Chemoenzymatic synthesis of immunogenic meningococcal group C polysialic acid-tetanus Hc fragment glycoconjugates.
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-MS(E) 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 acid-TetHc glycoconjugates contained much greater levels of polysaccharide-reactive IgG than the sera of control mice receiving unconjugated oligosialic acids. There was no apparent difference between glycoconjugates containing oligosaccharides of DP < 47 and DP < 100. These results suggest that chemoenzymatic synthesis may provide a viable method for making defined meningococcal vaccine candidates.
Laboratory of Bacterial Polysaccharides, CBER-FDA, 8800 Rockville Pike, Bethesda, MD, 20892, USA.
Pumtiwitt C
PC
McCarthy
Rina
R
Saksena
Dwight C
DC
Peterson
Che-Hung
CH
Lee
Yanming
Y
An
John F
JF
Cipollo
Willie F
WF
Vann
D000595:Amino Acid Sequence; D000818:Animals; D016123:Campylobacter jejuni / Q000276:immunology; D022401:Meningococcal Vaccines / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000276:immunology; D010446:Peptide Fragments / Q000737:chemistry* / Q000276:immunology; D012794:Sialic Acids / Q000737:chemistry* / Q000276:immunology; D013744:Tetanus Toxin / Q000737:chemistry* / Q000276:immunology; D018074:Vaccines, Conjugate / Q000737:chemistry* / Q000276:immunology
D022401:Meningococcal Vaccines; D010446:Peptide Fragments; D012794:Sialic Acids; D013744:Tetanus Toxin; D018074:Vaccines, Conjugate; C021319:polysialic acid; C040635:tetanus toxin fragment C