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  4. Studying the Structure of Carbohydrates in Order to Better Understand Bacterial Vaccines and Pathogens
  1. Science & Research (Biologics)

Studying the Structure of Carbohydrates in Order to Better Understand Bacterial Vaccines and Pathogens

Principal Investigator: John F. Cipollo
Office / Division / Lab: OVRR / DBPAP / LBP

General Overview

Many products currently regulated by CBER contain glycoconjugates as one or more of their primary components. Our laboratory studies vaccine glycoconjugates and glycoconjugate vaccines in order to help us to understand how to make better vaccines.

Glycoconjugates are made up of a carbohydrate bonded to either a fat molecule (glycolipid) or a protein (glycoprotein). They play many important roles in biological processes. During infection one component on the infecting microorganism needs to recognize a component on the host cell, a process in which at least one of these components is often a glycoconjugate. Therefore, scientists study glycoconjugates to determine if they can be the basis of new drugs or vaccines. In fact, the vaccines that protect against meningitis (Neisseria meningitidis), pneumonia, blood infections (Haemophilus influenzae type b), and pneumococcal bacteria (Streptococcus pneumoniae) are primarily or exclusively glycoconjugate in nature. These glycoconjugates represent part of the infectious bacteria's outer surface that are normally used to hide from its host. These vaccines help the human immune system to recognize these components on the bacterial surface and stop the infection before it starts--turning the bacteria's strength into its weakness.

Our laboratory uses very sensitive tools and methods which together are called mass spectrometry (MS) to rapidly determine the complex structure of glycoconjugates with high accuracy. The use of MS to analyze vaccine glycoconjugates is a relatively new field that is rapidly being adopted by the manufacturers of products that CBER reviews and licenses. Therefore, it is important for CBER to become familiar with this technology. We use this technique to analyze glycoconjugates and glycoconjugate vaccines and to help us discover molecules that hold potential for being new vaccines.

As part of our effort to understand the role of glycoconjugates in bacterial infections, we are using a wormlike organism called a nematode. We are also studying infection of this nematode with Coryneform bacteria, a large group of microorganisms that include, among other bacteria, those that cause diphtheria, as well as with Yersinia bacteria, one type of which causes plague. These studies provide a model system to 1) find the glycoconjugate molecule that the bacteria must latch onto in order to infect; 2) characterize the bacteria that bind to the glycoconjugates on the host; and 3) isolate the molecules on bacteria that bind to host glycoconjugates in order to determine if these molecules might be potential vaccines.

In addition, we are studying the glycoprotein hemagglutinin (HA), which is found on the surface of influenza viruses and is a key part of influenza vaccines. These vaccines are made by growing the viruses in eggs and then harvesting the viruses. Currently, however, several manufacturers are planning to grow these viruses in animal and insect cells. The HA glycoprotein made in these cells is somewhat different from HA made by viruses grown in eggs. Depending on the type of differences in the HA molecules from viruses grown in cells, there may be a change to the effectiveness of vaccines made from these glycoproteins. Therefore, it is important to develop new tests to analyze these glycoproteins, in order to measure the potency of animal and insect cell vs. egg-grown vaccines and ensure that the new vaccines will work properly.

Scientific Overview

Growing numbers of biologics licensed by CBER contain glycoconjugates. In the past, glycoconjugates were hard to analyze because they had complex structures and there was no technology powerful enough to effectively study them. In recent years, industry has adopted mass spectrometry (MS) to analyze these compounds; therefore, it is important that CBER have expertise in the area of glycoconjugate MS.

Our laboratory is developing MS-based techniques to analyze vaccine-based glycoconjugates and discover new vaccine candidates. Major projects in the laboratory include 1) development of Caenorhabditis elegans as a model host for bacterial infection and vaccine candidate discovery; and 2) an MS-based approach to monitor influenza hemagglutinin glycosylation. Additionally, glycoconjugates such as bacterial lipid oligosaccharides and polysaccharides of current interest in licensed vaccines and vaccine research are studied in this group.

C. elegans is infected by nearly 40 human pathogens or their close relatives, many of which require host cell surface glycoconjugates for infection. We use wild-type and glycosylation-deficient nematode strains in MS- based strategies to identify 1) host glycoconjugate receptors required for bacterial adhesion; and 2) bacterial lectins required for adhesion. There are three aims to these studies:

1. Compare the glycoconjugates of infection resistant mutants with those of their parent strain. This will reveal bacterial adhesin ligand candidates that might be involved in the early stages of host recognition. We will study nematode lectins in situ to localize glycoconjugates in the worm and conduct adhesion-inhibition studies using glycosides to study the specificity of the carbohydrate-dependant interaction between C. elegans and the model Coryneform bacteria M. nematophilum.

2. Construct a C. elegans glycan array in collaboration with Dr. David Smith's group at Emory University. We will use the array to screen pathogenic bacteria and purified bacterial lectins for binding to C. elegans glycans.

3. Design and construct in collaboration with Dr. Nicola Pohl (Iowa State University) affinity proteomics reagents for identifying bacterial lectins.

A second major project uses MS-based strategies to analyze glycosylation of hemagglutinin (HA), a glycoprotein that is a major antigen in influenza vaccines. Currently, several manufactures are changing from embryonated eggs to other cell types (e.g., MDCK, Vero, and Baculovirus-Sf9) as a cellular source for vaccine production. These alternative cell types will produce HA with different glycosylation patterns on their glycoproteins that may change antigenicity and vaccine efficacy. Such changes pose challenges for potency testing: new reagents must be made that are appropriate across different cellular platforms. Therefore, we are currently developing methods to analyze the altered glycosylation patterns present on hemagglutinin that will be adaptable to other glycoprotein molecules in influenza and other vaccine products.

Publications

  1. Front Mol Biosci 2023 Dec 18;10:1350141
    Editorial: Glycoconjugate antigen processing and immune response.
    Cipollo JF, Stefanetti G
  2. Front Mol Biosci 2023 Jun 13;10:1207670
    Glycosylation of H4 influenza strains with pandemic potential and susceptibilities to lung surfactant SP-D.
    Parsons LM, Zoueva O, Grubbs G, Plant E, Jankowska E, Xie Y, Song H, Gao GF, Ye Z, Khurana S, Cipollo JF
  3. Proteomics 2023 Mar;23(5):e2200320
    Assign-MALDI--a free software for assignment of MALDI-TOF MS spectra of glycans derivatized using common and novel labeling strategies.
    Parsons LM, Cipollo JF
  4. J Infect Dis 2023 Feb 15;227(4):583-91
    Antigen discovery for next-generation pertussis vaccines using immunoproteomics and transposon directed insertion sequencing.
    Gregg KA, Wang Y, Warfel J, Schoenfeld E, Jankowska E, Cipollo JF, Mayho M, Boinett C, Prasad D, Brickman TJ, Armstrong SK, Parkhill J, Da Silva Antunes R, Sette A, Papin JF, Wolf R, Merkel TJ
  5. Proteomics 2022 Dec;22(23-24):e2200156
    Recent advances in demystifying O-glycosylation in health and disease.
    Li J, Guo B, Zhang W, Yue S, Huang S, Gao S, Ma J, Cipollo JF, Yang S
  6. J Proteome Res 2022 Oct 7;21(10):2341-55
    Comprehensive N- and O-glycoproteomic analysis of multiple Chinese hamster ovary host cell lines.
    Wang Q, Wang T, Wu WW, Lin CY, Yang S, Yang G, Jankowska E, Hu Y, Shen RF, Betenbaugh MJ, Cipollo JF
  7. Sci Rep 2022 Aug 19;12(1):14173
    Altered sialidase expression in human myeloid cells undergoing apoptosis and differentiation.
    Hyun SW, Feng C, Liu A, Lillehoj EP, Trotta R, Kingsbury TJ, Passaniti A, Lugkey KN, Chauhan S, Cipollo JF, Luzina IG, Atamas SP, Cross AS, Goldblum SE
  8. mBio 2022 Apr;13(2):e0298321
    Aberrant cellular glycosylation may increase the ability of influenza viruses to escape host immune responses through modification of the viral glycome.
    Alymova IV, Cipollo JF, Parsons LM, Music N, Kamal RP, Tzeng WP, Goldsmith CS, Contessa JN, Hartshorn KL, Wilson JR, Zeng H, Gansebom S, York IA
  9. Biotechnol Bioeng 2022 Jan;119(1):102-17
    The interplay of protein engineering and glycoengineering to fine-tune antibody glycosylation and its impact on effector functions.
    Wang Q, Wang T, Zhang R, Yang S, McFarland K, Chung CY, Jia H, Wang LX, Cipollo JF, Betenbaugh MJ
  10. Glycobiology 2021 Dec;31(12):1737
    The interplay of protein engineering and glycoengineering to fine tune antibody glycosylation and its impact on effector functions.
    Wang Q, Cipollo JF, Betenbaugh MJ
  11. J Virol 2021 Nov;95(24):e0116021
    Design of the recombinant influenza neuraminidase antigen is crucial for its biochemical properties and protective efficacy.
    Gao J, Klenow L, Parsons L, Malik T, Phue JN, Gao Z, Withers SG, Cipollo J, Daniels R, Wan H
  12. Front Chem 2021 Nov 19;9:767448
    Site-specific glycosylation patterns of the SARS-CoV-2 spike protein derived from recombinant protein and viral WA1 and D614G strains.
    Tian Y, Parsons LM, Jankowska E, Cipollo JF
  13. Front Chem 2021 Sep 24;9:735558
    A linkage-specific sialic acid labeling strategy reveals different site-specific glycosylation patterns in SARS-CoV-2 spike protein produced in CHO and HEK cell substrates.
    Wang Q, Wang Y, Yang S, Lin C, Aliyu L, Chen Y, Parsons L, Tian Y, Jia H, Pekosz A, Betenbaugh MJ, Cipollo JF
  14. Glycoconj J 2021 Apr;38(2):145-56
    Improved online LC-MS/MS identification of O-glycosites by EThcD fragmentation, chemoenzymatic reaction, and SPE enrichment.
    Yang S, Wang Y, Mann M, Wang Q, Tian E, Zhang L, Cipollo JF, Ten Hagen KG, Tabak LA
  15. Metab Eng 2020 Sep;61:301-14
    Metabolic engineering challenges of extending N-glycan pathways in Chinese hamster ovary cells.
    Wang Q, Wang T, Yang S, Sha S, Wu WW, Chen Y, Paul JT, Shen RF, Cipollo JF, Betenbaugh MJ
  16. Anal Chem 2020 Aug 18;92(16):10946-51
    Optimization of O-GIG for O-glycopeptide characterization with sialic acid linkage determination.
    Yang S, Wu WW, Shen R, Sjogren J, Parsons L, Cipollo JF
  17. Nat Rev Chem 2020 Mar 17;4:229-42
    Glycomics studies using sialic acid derivatization and mass spectrometry.
    de Haan N, Yang S, Cipollo J, Wuhrer M
  18. J Virol 2020 Feb 14;94(5):e01951-19
    Influenza hemagglutinins H2, H5, H6, and H11 are not targets of pulmonary surfactant protein D: N-glycan subtypes in host-pathogen interactions.
    Parsons L, An Y, Qi L, White M, van der Woude R, Hartshorn K, Taubenberger JK, de Vries RP, Cipollo JF
  19. Biotechnol Bioeng 2020 Jan;117(1):157-66
    The impact of sialylation linkage type on the pharmacokinetics of recombinant butyrylcholinesterases.
    Chung CY, Wang Q, Yang S, Chough S, Seo Y, Cipollo JF, Balthasar JP, Betenbaugh MJ
  20. Mol Cell Proteomics 2020 Jan;19(1):11-30
    NIST interlaboratory study on glycosylation analysis of monoclonal antibodies: comparison of results from diverse analytical methods.
    De Leoz MLA, Duewer DL, Fung A, Liu L, Yau HK, Potter O, Staples GO, Furuki K, Frenkel R, Hu Y, Sosic Z, Zhang P, Altmann F, Gruber C, Shao C, Zaia J, Evers W, Pangelley S, Suckau D, Wiechmann A, Resemann A, Jabs W, Beck A, Froehlich JW, Huang C, Li Y, Liu Y, Sun S, Wang Y, Seo Y, An HJ, Reichardt NC, Ruiz JE, Archer-Hartmann S, Azadi P, Bell L, Lakos Z, An Y, Cipollo JF, Pucic-Bakovic M, Stambuk J, Lauc G, Li X, Wang PG, Bock A, Hennig R, Rapp E, Creskey M, Cyr T, Nakano M, et al
  21. J Virol 2020 Jan 6;94(2):e01363-19
    Three amino acid changes in avian coronavirus spike protein allows binding to kidney tissue.
    Bouwman KM, Parsons LM, Berends AJ, de Vries RP, Cipollo JF, Verheije MH
  22. Nat Microbiol 2019 Dec;4(12):2216-25
    The neuraminidase of A(H3N2) influenza viruses circulating since 2016 is antigenically distinct from the A/Hong Kong/4801/2014 vaccine strain.
    Wan H, Gao J, Yang H, Yang S, Harvey R, Chen YQ, Zheng NY, Chang J, Carney PJ, Li X, Plant E, Jiang L, Couzens L, Wang C, Strohmeier S, Wu WW, Shen RF, Krammer F, Cipollo JF, Wilson PC, Stevens J, Wan XF, Eichelberger MC, Ye Z
  23. Virulence 2019 Dec;10(1):1013-25
    Pmr-1 gene affects susceptibility of Caenorhabditis elegans to Staphylococcus aureus infection through glycosylation and stress response pathways' alterations.
    Schifano E, Ficociello G, Vespa S, Ghosh S, Cipollo JF, Talora C, Lotti LV, Mancini P, Uccelletti D
  24. Mass Spectrom Rev 2020 Jul;39(4):371-409
    Glycomics and glycoproteomics of viruses: mass spectrometry applications and insights toward structure-function relationships.
    Cipollo JF, Parsons LM
  25. Bioanalysis 2019 Jun;11(11):1039-43
    Bioanalysis for precision medicine.
    Yang S, Yang Z, Wang PG
  26. J Biol Chem 2019 May 10;294(19):7797-809
    Glycosylation of the viral attachment protein of avian coronavirus is essential for host cell and receptor binding.
    Parsons L, Bouwman KM, Azurmendi HF, de Vries RP, Cipollo JF, Verheije MH
  27. J virol 2019 Jan 4;93(2):e01693-18
    N-glycosylation of seasonal influenza vaccine hemagglutinins: implication for potency testing and immune processing.
    An Y, Parsons L, Jankowska E, Melnyk D, Joshi M, Cipollo JF
  28. Proteomics Clin Appl 2018 Sep;12(5):e1700075
    The glycoproteomics-mass spectrometry for studying glycosylation in cardiac hypertrophy and heart failure.
    Yang S, Chatterjee S, Cipollo J
  29. Anal Chem 2018 Jul 3;90(13):8261-9
    Deciphering protein O-glycosylation: solid-phase chemoenzymatic cleavage and enrichment.
    Yang S, Onigman P, Wu WW, Sjogren J, Nyhlen H, Shen RF, Cipollo J
  30. J Am Soc Mass Spectrom 2018 Jun;29(6):1273-83
    Identification of Sialic Acid Linkages on Intact Glycopeptides via Differential Chemical Modification Using IntactGIG-HILIC.
    Yang S, Wu WW, Shen RF, Bern M, Cipollo J
  31. Anal Chem 2018 Apr 17;90(8):5040-7
    Improving analytical characterization of glycoconjugate vaccines through combined high-resolution MS and NMR: application to Neisseria meningitidis serogroup B oligosaccharide-peptide glycoconjugates.
    Yu H, An Y, Battistel MD, Cipollo JF, Freedberg DI
  32. Glycobiology 2018 Apr 1;28(4):223-32
    A comprehensive Caenorhabditis elegans N-glycan shotgun array.
    Jankowska E, Parsons LM, Song X, Smith DF, Cummings RD, Cipollo JF
  33. Bioanalysis 2017 Dec;9(23):1839-44
    Matrix effects and application of matrix effect factor.
    Zhou W, Yang S, Wang PG
  34. Anal Chem 2017 Sep 5;89(17):9508-9517
    Solid-phase chemical modification for sialic acid linkage analysis: application to glycoproteins of host cells used in influenza virus propagation.
    Yang S, Jankowska E, Kosikova M, Xie H, Cipollo J
  35. ACS Chem Biol 2017 Jun 16;12(6):1665-73
    Glycan remodeling of human erythropoietin (EPO) through combined mammalian cell engineering and chemoenzymatic transglycosylation.
    Yang Q, An Y, Zhu S, Zhang R, Loke CM, Cipollo JF, Wang LX
  36. Anal Chem 2017 Jun 20;89(12):6330-5
    Modification of sialic acids on solid phase: accurate characterization of protein sialylation.
    Yang S, Zhang L, Thomas S, Hu Y, Li S, Cipollo J, Zhang H
  37. J Proteome Res 2017 Feb 3;16(2):398-412
    Glycosylation characterization of an influenza H5N7 hemagglutinin series with engineered glycosylation patterns: implications for structure-function relationships.
    Parsons LM, An Y, de Vries RP, de Haan CA, Cipollo JF
  38. Sci Rep 2016 Oct 31;6:36216
    Glycosylation changes in the globular head of H3N2 influenza hemagglutinin modulate receptor binding without affecting virus virulence.
    Alymova IV, York IA, Air GM, Cipollo JF, Gulati S, Baranovich T, Kumar A, Zeng H, Gansebom S, McCullers JA
  39. J Proteome Res 2015 Sep 4;14(9):3957-69
    Glycosylation analysis of engineered H3N2 influenza A virus hemagglutinins with sequentially added historically relevant glycosylation sites.
    An Y, McCullers JA, Alymova I, Parsons LM, Cipollo JF
  40. Pharm Bioprocess 2015;3(4):323-40
    Monitoring vaccine protein glycosylation: analytics and recent developments.
    Cipollo JF
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