The development of vaccines against fungi and other intracellular microbes is impeded in part by a lack of suitable adjuvants. While most current vaccines against infectious diseases preferentially induce production of antibodies, cellular immunity is essential for the resolution of fungal infections. Microbes such as fungi and Mycobacterium tuberculosis require Th17 and Th1 cells for resistance, and engage the C-type lectin receptors including Dectin-2. Herein, we discovered a novel Dectin-2 ligand, the glycoprotein Blastomyces Eng2 (Bl-Eng2). Bl-Eng2 triggers robust signaling in Dectin-2 reporter cells and induces IL-6 in human PBMC and BMDC from wild type but not Dectin-2-/- and Card9-/- mice. The addition of Bl-Eng2 to a pan-fungal subunit vaccine primed large numbers of Ag-specific Th17 and Th1 cells, augmented activation and killing of fungi by myeloid effector cells, and protected mice from lethal fungal challenge, revealing Bl-Eng2’s potency as a vaccine adjuvant. Thus, ligation of Dectin-2 by Bl-Eng-2 could be harnessed as a novel adjuvant strategy to protect against infectious diseases requiring cellular immunity.
Fungal disease remains a challenging clinical and public health problem. Despite medical advances, invasive fungal infections have skyrocketed over the last decade and pose a mounting health threat in immune-competent and -deficient hosts with worldwide mortality rates ranking 7th, even ahead of tuberculosis [1,2]. The development of safe, effective vaccines remains a major hurdle for fungi. Critical barriers to progress include the lack of defined fungal antigens (Ags) and suitable adjuvants that together exert protective immunity. Recent strides in our understanding of fungal immunity and discovery of fungal Ags have raised the prospect that vaccines against fungi can be developed to elicit lasting protective immunity if suitable adjuvants are available. Adaptive immunity is critical for the prevention and resolution of fungal infections. The contribution of antibodies to host defense is debated [3,4]. In contrast, Ag-specific CD4+ T cells play the major role in fungal resistance [4,5], as evidenced by the high incidence of life-threatening fungal infections in patients with impaired CD4+ T cells. CD4+ T cells confer resistance by secretion of T-helper 1 (Th1) and Th17 cytokines such as IFN-γ, TNF-α, GM-CSF, and IL-17A, which activate neutrophils, monocytes, macrophages and DCs for fungal clearance [3,4, 6,7]. Since CD4+ T cells are germane to host defense against fungi, the challenge is how best to elicit these T cells. The transition from innate to adaptive immunity is fostered by dendritic cells (DCs). These cells process and present Ag to naïve CD4+ T cells in the context of co-stimulatory factors (e. g. cell surface ligands and cytokines) that provide the combination of signals necessary to induce naive T cell activation and proliferation. During their interactions with DCs, naive T cells also become functionally specialized. Helper T cell polarization occurs as a result of the cytokines produced by DCs: Th1 polarization is associated with DC production of high levels of IL-12p70, and Th17 polarization is associated with DC production of IL-1β and IL-6. While vaccine Ags typically have little impact on the nature of the cytokines produced by DCs, the adjuvant can have a dramatic effect. Alum (aluminum hydroxide), which is the most commonly used adjuvant in current vaccine formulations, suppresses DC production of pro-inflammatory cytokines such as IL-12p70 [8], creating an environment that polarizes T cells towards a Th2 phenotype. Thus, a major weakness and central challenge in the field of vaccinology is the lack of adjuvants that drive Th1 and/or Th17 polarization and stimulate DCs to produce the appropriate cytokines. Pathways that can differentially activate DC cytokine profiles include toll-like receptors (TLRs), C-type lectin receptors (CLRs), co-stimulatory ligands such as CD40, and cytokine receptors. C-type lectins are important in fungal recognition by DCs and in inducing anti-fungal Th1 and Th17 responses [9,10,11]. Dectin-1 and Dectin-2 induce Th1/Th17 cells in response to Candida albicans [12,13,14,15] and Aspergillus fumigatus [16,17,18] infection. While Dectin-1 is dispensable, Dectin-2 is requisite for the development of protective Th1 and Th17 cells and vaccine resistance against dimorphic fungi [19]. Crude fractions of mannoproteins isolated from Malassezia pachydermatis as well as a lipoglycan (Man-LAM) of Mycobacterium tuberculosis [20] have been shown to trigger Dectin-2 signaling, however they have not been evaluated as vaccine adjuvants, and glycans and lipids may be difficult to express and scale. Here, we report the identification of a novel fungal Dectin-2 ligand from an attenuated vaccine strain of Blastomyces dermatitidis, Bl-Eng2. We tested whether the ligation of Dectin-2 effectively vaccinates mice against fungi. Our vaccination strategy was to ligate Dectin-2 with Bl-Eng2 and assess the adjuvant activity by combining it with the recently reported pan-fungal vaccine calnexin [21]. Fungal recombinant Bl-Eng2 was expressed and scaled efficiently, it stimulated IL-6 and IL-1β production in vitro and Th1 and Th17 cells in vivo and, when used as an adjuvant in combination with calnexin, it protected mice against pneumonia in a model of lethal pulmonary fungal infection.
To evaluate whether Bl-Eng2 is recognized by Dectin-2, we cloned and expressed the recombinant protein in Pichia pastoris. This eukaryotic expression system modifies recombinant proteins with both O- and N-linked glycosylation. Full-length Bl-Eng2 was fused to a N-terminal α-factor secretion signal and a C-terminal Myc-6×His tag (Fig 2B). Ni-NTA purified Bl-Eng2 showed a band of ~120 kDa on SDS-PAGE gel (Fig 2C), which falls within the size range determined in S1A and S1B Fig. Periodic acid-Schiff (PAS) based glyco-stain of Bl-Eng2 showed strong glycosylation (Fig 2C), which likely accounts for the discrepancy between predicted Mr of 57 kDa and apparent Mr of ~120 kDa. Gas chromatography (GC) analysis indicated that mannose is the major monosaccharide, and constitutes 82. 8% in glycan mass of Pichia-expressed Bl-Eng2 (Fig 2D). To verify Bl-Eng2 ligand activity, B3Z reporter cells expressing Dectin-2 or other distinct CLRs were incubated with recombinant Bl-Eng2. Bl-Eng2 elicited strong NFAT-lacZ signalling from Dectin-2 reporter cells, but not from the other CLR-expressing cells (Fig 3A), indicating a specific interaction between Dectin-2 and Bl-Eng2. Since Aspergillus Eng2 (Asp-Eng2) exhibits a high degree of similarity to Bl-Eng2 and contains a Ser/Thr-rich C terminus, we also tested whether Asp-Eng2 is recognized by Dectin-2. Asp-Eng2 and Bl-Eng2 were similarly recognized by Dectin-2 expressing reporter cells (S3A and S3B Fig), hence Eng2 from both fungal species are Dectin-2 ligands. To investigate whether Bl-Eng2 stimulates primary cells, we examined pro-inflammatory cytokine production from bone marrow–derived dendritic cells (BMDCs). BMDCs from wild type mice, but not Dectin-2-/- or Card9-/- mice, produced a strong IL-6 response when stimulated with recombinant Bl-Eng2, but not PDIA1 (Fig 3B), indicating ligand specificity for Dectin-2. Lack of stimulation of BMDCs from knockout mice also excludes the possibility of endotoxin contamination as the stimulus of IL-6 in wild type cells. Thus, Pichia-expressed Bl-Eng2 triggers a cytokine response in vitro that requires Dectin-2 and downstream Card9. These results together indicate that Bl-Eng2 appears to be a selective Dectin-2 ligand. Dectin-2 recognizes several fungi including C. albicans, A. fumigatus and Malassezia, which possess N- and O-linked mannan on their surface [17,18,23,24,25,26]. Thus, not surprisingly, there are two other Dectin-2 ligands described in the literature. They are Furfurman from Malassezia spp. [25] and Man-LAM from M. tuberculosis [20]. In addition to these ligands, by using B3Z reporter cells in the work here, we observed that MP98 from Cryptococcus neoformans [27] is also recognized by Dectin-2 (S3C Fig). MP98 also triggers IL-6 by BMDC in a Dectin-2- and concentration-dependent manner (S3D Fig). MP98 is a mannoprotein of Mr of 98 kDa with 103 Ser/Thr residues at the C-terminus that serve as potential O-linked glycosylation sites, and 12 putative N-linked glycosylation sites [27]. To begin to evaluate the relative potency of Dectin-2 ligands, we compared the ability of Bl-Eng2 and the other three Dectin-2 ligands to induce cytokine production by BMDCs. Bl-Eng2 induced the strongest IL-6 production by BMDCs when compared at equal molar and mass ratios to the other ligands (Fig 3C). These results suggest that Bl-Eng2 is relatively potent for triggering IL-6 and might be used as an adjuvant for vaccination to boost the development of Ag-specific T cell responses. A suitable adjuvant for vaccine formulation should ideally stimulate human accessory cells. To test this capacity, we assessed the effect of Bl-Eng2 on the function of human PBMCs. After stimulation with plate-coated Bl-Eng2, human PBMCs from five healthy subjects produced up to 17 ng/ml IL-6 and 9 ng/ml IL-1β as measured in the cell culture supernatants by ELISA (Fig 3D). These data suggest that recombinant Bl-Eng2 has the capacity to induce the production of Th17 cell priming cytokines by human antigen-presenting cells (APC) in vitro. To investigate whether Bl-Eng2 could be harnessed as a vaccine adjuvant, we performed preclinical studies in mice. We first tested whether Bl-Eng2 augments the development of vaccine Ag-specific T cells. To assess these T cell responses in vivo, we vaccinated mice with the pan-fungal Ag calnexin [21] and enumerated CD4+ T cell responses by TCR Tg 1807 cells, which are specific for calnexin [28]. Calnexin was suspended with incomplete freund’s adjuvant (mineral oil) and injected subcutaneously. The addition of Bl-Eng2 into the formulation sharply increased the frequency of IL-17 producing 1807 T cells (Fig 4A) and the number of activated (CD44+) and IL-17 and IFN-γ producing 1807 T cells, as measured by ex vivo stimulation with anti-CD3 and anti-CD28 mAb (Fig 4B and S4A Fig). Ex vivo stimulation with the vaccine Ag calnexin also yielded sharp increases in the amount of IL-17 produced by T cells from the draining lymph nodes (Fig 4D). Thus, Bl-Eng2 promoted the development of Th17 cells more so than Th1 cells. Addition of Bl-Eng2 to the vaccine also reduced lung CFU as early as four days after mice received a lethal experimental challenge, and did so in a concentration-dependent manner (S4B Fig). In a parallel group, at the time unvaccinated control mice were moribund (day18 post-infection), the addition of Bl-Eng2 to the vaccine reduced lung CFU by more than two logs (Fig 4C). Combining the vaccine with commercial alum as an adjuvant did not increase the frequency and numbers of cytokine producing T cells or reduce the fungal burden (Fig 4A–4D). However, combining Bl-Eng-2 together with Alum increased the adjuvancy of Alum as measured by the number of activated (CD44+), IL-17 and IFN-γ producing 1807 T cells and the reduction in lung CFU (S5 Fig). These results suggest that Bl-Eng-2 can work in concert with other (commercially available and FDA approved) adjuvants and augment vaccine efficacy. Bl-Eng2 failed to increase the development of Th17 and Th1 cells, the production ex vivo of IL-17 and IFN-γ, or reduce lung CFU in Dectin-2-/- mice, verifying that the adjuvant effect is Dectin-2-dependent in vivo (S4C–S4E Fig). Thus, Bl-Eng2 exhibits adjuvant-like properties by increasing the development of Ag-specific (1807) Th17 and Th1 cells and protecting mice from lethal pulmonary infection with B. dermatitidis. The studies above exploited TCR Tg T cells to sensitively report the ability of Bl-Eng2 to enhance development of calnexin Ag-specific Th17 and Th1 cells upon vaccination. However, adoptive transfer of these cells into mice artificially enhances the number of CD4+ T cell precursors in the animal. To investigate whether Bl-Eng2 also has the capacity to induce the development of endogenous calnexin-Ag specific CD4+ T cells and similarly protect animals, we vaccinated wild type mice in the absence of adoptive transfer. The formulation of Bl-Eng2 with the calnexin subunit vaccine again reduced lung CFU by over two logs vs. control mice vaccinated with calnexin in mineral oil alone (IFA), and by over 3 logs vs. mice that got IFA alone (Fig 4E). The addition of Bl-Eng2 to the calnexin vaccine formulation also increased survival significantly vs. control mice vaccinated with calnexin alone (Fig 4E). This is remarkable since the number of Ag-specific T cell precursors before vaccination was far lower in the absence than in the presence of transferred of naïve 1807 cells, indicating that Bl-Eng2 is a powerful adjuvant that drives the development of protective endogenous calnexin-specific CD4+ T cells (Fig 4F). To investigate the downstream myeloid effector mechanisms of Bl-Eng-2 adjuvancy we used red fluorescent B. dermatitidis yeast to report phagocytic uptake and fungal viability during cellular interactions with the murine leukocytes. The concept of using fluorescence to monitor microbial fate and investigate functional outcomes of individual microbial cell-host cell encounters has been introduced recently [29] and provides a powerful strategy to measure effector mechanisms in vivo. At day 4 post-infection, mice vaccinated with calnexin+Bl-Eng-2 and calnexin+Alum+Bl-Eng-2 showed increased activation and killing by neutrophils and alveolar macrophages vs. calnexin and calnexin+ Alum controls (Fig 5A–5C and S6 Fig). The increase in in vivo fungal killing by neutrophils and macrophages correlated with reduced numbers of DsRed+ yeast in the lung (Figs 5D and S6D) and CFU by plating (Figs 5E and S6D). Bl-Eng-2 mediated effects were observed in the presence of adoptively transferred 1807 T cells (Fig 5) and by endogenous CD4+ T cells without adoptive transfer (S6C and S6D Fig). Thus, the addition of Bl-Eng-2 augments the activation and killing by myeloid effector cells such as the neutrophils and alveolar macrophages in the lung.
We describe a novel ligand for Dectin-2: Bl-Eng2. Discovery of a potent CLR ligand may address a limitation of current vaccines: the lack of adjuvants that elicit protective cell-mediated immunity. The approach we took to identify Bl-Eng2 was based on prior work from our group and other laboratories. Dectin-2 recognizes and mediates host defense against several fungi including C. albicans, C. glabrata, A. fumigatus, Malassezia spp. , Coccidiodes posadasii, Histoplasma capsulatum and B. dermatitidis [17,18,19,23,24,25,26,30]. Additionally, Dectin-2-/- mice vaccinated with attenuated B. dermatitidis yeast fail to prime Ag-specific Th1 and Th17 cells or acquire vaccine resistance to pulmonary infection. Thus, Dectin-2 regulates innate recognition of the fungal vaccine, and the development of a protective cellular immune response [19]. Hence, we sought to identify the Dectin-2 ligand from the vaccine strain. We hypothesized that the ligand would prime APC to produce cytokines (e. g. IL-6) that are known to foster the development of Th17 cells that protect against lethal fungal challenge [6]. By using Dectin-2 reporter cells as a probe, we enriched and identified Bl-Eng2 by ConA binding, gel filtration and Mass spectrometry. The identification of Bl-Eng2 also led us to unveil the unappreciated role of Asp-Eng2 in binding Dectin-2. Both Eng2 proteins are bona fide Dectin-2 ligands since they trigger NFAT signaling in Dectin-2 reporter cells. Bl-Eng2 features a 45. 2% overall and 60. 1% GH16 domain sequence similarity to Eng2 from A. fumigatus (Asp-Eng2) and contains a Ser/Thr-rich C-terminus that both proteins have in common. Bl-Eng2 and Asp-Eng2 respectively harbor 68 and 74 potential O-linked glycosylation sites within their respective 134-aa and 234-aa long Ser/Thr-rich C-terminus, but display no consensus sites for N-linked glycosylation (Asn-X-Ser/Thr). In addition to the Eng2 glycoproteins, we now also establish here that MP98 from C. neoformans serves as a ligand for Dectin-2. Dectin-2 has been reported to recognize high mannose structures of fungi [23], such as α-1,2-mannan from C. albicans [13,14] and furfurman, which is a mannoprotein from Malassezia spp. [25]. Man-Lam from M. tuberculosis consists of four components: a mannosyl-phophatidyl-myo-inositol (MPI) anchor, a mannose backbone, an arabinan domain, and a α1,2-mannose cap [20]. MP98 from C. neoformans is a mannoprotein with a Mr of 98 kDa; it contains 12 possible N-linked glycosylation sites, and 103 Ser/Thr residues at the C-terminus that serve as potential O-linked glycosylation sites [27]. The minimal unit of Bl-Eng2 that confers ligand activity is uncertain. Since both mannosidase and proteinase K digestion of CWE starting material reduced Dectin-2 ligand activity, both the protein and glycan moieties of Bl-Eng2 may contribute to its action, perhaps explaining its superior stimulation of cytokine responses compared to the other ligands. We found that recombinant Bl-Eng2 elicits potent downstream functions. It induces the production of IL-6 by BMDC in a Dectin-2- and Card9-dependent manner. In addition, Bl-Eng2 induces the production of IL-6 and IL-1β by human PBMC, which may have strong implications for the translational aspect of our discovery. In comparison to previously described Dectin-2 ligands, Bl-Eng2 triggers superior cytokine production by murine BMDC. Ligand induced IL-6 production was >100 fold higher for Bl-Eng2 than the other Dectin-2 ligands: Furfurman from Malassezia spp. [25] and Mannose-capped lipoarabinomannan (Man-Lam) from M. tuberculosis [20] and MP98 from C. neoformans [27]. Bl-Eng2 induction of T cell priming cytokines by APCs efficiently promoted the development of calnexin Ag-specific Th17 cells (more so than Th1 cells), and recall of these cells to the lung upon fungal challenge of vaccinated mice. The large numbers of pro-inflammatory T cells sharply reduced lung CFU and increased survival after infection of Bl-Eng2 vaccinated vs. control mice. In comparison, combining commercial Alum with the calnexin subunit vaccine did not show an adjuvant effect. However, Bl-Eng-2 combined with Alum augmented its adjuvancy indicating that Bl-Eng-2 has the potential to improve T cell priming by the commercially available and FDA approved Alum. Thus, in our subunit vaccine model, Bl-Eng2-induced Dectin-2 signaling was associated with cellular immune responses that protected mice against lethal pulmonary fungal infection. Although not experimentally addressed in this manuscript, it is conceivable that Bl-Eng-2 can also augment the induction of CD4+ T cell-dependent antibody responses that promote host protection against fungi, especially when combined with Alum since the latter is known to stimulate both T and B cell immune responses [31]. It remains to be investigated whether antibody will be protective in our vaccine setting [32]. We previously reported that mice vaccinated with calnexin and other adjuvants (glucan particles engaging Dectin-1, Adjuplex, or the combination) were optimally protected when we adoptive transferred naïve 1807 cells to increase the pool of Ag-experienced CD4+ T cells [21]. Here, the addition of Bl-Eng2 to the same calnexin vaccine reduced lung CFU by more than two to three logs vs. control mice even without adoptive transfer of large numbers of naïve 1807 T cell precursors. These results imply that engagement of Dectin-2 by Bl-Eng2 may be better than engagement of Dectin-1 by glucan particles and other previously used adjuvants at expanding the pool of endogenous calnexin-specific CD4+ T cell precursors or that Bl-Eng2 induced individual Ag-experienced cells to produce larger amounts of effector cytokines. Thus, Bl-Eng2 may be a powerful vaccine adjuvant in situations where T cell precursors are low in number and adoptive transfer of naïve T cell precursors is either not feasible or too costly. In contrast to the protective effects of Bl-Eng2 vaccination, Man-Lam induced Dectin-2 responses that caused Th17 cell-mediated autoimmune disease pathology and EAE [20]. Man-Lam stimulation of Dectin-2 led to the development of MOG35-55 peptide-specific T cells that produced IL-17, IFN-γ and GM-CSF upon ex vivo stimulation. This could simply relate to model selection rather than adjuvant efficiency. Thus, it is unclear whether Man-Lam is capable of inducing protective T cell immunity in an infectious disease setting. Although C. neoformans MP98 and its glycan modifications also promoted T cell activation, the T-helper phenotype and functional role in resistance by primed T cells were not investigated [33,34]. In conclusion, among the few Dectin-2 ligands reported to date, or newly discovered here, Bl-Eng2 is the most potent at stimulating murine and human cells to produce cytokines known to foster the development of protective Th17 and Th1 cells e. g. IL-6 and IL-1β. The production of IL-17 and IFN-γ by Th17 and Th1 cells then promotes the activation and killing of fungi by myeloid effector cells such as neutrophils and alveolar macrophages [6]. Since Bl-Eng2 also greatly augments protective immunity mediated by a subunit vaccine, Bl-Eng2 could potentially be harnessed as an adjuvant for vaccination against infectious disease that requires cellular immunity for host defense. The structural basis underpinning Bl-Eng2 potency as an adjuvant will be important to investigate and understand so that those features can be harnessed for vaccine development in the fight against infectious disease due to intracellular pathogens.
Strains used were wild-type, virulent B. dermatitidis ATCC strain 26199, DsRed26199 [35] and strain #55, the isogenic, attenuated mutant lacking BAD1 [36]. B. dermatitidis was grown as yeast on Middlebrook 7H10 agar with oleic acid-albumin complex (Sigma) at 39°C. Inbred wild type C57BL/6 and congenic B6. PL-Thy1a/Cy (stock #00406) mice carrying the Thy 1. 1 allele were obtained from Jackson Laboratories, Bar Harbor, ME. Blastomyces-specific TCR Tg 1807 mice were generated in our lab and were backcrossed to congenic Thy1. 1+ mice as described elsewhere [28]. Dectin-2-/- [14] mice were bred at our facility. Mice were 7–8 weeks old at the time of these experiments. Mice were housed and cared for as per guidelines of the University of Wisconsin Animal Care Committee who approved all aspects of this work. Blastomyces dermatitidis yeast were harvested from 7H10 agar, washed with H2O, and sonicated for 3 min on ice. After centrifuging, the soluble extract was collected, passed through a 0. 45-μm pore-size filter and used as CWE. The protein level was measured with the Pierce BCA assay (Thermo Fisher Scientific). To enrich the mannosylated proteins, CWE was incubated with Concanavalin A (ConA) Sepharose resin (Sigma-Aldrich), and the bound fraction was eluted with methyl-α-D-mannopyranoside as described previously [21]. The ConA-enriched proteins were then applied to a size exclusion column of Ultragel AcA 44 resin (Pall) and eluted with PBS. The ConA enrichment and size exclusion fractions were assessed using SDS-PAGE and silver staining. Size exclusion fractions that contained Dectin-2 ligand activity were analyzed by mass spectrometry as previously described [21] at the Mass Spectrometry Facility, University of Wisconsin-Madison. Briefly, peptides were analyzed by nano-LC-MS/MS using the Agilent 1100 nanoflow system (Agilent Technologies) connected to a hybrid linear ion trap-orbitrap mass spectrometer (LTQ-Orbitrap XL, Thermo Fisher Scientific) equipped with a nanoelectrospray ion source. Bl-Eng2 was cloned and expressed in P. pastoris using standard recombinant techniques. Total RNA was extracted from B. dermatitidis yeast and transcribed to cDNA as previously described [37]. Using the cDNA as a template, the Bl-ENG2 coding sequence was amplified using KOD Hot Start DNA Polymerase (Toyobo) with primers 5′-GGCTCGAGAAAAGAGAGGCTGAAGCTAGGGCTACCAAGCTCGCGTT and 5′-GTTTCTAGACCGTACTTGTCATTTGTGGGGTATCCCG, and inserted in-frame into the XhoI/XbaI sites of the pPICZαA vector (Invitrogen). The resulting expression vector was then linearized with PmeI and transformed into Pichia pastoris strain X-33 (Invitrogen) by electroporation. Yeast colonies were screened for Bl-Eng2 protein expression by Western blot analysis using an anti-His antibody (Cell Signaling Technology). Bl-Eng2 protein secreted from methanol-induced yeast cells was purified using Ni-NTA agarose (Qiagen) according to the manufacturer' s protocol, and dialyzed against PBS. Purity of recombinant Bl-Eng2 was assessed by SDS-PAGE and silver staining. Bl-Eng2 protein glycosylation was assessed using the Pierce Glycoprotein Staining Kit (Thermo Fisher Scientific). Monosaccharide composition was determined by gas chromatography as described elsewhere [38]. B3Z/BWZ reporter cells expressing Dectin-2, Mincle, MCL and Dectin-1 have been described previously [19,39]. For B3Z/BWZ cell stimulation, 105 B3Z/BWZ cells per well in a 96-well plate were incubated for 18 h with heat-killed fungal cells or plate-coated ligands. β-galactosidase (lacZ) activity was measured in total cell lysates using CPRG (Roche) as a substrate. OD 560 was measured using OD 620 as a reference. Generation of bone marrow–derived dendritic cells (BMDCs) has been described previously [19]. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood collected over Ficoll-Paque Plus (GE). 1–2 × 105 BMDCs or 5 × 105 PBMCs per well in a 96-well plate were incubated with plate-bound Bl-Eng2. After 24 h, supernatants were collected and cytokine levels were measured by ELISA (R&D Systems or Biolegend) according to the manufacturer’s specifications. Prior to vaccination, mice received adoptively transferred naïve 1807 T cells [28] or not. Mice were vaccinated twice subcutaneously with 10μg recombinant calnexin and 10μg Bl-Eng2 formulated in incomplete Freund’s adjuvant (IFA), two weeks apart. Two weeks after the boost, mice were challenged with 2x104 26199 yeast and analyzed for lung T cell responses (at day 4 post-infection) and lung CFU (at day 4 or two weeks post-infection). 1807 T cell responses were detected with the congenic Thy1. 1 marker and endogenous, calnexin-specific T cells by tetramer [28]. T cells were detected using the following antibodies: tetramer-PE, CD4-BUV395, CD8-PeCy7, CD3-BV421, CD90. 2-BV785, CD44-BV650, Live-dead Near IR, IFN-γ-A488 and IL-17-A647. Lung cells were harvested at day 4 post-infection. Cells (0. 5 × 106 cells/ml) were stimulated for 5 hours with anti-CD3 (clone 145-2C11; 0. 1μg/ml) and anti-CD28 (clone 37. 51; 1μg/ml) in the presence of Golgi-Stop (BD Biosciences). Stimulation with fungal ligands yielded comparable cytokine production by transgenic T-cells compared to CD3/CD28 stimulation. After cells were washed and stained for surface CD4 and CD8 using anti-CD4 BV395, anti-CD8 PeCy7, and anti-CD44-FITC mAbs (Pharmingen), they were fixed and permeabilized in Cytofix/Cytoperm at 4°C overnight. Permeabilized cells were stained with anti-IL-17A PE and anti-IFN-γ Alexa 700 (clone XMG1. 2) conjugated mAbs (Pharmingen) in FACS buffer for 30 min at 4°C, washed, and analyzed by FACS. Cells were gated on CD4 and cytokine expression in each gate analyzed. The number of cytokine positive CD4+ T cells per lung was calculated by multiplying the percent of cytokine- producing cells by the number of CD4+ T cells in the lung. Bone marrow-derived dendritic cells (BMDCs) were obtained from the femurs and tibias of individual mice. Each bone was flushed with 10 ml of 1% FBS in RPMI through a 22G needle. Red blood cells were lysed followed by wash and re-suspension of cells in 10% FBS in RPMI medium. In a petri dish, 2 × 106 bone marrow cells were plated in 10 ml of RPMI containing 10% FBS plus penicillin-streptomycin (P/S) (HyClone), 2-mercaptoethanol and 20 ng/ml of rGM-CSF. The culture media were refreshed every three days and BMDCs were harvested after 10 days for in vitro co-culture assays. Ex vivo cell culture supernatants were generated using the brachial and inguinal draining lymph nodes harvested from mice 28 days post-vaccination and at day 4 post-infection, washed and resuspended in complete RPMI containing 10 μg/ml recombinant calnexin [21,40], and plated in 96-well plates at a concentration of 5 × 105 cells/well. Supernatants were collected from ex vivo co-cultures after three days of incubation at 37°C and 5% CO2 [6]. IFN-γ and IL-17 (R&D System) were measured by ELISA according to manufacturer specifications (detection limits, 0. 05 ng/ml and 0. 02 ng/ml, respectively). Mice were euthanized three days after challenge i. t. with 105 DsRed yeast and hearts were perfused with PBS to remove blood from the lungs to improve staining. Lungs were dissociated, digested and stained as described previously [35]. In summary, lungs were dissociated and digested in buffer containing collagenase D and DNase I. After erythrocyte lysis, cells were stained for myeloid cell markers and then fixed in Cytofix/Cytoperm (BD Biosciences, San Jose, CA). Cells were stained for 30 minutes at room temperature with 1 μg/ml Uvitex-2B (Polysciences, Warrington, PA) diluted in BD perm/wash buffer and then subsequently washed with BD perm/wash buffer and fixed with 2% paraformaldehyde. Differences in the number of cells and lung CFU were analyzed using Wilcoxon rank and Mann Whitney test for non-parametric data or a T-test if data were normally distributed. A Bonferroni adjustment was used to correct for multiple tests. A value of P < 0. 05 is considered significant. Studies with human peripheral blood mononuclear cells were approved by University of Wisconsin-Madison IRB (protocol 2014–1167 CR002) and patients provided informed written consent. The animal studies performed were governed by protocols M00969 as approved by the IACUC committees of the University of Wisconsin-Madison Medical School. Animal studies were compliant with all applicable provisions established by the Animal Welfare Act and the Public Health Services (PHS) Policy on the Humane Care and Use of Laboratory Animals.
Despite several million new systemic fungal infections annually worldwide, there are no commercial vaccines available. The lack of appropriate adjuvants is one major impediment to developing safe and effective vaccines against infections with fungal pathogens. Current vaccines against infectious diseases preferentially induce protective antibodies, driven by adjuvants such as alum. While clonally-derived and adoptively transferred monoclonal antibodies may confer protection against fungi, the contribution of antibody to host defense is likely to be limited. Animal studies and clinical observations implicate cellular immunity as an essential component of the resolution of fungal infections. We found a promising adjuvant that augments cell mediated immune responses and vaccine-induced protection against fungal infection. We anticipate that our discovery will be a useful adjuvant for vaccination with non-replicating and safe subunit vaccines against many microbial pathogens that require protective cell mediated immune responses.
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Ligation of Dectin-2 with a novel microbial ligand promotes adjuvant activity for vaccination