Vaccine-Induced Protection in Two Murine Models of Invasive Pulmonary Aspergillosis.

Life-threatening, invasive fungal infections (IFIs) cause over 1.5 million deaths worldwide and are a major public health concern with high mortality rates even with medical treatment. Infections with the opportunistic fungal pathogen, Aspergillus fumigatus are among the most common. Despite the growing clinical need, there are no licensed vaccines for IFIs. Here we evaluated the immunogenicity and protective efficacy of an A. fumigatus recombinant protein vaccine candidate, AF.KEX1, in experimental murine models of drug-induced immunosuppression. Immunization of healthy mice with AF.KEX1 and adjuvant induced a robust immune response. Following AF.KEX1 or sham immunization, mice were immunosuppressed by treatment with either cortisone acetate or hydrocortisone and the calcineurin inhibitor, tacrolimus. To test vaccine efficacy, immunosuppressed mice were intranasally challenged with A. fumigatus conidia (Af293) and weight and body temperature were monitored for 10 days. At study termination, organism burden in the lungs was evaluated by quantitative PCR and Gomori's methanamine silver staining. In both models of immunosuppression, AF.KEX1 vaccinated mice experienced decreased rates of mortality and significantly lower lung organism burden compared to non-vaccinated controls. The lung fungal burden was inversely correlated with the peak anti-AF.KEX1 IgG titer achieved following vaccination. These studies provide the basis for further evaluation of a novel vaccine strategy to protect individuals at risk of invasive aspergillosis due to immunosuppressive treatments.

NDV-3A vaccination prevents C. albicans colonization of jugular vein catheters in mice.

NDV-3A, a novel fungal vaccine undergoing clinical trials, contains a recombinant version of the Candida albicans rAls3 N-terminus protein (rAls3p-N) in aluminum hydroxide. In a Phase 1b/2a clinical trial, NDV-3A protected women from recurrent vulvovaginal candidiasis. Here, we reveal that active immunization in mice with NDV-3A induces high titers of anti-rAls3p-N antibodies that interfere with C. albicans ability to adhere to and invade endothelial cells, and form biofilm in vitro. Anti-rAls3p-N antibodies also significantly inhibit yeast dispersal from the hyphal layers of biofilms. Compared to placebo, NDV-3A vaccination inhibited C. albicans dissemination to kidneys and prevented colonization of central venous catheters in mice. Overall, these preclinical studies suggest that NDV-3A may serve as an immunotherapeutic strategy for prevention of infections on indwelling medical devices.

Fungal vaccines, mechanism of actions and immunology: A comprehensive review.

Fungal infections include a wide range of opportunistic and invasive diseases. Two of four major fatal diseases in patients with human immunodeficiency virus (HIV) infection are related to the fungal infections, cryptococcosis, and pneumocystosis. Disseminated candidiasis and different clinical forms of aspergillosis annually impose expensive medical costs to governments and hospitalized patients and ultimately lead to high mortality rates. Therefore, urgent implementations are necessary to prevent the expansion of these diseases. Designing an effective vaccine is one of the most important approaches in this field. So far, numerous efforts have been carried out in developing an effective vaccine against fungal infections. Some of these challenges engaged in different stages of clinical trials but none of them could be approved by the United States Food and Drug Administration (FDA). Here, in addition to have a comprehensive overview on the data from studied vaccine programs, we will discuss the immunology response against fungal infections. Moreover, it will be attempted to clarify the underlying immune mechanisms of vaccines targeting different fungal infections that are crucial for designing an effective vaccination strategy.

CBLB Constrains Inactivated Vaccine-Induced CD8+ T Cell Responses and Immunity against Lethal Fungal Pneumonia.

Fungal infections in CD4+ T cell immunocompromised patients have risen sharply in recent years. Although vaccines offer a rational avenue to prevent infections, there are no licensed fungal vaccines available. Inactivated vaccines are safer but less efficacious and require adjuvants that may undesirably bias toward poor protective immune responses. We hypothesized that reducing the TCR signaling threshold could potentiate antifungal CD8+ T cell responses and immunity to inactivated vaccine in the absence of CD4+ T cells. In this study, we show that CBLB, a negative regulator of TCR signaling, suppresses CD8+ T cells in response to inactivated fungal vaccination in a mouse model of CD4+ T cell lymphopenia. Conversely, Cblb deficiency enhanced both the type 1 (e.g., IFN-γ) and type 17 (IL-17A) CD8+ T cell responses to inactivated fungal vaccines and augmented vaccine immunity to lethal fungal pneumonia. Furthermore, we show that immunization with live or inactivated vaccine yeast did not cause detectable pathologic condition in Cblb-/- mice. Augmented CD8+ T cell responses in the absence of CBLB also did not lead to terminal differentiation or adversely affect the expression of transcription factors T-bet, Eomes, and RORγt. Additionally, our adoptive transfer experiments showed that CBLB impedes the effector CD8+ T cell responses in a cell-intrinsic manner. Finally, we showed that ablation of Cblb overcomes the requirement of HIF-1α for expansion of CD8+ T cells upon vaccination. Thus, adjuvants that target CBLB may augment inactivated vaccines and immunity against systemic fungal infections in vulnerable patients.

Viable spores of Coccidioides posadasii Δcps1 are required for vaccination and provide long lasting immunity.

Coccidioidomycosis is a systemic fungal infection for which a vaccine has been sought for over fifty years. The avirulent Coccidioides posadasii strain, Δcps1, which is missing a 6 kb gene, showed significant protection in mice. These studies explore conditions of protection in mice and elucidate the immune response. Mice were vaccinated with different doses and viability states of Δcps1 spores, challenged with virulent C. posadasii, and sacrificed at various endpoints, dependent on experimental objectives. Tissues from vaccinated mice were harvested for in vitro elucidation of immune response. Vaccination with viable Δcps1 spores was required for protection from lethal challenge. Viable spore vaccination produced durable immunity, lasting at least 6 months, and prolonged survival (≥6 months). The C. posadasii vaccine strain also protected mice against C. immitis (survival ≥ 6 months). Cytokines from infected lungs of vaccinated mice in the first four days after Cp challenge showed significant increases of IFN-γ, as did stimulated CD4+ spleen cells from vaccinated mice. Transfer of CD4+ cells, but not CD8+ or B cells, reduced fungal burdens following challenge. IFN-γ from CD4+ cells in vaccinated mice indicates a Th1 response, which is critical for host control of coccidioidomycosis.

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.

An immunoproteomic approach revealing peptides from Sporothrix brasiliensis that induce a cellular immune response in subcutaneous sporotrichosis.

Sporothrix brasiliensis is the most virulent fungus of the Sporothrix complex and is the main species recovered in the sporotrichosis zoonotic hyperendemic area in Rio de Janeiro. A vaccine against S. brasiliensis could improve the current sporotrichosis situation. Here, we show 3 peptides from S. brasiliensis immunogenic proteins that have a higher likelihood for engaging MHC-class II molecules. We investigated the efficiency of the peptides as vaccines for preventing subcutaneous sporotrichosis. In this study, we observed a decrease in lesion diameters in peptide-immunized mice, showing that the peptides could induce a protective immune response against subcutaneous sporotrichosis. ZR8 peptide is from the GP70 protein, the main antigen of the Sporothrix complex, and was the best potential vaccine candidate by increasing CD4+ T cells and higher levels of IFN-γ, IL-17A and IL-1ß characterizing a strong cellular immune response. This immune environment induced a higher number of neutrophils in lesions that are associated with fungus clearance. These results indicated that the ZR8 peptide induces a protective immune response against subcutaneous sporotrichosis and is a vaccine candidate against S. brasiliensis infection.

Protective effect of antigen delivery using monoolein-based liposomes in experimental hematogenously disseminated candidiasis.

UNLABELLED: We evaluated the potential of a liposomal antigen delivery system (ADS) containing Candida albicans cell wall surface proteins (CWSP) in mediating protection against systemic candidiasis. Treatment of bone-marrow-derived dendritic cells with CWSP-loaded dioctadecyldimethylammonium bromide:monoolein (DODAB:MO) liposomes enhanced and prolonged their activation comparatively to free antigen, indicating that liposome-entrapped CWSP were released more sustainable. Therefore, we immunized mice with CWSP either in a free form or loaded into two different DODAB:MO liposome formulations, respectively designated as ADS1 and ADS2, prior to intravenous C. albicans infection. Immunization with ADS1, but not with ADS2, conferred significant protection to infected mice, comparatively to immunization with CWSP or empty liposomes as control. ADS1-immunized mice presented significantly higher serum levels of C. albicans-specific antibodies that enhanced phagocytosis of this fungus. In these mice, a mixed cytokine production profile was observed encompassing IFN-γ, IL-4, IL-17A and IL-10. Nevertheless, only production of IL-4, IL-17 and IL-10 was higher than in controls. In this study we demonstrated that DODAB:MO liposomes enhance the immunogenicity of C. albicans antigens and host protection in a murine model of systemic candidiasis. Therefore, this liposomal adjuvant could be a promising candidate to assess in vaccination against this pathogenic fungus. STATEMENT OF SIGNIFICANCE: This work describes the immunomodulation capacity of the previously validated antigen delivery system (ADS) composed by dioctadecyldimethylammonium bromide (DODAB) and monoolein (MO) lipids incorporating the cell wall surface proteins (CWSP) from C. albicans. Here, we not only present the ability of this system in facilitating antigen uptake by DCs in vitro, but also that this system induces higher levels of pro-inflammatory cytokines and opsonizing specific IgG antibodies in serum of mice immunized subcutaneously. We show that the ADS are efficient nanocarrier and modulate the immune response against intravenous C. albicans infection favoring mouse protection. In sum, we show that the incorporation of C. albicans antigens in DODAB:MO nanocarries are a promising vaccine strategy against C. albicans fungal infection.

Synthetic p55 tandem DNA vaccine against Pneumocystis carinii in rats.

Pneumocystis spp. are opportunistic fungal pathogens that are closely associated with severe pneumonia and pulmonary complications in patients with impaired immunity. In this study, the antigenic epitopes of the gene encoding the 55 kDa antigen fragment of Pneumocystis (p55), which may play an important role in Pneumocystis pneumonia, were analyzed. A gene containing tandem variants of the p55 antigen was synthesized and named the tandem antigen gene (TAG). TAG's potential as a DNA vaccine was assessed in immunosuppressed rats. Immunization with p55-TAG DNA vaccine significantly reduced both the pathogen burden and lung-weight to body-weight ratios. Additionally, p55-TAG vaccination in immunosuppressed rats elicited both cell-mediated and humoral immunity.

Structural characterization of low molecular weight polysaccharide from Astragalus membranaceus and its immunologic enhancement in recombinant protein vaccine against systemic candidiasis.

Structure and immunologic enhancement of low molecular weight polysaccharide (LMW-ASP) isolated from the root of Astragalus membranaceus (Fisch) Bge. Were detected in recombinant protein vaccine. Structure analysis of LMW-ASP revealed that LMW-ASP (Mw=5.6kDa) was an acid heteropolysaccharide, which consisted of Glc, Gal, Ara, Xyl and GalA in ratio of 10.0：1.3：1.7：1.0：0.9. Recombinant protein (rP-HSP90C) contained epitope C (LKVIRK) from heat shock protein 90 (HSP90) of Candida albicans was used as a vaccine. The results indicated that LMW-ASP significantly promoted specific antibody titers IgG, IgG1, IgG2b, and IL-2, IL-4, IL-10, IL-12 in sera of mice immunized with rP-HSP90C (p<0.05). It was also found LMW-ASP improved DTH response in HSP90C-injceted mice. More importantly, the mice immunized with rP-HSP90C/LMW-ASP had fewer CFU (colony forming unites) in the kidneys compared to the mice immunized with rP-HSP90C (p<0.05). Therefore, LMW-ASP could be exploited into the novel adjuvant to enhance the efficacy of recombinant protein vaccine.

Protection against Paracoccidioides brasiliensis infection in mice treated with modulated dendritic cells relies on inhibition of interleukin-10 production by CD8+ T cells.

Paracoccidioidomycosis is a systemic infection prevalent in Latin American countries. Disease develops after inhalation of Paracoccidioides brasiliensis conidia followed by an improper immune activation by the host leucocytes. Dendritic cells (DCs) are antigen-presenting cells with the unique ability to direct the adaptive immune response by the time of activation of naive T cells. This study was conducted to test whether extracts of P. brasiliensis would induce maturation of DCs. We found that DCs treated with extracts acquired an inflammatory phenotype and upon adoptive transfer conferred protection to infection. Interestingly, interleukin-10 production by CD8(+) T cells was ablated following DC transfer. Further analyses showed that lymphocytes from infected mice were high producers of interleukin-10, with CD8(+) T cells being the main source. Blockage of cross-presentation to CD8(+) T cells by modulated DCs abolished the protective effect of adoptive transfer. Collectively, our data show that adoptive transfer of P. brasiliensis-modulated DCs is an interesting approach for the control of infection in paracoccidioidomycosis.

Anti-beta-glucan-like immunoprotective candidacidal antiidiotypic antibodies.

Mycoses, candidiasis in particular, are relatively common opportunistic infections still characterized by an unacceptable high mortality rate. Furthermore, they are often complicated by resistance or refractoriness to the existing antimicrobial agents. In recent years new effective therapeutic and large-scale preventative strategies have been proposed by exploiting the identification of fungal beta-glucans as target of antifungal agents such as echinocandins, yeast killer toxins and protective antibodies. Anti-beta-glucan antibodies are detectable in animal and human sera. When elicited by glucan-based vaccines they can exert a fungicidal protective activity. Beta-glucan cell wall killer toxin receptors can elicit fungicidal protective antibodies following natural and experimental infections. When used as an immunogen a killer toxin-neutralizing monoclonal antibody (beta-glucan-like) is able to elicit a significant anticandidal protection mediated by anti-idiotypic anti-beta-glucan-like candidacidal antibodies. Polyclonal, monoclonal and recombinant anti-beta-glucan-like antibodies and peptide mimotopes are able to exert an in vitro and/or in vivo microbicidal activity against eukaryotic and prokaryotic killer toxin receptor-bearing pathogenic microorganisms. Implications and perspectives for transphyletic anti-infectious control strategies, as immunoprevention and immunotherapy, are discussed.

Sialoglycotherapeutics in protozoal diseases.

The manipulation of glycosylation, mainly sialylation, holds enormous potential for understanding the biological functions of glycoproteins and glycolipids to treat many diseases. The existing knowledge in the field of glycobiology is exploited by glycotherapeutics for combating protozoan diseases. This review focuses on the development of novel glycobiological therapeutic strategies in the field of protozoan infections.

Immunization with radioattenuated yeast cells of Paracoccidioides brasiliensis induces a long lasting protection in BALB/c mice.

Paracoccidioides brasiliensis is the fungus agent of paracoccidioidomycosis, a chronic systemic disease prevalent in Latin America. The aim of the present work was to evaluate the protection elicited by the immunization of BALB/c mice with radioattenuated yeast cells of P. brasiliensis. The immunization promoted a long lasting protection against highly infective yeast forms of P. brasiliensis. A 99.5% decrease in CFUs recovery was verified 90 days post challenge. At the same time the levels of IgG2a and IFN-gamma were high while a very low production of IL-10 and IL-5 was verified, suggesting that a Th1 pattern was dominant. This work shows the potential of radioattenuated yeast cells for the development of vaccines against fungi infections.

Evaluation of two homologous proline-rich proteins of Coccidioides posadasii as candidate vaccines against coccidioidomycosis.

Evaluation of the protective efficacy of recombinant T-cell-reactive proteins of Coccidioides posadasii in a murine model of coccidioidomycosis has led to the discovery of potential vaccines against this respiratory disease. A recombinant proline-rich antigen (rAg2/Pra) has been reported to be a leading vaccine candidate. However, contradictory results exist on the protection afforded by this antigen. Subcutaneous vaccination of either C57BL/6 or BALB/c mice with rAg2/Pra plus adjuvant followed by intraperitoneal challenge with C. posadasii resulted in a significant reduction of the fungal burden at 12 to 14 days postchallenge compared to that in nonvaccinated animals. Use of the same vaccination protocol followed by intranasal (i.n.) challenge of C57BL/6 mice with an equal number of organisms culminated in chronic pulmonary infection or death over a 90-day period. Early studies of Ag2/Pra suggested that it is a component of an immunogenic complex. We reveal in this study that C. posadasii produces a homolog of the reported proline-rich antigen, designated Prp2, which shows 69% protein sequence identity and 86% similarity to Ag2/Pra. Protection against i.n. challenge of C57BL/6 mice was evaluated by vaccination with the single bacterially expressed homolog, rAg2/Pra, or rPrp2 in combination with rAg2/Pra, each in the presence of the same adjuvant. The combined vaccine provided significantly better protection than either of the single recombinant protein vaccines. Results of enzyme-linked immunospot assays of the immunized mice revealed that the two proline-rich homologs contain unique T-cell epitopes. In combination, the recombinant proteins stimulate a more heterogeneous and protective T-cell repertoire than the monovalent vaccines.

Polysaccharide-containing conjugate vaccines for fungal diseases.

The recognition that antibodies are effective against fungal pathogens has spawned interest in developing vaccines that elicit antibody-mediated protection. Recently, a novel polysaccharide-protein conjugate vaccine that uses the algal antigen laminarin was shown to elicit antibodies to beta-glucan in fungal cell walls and to mediate protection against both experimental candidiasis and aspergillosis. Remarkably, vaccine-induced antibodies manifested direct antifungal effects, suggesting that vaccine efficacy might not require cellular or other components of the immune system. The description of a vaccine that could protect against various fungal pathogens opens exciting new dimensions in the search for approaches to control fungal diseases.

Vaccine immunity to pathogenic fungi overcomes the requirement for CD4 help in exogenous antigen presentation to CD8+ T cells: implications for vaccine development in immune-deficient hosts.

Systemic fungal infections with primary and opportunistic pathogens have become increasingly common and represent a growing health menace in patients with AIDS and other immune deficiencies. T lymphocyte immunity, in particular the CD4+ Th 1 cells, is considered the main defense against these pathogens, and their absence is associated with increased susceptibility. It would seem illogical then to propose vaccinating these vulnerable patients against fungal infections. We report here that CD4+ T cells are dispensable for vaccine-induced resistance against experimental fungal pulmonary infections with two agents, Blastomyces dermatitidis an extracellular pathogen, and Histoplasma capsulatum a facultative intracellular pathogen. In the absence of T helper cells, exogenous fungal antigens activated memory CD8+ cells in a major histocompatibility complex class I-restricted manner and CD8+ T cell-derived cytokines tumor necrosis factor alpha, interferon gamma, and granulocyte/macrophage colony-stimulating factor-mediated durable vaccine immunity. CD8+ T cells could also rely on alternate mechanisms for robust vaccine immunity, in the absence of some of these factors. Our results demonstrate an unexpected plasticity of immunity in compromised hosts at both the cellular and molecular level and point to the feasibility of developing vaccines against invasive fungal infections in patients with severe immune deficiencies, including those with few or no CD4+ T cells.

Interplay between protective and inhibitory antibodies dictates the outcome of experimentally disseminated Candidiasis in recipients of a Candida albicans vaccine.

Mice immunized with heat-inactivated, whole yeast-form cells (Y cells) of Candida albicans developed intense, specific humoral and cell-mediated immune responses. However, they were modestly protected against a lethal challenge by the fungus, and their sera did not confer passive protection upon nonimmunized animals. Surprisingly, this immune serum conferred an elevated degree of passive protection to normal and SCID mice when preadsorbed on whole C. albicans cells. After adsorption, no antibodies specific to mannoprotein (MP)-rich extracts or secretions were detected by indirect enzyme-linked immunosorbent assay and no serum reaction with the fungal cell surface was seen in immunofluorescence assays. However, this serum had totally preserved the level of other antibodies, in particular those reacting with beta-1,3 and beta-1,6 glucan (GG). The hypothesis that anti-GG antibodies contributed to the passive protection was suggested by the following circumstantial evidence: (i) mice immunized with C. albicans cells treated with dithiothreitol and protease (YDP cells), which exposed GG on their surfaces and generated anti-GG but not anti-MP antibodies, were substantially protected against a lethal fungus challenge; (ii) the sera, and their immunoglobulin fractions, of mice immunized with YDP cells transferred protection to nonimmune animals; and (iii) this passive protection was substantially abolished by preadsorption on GG but not on intact cells. Overall, our findings demonstrate that some anti-Candida antibodies can block the protective potential of immune serum, a potential to which anti-GG antibodies appear to contribute. Our observations may also help explain why subjects with elevated anti-Candida antibody titers, inclusive of anti-MP and anti-GG antibodies, remain nonetheless susceptible to invasive candidiasis.

Purification and characterization of a second immunoreactive mannoprotein from Cryptococcus neoformans that stimulates T-Cell responses.

Although T-cell responses are known to be critical for effective host defenses against the fungal pathogen Cryptococcus neoformans, the antigens that stimulate protective responses are poorly characterized but are thought to be comprised, at least in part, of mannoproteins. Recently, we created a panel of murine CD4(+)-T-cell hybridomas that react with C. neoformans antigens. A mannoprotein antigen, MP98, that stimulated one of the hybridomas was purified, and the gene encoding MP98 was cloned. In the present study, the cryptococcal antigen, MP88, that stimulated a second T-cell hybridoma, X5A3, to secrete interleukin-2 was characterized. MP88 was purified from supernatants of glass bead-disrupted C. neoformans by anion-exchange and hydrophobic interaction chromatography. A single band with an apparent molecular mass of 88 kDa was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to partial internal amino acid sequencing. The gene encoding MP88 was cloned and sequenced. MP88 features a C-terminal serine/threonine-rich region, which presumably serves as a site for extensive O glycosylation, followed by a putative glycosylphosphatidylinositol anchor site. A search of C. neoformans genomic databases revealed that MP88 shares this feature with at least 11 other genes, including MP98. The mannoprotein nature of MP88 was established based upon the capacity of (i) the mannoprotein fraction of C. neoformans supernatants to stimulate X5A3 and (ii) mannosylated ligands to competitively inhibit this stimulation. Thus, a second cryptococcal mannoprotein has been identified which stimulates T-cell responses and is a vaccine candidate.