Pharmaceutical compositions and methods for treating fungal infections

Abstract

This invention describes pharmaceutical compositions and their methods of use utilizing inactivated F-box protein 1 deletion (fbp1Δ) mutant fungal cells and adjuvant ODN 2395. The pharmaceutical compositions can be administered by intranasal (IN) inhalation, subcutaneous (SC) injection, or intramuscular (IM) injection. Vaccination with a pharmaceutical composition confers host immunity to fungal infection in different host backgrounds and in different aged hosts.

Description

PHARMACEUTICAL COMPOSITIONS AND METHODS FOR TREATING FUNGAL INFECTIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 513,029, filed July 11, 2023, which is incorporated herein by reference in its entirty.STATEMENT REGARDING FEDERAL FUNDING

[0002] This invention was made with government support under grant number U01 HL 150852 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (SeqList-096747-00490.xml; Size: 46,977 bytes; and Date of Creation: July 9, 2024) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0004] This invention relates to pharmaceutical compositions and their methods of use. The pharmaceutical compositions utilize inactivated fbplA deletion mutant cells combined with an adjuvant and are effective to provide immunity in a host against infection by, e.g. a fungus.BACKGROUND

[0005] Invasive fungal infections represent a significant public health concern, particularly in developing countries and those in tropical regions. Invasive fungal infections kill over 1.5 million people worldwide annually, and the number is expected to grow. Certain groups of individuals remain particularly susceptible to invasive fungal infection, including immunocompromised individuals, such as individuals infected with HIV or undergoing certain types of chemotherapy, as well as children and the elderly. One fungal species of particular interest is Cryptococcus neoformans, a basidiomycetous yeast pathogen, as it infects the centralnervous system (CNS) and is the leading cause of fungal meningitis in immunocompromised populations worldwide, with roughly 1 million cases that account for approximately 600,000 deaths annually. Thus, C. neoformans represents a significant percentage of the total annual deaths caused by invasive fungal infections. Treatment for infection by C. neoformans is often accomplished with anti-fungal medication, such as fluconazole, but treatment is not always effective, especially in the immunocompromised people. Existing small moleucle-based antifungal drugs are expensive and toxic, leading to side effects. Therefore, there is a unmet need to develop better treatment stratgies for fungal infections.

[0006] To date, only a few C. neoformans mutants have been found to be able to elicit protection against challenges with the highly virulent H99 strain of C. neoformans. The first strain developed in the H99 background was done by enforcing expression of murine IFN-y in the yeast cells (yH99). Vaccination studies with yH99 have provided critical proof-of-principle evidence for the importance of type 1 immunity in protection as well as for the capacity of the host immune response to overcome virulence mechanisms of H99 given the proper inflammatory conditioning. Studies with simplified vaccines based on beta-glucan particles loaded with cryptococcal antigenic extracts have also been found to induce robust Thl and Th 17 responses that correlate with vaccine-mediated protection. However, no commercially available safe and reliable vaccine currently exists for C. neoformans. Because of this, there is an urgent need for such.SUMMARY OF THE INVENTION

[0007] The present invention relates to fungal immunogenic compositions such as pharmaceutical compositions (e.g., vaccine compositions) and methods of use thereof. The experiments descrbed in the Examples show that vaccination by a combination of inactivated F- box protein 1 deletionmutant heat-killed Cryptococcus neoformans cells and adjuvant ODN 2395 (the combination referred to herein interchangeably as “HK-fbpl+ODN”, “HK- fbpl / ODN 2395”, “ODN 2395 adjuvanted HK-fbpl vaccine” and “the HK-fbpl+ODN2395 adjuvant combination”) surprisingly confers host immunity to fungal infection in different host backgrounds and in different aged hosts, for example but not necessarily limited to, infection by C. neoformans. Also as shown in the Examples, mice vaccinated by the pharmaceutical compositions described herein (containing inactivated f mutant Cryptococcus neoformanscells and adjuvant ODN 2395) demonstrated pro-inflammatory responses against virulent H99 challenge.

[0008] In embodiments, the present invention is directed to an immunogenic composition such as a pharmaceutical composition (a vaccine composition). In some embodiments, the present invention is directed to a method of treating a fungal infection in a subject (e.g., human) in need thereof. In all embodiments, the pharmaceutical composition contains a combination of inactivated foplA deletion mutant fungal cells (also referred to herein as “inactivated fop Ik deletion mutant”) and adjuvant ODN 2395 or a variant thereof.

[0009] In embodiments, the present invention is directed to a pharmaceutical composition including: i) inactivated F-box protein 1 deletion (foplA) mutant Cryptococcus neoformans cells, wherein the inactivated foplA mutant cells are heat-killed, and ii) adjuvant ODN 2395. In embodiments, adjuvant ODN 2395 comprises the sequence of SEQ ID NO: 50. In embodiments, adjuvant ODN 2395 consists essentially of the sequence of SEQ ID NO: 50. In embodiments, adjuvant ODN 2395 consists of the sequence of SEQ ID NO: 50. Variants of ODN 2395 are also encompassed by the pharmaceutical compositions described herein. The pharmaceutical composition can further include at least one pharmaceutically acceptable carrier or excipient. In embodiments, the pharmaceutical composition contains a heat-killed foplA deletion mutant C. neoformans cell. In embodiments, the foplA is a partial deletion, e.g., of an F-box domain. The partial deletion can occur, for example, by homologous recombination. In other embodiments, the foplA is a full deletion. The full deletion can occur by homologous recombination.

[0010] In embodiments, the present invention is directed to a method of stimulating a host immune system or an immune response in a host. In some embodiments, the host is an immunocompromised host. In some embodiments, the immunocompromised host is infected with human immunodeficiency virus (HIV). The methods described herein are directed to administration of a pharmaceutical composition (a vaccine composition) containing inactivated foplA deletion mutant fungal cell and adjuvant ODN 2395. In embodiments, adjuvant ODN 2395 comprises the sequence of SEQ ID NO: 50. In embodiments, adjuvant ODN 2395 consists essentially of the sequence of SEQ ID NO: 50. In embodiments, adjuvant ODN 2395 consists of the sequence of SEQ ID NO: 50. Variants of ODN 2395 are also encompassed by the methods described herein. In embodiments, the inactivated foplA deletion mutant fungal cell is a C.neoformans cell. In embodiments, the method is directed to administration of a pharmaceutical composition as described herein (e.g., a vaccine) containing a heat-killeddeletion mutant C. neoformans cell and adjuvant ODN 2395. In some embodiments, the fbplA deletion mutant is a partiaA deletion mutant. In some embodiments, thedeletion mutant is generated by homologous recombination. In some embodiments, the partiaA deletion mutant is generated by homologous recombination. In some embodiments, the partialdeletion mutant has an F-box domain deleted. In some embodiments, the pharmaceutical composition is administered with at least one pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition is administered more than once (e.g., 2, 3, 4, etc. administrations). In the methods, the pharmaceutical composition can be administered at one or more timepoints (e.g., one, two, three, etc. timepoints).

[0011] In embodiments, the present invention is directed to a method of treating or preventing a fungal infection in a subject in need thereof. In some embodiments, the subject is an immunocompromised subject. In some embodiments, the immunocompromised subject is infected with HIV. In embodiments, the method generally comprises administering one or more doses (e.g., 1, 2, 3, 4, etc. doses) of a pharmaceutical composition (e.g., a vaccine formulation) comprising an inactivatedy&pJJ deletion mutant and adjuvant ODN 2395 according to any aspect of the present disclosure to the subject in need thereof. Typically, a method of treating a fungal infection in a subject (e.g., human) in need thereof includes administering to the subject a therapeutically effective amount of a pharmaceutical composition including: i) inactivated F-box protein 1 deletion (fbplA) mutant Cryptococcus neoformans cells, wherein the inactivated fbplA mutant cells are heat-killed, and ii) adjuvant ODN 2395. In embodiments, adjuvant ODN 2395 comprises the sequence of SEQ ID NO: 50. In embodiments, adjuvant ODN 2395 consists essentially of the sequence of SEQ ID NO: 50. In embodiments, adjuvant ODN 2395 consists of the sequence of SEQ ID NO: 50. The pharmaceutical composition can further include a pharmaceutically acceptable carrier or excipient. In the methods, the pharmaceutical composition can be administered by intranasal (IN) inhalation, subcutaneous (SC) injection, or intramuscular (IM) injection. The inactivatedA deletion mutant is an inactivateddeletion mutant according to any aspect of the present disclosure (e.g., the can be a fulldeletion or a partial deletion, the partial deletion can be of an F-box domain and can occur by homologous recombination, a full deletion can occur by homologous recombination). In themethod, the pharmaceutical composition can be administered once or more than once (e.g., two, three, four, etc. administrations). In other words, the pharmaceutical composition can be administered at one or more timepoints (e.g., one, two, three, etc. timepoints). In some embodiments, the fungal infection comprises an infection with virulent C. neoformans. In some embodiments, the virulent C. neoformans strain comprises strain H99. In some embodiments, the fungal infection comprises an infection with C. albicans, A. fumigatus, or C. gattii. In some embodiments, the method includes administering at least one (e.g., 1, 2, 3) antifungal agent to the subject in need thereof. In some embodiments, the at least one antifungal agent comprises one of azole antifungals, echinocandins, polyenes, or other antifungal agents.

[0012] In embodiments, the present invention is directed to the use of an inactivated fbplA deletion mutant and adjuvant ODN 2395 for the manufacture of a medicament for the treatment or prevention of a fungal infection. The inactivated fbplA deletion mutant is an inactivated JbplA deletion mutant according to any aspect of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 represents a method for generating / ^ / ; / deletion (knockout) mutants.

[0014] Figures 2A, 2B, 2C and 2D represents that the deletion of Fbpl does not affect the expression of known virulence factors in C. neoformans. Figure 2A represents GXM secretion was measured after H99 and strains were grown on capsule inducible minimummedium for three days. A typical yeast cell and the capsule of each strain was shown at the top following Indian ink staining. Figure 2B represents ID proton nuclear magnetic resonance (NMR) spectroscopy detection of the GXM structure produced by H99 and fbplA cells. The spectrum peaks indicate the M2 mannosyl triad structure reporter groups. Figure 2C represents cell cultures of H99 and fbplA were incubated with FITC labeled Concanavalin A (ConA-FITC) and the fluorescence quantified using flow cytometry. The bar graph represents the average signal intensity. Figure 2D represents chitin and chitosan production was compared between H99 and fbplA cells grown on YPD overnight.

[0015] Figures 3 A, 3B, 3C, 3D, 3E, 3F, 3G and 3H represent mice infected with fbplA display enhanced inflammatory responses and survival. C57B1 / 6J mice were infected intranasally (IN) with 105H99 or 106fbplA. Figure 3A represents colony forming units (CFU) of total fungal burden in the lung. Data shown is mean ± SEM for 5-12 mice per group per timepoint cumulative of two independent experiments. Figure 3B represents survival rates after infection in C57B1 / 6J. Data shown for each graph is for 8 mice per group and cumulative of two independent experiments. Figure 3C and Figure 3D represent fungal burden as examined by CFU recovered in spleen (Figure 3C) and brain (Figure 3D). Data shown is mean ± SEM for 5- 12 mice per group per time point cumulative of two independent experiments. Figures 3E through 3H represent cellular infiltration to the lung was analyzed by flow cytometry. Each symbol represents one mouse, data is cumulative if two independent experiments. Each cell population was identified as CD45+DAPf live leukocytes, neutrophils were gated as CD1 lb+Ly6C+Ly6G+(Figure 3E), monocytes were gated as CD1 lb+Ly6C+Ly6G' (Figure 3F), CD4+T-cells were gated as Thyl .2+CDl lb’CD4+CD8’ (Figure 3G) and CD8+T-cells were gated as Thyl .2+CD1 lb'CD4 CD8+(Figure 3H). Statistical analysis, Mann- Whitney test. * p<0.05,** p<0.01 . Statistical analysis of survival curve was done by Log Rank (Mantel-Cox) test, *** p<0.001.

[0016] Figures 4A, 4B, 4C, 4D, 4E, 4F and 4G represent additional data regarding survival patterns and CD4+T-cell responses. Figure 4A represents survival pattern in A / J mice infected with \Q6fi>plA. Figure 4B represents that vaccinated mice that survivedwithout symptoms for more than 60 days contained a significant number of CD4+T-cells that remained in the airways. Figures 4C and 4D represent airway CD4+T-cells recovered from vaccinated mice rapidly produced IFN-y (Figure 4D) and IL-17A (Figure 4C) upon restimulation. Figures 4E through 4G represent Cryptococcus-specific CD4+T-cell responses were sustained in the MLN of vaccinated mice, for IL- 12 (Figure 4E), IFN-y (Figure 4F) and IL-17A (Figure 4G).

[0017] Figures 5A, 5B, SC, 5D, 5E, 5F, 5G, 5H, 51 and 5K represent enhanced Thl and Th 17 CD4+T-cells responses in mice infected with. Figures 5A through 5F represent cryptococcus-specific CD4+T-cell responses were examined in lung-draining lymph node (MLN) at days 3, 7 and after infection with H99 or CD4+T-cells were purified from theMLN of infected mice and stimulated with antigen-presenting cells in the presence or absence of C. neoformans (Cn) sonicated antigens. Data shown is for cytokines secreted to the supernatant after 72 hours of in vitro re-stimulation. No cytokines were detected in samples cultured without Cn antigen. Each symbol represents one mouse. Data shown is cumulative of three independent experiments with 4-5 mice per group. IL-2 (Figure 5A), IFN-g (Figure 5B), IL-17A (Figure5C), IL-4 (Figure 5D), IL-5 (Figure 5E) and IL- 13 (Figure 5F) cytokines were measured by ELISA. Figure 5G represents a FACS plots of CD4+T-cells (top panel) and CD8+T-cells (bottom panel) in the bronchoalveolar lavage fluid (BALF). Figures 5H through 5K represent cytokine expression analyzed by intracellular cytokine staining (ICCS). Each symbol represents one mouse. Data is cumulative of 4 independent experiments with 5 mice per group. H-J) Plots shown are for cytokine production in CD4+ T-cells gated as Thy 1 .2+CD4+CD8’ T-cells.Frequency of IFN-y (Figure 5H) and IL-17A-(Figure 51) and TNF (Figure 5J)-producing CD4+T-cells in BALF were analyzed as shown in Figure 5G. Figure 5K represents frequency of IFN-g-producing Thyl .2+CD4’CD8+T-cells. **** p<0.0001, *** p<0.001,** p<0.01, * p<0.05, as determined by Mann-Whitney test.

[0018] Figure 6A, 6B, 6C, 6D, 6E and 6E represent that lymphocytes are required for long-term protection against infection with JbplA. Lymphocyte deficient mice (RAG’7’ dashed lines) and control mice (solid lines) were infected (i.n.) with 105H99 or 106JbplA . Survival data shown is cumulative of two independent experiments with 5-7 mice per group. Figure 6A represents survival rates of RAG’ and wild type control mice after infection with JbplA. Figure 6B represents survival rates of RAG’ and wild type control mice after infection with H99. Figure 6C through 6E represent colony forming units in lung (Figure 6C), brain (Figure 6D) and spleen (Figure 6E) of RAG’ ’ and WT mice infected with JbplA. Each symbol represents one mouse. Data shown is cumulative of two independent experiments. **** p<0.0001, ***as determined by by Log Rank (Mantel-Cox) test (Figure 6A, 6B) or Mann- Whitney test (Figures 6C through 6E).

[0019] Figure 7A, 7B, 7C, 7D, 7E and 7F represent effective maturation of CCR2+Ly6C+monocytes into mo-DCs after Fbpl infection. Mo-DC differentiation was analyzed at day 3 after i.n. infection with H99 or JbplA. Data shown is cumulative of two independent experiments with 4-5 mice per group and is depicted as mean ± SEM. Figure 7A represents a FACS profile of Ly6C+monocyte maturation into mo-DCs (defined as CD45+CD1 lb+Ly6C+Ly6G’CDl lc+ClassII+) in H99 or JbplA infected mice. Figure 7B represents percent of mo-DCs (CD1 lc+ClassII+) in monocyte gate (CD45+CD1 lb+Ly6ChlLy6G ) in mice infected with H99 or JbplA. Figure 7C represents total number of mo-DC recruited to the lung. Figure 7D through 7F represent chemokine expression in lung tissue as analyzed by qRT-PCR; CCL2 (Figure 7D), CCL7 (Figure 7E), and CCL12 (Figure 7F). The expression ofeach CCR2 ligand was examined using taq-man probes. Differential gene expression was calculated relative to GAPDH using the AACT method. ** p<0.01 as determined by Mann- Whitney test.

[0020] Figure 8A, 8B, 8C, 8D, 8E and 8F represent that depletion of CCR2+inflammatory monocytes impairs CD4+T activation and leads to mortality of fbpl A infected mice. CCR2-depleter mice and control (DTR negative) littermates were treated with diphtheria toxin (DT) before and after infection withA as illustrated in Figure 8A. Figure 8B represents survival curve off p - infected CCR2-depleted (dashed) and control littermates (solid). Data shown is cumulative of three independent experiments with 4-5 mice per group. **** p<0.0001 as determined by Log Rank (Mantel-Cox) test. Figure 8C represents total number of CD4+ T-cells recovered from the BALF of-infected CCR2-depleted and control littermates at day 6 after infection. Each symbol represents one mouse. Data shown is cumulative of two experiments with 4-5 mice per group *** p<0.001 as determined by Mann- Whitney test. Figure 8D represents a FACS profile of cytokine production by CD4+T-cells recovered from the BALF o-infected CCR2 -depleted or control littermates. Plots are gated on Thyl ,2+CD4+CD8‘ lymphocytes in BALF. Figure 8E represents colony forming units in lung tissue of -infected CCR2-depleted or control littermates at day 6 after infection.Data is cumulative of two independent experiments with 3-4 mice per group. *** p<0.001 as determined by Mann-Whitney test. Figure 8F represents that cytokine gene transcription in lung tissue was examined at day 6 after infection with fbpl A in CCR2 -depleted and control littermates (striped). Control B6 mice infected with H99 were also analyzed as a control population. Differential gene expression relative to GAPDH was examined by qRT-PCR using cytokinespecific Taq-Man probes and calculated using the AACt method. Data shown is cumulative of two independent experiments with 4 mice per group and is depicted as mean ± SEM.** p<0.01 as determined by Mann-Whitney test.

[0021] Figure 9 represents that vaccination with inactivated (heat-killed) fbpl A mutants confers protection from infection with virulent H99 yeast. 25 A / Jcr mice were used; 5 were naive control, 10 infected with livedeletion mutant (106per mouse), and 10 infected with heat-killed deletion mutant (0.5x108per mouse). For the mice infected with heat-killed deletion mutant, a booster inoculation was given after 24 days. After 30 days, mice fromeach group (naive, deletion mutant, and heat-killed fbplA deletion mutant) wereinoculated with H99 strain of C. neoformans (106per mouse). Naive mice succumbed to infection around 25 days post-infection with H99, as well as mice inoculated with live fbplA deletion mutant. Mice inoculated with heat-killed fbplA deletion mutant showed full immunity (100% effective) to virulent H99, as none of the mice succumbed to infection, even more than 60 days post infection by H99. Only the inactivated fbplA deletion mutant was effective at providing immunity.

[0022] Figure 10 represents histopathology results (H&E staining / GMS silver staining) of the vaccination experiments described in Figure 9. The results indicated minimal fungal burden and inflammation in those mice vaccinated with inactivated fbplA deletion mutant.

[0023] Figure 11 represents cytokine production by CD4+ T-cells recovered by the surviving mice from the vaccination experiments described in Figure 9, for interleukin- 17 (IL- 17) and IFNy production.

[0024] Figure 12 represents vaccination experiments with the conditions set forth in Figure 9 but with heat-killed H99 wild type strains instead of live fbplA deletion mutants. The results showed that heat-killed H99 strains did not provide any protection against subsequent infection by live H99, whereas heat-killed fbplA deletion mutants did. The results show that an effective vaccine composition must include inactivated fbplA deletion mutants, either full or partial (e.g. F-box knockouts).

[0025] Figures 13A, 13B, 13C, 13D and 13E represent CCR2+ monocyte / mo-DC recruitment to lungs as well as CD4+ T-cell responses measured. Figures 13A and 13B represent CCR2+ monocyte and mo-DC recruitment to lung Day 3 after challenge. Figure 13A represents total number of CCR+Ly6C+ monocytes in lung. Figure 13B represents total number of CCR2+Ly6C+ CD11 c+MHCII+mo-DC in lung. Figures 13C through 13E represent CD4+ T-cell responses in MLN at day 7 after challenge, for IL- 12 (Figure 13C), IFN-y (Figure 13D), and IL-17A (Figure 13E).

[0026] Figures 14A, 14B, 14C, 14D, 14E, 14F, 14G, 14H and 41 represent additional vaccination experiments, further illustrating that vaccination with inactivated fbplA confers protection against H99 infection. Mice were vaccinated with heat-killed fbplA yeast i.n. on days -32 and -7. At day 0 vaccinated and unvaccinated controls were infected with 104virulent H99. Figure 14A represents percent survival after H99 infection in naive mice or mice vaccinatedwith Data shown is cumulative of three independent experiments with a total of 18mice per group on the C57B1 / 6J background. Figure 14B represents percent survival after H99 infection in naive mice or mice vaccinated withData shown is cumulative of three independent experiments with a total of 15 unvaccinated and 21 vaccinated mice on theA / Jcr background. Figure 14C represents percent survival after H99 infection in naive mice or mice vaccinated with HK-H99 a total of 10 mice per group were analyzed in two independent experiments. Figure 14D represents percent survival after H99 infection in naive mice or RAG- / - that were unvaccinated and RAG- / - that were vaccinated withy p . Data shown is for 5- 10 mice per group and is cumulative of two independent experiments. Figure 14E represents total number of CD4+T-cells recovered in the BALF of naive mice or HK-ybpU-vaccinated B6 mice that survived for 72 days after H99 challenge. Each symbol represents one mouse. Figure 14F represents a FACS plot of intracellular cytokine production by CD4+T-cell recovered from BALF of vaccinated B6 mice 72 days after H99 challenge. Percent IFN-y and IL-17A-producing CD4+T-cells in vaccinated mice as analyzed in FACS shown. Each symbol represents one mouse. Data shown is cumulative of 3 independent experiments. Figures 14G through 141 represent that CD4+T-cells were isolated from the lung-draining lymph node of JbplA- vaccinated that survived an H99 challenge for 72 days. Cytokine secretion (IL-1, FIG. 14G;IFN-y, FIG. 14H; IL-17A, FIG. 141) in the presence or absence of Cn antigens was examined by ELISA as described in materials and methods. Data shown is cumulative of three independent experiments with 4-5 mice per group and is depicted as mean ± SEM. **** p<0.0001, *** p<0.001, ** p<0.01 as determined by Log Rank (Mantel-Cox) test (Figure 14A, 14B) or Mann- Whitney test (Figure 14E, 14G through 141).

[0027] Figure 15 represents the results of cross-protection tests using inactivated JbplA deletion mutants. A / Jcr mice were vaccinated with heat-killed JbplA deletion mutant cells. The results indicated that, in addition to protection against H99 strain, the inactivated JbplA deletion mutants can protect mice from rechallenge of highly virulent C. gattii strain R265.

[0028] Figure 16A, 16B, 16C, 16D show an animal survival curve and fungal CFU following HK-fbpl treatment.

[0029] Figure 17 is a series of graphs showing T cell differentiation in survived animal at the endpoint of the experiment (day 70 post infection).

[0030] Figure 18 is a scheme of vaccination strategy for both single HK-fbpl vaccine and the HK-fbpl +ODN2395 adjuvant combination immunization.

[0031] Figure 19A, 19B and 19C are a series of graphs showing that mice vaccinated with a combination of low dose HK-fbpl and adjuvant ODN 2395 are protected from H99 challenge in an inhalation model. Statistical analysis was done by Log-rank (Mantel-Cox) Test. *P<0.05, **P<0.01, ***P<0.0001. IN HK-fbpl A 5x107: N=8, survival rate =100%; IN HK- fbpl A Ixl07+Odn: N=7 (1 sacrificed on Day 50 due to conjunctivitis), survival rate = 71.4%; IN HK-fbplA 1x107: N=8; IN Odn only: N=8; Unvaccinated: N=8

[0032] Figure 20A, 20B and 20C are a series of graphs showing that the combination vaccination as described herein also improves vaccine efficacy in a subcutaneous (SC) model. Fig. 20A: The animal survival rate following two doses vaccination via SC route and challneg with virulent H99 strain. Fig. 20B: The overtime change of animal body weights of tested anmals. Fig. 20C: The fungal burden at the edn point of experiment of animals vaccinated (SC) vs. non vaccinated. SC HK-fbplA 5x107: N=8; SC HK-fbpl A 5xl07+Odn: N=8, survival rate = 50% (Day65), 37.5% (Day75); SC Odn only: N=7 (1 sacrificed after 2nd vaccination due to bacterial infection); Unvaccinated: N=8

[0033] Figure 21A, 21B and 21C are a series of graphs showing that the combination vaccination also improves vaccine efficacy in an intramuscular (IM) injection model. Fig. 21A: The animal survival rate following two doses vaccination via IM route and challneg with virulent H99 strain. Fig. 21B: The overtime change of animal body weights of tested anmals. Fig. 21C: The fungal burden at the edn point of experiment of animals vaccinated (IM) vs. non vaccinated. IM HK-fbplA 5x107: N=8; IM HK-fbplA 5xl07+Odn: N=8, current survival rate = 62.5%; IM Odn only: N=8; Unvaccinated: N=8

[0034] Figure 22A and 22B are a pair of graphs showing that a combination vaccine strategy with another adjuvant did not show improved protection in either subcutaneous injection (SC) or intramuscular injection (IM) vaccination models.

[0035] Figure 23A, 23B and 23C are a vaccine study model and a pair of graphs showing that the HK-fbpl vaccine is highly stable. The data demonstrates that both fresh prepared HK-fbpl vaccine (vaccine-new) and the vaccine stored in freezer for one year (vaccine- old) had the same full protection in mice against challenge infection.

[0036] Figure 24A, 24B and 24C are a series of graphs showing that HK-fbpl+ODN vaccine induces increased T cell differentiation in both inhalation (IN) model and subcutaneous (SC) immunization model. The HK-fbpl vaccine using IN model in high inoculum (IN HK-fbpl 5x107CFU) has similar CD4 (Fig. 24A) and CD8 (Fig. 24B) T cell differentiation as the low dosage (lx 107CFU) HK-fbpl in combination with ODN 2395 (IN HK-fbpl lxl07+odn), or SC model in combination (SC HK-fbpl 5xl07+odn). In contrast, the Eosinophis production is reduced in the same setting (Fig. 24C). These data indicate an induced Thl immune response.DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention is based at least in part on unexpected discoveries that: vaccination by inactivated fbplA deletion mutant (including partial deletion mutant) fungal cells surprisingly conferred host immunity to fungal infection, for example but not necessarily limited to, infection by C. neoformans', and using a novel combination of inactivated fbplA deletion mutant fungal cells (heat-killed fbp 1 ) and adjuvant ODN 2395 was effective in different hosts and different-aged hosts in all three vaccination methods: IN inhalation, SC injection and IM injection. Examples 1-7 show that IN vaccination with inactivated (heat-killed) fbplA mutants is highly protective against invasive fungal infections in animal models (e.g., Example 4 illustrates protection against infection with virulent C. neoformans (strain H99) and Example 5 illustrates protection against other pathogenic fungal species, e.g.. C. gattii). In the experiments described in Examples 8-11, a new vaccine strategy that combines inactivated fbplA deletion mutant fungal cells and adjuvant ODN 2395 was tested. Unexpectedly, it was found that using this novel combination of inactivated fbplA deletion mutant fungal cells and ODN 2393 as a combination vaccine strategy works in all three vaccination methods: IN inhalation, SC injection and IM injection models. The results described in Examples 8-11 show that: mice vaccinated with the combination vaccine of heat-killed fbplA deletion mutant fungal cells and adjuvant ODN 2395 (HK-fbpl / ODN 2395) showed strong protection against virulent Cryptococcus neoformans challenge; the combination vaccine of HK-fbpl / ODN 2395 provided protection in different host backgrounds and in hosts of different ages; and that in mice protected with the combination vaccine of HK-fbpl / ODN 2395, pro-inflammatory responses against virulent H99 challenge were observed. The data presented as Figure 19 and described in Example 8 demonstrate that the combination of inactivated fbplA deletion mutant fungal cells and adjuvant ODN 2395 is superior to inactivated fbplA deletion mutant fungal cells alone. For example, apharmaceutical composition including inactivated fop Id deletion mutant fungal cells (heat-killed fbpl) and adjuvant ODN 2395 as described herein is effective even with 5x smaller doses compared to inactivated fop Id deletion mutant fungal cells alone. These new pharmaceutical compositions and methods described herein include a vaccine strain combined with an adjuvant (ODN 2395) to boost the host immune system to control and kill the pathogens in infected patients. In contrast to all existing antifungal drugs which are small molecule compounds that target the pathogens rather than modulating host immunity, the pharmaceutical compositions and methods described herein help control infection by modulating host immunity.

[0038] Adjuvant ODN 2395 is a commercially available Class C CpG oligonucleotide (ODN) with a preference for the human and mouse Toll-like receptor 9 (TLR9). ODN 2395 is a synthetic oligodeoxynucleotide containing unmethylated CpG motifs (CpG ODNs), is also referred to as CpG ODN 2395, and is commercially available (InvivoGen, CAT# vac-2395-1). The sequence of ODN 2395 is 5’-tcgtcgttttcggcgcgcgccg-3 (SEQ ID NO: 50). In this sequence, the bases are phosphorothioate (nuclease resistant), and the palindrome is underlined. Variants of ODN 2395 are also encompassed by the pharmaceutical compositions and methods described herein. An ODN 2395 variant is any oligonucleotide sequence having a degree of identity that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or greater sequence identity with SEQ ID NO: 50 and that is functionally similar or equivalent to SEQ ID NO: 50, that is, retains the ability to act as an adjuvant in the pharmaceutical compositions described herein for conferring host immunity to a fungal infection.

[0039] Fbpl is a Grrl-like F-box protein (F-box protein 1) found in C. neoformans as well as a number of other fungal organisms, including pathogens. Fbp1 is important for fungal sexual reproduction (fungal sporulation), and is essential for virulence in C. neoformans.Specifically, Fbpl is required for fungal proliferation in macrophages, after phagocytosis. The present disclosure identifies Fbpl as a control point for how C. neoformans, e.g. H99 strain of C. neoformans, interacts with the host immune system. Cryptococcus Fbpl shares sequence and structural similarity with Grrl in S. cerevisiae and C. albicans. Both Grrl proteins in the two yeasts are important for cell morphology, and grrl mutants produce pseudohyphae. This isconsistent with the observation that Grrl in other plant-pathogenic fungi play a role in growth and fungal infectivity. F-box proteins are characterized by an F-box domain, first identified in human cyclin F. Fbpl contains an F-box domain and 12 leucine-rich repeats (LRR). F-box proteins are exchangeable subunits that facilitate the capacity of SCF to specifically target a number of structurally and functionally diverse substrates.

[0040] The ubiquitin proteasome system is crucial for the controlled turnover of proteins in eukaryotic cells and thus plays an important role in the regulation of diverse cellular activities. As part of the SCFFbplE3 ligase complex,contributes to the specificity of UPS degradation process by targeting specific proteins for ubiquitination. Similar to other F-Box proteins, it is possible that C. neoformans Fbpl targets multiple proteins for degradation. Therefore the observed phenotype ofA could be due to the aggregate function of altered turn over of multiple proteins. Alternatively, the effect of fbpl A on the host immune response could be due to the regulation of just one factor. A thorough analysis of Fbpl targets so far failed to identify Znf2 or chitin deacetylases (CDA) as targets of Fbpl regulation (data not shown). Thus, Fbpl may act independently of Znf2 and CDA genes to regulate the immunogenicity of H99. Given that Znf2 and Fbpl both have multiple, yet to be defined targets, it could be possible that there is a convergence of these two immunogenic strains on a common downstream target. Fbpl physically interacts with Skpl of the SCFFbplE3 ligase complexes to take part in protein ubiquitination and degradation. The E3 ubiquitin ligases are key enzymes in the UPS-mediated ubiquitination and degradation process. They function as the substrate recognition modules of the system and determine the specificity of protein degradation. The SCF E3 ligase complexes bind to substrates that contain specific recognition signals for ubiquitin-protein ligation and degradation, thereby determining the specificity of the regulatory system. Without wishing to be bound by theory, SCF(Fbpl) E3 ligase may regulate fungal pathogenesis through a mechanism that is independent of the classical virulence factors. As shown in Example 2 below, inositol phosphosphingolipid-phospholipase Cl (Iscl) is a substrate of Fbpl, and Iscl is required for fungal virulence, which constitutes a novel regulatory connection between the SCF(Fbpl) E3 ligase complex and inositol sphingolipid biosynthesis in C. neoformans.

[0041] Fbpl is coded for by the FBP1 gene in c. neoformans. Fbpl -knockout (KO) C. neoformans are avirulent, blocking basidospore production, as shown in Example 1 below. However, Fbpl does not regulate cell morphology in C. neoformans, as fbpl A mutants producednormal yeast cells when grown in liquid medium. Surprisingly, Fbpl is essential for fungal virulence yet is not involved in regulation of any of these well-studied virulence factors, an indication that additional virulence-controlling mechanisms are involved, as fbplA knockouts produce normal virulence factors, including melanin, capsule, and the ability to grow at body temperature. Although Fbpl is not involved in the development of virulence factors, it is important for maintaining cell membrane integrity. Many enzymes important for either cell wall construction or cell wall maintenance have been found to be critical for maintaining cell integrity, such as a-l,3-glucan synthase, the chitinase gene family and chitin / polysaccharide deacetylases, which control chitosan biosynthesis, and the KRE family, which is involved in p- 1,6-glucan synthesis. However, most of these genes are important for cell wall integrity. The effect of Fbpl on cell integrity is more specific to cell membranes, as it is sensitive to only SDS, as shown in Example 1 below. Without wishing to be bound by theory, Fbpl may target certain membrane proteins that are important for membrane integrity for degradation. Fbpl KO C. neoformans show defects in intracellular proliferation after phagocytosis, and cannot disseminate to other organs following pulmonary infection, as shown in Example 2 below.

[0042] As shown in Examples 1 and 2 infra, generation o KO mutants can occurvia homologous recombination methods, for example, by replacing the wild type genome with a knock-out cassette, for example (but not necessarily) one containing a gene coding for a marker protein, for example (but not necessarily) a NEO marker as used in Example 1 infra, and is illustrated in FIG. 1. One skilled in the art will recognize that there are numerous knock-out cassettes which may be used, for example those containing NAT markers as also disclosed in Example 1 , and that the thrust is to generate C. neoformans (for example) mutants lacking expression o such that the mutants are suitable for incorporation into a vaccine composition of the present invention as discussed herein. Furthermore, it is possible to create partia knockouts that are functionally similar to full fbpl A knockout mutants. Forexample, knocking out just the F-box domain of Fbpl, as shown in Example 1, may result in a phenotype similar to the fbpl A null mutant (full fbpl knockout). The method utilized for partial deletion knockouts is practically identical to that to create full length fbpl A KO mutants, in that the method utilizes homologous recombination with an expression cassette, only the region of genomic DNA that is replaced with, e.g. a marker (such as the NAT marker utilized in Example 1 infra'), is limited to a portion of the complete gene, for example, but not necessarily, the regioncoding for the F-box domain of Fbpl . One of ordinary skill in the art will appreciate that there are other domains of Fbpl which may be knocked out which still render the partial KO mutant phenotypically same or similar to the full-length KO mutant, and that the invention as directed to partial deletion / knockout mutants is not limited to just those partial deletion mutants that knock out the F-box domain of Fbpl, so long as the partial KO mutants are suitable for incorporation into a vaccine composition of the present invention.

[0043] Example 3 infra examines the underlying mechanisms that contribute to the in vivo hypovirulent phenotype of the .fbpl A deletion mutants. It was found that infection with fbpl A mutants induced a robust inflammatory response and enhanced activation of innate and adaptive immune responses as compared to the parental H99 strain. Long-term protection from fbpl A infection was dependent on the activation of adaptive immune responses since lymphocyte-deficient mice (RAG'' ) were not able to contain infection with mutants andsuccumbed to infection. The activation of protective immunity against fbpl A mutants was also facilitated by increased innate responses. Infection with fbpl A induced a more robust recruitment of Ly6C+monocytes and their maturation into dendritic cells (mo-DCs) as compared with H99. Selective depletion of CCR2+cells in CCR2-DTR mice resulted in the abrogation of protective immunity and rapid mortality o-infected mice. Example 4 infra illustrates that vaccination with inactivated (heat-killed) fbpl A deletion mutants results in the induction of immune responses that protect the host against a lethal challenge with the parental H99 strain. Without wishing to be bound by theory, it is believed that Fbpl acts as a virulence factor that shapes the immunogenicity of C. neoformans. A disruption in this Fbpl -controlled pathway is sufficient to induce robust innate and adaptive immune responses that protect the host from infection and can be harnessed in the vaccination strategies of the present disclosure.

[0044] The enhanced immunogenicity and vaccine potential of fbpl A deletion mutants in the pharmaceutical compositions described herein are believed to be dependent on the increased recruitment of CCR2+Ly6C+monocytes. Without wishing to be bound by theory, it is believed that an intact Fbpl -regulated pathway functions to inhibit the optimal recruitment and maturation of CCR2+Ly6C+monocytes. The importance of this recruitment is demonstrated by the increased susceptibility of CCR2-depleted mice to infection. CCR2+monocytes areprecursors to monocyte-derived macrophages and dendritic cells (mo-DCs) that are important for control of multiple infections. Previous studies have shown that mo-DCs are crucial for thepriming and Thl differentiation of Aspergillus-fumigatus-s^ecific CD4+T-cell responses. Similarly, CCR2+monocytes and their derivative cells are required for the induction of protective immunity to Blastomyces dermatitidis and other clinically important fungal pathogens. Inhibition of CCR2+monocyte recruitment has been shown to be a crucial mechanism of virulence exploited by B. dermatitidis. The present disclosure shows that inhibition of CCR2+monocyte recruitment is similarly an important point of regulation by Fbpl -controlled targets that thus help shape the virulence of C. neoformans. Without wishing to be bound by theory, inhibition of CCR2+monocyte recruitment might thus be a common mechanism of virulence exploited by pathogenic fungi. Accordingly, as also illustrated by Example 4 and FIG. 15, inactivated fbpl A deletion mutants may exhibit cross-protectivity against different pathogenic fungal species.

[0045] In addition to acting as precursors of DCs for the activation of adaptive immunity, CCR2+ monocytes can give rise to direct effectors of innate-mediated control of fungal growth. The more rapid mortality of CCR2-depleted mice as compared with lymphocyte-deficient (RAG'A) in response to fbpl A infection is likely due to additional contributions of CCR2+monocytes as innate antifungal effectors. Protective immune responses to infection with C. neoformans strain 52D have been shown to be critically dependent on CCR2+monocytes and their derivative macrophages and DCs. Without wishing to be bound by theory, it might be possible that differential targeting of CCR2+monocyte recruitment by distinct C. neoformans isolates could underlie their variable virulence whereby more virulent strains like H99 inhibit CCR2 influx while less virulent ones like 52D do not. The inhibition of CCR2+monocyte recruitment and maturation is thus a potential virulence mechanism that can help fungal pathogens to escape both innate and adaptive immune-mediated control of infection. The results of Example 3 suggests that an Fbpl -regulated target / s is involved in regulation of such a mechanism of virulence in C. neoformans.

[0046] As shown in Example 4 infra, inactivated fbpl A deletion mutants are effective to prevent infection against fungal infection, particularly infection by C. neoformans. Surprisingly, live fbpl A deletion mutants were not effective vaccine compositions, whereas inactivated fbpl A deletion mutants were; see also FIG. 9. One of ordinary skill in the art will understand that although the Examples include in vivo studies on mice, that because the similarities of adaptive immunity between rodents and humans, the vaccine compositions of the present invention aresuitable to prevent infection in humans and other mammals. The link between in vivo rodent studies and how such translates to use in humans is further discussed in Caminci P., “Genomics: Mice in the ENCODE spotlight,” Nature 515, 346-347 (20 November 2014), hereby incorporated by reference in its entirety. The fbplA deletion mutant may be either a full fbplA knockout or only a partial knockout as described herein. The fbplA deletion mutant may be inactivated by heat (i.e. heat-killed) or by other means. As described herein, an inactivated vaccine (i.e. containing inactivated fbplA deletion mutant) refers to a vaccine comprising fbplA deletion mutants that have been killed (and adjuvant ODN 2395), as opposed to live / attenuated vaccines. Inactivation preferably comprises heat-killing the fbplA deletion mutants, but is considered to embody any method of killing the fungal cells without breaking them, so the cell surface of the comprising fbplA deletion mutants remains intact. In addition to heat-killing such potential methods include, but are not limited to, irradiation (e.g. with gamma irradiation or similar methods) and chemical sterilization (e.g. with formaldehyde or other chemicals that leave the cell surface intact).

[0047] The present pharmaceutical compositions may be used to immunize and treat a subject (e.g., a human). The pharmaceutical compositions can be used as, or for the preparation of, a vaccine (e.g., a prophylactic and / or therapeutic vaccine). Thus, within the scope of this invention is an immunogenic or vaccine composition that contains a pharmaceutically acceptable carrier and a therapeutically effective amount of inactivated F-box protein 1 deletion (fbplN) mutant Cryptococcus neoformans cells and adjuvant ODN 2395. The pharmaceutically acceptable carriers used in the compositions can be selected on the basis of the mode and route of administration, and standard pharmaceutical practice. A therapeutically effective amount of a pharmaceutical composition is one sufficient to induce an immune response in a subject sufficient to prevent or treat a fungal infection in the subject.[004S] The present disclosure additionally relates to methods of preventing or treating a fungal infection in an individual or subject in need thereof (e.g., a human with a fungal infection). The method generally comprises administering to the subject in need thereof one or more (e.g., 1, 2, 3, 4) doses of a therapeutically effective amount of a pharmaceutical composition comprising inactivated fbplA mutant Cryptococcus neoformans cells and ODN 2395. In a typical embodiment, the fungal infection is infection with a virulent strain of C. neoformans, although the fungal infection is not limited as such as the pharmaceuticalcompositions (e.g., vaccines) of the present invention may be used to prevent infection by other fungal species than by C. neoformans. For example, in some embodiments, the fungal species comprises C. albicans, A. fumigatus or C. gatti. However, the invention is not limited as such. For example, the fungal species may comprise any of the following fungal species. Absidia corymbifera or ramose, Achorion gallinae, Actinomadura spp., Actinomyces spp., Ajellomyces dermatididis, Aleurisma brasiliensis, Allersheria boydii, Arthroderma spp., Aspergillus spp. (including but not limited to A. fumigatus, A.flavus, and A. lent ulus)', Basidiobolus spp, Blastomyces spp, Cadophora spp, Candida spp (including but not limited to C. albicans, C. krusei, C. galbrata, C. rugose, C parapsilosis, C. tropicalis, and C. dubliniensis), Cercospora apii, Chrysosporium spp, Cladosporium spp, Cladothrix asteroids, Coccidioides immitis, Cryptococcus neoformans (including strain H99), Cunninghamella elegans, Dematium wernecke, Discomyces israelii, Emmonsia spp, Emmonsiella capsulate, Endomyces geotrichum, Entomophthora coronate, Epidermophyton floccosum, Filobasidiella neoformans, Fonsecaea spp., Geotrichum candidum, Glenospora khartoumensis, Gymnoascus gypseus, Histoplasma spp, Hormiscium dermatididis, Hormodendrum spp, Keratinomyces spp, Langeronia soudanense, Leptosphaeria senegalensis, Lichtheimia corymbifera, Lobmyces loboi, Loboa loboi, Lobomycosis, Madurella spp., Malassezia furfur, Microsporum spp (including but not limited to “ringworm”), Monilia spp, Mucor spp., Nannizzia spp., Neotestudina rosatii, Nocardia spp., Oidium albicans, Oospora lactis, Paracoccidioides brasiliensis, Petriellidium boydii, Phialophora spp., Piedraia hortae, Pityrosporum furfur, Pneumocystis spp. (including but not limited to P. carinii / P. jirovecii), Pullularia gougerotii, Pyrenochaeta romeroi, Rhinosporidium seeberi, Sabouraudites, Sartorya fumigate, Sepedonium, Sporotrichum spp., Tinea spp. (also including but not limited to “ringworm”), Torula spp, Trichophyton spp (also including but not limited to “ringworm”), Trichosporon spp, Zopfia rosatii, and combinations thereof.

[0049] The pharmaceutical compositions (e.g., vaccine formulations) may be administered (e.g., but not necessarily, co-administered) with one or more antifungal agents. The antifungal agents may include but are not limited to azole antifungals, echinocandins, polyenes, or other antifungal agents. Specific antifungal agents include, but are not limited to, anidulafungin, caspofungin, clotrimazole, econazole nitrate, miconazole, terbinafine, fluconazole, ketoconazole, amphotericin (including amphotericin B), flucytosine, itraconzaole,micafungin, posconazole, isavuconazole, voriconazole, nystatin, girseofulvin, and combinations thereof.

[0050] The pharmaceutical compositions (e.g., vaccine formulations) described herein may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the antigens of the invention into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0051] For injection, pharmaceutical compositions (e.g., vaccine preparations) may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, phosphate buffered saline, or any other physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the pharmaceutical compositions described herein may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0052] The amount of a pharmaceutical composition administered depends, for example, on the particular antigen in the composition, whether an adjuvant is co-administered with the antigen, the type of adjuvant co-administered, the mode and frequency of administration, and the desired effect (e.g., protection or treatment), as can be determined by one skilled in the art. Determination of a therapeutically effective amount of the pharmaceutical composition (e.g., vaccine formulation) for administration is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein. An effective dose can be estimated initially from in vitro and in vivo assays. For example, a dose can be formulated in animal models to achieve an induction of an immune response using techniques that are well known in the art. In the rodent experiments described herein, 5x 10A7 cells of inactivated fbpl / \ mutant Cryptococcus neoformans cells and about 20 pg of adjuvant ODN 2395 were used and found to be therapeutically effective. One having ordinary skill in the art could readily optimize administration to humans based on results described herein. Dosage amount and interval may be adjusted individually. For example, when used as a vaccine in humans, the pharmaceutical compositions of the invention may be administered in about 1 to about 3 doses (e.g., 1, 2, 3, 4 doses) for a 1-36 week (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 week) period. Preferably, 1 or 2 doses are administered, at intervals of about 3 weeks (e.g., about 2.5, 3, 3.5 weeks) to about 4 months (eg., about 3.5, 4.0, 4.5 months), and booster vaccinations may be given periodically thereafter. A suitable dose is an amount of the vaccine formulation that, when administered as described above, is capable of raising or promoting an immune response in an immunized subject (e.g., human) sufficient to treat a fungal infection in a subject in need thereof or protect the subject from an infection, and in some embodimens, for at least 4 to 12 months. In general, for human administration, the amount of the inactivated F-box protein 1 deletion (fbplK) mutant Cryptococcus neoformans cells present in a dose ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg, and preferably from about 100 pg to about 1 pg (e.g., about 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pg). In general, the amount of ODN 2395 adjuvant present in a dose ranges from about 125 pg to about 1000 pg (e.g., about 124, 125, 200, 225, 300, 325, 400, 425, 500, 525, 600, 625, 700, 725, 800, 825, 900, 925, 950, 999, 1000, 10001 pg). In embodiments, the amount of ODN 2395 adjuvant present in a dose ranges from about 500 pg to about 600 pg (about 499, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 599, 600, 601 pg). Suitable dose range will vary with the route of injection and the size of the subject, but will typically range from about 0.1 mL to about 5 mb. Sera can be taken from the subject for testing the immune response or antibody production elicited by the composition against the antigen. Methods of assaying antibodies against a specific antigen are well known in the art. Additional boosters can be given as needed. By varying the amount of the composition and frequency of administration, the protocol can be optimized for eliciting a maximal production of the antibodies.

[0053] A pharmaceutical composition of this invention can be administered parenterally (e.g., intravenous, intramuscular, or subcutaneous injection), orally, and nasally (IN inhalation). A sterile injectable composition can be a solution or suspension in a non-toxic parenterally acceptable diluent, solvent, or carrier. Such solutions include, but are not limited to, 1,3- butanediol, mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, fixed oils can be conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or diglycerides). Fatty acid, such as, but not limited to, oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as, but not limited to, olive oil or castor oil, polyoxyethylated versionsthereof. These oil solutions or suspensions also can contain a long chain alcohol diluent or dispersant such as, but not limited to, carboxymethyl cellulose, or similar dispersing agents. Other commonly used surfactants, such as, but not limited to, Tweens or Spans or other similar emulsifying agents or bioavailability enhancers, which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms also can be used for the purpose of formulation.

[0054] As used herein, the terms “antigenic agent,” “antigen,” or “immunogen” mean a substance that induces a specific immune response in a host animal. As used herein, the term “immunogenic” refers to a capability of producing an immune response in a host animal against an antigen or antigens, e.g. heat-killed fbplA C. neoformans cells. This immune response forms the basis of the protective immunity elicited by a vaccine against a specific infectious organism. An “immune response” refers to a response elicited in an animal (e.g., a human), which may refer to cellular immunity; humoral immunity or both.

[0055] The terms “co-administration,” “co-administered,” and “in combination with” as used herein may refer to the administration of at least two agents or therapies to a subject. In some embodiments, the co-administration of two or more agents / therapies is concurrent. In other embodiments, a first agent / therapy is administered prior to a second agent / therapy. Those of skill in the art understand that the formulations and / or routes of administration of the various agents / therapies used may vary.

[0056] As used herein, the term “pharmaceutical composition” may refer to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo. Pharmaceutical compositions described herein at least include inactivated F-box protein 1 deletion (fbpltx) mutant Cryptococcus neoformans cells, wherein the inactivated fbplts mutant cells are heat-killed, and adjuvant ODN 2395. A “pharmaceutically acceptable carrier,” after administered to or upon a subject, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition must be “acceptable” also in the sense that it is compatible with the active ingredient, e.g. heat-killed fbpl-KO C. neoformans cells, and can be capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as adosage form. Specific examples of physiologically acceptable carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including, but not limited to, ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as, but not limited to, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as, but not limited to, polyvinylpyrrolidone (PVP); amino acids such as, but not limited to, glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including, but not limited to, glucose, mannose, or dextrins; chelating agents such as, but not limited to, EDTA; sugar alcohols such as, but not limited to, mannitol or sorbitol; salt-forming counterions such as, but not limited to, sodium; and / or nonionic surfactants such as, but not limited to, TWEEN.; polyethylene glycol (PEG), and PLURONICS.Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington: The Science and Practice of Pharmacy (Remington the Science and Practiice of Pharmacy) Twenty-First Edition (2005), hereby incorporated by reference in its entirety.

[0057] As used herein, a “patient” or “subject” may refer to a biological system to which a treatment can be administered. A biological system can include, for example, an individual cell, a set of cells (e.g., a cell culture), an organ, a tissue, or a multi-cellular organism. For example, a “patient” or “subject” may refer to human and non-human animals. Examples of a non-human animal include all vertebrates, e.g., mammals, such as non-human mammals, non- human primates (particularly higher primates), dog, rodent (e.g., mouse or rat), guinea pig, cat, and rabbit, and non-mammals, such as birds, amphibians, reptiles, etc. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental, non-human animal or animal suitable as a disease model. The term “animal” includes all vertebrate animals including humans. In particular, the term “vertebrate animal” includes, but not limited to, humans, canines (e.g., dogs), felines (e.g., cats); equines (e.g., horses), bovines (e.g., cattle), porcine (e.g., pigs), as well as in avians. The subject may or may not be an immunocompromised subject, e.g. a subject infected with human immunodeficiency virus (HIV) or suffering from another condition or pathology that results in reduced ability to fight infection.

[0058] As used herein, the terms “treating” or “treatment” of a disease refers to executing a protocol, which may include administering one or more drugs to a patient (human orotherwise), in an effort to alleviate signs or symptoms of the disease (e.g., a fungal infection). Alleviation can occur prior to signs or symptoms of the disease appearing as well as after their appearance. In addition, “treating” or “treatment” does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient. Thus, “treating” or “treatment” additionally includes “preventing” or “prevention” of disease. The terms “prevent” or “preventing” or “prevention” refer to prophylactic and / or preventative measures, wherein the object is to prevent or slow down the targeted pathologic condition or disorder. These terms are not limited solely to a situation in which the patient experiences no aspect of the condition whatsoever. For example, a treatment will be said to have “prevented” the condition if it is given during exposure of a patient to a stimulus that would have been expected to produce a given manifestation of the condition, and results in the patient's experiencing fewer and / or milder symptoms of the condition than otherwise expected. For example, a treatment can “prevent” infection by resulting in the patient's displaying only mild overt symptoms of the infection; it does not imply that there must have been no cellular damage caused by the infecting fungal organism.

[0059] As used herein, an “effective amount” and “therapeutically effective amount” refers to the amount of an active compound / agent that is required to confer a therapeutic effect on a treated subject (e.g., induce an immune response, treat a fungal infection). Effective doses will vary, as recognized by those skilled in the art, depending on the types of conditions treated, route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatment. A therapeutically effective amount to treat or inhibit a fungal infection is an amount that will cause a reduction in one or more of the manifestations of fungal infection, such as amount of fungus present in the host organism and mortality as compared to untreated control subjects.

[0060] As used herein, the term “parenteral” refers to subcutaneous (SC), intracutaneous, intravenous, intramuscular (IM), intraarticular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion technique.

[0061] As used herein, the terms “purified” or “isolated” antibody, peptide, polypeptide, or protein may refer to a peptide, polypeptide, or protein, as used herein, may refer to a peptide, polypeptide, or protein that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated. The polypeptide / protein can constitute at least 10% (i.e., anypercentage between 10% and 100%, e.g., 20%, 30%, 40%, 50%, 60%, 70 %, 80%, 85%, 90%, 95%, and 99%) by dry weight of the purified preparation. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0062] Where a value of ranges is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0063] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entireties.

[0064] As used herein and in the appended claims, the singular forms “a”, “and” and “the” include plural references unless the context clearly dictates otherwise.

[0065] The term “about” refers to a range of values which would not be considered by a person of ordinary skill in the art as substantially different from the baseline values. For example, the term “about” may refer to a value that is within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value, as well as values intervening such stated values. Context in individual circumstances will dictate to what range of values the term “about” refers. Accordingly, the term “about” may or may not refer to different ranges of values throughout this disclosure.

[0066] Publications disclosed herein are provided solely for their disclosure prior to the filing date of the present invention. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further,the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0067] Each of the applications and patents cited in this text, as well as each document or reference, patient or non-patient literature, cited in each of the applications and patents (including during the prosecution of each issued patent; “application cited documents”), and each of the PCT and foreign applications or patents corresponding to and / or claiming priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference in their entirety. More generally, documents or references are cited in this text, either in a Reference List before the claims; or in the text itself; and, each of these documents or references (“herein-cited references”), as well as each document or reference cited in each of the herein-cited references (including any manufacturer's specifications, instructions, etc.), is hereby expressly incorporated herein by reference.

[0068] The following non-limiting examples serve to further illustrate the present invention.EXAMPLES1. Fbpl regulates sexual reproduction and virulence in Cryptococcus neoformans

[0069] This example studies the function of F-box protein Fbp 1 , includingy&p / zf mutants. This example shows that the F-box protein Fbpl is essential for fungal sporulation and virulence in C. neoformans.Materials and Methods

[0070] Strains of C. neoformans and S. cerevisiae were grown at 30°C on yeast extract- peptone-dextrose (YPD) agar medium and synthetic (SD) medium. J774 murine macrophages were grown in 10-cm petri dishes in liquid Dulbecco modified Eagle's (DME) medium with 10% fetal calf serum (FCS) (ATL Biologicals, GA), 10% NCTC-109 (Gibco), and 1% nonessential amino acids (MP Biomedicals, OH). Modified MS medium (Murashige and Skoog medium) was used for mating and sporulation assays.

[0071] To test the expression of Fbpl during mating and how Fbpl is expressed in response to glucose, fbpl expression was measured at mRNA levels throughout the mating process and under conditions with or without glucose via quantitative real-time PCR (qRT-PCR).Cultures of C. neoformans var. grubii wild-type strain H99 and its near congenic strain KN99a were grown overnight on YPD liquid medium with shaking. Cells from an H99 overnight culture were collected and washed with distilled H2O (dH2O) and then resuspended in medium with 2% glucose (YPD) or 2% galactose (YPG) and incubated for 2 h. Cells were then collected and washed with dH2O. The cells collected from YPD were resuspended in YPG, while the cells collected from YPG were resuspended in YPD. Both cultures were incubated for 2 h. Total RNAs were prepared from the cells with each treatment, and cDNA was synthesized as described below.

[0072] Mating was performed by mixing H99 and KN99a, which were then co-cultured on V8 medium (pH 5.0). Mating mixtures were collected from agar surfaces using cell scrapers after 6, 24, 48, 72, or 96 h of incubation. The collected cells were washed with dH2O, and the pellets were used for total RNA extraction. Total RNAs were extracted using Trizol reagent (Invitrogen) and purified with the Qiagen RNeasy cleanup kit (Qiagen) following the manufacturer's instructions. The purified RNAs were used as templates for PCR amplification with primers of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) to determine potential genomic DNA contamination. Purified RNAs were quantified using a Nanodrop spectrometer (Thermo Scientific).

[0073] First-strand cDNAs were synthesized using a Superscript III cDNA synthesis kit (Invitrogen) following the manufacturer's instructions. Expression of fbpl and gapdh was analyzed using SYBR advantage QPCR premix reagents (Stratagene). Primer efficiency was determined by serially diluting the cDNA and monitoring DNA amplification by real-time PCR. Gene expression levels were normalized using the endogenous control gene GAPDH, and the relative levels were determined using the comparative threshold cycle (CT) method. Real-time PCRs were performed using an Mx4000 QPCR system (Stratagene). The specificity of the PCR was further verified by subjecting the amplification products to agarose gel electrophoresis and sequencing them.

[0074] The fbpl A mutants were generated in both H99 and KN99a strain backgrounds by overlap PCR. See FIG. 1. The 5' and 3' regions of each fbpl gene were amplified from H99 genomic DNA with primers CX278 / CX279 (SEQ ID NO: 20 and SEQ ID NO: 21), and CX280 / CX281 (SEQ ID NO: 22 and SEQ ID NO: 23), respectively. The dominant selectable markers (NEOr) were amplified with the Ml 3 primers, M13F (SEQ ID NO: 1) and M13R (SEQID NO: 2), from plasmid pJAFl . Each target gene replacement cassette was generated by overlap PCR with primers CX278 (SEQ ID NO: 20) and CX281 (SEQ ID NO: 23). The purified overlap PCR products were precipitated onto 10-p.L gold microcarrier beads (0.6 gm; Bio-Rad), and strain H99 or KN99a was biolistically transformed. Stable transformants were selected on YPD medium containing G418 (200 mg / liter). To screen for mutants of the fbpl gene, diagnostic PCR was performed by analyzing the 5' junction of the disrupted mutant alleles with primers CX284 (SEQ ID NO: 26) and JH8994 (SEQ ID NO: 29). Positive transformants identified by PCR screening were further confirmed by Southern blot analysis.

[0075] To generate complemented strains of fbpl mutants, a genomic DNA fragment that contains a 1.5-kb upstream promoter region, the fbpl open reading frame (ORF), and its 500-bp downstream region was amplified in a PCR using primers CX285 (SEQ ID NO: 27) and CX286 (SEQ ID NO: 28). This PCR fragment was fused with the NATrselective marker gene at its C terminus in an overlap PCR using primers CX285 (SEQ ID NO: 27) and M13R (SEQ ID NO: 2). The overlap PCR product was biolistically transformed in both a and a mating-type fbpl mutant strains. Mating assays were performed to identify transformants that complemented the fbpl phenotype.

[0076] To generate strains expressing Fbpl lacking the F-box domain, the F-box domain sequence was deleted by amplifying two fragments from H99 genomic DNA with primers CX225 / CX198 (SEQ ID NO: 30 and SEQ ID NO: 16) and CX199 / CX231 (SEQ ID NO: 17 and SEQ ID NO: 47) respectively. Overlap PCR was performed by using the mixture of two fragments as a template with primers CX225 (SEQ ID NO: 30) and CX231 (SEQ ID NO: 47). The amplified overlap PCR product contains the fbpl gene lacking the F-box domain sequence and was cloned into vector pJAFl 3, which contains the NAT selective marker, to generate the construct pCUX58. pCUX58 was introduced and expressed in the fbpl A mutant background by biolistic transformation. The expression of Fbpl lacking an F-box domain was confirmed by Western blot analysis.

[0077] Melanin production was assayed by inoculating C. neoformans strains into 2 mL YPD liquid medium and incubating them overnight at 30°C. Five microliters of each overnight culture with series dilutions was placed on 1-3,4-dihydroxyphenylalanine (1-DOPA) agar medium (13 mM glycine, 15 mM glucose, 29.4 mM KH2PO4, 10 mM MgSO4 7 H2O, 3 yM thiamine, 5pM d-biotin, 2% agar, 1 mM 1-DOPA, pH 5.5). The agar plates were incubated at 30 °C or 37 °C for 2 days, and the pigmentation of fungal colonies was assessed.

[0078] To examine capsule production, yeast cells were grown in YPD broth overnight at 37°C with constant agitation, washed 3 times with phosphate-buffered saline (PBS), and resuspended at a concentration of 1 x 106cells / mL in various media: DME medium, PBS, or YPD broth. Cells were added to 6-well plates and incubated with or without the addition of 10% heat- inactivated FCS and in the absence or presence of 10% CO2 for 24 h. The capsule size was visualized by adding a drop of India ink to the cell suspensions and observing them on an Olympus (Melville, NY) AX70 microscope using a 100* objective. The cells were analyzed using the Image! 1 .40g software by measuring the distance from the cell wall to the capsule edge (India ink exclusion zone). The average and standard deviation from 15 to 30 cells were calculated for each condition tested.

[0079] In a mating assay, C. neoformans cells of opposite mating types were mixed and cocultured on V8 or MS agar medium at 25 °C in the dark. Mating filaments and basidiospore formation were examined and recorded by photography using the Olympus CX41 light microscope after incubation for 7 to 10 days. Mating results were also examined using scanning electron microscopy (SEM).

[0080] DAPI (4',6-diamidino-2-phenylindole) staining was performed with minor modification. Cell cultures and mating filaments were fixed with formaldehyde (9.3%) for 10 min. The fixed cells were washed twice with PBS, permeabilized with an equal volume of PBS buffer containing 1% Triton X-100 for 5 min, washed twice again with PBS, and resuspended in PBS. For DAPI staining, equal volumes of cell suspension and DAPI mixture (20 ng / mL DAPI, 1 mg / mL antifade, 40% glycerol) were mixed and observed with a Nikon fluorescence microscope.

[0081] Trypan blue staining was performed. Briefly, strains were inoculated in 5 mL YPD liquid medium and grown for 2 days at 30°C. Yeast cells were mixed with an equal volume of 0.4% trypan blue solution and viewed through an Olympus CX41 light microscope. The percentages of cells that were able to take up dye (dark blue) were calculated by counting at least 500 yeast cells for each strain.

[0082] Yeast two-hybrid interaction assays were performed. The ft>pl full-length cDNA and partial cDNA lacking the F-box domain, full-length cDNAs of skpl homologues in strainH99 (Cnskpl ) and S. cerevisiae (Sc skpl ), and crkl cDNA were cloned into the bait vector pGBKT7 and fused with the BD domain. cDNAs of fbpl, Cnskpl , and Scskpl were also cloned into the prey vector pGADT7 and fused with the AD domain. All inserted cDNA sequences were confirmed by sequencing. Both bait constructs and prey constructs were cotransformed into the yeast strain PJ69-4A. Transformants growing on SD medium lacking histidine or adenine were considered positive interactions. The expression of the LacZ gene in these transformations was quantified by P-galactosidase enzyme activity assays using chlorophenol red-P-D-galactopyranoside (CPRG) (Calbiochem, San Diego, CA) as a substrate.

[0083] Phagocytosis assays were performed in 96-well plates, using J774 macrophages at a concentration of 2.5 x 104cells / well that were allowed to double overnight at 37 °C in activating medium (DME medium, 50 U / mL of gamma interferon [IFN-y], 1 pg / mL of lipopolysaccharide [LPS]). PBS-washed fungal cells were opsonized with 20% mouse complement (Pel-Freez, AK) and added to the macrophages at an effector-to-target ratio of 1 :2. Phagocytosis was allowed to occur for 2 h at 37 °C in 10% CO2. The cells were then washed three times with PBS and fixed with methanol at -20 °C for 30 min. Giemsa stain was added to the wells at a dilution of 1 :20, and the plates were incubated at room temperature for 30 min. The wells were washed once with PBS and analyzed using an inverted microscope. For each well, 3 different fields were counted, for a total of at least 100 macrophages. The percent phagocytosis was determined by dividing the number of macrophages that contained C. neoformans by the total number of macrophages counted.

[0084] To check macrophage antifungal activity, phagocytosis was allowed to occur for 3 h and 24 h. After each time interval, medium was removed from the well and transferred to an Eppendorf tube containing sterile PBS. Macrophages were lysed by adding sterile dH2O to each well and incubating the plate for 45 min at room temperature. Fluid was transferred to each respective tube after resuspension. Fungal cells were counted, appropriate dilutions were made, and approximately 200 cells were plated on YPD agar plates. The colonies on each plate were counted after 2 days of incubation at 37 °C to determine the actual number of live cells per well.

[0085] Yeast strains were grown at 30 °C overnight, and the cultures were washed twice with PBS buffer and re-suspended at a final concentration of 2 * 106CFU / ml (colony forming units / mL). Groups of 10 female A / Jcr mice (NCI, Frederick, MD) were intranasally (IN) infected with 105cells of each yeast strain. Over the course of the experiments, animals thatappeared moribund or in pain were sacrificed by CO2 inhalation. Survival data from the murine experiments were statistically analyzed between paired groups using the log-rank test with PRISM version 4.0 (GraphPad Software, San Diego, CA) (P values of <0.001 were considered significant).

[0086] Infected animals were sacrificed at the endpoint of the experiment according to the University of Medicine and Dentistry of New Jersey (UMDNJ) lACUC-approved animal protocol. For mice infected by the fbpl mutant strain, the experiment was terminated 60 days post infection. To compare the fungal burdens and host inflammatory responses, lungs and brains from mice infected by H99, the fbpl mutant, or the complemented strain of the fbpl mutant were isolated at 10 or 20 days post infection, fixed in 10% formalin solution, and sent to the AML laboratory for section preparation (AML Laboratories, Inc. Rosedale, MD). Lungs and brains infected by the fbpl mutant were also isolated at the endpoint of the experiment (60 days post infection). Tissue slides were stained with hematoxylin and eosin (H&E) and examined by light microscopy. Infected lungs, brains, and spleens were also isolated and homogenized using a homogenizer in PBS buffer. Resuspensions were diluted, 100 pL of each dilution was spread on YPD medium with ampicillin and chloramphenicol, and colonies were determined after 3 days of incubation at 30 °C.Results

[0087] Fbpl contains 928 amino acids (SEQ ID NO: 48) with an F-box domain (SEQ ID NO: 49) and an LRR domain containing at least 12 LRRs. Fbpl shows sequence similarity to several F-box proteins reported in fungi, including Grrl in S'. cerevisiae and C. albicans, GrrA in A. nidulans, and Fbpl in F. graminearum. Fbpl showed 19% protein sequence identity and 33% sequence similarity with Grrl, suggesting it could also be an important nutrient regulator. Due to the importance of nutrient sensing and its regulation in both the development and pathogenesis of C. neoformans, the function of Fbpl was investigated.

[0088] Mating between H99 and KN99a was performed on V8 mating medium, and mating mixtures were collected from plates after incubation for 0, 6, 24, 48, 72, and 96 h. RNAs were purified, and qRT-PCR was performed. The results indicate that the expression of fbpl was significantly downregulated during mating, especially after 72 h, compared to expression at the 0-h time point, an indication that the fbpl plays a role in later stages of sexual reproduction after cell fusion. qRT-PCR results showed that fbpl expression was moderately upregulated inresponse to the glucose starvation caused by switching cultures from YPD (2% glucose) to YPG (2% galactose) for 2 h, indicating that expression of fbpl is subject to glucose repression.

[0089] In S. cerevisiae, Grrl functions as a repressor of glucose repression and is also involved in cell cycle regulation. Overnight cultures of grrl mutants grown on YPD quickly flocculated without being shaken and mutant cells were elongated. To examine whether Fbpl is an ortholog of Grrl, the pGBKT7 vector expressing Fbpl or Grrl was transformed into a grrl mutant strain. Reintroducing the GRR1 gene complements the grr 1 mutant on cell morphology. However, no morphological change was observed when Fbpl was expressed in a. grrl mutant strain, which was confirmed by RT-PCR (not shown), indicating Fbpl could not complement the function of Grrl in S. cerevisiae. Because cell morphology was not altered in C. neoformans jbpl mutants, Fbpl has different functions than Saccharomyces Grrl.

[0090] Hxt3 is a glucose transporter, and its expression is induced by either low or high glucose concentrations in S. cerevisiae in a Grrl -dependent manner. A homologue of HXT3 in C. neoformans(CnHXT3‘, CNAG 03372.2) was identified. The expression of CnHXT3 under different glucose conditions was measured by qRT-PCR and compared between wild-type H99 and the fbpl strain. When cells grown on medium without glucose (YPG, 2% galactose) were switched to glucose-rich medium (YPD, 2% glucose) for 2 h to generate glucose induction conditions, expression of CnHXT3 was higher in the fbpl mutant background and was downregulated in the H99 background, n the other hand, when cells grown on YPD were switched to medium without glucose (YP, no glucose; YP0.1, 0.1% glucose; or YPG, 2% galactose) to generate glucose starvation conditions, CnHXT3 expression was not significantly altered in the wild-type strain but was upregulated in fbpl mutants. These results indicated that CnHXT3 expression was repressed by a high glucose concentration and induced by glucose starvation. Fbpl negatively regulates CnHXT3 expression under these conditions.

[0091] fbpl deletion mutants were generated in both C. neoformans H99 and KN99a strain backgrounds. The development of dikaryotic hyphae and basidiospores was examined in both fbpl unilateral mating (fbplxwild type) and bilateral mating (fbpl * fbpl). No obvious phenotypic changes were observed in fbpl unilateral-mating assays. The bilateral mating between fbpl mutants failed to produce basidiospores, even though it produced normal dikaryotic hyphae, indicating that Fbpl is essential for sporulation. As the F-box domain of an F-box protein is important for its function the role of the F-box domain in Fbpl function wasinvestigated by generating strains expressing Fbpl lacking the F-box domain (FbplAFB), which contains a 6><His tag at the C-terminal end. The expression of FbplAFBin fbpl mutants was confirmed by Western blot analysis using anti-His antibody. The mating results showed that bilateral mating between strains expressing Fbpl4FB(Fbpl4FBx Fbpl41'13) could not produce spores, similar to the bilateral mating of fbpl mutants, indicating the F-box domain is essential for Fbpl function in mating.

[0092] The development of fungal nuclei at different stages of sexual reproduction by DAPI staining was investigated to understand why fbpl mutants fail to produce spores. A single nucleus in each yeast cell was observed in both the wild-type and the fbpl mutant cultures, and two separated nuclei can be observed in each dikaryotic hypha produced from bilateral-mating mixtures after cell fusion. A single fused nucleus could be observed in each young basidium of both the wild type and fbpl mutants after mating mixtures were incubated for 3 days, indicating that both strains undergo normal nuclear fusion to produce basidia during mating. However, nuclei in the bilateral mating of fbpl mutants failed to undergo meiosis after fusion, and only a single nucleus was observed in each mature basidium afterl4 days of incubation, while all basidia from wild-type mating produced four nuclei. This observation suggested that Fbpl is important for regulating meiosis during mating, which could explain the defect of sporulation in bilateral mating of fbpl mutants. Because fbpl mutants have a normal growth rate and also have normal nuclear division when grown in rich medium, Fbpl may not be involved in the cell cycle during mitotic division; rather, it plays a role only in regulating meiosis. To confirm these observations, fluorescence-activated cell sorter (FACS) analysis was performed for both H99 and fbpl mutants cultured in YPD liquid medium and minimal medium. No difference was observed between the wild-type strain and the fbpl mutant, which confirmed that Fbpl is dispensable for mitotic cell division in C. neoformans.

[0093] In C. neoformans, several virulence factors that are important for fungal virulence have been well characterized in vitro, including production of a polysaccharide capsule and melanin, as well as the ability to grow at mammalian body temperature. The development of these virulence factors inmutants was examined in vitro, fbpl mutants produced normal melanin on both Niger seed agar and L-DOPA agar, produced regular capsules on DME medium, and also showed normal growth at 37 °C, indicating that Fbpl is not important for development of these virulence factors in vitro.

[0094] The capsule size of C. neoformans can be further induced by the presence of high CO2concentrations and serum. The capsule enlargement under such inducible conditions is similar to what has been observed in vivo during infection, since 10% CO2and serum are part of the physiological condition in mammals. The ability of capsule to grow under such conditions is important for fungal pathogenicity. To test whether Fbpl plays a role in capsule growth under such inducible conditions, capsule sizes of fbpl mutants were examined after they were treated with 10% CO2and 10% heat-inactivated FCS. Yeast cells were grown on different culture media, including rich medium (YPD), PBS buffer, and DME medium, and the cultures were treated with 10% CO2, 10% FCS, or both. The results showed that in every medium tested, the capsule sizes of both H99 and fbpl mutants were significantly enlarged in the presence of 10% CO2or 10% CO2 plus 10% FCS. However, there was no significant difference between H99 and mutants, indicating Fbpl is dispensable for capsule growth under these inducible conditions.

[0095] The virulence of fbpl mutants were examined. Consistent with previous studies, mice infected by the H99 wild-type strain were terminated around 18 to 22 days post inoculation due to lethal infection. However-infected mice stayed healthy and continued to gain body weight even after 60 days post infection. A complemented strain of the fbpl mutant developed lethal infection in mice around 24 days, which confirmed that the avirulent or hypovirulent phenotype in mutants is caused by the deletion of the FBP1 gene.

[0096] The fungal burdens of infected mice were evaluated at the endpoint of the infection experiments. Lungs, spleens, and brains from three mice infected by each fungal strain were isolated, and the fungal loads in these organs were measured as yeast CFU per gram fresh organ. The results showed that when mice infected by the wild-type strain, H99, were sacrificed around 20 days, 109, 105, and 106CFU were isolated in mouse lung, brain, and spleen, respectively. Mice inoculated with fbpl mutant cells were sacrificed 60 days post inoculation, and mouse organs were isolated to examine the fungal burden. No yeast cells were recovered in fbpl -infected spleen and brain, and only an average of 103yeast CFU were observed in each gram of lung.

[0097] Fungal lesion development in lung and brain was also visualized in H&E-stained slides. At either 10 or 20 days post-infection, both the wild-type H99 and the complemented strain caused severe damage in infected lungs, with abundant yeast cells containing big capsules.In contrast, only very limited damage was produced in the fbpl mutant-infected lung, with very few yeast cells observed at both time points. There was no obvious difference between brains infected by any of the three strains at 10 days post infection. However, at 20 days post infection, severe organ damage with visible lesion development was observed in brains infected by H99 or the complemented strain, while no detectable damage or lesion was detected in brains infected by the fbpl mutant. Even at 60 days post infection, the jbpl infection caused only very limited damage in lungs and did not cause any lesion development in brains. These results demonstrate that Fbpl is essential for the development of cryptococcosis.

[0098] C. neoformans can proliferate inside macrophages as an intracellular pathogen. To evaluate whether the avirulent phenotype of fbpl mutants could be due to increased susceptibility to macrophage killing during fungus-macrophage interactions, the sensitivity of \hzfbpl mutant to phagocytotic killings in the J774 murine macrophage cell line was tested. In these studies, no significant difference was observed in the internalization of yeast cells in macrophages. The macrophage antifungal activity assay also showed that the number of CFU recovered from the fbpl mutant is similar to that from the wild-type strain H99 after coinoculation with activated macrophages in DME medium for either 3 h or 24 h, suggesting that the fbpl mutant strain is equally resistant to macrophage killing, fbpl mutants appeared to be less viable when incubated in DME tissue culture medium alone or co-incubated with inactivated macrophages for 24 h, suggesting that the fbpl mutant grows better inside the macrophage, perhaps by obtaining nutrients from host cells.

[0099] The growth of fbpl mutants was tested under osmotic stress (1 .5 M NaCl, 1 M or2.5 M sorbitol) and oxidative stress (2.5 mM H2O2), but no growth defect was observed. The cell integrity of fbpl mutants was examined by applying several chemicals that target cell integrity, such as SDS, CFW, and Congo red. SDS disrupts the plasma membrane and lyses cells with a defective membrane, while CFW specifically binds to chitins and Congo red binds to p-1, 4- glucans of the cell wall to disrupt cell wall integrity. The results showed that fbpl mutant cells were hypersensitive to 0.05% SDS, but not CFW or Congo red, an indication that fbpl mutants have cell membrane defects. Expressing Fbpl lacking the F-box domain (FbplAFB) in an fbpl mutant background could not rescue the growth defect on YPD with SDS, further supporting the idea that the F-box domain is essential for the function of Fbpl . Because trypan blue only stains cells with membrane defects or dead cells, wild-type H99, / ip7 mutants, andstrains expressing FbplAFBwere stained with trypan blue after being cultured at 30°C for 48 h in YPD liquid medium with shaking. The results showed that while approximately 0.7% of H99 cells were heavily stained, over 12% omutant cells and FbplAFBcells were stained under the same conditions. These results further indicate that Fbpl is important for maintaining cell membrane integrity.

[0100] F-box proteins involved in SCF E3 ligase function regulate cellular processes mostly through substrate proteins. To examine whether Fbpl is a subunit of an SCF E3 ubiquitin ligase complex, protein-protein interaction assays were performed between Skp 1 in S'. cerevisiae (ScSkpl) and Fbpl in a yeast two-hybrid system. A homologue of ScSkpl was identified in C. neoformans, CnSkpl (CNAG_00829.2). Interactions between CnSkpl and Fbpl were also performed. Fbpl, ScSkpl, and CnSkpl were fused with both the activation domain (AD) and the binding domain (BD). Constructs containing Skpl homologues and Fbpl were cotransformed into the yeast strain pJ69-4A, and interacting transformants were screened on SD dropout medium (SD-Trp-Leu-His and SD-Trp-Leu-His-Ade). The expression of the LacZ gene was determined by detecting the P-galactosidase enzyme activity. The results showed that both ScSkpl and CnSkpl interacted with Fbpl in a yeast two-hybrid protein-protein interaction system. Deletion of the F-box domain in Fbpl eliminated its interaction with either ScSkpl or CnSkpl, indicating that the F-box domain is essential for these interactions.

[0101] Because Fbpl is essential for sporulation, homologues of Grrl substrates in S. are involved in meiosis regulation were searched for. In 5. cerevisiae, theserine / threonine kinase Ime2 is a key regulator of meiosis, and its degradation is Grrl dependent. Crkl was identified as a Cdk-related kinase that shares sequence homology with both Ime2 in S. cerevisiae and Crkl in Ustilago maydis. Crkl interacts with Fbpl in the yeast two-hybrid system and could potentially be a substrate of Fbpl. In the yeast two-hybrid protein-protein assays, besides the positive control, only colonies expressing both Fbpl and Crkl grew on SD- Trp-Leu-His-Ade medium, indicating a strong interaction between the two protein^. This strong interaction was also evident in the high P-galactosidase enzyme activities.2. Fbpl-mediated ubiquitin-proteasome pathway controls Cryptococcus neofortnans virulence by regulating intracellular growthMaterials and Methods

[0102] C. neoformans strains were grown at 30°C on yeast extract-peptone-dextrose (YPD) agar medium and synthetic (SD) medium. Strains containing genes controlled by the CTR4 promoter were grown in YPD medium supplemented with either 25 pM CuSO4and 1 mM ascorbic acid or 200 pM bathocuproinedisulfonic acid (BCS). The pCTR4-2 plasmid was obtained from Washington University, St. Louis, MO, while the pCN19 plasmid was obtained from Duke University. The mutant and its complemented strain were kindly provided byMaurizio Del Poeta at Stony Brook University. The macrophage-like murine cell line J774 was grown in 10-cm petri dishes in liquid Dulbecco modified Eagle's medium (DMEM) with 10% heat-inactivated fetal calf serum (FBS) (ATL Biologicals), 10% NCTC-109 (Gibco), and 1% nonessential amino acids (MP Biomedicals).

[0103] For SDS sensitivity testing, SDS was added to YPD to a final concentration of 0.2% (wt / vol), and 2x serial dilutions were prepared in 96-well plates. Cells of H99, the fbplUs mutant, and its complemented strain were added to the wells to a final optical density at 600 nmof 0.01 . The plates were kept at 30°C, and the OD6oo was measured every 24 h. For inhibitor assays, MG 132 (Sigma-Aldrich) or PS-341 (LC Laboratories) was added to the YPD liquid medium with 0.025% SDS to a final concentration of 100 pM or 200 pM, respectively. Twofold serial dilutions were prepared in 96-well plates. The wild-type H99 strain was added to the well containing proteasome inhibitors (MG 132 or PS- 134) and SDS to a final OD600of 0.01 . The fbplk. mutant was added to YPD with 0.025% SDS only to a final OD600of 0.01 . The growth of cultures was determined by measuring the OD600every 24 h.

[0104] Macrophage-like J774 cells were cultured in DMEM with 10% heat-inactivated FBS at 37°C with 5% CO2. A total of 5 * 104J774 cells in 0.5 ml fresh DMEM were added to each well of a 48-well culture plate and incubated at 37°C in 5% CO2overnight. To activate macrophage cells, 50 units / ml gamma interferon (IFN-y; Invitrogen) and 1 pg / ml lipopolysaccharide (LPS; Sigma) were added to each well. C. neoformans overnight cultures were washed with phosphate-buffered saline (PBS) twice and opsonized with 20% mouse complement. A total of 2 x 105Cryptococcus cells were added to each well (yeast / J774 cellratio, 4:1). To assess intracellular proliferation of C. neoformans, non-adherent extracellular yeast cells were removed by washing with fresh DMEM after 2 h of co-incubation, and cultures were incubated for another 0, 2, or 22 h. At the indicated time points, the medium in each well was replaced by distilled water (dH2O) to lyse macrophage cells for 30 min at room temperature. The lysate was spread on YPD plates, and the number of CFU was counted to determine intracellular proliferation.

[0105] For time-lapse movie production, activated macrophage cells (2 x 105) and opsonized Cryptococcus cells (2 x 10s) were co-incubated in a 30-mm MatTek glass-bottom dish (coated). After 2 h of incubation, the culture was washed with fresh DMEM twice to remove detached yeast cells and replaced by 2 ml fresh DMEM. Seventeen-hour time-lapse movies with 2 min per frame were taken using a Nikon Eclipse AIRS confocal microscope. A total of 15 different views were taken for every 2 min. Resulting movies were analyzed using the software Nis Elements Viewer (Nikon). All statistical analysis was undertaken using the Student t test. P values of <0.001 were considered statistically significant.

[0106] Cryptococcus cells from overnight culture or mating cultures were collected. Total RNA extraction and first-strand cDNA synthesis were performed. Expression of ISC 1 and GAPDH was analyzed using SYBR advantage QPCR premix reagents (Clontech). Gene expression levels were normalized using the endogenous control gene GAPDH, and the relative levels were determined using the comparative threshold cycle (CT) method. Quantitative realtime PCRs (qRT-PCRs) were performed using an Mx4000 QPCR system (Stratagene).

[0107] The fbpl full-length cDNA was amplified with primers CX225 (SEQ ID NO: 30) and CX443 (SEQ ID NO: 31). The fbpl cDNA lacking the F-box domain was amplified by overlap PCR using primers CX198-CX199 (SEQ ID NO: 16 and SEQ ID NO: 17) and CX225- CX443 (SEQ ID NO: 30 and SEQ ID NO: 31). Both fragments were cloned into the BamHI / Notl sites of a vector containing the Cryptococcus actin promoter and a Flag epitope, generating plasmids pCXU115 and pCXUl 17, which contain FBPl :Flag and FBPlAF:Flag fusions, respectively. The above plasmids were biolistically transformed into the strain to generate strains CUX138 and CUX135, which express Fbpl :Flag andp g proteins, respectively. The ISC1 cDNA was amplified with primers CX551 (SEQ ID NO: 37) and CX552 (SEQ ID NO: 38) and cloned into the BamHI sites of pCTR4-2 vector by use of an In-Fusion HD cloning kit (Clontech), generating the Iscl -hemagglutinin (HA) fusion plasmidpCXU170. To test the stability of Iscl in the wild-type and mutant strain backgrounds,pCXU170 and pCXU108 were biolistically transformed into the wild-type strain and an fmutant to generate strains CUX160 plus CUX167 and CUX1 18 plus CUX1 19, respectively.

[0108] To test the stability of Iscl and Crkl in the wild-type and mutant strainbackgrounds, Iscl :HA-tagged strains CUX167 and CUX168 and Crkl :HA-tagged strains CUX118 and CUX119 were grown to mid-logarithmic phase in YPD, transferred to YPD with 25 μM CuSO4and 1 mM ascorbic acid, and further incubated for the indicated amount of time. Protein extracts were prepared. Iscl :HA was detected by Western blotting using a monoclonal anti-HA antibody (GenScript).

[0109] The purification of Flag-tagged Fbpl was done using EZview Red anti-Flag M2 affinity gel (Sigma-Aldrich). Cells were grown in YPD, harvested and washed with ice-cold water, and transferred into 2-ml bead-beating tubes containing 600 pl acid-washed glass beads. The proteins were extracted in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) by lysing cells at 4 °C with glass beads (four times for 20 s each) in a FastPrep FP120 apparatus (MP Biomedical). The protein extracts were collected after 15 min of centrifugation at 14,000 rpm at 4 °C. Flag-tagged proteins were affinity purified under native conditions. Protein elutions were prepared by using SDS-PAGE sample buffer without reducing agents such as 2-mercaptoethanol or dithiothreitol (DTT).

[0110] To test the interaction between Fbpl and Iscl in vivo, Flag-tagged proteins were purified from strains CUX160, CUX140, and CUX141 by using anti-Flag affinity gel and then analyzed by immunoblotting with anti-Flag and anti-HA antibodies, respectively. To identify the proteins interacting with Fbpl, the Flag-tagged strains CUX138 and CUX135 were cultured in YPD medium, and the Flag-tagged proteins were purified using anti-Flag affinity gel and sent to the Center for Advanced Proteomics Research at Rutgers for mass spectrometry (liquid chromatography-tandem mass spectrometry [LC-MS / MS]) analysis.

[0111] Yeast strains were grown at 30°C overnight, and cultures were washed twice with PBS and re-suspended at a final concentration ofL. Groups of 10 female A / Jcr mice (NCI-Frederick) were infected intranasally with 105yeast cells of each strain. For the intravenous injection model, mice were inoculated with 5 x 104cells via tail vein injection. Over the course of the experiments, animals that appeared moribund or in pain were sacrificed by CO2inhalation. Survival data from the murine experiments were statistically analyzed betweenpaired groups by using the log rank test in Prism 4.0 (GraphPad Software) (P values of <0.01 were considered significant).

[0112] Infected animals were sacrificed at designated time points and the endpoint of the experiment according to the Rutgers Institutional Animal Care and Use Committee (IACUC)- approved animal protocol. Infected lungs and brains were isolated, fixed in 10% formalin solution, and sent to the Rutgers Histology Core Facility. Tissue slides were stained with hematoxylin and eosin (H&E) and examined by light microscopy. Infected lungs, brains, and spleens were also isolated and homogenized in lx PBS by use of a homogenizer. Resuspensions were diluted, 100 pl of each dilution was spread on YPD medium with ampicillin and chloramphenicol, and numbers of colonies were determined after 3 days of incubation. All statistical analyses were undertaken using the Student / test. P values of <0.05 were considered statistically significant.Results

[0113] Example 1 supra shows that jbplts. null mutant is hypovirulent in a murine model of systemic infection. Mice infected by the mutant strain are asymptomatic even after 60 days post-infection, in contrast to the average survival rate of 20 to 25 days when infected by wildtype H99. No yeast cells were recovered from the brains or spleens of mice that were infected by the mutant, and only ~300 yeast CFU in each lung were recovered at 60 days postinfection, compared to ~108CFU in wild-type infection at the endpoint of the infection. To better understand the dynamic of the fbplts mutant-host interaction during the infection process, fungal burdens in infected lungs were examined at 3, 7, 15, and 50 days post-inoculation. The results showed that fungal cells in fbplfs mutant-infected lungs remained at a persistently low level (~103CFU / g fresh lung) throughout the infection process. Fungal lesion development in the lung was also visualized in H&E-stained slides. Wild-type strain H99 caused severe damage in infected lungs, with abundant yeast cells, as early as 3 days post-inoculation. In contrast, lungs infected by the f mutant showed little damage, with very few yeast cells observed atdifferent time points and predominantly localized intracellularly.

[0114] Studies with animal models in vivo demonstrated that the fbplts mutant cannot disseminate to infect other organs following pulmonary infection with 10scells. To better understand the role of Fbpl in fungal dissemination, a murine intravenous injection model of cryptococcosis was applied to investigate whether the mutant simply cannot leave the lung or canleave the lung but cannot cross the blood-brain barrier (BBB) to cause central nervous system (CNS) infection. The results showed that the fbplk mutant still caused lethal infection but had significant virulence attenuation compared to the wild type and the complemented strain (foplk strain plus FBP1). All mice (n = 5) infected by H99 or the complemented strain died at around 7 to 8 days post-injection. Three mice infected by the mutant died at 15 to 25 days post-injection, while the other twomutant-infected mice were still alive and had no sign of disease even at 37 days post-injection, when this experiment was terminated. Because both mouse groups infected by H99 or the complemented strain were terminated at around 7 days, organs were isolated from three mice infected by the mutant at 7 days post-infection for comparison.The analysis of yeast CFU counts and lesion development in infected brains showed that the fo mutant could still cause infection in the brain, but the numbers of CFU were significantly reduced compared to those in mice infected by the wild-type strain. At the endpoint for foplA mutant-infected mice, three sick mice contained comparable numbers of CFU in both brains and lungs, while the two remaining, non-symptomatic mice at 37 days post-infection contained significantly smaller fungal burdens in both brains and lungs. The results indicate that Fbpl is required but not essential for dissemination from the bloodstream to the brain. Therefore, foplA mutants could not leave the infected lung when the mice were infected via nasal inhalation.

[0115] Because the CFU results showed that th mutant remains at a persistentlevel in infected lungs throughout the course of intranasal infection, it was hypothesized that foplA mutants may have a defect in proliferation inside macrophages and that extracellular fungal cells may not be able to grow in the hostile host environment. To investigate how deletion of the FBP1 gene influences the interaction with host cells macrophageinteraction assays were performed by using the murine J774 macrophage-like cell line in 48-well plates. Two hours after co-incubation of opsonized Cryptococcus cells and activated macrophages, non-adherent extracellular yeast cells were removed by washing with fresh medium and incubated for another 0, 2, or 22 h before macrophages were lysed by H2O. The results showed that after 2 h of incubation the number of yeast CFU recovered from macrophages co-incubated with the mutant was comparable to that for cells co-incubatedwith the wild type or the complemented strain, suggesting a similar level of phagocytosis, which is consistent with Example 1 supra. However, after 4 h of incubation, significantly fewer CFUwere recovered from the fbplfs mutant-interacting macrophages (P < 0.001), i.e., only 36% of the CFU recovered from macrophages infected by the wild type. The ratio was reduced to 1:26 after 24 h of incubation. Surprisingly, while a significantly larger number of CFU was recovered from macrophages infected by the wild type after 24 h, recovered mutant cells remain at a persistently low level. By testing the fungal growth rate in DMEM without macrophages, it was found that both the wild-type and the mutant had similar growth in this medium. These results suggest that the fbpl A mutant proliferates very slowly once it is engulfed by macrophages, which, without wishing to be bound by theory, could be one reason why the fbpl Is mutant cannot leave the lung and disseminate to the brain in the mouse systemic infection model.

[0116] To better understand the role of Fbpl in Cryptococcus-macrophage interaction, the numbers of extracellular CFU of both the wild type and the fbplN mutant were measured after co-incubation with activated macrophages for 2, 4, and 24 h. The results showed that similar number of extracellular CFU were recovered between the wild type and the fbplh. mutant after 2 and 4 h of incubation. However, the number of CFU of the fbpl A mutant was only 57% that for the wild type, on average, after 24 h of co-incubation. Because the wild type and the fbplk mutant had similar growth rates in DMEM, the difference in numbers of extracellular CFU between these two strains in cultures with activated macrophages may have been caused by chemicals secreted by macrophages. To test this possibility, the growth assay was performed on these strains with a spent medium from an activated macrophage culture grown under the same conditions. The results showed that the number of CFU recovered by the fbpl\ mutant was —63% of that for the wild-type strain, on average, after 24 h of incubation, which supports the hypothesis that compounds secreted by activated macrophages likely can inhibit the fbplts mutant's growth during coincubation, in addition to the intracellular growth arrest of the mutant. Because the difference in intracellular growth between the wild type and the fbpl A mutant was >25-fold, compared to an ~2-fold difference in the growth in extracellular medium, it was concluded that Fbpl plays a critical role in fungal intracellular proliferation in macrophages. This notion was confirmed by time-lapse movies showing the replication of yeast cells inside macrophages in real time. Much slower proliferation of the mutant cells than the wild-type H99 cells inside macrophages was observed.

[0117] Example 1, supra, showed a direct interaction between Fbpl and the Skpl homolog in C. neoformans in a yeast two-hybrid system, indicating that Fbpl is part of an SCFE3 ligase complex. To further investigate Fbpl function, an immunoprecipitation (IP) assay was performed to pull down Fbp 1 -interacting proteins. An FBP1 overexpression construct tagged with a Flag epitope at the carboxy terminus of the Fbpl protein (Fbpl :Flag) was generated, in which Fbpl expression is under the control of theCryptococcus actin promoter. Using the same strategy, the Flag tag was also fused to an Fbpl construct lacking the F-box domain (FbplAF:Flag). The Fbpl :Flag and constructs were expressed in anfbplB. mutantbackground. Fbpl -associating proteins were purified by immunoprecipitation with an anti-Flag monoclonal antibody (mAb). As a control, a strain expressing a Flag tag fused to the Ga protein Gpal (Gpal . ’Flag), which is functionally unrelated to Fbpl, was also generated, and the same immunoprecipitation method was used to purify Gpal :Flag-binding proteins as a control. Protein pulldown results were analyzed by LC-MS / MS analysis.

[0118] Based on the LC-MS / MS analysis of the IP results, 21 proteins were identified that were observed only in the strains expressing the Fbpl rFlag or FbplAF'.Flag construct, not in the Gpal :Flag-expressing strain, suggesting that they may specifically interact with Fbpl . Among them were a Skpl homologue and a Cdc53 / Cullin homologue, and both of them were pulled down only by Fbpl .‘Flag, not by FbplAF:Flag, indicating that Fbpl likely associates with Skpl and Cdc53 through the F-box domain to form an SCF E3 ligase complex in C. neoformans.

[0119] Example 1 supra showed that Fbpl is required for cell membrane integrity and that both the mutant and strains expressing the Fbpl allele lacking the F-box domain arehypersensitive to SDS. To test whether SDS sensitivity is regulated by the SCF(Fbpl) E3 ligase function, the effects of proteasome inhibitors on Cryptococcus SDS sensitivity was investigated. Fungal growth rates of the wild-type H99 strain, the fbplts mutant, and its complemented strain on YPD medium containing 0.025% SDS were determined in the absence or presence of the proteasome inhibitor MG132 or PS-341 . MG132 is a cell-permeative proteasome inhibitor which can reduce the degradation of ubiquitin-conjugated proteins by the 26S complex without affecting its ATPase or isopeptidase activity. PS-341 is a potent and reversible proteasome inhibitor that functions to degrade intracellular polyubiquitinated proteins. In these assays, the fbplk mutant had growth defects, while the wild type and the complemented strain exhibited normal growth on YPD agar medium containing 0.025% SDS. Neither 25 pM MG132 nor 100 pM PS-341 alone affected the growth of the wild-type strain or the mutant. Surprisingly, when grown on YPD medium containing both SDS and a proteasome inhibitor (either MG 132 or PS-341), the wild type and the complemented strain showed a significant growth defect that was similar to the growth rate of the Jbplk mutant in YPD with SDS alone. These results indicate that treatments with proteasome inhibitors lead to the SDS hypersensitivity of wild type Cryptococcus, which mimics that of the foplts mutant, strongly suggesting that Fbpl is part of the ubiquitin-proteasome system and likely regulates cell membrane integrity through its E3 ligase function. Therefore, without wishing to be bound by theory, certain substrates of Fbpl may also participate in regulation of cell membrane integrity.

[0120] Because E3 ligases usually interact with phosphorylated substrates for ubiquitination and eventual degradation, other proteins that were pulled down by Fbpl in the immunoprecipitation analysis were analyzed to identify potential Fbpl substrates. Besides the SCF(Fbpl) E3 ligase components, 18 other proteins interacted with both Fbpl :Flag and FbplAF:Flag in the IP pulldown and LC-MS / MS analyses, which were selected as potential substrate candidates. The protein sequence of an E3 ligase substrate usually contains the PEST domain, a sequence rich in proline (P), glutamic acid (E), serine (S), and threonine (T), which is a signature of the short-half-life proteins that are commonly targeted for degradation by the ubiquitin-proteasome system. Among the 18 proteins identified, 12 possessed putative PEST domains in their protein sequences, based on the ePESTFind program, including the inositol phosphorylsphingolipid phospholipase Cl Iscl and the iron regulator Cirl.

[0121] Iscl in C. neoformans is an enzyme involved in inositol sphingolipid metabolism, as it stimulates the activity to break down inositol phosphorylceramide (IPC) into phytoceramide and phosphorylinositol. Iscl has been found to be required for intracellular growth of Cryptococcus in macrophages, a function shared by Fbpl . Iscl has three putative PEST domains, suggesting that it could be a target of the ubiquitin-proteasome pathway. Cirl is an iron-responsive transcription factor that controls the regulation of genes for iron acquisition and the known major virulence factors of the pathogen, including capsule and melanin production and the ability to grow at body temperature (37°C). Cirl is degraded by a ubiquitin-proteasome system, but the nature of the potential E3 ligase remains unknown. Cirl contains two putative PEST domains. Therefore, Iscl and Cirl may be substrates of the SCF(Fbpl) E3 ligase, however, the involvement of Cirl in the development of major virulence factors is different from the Fbpl function.

[0122] Because a substrate of Fbpl will likely be accumulated in an jbplts mutant background due to a lack of proper ubiquitination and degradation, ISC1 overexpression strains were generated in which ISC1 expression is under the control of the Cryptococcus actin promoter. Compared to its expression in the wild type, the expression of ISC J in the overexpression strains was increased over 300-fold as detected by qRT-PCR. To fully evaluate the function of Iscl, a isclts. mutant for this study was utilized. The potential role of Iscl in regulating cell membrane integrity was examined by observing the SDS sensitivity phenotype of the ISC1 overexpression strains and the isclk. mutant cells. All overexpression strains were sensitive to SDS treatment, as they had growth defects in medium containing 0.025% SDS.These strains grew normally in the presence of the cell wall-destabilizing agents calco fluor white (CFW) and Congo red, which is consistent with phenotypes of the jbplb. mutant. The isclls. mutant was sensitive to SDS as well. The SDS sensitivity phenotype of both null mutants and overexpression strains of Iscl suggests that the expression of Iscl is tightly regulated and that proper expression is necessary for its normal cellular function. The phenomenon that deletion or overexpression of a target gene leads to similar phenotypes has also been documented in other organism.

[0123] To further examine the potential interaction between Iscl and Fbpl, an Iscl expression construct in the vector pCTR4-2 was generated in which Iscl was fused with an HA tag at its C terminus and was under the control of an inducible CTR4 promoter that is induced by BCS and repressed by copper. The construct expressing the Iscl :HA fusion protein was introduced into an Fbpl :Flag overexpression strain and the fbplk mutant background. The total protein from the strain expressing both Fbpl :Flag and Iscl :HA was purified using EZview Red anti-Flag M2 affinity gel and immunoblotted with anti-Flag and anti-HA antibodies. Both Flag and HA signals were detected from the co-IP product, demonstrating that Iscl interacts with Fbpl in C. neoformans.

[0124] To evaluate the hypothesis that Iscl is an Fbpl substrate, it was examined whether the stability of the Iscl protein is dependent on SCF(Fbpl) E3 ligase function. The Iscl :HA fusion construct was expressed in the wild-type H99 strain and the fopl\ mutant, and the stability of the Iscl :HA protein was examined in these strain backgrounds. Strains expressing the Iscl :HA fusion protein were first grown in YPD medium with 200 pM BCS to induce the CTR4 promoter and then washed with PBS. Washed cultures were transferred toYPD containing 25 p.M CuSO4and 1 mM ascorbic acid to block the transcription of ISC1:HA. Cells were collected after 0, 1, 2, and 4 h of incubation, and the abundance of the Iscl :HA protein was measured by Western blotting. In these assays, the Iscl :HA protein was degraded in a time-dependent manner over the period examined (0 to 4 h) in the wild-type background, but it was relatively stable in the fbpl Is mutant, indicating that the stability of Iscl :HA is dependent on Fbpl .

[0125] To test whether Iscl accumulated in the fbplfs mutant background, another Iscl :HA fusion construct in which ISC1 was controlled by its native promoter was made and transformed into H99 and the fbpl A mutant background. The abundance of the Iscl :HA protein was measured by Western blotting. The signal of Iscl :HA in the fbpl A mutant background was much stronger than that in the wild-type strain, but it was weaker than that in strains expressing Iscl :HA under the control of the CTR4 promoter. The presence of a single ectopic copy of thelSCl.HA gene in these strains was confirmed by Southern blotting. This result demonstrates that Iscl is stabilized in the fbpl / S mutant background, suggesting that Iscl is a substrate of Fbpl in C. neoformans.

[0126] Iscl has been reported to play a key role in protecting C. neoformans from the intracellular environment of macrophages and is important for fungal dissemination to the central nervous system and the development of meningoencephalitis. Since Fbpl is also required for s-macrophage interaction and for fungal dissemination, the connection betweenthe Fbpl E3 ligase and Iscl was further investigated. As an Fbpl substrate, the Iscl protein is stabilized in an fbpl Is mutant background. Therefore, the role of Iscl in fungal virulence was investigated by using both the iscl Is mutant and its overexpression strain.

[0127] In a murine inhalation model of cryptococcosis, virulence attenuation was observed in both the iscl / S mutant and its overexpression strain. All mice infected with 105yeast cells of wild-type strain H99 had a median survival time of 20 days due to lethal infection. In contrast, both the iscl Is mutant and the ISC1 overexpression strain showed significant virulence attenuation, with median survival times of 29 and 25 days, respectively. To test whether an iscl IS mutation can partially rescue the hypovirulence of the fbpl Is mutant, an iscl A fbpl Is double mutant was generated. Mice infected by the iscl A fbpl A double mutant developed lethal infections by 40 days post inoculation indicating that the iscl / S mutation partially rescued the virulence attenuation of the fbpl Is mutant.

[0128] To better understand the virulence attenuation of the Ise 1 -related strains, the disease progression was investigated by examining the fungal burdens of infected lungs and brains in a time course study. Mouse lungs and brains infected by the wild type, the ISC1 overexpression strain, or the iscl Is mutant were isolated at 3, 7, and 14 days post infection. The results showed that lungs infected by either the iscl Is mutant or the ISC1 overexpression strain showed around 10 times fewer CFU at 7 and 14 days post infection than the H99-infected lungs, but the difference was not significant at 3 days post infection. Brains infected by the Ac / A mutant showed significantly reduced fungal burdens throughout the infection process, showing that Iscl is required for dissemination. Fungal burdens in brains infected by the ISC1 overexpression strain were also reduced, but to a lesser extent, consistent with the survival curve for infected mice. These outcomes suggest that the Iscl protein level may be tightly regulated by the Fbpl E3 ligase, and levels that are too high or too low could lead to virulence attenuation.

[0129] To better understand how Fbpl may regulate Iscl function in vivo, the macrophage interaction in the iscl Is mutant and its overexpressed strain wastested. The results found that both strains showed reduced intracellular growth, a phenotype observed in the fbpl Is mutant as well, with the ZSC / overexpression strain showing a lesser defect than the null mutant, he iscl A fbpl / S double mutant also showed a significant intracellular growth defect, but it was much less severe than that of the fbpl Is single mutant, which is consistent with the conclusion that Iscl is a downstream target of Fbpl.

[0130] A growth assay of the wild type and the fbpl IS mutant under either nitrosative or oxidative stress conditions. Compared to the wild-type strain, the iscl IS mutant, fbpl A mutant, iscl A fbpl Is double mutant, and ISC1 overexpression strains all had growth defects under nitrosative stress conditions, although the sensitivities of the fbpl Is mutant and the ISC1 overexpression strain were not as high as that of the iscl IS mutant. Oxidative stress response assays showed a less conclusive outcome. The iscl Is mutant and iscl A fbpl Is double mutant showed high sensitivity to 5 mM H2O2at low pH (pH 4.0), but the fbpl Is and ISC1 overexpression strains did not show significant differences compared to wild-type strain H99. The function of Iscl in the nitrosative stress response is regulated by the Fbpl E3 ligase, but its function in the oxidative stress response may be regulated by some other upstream factors.3. The F-box protein Fbpl shapes the immunogenic potential of Cryptococcus neoformansMaterials and MethodsMice

[0131] Age and sex matched mice of the A / Jcr and C57BL / 6 genetic backgrounds were obtained from the Jackson Laboratories. CCR2-depleter mice (CCR2-DTR) in the B6 background were generated. For studies with CCR2-depleter sex and age-matched littermates were used as control mice. RAG1- / - lymphopenic mice were purchased from the Jackson Laboratories. All mouse strains were maintained and bred at the Rutgers-NJMS Cancer Center Research Animal Facility under specific pathogen-free conditions. Animal studies were performed following biosafety level 2 (BSL-2) protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Rutgers University under protocol 15041D. The studies performed were governed by protocol 15041D as approved by the IACUC committee of New Jersey 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.C. neoformans strains and growth conditions

[0132] C. neoformans var. grubii (serotype A) H99 and its isogenic mutant fbpl A deletion mutants were generated as described in the above Examples. Strains were grown at 30 °C on yeast extract-peptone-dextrose (YPD) agar medium overnight and the next day were washed with PBS three times before inoculum preparation.Capsule production and GXM purification

[0133] To examine capsule production, 5 pL of overnight cultures were inoculated on minimum medium (MM) and incubated at 30 °C for three days. Capsule was visualized with India ink negative staining and observed with a lOOx Olympus CX41 equipped with an Infinity digital camera (Olympus, NJ). Secreted total polysaccharides was purified from 500 mL YPD culture of each strain using the CTAB precipitation method. The amount of the total GXM was determined by the phenol sulfuric method. Purified GXM was sent to the Comprehensive Carbohydrate Research Center at the University of Georgia for 1 dimensional proton nuclear magnetic resonance (NMR) spectroscope analysis. The two-pair T-test method was used to determine the statistical significance of the difference between samples.Measurement of cell surface mannoprotein, chitin and chitosan levels

[0134] Mannoprotein staining proceeded according to the following. Briefly, fungal cells were grown on YPD overnight, re-suspended in PBS supplemented with 0.5% gelatin at a final OD60 of 0.5 for 30 min. On hundred microliter of each suspension was co-incubated with 10 uL of ConA-FITC (Sigma, 2.5 mg / ml stock) for 30 min at room temperature. The extent of ConA- FITC binding was determined using flow cytometry.Infections, CCR2+depletion, and vaccinations

[0135] All infections were performed by intranasal (IN) inoculation. Mice were anesthetized with 100 pL of Ketamine (12.5 mg / mL) and Xylazine (1 mg / mL) prior to inoculum instillation into the nostrils. Varying inoculum doses ranging from IxlO4to IxlO6of H99 and fop 14 deletion mutants were used in the study. For survival experiments, mice were monitored daily for the development of disease symptoms and euthanized according to IACUC guidelines. For analysis of parameters of host immunity, lungs, bronchoalveolar lavage fluid (BALF) and mediastinal lymph nodes (MLNs) were harvested and processed as previously described. The effective depletion of CCR2+cells was achieved by intraperitoneal administration of diphtheria toxin. For vaccination experiments, fopl4 deletion mutants were heat-killed by incubation at 75 °C for 40 minutes. The inoculum was prepared at a concentration of IxlO8cells per 50 pL. Mice were immunized and boosted at days -31 and -7 days prior to infection. At day 0, mice were challenged intranasally (IN) with IxlO4live H99 per mouse.Histology

[0136] For histological examination, lungs were perfused with 10 ml of PBS to remove blood and fixed in 10% buffered formalin. Fixed lung tissue was paraffin embedded and stained by H&E at the Histology Core Facility (Rutgers-NJMS).Lung processing

[0137] Single cell suspensions from lung tissue lung samples were minced in PBS with 3 mg / ml collagenase type IV (Worthington), and were incubated at 37 °C for 45 min. After digestion, lung suspensions underwent RBC lysis. Total lung cells were counted for each sample. Lung cell suspensions were used for RNA extraction and flow cytometry as described herein, as well as for CFU determination by plating serial dilutions.T-cell responses in airways

[0138] Bronchoalveolar lavage fluid (BALF) was harvested in PBS and plated in a 96- well round-bottom plate in RPMI containing 10% fetal calf serum (FCS), Penicillin- Streptomycin (2200 U / ml, Gibco™) and Gentamicin sulfate solution (1 mg / mL). BALF cells were re-stimulated using Leukocyte Activation Cocktail, with BD GolgiPlug™ (BD Biosciences) according to the manufacturer’s instructions. Six hours after activation, BALF cells were surface stained and fixed in 1% paraformaldehyde overnight, then intracellular strained, and finally analyzed by flow cytometry as described herein.T-cell assays from MLNs

[0139] Total lymphocyte suspension was prepared from the MLNs by utilizing the frosted ends of a glass slide and counted. Cells from the same experimental group were pooled, and CD4+T-cells were isolated with a negative-sorting CD4+isolation kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions and were consistently >90% pure. CD4+T-cells (2x105) were cultured with T-cell-depleted APCs (3x105) in RPMI containing 10% fetal calf serum (FCS), Penicillin-Streptomycin (2200 U / ml, Gibco™) and Gentamicin sulfate solution (1 mg / mL), plated in flat-bottom 96-well plates and incubated at 37 °C with 5% CO2. To prepare APCs, splenocyte suspension of a naive syngeneic mouse was prepared similarly to MLNs, and then depleted of T-cells by incubation with an anti-Thyl .2 antibody and rabbit complement (Low Tox; Cedarlane Labs, Hornby, Ontario, Canada) at 37 °C for 45 min. For antigen stimulation, live or sonicated H99 were added in a MOI of 1 :1 .5 (APC:yeast). All cultures were made in presence of the fungal growth inhibitor voriconazole at a final concentration of 0.5 mg / mL. After 72 h post-culture initiation, supernatants were collected for cytokine analysis by ELISA. For IL-2 and TNF-a, ELISA kits from BD-OptEIATM were used, and for IFN-y and IL-17A (homodimer), ELISA kits were purchased from eBioscience.Flow cytometry

[0140] Single cell suspensions were stained for monocytes [CD45 (30-F11 APC-Cy7), CD1 lb (MI / 70 PerCP Cy5.5) and Ly6C (AL-21 PE)], Mo-DCs [CD45 (30-F11 APC-Cy7), CD1 lb (MI / 70 PerCP Cy5.5), Ly6C (AL-21 PE), CD11c (N418 Pacific Blue) and MHC Class II I-A / I-E (M5 / 11.415.2 Alexa Fluor 700)], neutrophils [CD45 (30-F11 APC-Cy7), CD1 lb (MI / 70 PerCP Cy5.5), Ly6C (AL-21 PE) and Ly6G (1 A8 APC)], CD4+T-cells [CD45 (30-F11 APC- Cy7), CD4 (RM4-5 Pacific Blue)], CD8+T-cells [CD45 (30-F1 1 APC-Cy7), CD8a (53-6.7FITC), B cells [CD45 (30-F1 1 APC-Cy7) and B220 (RA3-6B2 APC-Cy7)]. All antibodies used for lung staining and MLNs were from BD Biosciences. BALFs were cell surface stained for T- cells with Thy 1.2 (53-2.1 PE-Cy7) and CD4 (RM4-5 Pacific Blue) and ICCS for IFN-y (XMG1.2 PE), IL-17A (eBiol7B7 APC), IL- 13 (eBiol3A AlexaFlour 488) and TNF-a (MP6- XT22 Alexa Flour 700) following standard procedures. Most antibodies and reagents for cell surface and ICCS were from BD Biosciences, except for IL-17A that were obtained from eBioscience, Inc. All samples were analyzed using a BD LSRII Flow Cytometer and FlowJo software (Tree Star, Inc).RNA extraction and qRT-PCR

[0141] Total RNA from lungs was extracted with Trizol (Invitrogen). Relative mRNA levels were determined by qRT-PCR. One microgram of total RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Taq Man Fast Universal PCR Master Mix (2x) No Amp and TaqMan probes (Applied Biosystems) for each gene were used and normalized to GAPDH. Gene expression was calculated using AACt method relative to naive sample.ResultsDeletion of Fbpl does not affect the expression of known virulence factors in C. neoformans

[0142] Examples 1 and 2 supra illustrate that deletion of Fbpl in the H99 strain of C. neoformans resulted in virulence attenuation in vivo without affecting the production of the primary virulence factors, such as melanin, capsule and thermotolerance in vitro. To further examine potential effects on known virulence factors a detailed characterization was performed on the secretion of the primary capsule polysaccharide, glucuronoxylomannan (GXM), by fbpl A deletion mutants as compared to the parental strain and determined that both strains secreted comparable amounts (FIG. 2A). The capsule composition was also tested in detail and no differences in size or structure (FIG. 2B) were found. Similarly, there was no observation of any differences in the amount of surface mannoproteins expressed by fbpl A deletion mutants based on the ConA-FITC binding intensity (FIG. 2C). Recent studies have suggested that changes in the amount of chitin and chitosan impact the virulence of C. neoformans. Accordingly, it was also tested whether expression of these carbohydrates was affected in the fbpl A strain. It was found that there were no significant differences in the amount of chitin or chitosan expressed byfbplA as compared to H99 (FIG. 2D). Altogether, these results, together with the findings in Examples 1 and 2, suggest that the attenuated virulence in vivo upon infection with fbplA cannot be explained by measurable changes in the production of known virulence factors. Therefore, it was examined whether infection with fbplA impacts the development of host immunity as compared to the parental strain H99 and to define host factors that might confer protection from infection with fbplA.Infection with fbplA induces a robust inflammatory response in the host.

[0143] In the previous Examples, an infection dose of 105H99 and fbplA in mice of the A / J background was used and it was found that these mice survived an infection with fbplA for over 60 days. Although the initial inoculum was the same, H99 grew several log higher than fbplA and disseminated to the brain and spleen while fbplA remained in the lung. This Example sets out to determine whether increasing the infection dose of fbplA would overcome this attenuated phenotype. It additionally examines whether attenuation of fbplA was maintained in mice of a different genetic background, because while not wishing to be bound by theory, several previous studies reported substantial intrinsic differences among mouse strains in terms of their susceptibility to cryptococcosis. Therefore, it was chosen to test mice in the C57B1 / 6J (B6) background since this is a commonly studied strain for analyzes of host immunity and there are a significant number of immune gene-deficient mice in this background. Infection of B6 mice with 105H99 and 1 if fbplA deletion mutants resulted in equal seeding of yeast cells in the lung at 24 hours after infection (FIG. 3A). All B6 mice infected with H99 succumbed to infection by 20 days after inoculation while fbplA infected mice survived for over 50 days (FIG. 3B). A similar survival pattern was observed in A / J mice infected with \ (f fbplA (FIG. 4A). Therefore, increasing the infection dose or changing the host genetic background did not overcome the attenuation of fbplA. It was observed that the fungal burden of fbplA yeast remained constant in the lung of infected mice during the observation period (FIG. 3A). In contrast, H99 continued to grow and by day 14 after infection the pulmonary fungal burden of H99 had increased to several log higher as compared to the initial number of yeast seeded in the lung at 24 hrs after inoculation (FIG. 3A). H99 was also able to quickly escape from the lung and viable CFU were recovered as early as day 7 after infection from the spleen and brain (FIG. 3C, 3D). In contrast, fbplA deletion mutants remained in the lung and there was no detection of viable yeast in thespleen or brain of infected mice (FIG. 3A, 3C, 3D). Thus, virulent H99 was able to rapidly overcome the host and disseminates to extra pulmonary sites while fbplA remained in the lung. Based on these observations it was hypothesized that changes in the host immune response were central to the pulmonary containment of fbplA and the long-term survival of mice infected with fbplA as compared to H99. Several previous studies have documented the critical importance of host immunity as determinants of outcomes from infection with C. neoformans mutants. As a first step, the recruitment of immune cells to the lung was examined by flow cytometric analysis. It was observed that infection with fbpl A induced a significant increase in the number of innate and adaptive immune cells that were recruited to the lung (FIG. 3E, 3F, 3G, 3H). Further observations included increased frequencies in the number of neutrophils and monocytes (FIG. 3E, 3F) as well as increased numbers of pulmonary CD4+and CD8+T-cells (FIG. 3G, 3H). In aggregate, these observations suggest that host immune cells are more robustly recruited to the lung of mice infected with yeast lacking Fbpl as compared to the parental strain H99.Mice infected with / &z>7zf display enhanced differentiation of Th 1, Th 17 and Th 1 / Th 17 CD4+T-cell responses.

[0144] Given the importance of CD4+T-cells in defense against cryptococcosis, the differentiation of Cryptococcus-specific CD4+T-cells in mice infected with Fbpl -deficient or sufficient yeast was examined. The cytokine profile of CD4+T-cells recovered from the airways and lung-draining lymph node (MLN) of mice infected with fbplA to H99 was compared. Peak CD4+T-cell responses were detected at day 7 after infection as measured by expansion in MLN (IL-2 production, FIG. 5A) and recruitment to the lung. Infection with H99 and fbpl A induced comparable CD4+T-cell activation in the MLN as examined by IL-2 production after ex vivo restimulation (FIG. 5A). It was observed that infection with fbplA induced enhanced differentiation of IFN-y secreting Thl cells (FIG. 5B) and Il-17A-secreting-Thl7 CD4+T-cells (FIG. 5C). Increased differentiation of Thl and Thl 7 cells after fbplA infection was accompanied with decreased differentiation of IL-4 (FIG. 5D), IL-5 (FIG. 5E) and IL- 13 (FIG. 5F)-producing Th2 cells as compared to H99. This suggests that infection with fbplA skews CD4+T-cell differentiation towards Thl and Thl 7 responses and diminished Th2. At days 7 and 14 mice infected with fbplA also displayed enhanced frequencies of IFN-g+, IL17+and TNF+CD4+that infiltrated the airways as compared to mice infected with H99 (FIG. 5G, 5H, 51, 5J).Increased frequencies of IFN-y-producing CD8+T-cells were also present in the airways of yZ> / ?7zl-infected mice (FIG. 5G, 5K). Collectively, these findings indicate that infection with JbplA induces enhanced recruitment of innate and adaptive immune cells as well as increased induction of Thl and Thl7 responses and lower Th2 CD4+T-cell responses. Previous studies have shown that Th2 responses against C. neoformans are detrimental while increased Thl responses are protective. Therefore, the long-term survival observed in / &p7zl-infected mice might be explained at least in part by enhanced induction of protective host immune responses that help containLymphocytes are required to maintain the long-term survival of mice infected with C. neoformans fbpl A.

[0145] The results indicate infection with JbplA deletion mutants induces an enhanced activation of immune responses including increased differentiation of Thl and Th 17 cells as well as increased production of IFN-y by CD8+T-cells (FIG. 5). Therefore, while not wishing to be bound by theory, enhanced adaptive immunity against infection with JbplA deletion mutants might be an important protective component of the host response. In order to test the importance of lymphocytes in this response RAG-17' mice that lack mature B and T-cells were infected with JbplA and the parental H99 strain. Remarkably, 100% of mice lacking lymphocytes succumbed to infection with JbplA while 85% of normal control mice survived the infection for over 76 days after infection (FIG. 6A). In contrast, mice infected with the parental H99 strain died within 21 days after infection whether they had an intact lymphoid compartment or not (FIG. 6B). Analysis of fungal burden in yS / >7J-infected RAG- / - mice at the time of euthanasia showed increased fungal burden in the lung as well as dissemination of JbplA yeast cells to the spleen and brain (FIG. 6C, 6D, 6E). Altogether these findings indicate that an intact lymphoid compartment is required for the protection and long-term survival of mice infected with JbplAEnhanced recruitment and maturation of CCR2+Ly6C+monocytes in mice infected with fbpl A mutants

[0146] Although 100% of fbpl -injected RAG- / - succumbed to infection there was a delay in mortally of 30 days in average as compared to H99 infected mice (FIG. 6A, 6B). This observation suggested that immune cells, other than lymphocytes, can also help contain infectionwith fbpl A The analysis of immune cell infiltration to the lung of / &p7zl-infected mice showed significant increases in the number of recruited monocytes (FIG. 3F). Previous studies observed that that CCR2+Ly6C+monocytes are important precursors of mo-DCs that orchestrate the development of Thl CD4+T-cell responses to pulmonary fungal infection. Moreover, CCR2+monocytes have been previously shown to be important innate cells that contribute to defense against infection with C. neoformans. In addition, studies in pulmonary B. dermatitidis infection have demonstrated that inhibition of CCR2+Ly6C+influx is a mechanism of virulence employed by other fungi. Importantly, blockade of CCR2+Ly6C+influx by B. dermatitidis results in impaired immunity while robust recruitment correlates with induction of protective immunity. It was thus hypothesized that the Fbpl -regulated mechanism of virulence in C. neoformans might similarly affect the recruitment of Ly6C+monocytes. To test this hypothesis the differentiation of monocytes into mo-DCs was examined at day 3 after C. neoformans infection with H99 or fbpl A. Infection with Fbpl -deficient yeast resulted in significantly increased influx of monocytes (FIG. 3F) as well as their maturation into CD1 lc+ClassII+mo-DCs (FIG. 7A, 7B, 7C) as examined by percent CD1 lc+ClassII+ cells among monocytes (FIG. 7A, 7B) and by total number of mo-DCs recruited to the lung (FIG. 7C). Increased influx Ly6C+monocytes in fbpl J-infected mice correlated with higher production of CCR2 ligands CCL2, CCL7 and CCL12 (FIG. 7D, 7E, 7F). These observations suggest that increased recruitment of CCR2+Ly6C+ monocytes and their differentiation into mo-DCs is a potentially important innate mechanism of protection in mice infected with fbpl A.CCR2+monocytes are required for the activation of C. neoformans- CD4+T-cellsand for the survival of fbpl A- infected mice

[0147] In order to test the potential contributions of CCR2+monocytes and their derivative cells to host-mediated defense against infection with fbpl A the CCR2-depleter mouse strain was employed. This strain permits selective and temporal removal of CCR2+monocytes upon diphteria toxin (DT) administration. CCR2-depleter and control littermates were infected with fbpl A yeast and treated with DT as depicted in FIG. 8A. Removal of CCR2+monocytes resulted in rapid mortality of mice (FIG. 8B) as compared to control, monocyte-sufficient mice. In the absence of CCR2+monocytes and mo-DCs there was minimal recruitment of CD4+T-cells to the airways (FIG. 8C), and the few cells that infiltrated the airways failed to differentiate into IFN-y or IL-17A producing cells (FIG. 8D). Depletion of CCR2+monocytes and theirderivative cells also resulted in a failure to induce the activation of Cryplococcus-specific CD4+T-cells responses in the MLN. Removal of CCR2+monocytes and impaired CD4+T-cell responses were also accompanied by a failure to contain fungal growth in the lung (FIG. 8E). The expression of protective cytokines in the lung was also significantly diminished in JbplA- infected mice that were depleted of CCR2+monocytes (FIG. 8F). Altogether, these observations suggest that CCR2+ cells are important innate cells in the containment of infection with JbplA and that in their absence fbplA manifests as a virulent strain.4. Vaccination with inactivated (heat-killed) fbplA mutants confers protection from infection with virulent H99 yeast.Initial vaccination studies

[0148] 25 A / Jcr mice were ordered for vaccination studies. 5 were chosen as naive control, 10 were chosen to be infected with live JbplA deletion mutant ( 106per mouse), and 10 were chosen to be infected with heat-killed JbplA deletion mutant (0.5x108per mouse). For the mice infected with heat-killed fbplA deletion mutant, a booster inoculation was given after 24 days. After 30 days, mice from each group (naive, jbplA deletion mutant, and heat-killed JbplA deletion mutant) were inoculated with H99 strain of C. neoformans (106per mouse). The naive mice succumbed to infection around 25 days post-infection with H99, as well as those mice inoculated with live JbplA deletion mutant. Surprisingly, those mice which were inoculated with heat-killed JbplA deletion mutant showed full immunity to H99, as none of the mice succumbed to infection, even more than 60 days post infection by H99 (FIG. 9). This is surprising because although Fbpl has been shown to be essential for virulence (discussed supra), live JbplA deletion mutants do not confer immunity to infection whereas inactivated (z'.e. heat-killed) JbplA deletion mutants confer full immunity. Colony forming units (CFUs) were checked in lungs and brains of mice from each group, and H&E / GMS silver staining was conducted on lung tissue. The results showed that while there were no fungal cells in the brains of the surviving mice, there were trace amounts of H99 cells recovered from 5 of the 10 mice inoculated with heat-killed JbplA deletion mutant. This indicated that H99 cells were contained or cleared by the mice vaccinated by heat-killed JbplA deletion mutants. The histopathology results (H&E staining / GMS silver staining) indicated minimal fungal burden and inflammation (FIG. 10) especially as compared to those mice infected by H99 in the above experiments. Cytokineproduction was measured from CD4 cells recovered by the surviving mice, for interleukin- 17 (IL- 17) and IFNy (FIG. 11). Vaccination studies were repeated as described above but including heat-killed H99 wild type strains instead of live fbplA deletion mutants. The results showed that heat-killed H99 strains did not provide any protection against subsequent infection by live H99, whereas heat-killed fbpl zf deletion mutants did (FIG. 12). This shows that an effective vaccine composition must include inactivated (e.g. heat-killed) fbpl A deletion mutants, either full or partial (e.g. F-box knockouts), as live fbpl A deletion mutants do not offer immunity, nor does heat-killed H99, only inactivated fbplA deletion mutants.Additional vaccination studies

[0149] Additional vaccination studies were undertaken with the protocol set forth in the methods section of Example 3 supra. Mice challenged with live or heat-killed fbpl A displayed equal recruitment of CCR2+Ly6C+ monocytes (FIG. 13A, 13B) and developed comparably robust Thl and Th 17 responses (FIG. 13C, 13D, 13E). Mice were thus immunized according to the vaccination strategy employed by Zhai, et al., Development of protective inflammation and cell-mediated immunity against Cryptococcus neoformans after exposure to hyphal mutants. mBio. 2015;6(5):e01433-15, hereby incorporated by reference in its entirety, to successfully protect mice against H99 challenge. Briefly, mice were immunized with heat-killed (HK)at days -32 and -7 and on day 0 they were infected with 104virulent H99. The protective efficacy of vaccination in both A / Jcr and C57B1 / 6J genetic backgrounds was tested. Consistent with the vaccination experiment described above, vaccination with inactivated fbplA deletion mutants conferred significant protection to C57B1 / 6J mice (FIG. 14A) and A / Jcr (FIG. 14B). This is in contrast to the 100% mortality of H99-challenged mice that did not receive vaccination. Consistent with previous findings, vaccination with HK-H99 was unable to confer protection from infection with live H99 (FIG. 14C). HKvaccinated mice that survived without symptoms for more than 60 days contained a significant number of CD4+T-cells that remained in the airways (FIG. 14E and FIG. 4B). Moreover, airway CD4+T-cells recovered from vaccinated mice rapidly produced IFN-y and IL-77 A upon re-stimulation (FIG. 14F and FIG. 4C, 4D). Cryptococcus-spectifi cD4+T-cell responses were also sustained in the MLN of vaccinated mice (FIG. 14G, 14H, 141 and FIG. 4E, 4F, 4G). These observations suggest that enhanced adaptive immune responses might be responsible for protection inp vaccinated mice. To test the importance of adaptive immunity in vaccine-mediated protectionRAG- / - mice were vaccinated with HK-yJ / ?7zl prior to a challenge with virulent H99. It was found that vaccination with HK- / hp7zl could not protect lymphocyte-deficient mice from a challenge with virulent H99 (FIG. 14D) thus demonstrating that lymphocytes are responsible for vaccine-mediated protection in this model. Altogether, these findings demonstrate that the development of a robust host immune response can overcome the pathogenicity of H99.

[0150] In aggregate, the vaccination results are consistent with the interpretation that the Fbpl -regulated pathway shapes the immunogenicity C. neoformans. The combined results of Example 3 and 4 illustrate that host immune responses are critical for the in vivo hypovirulent phenotype ofyeast. The data indicates that the long-term survival of mice infected with (FIG. 3) is dependent on the activation of protective adaptive immunity (FIG. 5 and FIG. 6) and by increased recruitment and differentiation of CCR2+Ly6C+monocytes (FIG.7 and FIG. 8). It was found that enhanced innate and adaptive immune responses cooperate to help protect the host from infection withSurprisingly, it was discovered that mice which were inoculated with heat-killeddeletion mutant showed full immunity to H99 whereas live deletion mutants do not confer immunity to infection with H99. Furthermore, heat-killedH99 does not confer immunity. Importantly, this protective immune response to inactivated (e.g. heat-killed) deletion mutants can be harnessed in vaccination strategies to prevent mortality against challenge with the virulent parental strain H99 (FIG. 14).5. Vaccination with inactivated (heat-killed)mutants confers cross-protection against infection with other fungal species

[0151] Vaccination studies were undertaken utilizing the protocol set forth in Example 4 supra with the following modifications. Briefly, mice were immunized with heat-killed (HK) fbpl A deletion mutants. After one month, the mice were infected with virulent Cryptococcus gattii (C. gattii) strain R265, a virulent sibling species of C. neoformans that often causes infection in immunocompetent individuals, or with C. neoformans strain H99. Mice not vaccinated (naive) were also infected with both virulent strains R265 and H99 as control. The results are indicated in FIG. 15. Briefly, by 26 days post infection, 100% of the naive mice had died, compared to 0% of the mice infected with H99 and 0% of the mice infected with R265. By day 43, survival among mice infected with H99 was still 100% and for mice infected with R265 the survival rate was approximately 60%. The results thus indicate that heat-killed (HK) fbpl Adeletion mutants confer significant cross-protection against infection with fungus other than C. neoformans (C. gattii in this instance).

[0152] Additional vaccination studies are undertaken. Briefly, mice are immunized with heat-killed and are infected with one of i) virulent Candida albicans (C. albicans)strain ATCC90028 or ii) virulent Aspergillus fumigatus (A. fumigatus) strain ATCC13073. Mice re-challenged with virulent Cryptococcus neoformans strain H99 are used as a control. Mouse survival rates are monitored to determine protection. Results indicate vaccination with inactivated (heat killed) fbplA deletion mutants confer significant protection against infection with C. albicans. Results indicate vaccination with inactivated (heat killed) fbplA deletion mutants confers significant protection against infection with A. fumigatus. Vaccination with inactivated (heat killed) fbplA deletion mutants confer significant protection against infection with virulent H99.6. Vaccination with inactivated (heat-killed) fbplA mutants confer protection against infection in immune deficient animals

[0153] Vaccination studies are undertaken utilizing the protocol set forth in Example 4 supra with the following modifications. Briefly, mice are immunized with heat-killed (HK) fbplA at days -32 and -7. Additionally at day -7, mice are intraperitoneally injected with 200 pg anti-CD4 antibody (GK1.5). Mice continue to be intraperitoneally injected with 200 pg anti- CD4 antibody on a weekly basis throughout the experiment. Depletion of CD4+T-cells in mice is determined by anti-CD4 antibody staining and flow cytometry analysis. At day 0, mice are infected with one of the following: i) virulent Cryptococcus neoformans strain H99; ii) parental Cryptococcus gattii (C. gattii) strain R265; iii) virulent Candida albicans (C. albicans) strain ATCC90028; iv) virulent Aspergillus fumigatus (A. fumigatus) strain ATCC 13073. Results indicate vaccination with inactivated (heat killed) fbplA deletion mutants confer significant protection against infection with virulent H99. Results indicate vaccination with inactivated (heat killed) fbplA deletion mutants confer significant protection against infection with C. gattii strain R265. Results indicate vaccination with inactivated (heat killed) fbplA deletion mutants confer significant protection against infection with C. albicans. Results indicate vaccinationwith inactivated (heat killed) foplA deletion mutants confer significant protection against infection with A. fumigatus.

[0154] TABLE 1 — Primers utilized in the Examples

[0155] TABLE 2 — Fungal strains and plasmids utilized in the ExamplesFull length sequence (928 AA) of FBP1 protein in C. neoformans. F-box domain is underlined. The F-box domain precedes the 12 LRRs; see also Fig. 1 of Liu et al. Eukaryot Cell. 2011 Jun; 10(6):791-802, hereby incorporated by reference in its entirety.LPPEILIQIFRYLPGNKDLLSVLLVSRFWCLCAFSLLWYKPTLPTITQL (SEQ ID NO: 49) Sequence of F -box domain.7. Therapeutic treatment of Cryptococcus infection with HK-fbpl vaccine

[0156] The HK-fbpl Cryptococcus vaccine described herein is a novel therapeutic to treat Cryptococcus infection. The data shown in Figure 16 and Figure 17 demonstrate that mice infected by Cryptococcus neoformans and subsequentially treated with HK-fbpl cells can fully recover from infection with no fungal dissemination detected, while mice without treatment all die within three weeks with disseminated ciyptococcosis (Figure 16B). Therefore, the HK-fbpl may be used as a therapeutic vaccine for patients with cryptococcosis. Further, the immune response in mice post-treatment was evaluated, and the accumulation of T cell response and Thl cytokines was observed (Figure 17).8. Development of a novel vaccine combination strategy against Cryptococcus infection

[0157] In the Examples above, vaccine HK-fbpl that is highly protective against invasive fungal infections in animal models was demonstrated to work. The HK-fbpl vaccine was shownto work by intranasal (IN) inhalation. In the experiments described herein, a new vaccine strategy that combines HK-fbpl and a commercial adjuvant ODN 2395 was tested, referred to below as “combination strategy” and “combination vaccination strategy”. Surprisingly, it was found that using a novel combination of HK-fbpl and ODN 2393, the new combination vaccine strategy, works in all three vaccination methods: intranasal (IN) inhalation, SC injection and IM injection models. Mice vaccinated with two doses of HK-fbpl / ODN 2395 combination showed strong protection against virulent Cryptococcus neoformans challenge.Materials and Methods

[0158] ODN 2395: a synthetic oligodeoxynucleotide containing unmethylated CpG motifs (CpG ODNs). CpG ODNs are recognized by TLR9, which is expressed exclusively on human B cells and plasmacytoid dendritic cells (pDCs), thereby inducing Th 1 -dominated immune responses. It was purchased from InvivoGen (San Diego, CA). The AS03 adjuvant was also tested using this strategy but did not show an obvious protection. So the protection is due to a unique combination vaccination.

[0159] Ethics statement on animal use: Female mice with an average weight of 20 - 25 g were used throughout these studies. BALB / c mice were purchased from the Jackson Laboratories. Animal studies were performed at Rutgers University Newark campus animal facility. All studies were conducted following biosafety level 2 (BSL-2) protocols and procedures approved by the Institutional Animal Care and Use Committee (IACUC) and Institutional Biosafety Committee of Rutgers University under protocol 999901066. 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.

[0160] Vaccination strategy: C. neoformans fbplts mutant strain was heat-killed following a previously described procedure (Zhai et al., mBio Vol. 6, No. 5, 2015). Briefly, fungal cells from YPD overnight cultures were precipitated and washed twice with sterile PBS. The cell suspension with the correct concentration was then aliquoted into Eppendorf tubes and heated on a hot plate at 75°C for 90 min. The viability of the cells following heat treatment was examined by plating the processed cell suspension on YPD agar plates; no colonies were recovered after incubation at 30°C for 3 days. Adjuvant CpG ODN 2395 (InvivoGen, CAT# vac- 2395-1) was dissolved following manufacturer’s instruction. Heat-killed 5 x 107or 1 x 107fbplA cells (HK-fbpl vaccines) were prepared in 50 pl 1 x PBS. Adjuvanted fbplA vaccines (HK-fbpl / ODN 2395) were prepared by adding 10 pl (2 mg / ml) ODN 2395 after aspirating the same volume of supernatant from the 50 pl cell suspension in 1 x PBS. ODN 2395 vaccines were prepared in 50 pl (0.4 mg / ml) sterile endotoxin-free physiological water. All vaccines were thoroughly mixed before immunization. Mice were vaccinated IN, SC, or IM at day -42 unless otherwise specified. Each group of 8~10 mice were vaccinated again with the same type and dose at day -12. A group of unvaccinated mice served as a control. The vaccinated groups and unvaccinated control group were challenged with 1 x 104live H99 cells via intranasal inoculation at day 0. All animals were weighed and monitored daily for disease progression, and moribund mice were euthanized. All survivors were euthanized on day 65 after challenge with live H99 cells unless otherwise specified.

[0161] Infection with Cryptococci: To prepare fungal cells for infection, overnight cultures of C. neoformans H99 were washed three times with 1 x PBS buffer and the concentration of yeast cells was determined by hemocytometer counting. The final fungal concentration was adjusted with 1 x PBS to 2 x 105cell / ml. Each mouse was infected intranasally with 1 x 104H99 cells in a 50 pl volume after being anesthetized with a mix of Ketamine (12.5 mg / mL) and Xylazine (1 mg / mL). After infection, animals were weighed daily and monitored twice daily for progression of disease, including weight loss, gait changes, labored breathing, and fur ruffling. Over the course of the experiments, animals that appeared moribund or in pain were euthanized by CO2 inhalation. Survival data from the murine experiments were statistically analyzed between paired groups using the Log Rank (Mantel-Cox) test with PRISM version 9.5 (GraphPad Software) (P values < 0.05 were considered statistically significant). The change in body weight of each animal was calculated as follows: [(weight on day X - weight on day 0) / weight on day 0] x 100%. The resulting data were plotted against time. To compare the fungal burdens, infected lungs, brains, and spleens were isolated and homogenized (Ultra-Trra T8, IKA) in 3 ml cold 1 x PBS buffer for 1 minute for each type of organ. The tissue suspensions were serially diluted and plated onto YPD agar medium with ampicillin and chloramphenicol, and colonies were counted after 3 days of incubation at 30°C.

[0162] Histopathology: Infected animals were sacrificed at the endpoint of the experiment according to the Rutgers University IACUC approved animal protocol. To compare the fungal burdens and host inflammatory responses, the lungs, brains, and spleens were dissected and fixed in 10% formalin solution for section preparation at Rutgers UniversityHistology Core Facility. Tissue slides were treated either with hematoxylin and eosin (H&E) staining for bronchus- associated lymphoid tissue or with Grocott’s methenamine silver (GMS) staining for fungal morphology observation in vivo.

[0163] Lung processing: Single cell suspensions of pulmonary cells were prepared for flow cytometric analysis as previously described. In brief, lung tissue was minced in 5 ml of 1 x PBS containing 3 mg / ml collagenase type IV (Worthington). Samples were incubated at 37°C for 45 min and washed with 1 x PBS three times. After digestion, residual RBCs were removed using RBC lysis buffer (155 mM NH4CI and 10 mM NaHCCh, PH 7.2). Lung cell suspensions were used for flow cytometry. Lungs single cell suspensions were stained for monocytes [CD45 (30-F11 BUV395), CD1 lb (MI / 70 PerCP Cy5.5) and Ly6C (AL-21 PE)], Mo-DCs [CD45 (30- F11 BUV395), CD1 lb (MI / 70 PerCP Cy5.5), Ly6C (AL-21 PE), CD11c (HL3 BV510) and MHC Class II I-A / I-E (M5 / 11.415.2 BV71 1)], neutrophils [CD45 (30-F1 1 APC-Cy7), CD1 lb (MI / 70 PerCP Cy5.5), Ly6C (AL-21 PE) and Ly6G (1A8 APC)], CD4 T cells [CD45 (30-F11 BUV395), CD4 (RM4-5 BV421)], CD8 T cells [CD45 (30-F1 1 BUV395), CD8a (53-6.7 BV711). All antibodies used for lung staining were from BD Biosciences. All samples were analyzed using BD LSRFortessa™ Flow Cytometer and FlowJo software (Tree Star, Inc).

[0164] Intracellular cytokine staining of T cells harvested in BALF and Flow Cytometry: For analyzing host immune responses, bronchoalveolar lavage fluid (BALF) samples were harvested at the endpoint after inoculation. BALF was collected in 3 ml of 1 x PBS buffer using a catheter inserted into the trachea of animal post-euthanasia, and airway-infiltrating cells were lavaged with ~ 1 mL of 1 x PBS at a time to a total volume of 5 mL. RBCs were removed using RBC lysis buffer. BALF cells were then plated in a 96-well round-bottom plate and restimulated using BD-Leukocyte Activation Cocktail containing BD GolgiPlug™ (BD Biosciences) according to the manufacturer’s instructions. Six hours after activation, BALF cells were surface-stained with fluorescently labeled antibodies against Thyl .2, CD4 and CD8. Samples were fixed in 1 % paraformaldehyde overnight. Prior to intracellular staining the samples were permeabilized with 1 x BD Perm / Wash buffer according to the manufacturer’s instructions. Intracellular cytokine staining (ICCS) was done using fluorescently labeled antibodies against IFN-y, IL-17A, TNF-a and IL13 diluted in 1 x BD Perm / Wash for 30 minutes on ice. Samples were immediately washed and analyzed by flow cytometry as described below. BALFs were cell surface stained for T cells with Thy 1.2 (53-2.1 PE-Cy7), CD4 (RM4-5 BV421) CD8a (53-6.7BV71 1) and ICCS for IFN-y (XMG1.2 PE), IL-17A (eBiol7B7 APC), TNF-a (MP6-XT22 BV711) and IL-13 (eBiol3A FITC) following standard procedures. Most antibodies and reagents for cell surface and ICCS were from BD Biosciences, except IL-17A and IL-13 which were obtained from eBioscience, Inc.

[0165] CD4+T cell isolation and CD4+T cell recall response: Antigen-presenting cells (APCs) were prepared from the spleen of syngeneic, uninfected donor mice. Splenic cell suspensions were depleted of T cells by antibody complement-mediated lysis. Splenic cells were incubated with anti -Thy 1.2 antibodies and rabbit complement (Low Tox; Cedarlane Labs) at 37 °C for 1 hour. Lung-draining lymph nodes (MLNs) were collected and placed in a 10ml of 1 x PBS. Total lymphocyte cell suspensions were prepared by gently releasing the cells into the 1 x PBS by applying pressure to the lymph nodes with the forested ends of two glass slides.Repeated pressure was applied until the tissue was reduced to the smallest size possible. Samples were collected and processed in the same way individually. For CD4 T cell isolation, individual samples from each group were pooled (5 mice). CD4+T cells were purified using a negativesorting CD4+isolation kit (Miltenyi Biotec, Inc.). CD4+T cell isolation was done following the manufacturer’s instructions and were consistently found to be > 90% pure, as assessed by flow cytometry. Purified CD4+T cells (2x105) were cultured with T cell-depleted antigen-presenting cells (APCs; 3x105) in RPMI containing 10% fetal calf serum (FCS), Penicillin-Streptomycin (2200 U / ml, Gibco™) and Gentamicin sulfate solution (1 mg / ml). The cultures were plated in flat-bottom 96-well plates and incubated at 37°C with 5% CO2 for 72 hours. To measure Cryptococcus-specific CD4+ T cells responses, CD4-antigen-presenting cell cultures were incubated with sonicated (Qsonica Sonicator Q55) H99 yeasts as a source of fungal antigens.The amount of antigen used was adjusted to a multiplicity of infection of 1 :1.5 (antigen- presenting cell :y east ratio). The fungal growth inhibitor voriconazole was used at a final concentration of 0.5 mg / ml to prevent any fungal cell outgrowth during the culture period. After 72 hours of culture at 37 °C with 5% CO2, supernatants were collected for cytokine analysis by ELISA (IL-2, IL-5, IL-17A, IFN-y ThermoFisher) following manufacturer’s instructions.Results

[0166] Figure 18 is a Scheme of a vaccination strategy as described herein. Eight week- old Balb / c mice were vaccinated twice with the HK-fbpl vaccine with or without addition of ODN 2395 adjuvant. Two weeks after the second vaccination, mice were challenged with lx 104cells of virulent wild type strain H99. Challenged mice were observed for 65 days before end the experiment, survival mice were sacrificed for detail immune analysis and fungal burden measurement.

[0167] As shown in Figure 19A-19C, combining HK-fbpl vaccine with ODN 2395 adjuvant significantly improves mice protection against WT H99 challenge. Mice vaccinated with a combination of low dose HK-fbpl and adjuvant ODN 2395 were protected from H99 challenge in an inhalation model. The low dose (IxlO7CFU) HK-fbpl vaccination, HK-fbpl alone or ODN alone did not provide protection against H99 challenge and mice die at the similar rate as non-vaccinated ones (Figure 19A). However, in the presence of 20 pg ODN 2395 adjuvant, over 70% immunized mice showed protection against H99 challengem indicating that the unique combination of HK-fbpl and ODN 2395 adjuvant significantly enhanced vaccine efficacy in this intranasal inhalation vaccination model. Figure 19B shows changes in mouse body weight after challenge infection in each group. All living mice for each group were weighed daily and presented. The protected mice showed overall weight gain following challenge infection, further confirming the mice are normal and healthy. Figure 19C shows fungal burden in the lungs, brains, and spleens of the surviving mice at the end of the experiment. The endpoint fungal burden of unvaccinated mice is shown as a control. Mice vaccinated with high dose (5xl07HK-fbpl) or low dose with ODN 2395 (lx 107HK-fbpl) survived at the end point. Fungal burden in their lungs, brains and spleens were measured. While significantly less CFUs were detected in mouse lungs, there is no CFU in spleens and brains, suggesting there was no dissemination. This result further demonstrates that the combination vaccination strategy is highly effective. Statistical analysis was done by Log-rank (Mantel-Cox) Test. *P<0.05, **P<0.01, ***P<0.0001.

[0168] As shown in Figure 20A-20C, combination vaccination also improves vaccine efficacy in a SC model. ODN 2395 adjuvanted HK-fbpl vaccine showed significant protection in an SC injection vaccination route. Following the same vaccination strategy using the subcutaneous administration route, it was found that mice vaccinated with HK-fbpl in combination of ODN 2395 were significantly protected against H99 challenge, while HK-fbpl vaccine alone or ODN 2395 adjuvant alone did not induce any protection (Figure 20A). This data indicates this novel combination vaccination strategy works in a subcutaneous route. Changes in mouse body weight after challenge infection in each group were observed (Figure20B). All living mice for each group were weighed daily and presented. The protected mice showed overall weight gain following challenge infection, further confirming the mice are normal and healthy. Fungal burden in the lungs, brains, and spleens of the surviving mice at the end of the experiment were measured (Figure 20C). The endpoint fungal burden of unvaccinated mice is shown as a control. A significant number of mice vaccinated with combination of HK- fbpl and ODN 2395 survived at the end point. Fungal burden in their lungs, brains and spleens were measured. While significantly less CFUs were detected in mouse lungs, there is no or only few CFU in spleens and brains, suggesting there was minimal dissemination. This result further demonstrates that the combination vaccination strategy is highly effective.

[0169] As shown in Figure 21A-21C, combination vaccination also improves vaccine efficacy in an IM injection model. ODN 2395 adjuvanted HK-fbpl vaccine showed significant protection in the IM injection vaccination route. Following the same vaccination strategy using the IM administration route, it was found that mice vaccinated with HK-fbpl in combination of ODN 2395 were significantly protected against H99 challenge with over 62%, while HK-fbpl vaccine alone or ODN 2395 adjuvant alone did not induce any protection. This data indicates this novel combination vaccination strategy works in the IM injection route. Changes in mouse body weight after challenge infection in each group were observed (Figure 21B). All living mice for each group were weighed daily and presented. The protected mice showed overall weight gain following challenge infection, further confirming the mice are normal and healthy. Fungal burden in the lungs, brains, and spleens of the surviving mice at the end of the experiment was measured (Figure 21C). The endpoint fungal burden of unvaccinated mice is shown as a control. A significant number of mice vaccinated with combination of HK-fbpl and ODN 2395 survived at the end point. Fungal burden in their lungs, brains and spleens were measured. While significant less CFUs were detected in mouse lungs, there is no or only few CFU in spleens and brains, suggesting there was minimal dissemination. This result further demonstrates that the combination vaccination strategy is highly effective.

[0170] As shown in Figure 22A and Figure 22B, combination vaccine strategy with another adjuvant did not show improved protection. AS03 adjuvanted HK-fbpl vaccine did not show significant protection in either the SC or IM injection vaccination route. To assess whether adding other adjuvants to HK-fbpl may also provide enhanced protection, HK-fbpl was combined with a commonly used adjuvant AS03 and tested in all three murine vaccinationmodels, intranasal inhalation model, subcutaneous injection model and intramuscular injection model. The data showed that none of these vaccination strategies provided significant protection against H99 challenge. This result indicates that the ODN 2395 combination with HK-fbpl vaccine strategy is specific and novel, not due to some non-specific adjuvant induced immunity.

[0171] As shown in Figure 23A-23C, the HK-fbpl vaccine is highly stable. To test whether the HK-fbpl vaccine is stable and safe for long term storage, the vaccine efficacy between fresh prepared HK-fbpl vaccine and the old vaccine that has been stored in -20 degree for over one year was compared. The same inhalation vaccination strategy was used for this experiment, and the data showed that both vaccines had full protection against H99 challenge. This data demonstrates that the HK-fbpl vaccine is stable and safe for long-term storage in the freezer.

[0172] As shown in Figure 24A-24C, the HK-fbpl+ODN vaccine induces increased T cell differentiation in both inhalation model and subcutaneous immunization model. At the endpoint of the vaccine experiment lungs from survival mice were isolated and both CD4+ (Fig. 24A) and CD8+ (Fig. 24B) T cell population were measured using the flow cytometry. The data showed that both CD4 and CD8 T cell populations were significantly increased in both the inhalation model and the subcutaneous vaccination model. In addition, a significant reduction of Eosinophils population was observed in the same lung tissues of different vaccination methods (Fig. 24C and Fig. 24D), indicating a suppression of detrimental Th2 response.

[0173] Overall, this data demonstrates the development of a novel vaccine combination by adding an adjuvant ODN 2395 to the HK-fbpl vaccine. This combinational vaccine showed unexpected high efficacy in multiple vaccination routes, intranasal inhalation, subcutaneous injection and intramuscular injection. This new invention significantly advances vaccine development and could speed up the potential vaccine commercialization.9. The HK-fbpl+ODN vaccine provides protection in different host backgrounds.

[0174] As shown in Figure 25, the HK-fbpl+ODN vaccine provided protection in different host backgrounds. The vaccine efficacy of this combination vaccine (ODN2395 + HK- fbpl) was compared in both Balb / c and C57BL / 6J mouse backgrounds in the IM injection vaccination model. The data in Figure 25 shows that while Balb / c mice immunized with thecombination vaccine had -75% protection, the C57BL / 6j mice had full protection. At least ten mice for each group were used for the experiment. “****” P<0.0001. This data indicates that the ODN adjuvanted vaccine (combination vaccine) provides protection in different host backgrounds.10. The HK-fbpl+ODN vaccine provides protection in hosts at different ages.

[0175] As shown in Figure 26, the HK-fbpl+ODN vaccine provided protection in hosts at different ages. The combination IM vaccination strategy was tested in both young mice (8 weeks old) and old mice (6-8 months old) to compare their potential protection. At least ten mice for each group were used for the experiment. “****” PO.OOOl. The data of Figure 26 shows that both mice with different ages immunized IM by the combination vaccine were fully protected in the C57BL / 6J mouse background.10. The HK-fbpl+ODN vaccine-protected mice display pro-inflammatory responses against virulent H99 challenge.

[0176] As shown in Figure 27A-27G, the HK-fbpl+ODN vaccine-protected mice displayed pro-inflammatory responses against virulent H99 challenge. Balb / c mice were vaccinated IM and challenged with lx 10A4 H99 CFU following the IM vaccination strategy. At the endpoint of the vaccination experiment, cytokine producing CD4 T cells were isolated from BALF and the production of cytokines such as IFN-g, TNF-a and IL-17A were determined by antibody staining and measured in a Flow cytometry. The data of Figure 27 show that in comparison with unvaccinated mice, the vaccinated mice produced significantly increased pro- inflammatory cytokines, e.g., IFN-y, TNF-a and IL-17A (Figure 27A-27C). A T cell recall experiment also showed that the production of IL-2, IL-17A and IFN-y significantly increased in an ELISA assay (Fig. 27D-27G).

[0177] The foregoing examples and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the scope of the invention, and all such variationsare intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entireties.

Claims

CLAIMS1. A pharmaceutical composition comprising: i) inactivated F-box protein 1 deletionmutant Cryptococcus neoformans cells, wherein the inactivated fttplA mutant cells are heat-killed, and ii) adjuvant ODN 2395.

2. The pharmaceutical composition of claim 1 , further comprising a pharmaceutically acceptable carrier.

3. The pharmaceutical composition of claim 1, wherein the fbplA is a partial deletion.

4. The pharmaceutical composition of claim 3, wherein the partial deletion is of an F-box domain.

5. The pharmaceutical composition of claim 3, wherein the partial deletion occurs by homologous recombination.

6. The pharmaceutical composition of claim 1, wherein the fbplA is a full deletion.

7. The pharmaceutical composition of claim 1, wherein the occurs by homologousrecombination.

8. A method of treating a fungal infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 1.

9. The method of claim 8, further comprising administering at least one antifungal agent to the subject.

10. The method of claim 8, wherein the subject is a human.

11. The method of claim 8, wherein the pharmaceutical composition is administered at two timepoints.

12. The method of claim 8, wherein the pharmaceutical composition is administered by intranasal inhalation.

13. The method of claim 8, wherein the pharmaceutical composition is administered by subcutaneous injection.

14. The method of claim 8, wherein the pharmaceutical composition is administered by intramuscular injection.

15. The method of claim 8, wherein the fungal infection comprises an infection with a virulent strain of C. neoformans.

16. The method of claim 8, wherein the fungal infection comprises an infection with C. albicans, A. fumigatus, or C. gattii.

17. The method of claim 8, wherein the subject is an immunocompromised subject.

18. The method of claim 17, wherein the immunocompromised subject is infected with human immunodeficiency virus (HIV).

19. The method of claim 8, wherein the JbplA is a full deletion.

20. The method of claim 8, wherein the JbpA is a partial deletion.

21. The method of claim 20, wherein the partial deletion is of an F-box domain.

22. The method of claim 20, wherein the partial deletion occurs by homologous recombination.

23. The method of claim 8, wherein the JbpA occurs by homologous recombination.

24. The method of claim 8, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

25. The pharmaceutical composition of claim 1, wherein adjuvant ODN 2395 comprises the sequence of SEQ ID NO: 50.

26. The pharmaceutical composition of claim 1, wherein adjuvant ODN 2395 consists essentially of the sequence of SEQ ID NO: 50.

27. The pharmaceutical composition of claim 1, wherein adjuvant ODN 2395 consists of the sequence of SEQ ID NO: 50.

28. The method of claim 8, wherein adjuvant ODN 2395 comprises the sequence of SEQ ID NO: 50.

29. The method of claim 8, wherein adjuvant ODN 2395 consists essentially of the sequence of SEQ ID NO: 50.

30. The method of claim 8, wherein adjuvant ODN 2395 consists of the sequence of SEQ IDNO: 50.

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