EP0465561A1 - Avirulent microbes and uses therefor - Google Patents

Abstract

The invention relates to a vertebrate or invertebrate immunization vaccine comprising an avirulent derivative of S. choleraesuis. Said derivatives are incapable of producing a functional adenylate cyclase and / or a cyclic AMP receptor protein. The invention also relates to a vaccine for immunizing a vertebrate and an invertebrate, comprising a virulent derivative of a pathogenic microbe, said derivative being incapable of producing a functional adenylate cyclase and / or a receptor protein. Cyclic AMP, while being able to express a recombinant gene derived from a pathogenic germ of said vertebrate, in order to produce an antigen capable of causing an immune response in said vertebrate against said pathogenic germ.

Description

AVIRULENT MICROBES AND USES THEREFOR

Field of the Invention

This invention relates to avirulent microbes, their method of preparation, and their use in vaccines.

Background of the Invention

Salmonellosis of swine is one of the most

economically important of the enteric and septicemic diseases affecting young pigs, and has been described as a significant health problem in swine. Although many serotypes of Salmonella have been isolated from pigs, S. choleraesuis var. kunzendorf and S. typhimurium are the two most frequently isolated serotypes associated with clinical salmonellosis in swine, Wilcock, B.P., in

DISEASES OF SWINE, pp. 508-519 (Leman, A.D., et al., eds., 1986). S. choleraesuis is host-adapted to swine and is often the etiologic agent of fatal septicemic disease with little involvement of the intestinal tract. This S.

choleraesuis reservoir in swine is a concern not only because of its disease-causing potential for pigs, but also because of its public health significance for humans.

The disease caused by S. choleraesuis manifests in many clinical signs. The organism is inherently invasive, and does not require the massive luminal

proliferation required by S. typhimurium. Lesions and necrosis occur in the submucosa and lamina propria of the gut. Mortality is high, and the duration and severity of the disease is unpredictable. Currently there is little information on vaccine use for control of swine salmonellosis. A rough variant of S. choleraesuis was used by Smith, H.W., J. Hyg.

63: 117-135 (1965), to demonstrate protection after challenge with virulent S. choleraesuis. However, the animals developed fever, sublethal disease, and became shedders of the bacteria. The Smith strain is commercially available in Europe as Suscovax™, which is manufactured and

distributed by Wellcome Laboratories. Hanna, J., et al., Vet. Microbiol. 3: 303-309 (1979), reported use of Smith's live attenuated S. choleraesuis vaccine by intramuscular route in pregnant sows. The piglets, after birth, had high titers of circulating maternally-derived antibodies, and resisted intranasal challenge. Although it has been reported that galE mutants of S. typhimurium are avirulent and immunogenic, in contrast, S. choleraesuis strains with galE mutations remain as virulent in mice as the wild-type gal⁺ parent. S. choleraesuis auxotrophs with requirements for aromatic amino acids due to an aroA mutation have reduced virulence in mice, but were unable, even after three immunizing doses, to induce protective immunity. At present, the only S. choleraesuis vaccine licensed for use in the United States of America is a killed bacterial bactrin, which is not particularly effective in inducing protective immunity.

Applicant has discovered a new method of

protecting against virulent infections with a vaccine employing transposon-induced avirulent mutants of virulent agents in which the impairment leading to avirulence can- not be repaired by diet or by anything supplied by an animal host. Applicant's initial work, including a method for creating an avirulent microbe by the introduction of deletion mutations in the adenylate cyclase gene (cya) and the cyclic AMP receptor protein gene (crp) of Salmonella typhimurium is described in EPO Pub. No. 315,682

(published 17 May 1989), and PCT Pub. No. WO 88/09669 (published 15 December 1988). The disclosure of these patent applications, as well as any corresponding national patent applications, are incorporated herein by reference. These applications teach, inter alia, recombinant DNA

.methods of inactivating cya and crp genes in S.

typhimurium, introducing thereby a recombinant gene for a heterologous pathogen in the avirulent S. typhimurium, and thereby producing a new antigen capable of inducing an immune response, introducing a recombinant gene for a product capable of suppressing, modulating, or augmenting an immune response, and vaccines and methods of

vaccination utilizing the avirulent S. typhimurium

constructs. Brief Description of the Invention

The present invention is based, in part, on new avirulent S. choleraesuis derivatives that are not

disclosed in EPO Pub. No. 315,682. Included within the invention is the application of these new S. choleraesuis derivatives in, inter alia, commercial vaccines, methods of stimulating the immune system to respond' to an immunogenic antigen of S. choleraesuis, and methods of stimulating the immune system to respond to an immunogenic antigen of a pathogen. The strains provided herein are directly and indirectly suitable for the production of commercial vaccines to prevent diseases caused by S.

choleraesuis, and other enteric bacteria with which antibodies to S. choleraesuis cross react. These strains are also useful as carrier microorganisms for the production of expression products encoded on recombinant genes in the bacterial cells.

Accordingly, one embodiment of the invention is a vaccine for the immunization of an individual comprising an avirulent derivative of pathogenic S. choleraesuis, said derivative being substantially incapable of producing functional adenylate cyclase due to a mutation in a cya gene.

Another embodiment of the invention is a method for stimulating the immune system to respond to an im- munogenic antigen of S. choleraesuis comprising

administering to said individual an avirulent derivative of pathogenic S. choleraesuis, said derivative being substantially incapable of producing functional adenylate cyclase due to a mutation in a cya gene.

Still another embodiment of the invention is a method for stimulating the immune system to respond to an immunogenic antigen of a pathogen comprising administering to said individual an avirulent derivative of pathogenic S. choleraesuis, said derivative being substantially in- capable of producing functional adenylate cyclase and cyclic AMP receptor protein (CRP) and being capable of expressing a recombinant gene encoding the immunogenic antigen, to produce an antigen capable of inducing an immune response in said vertebrate against said pathogen.

Another embodiment of the invention is an isolated avirulent strain of S. choleraesuis which is substantially incapable of producing functional adenylate cyclase.

Still another embodiment of the invention is a vaccine for the immunization of an individual comprising an avirulent derivative of pathogenic S. choleraesuis, said derivative being substantially incapable of producing functional cyclic AMP receptor protein (CRP) due to a mutation in a crp gene.

Yet another embodiment of the invention is a method for stimulating the immune system to respond to an immunogenic antigen of S. choleraesuis comprising

administering to said individual an avirulent derivative of pathogenic S. choleraesuis, said derivative being substantially incapable of producing functional CRP due to a mutation in a crp gene. Another embodiment of the invention is a method for stimulating the immune system to respond to an immunogenic antigen of a pathogen comprising administering to said individual an avirulent derivative of pathogenic S. choleraesuis, said derivative being substantially incapable of producing functional CRP and being capable of expressing a recombinant gene encoding the immunogenic antigen, to produce an antigen capable of inducing an immune response in said vertebrate against said pathogen.

Yet another embodiment of the invention is an isolated avirulent strain of S. choleraesuis which is substantially incapable of producing functional CRP due to a mutation in a crp gene.

Still another embodiment of the invention is a vaccine for the immunization of an individual comprising an avirulent derivative of pathogenic S. choleraesuis, said derivative being substantially incapable of producing functional adenylate cyclase and CRP due to a mutation in the cya and crp genes.

Yet another embodiment of the invention is a method for stimulating the immune system to respond to an immunogenic antigen of S. choleraesuis comprising

administering to said individual an avirulent derivative of pathogenic S. choleraesuis, said derivative being substantially incapable of producing functional adenylate cyclase or CRP due to a mutation in a cya gene and a crp gene.

Another embodiment of the invention is a method for stimulating the immune system to respond to an immunogenic antigen of a pathogen comprising administering to said individual an avirulent derivative of pathogenic S. choleraesuis, said derivative being substantially incapable of producing functional adenylate cyclase and CRP and being capable of expressing a recombinant gene encoding the immunogenic antigen, to produce an antigen capable of inducing an immune response in said vertebrate against said pathogen.

Still another embodiment of the invention is an isolated avirulent strain of S. choleraesuis which issubstantially incapable of producing functional adenylate cyclase and CRP due to mutations in the cya and crp genes.

Another embodiment of the invention is a strain selected from the group of strains ATCC 53647, ATCC 53648, ATCC 67923, ATCC 53885, ATCC 67922, and mutants thereof, and derivatives thereof.

Detailed Description of the Invention

Definitions;

"Vaccine," as used herein, means an agent used to stimulate the immune system of a living organism so that protection against future harm is provided . " Immunization" refers to the process of inducing a continuing high level of antibody and/or cellular immune response in which T-lymphocytes can either kill a pathogen and/or activate other cells (e.g., phagocytes) to do so in an organism, which is directed against a pathogen or antigen to which the organism has been previously exposed.

Although the phrase "immune system" can encompass

responses of unicellular organisms to the presence of foreign bodies, e.g., interferon production, in this application the phrase is restricted to the anatomical features and mechanisms by which a multicellular organism produces antibodies against an antigenic material which invades the cells of the organism or the extracellular fluid of the organism. The antibody so produced may belong to any of the immunological classes, such as immunoglobulins A, D, E, G or M. Of particular interest are vaccines which stimulate production of immunoglobulin A (IgA) since this is the principle immunoglobulin produced by the secretory system of warm-blooded animals, although vaccines of the invention are not limited to those which stimulate IgA production. For example, vaccines of the nature described herein are likely to produce a broad range of other immune responses in addition to IgA formation, for example, cellular and humoral immunity.

Immune response to antigens is well studies and widely reported. A survey of immunology is given in Barrett, James, T., Textbook of Immunology: Fourth Edition, C.V. Mosby Co., St. Louis, MO (1983), the entire of which is herein incorporated by reference.

The term "vertebrate," as used herein, means any member of the subphylum Vertebrata, a primary division of the phylum Chordata that includes the fishes, amphibians, reptiles, birds, and mammals, all of which are characterized by a segmented bony or cartilaginous spinal column. All vertebrates have a functional immune system and respond to antigens by producing antibodies. Thus, all vertebrates are capable of responding to vaccines.

Although vaccines are most commonly given to mammals, such as humans or dogs (rabies vaccine), vaccines for commercially raised vertebrates of other classes, such as the fishes and birds if of the nature described herein, are within the scope of the present invention.

By "invertebrate" is meant any member of the Animal Kingdom, excluding the vertebrates. Such animals constitute the Division Invertebrata and have no backbone or spinal column. This classification includes all animals except fishes, amphibians, reptiles, birds and mammals. Many invertebrates are capable of eliciting a primitive immune response to antigenic stimulation and are susceptible to the same microorganisms which infect vertebrates and which are disclosed herein in accordance with this invention. Examples of such invertebrates are shellfish and mollusks and other related animals.

Although the use of vaccines in the protection of invertebrate animals have hitherto before not been well documented, one skilled in the art will recognize the applicability of the subject invention to said invertebrates by use of their primitive immune systems. For example, and in accordance with this invention, the susceptibilityof shellfish to infection by S. choleraesuis will allow the introduction of avirulent strains of S. choleraesuis species and thereby provide potential for the primitive immune system to respond. Therefore, the use of an avirulent derivative of a pathogenic microbe that is capable of infecting an invertebrate to stimulate a response from an immune system present in the invertebrate against a pathogen is within the scope of this invention.

An "individual" treated with a vaccine of the invention, as used herein, is defined to include all vertebrates, such as mammals, including domestic animals and humans, and various species of birds, including domestic birds, particularly those of agricultural

importance. In addition, mollusks and certain other invertebrates have a primitive immune system, and are included as "individuals."

The term "avirulent", as used herein, does not mean that a microbe of that genus or species cannot ever function as a pathogen, but that the particular microbe being used is avirulent with respect to the particular animal being treated. The microbe may belong to a genus or even a species that is normally pathogenic but must belong to a strain that is avirulent. "Pathogenic", as used herein, means capable of causing disease or impairing normal physiological functioning. An "avirulent strain" is incapable of inducing the full set of symptoms of the disease that is normally associated with its virulent pathogenic counterpart. The term "microbes", as used herein, includes bacteria, protozoa, and unicellular fungi.

Derivatives of avirulent S. choleraesuis

microbes are also contemplated to be within the scope of this invention. By "derivative" is meant sexually or asexually derived progeny and mutants of the avirulent strains including single or multiple base substitutions, deletions, insertions or inversions which retain the in-ability to produce functional adenylate cyclase and cAMP receptor protein with or without naturally occurring virulence plasmids. For example, strains such as Chi4062 and Chi4064 carry the qyrA mutation conferring nalidixic acid resistance which has been used herein as a convenient marker to follow strains following oral inoculation.

However, drug resistance is not a desirable attribute for strains to be used as vaccines. Thus, the gyrA+ mutation can be easily removed by transducing the qyrA+ (conferring sensitivity to nalidixic acid) gene into strains by

selecting for inheritance of a closely linked Tn10 and then removing Tn10 by selection for fusaric acid resistance.

The term "recombinant gene", as used in this application, refers to genetic material that has been transferred from one organism into a second in such a manner that reproduction of the second organism gives rise to descendants containing the same genetic material.

Techniques for transferring genetic material from a first organism to a second organism that normally does not exchange genetic material with the first organism have become widely available as the result of rapidly expanding recombinant DNA technology.

The term "gene" is used herein in its broadest sense to represent any biological unit of heredity. It is not necessary that the recombinant gene be a complete gene as present in the parent organism, which was capable of producing or regulating the production of a macromolecule, for example, a functioning polypeptide. It is only necessary that the gene be capable of serving as the template in the production of an antigenic product. The product will not necessarily be found in that exact form in the parent organism. For example, a functional gene coding for a polypeptide antigen comprising 100 amino acid residues may be transferred in part into a carrier microbe so that a peptide comprising only 75, or even 10, aminoacid residues is produced by the cellular mechanism of the host cell. However, if this gene product is an antigen that will cause formation of antibodies against a similar antigen present in the parent organism, the gene is considered to be within the scope of the term "gene" as defined herein. Alternatively, if the amino acid sequence of a particular antigen or fragment thereof is known, it is possible to chemically synthesize the DNA fragment or analog thereof by means of automated gene synthesizers or the like and introduce said DNA sequence into the appropriate expression vector. At the other end of the spectrum is a long section of DNA coding for several gene products, one or all of which can be antigenic. Thus, a gene as defined and claimed here is any unit of heredity capable of producing an antigen. The gene may be of chromosomal, plasmid, or viral origin.

The term "gene expression", as used here means that the information inherent in the structure of the gene is transformed into a physical product in the form of an RNA molecule, polypeptide or other biological molecule by the biochemical mechanisms of the cell in which the gene is located. The biological molecule so produced is called the gene product. The term "gene product" as used herein refers to any biological product or products produced as a result of the biochemical reactions that occur under the control of a gene. The gene product may be, for example, an RNA molecule, a peptide, or a product produced under the control of an enzyme or other molecule that is the initial product of the gene, i.e., a metabolic product. For example, a gene may first control the synthesis of an RNA molecule which is translated by the action of

ribosomes into an enzyme which controls the formation of glycans in the environment external to the original cell in which the gene was found. The RNA molecule, the

enzyme, and the glycans are all gene products as the term is used here. Any of these as well as many other types of -gene products, such as glycoproteins and polysaccharides, will act as antigens if introduced into the immune system of an animal. Protein gene products, including

glycoproteins and lipoproteins, are preferred gene

products for use as antigens in vaccines.

The term "allergens", as used herein, means substances that cause allergic reaction, in this case in the animal which will be vaccinated against them. Many different materials may be allergens, such as animal dander and pollen, and the allergic reaction of individual animals will vary for any particular allergen. It is possible to induce tolerance to an allergen in an animal that normally shows an allergic response. The methods of inducing tolerance are well-known and generally comprise administering the allergen to the animal in increasing dosages. Further discussion of tolerance induction is given in the Barrett textbook previously cited.

The Invention:

This invention is predicated on the discovery that certain mutations can render a microbe avirulent without substantially affecting its immunogenicity. More specifically, this invention is made possible by microbial vaccines in which the microbe carries the deletion (delta) mutations delta-cya and delta-crp. These deletions eliminate the ability to synthesize adenylate cyclase (ATP pyrophosphate lyase (cyclizing) EC 4.6.1.1) and the cyclic AMP receptor protein (CRP), respectively, as described in EPO Pub. No. 315,682.

EPO Pub. No. 315,862 described how the elimination of cyclic-3',5'-adenosine monophosphate (cAMP), adenylate cyclase and the cyclic AMP receptor protein through delta-cya and delta-crp mutants rendered S.

typhimurium avirulent. The procedure for obtaining those mutants was followed in the current invention, and applied to a new strain, S. choleraesuis, to obtain avirulent mutants thereof which can be used as the immunogenic component of a vaccine to induce S. choleraesuis immunity.

In another embodiment of the invention, the avirulent S. choleraesuis derivative can be used as a carrier bacteria to deliver selected antigens to the gutassociated lymphoid tissue (GALT), for example to the

Peyer's patches of the ileum. Methods for expression of a recombinant gene from a pathogenic organism in S.

typhimurium to induce antibody production against the pathogen in the host were reported in EPO Pub. No.

315,682. That publication also explains how the carrier microbe may present the recombinant pathogen-derived antigen to the host.

Recombinant DNA techniques, whereby genetic material from one organism is transferred to and becomes a permanent part of the genetic material of a second organism, are now sufficiently well known and widespread so as to be considered routine. Usually, a small piece of DNA from the parent organism is obtained either from a plasmid or a parent chromosome. A plasmid (also called an

extrachromosomal element) is a hereditary unit that is physically separate from the chromosome of the cell. The DNA may be of any size and is often obtained by the action of a restriction endonuclease enzyme which acts to split DNA molecules at specific basepair sites. Following ligation to plasmid, phage or cosmid vectors, the new recombinant molecules may be transferred into a host cell by various means such as transformation (uptake of naked DNA from the external environment, which can be artificially induced by the presence of various chemical agents, such as calcium ions) or transduction (recombinant DNA packaged and introduced within a phage such as transducing phage or cosmid vectors). Recombinant DNA in the carrier cell may continue to exist as a separate piece of DNA or it may insert into the host cell chromosome and be reproduced with the chromosome during cell division. This invention sometimes utilizes transposons as the transferred

material. Transposons are highly movable pieces of DNA that insert in DNA and may also be excised. The excision may carry off surrounding genetic material, causing

deletion mutations. Presence or absence of transposons is then monitored by antibiotic-residue genetic markers.

Selection for organisms that have received transferred genetic material is performed by a "shotgun" approach when pathogen-derived antigens are desired. The "shotgun" approach is described in EPO Pub. No. 315,682, and in detail in Maniatis, T., et al., Molecular Cloning, Cold Spring Harbor Laboratories (1982), which are herein incorporated by reference. The techniques of gene

transfer are not considered to be part of this invention, and any method capable of producing recombinant organisms comprising genes from an organism that are expressed in avirulent microbes will suffice. In cases where the species normally exchange genetic information, classical methods of gene transfer such as conjugation, transformation or transduction may be employed.

In another embodiment, when the immunogenic component of the vaccine is an allergen of the host such a vaccine may be used in an exposure regimen designed to specifically desensitize an allergic host.

Yet another embodiment of this invention

provides a vaccine for the immunization of a vertebrate or invertebrate animal comprising a live avirulent derivative of S. choleraesuis incapable of producing functional adenylate cyclase and cAMP receptor protein, and capable of expressing a recombinant gene derived from an organism that is a pathogen of or that produces an allergen of said animal. In an embodiment which contemplates all of the above, a subject of the invention is avirulent strains of S. choleraesuis, which carry mutations in the cya and/or crp genes.

Administration and Use:

In order for a vaccine to be effective in producing antibodies, antigenic material must be released in such a way that the antibody-producing mechanism of the vaccinated animal can come into play. Therefore, the S. choleraesuis carrier of the gene product must be properly introduced into the animal. In order to stimulate a preferred response of the GALT or bronchus-associated (BALT) cells as discussed previously, introduction of the microbe or gene product directly into the gut or bronchus is preferred, such as by oral administration, gastric intubation or in the form of aerosols, although other methods of administering the vaccine, such as intravenous, intramuscular, subcutaneous injection or intramammary or intrapenial or vaginal administration, are possible.

Lastly, the host organism itself can serve as a source of genetic material when immunoregulatory genes or genes for other pharmacologically active substances are being expressed by the vectors.

Administration of a live vaccine of the type disclosed above to an animal may be by any known or standard technique. These include oral ingestion, gastric intubation, or broncho-nasal spraying. All of these methods allow the live vaccine to easily reach the GALT or BALT cells and induce antibody formation and are the preferred methods of administration. Other methods of administration, such as intravenous injection, that allow the carrier microbe to reach the animal's blood stream may be acceptable. Intravenous, intramuscular or intramammary injection are also acceptable with other embodiments of the invention, as is described later. The dosages required will vary with the

antigenicity of the gene product and need only be an amount sufficient to induce an immune response typical of existing vaccines. Routine experimentation will easily jestablish the required amount. Multiple dosages used as needed to provide the desired level of protection.

The pharmaceutical carrier in which the vaccine is suspended or dissolved may be any solvent or solid or encapsulated in a material that is nontoxic to the

inoculated animal and compatible with the carrier organism or antigenic gene product. Suitable pharmaceutical carriers include liquid carriers, such as normal saline and other nontoxic salts at or near physiological concentrations, and solid carriers, such as talc or sucrose and which can also be incorporated into feed for farm animals. Adjuvants may be added to enhance the antigenicity if desired. When used for administering via the bronchial tubes, the vaccine is preferably presented in the form of an aerosol.

Immunization with a pathogen derived gene product can also be used in conjunction with prior immunization with the avirulent derivative of a pathogenic microorganism acting as a carrier to express the gene product specified by a recombinant gene from a pathogen. Such parenteral immunization can serve as a booster to enhance expression of the secretory immune response once the secretory immune system to that pathogen-derived gene product has been primed by immunization with the carrier microbe expressing the pathogen derived gene product to stimulate the lymphoid cells of the GALT or BALT. The enhanced response is known as a secondary, booster, or anamnestic response and results in prolonged immune protection of the host. Booster immunizations may be repeated numerous times with beneficial results.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific example which is provided herein for purposes of illustration only and is not intended to be limiting unless otherwise specified.

Example

This example illustrates the construction of delta-cya, delta-crp, and delta-cya delta-crp derivatives of S. choleraesuis, the virulence properties of the mutants after peroral (p.o.) inoculation, and the

immunogenicity of these derivatives.

Bacterial strains.

The S. choleraesuis strains used in this Example are listed in Table 1. Bacterial strains were grown at 37°C in L broth and on L agar (Lennox, Virology 1: 190-206

(1965); Luria and Burrous, J. Bacteriol. 74:461-476

(1957)), Penassay agar (Difco antibiotic media #3 + 1.5% BBL agar, Becton Dickinson Microbiology Systems,

Cockeysville, MD) and MacConkey Base agar (Difco

Laboratories) with 1% final concentration of an appropriate carbohydrate. Media were supplemented with MgSO₄ (10 mM), CaCl₂ (5 mM), tetracycline (12.5 micrograms/ml) and ampicillin (100 micrograms/ml) when required. Synthetic media were minimal liquid and minimal agar supplemented with nutrients at optimal levels as previously described (Curtiss, J. Bacteriol. 89 : 28-40 (1965)). Buffered saline with gelatin (BSG) was used as a diluent (Curtiss, 1965, supra).

The pertinent references in Table 1 are the following:

Zinder and Lederberg, J. Bacteriol. 64:679 (1952). Hoiseth and Stocker, Nature 291:238 (1981).

Gulig and Curtiss, Infect. Immun. 55:2891 (1987).

Postma et al., J. Bacteriol. 168:1107 (1986).

Curtiss and Kelly, Infect. Immun. 55:3035 (1987) Gulig and Curtiss, Infect. Immun. 56: 3262 (1988).

Genetic manipulations.

Transductions were performed with bacteriophages P1L4 or P22 HT int with standard methods and media, as

described in Curtiss, MANUAL OF METHODS FOR GENERAL

BACTERIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, p. 243 (Gerhardt et al., eds., 1981), Schmeiger, Mol. Gen. Genet. 119:75 (1972), and Davis et al., A MANUAL FOR GENETIC ENGINEERING: ADVANCED BACTERIAL GENETICS (Cold Spring

Harbor Laboratory, Cold Spring Harbor, N.Y., 1980). The methods and media described by Maloy and Nunn, J.

Bacteriol. 145:1110 (1981) were used for fusaric acid selection for deletion mutations in strains with Tn10 insertions. Transformations were performed by the method of Dagert and Ehrlich, Gene 6:23 (1979). The plasmid, pSD110, which carries the crp and Amp genes from E. coli, which was described in Schroeder and Dobrogosz, J.

Bacteriol. 167:616 (1986), was generously provided by C. Schroeder. Curing of the virulence plasmid was

facilitated by use of pYA2028 which has the Inc/Par region of the virulence plasmid cloned into the high copy number plasmid pUC18, as described below.

Animal infectivity studies.

Female BALB/c mice (Harlan Sprague-Hawley,

Indianapolis, IN) were used for all infectivity and immunization studies. Seven-week-old mice were held for one week in a quarantined room prior to being used in

experiments. Animals were housed in Nalgene filter- covered cages with raised wire floors. Food and water were given ad libitum. The animal room was maintained at 22°C to 23°C with 12 h of illumination daily.

Virulence of S. choleraesuis strains was determined after p.o. inoculation. Bacteria for inoculation in mice were grown overnight as static cultures at 37ºC in L broth. All cultures were diluted 1:20 into prewarmed L broth and aerated at 37ºC for approximately 5 h to an optical density at 600 nm of about 0.8 to 1.0. The cells were concentrated 50-fold by centrifugation at 8,000 x g for 10 min at room temperature, followed by suspension in BSG. Dilutions were plated on MacConkey agar with 1% maltose to verify the Cya or Crp phenotype and to enumerate cells.

Prior to p.o. inoculations, mice were deprived of food and water for 4 h before infection. They were then given 30 microliters of 10% (wt/vol) sodium bicarbonate 5- 10 min before being fed a 20 microliter aliquot of S.

choleraesuis cells suspended in BSG. Food and water were returned 30 min after the inoculation. Data on morbidity and mortality of mice were collected daily. Evaluation of protective immunity.

Groups of five mice/cage were perorally immunized with various doses of avirulent mutants and then challenged 30 days later with various doses of the wild-type, virulent parent, Chi3246. Morbidity and mortality conditions were observed for at least 60 days.

Construction of S. choleraesuis strains with cya and crp mutations.

The highly virulent strain Chi3246 was used as the parent in the construction of all the vaccine strains used in these studies (the peroral LD₅₀ value for Chi3246 is presented in Table 2, infra.)

The strains which were constructed which were derived from Chi3246 are shown in Table 1. Also shown in Table 1 are the relevant genotypes of the strains, and a description of the method by which the strain was derived, utilizing the methods for transposon insertion via

transduction, transposon deletion, as well as the selection methods described in Example 1 of EPO Pub. No.

315,682, with the modifications described below.

Introduction of cya::Tn10, crp::Tn10, delta-cya-12, and delta-crp-11 from the S. typhimurium strains PP1002, PP1037, Chi3615, and Chi3623, respectively (see Table 1), into S. choleraesuis strain Chi3246 was facilitated by P1L4 transduction (via intermediate S. typhimurium hosts Chi3385 and Chi3477) and transformation with pSD110. S. choleraesuis strain Chi3246 which is P22HTint resistant is, however, P1L4 immune. Thus, P1L4 or P1 clr clm

bacteriophage can adsorb to and inject DNA into the cells of all S. choleraesuis strains used in this example, but are unable to replicate their DNA in these bacterial cells.

Since delta-cya-12 zid-62::Tn10, delta-crp-11

zhb::Tn10, crp773::Tn10 and cya::Tn10 mutations are in S. typhimurium strains Chi 3711, Chi3773, PP1037 and PP1002, respectively (see Table 1), it was necessary to move the mutations from the smooth-LPS S. typhimurium background into an intermediate Salmonella host in which both

P22HTint and P1L4 could be propagated. The bacteriophagesP22HTint and P1L4 are specific for strains with smooth and rough LPS coats, respectively. The hosts, Chi3385 and Chi3477, are restriction-deficient, modification- proficient galE S. typhimurium strains. Growth of Chi3385 and Chi3477 in media with low concentrations of galactose permits synthesis of UDP-galactose, resulting in normal levels of LPS side chains; these conditions are essential for attachment and infection by P22HTint. Growth of

Chi3385 and Chi3477 in media containing glucose and lacking galactose permits synthesis of a rough or incomplete LPS, and enables the adsorption and replication of P1L4 or P1 clr clm in these rough strains. By these means, the S. choleraesuis strains Chi3492, Chi3751, Chi3755, Chi3759, Chi3820, and Chi3858 were constructed. In the latter two strains, the Tn10 linked to the delta-cya or delta-crp mutation was eliminated by selection for fusaric acid resistance to yield Chi3860 and Chi3659, respectively.

In order to determine whether different deletion mutations generated by the excision of Tn10 from cya or crp exhibit different levels of virulence and/or

immunogenicity, two different deletion mutants of cya and crp were constructed in S. choleraesuis Chi3246. Two deletions were isolated in, and transduced from S.

typhimurium as described above, and are represented by S.. choleraesuis strains Chi3859 (delta-cya-12) and Chi3860 (delta-crp-11). The other two deletion mutations were isolated by excision of Tn10 inserted into the S.

choleraesuis cya and crp genes in Chi3492 and Chi3751, respectively, using selection for fusaric acid resistance to yield Chi3752 (delta-crp-19) and Chi3753 (delta-cya- 24), respectively. Construction of a S. choleraesuis strain with

deletion mutations in both cya and crp genes was

facilitated by using pSD110, which encodes the E. coli crp⁺ gene. pSD110 was thus transduced into Chi3752

(Table 1), and this was followed by transducing in the delta-cya-12 mutation, which is closely linked to zid- 62::Tn10 by selection for tetracycline resistance and screening for inability to ferment maltose, which is indicative of the introduction of the cya mutation. Following selection of Chi3759 for fusaric acid resistance to eliminate Tn10, pSD110 was cured by growth at 43ºC to yield Chi3781 (See Table 1). The serotype of all

constructs was confirmed by agglutination with Salmonella group C₁ O-antigen antisera.

Phenotypic analysis of cya and crp mutants.

The cya mutants (Chi3492, Chi3753, Chi3859), the crp mutants (Chi3751, Chi3752, Chi3820) and the cya crp mutant (Chi3781) were subjected to phenotypic analysis. These strains failed to ferment maltose, mannitol, sorbitol, and melibiose, and slowly fermented galactose. The phenotypes were as expected based on known requirement for cAMP, and for CRP for catabolic activities. The requirements for cAMP, and for CRP for regulation of gene expression are described in the following references. Perlman and

Pastan, Biochem. Biophys. Res. Comm. 37:151 (1969); Pastan and Perlman, Science 169:339 (1970); Schwartz and

Beckwith, THE lac OPERON (1970, Zipser and Beckwith, eds.); Pastan and Adhya, Bacteriol. Rev. 40:527 (1976); and Scholte and Postma, J. Bacteriol. 141:757 (1980).

Construction of virulence plasmid-cured derivatives of S. choleraesuis and of its delta-cya and/or delta-crp strains.

We have discovered that the Inc/Par region of the virulence plasmid of S. typhimurium, which encodes in compatibility and partition functions, hybridizes to and exhibits incompatibility with the virulence plasmid of S. choleraesuis. Based upon this finding, pYA2028, which contains the Inc/Par region of the virulence plasmid of S.typhimurium, high copy number pUC vector was used to facilitate the curing of the virulence plasmid from S.

choleraesuis. Introduction of pYA2028 into S.

choleraesuis was accomplished using essentially either the transformation methods described in Dagert and Ehrlich 1979, supra, or the electroporation methods of Feidler and Worth, Anal. Biochem. 170:38 (1988). Transformants were selected for, and maintained by, the inclusion of

ampicillin in the medium. After several generations of growth, the virulence plasmid is lost due to incompatibility exclusion. Growth of the cured S. choleraesuis strain for subsequent cycles in media without ampicillin leads to a rapid loss of pYA2028. The resulting cells lack both the virulence plasmid and pYA2028. Chi3903 represents a virulence plasmid-cured derivative of the wild-type S. choleraesuis strain Chi3246. Loss of the virulence plasmid reduces the ability of Salmonella to reach and/or colonize deep tissues such as the liver and spleen, but is without effect in terms of colonization of the intestinal tract and the GALT. Elimination of the virulence plasmid thus reduces virulence without diminishing immunogenicity. Removal of the virulence plasmid from the double cya crp mutants would follow the same

procedure. Virulence of cya and crp mutant strains in mice.

In order to study the virulence of the S.

choleraesuis strains listed in Table 1, groups of mice were orally inoculated with 100-fold varying doses of each strain. The results are shown in Table 2. The p.o. LD₅₀ of the parental wild-type strain (Chi3246), which was about 1 x 10⁵ CFU, was determined by the method of Reed and Muench (1938). This LD₅₀ is much lower than those of the respective cya and crp strains.

The data in Table 2 show that the mutant strains are avirulent at at least 6,000 times the LD₅₀ of the parentalwild-type strain, Chi3246. Mice orally infected with 10⁹ CFU of Chi3751 (crp::Tn10) and Chi3752 (delta-crp) became scruffy, lethargic, inappetent, and some died. However, all mice infected with the same dose of Chi3492

(cya::Tn10) and Chi3753 (delta-cya-24) became slightly ill, recovered, and survived the dose. This dose

represents 12,500 times the LD₅₀ of the wild-type parent strain. It is therefore evident that the cya::Tn10 and delta-cya mutants are more avirulent than the crp::Tn10 and delta-crp mutants. This result is somewhat surprising since the opposite has been found with S. typhimurium delta-cya and delta-crp infections in mice.

The results also showed that there were no obvious differences in the health of the animals inoculated with delta-crp-19 compared to delta-crp-11 strains, and delta- cya-12 as compared to delta-cya-24 strains.

Virulence of the strains with both delta-cya and delta-crp may also be established using the above

described procedure. Immunogenicity of cya and crp mutant strains.

The ability of the different S. choleraesuis mutants to induce immunity to subsequent oral challenge with the wild-type virulent parent, Chi3246, was examined. Mice were inoculated p.o. with the S. choleraesuis delta-cya and delta-crp strains shown in Table 3, at the indicated doses. Thirty days after immunization with the attenuated strains, mice were challenged p.o. with Chi3246. Morbidity and mortality were observed daily for 30 days post- challenge. The results in Table 3 reveal the apparent differences in the degrees of immunogenicity of the different strains. Mice immunized with 10 ⁷, 10⁸, or 10⁹ CFU of the somewhat more virulent strains with mutations in crp exhibited better health and a higher rate of survival after oral challenge with Chi3246 than did mice immunized with the less virulent cya mutant strains. Thus, animalsimmunized with 10⁹ CFU of the cya mutants

had a rate of survival below 50% whereas 100% of the mice immunized with 10⁹ cells of the crp strain survived chal- lenge; in fact, those immunized with 10 ⁷ or 10⁸ cells of the crp strain survived challenge with 10⁹ cells of the wild-type strain.

The immunogenicity of cya crp mutants of S.

choleraesuis is determined by immunizing the mice with a high, but sublethal dose (8 x 10⁸ CFU) of the attenuated strains. Thirty days later, survivors are challenged with Chi3246 at 10¹, 10², and 10³ times the LD₅₀ value of

Chi3246. Morbidity and mortality conditions are observed as described above.

Construction of asd cya crp mutants of S.

choleraesuis

Salmonella vaccine strains can serve as carriers to deliver a foreign antigen to the GALT of an animal host by introduction of a gene encoding the antigen into the vaccine strains. Nakayama et al., Bio/Tech 6:693 (1988) described a unique system where an Asd⁺ expression-cloning vector was constructed for the purpose of high-level stable expression of foreign antigen genes in delta-cya delta-crp delta-asd S. typhimurium. The avirulent properties of the delta-cya delta-crp mutations have been consistently proven with doses administered to mice at approximately 1000 times the LD₅₀ of the wild-type parent in all Salmonella species previously tested. These avirulent strains have also stimulated a protective immune response in the immunized animals as demonstrated by challenge with approximately 1000 times the wild-type parent. Repeated animal experiments have confirmed that the S. choleraesuis delta-cya and delta-crp strains are avirulent and immunogenic in mice. It can be deduced from earlier results obtained from studies on avirulence and

immunogenicity that a S. choleraesuis construct with delta-cya and delta-crp mutations would be avirulent and immunogenic. The addition of the delta-asd mutation to the S. choleraesuis delta-cya delta-crp Chi3781 is

facilitated by bacteriophage P22 HT int transduction to a restriction-deficient, modification-proficient intermediate S. typhimurium with subsequent propagation of

bacteriophage plL4 on the intermediate S. typhimurium host and final transduction of delta-asd into the S .

choleraesuis vaccine strain.

S. typhimurium Chi3656 is grown in L broth containing 5 mM CaCl₂, and infected with P1L4 to propagate a high titer lysate. The PlL4(Chi3656) lysate is then used to transduce S. choleraesuis delta-cya delta-crp Chi3781;

transductants are screened for by tetracycline resistance. A portion of the tetracycline-resistant transductants are screened for the Asd- phenotype. As a final step, selection on fusaric acid media is performed to identify a tetracycline-sensitive derivative of the S. choleraesuis delta-cya delta-crp delta-asd strain. Additional

characterization of the final construct is completed by verifying the markers and presence of a complete LPS coat, and by the nonacquisition of additional auxotrophic phenotypes.

Deposits of Strains

The following listed materials are on deposit under the terms of the Budapest Treaty, with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland. The accession number indicated was assigned after successful viability testing, and the requisite fees were paid. Strain Deposit Date ATCC No. Chi4062 July 15, 1987 53647 Chi4064 July 15, 1987 53648

Chi3781 delta-crp-19 March 29, 1989 67923 delta-cya-12

Chi3903 (wild-type, 50kb March 29, 1989 53885 plasmid-cured)

Chi3246 (wild-type) March 29, 1989 67922

Commercial Utility

The strains provided herein are directly and indirectly suitable for the production of commercial vaccines to prevent diseases caused by S. choleraesuis, and other enteric bacteria with which antibodies to S.

choleraesuis cross react. These strains are also useful as carrier microorganisms for the production of expression products encoded on recombinant genes in the bacterial cells.

Claims

1. A vaccine for the immunization of an

individual comprising an avirulent derivative of

pathogenic S. choleraesuis, said derivative being

substantially incapable of producing functional adenylate cyclase due to a mutation in the cya gene, or

substantially incapable of producing functional cyclic AMP receptor protein (CRP) due to a mutation in the crp gene, or substantially incapable of both.

2. A vaccine according to claim 1, wherein said avirulent derivative is capable of expressing a recombinant gene derived from an agent which is pathogenic to said individual, to produce an immunogenic antigen capable of inducing an immune response in said vertebrate against said pathogen.

3. A method for stimulating the immune system to respond to an immunogenic antigen of S. choleraesuis comprising administering to said individual a vaccine according to claim 1.

4. A method for stimulating the immune system to respond to an immunogenic antigen of a pathogen

comprising administering to said individual a vaccine according to claim 2.

5. An isolated avirulent strain of S.

choleraesuis which is substantially incapable of producing functional adenylate cyclase, functional CRP, or both.

6. The isolated avirulent strain of S.

choleraesuis of claim 5 which is capable of expressing a recombinant gene derived from an agent which is pathogenic to said individual, to produce an antigen capable of inducing an immune response in said vertebrate against said pathogen.

7. A strain according to claim 6, wherein theS. choleraesuis contains a chromosomal mutation which is lethal, balanced by a vector gene which complements the lethal mutation to constitute a balanced lethal host vector system.

8. A strain according to claim 7, wherein cells of the strain:

a) lack a functioning native chromosomal gene encoding beta-aspartate semialdehyde dehydrogenase ( asd);

b) have present a recombinant gene encoding a functional Asd polypeptide which complements the

chromosomal asd mutation, but which cannot replace the defective chromosomal gene by recombination;

c) have a physical linkage between the recombinant genes encoding the functional Asd polypeptide and the immunogenic antigen, wherein the loss of the recombinant gene encoding the functional Asd polypeptide causes the cells to lyse when the cells are in an environment in which the lack of functional Asd causes the cells to lyse.

9. An isolated avirulent strain according to claim 5, 6, 7 or 8 which lacks a 50 kb virulence plasmid.

10. A strain selected from the group of strains ATCC 53647, ATCC 53648, ATCC 67923, ATCC 53885, ATCC

67922, and mutants thereof, and derivatives thereof.