Amphoteric Mannan as an Immune Response Modifier. New Model Immunobiologically Active Candida albicans Mannan-Based Formula.

BACKGROUND: Currently, the incidence and prevalence of serious fungal infections is increasing, especially in immunosuppressed individuals. The co-administration of antibiotic and immunosuppressive therapies has driven the emergence of new multidrug-resistant fungal pathogens. Their significant increase and their ability to form biofilms is associated with rising morbidity and mortality. Research into novel synthetically prepared immunomodulators as potential immune response modifiers and prospective participants in drug delivery systems is of interest. Microbial polysaccharides with zwitterionic charge motifs were shown to be promising candidates. METHODS: Native and ultrasonically treated mannan from the yeast Candida albicans were chemically modified to contain both positive and negative charges in a nearly equimolar ratio mimicking the zwitterionic polysaccharides. RAW 264.7 macrophages and Balb/c mice were subjected as in vitro and in vivo models. Macrophage exposure to the set of amphoteric derivatives of mannan induced a release of Th1, Th2, Th17, and Treg cytokine signature patterns. The functionality of the exposed macrophages was assayed by cell proliferation and phagocytosis. RESULTS: The Th1 and Th17 dominance was over Th2. The phagocytosis and respiratory burst, together with the viability based on cell proliferation supported the bioavailability of formulas. Mouse immunization induced humoral immune responses with high titers of the IgM isotype with the IgM/IgG shift. CONCLUSION: Our study demonstrated the immunobiological activities of amphoteric derivatives of mannan from Candida albicans. Amphoteric derivatives can be considered as bioavailable formulas with an effective immunomodulatory potency, prospectively applied as a subunit formula in the design of a mannan-based platform for drug and vaccine delivery systems.

Isolation, Purification, Characterization and Direct Conjugation of the Lipid†A-Free Lipopolysaccharide of Vibrio cholerae O139.

The lipopolysaccharide (LPS) of Vibrio cholerae O139, strain CIRS245, was isolated conventionally, and the lipid A was removed by mild acid hydrolysis (0.1 m NaOAc buffer containing 1 % SDS, pH 4.2, 95 °C, 8 h). The crude product was a complex mixture consisting mainly of constituent fragments of the O-specific polysaccharide-core (OSPc). The OSPc was only a minor component in the mixture. Two-stage purification of the crude OSPc by HPLC gave pure OSPc fragment of the LPS, as shown by NMR spectroscopy, analytical HPLC and ESI-MS. This material is the purest OSPc fragment of the LPS from Vibrio cholerae O139 reported to date. The purified OSPc was readily converted to the corresponding methyl squarate derivative and the latter was conjugated to BSA. The conjugate, when examined by ELISA, showed immunoreactivity with sera from patients in Bangladesh recovering from cholera caused by V. cholerae O139, but not O1.

NMR comparison of hyphal and yeast Candida albicans serotype B mannans.

A change from a globular to a filamentous hyphal form is an important feature in the pathogenicity of yeasts. Such a dimorphism while infecting a host organism is thought to be also accompanied in the case of Candida albicans spp. by a structural rearrangement of surface mannan antigen. The presented work brings new insights into the molecular structural changes of mannan C. albicans serotype B based on NMR experimental data. 1H and 13C signal identification of the anomeric region and the assignment of their linkage type is presented here. 2D deconvolution of the HSQC spectra facilitated accurate integration of all anomeric cross-peaks. Analysis of the differences in the integrals led to the proposal that C. albicans serotype B hyphal mannan side chains have the shortened structural moieties: Manα1-2Manα1- and Manα1-3 [Manα1-6] Manα1-2Manα1-. These represent the dominant structures important for construction of a saccharide-based prospective anti-candida vaccine.

One-pot preparation of labelled mannan-peptide conjugate, model for immune cell processing.

An efficient method for preparation of fluorescently labelled mannan-peptide glycoconjugates has been developed. After selective Dess-Martin periodinane oxidation of mannan, it was conjugated to the fluorescent label alone and a peptide with the label via reductive amination. Prepared glycoconjugates were characterised by HPSEC, FTIR-ATR and UV-VIS spectroscopy. Finally, the fluorescently labelled mannan and mannan-peptide conjugate were used for microscopic visualization of their accumulation in intracellular organelles of RAW 264.7 cells.

Immunological basis of anti-Candida vaccines focused on synthetically prepared cell wall mannan-derived manno-oligomers.

The increasing incidence of diseases caused by Candida species and complications in individuals with impaired immunity require new strategies for candidiasis treatment and prevention. The available therapies are often of limited effectiveness in immunocompromised patients, resulting in treatment failures, chronic infections and high mortality rates. Research directed at identifying the composition of an effective vaccine is required. Mannan forms the outermost layer of the Candida cell wall and has an essential role in modulation of anti-Candida host immune responses. Therefore, Candida cell wall mannan and synthetically prepared manno-oligomer-based glycoconjugates are the foci of attention in vaccine candidate development. Almost all of the existing human vaccines mediate protection through neutralizing antibodies. Th1-based and/or Th17-based cellular immune responses, rather than antibody-mediated immunity, mediate protection against candidiasis. Findings of published studies indicate that analysis of cellular immune responses as well as antibody responses is necessary when assessing the immunomodulatory properties of manno-oligomer-based glycoconjugates that are potential anti-Candida vaccine candidates.

Preparation and immunogenicity of conjugate based on hydrazine-treated lipopolysaccharide antigen of Vibrio cholerae O139.

A glycoconjugate construct was based on attachment of V. cholerae O139 hydrazine-treated lipopolysaccharide (LPS) to carboxylated bovine serum albumin (CBSA) via its amino group. The immunological properties of the glycoconjugate were tested using BALB/c mice, injected subcutaneously without any adjuvant three times at 2 weeks interval. The immunogenicity of the conjugate was estimated by enzyme-linked immunosorbent assay, testing of anti-LPS IgG, IgM, and IgA antibodies. The conjugate elicited a statistically significant increase of LPS-specific IgG levels in mice (p < 0.001). The specific anti-LPS IgG and IgA response after the second booster dose was significantly higher compared with reference and unconjugated detoxified LPS response. Antibodies elicited by the dLPS-CBSA conjugate were vibriocidal.

Synthetically prepared glycooligosaccharides mimicking Candida albicans cell wall glycan antigens--novel tools to study host-pathogen interactions.

The immunobiological efficacy of synthetically prepared mannooligosaccharides and a glucooligosaccharide mimicking the structure of Candida albicans cell wall glycans was assessed in vivo and in vitro to exploit immune responses. The exposure of mice splenocytes to BSA-based conjugates of synthetic oligomannosides and oligoglucoside revealed intense influence on T-cell subset polarization. The conjugates biased the immune responses towards Th1 and Th17 with respect to the prevalence of interferon-gamma (IFN-γ) and interleukin (IL)-17 (IL-17) over IL-4 and IL-10 levels. The inflammatory activity of the conjugates has been evaluated based on the induction of pro-inflammatory cytokines. Postvaccination, antimannooligosaccharide and antiglucooligosaccharide antisera were subjected to an evaluation of the structure-immunomodulation activity relationship. Clinical isolates of C. albicans CCY 29-3-32 and C. albicans CCY 29-3-164 were applied to study interactions between Candida cells and anti-oligosaccharide antibodies. In situ recognition of parietal oligomannosyl and oligoglucosyl sequences in C. albicans cell wall by the antisera raised against BSA-based conjugates of synthetic oligomannosides and oligoglucoside revealed the effective recognition of specific distribution of natural oligosaccharide sequences in the cell wall of C. albicans serotype A. With respect to these results, it can be concluded that new, synthetically prepared oligosaccharides mimicking Candida cell wall structures represent prospective immunobiologically effective components for further immunopharmacologically relevant Candida vaccine design.

Molar-mass-dependent antibacterial activity of cationic dextran derivatives against resistant nosocomial pathogens.

The rise of various multidrug-resistant bacteria has created a need for new biocompatible and biodegradable antibacterial compounds. Cationic polysaccharides are promising candidates for this role. Therefore, cationic derivatives of commercial dextrans with molar masses of 11 kDa, 76 kDa, 411 kDa, and 1500-2500 kDa and various degrees of substitution (DSQ 0.34-0.52) were prepared and their antimicrobial properties against four gram-negative nosocomial bacteria were tested. As expected, a higher DSQ led to higher efficiency. The best antimicrobial properties were found for derivatives of 411 kDa, followed by 76 kDa and 1500-2000 kDa dextrans. This indicates that there is a certain optimum molar mass with the best antimicrobial properties. However, as molar mass increased, the biocompatibility of cationic dextran steadily decreased, with increased hemagglutination and toxicity being seen for human cells. The derivatives of 76 kDa dextran with higher DSQ (0.40-0.52) were the best antimicrobial agents suitable for further clinical testing.

Comparison of O-specific polysaccharide responses in patients following infection with Vibrio cholerae O139 versus vaccination with a bivalent (O1/O139) oral killed cholera vaccine in Bangladesh.

Cholera caused by Vibrio cholerae O139 emerged in the early 1990s and spread rapidly to 11 Asian countries before receding for unclear reasons. Protection against cholera is serogroup-specific, which is defined by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS). V. cholerae O139 also expresses the OSP-capsule. We, therefore, assessed antibody responses targeting V. cholerae O139 OSP, LPS, capsule, and vibriocidal responses in patients in Bangladesh with cholera caused by V. cholerae O139. We compared these responses to those of age-gender-blood group-matched recipients of the bivalent oral cholera vaccine (OCV O1/O139). We found prominent OSP, LPS, and vibriocidal responses in patients, with a high correlation between these responses. OSP responses primarily targeted the terminal tetrasaccharide of OSP. Vaccinees developed OSP, LPS, and vibriocidal antibody responses, but of significantly lower magnitude and responder frequency (RF) than matched patients. We separately analyzed responses in pediatric vaccinees born after V. cholerae O139 had receded in Bangladesh. We found that OSP responses were boosted in children who had previously received a single dose of bivalent OCV 3 yr previously but not in vaccinated immunologically naïve children. Our results suggest that OSP-specific responses occur during cholera caused by V. cholerae O139 despite the presence of capsules, that vaccination with bivalent OCV is poorly immunogenic in the short term in immunologically naïve individuals, but that OSP-specific immune responses can be primed by previous exposure, although whether such responses can protect against O139 cholera is uncertain. IMPORTANCE Cholera is a severe dehydrating illness in humans caused by Vibrio cholerae serogroups O1 or O139. Protection against cholera is serogroup-specific, which is defined by the O-specific polysaccharide (OSP) of V. cholerae LPS. Yet, little is known about immunity to O139 OSP. In this study, we assessed immune responses targeting OSP in patients from an endemic region with cholera caused by V. cholerae O139. We compared these responses to those of the age-gender-blood group-matched recipients of the bivalent oral cholera vaccine. Our results suggest that OSP-specific responses occur during cholera caused by V. cholerae O139 and that the OSP responses primarily target the terminal tetrasaccharide of OSP. Our results further suggest that vaccination with the bivalent vaccine is poorly immunogenic in the short term for inducing O139-specific OSP responses in immunologically naïve individuals, but OSP-specific immune responses can be primed by previous exposure or vaccination.

Vaccination with mannan protects mice against systemic aspergillosis.

Invasive aspergillosis is a major cause of mortality in immunocompromised patients and therapeutic options are often limited, thus a vaccine would be desirable. We presently studied acid-stable cell-wall mannan (α-1, 6-linked backbone highly branched with α-1, 2; α-1, 3; and ß-1, 2-linked manno-oligomers) derived from C. albicans, with or without conjugation to bovine serum albumin (BSA), as a vaccine against systemic aspergillosis. Mice were vaccinated subcutaneously with mannan or mannan-BSA conjugate weekly 3 times, ending 2 weeks prior to infection with A. fumigatus conidia. Results showed that the protection induced by mannan is dose-dependent; 12 mg unconjugated mannan alone or > 0.3 mg mannan-BSA consistently enhanced survival (P < 0.05). Fungal burdens in brains and kidneys were reduced after > 0.3 mg of mannan-BSA (all P < 0.05). Mannan-induced protection was improved about 40-fold by conjugation of BSA to mannan. Mannan-BSA (500 kDa) was more protective than 40 kDa mannan-BSA. Mannan is a candidate for a cross-protective conjugate fungal vaccine.

Fragmentation analysis of O-specific polysaccharide from bacteria Vibrio cholerae O139 by MALDI-TOF and LC/ESI-MS/MS.

Cholera is a life-threatening diarrhoeal disease caused by ingestion of Vibrio cholerae. There are at least 200 serogroups of V. cholerae but only two of them are causing epidemics - O1 and O139 serogroups. Fragmentation analysis of O-antigen, also known as O-specific polysaccharide (OSP), from lipopolysaccharide (LPS) is important to obtain new information about its structure, such as fragmentation patterns and fragment structures. In the present study, OSP and core (OSPc) structure from V. cholerae O139 was studied using matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) and direct injection electrospray ionization (ESI)-MS methods. MALDI-TOF analysis was performed in positive-ion reflectron mode, while ESI-MS was performed in negative ionization mode. ESI-MS analysis was followed by ESI-MS/MS analysis. Using this analytical approach, we managed to obtain two possible fragmentation pathways of OSP from V. cholerae O139. Mutual sign of these two pathways is shortening the length of the oligosaccharide by neutral loss of monosaccharide residues. Additionally, liquid chromatography-MS analysis was performed to separate depicted molecular forms of OSPc.

B cells - ontogenesis and immune memory development.

Functional diversity in the distinct developmental stages as well as anti-pathogen effectiveness and memory functionality make B cells unique and attractive object of physiology studies of the immune system. B cells are produced throughout the life of an organism, originate from the hematopoietic stem cells before birth and continue to differentiate to terminal stages. Over the past decade, there has been considerable progress in the research of all B cell intermediates and developmental processes. In this review, we will try to bring brief and comprehensive description of the current understanding of this fascinating topic.

Defining Polysaccharide-Specific Antibody Targets against Vibrio cholerae O139 in Humans following O139 Cholera and following Vaccination with a Commercial Bivalent Oral Cholera Vaccine, and Evaluation of Conjugate Vaccines Targeting O139.

Cholera caused by Vibrio cholerae O139 could reemerge, and proactive development of an effective O139 vaccine would be prudent. To define immunoreactive and potentially immunogenic carbohydrate targets of Vibrio cholerae O139, we assessed immunoreactivities of various O-specific polysaccharide (OSP)-related saccharides with plasma from humans hospitalized with cholera caused by O139, comparing responses to those induced in recipients of a commercial oral whole-cell killed bivalent (O1 and O139) cholera vaccine (WC-O1/O139). We also assessed conjugate vaccines containing selected subsets of these saccharides for their ability to induce protective immunity using a mouse model of cholera. We found that patients with wild-type O139 cholera develop IgM, IgA, and IgG immune responses against O139 OSP and many of its fragments, but we were able to detect only a moderate IgM response to purified O139 OSP-core, and none to its fragments, in immunologically naive recipients of WC-O1/O139. We found that immunoreactivity of O139-specific polysaccharides with antibodies elicited by wild-type infection markedly increase when saccharides contain colitose and phosphate residues, that a synthetic terminal tetrasaccharide fragment of OSP is more immunoreactive and protectively immunogenic than complete OSP, that native OSP-core is a better protective immunogen than the synthetic OSP lacking core, and that functional vibriocidal activity of antibodies predicts in vivo protection in our model but depends on capsule thickness. Our results suggest that O139 OSP-specific responses are not prominent following vaccination with a currently available oral cholera vaccine in immunologically naive humans and that vaccines targeting V. cholerae O139 should be based on native OSP-core or terminal tetrasaccharide. IMPORTANCE Cholera is a severe dehydrating illness of humans caused by Vibrio cholerae serogroup O1 or O139. Protection against cholera is serogroup specific, and serogroup specificity is defined by O-specific polysaccharide (OSP). Little is known about immunity to O139 OSP. In this study, we used synthetic fragments of the O139 OSP to define immune responses to OSP in humans recovering from cholera caused by V. cholerae O139, compared these responses to those induced by the available O139 vaccine, and evaluated O139 fragments in next-generation conjugate vaccines. We found that the terminal tetrasaccharide of O139 is a primary immune target but that the currently available bivalent cholera vaccine poorly induces an anti-O139 OSP response in immunologically naive individuals.

Importance of α- and ß/α-linked mannooligosaccharides in antibody response against C. dubliniensis.

A conjugate of C. dubliniensis cell-wall mannan and human serum albumin (HSA) induced significant level of anti-mannan IgGs in sera of immunized rabbits, whereas mannan alone was not immunogenic. Binding affinities of anti-mannan IgGs induced by the conjugate were evaluated by inhibition ELISA (iELISA) using mannooligosaccharides (dimer-octamer), derived from the side chains of C. dubliniensis mannan, as the inhibitors. Inhibition power of the mannooligosaccharides increased exponentially with their size, with dimer being the weakest (IC(50) = 4 mmol/L) and heptamer/octamer the strongest inhibitors (IC(50) = 0.01 mmol/L). In addition, the mannooligosaccharides proved effective as inhibitors against antiserum obtained from rabbits immunized with C. dubliniensis heat-killed cells, demonstrating a high correlation in the IC(50) values with anti-conjugate serum (Pearson's correlation coefficient r = 0.98; P < 0.01). These findings suggest that a) the mannooligosaccharides comprising the side chains of C. dubliniensis mannan may represent relevant points of interaction with host immune system during infection and b) anti-mannan antibodies induced by the two antigens (the mannan conjugate and the yeast) are of similar specificities.

Anti-staphylococcal activity of quaternized mannan from the yeast Candida albicans.

Global increase of antibiotic-resistant pathogens as well as elevated content of drug residues in the foodstuffs and the environment urgently calls for new biocompatible antimicrobial biomaterials. Yeast mannans represent readily available source of biodegradable materials for tailor-made derivatives that could be effective in biomedical applications. Here, antimicrobial properties of quaternized mannans (DSQ 0.12, 0.24, 0.30, 0.62) from Candida albicans against clinical multi-resistant strains of Staphylococcus aureus are confronted with possible cytotoxicity against human cells. As expected, both effects increase with increasing degree of quaternization. However, it is possible to define the "window", at quaternized mannan with DSQ 0.30 with good anti-microbial effectiveness and low cytotoxicity. This derivative exhibit minimum inhibitory (MIC) and minimum bactericidal (MBC) concentration from 62.5 to 250 µg/mL and demonstrate good biofilm inhibition effect. Also acceptable values were obtained in hemagglutination and hemolytic activity assays and also in cytotoxicity tests on human fibroblasts.

Stability of cationic and amphoteric derivatives of mannan from the yeast Candida albicans.

Infection with Candida albicans can prove lethal in immuno-compromised patients. It is imperative to develop a vaccine against this common organism. The amphoteric derivatives of the mannan component of the Candida cell wall may present a prospective target for the development of such a vaccine; however, the radical processing by antigen-presenting cells of the immune system is not fully understood. In this work a set of tailor-made cationic and amphoteric derivatives of three different degrees of quaternization (DSQ 0.14-0.38) has been prepared by chemical modification of ultrasonically-treated mannan and three carboxymethylated mannan derivatives (DSCM 0.13-0.32). These were exposed to free-radical attack by OH, generated in situ by the Fenton reaction. Potential changes in composition, DSQ, and molar mass distribution due to free-radical degradation were monitored by elemental analysis, NMR and FTIR spectroscopies, and size exclusion chromatography. A protective effect of quaternization against OH degradation was found. Non-isothermal thermogravimetric analysis found that the thermal stability of this mannan was also improved by chemical modification.

Immune responsiveness of a novel peptidoglycan conjugate prepared from surface Candida immunogens: mannan and CR3-related protein.

Candida albicans expresses a CR3-related protein (CR3-RP) antigenically, structurally and functionally related to human adhesion glycoprotein, also known as Mac-l, the iC3b receptor, or complement receptor type 3. The purpose of the present study was to analyze the immunogenic properties of a novel CR3-RP glycoconjugate in a rabbit model. Cell-mediated responses revealed immunoenhancement triggered by CR3-RP glycoconjugate with respect to expression of IL-2 receptor subunit CD25 on B-lymphocytes and inductive increase of the CD4(+)/CD8(+) ratio compared with unconjugated cell wall mannan (P<0.001). Active immunization with the CR3-RP glycoconjugate resulted in an effective IgM-IgG isotypic switch even after the second booster. Altogether, it could be assumed that the novel peptide glycoconjugate is a prospective antigenic candidate for further Candida vaccine design.

Growth inhibition of Candida albicans in the presence of antiserum elicited in rabbits by mannan-protein conjugate.

Antifungal properties of rabbit antiserum prepared by immunization are reported. The immunization was done by a chemically prepared conjugate consisting of Candida albicans (serotype A) surface mannan and human serum albumin. Addition of rabbit antiserum to D-glucose medium inoculated with C. albicans effectively inhibited its growth. Moreover, C. albicans cells treated with rabbit antiserum revealed the entire loss of viability (expressed as decreased mitochondrial dehydrogenase activity). No growth of treated cells on an agar plate was observed. The results confirmed that the mannan-protein conjugate could be considered as an effective component of perspective vaccine.

Cell and antibody mediated immunity induced by vaccination with novel Candida dubliniensis mannan immunogenic conjugate.

Antigen-specific humoral response, as well as the induction of cellular immunity generated by Candida dubliniensis mannan-human serum albumin (HSA) conjugate, a novel proposed immunogenic structure for subcellular vaccine, were evaluated in rabbits. Mannan-HSA conjugate-induced specific IgG and IgA increased significantly after boosters (IgG: P<0.001 and IgA: P<0.01). Mannan-HSA conjugate up-regulation of cell-surface expression of B-lymphocyte and granulocyte activation antigens CD25 and CD11b indicated the effective activation. Immunogenic effect of conjugate on T lymphocytes was demonstrated via inductive increase of CD4+ T lymphocyte subset and CD4+/CD8+ ratio and via induction of T(H)1 cytokines. Immunogenic effectiveness of mannan-HSA conjugate at a dose of 0.25 mg of mannan antigenic moiety overcame that of the mannan alone and of yeast whole cells, thus promising further application in Candida vaccine development.