WO1994006911A2 - Antigenes de mycoplasma pulmonis, et procedes et compositions destines a etre utilises dans le clonage et la vaccination - Google Patents

Antigenes de mycoplasma pulmonis, et procedes et compositions destines a etre utilises dans le clonage et la vaccination Download PDF

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Publication number
WO1994006911A2
WO1994006911A2 PCT/US1993/008744 US9308744W WO9406911A2 WO 1994006911 A2 WO1994006911 A2 WO 1994006911A2 US 9308744 W US9308744 W US 9308744W WO 9406911 A2 WO9406911 A2 WO 9406911A2
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antigen
pulmonis
dna
coli
recombinant
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WO1994006911A3 (fr
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Wayne C. Lai
Raymond J. Macdonald
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/30—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • 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
  • Mycoplasma pulmonis is an important rodent pathogen which primarily affects the respiratory and urogenital systems. M. pulmonis is the causative agent of
  • 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
  • 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.
  • 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).
  • 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.
  • complications arising from vaccination with viable wild-type M. pulmonis organisms include
  • subclinical infection can be detected in tissue such as the liver and spleen (Charles &. Dougan, 1990).
  • 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.
  • 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
  • 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 .
  • the P1 gene of M. pneumoniae has 21 TGA codons (Su et al ., 1987;
  • 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 M. pulmonis antigen
  • 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.
  • 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
  • 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
  • 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.
  • 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.
  • 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
  • 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.
  • the inducible immunizing agent will contain a genetic construct wherein the immunogen-encoding DNA is
  • 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.
  • the immunizing agent 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.
  • 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.
  • Important aspects of the present invention concern isolated DNA segments and recombinant vectors encoding a M. pulmonis antigen, and recombinant host cells
  • 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.
  • 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
  • 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.
  • 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.
  • metallothionein promoter which is induced by zinc or copper (Zn/Cu);
  • tryptophan operon induced by the amino acid tryptophan
  • the ara operon induced by the sugar L-arabinose
  • 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.
  • inducer molecules such as lactose, and more particularly, the synthetic inducer IPTG.
  • 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,
  • the cloned antigen contains one or more codons, such as TGA tryptophan codons in M.
  • 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.
  • biologically functionally equivalent proteins or peptides may be created, for example, via the application of recombinant DNA
  • 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
  • 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:
  • 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).
  • amino acid 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
  • 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.
  • 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);
  • 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.
  • 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.
  • the deliberate modification or 'genetic engineering' of antigen sequences is contemplated by certain aspects of the present invention.
  • 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.
  • 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.
  • 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
  • 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.
  • a codon for tyrosine UAC or UAU
  • 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
  • polypeptides may encode larger polypeptides which nevertheless include sequences encoding an
  • 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.
  • 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.
  • 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 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.
  • 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.
  • probes may be used in a variety of assays for detecting the presence of complementary sequences in a given sample.
  • they may be in the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
  • nucleotides in length allows the formation of a duplex molecule that is both stable and selective.
  • 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.
  • 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
  • nucleotide sequences of the invention may be used for their ability to selectively form duplex
  • nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization.
  • an appropriate means such as a label
  • radioactive, enzymatic or other ligands such as avidin/biotin, which are capable of giving a
  • an enzyme tag such as a urease, alkaline phosphatase or peroxidase, instead of
  • 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.
  • hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase.
  • the test DNA or RNA is adsorbed or
  • 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 , shigella ⁇ , BCG, Citrobacter faundii , Neisseria
  • gonococcus 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.
  • eucaryotic cells such as yeast, retrovirus, vaccinia, baculovirus, adenovirus, pox virus, hematopoietic stem cells or muscle cells, is also preferred.
  • 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.
  • the term 'inducible' means that the coding sequence of the exogenous DNA is
  • invention is not limited to inducible host cells inccrporating M. pulmonis antigen DNA, but rather
  • 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.
  • Zn/Cu zinc or copper
  • 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.
  • an antigen such as an M. pulmonis antigen
  • a recombinant host cell 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.
  • 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.
  • 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
  • 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
  • M. pulmonis antigen or fusion protein, such as
  • ß-galactosidase-antigen fusion protein purified relative to its natural state.
  • 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
  • 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.
  • the present invention concerns antibodies, and particularly, monoclonal
  • mAb monoclonal antibody
  • 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
  • Resultant hybridomas may then be screened for antibody production, for example, by
  • 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
  • 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
  • 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.
  • 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
  • prokaryotic host cells for use in accordance herewith include, for example, salmonella, shigella, BCG,
  • a preferred combination for use in an inducible vector is to employ a lac operon genetic construct and the inducer IPTG.
  • 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.
  • 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.
  • a parenteral route for example, intranasally, subcutaneously, or most preferably, intravenously.
  • the doses of inducible host cells suitable for vaccination of ail animals will range from about 1 ⁇ 10 1 CFU to about 1 ⁇ 10 10 CFU.
  • the dosage of the lysogenic host cells may be varied over such a wide range as E. coli multiply every 20-30 minutes.
  • 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.
  • 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.
  • the inducer molecule will be IPTG, which may be straightforwardly supplied to the animal in the drinking water.
  • 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 .
  • 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.
  • 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.
  • pharmacologically acceptable vehicles 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.
  • 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.
  • 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,
  • enterotoxemia pseudorabies, Newcastle disease and AIDS.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • the DNA includes a codon intended to encode an amino acid and which codon is interpreted as a stop codon in E. coli .
  • other examples include yeast mitochondria and neurospora mitochondria.
  • mitochondria During evolution, mitochondria have evolved to utilize a slightly different genetic code from that generally employed by eukaryotic and prokaryotic cells.
  • 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; Schunberger &
  • 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
  • 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
  • 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 ⁇ gt11 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).
  • 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.
  • other screening methods may also be
  • 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.
  • 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 ( ⁇ gt11) phage. This was used in turn to infect E. coli and resulted in the expression of
  • 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
  • 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).
  • FIG. 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,
  • FIG. 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 10 Strategy to construct a lysogenic E. coli .
  • a small fragment of Mycoplasma pulmonis DNA was ligated in the lacZ portions of a ⁇ gt11 phage which in turn
  • 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.
  • 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;
  • 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.
  • retroviral systems 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.
  • 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).
  • 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
  • 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.
  • the cell-mediated immunity play a significant role in preventing mycoplasma infection.
  • 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
  • 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
  • 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
  • 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 have 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).
  • 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.
  • the M. pulmonis antigen-encoding DNA was inserted into the lacZ portion of the universal expression vector lambda gt 11 ( ⁇ gt11). 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.
  • recombinant protein or antigen can be manipulated
  • the whole-animal control system has been designed to stimulate antigen production using a non-toxic inducer.
  • 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
  • IPTG isopropyl- ⁇ -D thiogalactopyranoside
  • an IPTG dose of from about 0.001mM to about 10mM will be suitable to induce fusion protein synthesis in vaccinated animals.
  • 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
  • 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 agents
  • 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
  • viruses such as vaccinia virus.
  • 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
  • promoters and inducers include, for example, the metallothionein promoter and Zn/Cu, the trp operon and tryptophan, or the ara operon and L-arabinose.
  • 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.
  • 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.
  • the present invention represents a marked advancement in the art of
  • 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
  • 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
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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. ⁇ g 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
  • Mycoplasma pellets were used either for DNA extraction, or as an antigen source for polyclonal rabbit antiserum.
  • E. coli Y1090 (Promega Biotech ⁇ lac U169 proA+ ⁇ lon 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 MgSO 4 /PBS as the competent cells.
  • 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) 30 ⁇ g/ml at 37°C overnight to selected for NA-resistance (Hane et al . , 1969).
  • NA Nalidixic acid
  • NA-resistant E. coli C600 was grown in 0.2% maltose at 37°C overnight, spun and resuspended in 10mM MgCl 2 /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
  • Mycoplasma pulmonis were produced by inoculating 1 ⁇ 10 10 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
  • 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 ⁇ 10 11 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 ⁇ 10 11 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).
  • 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 20 ⁇ g 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.
  • SDS sodium dodecyl sulfate
  • the ⁇ gt11 expression library was constructed as described by Huynh (Huynh et al . , 1985) with 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
  • Recombinant phage (1 ⁇ l) was plated with 100 ⁇ l 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 ⁇ gt11, 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
  • 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
  • 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
  • the positive recombinant clones were selected from the master plates by using a sterile pasteur pipette to remove an agar plug containing phage particles
  • agar plug was incubated in 1ml of SM buffer (0.15M NaCl, 8mM MgSO 4 , 1mM Tris-HCl pH 8.0) at 4°C overnight. Positive clones from this solution were then replated and
  • 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 ⁇ gt11 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 ⁇ gt11-MP DNA yielded 1.6 ⁇ 10 5 PFU/2 ⁇ g 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 ⁇ 10 5 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.
  • 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 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).
  • 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.
  • 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.
  • 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 ⁇ gt11 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.
  • PCR polymerase chain reaction
  • the size of inserted MP DNA was rapidly analyzed and isolated by electrophoresis in 1% agarose or 5% polyacrylamide gels.
  • Total recombinant phage DNA was extracted by using plate lysate techniques (Sambrook et al . , 1989) and
  • 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.
  • Example I 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 ⁇ -galactosidase control band. None of the proteins from a control ⁇ gt11 lysate reacted with the antibody. Thus, distinct mycoplasma proteins were synthesized in E. coli infected with the recombinant phage.
  • 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 ⁇ gt11, the recombinant DNA preparations were digested with EcoR1 endonuclease. This digestion resulted in the cleavage of the ⁇ gt11 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.
  • PCR polymerase chain reaction
  • M. pneumoniae organisms that lack Pl are examples of M. pneumoniae organisms that lack Pl.
  • ⁇ -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).
  • the nucleotide sequence of the L150 fragment was determined (Fig. 4) and deposited with GenBank (Accession number, M76406).
  • GenBank accesion number, M76406
  • the first TGA stop codon was located at 76 bp followed by two in-frame TAA stop codons 112 and
  • the first 75 bp sequence could encode for a protein of 25 amino acids with a calculated
  • mice 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.
  • the fusion protein band was excised and
  • 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.
  • 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
  • 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
  • the animals were fed low dose of IPTG (0.1mM) in their drinking water for 2-3 days after each inoculation.
  • IPTG 0.1mM
  • the bacteria colonies were plated out individually using the toothpick method on two
  • 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 ⁇ 10 3 , 5 ⁇ 10 4 , and 1 ⁇ 10 6 ) 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);
  • 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).
  • spleen cells (4 ⁇ 10 5 /ml) were stimulated with 30 ⁇ g MP antigen or 2.5 ⁇ g concanavalin A (non-specific T cell nitrogen) per well of microtiter plates.
  • 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.
  • mice Twenty-two mice were then immunized with 10-20 ⁇ g 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
  • Titer is expressed as the reciprocal of the highest dilution giving a positive reaction in IFA or ELISA tests.
  • 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 ⁇ 10 7 and 1 ⁇ 10 8 CFU/ml respectively in group 2) of the mice, following oral inoculation at 40 and 50 days (even sever months after inoculation in another
  • 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 ⁇ 10 3 CFU dose, i.e., no MP organisms were recovered from tracheolung lavages
  • 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
  • mice receiving the oral vaccination were protected as well as group 5 mice when the challenge dose was 1 ⁇ 10 3 CFU (Fig. 7), but there was less protection if the challenged dose was higher (5 ⁇ 10 4 or 10 6 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
  • mice 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,
  • 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.
  • 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
  • the second application described herein is the production of a highly purified epitope of interest.
  • the antigen of MP has not previously been selectively
  • 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.
  • 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.
  • Expression Vector Coexpression of Beta- Galactosidase Provides Visual Screening of
  • mice against tetanus by use of a live attenuated Salmonella carrier.
  • Mycoplasma pneumoniae infection Role of a surface protein in the attachment organelle. Science
  • Mycoplasma pulmonis depresses humoral and cell- mediated response in mice. Lab Anim. Sci. 39:11-15. Lai, W. C, M. Bennett, Y. S. Lu, and S. P. Pakes. 1990. Biological evaluation of Mycoplasma pulmonis temperature-sensitivemutants for use as possible rodent vaccines. Infect. Immun. 58:2289-2296.
  • Vaccines '88 Modern Approaches to New Vaccines, (R.M. Chanock, R.A. Lerner, F. Brown, and H. Ginsberg, eds.), p. 384. Cold Spring Harbor Lab., Cold Spring Harbor, New York.
  • mice pulmonis-induced respiratory disease of mice.

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Abstract

L'invention se rapporte au clonage, au séquençage et à l'expression d'un antigène de M. pulmonis, et au développement de ces systèmes de clonage et de vaccination. L'on décrit une stratégie qui permet le clonage d'antigènes en dépit de la présence de codons qui provoqueraient normalement l'arrêt de la transcription dans E. coli. Grâce à ce procédé, on a cloné et produit sous forme de protéine de fusion un antigène de M. pulmonis, et on a identifié l'épitope principal. La transfection en E. coli lytique et lysogénique a entraîné la production du produit. Cet antigène s'est révélé déclencher la production d'anticorps chez les souris, et notamment l'IgG et l'IgA dans le lavage de la trachée. Des E. coli lysogéniques transfectés ont été utilisés pour la vaccination. On peut réguler la production de l'immunogène in vivo par l'introduction contrôlée de l'inducteur IPTG. Ce procédé de vaccination régulée, utilisant des agents d'immunisation pouvant être déclenchés, peut être généralement appliqué à de nombreux organismes et maladies.
PCT/US1993/008744 1992-09-16 1993-09-15 Antigenes de mycoplasma pulmonis, et procedes et compositions destines a etre utilises dans le clonage et la vaccination Ceased WO1994006911A2 (fr)

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AU49252/93A AU4925293A (en) 1992-09-16 1993-09-15 Mycoplasma pulmonis antigens and methods and compositions for use in cloning and vaccination

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US94581092A 1992-09-16 1992-09-16
US07/945,810 1992-09-16

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WO1994006911A2 true WO1994006911A2 (fr) 1994-03-31
WO1994006911A3 WO1994006911A3 (fr) 1997-03-06

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996031613A1 (fr) * 1995-04-07 1996-10-10 Board Of Regents, The University Of Texas System Immunisation par banque d'expressions
US6358742B1 (en) 1996-03-25 2002-03-19 Maxygen, Inc. Evolving conjugative transfer of DNA by recursive recombination

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7390619B1 (en) 1998-02-11 2008-06-24 Maxygen, Inc. Optimization of immunomodulatory properties of genetic vaccines

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0474891A1 (fr) * 1990-09-08 1992-03-18 BEHRINGWERKE Aktiengesellschaft Vecteurs pour l'expression d'antigènes malariales sur la surface de souches vaccinales de Salmonella

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996031613A1 (fr) * 1995-04-07 1996-10-10 Board Of Regents, The University Of Texas System Immunisation par banque d'expressions
US6358742B1 (en) 1996-03-25 2002-03-19 Maxygen, Inc. Evolving conjugative transfer of DNA by recursive recombination
US6387702B1 (en) 1996-03-25 2002-05-14 Maxygen, Inc. Enhancing cell competence by recursive sequence recombination
US6391552B2 (en) 1996-03-25 2002-05-21 Maxygen, Inc. Enhancing transfection efficiency of vectors by recursive recombination
US6482647B1 (en) 1996-03-25 2002-11-19 Maxygen, Inc. Evolving susceptibility of cellular receptors to viral infection by recursive recombination

Also Published As

Publication number Publication date
WO1994006911A3 (fr) 1997-03-06
AU4925293A (en) 1994-04-12

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