A glucan-particle based tularemia subunit vaccine induces T-cell immunity and affords partial protection in an inhalation rat infection model.

Tularemia is a zoonotic disease caused by the facultative intracellular gram-negative bacterium Francisella tularensis. F. tularensis has a very low infection dose by the aerosol route which can result in an acute, and potentially lethal, infection in humans. Consequently, it is classified as a Category A bioterrorism agent by the US Centers for Disease Control (CDC) and is a pathogen of concern for the International Biodefence community. There are currently no licenced tularemia vaccines. In this study we report on the continued assessment of a tularemia subunit vaccine utilising ß-glucan particles (GPs) as a vaccine delivery platform for immunogenic F. tularensis antigens. Using a Fischer 344 rat infection model, we demonstrate that a GP based vaccine comprising the F. tularensis lipopolysaccharide antigen together with the protein antigen FTT0814 provided partial protection of F344 rats against an aerosol challenge with a high virulence strain of F. tularensis, SCHU S4. Inclusion of imiquimod as an adjuvant failed to enhance protective efficacy. Moreover, the level of protection afforded was dependant on the challenge dose. Immunological characterisation of this vaccine demonstrated that it induced strong antibody immunoglobulin responses to both polysaccharide and protein antigens. Furthermore, we demonstrate that the FTT0814 component of the GP vaccine primed CD4+ and CD8+ T-cells from immunised F344 rats to express interferon-γ, and CD4+ cells to express interleukin-17, in an antigen specific manner. These data demonstrate the development potential of this tularemia subunit vaccine and builds on a body of work highlighting GPs as a promising vaccine platform for difficult to treat pathogens including those of concern to the bio-defence community.

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

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

Efficient and Scalable Process to Produce Novel and Highly Bioactive Purified Cytosolic Crystals from Bacillus thuringiensis.

Bacillus thuringiensis (Bt) is a Gram-positive soil bacterium that is widely and safely applied in the environment as an insecticide for combatting insect pests that damage crops or are disease vectors. Dominant active ingredients made by Bt are insect-killing crystal (Cry) proteins released as crystalline inclusions upon bacterial sporulation. Some Bt Cry proteins, e.g., Cry5B (formally Cry5Ba1), target nematodes (roundworms) and show exceptional promise as anthelmintics (cures for parasitic nematode diseases). We have recently described inactivated bacteria with cytosolic crystal(s) (IBaCC) in which bioactive Bt Cry crystals (containing Cry5B) are fully contained within the cytosol of dead bacterial ghosts. Here, we demonstrate that these IBaCC-trapped Cry5B crystals can be liberated and purified away from cellular constituents, yielding purified cytosolic crystals (PCC). Cry5B PCC contains ~95% Cry5B protein out of the total protein content. Cry5B PCC is highly bioactive against parasitic nematode larvae and adults in vitro. Cry5B PCC is also highly active in vivo against experimental human hookworm and Ascaris infections in rodents. The process was scaled up to the 100-liter scale to produce PCC for a pilot study to treat two foals infected with the ascarid Parascaris spp. Single-dose Cry5B PCC brought the fecal egg counts of both foals to zero. These studies describe the process for the scalable production of purified Bt crystals and define a new and attractive pharmaceutical ingredient form of Bt Cry proteins. IMPORTANCE Bacillus thuringiensis crystal proteins are widely and safely used as insecticides. Recent studies have shown they also can cure gastrointestinal parasitic worm (nematode) infections when ingested. However, reproducible, scalable, and practical techniques for purifying these proteins have been lacking. Here, we address this severe limitation and present scalable and practical methods for large-scale purification of potently bioactive B. thuringiensis crystals and crystal proteins. The resultant product, called purified cytosolic crystals (PCC), is highly compatible with ingestible drug delivery and formulation. Furthermore, there are growing applications in agriculture and insect control where access to large quantities of purified crystal proteins is desirable and where these methods will find great utility.

Protection of Mice against Experimental Cryptococcosis by Synthesized Peptides Delivered in Glucan Particles.

The high global burden of cryptococcosis has made development of a protective vaccine a public health priority. We previously demonstrated that a vaccine composed of recombinant Cryptococcus neoformans chitin deacetylase 2 (Cda2) delivered in glucan particles (GPs) protects BALB/c and C57BL/6 mice from an otherwise lethal challenge with a highly virulent C. neoformans strain. An immunoinformatic analysis of Cda2 revealed a peptide sequence predicted to have strong binding to the major histocompatibility complex class II (MHC II) H2-IAd allele found in BALB/c mice. BALB/c mice vaccinated with GPs containing a 32-amino-acid peptide (Cda2-Pep1) that included this strong binding region were protected from cryptococcosis. Protection was lost with GP-based vaccines containing versions of recombinant Cda2 protein and Cda2-Pep1 with mutations predicted to greatly diminish MHC II binding. Cda2 has homology to the three other C. neoformans chitin deacetylases, Cda1, Cda3, and Fpd1, in the high-MHC II-binding region. GPs loaded with homologous peptides of Cda1, Cda3, and Fpd1 protected BALB/c mice from experimental cryptococcosis, albeit not as robustly as the Cda2-Pep1 vaccine. Finally, seven other peptides were synthesized based on regions in Cda2 predicted to contain promising CD4+ T cell epitopes in BALB/c or C57BL/6 mice. While five peptide vaccines significantly protected BALB/c mice, only one protected C57BL/6 mice. Thus, GP-based vaccines containing a single peptide can protect mice against cryptococcosis. However, given the diversity of human MHC II alleles, a peptide-based Cryptococcus vaccine for use in humans would be challenging and likely need to contain multiple peptide sequences. IMPORTANCE Cryptococcosis, due to infection by fungi of the Cryptococcus neoformans species complex, is responsible for substantial morbidity and mortality in immunocompromised persons, particularly those with AIDS. Cryptococcal vaccines are a public health priority yet are not available for human use. We previously demonstrated mice could be protected from experimental cryptococcosis with vaccines composed of recombinant cryptococcal proteins encased in hollow highly purified yeast cell walls (glucan particles). In this study, we examined one such protective protein, Cda2, and using bioinformatics, we identified a region predicted to stimulate strong T cell responses. A peptide containing this region formulated in glucan particle-based vaccines protected mice as well as the recombinant protein. Other peptide vaccines also protected, including peptides containing sequences from proteins homologous to Cda2. These preclinical mouse studies provide a proof of principle that peptides can be effective as vaccines to protect against cryptococcosis and that bioinformatic approaches can guide peptide selection.

Recombinant Paraprobiotics as a New Paradigm for Treating Gastrointestinal Nematode Parasites of Humans.

Gastrointestinal nematodes (GINs) of humans, e.g., hookworms, negatively impact childhood growth, cognition, nutrition, educational attainment, income, productivity, and pregnancy. Hundreds of millions of people are targeted with mass drug administration (MDA) of donated benzimidazole anthelmintics. However, benzimidazole efficacy against GINs is suboptimal, and reduced/low efficacy has been seen. Developing an anthelmintic for human MDA is daunting: it must be safe, effective, inexpensive, stable without a cold chain, and massively scalable. Bacillus thuringiensis crystal protein 5B (Cry5B) has anthelmintic properties that could fill this void. Here, we developed an active pharmaceutical ingredient (API) containing B. thuringiensis Cry5B compatible with MDA. We expressed Cry5B in asporogenous B. thuringiensis during vegetative phase, forming cytosolic crystals. These bacteria with cytosolic crystals (BaCC) were rendered inviable (inactivated BaCC [IBaCC]) with food-grade essential oils. IBaCC potency was validated in vitro against nematodes. IBaCC was also potent in vivo against human hookworm infections in hamsters. IBaCC production was successfully scaled to 350 liters at a contract manufacturing facility. A simple fit-for-purpose formulation to protect against stomach digestion and powdered IBaCC were successfully made and used against GINs in hamsters and mice. A pilot histopathology study and blood chemistry workup showed that five daily consecutive doses of 200 mg/kg body weight Cry5B IBaCC (the curative single dose is 40 mg/kg) was nontoxic to hamsters and completely safe. IBaCC is a safe, inexpensive, highly effective, easy-to-manufacture, and scalable anthelmintic that is practical for MDA and represents a new paradigm for treating human GINs.

A new paraprobiotic-based treatment for control of Haemonchus contortus in sheep.

Haemonchus contortus is a critical parasite of goats and sheep. Infection by this blood-feeding gastrointestinal nematode (GIN) parasite has significant health consequences, especially in lambs and kids. The parasite has developed resistance to virtually all known classes of small molecule anthelmintics used to treat it, giving rise in some areas to multidrug resistant parasites that are very difficult to control. Thus, new anthelmintics are urgently needed. Bacillus thuringiensis (Bt) crystal protein 5B (Cry5B), a naturally occurring protein made by a bacterium widely and safely used around the world as a bioinsecticide, represents a new non-small molecule modality for treating GINs. Cry5B has demonstrated anthelmintic activities against parasites of monogastric animals, including some related to those that infect humans, but has not yet been studied in a ruminant. Here we show that H. contortus adults are susceptible to Cry5B protein in vitro. Cry5B produced in its natural form as a spore-crystal lysate against H. contortus infections in goats had no significant efficacy. However, a new Active Pharmaceutical Ingredient (API) paraprobiotic form of Cry5B called IBaCC (Inactivated Bacterium with Cytosolic Crystals), in which Cry5B crystals are encapsulated in dead Bt cell wall ghosts, showed excellent efficacy in vitro against larval stages of H. contortus and relative protein stability in bovine rumen fluid. When given to sheep experimentally infected with H. contortus as three 60 mg/kg doses, Cry5B IBaCC resulted in significant reductions in fecal egg counts (90%) and parasite burdens (72%), with a very high impact on female parasites (96% reduction). These data indicate that Cry5B IBaCC is a potent new treatment tool for small ruminants in the battle against H. contortus.

Antigen discovery unveils resident memory and migratory cell roles in antifungal resistance.

Priming at the site of natural infection typically elicits a protective T cell response against subsequent pathogen encounter. Here, we report the identification of a novel fungal antigen that we harnessed for mucosal vaccination and tetramer generation to test whether we can elicit protective, antigen-specific tissue-resident memory (Trm) CD4+ T cells in the lung parenchyma. In contrast to expectations, CD69+, CXCR3+, CD103- Trm cells failed to protect against a lethal pulmonary fungal infection. Surprisingly, systemic vaccination induced a population of tetramer+ CD4+ T cells enriched within the pulmonary vasculature, and expressing CXCR3 and CX3CR1, that migrated to the lung tissue upon challenge and efficiently protected mice against infection. Mucosal vaccine priming of Trm may not reliably protect against mucosal pathogens.

Protection of mice against experimental cryptococcosis using glucan particle-based vaccines containing novel recombinant antigens.

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 GP-vaccines, 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 new proteins which, when administered as GP-vaccines, protect BALB/c and/or C57BL/6 mice against cryptococcal infection. With these results, we expand the pool of novel protective antigens to eleven proteins and demonstrate the potential for selection of highly transcribed extracellular proteins as vaccine targets. These screens highlight the efficacy of GP-subunit vaccines and identify promising antigens for further testing in anti-cryptococcal, multi-epitope vaccine formulations.

Bacillus thuringiensis Cry5B is Active against Strongyloides stercoralis in vitro.

Strongyloidiasis, caused by Strongyloides stercoralis infection, is an important neglected tropical disease that causes significant public health problems in the tropics and subtropics. The disease can persist in hosts for decades and may be life-threatening because of hyperinfection and dissemination. Ivermectin (mostly) and albendazole are the most common anthelmintics used for treatment. Albendazole is suboptimal for this parasite, and although ivermectin is quite effective in immunocompromised patients, a multiple-course regimen is required. Furthermore, reliance on a single drug class for treating intestinal nematodes is a recipe for future failure. Therefore, it is important to discover new anthelmintics to treat or prevent human strongyloidiasis. One promising candidate is the Bacillus thuringiensis crystal protein Cry5B. Cry5B is highly potent against parasitic nematodes, for example, hookworms and Ascaris suum. Here, we investigated the potential of Cry5B against S. stercoralis. Multiple stages of S. stercoralis, including the first larval stage (L1s), infective stage (iL3s), free-living adult stage, and parasitic female stage, were all susceptible to Cry5B as indicated by impairment of motility and decreased viability in vitro. In summary, Cry5B demonstrated strong potential as an effective anthelmintic for treatment and transmission control of human strongyloidiasis, justifying further experiments to investigate in vivo therapeutic efficacy.

Maize-Produced Ag2 as a Subunit Vaccine for Valley Fever.

Coccidioides is the causative agent of San Joaquin Valley fever, a fungal disease prevalent in the semiarid regions of the Americas. Efforts to develop a fungal vaccine over the last 2 decades were unsuccessful. A candidate antigen, Antigen 2 (Ag2), is notoriously difficult to express in Escherichia coli, and this study sought to accumulate the antigen at high levels in maize. Transformed maize lines accumulated recombinant Ag2 at levels >1 g/kg. Mice immunized with this antigen and challenged with live Coccidioides arthroconidia showed a reduction in the fungal load when Ag2 derived from either E. coli or maize was loaded into glucan chitin particles. A fusion of Ag2 to dendritic cell carrier peptide (DCpep) induced a T-helper type 17 response in the spleen when orally delivered, indicative of a protective immune response. The maize production platform and the glucan chitin particle adjuvant system show promise for development of a Coccidioides vaccine, but further testing is needed to fully assess the optimal method of administration.

Protection induced by a Francisella tularensis subunit vaccine delivered by glucan particles.

Francisella tularensis is an intracellular pathogen causing the disease tularemia, and an organism of concern to biodefence. There is no licensed vaccine available. Subunit approaches have failed to induce protection, which requires both humoral and cellular immune memory responses, and have been hampered by a lack of understanding as to which antigens are immunoprotective. We undertook a preliminary in silico analysis to identify candidate protein antigens. These antigens were then recombinantly expressed and encapsulated into glucan particles (GPs), purified Saccharomyces cerevisiae cell walls composed primarily of ß-1,3-glucans. Immunological profiling in the mouse was used to down-selection to seven lead antigens: FTT1043 (Mip), IglC, FTT0814, FTT0438, FTT0071 (GltA), FTT0289, FTT0890 (PilA) prior to transitioning their evaluation to a Fischer 344 rat model for efficacy evaluation. F344 rats were vaccinated with the GP protein antigens co-delivered with GP-loaded with Francisella LPS. Measurement of cell mediated immune responses and computational epitope analysis allowed down-selection to three promising candidates: FTT0438, FTT1043 and FTT0814. Of these, a GP vaccine delivering Francisella LPS and the FTT0814 protein was able to induce protection in rats against an aerosol challenge of F. tularensis SchuS4, and reduced organ colonisation and clinical signs below that which immunisation with a GP-LPS alone vaccine provided. This is the first report of a protein supplementing protection induced by LPS in a Francisella vaccine. This paves the way for developing an effective, safe subunit vaccine for the prevention of inhalational tularemia, and validates the GP platform for vaccine delivery where complex immune responses are required for prevention of infections by intracellular pathogens.

Glucan-Chitin Particles Enhance Th17 Response and Improve Protective Efficacy of a Multivalent Antigen (rCpa1) against Pulmonary Coccidioides posadasii Infection.

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 Coccidioides polypeptide antigen (rCpa1) that consists of three previously identified antigens (i.e., Ag2/Pra, Cs-Ag, and Pmp1) and five pathogen-derived peptides with high affinity for human major histocompatibility complex class II (MHC-II) molecules. The purified rCpa1 was encapsulated into four types of yeast cell wall particles containing ß-glucan, mannan, and chitin in various proportions or was mixed with an oligonucleotide (ODN) containing two methylated dinucleotide CpG motifs. This multivalent antigen encapsulated into glucan-chitin particles (GCP-rCpa1) showed significantly greater reduction of fungal burden for human HLA-DR4 transgenic mice than the other adjuvant-rCpa1 formulations tested. Among the adjuvants tested, both GCPs and ß-glucan particles (GPs) were capable of stimulating a mixed Th1 and Th17 response. Mice vaccinated with GCP-rCpa1 showed higher levels of interleukin 17 (IL-17) production in T-cell recall assays and earlier lung infiltration by activated Th1 and Th17 cells than GP-rCpa1-vaccinated mice. Both C57BL/6 and HLA-DR4 transgenic mice that were vaccinated with the GCP-rCpa1 vaccine showed higher survival rates than mice that received GCPs alone. Concurrently, the GCP-rCpa1 vaccine stimulated greater infiltration of the injection sites by macrophages, which engulf and process the vaccine for antigen presentation, than the GP-rCpa1 vaccine. This is the first attempt to systematically characterize the presentation of a multivalent coccidioidomycosis vaccine encapsulated with selected adjuvants that enhance the protective cellular immune response to infection.

Bacillus thuringiensis Cry5B protein as a new pan-hookworm cure.

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.

Vaccination with an alkaline extract of Histoplasma capsulatum packaged in glucan particles confers protective immunity in mice.

Infection with the dimorphic fungus, Histoplasma capsulatum, occurs world-wide, but North and South America are regions of high endemicity. Interventions to mitigate exposure and consequent disease are limited to remediating a habitat harboring the fungus. The development of a vaccine to prevent infection or lessen its severity is an important advance in disease prevention. Accordingly, we prepared an alkaline extract from the yeast phase of Histoplasma and encased it in glucan particles that act as an adjuvant and delivery vehicle. Immunization of C57BL/6 mice with this encapsulated extract decreased the number of CFUs in lungs and spleens at days 7 and 14 following intranasal infection. Moreover, this vaccine conferred protection against a lethal challenge with the fungus. Cytokine assessment in lungs at a time when the CFUs were similar between controls and vaccinated groups revealed increased quantities of interferon-γ and interleukin-17 in vaccine recipients. This finding was supported by increased generation of both Th1 and Th17 cells in lungs and draining lymph nodes of vaccinated mice compared to controls. Neutralization of interferon-γ or interleukin-17 blunted the effectiveness of vaccination. To identify the proteins comprising this extract, liquid chromatography tandem mass spectrometry was performed. Thus, an H. capsulatum alkaline extract packaged in glucan particles confers protection in an interferon-γ and interleukin-17-dependent manner. Discovery of a single protein or a few proteins in this admixture that mediate protective immunity would represent significant progress in efforts to prevent histoplasmosis.

Vaccination with Recombinant Cryptococcus Proteins in Glucan Particles Protects Mice against Cryptococcosis in a Manner Dependent upon Mouse Strain and Cryptococcal Species.

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.IMPORTANCE The encapsulated fungi Cryptococcus neoformans and Cryptococcus gattii are responsible for nearly 200,000 deaths annually, mostly in immunocompromised individuals. An effective vaccine could substantially reduce the burden of cryptococcosis. However, a major gap in cryptococcal vaccine development has been the discovery of protective antigens to use in vaccines. Here, six cryptococcal proteins with potential as vaccine antigens were expressed recombinantly and purified. Mice were then vaccinated with glucan particle preparations containing each antigen. Of the six candidate vaccines, four protected mice from a lethal cryptococcal challenge. However, the degree of protection varied as a function of mouse strain and cryptococcal species. These preclinical studies identify cryptococcal proteins that could serve as candidate vaccine antigens and provide a proof of principle regarding the feasibility of protein antigen-based vaccines to protect against cryptococcosis.

A novel viral RNA helicase with an independent translation enhancement activity.

RNA helicases have not been identified among negative sense RNA viruses. In this study, it is shown that Nonstructural protein (NSs) of Groundnut bud necrosis virus (GBNV) acts as a Mg(2+) - and ATP-dependent bipolar RNA helicase. Biophysical and biochemical analysis of the deletion mutants (NΔ124 NSs, CΔ80 NSs) revealed that both the N- and C-terminal residues are required for substrate binding, oligomerization and helicase activity, but are dispensable for ATPase activity. Interestingly, NSs could enhance the translation of RNA (~ 10-fold) independent of its helicase activity. This is the first report of a RNA helicase from negative strand RNA viruses.

Intracellular delivery of antibodies by chimeric Sesbania mosaic virus (SeMV) virus like particles.

The therapeutic potential of antibodies has not been fully exploited as they fail to cross cell membrane. In this article, we have tested the possibility of using plant virus based nanoparticles for intracellular delivery of antibodies. For this purpose, Sesbania mosaic virus coat protein (CP) was genetically engineered with the B domain of Staphylococcus aureus protein A (SpA) at the ßH-ßI loop, to generate SeMV loop B (SLB), which self-assembled to virus like particles (VLPs) with 43 times higher affinity towards antibodies. CP and SLB could internalize into various types of mammalian cells and SLB could efficiently deliver three different monoclonal antibodies-D6F10 (targeting abrin), anti-α-tubulin (targeting intracellular tubulin) and Herclon (against HER2 receptor) inside the cells. Such a mode of delivery was much more effective than antibodies alone treatment. These results highlight the potential of SLB as a universal nanocarrier for intracellular delivery of antibodies.

Structural studies on chimeric Sesbania mosaic virus coat protein: Revisiting SeMV assembly.

The capsid protein (CP) of Sesbania mosaic virus (SeMV, a T=3 plant virus) consists of a disordered N-terminal R-domain and an ordered S-domain. Removal of the R-domain results in the formation of T=1 particles. In the current study, the R-domain was replaced with unrelated polypeptides of similar lengths: the B-domain of Staphylococcus aureus SpA, and SeMV encoded polypeptides P8 and P10. The chimeric proteins contained T=3 or larger virus-like particles (VLPs) and could not be crystallized. The presence of metal ions during purification resulted in a large number of heterogeneous nucleoprotein complexes. N∆65-B (R domain replaced with B domain) could also be purified in a dimeric form. Its crystal structure revealed T=1 particles devoid of metal ions and the B-domain was disordered. However, the B-domain was functional in N∆65-B VLPs, suggesting possible biotechnological applications. These studies illustrate the importance of N-terminal residues, metal ions and robustness of the assembly process.

Demonstration of helicase activity in the nonstructural protein, NSs, of the negative-sense RNA virus, groundnut bud necrosis virus.

The nonstructural protein NSs, encoded by the S RNA of groundnut bud necrosis virus (GBNV) (genus Tospovirus, family Bunyaviridae) has earlier been shown to possess nucleic-acid-stimulated NTPase and 5' α phosphatase activity. ATP hydrolysis is an essential function of a true helicase. Therefore, NSs was tested for DNA helicase activity. The results demonstrated that GBNV NSs possesses bidirectional DNA helicase activity. An alanine mutation in the Walker A motif (K189A rNSs) decreased DNA helicase activity substantially, whereas a mutation in the Walker B motif resulted in a marginal decrease in this activity. The parallel loss of the helicase and ATPase activity in the K189A mutant confirms that NSs acts as a non-canonical DNA helicase. Furthermore, both the wild-type and K189A NSs could function as RNA silencing suppressors, demonstrating that the suppressor activity of NSs is independent of its helicase or ATPase activity. This is the first report of a true helicase from a negative-sense RNA virus.