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Prevention of hematogenously disseminated candidiasis and mucocutaneous disease, including Candida vaginitis, through immunological approaches is appealing for the following reason. A long-acting and safe vaccine that protects against both serotypes of Candida albicans and other important species, such as C. tropicalis and C. glabrata, should significantly reduce the incidence of various forms of candidiasis by these etiologic agents. Through extensive experimentation on protective responses in experimental animals against Candida mannan components, others and we have evidence that antibodies specific for short-chain β-linked oligomannosides are protective against candidiasis. Although the mechanism of protection against vaginal infection requires further investigation, experimentally the ability of antibody to rapidly deposit high amounts of complement factor C3 onto the yeast cell wall is requisite for enhancing resistance against disseminated candidiasis.

Increasing the growth temperature from 28 to 37 degrees C reduced the expression of beta-1,2-oligomannoside epitopes on mannoproteins of Candida albicans serotypes A and B. In contrast, beta-1,2-mannosylation of phospholipomannan (PLM) remained constant despite a slight decrease in the relative molecular weight (M(r)) of this compound. At all growth temperatures investigated, serotype A PLM displayed an M(r) and an antigenicity different from those of serotype B PLM when they were tested with a panel of monoclonal antibodies.

ABSTRACTIncreasing the growth temperature from 28 to 37°C reduced the expression of β-1,2-oligomannoside epitopes on mannoproteins of Candida albicansserotypes A and B. In contrast, β-1,2-mannosylation of phospholipomannan (PLM) remained constant despite a slight decrease in the relative molecular weight (Mr) of this compound. At all growth temperatures investigated, serotype A PLM displayed an Mrand an antigenicity different from those of serotype B PLM when they were tested with a panel of monoclonal antibodies.

We previously reported that a liposome-mannan vaccine (L-mann) of Candida albicans induces production of mouse antibodies that protect against disseminated candidiasis and vaginal infection. Immunoglobulin M (IgM) monoclonal antibody (MAb) B6.1, specific for a C. albicans cell surface beta-1,2-mannotriose, protects mice against both infections. Another IgM MAb, termed B6, which is specific for a different cell surface mannan epitope, does not protect against disseminated candidiasis. The B6.1 epitope is displayed homogeneously over the entire cell surface, compared to a patchy distribution of the B6 epitope. To determine if protection is restricted to an IgM class of antibody, we tested an IgG antibody. MAb C3.1 was obtained from L-mann-immunized mice. By results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, capture enzyme-linked immunosorbent assay, and immunodiffusion tests, MAb C3.1 is an IgG3 isotype. By epitope inhibition assays, we determined that MAb C3.1 is specific for same mannotriose as MAb B6. 1. As expected by the results of the inhibition assays, immunofluorescence microscopy showed that the C3.1 epitope is distributed on the yeast cell surface in a pattern identical to that of the B6.1 epitope. Kidney CFU and mean survival times of infected mice pretreated with MAb C3.1 indicated that the antibody enhanced resistance of mice against disseminated candidiasis. Mice in pseudoestrus that were given MAb C3.1 prior to vaginal infection developed fewer vaginal Candida CFU than control animals that received buffered saline instead of the antibody. The finding that an IgG3 antibody is protective is consistent with our hypothesis that epitope specificity and complement activation are related to the ability of an antibody to protect against candidiasis.

Serum-free culture filtrates of six Candida species and Saccharomyces cerevisiae were found to contain chemoattractants for human polymorphonuclear leukocytes (PMNs) and a mouse macrophage-like cell line, J774. The chemotactic factors differed for the PMN and J774 cells, however, in terms of heat stability, kinetics of liberation by the yeast cells, and divalent cation requirements for production. The chemoattractant in Candida albicans culture filtrates appeared to act through the formyl peptide receptor (FPR) of PMNs, since it was found to induce chemotaxis of Chinese hamster ovary (CHO) cells that were expressing the human FPR but did not induce chemotaxis of wild-type CHO cells. The C. albicans culture filtrates also induced migration of PMNs across confluent monolayers of a human gastrointestinal epithelial cell line, T84; migration occurred in the basolateral-to-apical direction but not the reverse direction, unless the epithelial tight junctions were disrupted. J774 cells did not migrate toward the formylated peptide (fMet-Leu-Phe; fMLF), and chemotaxis toward the C. albicans culture filtrate was not inhibited by an FPR antagonist (t-butoxycarbonyl-Met-Leu-Phe), suggesting that a different receptor mediated J774 cell chemotaxis. In conclusion, we have identified a receptor by which a non-serum-dependent chemotactic factor (NSCF) produced by C. albicans induced chemotaxis of PMNs. Additionally, we have shown that NSCF was active across epithelial monolayers. These findings suggest that NSCFs produced by C. albicans and other yeast species may influence host-pathogen interactions at the gastrointestinal tract mucosal surface by inducing phagocytic-cell infiltration.

This study was conducted to define adhesive characteristics of the acid-stable moiety of the Candida albicans phosphomannoprotein complex (PMPC) on adherence of this fungus to marginal zone macrophages of the mouse spleen. Complete digestion of the acid-stable moiety (Fr.IIS) of the C. albicans PMPC with an alpha-mannosidase or hydrolysis with 0.6 N sulfuric acid destroyed adhesin activity, as determined by the inability of the soluble digests to inhibit yeast cell adherence to the splenic marginal zone. Fr.IIS adhesin activity was decreased following digestion with an alpha-1,2-specific mannosidase. Oligomannosyls consisting of one to six mannose units, which were isolated from the acid-stable part of the PMPC, did not inhibit yeast cell binding and thus do not function alone as adhesin sites in the PMPC. To gain more insight into the minimum requirements for adhesin activity, PMPCs were isolated from a Saccharomyces cerevisiae wild-type strain and from mutant strains mnn1, mnn2, and mnn4; the PMPCs were designated scwt/Fr.II, scmn1/Fr.II, scmn2/Fr.II, and scmn4/Fr.II, respectively. S. cerevisiae scmn2/Fr.II lacks oligomannosyl side chain branches from the outer core mannan, and scmn2/Fr.II was the only PMPC without adhesin activity. S. cerevisiae scwt/Fr.II, scmn1/Fr.II, and scmn4/Fr.II showed adhesin activities less than that of C. albicans Fr.II. These three S. cerevisiae PMPCs are generally similar to Fr. IIS, except that the S. cerevisiae structure has fewer and shorter side chains. Immunofluorescence microscopy show that the acid-stable part of the PMPC is displayed homogeneously on the C. albicans yeast cell surface, which would be expected for a surface adhesin. Our results indicate that both the mannan core and the oligomannosyl side chains are responsible for the adhesin activity of the acid-stable part of the PMPC.

We previously reported that a vaccine composed of liposome-mannan complexes of Candida albicans (L-mann) stimulates mice to produce protective antibodies against disseminated candidiasis. An immunoglobulin M (IgM) monoclonal antibody (MAb), B6.1, specific for a beta-1,2-mannotriose in the complexes protects against the disease, whereas MAb B6 does not. In the present study, the vaccine and MAbs B6.1 and B6 were tested for the ability to protect against Candida vaginal infection, established by intravaginal (i.vg.) inoculation of yeast cells in mice maintained in pseudoestrus. Fungal CFU in each vagina was determined to assess the severity of infection. Mice vaccinated before infection developed about 62% fewer vaginal CFU than nonimmunized controls. Naive mice that received polyclonal antiserum (from vaccinated mice) i.vg. before infection had 60% fewer CFU than controls. The serum protective factor was stable at 56 degreesC, but C. albicans cells absorbed this factor. Mice given MAb B6.1 i.vg. after infection was established had fewer Candida CFU in vaginal tissue than control mice given buffer instead of antibody. MAbs B6.1 and B6 given intraperitoneally before infection protected mice, but MAbs preabsorbed with yeast cells did not. MAb B6.1 also protected against C. tropicalis vaginal infection, but MAb B6 did not. The protective activities of MAbs B6.1 and B6 appeared to be specific because an irrelevant IgM carbohydrate-specific MAb and an irrelevant IgG protein-specific MAb were not protective; also, MAb B6.1 did not affect development of vaginal chlamydial infection. These studies show that an appropriate antibody response, or administration of protective antibodies, can help the host to resist Candida vaginal infection.

Germ tube formation by Candida albicans is at least partially controlled by a product(s) of the yeast phase of the organism which is released from cells upon incubation at 37 degrees C in tissue culture medium or fetal calf serum. This germination regulatory substance is stable under conditions of lyophilization and heating of 70 degrees C, but becomes inactivated at pH values of 4.0 and 9.5. A germination regulatory substance was produced by both strains of C. albicans tested and by a strain of C. tropicalis. Production does not appear to be a universal characteristic of yeasts because the factor could not be recovered from either Cryptococcus laurentii or Candida parapsilosis. Previously described C. albicans germination inhibitors such as cysteine, tryptophol, and phenylethyl alcohol appear not to be the substance described here. Because of the ability of the factor to influence C. albicans morphology, we have designated it morphogenic autoregulatory substance.

Mouse phagocytic cells in in vivo diffusion chambers had either candidacidal or candidastatic activity depending on the kind of phagocyte studied, the type of mouse whence the phagocytes came, and whether the chamber inoculum of Candida albicans consisted primarily of yeasts or hyphae. Killing of C. albicans occurred when yeasts were placed into chambers with membranes with 3.0-micrometer pores and implanted intraperitoneally into normal mice or thymus-deficient (nude) mice. Although C. albicans remains in chambers with 3.0-micrometer pores, host phagocytic cells can migrate into the chambers. Killing also occurred when yeasts were combined with normal or nude mouse neutrophils in chambers made with membranes with 0.45-micrometer pores, which restrict migration of host cells, but not diffusion of soluble factors. Populations of cells rich in macrophages were candidastatic for yeasts when the phagocytes came from normal mice but candidacidal when obtained from nude mice. Results of gradient fractionations of peritoneal exudates indicated that more than one cell type may be responsible for candidacidal activity by nude mouse macrophage-rich cells. Hyphal-phase cells of C. albicans appear to be more resistant than yeast-phase cells to killing by normal and nude mouse phagocytic cells.

Conditions were established under which membrane diffusion chambers surgically implanted into mice could be used to study interactions between host defense factors and Candida albicans within the chambers. Depending on the size of membrane pores, soluble host substances and phagocytic cells entered the chambers during the first 24 h after chamber implantation. By 7 days in vivo, the membranes of chambers appeared impermeable to these host factors. Mouse phagocytic cells were found to be functional within the in vivo chambers whether the cells emigrated to the chambers on their own accord or were placed there before chamber implantation. Opsonic factors such as antibody and complement appeared to be biologically functional within the in vivo chambers. Conditions suitable for harvesting C. albicans from the chambers also were determined.

Candida albicans was found to produce a substance that was chemotactically active for guinea pig polymorphonuclear neutrophils. The chemotactic factor was detected in culture filtrates of organisms grown under aeration and incubated at 37 degrees C for at least 12 h. Nutrients found to be essential for the production of chemotactic factor included glucose, yeast extract, and a mixture of amino acids. Several strains of C. albicans isolated from humans were tested, and varying degrees of chemotactic activity were found to be associated with the culture filtrates. Only one of the eight isolates did not produce a measurable amount of chemotactic activity. Culture filtrates remained chemotactically active after several cycles of freezing and thawing and after heating at 90 degrees C for 10 min. Substantial evidence is presented that the chemotactic activity is not dependent upon activation of complement.

Candida albicans may immunopotentiate antibody responses in mice to antigens unrelated to the fungus. This effect occurred best with cell-associated, rather than soluble, antigens. When dead yeasts, cell walls, or a water-soluble candidal polysaccharide were used, immunopotentiation was most dramatic when the antigen and fungal materials were given concomitantly via an intraperitoneal injection. However, mice infected with viable yeasts several days before antigen administration also developed heightened responses to the antigen. The mechanism of the C. albicans-induced adjuvanticity was not defined, but the effect seemed to correlate with induction of inflammation. The presence of C. albicans or other inflammatory agents in the peritoneal cavity caused a more rapid uptake of particulate antigen by the liver. The relationship between this event and immunopotentiation is not known. These studies demonstrate that C. albicans may have profound effects on host immune responses. Because immunological aberrations are commonly found in patients with candidiasis it may be important to determine whether some of these aberrations result from, rather than precede candidiasis.

Mice varied in their ability to make detectable antibody responses to cell surface determinants of Candida albicans depending upon the antigen preparation and the immunization schedule used. Immunoglobulin M (IgM) appeared to be the major class of antibody responsible for the C. albicans-agglutinating activity of the immune sera. Various inbred strains of mice injected with a ribosomal fraction from C. albicans produced a low titer (average, 4 to 8) of yeast cell agglutinins and a higher titer (64 to 512) of IgE antibodies detected by passive cutaneous anaphylaxis (PCA) in rats. The two kinds of antibodies appeared to be specific for different antigens because the agglutinin, but not IgE, could be removed by absorbing the serum with a polysaccharide from the cell wall of C. albicans, but the polysaccharide did not provoke the PCA reaction. C. albicans-specific IgE antibodies showed cross-reactivity (PCA) with ribosomal antigens from a strain of C. albicans and C. tropicalis, but PCA reactions could not be elicited with similar antigen preparations from other yeast species. IgE responses were also detected in over 20% of the mice infected intravenously or intraperitoneally with live C. albicans.

Variability in the expression of two different cell surface carbohydrate determinants was examined with two agglutinating immunoglobulin M monoclonal antibodies (H9 and C6) and immunoelectron microscopy during growth of three strains of Candida albicans. A single strain of Candida parapsilosis did not express either antigen at any time during growth. Antigens were detected on the surface of C. albicans by agglutination tests with either H9 or C6 over a 48-h growth period. The difference in specificities of the monoclonal antibodies was demonstrated by Ouchterlony double-diffusion tests with solubilized antigens and by variabilities in the reactivity of the agglutinins among yeast strains. The antigenic determinants were isolated by specific immunoprecipitation and protease digestion and characterized by methods including high-pressure liquid chromatography, gas-liquid chromatography, and mass spectroscopy with both chemical and electron ionization. These determinants both contain mannose and glucose. In the case of antigen H9, an additional carbohydrate was detected with gas chromatography and mass spectroscopy. The location of antigens on individual cells was determined by indirect labeling of the determinants, first reacting cells with H9 or C6 followed by goat anti-mouse antibody conjugated with 20-nm colloidal gold particles. Transmission electron microscopy was used to examine cells. The antigens that were reactive with the monoclonal antibodies were associated with a flocculent surface layer. Expression of this layer and expression of the antigens is a dynamic process which is growth phase and strain dependent. The antigens were not expressed on very young cells and disappeared from the cell surface of most C. albicans strains with age. The use of monoclonal antibody to cell surface determinants may allow characterization of cell surface antigens of C. albicans and be helpful in establishing receptors which mediate adherence.