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Taxonomic group: bacteria / Firmicutes (Phylum: Firmicutes)
Associated disease: infection due to Streptococcus pneumoniae [ICD11: XN3PW

]

NCBI PubMed ID: 34838818
Publication DOI: 10.1016/j.jbc.2021.101453
Journal NLM ID: 2985121R
Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
Correspondence: F.Y. Avci <avci

uga.edu>
Institutions: Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, USA, Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA, Center for Molecular Medicine, University of Georgia, Athens, Georgia, USA, Fina Biosolutions, LLC, Rockville, Maryland, USA

In the preparation of commercial conjugate vaccines, capsular polysaccharides (CPSs) must undergo chemical modification to generate the reactive groups necessary for covalent attachment to a protein carrier. One of the most common approaches employed for this derivatization is sodium periodate (NaIO4) oxidation of vicinal diols found within CPS structures. This procedure is largely random and structurally damaging, potentially resulting in significant changes in the CPS structure and therefore its antigenicity. Additionally, periodate activation of CPS often gives rise to heterogeneous conjugate vaccine products with variable efficacy. Here, we explore the use of an alternative agent, galactose oxidase (GOase) isolated from Fusarium sp. in a chemoenzymatic approach to generate a conjugate vaccine against Streptococcus pneumoniae. Using a colorimetric assay and NMR spectroscopy, we found that GOase generated aldehyde motifs on the CPS of S. pneumoniae serotype 14 (Pn14p) in a site-specific and reversible fashion. Direct comparison of Pn14p derivatized by either GOase or NaIO4 illustrates the functionally deleterious role chemical oxidation can have on CPS structures. Immunization with the conjugate synthesized using GOase provided a markedly improved humoral response over the traditional periodate-oxidized group. Further, functional protection was validated in vitro by measure of opsonophagocytic killing and in vivo through a lethality challenge in mice. Overall, this work introduces a strategy for glycoconjugate development that overcomes limitations previously known to play a role in the current approach of vaccine design.

Streptococcus pneumoniae, capsular polysaccharide, pneumonia, glycoconjugate vaccine, vaccine development, galactose oxidase

Structure type: polymer chemical repeating unit
Location inside paper: Fig. 1
Compound class: O-polysaccharide, O-antigen, CPS
Contained glycoepitopes: IEDB_130646,IEDB_130697,IEDB_135813,IEDB_136044,IEDB_137340,IEDB_137472,IEDB_137776,IEDB_1391966,IEDB_1392542,IEDB_140108,IEDB_140110,IEDB_140122,IEDB_141794,IEDB_141807,IEDB_142351,IEDB_142487,IEDB_142488,IEDB_143634,IEDB_146664,IEDB_149138,IEDB_149139,IEDB_149141,IEDB_149142,IEDB_149143,IEDB_149144,IEDB_149145,IEDB_149147,IEDB_149148,IEDB_149150,IEDB_149151,IEDB_151531,IEDB_190606,IEDB_983931,SB_145,SB_165,SB_166,SB_173,SB_187,SB_192,SB_195,SB_30,SB_6,SB_7,SB_88

Methods: 1H NMR, NMR-2D, ELISA, immunization, conjugation, enzymatic activity assay, opsonophagocytic killing assay, Dot blot assay
Comments, role: published polymerization frame was shifted for conformity with other records

NCBI Taxonomy refs (TaxIDs): 1313
Reference(s) to other database(s): GTC:G52755VD, GlycomeDB:2039, CCSD:11172, CBank-STR:12723
Show glycosyltransferases

There is only one chemically distinct structure:

[*]O[C@@H]1[C@@H](CO)O[C@@H](OC[C@H]2O[C@@H](O[C@@H]3[C@@H](O)[C@H]([*])O[C@H](CO)[C@@H]3O)[C@H](NC(C)=O)[C@@H](O)[C@@H]2O[C@@H]2O[C@H](CO)[C@H](O)[C@H](O)[C@H]2O)[C@H](O)[C@H]1O

SVG MW SMILES 3D mol Ionize

Taxonomic group: bacteria / Firmicutes (Phylum: Firmicutes)
Associated disease: infection due to Streptococcus pneumoniae [ICD11: XN3PW

]

NCBI PubMed ID: 34061603
Publication DOI: 10.1128/mBio.00800-21
Journal NLM ID: 101519231
Publisher: Washington, DC: American Society for Microbiology
Correspondence: Fikri Y. Avci <avci

uga.edu>
Institutions: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA, Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA, Division of Infectious Diseases, Department of Medicine, Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, New York, USA

Capsular polysaccharides (CPSs) are major virulence factors that decorate the surfaces of many human bacterial pathogens. In their pure form or as glycoconjugate vaccines, CPSs are extensively used in vaccines deployed in clinical practice worldwide. However, our understanding of the structural requirements for interactions between CPSs and antibodies is limited. A longstanding model based on comprehensive observations of antibody repertoires binding to CPSs is that antibodies expressing heavy chain variable gene family 3 (VH3) predominate in these binding interactions in humans and VH3 homologs in mice. Toward understanding this highly conserved interaction, we generated a panel of mouse monoclonal antibodies (MAb) against Streptococcus pneumoniae serotype 3 CPS, determined an X-ray crystal structure of a protective MAb in complex with a hexasaccharide derived from enzymatic hydrolysis of the polysaccharide, and elucidated the structural requirements for this binding interaction. The crystal structure revealed a binding pocket containing aromatic side chains, suggesting the importance of hydrophobicity in the interaction. Through mutational analysis, we determined the amino acids that are critical in carbohydrate binding. Through elucidating the structural and functional properties of a panel of murine MAbs, we offer an explanation for the predominant use of the human VH3 gene family in antibodies against CPSs with implications in knowledge-based vaccine design. IMPORTANCE Infectious diseases caused by pathogenic bacteria are a major threat to human health. Capsular polysaccharides (CPSs) of many pathogenic bacteria have been used as the main components of glycoconjugate vaccines against bacterial diseases in clinical practice worldwide, with various degrees of success. Immunization with a glycoconjugate vaccine elicits T cell help for B cells that produce IgG antibodies to the CPS. Thus, it is important to develop an in-depth understanding of the interactions of carbohydrate epitopes with the antibodies. Structural characterization of the ligand binding of polysaccharide-specific antibodies laid out in this study may have fundamental biological implications for our comprehension of how the humoral immune system recognizes polysaccharide antigens, and in future knowledge-based vaccine design.

Streptococcus pneumoniae, capsular polysaccharide, monoclonal antibodies, monoclonal antibody, conjugate vaccines, glycoconjugate vaccine, carbohydrate antigens, VH3 gene family

Structure type: polymer chemical repeating unit
Location inside paper: p. e0080021-2, repeating unit
Compound class: EPS, CPS, glucan, polysaccharide
Contained glycoepitopes: IEDB_115136,IEDB_140630,IEDB_142488,IEDB_146664,IEDB_149553,IEDB_423153,IEDB_983931,SB_192

Methods: ELISA, enzymatic hydrolysis, MALDI-TOF MS, antibody binding, crystallization, RNA sequencing, biotinylation, monoclonal antibody generation, opsonophagocytic killing assay, agglutination assay
3D data: 3D data

NCBI Taxonomy refs (TaxIDs): 1313
Reference(s) to other database(s): GTC:G48944PP, GlycomeDB:25551, CCSD:45515, CBank-STR:3690
Show glycosyltransferases

There is only one chemically distinct structure:

[*]O[C@H]1[C@H](O)[C@@H](C(=O)O)O[C@@H](O[C@@H]2[C@@H](CO)O[C@@H]([*])[C@H](O)[C@H]2O)[C@@H]1O

SVG MW SMILES 3D mol Ionize