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Patent 2347849 Summary

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(12) Patent: (11) CA 2347849
(54) English Title: OMP85 PROTEINS OF NEISSERIA GONORRHOEAE AND NEISSERIA MENINGITIDIS, COMPOSITIONS CONTAINING SAME AND METHODS OF USE THEREOF
(54) French Title: PROTEINES OMP85 DE NEISSERIA GONORRHOEAE ET DE NEISSERIA MENINGITIDIS, COMPOSITIONS RENFERMANT LESDITES PROTEINES ET METHODES D'UTILISATION CORRESPONDANTES
Status: Deemed expired
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 15/31 (2006.01)
  • A61K 39/095 (2006.01)
  • C07K 14/22 (2006.01)
  • C07K 16/12 (2006.01)
  • C12N 15/62 (2006.01)
  • G01N 33/53 (2006.01)
  • G01N 33/68 (2006.01)
  • A61K 39/00 (2006.01)
  • C12Q 1/68 (2006.01)
(72) Inventors :
  • JUDD, RALPH C. (United States of America)
  • MANNING, D. SCOTT (United States of America)
(73) Owners :
  • THE UNIVERSITY OF MONTANA (United States of America)
(71) Applicants :
  • THE UNIVERSITY OF MONTANA (United States of America)
(74) Agent: GOWLING WLG (CANADA) LLP
(74) Associate agent:
(45) Issued: 2013-06-25
(86) PCT Filing Date: 1998-10-22
(87) Open to Public Inspection: 2000-04-27
Examination requested: 2003-10-02
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/US1998/022352
(87) International Publication Number: WO2000/023595
(85) National Entry: 2001-04-18

(30) Application Priority Data: None

Abstracts

English Abstract




Nucleic acid and amino acid sequences of the Omp85 proteins of N. gonorrhoeae
and N. meningitidis, and fragments thereof are useful in vaccine compositions,
therapeutic compositions and diagnostic compositions for use in the
prevention, treatment and diagnosis of non-symptomatic gonococcal infection or
symptomatic disease and non-symptomatic meningococcal infection and
symptomatic disease. Antibodies are developed to these proteins and also
useful in the compositions and methods described herein.


French Abstract

L'invention concerne des séquences d'acides nucléiques et d'acides aminés de protéines de surface externe de 85 kDa (Omp85) provenant de N. gonorrhoeae et N. meningitidis, et leurs fragments, lesquelles protéines sont utiles dans des compositions de vaccins, de traitement et de diagnostic destinées à être utilisées pour la prévention, le traitement et le diagnostic des gonococcies non symptomatiques et des maladies symptomatiques. L'invention concerne aussi la mise au point d'anticorps de ces protéines, utiles également dans les compositions et méthodes précitées.

Claims

Note: Claims are shown in the official language in which they were submitted.




CLAIMS

1. An immunogenic composition comprising an isolated polypeptide comprising
an
amino acid sequence having 95% or greater sequence identity with the amino
acid
sequence of SEQ ID NO:4, and a pharmaceutically acceptable carrier, wherein
said
polypeptide in said composition induces antibodies in a mammal that bind to
said amino
acid sequence of SEQ ID NO:4 and that interfere with adherence of a Neisseria
pathogen
as measured by the gonococcal cell adherence assay.
2. The composition according to claim 1, wherein said composition comprises
a
second polypeptide or protein.
3. The composition according to claim 2, wherein said polypeptide is fused
to said
second polypeptide or protein.
4. The composition according to claim 2, wherein said second polypeptide or
protein
is an antigen from a pathogenic bacterial species that is heterologous or
homologous to
Neisseria gonorrhoeae or Neisseria meningitidis.
5. The composition of claim 1, further comprising an adjuvant.
6. The composition according to claim 1, wherein said composition induces
antibodies which recognize a protein in multiple Neisseria gonorrhoeae strains
and
Neisseria meningitidis strains, said protein appearing as a reactive band of
approximately
85 kD on a Western blot.
7. The composition according to claim 6, wherein said Neisseria
meningitidis strains
are selected from the group consisting of N meningitidis HH, N. meningitidis
MP78, N
meningitidis MP3, and N meningitidis MP81.
8. The composition according to claim 1, wherein said polypeptide contains
one to
four conservative amino acid replacements in the amino acid sequence of SEQ ID
NO: 4
54




9. An immunogenic composition comprising an isolated polypeptide and a
pharmaceutically acceptable carrier, said polypeptide comprising amino acids 1
through
178 of SEQ ID NO:4, amino acids 22 through 178 of SEQ ID NO:4, or a sequence
having at least 95% sequence identity thereto, wherein said polypeptide in
said
composition induces antibodies in a mammal that bind to said amino acid
sequence of
SEQ ID NO:4 and that interfere with the adherence of a Neisseria pathogen as
measured
by the gonococcal cell adherence assay.
10. The composition of claim 1 or 9 wherein said isolated polypeptide is in
a purified
form.
11. The composition of claim 1 or 9 wherein said Neisseria pathogen is
Neisseria
gonorrhoeae or Neisseria meningitidis.
12. An immunogenic composition comprising an isolated polypeptide
comprising an
amino acid sequence having 95% or greater sequence identity with the amino
acid
sequence of SEQ ID NO:4, and a pharmaceutically acceptable carrier, wherein
said
polypeptide in said composition induces antibodies in a mammal that bind to
said amino
acid sequence of SEQ ID NO:4 and that interfere with adherence of Neisseria
gonorrhoeae as measured by the gonococcal cell adherence assay, wherein said
isolated
polypeptide is in a purified form.
13. An immunogenic composition comprising the polypeptide of SEQ ID NO:4,
and a
pharmaceutically acceptable carrier, for use in a vaccine.
14. The composition according to claim 13, wherein said polypeptide is
fused to a
second polypeptide or protein.
15. The composition according to claim 14, wherein said second polypeptide
or
protein is an antigen from a pathogenic bacterial species that is heterologous
or
homologous to Neisseria gonorrhoeae or Neisseria meningitidis.
16. A method of producing an immunogenic composition comprising isolating a
55



recombinant polypeptide comprising amino acids 1 through 178 of SEQ ID NO:4,
amino
acids 22 through 178 of SEQ ID NO: 4, or a sequence having at least 95%
sequence
identity thereto, and providing said polypeptide with a pharmaceutically
acceptable
carrier, wherein said composition induces antibodies in a mammal that bind to
said
epitope of SEQ ID NO:4 and that interfere with adherence of a Neisseria
pathogen as
measured by the gonococcal cell adherence assay.
17. The method of claim 16, wherein said composition comprises a second
polypeptide or protein.
18. The method according to claim 17, wherein said second polypeptide or
protein is
an antigen from a pathogenic bacterial species that is heterologous or
homologous to
Neisseria gonorrhaeae or Neisseria meningitidis.
19. The method according to claim 16, wherein said composition further
comprises an
adjuvant.
20. The method according to claim 18, wherein said second polypeptide or
protein is
an antigen from a pathogenic bacterial species that is homologous to Neisseria

meningitidis.
56

Description

Note: Descriptions are shown in the official language in which they were submitted.


CA 02347849 2001-04-18
WO 00/23595
PCT/US98/22352 -
Omp85 proteins of Neisseria gonorrhoeae and Neisseria meningititlis,
Compositions Containing Same and Methods of Use Thereof
This invention has been supported by National Institutes of Health Grant Nos.
M2I235 and AI37777. The United States Government has an interest in this
invention.
Field of the Invention
This invention relates generally to the cloning and identification of novel
outer
membrane proteins of several strains of Neisseria, and more specifically to
proteins
useful in the prevention, therapy and/or diagnosis of infection and diseases
in mammals
caused by these strains.
Background of the Invention
The pathogenic Neisseriae cause several important non-symptomatic infection
and symptomatic disease in humans. Neisseria gottorrhoeae is the agent of non-
symptomatic gonococcal infection or symptomatic disease, i.e., gonorrhea.
Neisseria
meningitidis is the agent of a rapidly progressive spinal meningitis, which
may also
have a non-symptomatic infective stage. The surface of such pathogens provide
crucial interfaces for interactions between the pathogen and the host. Many
bacterial
virulence factors are outer membrane proteins, and surface exposed proteins
are the
primary targets recognized and attacked by the host's immune system. Thus, the
role
of outer membrane proteins is of particular importance in understanding the
pathogenesis of these organisms. The most abundant and immunodominant outer
membrane proteins of the pathogenic Neisseriae have been studied extensively
[Sparling P. IF. eta!, Gun. Invest,, 5.2: 1699-1705 (1992)]. For example, it
is known
that the immunodominant components of the gonococcal surface are antigenically

variant, suggesting that this organism is capable of adapting to varying host
1

CA 02347849 2001-04-19
environments while avoiding host immune responses. Although the major
gonococcal
surface proteins have been extensively studied, little is known about less
abundant
proteins and their contribution to pathogenesis.
Two-dimensional electrophoresis (IEF and SDS-PAGE) of labeled, e.g.,
ralioiodinated or biotinylated, gonococcal surface proteins suggested that
numerous
3 (>20) of the less abundant gonococcal outer membrane proteins remained
uncharacterized (unpublished observations). Among these might be proteins
which
play an important role in infection.
For example, surface-exposed outer membrane proteins of other
microorganisms, e,g,, Haernophilus influenzae D15 surface antigen (D-15-Ag)
and the
Pasteurella multocida 0ma87 have been found to be useful in eliciting
antibodies that
were protective against infectious challenge in animal models. The Omp85-like
D-15-Ag was conserved in both non-typeable and typeable strains of H.
influenzae and
was recognized by convalescent patient sera; affinity-purified anti-D-15-Ag
serum was
protective in the rat pup model [Thomas, W. R., et al, Infect. Immun,, 58:1909-
1913
(1990); Flack, F. S. et al, Gene, 156:97-99 (1995)1 E. ir?fluenzae serotypes a-
f,
nontypeable H. influenzae and Haemophilia parainfluenzae all expressed
proteins
similar to the D-15-Ag, as demonstrated by immunoblot analysis. Antibodies to
recombinant D-15-Ag protected against H. iqfluenzae type b and type a
bacteremia in
the infant rat model [Loosmore, S. M. et al, Infect., Tipmun., 65:3701-3707
(1997)],
Like H. influenzae D-15-Ag, the Oma87 of P. multocida was highly conserved
among strains and was recognized by protective antibody; it was present in all
16
serotypes of?. tnultocida and was recognized by convalescent animal sera.
Antibodies
raised to recombinant Oma87 were protective against homologous challenge in
the
mouse model [Ruffolo, C. G. et al., Infect. Immun,, 64:3161-3167 (1996)].
Despite
the several publications describing the immunological properties of D-15-Ag
and
Oma87, the function of these proteins remains unknown.
The nucleotide and amino acid sequences of an Omp85 protein for Neissericz
gonorrhoeae and Neisserta meningiditis are illustrated in Manning et al,
"Neisseria
gonorrhoeae outer membrane protein (omp85) gene, complete cds' EMPRO
9
ArviEr 179

CA 02347849 2001-04-19
DATABASE; Accession No. U81959, 22 February 1997, XP002107049 and in
Manning et al, "Neiss.eria gonorrhoeae outer membrane protein (omp85) gene,
complete cds' EivEPRO DATABASE; Accession No, AF021245, 3 October 1997,
XP002107050, respectively. Neither reference suggests anything at all about
any uses
of these protein and gene sequences, and merely suggests that the proteins are
similar
to H ilybenzae D-15-Ag and P. muliocida Oma87.
International Patent Application W092/03467 (Univ. N. Carol.), published 5
March 3992 refers to iron-regulated transferrin binding proteins from
Neisseria
gonorrhoeae and Neisseria meningiditis. These proteins are indicated as
important in
the transferrin receptor function. Antibodies to these proteins are disclosed
for use in
inhibiting transferring receptor function.
European Patent Application No. 0,474,313 (CIGB), published 11 March 1992
refers to a 641cD outer membrane protein isolated from N. meningitidis, and.
its
potential use as a diagnostic or vaccine. As stated in this application, there
are many
immunological types of such Omp proteins.
There remains a need in the art for the development of proteins from
Neisseriae
which are useful in research, diagnosis and treatment of the infections,
especially non-
symptomatic infections, and the diseases caused by these pathogens.
25
2a
ArtlIENDEO SHEET

CA 02347849 2001-04-18
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PCT/US98/22352 _
Summary of the Invention
In one aspect, the invention provides an isolated outer membrane protein of N
gonorrhoeae having an apparent molecular weight of 85 kDa and characterized by
an
amino acid sequence of SEQ ID NO. 2, a fragment, an analog or a homolog
thereof.
In another aspect, the invention provides a nucleic acid sequence encoding the
0mp85 of N. gonorrhoeae or a fragment thereof.
In still another aspect, the invention provides a nucleic acid molecule
comprising a nucleic acid sequence encoding the Omp85 of N. gonorrhoeae or a
fragment thereof under the control of suitable regulatory sequences which
direct
expression of said 0mp85 protein or fragment in a selected host cell.
In yet a further aspect, the invention provides a host cell transformed with
the
above described nucleic acid molecule.
In still a further aspect, the invention provides a method of recombinantly
expressing the 0mp85 of N. gonorrhoeae or a fragment thereof comprising the
steps
of culturing a recombinant host cell transformed with a nucleic acid sequence
encoding
said protein or fragment under conditions which permit expression of said
protein or
peptide.
In another aspect, the invention provides a method for preparing an 0mp85
protein of N. gonorrhoeae or fragment thereof comprising chemically
synthesizing said
protein or fragment.
In yet another aspect, the invention provides a diagnostic reagent comprising
a
nucleic acid sequence encoding 0mp85 of N. gonorrhoeae, isolated from cellular

materials with which it is naturally associated, a sequence complementary
thereto, a
fragment thereof, a sequence which hybridizes thereto under stringent
conditions, an
allelic variant thereof, a mutant thereof, or a sequence encoding 0mp85 or a
fragment
thereof fused to a sequence encoding a second protein, and a detectable label
which is
associated with said sequence.
In still another aspect, the invention provides an isolated antibody which is
directed against 0mp85 of N. gonorrhoeae or a fragment thereof.
3

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PCT/US98/22352 _
In a further aspect, the invention provides an anti-idiotype antibody specific
for
the antibody described above.
In another aspect, the invention provides a diagnostic reagent comprising the
antibody or anti-idiotype antibody described above and a detectable label.
In yet another aspect, the invention provides a vaccine composition comprising
an effective amount of a Omp85 protein of N. gonorrhoeae, a fusion protein or
fragment thereof and a pharmaceutically acceptable carrier. This composition
can also
include at least one other antigen or fragment thereof.
In another aspect, the invention provides a vaccine composition comprising an
effective amount of a nucleic acid sequence encoding the Omp85 protein of N.
gonorrhoeae, a fusion protein, or a fragment thereof and a suitable nucleic
acid
delivery vehicle. This vaccine composition may also be polyvalent.
In still a further aspect, the invention provides a method of vaccinating a
human
or animal against gonococcal infection or disease comprising administering to
said
human or animal a composition comprising an effective amount of at least one
of the
compositions described above.
Another aspect of the present invention includes a method for diagnosing
gonococcal infection or disease in a human or animal comprising the steps of
contacting an 0mp85 antigen optionally associated with a detectable label or a

homolog thereof with a biological sample from a human subject to be diagnosed,
wherein the presence of naturally occurring antibodies to N. gonorrhoeae in
said
sample permits the formation of an antigen-antibody complex, and analyzing
said
sample for the presence of said complex, which indicates infection with N.
gonorrhoeae.
Still another aspect of the invention provides a method for diagnosing
gonococcal infection or disease in a human or animal comprising the steps of:
contacting an 0mp85 antibody, optionally associated with a detectable label,
with a
biological sample from a human subject to be diagnosed, wherein the presence
of
naturally occurring N gonorrhoeae 0mp85 in said sample permits the formation
of an
4

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PCT/US98/22352 _
antigen-antibody complex, and analyzing said sample for the presence of said
complex,
which indicates infection with N. gonorrhoeae
Yet a further aspect of the invention provides a method for diagnosing
gonococcal infection or disease in a human or animal comprising the steps of:
employing a nucleic acid sequence encoding all or a portion of an 0mp85
antigen or an
0pm85 antibody, optionally associated with a detectable label, as a probe
which, when
in contact with a biological sample from a human subject to be diagnosed,
enables
detection of infection by hybridization or amplification of nucleic acid
sequences of N.
gonorrhoeae 0mp85 in said sample.
Yet a further aspect of the invention includes a therapeutic composition
useful
in treating humans or animals with non-symptomatic gonococcal infection or
symptomatic disease comprising at least one anti-N gonorrhoeae 0mp85 antibody
and
a suitable pharmaceutical carrier.
In still another aspect, the invention includes a method for treating non-
symptomatic gonococcal infection or symptomatic disease in a mammalian host
comprising administering an effective amount of the therapeutic composition
described
above.
In yet another aspect, the invention provides a kit for diagnosing infection
with
N. gonorrhoeae in a human or animal comprising an 0mp85 protein or fragment
thereof or an anti-Omp85 antibody or a nucleic acid sequence encoding the
protein or
antibody as described above.
In another aspect, the invention provides a method of identifying compounds
which specifically bind to 0mp85 of N. gonorrhoeae or a fragment thereof,
comprising
the steps of contacting said 0mp85 protein or fragment with a test compound to

permit binding of the test compound to 0mp85; and determining the amount of
test
= 25 compound which is bound to 0mp85
In still another aspect, the invention provides a compound identified by the
above method.
5

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PCT/US98/22352 _
In one aspect, the invention provides an isolated outer membrane protein ofN
meningitidis having an apparent molecular weight of 85kDa and characterized by
an
amino acid sequence of SEQ ID NO: 4, a fragment, an analog or a homolog
thereof.
In another aspect, the invention provides a nucleic acid sequence encoding the

0mp85 of N. meningitidis or a fragment thereof.
In still another aspect, the invention provides a nucleic acid molecule
comprising a nucleic acid sequence encoding the 0mp85 of N. meningitidis or a
fragment thereof under the control of suitable regulatory sequences which
direct
expression of said Omp 85 or fragment in a selected host cell.
In yet a further aspect, the invention provides a host cell transformed with
the
above described nucleic acid molecule.
In still a further aspect, the invention provides a method of recombinantly
expressing the 0mp85 of N meningitidis or a fragment thereof comprising the
steps of
culturing a recombinant host cell transformed with a nucleic acid sequence
encoding
said protein or fragment under conditions which permit expression of said
protein or
peptide.
In another aspect, the invention provides a method for preparing an 0mp85
protein of N. meningitidis or fragment thereof comprising chemically
synthesizing said
protein or fragment.
In yet another aspect, the invention provides a diagnostic reagent comprising
a
nucleic acid sequence encoding 0mp85 of N. meningitidis, isolated from
cellular
materials with which it is naturally associated, a sequence complementary
thereto, a
fragment thereof, a sequence which hybridizes thereto under stringent
conditions, an
allelic variant thereof, a mutant thereof, or a sequence encoding 0mp85 or a
fragment
thereof fused to a sequence encoding a second protein, and a detectable label
which is
. 25 associated with said sequence
In still another aspect, the invention provides an isolated antibody which is
directed against 0mp85 of N. meningitidis or a fragment thereof.
In a further aspect, the invention provides an anti-idiotype antibody specific
for
the antibody described above
6

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PCT/US98/22352 _
In another aspect, the invention provides a diagnostic reagent comprising the
antibody or anti-idiotype antibody described above and a detectable label.
In yet another aspect, the invention provides a vaccine composition comprising

an effective amount of a 0mp85 protein of N. meningitidis, a fusion protein or

fragment thereof and a pharmaceutically acceptable carrier. This composition
can also
include at least one other antigen or fragment thereof.
In another aspect, the invention provides a vaccine composition comprising an
effective amount of a nucleic acid sequence encoding the 0mp85 protein of N.
meningitidis, a fusion protein, or a fragment thereof and a suitable nucleic
acid delivery
vehicle. This vaccine composition may also be polyvalent.
In still a further aspect, the invention provides a method of vaccinating a
human
or animal against non-symptomatic meningococcal infection and symptomatic
disease
comprising administering to said human or animal a composition comprising an
effective amount of at least one of the compositions described above in either
a
pharmaceutically acceptable carrier or a nucleic acid delivery system.
Another aspect of the present invention includes a method for diagnosing non-
symptomatic gonococcal infection or symptomatic disease in a human or animal
comprising the steps of contacting an 0mp85 antigen optionally associated with
a
detectable label or a homolog thereof with a biological sample from a human
subject to
be diagnosed, wherein the presence of naturally occurring antibodies to N
meningitidis
in said sample permits the formation of an antigen-antibody complex, and
analyzing
said sample for the presence of said complex, which indicates infection with
N.
meningitidis.
Still another aspect of the invention provides a method for diagnosing non-
symptomatic meningococcal infection and symptomatic disease in a human or
animal
comprising the steps of contacting an 0mp85 antibody, optionally associated
with a
detectable label, with a biological sample from a human subject to be
diagnosed,
wherein the presence of naturally occurring N. meningitidis 0mp85 in said
sample
permits the formation of an antigen-antibody complex, and analyzing said
sample for
the presence of said complex, which indicates infection with N. meningitidis.
7

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Yet a further aspect of the invention provides a method for diagnosing non-
symptomatic meningococcal infection and symptomatic disease in a human or
animal
comprising the steps of: employing a nucleic acid sequence encoding all or a
portion
of an 0mp85 antigen or an 0pm85 antibody, optionally associated with a
detectable
label, as a probe which, when in contact with a biological sample from a human
subject
to be diagnosed, enables detection of infection by hybridization or
amplification of
nucleic acid sequences ofN meningitidis 0mp85 in said sample.
Yet a further aspect of the invention includes a therapeutic composition
useful
in treating humans or animals with non-symptomatic meningococcal infection and

symptomatic disease comprising at least one anti-N meningitidis Omp85 antibody
and
a suitable pharmaceutical carrier.
In still another aspect, the invention includes a method for treating non-
symptomatic meningococcal infection and symptomatic disease in a mammalian
host
comprising administering an effective amount of the therapeutic composition
described
above.
In yet another aspect, the invention provides a kit for diagnosing infection
with
N. meningitidis in a human or animal comprising an 0mp85 protein or fragment
thereof or an anti-0mp85 antibody or a nucleic acid sequence encoding the
0mp85
protein or antibody, as described above.
In another aspect, the invention provides a method of identifying compounds
which specifically bind to 0mp85 of N meningitidis or a fragment thereof,
comprising
the steps of contacting said 0mp85 protein or fragment with a test compound to

permit binding of the test compound to 0mp85; and determining the amount of
test
compound which is bound to Omp85.
In still another aspect, the invention provides a compound identified by the
above method.
Other aspects and advantages of the present invention are described further in
the following detailed description of the preferred embodiments thereof,
reference
being made to the accompanying figures.
8

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Brief Description of the Drawings
Fig. 1 is a photograph of a sodium dodecyl sulfate polyacrylamide
electrophoretic gel (SDS-PAGE) illustrating the identification of recombinant
Omp85
produced by E. colt DH5a/pOmp85, as described in Example 2. Bacterial cell
lysates
were separated by SDS-PAGE, stained with Coomassie Brilliant blue (CBB) or
transferred to membranes and probed with anti-GC-OM serum. From left to right
are
E. colt DH5a, E. colt DH5a/pOmp85, and N. gonorrhoeae strain FA19. The
location
of 0mp85 is indicated. Prestained molecular mass markers (MW), were indicated
in
kilodaltons (kDa).
Fig. 2 illustrates the DNA sequence encoding an open reading frame of the N
gonorrhoeae omp85 [SEQ ID NO: 1] and the corresponding deduced amino acid
sequence of Omp85 (Genbank accession #U81959) [SEQ ID NO: 2], which is
preceded by an untranslated 5' sequence [SEQ ID NO: 7], and followed by an
untranslated 3' sequence [SEQ ID NO: 8]. The nucleotide sequence begins with
the
termination codon of a preceding open reading frame (ORF) that is similar to
that of
the H. influenzae hypothetical protein HI0918 and ends with the initiation
codon of a
downstream gene similar to ompH of S. typhimurium [Kosk P. eta!, J. Biol.
Chem.,
264: 18973-18980 (1989)]. The nucleotides of the omp85 open reading frame are
numbered on the left. A ribosome binding site (underlined) precedes the
initiation
codon of the omp85 ORF. The omp85 ORF was preceded and followed by
rho-independent transcriptional termination sequences (indicated by lines with
arrows).
The 0mp85 precursor polypeptide was composed of 792 amino acids. The amino
acid
sequence is numbered on the right. A putative signal peptide cleavage site was

identified (indicated by arrowhead) [Von Heijne, G., Nucl. Acids Res.,
.1.4:4683-4690
(1986)]. Cleavage at this site would produce a mature protein having a
predicted
molecular weight of 85,842 Da.
Fig. 3 is a photograph of a Western blot that illustrates the identification
of
0mp85 in N. gonorrhoeae strains FA19, FA635, FA1090, JS I, F62 and MS11LosA
and N meningitidis strains MP78, MP3, MP81 and HH by Western blot analysis
with
anti-GC-OM serum. E. colt DH5a and E. colt DH5a/pOmp85 were used as negative
9

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and positive controls. Prestained molecular mass markers (MW) were indicated
in
kDa.
Fig. 4 is a photograph of a Southern blot that illustrates the identification
of
omp85 in genomic DNA from N. gonorrhoeae FA19 and N. meningitidis strains MP3,

MP73, MP81 and HH digested with restriction endonucleases (Hind!, EcoRI, Psi!,
Clap and probed with a 688 bp fragment of gonococcal omp85 . This fragment was
used as a positive control in the first lane. Molecular weight markers are
indicated on
the left in kilobase pairs. E. coli DH5a was used as a negative control.
Fig. 5 illustrates the amino acid sequence of N. meningitidis 0mp85 (Genbank
accession #AF021045) [SEQ ID NO: 4] compared to that of N. gonorrhoeae 0mp85
[SEQ ID NO: 2]. On the top line is the N. meningitidis 0mp85 amino acid
sequence.
Below it are the amino acids that are different in the N gonorrhoeae 0mp85.
Amino
acids that are absent in the gonococcal 0mp85 are indicated by stars. Amino
acids
that are identical in the 0mp85 homologs of N meningitidis, N. gonorrhoeae, H.

influenzae (D-15-Ag) and P. ntultocida (0ma87) are underlined. The amino acids
of
the meningococcal 0mp85 are numbered on the right.
Fig. 6 is a photograph of a Western blot that illustrates the identification
of
0mp85 in N. gonorrhoeae strains FA19, FA635, FA1090, JS1, F62 and MS11LosA
and N. meningitidis strains MP78, MP3, MP81 and HH by Western blot analysis
with
anti-Omp85, as described in Example 7. E. colt DH5a, E. coli DH5a/pOmp85 and
E.
con DH5a,/pMCOmp85 were used as negative and positive controls. Prestained
molecular mass markers (MW) were indicated in kDa.
Fig. 7A is a photograph of a Western blot that illustrates the distribution of

0mp85 in pathogenic and commensal Neisseriae (relationship areas A, B, C, and
D)
and related Gram negative bacteria: N gonorrhoeae FA19 (A), Neisseria
phatyngis
(A), Neisseria cinerea (A), Neisseria lactamica (B), Neisseria nntcosae (B),
Neisseria
flavescens (C), Neisseria anima/is (C), Neisseria denitrificans (C), Moraxella

catarrhalis (D), Klebsiella pnettmoniae, Pseudomoncts aeruginosa, and N
meningitidis HH (A), as described in Example 7. E. colt DH5a and E. colt

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DH5a/pOmp85, were used as negative and positive controls. Prestained molecular

mass markers (MW) were indicated in kDa .
Fig. 7B is a photograph of a Western blot that illustrates the distribution of

Omp85 in N. gonorrhoeae FA19, Salmonella typhimurium, Shigella flexneri, E.
colt
strains 35150 (enterohemorrhagic EHEC), 35401 (enterotoxigenic - ETEC), 43887
(enteropathogenic - EPEC), 43892 (enteroinvasive - EIEC) and N. meningitidis,
as
described in Example 7. E. coil DH5a and E. colt DH5a/pOmp85 were used as
negative and positive controls. Prestained molecular mass markers (MW) were
indicated in kDa .
Fig. 8 is a bar graph showing the results of a gonococcal cell adherence assay
performed with no antibody (black bars), and with Fab fragments prepared from
antisera to: Msl 1 0mp85, MS11 hyperimmune sera to bovine serum albumin
(MS11BSA), MS11 hyperimmune serum to normal rabbit serum (MS11NRS), FA19
0mp85, FA19BSA and FA19NRS at concentrations of 1, 10 and 100 1g/ml (see key).

The assay was performed as described in Example 8.
Detailed Description of the Invention
The present invention provides novel, characterized, outer surface membrane
proteins, referred to as 0mp85, from N gonorrhoeae and N. meningitidis. These
novel antigens, fragments thereof, antibodies developed thereto, the nucleic
acid
sequences encoding same, and the use of such antigens, antibodies and nucleic
acid
sequences provide diagnostic, therapeutic and prophylactic compositions and
methods
for the treatment or prevention of gonococcal and meningococcal infections,
particularly non-symptomatic infections, and diseases.
= 25 L The 0mp85 Antigens of the Invention
To identify previously the previously uncharacterized N gonorrhoeae outer
membrane proteins, an N. gonorrhoeae genomic library was screened with an
antiserum raised against purified isolated gonococcal outer membranes. The
gonococcal gene, omp85, was identified that encodes a 792 amino acid outer
11

CA 02347849 2008-05-30
membrane protein, 0mp85, of N. gonorrhoeae having an apparent molecular weight
of
85kDa and characterized by the amino acid sequence of Fig. 2 and SEQ ID NO: 2.

0mp85 has a typical signal peptide and a carboxyl-terminal phenylalanine
characteristic
of outer membrane proteins. Southern analysis demonstrated that the omp85 gene
was
present as a single copy in N. gonorrhoeae and N. meningitidis. PCR
amplification
was used to obtain a clone of the N meningitidis omp85 homolog. The genes
encoding the N. gonorrhoeae and N. meningitidis Omp85 proteins, have been
cloned
and sequenced. The omp85 gene and its product in both N. gonorrhoeae and N.
meningitidis are characterized in Figs 2 and 5 below [SEQ ID NOS: 1-4].
The nucleic acid sequences encoding the 0mp85 proteins and the structures of
the proteins themselves are described below. Where in this specification,
protein
and/or DNA sequences are defined by their percent homologies or identities to
identified sequences, the algorithms used to calculate the percent identities
or percent
similarities include the following: the Smith-Waterman algorithm [J. F.
Collins et al,
1988, Comput. Appl. Biosci., 4:67-72; J. F. Collins et al, Molecular Sequence
Comparison and Alignment, (M. J. Bishop et al, eds.) In Practical Approach
Series:
Nucleic Acid and Protein Sequence Analysis XVIII, IRL Press: Oxford, England,
UK
(1987) pp.417], and the BLAST and FASTA programs [E. G. Shpaer et al, 1996,
Genomics, 38:179-191], including the BLAST2 program [S. D. Altschul et
al,J.Mol.
Biol., 2.li:403-407 (1990)1.
A. Nucleic Acid Sequences
The present invention provides bacterial nucleic acid sequences
encoding omp85 sequences of N. gonorrhoeae and N. meningitidis. The nucleic
acid
sequences of this invention are isolated from cellular materials with which
they are
naturally associated. The DNA sequence of the N. gonorrhoeae omp85 [SEQ ID NO:
- 25 1] and the corresponding deduced amino acid sequence of Omp85 (Genbank
accession
#U81959) [SEQ ID NO: 2] were obtained as described in the Example 2 and in
Fig. 2.
The nucleotide sequence begins with the termination codon of a preceding ORF
that is
similar to that of the H. influenzae hypothetical protein HI0918 and ends with
the
initiation codon of a downstream gene similar to ompH of S. ophinnirium [Kosk
P. et
12

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a!, J. Biol. Chem., 264: 18973-18980 (1989)]. A ribosome binding site precedes
the
initiation codon of the omp85 ORF. The omp85 ORF was preceded and followed by
rho-independent transcriptional termination sequences. The 0mp85 precursor
polypeptide was composed of 792 amino acids. A putative signal peptide
cleavage site
was identified (indicated by arrowhead) [Von Heijne, G., Nucl. Acids Res.,
1A:4683-
4690 (1986)]. Cleavage at this site produces a mature protein having a
predicted
molecular weight of 85,842 Da.
The DNA sequence of the N. meningitidis omp85 [SEQ ID NO: 3] and the
corresponding deduced amino acid sequence of 0mp85 (Genbank accession
#AF021045) [SEQ ID NO: 4] were obtained as described in Example 3. Fig. 5
shows
the comparison between the sequences of the two 0mp85 proteins, as well as
their
similarities to the Omp85 homologs of N. meningitidis, N. gonorrhoeae, H.
influenzae
(D-15-Ag) and P. multocida (0ma87).
In addition to the full-length nucleic acid sequences encoding the 0mp85
proteins provided herein, the specification also includes fragments of these
omp85
genes. Preferably, such fragments are characterized by encoding a biologically
active
portion of 0mp85, e.g., an epitope. Alternatively, other non-epitopic
fragments may
be useful as probes in diagnostic or research use. Generally, these
oligonucleotide
fragments are at least 10, or at least 15 consecutive nucleotides in length.
However,
oligonucleotide fragments of varying sizes may be selected as desired. Such
fragments
may be used for such purposes as performing polymerase chain reaction (PCR),
e.g.,
on a biopsied tissue sample. For example, useful fragments of omp85 DNA and
corresponding sequences comprise sequences occurring between nucleotides 2161
through 2208 of SEQ ID NO: 1 and nucleotides 2161 and 2218 of SEQ ID NO: 3.
Other usefiil fragments may be readily obtained by one of skill in the art by
resort to
conventional DNA sequencing techniques applied to the sequences disclosed
herein.
The DNA sequences of SEQ ID NOS: 1 and 3 permit one of skill in the
art to readily obtain the corresponding anti-sense strands of these DNA
sequences.
Further, using known techniques, one of skill in the art can readily obtain
additional
genomic and cDNA sequences which flank the illustrated DNA sequences or the
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corresponding RNA sequences, as desired. Similarly the availability of SEQ ID
NOS:
1 and 3 of this invention permits one of skill in the art to obtain other
species 0mp85
analogs, and fragments thereof, by use of the nucleic acid sequences of this
invention
as probes in a conventional technique, e.g., polymerase chain reaction.
Allelic variants
of these sequences within a species (i.e., sequences containing some
individual
nucleotide differences from a more commonly occurring sequence within a
species, but
which nevertheless encode the same protein or a protein with the same
function) such
as other variants of 0mp85 [SEQ ID NOS: 2 and 4] may also be readily obtained
given
the knowledge of these nucleic acid sequences provided by this invention.
The present invention further encompasses nucleic acid sequences
capable of hybridizing under stringent conditions [see, J. Sambrook et al,
Molecular
Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory (1989)] to
the
sequences of SEQ ID NOS: 1 and 3, their anti-sense strands, or biologically
active
fragments thereof. An example of a highly stringent hybridization condition is

hybridization at 2XSSC at 65 C, followed by a washing in 0.1XSSC at 65 C for
an
hour. Alternatively, an exemplary highly stringent hybridization condition is
in 50%
formamide, 4XSSC at 42 C. Moderately high stringency conditions may also prove

useful, e.g;., hybridization in 4XSSC at 55 C, followed by washing in 0.1XSSC
at
37 C for an hour. An alternative exemplary moderately high stringency
hybridization
condition is in 50% formamide, 4XSSC at 30 C.
According to the invention, the nucleic acid sequences may be
modified. Utilizing the sequence data of SEQ ID NOS: 1 and 3, it is within the
skill of
the art to obtain other synthetically or recombinantly-prepared polynucleotide

sequences, or modified polynucleotide sequences, encoding the full-length
proteins or
useful fragments of the invention. For example, one of skill may employ
preferred or
"preference" codons for expression of the sequence in selected host cells;
thus SEQ ID
NOS: 1 and 3 may be modified to contain different nucleotide triplets which
encode
the same amino acid as encoded by SEQ ID NOS: 1 and 3. Such modifications made

at the nucleic acid level include, for example, modifications to the
nucleotide sequences
which are silent or which change the amino acids, e.g. to improve expression
or
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secretion of the protein. Also included are allelic variations, caused by the
natural
degeneracy of the genetic code.
Also encompassed by the present invention are mutants of the omp85
sequences, including 5', 3' or internal deletions, which encode proteins that
substantially retain the antigenicity of the full-length 0mp85 or other
proteins or
fragments. Such a truncated, or deletion, mutants may be expressed by modified
nucleic acid sequences for the purpose of affecting the activity of the full-
length or
wild-type protein.
As described in more detail below, these nucleic acid sequences are
useful for a variety of diagnostic, prophylactic and therapeutic uses.
Advantageously,
the nucleic acid sequences are useful in the development of diagnostic probes
and
antisense probes for use in the detection and diagnosis of infections,
particularly non-
symptomatic infection, and diseases caused by these Neisseriae pathogens and
by
related pathogens discussed above by utilizing a variety of known nucleic acid
assays,
e.g., Northern and Southern blots, polymerase chain reaction (PCR), and other
assay
techniques known to one of skill in the art. The nucleic acid sequences of
this
invention are also useful in the production of 0mp85 proteins and homologs as
well as
a variety of fusion or other synthetic proteins
The nucleotide sequences of the invention may be readily synthesized or
may be isolated by conventional uses of polymerase chain reaction or cloning
techniques such as those described herein and in conventional texts such as
Sambrook
et al, cited above. For example, the nucleic acid sequences of the antigen of
this
invention may be prepared or isolated from genomic libraries using DNA primers
and
probes and PCR techniques These sequences, fragments thereof, modifications
thereto and the full-length sequences may be constructed recombinantly using
conventional genetic engineering or chemical synthesis techniques or PCR, and
the like
by utilizing the information provided herein. Further, such nucleic acid
sequences may
be conventionally labeled for diagnostic use Alternatively for use as
therapeutic or
vaccine components, the nucleic acid sequences of this invention may be
admixed with
a variety of pharmaceutically acceptable carriers, e.g., saline, liposomes,
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_
incorporated into nucleic acid molecules, e.g., plasmids under the regulatory
control of
sequences which direct expression of the encoded protein in a selected host
cell. The
nucleic acid sequences may also be delivered to a host as "naked" DNA or in a
gene
delivery vehicle, such as a recombinant virus, all as described in detail
below.
B. Protein Sequences
The present invention also provides 0mp85 proteins and peptides of
this invention. These proteins are free from association with other
contaminating
proteins or materials with which they are found in nature. The Neisseriae
0mp85
antigen has a relative molecular mass of 85kDa as measured by Western
immunoblot
(See Example 2 and Fig. 2). In one embodiment, the invention provides a
gonococcal
0mp85 antigen [SEQ ID NO:2] podypeptide of about 792 amino acids, with a
signal
peptide, having a predicted molecular weight of 85,842 daltons.
The meningococcal omp85 was found to encode a 797 amino acid
polypeptide with a predicted molecular weight of 88.5 kDa (Fig. 5). Between
amino
acid residues 720 and 745, the menigococcal 0mp85 varied substantially from
gonococcal 0mp85, including the insertion of five additional amino acids. The
deduced
amino acid sequence [SEQ ID NO: 4] of N. meningitidis 0mp85 was revealed by
sequence analysis to be 95% identical to N. gonorrhoeae 0mp85 and 98% similar
to
gonococcal 0mp85 using the BLAST2 algorithm.
The similarities of these two 0mp85 proteins to proteins of other
microorganisms provides evidence of an immunological role played by these
proteins,
as well as other potential roles. The D-15 protective surface antigen (D-15-
Ag) of
Haemophilus influenzae and the 0ma87 of Pasteurella multocida are the only
bacterial proteins, which have been previously described, that are similar to
the 0mp85
amino acid sequence [SEQ ID NO: 2]. This similarity suggested that these
proteins
played an important role in host-pathogen interactions and have an important
function
in pathogenesis. The importance of these 0mp85 proteins in pathogenesis and
immunobiology was demonstrated by the fact that antibody to similar proteins
in H.
influenzae and P. multocida were protective. The similarities suggest that the
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Neisseriae 0mp85 proteins are likely important immunological targets of the
host
immune response.
Western blot analysis demonstrated proteins similar to 0mp85 in all of
the Neisseriae tested with anti-0mp85 and three Neisseriae relationship areas.
Area A
contains the frank pathogens N. gonorrhoeae and N meningitidis and
opportunistic
organisms known to cause severe human diseases such as N. pharyngis and N
cinerea.
Area B contains species, such as N. mucosae and N. lactamica, typically found
in the
human nasopharynx which are able to cause opportunistic infections in
debilitated
hosts. Area C consists of commensal/saprophytic organisms, such as N.
flavescens, N
animalis and N denitricans, which generally do not cause human infections.
All Neisseriae species colonize mucosa! surfaces. The presence of
0mp85-like proteins in numerous pathogenic and commensal organisms suggests it

may be involved in establishing or maintaining colonization. The
identification of
0mp85 proteins in a number of pathogenic and commensal organisms provides
evidence that the 0mp85 proteins provide functions involved in establishing or
maintaining colonization.
Database searches identified genes in a number of pathogens which
encode hypothetical proteins similar to 0mp85, including genes in B. abortus,
H.
pylori, and B. burgdorferi . The proteins encoded by these genes have not yet
been
characterized. A search of the 0mp85 amino acid sequence against the GenBank
data
base resulted in the identification of a cyanobacterium protein [Kaneto T. et
al, DNA
Res., 1:109-136 (1996)] with 35% similarity to the gonococcal 0mp85. The
cyanobacterium protein was named IAP75 because of its similarity to the 75 kDa

chloroplast import associated protein, IAP75 [Schnell DJ et al, Science, 266:
1007-101 1 (1994)]. The chloroplast IAP75 was located in the outer membrane of
chloroplasts and was one of four outer membrane components of a complex that
transports polypeptides. This suggested that 0mp85 might be part of a similar
transport complex.
The sequences of other proteins from the chloroplast import associated
complex were searched against the Gonococcal Genome Sequencing Project Data
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Base (Dyer and Rowe, 1997). Sequences similar to the chloroplast IAP34 protein

[Kessler F. eta!, .Scieoce, 266.: 1035-1039 (1994)] were identified in the
gonococcal
genome. 1AP34 is believed to be a GTP-binding protein and the sequences of
highest
similarity to the gonococcal homolog were in regions identified as GTP-binding
protein
motifs. Surface-crosslinkage studies were performed to determine if 0mp85
might
participate in a system analogous to the 1AP complex (data not shown). Studies
using
the reversible crosslinker DTBP, which crosslinks proteins that are within
11.9A of
each other [Newhall WJ. eta!, Infect. Immun., 2s: 785-791 (1980)], showed that

Omp85 crosslinked with up to five other outer membrane proteins, one of ¨34
kDa
(IAP34 homolog) (data not shown) These data, which confirmed 0mp85 was exposed
on the bacterial surface, support the role for 0mp85 of participating in a
complex
analogous with the chloroplast IAP complex. Further characterization of
proteins
associated with 0mp85 in the outer membrane may provide evidence of additional

biological functions of these 0mp85 proteins.
One of skill in the art using conventional techniques, such as PCR, may
readily use the 0mp85 sequences provided herein to identify and isolate other
similar
proteins. Such methods are routine and not considered to require undue
experimentation, given the information provided herein
Antigens of this invention may be characterized by immunological
measurements including, without limitation, Western blot, macromolecular mass
determinations by biophysical determinations, such as SDS-PAGE/staining, high
pressure liquid chromatography (HPLC) and the like, antibody recognition
assays, T-
cell recognition assays, major histocompatibility complex (MHC) binding
assays, and
assays to infer immune protection or immune pathology by adoptive transfer of
cells,
proteins or antibodies.
The 0mp85 outer membrane antigens of this invention (as well as its
naturally occurring variants or analogs in other species) may be isolated in
the form of
a complete intact protein, or a polypeptide or fragment thereof In one
embodiment,
Omp85 is isolated by immunoblot procedures according to its respective
molecular
mass, as described below in the examples. Such isolation provides the antigen
in a
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form substantially free from other proteinaceous and non-proteinaceous
materials of
the microorganism. The molecules comprising the polypeptides and antigens of
this
invention may be isolated and further purified using any of a variety of
conventional
methods including, but not limited to: liquid chromatography such as normal or

reversed phase, using HPLC, FPLC and the like; affinity chromatography (such
as with
inorganic ligands or monoclonal antibodies); size exclusion chromatography;
immobilized metal chelate chromatography; gel electrophoresis; and the like.
One of
skill in the art may select the most appropriate isolation and purification
techniques
without departing from the scope of this invention.
Alternatively, the amino acid sequences of the proteins of this invention
may be produced recombinantly following conventional genetic engineering
techniques
[see e.g., Sambrook et al, cited above and the detailed description of making
the
proteins below].
1. Analogs/Modified Antigens
Also included in the invention are analogs, or modified versions,
of the 0mp85 protein or fragments provided herein. Typically, such analogs
differ
from the specifically identified proteins by only one to four codon changes.
Examples
include polypeptides with minor amino acid variations from the illustrated
partial
amino acid sequence of, for example, the gonococcal 0mp85 (SEQ ID NO: 2), in
particular, conservative amino acid replacements. Conservative replacements
are those
that take place within a family of amino acids that are related in their side
chains and
chemical properties. Also provided are homologs of the proteins of the
invention
which are characterized by having at least 80% identity with SEQ ID NO:2 or
SEQ ID
NO: 4. Also included in this invention are homologs having at least 85%
identity with
SEQ ID NO: 2 or SEQ ID NO: 4. Homologs having at least 90% identity with
either
SEQ ID NO: 2 or SEQ ID NO: 4 are also encompassed by this invention. Homologs
having at least 95% identity with either SEQ ID NO: 2 or SEQ ID NO: 4 are also

encompassed by this invention. Also provided are homologs of the proteins of
the
invention which are characterized by having at least 85% homology with SEQ ID
NO:2 or SEQ ID NO: 4. Also included in this invention are homologs having at
least
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90% homology with SEQ ID NO: 2 or SEQ ID NO: 4. Homologs having at least 95%
homology with either SEQ ID NO: 2 or SEQ ID NO: 4 are also encompassed by this

invention. Homologs having at least 99% homology with either SEQ ID NO: 2 or
SEQ ID NO: 4 are also encompassed by this invention. The algorithms used for
these
calculations are identified above. Based on the sequence information provided
herein,
one of skill in the art can readily obtain full-length homologs and analogs
from other
bacterial species.
An antigen of the present invention may also be modified to
increase its immunogenicity. For example, the antigen may be coupled to
chemical
compounds or immunogenic carriers, provided that the coupling does not
interfere
with the desired biological activity of either the antigen or the carrier. For
a review of
some general considerations in coupling strategies, see Antibodies. A
Laboratory
Manual, Cold Spring Harbor Laboratory, ed. E. Harlow and D. Lane (1988).
Useful
immunogenic carriers known in the art, include, without limitation, keyhole
limpet
hemocyanin (ICLH); bovine serum albumin (BSA), ovalbumin, purified protein
derivative of tuberculin (PPD); red blood cells; tetanus toxoid; cholera
toxoid; agarose
beads; activated carbon; or bentonite. Useful chemical compounds for coupling
include, without limitation, dinitrophenol groups and arsonilic acid. One of
skill in the
art may readily select other appropriate immunogenic carriers or coupling
agents.
The antigen may also be modified by other techniques, such as
denaturation with heat and/or SDS.
Fragments/Deletion Mutants
Further encompassed by this invention are additional fragments
of the 0mp85 polypeptides and peptides identified herein. Such fragments are
desirably characterized by having a biological activity similar to that
displayed by the
complete protein, including, e.g., the ability to induce antibodies which can
interfere
with the binding of the pathogen to its cellular targets (see Example 8).
These
fragments may be designed or obtained in any desired length, including as
small as
about 5-8 amino acids in length up to fragments encompassing just short of the
entire
protein. Such fragments may represent consecutive amino acids in the protein

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sequence or they may represent conformational sites of the protein. Such a
fragment
may represent an epitope or conformational epitope of the protein.
The 0mp85 proteins [SEQ ID NOS:2 and 4] of the invention
may be modified to create deletion mutants, for example, by truncation at the
amino or
carboxy termini, or by elimination of one or more amino acids. Deletion
mutants are
also encompassed by this invention, as are the DNA sequences encoding them.
In yet another embodiment, the Omp85 peptides or polypeptides
of this invention may be in the form of a multiple antigenic peptide ("MAP",
also
referred to as an octameric lysine core peptide) construct. Such a construct
may be
designed employing the MAP system described by Tam, Proc. Natl. Acad. Sci.
USA,
85:5409-5413 (1988). This system makes use of a core matrix of lysine residues
onto
which multiple copies of the same protein or peptide of the invention are
synthesized
as described [D. Posnett et al., J. Biol, Chem., 263(4):1719-1725 (1988); J.
Tam,
"Chemically Defined Synthetic Immunogens and Vaccines by the Multiple Antigen
Peptide A.pproach", Vaccine Research and Developments, Vol. 1, ed. W. Koff and
H.
Six, pp. 51-87 (Marcel Deblau, Inc., New York 1992)1 Each MAP contains
multiple
copies of only one peptide.
Still other modified fragments of Omp85 may be prepared by
any number of now conventional techniques to improve production thereof, to
enhance
protein stability or other characteristics, e g. binding activity or
bioavailability, or to
confer some other desired property upon the protein. Other useful fragments of
these
polypeptides may be readily prepared by one of skill in the art using known
techniques,
such as deletion mutagenesis and expression.
iii. Fusion or Multimeric Proteins and Compositions
The 0mp85 protein of the present invention, or fragments of it,
may also be constructed, using conventional genetic engineering techniques as
part of a
larger and/or multimeric protein or protein compositions Antigens of this
invention
may be in combination with outer surface proteins or other proteins or
antigens of
other pathogens, such as those identified above, or various fragments of the
antigens
described herein may be in combination with each other. In such combination,
the
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antigen may be in the form of a fusion protein. The antigen of the invention
may be
optionally fused to a selected polypeptide or protein derived from other
microorganisms. For example, an antigen or polypeptide of this invention may
be
fused at its N-terminus or C-terminus to a polypeptide from another pathogen
or to
more than one polypeptide in sequence. Polypeptides which may be useful for
this
purpose include polypeptides identified by the prior art.
Still another fusion protein of this invention is provided by
expressing the DNA molecule formed by the omp85 DNA sequence or a fragment
thereof fused to DNA fragments that are homologous (between about 25-95%
identity) to 0mp85. One example of such a protein comprises the amino acid
sequence of SEQ ID NO: 2 to which is fused amino acid fragments that are up to
95%
identical to that sequence, e.g., from SEQ ID NO: 4, or from any of the above-
described homologous proteins. These fragments may be inserted in any order
and
may contain repeated sequences. The fused fragments may produce a large DNA
molecule which expresses a protein which may stimulate a variety of antibody
specificities.
These fusion proteins comprising multiple polypeptides of this
invention are constructed for use in the methods and compositions of this
invention.
These fusion proteins or multimeric proteins may be produced recombinantly, or
may
be synthesized chemically. They also may include the polypeptides of this
invention
fused or coupled to moieties other than amino acids, including lipids and
carbohydrates. Further, antigens of this invention may be employed in
combination
with other vaccinal agents described by the prior art, as well as with other
species of
vaccinal agents derived from other microorganisms. Such proteins are useful in
the
prevention, treatment and diagnosis of diseases caused by a wide spectrum of
Neisseriae isolates.
A protein composition which may be a preferred alternative to
the fusion proteins described above is a cocktail (i.e., a simple mixture)
containing
different Omp85 proteins or fragments, optionally mixed with different
antigenic
proteins or peptides of other pathogens. Such mixtures of these proteins or
antigenic
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fragments thereof are likely to be useful in the generation of desired
antibodies to a
wide spectrum ofNeisseriae isolates.
iv. Salts
An antigen of the present invention may also be used in the form
of a pharmaceutically acceptable salt. Suitable acids and bases which are
capable of
forming salts with the polypeptides of the present invention are well known to
those of
skill in the art, and include inorganic and organic acids and bases.
III. Methods of Making Antigens and Nucleic Acid Sequences of the Invention
Expression In Vitro
To produce recombinant 0mp85 or peptide fragments of this invention,
the DNA sequences of the invention are inserted into a suitable expression
system.
Desirably, a recombinant molecule or vector is constructed in which the
polynucleotide
sequence encoding the selected protein, e.g., 0mp85 is operably linked to a
heterologous expression control sequence permitting expression of the protein.
Numerous types of appropriate expression vectors are known in the art for
protein
expression, by standard molecular biology techniques. Such vectors are
selected from
among conventional vector types including insects, e.g., baculovirus
expression, or
yeast, fungal, bacterial or viral expression systems. Other appropriate
expression
vectors, of which numerous types are known in the art, can also be used for
this
purpose. Methods for obtaining such expression vectors are well-known. See,
Sambrook et al, Molecular Cloning. A Laboratory Manual, 2d edition, Cold
Spring
Harbor Laboratory, New York (1989); Miller et at, Genetic Engineering, 8 277-
298
(Plenum Press 1986) and references cited therein.
Suitable host cells or cell lines for transfection by this method include
.25 bacterial cells. For example, the various strains of E. co/i, e.g.,
HB101, MC1061, and
strains used in the following examples, are well-known as host cells in the
field of
biotechnology. Various strains of B. Psetidomonas, Sireptomyces, and
other
bacilli and the like are also be employed in this method.
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Mammalian cells, such as human 293 cells, Chinese hamster ovary cells
(CHO), the monkey COS-1 cell line or murine 313 cells derived from Swiss, Balb-
c or
NIH mice are used. Another suitable mammalian cell line is the CV-1 cell line.
Still
other suitable mammalian host cells, as well as methods for transfection,
culture,
amplification, screening, production, and purification are known in the art.
[See, e.g.,
Gething and Sambrook, Nature, 293:620-625 (1981), or alternatively, Kaufman et
al,
Mol. Cell. Biol., 5(7):1750-1759 (1985) or Howley et al, U. S. Patent
4,419,446].
Many strains of yeast cells known to those skilled in the art are also
available as host cells for expression of the polypeptides of the present
invention.
Other final cells may also be employed as expression systems. Alternatively,
insect
cells such as Spodoptera fruppedera (Sf9) cells may be used.
Thus, the present invention provides a method for producing
recombinant 0mp85 proteins, which involves transfecting, e.g., by conventional
means
such as electroporation, a host cell with at least one expression vector
containing a
polynucleotide of the invention under the control of a transcriptional
regulatory
sequence. The transfected or transformed host cell is then cultured under
conditions
that allow expression of the protein The expressed protein is recovered,
isolated, and
optionally purified from the cell (or from the culture medium, if expressed
extracellularly) by appropriate means known to one of skill in the art.
For example, the proteins are isolated in soluble form following cell
lysis, or extracted using known techniques, e.g., in guanidine chloride. If
desired, the
proteins or fragments of the invention are produced as a fusion protein. Such
fusion
proteins are those described above. Alternatively, for example, it may be
desirable to
produce fusion proteins to enhance expression of the protein in a selected
host cell, to
improve purification, or for use in monitoring the presence of the desired
protein, e.g.,
0mp85, in tissues, cells or cell extracts. Suitable fusion partners for the
proteins of the
invention are well known to those of skill in the art and include, among
others, p-
galactosidase, glutathione-S-transferase, poly-histidine and maltose binding
protein.
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B. Expression In Vivo
Alternatively, where it is desired that the 0mp85 (whether full-length or
a fragment) be expressed in vivo, e.g., to induce antibodies, or alternatively
where the
omp85 is to be employed as a DNA vaccine, an appropriate vector for delivery
is
readily selected by one of skill in the art. Exemplary vectors for in vivo
gene delivery
are readily available from a variety of academic and commercial sources, and
include,
e.g., adeno-associated virus [International patent application No.
PCT/US91/03440],
adenovirus vectors [M. Kay et al, Proc. Natl. Acad. Sci. USA, 91:2353 (1994);
S.
Ishibashi et al, J. Clin. Invest., 92:883 (1993)], or other viral vectors,
e.g., various
poxviruses, vaccinia, etc. Methods for insertion of a desired gene, e.g.,
0mp85, and
obtaining in vivo expression of the encoded protein, are well known to those
of skill in
the art.
=
IV. Antibodies of the Invention
The present invention also provides antibodies capable of recognizing and
binding the isolated, or modified, or multimeric antigens of this invention,
including
antibodies derived from mixtures of such antigens or fragments thereof.
Certain of the
antibodies of this invention may be specific to the N. gonorrhoeae or N.
meningitidis
0mp85 proteins, by binding to epitopes on the proteins which differ from the
former
species to the latter species. For example, an antibody specific for N.
gonorrhoeae
may bind an epitope on SEQ ID NO: 2 which is not present in SEQ ID NO: 4, or
vice
versa. Thus, an N. gonorrhoeae 0mp85-specific antibody is defined herein as an

antibody that binds an 0mp85 antigen of N. gonorrhoeae only. An N meningitidis

0mp85-specific antibody is defined herein as an antibody that binds an 0mp85
antigen
of N. meningitidis only. Alternatively, certain antibodies to these proteins
may bind
an epitope present on both the N gonorrhoeae and N meningitidis 0mp85
proteins.
Still other antibodies of this invention may bind an epitope on N gonorrhoeae
and N.
meningitidis 0mp85, and the same epitope on the other homologous proteins in
homologous or heterologous species of bacteria having homologous proteins

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(described in Part I, B above). All of these antibodies are encompassed by
this
invention.
These antibodies are useful in diagnosis of gonococcal and meningococcal
infection (non-symptomatic) as well as symptomatic diseases, caused by N
gonorrhoeae,N meningitidis or other Neisseriae species, and in therapeutic
compositions for treating humans and/or animals that test positive for
infection, or,
prior to testing, exhibit symptoms of such diseases. The antibodies are useful
in
diagnosis alone or in combination with antibodies to other antigens of this
invention, as
well as antibodies to other known antigens from homologous or completely
heterologous species of microorganism These antibodies are also useful in
passive
vaccine compositions, which vaccines may also be polyvalent, by containing
antibodies
to antigens of other microorganisms as well as antibodies to the 0mp85
proteins of
this invention.
The antibodies of this invention are generated by conventional means utilizing

the isolated, recombinant or modified antigens of this invention, or mixtures
of such
antigens or antigenic fragments. For example, polyclonal antibodies are
generated by
conventionally stimulating the immune system of a selected animal or human
with the
isolated antigen or mixture of antigenic proteins or peptides of this
invention, allowing
the immune system to produce natural antibodies thereto, and collecting these
antibodies from the animal or human's blood or other biological fluid.
For example, an antibody according to the invention is produced by
administering to a vertebrate host the antigen or antigenic composition of
this
invention, e.g., 0mp85. Preferably a recombinant version of 0mp85 (rOmp85) or
an
0mp85 MAP is used as an immunogen. A suitable polyclonal antibody against the
0mp85 antigen may be generated as antisera, such as the 0mp85 antisera
employed in
the examples herein.
Thus, an antibody of the invention is isolated by affinity purifying antiserum

generated during an infection of a mammal, e.g., a mouse, with N. gonorrhoeae
or N
meningitidis, using as immunoabsorbant the 0mp85 antigen identified herein.
Similarly, an antibody of the invention is isolated by immunizing mice with a
purified,
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recombinant antigen of this invention, or a purified, isolated 0mp85 protein
of native
origin.
Monoclonal antibodies (MAbs) directed against 0mp85 are also generated.
Hybridorna cell lines expressing desirable MAbs are generated by well-known
conventional techniques, e.g. Kohler and Milstein and the many known
modifications
thereof. Similarly desirable high titer antibodies are generated by applying
known
recombinant techniques to the monoclonal or polyclonal antibodies developed to
these
antigens [see, e.g., PCT Patent Application No. PCT/GB85/00392; British Patent

Application Publication No. GB2188638A; Amit et at., Science, 233:747-753
(1986);
Queen et at., Proc. Nat'l. Acad. Sci USA, L6:10029-10033 (1989); PCT Patent
Application No. W090/07861; and Riechmann et at., Nature, 332:323-327 (1988);
Huse et at, Science, 246:1275-1281 (1988)a].
Given the disclosure contained herein, one of skill in the art may generate
chimeric, humanized or fully human antibodies directed against 0mp85 or
antigenic
fragments thereof by resort to known techniques by manipulating the
complementarity
determining regions of animals or human antibodies to the antigen of this
invention.
See, e.g., E. Mark and Padlin, "Humanization of Monoclonal Antibodies",
Chapter 4,
The Handbook of Experimental Pharmacology, Vol. 113, The Pharmacology of
Monoclonal Antibodies, Springer-Verlag (June, 1994).
Alternatively, the antigens are assembled as multi-antigenic complexes [see,
e.g., European Patent Application 0339695, published November 2, 1989] or as
simple
mixtures of antigenic proteins/peptides and employed to elicit high titer
antibodies
capable of binding the selected antigen(s) as it appears in the biological
fluids of an
infected animal or human.
Further provided by the present invention are anti-idiotype antibodies (Ab2)
and anti-anti-idiotype antibodies (Ab3). Ab2 are specific for the target to
which anti-
0mp85 antibodies of the invention bind and Ab3 are similar to 0mp85 antibodies

(Abl) in their binding specificities arid biological activities [see, e.g., M.
Wettendorff et
al., "Modulation of anti-tumor immunity by anti-idiotypic antibodies." in
Idiotypic
Network and Diseases, ed. by J. Cerny and J. Hiernaux J, Am. Soc. Microbiol.,
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Washington DC: pp. 203-229, (1990)]. These anti-idiotype and anti-anti-
idiotype
antibodies are produced using techniques well known to those of skill in the
art. Such
anti-idiotype antibodies (Ab2) can bear the internal image of 0mp85 or
fragments
thereof and are thus useful for the same purposes as 0mp85 or the fragments.
In general, polyclonal antisera, monoclonal antibodies and other antibodies
which bind to the selected antigen (Abl) are useful to identify epitopes of
0mp85 to
separate 0mp85 and analogs thereof from contaminants in living tissue (e.g.,
in
chromatographic columns and the like), and in general as research tools and as
starting
material essential for the development of other types of antibodies described
above.
Anti-idiotype antibodies (Ab2) are useful for binding the same target and thus
may be
used in place of the original antigen, e.g., 0mp85, to induce an immune
response. The
Ab3 antibodies are useful for the same reason the Abl are useful. Other uses
as
research tools and as components for separation of 0mp85 from other
contaminants,
for example, are also contemplated for the above-described antibodies.
For use in diagnostic assays, the antibodies are associated with conventional
labels which are capable, alone or in concert with other compositions or
compounds,
of providing a detectable signal. Where more than one antibody is employed in
a
diagnostic method, the labels are desirably interactive to produce a
detectable signal.
Most desirably, the label is detectable visually, e.g. colorimetrically. A
variety of
enzyme systems have been described in the art which will operate to reveal a
colorimetric signal in an assay. As one example, glucose oxidase (which uses
glucose
as a substrate) releases peroxide as a product. Peroxidase, which reacts with
peroxide
and a hydrogen donor such as tetramethyl benzidine (TMB) produces an oxidized
TMB that is seen as a blue color. Other examples include horseradish
peroxidase
(HRP) or alkaline phosphatase (AP), and hexokinase in conjunction with glucose-
6-
phosphate dehydrogenase which reacts with ATP, glucose, and NAD+ to yield,
among
other products, NADH that is detected as increased absorbance at 340 nm
wavelength.
Other label systems that may be utilized in the methods of this invention are
detectable
by other means, e.g., colored latex microparticles [Bangs Laboratories,
Indiana] in
which a dye is embedded may be used in place of enzymes to form conjugates
with the
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antibodies and provide a visual signal indicative of the presence of the
resulting
complex in applicable assays. Still other labels include fluorescent
compounds,
radioactive compounds or elements. Detectable labels for attachment to
antibodies
useful in diagnostic assays of this invention may be easily selected from
among
numerous compositions known and readily available to one skilled in the art of
diagnostic assays. The methods and antibodies of this invention are not
limited by the
particular detectable label or label system employed.
V. Diagnostic Methods and Assays
The present invention also provides methods of diagnosing infections and
diseases caused by infection with N gonorrhoeae, N meningitidis or possibly
other
species of pathogen which have homologous proteins to the 0mp85 proteins of
this
invention. These diagnostic methods are useful for diagnosing humans having
non-
symptomatic infection or exhibiting the clinical symptoms of gonococcal or
meningococcal disease, or possibly any of the other diseases caused by
homologous
bacterial species.
In one embodiment, this diagnostic method involves detecting the presence of
naturally occurring anti-0mp85 antibodies which are produced by the infected
human
or animal patient's immune system in its biological fluids, and which are
capable of
binding to the antigens of this invention or combinations thereof. This method
comprises the steps of incubating a 0mp85 antigen or antigenic fragment of
this
invention with a sample of biological fluid or tissue from the patient.
Antibodies
present in the fluids as a result of bacterial infection will form an antibody-
antigen
complex with the antigen. Subsequently the reaction mixture is analyzed to
determine
the presence or absence of these antigen-antibody complexes. The step of
analyzing
the reaction mixture can comprise detecting a label associated with the Omp85
antigen,
or contacting the reaction mixture with a labeled specific binding partner for
the
antibody or antibody.
In one embodiment of the method, purified antigen, fragment or mixture of
antigens is electro- or dot-blotted onto nitrocellulose paper. Subsequently,
the
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biological fluid (e.g. serum or plasma) is incubated with the blotted antigen,
and
antibody in the biological fluid is allowed to bind to the antigen(s). The
bound antibody
is then detected by standard immunoenzymatic methods.
In another embodiment of the method, latex beads are conjugated to the
antigen(s) of this invention. Subsequently, the biological fluid is incubated
with the
bead/protein conjugate, thereby forming a reaction mixture. The reaction
mixture is
then analyzed to determine the presence of the antibodies.
In another embodiment, the diagnostic method of the invention involves
detecting the presence of the naturally occurring 0mp85 itself in its
association with
the Neisseriae pathogen in the biological fluids or tissue of an animal or
human
infected by the pathogen. This method includes the steps of incubating an
antibody of
this invention (e.g. produced by administering to a suitable human and/or
animal an
antigen of this invention preferably conventionally labeled for detection)
with a
biological sample from a human or an animal to be diagnosed. In the presence
of
infection of the human or animal patient, an antigen-antibody complex is
formed
(specific binding occurs). Subsequently, excess labeled antibody is optionally
removed, and the reaction mixture is analyzed to determine the presence or
absence of
the antigen-antibody complex and the amount of label associated therewith.
Assays employing a protein antigen of the invention can be heterogenous (i.e.,

requiring a separation step) or homogenous. If the assay is heterogenous, a
variety of
separation means can be employed, including centrifugation, filtration,
chromatography, or magnetism.
One preferred assay for the screening of blood products or other physiological

or biological fluids is an enzyme linked immunosorbant assay, i.e., an ELISA.
Typically in an ELISA, the isolated antigen(s) of the invention is adsorbed to
the
surface of a microtiter well directly or through a capture matrix (i.e.,
antibody).
Residual protein-binding sites on the surface are then blocked with an
appropriate
agent, such as bovine serum albumin (BSA), heat-inactivated normal goat serum
(NGS), or BLOTTO (a buffered solution of nonfat dry milk which also contains a

preservative, salts, and an antifoaming agent). The well is then incubated
with a

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biological sample suspected of containing specific anti-N gonorrhoeae or N.
meningitidis antibody. The sample can be applied neat, or more often, it can
be
diluted, usually in a buffered solution which contains a small amount (0.1-
5.0% by
weight) of protein, such as BSA, NGS, or BLOTTO. After incubating for a
sufficient
length of time to allow specific binding to occur, the well is washed to
remove
unbound protein and then incubated with labeled anti-human immunoglobulin (a
HuIg)
or labeled antibodies to other species, e.g., dogs. The label can be chosen
from a
variety of enzymes, including horseradish peroxidase (HRP), P-galactosidase,
alkaline
phosphatase, and glucose oxidase, as described above. Sufficient time is
allowed for
specific binding to occur again, then the well is washed again to remove
unbound
conjugate, and the substrate for the enzyme is added. Color is allowed to
develop and
the optical density of the contents of the well is determined visually or
instrumentally.
Further, MAbs or other antibodies of this invention which are capable of
binding to the antigen(s) can be bound to ELISA plates. In another diagnostic
method,
the biological fluid is incubated on the antibody-bound plate and washed.
Detection of
any antigen-antibody complex, and qualitative measurement of the labeled MAb
is
performed conventionally, as described above.
Other useful assay formats include the filter cup and dipstick. In the former
assay, an antibody of this invention is fixed to a sintered glass filter to
the opening of a
small cap. The biological fluid or sample (5 ml) is worked through the filter.
If the
antigen is present (i.e., N. gonorrhoeae infection), it will bind to the
filter which is then
visualized through a second antibody/detector. The dipstick assay involves
fixing an
antigen or antibody to a filter, which is then dipped in the biological fluid,
dried and
screened with a detector molecule.
Other diagnostic assays can employ the omp85 gene sequences or fragments of
this invention as nucleic acid probes or an anti-sense sequences, which can
identify the
presence of infection in the biological fluid by hybridizing to complementary
sequences
produced by the pathogen in the biological fluids. Such techniques, such as
PCR,
Northern or Southern hybridizations etc. are well known in the art. For this
purpose,
the nucleic acid sequences or fragments of this invention may be
conventionally
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labelled by well known techniques, possibly employing one or more of the
labels
described above with reference to use of the antibodies, or with labels more
suited for
attachment to nucleic acids. Selection of such labels is a routine matter and
does not
limit this invention.
It should be understood by one of skill in the art that any number of
conventional protein assay formats, particularly immunoassay formats, or
nucleic acid
assay formats, may be designed to utilize the isolated antigens and antibodies
or their
nucleic acid sequences or anti-sense sequences of this invention for the
detection of N.
gonorrhoeae or N. meningilidis infection (as well as infection with other
bacteria
characterized by antigens homologous to the 0mp85 antigens of this invention)
in
animals and humans. This invention is thus not limited by the selection of the
particular assay format, and is believed to encompass assay formats which are
known
to those of skill in the art.
VL Diagnostic Kits
For convenience, reagents for ELISA or other assays according to this
invention may be provided in the form of kits. Such kits are useful for
diagnosing
bacterial infection in a human or an animal sample. Such a diagnostic kit
contains an
antigen of this invention and/or at least one antibody capable of binding an
antigen of
this invention, or the nucleic acid sequences encoding such 0mp85 antigens or
antibodies or their anti-sense sequences. Alternatively, such kits may contain
a simple
mixture of such antigens or nucleic acid sequences, or means for preparing a
simple
mixture.
These kits can include microtiter plates to which the antigenic proteins or
antibodies or nucleic acid sequences of the invention have been pre-adsorbed,
various
diluents and buffers, labeled conjugates for the detection of specifically
bound antigens
or antibodies, or nucleic acids and other signal-generating reagents, such as
enzyme
substrates, cofactors and chromogens. Other components of these kits can
easily be
determined by one of skill in the art. Such components may include polyclonal
or
monoclonal capture antibodies, antigen of this invention, or a cocktail of two
or more
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of the antibodies, purified or semi-purified extracts of these antigens as
standards,
MAb detector antibodies, an anti-mouse or anti-human antibody with indicator
molecule conjugated thereto, an ELISA plate prepared for absorption, indicator
charts
for colorimetric comparisons, disposable gloves, decontamination instructions,

applicator sticks or containers, and a sample preparator cup. Such kits
provide a
convenient, efficient way for a clinical laboratory to diagnose N. gonorrhoeae
or N.
meningiditus infection
Vii. Therapeutic and Vaccine Compositions
Protein-Containing Therapeutic and Vaccine Compositions
The antigens and antibodies of the invention, alone or in combination
with other antigens and antibodies of, or directed to, other pathogenic
microorganisms
may further be used in therapeutic compositions and in methods for treating
humans
and/or animals with non-symptomatic infection or symptomatic disease caused by
N.
gonorrhoeae, N. meningitidis or the other pathogens identified above.
For example, one such therapeutic composition may be formulated to
contain a carrier or diluent and one or more of the anti-0mp85 antibodies of
the
invention. In compositions containing the 0mp85 antigen or antibodies thereto,
i.e.,
protein components, suitable pharmaceutically acceptable carriers may be
employed
that facilitate administration of the proteins but are physiologically inert
and/or
nonharmful.
A variety of such pharmaceutically acceptable protein carriers, and/or
components suitable for administration therewith may be selected by one of
skill in the
art. For example, pharmaceutical carriers include, without limitation, sterile
saline,
lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut
oil, olive oil,
sesame oil, and water. Additionally, the carrier or diluent may include a time
delay
material, such as glycerol monostearate or glycerol distearate alone or with a
wax. In
addition, slow release polymer formulations can be used. Liposomes or
liposomal-like
vehicles may also be employed.
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Optionally, these compositions may also contain conventional
pharmaceutical ingredients, such as preservatives, or chemical stabilizers.
Suitable
ingredients which may be used in a therapeutic composition in conjunction with
the
antibodies include, for example, casamino acids, sucrose, gelatin, phenol red,
N-Z
amine, monopotassium diphosphate, lactose, lactalbumin hydrolysate, and dried
milk.
Alternatively, or in addition to the antigens or antibodies of the
invention, other agents useful in treating the disease in question, e.g.,
antibiotics or
immunostimulatory agents and cytokine regulation elements, are expected to be
useful
in reducing or eliminating disease symptoms. Such agents may operate in
concert with
the therapeutic compositions of this invention. The development of therapeutic
compositions containing these agents is within the skill of one in the art in
view of the
teachings of this invention.
Additionally, the therapeutic compositions may be polyvalent. Such
compositions may contain therapeutic components from other bacterial or viral
pathogens, e.g., components of bacterial species homologous to Neiserriae or
heterologous thereto and/or antigens from a disease-causing virus. Depending
upon
the compatibility of the components, the 0mp85 antibodies or antigens of this
invention may thus be part of a multi-component therapeutic composition
directed at
more than a single disease.
As a further embodiment of this invention, a therapeutic method
involves treating a human or an animal for infection with N gonorrhoeae or N.
meningitidis by administering an effective amount of such a therapeutic
composition.
An "effective amount" of a proteinaceous composition may be between about 0.05
to
about 1000 g/ml of an antibody or antigen of the invention. A suitable dosage
may
be about 1.0 ml of such an effective amount. Such a composition may be
administered
1 - 3 times per day over a 1 day to 12 week period. However, suitable dosage
adjustments for protein or nucleic acid containing compositions may be made by
the
attending physician or veterinarian depending upon the age, sex, weight and
general
health of the human or animal patient. Preferably, such a composition is
administered
parenterally, preferably intramuscularly or subcutaneously. However, it may
also be
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formulated to be administered by any other suitable route, including orally or
topically.
The selection of the route of delivery and dosage of such therapeutic
compositions is
within the skill of the art.
In another embodiment, the 0mp85 antigens, antibodies, and fragments
of the invention, alone or in combination with other antigens, antibodies, and
fragments
from other microorganisms, may further be used in compositions directed to
induce a
protective immune response in a subject to the pathogen. These components of
the
present invention are also useful in methods for inducing a protective immune
response
in humans and/or animals against infection with N gonorrhoeae, N rneningitidis
or the
other pathogens identified above.
In one embodiment, an outer membrane protein antigen-based vaccine
for the prevention of non-symptomatic gonococcal infection or symptomatic
disease,
non-symptomatic meningococcal infection and symptomatic disease, and other
diseases
in humans and other animals is provided which contains an effective amount of
the
0mp85 antigen of this invention, and a pharmaceutically acceptable carrier or
diluent.
This vaccine composition may contain one or more of the isolated, recombinant,
modified or multimeric forms of the 0mp85 antigen of the invention, or
mixtures
thereof. Similarly, salts of the antigenic proteins may be employed in such
compositions.
In another embodiment of this invention, a polyvalent vaccine
compositions may include not only the 0mp85 antigen or immunogenic fragment
thereof, but may also include antigens from other disease-causing agents. Such
other
agents may be antigens from other Neisseriae strains. Such other agents may be

antigens from completely distinct bacterial pathogens or from viral pathogens.

Combinations of the antigen(s) of this invention with other antigens or
fragments
thereof are also encompassed by this invention for the purpose of inducing a
protective
immune response in the vaccinated subject to more than a single pathogen. The
selection of these other vaccine components is not a limitation of the present
invention,
and may be left to one of skill in the art.

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Such proteinaceous vaccines may include exemplary carriers as
described above for therapeutic compositions. Optionally, the vaccine
compositions
may optionally contain adjuvants, preservatives, chemical stabilizers, as well
as other
conventionally employed vaccine additives. Typically, stabilizers, adjuvants,
and
preservatives are optimized to determine the best formulation for efficacy in
the target
human or animal. Suitable exemplary preservatives include chlorobutanol,
potassium
sorbate, sorbic acid, sulfur dioxide, propyl gallade, the parabens, ethyl
vanillin,
glycerin, phenol, and parachlorophenol.
With regard to the adjuvant, one or more of the above described
vaccine components may be admixed or adsorbed with a conventional adjuvant.
The
adjuvant is used to attract leukocytes or enhance an immune response. Such
adjuvants
include, among others, RIBI adjuvant, mineral oil and water, aluminum
hydroxide,
AMPHIGEN*adjuvant, ADJUVAX adjuvant, AVRIDINeadjuvant, L121/squalene, D-
lactide-polylactide/glycoside, pluronic plyois, muramyl di peptide; killed
Bordetella,
and saponins, such as Quil A.
The invention thus also encompasses a prophylactic method entailing
administering to an animal or human an effective amount of such a composition.
The
protein antigenic compositions are administered in an "effective amount", that
is, an
amount of antigen that is effective in a route of administration to provide a
vaccinal
benefit, i.e., protective immunity. Suitable amounts of the antigen can be
determined
by one of skill in the art based upon the level of immune response desired. In
general,
however, the vaccine composition contains between 1 ng to 1000 mg antigen, and

more preferably, 0.05 pg to 1 mg per ml of antigen. Suitable doses of the
vaccine
composition of the invention can be readily determined by one of skill in the
art.
Generally, a suitable dose is between 0.1 to 5 ml of the vaccine composition.
Further,
depending upon the human patient or the animal species being treated, i.e. its
weight,
age, and general health, the dosage can also be determined readily by one of
skill in the
art.
In general, the vaccine can be administered once; and optionally
boosters can be administered periodically thereafter. The vaccine may be
administered
36
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by any suitable route. However, parenteral administration, particularly
intramuscular,
and subcutaneous, is the preferred route. Also preferred is the oral route of
administration. Routes of administration may be combined, if desired, or
adjusted.
In still another vaccine embodiment, the invention includes a
composition which delivers passive protection against infection by the
pathogen. For
this composition, the antibodies against the 0mp85 proteins disclosed herein
are useful
to provide to the subject a short-term, passive immune protection against
infection.
These passive immunity vaccine compositions may contain antibodies to other
pathogens and suitable vaccine additives as described above, e.g., adjuvants,
etc.
These compositions may be administered in dosages similar to those described
above
for the compositions which actively induce immune protection in the vaccinated
subject.
Nucleic Acid Containing Compositions
The nucleic acid sequences or anti-sense sequences of the invention,
alone or in combination with other nucleic acid sequences encoding antigens or
antibodies of, or directed to other pathogenic microorganisms may further be
used in
therapeutic compositions and in methods for treating humans and/or animals
with the
disease caused by infection with N. gonorrhoeae, N. meningitidis or the other
pathogens identified above. In another embodiment, the nucleic acid sequences
of this
invention, alone or in combination with nucleic acid sequences encoding other
antigens
or antibodies from other pathogenic microorganisms, may further be used in
compositions directed to actively induce a protective immune response in a
subject to
the pathogen. These components of the present invention are useful in methods
for
inducing a protective immune response in humans and/or animals against
infection with
N. gonorrhoeae, N. meningitidis or the other pathogens identified above.
For use in the preparation of the therapeutic or vaccine compositions,
nucleic acid delivery compositions and methods are useful, which are known to
those
of skill in the art. The omp85 sequences or fragments thereof (or anti-sense
sequences
as desired) may be employed in the methods of this invention or in the
compositions
described herein as DNA sequences, either administered as naked DNA, or
associated
37

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with a pharmaceutically acceptable carrier and provide for in vivo expression
of the
0mp85 protein or peptide or provide for production of anti-sense sequences
which can
bind to the 0mp85 protein in the subject due to infection. So-called 'naked
DNA' may
be used to express the Omp85 protein or peptide fragment (or anti-sense
sequences)
in vivo in a patient. [See, e.g., J. Cohen, Science, 259:1691-1692 (March 19,
1993);
E. Fynan et al, Proc. Natl. Acad. Sci., 90: 11478-11482 (Dec. 1993); J. A.
Wolff et al,
Biotechniques, 11:474-485 (1991) which describe similar uses of 'naked DNA'].
For example, "naked" omp85 DNA (or anti-sense
sequences) associated with regulatory sequences may be administered
therapeutically
or as part of the vaccine composition e.g., by injection.
Alternatively, omp85 DNA or anti-sense DNA may be administered as
part of a vector or as a cassette containing the Omp85-encoding DNA sequences
or
fragments or anti-sense sequences thereof operatively linked to a promoter
sequence
and other plasmid sequences. Briefly, the DNA encoding the 0mp85 protein (or
anti-
sense sequence) or desired fragment thereof may be inserted into a nucleic
acid
cassette. This cassette may be engineered to contain, in addition to the omp85
sequence to be expressed (or anti-sense sequence), other optional flanking
sequences
which enable its insertion into a vector. This cassette may then be inserted
into an
appropriate DNA vector downstream of a promoter, an mRNA leader sequence, an
initiation site and other regulatory sequences capable of directing the
replication and
expression of that sequence in vivo. This vector permits infection of
vaccinate's cells
and expression of the omp85 (or anti-sense sequence) in vivo.
Numerous types of appropriate vectors are known in the art for protein
expression and may be designed by standard molecular biology techniques. Such
vectors are selected from among conventional vector types including insects,
e.g.,
- 25 baculovirus expression, or yeast, fungal, bacterial or viral
expression systems.
Methods for obtaining such vectors are well-known. See, Sambrook et al,
Molecular
Cloning. A Laboratory Manual, 2d edition, Cold Spring Harbor Laboratory, New
York (1989); Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986)
and
references cited therein. Recombinant viral vectors, such as retroviruses or
38

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adenoviruses, are preferred for integrating the exogenous DNA into the
chromosome
of the cell.
Also where desired, the regulatory sequences in such a vector which
control and direct expression of the omp85 gene product or anti-sense sequence
in the
transfected cell include an inducible promoter. Inducible promoters are those
which
"turn on" expression of the gene when in the presence of an inducing agent.
Examples
of suitable inducible promoters include, without limitation, the sheep
metallothionine
(MT) promoter, the mouse mammary tumor virus (MMTV), the tet promoter, etc.
The inducing agents may be a glucocorticoid such as dexamethasone, for, e.g.,
the
MMTV promoter, or a metal, e.g., zinc, for the MT promoter; or an antibiotic,
such as
tetracycline for tet promoter. Still other inducible promoters may be selected
by one of
skill in the art, such as those identified in International patent application
W095/13392, published May 18, 1995. The identity of the inducible promoter is
not
a limitation of this invention.
When omp85 nucleic acid sequences or anti-sense sequences are
employed as the therapeutic agent or vaccine agent as 'naked DNA' operatively
linked
to a selected promoter sequence, rather than the protein itself, the amounts
of DNA to
be delivered and the routes of delivery may parallel the protein amounts for
vaccine or
therapeutic delivery described above and may also be determined readily by one
of skill
in the art.
Thus, as one preferred example, a therapeutic composition may be
formulated to contain a carrier or diluent and one or more plasmid or DNA
molecule
or recombinant virus containing a nucleic acid sequence which is anti-sense to
the
omp85 gene sequence [SEQ ID NO: 1 or 3], a fragment thereof, under control of
suitable sequences regulating the expression thereof. In compositions
containing the
anti-sense omp85 nucleic acid sequences, vehicles suitable for delivery of DNA
may be
employed.
Additionally, these therapeutic compositions may be polyvalent. Such
compositions may contain therapeutic components which are anti-sense sequences
of
other bacterial or viral origin, e.g., the anti-sense sequence of components
of bacterial
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species homologous to Neiserriae or heterologous thereto and/or anti-sense
sequence
derived from antigens from a disease-causing virus. Depending upon the
compatibility
of the components, the anti-sense sequences to the 0mp85 antigens of this
invention
may thus be part of a multi-component therapeutic composition directed at more
than
a single disease.
As a further embodiment of this invention, a therapeutic method
involves treating a human or an animal for infection with N. gonorrhoeae or N.

meningitidis by administering an effective amount of such a therapeutic
nucleic-acid
containing composition. An "effective amount" of a nucleic acid composition
may be
calculated as that amount capable of expressing in vivo the above effective
amounts of
exogenously delivered proteins. Such amounts may be determined by one of skill
in
the art. Preferably, such a composition is administered parenterally,
preferably
intramuscularly or subcutaneously. However, it may also be formulated to be
administered by any other suitable route, including orally or topically. The
selection of
the route of delivery and dosage of such therapeutic compositions is within
the skill of
the art.
As another example, a vaccine composition of this invention may be a
DNA vaccine, which includes the omp85 DNA sequence [SEQ ID NOS: 1 or 31 or a
fragment thereof which encodes an immunogenic protein or peptide, optionally
under
the control of regulatory sequences. In one embodiment, this vaccine
composition may
contain a nucleic acid sequence that encodes one or more of the isolated,
recombinant,
modified or multimeric forms of the 0mp85 antigen of the invention, or
mixtures
thereof
In another embodiment of this invention, polyvalent vaccine
compositions may include not only the nucleic acid sequence encoding the 0mp85
antigen or an immunogenic fragment thereof, but may also include nucleic acid
sequences encoding antigens from other disease-causing agents. Such other
agents
may be antigens from other Neisseriae strains. Such other agents may be
nucleic acid
sequences encoding antigens from completely distinct bacterial pathogens or
from
viral pathogens. Combinations of the antigen-encoding sequences of this
invention with

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other nucleic acid sequences encoding antigens or fragments thereof from other

pathogens are also encompassed by this invention for the purpose of inducing a

protective immune response in the vaccinated subject to more than a single
pathogen.
The selection of these other vaccine components is not a limitation of the
present
invention, and may be left to one of skill in the art.
Such "DNA" or nucleic acid vaccines may include exemplary carriers
as described above for therapeutic compositions and, where suitable, the
components
or additives described above with reference to the proteinaceous compositions,
e.g.
adjuvants, preservatives, chemical stabilizers, etc. as well as other
conventionally
employed vaccine additives. Additives suitable for use in nucleic acid
compositions are
known to those of skill in the art, including certain lipids and liposomes,
among other
known components.
Generally, a suitable nucleic acid-based treatment contains between
1x10-3 plaque forming unit (pfu) to 1x1012 pfu per dose, if a virus is the
delivery
vector. Otherwise, the dosage is adjusted to provide the same amount of
expressed
protein as is provided by the protein vaccines. However, the dose, timing and
mode of
administration of these compositions may be determined by one of skill in the
art.
Such factors as the age, and physical condition of the vaccinate may be taken
into
account in determining the dose, timing and mode of administration of the
immunogenic or vaccine composition of the invention.
VIII Drug Screening and Development
The proteins, antibodies and polynucleotide sequences of the present invention

may also be used in the screening and development of chemical compounds or
proteins
which have utility as therapeutic drugs or vaccines for the treatment or
diagnosis or
prevention of diseases caused by infection with N. gonorrhoeae or N.
meningitidis,
and possibly for the other microorganisms having homologous proteins to 0mp85.
As
one example, a compound capable of binding to 0mp85 and preventing its
biological
activity may be a useful drug component for the treatment or prevention of
such non-
symptomatic gonococcal infection or symptomatic diseases as gonorrhea and non-
41

CA 02347849 2008-05-30
symptomatic meningococcal infection and symptomatic disease, e.g., spinal
meningitis,
among others. The methods described herein may also be applied to fragments of

Omp85.
Suitable assay methods may be readily determined by one of skill in the art.
Where desired, and depending on the assay selected, the selected antigen(s),
e.g.,
0mp85 or fragment thereof, may be immobilized directly or indirectly (e.g.,
via an
Omp85 antibody) on a suitable surface, e.g., in an ELISA format. Such
immobilization surfaces are well known. For example, a wettable inert bead may
be
used. Alternatively, the selected antigen, e.g., Omp85, may be used in
screening assays
which do not require immobilization, e.g., in the screening of combinatorial
libraries.
Assays and techniques exist for the screening and development of drugs capable
of
binding to an antigen of this invention, e.g., Omp85. These include the use of
phage
display system for expressing the antigenic protein(s), and using a culture of

transfected E. coil or other microorganism to produce the proteins for binding
studies
of potential binding compounds. See, for example, the techniques described in
G.
Cesarini, FEBS Letters, 307(1):66-70 (July 1992); H. Gram et al. , J. Immunol.
Meth.,
161:169-176 (1993); C. Summer etal., Proc. Natl. Acad. Sci,, USA, H:3756-3760
(May 1992).
Other conventional drug screening techniques may be employed using the
proteins, antibodies or polynucleotide sequences of this invention. As one
example, a
method for identifying compounds which specifically bind to a protein of this
invention, e.g., Omp85, can include simply the steps of contacting a selected
0mp85
protein with a test compound to permit binding of the test compound to 0mp85;
and
determining the amount of test compound, if any, which is bound to the 0mp85
protein. Such a method may involve the incubation of the test compound and the
0mp85 protein immobilized on a solid support. Similar methods may be employed
for
one or more of the cassette string proteins.
Typically, the surface containing the immobilized ligand is permitted to come
into contact with a solution containing the protein and binding is measured
using an
appropriate detection system. Suitable detection systems include the
streptavidin horse
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radish peroxidase conjugate, direct conjugation by a tag, e.g., fluorescein.
Other
systems are well known to those of skill in the art. This invention is not
limited by the
detection system used.
Another method of identifying compounds which specifically bind to Omp85 or
another protein of this invention can include the steps of contacting the
protein, e.g.,
0mp85, immobilized on a solid support with both a test compound and the
protein
sequence which is a receptor for 0mp85 to permit binding of the receptor to
the
0mp85 protein; and determining the amount of the receptor which is bound to
the
0mp85 protein. The inhibition of binding of the normal protein by the test
compound
thereby indicates binding of the test compound to the 0mp85 protein.
Still other conventional methods of drug screening can involve employing a
suitable computer program to determine compounds having similar or
complementary
chemical structures to that of the Omp85 proteins [SEQ ID NOS: 2 and 4], and
screening those compounds for competitive binding to 0mp85. Such programs
include the GRID program available from Oxford University, Oxford, UK. [P. J.
Goodford, "A Computational Procedure for Determining Energetically Favorable
Binding Sites on Biologically Important Macromolecules", J. Med. Chem., 21:849-

857 (1985)]; the MCSS program available from Molecular Simulations,
Burlington,
MA [A. Miranker and M. Karplus, "Functionality Maps of Binding Sites: A
Multiple
Copy Simultaneous Search Method", Proteins: Structure, Function and Genetics,
11:29-34 (1991)]; the AUTODOCK program available from Scripps Research
Institute, La Jolla, CA [D S. Goodsell and A. J. Olsen, "Automated Docking of
Substrates to Proteins by Simulated Annealing", Proteins: Structure. Function,
and
Genetics, a:195-202 (1990)]; and the DOCK program available from University of

California, San Francisco, CA [1. D. Kuntz et al, "A Geometric Approach to
Macromolecule-Ligand Interactions", J. Mol, Biol., 161:269-288 (1982)].
Additional
commercially available computer databases for small molecular compounds
include
Cambridge Structural Database, Fine Chemical Database. and CONCORD database
[for a review see Rusinko, A., Chem. Des. Auto. News, 8:44-47 (1993)].
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Thus, the invention provides a method of identifying a pharmacomimetic of
0mp85 of N. gonorrhoeae or N. meningifidis by using a combination of steps
including identifying a compound which binds to 0mp85 by screening said 0mp85
against a battery of compounds. Computer modelling of the three dimensional
structure of 0mp85 or of the previously identified binding compound permits
the
identification of a compound with the same three dimensional structure as
either
0mp85 or its binding compound. The compound selected from these tests is then
screened for the biological activity of 0mp85 or of a compound that binds
0mp85,
such as the development of antisera effective in the assay of Example 8 or
competitive
effect in that assay, respectively.
Thus, through use of such methods, the present invention is anticipated to
provide compounds capable of interacting with 0mp85 or portions thereof, and
either
enhancing or decreasing its biological activity, as desired. Such compounds
are
believed to be encompassed by this invention.
The following examples are provided to illustrate the invention and do not
limit
the scope thereof. One skilled in the art will appreciate that although
specific reagents
and conditions are outlined in the following examples, modifications can be
made
which are meant to be encompassed by the spirit and scope of the invention.
EXAMPLE 1: Bacterial Strains. Cells. Methods and Culture Conditions
N. gonorrhoeae strains FA19, FA635, FA1090, JS1, F62 and MS11LosA used
in the examples below were provided by Dr. William M. Shafer (Emory
University,
Atlanta, GA) Dr. John Swanson (Rocky Mountain Laboratories, Hamilton, MT) or
the
American Type Culture Collection (Rockville, MD). N. meningitidis strains
Is/fP78,
MP3, MP81, and HR were provided by Dr. Mark S. Peppier (University of Alberta,
Edmonton, Alberta, Canada) and Dr. Zell McGee (University of Utah, Salt Lake
City,
UT). All other strains were acquired from the American Type Culture
Collection.
Moraxella catarrhalis (ATCC# 8193) and Neisserial strains were grown on
clear gonococcal typing media [Swanson J., Infect. Immun., 19 320-331 (1978)].
E.
coil and all other Gram negative strains were grown an Luria broth.
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E. coil XL1-Blue and SOLR cells were obtained from Stratagene Cloning
Systems (La Jolla, CA). DNA fragments were purified from agarose gels using
Gene
Clean II (Bio101, La Jolla, CA). Plasmids were purified using the QIAprep
quick spin
kit (Qiagen, Chatsworth, CA). Immunoblotting was done with Millipore (Bedford,

MA) Immobilon PVDF. Unless specified otherwise in the examples below, all
reagents
were obtained from Sigma Chemical Co. (St. Louis, MO).
EXAMPLE 2: Cloning and Immunological Screening of a Gonococcal Genomic
Library
A genomic DNA library was produced by purifying N. gonorrhoeae strain
FA19 genomic DNA and partially digesting the DNA into fragments with the
restriction endonuclease Tsp509 [New England Biolabs]. These DNA fragments
were
ligated with T4 DNA ligase [New England Biolabs] into the EcoRI restriction
site of
the lambda Zap II bacteriophage vector [Stratagene Cloning Systems, La Jolla,
CA].
The ligated phage DNA was packaged, and plated on E. coil DH5a [Gibco BRL,
Gaithersburg, MD] host cells The resulting library was screened
immunologically for
the expression of gonococcal surface proteins according to protocols provided
with
the Lambda ZapII vector system. The plaques from the library were screened
with
anti-GC-OM, an antiserum raised to isolated gonococcal outer membranes.
Plasmid DNA was rescued from phage producing immunoreactive plaques.
The plasmid, pDR4, contained a 2.6 kbp gonococcal DNA insert. The gonococcal
DNA in pDR4 was subcloned into pUP1 [Elkins C., J. Bacteriol.,173: 3911-3913
(1991), generously provided by Dr. Chris Elkins (University of North Carolina,
Chapel
Hill, NC)] yielding pOmp85.
The cloned gonococcal DNA fragment in pOmp85 was characterized by
restriction enzyme analysis. The fragment contained three internal Hind!
restriction
sites which allowed the subcloning of three fragments into pBluescript
[Stratagene
Cloning Systems, La Jolla, CA]. These fragments and pDR4 were sequenced by the

University of Montana Molecular Biology Facility. A gene-walking strategy was
used
to sequence those regions which could not be sequenced with universal vector
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The entire length of the gonococcal DNA fragment was sequenced at least twice
and
most of the DNA was sequenced from both strands. The deduced amino acid
sequence
and comparisons of 0mp85 homologs were obtained with the MacVector software
package (Eastman Chemical Co., New Haven, CT).
The 2.6 kb gonococcal DNA in pOmp85 was found to contain a large open
reading frame (ORF) which encoded a polypeptide of 792 amino acids with a
predicted
molecular weight of 87.8 IcDa (Fig. 2) [SEQ ID NO:2]. The polypeptide
contained a
putative signal peptide [Von Heijne G., Nuc. Acids Res., 14: 4683-4690
(1986)].
Removal of this signal peptide yielded a mature polypeptide with an 85.8 IcDa
predicted molecular weight. The polypeptide also possesses a carboxyl-terminal
EXAMPLE 3: Cloning and Sequencing of Meningoco_ccal omp85
The meningococcal omp85 was obtained by PCR amplification using the
Boehringer Mannheim (Indianapolis, IN) Expand High Fidelity PCR System. The
46

CA 02347849 2008-05-30
meningococcal omp85 was obtained essentially as described for the gonococcal
omp85.
The meningococcal omp85 was found to encode a 797 amino acid polypeptide
with a predicted molecular weight of 88.5 kDa (Fig. 5). The meningococcal
0mp85
was 95% identical and 98% similar to gonococcal Omp85. Between amino acid
residues 720 and 745, the menigococcal 0mp85 varied substantially from
gonococcal
0mp85, including the insertion of five additional amino acids.
EXAMPLE 4: Presence of 0mp85 in Strains of N. gonorrhoeae and N. meningitidis
=
A. Western Analysis
The Western blot analyses were performed as follows. Bacterial
proteins were separated by SDS-PAGE (12.5%) [Laemmli UK, Nature, 227: 680-695
(1970)]. The separated proteins were electrophoretically transferred onto PVDF
as
previously described [Judd RC., Anal. Biochem., l73: 307-316 (1988)]. Blotting
was
performed in 20m.M sodium phosphate buffer, pH 8.0 for 2 hr at 600 mA. The
PVDF
membrane was blocked for 1 hr with PBS Tween*at room temperature, incubated
overnight in anti-GC-OM or anti-0mp85 sera at 4 C, washed several times,
incubated
with protein A-horseradish peroxidase conjugate (Boehringer Mannheim,
Indianapolis,
IN) and developed with 4-chloro-1-naphthol.
Western blot analysis of the proteins from E. coil DH5a/pOmp85 were
performed by separating bacterial cell lysates by SDS-PAGE, staining with
Coomassie
Brilliant blue (CBB) or transferring the lysates to membranes and probing with
anti-GC-OM serum. The results revealed expression of an approximately 85 kDa
polypeptide that was reactive with the anti-GC-OM serum (Fig. 1).
Anti-GC-OM serum was reacted in Western blot analysis with total cell
=
-25 proteins from six representative strains of N. gonorrhoeae and four
strains of N.
meningitidis. Whole cell lysates of E. coli DH5a, E. coil DH5a/pOmp85, and N.
gonorrhoeae strains FA19, FA635, FA1090, JSI, F62 and MS11LosA and N.
meningitidis strains MP78, MP3, MP81 and 1111 were separated by 12.5%
SDS-PAGE, blotted and probed with the anti-GC-OM serum. An immunoreactive,
47
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-
approximately 85 lcDa protein band was detected in all strains of N.
gonorrhoeae and
N. meningitidis (Fig. 3), but not in the control E. coli DH5a.
B. Southern Analysis
The Southern analyses are performed as follows. Chromosomal DNA
was obtained by phenol extraction [Moore D., "Preparation and analysis of
DNA", in:
Ausubel FM, et al, eds. Current Protocols in Molecular Biology. New York: John
Wiley and Sons, (1997): 2.1.1-2.1.3] and then digested with various
endonucleases.
Digested DNA was electrophoretically separated on a 1% agarose gel and the DNA

transferred to nitrocellulose with the Bio-Rad Model 785 Vacuum Blotter
according to
instructions and Southern [Southern E., J. Mol. Biolõ 98: 503-510 (1975)].
Probe
DNA was extracted from agarose gels using Bio 101 glassmilk (Vista, CA). The
probe was labeled and the blot probed with the Amersham ECL system (Arlington
Heights, IL).
A Southern blot (Fig. 4) illustrated the identification of omp85 in N.
gonorrhoeae and N. meningitidis by Southern analysis. Genomic DNA from E. coli
DH5a, N gonorrhoeae FA19 and N meningitidis strains MP3, MP73, Iv1P81 and 11H
were digested with restriction endonucleases EcoRI, Ps/I, Cla1). Blots of
the
separated DNA digests were probed with a 688 bp fragment of gonococcal omp85
that
extended from the most 3' Hindi site of omp85 to a Hindi site in the vector
near the
3' end of the cloned gene. This fragment was used as a positive control.
In N gonorrhoeae strain FA 19 and in all of the N. meningitidis strains,
the omp85 probe hybridized with a single DNA band. A single band was also
identified in gonococcal strains FA635, FA1090, JS I, F62, and MS 11 (data not

shown). These results suggested that omp85 was conserved as a single copy in
pathogenic Neisseria. Sequence data indicated that Hinc11 cleaved omp85
internally at
three sites. The probe hybridized to a fragment larger than itself in the
Hindi digest of
gonococcal DNA (FA19 - Hindi). The genomic fragment resulted from an internal
and external Hindi cleavage; the probe lacked the flanking gonococcal sequence

containing the external lfincll site. The genomic HincII band that hybridized
to the
probe was probably derived from a single copy of the omp85 gene since it is
unlikely
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that duplicate copies of the gene would have identically-located Hindi sites,
generating fragments of identical size.
The enzyme Pstl cleaved 309 base pairs from the 5' end of omp85. The
single fragment ofPstI-digested gonococcal genomic DNA (FA19 - Ps/I) which
hybridized with the probe probably contained a single copy of the gene. It is
possible,
but unlikely, that the PstI fragments contained approximately 2 kbp segments
of
omp85 in inverted orientations at each end of the approximately 8 kbp
fragment.
Sequence data indicated that the enzymes Clal and EcoRI did not cleave within
omp85. The omp85 probe hybridized to single bands of similar size in the Clal
digested
DNA of both N gonorrhoeae and N meningnidis. These bands suggested a single
copy of omp85, since it is unlikely that bands this small (<6 kbp) contained
two copies
of the approximately 2.3 kbp gene and it is unlikely that the bands represent
fragments
of identical size from duplicate copies of the gene. Similar results were
obtained for
gonococcal strains FA635, FA1090, JS1, F62 and MS II (data not shown). These
results support the conclusion that omp85 is conserved as a single copy in
pathogenic
Neisseria.
EXAMPLE 5: Similarity to Known Proteins
The non-redundant Genbank CDS database was searched [Altchul SD. et al,
Mol. Biolõ 215: 403-407 (1990)] for proteins similar to gonococcal 0mp85. The
H.
influenzae D-15-Ag was 31.5% identical and 61.4% similar (identical plus
conserved)
to gonococcal 0mp85. The P. multocida 0ma87 was 31.6% identical and 61.3%
similar to 0mp85. Several hypothetical proteins with similarity to 0mp85 were
identified. A Brucella abortus hypothetical protein (Genbank accession
#1.151683,
Bearden, et al., unpublished) was 24.3% identical and 54.2% similar. A
hypothetical
E. coil protein (Genbank accession #U70214, Schramm, et al., unpublished) was
33%
identical and 62% similar to 0mp85. The gonococcal 0mp85 was also similar to
hypothetical proteins of Helicobacter pylori (Genbank assession # AE001178 -
[Tomb
JF. et al., Nature, 388: 539-547 (1997)]), 23% identical and 51% similar, and
Borreha
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burgdoileri (Genbank assession #AE001178 - [Fraser CM. et al, Nature, 390:
580-586 (1997)]), 18% identical and 46% similar.
EXAMPLE 6: Gene Organization and Flanking Genes
Several hundred base pairs of the DNA flanking the omp85 ORFs of both N
gonorrhoeae and N. meningitidis were sequenced. For both N. gonorrhoeae and N.
meningitidis, a gene similar to ompH of Salmonella Ophimurium [Kosk P. et al,L

Biol. Chem., 264:18973-18980 (1989)]was identified approximately 65 base pairs
3' of
the omp85 ORF. A gene similar to onipH has also been identified in the same
relative
position in H. influenzae [Fleischmann, RD et al, Science, 269: 496-512
(1995)] and
P. multocida. In H. influenzae, a gene encoding a hypothetical protein,
H10918, was
located 5' of the D-15-Ag gene [Fleischmann, RD eta!, Science, 269: 496-512
(1995)]. A gene homologous to the H10918 gene was identified at the same
relative
position in N gonorrhoeae. These results indicated that the gene arrangement
around
omp85 homologs was notably conserved.
EXAMPLE 7: Presence in Neisseriae and Other Species
Western blot analysis was used to determine if proteins similar to 0mp85 were
produced by commensal Neisseriae and by other Gram negative species. To allow
more specific immunological analysis, an 0mp85-specific polyvalent rabbit sera
was
produced through use of a fusion protein in which the first 200 amino acids of
the
gonococcal 0mp85 were genetically fused to maltose binding protein (MBP).
A. Production of a MBP/Omp85 fusion protein
Fragments of gonococcal 0mp85 were genetically fused to MBP,
affinity purified and used to produce 0mp85-specific antiserum. Tsp5091
digested
-25 omp85 from pOmp85 was ligated into the EcoRI digested, maltose binding
protein
fusion vector, pMAL-c2 [New England Biolabs, Beverly, MA]. Sequence analysis
had
revealed that this could result in the fusion of several different omp85
fragments in
frame with malE in pMAL-c2. The ligated DNA was transformed into E. coli DH5a
and the transformants were screened for the expression of 0mp85 antigens with
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anti-OM serum. A number of immunoreactive clones were identified and
characterized. The plasmid in the most immunoreactive of these was designated
pM04 and determined by sequence analysis to express a fusion of MBP with the
first
181 amino acids of 0mp85. The MBP/0mp85 fusion protein was affinity purified
from E. coli DH5a/pM04 as previously described [Marchion DC et al,Moi.
Microbiol., 231-240 (1997)].
B. Raising of Anti-sera
Purified MBP/Omp85 was used to raise an anti-0mp85 sera in New
Zealand white rabbits. The rabbits were initially immunized with 1 mg of
protein in
Freund's complete adjuvant administered subcutaneously. Two weeks later 1 mg
was
administered subcutaneously in Freund's incomplete adjuvant. The rabbits then
received intravenous injections of 0.1 mg every two weeks for eight weeks Sera
was
collected and absorbed (1:1) with a lysed culture of E. co/i/pMal-c2 induced
with 1
mM IPTG for 1 hour. The resulting rabbit serum was designated anti-0mp85.
C'. Western Analysis of representative strains of N. gonorrhoeae and N.
meningitidis
The anti-0mp85 serum was used to probe Western blots containing cell
lysates of E. coli DH5a, E. coli DH5a/pOmp85 and representative strains of N.
gonorrhoeae and N meningitidis (Fig. 6). Whole cell lysates of E. coil DH5a,
E. coli
DH5a/pOmp85, N. gonorrhoeae strains FA19, FA635, FA1090, JS I, F62 and
MS11LosA, N meningitidis strains NTP78, M1P3, MP81 and HH, and E. coil
DH5a/pMCOmp85 were separated by 12.5% SDS-PAGE, blotted and probed with
the anti-0mp85 serum.
The anti-0mp85 serum reacted with the recombinant 0mp85 produced
by E. coli 1)H5a/pOmp85 and with proteins of approximately 85 IcDa in all of
the N
- 25 gonorrhoeae and N. meningilidis strains tested. These results
indicated that 0mp85
was conserved in the pathogenic Neisseriae. Presorption of the antiserum
removed the
majority of non-specific antibodies, but complete removal of all reactive
antibody is not
possible, thus, some reactive bands can be seen in the E co/i lanes of Fig. 6.
Reactive
bands in the 351cDa-42k1)a range in N. gonorrhoeae lanes are Por proteins,
which non-
51

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specifically bind protein-A. Meningococcal Pors do not bind protein-A as
strongly
(data not shown).
D. Western Analysis of Commensal Nesseriae and Other Gram
Negative
Species
The anti-0mp85 was also used to probe Western blots containing cell
lysates of commensal Nesseriae and several other Gram negative species. Whole
cell
lysates of E. coil DH5a, E. coil DH5a/pOmp85, N. gonorrhoeae FA19 (A),
Neisseria
pharyngis (A), Neisseria cinerea (A), Neisseria lactamica (B), Neisseria
nnicosae
(B), Neisseria flavescens (C), Neisseria animalis (C), Neisseria denitrificans
(C),
Moraxella catarrhalis (D), Klebsiella pneumoniae, Pseudomonas aeruginosa, and
N.
meningitidis WI (A) were separated in a 12.5% SDSaPAGE gel, blotted and probed
with anti-0mp85. In a separate experiment, whole cell lysates of E. coil DH5a,
E. coil
DH5a1pOmp85, N. gonorrhoeae FA19, Salmonella typhimurium, Shigella flexneri,
E.
coil strains 35150 (enterohemorrhagic - EHEC), 35401 (enterotoxigenic - ETEC),

43887 (enteropathogenic - EPEC), 43892 (enteroinvasive - EIEC) and N
meningitidis
were separated in a 12.5% SDS-PAGE gel, blotted and probed with anti-0mp85.
Two SDS-PAGE (Figs. 7A and 7B) illustrate the distribution of 0mp85
in pathogenic and commensal Neisseria (relationship areas A, B, C, and D) and
related
Gram negative bacteria Omp85 homologs were identified in all of the commensal
Neisserial species tested. This suggested that 0mp85 was conserved among all
the
Neisserial species. The anti-0mp85 serum failed to identify any 0mp85 homologs
in
Moraxella catarrhalis which is closely related to the Neisseriae [Rossau R. et
al, Jnt.
J. Syst. Bacteria, 19: 185-198 (1989)1. Southern analysis confirmed the
absence of an
omp85 homolog in this species (data not shown). The serum failed to identify
any
0mp85 homologs in Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella
typhimurium, Shigella flexneri and four pathogenic strains of E. coh tested.
EXAMPLE 8: Gonococcal Cell Adherence Assay
Gonococcal cell adherence assays were performed to evaluate the effect of
antiserum specific for outer membrane protein 85 (0mp85) on the ability of
52

CA 02347849 2009-02-26
gonococcal strains MSI1LOSA (MS11) and FA.19 to bind to Chang epithelial
cells.
Fab fragments were prepared from antiserum to the first 178 amino acids of
Omp85
[SEQ ID NO: 2], hyperimmune antiserum to bovine serum albumin (BSA) and normal

rabbit serum (NRS) and added at 1 pg, 10 gs or 100 ggs per ml to wells
containing a
confluent layer of Chang conjunctiva cells. Approximately 2.5 x 105 bacteria
(N.
gonorrhoeae strain MS11 or FA19) were added to each well and allowed to adhere
for
3 hours. Following fixation and immunogold/silver staining, the number of
adherent
gonococci was determined for 22 cells. The lowest and highest numbers were
discarded and the average number of bacteria/cell were determined. The
resulting data
is reported in the bar graph of Fig. 8, indicating that 0mp85-specific
antibody was able
to bind to the surface of the bacteria and interfere with the ability of the
bacteria to
adhere to the Chang epithelial cells.
53
A ik..4rAir)Fn SHEET

CA 02347849 2001-04-18
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SEQUENCE LISTING
<110> Judd, Ralph C.
Manning, Scott D.
The University of Montana
<120> Omp85 proteins of Neisseria gonorrhoeae and Neisseria
meningitidis, Compositions Containing Same and Methods
of Use Thereof
<130> UM/SBC147PCT
<140>
<141>
<160> 8
<170> PatentIn Ver. 2.0
<210> 1
<211> 2379
<212> DNA
<213> Neisseria gonorrhoeae
<220>
<221> CDS
<222> (1)..(2376)
<400> 1
atg aaa ctg aaa cag att gcc tcc gca ctg atg atg ttg ggc ata tcg 48
Met Lys Leu Lys Gin Ile Ala Ser Ala Leu Met Met Leu Gly Ile Ser
1 5 10 15
cct ttg gca ttt gcc gac ttc acc atc caa gac atc cgt gtc gaa ggc 96
Pro Leu Ala Phe Ala Asp Phe Thr Ile Gin Asp Ile Arg Val Glu Gly
20 25 30
ttg cag cgt acc gag ccg agc acc gta ttc aac tac ctg ccc gtc aaa 144
Leu Gin Arg Thr Glu Pro Ser Thr Val Phe Asn Tyr Leu Pro Val Lys
35 40 45
gtc ggc gac acc tac aac gac aca cac ggc agt gcc atc atc aaa agc 192
Val Gly Asp Thr Tyr Asn Asp Thr His Gly Ser Ala Ile Ile Lys Ser
50 55 60
ctg tac gcc acc ggt ttc ttt gac gac gta cga gtc gaa act gcg gac 240
Leu Tyr Ala Thr Gly Phe Phe Asp Asp Val Arg Val Glu Thr Ala Asp
65 70 75 80
1

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ggg ctg ctt ctg ctg acc gtt atc gta tgc cct acc atc ggc tcg ctc 288
Gly Leu Leu Leu Leu Thr Val Ile Val Cys Pro Thr Ile Gly Ser Leu
85 90 95
aac atc acc ggc gcc aaa atg ctg cag aac gac gcc atc aag aaa aac 336
Asn Ile Thr Gly Ala Lys Met Leu Gin Asn Asp Ala Ile Lys Lys Asn
100 105 110
ctc gaa tcg ttc ggg ctg gcg cag tcg caa tac ttt aat cag gcg aca 384
Leu Glu Ser Phe Gly Leu Ala Gin Ser Gin Tyr Phe Asn Gin Ala Thr
115 120 125
ctc aac cag gca gtc gcc ggc ctg aaa gaa gaa tat ctc ggg cgc ggc 432
Leu Asn Gin Ala Val Ala Gly Leu Lys Glu Glu Tyr Leu Gly Arg Gly
130 135 140
aaa ctc aat atc caa atc acg ccc aaa gta acc aaa ctc gcc cgc aac 480
Lys Leu Asn Ile Gin Ile Thr Pro Lys Val Thr Lys Leu Ala Arg Asn
145 150 155 160
cgc gtc gac ate gac atc acg att gac gag ggc aaa tcc gcc aaa atc 528
Arg Val Asp Ile Asp Ile Thr Ile Asp Glu Gly Lys Ser Ala Lys Ile
165 170 175
acc gac atc gaa ttt gaa ggc aac caa gtc tat tcc gac cgc aaa ctg 576
Thr Asp Ile Glu Phe Glu Gly Asn Gin Val Tyr Ser Asp Arg Lys Leu
180 185 190
atg egg cag atg tcg ctg acc gaa ggc ggc att tgg aca tgg ctg aca 624
Met Arg Gin Met Ser Leu Thr Glu Gly Gly Ile Trp Thr Trp Leu Thr
195 200 205
cga age gac egg ttc gac cgc cag aaa ttc gcc caa gac atg gaa aaa 672
Arg Ser Asp Arg Phe Asp Arg Gin Lys Phe Ala Gin Asp Met Glu Lys
210 215 220
gta acc gac ttc tac cag aac aac ggc tac ttc gat ttc cgt atc ctc 720
Val Thr Asp Phe Tyr Gin Asn Asn Gly Tyr Phe Asp Phe Arg Ile Leu
225 230 235 240
gat acc gac atc caa acc aac gaa gac aaa acc agg cag acc atc aaa 768
Asp Thr Asp Ile Gin Thr Asn Glu Asp Lys Thr Arg Gin Thr Ile Lys
245 250 255
atc acc gtc cac gaa ggc gga cgt ttc cgc tgg ggc aaa gtg tcg att 816
Ile Thr Val His Glu Gly Gly Arg Phe Arg Trp Gly Lys Val Ser Ile
260 265 270
2

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gaa ggc gac acc aac gaa gtc ccc aag gcc gaa ctg gaa aaa ctg ctg 864
Glu Gly Asp Thr Asn Glu Val Pro Lys Ala Glu Leu Glu Lys Leu Leu
275 280 285
acc atg aag ccc ggc aaa tgg tac gaa cgc cag cag atg acc gcc gtt 912
Thr Met Lys Pro Gly Lys Trp Tyr Glu Arg Gin Gin Met Thr Ala Val
290 295 300
ttg ggt gag att cag aac cgc atg ggc tcg gca ggc tac gca tac agc 960
Leu Gly Glu Ile Gin Asn Arg Met Gly Ser Ala Gly Tyr Ala Tyr Ser
305 310 315 320
gaa atc agc gta cag ccg ctg ccg aac gcc gga acc aaa acc gtc gat 1008
Glu Ile Ser Val Gin Pro Leu Pro Asn Ala Gly Thr Lys Thr Val Asp
325 330 335
ttc gtc ctg cac atc gaa ccg ggc aga aaa atc tac gtc aac gaa atc 1056
Phe Val Leu His Ile Glu Pro Gly Arg Lys Ile Tyr Val Asn Glu Ile
340 345 350
cac atc acc ggc aac aac aaa acc cgc gac gaa gtc gtg cgc cgc gaa 1104
His Ile Thr Gly Asn Asn Lys Thr Arg Asp Glu Val Val Arg Arg Glu
355 360 365 '
ttg cgc caa atg gaa tcc gcg cct tac gac acc tcc aag ctg caa cgc 1152
Leu Arg Gln Met Glu Ser Ala Pro Tyr Asp Thr Set Lys Leu Gin Arg
370 375 380
tcc aaa gag cgc gtc gag ctt ttg ggc tac ttc gac aac gta cag ttt 1200
Ser Lys Glu Arg Val Glu Leu Leu Gly Tyr Phe Asp Asn Val Gin Phe
385 390 395 400
gat gcc gtc ccg ctt gcc ggt acg ccc gac aaa gtc gat ttg aac atg 1248
Asp Ala Val Pro Leu Ala Gly Thr Pro Asp Lys Val Asp Leu Asn Met
405 410 415
agc ctg acc gaa cgt tcc acc ggc tcg ctc gac ttg agc gcg ggc tgg 1296
Ser Leu Thr Glu Arg Ser Thr Gly Ser Leu Asp Leu Ser Ala Gly Trp
420 425 430
gtt cag gat acc ggc ttg gtc atg tcc gcc ggc gta tcg cag gac aac 1344
Val Gin Asp Thr Gly Leu Val Met Ser Ala Gly Val Ser Gin Asp Asn
435 440 445
ctg ttc ggt acg ggc aag tcg gcc gcc ctg cgc gcc tcg cga agc aaa 1392
Leu Phe Gly Thr Gly Lys Ser Ala Ala Leu Arg Ala Ser Arg Ser Lys
450 455 460
3

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acc acg ctc aac ggc tcg ctg tcg ttt acc gac ccg tac ttc acg gca 1440
Thr Thr Leu Asn Gly Ser Leu Ser Phe Thr Asp Pro Tyr Phe Thr Ala
465 470 475 480
gac ggg gtc agc ctg ggc tac gat att tac gga aaa gcc ttc gac ccg 1488
Asp Gly Val Ser Leu Gly Tyr Asp Ile Tyr Gly Lys Ala Phe Asp Pro
485 490 495
cgc aaa gca tcg acc age gtc aaa caa tat aaa acc acc acc gcc ggc 1536
Arg Lys Ala Ser Thr Ser Val Lys Gin Tyr Lys Thr Thr Thr Ala Gly
500 505 510
ggc ggc gta agg atg ggt atc ccc gtt acc gaa tac gac cgc gtc aat 1584
Gly Gly Val Arg Met Gly Ile Pro Val Thr Glu Tyr Asp Arg Val Asn
515 520 525
ttc ggg ctg gcg gcg gaa cac ctg ace gtc aac acc tac aac aaa gca 1632
Phe Gly Leu Ala Ala Giu His Leu Thr Val Asn Thr Tyr Asn Lys Ala
530 535 540
ccc aaa cgc tat gcc gac ttt atc aaa caa tac ggc aaa acc gac ggc 1680
Pro Lys Arg Tyr Ala Asp Phe Ile Lys Gin Tyr Gly Lys Thr Asp Gly
545 550 555 560
gca gac ggc agc ttc aaa ggc ctg ctg tac aaa ggc act gtc ggc tgg 1728
Ala Asp Gly Ser Phe Lys Gly Leu Leu Tyr Lys Gly Thr Val Gly Trp
565 570 575
ggg cgc aac aag acc gac agc gcc tta tgg ccg acg cgc ggc tac ctg 1776
Gly Arg Asn Lys Thr Asp Ser Ala Leu Trp Pro Thr Arg Gly Tyr Leu
580 585 590
acc ggc gta aat gcc gaa atc gcc ctg ccc ggc agc aaa ctg caa tac 1824
Thr Gly Val Asn Ala Glu Ile Ala Leu Pro Gly Ser Lys Leu Gln Tyr
595 600 605
tac tee gcc ace cac aac caa acc tgg ttc ttc ccc tta agc aaa acc 1872
Tyr Ser Aia Thr His Asn Gin Thr Trp Phe Phe Pro Leu Ser Lys Thr
610 615 620
ttc acg ctg atg ctc ggc ggc gaa gtc ggc att gcg ggc ggc tac ggc 1920
Phe Thr Leu Met Leu Gly Gly Glu Val Gly Ile Ala Gly Gly Tyr Gly
625 630 635 640
aga acc aaa gaa atc ccc ttc ttt gaa aac ttc tac ggc ggc ggc ctg 1968
Arg Thr Lys Glu Ile Pro Phe Phe Glu Asn Phe Tyr Gly Gly Gly Leu
645 650 655
4

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ggt tcg gtg cgc ggc tac gaa agc ggc acg ctc ggc ccg aaa gtg tat 2016
Gly Ser Val Arg Gly Tyr Glu Ser Gly Thr Leu Gly Pro Lys Val Tyr
660 665 670
gac gaa tac ggc gaa aaa atc agc tac ggc ggc aac aaa aaa gcc aac 2064
Asp Glu Tyr Gly Glu Lys Ile Ser Tyr Gly Gly Asn Lys Lys Ala Asn
675 680 685
gtc tcc gcc gag ctg ctc ttc ccg atg ccc ggt gcg aaa gac gca cgc 2112
Val Ser Ala Glu Leu Leu Phe Pro Met Pro Gly Ala Lys Asp Ala Arg
690 695 700
acc gtc cgc ctg agc ctg ttt gcc gac gca ggc agc gtg tgg gac ggc 2160
Thr Val Arg Leu Ser Leu Phe Ala Asp Ala Gly Ser Val Trp Asp Gly
705 710 715 720
aga acc tat acc gcc gcc gaa aac ggt aac aac aaa tcg gtt tac tcg 2208
Arg Thr Tyr Thr Ala Ala Glu Asn Gly Asn Asn Lys Ser Val Tyr Ser
725 730 735
gaa aac gcg cat aaa tcc acc ttt acc aac gaa ttg cgc tat tcc gcc 2256
Glu Asn Ala His Lys Ser Thr Phe Thr Asn Glu Leu Arg Tyr Ser Ala
740 745 750
ggc ggc gcg gtt acc tgg ctc tcg cct ttg ggc ccg atg aaa ttc atc 2304
Gly Gly Ala Val Thr Trp Leu Ser Pro Leu Gly Pro Met Lys Phe Ile
755 760 765
tac gcc tac ccg ctg aag aaa aaa ccg gaa gac gaa atc caa cgc ttc 2352
Tyr Ala Tyr Pro Leu Lys Lys Lys Pro Glu Asp Glu Ile Gin Arg Phe
770 775 780
caa ttc cag ctc ggc acg acg ttc taa 2379
Gin Phe Gin Leu Gly Thr Thr Phe
765 790
<210> 2
<211> 792
<212> PRT
<213> Neisseria gonorrhoeae
<400> 2
Met Lys Leu Lys Gin Ile Ala Ser Ala Leu Met Met Leu Gly Ile Ser
1 5 10 15
Pro Leu Ala Phe Ala Asp Phe Thr Ile Gin Asp Ile Arg Val Glu Gly

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20 25 30
Leu Gin Arg Thr Glu Pro Ser Thr Val Phe Asn Tyr Leu Pro Val Lys
35 40 45
Val Gly Asp Thr Tyr Asn Asp Thr His Gly Ser Ala Ile Ile Lys Ser
50 55 60
Leu Tyr Ala Thr Gly Phe Phe Asp Asp Val Arg Val Glu Thr Ala Asp
65 70 75 80
Gly Leu Leu Leu Leu Thr Val Ile Val Cys Pro Thr Ile Gly Ser Leu
85 90 95
Asn Ile Thr Gly Ala Lys Met Leu Gin Asn Asp Ala Ile Lys Lys Asn
100 105 110
Leu Glu Ser Phe Gly Leu Ala Gin Ser Gin Tyr Phe Asn Gin Ala Thr
115 120 125
Leu Asn Gin Ala Val Ala Gly Leu Lys Glu Glu Tyr Leu Gly Arg Gly
130 135 140
Lys Leu Asn Ile Gin Ile Thr Pro Lys Val Thr Lys Leu Ala Arg Asn
145 150 155 160
Arg Val Asp Ile Asp Ile Thr Ile Asp Glu Gly Lys Ser Ala Lys Ile
165 170 175
Thr Asp Ile Glu Phe Glu Gly Asn Gin Val Tyr Ser Asp Arg Lys Leu
180 185 190
Met Arg Gin Met Ser Leu Thr Glu Gly Gly Ile Trp Thr Trp Leu Thr
195 200 205
Arg Ser Asp Arg Phe Asp Arg Gin Lys Phe Ala Gin Asp Met Glu Lys
210 215 220
Val Thr Asp Phe Tyr Gin Asn Asn Gly Tyr Phe Asp Phe Arg Ile Leu
225 230 235 240
Asp Thr Asp Ile Gin Thr Asn Glu Asp Lys Thr Arg Gin Thr Ile Lys
245 250 255
Ile Thr Val His Glu Gly Gly Arg Phe Arg Trp Gly Lys Val Ser Ile
260 265 270
Glu Gly Asp Thr Asn Glu Val Pro Lys Ala Glu Leu Glu Lys Leu Leu
6

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275 280 285
Thr Met Lys Pro Gly Lys Trp Tyr Glu Arg Gin Gin Met Thr Ala Val
290 295 300
Leu Gly Glu Ile Gin Asn Arg Met Gly Ser Ala Gly Tyr Ala Tyr Ser
305 310 315 320
Glu Ile Ser Val Gin Pro Leu Pro Asn Ala Gly Thr Lys Thr Val Asp
325 330 335
Phe Val Leu His Ile Glu Pro Gly Arg Lys Ile Tyr Val Asn Glu Ile
340 345 350
His Ile Thr Gly Asn Asn Lys Thr Arg Asp Glu Val Val Arg Arg Glu
355 360 365
Leu Arg Gin Met Glu Ser. Ala Pro Tyr Asp Thr Ser Lys Leu Gin Arg
370 375 380
Ser Lys Glu Arg Val Glu Leu Leu Gly Tyr Phe Asp Asn Val Gin Phe
385 390 395 400
Asp Ala Val Pro Leu Ala Gly Thr Pro Asp Lys Val Asp Leu Asn Met
405 410 415
Ser Leu Thr Glu Arg Ser Thr Gly Ser Leu Asp Leu Ser Ala Gly Trp
420 425 430
Val Gin Asp Thr Gly Leu Val Met Ser Ala Gly Val Ser Gin Asp Asn
435 440 445
Leu Phe Gly Thr Gly Lys Ser Ala Ala Leu Arg Ala Ser Arg Ser Lys
450 455 460
Thr Thr Leu Asn Gly Ser Leu Ser Phe Thr Asp Pro Tyr Phe Thr Ala
465 470 475 480
Asp Gly Val Ser Leu Gly Tyr Asp Ile Tyr Gly Lys Ala Phe Asp Pro
485 490 495
Arg Lys Ala Ser. Thr Ser Val Lys Gin Tyr Lys Thr Thr Thr Ala Gly
500 505 510
Gly Gly Val Arg Met Gly Ile Pro Val Thr Glu Tyr Asp Arg Val Asn
515 520 525
Phe Gly Leu Ala Ala Glu His Leu Thr Val Asn Thr Tyr Asn Lys Ala
7

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530 535 540
Pro Lys Arg Tyr Ala Asp Phe Ile Lys Gin Tyr Gly Lys Thr Asp Gly
545 550 555 560
Ala Asp Gly Ser Phe Lys Gly Leu Leu Tyr Lys Gly Thr Val Gly Trp
565 570 575
Gly Arg Asn Lys Thr Asp Ser Ala Leu Trp Pro Thr Arg Gly Tyr Leu
580 585 590
Thr Gly Val Asn Ala Glu Ile Ala Leu Pro Gly Ser Lys Leu Gin Tyr
595 600 605
Tyr Ser Ala Thr His Asn Gin Thr Trp Phe Phe Pro Leu Ser Lys Thr
610 615 620
Phe Thr Leu Met Leu Gly Gly Glu Val Gly Ile Ala Gly Gly Tyr Gly
625 630 635 640
Arg Thr Lys Glu Ile Pro Phe Phe Glu Asn Phe Tyr Gly Gly Gly Leu
645 650 655
Gly Ser Val Arg Gly Tyr Glu Ser Gly Thr Leu Gly Pro Lys Val Tyr
660 665 670
Asp Glu Tyr Gly Glu Lys Ile Ser Tyr Gly Gly Asn Lys Lys Ala Asn
675 680 685
Val Ser Ala Glu Leu Leu Phe Pro Met Pro Gly Ala Lys Asp Ala Arg
690 695 700
Thr Val Arg Leu Ser Leu Phe Ala Asp Ala Gly Ser Val Trp Asp Gly
705 710 715 720
Arg Thr Tyr Thr Ala Ala Glu Asn Gly Asn Asn Lys Ser Val Tyr Ser
725 730 735
Glu Asn Ala His Lys Ser Thr Phe Thr Asn Glu Leu Arg Tyr Ser Ala
740 745 750
Gly Gly Ala Val Thr Trp Leu Ser Pro Leu Gly Pro Met Lys Phe Ile.
755 760 765
Tyr Ala Tyr Pro Leu Lys Lys Lys Pro Glu Asp Glu Ile Gin Arg Phe
770 775 780
Gin Phe Gin Leu Gly Thr Thr Phe
6

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785 790
<210> 3
<211> 2394
<212> DNA
<213> Neisseria meningitidis
<220>
<221> CDS
<222> (1)..(2391)
<400> 3
atg aaa ctg aaa cag att gct tee gca ctg atg atg ttg ggc ata tcg 48
Met Lys Leu Lys Gin Ile Ala Ser. Ala Leu Met Met Leu Gly Ile Ser
1 5 10 15
cct ttg gca ttt gcc gac ttc acc atc caa gac ate cgt gtc gaa ggc 96
Pro Leu Ala Phe Ala Asp Phe The Ile Gin Asp Ile Arg Val Glu Gly
20 25 30
ttg cag cgt acc gag ccg age ace gta ttc aac tac ctg ccc gtc aaa 144
Leu Gin Arg Thr Glu Pro Ser The Val Phe Asn Tyr Leu Pro Val Lys
35 40 45 ,
gtc ggc gat acc tac aac gac aca cac ggc agt gee ate ate aaa age 192
Val Gly Asp Thr Tyr Asn Asp Thr His Gly Ser Ala Ile Ile Lys Ser
50 55 60
ctg tac gee acc ggt ttc ttt gac gac gta cgc gtc gaa act gcg gac 240
Leu Tyr Ala Thr Gly Phe Phe Asp Asp Val Arg Val Glu The Ala Asp
65 70 75 80
ggg cag ctc ctg ctg acc gtt ate gaa cgc ccc acc ate ggc tcg etc 288
Gly Gin Leu Leu Leu The Val Ile Glu Arg Pro Thr Ile Gly Ser Leu
85 90 95
aac ate acc ggc gca aaa atg ctg caa aac gac gee att aag aaa aac 336
Asn Ile Thr Gly Ala Lys Met Leu Gin Asn Asp Ala Ile Lys Lys Asn
100 105 110
etc gaa tcg ttc ggg ctg gcg cag tcg caa tac ttt aat cag gcg aca 384
Leu Glu Ser Phe Gly Leu Ala Gin Ser Gin Tyr Phe Asn Gin Ala Thr
115 120 125
etc aat cag gca gtc gee ggc ctg aaa gaa gaa tac etc ggg cgc ggc 432
Leu Asn Gin Ala Val Ala Gly Leu Lys Glu Glu Tyr Leu Gly Arg Gly
130 135 140
9

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aaa ctc aat atc caa atc acg ccc aaa gta acc aaa ctc gcc cgc aac 480
Lys Leu Asn Ile Gin Ile Thr Pro Lys Val Thr Lys Leu Ala Arg Asn
145 150 155 160
cgc gtc gac atc gac at: acg att gac gag ggc aaa tcc gcc aaa atc 528
Arg Val Asp Ile Asp Ile Thr Ile Asp Glu Gly Lys Ser Ala Lys Ile
165 170 175
acc gac atc gaa ttt gaa ggc aac caa gtc tat tcc gac cgc aaa ctg 576
Thr Asp Ile Glu Phe Glu Gly Asn Gin Val Tyr Ser Asp Arg Lys Leu
180 185 190
atg cgg cag atg tcg ctg acc gaa ggc ggc att tgg aca tgg ctg aca 624
Met Arg Gin Met Ser Leu Thr Glu Gly Gly Ile Trp Thr Trp Leu Thr
195 200 205
cga agc aac caa ttc aac gag cag aaa ttt gcc caa gac atg gaa aaa 672
Arg Ser Asn Gin Phe Asn Glu Gin Lys Phe Ala Gin Asp Met Glu Lys
210 215 220
gta acc gac ttc tac cag aac aac ggc tac ttc gat ttc cgt atc ctc 720
Val Thr Asp Phe Tyr Gin Asn Asn Gly Tyr Phe Asp Phe Arg Ile Leu
225 230 235 240
gat acc gac atc caa acc aac gaa gac aaa acc aag cag acc atc aaa 768
Asp Thr Asp Ile Gin Thr Asn Glu Asp Lys Thr Lys Gin Thr Ile Lys
245 250 255
atc acc gtc cac gaa ggc gga cgt ttc cgt tgg ggc aaa gtc tcc atc 816
Ile Thr Val His Glu Gly Gly Arg Phe Arg Trp Gly Lys Val Ser Ile
260 265 270
gaa ggc gac acc aac gaa gtc ccc aaa gcc gaa ctg gaa aaa ctg ctg 864
Glu Gly Asp Thr Asn Glu Val Pro Lys Ala Glu Leu Glu Lys Leu Leu
275 280 285
acc atg aag ccc ggc aaa tgg tac gaa cgc cag cag atg acc gcc gtt 912
Thr Met Lys Pro Gly Lys Trp Tyr Glu Arg Gin Gin Met Thr Ala Val
290 295 300
ttg ggt gag att cag aac cgc atg ggc tcg gca ggc tac gca tac agc 960
Leu Gly Glu Ile Gin Asn Arg Met Gly Ser Ala Gly Tyr Ala Tyr Ser
305 310 315 320
gaa atc agc gta cag ccg ctg cca aac gcc gaa acc aaa acc gtc gat 1008
Glu Ile Ser Val Gin Pro Leu Pro Asn Ala Glu Thr Lys Thr Val Asp
325 330 335

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ttc gtc ctg cac atc gaa ccg ggc cgg aaa ate tac gtc aac gaa ate 1056
Phe Val Leu His Ile Glu Pro Gly Arg Lys Ile Tyr Val Asn Glu Ile
340 345 350
cac ate ace ggc aac aac aaa ace cgc gac gaa gtc gtg cgc cgc gaa 1104
His Ile Thr Gly Asn Asn Lys Thr Arg Asp Glu Val Val Arg Arg Glu
355 360 365
ttg cgc caa atg gaa tcc gcg cct tac gac ace tcc aag ctg caa cgc 1152
Leu Arg Gin Met Glu Ser Ala Pro Tyr Asp Thr Ser Lys Leu Gin Arg
370 375 380
tcc aaa gag cgc gtc gag ctt ttg ggc tac ttc gac aac gta cag ttt 1200
Ser Lys Glu Arg Val Glu Leu Leu Gly Tyr Phe Asp Asn Val Gin Phe
385 390 395 400
gat gee gtc ccg ctt gcc ggc aca ccc gac aaa gtc gat ttg aac atg 1248
Asp Ala Val Pro Leu Ala Gly Thr Pro Asp Lys Val Asp Leu Asn Met
405 410 415
age ctg acc gaa cgt tcc ace ggc tcg etc gac ttg age gcg ggc tgg 1296
Ser Leu Thr Glu Arg Set Thr Gly Ser Leu Asp Leu Ser Ala Gly Trp
420 425 430
gta cag gat ace ggc ctg gtc atg tcc gca ggc gtt tcc caa gac aac 1344
Val Gin Asp Thr Gly Leu Val Met Ser Ala Gly Val Ser Gin Asp Asn
435 440 445
ctg ttc ggt acg ggc aag tcg gee gee ctg cgc gee tea cga age aaa 1392
Leu Phe Gly Thr Gly Lys Ser Ala Ala Leu Arg Ala Ser Arg Ser Lys
450 455 460
ace acg etc aac ggc tcg ctg tcg ttt ace gac ccg tac ttc acg gca 1440
Thr Thr Leu Asn Gly Ser Leu Ser Phe Thr Asp Pro Tyr Phe Thr Ala
465 470 475 480
gac ggg gtc age ctg ggc tac gat gtt tac gga aaa gee ttc gac ccg 1488
Asp Gly Val Ser Leu Gly Tyr Asp Val Tyr Gly Lys Ala Phe Asp Pro
485 490 495
cgc aaa gca tcg ace age ate aaa caa tat aaa ace ace acg gca ggc 1536
Arg Lys Ala Ser Thr Ser Ile Lys Gin Tyr Lys Thr Thr Thr Ala Gly
500 505 510
gca ggc ate cgc atg age gtg cct gtt ace gaa tac gac cgc gtg aat 1584
Ala Gly Ile Arg Met Ser Val Pro Val Thr Glu Tyr Asp Arg Val Asn
515 520 525
11

CA 02347849 2001-04-18
WO 00/23595
PCT/US98/22352
ttc ggt ttg gtg gca gaa cac ctg acc gtc aac acc tac aac aaa gcg 1632
Phe Gly Leu Val Ala Glu His Leu Thr Val Asn Thr Tyr Asn Lys Ala
530 535 540
ccc aaa cac tat gcc gac ttt atc aag aaa tac ggc aaa acc gac ggc 1680
Pro Lys His Tyr Ala Asp Phe Ile Lys Lys Tyr Gly Lys Thr Asp Gly
545 550 555 560
aca gac ggc agc ttc aaa ggc tgg ctg tac aaa ggt acc gtc ggc tgg 1728
Thr Asp Gly Ser Phe Lys Gly Trp Leu Tyr Lys Gly Thr Val Gly Trp
565 570 575
ggg cgc aac aaa acc gac agc gcg tta tgg ccg acg cgc ggc tac ctg 1776
Gly Arg Asn Lys Thr Asp Ser Ala Leu Trp Pro Thr Arg Gly Tyr Leu
580 585 590
acg ggc gtg aac gcc gaa atc gcc ctg ccc ggc agc aaa ctg caa tac 1824
Thr Gly Val Asn Ala Glu Ile Ala Leu Pro Gly Ser Lys Leu Gin Tyr
595 600 605
tac tcc gcc acc cac aac caa acc tgg ttc ttc ccc tta agc aaa acc 1872
Tyr Ser Ala Thr His Asn Gin Thr Trp Phe Phe Pro Leu Ser Lys Thr
610 615 620
ttc acg ctg atg ctc ggc ggc gaa gtc ggc att gcg ggc ggc tac ggc 1920
Phe Thr Leu Met Leu Gly Gly Glu Val Gly Ile Ala Gly Gly Tyr Gly
625 630 635 640
aga acc aaa gaa atc ccc ttc ttt gaa aac ttc tac ggc ggc ggc ctg 1968
Arg Thr Lys Glu Ile Pro Phe Phe Glu Asn Phe Tyr Gly Gly Gly Leu
645 650 655
ggt tcg gtg cgc gga tac gaa agc ggc acg ctc ggt ccg aaa gtg tat 2016
Gly Ser Val Arg Gly Tyr Glu Ser Gly Thr Leu Gly Pro Lys Val Tyr
660 665 670
gac gaa tac ggc gaa aaa atc agc tac ggc ggc aac aaa aaa gcc aac 2064
Asp Glu Tyr Gly Glu Lys Ile Ser Tyr Gly Gly Asn Lys Lys Ala Asn
675 680 685
gtc tcc gcc gag ctg ctc ttc ccg atg cct ggc gcg aaa gac gcg cgc 2112
Val Ser Ala Glu Leu Leu Phe Pro Met Pro Gly Ala Lys Asp Ala Arg
690 695 700
acc gtc cgc ctg agc ctg ttt gcc gac gca ggc agc gtg tgg gac ggc 2160
Thr Val Arg Leu Ser Leu Phe Ala Asp Ala Gly Ser Val Trp Asp Gly
705 710 715 720
12

CA 02347849 2001-04-18
WO 00/23595
PCT/US98/22352
aaa acc tac gac gac aac agc agt tcc gcg acc ggc ggc agg gtt caa 2208
Lys Thr Tyr Asp Asp Asn Ser Ser Ser Ala Thr Gly Gly Arg Val Gin
725 730 735
aac att tac ggc gcc ggc aat acc cat aaa tcc acc ttt acc aac gaa 2256
Asn Ile Tyr Gly Ala Gly Asn Thr His Lys Ser Thr Phe Thr Asn Glu
740 745 750
ttg cgc tat tcc gcc ggc ggc gcg gtt acc tgg ctc tcg cct tta ggc 2304
Leu Arg Tyr Ser Ala Gly Gly Ala Val Thr Trp Leu Ser Pro Leu Gly
755 760 765
ccg atg aaa ttc agg tac gcc tac ccg ctg aag aaa aaa ccg gaa gac 2352
Pro Met Lys Phe Arg Tyr Ala Tyr Pro Leu Lys Lys Lys Pro Glu Asp
770 775 780
gaa atc caa cgc ttc caa ttc caa ctc ggc acg acg ttc taa 2394
Glu Ile Gin Arg Phe Gin Phe Gin Leu Gly Thr Thr Phe
785 790 795
<210> 4
<211> 797
<212> PRT
<213> Neisseria meningitidis
<400> 4
Met Lys Leu Lys Gin Ile Ala Ser Ala Leu Met Met Leu Gly Ile Ser
1 5 10 15
Pro Leu Ala Phe Ala Asp Phe Thr Ile Gin Asp Ile Arg Val Glu Gly
20 25 30
Leu Gin Arg Thr Glu Pro Ser Thr Val Phe Asn Tyr Leu Pro Val Lys
35 40 45
Val Gly Asp Thr Tyr Asn Asp Thr His Gly Ser Ala Ile Ile Lys Ser
50 55 60
Leu Tyr Ala Thr Gly Phe Phe Asp Asp Val Arg Val Glu Thr Ala Asp
65 70 75 80
Gly Gin Leu Leu Leu Thr Val Ile Glu Arg Pro Thr Ile Gly Ser Leu
85 90 95
Asn Ile Thr Gly Ala Lys Met Leu Gin Asn Asp Ala Ile Lys Lys Asn
100 105 110
13

CA 02347849 2001-04-18
WO 00/23595
PCT/US98/22352
Leu Glu Ser Phe Gly Leu Ala Gin Ser Gin Tyr Phe Asn Gin Ala Thr
115 120 125
Leu Asn Gin Ala Val Ala Gly Leu Lys Glu Glu Tyr Leu Gly Arg Gly
130 135 140
Lys Leu Asn Ile Gin Ile Thr Pro Lys Val Thr Lys Leu Ala Arg Asn
145 150 155 160
Arg Val Asp Ile Asp Ile Thr Ile Asp Glu Gly Lys Ser Ala Lys Ile
165 170 175
Thr Asp Ile Glu Phe Glu Gly Asn Gin Val Tyr Ser Asp Arg Lys Leu
180 185 190
Met Arg Gin Met Ser Leu Thr Glu Gly Gly Ile Trp Thr Trp Leu Thr
195 200 205
Arg Ser Asn Gin Phe Asn Glu Gin Lys Phe Ala Gin Asp Met Glu Lys
210 215 220
Val Thr Asp Phe Tyr Gin Asn Asn Gly Tyr Phe Asp Phe Arg Ile Leu
225 230 235 240
Asp Thr Asp Ile Gin Thr Asn Glu Asp Lys Thr Lys Gin Thr Ile Lys
245 250 255
Ile Thr Val His Glu Gly Gly Arg Phe Arg Trp Gly Lys Val Ser Ile
260 265 270
Glu Gly Asp Thr Asn Glu Val Pro Lys Ala Glu Leu Glu Lys Leu Leu
275 280 285
Thr Met Lys Pro Gly Lys Trp Tyr Glu Arg Gin Gin Met Thr Ala Val
290 295 300
Leu Gly Glu Ile Gin Asn Arg Met Gly Ser Ala Gly Tyr Ala Tyr Ser
305 310 315 320
Glu Ile Ser Val Gin Pro Leu Pro Asn Ala Glu Thr Lys Thr Val Asp
325 330 335
Phe Val Leu His Ile Glu Pro Gly Arg Lys Ile Tyr Val Asn Glu Ile
340 345 350
His Ile Thr Gly Asn Asn Lys Thr Arg Asp Glu Val Val Arg Arg Glu
355 360 365
14

CA 02347849 2001-04-18
WO 00/23595
PCT/US98/22352
Leu Arg Gin Met Glu Ser Ala Pro Tyr Asp Thr Ser Lys Leu Gin Arg
370 375 380
Ser Lys Glu Arg Val Glu Leu Leu Gly Tyr Phe Asp Asn Val Gin Phe
385 390 395 400
Asp Ala Val Pro Leu Ala Gly Thr Pro Asp Lys Val Asp Leu Asn Met
405 410 415
Ser Leu Thr Glu Arg Ser Thr Gly Ser Leu Asp Leu Ser Ala Gly Trp
420 425 430
Val Gin Asp Thr Gly Leu Val Met Ser Ala Gly Val Ser Gin Asp Asn
435 44D 445
Leu Phe Gly Thr Gly Lys Ser Ala Ala Leu Arg Ala Ser Arg Ser Lys
450 455 460
Thr Thr Leu Asn Gly Ser Leu Ser Phe Thr Asp Pro Tyr Phe Thr Ala
465 470 475 480
Asp Gly Val Ser Leu Gly Tyr Asp Val Tyr Gly Lys Ala Phe Asp Pro
485 490 , 495
Arg Lys Ala Ser Thr Ser Ile Lys Gin Tyr Lys Thr Thr Thr Ala Gly
500 505 510
Ala Gly Ile Arg Met Ser Val Pro Val Thr Glu Tyr Asp Arg Val Asn
515 520 525
Phe Gly Leu Val Ala Glu His Leu Thr Val Asn Thr Tyr Asn Lys Ala
530 535 540
Pro Lys His Tyr Ala Asp Phe Ile Lys Lys Tyr Gly Lys Thr Asp Gly
545 550 555 560
Thr Asp Gly Ser Phe Lys Gly Trp Leu Tyr Lys Gly Thr Val Gly Trp
565 570 575
Gly Arg Asn Lys Thr Asp Ser Ala Leu Trp Pro Thr Arg Gly Tyr Leu
580 585 590
Thr Gly Val Asn Ala Glu Ile Ala Leu Pro Gly Ser Lys Leu Gin Tyr
595 600 605
Tyr Ser Ala Thr His Asn Gin Thr Trp Phe Phe Pro Leu Ser Lys Thr
610 615 620

CA 02347849 2001-04-18
WO 00/23595
PCT/US98/22352
Phe Thr Leu Met Leu Gly Gly Glu Val Gly Ile Ala Gly Gly Tyr Gly
625 630 635 640
Arg Thr Lys Glu Ile Pro Phe Phe Glu Asn Phe Tyr Gly Gly Gly Leu
645 650 655
Gly Ser Val Arg Gly Tyr Glu Ser Gly Thr Leu Gly Pro Lys Val Tyr
660 665 670
Asp Glu Tyr Gly Glu Lys Ile Ser Tyr Gly Gly Asn Lys Lys Ala Asn
675 680 685
Val Ser Ala Glu Leu Leu Phe Pro Met Pro Gly Ala Lys Asp Ala Arg
690 695 700
Thr Val Arg Leu Ser Leu Phe Ala Asp Ala Gly Ser Val Trp Asp Gly
705 710 715 720
Lys Thr Tyr Asp Asp Asn Ser Ser Ser Ala Thr Gly Gly Arg Val Gin
725 730 735
Asn Ile Tyr Gly Ala Gly Asn Thr His Lys Ser Thr Phe Thr Asn Giu
740 745 750
Leu Arg Tyr Ser Ala Gly Gly Ala Val Thr Trp Leu Ser Pro Leu Gly
755 760 765
Pro Met Lys Phe Arg Tyr Ala Tyr Pro Leu Lys Lys Lys Pro Glu Asp
770 775 780
Glu Ile Gin Arg Phe Gln Phe Gin Leu Gly Thr Thr Phe
785 790 795
<210> 5
<211> 23
<212> DNA
<213> PCR primer
<400> 5
cggaattcat gaaactgaaa cag 23
<210> 6
<211> 18
<212> DNA
<213> PCR primer
16

CA 02347849 2001-04-18
WO 00/23595
PCT/US98/22352
<400> 6
ttgcagtttt tgcaattc 18
<210> 7
<211> 59
<212> DNA
<213> Neisseria gonorrhoeae
<400> 7
tagattttac gtttcggaat gcagtctgaa accgcattcc gcaccacaag gaacttacg 59
<210> 8
<211> 67
<212> DNA
<213> Neisseria gonorrhoeae
<400> 8
cccgcaaatg ccgtctgaag cccttgagac ggcatttcgc ggcaacatcc gaaggagttt 60
taccatg 67
17

Representative Drawing

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Administrative Status

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Administrative Status

Title Date
Forecasted Issue Date 2013-06-25
(86) PCT Filing Date 1998-10-22
(87) PCT Publication Date 2000-04-27
(85) National Entry 2001-04-18
Examination Requested 2003-10-02
(45) Issued 2013-06-25
Deemed Expired 2014-10-22

Abandonment History

There is no abandonment history.

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $300.00 2001-04-18
Maintenance Fee - Application - New Act 2 2000-10-23 $50.00 2001-04-18
Registration of a document - section 124 $100.00 2001-09-14
Maintenance Fee - Application - New Act 3 2001-10-22 $100.00 2001-10-04
Maintenance Fee - Application - New Act 4 2002-10-22 $100.00 2002-10-08
Request for Examination $400.00 2003-10-02
Maintenance Fee - Application - New Act 5 2003-10-22 $150.00 2003-10-10
Maintenance Fee - Application - New Act 6 2004-10-22 $200.00 2004-10-05
Maintenance Fee - Application - New Act 7 2005-10-24 $200.00 2005-10-13
Expired 2019 - Corrective payment/Section 78.6 $50.00 2006-03-03
Maintenance Fee - Application - New Act 8 2006-10-23 $200.00 2006-10-11
Maintenance Fee - Application - New Act 9 2007-10-22 $200.00 2007-10-03
Maintenance Fee - Application - New Act 10 2008-10-22 $250.00 2008-10-01
Maintenance Fee - Application - New Act 11 2009-10-22 $250.00 2009-10-07
Maintenance Fee - Application - New Act 12 2010-10-22 $250.00 2010-10-18
Maintenance Fee - Application - New Act 13 2011-10-24 $250.00 2011-10-06
Maintenance Fee - Application - New Act 14 2012-10-22 $250.00 2012-10-05
Final Fee $300.00 2013-04-05
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
THE UNIVERSITY OF MONTANA
Past Owners on Record
JUDD, RALPH C.
MANNING, D. SCOTT
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Description 2001-04-19 70 3,291
Description 2001-04-18 70 3,290
Claims 2001-04-18 13 414
Abstract 2001-04-18 1 42
Cover Page 2001-07-19 1 35
Drawings 2001-04-18 8 638
Description 2001-04-19 71 3,317
Claims 2001-04-19 20 647
Claims 2008-05-30 3 80
Description 2008-05-30 71 3,287
Description 2009-02-26 71 3,284
Claims 2009-02-26 3 108
Claims 2011-01-18 3 106
Claims 2012-05-25 3 110
Cover Page 2013-05-30 1 37
Correspondence 2001-07-05 1 26
Assignment 2001-04-18 5 164
PCT 2001-04-18 7 240
Prosecution-Amendment 2001-04-18 1 19
Prosecution-Amendment 2001-04-18 3 75
Correspondence 2001-08-01 1 27
Assignment 2001-09-14 3 160
Correspondence 2001-10-31 1 25
Assignment 2001-11-27 1 31
Correspondence 2001-11-27 1 31
Prosecution-Amendment 2003-10-02 1 36
Prosecution-Amendment 2003-10-14 2 44
Fees 2001-04-18 2 53
Prosecution-Amendment 2006-03-03 2 66
Correspondence 2006-03-17 1 18
Prosecution-Amendment 2006-07-14 1 34
PCT 2001-04-19 9 339
Prosecution-Amendment 2007-11-08 8 422
Correspondence 2008-04-25 1 14
PCT 2001-04-19 32 1,124
PCT 2008-05-13 1 21
Prosecution-Amendment 2008-05-30 13 562
Prosecution-Amendment 2008-08-27 5 246
Prosecution-Amendment 2011-07-15 2 47
Prosecution-Amendment 2009-02-26 14 585
Prosecution-Amendment 2010-07-19 4 172
Prosecution-Amendment 2011-01-18 8 349
Prosecution-Amendment 2011-11-25 2 118
Prosecution-Amendment 2012-05-25 6 241
Correspondence 2013-04-05 2 52
Prosecution-Amendment 2012-12-11 1 38

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