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

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

Fungal vaccines and adjuvants: a tool to reveal the interaction between host and fungi.

Fungal infections are incurring high risks in a range from superficial mucosal discomforts (such as oropharyngeal candidiasis and vulvovaginal candidiasis) to disseminated life-threatening diseases (such as invasive pulmonary aspergillosis and cryptococcal meningitis) and becoming a global health problem in especially immunodeficient population. The major obstacle to conquer fungal harassment lies in the presence of increasing resistance to conventional antifungal agents used in newly clinically isolated strains. Although recombinant cytokines and mono-/poly-clonal antibodies are added into antifungal armamentarium, more effective antimycotic drugs are exceedingly demanded. It is comforting that the development of fungal vaccines and adjuvants opens up a window to brighten the prospective way in the diagnosis, prevention and treatment of fungal assaults. In this review, we focus on the progression of several major fungal vaccines devised for the control of Candida spp., Aspergillus spp., Cryptococcus spp., Coccidioides spp., Paracoccidioides spp., Blastomyces spp., Histoplasma spp., Pneumocystis spp. as well as the adjuvants adopted. We then expound the interaction between fungal vaccines/adjuvants and host innate (macrophages, dendritic cells, neutrophils), humoral (IgG, IgM and IgA) and cellular (Th1, Th2, Th17 and Tc17) immune responses which generally experience immune recognition of pattern recognition receptors, activation of immune cells, and clearance of invaded fungi. Furthermore, we anticipate an in-depth understanding of immunomodulatory properties of univalent and multivalent vaccines against diverse opportunistic fungi, providing helpful information in the design of novel fungal vaccines and adjuvants.

[Innovative therapies for treatment of invasive fungal diseases]. / Invasive Mykosen ­ Innovative Therapien.

Invasive fungal diseases (IFD) are difficult to treat and pose a significant threat to immunocompromised individuals. Current antifungal agents face limitations, including antifungal resistance and adverse effects. This review aims to give a comprehensive overview of emerging treatment strategies.Novel drugs in development are Ibrexafungerp, an orally available triterpenoid inhibiting glucan synthesis, and Rezafungin representing the echinocandins with extended half-life and improved tissue penetration, both recently licensed for certain indications. Fosmanogepix targets glycosylphosphatidylinositol biosynthesis, while Olorofim, an orotomide, inhibits fungal nucleic acid synthesis, both currently assessed in advanced clinical trials.Immunotherapeutic approaches include immune checkpoint inhibitors to enhance immune response in immunosuppressed individuals and fungal-specific allogeneic CAR-T cell therapy. For prophylactic purpose in high-risk populations to develop IFD, monoclonal antibodies against different virulence factors of Candida spp. have been discovered but are not yet seen in clinical trials. Vaccines against distinct fungal antigens as well as pan fungal vaccines to prevent IFD are under development in preclinical stages, notably for Candida spp., Cryptococcus spp., and Aspergillus spp., however, their clinical value is still discussed.In summary, major advances to treat IFD have been observed, but challenges for their establishment in the clinical routine persist.

Vaccine Strategies for Cryptococcus neoformans.

Cryptococcus neoformans infections are a major worldwide concern as current treatment strategies are becoming less effective in alleviating the infection. The most extreme and fatal cases are those of immunocompromised individuals. Clinical treatments for cryptococcosis are limited to a few classes of approved drugs, and due to a rise in drug resistance, these drugs are becoming less effective. Therefore, it is essential to develop innovative ways to control this infection. Vaccinations have emerged as a safe, viable, and cost-effective solution to treat a number of diseases over the years. Currently, there are no clinically available vaccines to treat cryptococcal infections, but a number of studies have shown promising results in animal models. Here, we present step-by-step experimental protocols using live-attenuated or heat-killed C. neoformans cells as a vaccination strategy in a preventive or in a therapeutic murine model of cryptococcosis.

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.

A chemically induced attenuated strain of Candida albicans generates robust protective immune responses and prevents systemic candidiasis development.

Despite current antifungal therapy, invasive candidiasis causes >40% mortality in immunocompromised individuals. Therefore, developing an antifungal vaccine is a priority. Here, we could for the first time successfully attenuate the virulence of Candida albicans by treating it with a fungistatic dosage of EDTA and demonstrate it to be a potential live whole cell vaccine by using murine models of systemic candidiasis. EDTA inhibited the growth and biofilm formation of C. albicans. RNA-seq analyses of EDTA-treated cells (CAET) revealed that genes mostly involved in metal homeostasis and ribosome biogenesis were up- and down-regulated, respectively. Consequently, a bulky cell wall with elevated levels of mannan and ß-glucan, and reduced levels of total monosomes and polysomes were observed. CAET was eliminated faster than the untreated strain (Ca) as found by differential fungal burden in the vital organs of the mice. Higher monocytes, granulocytes, and platelet counts were detected in Ca- vs CAET-challenged mice. While hyper-inflammation and immunosuppression caused the killing of Ca-challenged mice, a critical balance of pro- and anti-inflammatory cytokines-mediated immune responses are the likely reasons for the protective immunity in CAET-infected mice.

Immunogenicity and efficacy of CNA25 as a potential whole-cell vaccine against systemic candidiasis.

Disseminated fungal infections account for ~1.5 million deaths per year worldwide, and mortality may increase further due to a rise in the number of immunocompromised individuals and drug-resistance fungal species. Since an approved antifungal vaccine is yet to be available, this study explored the immunogenicity and vaccine efficacy of a DNA polymerase mutant strain of Candida albicans. CNA25 is a pol32ΔΔ strain that exhibits growth defects and does not cause systemic candidiasis in mice. Immunized mice with live CNA25 were fully protected against C. albicans and C. parapsilosis but partially against C. tropicalis and C. glabrata infections. CNA25 induced steady expression of TLR2 and Dectin-1 receptors leading to a faster recognition and clearance by the immune system associated with the activation of protective immune responses mostly mediated by neutrophils, macrophages, NK cells, B cells, and CD4+ and CD8+ T cells. Molecular blockade of Dectin-1, IL-17, IFNγ, and TNFα abolished resistance to reinfection. Altogether, this study suggested that CNA25 collectively activates innate, adaptive, and trained immunity to be a promising live whole-cell vaccine against systemic candidiasis.

Semisynthetic Glycoconjugate Vaccine Candidates against Cryptococcus neoformans.

Cryptococcus neoformans is a fungus classified by the World Health Organization as a critically important pathogen, which poses a significant threat to immunocompromised individuals. In this study, we present the chemical synthesis and evaluation of two semisynthetic vaccine candidates targeting the capsular polysaccharide glucuronoxylomannan (GXM) of C. neoformans. These semisynthetic glycoconjugate vaccines contain an identical synthetic decasaccharide (M2 motif) antigen. This antigen is present in serotype A strains, which constitute 95% of the clinical cryptococcosis cases. This synthetic oligosaccharide was conjugated to two proteins (CRM197 and Anthrax 63 kDa PA) and tested for immunogenicity in mice. The conjugates elicited a specific antibody response that bound to the M2 motif but also exhibited additional cross-reactivity toward M1 and M4 GXM motifs. Both glycoconjugates produced antibodies that bound to GXM in ELISA assays and to live fungal cells. Mice immunized with the CRM197 glycoconjugate produced weakly opsonic antibodies and displayed trends toward increased median survival relative to mice given a mock PBS injection (18 vs 15 days, p = 0.06). These findings indicate promise, achieving a successful vaccine demands further optimization of the glycoconjugate. This antigen could serve as a component in a multivalent GXM motif vaccine.

Design of a cryptococcus neoformans vaccine by subtractive proteomics combined with immunoinformatics.

The emergence of Cryptococcus neoformans has posed an undeniable burden to many regions worldwide, with its strains mainly entering the lungs through the respiratory tract and spreading throughout the body. Limitations of drug regimens, such as high costs and limited options, have directed our attention toward the promising field of vaccine development. In this study, the subtractive proteomics approach was employed to select target proteins from databases that can accurately cover serotypes A and D of the Cryptococcus neoformans. Further, two multi-epitope vaccines consisting of T and B cell epitopes were demonstrated that they have good structural stability and could bind with immune receptor to induce desired immune responses in silico. After further evaluation, these vaccines show the potential for large-scale production and applicability to the majority of the population of the world. In summary, these two vaccines have been theoretically proven to combat Cryptococcus neoformans infections, awaiting further experimental validation of their actual protective effects.

A novel Candida albicans Als3, Hwp1 and Met6 derived complex peptide protects mice against hematogenously induced candidiasis.

Candida albicans can cause superficial or systemic infections in humans, particularly in immunocompromised individuals. Vaccination strategies targeting specific antigens of C. albicans have shown promise in providing protection against invasive candidiasis. This study aimed to evaluate the immuno-protective capacity of a KLH conjugated complex peptide, 3P-KLH, containing epitopes from C. albicans antigens Als3, Hwp1, and Met6 in a murine model of hematogenously induced candidiasis. Mice immunized with 3P-KLH raised a specific antibody response, and protection against C. albicans infection was assessed. Immunized mice exhibited significantly lower fungal load in their kidneys compared to the control group. Moreover, 37.5 % of immunized mice survived 21 days after the infection, while all control animals died within the first nine days. These findings suggest that the 3P-KLH complex peptide, targeting C. albicans key antigens, elicits a protective immune response and reduces the severity of systemic Candida infection. In addition, the high binding affinity of the selected epitopes with MHC II alleles further supports the potential immunogenicity of this peptide in humans. This research provides insights into the development of novel immunotherapeutic approaches against invasive candidiasis.

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

The global burden of infections due to the pathogenic fungus Cryptococcus is substantial in persons with low CD4+ T-cell counts. Previously, we deleted three chitin deacetylase genes from Cryptococcus neoformans to create a chitosan-deficient, avirulent strain, designated as cda1∆2∆3∆, which, when used as a vaccine, protected mice from challenge with virulent C. neoformans strain KN99. Here, we explored the immunological basis for protection. Vaccine-mediated protection was maintained in mice lacking B cells or CD8+ T cells. In contrast, protection was lost in mice lacking α/ß T cells or CD4+ T cells. Moreover, CD4+ T cells from vaccinated mice conferred protection upon adoptive transfer to naive mice. Importantly, while monoclonal antibody-mediated depletion of CD4+ T cells just prior to vaccination resulted in complete loss of protection, significant protection was retained in mice depleted of CD4+ T cells after vaccination but prior to challenge. Vaccine-mediated protection was lost in mice genetically deficient in interferon-γ (IFNγ), tumor necrosis factor alpha (TNFα), or interleukin (IL)-23p19. A robust influx of leukocytes and IFNγ- and TNFα-expressing CD4+ T cells was seen in the lungs of vaccinated and challenged mice. Finally, a higher level of IFNγ production by lung cells stimulated ex vivo correlated with lower fungal burden in the lungs. Thus, while B cells and CD8+ T cells are dispensable, IFNγ and CD4+ T cells have overlapping roles in generating protective immunity prior to cda1∆2∆3∆ vaccination. However, once vaccinated, protection becomes less dependent on CD4+ T cells, suggesting a strategy for vaccinating HIV+ persons prior to loss of CD4+ T cells. IMPORTANCE: The fungus Cryptococcus neoformans is responsible for >100,000 deaths annually, mostly in persons with impaired CD4+ T-cell function such as AIDS. There are no approved human vaccines. We previously created a genetically engineered avirulent strain of C. neoformans, designated as cda1∆2∆3∆. When used as a vaccine, cda1∆2∆3∆ protects mice against a subsequent challenge with a virulent C. neoformans strain. Here, we defined components of the immune system responsible for vaccine-mediated protection. We found that while B cells and CD8+ T cells were dispensible, protection was lost in mice genetically deficient in CD4+ T cells and the cytokines IFNγ, TNFα, or IL-23. A robust influx of cytokine-producing CD4+ T cells was seen in the lungs of vaccinated mice following infection. Importantly, protection was retained in mice depleted of CD4+ T cells following vaccination, suggesting a strategy to protect persons who are at risk of future CD4+ T-cell dysfunction.

Designing an mRNA Vaccine against P. jirovecii Involved in Fatal Pneumonia Infections via Comparative Proteomics and Reverse Vaccinology Approaches.

BACKGROUND: Pneumocystis jirovecii is the most emerging life-threating health problem that causes acute and fatal pneumonia infection. It is rare and more contagious for patients with leukemia and immune-deficiency disorders. Until now there is no treatment available for this infection therefore, it is needed to develop any treatment against this pathogen. METHODS: In this work, we used comparative proteomics, robust immune-informatics, and reverse vaccinology to create an mRNA vaccine against Pneumocystis jirovecii by targeting outer and transmembrane proteins. Using a comparative subtractive proteomic analysis of two Pneumocystis jirovecii proteomes, a distinct non-redundant Pneumocystis jirovecii (strain SE8) proteome was chosen. Seven Pneumocystis jirovecii transmembrane proteins were chosen from this proteome based on hydrophilicity, essentiality, virulence, antigenicity, pathway interaction, protein-protein network analysis, and allergenicity. OBJECTIVE: The reverse vaccinology approach was used to predict the immunogenic and antigenic epitopes of major histocompatibility complex (MHC) I, II and B-cells from the selected proteins on the basis of their antigenicity, toxicity and allergenicity. These immunogenic epitopes were linked together to construct the mRNA-based vaccine. To enhance the immunogenicity, suitable adjuvant, linkers (GPGPG, KK, and CYY), and PRDRE sequences were used. RESULTS: Through predictive modeling and confirmation via the Ramachandran plot, we assessed secondary and 3D structures. The adjuvant RpfE was incorporated to enhance the vaccine construct's immunogenicity (GRAVY index: -0.271, instability index: 39.53, antigenicity: 1.0428). The physiochemical profiling of vaccine construct was predicted it an antigenic, efficient, and potential vaccine. Notably, strong interactions were observed between the vaccine construct and TLR-3/TLR-4 (-1301.7 kcal/mol-1 and -1374.7 kcal/mol-1). CONCLUSIONS: The results predicted that mRNA-based vaccines trigger a cellular and humoral immune response, making the vaccine potential candidate against Pneumocystis jirovecii and it is more suitable for in-vitro analysis and validation to prove its effectiveness.

Liposomal Fba and Met6 peptide vaccination protects mice from disseminated candidiasis.

Epitopes from the Candida cell surface proteins Fba and Met6 are putative vaccine targets for invasive candidiasis. Here, we describe a Candida vaccine approach in which short peptides derived from Fba and Met6 are used in spontaneous nanoliposome antigen particle (SNAP) format. SNAP was enabled by the interaction of cobalt porphyrin phospholipid in liposomes with three histidine residues on the N-terminus of synthetic short peptide immunogens from Fba (F-SNAP), Met6 (M-SNAP), or bivalent Fba and Met6 (FM-SNAP). Liposomes were adjuvanted with synthetic monophosphoryl lipid and QS-21. In mice, immunization with F-SNAP, M-SNAP, or FM-SNAP induced antigen-specific IgG responses and mixed Th1/Th2 immunity. The duplex FM-SNAP vaccine elicited stronger antibody responses against each peptide, even at order-of-magnitude lower peptide dosing than a comparable adjuvanted, conjugate vaccine. Enzyme-linked immunosorbent spot analysis revealed the induction of antigen-specific, cytokine-producing T cells. Compared to F-SNAP or M-SNAP, higher production of TNFα, IL-2, and IFNγ was observed with re-stimulation of splenocytes from bivalent FM-SNAP-immunized mice. When vaccinated BALB/c mice were challenged with Candida auris, analysis of the fungal burden in the kidneys showed that SNAP vaccination protected from disseminated candidiasis. In a lethal fungal exposure model in A/J mice, F-SNAP, M-SNAP, and FM-SNAP vaccination protected mice from candidiasis challenge. Together, these results show that further investigation into the SNAP adjuvant platform is warranted using Fba and Met6 epitopes for a pan-Candida peptide vaccine that provides multifaceted protective immune responses. IMPORTANCE: This study introduces a promising vaccine strategy against invasive candidiasis, a severe fungal infection, by targeting specific peptides on the surface of Candida. Using a novel approach called spontaneous nanoliposome antigen particle (SNAP), we combined peptides from two key Candida proteins, Fba and Met6, into a vaccine. This vaccine induced robust immune responses in mice, including the production of protective antibodies and the activation of immune cells. Importantly, mice vaccinated with SNAP were shielded from disseminated candidiasis in experiments. These findings highlight a potential avenue for developing a broad-spectrum vaccine against Candida infections, which could significantly improve outcomes for patients at risk of these often deadly fungal diseases.

Paracoccidioidoyycosis: challenges in the develpment of a vaccine against an eendemic mycosis in the americas / Paracoccidioidomicose: desafios no desenvolvimento de uma vacina contra micose endêmica nas Américas

SUMMARYParacoccidioidomycosis (PCM), caused by Paracoccidioides spp, is an important endemic mycosis in Latin America. There are two recognized Paracoccidioides species, P. brasiliensis and P. lutzii, based on phylogenetic differences; however, the pathogenesis and disease manifestations of both are indistinguishable at present. Approximately 1,853 (~51,2%) of 3,583 confirmed deaths in Brazil due to systemic mycoses from 1996-2006 were caused by PCM. Antifungal treatment is required for patients with PCM. The initial treatment lasts from two to six months and sulfa derivatives, amphotericin B, azoles and terbinafine are used in clinical practice; however, despite prolonged therapy, relapses are still a problem. An effective Th1-biased cellular immune response is essential to control the disease, which can be induced by exogenous antigens or modulated by prophylactic or therapeutic vaccines. Stimulation of B cells or passive transference of monoclonal antibodies are also important means that may be used to improve the efficacy of paracoccidioidomycosis treatment in the future. This review critically details major challenges facing the development of a vaccine to combat PCM.

RESUMOA paracoccidioidomicose (PCM), causada por Paracoccidioides spp, é importante micose endêmica na América Latina. Com base em diferenças filogenéticas, existem duas espécies reconhecidas de Paracoccidioides, P. brasiliensis e P. lutzii, no entanto, a patogênese e as manifestações clínicas de ambas são indistinguíveis atualmente. Aproximadamente 1853 (~51,2%) de 3583 mortes confirmadas, atribuídas a micoses sistêmicas de 1996-2006, no Brasil foram causadas por PCM. Tratamento antifúngico é necessário para pacientes com PCM. O tratamento inicial dura de dois a seis meses e derivados de sulfa, anfotericina B, azóis e terbinafina são utilizados na prática clínica; no entanto, apesar da terapêutica prolongada, as recaídas ainda são um problema. Uma resposta imune celular eficaz, tendendo a Th1, é essencial para controlar a doença que pode ser induzida por antígenos exógenos, ou moduladas por vacinas profiláticas ou terapêuticas. A estimulação de células B ou a transferência passiva de anticorpos monoclonais também são meios importantes que podem ser utilizados para melhorar a eficácia do tratamento da paracoccidioidomicose no futuro. Esta revisão detalha criticamente os principais desafios que o desenvolvimento de uma vacina para combater a PCM enfrenta.

New insights on the development of fungal vaccines: from immunity to recent challenges

Fungal infections are emerging as a major problem in part due to high mortality associated with systemic infections, especially in the case of immunocompromised patients. With the development of new treatments for diseases such as cancer and the acquired immune deficiency syndrome pandemic, the number of immunosuppressed patients has increased and, as a consequence, also the number of invasive fungal infections has increased. Several studies have proposed new strategies for the development of effective fungal vaccines. In addition, better understanding of how the immune system works against fungal pathogens has improved the further development of these new vaccination strategies. As a result, some fungal vaccines have advanced through clinical trials. However, there are still many challenges that prevent the clinical development of fungal vaccines that can efficiently immunise subjects at risk of developing invasive fungal infections. In this review, we will discuss these new vaccination strategies and the challenges that they present. In the future with proper investments, fungal vaccines may soon become a reality.