Nucleic acids and polypeptides specific of the neisseria...

Details Nucleic acids and polypeptides specific of the neisseria... Nucleic acids and polypeptides specific of the neisseria...

2001-08-16

2004-12-28

Smith, Lynette R. F. (Department: 1645)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Bacterium or component thereof or substance produced by said...

C424S249100, C424S190100, C424S185100, C424S184100, C530S300000, C530S350000, C536S023100, C536S023700, C536S024100, C536S024320, C435S069100, C435S069300, C435S243000, C435S252300

Reexamination Certificate

active

06835384

ABSTRACT:

The present invention relates to nucleic acids encoding polypeptides specific for pathogenic strains of the
Neisseria
genus, in particular which are useful for preventing or treating a
Neisseria meningitidis
infection.
In general, meningitis is either of viral origin or of bacterial origin. The bacteria mainly responsible are: type b
Haemophilus influenzae, Neisseria meningitidis
and
Streptococcus pneumoniae
. The
Neisseria meningitidis
species is subdivided into serogroups according to the nature of the capsular polysaccharides. Although about a dozen serogroups exist, 90% of meningitis cases can be attributed to three serogroups: A, B and C.
Effective vaccines based on capsular polysaccharides exist for preventing meningitis caused by
Neisseria meningitidis
serogroups A and C. These polysaccharides, unmodified, are only slightly immunogenic, or not at all, in children under the age of two, and do not induce any immune memory. However, these drawbacks can be overcome by conjugating these polysaccharides to a carrier protein.
On the other hand, the polysaccharide of
Neisseria meningitidis
serogroup B is non-immunogenic, or relatively non-immunogenic in humans, whether or not it is in a conjugated form. Thus, it appears to be highly desirable to seek a vaccine against meningitis caused by
Neisseria meningitidis
, in particular
Neisseria meningitidis
serogroup B, other than a vaccine based on polysaccharide.
To this end, various proteins of the external membrane of
N. meningitidis
have already been proposed, such as the membrane-bound receptor for human transferrin (WO 90/12591 and WO 93/06861).
Neisseria meningitidis
is genetically very close to
Neisseria gonorrhoeae
and
Neisseria lactamica. N. gonorrhoeae
is especially responsible for infections located in the urogenital tract. It colonizes the genital mucous membrane, crosses the epithelium and then invades the sub-epithelium, where it multiplies and is responsible for a severe inflammatory reaction. On the other hand,
N. lactamica
is considered to be a nonpathogenic species.
Sequences present in
N. gonorrhoeae
and
N. meningitidis
, but absent from
N. lactamica
, have been disclosed in patent application WO 98/02547, but this prior patent application does not locate or identify the coding sequences.
The authors of the present invention have now managed to identify some of these genes by searching, in the meningococcal genome, for the open reading frames specific for pathogenic strains of the
Neisseria
genus, using the following strategy:
Some of the sequences disclosed in patent application WO 98/02547 (referred to, in said prior application, as SEQ ID Nos 66, 67, 69, 70, 72 to 96, 98 and 99) were positioned on the sequence of the genome of the
N. meningitidis
serogroup B strain (ATCC 13090), available from the Pathoseq® bank of Incyte Pharmaceuticals, and also on the sequence of the genome of the
Neisseria meningitidis
strain Z2491 (Sanger Centre). This made it possible to identify, in the
N. meningitidis
genome which has 2.3 mega bases, 19 contigs representing 220000 base pairs.
The authors of the present invention then analysed, for each of the 19 contigs, the presence of open reading frames (ORFs) containing at least 100 amino acids (and, by definition, bordered by an initiation codon and a stop codon), using the Gene Jockey II sequence processor® program (Biosoft). This analysis made it possible to select approximately 400 candidate ORFs.
The sequences of each of these ORFs were then analysed using the Codon Use® program (Conrad Halling), which takes into account the codon use frequency in
N. meningitidis
. Only the ORFs with sequences having a maximum frequency of use of these codons were selected. At the end of this analysis, 197 candidate ORFs were selected.
The ORFs selected using this double analysis were subjected to a homology search through all of the available banks, using the BLASTX® program, from the access to the Pathoseq® bank of Incyte Pharmaceuticals. This homology search made it possible to exclude the ORFs encoding, a priori, cytoplasmic or periplasmic proteins, in particular metabolism proteins. The ORFs were also subjected to analysis of possible protein motifs, using the DNA Star Protean® program (Lasergene software).
The authors of the present invention then investigated whether the ORFs selected at the end of the previous step (118 in number) were effectively absent from
N. lactamica
, as predicted by the application of the prior art WO 98/02547.
To this end, a PCR amplification was carried out. This amplification proved to be ineffective for 78 of the 118 ORFs tested. Only the ORFs for which the amplification in
N. lactamica
was negative (sequences named “lactamical
−
”) were selected. In order to verify that these negative results were not “false negatives”, the lactamica
−
sequences selected were subjected to a control by dot blot. At the end of this step, only 23 ORFs were confirmed
N. meningitidis
+
/N. lactamica
−
.
Finally, these 23 ORFs were repositioned in their entirety on the
N. meningitides
ATCC13090 genome. This made it possible to demonstrate that three ORFs previously eliminated on the basis of their putative protein function appeared to be located close to, or were even framed by, some of the 23
N. meningitidis
+
/N. lactamica
−
ORFs. These three ORFs (SEQ ID Nos 29, 35 and 37) were reintroduced into the study, and it was proven that they were also
N. meningitidis
+
/N. lactamica
−
.
The authors of the present invention then attempted to discover whether the ORFs identified using the genome of the
N. meningitidis
serogroup B strain ATCC 13090 were also present in the genomes of
N. meningitidis
serogroup A Z2491 (Sanger Centre) and of
N. gonorrhoeae
FA1090 (Advanced Centre of Genome Technology, Oklahoma University). Then, they compared the sequences derived from these various genomes, with multiple alignment (Clustal, Infobiogen). This made it possible to redefine, for some of the ORFs, the most probable position of the initiation codon and translation stop codon. The sequences of the open reading frames derived from the strain ATCC13090 are given in the SEQ ID Nos 1-51 (odd numbers) and the amino acid sequences which are deduced therefrom are given in the SEQ ID Nos 2-52 (even numbers).
A subject of the present invention is, therefore, a nucleic acid in isolated form encoding a polypeptide, or an antigenic fragment thereof, excluding the nucleic acids disclosed in SEQ ID Nos 70, 73, 74, 77, 80, 81, 87, 88, 89, 94, 95 and 98 of application WO 98/02547 (sequences attached to the present description and numbered SEQ ID Nos 70A, 73A, 74A, 77A, 80A, 81A, 87A, 88A, 89A, 94A, 95A and 98A so as to distinguish them from the sequences of the invention); said polypeptide having an amino acid sequence which is identical or homologous to a sequence selected from those of group II; group II consisting of the sequences shown in SEQ ID Nos 2-52 (even numbers) and the sequence SEQ ID No. 53.
Preferably, said nucleic acid can have a nucleotide sequence selected from those of group I, group I consisting of the sequences shown in SEQ ID Nos 1-51 (odd numbers).
The term “nucleic acid” includes and means equally ORF, gene, polynucleotide, DNA and RNA. The term “nucleic acid in isolated form” means a nucleic acid separated from the biological environment in which it is found under natural conditions. For example, a DNA molecule exists under natural conditions when it is integrated into a genome or when it forms part of a library of genes. In that case, it cannot be in isolated form. On the other hand, the same molecule separated from the genome by cloning (for example subsequent to a PCR amplification) should be considered as being in isolated form. Typically, a DNA molecule in isolated form does not contain the coding regions which are contiguous with it in 5′ and 3′ in the genome from which it is derived. The nucleic acids in isolated form can be integrated into vectors (for example plasmids, or

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