AU5432690A - Bordetella vaccines - Google Patents

Description

BORDETELLA VACCINES

Technical Field

The present invention relates generally to vaccine 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.

Background of the Invention

The genus Bordetella is composed of four species, B. pertussis, B. parapertussis, B. bronchiseptica, 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 infection, birds present with depression, loss of appetite, weight loss, snicking (coughing), moist rales, ucous accumulation in the external nares, dyspnea, and vocal alterations (3, 18, 21, 34, 37). Younger birds are highly susceptible to infection while older birds are refractory (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 tropis for the ciliated tracheal epithelial cells (2, 41, 5). 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 fimbriae) , 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 of cells in _in vitro cell assays (42). However, FHA~ cells bind well to HeLa cells ₍46₎ and B^ pertussis mutants which lack filamentous hemagglu- tinin do not exhibit decreased virulence as compared with wild-type virulent B _ pertussis when infected intranasally into infant mice (45). Furthermore, B^ bronchiseptica, B. parapertussis, and B^ avium do not produce pertussis toxin (20), and B^ avium and most strains of B. bronchiseptica do not produce FHA.

There is also controversy over the role of the agglutinogens in adherence of Bordetella. Aτjglutinogens 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_^ pertussis to HeLa cells (35, 32). Conversely, B pertussis 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. aviu . 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 involved 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. Transposon mutagenesis with TnphoA can be used to identify bacterial cell surface proteins. Transposition 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 wild-type protein (27). Therefore, when TnphoA is inserted into a gene for an outer membrane protein, a periplasmic protein, or a secreted protein, alkaline phosphatase activity is 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 containing the chromo- genie 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 into 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 necessarily adhesins, might also be useful for treating or preventing Bordetella infections. Specifically, these proteins, or antibodies thereto, might block attachment of colonization antigens to their receptors and thereby prevent infection. Bordetella outer membrane proteins and putative adhesion antigens can also be identified, as described herein, using antibodies raised against crude extracts of Bordetella outer membrane proteins. These antibodies can be used to screen gene libraries to identify clones which produce proteins reactive with the same. Avirulent 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, 1988, and in Curtiss and Kelly (13), the disclosures of which are hereby incorporated by reference. Representative are mutants of Salmonella spp. which carry deletion mutations that impair the ability of the bacterium to synthesize adenylate cyclase (ATP pyrophosphate lyase (cyclizing) EC 4.6.1.1) (cya) and the cyclic AMP receptor 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 shape 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 the desired antigen will survive. The use of carrier avirulent microbes to administer an outer membrane antigen of Bordetella species could result in an effective vaccine against Bordetella-induced diseases . -6-

Disclosure 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 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 is derived from B^ aviu . 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, the 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 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 polyacrylamide gel electrophoresis and Western immunoblot analysis. These proteins are expressed in an avirulent derivative of S. typhimurium which is substantially incapable of producing functional adenylate cyclase, functional cyclic AMP receptor protein, and functional beta-aspartic semialde ye 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 method 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 expression of a Bordetella sp. outer membrane protein comprising an aviru- lent derivative of a pathogenic microbe. The derivative is substantially incapable of producing functional adeny- late cyclase 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 46 kDa comprising an avirulent derivative of S _ typhimurium. The avirulent-'derivative is 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 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 S^ 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 fragment thereof 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 (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 further 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 Figures Figure 1 depicts the restriction map of the Xhol

DNA fragment cloned into the Xhol restriction site of pYA2402. B=BamHI; Bq=-BqlII; Cla-Clal; E = EcoRI; H=HindIII; Sal=SalI; S = Sstl; P = Pstl; X = Xhol; Xb=XbaI; ///Λ =DNA from B^ avium GOBL124; I 1= fragment cut from pYA2402 and ligation with pCP13 cut with Bglll. 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 that did not react with probes isolated from the nonad- herent mutants. Lane C shows the profile of LE392 with cos id pYA2402. Lanes D, E and F depict the protein profiles of LE392 strains with recombinant 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 surrounding the bacteria and the arrow indicates part of a flagellu (magnification x 30,000).

Figure 6 depicts the results of an immunoblot analysis performed on bacterial 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 with 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 Pst_l 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 LE392 with cosmid pYA2320. Lanes 3 and 4 show the profile of E__j_ coli chi6097 and S_ typhimurium chi3987, respectively, both carrying pYA2336. Lanes 5 and 6 depict the profile of E_^ coli chi6097 and S_ typhimurium chi3987, respectively, both carrying pYA292.

Detailed Description of the Invention

The practice of the present invention will employ, unless otherwise indicated, conventional tech- niques of cell culture, molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g. , Maniatis, et al. , Molecular Cloning: A Laboratory Manual (1982); DNA Cloning (1985) 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. Rodriguez, 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. The term "epitope" refers to the site on an antigen or hapten to which a specific antibody molecule binds. The term is also used interchangeably with "antigenic determinant" or "antigenic determinant site." An epitope will normally include 3 amino acids necessary for recognition in spatial confirmation, more usually 5 amino acids, and most usually 8-10 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 phrase "immune 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 the 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 immunoglobulins A, D, E, G or M. Of particular interest are vaccines which stimulate production of immunoglobulin A (IgA) since this - 13-

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 Barrett, James T. , Textbook of Immunology: 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 production of secretory IgA (SlgA). (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.

By "avirulent derivative of a microbe" is meant an organism which is 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 species that is normally pathogenic but must belong to a strain that is avirulent. By "pathogenic" 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 with its virulent pathogenic counter¬ part. The term "microbe" as used herein includes bacteria, protozoa, and unicellular fungi.

A "carrier microbe" is an avirulent microbe as defined above which contains and expresses a recombinant gene encoding a protein of interest such as an outer membrane adhesion antigen, or outer membrane protein from Bordetella sp.

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 46 kDa protein, and a 50 kDa protein, as determined by SDS gel electrophoresis and described further below. Outer membrane adhesion antigens are by definition also outer membrane proteins. Outer membrane proteins may or may not be adhesion antigens. Antibodies 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. 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 other cellular components and proteins, and thus purified, when at least 30% by weight of the total A + B present 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 organism 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) linked through peptide bonds. Thus, the term "polypeptide" 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 cells transformed by an exogenous DNA construct encoding the desired polypeptide.

A "recombinant gene" is an identifiable segment of polynucleotide within a larger polynucleotide molecule that is not found in association with the larger molecule in nature.

A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autono- mous unit of DNA replication i_^n vivo; i.e., capable of replication under its own control. •16-

A "vector" is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. A DNA "coding sequence" is a DNA sequence which is transcribed and translated into a polypeptide n 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. A transcription termination sequence will usually be located 3' to the coding 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. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA 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 sites, polyadenyl- ation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the transcription and translation of a coding sequence in a host cell.

A 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 coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence. "Recombinant host cells", "host cells", "cells" and other such terms denoting microorganisms are used interchangeably, 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 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 accidental 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 related organism in order to identify the clone within the library which represents the desired σene. 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 found 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 DNA, 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 B__;_ avium 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 provides a method for identifying the antigens of the present invention. TnphoA can be introduced into Bordetella cells by mating with an E_^ coli donor as described in detail below. When TnphoA 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 protein transported across the cytoplasmic membrane. Alkaline phosphatase is normally absent from Bordetella spp. 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-p_- toluidine phosphate (XP), which is incorporated into the medium. The mutant strains can be screened lor their ability to attach to tracheal epithelial cells in jln vitro assays using 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 digested using conventional restriction enzymes. The restriction fragments can be separated using agarose gels . Since TnphoA 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 mutants 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 the outer membrane adhesion proteins from the various Bordetella species share a high degree of homology. Thus, a plasmid containing a Tnpho_A fragment bearing a gene encoding for one such protein will likely be useful for screening other Bordetella species -20-

for similar adhesion proteins. Furthermore, polyclonal antibodies (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.

Additionally, crude preparations of Bordetella outer membrane proteins can be used to raise antibodies which can in turn be used for recombinant expression screening. Thus, clones expressing proteins reactive with these antibodies can be identified and these proteins further characterized 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 of the various methods known to those skilled in the art. For example, the amino acid sequences of the subject 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 determined 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.g. , 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 oliσonucleotides are synthesized by any appropriate 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 preparation of large families of probes relatively straightforward. While the exact length of the probe employed is not critical, generally it is recognized in the art that probes from about 14 to about 20 base pairs are usually effective. Longer probes of about 25 to about 60 base pairs are also used.

The selected oligonucleotide probes are labeled with a marker, such as a radionucleotide or biσtin 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 ssDN_A 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 /12086

-22-

evolutionarily close or distant species. The selection of the appropriate conditions is within the skill of the art. See generally. Nucleic Acid Hybridization, supra. The basic requirement is that hybridization conditions be of sufficient stringency so that selective hybridization occurs; i.e., hybridization is due to a sufficient degree of nucleic acid homology (e.g., at least about 75%), as opposed to nonspecific binding. Once a clone from the screened library has been identified by positive hybridi- zation, it can be confirmed by restriction enzyme analysis and DNA sequencing"that the particular library insert contains a gene for the desired protein.

Alternatively, 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.g., Edge, Nature (1981) 292:756; Na bair 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 for cloning and host cells which they can transform include the bacteriophage lambda (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGVUOβ (gram-negative bacteria), pLAFRl (gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria), pHV14 (E__;_ coli and Bacillus subtilis) , pBD9 (Bacillus) , pIJ61 (Streptomyces) , pUC6 (Streptomyces) , YIp5 fSaccharomvces) , YCpl9 (Saccharo- myces) and bovine papilloma virus (mammalian cells). See, generally, 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 promoter, ribosome binding 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 containing this expression construction. 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 known promoters that function in gram negative bacteria such as the tac or trp promoters . The outer membrane antigens, when present in a carrier microbe, will normally be expressed under the control of a promoter that only allows expression .in vivo in the immunized host. However, if production of the protein is desired in bulk, outside of the intended recipient, in addition to control sequences, 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 may also 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 fusion protein. See, e.g. , U.S. Patent Nos . 4,431,739; 4,425,437. 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 transcribed 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 of the sequences encoding the particular antigen 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 di±ectly 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 secretory signal, if any. Leader sequences can be removed by the bacterial host in post-translational processing. See, e.g. , U.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 sequences, such as site-directed mutagenesis, are well known to those skilled in the art. See, e.g. , Maniatis, T. , et al. , supra; DNA Cloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra. A number of procaryotic expression vectors are known in the art. See, e.g. , U.S. Patent Nos. 4,440,859; 4,436,815; 4,431,740; 4,431,739; 4,428,941; 4,425,437; 4,418,149; 4,411,994; 4,366,246; 4,342,832; see also U.K. Patent Applications GB 2,121,054; GB 2,008,123; GB 2,007,675; 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 expressed. The particular

Bordetella protein is then isolated from the host cells and purified. If the expression 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 of the art.

The antigens of the present invention can also be isolated from Bordetella cultures using standard protein purification procedures well known in the art. See, e.g., 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- phσretic techniques, centrif gation, dialysis, and precipitation. 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. Chemical 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 fragments 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 fragment, 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 of 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 techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfeetion with Epstein-Barr virus. See, e.g. , 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 of monoclonal antibodies produced against the antiαen of interest, or fragment thereof, can be screened for various properties; i.e., for isotype or epitope affinity, etc. Monoclonal 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 generalized secretory immune response as well as humoral and cellular immune responses as described above.

Recombinant plasmids containing genes for the subject 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 adhesion 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. Expression of the recombinant gene encoding the desired antigen may be 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, 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 genus Salmonella such as S_^ typhimurium, S. typhi, S^ paratyphi, S. gallinarum, 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 whereas

S. typhimurium, S. typhi and S^ paratyphi 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 administration, 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 via the 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. 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. I_n 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 periplaεm without lysis is also possible. Alternatively, 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 the 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 Bordetella 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 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 heat 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 free amino groups of the active polypeptides ) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic 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 orqanic bases as isopropylamine, trimethyla ine, 2-ethylamino ethanol, histidine, procaine, and the like. Typically, the antigens, when used without an avirulent carrier, are administered as aerosols or intranasally. Intranasal formulations for 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 subject proteins by the nasal mucosa.

Injection with a pathogen-derived gene product can also be used in conjunction with prior oral, intra- nasal, gastric or aerosol immunization. Such parenteral immunization 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 BALT. 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 injectables, 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 be emulsified 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 , _nor /12086

-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. Adjuvants may include for example, mura yl 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, e.g. , 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 subject is immunized by administration of the parti¬ cular antigen or fragment 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 10 -1 x 10 recombinant avirulent bacteria/ immunized subject. Initial doses of vaccine using the protein without the avirulent microbe could be 0.001-1 mg antigen/kg body weight. Moreover, the subject may be administered increasing amounts or multiple dosages as required to maintain a state of immunity to Bordetella

^SPP-

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

Deposits of Strains Useful in Practicing 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 one determined by the Commis¬ sioner to be entitled thereto under 37 CFR 1.14 and 35 USC 122. All restriction on availability of said cultures to the public will 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 request 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 description.

-34-

Strain Deposit Date ATCC 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 67.921 pYA2336 in chi3987 March 22, 1990 pYA2326 in LE392 March 22, 1990 pYA2333 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 , Media 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 37°C 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 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-KpnT digested, T4 DNA polymerase-treated DNA fragment conferring spectino- ycin resistance from pUCD2 to Sail-digested, Klenow- treated pCPl3.

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.

Reagents . 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 Containing Antibodies Against 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 37°C. The cells were pelleted, resuspended in 50 ml PBS, pH 7.5, and sonicated with a Heat Systems-Ultrasonics 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 4 C. The supernatant fluid was removed and membranes were pelleted by centrifugation at 19,000 rpm for one hour at 4 C using an SS34 rotor. The 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 were pelleted by centrifugation at 19,000 for one hour using an SS34 rotor. The pelleted proteins were suspended in two ml of PBS, 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 B. 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 -20 C, or mixed with an equal volume of 100% glycerol and stored at -20°C.

Convalescent Turkey Sera. Four-day-old Nicholas turkey poults were contact-in ected by exposure to a 30-day-old, B^ avium GOBL124 infected turkey (which had been infected at 2 days of age) . The turkey poults were anesthetized and exsanguinated forty days post-exposure to obtain convalescent sera which was used to identify

E. coli clones which produced proteins reactive with the convalescent turkey sera. Alternatively, antisera against B. avium was produced by infecting one-day-old turkeys

7 intranasally with 1.5 x 10 bacteria and collecting blood

1 month poεtinfection.

Western immunoblots. Whole bacterial cells were denatured by boiling in sample buffer (Tris-HCl 50 mM pH 6.8, glycerol 10%, SDS 1%, 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 with horseradish peroxidase-conjugated goat anti-turkey immunoglobulin, and developed with 4, chloro- 1-naphthol.

2 • Mutagenesis of B. avium

TnphoA was mobilized into B__;_ avium by mating with the E _ 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, 75 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 37°C, 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 periplas of the cell, and since the promoter and the signal sequence of the phoA gene are missing in TnphoA, blue colonies are those in which the transposition and the fusion of phoA have occurred in frame with a promoter and a signal sequence of an exported protein of B^ avium.

About 0.2% of the transconjugants were blue, thus having phoA associated 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 clones 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), TnpjhoA-induced mutants 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

9 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 Na₂HP0₄ pH 7.4) were incubated for 30 minutes at 3 C 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 37°C on a rotating wheel

(30 rpm). Serial dilutions of the suspended bacteria were then plated in BHI agar and incubated 24 hours at 37°C to determine the number of adherent bacteria. The assays were made in triplicate for each strain.

4. Colonization 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

7 9 either 1.5 x 10 or 1.5 x 10 colony- orming units (C_FU₎ with each of the strains, respectively. Two weeks la_ter, the animals were killed by CO,, asphyxiation, the tracheas aseptically removed and homogenized in 3 ml of buffer B with an OMNI- ixer, DuPont, Wilmington, DE. Dilutions were then plated on BH agar containing 25 ug/ml tetra- cycline and 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 TnphoA-generated mutants showed the same adhe- sion efficiency as the wild-type strain. Table 1 also gives the results of infection of turkeys with those same strains. Although no mutants were reisolatable from the

7 turkeys when 1.5 x 10 CFU were given to the turkeys, bacteria were reisolated from all but one mutant when the

9 original inoculum was 1.5 x 10 CFU. These recovered bacteria were as adherent as the wild-type strain in the in vitro assay for adherence to tracheal rings .

Table 1

Adherence and colonization by wild-type . and TnphoA-induced B. avium mui ants

CFU (log) RECOVERED FROM TRACH

5 STRAIN PHENOTYPE ADHESION TO 2 WEEKS AFTER INOCULATION"

NUMBER TRACHEAL RINGS³

1.5 x 10' 1.5 x 10⁹ inoculum inoculum

GOBL124 Wild-type 100 8.4+0.5 8.7+0.3

0 STL6 Adh~^C 10.1+1 ND^d ND

STL167 Adh^" 7.6+1.7 ND^{d ■} 8.5+0.4^® ΞTL258 Adh^" 8.8+1.4 ND^d 3.7+0.3^® STL389 Adh^" 0.9+0.3 ND^d 8.0+0.3^®

15 _a g

5x10 CFU in phosphate buffer were incubated 30 minutes at 37 with tracheal rings from one-week-old turkeys. After washes, t bacteria still attached were recovered by incubating the trache with 1% Triton X-100. Measurements were made in triplicate.

Tracheas removed, crushed, and bacteria enumerated. : nonadherent phenotype

ND : none detected.

Bacteria recovered from turkeys were proficient in colonizing tracheal rings.

25

5. Electron Microscopy

The mutant and wild-type bacteria were examined for pili using electron microscopy. A drop of BHI-grown bacteria was applied to a 300-mesh Formvar-coated copper

30 grid and allowed to stand for 1 minute. The grid was washed with 3 drops of water, the excess water remove_d 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

35 with filter paper and the grid air dried. The bac_terial preparations were examined with a Hitachi H-600 trans- -41-

mission electron microscope. The four nonadherent mutants and the wild-type strain looked exactly 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. Identification of the 46 kDa Outer Membrane Adhesion Protein a. Cloning of the DNA sequences flanking 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 B_ avium DNA is included 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 Ba HI- digested plasmid pACYC184 (59) using standard methods. E . coli HB101 was transformed with these ligations and kana ycin-resistant colonies selected. b. Construction of a cos id library of wild- type B. avium. Chromosomal DNA from B^ avium GOBL124 was partially digested with Xhol, 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 Xhol . pCP13 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 i_n vitro packaging extracts (Packagene^w , Promega Biotec, Madison, WI ₎ . The packaged cosmids were adsorbed to E__;_ coli LE392 for 20 minutes at 37°C, LB broth was added, and incubation was continued for 45 minutes before the bacterial culture was 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 n vitro nick-translation kit, Bethesda Research Labora¬ tories, Gaithersburg, MD, and used as probes in n situ colony hybridization assays with the library to detect E. 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 pACYC184 ere used to hybridize with the cosmid library of wild-type B__;_ avium in E^ coli. The four probes reacted with the same cosmid clones. All these cosmids shared a common 17.6 kb Xhol DNA fragment 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 bacterial 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^ bronchiseptic . Therefore, it is likely that all these species possess a virulence factor identical or similar to the 46 kDa adhesion pro_tein identified in B. avium. Genes coding for this factor, or other related antigens, 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 J _L avium. Immune serum raised against the B^ avium adhe¬ sion, as described above, can be used in Western immunoblots 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 swine which 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 Antibody Against 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,

Hindlll, or Xhol, size-fractionated by sucrose gradient centrifugation, and 25-27 kb Sau3a, Hindlll , or Xhol- generated DNA fragments were selected for ligation. The size-fractionated, Sau3a-digested B. avium DNA was ligated to BamHI-digested cosmid pYA2329 DNA, while the size- fractionated, Hindlll and Xhol-digested B. avium DNA was ligated to Hindlll and Xhol-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 spectino ycin (to select for recombinant cosmids with the pYA2329 vector) . /12086

-44-

Identification of E. coli Recombinant Cosmid

Clones Which Produce Proteins Which React With Antibody Against 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^ coli 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 containing 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-l-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 proteins by electrotransfer to nitrocellulose 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 E. coli LE392 clones was isolated from the Xhol-generated cosmid library. This clone produced a 21 kDa protein, and the plasmid was designated pYA2320. Recombinant clone pYA2326 was isolated from the Hindi11-genera ed cosmid library and specified a 40 kDa protein. Three recombinant clones, designated pYA2337, ρYA2338, and pYA2339, were isolated from the Sau3a-generated cosmid library and expressed in LE392, 37 kDa, 43 kDa, and 46 kDa proteins, respectively. One recombinant cosmid clone from 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 outer membrane adhesion proteins . d. Characterization of Cosmid Clone pYA2320. pYA2320 DNA was isolated, digested with Ba HI-Pstl , ligated to BamHI-Pstl digested pYA2329, and the reco - 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-Pstl DNA fragment. Western immunoblots of whole cell proteins from LE392 which contained pYA2332 indicated that this clone produced the 21 kDa protein. This information indicated that the 6 kb BamHI-Pstl 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 promoter of the cloned gene. The 6 kb BamHI-Pstl fragment was ligated to similarly digested ρUC8-l DNA and transformed into E. coli JM109 for restriction endonuclease mapping and further manipulation. This E__;_ coli clone was designated pYA2340. Restriction endonuclease mapping of the plasmid DNA from pYA2340 was performed as described in Maniatis et al . , supra.

8. Maintenance of E. coli Recombinant Clones

_______ coli recombinant clones which produced

B. avium proteins identified by reactivity with antibody against B__;_ avium outer membrane proteins or by reactivity with convalescent sera from B__;_ avium outer membrane proteins were stored at -70 C as follows. E^ coli clones were grown at 37 C overnight in LB containing the appro¬ priate antibiotic, mixed with an equal volume of 80% glycerol in water, and frozen at -70°C

Production of Recombinant Vaccine Strains of

Salmonella

Salmonella spp. can be rendered avirulent by the deletion of cya and crp genes which code for adenylate cyclase and cAMP receptor protein, respectively. These 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 copending application serial no. 251,304. Chi4064 has been shown to be avirulent for one-day-old chickens and is able to colonize the intestinal wall for several weeks . Such strains colonizing the GALT have 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. Construction of a Recombinant Vaccine Strain of Salmonella Expressing the 21 kDa Antigen The 6 kb BamHI-Pstl B. avium DNA fragment from pYA2332 was ligated to BamHI-Pstl-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 log-phase chi3730 (pYA2336) with bacteriophage P22 Hlint at a multiplicity of infection 0.01 as described in Davis, Botstein and Roth, Advanced Bacterial Genetics (Cold Spring Harbor Laboratories) . After growth for 12-15 hours, chloroform lysis of the infected bacteria, and centrifugation to remove cellular debris, the bacteriophage lysate was harvested and titered. S. ty^phimurium chi3987 was transduced with the P22 Hlint lysate generated above by infection of log-phase cells with an m.o.i. of 0.5, and subsequent selection of 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 Turkeys Using Recombinant Salmonella Strains

The recombinant Salmonella strains 'described in C.9 and CIO 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

9 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 immunized with avirulent Salmonella expressing B^ avium outer membrane antigens to permit egg transfer of maternal antibodies. Such treatment would augment immunity to E^ 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 preferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the invention as defined by the appended claims.

5 References

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15

6 Bienenstock, J., et al . , Adv Exp Med Biol (1978) 107:53-59.

7 Boycott, B.R., et al . , Avian Dis (1984) 28:1110- 1114.

8 Bradley, D.E., et al. , J Bact (1980) 143:1466-

20 1470.

9 Brennan, M. , et al . , Abstr Annu Meet Am Soc Microbiol (1988) B2 , p.30.

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25

11 Chang, A.C.Y., et al. , J Bacteriol (1978) 134:1141-1166.

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(1984) pp. 69-105, R. Germanier (ed.), Academic Press, Inc., NY.

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Claims (20)

-52-Claims
1. Purified Bordetella spp. outer membrane protein.
2. Purified B__;_ avium outer membrane protein,
3. 3. The protein of claims 1 or 2 wherein the protein has a molecular mass of about 21 kDa, 37 kDa, 40 kDa, 43 kDa, 46 kDa, or 50 kDa.
4. 4. A vaccine composition comprising one or more outer membrane proteins of Bordetella spp. formulated in a pharmaceutically acceptable vehicle.
5. 5. A vaccine composition comprising one or more B. avium proteins wherein the protein has a molecular mass of 21 kDa, 37 kDa, 40 kDa, 43 kDa, 46 kDa or 50 kDa, said one or more proteins formulated in a pharmaceutically acceptable vehicle.
6. 6. The vaccine composition of claim 4 further comprising an avirulent bacterium substantially incapable of producing functional adenylate cyclase and functional cyclic TAMP receptor protein or a bacterium substantially incapable of producing functional adenylate cyclase, functional cyclic AMP receptor protein, and further substantially incapable of producing functional beta- aspartic semialdehyde dehydrogenase and wherein said microbe comprises a vector carrying a gene encoding beta- aspartic semialdehyde dehydrogenase or a functional fragment thereof linked to one or more genes encoding said one or more outer membrane proteins .
7. 7. The vaccine composition of claim 6 wherein said avirulent microbe is a member of the genus Salmonella.
8. 8. A method of preventing or ameliorating upper respiratory disease in fowl comprising administering to said fowl an effective amount of a vaccine composition according to any one of claims 4, 5, 6, or 7.^'
9. 9. A carrier microbe for the expression of a

   Bordetella sp. outer membrane protein comprising an avirulent derivative of a pathogenic microbe, said derivative being substantially incapable of producing functional adenylate cyclase and functional cyclic AMP receptor protein while being capable of expressing a recombinant gene encoding said outer membrane protein.
10. 10. The carrier microbe of claim 9 wherein said microbe is further substantially incapable of producing functional beta-aspartic semialdehyde dehydrogenase and said recombinant gene is introduced into said carrier microbe using a vector carrying a gene encoding beta- aspartic semialdehyde dehydrogenase or a functional frag¬ ment thereof linked to a gene encoding said outer membrane protein.
11. 11. The carrier microbe of claim 10 wherein said outer membrane protein is from B^ avium.
12. 12. A carrier microbe for the expression of

    B. avium protein having a molecular mass of 21 kDa, 37 kDa, 40 kDa, 43 kDa, 46 kDa, or 50 kDa, said carrier microbe comprising an avirulent derivative of a pathogenic microbe, said derivative being substantially incapable of producing functional adenylate cyclase, functional cyclic AMP receptor protein and functional beta-aspartic semialdehyde dehydrogenase, wherein said carrier microbe comprises a vector carrying a gene encoding beta-aspartic semialdehyde dehydrogenase or a functional fragment thereof linked to one or more genes encoding said one or more 13^ avium proteins .
13. 13. The carrier microbe of any one of claims 9, 10, 11 or 12 wherein said microbe is a member of the genus Salmonella.
14. 14. The carrier microbe of claim 9 wherein said microbe is chi4064 (ATCC 53648).
15. 15. The carrier microbe of claim 10 wherein said microbe is chi4072 (ATCC 67538) or chi6987 (ATCC ) •
16. 16. A DNA construct comprising 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 at least one epitope of a B^ avium outer membrane protein; and

    (b) control sequences that are operably linked to said coding sequence whereby said coding sequence can be transcribed and translated in a host cell, and at least one of said DNA coding sequences or said control sequence is heterologous to said host cell.
17. 17. A host cell stably transformed by a DNA construct according to claim 16.
18. 18. A method of producing a recombinant polypeptide comprising: (a) providing a population of host cells according to claim 17; and (b) growing said population of cells under conditions whereby the polypeptide encoded by said expression cassette is expressed.
19. 19. Purified polyclonal antibodies specific for a Bordetella sp. outer membrane protein or a B^ avium outer membrane adhesion protein.
20. 20. Monoclonal antibodies specific for a Bordetella sp. outer membrane protein or a B^ avium outer membrane protein.