IE84199B1 - Avirulent salmonella microbes comprising a mutation in the CDT gene and uses therefor - Google Patents

Description

PATENTS ACT, 3912/91 AVIRULENT SALMONELLA MICROBES COMPRISING A MUTATION IN THE CDT GENE AND USES THEREFOR WASHINGTON UNIVERSITY This invention relates to avirulent microbes, their method of preparation, and their use in vaccines.

Background of the Invention Typhoid fever, which is caused by Salmonella typhi, remains an important public health problem for residents in the less developed world, for travelers from industrialized countries who visit endemic areas, and for clinical microbiologists in laboratories which conduct proficiency tests. The currently licensed parenteral killed whole cell typhoid vaccines are protective but cause marked systemic and local adverse reactions at an unacceptably high frequency (Levine, Typhoid fever vaccines, in Plotkin SA, Mortimer EA Jr. (eds): VACCINES.

Philadelphia, WB Saunders, 1988, pp. 333-361).

Alternative vaccines include the recently licensed live oral vaccine strain Ty21a and the experimental parenteral Vi polysaccharide vaccine.

The advantage of an oral vaccine is the delivery of replicating organisms to the mucosal immune system where local responses are maximally stimulated. protection against experimental challenge with pathogenic The main drawbacks to the use of Ty21a as a cholerae candidate carrier strain include its limited immunogenicity, a lack of precise information on the molecular basis of its attenuation and practical difficulties in bacterial genetic manipulation of the strain (e.g., in transformation, electroporation, and recombination frequency). It also exhibits very poor viability after reconstitution of lyophilized cultures.

Applicant has discovered new methods of protecting against virulent infections with vaccines employing transposon-induced avirulent mutants of virulent agents in which the impairment leading to avirulence cannot be repaired by diet or by anything supplied by an animal host. Some of Applicant's initial work, including a method for creating an avirulent microbe by the introduction of deletion mutations in the adenylate cyclase gene (gya) and the cyclic AMP receptor protein gene (grp) 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).

Applicant has also provided methods for producing other types of avirulent mutant cells which are desirable as carrier cells for the expression of recombinant antigens.

These cells are characterized by a lack of a functioning native gene encoding an enzyme which is essential for cell survival, wherein the enzyme catalyses a step in the biosynthesis of an essential cell wall structural component and the presence of a first recombinant gene encoding an enzyme which is a functional replacement for the native enzyme, wherein the first recombinant gene cannot replace the defective chromosomal gene. In these cells, the first recombinant gene is structurally linked to a second recombinant gene encoding a desired product.

Loss of the first recombinant gene causes the cells to lyse. These methods are described in WO 89/03427 (published 20 April 1989).

Brief Description of the Invention The present invention is based, in part, on new Included within the invention is Accordingly, one embodiment of the invention is an immunogenic composition for the immunization of an still another embodiment of the invention is an immunogenic composition for the immunization of an individual comprising an avirulent derivative of Salmonella with a mutation in a 9g; gene.

Another embodiment of the invention is an isolated avirulent strain of Salmonella which contains a mutation in a ggt gene.

Still another embodiment of the invention is an isolated strain selected from the group of strains X3958, X4323, X3925, X3927, X4297, X4346, X3940, X4073, and derivatives thereof. comprising preparing an immunogenic composition by suspending the strain in a physiologically acceptable excipient.

Still another embodiment of the invention is a method of utilizing a strain of avirulent Salmonella which is comprised of a mutation in a ggt gene, the method comprising preparing an immunogenic composition by suspending the strain in a physiologically acceptable excipient.

Another embodiment of the invention is an Brief Description of the Drawings Figure 1.A. is a graph which shows the recovery of CFU from the Peyer’s patches of 8 week old BALB/c mice at specified times after peroral inoculation with 9 x 103 CFU of X3522 (A[grp-gygg]-lg), 1 x 10’ CFU of X3737 (psD11o*/A[g;p-gygg]-;g) and 1 x 10’ CFU of X3339 (wild tYP9)- The results are given as geometric means i standard Three mice were sacrificed for each time point. deviations.

Figure 1.B. is a graph which shows the recovery of CFU from the spleens of 8-week-old BALB/c female mice at specified times after peroral inoculation with 9 x 108 CFU of X3522 (A[g;p-gysg]-lg), 1 x 10’ CFU of X3737 (psD11o*/A[g;p-gygg]-lg) and 1 x 10’ CFU of X3339 (wild type). Three mice were sacrificed for each time point.

The results are given as geometric means 1 standard deviations.

Figure 2 is a partial restriction map of pYA1077.

Xgtll clone L14 was subcloned into the EQQRI site of The 1.0 kb N. lgpgag insert DNA fragment from pYA292. There is a single asymmetrical SalI site within the M. leprae insert DNA. There are no sites within the N. leprae insert DNA for the following restriction endonucleases: ggmfil, fiigdlll, ggtl, and zbgl. reacted with pooled sera from 21 lepromatous leprosy The proteins on the nitrocellulose filters were patients. Lane 1, molecular size markers (sizes are indicated to the left of the blot); Lane 2, proteins immunologically reactive protein specified by pYA1075 is the lacz promoter as it is in pYA1077). slightly larger than that specified by pYA1077 because it is a fusion protein with the alpha region of B- galactosidase. strains harboring pYA292, pYA1077 and pYA1078.

°C in human sera.

Modes for Carrying Out 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 relates to microbial vaccines in which the microbe carries the deletion (open triangle or delta) mutations Agya and/or Agrp eliminating the ability to synthesize adenylate cyclase (ATP pyrophosphate lyase (cyclizing) EC 4.6.1.1) and the cyclic AMP receptor protein (CRP), respectively.

Cyclic-3'5’-AMP (CAMP) and the cyclic AMP receptor protein are necessary for the transcription of a large number of genes and operons concerned with the transport and breakdown of a large number of catabolites.

Evidence has been provided that shows that systems used for transporting fuel/carbon sources are all under positive control by cAMP, as are several amino acid permeases. In addition to its very important role in catabolism, the CAMP concentration in cells also influences lysogenization by temperate phages, synthesis of fimbriae, synthesis of flagella and synthesis of at Although cAMP is present in mammalian cells, the concentrations present in least one outer membrane protein. macrophages and other cells in which Salmonella can invade and multiply are below the concentration of 0.1 to 1.0 mM cAMP necessary to allow Agyg mutants to exhibit a wild-type phenotype in yitrg. Furthermore, the inclusion of the Agrp mutation would essentially abolish any benefit that could accrue from uptake of CAMP in yitrg or in vivo by such Acya mutants.

Transposons can be added to a bacterial The characteristics of transposon insertion and deletion have been reviewed in (1977), J. Mol. Biol. ;ig:125. For example, the transposon Tnlg, which confers resistance to Kleckner et al. tetracycline (and sensitivity to fusaric acid) can be used to create Acya and Acrp mutations in a variety of bacterial species, including, for example, E. the Examples, infra. typhi strains are shown in Once rendered avirulent by the introduction of the Agyg and/or Agrp mutations, the microbes can serve as the immunogenic component of a vaccine to induce immunity against the microbe.

Mutants with the A(g;p-ggt) mutation containing a g;p+ In addition, the cdt mutation can typhimurium described in the Examples into other mutagenesis which are known in the art. :1189 (1974)). S. tvphimurium-E. coli hybrids have also been shown to colonize Peyer's patches in mice (Hohmann, A.W., et al., Infect. and immun. ;;:763 (1973)). recombinant gene from a pathogenic organism, antibodies If these carrier bacteria contain and express a against the antigenic gene product produced from the pathogen will be induced. With the advent of recombinant DNA techniques, it now becomes possible to develop totally unique vaccines in which specific antigens are produced, not by the etiologic agent, but by another host strain of bacteria capable of expressing the gene for that antigen. It is also possible, when antigens might cross-react with an antigen of the mammalian host and thus potentiate the induction of autoimmunity, to use recombinant DNA techniques to alter the gene so that the affecting cross-reacting antigenic determinant is not produced. Thus, recombinant DNA techniques can be employed to develop vaccines that do not have any material capable of cross-reacting with vertebrate host antigens or capable to eliciting an autoimmune state.

Methods of preparing organisms, particularly Salmonella, which can function as carrier bacteria are discussed in WO 89/03427 (published 20 April 1989), and in U.S. Serial No. 07/251,304, filed 3 October 1988, which is commonly owned.

Generally, the salmonella are treated to cause a mutation in a chromosomal gene which encodes an enzyme that is essential for cell survival, wherein this enzyme catalyzes a step in the biosynthesis of an essential cell wall structural component. An extrachromosomal genetic element, for example, a recombinant vector, is introduced into the mutant cell. This genetic element contains a first recombinant gene which encodes an enzyme which is a functional replacement for the native enzyme, but the first recombinant gene cannot replace the defective chromosomal gene. The first recombinant gene is structurally linked to a second recombinant gene encoding a desired product, which is to be expressed in the carrier microorganism. Loss of the first recombinant gene causes the cells to lyse when the cells are in an environment where a product due to the expression of the first recombinant gene is absent. ggg gene. introducing a deletion mutation mutagenesis is shown in the Examples. Also shown in the Examples, is the construction of a genetic element which carries the functional replacement for the ggg gene, linked to a gene encoding an antigen which is to be uphi- It is apparent that the present invention has wide applicability to the development of effective vaccines against bacterial, fungal, parasite or viral disease agents where local immunity is important and might be a first line of defense. some examples are vaccines for the control of pneumonic plague caused by Yersinia pestis, of gonorrhea caused by Neisseria onorrhoeae, of syphilis caused by Treponema allidum, and of venereal diseases as well as eye infections caused by Chlamydia trachomatis. Species of streptococci from both group A and group B, such as those species that cause sore throat or heart disease, Neisseria meninqitidis, Mvcoplasma pneumoniae, Haemophilus influenzae, Bordetella pertussis, Mycobacterium tuberculosis, Mvcobacterium leprae, Bordetella gyipp, Escherichia coli, Streptococcus ggpi, Streptococcus neumoniae, Brucella abortus, Pasteurella hemolytica, Vibrio cholerae, Shigella species, and Legionella pneumophila are additional examples of bacteria within the scope of this invention from which genes could be obtained. Viral vaccines, such as those produced against influenza viruses, are also encompassed by this invention. Viral vaccines can also be produced against other viruses, either DNA or RNA viruses, for example from the classes Papovirus, Adenovirus, Herpesvirus, Poxvirus, Parvovirus, Reovirus, Picornavirus, Myxovirus, Paramyxovirus, or Retrovirus. Vaccines to protect against infection by pathogenic fungi, protozoa and parasites are also contemplated by this invention.

In a further 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.

In one of its embodiments, the invention can be described as a vaccine for the immunization of a vertebrate animal or invertebrate comprising a live avirulent derivative of a pathogenic microbe said derivative being incapable of producing functional adenylate cyclase and cAMP receptor protein while being capable of expressing a recombinant gene derived from an organism that is a pathogen of or that produces an allergen of said animal.

In yet another embodiment the avirulent microbes of this invention may be used as vectors for the synthesis of various host proteins. Because the avirulent microbes of this invention are able to traverse a variety of immunocompetent structures including GALT, mesenteric lymph nodes and spleen after introduction into the host, such microbes may be used to target a variety of immunoregulatory products. Accordingly, one or more genes encoding immunoregulatory proteins or peptides may be recombinantly introduced into the avirulent microbes such that when the microbes taking up residence in the appropriate immunocompetent tissue are capable of expressing the recombinant product to suppress, augment or modify the immune response in the host. Examples of immunoregulatory molecules include but are not limited to: colony stimulating factors (macrophage, granulocyte, -14.. or mixed), macrophage chemotoxin, macrophage inhibition factor, leukocyte inhibitory factors, lymphotoxins, blastogenic factor, interferon, and interleukins.

Still another embodiment of the subject invention is the use of the avirulent microbes contemplated herein to deliver and produce pharmacologically active products that might stimulate or suppress various physiological functions (i.e., growth rate, blood pressure, etc.).

By immunogenic agent is meant an agent used to stimulate the immune system of a living organism, so that one or more functions of the immune system are increased and directed towards the immunogenic agent. Immunogenic agents include vaccines. Immunogenic agents can be used in the production of antibodies, both isolated polyclonal antibodies and monoclonal antibodies, using techniques known in the art.

By vaccine is meant 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 the pathogen and/or activate other cells (e.g., phagocytes) to do so in an organism, which is directed against a pathogen or antigen to which the organism has been previously exposed.

Although the phrase "immune system" can encompass responses of unicellular organisms to the presence of foreign bodies, e.g., interferon production, in this application the phrase is restricted to the anatomical features and mechanisms by which a multi-cellular organism produces antibodies against an antigenic material which invades the cells of the organism or the extra-cellular 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 studied and widely reported. A survey of immunology is given in Barrett, James, T., Textbook of Immunology: Fourth Edition, C.V. Mosby Co., St. Louis, MO (1983L A vertebrate is any member of the subphylum Vertebrata, a primary division of the phylum Chordata that includes the fishes, amphibians, reptiles, birds, and mammals, all of which are characterized by a segmented bony or cartilaginous spinal column. All vertebrates have a functional immune system and respond Thus, all vertebrates are capable of responding to vaccines. to antigens by producing antibodies.

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.

An invertebrate is 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 illiciting 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. Exemplary of such invertebrates are shellfish and molluses 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 susceptibility of shellfish to infection by Salmonella will allow the introduction of avirulent strains of Salmonella species and thereby provide potential for the primitive immune system to respond. Therefore, it is within the scope of this invention, 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 said invertebrate against a pathogen.

An "individual" treated with a vaccine of the invention is defined herein as including all vertebrates, for example, mammals, including domestic animals and humans, 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 an "individual". one embodiment of the invention is the use of an avirulent derivative of a pathogenic microbe that attaches to, invades and persists in the GALT or BALT as a carrier of the gene product which is used for stimulating antibody response against a pathogen or allergen. Avirulent does not mean that a microbe of that genus or species cannot ever function as a pathogen, but that the particular microbe being used is avirulent with respect to the particular animal being treated. The microbe may belong to a genus or even a species that is normally pathogenic but must belong to a strain that is avirulent. By pathogenic is meant capable of causing disease or impairing normal physiological functioning.

Avirulent strains are incapable of inducing a full suite of symptoms of the disease that is normally associated with its virulent pathogenic counterpart. Microbes as used herein include bacteria, protozoa, and unicellular fungi.

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

In this application, 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 descendents containing the same genetic material is referred to as a recombinant gene. The term gene is being used here 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 used as a guide in the production of an antigenic product.

The product may be one that was not 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, amino acid 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 in the present invention. 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 At the other end of the spectrum is a long section of DNA coding for several gene appropriate expression vector. 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.

In order for the gene to be effective in eliciting an immune response, the gene must be expressed.

Expression of a gene means that the information inherent in the structure of the gene (the sequence of DNA bases) 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 here 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 glycan 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.

In order for a vaccine to be effective in producing antibodies, the antigenic material must be released in such a way that the antibody-producing mechanism of the vaccinated animal can come into play.

Therefore, the microbe carrier of the gene product must be introduced into the animal. In order to stimulate a preferred response of the GALT or 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.

When the avirulent microbe is used as a carrier microbe, and once the carrier microbe is present in the animal, the antigen needs to become available to the animal's immune system. This may be accomplished when the carrier microbe dies so that the antigen molecules are released. Of course, the use of "leaky" avirulent mutants that release the contents of the periplasm without lysis is also possible. Alternatively, a gene may be selected that controls the production of an antigen that will be made available by the carrier cell to the outside environment prior to the death of the cell. microbe that will persist in the vaccinated animal, for In this way, it is possible to use a viable example in its Peyer's patches, and continue to produce antigen, thereby continually inducing antibody formation.

A preferred gene product under these circumstances is a product that is transferred through the cell membrane into the external environment or a product that becomes attached to or embedded in the external membrane so that all or part of the gene product is exposed to the environment. Typical of this latter type of gene product are antigens normally found on the surface of the If these antigens are transported to the cell surface in a normal organism against which protection is desired. manner, antibody formation against the antigens will be enhanced.

The use of pathogens to deliver antigens from other pathogens to the GALT or BALT would be inappropriate if it were not for the fact that such pathogens can be rendered avirulent while retaining ability to colonize Peyer's patches or the BALT.

The organism from which the recombinant gene is derived may be any pathogen of the animal being vaccinated or may be an organism that produced an allergen or other antigen of the animal. Allergens are 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. 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-ocular 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.

Since preferred methods of administration are oral ingestion, aerosol spray and gastric intubation, -22.. preferred carrier microbes are those that belong to species that attach to, invade and persist in any of the lymphoepithelial structures of the intestines or of the bronchii of the animal being vaccinated. These strains are preferred to be avirulent derivatives of enteropathogenic strains produced by genetic manipulation Strains that attach to, invade and persist in Peyer's patches and thus directly of enteropathogenic strains. stimulate production of IgA are most preferred. In animals these include specific strains of Salmonella, and Salmonella-E; 99;; hybrids that home to the Peyer's patches.

Recombinant DNA techniques are now sufficiently well known and widespread so as to be considered routine.

In very general and broad terms, this method consists of transferring the genetic material, or more usually part of the genetic material, of one organism into a second organism so that the transferred genetic material becomes a part of the genetic material of the organisms to which it is transferred. This usually consists of first obtaining a small piece of DNA from the parent organism 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. obtained by the action of a restriction endonuclease The DNA may be of any size and is often enzyme which acts to split DNA molecules at specific basepair sites. Following ligation to plasmid, phage or cosmid vectors to form recombinant molecules the 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), including electroporation. other methods such as transduction are also suitable, wherein the recombinant DNA is packaged within a phage Once the recombinant DNA is in the carrier cell, it may continue such as transducing phage or cosmid vectors. to exist as a separate piece (generally true of complete transmitted plasmids) or it may insert into the host cell chromosome and be reproduced with the chromosome during cell division.

Although transferring genetic material is relatively straightforward, predicting which transfers will result in expressed genes is not yet possible. This selection process, however, does not present any difficulty to the present invention. Since the host microbe must express the transferred gene and thereby produce an antigen, a "shotgun" approach works well.

Antibodies are first produced against the desired antigen, for example, fragments of cell membranes from DNA from the organism that is the source of the antigen is cleaved pathogenic microbes, by standard techniques. into multiple fragments by endonucleases, and the fragments are inserted randomly into carrier microbes that express antigens from the pathogen can be easily identified by their reaction with antibody against pathogen antigens. Antigen-expressing microbes can be selected and cloned to give the desired recombinant organism. Shotgun cloning is well known and is described in detail in Maniatis, T., et al., Molecular Cloning, Second Edition, Cold Spring Harbor Laboratories (1989).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 more classical methods of gene transfer may be employed such as conjugation, transformation or transduction.

Derivatives of avirulent 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 inability to produce functional adenylate cyclase and/or CAMP receptor protein and/or the expression of the 9g; gene, with or without naturally occurring virulence plasmids. For example, strains such as 4062 and 4064 carry the gyrg mutation conferring nalidixic acid resistance which has been used herein as a convenient marker to follow strains through the animal following oral inoculation. However, drug resistance is not a desirable attribute for strains to be used as vaccines. Thus, the gyrg mutation can be easily removed by transducing the wild-type gyrgi (conferring sensitivity to nalidixic acid) gene into strains by selecting for inheritance of a closely linked Tnlg and then removing Tnlg by transduction with a phage lysate propagated on the parent strain carrying the gyrg: allele with selection for fusaric acid resistance.

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 establish the required amount. Multiple dosages are used as needed to provide the desired level of protection.

The pharmaceutical carrier or excipient in which the vaccine is suspended or dissolved may be any solvent or solid or encapsulated in a material that is non-toxic to the inoculated animal and compatible with the carrier organism or antigenic gene product. Suitable pharmaceutical carriers are known in the art, and for nltxuovulnn-—- _-_ example, include liquid carriers, such as normal saline and other non-toxic 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. Suitable pharmaceutical carriers and adjuvants and the preparation of dosage forms are described in, for example, Remington's Pharmaceutical Sciences, 17th Edition, (Gennaro, Ed., Mack Publishing Co., Easton, Pennsylvania, 1985).

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. known as a secondary, booster, or anamnestic response and The enhanced response is 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 examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.

EXAMPLES Example 1 This example describes the isolation of avirulent microbes by the introduction of deletion mutations affecting CAMP synthesis and utilization and the identification of strains with mutations conferring stability of phenotype, complete avirulence and high immunogenicity.

Bacterial strains. The Escherichia ggli and Salmonella typhimurium strains used are listed in Table 1.A. and B. suspended in 1% Bacto—peptone containing 5% glycerol and They were maintained as frozen cultures fast-frozen in dry ice-ethanol for storage in duplicate at —70°C and also suspended in 1% Bacto-peptone containing 50% glycerol for storage at -20°C for routine use.

Complex media for routine cultivation were L broth (Lennox, Virology 1:190-206, (1965)) and Bacteriol. 1g:461—476 Difco agar was added to Luria broth at 1.2% for Media.

(Davis et al., "A Man. for Genet. Eng.-Adv.Bacterial -27..

Genetics".

Harbor, NY, Cold Spring Harbor Laboratory, Cold Spring (1979)). strain was diluted 1:20 into prewarmed Luria broth, grown An overnight culture of the donor for 60 minutes with shaking at 37°C and then infected with P22HTint at a multiplicity of 0.01., The infection mixture was shaken overnight for approximately 15 h, chloroform added and allowed to shake an additional 10 min at 37°C, and the suspension centrifuged (sorvall RCSC, SS-34 rotor, 7,000 rpm, 10 min) to remove bacterial debris. The supernatant fluid containing the phage (ca. 101°/ml) was stored at 4°C over chloroform. Tetracycline to a concentration of 12.5 pg/ml was used to select for transduction of Tn10 insertions and Tnlo-induced mutations. with Tn10-induced mutations were grown overnight in L Strains broth containing 12.5 mg tetracycline/ml at 37°C to approximately 5 x 108 CFU/ml. Cultures were then diluted 1:40 into prewarmed L broth without tetracycline and aerated at 37°C to a titer of about 2 X 109 CFU/ml.

Suitable numbers of cells (i.e. 107-108) diluted in BSG were plated on fusaric acid-containing medium and incubated 48 h at 37°C. were purified on the same selective medium.

Fusaric acid-resistant isolates Single isolates were picked, grown and tested for tetracycline sensitivity on Penassay agar with and without 12.5 pg tetracycline/ml.

Female BALB/c mice (6 to 8 weeks old) (Sasco, Omaha, NB) were used for infectivity and/or Mice. immunization experiments. Animals were held for one week in a quarantined room prior to being used in experiments.

Experimental mice were placed in Nalgene filter-covered typhimurium strains was determined following peroral (p.o.) or intraperitoneal (i.p.) inoculation.

Food and water were given ad The animal room was maintained at 22-23°C with They were then given 30 ml (w/v) sodium bicarbonate using a Pipetman P200 Evaluation of protective immunity.

Morbidity and mortality of mice were In initial route. Subsequently, groups of mice were perorally immunized with various doses of a virulent mutants and then challenged with various doses of virulent wild-type parent cells at various times after the initial immunization. Morbidity and mortality were observed throughout the experiment and for a least 30 days after challenge with the wild-type parent.

The resulting for a maltose-negative phenotype. The phage-bacteria infection mixtures were incubated for 20 min at 37°C before 100 pl samples were spread onto Macconkey agar (Difco Laboratories, Detroit, MI) containing 1% maltose (final concentration) supplemented with 12.5 pg tetracycline/ml. After approximately 26 h incubation at MONROSDA . WD 1'3 °C, a tetracycline-resistant, maltose-negative colony resulting from the P22HT;gt (PP1037) ~ X3339 infection and a tetracycline-resistant, maltose-negative colony resulting from the P22HT;n; (PP1002) ~ x3339 infection were picked into 0.5 ml BSG and streaked onto the same selective media. designated x3604 (gy_::Tn;Q) and x3605 (g;p;11_::Tn;Q) (Table 1.A.).

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Strains to be orally administered as live vaccines must have complete stability with regard to both their when 50-fold concentrated cultures and various dilutions (~1o’, 107, 105, 103 CFU/plate) of each of the ten independent Agyg mutants and each of the ten independent avirulence and their immunogenic attributes.

Agrp mutants were plated on minimal agar media (supplemented with 22 pg cysteine/ml and 22 pg arginine/ml) containing 0.5% maltose, melibiose, xylose, glycerol, or rhamnose that should not support their growth, revertants and mutants were not detected. One set of duplicate plates were UV-irradiated (5 joules/meterz/sec) and incubated at 37°C in the dark.

The other set of plates was incubated at 37°C with illumination. Revertants and mutants were not detected after a 48 h growth period. An investigation was also conducted as to whether tetracycline-resistant revertants/mutants could be recovered from the fusaric acid resistant Agyg and Agrp mutants at frequencies higher than could be observed for the tetracycline- sensitive wild-type parental strain. In all cases, such tetracycline—resistant revertants/mutants were not observed.

Virulence and immunogenicity of Acrp and Acva The resulting ten Agrp and ten Agya mutants were screened in BALB/c mice by peroral inoculation to mutants. determine the lowest virulence and disease symptomology as revealed by the appearance of the coat (scruffy versus smooth), appetite, and activity (high or low). Five mice per group were p.o. inoculated with ~10’ CFU of each of Animals were scored based on the above criteria and on day 30 of the independent cya or ggp deletion mutants. the experiment the survivors were challenged with 108 CFU of the wild-type virulent parent strain X3339. In three of the twenty groups infected with the gya or grp deletion mutants, five of five mice survived the initial infection with the Acya-12, Acrp-11 and Acrp-10 mutants and were also completely protected against 10‘ LD5os of the wild-type challenge. one group in particular, the Acrp-10 mutant, was unequalled in avirulence, immunogenicity and stability. After repeating these experiments, mice never appeared affected by any dose given p.o. or i.p. of the Acrp-10 mutant (see Example 3, Table 6).

Properties of selected mutant strains. x3615, x3622 and X3623 with the Ac a-12, Acrp-10 and Agrpgll mutations, respectively, were judged to be least virulent, highly immunogenic and extremely stable phenotypically and genotypically. Data on the phenotypic properties of these strains is given in Table 2. Table 3 presents data on the avirulence and immunogenicity of these strains in comparison to results with the virulent wild-type parent x3339 and strains x3604 and x3605 with the gyg::Tn;g and crp-77 ::Tn;Q mutations, respectively.

In addition to requiring histidine, which is due to the gisg mutation in the parental X3339, the Acrp—10 mutation imposed on X3622 requirements for the amino acids arginine and cysteine. The bases for this observation and further analysis of the properties of the Acrp-10 mutation are given in Example 3. .m0u:om conumo uo »m.o + 4: :0 SHSOHU Ucm mom Hmd Ucm mwvmfi HMUM wmmm xmxcooumz co COHUMHCUEHNLQ ucmumwmwmflm u0>wuHm:mmum Mdflfizmmm wmwnmowuwuommn + + a + u\+ n n 1 u u n m Aflummwd nmwnx u u I + u\+ a a u r n u m o u o< mnenx + . + u + :\+ u u n u u a m Ndnmamd maonx + + I + + + + + + + + m mmau UH«3 mnnnx mac mu¢ ma: uaw How am: mzm Hum oca Au: Hm: umnm mmxuocwm >£mohuox=< awm: vac cowumucmeuwu wuwuvhsonumu Una cwmuum mxm aoa x m.m mxm aoa x o.v H u 04 nmmmx mxm nod x ~.n mxm mom x m.a o I u< mmomx mxm aoa x m.n mxm . «OH x ~.~ MAHmNm< mamnx mxm sou x m.m mxm . nod x m.o mwcanuuwmnmmm moenx mxv «on x m.m mxm «ca x ~.m mdcanumam «coax . mxm coa x 0.0 II II mama cHw3 mnnnx ~muou\m>«H Ashe. mmon Hmuou\0>wH «buoy wmoo wamuocwm HUQESC Hn>u>u:m Hm>«>h:m u:m>wawm cwmuum Example 2 This example describes the construction of avirulent microbes by the introduction of deletion mutations affecting CAMP synthesis and utilization and the characterization of strains with two deletion mutations for stability of phenotype, complete avirulence and high immunogenicity.

Bacterial strains. The Escherichia 99;; and Salmonella typhimurium strains used are listed in Table .A. and B.

The maintenance and storage of these strains are as described in Example 1.

Media. enumeration and identification of bacteria are as Complex media for routine cultivation, described in Example 1.

Transduction and fusaric acid selection for Construction of S. typhimurium strains with Acya-12 and Acrp-11 deletion mutations. The best vaccine plasmids), and colicin sensitivity.

The strategy consists of mobilizing deletions of (Acrp-11 zhc-1431::Tn;Q) was used to transduce the virulent strains to tetracycline resistance with screening for Mal". The phage-bacteria infection mixtures were incubated for 20 min at 37°C before 100 pl samples were spread onto Macconkey agar (Difco Laboratories, Detroit, MI) containing 1% maltose (final concentration) supplemented with 12.5 pg tetracycline/ml.

After approximately 26 h incubation at 37°C, tetracycline resistant Mal" transductants were picked and purified onto the same medium. The resulting 798 derivative was designated X3825 and the UK—1 derivative was designated X3828. Strains X3773, X3825 and X3828 have the genotype Acrp-11 zhc-1431::Tn;Q (Table 1.B.). grown in L broth + 12.5 pg tetracycline/ml and each were diluted 1:10 into buffered saline with gelatin (BSG), 100 These strains were pl of each were spread onto fusaric acid-containing (FA) media (Maloy and Nunn, 1981) and the plates were incubated approximately 36 h at 37°C. Fusaric acid-resistant colonies of each strain were picked into 0.5 ml BSG and purified onto FA media. acid-resistant colonies were picked into L broth and Purified fusaric grown at 37°C to turbidity and checked for loss of Tnlg (tetracycline sensitivity), presence of complete LPS and auxotrophy. The new strains were designated X3876 (798) and X3954 (UK-1) which both have the genotype Acrp—11 A[zhc-1431::Tnlg] and X3623 (SL1344 Acrp-11 was originally isolated as described in Example 1) (Table 1.13.).

Selection was made on Macconkey agar + 1% After 26 h, an ampicillin-resistant, Mal+ colony of each strain was picked and purified on Macconkey agar + 1% maltose agar + pg ampicillin/ml and designated X3938 (798) and X3961 (UK-1) which both have the genotype Acrp-11 A[;gg;;g;;::Tn;g] psD11o* and X3774 (SL1344) which has the genotype Acrp-11 pSD110+. containing media and incubated approximately 36 h at 37°C. were picked and purified onto FA medium.

Fusaric acid-resistant colonies of each strain Purified FA-resistant colonies were picked into L broth, grown to turbidity and then checked for loss of Tnlg (tetracycline The pSDl10 plasmid was usually lost spontaneously from the strains sensitivity), complete LPS and auxotrophy. during this process to result in ampicillin sensitivity, except for the SL1344 derivative which involved two steps to eliminate pSD110. The final strains were designated Genotvpic and phenotvpic stability of avirulent mutants. Methods for determining stability of genetic traits are as described in Example 1. All genotypic and phenotypic traits due to the Agyg Aggp mutations were completely stable except motility. Although synthesis of functional flagella and display of motility is dependent on wild-type gyg and ggp gene functions, a suppressor mutation in the gig (constitutive flagellar synthesis) gene can easily be selected to cause flagella synthesis and motility to be independent of gyg and ggp gene variants were readily selected during the strain functions. construction process. Since immunity to flagellar antigens may be protective, motile variants of all vaccine strains were selected.

Fermentation of sugars and growth on various carbon sources of the double mutant strains were identical to strains with only Agyg or Aggy as listed in Table 2. published reports of the requirement for cyclic AMP and The phenotypes were as expected based on the cyclic AMP receptor protein for catabolic activities.

At each step in the construction following selection of a fusaric acid—resistant tetracycline- sensitive derivative, an investigation as to whether tetracycline~resistant revertants/mutants could be recovered at frequencies higher than could be observed for the parental tetracyc1ine—sensitive wild-type strain was conducted. In all cases, such tetracycline-resistant revertants/mutants were not observed.

.HHw xanmwoauoclaaw .CHU—JE UAHUWMU O05 Nah: HE m.C CH 6wum.>«HmU WHHUUIQHD mHHH %HQUMH@UOE|0UMHOUOE ummcmmwfi mo wcvwm wmnmwowuoc O:I>£uHmw:I cuss: owv >:uHmm: exm noaxn ma wflummoq mwumuod nmmnx cuss: aw: xnuammz wxe noaxm nan flqnmmma manmxmq mmmnx Amman aw mam noaxa xnuamon oaxoa ,ao~xa om Hmlmmod m~am»o< mnovx mmuo: noaxa mumumuoe oaxm aoaxm om qfl-muo< NAIMNUQ mmmnx mmsoe coax» xcuaumc mxm ecaxm om -nmum< nmunx muses ooaxw azuammn mxm aoaxm om ~4nm»o< maunx amflumawnmam 4w cwmfiuo omen azuamwz AMUOB .amo. coauua mmauocoo umnazz mnau max» \w>wH mmoo I:Uo:H u:m>0a0m Cwmuum IUHM3 Ivnwi au>A> mcwuma no muzom .xouQm¢ Iusm I:0o:H Effectiveness of immunization with avirulent mutants. remained healthy and ate and grew normally. oaxoa aoa x m mod x H waumuod mHnm»u< mmovx mxm cod x 5 non x N qmummud Mwlmfiod mmmnx mxm ecu x n cog x m nHumuu< nmmnx _ mxm ooa x m aon x w mmum»o< mawmx _amuou cumuum cwmuum 0Q>uocmw uwnfiiz \m>wH ovcmaamcu m:«nw:5EEH u:a>m~wm cfiauum ~m>«>u:m mmhulvawx mo ADMUV mmoa uo .:mu. mmoo mcwnuuw ucwhmm u:wH:uw> 0Q>UlUHw3 cum: wucvqqmnw Mmflwmud Ucwuowuoum cw muflmusfl HHIQHOQ Hoxvcm Ndlmfiod O 5 2 2 Example 3 This Example demonstrates the isolation of an avirulent microbe that possesses a deletion mutation encompassing the crp gene and an adjacent gene which also governs virulence of Salmonella.

Bacterial strains. The Escherichia coli and Salmonella tvphimurium strains used are listed in Table 1A and B. are as described in Example 1.

The maintenance and storage of these strains Media. Complex media for routine cultivation, enumeration and identification of bacteria are as described in Example 1.

Transduction and fusaric acid selection for loss of Tnlo. The media and methods are as described in typhimurium strains were determined as described in Example 1.

Isolation of S. typhimurium strain with the Acrp-10 mutation. As described in Example 1, one of ten Aggy mutations isolated in X3605 conferred auxotrophy for arginine (due to deletion of grgg) and cysteine (due to The mutation in the S. typhimurium auxotrophy for cysteine. A group of five BALB/c mice orally infected with 109 X3622 cells remained healthy and was totally unaffected (Table 3). mice gained high-level immunity to oral challenge with " parental X3339 cells (Table 3).

Furthermore, these A series of strains was constructed to independently evaluate each of the phenotypic characteristics of X3622. The plasmid, pSD110, carrying -62..

An L broth culture of X3622 was typhimurium Selection was (Schroeder and Dobrogosz, Q. designated X3731 and X3774, respectively. invasive strains isolated from a moribund horse and pig, the Acrp—10 mutation. screened for the Mal", Arg' and Cys' phenotypes and the resulting 798 derivative designated X4246 and the X3761 (UK-1) derivative designated X4248 (Table 1).

L broth grown cultures of X4246 typhimurium X3670, which contains the plasmid pSD11O (Table 1). maltose + 100 pg ampicillin/ml + 12.5 pg tetracycline/ml.

Selection was made on Macconkey agar + 1% After 26 h, an ampicillin, Mal+ colony of each strain was picked and purified on the same medium and designated X4247 (798) and X4262 (UK-1) which both have the genotype psD11o*/Acrp-10 zhc-1431::Tn;g.

Virulence of the S. typhimurium X3622, X3731.

X3737. X3774. X3910. X4063 and X4071. data on morbidity and mortality of mice infected Strain X3737 was LDSO dose for the wild-type X3339 parent strain. Mice never appeared ill throughout the 30-day observation period. As a control for this experiment, the crp- 773::Tn;Q mutation in X3605 was complemented by pSD110 to deletion of the grp gene and was not conferred by gene. >uu:uom m ~\o aoa H mwcaunmmm mocvx >uu:uom ma mxo pea H mwceuummxm oamnx >uu:uom ma m\n «ca n wwumumq +oHHGmQ cnnnx mw-mww»m-mmm.< anuammz n mxm .ooa m +oHaomm nnnnx mwcauumwmnmum auusuom m m\a noa H +oHHomm Hnnnx >:uHmm: : mxm «ea c m4-.mm»m-mmm_< mmunx huusuom 5 mxm «ca m @m%uIfiHw3 mnnnx nnuanmz dnummc uo ~3ou\m>3 5.3. mmou wmzuocwo uwnfisc >56 cam: Hm>w>u=m mcwumasoocu u:m>m~mm :.._.m.uum .HHm xanmmoauocuzuusuom uaaw >~mumumUoEImunhwfloE uommwmwo uo mcmww ounmuowuoc oclxnuamonn umwu umzu mdmaficm mo. >uu5.uUm m to aoa x H dd: Imum Hnovx nuuumm qnummfl uo amuou\w>«H Annoy omov wmauocww HUQEDC 2 ant cum: ~u>w>u:m mcwumasuocm u:m>wamm cwmhum Effectiveness of immunization with x3622, highly immunogenic. m4u_owNm«mmm_< mxm aoa x e.m om aoa x m.o +oHHama mmmmx mw-.mmxm-mmm.< mxm ooa x e.m on aoa x m.m +oHaomm nnnnx mxn con x v.H ma «ca x o.m _ nxn «ca x v.H ma . you x o.m M mxm ooa x ¢.H mu . ecu x o.m _ mxm ooa x m.m om eon x ~.v mxm com x ~.n om aon x m.H mxm aoa x w.n on ecu x ~.w mw:.mm»m-mmm.< wnwnx auuou\m>«H cwmuum cowunn cwmuum omauocwv uwnasc an>w>u=m omxulvawz IHCDEEH OCHNHCDEEM no . u:u>mamm camuum uo Aamo. «moo we musox Ammo. wmoo u.3 N 0 H3.H..n>M EUH h NHDUH . 3 USU q4-.mm»m-mmm. aoa x v.m om . «ca x m.H +oaaomm nomvx m4-.mm»uamum.< ~\~ cod x m.m on men x o.m +oaaamm nvmvx amuou\w>w.n cwmuum cowumn cwmhum wnuxnuocwmu uwnfisc an>«>u:m umaulflawa IHCJEEH wcwnwcafifiw uo ucm>0a0m camuum mo .:mu. mmoo HO WU30Nm .:mo. omoo ..n.u:oo.

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Isolation of S. tvphimurium strain with the Acrp-14 mutation. crp-773::Tn;g generated the deletion of genes extending from argg through gygg, another strategy was designed to Since an imprecise excision event of for histidine and cysteine, but not arginine. lysate grown on X3670 to introduce pSD11O carrying the wild-type g;p+ gene. An ampicillin-resistant, maltose-positive transductant was picked and purified on the same medium and the resulting strain was designated X3955.

Virulence of S. typhimurium pSD1l0+[A[crp-cysG|—14 X3955. Table 7 shows morbidity typhimurium X3955. for mice that received approximately 109 CFU. for oral inoculation of 8-week-old female BALB/c mice as described in Example 1. Animals were sacrificed 1, 3, 5 and 7 days after p.o. inoculation with 9.4 x 103 CFU (X3622), 1.2 x 109 CFU (X3737) or 1.1 x 10’ CFU (X3339).

Three mice per group were randomly selected, euthanized and tissue samples collected. The spleen, Peyer's patches, a 10-cm section of the ileum and the small intestinal contents from each mouse were placed in polypropylene tubes with BSG, homogenized with a Brinkmann tissue homogenizer and placed on ice. Undiluted or diluted samples (100 pl) were plated directly on Macconkey agar +1% lactose + 50 pg streptomycin/ml (x3339 and X3737) and Macconkey agar + 1% maltose + 50 pg streptomycin/ml (x3622) and the plates were incubated for 26 h 37°C. determined for each time period and the geometric mean Titers in the perspective tissues were calculated for 3 mice per group at each time of sampling. decrease growth rate as does the grp mutation. diminishing their immunogenicity. pullorum (both infect poultry), as well as non-host Example 4 This example describes the construction of avirulent microbes by the introduction of deletion mutations affecting CAMP synthesis and utilization and an adjacent gene which also governs virulence of Salmonella by affecting colonization of deep tissues and the characterization of strains with two deletion mutations for stability of phenotype, complete avirulence and high immunogenicity.

Bacterial strains. The Escherichia coli and Salmonella typhimurium strains used are listed in Table 1.A. and B. are as described in Example 1. enumeration and identification of bacteria are as The maintenance and storage of these strains Complex media for routine cultivation, described in Example 1.

Transduction and fusaric acid selection for loss of Tn10. The media and methods are as described in Example 1. negative phenotype. used for this purpose. zid-62::Tn1Q mutations. The resulting P22HTigt lysates were then used to transduce the genetic traits into the wild-type recipient strains X3339, 798 and X3761.

P22HTigt propagated on g. typhimurium X3712 (A[g;p-gygg]-19 zhc-1431::Tn;g) was used to transduce the virulent strains to tetracycline resistance with X3777 and X3779 were grown in L broth + 12.5 ug tetracycline/ml and each were diluted 1:10 into buffered saline with gelatin (BSG), 100 pl of each were spread onto fusaric acid-containing (FA) media (Maloy and Nunn, 1981) and the plates were incubated approximately 36 h at 37°C. were picked into 0.5 ml BSG and purified onto FA medium.

Fusaric acid-resistant colonies of each strain since the phenotype of Cya' and Crp' mutants are the same (Mal', Stl', Mtl', etc.), the plasmid, pSD110, carrying the cloned crp+ gene and conferring maltose + 100 pg ampicillin/ml. pl samples of each culture spread onto fusaric acid- containing media and incubated approximately 36 h at 37°C. Fusaric acid-resistant colonies of each strain were picked and purified onto FA medium. Purified FA-resistant colonies were picked into L broth, grown to turbidity and then checked for loss of Tnlg (tetracycline The pSD110 plasmid was usually lost spontaneously from the strains during this process to result in ampicillin sensitivity, except for the SL1344 and UK-1 derivatives which involved two steps to eliminate pSD110. sensitivity), complete LPS and auxotrophy. mutants. Methods for determining stability of genetic sensitivity by the Luria soft agar overlay technique.

Fermentation of sugars and growth on various carbon sources of the double mutant strains were identical to strains with only Agyg or Agrp as listed in Table 2. The phenotypes were as expected based on published reports of the requirement for cyclic AMP and the cyclic AMP receptor protein for catabolic activities.

At each step in the construction following selection of a fusaric acid-resistant tetracycline- sensitive derivative, an investigation as to whether tetracycline-resistant revertants/mutants could be recovered at frequencies higher than could be observed for the tetracycline-sensitive wild-type parental strain was conducted. In all cases, such tetracycline-resistant revertants/mutants were not observed.

Example 5 This Example describes the construction of avirulent microbes by the introduction of deletion mutations affecting cAMP synthesis and utilization and the characterization of strains with two deletion mutations for stability of phenotype and complete avirulence.

Bacterial strains. The Salmonella typhimurium and S. typhi strains used are listed in Table 1.B. and C.

The maintenance and storage of these strains are as described in Example 1.

Media. enumeration and identification of bacteria are as Complex media for routine cultivation, described in Example 1.

Transduction and fusaric acid selection for loss of Tn10.

Example 1.

Genetic stability of avirulent mutants.

Methods for determining stability of genetic traits are The media and methods are as described in as described in Example 1. -78..

Mice. Female CFW-1 mice (18-20 g) (Charles River, Wilmington, MA) were used for all infectivity experiments.

Animals were held for one week in a quarantined room prior to being used in experiments.

Experimental mice were placed in Nalgene filter—covered cages with wire floors. Food and water were given ad libitum. The animal room was maintained at 22-23°C with a period of 12 h illumination. suspension was prepared by autoclaving 10 min 121°F (15 The mucin using classical genetic methods similar to those Their maltose-negative phenotype.

Neither insertion alone affects virulence of S. typhimurium.

Transduction of the gene deletions with the purpose. tetracycline resistance with screening for Mal’. The phage—bacteria infection mixtures were incubated for 20 min at 37°C before 100 pl samples were spread onto Macconkey agar (Difco Laboratories, Detroit, MI) containing 1% maltose (final concentration) supplemented with 12.5 pg tetracycline/ml. After approximately 26 h incubation at 37°C, tetracycline—resistant Mal" transductants were picked and purified onto the same medium. The resulting Ty2 derivative was designated X3853, the ISP1820 derivative designated X4298 and the ISP2822 derivative designated X3852. All of these strains have the genotype Acrp-11 zhc-1431::Tn;Q (Table 1.c.). broth + 12.5 pg tetracycline/ml and each were diluted :10 into buffered saline with gelatin (BSG), 100 pl of Strains X3852, X3853 and X4298 were grown in L each were spread onto fusaric acid-containing (FA) media (Maloy and Nunn, 1981) and the plates were incubated approximately 36 h at 37°C. Fusaric acid-resistant colonies of each strain were picked into 0.5 ml BSG and purified onto FA medium. Purified fusaric acid-resistant colonies were picked into L broth and grown at 37°C to turbidity and checked for loss of Tn;Q (tetracycline sensitivity), presence of complete LPS and Vi antigen and auxotrophy for cysteine and tryptophan (two amino acids required by all the parent strains). The new strains were designated X3877 (ISP2822), X3878 (Ty2) and X4299 (ISP1820) which all have the genotype Acrp-11 A[zhc-1431::Tn;Q] (Table 1.C.). typhimurium X3670, which contains the plasmid pSD1 (Table 1.B.). selection was made on Macconkey agar + 1% maltose + 100 pg ampicillin/ml. After 26 h, an ampicillin-resistant, Mal+ colony of each strain was picked and purified on Macconkey agar + 1% maltose agar + 100 pg ampicillin/ml and designated X3879 (ISP2822), X3880 (Tyz) and X4300 (ISP1820) which all have the genotype Ag;p;1; A[zhc-1431::Tn;Q] pSD110+.

The resulting Cultures of X3921, X3922 and X4316 were grown in incubated approximately 36 h at 37°C. Fusaric acid—resistant colonies of each strain were picked and purified onto FA medium. Purified FA-resistant colonies were picked into L broth, grown to turbidity and then checked for loss of Tnlg (tetracycline sensitivity), complete LPS, Vi antigen and auxotrophy for cysteine and tryptophan. The pSD110 plasmid was usually spontaneously -82..

Detroit, MI) and by ViII bacteriophage sensitivity by the Luria soft agar overlay technique. Synthesis of flagella were isolated as flagellated and motile whereas strain gene (Silverman and Simon, J. (1974), were selected in motility agar. x4323 was selected as a flagel1a—positive motile derivative of x4222.

Table 8 lists the phenotypic properties of all the mutant strains and their parents with regard to fermentation of sugars and growth on various carbon sources, LPS profile, Vi antigen and mean generation time. The phenotypes are as expected based on published reports of the requirement for cyclic AMP and the cyclic AMP receptor protein for catabolic activities. nmmmx I + + + + + + + I + + + + I mmnmx WNOMN I + + + + + + + + + + + mvnnx wmhuocmzm QWOCHQMHM UUMHHHU GMOEEMSH 0WOHQHHwE HOUHCCME HOUHQHOM mmO03HO wm.o wm.o wm.o wm.o wm.o »m.o wm.o MWOUSHO wa mmoawn wa QWOCCME NH QMOCHQMHM WH GDMHUHU wa NWCCEMCH N H mmoflnflawe wa HONHCCME NH Houwnuom .omH wmouamfi »H + H004 mwmm wmxcouomz + + + + + + + + + + + + + + + + Hmww amfiwcwz mcwmuum HSQNU 4W we mwwuhmmohm £u3oHm UCM COHHMPCQEHGM O .|_ .|_ .m mflmfifi Acamuum Hw>aamV QUUHQEOU 0#mHQEoU mumamfioo wumamfioo mommnmom an OHHMOHQ mmq M m M K HQM M M M M .H m m M M eqam m m m m ucflemmmm m m m m onxflawm m m w m HHH> n%U«>Huam:wm mwmnmoauouomm I I I I kmmmm COfl#M#CWEHOM UHOUCH IU5m Iodm Iozm Iosm.

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The other set of plates was incubated Revertants and mutants were not detected after a 48 h growth period. An investiga- tion was also conducted as to whether tetracycline- resistant revertants/mutants could be recovered at frequencies higher than could be observed for the parental strain. In all cases, such tetracycline- resistant revertants/mutants were not observed.

Virulence of mutant strains for mice. typhi is man. Therefore, hog typhi vaccine candidates in this model system.

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Example 6 This Example demonstrates the construction of an avirulent microbe by the introduction of deletion mutations affecting cAMP synthesis and utilization and an adjacent gene which governs virulence of Salmonella by affecting colonization of deep tissues.

The maintenance and storage of these strains are as typhi strains used are listed in Table 1.B. and C. described in Example 1.

Media. enumeration and identification of bacteria are as Complex media for routine cultivation, described in Example 1.

Transduction and fusaric acid selection for loss of Tnlg.

Example 1.

Genetic stability of avirulent mutants.

Methods for determining stability of genetic traits are The media and methods are as described in invasive for humans. would seem prudent to consider adding an additional attenuating mutation to further enhance safety without compromising immunogenicity. The properties of the S. typhimurium strains The wild-type, virulent Ty2 (type E1), ISP182O (type 46) and ISP2822(type E1) strains were genetically modified as described below, using classical genetic -90.. methods similar to those described in Curtiss and Kelly (1987). ISP1820 and ISP2822 were recently isolated during a typhoid epidemic in Chile and are likely to be more invasive than the standard laboratory Ty2 strain of vaccine strains that could be more efficacious than those linked to gya were used for this purpose. -91.. containing 1% maltose (final concentration) supplemented with 12.5 pg tetracycline/ml. After approximately 26 h incubation at 37°C, tetracycline-resistant Mal‘ transductants were picked and purified onto the same medium. The resulting ISP2822 derivative was designated X3791, the Ty2 derivative was designated X3792, and the All of these strains have the genotype A[g;p-gygg]-lg zhc-1431::Tn;g ISP1820 derivative was designated X4324. and were auxotrophic for cysteine, tryptophan and Strains X3791, X3792 and X4324 Each cultures of X3802, X3803 and X4325 were transduced with Strains X3824, X3845, and X4331 were grown in L broth + 100 pg ampicillin/ml and were each independently transduced with P22HTigt propagated on X3711 to introduce the linked Acya-12 and zid-62::Tn;Q mutations. The transduction mixtures were plated on Macconkey agar + 1% maltose + 100 pg ampicillin/ml + 12.5 pg tetracycline/ml.

Ampicillin-resistant (pSD110+), tetracycline-resistant (zid-62::Tn;Q), Mal" (Agyg) colonies were picked and purified on Macconkey agar + 1% maltose + 100 pg ampicillin/ml + 12.5 pg tetracycline/ml. Purified colonies were picked into L broth, grown to turbidity and the strains checked for complete LPS, Vi antigen and auxotrophy for cysteine and tryptophan. The resulting strains were designated X3919 (Tyz), X3920 (ISP2822) and X4340 (ISP1820) which all have the genotype A[g;p-gygg]-lg A[zhc-1431::Tn;g] pSD110+ Acya-12 zid-62::Tn;Q. Cultures of X3919, X3920 and X4340 were grown in L broth + 100 pg ampicillin/ml + 12.5 pg tetracycline/ml to turbidity, diluted 1:10 into BSG, and 100 pl samples of each culture spread onto fusaric acid- containing media and incubated approximately 36 h at 37°C. were picked and purified onto FA medium.

FA-resistant colonies were picked into L broth, grown to turbidity and then checked for loss of Tnlg (tetracycline Fusaric acid-resistant colonies of each strain Purified sensitivity), complete LPS, Vi antigen and auxotrophy for The pSD110 plasmid was usually spontaneously lost from the strains during cysteine, arginine and tryptophan. this process to result in ampicillin sensitivity. The final strains were designated X3924 (Tyz), X3925 (ISP2822) and X4345 (ISP1820) which all have the genotype A[g;p-gysg]-19 A[;hg;14;;::Tn1g] Agyg;1; A[zid-62::Tn1Q] (Table 1.C.). confirmed by slide agglutination with antisera to Vi (Difco Laboratories, Detroit, MI) and by ViII Genetic stability of avirulent mutants. .5% maltose, melibiose, xylose, glycerol, or rhamnose that should not support their growth, revertants and mutants were not detected. One set of duplicate plates was UV-irradiated (5 joules/meter:/sec) and incubated at 37°C in the dark. The other set of plates was incubated at 37°C with illumination. Revertants and mutants were not detected after a 48 h growth period. An investigation was also conducted as to whether tetracycline-resistant revertants/mutants could be recovered at frequencies higher than could be observed for the parental strain. In all cases, such tetracycline-resistant revertants/mutants were not observed.

Example 7 enumeration and identification of bacteria are as Complex media for routine cultivation, described in Example 1.

Transduction and fusaric acid selection for loss of Tn10.

The media and methods are as described in was genetically modified as described below, using classical genetic methods similar to those described in Curtiss and Kelly (1987) and Nakayama, Kelly and Curtiss (1988). containing the Acya-12 Acrp-11 mutations was described in balanced—lethal host-vector combinations are stable for vector possessing the wild-type asd+ gene. plasmid results in DAPless death and cell lysis. several weeks in the immunized animal host and elicit strong immune responses against the cloned gene product as well as against Salmonella.

The construction strategy consists of mobilizing the AasdA1 mutation that has been isolated and X4346 via P22HTint transduction with selection for Lxphimurium x3520 containing the AasdA1 and zhf-4::Tn;g mutations. tetracycline/ml and was diluted 1:10 into buffered saline The resulting ISP1820 derivatives were with gelatin (BSG), 100 pl was spread onto fusaric acid-containing (FA) + 50 pg DAP/ml medium (Maloy and Nunn, 1981) and the plates were incubated approximately 36 h at 37°C. picked into 0.5 ml BSG and purified onto FA + 50 pg DAP/ml media. were picked into L broth + 50 pg DAP/ml and grown at 37°C Fusaric acid-resistant colonies were Purified fusaric acid-resistant colonies to turbidity and checked for loss of Tnlg (tetracycline sensitivity), presence of complete LPS and Vi antigen and auxotrophy for cysteine, tryptophan, methionine, threonine and DAP on minimal media. The new strains were designated X4297 (Ty2), which has the genotype Ag;p;;l Asd' derivatives of the wild-type parent sample was spread onto fusaric acid containing + 50 pg identified by immunological screening of a kgt11::n. kDa, both of which react very strongly with antibodies in the pooled LL patients’ sera (Sathish et al., 1990). 14 out of the 21 LL patients’ sera when the sera were tested individually (Clark-Curtiss, Thole, Sathish, Bosecker, Sela, de Carvalho and Esser, Res. in Microbiology, in press).

The 1.0 kb u. leprae insert DNA fragment was These proteins also react with antibodies in patients’ sera.

In addition, expression of immunologically reactive proteins from pYA1077 was also shown in X4417, X4435, X4455, and X4457. of proteins produced by Agtllzu. pYA292, pYA1077 and pYA1078. nitrocellulose filter were reacted with pooled sera from llnlvhnrnn ran 1 A Example 8 This example provides a procedure for testing The Individuals Studied. studied are volunteers who are healthy adult humans aged The individuals -39 years. The prospective volunteers are screened before the study. The screening procedure includes: 1. medical history 2. physical examination 3. electrocardiogram 4. urinalysis . complete blood count 6. blood chemistries (BUN, creatinine, fasting blood glucose 7. Serum Na+, Cl", K+, HCO3 8. VDRL 9. Hepatitis B surface antigen . HIV antibody by ELISA . Pregnancy test (females) . Liver function tests (SPGT) . Psychological examination and interviews.

The Volunteers to participate in the study are selected on the basis of general good health and have: . no clinically significant history of gall bladder disease, immunodeficiency, cardiovascular disease, respiratory disease, endocrine disorder, liver disease including a history of hepatitis, renal and bladder disease, enlarged prostate, glaucoma, gastrointestinal disease, disorder of reticuloendothelial system, neurologic illness, psychiatric disorder requiring hospitalization, drug or alcohol abuse; . normal and regular bowel habits falling within the limits defined for a normal population: at least 3 stools per week and less than 3 stools per day without frequent use of laxatives or antidiarrheal agents; . absence of allergy to amoxicillin or ciprofloxacin; . no history of any antibiotic therapy during the 7 days before vaccination; . a negative pregnancy test (females); . a negative HIV antibody test.

The Volunteers are admitted to an Isolation Ward, and informed, witnessed, written consent is obtained.

Study design. Groups of 22 volunteers are studied. Baseline blood and intestinal fluid specimens are collected. After a two-day period of acclimatization on the ward, the fasting volunteers are randomly allowed to ingest with bicarbonate buffer a single oral dose containing 5 X 105 of either the Agyg Aggy derivative of Ty2, ISP1820 or ISP2822. The volunteers are observed for the next 15 days for adverse reactions (fever, malaise, chills, vomiting, diarrhea)(the usual incubation period of typhoid fever is 8-12 days). Serial blood and stool cultures are obtained. In addition, any volunteer who has a temperature elevation to 100.8° F has blood samples drawn at the time the observation is made; if the temperature remains elevated at this level for 12 hours, therapy is initiated with oral amoxicillin (1.0 gram every 6h) and oral ciprofloxacin (750 mg every 12h for 10 days). Duodenal fluid cultures are also obtained during the period of observation on days 7, 10, and 13.

Animal tests. The LD5°s for the parent strains and attenuated derivatives in mice by intraperitoneal inoculation with hog gastric mucin as adjuvant are also determined.

Preparation of the vaccine inocula. Stock suspension is inoculated onto trypticase soy agar plates, colonies are picked and suspended in saline. typhi O and H antisera are performed before use. Replica spread plate quantitative cultures are made of the inocula before and after vaccination to confirm viability and inoculum size.

Inoculation of Volunteers. The vaccine is administered by the oral route with NaHCO3. Volunteers are NPO for 90 minutes before vaccination. Two grams of NaHCO3 are dissolved in 5 ounces of distilled water.

Volunteers drink 4 ounces of the bicarbonate water; one minute later the volunteers ingest the vaccine suspended in the remaining 1 ounce of bicarbonate water.

Volunteers take no food or water for 90 minutes after inoculation.

Procedures for Specimen Collection.

Stool specimens. number, consistency, and description of all stools passed A record is kept of the by volunteers. A specimen of every stool (or rectal swab if stool is not passed) is collected for culture. The volume of the specimen is measured. Stools are graded on a five point system: grade 1-firm stool (normal) grade 2-soft stool (normal) grade 3-thick liquid (abnormal) grade 4-opaque watery (abnormal) grade 5-rice water (abnormal).

Phlebotomy. is obtained before and 8, 21, 28, 60, and 180 days after Serum for antibody determinations vaccination. Heparinized blood for lymphocyte separations for antibody-secreting cell assays is collected on days 0, 4, 7, and 10. Mononuclear cells collected on days 0, 28, 60, and 180 days are used to assess lymphocyte proliferative responses to Salmonella and control antigens. Lastly mononuclear cells from days 0, 28, 60, and 180 are also used in the antibody- organisms. Blood (5 ml) is obtained for culture on days 3, 4, 7, 8, 10, 12, and 15 during the post-vaccination observation period to detect vaccine organisms. An additional specimen of serum and mononuclear cells are obtained 180 days after primary vaccination.

Jeiunal fluid aspiration. Before oral vaccination and immediately before discharge (day 15), volunteers swallow polyvinyl chloride intestinal tubes to a distance of 130 cm from the mouth to collect intestinal fluid for measurement of local SIgA antibody. Ten mg of metoclopramide is given orally after ingestion of the tube to accelerate its passage from the stomach through the pylorus into the small intestine. Placement of the tubes in the jejunum is verified by distance (130 cm), color (yellow-green), and pH (6) of aspirated fluid.

Approximately 100 ml of jejunal fluid is removed at each intubation.

Gelatin String Capsules. rates of intestinal colonization with each vaccine In order to determine strain, gelatin string capsules (Entero-Test) are ingested by volunteers three times during the period of hospitalization.

The volunteer is NPO from 6 A.M. water is used to moisten the mouth and throat. The A swallow of capsule, with a portion of the string pulled out, is swallowed with water while holding the loop of the nylon string. The line is secured to the face, and left in place for 4 hours. The volunteers are allowed to drink water ad lib, but are not allowed other food or beverages. After 4 hours, the line is withdraw, the distal section saturated with bile stained mucus is cut and placed in a sterile petri dish, which is labeled for identification. The strings are then cultured for microorganisms, using the same method as with the stool specimens. ~105- Tonsillar Cultures. In order to detect possible invasion of tonsillar lymph tissue after vaccination, serial tonsillar cultures are obtained on days 3, 4, 7, 8, 10, 12, and 15.

Bacteriological Analysis. swabs, and the distal 15 cm of bile-stained duodenal Stools, rectal string from the ingested gelatin capsule is inoculated into selenite F enrichment broth. Tonsillar swabs are inoculated into GN broth.

°C, subcultures are made onto Salmonella-Shigella agar After overnight incubation at Blood cultures (5 ml) are inoculated into 50 ml of supplemented brain heart infusion broth.

Immunological Analysis. specimens are tested for IgA, IgM, and IgG antibodies to Sera and jejunal fluid H antibody is also measured by Virginia shares an identical flagellar antigen with g.

HEM)- Peripheral blood mononuclear cells are collected and separated for studies of specific responses to Salmonella antigens. These include the following. method of Kantele et al.

Levine et al., supra.

It is expected that excretion of the vaccine strain would cease Excretion of the Vaccine Strain. within 1 week after a dose of vaccine. If excretion continues for 7 or more days, the volunteer who continues to excrete is given a dose of ciprofloxacin (750 mg every 12 hours). Negative cultures for 22 consecutive days are required for discharge.

Example 9 This example demonstrates the safety and strains. Volunteers were closely monitored on an Isolation Ward for 15 days (first study) or 24 days (second study). Vital signs were measured every six All stools from each volunteer were collected in plastic containers, hours during the period of observation. examined, graded on a five-point scale, and the volume measured if the stool was loose. Volunteers were interviewed daily by a physician and asked about symptoms. Fever was defined as oral temperature 3 38.2°C; diarrhea was defined as two or more loose stools within 48 hours totalling at least 200 ml in volume or a single loose stool 3 300 ml in volume. Antibiotic therapy was given to volunteers who developed fever or positive blood cultures.

In order to prepare the vaccine, stock cultures of X3927 which had been maintained on trypticase soy broth with 15% glycerol at -70°C were thawed and grown on After incubation at 37°C, 20-30 typical colonies of the vaccine strain were picked from supplemented aro agar. aro agar, suspended in saline, and inoculated again onto aro agar. After overnight incubation at 37°C, the bacteria were harvested with 3 ml of sterile phosphate buffered saline (PBS) and the concentration of bacteria Dilutions of the suspensions were made in PBS to achieve the desired was standardized turbidimetrically. volunteers drank 120 ml to neutralize gastric acid. one minute later, volunteers drank the vaccine suspended in the remaining 30 ml of bicarbonate solution. Volunteers had nothing to eat or drink for 90 minutes before and after vaccination.

On days 7, 10, and 13 after vaccination, fasting volunteers swallowed gelatin capsules containing string devices to collect samples of bile-stained duodenal fluid. and the color and pH of the distal 15 cm were recorded.

After 4 hours, the strings were removed Duodenal fluid was squeezed from the end of the string and cultured as above.

Blood for culture of the vaccine organisms was systematically collected on days 4, 5, 7, 8, 10, 12, and after vaccination and again if fever occurred. Five ml of blood was inoculated into 50 ml of supplemented aro broth.

In addition, tonsillar cultures were obtained on days 1, 2, 4, 5, 7, 8, 10, 12 and 15 to detect the vaccine strain. Swabs applied to the tonsils were inoculated into Gram Negative broth with supplements for 24 hours and then onto supplemented salmonella-shigella agar.

In order to determine the immunological response, the following procedures were followed. Serum samples were obtained before and on days 7, 21, 28, and 60 after vaccination. Jejunal fluids were collected before and on day 14 after vaccination, as described in The total IgA content of the fluids were measured by ELISA and each specimen was standardized to Antibodies to S. typhi lipopolysaccharide (LPS), H, and Vi antigens were Example 8. contain 20 mg of IgA per 100. measured in serum and jejunal fluids.

IgG antibody to LPS 0 antigen was detected by ELISA. and post-vaccination sera tested at a 1:100 dilution was A rise in net optical density 3 0.20 between pre- considered a significant rise. The positive control serum used with each microtiter plate contained a high level of LPS 0 antibody and represented a pool of sera from 12 healthy Chileans who had strong IgG LPS 0 antibody responses after immunization with Ty21a vaccine.

IgA antibody to LPS 0 antigen was measured using two- fold dilutions of serum, starting with a 1:25 dilution.

An IgA titer was considered significant if a 4-fold rise occurred between pre- and post-vaccination procedures.

The Widal tube agglutination test for H antibody was performed using Salmonella Virgina which shares the flagellar antigen d with g. typhi, but no other antigens.

Vi antibody was measured in serum and jejunal fluid by ELISA; a 4-fold rise was considered significant. binding of antibody secreted by lymphocytes was measured NC) were added to antigen-coated plates. by the change in optical density produced by the reaction of the substrate with bound anti-IgA conjugate.

Significant responses to LPS, H, and Vi antigens were determined using the differences in 0.D. pllus 3 S.D. generated from pre-immunization and day 4 cells taken In the ELISPOT assay, specific IgA secreted by individual from volunteers participating in these studies. lymphocytes was detected by adding an agarose overlay to each well and counting colored spots produced by reaction of the substrate with bound anti-human IgA conjugate.

Detection of 3 4 spots per well after vaccination was defined as a positive response; this number is based on the mean number of spots counted before vaccination plus 2 S.D.

The results obtained were the following.

The clinical signs and symptoms of volunteers after vaccination were evaluated in a double-blind fashion. One of 12 volunteers who received strain X3927 This volunteer developed fever with a maximum This volunteer had severe abdominal cramps, malaise, anorexia, had fever. temperature of 40.1°C on day 22 after vaccination. headache, and vomiting on days 4-13, but his fever did not begin until day 22. His symptoms then included dizziness, muscle and body aches, constipation, insomnia, and cough productive of brown sputum. Another volunteer in this group had malaise, cramps, headache, and nausea during the inpatient surveillance period.

The bacteriology studies showed that one of six volunteers who received 5 x 10‘ and one of six volunteers who received 5 x 105 cfu of X3927 had positive blood cultures. These occurred on days 15 and days 8 and 12, respectively. Neither of these volunteers had any symptoms. one of the 12 volunteers who received X3927 had one colony of vaccine organisms detected in the stool None of these volunteers had positive The X3927 isolates recovered from the blood and the stool of on day 1. tonsillar or duodenal string cultures. volunteers retained all expected phenotypes associated with the presence of Agya Aggy mutations.

The degree of attenuation conferred by deletions in the cyclic AMP regulatory pathway cannot be strictly measured without simultaneous challenge of volunteers with mutant and parent strains. However, based on historical experience with volunteers given similar doses of wild type strains, it is likely that the typh;. When wild- typhi strain Ty2 was fed to six volunteers at a typhoid fever (defined as temperature 103°F for >36 hours) or infection (defined as low grade fever, significant serologic response, positive blood culture, typhi for > 5 days. among the 12 volunteers reported herein who received the enteric infection with the vaccine.

Example 10 These occurred on day 15 and days 8 and protective immunity in the majority of those immunized, be less likely to induce vaccinemia. and ISP1820 (Type 46) strains have been genetically modified using classical genetics by similar methods described in Curtiss and Kelly ((1987), Infect. Immun. 55:3035-3043), and described in Example 1. Salmonella typhimurium deletion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and Infect. Immun. 55:3035-3043.(1). The strategy consists of facilitating transduction of immunogenic. incubated approximately 36 h at 37°C.

Samples of 100 pl of each strain were spread onto Fusaric acid- resistant colonies of each strain were picked into 0.5 ml BSG and purified by streaking onto FA media. Purified fusaric acid-resistant colonies were picked into Luria broth and grown at 37°C to turbidity and checked for loss of Tnlg (tetracycline sensitivity), complete LPS, Vi antigen and auxotrophy for arginine, cysteine and tryptophan. The new strains were designated X3803 (Ty2) and X4325 (ISP1820) which have the genotype Acrp-10 A[zhc-1431::Tn;Q]. maltose + 100 pg ampicillin/ml.

Selection was made on Macconkey agar + 1% After 26 h, an ampicillin-resistant, Mal+ colony of each strain was picked and purified on Macconkey agar + 1% maltose agar and designated X3824 (ty2) and X4331 (ISP1820) which have the genotype Acrp-10 A[zhc-1431::TnlQ] pSD1l0+.

Strains X3824 and X4331 were grown in L broth + 100 pg ampicillin/ml and were each independently transduced with P22HTig; propagated on X3712 to introduce the Acya-12 and the linked zid—62::Tn;Q mutations.

Selection for a maltose negative, tetracycline resistance, ampicillin resistance phenotype was made on Macconkey agar + 1% maltose + 100 pg ampicillin/ml + 12.5 Ampicillin-resistant (pDS110+), tetracycline-resistant (zid-62::Tn;Q), Mal" (Agya) pg tetracycline/ml. colonies were picked and purified onto Macconkey agar + % maltose + 100 pg ampicillin/ml + 12.5 ug tetracycline/ml. Purified colonies were picked into Luria broth, grown to turbidity and the strains checked for complete LPS, Vi antigen and auxotrophy for arginine, cysteine and tryptophan. Isolates of the correct phenotype were designated X3919 (Tyz) and X4340 (ISP1820) which have the genotype Aggpglg A[ghg;;4;;::Tn;Q] pSD110+ Acya-12 zid-62::Tn;g. Cultures of X3919 and X4340 were grown in L broth + 100 pg ampicillin/ml + 12.5 pg tetracycline/ml to turbidity, diluted 1:10 into BSG, and 100 ul samples of each culture spread onto fusaric- containing media and incubated approximately 36 h at 37°C. were picked and purified onto FA media.

Fusaric acid-resistant colonies of each strain Purified FA- resistant colonies were picked into Luria broth, grown to turbidity and then checked for loss of Tnlg (tetracycline sensitivity), complete LPS, Vi antigen and auxotrophy for The pSD110 plasmid was spontaneously lost during growth of the strains in arginine, cysteine and tryptophan. the absence of ampicillin. The final strains which were ampicillin-sensitive and plasmid—free were designated X3924 (ty2) and X4345 (ISP1820) which have the genotype Acrp-10 A[zhc-1431::Tn;g] Acya-12 A[zid—62::Tn;g]. synthesis of flagella with display of motility is Since partially dependent upon functional gya and grp genes and since flagella are important antigens, we selected derivatives of X3924 and X4346 that possess a suppressor mutation (gfg) that permits flagella synthesis and function to be independent of the gya and ggp gene functions. X4073 was selected as a flagella-positive derivative of X3924, and X4346 was selected as a Table 10 lists the wild-type parent strains and their Agya Aggy flagella-positive derivative of X4345. derivatives.

Strains x4073 and x4346 can easily be distinguished from their wild-type parents by the following phenotypic characteristics: the inability to ferment or grow on the carbon sources maltose, mannitol, sorbitol, melibiose and xylose, inability to produce H25, increased generation time, and the significantly increased murine LDSO values.

Table 10 Bacterial Strains Type E1, wild type, Vi+.

Received from L. Baron, Walter Reed Army Institute of Research, Washington, DC, as Ty2.

Type 46, wild type, Vi+.

Received from M. Levine, Center for Vaccine Development, Baltimore, MD, as ISP1820. 1983 isolate from a Chilean patient.

X3769. X4073 and X4346 Cells of each strain were picked from agar Growth conditions for x3744.

Cultures were incubated as when the medium into 2 ml Luria broth. static cultures at 37°C for approximately 14 h. slowly than their wild-type parents. x3744 X4346 x3769 X4073 Macconkey Base Agar + % maltose + - + - 1% sorbitol + - + - 1% mannitol + - + - 1% melibiose + - + - 1% rhamnose - - - - 1% citrate - - - - 1% arabinose - - - - 1% mannose + + + + 1% xylose + - + - 1% glucose + + + + Minimal agar‘ + 0.5% glucose + + + + 0.5% sorbitol + - + - 0.5% mannitol + - + - 0.5% melibiose + - + - .5% rhamnose - - 0.5% citrate — - 0.5% arabinose - - 0.5% mannose + + 0.5% xylose + - ‘Minimal media recipe attached; supplements L—arginine Hcl 22 pg/ml, L-cysteines Hcl 22 L-tryptophan 20 pg/ml. include uq/ml, Table 11 (cont’d) Phenotype x3744 X4346 X3769 X4073 Triple Sugar Iron media - H28 + - + - production alkaline slant = Lac" Lac" Lac" Lac" Glu+ Glu+ G1u+ Glu+ Suc Suc' Suc Suc Indole fermentation assay - - - - Bacteriophage sensitivity" LPS profile by SDS-PAGE (silver comp. comp. comp. comp. stain) (comp. = complete) Motilityd + + + + Colicin(s) production - - — - ° phage sensitivity was assayed by soft agar overlay technique of by transduction. S = sensitive; R = resistant. d Motility determined by stabbing a loopful of a standing—overnight Luria broth culture into media containing 1.0% casein, 0.5% NaCl, 0.5% Difco agar and 50 pg/mg triphenyl-tetrazoleum chloride; incubation at °C and motility recorded at 24 and 48 h.

Table 11 (cont'd) MGT° Plasmid content Auxotrophy MIC Tetracycline Streptomycin Ampicillin Gentamicin Chloramphenicol Neomycin Rifampicin Nalidixic acid Spectinomycin Kanamycin Phenotvpe X3744 x4346 X3769 x4073 .6 DOHE Cys' Trp Arg <2 <2 <2 DONE Cys Trp Arg <2 <2 <2 <2 DONE Cys' Trp' <2 16 <2 <2 <2 .5 none Cys' Trp' Arg+ <2 <2 <2 ° Mean Generation Time (min.) = determined in Luria broth with aeration (150 rpm New Brunswick platform shaker) at °C. f Minimal Inhibitory Concentrations (pg/ml) of antibiotics were determined by streaking standing- overnight cultures of each strain onto agar containing defined concentrations of antibiotics.

Table 11 (cont'd) Phenotype xglgg 4346 X3769 x4073 Agglutination with Difco antisera to: flagellar antigen H:1 flagellar antigen H:2 Group D factor 9 Group D factor 12 Group D (0-1,9,12) +++++ +++++ +++++ +++++ Growth characteristics on agar media Strains were grown in Luria broth as standing- overnight cultures at 37°C, diluted in buffered saline and gelatin (BSG) and plated on Macconkey agar containing 1% maltose to achieve isolated colony-forming units (cfu). size and color.

All colonies of a given strain appear uniform in Due to the slower growth rates of A91; ggp strains compared to their wild-type parents, growth on Macconkey media takes ~36+ h at 37°C before colonies of X4073 and x4346 are easily visible.

Stability of mutant phenotypes Fifty-fold concentrated cultures and various dilutions (~ 10’, 107, 105, 103 cfu/plate) of X4073 and X4346 were plated on minimal agar media (supplemented with 22 ug L-arginine/ml, 22 pg L-cysteine/ml and 20 pg L-tryptophan/ml) containing either 0.5% maltose, melibiose, xylose, glycerol, or rhamnose that should not support their growth. one set of duplicate plates were UV—irradiated (S joules/meterz/sec) and incubated at 37°C in the dark. The other set was incubated at 37°C with illumination.

No revertants and/or mutants were detected after a 48 h growth period.

Storage of strains Each strain was maintained in a 1% peptone- % glycerol suspension and stored at -70°C.

Preparation of inoculum for animal experimentation The following is a standardized protocol for growth and suspension of each vaccine strain and its parent for intraperitoneal (i.p.) inoculation of mice.

Female CFW mice (18-20 g) (Charles River, Wilmington, MA) were used for determining LD5o values of The cell suspensions were then administered i.p.

See Table 12 for results on parents. type and Acrp-11 Acrp-10 strains I LD501 Strain No. Genotype CFU X3744 ISP1820 wild type 32 x4299 Ag;p;;; A[gQg;;A;1::Tn;Q] <600 X4300 Ag;p;;; A[zhc-1431::Tn;Q]/ 107 psD11o*’ X4323 Ag;p;11 A[zhc—1431::Tn1g] >2.3 x 103 Agygggg A[zid-62::Tn;Q] X4325 Ag;p;1g A[;gg;14;1::Tn1g] >3.2 x 105 X4331 Ag;g;1g A[zhc—1431::Tn;g]/ >2.3 x 105 psD11o* X4346 Ag;p;1g A[zhc-1431::Tn1g] 4.4 x 105 Agyg;;g A[zid-62::Tn;Q] X3769 Ty2 wild type 54 X3373 Ag;g;11 A[zhc-1431::Tn1g] 1.0 x 103 X3880 Acrp-11 A[zhc-1431::Tn;g]/ <19 psD11o* X3303 Acrp-10 A[zhc-1431::Tn1g] 1.5 x 105 X3824 Acrp-10 A[zhc-1431::Tn1g]/ >1.9 x 105 psD11o* X4073 Acrp-10 A[zhc-1431::Tn1g] >1.o x 105 collected over a 72 h period. 2 pSD110 (Schroeder, C.J., and W.J. Dobrogosz. 1986. J.

L050 calculated by method of Reed and Muench (1938.

Am. J. Hyg. ;1:493-497.) Morbidity and mortality data Bacteriol. 167:616—622) is a pBR322 derivative containing plasmid to complement a crp mutation in S. choleraesuis, Previous virulence assays have shown this Mammalian cell culture adherence and invasion assa s _______________________________________________X_ Data on the ability of Acrp-10 Acya-12 and Adherence and invasion of CHO cell monolayers Strain Percent Percent No. Genotype adherencelinvasionz X3744 wild type 43.5~6.5 34.2~3.3 Acya-12 A[zid-62::Tn;g] Percentage of inoculum adhered to cells after incubation for 2 h.

Percentage of inoculum recovered from CHO cells 2 h after incubation in 100 pg gentamicin/ml.

Values are mean ~ SD of triplicate samples.

Growth and persistence of mutants in normal human sera as ggmpared to wild-type parents Figure 5 shows the net growth and/or persistence of each strain over a 24-h period at 37°C.

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. Access to said cultures will be available during pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR 1.14 and 35 USC 122.

All restriction on availability of said cultures to the public will be irrevocably removed upon the granting of a patent based upon the application. Moreover, the designated deposits will be maintained for a period of thirty (30) years from the date of deposit, or for five (5) years after the last request for the deposit, or for the enforceable life of the U.S. patent, whichever is longer. Should a culture become nonviable or be inadvertently destroyed, or, in the case of plasmid- containing strains, lose its plasmid, it will be replaced with a viable culture(s) of the same taxonomic description. The deposited materials mentioned herein are intended for convenience only, and are not required to practice the present invention in view of the description herein, and in addition, these materials are incorporated herein by reference.

Strain Deposit Date ATCC No.

X3958 November 2, 1990 55224 X4323 November 2, 1990 55115 X3926 November 2, 1990 55112 X3927 November 2, 1990 55117 X4297 November 2, 1990 55111 X4346 November 2, 1990 55113 X3940 November 2, 1990 55119 X4073 November 2, 1990 55118 ISP2822 November 2, 1990 55114 ISPl820 November 2, 1990 55116 X4417 November 6, 1991 55249 X4435 November 6, 1991 55250 X4073 November 6, 1991 55248 (X4073 is a Replacement Culture) also useful as carrier microorganisms for the production These strains are

Claims (1)

1.Claims An isolated avirulent strain of Salmonella comprising a mutation in the cdt gene located between the crp and cysG genes wherein the cdt gene governs virulence of Salmonella and said mutation diminishes the ability of the strain to effectively colonize deep tissues. An isolated avirulent strain of Salmonella according to claim 1, further comprising a mutation in a gene selected from the cya and crp genes. An isolated avirulent strain of Salmonella according to claim 1, further comprising a mutation in a cya gene and a mutation in a crp gene. An isolated avirulent strain of Salmonella of any of claims 1-3 for the immunization of an individual, 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 individual against said pathogen. A strain according to any of claims 1-4, wherein the Salmonella comprises a chromosomal mutation which is lethal, balanced by a vector gene which complements the lethal mutation to constitute a balanced—lethal host—vector system. A strain according to any of strains 1-5, 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 the lack of functional Asd causes the cells to lyse. An isolated strain of S. typhi comprising a mutation in the cdt gene located between the crp and cysG genes wherein the cdt gene governs virulence of Salmonella and said mutation diminishes the ability of the strain to effectively colonize deep tissues. An isolated strain selected from the group of strains X4073 (ATCC #55248), X4346 (ATCC #55113), and X4417 (ATCC #55249), and derivatives thereof which carry the A[crp—cysG] — 10 mutation. An immunogenic composition for the immunization of an individual comprising an avirulent derivative of Salmonella comprising a mutation in the cdt gene located between the crp and cysG genes wherein the cdt gene governs virulence of Salmonella and said mutation diminishes the ability of the avirulent derivative of Salmonella to effectively colonize deep tissues. An immunogenic composition for the immunization of an individual according to claim 9, wherein said avirulent derivative 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 individual against said pathogen. An immunogenic composition according to claim 9, or 10, wherein the avirulent derivative also has a mutation in a crp gene. An immunogenic composition according to claim 9 or 10. wherein the avirulent derivative also has a mutation in a cya gene. An immunogenic composition according to claim ll, wherein the avirulent derivative also has a mutation in a cya gene. An immunogenic composition comprising the strain of any of claims 1-8 and a pharmaceutically acceptable excipient. The use of the immunogenic composition according to any of claims 10—14 for the production of a vaccine capable of stimulating the immune system of an individual to respond to an immunogenic antigen of a pathogen when said vaccine is administered to said individual. The use of an isolated strain according to either of claims 7 or 8 for the production of a vaccine capable of stimulating the immune system of an individual to respond to an immunogenic antigen of S. typhi when said vaccine is administered to said individual. A method of utilizing a strain of avirulent Salmonella, the strain having a mutation in the cdt gene located between the crp and cysG genes wherein the cdt gene governs virulence of Salmonella and said mutation diminishes the ability of the strain to effectively colonize deep tissues, for the preparation of an immunogenic composition the method comprising suspending the strain in a physiologically acceptable excipient. A method of utilizing a strain of avirulent Salmonella according to claim 17, wherein the strain further comprises a mutation in a cya or a crp gene. A method of utilizing a strain of avirulent Salmonella according to claim 17, wherein the strain further comprises a mutation in a cya gene and a mutation in a crp gene.