Disease-associated Carbohydrate Antigen And Antibody:Preparation,Analysis And Immunological Applications

Carbohydrate is not only the important structural constituent and source of energy,but also a significant participant in the biological process.Herein,carbohydrate antigen and anti-carbohydrate antibody show close relationships with a series of pathological processes like pathogen infection and cell cancerization.Therefore,study of disease-associated carbohydrate antigens and anti-carbohydrate antibodies give important benefits towards diagnosis,analysis and therapy of diseases.Therefore,this thesis focuses on disease-associated carbohydrate antigen and antibody:preparation,analysis and immunological application.Specifically,the thesis is divided into three research projects:Part One is the preparation and analysis of antibodies against xenoantigen L-Rha;Part Two is the preparation and immunological application of L-Rha modified melanoma tumor antigen;Part Three is the preparation and application of DNA encoded glycan library.Part 1 Specificity and bactericidal activity of human anti-L-Rha antibodiesL-Rha exists widely in plants and bacteria.In bacteria,L-Rha is commonly bound to other sugars or lipids to form capsule polysaccharides(CPS),lipopolysaccharides(LPS)or glycolipid.The biosynthetic pathway of L-Rha does not exist in human.As xenoantigen,L-Rha is strongly against by high level of natural occurring anti-L-Rha antibodies in human sera.Despite knowledge about the role of natural occurring antibodies in pathogen elimination,there is lack of clarity specific to anti-L-Rha antibodies.In this part,we evaluated the specificity and bactericidal activity of anti-LRha antibodies.Besides,we analyzed L-Rha distribution in bacterial serotypes and outbreak frequencies of different bacterial serotypes.Specifically,we firstly purified anti-L-Rha antibodies(including IgG and IgM,mainly IgG)from healthy human sera.Besides,we purified anti-L-Rha IgG from human umbilical cord sera,which mean that infant could acquire anti-L-Rha IgG from its mother.Secondly,via interaction with various monosaccharides,anti-L-Rha antibodies bound specifically with L-Rha or its derivatives and its analogs(e.g.,L-Ara),but not D-Rha and other D-monosaccharides.Via interaction with several bacterial LPS,anti-L-Rha antibodies bound specifically with O-antigen had L-Rha or its derivatives at the non-reducing end.Besides,via complement-mediated bactericidal activity assay,anti-L-Rha antibodies specifically kill bacteria whose O-antigen had L-Rha or its derivatives at the non-reducing end.Lastly,the distribution of L-Rha or its derivatives in bacterial serotypes has correlation with the outbreak frequencies of different bacteria serotypes.The results in this part show that anti-L-Rha antibodies are broad-spectrum antibacteria antibodies,which plays important roles in bacteria elimination and normal immunity.The level of anti-L-Rha antibodies in blood could be a standard of human immunity level.Meanwhile,the structure analysis of bacterial surface polysaccharides(see if it contains L-Rha or its derivatives)has values towards pathogenicity or infectivity of bacteria.Besides,some bacteria whose O-antigen have L-Rha or its derivatives at the non-reducing end commonly appear as opportunistic pathogens,which show pathogenicity towards human with impaired immune system(probably weak or inactive anti-L-Rha antibodies).Therefore,anti-L-Rha antibodies might be used as drug to treat bacterial infections due to lack of anti-L-Rha antibodies.Besides anti-L-Rha antibodies,many other natural occurring anti-glycan antibodies also exist in human bodies.Systematic studies of the relationships between natural occurring antiglycan antibodies and pathogenicity of bacteria,have significance to prevent and treat bacterial infections.Part 2 Anti-tumor activity of L-Rha modified melanoma antigen MAGE-A3Melanoma antigen(MAGE)is highly expressed in many types of tumor cells.Herein,MAGE-A3 is widely expressed in a large proportion of tumor types such as melanoma,myeloma,bladder carcinoma and lung carcinoma.MAGE-A3 plays a definite role in a tumor-killing effect by inducing specific cytotoxic T cells(CTLs).MAGE-A3 is drawing many interests for tumor immunotherapy.However,tumor regression responses were observed in less than 30%melanoma patients vaccinated with MAGE-A3 peptide or MAGE-A3 recombinant protein.In addition,the clinical study of MAGE-A3 was recently suspended due to relatively weak patient responses.The reason is that MAGE-A3 is autoantigen with weak immunogenicity,which can not induce enough CTLs.Thus,there is an urgent need to increase the immunogenicity of MAGE-A3 associated tumor vaccines to enhance its anti-tumor activity.One avenue for boosting the immune responses to tumor antigens of interest involves linking of xenoantigens to them.By injecting xenoantigen-tumor antigen,the xenoantigen binds to abundant natural occurring antibodies existing in body and facilitates the uptake of the tumor antigen by antigen presenting cells(APCs).In this part,xenoantigen L-Rha was conjugated to truncated MAGE-A3 to obtain L-Rha modified tMAGE-A3.Furthermore,the anti-tumor activity of Rha-tMAGE-A3 was investigated in the presence of anti-L-Rha antibodies in vitro.Specially,we firstly expressed truncated MAGE-A3(tMAGE-A3),which could bind with commercial anti-MAGE-A3 antibodies,demonstrating that our selected tMAGE-A3 retained the immunoreactivity of MAGE-A3.Besides,Rha-tMAGE-A3 could also bind with anti-MAGE-A3 antibodies,demonstrating that L-Rha modification did not cover the antigen recognition sites of MAGE-A3.Secondly,NHSactivated L-Rha could efficiently conjugate with any peptides or proteins with free amino groups.In this study,L-Rha had a loading ratio of 9 on Rha-tMAGE-A3,with loading efficiency of 75%.Our previous studies showed that L-Rha could conjugate with tumor cells on surface.Thus,this method showed feasibility.Lastly,by anti-L-Rha antibody-mediated phagocytosis by macrophage cells,Rha-tAMGE-A3 could be better recognized and presented by APCs,but tMAGE-A3 can not.Besides,we found that Rha-tMAGE-A3 stimulated PBMCs could kill melanoma cells A3 75 efficiently in the presence of anti-L-Rha antibodies.The results in this part show that Rha-tMAGE-A3 has enhanced immunogenicity and anti-tumor activity.As immune enhancer,L-Rha shows significant values in tumor immunotherapy.Part 3 Preparation and application of DNA encoded glycan libraryThe interactions between glycan and glycan binding protein(GBP)play important roles in diverse cellular functions such as molecular recognition,adhesion,inflammation.Expression patterns of glycans are widely altered in cancer,retrovirus infection,atherosclerosis,thrombosis,diabetes,arthritis and other diseases.Concentration of related anti-glycan antibodies might also change accordingly.Therefore,study of glycan-GBP interaction means a lot for diagnosis and therapy of diseases.The methods to study the interactions between glycan antigen and GBP are limited,and all those methods including ELISA,glycan microarray fix the glycan antigens to the solid phase,which sometimes interfere its binding or interaction with GBP.Meanwhile,each glycan microarray chip can only test one sample at one time.Besides,some very tiny samples even cannot be detected by glycan microarray.Recently,DNA-labeled chemicals are used to detect very tiny samples by DNA amplification.Besides,DNA encoded library(DEL)technology is widely used for high-throughput and high-content analyses of target chemicals by many pharmaceutical companies.In this part,via combination of DNA labeled chemicals and DEL,we developed a novel method to detect glycan-GBP interaction---DNA encoded glycan library(DEGL),which can provide very sensitive,high-throughput and high-content analysis.Specially,each glycan antigen in DEGL was linked with a unique piece of DNA.After screening of DEGL with GBP,and then sequencing binding glycan-DNA via next generation sequencing(NGS)technologies,the glycan structures of binding glycan-DNA could be acquired.Specifically,we firstly synthesized 12 glycan antigens,and linked each glycan antigen with a unique piece of DNA,to obtain monovalent glycan-DNA(one glycan antigen linked with a piece of DNA)and multivalent glycan-DNA(three glycan antigens linked with a piece of DNA).By qPCR,monovalent blood type glycan antigen-DNA could not only detect commercial blood type antibodies,but also identify blood types in blood and saliva with 100%accuracy.Besides,monovalent-DNA or multivalent-DNA could detect anti-Globo H(VK9)antibodies and lectins HPA,ECA.The results above proved that,glycan antigen-DNA could not only detect standard antibodies,but also complicated and low antibody content samples like sera and even saliva.Further,monovalent glycan-DNA were mixed to prepare monovalent-DEGL.Similarly,multivalent glycan-DNA were mixed to prepare multivalent-DEGL.Then,monovalent-DEGL or multivalent-DEGL were used to react with antibodies(antiGlobo H antibodies,anti-L-Rha antibodies)and lectins(HPA,ECA),and analyzed by NGS.The results above proved that,multivalent-DEGL has higher enrichment folds(or higher resolution)than monovalent-DEGL.Besides,multivalent-DEGL could detect weak binding signals(glycan antigen BB3 and anti-Globo H antibodies)that could not be found by methods like ELISA by significant signal amplification.The preparation of DEGL was quite different from traditional DEL preparation methods(based on Split&Pool).Herein,chemoenzymatic synthesis of glycan antigens,encoding logic of sugar-encoding DNA,conjugation between glycan antigen and DNA(click chemistry),purification(HPLC)and detection(ELISA,MALDI-TOF,bioanalyzer,qPCR,NGS)of glycan-DNA and other novel methods provided complete solutions towards DEGL preparation.The results in this part show that,DEGL,especially multivalent-DEGL,can be successfully applied into the detection of antibodies,lectins and complicated samples with very sensitive,high-throughput and high-content analysis.Next,via the DEGL preparation methods provided in this part,DEGL would have widespread applications by increasing the glycan antigen number of DEGL.Predictably in future,DEGL will has huge values in drug discovery and disease diagnosis.In conclusion,this thesis 1)explores the specificity and bactericidal activity of anti-L-Rha antibodies,and finds its relationships with bacterial pathogenicity under the preparation and analysis of anti-L-Rha antibodies;2)provides a new vaccine design idea for tumor(including melanoma)treatment under the preparation and immunological application of L-Rha-modified melanoma antigen;3)provides a new technology to detect the glycan antigen-GBP interactions under the preparation and application of DEGL.All results above have significant merits for diagnosis,analysis and treatment of diseases.