Neisseria meningitidis serogroup a capsular polysaccharide acetyltransferase, methods and compositions

a technology of capsular polysaccharide and acetyltransferase, which is applied in the field of molecular biology, can solve the problems of poor immunogenicity in young children, and achieve the effects of improving immunogenic composition, complete acetylation, and stronger immune response results

Inactive Publication Date: 2006-04-06
EMORY UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011] The present invention also provides for improved immunogenic compositions comprising capsular polysaccharides of N. meningitidis, where the improvement comprises more complete acetylation of the capsular polysaccharides than is currently possible in the absence of the enzymatic acetylation by using the acetyltransferase of the present invention, especially those from Serogroup A N. meningitidis, with the result that a stronger immune response results. The immunogenic compositions of the present invention can comprise a pharmaceutically acceptable carrier and optionally can further comprise at least one immunological adjuvant or cytokine. These immunogenic compositions are useful as vaccines and as vaccine components. Desirably, the CPS is 90-95% acetylated for eliciting a robust immune response

Problems solved by technology

The constant challenge of epidemics.
However, because of poor immunogenicity in young children and short-lived immunity (Zollinger, W. D. and Moran, E.

Method used

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  • Neisseria meningitidis serogroup a capsular polysaccharide acetyltransferase, methods and compositions
  • Neisseria meningitidis serogroup a capsular polysaccharide acetyltransferase, methods and compositions
  • Neisseria meningitidis serogroup a capsular polysaccharide acetyltransferase, methods and compositions

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example 1

Materials and Bacterial Strains

[0135] Bacterial strains, plasmids and primers used in this study are described in Table 1. The serogroup A meningococcal strains were originally isolated during an outbreak in Nairobi, Kenya in 1989 (12) and were provided by the Centers for Disease Control and Prevention (CDC), Atlanta, Ga. Strain F8229 (CDC1750) is encapsulated and was isolated from the cerebrospinal fluid of a patient with meningitis. Strain F8239 (CDC16N3) is an unencapsulated variant originally isolated as a serogroup A strain from the pharynx of an asymptomatic carrier. These strains belong to clonal group III-I and are closely related to strains that have caused epidemics in Saudi Arabia, Chad, Ethiopia and other parts of the world.

[0136] Monoclonal antibody 14-1-A (13) against meningococcal serogroup A capsular polysaccharide was provided by Dr. Wendell Zollinger, Walter Reed Army Institute of Research.

[0137] Restriction enzymes were purchased from New England Biolabs (Bever...

example 2

Growth Conditions

[0138] Meningococcal strains were grown with 3.5% CO2 at 37° C. on GC base agar (Difco, Detroit, Mich.), supplemented with 0.4% glucose and 0.68 mM Fe (NO3)3, or in GC broth containing the same supplements and 0.043% NaHCO3. BHI medium (37 g / liter brain heart infusion) with 1.25% fetal bovine serum was used when kanamycin selection was required. Antibiotic concentrations (in μg / ml) used for E. coli strains were ampicillin, 100, kanamycin, 50, and erythromycin, 300; and for N. meningitidis were kanamycin, 80, spectinomycin, 60, erythromycin, 3. E. coli DH5α strain, cultured on Luria Bertani (LB) medium, was used for cloning and propagation of plasmids. Meningococci were transformed by the procedure of Janik et al. (14). E. coli strains were transformed by electroporation (Gene-pulser Bio-Rad, Hercules, Calif., according to the manufacturer's protocol).

example 3

Construction of Meningococcal mynC Nonpolar Mutant NmA001

[0139] An internal 745-bp fragment of mynC, produced by PCR amplification using primers SE57 and SE61 (11) and the chromosomal DNA of strain F8229 as a template, was cloned into pCR2.1 to yield pGS201. The aphA-3 fragment obtained from pUC18K with EcoR I and HinC II digestion and filled in with Klenow polymerase was inserted into the unique Sspl site of mynC in pGS201 to generate pGS202. The correct orientation of aphA-3 was confirmed by colony PCR and direct sequencing analysis of pGS202. A ScaI-linearized pGS202 plasmid was used to transform serogroup A meningococcal strain F8229 to generate NmA001. The correct homologous recombination of the aphA-3 cassette into the mynC coding sequence was confirmed by PCR with cassette-specific primers and chromosomal-specific primers.

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Abstract

Provided are recombinant DNA molecules that do not occur in nature encoding an O-acetyltransferase, vectors that direct expression of an O-acetyltransferase, recombinant host cells which express an O-acetyltransferase, methods for recombinant production of an O-acetyltransferase, methods for acetylating capsular polysaccharides, especially those of a Serogroup A Neisseria meningitidis using a recombinant O-acetyltransferase, and immunogenic compositions comprising the acetylated capsular polysaccharide.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims benefit of U.S. Provisional Application No. 60 / 600,862, filed Aug. 11, 2004, which application is incorporated by reference herein.ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT [0002] This invention was made, at least in part, with funding from the National Institutes of Health (Grant No. AI40247) and the Department of Energy (Grant No. DE-FG02-93ER20097). Accordingly, the United States Government has certain rights in this invention.BACKGROUND OF THE INVENTION [0003] The field of this invention is the area of molecular biology, in particular as related to recombinant expression of an acetyltransferase of Serogroup A Neisseria meningitidis, and immunogenic compositions, especially immunogenic compositions comprising fully acetylated capsule of Neisseria meningitidis, Serogroup A. [0004]Neisseria meningitidis is a leading worldwide cause of meningitis and rapidly fatal sepsis in otherwise health individuals (Apicella...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/095C07H21/04C12P21/06C12N9/10C12N15/74C12P19/04C12N1/21
CPCA61K39/00A61K39/095C12N9/1029C12P19/44A61P31/04
Inventor STEPHENS, DAVIDGUDLAVALLETI, SESHUTZENG, YIH-LINGDATTA, ANUPCARLSON, RUSSELL
Owner EMORY UNIVERSITY
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