WO1994006911A2 - Mycoplasma pulmonis antigens and methods and compositions for use in cloning and vaccination - Google Patents

Abstract

The cloning, sequencing and expression of an M. pulmonis antigen, and the development of cloning and vaccination systems are disclosed. A strategy is described which allows the cloning of antigens despite the presence of codons which would normally cause transcription arrest in E. coli. Using this method, an M. pulmonis antigen was cloned and produced as a fusion protein, and the major epitope was identified. Transfection into lytic and lysogenic E. coli resulted in the production of the product. The antigen was shown to elicit antibody production in mice, including IgG and IgA production in the tracheolong lavage. Transfected lysogenic E. coli were used for vaccination. The production of the immunogen can be regulated in vivo by controlled feeding with the inducer, IPTG. This method of controlled vaccination, employing inducible immunizing agents, is proposed to be generally applicable to a wide range of organisms and diseases.

Description

DESCRIPTION

MYCOPLASMA PULMONIS ANTIGENS AND METHODS AND COMPOSITIONS FOR USE IN CLONING AND VACCINATION

BACKGROUND OF THE INVENTION

The government owns rights in the present invention pursuant to NIH grant RR00890.

1. Field of the Invention

The present invention relates generally to the fields of molecular biology and disease prevention. The invention particularly concerns the cloning, sequencing and expression of antigens from M. pulmonis and the development of a novel vaccination system employing such antigens. Encompassed within the present invention are methods of cloning and methods and compositions of vaccine preparation and regulation, which will be

generally applicable to a wide range of organisms and diseases. 2. Description of the Related Art

Mycoplasma pulmonis is an important rodent pathogen which primarily affects the respiratory and urogenital systems. M. pulmonis is the causative agent of

mycoplasmosis, a widespread disease within rodent

colonies. The maintenance of pathogen- free rats and mice is of vital importance to scientific research and

development. Unfortunately, the establishment and maintenance of mycoplasma-fee rats has proven difficult in many biomedical institutions, partially due to

economic or space limitations. When an animal is exposed to mycoplasmas, the organisms often persist in the respiratory tract for prolonged periods. For example, carriage of M.

pneumoniae in the upper respiratory tracts of humans may persist for several weeks (Grayston et al ., 1967), while in mice inoculated intranasally with M. pulmonis, both the pneumonic lesions and the mycoplasmas persist for several months (Lindsey et al ., 1971). This tends to suggest that the host's immune response may be

inefficient. However, persistently infected animals are resistant to subsequent intranasal challenge with the same organism. Thus, mice inoculated intranasally with small numbers of M. pulmonis organisms do not develop lung lesions, although mycoplasmas can be isolated from their lungs for at least several weeks postinoculation. Following intranasal challenge with a large dose of M. pulmonis, which produces severe pneumonia in control animals, mice previously inoculated with small numbers of mycoplasmas develop much less severe pneumonia and fewer organisms are isolated from their lungs than from those of controls (Taylor et al ., 1977). Although reinfection of previously infected animals can occur, such

experiments offer hope for the eventual development of a successful vaccine.

Rats vaccinated with killed M. pulmonis have been found to be only partially protected from challenge. A number of potential vaccines consisting of live organisms have been reported to elicit better protection than vaccines employing killed organisms or cell extracts. However, complications arising from vaccination with viable wild-type M. pulmonis organisms include

polyarthritis and genital tract colonization (Cassell & Davis, 1978), which make this an unsuitable method to combat mycoplasmosis. More recently, attenuated mutants of Salmonella spp. and E. coli have been used as delivery systems to

stimulate immunity to cloned heterologous immunogens (Hoiseth & Stocker, 1981). Mucosal, humoral, and cell-mediated immunity to antigens expressed by these strains can be induced by oral or intravenous immunization

(Fairweather et al ., 1990; Guzman et al ., 1991).

However, despite the mutants' inability to multiply extensively, they do establish a self -limiting,

subclinical infection and can be detected in tissue such as the liver and spleen (Charles &. Dougan, 1990).

The development of a mycoplasmosis vaccine has been further hampered by the lack of an effective purified antigen. The production of large amounts of antigen(s), for example, for use in vaccine development, generally requires that the antigen first be cloned using

recombinant DNA technology. The cloned DNA can then be expressed in a recombinant host cell, such as E. coli , enabling large quantities of antigen to be produced and subsequently purified.

The cloning and production of a useful antigen from M. pulmonis has proven to be particularly difficult. A significant problem encountered is the fact that

Mycoplasma are very unusual in utilizing the codon TGA to code for the amino acid tryptophan (Inamine et al ., 1990; Yamao et al ., 1985), rather than to signal termination of translation, as is the usual role of TGA in most

bacteria. Thus, the appearance of TGA in the coding sequence of a gene, as occurs naturally in Mycoplasma, would unfortunately result in the premature termination of translation when the gene is placed in most common cloning hosts such as E. coli . For example, the P1 gene of M. pneumoniae has 21 TGA codons (Su et al ., 1987;

Inamine et al ., 1988), all of which would arrest

translation in E. coli . This feature has complicated efforts to clone and express mycoplasma genes in E . coli (Dallo et al ., 1988; Frydenbert et al ., 1987). As the occurrence of the TGA codons renders the production of a long chain

polypeptides highly improbable, the screening of an expression library with antibodies to detect antigenic protein products has been virtually impossible.

M. pulmonis remains one of the most important and frequently-encountered organisms in both conventionally maintained and specific-pathogen-free rodent colonies. The development of an effective vaccine against

mycoplasmosis would therefore be of significant

importance to the scientific community. The recombinant cloning of M. pulmonis antigen(s) would constitute a major step towards mycoplasmosis vaccine development, however, the problems created by the unusual

translational machinery of M. pulmonis need to be

overcome before significant progress can be made in this area.

SUMMARY OF THE INVENTION The current invention overcomes several of the obstacles present in the prior art through the molecular cloning of an M. pulmonis antigen, its expression in a recombinant host cell, and the development of an

effective vaccine employing recombinant antigens housed within inducible immunizing agents. The present

invention further provides a method for cloning an antigen, and particularly, an antigen from an organism or organelle in which a codon intended to encode an amino acid codon is interpreted by E . coli as a stop codon. Also disclosed is a novel and particularly advantageous method of controlled vaccination, through regulated antigen production, which will be generally applicable to a wide range of organisms and diseases.

A novel cloning strategy is disclosed which allows the cloning of proteins despite the presence of codons which would normally cause transcription arrest in

E . coli . This strategy is considered to be particularly appropriate for cloning antigens from Mycoplasma, such as M. pulmonis, and from the mitochondria of several

species. This method, which utilizes the fragmentation of DNA into pieces smaller than the expected size of the gene to be cloned, allows an antigen or antigenic

fragment to be obtained by expression cloning. The DNA may then be sequenced and oligonucleotide probes prepared and used to identify the entire gene by techniques other than expression screening, such as hybridization. In turn, the entire coding region of DNA may be sequenced and analyzed, and any E. coli stop codons identified. Such codons may then be modified allowing the gene to be expressed in E. coli , or other recombinant host cells, and thus enabling the production of the recombinant protein.

A particularly important aspect of the present invention is the preparation of inducible immunizing agents and the utilization of such agents in controlled vaccination regimens. As used herein, the term

"inducible immunizing agent" is intended to refer to a vehicle, such as a recombinant host cell or recombinant virus, suitable for administration to an animal, which vehicle is capable of being induced to express an

immunogen. Such inducible immunizing agents include, for example, eukaryotic or prokaryotic host cells, such as E. coli , and preferably, lysogenic E. coli , or viruses, such as vaccinia virus.

In any event, whether cellular or viral in origin, the inducible immunizing agent will contain a genetic construct wherein the immunogen-encoding DNA is

positioned so as to be under the control of an inducible promoter that is capable of being induced by a preferably non-toxic inducing agent, such as IPTG. The construct may then be introduced into a host cell or virus, using recombinant technology, to create an inducible immunizing agent for use in vaccination regimens. Once administered to an animal to be immunized, the immunizing agent may be induced to express the immunogen by administering the inducer to the animal. Using this new and advantageous method, immunogen production can thus be regulated in vivo in response to inducer administration. It is envisioned that this method of controlled vaccination will be generally applicable to a wide range of organisms and diseases.

DNA Segments and Recombinant Vectors

Important aspects of the present invention concern isolated DNA segments and recombinant vectors encoding a M. pulmonis antigen, and recombinant host cells

expressing such an antigen. The creation, through recombinant DNA technology, of such M. pulmonis antigen-expressing recombinant host cells is particularly

important for vaccine development.

As used herein, the term "DNA segment" in intended to refer to a DNA molecule which has been isolated free of total genomic DNA. Therefore, a DNA segment encoding an M. pulmonis antigen refers to a DNA segment which contains such coding sequences and yet is isolated away from total M. pulmonis DNA. Included within the term "DNA segment", are DNA segments which may be employed in the preparation of vectors, as well as the vectors themselves, including, for example, plasmids, cosmids, phage, viruses, and the like. In preferred embodiments, the invention concerns isolated DNA segments or recombinant vectors which encode an M. pulmonis antigen that includes within its amino acid sequence an amino acid sequence essentially as set forth in seq id no:2, corresponding to L150a, or a biologically functional equivalent thereof. In these embodiments, isolated DNA segments or recombinant vectors encoding the antigen, termed 3E12F4 MPAg, will include a nucleic acid sequence essentially as set forth in seq id no:1, or a biologically functional equivalent thereof.

A major epitope of the cloned antigen has been found to reside within the first 25 amino acids of the

polypeptide, therefore, even more preferred DNA segments or vectors are those which encode an M. pulmonis antigen which includes an amino acid sequence essentially as set forth in residues 1 through 25 of seq id no:2, or its a biological functional equivalent. Such an isolated DNA segment or vector will therefore include the nucleic acid sequence represented by residues 1 through 75 of seq id no:1, or a biologically functional equivalent thereof.

Preferred recombinant vectors for use in accordance with certain aspects of the present invention are those which include an inducible promoter, i.e., those in which transcription of the coding sequence is stimulated by an inducer. Many such recombinant vectors, including a variety of inducible promoters, will be known to those of skill in the art in light of the present disclosure.

These include, for example, the metallothionein promoter, which is induced by zinc or copper (Zn/Cu); the

tryptophan operon, induced by the amino acid tryptophan; the ara operon, induced by the sugar L-arabinose; and the lac operon which may be switched cn in response to a variety of inducer molecules, such as lactose, and more particularly, the synthetic inducer IPTG. As will be generally known to those of skill in the art, the recombinant vectors and DNA segments of the present invention may exhibit certain alterations or modifications in the coding region, and nonetheless encode an M. pulmonis antigen in accordance herewith. This is an important aspect of the invention,

particularly where the cloned antigen contains one or more codons, such as TGA tryptophan codons in M.

pulmonis , which are interpreted by E. coli as stop codons.

Accordingly, it will be understood that these aspects of the invention are not limited to the

particular nucleic and amino acid sequences of seq id no:1 and seq id no:2. DNA segments prepared in

accordance with the present invention may also encode biologically functional equivalent proteins or peptides which have variant amino acids sequences. Such sequences may arise as a consequence of codon redundancy and functional equivalency which are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, biologically functionally equivalent proteins or peptides may be created, for example, via the application of recombinant DNA

technology, in which changes in the protein structure are deliberately engineered.

Biologically Functional Equivalents Modifications and changes may be made in the

structure of the M. pulmonis antigen and still obtain a molecule having like or otherwise desirable

characteristics. For example, certain amino acids may be substituted for other amino acids within the protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antibodies or receptors. Since it is the interactive capacity and nature of a protein that defines that protein's

biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like properties.

Thus, various changes may be made in the sequence of an M. pulmonis antigen (or underlying DNA) without

appreciable loss of its biological utility. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are:

isoleucine (+4.5); valine (+4.2); leucine (+3.8);

phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3);

proline (-1.6); histidine (-3.2); glutamate (-3.5);

glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is believed that the relative hydropathic

character of the amino acid determines the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, antibodies, receptors, enzymes, substrates, and the like. It is known in the art that an amino acid may be substituted by another amino acid having a similar hydropathic index and still obtain a biological

functionally equivalent protein. In such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

Substitution of like amino acids can also be made on the basis of hydrophilicity. This is particularly suitable for the production of a biologically equivalent protein or peptide for use in immunological embodiments, such as is contemplated by the present invention. U.S. Patent 4,554,101, incorporated herein by reference, teaches that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with a biological property of the protein.

As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 ± 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5);

leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);

phenylalanine (-2.5); tryptophan (-3.4). It is

understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and importantly for the present invention, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

In addition to any naturally-occurring redundancy or equivalency, the deliberate modification or 'genetic engineering' of antigen sequences is contemplated by certain aspects of the present invention. As previously discussed, the modification of an antigen sequence is envisioned to be particularly useful where the cloned antigen contains one or more codons which are interpreted by E. coli as stop codons. In these cases, for example, where TGA is used within an M. pulmonis antigen sequence to encode the amino acid tryptophan, the specific codon may be deliberately changed to allow continued read-through and production of a longer length polypeptide or protein. Longer length proteins may be desired, for example, to increase antigenicity or stability of the resultant polypeptide, to increase the ease of its purification, or to allow fusion or coupling to further sequences.

In such embodiments, the stop codon may be changed to another codon which will nonetheless result in the incorporation of the same amino acid into the resultant polypeptide. It is contemplated that codons may be changed in the third base or "wobble position", and that base changes may also be made in the second or first positions. Appropriate codons may be chosen from a consideration of the known codons and tRNAs utilized by E. coli . For example, in the case of tryptophan, the codon UGG may be used. The designation of codon triplets and the amino acids encoded thereby will be generally known to those of skill in the art and are well

documented in the literature, for example, see Sambrook et al ., (1989) incorporated herein by reference.

Alternatively, a stop codon in the cloned DNA may be engineered so that, when expressed in E. coli , it will encode a different amino acid from that encoded by the organism or organelle from which the DNA was originally obtained. One may particularly choose to replace the stop codon with a triplet which encodes a biologically functional equivalent amino acid. For example, in the present case, a codon for tyrosine (UAC or UAU) could be employed to replace the 'stop codon' which was originally intended to encode the amino acid tryptophan.

The recombinant vectors and isolated DNA segments of the present invention may include solely the nucleic acid sequence of seq id no:1, or residues 1 through 75

thereof, or alternatively, may encode larger polypeptides which nevertheless include sequences encoding an

M. pulmonis antigen. In this regard, the DNA segments may include further coding regions such that the cloned antigen will be produced as a fusion protein, that is, the antigen may be linked to a portion of another

polypeptide without significantly altering its antigenic or other properties.

Techniques such as these are commonly employed in the art of molecular biology, and antigen-protein fusions with, for example, ß-galactosidase or ubiquitin, could be employed. A cloned antigen may be fused with a protein which exhibits a particular binding affinity, to enable the rapid and efficient purification of the fusion protein by affinity chromatography. Furthermore, any one of a variety of DNA sequences which encode a protease-sensitive polypeptide segment could be employed in the fusion protein DNA constructs, to allow the subsequent release of the antigen from the remainder of the fusion protein.

DNA Probes and Primers

The DNA segments of the present invention are contemplated to have utility in a variety of embodiments. They can be used in the preparation of nucleic acid probes or primers, which can, for example, be used in the identification and cloning of related genes or sequences, or in the study of antigen expression, and the like.

Importantly, the M. pulmonis antigen-encoding DNA

segments can be used in the recombinant production of the antigen, its expression in E. coli , and to create novel antigen-presenting vehicles for use in use in vaccination protocols.

Turning firstly to the use of primers and probes. It is contemplated that oligonucleotide fragments

corresponding to the sequence of seq id no:1 for

stretches of between about 10 and about 30 basepairs in length will find particular utility, with even longer sequences, e.g., 50, 100, 200, or even up to full length, also being of use in certain embodiments. The probes may be used in a variety of assays for detecting the presence of complementary sequences in a given sample.

Alternatively, they may be in the preparation of mutant species primers, or primers for use in preparing other genetic constructions.

The use of a hybridization probe of about 10

nucleotides in length allows the formation of a duplex molecule that is both stable and selective. However, molecules having complementary sequences over stretches greater than 10 bases in length are generally preferred because the stability and selectivity of the resultant hybrid is increased, and the quality and degree of specific hybrid molecules obtained is thus improved.

Accordingly, one will generally prefer to design nucleic acid molecules having gene-complementary stretches of 15 to 20 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR technology of U.S. Patent 4,603,102

(incorporated herein by reference) or by introducing selected sequences into recombinant vectors for

recombinant production.

The nucleotide sequences of the invention may be used for their ability to selectively form duplex

molecules with complementary stretches of Mycoplasma antigen genes or cDNAs. Depending on the application envisioned, one may desire to employ varying conditions of hybridization to achieve varying degrees of

selectivity of probe towards target sequence. For applications requiring high selectivity, one will

typically desire to employ relatively stringent

conditions to form the hybrids, e.g., one will select relatively low salt and\or high temperature conditions, such as provided by 0.02M-0.15M NaCl at temperatures of

50°C to 70°C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand and are thus particularly suitable for isolating further genes encoding M. pulmonis antigens.

Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template, or where one seeks to isolate antigens from more distantly related species, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such as 0.15M-0.9M salt, at

temperatures ranging from 20°C to 55°C. Cross-hybridizing species can be readily identified as

positively hybridizing signals with respect to control hybridizations. It will be generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated to achieve a variety of aims.

In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of

appropriate indicator means are known in the art,

including radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a

detectable signal. In preferred embodiments, one will likely desire to employ an enzyme tag such a urease, alkaline phosphatase or peroxidase, instead of

radioactive or other environmental undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or

otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label. Recombinant Host Cells and Immunizing Agents

Further embodiments of the present invention concern recombinant host cells containing an M. pulmonis DNA segment. The DNA segment will preferably be in the form of a vector containing a promoter, and more preferably, will contain an inducible promoter, such that the

recombinant host cell may be induced to express an M. pulmonis antigen. Recombinant host cells will have utility in various embodiments, such as in the production of recombinant proteins, and particularly, for use in vaccination schemes.

Preferred cells for use as host cells are

procaryotic cells, including, for example, salmonella , shigellaε, BCG, Citrobacter faundii , Neisseria

gonococcus , Streptomyces reticuli , and E. coli .

Procaryotic cells in general, and E. coli in particular, are preferred because their genetics is well developed, the expression of recombinant proteins can generally be optimized, and, because they are ideal as antigen-expression vehicles for use in vaccination protocols. However, the use of eucaryotic cells, such as yeast, retrovirus, vaccinia, baculovirus, adenovirus, pox virus, hematopoietic stem cells or muscle cells, is also

contemplated.

The preparation of recombinant host cells which include an inducible construct is particularly important as such cells are contemplated for use as inducible immunizing agents in the novel vaccination regimen of the present invention. As such, the term 'inducible' means that the coding sequence of the exogenous DNA is

positioned adjacent to and under the control of a

promoter which is capable of being stimulated by an inducer. It will be understood that the present

invention is not limited to inducible host cells inccrporating M. pulmonis antigen DNA, but rather

includes host cells capable of being induced to express any protein against which one ultimately desires to generate an immune response.

Any one of a variety of genetic constructs and inducer molecules may be employed in accordance herewith. These include, for example, the metallothionein promoter and zinc or copper (Zn/Cu); the tryptophan operon and tryptophan; the ara operon and L-arabinose; the estrogen promoter and estrogen; mouse mammary tumor virus and glucocorticoid; the lac operon and IPTG; and the like. The construction and use of these and numerous other such inducible recombinant vectors will be known to those of skill in the art in light of the present disclosure.

To express an antigen, such as an M. pulmonis antigen, in a recombinant host cell, one would introduce into the cell a recombinant DNA segment encoding the antigen and culture the transformed cell to express the antigen. In preferred embodiments, it is contemplated that the recombinant host cell prepared would contain an inducible antigen-encoding vector, and that antigen expression may thus be simply achieved by contacting the host cell with the appropriate inducer molecule. A host cell which incorporates such an inducible construct will not generally express the antigen until stimulated to do so by the inducer. This confers a distinct advantage to the use of such inducible cells in vaccination protocols.

Further aspects of the present invention concern recombinant viruses for use as inducible immunizing agents in vaccination schemes. To prepare such an inducible recombinant virus, one would firstly prepare a genetic construct in which the coding sequence of the protein against which one desires to generate an immune response is under the control of an inducible promoter, and secondly, introduce this construct into the virus. Appropriate methods for the introduction of exogenous DNA into a viral genome will be known to those of skill in the art, for example, see Fenner (1985) and Mackett et al . (1985). Viruses contemplated for use in this manner include those which have previously been employed in non- inducible vaccination, such as, for example, herpes virus (Lowe et al ., 1987), adenovirus (Levrero et al . , 1988), and pox viruses such as vaccinia virus

(Esposito & Murphy, 1989). Vaccinia viral vectors are particularly preferred because they are stable, produce long-lived immunity, and because they can be simply and inexpensively produced and administered. Purified Antigens

Further aspects of the present invention concern an M. pulmonis antigen, or fusion protein, such as

ß-galactosidase-antigen fusion protein, purified relative to its natural state. As used herein, the term purified "purified relative to its natural state" is intended to refer to an M. pulmonis antigen or antigen fusion protein from which has been removed various non-antigen

components, and which composition substantially retains its antigenicity. Of course, it will not be necessary to obtain a highly a purified antigen preparation. Indeed, any preparation in which the antigen is purified relative to its natural state may have utility, for example, as an immunogen in antibody production, as an antigen in assays to detect the presence of serum antibodies, or in further immunological embodiments such as in immunoprecipitation to remove antibodies from a given sample.

Any one of a variety of methods may be employed to purify an M. pulmonis antigen or antigen fusion protein relative to its natural state. For example, one may simply and advantageously fractionate the recombinant host cells and obtain the antigen-containing fraction. Alternatively, the antigen may be further purified, either from whole or fractionated cells, using any of the techniques generally known to those of skill in the art. These may include, for example, differential solubility under a variety of conditions, pH- or heat-stability, and a wide range of chromatographic techniques which separate proteins based upon properties such as size, charge, hydrophilicity, and the like. Such chromatographic techniques include, for example, gel filtration, anion exchange, cation exchange, and immuno- or affinity-chromatography.

In further embodiments, the present invention concerns antibodies, and particularly, monoclonal

antibodies directed against the M. pulmonis antigen of the present invention. One such monoclonal antibody (mAb) has been generated and termed Mab 3E12F4.

Techniques for generating monoclonal antibodies will be known to those of skill in the art in light of the present disclosure. Generally, an experimental animal, such as a mouse, would be immunized with the antigen to stimulate antibody production. Mice producing reasonable titers of circulating antibodies would later be

sacrificed and spleen cells removed for cell fusion.

Mouse myeloma cell lines which are sensitive to selective media, such as HAT media, are proposed to be particularly useful for hybridization, which may be carried out using any appropriate method, such as polyethylene glycol

(PEG)-mediated fusion. Resultant hybridomas may then be screened for antibody production, for example, by

employing ELISAs or immunodot procedures.

Vaccination Protocols

In yet further important embodiments, the present invention provides novel methods of vaccinating an animal, including both laboratory and farm animals and human subjects, against a disease. These aspects of the invention are particularly exemplified by methods and compositions for vaccinating an animal against mycoplasma infection. The method, which will be generally

applicable to a wide range of organisms and diseases, is believed to be particularly advantageous as it adds a new dimension to the control of vaccination through regulated antigen production. Specifically, the method can be carried out with a single administration step and yet allows for antigen production to be regulated in response to compounds administered orally.

The vaccination method of the present invention employs an inducible immunizing agent, for example, a recombinant prokaryotic or eukaryotic host cell, or a recombinant virus, such as vaccinia virus, which is capable of being induced to express an antigen

(immunogen) against which an immune response is desired. As used herein, an inducible immunizing agent which is "capable of being induced to express an antigen" refers to an immunizing agent incorporating an antigen-encoding DNA segment, where expression of the antigen can be stimulated by exposing the cellular or viral agent to an inducer molecule.

The use of recombinant prokaryotic cells, such as E . coli , and more preferably, lysogenic E. coli , as inducible immunizing agents is preferred in certain embodiments. In such cases, it is important to note that as antigen-encoding DNA will be incorporated into the genome of the host cell which is colonized in a

vaccinated animal, this DNA will continually be presented in their offspring. In addition to E. coli , other prokaryotic host cells for use in accordance herewith include, for example, salmonella, shigella, BCG,

Neisseria, Streptomyces, and the like. A preferred combination for use in an inducible vector is to employ a lac operon genetic construct and the inducer IPTG. However, any one of a variety of other constructs and inducers could be employed, such as the metallothionein promoter and Zn/Cu, the trp operon and tryptophan, or the ara operon and L-arabinose.

Naturally, one will wish to ensure that the properties of the chosen inducer render it suitable for use in the animal to be vaccinated. In particular, it is

contemplated that one will choose to use an inducer that is generally non-toxic, that can be absorbed by the animal in question and circulated in the body, and preferably, an inducer that is non- or slowly-metabolized by the particular animal.

The vaccination method of the present invention comprises, firstly, administering to an animal a cellular or viral immunizing agent which is capable of being induced to express an antigen, as discussed above. The inducible immunizing agent may be administered to the animal orally, or more preferably, via a parenteral route, for example, intranasally, subcutaneously, or most preferably, intravenously. In embodiments employing lysogenic E. coli , it is contemplated that the doses of inducible host cells suitable for vaccination of ail animals will range from about 1×10¹ CFU to about 1×10¹⁰ CFU. The dosage of the lysogenic host cells may be varied over such a wide range as E. coli multiply every 20-30 minutes. This high multiplication rate allows the minimum dosage, of 1×10¹ CFU, to be used optimally in vaccination. However, if a rapid initial response is desired, one may wish to employ a higher vaccinating dose. The second stage of the novel vaccination scheme is the antigen-expression stage. Antigen expression is stimulated by administering to the animal an inducer in an amount effective to induce the immunizing agent to express the antigen. It is preferred, but not essential, that the inducer be administered orally, as this is the simplest and least invasive method. In exemplary

embodiments, the inducer molecule will be IPTG, which may be straightforwardly supplied to the animal in the drinking water.

Where a lac operon construct and an IPTG inducer combination are used, it is contemplated that an IPTG dose of from about 0.001mM to about 10mM will be suitable to induce fusion protein synthesis in vaccinated animals. This wide dose range is possible as IPTG is non-toxic, non-metabolized, and its small molecular weight (238Da) allows it to be easily absorbed and easily excreted in the urine or feces of the animal, thus preventing bodily IPTG accumulation. The minimum dose of IPTG required to stimulate antigen expression will be in the order of about 0.1mM, which had been used in vitro and in vivo to stimulate expression of fusion proteins in lysogenic E. coli .

Only on administration of the inducer will the inducible immunizing agent be stimulated to express the antigen. This is an important feature of the invention which enables antigen production, and hence antibody generation by the vaccinated animal, to be closely regulated. This has particular advantages over

presently-used vaccination protocols as several booster injections of immunogens are no longer needed.

Furthermore, the inducible control element of the

invention prevents continuous antigen production which may otherwise have rendered the vaccinated animal

tolerant to the immunogen and prevented subsequent immune responses on exposure to the pathogen in question. It is contemplated that long term immunity may be generally be acquired by administering the inducer at time intervals of from about 2 weeks to about 24 weeks or longer.

It is contemplated that the inducible immunizing agents of the present invention may be administered to an animal either alone, or within a pharmacological

composition. Various pharmacologically acceptable vehicles could be employed in accordance herewith, such as, for example, oils, emulsions, or aqueous sterile buffers. The precise compositions and use of such pharmaceutical vehicles will be known to those of skill in the art in light of the present disclosure.

Although exemplified by vaccination against

mycoplasmosis in rodents, the compositions and methods for vaccination disclosed herein will be appropriate for use to prevent Mycoplasma infections in other animals, such as, for example, in chicken, cattle, swine, and humans. Furthermore, the method of the present invention is contemplated to be of use in stimulating both humoral and cellular immune responses against a myriad of other pathogens and thus will be of use in combatting many diseases. The methods of the present invention can be used against a wide variety of diseases such as, for example, foot and mouth disease, malaria, cholera, polio,

salmonellosis, rubella, tuberculosis, hepatitis,

enterotoxemia, pseudorabies, Newcastle disease and AIDS. In fact, this invention is suitable for use against any disease, including cancer, in which the generation of an immune response would be desirable or advantageous. The present invention may be employed in conjunction with antigens or epitopes, including tumor markers, which have already been identified as important to the particular disease in question. Alternatively, novel antigens or epitopes may be identified and their genes isolated by employing standard techniques known to those of skill in the art of molecular biology. In any event, DNA encoding the antigen or epitope against which the immune response is desired may be expressed in an inducible immunizing agent, which would then be administered to an animal or human subject. Antigen expression may then be regulated simply by feeding the vaccinated host with an inducer molecule. In yet further embodiments, inducible immunizing agents may be prepared which are capable of being induced to express more than one antigen or antigenic fragment. Such agents could therefore be employed as multivalent vaccines to stimulate immune responses against various components of the same pathogen, or against different disease-causing organisms. In such cases, the subsequent administration of an inducer would stimulate the

expression of more than one antigen, and wide-ranging immunity against a range of pathogens could be achieved following only a single vaccination event. In an added dimension, inducible immunizing agents could be created which contain two or more vectors each of which is capable of being induced by distinct inducer molecule. This would allow the immune responses of the vaccinated animal to different immunogens to be separated in time.

Molecular Cloning

In still further important embodiments, the present invention provides method of cloning a protein, and particularly, an antigen or antigenic fragment thereof. The inventors developed the method in response to the difficulties previously encountered in attempts to clone antigens from M. pulmonis, which is known to utilize certain codons which are interpreted as stop signals in E. coli . The method is thus contemplated to be

particularly suitable for cloning antigens from organisms, or organelles, in which the DNA includes a codon intended to encode an amino acid and which codon is interpreted as a stop codon in E. coli . In addition to Mycoplasma, other examples include yeast mitochondria and neurospora mitochondria.

During evolution, mitochondria have evolved to utilize a slightly different genetic code from that generally employed by eukaryotic and prokaryotic cells. Of relevance to the present invention, mitochondria from yeast, neurospora and human cells use the codon UGA, commonly recognized as a stop codon, to encode the amino acid tryptophan (Borst, 1981; Breitenberger &

Rajbhandary, 1985; Anderson et al ., 1981). The novel cloning method embodied by the present invention is therefore also envisioned to be suitable for cloning mitochondrial proteins or fragments thereof. This method will be particularly suitable for cloning mitochondrial antigens, for example, antigens known to be associated with autoimmune diseases such as syphilis or myocarditis

(Berg & Klein, 1989).

To clone a protein, antigen, or fragment thereof, in accordance with the present invention, one would first obtain a sample of DNA suspected to encode the protein, and fragment the DNA into small pieces. Fragmentation by random shearing rather than restriction endonuclease cleavage is preferred to ensure that the correct reading frame will be included after insertion into a suitable expression vector. It is envisioned that the size of the DNA fragments one would wish to produce will depend upon several factors, such as, for example, the source of the DNA, an estimate of the frequency of the unusual codon, and the size of DNA fragment ultimately desired. It is contemplated that the size of the small fragments

generated should be smaller than the expected size of the gene to be cloned. In exemplary embodiments, fragments of between about 100 and about 600 base pairs long are preferred.

A DNA library should then be generated from the small DNA pieces, preferably using a suitable phage-based system, such as by employing the vector λgt11 to express polypeptides in E. coli . Phage-based libraries are generally preferred because of the large numbers of recombinants that may be prepared and screened will relative ease. Suitable techniques for use in generating an expression library will be known to those of skill in the art in light of the present disclosure.

The expression library can then be screened for the presence of the desired protein. The most suitable screening method is considered to be screening for immunological reactivity using antibodies. Epitopes recognized by antibodies generally involve about 10 amino acids or less (Wiley et al ., 1981; Green et al ., 1982). As the present cloning method is designed to create DNA fragments encoding proteins of generally between about 30 and about 200 amino acids long, it is particularly suitable for cloning antigenic proteins, or the main immunogenic regions or epitopes thereof. A further and important advantage of this method is, therefore, that it represents a labor- and cost-effective method identifying the most immunologically important regions and epitopes of an antigenic protein. However, other screening methods may also be

employed to identify the desired protein or fragment thereof. The use of alternative screening methods will allow the cloning of proteins other than natural antigens or proteins against which antibodies have previously been raised. Screening methods which may be employed include further methods based upon the ability of a labelled molecule other than an antibody to bind to the desired protein. Suitable molecules include, for example, receptors, hormones, substrates, inhibitors, and the like, which incorporate a label such as a fluorescent or radioactive label or are conjugated to an enzyme allowing subsequent detection. It is further envisioned that an active domain or region of an enzyme may be cloned by employing a direct enzyme assay screening method to detect a product of the enzymatic reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Strategy to construct a recombinant DNA expression library. To substantially reduce the presence of TGA stop codons in the M. pulmonis DNA fragments, the genomic DNA was fragmented into very small pieces, and then cloned into a unique EcoR1 site of the lac Z gene in lambda gt11 (λgt11) phage. This was used in turn to infect E. coli and resulted in the expression of

β-galactosidase fusion proteins.

Figure 2. SDS PAGE and Western blot analysis of

recombinant fusion protein. Duplicate 7.5% SDS

polyacrylamide gels were stained with Coomassie brilliant blue or transferred to nitrocellulose by Western blotting and stained with preabsorbed monospecific polyclonal rabbit anti-M. pulmonis antibody. (A) SDS-PAGE loaded with protein marker (left lane) or duplicate of extracts from IPTG-induced (panel A lane 1 and 2) or uninduced (lane 3 and 4). IPTG induced a significant amount of fusion protein product. Only IPTG induced cells (left two lanes of panel B), not uninduced cells (right two lanes), produced M. pulmonis antigen which reacted with preabsorbed monospecific rabbit anti-M. pulmonis

antibody. The secondary antibodies employed was alkaline phosphatase-conjugated goat anti-rabbit IgG. Figure 3. Identification of M. pulmonis DNA used in transfection of E. coli by λgt11 phage. The recombinant DNA extracted from a positive clone was amplified by PCR. The M. pulmonis DNA insert is near the 615 bp position. The DNA molecular size markers, which are multiples of 123 bp, are in the left lane.

Figure 4. The nucleotide sequence of the inserted M.

pulmonis DNA. The deduced amino acid sequence is shown below the nucleotide sequence. Only the nucleotides (nt) are numbered, with 1 being the first nt of the inserted M. pulmonis DNA coding sequence. There are two

tryptophan residues which correspond to the TGA codons located at 76 and 523 nt and two stop codon TAA located at 102 and 240 nt. The restriction sites are represented as follows, the nt position in parenthesis referring to the first 5'-base in the recognition sequence: ACC1(58), AHA2(49), BCL1(122), ECOR1(258), HINC2(375), HPA1(375), MLU1(117), PVU2(519), SNAB1(41), and XCA1(58).

Figure 5. Serum antibody responses IgG and IgM after immunization with fusion protein and lysogenic E. coli given orally or intravenously. Values are significantly different from those of the control (OD=0.05) at

P < 0.05. All vaccines elicited a good antibody titer, however the fusion protein and lysogenic/IV elicited both IgG & IgM titers higher than lysogenic/oral

administration. Figure 6. Immune response in tracheolung lavage; IgG and IgA antibody responses after immunization with fusion protein, lysogenic/oral and lysogenic/IV. All vaccines produced a high antibody titer in their tracheolung lavage. The highest antibody response was observed in lysogenic/IV groups. Figure 7. Isolation of M. pulmonis from the respiratory tract of mice vaccinated with fusion protein,

lysogenic/oral and lysogenic/IV then challenged by various doses of wild-type MPT2. The greatest protection was observed in all groups with the dose of 1 × 10³

CFU/mouse. The non-vaccinated mice of groups 6, 7, 9, and 10 had large numbers of MP in their respiratory tract at all challenging doses. Data from groups 6, 9, and 10 were similar to group 7, group 7 data is presented.

Figure 8. Histopathologic lesions in mice vaccinated with fusion protein, lysogenic/IV and lysogenic/oral then challenged with various doses of MPT2. Significantly less lung lesion were observed in ail groups compared to the control (group 8). The maximum lung lesion is equal to 1.

Figure 9. Cell -mediated immune response in mice

vaccinated with fusion protein, lysogenic/IV, and

lysogenic/oral then challenged by wild-type MPT2

(lymphocyte transformation). Stimulation indices were calculated by dividing the mean count per minute of antigen or mitogen stimulated cultures by the mean count per minute of control cultures. There was a significant difference (P < 0.05) between control (group 8) and all vaccinated groups.

Figure 10: Strategy to construct a lysogenic E. coli . A small fragment of Mycoplasma pulmonis DNA was ligated in the lacZ portions of a λgt11 phage which in turn

integrated into the chromosome of E. coli. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The development of various vaccines is a good example of a successful and cost-effective scientific accomplishment. Vaccination has been mainly responsible for the eradication of smallpox and for the control of yellow fever, poliomyelitis and German measles in the human population, and of Newcastle, foot and mouth, and Marek's diseases in domestic animals. The art of deliberate immunization against infections has been practiced for centuries by the mechanism of protective immunity but were not fully appreciated until the advent of modern immunology. With the discoveries of newer technologies and greater understanding of the molecular biology of pathogens, the conventional empirical

approaches to vaccine development have given way to more rational vaccine design. Recent advances in

biotechnology have brought exciting prospects for the development of new vaccines and improvements of existing ones.

Biotechnologicai advances in vaccine manufacture involve two methods: One is to synthesis antigens chemically. The other is to force harmless, easy-to-grow, bacteria or yeast to produce antigens through genetic engineering. The synthetic approach involves making antigens in the test tube from simple chemical building blocks. Genetic engineering harnesses living organisms to mass produce antigen vicariously. One of the greatest advantages of genetic engineering is that, potentially, antigen-encoding DNA can be engineered into living microbes which could grow inside the host being immunized, thus making a genetically engineered live alternative vaccine with all the advantages of dosage and duration of antigen stimulation. The vaccinia virus has been successfully engineered to act as a carrier for several antigens opening up an entire new potential (Falkner & Moss, 1988; Davison & Moss, 1990; Chakrabarti et al . , 1985; Kieny et al ., 1984; Lyons et al ., 1990). Vaccinia virus-based vaccines have been proposed for use against rabies, lassa fever and influenza. Further examples of viral vaccines include the use of recombinant BCG (Aldovini & Young, 1991;

Stover et al ., 1991), and Salmonella (Schodel & Will, 1990; Cohen et al ., 1990; Bowen et al ., 1990; Salas-Vidal et al ., 1990) as carriers.

Retroviral systems have also been employed to express proteins in animal hosts, following retroviral infection of epithelial cells in the respiratory or gastro-intestinal tracts. Retroviral vectors allow for high efficiency gene transfer into replicating cells and the precise integration of the transferred genes into cellular DNA. However, there are distinct disadvantages in retroviral systems. Firstly, retrovirally infected cells stimulate cellular immune responses, including cytotoxic T cell and activated macrophage responses, which will eventually destroy retrovirus- infected cells because they carry foreign protein on their cell-surface. Secondly, introduction of DNA in retroviral vectors results in the ultimate production of virions that carry vector RNA and can infect target cells, but which do not spread after infection (Miller, 1992). Thirdly,

retroviral vectors are apparently unable to infect non-dividing cells and cannot be made synthetically but must be produced by cultured cells.

M. pulmonis is a wide-spread pathogen that causes the disease mycoplasmosis within rodent colonies. An effective vaccine against M. pulmonis is highly

desirable, as this would allow the maintenance of

mycoplasmosis-free rats and mice in scientific and medical research establishments. To prevent infection, it is desirable to develop vaccines which stimulate not only humoral immunity but also cell-mediated immunity. The humoral immunity included systemic serum antibodies and local IgG and IgA antibodies in the respiratory tract. Such secretory antibodies may interfere with the early events of bacterial attachment and colonization in mouse models. However, in the case of rat models, the cell-mediated immunity play a significant role in preventing mycoplasma infection.

With regard to mycoplasma infections specifically, various strategies have been employed in recent years in an attempt to create an effective vaccine. Salmonella spp. and E. coli mutants which express foreign antigens have been used to promote immunity (Hoiseth & Stocker, 1981), but have also been found to establish a

subclinical infection (Charles & Dougan, 1990). Delivery systems based on E. coli offer more flexibility than Salmonella spp . , firstly, because the genetics of

Salmonella is not as well developed as that of E. coli , and secondly, as it is extensively used to optimize expression of recombinant antigens in E. coli . It was reasoned by the inventor that an E . coli -based antigen-delivery system, used in either an oral or intravenous immunization protocol, would be promising for vaccine development if an effective M. pulmonis antigen was available. Unfortunately, the development of a mycoplasmosis vaccine has been particularly hampered by the lack of such an antigen. The production of a useful antigen from M. pulmonis has been found to be

particularly difficult due to differences in the genetic code between E. coli and M. pulmonis . Mycoplasma utilize the common stop codon, TGA, to code for the amino acid tryptophan (Inamine et al ., 1990; Yamao et al ., 1985). This renders any sizable portion of Mycoplasma DNA untranslatable in E. coli , and the creation an antibody-screening of the expression library using conventional molecular biological techniques is therefore precluded. The present invention arose in part out of an advantageous strategy which allowed both the cloning of an M. pulmonis antigen and the development of an

effective vaccine. The P1 gene of M. pneumoniae has 21 TGA stop codons (Inamine et al . , 1988; Su et al ., 1987) and the antigenic epitope is a very small, 39 bp portion of this gene (Dallo et al ., 1988). The inventors

reasoned that one could randomly fragment genomic M.

pulmonis DNA into fragments small enough to avoid the presence of a TGA stop codon, and yet large enough to encode an antigenic epitope. As demonstrated below, the cloning and expression of an M. pulmonis antigen in E. coli was highly successful, and led to the development of an effective vaccine. The M. pulmonis antigen-encoding DNA was inserted into the lacZ portion of the universal expression vector lambda gt 11 (λgt11). The antigen was then expressed as a fusion protein in lysogenic wild-type E. coli which were used as a novel M. pulmonis vaccine delivery system. One potential drawback to be considered was the

possibility that continuous synthesis of the fusion protein might promote host tolerance of the vaccine.

There was thus a need for a further method to allow close control of in vivo antigen production.

To address this need, a system was developed to regulate antigen generation within the vaccinated host animal. Using this systemm, the production of a

recombinant protein or antigen can be manipulated

according to the particular genetic construct utilized. The whole-animal control system has been designed to stimulate antigen production using a non-toxic inducer. In the present case, the ß-galactosidase-antigen fusion protein is under the regulatory control of elements of the lac operon, and antigen production can thus be stimulated by the addition of the inducer IPTG

(isopropyl-β-D thiogalactopyranoside). Indeed, it was found that the oral administration of a low dose of IPTG to vaccinated animals was a particularly effective way to promote antigen production and to stimulate antibody generation in a controlled manner. IPTG has a small molecular weight (238 Da) and can thus be absorbed and circulated in the body but it is non-toxic and non-metabolized in the animal host (Stryer, 1988).

Where a lac operon construct and an IPTG inducer combination are used, it is contemplated that an IPTG dose of from about 0.001mM to about 10mM will be suitable to induce fusion protein synthesis in vaccinated animals. In using a metallothionein promoter with a zinc (Zn) or copper (Cu) diet, gene expression may be stimulated by feeding with a diet containing cupric and zinc carbonate. It is contemplated that doses of cupric and zinc

carbonate in the order of 1-36mg/kg and 5-180 mg/kg, respectively, will be appropriate in such embodiments (Blalock et al . , 1988).

It must be noted, however, that the novel

vaccination strategy disclosed herein is not limited to the use of E. coli , nor to the use of the lac operon and IPTG. Indeed, a variety of agents, herein termed

"inducible immunizing agents", are contemplated for use with the present invention. These include other

procaryotic cells, such as salmonella, shigella, BCG, Neisseria and Streptomyces; eukaryotic cells, such as yeast and any other eukaryotic cell that is useful in expression of the particular, selected recombinant gene; and also viruses such as vaccinia virus. Likewise, the cellular or viral inducible immunizing agents may contain any antigen-encoding genetic construct that is capable of being induced by a inducer molecule. Suitable

combinations of promoters and inducers include, for example, the metallothionein promoter and Zn/Cu, the trp operon and tryptophan, or the ara operon and L-arabinose. One would, of course, generally chose an inducer molecule which is non-toxic in the animal to be vaccinated, and preferably, one with a relatively long half life. The novel and controlled vaccination strategy disclosed herein is proposed to be universally suitable for use against an extensive variety of diseases. As the antigen-expression vehicle of the present invention is non-invasive and non-virulent, the methods are suitable for use not only in animals, but also in human subjects. Diseases which can be combatted in this manner include, for example, foot and mouth disease, malaria, cholera, polio, salmonellosis, rubella, tuberculosis, hepatitis, enterotoxemia, pseudorabies, Newcastle disease, AIDS and cancer. As discussed above, the present invention represents a marked advancement in the art of

vaccination. For instance, although recombinant

hepatitis B vaccines produced in yeast and E. coli pili vaccine are commercially available, those vaccines are non-living vaccines which will be metabolized, requiring repeat inoculations to maintain the immune response.

The present invention is also envisioned to be of use in the expression of eukaryotic DNA segments and in generating long-lasting and controllable immunity against eukaryotic antigens. Different glycosylation events in eukaryotic and prokaryotic expression systems have been considered to be a possible drawback to vaccine

production in bacteria, particularly as the important antigens of known pathogens are generally glycosylated. However, the importance of glycosylation in stimulating protective immune responses has yet to be established. The expression of hepatitis B virus antigens in

salmonella (Schodel & Will, 1990), and retrovirus antigen in E. coli (Arias et al . , 1986) has been reported.

Therefore, vaccination against both prokaryotic and eukaryotic antigens is contemplated to be possible using the present invention.

The novel approaches to cloning and vaccination described herein are particularly important in several respects. Firstly, the production of highly purified antigens and specific epitopes of interest is now

possible. M. pulmonis antigens have not previously been selectively produced and purified from cultures of the organisms. The developments encompassed in the present invention allow recombinant DNA technology to be employed to produce large quantities of highly purified antigens. Furthermore, the method developed to overcome the

problems caused by the M. pulmonis stop codons represents a generally applicable advance in molecular biological techniques. This method can be used to clone useful fragments, such as antigenic fragments, from any organism or organelle in which a particular codon is interpreted as a stop signal in E. coli . Also, the recombinant DNA technology can be further developed for vaccine generation against species of mycoplasma which cause disease in animals and in humans. The analysis and development of vaccines from pathogenic mycoplasma has previously been hindered by the necessity of using highly enriched media to grow relatively small quantities of these fastidious organisms. The present invention, allowing the production and expression of M. pulmonis antigen(s) in recombinant host cells, overcomes this problem.

Importantly, the novel administration of an inducer molecule to stimulate antigen, and hence antibody, production in vivo results in a vaccine that is safe, effective, non-pathogenic, and controllable. Again, it must be stressed that this aspect of the invention is widely applicable to any vaccine for use in combatting a variety of diseases in both animals and human patients.

Another possible benefit of this new approach is as a novel research tool. Antigenic epitopes of interest from M. pulmonis can be selectively isolated and used to transform a host such as E. coli , where their expression can then be controlled with great flexibility. One then has a potentially powerful tool for analysis of antigen presentation. This approach would allow, for example, the population dynamics of a single foreign epitope to be examined following infection of the host with the

nonpathogenic E. coli . After the bacteria become

established in the host, the administration of IPTG allows the presentation of the experimental epitope to the immune system via a live organism without many of the confounding factors now commonly used such as adjuvant, hapten carrier, or denaturing agents and conditions. The co-administration of a naturally infective dose and IPTG could produce a dose response in the host that would be unique to the specific protein of interest. The

transformed E. coli with the unique method of controlling protein expression in vivo offers a new method of

approaching molecular pathology and antigen presentation.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the

techniques disclosed in the examples which follow

represent techniques found to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE I

CLONING OF AN M. PULMONIS ANTIGEN

A. Materials and Methods 1. Organisms and Growth. Conditions

Mycoplasma pulmonis CT strain (also called T2), originally isolated from mouse lungs with high virulence to mice (Davis et al . , 1985), was grown at 37°C in 1 liter of Chalquest's broth medium. The Chalquest's medium was prepared by adding 21.0g PPLO broth without crystal violet and 5. μg glucose to 900ml of deionized water, the mixture was boiled briefly, and 10ml of 5% trypticase-peptone was added. The solution was then autoclaved and allowed to cool in a 56°C water bath. The final ingredients were then added in the following order: 10ml 1% NAD, 100ml heat -inactivated mycoplasma-free pig serum and 2ml of potassium penicillin at 1,000,000 units/ml. After 72 hours, the mycoplasma were washed three times with phosphate-buffered saline (PBS, 145mM

NaCl, 5.8mM NaH₂PO₄, 20mM Na₂HPO₄, pH 7.4) and pelleted at 17,000 g for 20 minutes at 4°C. Mycoplasma pellets were used either for DNA extraction, or as an antigen source for polyclonal rabbit antiserum.

E. coli Y1090 (Promega Biotech Δlac U169 proA+ Δlon araD139 strA sup F hsd R- hsd M⁺ pMC9) were grown in LB broth with 0.2% maltose at 37 °C overnight, spun and resuspended in one-third of original culture volume of 10mM MgSO₄/PBS as the competent cells. E. coli K-12 C600 (ATCC #23724 F- supE44 lacY1 thr-1 leuB6 mcrB thi-1 tonA21 lambda) was grown in LB broth containing Nalidixic acid (NA) 30μg/ml at 37°C overnight to selected for NA-resistance (Hane et al . , 1969).

2. Generation of Lysogen of E. coli-λgt11

NA-resistant E. coli C600 was grown in 0.2% maltose at 37°C overnight, spun and resuspended in 10mM MgCl₂/LB, lysogenized with the positive recombinant phage at a ratio 10:1 (10 phages to 1 bacterium) at 32°C for 30 minutes and plated out on NA incorporated LB agar medium at 32°C overnight. The next day, 500 single colonies were selected and placed on two NA-containing LB agar plates-. One plate was incubated at 32°C and the other at 42°C. Only colonies that grew at 32°C but not at 42°C were selected as lysogen candidates (Fig. 10). The lysogen candidates were further tested for temperature-dependency by 2 more consecutive single-colony

isolations. Only genetically stable isolates were inoculated into mice orally or intravenously.

3. Antiserum Preparation

Hyperimmune rabbit sera directed against whole

Mycoplasma pulmonis were produced by inoculating 1 × 10¹⁰ C.F.U of MP organisms with complete Freund's adjuvant subcutaneously into 2 rabbits. This was done in three doses at 2-wewk intervals; 2nd and 3rd doses were

combined with incomplete Freund's adjuvant. One week following the final inoculation the rabbits were bled from the ear vein. The sera was collected and pooled, and stored at -20°C. Monospecific MP antigen was

purified using monoclonal antibody coupled to a

Sepharose-4B chromatography column (Lai et al . , 1991). The hyperimmune poiycional sera, monospecific antisera, and pre-immune rabbit sera were absorbed extensively with E. coli Y1090 (intact cells and lysates) before being used in immunological screening of the clone bank. Test sera were incubated at 4°C overnight with intact E. coli Y1090 at a concentration of 1×10¹¹ E. coli cells per 1ml of serum. Aggregations were pelleted by centrifugation at 10,000g for 20 minutes at 4°C.

Absorbed sera were then incubated at 4°C overnight with a lysate suspension of 1×10¹¹ E. coli cells coated on nitrocellulose paper per 1ml of serum. E. coli Y1090 lysates were prepared by passing cells which were washed and suspended in PBS containing 1mM phenylmethylsulfonyl fluoride, through a French pressure cell twice at 15,000 lb/in (Trevino et al . , 1986).

DNA Extraction

Pellets of M. pulmonis were suspended in 2.7ml of PBS, lysed by the addition of 0.3ml of 10% sodium dodecyl sulfate (SDS), and incubated with 20μg of RNAase for 30 minutes at 37°C. Preparations were extracted three times with an equal volume of redistilled phenol (equilibrated with 100mM Tris, 10mM EDTA (TE)) buffer followed by extraction once with chloroform. The DNA was

precipitated with 100% ethanol, dried in a spin vac and resuspended in 50μi of TE (Trevino et al . , 1986).

Construction of Genomic Library

The λgt11 expression library was constructed as described by Huynh (Huynh et al . , 1985) with the

following modifications. The mycoplasmal DNA was

sonicated for 4 minutes at 30 second intervals into 200-500 base pair fragments (bp) to reduce TGA stop codon inclusion. To remove endogenous EcoR1 sites, the

fragment DNA was then methylated with EcoR1 methylase (BRL, Bethesda Research Laboratories, Inc., Gaithersburg, MD). The sheared ends were made flush with T4 DNA polymerase (BRL), and EcoR1 linkers (BRL) were ligated to the blunt ends. After digestion with EcoR1, excess linkers were removed by spun-column chromatography

(Sambrook et al . , 1989). These DNA fragments were collected and precipitated in 100% cold ethanol, spundried, and reconstituted in 10μl of TE buffer. The DNA was kinased and ligated into EcoR1-digested,

dephosphorylated λgt11 DNA (Promega Biotech, Madison, WI) at a molar ratio 1:3 (1mM of λgt11 phage DNA arms to 3mM of Mycoplasma DNA). The DNA was ligated overnight at 14°C with T4 DNA ligase (BRL). Recombinant DNA was packaged to produce viable phage with a lambda (λ) in vitro packaging system (Gigapack, Strategene Razolla, CA). 6. Titration of Recombinant Clones and Efficiency of Cloning

The total number of recombinant clones was

calculated by a 10-fold serial dilution of the

recombinant plaque-formifg phages on an E. coli Y1090-incorporated LB agar plate (100μl of competent cells of E . coli Y1090 thoroughly mixed with 5ml of 0.75g% agar in LB broth at 50°C, and poured onto LB agar plates

[15×100mm] and allowed to solidify.) Five microliters of each serial dilution, in triplicate, was inoculated onto an LB agar plate. After incubation at 37°C overnight, the number of plaques were counted and expressed as plaque-forming unit (PFU) per ml. The probability of cloning any one region was calculated by the formula P=1- (1-f)^N (Clarke & Carbon, 1976), where N = total number of plaques formed; P = probability; and f = size of

fragments (regions) divided by the total genome size. That is, mycoplasma DNA sonicated to ~500bp, and a total genome size [Mycoplasma] of 750kbp would produce an "f" value of f=0.5kbp/750kbo=6.67×10^-4. 7. immunological Screening of the Clone Bank

Recombinant phage (1μl) was plated with 100μl of competent cells ( E. coli Y1090) in top agar (0.75g% agar in LB broth) at 50°C to produce approximately 400-500 plaques per plate. The plates were first incubated at 42°C for 3-4 hours to inactivate C1857 suppressor gene present in λgt11, then removed and carefully overlayed with a dry nitrocellulose filter disk which had been saturated previously in 10mM IPTG. The plates were then incubated for another 3-5 hours at 37°C. The plates were removed to room temperature, quickly marked for

orientation and the filters carefully removed. Plates were chilled at 4°C for 20 minutes if the top agar showed a tendency to adhere to the filter.

The filters were then placed in TBST (10mM Tris-HCl, pH 8.0, 150mM Nacl, 0.05% Tween 20) and rinsed briefly to remove any remnants of agar. Filter disks were blocked in two changes of 0.25% BSA (bovine serum albumin) in TBST for 15 minutes each. The filter disks were

incubated in TBST with a 1:100 dilution of pre-absorbed rabbit anti-MP sera or monospecific rabbit antisera at 37°C for 1-2 hours or 4°C overnight. The filters were then washed in TBST 3 times for 5 to 10 minutes each, and transferred to TBST containing a 1:7500 dilution of goat anti-rabbit antibody conjugated with alkaline phosphatase (Protoblot, Immunoscreening system, Promega, Madison, WI) at 37°C for 30 minutes. Following one rinse, the filter was developed with a substrate solution containing nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in AP buffer (100mM Tris-Cl pH 9.5, 100mM NaCl, 5mM

MgCl₂) for 10-20 minutes. Positive clones appeared as purple plaques on the filters, indicating antibody binding (Promega. 1987). 8. Plaque Purification

The positive recombinant clones were selected from the master plates by using a sterile pasteur pipette to remove an agar plug containing phage particles

corresponding to the signal on the filter disk. The agar plug was incubated in 1ml of SM buffer (0.15M NaCl, 8mM MgSO₄, 1mM Tris-HCl pH 8.0) at 4°C overnight. Positive clones from this solution were then replated and

rescreened with monospecific rabbit antibody probe until virtually all the plaques on the plate produced a strong signal.

B, Results

A recombinant DNA expression library, with a limited frequency of TGA stop codons present in both ends of the MP fragments, was constructed as outlined in Figure 1. Briefly, MP genomic DNA was first randomly sheared into very small pieces which were cloned into the lac Z gene of λgt11 DNA, the unique EcoR1 site of which had been digested with the restriction enzyme and dephosphorylated to decrease the possibility of reforming the original vector. Packaging of the recombinant λgt11-MP DNA yielded 1.6 × 10⁵ PFU/2 μg of DNA when plated on the E. coli lytic strain Y1090.

The mycoplasma genome consists of approximately 750 Kb of DNA (Yamao et al . , 1985). Assuming that random fragmentation results in the generation of random DNA fragments, and that the average cloned fragment is approximately 0.4 kb, then it can be calculated that approximately 10,000 clones would be needed to have a 99% probability of including any given portion of the

mycoplasma genome within the clone bank (Clarke & Carbon, 1976). The library generated consisted of 1.6 × 10⁵ independent clones containing M. pulmonis DNA fragments (80 genome equivalents). Therefore, the yield of recombinant phage should contain the entire mycoplasma genome many times over, allowing for the expression of most M. pulmonis proteins.

The resultant expression library of β-galactosidase fusion proteins was screened using pre-absorbed

polyclonal anti-MP serum. It was found that 0.03% of the clones containing MP DNA inserts produced proteins which were immunologically reactive, with the reactivity among specific clones ranging from very intense color reactions to very light spots.

Forty-eight of the positive clones were spotted in duplicate onto fresh E. coli lawns. Plaques were again screened with pre-absorbed anti-MP serum and with the monospecific antiserum to purified antigen. Only four, termed L111, L112, L113, and L150, reacted with

monospecific antiserum. Reactivity among positive duplicate plaques was equally intense, while the negative plaques were nonreactive. Pre-immune rabbit serum absorbed with E. coli was nonreactive with the genomic library. It is contemplated that immunoscreening of the recombinant phage library with a mycoplasmacidal

monoclonal antibody (Lai et al ., 1991) should identify genomic sequences encoding this protective antigen.

Mycoplasmacidal monoclonal antibodies have been produced by immunization of mice with viable M. pulmonis followed by fusion of spleen cells with myeloma SP 2/0 cells to generate hybridomas (Lai et al . , 1991; incorporated herein by reference). EXAMPLE II

ANALYSIS OF CLONED M. PULMONIS DNA AND ANTIGEN

A. Materials and Methods

1. Gel Electrophoresis and Blotting Analysis of the

Clones

The clone (L150) at second screening was 100% positive to monospecific antisera, and was infected into Y1090 E. coli and grown in 500ml NzCYM media at 37°c with good aeration in a shaker until the culture reached an optical density at 600 nm of 0.5, at which time, the temperature was increased to 42°C. After 20 minutes 0.1M IPTG was added to a final concentration of 1mM, and the cultures were incubated for an additional 2 to 3 hours at 37°C. The cells were harvested by centrifugation at 7000 rpm for 20 minutes at 4°C, and resuspended in 1/25 of the original culture volume in 10mM Tris, 1mM EDTA, 2mM phenylmethylsulfonyl fluoride (PMSF), and an equal volume of 2X sample buffer (0.125M Tris, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.02% bromophenol blue) was added. In addition, 1 ug/ml of pepstatin A and leupeptin was added to inhibit proteolytic enzyme activity which might potentially digest fusion proteins.

Samples were boiled for 5 minutes and 100 to 150μl per lane were loaded on two 7.5%, 1.5mm polyacrylamide gels. After electrophoresis (60 to 90mA), one gel was stained with coomassie blue; the other gel was

transferred to nitrocellulose paper (Towbin et al . ,

1979). After protein transfer, the nitrocellulose was blocked with 0.25% BSA and processed by the same method described in the immunoiogicai screening of the clone bank. The solubilized recombined protein was also run with counter-immunoelectrophoresis (Cho & Ingram 1972). The recombinant protein was placed into the cathodic well and the absorbed rabbit anti-MP or monospecific antiserum was placed into the anodic well. The distance between the two wells was 0.5cm; electrophoretic conditions were 20mA for one hour.

2. DNA Analysis of Recombinant Clones

The recombinant phage DNA of a typical positive clone such as L150 was isolated by a rapid, moderate-scale procedure (Bellomy & Reard, Jr. 1989). The

inserted MP DNA in λgt11 was rapidly amplified directly from bacteriophage plaques using the polymerase chain reaction (PCR) (Dorfman et al . , 1991) with forward 5'-GGTGGCGACGAATAATGGAGCCCG-3' and reverse 5'-TTGACACCAGACCAACTGGTAATG-3' oligonucleotide primers corresponding to vector DNA sequences flanking the EcoR1 cloning site. The size of inserted MP DNA was rapidly analyzed and isolated by electrophoresis in 1% agarose or 5% polyacrylamide gels.

DNA Sequencing

Total recombinant phage DNA was extracted by using plate lysate techniques (Sambrook et al . , 1989) and

Qiazen column purification (Qiazen Inc., Chatsworth, CA). Nucleotides were sequenced by the dideoxy chain

termination method (Biggin et al . , 1983; Sanger et al . , 1977) using Sequenase (U.S. Biochemical, Cleveland, OH) and using the primers described in the previous section. To sequence further into the 529 bp fragment, a second set of primers was synthesized using the initial

sequencing data. Both strands of the entire insert were sequenced. Sequencing reactions were resolved on 6% polyacrylamide field gradient gels (Olsson et al . , 1984) cast with wedge spacer using a bio-rad sequencer.

Nucleic acid and protein computer analyses were performed using the Microgenie Program (Beckman Instrument, Inc., Palo Alto, CA).

B. Results

1. Electrophoretic Analysis of Immunologically Reactive Proteins

To further characterize the MP protein-producing clones identified in Example I, a liquid culture method was used to prepare proteins from the highly reactive clone termed L150. These were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting (Fig. 2). Absorbed monospecific antiserum were found to react with a wide fusion protein band which was distinct from, and slightly above, the β-galactosidase control band. None of the proteins from a control λgt11 lysate reacted with the antibody. Thus, distinct mycoplasma proteins were synthesized in E. coli infected with the recombinant phage.

2. Analysis of Cloned MP DNA Inserts

DNA analyses of the recombinants were performed to examine the size and possible relatedness of the cloned inserts. Because of the single EcoR1 site used as the cloning site in λgt11, the recombinant DNA preparations were digested with EcoR1 endonuclease. This digestion resulted in the cleavage of the λgt11 phage arms (24.1 kbp and 19.6 kbp) away from the complete MP DNA insert. However, since the size of the MP DNA insert was very small, it was difficult to detect on a 1% agarose gel.

The polymerase chain reaction (PCR) was therefore employed using forward and reverse oligonucleotide primers corresponding to λgt11 vector DNA sequence flanking the EcoR1 clone sites of the clones. The amplified inserts products were run on ethidium bromide stained 1% agarose gel when the MP DNA inserts could be readily seen near the 615 bp position (Fig. 3). As the primers used were 37 bp upstream and 46 bp downstream, and as 10 bp of EcoR1 linker must be included on each end of the insert, the actual length of the insert would be 529 bp.

Recently it has been reported that the epitope of the P1 protein in M. pneumoniae contains only 13 amino acids and represents 39 nucleotides of the Pl gene which occur only once in the genome (Dallo et al . , 1988). This Pl protein has been designated the major adhesive

molecule mediating the attachment of M. pneumoniae to respiratory epithelium (Baseman et al . , 1982; Hu et al . , 1982). M. pneumoniae organisms that lack Pl are

avirulent (Baseman et al . , 1982; Krause et al . , 1983). Partially overlapping B cell and T cell epitopes are present within the 13 amino acid segment (Jacobs et al . , 1990), and this segment reacts with Pl cytoadherence blocking monoclonal antibodies and with acute and

convalescent phase sera from M. pneumoniae-infected individuals (Dallo et al . , 1988).

A similar example has been found in the VP1 gene of Foot and Mouth disease, in which 20 amino acids located between numbers 141 to 160 could elicit a protective antibody in cattle and pigs sufficient to protect them against the disease (Bachrach, 1982). In light of these facts, it seemed likely that the cloned M. pulmonis antigen encoded by the 529 bp DNA segment would contain a particularly useful epitope.

Furthermore, the inventors reasoned that β-galactosidase, of molecular weight 114 kilodaltons, may act as a carrier to enhance the animal's immune response to this epitope, as was reported by Altman and Dixon (1989). This

information prompted further studies, the results of which are reported in Example III, which clearly

demonstrated the effectiveness of the cloned antigen as a vaccine component. 3. Nucleotide Sequencing of the Insert Fragments

The nucleotide sequence of the L150 fragment was determined (Fig. 4) and deposited with GenBank (Accession number, M76406). The first TGA stop codon was located at 76 bp followed by two in-frame TAA stop codons 112 and

238 bp downstream. The first 75 bp sequence could encode for a protein of 25 amino acids with a calculated

molecular weight of 3090. β-galactosidase has molecular weight of 114 KDa. Therefore, the total fusion protein would have a molecular weight of about 117 kDa, which is in agreement with the data from SDS-PAGE (Fig. 2).

Following the initial cloning, as reported above, the determination of corresponding DNA sequences should provide direct assessment of protein structural features pertinent to the function of these components. Further investigation and analyses along these lines may also allow the genomic structures and mechanisms underlying mycoplasmal antigenic variation to be elucidated.

EXAMPLE III VACCINATION OF ANIMALS AGAINST MYCOPLASMOSIS A. Materials and Methods

Mice

MP-free BALB/c female mice were purchased from

Harlan Sprague-Dawley, Inc. (Indianapolis, Indiana). The care of the animals was in accordance with institutional guidelines, and the mice were maintained in a specific-pathogen-free environment.

2. Immunization with Fusion Proteins

One (L150) of the four positive clones (L111, L112, L113, and L150) which identified with the monospecific antiserum was used to infect Y1090 E. coli , and IPTG was added to induce β-galactosidase fusion protein synthesis. The fusion protein was purified in a 7.5% polyacrylamide gel, fixed and stained with Coomassie blue to determine the size of the β-galactosidase fusion protein. The total size of the fusion protein was found to be 117 kDa. The β-galactosidase portion of the fusion protein

accounts for 114 kDa.

The fusion protein band was excised and

electroeluted (Hunkapiller et al . , 1983). 10-20μg of the fusion protein was dissolved in 0.1 ml elution buffer and combined with an equal volume of Freund's adjuvant. The mixture was injected subcutaneously into 22 mice on three occasions at intervals of two weeks. CFA was used in the first injection and IFA was used in the second and third injections. The fourth and the last vaccination involved intranasal inoculation of 10-20μg protein. One week later, three mice were killed by ether inhalation, and the sera and tracheolung lavages were collected for immunofluorescent antibody (IFA) and enzyme linked immunosorbent (ELISA) assays (Lai et al . , 1990). Spleen cells were harvested for the lymphocyte transformation test to measure T cell-mediated immunity (Naot et al . , 1982; Lai et al . , 1989). Values are expressed as

stimulation indices, ³H-thymidine incorporation, cpm, test divided by control (media alone). The remainder of the 19 vaccinated mice were divided into three subgroups (7, 6 and 6 in each subgroup) and challenged with 1 × 10³, 5 × 10⁴ and 1 × 10⁶ C.F.U. of MPT2 and killed two weeks later (Table 1).

TABLE 1

Experimental Design For Various Vaccines And Treatments

Group No. Vaccine/ VaccinaIPTG No. Mice Non-challenge No. Mice Used for M.

Mice Route tion in Used pulmoni s Challenge

Schedule DrinkFor

ing BacterWater ial

IsolaNo

tion/ Mice for

Dot Blot Ab

Analysis Analysis

No. Mice No. Mice

Lymphofor Ab 1×10³ 1×10⁴ 1×10⁶ cyte Analysis CFU CFU CFU

Transformation

1 22 Fusion 5xtwo - 0 3 3 19 7 6 6

Protein/ week

SC, IN interval

2 5 Lysogen/ Day 0 - 5 (1 5 5 N/A^a N/A N/A N/A

Oral every 10

days)

3 5 Lysogen/ Day 0 - 5 (1 5 5 N/A N/A N/A N/A

IV every 10

days)

4 23 Lysogen/ Days 0, + 2 2 2 21 7 7 7

Oral 14, 28

5 15 Lysogen/ Days 0, + 2 2 2 13 5 4 4

IV 14, 28

TABLE 1 ( CONTINUED)

Experimental Design For Various Vaccines And Treatments

Group No. Vaccine/ VaccinaIPTG No. Mice Non-challenge No. Mice Used for M.

Mice Route tion in Used pulmonis Challenge

Schedule DrinkFor

ing BacterWater ial

IsolaNo

tion/ Mice for

Dot Blot Ab

Analysis Analysis

No. Mice No. Mice

Lymphofor Ab 1×10³ 1×10⁴ 1×10⁶ cyte Analysis CFU CFU CFU Ti ansformation

6 15 Lysogen Days 0, + 2 2 2 13 5 4 4 without 14, 28

insert

7 15 - - - - - 0 0 0 15 5 5 58 5 - - -- - 5 5 5 N/A N/A N/A N/A9 15 Lysogen/ Days 0, - 15 5 5 10 4 3 3

Oral 14, 28

10 15 Lysogen/ Days 0, - 15 5 5 10 4 3 3

IV 14, 28

Not assayed

3. Immunization with Lysogen of E. coli λgt11

Live vaccines have been reported to elicit better protection than killed organisms (Cassell & Davis 1978). The inventor therefore cloned the protective MP antigen-encoded DNA into lambda gt11, induced lysogeny in a wild-type Escherichia coli C600 strain, and colonized mice with the bacteria. For use in immunization, an overnight broth culture of bacteria was diluted to an O.D. 600 of 0.4 with LB broth containing Nalidixic Acid, grown at 32°C (with shaking) to an O.D. 600 of 0.9, and IPTG was added to a final concentration of 1.0 mM. The culture was

centrifuged and the bacteria were resuspended in PBS to an appropriate optical density (O.D. 600 of 1 = 2 × 10 C.F. U/ml). Mice were orally inoculated with 0.2ml bacterial suspension by using feeding needle, or were intravenously injected with 0.2ml bacterial suspension per tail vein. Groups of mice were immunized as

indicated in Table 2.

The animals (except those in groups 9 and 10 of Table 2) were fed low dose of IPTG (0.1mM) in their drinking water for 2-3 days after each inoculation. One mouse each from groups 2 and 3 were was sacrificed every 10 days after inoculation, samples from upper intestine, caecum, colon, lung, spleen, liver were collected, and plated on blood agar media. The bacteria colonies were plated out individually using the toothpick method on two

LB agar media containing nalidixic acid, one incubated at 32°C and the other at 42°C.

IPTG imprinted nitrocellulose paper was blotted from the 32°C plate and screened using the same methods described in the immunoiogicai screening of the clone bank. Serum was also collected for antibody titer by IFA. Lymphocyte-transformation assays of spleen ceils of control mice (group 8) and vaccinated, but not challenged mice (group 3, 4), were conducted as described by Naot et al . (1982) and Lai et al . (1989), using purified MP antigen. Two mice each from groups 4, 5 and 6, and five mice each from group 8, 9, and 10 were killed and the sera and tracheolung lavages were collected for IFA and ELISA titers. Group 7 and the rest of vaccinated animal in groups 4, 5, 6, 9 and 10 were divided into three subgroups (Table 1) and challenged with various doses of MP (1 × 10³, 5 × 10⁴, and 1 × 10⁶) at day 38, and killed on day 52.

The criteria for evaluation of the vaccine efficacy are based on: (1) Microbiological evaluation to compare the number of M. pulmonis recovered from vaccinated-challenged (group 1, 4, 5, 6, 9, and 10) with numbers from nonvaccinated-challenged group (group 7);

(2) Pathological evaluation (Lai et al . , 1990), to compare histopathological change in lungs of vaccinated-challenged with nonvaccinated-challenged group (group 7);

(3) Serological evaluation to compare serum and

tracheolung lavages antibody titers with vaccinated alone (groups 1, 2, 3, 4, 5, 6, 9, and 10) and control normal mice (group 8); and (4) Proliferation responses of lymphocytes to MP antigen to compare vaccinated groups 2 and 3, and vaccinated nonchallenged group 4, 5, 6, 9, and 10 with normal control mice (group 8). Thus, spleen cells (4 × 10⁵/ml) were stimulated with 30 μg MP antigen or 2.5 μg concanavalin A (non-specific T cell nitrogen) per well of microtiter plates. ³H-thymidine

incorporation (cpm) 72 hours later was the measure of Droliferation (Lai et al . , 1989). B . Results

Immunogenicity of Antigen Generated from Recombinant Phage Clones

The specificity of mycoplasma antigens expressed from cloned genomic fragments was firstly examined in vi tro by counter immunoelectrophoresis. A clear white precipitation line was formed between the fusion protein well and the absorbed polyclonal rabbit anti-MP well.

Twenty-two mice were then immunized with 10-20 μg of the fusion proteins. The mice produced high levels of IgG and IgM antibodies in their sera (Fig. 5) and IgG and IgA antibodies in their tracheolung lavages by IFA and

ELISA (Fig 6; Table 2). These results demonstrated that mycoplasmal antigens generated from a recombinant phage were able to induce specific antibodies in naive animals. The vaccinated mice were also shown to be completely protected against a challenging dose of 1 × 10³ C.F.U, and there was still significant protection against higher challenging doses (Fig. 7). These results demonstrated that mycoplasmal antigens generated from a recombinant phage were able to induce specific antibodies in naive animals.

TABLE 2

Humoral Immune Response (IFA and ELISA) In Mice Vaccinated

With Fusion Protein Or Lysogen And Challenged With M. Pulmonis

Group TreatChallIFA ELISA

ment enged

Tracheolung lavages Serum

Serum IgA IgG IgM IgG

PreMean PreMean PreMean PreMean Pre Mean vatiter^b vatiter^b vatiter^b vatiter^b vatiter^b lence^a lence^a lence^a lence^a lence^a

1 Fusion - 3/3 1000=0 3/3 100±0 3/3 83±24 3/3 1000±0 3/3 1000±

Protein + 19/19 10,000±0 19/19 190±278 19/19 121±89 19/19 1842± 19/19 424

1630 5947±

424

2 Lysogen - 5/5 100±0 N/A N/A N/A N/A N/A N/A N/A N/A

/Oral

3 Lysogen - 5/5 10,000=0 N/A N/A N/A N/A N/A N/A N/A N/A

/IV

4 Lysogen - 2/2 1000±0 2/2 100±0 2/2 100±0 2/2 1000±0 2/2 750±

/Oral + 21 /21 10,000=0 21/21 219±311 21/21 241±313 21/21 1159± 21/21 250

844 3213 ±

3976

5 Lysogen - 2/2 10,000±0 2/2 55±45 2/2 100±0 2/2 1000±0 2/2 1000±0

/IV + 13/13 10,000±0 13/13 61±45 13/13 133±110 13/13 2500± 13/13 4583±

3354 3569

6 Lysogen - 0/2 0 0/2 0 0/2 0 0/2 0 0/2 0

without + 13/13 1000±0 13/13 155±32 13/13 100±21 13/13 890± 13/13 2500± Insert 110 310

TABLE 2 (CONTINUED)

Humoral Immune Response (IFA and ELISA) In Mice Vaccinated

With Fusion Protein Or Lysogen And Challenged With M. Pulmonis

Group TreatChallIFA ELISA

ment enged

Tracheolung lavages Serum

Serum IgA IgG IgM IgG

PreMean PreMean PreMean PreMean PreMean vatiter^b vatiter^b vatiter^b vatiter^b vatiter^b lence^a lence^a lence^a lence^a lence^a

7 Non- + 15/15 1000±0 15/15 145±33 15/15 105±17 15/15 1000±0 15/15 3000± vaccina 260 ted

8 Non- - 0/5 0 0/5 0 0/5 0 0/5 0 0/5 0 vaccina

ted

9 Lysogen^c - 0/5 0 0/5 0 0/5 0 0/5 0 0/5 0

/Oral + 10/10 1000±0 10/10 130±41 10/10 150±31 10/10 505±20 10/10 2010±

305

10 Lysogen - 0/5 0 0/5 0 0/5 0 0/5 0 0/5 0 d/IV + 10/10 1000±0 10/10 100±25 10/10 121±51 10/10 862± 10/10 3000±

720 1050 ^a Number positive/total number animals.

b Mean ± standard deviation value presented. Titer is expressed as the reciprocal of the highest dilution giving a positive reaction in IFA or ELISA tests.

c Mice vaccinated with lysogen orally or intravenously but no feeding IPTG in drinking water.

d N/A, not assayed.

2. Recovery, Immunogenicity and Ability to Vaccinate, of Lysogenic E. coli

E. coli C600 were unable to multiply extensively int he host, but did establish colonization in tissues such as lung, cecum or colon. A large number of transformed lysogenic E. coli were recovered from the cecum and colon (1 × 10⁷ and 1 × 10⁸ CFU/ml respectively in group 2) of the mice, following oral inoculation at 40 and 50 days (even sever months after inoculation in another

experiment) as demonstrated in blood agar by Western blot analysis. However, no bacteria were isolated from spleen, liver or lung in the group of animals that were inoculated orally. In contrast, animals inoculated intravenously yielded large numbers of bacteria from the lung (1 × 10⁷ CFU/ml) and a smaller number from the spleen and liver (1 × 10³ and 1 × 10⁴ CFU/ml,

respectively, group 3). No bacteria were found in the cecum or colon of intravenously inoculated animals. The serum antibody titers (IFA) were greater than 1:10,000 in IV injected animals versus 1:100 in orally inoculated animals. There were no MP antibody titers found in group 6 animals which were vaccinated with E. coli lysogenized with lambda gtll without an insert. Antibodies against ß-galactosidase were measured with ELISA using ß-gal as an antigen. They were found in all mice in group 2 and

3, and the vaccinated-nonchallenged groups 4, 5 and 6. This indicated that the ß-gal protein had been expressed in vivo and could elicit antibody production. However, no antibodies against ß-galactosidase were found in groups 9 and 10. The vaccinated but unchallenged mice (groups 2, 3, 4, 5, 6, 9, and 10) showed no

histopathologic changes in the lung. This result

indicates that the transformed lysogenic E. coli are no: pathogenic for mice. The vaccinated BALB/c mice (groups 4, 5, 6, 9, and 10) and nonvaccinated (group 7) were divided into three subgroups and were challenged with various doses of highly virulent MPT2. The MPT2 results are shown in Figures 3, 4, and 5. All vaccinated mice in groups 1, 4, and 5 were protected at the 1 × 10³ CFU dose, i.e., no MP organisms were recovered from tracheolung lavages

(Figure 7) and no histopathological changes were observed in lung sections (Figure 8). These data indicate that all mice were protected at the 'natural infective dose. Significant protection (p < 0.05) was also observed even with the highest challenge dose of lxlO⁶ CFU of MP. In contrast, the groups 6, 7, 9, and 10 animals were not protected against any challenging dose of MP. Data from groups 6, 9, and 10 were similar to that of group 7 which is presented in Figure 7.

The group 5 mice injected IV with lysogenized

E. coli demonstrated the greatest protection against mycoplasma colonization at all challenge doses (Figures 7 and 8). They also developed the highest serum

immunoglobulin levels as determined by ELISA (Table 2). The group 4 mice receiving the oral vaccination were protected as well as group 5 mice when the challenge dose was 1 × 10³ CFU (Fig. 7), but there was less protection if the challenged dose was higher (5 × 10⁴ or 10⁶ CFU) (Fig. 7). They produced more IgA and IgG antibodies in traceolung lavages than group 5 mice. Histopathologic lesions were calculated by a non-parametric grading index score (lai et al . , 1990). Zero is equal to no lesions and one would equal total

pneumonic evolvement with increments of 0.1. The average of five trials is shown in Fig. 8. As can be seen from the figure ail the groups were protective with the greatest protective capability obtained by the

lysogenic/IV method (group 5).

All three groups of vaccinated mice (1, 4 and 5) are capable of eliciting a cellular mediated immunity as judged by the stimulated index. Stimulated index is calculated by dividing the test CPM by the control CPM (Fig. 9). Group 8 is the control group. It is known that specialized cells present in the gut are associated with the uptake of macromolecules (Neutra et al . , 1987). There is also a mucosal

immunoiogicai network with a sub-population of lymphoid cells that can immigrate from the intestine to other distant mucosae, such as the respiratory tract,

(McDermott & Bienenstock, 1979). These facts, taken together with the experimental evidence of immune

response in serum, salivary gland and lung after oral immunization (Czerkinsky et al . , 1991), suggested that live peroral vaccines may be good candidates for the prevention of murine respiratory mycoplasmosis.

The results of the approach described herein suggest various applications. The first is to produce selected mycoplasma antigens in large quantities by using a bacterial host. Prior to this invention, analysis and development of vaccines from pathogenic mycoplasma has been hindered by the necessity of using highly enriched media to grow relatively small quantities of these fastidious organisms. Therefore, the inventors reasoned that the identification and large-scale production of specific antigens for vaccines to control mycoplasmal infections would be ideal using recombinant DNA

techniques.

The second application described herein is the production of a highly purified epitope of interest. The antigen of MP has not previously been selectively

produced and purified from cultures of the organisms. However, as shown herein, recombinant DNA technology using E. coli can produce large quantities of highly purified epitopes recognized by monoclonal antibodies. This approach is useful to generate vaccines against species of mycoplasma which cause disease in other animals and in humans. Many of the earlier mycoplasma vaccines were inadequate. To achieve an adequate titer, a live vaccine is apparently required. The use of temperature-sensitive mutants (Lai et al . , 1990) led to the objection that reversion to the wild type is always possible. This new method of the invention overcomes these objections by offering a vaccine that is safe, effective, non-pathogenic, and controllable. Moreover it can be given orally, which is an advantage for large scale immunity, including herds of animals and even the human population. This approach allows the observation of the

population dynamics of a single foreign epitope after infecting the host with the nonpathogenic E. coli . After the bacteria becomes established in the host the

administration of IPTG allows the resentation of the experimental epitope to the immune system via a live organism without many of the confounding factors now commonly used such as adjuvant, hapten carrier, or denaturing agents and conditions. The coadministration of a naturally infective dose and IPTG is envisioned to produce a dose response in the host that would be unique to the specific protein of interest. The transformed E. coli and the unique method of controlling protein expression in vivo offers a new method of studying molecular pathology and antigen presentation issues in infectious disease. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT:

NAME: BOARD OF REGENTS, THE UNIVERSITY OF

TEXAS SYSTEM STREET: 201 West 7th Street

CITY: Austin

STATE: Texas

COUNTRY: United States of America POSTAL CODE: 78701

TELEPHONE NO: (512)499-4462

TELEFAX: (512)499-4523

(ii) TITLE OF INVENTION:

MYCOPLASMA PULMONIS ANTIGENS AND METHODS AND COMPOSITIONS FOR USE IN CLONING AND VACCINATION (iii) NUMBER OF SEQUENCES: 6

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: ARNOLD, WHITE & DURKEE

(B) STREET: P.O.BOX 4433

(C) CITY: HOUSTON

(D) STATE: TX

(E) COUNTRY: USA

(F) ZIP: 77210 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: FLOPPY DISK

(B) COMPUTER: IBM PC COMPATIBLE

(C) OPERATING SYSTEM: PC-DOS/MS-DOS;

FORMAT: ASKII

(D) SOFTWARE: WORDPERFECT 5.1 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: Unknown

(B) FILING DATE: 13 September 1993

(C) CLASSIFICATION: UNKNOWN

(vii) PREVIOUS APPLICATION DATA:

(A) APPLICATION NUMBER: 07/945,810

(B) FILING DATE: 16 SEPTEMBER 1992

(C) CLASSIFICATION: UNKNOWN

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: PARKER, DAVID L.

(B) REGISTRATION NUMBER: 32,165

(C) REFERENCE/DOCKET NUMBER: UTFD309PCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 512-320-7200

(B) TELEFAX: 512-474-7577 (2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 544 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO :1:

AGCCCGTCAG TATCGTTAAA CAGATGGACA TTCTGCGACA CCAGAGCAAC CTGGTTACGT 60 AACGACGCCA GGGTATACTC GCGCAGATCG TGACCATCCA TCAGGATTTC GCCTTCATCA 120

ATATCGTAAA AACGCGTGAT CAGGCTGGCG ATGGTTGATT TACCCGAACC AGAGCGTCCA 180

ACCAGAGCAA CCGTCTTCCC TGCCGGAATT TTCAGGTTGA TGTTACGCAA TGCAGGTACG 240

TCACGTCCCG GATAAGTAAA GGTGACATTG CGGAATTCCA CGTCGCCAGT CGCACGCTCG 300

ATCACGCGCT TACCTTCATC TTTCTCCTGC TCACTGTCCA GAATGGTAAA CAGCGTCTGA 360 CAAGCCGCCA TACCGCGCTG GAACTGGGCG TTAACGTTGG TCAGCGATTT CAGCGGACGC 420

ATCAGTGCAA TCATTGAAGA GAAAACAACG GTAATCGTAC CGGCAGTCAG GCTATCCATG 480

ACACTTGGGA AGCTCGCCGC ATACAGAACA AACGCCAGCG CCAAAGAGGC GATCAGCTGA 540 ATGA 544

(3) INFORMATION FOR SEQ ID NO:2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 37 amino acid residues

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Leu Asn Arg Trp Thr Phe Cys Asp Thr Arg Ala Thr Trp Leu Arg Asn 1 5 10 15

Asp Ala Arg Val Tyr Ser Arg Arg Ser Trp Pro Ser Ile Arg Ile Ser

20 25 30

Pro Ser Ser Ile Ser

35 (4) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 amino acid residues

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Lys Arg Val Ile Arg Leu Ala Met Val Asp Leu Pro Glu Pro Glu Arg 1 5 10 15

Pro Thr Arg Ala Thr Val Phe Pro Ala Gly Ile Phe Arg Leu Met Leu

20 25 30 Arg Asn Ala Gly Thr Ser Arg Pro Gly

35 40

(5) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 96 amino acid residues

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Val Lys Val Thr Leu Arg Asn Ser Thr Ser Pro Val Ala Lys Ser Ile 1 5 10 15

Thr Arg Leu Pro Ser Ser Phe Set Cys Ser Leu Ser Arg Met Val Asn

20 25 30 Ser Val Trp Gln Ala Ala Ile Pro Arg Trp Asn Trp Ala Leu Thr Leu

35 40 45

Val Ser Asp Phe Ser Gly Arg Ile Ser Ala Ile Ile Glu Glu Lys Thr

50 55 60 Thr Val Ile Val Pro Ala Val Arg Leu Ser Met Thr Leu Gly Lys Leu

65 70 75 80

Ala Ala Tyr Arg Thr Asn Ala Ser Ala Lys Glu Ala Ile Ser Trp Met

85 90 95

(6) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

GGTGGCGACG AATAATGGAG CCCG

24

(7) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

TTGACACCAG ACCAACTGGT AATG

24

Claims

CLAIMS 1. A DNA segment encoding an M. pulmonis antigen.

2. The DNA segment of claim 1, wherein the encoded M. pulmonis antigen includes an M. pulmonis antigen amino acid sequence essentially as set forth in seq id no:2, or a biologically functional equivalent thereof.

3. The DNA segment of claim 2, wherein the encoded M. pulmonis antigen includes an M. puimonis antigen amino acid sequence essentially as set forth in residues 1 through 25 of seq id no:2, or a biologically functional equivalent thereof.

4. The DNA segment of claim 2, defined as comprising the M. pulmonis antigen-encoding nucleic acid sequence as set forth in seq id no:1, or a biologically functional equivaient thereof.

5. The DNA segment of claim 4, defined as comprising the M. pulmonis antigen-encoding nucleic acid sequence as set forth in residues 1 through 75 of seq id no:1, or a biologically functional equivalent thereof.

6. The DNA segment of claim 1, wherein antigen coding DNA sequences are positioned adjacent to and under the control of a promoter.

7. The DNA segment of claim 6, wherein antigen coding sequences are under the control of an inducible promoter.

8. The DNA segment of claim 7, wherein the promoter is one that is inducible by IPTG, zinc, copper, tryptophan, or L-arabinose.

9. The DNA segment of claim 7, wherein the inducible promoter is a lacZ, metallothionein, tryptophan, or ara promoter.

10. The DNA segment of any one of claims 1-9, further defined as a recombinant vector.

11. A recombinant host cell incorporating a recombinant DNA segment comprising the DNA segments of any of claims 1 through 10.

12. The recombinant host cell of claim 11, wherein the host cell is salmonella, shigella, BCG, citrobacter, neisseria, streptomyces, or E. coli .

13. The recombinant host cell of claim 12, wherein the host ceil is E. coli .

14. A recombinant virus incorporating a recombinant DNA segment comprising the DNA segments of any of claims 1 through 10.

15. The recombinant virus of claim 14, wherein the virus is vaccinia virus.

16. A nucleic acid segment which comprises at least a ten nucleotide long stretch which corresponds to the nucleic acid sequence shown in seq id no:1.

17. The nucleic acid segment of claim 16, further defined as comprising at least a twenty nucleotide long stretch which corresponds to the nucleic acid sequence of seq id no:1.

18. The nucleic acid segment of claim 17, further defined as comprising at least a thirty nucleotide long stretch which corresponds to the nucleic acid sequence of seq id no:1.

19. The nucleic acid segment of claim 16, further defined as comprising a nucleic acid fragment of up to 200 basepairs in length.

20. The nucleic acid segment of claim 19, further defined as comprising a nucleic acid fragment of up to 100 basepairs in length.

21. The nucleic acid segment of claim 20, further defined as comprising a nucleic acid fragment of up to 50 basepairs in length.

22. The nucleic acid segment of claim 15, further defined as a DNA segment.

23. A method for expressing an M. pulmonis antigen in a recombinant host cell comprising:

(a) preparing a recombinant host cell in

accordance with claim 11; and

(b) culturing the transformed host cell to

express the M. pulmonis antigen.

24. The method of claim 23, wherein the transformed host cell is stimulated to express the M. pulmonis antigen by contacting said host cell with an inducer.

25. A method for preparing an M. pulmonis antigen, comprising:

(a) expressing an M. pulmonis antigen in a

recombinant host cell in accordance with claim 23; and

(b) purifying said M. pulmonis antigen from said recombinant host cell relative to its natural state.

26. An M. pulmonis antigen, prepared by the process of claim 25.

27. An M. pulmonis antigen including within its amino acid sequence an amino acid sequence essentially as set forth in seq id no:2, or a biologically functional equivalent thereof.

28. The M. pulmonis antigen of claim 27, wherein the M. pulmonis antigen includes within its amino acid sequence an amino acid sequence essentially as set forth in residues 1 through 25 of seq id no:2, or a

biologically functional equivalent thereof.

29. The M. pulmonis antigen of claim 27, further defined as a fusion protein.

30. The M. pulmonis antigen of claim 29, further defined as a ß-galactosidase fusion protein.

31. An antibody directed against the protein of claim 27.

32. The antibody of claim 31, wherein the antibody is a monoclonal antibody.

33. A pharmaceutical composition comprising an M.

pulmonis antigen in a pharmacologically acceptable vehicle.

34. The pharmaceutical composition of claim 33, wherein the M. pulmonis antigen is expressed by a recombinant host cell or a recombinant virus.

35. A method of cloning a gene encoding a protein comprising:

(a) obtaining a sample of DNA suspected to

encode the protein;

(b) fragmenting said DNA into pieces smaller than the expected size of the gene; (c) generating an expression library from said small DNA pieces; and

(d) screening said expression library for the presence of the protein.

36. The method of claim 35, wherein the expression library is screened with an antibody.

37. The method of claim 36, wherein the protein is an antigenic peptide fragment.

38. The method of claim 35, wherein the DNA sample includes a codon intended to encode an amino acid, which codon is interpreted as a stop codon in the host cell.

39. The method of claim 38, wherein the codon intended to encode an amino acid is intended to encode tryptophan.

40. The method of claim 39, wherein the codon intended to encode tryptophan is TGA.

41. The method of claim 38, wherein the DNA sample is genomic DNA.

42. The method of claim 41, wherein the genomic DNA sample is obtained from M. pulmonis .

43. The method of claim 38, wherein the DNA sample is obtained from mitochondria.

44. The method of claim 35, wherein the small DNA pieces are in the order of between about 100 base pairs long to about 600 base pairs long.

45. The method of claim 35, wherein the expression library is generated using the vector λgt11 and E. coli .

46. A method of vaccinating an animal, comprising:

(a) preparing an inducible immunizing agent which is capable of being induced to express an antigen against which an immune response is desired;

(b) administering to the animal said inducible immunizing agent; and

(c) administering to the animal an inducer m an amount effective to induce the

immunizing agent to express the antigen.

47. The method of claim 46, wherein the inducible immunizing agent is a recombinant host cell.

48. The method of claim 47, wherein the recombinant host cell is salmonella, shigella, BCG, citrobacter neisseria streptomyces, or E. coli .

49. The method of claim 48, wherein the recombinant host cell is E. coli .

50. The method of claim 49, wherein the E. coli is lysogenic E. coli .

51. The method of claim 46, wherein the inducible immunizing agent is a recombinant virus.

52. The method of claim 51, wherein the recombinant virus is vaccinia virus.

53. The method of claim 46, wherein the inducer is zinc, copper, tryptophan, L-arabinose, or IPTG.

54. The method of claim 53, wherein the inducer is IPTG.

55. The method of claim 46, wherein the inducer is repeatedly administered at time intervals of from about 2 weeks to about 24 weeks.

56. The method of claim 46, wherein the inducible immunizing agent is capable of being induced to express two or more antigens against which an immune response is desired.

57. The method of claim 47, further defined as a method for vaccinating against mycoplasmosis, wherein the recombinant host cell is prepared in accordance with claim 11.