar
Muleme, M. et al. A longitudinal study of serological responses to Coxiella burnetii and shedding at kidding among intensively-managed goats supports early use of vaccines. Vet. Res. 48, 50 (2017).
Article PubMed PubMed Central Google Scholar
Rousset, E. et al. Efficiency of a phase 1 vaccine for the reduction of vaginal Coxiella burnetii shedding in a clinically affected goat herd. Clin. Microbiol. Infect. 15(Suppl 2), 188–189 (2009).
Article CAS PubMed Google Scholar
Astobiza, I. et al. Coxiella burnetii shedding and environmental contamination at lambing in two highly naturally-infected dairy sheep flocks after vaccination. Res. Vet. Sci. 91, e58–e63 (2011).
Article CAS PubMed Google Scholar
Jansen, W. et al. Belgian bulk tank milk surveillance program reveals the impact of a continuous vaccination protocol for small ruminants against Coxiella burnetii. Transbound. Emerg. Dis. 69, e141–e152 (2022).
Article PubMed Google Scholar
Bauer, B. U. et al. Humoral immune response to Q fever vaccination of three sheep flocks naturally pre-infected with Coxiella burnetii. Vaccine 39, 1499–1507 (2021).
Article CAS PubMed Google Scholar
Coffman, R. L., Sher, A. & Seder, R. A. Vaccine adjuvants: putting innate immunity to work. Immunity 33, 492–503 (2010).
Article CAS PubMed PubMed Central Google Scholar
Katayama, S., Oda, K., Ohgitani, T., Hirahara, T. & Shimizu, Y. Influence of antigenic forms and adjuvants on the IgG subclass antibody response to Aujeszky’s disease virus in mice. Vaccine 17, 2733–2739 (1999).
Article CAS PubMed Google Scholar
Borja-Cabrera, G. P. et al. Effective immunotherapy against canine visceral leishmaniasis with the FML-vaccine. Vaccine 22, 2234–2243 (2004).
Article CAS PubMed Google Scholar
Haçariz, O. et al. The effect of Quil A adjuvant on the course of experimental Fasciola hepatica infection in sheep. Vaccine 27, 45–50 (2009).
Article PubMed Google Scholar
Sun, H.-X., Xie, Y. & Ye, Y.-P. Advances in saponin-based adjuvants. Vaccine 27, 1787–1796 (2009).
Article CAS PubMed Google Scholar
Villa-Mancera, A., Reynoso-Palomar, A., Utrera-Quintana, F. & Carreón-Luna, L. Cathepsin L1 mimotopes with adjuvant Quil A induces a Th1/Th2 immune response and confers significant protection against Fasciola hepatica infection in goats. Parasitol. Res. 113, 243–250 (2014).
Article PubMed Google Scholar
Rahman, M. et al. QuilA-adjuvanted T. gondii lysate antigens trigger robust antibody and IFNγ+ T cell responses in pigs leading to reduction in parasite DNA in tissues upon challenge infection. Front. Immunol. 10, 2223 (2019).
Article CAS PubMed PubMed Central Google Scholar
Narasaki, C. T. & Toman, R. Lipopolysaccharide of Coxiella burnetii. Adv. Exp. Med. Biol. 984, 65–90 (2012).
Article CAS PubMed Google Scholar
Beare, P. A., Jeffrey, B. M., Long, C. M., Martens, C. M. & Heinzen, R. A. Genetic mechanisms of Coxiella burnetii lipopolysaccharide phase variation. PLoS Pathog. 14, e1006922 (2018).
Article PubMed PubMed Central Google Scholar
Beare, P. A. et al. Genetic diversity of the Q fever agent, Coxiella burnetii, assessed by microarray-based whole-genome comparisons. J. Bacteriol. 188, 2309–2324 (2006).
Article CAS PubMed PubMed Central Google Scholar
Kishimoto, R. A., Rozmiarek, H. & Larson, E. W. Experimental Q fever infection in congenitally athymic nude mice. Infect. Immun. 22, 69–71 (1978).
Article CAS PubMed PubMed Central Google Scholar
Guigno, D. et al. Primary humoral antibody response to Coxiella burnetii, the causative agent of Q fever. J. Clin. Microbiol. 30, 1958–1967 (1992).
Article CAS PubMed PubMed Central Google Scholar
Zhang, G. et al. Mechanisms of vaccine-induced protective immunity against Coxiella burnetii infection in BALB/c mice. J. Immunol. 179, 8372–8380 (2007).
Article CAS PubMed Google Scholar
Singh, M. & O’Hagan, D. T. Recent advances in veterinary vaccine adjuvants. Int. J. Parasitol. 33, 469–478 (2003).
Article CAS PubMed Google Scholar
Cox, E., Verdonck, F., Vanrompay, D. & Goddeeris, B. Adjuvants modulating mucosal immune responses or directing systemic responses towards the mucosa. Vet. Res. 37, 511–539 (2006).
Article CAS PubMed Google Scholar
Burakova, Y., Madera, R., McVey, S., Schlup, J. R. & Shi, J. Adjuvants for animal vaccines. Viral Immunol. 31, 11–22 (2018).
Article CAS PubMed Google Scholar
Sander, V. A., Corigliano, M. G. & Clemente, M. Promising plant-derived adjuvants in the development of coccidial vaccines. Front. Vet. Sci. 6, 20 (2019).
Article PubMed PubMed Central Google Scholar
Kumaresan, V., Alam, S., Zhang, Y. & Zhang, G. The feasibility of using Coxiella burnetii avirulent nine mile phase II viable bacteria as a live attenuated vaccine against Q fever. Front. Immunol. 12, 754690 (2021).
Article CAS PubMed PubMed Central Google Scholar
Turco, J., Thompson, H. A. & Winkler, H. H. Interferon-gamma inhibits growth of Coxiella burnetii in mouse fibroblasts. Infect. Immun. 45, 781–783 (1984).
Article CAS PubMed PubMed Central Google Scholar
Howe, D., Barrows, L. F., Lindstrom, N. M. & Heinzen, R. A. Nitric oxide inhibits Coxiella burnetii replication and parasitophorous vacuole maturation. Infect. Immun. 70, 5140–5147 (2002).
Article CAS PubMed PubMed Central Google Scholar
Andoh, M. et al. T cells are essential for bacterial clearance, and gamma interferon, tumor necrosis factor alpha, and B cells are crucial for disease development in Coxiella burnetii infection in mice. Infect. Immun. 75, 3245–3255 (2007).
Article CAS PubMed PubMed Central Google Scholar
Zamboni, D. S. & Rabinovitch, M. Nitric oxide partially controls Coxiella burnetii phase II infection in mouse primary macrophages. Infect. Immun. 71, 1225–1233 (2003).
Article CAS PubMed PubMed Central Google Scholar
Dellacasagrande, J., Capo, C., Raoult, D. & Mege, J. L. IFN-gamma-mediated control of Coxiella burnetii survival in monocytes: the role of cell apoptosis and TNF. J. Immunol. 162, 2259–2265 (1999).
Article CAS PubMed Google Scholar
Marciani, D. J. Vaccine adjuvants: role and mechanisms of action in vaccine immunogenicity. Drug Discov. Today 8, 934–943 (2003).
Article CAS PubMed Google Scholar
Cowley, S. C. et al. CD4-CD8- T cells control intracellular bacterial infections both in vitro and in vivo. J. Exp. Med. 202, 309–319 (2005).
Article CAS PubMed PubMed Central Google Scholar
Raju Paul, S. et al. Natural exposure- and vaccination-induced profiles of ex vivo whole blood cytokine responses to Coxiella burnetii. Front. Immunol. 13, 886698 (2022).
Article PubMed PubMed Central Google Scholar
Capo, C. et al. Upregulation of tumor necrosis factor alpha and interleukin-1 beta in Q fever endocarditis. Infect. Immun. 64, 1638–1642 (1996).
Article CAS PubMed PubMed Central Google Scholar
Honstettre, A. et al. Dysregulation of cytokines in acute Q fever: role of interleukin-10 and tumor necrosis factor in chronic evolution of Q fever. J. Infect. Dis. 187, 956–962 (2003).
Article CAS PubMed Google Scholar
Kremers, M. N. T. et al. Correlations between peripheral blood Coxiella burnetii DNA load, interleukin-6 levels, and C-reactive protein levels in patients with acute Q fever. Clin. Vaccin. Immunol. 21, 484–487 (2014).
Article CAS Google Scholar
Penttila, I. A. et al. Cytokine dysregulation in the post-Q-fever fatigue syndrome. QJM 91, 549–560 (1998).
Article CAS PubMed Google Scholar
Ghigo, E., Imbert, G., Capo, C., Raoult, D. & Mege, J.-L. Interleukin-4 induces Coxiella burnetii replication in human monocytes but not in macrophages. Ann. NY Acad. Sci. 990, 450–459 (2003).
Article CAS PubMed Google Scholar
Ghigo, E., Capo, C., Raoult, D. & Mege, J. L. Interleukin-10 stimulates Coxiella burnetii replication in human monocytes through tumor necrosis factor down-modulation: role in microbicidal defect of Q fever. Infect. Immun. 69, 2345–2352 (2001).
Article CAS PubMed PubMed Central Google Scholar
Ghigo, E. et al. Link between impaired maturation of phagosomes and defective Coxiella burnetii killing in patients with chronic Q fever. J. Infect. Dis. 190, 1767–1772 (2004).
Article CAS PubMed Google Scholar
Meghari, S. et al. Persistent Coxiella burnetii infection in mice overexpressing IL-10: an efficient model for chronic Q fever pathogenesis. PLoS Pathog. 4, e23 (2008).
Article PubMed PubMed Central Google Scholar
Mantovani, A. et al. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol. 25, 677–686 (2004).
Article CAS PubMed Google Scholar
Benoit, M., Barbarat, B., Bernard, A., Olive, D. & Mege, J.-L. Coxiella burnetii, the agent of Q fever, stimulates an atypical M2 activation program in human macrophages. Eur. J. Immunol. 38, 1065–1070 (2008).
Article CAS PubMed Google Scholar
Gilkes, A. P. et al. Tuning subunit vaccines with novel TLR triagonist adjuvants to generate protective immune responses against Coxiella burnetii. J. Immunol. 204, 611–621 (2020).
Article CAS PubMed Google Scholar
Long, C. M. Q fever vaccine development: current strategies and future considerations. Pathogens 10, 1223 (2021).
Article CAS PubMed PubMed Central Google Scholar
Elliott, A., Schoenlaub, L., Freches, D., Mitchell, W. & Zhang, G. Neutrophils play an important role in protective immunity against Coxiella burnetii infection. Infect. Immun. 83, 3104–3113 (2015).
Article CAS PubMed PubMed Central Google Scholar
Ledbetter, L., Cherla, R., Chambers, C., Zhang, Y. & Zhang, G. Eosinophils affect antibody isotype switching and may partially contribute to early vaccine-induced immunity against Coxiella burnetii. Infect. Immun. 87, e00376-19 (2019).
Article PubMed PubMed Central Google Scholar
Gregory, A. E., van Schaik, E. J., Russell-Lodrigue, K. E., Fratzke, A. P. & Samuel, J. E. Coxiella burnetii intratracheal aerosol infection model in mice, guinea pigs, and nonhuman primates. Infect. Immun. 87, e00178-19 (2019).
Article PubMed PubMed Central Google Scholar
Read, A. J., Erickson, S. & Harmsen, A. G. Role of CD4+ and CD8+ T cells in clearance of primary pulmonary infection with Coxiella burnetii. Infect. Immun. 78, 3019–3026 (2010).
Article CAS PubMed PubMed Central Google Scholar
Matthiesen, S. et al. Coxiella burnetii-infected NK cells release infectious bacteria by degranulation. Infect. Immun. 88, e00172-20 (2020).
Article PubMed PubMed Central Google Scholar
Parker, M. E. & Ciofani, M. Regulation of γδ T cell effector diversification in the thymus. Front. Immunol. 11, 42 (2020).
Article CAS PubMed PubMed Central Google Scholar
Leone, M. et al. Coxiella burnetii infection in C57BL/6 mice aged 1 or 14 months. FEMS Immunol. Med. Microbiol. 50, 396–400 (2007).
Article CAS PubMed Google Scholar
Schoffelen, T. et al. Early cytokine and antibody responses against Coxiella burnetii in aerosol infection of BALB/c mice. Diagn. Microbiol. Infect. Dis. 81, 234–239 (2015).
Article CAS PubMed Google Scholar
Kohl, L. et al. MyD88 is required for efficient control of Coxiella burnetii infection and dissemination. Front. Immunol. 10, 165 (2019).
Article CAS PubMed PubMed Central Google Scholar
Pennings, J. L. A. et al. Dysregulation of serum gamma interferon levels in vascular chronic Q fever patients provides insights into disease pathogenesis. Clin. Vaccin. Immunol. 22, 664–671 (2015).
Article CAS Google Scholar
Raijmakers, R. P. H. et al. A possible link between recurrent upper respiratory tract infections and lower cytokine production in patients with Q fever fatigue syndrome. Eur. J. Immunol. 49, 1015–1022 (2019).
Article CAS PubMed PubMed Central Google Scholar
Mezouar, S. et al. T-Bet controls susceptibility of mice to Coxiella burnetii infection. Front. Microbiol. 11, 1546 (2020).
Article PubMed PubMed Central Google Scholar
Schneider, T. et al. The number and proportion of Vgamma9 Vdelta2 T cells rise significantly in the peripheral blood of patients after the onset of acute Coxiella burnetii infection. Clin. Infect. Dis. 24, 261–264 (1997).
Article CAS PubMed Google Scholar
Clemente, T. M. et al. Coxiella burnetii blocks intracellular interleukin-17 signaling in macrophages. Infect. Immun. 86, e00532-18 (2018).
Article PubMed PubMed Central Google Scholar
Mori, M., Mertens, K., Cutler, S. J. & Santos, A. S. Critical aspects for detection of Coxiella burnetii. Vector Borne Zoonotic Dis. 17, 33–41 (2017).
Article PubMed Google Scholar
Omsland, A., Cockrell, D. C., Fischer, E. R. & Heinzen, R. A. Sustained axenic metabolic activity by the obligate intracellular bacterium Coxiella burnetii. J. Bacteriol. 190, 3203–3212 (2008).
Article CAS PubMed PubMed Central Google Scholar
Omsland, A. et al. Host cell-free growth of the Q fever bacterium Coxiella burnetii. Proc. Natl. Acad. Sci. USA 106, 4430–4434 (2009).
Article CAS PubMed PubMed Central Google Scholar
Tomaiuolo, S. et al. Phylogeography of human and animal Coxiella burnetii strains: genetic fingerprinting of Q fever in Belgium. Front. Cell Infect. Microbiol. 10, 625576 (2020).
Article PubMed Google Scholar
Mori, M. et al. In vitro and in vivo infectious potential of Coxiella burnetii: a study on Belgian livestock isolates. PLoS ONE 8, e67622 (2013).
Article CAS PubMed PubMed Central Google Scholar
Cohen, J. Statistical Power Analysis for the Behavioral Sciences (Lawrence Erlbaum Associates, 1988).
Najafpanah, M. J., Sadeghi, M. & Bakhtiarizadeh, M. R. Reference genes selection for quantitative real-time PCR using RankAggreg method in different tissues of Capra hircus. PLoS ONE 8, e83041 (2013).
Article PubMed PubMed Central Google Scholar
Zhang, Y. et al. Reference gene screening for analyzing gene expression across goat tissue. Asian-Australas. J. Anim. Sci. 26, 1665–1671 (2013).
Article CAS PubMed PubMed Central Google Scholar
Manjunath, S. et al. Identification of suitable reference gene in goat peripheral blood mononuclear cells (PBMCs) infected with peste des petits ruminants virus (PPRV). Livest. Sci. 181, 150–155 (2015).
Article Google Scholar
Puech, C., Dedieu, L., Chantal, I. & Rodrigues, V. Design and evaluation of a unique SYBR Green real-time RT-PCR assay for quantification of five major cytokines in cattle, sheep and goats. BMC Vet. Res. 11, 65 (2015).
Article PubMed PubMed Central Google Scholar
Sahu, A. R. et al. Selection and validation of suitable reference genes for qPCR gene expression analysis in goats and sheep under Peste des petits ruminants virus (PPRV), lineage IV infection. Sci. Rep. 8, 15969 (2018).
Article PubMed PubMed Central Google Scholar
Pfaffl, M. W., Tichopad, A., Prgomet, C. & Neuvians, T. P. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations. Biotechnol. Lett. 26, 509–515 (2004).
Article CAS PubMed Google Scholar
Andersen, C. L., Jensen, J. L. & Ørntoft, T. F. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 64, 5245–5250 (2004).
Article CAS PubMed Google Scholar
Hellemans, J., Mortier, G., De Paepe, A., Speleman, F. & Vandesompele, J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 8, R19 (2007).
Article PubMed PubMed Central Google Scholar

Download references

Acknowledgements

The authors acknowledge the interesting and fruitful discussions within the Vetimmune initiative group (Sciensano, BE), as well as the precious advice from Prof. B. Goddeeris (KU Leuven, BE), Prof. C.L. Baldwin (University of Massachusetts, USA), Prof. P.A. Beare (NIH, USA) and Dr. Ana Santos (Instituto Nacional de Saúde Dr Ricardo Jorge, PT). Gratitude is also due to T. Fancello for his important technical assistance. The authors thank (listed in alphabetical order) R. Bakinahe Ntamukunzi, D. Desqueper, P. Michel, M. Rhabi, A. Soares and P. Vanoorenberghe for their excellent technical support. We also thank B. De Bock, W. Van Campe, A. Bouquiaux, L. Mostin, and all the colleagues from the experimental center in Machelen (Sciensano, BE). S.T. is supported by the NRC grant, financed by the Belgian Ministry of Social Affairs through a fund of the Health Insurance System. This work was partly supported by Sciensano through the internal project VETIMMUNE. The work of M.M. is dedicated to Scibetta G. (†30/3/2020).

Author information

Authors and Affiliations

Bacterial Zoonoses Unit, Veterinary Bacteriology, Infectious Diseases in Animals Scientific Directorate, Sciensano, Brussels, Belgium
Sara Tomaiuolo, Wiebke Jansen, Susana Soares Martins & Marcella Mori
National Reference Centre for Coxiella burnetii and Bartonella, Brussels, Belgium
Sara Tomaiuolo, Wiebke Jansen & Marcella Mori
Laboratory of Immunology, Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
Sara Tomaiuolo, Bert Devriendt & Eric Cox

Authors

Sara Tomaiuolo
View author publications
You can also search for this author in PubMed Google Scholar
Wiebke Jansen
View author publications
You can also search for this author in PubMed Google Scholar
Susana Soares Martins
View author publications
You can also search for this author in PubMed Google Scholar
Bert Devriendt
View author publications
You can also search for this author in PubMed Google Scholar
Eric Cox
View author publications
You can also search for this author in PubMed Google Scholar
Marcella Mori
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization: M.M., W.J., S.T.; methodology: M.M., W.J., S.T., B.D., E.C.; software: S.T., S.S.M.; formal analysis: S.T., S.S.M.; investigation: W.J., S.T., S.S.M., M.M.; data curation: S.T., M.M.; writing—original draft preparation: S.T.; writing—review and editing: S.T., M.M., B.D., W.J., E.C.; supervision: M.M., B.D., E.C.; project administration and funding acquisition: M.M.

Corresponding author

Correspondence to Marcella Mori.

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethics statement

Animal experiments were approved by the ethics committee of Sciensano under the file numbers 20160323 (mouse infection), 20190704-01 (goat vaccination), and 20190704-02 (goat challenge).

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Info

REPORTING SUMMARY

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Tomaiuolo, S., Jansen, W., Soares Martins, S. et al. QuilA® adjuvanted Coxevac® sustains Th1-CD8⁺-type immunity and increases protection in Coxiella burnetii-challenged goats. npj Vaccines 8, 17 (2023). https://doi.org/10.1038/s41541-023-00607-z

Download citation

Received: 26 July 2022
Accepted: 24 January 2023
Published: 14 February 2023
DOI: https://doi.org/10.1038/s41541-023-00607-z
Springer Nature Limited

This article is cited by

Q fever immunology: the quest for a safe and effective vaccine
- Gayathri Sam
- John Stenos
- Bernd H. A. Rehm
npj Vaccines (2023)