Note: Descriptions are shown in the official language in which they were submitted.
-1- 20~3371
_0RDETELLA V~CC I NES
Technical Field
The present invention relates generally to
vaccLne compositions and methods of administering the
same. More particularly, the present invention pertains
to Bordetella adhesion antigens and other outer membrane
proteins for use in stimulating immunity against
Bordetella infections.
- 25 Backqround of the Invention
The genus Bordetella is composed of four species,
B. ~ertussis, B. parapertussis, B. bronchise~tica, and
B avium. B. avium causes an upper respiratory tract
disease in turkeys and chickens which mimics B. pertussis
and B. parapertussis infection of humans (21, 23, 37, 38),
as well as resembling rhinitis in~swine caused by
B. bronchiseptica. B. avium infection induces symptoms in
birds consistent with those exhibited by children with
whooping cough (26). Thus, five to seven days after
; 35 infection, birds present with depression, 105s of
~ appetite, weight loss, snicking (coughing), moist rales,
-2- 2~3~
mucous accumulation in the external nares, dyspnea, and
vocal alterations ~3, 18, 21, 34, 37). Yollnger birds are
highly susceptible to infection while older birds are
rsfractory (36). Clinical symptoms may last from two
weeks to several months (7, 34) and secondary respiratory
infections with viral, bacterial, and fungal pathogens are
a major cause of morbidity and mortality (3, 4, 17, 21,
34). Thus, the disease causes economic losses to the
farming industry. To date no known universally effective
vaccine for B. avium exists.
All the Bordetella species exhibit specific
tropism for the ciliated tracheal epithelial cells (2, 41,
S). These cells are the only cells targeted by the
bacteria. B. avium produces histopathological changes in
the trachea which are identical to those seen in tracheas
from humans and hamsters infected with B. pertussis (2, 3,
17, 18, 19, 12, 33, 34). Following adherence and
colonization of the bacteria to the ciliated tracheal
epithelial cells, tracheas from B. avium-infected birds
and B. pertussis-infected hamsters exhibit irregularities
of the tracheal surface, mucous accumulation, leukocytic
infiltration, and a progressive loss of the ciliated
tracheal cells.
Since all Bordetella adhere to the tracheal
epithelial cells, surface structures common to all
Bordetella species are probably involved in adherence and
might be useful antigens in a vaccine. Proteins which
have been implicated as adhesins of B. pertussis include
filamentous hemagglutinin (FHA), agglutinogens (which may
or may not be ~imbriae)~ and pertussis toxin (PT). (For
a general review, see, e.g., 44, 9). FHA and PT
B. pertussis mutants lack adherence to human ciliated
epithelial cells and antibody to FHA blocks adherence of
B. pertussis to a variety o~ cells in ln vitro cell assays
(42). However, FHA cells bind well to HeLa cells (46)
and _ ~ tussis mutants which lack filamentous hemagglu-
--3--
tinin do not exhibit decreased virulence as compared withwild-type virulent B. pertussls when infected intranasally
into infant mice (45). Furthermore, B. bronchiseptica,
B. par~pertussis, and B. avium do not produce pertussis
toxin (20), and B. avium and most strains of
B. bronchLseptica do not produce FHA.
There is also controversy over the role of the
agglutinogens in adherence of Bordetella. Agglutinogens
2, 3, and 6 of B. pertussis are fimbrial antigens (for
review see 44). Monoclonal antibody to type 2 fimbriae
inhibits attachment of the homologous serotype to Vero
cells (16) while antibody to agglutinogen 1 and 2 inhibit
attachment of B. per~ussis to HeLa cells (35, 32).
Conversely, B. ~ertussis which lack fimbriae may adhere to
nonciliated WiDr (43) and human ciliated cells. Further-
more, some fimbriated strains lack adherence (42).
Fimbriae are also present on the surface of
B. avium. As with B. pertussis, it has been suggested
-
that fimbriae of B. avium are involved in adherence since
antibody against fimbriae blocks adherence of B. avium to
turkey tracheal explants (22). However, conclusive
evidence for fimbriae-mediated adherence in B. avium has
not been demonstrated. Thus, confusion exists as to the
identity of the specific proteins in~olved in adherence of
the bacteria to the ciliated tracheal epithelial cells.
Possibly there are several adhesion antigens
which might act independently or in an additive manner.
If independent, elimination of one adhesin due to mutation
would not prevent attachment. If additive, elimination of
one adhesion antigen by mutation would decrease attachment
proportional to the relative importance of the adhesion.
In either case, antibodies against the adhesin might
prevent attachment due to steric properties of the
antibody-antigen interaction which might effectively block
other adhesions from interacting with their receptors.
-4- ~ 7~
Transposon mutagenesis with Tn~A can be used to
identify bacterial cell surface proteins. Transposi-tion
of TnphoA (27) into a gene results in a protein fusion
between the N-terminal portion of the wild-type protein
and alkaline phosphatase with the concomitant loss of
expression of the sequences downstream of the insertion.
These fused proteins are localized to the site specified
by the ~ild-type protein (27). Therefore, when Tn~ is
inserted i.nto a gene for an outer membrane protein, a
periplasmic protein, or a secreted protein, alkaline
phosphatase activity i8 expressed on the bacterial cell
(27). In E. coli these insertions are readily identified
by the blue color exhibited when colonies are plated on
modified Neidhardt's MOPS medium containin~ the chromo~
genic alkaline phosphatase substrate 5-bromo-4-chloro-3-
indoyl-phosphate-~-toluidene salt (XP) as the sole
phosphate. Only alkaline phosphatase present outside the
cytoplasmic membrane shows activity; hence insertions in-to
genes for cytoplasmic proteins do not result in blue
colonies. Thus, TnphoA mutagenesis provides an effective
means to identify colonization antigens (i.e., adhesion
antigens) which must be located on the surface of
bacteria. Such TnphoA fusions, however, can inactivate
the gene, and if the gene specifies a protein essential
for bacterial viability, the transposon-induced mutant
will be lost. Since, Bordetella species lack phospha-
tases, TnphoA mutagenesis can be used as a reliable method
of identifying colonization antigens for use in vaccines
against the same.
Other outer membrane proteins, not necessarlly
adhesins, might also be useful for treating or preventing
Bordetella infections. Specifically, these proteins, or
antibodies thereto, might bloc~ attachment of co]onization
antigens to their receptors and thereby prevent infection.
Bordetella outer membrane proteins and putative
adhesion antigens can also be identified, as described
5 2~13~71
herein, using antibodies raised against crude extracts of
Bordet_lla outer membrane proteins. These antibodies can
be us~d to screen gene libraries to identify clones which
produce proteins reactive with the same.
~virulent microbes have been used as carriers in
vaccine compositions. These strains are developed by the
introduction of mutations that cause the bacteria to be
substantially incapable of survival in a host. That is,
these avirulent strains do not survive in a manner or for
a duration that would cause impairment or a disease state
in the host. Such mutants are disclosed in commonly owned
copending application serial no. 251,304, filed on
October 3, 1~88, and in Curtiss and Kelly (13), the
disclosures of which are hereby incorporated by reference.
Representative are mutants of Salmonella ~
which carry deletion mutations that impair the ability of
the bacterium to synthesize adenylate cyclase (~TP
pyrophosphate lyase (cyclizing) EC 4.6.1.1) (~y~ and the
cyclic AMP raceptor protein (crp). Mutants carrying
either a point mutation or deletion of the gene encoding
beta-aspartic semialdehyde (asd) have also been developed.
This enzyme is found in the meso-diaminopimelic acid
(DAP)-synthesis pathway. DAP is an essential component of
peptidoglycan which imparts shapa and rigidity to the
bacterial cell wall. Bacteria carrying asd mutations can
only survive in carefully controlled laboratory envi-
ronments. Thus, a recombinant vector encoding both asd
(an Asd vector) and the antigen of interest, can be
placed into an Asd carrier cell. Only those cells
encoding tha desired antigen will survive. The use of
carrier avirulent microbes to administer an outer membrane
antigan of Bordetella species could result in an effective
vaccine against Bordetella-induced diseases.
-6- 2~13~
Di~closure of the Invention
The present invention is based on the identifi-
cation and isolation of colonization antigens and outer
membrane proteins important in the adhesion of Bordetella
5 spp. to tracheal epithelial cells. The antigens can be
used in a vaccine composition to protect subjects from
various infections. Furthermore, the vaccine compositions
can be produced using recombinant DNA technology. Based
on these discoveries, the present invention can take
several embodiments.
In one embodiment, the present invention is
directed to a purified Bordetella sp outer membrane
protein. In another embodiment, the purified outer
membrane protein i5 derived ~rom B. avium.
In another embodiment, the invention is directed
to purified B. avium protein selected from the group
consisting of a 21 kDa protein, a 37 kDa protein, a 40 kDa
protein, a 43 kDa protein, a 46 kDa protein, and a 50 kDa
protein, as determined by SDS polyacrylamide gel
electrophoresis and Western immunoblot analysis
In yet another embodiment, the subject invention
is directed to a vaccine composition including one or more
outer membrane proteins of Bordetella sp. formulated in a
pharmaceutically acceptable vehicle.
In another embodiment, the vaccine composition
comprises one or more B. avium proteins selected from the
group consisting of a 21 kDa protein, a 37 kDa protein, a
40 kDa protein, a 43 kDa protein, a 46 kDa protein, and a
50 kDa protein, as determined by SDS polyacrylamide gel
electrophoresis and Western immunoblot analysis, the one
or more proteins formulated in a pharmaceutically
acceptable vehicle.
In still another embodiment of the subject
invention, khe vaccine composition comprises an adhesion
antigen of B avium with a molecular mass of about 46 kDa.
The antigen is expressed in an avirulent derivative of
-7~ 3~7~
S. typhimurium which is substantially incapable of
-
producing functional adenylate cyclase, functional cyclic
AMP receptor protein, and functional beta-aspartic semi-
aldehyde dehydrogenase. The avirulent derivative
comprises a vector carrying a gene encoding beta-aspartic
semialdehyde dehydrogenase or a functional fragment
thereof and a gene encoding the adhesion antigen.
In another embodiment, the subject invention is
directed to a vaccine composition comprising one or more
proteins of B. avium. The protein is selected from the
__
group consisting of a 21 kDa protein, a 37 kDa protein, a
40 kDa protein, a 43 kDa protein, a 46 kDa protein, and a
50 kDa protein, as determined by SDS pol~acrylamide gel
electrophoresis and Wes-tern immunoblot analysis. These
proteins are expressed in an avirulent derivative of S.
typhimurium which is substan-tially incapable of producing
functional adenylate cyclase, functional cyclic AMP
receptor protein, and functional beta-aspartic semialdehye
dehydrogenase. The avirulent derivative comprises a
vector carrying a gene encoding beta-aspartic semialdehyde
dehydrogenase or a functional fragment thereof linked to
one or more genes encoding one or more B. avium proteins.
In another embodiment, the present invention is
directed to a me~hod for preventing or ameliorating upper
respiratory disease in a vertebrate subject comprising
administering to the subject an effective amount of a
vaccine composition containing an outer membrane protein
of Bordetella sp. formulated in a pharmaceutically
acceptable vehicle. In preferred embodiments, the protein
is derived from B. avium and the subject to which the
vaccine composition is administered is fowl.
In yet another embodiment, the subject invention
is directed to a carrier microbe for the expressiorl of a
Bordetella sp. outer membrane protein comprising an aviru-
lent derivative of a pathogenic microbe. The derivativeis substantially incapable of producing functional adeny-
-8- ~13~
late cyclas~ and functional cyclic AMP receptor protein
while being capable of expressing a recombinant gene
encoding the outer membrane protein.
In still a further embodiment, the present
invention is directed to a carrier microbe for the
expression of a B. avium adhesion antigen having a
molecular mass of about ~6 kDa comprising an avir~llent
derivative of S. typhimurlum. The avirul~nt derivative is
substantially incapable of producing functional adenylate
cyclase, functi.onal cyclic AMP receptor protein and
functional beta-aspartic semialdehyde dehydrogenase. The
carrier microbe comprises a vector carrying a gene
encoding beta-aspartic semialdehyde dehydrogenase or a
functional fragment thereof and a gene encoding the
adhesion antigen.
In another embodiment, the invention is directed
to a carrier microbe for the expression of a B. avium
protein. The protein is selected from the group
consisting of a 21 kDa protein, a 37 kDa protein, a 40 kDa
protein, a 43 kDa protein, a 46 kDa protein, and a 50 kDa
protein, as determined by SDS gel electrophoresis and
Western immunoblot analysis. The carrier microbe
comprises an avirulent derivative of _ typhimurium
substantially incapable of producing functional adenylate
cyclase, functional cyclic AMP receptor protein and
functional beta-aspartic semialdehyde dehydrogenase. The
carrier microbe comprises a vector carrying a gene
encoding beta-aspartic semialdehyde dehydrogenase or a
functional fraym~nt t~ereof linked to one or more genes
encoding one or more B. avium proteins.
In further embodiments, the subject invention is
directed to DNA constructs including an expression
cassette comprised of:
(a) a DNA coding sequence for a polypeptide
containing at least one epitope of a Bordetella sp. outer
membrane protein or a B. avium outer membrane protein; and
9 ~3~
(b) control sequences that are operably linked
to the coding sequence whereby the coding sequence can be
transcribed and translated in a host cell, and at least
one of the DNA coding sequences or the control sequence is
heterologous to the host cell.
In still fur-ther embodiments of the subject
invention, methods for producing these proteins recombi-
nantly, as well as purified polyclonal and monoclonal
antibodies specific for these outer membrane proteins are
disclosed.
Further embodiments of the present invention will
readily occur to those of ordinary skill in the art.
Brief Description_of the Fiqures
Figure 1 depicts the restriction map of the XhoI
DNA fragment cloned into the XhoI restriction site of
pYA2402. B=BamHI; Bg=~g~II; Cla-ClaI; E = EcoRI;
H=HindIII; Sal=SalI; S = SstI; P = PstI; X = XhoI;
Xb=XbaI; ~/////~ =DNA from B. avium GOBL124; l ¦= fragment
cut from pYA2402 and ligation with pCP13 cut with BglII.
There are two possible orientations.
Figure 2 is a map showing the significant
features of the Asd vector, pYA292.
Figure 3 shows the results of an immunoblot
analysis performed on bacterial lysates from mutant and
parental strains of B avium. Lane A depicts the protein
profile of STL389. Lane B shows the protein profile of
STL258. Lane C shows the protein profile of STL167. Lane
D depicts the protein profile of STL6. Lane E shows the
profile of parental strain GOBL124. Lane F represents
molecular weight standards.
Figure 4 shows the results of an immunoblot of
bacterial lysates from clones of E. coli LE392 carrying
cosmid pCP13 with GOBL124 DNA inserts. Lane A depicts the
protein profile of LE392. Lane B shows the protein
profile of LE392 containing a pCP13 clone with an insert
2~337~
that did not react with probes isolated from the nonad-
herent mutants. Lane C shows the profile of LE392 with
cosmid pYA2402. Lanes D, E and F depict the protein
profiles of LE392 strains with recombinan-t inserts
reacting with probes isolated from the nonadherent
mutants. The last lane represents molecular weight
standards.
Figure 5 is an electron micrograph of nonadherent
mutant strain STL258. Pili can be seen surroundLng the
bacteria and the arrow indicates part of a flagellum
(magnification x 30,000).
Figure 6 depicts the results of an immunoblot
analysis performed on bact~rial lysates from clones of
E. coli LE392 expressing B. avium outer membrane proteins.
The lysates were probed with antibodies raised against
crude extracts of B. avium outer membrane proteins. Lane
1 represents molecular weight standards. Lane 2 shows the
profile of B. avium GOBL124. Lanes 3-8 depict the protein
profiles of LE392 with cosmids pYA2320, pYA2337, pYA2338,
pYA2339, pYA2326 and pYA2329, respectively. Lane 9
represents molecular weight standards.
Figure 7 shows the results of an immunoblot
analysis performed on bacterial lysates probed with
convalescent sera from B. avium infected turkeys. Lane 1
shows the protein profile of B. avium GOBL124. Lanes 2-4
depict the protein profiles of LE392 ~ith cosmids pYA2320,
pYA2333 and pYA2329, respectively. Lane 5 represents
molecular weight standards.
Figure 8 is a map showing the significant
features of plasmid pYA2336 containing a 6kb BamHI to PstI
B. avium fragment specifying the 21 kDa protein.
Figure 9 depicts the results of an immunoblot
analysis performed on bacterial lysates probed with
antibodies raised against crude extracts of B~ avium outer
membrane proteins. Lane 1 shows the protein profile of
B. avium GOBL124. Lane 2 depicts the protein profile of
2~ ~3:~7~
LE392 with cosmid pY~2320. Lanes 3 and 4 show the profile
of E. coli chi6097 and S. typhimurium chi3987,
respectively, both carrying p~A2336. Lanes 5 and 6 depict
the profile of E. coli chi6097 and S. typhimurlum chi3987,
S respectively, both carrying pYA292.
Detail.ed Description of the Invention
The practice of the present i.nvention will
employ, unless otherwise indicated, conventional tech-
niques of cell culture, molecular biology, microbiology,recombinant DNAr and immunology, which are within the
skill of the art. Such techniques are explained fully in
the literature. See, e.q., Maniatis, et al., Molecular
Cloning: A Laboratory Manual ~1982); DNA Cloning (198S)
Vols. I and II, D.N. Glover (ed.); Nucleic Acid Hybridi-
zation (1984), B.D. Hames, et al. (eds.); Perbal, B., A
Practical Guide to Molecular Cloning (1984); Methods in
Enzymology (the series), Academic Press, Inc.; Vectors: A
Survey of Molecular Cloning Vectors and Their Uses (1987),
R.L. Rodriquez, et al., (eds.), Butterworths; and Miller,
J.H., et al., Experiments in Molecular Genetics (1972)
Cold Spring Harbor Laboratory.
All patents, patent applications, and publi-
cations mentioned herein, whether supra or infra, are
hereby incorporated by reference in their entirety.
A. Definitions
An "antigen" refers to a molecule containing one
or more epitopes that will stimulate a host's immune
system to make a secretory, humoral and/or cellular
antigen-specific response. The term is also used inter-
changeably with "immunogen.
A "hapten" is a molecule containing one or more
epitopes that does not itself stimulate a host's immune
system to make a secretory, humoral or cellular response.
-12- 2~
The term ~'epitope" refers to the site on an
antigen or hapten to which a specific antibody molecule
'oinds. The term is also used interchangeably ~ith
"anti~enic determinant or "anti~enic determinant site."
S An epitope will normally include 3 amino acids necessary
for recognition in spatial confirmation, more usually
S amino acids, and most usually 8-lO amino acids.
An "immunological response" to a composition or
vaccine is the development in the host of a cellular and/
or antibody-mediated immune response to the composition or
vaccine of interest. Usually, such a response consists of
-the subject producing antibodies, B cells, helper T cells,
suppressor T cells, and/or cytotoxic T cells directed
specifically to an antigen or antigens included in the
composition or vaccine of interest.
By ~vaccine composition" is meant an agent used
to stimulate the immune system of a living organism so
that protection against future harm is provided. "Immuni-
zation" refers to the process of inducing a continuing
high level of antibody and/or cellular immune response in
which T-lymphocytes can either kill the pathogen and/or
activate other cells (e.g., phagocytes) to do so in an
organism, which is directed against a pathogen or antigen
to which the organism has been previously exposed.
Although the ph~ase "immunè system" can encompass
responses of unicellular organisms to the presence of
foreign bodies, e.g., interferon production, in this
application the phrase is restricted to ~he anatomical
features and mechanisms by which a multicellular organism
produces antibodies against an antigenic material which
invades the cells of the organism or the extracellular
fluid of the organism. The antibody so produced may
belong to any of the immunological classes, such as
immunoqlobulins A, D, E, G or M.
Of particular interest are vaccines which
stimulate production of immunoglobulin A (IgA) since this
-13- 20~
is the principle immunoglobulin produced by the secretory
system of warm-blooded animals. Immune response to
antigens is well studied and widely reported. A survey of
immunology is given in Barre~t, James T., Textbook of
Immunoloqy: Fourth Edition, C.V. Mosby Co., St. Louis, MO
(1983). Avian species have a mucosal immune network
consisting of gut~associated lymphoid tissue (termed GALT
or Peyer's patches), bronchial-associated lymphoid tissue
(BALT), and the harder gland, located ventrally and
posteriomedially to the eyeball. Presentation of antigen
to these tissues triggers proliferation and dissemination
of committed B cells to the secretory tissues and glands
in the body, with the ultimate produc-tion of secretory Ig~
(SIgA~. (6, 10, 25, 28, 47, 15, 30, 31). SIgA serves to
block the colonization and invasion of specific surface
antigens that colonize on, and pass through, a mucosal
surface. (39, 48).
A ~vertebrate" is any member of the subphylum
Vertebrata, a primary division of the phylum Chordata that
includes the fishes, amphibians, reptiles, birds, and
mammals, all of which are characterized by a segmented
bony or cartilaginous spinal column. All vertebrates have
a functional immune system and respond to antigens by
producing antibodies.
By "fowl" is meant domestic, wild and game birds
such as cocks and hens including chickens, turkeys and
other gallinaceous birds as well as other avian species.
The definition encompasses birds of all ages.
B~ l'avirulent derivative of a microbe~ is meant
an organism which i5 substantially incapable of causing
disease in a host being treated with the particular
avirulent microbe. Avirulent does not mean that a microbe
of that genus or species cannot ever function as a
pathogen, but that the particular microbe being used is
avirulent with respect to the particular animal being
treated. The microbe may belong to a genus or even a
-14- 2~ 3~7~
species that is normally pathogenic but must belong to a
strain that is avirulent. By "pathogenic~l is meant
capable of causing disease or impairing normal physio-
logical functioning. Avirulent strains are incapable of
inducing a full suite of symptoms of the disease that is
normally associated wlth its virulent pathogenic counter-
part. The term "microbe" as used hereLn includes
bacteria, protozoa, and unicellular fungi.
~ ~carrier microbs~ is an avirulent microbe as
defined above which contains and e~presses a recombinant
gene encoding a protein of interest such as an outer
membrane adhesion antigen, or outer membrane protein from
Bordetella 9p.
An ~'outer membrane adhesion antigen" is an
antigen that is localized on the outer membrane of the
bacterial cell and is involved in the adherence of a
pathogenic bacterium to target host cells. In Bordetella
sp., the targeted host cells are tracheal epithelial
cells. Exemplary of one such adhesion antigen is a
protein having a molecular mass of about 46 kDa found in
B. avium. An "outer membrane protein" as defined herein
is a protein reactive with antibodies raised against crude
extracts of outer membrane proteins isolated from B. avium
by the procedure described in the Experimental section.
These outer membrane proteins include, but are not limited
to, a 21 kDa protein, a 37 kDa protein, a 40 kDa protein,
a 43 kDa protein, a ~6 kDa protein, and a 50 kDa protein,
as detarmined by SDS gel electrophoresis and described
fur~her below. Outer membrane adhesion antigens are by
definition also outer membrane proteins. Outer membrane
proteins may or may not be adhesion antigens. ~ntibodies
raised against these proteins may directly prevent
Bordetella infection. Alternatively, these proteins, or
antibodies raised thereto, may block the effects of
colonization antigens by interfering with the attachment
of these antigens to cell surface receptors.
-15- 2~.~3~
A "purified protein or antigen~ is one substan-
tially free of other materials. For example, protein A is
substantially free of B where B is a mixture of o-ther
cellular components and proteins, and thus purified, when
at least 30% by weight of the total A ~ B presen~ is A.
Preferably, A comprises at least about 50% by weight of
the total A -~ B present, more preferably at least 75~, and
most preferably 90-95% or even 99~ by weight. "Purified~
does not, however refer to the method by which the protein
is derived. Thus, a purified protein can be one produced
by recombinant techniques, synthetically produced, or
isolated directly from an or~anism in which the protein is
found in nature.
The term "polypeptide" is used in its broadest
sense, i.e., any polymer of amino acids (dipeptide or
greater) linXed through peptide bonds. Thus, the term
l~polypep~ide~' includes proteins, oligopeptides, protein
fragments, analogs, muteins, fusion proteins and the like.
The term includes native and recombinant proteins.
"Native" proteins or polypeptides refer to
proteins or polypeptides recovered from a source occurring
in nature. Thus, the term "native Bordetella protein or
polypeptide~' would include naturally occurring Bordetella
proteins and fragments thereof.
"Recombinant" polypeptides refer to polypeptides
expressed from a recombinant gene; i.e., produced from
c~alls transformed by an exogenous DNA construct encoding
the desired polypeptide.
A "recombinant ~ene~ is an identifiable segment
of polynucleotide within a larger polynucleotide molecule
that is not found in association with the larger molecule
in nature.
A "repliconl' is any genetic element (e.g.,
plasmid, chromosome, virus) that functions as an autono-
mous unit of DNA replication ln vivo; i.e., capable ofreplication under its own control.
-16- 2~3.37~
.
A ~vector~ is a replicon, such as a plasmid,
phage, or cosmid, to which another DN~ segment may be
attached so as to bring about the replication of the
attached segment.
A DNA "coding sequence" is a DN~ sequence which
is transcribed and translated into a polypeptide in vivo
when placed under the control of appropriate regulatory
sequences. The boundaries of the coding sequence are
determined by a start codon at the 5' (amino) -terminus and
a translation stop codon at the 3' (carboxy) terminus. A
coding sequence can include, but is not limited to,
procaryotic sequences, cDNA from eucaryotic mRNA, genomic
DNA sequences from eucaryotic (e.g., mammalian) DNA, and
even synthetic DNA sequences. ~ transcription termination
sequence will usually be located 3~ to the codin~
sequence.
A "promoter sequence" is a DNA regulatory region
capable of binding RNA polymerase in a cell and initiating
transcription of a downstream (3' direction) coding
sequence. For purposes of defining the present invention,
the promoter sequence is bound at the 3' terminus by the
translation start codon (ATG) of a coding sequence and
extends upstream (5' direction) to include the minimum
number of bases or elements necessary to initiate trans-
cription at levels detectable above background. Withinthe promoter sequence will be found a transcription
initiation site (conveniently defined by mapping with
nuclease Sl), as well as protein binding domains
(consensus sequences) responsible for the binding of ~NA
polymerase. Eucaryotic promoters will often, but not
always, contain "TATA~ boxes and "CAT" boxes. Procaryotic
promoters contain Shine-Dalgarno sequences in addition to
the -10 and -35 consensus sequences.
DNA "control sequences" refers collectively to
promoter sequences, ribosome binding sit~s, polyadenyl-
ation signals, transcription termination sequences,
-17~ 3 3vr~
upstream regulatory domains, enhancers, and ~he like,
which collectively provide for the transcription and
translation of a coding sequence in a host cell.
~ coding sequence is "operably linked to" or
~under the control of" control sequences in a cell when
RNA polymerase will bind the promoter sequence and
transcribe the codinq sequence into mRNA, which is then
translated into the polypeptide encoded by the coding
se~uence.
"Recombinant host cells", "host cells", "cells"
and other such terms denoting microorganisms are used
interchangea~ly, and refer to cells which can be, or have
been, used as recipients for recombinant vectors or other
transferred DNA, and include the progeny of the original
lS cell transfected. It is understood that the progeny of a
single parental cell may not necessarily be completely
identical in genomic or total DNA complement as the
original parent, due to acciden-tal or deliberate mutation.
Progeny of the parental cell include those cells which are
sufficiently similar to the parent to be characterized by
the relevant property, for example, the substitution of a
native gene encoding an essential enzyme with a cloned
gene linked to a structural gene encoding a desired gene
product.
A "clone" is a population of cells derived from a
single cell or common ancestor by cell division. A "cell
line" is a clone of a primary cell that is capable of
stable growth in vitro for many generations.
A "gene library" is a collection of cloned genes,
generally comprising many or all of the genes from a
particular species. Libraries are made by treating DNA
with selected restriction endonucleases, followed by
cloning the fragments into a suitable vector. Gene
libraries can be searched using a homologous sequence of
DNA from a rela~ed organism in order to identify the clone
within the library which represents the desired gene.
2~1 3~7~
-18-
A "heterologous~ region of a DNA construct is an
identifiable segment of DNA within or attached to another
DNA molecule that is not found in association with the
other molecule in nature. Thus, when the heterologous
region encodes a bacterial gene, the gene will usually be
flanked by DNA that does not flank the bacterial gene in
the genome of the source bacteria. Another example of the
heterologous coding sequence is a construct where the
coding sequence itself is not foun~ in nature (e.g.,
synthetic sequences having codons different from the
native gene). Allelic variation or naturally occurring
mutational events do not give rise to a heterologous
region of DN~, as used herein.
"Transformation", as used herein, refers to the
insertion of an exogenous polynucleotide into a host cell,
irrespective of the method used for the insertion, for
example, direct uptake, transduction, or conjugation. The
exogenous polynucleotide may be maintained as a plasmid,
or alternatively, may be integrated within the host
genome.
B. General Methods
The present invention involves the discovery of
outer membrane adhesion proteins of Bordetella spp., and
proteins capable of reacting with antibodies raised
against crude extracts from _ ~vium containing outer
membrane proteins, for use in vaccine compositions against
Bordetella-induced diseases. Since adhesion proteins are
present on the bacterial outer membrane, transposon
mutagenesis using TnphoA pro~ides a method for identifying
the antlgens of the present invention. TnphoA can be
introduced into Bordetella cells by mating with an E. coli
donor as described in detail below. When Tn~ is
transposed into the Bordetella chromosome, kanamycin
resistance is always imparted and alkaline phosphatase may
be produced if the TnphoA inserts into a gene encoding a
-19- 2~ 7~
protein transported across the cytoplasmlc membrane.
Alkaline phosphatase is normally absent from ~ordetella
Thus, transposon-induced mutants can be selected on
kanamycin~containing medium and expression of the alkaline
phosphatase gene can be detected by hydrolysis of the
chromogenic substrate 5-bromo-4-chloro-3-indoyl-~-
toluidine phosphate (XP), which is incorporated into the
medium. The mutant strains can be screened for their
ability to attach to tracheal epithelial cells in Ln vitro
assays usin~ chicken and turkey tracheal rings. Coloni-
zation using both adherent and nonadherent strains can
also be studied in vivo by inoculating appropriate animals
with the various strains as described below.
The chromosomal DNA from mutant strains demon-
strating impaired adhesiveness can be partially digestedusing conventional restriction enzymes. The restriction
fragments can be separated using agarose gels. Since
Tnpho~ is approximately 7.5 kilobases in length, fragments
larger than 7.5 kb in length are cut from the gel,
recovered by electroelution and ligated to a suitably
digested plasmid (see below). E. coli can be transformed
using these constructs and colonies demonstrating
kanamycin-resistance selected. Plasmids containing the
DNA isolated from the mutant~ can be used as probes to
detect the presence of the adhesion genes in gene
libraries made from wild-type Bordetella species.
Western immunoblots can be performed on bacterial
lysates from the mutant and wild-type strains and adhesion
antigens identified as described in the experimental
section. These proteins can be isolated using conven-
tional techniques.
It is likely that ~he outer membrane adhesion
proteins from the various Bordetella species share a high
degree of homology. Thus, a plasmid containing a TnphoA
fragment bearing a gene encoding for one such protein will
likely be useful for screening other Bordetella species
2 ~ 7~
-20-
for similar adhesion proteins. Furthermore, polyclonal
anti~odies (discussed more fully below) directed against
one Bordetella outer membrane adhesion antigen, would
likely be cross-reactive with outer membrane adhesion
antigens from other Bordetella species.
Ad~itionally, crude preparations of Bordetella
outer membrane proteins can be used to raise antibodies
which can in turn be u~ed for recombinant expression
screening. Thus, clones expressing proteins reactive with
these antibodies can be identified and these proteins
further chaxacterized and tested for their ability to
attach to tracheal epithelial cells as described above.
These proteins are also likely to be adhesion antigens.
The isolated proteins can be sequenced by any o~
the various methods known to those skilled in the art.
For example, the amino acid sequences of the su~ject
proteins can be determined from the purified proteins by
repetitive cycles of Edman degradation, followed by amino
acid analysis by HPLC. Other methods of amino acid
sequencing are also known in the art.
The amino acid sequences datermined by the above
method may be used to design oligonucleotide probes which
contain the codons for a portion of the determined amino
acid sequences which can be used to screen DNA libraries
for genes encoding the subject proteins. The basic
strategies for preparing oligonucleotide probes and DNA
libraries, as well as their screening by nucleic acid
hybridization, are well known to those of ordinary skill
in the art. See, e.q., DNA Cloning: Vol. I, supra;
Nucleic Acid Hybridization, supra; Oligonucleotide
Synthesis, supra; Maniatis, T., et al., supra.
First, a DNA library is prepared. The library
can consist of a genomic DNA library from Bordetella spp.
Once the library is constructed, oligonucleotides to probe
the library are prepared and used to isolate the gene
encoding the outer membrane protein. The oligonucleotides
-21- 20~ ~7~
are synthesized by any apprvpriate method. The particular
nucleotide sequences selected are chosen so as to corres-
pond to the codons encoding a known amino acid sequence
from the desired Bordetella antigen. Since the genetic
code is degenerate, it will often be necessary to
synthesize several oligonucleotides to cover all, or a
reasonable number, of the possible nucleotide sequences
which encode a particular region of the protein. Thus, it
is generally preferred in selecting a region upon which to
base the probes, that the region not contain amino acids
whose codons are highly degenerate. In certain circum-
stances, one of skill in the art may find it desirable to
prepare probes that are fairly long, and/or encompass
regions of the amino acid sequence which would have a high
degree of redundancy in corresponding nucleic acid
sequences, particularly if this lengthy and/or redundant
region is highly characteristic of the protein of inter-
est. It may also be desirable to use two probes (or sets
of probes), each to different regions of the gene, in a
single hybridization experiment. Automated oligonucleo-
tide synthesis has made the preparatLon of large families
of probes relatively straightforward. While the exact
length of ths probe employed is not critical, generally it
is recognized in the art that probes from about 14 to
about 20 base pairs are usually efective. Longer probes
of about 25 to about 60 base pairs are also used.
The sel~cted oligonucleotide probes are labeled
with a marker, such as a radionucleotide or biotin using
standard procedures. The labeled set of probes is then
used in the screening step, which consists of allowing the
single-stranded (ss) probe to hybridize to isolated ssDNA
from the library, according to standard techniques.
Either stringent or permissive hybridization conditions
could be appropriate, depending upon several factors, such
as the length of the probe and whether the probe is
derived from the same species as the library, or an
-22- 2 ~1 3^S 7~
evolutionarily close or distant species. The selection of
the approprlate conditions is within the skill of the art.
ee qenerally, Nucleic Acid Hybridizationl supra. The
basic requirement is that hybridization conditions be of
sufficient stringency so that selective hybridization
occurs; i.e., hybridlzation is due to a sufficient degree
of nucleic acid homology (e.g., at least about 75%), as
oppos~d to nonspecific binding. Once a clone from the
screened library has been identified by positive h~bridi-
zation, it can be confirmed by restriction enzyme analysisand DNA sequencing that the particular library insert
contains a gene for the desired protein.
~ lternatively, DNA sequences encoding the
proteins of interest can be prepared synthetically rather
than cloned. The DNA sequence can be designed with the
appropriate codons for the particular Bordetella amino
acid sequence. In general, one will select preferred
codons for the intended host if the sequence will be used
for expression. The complete sequence is assembled from
overlapping oligonucleotides prepared by standard methods
and assembled into a complete coding sequence. See, e.q.,
Edge, Nature (1981) 292:756; Nambair et al., Science
(1984) 223:1299; Jay et al., J Biol Chem (1984) 259:6311.
Once a coding sequence for the desired protein
has been prepared or isolated, it can be cloned into any
suitable vector or replicon. Numerous cloning vectors are
known to those of skill in the art, and the selection of
an appropriate cloning vector is a matter of choice.
Examples of recombinant DNA vectors ~or cloning and host
cells which they can transform include the bacteriophage
lambda (E. coli), pBR322 (E. coli), pACYC177 (E. coli),
pKT230 (gram-negative bacteria), pGV1106 (gram-negative
bacteria), pL~FRl (gram-negative bacteria), pME290 (non-E.
coli gram-negative bacteria), pH~14 (E. coli and Bacillus
subtilis), pBD9 (Bacillus), pI~r6l (StreptomYces), pUC6
(Streptomvces), YIpS (SaccharomYces), YCpl9 (Saccharo-
2~3~7~
-23-
myces~ and bovine papilloma virus (mammalian cells). See,
~enerally, DNA Cloning: Vols. I ~ II, supra; Maniatis, T.,
et al., supra; Perbal, B., supra.
The coding sequence for the Bordetella outer
membrane protein of interest can be placed under the
control of a promo~er, ribosome blnding site (for bacte-
rial expression) and, optionally, an operator (collec-
tively referred to herein as "control" elements), so that
the DNA sequence encoding the protein is transcribed into
RNA in the host cell transformed by a vector containinq
this expression const~uction. The coding sequence may or
may not contain a signal peptide or leader sequence. The
full-length Bordetella proteins of the present invention
can be expressed using, for example, a native Bordetella
promoter or other well ~nown promoters that function in
gram negati~e bacteria such as the tac or trp promo-ters.
The outer membrane antigens, when present in a
carrier microbe~ will normally be expressed under the
control of a promoter that only allows expression ln vivo
in the immunized host. However, if production of the
protein is desired in bulk, outside of the intended
recipient, in addition to control se~uences, it may be
desirable to add regulatory sequences which allow for
regulation of the expression of the bacterial antigen
sequences relative to the growth of the host cell.
Regulatory sequences are known to those of skill in the
art, and examples include those which cause the expression
of a gene to be turned on or off in response to a chemical
or physical stimulus, including the presence of a regula-
tory compound. Other types of regulatory elements mayalso be present in the vector, for example, enhancer
sequences. The subject proteins can also be expressed in
the form of a fusion protein, wherein a heterologous amino
acid sequence is expressed at the N-terminal end of the
3S fusion protein. See, e.q., U.S. Patent Nos. 4,431,739;
4,425,437.
-24- 2~3 3~
An expression vector is constructed so that the
particular coding sequence is located in the vector with
the appropriate regulatory sequences, the positioning and
orientation of the coding sequence with respect to the
control sequences being such that the coding sequence is
transcribèd under the "control" of the control sequences
(i.e., RNA polymerase which binds to the DNA molecule at
the control sequences transcribes the coding sequence).
Modification o~ the sequences encoding the particular
antig~n of interest may be desirable to achieve this end.
For example, in some cases it may be necessary to modify
the sequence so that it may be attached to the control
sequences with the appropriate orientation; i.e., to
maintain the reading frame. The control sequences and
other regulatory sequences may be ligated to the coding
sequence prior to insertion into a vector, such as the
cloning vectors described above. Alternatively, the
coding sequence can be cloned directly into an expression
vector which already contains the control sequences and an
appropriate restriction site.
In some cases, it may be desirable to add leader
sequences which cause the secretion of the polypeptide
from the host organism, with subsequent cleavage of the
secratory signal, if any. Leader sequences can be remo~ed
2S by the bacterial host in post-translational processing.
See, e.q., V.S. Patent Nos. 4,431,739; 4,425,437,
4,338,397. It may also be desirable to produce mutants or
analogs of the antigen of interest. Mutants or analogs
may be prepared by the deletion of a portion of the
sequence encoding the antigen, by insertion of a sequence,
and/or by substitution of one or more nucleotides within
the sequence. For example, proteins used to immunize a
host may contain epitopes that stimulate helper cells as
well as epitopes that stimulate suppressor cells. Thus,
deletion or modification of these latter nucleotides would
be desirable. Techniques for modifying nucleotide
-25- 2~.3~7~
sequences, such as site-directed mutagenesis, are well
known to those skilled in the art. See, ~ , Maniatis,
T., et al., supra; DN~ Cloning, Vols. I and II, supra;
Nucleic ~cid Hybridization, supra.
A number of procaryotic expression vectors are
known in the art. See, e.q., U.S. Patent Nos. 4,440,859;
4,436,815; 4,431,740; 4,431,739; 4,428,941; ~,425,437;
4,~18,149; 4,~11,994; 4,366,246; 4,342,832; see also U.K.
Patent Applications GB 2,121,05~; GB 2,008~123; GB
2,007,~75; and European Patent Application 103,395.
Depending on the expression system and host
selected, the proteins of the present invention are
produced by growing host cells transformed by an expres-
sion vector described above under conditions whereby the
protein of interest is e~pressed. The particular
Bordetella protein is then isolated from the host cells
and purified. If the expre~sion system secretes the
protein into growth media, the protein can be purified
directly from the media. If the protein is transported to
the periplasmic space, it can be released to the medium by
cold osmotic shock, a technique well known in the art. If
` the protein is not secreted or transported to the peri-
- plasmic space, it is isolated from cell lysates. The
selection of the appropriate growth conditions and
recovery methods are within the skill o~ the art.
I'he anti~ens of the present invention can also be
isolated from Bordetella cultures using standard protein
purification procedures well known in the art. See,
Protein Purification Principles and Practice (1987) 2d
ed., R.K. Scopes (ed.). Such techniques include gel
filtration chromatography, ion exchange chromatography,
affinity chromatography, immunoadsorbent chromatography,
polyacrylamide gel electrophoresis and other electro-
phoretic t~chniques, centrifugation, dialysis, and
precipitationO
-26- 2~3~
The antigens of the present invention may also be
produced by chemical synthesis such as solid phase peptide
synthesis, using known amino acid sequences or amino acid
sequences derived from the DNA sequence of the gene of
interest. Such methods are known to those skilled in the
art. Chemic~l synthesis of peptides may be preferable if
a small fragment of the antigen in question is capable of
raising an immunological response in the subject of inter-
est.
The proteins of the present invention or their
fra~ments can be used to produce antibodies, both poly-
clonal and monoclonal. If polyclonal antibodies are
desired, a selected bird or mammal, (e.g., chicken,
turkey, mouse, rabbit, goat, horse, etc.) is immunized
with an antigen of the present invention, or its ~ragment,
or a mutated antigen. Serum from the immunized animal is
collected and treated according to known procedures. If
serum containing polyclonal antibodies to the protein of
interest contains antibodies to other antigens, the
polyclonal antibodies can be purified by immunoaffinity
chromatography, using known procedures.
Monoclonal antibodies to the proteins o~ the
present invention, and to the fragments thereof, can also
be readily produced by one skilled in the art. The
general methodology for making monoclonal antibodies by
hybridomas is well known. Immortal antibody-producing
cell lines can be created by cell fusion, and also by
other techni~ues such as direct transformation of B
lymphocytes with oncogenic DNA, or transfection with
Epstein-Barr virus. See, e.~., Schreier, M., et al.,
Hybridoma Techniques (1980); Hammerling et al., Monoclonal
Antibodies and T-cell Hybridomas (1981); Kennett et al.,
Monoclonal Antibodies (1980); see also U.S. Patent Nos.
4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,452,570;
4,466,917; 4,472,500, 4,491,632 and 4,493,890. Panels o~
monoclonal antibodies produced against the antigen of
-27- 2~13~7~
interest, or fragment thereof, can be screened for various
properties; i.e., for isotype or epitope affinity, etc.
~onoclonal antibodies are useful in purification, using
immunoaffinity techniques, of the antigens which they are
directed against.
The outer membrane proteins of the present
invention, produced as described above, can be used to
immunize subjects against Bordetella-induced diseases.
Avirulent carrier microbes can be used to administer the
present antigens. This method of administration is
particularly suitable since appropriate carrier microbes
can invade and proliferate in -the cells of the GALT and
BALT. Delivery of an antigen to the BALT or the GALT
elicits a generaliæed secretory immune response as well as
humoral and cellular immune responses as described above.
~ ecombinant plasmids containing genes for the
sub~ect proteins can be introduced into one of several
avirulent strains of bacteria containing mutations for
genes necessary for long-term survival in the targeted
host. Particularly useful are the cya, crp and asd
mutants described above, however, other avirulent microbes
will also find use with the present invention. If Asd
mutants are used, the adhe~ion antigen of interest is
transferred to the carrier microbe using a vector encoding
both the adhesion antigen and asd. Thus, only those
carrier microbes containing the desired adhesion antigen
will survive and these microbes can be selected for
further use. Figure 2 depicts a map of pYA292 Asd , a
vector into which a gene encoding the desired adhesion
antigen can be cloned. This vector can then be trans-
ferred into an Asd carrier microbe. E~pression of the
recombinant gene encoding the desired antigen may ~e
dependent on a control sequence linked to the asd gene.
This linkage may result from the orientation of the two
genes in the vector so that both genes could be, for
example, under the control of the same control elements,
-28- 2013~7~
. . .
i.e., the same promoter and operator. Methods of
constructing vectors with these characteristics are known
in the art using recombinant DNA technology and are
discussed more fully in copending patent application
Serial No. 251,304.
Useful avirulent microbes include, but are not
limited to, mutant derivatives of Salmonella and E. coli-
Salmonella hybrids. Preferred microbes are members of the
~enus Salmonella such as S. tYphimurium, S. typhi,
_
S. paratyphi/ S. qallinarum, S. pullorum, S. enteritidis,
S. choleraesuis, S. arizona, or S. dublin. Avirulent
derivatives of S . typhimurium and S. enteritidis find
broad use among many hosts. Avirulent derivatives of
S. gallinarum, S. pullorum and S. arizona may be particu-
larly useful for immunizing avian species whereasS. typhimurium, S. ~y~ and S. paratvphi are preferred
for use in humans. S. choleraesuis is preferably used to
immunize swine while S. dublin finds use in cattle. These
avirulent Salmonella strains are able to colonize the GALT
and BALT, where they persist for weeks, but are not patho-
genic to the host organism. The creation of such mutants
is described in copending patent application Serial
No. 251,304 and in Curtiss and Kelly ~13).
In order to stimulate a preferred response of the
GALT or BALT, introduction of the microbe or gene product
directly into the gut or bronchus is preferred, such as by
oral adminis~ration, intranasal administration, gastric
intubation or in the form of aerosols, as well as air sac
inoculation (in birds only), and intratracheal inocula-
tion. Other suitable methods include administration viathe conjuctiva to reach the Harder gland and intramammary
inoculation. Other methods of administering the vaccine,
such as intravenous, intramuscular, or subcutaneous injec-
tion are also possible, and used principally to stimulate
a secondary immune response, as described further below.
~ -29- 2~ 7~
A particularly useful mode of administration in
avian species is via drinking water. Thus, the carrier
microbes, expressing the Bordetella outer membrane antigen
of interest, can be placed directly into water given to
these animals. In ovo administration can also be
accomplished by inoculating avian eggs before they hatch,
generally at approximately 18 days.
Generally, when carrier microbes expressing the
Bordetella antigens are administered to humans or other
mammals, they will be coated or encapsulated with a
suitable gelatin-like substance, known in the art.
Once the carrier microbe is present in the
animal, the antigen needs -to become available to the
animal's immune system. This may be accomplished when the
carrier microbe dies so that the antigen molecules are
released. Of course, the use of "leaky" avirulent mutants
that release the contents of the periplasm without lysis
is also possible. ~l-ternatively, a gene may be selected
that controls the production of an antigen that will be
made available by the carrier cell to the outside envi-
ronment prior to the death of the cell.
Subjects can also be immunized with the antigens
of the present invention by administration of tho protein
of interest, or a fragment thereof, or an analog thereof
without a carrier microbe. If the fragment or analog is
used, it will include the amino acid sequence of one or
more epitopes which interact with the immune system to
immunize the subject to that and structurally similar
epitopes. Prior to immunization, it may be desirable to
increase the immunogenicity of the particular sordetella
protein, or an analog of the protein, or particularly
fragments of the protein. This can be accomplished in any
one of several ways known to those of skill in the art.
For example, the antigenic peptide may be administered
linked to a carrier. For example, a fragment may be
conjugated with a macromolecular carrier. Suitable
-30- 2~3 ^3 ~
carriers are typically large, slowly metabolized macro-
molecules such as: proteins; polysaccharides, such as
sepharose, agarose, cellulose, cellulose beads and the
like; polymeric amino acids such as polyglutamic acid,
polylysine, and the like; amino acid copolymers; and
inactive virus particles. Especially useful protein
substrates are keyhole limpet hemocyanin, immunoglobulin
molecules, thyroglobulin, the beta subunit of hea-t labile
toxin of E. coli or beta subunit of cholera toxin, and
other proteins well known to those skilled in the art.
The protein substrates may be used in their
native form or their functional group content may be
modified by, for example, succinylation of lysine residues
or reaction with Cys-thiolactone. A sulfhydryl group may
also be incorporated into the carrier (or antigen) by, for
example, reaction of amino functions with 2-iminothiolane
or the N-hydroxysuccinimide ester of 3-(4-dithiopyridyl
propionate. Suitable carriers may also be modified to
incorporate spacer arms (such as hexamethylene diamine or
other bifunctional molecules of similar size) for attach-
ment of peptides.
Furthermore, the Bordetella antigens (or
complexes thereof), when administered without the carrier
microbe, may be formulated into vaccine compositions in
either neutral or salt forms. Pharmaceutically acceptable
salts include the acid addition salts (formed with the
~ree amino groups of the active polypeptides) and which
are formed with inorganic acids such as, for example,
hydrochloric or phosphoric acids, or such orqanic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts
formed from free carboxyl groups may also be derived from
inorganic bases such as, for example, sodium, potassium,
ammonium, calcium, or ferric hydroxides, and such organic
bases as isopropylamine, trimethylamine, 2-ethylamino
ethanol, histidine, procaine, and the like.
~1 3~7~
-31-
Typically, the antigens, when used without an
avi~ulent carrier, are administered as aerosols or
intranasally. Intranasal formulations Eor mammalian
subjects will usually include vehicles that neither cause
irritation to the nasal mucosa nor significantly disturb
ciliary function. Diluents such as water, aqueous saline
or other known substances can be employed with the subject
invention. The nasal formulations may also contain
preservatives such as but not limited to chlorobutanol and
benzalkonium chloride. A surfactant may be present to
enhance absorption of the sub~ect proteins by the nasal
mucosa.
Injec~ion with a pathogen-derived gene product
can also be used in conjunction with prior oral, intra-
nasal, gastric or aerosol immunization. Such parenteralimmunization can serve as a booster to enhance expression
of the secretory immune response once the secretory immune
system to that pathogen-derived gene product has been
primed by immunization with the carrier microbe expressing
the pathogen-derived gene product to stimulate the
lymphoid cells of the GALT or B~LT. The enhanced response
is known as a secondary, booster, or anamnestic response
and results in prolonged immune protection of the host.
Booster immunizations may be repeated numerous times with
beneficial results.
When the vaccines are prepared as in~ectables,
such as for boosters, they can be made either as liquid
solutions or suspensions; solid forms suitable for solu-
tion in, or suspension in, liquid vehicles prior to injec-
tion may also be prepared. The preparation may also beemulsified or the active ingredient encapsulated in
liposome vehicles. The active immunogenic ingredient is
often mixed with vehicles containing excipients which are
pharmaceutically acceptable and compatible with the active
ingredient. Suitable vehicles are, for example, water,
saline, dextrose, glycerol, ethanol, or the like, and
2 ~
-32-
combinations thereof. In addition, if desired, the
vehicle may contain minor amounts of auxiliary substances
such as wetting or emulsifying agents, pH buffering
agents, or adjuvants which enhance the effectiveness of
the vaccine. Ad~uvants may include for example, muramyl
dipeptides, avridine, aluminum hydroxide, oils, saponins
and other substances known in the art. Actual methods of
preparing such dosage forms are known, or will be
apparent, to those skilled in the art. See, ~
Remington's Pharmaceutical Sciences, Mack Publishing
Company, Easton, PA, 15th ed., 1975. The composition or
formulation to be administered will, in any event, contain
a quantity of the protein adequate to achieve the desired
immunized state in the subject being treated.
The quantity of antigen to be administered
depends on the subject to be treated, the capacity of the
subject's immune system to synthesize antibodies, and the
degree of protection desired. Effective dosages can be
readily established by one of ordinary skill in the art
through routine trials establishing dose response curves.
The sub~ect is immuni~ed by administration of the parti-
cular antigen or ~ragment thereof, or analog thereof,
either with or without a carrier microbe, in at least one
dose. Typical doses using the carrier microbe are on the
order of 1 x 106-1 x 101 recombinant avirulent bacteria/
immunized subject. Initial doses of vaccine using the
protein without the avirulent microbe could be 0.001-1 mg
antigen/kq body weight. Moreover, the subject may be
administered increasing amounts or multiple dosages as
required to maintain a state of immunity to sordetella
spp .
The above disclosure generally describes the
present invention. A more complete understanding can be
obtained by re~erence to the following specific examples,
which are offered ~or illustrative purposes only, and are
33
not intended to limit the scope of the present invention
in any way.
Deposits of Strains Useful in Practicinq the Invention
A deposit of biologically pure cultures of the
following strains were made with the American Type Culture
Collection, 12301 Parklawn Drive, Rockville, Maryland.
The accession number indicated was assigned after success-
ful viability testing, and the requisite fees were paid.
Access to said cultures will be available during pendency
of the patent application to ona determined by the Commis-
sioner to be entitled thereto under 37 CFR 1.1~ and 35 USC
122. All restriction on availability of said cultures to
the public ~ill be irrevocably removed upon the granting
of a patent based upon the application. Moreover, the
designated deposits will be maintained for a period of
thirty (30) years from the date of deposit, or for five
(5) years after the last re~uest for the deposit; or for
the enforceable life of the U.S. patent, whichever is
longer. Should a culture become nonviable or be inadvert-
ently destroyed, or, in the case of plasmid-containing
strains, loose its plasmid, it will be replaced with a
viable culture(s) of the same taxonomic descrip-tion.
-34- 2 ~ 7 ~
Strain Deposit Date ATC~ No.
_
chi4064 July 15, 1987 53648
chi4072 Oct. 6, 1987 67538
pYA292 Asd
in chi6097 Sept. 26, 1988 67813
pYA2402
in chi6121 March 28, 1989 67921
pYA2336
in chi3987 March 22, 1990
pYA2326
in LE392 March Z2, 1990
pY~2333
in LE 392 March 22, 1990 -----
pYA2337
in LE 392 March 22, 1990
pYA2338
in LE 392 March 22, 1990 -----
pYA2339
in LE 392 March 22, 1990 ----~
C. Experimental
1. Materials and Methods
The following materials and methods were
utilized unless otherwise noted.
Bacterial Strains~edia and Vectors. B. avium
_
GOBL124 was isolated from strain 197 obtained from Y.M.
Saif ~OH~. This strain was found to be virulent for
turkey poults, agglutinized guinea pig red blood cells,
and was identified as B. avium by oxidative alkalinization
of substrates. GOBL124 was grown at 37C for 24 hours on
Bordet-Gengou agar supplemented with 15% defibrinated
sheep blood (BGB agar) prior to the in vitro adherence
assay in order to maximize adhesion (1), or, otherwise, on
brain heart infusion medium (BHI). GOBL124 was the source
2 0 i ~ ~ 7 ~
of chromosomal DNA used in the various vectors. E. coli
strains were grown on LB medium (1% tryptone, 0.5% yeast
extract, 0.5% NaCl, 0.1~ glucose, 1.5% agar). Media were
supplemented with appropriate antibiotics. The suicide
vector pRT733, a derivative of pJM703.1 (29) carrying
TnphoA was provided in its donor E. coli strain SM10-
lambda-pir by J.J. Mekalanos (29). Cosmid pYA2329 was
-
constructed by ligating the 1.8 kb SacII-KpnI digested, T4
DNA polymerase-treated DNA fragment conferring spectino-
mycin resistance from pUCD2 to SalI-digested, Klenow-
treated pCP13.
Animals. Fertile Nicholas turkey eggs were
obtained from Cargill, Inc., Elgin, MO, and fertile White
Leghorn chicken eggs were obtained from SPAFAS, Inc.,
Roanoke, IL. They were incubated in a model no. 21
Humidaire incubator until they hatched.
Reaqents. Reagents were from Sigma, St. Louis,
MO, unless otherwise stated. Bordet-Gengou agar and
reagents for LB and BHI media were from Difco Labora-
tories, Detroit, MI. Sheep blood was from Brown Labora-
tories, Topeka, KS. Restriction enzymes and T4 DNA ligase
were from International Biotechnologies, Inc., New Haven
CT, and were used according to the recommendations of the
manufacturer. Horseradish peroxidase-conjugated goat
anti-turkey immunoglobulin was from ICN Immunochemicals,
Lisle, IL.
Polyclonal Rabbit Sera Contalni-n~LA t_bodies
Aqainst B. avium Outer Membrane Proteins. Polyclonal
rabbit sera was obtained by injecting New Zealand White
rabbits intradermally with purified outer membrane
proteins of B. avium GOBL124. B. avium GOBL124 outer
membrane proteins were prepared according to the method of
Rapp, et al. (49) with some modifications. B. avium
GOBL124 was grown by shaking overnight to a titer of
9.9 x 108/ml in one liter of SSM-S media at 37C. The
cells were pelleted, resuspended in S0 ml PBS, pH 7.5, and
2 ~ 7 ~
-36-
sonicated with a Heat Systems-Vltrasonics Inc. Model W185D
sonifier cell disruptor, for 4 bursts of 5 minutes each at
a setting of 40 Watts with 50% output. The sonicate was
incubated on ice for 5 min. between each burst. The cell
debris was pelleted at 2000 x g for 20 min at 4C. The
supernatant fluid was removed and membranes were pelleted
by centrifugation at 19,000 rpm for one hour at 4C using
an SS34 rotor. 'rhe pellet was resuspended in 5 ml PBS,
pH 7.5 and the protein concentration determined to be
4 mg protein/ml. One volume of 2~ sodium lauryl sarco-
sinate in PBS, pH 7.5 was added to the suspended membranes
to obtain a final concentration of 1% sodium lauryl
sarcosinate. The solution was stirred at room temperature
for 30 minutes and the insoluble outer membrane proteins
15 were pelleted by centrifugation at 19,000 for one hour
using an SS34 rotor. The pelleted proteins were suspended
in two ml of P~S, pH 7.5 and the protein concentration
determined to be 2.5 mg protein/ml.
Rabbits were immunized intradermally with 500 ug
of the purified outer membrane protein (OMP) preparation
in an equal volume of complete Freund's adjuvant. Two
weeks later, the rabbits were boosted intradermally with
500 ug of the OMP preparation in an equal volume of
Freund's incomplete adjuvant. Rabbits were bled for a
small volume of blood and the serum obtained was used for
detection of E. coli recombinant clones which produce
~. avium outer membrane proteins as described below.
Rabbits were periodically boosted with 500 ng of the OMP
preparation in incomplete Freund's adjuvant, bled for
larger volumes of blood (40 ml) and the serum collected.
Serum was either frozen at -20C, or mixed with an equal
volume of 100% glycerol and stored at -20C.
Convalescent Turkey Sera. Four-day-old Nicholas
turkey poults were contact-infected by exposure to a
30-day-old, ~. avium GOBL124 infected turkey (which had
been infected at 2 days of age). The turkey poults were
` _37_ 2~13~
anesthetized and exsanguinated forty days post-exposure to
obtain convalescent sera which was used to identify
E. coli clones which produced proteins reactive with ~he
convalescent turkey sera. Alternatively, antisera against
B. avium was produced by infecting one-day-old turkeys
intranasally with 1.5 x 107 bacteria and collecting blood
1 month postinfection.
Western immunoblots. Whole bacterial cells were
denatured by boiling in sample bu~fer (Tris-HCl 50 mM pH
6.8, glycerol 10%, SDS l~, 2 beta-mercaptoethanol 1%,
Bromophenol Blue 0.01~) and were electrophoresed through
sodium dodecyl sulfate-10~ polyacrylamide gels as
described by Laemmli (24). Gels were transferred to
nitrocellulose using established methods (40). Filters
were saturated with buffer A (50 mM Tris, 150 mM NaCl, 3%
bovine serum albumin, pH 8), incubated with anti-B. avium
sera from convalescing turkeys or antibody against
B. avium outer membrane proteins, washed in buffer A,
incubated wi-th horseradish peroxidase-conjugated goat
anti-turke~ immunoglobulin, and developed with 4, chloro-
1-naphthol.
2. Mutaqenesis of B. avium
TnphoA was mobilized into _ avium by mating
with the _ coli strain SMlO-lambda-pir donor of pRT733 by
the plate method of Bradley et al. (8). After 4 hours,
the bacteria were spread on BHI agar supplemented with
25 ug/ml tetracycline, 150 ug/ml streptomycin, 7~ ug/ml
kanamycin, and the chromogenic substrate for alkaline
phosphatase: 5-bromo-4-chloro-3~indoyl phosphate at
40 mg/ml. After 5 days at 37C, blue colonies of B. avium
were collected. Since alkaline phosphatase activity can
be expressed only when the enzyme is located in the outer
membrane or in the periplasm of the cell, and since the
promoter and the signal sequence of the phoA gene are
missing in TnE~, blue colonies are ~hose in which the
2~13~7~
-38-
transposition and the fusion of ~ have occurred in
frame with a promoter and a signal sequence of an exported
protein of 3. avium.
About 0.2~ of the transconjugants were blue,
thus having phoA a~socia~ed with a promoter and a signal
sequence for a gene encoding a protein localized either in
the other membrane or in the periplasm of the bacteria.
A total of 487 blue clorles were collected after several
independent experiments.
3 In Vitro Assay for the B. avium Adhesion to
.
Tracheal Cells
.
Since B. avium adhesion is very specific to
ciliated epithelial cells (2), TnphoA-induced mutant~ were
screened for their ability to attach to these cells using
the following assay. Tracheas were aseptically removed
from one-week-old chickens or turkeys and cut in 2 mm
length rings. 5 x 10 B. avium bacteria grown on BGB agar
and suspended in 1 ml of buffer B (150 mM NaCl, 2.5 mM
KCl, 10 mM Na2HPO4 pH 7.4) were incubated for 30 minutes
at 3C with the tracheal rings. The rings were then
washed 5 times with buffer B to remove nonadherent
bacteria. The adherent bacteria were then recovered by
incubating the tracheal rings for 5 minutes in 100 ul of
1% Triton X-100 in buffer B at 37C on a rotating wheel
(30 rpm). Serial dilutions of the suspended bacteria were
then plated in BHI agar and incubated 24 hours at 37C to
determine the number of adherent bacteria. The assays
were made in triplicate for each strain.
4. Coloniæation in Vivo
The parental strain and the nonadherent mutants
selected by the above method were also tested in vivo by
inoculating five one-day-old turkeys intranasally with
either 1.5 x 10 or 1.5 x 10 colony-forming units (CFU)
with each of the strains, respectively. Two weeks later,
2~13~
-39-
the animals were killed by CO2 asphyxia-tion, the tracheas
aseptically removed and homogenized in 3 ml of buffer B
with an OMNI-mixer, DuPont, Wilmington, DE. Dilutions
were then plated on BH agar containing 25 ug/ml tetra-
cycline ancl 150 ug/ml streptomycin.
Results are given in Table 1 for the adhesion to
isolated tracheal rings of the wild--type strain and four
nonadherent (Adh ) mutants named STL 6, 167, 258, and 389.
The other Tn~ -generated mutants showed the same adhe-
sion efficiency as the wild-type stxain. Table l also
gives the results of infection of turkeys with those same
strains. Although no mutants were reisolatable from the
turkeys when 1.5 x 107 CFU were given to the turkeys,
bacteria were reisolated from all but one mutant when the
original inoculum was 1.5 x 10 CFU. These recovered
bacteria wexe as adherent as the wild-type strain in the
in vitro assay for adherence to tracheal rings.
-40- 201 3571
Table 1
Adherence and colonization b
wild-type and Tnpho~-induced ~. avium mutants
CFU (log) RECOVERED FROM TRACHEA
STRAIN PHENOTYPE ADHESION TO 2 WEEXS AFTER INOCULATIONb
NUMBER TRACHE~L RINGSa
1.5 x 107 1.5 x 109
inoculum inoculum
. . _ . . .
GO~L124 Wilcl-type 100 8.4+0.5 8.7~0.3
STL6 Adh-C 10.1+1 NDd ND
STL167 Adh 7.6+1.7 NDd 8.5+0.4
STL258 Adh 8.8~1.4 NDd 3~7~0.3e
STL389 Adh 0 9~0'3 NDd 8.0~0.3e
.
aSx109 CFU in phosphate buffer were incubated 30 minutes at 37C
with tracheal rings from one-week-old turkeys. After washes, the
bacteria still attached were recovered by incubating the trachea
with 1% Triton X-100. Measurements were made in triplicate.
bTracheas removed, crushed, and bacteria enumera-ted.
CAdh : nonadherent phenotype
dND : none detected.
eBacteria recovered from turkeys were proficient in colonizing
tracheal rings.
5. Electron Microscopv
The mutant and wild-type bacteria were examined
for pili using electron microscopy. A drop of ~HI-grown
bacteria was applied to a 300-mesh Formvar-coated copper
grid and allowed to stand for 1 minute. The grid was
washed with 3 drops of water, the excess water removed
with filter paper, and the grid air dried. The grid was
then covered with 2% uranyl acetate (Urac) for 30 seconds
and then rinsed with 0.2% Urac. Excess fluid was removed
with filter paper and the grid air dried. The bacterial
preparations were examined with a Hitachi H-600 trans-
-41- 2 Q ~
mission electron microscope. The four nonadherent mutants
and the wild-type strain looked e~actly alike. More
precisely, pili and flagella can easily be seen as shown
in Figure 5 for mutant STL258. This indicates -that the
inactivated protein is probably not the pilin.
6. Identiflcation of the 46 kDa Outer Membrane
Adhesl _ Protein
a. Cloninq of the DNA sequences flankinq TnphoA
insertions. TnphoA insertions tag a gene with kanamycin
resistance. Thus, kanamycin resistance can be used to
select clones containing the TnphoA insert. Chromosomal
DNA from B. avium TnphoA-induced mutants was partially
digested with Sau3A and the fragments separated by size on
an agarose gel. Since TnphoA is about 7.5 kilobases (kb)
in length, cloning larger fragments insures that the
surrounding _ avium DNA is include~ in clones -that are
selected for kanamycin resistance. Fragments in the size
range of 10 to 20 kb were cut from the gel, recovered from
the agarose by electroelution, and ligated to BamHI-
digested plasmid pACYC184 (59) using standard methods.
E. coli HB101 was transformed wi-th these ligations and
kanamycin-resistant colonies selected.
b. Construction of a cosmid librarv of wild-
_ _ _ _
tYpe B. avium. Chromosomal DNA from Bo avium GOBL124 was
partially digested with XhoI, and separated by size on a
10 to 40~ sucrose gradient. Fragments in the size range
of 13 to 27 kb were then ligated with cosmid pCP13 (14)
cut with XhoI. pCPl3 is a broad-host-range cosmid cloning
vector with a relatively low copy number of 5-8 per
chromosome. The resulting ligation was packaged into
phage particles by using lambda n vitro packaging
extracts (Packagene~, Promega Biotec, Madison, WI). The
packaged cosmids were adsorbed to E. coli LE3~2 for
20 minutes at 37C, LB broth was added, and incubation was
continued for ~5 minutes before the bacterial culture was
2~13~7~
-42-
spread on LB plates supplemented with 15 ug/ml tetra-
cycline.
Plasmids containing the DNA isolated from the
mutants were 32P-labeled by nick-translation with an in
vitro nick-translatlon kit, Bethesda Research Labora-
tories, Galthersburg, MD, and used as probes in ln situ
colony hybridization assays with the library to detect
_ coli clones containing gene inserts derived from the
wild-type strain. Specifically, four probes obtained by
cloning the TnphoA mutated sequences from each mutant in
pACYC1~4 were used to hybridlze with the cosmid library o~
wild-type B. avium ln E. coli. The four probes reacted
with the same cosmid clones. All these cosmids shared a
common 17.~ kb XhoI DNA fragmen-t which map is depicted in
Figure 1. One of these pCP13 clones was designated
pYA2402.
c. Identification of the protein missing in
Adh mutants. To identify the adhesion protein missing
from the nonadherent mutants, immunoblot analysis was
performed on bacteri~l lysates from the mutant and
parental strains by the method described above. A
46-kilodalton (kDa) protein produced by the wild-type
B. avium strain is clearly missing in the four mutants as
shown in Figure 3. On the other hand, Figure 4 shows that
this protein is expressed in E. coli LE392 carrying the
relevant cosmid clones isolated from the library, speci-
fied by plasmid pYA2402.
The DNA from Bordetella species share a high
degree of homology. Furthermore, avian rhinotracheitis
caused by B. avium is physiopathologically similar to
whooping cough caused in humans by B. pertussis and
B. parapertussis, as well as porcine atrophic rhino-
tracheitis caused by B. bronchisep~ica. Therefore, it is
likely that all these species possess a virulencs factor
identical or similar to the 46 kDa adhesion protein
identified in B. avium.
2~13~
-43-
Genes coding for -this factor, or other related
antigenq, can be investigated in other Bordetella species
using Southern blot hybridizations of their genomic DNAs
using an internal portion of the adhesion gene cloned from
B. avium. Immune serum raised aga.inst the B. avium adhe-
sion, as described above, can be used in Western
i~nunoblots to assess the production of a related protein.
If similar adhesions are found, the presence of antibodies
to these proteins can be examined in sera from conva-
lescing humans who have had whooping cough and from swinewhich have had atrophic rhinitis. These antigens can be
used in vaccine compositions to confer immunity to the
various Bordetella species.
7. Identification Proteins Which React With Antlbody
.
Aaainst B. avium Outer Membrane Proteins
_
a. Construction of B. avium Gene Libraries in
E. coli. Chromosomal DNA was isolated from B. avium
GOBL124 by the method of Hull et al. (50). B. avium
GOBL124 DNA was partially digested with either Sau3a,
HindIII, or XhoI, slze-fractionated by sucrose gradient
centrifugation, and 25~27 kb Sau3a, HindIII, or XhoI-
generated DNA fragments were selected for ligation. The
size-fractionated, Sau3a-digested B. avium DNA was ligated
to BamHI-digested cosmid pY~2329 DNA, while the size-
fractionated, HindIII and XhoI-digested B. avium DNA was
ligated to HindIII and XhoI-digested pCP13 DNA, respec-
tively. The ligated DNA was packaged into Packagene
lambda-head and tail proteins supplied by Promega, and
transfected into E. coli LE392 using the Promega protocol.
Recombinant E. coli clones were plated onto LB agar
containing either 50 ug/ml tetracycline (to select for
recombinant cosmids with the pCP13 vector) or 50 ug/ml
spectinomycin (to select for recombinant cosmids with the
pYA2329 vector).
_44_ 2~3~7~
b. Identification of E. coli Recombinant Cosmid
Clones Which Produce Proteins Which React With AntibodY
Aqainst B. avium Outer Membrane Proteins and Convalescent
Turkey Sera. Recombinant E. coli clones were tested for
reactivity with antibody against B. avium outer membrane
proteins and by reaction with convalescent sera from
B. avium-infected turkeys by the colony immunoblot assay.
Briefly, recombinant E. coll clones were plated onto LB
agar containing 50 ug/ml tetracycline. Colonies were
blotted with nitrocellulose filter paper, lysed by
exposure to chloroform fumes, and reacted for 4 hours with
rabbit antibody against B. avium outer membrane proteins.
After washing to remove residual primary antibody, the
filter paper containlng the lysed recombinant clones was
reacted for 4-5 hours with horseradish peroxidase labeled-
goat-anti-rabbit IgG (affinity purified) from ICN.
Following incubation, the nitrocellulose filters were
developed with 4-chloro-1-napthol substrate and the
results were photographed.
An identical protocol was used for identifi-
cation of E. coli recombinant clones which produce
B. avium proteins which react with convalescent turkey
sera, except that convalescent turkey sera was used as the
primary antibody and horseradish peroxidase-labeled-goat-
antiturkey IgG antibody from Kappel was used as the
secondary antibody.
c. Western Immunoblot of B. avium Proteins
Produced by E. coli Recombinant Clones. After initial
identification of E. coli LE392 clones by colony
immunoblot assays, the positive E. coli clones were
analyzed by SDS-polyacrylamide electrophoresis of boiled
whole cell protains by electrotransfer to nitrGcellulose
paper and Western immunoblot analysis as described in
Materials and Methods. Five recombinant cosmid clones
were identified by reactivity with antibody against
B. avium outer membrane proteins (Figure 6). One of these
2 ~
-45-
E. coli LE392 clones was isolated from the XhoI-generated
cosmid library. This clone produced a 21 kDa protein, and
the plasmid was designated pYA2320. Recombinant clone
pY~2326 was isolated from the HindIII-generated cosmid
library and specified a 40 kDa protein. Three recombinant
clones, designated pYA2337, pY~2338, and pYA2339, were
isolated from the Sa-l3a-generated cosmid library and
expressed in LE392, 37 kDa, 43 kDa, and 46 kDa proteins,
respectively.
One recombinant cosmid clone f.rom the Sau3a-
generated library was identified that expressed a 50 kDa
protein in LE392 that reacted with convalescent turkey
sera and was designated pYA2333 (Figure 7).
These proteins can be further tested for
adhesion to tracheal cells as described in Example C.3 to
determine whether they are o~ter membrane adhesion
proteins.
d. Characterization of Cosmid Clone pYA2320.
pYA2320 DNA was isolated, di~ested Wit]l BamHI-PstI,
ligated to BamHI-PstI digested pYA2329, and the recom-
binants transformed into E. coli LE392. Transformants
were screened by colony immunoblot assay with antibody
against B. avium OMPs to identify the E. coli subclone
which produced the 21 kDa protein. One transformant was
identified by the colony immunoblot assay and restriction
endonuclease analysis revealed that this recombinant
clone, designated pYA2332, contained a 6 kb BamHI-PstI DNA
fragment. Western immunoblots of whole cell proteins from
LE392 which contained pYA2332 indicated that this clone
30 produced the 21 kDa prot~in. This information indicated
that the 6 kb BamHI-PstI DNA fragment encodes the entire
gene for the 21 kDa protein and suggests that expression
of the gene in E. coli is directed by the promo-ter of the
cloned gene. The 6 kb BamHI-PstI fragment was llgated to
similarly digested pUC8-1 DNA and transformed into E. coli
JM109 for restriction endonuclease mapping and fur-ther
2 0 1 3 ~ r~ ~
-46-
manipulation. This E. coli clone was designated pYA2340.
Restriction endonuclease mapping of the plasmid DNA from
p~A2340 was performed as described in Maniatis et al.,
supra.
8. Maintenance of E. coli Recombinant Clones
_
E. col_ recombinant clones which produced
B. avium proteins identified by reactivity with antibody
against B. avium outer membrane proteins or by reactivity
10 with convalescent sera from B. avium outer membrane
proteins were ~tored at -70C as follows. E. coli clones
were grown at 37C overnight in LB containing the appro-
priate antibiotic, mixed with an equal volume of 80%
glycerol in water, and frozen at -70C.
9. Production of Recombinant vaccine Strains O.e
Salmonella
Salmonella spp. can be rendered avirulent by the
deletion of ~y~ and crp genes which code for adenylate
cyclase and cAMP receptor protein, respectively. rrhese
two proteins are necessary for the transcription of a
large number of genes and operons concerned with the
transport and breakdown of a large number of catabolites.
Furthermore, the gene coding for beta-aspartic semi-
aldehyde dehydrogenase (the asd gene) can be deleted.This enzyme is used in the synthesis of diaminopimelic
acid (DAP), a constituent of bacterial cell walls.
Mutants that fail to synthesize DAP cannot survive in DAP-
less media. Several Salmonella strains have been
developed containing these deletion mutations. Particu-
larly useful are strains chi4064 and chi4072, both
described in Curtiss and Kelly (13) and in cope~ding
application serial no. 251,304. Chi4064 has been shown to
be avirulent for one-day-old chickens and is able to
35 colonize the intestinal wall for several weeks.
2~13~
-~7-
Such strains colonizing the GALT havs also been
shown to give rise to a systemic immune response, produc-
ing secretory immunoglobulin A (SIgA), at several mucosal
surfaces, directed against the cloned antigen.
In order to introduce the B. avium adhesion
antigens into an avirulent Salmonella strain, the antigen
can be cloned into the Asd vector, pYA292, using standard
techniques. This vector is described in copending patent
application serial no. 251,304 and illustrated in Figure
2. This vector can be introduced into the avirulent
strain chi4072 described.
10. C truction of a Recombinant Vaccine Strain of
Salmonella Expressinq the 21 kDa Antiqen
The 6 kb BamHI-PstI B. avium DNA fragment from
pYA2332 was ligated to BamHI-PstI-digested pYA292 and
transformed into E. coli chi6097. The resulting
transformant, containing a recombinant plasmid designated
pYA2336 (Figure 8), was found to produce the 21 kDa
protein when analyzed by Western immunoblot analysis with
antibody against B. avium OMPs (Figure 9). Isolated
pYA2336 DNA was transformed into Salmonella typhimurium
chi3730. The resulting transformant, designated chi3730
(pYA2336) was also found to produce the 21 kDa protein
when analyzed by Western immunoblot analysis, as described
above. A bacteriophage P22 HTint lysate was produced by
infecting lo~-phase chi3730 ~pYA2336) with bacteriophage
P22 HIint at a multiplicity of infection 0.01 as described
in Davisr Botstein and Roth, Advanced Bacterial Genetics
(Cold Spring Harbor Laboratories). After growth for 12-15
hours, chloroform lysis of the infected bacteriar and
centrifugation to remove cellular debris, the
bacteriophage lysate was harvested and titered. S.
typhimurium chi3987 was transduced with the P22 HIint
lysate generated above by infection of log-phase cells
with an m.o.i. of 0.5, and subsequent selection of
2~13~
-48-
transductants on LB agar. Transductants were found to
produce the 21 kDa protein when analyzed by Western
immunoblot analysis with antibody against B. avium OMPs
(Figure 9).
11. Immunization of Chickens and Turkey~ Using
Recombinant Salmo ella Strains
The recombinant Salmonella strains described in
-
C.9 and C.10 can be used to confer immunity in chickens
and turkeys as follows. One- to three-day-old poults are
immunized perorally and intranasally by including the
recombinant avirulent microbe in drinking water. Serum
and respiratory secretions are monitored to determine the
type [IgY (IgG, 7S Ig), IgM and IgA (IgB)] and quantity of
the antibody response. Immunized and nonimmunized chicken
and turkey poults are compared for weight loss to monitor
possible side-effects of the vaccine. In addition,
chickens and turkey poults are challenged by intranasal
inoculation with 10 virulent B. avium or by exposure to
infected birds. Challenged chicken and turkey poults are
observed for disease, necropsied, and examined for
tracheal tissue damage and changes in titers of the
infecting challenged organism.
If a mucosal immune response against B. avium is
inadequate to protect very young chickens and turkeys,
breeding hens can be immuniæed with avirulent Salmonella
expressing B. avium outer membrane antigens to permit egg
transfer of maternal antibodies. Such treatment would
augment immunity to B. avium while the young chicks or
turkey poults are maturing. A protective peroral immuni-
zation employing the same recombinant carrier may also be
administered during maturation.
Thus, Bordetella outer membrane antigens,
vaccines containing these antigens and methods of adminis-
tering the same are disclosed. Although preferredembodiments of the subject invention have been described
_49- 2~3~7~
in some detail, it is understood that obvious variations
can be made without departing rom the spirit and the
scope of the invention as deflned by the appended claims.
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