U.S. patent application number 11/929892 was filed with the patent office on 2008-05-29 for proteinase k resistant surface protein of neisseria meningitidis.
This patent application is currently assigned to ID BIOMEDICAL CORPORATION. Invention is credited to Bernard R. Brodeur, Josee Hamel, Denis Martin, Clement Rioux.
Application Number | 20080124353 11/929892 |
Document ID | / |
Family ID | 56289803 |
Filed Date | 2008-05-29 |
United States Patent
Application |
20080124353 |
Kind Code |
A1 |
Brodeur; Bernard R. ; et
al. |
May 29, 2008 |
PROTEINASE K RESISTANT SURFACE PROTEIN OF NEISSERIA
MENINGITIDIS
Abstract
The identification of a highly conserved, immunologically
accessible antigen at the surface of Neisseria facilitates
treatment, prophylaxis, and diagnosis of Neisseria diseases. This
antigen is highly resistant to Proteinase K and has an apparent
molecular weight of 22 kDa on SDS-PAGE. Specific polynucleotides
encoding proteins of this class have been isolated from three
Neisseria meningitidis strains and from one Neisseria gonorrhoeae
strain. These polynucleotides have been sequenced, and the
corresponding full-length amino acid sequences of the encoded
polypeptides have been deduced. Recombinant DNA methods for the
production of the Neisseria surface protein, and antibodies that
bind to this protein are also disclosed.
Inventors: |
Brodeur; Bernard R.;
(Sillery, CA) ; Martin; Denis; (St.
Augustin-de-Des Maures, CA) ; Hamel; Josee; (Sillery,
CA) ; Rioux; Clement; (Ile Bizard, CA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE, SUITE 5400
SEATTLE
WA
98104
US
|
Assignee: |
ID BIOMEDICAL CORPORATION
Laval
CA
|
Family ID: |
56289803 |
Appl. No.: |
11/929892 |
Filed: |
October 30, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11582527 |
Oct 16, 2006 |
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11929892 |
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09684883 |
Oct 6, 2000 |
7273611 |
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11582527 |
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08913362 |
Nov 13, 1997 |
6287574 |
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PCT/CA96/00157 |
Mar 15, 1996 |
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09684883 |
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08406362 |
Mar 17, 1995 |
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08913362 |
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60001983 |
Aug 4, 1995 |
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Current U.S.
Class: |
424/190.1 ;
435/68.1; 435/69.1; 435/71.2 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 39/095 20130101; A61K 39/00 20130101; A61P 31/04 20180101;
C07K 14/22 20130101; A61K 2039/522 20130101; A61K 38/00
20130101 |
Class at
Publication: |
424/190.1 ;
435/71.2; 435/68.1; 435/69.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C12P 21/04 20060101 C12P021/04; A61P 43/00 20060101
A61P043/00 |
Claims
1. A method of isolating a polypeptide comprising: a) isolating a
culture of Neisseria meningitidis bacteria; and b) isolating an
outer membrane portion from the culture of the bacteria, wherein
the polypeptide comprises (i) an amino acid sequence at least 90%
identical to the amino acid sequence set forth in SEQ ID NO:2, SEQ
ID NO:4, or SEQ ID NO:6, wherein the polypeptide is capable of
eliciting an antibody that specifically binds to a protein
consisting of the amino acid sequence set forth in SEQ ID NO:2, SEQ
ID NO:4, or SEQ ID NO:6; or (ii) the amino acid sequence set forth
in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
2. The method according to claim 1 further comprising isolating the
polypeptide from the outer membrane portion.
3. The method of claim 1, further comprising treating the outer
membrane portion with proteinase K.
4. The method of claim 1 wherein the polypeptide is substantially
purified from other N. meningitidis proteins.
5. The method of claim 1 wherein the polypeptide is a recombinant
polypeptide.
6. The method of claim 1 wherein the polypeptide is capable of
inducing an immunological response to N. meningitidis.
7. The method of claim 1 wherein the polypeptide comprises a
polypeptide fragment of the amino acid sequence set forth in any
one of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, wherein the
polypeptide fragment has at least one immunogenic epitope, and
wherein the polypeptide fragment is capable of eliciting an
antibody that specifically binds to a protein consisting of the
sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6, and SEQ ID NO:8.
8. The method according to claim 7 wherein the polypeptide fragment
comprises the amino acid sequence set forth at (a) residue 31 to
residue 55 of SEQ ID NO:2; (b) residue 51 to residue 86 of SEQ ID
NO:2; or (c) residue 110 to residue 140 of SEQ ID NO:2.
9. The method according to claim 7 wherein the polypeptide fragment
comprises the amino acid sequence set forth in any one 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.
10. A method of manufacturing a vaccine comprising (a) isolating
the polypeptide according to the method of claim 1 or claim 7; and
(b) formulating the polypeptide with a pharmaceutically acceptable
excipient.
11. The method according to claim 10 further formulating the
polypeptide with a pharmaceutically acceptable adjuvant.
12. The method according to claim 10 wherein the vaccine is
formulated in a suitable vehicle.
13. A method for producing a recombinant polypeptide wherein the
recombinant polypeptide comprises (a) the amino acid sequence set
forth from amino acid residue 31 to amino acid residue 55 of SEQ ID
NO:2; (b) the amino acid sequence set forth from amino acid residue
51 to amino acid residue 86 of SEQ ID NO:2; or (c) the amino acid
sequence set forth from amino acid residue 110 to amino acid
residue 140 of SEQ ID NO:2, said method comprising culturing a host
cell that comprises an expression vector, wherein the expression
vector comprises a polynucleotide that encodes the recombinant
polypeptide, and wherein the polynucleotide is operatively linked
to one or more expression control sequences.
14. The method according to claim 13 wherein the recombinant
polypeptide at least 90% identical to the amino acid sequence set
forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8.
15. The method according to claim 13 wherein the recombinant
polypeptide is capable of inducing an immunological response
against Neisseria.
16. The method according to claim 13 wherein the recombinant
polypeptide is capable of eliciting an antibody that specifically
binds to a protein consisting of the amino acid sequence set forth
in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8.
17. The method according to claim 13 wherein the host cell is a
eukaryotic cell or a bacteria cell.
18. The method according to claim 13 wherein the host cell is a
bacterial cell.
19. The method according to claim 13 wherein the one or more
expression control sequences is heterologous.
20. The method according to claim 18 further comprising (a)
isolating a culture of the bacterial cell; and (b) isolating an
outer membrane portion from said bacterial cell culture.
21. The method according to claim 20 further comprising isolating
the recombinant polypeptide from the outer membrane portion.
22. A method of manufacturing a vaccine comprising (a) producing a
recombinant polypeptide according to the method of claim 13; and
(b) formulating the recombinant polypeptide with a pharmaceutically
acceptable excipient.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/582,527 filed Oct. 16, 2006, which is a
continuation of U.S. patent application Ser. No. 09/684,883, filed
Oct. 6, 2000, now issued as U.S. Pat. No. 7,273,611 on Sep. 25,
2007; which is a continuation application of U.S. patent
application Ser. No. 08/913,362, filed Nov. 13, 1997, now issued as
U.S. Pat. No. 6,287,574; which is the National Stage of
International Application No. PCT/CA96/00157, filed Mar. 15, 1996,
which is a continuation of U.S. patent application Ser. No.
08/406,362, filed Mar. 17, 1995, now abandoned, and which claims
the benefit of U.S. Provisional Patent Application No. 60/001,983,
filed Aug. 4, 1995, all of which are incorporated herein by
reference in their entireties.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is
484112.sub.--417C6--SEQUENCE_LISTING.txt. The text file is 25 KB,
was created on Oct. 30, 2007, and is being submitted electronically
via EFS-Web.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] 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.
[0005] 2. Description of the Related Art
[0006] 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)].
[0007] 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)].
[0008] 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.
[0009] 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.
[0010] 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)].
[0011] 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)].
[0012] 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.
[0013] 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.
BRIEF SUMMARY OF THE INVENTION
[0014] 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.
[0015] 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.
[0016] The present invention further provides methods for isolating
novel Neisseria meningitidis surface antigens and antibodies
specific thereto.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1A-1C 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.
[0018] FIG. 2 is a photograph of a Coomassie Blue stained 14%
SDS-PAGE gel displaying a-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.
[0019] 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).
[0020] 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.
[0021] FIGS. 4A & 4B are photographs 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.
[0022] 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.
[0023] FIGS. 6A-6C are photographs of stained 14% SDS-PAGE gels and
their corresponding Western immunoblot 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 immunoblotting of a duplicate gel using
monoclonal antibody Me-5.
[0024] 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).
[0025] FIG. 8A-8B 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.
[0026] FIG. 9A-9B 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.
[0027] FIG. 10A-10B 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.
[0028] FIG. 11A-11C 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.
[0029] 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.
[0030] 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
pN.sub.22O.sub.2 or with control plasmid pWKS30. The development of
the specific humoral response was analyzed by ELISA using outer
membrane preparations obtained from Neisseria meningitidis strain
608B (B:2a:P1.2) as coating antigen.
[0031] FIG. 14 represents the time course of the immune response to
the recombinant 22 kDa 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
(1276) of affinity purified 22 kDa 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 analyzed by ELISA using outer
membrane preparations obtained from Neisseria meningitidis strain
608B (B:2a:P1.2) as coating antigen.
[0032] 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).
[0033] 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; cl857, bacteriophage A cl857 temperature-sensitive
repressor gene; .lamda.PL, bacteriophage .lamda. transcription
promoter; T1 transcription terminator. The direction of
transcription is indicated by the arrows. BglIII and BamH1 are the
restriction sites used to insert the 22 kDa gene in the p629
plasmid.
DETAILED DESCRIPTION OF THE INVENTION
[0034] 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.
[0035] 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.
[0036] 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".
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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).
[0047] 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.
[0048] A Neisseria meningitidis 22 kDa surface protein according to
the invention may be further characterized by one or more of the
following features:
[0049] (1) it has an approximate molecular weight of 22 kDa as
evaluated on SDS-PAGE gel;
[0050] (2) its electrophoretic mobility on SDS-PAGE gel is not
modified by treatment with reducing agents;
[0051] (3) it has an isoelectric point (pl) in a range around pl 8
to pl 10;
[0052] (4) it is highly resistant to degradation by proteolytic
enzymes such as a-chymotrypsin, trypsin and proteinase K;
[0053] (5) periodate oxidation does not abolish the specific
binding of antibody directed against the Neisseria meningitidis 22
kDa surface protein;
[0054] (6) it is a highly conserved antigen;
[0055] (7) it is accessible to antibody at the surface of intact
Neisseria meningitidis organisms;
[0056] (8) it can induce the production of bactericidal
antibodies;
[0057] (9) it can induce the production of antibodies that can
protect against experimental infection;
[0058] (10) it can induce, when injected into an animal host, the
development of an immunological response that can protect against
Neisseria meningitidis infection.
[0059] 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:
[0060] (a) the DNA sequence of FIG. 1 (SEQ ID NO:1);
[0061] (b) the DNA sequence of FIG. 8 (SEQ ID NO:3);
[0062] (c) the DNA sequence of FIG. 9 (SEQ ID NO:5);
[0063] (d) the DNA sequence of FIG. 10 (SEQ ID NO:7);
[0064] (e) analogues or derivatives of the foregoing DNA
sequences;
[0065] (f) DNA sequences degenerate to any of the foregoing DNA
sequences; and
[0066] (g) fragments of any of the foregoing DNA sequences;
[0067] wherein said sequences encode a product that displays the
immunological activity of the Neisseria meningitidis 22 kDa surface
protein.
[0068] Such fragments are preferably peptides as depicted in FIG.
15 (SEQ ID NO:9, through SEQ ID NO:26).
[0069] 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:
[0070] (a) the DNA sequence of FIG. 1 (SEQ ID NO:1);
[0071] (b) analogues or derivatives of the foregoing DNA
sequences;
[0072] (c) DNA sequences degenerate to any of the foregoing DNA
sequences; and
[0073] (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.
[0074] 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.
[0075] 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.
[0076] The Neisseria meningitidis surface proteins of this
invention may be isolated by a method comprising the following
steps:
[0077] a) isolating a culture of Neisseria meningitidis
bacteria,
[0078] b) isolating an outer membrane portion from the culture of
the bacteria; and
[0079] c) isolating said antigen from the outer membrane
portion.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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.
[0084] 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.
[0085] 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).
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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:
[0099] a) isolating the biological sample from a patient;
[0100] b) incubating an anti-Neisseria meningitidis 22 kDa surface
protein antibody or fragment thereof with the biological sample to
form a mixture; and
[0101] c) detecting specifically bound antibody or bound fragment
in the mixture which indicates the presence of Neisseria
meningitidis antigen.
[0102] Preferred antibodies in the foregoing diagnostic method are
Me-i and Me-7.
[0103] 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:
[0104] a) isolating the biological sample from a patient;
[0105] b) incubating a Neisseria meningitidis surface protein of
this invention or fragment thereof with the biological sample to
form a mixture; and
[0106] 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.
[0107] 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:
[0108] a) isolating the biological sample from a patient;
[0109] b) incubating a DNA probe having a DNA sequence of this
invention with the biological sample to form a mixture; and
[0110] c) detecting specifically bound DNA probe in the mixture
which indicates the presence of Neisseria bacteria.
[0111] 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).
[0112] A more preferred DNA probe has the 525 base pair sequence of
FIG. 1 (SEQ ID NO:1).
[0113] 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.
[0114] 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).
[0115] 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).
[0116] Another diagnostic method for the detection of Neisseria
meningitidis in a patient comprises the steps of:
[0117] a) labeling an antibody of this invention or fragment
thereof with a detectable label;
[0118] b) administering the labeled antibody or labeled fragment to
the patient; and
[0119] c) detecting specifically bound labeled antibody or labeled
fragment in the patient which indicates the presence of Neisseria
meningitidis.
[0120] 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.
[0121] 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.
[0122] 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.
[0123] 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
[0124] 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.
[0125] 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.
[0126] Example 2 describes the preparation of monoclonal antibodies
specific for Neisseria meningitidis 22 kDa surface protein.
[0127] Example 3 describes the preparation of Neisseria
meningitidis recombinant 22 kDa surface protein.
[0128] Example 4 describes the use of DNA probes for the
identification of organisms expressing the Neisseria meningitidis
22 kDa surface protein.
[0129] Example 5 describes the use of an anti-Neisseria
meningitidis 22 kDa surface protein monoclonal antibody to protect
mice against Neisseria meningitidis infection.
[0130] Example 6 describes the use of purified recombinant 22 kDa
surface protein to induce a protective response against Neisseria
meningitidis infection.
[0131] 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.
[0132] Example 8 describes the immunological response of rabbits
and monkeys to the 22 kDa Neisseria meningitidis surface
protein.
[0133] Example 9 describes the procedure used to map the different
immunological epitopes of the 22 kDa Neisseria meningitidis surface
protein.
[0134] Example 10 describes the induction by heat of an expression
vector for the large scale production of the 22 kDa surface
protein.
[0135] Example 11 describes a purification process for the 22 kDa
surface protein when produced by recombinant technology.
[0136] 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
[0137] 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.
[0138] 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
.alpha.-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.
[0139] 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.
[0140] 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.
[0141] 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) (SEQ ID NO: 31) 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.
[0142] 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
[0143] 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.
[0144] 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-1, 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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] 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.
[0152] 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.
[0153] 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 BL21 (DE3) with a plasmid vector pNP2202 containing the gene
coding for the Neisseria meningitidis 22 kDa surface protein.
TABLE-US-00001 TABLE 1 Reactivity of the monoclonal antibodies with
Neisseria isolates Number of Neisseria isolates recognized by the
monoclonal antibodies Serogroup of Neisseria meningitidis Moraxella
Neisseria Neisseria Iso- A B C 29% W135 X Y Z NS.sup.1 Total
catarrhalis gonorrhoeae lactamica Name type (19) (23) (13) (1) (5)
(1) (2) (2) (4) (71) (5) (16) (5) Me-1 IgG2a 19 22 13 1 6 1 2 2 3
69 0 0 1 (k) Me-2 IgG2a 19 20 13 1 6 0 2 2 4 67 0 2 0 (k) Me-3 IgG3
19 22 13 1 6 1 2 2 3 69 0 2 4 (k) Me-5 IgG2a 19 22 13 1 6 1 2 2 3
69 0 2 0 (k) Me-6 IgG1 19 23 13 1 6 1 2 2 3 70 0 2 4 (k) Me-7 IgG2a
19 23 13 1 6 1 2 2 4 71 5 2 4 (k)
Example 3
Molecular Cloning, Sequencing of the Gene, High Yield Expression
and Purification of the Neisseria Meningitidis 22 kDa Surface
Protein
A. Molecular Cloning
[0154] 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.
[0155] 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
[0156] 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 pl of 9.93. The nucleotide and
deduced amino acid sequences are presented in FIG. 1 (SEQ ID NO:1;
SEQ ID NO:2).
[0157] 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 (SEQ ID NO: 32) 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).
[0158] 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
[0159] 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).
[0160] 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.
[0161] 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.times.g for
10 minutes at 4.degree. C. The culture supernatant was filtered
onto a 0.22 mm membrane (Millipore, Bedford, 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.times.g 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 1 M 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.
[0162] 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
a-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.
[0163] 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.
[0164] Important data were presented in Example 3 and can be
summarized as follows:
[0165] 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);
[0166] 2) N-terminal sequencing of the native protein confirmed
that the Neisseria meningitidis 22 kDa gene was indeed cloned;
[0167] 3) this protein was not described previously;
[0168] 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;
[0169] 5) it is possible to obtain the recombinant protein free of
other Neisseria meningitidis molecules and almost free of
components produced by Escherichia coli;
[0170] 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
[0171] 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
[0172] 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.
[0173] 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.
[0174] Stringency Washes
[0175] 1. Wash the membranes twice for 5 min in ample 2.times.SSC,
0.1% SDS min at room temperature with gentle agitation.
[0176] 2. Transfer the membranes to 0.5.times.SSC, 0.1% SDS and
wash twice for 15 min at 68.degree. C. with gentle agitation.
[0177] 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).
[0178] The DNA probe also detected the gene coding for the
Neisseria meningitidis 22 kDa surface protein in all Neisseria
gonorrhoeae strains tested.
[0179] 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).
[0180] 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.
[0181] 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.
[0182] 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).
[0183] 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-US-00002 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.1The
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.
[0184] 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
[0185] 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-I 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.
[0186] 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
meningitidis 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-US-00003 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) Monoclonal
Number of living mice after challenge Antibodies 24 hr 72 hrs % of
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
[0187] 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
[0188] 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
BL21(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).
[0189] 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
antigens that might be present in small amounts after purification
do not contribute to the observed protection against Neisseria
meningitidis.
TABLE-US-00004 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 purified 20/20 16/20 80 22 kDa
protein 10 .mu.g of BSA 17/19 8/19 42 2 20 .mu.g of purified 9/10
8/10 8/10 80 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
[0190] The injection of purified recombinant 22 kDa surface protein
greatly protected the immunized mice against the development of a
lethal infection by Neisseria meningitidis.
[0191] 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
[0192] The 2.75 kb c/al 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.
[0193] 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) (SEQ ID NO: 33).
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.
[0194] 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) (SEQ ID NO: 34). 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.
[0195] 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)(SEQ
ID NO: 33). 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.
[0196] 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.
[0197] 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
[0198] Rabbits and monkeys were immunized with the recombinant 22
kDa protein to assess the antibody response in species other than
the mouse.
[0199] a) Rabbits
[0200] Male New Zealand rabbits were immunized with outer membrane
preparations obtained from E. coli strain JM109 with the plasmid
pN.sub.22O.sub.2 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 was 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 peroxidase-labeled Donkey anti-rabbit
immunoglobulins (Jackson ImmunoResearch Laboratories, West Grove,
Pa.) were used for these assays.
[0201] 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.
[0202] b) Monkeys
[0203] Two Macaca fascicularis (cynomolgus) monkeys were
respectively immunized with two injections of 100 .mu.g (K28) and
200 .mu.g (1276) of affinity purified recombinant 22 kDa protein
per injection. The methods used to produce and purify the protein
from E. coli strain BL2IDe3 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 peroxidase-labeled Goat anti-human
immunoglobulins (Jackson ImmunoResearch Laboratories, West Grove,
Pa.) were used for these assays.
[0204] 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 1276 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
[0205] 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
[0206] 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.
[0207] 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.
[0208] 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 (.lamda.=k=410 nm) was read
spectrophotometrically with a microplate reader.
[0209] 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.
[0210] 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.
[0211] 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.
[0212] 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
Heat-Inducible Expression Vector for the Large Scale Production of
the 22 kDa Surface Protein
[0213] 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
.lamda. cl857 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 cl857 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.
[0214] 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.
[0215] 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
[0216] 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
(.lamda.=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.times.g 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.times.g 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.
[0217] 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
[0218] 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.
[0219] 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 diphtheria. 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.
[0220] The composition would be administered to the patient in any
of a number of pharmaceutically acceptable forms including
intramuscular, intradermal, subcutaneous or topic. Preferably, the
vaccine will be administered intramuscularly.
[0221] 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.
[0222] 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.
[0223] The anti-22 kDa surface protein antibodies of this invention
are useful for passive immunotherapy and immunoprophylaxis of
humans infected with Neisseria meningitidis 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.
[0224] 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).
[0225] 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 CWU 1
1
341830DNANeisseria
meningitidisCDS(143)...(667)sig_peptide(143)...(199)mat_peptide(200)...(6-
67) 1tcggcaaagc agccggatac cgctacgtat cttgaagtat tgaaaatatt
acgatgcaaa 60aaagaaaatt taagtataat acagcaggat tctttaacgg attcttaaca
atttttctaa 120ctgaccataa aggaaccaaa at atg aaa aaa gca ctt gcc aca
ctg att gcc 172 Met Lys Lys Ala Leu Ala Thr Leu Ile Ala -15 -10ctc
gct ctc ccg gcc gcc gca ctg gcg gaa ggc gca tcc ggc ttt tac 220Leu
Ala Leu Pro Ala Ala Ala Leu Ala Glu Gly Ala Ser Gly Phe Tyr -5 1
5gtc caa gcc gat gcc gca cac gca aaa gcc tca agc tct tta ggt tct
268Val Gln Ala Asp Ala Ala His Ala Lys Ala Ser Ser Ser Leu Gly Ser
10 15 20gcc aaa ggc ttc agc ccg cgc atc tcc gca ggc tac cgc atc aac
gac 316Ala Lys Gly Phe Ser Pro Arg Ile Ser Ala Gly Tyr Arg Ile Asn
Asp 25 30 35ctc cgc ttc gcc gtc gat tac acg cgc tac aaa aac tat aaa
gcc cca 364Leu Arg Phe Ala Val Asp Tyr Thr Arg Tyr Lys Asn Tyr Lys
Ala Pro40 45 50 55tcc acc gat ttc aaa ctt tac agc atc ggc gcg tcc
gcc att tac gac 412Ser Thr Asp Phe Lys Leu Tyr Ser Ile Gly Ala Ser
Ala Ile Tyr Asp 60 65 70ttc gac acc caa tcg ccc gtc aaa ccg tat ctc
ggc gcg cgc ttg agc 460Phe Asp Thr Gln Ser Pro Val Lys Pro Tyr Leu
Gly Ala Arg Leu Ser 75 80 85ctc aac cgc gcc tcc gtc gac ttg ggc ggc
agc gac agc ttc agc caa 508Leu Asn Arg Ala Ser Val Asp Leu Gly Gly
Ser Asp Ser Phe Ser Gln 90 95 100acc tcc atc ggc ctc ggc gta ttg
acg ggc gta agc tat gcc gtt acc 556Thr Ser Ile Gly Leu Gly Val Leu
Thr Gly Val Ser Tyr Ala Val Thr 105 110 115ccg aat gtc gat ttg gat
gcc ggc tac cgc tac aac tac atc ggc aaa 604Pro Asn Val Asp Leu Asp
Ala Gly Tyr Arg Tyr Asn Tyr Ile Gly Lys120 125 130 135gtc aac act
gtc aaa aac gtc cgt tcc ggc gaa ctg tcc gtc ggc gtg 652Val Asn Thr
Val Lys Asn Val Arg Ser Gly Glu Leu Ser Val Gly Val 140 145 150cgc
gtc aaa ttc tga tatgcgcctt attctgcaaa ccgccgagcc ttcggcggtt 707Arg
Val Lys Phe * 155ttgttttctg ccaccgcaac tacacaagcc ggcggttttg
tacgataatc ccgaatgctg 767cggcttctgc cgccctattt tttgaggaat
ccgaaatgtc caaaaccatc atccacaccg 827aca 8302174PRTNeisseria
meningitidisSIGNAL(1)...(19) 2Met Lys Lys Ala Leu Ala Thr Leu Ile
Ala Leu Ala Leu Pro Ala Ala -15 -10 -5Ala Leu Ala Glu Gly Ala Ser
Gly Phe Tyr Val Gln Ala Asp Ala Ala 1 5 10His Ala Lys Ala Ser Ser
Ser Leu Gly Ser Ala Lys Gly Phe Ser Pro 15 20 25Arg Ile Ser Ala Gly
Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val Asp30 35 40 45Tyr Thr Arg
Tyr Lys Asn Tyr Lys Ala Pro Ser Thr Asp Phe Lys Leu 50 55 60Tyr Ser
Ile Gly Ala Ser Ala Ile Tyr Asp Phe Asp Thr Gln Ser Pro 65 70 75Val
Lys Pro Tyr Leu Gly Ala Arg Leu Ser Leu Asn Arg Ala Ser Val 80 85
90Asp 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 Asp110 115 120 125Ala Gly Tyr Arg Tyr Asn Tyr Ile Gly Lys Val
Asn Thr Val Lys Asn 130 135 140Val Arg Ser Gly Glu Leu Ser Val Gly
Val Arg Val Lys Phe 145 150 1553710DNANeisseria
meningitidisCDS(116)...(643)sig_peptide(116)...(172)mat_peptide(173)...(6-
43) 3gtatcttgag gcattgaaaa tattacaatg caaaaagaaa atttcagtat
aatacggcag 60gattctttaa cggattctta accatttttc tccctgacca taaaggaatc
aagat atg 118 Metaaa aaa gca ctt gcc gca ctg att gcc ctc gcc ctc
ccg gcc gcc gca 166Lys Lys Ala Leu Ala Ala Leu Ile Ala Leu Ala Leu
Pro Ala Ala Ala -15 -10 -5ctg gcg gaa ggc gca tcc ggc ttt tac gtc
caa gcc gat gcc gca cac 214Leu Ala Glu Gly Ala Ser Gly Phe Tyr Val
Gln Ala Asp Ala Ala His 1 5 10gcc aaa gcc tca agc tct tta ggt tct
gcc aaa ggc ttc agc ccg cgc 262Ala Lys Ala Ser Ser Ser Leu Gly Ser
Ala Lys Gly Phe Ser Pro Arg15 20 25 30atc tcc gca ggc tac cgc atc
aac gac ctc cgc ttc gcc gtc gat tac 310Ile Ser Ala Gly Tyr Arg Ile
Asn Asp Leu Arg Phe Ala Val Asp Tyr 35 40 45acg cgc tac aaa aac tat
aaa caa gtc cca tcc acc gat ttc aaa ctt 358Thr Arg Tyr Lys Asn Tyr
Lys Gln Val Pro Ser Thr Asp Phe Lys Leu 50 55 60tac agc atc ggc gcg
tcc gcc att tac gac ttc gac acc caa tcc ccc 406Tyr Ser Ile Gly Ala
Ser Ala Ile Tyr Asp Phe Asp Thr Gln Ser Pro 65 70 75gtc aaa ccg tat
ctc ggc gcg cgc ttg agc ctc aac cgc gcc tcc gtc 454Val Lys Pro Tyr
Leu Gly Ala Arg Leu Ser Leu Asn Arg Ala Ser Val 80 85 90gac ttt aac
ggc agc gac agc ttc agc caa acc tcc acc ggc ctc ggc 502Asp Phe Asn
Gly Ser Asp Ser Phe Ser Gln Thr Ser Thr Gly Leu Gly95 100 105
110gta ttg gcg ggc gta agc tat gcc gtt acc ccg aat gtc gat ttg gat
550Val Leu Ala Gly Val Ser Tyr Ala Val Thr Pro Asn Val Asp Leu Asp
115 120 125gcc ggc tac cgc tac aac tac atc ggc aaa gtc aac act gtc
aaa aat 598Ala Gly Tyr Arg Tyr Asn Tyr Ile Gly Lys Val Asn Thr Val
Lys Asn 130 135 140gtc cgt tcc ggc gaa ctg tcc gcc ggc gta cgc gtc
aaa ttc tga 643Val Arg Ser Gly Glu Leu Ser Ala Gly Val Arg Val Lys
Phe * 145 150 155tatacgcgtt attccgcaaa ccgccgagcc tttcggcggt
tttgttttcc gccgccgcaa 703ctacaca 7104175PRTNeisseria
meningitidisSIGNAL(1)...(19) 4Met Lys Lys Ala Leu Ala Ala Leu Ile
Ala Leu Ala Leu Pro Ala Ala -15 -10 -5Ala Leu Ala Glu Gly Ala Ser
Gly Phe Tyr Val Gln Ala Asp Ala Ala 1 5 10His Ala Lys Ala Ser Ser
Ser Leu Gly Ser Ala Lys Gly Phe Ser Pro 15 20 25Arg Ile Ser Ala Gly
Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val Asp30 35 40 45Tyr Thr Arg
Tyr Lys Asn Tyr Lys Gln Val Pro Ser Thr Asp Phe Lys 50 55 60Leu Tyr
Ser Ile Gly Ala Ser Ala Ile Tyr Asp Phe Asp Thr Gln Ser 65 70 75Pro
Val Lys Pro Tyr Leu Gly Ala Arg Leu Ser Leu Asn Arg Ala Ser 80 85
90Val Asp Phe Asn Gly Ser Asp Ser Phe Ser Gln Thr Ser Thr Gly Leu
95 100 105Gly Val Leu Ala Gly Val Ser Tyr Ala Val Thr Pro Asn Val
Asp Leu110 115 120 125Asp Ala Gly Tyr Arg Tyr Asn Tyr Ile Gly Lys
Val Asn Thr Val Lys 130 135 140Asn Val Arg Ser Gly Glu Leu Ser Ala
Gly Val Arg Val Lys Phe 145 150 1555850DNANeisseria
meningitidisCDS(208)...(732)sig_peptide(208)...(264)mat_peptide(265)...(7-
32) 5cacccatccg ccgcgtgatg ccgccaccac catttaaagg caacgcgcgg
gttaacggct 60ttgccgtcgg caaagcagcc ggataccgct acgtatcttg aagtattaaa
aatattacga 120tgcaaaaaga aaatttaagt ataataaagc agaattcttt
aacggattct taacaatttt 180tctaactgac cataaaggaa ccaaaat atg aaa aaa
gca ctt gcc aca ctg att 234 Met Lys Lys Ala Leu Ala Thr Leu Ile
-15gcc ctc gct ctc ccg gcc gcc gca ctg gcg gaa ggc gca tcc ggc ttt
282Ala Leu Ala Leu Pro Ala Ala Ala Leu Ala Glu Gly Ala Ser Gly
Phe-10 -5 1 5tac gtc caa gcc gat gcc gca cac gca aaa gcc tca agc
tct tta ggt 330Tyr Val Gln Ala Asp Ala Ala His Ala Lys Ala Ser Ser
Ser Leu Gly 10 15 20tct gcc aaa ggc ttc agc ccg cgc atc tcc gca ggc
tac cgc atc aac 378Ser Ala Lys Gly Phe Ser Pro Arg Ile Ser Ala Gly
Tyr Arg Ile Asn 25 30 35gac ctc cgc ttc gcc gtc gat tac acg cgc tac
aaa aac tat aaa gcc 426Asp Leu Arg Phe Ala Val Asp Tyr Thr Arg Tyr
Lys Asn Tyr Lys Ala 40 45 50cca tcc acc gat ttc aaa ctt tac agc atc
ggc gcg tcc gcc att tac 474Pro Ser Thr Asp Phe Lys Leu Tyr Ser Ile
Gly Ala Ser Ala Ile Tyr55 60 65 70gac ttc gac acc caa tcg ccc gtc
aaa ccg tat ctc ggc gcg cgc ttg 522Asp Phe Asp Thr Gln Ser Pro Val
Lys Pro Tyr Leu Gly Ala Arg Leu 75 80 85agc ctc aac cgc gcc tcc gtc
gac ttg ggc ggc agc gac agc ttc agc 570Ser Leu Asn Arg Ala Ser Val
Asp Leu Gly Gly Ser Asp Ser Phe Ser 90 95 100caa acc tcc acc ggc
ctc ggc gta ttg gcg ggc gta agc tat gcc gtt 618Gln Thr Ser Thr Gly
Leu Gly Val Leu Ala Gly Val Ser Tyr Ala Val 105 110 115acc ccg aat
gtc gat ttg gat gcc ggc tac cgc tac aac tac atc ggc 666Thr Pro Asn
Val Asp Leu Asp Ala Gly Tyr Arg Tyr Asn Tyr Ile Gly 120 125 130aaa
gtc aac act gtc aaa aac gtc cgt tcc ggc gaa ctg tcc gcc ggt 714Lys
Val Asn Thr Val Lys Asn Val Arg Ser Gly Glu Leu Ser Ala Gly135 140
145 150gtg cgc gtc aaa ttc tga tatgcgcctt attctgcaaa ccgccgagcc
762Val Arg Val Lys Phe * 155ttcggcggtt ttgttttctg ccaccgcaac
tacacaagcc ggcggttttg tacgataatc 822ccgaatgctg cggcttctgc cgccctat
8506174PRTNeisseria meningitidisSIGNAL(1)...(19) 6Met Lys Lys Ala
Leu Ala Thr Leu Ile Ala Leu Ala Leu Pro Ala Ala -15 -10 -5Ala Leu
Ala Glu Gly Ala Ser Gly Phe Tyr Val Gln Ala Asp Ala Ala 1 5 10His
Ala Lys Ala Ser Ser Ser Leu Gly Ser Ala Lys Gly Phe Ser Pro 15 20
25Arg Ile Ser Ala Gly Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val Asp30
35 40 45Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala Pro Ser Thr Asp Phe Lys
Leu 50 55 60Tyr Ser Ile Gly Ala Ser Ala Ile Tyr Asp Phe Asp Thr Gln
Ser Pro 65 70 75Val Lys Pro Tyr Leu Gly Ala Arg Leu Ser Leu Asn Arg
Ala Ser Val 80 85 90Asp Leu Gly Gly Ser Asp Ser Phe Ser Gln Thr Ser
Thr Gly Leu Gly 95 100 105Val Leu Ala Gly Val Ser Tyr Ala Val Thr
Pro Asn Val Asp Leu Asp110 115 120 125Ala Gly Tyr Arg Tyr Asn Tyr
Ile Gly Lys Val Asn Thr Val Lys Asn 130 135 140Val Arg Ser Gly Glu
Leu Ser Ala Gly Val Arg Val Lys Phe 145 150 1557810DNANeisseria
gonorrhoeaeCDS(241)...(765)sig_peptide(241)...(297)mat_peptide(298)...(76-
5) 7ccccgccttt gcggtttttt ccaaaccgtt tgcaagtttc acccatccgc
cgcgtgatgc 60cgccgtttaa gggcaacgcg cgggttaacg gatttgccgt cggcaaagca
gccggatgcc 120gccgcgtatc ttgaggcatt gaaaatatta cgatgcaaaa
agaaaatttc agtataatac 180ggcaggattc tttaacggat tattaacaat
ttttctccct gaccataaag gaaccaaaat 240atg aaa aaa gca ctt gcc gca ctg
att gcc ctc gca ctc ccg gcc gcc 288Met Lys Lys Ala Leu Ala Ala Leu
Ile Ala Leu Ala Leu Pro Ala Ala -15 -10 -5gca ctg gcg gaa ggc gca
tcc ggc ttt tac gtc caa gcc gat gcc gca 336Ala Leu Ala Glu Gly Ala
Ser Gly Phe Tyr Val Gln Ala Asp Ala Ala 1 5 10cac gcc aaa gcc tca
agc tct tta ggt tct gcc aaa ggc ttc agc ccg 384His Ala Lys Ala Ser
Ser Ser Leu Gly Ser Ala Lys Gly Phe Ser Pro 15 20 25cgc atc tcc gca
ggc tac cgc atc aac gac ctc cgc ttc gcc gtc gat 432Arg Ile Ser Ala
Gly Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val Asp30 35 40 45tac acg
cgc tac aaa aac tat aaa gcc cca tcc acc gat ttc aaa ctt 480Tyr Thr
Arg Tyr Lys Asn Tyr Lys Ala Pro Ser Thr Asp Phe Lys Leu 50 55 60tac
agc atc ggc gcg tcc gtc att tac gac ttc gac acc caa tcg ccc 528Tyr
Ser Ile Gly Ala Ser Val Ile Tyr Asp Phe Asp Thr Gln Ser Pro 65 70
75gtc aaa ccg tat ttc ggc gcg cgc ttg agc ctc aac cgc gct tcc gcc
576Val Lys Pro Tyr Phe Gly Ala Arg Leu Ser Leu Asn Arg Ala Ser Ala
80 85 90cac ttg ggc ggc agc gac agc ttc agc aaa acc tcc gcc ggc ctc
ggc 624His Leu Gly Gly Ser Asp Ser Phe Ser Lys Thr Ser Ala Gly Leu
Gly 95 100 105gta ttg gcg ggc gta agc tat gcc gtt acc ccg aat gtc
gat ttg gat 672Val Leu Ala Gly Val Ser Tyr Ala Val Thr Pro Asn Val
Asp Leu Asp110 115 120 125gcc ggc tac cgc tac aac tac gtc ggc aaa
gtc aac act gtc aaa aac 720Ala Gly Tyr Arg Tyr Asn Tyr Val Gly Lys
Val Asn Thr Val Lys Asn 130 135 140gtc cgt tcc ggc gaa ctg tcc gcc
ggc gtg cgc gtc aaa ttc tga 765Val Arg Ser Gly Glu Leu Ser Ala Gly
Val Arg Val Lys Phe * 145 150 155tatacgcgtt attccgcaaa ccgccgagcc
ttcggcggtt ttttg 810 8174PRTNeisseria gonorrhoeaeSIGNAL(1)...(19)
8Met Lys Lys Ala Leu Ala Ala Leu Ile Ala Leu Ala Leu Pro Ala Ala
-15 -10 -5Ala Leu Ala Glu Gly Ala Ser Gly Phe Tyr Val Gln Ala Asp
Ala Ala 1 5 10His Ala Lys Ala Ser Ser Ser Leu Gly Ser Ala Lys Gly
Phe Ser Pro 15 20 25Arg Ile Ser Ala Gly Tyr Arg Ile Asn Asp Leu Arg
Phe Ala Val Asp30 35 40 45Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala Pro
Ser Thr Asp Phe Lys Leu 50 55 60Tyr Ser Ile Gly Ala Ser Val Ile Tyr
Asp Phe Asp Thr Gln Ser Pro 65 70 75Val Lys Pro Tyr Phe Gly Ala Arg
Leu Ser Leu Asn Arg Ala Ser Ala 80 85 90His Leu Gly Gly Ser Asp Ser
Phe Ser Lys Thr Ser Ala Gly Leu Gly 95 100 105Val Leu Ala Gly Val
Ser Tyr Ala Val Thr Pro Asn Val Asp Leu Asp110 115 120 125Ala Gly
Tyr Arg Tyr Asn Tyr Val Gly Lys Val Asn Thr Val Lys Asn 130 135
140Val Arg Ser Gly Glu Leu Ser Ala Gly Val Arg Val Lys Phe 145 150
155916PRTNeisseria meningitidis 9Met Lys Lys Ala Leu Ala Thr Leu
Ile Ala Leu Ala Leu Pro Ala Ala 1 5 10 151015PRTNeisseria
meningitidis 10Leu Ala Leu Pro Ala Ala Ala Leu Ala Glu Gly Ala Ser
Gly Phe 1 5 10 151115PRTNeisseria meningitidis 11Gly Ala Ser Gly
Phe Tyr Val Gln Ala Asp Ala Ala His Ala Lys 1 5 10
151215PRTNeisseria meningitidis 12Ala Ala His Ala Lys Ala Ser Ser
Ser Leu Gly Ser Ala Lys Gly 1 5 10 151315PRTNeisseria meningitidis
13Gly Ser Ala Lys Gly Phe Ser Pro Arg Ile Ser Ala Gly Tyr Arg 1 5
10 151415PRTNeisseria meningitidis 14Ser Ala Gly Tyr Arg Ile Asn
Asp Leu Arg Phe Ala Val Asp Tyr 1 5 10 151516PRTNeisseria
meningitidis 15Phe Ala Val Asp Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala
Pro Ser Thr 1 5 10 151615PRTNeisseria meningitidis 16Tyr Lys Ala
Pro Ser Thr Asp Phe Lys Leu Tyr Ser Ile Gly Ala 1 5 10
151715PRTNeisseria meningitidis 17Tyr Ser Ile Gly Ala Ser Ala Ile
Tyr Asp Phe Asp Thr Gln Ser 1 5 10 151815PRTNeisseria meningitidis
18Phe Asp Thr Gln Ser Pro Val Lys Pro Tyr Leu Gly Ala Arg Leu 1 5
10 151915PRTNeisseria meningitidis 19Leu Gly Ala Arg Leu Ser Leu
Asn Arg Ala Ser Val Asp Leu Gly 1 5 10 152015PRTNeisseria
meningitidis 20Ser Val Asp Leu Gly Gly Ser Asp Ser Phe Ser Gln Thr
Ser Ile 1 5 10 152115PRTNeisseria meningitidis 21Ser Gln Thr Ser
Ile Gly Leu Gly Val Leu Thr Gly Val Ser Tyr 1 5 10
152215PRTNeisseria meningitidis 22Thr Gly Val Ser Tyr Ala Val Thr
Pro Asn Val Asp Leu Asp Ala 1 5 10 152315PRTNeisseria meningitidis
23Val Asp Leu Asp Ala Gly Tyr Arg Tyr Asn Tyr Ile Gly Lys Val 1 5
10 152415PRTNeisseria meningitidis 24Tyr Ile Gly Lys Val Asn Thr
Val Lys Asn Val Arg Ser Gly Glu 1
5 10 152514PRTNeisseria meningitidis 25Val Arg Ser Gly Glu Leu Ser
Val Gly Val Arg Val Lys Phe 1 5 102625PRTNeisseria meningitidis
26Phe Ala Val Asp Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala Pro Ser Thr 1
5 10 15Asp Phe Lys Leu Tyr Ser Ile Gly Ala 20 252730DNAArtificial
SequenceOligonucleotide primer 27taatagatct atgaaaaaag cacttgccac
302827DNAArtificial SequenceOligonucleotide primer 28attagatctt
cagaatttga cgcgcac 2729528DNAUnknownConsensus sequence 29atgaaaaaag
cacttgccrc actgattgcc ctcgchctcc cggccgccgc actggcggaa 60ggcgcatccg
gcttttacgt ccaagccgat gccgcacacg cmaaagcctc aagctcttta
120ggttctgcca aaggcttcag cccgcgcatc tccgcaggct accgcatcaa
cgacctccgc 180ttcgccgtcg attacacgcg ctacaaaaac tataaacaag
ycccatccac cgatttcaaa 240ctttacagca tcggcgcgtc cgycatttac
gacttcgaca cccaatcscc cgtcaaaccg 300tatytcggcg cgcgcttgag
cctcaaccgc gcytccgycs acttkrrcgg cagcgacagc 360ttcagcmaaa
cctccrycgg cctcggcgta ttgrcgggcg taagctatgc cgttaccccg
420aatgtcgatt tggatgccgg ctaccgctac aactacrtch gcaaagtcaa
cactgtcaaa 480aaygtccgtt ccggcgaact gtccgycggy gtrcgcgtca aattctga
52830175PRTUnknownConsensus sequence 30Met Lys Lys Ala Leu Ala Xaa
Leu Ile Ala Leu Ala Leu Pro Ala Ala 1 5 10 15Ala Leu Ala Glu Gly
Ala Ser Gly Phe Tyr Val Gln Ala Asp Ala Ala 20 25 30His Ala Lys Ala
Ser Ser Ser Leu Gly Ser Ala Lys Gly Phe Ser Pro 35 40 45Arg 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 Lys65 70 75
80Leu Tyr Ser Ile Gly Ala Ser Ala Ile Tyr Asp Phe Asp Thr Gln Ser
85 90 95Pro Val Lys Pro Tyr Leu Gly Ala Arg Leu Ser Leu Asn Arg Ala
Ser 100 105 110Val Asp Leu Gly Gly Ser Asp Ser Phe Ser Gln Thr Ser
Xaa Gly Leu 115 120 125Gly Val Leu Ala Gly Val Ser Tyr Ala Val Thr
Pro Asn Val Asp Leu 130 135 140Asp Ala Gly Tyr Arg Tyr Asn Tyr Ile
Gly Lys Val Asn Thr Val Lys145 150 155 160Asn Val Arg Ser Gly Glu
Leu Ser Ala Gly Val Arg Val Lys Phe 165 170 175319PRTNeisseria
meningitidis 31Glu Gly Ala Ser Gly Phe Tyr Val Gln 1
53210PRTNeisseria meningitidis 32Glu Gly Ala Ser Gly Phe Tyr Val
Gln Ala 1 5 103319PRTNeisseria meningitidis 33Met Lys Lys Ala Leu
Ala Ala Leu Ile Ala Leu Ala Leu Pro Ala Ala 1 5 10 15Ala Leu
Ala3419PRTNeisseria meningitidis 34Met Lys Lys Ala Leu Ala Thr Leu
Ile Ala Leu Ala Leu Pro Ala Ala 1 5 10 15Ala Leu Ala
* * * * *