U.S. patent application number 10/288978 was filed with the patent office on 2003-05-29 for chaperone and adhesin proteins; vaccines, diagnostics and method for treating infections.
Invention is credited to Auguste, Christine Gale, Hultgren, Scott J., Langermann, Solomon, Pinkner, Jerome S..
Application Number | 20030099665 10/288978 |
Document ID | / |
Family ID | 26767901 |
Filed Date | 2003-05-29 |
United States Patent
Application |
20030099665 |
Kind Code |
A1 |
Langermann, Solomon ; et
al. |
May 29, 2003 |
Chaperone and adhesin proteins; vaccines, diagnostics and method
for treating infections
Abstract
The present invention provides bacterial immunogenic agents for
administration to humans and non-human animals to stimulate an
immune response. It particularly relates to the vaccination of
mammalian species with heteropolymeric protein complexes as a
mechanism for stimulating production of antibodies that protect the
vaccine recipient against infection by pathogenic bacterial
species. In another aspect the invention provides antibodies
against such proteins and protein complexes that may be used as
diagnostics and/or as protective/treatment agents for pathogenic
bacterial species. A novel vector for expressing the FimC-H complex
at optimal levels is also disclosed.
Inventors: |
Langermann, Solomon;
(Baltimore, MD) ; Hultgren, Scott J.; (Ballwin,
MO) ; Pinkner, Jerome S.; (St. Louis, MO) ;
Auguste, Christine Gale; (Germantown, MD) |
Correspondence
Address: |
CARELLA, BYRNE, BAIN, GILFILLAN,
CECCHI, STEWART & OLSTEIN
6 Becker Farm Road
Roseland
NJ
07068
US
|
Family ID: |
26767901 |
Appl. No.: |
10/288978 |
Filed: |
November 6, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10288978 |
Nov 6, 2002 |
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09298494 |
Apr 23, 1999 |
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6500434 |
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60082824 |
Apr 23, 1998 |
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Current U.S.
Class: |
424/190.1 ;
424/164.1 |
Current CPC
Class: |
Y02A 50/474 20180101;
Y02A 50/30 20180101; A61K 39/0258 20130101 |
Class at
Publication: |
424/190.1 ;
424/164.1 |
International
Class: |
A61K 039/40; A61K
039/02 |
Claims
What is claimed is:
1. A vaccine against bacterial infections comprising a complex of a
bacterial chaperone protein with either an adhesin protein or an
immunogenic fragment of said adhesin protein.
2. A vaccine according to claim 1, wherein said vaccine is for
urinary tract infections.
3. A vaccine according to claim 2, wherein said vaccine is for
bladder infections.
4. A vaccine according to claim 3, wherein said vaccine is for
bladder infections caused by E. coli.
5. A vaccine according to claim 1, wherein said immunogenic
fragment of said adhesin protein is a mannose-binding fragment.
6. A vaccine according to claim 1 wherein said adhesin protein is
FimH, and said fragment is a mannose-binding fragment of FimH.
7. A vaccine according to claim 4, wherein said chaperone protein
is FimC.
8. A vaccine against bacterial infections comprising the bacterial
adhesin protein FimH or a mannose-binding fragment of FimH.
9. An antibody raised against a complex of a bacterial chaperone
protein with either an adhesin protein or an immunogenic fragment
of said adhesin protein.
10. An antibody according to claim 9, wherein said antibody is an
antibody that will detect urinary tract infections.
11. An antibody according to claim 9, wherein said antibody is
effective for the prevention and/or treatment of urinary tract
infections.
12. An antibody according to claim 9, wherein said antibody is
effective for the prevention and/or treatment of urinary tract
infections caused by E. coli bacteria.
13. An antibody according to claim 9, wherein said immunogenic
fragment of said adhesin protein is a mannose-binding fragment.
14. A antibody according to claim 9 wherein said adhesin protein is
FimH, and said fragment is a mannose-binding fragment of FimH.
15. An antibody according to claim 7, wherein said chaperone
protein is FimC.
16. An antibody raised against either the bacterial adhesin protein
FimH or an immunogenic mannose-binding fragment of FimH.
17. A method for preventing and or treating UTIs in a host
comprising immunizing said host with a member selected from the
group consisting of: (a) a vaccine according to claim 1, and (b) at
least one antibody raised against a complex of a bacterial
chaperone protein with either an adhesin protein or an immunogenic
mannose-binding fragment of said adhesin protein.
18. A method for preventing and or treating enterobacterial
infections in a host comprising immunizing said host with a member
selected from the group consisting of: (a) a vaccine according to
claim 7, and (b) at least one antibody raised against a complex of
a FimC with either FimH or an immunogenic mannose-binding fragment
of FimH.
19. The method according to claim 18 wherein the enterobacterial
infection is a urinary tract infection (or UTI).
20. A method for preventing and or treating an enterobacterial
infection in a host comprising immunizing said host with a member
selected from the group consisting of: (a) a vaccine according to
claim 8, and (b) at least one antibody raised against FimH or an
immunogenic mannose-binding fragment of FimH.
21. The method according to claim 20 wherein the enterobacterial
infection is a urinary tract infection (or UTI).
22. A method according to claim 21 wherein the UTI is caused by E.
coli bacteria.
23. A method for expressing a protein complex in a bacterial cell
comprising inserting into said cell a vector containing the protein
complex genes, wherein each said gene has a lac promoter attached
in 5' orientation with respect to each said gene.
24. The method of claim 23 wherein the protein complex is an
adhesin/chaperone complex.
25. The method of claim 23 wherein said bacterial cell is an
Escherichia coli cell.
26. The method according to claim 23 wherein the vector is a
modified pUC19 vector.
Description
[0001] This application claims the priority of Provisional
Application Serial No. 60/082,824, filed Apr. 23, 1998.
BACKGROUND OF THE INVENTION
[0002] This invention relates generally to the field of bacterial
antigens and their use, for example, as immunogenic agents in
humans and animals to stimulate an immune response. More
specifically, it relates to the vaccination of mammalian species
with heteropolymeric protein complexes as a mechanism for
stimulating production of antibodies that protect the vaccine
recipient against infection by pathogenic bacterial species,
including all types of enterobacteriacea.
[0003] In a particular aspect, the present invention relates to the
prevention and treatment of urinary tract infections such as
cystitis and/or pyelonephritis that are caused by pilus-carrying
bacteria (uropathogenic bacteria). In this regard, certain types of
Escherichia coli are of particular interest since they are the
predominant etiologic agent of urinary tract infections (UTIs).
[0004] UTIs provide one example of a disease process that is
mediated (or assisted) by the attachment to bacteria to cells. E.
coli is the most common pathogen of the urinary tract, accounting
for greater than 85% of cases of asymptomatic bacteriuria, acute
cystitis and acute pyelonephritis, as well as greater than 60% of
recurrent cystitis, and at least 35% of recurrent pyelonephritis
infections. Furthermore, approximately 25%-30% of women experience
a recurrent E. coli urinary tract infection within the first 12
months following an initial infection: after a second or third
infection the rate of recurrence increases to 60%-75%. Given the
high incidence, continued persistence, and significant expense
associated with E. coli urinary tract infections, there is a need
for a prophylactic vaccine to reduce susceptibility to this
disease.
[0005] While many factors contribute to the acquisition and
progression of E. coli urinary tract infections, it is widely
accepted that colonization of the urinary epithelium is a
prerequisite to infection. In a typical course of E. coli urinary
tract infection, bacteria originate from the bowel, ascend into the
bladder, and adhere to the bladder mucosa where they multiply and
establish an infection (cystitis) before ascending into the ureters
and kidneys. Disruption or prevention of pilus-mediated attachment
of E. coli to urinary epithelia may prevent or retard the
development of urinary tract infections. In this regard, a number
of studies have pointed to a role for pili in mediating attachment
to host uroepithelial cells.
[0006] To initiate infection bacterial pathogens must first be able
to colonize an appropriate target tissue of the host. For many
pathogens this tissue is located at a mucosal surface. Colonization
begins with the attachment of the bacterium to receptors expressed
by cells forming the lining of the mucosa. Attachment is mediated
via proteins on the bacterium that bind specifically to cellular
receptors. These proteins, or adhesins, are expressed either
directly on the surface of the bacterium, or more typically, as
components of elongated rod-like protein structures called pili,
fimbriae or fibrillae.
[0007] Type 1 pili are thought to be important in initiating
colonization of the bladder and inducing cystitis, whereas P pili
are thought to play a role in ascending infections and the ensuing
pyelonephritis.
[0008] Such pili are heteropolymeric structures that are composed
of several different structural proteins required for pilus
assembly. Two types of pili are of particular interest: P pili and
type 1 pili. P pili-carrying bacteria recognize and bind to the
gal.alpha.(1-4)gal moiety present in the globoseries of glycolipids
on kidney cells in mammals. Type 1 pili-carrying bacteria recognize
and bind to D-mannose in glycolipids and glycoproteins of bladder
epithelial cells.
[0009] PapG, the adhesin protein in P pili bacteria that mediates
the specific interaction of the pilus with receptors on the surface
of host cells, is found at the distal end of the tip fibrillum. Its
periplasmic chaperone protein is PapD which is highly conserved
across strains of E. coli. (Hultgren et al., Proc. Natl. Acad. Sci.
USA 86:4357 (1989); EMBO Journal 15:3792-3805 (1996).
[0010] With regard to type 1 pili, tip adhesins and other ancillary
subunits also have been identified. FimH is the D-mannose-binding
adhesin that promotes attachment of type 1 piliated bacteria to
host cells via mannose-containing glycoproteins on eukaryotic cell
surfaces. FimC is its periplasmic chaperone protein. FimH is also
highly conserved not only among uropathogenic strains of E. coli,
but also among a wide range of gram-negative bacteria. For example,
all Enterobacteriacea produce FimH. Thus, vaccines incorporating
the FimH antigen should exhibit a broad spectrum of protection.
[0011] Chaperone proteins are a class of proteins in gram-negative
bacteria that are involved in the assembly of pili by mediating
such assembly, but are not incorporated into the structure. PapD is
the periplasmic chaperone protein mediating the assembly of pili
for P piliated bacteria and FimC is the periplasmic chaperone
protein that mediates assembly of type 1 pili in bacteria.
[0012] Antibodies directed against purified whole type 1 or P pili
protect against cystitis and pyelonephritis, respectively, in both
murine and primate models for these diseases. Abraham et al.,
Infect Immun. 48:625 (1985), Roberts et al., Proc. Natl. Acad. Sci.
(USA) 91:11889 (1994), O'Hantey et al., J. Clin. Invest. 75: 347
(1985). However, such protection is limited to either homologous E.
coli strains from which the pili used as immunogens were derived,
or to a small subset of serologically cross-reactive heterologous
strains. Therefore, vaccines composed predominantly of the major
structural proteins of pili (i.e., PapA or FimA) appear to be of
limited value because antibodies developed against these highly
variable proteins are specific for the strains used for
immunization.
BRIEF SUMMARY OF THE INVENTION
[0013] In one aspect the present invention relates to a vaccine for
treating or preventing bacterial infections which utilizes as an
immunogen a complex of a bacterial periplasmic chaperone protein
with a bacterial adhesin protein. Preferably, the adhesin protein
is a pilus adhesin protein. In one embodiment, the periplasmic
chaperone protein and pilus adhesin protein are from E. coli; for
example, a member selected from the complexes PapD/PapG and
FimC/FimH.
[0014] In a particular aspect, the invention relates to vaccines
formulated from type 1 pilus-associated adhesins (or from
mannose-binding fragments thereof) or from complexes of chaperone
proteins (including PapD-like chaperones) and pilus-associated
adhesins for the treatment and/or prophylaxis of diseases caused by
pathogenic species of gram-negative bacteria, such as Escherichia
coli (E. coli). For example, it relates to treatment and/or
prophylaxis of urinary tract infections caused by E. coli with
vaccines formulated from at least one of (1) a fragment of the
pilus-associated adhesin FimH that retains mannose binding
capability (alone or complexed with its chaperone FimC), (2) the
pilus-associated adhesin PapG protein complexed with its
periplasmic chaperone protein PapD or (3) the full-length
pilus-associated adhesin FimH (alone or in a complex with its
chaperone protein FimC). This invention also relates generally to
the use of heteropolymeric protein complexes to raise antibodies in
non-human mammalian species useful, for example, as diagnostic
reagents and vaccines.
[0015] In yet another aspect, the present invention relates to the
production of essentially full-length bacterial adhesin proteins in
a recombinant host (in E. coli, another bacterial species, a
bacterial species with one or more disabled proteases, or a
non-bacterial production vector or host cell) or by synthesis. Such
recombinant or synthetic methods permit the production of the
full-length adhesin protein in the presence or absence of its
chaperone protein and when chaperones are absent preferably in the
absence of proteases that will shorten its length or start to break
it down. Even more preferable is the production of essentially
full-length FimH or a mannose binding analog or variant for use as
a vaccine. If such recombinant production is performed in a host
which is capable of producing an usher protein, the recombinant
production is under conditions which eliminate usher
production.
[0016] Using novel methods disclosed herein, it is possible to
recombinantly introduce into a bacterial cell the FimH and FimC
genes using a single vector, commonly a plasmid but in no way
limited thereto.
[0017] In a yet further aspect, the present invention relates to
the production of mannose-binding fragments of bacterial adhesin
proteins in a recombinant host (in E. coli, another bacterial
species, or a non-bacterial production vector or host cell) or by
synthesis. Preferably, when such protein fragments are expressed in
a bacterial host that produces an usher protein they are expressed
under such conditions that their usher protein is not expressed.
Such recombinant or synthetic methods permit the production of
mannose-binding adhesin protein fragments in the absence of the
their chaperone protein or as a complex with their chaperone
protein that can later be separated from the chaperone protein.
Preferably, such fragments are produced under conditions that avoid
shortening of their length by cleavage or break down by
proteases.
[0018] In another aspect, the proteins are produced with a
histidine label (or other suitable label) such that the full-length
proteins or fragments can be isolated due to their label.
[0019] In a further aspect, the present invention relates to the
production of a periplasmic chaperone protein in a complex with an
essentially full-length bacterial adhesin protein or appropriate
fragment thereof in a recombinant host (in E. coli, another
bacterial species, a bacterial species with one or more disabled
proteases, or a non-bacterial production vector or host cell) or by
synthesis or by recovering from a natural source. Even more
preferable is the production of the periplasmic chaperone protein
FimC complexed with essentially full-length FimH or a mannose
binding analog or variant for use as a vaccine.
[0020] In another aspect the present invention relates to a method
of prophylaxis and/or treatment of diseases that are mediated by
pili-bearing bacteria that have adhesin proteins. In particular,
the invention relates to a method for the prophylaxis and/or
treatment of infectious diseases that are mediated by type 1 pili
adhesin proteins. In a still further preferred aspect, the
invention relates to a method for the prophylaxis and/or treatment
of UTIs in humans, particularly in women or children.
[0021] In still another aspect the present invention relates to a
method of using one or more antibodies (monoclonal, polyclonal or
sera) to either a periplasmic chaperone protein or fragment thereof
complexed with an adhesin protein or an adhesin protein (alone) for
the prophylaxis and/or treatment of diseases that are mediated by
pili-bearing bacteria that have adhesin proteins. In particular,
the invention relates to a method for the prophylaxis and/or
treatment of infectious diseases that are mediated by type 1 pili
adhesin proteins. In a still further preferred aspect, the
invention relates to a method for the prophylaxis and/or treatment
of UTIs in humans, particularly in women or children, by utilizing
antibodies to either the periplasmic chaperone protein FimC
complexed with the adhesin protein FimH (anti-FimC-H) or the
adhesin protein FimH alone (anti-FimH).
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIGS. 1A and 1B show data results of immunoglobulin G (IgG)
titer to FimHt adhesin and whole type 1 pili, respectively, up to
78 weeks post immunization with purified adhesin, adhesin-chaperone
complex or whole type 1 pili, further described in Example 1.
[0023] FIGS. 2A-2C report the data for in vitro binding of type
1-piliated E. coli to human bladder epithelial cells and inhibition
by anti-FimH. FIG. 2A shows Type 1-piliated [HB101/pSH2 (black
bars) and non-piliated (cross-hatched)] bacteria which were
directly labeled with FITC and tested for their ability to bind to
bladder cells at various dilutions. FIGS. 2B and 2C, respectively,
illustrate the ability of anti-FimH and anti-FimC-H antibodies to
inhibit the binding of such type 1-piliated E. coli to human
bladder epithelial cells.
[0024] FIGS. 3A-3E report the data in the C3H/HaJ murine cystitis
model for protection of anti-FimH or anti-FimC-H antibodies in vivo
against bladder and kidney infections. FIGS. 3A and 3B show the
results of in vivo CFU/bladder counts of type 1-pilated FimH+ E.
coli (NU14) type 1-pilated FimH- E coli (NU14-1) at various
dilutions in unprotected mice. FIG. 3C shows the CFU/bladder in
vivo results for mice challenged with NU14 E coli after having been
injected with (histograms from left to right, respectively)
adjuvant alone, FimHt 30 .mu.g, FimHt 15 .mu.g, FimHt 3 .mu.g,
FimHt 0.05 .mu.g and FimC 30 .mu.g. FIG. 3D shows the protective
ability of anti-FimHt in mice injected with FimHt against the
progression of infection from the bladder to the kidney as
contrasted with naive mice who do not produce anti-FimHt. FIG. 3E
shows the protective effects of anti-FimHt antisera in mice
challenged with NU14 bacteria. The anti-FimC-H appeared to give
slightly better protection than FimHt.
DETAILED DESCRIPTION OF THE INVENTION
[0025] It is an object of the present invention to utilize as
immunogenic composition for a vaccine (or to produce antibodies for
use as a diagnostic or as a passive vaccine) comprising a bacterial
adhesin protein or a complex of a bacterial periplasmic chaperone
protein and such a bacterial adhesin protein. In one embodiment,
proteins (naturally or recombinantly produced, as well as
functional analogs) from bacteria that produce type 1 pili are
contemplated. Even more particularly, E coli proteins are
contemplated.
[0026] A particularly preferred embodiment of such an immunogenic
composition is for use as a vaccine (or as an immunogen for
producing antibodies useful for diagnostics or vaccines) wherein
the active component of the immunogenic composition is a member
selected from mannose-binding fragments of FimH adhesin protein
(alone or complexed with a periplasmic chaperone protein), the PapG
adhesin protein complexed with its chaperone protein PapD or the
full-length FimH adhesin protein (alone or complexed with FimC). Of
course, any adhesin assembled by the chaperone/usher pathway could
be prepared and utilized as a vaccine according to the present
invention.
[0027] In another aspect of the invention, such an immunogenic
composition may be utilized to produce antibodies to diagnose
urinary tract infections, or to produce vaccines for prophylaxis
and/or treatment of such infections as well as booster vaccines to
maintain a high titer of antibodies against the immunogen(s) of the
immunogenic composition.
[0028] While other antigens have been utilized to produce
antibodies for diagnosis and for the prophylaxis and/or treatment
of bacterial urinary tract infections, there is a need for improved
or more efficient vaccines. Such vaccines should have an improved
or enhanced effect in preventing bacterial infections mediated by
adhesins and pili.
[0029] There is a need for improved antigenic compositions
comprising adhesins for stimulating high-titer specific antisera to
provide protection against infection by pathogenic bacteria and
also for use as diagnostic reagents.
[0030] In one aspect, the present invention is directed to an
immunogenic composition comprising a purified complex of a
periplasmic chaperone protein and a chaperone-binding protein. The
chaperone-binding protein is maintained in the complex in an
immunogenic form capable of inducing an immune response when
appropriately introduced into a human or other mammalian species.
Adhesins are suitable chaperone-binding proteins for use in these
immunogenic compositions.
[0031] In this specification, the terms "pili", "fimbriae," and
"fibrillae" are used to refer to heteropolymeric protein structures
located on the extracellular surface of bacteria, most commonly
gram-negative bacteria. Typically these structures are anchored in
the outer membrane. Throughout this specification the terms pilus,
pili, fimbriae, and fibrilla will be used interchangeably.
[0032] A "periplasmic chaperone" is defined as a protein localized
in the periplasm of bacteria that is capable of forming complexes
with a variety of chaperone-binding proteins via recognition of a
common binding epitope (or epitopes). Chaperones perform several
functions. They serve as templates upon which proteins exported
from the bacterial cell into the periplasm fold into their native
conformations. Association of the chaperone-binding protein with
the chaperone also serves to protect the binding proteins from
degradation by proteases localized within the periplasm, increases
their solubility in aqueous solution, and leads to their
sequentially correct incorporation into an assembling pilus.
[0033] According to the present invention, it has been found that
FimH or fragments thereof that retain mannose binding capability
(alone or complexed with FimC) are superior immunogens to the
protein PapG for prevention of bladder infections due to E. coli. A
superior immunogen is defined as an antigen that will stimulate a
greater immune response, or a response that will last longer than
the immune response to another antigen utilized in the same
immunization protocol, or an antigen that will confer better
protection against bladder infections (e.g., FimH, FimC-H or
PapD/PapG).
[0034] The protein fragments and proteins of the invention are
useful immunogens for preparing vaccine compositions that stimulate
the production of antibodies that can confer immunity to pathogenic
species of bacteria. Further, preparation of vaccines containing
purified proteins as antigenic ingredients are well known in the
art.
[0035] Generally, vaccines are prepared as injectables, in the form
of aqueous solutions or suspensions. Vaccines in an oil base are
also well known such as for inhaling. Solid forms which are
dissolved or suspended prior to use may also be formulated.
Pharmaceutical carriers are generally added that are compatible
with the active ingredients and acceptable for pharmaceutical use.
Examples of such carriers include, but are not limited to, water,
saline solutions, dextrose, or glycerol. Combinations of carriers
may also be used.
[0036] Vaccine compositions may further incorporate additional
substances to stabilize pH, or to function as adjuvants, wetting
agents. or emulsifying agents, which can serve to improve the
effectiveness of the vaccine.
[0037] Vaccines are generally formulated for parenteral
administration and are injected either subcutaneously or
intramuscularly. Such vaccines can also be formulated as
suppositories or for oral administration, using methods known in
the art.
[0038] The amount of vaccine sufficient to confer immunity to
pathogenic bacteria is determined by methods well known to those
skilled in the art. This quantity will be determined based upon the
characteristics of the vaccine recipient and the level of immunity
required. Typically, the amount of vaccine to be administered will
be determined based upon the judgment of a skilled physician. Where
vaccines are administered by subcutaneous or intramuscular
injection, a range of 50 to 500 .mu.g purified protein may be
given.
[0039] The term "patient in need thereof" refers to a human that is
infected with, or likely, to be infected with, pathogenic bacteria
that produce pili and/or chaperone-binding proteins, preferably E.
coli bacteria (however a mouse model can be utilized to simulate
such a patient in some circumstances).
[0040] In addition to use as vaccines, such pili adhesin proteins,
mannose-binding adhesin protein fragments, and complexes of
periplasmic chaperone proteins and such pili adhesin proteins can
be used as immunogens to stimulate the production of antibodies for
use in passive immunotherapy, for use as diagnostic reagents, and
for use as reagents in other processes such as affinity
chromatography.
[0041] In an aspect of the invention complexes comprising the E.
coli chaperone FimC and the FimH adhesin associated with type 1
pili may be recovered from E. coli. These complexes are found in
relatively great amounts in recombinant E. coli strains which
express the FimC protein at levels in excess of those produced in
wild type strains. A suitable recombinant strain is C600/pHJ9205, a
strain in which expression of FimC has been put under control of
the arabinose promoter. Those skilled in the art will recognize
that other promoter sequences that can be regulated easily may also
be used.
[0042] Because FimC is a periplasmic chaperone, proteins isolated
from the periplasm of E. coli serve as the starting material for
the purification. An extract of periplasm is obtained by exposing
the bacteria to lysozyme in the presence of a hypertonic sucrose
solution. The elution of periplasmic proteins so obtained are
separated from cellular proteins by centrifugation.
[0043] The FimC-H complexes are purified from the crude mixture of
periplasmic proteins. One method for achieving this purification
takes advantage of the affinity of the FimH adhesin for D-mannose
residues. Because of this affinity. FimC-H complexes will bind to
mannose which has been covalently attached to a suitable
chromatography resin, such as Sepharose. After binding reaches
equilibrium the beads are washed to remove unbound, contaminating
proteins. Bound FimC-H complexes may then be eluted from the beads
using a solution containing mannose-residues, such as, for example.
methyl-.alpha.-d-mannopyranoside. To achieve further purification
of the complexes, an additional chromatography step, such as ion
exchange chromatography, may be used. Alternatively, FimC-H
complexes can be purified using conventional protein purification
methods.
[0044] In a similar manner, FimH fragments that are recombinantly
produced either by having E. coli produce the full-length FimH
protein and then fragmenting the protein or are produced
recombinantly may be isolated by the above mannose-binding affinity
purification. Thus, only fragments of the FimH protein that retain
mannose binding are isolated. Preferably, the mannose-binding
fragments have a label such as a his-tag included and may be
purified by methods such as Nickel chromatography (see examples
below).
[0045] The present invention provides for a recombinant production
or synthesis of adhesin proteins of pilus-bearing bacteria adhesin
proteins in the presence or absence of its chaperone protein for
use as a vaccine (or as an immunogen to produce antibodies for
diagnostic or therapeutic purposes). In particular an adhesin
protein may be individually expressed or co-expressed with its
corresponding periplasmic chaperone protein to make a complex of
the co-expressed proteins. Preferably, the adhesin protein is a
substantially full-length FimH protein, or an analog or derivative
thereof which maintains mannose binding capability. In this regard
cDNA, RNA and genomic sequences for such chaperone and adhesin
proteins are known. See Tables 1 and 2, below.
[0046] Moreover, the known sequences of chaperone and adhesin
proteins as referred to in Tables 1 and 2 may be utilized as probes
to retrieve other polynucleotides (such as from other species) that
encode the same or similar proteins which polynucleotides may then
be utilized for such recombinant expression. For example, it is
well-known in the art that FimH is highly conserved among various
bacterial species (not just among E. coli).
[0047] Tables 1 and 2 are as follows:
1TABLE 1 BACTERIAL CHAPERONE PROTEINS AND THEIR SOURCE Gen Bank
Chaperone Protein Accession No. Organism PapD X61239 E. coli FaeE
X56003 E. coli FanE X56001 E. coli Sfae 227911 E. coli MrkB M55912
K. pneumonia HifB X66606 H. influenzae FimC Z37500 E. coli MrpD
Z32686 P. mirabilis FocC Z46635 E. coli FimB X64876 B. pertussis
PefD L08613 S. typhimurium PmfD Z35428 P. mirabilis LpfB U18559 S.
typhimurium FasB U50547 E. coli HafB U54780 H. influenzae AftB
L77091 P. mirabilis F17D AF022140 E. coli EcpD L00680 E. coli YehC
AE000300 ? YraI AE000395 E. coli RalE U84144 Rabbit EPEC ClpE
L05180 E. coli CssC U04844 E. coli MyfB Z21953 Y. enterocolitica
PsaB M86713 Y. pestis CS3-1 X16944 E. coli CaflM X61996 Y. pestis
NfaE S61968 E. coli SefB L11009 S. enteritidis AggD U12894 E. coli
AfaB X76688 E. coli
[0048]
2TABLE 2 BACTERIAL ADHESIN PROTEINS AND THEIR SOURCE Adhesin
Accession Protein No. Organism PagG 42391 Escherichia coli PrsG
1172645 Escherichia coli SfaS 134449 Escherichia coli FimH 1361011
Escherichia coli HifE 642038 Haemophilus influenza HifE 1170264
Haemophilus influenza FocH 239711 Escherichia coli FimD 480043
Bordetella pertussis FimD 416479 Bordetella bronchiseptica BB171
PmfE 1709671 Proteus mirabalis LpfD 1361301 Salmonella
typhimurium
[0049] The chaperone and adhesin proteins as well as the sources
for their genes (or cDNA) are listed in the above tables for
illustrative purposes only. For example, the polynucleotides
encoding FimH, FimC-H complex and FimH mannose-binding fragments
may vary and the encoded proteins also vary from organism to
organism and from species to species. While the FimH protein is
highly conserved across different species (e.g., E. coli,
Salmonella and Klebsiella) and in various strains of E. coli, the
amino acid sequences of FimH do vary to some extent. However, such
variant amino acid sequences (as well as their analogs and
mannose-binding fragments) are contemplated within the meaning of
the terms "FimH" and "a FimH mannose-binding fragment" when such
terms are utilized in this application, for example. The meanings
of such terms are not limited to the particular amino acid
sequences shown or to the particular species of E. coli
specifically described in the specification or utilized in the
examples.
[0050] The polynucleotides encoding the proteins above and in the
above tables may also have the coding sequence fused in frame to a
marker sequence which allows for purification of the polypeptides
of the present invention. The marker sequence may be, for example,
a hexa-histidine tag supplied by a pQE-9 vector to provide for
purification of the mature polypeptides fused to the marker in the
case of a bacterial host, or, for example, the marker sequence may
be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7
cells, is used. The HA tag corresponds to an epitope derived from
the influenza hemagglutinin protein (Wilson, I., et al., Cell,
37:767 (1984)).
[0051] In a preferred aspect, the invention provides for
recombinant production of such proteins, chaperone/adhesin
complexes and mannose-binding fragments of such proteins in an E.
coli species host. A preferred host is a species of such bacteria
that can be cultured under conditions such that the usher gene (if
present) is not expressed. Further preferred is a host species that
is missing the usher gene or has a defective usher gene. Even
further preferred is a host which is missing the pilus proteins
other than the adhesin protein (and preferably produces its
chaperone). When an adhesin protein or a mannose binding fragment
of such adhesin protein is to be produced in the absence of its
chaperone protein (or to be separated from the chaperone after
production), the adhesin protein (or fragment) may be permitted to
become properly folded in the presence of its chaperone protein and
is then separated from the chaperone protein.
[0052] The present invention also relates to vectors which include
polynucleotides encoding one or more of the adhesin or chaperone
proteins of the present invention, host cells which are genetically
engineered with vectors of the invention and the production of such
adhesin proteins and/or chaperone proteins by recombinant
techniques in an isolate and substantially immunogenically pure
form.
[0053] Host cells are genetically engineered (transduced or
transformed or transfected) with the vectors comprising a
polynucleotide encoding a chaperone, adhesin protein, mannose
binding fragment of an adhesin protein, or the like of this
invention which may be, for example, a cloning vector or an
expression vector. The vector may be, for example, in the form of a
plasmid, a viral particle, a phage, etc. The engineered host cells
can be cultured in conventional nutrient media modified as
appropriate for activating promoters, selecting transformants or
amplifying the polynucleotides which encode such polypeptides. The
culture conditions, such as temperature, pH and the like, are those
previously used with the host cell selected for expression, and
will be apparent to the ordinarily skilled artisan.
[0054] Vectors include chromosomal, nonchromosomal and synthetic
DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage
DNA; baculovirus; yeast plasmids; vectors derived from combinations
of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus,
fowl pox virus, and pseudorabies. However, any other vector may be
used as long as it is replicable and viable in the host.
[0055] The appropriate DNA sequence may be inserted into the vector
by a variety of procedures. In general, the DNA sequence is
inserted into an appropriate restriction endonuclease site(s) by
procedures known in the art. Such procedures and others are deemed
to be within the scope of those skilled in the art.
[0056] The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequence(s) (promoter)
to direct mRNA synthesis. As representative examples of such
promoters, there may be mentioned: LTR or SV40 promoter, the E.
coli. lac or trp, the phage lambda P.sub.L promoter and other
promoters known to control expression of genes in prokaryotic or
eukaryotic cells or their viruses. The expression vector also
contains a ribosome binding site for translation initiation and a
transcription terminator. The vector may also include appropriate
sequences for amplifying expression.
[0057] In addition, the expression vectors preferably contain one
or more selectable marker genes to provide a phenotypic trait for
selection of transformed host cells such as dihydrofolate reductase
or neomycin resistance for eukaryotic cell culture, or such as
tetracycline or ampicillin resistance in E. coli.
[0058] In accordance with the present invention, optimal expression
of a FimH-C complex has been achieved using a newly constructed
single vector containing the FimH and FimC genes but having the
advantage that each gene is under its own separate lac promoter.
Thus, one lac promoter is 5' with respect to FimC while the second
lac promoter is 5' to the FimH gene. This plasmid was successfully
constructed using the common plasmid pUC19 as a background vector
[Yannish-Perron, C., Vierira, J. and Messing, J., Gene, 33:103-119
(1985)]. This new plasmid, when used to transform the host E. coli
strain BL21 [as described in Phillips, T. A., Van Bogelen, R. A.,
and Neidhart, F. C., J. Bacteriol. 159:283-287 (1984)] and then
induced using IPTG at the mid-logarithmic stage of growth, gives
maximal expression of the FimCH complex in the bacterial
periplasmic space. This material is then extracted and purified by
methods well known in the art, including those described
herein.
[0059] The vector containing the appropriate DNA sequence as
hereinabove described, as well as an appropriate promoter or
control sequence, may be employed to transform an appropriate host
to permit the host to express the proteins.
[0060] As representative examples of appropriate hosts, there may
be mentioned: bacterial cells, such as E. coli, Streptomyces,
Salmonella typhimurium; fungal cells, such as yeast; insect cells
such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO,
COS or Bowes melanoma; adenoviruses; plant cells, etc. The
selection of an appropriate host is deemed to be within the scope
of those skilled in the art from the teachings herein.
[0061] More particularly, the present invention also includes
recombinant constructs comprising one or more of the sequences as
broadly described above. The constructs comprise a vector, such as
a plasmid or viral vector, into which a sequence of the invention
has been inserted, in a forward or reverse orientation. In a
preferred aspect of this embodiment, the construct further
comprises regulatory sequences, including, for example, a promoter,
operably linked to the sequence. Large numbers of suitable vectors
and promoters are known to those of skill in the art, and are
commercially available. The following vectors are provided by way
of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen, Inc.), pbs,
pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,
pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540,
pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG
(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any
other plasmid or vector may be used as long as they are replicable
and viable in the host.
[0062] Promoter regions can be selected from any desired gene using
CAT (chloramphenicol transferase) vectors or other vectors with
selectable markers. Two appropriate vectors are pKK232-8 and pCM7.
Particular named bacterial promoters include lac, lacZ, T3, T7,
gpt, lambda P.sub.R, P.sub.L and TRP. Eukaryotic promoters include
CMV immediate early, HSV thymidine kinase, early and late SV40,
LTRs from retrovirus, and mouse metallothionein-I. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art.
[0063] In a further embodiment, the present invention relates to
host cells containing the above-described constructs. The host cell
can be a higher eukaryotic cell, such as a mammalian cell, or a
lower eukaryotic cell, such as a yeast cell, or the host cell can
be a prokaryotic cell, such as a bacterial cell. Introduction of
the construct into the host cell can be effected by calcium
phosphate transfection, DEAE-Dextran mediated transfection, or
electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods
in Molecular Biology, (1986)).
[0064] The constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. Alternatively, the polypeptides of the invention can be
synthetically produced by conventional peptide synthesizers.
[0065] Mature proteins can be expressed in mammalian cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the present invention. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are described by
Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which
is hereby incorporated by reference.
[0066] Transcription of the DNA encoding the polypeptides of the
present invention by higher eukaryotes is increased by inserting an
enhancer sequence into the vector. Enhancers are cis-acting
elements of DNA, usually about from 10 to 300 bp that act on a
promoter to increase its transcription. Examples including the SV40
enhancer on the late side of the replication origin bp 100 to 270,
a cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus
enhancers.
[0067] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived
from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), .alpha.-factor, acid phosphatase, or heat shock
proteins, among others. The heterologous structural sequence is
assembled in appropriate phase with translation initiation and
termination sequences. Optionally, the heterologous sequence can
encode a fusion protein including an N-terminal identification
peptide imparting desired characteristics, e.g., stabilization or
simplified purification of expressed recombinant product.
[0068] Useful expression vectors for bacterial use are constructed
by inserting a structural DNA sequence encoding a desired protein
together with suitable translation initiation and termination
signals in operable reading phase with a functional promoter. The
vector will comprise one or more phenotypic selectable markers and
an origin of replication to ensure maintenance of the vector and
to, if desirable, provide amplification within the host. Suitable
prokaryotic hosts for transformation include E. coli, Bacillus
subtilis, Salmonella typhimurium and various species within the
genera Pseudomonas, Streptomyces, and Staphylococcus, although
others may also be employed as a matter of choice.
[0069] As a representative but nonlimiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA).
These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed.
[0070] Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, the
selected promoter is induced by appropriate means (e.g.,
temperature shift or chemical induction) and cells are cultured for
an additional period.
[0071] Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract
retained for further purification.
[0072] Microbial cells employed in expression of proteins can be
disrupted by any convenient method, including freeze-thaw cycling,
sonication, a french press, mechanical disruption, or use of cell
lysing agents, such methods are well know to those skilled in the
art.
[0073] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts,
described by Gluzman, Cell, 23:175 (1981), and other cell lines
capable of expressing a compatible vector, for example, the C127,
3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors
will comprise an origin of replication, a suitable promoter and
enhancer, and also any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences. DNA sequences derived from the SV40
splice, and polyadenylation sites may be used to provide the
required nontranscribed genetic elements.
[0074] The polypeptides can be recovered and/or purified from
recombinant cell cultures by well-known protein recovery and
purification methods. Such methodology may include ammonium sulfate
or ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Protein
refolding steps can be used, as necessary, in completing
configuration of the mature protein. In this respect, chaperones
may be used in such a refolding procedure. Finally, high
performance liquid chromatography (HPLC) can be employed for final
purification steps.
[0075] The polypeptides that are useful as immunogens in the
present invention may be a naturally purified product, or a product
of chemical synthetic procedures, or produced by recombinant
techniques from a prokaryotic or eukaryotic host (for example, by
bacterial, yeast, higher plant, insect and mammalian cells in
culture). Depending upon the host employed in a recombinant
production procedure, the polypeptides of the present invention may
be glycosylated or may be non-glycosylated. Particularly preferred
immunogens are FimH adhesin protein or mannose-binding fragments
thereof since FimH is highly conserved among many bacterial
species. Therefore, antibodies against FimH (or its mannose-binding
fragments) should bind to FimH of other bacterial species (in
addition to E. coli) and vaccines against E. coli FimH (or FimH
mannose-binding fragments) should give protection against other
bacterial infections in addition to E. coli infections (for
example, against other enterobacteriacea infections).
[0076] Procedures for the isolation of a periplasmic chaperone
protein complexed with an adhesin protein are known in the art, as
an example see Jones et al., Proc. Natl. Acad. Sci. (USA)
90:8397-8401 (1993). Further, the individually expressed adhesin
proteins may be isolated by recombinant expression/isolation
methods that are well-known in the art. Typical examples for such
isolation may utilize an antibody to the protein or to a His tag or
cleavable leader or tail that is expressing as part of the protein
structure.
[0077] The polypeptides, their fragments or other derivatives, or
analogs thereof, or cells expressing them can be used as an
immunogen to produce antibodies thereto. These antibodies can be,
for example, polyclonal or monoclonal antibodies. The present
invention also includes chimeric, single chain, and humanized
antibodies, as well as Fab fragments, or the product of an Fab
expression library. Various procedures known in the art may be used
for the production of such antibodies and fragments.
[0078] Antibodies generated against the polypeptides corresponding
to a sequence of the present invention can be obtained by direct
injection of the polypeptides into an animal or by administering
the polypeptides to an animal, preferably a nonhuman. The antibody
so obtained will then bind the polypeptides itself. In this manner,
even a sequence encoding only a fragment of the polypeptides can be
used to generate antibodies binding the whole native
polypeptides.
[0079] For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line cultures
can be used. Examples include the hybridoma technique (Kohler and
Milstein, 1975, Nature, 256:495-497), the trioma technique, the
human B-cell hybridoma technique (Kozbor et al., 1983, Immunology
Today 4:72), and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
[0080] Techniques described for the production of single chain
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce
single chain antibodies to immunogenic polypeptide products of this
invention. Also, transgenic mice may be used to express humanized
antibodies to immunogenic polypeptide products of this
invention.
[0081] In order to facilitate understanding of the above
description and the examples which follow below certain frequently
occurring methods and/or terms will be described.
[0082] "Plasmids" are designated by a lower case p preceded and/or
followed by capital letters and/or numbers. The starting plasmids
herein are either commercially available, publicly available on an
unrestricted basis, or can be constructed from available plasmids
in accord with published procedures. In addition, equivalent
plasmids to those described are known in the art and will be
apparent to the ordinarily skilled artisan.
[0083] "Digestion" of DNA refers to catalytic cleavage of the DNA
with a restriction enzyme that acts only at certain sequences in
the DNA. The various restriction enzymes used herein are
commercially available and their reaction conditions, cofactors and
other requirements were used as would be known to the ordinarily
skilled artisan. For analytical purposes, typically 1 .mu.g of
plasmid or DNA fragment is used with about 2 units of enzyme in
about 20 .mu.l of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 .mu.g of DNA
are digested with 20 to 250 units of enzyme in a larger volume.
Appropriate buffers and substrate amounts for particular
restriction enzymes are specified by the manufacturer. Incubation
times of about 1 hour at 37.degree. C. are ordinarily used, but may
vary in accordance with the supplier's instructions. After
digestion the reaction is electrophoresed directly on a
polyacrylamide gel to isolate the desired fragment.
[0084] Size separation of the cleaved fragments is performed using
8 percent polyacrylamide gel described by Goeddel, D. et al.,
Nucleic Acids Res., 8:4057 (1980).
[0085] "Oligonucleotides" refers to either a single stranded
polydeoxynucleotide or two complementary polydeoxynucleotide
strands which may be chemically synthesized. Such synthetic
oligonucleotides have no 5' phosphate and thus will not ligate to
another oligonucleotide without adding a phosphate with an ATP in
the presence of a kinase. A synthetic oligonucleotide will ligate
to a fragment that has not been dephosphorylated.
[0086] "Ligation" refers to the process of forming phosphodiester
bonds between two double stranded nucleic acid fragments (Maniatis,
T., et al., Id., p. 146). Unless otherwise provided, ligation may
be accomplished using known buffers and conditions with 10 units to
T4 DNA ligase ("ligase") per 0.5 .mu.g of approximately equimolar
amounts of the DNA fragments to be ligated.
[0087] "FimHt" in the mouse in vivo experiments below refers to a
naturally occurring FimH adhesin protein truncate corresponding to
the NH.sub.2-terminal two-thirds of the FimH protein which was
purified away from complexes of FimC and FimH (FimC-H)). Such FimHt
has mannose-binding capability. However, in other locations in the
specification and Figures, the terms "FimHt" or "FimH truncate" may
refer to either a naturally occurring protein truncate having
mannose-binding capability or to a labelled (such as His-tag) or
unlabelled recombinant fragment of FimH that has mannose-binding
capability.
[0088] Unless otherwise stated, transformation was performed as
described in the method of Graham, F. and Van der Eb, A., Virology,
52:456-457 (1973).
[0089] The present invention will be further described with
reference to the following examples; however, it is to be
understood that the present invention is not limited to such
examples. All parts or amounts, unless otherwise specified, are by
weight.
EXAMPLE 1
[0090] Immunogenicity of Adhesin, Adhesin/Chaperone and Whole
Pili
[0091] The immunogenicity of purified adhesin, adhesin-chaperone
complex or whole type 1 pili proteins were assessed by measuring
immunoglobulin G (IgG) titer to FimHt adhesin (a naturally
occurring FimH truncate corresponding to the NH.sub.2-terminal
two-thirds of the FimH protein which was purified away from
complexes of FimC and FimH (FimC-H)) and whole type 1 pili,
respectively, up to 78 weeks post immunization.
[0092] C3H/HeJ mice, five mice per group were immunized on day 0
(primary immunization) [in Freund's adjuvant (CFA)] and booster
immunization (week 4) [in incomplete Freund's adjuvant (IFA)] with
one of the three antigens: purified adhesin (FimHt),
adhesin-chaperone complex (FimC-H) or whole type 1 pili. Samples
from individual mice treated identically were pooled for
serological analysis and diluted 1:100 before serial dilution.
Antibody responses were assessed by an ELISA with purified FimHt or
whole pili as the capture antigens. Titers reflect the highest
dilution of serum reacting twice as strongly as a comparable
dilution of preimmune sera obtained from the same mice. The purity
of the protein preparations of the capture antigens was 95% pure
for whole type 1 pili and FimHt to 98 to 99% purity for FimC-H. In
all cases the protein preparations were free of any
lipopolysaccharide contaminants. Data for immune responses of such
mice to FimHt adhesin (FIG. 1A) and whole type 1 pili (FIG. 1B) of
such mice are reported in FIGS. 1A and 1B as FimHt (squares),
FimC-H (circles) or whole type 1 pili (triangles).
[0093] As shown in FIGS. 1A and 1B, both FimHt and FimC-H induced
strong, long-lasting immune responses to isolated FimHt and to FimH
associated with whole type 1-pilus organelles. The responses
persisted more than 30 weeks, and booster immunizations with FimHt
or FimC-H increased responsiveness. In contrast, type 1 pili
elicited poor anti-FimH responses even though mice developed strong
responses to whole pilus rods. Immunization studies in rabbits
demonstrated similar immunogenicity profiles to those seen in mice.
Antisera to FimHt and to FimC-H bound to recombinant type 1+/FimH+
E. coli strains (ORN103/pSH2) but not to the type 1+/FimH-
isogeneic mutant (ORN103/pUT2002) as determined by indirect
immunofluorescence and flow cytometric analysis. Antibody to the
whole pilus bound both ORN103/pSH2 and ORN103/pUT2002, as
expected.
[0094] Comparable immune responses (unreported) to the three
antigens FimHt, FimC-H and whole type 1 pili were seen in BALB/C
and C57/BL6 strains of mice.
EXAMPLE 2
Role of FimH in E. coli Binding Human Bladder Cells
[0095] The ability type 1-piliated E. coli to bind to human and
mouse epithelial tissues was investigated in the absence and
presence of FimH. The presence of a receptor for binding of type 1
pili to human bladder mucosa that is recognized by FimH was
verified by determination of superior binding results when the FimH
protein is present. FimH is the adhesin that confers
mannose-specific binding activity to type 1 pili since adding
mannose results in the inhibition of such binding.
[0096] In situ binding of E. coli to human and mouse tissues
elucidates the binding role of FimH in the adhesin of type 1 pili
to such tissues. A NU14 E. coli (serotype) cystitis isolate from
UTI patents bound to the luminal surface of both mouse and human
bladder epithelium. However, the presence of mannose completely
blocked this binding.
[0097] Tests were then performed to determine that FimH is
responsible for such binding. A mutant form of E. coli was provided
that did not produce FimH protein. A chloramphenicol cassette
recombined with the fimH gene in the chromosome of strain NU14
creates a fimH- mutant NU14-1 (i.e., bacteria that does not produce
FimH). Binding assays confirm that such mutant fails to bind the
human and mouse bladder tissues while the wild type bacteria
maintains the ability to bind such tissues. Further the mutant can
then be modified further genetically to provide regulated
restoration of such gene and its expression restored the ability of
the bacterial pili to bind to the human and mouse bladder
walls.
[0098] Thus, FimH is responsible for the adhesin binding of type 1
pili to bladder epithelial cells. According, antibodies to such
protein should block the binding of type 1 pili to such cells and
result in prevention or treatment of UTIs that are caused by type
1-piliated E. coli.
EXAMPLE 3
Standard Ability of E. coli to Bind Human Bladder Cells
[0099] The ability type 1-piliated E. coli to bind to human bladder
cells epithelial cells was investigated in vitro by a flow
cytometric method modified from a procedure originally developed to
evaluate Rickettsia-cell attachment. In vitro binding of type
1-pilated E. coli to human bladder (J-82 (ATCC HTB1) in the absence
of anti-binding antibodies was assayed to obtain standard binding
rates for particular bacteria.
[0100] The ability of Type 1-piliated E. coli of HB101/pSH2 (and
strain ORN103/pSH2) and E. coli NU14 to bind to bladder cells from
the human bladder epithelial cell line J82 was measured as
follows.
[0101] Type 1-piliated, NU14 and nonpiliated [HB 101
(cross-hatched)] bacteria were directly labeled with FITC and
tested for their ability to bind to bladder cells. The labelled
bacteria were first assayed for the expression of type 1 pili. Such
expression of type 1 pili was confirmed by hemagglutination of a 3%
solution of guinea pig erythrocytes and inhabitation of
hemagglutination by a 10 mM solution of
.alpha.-methylmannoside.
[0102] Next the labeled bacteria were incubated with
2.times.10.sup.6 J82 (ATCC HTB1) bladder cells at the
bacteria:bladder cell concentration ratios of 1000:1 to 62.5:1.
Samples were assayed by flow cytometry in a FACStar PLUS (Becton
Dickinson). Mean channel fluorescence was used as an indicator of
FITC-labeled bacteria bound for J82 cells. The threshold for
positivity was set for each experiment by flow cytometric analysis
of J82 cells that were incubated with PBS only. For evaluation of
FITC labeling of bacteria, gates were set with non-FITC-labeled
bacteria. Lysis II software (Becton Dickinson immunocytometry
Systems) was used for analysis of data.
[0103] FIG. 2A shows the bladder cell binding results for such type
1-piliated [HB101/pSH2 (black bars) and NU14 (striped bars)] and
nonpiliated [HB 101 (cross-hatched)] bacteria.
EXAMPLE 4
Ability of Antibodies to Inhibit Binding of E. coli Species to
Human Bladder Cells
[0104] The ability of anti-FimHt antibodies to inhibit the binding
of type 1-piliated E. coli to human bladder cells epithelial cells
was investigated in vitro by the flow cytometric method described
in Example 2. In vitro binding of type 1-pilated E. coli to human
bladder (J-82 (ATCC HTB1) in the presence of anti-FimHt antibodies
was assayed for as set forth below.
[0105] High titer anti-FimHt or anti-FimC-H antibodies were
obtained as antisera from bleeds of mice at 6 to 9 weeks post
initial immunization as set forth in Example 1. About
2.5.times.10.sup.8 HB101/pSH2 or NU 14 bacteria incubated for 30
min at 37.degree. C. with such high-titer anti-FimHt or anti-FimC-H
(from bleeds at 6 to 9 weeks) at 1:50, 1:100, or 1:200 dilutions in
PBS or with PBS alone. After incubation with the antisera,
2.times.10.sup.8 J82 cells were added and allowed to mix with the
bacteria for 30 min. at 37.degree. C. The remainder of the
adherence assay and evaluation by flow cytometry were carried out
as described in Example 3. J82 bladder cells that were sorted from
the flow cytometric adherence assay were also analyzed by
fluorescent microscopy. The number of fluorescent bacteria attached
to 40 bladder cells was visually quantitated. Adherence values
(mean number of bacteria.+-.SD per cell) for two representative
cystitis isolates, NU14 and EC72, were 35.2.+-.4.9 bacteria/cell,
respectively, in the absence of FimH-specific antibody; incubation
with a 1:50 dilution of anti-FimH-specific antibody; incubation
with a 1:50 dilution of anti-FimH completely blocked attachment.
Furthermore, the same examination of samples taken from bacteria
incubated with different dilutions of anti-FimHt confirmed the
dose-dependency of the phenomenon.
[0106] The abilities of the anti-FimHt and anti-FimC-H antibodies
(antibodies to the truncated adhesin protein and to the adhesin
protein FimH complexed with its chaperone FimC, respectively) to
prevent bacterial binding to such bladder cells are set forth in
FIGS. 2B and 2C, respectively.
[0107] At the antibody dilutions 1:50, 1:100 and 1:200 in PBS each
of the antibody (antisera) dilutions strongly inhibited the ability
of type 1 piliated bacteria to bind to the human bladder cells as
verified by the PBS control solution which had no antibodies
present.
EXAMPLE 5
Effects of Antibody Titer on Antibody Inhibition of E. coli Species
Binding to Human Bladder Cells
[0108] The procedures for antibody inhibition of bacterial binding
as set forth in Example 4 were followed except that the antibodies
to FimHt and FimC-H were obtained at various time periods and at
various titer levels as set forth in Example 1.
[0109] The results observed (not shown) establish that there is a
direct relationship between the antibody titer to FimH and the
ability of the antisera to block microbial attachment (functional
inhibitory titer). Furthermore, as titer to FimH dropped or
levelled off in the FimHt-immunized and FimCH-immunized mice, the
concomitant functional inhibitory titers dropped as well. However,
as titers increased after a booster immunization at week 42, the
corresponding functional inhibitory titers were elevated.
[0110] Utilizing such results (not shown), the functional
inhibitory titers of anti-FimHt was determined for a panel of
primary clinical isolates from women and children with active
urinary tract infections. Antisera raised to FimHt and FimC-H
blocked attachment of 49 out of 52 (roughly 94%) primary clinical
E. coli UTI isolates induced to express type 1 pili (results not
shown). A subset of strains induced to express P pili, S pili, or
both in addition to type 1 pili were also inhibited from attachment
to the bladder epithelial cells by anti-FimHt antibodies.
EXAMPLE 6
Comparative Example
Effects of Antibodies to Whole Type 1 Pili Inhibition of E. coli
Species Binding to Human Bladder Cells
[0111] The antibody inhibition of bacteria binding procedures of
Example 5 were followed with antibodies to whole type 1 pili
(obtained as set forth in Example 1), in purified form ORN103/pSH2,
were poor inhibitors of bacterial binding to bladder epithelial
cells; antisera to whole type 1 pili blocked <50% of the
clinical isolates even at a 1:50 dilution of antiserum. Such is
presumably due to the variation in antigenicity of FimA among
clinical isolates and the inability of whole type 1 pili to elicit
significant anti-FimH responses. Preimmune sera and antisera from
mice given adjuvant alone or antisera from mice immunized with a
control FimC chaperone protein did not inhibit binding.
EXAMPLE 7
Immunization Effects of FimH and FimC-H to Inhibit E. coli Species
Binding in Mouse Bladder
[0112] A C3H/HaJ murine cystitis model was utilized to demonstrate
the effectiveness of immunization with a FimHt or FimC-H vaccine
(see generally the immunization procedures in Example 1,
above).
[0113] As a control to assess UTI levels in the bladders of
unprotected mice, ten to 15 week old C3H/HeJ mice were anesthetized
with methoxyflurane and challenged with various doses of
streptomycin-resistant, type 1-piliated E. coli NU14 (an isolate
from a clinical human UTI). The values that were measured represent
the final challenge doses the mice received of the NU14. The
colony-forming units per bladder that are measured represent the
mean.+-.SD for each group (N=10, group of ten mice) 2 days after
the challenge with NU14.
[0114] Intraurethral inoculation of C3H mice with 5.times.10.sup.7
type 1-piliated E. coli (strain NU14) resulted in a highly
reproducible colonization of the mouse bladder in unvaccinated mice
(See FIG. 3A). Piliated bacteria persisted in the bladder for at
least 7 days [10.sup.4 colony-forming units (CFU)/bladder] and
produced ascending infection into the kidney.
[0115] The role of mannose-binding type 1 pili in bladder
colonization was investigated by testing the effect of the fimH-
mutation (see Example 2, above) as compared to the fimH+ wild-type
in the murine model. Ten to 15-week old C3H/HeJ mice were
anesthetized and challenged with 10.sup.7 and 10.sup.5 CFU of
either E. coli NU14 (type 1+/FimH+) or NU14-1(fimH-). The data
measured represents the mean.+-.SD for each group (N=10, group of
ten mice) 2 days variance was used in the calculation as well as
the Dunnet's t test for multiple comparisons. Inoculation with the
fimH- mutant, NU14-1, resulted in little or no colonization of the
mouse bladder (FIG. 3B), supporting the indication in Example 2
(above) that FimH plays a critical role in colonization of the
bladder.
[0116] The protective effects against E. coli bladder infections in
immunized mice provided by in vivo anti-FimHt and anti-FimC-H (mice
immunized with FimHt and FimC-H as set forth in Example 1) as
contrasted with in vivo anti-FimC (mice immunized with FimC alone
by following the procedures and protocol as set forth in Example 1)
were assayed for as follows.
[0117] Four groups of C3H/HeJ mice were immunized on day 0 and
boosted at week 4 with purified FimHt protein (doses ranging from
0.6 to 30 .mu.g in CFA for the initial immunization and IFA for the
booster), FimC or the adjuvant alone, as generally set forth in
Example 1. At week (9) nine after immunization (high titer, see
Example 1 data reported in FIG. 1A) the urine of the immunized mice
was assayed for the presence or absence of the antibody
corresponding to the immunization. Mice that received the FimHt
vaccine had significant amounts of IgG to FimHt in their urine,
whereas unimmunized mice, or mice vaccinated with FimC alone, did
not have any measurable amounts of anti-FimHt in urinary
secretions.
[0118] The protective effects of such in vivo antibodies resulting
from such vaccination were assessed concurrently with the
measurement of antibodies in the urinary secretions as set forth
below. Such assessment is helpful in determining the effectiveness
of such vaccine for the prevention of UTIs.
[0119] The average number of colony-forming units per bladder for
each group of 10 mice was evaluated in the same manner as indicated
for the control group of unprotected mice above. FimC-immunized
mice were also included as a negative control along with naive
mice. [P-0.0001 comparing the 30-, 15- and 3-.mu.g doses of FimHt
with the nave data; P=0.205 for the FimC negative control at the
highest (30 .mu.g) dose].
[0120] The C3H mice that were immunized with the various vaccines
as set forth above were challenged with 5.times.10.sup.7 CFU of
type 1-piliated E. coli (NU14 isolate, see above) at week 9 after
immunization (high titer, see Example 1 data reported in FIG. 1A).
Animals vaccinated with FimHt exhibited from a 100- to 1000-fold
reduction in the number of organisms recovered from the bladders as
compared with the control mice that were immunized with only
adjuvant or with only FimC. The CFUs for such mice are set forth in
FIG. 3C. Protection with the FimHt vaccine was seen as late as 29
weeks after immunization, the latest time point tested.
[0121] Such mice were provided with additional vaccination at 51
weeks and rechallenged at 54 weeks. Protection was also seen at
such 54 week period.
[0122] The above procedures were repeated with FimC-H as the
vaccine instead of FimHt. Similar protective results were seen with
the FimC-H vaccine (not reported) as were observed for FimHt at 29
weeks. Such mice were provided with additional vaccination at 51
weeks and rechallenged at 54 weeks. Protection was also seen at
such 54 week period.
[0123] To access the in vivo protective effects against ascending
UTI infections in the mouse model, the above immunization
procedures were repeated with two groups of C3H/HeJ mice. Such mice
were inoculated on day 0 with 15 .mu.g CFA and boosted at week 4
with purified FimHt protein (15 .mu.g IFA) or with CFA/IFA adjuvant
alone. At week 9 post immunization the presence of antibodies was
assessed by the above described urine test. At the time of such
urine test the mice were challenged with 5.times.10.sup.7 CFU of
type 1-piliated E. coli strain.
[0124] The average number of CFUs were assessed as set forth above,
except that the CFUs were assessed per kidney (instead of per
bladder) for each group of 10 challenged mice. The average number
of CFUs was evaluated as indicated above 7 days after the
intraurethral challenger (P=0.295) (see FIG. 3D, where solid
histogram is mice administered anti-FimHt and cross-hatched
histogram is naive mice). Such data indicates that such vaccination
blocked an ascending infection into the kidney over a 7-day
period.
EXAMPLE 8
Protective Effects of Antibody Sera in Non-Immunized Mice for
Inhibiting E. coli Binding in Mouse Bladder
[0125] To confirm the protective effects that were observed in
Example 7 were mediated by anti-FimHt, naive C3H mice were
challenged with 5.times.10.sup.7 type 1-piliated E. coli strain
NU14 after passive, intraperitoneal administration of hyperimmune
mouse FimHt antisera (or FimC-H antisera) raised to either the
FimHt protein or the FimC-H complex. In each cases the sera
containing anti-FimHt or anti FimC-H resulted in a 100- to 150-fold
reduction in the number of organisms recovered from the bladder 2
days after challenge (See FIG. 3E). Sera from mice that received
only adjuvant did not have any protective effects at all against
such bladder infections or ascending UTIs.
EXAMPLE 9
Protective Effects in FimH and FimC-H Immunized Neutropenic Mice
Against E. coli UTIs in Mouse Model
[0126] Non-immunized and FimH-immunized mice were rendered
neutropenic before intraurethral challenge with type 1-piliated E.
coli to determine whether protection was neutrophil dependent. The
assays for the number of organisms in the mice bladders were
determined as set forth above in Example 8.
[0127] As control groups, mice that were nonimmunized and
neutropenic showed a 100-fold increase in the number of organisms
in the bladder relative to immunocompetent, nonimmunized mice
(10.sup.6 CFU/bladder compared with 10.sup.4 CFU/bladder).
[0128] Immunization with FimH vaccine reduced colonization in both
neutropenic and non-neutropenic mice to equivalent levels of
10.sup.2 CFU/bladder. Thus, the absence of neutrophils did not
impede the antibacterial activities of the FimH vaccine in
vivo.
EXAMPLE 10
Recombinant Expression and Purification of FimCH by Cation and
Hydrophobic Interaction Chromatography
[0129] Polynucleotides encoding FimC (in a plasmid vector with an
arabinose promoter and spectinomycin resistance; obtained by
inserting an FimC gene into a plasmid vector such as are
commercially available) and FimH (pHJ20-FimH-pMMB66; IPTG promoter
and ampicillin resistance; FimH gene inserted into a commercially
available plasmid vector by standard techniques, equivalents are
readily available), respectively, were inserted into an E. coli
strain (C600/pHJ9205/pHJ20) under standard calcium chloride
conditions. A stock culture of the E. coli strain with its inserts
was then obtained for use as a fermentation inoculate.
[0130] The E. coli host cells of the stock culture were grown under
conditions such that the type 1 usher protein was not expressed. In
particular, 16 liters of media (11 liters LB Millers and 5 liters
BHI; commercially available) was autoclaved and placed in a 20
liter fermentor (New Brunswick Bioflow IV). Culturing conditions of
the fermentor are a constant temperature of 37.degree. C., pH 7.4,
agitation 500 rpm and DO.sub.2 90%-25%. The media was inoculated
with 500 ml of stock culture to provide a starting optical density
(O.D.) of 0.1. At about 2-3 hours and an O.D. of 0.6, 0.2%
arabinose was added. Then at an O.D. of 1.2, 0.25 mM IPTG was
added.
[0131] After the IPTG was added, the culture was maintained for
about 1 hour and the E. coli host cells were harvested in CEPA LE
continuous flow centrifuge. The dry weight of the pellet was a
yield of about 100-150 grams of cells.
[0132] The pellet was placed in a container on ice and to prepare
the periplasm there was added per gram of cells: 4 ml 20%
sucrose/20 mM Tris 8.0; 200 .mu.l EDTA 8.0; and 40 .mu.l lysozyme
(10 mg/ml). After such addition the mixture was maintained on ice
for about 20 minutes. Then 160 .mu.l of 0.5M MgCl.sub.2 was added
to the mixture and the mixture was centrifuged at 12,000 g. The
supernatant was brought to 75% NH.sub.4SO.sub.4 and then
centrifuged for at least 2 hours (preferably overnight). The
NH.sub.4SO.sub.4 pellet was then centrifuged at 12,000 g for 30
minutes and dialyzed against 20 mM KMES pH 6.8.
[0133] FimCH was purified by chromatography, cation exchange
chromatography followed by hydrophobic interaction chromatography.
A sample obtained by the above procedures was injected on Pharmacia
XK16/10 15S Source Sepharose Column at a rate of 2 ml/min, A: 20 mM
KMES pH 6.8 B: 20 mM KMES 6.8/0.6M NaCl. The run gradient was O-30%
over 100 ml, 30-100% over 20 ml. The FimCH was eluted at around 60
mM NaCl, and FimC was eluted at around 75 mM NaCl. The respective
fractions were pooled from individual runs and then further
purified by dialysis into 50 mM NaPhosphate pH 7.0/0.6M
NH.sub.4SO.sub.4. The FimCH thus obtained was then further purified
by hydrophobic interaction chromatography by injecting samples on
to Pharmacia HR10/10 Butyl Sepharose 4FF Column at a rate of 2
milliliters per minute: A:50 mM NaPhosphate pH 7.0/0.6M
NH.sub.4SO.sub.4' and B:50 mM Naphosphate pH 7.0. FimCH was eluted
from 180-300 mM NH.sub.4SO.sub.4 and dialyzed back to 20 mM KMES pH
6.8. The yields from such procedure were an average of 26 mg FimCH
per 50 liters.
EXAMPLE 11
Recombinant Expression and Purification of FimCH and FimHt by
Affinity and Cation Exchange Chromatography
[0134] The fermentation and periplasm preparation with the E. coli
host cells of Example 10 were conducted essentially as set forth in
Example 10 except that the NH.sub.4SO.sub.4 pellet was centrifuged
at 12,000 g for 30 minutes and dialyzed against 1.times.PBS
fully.
[0135] Affinity chromatography was utilized to separate a mixture
of FimCH and FimHt from the filtered dialysate. In particular,
mannose-sepharose beads were added to the filtered dialysate at
1:25 by volume followed by rocking for at least two hours
(preferably overnight) in a 50 ml conical tube. The mixture was
then centrifuged at 2000 g with no brake and the supernatant was
pulled off. This was repeated with 5.times.washing with
1.times.PBS. Then one volume of 15%
methyl-.alpha.-D-mannopyranoside was added and rocked at 4.degree.
C. for at least two hours (preferably overnight). The mixture was
gently spun by centrifugation and the supernatant containing both
the FimCH and FimHt as a mixture was pulled off and dialyzed into
20 mM KMES pH 6.5. The FimCH and FimHt were then separated from one
another by cation exchange chromatography of the dialyzate. Samples
of the dialyzate were injected onto a Pharmacia XK16/10 15S Source
Sepharose Column at a rate of 2 milliliters per minute; A: 20 mM
KMES pH 6.8 B: 20 mM KMES 6.8/1M NaCl. The FimHt was collected in
the flow through at a run gradient of O-30% over 100 ml, 30-100%
over 20 ml. The FimCH was eluted around 100 mM NaCl. Further
processing of the respective FimHt and FimCH was done by a final
dialysis into 20 mM HEPES 7.0 for crystallography and assays. The
yield was an average of 5 mg/50 liters.
EXAMPLE 12
Recombinant Expression and Purification of His-tag FimH Truncates
by Nickel and Anion/Cation Chromatography
[0136] A polynucleotides encoding either FimH or a fragment of FimH
(cloned into pMMB91 plasmid as EcoRI/BamHI with IPTG promoter,
His-tag and Kanamycin resistance; commercially available plasmid
vector, other readily obtainable functionally equivalent vectors
may also be used) was inserted into an E. coli strain HB101
(commercially available) under standard calcium chloride
conditions. Such host cells were designated as HB101/T1-T7
(depending upon the polynucleotide inserted into the E. coli
strain) and lack an usher protein, thus not needing a chaperone
protein except in the case of T7 which is the full-length FimH and
may need to have FimC also inserted for proper folding. A stock
culture of each E. coli strain with its insert was then obtained
for use as a fermentation inoculate.
[0137] The His-tag FimH truncates and His-tag full-length FimH are
as follows: FimHt1 (T1=SEQ ID NO:2, amino acids 1 to 161), FimHt2
(T2=SEQ ID NO:2, amino acids 1 to 181), FimHt3 (T3=SEQ ID NO:2,
amino acids 1 to 186), FimHt4 (T4=SEQ ID NO:2, amino acids 1 to
196), FimHt5 (T5=SEQ ID NO:2, amino acids 1 to 207) and FimHt6
(T6=SEQ ID NO:2, amino acids 1 to 223), and FimHt7 (T7=SEQ ID NO:2,
amino acids 1 to 300, also referred to as full-length FimH, and
encoded by residues 1-900 of SEQ ID NO.: 1) are all produced by the
procedures set forth below. For illustrative purposes T2, T3, T4
and T5 were produced as follows.
[0138] The E. coli host cells (containing an individual truncate)
of the stock culture were grown under the following conditions.
Sixteen liters of media (11 liters LB Millers and 5 liters BHI;
commercially available) were autoclaved and placed in a 20 liter
fermentor (New Brunswick Bioflow IV). Culturing conditions of the
fermentor are a constant temperature of 37.degree. C., pH 7.4,
agitation 500 rpm and DO.sub.2 100%-25%. The media was inoculated
with 500 ml of stock culture and Kanamycin. At an O.D. of 1.0-1.5,
0.25 mM IPTG was added.
[0139] After the IPTG was added, the culture was maintained for
about 1 hour and the E. coli host cells were harvested in CEPA LE
continuous flow centrifuge. The dry weight of the pellet was a
yield of about 50-75 grams of cells.
[0140] The pellet was placed in a container on ice and to prepare
the periplasm there was added per gram of cells: 4 ml 20%
sucrose/20 mM Tris 8.0; 200 .mu.l EDTA 8.0; and 40 .mu.l lysozyme
(10 mg/ml). After such addition the mixture was maintained on ice
for about 20 minutes. Then 160 ul of 0.5M MgCl.sub.2 was added to
the mixture and the mixture was centrifuged at 12,000 g. The
supernatant was brought to 75% NH.sub.4SO.sub.4 and then
centrifuged for at least 2 hours (preferably overnight). The
NH.sub.4SO.sub.4 pellet was then centrifuged at 12,000 g for 30
minutes and dialyzed against 20 mM KMES pH 6.1 for T2 and 20 mM
Tris pH 8.4 for each of T3-T5.
[0141] Each of the T2 and T3-T5 samples was purified by nickel
chromatography, followed by anion (T2) and Cation (T3-T5) Exchange
chromatography, as required. Ni-NTA Agarose Beads (Qiagen) were
added at 1:25 by volume to the dialysate followed by rocking for at
least two hours (preferably overnight) in a conical tube. The
mixture was then centrifuged at 2000 g with no brake and the
supernatant was pulled off. This was repeated with 5.times.washing
with 20 mM Tris pH 8.0. Then one bead volume of 100 mM EDTA in 20
mM Tris pH 8.0 was added and rocked for at least two hours
(preferably overnight). The mixture was gently spun by
centrifugation and the supernatant containing the one of the FimH
truncate T2-T5 was pulled off. If the supernatant contained T2 it
was dialyzed into 20 mM KMES pH 6.1, but when the supernatant
contained one of the T3-T5 truncates it was dialyzed into 20 mM
Tris pH 8.4.
[0142] The T2 was purified by anion exchange chromatography of the
dialyzate. Samples of the dialyzate were injected onto a Pharmacia
Source 15Q column at a rate of 1.5 milliliters per minute; A:20 mM
Tris pH 8.4 B: 20 mM Tris pH 8.4/1M NaCl. The run gradient of 0-40%
over 80 ml, 40-100% over 20 ml was utilized. The truncate was
eluted at around 100-200 mM salt. Further purification was done by
a final dialysis into 20 mM Tris pH 8.0. The yield was an average
of 3-5 mg/50 liters.
[0143] Each of the T3-T5 truncates was purified by anion exchange
chromatography of the dialyzate. Samples of the dialyzate were
injected onto a Pharmacia Source S 10/10HR column at a rate of 1.5
milliliters per minute; A: 20 mM KMES pH6.1 B: 20 mM KMES 6.1/1M
KCL. The run gradients of 0-40% over 80 ml and 40-100% over 20 ml
were utilized. The individual truncate being processed was eluted
at around 100-200 mM salt. Further purification was done by a final
dialysis into 20 mM Tris pH 8.0. The yield was an average of 3-5
mg/50 liters.
[0144] The identity of the each of the protein fragments T2-T5 was
confirmed by Western Blot for the truncate fragment lengths (after
cleavage of the signal sequence) 160, 165, 175 and 186,
respectively (data not shown).
EXAMPLE 13
Recombinant Expression and Purification of Full-Length His-tag FimH
by Nickel and Anion/Cation Chromatography
[0145] A polynucleotide encoding FimH as in Example 12, above,
(cloned into pMMB91 plasmid as EcoRI/BamHI with IPTG promoter,
His-tag and Kanamycin resistance; commercially available plasmid
vector, other readily obtainable functionally equivalent vectors
may also be used) and a polynucleotide encoding FimC (as in Example
10, above) can be co-inserted into the same E. coli strain
described in Example 12, above, essentially as in Examples 10 and
12. The procedures set forth in Example 12 are available to isolate
the FimCH complex. FimH is then isolated from the FimCH
complex.
EXAMPLE 14
Recombinant Expression and Purification of PapD/PapG (PapDG) by
Gal.alpha.(1-4)Gal and Anion Exchange Chromatography
[0146] Polynucleotides encoding PapD/PapG (PapDG) in a plasmid
vector (commercially available pMMB91 with a tac promoter and
Kanamycin resistance having the PapD and PapG genes inserted, thus
by standard methods; other plasmid vectors are commercially
available), were inserted into an E. coli strain C600 under
standard calcium chloride conditions to produce strain C600/pJP1. A
stock culture of the E. coli strain with its inserts was then
obtained for use as a fermentation inoculate.
[0147] The E. coli host cells of the stock culture were cultured in
16 liters of media (11 liters LB Millers and 5 liters BHI;
commercially available) was previously autoclaved and placed in a
20 liter fermentor (New Brunswick Bioflow IV). Culturing conditions
of the fermentor are a constant temperature of 37.degree. C., pH
7.4, agitation 500 rpm and DO.sub.2 90%-25%. The media was
inoculated with 500 ml of stock culture to provide a starting
optical density (O.D.) of 0.1. At about 2-3 hours and an O.D. of
0.6 was observed. Then at an O.D. of 1.2, 0.25 mM IPTG was
added.
[0148] After the IPTG was added, the culture was maintained for
about 1 hour and the E. coli host cells were harvested in CEPA LE
continuous flow centrifuge. The dry weight of the pellet was a
yield of about 75-100 grams of cells.
[0149] The pellet was placed in a container on ice and to prepare
the periplasm there was added per gram of cells: 4 ml 20%
sucrose/20 mM Tris 8.0; 200 .mu.l EDTA 8.0; and 40 .mu.l lysozyme
(10 mg/ml). After such addition the mixture was maintained on ice
for about 20 minutes. Then 160 .mu.l of 0.5M MgCl.sub.2 was added
to the mixture and the mixture was centrifuged at 12,000 g. The
supernatant was brought to 75% NH.sub.4SO.sub.4 and then
centrifuged for at least 2 hours (preferably overnight). The
NH.sub.4SO.sub.4 pellet was then centrifuged at 12,000 g for 30
minutes and dialyzed against 1.times.PBS fully.
[0150] PapDG was purified by chromatography, affinity
chromatography followed by anion exchange chromatography. In
particular, Gal.alpha.(1-4)Gal-sepharose beads were added to the
filtered dialysate at 1:25 by volume followed by rocking for at
least two hours (preferably overnight) in a 50 ml conical tube. The
mixture was then centrifuged at 2000 g with no brake and the
supernatant was pulled off. This was repeated with 5.times.washing
with 1.times.PBS. Then one volume of 5 mg/ml Gal.alpha.(14)Gal was
added and rocked at 4.degree. C. for at least two hours (preferably
overnight). The mixture was gently spun by centrifugation and the
supernatant containing PapDG was pulled off and dialyzed into 20 mM
Tris pH 8.0.
[0151] Samples of the dialyzate obtained by the above procedures
were injected on Pharmacia XK16/10 15Q Source Column at a rate of 2
ml/min, A: 20 mM Tris pH 8.5 B: 20 mM Tris pH 8.6/0.6M NaCl. The
run gradient was O-30% over 100 ml, 30-100% over 10 ml. PapDG was
eluted at around 100-200 mM NaCl. Further processing of the eluted
PapDG was done by dialysis with 20 mM Tris pH 8.0. The yields from
such procedure were an average of 2 mg PapDG per 50 liters.
[0152] The data from the above examples indicate that the binding
of FimH to a receptor that is exposed on the luminal surface of
both the human and mouse bladder epithelium is critical for
uropathogenic E. coli strains to colonize the bladder and cause
cystitis. Further immunization of mice to produce IgG to FimH in a
mouse model of UTIs blocks the colonization of type 1 piliated
bacteria in vivo. Therefore, such immunization protects mice
against a mucosal infection of the bladder and subsequent ascending
urinary tract infections.
[0153] Prevention of other types of bacteria from binding to
mucosal surfaces may also be possible by utilizing the
above-described vaccines due to significant cross-reactivity of
various bacteria species with regard to adhesin proteins.
[0154] Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, within the scope of the appended claims, the invention
may be practiced otherwise than as particularly described.
Sequence CWU 1
1
2 1 957 DNA Escherichia coli 1 atgaaacgag ttattaccct gtttgctgta
ctgctgatgg gctggtgcgt aaatgcctgg 60 tcattcgcct gtaaaaccgc
caatggtacc gctatcccta ttggcggtgg cagcgccaat 120 gtttatgtaa
accttgcgcc cgtcgtgaat gtggggcaaa acctggtcgt ggatctttcg 180
acgcaaatct tttgccataa cgattatccg gaaaccatta cagactatgt cacactgcaa
240 cgaggctcgg cttatggcgg cgtgttatct aatttttccg ggaccgtaaa
atatagtggc 300 agtagctatc catttcctac caccagcgaa acgccgcgcg
ttgtttataa ttcgagaacg 360 gataagccgt ggccggtggc gctttatttg
acgcctgtga gcagtgcggg cggggtggcg 420 attaaagctg gctcattaat
tgccgtgctt aggttgcgac agaccaacaa ctataacagc 480 gatgatttcc
agtttgtgtg gaatatttac gccaataatg atgtggtggt gcctactggc 540
ggctgcgatg tttctgctcg tgatgtcacc gttactctgc cggactaccg tggttcagtg
600 ccaattcctc ttaccgttta tcgtgcgaaa agccaaaacc tggggtatta
cctctccggc 660 acacacgcag atgcgggcaa ctcgattttc accaataccg
cgtcgttttc acctgcacag 720 ggcgtcggcg tacagttgac gcgcaacggt
acgattattc cagcgaataa cacggtatcg 780 ttaggagcag tagggacttc
ggcggtgagt ctgggattaa cggcaaatta tgcacgtacc 840 ggagggcagg
tgactgcagg gaatgtgcaa tcgattattg gcgtgacttt tgtttatcaa 900
taaagaaatc acaggacatt gctaatgctg gtacgcaata ttacctgaag ctaaaaa 957
2 300 PRT Escherichia coli 2 Met Lys Arg Val Ile Thr Leu Phe Ala
Val Leu Leu Met Gly Trp Ser 1 5 10 15 Val Asn Ala Trp Ser Phe Ala
Cys Lys Thr Ala Asn Gly Thr Ala Ile 20 25 30 Pro Ile Gly Gly Gly
Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val 35 40 45 Val Asn Val
Gly Gln Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe 50 55 60 Cys
His Asn Asp Tyr Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln 65 70
75 80 Arg Gly Ser Ala Tyr Gly Gly Val Leu Ser Asn Phe Ser Gly Thr
Val 85 90 95 Lys Tyr Ser Gly Ser Ser Tyr Pro Phe Pro Thr Thr Ser
Glu Thr Pro 100 105 110 Arg Val Val Tyr Asn Ser Arg Thr Asp Lys Pro
Trp Pro Val Ala Leu 115 120 125 Tyr Leu Thr Pro Val Ser Ser Ala Gly
Gly Val Ala Ile Lys Ala Gly 130 135 140 Ser Leu Ile Ala Val Leu Ile
Leu Arg Gln Thr Asn Asn Tyr Asn Ser 145 150 155 160 Asp Asp Phe Gln
Phe Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val 165 170 175 Val Pro
Thr Gly Gly Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr 180 185 190
Leu Pro Asp Tyr Arg Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys 195
200 205 Ala Lys Ser Gln Asn Leu Gly Tyr Tyr Leu Ser Gly Thr His Ala
Asp 210 215 220 Ala Gly Asn Ser Ile Phe Thr Asn Thr Ala Ser Phe Ser
Pro Ala Gln 225 230 235 240 Gly Val Gly Val Gln Leu Thr Arg Asn Gly
Thr Ile Ile Pro Ala Asn 245 250 255 Asn Thr Val Ser Leu Gly Ala Val
Gly Thr Ser Ala Val Ser Leu Gly 260 265 270 Leu Thr Ala Asn Tyr Ala
Arg Thr Gly Gly Gln Val Thr Ala Gly Asn 275 280 285 Val Gln Ser Ile
Ile Gly Val Thr Phe Val Thr Gln 290 295 300
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