Yeast Antigens Research Articles

Assembly and expression of a synthetic gene encoding the antigen Pfs48/45 of the human malaria parasite Plasmodium falciparum in yeast

Pfs48/45 is an important transmission-blocking vaccine candidate antigen of the human malaria parasite Plasmodium falciparum. This study was aimed at synthesis of recombinant Pfs48/45 containing conformation-constrained epitopes of the native antigen in yeast. Since in the yeast Saccharomyces cerevisiae induction of gene-expression led to prematurely terminated transcripts, an entirely synthetic gene of higher GC content was assembled. Replacement of the AT rich natural gene by the synthetic gene relieved the observed premature transcription termination. Nevertheless, recombinant protein expression could not be detected. In contrast, in the yeast Pichia pastoris low levels of recombinant Pfs48/45 were produced upon induction of synthetic gene expression. The recombinant protein was shown to be disulphide-bridge constrained, but was not recognised by transmission-blocking antibodies and did not induce transmission-blocking sera in mice.

Respiratory and immunological findings in brewery workers.

Occupational exposure of brewery workers to organic dusts such as hops, barley, and brewery yeast has the potential to change respiratory function and immunological status. Ninety-seven male workers employed in a brewery plant were studied. The mean age of the workers in this plant was 40 years, the mean duration of their employment was 16 years. In addition, a group of 76 unexposed workers was studied as a control. Respiratory symptoms were recorded. Lung function was measured by recording maximum expiratory flow-volume (MEFV) curves. Immunological testing was performed on all brewery workers and some control volunteers using skin prick testing with hops, barley, and yeast antigens as well as other nonoccupational allergens, and by determining total serum IgE levels. There was a significantly higher prevalence of most of the chronic respiratory symptoms in brewery workers compared to controls (P < 0.01). Occupational asthma, however, was recorded in only 2 (2.1%) of the brewery workers. Logistic regression analysis showed that smoking was the major studied factor responsible for the high prevalence of chronic respiratory symptoms in workers. A large number of brewery workers complained of acute symptoms that developed during the work shift. Lung function tests were decreased compared to predicted. Multivariate analysis of these respiratory function parameters suggested the importance of workplace exposure in explaining lung function abnormalities. Significantly higher prevalences of positive skin prick tests were recorded in 37 brewery workers for molds, hops, and barley than in controls. Increased serum levels of total IgE were documented in 34/97 (45.1%) brewery workers and in 1/76 (2.7%) of the control workers (P < 0.01). However, workers with positive skin prick tests had prevalences of chronic respiratory symptoms and lung function changes similar to those of workers with negative skin prick tests. Our data suggest that both smoking and dust exposure in the brewery industry may be responsible for the development of respiratory impairment and immunological reactions.

Identification and purification of specific Penicillium marneffei antigens and their recognition by human immune sera.

Disseminated infection with the dimorphic pathogenic fungus Penicillium marneffei is increasingly seen among patients with AIDS in southeast Asian countries. Previous studies have demonstrated the presence of humoral immune responses to this fungus in patient sera; we have confirmed this work using sera from P. marneffei-infected patients (n = 21) to develop Western blots of P. marneffei cytoplasmic yeast antigen (CYA). P. marneffei CYA was then partially purified by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Immunoenzyme development of the Western blots with pooled sera from patients with P. marneffei infection and with pooled sera from patients with aspergillosis (n = 20), candidiasis (n = 10), cryptococcosis (n = 9), and histoplasmosis (n = 11) revealed three antigens with relative molecular masses of 61, 54, and 50 kDa. These antigens were specifically recognized by the pooled sera from the P. marneffei-infected patients. The 61- and 54-kDa antigens were subsequently purified to homogeneity by preparative gel electrophoresis, and the 50-kDa antigen was partially purified by the same technique. N-terminal amino acid sequencing revealed that the 61-kDa antigen had a strong homology (87% identity) with the antioxidant enzyme catalase. The three antigens were then subjected to SDS-PAGE and Western blotting and to immunoenzyme development with individual patient sera; sera from 86% of P. marneffei-infected patients recognized the 61-kDa antigen, sera from 71% recognized the 54-kDa antigen, and sera from 48% recognized the 50-kDa antigen. These specifically recognized antigens are the first to be purified from P. marneffei and can be used either singly or in combination to detect antibody responses in a large percentage of individuals infected with P. marneffei.

Release of Candida albicans yeast antigens upon interaction with human neutrophils in vitro.

Candida albicans is the leading cause of invasive candidosis. As conventional tests do not reliably detect invasive infection, attention has turned to the detection of C. albicans antigens circulating in blood. As antigen tests for invasive candidosis could be improved if C. albicans antigens released upon phagocytosis were defined, this study was undertaken to characterise antigens released during the interaction of yeasts and human neutrophils in vitro. An enzyme immunoassay developed previously to detect what were believed to be predominantly C. albicans cytoplasmic antigens in patients with invasive candidosis was used to follow the neutrophil-mediated release of yeast antigens. Serum opsonisation enhanced antigen release, which was rapid and essentially complete by 1 h. When fresh C. albicans yeasts were added to medium from cultures of neutrophils plus yeasts or neutrophils plus latex beads, additional yeast antigens were released. Medium from neutrophils plus yeasts or from yeasts alone had similar immunoblot patterns with rabbit antibodies to a C. albicans cytoplasmic antigen preparation, with the reactive antigens generally being of higher mol. wt than the reactive antigens in the antigen mixture used for preparation of the antiserum. The two supernates also had similar immunoblot patterns with rabbit anti-C. albicans cell-wall mannan antibodies. These results suggest that yeast surface antigens are released quickly during phagocytosis by neutrophils. Detection of such yeast surface antigens, possibly together with selected yeast cytoplasmic antigens, should improve the sensitivity of C. albicans antigen assays.

Molecular cloning and characterization of a recombinant Histoplasma capsulatum antigen for antibody-based diagnosis of human histoplasmosis.

Immunological cross-reactivity among fungi has hampered the development of specific serodiagnostic assays for histoplasmosis. We report the molecular cloning and characterization of a Histoplasma capsulatum cDNA (GH17) that encodes an antigen with immunodiagnostic potential. GH17 is an 810-bp cDNA which encodes a protein of 211 amino acid residues. The GH17 sequence has almost no significant homology with other sequences in GenBank. Southern blot analysis suggests that GH17 is confined to a single location in the genomic DNA of H. capsulatum. Immunoblots indicated that the protein product of GH17 (expressed as a 140-kDa beta-galactosidase fusion protein) was recognized by antibodies in 18 of 18 sera from histoplasmosis patients, but not by antibodies in sera from patients or animals infected with other fungi. GH17 was expressed in a prokaryotic expression vector, pPROEX-1, and recombinant protein was purified by preparative electrophoresis. Antibodies raised to this protein bound to a 60-kDa native antigen in immunoblots of H. capsulatum yeast antigen extract. These results suggest that GH17 encodes an H. capsulatum antigen that may be useful for the diagnosis of histoplasmosis in humans.

Rapid Detection of Microbial Contaminants in Fruit Juice by Enzyme-linked Immunosorbent Assay

We examined immunological methods for the detection of microbial contaminants in fruit juice. Polyclonal antibodies against native yeast of 7 species, including Candida, Cryptococcus, Rhodotorula and Saccharomyces, and the monoclonal antibody against native C. intermedia, which was named mAb44 (IgG2a subclass), were prepared for use as probes in enzyme-linked immunosorbent assay (ELISA). These antibodies reacted with yeast cell-surface antigens that are thought to consist of protein moieties. The cross-reactivities between the antibodies and yeast antigens of 12 species were investigated by immunofluorescence and immunoblotting methods. C. intermedia polyclonal antibody showed extensive cross-reactivities with yeast antigens of 11 species. ELISA was carried out using C. intermedia polyclonal antibody and mAb44. Yeast cells (104-107) suspended per 100μl of fruit juice or PBS could be quantitatively measured using polyclonal antibody, although quantitative values were not obtained in the case of mAb44. When more than 103 per 100μl yeast cells were present, they were detectable by using mAb44 as well as polyclonal antibody. These results show that ELISA is a valuable method to detect microbial contaminants in fruit juice.

Immuno-assay techniques for detecting yeasts in foods

A brief literature review on immuno-assay of yeast cell wall antigens is gien. Special attention is paid to extracellular, Thermostable yeast antigens (EPS), which are released to the growth medium by many yeast species. The EPS of Saccharomyces cerevisiae and of Stephanoascus ciferrii (syn. Candida ciferrii) could be specifically and sensitively detected by a sandwich ELISA, using an IgG raised in rabbits immunized with the EPS of these yeasts. The EPS ELISA of three basidiomycetous yeasts tested was not specific, that of Geotrichum candidum was genus-specific but was not sensitive. The EPS of Zygosaccharomyces bailii could be detected in a highly specific competitive ELISA but not in a sandwich ELISA or in a latex agglutination test.

Variability in commercial histoplasma complement fixation antigens

Using Immuno-Mycologics (IMMY; Norman, Okla.) histoplasmal yeast (HY) and mycelial (HM) antibody complement fixation test antigens, we retested 1,386 samples that were initially tested with Meridian Diagnostics, Inc. (Cincinnati, Ohio), antigens. Histoplasma antibody was identified (greater than or equal to 1:16) in 20% of HY and 5% of HM samples reported to have titers of less than 1:8 with Meridian reagents. IMMY titers were at least fourfold higher than Meridian titers in 39% of HY and 54% of HM samples that initially had titers of greater than or equal to 1:8 with Meridian antigens. Because 30 of 58 (52%) samples from confirmed cases of histoplasmosis yielded negative results with Meridian antigens and positive results upon retesting with IMMY antigens, we concluded that the Meridian antigens had less reactivity with human histoplasmal antibody.

Nutritional Value of Yeast for Dermatophagoides pteronyssinus (Acari: Epidermoptidae) and the Antigenic and Allergenic Composition of Extracts During Extended Culturing

Cultures of European house dust mite, Dermatophagoides pteronyssinus (Trouessart), were evaluated with respect to yeast nutritional value, together with an assessment of the antigenic and allergenic composition. CIE analysis of extracts illustrated that yeast contributes many antigens throughout the rearing period. However, there is an indication of the consumption of only one particular yeast antigen by the mites. Total protein content profile of the mite cultures is shown in addition to the total allergen activity (RAST determinations). The allergenic activity of the extracts steadily increased during the mite culturing period; this may be due to the accumulation of fecal particles containing the major allergen.

Detection of antibody responses and delayed dermal hypersensitivity with Blastomyces dermatitidis yeast and mycelial lysate antigens

Yeast cell lysate and mycelial lysate antigens prepared from one strain (T-58) of Blastomyces dermatitidis were evaluated with respect to the detection of antibodies and delayed dermal hypersensitivity. Comparable ELISA sensitivity values were evidenced with the two antigens when assayed against serum specimens from dogs with blastomycosis, sera from non-infected dogs residing in endemic and non-endemic areas for blastomycosis and sera from rabbits that were hyperimmunized with B. dermatitidis antigens. Specificity determinations with anti-Histoplasma capsulatum rabbit sera indicated that both reagents exhibited only minimal cross-reactivity; the mycelial antigen was slightly more specific than the yeast phase reagent. Similar sensitivity and specificity results were experienced when the two antigens were used to detect delayed dermal hypersensitivity in guinea pigs previously sensitized with B. dermatitidis or H. capsulatum.

Serological potential for fungal identification

POLONELLI, L. & MORACE, G., 1989. Serological potential for fungal identification. Specific antigens are valuable for the identification of fungal cultures. Early attempts to immunoidentify fungi were hampered by heterogeneity of antigens, antibody preparations and use of improper serological procedures. In recent years, the double diffusion exoantigcn technique has proved to be the most effective method for immunological identification of mycelial fungus cultures. Additional advances in perfecting methods occurred with the adoption of improved reference antisera obtained either through absorption or by immunizing animals with selected immunoelectrophoretic arcs or precipitin bands (reference antigens). Preliminary studies have shown that serodiagnostically important antigens may be used for accurately and rapidly identifying hyaline as well as dematiaceous fungi. Agglutination techniques consisting oflatex particles sensitized with rabbit anti-Cryptococcus neoformans globulin or Candida monospecific antisera permit the detection of specific yeast antigens in a few minutes. In spite of the great success obtained with the antigen test methods, some limitations in these procedures are apparent. The major problem derives from the occurrence of extensive cross reactions among congeneric species. Hybridoma technology permits the production of uniform and standardized antisera (monoclonal antibodies) reacting with species-specific or strain-specific antigenic determinants (Western blotting technique) and the availability of functional pure epitopes (affinity chromatography). The current value and limitations as well as further avenues for the advance of the different procedures are reported.

Inhalant allergies to fungi: reactions to bakers' yeast (Saccharomyces cerevisiae) and identification of bakers' yeast enolase as an important allergen.

Forty-seven subjects diagnosed as having inhalant allergies to fungi were tested for allergic sensitivity to bakers' yeast. Skin prick tests with yeast extract showed that 35 subjects responded with wheal reactions that were at least 3 mm while 32 subjects were regarded as clearly RAST-positive to bakers' yeast antigens. Skin and RAST testing with purified enolase from bakers' yeast and comparisons with the whole yeast extract showed that the enzyme is a major allergenic component of the extract. This conclusion was supported by results of electroblotting studies. RAST inhibition experiments demonstrated allergenic cross-reactivity between bakers' yeast, bakers' yeast enolase and Candida albicans.

Lack of Induction of IgE and IgG Antibodies to Yeast in Humans Immunized with Recombinant Hepatitis B Vaccines

Yeast-derived hepatitis B vaccines (partially purified or highly purified) at different dosages and a plasma-derived hepatitis B vaccine (control) were injected into 50 young volunteers 3 times at monthly intervals. Before and 4 weeks after this series of immunizations, blood samples were drawn and tested for presence of IgE and IgG antibodies against yeast antigens. No rise in IgE antibodies against Saccharomyces cerevisiae antigens nor in IgG antibodies against Candida albicans antigens was found. Together with former clinical results this underlines the experience that type I and type III reactions against putative yeast contaminants apparently play no major role after immunization with recombinant hepatitis B vaccines.

Lack of antibody induction following skin testing with histolyn-CYL: ELISA determinations with Histoplasma capsulatum yeast and mycelial phase antigens.

No significant increases in antibody levels (IgG, IgM) were detected when serum specimens obtained from individuals exhibiting a positive skin test to the Histoplasma capsulatum yeast phase reagent, Histolyn-CYL, were assayed by the indirect enzyme-linked immunosorbent assay (ELISA) using yeast and mycelial phase antigens.

Variation in complement fixation test results with three Histoplasma capsulatum yeast phase antigens

Antigens derived from three different yeast strains of Histoplasma capsulatum (GW and VC used by the Veterans Administration Serology Reference Laboratory and the Centers for Disease Control reference antigen, A811) were compared with each other to determine their equivalency. Results from 200 sera, tested concurrently by the Serology Reference Laboratory and the University of Kentucky Mycoserology Laboratory, indicated that the VC yeast antigen gave better correlation to the A811 antigen than did the GW antigen.

Multiple chemical forms of hepatitis B surface antigen produced in yeast.

Hepatitis B surface antigen (HBsAg) has been extracted from yeast cells that produce HBsAg. These cells contain the gene for surface antigen carried on a plasmid that replicates in the cells. Analysis of the yeast-derived HBsAg by sucrose gradient centrifugation and by polyacrylamide gel electrophoresis shows that the antigen that is initially released from yeast cells is a high molecular weight aggregate of the fundamental Mr 25,000 subunit. Unlike HBsAg derived from human plasma, the yeast antigen is held together by noncovalent interactions and can be dissociated in 2% NaDodSO4 without the use of reducing agents. During in vitro purification of the yeast antigen, some disulfide bonds form spontaneously between the antigen subunits, resulting in a particle composed of a mixture of monomers and disulfide-bonded dimers. Treatment with 3 M thiocyanate converts the 20-nm particles into a fully disulfide-bonded form that is not disrupted in NaDodSO4 unless a reducing agent is added. This disulfide-bonded particle resembles the naturally occurring, plasma-derived surface antigen particle, and the in vitro formed particle has been used to prepare a vaccine for humans against hepatitis B virus infection.

Cellular and humoral immune responses to Candida albicans in subcutaneously infected mice.

Pure mycelial and yeast cultures of Candida albicans were produced in a low sulphate medium. Groups of mice were injected subcutaneously with increasing doses of viable or heat-killed mycelial or yeast cells and the kinetics of delayed-type hypersensitivity (DTH), anti-mycelial and anti-yeast antibodies were studied. Both the dose and the morphological phase of C. albicans showed an influence on the development of the DTH, but the viability is the factor which showed the highest influence on this reaction, since on the one hand mice infected with viable yeast or mycelial cells developed higher DTH levels than mice injected with heat killed cells, and on the other hand this factor seems to play an important role in the kinetics of DTH response. The enzyme-linked immunosorbent assay has been adapted to detect antibodies to yeast and mycelial phase cytoplasmic antigens of C. albicans. In contrast with the DTH reactions, neither dose, morphological phase nor viability played an important role on the antibody titer developed. However, the use of mycelial cytoplasmic antigens seems to be better than the yeasts to detect anti-Candida antibodies over the last days studied.

Significance of serum antibodies to Histoplasma capsulatum in endemic areas.

To evaluate the specificity of serologic tests for histoplasmosis in an endemic area, we studied sera from 104 consecutive healthy blood donors, testing for antibodies to either histoplasmin or Histoplasma yeast antigen, using complement fixation (CF), radioimmunoassay (RIA), and radial immunodiffusion (ID). Twenty-five subjects (24%) had CF antibody titers of 1:8 or 1:16 to one or both antigens; none had titers above 1:16. Nine subjects (9%) had RIA antibody titers of 1:8 or higher. No titers above 1:8 were found using the yeast antigen; five subjects had titers of greater than 1:16 to histoplasmin using RIA. No precipitin bands were found in any of these subjects. The results of four previous studies in endemic populations yielded similar or lower incidences of positive tests. CF antibody titers to either antigen, RIA antibody titers to yeast antigen of 1:32 or greater, or precipitin bands are rare in persons who live where histoplasmosis is endemic.

Quantitative relationship between yeasts and bacteria in denture‐induced stomatitis

Quantitative cultural studies of yeasts and bacteria were made from 7-day-old denture plaque accumulate on pieces of self-adhesive tape stuck on the fitting surface of the maxillary denture in 17 edentulous subjects with healthy oral mucosa and in 27 patients affected with denture-induced stomatitis. Significantly higher numbers of yeasts and bacteria were cultured in the stomatitis patients than in the controls. This indicates that the rate of plaque formation is increased in patients with denture-induced stomatitis. Yeasts usually constituted less than 1% of the anaerobic bacterial counts, but the percentage of yeasts was significantly higher in the stomatitis patients than in the controls. There was a significant correlation between initially higher yeast counts and improvement of the clinical condition of the palatal mucosa following antimycotic treatment. In some patients only bacteria were grown and antimycotic treatment had no effect. The study supported the contention that yeast antigens and toxins of denture plaque are significant factors in initiation and maintenance of denture-induced stomatitis. However, bacteria may also be involved as pathogens.

Factors Relating to Recurrence of Chronic Pulmonary Histoplasmosis Following Treatment with Amphotericin B

A retrospective analysis was made of cases of Chronic Pulmonary Histoplasmosis during the admission in which patients received amphotericin B to determine if there were features related to recurrence of the disease. There was significance at the p⩽0.05 level in that the nonrecurrent cases tended to have a lower initial complement fixation titers using mycelial antigen; had initial complement fixation tests which were likely to result in no reaction using either mycelial or yeast antigen; and had more rapid drops in the complement fixation values using the yeast antigen. Although the present evaluation does not suggest an absolute minimum effective dose of amphotericin B, it would seem plausible to administer a course of at least 2300mg if the initial complement fixation test was nonreactive but if the drug was tolerated well by the patient and the initial complement fixation test was reactive, the goal should be at least 2500mg.