Glycoconj J (2013) 30:857–870
Chemoenzymatic synthesis of immunogenic meningococcal
group C polysialic acid-tetanus Hc fragment glycoconjugates
Pumtiwitt C. McCarthy & Rina Saksena &
Dwight C. Peterson & Che-Hung Lee & Yanming An &
John F. Cipollo & Willie F. Vann
Received: 28 March 2013 / Revised: 25 July 2013 / Accepted: 28 July 2013 / Published online: 16 August 2013
# Springer Science+Business Media New York (outside the USA) 2013
Abstract Vaccination with meningococcal glycoconjugate
vaccines has decreased the incidence of invasive meningitis
worldwide. These vaccines contain purified capsular polysaccharides attached to a carrier protein. Because of derivatization chemistries used in the process, conjugation of polysaccharide to protein often results in heterogeneous mixtures.
Well-defined vaccines are needed to determine the relationship between vaccine structure and generated immune response. Here, we describe efforts to produce well-defined
vaccine candidates by chemoenzymatic synthesis. Chemically
synthesized lactosides were substrates for recombinant sialyltransferase enzymes from Camplyobacter jejuni and Neisseria
meningitidis serogroup C. These resulting oligosialic acids
have the same α(2-9) sialic acid repeat structure as Neisseria
polysaccharide capsule with the addition of a conjugatable
azide aglycon. The degree of polymerization (DP) of carbohydrate products was controlled by inclusion of the inhibitor
CMP-9-deoxy-NeuNAc. Polymers with estimated DP<47
(median DP 25) and DP<100 (median DP 51) were produced.
The receptor binding domain of the tetanus toxin protein
(TetHc) was coupled as a carrier to the enzymatically synthesized oligosialic acids. Recombinant TetHc was derivatized
with an alkyne squarate. Protein modification sites were determined by trypsin proteolysis followed by LC/MS-MSE
analysis of peptides. Oligosialic acid azides were conjugated
to modified TetHc via click chemistry. These chemoenzymatically prepared glycoconjugates were reactive in immunoassays with specific antibodies against either group C polysaccharide or TetHc. Sera of mice immunized with oligosialic
Electronic supplementary material The online version of this article
(doi:10.1007/s10719-013-9490-x) contains supplementary material,
which is available to authorized users.
P. C. McCarthy : R. Saksena : D. C. Peterson : C.
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