Lung eosinophils elicited during allergic and acute aspergillosis express RORγt and IL-23R but do not require IL-23 for IL-17 production.

Exposure to the mold, Aspergillus, is ubiquitous and generally has no adverse consequences in immunocompetent persons. However, invasive and allergic aspergillosis can develop in immunocompromised and atopic individuals, respectively. Previously, we demonstrated that mouse lung eosinophils produce IL-17 in response to stimulation by live conidia and antigens of A. fumigatus. Here, we utilized murine models of allergic and acute pulmonary aspergillosis to determine the association of IL-23, IL-23R and RORγt with eosinophil IL-17 expression. Following A. fumigatus stimulation, a population of lung eosinophils expressed RORγt, the master transcription factor for IL-17 regulation. Eosinophil RORγt expression was demonstrated by flow cytometry, confocal microscopy, western blotting and an mCherry reporter mouse. Both nuclear and cytoplasmic localization of RORγt in eosinophils were observed, although the former predominated. A population of lung eosinophils also expressed IL-23R. While expression of IL-23R was positively correlated with expression of RORγt, expression of RORγt and IL-17 was similar when comparing lung eosinophils from A. fumigatus-challenged wild-type and IL-23p19-/- mice. Thus, in allergic and acute models of pulmonary aspergillosis, lung eosinophils express IL-17, RORγt and IL-23R. However, IL-23 is dispensable for production of IL-17 and RORγt.

Structural basis of Blastomyces Endoglucanase-2 adjuvancy in anti-fungal and -viral immunity.

The development of safe subunit vaccines requires adjuvants that augment immunogenicity of non-replicating protein-based antigens. Current vaccines against infectious diseases preferentially induce protective antibodies driven by adjuvants such as alum. However, the contribution of antibody to host defense is limited for certain classes of infectious diseases such as fungi, whereas animal studies and clinical observations implicate cellular immunity as an essential component of the resolution of fungal pathogens. Here, we decipher the structural bases of a newly identified glycoprotein ligand of Dectin-2 with potent adjuvancy, Blastomyces endoglucanase-2 (Bl-Eng2). We also pinpoint the developmental steps of antigen-specific CD4+ and CD8+ T responses augmented by Bl-Eng2 including expansion, differentiation and tissue residency. Dectin-2 ligation led to successful systemic and mucosal vaccination against invasive fungal infection and Influenza A infection, respectively. O-linked glycans on Bl-Eng2 applied at the skin and respiratory mucosa greatly augment vaccine subunit- induced protective immunity against lethal influenza and fungal pulmonary challenge.

Central Role of IL-23 and IL-17 Producing Eosinophils as Immunomodulatory Effector Cells in Acute Pulmonary Aspergillosis and Allergic Asthma.

Aspergillus fumigatus causes invasive pulmonary disease in immunocompromised hosts and allergic asthma in atopic individuals. We studied the contribution of lung eosinophils to these fungal diseases. By in vivo intracellular cytokine staining and confocal microscopy, we observed that eosinophils act as local sources of IL-23 and IL-17. Remarkably, mice lacking eosinophils had a >95% reduction in the percentage of lung IL-23p19+ cells as well as markedly reduced IL-23 heterodimer in lung lavage fluid. Eosinophils killed A. fumigatus conidia in vivo. Eosinopenic mice had higher mortality rates, decreased recruitment of inflammatory monocytes, and decreased expansion of lung macrophages after challenge with conidia. All of these functions underscore a potential protective role for eosinophils in acute aspergillosis. Given the postulated role for IL-17 in asthma pathogenesis, we assessed whether eosinophils could act as sources of IL-23 and IL-17 in models where mice were sensitized to either A. fumigatus antigens or ovalbumin (OVA). We found IL-23p19+ IL-17AF+ eosinophils in both allergic models. Moreover, close to 95% of IL-23p19+ cells and >90% of IL-17AF+ cells were identified as eosinophils. These data establish a new paradigm in acute and allergic aspergillosis whereby eosinophils act not only as effector cells but also as immunomodulatory cells driving the IL-23/IL-17 axis and contributing to inflammatory cell recruitment.

Recognition of Aspergillus fumigatus hyphae by human plasmacytoid dendritic cells is mediated by dectin-2 and results in formation of extracellular traps.

Plasmacytoid dendritic cells (pDCs) were initially considered as critical for innate immunity to viruses. However, our group has shown that pDCs bind to and inhibit the growth of Aspergillus fumigatus hyphae and that depletion of pDCs renders mice hypersusceptible to experimental aspergillosis. In this study, we examined pDC receptors contributing to hyphal recognition and downstream events in pDCs stimulated by A. fumigatus hyphae. Our data show that Dectin-2, but not Dectin-1, participates in A. fumigatus hyphal recognition, TNF-α and IFN-α release, and antifungal activity. Moreover, Dectin-2 acts in cooperation with the FcRγ chain to trigger signaling responses. In addition, using confocal and electron microscopy we demonstrated that the interaction between pDCs and A. fumigatus induced the formation of pDC extracellular traps (pETs) containing DNA and citrullinated histone H3. These structures closely resembled those of neutrophil extracellular traps (NETs). The microarray analysis of the pDC transcriptome upon A. fumigatus infection also demonstrated up-regulated expression of genes associated with apoptosis as well as type I interferon-induced genes. Thus, human pDCs directly recognize A. fumigatus hyphae via Dectin-2; this interaction results in cytokine release and antifungal activity. Moreover, hyphal stimulation of pDCs triggers a distinct pattern of pDC gene expression and leads to pET formation.

Chitin recognition via chitotriosidase promotes pathologic type-2 helper T cell responses to cryptococcal infection.

Pulmonary mycoses are often associated with type-2 helper T (Th2) cell responses. However, mechanisms of Th2 cell accumulation are multifactorial and incompletely known. To investigate Th2 cell responses to pulmonary fungal infection, we developed a peptide-MHCII tetramer to track antigen-specific CD4+ T cells produced in response to infection with the fungal pathogen Cryptococcus neoformans. We noted massive accruement of pathologic cryptococcal antigen-specific Th2 cells in the lungs following infection that was coordinated by lung-resident CD11b+ IRF4-dependent conventional dendritic cells. Other researchers have demonstrated that this dendritic cell subset is also capable of priming protective Th17 cell responses to another pulmonary fungal infection, Aspergillus fumigatus. Thus, higher order detection of specific features of fungal infection by these dendritic cells must direct Th2 cell lineage commitment. Since chitin-containing parasites commonly elicit Th2 responses, we hypothesized that recognition of fungal chitin is an important determinant of Th2 cell-mediated mycosis. Using C. neoformans mutants or purified chitin, we found that chitin abundance impacted Th2 cell accumulation and disease. Importantly, we determined Th2 cell induction depended on cleavage of chitin via the mammalian chitinase, chitotriosidase, an enzyme that was also prevalent in humans experiencing overt cryptococcosis. The data presented herein offers a new perspective on fungal disease susceptibility, whereby chitin recognition via chitotriosidase leads to the initiation of harmful Th2 cell differentiation by CD11b+ conventional dendritic cells in response to pulmonary fungal infection.

Functional specialization among members of Knickkopf family of proteins in insect cuticle organization.

Our recent study on the functional analysis of the Knickkopf protein from T. castaneum (TcKnk), indicated a novel role for this protein in protection of chitin from degradation by chitinases. Knk is also required for the laminar organization of chitin in the procuticle. During a bioinformatics search using this protein sequence as the query, we discovered the existence of a small family of three Knk-like genes (including the prototypical TcKnk) in the T. castaneum genome as well as in all insects with completed genome assemblies. The two additional Knk-like genes have been named TcKnk2 and TcKnk3. Further complexity arises as a result of alternative splicing and alternative polyadenylation of transcripts of TcKnk3, leading to the production of three transcripts (and by inference, three proteins) from this gene. These transcripts are named TcKnk3-Full Length (TcKnk3-FL), TcKnk3-5' and TcKnk3-3'. All three Knk-family genes appear to have essential and non-redundant functions. RNAi for TcKnk led to developmental arrest at every molt, while down-regulation of either TcKnk2 or one of the three TcKnk3 transcripts (TcKnk3-3') resulted in specific molting arrest only at the pharate adult stage. All three Knk genes appear to influence the total chitin content at the pharate adult stage, but to variable extents. While TcKnk contributes mostly to the stability and laminar organization of chitin in the elytral and body wall procuticles, proteins encoded by TcKnk2 and TcKnk3-3' transcripts appear to be required for the integrity of the body wall denticles and tracheal taenidia, but not the elytral and body wall procuticles. Thus, the three members of the Knk-family of proteins perform different essential functions in cuticle formation at different developmental stages and in different parts of the insect anatomy.

Spectrum and mechanisms of inflammasome activation by chitosan.

Chitosan, the deacetylated derivative of chitin, can be found in the cell wall of some fungi and is used in translational applications. We have shown that highly purified preparations of chitosan, but not chitin, activate the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in primed mouse bone marrow-derived macrophages (BMMΦ), inducing a robust IL-1ß response. In this article, we further define specific cell types that are activated and delineate mechanisms of activation. BMMΦ differentiated to promote a classically activated (M1) phenotype released more IL-1ß in response to chitosan than intermediate or alternatively activated macrophages (M2). Chitosan, but not chitin, induced a robust IL-1ß response in mouse dendritic cells, peritoneal macrophages, and human PBMCs. Three mechanisms for NLRP3 inflammasome activation may contribute: K(+) efflux, reactive oxygen species, and lysosomal destabilization. The contributions of these mechanisms were tested using a K(+) efflux inhibitor, high extracellular potassium, a mitochondrial reactive oxygen species inhibitor, lysosomal acidification inhibitors, and a cathepsin B inhibitor. These studies revealed that each of these pathways participated in optimal NLRP3 inflammasome activation by chitosan. Finally, neither chitosan nor chitin stimulated significant release from unprimed BMMΦ of any of 22 cytokines and chemokines assayed. This study has the following conclusions: 1) chitosan, but not chitin, stimulates IL-1ß release from multiple murine and human cell types; 2) multiple nonredundant mechanisms appear to participate in inflammasome activation by chitosan; and 3) chitin and chitosan are relatively weak stimulators of inflammatory mediators from unprimed BMMΦ. These data have implications for understanding the nature of the immune response to microbes and biomaterials that contain chitin and chitosan.

Retroactive maintains cuticle integrity by promoting the trafficking of Knickkopf into the procuticle of Tribolium castaneum.

Molting, or the replacement of the old exoskeleton with a new cuticle, is a complex developmental process that all insects must undergo to allow unhindered growth and development. Prior to each molt, the developing new cuticle must resist the actions of potent chitinolytic enzymes that degrade the overlying old cuticle. We recently disproved the classical dogma that a physical barrier prevents chitinases from accessing the new cuticle and showed that the chitin-binding protein Knickkopf (Knk) protects the new cuticle from degradation. Here we demonstrate that, in Tribolium castaneum, the protein Retroactive (TcRtv) is an essential mediator of this protective effect of Knk. TcRtv localizes within epidermal cells and specifically confers protection to the new cuticle against chitinases by facilitating the trafficking of TcKnk into the procuticle. Down-regulation of TcRtv resulted in entrapment of TcKnk within the epidermal cells and caused molting defects and lethality in all stages of insect growth, consistent with the loss of TcKnk function. Given the ubiquity of Rtv and Knk orthologs in arthropods, we propose that this mechanism of new cuticle protection is conserved throughout the phylum.

Relative contributions of dectin-1 and complement to immune responses to particulate ß-glucans.

Glucan particles (GPs) are Saccharomyces cerevisiae cell walls chemically extracted so they are composed primarily of particulate ß-1,3-D-glucans. GPs are recognized by Dectin-1 and are potent complement activators. Mice immunized with Ag-loaded GPs develop robust Ab and CD4(+) T cell responses. In this study, we examined the relative contributions of Dectin-1 and complement to GP phagocytosis and Ag-specific responses to immunization with OVA encapsulated in GPs. The in vitro phagocytosis of GPs by bone marrow-derived dendritic cells was facilitated by heat-labile serum component(s) independently of Dectin-1. This enhanced uptake was not seen with serum from complement component 3 knockout (C3(-/-)) mice and was also inhibited by blocking Abs directed against complement receptor 3. After i.p. injection, percent phagocytosis of GPs by peritoneal macrophages was comparable in wild-type and Dectin-1(-/-) mice and was not inhibited by the soluble ß-glucan antagonist laminarin. In contrast, a much lower percentage of peritoneal macrophages from C3(-/-) mice phagocytosed GPs, and this percentage was further reduced in the presence of laminarin. Subcutaneous immunization of wild-type, Dectin-1(-/-), and C3(-/-) mice with GP-OVA resulted in similar Ag-specific IgG(1) and IgG(2c) type Ab and CD4(+) T cell lymphoproliferative responses. Moreover, while CD4(+) Th1 and Th2 responses measured by ELISPOT assay were similar in the three mouse strains, Th17 responses were reduced in C3(-/-) mice. Thus, although Dectin-1 is necessary for optimal phagocytosis of GPs in the absence of complement, complement dominates when both an intact complement system and Dectin-1 are present. In addition, Th-skewing after GP-based immunization was altered in C3(-/-) mice.

Knickkopf protein protects and organizes chitin in the newly synthesized insect exoskeleton.

During each molting cycle of insect development, synthesis of new cuticle occurs concurrently with the partial degradation of the overlying old exoskeleton. Protection of the newly synthesized cuticle from molting fluid enzymes has long been attributed to the presence of an impermeable envelope layer that was thought to serve as a physical barrier, preventing molting fluid enzymes from accessing the new cuticle and thereby ensuring selective degradation of only the old one. In this study, using the red flour beetle, Tribolium castaneum, as a model insect species, we show that an entirely different and unexpected mechanism accounts for the selective action of chitinases and possibly other molting enzymes. The molting fluid enzyme chitinase, which degrades the matrix polysaccharide chitin, is not excluded from the newly synthesized cuticle as previously assumed. Instead, the new cuticle is protected from chitinase action by the T. castaneum Knickkopf (TcKnk) protein. TcKnk colocalizes with chitin in the new cuticle and organizes it into laminae. Down-regulation of TcKnk results in chitinase-dependent loss of chitin, severe molting defects, and lethality at all developmental stages. The conservation of Knickkopf across insect, crustacean, and nematode taxa suggests that its critical roles in the laminar ordering and protection of exoskeletal chitin may be common to all chitinous invertebrates.

Fluorescence and Biochemical Assessment of the Chitin and Chitosan Content of Cryptococcus.

The cell wall of the fungal pathogens Cryptococcus neoformans and C. gattii is critical for cell wall integrity and signaling external threats to the cell, allowing it to adapt and grow in a variety of changing environments. Chitin is a polysaccharide found in the cell walls of fungi that is considered to be essential for fungal survival. Chitosan is a polysaccharide derived from chitin via deacetylation that is also essential for cryptococcal cell wall integrity, fungal pathogenicity, and virulence. Cryptococcus has evolved mechanisms to regulate the amount of chitin and chitosan during growth under laboratory conditions or during mammalian infection. Therefore, levels of chitin and chitosan have been useful phenotypes to define mutant Cryptococcus strains. As a result, we have developed and/or refined various qualitative and quantitative methods for measuring chitin and chitosan. These techniques include those that use fluorescent probes that are known to bind to chitin (e.g., calcofluor white and wheat germ agglutinin), as well as those that preferentially bind to chitosan (e.g., eosin Y and cibacron brilliant red 3B-A). Techniques that enhance the localization and quantification of chitin and chitosan in the cell wall include (i) fluorescence microscopy, (ii) flow cytometry, (iii) and spectrofluorometry. We have also modified two highly selective biochemical methods to measure cellular chitin and chitosan content: the Morgan-Elson and the 3-methyl-2-benzothiazolone hydrazine hydrochloride (MBTH) assays, respectively.

Immune evasion by Cryptococcus gattii in vaccinated mice coinfected with C. neoformans.

Cryptococcus neoformans and C. gattii, the etiologic agents of cryptococcosis, cause over 100,000 deaths worldwide every year, yet no cryptococcal vaccine has progressed to clinical trials. In preclinical studies, mice vaccinated with an attenuated strain of C. neoformans deleted of three cryptococcal chitin deacetylases (Cn-cda1Δ2Δ3Δ) were protected against a lethal challenge with C. neoformans strain KN99. While Cn-cda1Δ2Δ3Δ extended the survival of mice infected with C. gattii strain R265 compared to unvaccinated groups, we were unable to demonstrate fungal clearance as robust as that seen following KN99 challenge. In stark contrast to vaccinated mice challenged with KN99, we also found that R265-challenged mice failed to induce the production of protection-associated cytokines and chemokines in the lungs. To investigate deficiencies in the vaccine response to R265 infection, we developed a KN99-R265 coinfection model. In unvaccinated mice, the strains behaved in a manner which mirrored single infections, wherein only KN99 disseminated to the brain and spleen. We expanded the coinfection model to Cn-cda1Δ2Δ3Δ-vaccinated mice. Fungal burden, cytokine production, and immune cell infiltration in the lungs of vaccinated, coinfected mice were indicative of immune evasion by C. gattii R265 as the presence of R265 neither compromised the immunophenotype established in response to KN99 nor inhibited clearance of KN99. Collectively, these data indicate that R265 does not dampen a protective vaccine response, but rather suggest that R265 remains largely undetected by the immune system.

Protection against experimental cryptococcosis elicited by Cationic Adjuvant Formulation 01-adjuvanted subunit vaccines.

The fungal infection, cryptococcosis, is responsible for >100,000 deaths annually. No licensed vaccines are available. We explored the efficacy and immune responses of subunit cryptococcal vaccines adjuvanted with Cationic Adjuvant Formulation 01 (CAF01). CAF01 promotes humoral and T helper (Th) 1 and Th17 immune responses and has been safely used in human vaccine trials. Four subcutaneous vaccines, each containing single recombinant Cryptococcus neoformans protein antigens, partially protected mice from experimental cryptococcosis. Protection increased, up to 100%, in mice that received bivalent and quadrivalent vaccine formulations. Vaccinated mice that received a pulmonary challenge with C. neoformans had an influx of leukocytes into the lung including robust numbers of polyfunctional CD4+ T cells which produced Interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin (IL)-17 upon ex vivo antigenic stimulation. Cytokine-producing lung CD8+ T cells were also found, albeit in lesser numbers. A significant, durable IFNγ response was observed in the lungs, spleen, and blood. Moreover, IFNγ secretion following ex vivo stimulation directly correlated with fungal clearance in the lungs. Thus, we have developed multivalent cryptococcal vaccines which protect mice from experimental cryptococcosis using an adjuvant which has been safely tested in humans. These preclinical studies suggest a path towards human cryptococcal vaccine trials.

Chitosan-Deficient Cryptococcus as Whole-Cell Vaccines.

Creating a safe and effective vaccine against infection by the fungal pathogen Cryptococcus neoformans is an appealing option that complements the discovery of new small molecule antifungals. Recent animal studies have yielded promising results for a variety of vaccines that include live-attenuated and heat-killed whole-cell vaccines, as well as subunit vaccines formulated around recombinant proteins. Some of the recombinantly engineered cryptococcal mutants in the chitosan biosynthesis pathway are avirulent and very effective at conferring protective immunity. Mice vaccinated with these avirulent chitosan-deficient strains are protected from a lethal pulmonary infection with C. neoformans strain KN99. Heat-killed derivatives of the vaccination strains are likewise effective in a murine model of infection. The efficacy of these whole-cell vaccines, however, is dependent on a number of factors, including the inoculation dose, route of vaccination, frequency of vaccination, and the specific mouse strain used in the study. Here, we present detailed methods for identifying and optimizing various factors influencing vaccine potency and efficacy in various inbred mouse strains using a chitosan-deficient cda1Δcda2Δcda3Δ strain as a whole-cell vaccine candidate. This chapter describes the protocols for immunizing three different laboratory mouse strains with vaccination regimens that use intranasal, orotracheal, and subcutaneous vaccination routes after the animals were sedated using two different types of anesthesia.

Immunological correlates of protection mediated by a whole organism Cryptococcus neoformans vaccine deficient in chitosan.

The global burden of infections due to the pathogenic fungus Cryptococcus is substantial in persons with low CD4 + T cell counts. Previously, we deleted three chitin deacetylase genes from C. neoformans to create a chitosan-deficient, avirulent strain, designated cda1Δ2Δ3Δ which, when used as a vaccine, protected mice from challenge with virulent C. neoformans strain KN99. Here, we explored the immunological basis for protection. Vaccine-mediated protection was maintained in mice lacking B cells or CD8 + T cells. In contrast, protection was lost in mice lacking α/ß T cells or CD4 + T cells. Moreover, CD4 + T cells from vaccinated mice conferred protection upon adoptive transfer to naive mice. Importantly, while monoclonal antibody-mediated depletion of CD4 + T cells just prior to vaccination resulted in complete loss of protection, significant protection was retained in mice depleted of CD4 + T cells after vaccination, but prior to challenge. Vaccine-mediated protection was lost in mice genetically deficient in IFNγ, TNFα, or IL-23p19. A robust influx of leukocytes and IFNγ- and TNFα-expressing CD4 + T cells was seen in the lungs of vaccinated and challenged mice. Finally, a higher level of IFNγ production by lung cells stimulated ex vivo correlated with lower fungal burden in the lungs. Thus, while B cells and CD8 + T cells are dispensable, IFNγ and CD4 + T cells have overlapping roles in generating protective immunity prior to cda1Δ2Δ3Δ vaccination. However, once vaccinated, protection becomes less dependent on CD4 + T cells, suggesting a strategy for vaccinating HIV + persons prior to loss of CD4 + T cells. Importance: The fungus Cryptococcus neoformans is responsible for >100,000 deaths annually, mostly in persons with impaired CD4 + T cell function such as AIDS. There are no approved human vaccines. We previously created a genetically engineered avirulent strain of C. neoformans , designated cda1Δ2Δ3Δ . When used as a vaccine, cda1Δ2Δ3Δ protects mice against a subsequent challenge with a virulent C. neoformans strain. Here, we defined components of the immune system responsible for vaccine-mediated protection. We found that while B cells and CD8 + T cells were dispensible, protection was lost in mice genetically deficient in CD4 + T cells, and the cytokines IFNγ, TNFα, or IL-23. A robust influx of cytokine-producing CD4 + T cells was seen in the lungs of vaccinated mice following infection. Importantly, protection was retained in mice depleted of CD4 + T cells following vaccination, suggesting a strategy to protect persons who are at risk for future CD4 + T cell dysfunction.

One Step Purification-Vaccine Delivery System.

Glucan particles (GPs) are hollow, porous 3-5 µm microspheres derived from the cell walls of Baker's yeast (Saccharomyces cerevisiae). Their 1,3-ß-glucan outer shell allows for receptor-mediated uptake by macrophages and other phagocytic innate immune cells expressing ß-glucan receptors. GPs have been used for the targeted delivery of a wide range of payloads, including vaccines and nanoparticles, encapsulated inside the hollow cavity of GPs. In this paper, we describe the methods to prepare GP-encapsulated nickel nanoparticles (GP-Ni) for the binding of histidine (His)-tagged proteins. His-tagged Cda2 cryptococcal antigens were used as payloads to demonstrate the efficacy of this new GP vaccine encapsulation approach. The GP-Ni-Cda2 vaccine was shown to be comparable to our previous approach utilizing mouse serum albumin (MSA) and yeast RNA trapping of Cda2 in GPs in a mouse infection model. This novel GP-Ni approach allows for the one-step binding of His-tagged vaccine antigens and encapsulation in an effective delivery vehicle to target vaccines to antigen-presenting cells (APCs), antigen discovery, and vaccine development.

An efficient (nano) silica - In glucan particles protein encapsulation approach for improved thermal stability.

Glucan particles (GPs) are hollow, porous microspheres derived from Saccharomyces cerevisiae (Baker's yeast). The hollow cavity of GPs allows for efficient encapsulation of different types of macromolecules and small molecules. The ß-1,3-D-glucan outer shell provides for receptor-mediated uptake by phagocytic cells expressing ß-glucan receptors and uptake of particles containing encapsulated proteins elicit protective innate and acquired immune responses against a wide range of pathogens. A limitation of the previously reported GP protein delivery technology is limited protection from thermal degradation. Here we present results of an efficient protein encapsulation approach using tetraethylorthosilicate (TEOS) to lock protein payloads in a thermostable silica cage formed in situ inside the hollow cavity of GPs. The methods for this improved, efficient GP protein ensilication approach were developed and optimized using bovine serum albumin (BSA) as model protein. The improved method involved controlling the rate of TEOS polymerization, such that the soluble TEOS-protein solution was able to be absorbed into the GP hollow cavity before the protein-silica cage polymerized and becomes too large to transverse across the GP wall. This improved method provided for >90% GP encapsulation efficiency, increased thermal stabilization of GP ensilicated BSA, and was shown to be applicable for encapsulation of proteins of different molecular weights and isoelectric points. To demonstrate the retention of bioactivity of this improved method of protein delivery, we evaluated the in vivo immunogenicity of two GP ensilicated vaccine formulations using (1) ovalbumin as a model antigen and (2) a protective antigenic protein from the fungal pathogen Cryptococcus neoformans. The results show that the GP ensilicated vaccines have a similar high immunogenicity as our current GP protein/hydrocolloid vaccines, as evidenced by robust antigen-specific IgG responses to the GP ensilicated OVA vaccine. Further, a GP ensilicated C. neoformans Cda2 vaccine protected vaccinated mice from a lethal pulmonary infection of C. neoformans.

Immunological correlates of protection following vaccination with glucan particles containing Cryptococcus neoformans chitin deacetylases.

Vaccination with glucan particles (GP) containing the Cryptococcus neoformans chitin deacetylases Cda1 and Cda2 protect mice against experimental cryptococcosis. Here, immunological correlates of vaccine-mediated protection were explored. Studies comparing knockout and wild-type mice demonstrated CD4+ T cells are crucial, while B cells and CD8+ T cells are dispensable. Protection was abolished following CD4+ T cell depletion during either vaccination or infection but was retained if CD4+ T cells were only partially depleted. Vaccination elicited systemic and durable antigen-specific immune responses in peripheral blood mononuclear cells (PBMCs), spleens, and lungs. Following vaccination and fungal challenge, robust T-helper (Th) 1 and Th17 responses were observed in the lungs. Protection was abrogated in mice congenitally deficient in interferon (IFN) γ, IFNγ receptor, interleukin (IL)-1ß, IL-6, or IL-23. Thus, CD4+ T cells and specific proinflammatory cytokines are required for GP-vaccine-mediated protection. Importantly, retention of protection in the setting of partial CD4+ T depletion suggests a pathway for vaccinating at-risk immunocompromised individuals.

Dysregulated Pulmonary Inflammatory Responses Exacerbate the Outcome of Secondary Aspergillosis Following Influenza.

Inhalation of airborne conidia of the ubiquitous fungus Aspergillus fumigatus commonly occurs but invasive aspergillosis is rare except in profoundly immunocompromised persons. Severe influenza predisposes patients to invasive pulmonary aspergillosis by mechanisms that are poorly defined. Using a post-influenza aspergillosis model, we found that superinfected mice had 100% mortality when challenged with A. fumigatus conidia on days 2 and 5 (early stages) of influenza A virus infection but 100% survival when challenged on days 8 and 14 (late stages). Influenza-infected mice superinfected with A. fumigatus had increased levels of the pro-inflammatory cytokines and chemokines IL-6, TNFα, IFNß, IL-12p70, IL-1α, IL-1ß, CXCL1, G-CSF, MIP-1α, MIP-1ß, RANTES and MCP-1. Surprisingly, on histopathological analysis, superinfected mice did not have greater lung inflammation compared with mice infected with influenza alone. Mice infected with influenza had dampened neutrophil recruitment to the lungs following subsequent challenge with A. fumigatus , but only if the fungal challenge was executed during the early stages of influenza infection. However, influenza infection did not have a major effect on neutrophil phagocytosis and killing of A. fumigatus conidia. Moreover, minimal germination of conidia was seen on histopathology even in the superinfected mice. Taken together, our data suggest that the high mortality rate seen in mice during the early stages of influenza-associated pulmonary aspergillosis is multifactorial, with a greater contribution from dysregulated inflammation than microbial growth. Importance: Severe influenza is a risk factor for fatal invasive pulmonary aspergillosis; however, the mechanistic basis for the lethality is unclear. Utilizing an influenza-associated pulmonary aspergillosis (IAPA) model, we found that mice infected with influenza A virus followed by A. fumigatus had 100% mortality when superinfected during the early stages of influenza but survived at later stages. While superinfected mice had dysregulated pulmonary inflammatory responses compared to controls, they had neither increased inflammation nor extensive fungal growth. Although influenza-infected mice had dampened neutrophil recruitment to the lungs following subsequent challenge with A. fumigatus , influenza did not affect the ability of neutrophils to clear the fungi. Our data suggest that the lethality seen in our model IAPA is multifactorial with dysregulated inflammation being a greater contributor than uncontrollable microbial growth. If confirmed in humans, our findings provide a rationale for clinical studies of adjuvant anti-inflammatory agents in the treatment of IAPA.

Dysregulated pulmonary inflammatory responses exacerbate the outcome of secondary aspergillosis following influenza.

IMPORTANCE: Severe influenza is a risk factor for fatal invasive pulmonary aspergillosis; however, the mechanistic basis for the lethality is unclear. Utilizing an influenza-associated pulmonary aspergillosis (IAPA) model, we found that mice infected with influenza A virus followed by Aspergillus fumigatus had 100% mortality when superinfected during the early stages of influenza but survived at later stages. While superinfected mice had dysregulated pulmonary inflammatory responses compared to controls, they had neither increased inflammation nor extensive fungal growth. Although influenza-infected mice had dampened neutrophil recruitment to the lungs following subsequent challenge with A. fumigatus, influenza did not affect the ability of neutrophils to clear the fungi. Our data suggest that the lethality seen in our model of IAPA is multifactorial with dysregulated inflammation being a greater contributor than uncontrollable microbial growth. If confirmed in humans, our findings provide a rationale for clinical studies of adjuvant anti-inflammatory agents in the treatment of IAPA.