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Infection and Immunity, May 2000, p. 2525-2534, Vol. 68, No. 5
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Identification of a Haemophilus influenzae 5'-Nucleotidase Protein: Cloning of the nucA Gene and Immunogenicity and Characterization of the NucA Protein

Robert J. Zagursky,* Peggy Ooi, Kevin F. Jones,dagger Michael J. Fiske, Robert P. Smith, and Bruce A. Green

Wyeth-Lederle Vaccines, West Henrietta, New York

Received 5 January 2000/Returned for modification 2 February 2000/Accepted 14 February 2000

We report on the identification of a surface-exposed, highly conserved, immunogenic nontypeable Haemophilus influenzae (NTHi) protein, which elicits cross-reactive bactericidal antibodies against NTHi. The protein was extracted from NTHi strain P860295 with KSCN and purified; it migrated as a single band on a sodium dodecyl sulfate-polyacrylamide gel with an apparent molecular mass of 63 kDa. Mouse antiserum generated against the purified protein was reactive on whole-cell enzyme-linked immunosorbent assay (ELISA) with seven NTHi strains and type b Eagan and Whittier strains and exhibited bactericidal activity to homologous and heterologous NTHi strains. However, the protein is made in small amounts in NTHi as corroborated by immunoelectron microscopy. To further study this protein, we cloned, sequenced, and expressed it recombinantly in Escherichia coli. The recombinant protein is localized in the periplasm of E. coli and has been purified to homogeneity. Both the recombinant and native proteins possess 5'-nucleotidase activity; hence, the protein has been called NucA. Mouse antiserum directed against the recombinant NucA protein was reactive on Western immunoblots and whole-cell ELISA with all H. influenzae strains tested including Eagan and was bactericidal for two heterologous strains tested. The antiserum also resulted in a log reduction in bacteremia, in an infant-rat protection study with H. influenzae type b as the challenge strain. These features suggest that NucA is a potential subunit vaccine candidate against NTHi disease.

* Corresponding author. Mailing address: Wyeth-Lederle Vaccines, 211 Bailey Rd., West Henrietta, NY 14586. Phone: (716) 273-7619. Fax: (716) 273-7515. E-mail: zagursrj{at}war.wyeth.com.

dagger Present address: SIGA Research Laboratories, Corvallis, OR 97333.

Infection and Immunity, May 2000, p. 2525-2534, Vol. 68, No. 5
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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