RESUMO
A rhamnose targeting strategy for generating effective anticancer vaccines was successful in our previous studies. We showed that by utilizing natural anti-rhamnose antibodies, a rhamnose-containing vaccine can be targeted to antigen-presenting cells, such as dendritic cells. In this case, rhamnose (Rha) was linked directly to the liposomes bearing the antigen. However, in the current approach, we conjugated a multivalent Tri-Rha ligand with the antigen itself, making it a single component vaccine construct, unlike the previous two-component vaccine construct where Rha cholesterol and Mucin1 (MUC1) antigen were both linked separately to the liposomes. Synthesis required the development of a linker for coupling of the Rha-Ser residues. We compared those two systems in a mouse model and found increased production of anti-MUC1 antibodies and more primed antigen-specific CD4+ T cells in both of the targeted approaches when compared to the control group, suggesting that this one-component vaccine construct could be a potential design used in our MUC1 targeting mechanisms.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Vacinas Anticâncer , Células Dendríticas/imunologia , Mucina-1 , Ramnose , Animais , Vacinas Anticâncer/química , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/farmacologia , Feminino , Lipossomos , Camundongos , Mucina-1/química , Mucina-1/imunologia , Mucina-1/farmacologia , Ramnose/química , Ramnose/imunologia , Ramnose/farmacologiaRESUMO
Generation of a CD8(+) response to extracellular antigen requires processing of the antigen by antigen presenting cells (APC) and cross-presentation to CD8(+) T cell receptors via MHC class I molecules. Cross-presentation is facilitated by efficient antigen uptake followed by immune-complex-mediated maturation of the APCs. We hypothesize that improved antigen uptake of a glycopeptide sequence containing a CD8(+) T cell epitope could be achieved by delivering it on a liposome surface decorated with an immune complex-targeting ligand, an l-Rhamnose (Rha) epitope. We synthesized a 20-amino-acid glycopeptide TSAPDT(GalNAc)RPAPGSTAPPAHGV from the variable number tandem repeat region of the tumor marker MUC1 containing an N-terminal azido moiety and a tumor-associated α-N-acetyl galactosamine (GalNAc) at the immunogenic DTR motif. The MUC1 antigen was attached to Pam3Cys, a Toll-like receptor-2 ligand via copper(I)-catalyzed azido-alkyne cycloaddition (CuAAc) chemistry. The Rha-decorated liposomal Pam3Cys-MUC1-Tn 4 vaccine was evaluated in groups of C57BL/6 mice. Some groups were previously immunized to generate anti-Rha antibodies. Anti-Rha antibody expressing mice that received the Rha liposomal vaccine showed higher cellular immunogenicity compared to the control group while maintaining a strong humoral response.
Assuntos
Imunoconjugados/farmacologia , Mucina-1/química , Ramnose/imunologia , Vacinas/imunologia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Proliferação de Células , Técnicas de Química Sintética , Colesterol/química , Epitopos/genética , Epitopos/imunologia , Feminino , Imunoconjugados/química , Interferon gama/metabolismo , Lipossomos/química , Camundongos Endogâmicos C57BL , Mucina-1/imunologia , Engenharia de Proteínas/métodosRESUMO
Immunotherapy is a promising strategy for targeting tumors. One emerging approach is to harness the immune effector functions of natural antibodies to destroy tumor cells. Dinitrophenyl (DNP) and the galactose-α-1,3-galactose (αGal) epitope are two haptens that bind endogenous antibodies. One potential alternative is the deoxysugar L-rhamnose. We compared these candidates by using a biosensor assay to evaluate human sera for endogenous antibody concentration, antibody isotype distribution, and longevity of antibody-hapten interactions. Antibodies recognizing α-rhamnose are of equal or greater abundance and affinity as those recognizing αGal. Moreover, both rhamnose and αGal epitopes are more effective than DNP at recruiting the IgG antibody subtype. Exposure of tumor cells to rhamnose-bearing glycolipids and human serum promotes complement-mediated cytotoxicity. These data highlight the utility of α-rhamnose-containing glycoconjugates to direct the immune system to target cells.
Assuntos
Anticorpos/imunologia , Glicoconjugados/química , Glicoconjugados/farmacologia , Neoplasias/terapia , Ramnose/análogos & derivados , Ramnose/farmacologia , Linhagem Celular Tumoral , Galactose/química , Galactose/imunologia , Glicoconjugados/imunologia , Haptenos/química , Haptenos/imunologia , Humanos , Imunoglobulina G/imunologia , Imunoterapia , Neoplasias/imunologia , Ramnose/imunologia , Ressonância de Plasmônio de SuperfícieRESUMO
An α-L-rhamnosyl ceramide (1, α-L-RhaCer) has been prepared that was recognized by anti-L-rhamnose (anti-Rha) antibodies. During these studies we explored the use of an α-L-rhamnosyl thioglycoside and a trichloroacetimidate as a glycosyl donors. Subsequently, the acceptors desired for glycosylation, 3-O-benzoylazidosphingosine or 3-O-alloxycarbonylsphingosine, were prepared from D-xylose. The thioglycoside donor, 2,3,4-tri-O-acetyl-1-(4-tolyl)thio-α-L-rhamnopyranoside, and the trichloroacetimidate donor, 2,3,4-tri-O-acetyl-1-(2,2,2-trichloroethanimidate)-α-L-rhamnopyranoside, were synthesized in 50% and 78% yield overall, respectively. The synthesis of the glycosylation acceptor employed an addition-fragmentation olefination that was successfully carried out in 53% yield. With the successful synthesis of key intermediates, α-L-RhaCer (1) was prepared without any insurmountable obstacles. Anti-Rha antibodies were prepared in BALB/c mice by immunizing them with rhamnose-ovalbumin (Rha-Ova) with Sigma Adjuvant System (SAS) and the anti-L-Rha antibodies were isolated from the blood sera. Liposomes and EL4 tumor cells were used as model systems to demonstrate the ability of 1 to insert into a lipid bilayer. The interaction of the liposomes or the EL4 cells with α-L-RhaCer (1) and anti-Rha antibodies were investigated by fluorescence microscopy and flow cytometry, respectively, to confirm the ability of glycolipid 1 to be displayed on the tumor cell surface as well as the ability to be recognized by anti-Rha antibodies.
Assuntos
Anticorpos/imunologia , Manose/análogos & derivados , Ramnose/imunologia , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Linfoma/imunologia , Linfoma/metabolismo , Manose/síntese química , Manose/química , Manose/imunologia , Camundongos , Estrutura Molecular , Ramnose/químicaRESUMO
Adjuvant is an integral part of all vaccine formulations but only a few adjuvants with limited efficacies or application scopes are available. Thus, developing more robust and diverse adjuvants is necessary. To this end, a new class of adjuvants having α- and ß-rhamnose (Rha) attached to the 1- and 6'-positions of monophosphoryl lipid A (MPLA) was designed, synthesized, and immunologically evaluated in mice. The results indicated a synergistic effect of MPLA and Rha, two immunostimulators that function via interacting with toll-like receptor 4 and recruiting endogenous anti-Rha antibodies, respectively. All the tested MPLA-Rha conjugates exhibited potent adjuvant activities to promote antibody production against both protein and carbohydrate antigens. Overall, MPLA-α-Rha exhibited better activities than MPLA-ß-Rha, and 6'-linked conjugates were slightly better than 1-linked ones. Particularly, MPLA-1-α-Rha and MPLA-6'-α-Rha were the most effective adjuvants in promoting IgG antibody responses against protein antigen keyhole limpet hemocyanin and carbohydrate antigen sTn, respectively.
Assuntos
Lipídeo A , Ramnose , Lipídeo A/análogos & derivados , Lipídeo A/química , Lipídeo A/farmacologia , Lipídeo A/imunologia , Animais , Ramnose/química , Ramnose/imunologia , Ramnose/farmacologia , Camundongos , Adjuvantes de Vacinas/química , Adjuvantes de Vacinas/farmacologia , Feminino , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/síntese química , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/sangue , Camundongos Endogâmicos BALB C , Hemocianinas/química , Hemocianinas/imunologiaRESUMO
The complement system is part of our first line of defense against invading pathogens. The strategies used by Enterococcus faecalis to evade recognition by human complement are incompletely understood. In this study, we identified an insertional mutant of the wall teichoic acid (WTA) synthesis gene tagB in E. faecalis V583 that exhibited an increased susceptibility to complement-mediated killing by neutrophils. Further analysis revealed that increased killing of the mutant was due to a higher rate of phagocytosis by neutrophils, which correlated with higher C3b deposition on the bacterial surface. Our studies indicated that complement activation via the lectin pathway was much stronger on the tagB mutant compared with wild type. In concordance, we found an increased binding of the key lectin pathway components mannose-binding lectin and mannose-binding lectin-associated serine protease-2 (MASP-2) on the mutant. To understand the mechanism of lectin pathway inhibition by E. faecalis, we purified and characterized cell wall carbohydrates of E. faecalis wild type and V583ΔtagB. NMR analysis revealed that the mutant strain lacked two WTAs with a repeating unit of â6)[α-l-Rhap-(1â3)]ß-D-GalpNAc-(1â5)-Rbo-1-P and â6) ß-D-Glcp-(1â3) [α-D-Glcp-(1â4)]-ß-D-GalpNAc-(1â5)-Rbo-1-Pâ, respectively (Rbo, ribitol). In addition, compositional changes in the enterococcal rhamnopolysaccharide were noticed. Our study indicates that in E. faecalis, modification of peptidoglycan by secondary cell wall polymers is critical to evade recognition by the complement system.
Assuntos
Parede Celular/imunologia , Lectina de Ligação a Manose da Via do Complemento/imunologia , Enterococcus faecalis/imunologia , Ácidos Teicoicos/imunologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Sequência de Carboidratos , Parede Celular/química , Parede Celular/metabolismo , Complemento C3b/imunologia , Complemento C3b/metabolismo , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Humanos , Lipoproteínas/genética , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Espectroscopia de Ressonância Magnética , Lectina de Ligação a Manose/imunologia , Lectina de Ligação a Manose/metabolismo , Dados de Sequência Molecular , Mutação , Neutrófilos/imunologia , Neutrófilos/metabolismo , Oligossacarídeos/imunologia , Oligossacarídeos/metabolismo , Peptidoglicano/imunologia , Peptidoglicano/metabolismo , Fagocitose/imunologia , Polímeros/metabolismo , Polissacarídeos/imunologia , Polissacarídeos/metabolismo , Ligação Proteica , Coelhos , Ramnose/imunologia , Ramnose/metabolismo , Ácidos Teicoicos/metabolismoRESUMO
MUC1 variable number tandem repeats (VNTRs) conjugated to tumor-associated carbohydrate antigens (TACAs) have been shown to break self-tolerance in humanized MUC1 transgenic mice. Therefore, we hypothesize that a MUC1 VNTR TACA-conjugate can be successfully formulated into a liposome-based anticancer vaccine. The immunogenicity of the vaccine should be further augmented by incorporating surface-displayed l-rhamnose (Rha) epitopes onto the liposomes to take advantage of a natural antibody-dependent antigen uptake mechanism. To validate our hypothesis, we synthesized a 20-amino-acid MUC1 glycopeptide containing a GalNAc-O-Thr (Tn) TACA by SPPS and conjugated it to a functionalized Toll-like receptor ligand (TLRL). An l-Rha-cholesterol conjugate was prepared using tetra(ethylene glycol) (TEG) as a linker. The liposome-based anticancer vaccine was formulated by the extrusion method using TLRL-MUC1-Tn conjugate, Rha-TEG-cholesterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a total lipid concentration of 30 mM. The stability, homogeneity, and size characterization of the liposomes was evaluated by SEM and DLS measurements. The formulated liposomes demonstrated positive binding with both anti-Rha and mouse anti-human MUC1 antibodies. Groups of female BALB/c mice were immunized and boosted with a rhamnose-Ficoll (Rha-Ficoll) conjugate formulated with alum as adjuvant to generate the appropriate concentration of anti-Rha antibodies in the mice. Anti-Rha antibody titers were >25-fold higher in the groups of mice immunized with the Rha-Ficoll conjugate than the nonimmunized control groups. The mice were then immunized with the TLRL-MUC1-Tn liposomal vaccine formulated either with or without the surface displaying Rha epitopes. Sera collected from the groups of mice initially immunized with Rha-Ficoll and later vaccinated with the Rha-displaying TLRL-MUC1-Tn liposomes showed a >8-fold increase in both anti-MUC1-Tn and anti-Tn antibody titers in comparison to the groups of mice that did not receive Rha-Ficoll. T-cells from BALB/c mice primed with a MUC1-Tn peptide demonstrated increased proliferation to the Rha-liposomal vaccine in the presence of antibodies isolated from Rha-Ficoll immunized mice compared to nonimmune mice, supporting the proposed effect on antigen presentation. The anti-MUC1-Tn antibodies in the vaccinated mice serum recognized MUC1 on human leukemia U266 cells. Because this vaccine uses separate rhamnose and antigenic epitope components, the vaccine can easily be targeted to different antigens or epitopes by changing the peptide without having to change the other components.
Assuntos
Mucina-1/química , Mucina-1/imunologia , Ramnose/síntese química , Ramnose/imunologia , Animais , Vacinas Anticâncer/síntese química , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Glicopeptídeos/síntese química , Glicopeptídeos/imunologia , Humanos , Imunização/métodos , Lipossomos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Carbohydrates are generally considered to be poorly immunogenic. Therefore, new approaches for enhancing their immunogenicity are important for the development of carbohydrates as vaccine components. We hypothesized that conjugation of an l-rhamnose (Rha) moiety to a carbohydrate antigen would enhance the antigenicity of the antigen in mice possessing anti-Rha antibodies via an antibody-dependent antigen uptake mechanism. To explore this hypothesis, we synthesized a single-molecule three-component vaccine containing the GalNAc-O-Thr (Tn) tumor-specific antigen, a 20 amino acid helper T-cell epitope (YAF) derived from an outer-membrane protein of Neisseria meningitides, and a Rha moiety. The vaccine was synthesized by automated Fmoc-based solid-phase peptide synthesis and deacetylated by brief treatment with NaOMe. Groups of female BALB/c mice were immunized and boosted with Rha-ovalbumin (Rha-OVA) formulated with either TiterMax Gold or Sigma Adjuvant System for a period of 35 days in order to determine optimal conditions for generating anti-Rha titers in mice. Anti-Rha antibody titers were >100 fold higher in groups of mice immunized with Rha-OVA than in the control groups. Mice producing anti-Rha were challenged with Rha-YAF-Tn or YAF-Tn. Sera collected from the groups initially immunized with Rha-OVA and later challenged with Rha-YAF-Tn showed a 2-fold increase in anti-Tn titer at 1/100 serum dilution relative to mice not immunized with Rha-OVA. An in vitro T-cell proliferation study using cells primed with either Rha-YAF-Tn or YAF-Tn was done to examine possible differences in antigen uptake and presentation due to anti-Rha antibody and chemical modification. Proliferation of T cells was stimulated by a 10-fold lower antigen concentration in the presence of Rha antibodies. The results strongly suggest that T cells present in the spleen were presented with higher concentrations of Rha-YAF-Tn as a result of the presence of the anti-Rha antibodies.
Assuntos
Anticorpos/imunologia , Ramnose/imunologia , Vacinas/síntese química , Vacinas/imunologia , Sequência de Aminoácidos , Animais , Antígenos Glicosídicos Associados a Tumores/metabolismo , Proliferação de Células , Técnicas de Química Sintética , Epitopos/imunologia , Feminino , Hemocianinas/metabolismo , Camundongos , Dados de Sequência Molecular , Ramnose/química , Soroalbumina Bovina/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Receptores Toll-Like/metabolismo , Vacinas/química , Vacinas/metabolismoRESUMO
Rhamnose and sTn antigen were co-conjugated to bovine serum albumin (BSA), a weakly immunogenic carrier protein, for cancer vaccine development. The immune responses against sTn have been significantly augmented with the involvement of Rha-specific antibodies to enhance antigen uptake.
Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Ramnose/imunologia , Soroalbumina Bovina/imunologia , Animais , Reações Antígeno-Anticorpo , Antígenos Glicosídicos Associados a Tumores/genética , Bovinos , Humanos , Imunidade , Células MCF-7 , Camundongos , Conformação Molecular , Ramnose/químicaRESUMO
Utilizing natural antibodies to augment vaccine immunogenicity is a promising approach toward cancer immunotherapy. Anti-rhamnose (anti-Rha) antibodies are some of the most common natural anti-carbohydrate antibodies present in human serum. Therefore, rhamnose can be utilized as a targeting moiety for a rhamnose-containing vaccine to prepare an effective vaccine formulation. It was shown previously that anti-Rha antibody generated in mice binds effectively with Rha-conjugated vaccine and is picked up by antigen presenting cells (APCs) through stimulatory Fc receptors. This leads to the effective uptake and processing of antigen and eventually presentation by major histocompatibility complex (MHC) molecules. In this article, we show that natural human anti-Rha antibodies can also be used in a similar mechanism and immunogenicity can be enhanced by targeting Rha-conjugated antigens. In doing so, we have purified human anti-Rha antibodies from human serum using a rhamnose affinity column. In vitro, human anti-Rha antibodies are shown to enhance the uptake of a model antigen, Rha-ovalbumin (Rha-Ova), by APCs. In vivo, they improved the priming of CD4+ T cells to Rha-Ova in comparison to non-anti-Rha human antibodies. Additionally, increased priming of both CD4+ and CD8+ T cells toward the cancer antigen MUC1-Tn was observed in mice that received human anti-Rha antibodies prior to vaccination with a rhamnose-modified MUC1-Tn cancer vaccine. The vaccine conjugate contained Pam3CysSK4, a Toll-like receptor (TLR) agonist linked via copper-free cycloaddition chemistry to a 20-amino-acid glycopeptide derived from the tumor marker MUC-1 containing the tumor-associated carbohydrate antigen α- N-acetyl galactosamine (GalNAc). The primed CD8+ T cells released IFN-γ and killed tumor cells. Therefore, we have confirmed that human anti-Rha antibodies can be effectively utilized as a targeting moiety for making an effective vaccine.
Assuntos
Anticorpos/imunologia , Vacinas Anticâncer/imunologia , Imunogenicidade da Vacina , Ramnose/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos Glicosídicos Associados a Tumores/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mucina-1/imunologia , Neoplasias/imunologia , Neoplasias/prevenção & controle , Ovalbumina/imunologiaRESUMO
Installation of an antibody-recruiting moiety on the surface of disease-relevant cells can lead to the selective destruction of targets by the immune system. Such an approach can be an alternative strategy to traditional chemotherapeutics in cancer therapy and possibly other diseases. Herein we describe the development of a new strategy to selectively label targets with an antibody-recruiting moiety through its covalent and stable installation, complementing existing methods of employing reversible binding. This is achieved through selective delivery of 1,3,4- O-acetyl- N-azidoacetylmannosamine (Ac3ManNAz) to folate receptor-overexpressing cells using an Ac3ManNAz-folate conjugate via a cleavable linker. As such, Ac3ManNAz is converted to cell surface glycan bearing an azido group, which serves as an anchor to introduce l-rhamnose (Rha), a hapten, via a click reaction with aza-dibenzocyclooctyne (DBCO)-Rha. We tested this method in several cell lines including KB, HEK-293, and MCF7 and were able to demonstrate the following: 1) Rha can be selectively installed to the folate receptor overexpressing cell surface and 2) the Rha installed on the target surface can recruit anti-rhamnose (anti-Rha) antibodies, leading to the destruction of target cells via complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP).
Assuntos
Imunidade Adaptativa/imunologia , Anticorpos/imunologia , Biomarcadores Tumorais/imunologia , Receptores de Folato com Âncoras de GPI/imunologia , Ramnose/imunologia , Azidas/química , Linhagem Celular Tumoral , Química Click , Ativação do Complemento/imunologia , Ciclo-Octanos/síntese química , Ciclo-Octanos/química , Células HEK293 , Haptenos , Hexosaminas/química , Humanos , Neoplasias/terapia , Fagocitose/imunologia , Ramnose/síntese química , Ramnose/químicaRESUMO
Recruitment of human endogenous antibodies to target and eliminate tumor cells is a promising therapeutic strategy in the biomedical field. Current antibody-recruiting molecules are typically bi-functional agents that utilize cell-surface receptor binding property for targeting. This approach has intrinsic limitations due to the heterogeneity of tumor cells and the limited number of receptors on the cell surface. Here we report a targeting strategy based on remodeling of cell surface glycans through metabolic engineering and bioorthogonal chemical ligation. In vitro cultured tumor cells and in vivo xenograft tumors were actively remodeled with rhamnose carbohydrate epitopes, which were capable of recruiting endogenous anti-rhamnose antibodies and activating complement-mediated cell cytotoxicity. This study highlights the therapeutic potential for modulating endogenous immune response through cell-surface glycan engineering.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos/imunologia , Polissacarídeos/imunologia , Ramnose/imunologia , Células A549 , Animais , Linhagem Celular Tumoral , Epitopos/imunologia , Células HeLa , Humanos , Lipossomos , Engenharia Metabólica , Camundongos , Camundongos Endogâmicos BALB C , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recruitment of antibodies in human immune systems for targeted destruction of tumor cells has emerged as an exciting area of research due to its low occurrence of side effects, high efficacy, and high specificity. The presence of large amounts of anticarbohydrate natural antibodies in human sera has prompted research efforts to utilize carbohydrate epitopes for immune recruitment. Here, we have developed a general strategy for specific targeted destruction of tumor cells based on rhamnose-functionalized liposomes. Tumor cells artificially decorated with rhamnose epitopes were subjected to complement-mediated cytotoxicity in vitro and showed delayed tumor growth in vivo. This study highlights the therapeutic potential for activation of endogenous immune response through cell-surface glycan engineering.
Assuntos
Proteínas do Sistema Complemento/imunologia , Lipossomos/uso terapêutico , Neoplasias/imunologia , Neoplasias/terapia , Ramnose/uso terapêutico , Células A549 , Animais , Anticorpos/imunologia , Citotoxicidade Imunológica , Epitopos/imunologia , Feminino , Células HeLa , Humanos , Imunoterapia , Lipossomos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/patologia , Ramnose/imunologiaRESUMO
The determination of the chemical nature of immunodeterminant groups of carbohydrate antigens has been achieved by a micro method coupling inhibition and double diffusion in agar. This method has been tested with antigens which react with anti-lactose, anti-galactose, anti-N-acetyl-glucosamine and antirhamnose antibodies. The analysis can be performed with as little as 10 micrograms of inhibitor, 0.2 microgram of antigen and 10 micrograms of antibody. The procedure has also been used for the identification of the determinant groups of 2 antigens with a phosphoglycan structure. The determinants of these antigens have been found to be N-acetyl-beta-glucosamine 1-phosphate and beta-glucose 1-phosphate. The glycosyl 1-phosphate units are novel types of antigenic determinants and antigens with such determinants should be useful for investigating the interactions of antigens with homologous antibodies. The specificity of monoclonal antibodies directed at an O-antigen has been determined by use of the coupled method.
Assuntos
Ágar , Epitopos/análise , Imunodifusão/métodos , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Ligação Competitiva , Epitopos/imunologia , Galactose/imunologia , Lactose/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , Ramnose/imunologia , Streptococcus/imunologia , Streptococcus pneumoniae/imunologiaRESUMO
The grouping antigens from five of the seven serological groups of lactobacilli have been defined with respect to the carbohydrate units responsible for serological specificity. When the antigens are polysaccharides, specificity is more absolute than with the teichoic acids where cross-reactions may occur because of the same or similar carbohydrate substituents, or more particularly because of the common "backbone" structure of the glycerol teichoic acids. This glycerol phosphate "backbone" accounts for a number of reports on the presence of a "common antigen" in gram-positive organisms and their culture fluids, with the amount of extracellular material being particularly high for S mutans strains.
Assuntos
Antígenos de Bactérias/classificação , Reações Cruzadas , Lactobacillus/imunologia , Boca/microbiologia , Animais , Membrana Celular/imunologia , Parede Celular/imunologia , Epitopos/classificação , Lactobacillus/ultraestrutura , Polissacarídeos Bacterianos/imunologia , Coelhos , Ramnose/imunologia , Ácidos Teicoicos/imunologiaRESUMO
The extracellular antigens of Actinobacillus actinomycetemcomitans Y4 (serotype b) contain a 37-kDa protein which is a major target for IgGs from patients suffering from severe alveolar bone loss. Since the 37-kDa protein has not been studied sufficiently, our investigation focused on its characteristics, e.g., its localization, specificity, and whether it directly stimulates macrophages to produce cytokines. The 37-kDa protein was purified from the culture supernatant of the Y4 strain by means of chromatofocusing and gel filtration. The 37-kDa protein is a unique glycoprotein which forms immune complexes with monoclonal antibodies against rhamnose-fucose polysaccharide. Patients with A. actinomycetemcomitans-associated periodontitis had higher antibody titers to the purified 37-kDa protein than healthy subjects (p < 0.001). Anti-37-kDa protein antibodies recognized a 37-kDa band in the cytosolic, ribosomal, and total membrane fractions from Y4 cells. Extracellular substances from other strains of A. actinomycetemcomitans (serotypes a and c) also reacted in the Western blots, but Haemophilus spp. or several periodontopathic bacteria did not. These results suggested that the 37-kDa protein is a cytosolic protein that is passed through the cell membrane, and its protein portion is specific for A. actinomycetemcomitans but common to serotypes. This protein induced Il-1 beta, Il-6, and TNF-alpha release from murine macrophages. The Il-6-inducing activity of the 37-kDa protein was higher than that of LPS. These findings suggested that the 37-kDa protein which is released from live cells plays a role in A. actinomycetemcomitans-associated periodontitis, as antigen inducing the release of inflammatory cytokines which are associated with alveolar bone loss.
Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Superfície/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Interleucina-1/biossíntese , Interleucina-6/biossíntese , Macrófagos/imunologia , Glicoproteínas de Membrana/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Aggregatibacter actinomycetemcomitans/classificação , Perda do Osso Alveolar/imunologia , Perda do Osso Alveolar/microbiologia , Animais , Anticorpos Antibacterianos/análise , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/imunologia , Antígenos de Bactérias/isolamento & purificação , Antígenos de Superfície/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Western Blotting , Membrana Celular/imunologia , Cromatografia em Gel , Citosol/imunologia , Epitopos , Feminino , Fucose/imunologia , Haemophilus/imunologia , Imunoglobulina G/imunologia , Interleucina-1/imunologia , Interleucina-6/imunologia , Focalização Isoelétrica , Lipopolissacarídeos/imunologia , Glicoproteínas de Membrana/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Periodontite/imunologia , Periodontite/microbiologia , Polissacarídeos/imunologia , Ramnose/imunologia , Ribossomos/imunologia , Sorotipagem , Fator de Necrose Tumoral alfa/imunologiaRESUMO
L-Rhamnose (6-deoxy-L-mannose) is a constituent carbohydrate unit of microbial, immunogenic heteroglycans and lipopolysaccharides, and often functions as the immunodeterminant group of such immunogens. Two types of anti-rhamnose antibody have now been isolated by affinity chromatography of immune sera obtained from rabbits immunized with vaccines of Streptococcus mutans, strain KI-R, and Streptococcus pneumoniae, type 32. The antibodies of one type were directed at a glycan of L-rhamnose, D-glucose, and D-galactose in the cell wall of S. mutans, and those of the other type, against a capsular glycan of L-rhamnose and D-glucose from S. pneumoniae. The two types of anti-rhamnose antibody were immunologically distinct, and showed no reciprocal cross-reactivity. Additional properties of the two types of antibody were determined; thus, both types of antibody were of the IgG class of immunoglobulins, both possessed molecular weights of 1.45 X 10(5), and both consisted of multiple or isomeric forms.
Assuntos
Especificidade de Anticorpos , Ramnose/imunologia , Streptococcus pneumoniae/imunologia , Parede Celular/imunologia , Cromatografia de Afinidade , Imunodifusão , Lipopolissacarídeos/imunologia , Peso MolecularRESUMO
Methyl 4-azido-4,6-dideoxy-3-O-benzyl-alpha-D-mannopyranoside and its analogous 3-O-(4-methoxybenzyl) derivative were methylated and the 2-O-methyl derivatives formed were converted into methyl 4-amino-4,6-dideoxy-2-O-methyl-alpha-D- mannopyranoside [sequence: see text]. Reaction of the latter with 3-deoxy-L-glycero-tetronolactone gave the methyl glycoside of 4,6-dideoxy-4-(3-deoxy-L-glycero- tetronamido)-2-methyl-alpha-D-mannopyranose [sequence: see text], the monosaccharide that is reported to be the terminal moiety of the O-specific polysaccharide of Vibrio cholerae O:1, serotype Ogawa. The unit cell packing of the compound, which crystallized as a monohydrate, differs from that of the previously described crystalline compound lacking the 2-O-methyl group. The unmethylated sugar is the terminal moiety of the O-specific polysaccharide of Vibrio cholerae O:1, serotype Inaba. The crystal structure of methyl 4,6-dideoxy-2-O- methyl-4-trifluoroacetamido-alpha-D-mannopyranoside [sequence: see text] is also described.
Assuntos
Antígenos O/química , Ramnose/análogos & derivados , Vibrio cholerae/química , Antígenos de Bactérias/química , Configuração de Carboidratos , Cristalografia por Raios X , Epitopos/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Antígenos O/imunologia , Ramnose/síntese química , Ramnose/química , Ramnose/imunologia , Sorotipagem , Vibrio cholerae/imunologiaRESUMO
In the sera of patients with recurrent rheumocarditis, and especially in cases of primary rheumatism, the level of antibodies to group A streptococcal polysaccharide (A-PS) has been found, according to the results of the enzyme immunoassay, to be considerably higher than in the sera of healthy donors. The level of antibodies to rhamnose determinants (RD) of A-PS has been determined by the inhibition of the immunoenzyme reaction with A-PS under the influence of a variant of group A streptococcus and rhamnose disaccharides with the bonds alpha 1-2 and alpha 1-3. In patients with recurrent rheumocarditis the level of antibodies to A-PS has been shown to be considerably higher than in healthy donors having these antibodies. In acute primary rheumatism a high level of antibodies to A-PS has been detected only in a few cases, and at the same time the prevalence of antibodies to the specific RD of A-PS, bound with beta-N-acetylglucosamine, is observed. In the sera of patients with recurrent rheumocarditis and donors having a high content of antibodies to the rhamnose site of A-PS antibodies, seemingly active against at least two RD, have been detected. In acute primary rheumatism an insignificant amount of antibodies to the rhamnose site of A-PS may probably cause the autoimmune process accompanying rheumatism. This suggestion is substantiated by the previously established capacity of these antibodies for inducing the suppression of cytotoxic cell reactions to microbial antigens.
Assuntos
Anticorpos Antibacterianos/sangue , Doadores de Sangue , Epitopos/imunologia , Polissacarídeos Bacterianos/imunologia , Ramnose/imunologia , Doenças Reumáticas/imunologia , Streptococcus pyogenes/imunologia , Doença Aguda , Adolescente , Adulto , Humanos , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Recidiva , Cardiopatia Reumática/imunologiaRESUMO
The data obtained for the first time in our studies indicate that the production of antibodies to group A streptococcal polysaccharide (A-PS), one of the cross-reacting streptococcal antigens, may suppress delayed hypersensitivity (DH) to microbial antigens. The existence of sharply pronounced correlation between the suppression of DH and the presence of antibodies to the rhamnose area of A-PS in the blood of BALB/c mice immunized with the pepsin-treated culture of group A streptococci has been shown. The suppression of DH is absent in the immunized animals of the same group whose blood contains antibodies to the determinant, specific for A-PS. As revealed in this study, the effect of the suppression of antigen-specific cytotoxicity linked with DH to BCG antigens can be reproduced by mixing lymph node cells taken from these two groups of the animals. The data thus obtained are possibly linked with the activation of nonspecific T suppressors in the production of antibodies to the rhamnose determinants of A-PS in the animals immunized with streptococci. The mechanism of the newly discovered phenomenon is discussed.