Introduction Staphylococcus aureus and Candida spp. are the second and third leading causes of bloodstream infections in hospitalized patients [1]. These organisms jointly cause at least 150,000 clinical bloodstream infections resulting billions of dollars of health-care expenditures and ∼40,000 deaths per year in the US alone [1]–[4]. Identification of immune mechanisms of protective adaptive immunity against these organisms is critical to lay the groundwork for development of active vaccine strategies against both organisms. We previously reported that vaccination with the recombinant N terminus of the candidal Als3p adhesin (rAls3p-N) with aluminum hydroxide (Al(OH)3) adjuvant improved the survival of mice subsequently infected intravenously with lethal inocula of Candida albicans or methicillin resistant Staphylococcus aureus (MRSA) [5]–[7]. The vaccine retained efficacy against both infections in B cell deficient animals but not T cell deficient animals [6],[7]. Furthermore, adoptive transfer of CD4+ T cells but not B220+ B cells or immune serum improved the survival of recipient mice infected with both organisms [6],[7]. Although T cells are necessary for rAls3p-N vaccine efficacy, lymphocytes are not capable of directly killing C. albicans or S. aureus in culture [8],[9]. Therefore, the downstream effectors of vaccination against both organisms have remained unclear. In contrast to lymphocytes, phagocytes kill C. albicans and S. aureus in vitro [8],[10],[11] and in vivo [12]–[16], especially when primed with pro-inflammatory cytokines such as interferon (IFN)-γ, which is produced by CD4+ lymphocytes. Therefore, we hypothesized that the end effectors of rAls3p-N vaccine-mediated protection against bloodstream infection caused by S. aureus and C. albicans were phagocytes primed by pro-inflammatory cytokines produced by vaccine-responsive lymphocytes. We sought to elucidate fundamental requirements of protective host immunity to bloodstream infection caused by S. aureus and C. albicans. Results CD4+ lymphocyte-derived IFN-γ was necessary for vaccine efficacy in mice infected with either organism We previously established that the rAls3p-N vaccine was not effective against C. albicans iv infection in IFN-γ-deficient mice [6]. We sought to determine if IFN-γ was similarly required for vaccine-mediated protection against S. aureus, and also to determine if CD4+ T cells were the required source of IFN-γ production to mediate vaccine efficacy against both organisms. IFN-γ-deficient mice or their wild-type, congenic controls were vaccinated with rAls3p-N plus Al(OH)3 (vaccinated) or Al(OH)3 alone (control), and boosted at three weeks. Two weeks following the boost, CD4+ splenic and lymph node lymphocytes from vaccinated or control donor mice were purified and cross-adoptively transferred to recipient mice (IFN-γ deficient donor cells were transferred to wild type recipient mice, and visa versa). As a negative control, vaccinated or control IFN-γ knockout mice were infected without undergoing adoptive transfer. Mice were infected via the tail-vein with C. albicans or MRSA the day following adoptive transfer. IFN-γ-deficient mice receiving immune CD4+ lymphocytes from vaccinated, wild type donor mice had improved survival after either infection, whereas wild type mice receiving immune CD4+ lymphocytes from IFN-γ-deficient, vaccinated donor mice did not have improved survival (Fig. 1). Cells from control donor mice were not effective at improving survival of recipient mice. Hence, IFN-γ produced by vaccine-primed CD4+ T cells was required for mediating adaptive immunity against both infections. 10.1371/journal.ppat.1000703.g001 Figure 1 CD4 derived IFN-γ is required for vaccine protection. Wild type Balb/c mice were infected with 2×105 C. albicans SC5314 or 2×107 S. aureus LAC; IFN-γ deficient mice on a Balb/c background were infected with 105 C. albicans or 107 S. aureus. A) Wild type, n = 8 mice per group, or IFN-γ deficient mice, n = 7 mice per group, were vaccinated and infected iv via the tail-vein 2 weeks after the booster dose. *p 150-fold lower in gp91 phox−/− mice vs. wild-type controls ( 50% mortality in humans despite treatment with antifungal therapy [4]. Hence, achievement of survival approaching 50% by vaccination alone is felt to reflect meaningful protection. Furthermore, the experiment in which 12.5% survival was seen in the vaccinated arm was an adoptive transfer experiment, in which immune cells from wild type mice were transferred into IL-17A-/- recipient mice. Thus, while IL-17A production from CD4+ immune T cells can transfer protection, production of IL-17A by other cell types may be required to achieve maximal protection. Specifically, we previously found that immune CD8+ T cells could transfer protection against S. aureus [7], and macrophages or dendritic cells can produce pro-inflammatory cytokines such as IFN-γ, suggesting that these cell types may play an adjunctive role and be required for full vaccine-mediated protection. We previously reported that cyclophosphamide-induced neutropenia did not completely abrogate vaccine-induced protection during subsequent disseminated candidiasis [53]. In contrast, in the current study, we did find total abrogation of protection against both candidal strains and against S. aureus. The prior study used a different but related vaccine immunogen, rAls1p-N, instead of rAls3p-N. As well, the prior study used Complete Freund's Adjuvant (CFA), not Al(OH)3. The greater efficacy of the former adjuvant may account for the residual efficacy found in neutropenic mice in the former study. S. aureus and C. albicans express adhesins on their cell surface which possess similar three dimensional shapes [54] and which bind to similar endovascular surfaces (e.g. endothelial cells and subendothelial matrix proteins) and medically relevant plastics [54],[55]. Given these similar virulence mechanisms, it is not surprising that the organisms also infect patients with similar risk factors, including post-operative and trauma patients, patients with central venous catheters, patients on hemodialysis, and patients with compromised phagocytic host defense mechanisms [4],[56],[57]. Finally, our data demonstrate that the host defends itself against both infections by similar mechanisms, and that adaptive immunity to both organisms required CD4+ T cell production of both IFN-γ and IL-17A. In summary, the rAls3p-N vaccine improved outcomes in mouse models of iv S. aureus and C. albicans infection by inducing upstream, pro-inflammatory, Th1, Th17, and Th1/17 lymphocytes, which enhanced recruitment and activation of neutrophils in infected tissues, thereby reducing tissue infectious burden. Thus, vaccination showed a potential to protect against both infections by targeting the microbes for enhanced destruction by innate effector cells, irrespective of neutralization of microbial virulence factors. Therefore, potential vaccine antigens need not be restricted to microbial virulence factors, and can be expanded to include any target antigen which results in a potent Th1 and/or Th17 immune response against the organisms. Methods Organisms and mouse strains C. albicans SC5314 was supplied by W. Fonzi (Georgetown University), and S. aureus LAC, a USA300 MRSA clinical isolate, was provided by Frank Deleo (NIAID/NIH). C. albicans 15563 is a clinical bloodstream isolate from a patient at Harbor-UCLA Medical Center which is also virulent in our murine model [50]. Candida was serially passaged three times in yeast peptone dextrose broth (Difco) at room temperature prior to infection. S. aureus was grown overnight at 37°C in BHI broth, and then passaged for 4 hours at 37°C in fresh BHI broth. Female Balb/c or C57BL/6 mice were obtained from Taconic Farms (Bethesda, MD). Congenic IL-17A deficient mice on a Balb/c background were obtained from Y. Iwakura (University of Tokyo) [58]. Vaccinated mice were infected via the tail vein with the appropriate inocula of C. albicans blastospores or S. aureus organisms in PBS, as previously described [7],[52]. In some experiments, mice were made neutropenic by treatment with 230 mg/kg cyclophosphamide 2 days prior to infection, a regimen which results in profound neutropenia for approximately one week [59],[60]. All procedures involving mice were approved by the Los Angeles Biomedical Research Institute animal use and care committee, following the National Institutes of Health guidelines for animal housing and care. Immunization protocols rAls3p-N (amino acids 17 to 432 of Als3p) was produced in Saccharomyces cerevisiae and purified by Ni-NTA matrix affinity purification as previously described [61]. Mice were immunized by subcutaneous (SQ) injection of 300 µg of rAls3p-N in 0.1% Al(OH)3 (Alhydrogel, Brenntag Biosector, Frederikssund, Denmark) in PBS. Control mice received adjuvant alone on the same schedule. Some mice were boosted at 21 days. Mice were infected two weeks following the boost. Adoptive transfer and passive immunization Serum and splenic lymphocytes were harvested from vaccinated or control mice, as we have previously described [62]. Lymph node lymphocytes were harvested from cervical and axillary lymph nodes, based on pilot studies with Evans Blue dye lymph node mapping demonstrating that SQ vaccination at the base of the neck drained primarily to these lymph nodes. For adoptive transfers, splenic and lymph node lymphocytes were pooled. CD4+ T lymphocytes were purified by use of the IMag system (BD Pharmingen), as we have described [6],[7]. Purified lymphocytes (5×106 per mouse) were administered iv to congenic, unvaccinated recipient mice. Transferred cell populations were ≥95% pure by flow cytometric analysis. Mice were infected via the tail-vein with C. albicans SC5314 24 h after lymphocyte adoptive transfer. Intracellular cytokine analysis and cytokine supernatant analysis Intracellular cytokines from lymphocytes were analyzed based on a modification of our previously described method [62]. In brief, cervical and axillary lymph nodes and spleens were dissected from vaccinated or control mice and passed through 70 µm filters. Cells were stimulated for 5 days with rAls3p-N (12.5 µg/ml) in complete media (RPMI 1640, 50 U/ml penicillin, 50 µg/ml streptomycin, 2 mM L-glutamine, 10% FBS, 5 µM 2-ME) in 96 well plates. PMA (50 ng/ml), ionomycin (1 µM), and monensin (10 µg/ml) were added during the final 6 hours of culture. Supernatant was harvested prior to adding monensin for analysis of cytokine content using Cytometric Bead Array Flex kits (BD Pharmingen, La Jolla, CA) or ELISA (for IL-6, TGF-β, and IL-13), per the manufacturer's instructions. Cells were stained on ice with PerCP-anti-CD4 and Alexa647-anti-CCR6 (BD Pharmingen, San Diego), or their isotype control antibodies. The cells were fixed and permeabilized as previously described [62]. Intracellular cytokines were stained with rat FITC-anti-mouse IFN-γ and PE-anti-IL-17, or their isotype controls (BD Pharmingen). Four-color flow cytometry was performed on a Becton-Dickinson FACScan instrument calibrated with CaliBRITE beads (Becton Dickinson, San Jose, CA) using FACSComp software as per the manufacturer's recommendations. Data for each sample were acquired until 10,000 CD4+ lymphocytes were analyzed. Th1 cells were defined as CD4+IFNγ+IL-17−, Th17 cells defined as CD4+IFN-γ−IL-17+, and Th1/17 cells defined CD4+IFNγ+IL-17+. Killing assay The killing assay for both C. albicans and S. aureus was modified based on our well-described assay [59],[60]. In brief, RAW murine macrophage cells or murine neutrophil cells were grown in DMEM plus 10% fetal bovine serum. Fresh murine neutrophils were harvested by dextran sedimentation of whole, heparanized blood, followed by centrifugation over Ficoll Hypaque at 500 g for 10 minutes. The RAW cells or neutrophils were added into 24 well plates, the media in the wells was aspirated and the RAW cells or fresh neutrophils were cultured for 4 hours in 10% conditioned media (from vaccinated or control splenic and lymph node lymphocytes exposed to rAls3p-N for 5 days) plus 90% complete media (RPMI + 10% FBS). The conditioned media was then aspirated, and the microorganisms added to the wells in fresh DMEM plus 10% fetal bovine serum. Microorganisms were added to the wells at a ratio of 20∶1 RAW cells to C. albicans, 5∶1 RAW cells to S. aureus, or 10∶1 fresh neutrophils to C. albicans or S. aureus. Media for the wells containing S. aureus contained no antibiotics. The cells were incubated at 37°C for 1 h, at which point 4% blood heart infusion (BHI) agar was directly added to the wells. Plates were incubated overnight at 37°C and colony forming units (CFUs) counted in each well. Killing was defined as the percent reduction CFUs in wells containing co-cultures of phagocytes cells and microorganisms compared to wells just containing microorganisms. Tissue burden, whole organ cytokines, myeloperoxidase (MPO), and histopathology On day 4 post-infection, kidneys (primary target organ) were harvested and homogenized in saline with protease inhibitors (pepstatin, leupeptin, and PMFS). For determination of infectious burden, organ homogenates were quantitatively cultured on Sabourad dextrose agar for C. albicans or tryptic soy agar for S. aureus. Whole organ cytokines were analyzed from kidney homogenates by ELISA (R&D Systems) or Cytometric Bead Array Flex kit for KC (BD Pharmingen, La Jolla, CA), per the manufacturer's instructions. MPO levels were determined by ELISA (Hycult Biotechnology, Uden, Netherlands) of whole organ homogenates. For histopathology, organs were fixed in zinc-buffered formalin, embedded in paraffin, sectioned, and stained with PAS for fungi and H&E and Gram stain for bacteria. Statistics The non-parametric Log Rank test was utilized to determine differences in survival times. The Wilcoxon Rank test was used to compare cytokines, MPO levels, and organ burden across groups. P<0.05 was considered significant. Supporting Information Figure S1 Chemotherapy-induced neutropenia ablated vaccine induced protection against a second C. albicans clinical isolate. Sixteen Balb/c mice per group were vaccinated with rAls3p-N plus Al(OH)3 or Al(OH)3 alone, and boosted three weeks later. Two weeks after the boost, half the mice were treated with cyclophosphamide. Two days later the mice were infected with C. albicans 15563 (7x105). *p<0.05 for vaccinated vs. control by Log Rank test. (1.28 MB TIF) Click here for additional data file. Figure S2 FACS plots for gating on Th1, Th17, and Th1/17 cells in draining lymph nodes. Shown here are representative FACS plots, corresponding to the data in Fig. 6 of the manuscript, demonstrating acquisition gates based on size (FSC), density (SSC), or expression of CD4 or CCR6 on the cell surface. (1.85 MB TIF) Click here for additional data file. Figure S3 Vaccination primed Th1, Th17, and Th1/17 cells in draining lymph nodes. FACS plots demonstrating analysis of cytokine expression among lymphocytes using the gates shown in Fig. S2. (1.52 MB TIF) Click here for additional data file.
