Development of a vaccine to protect against cryptococcosis is a priority given the enormous global burden of disease in at-risk individuals. Using glucan particles (GPs) as a delivery system, we previously demonstrated that mice vaccinated with crude Cryptococcus-derived alkaline extracts were protected against lethal challenge with Cryptococcus neoformans and Cryptococcus gattii. The goal of the present study was to identify protective protein antigens that could be used in a subunit vaccine. Using biased and unbiased approaches, six candidate antigens (Cda1, Cda2, Cda3, Fpd1, MP88, and Sod1) were selected, recombinantly expressed in Escherichia coli, purified, and loaded into GPs. Three mouse strains (C57BL/6, BALB/c, and DR4) were then vaccinated with the antigen-laden GPs, following which they received a pulmonary challenge with virulent C. neoformans and C. gattii strains. Four candidate vaccines (GP-Cda1, GP-Cda2, GP-Cda3, and GP-Sod1) afforded a significant survival advantage in at least one mouse model; some vaccine combinations provided added protection over that seen with either antigen alone. Vaccine-mediated protection against C. neoformans did not necessarily predict protection against C. gattii. Vaccinated mice developed pulmonary inflammatory responses that effectively contained the infection; many surviving mice developed sterilizing immunity. Predicted T helper cell epitopes differed between mouse strains and in the degree to which they matched epitopes predicted in humans. Thus, we have discovered cryptococcal proteins that make promising candidate vaccine antigens. Protection varied depending on the mouse strain and cryptococcal species, suggesting that a successful human subunit vaccine will need to contain multiple antigens, including ones that are species specific.

Heart rate (HR), hematocrit, hemoglobin, blood glucose, and plasma concentrations of lactate, cortisol, and testosterone were monitored in 10 male subjects (Division I, 20.3 +/- 2.5 yrs, VO2max: 58.5 +/- 9.4 ml.kg-1.min-1) during singles tennis and a treadmill test. During the on-court session, HR was 144.6 +/- 13.2 beats.min-1 for the 85 min of play. Plasma lactate rose 50% from a post-warmup value of 1.6 +/- 0.6 mmol.l-1 to 2.3 +/- 1.2 mmol.l-1 during play (p greater than 0.05). Blood glucose slightly decreased (8%, p greater than 0.05) from a pre-exercise value of 4.6 +/- 0.8 mmol.l-1 as a result of the 10-min warmup. This was followed by a 23% rise (p less than 0.05) from 4.2 +/- 1.0 mmol.l-1 to 5.2 +/- 0.6 mmol.l-1, measured after the first 30 min of play. Blood glucose subsequently remained steady at slightly above the pre-exercise value. Plasma cortisol rose (9%, p greater than 0.05) during the warmup and subsequently decreased (p less than 0.05) from a post-warmup value of 558.2 +/- 285.2 nmol.l-1 to 337.1 +/- 173.3 nmol.l-1 (a 40% decrease), and remained decreased during recovery. Plasma testosterone rose 22% (p less than 0.05) from pre-exercise to recovery (13.5 +/- 3.8 nmol.l-1 and 16.5 +/- 2.6 nmol.l-1, respectively). Although tennis is characterized by periods of high-intensity exercise, the overall metabolic response resembles prolonged moderate-intensity exercise.

Developing an effective and safe recombinant vaccine requires microbe-specific antigens combined with an adjuvant/delivery system to strengthen protective immunity. In this study, we designed and expressed a multivalent, recombinant polypeptide antigen (rCpa1) that consists of three previously identified antigens (i.e., Ag2/Pra, Cs-Ag and Pmp1) and 5 pathogen-derived peptides with high affinity for human MHC II molecules. The purified rCpa1 was encapsulated into four types of yeast cell-wall particles containing various compositions of β-glucan, mannan and chitin or mixed with an oligonucleotide (ODN) containing 2 methylated dinucleotide CpG motifs. This multivalent antigen encapsulated into glucan-chitin particles (GCP-rCpa1) showed a significantly elevated reduction of fungal burden for human HLA-DR4 transgenic mice compared to the other tested adjuvant-rCpa1 formulations. Among the tested adjuvants GCPs and GPs were both capable of stimulating a mixed Th1 and Th17 response. Mice vaccinated with GCP-rCpa1 showed elevated IL-17 production in T-cell recall assays and early lung infiltration of activated Th1 and Th17 cells compared to GP-rCpa1-vaccinated mice. Both C57BL/6 and HLA-DR4 transgenic mice that were vaccinated with GCP-rCpa1 vaccine increased surivial compared to the mice received GCPs alone. Concurringly, GCP-rCpa1 vaccine stimulated enhanced infiltration of macrophages to engulf and process the vaccine for antigen presentation in the injection sites compared to GP-rCpa1 injection. This is the first attempt to systematically characterize the presentation of a multivalent coccidioidomycosis vaccine encapsulated with selected adjuvants which enhance protective cellular immune response to infection.

Meningitis due to Cryptococcus neoformans is responsible for upwards of 180,000 deaths worldwide annually, mostly in immunocompromised individuals. Currently there are no licensed fungal vaccines, and even with anti-fungal drug treatment, cryptococcal meningitis is often fatal. Our lab previously demonstrated vaccination with recombinant cryptococcal proteins delivered in glucan particles (GPs) protects mice against an otherwise lethal infection. The aim of the present study was to discover additional cryptococcal antigens affording vaccine-mediated protection. Sixteen proteins, each with evidence of extracellularity, were selected for in vivo testing based on their abundance in protective alkaline extracts of an acapsular C. neoformans strain, their known immunogenicity, and/or their high transcript level during human infection. Candidate antigens were recombinantly expressed in E. coli, purified and loaded into GPs. BALB/c and C57BL/6 mice received three subcutaneous injections of GP-based vaccine, and survival was assessed for 84 days following a lethal orotracheal challenge with strain KN99. As with our six published GPvaccines, we saw differences in overall protection between mouse strains such that BALB/c mice typically demonstrated better survival than C57BL/6 mice. From these studies, we identified seven

Hookworms are intestinal nematode parasites that infect nearly half a billion people and are globally one of the most important contributors to iron-deficiency anemia. These parasites have significant impacts in developing children, pregnant women and working adults. Of all the soil-transmitted helminths or nematodes (STNs), hookworms are by far the most important, with disease burdens conservatively estimated at four million DALYs (Disability-Adjusted Life Years) and with productivity losses of up to US$139 billion annually. To date, mainly one drug, albendazole is used for hookworm therapy in mass drug administration, which has on average ∼80% cure rate that is lower (<40%) in some places. Given the massive numbers of people needing treatment, the threat of parasite resistance, and the inadequacy of current treatments, new and better cures against hookworms are urgently needed. Cry5B, a pore-forming protein produced by the soil bacterium Bacillus thuringiensis (Bt) has demonstrated good efficacy against Ancylostoma ceylanicum hookworm infections in hamsters. Here we broaden studies of Cry5B to include tests against infections of Ancylostoma caninum hookworms in dogs and against infections of the dominant human hookworm, Necator americanus, in hamsters. We show that Cry5B is highly effective against all hookworm parasites tested in all models. Neutralization of stomach acid improves Cry5B efficacy, which will aid in practical application of Cry5B significantly. Importantly, we also demonstrate that the anti-nematode therapeutic efficacy of Cry5B is independent of the host immune system and is not itself negated by repeated dosing. This study indicates that Bt Cry5B is a pan-hookworm anthelmintic with excellent properties for use in humans and other animals.