Chitosan-Deficient Cryptococcus as Whole-Cell Vaccines.

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

Epitope-Based Immunoinformatic Approach on Heat Shock 70 kDa Protein Complex of Cryptococcus neoformans var. grubii.

INTRODUCTION: Cryptococcosis is a ubiquitous opportunistic fungal disease caused by Cryptococcus neoformans var. grubii. It has high global morbidity and mortality among HIV patients and non-HIV carriers with 99% and 95%, respectively. Furthermore, the increasing prevalence of undesired toxicity profile of antifungal, multidrug-resistant organisms and the scarcity of FDA-authorized vaccines were the hallmark in the present days. This study was undertaken to design a reliable epitope-based peptide vaccine through targeting highly conserved immunodominant heat shock 70 kDa protein of Cryptococcus neoformans var. grubii that covers a considerable digit of the world population through implementing a computational vaccinology approach. MATERIALS AND METHODS: A total of 38 sequences of Cryptococcus neoformans var. grubii's heat shock 70 kDa protein were retrieved from the NCBI protein database. Different prediction tools were used to analyze the aforementioned protein at the Immune Epitope Database (IEDB) to discriminate the most promising T-cell and B-cell epitopes. The proposed T-cell epitopes were subjected to the population coverage analysis tool to compute the global population's coverage. Finally, the T-cell projected epitopes were ranked based on their binding scores and modes using AutoDock Vina software. Results and Discussion. The epitopes (ANYVQASEK, QSEKPKNVNPVI, SEKPKNVNPVI, and EKPKNVNPVI) had shown very strong binding affinity and immunogenic properties to B-cell. (FTQLVAAYL, YVYDTRGKL) and (FFGGKVLNF, FINAQLVDV, and FDYALVQHF) exhibited a very strong binding affinity to MHC-I and MHC-II, respectively, with high population coverage for each, while FYRQGAFEL has shown promising results in terms of its binding profile to MHC-II and MHC-I alleles and good strength of binding when docked with HLA-C∗12:03. In addition, there is massive global population coverage in the three coverage modes. Accordingly, our in silico vaccine is expected to be the future epitope-based peptide vaccine against Cryptococcus neoformans var. grubii that covers a significant figure of the entire world citizens.

Combination Adjuvants Enhance Recombinant Protein Vaccine Protection against Fungal Infection.

The development of effective vaccines against fungal infections requires the induction of protective, pathogen-specific cell-mediated immune responses. Here, we asked whether combination adjuvants based on delta inulin (Advax) formulated with Toll-like receptor (TLR) agonists could improve vaccine protection mediated by a fungal recombinant protein, Bl-Eng2 (i.e., Blastomyces endoglucanase 2), which itself harbors an immunodominant antigen and dectin-2 agonist/adjuvant. We found that Bl-Eng2 formulated with Advax3 containing TLR9 agonist or Advax8 containing TLR4 agonist provided the best protection against pulmonary infection with Blastomyces dermatitidis, being more effective than complete Freund's adjuvant or Adjuplex. Advax3 was most efficient in inducing gamma interferon (IFN-γ)- and interleukin-17 (IL-17)-producing antigen-specific T cells that migrated to the lung upon Blastomyces dermatitidis infection. Mechanistic studies revealed Bl-Eng2/Advax3 protection was tempered by neutralization of IL-17 and particularly IFN-γ. Likewise, greater numbers of lung-resident T cells producing IFN-γ, IL-17, or both IFN-γ and IL-17 correlated with fewer fungi recovered from lung. Protection was maintained after depletion of CD4+ T cells, partially reduced by depletion of CD8+ T cells, and completely eliminated after depletion of both CD4+ and CD8+ T cells. We conclude that Bl-Eng2 formulated with Advax3 is promising for eliciting vaccine-induced antifungal immunity, through a previously uncharacterized mechanism involving CD8+ and also CD4+ T cells producing IFN-γ and/or IL-17. Although no licensed vaccine exists as yet against any fungal disease, these findings indicate the importance of adjuvant selection for the development of effective fungal vaccines. IMPORTANCE Fungal disease remains a challenging clinical and public health problem. Despite medical advances, invasive fungal infections have skyrocketed over the last decade and pose a mounting health threat in immunocompetent and -deficient hosts, with worldwide mortality rates ranking 7th, even ahead of tuberculosis. The development of safe, effective vaccines remains a major hurdle for fungi. Critical barriers to progress include the lack of defined fungal antigens and suitable adjuvants. Our research is significant in identifying adjuvant combinations that elicit optimal vaccine-induced protection when formulated with a recombinant protective antigen and uncovering the mechanistic bases of the underlaying vaccine protection, which will foster the strategic development of antifungal vaccines.

Immunization with Pneumocystis carinii A121-85 antigen activates immune function against P. carinii.

BACKGROUND: Pneumocystis pneumonia (PcP), which is caused by Pneumocystis carinii, is a life-threatening infection that affects immunocompromised individuals. Unfortunately, chemoprophylaxis and dapsone are only effective for half of the patients with PcP, indicating that additional preventive methods are needed. We predicated the pneumocystis surface protein A12 sequence 1-85 by DNAStar software and BepiPred, and identified it as a potential vaccine candidate by bioresearch. METHODS: We used recombinant A121-85 as antigen to immunized mice and detected serum titer of IgG, expression of inflammatory factors by EILSA, qRT-PCR and flow cytometry. RESULTS: Our results showed that immunization with recombinant A121-85 increased the serum titer of IgG, promoted the secretion of T lymphocytes, increased the expression of inflammatory factors, and elevated lung inflammatory injury in mice. CONCLUSIONS: Our findings suggest that A121-85 is a potential vaccine target for preventing Pneumocystis carinii. The evaluation of A121-85-elicited antibodies in the prevention of PcP in humans deserves further investigation.

Epitope-Based Peptide Vaccine Design against Fructose Bisphosphate Aldolase of Candida glabrata: An Immunoinformatics Approach.

BACKGROUND: Candida glabrata is a human opportunistic pathogen that can cause life-threatening systemic infections. Although there are multiple effective vaccines against fungal infections and some of these vaccines are engaged in different stages of clinical trials, none of them have yet been approved by the FDA. AIM: Using immunoinformatics approach to predict the most conserved and immunogenic B- and T-cell epitopes from the fructose bisphosphate aldolase (Fba1) protein of C. glabrata. Material and Method. 13 C. glabrata fructose bisphosphate aldolase protein sequences (361 amino acids) were retrieved from NCBI and presented in several tools on the IEDB server for prediction of the most promising epitopes. Homology modeling and molecular docking were performed. RESULT: The promising B-cell epitopes were AYFKEH, VDKESLYTK, and HVDKESLYTK, while the promising peptides which have high affinity to MHC I binding were AVHEALAPI, KYFKRMAAM, QTSNGGAAY, RMAAMNQWL, and YFKEHGEPL. Two peptides, LFSSHMLDL and YIRSIAPAY, were noted to have the highest affinity to MHC class II that interact with 9 alleles. The molecular docking revealed that the epitopes QTSNGGAAY and LFSSHMLDL have the lowest binding energy to MHC molecules. CONCLUSION: The epitope-based vaccines predicted by using immunoinformatics tools have remarkable advantages over the conventional vaccines in that they are more specific, less time consuming, safe, less allergic, and more antigenic. Further in vivo and in vitro experiments are needed to prove the effectiveness of the best candidate's epitopes (QTSNGGAAY and LFSSHMLDL). To the best of our knowledge, this is the first study that has predicted B- and T-cell epitopes from the Fba1 protein by using in silico tools in order to design an effective epitope-based vaccine against C. glabrata.

The Pathogenesis of Aspergillus fumigatus, Host Defense Mechanisms, and the Development of AFMP4 Antigen as a Vaccine.

Aspergillus fumigatus is one of the ubiquitous fungi with airborne conidia, which accounts for most aspergillosis cases. In immunocompetent hosts, the inhaled conidia are rapidly eliminated. However, immunocompromised or immunodeficient hosts are particularly vulnerable to most Aspergillus infections and invasive aspergillosis (IA), with mortality from 50% to 95%. Despite the improvement of antifungal drugs over the last few decades, the therapeutic effect for IA patients is still limited and does not provide significant survival benefits. The drawbacks of antifungal drugs such as side effects, antifungal drug resistance, and the high cost of antifungal drugs highlight the importance of finding novel therapeutic and preventive approaches to fight against IA. In this article, we systemically addressed the pathogenic mechanisms, defense mechanisms against A. fumigatus, the immune response, molecular aspects of host evasion, and vaccines' current development against aspergillosis, particularly those based on AFMP4 protein, which might be a promising antigen for the development of anti-A. fumigatus vaccines.

The evaluation of attenuated Neospora caninum by long-term passages on murine macrophage cell line in prevention of vertical transmission in mice.

To date, there is no effective vaccine to prevent abortion or vertical transmission associated with neosporosis in cattle. In the present study, the efficacy of a live experimental vaccine of Neospora caninum attenuated (NCa) by long-term serial passages on a murine macrophage cell line was evaluated in the prevention of vertical transmission and abortion in the mouse model. Forty non-pregnant mice were randomly divided into four equal groups including non-immunized/challenged (injected with PBS); positive control (inoculated with un-attenuated NC-1 tachyzoites); immunized/challenged (inoculated with NCa attenuated strain) and immunized/non-challenged or vaccinated (inoculated with NCa) groups. Following pregnancy synchronization, both the immunized and control mice were challenged with virulent live NC-1 tachyzoites (2.5 × 106) in the mid-pregnancy stage. The number of abortions and post-natal pup mortalities was recorded. Serological, molecular, and histopathologic examinations were employed to evaluate the efficacy of the vaccine and the vertical transmission rates. Results indicated that the live attenuated N. caninum strain (NCa) could significantly reduce the risk of abnormal parturitions and fetal mortality in the vaccinated group (20 %) compared to the non-immunized/challenged group (80 %). Also, the NCa strain reduced the lesion score in the brain of the offspring (0.3 vs 1.9) compared to the non-immunized/challenged group (P < 0.05). The molecular assay showed a decrease in the parasite DNA detection rates from 83 % and 77 % in the non-immunized/challenged group to 27 % and 0 % in the vaccine group in the brain and liver tissues, respectively. While in the immunized/non-challenged group no parasite DNA was detected in the brain tissue samples of the pups. Serological analyses showed that NCa strain was able to stimulate the humoral immunity and create effective protection against neosporosis with a moderate systemic IFN-γ response. In conclusion, the NCa strain could significantly (P < 0.05) reduce the risk of vertical transmission and proved to be a safe vaccine while conferring significant levels of protection in the laboratory mice.

Vaccination with Secreted Aspartyl Proteinase 2 Protein from Candida parapsilosis Can Enhance Survival of Mice during C. tropicalis-Mediated Systemic Candidiasis.

The rising incidence of non-albicans Candida species globally, along with the emergence of drug resistance, is a cause for concern. This study investigated the protective efficacy of secreted aspartyl proteinase 2 (Sap2) in systemic C. tropicalis infection. Vaccination with recombinant Sap2 (rSap2) protein from C. parapsilosis enhanced survival of mice compared to rSap2 vaccinations from C. albicans (P = 0.02), C. tropicalis (P = 0.06), and sham immunization (P = 0.04). Compared to sham-immunized mice, the fungal CFU number was significantly reduced in organs of Sap2-parapsilosis-immunized mice. Histopathologically, increased neutrophilic recruitment was observed in Sap2-parapsilosis- and Sap2-tropicalis-immunized mice. Among different rSap2 proteins, Sap2-parapsilosis vaccination induced increased titers of Sap2-specific Ig, IgG, and IgM antibodies, which could bind whole fungus. Between different groups, sera from Sap2-parapsilosis-vaccinated mice exhibited increased C. tropicalis biofilm inhibition ability in vitro and enhanced neutrophil-mediated fungal killing. Passive transfer of anti-Sap2-parapsilosis immune serum in naive mice significantly reduced fungal burdens compared to those in mice receiving anti-sham immune serum. Higher numbers of plasma cells and Candida-binding B cells in Sap2-vaccinated mice suggest a role of B cells during early stages of Sap2-mediated immune response. Additionally, increased levels of Th1/Th2/Th17 cytokines observed in Sap2-parapsilosis-vaccinated mice indicate immunomodulatory properties of Sap2. Epitope analysis performed using identified B-cell epitopes provides a basis to understand differences in immunogenicity observed among Sap2-antigens and can aid the development of a multivalent or multiepitope anti-Candida vaccine(s). In summary, our results suggest that Sap2-parapsilosis vaccination can improve mouse survival during C. tropicalis infection by inducing both humoral and cellular immunity, and higher titers of Sap2-induced antibodies are beneficial during systemic candidiasis.

In silico Design of a Multivalent Vaccine Against Candida albicans.

Invasive candidiasis (IC) is the most common nosocomial infection and a leading cause of mycoses-related deaths. High-systemic toxicity and emergence of antifungal-resistant species warrant the development of newer preventive approaches against IC. Here, we have adopted an immunotherapeutic peptide vaccine-based approach, to enhance the body's immune response against invasive candida infections. Using computational tools, we screened the entire candida proteome (6030 proteins) and identified the most immunodominant HLA class I, HLA class II and B- cell epitopes. By further immunoinformatic analyses for enhanced vaccine efficacy, we selected the 18- most promising epitopes, which were joined together using molecular linkers to create a multivalent recombinant protein against Candida albicans (mvPC). To increase mvPC's immunogenicity, we added a synthetic adjuvant (RS09) to the mvPC design. The selected mvPC epitopes are homologous against all currently available annotated reference sequences of 22 C. albicans strains, thus offering a higher coverage and greater protective response. A major advantage of the current vaccine approach is mvPC's multivalent nature (recognizing multiple-epitopes), which is likely to provide enhanced protection against complex candida antigens. Here, we describe the computational analyses leading to mvPC design.

A Combination of Polybacterial MV140 and Candida albicans V132 as a Potential Novel Trained Immunity-Based Vaccine for Genitourinary Tract Infections.

Recurrent urinary tract infections (RUTIs) and recurrent vulvovaginal candidiasis (RVVCs) represent major healthcare problems with high socio-economic impact worldwide. Antibiotic and antifungal prophylaxis remain the gold standard treatments for RUTIs and RVVCs, contributing to the massive rise of antimicrobial resistance, microbiota alterations and co-infections. Therefore, the development of novel vaccine strategies for these infections are sorely needed. The sublingual heat-inactivated polyvalent bacterial vaccine MV140 shows clinical efficacy for the prevention of RUTIs and promotes Th1/Th17 and IL-10 immune responses. V132 is a sublingual preparation of heat-inactivated Candida albicans developed against RVVCs. A vaccine formulation combining both MV140 and V132 might well represent a suitable approach for concomitant genitourinary tract infections (GUTIs), but detailed mechanistic preclinical studies are still needed. Herein, we showed that the combination of MV140 and V132 imprints human dendritic cells (DCs) with the capacity to polarize potent IFN-γ- and IL-17A-producing T cells and FOXP3+ regulatory T (Treg) cells. MV140/V132 activates mitogen-activated protein kinases (MAPK)-, nuclear factor-κB (NF-κB)- and mammalian target of rapamycin (mTOR)-mediated signaling pathways in human DCs. MV140/V132 also promotes metabolic and epigenetic reprogramming in human DCs, which are key molecular mechanisms involved in the induction of innate trained immunity. Splenocytes from mice sublingually immunized with MV140/V132 display enhanced proliferative responses of CD4+ T cells not only upon in vitro stimulation with the related antigens contained in the vaccine formulation but also upon stimulation with phytohaemagglutinin. Additionally, in vivo sublingual immunization with MV140/V132 induces the generation of IgG and IgA antibodies against all the components contained in the vaccine formulation. We uncover immunological mechanisms underlying the potential mode of action of a combination of MV140 and V132 as a novel promising trained immunity-based vaccine (TIbV) for GUTIs.

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.

A Heat-Killed Cryptococcus Mutant Strain Induces Host Protection against Multiple Invasive Mycoses in a Murine Vaccine Model.

Cryptococcus neoformans is a fungal pathogen that infects the lungs and then often disseminates to the central nervous system, causing meningitis. How Cryptococcus is able to suppress host immunity and escape the antifungal activity of macrophages remains incompletely understood. We reported that the F-box protein Fbp1, a subunit of the SCF(Fbp1) E3 ligase, promotes Cryptococcus virulence by regulating host-Cryptococcus interactions. Our recent studies demonstrated that the fbp1Δ mutant elicited superior protective Th1 host immunity in the lungs and that the enhanced immunogenicity of heat-killed fbp1Δ yeast cells can be harnessed to confer protection against a subsequent infection with the virulent parental strain. We therefore examined the use of heat-killed fbp1Δ cells in several vaccination strategies. Interestingly, the vaccine protection remains effective even in mice depleted of CD4+ T cells. This finding is particularly important in the context of HIV/AIDS-induced immune deficiency. Moreover, we observed that vaccinating mice with heat-killed fbp1Δ induces significant cross-protection against challenge with diverse invasive fungal pathogens, including C. neoformans, C. gattii, and Aspergillus fumigatus, as well as partial protection against Candida albicans Thus, our data suggest that the heat-killed fbp1Δ strain has the potential to be a suitable vaccine candidate against cryptococcosis and other invasive fungal infections in both immunocompetent and immunocompromised populations.IMPORTANCE Invasive fungal infections kill more than 1.5 million people each year, with limited treatment options. There is no vaccine available in clinical use to prevent and control fungal infections. Our recent studies showed that a mutant of the F-box protein Fbp1, a subunit of the SCF(Fbp1) E3 ligase in Cryptococcus neoformans, elicited superior protective Th1 host immunity. Here, we demonstrate that the heat-killed fbp1Δ cells (HK-fbp1) can be harnessed to confer protection against a challenge by the virulent parental strain, even in animals depleted of CD4+ T cells. This finding is particularly important in the context of HIV/AIDS-induced immune deficiency. Moreover, we observed that HK-fbp1 vaccination induces significant cross-protection against challenge with diverse invasive fungal pathogens. Thus, our data suggest that HK-fbp1 has the potential to be a broad-spectrum vaccine candidate against invasive fungal infections in both immunocompetent and immunocompromised populations.

The NDV-3A vaccine protects mice from multidrug resistant Candida auris infection.

Candida auris is an emerging, multi-drug resistant, health care-associated fungal pathogen. Its predominant prevalence in hospitals and nursing homes indicates its ability to adhere to and colonize the skin, or persist in an environment outside the host-a trait unique from other Candida species. Besides being associated globally with life-threatening disseminated infections, C. auris also poses significant clinical challenges due to its ability to adhere to polymeric surfaces and form highly drug-resistant biofilms. Here, we performed bioinformatic studies to identify the presence of adhesin proteins in C. auris, with sequence as well as 3-D structural homologies to the major adhesin/invasin of C. albicans, Als3. Anti-Als3p antibodies generated by vaccinating mice with NDV-3A (a vaccine based on the N-terminus of Als3 protein formulated with alum) recognized C. auris in vitro, blocked its ability to form biofilms and enhanced macrophage-mediated killing of the fungus. Furthermore, NDV-3A vaccination induced significant levels of C. auris cross-reactive humoral and cellular immune responses, and protected immunosuppressed mice from lethal C. auris disseminated infection, compared to the control alum-vaccinated mice. The mechanism of protection is attributed to anti-Als3p antibodies and CD4+ T helper cells activating tissue macrophages. Finally, NDV-3A potentiated the protective efficacy of the antifungal drug micafungin, against C. auris candidemia. Identification of Als3-like adhesins in C. auris makes it a target for immunotherapeutic strategies using NDV-3A, a vaccine with known efficacy against other Candida species and safety as well as efficacy in clinical trials. Considering that C. auris can be resistant to almost all classes of antifungal drugs, such an approach has profound clinical relevance.

Immunization with Pneumocystis recombinant KEX1 induces robust and durable humoral responses in immunocompromised non-human primates.

Infection with the opportunistic fungal pathogen, Pneumocystis jirovecii causes life-threatening pneumonia in immunocompromised individuals. In addition to HIV-1 infected patients, individuals at risk of Pneumocystis infection include those receiving immunosuppressive therapies due to transplantation, cancer or autoimmune disease. Antibiotic treatment is not always successful, and it does not prevent obstructive lung disease after clearance of the pathogen. Therefore, it is essential to develop therapeutic alternatives that are more effective against PCP. We reported that Pneumocystis recombinant protein KEX1 induces protective immunity against the development of PCP in a non-human primate model of HIV-induced immunosuppression. In this study, we tested the immunogenicity KEX1 immunization of healthy rhesus macaques and the durability of these responses during drug-induced immunosuppression using tacrolimus (FK506) and methylprednisolone. We observed that vaccination with KEX1 prior to the start of the immunosuppressive regimen generated a robust and long-lasting antibody response that was maintained throughout the immunosuppressive treatment. Furthermore, boosting with KEX1 during immunosuppression induced recall of memory responses against recombinant KEX1. The durability of the anti-KEX1 response and the ability to induce a recall response during immunosuppressive therapy provide a proof-of-concept data supporting further investigation of the KEX1 as a prophylactic vaccine to prevent PCP in drug-induced immunosuppression. This approach provides fundamental knowledge for the elaboration of therapeutic and prophylactic alternatives for PCP in patients undergoing severe immunosuppressive therapy.

Prophylactic and therapeutic vaccines against sporotrichosis. Feasibility and prospects.

Sporotrichosis is an emergent subcutaneous mycosis of humans and some animals caused by dimorphic fungi of the genus Sporothrix. The disease occurs worldwide but is endemic or hyperendemic in tropical and subtropical areas. The epidemiology of the disease is changing dramatically, and it is now considered an important zoonosis with high morbidity rates, principally in Brazil, and an opportunistic infection in immunocompromised patients. Due to the limited options currently available to treat invasive fungal infections, including sporotrichosis, and the emergence of drug resistance and toxicity, the development of anti-Sporothrix vaccines has become an area of great interest. This work provides a brief analysis of the feasibility of the development of prophylactic and therapeutic vaccines against sporotrichosis, the main advances achieved to date, future challenges and prospects.

Cryptococcus neoformans Glucuronoxylomannan and Sterylglucoside Are Required for Host Protection in an Animal Vaccination Model.

Cryptococcus neoformans is an encapsulated fungal pathogen that causes meningoencephalitis. There are no prophylactic tools for cryptococcosis. Previously, our group showed that a C. neoformans mutant lacking the gene encoding sterylglucosidase (Δsgl1) induced protection in both immunocompetent and immunocompromised murine models of cryptococcosis. Since sterylglucosidase catalyzes degradation of sterylglucosides (SGs), accumulation of this glycolipid could be responsible for protective immunity. In this study, we analyzed whether the activity of SGs is sufficient for the protective effect induced by the Δsgl1 strain. We observed that the accumulation of SGs impacted several properties of the main polysaccharide that composes the fungal capsule, glucuronoxylomannan (GXM). We therefore used genetic manipulation to delete the SGL1 gene in the acapsular mutant Δcap59 to generate a double mutant (strain Δcap59/Δsgl1) that was shown to be nonpathogenic and cleared from the lung of mice within 7 days post-intranasal infection. The inflammatory immune response triggered by the Δcap59/Δsgl1 mutant in the lung differed from the response seen with the other strains. The double mutant did not induce protection in a vaccination model, suggesting that SG-related protection requires the main capsular polysaccharide. Finally, GXM-containing extracellular vesicles (EVs) enriched in SGs delayed the acute lethality of Galleria mellonella against C. neoformans infection. These studies highlighted a key role for GXM and SGs in inducing protection against a secondary cryptococcal infection, and, since EVs notoriously contain GXM, these results suggest the potential use of Δsgl1 EVs as a vaccination strategy for cryptococcosis.IMPORTANCE The number of deaths from cryptococcal meningitis is around 180,000 per year. The disease is the second leading cause of mortality among individuals with AIDS. Antifungal treatment is costly and associated with adverse effects and resistance, evidencing the urgency of development of both therapeutic and prophylactic tools. Here we demonstrate the key roles of polysaccharide- and glycolipid-containing structures in a vaccination model to prevent cryptococcosis.

Moonlighting proteins induce protection in a mouse model against Candida species.

In recent years, C. albicans and C. glabrata have been identified as the main cause of candidemia and invasive candidiasis in hospitalized and immunocompromised patients. In order to colonize the human host, these fungi express several virulence factors such as the response to oxidative stress and the formation of biofilms. In the expression of these virulence factors, the cell wall of C. albicans and C. glabrata is of fundamental importance. As the outermost structure of the yeast, the cell wall is the first to come in contact with the reactive oxygen species (ROS) generated during the respiratory outbreak, and in the formation of biofilms, it is the first to adhere to organs or medical devices implanted in the human host. In both processes, several cell wall proteins (CWP) are required, since they promote attachment to human cells or abiotic surfaces, as well as to detoxify ROS. In our working group we have identified moonlighting CWP in response to oxidative stress as well as in the formation of biofilms. Having identified moonlighting CWP in Candida species in response to two virulence factors indicates that these proteins may possibly be immunodominant. The aim of the present work was to evaluate whether proteins of this type such as fructose-bisphosphate aldolase (Fba1), phosphoglycerate kinase (Pgk) and pyruvate kinase (Pk), could confer protection in a mouse model against C. albicans and C. glabrata. For this, recombinant proteins His6-Fba1, His6-Pgk and His6-Pk were constructed and used to immunize several groups of mice. The immunized mice were infected with C. albicans or C. glabrata, and subsequently the liver, spleen and kidney were extracted and the number of CFU was determined. Our results showed that Pk confers immunity to mice against C. albicans, while Fba1 to C. glabrata. This data allows us to conclude that the moonlighting CWP, Fba1 and Pk confer in vivo protection in a specific way against each species of Candida. This makes them promising candidates for developing specific vaccines against these pathogens.

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.