Animal research protocols for studies with HLA-DR3 transgenic mice performed by EpiVax were reviewed and approved by TGA Sciences Incorporated Institutional Animal Care and Use Committee (P07-10R20-EV69, P07-10R20-EV71). Animal research protocols for guinea pig experiments were reviewed and approved by the Colorado State University Institutional Animal Care and Use Committee (14-5305A, 16-6844A). All animal experimental activities were conducted in full compliance with university, federal and international regulations and the standards of the DoD Animal Care and Use Review Office. Methods of euthanasia as described below were consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association (AVMA).

The human study was carried out in accordance with the recommendations of the Medical Ethical Committee Brabant (Tilburg, Netherlands). All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was reviewed and approved by the Medical Ethical Committee Brabant (Tilburg, Netherlands, NL51305.028.15).

Five milligrams of peptide for each epitope selected as defined above were prepared by Fmoc solid phase peptide synthesis (Twentyfirst Century Biochemicals, Marlborough, MA, United States) and HPLC purified to >90% purity (peak area). The peptide mass was verified by ESI-MS (QSTAR XL Pro, Qo-TOF) and the sequence of each peptide was verified by tandem mass spectrometry (CID MSMS).

Q fever exposed individuals were recruited from a cohort characterized in a previous large Q fever study conducted in the village of Herpen, the Netherlands (48), which experienced a high incidence of C. burnetii infection during the 2007–2010 Q fever outbreak (49). All subjects had been tested using a Q fever interferon-γ release assay (IGRA, Q-detect™) assay of cellular immunity during a previous study in which 80% of the adult population of Herpen were screened for Q fever (48). Individuals were invited to participate in the current study following preselection based on clinical history (disease, comorbidities, and medication) as well as IGRA and serological data generated during the previous study (48). To maximize the potential to detect C. burnetii epitope-specific T-cells, preference was given to donors with strong responses to whole heat-killed C. burnetii in the IGRA and without potentially confounding immune disorders. In addition, five individuals with known past symptomatic Q fever consented to participate. In total, 143 participants provided written informed consent. IGRA responses were re-assessed upon inclusion in October 2015. Volunteers who had no history of Q fever disease (48) and scored negative by immunofluorescence assay (50) as well as by IGRA in spring 2014 and upon inclusion into the present study in autumn 2015 were allocated to control group A (n = 26). Seven volunteers that were IGRA positive in 2014 but did not meet positive scoring thresholds anymore 1.5 years later upon inclusion into the present study were excluded from further analysis. The remaining 110 volunteers that were positive by IGRA in both 2014 and 2015 were subdivided based on past Q fever disease [either registered (notified) in the national surveillance system, or self-reported] into groups B (asymptomatic, n = 73) and C (symptomatic, n = 37).

Whole lithium-heparin anti-coagulated blood was stimulated with C. burnetii antigen (heat killed Cb02629, Wageningen Bioveterinay Research, lot 14VRIM014) in 96-well polypropylene plates (Greiner BioOne) by adding 180 μl blood to 20 μl C. burnetii antigen diluted in phenol red-free RPMI supplemented with Glutamax (2 mM), Gentamycin (5 μg/ml) and sodium pyruvate (1 mM, all ThermoFisher Scientific). A 1.5 % (v/v, final concentration) solution of PHA-M (ThermoFisher Scientific), was added to separate wells as a positive control. Medium only was added to the negative control wells. All stimulations were performed in duplicate. After 22 ± 1 h whole blood cultures were re-suspended and IFNγ concentrations were assessed in whole blood by ELISA, using the IFNγ Pelipair protocol (Sanquin) with minor modifications. The upper detection limit of IGRA under these conditions is 1,050 pg/ml. A subject was scored positive by IGRA if the C. burnetii-induced IFNγ production was ≥16 pg/ml above background and the ratio of the logarithmic value of background-subtracted C. burnetii and PHA responses {(log[C. burnetii]–log[neg control])/(log[PHA]-log[neg control])} was ≥0.4.

HLA typing was performed at the HLA laboratory at the Laboratory of Translational Immunology at the UMC Utrecht, the Netherlands. Genomic DNA was isolated from EDTA anti-coagulated blood within 48 h upon collection using the MagNA Pure Compact system (Roche Diagnostics). The DNA samples were typed for HLA-A, -B (5′-UTR-3′UTR), and -DRB1 (exon 2–3′-UTR) by Next Generation Sequencing (NGS). Firstly, HLA target sequences were generated by long-range PCR using the Qiagen LongRange PCR kit and HLA-A, -B, and -DRB1 primers as described previously (51). Library preparation was performed using the GenDX NGSgo®-LibrX and NGSgo®-IndX kits following the manufacturers' recommendations (GenDX). Pooled samples were sequenced on an Illumina MiSeq by 2 × 250 paired end reading using the MiSeq reagent kit v2 (500 cycles). Sequences were analyzed with the NGSEngine software (GenDX). The resulting HLA-A, HLA-B, and HLA-DRB1 alleles were assigned to supertype families as defined by (42, 52) and/or based on homology of the HLA binding pockets. For determining allelic frequencies, donors homozygous for a given HLA allele were counted once.

A combination of antigen-specific T-cell expansion culture and enzyme-linked immune spot assay (cultured ELISpot) was chosen as the primary assay for peptide screening, to achieve high sensitivity for detecting low frequency antigen-specific T-cell responses to C. burnetii peptides and facilitate detection of central memory T-cells (53). The protocol was adapted from Subbramanian et al. (54) and optimized at Innatoss using two reference peptide pools of HLA class I and class II peptides of CEF (Cytomegalovirus, Epstein-Barr virus, and influenza; JPT Peptide Technologies). A comparison of three media showed that RPMI medium supplemented with 10% FCS (HyClone) as a blocking and culture medium gave the best signal to background ratio in this ELISpot assay.

Female Dunkin-Hartley guinea pigs were sensitized by intranasal inoculation with 10⁶ genome equivalents of C. burnetii Nine Mile strain or saline in 100 μL volume, as described previously (55). Peptides were tested by intradermal challenge, delivered at day 42 post sensitization. HLA class II and I peptides were administered in pools of five, with each peptide being tested twice in two different pool preparations. Peptide pools were created with 2 μg of each peptide (10 μg peptide per pool in total) in 100 μL saline with 1% DMSO. Challenge with 2 μg COXEVAC® whole cell vaccine (Ceva Sante Animale, Libourne, France) was used as a positive control; negative controls consisted of saline or 1% DMSO injections. On day 7, animals were anesthetized (ketamine 40 mg/kg and xylazine 5 mg/kg, i.p.) and euthanized with beuthanasia (i.p.). Gross reactions were monitored daily and skin biopsies obtained at day 7 post-challenge were fixed, sectioned, and stained with hematoxylin and eosin. Histological reactions were scored by an experimenter blinded to the treatment group, using the criteria previously described (55). Briefly, a score of 0 indicates no inflammation, 1 indicates localized macrophage dominated inflammation, 2 macrophage dominated inflammation with limited tissue infiltrations, 3 lymphocytic inflammatory infiltrates extending into the deep dermis, 4 edema and increased pyogranulomatous inflammation extending deep into the subcutis, and 5 widespread pyogranulomatous inflammation including necrosis.

Two C. burnetii antigen sets were selected as the basis for immunoinformatic identification of HLA class I and II T-cell epitopes. The first set (for HLA class I epitope prediction) was comprised of 53 published substrates of the type IV secretion system (T4SS), which are translocated from C. burnetii to the host cytoplasm where they are expected to enter the class I antigen processing pathway and trigger CD8⁺ T cell responses (21, 23, 30–37). The second set (for both HLA class I and II epitope prediction) covered 40 sero-reactive C. burnetii antigens based on antibody responses in humans and mice, as well as evidence of processing and presentation to stimulate murine CD4⁺ T-cells (18, 26, 28, 29, 56). Using the EpiMatrix algorithm, 8,643 putative 9- and 10-mer T-cell epitopes predicted to bind to at least one of six HLA class I supertype alleles and 282 promiscuous epitope clusters, spanning 14–25 amino acids, predicted to bind HLA class II alleles covering >90% of the world-wide human population, were identified from the reference C. burnetii Nine Mile strain (Table 1).

The derived HLA class I epitopes and HLA class II promiscuous epitope clusters were then filtered to focus on sequences that (i) are conserved with other C. burnetii strains; (ii) have very high likelihood of binding human HLA alleles; (iii) exhibit low potential for cross-reactivity with peptides derived from the human proteome or microbiome based on the JanusMatrix algorithm (44); and (iv) do not present obvious issues for peptide synthesis or stability. Finally, 50 HLA class II epitope clusters (Table 2) and 65 HLA class I epitopes (Table 3) were selected for immune reactivity screening such that no source antigen was represented more than twice. Five epitopes were selected for each of the six HLA class I supertypes and for each antigen set (T4SS substrates and sero-reactive antigens), if possible giving preference to HLA class I ligands predicted to bind to at least two class I supertype alleles. An additional five HLA class I epitopes were specifically selected from the immunodominant C. burnetii antigen com1 (28, 56).

Epitope predictions were validated using HLA binding assays. Peptides representing the 50 promiscuous C. burnetii HLA class II epitope clusters were assayed in competition binding assays against each of the eight class II HLA supertype alleles (Figure 1). Each of the peptides bound as predicted to at least two HLA-DR alleles: 6% bound to two HLA-DR alleles, 2% to three alleles, 10% to four alleles, 4% to five alleles, 28% to six alleles, 24% to seven alleles, and 26% to all eight alleles. Independent of original binding predictions, amongst all 400 peptide-allele pairs tested, 6.5% of peptides bound with very high affinity (IC₅₀ < 0.1 μM), 16% of peptides bound with high affinity (IC₅₀ 0.1–1 μM), 30% bound with moderate affinity (IC₅₀ 1–10 μM), 25.5% bound with low affinity (IC₅₀ 10–100 μM), and 22% exhibited no detectable affinity for the HLA-DR tested (IC₅₀ >100 μM or no dose-dependent response). Overall, 81% of predicted binding events and 41% of predicted non-binding events were verified in vitro. Collectively, the agreement of computational predictions with binding assay results was 75% (χ², p < 0.001), ranging from 60 to 88% for individual alleles (Table S1), consistent with published studies using the same algorithms and assay conditions (57, 58).

HLA class I binding affinities were determined for the primary predicted HLA-A/B supertype allele for each peptide (Figure 2). Among the 65 peptide-allele pairs tested, 56% bound with high affinity (IC₅₀ < 5 μM), 21.5% with moderate affinity (IC₅₀ 5–50 μM), 7.5% with low affinity (IC₅₀ 50–1,000 μM), and 14% exhibited no detectable affinity for the HLA class I allele tested (IC₅₀ >1,000 μM or no dose-dependent response). All predicted A^*0201, A^*0301, and A^*2402 peptides, and 8/11 A^*0101 and B^*0702 peptides bound HLA as predicted. An assay using B^*4403 molecules was not available, but an assay using the related B^*4402 allele showed eight out of 11 B^*4403 peptides bound HLA as predicted. Based on these results, epitope prediction accuracy was 100% for A^*0201, A^*0301, and A^*2402 and 73% for A^*0101, B^*0702, and B^*4403. Overall, predictive accuracy for this set of HLA class I peptides was 89% (Table S1). Taken together, the binding assay results suggest that the predicted HLA class II epitope clusters and HLA class I epitopes represent a set of sequences with meaningful potential for stimulating C. burnetii-specific immune responses across a broad range of HLA types.

While peptide binding to HLA is necessary to induce a T-cell response, it is not sufficient. To determine whether the predicted C. burnetii HLA class II epitope clusters are capable of eliciting a de novo immune response via a cognate human HLA in vivo, the class II peptides were screened for immunogenicity in transgenic mice expressing human HLA-DR3 (tgHLA-DR3). The 50 HLA class II C. burnetii peptides were arranged into five groups of 10 peptides each based on predicted HLA-DR3 immunogenicity and three mice per group were subjected to heterologous DNA/DNA/peptide/peptide prime-boost immunizations. Peptide-specific responses of splenocytes from tgHLA-DR3 mice were assessed by ex vivo IFNγ ELISpot assay. Positive peptide-specific IFNγ responses in vivo were observed for 11 peptides, eight of which evoked responses in at least 2/3 mice (Table 4). All 11 of these immunogenic peptides were predicted to bind HLA-DR3, and all except one (p36) also showed binding to DRB1^*0301 in vitro. Therefore, altogether 10/21 peptides both predicted and confirmed to bind HLA-DR3 were immunogenic in vivo. None of the 13 peptides that were predicted not to bind HLA-DR3 were immunogenic. An odds ratio, calculated to determine the association of epitope prediction and immunogenicity, showed a statistically significant association for immunogenicity given an HLA-DR3 prediction (Fisher's exact test, p = 0.0229). No peptide-specific responses were detected in mock-immunized mice that received vehicle vaccine controls.

The transgenic mouse study provided a snapshot of peptide-specific immunogenicity for a single HLA class II allele. To assess antigenicity across multiple HLA types and determine whether the selected class I and II peptides are capable of recalling long-lasting IFNγ memory responses, we next turned to a unique cohort of individuals naturally exposed to C. burnetii. Subjects for this study were recruited from a well-characterized population in the village of Herpen in the Netherlands (48), which experienced a high incidence of C. burnetii infection during the 2007–2010 Q fever outbreak. Recruited subjects were categorized based on clinical Q fever history and recall responses to heat-killed C. burnetii in a whole blood IFNγ release assay (IGRA) (Table 5). Group A and B subjects had no clinical history of Q fever, while group C individuals had recovered from an acute episode of diagnosed Q fever. Group A controls had no IFNγ recall responses to C. burnetii. Group B (past asymptomatic infection) and group C (past symptomatic infection) subjects showed varying degrees of IFNγ recall responses to C. burnetii (both p < 0.0001 compared to group A controls by one-way ANOVA with Holm-Šídák multiple comparisons test), with no significant difference between the two groups (p = 0.9). HLA types were sufficiently diverse within the total cohort to have all desired HLA types represented in all three clinical groups (Tables S2, S3), with the exception of HLA-DR8, which is underrepresented particularly in group C, in accordance with this allele's expected low frequency in the Dutch population (42, 59, 60). Assessment of the binding potential of all peptides with respect to the HLA types of the 136 subjects in groups A-C showed no significant differences across groups, with 75–82% of all class I peptides and between 93 and 96% of all class II peptides predicted to be recognized by each group.

Two partially overlapping subgroups of the total cohort were selected based on a broad representation of HLA-A/HLA-B and HLA-DR supertypes and allocated to HLA class I and II peptide screening, respectively. The screening cohort for the 50 HLA class II peptides consisted of 21 group A control donors (IGRA-, no clinical disease), and 56 IGRA+ donors from groups B (asymptomatic, n = 33) and C (symptomatic, n = 23) (Table S2). The screening cohort for the 65 HLA class I peptides included 20 group A control donors (IGRA-, no clinical disease), and 57 IGRA+ donors from groups B (asymptomatic, n = 32) and C (symptomatic, n = 25) (Table S3). Care was taken to maintain a distribution of IGRA responses comparable to the total cohort (Tables S2, S3).

Human antigenic T-cell IFNγ recall responses against the predicted epitope clusters from C. burnetii were evaluated ex vivo using freshly isolated peripheral blood mononuclear cells (PBMCs) in a cultured ELISpot assay for detection of central memory T-cells. A cultured ELISpot assay was used to achieve high sensitivity for detecting low frequency antigen-specific T-cell responses. Amongst the 3,843 assessments made for the HLA class II peptides (50 peptides screened against 77 donors with some exceptions due to technical error or insufficient cell numbers), 307 yielded positive responses [stimulation index (SI) ≥2, Figures S1, S2]. These antigenic responses covered 44/50 HLA class II peptides. Only six peptides were not recognized by any subject (p9, p11, p34, p35, p40, and p49). For four of these peptides, a second peptide from the same respective source antigen induced recall responses by at least one IGRA+ subject. Therefore, responses were detected to all but two (CBU_2065 and CBU_0092/YbgF) of the 29 source antigens from which HLA class II epitope clusters were predicted and screened.

The cumulative HLA class II peptide response per subject was more frequent in IGRA+ compared to IGRA- subjects (Figure 3A). The majority of IGRA- individuals recognized none (10/21 donors) or only one or two peptides (8/21 donors). While only 14% (3/21) of IGRA- group A individuals recognized three or more peptides, this was true for 46% (26/56) IGRA+ group B and C donors (Figure 3B). Amongst IGRA+ individuals, there was a positive correlation between the cumulative number of recognized peptides per donor, and the IFNγ responses to whole heat-killed C. burnetii as assessed 12–17 months prior to peptide screening (Figure 3C). Of note, all but one individual that showed responses to three or more peptides had an IGRA response of at least 250 pg/ml IFNγ in this standardized assay (Figure 3D).

Of the 44 antigenic HLA class II peptides, 21 peptides were recognized by >10% of all IGRA+ individuals (at least 6/56, Figure 4A) and were hence considered highly antigenic. These 21 highly antigenic peptides accounted for 78.3% of all positive peptide responses of IGRA+ individuals that reached a SI≥2 (220/281). A large proportion of these responses was well above the cut-off of SI ≥2: Amongst responding IGRA+ individuals, responses to 13 of these 21 peptides showed a median SI between 3.0 and 4.8 (Figure 4B), and for 11 of these peptides >10% of all IGRA+ donors reached at least SI ≥3. Amongst IGRA- control subjects group A, these peptides elicited either no recall responses (11/21), or responses from only one or two individuals. Only for a single peptide (p21) did the recall response frequency reach a similar proportion in IGRA- (3/21; 14.3%) as in IGRA+ donors (8/56; 14.3%). Amongst the 21 highly antigenic peptides, there were five source proteins that were represented by two epitopes each (p14+p15, CBU_1835/protoporphyrinogen oxidase; p18+p19, CBU_1513/short chain dehydrogenase; p22+p23, CBU_1398/SucB, p37+p38, CBU_0718, p45+p46, CBU_0307/outer membrane protein). Notably, not only were all 11 of the tgHLA-DR3 mice immunogenic peptides also antigenic in at least one IGRA+ individual, but five of the tgHLA-DR3 immunogenic peptides were amongst the 21 highly reactive peptides in the human cohort (p2, p14, p16, p22, p31).

Amongst IGRA+ donors, group C of past symptomatic individuals had a higher proportion of high responders (recognizing >10 peptides) and a smaller proportion of non-responders to HLA class II epitope clusters than past asymptomatic subjects (Group B, Figure 5A). However, the two groups did not differ significantly in their cumulative peptide response per subject (Figure 5B). When comparing responses to individual peptides between group B and C, there was again a trend that responses particularly to the most antigenic peptides were more frequent amongst past symptomatic donors and a number of peptides were recognized only within this group by either a single (p3, p33) or multiple donors (p1, p6, p10) (Figure 5C). There were some peptides, however, that were recognized solely by past asymptomatic subjects (p25, p42, and p43, 3/33 each; p13, p28, p29, and p33, 1/33 each).

Recall responses to the 65 HLA class I peptides were also assessed using the cultured IFNγ ELISpot approach. In 4,794 assessments (65 peptides screened in 77 donors with some exceptions due to technical error or insufficient cell numbers), only 23 positive responses were detected (Figure S3). Four of these positive responses came from IGRA- group A control subjects. The remaining 19 responses were detected in IGRA+ subjects, with one individual recognizing 8 different peptides (Figures S3, S4). Of the 15 HLA class I peptides that were found to be antigenic in humans, ten peptides were recognized by only a single donor each and only five HLA class I peptides were recognized more than once: p53, p91, and p92 were each recognized by 2/57 donors, and two epitopes (p111 and p113) from com1 were recognized by 3/57 and 4/57 donors, respectively. Only two of the reactive peptides came from the T4SS data set (p53, p54), the remaining 13 were derived from sero-reactive source antigens. Albeit rare, HLA class I peptide responses were clearly detectable, with nearly half (11/23) of the positive responses reaching a stimulation index (SI) of ≥3 (Figures S3, S4), and all responses were consistent with predictions for primary or secondary HLA-A/B allele binding.

Due to reactogenicity issues observed with the only Q fever vaccine licensed for humans, all C. burnetii HLA class I and class II peptides examined in this study were screened for potential reactogenicity in a guinea pig model of exposure-primed delayed-type hypersensitivity (55, 61), with the goal of eliminating reactogenic peptides from further consideration for inclusion in a vaccine. In contrast to reactogenicity mediated by the whole cell phase I vaccine COXEVAC®, no gross reactions were noted at any of the negative control or peptide pool injection sites (Figure S5). Mild lymphocytic inflammation (score = 1) was noted in two HLA class II peptide pools; no overlap in peptides was noted between the two pools and no other pools with the same peptides showed histological changes. Therefore, these reactions were not likely to be considered a reactogenic response to C. burnetii peptides.

AG is a senior officer and shareholder and AS is an employee of Innatoss Laboratories B.V., which provides diagnostic screening for Q fever. ADG and WM are senior officers and shareholders, and LM, GR, and CB are employees of EpiVax, Inc., a company specializing in immunoinformatic analysis. Innatoss Laboratories B.V. and EpiVax, Inc., own patents to technologies utilized by associated authors in the research reported here. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.