U.S. patent number 6,287,574 [Application Number 08/913,362] was granted by the patent office on 2001-09-11 for proteinase k resistant surface protein of neisseria meningitidis.
This patent grant is currently assigned to BioChem Pharma Inc.. Invention is credited to Bernard R. Brodeur, Josee Hamel, Denis Martin, Clement Rioux.
United States Patent |
6,287,574 |
Brodeur , et al. |
September 11, 2001 |
Proteinase K resistant surface protein of neisseria
meningitidis
Abstract
A highly conserved, immunologically accessible antigen at the
surface of Neisseria meningitidis organisms. Immunotherapeutic,
prophylactic and diagnostic compositions and methods useful in the
treatment, prevention an diagnosis of Neisseria meningitidis
diseases. A proteinase K resistant Neisseria meningitidis surface
protein having an apparent molecular weight of 22 kDa, the
corresponding nucleotide and derived amino acid sequences (SEQ ID
NO: 1, NO:3, NO:5 and NO:7: SEQ ID NO: 2, NO:4, NO:6, and NO:8),
recombinant DNA methods for the production of the Neisseria
meningitidis 22 kDA surface protein, and antibodies that bind to
the Neisseria meningitidis 22 kDA surface protein.
Inventors: |
Brodeur; Bernard R. (Sillery,
CA), Martin; Denis (St-Augustin-de-Des Maures,
CA), Hamel; Josee (Sillery, CA), Rioux;
Clement (Ville-de-Cap-Rouge, CA) |
Assignee: |
BioChem Pharma Inc. (Quebec,
CA)
|
Family
ID: |
56289803 |
Appl.
No.: |
08/913,362 |
Filed: |
November 13, 1997 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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406362 |
Mar 17, 1995 |
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Current U.S.
Class: |
424/250.1;
424/184.1; 424/249.1; 530/300; 536/23.7; 530/350; 424/185.1;
424/190.1 |
Current CPC
Class: |
C07K
14/22 (20130101); A61P 43/00 (20180101); A61K
39/095 (20130101); A61P 31/04 (20180101); A61K
39/00 (20130101); A61K 2039/522 (20130101); A61K
38/00 (20130101) |
Current International
Class: |
C07K
14/22 (20060101); C07K 14/195 (20060101); A61K
38/00 (20060101); A61K 39/00 (20060101); A61K
039/095 () |
Field of
Search: |
;530/350,412,418,300
;424/249.1,250.1,184.1,185.1,190.1 ;536/23.7 |
Foreign Patent Documents
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0 273 116 |
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Jul 1988 |
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EP |
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94/05703 |
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Mar 1994 |
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WO |
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|
Primary Examiner: Graser; Jennifer
Attorney, Agent or Firm: Foley & Lardner
Parent Case Text
This is the National Stage of International application Ser. No.
PCT/CA96/00157, filed Mar. 15, 1996, which claims the benefit of
U.S. Provisional application Ser. No. 60/001,983, filed Aug. 4,
1995, and which is a continuation of U.S. application Ser. No.
08/406,362, filed Mar. 17, 1995, now abandoned.
Claims
We claim:
1. An isolated polypeptide comprising a sequence as set forth in
SEQ ID NO:2.
2. An isolated polypeptide comprising a sequence as set forth in
SEQ ID NO:4.
3. An isolated polypeptide comprising a sequence as set forth in
SEQ ID NO:6.
4. An isolated polypeptide comprising a sequence as set forth in
SEQ ID NO:8.
5. An isolated polypeptide comprising a sequence selected from the
group of sequences consisting of SEQ ID NO:9; SEQ ID NO:10, SEQ ID
NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ
ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,
SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID
NO:25, and SEQ ID NO:26.
6. An isolated polynucleotide encoding a polypeptide comprising a
sequence as set forth in SEQ ID NO:2.
7. An isolated polynucleotide encoding a polypeptide comprising a
sequence as set forth in SEQ ID NO:4.
8. An isolated polynucleotide encoding a polypeptide comprising a
sequence as set forth in SEQ ID NO:6.
9. An isolated polynucleotide encoding a polypeptide comprising a
sequence as set forth in SEQ ID NO:8.
Description
TECHNICAL FIELD OF THE INVENTION
This invention relates to a highly conserved, immunologically
accessible antigen at the surface of Neisseria meningitidis
organisms. This unique antigen provides the basis for new
immunotherapeutic, prophylactic and diagnostic agents useful in the
treatment, prevention and diagnosis of Neisseria meningitidis
diseases. More particularly, this invention relates to a proteinase
K resistant Neisseria meningitidis surface protein having an
apparent molecular weight of 22 kDa, the corresponding nucleotide
and derived amino acid sequences (SEQ ID NO:1 to SEQ ID NO:26),
recombinant DNA methods for the production of the Neisseria
meningitidis 22 kDa surface protein, antibodies that bind to the
Neisseria meningitidis 22 kDa surface protein and methods and
compositions for the diagnosis, treatment and prevention of
Neisseria meningitidis diseases.
BACKGROUND OF THE INVENTION
Neisseria meningitidis is a major cause of death and morbidity
throughout the world. Neisseria meningitidis causes both endemic
and epidemic diseases, principally meningitis and meningococcemia
[Gold, Evolution of meningococcal disease, p. 69, Vedros N. A., CRC
Press (1987); Schwartz et al., Clin. Microbiol. Rev., 2, p. S118
(1989)]. In fact, this organism is one of the most common causes,
after Haemophilus influenzae type b, of bacterial meningitis in the
United States, accounting for approximately 20% of all cases. It
has been well documented that serum bactericidal activity is the
major defense mechanism against Neisseria meningitidis and that
protection against invasion by the bacteria correlates with the
presence in the serum of anti-meningococcal antibodies
[Goldschneider et al., J. Exp. Med. 129, p. 1307 (1969);
Goldschneider et al., J. Exp. Med., 129, p. 1327 (1969)].
Neisseria meningitidis are subdivided into serological groups
according to the presence of capsular antigens. Currently, 12
serogroups are recognized, but serogroups A, B, C, Y, and W-135 are
most commonly found. Within serogroups, serotypes, subtypes and
immunotypes can be identified on outer membrane proteins and
lipopolysaccharide [Frasch et al., Rev. infect. Dis. 7, p. 504
(1985)].
The capsular polysaccharide vaccines presently available are not
effective against all Neisseria meningitidis isolates and do not
effectively induce the production of protective antibodies in young
infants (Frasch, Clin. Microbiol. Rev. 2, p. S134 (1989); Reingold
et al., Lancet, p. 114 (1985); Zollinger, in Woodrow and Levine,
New generation vaccines, p. 325, Marcel Dekker Inc. N.Y. (1990)].
The capsular polysaccharide of serogroups A, C, Y and W-135 are
presently used in vaccines against this organism. These
polysaccharide vaccines are effective in the short term, however
the vaccinated subjects do not develop an immunological memory, so
they must be revaccinated within a three-year period to maintain
their level of resistance.
Furthermore, these polysaccharide vaccines do not induce sufficient
levels of bactericidal antibodies to obtain the desired protection
in children under two years of age, who are the principal victims
of this disease. No effective vaccine against serogroup B isolates
is presently available even though these organisms are one of the
primary causes of meningococcal diseases in developed countries.
Indeed, the serogroup B polysaccharide is not a good immunogen,
inducing only a poor response of IgM of low specificity which is
not protective [Gotschlich et al., J. Exp. Med., p. 129, 1349
(1969); Skevakis et al., J. Infect. Dis., 149, p. 387 (1984);
Zollinger et al., J. Clin. Invest., 63, p. 836 (1979)].
Furthermore, the presence of closely similar, crossreactive
structures in the glycoproteins of neonatal human brain tissue
[Finne et al., Lancet, p. 355 (1983)] might discourage attempts at
improving the immunogenicity of serogroup B polysaccharide.
To obtain a more effective vaccine, other Neisseria meningitidis
surface antigens such as lipopolysaccharide, pili proteins and
proteins present in the outer membrane are under investigation. The
presence of a human immune response and bactericidal antibodies
against certain of these proteinaceous surface antigens in the sera
of immunized volunteers and convalescent patients was demonstrated
[Mandrell and Zollinger, Infect. Immun., 57, p. 1590 (1989);
Poolman et al., Infect. Immun., 40, p. 398 (1983); Rosenquist et
al., J. Clin. Microbiol., 26, p. 1543 (1988); Wedege and Froholm,
Infect. Immun. 51, p. 571 (1986); Wedege and Michaelsen, J. Clin.
Microbiol., 25, p. 1349 (1987)].
Furthermore, monoclonal antibodies directed against outer membrane
proteins, such as class 1, 2/3 and 5, were also reported to be
bactericidal and to protect against experimental infections in
animals [Brodeur et al., Infec. Immun., 50, p. 510 (1985); Frasch
et al, Clin. Invest. Med., 9, p. 101 (1986); Saukkonen et al.
Microb. Pathogen., 3, p. 261 (1987); Saukkonen et al., Vaccine, 7,
p. 325 (1989)].
Antigen preparations based on Neisseria meningitidis outer membrane
proteins have demonstrated immunogenic effects in animals and in
humans and some of them have been tested in clinical trials [Bjune
et al., Lancet, p. 1093 (1991); Costa et al., NIPH Annals, 14, p.
215 (1991); Frasch et al., Med. Trop., 43, p. 177 (1982); Frasch et
al., Eur. J. Clin. Microbiol., 4, p. 533 (1985); Frasch et al. in
Robbins, Bacterial Vaccines, p. 262, Praeger Publications, N.Y.
(1987); Prasch et al, J. Infect. Dis., 158, p. 710 (1988); Moreno
et al. Infec. Immun., 47, p. 527 (1985); Rosenqvist et al., J.
Clin. Microbiol., 26, p. 1543 (1988); Sierra et al., NIPH Annals,
14, p. 195 (1991); Wedege and Froholm, Infec. Immun. 51, p. 571
(1986); Wedege and Michaelsen, J. Clin. Microbiol., 25, p. 1349
(1987); Zollinger et al., J. Clin. Invest., 63, p. 836 (1979);
Zollinger et al., NIPH Annals, 14, p. 211 (1991)]. However, the
existence of great interstrain antigenic variability in the outer
membrane proteins can limit their use in vaccines [Frasch, Clin.
Microb., Rev. 2, p. S134 (1989)]. Indeed, most of these
preparations induced bactericidal antibodies that were restricted
to the same or related serotype from which the antigen was
extracted [Zollinger in Woodrow and Levine, New Generation
Vaccines, p. 325, Marcel Dekker Inc. N.Y. (1990)]. Furthermore, the
protection conferred by these vaccines in young children has yet to
be clearly established. The highly conserved Neisseria meningitidis
outer membrane proteins such as the class 4 [Munkley et al. Microb.
Pathogen., 11, p. 447 (1991)] and the lip protein (also called H.8)
[Woods et al., Infect. Immun., 55, p. 1927 (1987)] are not
interesting vaccine candidates since they do not elicit the
production of bactericidal antibodies. To improve these vaccine
preparations, there is a need for highly conserved proteins that
would be present at the surface of all Neisseria meningitidis
strains and that would be capable of eliciting bactericidal
antibodies in order to develop a broad spectrum vaccine.
The current laboratory diagnosis of Neisseria meningitidis is
usually done by techniques such as Gram stain of smear
preparations, latex agglutination or coagglutination, and the
culture and isolation on enriched and selective media [Morello et
al., in Balows, Manual of Clinical Microbiology, p. 258, American
Society for Microbiology, Washington (1991)]. Carbohydrate
degradation tests are usually performed to confirm the
identification of Neisseria meningitidis isolates. Most of the
described procedures are time-consuming processes requiring trained
personnel. Commercial agglutination or coagglutination kits
containing polyvalent sera directed against the capsular antigens
expressed by the most prevalent serogroups are used for the rapid
identification of Neisseria meningitidis. However, these polyvalent
sera often nonspecifically cross-react with other bacterial species
and for that reason should always be used in conjunction with Gram
stain and culture. Accordingly, there is a need for efficient
alternatives to these diagnostic assays that will improve the
rapidity and reliability of the diagnosis.
DISCLOSURE OF TEE INVENTION
The present invention solves the problems referred to above by
providing a highly conserved, immunologically accessible antigen at
the surface of Neisseria meningitidis organisms. Also provided are
recombinant DNA molecules that code for the foregoing antigen,
unicellular hosts transformed with those DNA molecules, and a
process for making substantially pure, recombinant antigen. Also
provided are antibodies specific to the foregoing Neisseria
meningitidis antigen. The antigen and antibodies of this invention
provide the basis for unique methods and pharmaceutical
compositions for the detection, prevention and treatment of
Neisseria meningitidis diseases.
The preferred antigen is the Neisseria meningitidis 22 kDa surface
protein, including fragments, analogues and derivatives thereof.
The preferred antibodies are the Me-1 and Me-7 monoclonal
antibodies specific to the Neisseria meningitidis 22 kDa surface
protein. These antibodies are highly bacteriolytic against
Neisseria meningitidis and passively protect mice against
experimental infection.
The present invention further provides methods for isolating novel
Neisseria meningitidis surface antigens and antibodies specific
thereto.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts the nucleotide and derived amino acid sequences of
the Neisseria meningitidis strain 608B 22 kDa surface protein (SEQ
ID NO:1; SEQ ID NO:2). Conventional three letter symbols are used
for the amino acid residues. The open reading frame extends from
the start codon at base 143 to the stop codon at base 667. The box
indicates the putative ribosome binding site whereas the putative
-10 promoter sequence is underlined. A 19-amino-acid signal peptide
is also underlined.
FIG. 2 is a photograph of a Coomassie Blue stained 14% SDS-PAGE gel
displaying .alpha.-chymotrypsin and trypsin digests of Neisseria
meningitidis strain 608B (B:2a:P1.2) outer membrane preparations.
Lane 1 contains the following molecular weight markers:
Phosphorylase b (97,400); bovine serum albumin (66,200); ovalbumin
(45,000); carbonic anhydrase (31,000); soybean trypsin inhibitor
(21,500); and lysozyme (14,400). Lane 2 contains undigested control
outer membrane preparation. Lane 3 contains a-chymotrypsin treated
preparation (2 mg of enzyme per mg of protein); lane 4 contains
trypsin treated preparation.
FIG. 3a is a photograph of a Coommasie Blue stained 14% SDS-PAGE
gel displaying proteinase K digests of Neisseria meningitidis
strain 608B (B:2a:P1.2) outer membrane preparations. Lanes 1, 3, 5,
and 7 contain undigested control. Lanes 2, 4, 6 and 8 contain outer
membrane preparations digested with proteinase K (2 IU per mg of
protein). Lanes 1 to 4 contain preparations treated at pH 7.2.
Lanes 5 to 8 contain preparations treated at pH 9.0. Lanes 1, 2, 5
and 6 contain preparations treated without SDS. Lanes 3, 4, 7 and 8
contain preparations treated in the presence of SDS. Molecular
weight markers are indicated on the left (in kilodaltons).
FIG. 3b is a photograph of a Coomassie Blue stained 14% SDS-PAGE
gel displaying the migration profiles of affinity purified
recombinant 22 kDa protein. Lane 1 contains molecular weight
markers: Phosphorylase b (97,400), bovine serum albumin (66,200),
ovalbumin (45,000), carbonic anhydrase (31,000), soybean trypsin
inhibitor (21,500) and lysozyme (14,400). Lane 2 contains 5 .mu.g
of control affinity purified recombinant 22 kDa protein. Lane 3
contains 5 .mu.g of affinity purified recombinant 22 kDa protein
heated at 100.degree. C. for 5 min. Lane 4 contains 5 .mu.g of
affinity purified recombinant 22 kDa protein heated at 100.degree.
C. for 10 min. . Lane 5 contains 5 .mu.g of affinity purified
recombinant 22 kDa protein heated at 100.degree. C. for 15 min.
FIGS. 4A & 4B is a photograph of Coomassie Blue stained 14%
SDS-PAGE gels and their corresponding Western immunoblots showing
the reactivity of monoclonal antibodies specific to the Neisseria
meningitidis 22 kDa surface protein. Outer membrane preparation
from Neisseria meningitidis strain 608B (B:2a:P1.2) (A) untreated;
(B) Proteinase K treated (2 IU per mg of protein)- Lane 1 contains
molecular weight markers and characteristic migration profile on
14% SDS-PAGE gel of outer membrane preparations. Lane 2 contains
Me-2; Lane 3 contains Me-3; lane 4 contains Me-5; lane 5 contains
Me-7; and lane 6 contains an unrelated control monoclonal antibody.
The molecular weight markers are phosphorylase b (97,400), bovine
serum albumin (66,200), ovalbumin (45,000), carbonic anhydrase
(31,000), soybean trypsin inhibitor (21,500) and lysozyme (14,400).
The immunoblot results shown in FIG. 4 for Me-2, Me-3, Me-5, Me-6
and Me-7 are consistent with the immunoblot results obtained for
Me-1.
FIG. 5 is a graphic depiction of the binding activity of the
monoclonal antibodies to intact bacterial cells. The results for
representative monoclonal antibodies Me-5 and Me-7 are presented in
counts per minute ("CPM") on the vertical axis. The various
bacterial strains used in the assay are shown on the horizontal
axis. A Haemophilus influenzae porin-specific monoclonal antibody
was used as a negative control. Background counts below 500 CPM
were recorded and were subtracted from the binding values.
FIGS. 6A-6C is a photograph of stained 14% SDS-PAGE gels and their
corresponding Western imunoblot demonstrating the purification of
the recombinant 22 kDa Neisseria meningitidis surface protein from
concentrated culture supernatant of Escherichia coli strain
BL21(DE3). FIG. 6(A) is a photograph of a Coomassie Blue and silver
stained 14% SDS-Page gel. Lane 1 contains the following molecular
weight markers: phosphorylase b (97,400), bovine serum albumin
(66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soybean
trypsin inhibitor (21,500) and lysozyme (14,400). Lane 2 contains
outer membrane protein preparation extracted from Neisseria
meningitidis strain 608B (serotype B:2a:p1.2)(10 mg). Lane 3
contains concentrated culture supernatant of Escherichia coli
BL21(DE3) (10 mg). Lane 4 contains affinity purified recombinant 22
kDa Neisseria meningitidis surface protein (1 mg). FIG. 6(B) is a
photograph of a Coomassie Blue stained 14% SDS-PAGE gel of
a-chymotrypsin, trypsin and proteinase K digests of affinity
purified recombinant 22 kDa Neisseria meningitidis surface protein.
Lane 1 contains the following molecular weight markers:
phosphorylase b (97,400), bovine serum albumin (66,200), ovalbumin
(45,000), carbonic anhydrase (31,000), soybean trypsin inhibitor
(21,500) and lysozyme (14,400). Lanes 2 to 5 contain purified
recombinant 22 kDa Neisseria meningitidis surface protein (2 mg)-
Lanes 7 to 10 contain bovine serum albumin (2 mg). Lanes 2 and 7
contain undigested protein ("NT"). Lanes 3 and 8 contain
.alpha.-chymotrypsin ("C") treated protein (2 mg of enzyme per mg
of protein). Lanes 4 and 9 contain trypsin ("T") treated protein (2
mg of enzyme per mg of protein). Lanes 5 and 10 contain proteinase
K ("K") treated protein (2 IU per mg of protein). FIG. 6(C) is a
photograph of the Western inmunoblotting of a duplicate gel using
monoclonal antibody Me-5.
FIG. 7 is a graphical depiction of the bactericidal activity of
protein A-purified anti-Neisseria meningitidis 22 kDa surface
protein monoclonal antibodies against Neisseria meningitidis strain
608B (B:2a:P1.2). The vertical axis of the graph shows the
percentage of survival of the Neisseria meningitidis bacteria after
exposure to various concentrations of monoclonal antibody (shown on
the horizontal axis of the graph).
FIG. 8 depicts the nucleotide and derived amino acid sequences of
the Neisseria meningitidis strain MCH88 22 kDa surface protein (SEQ
ID NO:3; SEQ ID NO:4). Conventional three letter symbols are used
for the amino acid residues. The open reading frame extends from
the start codon at base 116 to the stop codon at base 643.
FIG. 9 depicts the nucleotide and derived amino acid sequences of
the Neisseria meningitidis strain Z4063 22 kDa surface protein (SEQ
ID NO:5; SEQ ID NO:6). Conventional three letter symbols are used
for the amino acid residues. The open reading frame extends from
the start codon at base 208 to the stop codon at base 732.
FIG. 10 depicts the nucleotide and derived amino acid sequences of
the Neisseria gonorrhoeae strain b2, 22 kDa surface protein (SEQ ID
NO:7; SEQ ID NO:8). Conventional three letter symbols are used for
the amino acid residues. The open reading frame extends from the
start codon at base 241 to the stop codon at base 765.
FIG. 11 depicts the consensus sequence (SEQ ID NO:29) established
from the DNA sequences of the four strains of Neisseria and
indicates the substitutions or insertion of nucleotides specific to
each strain.
FIG. 12 depicts the consensus sequence (SEQ ID NO:30) established
from the protein sequences of the four strains of Neisseria and
indicates the substitutions or insertion of amino acid residues
specific to each strain.
FIG. 13 represents the time course of the immune response to the
recombinant 22 kDa protein in rabbits expressed as the reciprocal
ELISA titer. The rabbits were injected with outer membrane
preparations from E. coli strain JM109 with plasmid pN2202 or with
control plasmid pWKS30. The development of the specific humoral
response was analysed by ELISA using outer membrane preparations
obtained from Neisseria meningitidis strain 608B (B:2a:P1.2) as
coating antigen.
FIG. 14 represents the time course of the immune response to the
recombinant 22kDa protein in Macaca fascicularis (cynomolgus)
monkeys expressed as the reciprocal ELISA titer. The two monkeys
were respectively immunized with 100 .mu.g (K28) and 200 .mu.g
(I276) of affinity purified 22kDa protein per injection. The
control monkey (K65) was immunized with 150 .mu.g of unrelated
recombinant protein following the same procedure. The development
of the specific humoral response was analysed by ELISA using outer
membrane preparations obtained from Neisseria meningitidis strain
608B (B:2a:P1.2) as coating antigen.
FIG. 15 is a graphic representation of the synthetic peptides of
the invention (SEQ ID NO:2) as well as their respective position in
the full 22 kDa protein of Neisseria meningitidis strain 608B
(B:2a:P1.2).
FIG. 16 is a map of plasmid pNP2204 containing the gene which
encodes the Neisseria meningitidis 22 kDa surface protein 22 kDa,
Neisseria meningitidis 22 kDa surface protein gene; Ampi.sup.R,
ampicillin-resistance coding region; ColE1, origin of replication;
cI857, bacteriophage .lambda. cI857 temperature-sensitive repressor
gene; .lambda.PL, bacteriophage .lambda. transcription promoter; T1
transcription terminator. The direction of transcription is
indicated by the arrows. BglII and BamH1 are the restriction sites
used to insert the 22 kDa gene in the p629 plasmid.
DETAILED DESCRIPTION OF THE INVENTION
During our study of the ultrastructure of the outer membrane of
Neisseria meningitidis we identified a new low molecular weight
protein of 22 kilodaltons which has very unique properties. This
outer membrane protein is highly resistant to extensive treatments
with proteolytic enzymes, such as proteinase K, a serine protease
derived from the mold Tritirachium album limber. This is very
surprising since proteinase K resistant proteins are very rare in
nature because of the potency, wide pH optimum, and low peptide
bond specificity of this enzyme [Barrett, A. J. and N. D. Rawlings,
Biochem. Soc. Transactions (1991) 19: 707-715]. Only a few reports
have described proteins of prokaryotic origin that are resistant to
the enzymatic degradation of proteinase K. Proteinase K resistant
proteins have been found in Leptospira species [Nicholson, V. M.
and J. F. Prescott, Veterinary Microbiol. (1993) 36:123-138],
Mycoplasma species [Butler, G. H. et al. Infec. Immun. (1991)
59:1037-1042], Spiroplasma mirum [Bastian, F. O. et al. J. Clin.
Microbiol. (1987) 25:2430-2431] and in viruses and prions [Onodera,
T. et al. Microbiol. Immunol. (1993) 37:311-316; Prusiner, S. B. et
al. Proc. Nat. Acad. Sci. USA (1993) 90:2793-2797]. Herein, we
describe the use of this protein as a means for the improved
prevention, treatment and diagnosis of Neisseria meningitidis
infections.
Thus according to one aspect of the invention we provide a highly
conserved, immunologically accessible Neisseria meningitidis
surface protein, and fragments, analogues, and derivatives thereof.
As used herein, "Neisseria meningitidis surface protein" means any
Neisseria meningitidis surface protein encoded by a naturally
occurring Neisseria meningitidis gene. The Neisseria meningitidis
protein according to the invention may be of natural origin, or may
be obtained through the application of molecular biology with the
object of producing a recombinant protein, or fragment thereof.
As used herein, "highly conserved" means that the gene for the
Neisseria meningitidis surface protein and the protein itself exist
in greater than 50% of known strains of Neisseria meningitidis.
Preferably, the gene and its protein exist in greater than 99% of
known strains of Neisseria meningitidis. Examples 2 and 4 set forth
methods by which one of skill in the art would be able to test a
variety of different Neisseria meningitidis surface proteins to
determine if they are "highly conserved".
As used herein, immunologically accessible means that the Neisseria
meningitidis surface protein is present at the surface of the
organism and is accessible to antibodies. Example 2 sets forth
methods by which one of skill in the art would be able to test a
variety of different Neisseria meningitidis surface proteins to
determine if they are "immunologically accessible". Immunological
accessibility may be determined by other methods, including an
agglutination assay, an ELISA, a RIA, an immunoblotting assay, a
dot-enzyme assay, a surface accessibility assay, electron
microscopy, or a combination of these assays.
As used herein, "fragments" of the Neisseria meningitidis surface
protein include polypeptides having at least one peptide epitope,
or analogues and derivatives thereof. Peptides of this type may be
obtained through the application of molecular biology or
synthesized using conventional liquid or solid phase peptide
synthesis techniques.
As used herein, "analogues" of the Neisseria meningitidis surface
protein include those proteins, or fragments thereof, wherein one
or more amino acid residues in the naturally occurring sequence is
replaced by another amino acid residue, providing that the overall
functionality and protective properties of this protein are
preserved. Such analogues may be produced synthetically, or by
recombinant DNA technology, for example, by mutagenesis of a
naturally occurring Neisseria meningitidis surface protein. Such
procedures are well known in the art.
For example, one such analogue is selected from the recombinant
protein that may be produced from the gene for the 22 kDa protein
from Neisseria gonorrhoeae strain b2, as depicted in FIG. 10. A
further analog may be obtained from the isolation of the
corresponding gene from Neisseria lactamica.
As used herein, a "derivative" of the Neisseria meningitidis
surface protein is a protein or fragment thereof that has been
covalently modified, for example, with dinitrophenol, in order to
render it immunogenic in humans. The derivatives of this invention
also include derivatives of the amino acid analogues of this
invention.
It will be understood that by following the examples of this
invention, one of skill in the art may determine without undue
experimentation whether a particular fragment, analogue or
derivative would be useful in the diagnosis, prevention or
treatment of Neisseria meningitidis diseases.
This invention also includes polymeric forms of the Neisseria
meningitidis surface proteins, fragments, analogues and
derivatives. These polymeric forms include, for example, one or
more polypeptides that have been crosslinked with crosslinkers such
as avidin/biotin, gluteraldehyde or dimethylsuberimidate. Such
polymeric forms also include polypeptides containing two or more
tandem or inverted contiguous Neisseria meningitidis sequences,
produced from multicistronic mRNAs generated by recombinant DNA
technology.
This invention provides substantially pure Neisseria meningitidis
surface proteins. The term "substantially pure" means that the
Neisseria meningitidis surface protein according to the invention
is free from other proteins of Neisseria meningitidis origin.
Substantially pure Neisseria meningitidis surface protein
preparations can be obtained by a variety of conventional
processes, for example the procedure described in Examples 3 and
11.
In a further aspect, the invention particularly provides a 22 kDa
surface protein of Neisseria meningitidis having the amino acid
sequence of FIG. 1 (SEQ ID NO:2), or a fragment, analogue or
derivative thereof.
In a further aspect, the invention particularly provides a 22 kDa
surface protein of Neisseria meningitidis having the amino acid
sequence of FIG. 8 (SEQ ID NO:4), FIG. 9 (SEQ ID NO:6) or a
fragment, analogue or derivative thereof. Such a fragment may be
selected from the peptides listed in FIG. 15 (SEQ ID NO:9 to SEQ ID
NO:26).
In a further aspect, the invention provides a 22 kDa surface
protein of Neisseria gonorrhoeae having the amino acid sequence of
FIG. 10 (SEQ ID NO:8), or a fragment, analogue or derivative
thereof. As will be apparent from the above, any reference to the
Neisseria meningitidis 22 kDa protein also encompasses 22 kDa
proteins isolated from, or made from genes isolated from other
species of Neisseriacae such as Neisseria gonorrhoeae or Neisseria
lactamica.
A Neisseria meningitidis 22 kDa surface protein according to the
invention may be further characterized by one or more of the
following features:
(1) it has an approximate molecular weight of 22 kDa as evaluated
on SDS-PAGE gel;
(2) its electrophoretic mobility on SDS-PAGE gel is not modified by
treatment with reducing agents;
(3) it has an isoelectric point (pI) in a range around pI 8 to pI
10;
(4) it is highly resistant to degradation by proteolytic enzymes
such as .alpha.-chymotrypsin, trypsin and proteinase K;
(5) periodate oxidation does not abolish the specific binding of
antibody directed against the Neisseria meningitidis 22 kDa surface
protein;
(6) it is a highly conserved antigen;
(7) it is accessible to antibody at the surface of intact Neisseria
meningitidis organisms;
(8) it can induce the production of bactericidal antibodies;
(9) it can induce the production of antibodies that can protect
against experimental infection;
(10) it can induce, when injected into an animal host, the
development of an immunological response that can protect against
Neisseria meningitidis infection.
This invention also provides, for the first time, a DNA sequence
coding for the Neisseria meningitidis 22 kDa surface protein (SEQ
ID NO:1, NO:3, NO:5, and NO:7) . The preferred DNA sequences of
this invention are selected from the group consisting of:
(a) the DNA sequence of FIG. 1 (SEQ ID NO:1);
(b) the DNA sequence of FIG. 8 (SEQ ID NO:3);
(c) the DNA sequence of FIG. 9 (SEQ ID NO:5);
(d) the DNA sequence of FIG. 10 (SEQ ID NO:7);
(e) analogues or derivatives of the foregoing DNA sequences;
(f) DNA sequences degenerate to any of the foregoing DNA sequences;
and
(g) fragments of any of the foregoing DNA sequences;
wherein said sequences encode a product that displays the
immunological activity of the Neisseria meningitidis 22 kDa surface
protein.
Such fragments are preferably peptides as depicted in FIG. 15 (SEQ
ID NO:9, through SEQ ID NO:26).
Preferably, this invention also provides, for the first time, a DNA
sequence coding for the Neisseria meningitidis 22 kDa surface
protein (SEQ ID NO:1) More preferred DNA sequences of this
invention are selected from the group consisting of:
(a) the DNA sequence of FIG. 1 (SEQ ID NO:1);
(b) analogues or derivatives of the foregoing DNA sequences;
(c) DNA sequences degenerate to any of the foregoing DNA sequences;
and
(d) fragments of any of the foregoing DNA sequences;
wherein said sequences encode a product that displays the
immunological activity of the Neisseria meningitidis 22 kDa surface
protein.
Analogues and derivatives of the Neisseria meningitidis 22 kDa
surface protein coding gene will hybridize to the 22 kDa surface
protein-coding gene under the conditions described in Example
4.
For purposes of this invention, the fragments, analogues and
derivatives of the Neisseria meningitidis 22 kDa surface protein
have the "immunological activity" of the Neisseria meningitidis 22
kDa surface protein if they can induce, when injected into an
animal host, the development of an immunological response that can
protect against Neisseria meningitidis infection. One of skill in
the art may determine whether a particular DNA sequence encodes a
product that displays the immunological activity of the Neisseria
meningitidis 22 kDa surface protein by following the procedures set
forth herein in Example 6.
The Neisseria meningitidis surface proteins of this invention may
be isolated by a method comprising the following steps:
a) isolating a culture of Neisseria meningitidis bacteria,
b) isolating an outer membrane portion from the culture of the
bacteria; and
c) isolating said antigen from the outer membrane portion.
In particular, the foregoing step (c) may include the additional
steps of treating the Neisseria meningitidis outer membrane protein
extracts with proteinase K, followed by protein fractionation using
conventional separation techniques such as ion exchange and gel
chromatography and electrophoresis.
Alternatively and preferably, the Neisseria meningitidis surface
proteins of this invention may be produced by the use of molecular
biology techniques, as more particularly described in Example 3
herein. The use of molecular biology techniques is particularly
well-suited for the preparation of substantially pure recombinant
Neisseria meningitidis 22 kDa surface protein.
Thus according to a further aspect of the invention we provide a
process for the production of recombinant Neisseria meningitidis 22
kDa surface protein, including fragments, analogues and derivatives
thereof, comprising the steps of (1) culturing a unicellular host
organism transformed with a recombinant DNA molecule including a
DNA sequence coding for said protein, fragment, analogue or
derivative and one or more expression control sequences operatively
linked to the DNA sequence, and (2) recovering a substantially pure
protein, fragment, analogue or derivative.
As is well known in the art, in order to obtain high expression
levels of a transfected gene in a host, the gene must be
operatively linked to transcriptional and translational expression
control sequences that are functional in the chosen expression
host. Preferably, the expression control sequences, and the gene of
interest, will be contained in an expression vector that further
comprises a bacterial selection marker and origin of replication.
If the expression host is a eukaryotic cell, the expression vector
should further comprise an expression marker useful in the
expression host.
A wide variety of expression host/vector combinations may be
employed in expressing the DNA sequences of this invention. Useful
expression vectors for eukaryotic hosts include, for example,
vectors comprising expression control sequences from SV40, bovine
papilloma virus, adenovirus and cytomegalovirus. Useful expression
vectors for bacterial hosts include known bacterial plasmids, such
as plasmids from E. coli, including col E1, pCR1, pBR322, pMB9 and
their derivatives, wider host range plasmids, such as RP4, phage
DNAs, e.g., the numerous derivatives of phage lambda, e.g. NM989,
and other DNA phages, such as M13 and filamentous single stranded
DNA phages. Useful expression vectors for yeast cells include the 2
mu plasmid and derivatives thereof. Useful vectors for insect cells
include pVL 941.
In addition, any of a wide variety of expression control sequences
may be used in these vectors to express the DNA sequences of this
invention. Such useful expression control sequences include the
expression control sequences associated with structural genes of
the foregoing expression vectors. Examples of useful expression
control sequences include, for example, the early and late
promoters of SV40 or adenovirus, the lac system, the trp system,
the TAC or TRC system, the major operator and promoter regions of
phage lambda, the control regions of fd coat protein, the promoter
for 3-phosphoglycerate kinase or other glycolytic enzymes, the
promoters of acid phosphatase, e.g., Pho5, the promoters of the
yeast alpha-mating system and other sequences known to control
expression of genes of prokaryotic and eukaryotic cells or their
viruses, and various combinations thereof. The Neisseria
meningitidis 22 kDa surface protein's expression control sequence
is particularly useful in the expression of the Neisseria
meningitidis 22 kDa surface protein in E. coli (Example 3).
Host cells transformed with the foregoing vectors form a further
aspect of this invention. A wide variety of unicellular host cells
are useful in expressing the DNA sequences of this invention. These
hosts may include well known eukaryotic and prokaryotic hosts, such
as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi,
yeast, insect cells such as Spodoptera frugiperda (SF9), animal
cells such as CHO and mouse cells, African green monkey cells such
as COS 1, COS 7, BSC 1, BSC 40, and BMT 10, and human cells and
plant cells in tissue culture. Preferred host organisms include
bacteria such as E. coli and Bacillus subtilis and mammalian cells
in tissue culture.
It should of course be understood that not all vectors and
expression control sequences will function equally well to express
the DNA sequences of this invention. Neither will all hosts
function equally well with the same expression system. However, one
of skill in the art may make a selection among these vectors,
expression control sequences and hosts without undue
experimentation and without departing from the scope of this
invention. For example, in selecting a vector, the host must be
considered because the vector must replicate in it. The vectors
copy number, the ability to control that copy number, and the
expression of any other proteins encoded by the vector, such as
antibiotic markers, should also be considered.
In selecting an expression control sequence, a variety of factors
should also be considered. These include, for example, the relative
strength of the sequence, its controllability, and its
compatibility with the DNA sequences of this invention,
particularly as regards potential secondary structures. Unicellular
hosts should be selected by consideration of their compatibility
with the chosen vector, the toxicity of the product coded for by
the DNA sequences of this invention, their secretion
characteristics, their ability to fold the protein correctly, their
fermentation or culture requirements, and the ease of purification
from them of the products coded for by the DNA sequences of this
invention.
Within these parameters, one of skill in the art may select various
vector/expression control sequence/host combinations that will
express the DNA sequences of this invention on fermentation or in
large scale animal culture.
The polypeptides encoded by the DNA sequences of this invention may
be isolated from the fermentation or cell culture and purified
using any of a variety of conventional methods. One of skill in the
art may select the most appropriate isolation and purification
techniques without departing from the scope of this invention.
The Neisseria meningitidis surface proteins of this invention are
useful in prophylactic, therapeutic and diagnostic compositions for
preventing, treating and diagnosing diseases caused by Neisseria
meningitidis infection.
The Neisseria meningitidis surface proteins of this invention are
useful in prophylactic, therapeutic and diagnostic compositions for
preventing, treating and diagnosing diseases caused by Neisseria
gonorrhoeae, or Neisseria lactamica infection.
The Neisseria meningitidis surface proteins according to this
invention are particularly well-suited for the generation of
antibodies and for the development of a protective response against
Neisseria meningitidis diseases.
The Neisseria meningitidis surface proteins according to this
invention are particularly well-suited for the generation of
antibodies and for the development of a protective response against
Neisseria gonorrhoeae or Neisseria lactamica diseases.
In particular, we provide a Neisseria meningitidis 22 kDa surface
protein having an amino acid sequence of FIG. 1 (SEQ ID NO:2) or a
fragment, analogue, or derivative thereof for use as an immunogen
and as a vaccine.
In particular, we provide a Neisseria meningitidis 22 kDa surface
protein having an amino acid sequence of FIG. 1 (SEQ ID NO:2), FIG.
8 (SEQ ID NO:4), FIG. 9 (SEQ ID NO:6), or FIG. 10 (SEQ ID NO:8), or
a fragment, analogue, or derivative thereof for use as an immunogen
and as a vaccine.
Standard immunological techniques may be employed with the
Neisseria meningitidis surface proteins in order to use them as
immunogens and as vaccines. In particular, any suitable host may be
injected with a pharmaceutically effective amount of the Neisseria
meningitidis 22 kDa surface protein to generate monoclonal or
polyvalent anti-Neisseria meningitidis antibodies or to induce the
development of a protective immunological response against
Neisseria meningitidis diseases. Prior to injection of the host,
the Neisseria meningitidis surface proteins may be formulated in a
suitable vehicle, and thus we provide a pharmaceutical composition
comprising one or more Neisseria meningitidis surface antigens or
fragments thereof. Preferably, the antigen is the Neisseria
meningitidis 22 kDa surface protein or fragments, analogues or
derivatives thereof together with one or more pharmaceutically
acceptable excipients. As used herein, "pharmaceutically effective
amount" refers to an amount of one or more Neisseria meningitidis
surface antigens or fragments thereof that elicits a sufficient
titer of anti-Neisseria meningitidis antibodies to treat or prevent
Neisseria meningitidis infection.
The Neisseria meningitidis surface proteins of this invention may
also form the basis of a diagnostic test for Neisseria meningitidis
infection. Several diagnostic methods are possible. For example,
this invention provides a method for the detection of Neisseria
meningitidis antigen in a biological sample containing or suspected
of containing Neisseria meningitidis antigen comprising:
a) isolating the biological sample from a patient;
b) incubating an anti-Neisseria meningitidis 22 kDa surface protein
antibody or fragment thereof with the biological sample to form a
mixture; and
c) detecting specifically bound antibody or bound fragment in the
mixture which indicates the presence of Neisseria meningitidis
antigen.
Preferred antibodies in the foregoing diagnostic method are Me-i
and Me-7.
Alternatively, this invention provides a method for the detection
of antibody specific to Neisseria meningitidis antigen in a
biological sample containing or suspected of containing said
antibody comprising:
a) isolating the biological sample from a patient;
b) incubating a Neisseria meningitidis surface protein of this
invention or fragment thereof with the biological sample to form a
mixture; and
c) detecting specifically bound antigen or bound fragment in the
mixture which indicates the presence of antibody specific to
Neisseria meningitidis antigen.
One of skill in the art will recognize that this diagnostic test
may take several forms, including an immunological test such as an
enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay or a
latex agglutination assay, essentially to determine whether
antibodies specific for the protein are present in an organism.
The DNA sequences of this invention may also be used to design DNA
probes for use in detecting the presence of the pathogenic
Neisseria bacteria in a biological suspected of containing such
bacteria. The detection method of this invention comprises the
steps of:
a) isolating the biological sample from a patient;
b) incubating a DNA probe having a DNA sequence of this invention
with the biological sample to form a mixture; and
c) detecting specifically bound DNA probe in the mixture which
indicates the presence of Neisseria bacteria.
Preferred DNA probes have the base pair sequence of FIG. 1 (SEQ ID
NO:1), FIG. 8 (SEQ ID NO:3), FIG. 9 (SEQ ID NO:5), or FIG. 10 (SEQ
ID NO:7), or consensus sequence of FIG. 11 (SEQ ID NO:9).
A more preferred DNA probe has the 525 base pair sequence of FIG. 1
(SEQ ID NO:1).
The DNA probes of this invention may also be used for detecting
circulating Neisseria meningitidis nucleic acids in a sample, for
example using a polymerase chain reaction, as a method of
diagnosing Neisseria meningitidis infections. The probe may be
synthesized using conventional techniques and may be immobilized on
a solid phase, or may be labeled with a detectable label.
A preferred DNA probe for this application is an oligomer having a
sequence complementary to at least about 6 contiguous nucleotides
of the Neisseria meningitidis 22 kDa surface protein gene of FIG. 1
(SEQ ID NO:1), FIG. 8 (SEQ ID NO:3), FIG. 9 (SEQ ID NO:5), FIG. 10
(SEQ ID NO:7), or consensus sequence of FIG. 11 (SEQ ID NO:9).
A more preferred DNA probe for this application is an oligomer
having a sequence complementary to at least about 6 contiguous
nucleotides of the Neisseria meningitidis 22 kDa surface protein
gene of FIG. 1 (SEQ ID NO:1).
Another diagnostic method for the detection of Neisseria
meningitidis in a patient comprises the steps of:
a) labeling an antibody of this invention or fragment thereof with
a detectable label;
b) administering the labeled antibody or labeled fragment to the
patient; and
c) detecting specifically bound labeled antibody or labeled
fragment in the patient which indicates the presence of Neisseria
meningitidis.
For purification of any anti-Neisseria meningitidis surface protein
antibody, use may be made of affinity chromatography employing an
immobilized Neisseria meningitidis surface protein as the affinity
medium.
Thus according to another aspect of the invention we provide a
Neisseria meningitidis 22 kDa surface protein having an amino acid
sequence which includes the sequence of FIG. 1 (SEQ ID NO:2), FIG.
8 (SEQ ID NO:4), FIG. 9 (SEQ ID NO:6), or FIG. 10 (SEQ ID NO;8), or
portion thereof or an analogue thereof, covalently bound to an
insoluble support.
Thus according to a preferred aspect of the invention we provide a
Neisseria meningitidis 22 kDa surface protein having an amino acid
sequence which includes the sequence of FIG. 1 (SEQ ID NO:2), or
portion thereof or an analogue thereof, covalently bound to an
insoluble support.
A further feature of the invention is the use of the Neisseria
meningitidis surface proteins of this invention as immunogens for
the production of specific antibodies for the diagnosis and in
particular the treatment of Neisseria meningitidis infection.
Suitable antibodies may be determined using appropriate screening
methods, for example by measuring the ability of a particular
antibody to passively protect against Neisseria meningitidis
infection in a test model. One example of an animal model is the
mouse model described in the Examples herein. The antibody may be a
whole antibody or an antigen-binding fragment thereof and may in
general belong to any immunoglobulin class. The antibody or
fragment may be of animal origin, specifically of mammalian origin
and more specifically of murine, rat or human origin. It may be a
natural antibody or a fragment thereof, or if desired, a
recombinant antibody or antibody fragment. The term recombinant
antibody or antibody fragment means antibody or antibody fragment
which were produced using molecular biology techniques. The
antibody or antibody fragments may be of polyclonal, or
preferentially, monoclonal origin. It may be specific for a number
of epitopes associated with the Neisseria meningitidis surface
proteins but it is preferably specific for one. Preferably, the
antibody or fragments thereof will be specific for one or more
epitopes associated with the Neisseria meningitidis 22 kDa surface
protein. Also preferred are the monoclonal antibodies Me-1 and Me-7
described herein.
EXAMPLES
In order that this invention may be better understood, the
following examples are set forth. These examples are for purposes
of illustration only, and are not to be construed as limiting the
scope of the invention in any manner.
Example 1 describes the treatment of Neisseria meningitidis outer
membrane preparation with proteolytic enzymes and the subsequent
identification of the Neisseria meningitidis 22 kDa surface
protein.
Example 2 describes the preparation of monoclonal antibodies
specific for Neisseria meningitidis 22 kDa surface protein.
Example 3 describes the preparation of Neisseria meningitidis
recombinant 22 kDa surface protein.
Example 4 describes the use of DNA probes for the identification of
organisms expressing the Neisseria meningitidis 22 kDa surface
protein.
Example 5 describes the use of an anti-Neisseria meningitidis 22
kDa surface protein monoclonal antibody to protect mice against
Neisseria meningitidis infection.
Example 6 describes the use of purified recombinant 22 kDa surface
protein to induce a protective response against Neisseria
meningitidis infection.
Example 7 describes the identification of the sequence for the 22
kDa protein and protein-coding gene for other strains of Neisseria
meningitidis (MCH88, and Z4063), and one strain of Neisseria
gonorrhoeae.
Example 8 describes the immunological response of rabbits and
monkeys to the 22 kDa Neisseria meningitidis surface protein.
Example 9 describes the procedure used to map the different
immunological epitopes of the 22 kDa Neisseria meningitidis surface
protein.
Example 10 describes the induction by heat of an expression vector
for the large scale production of the 22 kDa surface protein.
Example 11 describes a purification process for the 22 kDa surface
protein when produced by recombinant technology.
Example 12 describes the use of 22 kDa surface protein as a human
vaccine.
EXAMPLE 1 Treatment Of Neisseria meningitidis Outer Membrane
Preparations With Proteolytic Enzymes And The Subsequent
Identification Of An Enzyme Resistant Neisseria meningitidis 22 kDa
Surface Protein
Several antigenic preparations derived from whole cell, lithium
chloride, or sarcosyl extracts were used to study the
ultrastructure of Neisseria meningitidis outer membrane. The outer
membrane of Gram-negative bacteria acts as an interface between the
environment and the interior of the cell and contains most of the
antigens that are freely exposed to the host immune defense. The
main goal of the study was the identification of new antigens which
can induce a protective response against Neisseria meningitidis.
One approach used by the inventors to identify such antigens, was
the partial disruption of the antigenic preparations mentioned
above with proteolytic enzymes. The antigenic determinants
generated by the enzymatic treatments could then be identified by
the subsequent analysis of the immunological and protective
properties of these treated antigenic preparations. To our surprise
we observed after electrophoretic resolution of Neisseria
meningitidis lithium chloride outer membrane extracts, that one low
molecular weight band, which was stained with Coomassie Brilliant
Blue R-250, was not destroyed by proteolytic enzyme treatments.
Coomassie Blue is used to stain proteins and peptides and has no or
very little affinity for the polysaccharides or lipids which are
also key components of the outer membrane. The fact that this low
molecular weight antigen was stained by Coomassie blue suggested
that at least part of it is made up of polypeptides that are not
digested by proteolytic enzymes, or that are protected against the
action of the enzymes by other surface structures. Moreover, as
demonstrated below the very potent enzyme proteinase K did not
digest this low molecular weight antigen even after extensive
treatments.
Lithium chloride extraction was used to obtain the outer membrane
preparations from different strains of Neisseria meningitidis and
was performed in a manner previously described by the inventors
[Brodeur et al., Infect. Immun., 50, p. 510 (1985)]. The protein
content of these preparations were determined by the Lowry method
adapted to membrane fractions [Lowry et al., J. Biol. Chem. 193, p.
265 (1951)]. Outer membrane preparations derived from Neisseria
meningitidis strain 608B (B:2a:P1.2) were treated for 24 hours at
37.degree. C. and continuous shaking with either a-chymotrypsin
from bovine pancreas (E.C. 3.4.21.1) (Sigma) or trypsin type 1 from
bovine pancreas (E.C. 3.4.21.4) (Sigma). The enzyme concentration
was adjusted at 2 mg per mg of protein to be treated. The same
outer membrane preparations were also treated with different
concentrations (0.5 to 24 mg per mg of protein) of Proteinase K
from Tritirachium album limber (Sigma or Boehringer Mannheim,
Laval, Canada) (E.C. 3.4.21.14). In order to promote protein
digestion by proteinase K, different experimental conditions were
used. The samples were incubated for 1 hour, 2 hours, 24 hours or
48 hours at 37.degree. C. or 56.degree. C. with or without shaking.
The pH of the mixture samples was adjusted at either pH 7.2 or pH
9.0. One % (vol/vol) of sodium dodecyl sulfate (SDS) was also added
to certain samples. Immediately after treatment the samples were
resolved by SDS-PAGE gel electrophoresis using the
MiniProteanII.RTM. (Bio-Rad, Mississauga, Ontario, Canada) system
on 14% (wt/vol) gels according to the manufacturer's instructions.
Proteins were heated to 100.degree. C. for 5 minutes with
2-mercaptoethanol and SDS, separated on 14% SDS gels, and stained
with Coomassie Brilliant Blue R-250.
FIG. 2 presents the migration profile on 14% SDS-PAGE gel of the
proteins present in outer membrane preparations derived from
Neisseria meningitidis strain 608B (B:2a:P1.2) after treatment at
37.degree. C. for 24 hours with .alpha.-chymotrypsin and trypsin.
Extensive proteolytic digestion of the high molecular weight
proteins and of several major outer membrane proteins can be
observed for the treated samples (FIG. 2, lanes 3 and 4) compared
to the untreated control (FIG. 2, lane 2). On the contrary, a
protein band with an apparent molecular weight of 22 kDa was not
affected even after 24 hours of contact with either proteolytic
enzyme.
This unique protein was further studied using a more aggressive
proteolytic treatment with Proteinase K (FIG. 3). Proteinase K is
one of the most powerful proteolytic enzymes since it has a low
peptide bond specificity and wide pH optimum. Surprisingly, the 22
kDa protein was resistant to digestion by 2 International Units
(IU) of proteinase K for 24 hours at 56.degree. C. (FIG. 3, lane
2). This treatment is often used in our laboratory to produce
lipopolysaccharides or DNA that are almost free of proteins.
Indeed, only small polypeptides can be seen after such an
aggressive proteolytic treatment of the outer membrane preparation.
Furthermore, longer treatments, up to 48 hours, or higher enzyme
concentrations (up to 24 IU) did not alter the amount of the 22 kDa
protein. The amount and migration on SDS-PAGE gel of the 22 kDa
protein were not affected when the pH of the reaction mixtures was
increased to pH 9.0, or when 1.0% of SDS, a strong protein
denaturant was added (FIG. 3, lanes 4, 6 and 8). The combined use
of these two denaturing conditions would normally result in the
complete digestion of the proteins present in the outer membrane
preparations, leaving only amino acid residues. Polypeptides of low
molecular weight were often observed in the digests and were
assumed to be fragments of sensitive proteins not effectively
digested during the enzymatic treatments. These fragments were most
probably protected from further degradation by the carbohydrates
and lipids present in the outer membrane. The bands with apparent
molecular weight of 28 kDa and 34 kDa which are present in treated
samples are respectively the residual enzyme and a contaminating
protein present in all enzyme preparations tested.
Interestingly, this study about the resistance of the 22 kDa
protein to proteases indicated that another protein band with
apparent molecular weight of 18 kDa seems to be also resistant to
enzymatic degradation (FIG. 3a). Clues about this 18 kDa protein
band were obtained when the migration profiles on SDS-PAGE gels of
affinity purified recombinant 22 kDa protein were analyzed (FIG.
3b). The 18 kDa band was apparent only when the affinity purified
recombinant 22 kDa protein was heated for an extended period of
time in sample buffer containing the detergent SDS before it was
applied on the gel. N-terminal amino acid analysis using the Edman
degradation (Example 3) clearly established that the amino acid
residues (E-G-A-S-G-F-Y-V-Q) identified on the 18 kDa band
corresponded to the amino acids 1-9 (SEQ ID NO:1). These results
indicate that the 18 and 22 kDa bands as seen on the SDS-PAGE is in
fact derived from the same protein. This last result also indicates
that the leader sequence is cleaved from the mature 18 kDa protein.
Further studies will be done to identify the molecular
modifications explaining this shift in apparent molecular weight
and to evaluate their impact on the antigenic and protective
properties of the protein.
In conclusion, the discovery of a Neisseria meningitidis outer
membrane protein with the very rare property of being resistant to
proteolytic digestion warranted further study of its molecular and
immunological characteristics. The purified recombinant 22 kDa
surface protein produced by Escherichia coli in Example 3 is also
highly resistant to proteinase K digestion. We are presently trying
to understand the mechanism which confers to the Neisseria
meningitidis 22 kDa surface protein this unusual resistance to
proteolytic enzymes.
EXAMPLE 2 Generation of Monoclonal Antibodies Specific for the 22
kDa Neisseria meningitidis Surface Protein
The monoclonal antibodies described herein were obtained from three
independent fusion experiments. Female Balb/c mice (Charles River
Laboratories, St-Constant, Quebec, Canada) were immunized with
outer membrane preparations obtained from Neisseria meningitidis
strains 604A, 608B and 2241C respectively serogrouped A, B and C.
The lithium chloride extraction used to obtain these outer membrane
preparations was performed in a manner previously described by the
inventors. [Brodeur et al., Infect. Immun. 50, p. 510 (1985)]. The
protein content of these preparations were determined by the Lowry
method adapted to membrane fractions [Lowry et al., J. Biol. Chem.
193, p. 265 (1951)]. Groups of mice were injected intraperitoneally
or subcutaneously twice, at three-week intervals with 10 mg of
different combinations of the outer membrane preparations described
above. Depending on the group of mice, the adjuvants used for the
immunizations were either Freund's complete or incomplete adjuvant
(Gibco Laboratories, Grand Island, N.Y.), or QuilA (CedarLane
Laboratories, Hornby, Ont., Canada). Three days before the fusion
procedure, the immunized mice received a final intravenous
injection of 10 mg of one of the outer membrane preparations
described above. The fusion protocol used to produce the hybridoma
cell lines secreting the desired monoclonal antibody was described
previously by the inventors [Hamel et al., J. Med. Microbiol., 25,
p. 2434 (1987)]. The class, subclass and light-chain type of
monoclonal antibodies Me-1, Me-2, Me-3, Me-5, Me-6 and Me-7 were
determined by ELISA as previously reported [Martin et al., J. Clin.
Microbiol., 28, p. 1720 (1990)] and are presented in Table 1.
The specificity of the monoclonal antibodies was established using
Western immmoblotting following the method previously described by
the inventors [Martin et al., Eur. J. Immunol. 18, p. 601 (1988)]
with the following modifications. Outer membrane preparations
obtained from different strains of Neisseria meningitidis were
resolved on 14% SDS-PAGE gels. The proteins were transferred from
the gels to nitrocellulose membranes using a semi-dry apparatus
(Bio-Rad). A current of 60 mA per gel (6.times.10 cm) was applied
for 10 minutes in the electroblot buffer consisting of 25 mM
Tris-HCl, 192 mM glycine and 20% (vol/vol) methanol, pH 8.3. The
Western immunoblotting experiments clearly indicated that the
monoclonal antibodies Me-l, Me-2, Me-3, Me-5, Me-6 and Me-7
recognized their specific epitopes on the Neisseria meningitidis 22
kDa protein (FIG. 4A). Analysis of the SDS-PAGE gels and the
corresponding Western immunoblots also indicated that the apparent
molecular weight of this protein does not vary from one strain to
another. However, the amount of protein present in the outer
membrane preparations varied from one strain to another and was not
related to the serogroup of the strain. Moreover, these monoclonal
antibodies still recognized their epitopes on the Neisseria
meningitidis 22 kDa surface protein after treatment of the outer
membrane preparation with 2 IU of proteinase K per mg of protein
(treatment described in Example 1, supra) (FIG. 4B). Interestingly,
the epitopes remained intact after the enzyme digestion thus
confirming that even if they are accessible in the membrane
preparation to the antibodies they are not destroyed by the enzyme
treatment. This latter result suggested that the mechanism which
explains the observed proteinase K resistance is most probably not
related to surface structures blocking the access of the enzyme to
the protein, or to the protection offered by the membrane to
proteins which are deeply embedded. While not shown in FIG. 4, the
results of the immunoblots for Me-1 were consistent with the
results for the other five monoclonal antibodies.
A series of experiments were performed to partially characterize
the Neisseria meningitidis 22 kDa surface protein and to
differentiate it from the other known meningococcal surface
proteins. No shift in apparent molecular weight on SDS-PAGE gel of
the Neisseria meningitidis 22 kDa surface protein was noted when
outer membrane preparations were heated at 100.degree. C. for 5
minutes, or at 37.degree. C. and 56.degree. C. for 30 minutes in
electrophoresis sample buffer with or without 2-mercaptoethanol.
This indicated that the migration of the 22 kDa surface protein,
when present in the outer membrane, was not heat- or
2-mercaptoethanol-modifiable.
Sodium periodate oxidation was used to determine if the monoclonal
antibodies reacted with carbohydrate epitopes present in the outer
membrane preparations extracted from Neisseria meningitidis
organisms. The method used to perform this experiment was
previously described by the inventors. [Martin et al., Infect.
Immun., 60, pp. 2718-2725 (1992)]. Treatment of outer membrane
preparations with 100 mM of sodium periodate for 1 hour at room
temperature did not alter the reactivity of the monoclonal
antibodies toward the Neisseria meningitidis 22 kDA surface
protein. This treatment normally abolishes the binding of
antibodies that are specific for carbohydrates.
Monoclonal antibody 2-1-CA2 (provided by Dr. A. Bhattacharjee.
Walter Reed Army Institute of Research, Washington, D.C.) is
specific for the lip protein (also called H.8), a surface antigen
common to all pathogenic Neisseria species. The reactivity of this
monoclonal antibody with outer membrane preparations was compared
to the reactivity of monoclonal antibody Me-5. The lip-specific
monoclonal antibody reacted with a protein band having an apparent
molecular weight of 30 kDa, while monoclonal antibody Me-5 reacted
with the protein band of 22 kDa. This result clearly indicates that
there is no relationship between Neisseria meningitidis 22 kDa
surface protein and the lip protein, another highly conserved outer
membrane protein.
To verify the exposure of the 22 kDa protein at the surface of
intact Neisseria meningitidis bacterial cells, a radioimmunoassay
was performed as previously described by the inventors [Proulx et
al., Infec. Immun., 59, p. 963 (1991)]. Six-hour and 18-hour
bacterial cultures were used for this assay The six monoclonal
antibodies were reacted with 9 Neisseria meningitidis strains (the
serogroup of the strain is indicated in parentheses on FIG. 5), 2
Neisseria gonorrhoeae strains ("NG"), 2 Moraxella catarrhalis
strains ("MC") and 2 Neisseria lactamica strains ("NL"). The
radioimmunoassay confirmed that the epitopes recognized by the
monoclonal antibodies are exposed at the surface of intact
Neisseria meningitidis isolates of different serotypes and
serogroups and should also be accessible to the proteolytic enzymes
(FIG. 5). The monoclonal antibodies bound strongly to their target
epitopes on the surface of all Neisseria meningitidis strains
tested. The recorded binding values (between 3,000 to 35,000 CPM),
varied from one strain to another, and with the physiological state
of the bacteria. A Haemophilus influenzae porin-specific monoclonal
antibody was used as a negative control for each bacterial strain.
Counts below 500 CPM were obtained and subsequently subtracted from
each binding value. With respect to the Neisseria meningitidis
strains tested in this assay, the results shown in FIG. 5 for
monoclonal antibodies Me-5 and Me-7 are representative of the
results obtained with monoclonal antibodies Me-1, Me-2, Me-3 and
Me-6. With respect to the other bacterial strains tested, the
binding activities shown for Me-7 are representative of the binding
activities obtained with other monoclonal antibodies that
recognized the same bacterial strain.
The antigenic conservation of the epitopes recognized by the
monoclonal antibodies was also evaluated. A dot enzyme immunoassay
was used for the rapid screening of the monoclonal antibodies
against a large number of bacterial strains. This assay was
performed as previously described by the inventors [Lussier et al.,
J. Immunoassay, 10, p. 373 (1989)]. A collection of 71 Neisseria
meningitidis strains was used in this study. The sample included 19
isolates of serogroup A, 23 isolates of serogroup B, 13 isolates of
serogroup C, 1 isolate of serogroup 29E, 6 isolates of serogroup
W-135, 1 isolate of serogroup X, 2 isolates of serogroup Y, 2
isolates of serogroup Z, and 4 isolates that were not serogrouped
("NS"). These isolates were obtained from the Caribbean
Epidemiology Centre, Port of Spain, Trinidad; Children's Hospital
of Eastern Ontario, Ottawa, Canada; Department of Saskatchewan
Health, Regina, Canada; Laboratoire de Sante Publique du Quebec,
Montreal, Canada; Max-Planck Institut fur Molekulare Genetik,
Berlin, FRG; Montreal Children Hospital, Montreal, Canada; Victoria
General Hospital, Halifax, Canada; and our own strains collection.
The following bacterial species were also tested: 16 Neisseria
gonorrhoeae, 4 Neisseria cinerea, 5 Neisseria lactamica, 1
Neisseria flava, 1 Neisseria flavescens, 3 Neisseria mucosa, 4
Neisseria perflava/sicca, 4 Neisseria perflava, 1 Neisseria sicca,
1 Neisseria subflava and 5 Moraxella catarrhalis, 1 Alcaligenes
feacalis (ATCC 8750), 1 Citrobacter freundii (ATCC 2080), 1
Edwarsiella tarda (ATCC 15947), 1 Enterobacter cloaca (ATCC 23355),
1 Enterobacter aerogenes (ATCC 13048), 1 Escherichia coli, 1
Flavobacterium odoratum, 1 Haemophilus influenzae type b (Eagan
strain), 1 Klebsiella pneumoniae (ATCC 13883), 1 Proteus rettgeri
(ATCC 25932), 1 Proteus vulgaris (ATCC 13315), 1 Pseudomonas
aeruginosa (ATCC 9027), 1 Salmonella typhimurium (ATCC 14028), 1
Serrati marcescens (ATCC 8100), 1 Shigella flexneri (ATCC 12022), 1
Shigella sonnei (ATCC 9290). They were obtained from the American
Type Culture Collection or a collection held in the Laboratory
Centre for Disease Control, Ottawa, Canada. The reactivities of the
monoclonal antibodies with the most relevant Neisseria strains are
presented in Table 1. One monoclonal antibody, Me-7, recognized its
specific epitope on 100% of the 71 Neisseria meningitidis strains
tested. This monoclonal antibody, as well as Me-2, Me-3, Me-5 and
Me-6 also reacted with certain strains belonging to other
Neisserial species indicating that their specific epitope is also
expressed by other closely related Neisseriaceae. Except for a
faint reaction with one Neisseria lactamica strain, monoclonal
antibody Me-1 reacted only with Neisseria meningitidis isolates.
Me-1 was further tested with another sample of 177 Neisseria
meningitidis isolates and was able to correctly identify more than
99% of the total Neisseria meningitidis strains tested. Besides the
Neisseria strains presented in Table 1, the monoclonal antibodies
did not react with any of the other bacterial species mentioned
above.
In conclusion, six monoclonal antibodies which specifically reacted
with the Neisseria meningitidis 22 kDa surface protein were
generated by the inventors. Using these monoclonal antibodies we
demonstrated that their specific epitopes are 1) located on a
proteinase K resistant 22 kDa protein present in the outer membrane
of Neisseria meningitidis, 2) conserved among Neisseria
meningitidis isolates, 3) exposed at the surface of intact
Neisseria meningitidis cells and accessible to antibody, and 4) the
reactivity of these monoclonal antibodies with the Neisseria
meningitidis 22 kDa surface protein is not modified by a treatment
with sodium periodate, suggesting that their specific epitopes are
not located on carbohydrates.
Although we found that the migration of the Neisseria meningitidis
22 kDa protein is moved to an apparent molecular weight of about 18
kDa when heated under stringent conditions, we observed that the
migration is not modified by 2-mercaptoethanol treatment.
We also demonstrated that the Neisseria meningitidis 22 kDa surface
protein has no antigenic similarity with the lip protein, another
low molecular weight and highly conserved protein present in the
outer membrane of Neisseria meningitidis.
As will be presented in Example 3, these monoclonal antibodies also
reacted with the purified, recombinant 22 kDa surface protein
produced after transformation of Escherichia coli strain BL2l (DE3)
with a plasmid vector pNP2202 containing the gene coding for the
Neisseria meningitidis 22 kDa surface protein.
TABLE 1 Reactivity of the monoclonal antibodies with Neisseria
isolates Number of Neisseria isolated recognized by the monoclonal
antibodies Serogroup of Neisseria meningitidis Numarella Neisseria
Neisseria A B C 29% W135 X Y Z NS.sup.1 Total catarrbalis
gonorrhoeae lactamica Name Isotype (19) (23) (13) (1) (5) (1) (2)
(2) (4) (71) (5) (14) (5) Me-1 1gG2a(k) 19 22 13 1 6 1 2 2 3 69 0 0
1 Me-2 1gG2a(k) 19 20 13 1 6 0 2 2 4 67 0 2 0 Me-3 1gG3(k) 19 22 13
1 6 1 2 2 3 69 0 2 4 Me-5 1gG2a(k) 19 22 13 1 6 1 2 2 3 69 0 2 0
Me-6 1gG1(k) 19 23 13 1 6 1 2 2 3 70 0 2 4 Me-7 1gG2a(k) 19 23 13 1
6 1 2 2 4 71 5 2 4 .sup.1 isolates not serogrouped
EXAMPLE 3 Molecular Cloning, Sequencing Of The Gene, High Yield
Expression And Purification Of The Neisseria meningitidis 22 kDa
Surface Protein
A. Molecular Cloning
A LambdaGEM-11 genomic DNA library from Neisseria meningitidis
strain 608B (B:2a:P1.2) was constructed according to the
manufacturer's recommendations (Promega CO, Madison, Wis.).
Briefly, the genomic DNA of the 608B strain was partially digested
with Sau 3AI, and fragments ranging between 9 and 23 Kb were
purified on agarose gel before being ligated to the Bam HI sites of
the LambdaGEM-11 arms. The resulting recombinant phages were used
to infect Escherichia coli strain LE392 (Promega) which was then
plated onto LB agar plates. Nineteen positive plaques were
identified after the immuno-screening of the library with the
Neisseria meningitidis 22 kDa surface protein-specific monoclonal
antibodies of Example 2 using the following protocol. The plates
were incubated 15 minutes at -20.degree. C. to harden the top agar.
Nitrocellulose filters were gently applied onto the surface of the
plates for 30 minutes at 4.degree. C. to absorb the proteins
produced by the recombinant viral clones. The filters were then
washed in PBS-Tween 0.02% (vol/vol) and immunoblotted as described
previously [Lussier et al., J. Immunoassay, 10, p. 373 (1989)].
After amplification and DNA purification, one viral clone,
designated clone 8, which had a 13 Kb insert was selected for the
subcloning experiments. After digestion of this clone with Sac I,
two fragments of 5 and 8 Kb were obtained. These fragments were
purified on agarose gel and ligated into the Sac I restriction site
of the low copy number plasmid pWKS30 [Wang and Kushner, Gene, 100,
p. 195 (1991)]. The recombinant plasmids were used to transform
Escherichia coli strain JM109 (Promega) by electroporation
(Bio-Rad, Mississauga, Ont., Canada) following the manufacturer's
recommendations, and the resulting colonies were screened with the
Neisseria meningitidis 22 kDa surface protein-specific monoclonal
antibodies of Example 2. Positive colonies were observed only when
the bacteria were transformed with the plasmid carrying the 8 Kb
insert. Western blot analysis (the methodology was described in
Example 2) of the positive clones showed that the protein expressed
by Escherichia coli was complete and migrated on SDS-PAGE gel like
the Neisseria meningitidis 22 kDa surface protein. To further
reduce the size of the insert, a clone containing the 8 Kb fragment
was digested with Cla I and a 2.75 Kb fragment was then ligated
into the Cla I site of the pWKS30 plasmid. Western blot analysis of
the resulting clones clearly indicated once again that the protein
expressed by Escherichia coli was complete and migrated on SDS-PAGE
gel like the native Neisseria meningitidis 22 kDa surface
protein.
After restriction analysis, two clones, designated pNP2202 and
pNP2203, were shown to carry the 2.75 Kb insert in opposite
orientations and were selected to proceed with the sequencing of
the gene coding for the Neisseria meningitidis 22 kDa surface
protein. The "Double Stranded Nested Deletion Kit" from Pharmacia
Biotech Inc. (Piscataway, N.J.) was used according to the
manufacturer's instructions to generate a series of nested
deletions from both clones. The resulting truncated inserts were
then sequenced from the M13 forward primer present on the pWKS30
vector with the "Taq Dye Deoxy Terminator Cycle Sequencing Kit"
using an Applied Biosystems Inc. (Foster City, Calif.) automated
sequencer model 373A according to the manufacturer's
recommendations.
B. Sequence Analysis
After the insert was sequenced in both directions, the nucleotide
sequence revealed an open reading frame consisting of 525
nucleotides (including the stop codon) encoding a protein composed
of 174 amino acid residues having a predicted molecular weight of
18,000 Daltons and a pi of 9.93. The nucleotide and deduced amino
acid sequences are presented in FIG. 1 (SEQ ID NO:1; SEQ ID
NO:2).
To confirm the correct expression of the cloned gene, the
N-terminal amino acid sequence of the native 22 kDa surface protein
derived from Neisseria meningitidis strain 608B was determined in
order to compare it with the amino 30 acid sequence deduced from
the nucleotide sequencing data. Outer membrane preparation derived
from Neisseria meningitidis strain 608B was resolved by
electrophoresis on a 14% SDS-PAGE gel and transferred onto a
polyvinylidine difluoride membrane (Millipore Products, Bedford
Mass.) according to a previously described method [Sambrook et al.,
Molecular Cloning; a laboratory manual, Cold Spring Harbor
Laboratory Press (1989)]. The 22 kDa protein band was excised from
the gel and then subjected to Edman degradation using the Applied
Biosystems Inc. (Foster City, Calif.) model 473A automated protein
sequencer following the manufacturer's recommendations. The amino
acid sequence E-G-A-S-G-F-Y-V-Q-A corresponded to amino acids 1-10
(SEQ ID NO:2) of the open reading frame, indicating that the
Neisseria meningitidis strain 608B, 22 kDa surface protein has a 19
amino acid leader peptide (amino acid residues -19 to -1 of SEQ ID
NO:2).
A search of established databases confirmed that the Neisseria
meningitidis strain 608B, 22 kDa surface protein (SEQ ID NO:2) or
its gene (SEQ ID NO:1) have not been described previously.
C. High Yield Expression And Purification of The Recombinant
Neisseria meningitidis 22 kDa Surface Protein
The following process was developed in order to maximize the
production and purification of the recombinant Neisseria
meningitidis 22 kDa surface protein expressed in Escherichia coli.
This process is based on the observation that the recombinant 22
kDa surface protein produced by Escherichia coli strain BL21(DE3)
[Studier and Moffat, J. Mol. Biol., 189, p. 113 (1986)] carrying
the plasmid pNP2202 can be found in large amounts in the outer
membrane, but can also be obtained from the culture supernatant in
which it is the most abundant protein. The culture supernatant was
therefore the material used to purify the recombinant 22 kDa
protein using affinity chromatography (FIG. 6A).
To generate an affinity chromatography matrix, monoclonal
antibodies Me-2, Me-3 and Me-5 (described in Example 2) were
immobilized on CNBr-activated sepharose 4B (Pharmacia Biotech Inc.,
Piscataway, N.J.) according to the manufacturer's instructions.
To prepare the culture supernatant, an overnight culture of
Escherichia coli strain BL21(DE3), harboring the plasmid pNP2202
was inoculated in LB broth (Gibco Laboratories, Grand Island, N.Y.)
containing 25 mg/ml of ampicillin (Sigma) and was incubated 4 hours
at 37.degree. C. with agitation. The bacterial cells were removed
from the culture media by two centrifugations at 10,000 Xg for 10
minutes at 4.degree. C. The culture supernatant was filtered onto a
0.22 mm membrane (Millipore, Bedfords, Mass.) and then concentrated
approximately 100 times using an ultra-filtration membrane (Amicon
Co., Beverly, Mass.) with a molecular cut off of 10,000 Daltons. To
completely solubilize the membrane vesicles, Empigen BB (Calbiochem
Co., LaJolla, Calif.)) was added to the concentrated culture
supernatant to a final concentration of 1% (vol/vol). The
suspension was incubated at room temperature for one hour, dialyzed
overnight against several liters of 10 mM Tris-HCl buffer, pH 7.3
containing 0.05% Empigen BB(vol/vol) and centrifuged at 10,000 Xg
for 20 minutes at 4.degree. C. The antigen preparation was added to
the affinity matrix and incubated overnight at 4.degree. C. with
constant agitation. The gel slurry was poured into a chromatography
column and washed extensively with 10 mM Tris-HCl buffer, pH 7.3
containing 0.05% Empigen BB (vol/vol). The recombinant 22 kDa
protein was then eluted from the column with 1M LiCl in 10 mM
Tris-HCl buffer, pH 7.3. The solution containing the eluted protein
was dialyzed extensively against several liters of 10 mM Tris-HCl
buffer, pH 7.3 containing 0.05% Empigen BB. Coomassie Blue and
silver stained SDS-Page gels [Tsai and Frasch, Analytical Biochem.,
119, pp. 19 (1982)] were used to evaluate the purity of the
recombinant 22 kDa surface protein at each step of the purification
process and representative results are presented in FIG. 6A. Silver
staining of the gels clearly demonstrated that the purification
process generated a fairly pure recombinant 22 kDa protein with
only a very small quantity of Escherichia coli
lipopolysaccharide.
The resistance to proteolytic cleavage of the purified recombinant
22 kDa surface protein was also verified and the results are
presented in FIG. 6B. Purified recombinant 22 kDa surface protein
was treated as described in Example 1 with .alpha.-chymotrypsin and
trypsin at 2 mg per mg of protein and with 2 IU of proteinase K per
mg of protein for 1 hour at 37.degree. C. with constant shaking. No
reduction in the amount of protein was observed after any of these
treatments. In comparison, partial or complete digestion depending
on the enzyme selected was observed for the control protein which
was in this case bovine serum albumin (BSA, Sigma). Furthermore,
longer periods of treatment did not result in any modification of
the protein. These latter results demonstrated that transformed
Escherichia coli cells can express the complete recombinant 22 kDa
surface protein and that this protein is also highly resistant to
the action of these three proteolytic enzymes as was the native
protein found in Neisseria meningitidis. In addition, the purified
recombinant 22 kDa surface protein which is not embedded in the
outer membrane of Escherichia coli is still highly resistant to the
action of the proteolytic enzymes.
We also verified the effect of the enzymatic treatments on the
antigenic properties of the recombinant 22 kDa protein. As
determine by ELISA and Western immunoblotting, the monoclonal
antibodies described in Example 2 readily recognized the
recombinant 22 kDa surface protein that was purified according to
the process described above (FIG. 6C). Moreover, the reactivity of
monoclonal antibody Me-5, as well as the reactivity of other 22 kDa
protein-specific monoclonal antibodies, with the purified
recombinant 22 kDa surface protein was not altered by any of the
enzyme treatments, thus confirming that the antigenic properties of
the recombinant 22 kDa protein seem similar to the ones described
for the native protein.
Important data were presented in Example 3 and can be summarized as
follows:
1) the complete nucleotide and amino acid sequences of the
Neisseria meningitidis 22 kDa surface protein were obtained (SEQ ID
NO:1; SEQ ID NO:2);
2) N-terminal sequencing of the native protein confirmed that the
Neisseria meningitidis 22 kDa gene was indeed cloned;
3) this protein was not described previously;
4) it is possible to transform a host such as Escherichia coli and
obtain expression of the recombinant Neisseria meningitidis 22 kDa
surface protein in high yield;
5) it is possible to obtain the recombinant protein free of other
Neisseria meningitidis molecules and almost free of components
produced by Escherichia coli;
6) the purified recombinant 22 kDa surface protein remains highly
resistant to the action of proteolytic enzymes such as
a-chymotrypsin, trypsin and proteinase K; and
7) the antigenic properties of the recombinant 22 kDa protein
compare to the ones described for the native Neisseria meningitidis
22 kDa surface protein.
EXAMPLE 4 Molecular Conservation Of The Gene Coding for the
Neisseria meningitidis 22 kDa Surface Protein
To verify the molecular conservation among Neisseria isolates of
the gene coding for the Neisseria meningitidis 22 kDa surface
protein, a DNA dot blot hybridization assay was used to test
different Neisseria species and other bacterial species. First, the
525 base pair gene coding for the Neisseria meningitidis 22 kDa
surface protein was amplified by PCR, purified on agarose gel and
labeled by random priming with the non radioactive DIG DNA labeling
and detection system (Boehringer Mannheim, Laval, Canada) following
the manufacturer's instructions.
The DNA dot blot assay was done according to the manufacturer's
instructions (Boehringer Mannheim). Briefly, the bacterial strains
to be tested were dotted onto a positively charge nylon membrane
(Boehringer Mannheim), dried and then treated as described in the
DIG System's user's guide for colony lifts. Pre-hybridizations and
hybridizations were done at 42.degree. C. with solutions containing
50% formamide (Sigma). The pre-hybridization solution also
contained 100 mg/ml of denatured herring sperm DNA (Boehringer
Mannheim) as an additional blocking agent to prevent non-specific
hybridization of the DNA probe. The stringency washes and detection
steps using the chemiluminescent lumigen PPD substrate were also
done as described in the DIG System's user's guide.
For the 71 Neisseria meningitidis strains tested the results
obtained with monoclonal antibody Me-7 and the 525 base pair DNA
probe were in perfect agreement. According to the results, all the
Neisseria meningitidis strains tested have the Neisseria
meningitidis 22 kDa surface protein gene and they express the
protein since they were all recognized by the monoclonal antibody,
thus confirming that this protein is highly conserved among the
Neisseria meningitidis isolates (Table 2).
The DNA probe also detected the gene coding for the Neisseria
meningitidis 22 kDa surface protein in all Neisseria gonorrhoeae
strains tested.
On the contrary, the monoclonal antibody Me-7 reacted only with 2
out of the 16 Neisseria gonorrhoeae strains tested indicating that
the specific epitope is somehow absent, inaccessible or modified in
Neisseria gonorrhoeae strains, or that most of the Neisseria
gonorrhoeae strains do not express the protein even if they have
the coding sequence in their genome (Table 2).
A good correlation between the two detection methods was also
observed for Neisseria lactamica, since only one strain of
Neisseria lactamica was found to have the gene without expressing
the protein (Table 2). This result could also be explained by the
same reasons presented in the last paragraph.
This may indicate that, although the 22 kDa is not expressed, or
not accessible on the surface of Neisseria gonorrhoeae strains, the
22 kDa protein-coding gene of the Neisseria gonorrhoeae and
Neisseria lactamica strains may be used for construction of
recombinant plasmids used for the production of the 22 kDa surface
protein or analogs. All such protein or analogs may be used for the
prevention, detection, or diagnosis of Neisseria infections. More
particularly, such infections may be selected from infections from
Neisseria meningitidis, Neisseria gonorrhoeae, and Neisseria
lactamica. Therefore, the 22 kDa surface protein or analogs, may be
used for the manufacture of a vaccine against such infections.
Moreover, the 22 kDa protein or analogs, may be used for the
manufacture of a kit for the detection or diagnosis of such
infections.
The results obtained with Moraxella catharralis strains showed that
out of the 5 strains tested, 3 reacted with monoclonal antibody
Me-7, but none of them reacted with the DNA probe indicating that
the gene coding for the Neisseria meningitidis 22 kDa surface
protein is absent from the genome of these strains (Table 2).
Several other Neisserial species as well as other bacterial species
(see footnote, Table 2) were tested and none of them were found to
be positive by any of the two tests. This latter result seems to
indicate that the gene for the 22 kDa surface protein is shared
only among closely related species of Neisseriacae.
TABLE 2 Reactivity of the 525 base pair DNA probe and monoclonal
antibody Me-7 with different Neisseria species Number of strains
identified by Neisseria species Monoclonal (number of strains
tested).sup.1 antibody Me-7 DNA probe Neisseria meningitidis (71)
71 71 Moraxella catharallis (5) 3 0 Neisseria gonorrhoeae (16) 2 16
Neisseria lactamica (5) 4 5 .sup.1 The following Neisserrial
species and other bacterial species were also tested with the two
assays and gave negative results: 1 Neisseria cinerea, 1 Neisseria
flava, 1 Neisseria flavescens, 2 Neisseria mucosa, 4 Neisseria
perflavalsicca, 1 Neisseria perflava, 1 N. sicca, 1 N. subflava, 1
Alcaligenes feacalis (ATCC 8750), 1 Bordetella pertussis (9340), 1
Bordetella #bronchiseptica, 1 Citrobacter freundii (ATCC 2080), 1
Edwarsiella tarda (ATCC 15947), 1 Enterobacter cloaca (ATCC 23355),
1 Enterobacter aerogenes (ATCC 13048), 1 Escherichia coli, 1
Flavobacterium odoratum, 1 Haemophilus influenzae type b (Eagan
strain), 1 Klebsiella pneumoniae (ATCC 13883), 1 Proteus rettgeri
(ATCC 25932), 1 Proteus vulgaris (ATCC #13315), 1 Pseudomonas
aeruginosa (ATCC 9027), 1 Salmonella typhimurium (ATCC 14028), 1
Serrati marcescens (ATCC 8100), 1 Shigella flexneri (ATCC 12022), 1
Shigella sonnei (ATCC 9290), and 1 Xanthomonas maltophila.
In conclusion, the DNA hybridization assay clearly indicated that
the gene coding for the Neisseria meningitidis 22 kDa surface
protein is highly conserved among the pathogenic Neisseria.
Furthermore, the results obtained clearly showed that this DNA
probe could become a valuable tool for the rapid and direct
detection of pathogenic Neisseria bacteria in clinical specimen.
This probe could even be refined to discriminate between the
Neisseria meningitidis and Neisseria gonorrhoeae.
EXAMPLE 5 Bacteriolytic And Protective Properties of The Monoclonal
Antibodies
The bacteriolytic activity of the purified Neisseria meningitidis
22 kDa surface protein-specific monoclonal antibodies was evaluated
in vitro according to a method described previously [Brodeur et
al., Infect. Immun., 50, p. 510 (1985); Martin et al., Infect.
Immun., 60, p. 2718 (1992)]. In the presence of a guinea pig serum
complement, purified monoclonal antibodies Me-l and Me-7
efficiently killed Neisseria meningitidis strain 608B. Relatively
low concentrations of each of these monoclonal antibodies reduced
by more than 50% the number of viable bacteria. The utilization of
higher concentrations of purified monoclonal antibodies Me-1 and
Me-7 resulted in a sharp decrease (up to 99%) in the number of
bacterial colony forming units. Importantly, the bacteriolytic
activity of these monoclonal antibodies is complement dependent,
since heat-inactivation of the guinea pig serum for 30 minutes at
56.degree. C. completely abolished the killing activity. The other
monoclonal antibodies did not exhibit significant bacteriolytic
activity against the same strain. The combined, representative
results of several experiments are presented in FIG. 7, wherein the
results shown for Me-7 are representative and consistent with the
results obtained for Me-1 The results shown for Me-2 are
representative and consistent with the results obtained for the
other monoclonal antibodies Me-3, Me-5 and Me-6.
A mouse model of infection, which was described previously by one
of the inventors [Brodeur et al, Infect. Immun., 50, p. 510 (1985);
Brodeur et al., Can. J. Microbial., 32, p. 33 (1986)] was used to
assess the protective activity of each monoclonal antibody.
Briefly, Balb/c mice were injected intraperitoneally with 600 ml of
ascitic fluid containing the monoclonal antibodies 18 hours before
the bacterial challenge. The mice were then challenged with one ml
of a suspension containing 1000 colony forming units of Neisseria
meningitdis strain 608B, 4% mucin (Sigma) and 1.6% hemoglobin
(Sigma). The combined results of several experiments are presented
in Table 3. It is important to note that only the bacteriolytic
monoclonal antibodies Me-1 and Me-7 protected the mice against
experimental Neisseria meningitidis infection. Indeed, the
injection of ascitic fluid containing these two monoclonal
antibodies before the bacterial challenge significantly increased
the rate of survival of Balb/c mice to 70% or more compared to the
9% observed in the control groups receiving either 600 ml Sp2/0
induced ascitic fluid or 600 ml ascitic fluid containing unrelated
monoclonal antibodies. Results have also indicated that 80% of the
mice survived the infection if they were previously injected with
400 .mu.g of protein A purified Me-7 18 hours before the bacterial
challenge. Subsequent experiments are presently being done to
determine the minimal antibody concentration necessary to protect
50% of the mice. Lower survival rates from 20 to 40% were observed
for the other Neisseria meningitidis 22 kDa surface
protein-specific monoclonal antibodies.
TABLE 3 Evaluation of the immunoprotective potential of the 22 kDa
surface protein-specific monoclonal antibodies against Neisseria
meningitidis strain 608B (B:2a:P1.2) Number of living mice after
Monoclonal challenge % of antibodies 24 h 72 h survival Me-1 29/30
23/30 76 Me-2 17/20 3/20 25 Me-3 5/10 2/10 20 Me-5 11/20 8/20 40
Me-7 10/10 7/10 70 purified Me-7 13/15 12/15 80 Control 31/100
9/100 9
In conclusion, the results clearly indicated that an antibody
specific for the Neisseria meningitidis 22 kDa surface protein can
efficiently protect mice against an experimental lethal challenge.
The induction of protective antibodies by an antigen is one of the
most important criteria to justify further research on potential
vaccine candidate.
EXAMPLE 6 Immunization With Purified Recombinant 22 kDa Surface
Protein Confers Protection Against Subsequent Bacterial
Challenge
Purified recombinant 22 kDa surface protein was prepared according
to the protocol presented in Example 3, and was used to immunize
Balb/c mice to determine its protective effect against challenge
with a lethal dose of Neisseria meningitidis 608B (B:2a:P1.2). It
was decided to use the purified recombinant protein instead of the
native meningococcal protein in order to insure that there was no
other meningococcal antigen in the vaccine preparation used during
these experiments. The mouse model of infection used in these
experiments was described previously by one of the inventors
[Brodeur et al., Infec. Immun., 50, p. 510 (1985); Brodeur et al.,
Can. J. Microbiol., 32, p. 33 (1986)]. The mice were each injected
subcutaneously three times at three-week intervals with 100 ml of
the antigen preparation containing either 10 or 20 .mu.g per mouse
of the purified recombinant 22 kDa surface protein. QuilA was the
adjuvant used for these experiments at a concentration of 25 .mu.g
per injection. Mice in the control groups were injected following
the same procedure with either 10 or 20 .mu.g of BSA, 20 .mu.g of
concentrated culture supernatant of Escherichia coli strain
BL2l(DE3) carrying the plasmid pWKS30 without the insert gene for
the meningococcal protein prepared as described in Example 3, or
phosphate-buffered saline. Serum samples from each mouse were
obtained before each injection in order to analyze the development
of the immune response against the recombinant protein. Two weeks
following the third immunization the mice in all groups were
injected intraperitoneally with 1 ml of a suspension containing
1000 colony forming units of Neisseria meningitidis strain 608B in
4% mucin (Sigma) and 1.6% hemoglobin (Sigma).
The results of these experiments are presented in Table 4. Eighty
percent (80%) of the mice immunized with the purified recombinant
22 kDa surface protein survived the bacterial challenge compared to
0 to 42% in the control groups. Importantly, the mice in the
control group injected with concentrated Escherichia coli culture
supernatant were not protected against the bacterial challenge.
This latter result clearly demonstrated that the components present
in the culture media and the Escherichia coli's antigens that might
be present in small amounts after purification do not contribute to
the observed protection against Neisseria meningitidis.
TABLE 4 Immunization With Purified Recombinant 22 kDa Surface
Protein Confers Protection Against Subsequent Bacterial Challenge
with Neisseria meningitidis 608B (B:2a:P1.2) strain. Number of
living mice after challenge % of Experiment Group 24 h 48 h 72 h
survival 1 10 .mu.g of 20/20 16/20 80 purified 22 kDa 10 .mu.g of
BSA 17/19 8/19 42 2 20 .mu.g of 9/10 8/10 8/10 80 purified 22 kDa
protein 20 .mu.g of 7/10 5/10 2/10 20 concentrated E. coli
supernatant 20 .mu.g of BSA 6/10 4/10 2/10 20 Phosphate 8/10 0/10
0/10 0 buffered saline
CONCLUSION
The injection of purified recombinant 22 kDa surface protein
greatly protected the immunized mice against the development of a
lethal infection by Neisseria meningitidis.
Antibodies according to this invention are exemplified by murine
hybridoma cell lines producing monoclonal antibodies Me-1 and Me-7
deposited in the American Type Culture Collection in Rockville,
Md., USA on Jul. 21, 1995. The deposits were assigned accession
numbers HB 11959 (Me-1) and HB 11958 (Me-7).
EXAMPLE 7 Sequence analysis of other strains of Neisseria
meningitidis and of Neisseria gonorrhoeae
The 2.75 kb claI digested DNA fragment containing the gene coding
for the 22 kDa surface protein was isolated from the genomic DNA of
the different strains of Neisseria meningitidis and Neisseria
gonorrhoeae as described in Example 3.
a) MCH88 strain: The nucleotide sequence of strain MCH88 (clinical
isolate) is presented in FIG. 8 (SEQ ID NO:3). From experimental
evidence obtained from strain 608B (Example 3), a putative leader
sequence was deduced corresponding to amino acid -19 to -1
(M-K-K-A-L-A-A-L-I-A-L-A-L-P-A-A-A-L-A). A search of established
databases confirmed that 22 kDa surface protein from Neisseria
meningitidis strain MCH 188 (SEQ ID NO:4) or its gene (SEQ ID NO:3)
have not been described previously.
b) Z4063 strain: The nucleotide sequence of strain Z4063 (Wang
J.-F. et al. Infect. Immun., 60, p.5267 (1992)) is presented in
FIG. 9 (SEQ ID NO:5). From experimental evidence obtained from
strain 608B (Example 3), a putative leader sequence was deduced
corresponding to amino acid -19 to -1
(M-K-K-A-L-A-T-L-I-A-L-A-L-P-A-A-A-L-A). A search of established
databases confirmed that 22 kDa surface protein from Neisseria
meningitidis strain Z4063 (SEQ ID NO:6) or its gene (SEQ ID NO:5)
have not been described previously.
c) Neisseria gonorrhoeae strain b2: The nucleotide sequence of
Neisseria gonorrhoeae strain b2 (serotype 1. Nat. Ref. Center for
Neisseria, LCDC, Ottawa, Canada) is described in FIG. 10 (SEQ ID
NO:7). From experimental evidence obtained from strain 608B
(Example 3), a putative leader sequence was deduced corresponding
to amino acid -19 to -1 (M-K-K-A-L-A-A-L-I-A-L-A-L-P-A-A-A-L-A). A
search of established databases confirmed that 22 kDa surface
protein from Neisseria gonorrhoeae strain b2 (SEQ ID NO:8) or its
gene (SEQ ID NO:7) have not been described previously.
FIG. 11 shows the consensus sequence established from the DNA
sequence of all four strains tested. The MCH88 strain showed an
insertion of one codon (TCA) at nucleotide 217, but in general the
four strains showed striking homology.
FIG. 12 depicts the homology between the deduced amino acid
sequence obtained from the four strains. There is greater than 90%
identity between all four strains.
Example 8 Immunological response of rabbits and monkeys to the 22
kDa Neisseria meningitidis surface protein
Rabbits and monkeys were immunized with the recombinant 22 kDa
protein to assess the antibody response in species other than the
mouse.
a) Rabbits
Male New Zealand rabbits were immunized with outer membrane
preparations obtained from E. coli strain JM109 with the plasmid
pN2202 or with the control plasmid pWKS30 (the strain and the
plasmids are described in Example 3). The lithium chloride
extraction used to obtain these outer membrane preparations was
performed in a manner previously described by the inventors
[Brodeur et al, Infect. Immun. 50, 510 (1985)]. The protein content
of these preparations were determined by the Lowry method adapted
to membrane fractions [Lowry et al, J. Biol. Chem. 193, 265
(1951)]. The rabbits were injected subcutaneously and
intramuscularly at several sites twice at three week intervals with
150 .mu.g of one of the outer membrane preparations described
above. QuilA, at a final concentration of 20% (vol./vol.)
(CedarLane Laboratories, Hornby, Ont., Canada), was the adjuvant
used for these immunizations. The development of the specific
humoral response was analyzed by ELISA using outer membrane
preparations extracted from Neisseria meningitidis strain 608B
(B:2a:P1.2) as coating antigen and by Western immunoblotting
following methods already described by the inventors [Brodeur et
al., Infect. Immun. 50, 510 (1985); Martin et al, Eur. J. Immunol.
18, 601 (1988)]. Alkaline phosphatase or peroxydase-labeled Donkey
anti-rabbit immunoglobulins (Jackson ImmunoResearch Laboratories,
West Grove, Pa.) were used for these assays.
The injection of E. coli outer membrane preparation containing the
22 kDa recombinant protein in combination with QuilA adjuvant
induced in the rabbit a strong specific humoral response of
1/32,000 as determined by ELISA (FIG. 13). The antibodies induced
after the injection of the recombinant 22 kDa protein reacted with
the purified recombinant 22 kDa protein, but more importantly they
also recognized the native protein as expressed, folded and
embedded in the outer membrane of Neisseria meningitidis. Western
Immunoblotting experiments clearly indicated that the antibodies
present after the second injection recognized on nitrocellulose
membrane the same protein band as the one revealed by Mab Me-2
(described in Example 2), which is specific for the 22 kDa
protein.
b) Monkeys
Two Macaca fascicularis (cynomolgus) monkeys were respectively
immunized with two injections of 100 .mu.g (K28) and 200 .mu.g
(I276) of affinity purified recombinant 22 kDa protein per
injection. The methods used to produce and purify the protein from
E. coli strain BL2lDe3 were described in Example 3. Alhydrogel, at
a final concentration of 20% (vol./vol.) (CedarLane Laboratories,
Hornby, Ont., Canada), was the adjuvant used for these
immunizations. The monkeys received two intramuscular injections at
three weeks interval. A control monkey (K65) was immunized with an
unrelated recombinant protein preparation following the same
procedures. The sera were analyzed as described above. Alkaline
phosphatase or Peroxydase-labeled Goat anti-human immunoglobulins
(Jackson ImmunoResearch Laboratories, West Grove, Pa.) were used
for these assays.
The specific antibody response of monkey K28 which was immunized
with 100 .mu.g of purified protein per injection appeared faster
and was stronger than the one observed for monkey I276 which was
injected with 200 .mu.g of protein (FIG. 14). Antibodies specific
for the native 22 kDa protein as detected by Western immunoblotting
were already present in the sera of the immunized monkeys twenty
one days after the first injection, but were absent in the sera of
the control monkey after two injections of the control antigen.
Conclusion
The data presented in Examples 2 and 5 clearly showed that the
injection of the recombinant 22 kDa protein can induce a protective
humoral response in mice which is directed against Neisseria
meningitidis strains. More importantly, the results presented in
this example demonstrate that this immunological response is not
restricted to only one species, but this recombinant surface
protein can also stimulate the immune system of other species such
as rabbit or monkey.
EXAMPLE 9 Epitope mapping of the 22 kDa Neisseria meningitidis
protein
Neisseria meningitidis 22 kDa surface protein was epitope mapped
using a method described by one of the inventors [Martin et al.
Infect. Immun (1991): 59:1457-1464]. Identification of the linear
epitopes was accomplished using 18 overlapping synthetic peptides
covering the entire Neisseria meningitidis 22 kDa protein sequence
derived from strain 608B (FIG. 15) and hyperimmune sera obtained
after immunization with this protein. The identification of
immunodominant portions on the 22 kDa protein may be helpful in the
design of new efficient vaccines. Furthermore, the localization of
these B-cell epitopes also provides valuable information about the
structural configuration of the protein in the outer membrane of
Neisseria meningitidis.
All peptides were synthesized by BioChem Immunosystems Inc.
(Montreal, Canada) with the Applied Biosystems (Foster City,
Calif.) automated peptide synthesizer. Synthetic peptides were
purified by reverse-phase high-pressure liquid chromatography.
Peptides CS-845, CS-847, CS-848, CS-851, CS-852 and CS-856 (FIG.
15) were solubilized in a small volume of 6M guanidine-HCl (J. T.
15 Baker, Ontario, Canada) or dimethyl sulfoxide (J. T. Baker).
These peptides were then adjusted to 1 mg/ml with distilled water.
All the other peptides were freely soluble in distilled water and
were also adjusted to 1 mg/ml.
Peptide enzyme-linked immunosorbent assays (ELISA) were performed
by coating synthetic peptides onto microtitration plates (Immulon
4, Dynatech Laboratories Inc., Chantilly, Va.) at a concentration
of 50 .mu.g/ml in 50 mM carbonate buffer, pH 9.6. After overnight
incubation at room temperature, the plates were washed with
phosphate-buffered saline (PBS) containing 0.05% (wt/vol) Tween 20
(Sigma Chemical Co., St.-Louis, Mo.) and blocked with PBS
containing 0.5% (wt/vol) bovine serum albumin (Sigma). Sera
obtained from mice and monkeys immunized with affinity purified
recombinant 22 kDa surface protein were diluted and 100 .mu.l per
well of each dilution were added to the ELISA plates and incubated
for 1 h at 37.degree. C. The plates were washed three times, and
100 .mu.l of alkaline phosphatase-conjugated goat anti-mouse or
anti-human immunoglobulins (Jackson ImmunoResearch Laboratories,
West Grove, Pa.) diluted according to the manufacturer's
recommendations was added. After incubation for 1 h at 37.degree.
C., the plates were washed and 100 .mu.l of diethanolamine (10%
(vol/vol), pH 9.8) containing p-nitro-phenylphosphate (Sigma) at 1
mg/ml was added. After 60 min., the reaction (.lambda.=k=410 nm)
was read spectrophotometrically with a microplate reader.
Mouse and monkey antisera obtained after immunization with affinity
purified recombinant 22 kDa protein (Example 8) were successfully
used in combination with eighteen overlapping synthetic peptides to
localize B-cell epitopes on the protein. These epitopes are
clustered within three antigenic domains on the protein.
The first region is located between amino acid residues 51 and 86.
Computer analysis using different algorithms suggested that this
region has the highest probability of being immunologically
important since it is hydrophilic and surface exposed. Furthermore,
comparison of the four protein sequences which is presented in FIG.
12 indicates that one of the major variation, which is the
insertion of one amino acid residue at position 73, is also located
in this region.
The antisera identified a second antigenic domain located between
amino acid residues 110 and 140. Interestingly, the sequence
analysis revealed that seven out of the fourteen amino acid
residues that are not conserved among the four protein sequences
are clustered within this region of the protein.
A third antigenic domain located in a highly conserved portion of
the protein, between amino acid residues 31 and 55, was recognized
only by the monkeys' sera.
EXAMPLE 10 Beat-inducible expression vector for the large scale
production of the 22 kDa surface protein
The gene coding for the Neisseria meningitidis 22 kDa surface
protein was inserted into the plasmid p629 [George et al.
Bio/technology 5: 600-603 (1987)]. A cassette of the bacteriophage
.lambda. cI857 temperature sensitive repressor gene, from which the
functional Pr promoter has been deleted, is carried by the plasmid
p629 that uses the PL promoter to control the synthesis of the 22
kDa surface protein. The inactivation of the cI857 repressor by a
temperature shift from 30.degree. C. to temperatures above
38.degree. C. results in the production of the protein encoded by
the plasmid. The induction of gene expression in E. coli cells by a
temperature shift is advantageous for large scale fermentation
since it can easily be achieved with modern fermentors. Other
inducible expression vectors usually require the addition of
specific molecules like lactose or
isopropylthio-.beta.-D-galactoside (IPTG) in the culture media in
order to induce the expression of the desired gene.
A 540 nucleotide fragment was amplified by PCR from the Neisseria
meningitidis strain 608B genomic DNA using the following two
oligonucleotide primers (SEQ ID NOS 27 & 28, respectively)
(OCRR8: 5'-TAATAGATCTATGAAAAAAGCACTTGCCAC-3' and OCRR9:
3'-CACGCGCAGTTTAAGACTTCTAGATTA-5'). These primers correspond to the
nucleotide sequences found at both ends of the 22 kDa gene. To
simplify the cloning of the PCR product, a Bgl II (AGATCT)
restriction site was incorporated into the nucleotide sequence of
these primers. The PCR product was purified on agarose gel before
being digested with Bgl II. This Bgl II fragment of approximately
525 base pairs was then inserted into the Bgl II and Bam HI sites
of the plasmid p629. The plasmid containing the PCR product insert
named pNP2204 was used to transform E. coli strain DH5.alpha.F'IQ.
A partial map of the plasmid pNP2204 is presented in FIG. 16. The
resulting colonies were screened with Neisseria meningitidis 22 kDa
surface-protein specific monoclonal antibodies described in Example
2. Western blot analysis of the resulting clones clearly indicated
that the protein synthesized by E. coli was complete and migrated
on SDS-PAGE gel like the native Neisseria meningitidis 22 kDa
surface protein. Plasmid DNA was purified from the selected clone
and then sequenced. The nucleotide sequence of the insert present
in the plasmid perfectly matched the nucleotide sequence of the
gene coding for the Neisseria meningitidis 22 kDa protein presented
in FIG. 1.
To study the level of synthesis of the 22 kDa surface protein, the
temperature-inducible plasmid pNP2204 was used to transform the
following E. coli strains: W3110, JM105, BL21, TOPP1, TOPP2 and
TOPP3. The level of synthesis of the 22 kDa surface protein and the
localization of the protein in the different cellular fractions
were determined for each strain. Shake flask cultures in LB broth
(Gibco BRL, Life Technologies, Grand Island, N.Y.) indicated that a
temperature shift from 30.degree. C. to 39.degree. C. efficiently
induced the expression of the gene. Time course evaluation of the
level of synthesis indicated that the protein appeared, as
determined on SDS-PAGE gel, as soon as 30 min after induction and
that the amount of protein increased constantly during the
induction period. Expression levels between 8 to 10 mg of 22 kDa
protein per liter were determined for E. coli strains W3110 and
TOPP1. For both strains, the majority of the 22 kDa protein is
incorporated in the bacterial outer membrane.
EXAMPLE 11 Purification of the Neisseria meningitidis 22 kDa
protein
Since the vast majority of the 22 kDa protein is found embedded in
the outer membrane of E. coli strains, the purification protocol
presented in this Example is different from the one already
described in Example 3 where a large amount of protein was released
in the culture supernatant. An overnight culture incubated at
30.degree. C. of either E. coli strain W3110 or TOPP1 harboring the
plasmid pNP2204 was inoculated in LB broth containing 50 .mu.g/ml
of Ampicillin (Sigma) and was grown at 30.degree. C. with agitation
(250 rpm) until it reached a cell density of 0.6 (.lambda.=600 nm),
at which point the incubation temperature was shifted to 39.degree.
C. for three to five hours to induce the production of the protein.
The bacterial cells were harvested by centrifugation at 8,000 xg
for 15 minutes at 4.degree. C. and washed twice in phosphate
buffered saline (PBS), pH 7.3. The bacterial cells were
ultrasonically broken (ballistic disintegration or mechanical
disintegration with a French press may also be used). Unbroken
cells were removed by centrifugation at 5,000 xg for 5 minutes and
discarded. The outer membranes were separated from cytoplasmic
components by centrifugation at 100,000 xg for 1 h at 10.degree. C.
The membrane-containing pellets were resuspended in a small volume
of PBS, pH 7.3. To solubilize the 22 kDa surface protein from the
membranes, detergents such as Empigen BB (Calbiochem Co., LaJolla,
Calif.), Zwittergent-3,14 (Calbiochem Co.), or .beta.-octyglucoside
(Sigma) were used. The detergent was added to the membrane fraction
at final concentration of 3% and the mixture was incubated for 1 h
at 20.degree. C. The non soluble material was removed by
centrifugation at 100,000 xg for 1 h at 10.degree. C.
The 22 kDa protein was efficiently solubilized by either three of
the detergents, however .beta.-octylglucoside had the advantage of
easily removing several unwanted membrane proteins since they were
not solubilized and could be separated from the supernatant by
centrifugation. To remove the detergent, the 22 kDa containing
supernatant was dialyzed extensively against several changes of PBS
buffer. Proteinase K treatment (as in Example 1) can be used to
further remove unwanted proteins from the 22 kDa surface protein
preparation. Differential precipitation using ammonium sulfate or
organic solvents, and ultrafiltration are two additional steps that
can be used to remove unwanted nucleic acid and lipopolysaccharide
contaminants from the proteins before gel permeation and
ion-exchange chromatography can be efficiently used to obtain the
purified 22 kDa protein. Affinity chromatography, as described in
Example 3, can also be used to purify the 22 kDa protein.
EXAMPLE 12 Use of 22 kDa surface protein As a Human Vaccine
To formulate a vaccine for human use, appropriate 22 kDa surface
protein antigens may be selected from the polypeptides described
herein. For example, one of skill in the art could design a vaccine
around the 22 kDa polypeptide or fragments thereof containing an
immunogenic epitope. The use of molecular biology techniques is
particularly well-suited for the Preparation of substantially pure
recombinant antigens.
The vaccine composition may take a variety of forms. These include,
for example, solid, semi-solid, and liquid dosage forms, such as
powders, liquid solutions or suspensions, and liposomes. Based on
our belief that the 22 kDa surface protein antigens of this
invention may elicit a protective immune response when administered
to a human, the compositions of this invention will be similar to
those used for immunizing humans with other proteins and
polypeptides, e.g. tetanus and diphteria. Therefore, the
compositions of this invention will preferably comprise a
pharmaceutically acceptable adjuvant such as incomplete Freund's
adjuvant, aluminum hydroxide, a muramyl peptide, a water-in-oil
emulsion, a liposome, an ISCOM or CTB, or a non-toxic B subunit
form cholera toxin. Most preferably, the compositions will include
a water-in-oil emulsion or aluminum hydroxide as adjuvant.
The composition would be administered to the patient in any of a
number of pharmaceutically acceptable forms including
intramuscular, intradermal, subcutaneous or topic. Preferrably, the
vaccine will be administered intramuscularly.
Generally, the dosage will consist of an initial injection, most
probably with adjuvant, of about 0.01 to 10 mg, and preferably 0.1
to 1.0 mg of 22 kDa surface protein antigen per patient, followed
most probably by one or more booster injections. Preferably,
boosters will be administered at about 1 and 6 months after the
initial injection.
A consideration relating to vaccine development is the question of
mucosal immunity. The ideal mucosal vaccine will be safely taken
orally or intranasally as one or a few doses and would elicit
protective antibodies on the appropriate surfaces along with
systemic immunity. The mucosal vaccine composition may include
adjuvants, inert particulate carriers or recombinant live
vectors.
The anti-22 kDa surface protein antibodies of this invention are
useful for passive immunotherapy and immunoprophylaxis of humans
infected with Neisseria meninigitidis or related bacteria such as
Neisseria gonorrhoeae or Neisseria lactamica. The dosage forms and
regimens for such passive immunization would be similar to those of
other passive immunotherapies.
An antibody according to this invention is exemplified by a
hybridoma producing MAbs Me-1 or Me-7 deposited in the American
Type Culture Collection in Rockville, Md., USA on Jul. 21, 1995,
and identified as Murine Hybridoma Cell Lines, Me-1 and Me-7
respectively. These deposits were assigned accession numbers HB
11959 (Me-1) and HB 11958 (Me-7).
While we have described herein a number of embodiments of this
invention, it is apparent that our basic embodiments may be altered
to provide other embodiments that utilize the compositions and
processes of this invention. Therefore, it will be appreciated that
the scope of this invention includes all alternative embodiments
and variations that are defined in the foregoing specification and
by the claims appended thereto; and the invention is not to be
limited by the specific embodiments which have been presented
herein by way of example.
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 30 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 830 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)
ORIGINAL SOURCE: (A) ORGANISM: Neisseria meningitidis (B) STRAIN:
608B (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 143..667 (ix)
FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATION: 143..199 (ix)
FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION: 200..667 (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:1 TCGGCAAAGC AGCCGGATAC CGCTACGTAT
CTTGAAGTAT TGAAAATATT ACGATGCAAA 60 AAAGAAAATT TAAGTATAAT
ACAGCAGGAT TCTTTAACGG ATTCTTAACA ATTTTTCTAA 120 CTGACCATAA
AGGAACCAAA AT ATG AAA AAA GCA CTT GCC ACA CTG ATT GCC 172 Met Lys
Lys Ala Leu Ala Thr Leu Ile Ala -19 -15 -10 CTC GCT CTC CCG GCC GCC
GCA CTG GCG GAA GGC GCA TCC GGC TTT TAC 220 Leu Ala Leu Pro Ala Ala
Ala Leu Ala Glu Gly Ala Ser Gly Phe Tyr -5 1 5 GTC CAA GCC GAT GCC
GCA CAC GCA AAA GCC TCA AGC TCT TTA GGT TCT 268 Val Gln Ala Asp Ala
Ala His Ala Lys Ala Ser Ser Ser Leu Gly Ser 10 15 20 GCC AAA GGC
TTC AGC CCG CGC ATC TCC GCA GGC TAC CGC ATC AAC GAC 316 Ala Lys Gly
Phe Ser Pro Arg Ile Ser Ala Gly Tyr Arg Ile Asn Asp 25 30 35 CTC
CGC TTC GCC GTC GAT TAC ACG CGC TAC AAA AAC TAT AAA GCC CCA 364 Leu
Arg Phe Ala Val Asp Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala Pro 40 45
50 55 TCC ACC GAT TTC AAA CTT TAC AGC ATC GGC GCG TCC GCC ATT TAC
GAC 412 Ser Thr Asp Phe Lys Leu Tyr Ser Ile Gly Ala Ser Ala Ile Tyr
Asp 60 65 70 TTC GAC ACC CAA TCG CCC GTC AAA CCG TAT CTC GGC GCG
CGC TTG AGC 460 Phe Asp Thr Gln Ser Pro Val Lys Pro Tyr Leu Gly Ala
Arg Leu Ser 75 80 85 CTC AAC CGC GCC TCC GTC GAC TTG GGC GGC AGC
GAC AGC TTC AGC CAA 508 Leu Asn Arg Ala Ser Val Asp Leu Gly Gly Ser
Asp Ser Phe Ser Gln 90 95 100 ACC TCC ATC GGC CTC GGC GTA TTG ACG
GGC GTA AGC TAT GCC GTT ACC 556 Thr Ser Ile Gly Leu Gly Val Leu Thr
Gly Val Ser Tyr Ala Val Thr 105 110 115 CCG AAT GTC GAT TTG GAT GCC
GGC TAC CGC TAC AAC TAC ATC GGC AAA 604 Pro Asn Val Asp Leu Asp Ala
Gly Tyr Arg Tyr Asn Tyr Ile Gly Lys 120 125 130 135 GTC AAC ACT GTC
AAA AAC GTC CGT TCC GGC GAA CTG TCC GTC GGC GTG 652 Val Asn Thr Val
Lys Asn Val Arg Ser Gly Glu Leu Ser Val Gly Val 140 145 150 CGC GTC
AAA TTC TGATATGCGC CTTATTCTGC AAACCGCCGA GCCTTCGGCG 704 Arg Val Lys
Phe 155 GTTTTGTTTT CTGCCACCGC AACTACACAA GCCGGCGGTT TTGTACGATA
ATCCCGAATG 764 CTGCGGCTTC TGCCGCCCTA TTTTTTGAGG AATCCGAAAT
GTCCAAAACC ATCATCCACA 824 CCGACA 830 (2) INFORMATION FOR SEQ ID NO:
2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 174 amino acids (B)
TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE: (A) ORGANISM: not provided (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:2 Met Lys Lys Ala Leu Ala Thr Leu Ile Ala
Leu Ala Leu Pro Ala Ala -19 -15 -10 -5 Ala Leu Ala Glu Gly Ala Ser
Gly Phe Tyr Val Gln Ala Asp Ala Ala 1 5 10 His Ala Lys Ala Ser Ser
Ser Leu Gly Ser Ala Lys Gly Phe Ser Pro 15 20 25 Arg Ile Ser Ala
Gly Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val Asp 30 35 40 45 Tyr Thr
Arg Tyr Lys Asn Tyr Lys Ala Pro Ser Thr Asp Phe Lys Leu 50 55 60
Tyr Ser Ile Gly Ala Ser Ala Ile Tyr Asp Phe Asp Thr Gln Ser Pro 65
70 75 Val Lys Pro Tyr Leu Gly Ala Arg Leu Ser Leu Asn Arg Ala Ser
Val 80 85 90 Asp Leu Gly Gly Ser Asp Ser Phe Ser Gln Thr Ser Ile
Gly Leu Gly 95 100 105 Val Leu Thr Gly Val Ser Tyr Ala Val Thr Pro
Asn Val Asp Leu Asp 110 115 120 125 Ala Gly Tyr Arg Tyr Asn Tyr Ile
Gly Lys Val Asn Thr Val Lys Asn 130 135 140 Val Arg Ser Gly Glu Leu
Ser Val Gly Val Arg Val Lys Phe 145 150 155 (2) INFORMATION FOR SEQ
ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 710 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Neisseria
meningitidis (B) STRAIN: MCH88 (ix) FEATURE: (A) NAME/KEY: CDS (B)
LOCATION: 116..643 (ix) FEATURE: (A) NAME/KEY: sig_peptide (B)
LOCATION: 116..172 (ix) FEATURE: (A) NAME/KEY: mat_peptide (B)
LOCATION: 173..643 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3
GTATCTTGAG GCATTGAAAA TATTACAATG CAAAAAGAAA ATTTCAGTAT AATACGGCAG
60 GATTCTTTAA CGGATTCTTA ACCATTTTTC TCCCTGACCA TAAAGGAATC AAGAT ATG
118 Met -19 AAA AAA GCA CTT GCC GCA CTG ATT GCC CTC GCC CTC CCG GCC
GCC GCA 166 Lys Lys Ala Leu Ala Ala Leu Ile Ala Leu Ala Leu Pro Ala
Ala Ala -15 -10 -5 CTG GCG GAA GGC GCA TCC GGC TTT TAC GTC CAA GCC
GAT GCC GCA CAC 214 Leu Ala Glu Gly Ala Ser Gly Phe Tyr Val Gln Ala
Asp Ala Ala His 1 5 10 GCC AAA GCC TCA AGC TCT TTA GGT TCT GCC AAA
GGC TTC AGC CCG CGC 262 Ala Lys Ala Ser Ser Ser Leu Gly Ser Ala Lys
Gly Phe Ser Pro Arg 15 20 25 30 ATC TCC GCA GGC TAC CGC ATC AAC GAC
CTC CGC TTC GCC GTC GAT TAC 310 Ile Ser Ala Gly Tyr Arg Ile Asn Asp
Leu Arg Phe Ala Val Asp Tyr 35 40 45 ACG CGC TAC AAA AAC TAT AAA
CAA GTC CCA TCC ACC GAT TTC AAA CTT 358 Thr Arg Tyr Lys Asn Tyr Lys
Gln Val Pro Ser Thr Asp Phe Lys Leu 50 55 60 TAC AGC ATC GGC GCG
TCC GCC ATT TAC GAC TTC GAC ACC CAA TCC CCC 406 Tyr Ser Ile Gly Ala
Ser Ala Ile Tyr Asp Phe Asp Thr Gln Ser Pro 65 70 75 GTC AAA CCG
TAT CTC GGC GCG CGC TTG AGC CTC AAC CGC GCC TCC GTC 454 Val Lys Pro
Tyr Leu Gly Ala Arg Leu Ser Leu Asn Arg Ala Ser Val 80 85 90 GAC
TTT AAC GGC AGC GAC AGC TTC AGC CAA ACC TCC ACC GGC CTC GGC 502 Asp
Phe Asn Gly Ser Asp Ser Phe Ser Gln Thr Ser Thr Gly Leu Gly 95 100
105 110 GTA TTG GCG GGC GTA AGC TAT GCC GTT ACC CCG AAT GTC GAT TTG
GAT 550 Val Leu Ala Gly Val Ser Tyr Ala Val Thr Pro Asn Val Asp Leu
Asp 115 120 125 GCC GGC TAC CGC TAC AAC TAC ATC GGC AAA GTC AAC ACT
GTC AAA AAT 598 Ala Gly Tyr Arg Tyr Asn Tyr Ile Gly Lys Val Asn Thr
Val Lys Asn 130 135 140 GTC CGT TCC GGC GAA CTG TCC GCC GGC GTA CGC
GTC AAA TTC TGATATACGC 650 Val Arg Ser Gly Glu Leu Ser Ala Gly Val
Arg Val Lys Phe 145 150 155 GTTATTCCGC AAACCGCCGA GCCTTTCGGC
GGTTTTGTTT TCCGCCGCCG CAACTACACA 710 (2) INFORMATION FOR SEQ ID NO:
4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 175 amino acids (B)
TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE: (A) ORGANISM: not provided (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:4 Met Lys Lys Ala Leu Ala Ala Leu Ile Ala
Leu Ala Leu Pro Ala Ala -19 -15 -10 -5 Ala Leu Ala Glu Gly Ala Ser
Gly Phe Tyr Val Gln Ala Asp Ala Ala 1 5 10 His Ala Lys Ala Ser Ser
Ser Leu Gly Ser Ala Lys Gly Phe Ser Pro 15 20 25 Arg Ile Ser Ala
Gly Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val Asp 30 35 40 45 Tyr Thr
Arg Tyr Lys Asn Tyr Lys Gln Val Pro Ser Thr Asp Phe Lys 50 55 60
Leu Tyr Ser Ile Gly Ala Ser Ala Ile Tyr Asp Phe Asp Thr Gln Ser 65
70 75 Pro Val Lys Pro Tyr Leu Gly Ala Arg Leu Ser Leu Asn Arg Ala
Ser 80 85 90 Val Asp Phe Asn Gly Ser Asp Ser Phe Ser Gln Thr Ser
Thr Gly Leu 95 100 105 Gly Val Leu Ala Gly Val Ser Tyr Ala Val Thr
Pro Asn Val Asp Leu 110 115 120 125 Asp Ala Gly Tyr Arg Tyr Asn Tyr
Ile Gly Lys Val Asn Thr Val Lys 130 135 140 Asn Val Arg Ser Gly Glu
Leu Ser Ala Gly Val Arg Val Lys Phe 145 150 155 (2) INFORMATION FOR
SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 850 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Neisseria
meningitidis (B) STRAIN: Z4063 (ix) FEATURE: (A) NAME/KEY: CDS (B)
LOCATION: 208..732 (ix) FEATURE: (A) NAME/KEY: sig_peptide (B)
LOCATION: 208..264 (ix) FEATURE: (A) NAME/KEY: mat_peptide (B)
LOCATION: 265..732 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5
CACCCATCCG CCGCGTGATG CCGCCACCAC CATTTAAAGG CAACGCGCGG GTTAACGGCT
60 TTGCCGTCGG CAAAGCAGCC GGATACCGCT ACGTATCTTG AAGTATTAAA
AATATTACGA 120 TGCAAAAAGA AAATTTAAGT ATAATAAAGC AGAATTCTTT
AACGGATTCT TAACAATTTT 180 TCTAACTGAC CATAAAGGAA CCAAAAT ATG AAA AAA
GCA CTT GCC ACA CTG 231 Met Lys Lys Ala Leu Ala Thr Leu -19 -15 ATT
GCC CTC GCT CTC CCG GCC GCC GCA CTG GCG GAA GGC GCA TCC GGC 279 Ile
Ala Leu Ala Leu Pro Ala Ala Ala Leu Ala Glu Gly Ala Ser Gly -10 -5
1 5 TTT TAC GTC CAA GCC GAT GCC GCA CAC GCA AAA GCC TCA AGC TCT TTA
327 Phe Tyr Val Gln Ala Asp Ala Ala His Ala Lys Ala Ser Ser Ser Leu
10 15 20 GGT TCT GCC AAA GGC TTC AGC CCG CGC ATC TCC GCA GGC TAC
CGC ATC 375 Gly Ser Ala Lys Gly Phe Ser Pro Arg Ile Ser Ala Gly Tyr
Arg Ile 25 30 35 AAC GAC CTC CGC TTC GCC GTC GAT TAC ACG CGC TAC
AAA AAC TAT AAA 423 Asn Asp Leu Arg Phe Ala Val Asp Tyr Thr Arg Tyr
Lys Asn Tyr Lys 40 45 50 GCC CCA TCC ACC GAT TTC AAA CTT TAC AGC
ATC GGC GCG TCC GCC ATT 471 Ala Pro Ser Thr Asp Phe Lys Leu Tyr Ser
Ile Gly Ala Ser Ala Ile 55 60 65 TAC GAC TTC GAC ACC CAA TCG CCC
GTC AAA CCG TAT CTC GGC GCG CGC 519 Tyr Asp Phe Asp Thr Gln Ser Pro
Val Lys Pro Tyr Leu Gly Ala Arg 70 75 80 85 TTG AGC CTC AAC CGC GCC
TCC GTC GAC TTG GGC GGC AGC GAC AGC TTC 567 Leu Ser Leu Asn Arg Ala
Ser Val Asp Leu Gly Gly Ser Asp Ser Phe 90 95 100 AGC CAA ACC TCC
ACC GGC CTC GGC GTA TTG GCG GGC GTA AGC TAT GCC 615 Ser Gln Thr Ser
Thr Gly Leu Gly Val Leu Ala Gly Val Ser Tyr Ala 105 110 115 GTT ACC
CCG AAT GTC GAT TTG GAT GCC GGC TAC CGC TAC AAC TAC ATC 663 Val Thr
Pro Asn Val Asp Leu Asp Ala Gly Tyr Arg Tyr Asn Tyr Ile 120 125 130
GGC AAA GTC AAC ACT GTC AAA AAC GTC CGT TCC GGC GAA CTG TCC GCC 711
Gly Lys Val Asn Thr Val Lys Asn Val Arg Ser Gly Glu Leu Ser Ala 135
140 145 GGT GTG CGC GTC AAA TTC TGATATGCGC CTTATTCTGC AAACCGCCGA
759 Gly Val Arg Val Lys Phe 150 155 GCCTTCGGCG GTTTTGTTTT
CTGCCACCGC AACTACACAA GCCGGCGGTT TTGTACGATA 819 ATCCCGAATG
CTGCGGCTTC TGCCGCCCTA T 850
(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 174 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: not
provided (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 Met Lys Lys Ala Leu
Ala Thr Leu Ile Ala Leu Ala Leu Pro Ala Ala -19 -15 -10 -5 Ala Leu
Ala Glu Gly Ala Ser Gly Phe Tyr Val Gln Ala Asp Ala Ala 1 5 10 His
Ala Lys Ala Ser Ser Ser Leu Gly Ser Ala Lys Gly Phe Ser Pro 15 20
25 Arg Ile Ser Ala Gly Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val Asp
30 35 40 45 Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala Pro Ser Thr Asp Phe
Lys Leu 50 55 60 Tyr Ser Ile Gly Ala Ser Ala Ile Tyr Asp Phe Asp
Thr Gln Ser Pro 65 70 75 Val Lys Pro Tyr Leu Gly Ala Arg Leu Ser
Leu Asn Arg Ala Ser Val 80 85 90 Asp Leu Gly Gly Ser Asp Ser Phe
Ser Gln Thr Ser Thr Gly Leu Gly 95 100 105 Val Leu Ala Gly Val Ser
Tyr Ala Val Thr Pro Asn Val Asp Leu Asp 110 115 120 125 Ala Gly Tyr
Arg Tyr Asn Tyr Ile Gly Lys Val Asn Thr Val Lys Asn 130 135 140 Val
Arg Ser Gly Glu Leu Ser Ala Gly Val Arg Val Lys Phe 145 150 155 (2)
INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 810 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A)
ORGANISM: Neisseria gonorrhoeae (B) STRAIN: b2 (ix) FEATURE: (A)
NAME/KEY: CDS (B) LOCATION: 241..765 (ix) FEATURE: (A) NAME/KEY:
sig_peptide (B) LOCATION: 241..297 (ix) FEATURE: (A) NAME/KEY:
mat_peptide (B) LOCATION: 298..765 (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:7 CCCCGCCTTT GCGGTTTTTT CCAAACCGTT TGCAAGTTTC ACCCATCCGC
CGCGTGATGC 60 CGCCGTTTAA GGGCAACGCG CGGGTTAACG GATTTGCCGT
CGGCAAAGCA GCCGGATGCC 120 GCCGCGTATC TTGAGGCATT GAAAATATTA
CGATGCAAAA AGAAAATTTC AGTATAATAC 180 GGCAGGATTC TTTAACGGAT
TATTAACAAT TTTTCTCCCT GACCATAAAG GAACCAAAAT 240 ATG AAA AAA GCA CTT
GCC GCA CTG ATT GCC CTC GCA CTC CCG GCC GCC 288 Met Lys Lys Ala Leu
Ala Ala Leu Ile Ala Leu Ala Leu Pro Ala Ala -19 -15 -10 -5 GCA CTG
GCG GAA GGC GCA TCC GGC TTT TAC GTC CAA GCC GAT GCC GCA 336 Ala Leu
Ala Glu Gly Ala Ser Gly Phe Tyr Val Gln Ala Asp Ala Ala 1 5 10 CAC
GCC AAA GCC TCA AGC TCT TTA GGT TCT GCC AAA GGC TTC AGC CCG 384 His
Ala Lys Ala Ser Ser Ser Leu Gly Ser Ala Lys Gly Phe Ser Pro 15 20
25 CGC ATC TCC GCA GGC TAC CGC ATC AAC GAC CTC CGC TTC GCC GTC GAT
432 Arg Ile Ser Ala Gly Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val Asp
30 35 40 45 TAC ACG CGC TAC AAA AAC TAT AAA GCC CCA TCC ACC GAT TTC
AAA CTT 480 Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala Pro Ser Thr Asp Phe
Lys Leu 50 55 60 TAC AGC ATC GGC GCG TCC GTC ATT TAC GAC TTC GAC
ACC CAA TCG CCC 528 Tyr Ser Ile Gly Ala Ser Val Ile Tyr Asp Phe Asp
Thr Gln Ser Pro 65 70 75 GTC AAA CCG TAT TTC GGC GCG CGC TTG AGC
CTC AAC CGC GCT TCC GCC 576 Val Lys Pro Tyr Phe Gly Ala Arg Leu Ser
Leu Asn Arg Ala Ser Ala 80 85 90 CAC TTG GGC GGC AGC GAC AGC TTC
AGC AAA ACC TCC GCC GGC CTC GGC 624 His Leu Gly Gly Ser Asp Ser Phe
Ser Lys Thr Ser Ala Gly Leu Gly 95 100 105 GTA TTG GCG GGC GTA AGC
TAT GCC GTT ACC CCG AAT GTC GAT TTG GAT 672 Val Leu Ala Gly Val Ser
Tyr Ala Val Thr Pro Asn Val Asp Leu Asp 110 115 120 125 GCC GGC TAC
CGC TAC AAC TAC GTC GGC AAA GTC AAC ACT GTC AAA AAC 720 Ala Gly Tyr
Arg Tyr Asn Tyr Val Gly Lys Val Asn Thr Val Lys Asn 130 135 140 GTC
CGT TCC GGC GAA CTG TCC GCC GGC GTG CGC GTC AAA TTC TGATATACGC 772
Val Arg Ser Gly Glu Leu Ser Ala Gly Val Arg Val Lys Phe 145 150 155
GTTATTCCGC AAACCGCCGA GCCTTCGGCG GTTTTTTG 810 (2) INFORMATION FOR
SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 174 amino
acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (vi) ORIGINAL SOURCE: (A) ORGANISM: not provided (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:8 Met Lys Lys Ala Leu Ala Ala Leu
Ile Ala Leu Ala Leu Pro Ala Ala -19 -15 -10 -5 Ala Leu Ala Glu Gly
Ala Ser Gly Phe Tyr Val Gln Ala Asp Ala Ala 1 5 10 His Ala Lys Ala
Ser Ser Ser Leu Gly Ser Ala Lys Gly Phe Ser Pro 15 20 25 Arg Ile
Ser Ala Gly Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val Asp 30 35 40 45
Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala Pro Ser Thr Asp Phe Lys Leu 50
55 60 Tyr Ser Ile Gly Ala Ser Val Ile Tyr Asp Phe Asp Thr Gln Ser
Pro 65 70 75 Val Lys Pro Tyr Phe Gly Ala Arg Leu Ser Leu Asn Arg
Ala Ser Ala 80 85 90 His Leu Gly Gly Ser Asp Ser Phe Ser Lys Thr
Ser Ala Gly Leu Gly 95 100 105 Val Leu Ala Gly Val Ser Tyr Ala Val
Thr Pro Asn Val Asp Leu Asp 110 115 120 125 Ala Gly Tyr Arg Tyr Asn
Tyr Val Gly Lys Val Asn Thr Val Lys Asn 130 135 140 Val Arg Ser Gly
Glu Leu Ser Ala Gly Val Arg Val Lys Phe 145 150 155 (2) INFORMATION
FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16
amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Neisseria
meningitidis (B) STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:9 Met Lys Lys Ala Leu Ala Thr Leu Ile Ala Leu Ala Leu Pro Ala
Ala 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL
SOURCE: (A) ORGANISM: Neisseria meningitidis (B) STRAIN: 608B (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:10 Leu Ala Leu Pro Ala Ala Ala Leu
Ala Glu Gly Ala Ser Gly Phe 1 5 10 15 (2) INFORMATION FOR SEQ ID
NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Neisseria meningitidis
(B) STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11 Gly Ala
Ser Gly Phe Tyr Val Gln Ala Asp Ala Ala His Ala Lys 1 5 10 15 (2)
INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM:
Neisseria meningitidis (B) STRAIN: 608B (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:12 Ala Ala His Ala Lys Ala Ser Ser Ser Leu Gly Ser Ala
Lys Gly 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL
SOURCE: (A) ORGANISM: Neisseria meningitidis (B) STRAIN: 608B (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:13 Gly Ser Ala Lys Gly Phe Ser Pro
Arg Ile Ser Ala Gly Tyr Arg 1 5 10 15 (2) INFORMATION FOR SEQ ID
NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Neisseria meningitidis
(B) STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14 Ser Ala
Gly Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val Asp Tyr 1 5 10 15 (2)
INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM:
Neisseria meningitidis (B) STRAIN: 608B (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:15 Phe Ala Val Asp Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala
Pro Ser Thr 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 16: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi)
ORIGINAL SOURCE: (A) ORGANISM: Neisseria meningitidis (B) STRAIN:
608B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16 Tyr Lys Ala Pro Ser
Thr Asp Phe Lys Leu Tyr Ser Ile Gly Ala 1 5 10 15 (2) INFORMATION
FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15
amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Neisseria
meningitidis (B) STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:17 Tyr Ser Ile Gly Ala Ser Ala Ile Tyr Asp Phe Asp Thr Gln Ser 1
5 10 15 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL
SOURCE: (A) ORGANISM: Neisseria meningitidis (B) STRAIN: 608B (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:18 Phe Asp Thr Gln Ser Pro Val Lys
Pro Tyr Leu Gly Ala Arg Leu 1 5 10 15 (2) INFORMATION FOR SEQ ID
NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Neisseria meningitidis
(B) STRAIN: 608B
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19 Leu Gly Ala Arg Leu Ser Leu
Asn Arg Ala Ser Val Asp Leu Gly 1 5 10 15 (2) INFORMATION FOR SEQ
ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Neisseria meningitidis
(B) STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20 Ser Val
Asp Leu Gly Gly Ser Asp Ser Phe Ser Gln Thr Ser Ile 1 5 10 15 (2)
INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM:
Neisseria meningitidis (B) STRAIN: 608B (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:21 Ser Gln Thr Ser Ile Gly Leu Gly Val Leu Thr Gly Val
Ser Tyr 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL
SOURCE: (A) ORGANISM: Neisseria meningitidis (B) STRAIN: 608B (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:22 Thr Gly Val Ser Tyr Ala Val Thr
Pro Asn Val Asp Leu Asp Ala 1 5 10 15 (2) INFORMATION FOR SEQ ID
NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Neisseria meningitidis
(B) STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23 Val Asp
Leu Asp Ala Gly Tyr Arg Tyr Asn Tyr Ile Gly Lys Val 1 5 10 15 (2)
INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM:
Neisseria meningitidis (B) STRAIN: 608B (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:24 Tyr Ile Gly Lys Val Asn Thr Val Lys Asn Val Arg Ser
Gly Glu 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL
SOURCE: (A) ORGANISM: Neisseria meningitidis (B) STRAIN: 608B (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:25 Val Arg Ser Gly Glu Leu Ser Val
Gly Val Arg Val Lys Phe 1 5 10 (2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi)
ORIGINAL SOURCE: (A) ORGANISM: Neisseria meningitidis (B) STRAIN:
608B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26 Phe Ala Val Asp Tyr
Thr Arg Tyr Lys Asn Tyr Lys Ala Pro Ser Thr 1 5 10 15 Asp Phe Lys
Leu Tyr Ser Ile Gly Ala 20 25 (2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(vi) ORIGINAL SOURCE: (A) ORGANISM: not provided (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:27 TAATAGATCT ATGAAAAAAG CACTTGCCAC 30 (2)
INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer" (vi) ORIGINAL SOURCE: (A)
ORGANISM: not provided (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28
ATTAGATCTT CAGAATTTGA CGCGCAC 27 (2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 528 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc =
"consensus" (vi) ORIGINAL SOURCE: (A) ORGANISM: not provided (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:29 ATGAAAAAAG CACTTGCCRC ACTGATTGCC
CTCGCHCTCC CGGCCGCCGC ACTGGCGGAA 60 GGCGCATCCG GCTTTTACGT
CCAAGCCGAT GCCGCACACG CMAAAGCCTC AAGCTCTTTA 120 GGTTCTGCCA
AAGGCTTCAG CCCGCGCATC TCCGCAGGCT ACCGCATCAA CGACCTCCGC 180
TTCGCCGTCG ATTACACGCG CTACAAAAAC TATAAACAAG YCCCATCCAC CGATTTCAAA
240 CTTTACAGCA TCGGCGCGTC CGYCATTTAC GACTTCGACA CCCAATCSCC
CGTCAAACCG 300 TATYTCGGCG CGCGCTTGAG CCTCAACCGC GCYTCCGYCS
ACTTKRRCGG CAGCGACAGC 360 TTCAGCMAAA CCTCCRYCGG CCTCGGCGTA
TTGRCGGGCG TAAGCTATGC CGTTACCCCG 420 AATGTCGATT TGGATGCCGG
CTACCGCTAC AACTACRTCH GCAAAGTCAA CACTGTCAAA 480 AAYGTCCGTT
CCGGCGAACT GTCCGYCGGY GTRCGCGTCA AATTCTGA 528 (2) INFORMATION FOR
SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 175 amino
acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: not
provided (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30 Met Lys Lys Ala
Leu Ala Xaa Leu Ile Ala Leu Ala Leu Pro Ala Ala 1 5 10 15 Ala Leu
Ala Glu Gly Ala Ser Gly Phe Tyr Val Gln Ala Asp Ala Ala 20 25 30
His Ala Lys Ala Ser Ser Ser Leu Gly Ser Ala Lys Gly Phe Ser Pro 35
40 45 Arg Ile Ser Ala Gly Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val
Asp 50 55 60 Tyr Thr Arg Tyr Lys Asn Tyr Lys Xaa Ala Pro Ser Thr
Asp Phe Lys 65 70 75 80 Leu Tyr Ser Ile Gly Ala Ser Ala Ile Tyr Asp
Phe Asp Thr Gln Ser 85 90 95 Pro Val Lys Pro Tyr Leu Gly Ala Arg
Leu Ser Leu Asn Arg Ala Ser 100 105 110 Val Asp Leu Gly Gly Ser Asp
Ser Phe Ser Gln Thr Ser Xaa Gly Leu 115 120 125 Gly Val Leu Ala Gly
Val Ser Tyr Ala Val Thr Pro Asn Val Asp Leu 130 135 140 Asp Ala Gly
Tyr Arg Tyr Asn Tyr Ile Gly Lys Val Asn Thr Val Lys 145 150 155 160
Asn Val Arg Ser Gly Glu Leu Ser Ala Gly Val Arg Val Lys Phe 165 170
175
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