Viability droplet digital polymerase chain reaction accurately enumerates probiotics and provides insight into damage experienced during storage.

Probiotics are typically enumerated by agar plate counting (PC) techniques. PC has several limitations including poor specificity, high variability, inability to enumerate dead cells, viable but non-culturable cells and cells in complex matrices. Viability droplet digital polymerase chain reaction (v-ddPCR) is an emerging enumeration technique with improved specificity, precision, and the ability to enumerate cells in varying states of culturability or in complex matrices. Good correlation and agreement between v-ddPCR and PC is well documented, but not much research has been published on the comparison when enumerating freeze-dried (FD) probiotics during storage. In this study, v-ddPCR utilizing PE51 (PE51-ddPCR), a combination of propidium monoazide (PMA) and ethidium monoazide (EMA), was evaluated as alternative enumeration technique to PC on blends of four FD probiotic strains over the course of a 3-month storage study with accelerated conditions. When PMA and EMA are combined (PE51), this study demonstrates agreement (bias = 7.63e+9, LOA = 4.38e+10 to 5.9e+10) and association (r = 0.762) between PC and v-ddPCR, at or above levels of an accepted alternative method. Additionally, v-ddPCR with individual dyes PMA and EMA provide insight into how they individually contribute to the viable counts obtained by PE51-ddPCR and provide a more specific physiological understanding of how probiotics cope with or experience damage during storage.

Virally-vectored vaccine candidates against white-nose syndrome induce anti-fungal immune response in little brown bats (Myotis lucifugus).

White-nose syndrome (WNS) caused by the fungus, Pseudogymnoascus destructans (Pd) has killed millions of North American hibernating bats. Currently, methods to prevent the disease are limited. We conducted two trials to assess potential WNS vaccine candidates in wild-caught Myotis lucifugus. In a pilot study, we immunized bats with one of four vaccine treatments or phosphate-buffered saline (PBS) as a control and challenged them with Pd upon transfer into hibernation chambers. Bats in one vaccine-treated group, that received raccoon poxviruses (RCN) expressing Pd calnexin (CAL) and serine protease (SP), developed WNS at a lower rate (1/10) than other treatments combined (14/23), although samples sizes were small. The results of a second similar trial provided additional support for this observation. Bats vaccinated orally or by injection with RCN-CAL and RCN-SP survived Pd challenge at a significantly higher rate (P = 0.01) than controls. Using RT-PCR and flow cytometry, combined with fluorescent in situ hybridization, we determined that expression of IFN-γ transcripts and the number of CD4 + T-helper cells transcribing this gene were elevated (P < 0.10) in stimulated lymphocytes from surviving vaccinees (n = 15) compared to controls (n = 3). We conclude that vaccination with virally-vectored Pd antigens induced antifungal immunity that could potentially protect bats against WNS.

Droplet Digital PCR Is an Improved Alternative Method for High-Quality Enumeration of Viable Probiotic Strains.

Traditional microbiological enumeration methods have long been employed as the standard evaluation procedure for probiotic microorganisms. These methods are labor intensive, have long-time to results and inherently have a high degree of variability - up to 35%. As clinical probiotic and microbiome science continues to grow and develop, it is increasingly important that researchers thoroughly define and deliver the targeted probiotic dose. Furthermore, to establish high quality commercial products, the same dosage level must be administered to consumers. An ISO method for the use of flow cytometry has been established which does speed up the time to results and reduce variability, but the method has not yet gained widespread adoption across the probiotic industry. This is possibly due to expertise needed to implement and maintain a new testing platform in an established quality system. In this study we compare enumeration using plate counts and flow cytometry to the use of droplet digital PCR (ddPCR), which in addition to giving faster time to results than plate count and less variability than both plate count and flow cytometry, has additional benefits such as strain-specific counts. Use of ddPCR gives the ability to design primers to target deletions and single base pair differences which will allow for strain profiling in microbiome analyses. We demonstrate that ddPCR probiotic enumeration results are positively correlated to both plate count and flow cytometry results and should be considered a viable, next generation enumeration method for the evaluation of probiotics.

Ligation of Dectin-2 with a novel microbial ligand promotes adjuvant activity for vaccination.

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.

Antifungal Tc17 cells are durable and stable, persisting as long-lasting vaccine memory without plasticity towards IFNγ cells.

Our understanding of persistence and plasticity of IL-17A+ memory T cells is clouded by conflicting results in models analyzing T helper 17 cells. We studied memory IL-17A+ CD8+ T-cell (Tc17) homeostasis, persistence and plasticity during fungal vaccine immunity. We report that vaccine-induced memory Tc17 cells persist with high fidelity to the type 17 phenotype. Tc17 cells persisted durably for a year as functional IL-17A+ memory cells without converting to IFNγ+ (Tc1) cells, although they produced multiple type I cytokines in the absence of residual vaccine antigen. Memory Tc17 cells were canonical CD8+ T cells with phenotypic features distinct from Tc1 cells, and were Ror(γ)thi, TCF-1hi, T-betlo and EOMESlo. In investigating the bases of Tc17 persistence, we observed that memory Tc17 cells had much higher levels of basal homeostatic proliferation than did Tc1 cells. Conversely, memory Tc17 cells displayed lower levels of anti-apoptotic molecules Bcl-2 and Bcl-xL than Tc1 cells, yet were resistant to apoptosis. Tc1 cells required Bcl-2 for their survival, but Bcl-2 was dispensable for the maintenance of Tc17 cells. Tc17 and Tc1 cells displayed different requirements for HIF-1α during effector differentiation and sustenance and memory persistence. Thus, antifungal vaccination induces durable and stable memory Tc17 cells with distinct requirements for long-term persistence that distinguish them from memory Tc1 cells.

MyD88 Shapes Vaccine Immunity by Extrinsically Regulating Survival of CD4+ T Cells during the Contraction Phase.

Soaring rates of systemic fungal infections worldwide underscore the need for vaccine prevention. An understanding of the elements that promote vaccine immunity is essential. We previously reported that Th17 cells are required for vaccine immunity to the systemic dimorphic fungi of North America, and that Card9 and MyD88 signaling are required for the development of protective Th17 cells. Herein, we investigated where, when and how MyD88 regulates T cell development. We uncovered a novel mechanism in which MyD88 extrinsically regulates the survival of activated T cells during the contraction phase and in the absence of inflammation, but is dispensable for the expansion and differentiation of the cells. The poor survival of activated T cells in Myd88-/- mice is linked to increased caspase3-mediated apoptosis, but not to Fas- or Bim-dependent apoptotic pathways, nor to reduced expression of the anti-apoptotic molecules Bcl-2 or Bcl-xL. Moreover, TLR3, 7, and/or 9, but not TLR2 or 4, also were required extrinsically for MyD88-dependent Th17 cell responses and vaccine immunity. Similar MyD88 requirements governed the survival of virus primed T cells. Our data identify unappreciated new requirements for eliciting adaptive immunity and have implications for designing vaccines.

Mannose Receptor Is Required for Optimal Induction of Vaccine-Induced T-Helper Type 17 Cells and Resistance to Blastomyces dermatitidis Infection.

We investigated how innate sensing by the mannose receptor (MR) influences the development of antifungal immunity. We demonstrate that MR senses mannan on the surface of attenuated Blastomyces dermatitidis vaccine yeast and that MR(-/-) mice demonstrate impaired vaccine immunity against lethal experimental blastomycosis, compared with wild-type control mice. Using naive Blastomyces-specific transgenic CD4(+) T cells, we found that MR regulates differentiation of naive T cells into T-helper type 17 (Th17) effector cells, which are essential in vaccine immunity against systemic dimorphic fungi. Thus, MR regulates differentiation of Th17 cells and is required to induce vaccine immunity against lethal pulmonary blastomycosis.

Intrinsic MyD88-Akt1-mTOR Signaling Coordinates Disparate Tc17 and Tc1 Responses during Vaccine Immunity against Fungal Pneumonia.

Fungal infections have skyrocketed in immune-compromised patients lacking CD4+ T cells, underscoring the need for vaccine prevention. An understanding of the elements that promote vaccine immunity in this setting is essential. We previously demonstrated that vaccine-induced IL-17A+ CD8+ T cells (Tc17) are required for resistance against lethal fungal pneumonia in CD4+ T cell-deficient hosts, whereas the individual type I cytokines IFN-γ, TNF-α and GM-CSF, are dispensable. Here, we report that T cell-intrinsic MyD88 signals are crucial for these Tc17 cell responses and vaccine immunity against lethal fungal pneumonia in mice. In contrast, IFN-γ+ CD8+ cell (Tc1) responses are largely normal in the absence of intrinsic MyD88 signaling in CD8+ T cells. The poor accumulation of MyD88-deficient Tc17 cells was not linked to an early onset of contraction, nor to accelerated cell death or diminished expression of anti-apoptotic molecules Bcl-2 or Bcl-xL. Instead, intrinsic MyD88 was required to sustain the proliferation of Tc17 cells through the activation of mTOR via Akt1. Moreover, intrinsic IL-1R and TLR2, but not IL-18R, were required for MyD88 dependent Tc17 responses. Our data identify unappreciated targets for augmenting adaptive immunity against fungi. Our findings have implications for designing fungal vaccines and immune-based therapies in immune-compromised patients.

C-type lectin receptors differentially induce th17 cells and vaccine immunity to the endemic mycosis of North America.

Vaccine immunity to the endemic mycoses of North America requires Th17 cells, but the pattern recognition receptors and signaling pathways that drive these protective responses have not been defined. We show that C-type lectin receptors exert divergent contributions to the development of antifungal Th17 cells and vaccine resistance against Blastomyces dermatitidis, Histoplasma capsulatum, and Coccidioides posadasii. Acquired immunity to B. dermatitidis requires Dectin-2, whereas vaccination against H. capsulatum and C. posadasii infection depends on innate sensing by Dectin-1 and Dectin-2, but not Mincle. Tracking Ag-specific T cells in vivo established that the Card9 signaling pathway acts indispensably and exclusively on differentiation of Th17 cells, while leaving intact their activation, proliferation, survival, and migration. Whereas Card9 signaling is essential, C-type lectin receptors offer distinct and divergent contributions to vaccine immunity against these endemic fungal pathogens. Our work provides new insight into innate immune mechanisms that drive vaccine immunity and Th17 cells.

Structure and function of a fungal adhesin that binds heparin and mimics thrombospondin-1 by blocking T cell activation and effector function.

Blastomyces adhesin-1 (BAD-1) is a 120-kD surface protein on B. dermatitidis yeast. We show here that BAD-1 contains 41 tandem repeats and that deleting even half of them impairs fungal pathogenicity. According to NMR, the repeats form tightly folded 17-amino acid loops constrained by a disulfide bond linking conserved cysteines. Each loop contains a highly conserved WxxWxxW motif found in thrombospondin-1 (TSP-1) type 1 heparin-binding repeats. BAD-1 binds heparin specifically and saturably, and is competitively inhibited by soluble heparin, but not related glycosaminoglycans. According to SPR analysis, the affinity of BAD-1 for heparin is 33 nM±14 nM. Putative heparin-binding motifs are found both at the N-terminus and within each tandem repeat loop. Like TSP-1, BAD-1 blocks activation of T cells in a manner requiring the heparan sulfate-modified surface molecule CD47, and impairs effector functions. The tandem repeats of BAD-1 thus confer pathogenicity, harbor motifs that bind heparin, and suppress T-cell activation via a CD47-dependent mechanism, mimicking mammalian TSP-1.

Interleukin 1 enhances vaccine-induced antifungal T-helper 17 cells and resistance against Blastomyces dermatitidis infection.

Vaccine-induced T-helper 17 (Th17) cells are necessary and sufficient to protect against fungal infection. Although live fungal vaccines are efficient in driving protective Th17 responses and immunity, attenuated fungi may not be safe for human use. Heat-inactivated formulations and subunit vaccines are safer but less potent and require adjuvant to increase their efficacy. Here, we show that interleukin 1 (IL-1) enhances the capacity of weak vaccines to induce protection against lethal Blastomyces dermatitidis infection in mice and is far more effective than lipopolysaccharide. While IL-1 enhanced expansion and differentiation of fungus-specific T cells by direct action on those cells, cooperation with non-T cells expressing IL-1R1 was necessary to maximize protection. Mechanistically, IL-17 receptor signaling was required for the enhanced protection induced by IL-1. Thus, IL-1 enhances the efficacy of safe but inefficient vaccines against systemic fungal infection in part by increasing the expansion of CD4(+) T cells, allowing their entry into the lungs, and inducing their differentiation to protective Th17 cells.

Fungi subvert vaccine T cell priming at the respiratory mucosa by preventing chemokine-induced influx of inflammatory monocytes.

Vaccinologists strive to harness immunity at mucosal sites of pathogen entry. We studied respiratory delivery of an attenuated vaccine against Blastomyces dermatitidis. We created a T cell receptor transgenic mouse responsive to vaccine yeast and found that mucosal vaccination led to poor T cell activation in the draining nodes and differentiation in the lung. Mucosal vaccination subverted lung T cell priming by inducing matrix metalloproteinase 2 (MMP2), which impaired the action of the chemokine CCL7 on egress of CCR2(+) Ly6C(hi) inflammatory monocytes from the bone marrow and their recruitment to the lung. Studies in Mmp2(-/-) mice, or treatment with MMP inhibitor or rCCL7, restored recruitment of Ly6C(hi) monocytes to the lung and CD4(+) T cell priming. Mucosal vaccination against fungi and perhaps other respiratory pathogens may require manipulation of host MMPs in order to alter chemokine signals needed to recruit Ly6C(hi) monocytes and prime T cells at the respiratory mucosa.

A TCR transgenic mouse reactive with multiple systemic dimorphic fungi.

Dimorphic fungi collectively account for 5-10 million new infections annually worldwide. Ongoing efforts seek to clarify mechanisms of cellular resistance to these agents and develop vaccines. A major limitation in studying the development of protective T cells in this group of organisms is the lack of tools to detect, enumerate, and characterize fungus-specific T cells during vaccination and infection. We generated a TCR transgenic mouse (Bd 1807) whose CD4(+) T cells respond to a native epitope in Blastomyces dermatitidis and also in Histoplasma capsulatum. In this study, we characterize the mouse, reveal its applications, and extend our analysis showing that 1807 cells also respond to the related dimorphic fungi Coccidioides posadasii and Paracoccidioides lutzii. On adoptive transfer into vaccinated wild-type mice, 1807 cells become activated, proliferate, and expand in the draining lymph nodes, and they differentiate into T1 effectors after trafficking to the lung upon lethal experimental challenge. Bd 1807 cells confer vaccine-induced resistance against B. dermatitidis, H. capsulatum, and C. posadasii. Transfer of naive 1807 cells at serial intervals postvaccination uncovered the prolonged duration of fungal Ag presentation. Using 1807 cells, we also found that the administration of vaccine only once induced a maximal pool of effector/memory CD4(+) cells and protective immunity by 4 wk after vaccination. The autologous adoptive transfer system described in this study reveals novel features of antifungal immunity and offers a powerful approach to study the differentiation of Ag-specific T cells responsive to multiple dimorphic fungi and the development of CD4(+) T cell memory needed to protect against fungal infection.

Vaccine-induced protection against 3 systemic mycoses endemic to North America requires Th17 cells in mice.

Worldwide rates of systemic fungal infections, including three of the major pathogens responsible for such infections in North America (Coccidioides posadasii, Histoplasma capsulatum, and Blastomyces dermatitidis), have soared recently, spurring interest in developing vaccines. The development of Th1 cells is believed to be crucial for protective immunity against pathogenic fungi, whereas the role of Th17 cells is vigorously debated. In models of primary fungal infection, some studies have shown that Th17 cells mediate resistance, while others have shown that they promote disease pathology. Here, we have shown that Th1 immunity is dispensable and that fungus-specific Th17 cells are sufficient for vaccine-induced protection against lethal pulmonary infection with B. dermatitidis in mice. Further, vaccine-induced Th17 cells were necessary and sufficient to protect against the three major systemic mycoses in North America. Mechanistically, Th17 cells engendered protection by recruiting and activating neutrophils and macrophages to the alveolar space, while the induction of Th17 cells and acquisition of vaccine immunity unexpectedly required the adapter molecule Myd88 but not the fungal pathogen recognition receptor Dectin-1. These data suggest that human vaccines against systemic fungal infections should be designed to induce Th17 cells if they are to be effective.

Spontaneous coronary vasospasm in KATP mutant mice arises from a smooth muscle-extrinsic process.

In the vasculature, ATP-sensitive potassium channels (KATP) channels regulate vascular tone. Mice with targeted gene disruptions of KATP subunits expressed in vascular smooth muscle develop spontaneous coronary vascular spasm and sudden death. From these models, it was hypothesized that the loss of KATP channel activity in arterial vascular smooth muscle was responsible for coronary artery spasm. We now tested this hypothesis using a transgenic strategy where the full-length sulfonylurea receptor containing exon 40 was expressed under the control of a smooth muscle-specific SM22alpha promoter. Two transgenic founder lines were generated and independently bred to sulfonylurea receptor 2 (SUR2) null mice to generate mice that restored expression of KATP channels in vascular smooth muscle. Transgenic expression of the sulfonylurea receptor in vascular smooth muscle cells was confirmed by detecting mRNA and protein from the transgene. Functional restoration was determined by recording pinacidil-based KATP current by whole cell voltage clamping of isolated aortic vascular smooth muscle cells isolated from the transgenic restored mice. Despite successful restoration of KATP channels in vascular smooth muscle, transgene-restored SUR2 null mice continued to display frequent episodes of spontaneous ST segment elevation, identical to the phenotype seen in SUR2 null mice. As in SUR2 null mice, ST segment elevation was frequently followed by atrioventricular heart block. ST segment elevation and coronary perfusion pressure in the restored mice did not differ significantly between transgene-negative and transgene-positive SUR2 null mice. We conclude that spontaneous coronary vasospasm and sudden death in SUR2 null mice arises from a coronary artery vascular smooth muscle-extrinsic process.