The incidence of life-threatening, hematogenously disseminated candidiasis, which is predominantly caused by Candida albicans, parallels the use of modern medical procedures that adversely affect the immune system. Limited antifungal drug choices and emergence of drug-resistant C. albicans strains indicate the need for novel prevention and therapeutic strategies. We are developing vaccines and Abs that enhance resistance against experimental candidiasis. However, the prevalence of serum anti-Candida Abs in candidiasis patients has led to the misconception that Abs are not protective. To explain the apparent discrepancy between such clinical observations and our work, we compared functional activities of C. albicans-specific protective and nonprotective mAbs. Both kinds of Abs are agglutinins that fix complement and are specific for cell surface mannan, but the protective Abs recognize β-mannan, and the nonprotective Ab is specific for α-mannan. By several indirect and direct measures, the protective mAbs more efficiently bind complement factor C3 to the yeast cell than do nonprotective Ab. We hypothesize that the C3 deposition causes preferential association of blood-borne fungi with host phagocytic cells that are capable of killing the fungus. We conclude from these results that the protective potential of Abs is dependent on epitope specificity, serum titer, and ability to rapidly and efficiently fix complement to the fungal surface. The mechanism of protection appears to be associated with enhanced phagocytosis and killing of the fungus.

Mannan is a major cell wall component found in Candida species. Natural antimannan antibody is present in sera from most normal adults, but its role in host resistance to hematogenously disseminated candidiasis is unknown. The purpose of this study was to develop recombinant human antimannan antibody and to study its protective function. A phage Fab display combinatorial library containing Fab genes from bone marrow lymphocytes was screened with Candida albicans yeast cells and chemically purified mannan. One antimannan Fab, termed M1, was converted to a full-length immunoglobulin G1 antibody, M1g1, and M1g1 was produced in CHO cells. The M1g1 epitope was found in C. albicans serotypes A and B, Candida tropicalis, Candida guilliermondii, Candida glabrata, and Candida parapsilosis. Its expression was active at both 23°C and 37°C and uniform over the cell surface. BALB/c mice passively immunized with M1g1 were more resistant than control mice to a lethal hematogenous infection by C. albicans, as evidenced by extension of survival in an M1g1 dose-dependent manner (P, 0.08 to <0.001) and by reduction in number of infection foci and their size in the kidney. In vitro studies found that M1g1 promoted phagocytosis and phagocytic killing of C. albicans yeast cells by mouse peritoneal macrophages and was required for activation of the mouse complement cascade. Thus, human antimannan antibody may have a protective role in host resistance to systemic candidiasis.

Candida albicans activates both the classical and alternative complement pathways. Previous studies found that immunoglobulin G (IgG) in normal human serum (NHS) mediates classical pathway initiation. The goal of this study was to determine the role of candidal mannan-specific human IgG antibodies in complement activation. Mannan was purified from the yeast cells, and naturally occurring antimannan IgG was isolated from pooled NHS or plasma samples by immunoaffinity chromatography. Early activation and binding of C3, characteristics of classical pathway activity, were abolished in yeast-or mannan-absorbed serum but could be restored to absorbed serum with added purified antimannan IgG in a dose-dependent manner. Microscopically, the immunofluorescence pattern of initial C3 binding was diffuse over the entire cell surface for yeast cells incubated in NHS or in mannan-absorbed NHS supplemented with antimannan IgG but was asynchronous and focal for yeast cells incubated in EGTA-treated or mannan-absorbed NHS. The antimannan IgG level in serum samples from 21 donors varied from 17 to 570 g/ml of serum compared to 220 g in pooled NHS samples. The rate of initial C3 binding to yeast cells correlated with the level of antimannan IgG in sera from different individuals (r 2 ‫؍‬ 0.94) and could be accelerated in sera containing lower amounts of antimannan IgG with exogenous antimannan IgG. These observations identify antimannan IgG as the initiator of classical pathway C3 deposition on C. albicans. Given the variability in the levels of antimannan antibodies in sera from different individuals, the presence or absence of these antibodies may be an important determinant of host resistance to disseminated candidiasis.

Candida albicans is a fungal pathogen that causes severe disseminated infections that can be lethal in immunocompromised patients. Genetic factors are known to alter the initial susceptibility to and severity of C. albicans infection. We developed a next-generation computational genetic mapping program with advanced features to identify genetic factors affecting survival in a murine genetic model of hematogenous C. albicans infection. This computational tool was used to analyze the median survival data after inbred mouse strains were infected with C. albicans, which provides a useful experimental model for identification of host susceptibility factors. The computational analysis indicated that genetic variation within early classical complement pathway components (C1q, C1r, and C1s) could affect survival. Consistent with the computational results, serum C1 binding to this pathogen was strongly affected by C1rs alleles, as was survival of chromosome substitution strains. These results led to a combinatorial, conditional genetic model, involving an interaction between C5 and C1r/s alleles, which accurately predicted survival after infection. Beyond applicability to infectious disease, this information could increase our understanding of the genetic factors affecting susceptibility to autoimmune and neurodegenerative diseases.Genetic factors are known to alter susceptibility to and severity of Candida albicans infection in mice (1, 3, 22) and humans (42). Therefore, characterizing genetic factors affecting host susceptibility to C. albicans infection is of great importance. Since systemic candidiasis in mice closely resembles the human disease, inbred mouse strains provide a useful experimental model for identification of host susceptibility factors. Although virtually all organs are infected, the kidney is the major target, and the histopathology of infected lesions is similar in mice and humans. Mutations in several immune response genes have been associated with susceptibility to chronic mucocutaneous candidiasis in human families (14,17,36,48), and several have been verified in murine models. Differences in survival after hematogenous C. albicans infection among inbred mouse strains have been associated with complement factor 5 (Hc or C5) alleles (1, 2, 4, 34). A 2-bp deletion polymorphism at the 5Ј end of the C5 transcript shifts its reading frame and causes ϳ50% of inbred strains to be C5 protein deficient (54). Disseminated candidiasis is rapidly fatal in C5-deficient strains because of uncontrolled fungal proliferation in most organs (34). Although C5 alleles make an important contribution, several previous analyses indicated that there are other genetic factors that affect the severity of tissue damage or survival after C. albicans infection (2, 38). However, no one has yet been able to identify these other genetic factors.Since its inception in 2004, haplotype-based computational genetic mapping (HBCGM) (30) has been used to identify the genetic basis for many biomedical trait differences among inbred mou...

The complement system has an important role in host resistance to systemic candidiasis but regulation of complement activation by C. albicans remains poorly defined. Previous studies have identified a requirement for naturally occurring antimannan IgG antibody in initiation of C3 opsonization of C. albicans through either the classical or alternative pathway. This study characterized antibodydependent initiation of the alternative pathway using the recombinant human monoclonal antimannan Fab fragment M1 and its full-length IgG1 antibody M1g1. Kinetic analysis of C3b deposition onto C. albicans with flow cytometry demonstrated the ability of M1g1 to restore the activity of either the classical or alternative pathway to the yeast-absorbed normal human serum, but the Fc-free M1 Fab restored only the activity of the alternative pathway. This Fc-independent, antimannan Fabmediated C3 deposition through the alternative pathway was also observed in a serum-free assay containing the six alternative pathway proteins and in C1q-or C2-depleted serum but not in factor B-depleted serum. M1-or M1g1-dependent alternative pathway initiation of C3b deposition occurred in an asynchronous manner at discrete sites that expanded to cover the entire cell surface over time as revealed with immunofluorescence microscopy, in contrast to a uniform appearance of initial C3 deposition through the classical pathway. Furthermore, antimannan Fab M1 promoted the assembly of the alternative pathway convertase on the cell surface seen as colocalization of C3 and factor B with immunofluorescence microscopy. Thus, human antimannan antibody has a distinct Fcindependent effector function in regulation of C3 deposition to C. albicans.