Cryptococcosis is caused by the fungal genus Cryptococcus. Cryptococcosis, predominantly meningoencephalitis, emerged with the HIV pandemic, primarily afflicting HIV-infected patients with profound T-cell deficiency. Where in use, combination antiretroviral therapy has markedly reduced the incidence of and risk for disease, but cryptococcosis continues to afflict those without access to therapy, particularly in sub-Saharan Africa and Asia. However, cryptococcosis also occurs in solid organ transplant recipients and patients with other immunodeficiencies as well as those with no known immunodeficiency. This article reviews innate and adaptive immune responses to C. neoformans, with an emphasis on recent studies on the role of B cells, natural IgM and Fc gamma receptor polymorphisms in resistance to cryptococcosis.

Cryptococcus neoformans is one of the most common causes of fungal disease in HIV-infected persons, but not all of those who are infected develop cryptococcal disease (CD). Although CD4+ T cell deficiency is a risk factor for HIV-associated CD, polymorphisms of phagocytic Fc gamma receptors (FCGRs) have been linked to CD risk in HIV-uninfected persons. To investigate associations between FCGR2A 131 H/R and FCGR3A 158 F/V polymorphisms and CD risk in HIV-infected persons, we performed PCR-based genotyping on banked samples from 164 men enrolled in the Multicenter AIDS Cohort Study (MACS): 55 who were HIV infected and developed CD and a matched control group of 54 who were HIV infected and 55 who were HIV uninfected. Using additive and allelic statistical models for analysis, the high-affinity FCGR3A 158V allele was significantly associated with CD status after adjusting for race/ethnicity (odds ratio [OR], 2.1; P = 0.005), as was the FCGR3A 158 VV homozygous genotype after adjusting for race/ethnicity, rate of CD4+ T cell decline, and nadir CD4+ T cell count (OR, 21; P = 0.005). No associations between CD and FCGR2A 131 H/R polymorphism were identified. In binding studies, human IgG (hIgG)-C. neoformans complexes exhibited more binding to CHO-K1 cells expressing FCGR3A 158V than to those expressing FCGR3A 158F, and in cytotoxicity assays, natural killer (NK) cells expressing FCGR3A 158V induced more C. neoformans-infected monocyte cytotoxicity than those expressing FCGR3A 158F. Together, these results show an association between the FCGR3A 158V allele and risk for HIV-associated CD and suggest that this polymorphism could promote C. neoformans pathogenesis via increased binding of C. neoformans immune complexes, resulting in increased phagocyte cargo and/or immune activation.

The role of B cells in host defense against fungi has been difficult to establish. We quantified and determined the molecular derivation of B-1a, B-1b and B-2 B-cell populations in C57BL/6 mice after pulmonary infection with Cryptococcus neoformans (CN). Total B-1 and B-2 cell numbers increased in lungs and peritoneal cavity (PerC) as early as day one post-infection, but lacked signs of clonal expansion. Labeled capsular (24067) and acapsular (Cap67) CN strains were used to identify CN-binding B-cell subsets by flow-cytometry. PerC B-1a B cells exhibited the most acapsular and capsular CN-binding in CN-infected mice and CN-selected B-1 B cells secreted laminarin- and CN-binding IgM. Single-cell PCR-based sequence analysis of B-1a, B-1b and B-2 cell immunoglobulin heavy chain variable region (VH) genes revealed increased usage of VH11 and VH12, respectively, in acapsular and capsular CN-selected B-1a cells. Germline VH segments were used with capsular CN-selected cells having less junctional diversity than acapsular CN-selected cells. Further studies in B-1 B cell-depleted mice showed that these mice had higher brain and lung fungal burdens and less alveolar macrophage phagocytosis of CN than control and B-1a B cell-reconstituted mice. Together, these results establish a mechanistic role for B-1 B cells in the innate B-cell response to pulmonary infection with CN and reveal that IgM-producing B-1a cells, which express germline VH genes, bind CN and contribute to early fungal clearance. Thus, B-1a B cells provide a first line of defense during pulmonary CN infection in mice.

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IgM and B-1 cell deficient mice exhibit early C. neoformans dissemination from lungs to brain, but a definitive role for B cells in conferring resistance to C. neoformans dissemination has not been established. To address this question, we developed an intranasal (i.n.) C. neoformans infection model in B and T cell deficient Rag1−/− mice and found they also exhibit earlier fungal dissemination and higher brain CFU than wild-type C57Bl/6 (wild-type) mice. To probe the effect of B cells on fungal dissemination, Rag1−/− mice were given splenic (intravenously) or peritoneal (intraperitoneally) B cells from wild-type mice and infected i.n. with C. neoformans 7 d later. Mice that received B cells had lung histopathology resembling wild type mice 14 d post-infection, and B-1, not B-2 or T cells in their lungs, and serum and lung IgM and IgG 21 d post-infection. Lung CFU were comparable in wild-type, Rag1−/−, and Rag1−/− mice that received B cells 21 d post-infection, but brain CFU were significantly lower in mice that received B cells than Rag1−/− mice that did not. To determine if natural antibody can promote immunity in our model, we measured alveolar macrophage phagocytosis of C. neoformans in Rag1−/− mice treated with naive wild-type IgM-sufficient or sIgM−/− IgM-deficient sera before infection. Compared to IgM-deficient sera, IgM-sufficient sera significantly increased phagocytosis. Our data establish B cells are able to reduce early C. neoformans dissemination in mice and suggest natural IgM may be a key mediator of early antifungal immunity in the lungs.

The importance of antibody immunity in protection against Cryptococcus neoformans remains unresolved. We measured serum C neoformans-specific and total antibody levels and peripheral blood B cell subsets of 12 previously healthy patients with cryptococcosis (cases) and 21 controls. Before and after adjustment for age, sex, and race, cryptococcal capsular polysaccharide immunoglobulin G was higher in cases than controls, whereas total B and memory B cell levels were lower. These associations parallel previous findings in patients with human immunodeficiency virus-associated cryptococcosis and suggest that B cell subset perturbations may also associate with disease in previously normal individuals with cryptococcosis.

Immunization of human volunteers with a single dose of pneumococcal surface protein A (PspA) stimulates broad cross-reactive Abs to heterologous PspA molecules that, when transferred, protect mice from fatal infection with Streptococcus pneumoniae. In this study, we report the molecular characterization of 36 mouse mAbs generated against the extracellular domain of PspA (PspA 3-286 ) from strain R36A. Abs to PspA 3-286 were encoded by diverse V H and V families/genes. The H chain CDR3 and L chain CDR3 lengths were 3-13 (7.8 ؎ 0.5) and 8 -9 (8.7 ؎ 0.2) codons, respectively. Unexpectedly, seven hybridomas expressed H chains that lack D H gene-derived amino acids. Nontemplate-encoded addition(s) were observed in the H chain expressed in six of these seven hybridomas; Palindromic addition(s) were absent. Absence of D H gene-derived amino acids did not prevent antiPspA 3-286 mAbs from attaining average relative avidity. Avidity maturation occurred during primary IgG anti-PspA 3-286 polyclonal Ab response in PspA 3-286 -and R36A-immunized mice. Compared with PspA 3-286 -immunized mice, the relative avidity of the primary polyclonal IgG Abs was higher in R36A immunized mice on days 72, 86, and 100. Two pairs of clonally related hybridomas were observed. D H genes expressed in the majority (75.9%) of the hybridomas used reading frame 3. Analysis of replacement/silent mutation ratio in the CDR and framework regions provided evidence for Ag-driven selection in 11 mAbs. Based on epitope localization experiments, the mAbs were classified into 12 independent groups. ELISA additivity assay indicated that members within a group recognized topographically related epitopes. This study provides molecular insights into the biology of D H -less Abs.

Rising incidence of non-albicans Candida species globally along with emergence of drug resistance is a cause of concern. This study investigated the protective efficacy of secreted aspartyl proteinase 2 (Sap2) in systemic C. tropicalis infection. Vaccination with 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, fungal CFUs were 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 naïve mice significantly reduced fungal burdens compared to mice receiving anti-sham-immune serum. Higher numbers of plasma cells and Candida-binding B-cells in Sap2-vaccinated mice suggests role of B-cells during early stage of Sap2-mediated immune response. Additionally, increased levels of Th1/Th2/Th17 cytokines observed in Sap2-parapsilosis vaccinated mice indicate towards 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 development of multivalent or multi-epitope anti-Candida vaccine/s. In summary, our results suggest that Sap2-parapsilosis vaccination can improve mice survival during C. tropicalis infection by inducing both humoral and cellular immunity, and higher titers of Sap2-induced antibodies are beneficial during systemic candidiasis.