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Title:  Invasion associated genes from Neisseria meningitidis serogroup B

United States Patent:  6,472,518

Issued:  October 29, 2002

Inventors:  Ribot; Efrain M. (Atlanta, GA); Stephens; David S. (Stone Mountain, GA); Raymond; Nigel (Wellington, NZ); Quinn; Frederick D. (Avondale Estates, GA)

Assignee:  Centers for Disease Control and Prevention, as represented by the (Atlanta, GA)

Appl. No.:  284926

Filed:  August 17, 1999

PCT Filed:  October 24, 1997

PCT NO:  PCT/US97/19424

371 Date:  August 17, 1999

102(e) Date:  August 17, 1999

PCT PUB.NO.:  WO98/17805

PCT PUB. Date: April 30, 1998

Abstract

Genes isolated from Neisseria memingitidis, as well as isolated nucleic acids, probes, expression cassettes, polypeptides, antibodies, immunogenic compositions, antisense nucleic acids, amplification mixtures, and new invasion deficient swains of Neisseria meningitidis are provided Methods of detecting Neisseria meningitidis and Neisseria meningitidis nucleic acids, and methods of inhibiting the invasion of mammalian cells by Neisseria meningitidis are also provided.

SUMMARY OF THE INVENTION

The invention provides nucleic acids and encoded polypeptides associated with invasion of Neisseria meningitidis. The polypeptides are used as diagnostic reagents as immunogenic reagents; and as components of vaccines. The nucleic acids are used as diagnostic reagents, as components of vectors and vaccines, and to encode the polypeptides of the invention. The invention also provides strains of Neisseria meningitidis which have an invasion deficient phenotype.

In one embodiment, the invention provides isolated nucleic acids encoding the polypeptides of the invention, including ORF 1 (SEQ ID NO:2), ORF 2 (ORF2 a (SEQ ID NO:4) and ORF2b (SEQ ID NO:5), two separate embodiments depending on alternate start sites for the ORF2 polypeptide), ORF 3 (SEQ ID NO:7) and, conservatively modified variations of each of the polypeptides. Exemplar nucleic acids include Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), and Seq 3 (SEQ ID NO:7) (see, FIGS. 5, 6, and 7 respectively). Other nucleic acids encoding the same polypeptides include those with silent codon substitutions relative to Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3) for Seq 3 (SEQ ID NO:6); as well as conservatively modified variations thereof.

Isolated nucleic acids which hybridize under stringent conditions to the exemplar nucleic acids Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), or Seq 3 (SEQ ID NO:6) are also provided. For example, a complementary nucleic acid to a sequence provided by Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), or Seq 3 (SEQ ID NO:6) hybridizes to Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), or Seq 3 (SEQ ID NO:6), respectively. Nucleic acids which include substantial subsequences complementary to Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), or Seq 3 (SEQ ID NO:6) also hybridize to Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), or Seq 3 (SEQ ID NO:6), respectively.

Isolated nucleic acids which hybridize under stringent conditions to Seq 4 (SEQ ID NO:8) are provided. Seq 4 (SEQ ID NO:8) is a genomic sequence which encodes Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), and Seq 3 (SEQ ID NO:6). Thus, complementary nucleic acids to sequences provided by Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), Seq 3 (SEQ ID NO:6), or Seq 4 (SEQ ID NO:8) all hybridize to Seq 4 (SEQ ID NO:8) under stringent conditions. Similarly, nucleic acids which include substantial subsequences of Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), Seq 3 (SEQ ID NO:6) or Seq 4 (SEQ ID NO:8) also hybridize to Seq 4 (SEQ ID NO:8). The isolated nucleic acids are optionally vector nucleic acids which comprise a transcription cassette. The transcription cassette optionally encodes a polypeptide. Typically, the portion of the transcription cassette which encodes the polypeptide hybridizes to Seq 4 (SEQ ID NO:8) under stringent conditions. Upon transduction of the transcription cassette into a cell, an mRNA which hybridizes to Seq 4 (SEQ ID NO:8) under stringent conditions is produced. The mRNA is translated in the cell into a polypeptide such as the ORF 1 (SEQ ID NO:2), ORF 2a (SEQ ID NO:4), ORF 2b (SEQ ID NO:5) or ORF 3 (SEQ ID NO:7) polypeptides.

Polypeptides encoded by nucleic acids which hybridize under stringent conditions to Seq 4 (SEQ ID NO:8), including Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), Seq 3 (SEQ ID NO:7) are provided herein. Exemplar polypeptides include ORF 1 (SEQ ID NO:1), ORF 2a (SEQ ID NO:4), ORF 2b (SEQ ID NO:5), or ORF 3 (SEQ ID NO:6).

Full length polypeptides of the invention, or antigenic epitopes derived from the full length polypeptides of the invention are optionally present in immunogenic compositions. The antigenic epitopes are optionally incorporated into fusion proteins which optionally include antigenic epitopes from related or unrelated proteins. The antigenic epitopes are optionally expressed on the surface or antigenic viral vectors.

The immunogenic compositions optionally comprise components to enhance immunogenicity, Such as an adjuvant. The compositions optionally include pharmaceutically acceptable excipients. When administered to a mammal, the immunogenic compositions optionally provide an immune response against antigenic epitopes which are included In the immunogenic compositions. In one preferred embodiment, administration of the immunogenic composition of the invention to a mammal inhibits invasion of the cells of the mammal by Neisseria meningitidis.

Antibodies which specifically bind to the polypeptides of the invention are provided. In a preferred embodiment, the antibodies bind to a polypeptide such as ORF 1 (SEQ ID NO:2), ORF 2a (SEQ ID NO:4), ORF 2b (SEQ ID NO:5), or ORF 3 (SEQ ID NO:7); without binding to the E coli FtsZ protein, or to the E coli UNK protein. Typically, the antibodies specifically bind to the ORF 1 (SEQ ID NO:2), ORF 2a (SEQ ID NO:4), ORF 2b (SEQ ID NO:5), or ORF 3 (SEQ ID NO:7) proteins.

The invention provides isolated Neisseria meningitidis diplococcus. The diplococcus has a reduced ability to invade tissue culture epithelial cells in vitro as compared to a wild-type Neisseria meningitidis diplococcus and the genome of the isolated Neisseria meningitidis diplococcus has a modification in the region of the genome corresponding to Seq 4 (SEQ ID NO:8). In one embodiment, the isolated Neisseria meningitidis diplococcus comprises a transposon insertion in the region of the genome corresponding to Seq 4 (SEQ ID NO:8).

The invention provides a variety of assays for detecting Neisseria meningitidis, including PCR assays, northern blots, Southern bloc, western blots and ELISA assays. For example, the invention provides PCR reaction mixtures using template nucleic acids which hybridize to Seq 4 (SEQ ID NO:8) under stringent conditions. The mixture has a primer pair which hybridizes to the template nucleic acid, wherein the primers, when hybridized to the template, serve as initiation sites for primer extension by a thermostable polymerase such as taq or vent DNA polymerase. The products of PCR amplification are detected by detecting the amplified nucleic acid products (amplicons) of the PCR reaction.

In several methods relying on nucleic acid hybridization, the detection of a Neisseria meningitidis nucleic acid in a biological sample is performed by contacting a probe nucleic acid to the sample and detecting binding of the nucleic acid to the Neisseria meningitidis nucleic acid. The probe hybridizes to Seq 4 (SEQ ID NO:8), or the complement thereof. Many assay formats are appropriate, including northern and Southern blotting.

In one embodiment, the invention provides methods of inhibiting the invasion of a mammalian cell by Neisseria meningitidis by expressing an anti-sense RNA molecule in the mammalian cell. The antisense RNA molecule hybridizes to a nucleic acid which hybridizes under stringent conditions to a nucleic acid encoded by Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), Seq 3 (SEQ ID NO:7), or Seq 4 (SEQ ID NO:8). Such anti sense molecules optionally comprise catalytic RNA ribonuclease domains, such as those derived from a ribozyme.

Claim 1 of 7 Claims

What is claimed is:

1. An isolated nucleic acid encoding a polypeptide selected from the group of polypeptides consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:7.
 


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