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  5. Studying the Structure of Carbohydrates in Order to Better Understand Bacterial Vaccines and Pathogens
  1. Biologics Research Projects

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.


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

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

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

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

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

Pharm Bioprocess 2015;3(4):323-40
Monitoring vaccine protein glycosylation: analytics and recent developments.
Cipollo JF

PLoS One 2014 Oct 8;9(10):e107250
Caenorhabditis elegans bacterial pathogen resistant bus-4 mutants produce altered mucins.
Parsons LM, Mizanur RM, Jankowska E, Hodgkin J, O Rourke D, Stroud D, Ghosh S, Cipollo JF

Microbes Infect 2014 Apr;16(4):356-61
Oral ingestion of Microbacterium nematophilum leads to anal-region infection in Caenorhabditis elegans.
Parsons LM, Cipollo J

Glycoconj J 2013 Dec;30(9):857-70
Chemoenzymatic synthesis of immunogenic meningococcal group C polysialic acid-tetanus Hc fragment glycoconjugates.
McCarthy PC, Saksena R, Peterson DC, Lee CH, An Y, Cipollo JF, Vann WF

Mol Cell Proteomics 2013 Oct;12(10):2935-51
Interlaboratory study on differential analysis of protein glycosylation by mass spectrometry: the ABRF glycoprotein research multi-institutional study 2012.
Leymarie N, Griffin PJ, Jonscher K, Kolarich D, Orlando R, McComb M, Zaia J, Aguilan J, Alley WR, Altmann F, Ball LE, Basumallick L, Bazemore-Walker CR, Behnken H, Blank MA, Brown KJ, Bunz SC, Cairo CW, Cipollo JF, Daneshfar R, Desaire H, Drake RR, Go EP, Goldman R, Gruber C, Halim A, Hathout Y, Hensbergen PJ, Horn DM, Hurum D, Jabs W, Larson G, Ly M, Mann BF, Marx K, Mechref Y, Meyer B, Moginger U, Neusubeta C, Nilsson J, Novotny MV, Nyalwidhe JO, Packer NH, Pompach P, Reiz B, Resemann A, Rohrer JS, Ruthenbeck A, Sanda M, Schulz JM, et al

J Proteome Res 2013 Aug 2;12(8):3707-20
Comparative Glycomics Analysis of Influenza Hemagglutinin (H5N1) Produced in Vaccine Relevant Cell Platforms.
An Y, Rininger JA, Jarvis DL, Jing X, Ye Z, Aumiller JJ, Eichelberger M, Cipollo JF

Bioanalysis 2011 Nov;3(21):2401-17
Platform for analysis of anthranilic acid N-glycan derivatives utilizing multipolarity mode LC-MS with hydrophilic interaction chromatography separation and ion trap MS/MS.
Jankowska E, Cipollo J

Anal Biochem 2011 Aug 1;415(1):67-80
An unbiased approach for analysis of protein glycosylation and application to influenza vaccine hemagglutinin.
An Y, Cipollo JF

J Biol Chem 2010 Jun 4;285(23):17662-72
The Caenorhabditis elegans bus-2 mutant reveals a new class of O-glycans affecting bacterial resistance.
Palaima E, Leymarie N, Stroud D, Mizanur RM, Hodgkin J, Gravato-Nobre MJ, Costello CE, Cipollo JF

Blood 2009 Mar 12;113(11):2578-86
Haptoglobin preserves the CD163 hemoglobin scavenger pathway by shielding hemoglobin from peroxidative modification.
Buehler PW, Abraham B, Vallelian F, Linnemayr C, Pereira CP, Cipollo JF, Jia Y, Mikolajczyk M, Boretti FS, Schoedon G, Alayash AI, Schaer DJ

J Biol Chem 2009 Feb 13;284(7):4616-25
The fine structure of Neisseria meningitidis lipooligosaccharide from the M986 strain and three of its variants.
Tsai CM, Jankowska-Stephens E, Mizanur RM, Cipollo JF

Appl Microbiol Biotechnol 2008 Oct;80(5):757-65
Bacterial CMP-sialic acid synthetases: production, properties, and applications.
Mizanur RM, Pohl NL

J Biol Chem 2008 Jun 27;283(26):18355-64
Unique Asn-linked oligosaccharides of the human pathogen Entamoeba histolytica.
Magnelli P, Cipollo JF, Ratner DM, Cui J, Kelleher D, Gilmore R, Costello CE, Robbins PW, Samuelson J

J Am Soc Mass Spectrom 2007 Oct;18(10):1799-812
A glycomics platform for the analysis of permethylated oligosaccharide alditols.
Costello CE, Contado-Miller JM, Cipollo JF