Note: Descriptions are shown in the official language in which they were submitted.
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MENINGOCOCCAL CLASS 1 OUTER-MEMBRANE PROTEIN VACCINE
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Descri tion
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Background_of the Invention
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Bacterial meningitis is an inflammatory disease
of the central nervous system caused by the growth
of bacteria in and adjacent to the leptomeninges.
Meningitis is an acute infectious disease which
affects children and young adults and is caused by
the Neisseria meninqitidis, amongst other agents
---------------------------
including other bacterial and viral pathogens.
Meningococci are subdivided into serological
groups depending on the presence of either capsular
or cell wall antigens. Currently recognized sero-
groups include A, B, C, D, W-135, X, Y, Z, and 29E
as segregated by seroagglutination. The polysac-
charides responsible for the serogroup specificity
of the groups A, B, C, X, W-135 and Y have been
purified.
The carrier rate for meningococci is much
higher than the incidence of the disease. Some
persons are temporary carriers, while others are
chronic carriers, discharging meningococci either
more or less continuously or in a sporadic fashion.
The meningococcal carrier state is an immunizing
process, and within two weeks of colonization,
production of antibodies to meningococci can be
identified. It appears that bactericidal antibodies
are directed against both the capsular polysaccha-
ride and other cell wall antigens.
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Studies have shown that meningococcal outer
membranes have three to five major proteins, with
the predominant 41,000Mr or 38,000Mr proteins
carrying the serotype specific determinants. There
is a considerable degree of interstrain hetero-
geneity in the profiles of the outer membrane
proteins on sodium dodecyl sulfate-polyacrylamide
electrophoretic gels (SDS-PAGE). As defined by
peptide mapping studies, the proteins comprise five
classes, designated 1 through 5, based upon common
peptide structures. Bactericidal monoclonal anti-
bodies have been produced against the 46,000 Mr
Class 1 proteins which are shared to some extent
among strains of different serotypes. (Frasch, C.E.
et al., (1985) pg. 633,"New Developments in
Meningococcal Vaccines", in G.K. Schoolnik et al.
(ed.) The Pathogenic-Neisseriae, American Society
for Microbiology, Washington, D.C.).
The capsular polysaccharides of groups A, C, W-
135 and Y meningococci have been used to develop
vaccines against the organism. Although these
vaccines have been effective in the short term, they
do not induce immunological memory and subjects must
be revaccinated within approximately 3 years to
maintain their resistance. The group B polysaccha-
ride is poorly immunogenic and successful vaccines
have not been produced. A possible explanation for
the low activity may be due to tolerance to the
group B polysaccharide induced by crossreactive
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antigens found in. human tissues such as the brain.
Furthermore, studies show that most of the bacter-
icidal antibodies in the convelescent sera of
patients who have had group B meningococcal disease
are directed against outer membrane proteins.
Vaccines for protecting against group B menin-
gococcal disease have been developed in which non-
covalent complexes of outer membrane proteins (OMP)
and group B polysaccharide were administered.
.Beuvery,.et al. (1983) Infect. Immun. 40:369-380.
However, the B polysaccharide is known to induce a
transient IgM antibody response, which does not
confer immunoprotection. Furthermore, there is
great antigenic diversity and variability in the
meningococci outer membrane proteins from strain to
strain. Additionally, lipopolysaccharides are
present in the OMP and exhibit antigenic variability
as well.
There is a need for safe and effective vaccines
against meningococcal disease which provide immunity
from infection, particularly in infants and the
elderly.
SummarY-of-the Invention
---------------------
This invention pertains to isolated outer
membrane vesicles (OMV's), to substantially purified
Class 1 outer membrane protein (OMP) of Neisseria
meningitidis, to fragments of the Class I OMP and to
oligopeptides derived from the Class I OMP which
contain continuous or discontinuous, immunogenic and
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protective B cell epitopes reactive with
bactericidal antibodies against N. meningitidis and
to the use of isolated OMV's, the meningococcal
Class I OMP, fragments or oligopeptides for
vaccination against N. menin~itidis.
The isolated OMV's, meningococcal Class I OMP,
fragments or oligopeptides derived therefrom can be
used in univalent or multivalent subunit vaccines
alone, in mixtures, or as chemical conjugates or
genetic fusions. In preferred vaccines, epitopes
from different epidemiologically relevant
meningococcal strains are used. In addition,
isolated OMV's, the Class I OMP, fragments or
oligopeptides can be used in conjunction (as
mixtures, fusions or conjugates) with other antigens
of N. meningitidis. For example, they can be used
in conjunction with capsular polymers or oligomers
(or fragments thereof) of N. meninqitidis or with
Class I outer membrane proteins (or epitopes
thereof) of different subtypes. In addition, they
can be used with antigens of other infectious
bacteria, viruses, fungi or parasites. Class I OMP
T cell epitopes also are defined and these can be
used in conjunction with other vaccine components to
enhance the protective immune response to the
vaccines.
This invention also pertains to the methods of
producing isolated OMV's, the Class I OMP, fragments
and oligopeptides and to various vaccine
formulations containing them. The isolated OMV's
Class I OMP can be produced by mutant meningococcal
strains which do not express the Class 2/3
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outer membrane protein. Fragments can be produced by
cyanogen bromide cleavage and subsequent purification.
Isolated OMV's, the Class I OMP, fragments or
oligopeptides can be produced by recombinant DNA
techniques, chemical synthesis or chemical or enzymatic
cleavage. These materials, in turn, can be conjugated
or fused to carrier peptides or proteins, to other
antigens of N. meningitidis or to antigens of other
microorganisms by chemical or genetic coupling
techniques to produce multivalent antigenic conjugates
and fusion peptides or proteins. They can be modified
for conjugation such as by the addition of amino acids
or other coupl:Lng groups. For vaccination, isolated
OMV's, the Class I OMP, fragments or oligopeptides, in
any of the forms described, can be formulated in
pharmaceutically acceptable vehicles with optional
additives such as adjuvants.
This invention also pertains to isolated nucleic
acids which encode Class I OMP, fragments or
oligopeptides. The nucleic acids can be incorporated
into appropriate expression systems for production of
isolated OMV's, Class I OMP, fragments or any
oligopeptides derived therefrom. These nucleic acids
can be modified as genetic fusions to contain sequences
encoding additional polypeptides useful in enhancing
the immune response to the vaccine formulation
containing the expressed fusion polypeptides. In
addition, Class I OMP of N. meningitidis is
homologous in amino acid sequence and structure to
porin proteins of other gram negative pathogens and
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thus the Class I OMP, fragments and oligopeptides of this
invention allow for the development of vaccines for other gram
negative pathogens.
More particularly, in one aspect the invention provides a
vaccine effective against meningococcal disease, comprising an
outer-membrane vesicle which comprises Class 1 outer-membrane
protein and which is isolated from Neisseria meningitidis
strain HIII-5 deposited as CBS 636.89.
In another aspect, the invention provides substantially
purified CNBr fragment of Class 1 outer-membrane protein of
Neisseria meningitidis, the fragment having a molecular weight
of 25 kD, wherein the Class 1 outer-membrane protein has an
amino acid sequence selected from the group consisting of:
DVSLYGEIKAGVEGRNIQAQLTEQPQVTNGVQGNQVKVTKAKSRIRTKISDFGSFIGFKGSE
DLGEGLKAVWQLEQDVSVAGGGASQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAINPWD
SNNDVASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYYTKDTNNNLT
LVPAVVGKPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSATSDEAKGTDP
LKNHQVHRLTGGYEEGGLNLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYA
HGFDLIERGKKGENTSYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRH
KF;
DVSLYGEIKAGVEGRNFQLQLTEPPSKSQPQVKVTKAKSRIRTKISDFGSFIGFKGSEDLGE
GLKAVWQLEQDVSVAGGGATQWGNRESFVGLAGEFGTLRAGRVANQFDDASQAIDPWDSNND
VASQLGIFKRHDDMPVSVRYDSPDFSGFSGSVQFVPIQNSKSAYTPAHYTRQNNTDVFVPAV
VGKPGSDVYYAGLNYKNGGFAGSYAFKYARHANVGRDAFELFLLGSTSDEAKGTDPLKNHQV
HRLTGGYEEGGLNLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLI
ERGKKGENTSYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKG
SEDLGDGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDP
WDSNNDVASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTKNTNNN
LTLVPAVVGKPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSGSDQAKGTD
PLKNHQVHRLTGGYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAH
GFDFIERGKKGENTSYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHK
F; and
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DVSLYGEIKAGVEGRNIQLQLTEPLQNIQQPQVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKA
VWQLEQDVSVAGGGATRWGNRESFVGLAGEFGTLRAGRVANQFDDASKAIDPWDSNNVVASQLGI
FKRMDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYTPAHFVQQTPQQSPTLVPAVVGKPGSDV
YYAGLNYKNGGFAGNYAFKYAKHANVGRDAFELFLLGSGSDEAKGTDPLKNHQVHRLTGGYEEGG
LNLALAAQLDLSENADKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIA
GVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF.
In another aspect, the invention provides a substantially
purified oligopeptide having an amino acid sequence selected from
the group consisting of YYTKNTNNNL, HFVQQTPQSQP, YYTKDTNNNL,
YTKDTNNNLT, TKDTNNNLTL, KDTNNNLTLV, AQAANGGASG, QAANGGASGQ,
AANGGASGQV, ANGGASGQVK, NGGASGQVKV, GGASGQVKVT, GASGQVKVTK,
ASGQVKVTKV, SGQVKVTKVT, PAHYTRQNNT, AHYTRQNNTD, HYTRQNNTDVF,
TRQNNTDVFV, RQNNTDVFVP, QNNTDVFVP, QNNTDVFVPA, NNTDVFVPAV,
ANVGRNAFELFLIGSATSDEAKG, NIQAQLTEQPQVTNGVQGN, TKISDFGSFIGFK,
VSVAGGGASQWGN, TLRAGRVANQFDDASQAIN, DSNNDVASQLGIFK, GGFSGFSG,
LSENGDKAKTKNSTTE and VPRISYAHGFDLIERGKKG.
In another aspect, the invention provides a genetic fusion
peptide or protein, comprising a peptide to which an anti-
meningococcal outer-membrane protein antibody binds, fused to a
carrier polypeptide, wherein the peptide is selected from the
group consisting of QPQVTNGVQGN, PPSKSQP, AQAANGGASG,
YYTKDTNNNLTL, YYTKNTNNNLTL, YYTKDTNNNL, YYTKNTNNNL, HFVQQTPQSQP
and HYTRQNNTDVF.
In another aspect, the invention provides a vaccine effective
against meningococcal disease, comprising an outer-membrane
vesicle isolated from Neisseria meningitidis strain HIII-5
deposited as CBS 636.89 that has been genetically modified to
express more than one subtype of meningococcal Class 1 outer-
membrane protein, the vesicle comprising meningococcal Class 1
outer-membrane proteins of more than one subtype.
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In still another aspect, the invention provides a vaccine
comprising a pharmaceutically acceptable vehicle and a fragment
of a meningococcal Class 1 outer-membrane protein obtained by use
of a reagent selected from the group consisting of:
a. cyanogen bromide,
b. endoproteinase Lys-C;
c. endoproteinase Arg-C; and
d. endoproteinase Glu-C;
wherein the meningococcal Class 1 outer-membrane protein has an
amino acid sequence selected from the group consisting of:
DVSLYGEIKAGVEGRNIQAQLTEQPQVTNGVQGNQVKVTKAKSRIRTKISDFGSFIGFKGSEDLG
EGLKAVWQLEQDVSVAGGGASQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAINPWDSNNDVA
SQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYYTKDTNNNLTLVPAVVGKP
GSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSATSDEAKGTDPLKNHQVHRLTGG
YEEGGLNLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENT
SYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNFQLQLTEPPSKSQPQVKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLK
AVWQLEQDVSVAGGGATQWGNRESFVGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLG
IFKRHDDMPVSVRYDSPDFSGFSGSVQFVPIQNSKSAYTPAHYTRQNNTDVFVPAVVGKPGSDVY
YAGLNYKNGGFAGSYAFKYARHANVGRDAFELFLLGSTSDEAKGTDPLKNHQVHRLTGGYEEGGL
NLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQII
AGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSED
LGDGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNND
VASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTKNTNNNLTLVPAVVG
KPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSGSDQAKGTDPLKNHQVHRLTG
GYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTS
YDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF, and
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DVSLYGEIKAGVEGRNIQLQLTEPLQNIQQPQVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKA
VWQLEQDVSVAGGGATRWGNRESFVGLAGEFGTLRAGRVANQFDDASKAIDPWDSNNVVASQLGI
FKRMDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYTPAHFVQQTPQQSPTLVPAVVGKPGSDV
YYAGLNYKNGGFAGNYAFKYAKHANVGRDAFELFLLGSGSDEAKGTDPLKNHQVHRLTGGYEEGG
LNLALAAQLDLSENADKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIA
GVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF.
In another aspect, the invention provides a vaccine comprising a
pharmaceutically acceptable vehicle and a peptide having an amino
acid sequence selected from the group consisting of QPQVTNGVQGN,
PPSKSQP, AQAANGGASG, YYTKDTNNNLTL, YYTKNTNNNLTL, YYTKDTNNNL,
YYTKNTNNNL, HFVQQTPQSQP and HYTRQNNTDVF.
In another aspect, the invention provides a vaccine effective
against Neisseria meningitidis, comprising a pharmaceutically
acceptable vehicle and a peptide having an amino acid sequence
selected from the group consisting of QPQVTNGVQGN, EAQAANGGASGQ,
YYTKDTNNNLTL and YYTKNTNNNLT.
In still another aspect, the invention provides a vaccine
comprising a pharmaceutically acceptable vehicle, and a conjugate
of Neisseria meningitidis Class 1 outer-membrane protein and
meningococcal polysaccharide selected from the group consisting
of A, B, W-135 and Y polysaccharide, wherein the Class 1 outer-
membrane protein has an amino acid sequence selected from the
group consisting of:
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DVSLYGEIKAGVEGRNIQAQLTEQPQVTNGVQGNQVKVTKAKSRIRTKISDFGSFIGFKGSEDLG
EGLKAVWQLEQDVSVAGGGASQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAINPWDSNNDVA
SQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYYTKDTNNNLTLVPAVVGKP
GSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSATSDEAKGTDPLKNHQVHRLTGG
YEEGGLNLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENT
SYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNFQLQLTEPPSKSQPQVKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLK
AVWQLEQDVSVAGGGATQWGNRESFVGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLG
IFKRHDDMPVSVRYDSPDFSGFSGSVQFVPIQNSKSAYTPAHYTRQNNTDVFVPAVVGKPGSDVY
YAGLNYKNGGFAGSYAFKYARHANVGRDAFELFLLGSTSDEAKGTDPLKNHQVHRLTGGYEEGGL
NLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQII
AGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSED
LGDGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNND
VASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTKNTNNNLTLVPAVVG
KPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSGSDQAKGTDPLKNHQVHRLTG
GYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTS
YDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF, and
DVSLYGEIKAGVEGRNIQLQLTEPLQNIQQPQVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKA
VWQLEQDVSVAGGGATRWGNRESFVGLAGEFGTLRAGRVANQFDDASKAIDPWDSNNVVASQLGI
FKRMDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYTPAHFVQQTPQQSPTLVPAVVGKPGSDV
YYAGLNYKNGGFAGNYAFKYAKHANVGRDAFELFLLGSGSDEAKGTDPLKNHQVHRLTGGYEEGG
LNLALAAQLDLSENADKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIA
GVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF.
In still another aspect, the invention provides an antigenic
conjugate, said conjugate comprising an antigenic carrier
polypeptide, and a meningococcal Class 1 outer-membrane protein,
wherein said Class 1 outer-membrane protein has an amino acid
sequence selected from the group consisting of:
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DVSLYGEIKAGVEGRNIQAQLTEQPQVTNGVQGNQVKVTKAKSRIRTKISDFGSFIGFKGSEDLG
EGLKAVWQLEQDVSVAGGGASQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAINPWDSNNDVA
SQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYYTKDTNNNLTLVPAVVGKP
GSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSATSDEAKGTDPLKNHQVHRLTGG
YEEGGLNLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENT
SYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNFQLQLTEPPSKSQPQVKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLK
AVWQLEQDVSVAGGGATQWGNRESFVGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLG
IFKRHDDMPVSVRYDSPDFSGFSGSVQFVPIQNSKSAYTPAHYTRQNNTDVFVPAVVGKPGSDVY
YAGLNYKNGGFAGSYAFKYARHANVGRDAFELFLLGSTSDEAKGTDPLKNHQVHRLTGGYEEGGL
NLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQII
AGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSED
LGDGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNND
VASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTKNTNNNLTLVPAVVG
KPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSGSDQAKGTDPLKNHQVHRLTG
GYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTS
YDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF; and
DVSLYGEIKAGVEGRNIQLQLTEPLQNIQQPQVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKA
VWQLEQDVSVAGGGATRWGNRESFVGLAGEFGTLRAGRVANQFDDASKAIDPWDSNNVVASQLGI
FKRMDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYTPAHFVQQTPQQSPTLVPAVVGKPGSDV
YYAGLNYKNGGFAGNYAFKYAKHANVGRDAFELFLLGSGSDEAKGTDPLKNHQVHRLTGGYEEGG
LNLALAAQLDLSENADKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIA
GVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF.
In another aspect, the invention provides a recombinant
microorganism which expresses a peptide selected from the group
consisting of QPQVINGVQCN, PPSKSQP, QAANOGASG, YYTKUINZRII,TL,
YY'I'fQVIDNNLTL,
YYTfOINNNL, YYTKNIMM, HFVQQTPQSQP and HYTRQDIIaI'DVF.
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In still another aspect, the invention provides a recombinant
microorganism capable of expressing a fusion protein comprising a
modified flagellin protein and an epitope which binds to
antibodies specific to a Neisseria meningitidis Class 1 outer-
membrane protein, wherein the epitope is defined by an amino acid
sequence selected from the group consisting of YYTKNTNNNL,
HFVQQTPQSQP, YYTKDTNNNL, YTKDTNNNLT, TKDTNNNLTL, KDTNNNLTLV,
AQAANGGASG, QAANGGASGQ, AANGGASGQV, ANGGASGQVK, NGGASGQVKV,
GGASGQVKVT, GASGQVKVTK, ASGQVKVTKV, SGQVKVTKVT, PAHYTRQNNT,
AHYTRQNNTD, HYTRQNNTDVF, TRQNNTDVFV, It~tTIIn7FVP, QWIDVFVP, QWIi7VFVPA,
NgIi7VFVPAV, ANVGRNAFELFLIGSATSDEAKG, NIQAQLTDQPQV'TNGVQGN, TKISDFGSFIGFK,
VSVACOC-A.SQWGQ, TLRAGRVANQFDDASQAIN, DSNN7VASQLGIFK, GGFSGFSG,
LSENGDKAKTKNSTTE and VPRISYAHGFDLIERGKKG.
In another aspect, the invention provides the Neisseria
meningitidis strain consisting of HIII-5, deposit number CBS
636.89.
In another aspect, the invention provides a strain of Neisseria
meningitidis which is transformed with a gene encoding a Class 1
outer-membrane protein of Neisseria meningitidis or a fusion
protein thereof, wherein the Class 1 outer-membrane protein has a
molecular weight of 42 kD to 46 kD and comprises an amino acid
sequence selected from the group consisting of:
DVSLYGEIKAGVEGRNIQAQLTEQPQVTNGVQGNQVKVTKAKSRIRTKISDFGSFIGFKGSEDLG
EGLKAVWQLEQDVSVAGGGASQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAINPWDSNNDVA
SQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYYTKDTNNNLTLVPAVVGKP
GSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFIGSATSDEAKGTDPLKNHQVHRLTGGY
EEGGLNLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENTS
YDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
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DVSLYGEIKAGVEGRNFQLQLTEPPSKSQPQVKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLK
AVWQLEQDVSVAGGGATQWGNRESFVGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLG
IFKRHDDMPVSVRYDSPDFSGFSGSVQFVPIQNSKSAYTPAHYTRQNNTDVFVPAVVGKPGSDVY
YAGLNYKNGGFAGSYAFKYARHANVGRDAFELFLLGSTSDEAKGTDPLKNHQVHRLTGGYEEGGL
NLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQII
AGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSED
LGDGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNND
VASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTKNTNNNLTLVPAVVG
KPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSGSDQAKGTDPLKNHQVHRLTG
GYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTS
YDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF; and
DVSLYGEIKAGVEGRNIQLQLTEPLQNIQQPQVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKA
VWQLEQDVSVAGGGATRWGNRESFVGLAGEFGTLRAGRVANQFDDASKAIDPWDSNNVVASQLGI
FKRMDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYTPAHFVQQTPQQSPTLVPAVVGKPGSDV
YYAGLNYKNGGFAGNYAFKYAKHANVGRDAFELFLLGSGSDEAKGTDPLKNHQVHRLTGGYEEGG
LNLALAAQLDLSENADKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIA
GVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF.
In still another aspect, the invention provides a recombinant
microorganism capable of expressing Neisseria meningitidis Class
1 outer-membrane proteins of more than one subtype, wherein at
least one of the Class 1 outer-membrane proteins has a molecular
weight of 42 kD to 46 kD and comprises an amino acid sequence
selected from the group consisting of:
DVSLYGEIKAGVEGRNIQAQLTEQPQVTNGVQGNQVKVTKAKSRIRTKISDFGSFIGFKGSEDLG
EGLKAVWQLEQDVSVAGGGASQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAINPWDSNNDVA
SQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYYTKDTNNNLTLVPAVVGKP
GSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSATSDEAKGTDPLKNHQVHRLTGG
YEEGGLNLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENT
SYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
CA 02007248 2007-03-28
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DVSLYGEIKAGVEGRNFQLQLTEPPSKSQPQVKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLK
AVWQLEQDVSVAGGGATQWGNRESFVGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLG
IFKRHDDMPVSVRYDSPDFSGFSGSVQFVPIQNSKSAYTPAHYTRQNNTDVFVPAVVGKPGSDVY
YAGLNYKNGGFAGSYAFKYARHANVGRDAFELFLLGSTSDEAKGTDPLKNHQVHRLTGGYEEGGL
NLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQII
AGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSED
LGDGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNND
VASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTKNTNNNLTLVPAVVG
KPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSGSDQAKGTDPLKNHQVHRLTG
GYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTS
YDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF; and
DVSLYGEIKAGVEGRNIQLQLTEPLQNIQQPQVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKA
VWQLEQDVSVAGGGATRWGNRESFVGLAGEFGTLRAGRVANQFDDASKAIDPWDSNNVVASQLGI
FKRMDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYTPAHFVQQTPQQSPTLVPAVVGKPGSDV
YYAGLNYKNGGFAGNYAFKYAKHANVGRDAFELFLLGSGSDEAKGTDPLKNHQVHRLTGGYEEGG
LNLALAAQLDLSENADKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIA
GVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF.
In another aspect, the invention provides an isolated nucleic
acid encoding a Neisseria rneningitidis Class 1 outer-membrane
protein, said protein having an amino acid sequence selected from
the group consisting of:
DVSLYGEIKAGVEGRNIQAQLTEQPQVTNGVQGNQVKVTKAKSRIRTKISDFGSFIGFKGSEDLG
EGLKAVWQLEQDVSVAGGGASQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAINPWDSNNDVA
SQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYYTKDTNNNLTLVPAVVGKP
GSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSATSDEAKGTDPLKNHQVHRLTGG
YEEGGLNLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENT
SYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
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DVSLYGEIKAGVEGRNFQLQLTEPPSKSQPQVKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLK
AVWQLEQDVSVAGGGATQWGNRESFVGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLG
IFKRHDDMPVSVRYDSPDFSGFSGSVQFVPIQNSKSAYTPAHYTRQNNTDVFVPAVVGKPGSDVY
YAGLNYKNGGFAGSYAFKYARHANVGRDAFELFLLGSTSDEAKGTDPLKNHQVHRLTGGYEEGGL
NLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQII
AGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSED
LGDGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNND
VASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTKNTNNNLTLVPAVVG
KPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSGSDQAKGTDPLKNHQVHRLTG
GYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTS
YDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF; and
DVSLYGEIKAGVEGRNIQLQLTEPLQNIQQPQVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKA
VWQLEQDVSVAGGGATRWGNRESFVGLAGEFGTLRAGRVANQFDDASKAIDPWDSNNVVASQLGI
FKRMDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYTPAHFVQQTPQQSPTLVPAVVGKPGSDV
YYAGLNYKNGGFAGNYAFKYAKHANVGRDAFELFLLGSGSDEAKGTDPLKNHQVHRLTGGYEEGG
LNLALAAQLDLSENADKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIA
GVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF.
In still another aspect, the invention provides an isolated
nucleic acid having a nucleotide sequence selected from the group
consisting of
GGCAGGAACTACCAGCTGCAATTGACTGAAGCACAAGCCGCTAACGGTGGAGCGAGCGGTCAGGT
AAAAGTTACTAAAGTTACTAAGGCCAAAAGCCGCATCAGGACGAAAATCAGT;
GGCAGGAACATCTAGGCGCAATTGACCGAGCAGCCCCAAGCAACTAACGGTGTGCAAGGCGGTCG
GCAAGGCAATCAGGTAACAGTTACTAAGGTCAAAAGCCGCATCAGGACGGAAATCAGC;
GGCAGGAACTTCCAGCTGCAGTTGACCGAACCGCCCTCAAAGAGTCAACCTCAGGTAAAAGTTAC
TAAGGCCAAAAGCCGCATCAGGACGAAAATCAGT;
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GGCAACAACATTCAGCTGCAATTGACCGAACCACCCTCAAAAGGTCAGACGGGCAATAAAGTTAC
TAAGGCCAAAAGCCGCATCAGGACGAAAATCAGT;
GGCAGGAACTACCAGCTGCAATTGACTGAACAACCCTCAAGAACTCAAGGTCAAACGAGCAATCA
GGTAAAAGTTACTAAGGCCAAAAGCCGCATCAGGACGAAAATCAGT; and
GGCAGGAACATCCAGCTGCAGTTGACCGAACCGCTCCCAAATATTCAACCTCAGGTTACTAAGCG
CAAAAGCCGCATCAGGACGAAAATCAGC.
In still another aspect, the invention provides an isolated
nucleic acid, having a nucleotide sequence selected from the
group consisting of
ACGCTGCGCGCCGGTCGCGTTGCGAATCAGTTTGACGATGCCAGCCAAGCCATTGATCCTTGGGA
CAGCAATAATGATGTGGCTTCGCAATTGGGTATTTTCAAACGCCACGACGACATGCCGGTTTCTG
TACGCTACGATTCCCCCGAATTTTCCGGTTTCAGCGGCAGCGTTCAATTCGTTCCGATCCAAAAC
AGCAAGTCCGCCTATACGCCGGCTTATTATACTAAGGATACAAACAATAATCTTACTCTCGTTCC
GGCTGTTGTCGGCAAGCCCGGATCGGATGTGTATTATGCCGGTCTGAATTACAAAAATGGCGGTT
TTGCCGGGAACTATGCCTTTAAATATGCGAGACACGCCAATGTCGGACGTAATGCTTTTGAGTTG
TTC;
ACGCTGCGCACCGGTCGCGTTGCGAATCAGTTTGACGATGCCAGCCAAGCCATTGATCCTTGGGA
CAGCAATAATGATGTGGCTTCGCAATTGGGTATTTTCAAACGCCACGACGATATGCCGGTTTCTG
TACGCTACGACTCTCCGGACTTTTCCGGTTTCAGCGGCAGCGTCCAATTCGTTCCGGCTCAAAAC
AGCAAGTCCGCCTATACGCCGGCTTATGTGGCGGTGGAAAATGGCGTAGCTAAAAAAGTTGCGGC
TGTTGTCGGCAAGCCCGGATCGGATGTGTATTATGCCGGTCTGAATTATAAGAATGGCGGTTTTG
CCGGGAACTATGCCTTTAAATATGCGAAACACGCCAATGTCGGACGTGATGCTTTTGAGTTGTTC
ACGCTGCNNNNNGGTCGCGTCGCGAATCAGTTTGACGATGCCAGCCAAGCCATTGATCCTTGGGA
CAGCAACAATGATGTGGCTTCGCAATTGGGTATTTTCAAACGCCACGACGATATGCCGGTTTCTG
TACGCTACGACTCTCCGGACTTTTCCGGTTTCAGCGGCAGCGTCCAATTCGTTCCGATCCAAAAC
AGCAAGTCCGCCTATACGCCGGCTCATAATACTAGGCAGAACAATGCTGATGTTTTCGTTCCGGC
TGTTGTCGGCAAGCCCGGATCGGATGTGTATTATGCCGGTCTGAATTACAAAAATGGCGGTTTTG
CCGGGCGCTATGCCTTTAAATATGCGAGACACGCCAATGTCGGACGTGATGCTTTTGAGTTGTTC
CA 02007248 2007-03-28
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CGCTACGACTCTCCGGACTTTTCCGGTTTCAGCGGCAGCGTCCAATTCGTTCCGGCCCAAAACAG
CAAATCCGCCTATACGCCGGCTACTTATACTGTGGATAGTAGTGGTGTTGTTACTCCCGTTCCTG
CTGTTGTCGGCAAGCCCGGATCGGATGTGTATTATGCCGGTCTGAATTACAAAAATGGCGGTTTT
GCCGGGAACTATGCCTTTAAATACGCGAAACACGCCAATGTGGGCCGTGATGCTTTTAATTTGTT
C;
CGCTACGACTCTCCGGACTTTTCCGGTTTCAGCGGCAGCGTCCAATTCGTTCCGGCTCAAAACAG
CAAGTCCGCCTATAAGCCGGCTTATGTGGATGAGAAGAAAATGGTTCATGCGGCTGTTGTCGGCA
AGCCCGGATCGGATGTGTATTATGCCGGTCTGAATTACAAAAATGGCGGTTTTGCCGGGAACTAT
GCCTTTAAATATGCGAAACACGCCAATGTGGGCCGTGATGCTTTTAATTTGTTC; and
ACGCTGCGCACCGGTCGCGTTGCAAATCAGTTTGACGATGCCAGCCAAGCCATTGATCCTTGGGA
CAGCAATAATGATGTGGCTTCGCAATTGGGTATTTTCAAACGCCACGACGATATGTCGGTTTCTG
TACGCTACGATTCCCCCGAATTTTCCGGTTTTAGCGGCAGCGTCCAATTCGTTCCGGCCCAAAAC
AGCAAGTCCGCCTATACGCCGGCTCATTTTGTTCAGAATAAGCAAAATCAGCGGCCTACTCTCGT
TCCGGCTGTTGTCGGCAAGCCGGGGTCGGATGTGTATTATGCCGGTCTGAATTACAAAAATGGCG
GTTTTGCCGGGAACTATGCCTTTAAATACGCGAAACACGCCAATGTGGGCCGTGATGCTTTTGAG
TTGTTC.
In another aspect, the invention provides an expression vector
comprising a nucleic acid encoding a Class 1 outer-membrane
protein of Neisseria meningitidis or a fusion protein thereof
comprising Class 1 outer-membrane protein of Neisseria
meningitidis under the control of transcription signals and
linked to appropriate translation signals for expression in a
suitable host cell, wherein the Class 1 outer-membrane protein
has a molecular weight of 42 kD to 46 kD and an amino acid
sequence selected from the group consisting of:
DVSLYGEIKAGVEGRNIQAQLTEQPQVTNGVQGNQVKVTKAKSRIRTKISDFGSFIGFKGSEDLG
EGLKAVWQLEQDVSVAGGGASQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAINPWDSNNDVA
SQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYYTKDTNNNLTLVPAVVGKP
GSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSATSDEAKGTDPLKNHQVHRLTGG
YEEGGLNLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENT
SYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
CA 02007248 2007-03-28
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DVSLYGEIKAGVEGRNFQLQLTEPPSKSQPQVKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLK
AVWQLEQDVSVAGGGATQWGNRESFVGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLG
IFKRHDDMPVSVRYDSPDFSGFSGSVQFVPIQNSKSAYTPAHYTRQNNTDVFVPAVVGKPGSDVY
YAGLNYKNGGFAGSYAFKYARHANVGRDAFELFLLGSTSDEAKGTDPLKNHQVHRLTGGYEEGGL
NLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQII
AGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSED
LGDGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNND
VASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTKNTNNNLTLVPAVVG
KPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSGSDQAKGTDPLKNHQVHRLTG
GYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTS
YDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF; and
DVSLYGEIKAGVEGRNIQLQLTEPLQNIQQPQVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKA
VWQLEQDVSVAGGGATRWGNRESFVGLAGEFGTLRAGRVANQFDDASKAIDPWDSNNVVASQLGI
FKRMDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYTPAHFVQQTPQQSPTLVPAVVGKPGSDV
YYAGLNYKNGGFAGNYAFKYAKHANVGRDAFELFLLGSGSDEAKGTDPLKNHQVHRLTGGYEEGG
LNLALAAQLDLSENADKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIA
GVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF.
In yet another aspect, the invention provides a method for
producing Neisseria meningitidis Class 1 outer-membrane protein,
said method comprising maintaining a host cell containing a
recombinant nucleic acid encoding Neisseria meningitidis Class 1
outer-membrane protein under conditions suitable for expression
thereof, whereby the Class 1 outer-membrane protein is expressed
and thereby produced, wherein the Neisseria meningitidis Class 1
outer-membrane protein has a molecular weight of 42 kD to 46 kD
and comprises an amino acid sequence selected from the group
consisting of:
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DVSLYGEIKAGVEGRNIQAQLTEQPQVTNGVQGNQVKVTKAKSRIRTKISDFGSFIGFKGSEDLG
EGLKAVfnTQLEQDVSVAGGGASQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAINPWDSNNDVA
SQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYYTKDTNNNLTLVPAVVGKP
GSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSATSDEAKGTDPLKNHQVHRLTGG
YEEGGLNLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENT
SYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNFQLQLTEPPSKSQPQVKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLK
AVWQLEQDVSVAGGGATQWGNRESFVGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLG
IFKRHDDMPVSVRYDSPDFSGFSGSVQFVPIQNSKSAYTPAHYTRQNNTDVFVPAVVGKPGSDVY
YAGLNYKNGGFAGSYAFKYARHANVGRDAFELFLLGSTSDEAKGTDPLKNHQVHRLTGGYEEGGL
NLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQII
AGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSED
LGDGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNND
VASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTKNTNNNLTLVPAVVG
KPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSGSDQAKGTDPLKNHQVHRLTG
GYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTS
YDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF; and
DVSLYGEIKAGVEGRNIQLQLTEPLQNIQQPQVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKA
VWQLEQDVSVAGGGATRWGNRESFVGLAGEFGTLRAGRVANQFDDASKAIDPWDSNNVVASQLGI
FKRMDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYTPAHFVQQTPQQSPTLVPAVVGKPGSDV
YYAGLNYKNGGFAGNYAFKYAKHANVGRDAFELFLLGSGSDEAKGTDPLKNHQVHRLTGGYEEGG
LNLALAAQLDLSENADKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIA
GVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF.
CA 02007248 2007-03-28
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In another aspect, the invention provides an isolated peptide
having an amino acid sequence selected from the group consisting
of: QPQVTNGVQGN, EAQAANGGASGQ, YYTKDTNNNLTL or YYTKNTNNNLT.
In still another aspect, the invention provides a fusion protein
comprising a flagellin protein having an amino acid sequence for
an epitope of a meningococcal Class 1 outer-membrane inserted
within it, wherein the amino acid sequence is selected from the
group consisting of:
YYTKNTNNNL, HFVQQTPQSQP, YYTKDTNNNL, YTKDTNNNLT, TKDTNNNLTL,
KDTNNNLTLV, AQAANGGASG, QAANGGASGQ, AANGGASGQV, ANGGASGQVK,
NGGASGQVKV, GGASGQVKVT, GASGQVKVTK, ASGQVKVTKV, SGQVKVTKVT,
PAHYTRQNNT, AHYTRQNNTD, HYTRQNNTDVF, TRQNNTDVFV, RQNNTDVFVP,
QNNTDVFVP, QNNTDVFVPA, NNTDVFVPAV, ANVGRNAFELFLIGSATSDEAKG,
NIQAQLTEQPQVTNGVQCV, TKISDFGSFIGFK, VSVAGGGA.SQVGN, TLRAG2VANQFDDASQAIN,
DMIDVASQIGIFK, GGFSGFSG, LSENGDKAKTKNSTTE and VPRISYAHGFDLIERGKKG.
In another aspect, the invention provides a Neisseria
meningitidis Class 1 outer-membrane protein which is
substantially free from Neisseria meningitidis Class 2 and Class
3 outer-membrane proteins, wherein the Class 1 outer-membrane
protein comprises an amino acid sequence selected from the group
consisting of:
DVSLYGEIKAGVEGRNIQAQLTEQPQVTNGVQGNQVKVTKAKSRIRTKISDFGSFIGFKGSEDLG
EGLKAVWQLEQDVSVAGGGASQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAINPWDSNNDVA
SQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYYTKDTNNNLTLVPAVVGKP
GSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSATSDEAKGTDPLKNHQVHRLTGG
YEEGGLNLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENT
SYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
CA 02007248 2007-03-28
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DVSLYGEIKAGVEGRNFQLQLTEPPSKSQPQVKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLK
AVWQLEQDVSVAGGGATQWGNRESFVGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLG
IFKRHDDMPVSVRYDSPDFSGFSGSVQFVPIQNSKSAYTPAHYTRQNNTDVFVPAVVGKPGSDVY
YAGLNYKNGGFAGSYAFKYARHANVGRDAFELFLLGSTSDEAKGTDPLKNHQVHRLTGGYEEGGL
NLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQII
AGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNYQLQLTEAQAAIJGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSED
LGDGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNND
VASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTKNTNNNLTLVPAVVG
KPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSGSDQAKGTDPLKNHQVHRLTG
GYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTS
YDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF; and
DVSLYGEIKAGVEGRNIQLQLTEPLQNIQQPQVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKA
VWQLEQDVSVAGGGATRWGNRESFVGLAGEFGTLRAGRVANQFDDASKAIDPWDSNNVVASQLGI
FKRMDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYTPAHFVQQTPQQSPTLVPAVVGKPGSDV
YYAGLNYKNGGFAGNYAFKYAKHANVGRDAFELFLLGSGSDEAKGTDPLKNHQVHRLTGGYEEGG
LNLALAAQLDLSENADKTKBTSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIA
GVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF.
In still another aspect, the invention provides a substantially
purified meningococcal Class 1 outer-membrane protein having an
amino acid sequence selected from the group consisting of:
DVSLYGEIKAGVEGRNIQAQLTEQPQVTNGVQGNQVKVTKAKSRIRTKISDFGSFIGFKGSEDLG
EGLKAVWQLEQDVSVAGGGASQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAINPWDSNNDVA
SQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYYTKDTNNNLTLVPAVVGKP
GSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSATSDEAKGTDPLKNHQVHRLTGG
YEEGGLNLALAAQLDLSENGDKAKTKIJSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENT
SYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
CA 02007248 2007-03-28
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DVSLYGEIKAGVEGRNFQLQLTEPPSKSQPQVKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLK
AVWQLEQDVSVAGGGATQWGNRESFVGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLG
IFKRHDDMPVSVRYDSPDFSGFSGSVQFVPIQNSKSAYTPAHYTRQNNTDVFVPAVVGKPGSDVY
YAGLNYKNGGFAGSYAFKYARHANVGRDAFELFLLGSTSDEAKGTDPLKNHQVHRLTGGYEEGGL
NLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQII
AGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSED
LGDGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNND
VASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTKNTNNNLTLVPAVVG
KPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSGSDQAKGTDPLKNHQVHRLTG
GYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTS
YDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF; and
DVSLYGEIKAGVEGRNIQLQLTEPLQNIQQPQVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKA
VWQLEQDVSVAGGGATRWGNRESFVGLAGEFGTLRAGRVANQFDDASKAIDPWDSNNVVASQLGI
FKRMDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYTPAHFVQQTPQQSPTLVPAVVGKPGSDV
YYAGLNYKNGGFAGNYAFKYAKHANVGRDAFELFLLGSGSDEAKGTDPLKNHQVHRLTGGYEEGG
LNLALAAQLDLSENADKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIA
GVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF.
In another aspect, the invention provides a vaccine comprising a
pharmaceutically acceptable vehicle and a Neisseria meningitidis
Class 1 outer-membrane protein(s) of one or more subtypes, said
Class 1 outer-membrane protein(s) being free of meningococcal
Class 2 and Class 3 outer-membrane protein, wherein at least one
of the Class 1 outer-membrane proteins comprises an amino acid
sequence selected from the group consisting of:
DVSLYGEIKAGVEGRNIQAQLTEQPQVTNGVQGNQVKVTKAKSRIRTKISDFGSFIGFKGSEDLG
EGLKAVWQLEQDVSVAGGGASQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAINPWDSNNDVA
SQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYYTKDTNNNLTLVPAVVGKP
GSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSATSDEAKGTDPLKNHQVHRLTGG
YEEGGLNLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENT
SYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
CA 02007248 2007-03-28
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DVSLYGEIKAGVEGRNFQLQLTEPPSKSQPQVKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLK
AVWQLEQDVSVAGGGATQWGNRESFVGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLG
IFKRHDDMPVSVRYDSPDFSGFSGSVQFVPIQNSKSAYTPAHYTRQNNTDVFVPAVVGKPGSDVY
YAGLNYKNGGFAGSYAFKYARHANVGRDAFELFLLGSTSDEAKGTDPLKNHQVHRLTGGYEEGGL
NLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQII
AGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSED
LGDGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNND
VASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTKNTNNNLTLVPAVVG
KPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSGSDQAKGTDPLKNHQVHRLTG
GYEEGGLNLALAAQLDLSENGDKTMSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTS
YDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF, and
DVSLYGEIKAGVEGRNIQLQLTEPLQNIQQPQVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKA
VWQLEQDVSVAGGGATRWGNRESFVGLAGEFGTLRAGRVANQFDDASKAIDPWDSNNVVASQLGI
FKRMDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYTPAHFVQQTPQQSPTLVPAVVGKPGSDV
YYAGLNYKNGGFAGNYAFKYAKHANVGRDAFELFLLGSGSDEAKGTDPLKNHQVHRLTGGYEEGG
LNLALAAQLDLSENADKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIA
GVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF.
In yet another aspect, the invention provides a vaccine effective
against a meningococcal disease, comprising a pharmaceutically
acceptable vehicle and a meningococcal Class 1 outer-membrane
protein which is free of Neisseria meningitidis Class 2 and Class
3 outer-membrane protein, wherein the Class 1 outer-membrane
protein comprises an amino acid sequence selected from the group
consisting of:
DVSLYGEIKAGVEGRNIQAQLTEQPQVTNGVQGNQVKVTKAKSRIRTKISDFGSFIGFKGSEDLG
EGLKAVWQLEQDVSVAGGGASQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAINPWDSNNDVA
SQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYYTKDTNNNLTLVPAVVGKP
GSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSATSDEAKGTDPLKNHQVHRLTGG
YEEGGLNLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENT
SYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
CA 02007248 2007-03-28
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DVSLYGEIKAGVEGRNFQLQLTEPPSKSQPQVKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLK
AVWQLEQDVSVAGGGATQWGNRESFVGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLG
IFKRHDDMPVSVRYDSPDFSGFSGSVQFVPIQNSKSAYTPAHYTRQNNTDVFVPAVVGKPGSDVY
YAGLNYKNGGFAGSYAFKYARHANVGRDAFELFLLGSTSDEAKGTDPLKNHQVHRLTGGYEEGGL
NLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQII
AGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSED
LGDGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNND
VASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTKNTNNNLTLVPAVVG
KPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSGSDQAKGTDPLKNHQVHRLTG
GYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTS
YDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF, and
DVSLYGEIKAGVEGRNIQLQLTEPLQNIQQPQVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKA
VWQLEQDVSVAGGGATRWGNRESFVGLAGEFGTLRAGRVANQFDDASKAIDPWDSNNVVASQLGI
FKRMDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYTPAHFVQQTPQQSPTLVPAVVGKPGSDV
YYAGLNYKNGGFAGNYAFKYAKHANVGRDAFELFLLGSGSDEAKGTDPLKNHQVHRLTGGYEEGG
LNLALAAQLDLSENADKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIA
GVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF.
In another aspect, the invention provides a vaccine against
Neisseria meningitidis comprising a peptide-carrier protein
conjugate, wherein the peptide has an amino acid sequence
selected from the group consisting of:
YYTKNTNNNL, HFVQQTPQSQP, YYTKDTNNNL, YTKDTNNNLT, TKDTNNNLTL,
KDTNNNLTLV, AQAANGGASG, QAANGGASGQ, AANGGASGQV, ANGGASGQVK,
NGGASGQVKV, GGASGQVKVT, GASGQVKVTK, ASGQVKVTKV, SGQVKVTKVT,
PAHYTRQNNT, AHYTRQNNTD, HYTRQNNTDVF, TRQNNTDVFV, RQNNTDVFVP,
QNNTDVFVP, QNNTDVFVPA, NNTDVFVPAV, ANVGRNAFELFLIGSATSDEAKG,
NIQP,QLTEQPQVINGVQGU, TKISDFGSFIGFK, VSVAGOGASQWGN, TLRAG2VANQFDDASQAIN,
DSTIIVDVASQLGIFK, GGFSGFSG, LSENMKAKTMSTTE and VPRISYAHGFDLIEI2GIQtG.
CA 02007248 2007-03-28
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In another aspect, the invention provides a vaccine against
Neisseria meningitidis comprising a peptide-carrier protein
conjugate, wherein the peptide has an amino acid sequence
selected from the group consisting of QPQVTNGVQGN, PPSKSQP,
QAANGGASGQV, PLQNIQQPQ, YYTKDTNNNLTL, HYTRQNNTDVF, YYTKNTNNNLTL
and HFVQQTPQQSPT.
In still another aspect, the invention provides an antigenic
composition comprising a pharmaceutically acceptable vehicle and
a conjugate of a peptide linked to a carrier protein, wherein the
peptide has amino acid sequence selected from the group
consisting of: YYTKNTNNNL, HFVQQTPQSQP, YYTKDTNNNL, YTKDTNNNLT,
TKDTNNNLTL, KDTNNNLTLV, AQAANGGASG, QAANGGASGQ, AANGGASGQV,
ANGGASGQVK, NGGASGQVKV, GGASGQVKVT, GASGQVKVTK, ASGQVKVTKV,
SGQVKVTKVT, PAHYTRQNNT, AHYTRQNNTD, HYTRQNNTDVF, TRQNNTDVFV,
RQNN'IDVFVP, QNNTDVFVP, QNVZTIVF'VPA, NNTDVFVPAV, IGSATSDEAKG,
NIQP.QLTEQPQV'INGVQCU, TKISDFGSFIGFK, VSVAGGCASQWM, TLRAC2VANQFDDASQAIlV,
DSNNDVASQLGIFK, GGFSGFSG, LSEN KAKTKNSTI'E and VPRISYAHGFDLIERGKKG.
In still another aspect, the invention provides an antigenic
composition comprising a pharmaceutically acceptable vehicle and
a hybrid protein, said protein comprising one or more peptides
fused internally or terminally to a Salmonella flagellin, wherein
the peptide has an amino acid sequence selected from the group
consisting of: YYTKNTNNNL, HFVQQTPQSQP, YYTKDTNNNL, YTKDTNNNLT,
TKDTNNNLTL, KDTNNNLTLV, AQAANGGASG, QAANGGASGQ, AANGGASGQV,
ANGGASGQVK, NGGASGQVKV, GGASGQVKVT, GASGQVKVTK, ASGQVKVTKV,
SGQVKVTKVT, PAHYTRQNNT, AHYTRQNNTD, HYTRQNNTDVF, TRQNNTDVFV,
RQNNTDVFVP, QNNTDVFVP, QNN'IflVFVPA, NV'MVF'VPAV, ANVGRIMF'ELFLIGSATSDEAKG,
NIQAQLTEQPQVTNGVQGN, TKISDFGSFIGFK, VSVAZ3GASQWQN, TLRAC2VANQFDDASQPAM,
DSNIIVDVASQIGIFK, GGFSGFSG, LSIINGDKAKTKNSTTE and VPRISYAHGFDLIERGKKG.
CA 02007248 2007-03-28
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In another aspect, the invention provides an antigenic
composition comprising a pharmaceutically acceptable vehicle and
a hybrid protein, said protein comprising one or more peptides
fused internally or terminally to a Salmonella flagellin, wherein
the peptide has an amino acid sequence consisting from the group
consisting of: QPQVTNGVQGN, PPSKSQP, QAANGGASGQV, PLQNIQQPQ,
YYTKDTNNNLTL, HYTRQNNTDVF, YYTKNTNNNLTL and HFVQQTPQQSPT.
In still another aspect, the invention provides an antigenic
composition which elicits antibodies against Neisseria
meningitidis, said composition comprising a pharmaceutically
acceptable vehicle and a meningococcal capsular polysaccharide or
oligosaccharide derived therefrom, conjugated to a hybrid protein
comprising a carrier protein and a peptide having an amino acid
sequence selected from the group consisting of: YYTKNTNNNL,
HFVQQTPQSQP, YYTKDTNNNL, YTKDTNNNLT, TKDTNNNLTL, KDTNNNLTLV,
AQAANGGASG, QAANGGASGQ, AANGGASGQV, ANGGASGQVK, NGGASGQVKV,
GGASGQVKVT, GASGQVKVTK, ASGQVKVTKV, SGQVKVTKVT, PAHYTRQNNT,
AHYTRQNNTD, HYTRQNNTDVF, TRQNNTDVFV, RQNNTDVFVP, QNNTDVFVP,
QNNTDVFVPA, NNTDVFVPAV, ANVGRNAFELFLIGSATSDEAKG,
NIQAQLTEQPQVTNGVQGN, TKISDFGSFIGFK, VSVACK3GASQWM, TLRAGf2VANQFDDASQAIlV,
DS=VASQLGIFK, GGFSGFSG, LSE[JGDKAECI'KNSTTE and VPRISYAHGFDLIERGKFCG.
In another aspect, the invention provides an antigenic
composition which elicits antibodies against Neisseria
meningitidis, said composition comprising a pharmaceutically
acceptable vehicle, and a meningococcal capsular polysaccharide
or oligosaccharide derived therefrom, wherein the polysaccharide
or oligosaccharide is conjugated to a hybrid protein comprising a
carrier protein and a peptide having an amino acid sequence
selected from the group consisting of: QPQVTNGVQGN, PPSKSQP,
QAANGGASGQV, PLQNIQQPQ, YYTKDTNNNLTL, HYTRQNNTDVF, YYTKNTNNNLTL
and HFVQQTPQQSPT.
CA 02007248 2007-03-28
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In still another aspect, the invention provides a vaccine
comprising one or more peptides, in liposomes or microspheres,
wherein the peptide has an amino acid sequence selected from the
group consisting of:
YYTKNTNNNL, HFVQQTPQSQP, YYTKDTNNNL, YTKDTNNNLT, TKDTNNNLTL,
KDTNNNLTLV, AQAANGGASG, QAANGGASGQ, AANGGASGQV, ANGGASGQVK,
NGGASGQVKV, GGASGQVKVT, GASGQVKVTK, ASGQVKVTKV, SGQVKVTKVT,
PAHYTRQNNT, AHYTRQNNTD, HYTRQNNTDVF, TRQNNTDVFV, RQNNTDVFVP,
QNNTDVFVP, QNNTDVFVPA, NNTDVFVPAV, ANVGRNAFELFLIGSATSDEAKG,
NIQIAQLTEQPQV'INGVQCN, TKISDFGSFIGFK, VSVACGGA~, TLRAGRVANQFDDASQAIN,
DSNNDVASQLGIFK, GGFSGFSG, LSENGDKAKTKNSTTE and VPRISYAHGFDLIERGKKG.
In another aspect, the invention provides a vaccine comprising
meningococcal Class 1 outer-membrane protein(s) of one or more
subtypes, wherein said proteins are substantially free from Class
2 and 3 outer membrane proteins, in liposomes or microspheres,
wherein at least one of the meningococcal Class 1 outer-membrane
proteins comprises an amino acid sequence selected from the group
consisting of:
DVSLYGEIKAGVEGRNIQAQLTEQPQVTNGVQGNQVKVTKAKSRIRTKISDFGSFIGFKGSEDLG
EGLKAVWQLEQDVSVAGGGASQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAINPWDSNNDVA
SQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYYTKDTNNNLTLVPAVVGKP
GSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSATSDEAKGTDPLKNHQVHRLTGG
YEEGGLNLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENT
SYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNFQLQLTEPPSKSQPQVKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLK
AVWQLEQDVSVAGGGATQWGNRESFVGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLG
IFKRHDDMPVSVRYDSPDFSGFSGSVQFVPIQNSKSAYTPAHYTRQNNTDVFVPAVVGKPGSDVY
YAGLNYKNGGFAGSYAFKYARHANVGRDAFELFLLGSTSDEAKGTDPLKNHQVHRLTGGYEEGGL
NLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQII
AGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
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DVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSED
LGDGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNND
VASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTKNTNNNLTLVPAVVG
KPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSGSDQAKGTDPLKNHQVHRLTG
GYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTS
YDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF, and
DVSLYGEIKAGVEGRNIQLQLTEPLQNIQQPQVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKA
VWQLEQDVSVAGGGATRWGNRESFVGLAGEFGTLRAGRVANQFDDASKAIDPWDSNNVVASQLGI
FKRMDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYTPAHFVQQTPQQSPTLVPAVVGKPGSDV
YYAGLNYKNGGFAGNYAFKYAKHANVGRDAFELFLLGSGSDEAKGTDPLKNHQVHRLTGGYEEGG
LNLALAAQLDLSENADKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIA
GVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF.
In another aspect, the invention provides an antigenic
composition comprising a pharmaceutically acceptable vehicle and
a Class 1 N. meningitidis outer-membrane protein, wherein said
Class 1 outer-membrane protein is substantially free from Class 2
or Class 3 outer-membrane proteins, and wherein said Class 1
outer-membrane protein comprises an amino acid sequence selected
from the group consisting of:
DVSLYGEIKAGVEGRNIQAQLTEQPQVTNGVQGNQVKVTKAKSRIRTKISDFGSFIGFKGSEDLG
EGLKAVWQLEQDVSVAGGGASQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAINPWDSNNDVA
SQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYYTKDTNNNLTLVPAVVGKP
GSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSATSDEAKGTDPLKNHQVHRLTGG
YEEGGLNLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENT
SYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNFQLQLTEPPSKSQPQVKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLK
AVWQLEQDVSVAGGGATQWGNRESFVGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLG
IFKRHDDMPVSVRYDSPDFSGFSGSVQFVPIQNSKSAYTPAHYTRQNNTDVFVPAVVGKPGSDVY
YAGLNYKNGGFAGSYAFKYARHANVGRDAFELFLLGSTSDEAKGTDPLKNHQVHRLTGGYEEGGL
NLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQII
AGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
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DVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSED
LGDGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNND
VASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTKNTNNNLTLVPAVVG
KPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSGSDQAKGTDPLKNHQVHRLTG
GYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTS
YDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF, and
DVSLYGEIKAGVEGRNIQLQLTEPLQNIQQPQVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKA
VWQLEQDVSVAGGGATRWGNRESFVGLAGEFGTLRAGRVANQFDDASKAIDPWDSNNVVASQLGI
FKRMDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYTPAHFVQQTPQQSPTLVPAVVGKPGSDV
YYAGLNYKNGGFAGNYAFKYAKHANVGRDAFELFLLGSGSDEAKGTDPLKNHQVHRLTGGYEEGG
LNLALAAQLDLSENADKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIA
GVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF.
In another aspect, the invention provides an antigenic
composition comprising a polysaccharide or oligosaccharide, and a
peptide having an amino acid sequence selected from the group
consisting of YYTKNTNNNL, HFVQQTPQSQP, YYTKDTNNNL, YTKDTNNNLT,
TKDTNNNLTL, KDTNNNLTLV, AQAANGGASG, QAANGGASGQ, AANGGASGQV,
ANGGASGQVK, NGGASGQVKV, GGASGQVKVT, GASGQVKVTK, ASGQVKVTKV,
SGQVKVTKVT, PAHYTRQNNT, AHYTRQNNTD, HYTRQNNTDVF, TRQNNTDVFV,
RQNNTDVFVP, QNNTDVFVP, QNNTDVFVPA, NNTDVFVPAV,
ANVGRNAFELFLIGSATSDEAKG, NIQAQLTEQPQVINGVQCN, TKISDFGSFIGFK,
VSVAGGGASQWCN, TLRAGRVANQFDDASQAIlN, DSTIIVDVASQLGIFK, GGFSGFSG,
LSE[JGDKAK=STTE and VPRISYAHGFDLIERGKKG.
In yet another aspect, the invention provides an antigenic composition,
said composition comprising a pharmaceutically acceptable vehicle
and a polysaccharide or oligosaccharide conjugated to a peptide
having an amino acid sequence selected from the group consisting
of YYTKNTNNNL, HFVQQTPQSQP, YYTKDTNNNL, YTKDTNNNLT, TKDTNNNLTL,
KDTNNNLTLV, AQAANGGASG, QAANGGASGQ, AANGGASGQV, ANGGASGQVK,
NGGASGQVKV, GGASGQVKVT, GASGQVKVTK, ASGQVKVTKV, SGQVKVTKVT,
PAHYTRQNNT, AHYTRQNNTD, HYTRQNNTDVF, TRQNNTDVFV, RQNNTDVFVP,
QNNTDVFVP, QNN'IDVFVPA, NN'lDVFVPAV, IGSATSDEAKG,
CA 02007248 2007-03-28
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NIQAQLTEQPQVTNWQCN, TKISDFGSFIGFK, VSVACGC''~ASQWCN, 'I'LRAQ2VANQFDDASQAIN,
DStZ]DVASQLGIFK, GGFSGFSG, LSE[JMKAKTTKDTSTIE and VPRISYAHGFDLIERGKKG.
In still another aspect, the invention provides a recombinant
microorganism that expresses a fusion protein comprising a
carrier protein and an immunogenic portion of subtype P1.15,
P1.7.16, or P1.2 meningococcal Class 1 outer-membrane protein,
wherein said meningococcal Class 1 outer-membrane protein
comprises an amino acid sequence selected from the group
consisting of:
DVSLYGEIKAGVEGRNIQAQLTEQPQVTNGVQGNQVKVTKAKSRIRTKISDFGSFIGFKGSEDLG
EGLKAVWQLEQDVSVAGGGASQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAINPWDSNNDVA
SQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYYTKDTNNNLTLVPAVVGKP
GSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSATSDEAKGTDPLKNHQVHRLTGG
YEEGGLNLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENT
SYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNFQLQLTEPPSKSQPQVKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLK
AVWQLEQDVSVAGGGATQWGNRESFVGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLG
IFKRHDDMPVSVRYDSPDFSGFSGSVQFVPIQNSKSAYTPAHYTRQNNTDVFVPAVVGKPGSDVY
YAGLNYKNGGFAGSYAFKYARHANVGRDAFELFLLGSTSDEAKGTDPLKNHQVHRLTGGYEEGGL
NLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQII
AGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSED
LGDGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNND
VASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTKNTNNNLTLVPAVVG
KPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSGSDQAKGTDPLKNHQVHRLTG
GYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTS
YDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF, and
CA 02007248 2007-03-28
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DVSLYGEIKAGVEGRNIQLQLTEPLQNIQQPQVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKA
VWQLEQDVSVAGGGATRWGNRESFVGLAGEFGTLRAGRVANQFDDASKAIDPWDSNNVVASQLGI
FKRMDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYTPAHFVQQTPQQSPTLVPAVVGKPGSDV
YYAGLNYKNGGFAGNYAFKYAKHANVGRDAFELFLLGSGSDEAKGTDPLKNHQVHRLTGGYEEGG
LNLALAAQLDLSENADKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIA
GVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF.
In still another aspect, the invention provides a recombinant
microorganism that is transformed with a gene that expresses a
Class 1 meningococcal outer-membrane protein comprising an amino
acid sequence selected from the group consisting of:
DVSLYGEIKAGVEGRNIQAQLTEQPQVTNGVQGNQVKVTKAKSRIRTKISDFGSFIGFKGSEDLG
EGLKAVWQLEQDVSVAGGGASQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAINPWDSNNDVA
SQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYYTKDTNNNLTLVPAVVGKP
GSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSATSDEAKGTDPLKNHQVHRLTGG
YEEGGLNLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENT
SYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNFQLQLTEPPSKSQPQVKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLK
AVWQLEQDVSVAGGGATQWGNRESFVGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLG
IFKRHDDMPVSVRYDSPDFSGFSGSVQFVPIQNSKSAYTPAHYTRQNNTDVFVPAVVGKPGSDVY
YAGLNYKNGGFAGSYAFKYARHANVGRDAFELFLLGSTSDEAKGTDPLKNHQVHRLTGGYEEGGL
NLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQII
AGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
DVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSED
LGDGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNND
VASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTKNTNNNLTLVPAVVG
KPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSGSDQAKGTDPLKNHQVHRLTG
GYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTS
YDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF, and
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DVSLYGEIKAGVEGRNIQLQLTEPLQNIQQPQVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKA
VWQLEQDVSVAGGGATRWGNRESFVGLAGEFGTLRAGRVANQFDDASKAIDPWDSNNVVASQLGI
FKRMDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYTPAHFVQQTPQQSPTLVPAVVGKPGSDV
YYAGLNYKNGGFAGNYAFKYAKHANVGRDAFELFLLGSGSDEAKGTDPLKNHQVHRLTGGYEEGG
LNLALAAQLDLSENADKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIA
GVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF.
In still another aspect, the invention provides a vaccine
comprising one or more fragments of meningococcal Class 1 outer-
membrane protein, said fragment comprising one or more amino acid
sequences selected from the group consisting of:
YYTKNTNNNL, HFVQQTPQSQP, YYTKDTNNNL, YTKDTNNNLT, TKDTNNNLTL,
KDTNNNLTLV, AQAANGGASG, QAANGGASGQ, AANGGASGQV, ANGGASGQVK,
NGGASGQVKV, GGASGQVKVT, GASGQVKVTK, ASGQVKVTKV, SGQVKVTKVT,
PAHYTRQNNT, AHYTRQNNTD, HYTRQNNTDVF, TRQNNTDVFV, RQNNTDVFVP,
QNNTDVFVP, QNNTDVFVPA, NNTDVFVPAV, ANVGRNAFELFLIGSATSDEAKG,
NIQAQLTEQPQVTNGVQGN, TKISDFGSFIGFK, VSVACOC'zASQWCN, TLRAGRVANQFDDASQAIN,
DSNNDVASQLGIFK, GGFSGFSG, LSENGDKAKTKNSTTE and VPRISYAHGFDLIERGKKG.
In another aspect, the invention provides a recombinant
microorganism that expresses a fusion protein comprising a
carrier protein and an immunogenic portion of a meningococcal
Class 1 outer-membrane protein, wherein the Class 1 outer-
membrane protein has an amino acid sequence selected from the
group consisting of:
DVSLYGEIKAGVEGRNFQLQLTEPPSKSQPQVKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLK
AVWQLEQDVSVAGGGATQWGNRESFVGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLG
IFKRHDDMPVSVRYDSPDFSGFSGSVQFVPIQNSKSAYTPAHYTRQNNTDVFVPAVVGKPGSDVY
YAGLNYKNGGFAGSYAFKYARHANVGRDAFELFLLGSTSDEAKGTDPLKNHQVHRLTGGYEEGGL
NLALAAQLDLSENGDKAKTKNSTTEIAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQII
AGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF;
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DVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSED
LGDGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNND
VASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPIQNSKSAYTPAYYTKNTNNNLTLVPAVVG
KPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAFELFLIGSGSDQAKGTDPLKNHQVHRLTG
GYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTS
YDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF; and
DVSLYGEIKAGVEGRNIQLQLTEPLQNIQQPQVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKA
VWQLEQDVSVAGGGATRWGNRESFVGLAGEFGTLRAGRVANQFDDASKAIDPWDSNNVVASQLGI
FKRMDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYTPAHFVQQTPQQSPTLVPAVVGKPGSDV
YYAGLNYKNGGFAGNYAFKYAKHANVGRDAFELFLLGSGSDEAKGTDPLKNHQVHRLTGGYEEGG
LNLALAAQLDLSENADKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIA
GVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF.
Finally, in another aspect the invention provides an antigenic
composition comprising a pharmaceutically acceptable vehicle and
a peptide having an amino acid sequence selected from the group
consisting of:
YYTKNTNNNL, HFVQQTPQSQP, YYTKDTNNNL, YTKDTNNNLT, TKDTNNNLTL,
KDTNNNLTLV, AQAANGGASG, QAANGGASGQ, AANGGASGQV, ANGGASGQVK,
NGGASGQVKV, GGASGQVKVT, GASGQVKVTK, ASGQVKVTKV, SGQVKVTKVT,
PAHYTRQNNT, AHYTRQNNTD, HYTRQNNTDVF, TRQNNTDVFV, RQNNTDVFVP,
QNNTDVFVP, QNNTDVFVPA, NNTDVFVPAV, ANVGRNAFELFLIGSATSDEAKG,
NIQAQLTEQPQV'ING\TQCN, TKISDFGSFIGFK, VSVAGGCASQWM, TLRAGRVANQFDDASQAIN,
DSNNDVASQLGIFK, GGFSGFSG, LSENGDKP,K'IIQNSTTE and VPRISYAHGFDLIERGKKG.
Brief-Description-of-the Fiqures
--------------------------------
Figure 1. Scheme for amplification of genes encoding
meningococcal Class I outer membrane protein by PCR (Polymerase
Chain Reaction).
CA 02007248 2007-03-28
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Figure 2. 5' gene sequences encoding VR1 (first variable
region) of Class I outer membrane proteins of several N.
meninqitidis subtypes.
Figure 3. 3' gene sequences encoding VR2 (second variable
region) of Class I outer membrane proteins of several N.
meninqitidis subtypes.
Figure 4. Epitope scanning by reaction of monoclonal
antibodies with solid phase decapeptides spanning the predicted
amino acid sequences of Class I proteins from strains P1.7,16,
P1.16 and P1.15. Adjacent decapeptides differ by five amino acid
residues. Annotations show the strain from which the sequence was
derived, the mAb used and its subtype specificity.
Figure S. Reaction of the monoclonal
antibodies with a series of overlapping decapeptides
corresponding to variable regions VR1 and VR2, with adjacent
peptides differing by a single amino acid residue. Annotations
show the strain from which the sequence was derived, the mAb used
and its subtype specificity.
CA 02007248 2006-06-15
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Figure 6. Construction of recombinant
flagellins expressing variable region epitopes of N.
menin~itidis Class I OMP subtype P1.6,16.
Figure 7. Structure of recombinant flagellins
expressing variable region epitopes of N.
meningitidis Class I OMP subtype P1.6,16.
----------------
Figure 8. Representative chromatogram of high
performance liquid chromatography of a recombinant
flagellin.
Figure 9. Representative analysis by SDS-PAGE
of recombinant flagellin.
Figure 10. Representative Western blot
analyses of a conjugate comprising an epitope of N.
meninqitidis Class I OMP conjugated to CRM197'
Figure 11. Putative conformation of N.
meninqitidis Class I OMP subtype P1.16.
Detailed Descrip---- tion of the Invention
------------------- ----------------------
This invention pertains to vaccines comprising
isolated OMV's, meningococcal Class 1 OMP, fragments
of the OMP (e.g., prepared by the application of
cyanogen bromide)and oligopeptides bearing epitopes
of the OMP; the preparation of isolated OMV's, pure
Class 1 outer-membrane proteins, using mutant
strains which do not express the Class 2/3 outer-
membrane protein; the preparation of isolated OMV's
pure Class 1 outer-membrane proteins with the aid of
cloned DNA in recombinant DNA expression vectors.
This invention also comprises the application of
genetic engineering with the object of producing
isolated OMV's, Class I OMP or portions thereof,
CA 02007248 2006-06-15
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genetic fusions of Class I OMP, portions or epitopes
thereof; and the preparation of multivalent Class 1
outer-membrane protein vaccine through peptide
synthesis, as the epitopes with a short peptide
chain can be synthetically prepared.
It has emerged that meningococcal Class 1
outer-membrane proteins induce a strong bactericidal
immune response to the strains containing the
appropriate subtype epitopes, irrespective of
whether these are from group A,B,C,W-135,and Y
strains. The polysaccharide vaccine can be enhanced
or replaced by a vaccine according to the invention
as a vaccine with broad, extensive action against
most serotypes, The protective bactericidal mono-
clonal antibodies specific for the Class 1 outer-
membrane protein react strongly with fragments that
have been split off and short synthetic peptides
which have been prepared using the amino acid
sequence of Class 1 outer-membrane proteins. Since
meningococcal disease is currently caused chiefly by
group B meningococci and because the Class 1 outer-
membrane proteins occurring in group B meningococci
also occur in group A, C, W-135 and Y meningococci,
vaccines of this invention which comprise one or
more Class 1 OMP epitopes derived from N.
meninqitidis group B should be effective in
preventing disease caused by group A, C, W-135 and
Y. Preferably, the preparation of such a vaccine
starts from at least two different immunogenic and
protective epitopes which have been selected on
9 2007248
epidemiological grounds. Vaccines according to the
invention comprise, for example, at least one protein
which is obtained either in OMV formulation or by
purification from mutant strains producing one or more
Class I OMP or at least two fragments prepared through
a cyanogen bromide fragmentation or at least two
synthetic peptides, chosen from about 10 major
epitopes, or products obtained by gene expression via
recombinant DNA technology, which contain the desired
epitopes. To maximize efficacy to a broad range. of
meningococcal strains, the greater number of different
protective epitopes in the vaccine the better. In
addition, the vaccines according to the invention may
advantageously contain meningococci A and C or
optionally W-135 and Y polysaccharides and/or
detergents. Preferably, the A and C polysaccharides
are covalently coupled to a protein or polypeptide
carrier. These carriers include, for example, isolated
OMV, the Class I OMP protein, T-helper epitopes,
bacterial toxins, toxoids, nontoxic mutants (CRM's),
recombinant Salmonella flagellin and viral particles
such as rotavirus VP6 protein, Hepatitis B surface
antigen or parvovirus VP1 and VP2 proteins. Both
Zwitterionogenic, cationogenic, anionogenic and
nonionogenic detergents can be used. ExamplesIx of
such detergents are Zwi.ttergent 3-10, Zwittergent 3--
14 (N-tetradecyl-N, N-dimethyl-3-ammonia-l-propane
sulphonate), Tween-20*, sodium deoxycholate, sodium
cholate and octylglucoside. The vaccines according
to the invention may also contain an adsorbent such
XTrade-mark
2007248
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as aluminium hydroxide, calcium phosphate; or
advantageously, aluminium phosphate. The fragments,
proteins, peptides can also be processed in immuno-
stimulating complexes (ISCOMS), liposomes or
microspheres for delivering and/or use as an
adjuvant or in connection with other adjuvants so
that greater immunogenicity is obtained.
This invention encompasses isolated OMV,
substantially pure meningococcal class 1 outer
membrane proteins (of any subtype) and fragments of
the proteins containing epitopes thereof. The
fragments can be any portions of the molecular
weight of 25kD or less which contain epitopes which
are bound by protective bactericidal antibodies
against N. meningitidis. These include proteolytic
fragments and synthetic oligopeptides which are
comprised of amino acid sequences which correspond,
at least in part, to epitopes of a Class I OMP.
The isolated. OMV's, Class I OMP, fragments or
epitope-containing oligopeptides derived therefrom
can be comprised of amino acid sequences which are
different, but essentially biologically equivalent
to-the natural sequences. These sequences can
include sequences in which functionally equivalent
amino acid residues are substituted for residues
within the sequerice resulting in a silent change.
For example, one or more amino acid residues within
the sequence can be substituted by another amino
acid of a similar polarity which acts as a func-
tional equivalent, resulting in a silent alteration.
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Substitutes for an amino acid within the sequence
may be selected from other members of the class to
which the amino acid belongs. For example, the
nonpolar (hydrophobic) amino acids include glycine,
alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan and methionine. The polar
neutral amino acids include serine, threonine,
cysteine, tyrosine, asparagine, and glutamine. The
charged (basic) amino acids include arginine, lysine
arid hi-sti"dine. The negatively charged (acidic)
amino-acids include aspartic and glutamic acids.
Additionally, isolated OMV's, the class I-0MP,
fragments or the oligopeptides can be modified for
conjugation to other molecules (e.g., by the
attachment of coupling groups such as the amino
acids cysteine and/or lysine or other linking groups
and/or -spac-er-~groups) including other class 1 OMP of
a different subtype, T cell epitopes, B cell
epitopes, carrier peptidesor proteins or
adjuvanting molecules.
As described in detail below, the Class I OMP.
fragtnents or olIgopep.t-i.des can be used in many
different forms (e.g., alone, in mixtures, or as
conjugates and genetic fusions produced from recom-
binant DNA vectors) in vaccines. For these pur-
poses, the materials can be produced by isolation
from N. meningitidis, by proteolytic digestion, by
chemical synthesis, or by expression as recombinant
molecules. The methods of production and use of the
isolated OMV's, the class I OMP and the fragments
and the oligopeptides of class 1 OMP are described
below.
CA 02007248 2006-06-15
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Protein modeling and structure analysis of the
Class I OMPs were performed using the principles for
several E. coli outer membrane proteins. (Vogel, H.
et al., J. Mol. Bio., 190:191 (1986); Ference, T. et
---------------
al., J. Mol. Bio., 201:493 (1988) and Tommassen, J.
in "Membrane Biogenesis", NATO ASI Series H16,
------------------------
pp. 351, Springer-Verlag, NY (1988)). The derived
amino acid sequences of the Class I OMPs were used
for the modeling studies and comparison. The amino
acid sequence homology was compared to other gram
negative bacterial porin proteins and similarity was
established for the protein structure. Exposed
surface loops and transmembrane structures were very
similar for these porin proteins. With the
information revealed concerning variable and
constant region protective epitopes of N.
meninqitidis and their structure, one can predict
based upon the amino acid sequence where protective
epitopes may reside for other pathogenic gram
negative bacteria to be evaluated and included in
vaccines for the same.
Production of isolated OMV's
----------------------------------
OMV's can be produced either from the culture
supernatant or from the bacterial cells after
fragmentation as described by Beuvery et al. (1983)
Infect. Immun. 40: 369-380. OMV's carrying proteins
from more than one meningococcus can be isolated
from strains manipulated to express heterologous
proteins.
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Production and Purification of Class I OMP and CNBr
fragmentsthereof
Class 1 and Class 3 outer membrane proteins can
be isolated as described by Beuvery, E.C. et al.,
Antonie wan Leeuvenhoek J. Microbiol. 52:232 (1986).
------- --- ----------- - --------- The production o:E substantially pure
Class I OMP
free of Class 2 or 3 OMP's is achieved by this
method using mutant meningococcal strains which do
not express Class 2/3 OMP. A preferred strain for
production of Class I OMP is the H1115 strain,
deposited as CBS 636.89.
Fragments can be produced by cyanogen bromide
cleavage as described by Teerlink T. et al., J. Exp.
Med. 166:63 (1987) for a gonococcal protein. The
N-terminal fragment is referred to as CB-1 and the
C-terminal fragment is referred to as CB-2. These
CNBr fragments can be purified via reverse phase
HPLC on a VydaxTM C4 or an AquaporTM R-300 column
using a water/acetonitrile gradient. Alternatively,
the fragment can be purified by multiple cold
trichloroacetic acid precipitations. These proce-
dures remove greater than 95% of interferring
contaminants (e.g., buffer salts, detergents and
fragment contaminants).
PreparationoffraZments_and_oligopeptides
containing_epitopesofclassIOMP
A. Preparation by proteolytic digestion
Oligopeptides containing epitopes reactive with
bactericidal antibodies against N. meninZitidis can
be produced by digestion of the class I OMP, CB-1 or
CB-2 fragments with proteinases such as endoLys-C,
14 -
2007248
endoArg-C, endoGlu-C and staphylococcins V8-protease.
The digested fragments can be purified by, for example,
high performance liquid chromatographic (HPLC)
techniques.
B. Preparation by chemical synthesis
Oligopeptides of this invetionb can be
synthesized by standard solid peptide synthesis
(Barany, G. and Merrifield, R.B., The Peptides 2:1-284,
Gross, E. and Meienhofer, J., Eds., Academic Press,.New
York) using tert-butyloxycarbonyl amino acids and
phenylacetamidomethyl resins (Mitchell, A.R. et al. J.
Org. Chem. 43:2845-2852 (1978)) or 9-fluor-
enylmethyloxycarbonyl amino acids on a polyamide
support (Dryland, A. and Sheppard, R.C., J. Chem. So.
Perkin Trans. I, 125-137 (1986)). Alternatively,
synthetic peptides can be prepared by pepscan synthesis
(Geysen, H.M. et al., J. Immunol. Methods 03:259
(1987); Proc. Natl. Acad. Sci. USA 81:3998 (1984)),
Cambridge Research Biochemicals, Cambridge, U.K. or by
standard liquid phase peptide synthesis. The deletion
or substitution of amino acids (and including
extensions and additions to amino acids) in other ways
which do not substantially detract from the
immunological properties of the oligopeptide.
C. Preparation by recombinant DNA techniques
The Class I OMP, fragments and oligopeptides
which exhibit epitopes of the Class I OMP can be
~~.
2007248
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produced by recombinant DNA techniques. In general,
these entail obtaining DNA sequences which encode
the desired OMP, [Barlow et al., (1989) Mol. Micro.,
3:131) fragment or oligopeptide sequences and intro-
ducing into an appropriate vector/host expression
system one or more similar or different DNA se-
quences of Class I OMP's where it is expressed. The
DNA cari consist of the gene encoding the Class I OMP
or any segment of the gene which encodes a function-
al epitope of the OMP. The DNA can be fused to DNA
encoding other antigens of N. meninRitidis (such as
other outer membrane proteins either of the same or
different class) or antigens of other bacteria,
viruses, parasites or fungi to create genetically
fused (sharing a common peptide backbone), multi-
valent antigens. For example, Class I OMP fragments
can be fused to another class 1 outer membrane
protein of a different subtype (or fragments or
epitopes thereof) of N. meningitidis to yield fusion
proteins comprising multiple class 1 outer membrane
protein subtype determinants.
Genetic engineering techniques can also be used
to characterize, modify and/or adapt the encoded
peptides or proteins. For example, site directed
mutagenesis to modify an OMP fragment in regions
outside the protective domains, for example, to
increase the solubility of the subfragment to allow
easier purification. DNA can also be manipulated to
effect superproduction of OMP fragments or combina-
tions thereof in various organisms.
20 '7248
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DNA encoding a Class I OMP, fragments or oligo-
peptides can be synthesized or isolated and sequenc-
ed as described 'by Barlow, A.K. et al. Infect.
Immune 55:2734-40 (1987) and Barlow, A.K. et al.,
Mol.Micro. 3:131 (1989). Class I OMP genes can be
amplified from bacterial DNA by the methods of
Mullis and Faloona, (1987) Method. Enzym.
155:335-350, using the primer sequences disclosed
'herein. Related DNA sequences for class 1 OMP of
different subtypes can be obtained by the procedures
described and the amino acid sequences deduced.
A variety of host-vector systems can be used.to
express the oligopeptides of this invention.
Primarily the vector system must be compatible with
the host cell used. Host-vector systems include but
are not limited to the following: bacteria trans-
formed with bacteriophage DNA, plasmid DNA or cosmid
DNA; microorganisms such as yeast containing yeast
vectors; mammalian cell systems infected with virus
(e.g., vaccinia virus, adenovirus, etc.); insect
cell systems inf:ected with virus (e.g., baculo-
virus). The expression elements of these vectors
vary in their strength and specificities. Depending
upon the host-vector system utilized, any one of a
-number oÃsuitable transcription and translation
elements can be used.
In order to obtain efficient expression of the
cloned DNA, a promoter must be present in the
expression vector. RNA polymerase normally binds to
the promoter and initiates transcription of a gene
200'7248
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or a group of linked genes and regulatory elements
(called an operon). Promoters vary in their
"strength", i.e., their ability to promote tran-
scription. It is desirable to use strong promoters
in order to obtain a high level of transcription
and, hence, a high level of DNA expression. Depend-
ing upon the host cell system any one of a number of
suitable promoters can be used. For instance, for
E. coli, its bacteriophages or plasmids, promoters
such as the lac promoter, trp promoter, recA pro-
moter, ribosomal RNA promoter, and PR or PL pro-
moters of coliphage lambda and others including but
not limited to lacUV5, ompF, bla, lpp and the like,
may be used to d:irect high levels of transcription
of adjacent DNA segments. Additionally, a hybrid
trp-1acUV5 (tac) promoter or other E. coli promoters
produced by recombinant DNA or other synthetic DNA
techniques may be used to provide for transcription
of the inserted DNA.
Bacterial host cells and expression vectors may
be chosen which inhibit the action of the promoter
unless specifically induced. In certain operons the
addition of -specific inducers is necessary for ef-
ficient transcription of the inserted DNA; for exam-
ple, the lac operon is induced by the addition of
lactose or IPTG (isopropylthio-beta-D-galactoside).
A variety of other operons, such as trp, etc., are
under different controls. The trp operon is induced
when tryptophan is absent in the growth media; and
the PL promoter of lambda can be induced by an
'2U0'7248
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increase in temperature in host cells containing a
temperature sensitive lambda repressor, e.g., cI857.
In this way, greater than 95% of the promoter-
-directed transcription may be inhibited in unin-
duced cells. Thus, expression of the recombinant
peptide or protein can be controlled. This is
important if the expression product of the DNA is
lethal or detrimental to the host cells. In such
cases, transformants may be cultured under condi-
tions such that the promoter. is not induced; then,
when the cells reach a suitable density in the
growth medium, the promoter can be induced for
production of the protein.
One such promoter/operator system is the "tac"
or trp-lac promoter/operator system (Russell and
Bennett, 1982, Gene 20:2312-243; DeBoer, European
Patent Application, 67, 540 filed May 18, 1982).
This hybrid promoter is constructed by combining the
-35 b.p. (-35 region) of the trp promoter and the
-10 b.p. (-10 region or Pribnow box) of the lac
promoter (the sequences of DNA which are the RNA
polymerase binding site). In addition to main-
taining the strong promoter characteristics of the
tryptophan promoter, tac is also controlled by the
lac repressor.
When cloning in a eukaryotic host cell, en-
hancer sequences (e.g., the 72 bp tandem repeat of
SV40 DNA orthe retroviral long terminal repeats or
LTRs, etc.) may be inserted to increase transcrip-
tional efficiency. Enhancer sequences are a set of
2007248
~~..
-19-
eucaryotic DNA elements that appear to increase
transcriptional efficiency in a manner relatively
independent of their position and orientation with
respect to a nearby gene. Unlike the classic
promoter elements (e.g., the polymerase binding site
and the Goldberg-Hogness "TATA" box) which must be
located immediately 5' to the gene, enhancer se-
quences have a remarkable ability to function
upstream from, within, or downstream from eucaryotic
genes; therefore, the position of the enhancer
sequence with respect to the inserted DNA is less
critical.
Specific initiation signals are also required
for efficient gene transcription and translation in
procaryotic cells. These transcription and transla-
tion initiation signals may vary in "strength" as
measured by the quantitiy of gene specific messenger
RNA and protein synthesized, respectively. The DNA
expression vector, which contains a promoter, may
also contain any combination of various "strong"
transcription and/or translation initiation signals.
For instance, efficient translation in E. coli
requires a Shine-Dalgarno (SD) sequence about 7-9
bases 5' to the initiation codon (ATG) to provide a
ribosome binding site. Thus, any SD-ATG combination
that can be utilized by host cell ribosomes may be
employed. Such combinations include but are not
limited to the SD-ATG combination from the cro gene
or the N gene of coliphage lambda, or from the E.
coli tryptophan E, D, C, B or A genes.
CA 02007248 2006-06-15
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Additionally, any SD-ATG combination produced by
recombinant DNA or other techniques involving
incorporation of synthetic nucleotides may be used.
Any of the methods described for the insertion
of DNA into an expression vector can he used to
ligate a promoter and other genetic control elements
into specific sites within the vector. N. meninqi-
tidis sequences for expression can be ligated into
an expression vector at a specific site in relation
to the vector promoter and control elements so that
when the recombinant DNA molecule is introduced into
a host cell the foreign genetic sequence can be
expressed (i,e., transcribed and translated) by the
host cell.
The recombinant DNA vector can be introduced
into appropriate host cells (bacteria, virus, yeast,
mammalian cells or the like) by transformation,
transduction or transfection (depending upon the
vector/host cell system). Host cells containing the
vector are selected based upon the expression of one
or more appropriate gene markers normally present in
the vector, such as ampicillin resistance or tetra-
cycline resistance in pBR322, or thymidine kinase
activity in eucaryotic host systems. Expression
vectors may be derived from cloning vectors, which
usually contain a marker function. Such cloning
vectors may include, but are not limited to the
following: SV40 and adenovirus, vaccinia virus
vectors, insect viruses such as baculoviruses, yeast
vectors, bacteriophage vectors such as lambda gt-WES-
00'72.48
-21-
lambda B, Charon 28, Charon 4A, lambda gt-l-lambda
BC, lambda gt-l-lambda B, M13mp7, M13mp8, M13mp9, or
plasmid DNA vectors such as pBR322, pAC105, pVA51,
pACYC177, pKH47, pACYC184, pUB110, pMB9, pBR325, Col
El, pSC101, pBR313, pML21,.RSF2124, pCR1, RP4,
pBR328 and the like.
Expression vectors containing the DNA inserts
can be.identified by three.general approaches: (1)
DNA-DNA hybridization using probes comprising se-
quences that are homologousto the inserted gene;
(2) presence or absence of "marker" gene functions
resistance to antibiotics, transformation
phenotype, thymidine kinase activity, etc.); and (3)
expression of inserted sequences based on the
physical immunological or functional properties of
the gene product.
Once a putative recombinant clone which ex-.
presses a desired Class I OMP amino acid sequence is
identified, the gene product can be analyzed as
follows. Immunological analysis is especially
important because the ultimate goal is to use the
gene products in vaccine formulations and/or as
antigens in.dia.gnostic immunoassays. The expressed
peptide or protein should be immunoreactive with
bactericidal antibodies against N. meningitidis.
This reactivity may be demonstrated by standard
immunological techniques, such as radioimmuno-
precipitation, radioimmune competition, ELISA or
immunoblots.
-
22
2007248
Once the gene product is identified as a Class I
OMP fragment or an oligopeptide containing a functional
epitope thereof, it can be isolated and purified by
standard methods including chromatography (e.g., ion
exchange, affinity, and sizing column chromatography),
centrifugation, differential solubility, or by any
other standard techniques for the purification of
proteins. Several techniques exist for purification of
heterologous protein from prokaryotic cells. See e.g.,
Olson, U.S. Patent No. 4,518,526, Wetzel, U.S. Patent
No. 4,599,197 and Hung et al., U.S. Patent No.
4,734,362. The purified preparation however produced
should be substantially free of host toxins which might
be harmful to humans. In particular, when expressed in
gram negative bacterial host cells such as E. coll or
Salmonella, the purified peptide, or protein should be
substantially free of endotoxin contamination.
Class I()MP, fragments and oligopeptides of this
invention can be formulated as univalent and
multivalent vaccines. These materials can be used as
produced or isolated by the methods described above.
They can be mixed, conjugated or fused with other
antigens, including B or T cell epitopes of other
antigens. In addition, they can be conjugated to a
carrier protein as described below for oligopeptides.
When a 3iaptenic oligopeptide is used (i.e., a
peptide which reacts with. cognate antibodies, but
CA 02007248 2006-06-15
-23-
cannot itself elicit an immune response), it can be
conjugated to an immunogenic carrier molecule.
Conjugation to an immunogenic carrier can render the
oligopeptide immunogenic. The conjugation can be
performed by standard procedures. Preferred carrier
proteins for the haptenic oligopeptides are toxins,
toxoids or any mutant crossreactive material (CRM)
of the toxin from tetanus, diphtheria, pertussis,
Pseudomonas, E. coli, Staphylococcus, and
Streptococcus. A particularly preferred carrier is
CRM197 of diphtheria toxin, derived from P.
diphtheriae strain C7(3 197) which produces CRM197
protein. This strain has ATCC accession no. 53281.
Alternatively, a fragment or epitope of the carrier
protein or other immunogenic protein can be used.
For example, the hapten can be coupled to a T cell
epitope of a bacterial toxin, toxoid or CRM. See
U.S. Patent Application Serial No. 150, 688, filed
February 1, 1988, entitled "Synthetic Peptides
Representing a T-Cell Epitope as a Carrier Molecule
For Conjugate Vaccines", the teachings of which are
incorporated herein. Other carriers include viral
particles composed of Rotavirus VP6, Hepatitis B
surface antigen or parvovirus VP1 and VP2.
The peptides or proteins of this invention can
be administered as multivalent subunit vaccines in
combination with antigens of N. meninqitidis or
antigens of other organisms. Some of the other
organisms include the pathogenic bacteria H.
influenzae, N. meninqitidis, B. catarrhalis, N.
24 -
2007248
gonorrhoeae, E. coli, S. pneumoniae, etc. For example,
they may be administered in conjunction with oligo- or
polysaccharide capsular components of N. meningitidis.
The capsular components can be derived from any of the
serological groups, including A, B', C, D, X, Y, Z, 29E
and W135.
Class 1 outer-membrane protei~,ns of different
subtypes can be used. These may be used in combination
to evoke bactericidal antibodies against N.
meningitisdis. For example, a fragment derived from
Class 1 outer-membrane protein of the P1.7.16 subtype
can be used together with outer-membrane proteins or
fragments of outer-membrane proteins of other subtypes,
such as P1.1; P1.1.16; P1.2; P1.6; P1.9; P1.15; P1.16;
or P1.4 (Abdillahi, H. et al. Micro. Pathog. 4:27
(1988)) or with meningococcal polysaccharides in
mixtures or as chemical conjugates. For combined
administration with epitopes of other outer membrane
proteins, they can be administered separately, as a
mixture or as a. conjugate or genetic fusion peptide or
protein. The conjugates can be formed by standard
techniques for coupling proteinaceous materials or
techniques for coupling saccharide polymers to
proteins. Fusions can. be expressed from fused gene
constructs prepared by recombinant DNA techniques as
described.
As mentioned, Class I OMP, fragment or any
oligopeptides derived therefrom can be used in
conjunction with antigens (e.g., polymer capsules or
saccharide units, envelope or surface proteins) of
õl ~
CA 02007248 2006-06-15
-25-
other pathogenic organisms (e.g. bacteria (encap-
sulated or nonencapsulated), viruses, fungi and
parasites). Additional examples of other organisms
include respiratory syncytial virus, rotavirus,
malaria parasites, and Cr~tococcus neoformans.
In formulating the vaccine compositions with
the peptide or protein, alone or in the various
combinations described, the immunogen is adjusted to
an appropriate concentration and formulated with any
suitable vaccine adjuvant. Suitable adjuvants in-
clude, but are not limited to: surface active sub-
stances, e.g., hexadecylamine, octadecylamine,
octadecyl amino acid esters, lysolecithin, dimethyl-
dioctadecylammonium bromide, methoxyhexadecylglyce-
rol, and pluronic polyols; polyamines, e.g., pyran,
dextransulfate, poly IC, carbopol; peptides, e.g.,
muramyl dipeptide and derivatives, dimethylglycine,
tuftsin; oil emulsions; and mineral gels, e.g.,
aluminum hydroxide, aluminum phosphate, etc.,
lymphokines and immune stimulating complexes
(ISCOMS). The immunogen may also be incorporated
into liposomes, microspheres, or conjugated to
polysaccharides and/or other polymers for use in a
vaccine formulation.
The vaccines can be administered to a human or
animal in a variety of ways. These include
intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, oral and intranasal
routes of administration.
CA 02007248 2006-06-15
-26-
Live vaccines
-----------------
The peptides and proteins of this invention can
be administered as live vaccines. To this end,
recombinant microorganisms are prepared that express
the peptides or proteins. The vaccine recipient is
inoculated with the recombinant microorganism which
multiplies in the recipient, expresses the Class I
OMP, fragment or oligopeptide thereof and evokes an
immune response to N. meninqitidis. Live vaccine
vectors include: adenovirus, cytomegalovirus and
preferably, pox viruses such as vaccinia (Paoletti
and Panicali, U.S. Patent No. 4,603,112) and atten-
uated Salmonella strains (Stocker, U.S. Patent No.
-------------
4,550,081 and Curtiss et al., Vaccine 6:155-160
(1988)). In addition, Class I OMP epitopes can be
incorporated into the flagella of attenuated
bacterial strains.
Live vaccines are particularly advantageous
because they lead to a prolonged stimulus which can
confer substantially long-lasting immunity. When the
immune response is protective against subsequent
N. meningitidis infection, the live vaccine itself
may be used in a preventive vaccine against N.
menin2i------.
-------tidis~
Multivalent live vaccines can be prepared from
a single or a few recombinant microorganisms that
express different epitopes of N. meningitidis (e.g.,
other outer membrane proteins from other subtypes or
epitopes thereof). In addition, epitopes of other
2007248
-27-
pathogenic microorganisms can be incorporated into
the vaccine. For example, a vaccinia virus can be
engineered to contain coding sequences for other
epitopes in addition to those of N. meningitidis.
Such a recombinant virus itself can be used as the
immunogen in a mulivalent vaccine. Alternatively, a
mixture of vaccinia or other viruses, each expres-
sing a different gene encoding for different epi-
topes of outer membrane proteins of N. meningitidis
and/or epitopes of other disease causing organisms
can be formulated as a multivalent vaccine.
An inactivated virus vaccine may be prepared.
Inactivated vaccines are "killed", i.e., infecti-
vity has been destroyed, usually by chemical treat-
ment (e.g., formaldehyde treatment). Ideally, the
infectivity of the virus is destroyed without
affecting the proteins which carry the immunogeni-
city of the virus. In order to prepare inactivated
vaccines, large quanitites of the recombinant virus
expressing the desired epitopes are grown in culture
to provide the n cessary quantity of relevant
antigens. A mixture of inactivated viruses express
different epitopes may be used for the formulation
of "multivalent" vaccines. In certain instances,
these "multivalent" inactivated vaccines may be
preferable to live vaccine formulation because of
potential difficulties arising from mutual in-
terference of live viruses administered together.
In either case, the inactivated virus or mixture of
viruses may be formulated in a suitable adjuvant in
CA 02007248 2006-06-15
-28-
order to enhance the immunological response to the
antigens. Suitable adjuvants include: surface
active substances, e.g., hexadecylamine, octadecyl
amino acid esters, octadecylamine, lysolecithin,
dimethyl-dioctadecylammonium bromide, N, N-diocta-
decyl-N', N'bis (2-hydroxyethyl-propane diamine),
methoxyhexadecylglycerol, and pluronic polyols;
polyamines, e.g., pyran, dextransulfate, poly IC,
carbopol; peptides, e.g., muramyl dipeptide and
derivatives thereof, dimethylglycine, tuftsin; oil
emulsions; and mineral gels, e.g., aluminum
hydroxide, aluminum phosphate, and lymphokines.
EXEMPLIFICATION
EXAMPLE 1: Monoclonal Antibodies Against Class I
------------ ---------------------------------------------
OMP's and TheirBiological_Activit~
------------------
Type specific monoclonal antibodies were
prepared against various meningococcal Class 1
outer-membrane proteins. These monoclonal anti-
bodies recognize the following subtypes: P1.1;
P1.2; P1.6; P1.7; P1.9; P1.10; P1.15; P1.16 and
P1.17 (now called P1.14). The monoclonal antibodies
are available as "Monoclonal Kit Serotyping Meningo-
cocci" from the RIVM, Bilthoven, The Netherlands.
All these monoclonal antibodies react with the SDS
(sodium dodecyl sulphate) denatured protein when
tested by Western blotting. It also emerged that a
number of these monoclonal antibodies reacted with a
29 - 2007248
25Kd CNBr fragment of the 42Kd Class 1 outer-membrane
protein (see below). This result implied that the
Class 1 outer-membrane protein epitopes are mainly, of
linear type and can therefore be copied with synthetic
peptides. The epidemiological results of tests carried
out by the Applicant's show that the described
monoclonal antibodies can subtype most of the group A,
B, C meningococci which suggests a limited
heterogenity. Each Class 1 outer-membrane proteiin
also appears to contain two individual type specific
epitopes (Abdi:Llahi and Poolman, Microb. Pathogen,
4:27-32 (1988); idem FEMS Microbiol. Immunol. 47:139-
144).
The purified Class 1 outer-membrane protein (see
below) subtype P1.7.16, originating from the culture of
the Class 2/3 free mutant (H1115) appeared to induce a
bactericidal antibody response of 1:64 serum dilution
in a dose of 2.5 g in mice. The monoclonal antibodies
against meningococci Class 1 outer-membrane proteins,
Class 2/3 outer-membrane proteins and
lipopolysaccharides were compared as to bactericidal
effect. The monoclonal ant'ibodies against the Class 1
outer-membrane proteins appeared to possess the
strongest bactericidal activity (see Table 1). The
bactericidal response was determined as per Poolman,
J.T. (f985), in Schoolnik, G.K. et al. Eds. 'The
Pathogenic Neisseriae' ASM Publications, Washington,
D.C:, page 562.
L ~. :
30 - 2007248
TABLE 1
Bactericidal activity of a collection of
monoclonal antibodies,, directed against the
Class 1 (Cl 1), Class 2/3 (Cl 2/3) and
lipopolysaccharide (LPS) of meningococci. (ND -
not determined).
Test strain Bactericidal activity of antibody pool
(ititre
strain (Gp:serotype: Cl 2/3 pool Cl 1 pool LPS pool
subtype:LPS type
3006 (B:26:P1.2:;L2) 1000 8000 ND
M981 (B:4Pl.-:L5) 10 ND 2000
M990 (B.6:P1.6:L/) 10 2000 ND
M978 (B:8:Pl.l:Ll.8) ND 8000 1000
M982 (B:9:P1.9L3.7) 500 2000 1000
H355 (B:15:P1.15:L1.8) 1000 8000 1000
H44/76 (B:15:P1.7.16:L3.7) 1000 8000 4000
~ '.
31 - 2007248
The bactericidal activity of these monoclonal
antibodies appears to correlate well with the in vivo
protective activity as measured in the rat meningitis
model of Saukkonen et al., Micro-bial Pathogen 3:261
(1987).
EXAMPLE lA: Construction of meningococcal strains
carrying multiple Class 1 genes
Replacement of chromosomal genes by clones,
slightly different versions has been described for
Neisseria gonorrhoeae (Stein, D.C., Clin. Microbiol.
Rev. 2 (Suppl.), S146-S149 (1989)). We have found that
this method can be applied to the Class 1 gene in
Neisseria meningitidis. This was done in the following
way:
(i) The Class 1 gene of strain 2996 (subtype P1.2)
was cloned into the vector pTZ19R, (Mead, D.A.
et al. Protein Engineering 1:67 (1986)). The
complete gene is located on a 2.2 kb XbaI
fragment that was ligated to XbaI digested
vector DNA.
(ii) The resulting plasmid was used for
transforination of strain H44/76 (subtype
P1.7.16). Cells of the acceptor strain were
incubated with plasmid DNA in the presence
of Mg2+ and normal meningoccal medium; they
were subsequently diluted and plated, and the
~
CA 02007248 2006-06-15
-32-
resulting colonies were tested for their
ability to bind P1.2-specific monoclonal
antibody. Such transformants were found with a
frequency of approximately 10-3. Further
characterization showed that replacement of the
H44/76 Class 1 gene had indeed occurred.
An essential feature of the method is the
presence of the donor gene on a circular
plasmid DNA molecule that is not able to
replicate in N. meningitidis, since the use of
linearized DNA yielded no transformants at all.
Construction of a strain with two Class 1 genes
was done by a modification of the method described
above. For this purpose, the P1.2 Class 1 gene was
inserted into a cloned Class 5 gene. The Class 5
gene family has two features which make it
particularly suitable for this construction.
(Meyer, T.F. and Van Putten, J.P.M., Clin.
Microbiol_-Rev_, 2 (Suppl.) S139-S145 (1989): (i)
there are four or five Class 5 genes present in the
meningococcal genome, and (ii) expression of these
genes is not necessary for growth under laboratory
conditions. A Class 5 gene was cloned from strain
H44/76 and the P1.2 gene was inserted into an SphI
site located in or very close to the Class 5 gene.
The resulting hybrid plasmid, pMC22, was used for
transformation of strain HIII5, a Class 3-deficient
mutant of H44.76. Colonies reacting with the
P1.2-specific monoclonal antibody were isolated and
'2Q0'7248
-33-
characterized. Out of 10 such transformants, nine
were found to have lost the P1.16 epitope of the
acceptor strain. This indicates that in all these
cases recombination has only occurred between the
Class 1 genes, resulting in subtype replacement.
However, one transformant was found which made both
Class l subtypes, i.e., P1.7,16 and P1.2, suggesting
that recombination between the Class 5. gene
sequences on plasmid and chromosome must have
occurred. This was confirme.d by Western blottingõ
which revealed the presence of both types of Class 1
protein and by Southern blotting, which demonstrated
the acquisition of a second Class l gene.
By continuirig this construction with other
Class 1 subtypes, it is possible to make a strain
with four or five different Class 1 genes. The same
Class 5 gene can be used in each subsequent
transformation step, the different Class 5 genes can
be clones and used separately. These recombinant
strains can be used to prepare mixtures of different
purified Class I OMPs.
EXAMPLE 1B: Purification of isolated OMV's from
---------- bacteriological culture
The purification is carried out according to
Beuvery et al. (1983) loc. cit.
This culture can be done with the desired wild
type strains, mutant meningococci strains without
Class 2/3 outer-membrane proteins and/or homologeous
2Q i 24$
-34-
and heterologeous recombinant microorganisms which
express one or more of the desired meningococci
Class 1 outer-membrane protein and/or epitopes by
overproducing vectors either through or not through
existing open reading frames and/or manipulated
reading frames so that fusion proteins or proteins
with exchanged epitopes can be prepared.
Readily available of wild strains are:
H44/76 (B:15:P1,7.16) (Holten E., Norway, deposited
as CBS 635-89); 187 (B:4:Pl,.7) (Etienne J., France);
M1080 (B:1:P1,1.7) (Frasch C., USA); Swiss4
(B:4:Pl,15) (Hirschel B., Switzerland); B2106I
(B:4:Pl,2) (Berger U., West-Germany); 395
(B:NT:Pl,9) (Jonsdottir K., Iceland): M990
(B:6:Pl,6) (Frasch C., USA); 2996 (B:2b:Pl,2) RIVM,
The Netherlands; M982 (B:9:Pl,9) (Frasch C., USA);
S3446 (B:14:P1,6;) (Frasch C., USA); H355
(B:15:P1,15) (Ho:Lten E., Norway); 6557 (B:17:Pl,17)
(Zollinger W., USA) and B40 (A:4:Pl,10) (Achtman M.,
West-Germany). An example of a Class 3 negative
mutant is HIII5 (B:-:Pl.16) deposit # CBS 636.89.
These strains were inoculated from precultures
at -70 C into shake flasks and transferred from
these into 40, 150 or 350 litre fermenter cultures.
The semisynthetic medium had the following composi-
tion: L-glutamic acid 1.3 g/l, L-cysteine.HC1 0.02
g/l, Na2HP04.2H20 10 g/l, KC1 0.09 g/l, NaCl 6 g/l,
NH4C1 1.25 g/l, MgSO4.7H20 0.6 g/l, glucose 5 g/l,
Fe(N03)3 100 M, yeast dialysate.
2007248
-35-
During culturing in the fermenter, the pH and
P02 were monitored and automatically regulated to a
pH of 7.0-7.2 and an air saturation of 10%. The
cells were grown to early stationary phase harvested
by means of centrifuging and washing with sterile
0.1 M NaCl and stored at -20 C or freeze-dried.
EXAMPLE 2: Purification of Class 1 outer-membrane
proteinsfrombacteriologicalculture
This culture can be done with the desired wild
type strains, mutant meningococci strains without
Class 2/3 outer-niembrane proteins and/or homologeous
and heterologeous recombinant microorganisms which
express one or more of the desired meningococci
Class 1 outer-menibrane protein and/or epitopes by
overproducing vectors either through or not through
existing open reading frames and/or manipulated
reading frames so that fusion proteins or proteins
with exchanged epitopes can be prepared.
Readily available of wild strains are:
H44/76 (B:15:P1,7.16) (Holten E., Norway, deposited
as CBS 635-89); 187 (B:4:Pl,7) (Etienne J., France);
M1080 (B:I:P1,l.7) (Frasch C., USA); Swiss4
(B:4:P1,15) (Hirschel B., Switzerland); B2106I
(B:4:Pl,2) (Berger U., West-Germany); 395
(B:NT:Pl,9) (Jonsdottir K., Iceland): M990
(B:6:P1,6) (Frasch C., USA); 2996 (B:2b:Pl,2) RIVM,
The Netherlands; M982 (B:4:P1,9) (Frasch C., USA);
S3446 (B:14:Pl,6) (Frasch C., USA); H355
2007248
-36-
(B:15:Pl,15) (Holten E., Norway); 6557 (B:17:Pl,17)
(Zollinger W., USA) and B40 (A:4:P1,10) (Achtman M.,
West-Germany). An example of a Class 3 negative
mutant is H1115 (B:-:P1.16) deposit # CBS 636.89.
These strains were inoculated from precultures
at -70 C into shake flasks and transferred from
these into 40, 150 or 350 litre fermenter cultures.
The semisynthetic medium had the following composi-
tion: L-glutamic acid 1:3 g/l, L-cysteine.HC1 0.02
g/l, Na2HP04.2H20 10 g/l, KC1 0.09 g/1, NaC1 6 g/l,
NH4C1 1.25 g/l, MgSO4.7H20 0.6 g/l, glucose 5 g/l,
Fe(N03)3 100 MY yeast dialysate.
During culturing in the fermenter, the pH and
P02 were monitored and automatically regulated to a
pH of 7.0-7.2 anci an air saturation of 10%. The
cells were grown to early stationary phase harvested
by means of centrifuging and washing with sterile
0.1 M NaCl and stored at -20 C or freeze-dried.
The bacterial mass was for example extracted.
with.the aid of 0.5 M CaCl2, 1% (w/v) Zwittergent
3-14 (Zw 3-14) and 0.14 M NaCl, pH 4.0, using 100 ml
per gram of freeze-dried bacterial mass. The
suspension was stirred for 1 hour at room
temperature and then centrifuged (1 hour, 3000 x g),
after which the supernatant was collected in a
sterile manner. 20% ethanol (v/v) was added to the
supernatant and after stirring for 30 min. the
product was centrifuged (30 min., 10,000x g), after
which the supernatant was collected aseptically.
The supernatant was then concentrated by means of
20 '7248
-37-
diafiltration in an Amicon Hollow Fiber System (HID
x 50, cut off 50,000) and CaCl 2 and ethanol were
removed. The concentrate was diluted with 0.1 M
sodium acetate, 25 mm EDTA, 0.05% Zw 3-14 having a
pH of 6.0 to the original volume and then
concentrated again by means of diafiltration. This
procedure was repeated five times. The pH of the
final concentrate was adjusted to a value of 4Ø
20% (v/v) ethanol. was added to the concentrate and,
after stirring for 30 min., the product was
centrifuged (30 min., 10,000 x g). The whole
proteins are purified with the aid of column chrom-
atography in the presence of detergent, for example
Zw 3-14. Often gel filtration over Sephacryl S-300
as well as the ion exchange over DEAE Sepharose is
applied (Beuvery et al. (1986) supra). The used
extraction method, detergents, column chromatography
are not the only applicable method yet only serve as
examples and must not be regarded as restrictive.
EXAMPLE_3: Preparaton andCharacterizationofClass
I_OMP._Peptide_Fragments
Cyanogen bromide was used to prepare fragments
of meningococci Class l outer-membrane proteins.
The purified Class 1 or mixtures of Class 1 or 3
outer-membrane proteins were taken up in 70% (v/v)
formic acid and treated with a 10-fold excess of
CNBr for 16 hours at room temperature. The CNBr and
the formic acid were removed by means of evaporation
- 38 - 2007248
and replaced by 0.2 M Tris.HC1, 6 M urea solution, pH
7.2. The supernatant was prepurified by means of gel
filtration over Sephacryl* 'S-200 and subsequently
purified with the aid of TSK-2000_% ge1 filtration via
HPLC (Bouvery et al., (1986) supra).
Enzymatic digestion of CB2 fragments
To further delineate the epitopes, the
meningococcal CB2 fragment was subjected to digestion
with EndoArg-C, EndoGlu-C or V-8 and the resulting
fragments isolated by HPLC. Briefly, 20 nMoles of CB2
fragment in 1 ml of 25 mM phosphate/0 1 mM tris buffer
(pH 8.0) containing 3M urea was digested at 37 C with
0.2 nMoles of EndoArg-C (1 mg/ml in distilled water) or
0.22 nMoles of EndoGlu-C or V-8 (1 mg/ml in distilled
water) for 14-18 hours. The resulting digested
fragments y#ere separated by reverse phase HPLC using a
Vydac-C4column and a trifluoroacetic acid-acetonitrile
gradient. The main peak eluted from the EndoArg-C
digestion had an apparent molecular weight of 7-9 Kdal
while the main peak observed following EndoGlu-C or V-8
had an apparent molecular weight of 4-6 Kdals. The
isolated peaks were subsequently shown by Western blot
to react to a pool of monoclonal antibodies (Adam I,
62-D12-8, MN5-C11G and MN14-C116).
*Trade-mark
2007248
. . f.+a"~w,.,,. . . . .
-39-
The P1.16 epitope appears to be present on the
C-terminal CNBr f'ragment of the Class 1 outer-mem-
brane protein of strain H44/76 (B:15: P1,7.16).
Further characterisation of the P1,16 epitope was
carried out through amino acid sequence determina-
tion of the 17Kd (N-terminal) and 25Kd (C-terminal)
_CNBr_fragments. The C-terminal 25Kd is further
fragmented with V8 protease, endoLysC, endoGlu-C and
endoArg-C. Fragnients which were positive with the
P1,16 monoclonal antibody were sequenced as far as
possible. The sequences which were obtained are as
follows:
N-terminus of whole protein:
DVSLYGEIKAGVEDRNYQLQLTEAQUAAGN...
N-terminus of 25Kd C-terminal CNBr
fragment (M) PVSVRYDSPEFSGFSGSVQFVPIONS-
KSAYTPAYYTKDTNNN...
Fragments which react with P1,16 monoclonal anti-
bodies were isolated using V8 protease and endoArg-C
fragmentation with a molecular weight of 7-9Kd and
4-6Kd respectively. The N-terminal sequences hereof
are as follows:
V8 7-9Kd fragment: FSGFSGSVQFVPIQNSK.SAYTPAYYTKDTN...
2007248
,~.
-40-
Arg-C 4-6Kd fragment: PVSVRYDSPEFSGFSGSVQFVPI-
QNSKSAYTPAYYTK...
EXAMPLE 4:::.-ONA Seguences of Class I OMP Genes
Amino acid sequences of Class I OMP were
deduced from the nucleotide sequence of the struc-
tural genes of four meningococci Class 1 OMP's with
various subtypes. Comparison with four amino acid
sequences enabled a prediction of the composition
and the location of these epitopes. Further, the
P1,7 and P1,16 epitopes were confirmed with the ai.d
of peptide synthesis and the demonstration of
binding of the respective monoclonal antibodies.
Class I OMP genes were cloned into lambda gtll
(as described for P1, 16 in Barlow et al., (1987)
Infect. Immun. 55: 2743-2740)and subcloned in M13
sequencing vectors and the DNA sequence was deter-
mined by standard chain termination dideoxynucleo-
tide techniques.
The complete derived amino acid sequence for
P1,16; P1,15, P1,7.16; and P1,2 proteins are as
follows:
20 30 40 50
P1.16: DVSLYGEIRAGVEGRNIQAQLTEQPQVTNGVQGNQV--KVTKAKSRIR'Y'KYS
*****~k********** * ~k**~kdr*it9e~t*tk~lr4ellr!* .
P1.15: DVSLYGEIKAGVEGRNFQLQLTEPP-SKSQP~--QV--KVTRAKSRIRTKIS
*********~~r*~~*~ ~ +~*,~* **,~****~***~~
P1.7.16; DVSLYGEIKgGVEGRNYQLQLTE.AQAANGGASGQVRVTRVTRARSRIRTRIS
~*~*~******~**** ~ **** *****~******,t
P1-2: DVSLYGEIKAGVEGRNIQLQLTEPLQNIQQPQ--------VTRARSRIRTRIS
2007248
=41-
60 70 80 90 100 110
DFGSFIGFKGSEI7LGEGLKAVWQI,EQDVSVAGGGASQWGNRESFIGLAGEFGTLRAGRVA
DFGSFIGFKGSEDLGEGLKAVWQLEQDVSVAGGGATQWGNRESFVGLAGEFGTLRAGRVA
******~***~,~,t.t* *+~rt***~.t*.~**~,~**,rtfi,~,~~~*,~ ************~*~
DFGSFIGFRGSEDLGDGLK.AVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVA
~***~*~***~~~~* ~*~***~****~~**~*** **r**** ~~*~*~~~~~,~**,~*
DFGSFIGFKGSEDLGEGLKAVWQLEQDVSVAGGGATRWGNRESFV'GLAGEFGTLRAGRVA
,~,~*~~~~,~~~~*~** *~r~*~****~~******~r,~ ~~~*t~* ***************
220 130 140 150 160 170
NQFDDASQAINPWDSNN-DVASQLGIFKRHDDM-PVSVRYDSPEFSGFSGSVQFVPAQNSKS
*~~***,r *+*********~~~**~*~*****x******x ~r**r~******** *****
NQFDDASQAIDPWDSNNDVASQLGIFKRF?DDMPVSVRYDSPDFSGFSGSVQFVPIQNSKS
~~~~~~~ ** ~*~**,~~**********~*******~~c*~r* ***********~ ***,**
NQFDDASQAIDPWDSNNDVASQLGIFFiRADDMPVSVRYDSPEFSGFSGSVQFVPIQNSKS
. t1t9ckfr~c.ic* ~cic ic**#*iFrkiiYeYrYc~c**kicttic**ir***t****
****.7k*t1r*7k*.* 7C*7t*t
NQFDDASKAIDPWDSNNVVASQLGIFKRMDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKS
*~~t**** ** ***x,rx~~c~~c~r*~*#*************** ***xc**~c~c*~** ~r****
180 190 200 210 220 230
AYKPAYYTKDTNNNLTLVPAVVGR.PGSDVYYAGLNYKNGGFAGNYAFKYAAFiANVGRNAF
*t ~r* ****~r**t*~************~*** ******f,r***** **
AYTPAHYTRQNNTDV-I'VPAVVGKPGSDVYYAGLNYRNGGFAGSYAPKYAREANVGRDAF
*{** Y~*ir~klc***ic4c*F#lcflr~k*~t~t'!t**tk*~c~k ***"*ic*dc*it~tlctk* *~fc
AYTPAYYTKNTNNNLTLVPAVVGKPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRNAF
*t ** ,r**~****~r*****~****~~e***~~ *~t~*~***~**** **
AYTPAHFVQQTPQQPTLVPAVVGKPGSDVYYAGLNYKNGGFAGNYAFKYAKBANVGRDAP
S =+-
~* ** *~*****~~~******~*****~*** x*#******~~*~ **
2007248
.~,
-42-
240 250 260 270 280 290
~LFLIGSATSDEAKGTDPLKNI3QVHRLTGGYEEGGLNLALAAQI.DLSENGDKAKTKNSTT
**** ** ** ***~*~************~c*,t*~************** * ~**~***
ELFLLGS--TSDFAKGTDPLKNHQVIiRLTGGYEEGGLNLALA.AQLDLSENGDR.A.KTRNSTT
,t**~ ** ,~* ~**~~*~********~*~******,t**~r~,t****~t,t* ~ *t****,t
ELFLIGS-GSDQAKGTDPL,KNHQVHRLTGGYEEGGLNLALAAQLDLSENGD--KTKNSTT
*~** ** ** ,~~***~~~~***~****~*~~***~~**~***~**~* * ,~x***,~x
ELFLLGS-GSDEARGTDPLKNHQVHRLTGGYEEGGLNLALAP,QLDLSENAD--RTKNSTT
*~~* *~ ** ~*****~~*****~,~**~*~~e,t*************** * *****,~*
300 3:L0 320 330 340 350
EIAATASYRFGNAVPRISYAgGEDLIERGRRGENTSYDQIIAGVDYDFSKRTSAIVSGAW
*~*~tf*******~~***t ~~**** **~*,t*~r*f*t~~*,t**~*~*~~****~***#*~ex
EZAATASYRFGNAVPRISYAHGFDLIERGKKGENTSYDQIIAGVDYDFSKRTSAIVSGAW
*********~**~***~~=~~,t#~* ~**~~******~*****~***,t**~r~********~
EIAATASYRFGNAVPRIS'YAHGFDFIERGKRG~NTSYDQIIAGVDYDFSKRTSAIVSGAW
*,~#~*x*********~~**~*~** *~~~~**~~*~**~********,~***~*x~~**~,~
EIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIAGVDYDFSRRTSAIVSGAW
~**~~~******~r***~~**~*** **t*~***~~~~*~*x~~*~~~****~~t*t~~*,t~
360 370
LKRNTGIGNYTQINAASVGLRHKF
***IC*ft*iI******ih~r~ef***airati~ . .
LKRNTGIGNYTQINA.ASVGLRHRF
*************,~*~*~~*~*~~
LKRNTGIGNYTQINAASVGLR3KF
***t****~r**f****~*,~*~~~,t
LRRNTGIGNYTQINAASV'GLRFiKF
*******,rw******~c*~r***~~~
+ Note this amino acid 15 is located between
A.A.S.184 and 185 of this sequence
2007248
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EXAMPLE 5: 'DNA,Seguencinp~of_ClassIoMPGenesfrom
differentN.meningitidisSerosubtypes
--------
The Polymerase Chain Reaction (PCR) technique
of Mullis and Faloona (Methods in Enzymol. 155:335-
-50, 1987) was used to amplify the entire Class I
OMP gene and specific fragments according to the
scheme shown in figure 1.
Primers were synthesized on an Applied Biosys-
tems 380B DNA synthesizer and used in standard PCR
30 cycle amplification reactions using Taq poly-
merase in a Thermal Cycler (Perkin-Elmer Cetus,
Norwalk, CT) according to the recommendations of the
Supplier. Amplified fragments of about 1300, 900
and 450bp were generated from each serosubtype
genomic DNA preparation from the primer combinations
shown in Figure 1. The primers used had the follow-
ing sequences:,._.
PR1: (41 bases with universal primer extension)
TGT AAA ACG ACG GCC AGT TTG AAC ACG TAT CGG GRG TTT GC
PR2: (42 bases with universal primer extension)
TGT AAA ACG ACG GCC AGT GGC GAA TTC GGT ACG CTG CGC GCC
PR3: (42 bases with universal primer extension)
TGT AAA ACG ACG GCC AGT CAT CAG GTA CAC CGC CTG ACG GGC
PR4: (40 bases with universal primer extension)
20 '7248
-44-
TGT AAA ACG ACG GCC AGT GCA GAT TGG CAG TCA GAT TGC A
PR5: (40 bases with universal primer extension)
TGT AAA ACG ACG GCC AGT GGG ATC GGT ACC TTT GGC TTG A
PR6: (40 bases with universal primer extension)
TGT AAA ACG ACG GCC AGT AAC TGA TTC GCA ACG CGA CCG G
FWD: (24 bases)
TTG AAG GAC GTA TCG GGT GTT TCG
REV: (23 bases)
GCA GAT TGG CAC; TCA GAT TGC TT
Excess single stranded template for sequencing
was synthesized in an 'asymmetric PCR' amplification
using 100x excess of primer carrying an 18 base
extension at the 5' end corresponding to the univer-
sal fluorescent sequencing primers used with an
Model 370A Automated DNA Sequencer (Applied Biosy-
stems, Foster City, CA). Taq polymerase was used in
a Standard dideoxynucleotide chain termination
sequencing reaction with the PCR generated single
stranded Class I gene fragments as templates.
Derived sequences for gene segments of strains
H44/76 (P1.7,16), M1080 (P1.1,7), H355 (P1.15), 6940
(P1.6), 6557 (P1.14), 870227 (P1.10) and B40 (P1.10)
are shown in Figures 2 and 3.
2007248
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EXAMPLE 6: Confi.rmation of Amino Acid Sequences of
ClassIOMPSubtYpe_Epitopes
From these gene sequences confirmed by direct
sequencing of Class I OMP genes, it was deduced that
the sequences corresponding to amino acids 24-34 and
176-187 of P1.16 are markedly variable in the four
Class I OMP sequences. Three amino acid sequences
N-terminal or C-terminal from these positions should
also be considered.for possible inclusion in these
epitopes to allow for maximizing epitope stability
presentation and unexpected insertions or deletions
in the native protein sequence. Further the DNA and
amino acid sequerices of other Class I OMPs should be
compared with the P1.7,16 sequence to allow for
maximum allignment and epitope prediction. The
first variable region epitope and second variable
region epitope are called VR1 and VR2 respectively.
These regions encode the subtype epitopes as was
confirmed with the aid of peptide synthesis and the
reaction of the peptides with P1.2; P1.7; P1.15 and
P1.16 specific monoclonal antibodies.
A complete set of overlapping decapeptides
staggered by 5 amino ac.i.ds were prepared using the
P1.16 protein sequence. The anti- P1.16 monoclonal
antibody reacted with the decapeptide YYTKDTNNNL
from P1.16 reacted as expected and no other
decapeptide. (Figure 4).
Of overlapping decapeptides provided with a one
(1) amino acid sequence shift in the region 24-34
20Q-'7248
-46-
and 176-187 of the Class I OMP of strains H44/76
(P1.7,16), MC50 (P1.16) and MC51 (P1.15) more than
one peptide reacted with the subtype specific
monoclonal antibody. In most cases one or more of
the group of these overlapping peptides reacted with
the subtype specific monoclonal antibody more
strongly than ottiers (Figure 5).
These peptides are designated as the VRl and
VR2 epitopes. In the P1.7,16 strain, the sequence
YYTKNTNNNL is present, the change D to N at residue
180 does have some effect on reducing antibody bind-
ing. The sequence HYTRQNNTDVF in P1.15 in the same
relative position in the protein as the P1.16
epitope and is responsible for binding to the
anti-Pl.15 monocaonal antibody. AQAANGGASG.shows
some binding and peptides 1-3 amino acids downstream
show far greater binding to the P1.7 monoclonal
antibody. Sequence HFVQQTPQSQP of VR2 is
responsible for binding to the anti-P1.2 monoclonal
antibody. It is probable that the sequences
QPQVTNGVQGN and PPSKSQP in the P1.16 and P1.15
proteins also represent epitopes.
Example_6A: Class I OMPConstant_Region_Epitope
Identification
Peptides forming surface loops were prepared
and conjugated to tetanus toxoid. A Biolynx 4170
automated peptide synthesizer (Pharmacia/LKB) was
used for continuous flow solid-phase synthesis with
47 - 2007248
the following exception. In the last cycle of the
synthesis SAMA-OPfp (0.5 mmol) (Drijfhout, J.W., Ph.D.
Thesis, Leiden, The Netherlands (1989)) was coupled in
the presence of 1-hydroxybenzotriazole (0.5 mmol) for
30 min., using a standard protocol with omission of the
piperidine-trea=tment (i.e., the "Fmoc-deblocking step"
which in this case would cause undesirable S-
deacetylation). These are referred to as SAMA-
peptides.
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The peptides and their surface region location uhich vere conjugated to TT are
as follows;
Name Peptide Region
176 185
LBV 017 XGGYYTKDTNNNL P1.16, loop 4
24 33
018 XGGAQAANGGASG P1.7, loop 1
276 291
024 XGGLSENGDKARTKNSTTE P1.16, loop 6
24$
025a XGGNAFELFLIGSATSDEAKG P1.16, loop 5
223
025b )MNVGRNAFELFLIGSATSDEAKG P1.16, loop 5
124 137
026 XGGDS1vNDVASQLQIFR P1.16, loop 3
627 XADLNTDAERVAVNTArIASPV Class 2, loop 5
329
028a XGGGKKGENTSYDQ Class 1, loop 7
317
028b XGGERGRRGENTSYDQ Class 1, loop 7
029 RGGVKDAGTYRAQGGRSKTATQ Class 2, loop 1
78 90
030 xGGwSVA.EGGASQVGrt P1.16, loop 2
352 366
031 XKRNTGZGNYTQINAA P1.16, loop 8
16 34
032 XGG.NIQAQLTEQPQV7'NGVQGN P1.16, loop 1
49 - 2007248
Conjugation of SAMA-peptides to tetanus toxoid
was performed as follows. A solution of N-succinimidyl
bromoacetate (4.7 mg, 10 mol) in DMF (100 l) was
mixed with a solution of tetanus toxoid (TT) (20mg) in
0.1 M. sodium phosphate buffer pH 7.8 (3.5 ml). After
1 h, 1.8 ml of the reaction mixture was subjected to
gel filtration using a Sephadex PD-10 column
(Pharmacia) equilibrated in 0.1 M sodium phosphate,
containing 5 mm EDTA (PE buffer) pH 6.1. The
bromoacetylated tetanus toxoid was eluted with the same
buffer and collected in 3.5 ml. The solution of
bromoactylated tetanus toxoid (1.2 ml) was added to the
SAMA peptide (4.5 mg. 3 mol) and deaerated with
helium. Next, 150 l of 0.2 M hydroxylamine (in PE
buffer, pH 6.1) was added. After 16h remaining
bromoacetyl groups were blocked by addition of 2-
aminoethanethiol hydrochloride (4 mol) in buffer, pH
6.1 (150 l). After a further period of 16 h, the
peptide-TT conjugate was purified by gel filtration
over a PD-10 column using PE buffer, pH 6.1, as the
aluant. The appropriate fractions were combined and
stored at 4 C.
To determine the immunological activity, 25 g
(total protein) per dose of a peptide-TT conjugate
was injected subcutaneously at weeks 0 and 4
into 6-8 week old NIH outbred mice. (Note: Vaccine
LBV 017-TT and LBV 018-TT were used at 10 g
total protein/dose.) Sera were collected 6 weeks
following the first dose and evaluated for antibody
,~~~
50 - 2007248
response in an ELISA assay (Beuvery, E.C. et al.
Infect. and Immun. 40:369-380 (1983)). The following
antigens were coated into the microtiter wells: outer-
membrane protein (OMP), purified Class I OMP (Poolman,
J.T. et al. Infect. and Immun. 57:1005 (1989)) and the
unconjugated peptides. Bactericidal activity (BC) of
sera was also measured (Poolman, J.T. et al. (1985)
supra).
The results are presented in Table 2 below.
,'~L ~
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TABLE 2
Bactericidal
Vaccine OMC Class 1 0MP Synth.Peptide Test
LBV 018-TT 1:900 (0.05)* 1:2700 ND <1:64
LBV 017-TT 1:900 (1) 1:900 ND <1:64
LBV 024-TT 1s100 1:100 1:900 (homol.) <1:64
LBV 025a-TT - 1:100 1:2700 (homol.) <1:64
LBV 025b-TT 1:2700 (4) 1:300 1:8100 (homol.) <1:64
LBV 026-TT - - - (homol.) <1:64
LBV 027-TT - 1:300 1:300 (homol.) <1:64
LBV 028a-TT 1:100 - 1:2700 (homol.) <1:64
LBV 028b-TT 1:100 1:100 1:900 (homol.) <1:64
LBV 029-TT - 1:100 1:8100 (homol.) <1:64
LBV 030-TT - 1:100 1:2700 (homol.) <1:64
LBV 031-TT .1:100 - (homol.) <1:64
LBV 032-TT - 1:100 1:900 (homol.) <1:64
* numbers in O indicate O.D. level showing
this tite.r
These data suggest that of the constant surface
loops tested of Class 1 and 2 OMPs of N.
meningitidis loop 5 appears to represent at least
one region that will produce antibodies which will
cross-react with Class l and Class 2 OMP of many
strains of N. meningitidis.
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EXAMPLE 7: Const:ruction-of recombinant flagellins
---------- -----=-----------------------
expressing_menin~ococcal_epitopes
To create hybrid flagella containing epitopes
from class I meningococcal epitopes, a series of
oligonucleotides was designed based on primary
protein sequence data and epitope mapping data. Two
oligonucleotides based on VR1 or VR2 epitopes of
outer membrane P1.7.16 were designed so that_ the.y
could be cloned in single or. multiple copies into a
cloning region within the gene for S. muenchen
flagellin. Translation termination signals were
included on the non-coding strand of the oligo-
nucleotide to facilitate screening by expression of
the cloned inserts.
The plasmid vector pPX1650 containing the
entire coding region and promoter regions for the
structural gene for flagellin H1-d of Salmonella
meunchen (deposited at the ATCC, accession #67685)
was modified to contain several unique cloning sites
suitable for the insertion of either oligonucleo-
tides or gene fragments in each of the three reading
frames of the flagellin gene (Figure 6). First,
pPX1650 was digested with EcoRV, which cleaves
pPX1650 twice, 48 base pairs apart, and religated to
yield a plasmid, pPX1651, which has a unique EcoRV
cloning site and which results in a 16 amino acid
deletion in the flagellin protein. pPX1651 was
identified by screening E_ coli recombinants on
Western blots probed with polyclonal antibody
CA 02007248 2006-06-15
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directed against Hl-d flagellin. pPX1651 was
identified amongst several candidates having
flagellins smaller than wild type flagellin (of
1650) and was verified by sequencing. Second,
pPX1651 was restricted with BamHI and religated
after filling out the overhanging ends with Klenow
enzyme to remove the unique BamHI restriction enzyme
site in the polylinker region of the vector. As a
final step, the resulting vector was digested with
EcoRV and the following oligonucleotide linker was
inserted:
5' ATG ATC CAT GGA TTC 3'
3' TAG TAG CTA CCT AAG 5'
Candidates were screened for the newly created BamHI
sites and several candidates having BamHI sites were
screened for orientation of the linker by double
strand DNA sequencing methodology. One candidate
having the linker in the above orientation was
retained as pPX1647:
5'.... GAT ATC ATC GAT GGA TTC ATC....
EcoRV Clal BamHI
Plasmid pPK1647 (Figure 7) was digested with
BamHI and either oligonucleotides for VR1 or VR2
were cloned into E. coli cells, Screening for
desired recombinants was accomplished by digesting
plasmid minilysate DNA with appropriate diagnostic
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restriction enzymes and screening for expression by
probing hybrid flagella for decreased mobility on
SDS-PAGE gels with specific flagellar antiserum
(Hl-d). A number of the resultant clones showed
decreased mobility on SDS-PAGE, indicating proper
insertion of one or more of the oligonucleotides for
VR1 or VR2. Seve:ral of each were retained for
analysis by DNA sequencing. Clone CB1-2 results
from tandem insertion of two copies of the VRi
oligonucleotide and clone CBl-4 results from inser-
tion of four oligonucleotides. Likewise CB2 P
contained a single insert of the VR2 oligonucleotide
and CB2 W showed the expected trimeric insert, CB2 P
clone contained a single base pair change which
resulted in a change from Leu to Phe in the expres-
sed VR2 fusion protein and was not retained for
further study. The recombinant flagellin clones in
E. coli were probed with monoclonal antibodies
(Abdillahi and Poolman, Microbiol. Pathogenesis
4:27-32, 1988; RIVM, The Netherlands) known to react
with either VR1 or VR2 epitopes. Monoclonals Adam-1
(P1.7) and Mn14-C11-6 (P1.7) react with hybrid
flagellin containing 2 or 4 tandem inserts of VR1,
but do not react with clones containing VR2. The
weaker reaction of both monoclonals with CB1-2 than
with CB1-4 is lilcely due to epitope density. By the
same token, monoclonals 62 (P1,16) and Mn5-cll-G
(P1.16) react with CB2 W clone, but not with the VR1
inserts. The CB2 P clone fails to react with either
VR=2 antibody, probably due to the Leu to Phe change.
CA 02007248 2006-06-15
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Each of these clones was transformed into an
aroA S. dublin strain (SL5927), having a TnlO
insertion in the Hl-d locus, to examine the
functioning of the hybrid flagella. Each of the four
clones resulted in motile bacteria; motility of the
transformants was inhibited by the corresponding
monoclonal antibody, including clone CB2 P,
indicating affinity of the VR2 monoclonal for the
epitope in intact flagella. This result indicates
that epitopes are exposed at the cell surface and
are accessible to antibody.
Hybrid flagellin containing both VR1 and VR2
epitopes were created by cleaving either CB1-2, CB1-
4, or CB2 W with BamHI and cloning the heterologous
epitope. Clones CB12-7 and CB12-10 result from the
in-frame insertion of a single copy of the VR2
oligonucleotide behind either 2 or 4 VR1 tandem
inserts, respectively; clone CB21-F arose from the
insertion of one copy of the VR1 epitope behind 3
tandem copies of VR2. CB12-7 and CB12-10 are
recognized only by VR1 monoclonal antibody and
CB21-F is recognized only by VR2 monoclonal. These
results, taken together with DNA analysis revealing
predicted sequences, indicate epitope density is too
low in the combined hybrids. To create a hybrid
flagellin with increased density of both VR1 and VR2
epitopes, CB12-10 was digested with BamHI and VR2
encoding oligonucleotides were inserted. Clone
12-10-6 contains two further tandem inserts of the
VR2 epitope, resulting in a hybrid flagellin
CA 02007248 2006-06-15
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molecule in which four tandem copies of VR1 are
followed by three copies of VR2. As is shown in
Figure 3a and b, three of the hybrid flagellin
vaccine candidates have the expected molecular
properties. The flagellin (pCB1 x 4) containing 4
copies of VR1 reacts with anti-Hl-d (anti-flagellin)
and anti-VR1 monoclonal antibodies, but not with
anti-VR2 monoclonal antibodies; the flagellin
(pCB2-W) containing 3 tandem copies of VR2 reacts
with anti-Hl-d and anti-VR2 antibodies, but not with
anti-VR1; the combined hybrid containing copies of
VR1 and 3 copies of VR2 reacts with both anti-VRl
and anti-VR2 monoclonal antibodies. The combined
hybrid specified motility when introduced into a
non-motile recipient S. dublin strain.
As a subunit vaccine, the goal is to obtain
suitable initial vaccine candidates in high quantity
and high purity. A suitable vaccine candidate can be
chosen from the above type constructions based on
reactivity to monoclonal antibodies and function of
flagella in non-motile Salmonella host strains. A
subunit flagellin vaccine may not need to retain all
functional aspects of a parental flagellin, but
should at least retain surface localization for
purification purposes. Several subunit flagellin
meningococcal vaccines were chosen from the above
described hybrid molecules based on reactivity to
monoclonal antibodies and implied surface localiz-
ation based on restoration of bacterial motility.
2007248
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Three flagellin vaccine candidates contained either
4 tandem inserts of VR1, 3 tandem inserts of VR2, or
4 VRl.inserts followed by 3 VR2 inserts. Because
flagellin is a major protein of Salmonella, it is
possible to easily purify sufficient material for
vaccination studies using techniques established for
flagella purification (Logan et al., J. Bacteriol.
169: 5072-5077, 1987).
EXAMPLE 8: Initial Purification of recombinant
---------- .
flaBL-llin molecules
The three hybrid flagellin vaccine candidates
and a wild type (derived from pPX1650) were inocula-
ted into four-liter baffled Fernbach flasks contain-
ing 1 liter of LB broth. Bacterial cultures were
incubated-.a.t _37 C w-ith shaking -(200 rpm) for -22-24
hr. Under these conditions of culturing, the bulk
of the flagella were sloughed from the bacterial
cell surface and were localized in the supernatant
culture medium. To obtain suitable material,
tlagella were isolated from 6-8 liters of culture
medium. To obtain purified flagellin preparations
for.vaccination studies, flagellar filaments were
harvested from bacterial culture supernatants by the
following procecLure: Ammonium sulfate was added to
culture supernatant so that final solution was 50%
saturated; the solution was stirred gently at 4 C
for several hours and the precipitated material was
collected by centrifugation in a GSA rotor at 5000
CA 02007248 2006-06-15
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rpm for 30 minutes. The collected ammonium-sulfate
precipitated material was reconstituted in PBS and
dialyzed against PBS at 4 C for 12-15 hrs. The
dialyzed material was subjected to high speed
centrifugation at 100,000 x g for 1 hour in an SW-27
rotor to pellet the flagellar filaments. The
pelleted material, which consisted primarily of
flagellin, was subjected to further purification by
the following method.
EXAMPLE 9: HPLC Purification of recombinant
------------- ----------------------------------------
flaqellins
To prepare highly purified flagellins,
Salmonella expressing the constructions, in particu-
lar pCB12-10-6, was grown as described above and the
cells pelleted at 10,000G. The culture supernatant
was then precipitated with 50% ammonium sulfate,
centrifuged at 10,000G and resuspended in 30 ml PBS.
The resuspended pellet was dialyzed against 10mM
Tris buffer (pH=8.0) containing GM urea, 1mM PMSF,
2mM NEM, and 5mM EDTA overnight at 4 C. Dialyzed
material was then passed over two DEAE Sepharose*
minicolumns (3.0 ml volume, 4,0 ml eluent over
each). The columns were eluted (5X) with 50mM NaCl
in 10mM tris (pH=8.0) containing 6M urea and then
with 1M NaCl in 10mM tris (pH=8.0) containing 6M
urea. The first four elution collections (20 ml) of
the 50mM NaCl were pooled and dialyzed against 1.0
liter 10mM acetate buffer (pH=4.0) in 6M urea at
*Trade-mark
CA 02007248 2006-06-15
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room temperature. The dialyzed fractions were then
loaded onto a TSK SP PW* cation exchange HPLC column
(75mm x 300mm). The column was eluted with a mobile
phase consisting of 10 mM acetate (pH=4.0)
containing 6 M urea. A gradient 0 - 300 mM NaCl was
established in 10 mM acetate (pH=4.0) containing 6 M
urea over the 5- 30 min interval. After 30 min the
gradient went from 300 mM to 1 M NaCl in 10 mM
acetate in 6 M urea over the next 5 min. The
flagellin construct was collected at approximately
24 min. which corresponds to about 200 mM NaCl. The
fraction was dialyzed against PBS and purity
determined on the material was established by
Western blots using anti-flagellin antibody. A
representative HPLC analysis and SDS-PAGE are shown
in Figures 8 and 9 respectively.
EXAMPLE 10: Preparation of meningococcal-flagellin
--------------
glycoconjugate
Group C meningococcal capsular polysaccharide
(GCM CPS: lot # 86 NM 01) was prepared essentially
according to Bundle et al. Bundle et al., J. Biol.
-Chem. 249: 4797-801, 1974).
Neisseria menin itidis strain Cli was obtained
----------- ------~ -------
from the Walter Reed Army Institute (Washington,
DC). The strain was precultured twice on sheep blood
agar plates, then used for the inoculation of a
liquid seed culture medium Neisseria chemically
defined medium, NCDM) Kenney et. al _, Bul l_- W. H_ 0_
*Trade-mark
CA 02007248 2006-06-15
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37 469-73, 1967). Finally, 40 1 of liquid medium
(NCDM) in a fermentor was inoculated with the liquid
preculture. The purity of the strain was checked at
each stage. After centrifugation, the supernatant
was precipitated by addition of Cetavlon to a final
concentration of 0.1%, and the insoluble complex re-
dissolved in cold 1 M calcium chloride (CaC12)
(Gotschlich et al ., J.Exp_Med_ 129:1349-65, 1969).
Ethanol (96%) was added to a final concentration of
25% (v/v). After 1 h, the suspension was
centrifuged (1 h, 50,000 g), the supernatant was
collected, and its ethanol concentration was
increased to 80% (v/v). After 1 h, centrifugation
(20 mi.n, 5,000 g) yielded a precipitate which was
washed successively with absolute ethanol, acetone,
and diethylether, and then dried in a vacuum
dessicator over phosphorus pentoxide (P205) to
constant weight. This crude CPS was stored at
-20 C.
In order to obtain a purer preparation, the CPS
was then dissolved in sodium acetate buffer (1.10
dilution of a saturated solution, pH 7.0) and
extracted four times with hot phenol (Westphal et
al., Z. Naturforsch. 7b:148-55, 1952). After
dialysis of the combined aqueous phases against
0.1 M CaC12, followed by centrifugation (3-5 h,
100,000 g), a final ethanol precipitation was
performed on the clear supernatant, and the
resulting precipitate washed with organic solvents
-61-
and dried, as described above. The pure CPS was
then stored at -'20 C.
At each stage of the purification process, the
CPS was analyzed for c.arbohydrate N-acetyl-
neuraminic acid, NANA) (Svennerhold, Biochim.
Biophys__Acta 24:604, 957), 0-acetyl (Hestrin, J.
Biol.Chem. 180:249, 1949), and protein (260 nm
detection) content, and its molecular weight checked
by gel filtration.
Group C meningococcal capsular polysaccharide
(GCM CPS) was simultaneously depolymerized and
activated via sodium periodate (NaI04) oxidation in
aqueous buffer (Anderson et al., J.Immunol.
137:1181-6, 1986; Eby et al., Pediat.Res. 20:308A,
1986, Anderson et al., J._Pediatr. 111(5):644-50,
-- --- -- -------
1987; Anderson, U.S._Pat. 4,762,713; 1988). The
reaction was monitored by high performance gel per-
meation chromatography (HPGPC) in aqueous eluent,
using ultraviolet (UV) and refractive index (RI)
detection. The reaction was stopped and the
activated oligosaccharides (GCM OS) were desalted by
low pressure gel permeation (GPC) in water, and then
lyophilized. A solution was then prepared in water
and subsequently frozen for temporary storage. GCM
OS and flagellin pCB12-10-6 were mixed in aqueous
neutral buffer and the conjugation was initiated by
addition of sodium cyanoborohydride (NaBH3CN)
(Anderson, U.S. Patent 4,762,713, 1988; U.S.-Patent
4,673,574, 1987; U.S. Patent 4,761,283, 1988). The
reaction was carried out for 5 days, while being
~-. :0 7248
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monitored by HPGPC. It was finally stopped by
dialysis/concentration on centrifugal microconcen-
trators. The final preparation was stored in the
cold, in the presence of thimerosal to prevent
bacterial growth. The resulting glycoconjugate not
only provides a mechanism to present the expressed
VR1 and VR2 meningococcal epitopes to the immune
system but also serves as a carrier molecule for the
presentation of a meningococcal oligosaccharide.
In preparation of the conjugate, the following
conditions were employed. Purified flagellin
pCB12-10-6 was dissolved in 15% sucrose (3.5 mg/ml)
and then stored at -20 C. GCM CPS (9.7 mg; final
concentration: 5 mg/ml) was oxidized by 100 mM NaIO 4
in 0.05 M sodium phosphate buffer (pH 6.2 - 6.5) at
RT, in the dark, with agitation. Aliquots (100 l)
were withdrawn at regular intervals, the reaction
stopped by addition of ethylene glycol (10 l), and
analysis was performed by HPGPC on Waters (Milford,
MA) UltrahydrogelTM 250 + 120 (2 columns coupled; 2
x 300 mm x 7.8 mm) in 0.2 M phosphate-saline buffer
(PBS; 0.2 M sodium phosphate, 0.9% NaCl, pH 7.8), at
a flow rate of 0.8 ml/min, using UV (206 nm) and RI
detection. After 2 h 30 min, the reaction was
stopped by addition of ethylene glycol (1/10 of the
reaction volume), and the GCM OS were desalted by
GPC on Bio-Rad (Richmond, CA) Bio-Ge1R P-2 (200-400
mesh, 30 cm x 1.5 cm) in water, at about 18 ml/h.
Fractions were collected (1.2 ml) and analyzed for
the presence of NANA the carbohydrate N-acetyl-
20 '7248
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neuraminic acid (NANA) (Barry et al., J._Gen.
Microbiol. 29:335-52, 1962) and aldehydes (Porro et
al., Anal. Biochem 118:301-306, 1981). Positive
fractions were pooled and lyophilized. Desalted GCM
OS (4.7 mg) were then dissolved in water (10 mg/ml)
and frozen at -20 C.
Both GCM OS and pCB12-10-6 solutions were
analyzed by HPGPC (UV at 206 and 280 nm respec-
tively) before being frozen, and prior to the
conjugation. No degradation occurred during stor-
age, as ascertained by the exact similarity of the
elution profiles.
GCM OS (2 mg; final concentration: 2.6 mg/ml)
and flagellin pCB12-10-6 (2.3 mg; final concentra-
tion: 3 mg/ml) were mixed in a polypropylene tube
in 0.4 M sodium phosphate buffer (pH 7.0), and
NaBH3CN was added (12 moles) to initiate the
conjugation (Anderson, U.S. Patent 4,762,713, 1988;
U.S. Patent 4,673,574; U.S. Patent 4,761,283). The
reaction mixture was left one day at RT, then 4 days
at 35 C, without agitation. The reaction was
monitored by HPCPG (UV at 280 nm) at different
stages, and finally stopped by dialysis/concentra-
tion on microconcentrators. The final preparation
was analyzed for NANA (Barry et al., J. Gen. Micro-
biol. 29:335-353, 1962) (0.09 mg at 0.12 mg/ml) and
protein (Lowry et al., J. Biol. Chem. 193:265-275,
1951) (1.12 mg; 1.45 mg/ml) content. It was then
stored at 4 C in the presence of thimerosal (0.01%,
w/v) to prevent bacterial growth.
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The conjugate preparation was also checked by
SDS-PAGE (silver nitrate stain) and Western blot
analyses. Several high molecular weight bands
appeared on the gel above the pure pCB12-10-6 band
and near the stacking well, the latter being an
evidence that cross-linking occurred during
conjugation. Western blot analyses showed that each
band was reactive with the antisera used (anti-GCM,
-VR1, and -VR2), proving covalency of the conjugate
bonds.
EXAMPLE 11: Conlu~ation_of_Meningococcal_peptides
to CRM and bovine serum albumin
---------------------------------------
Peptides designated as M20 and M21 were pro-
duced on an ABI model-peptide synthesizer by solid
phase synthesis using the tBoc chemistry and were
coupled to CRM197 (prepared as described by Ander-
sen, U.S. Patent No. 4,762,713) using a bifunctional
crosslinking agent, sulfosuccinimidyl (4-iodoacetyl)
amino benzoate (Sulfo SIAB; purchased from Pierce)
following the modification of a published procedure
(Weltman, J.K. et al_, (1983) Bio Techniques 1,
148-152). Briefly CRM197 was activated by sulfo
SIAB resulting in the formation of an amide bond
between SIAB and amino groups of CRM197. After the
removal of unreacted crosslinker from the activated
CRM197 by gel filtration, peptide (M20 or M21)
containing linking spacer (represented in underlined
letters) with carboxy terminal cysteine residue was
CA 02007248 2006-06-15
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mixed with activated CRM and incubated at room
temperature for 2-4 hours. Following the reaction,
the conjugated material was dialyzed extensively
against PBS at 4 C.
The sequence of M20 peptide (VR2 epitope) is as
follows:
H-Tyr-Tyr-Thr-Lys-Asp-Thr-Asn-Asn-Asn-Leu-Thr-Leu-
Val-Pro-Ala_G1y_Ala_Cys_OH
The sequence of M21(VR1 epitope) peptide is:
H-Ala-Gln-Ala-Ala-Asn-Gly-Gly-Ala-Ser-Gly-Gln-Val-
Lys_ Ala_ Gly_Ala_Cys_OH _
Conjugated materials were subjected to SDS
PAGE, transferred to PVDF membranes (Immobilon,
Millipore) and reacted with specific monoclonals
which recognize VR1 and VR2 epitopes. Figure l0a
and lOb show the western blot analysis of M20 and
M21 CRM197 conjugates, against a pool of VR1 and VR2
specific monoclonals (Adam I, G2-D12-8 (P1.7), MN5-
C11-G (P1.16) and MN14-Cll-6 (P1.7)).
In order to assay the antibody response to M20
and M21 peptide by enzyme linked immunoassay
procedure, BSA conjugates were prepared by using a
different bifunctional crosslinking agent, N-suc-
cinimidyl bromoacetate as described by Bernatowicz
and Matsueda (Anal. Biochem. 155, 95-102 (1986)).
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Covalent coupling of peptide to the protein was
confirmed by western blotting of electrophoresed
samples as described for CRM197 conjugates.
EXAMPLE 12:- Retention of T cellactivitY_bY_M20
andM21_CRM}q_,_conj.ugates
To determine whether conjugation of the VR1 and
VR2 epitopes to CRM197 adver,sely affect the T cell
recognition of the CRM197 protein a T cell proli-
ferative assay was performed as previously described
by Bixler and Atassi (Immunol. Commun. 12:593,
1983). Briefly, SJL/j mice were immunized with 50
g of native CRM197 emulsified in CFA. Seven days
later, lymph nodes were removed, cultured in RPMI
and challenged with various concentrations of
proteins (0.05-100.0 pg/ml) and peptides. After 3
days incubation, cultures were pulsed with [3H]-thy-
midine for 16 hours and then harvested for counting.
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TABLE 3
T.cell responses to meningococcal peptide-CRM197
conjugates.
In Vitro Challenge Maximum observea (3H) Incorporation
,ug/m ACPM SI
-------------------------------------------------------------
Diphtheria toxoid 5 27,510 57
CRM197 50 108,631 221
CRM197 - mock conjugate 100 116,326 236
M21-CRM197 100 182,499 370
M20-CRM197 10 89,972 183
CON A 1 34,316 70
LPS 50 61,579 126
Tetanus toxoid 10 515 2
Background (cpm) - 494 1
-------------------------------------------------------------
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As shown in Table 3, a comparison of CRM197
with the CRM197-mock conjugate shows that the
conjugation procedure by itself did not alter the T
cell recognition of the protein. The T cell respon-
ses induced by the M20 and M21-CRM197 conjugates
were essentially equivalent to or greater than the
response elicited by CRM197 itself indicating that
the recognition of the T cell epitopes on the CRM197
is not adversely affected by the peptide
conjugation. The responses to the control materials
Con A, LPS and tetanus toxoid were as expected.
-EXAMPLE-13: -Immunoqenicit of--con'-u~ate -and
------------ --------------~'-------~--------
recombinant meningococcal B vaccines
--------------------------------------------
Recombinant flagellin expressing the
meningococcal VR1 and/or VR2 epitopes were prepared
and purified as described in Examples 7, 8 and 9.
In addition, synthetic peptides representing the
meningococcal epitopes VR1 and VR2 were synthesized,
covalently coupled to the carrier molecule CRM197
and purified as in Example 12. Vaccines were
formulated with each of these materials at protein
concentrations of 10 or 100 g/ml for each of the
components. The vaccine compositions also included
aluminum phosphate at 1 mg/ml or except as noted
were compounded with Freund's complete adjuvant or
without supplemental material.
To evaluate immunogenicity, outbred Swiss
Webster mice were immunized intramuscularly at weeks
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0 and 2 with 1 or 10 g protein/dose. Sera were
collected at two week intervals, pooled for assay,
and screened for antibody activity by ELISA to outer
membrane complex (OMC), purified OMP (P1.16), VR1
peptide coupled to bovine serum albumin (M21-BSA),
VR2 peptide coupled to BSA (M20-BSA), wildtype
flagellin, and to CRM197. The results of the ELISA
performed on sera obtained at 6 weeks are shown in
Table 4.
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TABLE4
Immunogenicity_of recombinnnt or CRM197 conjugate_vaccinee_
containing the meningococcal P1.16*OMP epitopea VR1 and VR2. ------------------
----------- ---------- -------------------
DOSE ELISA TITERS.4 WEEKS AFTER SECONDARY BOOSTi
Ng OMC P1.16 M21-BSA M20-BSA FLAGELLIN CRM
-----------------------------------------------------------------
pPx1650 (control wildtype flagellin)2
1 <150 <100 171 100 427,781 ND
<150 100 154 <100 468,385 ND
pCB1-4
1 532 4,376 4,525 ND 787,120 ND
10 2,034 12,387 17,565 ND 887,861 ND
pCB2-W
1 150 308 ND 501 263,143 ND
10 1,350 12,190 ND 5,476 1,493,216 ND
pCB12-10-6
1 615 3,374 4,651 824 299,889 ND
10 1,423 3,666 3,882 2,253 497,622 ND
pCB12-10-6 without aluminum phosphate
1 409 739 505 597 139,147 ND
10 450 1,533 817 1,611 358,033 ND
M20-CRM197
1 <150 <100 217 <100 ND 42.27
10 50 <100 150 <100 ND 95.31
M21-CRM197
1 68 249 10,494 100 ND 17.41
10 110 31.1 26,807 191 ND 20.92
MIXTURE OF M20 AND M21 CONJUGATES
1 50 100 40,000 187 ND 37.32
10 50 227 15,539 132 ND 184.27
OMP P1.16
1 12,630 17,714 100 764 ND ND
10 23,178 67,565 162 3,276 ND ND
pCB1-4 in CFA
10 1,665 10,606 19,945 ND 1,841,852 ND
pCB2-W in CFA
10 1,157 6,869 ND 17,749 1,217,063 ND
-------------------------------------------------------------------
1 All pre-bleed values at or below the lower limit of assay of 1/100
C~ilution.
' All vaccines were formulated with 1 mg/ml aluminum phosphate except
as noted.
2Q0'724$
..~.
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Alternatively, the various vaccines were
evaluated for immunogenicity in 6-8 week old NIH
outbred mice. Ttie mice were immunized with 100 g
(total protein)/dasesubcutaneously on week 0 and 4
with vaccine and sera was collected on week 6. The
sera were evaluated in an ELISA assay and using
antigens as described in Example 6. Bactericidal
activity was measured as in Example 6. The results
are found in Tabae 5.
TABLE S
ELISA (titer > 0.5 OD)
Bactericidal
Vaccine OHC Class 1 0MP Synth. Peptide Test
FLAGELLIN
<1:64
pi654
pCB12.10.6 - 1:900 - <1:64
PCB2_Q - 1:300 1:100 <1:64
pGB1-4 1:300 (.25) 1:2700 - <1:64
CRli197 - - - <1:64
H20-CRH197 1:00 1:8100 - <1:64
K21-CRM197 1:300 (.125) 1:8100 - <1:64
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The recombinant flagellins containing either a
VR1, VR2 or a cassette of both VR1 and VR2 were
effective in eliciting an antibody response which
was cross-reactive to the purified P1.16 and to a
lesser extent to OMC. Sera from animals immunized
with 10 g of either pCB1-4 or pCB2-w induced
antibodies which bound to their respective peptide-
-BSA conjugates as well as cross reacted with the
P1.16 and OMC. Similar results were obtained with
the constructed pCB12-10-6 which contains both
meningococcal epitopes. In addition, each con-
struction induced significant anti-flagellin titers
as well. In contrast, the control wildtype fla-
gellin only induced an antibody response to fla-
gellin itself. Sera collected prior to immunization
showed no pre-existing response to the materials
being evaluated.
The data also demonstrate the benefits of
formulating the recombinant flagellins with alum or
other adjuvants such as CFA. The construction
pCB12-10-6 was formulated with and without the
addition of aluminum phosphate. As shown in Table
2, pCB12-10-6 alone was capable of inducing an
antibody response which reacts to the peptide con-
jugates as well as to the purified P1.16 as well as
to OMC. In comparison, the same construction when
formulated with alum was able to elicit greater
antibody response at an equivalent dose. Similarly,
the recombinant flagellins pCB1-4 and pCB2-w were
also formulated with CFA. Again, equivalent or
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higher antibody titers were observed in the presence
of CFA.
The results of the immunogenicity studies with
the meningococcal VR1 and VR2 conjugates are also
shown in Table 4. Both the M20 and the M21-CRM197
conjugates as well as a mixture containing equal
amounts of both conjugates were capable of inducing
an anti-CRM197 response as well as an anti-Class I
OMP response.
These preliminary data indicate a Class I OMP
variable region epitope either chemically conjugated
to a carrier or genetically fused to a carrier
elicits an immune response. New epitope-carrier
conjugates can be made using standard techniques to
enhance the immune response to the vaccine, for
example, use of 1) larger epitopes, 2) peptides with
multiple epitbpe repeats andJor 3) different
carriers.
EXAMPLE 14: Preparation of Meningococcal-human
-------------- ---------------- ------------
serum albumin glycoconjuqate
-----------------
GCM CPS was depolymerized by acid hydrolysis
and GCM OS obtained were subsequently activated via
NaIO 4 oxidation in aqueous buffer. The reactions
were monitored by HPGPC in aqueous eluent, using W
and RI detection. The reactions were each followed
by GPC desalting in water. GCM OS and human albumin
(HA) were mixed and conjugated essentially as
described in Example 10 for the meningococcal-
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flagellin glycoconjugate. The final preparation was
stored in the cold, in the presence of thimerosal to
prevent bacterial growth.:,:.
In preparation of the conjugate, the following
experimental conditions were employed. Human
albumin (HA; SigmaR, St. Louis, MO) was dissolved in
15% sucrose (10 mg/ml) and then stored at -20 C.
GCM CPS (lot # 86 NM 01; 106 mg; final concen-
tration: 10 mg/ml was hydrolyzed in 0.1 N HC1 at
50 C with agitation. Aliquo.ts (25 l) were with-
drawn at regular intervals, the reaction stopped by
addition of sodium hydroxide (NaOH) and analysis was
performed by HPGPC as described. After 3 h 40 min.,
the reaction was stopped by addition of NaOH, and
the GCM OS were ciesalted by GPC. Fractions were
collected (1.2 ml) and analyzed as described before.
Positive fractions were pooled and lyophilized.
Desalted GCM OS (89 mg) were then stored at -20 C.
Activated OS were prepared by oxidation of GCM OS
(11.8 mg; final concentration: 5mg/ml) with 2 mM
NaIO4 in 0.05 M sodium _phosphate buffer (pH 6.2-6.5)
at RT, in the dark, with agitation. The reaction
w a s s t o p p e-d.- a f=t-e'r %-3=0 m-i=n. ~b-y- ~a:d.d i t i o.n_. o,f e t
h.y l e n e
glycol. HPGPC analyzes showed no degradation of the
molecular weight of the OS during activation.
Desalting and colorimetric anaylzes were then
performed as described above. The resulting
activated GCM OS (8.8 mg) were dissolved in water
(10 mg/ml) and frozen at -20 C.
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,__Both GCM-OS and HA sol.utions were analyzed by
HPGPC (UV at 206 and 280 nm respectively) before
being frozen, and prior to the conjugation. No
degradation occurred during storage, as ascertained
by the exact similarity of the elution profiles.
GCM OS (6 mg; final concentration: 2.5 mg/ml)
and HA (12 mg; final concentration: 5mg/ml) were
mixed in a polypropylene tube in 0.4 M sodium
phosphate buffer (pH 7.0), and NaBH3CN was added (60
moles) to initiate the conjugation (Anderson, U.S.
Patent 4,762,713, 1988; U.S. Patent 4,673,574, 1987;
U.S. Patent 4,761,283, 1988). The reaction mixture
was left one day at RT, then 4 days 15 35 C, without
agitation. The reaction was monitored by HPGPC (UV
at 280 nm) at different stages, and finally stopped
by dialysis/concentration on microconcentrators.
The final preparation was analyzed for NANA (Barry
et a1., J.Gen.Microbiol. 29P335-51, 1962) (2.07 mg
at 0.86 mg/ml) and protein (Lowry et al., J.Biol.
Chem. 193265-75, 1951) (9.51 mg at 3.96 mg/ml)
content. It was then stored at 4 C in the presence
of thimerosal (0.01$, w/v) to prevent bacterial
growth.
The conjugate preparation was also checked by
SDS-PAGE (silver nitrate stain) and Western blot
analyzes. A diffuse band appeared on the gel which
covered a significantly wider molecular weight range
than the pure HA. Western blot analyzes showed that
this band was reactive with the antiserum used
20 '7248
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(anti-GCM), proving covalency of the conjugate
bonds.
EXAMPLE_15: Immunogen.icitY_of-meningococcal
------- --- ------ - - -----
oliRosaccharide_recombinant_flagellin
vaccines
A meningococcal oligosaccharide-recombinant
flagellin vaccine was prepared as described above
and formulated at: 100 g pro.tein/ml. Vaccine
compositions were also prepared which contained
aluminum phosphate (1 mg/ml) or complete Freund's
adjuvant in addition to the glycoconjugate.
To evaluate the immunogenicity, outbred Swiss
Webster mice were immunized intramuscularly with 10
pg protein at week 0 and 2. Sera were collected at
weeks 0, 2 and then weekly intervals thereafter to 6
weeks. After collection, pooled sera samples were
assayed for antibody activity by ELISA to
meningococcal C oligosaccharide conjugate to human
serum albumin, OMC, P1.16, CB1 and CB2-BSA con-
jugates and flagellin.
The MenC-CB12-10-6 glycoconjugate was effective
at eliciting an immune response which was reactive
with both the oligosaccharide and the meningococcal
B.OMP epitopes expressed in the recombinant
flagellin. As shown in Table 5B, as little as three
weeks into the study, mice immunized with 1 pg of
MenC-CB12-10-6 conjugate in complete Freund's
adjuvant had detectable antibody to MenC-HSA, OMP
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and to both the CB1 and CB2 epitopes. Further, all
of the MenC-CB12-10-6 preparations, regardless of
adjuvant, elicited antibody response to MenC-HSA
which were greater than the response observed
following immunization with MenC-CRM197.
Table 5B. Immunogenicity of Meningococcal C-
recombinant flagellin vaccine one week after
secondary immunization.
-------------------------=-----------_-----------------------------------
ELISA TITERS!
IMMtJNOGEN Dose MenC-HSA OMP CB1-BSA CB2-BSA FLAGELLIN
-----------------------------------------------------------------------
MenC-CB12-10-6 -CFA 10 24,530 608 5,240 432 541,467
1 5,069 5,614 5,375 12,685 526,593
alum2 10 11,845 253 835 673 472,766
1 4,415 136 242 244 214,263
None 10 11,497 920 626 2,382 233,307
1 4,920 483 1,123 1,210 135,625
MenC-CRrt197 alum 10 4,905 ND ND ND ND
1 8,505 ND ND ND ND
OMP (P1.1-6) alum 10 ND 12,907 <100 <100 ND
I ND 10,405 <100 3,377 ND
------------------------------------------------------------------------
1 Titers for initial prebleed samples (week 0) samples were <100.
2 Aluminum phvsphate was used as adjuvant at 1 mg/ml.
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EXAMPLE 16: T-cell-epitopes_of-Class I OMPand
----------- ------- ----------- -
their identification
--------------------
An effective vaccine must contain one or more
T-cell epitopes. T-cell epitopes within a protein
can be predicted as described by Margalit et al., J.
Immunol_ 138:2213, (1987) or Rothbard and Taylor,
EMBO_J. 7:93.,.(1988). These predictive methods were
applied to the amino acid sequence of the Class I
OMP of N. meningitidis strains P1.7,16, P1.16 and
P1.15. The segments of the seque.nce containing_
potential T cell epitopes identified__by_these
methods are shown in Tables 6 and 7. The predicted
peptides were synthesized by standard FMOC
procedures, purified by standard methods and were
identified as shown in Table 8.
To determine which of the predicted peptides
actual contain T cell epitopes, their capacity to
stimulate human peripheral blood lymphocytes (PBL)
was tested by lymphocyte proliferative assay.
Briefly, peripheral blood was collected from HLA
typed normal volunteers or from volunteers who were
previously immunized with MPC-2 (Poolman, J.T. et
al., Antonievan Leeuwenhoek, 53:413-419, 1987)
--- ------- --------------- which contained P1.16, 15, Class 4 OMP and Group C
polysaccharide. Lymphocytes were isolated from the
peripheral blood by isolation on Ficoll-Hypaque
(Pharmacia Fine Chemicals AB, Uppsala, Sweden) and
cultured at 1 X 105 cells/well in supplemented RPMI
1640 (Gibco Laboratories, Paisly, Scotland)
CA 02007248 2006-06-15
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containing 10% heat-inactivated pooled human AB
serum. Cultures were challenged with various
concentrations of the predicted T cell epitopes
(0.05 - 10 g/ml). After in vitro challenge, the
cultures were incubated for six days and then pulsed
(18 hours) with 0.5 Ci of [3H]-thymidine. Cultures
were then harvested and counted by liquid
scintillation. Data are expressed as stimulation
indices which were calculated as a ratio of the CPM
obtained in the presence of antigen to the CPM
obtained in the absence of antigen.
As shown in Table 9, 10 of the 16 predicted
peptides showed some capacity to stimulate T-cells.
These include the peptides identified at 16-34,
47-59, 78-90, 103-121, 124-137, 151-158, 176-185,
223-245, 276-291 and 304-322. In some instances,
peptides stimulated a response in both immunized as
well as non-immune individuals. The response in the
non-immune individuals may be attributed to a
previous asymptomatic infection.
In the case of the T cell epitope identified as
region 176-185, enhancement of the T cell response
was observed following addition of the monoclonal
antibody MN5C11G (P1.16). Briefly, PBL were
challenged in vitro with a synthetic peptide
containing the region 175-185 or with this peptide
mixed with varying dilutions of MN5C11G. As shown
in Table 10, enhancement of the T cell response was
observed following addition of MN5C11G indicating
that monoclonal antibody recognized a B cell epitope
2007248
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within the region 176-185 and facilitates the
presentation of the peptide to the immune system.
Thus, it was established that the T and B cell
epitopes either coincide or are found on contiguous
sequences within the Class I OMP.
In several cases, T cell lines and clones were
established from individuals responding to various
peptides. Briefly, T cell lines were obtained by
culturing isolated lymphocytes in 24 well plates at
1 x 106 cells/ml. The culture medium, supplemented
RPMI-1640 with 10% human serum, also contained 12
U/ml recombinant IL-2 (Boehringer). In addition, 5.
x 104 homologous, irradiated (3,000R) antigen
presenting cells (APC) were also added to each well.
In some cases, APC were obtained from HLA compatible
donors. From the lines, T cell clones were isolated
by limiting dilution at a frequency of 0.5
cells/well. Cloiies were maintained by bi-weekly
stimulation with antigen in the presence of
irradiated APC and IL-2 (2 U/ml). Clones were
tested by lymphocyte proliferation assay essentially
as described above except that clones were cultured
at 1 x 104 cells/well in the presence of irradiated
APC.
Clones obtained as described were challenged in
vitro with OMP from 7 different strains of
meningococci. As shown in Table 11, the clones
recognized a T cell epitope or epitopes common to
the seven OMPs examined. Although the reactivity of
these clones to the various peptides remains to be
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determ.ined, the data, neverthel.es.s,: does indicate
the commonality of T cell epitopes among the various
strains. Now that these clones have- been
established and identified their peptide reactivity
will indicate T-cell epitopes for vaccine use.
Table 6:. ANALYSIS OF THE SEQUENCE OF N. MENINGITIDIS P1.16 OMP
FOR THE PRESENCE OF AMPHIPATHIC a-HELICIES ACCORDING
TO THE METHOD OF MARGALIT ET AL. (J. IMMUNOL. 138:2213, 1987)
----------------------------------------------------------------------
MID POINTS '
OF BLOCKS ANGLES AS
---------------------------------------------------------------------..
P 47-50 85-105 9.4
69-74 105-135 16.0
K 79-88 90-120 23.0
127-135 100-120 22.4
* 199-202 90-120 8.4
P 208-211 85-95 8.7
260-263 90-125 8.8
P 265-269 90-120 11.3
274-277 105-120 9.8
297-300 100-135 9.1
P 320-324 80-100 10.9
* 338-342 105-135 12.3
* 346-351 80-115 11.9
* 376-379 85-120 9.5
-----------------------------------------------------------------------
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Table 7. PRESENCE OF MOTIFS (UNDERLINED REGIONS) REPRESENTING
POTENTIAL. T CELL EPITOPES:- WITHTN.THE SEQUENCES OF N=.'MENINGITIDIS
P1.16 OMP AS DETECTED BY THE METHOD OF
ROTHBARD AND TAYLOR (EMBO J 7:93, 1988).
M R K K L T A L V L S A L P L A A V A D V S L Y G E I K A G V E G R N I
Q A Q L T E Q P Q V T N G V Q G N Q V K V T K A K S R I R T K I S DF G
S F I G F K G S E D L G E G L K A V W Q L E Q D V S V A G G G A S Q W G
N R E S F I G L A G E F G T L R A G R V A N Q F D D A S Q A I N P W D S
N N D V A S Q L G I f K R H D D M P V S V R Y D S P E F S G F S G S V Q
F V P A Q N S K S A Y K P A Y Y T K D T N N N L T L V P A V V G K P G S
D V Y Y A G L N Y K N G G F A G N Y A F K Y A R H A N V G R N A F E L F
L I G S A T S D E A K G T D P L K N H Q V H R L T G G Y E E G G L N L A
L A A Q L D L S E N G D K A K T K N S T T.E I A A T A S Y R F G N A V P
R I S Y A H G F D L I E R G K K G E N T S Y D Q I I A G V D Y D F S K R
T S A I V S G A 1+T L K R N T G I G N Y T Q I N A A S V G L R H K F
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TABLE 8. SUMMARY OF PREDICTED T CELL EPITOPES SYNTHESIZED.
-------------------------------------------------------------
RESIDUE NO. SEQUENCE
------------------------------------------------------------
S.. 16-34 NIQAQLTEQPQVTNGVQGI+1
2. 47-59 TKISDFGSFIGFK
3. 57-71 GFKGSEDLGEGLRAV
4. 78-90 VSVAGGGASQWGN
. 103-121 TLRAGRVANQFDDASQAI2it
6. 124-137 DSNNDVASQLGIFR
7. 151-158 GGFSGFSG
8. 176-185 YYTKDTNNNL
9. 190-202 AVVGKPGSDVYYA
10. 215-2 2 8 YAFKYARNAHVGRN
11. 223-245 ANVGRNAFELFLIGSATSDEAKG
12. 241-261 DEAKGTDPLKNHQVHRLTGGY
13. 276-291 LSENGDKAKTKNSTTE
14. 304-322 VPRISYAHGFDLIERGKKG
15. 317-329 ERGKKGENTSYDQ
16. 352-366 KRNTGIGNYTQINAA
------------------------------------------------------------
200'7248
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Table 9. SUMMARY OF LYMPHOCYTE RESPONSES TO MENINGOCOCCAL SYNTHETIC
-P=EPT'3DES- IN -HL-A - TYPED VOLUNTEERS.
RESPONSE TO SYNTHETIC PEPTIDE
VOLUNTEER/HLA TYPE 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
---------------------------------------------------------------------------
IMMUNIZED VOLUNTEERS
1. DR4,W10,W53 - - + - - - - -
2. DR3,W52 - + + - +
3. DR3,7,W52,W53 - - - + - - - - - + -
4. DR2,W6,W13,W15,W52 + - - + - - -
5. DR5,W11,7,W52,W53 + - + - - + - + +
6. DR5,W11,W10,W52
NON-IMMUNIZED CONTROL VOLUNTEERS
7. NOT TYPED - - - + - + - - - - + - - -
8. DRW13,W6,W52 - - - - - - - - - - - - - - - -
9. DR2,W15,4,w53 - - - - - - - - - -
10. NOT TYPED - - - - - - - - - - - - - - -
11. NOT TYPED - - - + - - - - -
12. DR2,W15,3,W52 - + - - - - - - - + - - - - -
13. DR5,W11,W52 - - - - - + - - - - - - - -
14. DR3,7,W52,W53 - - - - + - - - - - + - -
15. DR3,4,W52,W53 - - - - + - - - - - - - - - -
16. DR3,w12;5,W52
17. DR2,w15,7,W53 +
18. DR1,3,W52
19. DR3,4,W52,W53 + +
20. DR1,7,W53 -
21. DR4,W8,W52,W53
22. DR1,W13,W6,W52
23. DR2,W16,5,W11,W52 +
24. DR5,W11,W6,W13,W52 - -
25. DR1,3 W52 +
26. DR1,W6,W13,W52 + +
27. DRW6,W13,W52
28. DRW6,W13,W52 +
---------------------------------------------------------------------------
The responses were scored as follows -, SI<2; , 2<SI<3 and +, SI > 3.
20Q-'7248
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Table 10. PRESENTATION OF A SYNTHETIC PEPTIDE TO PERIPHERAL BLOOD
LYMPHOCYTES IS ENHANCED BY A MONOCLONAL ANTIBODY RECOGNIZING REGION
179-184 OF MENINGOCOCCAL CLASS I OMP.
----------------------------------------------------------------------
__.
IN VITRO CHALLENGE CPM
----------------------------------------------------------------------
GGYYTKDTNNNL 3,017
GGYYTKDTNNNL* + MN5C11G (1.200) 22,836
GGYYTKDTNNNL + MN5C11G (1:1000) 12,608
MEDIA 330
----------------------------------------------------------------------
* Underline region indicates sequence recognized by monoclonal antibody
MN5C11G.
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Table 11. RECOGNITION OF OMP FROM DIFFERENT MENINGOCOCCAL STRAINS
BY HUMAN T-CELL CLONES
--------------------=-----------------------------------------
RESPONSE OF HUMAN T CELL CLONES (CPM X 10-3)
STRAIN SUBTYPE 5-5 5-7 5-9 5-12 S- 13 - 5-15
------------------------------------- - -------------------------------
H44-76 P1.16 6.0 1.2 6.8 2.6 2.3 9.5 1.5
SWISS 4 P1.15 4.9 1.0 10.1 6.9 3.6 10.5 1.4
395 P1.9 5.2 1.5 4.8 1.5 6.1 13.1 1.4
2996 P1.2 5.4 1.0 3.7 2.3 3.4 11.8 1.0
M990 P1.6 3.6 0.4 3.5 2.5 0.9 4.7 0.6
187 P1.1 4.4 0.7 4.5 3.1 1.6 6.2 1.4
6557 P1.17 3.7 2.0 8.2 4.2 1.7 6.2 0.8
MEDIA -- <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1
-----------------------------------------------------------------------
CA 02007248 2006-06-15
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EXAMPLE 17: Construction of Protein Model for
-------------- -----------------------------------------
Membrane Topoloqy of-Class I OMP and
Comparison-to Other-Pathoqenic-Gram
Neqative Porin Proteins for Vaccine
-------------------------------------------
Development
-------------
A model was constructed using the principles
recognized for the structure of several Escherichia
coli outer membrane proteins (Vogel, H. et al.
(1986) supra Ferenci, T. et al-._ (1988) supra; and
Tommassen, J. (1988) supra). The central assumption
is that protein segments spanning the outer membrane
form beta-sheets. Specifically, in the case of
Class 1 protein, the division in exposed and trans-
membrane segments was arrived at in the following
way:
1. A comparison of the amino acid sequence of
Class 1 protein (subtype P1.16) with those of
the gonococcal PIA and PIB proteins
(Carbonetti, N.H. et al. (1967) PNAS 84:9084;
Carbonetti, N.H. et al., (1988) PNAS 85:6841;
and Gotschlich, E.C. et al. (1987) PNAS 84:
8135) reveals 34% identity. In the model, the
variable sequences form the surface-exposed
parts, whereas the conserved regions are placed
mostly in the outer membrane and periplasm.
Thus, the latter two areas consist of 58% of
residues that are conserved among all proteins,
CA 02007248 2006-06-15
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2. The hydrophilic maxima observed in a hydropathy
profile (Kyte, J. et al. (1982) J.-Mol_- Biol _
157:105) to correspond to exposed regions.
3. The transmembrane segments should preferen-
tially be able to form amphipathic beta-strands
of 9-12 residues, with at least one side
consisting entirely of hydrophobic residues.
These conditions are met in 12 of the 16
membrane-spanning segments.
4. The number of residues at the periplasmic side
is minimized.
Figure 11 shows the model for the folding of
Class 1 protein in the outer membrane. The sequence
shown is for subtype P1.16. The top part of the
figure shows the surface-exposed regions, whereas
the central part indicates the presumed trans-
membrane segments, whose length is set at ten.
Amino acid are shown alternating where they can form
an amphipathic beta-strand. This model contains
eight surface loops, whereby the first and the
fourth loop contain the type-specific and protective
variable region epitopes. These epitopes, as has
been shown when formulated into a vaccine, can
elicit a protective immune response. Loop 5 is
constant and has been shown to elicit cross-reactive
antibodies to other OMPs and is useful for vaccine
formulation.
CA 02007248 2006-06-15
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The one or two variable epitope regions of the
individual proteins are located on so called surface
loops of these membrane proteins. Such porin outer-
membrane proteins contain more than two surface
loops. This implicates that there are surface loops
which have near identical amino acid sequence in the
different Class 1 outer-membrane proteins as well.
This opens the way to use of common peptides of the
Class 1 outer-membrane protein for vaccine
objectives as well. More especially a schematic
two-dimensional model of the meningococci Class 1
outer-membrane protein P1,16 is illustrated in
Figure 11. This model contains eight surface loops,
whereby the first and the fourth loop contain the
type specific epitopes as shown on the basis of
strain subtyping results. The fifth surface loop
represents the constant region described above.
Antibody to the constant region of loop 5 appears to
react with N. meninqitidis OMP complex. The amino
acid sequence of Class I OMP, as derived, was
compared to the Class OMP of N. meninqitidis
(Murakami, K. et al., (1989), Infect. Immun.,
57:2318) and the porin PIA and PIB proteins of N.-
qonorrhoeae. With similar principles as used for
the Class I OMP modeling, the sequences were aligned
as follows:
2007248
~~...
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LOOP
1
Class I .. DVSLYGEIKAG VEGRNIQAQLTEQp~VTNGVQGN
Class IB DVTLYGAIKAG V------------- QTYRSVEHT
tk tk k~e ir !r ~t ic ~ ~c
Class IA DVTLYGTIKAG VEGRNIQAQLTEQPETSRSVAUg
** *** **xoT
Class II DVTLYGTIKAGV EGRNIQAQLTEQPEVSRVKDAG
~~ ~~* ***~c*
QVKVTKAKSRIRTKI SDFGSTIGFKGSEDLGEGL
* ,t ** ~~~~~ ***,t ** -
DGKVSRVET--GSEI ADFGSKIGFKGQEDLGNGL
GAQADRVKT--A.TEI ADLGSRIGFKGQEDLGNGL
TYKAQGGKSKTATQ IADFGSKIGFKGQEDLGNGL
* * ~~ **~** ** * ~~
LOOP 2
KAVWQLEQD VSVAGGGASQWGN FtESFIGLAGEFG
KAVWQLEQG ASVAGTNTG-WGN ftQSFVGLRGGFG
** ***~rx * ~r,~* ~* t~ * t*
KAYWQLEQK AYVSGTDTG-WGN RQSFIGLKGGFG
** ***** * *** ~* ,r* * *#
KAIWQLEQ KASIAGTNSG-WGNRQSFIGLRGGFG
,r,t *~e*** * ~*f ~~ ~~ ~k ,t*
- L4oP 3
TLRAGRVANQFDDIASQAINFWIISNNDVASQLQI-
* *
TIRAGSLNSPLF{N TGANVNAWESGKFTGNVI,EIS
* *
KVRVGRLNSYLKD TGGFNPWEGKSYYLPLSNIAQ
TVRPsGNLNTVLKDaGDNVNAWESGSNTEDVLGLG
* *
2007248
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LOOP 4
-FKRSDD MPV SVRYDSPEFSGFSGSVQFV PAQNS
~x***~~ ~~ ,~ * ~~t
GMAQREB RYL SVRYDSPEFAGFSGSVQY'A PKDNS
PEERPiV-- -- SVRYDSPEFAGFR:AV-QYV PNDNS
~ ****~** ~,~ * ~ ~,~
TIGRVESREI SVR.YDSPVFAGFaGSVQ YVPRDNA
* ~c ~c k x +~ dt dr ,t 9r * atr ~s
KSAYRPAYYTKDTDINNLTLVPAVVGKPGSDVYYA
I*
GS--------------------- --_NG ESYHV
~
ESYHA
ND---------,-- ---VDKYKHTKSSR ESYEiA
*
LOOP S
GLNYKNGGFAGNYAFKYA RHANVGRNAFELFLIG
GLNYQNSGFFAQYAGLFQ RYGEGTKK---. IE
GFI3YKNSGFFVQYAGFYK RHSYTTEKHFELFL--
*
GLKYENAGFFGQYA GSFARYADLNTD-----AE
,t ** * t * *.~
N
SATSDEAKGTIPPLKFi QVHRLTGGYEEGGLNLALA
DDQTYSIPSLFVERL QVHRLVGGYDNNALYVSVA
~~*** ~~t~ ,~ *
------L QVHRLVGGYDHDALYASVA
~~*** *~e~c * *
RVAVNTANAHPVKDY QVHAVVAGYDANDLYVSVA
**~** w,t* * #
2007248
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LOOP 6
AQLD LSE--NGDFCAKI'KNSTTE zAATASYRFGNA
AQQQ DYGAMSGNSQTE VAATAAYRFGNV
~ ~ ~*** *****
VQQQ DAKLTPTRND=NSFiNSQTE VAAATAAYRFGNV
GQ YEAAKNNEVGSTKGKKHQTQ VAATAAYRFGNV
LOOP 7
VPRISYAEGFDLI ERGRRGENTSYDQ IIAGVDYD
.. . .A 1
7C 7t 7C 3q....
TPRVSYAHGFKGT'VDSANbNT-YDQ VVVG.AEYD
~r* ****** ~r * * *~
TPRVSY'AgGFKGS VYDADNDNT-YDQ VVVGAEYD
** ~*,~*,~* ~ ~ # **
TPRVSYAHGF IC'AKVNGVKDANYQQDQ VIVGADYD
** *~r~r*** ~ ~r * **
LOOP 8
FSKRTvAIVSGAWL KRNTGIGNYTQIN AASVGLRHKF
FSKRTSALVSAGWL QGGKGA.DKIV-ST ASAVVLRHRF
FSKRTSALVSAGWLJQRGKGTEKFV-AT~VGGVGLRHKF
******* ** * * L * * *****
e
FSKR''SALVSAGWL KQGKGTGKVEQ-TASM VGLRFiKF
~.~ -93-
20Q1240
Structural similarities are indicated with
transmembrane and surface loop regions. With the
information now available for Class I OMP and
information based on surface loop size, location,
intraspecies amino acid homology or heterology of
the loop regions of the particular porin protein,
predictions.of epitopes for incorporation into
vaccines for other pathogenic gram negative bacteria
including N gonorrohoeae are possible. Using the
same methods employed for Class I OMP, these epi-
topes can be evaluated for vaccine purposes.
Deposit of Microorganisms
The N. meningitidis strain H4476 (B:15:P1.7,16)
was deposited on December 12, 1989 in the Centraal
Bureau voor Schimmelculturen (CBS), Baarn, The
Netherlands and has deposit number CBS 635.89. The
N. meningitidis Class 2/3 OMP deficient mutant
HIII-5 was deposited on December 12, 1989 in the
CBS, Baarn, The Netherlands and has deposit number
CBS 636.89.
Equivalents
Those skilled in the art will recognize, or be
able to ascertain using no more than routine experi-
mentation, many equivalents to the specific embodi-
ments of the invention described herein. Such
equivalents are intended to be encompassed by the
following c:Laims.
...
