Coccidioides species are fungal pathogens that can cause a widely varied clinical manifestation from mild pulmonary symptom to disseminated, life-threatening disease. We have previously created a subunit vaccine by encapsulating a recombinant coccidioidal Ag (rCpa1) in glucan–chitin particles (GCPs) as an adjuvant-delivery system. The GCP-rCpa1 vaccine has shown to elicit a mixed Th1 and Th17 response and confers protection against pulmonary coccidioidomycosis in mice. In this study, we further delineated the vaccine-induced protective mechanisms. Depletion of IL-17A in vaccinated C57BL/6 mice prior to challenge abrogated the protective efficacy of GCP-rCpa1 vaccine. Global transcriptome and Ingenuity Pathway Analysis of murine bone marrow–derived macrophages after exposure to this vaccine revealed the upregulation of proinflammatory cytokines (TNF-α, IL-6, and IL-1β) that are associated with activation of C-type lectin receptors (CLR) Dectin-1– and Dectin-2–mediated CARD9 signaling pathway. The GCP formulation of rCpa1 bound soluble Dectin-1 and Dectin-2 and triggered ITAM signaling of corresponding CLR reporter cells. Furthermore, macrophages that were isolated from Dectin-1−/−, Dectin-2−/−, and CARD9−/− mice significantly reduced production of inflammatory cytokines in response to the GCP-rCpa1 vaccine compared with those of wild-type mice. The GCP-rCpa1 vaccine had significantly reduced protective efficacy in Dectin-1−/−, Dectin-2−/−, and CARD9−/− mice that showed decreased acquisition of Th cells in Coccidioides-infected lungs compared with vaccinated wild-type mice, especially Th17 cells. Collectively, we conclude that the GCP-rCpa1 vaccine stimulates a robust Th17 immunity against Coccidioides infection through activation of the CARD9-associated Dectin-1 and Dectin-2 signal pathways.
Coccidioidomycosis, or San Joaquin Valley Fever, is caused by inhalation of spores produced by Coccidioides immitis and C. posadasii (1). Coccidioidomycosis displays a broad spectrum of disorders from self-limited flu-like symptoms to progressive pulmonary destruction and life-threatening dissemination (2). Approximately 17–29% of community-acquired pneumonia in endemic areas are caused by Coccidioides infection. These fungal pathogens infect both immunocompromised and immunocompetent individuals, posing a great risk to over 30 million people (∼10% of the United States population) who live in the endemic regions (3). Historically, the endemic areas of coccidioidomycosis are located on the Western hemisphere including southwest United States, Central America, and certain parts of the South America. An estimated 150,000 new infections occur each year in the United States. Recent reports show the geographic distribution of Coccidioides species has expanded as far north as Washington State (4–6). Because of the significant cost of health care and impact in livelihood, Coccidioides poses a major health threat to the public.
Evidence of acquiring long-term memory immunity against coccidioidomycosis has been observed in individuals who recover from symptomatic Coccidioides infections and yet remain skin-test positive (7, 8). Therefore, it is possible to develop an effective vaccine with long-term memory against coccidioidomycosis. Vaccine candidates against Coccidioides infection come in many forms, including live-attenuated strains, formalin-killed spherules, and subunit vaccines (3, 9). Although the understanding of protective immunity to Coccidioides infection remains incomplete, animal models and clinical data suggest that functional CD4+ T cell responses characterizing the expression of Th1- and Th17-type cytokines are essential for protection against this fungal disease (3, 9–12). Typically, Th1 cells express IFN-γ, whereas Th17 cells produce IL-17. Recent data also support the role of IL-17 and Th17 cells in the protective immunity conferred by a live-attenuated vaccine against Coccidioides infection in mice (11, 13, 14). Unfortunately, there is no approved adjuvant available for the formulation of subunit vaccines that are able to induce a Th17 response.
Several experimental adjuvants have recently been demonstrated to stimulate IL-17 production and to augment vaccine-mediated immunity against pulmonary Mycobacterium tuberculosis infections (15–17). Glucan particles (GPs) that are primarily composed of β-1,3-D-glucans derived from Saccharomyces cerevisiae cell wall have been shown to elicit a mixed Th1 and Th17 response against fungal infections (18, 19). Our laboratory has created a recombinant Coccidioides polypeptide Ag (rCpa1; GenBank no. KY883768) consisting of previously reported coccidioidal Ags (Ag 2/proline-rich protein, Coccidioides-specific Ag, and Pmp1) and five peptides with high affinity to human MHC class II molecules (10). The rCpa1 is encapsulated by Rhodotorula mucilaginosa yeast-derived glucan–chitin particles (GCPs) that allow for the effective delivery of this vaccine to APCs and subsequent activation of a protective memory response (10, 20). We also show that the GCP adjuvant elicits elevated infiltration of macrophages to the vaccination sites, and it activates a superior memory Th17 response in the lungs of C57BL/6 and HLA-DR4 transgenic mice against a potentially lethal challenge with C. posadasii compared with the GP adjuvant (10). Concurringly, the GCP-rCpa1 vaccine has improved protective efficacy in reduction of CFUs in the lungs and spleen of Coccidioides-infected mice compared with the GP-rCpa1 vaccine.
In this study, we investigated vaccine-induced immune mechanisms of the GCP-rCpa1 vaccine. Depletion of IL-17A in vaccinated mice reduced protective efficacy of GCP-rCpa1 against Coccidioides infection. We applied global transcriptome analysis of murine bone marrow–derived macrophages (BMMs) after exposure to the GCP-rCpa1 vaccine. Our results showed that both Dectin-1 and Dectin-2 C-type lectin receptors (CLRs) interact with GCPs to activate protective immune response against pulmonary coccidioidomycosis. Furthermore, caspase recruitment domain family member 9 (CARD9), a downstream immune adaptor of Dectin-1 and Dectin-2, was also required for activation of protective immunity. Overall, our data revealed that CLRs-CARD9–mediated Th17 immunity is essential for the GCP-rCpa1 vaccine to confer protection against pulmonary coccidioidomycosis in mice.
Materials and Methods
Virulent clinical isolated C. posadasii, C735, was used in this study. The saprobic phase was grown on glucose yeast extract agar (1% glucose, 0.5% yeast extract, and 1.5% agar) to produce spores as previously reported (10). All culturing and preparatory procedures, which involved live cells of C. posadasii, were conducted in a biosafety level 3 laboratory located at The University of Texas at San Antonio (UTSA).
Dectin-1−/− (21), Dectin-2−/− (22), and CARD9−/− (23) male and female mice (8–10 wk old) on C57BL/6 genetic background were bred in-house. Wild-type (WT) C57BL/6 (H-2b) sex- and age-matched mice were purchased from the National Cancer Institute/Charles River Laboratories. All animal experiments were approved by the Institutional Animal Care and Use Committee at UTSA according to National Institutes of Health and ABLS3 laboratory guidelines for housing and care of laboratory animals.
Vaccination protocol, animal challenge, and evaluation of protection
Mice were s.c. immunized twice in the abdominal region at a 2-wk interval (10). Mice were challenged intranasally with a suspension of 80–100 viable spores of C. posadasii in 35 μl of PBS at 4 wk after completion of the immunization protocol, as previously described (24). Mice were euthanized at 14 d postchallenge (dpc) for assessing fungal burden as previously described (14, 24, 25).
Neutralization of IL-17A in WT mice
Vaccinated and control C57BL/6 mice were injected with 250 μg of anti–IL-17A mAb (clone 17F3; Bio X Cell) or normal rat IgG isotype control (Sigma-Aldrich, St. Louis, MO) by the i.p. route as previously described (13, 26). The treatment was conducted at 24 h prior to challenge and every 3 d after an intranasal challenge with C. posadasii spores. Mice were euthanized at 13 dpc, and fungal burden was analyzed.
BMMs were prepared from femurs and tibiae of mice as previously described (27). Briefly, bone marrow cells were washed, counted, and plated at a concentration of 4 × 105 cells/ml in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine, 100 U penicillin/ml, 100 μg of streptomycin/ml, 50 mM 2-ME, and 20 ng/ml of murine M-CSF (PeproTech). Cells were incubated at 37°C and 5% CO2, half of the medium replaced every 3 d, and cells harvested on day 7. BMMs were washed and incubated with macrophage detachment solution DXF (PromoCell) for 40 min at 4°C. Macrophages were collected off the bottom of the petri dish plate. Purity for BMMs expressing F4/80 (BM8; eBioscience) was checked by flow cytometry, and it was routinely >95%.
RNA sequencing analysis
BMMs (1 × 106 cells/ml) were incubated with GCP-rCpa1 at a ratio of 1:50 (1 macrophage/50 particles) or were treated with PBS (the same volume as particles used) as a control for 12 h with 5% CO2. Total RNA was isolated from BMMs using the Qiagen RNeasy Kit. Preparation of sequencing libraries and sequencing analysis using Illumina HiSeq 3000 platform (50-bp single-read module) were conducted at the Genomics Core of The University of Texas Health San Antonio. RNA sequencing (RNA-seq) analysis module in CLC Genomics workbench was used to align the sequence reads with confidential length (>50 bp) to mouse GRCm38 (mm10) reference genome (28). Differential gene expression was analyzed after eliminating lowly expressed gene (counts per million <1 in more than three samples across the dataset) using edgeR package. Gene Ontology (GO) analysis were performed using DAVID and then visualized by GO plot (29). A heat map showed top 50 differentially expressed genes of the GCP-rCpa1–treated macrophages compared with controls was created with Euclidean distance method using Heatmap.2 function implemented in the gplots R package. The causal network analysis was performed using Ingenuity Pathway Analysis (IPA) software (QIAGEN) (30, 31).
Quantitative RT-PCR analysis and cytokine ELISAs
BMMs were incubated with GCP-rCpa1 (1:50 ratio) or treated with PBS as a control for 12 and 24 h for quantitative RT-PCR (qRT-PCR) and cytokine analyses, respectively. cDNA samples were synthesized from 1 μg of total RNA isolated from each sample of BMMs using oligo(dT) primer and SuperScript III Reverse Transcriptase (Invitrogen). The cDNA samples were used for qRT-PCR using SYBR Green detection method with gene-specific primers. Mouse β-actin gene expression was used as an internal control. Results of real-time PCR data were derived from comparative threshold cycle methods to detect relative gene expression as described (32). For cytokine assays, BMMs were spun down at 3500 rpm for 3 min, and supernatant samples were stored in −80°C with Halt Protease and Phosphatase Inhibitor (Life Technologies). Cytokine concentrations were measured using Bio-Rad Laboratories’ Bio-Plex Mouse Cytokine 23-Plex (catalog no. M60009RDPD) as described (27).
CLR reporter assay
Constructs of B3Z/BWZ reporter cells expressing Dectin-1, Dectin-2, Dectin-3, and Mincle have been described previously (33, 34). For B3Z/BWZ cell stimulation, 1 × 105 B3Z/BWZ cells per well in a 96-well plate were incubated for 18 h with GCPs or plate-coated ligands. β-Galactosidase (lacZ) activity was measured in total cell lysates using chlorophenol red-β-d-galactopyranoside (Roche) as a substrate. OD560 was measured using OD620 as a reference.
Dectin-1 and Dectin-2 binding assays
GCPs were incubated at 2 × 106 particles per well with either 20 μg/ml Fc–Dectin-1, Fc–Dectin-2, or human IgG Fc (negative control) in 100 μl of binding buffer (20 mM Tris-HCl, 150 nM NaCl, 10 mM CaCl2, and 0.05% Tween 20 [pH 7.4]) as previously described (33). The plate was incubated overnight at 4°C to prevent nonspecific binding. The particles were then washed three times in binding buffer and incubated with a 1:100 dilution of PE anti-human IgG1 Fc Ab (Jackson ImmunoResearch Laboratories) for 30 min at 4°C. Following incubation, the samples were washed three times in binding buffer and fixed in 500 μl of 2% ultrapure formaldehyde. Data were acquired using a BD LSR II flow cytometer (BD Biosciences) and analyzed with FlowJo software. Aliquots of samples were spun down and resuspended in 30 μl of PBS for imaging analysis using a Leica fluorescent microscope.
Pulmonary leukocytes were isolated from vaccinated and control Dectin-1−/− (ΔD1), Dectin-2−/− (ΔD2) and CARD9−/− (ΔC9) mice at 14 dpc (four mice per group) as previously reported (11). A standard flow cytometry methodology was employed for direct mAb labeling and enumeration of selected pulmonary immune T cell phenotypes using an FACSCalibur cytometer (11). Permeabilized leukocytes were stained with a mixture of fluorochrome-conjugated Abs for detecting IFN-γ, IL-17A, CD4, and CD8 molecules. Data were analyzed using FlowJo software version 10.
Student t test was used to analyze results between GCP adjuvant versus GCP-rCpa1–vaccinated groups for cytokine concentrations and calculations of cell numbers of lung-infiltrated immune cells. Student–Newman–Keuls test, a type of ANOVA statistical analysis for multiple comparisons of three or above independently treated groups was used as previously reported (35). The differences in fungal burdens (CFUs) between two groups were analyzed by the Mann–Whitney U ranking test (11). When comparing fungal burden among three and more groups of mice, the Kruskal–Wallis test, a nonparametric ranking method, was used as previously reported (11). A p value ≤0.05 was considered statistically significant.
IL-17A is essential for GCP-rCpa1–induced protective immunity
Vaccine toxicity may interfere with immune response and protective efficacy. Our previous histopathological analysis revealed minimal advisory effect on the s.c. vaccination sites and each of the recruited APC (i.e., macrophage and dendritic cells) could engulf one GCP particle (10). We further investigated vaccine cytotoxicity by incubation of human hepatocellular carcinoma (Hep2G) cells with the GCP particles loaded with mouse serum albumin and rCpa1 (GCP-rCpa1) at a series of doses from 1:1 to 1:1200 ratios (Supplemental Fig. 1). Viability curves were used to calculate a 50% cytotoxic concentration that was defined as the ratio of Hep2G cells to particles leading to 50% cell death. The 50% cytotoxic concentration values were calculated to be 1150 and >1200 for GCP-rCpa1 and GCP particles loaded with mouse serum albumin, respectively. In addition, the body weights of the vaccinated mice increased over the course of the study comparable to the nonvaccinated mice (data now shown). These data suggested that both GCP and rCpa1 had minimal cytotoxicity, and the GCP-rCpa1 vaccine was suitable for both in vitro and in vivo immunological assays.
We investigated whether IL-17A is essential for the GCP-rCpa1–induced immunity against pulmonary coccidioidomycosis. Depletion of IL-17A in vaccinated and nonvaccinated WT mice was conducted using a mAb (clone 17F3), as described in 2Materials and Methods. Mice vaccinated with GCP-rCpa1 and treated with anti–IL-17A Ab demonstrated a significant increase in fungal burden in the Coccidioides-infected lungs compared with isotype-treated mice (Fig. 1A; #p < 0.05). Similarly, CFUs were also significantly increased in the spleens of mice that were treated with anti–IL-17 Ab (Fig. 1B). These data suggest that IL-17–mediated immunity is required for GCP-rCpa1–induced immunity against Coccidioides infection.
Profile transcriptomes of macrophages after exposure to the GCP-rCpa1 vaccine
RNA-seq analysis was conducted in C57BL/6 mouse–derived BMMs that were incubated with GCP-rCpa1 vaccine (macrophage/particle ratio = 1:50) for 12 h. Aliquots of macrophages without stimulation served as a control. The RNA-seq data have been deposited in National Center for Biotechnology Information’s Gene Expression Omnibus and is accessible through Gene Expression Omnibus accession number GSE133140 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE133140). Approximately 37–54 millions of sequences per sample (∼80% of total reads) were mapped to the GRCm38 (mm10) mouse genome. The global gene expression differences between vaccine-treated and control group were visualized using volcano plot (Fig. 2A). Overall, 1954 genes exhibited significant difference greater than 2-fold (log2 fold change > 1 or <−1 and false discovery rate [FDR] < 0.05). Among them, 941 genes were downregulated, and 1013 were upregulated genes (Fig. 2A). We focused on the upregulated genes in response to the vaccine stimulation. The GO terms that were associated with the set of upregulated genes included inflammatory response, innate immune response, cytokine secretion, Ag processing and presentation, and cellular response to IFN-γ, IL-1β, and IL-6 (Supplemental Fig. 2). qRT-PCR analysis showed that the expression amounts of IL-1β and IL-6 genes were indeed elevated in the vaccine-treated macrophages (Fig. 2B). Hierarchical cluster from three independent experiments demonstrates the top 50 significantly increased genes in vaccine-treated BMMs compared with untreated cells (Fig. 2C).
Both Dectin-1 and Dectin-2 recognize GCP-rCpa1 vaccine
We used the IPA module of regulator effects to identify upstream receptors that could involve in the upregulation of these genes. Results showed that TLR1, TLR2, Dectin-1, and Dectin-2 were upstream of the upregulated genes (z-score > 2). Proinflammatory genes that were downstream of Dectin-1 (Clec7a), including IL-1B, TNF, IL-12A, IL-12B, IL-6, CXCL2, CCL22, PG-endoperoxide synthase 2 (PTGS2), and colony stimulating factor 3 (CSF3) were upregulated in vaccine-treated macrophages compared with unstimulated controls (Fig. 3A). Dectin-2 (Clec6a or Clec4n)–associated downstream genes IL-1B, IL-6, IL-12B, and TNF were also elevated in the treated macrophages (Fig. 3B). Both Dectin-1– and Dectin-2–mediated pathways are associated with increased differentiation and polarization of Th17 and Th1 cells. The GCP adjuvant is derived from fungal cell wall–containing polysaccharides, which serve as potential ligands for CLRs. Therefore, we focused on CLRs, including Dectin-1, Dectin-2, Dectin-3, and Mincle, in this study. We employed T hybridoma cells expressing NFAT-lacZ galactosidase reporter of ITAM signaling (33). In response to GCP-rCpa1 vaccine stimulation, lacZ activity was increased in T hybridoma cells expressing Dectin-1 and Dectin-2 in a dose-dependent manner (Fig. 3C). In contrast, reporter cells expressing Dectin-3 or Mincle did not increase lacZ activity. T hybridoma cells expressing the signaling adaptor FcRγ alone did not increase lacZ activity. Thus, Dectin-1 and Dectin-2, but not Dectin-3 or Mincle, recognized the GCP-rCpa1 vaccine and triggered downstream ITAM signaling through FcRγ.
Consequently, we sought to determine if Dectin-1 and Dectin-2 receptors bind to GCPs using soluble fusion proteins, Fc–Dectin-1 and Fc–Dectin-2, which were created by fusing carbohydrate recognition domain (CRD) of C-type lectin to the Fc fragment of human IgG1 (33, 36). Binding of GCP particles with Fc–Dectin-1 and Fc–Dectin-2, but not Fc fragment alone, was detected by fluorescent microscopy (Fig. 3D). Binding affinity was also validated by FACS analysis, in which Dectin-1 and Dectin-2 had greater affinity compared with Fc alone (Fig. 3E).
Dectin-1, Dectin-2, and CARD9 are essential for macrophages to produce inflammatory cytokines after exposure to the GCP-rCpa1 vaccine
BMMs were prepared from C57BL/6 (WT) and deficient mice that lack expression of Dectin-1 (ΔD1) and Dectin-2 (ΔD2). CARD9 is an intracellular immune adaptor downstream of Dectin-1 and Dectin-2 receptors. Thus, BMMs were also prepared from CARD9-deficient mice (ΔC9) for comparison. BMMs from each strain of mice were separately incubated with GCP-rCpa1 or PBS. Amounts of Th1-type (TNF-α, IL-12/p40, and IL-12/p70), Th2-type (IL-4, IL-5, and IL-13), and Th17-type (IL-1α, IL-1β, and IL-6) cytokines were determined and compared among BMMs prepared from these four strains of mice (Fig. 4). WT BMMs that were incubated with the vaccine elicited a mixed Th1-, Th2-, and Th17-type response, as evident by increased production of TNF-α, IL-12, IL-13, IL-1α, IL-1β, and IL-6 compared with the unstimulated macrophages (*p < 0.05). Notably, BMMs prepared from ΔD1, ΔD2, and ΔC9 mice produced reduced amounts of TNF-α, IL-12, IL-13, and IL-6 compared with those of WT mice (*p < 0.05). Interestingly, the vaccine-treated BMMs isolated from ΔC9 mice significantly increased IL-5 production compared with BMMs of WT mice. Taken together, these data suggest that Dectin-1, Dectin-2, and CARD9 molecules are essential for macrophages to respond to the GCP-rCpa1 vaccine in a mixed Th1 and Th17 manner.
CARD9-deficient mice showed increased fungal burden and reduced Th17 activation in response to GCP-rCpa1 vaccination
We evaluated whether CARD9 molecule was required for the GCP-rCpa1 vaccine to induce protective immunity against Coccidioides infection. CARD9−/− mice and WT littermates (n = 10 per group) were s.c. vaccinated twice with the GCP-rCpa1 vaccine. Mice immunized with GCP adjuvant alone served as controls. All mice were intranasally challenged with ∼80 viable C. posadasii spores, followed by the assessment of fungal burden in the lungs and spleen at 14 dpc. As expected, ΔC9 mice that were vaccinated with the GCP-rCpa1 vaccine had significantly elevated CFUs in their lungs and spleen compared with vaccinated WT mice (Fig. 5A; #p < 0.05). We further evaluated T cells that were activated and recruited to the Coccidioides-infected lungs of vaccinated and control mice at 7 and 14 dpc. Gating strategies for subsets of CD4+ Th cells (Th1 and Th17) and CD8+ cytotoxic T (Tc) cells (Tc1 and Tc17) were based on their differentiation expression of CD45, CD4, CD8, IFN-γ, and IL-17A as shown in Supplemental Fig. 3. Concurringly, percentages and total numbers of Th17 cells in the lungs of vaccinated ΔC9 mice were significantly reduced compared with vaccinated WT mice (Fig. 5B, 5C; #p < 0.05). In contrast, percentages and total numbers of Th1 cells in the lungs of vaccinated ΔC9 were not significantly reduced compared with WT mice. These results demonstrate that CARD9 is necessary for the GCP-rCpa1–mediated activation of Th17 cells and protective immunity against pulmonary coccidioidomycosis in mice.
Dectin-1– and Dectin-2–deficient mice also displayed increased fungal burden and reduced Th17 response after vaccination with the GCP-rCpa1 vaccine
We further investigated the impact of Dectin-1 and Dectin-2 in the development of protective immune response to GCP-rCpa1 vaccination using ΔD1 and ΔD2 mice. WT mice, which were immunized with GCP-rCpa1 or GCP alone, served as controls. These three strains of mice (WT, ΔD1, and ΔD2) that were vaccinated with the GCP-rCpa1 vaccine displayed significantly reduced CFUs in their lungs and spleens compared with their corresponding control mice of the same strain (Fig. 6A, Mann–Whitney U test; *p < 0.05). Importantly, the vaccinated ΔD1 and ΔD2 mice showed elevated CFUs in their lungs and spleens compared with the vaccinated WT mice (Fig. 6A; #p < 0.05). These results indicate that Dectin-1 and Dectin-2 receptors are required for the GCP-rCpa1 vaccine to induce protective immune responses against pulmonary C. posadasii infection. We further detected that all three strains of vaccinated mice (WT, ΔD1, and ΔD2) had significantly elevated percentages and total numbers of Th17 cells in the gated CD4+ T cell populations at 7 and 14 dpc compared with nonvaccinated mice (Fig. 6B, 6C). Importantly, the total numbers of Th17 cells of vaccinated ΔD1and ΔD2 mice were significantly reduced compared with vaccinated WT mice (Fig. 6B, 6C). Contrarily, the total numbers of Th1 cells in the lungs were comparable between vaccinated and control mice of all three strains (Fig. 6C). Assessment of percentages and total numbers of CD8+ T cells (Tc1 and Tc17) subsets in the lungs of vaccinated and control mice showed comparable results among these three strains of mice (data not shown). These results suggested that both Dectin-1 and Dectin-2 were required for the activation of Th17 cells that were associated with GCP-rCpa1–induced protection against pulmonary coccidioidomycosis (Fig. 7).
Vaccination has been shown historically to be one of the most effective methods for controlling infectious diseases (37). Although the majority of clinical vaccines today contain whole cells of attenuated, live, or killed organisms, subunit vaccines designed on a defined Ag or Ags are an attractive prospect for a number of reasons (38). First, subunit Ags offer enhanced safety profiles, and they eliminate the need of attenuated live organisms or killed whole cells. Second, subunit Ags tend to have lower reactogenicity than whole-cell vaccines, which may lead to severe inflammation at the vaccination sites or systemic symptoms (25). Third, subunit Ags can be fully characterized to ensure vaccine reproducibility and quality. The overall purity and simplicity of subunit Ags, however, comes at a price. Subunit Ags require an adjuvant to enhance immunoreactivity. The adjuvant components constitute a diverse group of compounds, which are typically categorized as either immunostimulators or delivery systems (39). We have previously created a subunit vaccine that is composed of a recombinant multivalent Ag (rCpa1) and yeast-derived GCPs against pulmonary coccidioidomycosis (10). GCPs serve as a combination adjuvant and vaccine delivery system similar to the previously reported pure GPs (18, 19, 40). Both GPs and GCPs can stimulate a mixed Th1 and Th17 immunity, whereas GCPs elicit an augmented Th17 response compared with GPs (10). The GCP-rCpa1 vaccine has low cytotoxicity (Supplemental Fig. 1) and shows minimal advisory effect upon s.c. immunization in our previous report (10). Vaccination of mice with this subunit vaccine achieves comparable protection generated by an attenuated live vaccine ΔT against coccidioidomycosis (10). Combination of low cytotoxicity with defined Ag composition, a Th17-boosting adjuvant, and a marked protective efficacy provide the advantage of the GCp-rCpa1 subunit vaccine over currently available experimental coccidioidal vaccines. Accumulated evidences have demonstrated the importance of Th17 immunity in protection against various fungal infections (41, 42). Furthermore, with the increase of clinical anti–IL-17 treatments against autoimmune diseases (e.g., psoriasis), more individuals may become susceptible to fungal infections (43, 44). These studies shed light on the need to elucidate the role of Th cells in vaccine designs against fungal infections. In this study, we have extended our previous findings to show that GCP-rCpa1–induced protection and enhancement of Th17 responses are mediated through CARD9-associated Dectin-1 and Dectin-2 molecules, as illustrated in Fig. 7. This conclusion is supported by both in vitro and in vivo experiments using BMMs and T hybridoma reporter cells and a murine model of pulmonary coccidioidomycosis, respectively. Macrophages that are isolated from Dectin-1, Dectin-2, or CARD9 knockout mice produced fewer IL-12, IL-1, and IL-6 cytokines in response to GCP-rCpa1 stimulation. These macrophage cytokines are required for shaping the differentiation and development of Th1 and Th17 cells. Furthermore, protective efficacy and the activation of Th17 cells responding to vaccination with GCP-rCpa1 are also significantly reduced in Dectin-1, Dectin-2 (Fig. 6), or CARD9 (Fig. 5) knockout mice. We have previously reported that GCP-rCpa1 vaccination stimulated mixed Th17 and Th1 responses by immune CD4+ cell recall with rCpa1 via the assessment of pulmonary Th cells in vaccinated mice following pulmonary challenge with Coccidioides spores (10). Although there is a much stronger and sustained Th17 response compared with Th1, our RNA-seq analysis did reveal cellular response to TNF and IL-12 in BMMs stimulated by GCP-rCpa1, implying subsequent Th1 immunity could be generated. Investigation of the role of Th1 immunity in GCP-rCpa1–mediated protection against coccidioidal infection is currently underway.
Various T cell subsets with different cytokine production properties and functionalities have been identified as protective immune responses against fungal infections (45). IFN-γ–producing Th1 cells promote clearance of Histoplasma capsulatum and Cryptococcus neoformans infections in the diseased lungs (46, 47). Likewise, IL-17–producing Th17 cells are an essential arm of protective immunity against Blastomyces dermatitidis infection (13, 33). Finding the optimal combination of T cell subpopulations to be induced by vaccines against Coccidioides infection remains elusive (9). We have previously reported that s.c. vaccination of IFN-γ and IL-4R knockout mice with an attenuated, live vaccine (ΔT) derived from C. posadasii C735 isolate still-induced protective immunity against pulmonary challenge with an autologous virulent isolate (11). In contrast, fungal burden, clearance, and survival are significantly compromised in mice defective in IL-17A or IL-17R (11). Similarly, we demonstrated in this study that depletion of IL-17A in mice reduces protective efficacy of the GCP-rCpa1 vaccine against a pulmonary challenge with C. posadasii C735 isolate. Interestingly, an attenuated live vaccine (ΔCps1) created from C. posadasii Silveira isolate elicits a Th1-biased response that confers protection against a pulmonary challenge with the autologous isolate or C. immitis RS isolate (48, 49). The genetic background of the attenuated live vaccines and the virulent isolates used for challenge in those experiments may attribute to the differences in immune responses.
The identification and development of novel adjuvants capable of inducing Th17 or Th1/Th17 adaptive responses for enhancing protective efficacy of recombinant protein Ags will provide new opportunities to advance vaccines against bacterial and fungal pathogens. Several infectious diseases of significant public health concerns could benefit from a Th17-inducing adjuvant system including M. tuberculosis, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumonia, Candida albicans, and Aspergillus fumigatus (17, 45, 50). Cationic adjuvant formulation 1 (CAF01) and derivatives of trehalose diesters that can bind to Mincle receptor on macrophages were used with subunit vaccines against M. tuberculosis (16, 51). A nanoemulsification adjuvant made of soybean oil with cetylpyridinium chloride, Tween 80, and ethanol in water has been shown to induce a Th1- and Th17-mixed immune response against mucosal viral infection (15). Yeast cell wall–derived particles (GPs and GCPs) are among various adjuvants currently under evaluation with subunit vaccines to stimulate a mixed Th1 and Th17 response against fungal infections (10, 19, 40). The use of GCPs as an adjuvant and vaccine delivery vehicle are ideal for fungal infections, which typically rely on IL-17 and IFN-γ for clearance (10, 13, 40, 45, 52). These particles can be packaged with multiple Ags as well as other immunomodulatory molecules, such as TLR agonists, DNA, and RNA, among others. GCPs will be mainly recognized and internalized by APC because they typically bare pattern recognition receptors.
Recent reviews have highlighted the importance of CLRs in shaping immune responses to fungal infections (53, 54). Interaction of CLRs with fungal carbohydrates induces intracellular activation of signals that trigger responses ranging from cytokine production to induction of adaptive immunity. Our data show that immune responses to GCPs are dependent on Dectin-1 and Dectin-2, but not Dectin-3 and Mincle. GCP particles are composed of ∼50–60% β-glucans, 20–30% chitin/chitosan, and <1% of mannans (20). The major GCP component, β-glucan, is mainly recognized by Dectin-1, a member of CLRs expressed on the surface of macrophages and dendritic cells. Dectin-1 is essential for recognition and activation of protective immunity against Coccidioides (33, 55–57). Dectin-1–deficient mice reduced expression of Th1 and Th17 cytokines required for vaccine immunity against pulmonary Coccidioides infection (26, 33). Similarly, human Dectin-1 deficiency predisposes patients to vulvovaginal candidiasis and onychomycosis. Mannans in GCPs are likely recognized by Dectin-2 to stimulate an adaptive immunity against fungal infections (53). Our data are in agreement with the role of Dectin-2 in activation of adaptive Th1 and Th17 immunity against pulmonary coccidioidomycosis, as shown for the live-attenuated ΔT vaccine (33). Although Dectin-2 is required for development of adaptive immune response to Coccidioides infection, it is not essential for inducing innate immunity against this disease (58).
Proteomic analysis has shown that R. mucilaginosa yeasts used to prepare GCP adjuvant-express chitin deacetylase. The enzyme converts chitin to chitosan, the deacetylated form of chitin (59). Binding assays using a chitin binding dye, calcofluor white, suggest that chitin may be more accessible in fungal cell wall of R. mucilaginosa than that of Cryptococcus species (60). The receptors on myeloid cells that bind chitin or chitosan have yet been definitively identified, whereas a study shows that fungal chitin can induce proinflammatory cytokines such as IL-6 and TNF-α via TLR2 signaling in murine and human monocytes (61). In contrast, a prior study showed that chitosan, but not chitin, can stimulate the NLRP3 inflammasomes in activated BMMs to enhance IL-1β release (62). IL-1β–mediated response is essential for vaccine immunity against pulmometry coccidioidomycosis (35, 63). Our data have also revealed that GCPs stimulate murine BMMs to produce and secrete IL-1β (Figs. 2, 4), although the ratio of chitosan to chitin in GCPs is still under investigation.
CARD9 is a critical intracellular adaptor protein that operates downstream of several ITAM-associated CLRs, including Dectin-1, Dectin-2, Dectin-3, and Mincle receptors (64). CARD9 complex regulates the activation of NF-кB pathway that triggers the transcription of IL-6 and IL-1β, which further drives the activation of Th17 response as illustrated in Fig. 7. CARD9 is mainly expressed in myeloid and epithelial cells (23). Mice deficient in CARD9 expression have increased susceptibility to fungal infections. CARD9 is also necessary for resistance to Coccidioides infection (26). CARD9−/− mice tend to be more susceptible to Coccidioides infection compared with Dectin-1−/− and Dectin-2−/− mice, suggesting that these two CLRs have additive or synergistic effect in recognition of GCPs. It is also possible that other CLRs upstream of CARD9 are involved in the response to GCP stimulation. Furthermore, TLR2 and TLR6 upstream of MyD88 are also predicted to respond to GCP stimulation in our global gene analysis. These receptors may recognize GCPs by interacting with specific linages of β-glucan and chitin/chitosan that have not been identified yet. Further studies are underway to delineate the roles of TLRs in response to this novel Coccidioides subunit vaccine.
We thank Dr. Sandra Cardona, manager of the Cell Analysis Core at UTSA, for excellent technical assistance in flow cytometry analysis.
This work was supported by National Institute of Allergy and Infectious Diseases, National Institutes of Health Grants AI135005 (to C.-Y.H.), AI093553 (to M.W.), AI035681 (to B.S.K.), and AI040996 (to B.S.K. and M.W.).
The sequences presented in this article have been submitted to the National Center for Biotechnology Information’s Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE133140) under accession number GSE133140.
The online version of this article contains supplemental material.
Abbreviations used in this article:
bone marrow–derived macrophage
caspase recruitment domain family member 9
C-type lectin receptor
false discovery rate
Ingenuity Pathway Analysis
recombinant Coccidioides polypeptide Ag
The University of Texas at San Antonio
The authors have no financial conflicts of interest.