MXPA01005449A - Plasmid maintenance system for antigen delivery - Google Patents

Abstract

The present invention relates generally to a Plasmid Maintenance System for the stabilization of expression plasmids encoding foreign antigens, and methods for making and using the Plasmid Maintenance System. The invention optimizes the maintenance of expression plasmids at two independent levels by:(1) removing sole dependence on balanced lethal maintenance functions;and (2) incorporating at least one plasmid partition function to prevent random segregation of expression plasmids, thereby enhancing their inheritance and stability. The Plasmid Maintenance System may be employed within a plasmid which has been recombinantly engineered to express a variety of expression products.

Description

PLASMID MAINTENANCE SYSTEM FOR THE DELIVERY OF ANTIGENS 1. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention generally relates to expression plasmids, stabilized by the Plasmid Maintenance System D (as defined herein), capable of expressing a protein or peptide, such as an antigen, for use in a live vector vaccine, and methods to obtain and use stabilized plasmids. The invention optimizes the maintenance of expression plasmids at two independent levels, by: (1) removing the unique dependency in lethal, balanced, catalytic maintenance systems; and (2) incorporate a plasmid cleavage system to prevent random segregation of expression plasmids, thereby increasing the stability inheritance. 1. 2 Description of the Related Art See below in a discussion of the pertinent art the present invention. 1. 2.1 Live Bacterial Vectors Vaccines Live bacterial vector vaccines deliver antigens to a host immune system, by expressing the antigens of the genetic material contained within a live bacterial vector. The genetic material is typically a replicon, such as a plasmid. The antigens may include a wide variety of proteins and / or peptides of bacterial, viral, parasite or other origin. «Among the live bacterial vectors, currently under investigation, are attenuated enteric pathogens (for example Salmonella typhi, Shigella, Vibri cholerae), commensals (for example Lactobacillus, Streptococcus gordonii) and license vaccine strains (eg BCG). The . Typhi is a particularly attractive strain for the human vaccine. 1. 2.2 Salmonella typhíAtenuada, as a live vector strain S. typhi is a well-tolerated live vector, which can deliver multiple immunological antigens not related to the human immune system. The live vectors of S. typhi have been shown to produce antibodies and a cellular immune response to an expressed antigen. Examples of antigens successfully delivered by S. typhi include the non-toxigenic, yet highly immunogenic fragment C of the tetanus toxin and the circumsporozoid protein of Plasmodium falciparum malaria. S. typhi is characterized by the enteric routes of infection, a quality that allows the delivery of oral vacun. S. typhi infects monocytes and macrophages and, therefore, target antigens to professional APCs. The expression of an antigen by S. typh generally requires the incorporation of a recombinant plasmid encoding the antigen. Consequently, the stability of the plasmid is a key factor in the development of attenuated S. typhi vaccines, of high quality, with the ability to consistently express foreign antigens. Candidates of the attenuated S. typhi vaccine for use in humans, must possess at least two separate and well-defined mutations, which cause, independently, attenuation, since the chance of the reversal i vivo of such double mutants, will be priceless. The candidate of the attenuated vaccine of S. typhi CVD908 possesses such properties. CVD908 contains two deletion mutations that are not reversed within the aroC and aroD genes.

These two enzymes encode critical genes in the biosynthetic pathway that leads to the synthesis of corismato, the key precursor required for the synthesis of aromatic amino acids, phenylalanine, tyrosine and tryptophan. Corismate is also required for the synthesis of p-aminobenzoic acid; after its conversion to tetrahydrofolate, the p-aminobenzoic acid is converted to purine nucleotides ATP and GTP. 1. 2.3 Instability of plasmids Bacterial cells without plasmids tend to accumulate more rapidly than cells carrying plasmids. One reason for this increased regimen is that the transcription and translation of plasmid genes imposes a metabolic load that decreases cell growth and supplies cells without plasmids, a competitive advantage. Also, the products of foreign plasmid genes are sometimes toxic to the host cell. The stable inheritance of the plasmids is convenient in the field of bacal live vector vaccines, attenuated, to ensure the production of successful continuous antigen, as in commercial operations of the bioreactor, in order to prevent this bio- reactor take over the cells without plasmids. The stable inheritance of a plasmid generally requires that: (1) the plasmid must duplicate once each generation, (2) the deviations of the number of copies must be corrected quickly before cell division, and (3) in cell division, the products of the duplication of plasmids must be distributed in both daughcells. Although the chromosomal integration of foreign gene integration increases the stability of such sequences, the genetic manipulations involved can hinder the drop in helogous gene copy numbers, resulting in the production of insufficient levels of helogous antigen to ensure an immune response. optimum The introduction of helogous genes in plasmids of multiple copies maintained within a live vector cep, is a natural solution to the problem of number of copies; The genetic manipulation of such plasmids for the controlled expression of such heterologous gene is direct. However, the resulting plasmids may become unstable in vivo, resulting in the loss of these foreign genes. 1. 2.4 Plasmid Stabilization Systems In nature, bacterial plasmids are often stably maintained, although they are usually present with very low copy numbers. The stable inheritance of plasmids from smaller copy numbers, which occur naturally, may depend on the presence of certain genetic systems that actively prevent the appearance of plasmid-free progeny. A recent review of plasmid maintenance systems can be found in Jansen et al., Molecular Microbiol 17-205 210, 1995 (incorporated herein by reference. 1. 2.5 Resistance to Antibiotics A resource to maintain the plasmids and to supply a gene of antibiotic resistance in the plasmid and to grow the cells in a medium enriched with antibiotics. However, the method is subject to a number of difficulties. The approach of antibiotic resistance is expensive, requires the use of expensive antibiotics and, more importantly, the use of antibiotics in conjunction with the in vivo administration of vaccine vectors and disapproved by the Food and Drug Administration of the United States. In large-scale production applications, the use of antibiotics may impose other limitations. With respect to commercial bio-reactors, the mechanism of resistance to antibiotics can degrade antibiotics allowing a substantial population of cells without plasmids to persist in culture. Said cells without plasmids are unproductive and decrease the production of the bioreactor. Therefore, there is a need in the art for a plasmid maintenance system, designed specifically for use in live bacterial vector vaccines, which do not depend on antibiotic resistance and preferably are also useful in commercial bioreactor applications. . 1. 2.6 Segregation Plasmid Maintenance Functions Stable plasmids, with minor copy number, typically employ a division function that actively distributes plasmid copies between daughter cells. Exemplary functions of division include, without limitation, the systems of pSClOl, factor F, profago Pl, and drug resistance plasmids IncFII. Such functions are referred to herein as "SEG" functions. 1. 2.7 Disposal Functions (PSK), after segregation The maintenance functions of naturally occurring PSK plasmids typically employ a two-component toxin-antitoxin system and generally operate as follows: The plasmid encodes both a toxin and an antitoxin . Antitoxins are less stable than toxins, which tend to be very stable. In a daughter cell without plasmid, toxins and antitoxins do not occur; however, antitoxins, less stable, degrade rapidly, thus releasing toxins and destroying the cell. The toxins are generally small proteins, the antitoxins are either small proteins (protein system, such as pho-doc), or antisense RNAs, which bind to the mRNAs that encode the toxin, preventing synthesis (antisense systems, such as hok-sok). Balanced lethal systems, discussed below in Section 1.2.7.3, are an example of a function of the artificial PSK. 1. 2.7.1 Protein Maintenance System: The phd-doc System In functions of the protein PSK, both the toxin and the antitoxin are synthesized from operons in which the gene encoding the antitoxin is upstream of the gene encoding the toxin. These operons self-regulate the levels of transcription, and the synthesis of the encoded proteins is translationally coupled. The antitoxin is generally synthesized in excess to ensure the action of the toxin is blocked. Unstable antitoxins are constantly degraded by host-encoded proteases, which require a constant synthesis of antitoxin to protect the cell. In the loss of the plasmid, the antitoxins are no longer produced and the existing antitoxins degrade rapidly, allowing the toxin to destroy the host cell. The phd-doc system is an example of a protein PSK function. The phd-doc system occurs naturally within the bacteriophage Pl of temperature, which lysogenizes the Escherichia coli, as a plasmid of -10 kb. This maintenance site codes for two small proteins; the protein Doc of the toxic amino acid 126, causes the death in the cure ("death on curing") of the plasmid by an unknown mechanism, and the PhD antitoxin of amino acid 73, prevents the death of the host ("p_revents hos death"), presumably by joining and blocking the action of the Doc. The Phd and Doc are coded by a sun transcript in which the ATG start codon of the downstream gene overlaps the TGA stop codon of the upstream gene phd by a base. The expression of these two proteins is, therefore, translationally coupled, with the synthesis of Phd exceeding the synthesis of the toxic Doc protein. In addition, the transcription of this operon is self-regulated at the level of transcription through the binding of a complete Phd-Doc protein to a site that blocks the access of the RNA polymerase to the promoter of the operon according to concentrations of both proteins. they reach their critical level. Although Doc seems to be relatively resistant to proteolytic attack, PhD is highly susceptible to splitting. The mechanism of the PKS of a plasmid encoded phd-doc site is, therefore, activated when the bacterium spontaneously loses this resident plasmid, which leads to the degradation of the Phd antitoxin and the subsequent activation of the Doc toxin, which causes the death of the cell. 1. 2.7.2 Antisensible Maintenance System: The hok-sok System Antisense maintenance systems, the antitoxins are antisense RNAs that inhibit the translació of the mRNAs that encode the toxin. Like the antitoxin peptides, the antisense RNAs are less stable than the mRNA that encodes the toxin. The loss of plasmid allows existing antitoxins to degrade, thus allowing the synthesis of the toxin, which destroys the host cell. An example of the antisense maintenance system is the hok-sok system, encoded by the B-site plasmid Rl. The system is comprised of three genes: hok, sok and mok. The hok is a protein associated with the membrane, which irreversibly damages the cell membrane, killing the host cells. The expression of Hok from the mRNA of ho leads to a loss of cell membrane potential, stops respiration, changes in cell morphology, cell death. The gene sok encodes a trans-active RNA, which blocks the translation of the hok mRNA, thus preventing the Hok from killing the host cells. The sok RNA is less stable than the hok mRNA, and is expressed from a relatively weak promoter. (Gerdes et al., Annu. Rev. Genet., 31: 1 31, 1997) incorporated herein. The mechanism by which the AR of sok blocks the translation of Hok in the cells that contain plasmid, becomes evident only after the identification of mok ("modulation of Jtilling") (Modulació de Muerte), a third gene in the site (parB). The open reading frame of mok overlaps with hok, and it is necessary to pair the expression and regulation of hok translation. The sok antisense RNA forms a duplex with the 5 'end of the mok-hok message that makes the binding site of the mok ribosome inaccessible to the ribosomes and which promotes the cleavage of RNase III and the degradation of mARV. In the absence of mok translation, the hok does not express the message intact, although its own ribosome binding site is not directly obscured by sok RNA. When the plasmid-free cell is formed, the unstable sok RNA decays much more rapidly than the stable mok-hok message. When the protection provided by sok is lost, Mok and Hok move and the cell dies. One limitation of the hok-sok system is that a significant number of cells without plasmids may arise when the hok-sok system is inactivated with the open reading frame of Hok. 1. 2.7.3 Balanced Lethal Systems In a balanced lethal system (a PSK function), a chromosomal gene that encodes an essential structural protein or enzyme is either deleted from the bacterial chromosome or mutated so that the gene can no longer operate. The gene removed or damaged is then replaced by a plasmid comprising a gene that operates completely. The loss of the plasmid results in an insufficiency of the essential protein and the death of the cells without plasmids. A balanced lethal system has been used successfully in S. typhimurium, based on the expression of gene asd, which codes for β-aspartate dehydrogenase semialadehyde (Asd). Asd is a critical enzyme involved in the synthesis of L-aspartic-β-semialdehyde, which is an essential precursor for the synthesis of the amino acids L threonine (and L-isoleucine), L-methionine and L-lysine, as well as diaminopimelic acid, a key structure component, essential in the formation of the cell wall and Gram-negative bacteria. The loss of midas squares encoding Asd will be lethal for any bacteria unable to synthesize the Asd of the chromosome, and will result in bacterial lysis due to the inability to properly assemble the peptidoglycan layer of its cell wall.

The asd system (a function of PSK) has been successfully employed in strains of the live vector based on attenuated S. typhimurium, for immunization in ratone with a variety of prokaryotic and eukaryotic antigens, including such diverse antigens as the fragment Cd detoxified tetanus toxin and LT enterotoxin, viral peptides of synthetic hepatitis B and gamete-specific antigen, such as the SP10 antigen of human sperm. Murine mucosal immunization with these vectors of live vectors has produced significant immune responses that involve the responses of serum IgG and secretory IgA on mucosal surfaces. The asd system has recently been introduced with attenuated Salmonella typhi vaccine strains in an attempt to increase the stability of synthetic hepatitis B viral peptides, which express plasmids. However, when volunteers were immunized with these live vecto strains, no immune response was detected in the ajen antigen. In fact, until now, very few reports have documented an immune response to the plasmid-based expression of an antigen foreign to the stabilized plasmids, after the human vaccine with a live vector of attenuated S. typhi. In one report, the Ty21a vaccine layer was auxotrophic by thymine, selecting in the presence of trimethoprim for an undefined mutation in the thyA gene, which encodes thymidylate synthetase. Although, in some cases, the failure of the live vector strain may have resulted from the over-attenuation of the strain itself, it seems likely that the current destruction system for the plasmids will suffer additional limitations. In those situations where the chromosomal copy of the gene has been inactivated, rather than removed it may allow restoration of the chromosomal copy by means of homologous recombination with the copy of the plasmid-carrying gene, if the bacterial strain used is in the recombination Balanced lethal systems, based on the production of the catalytic enzyme, are subject to a number of important deficiencies. In particular, since the complementation of the deletion of the chromosome gene requires only one copy of the gene, it is inherently difficult to maintain more than a few copies of a plasmid d expression. The plasmid-free host strain can grow in a special medium to chemically complement the existing metabolic deficiency. Also, cells without plasmids can also benefit from the effects of "cross-feeding" when a growth factor that can diffuse is limited in growth. Therefore there is a need in the art for a Plasmida Maintenance System, which only relies on the balanced lethal system particularly for use in live vector vaccines of bacteria. 2. SUMMARY OF THE INVENTION The present invention generally relates to a stabilized expression plasmid, comprising a Plasmid Maintenance System, and a nucleotide sequence encoding a protein or peptide, such as a foreign antigen, and methods for obtaining and use such stabilized expression plasmids. The Plasmid Maintenance System of the present invention optimizes viability using expression plasmids with smaller copies, stabilized, capable of expressing high levels of heterologous antigen in response to an environmental signal that will probably be found in vivo, after the organisms of vaccine have reached an appropriate ecological nich.

In a particular aspect, the stabilized expression plasmid is employed in a live vector vaccine of Salmonella typhi, such as strain CVD908-htrA. The invention optimizes the maintenance of expression plasmids at two independent levels by (1) remove the only dependence on balanced lethal maintenance systems; and (2) incorporate a plasmid cleavage system, to prevent the random segregation of expression plasmids, thereby increasing their inheritance and stability. In one aspect of the invention, the stabilized expression plasmid is recombinantly engineered to express one or more antigens, preferably one or more Shiga toxin antigens (Stx2) or their substantial counterparts, such as the pentamers of the Shiga toxin subunit. or a genetically detoxified Stx. The stabilized expression plasmid preferably comprises one or more non-catalytic plasmid maintenance functions. In another aspect, the expression plasmid comprises a Plasmid Maintenance System which comprises at least one function of the PSK and at least one function of SEG. For example, the Plasmida Maintenance System may comprise a two-component Plasma Maintenance System, which includes a PS function and a SEG function. Alternatively, the Plasmid Maintenance System may comprise a three-component Plasma Maintenance System, which includes a PSK function, a SEG function and a PSK function. In a preferred alternative, the Plasmid Maintenance System comprises the hok-sok + par + parA + phd-doc; e that any of the functions indicated may be replaced by its substantial counterpart. Plasmid Maintenance Systems can be incorporated into multiple-copy expression plasmids, which encode one or more proteins or peptides of interest. Such multi-copy expression plasmids produce a gene dose effect that increases the level of expression of the protein or peptide of interest. When the Plasmid Maintenance System is to be used in a live bacterial vector vaccine, the protein or peptide of interest is one or more foreign antigens. In one aspect, the expression plasmid is a vaccine expression plasmid, comprising a Plasmid Maintenance System and at least one antigen, eg, at least one Shiga toxin 2 antigen (Stx2) and / or its homologue. substantial Where the antigen is an antigen of Shiga toxin 2, this antigen can, for example, be either a pentamer of the B subunit or a Stx 2 genetically detoxified.

In another aspect, the expression plasmid comprises a Plasmid Maintenance System which incorporates the balanced lethal system ssb and the ss site of the live bacterial vector has been inactivated using a suicidal vector, comprising a responsive origin to the temperature. In one aspect, the live bacterial vector is S. typhi and the suicide vector is used to inactivate the sit ssab of S. typhi. In one aspect, the suicide vector is derived from pSClOl, which carries sacB, described herein. In another aspect, the present invention provides a Plasmid Maintenance System in the control mechanisms of antisense RNA, which only synthesize lethal proteins after the loss of the plasmid has occurred. In one aspect, the expression plasmid comprises a series of expression plasmids each with self-contained genetic cassettes, which encode the regulated expression of a heterologous antigen, an origin of response a selectable marker to recover the plasmid. In one aspect, the expression plasmid comprises a Plasmid Maintenance System that incorporates a PSK function based on the ssb gene. In a related aspect, the mutated alleles, such as ssb-1, described herein, are incorporated into the expression plasmids to increase the plasmids of larger copy numbers by the over-expression of the SSB1 type proteins, to form the tetramers Biologically required assets of SSB. In another aspect, the expression plasmid comprises a promoter. The promoter is preferably an inducible promoter, such as the ompC promoter. In one aspect, the inducible promoter is the mutated Pompci or the PomC3 promoter / described herein. In one aspect, the expression plasmid of the present invention comprises a plasmid inheritance site (or division); a response source selected to supply the number of copies that effectively stabilizes a given antigen; a function of the PSK; and a nucleotide sequence, which encodes an antigen and a promoter that ultimately controls the translation of the antigen and has a resistance that is selected to enhance antigen production without killing the cell. The present invention also provides a method of using the expression plasmid, which comprises transforming a bacterial cell using said expression plasmid, culturing the bacterial cell to produce the peptide protein (e.g., the antigen) and / or administering a transformed cell or the cell culture to a subject. Where the transformed bacterial cells are administered to a subject, they are administered in an amount necessary to produce an immune response that confers immunity to the subject for the protein or peptide. This subject preferably a human, but may also be another animal, such as a dog, horse or chicken. In one aspect, an provided expression plasmid, which comprises at least 3 independently functioning expression cassettes, wherein a cassette encodes a protein or peptide of interest and the remaining cassettes each encode a different Plasmid Maintenance Function. In one aspect, an provided expression plasmid, which encodes (1) a test antigen operably linked to a promoter and (2) a Plasmid Maintenance System. In another aspect, a cassette d is provided for expression of the regulated test antigen, which operates so that the induction of antigen expression is increased, a metabolic load is placed on the bacteria, which leads phenotypically to the instability of the plasmid. , ie a selective advantage is created for all bacteria that can spontaneously lose the offensive plasmid. The test antigen may be the green fluorescent protein (GFPuv). The expression cassette encoding the test antigen can also comprise an inducible promoter, such as the ompC promoter, positioned so that the inducible promoter finally drives the translation of the test antigen. In one aspect, a method for obtaining an expression plasmid is provided, which comprises synthesizing an expression plasmid that includes at least 3 independently functioning expression cassettes, in which a cassette encodes a protein or peptide of interest and the remaining cassettes each encode a different function of Plasmids Maintenance. In one aspect, a method to classify what Plasmid Maintenance Systems is provided, which comprises: supplying an expression cassette, the qua encoding a protein or peptide of interest, and at least other expression cassettes, each encoding and capable of expressing in the live bacterial host vector, a different Plasmida Maintenance Function; insert the three expression cassettes into a single expression plasmid; transform a living bacterial vector with the single plasmid d expression; cultivate the transformed bacterial live vector, determine the rate of introduction of cells if plasmids in the culture. In one aspect, the present invention comprises an attenuated bacterial live vector vaccine, comprising an attenuated bacterial live vector that has been transformed with the stabilized expression plasmid, comprising a Plasmid Maintenance System, preferably a plasmid maintenance system. not catalytic. In one aspect, the present invention comprises an attenuated bacterial live vector vaccine, comprising an attenuated bacterial live vector, which has been transformed with the expression plasmid comprising a Plasmid Maintenance System which incorporates at least one PSK and at least one SEG system. The attenuated bacterial viv vector, for example, is S. typhi CV908-htrA. The present invention also provides a method for vaccinating a subject, comprising administering to the subject an amount of a living bacterial vector vaccine sufficient to produce an enhanced immune response. The present invention also provides a method for preventing a disease by vaccinating a subject, using an amount of such a living bacterial vector sufficient to produce a protective immune response to one or more pathogens of the disease. The subject is preferably a human, but can also be another animal, such as a horse, pig cow. For example, the present invention provides APRA method for preventing hemolytic uremic syndrome / HUS) caused by Shiga toxin 2 that produces enterohemorrhagic Escherichia coli, by administering to a subject a quantity of a living bacterial vector transformed with a plasmid. stabilized that encodes at least the antigen of Shiga toxin 2. In another aspect, the present invention provides a method for classifying the Plasmid Maintenance Systems for efficacy, this method comprises supplying expression plasmids, comprising the Plasmid Maintenance Systems, described herein, encoding a protein or peptide of interest, said expression plasmids have copy numbers varying from a low copy number (for example ~ 5 copies per cell) to a mean copy number (for example ~ 15 copies per cell) up to a high copy number (for example - 6 copies per cell); transform live bacterial vectors with said expression plasmids; and to test the rate of introduction of cells without plasmids and / or the regimen of growth of cells containing plasmids. The modified origins of response can be the sources of response of plasmids pSClOl (low copy number), pACC184 (average number of copies) and pAT15 (high copy number). Plasmid response cassettes, which function independently, can be used to test the efficiency of one more plasmid stabilization systems, as the number of copies increases.

In one aspect, the present invention provides expression plasmids stabilized for use in live attenuated S. typhi vectors, containing a selectable marker, which can be easily replaced by a non-drug resistant site or by a gene that encodes an acceptable drug resistance marker, such as the aph, which encodes the resistance to aminoglycosides, kanamycin and neomycin. The Plasmid Maintenance Systems of the present invention provide improved stability of recombinant plasmids, overcoming the previous problems of plasmid instability, for example, in a bio reactor, and the uses of live vector vaccines. The plasmids of the present invention are specifically adapted to vaccine applications, although such plasmids are also useful in the production of large scale proteins. The plasmids of the present invention are a major improvement over the prior art, in that they overcome the problems associated with the acquisition of cells if plasmids and the instability of plasmids and have a wide range utility in the fields, such as protein production. commercial and production d live bacterial, attenuated vector vaccines.

There has been a need for the solution to the problems of acquisition of plasmid-free cells and the stability of plasmids, associated with the field of vaccine delivery and protein production. The present invention solves this need that has existed for some time. 3. DEFINITIONS The term "Maintenance System" Plasmids "(" PMS "), as used herein, refers to a nucleotide sequence, which comprises at least one function of elimination after segregation (" PSK "), and at least one system is split or segregated (" SEG ") and which includes, optionally, any other Plasmida Maintenance Function The term" Plasmid Maintenance Function "is used here to refer to any function that enables the stability of plasmids, associated with a PMS. nucleotide sequences, which occur naturally, that encode the functions of plasmid maintenance, as do the nucleotide sequences, which are substantially homologous to such maintenance functions of plasmids, which occur naturally, and which retain the function exhibited by the function of naturally occurring plasmid maintenance, the term "Elimination System After segregation" (PSK) is used here to refer to any func ion that results in the death of any newly divided bacterial cell, which does not inherit the plasmid of interest, and specifically includes balanced lethal systems, such as asd or ssb, protein systems such as phd-doc and antisense systems, such as hok- sok The term includes nucleotide sequences, which occur naturally, which encode such PSKs, as well as nucleotide sequences that are substantially homologous to such nucleotide sequences, occurring naturally and retaining the function exhibited by the d nucleotide sequences, which occur naturally, corresponding. The term "substantially homologous" or "substantial homolog" with reference to a sequence of nucleotides or an amino acid sequence, indicates that the nucleic acid sequence has sufficient homology, and comparison with a reference sequence (eg, a native sequence) to allow the sequence to execute the same basic function as the corresponding reference sequence; a substantially homologous sequence and typically at least 70 percent sequentially identical in comparison to the reference sequence, typically at least 85 percent sequentially identical, preferably at least 95 percent identical sequentially, and most preferably around 96.97, 98 or 99 percent identical sequentially, compared to the reference sequence. It will be appreciated that throughout the specification, where reference is made to specific nucleotide sequences and / or d-amino acid sequences, such as the nucleotide sequences and / or amino acid sequences, they can be replaced by the substantially homologous sequences. The terms "Segregation System" and / "Division System" (both referred to herein as "SEG") are used here interchangeably to refer to any function that improves the stability of the plasmid that operates to increase the frequency of successful delivery. of a plasmid to each bacterial cell recently divided, and comparison with the frequency of delivery of a corresponding plasmid, without such a SEG system. SE systems include, for example, equal division systems, pair split systems, and even sites of pSClOl. The term includes naturally occurring nucleotide sequences that encode such SEG systems, as well as nucleotide sequences that are substantially homologous to such naturally occurring nucleotide sequences and that retain the function exhibited by the nucleotide sequences. , which occur naturally corresponding. The term "detoxified" is used herein to describe a toxin that has one or more point mutations that significantly reduce the toxicity of said toxin., in compassion to a corresponding toxin without such point mutations. The term "immunologically effective" is used herein to refer to an immune response that confers an immunological cellular memory on the subject, with the effect that a secondary response (to the same or a similar toxin) is characterized by one or more of the next: shorter delay phase in compassion with the delay phase resulting from a corresponding exposure in the absence of immunization; antibody production that continues for a longer period than antibody production for a corresponding exposure in the absence of immunization; a change in the type and quality of antibody, produced in sympathy with the type and quality of antibody produced from such exposure in the absence of the immunization; a displacement in the class response, with IgG antibodies that appear in higher concentrations and with greater resistance than IgM; an increased average affinity (binding constant) of the antibodies for the antigen, compared to the average affinity of the antibodies for the antigen from such exposure, in the absence of the immunization; and / or other features known in the art that distinguish a secondary immune response. 4. BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1C: genetic maps of the exemplary pGEN expression plasmids d (pGEN2, pGEN3 and pGEN4) of the present invention, - Figures 2A-2D: genetic maps of exemplary oriel-based expression plasmids ( pJN72, pJN51, pJNlO pJN12) of the present invention. Figure 3: flow cytometry histograms of the GFP fluorescence for the expression vectors carrying CVD 908-htrA with the elimination system after the hok-sok segregation; Figures 4A-5B: sequence 1-4199 of nucleotides d pGEN2; Figure 5: 1201-2400 nucleotide sequence pGEN3 showing the sequence of oril5A; Figure 6: sequence 1201-3850 of nucleotides d pGEN4, showing the sequence of orilOl; Figures 7A-7E: gene maps of the expression plasmids d of pGEN based on oril5A, exemplary (pGEN91, pGENlll, pGEN121, pGEN193 and pGEN222) of the present invention; Figure 8: Flow cytometry histograms of the GFP fluorescence for the expression plasmids pGEN91 pGENlll, pGEN121, pGEN19 and pGEN222. 8. DETAILED DESCRIPTION OF THE INVENTION Live bacterial vector vaccines employ a live bacterial vector to express genes encoding antigens protective of bacterial, viral, parasitic pathogens. Bacterial protective antigens are preferably non-native to the bacterial living vector, ie, heterologous. The live bacterial vector vaccine is administered to a host, thereby exposing the expressed antigen to the host immune system, eliciting an immune response of an appropriate character to confer immunity to the host. In order to achieve enhanced immunogenicity, the plasmids, which express such protective antigens must be stabilized. According to the knowledge of the inventor, no Plasmid Maintenance System based on S. typhi, currently available, takes advantage of the naturally occurring, known cleavage mechanisms to improve the stability of multiple copy plasmids in other strains. The present invention provides a non-catalytic Plasmid Maintenance System for the stabilization of expression plasmids encoding foreign antigens in a live vector vaccine strain of S. typhi. In one aspect, the layer of S. typhi is the CVD 908 htrA. In another aspect, the present invention improves and / or optimizes the maintenance of the expression plasmids by the provision of the Plasmid Maintenance Systems that operate on two independent levels: (1) remove the only dependence on the balanced catalyst catalytic maintenance systems; and (2) incorporate a plasmid division system, which will prevent the random segregation of expression plasmids, thus increasing inheritance and stability. A critical reason for seeking this particular approach is that this method of improving and maintaining plasmids does not involve additional manipulations of the live vector strain and, therefore, can improve the immunogenicity of heterologous antigens expressed within any live vector strain. The Plasmid n Catalytic Maintenance System of the present invention improves the stability of multiple copy expression plasmids with a live bacterial vector vaccine, such as CVD908-htrA.

In one aspect, the present invention incorporates the function of naturally occurring PSK, hok-sok, of the pR1 antibiotic resistance factor or its substantial counterpart without the multiple copy expression plasmids. The hok-sok system is a silent addiction system based on the control mechanisms of antisense RNA, which only result in the synthesis of lethal proteins after the loss of the plasmid. The present invention also provides a Plasmid Maintenance System comprising a PSK function based on the complementation in which the chromosomal gene ssb, which encodes the single-cord, non-catalytic, essential binding protein required for the response of the DNA, is specifically suppressed and inserted into a plasmid of expression of multiple copies. The present invention also provides a Plasmid Maintenance System comprising an expression plasmid encoding at least one SE site and at least one PSK function. . 1 Suicidal vectors Heterologous antigens can be expressed within living vector strains, such as CVD908-htrA, from genes residing either in the plasmids or integrated within the chromosome. One technique for integrating these genes into the host chromosome involves the use of "temperature-sensitive suicide vectors, such as pIB307, which contain a temperature responsive origin of pSClO (oril01ts)." The present invention provides a vecto suicide. improved for use in CVD908 and CVD908-htrA derived from pIB307, which allows the easier construction of mutagenesis cassettes to alter the living vector chromosome.The integration of these suicide vectors in the chromosome by homologous recombination results from the inactivation of the temperature of the plasmid response protein, RepA, a protein essence to the d orilOl function The spontaneous resolution of the resultant unstable merodiploid intermediates is detected by the counter-selection for the loss of the sac gene contained in the suicide vector The gene contained in all plasmids excised encodes the levansucrase enzyme, which is lethal when e expresses within the cytoplasm of enteric bacteria, which include S. typhi, which grows in the presence of sucrose. Since the resolving merodiploids are selected by incubation in the presence of 10% sucrose, the cut plasmids will kill the host bacteria unless they heal spontaneously.

This system was successfully used to integrate a kanamycin resistance cassette into the? AroC101 site of CVD908. However, these experiments were successful because the gene is mobilized on the S typhi chromosome, encoded to a selectable drog resistance marker. Using these early vectors, the replacement of the kanamycin resistance cassette with a n-selectable marker was not successful because, although the incoming marker can be integrated into the chromosome with a merodiploid, the resolution of the merodiploid d replace the resistance gene The drug was never detected. The present invention also provides a method for using such suicide vectors to inactivate the siti ssb of attenuated Salmonella typhi strains, such as CVD908-htrA. The present invention allows such suicide vectors to efficiently mobilize proteins or peptides expressing genes of interest, such as heterologous antigens, on the chromosome of S. typhi CVD908 htrA in two steps. For example, the present invention introduced a cassette of SacB-aph at the site, which was then selected using kanamycin. The generation of this S. typhi cep CVD908-htrA? AroC1019:: sacB-aph, produced a valuable intermediate cep in which, in theory, any structural gene can be efficiently inserted into the C-ring site by marker exchange. The sacB gene was used as a counter-selectable marker by merodiploid raisins in the presence of 10% sucrose to select the sacB-aph cassette replacement with the incoming antigen cassette, since the resolution of the merodiploid, in the presence of sucrose, will result in the loss of the sacB gene, in order to produce viable progeny. This intermediate strain was used to efficiently integrate the non-toxigenic mutant LT-K63 of the heat-unstable enterotoxin of E. coli, which creates CVD908? AroC1019:: LT-K63. . 2 Plasmid-based Expression of Heterologous Antigens Although the chromosomal integration of foreign gene confers stability to such sequences, the genetic manipulations involved can be difficult, the drop in the copy number of the heterologous gene sometimes results in the production of insufficient levels d heterologous antigen to ensure an optimal immune response. In contrast, the stability of the plasmid is a complex phenomenon that depends on multiple factors that include (1) the number of copies of the plasmid; (2) l expression, appropriately regulated, of the genes contained within the plasmid; and (3) selective pressure to ensure proper segregation and inheritance of the plasmid. To ensure stability, plasmids must be duplicated in a regulated manner, to prevent the number of copies from rising to lethal levels. In addition, plasmids must be segregated during the division of a growth bacterium to ensure that each daughter cell receives at least one copy of the plasmid. Segregation can be a random, passive, or active process that involves the synthesis of the novel proteins that they help in the segregation of plasmid and heredity. The successful inheritance of randomly segregating plasmids depends on a sufficiently high copy number of randomly distributed plasmids, within a pair-dividing bacterium to virtually guarantee the inheritance of at least one plasmid for each daughter cell. Plasmid cloning vectors, commonly used, which include pBR322 derivatives of number d medium copies and pUC plasmids of high copy number, or are inherited by random segregation. Active segregation involves the synthesis of proteins that are proposed to bind such plasmids and coordinate with the membranes of the dividing bacteria to ensure that each daughter cell receives at least one plasmid copy. Plasmids employing such active division systems are typically plasmids with very low copy numbers, such as the F sex factor of E. coli or the R factors of antibiotic resistance, ta as pRl and pRK2. The present invention exploits the functions of SEG, which occur naturally, to increase the inheritance of multiply-expressing plasmids, which would otherwise be inherited by random segregation, to increase the stability of these plasmids. The present invention also takes advantage of naturally occurring genetic systems in which daughter cells that do not successfully inherit a plasmid d expression will be removed and removed from the growth population, i.e., PSK functions. The incorporation of more than one category of the plasmid stabilization function is referred to herein as a Plasmid Maintenance System. For example, the incorporation of both a SEG function, such as a division site, and a PSK function in a simple expression plasmid provides a Plasmida Maintenance System. It should be noted that a gene that confers resistance to a bactericidal antibiotic, such as the aph, which encoded the resistance to kanamycin and neomycin, is also considered a PSK function, like the balanced leta system, based on asd. . 3 Balanced Lethal Systems One method of ensuring the inheritance of expression plasmids involves the construction of a PS system or its substantial counterpart, referred to as a balanced letal system, for the plasmids expressing the heterologous antigen. In a balanced lethal system based on plasmid, the plasmids that respond in the cytoplasm of the bacterium express a critical protein required by the bacterium to grow and duplicate. The loss of this plasmids removes the ability of the bacterium to express the critical protein and the results in the death of the cell. The asd system has recently been introduced in attenuated S. typhi vaccine strains in an attempt to increase the stability of the plasmids expressing the synthetic hepatitis B viral peptides. However, when the volunteers were immunized with these live vector strains, no response was detected immune to the foreign antigen. See Tacket et al., Infection an Immuni ty, 65: 3381, 1997 (incorporated herein by reference). In fact, to date, few reports have documented an immune response to plasmid-based expression of a non-plasmid antigen (stabilized or otherwise) after vaccination of humans with a live vector of attenuated S. typhi . Although, in some cases, the failure of the live vector strains may have resulted from the over-attenuation of the strain itself, the conclusion of the inventor is that the PSK functions currently used for the plasmids suffer from additional limitations, in particular segregation limitations and the limitations of the catalytic activity. The present invention provides improved expression plasmids comprising increased segregation capacitance by incorporating at least one division system together with at least one PSK system. . 4 Limitations of Segregation A limitation of the functions of the maintenance of the plasmid, such as the function asd (as well as the thyA function) is that they do not increase the inheritance of the resident plasmids, which continues to segregate randomly with or without the presence of the function asd. Therefore, if resident expression plasmids that carry ace genes are inherently unstable, they are not lost, regardless of the bacteri requirement for Asd. The inherent stability of plasmid d asd expression can be defined by the strains that carry growth plasmids, in the presence of DAP, which removes the selective pressure that ensures that all viable bacteria contain the expression plasmid. If a given plasmid is inherently unstable, it will lose the bacteria at a high rate and such bacteria without plasmid will undergo lysis in the absence of growth supplements; The general result of this effect will be a population of bacteria that grows much slower than the wild type unaltered strains. The present invention improves the stability of the plasmid by the incorporation of a SEG function, ta as a cleavage site, or a substantial homolog of a SEG function, on the expression plasmid to increase the inheritance of such plasmids by bacteria that It is actively divided. The site of division occurs naturally in the virulence plasmids of S. typhimurim. Tingle Curtiss, Journal of Bacteriology, 172: 5266, 199 (incorporated herein by reference) reported that such division sites are well conserved among the virulence plasmids of S. typhimurium, and that when a fragment of restriction of 3.9 kb, which encodes this site, s enter the plasmid pACYC184 number of minor copies (-15 copies per cell), the stability of the observed plasmid increases from 34% to 99% of cells containing plasmids after 50 generations. The nucleotide sequence of this site was determined later by Cerin Hackett, Plamis 30:30, 1993 (incorporated herein by reference). (Access Number of GenBank M97752). . 5 Limitations of Catalytic Activity Another potential limitation of a plasmid maintenance function, just as the asd function (as well as the thyA system) depends on an enzyme with catalytic activity. Since complementation with only a single copy of the asad gene is sufficient to remove auxotrophy, it is unclear why all copies of a multiple-copy plasmid should remain stable, especially if they encode a problematic heterologous antigen especially that inhibits growth. of the bacteria. In addition, although expression plasmids of larger copy numbers may express appreciable levels of a heterologous antigen given in vi tro, such plasmids may not be maintained at the expected copy numbers, in vivo due to toxicity and may in fact , are present in much smaller numbers of copies, which will be expected to reduce any specific immune response observed for the heterologous antigen. Therefore, the present invention thus provides the maintained stability of plasmids with low and medium copy number, to express heterologous antigens. . 6 PSK Function of Non-Catalytic ssb The potential limitation of catalytic activity, associated with balanced lethal systems, is directed here, although the use of plasmids expressing the single-stranded binding protein (SSB) of S. typhi to trans-complement a lethal mutation in another way, and introduced into the chromosomal ssb gene. The biochemistry and metabolic roles of the SSB protein of E. coli has been extensively reviewed by Lohman et al., Annual Reviews i Biochemistry 63: 527, 1994 and Chase et al., Annual Reviews i Biochemistry 55: 103, 1986 ( whose descriptions are incorporated herein by reference). The SSB is a catalytic 177 n amino acid protein, with a relative molecular weight of 19 kDa, which binds with high affinity to a single-stranded DNA ("ssDNA") and plays an essential role as an accessory protein in the response of the DNA, recombination and repair. The biologically relevant form of the SSB involved in the binding to ssDNA is a tetramer, which binds in two modes to the ssDNA, associating intimately with an average of either 3 bases (SSB35 binding mode) or 65 bases (SSB65 binding mode) ). The specific conditions that control the preferred mode of binding are complex and depend on the concentration of the surrounding monovalent and divalent salts, pH, as well as the amount of the SS protein present. Under given conditions, the high concentrations of the SSB favor the SB35 binding mode, with lower SAP concentrations, which favor the SSB6s mode. However, it should be emphasized that in both joining modes, the required conformation of the SSB is a tetramer. Mutations of the temperature-sensitive point, occurring spontaneously, within the ssb gene have now been characterized at the biochemical, physiological and nucleotide levels, one such mutant, ssb-1, contains the mutation of point His 55 to Tyr, and h encountered will be unable to assemble properly tetramers at temperatures not allowed and natural expression levels. These mutant strains exhibit lethal temperature-sensitive defects in the DNA recombination response. The segregation frequencies of the plasmids bearing ssb, which complements the chromosomal ssb mutations in the E. coli bacteria, were examined by Porter et al. Bio / Technology 8:47, 1990 (incorporated herein by reference). They observed that in experiments involving bio-reactors, the frequency of segregation in strains carrying plasmids believed in continuous culture under non-selective conditions, for 150 hours, was less than x 10"7, this frequency of segregation was independent of number of copies, since both plasmids pACYC184 of smaller number of copies as plasmids pUC19 of high copy number, were kept at the same frequency However, it should be noted that the plasmids involved are expressed only in a resistance marker to The drug, in addition to the SSB protein, The present invention provides an improved plasmid maintenance system, which incorporates a dividing site, such as that present in pSClOl, or a substantial homologue of such a dividing site and can also incorporate a active division system, the substantial homolog, such as that described above for the virulence plasmid of S. typhimurium. The ion eliminates the dependence of the catalytic enzymes to confer the stability of the plasmid. In one aspect, mutated alleles, similar to ssb-1, are introduced into the expression plasmids to increase the larger copy number plasmids by over-expression of SSB1-type proteins, to form the biologically required active tetramers of SSB. In another aspect of the present invention, a PSK function is provided which involves an inactive plasmid addiction system based on the antisense AR control mechanisms that only synthesize lethal proteins, after the loss of plasmids has occurred. . 7 Plasmids of Expression and Self-Contained Genetic Cassettes The present invention also comprises a series of expression plasmids, which are referred to herein as plasmids gene. These pGEN plasmids comprise self-contained genetic cassettes that encode the regulated expression of a heterologous antigen, a replication origin, a selectable marker to recover the plasmid. This series of vectors has been specifically designed to test whether any Plasmid Maintenance System can increase the stability of plasmids, for example within a history of attenuated S. typhi vaccine. The basic structure of these vectors is represented in Figure 1 and the sequence of the compound gene for the vector pGEN 2 is represented in Figure 4; Figures 5 and 6 show specific compound sequences for the response origins in pGEN3 and pGEN4, respectively. It is critical to note that pGEN s plasmids are designed to comprise 3 independent genetic cassettes. These cassettes have been constructed in mod so that individual components can be optimized by replacement as necessary. Therefore, in addition to the various Plasmida Maintenance Systems described here, the cassettes can test other promising systems now in existence or that may become available in the future. In addition, optimized plasmids can be adapted to express heterologous protective antigens within attenuated vaccine strains, for immunization of humans. Plasmids pGEN provide a cassette of regulated test antigen expression, which operates so that the induction of antigen expression is increased, a metabolic load is placed on the bacteria, which leads phenotypically to the instability of the plasmid, is say, a selective advantage is created for all bacteria that can spontaneously lose aggressive plasmid. Thus, one aspect of the present invention provides a conditionally unstable plasmid that can be examined for stability as plasmid maintenance systems are incorporated. In a preferred mode, the regulated test antigen expression cassette contained within the pGEN plasmids comprises the inducible omp promoter, the substantial homologue, driving the expression of a detectable protein, such as the fluorescent protein verd optimized at the codon (GFPuv, available from Clontech), over-expression of which is toxic to E. coli and S. typhi. The present invention also comprises a seri of plasmid replicons having copy numbers qu ranging from a low copy number (i.e. - up to -10, preferably -5 copies per cell) up to a number d copies (i.e., - 11 to -25, preferably -15 copies per cell) up to a high copy number (i.e., -26 to -60 copies per cell). To achieve this, the replication origins of the well characterized pSClOl plasmids, pACYC184 and pAT153, have been modified using polymerase chain reaction (PCR) techniques to create plasmid response cassettes, which function independently. These response cassettes allow testing of the efficiency of a plasmid maintenance system as the number of copies increases. The present invention also comprises selectable expression plasmids, for use in live attenuated S. typhi vectors. These expression plasmids contain a selectable marker, which can ultimately be replaced either by a non-drug resistant site, such as ssb, or by a gene encoding an acceptable marker of drug resistance, such as aph, which encodes the resistance to the aminoglycosides of kanamycin and neomycin.

To achieve this, the resistance cassettes that encode the resistance to carbenicillin and tetracycline have been constructed, with the transcription being efficiently terminated by a mB T1T2 terminator. A detailed description of the individual components comprising the expression and replication cassettes is provided. The specific components of the Plasmida Maintenance System can be systematically inserted into the basic expression replicons, to evaluate any individual or synergistic influence of these functions on the stability of plasmid in the presence and absence of selection. For example, an elimination function after segregation (eg hok-sok sites) can be inserted as an EcoRI-Xbal cassette, such as flank transcription from the surrounding site, such as the antigen and selection cassettes. , it is divergent and the wild type transcription levels, which control the lethality of this site, are not significantly perturbed (Figur 7B, pGEN 111). Similarly, the passive splitting site can be inserted as a fragment of BamHl-Bg / II, between the replication origin and the selection cassettes.

(Figure 7C, pGEN 121). Interestingly, in the work leading to the present invention, it was observed that the pair-site orientation increases the synthesis of GFPuv in the solid medium when inserted in the natural orientation found within the pILClO orilOl; this orientation was adapted for all expression plasmids. The active cleavage site is preferably the parA site, constructed as an Xhol-EcoRI cassette, from the same resistance plasmid pRl of which it adapts and hok-sok. To preserve the natural transcription levels and regulation within this site, the cassette is preferably placed within an area of the plasmid d expression, such that flank transcription progresses away from parA (Figures 7D and 7E, pGEN193 pGEN222) . . 8 Components of Antigen Expression and Cassettes d Replica . 8.1 Promoter Those skilled in the art will appreciate that a wide variety of components, known in the art, can be included in the expression cassettes of the present invention, which include a wide variety of transcription signals, such as promoters and other sequences. that regulate the binding of the RNA polymerase to the promoter. The operation of promoters is well known in the art described in Doi, Gene Expression Regulation, Moder Microbial Genetics, pages 15-39 (1991) (the entire description is incorporated herein by reference). The resulting description uses the ompC promoter as an example and does not mean a limit in the invention. The promoter is preferably an environmentally regulatable promoter, controlled by a biologically relevant signal, such as osmolarity. In a preferred mod, the promoter is an ompC promoter. The omp gene encodes a porin protein that is inserted as a trimer in the outer membrane of a bacterial cell. The expression and control of ompC is complex and has recently been reviewed in considerable detail in Pratt et al., Molecular Microbiology 20: 911, 1996 and Egger et al., Genes Cells 2: 167, 1997 (the description of which is incorporated herein by reference). ). The synthesis of the OmpC protein is finally controlled at the level of transcription by the osmolarity of the surrounding environment, so that increases in osmolarity are accompanied by increases in the transcription of ompC. However, increases in osmolarity do not directly increase mediately in the transcription of ompC. Rather, the bacterium detects the surrounding osmolarity using a two-component signal transduction system, encoded by the ompB operon. This operon is composed of two genes transcribed in the order envZ-ompR. The envZ gene encodes an amino acid protein (a. A.) That contains two transmembrane regions, which are inserted into the bacterial inner membrane (perhaps a dimer) with a N 118 a terminal. to. domain of osmotic detection, which extends into the periplasmic space and a terminal C 270 a. a, a catalytic domain that extends into the cytoplasm. The catalytic domain of the N terminus possesses both kinase and phosphotase activities, which are modulated by osmolarity, as the osmolaride increases, the activity of the kinase predominates, and as osmolarity falls, the activity of the phosphatase predominates. The 55 residue of the phosphorilate aspartic acid of EnvZ kinase activity of 239 a. to. of OmpR cytoplasmic protein, which creates the OmpR-P. It is the modified protein of OmpR-P that binds to the ompC promoter and activates the transcription by the RNA polymerase; therefore, as osmolarity increases, the kinase activity of EnvZ increases and produces higher levels of OmpR-P, which, in turn, leads to higher transcription of OmpC. The OmpR-binds to a region of the -41 bases that expand the ompC promoter (relative to the transcriptional start site of +1) to -102, with the initial binding of the OmpR-P to the bases -78 up -102, being followed by the additional binding to bases extending to -41, as the concentration of OmpR-P increases with osmolarity.

In addition, the OmpR-P has been shto bind to an upstream region rich in AT, which extends back to the -405 base which further increases the transcription of ompC. In a preferred molarity, the ompC promoter fragment of E. coli expands nucleotides +70 to -389. This promoter can direct transcription within attenuated S. typhi strains of an antibiotic resistance gene, such as the kanamycin resistance gene in an osmotically sensitive manner. For example, our experiments have shthat when the concentration of NaCl in a liquid growth medium has increased from 0 mM to 300 mM, the resistance to kanamycin increases from 0 μg / ml up to > 800 μg / ml. . 8.2 Origin of Replica Due to the various degrees of toxicity associated with different heterologous antigens (ie, higher toxicity for antigens derived from parasitic organisms, such as Plasmodium falciparum vs. virtually no toxicity to fragment C of tetanus toxin) ), the present invention provides vector vaccines that preferably express such antigens from low or medium copies plasmids. It will be appreciated by the experts in the field that the selection of a replication origin will depend on the degrees of toxicity, ie the number of copies should decrease as the toxicity of the bacterium strain increases. In a preferred mode the Plasmid Maintenance Systems used with the ability to stabilize the replicone of low to medium copy numbers. It is preferable that the replication source confers an average number of copies that is between about 2 75. In a preferred mode, the replication origin is selected to confer an average number of copies that is between 5 and 50. More preferably, the range is from about 5 to 30. Optimally, the range is from about 15 to 20. In one aspect, the replication origin is from pSClOl, which confers a copy number of about 5 ppo genome equivalent. The origin site specifies the synthesis of a base transcriptionist named RNA1 and the synthesis of a base 110 antisense RNA transcript, named AR II. As RNA I is synthesized, the proximal 5 'region of the transcriber adopts a stem-loop structure, composed of 3 domains that can hybridize to a stem-loop structure formed by RNA II, resulting in an RNA structure. Double-stranded RNA that forms and causes it to abort the plasmid response. As the synthesis of RNA I, which generates the full-length base 555 transcript, continues, a rearrangement of the secondary structure of the transcript destroys the initial 3-stem-loop structure, to form an alternative stem-loop configuration, which n already hybridizes to RNA II. The formation of this alternative structure allows the transcription to hybridize to the DNA strand of the plasmid itself, which forms a RNA-DNA complex that is seized by RNAse endogenous to the activation syntesis of the plasmid's first strand of DNA and the response of plasmid. The response of plasmid, therefore, is controlled by the synthesis of RNA I, which undergoes a cascade of structural configurations that lead to the initiation of the response. The necessary progression of the RNA I doubling cascade (and the start of the resulting response) is interrupted by the competition of domains with RNA II. This mechanism is essentially the same in plasmids containing either oriEl or oril5A. The reason that these two types of plasmids can coexist within the same bacterium is due to the sequence divergence within the region d hybridization between RNA I and RNA II, such that oril5A RNA I does not hybridize to RNA I from origin.; This sequence divergence also affects the stability of RNA I: the RNA I hybrid that accounts for the difference in the number of copies between the plasmids that carry the origin of response oriEl or the ori5A. The structural organization of the engineering origins of the response cassettes for pSClOl (orilOl; -5 copies per genome equivalent), pACYC184 (d oril5A derivative; ~ 15 copies per genome equivalent) and pAT153 (derived from oriEl; -60 copies per genome equivalent) are analogous in structure and function. . 8.3 Protein or Expressed Peptide When the expression cassette is used to classify the Plasmid Maintenance Systems, preferably they express a protein or peptide if metabolic activity. A preferred protein is the green fluorescent protein (GFP) of the bio-luminescent jellyfish, Aequorea victoria, an amino acid protein 238 which undergoes a post-translational modification in which the 3 internal amino acids (65Ser-Tyr-Gly67) are involved in a cyclization and oxidation reaction. The resulting fluorophore emits maximum blue-green light at a wavelength of 509 nm, on irradiation with long-wave ultraviolet light, at a length of 395 nm. In addition, the fluorescence activity is remarkably constant over a wide pH range of 5.5 to 12, and at temperatures up to 70 ° C.

Since GFP does not have catalytic activity the level of fluorescence observed within the individual bacteri that expresses GFP, can provide a direct indication of transcription levels of the gf gene carried by each bacterium. The expression of the GF protein has now been quantified in a variety of prokaryotic and eukaryotic cells, and does not require additional cofactore or A enzymes. victory The formation of fluorophore is apparently dependent on either a ubiquitous enzyme and cofactors, or is a autocatalytic event. Individual bacteria expressing GFP can be quantified either alone or within macrophages, epithelial cell line and infected animal tissues using flow cytometry. The fluorescence of GFP is absolutely dependent on residues 2-232 of the denatured protein. However, the fusion of the active protein domains, biologically if related, to the N-terminus of the GFP, has still resulted in fusion proteins with the expected heterologous biological activity, which continues to fluoresce as well. It has been confirmed by sequencing analysis (Clontech) that the gfp allele, here preferred (ie gfpuv) expresses a GFP mutant (GFPuv) containing amino acid substitutions (does not involve the fluorophore) that increases fluorescence 18 times over that of the wild-type CFP. In addition, the 5 arginine codons, rarely used, have been optimized for the efficient expression of GFP in E. coli. Since the compassion of the expression levels of several heterologous proteins in E. coli CVD908 has demonstrated equivalent or superior expression within CVD908, gfpuv is expected to function efficiently in CVD908-htrA. A coding sequence is inserted in a correct relation to a promoter, where the promoter and the coding sequence are so related that the promoter drives the expression of the coding surface, so that the encoded peptide or protein is finally produced. It will be understood that the coding sequence must also be in correct relationship with any other regulatory sequence that may be present. . 8.4 Heterologous Antigen The expression plasmids of the present invention preferably express an antigen for presentation to a host to produce an immune response that results in immunization and protection from diseases. While the Shiga toxins are presented here as examples of antigens, usefully expressed by the vaccine expression plasmids described herein, the invention has a broad scope and encompasses the expression of any antigen that does not destroy the living bacterial vector and that produces an immune response. when the live bacterial vector, which contains the expression plasmids, is administered to a host, i.e. to a human or other animal. The vaccine expression plasmids provided herein are used to genetically transform the attenuated bacterial strains, preferably the strains used by the human vaccine and more preferably used to transform the attenuated S. typhi vaccine strains, such as CVD908-htrA, and preferably encode any of the B subunit of stx2 or a detoxified Stx holotoxin. A subset of STEC more frequently referred to as enterohemorrhagic E. coli (EHEC) is able to use severe clinical syndromes, which include haemorrhagic colitis, haemolytic uraemic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP), in a small proportion of infected individuals, in addition to causing bloody diarrhea in the majority of cases. Hemorrhagic colitis is characterized by copious, bloody diarrhea, usually without fever or with only a low grade fever, and a small relative number of fecal leukocytes demonstrable in stools of diarrhea. These characteristics make a difference from the hemorrhagic colitis of dysentery caused by Shigella, which is typically of scarce evacuations of blood and mucus, preceded by high fever and with a large number of fecal leukocytes visible by microscopy. HUS, a potentially fatal disease, which most often affects young children, but can afflict individuals of any age, is characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia, uremia. Currently in North America, HUS is the most frequent cause of acute renal failure in infants and young children. In a study by Siegler et al, of 288 patients treated with post-adrenal HUS in UTA, USA, from 1970-1994, severe diseases (defined as anuria that lasted more than 7 days, oliguria lasting more than 14 days, or structural damage). extra-renal such as an attack) occurred in 25% of cases and was associated with children under two years of age; about one third of these severe cases of HUS resulted in death (5%) or severe sequelae that include end-stage renal disease (5%) or chronic brain damage (3-5%) with less severe chronic problems, which involve hypertension, proteinuria or azotemia. TTP, which most often affects adults, is characterized by neurological complications, such as seizures. In addition to thrombocytopenia, hemolytic anemia kidney disease. By far, the most common EHEC serotype is e 0157: H7, However, other EHEC serotypes also cause HUS and hemorrhagic colitis, which include 026: H11, OHl: H8 a number of others. The EHEC strains associated with HUS always produce one or more Shiga toxins and carry a plasmid d virulence of 60 Mda. In addition, most also harbor an island of chromosomal pathogenicity (named LEE) that has a set of genes that encode the ability to bind suppress. It is well accepted that the Shiga toxins elaborated by EHEC play a key role in the pathogenesis of hemorrhagic colitis and HUS. As described in detail below, the family of Shiga toxin is comprised of two groups of toxins. Stxl (which is essentially identical to the cytotoxin / neurotoxin / enterotoxin, produced by type 1 d Shigella dysenteriae, the Shiga bacillus) and Srx2 (which is immunologically distinct from Stxl and has diverse related variants). In E.U.A., the overwhelming majority of EHEC associated with HUS cases express Stx2 alone or in conjunction with Stxl. The most important reserve of EHE infection is cattle. The most important simple way of transmitting the EHEC to humans is through the consumption of contaminated cattle, with low cooking, often with ground meat. Less commonly, a variety of other food vehicles and other modes of transmission have been framed. Most notably, EHEC is one of the enteric bacterial pathogens that, like Shigella, can be transmitted by direct contact or contact with contaminated domes. There is a great anticipation and optimism in the majority of epidemiologists, that the irradiation of carn sold in E.U.A., will drastically reduce the transmission of EHEC to humans, since it would reduce the important mod of simpler transmission. However, certain risk groups exposed other modes of transmission of the EHEC will not benefit from this intervention. For example, the exposure of the slaughterhouse workers to the EHEC, a hazardous occupation, occurs at a point where the meat is in the process cycle where the irradiation will be used. For such special groups, such as those for which the risk remains even after irradiation of the carn to reach the common site, anti-EHEC vaccines may be useful. The present invention provides vaccines against EHEC, useful in the prevention of infection (in animal or human deposits) and to prevent the severe complications of EHEC infection by stimulating Shiga neutralization.

Studies with attenuated VAbrio cholerae 01 expressing the Stxl B subunit have demonstrated the feasibility of producing Shiga antitoxin neutralized by mucosal immunization with live vectors. However, since virtually all EHEC associated with HUS cases, in. EUA, express Stx2 alone or together with Stxl, it is preferable that a vaccine be used to prevent severe complications of EHE infection by evoking antibodies that Neutralize the toxin should stimulate the anti-Stx2 just like the Stxl. It is within the broad scope of the present invention to provide a stabilized plasmid system for expressing the Stx2 antigen, alone or in conjunction with Stxl, in a live attenuated S. typhi vector. Other antigens that can be delivered suitably, according to the compositions and methods of the present invention, include, for example, those for hepatitis B, Haemophilus influenzae type b, acellual pertussis hepatitis (acP) / varicella rotavirus, Streptococcus pneumoniae (pneumococal) ) and Neisseria meningi tidi (meningococal). See Ellis et al., Advances in Pharm. , 39: 393-423, 1997 (incorporated herein by reference). In one aspect, the antigens encoded by the expression plasmids of the present invention are cancer vaccine.

In another aspect, the antigens encoded by these plasmids are designed to elicit an immune response to autoantigens, B cell receptors and / or T cell receptors, which are involved in autoimmune or immunological diseases. For example, when inappropriate immune responses are enlisted against body tissues or environmental antigens, vaccines of the present invention can immunize against self antigens, B cell receptors and / or T cell receptors, modulate the responses The diseases. For example, such techniques may be effective in treating myasthenia gravis, lupus erythematosis, rheumatoid, arthritis, multiple sclerosis, allergies, and asthma. . 8.4.1 The Toxin Family of Shiga Conradi, in 1903, first reported that S. dysenteriae 1 produced a potent exotoxin. Because the injection of this toxin led to the paralysis of the hind leg of rabbits, it was originally called a neurotoxin. Following this toxin, the Shiga toxin was shown to be lethal to certain cells in the tissue culture (ie, it was a cytotoxin). Vicari et al., And then Keusch et al., Demonstrated that it is also functional as an enterotoxin.

Scientists now recognize the existence of a family of Shiga cytotoxins that inhibit the synthesis of proteins, which lead to the death of cells in susceptible cells. For many years, after the revelation that such toxins were produced by certain strains of E. col besides the original Shiga toxin, produced by Shigell dysenteriae type 1, the nomenclature of this family of toxins was unclear. Since early reports described the activity of these toxins in Vero cells (a cell line derived from African green monkey kidney epithelial cells), many researchers named it verotoxins. Others refer to these toxins expressed in E. coli as Shiga-type toxins. Protein toxins are collectively named here as Shiga toxins (Stx) and the genes coding for these toxins are designated as stx with the subscripts denoting the group and variant [ie, stxx, for the Shiga toxin produced by E. coli , which is essentially identical to that of Shigella dysenteriae type 1 (stx) stx2, stx2c, stx2d, stx2e, for the antigenically distinct group of related toxins]. The structure, biochemistry and antigenicity of Shiga toxins are well described in Melton-Celsa et al., Escherichia coli 0157-H7 and other Shiga toxins that produce strains of E. coli, 1998; Takeda, Bacteria Toxins and Virulence Factors in Disease, 1995; Gyles, Canadian J. of Microbiology, 38: 734, 1992 and O'Brien et al., Current Topic in Microbiology and Immunology, 180: 165, 1992 (the descriptions of which are incorporated herein by reference). These Shiga cytoxins are composed of a sun catalytic subunit A of approximately 32 kDa, non-covalently associated with a pentameric receptor that binds a subunit domain of approximately 7.7 kDa B. These subunits are encoded by a single operon operon d stxA-stxB; the transcription of the stx and stxx operons are regulated by iron, in both S. dysenteriae and E. coli, but no environmental control signal has yet to be determined by the Stx2 operon. None of these toxins is encoded in a plasmid; rather they are encoded by phages (Stl, Stx2, Stx2c and Stx2d) or are coded chromosomally (Stx, Stx2e). As mentioned before, all members of the Shiga toxin family are cytolytic toxins, which inhibit protein synthesis, within susceptible cells, by blocking the binding of the aminoacyl-tRNA-dependent elongation factor-1 to ribosomes. For all toxins identified from human infections, the penetration of susceptible cells by endocytosis, followed by the binding of holotoxin to the necessary cell surface of the glycolipid receptor of the globotriasosyl ceramide (Gb3), the trafficking of the toxin to the apparatus Golgi and the endoplasmic reticulum, followed by release into the cytoplasm. Shiga toxins are N-glycosidase from RNA that shed a single adenine from RNA 28 of the eukaryotic 60S ribosomal subunit, thus inactivating the 60S subunit and ultimately leading to cell death. There are six prototypical members of the Shiga toxin family; STx, Stxl, Stx2, Stx2c, Stx2d and Stx2e, which differ immunologically and in the activity of the toxin. Significant details have been included here to provide the background for understanding the meaning of the point mutations, discussed below that are required for genetically detoxified holotoxins. Members of the Shiga toxin family differ from each other in 3 fundamental ways, as recently summarized by Melton-Celsa et al., Escherichi coli 0157: H7 and other strains of E. coli, which produce the Shi ga toxin, 1998 (1) Immunologically: Shiga s toxin family consists of two serogroups, Stx / Stxl and Stx2; Antiserum excited against Stx / Stxl does not neutralize the serogroup members of Stx2, as judged by the assay of cytotoxicity of Vero cells. (2) Structurally: The Stx and Stxl are essentially identical, differing in a single amino acid in the 45th position of the mature subunit A, and the crystal structure for the Stx holotoxin has been resolved. The Stx2 prototype is only 55% homologous in the residues of the mature A subunit of Stx / Stxl and 57% homologous to the mature B subunit, which explains why the antiserum excited against Stx / Stxl does not neutralize the Stx2 group members. , Within the Stx2 group, Stx2e is distantly related, comprising 93% of the amino acid homology, to the mature A subunit of Stx2 and 84% d homology to the mature B subunit; Stx2c and Stx2d are mu similar to Stx2, sharing 99-100% homology in the residues of the mature A subunit and 97% homology in the residues of the mature B subunit. (3) Cytotoxicity: The Stx2 is among the most lethal of the Shiga toxins, with an LD50 for mice, injected intraperitoneally, from 0.5 to 2 ng. The LD50 for the Stxl Stx2 is 200-400 ng and 1 to 5 ng for the Stx2d; However, Stx2d is unusual in that this toxin can become activated by murine intestinal mucus, to increase the toxicity of the toxin, decreasing LD50 0.5 ng. . 8.5 Specific Mutagenesis of the Shiga Toxin Site. In one aspect, the invention provides a genetically engineered Shiga toxin. Detoxification is achieved by site-specific mutagenesis, which introduces two well-defined and well-defined point mutations by altering the critical residues within the catalytic site of the A subunit. The invention also introduces additional, well-defined and well-defined point mutations within subunit B, to alter the critical residues within the primary binding site (i.e., SITE I) that resides within the cleft formed by the adjacent subunits of the pentameric holotoxin ring. Previous attempts have been made to alter the lower binding affinity of SITE II. However, this binding site has been defined only from molecular modeling studies, and is not extensively supported by mutation studies that favor the binding of SITE I of the GB3 receptor. Even if SITE II is an alternative low affinity binding site, which allows the entry of our mutant holotoxin into susceptible cells, inactivation of the catalytic domain will still prevent the death of the cell. Based on the amino acid sequence alignments, X-ray crystallography studies, molecular modeling studies, the essential amino acids have been identified, which comprise the active site within the catalytic A subunit of Stx, just as those residues that comprise the SITE I of union within the pentamer of the subunit of Stx / STxl. The invention concludes that the essential amino acids to the active site are selected from the group consisting of Tyr 77, Tyr 114, Gl 167, Arg 170, and Trp 203. The residues are believed to be required by the binding of the receptor to the slits formed by adjacent B subunits, include Lys 13, Asp 16, Asp 18, Thr 21, Glu 28, Phe 30, Gly 60, and Glu 65. These site predictions are consistent with functional studies and in vivo experiments using simple mutations and defined doubles, within the individual domains of holotoxin, introduced by site-specific mutagenesis. A summary of such mutations is presented in Table 1. Based on this data and crystallographic predictions, it is within the broad practice of the invention to provide expression plasmids that encode the Shiga toxins, which have specific sets of point mutations. within both subunits A and B, to create the nontoxic mutant of the Stx2 holotoxins, for use in vaccines, such as expression within live attenuated S. typh vectors, such as CVD908-htrA. . 9 Pharmaceutical Formulations It is considered that vaccines of the bacterial viv vector of the present invention will be administrated and pharmaceutical formulations for use in vaccines of individuals, preferably humans. Such pharmaceutical formulations can include pharmaceutically effective carriers and, optionally, can include other ingredients, such as various adjuvants, known in the art. The carrier or carriers must be pharmaceutically acceptable in the sense that they are compatible with the therapeutic ingredients and do not unduly harm their recipient. The therapeutic ingredient or ingredient is provided in an amount and frequency necessary to achieve the desired immunological effect. The mode of administration and dosage forms will affect the therapeutic amounts of the compounds, which are convenient and effective for the vaccine application. The live bacterial vector materials are delivered in an amount capable of producing an immune reaction which is effective to increase the patient's immune response to the expressed mutant holotoxin or other desired heterologous antigens. An effective amount and immunological form is an amount which confers an increased ability to prevent, retard or reduce the severity of the attack of a disease, in comparison with such abilities in the absence of this immunization. It will be readily apparent to one skilled in the art that this amount will vary based on factors, such as the weight and health of the recipient, the type of protein or peptide that is expressed, the type of infection organism that is combated the mode of administration. of the compositions. The modes of administration may comprise the use of any suitable means and / or methods for the delivery of the bacterial live vector vaccines in an immuno-stimulative manner. Methods of delivery may include, if limited, methods of parenteral administration, such as subcutaneous injection (SC), intravenous injection.

(IV), transdermal, intramuscular (IM), intradermal (ID), as well as non-parenteral, for example, the oral, nasal intravaginal, pulmonary, ophthalmic and / or rectal administration. The dosage regimen and dosage forms suitable for the viv bacterial vector vaccine compositions of the present invention can be readily determined by those of ordinary skill in the art, if undue experimentation, by the use of the techniques of determining the conventional antibody titre and conventional biocompatibility and bioefficacy protocols. Among other things, the dosage regimen and the appropriate dosage forms depend on the particular antigen used, the desired therapeutic effect and the desired period of time of the bioactivity.

The bacterial live vector vaccines of the present invention can be administered in useful form to a host animal with any other suitable pharmacological and physiological active agents, for example antigenic and / or other biologically active substances. The formulations of the present invention can, for example, be presented as discrete units, such as capsules, tablets or lozenges, each containing a predetermined amount of the vector delivery structure, or as a suspension. 8. EXAMPLES An isogenic series of expression plasmids, composed of individual cassettes, was constructed for use in live bacterial vector vaccines, such as E. coli.

Salmonella With the exception of the ribosomal binding sites (RBS), the initiation of transcription termination, which controls the key genetic site, the response of the plasmid or the expressed coding proteins, are contained within the defined restriction fragments, as illustrated by the representative plasmids diagram of pGEN 2, seen in Figure IA.

The basic structure of these primer expression plasmids will be highlighted and then the data demonstrating the function of each site within the attenuated vaccine strain CVD908-htrA will be summarized. . 1 Structure pGEN The transcription of any heterologo antigen that will be expressed within DVC908-htrA, is controlled primarily by an inducible promoter contained in an EcoRI-Bg / ll cassette. Since plasmid d expression was initially modeled after pTETnirl5, the early versions carried the nirl5 promoter, anaerobically activated (Pniris) • However, this promoter has been replaced with a Pompe promoter, osmotically controlled, tightly regulated, which is easy to manipulate in vi tro, varying the NaCl concentration. The heterologous antigens are contained in a Bg / II-Avrll cassette, flanked by an optimized RBS at the proximal 5 'end and a trpA transcriptional terminator at the 3' distal end of this cassette. The origin of replicate for these expression plasmids has been designated as an AvrII-Bg / lI cassette, and is protected from transcription through reading, which originates in the flanking regions. These cassettes carry an extremely efficient derivative of the T1T2 transcriptional terminator in a terminal with the trpA transcriptional terminator from the heterologous antigen cassette at the opposite end of the response cassette. The flanking Bg / II and Spel sites (see Figure 2) between the response cassette and the selection cassette are attempted for the insertion of a plasmid maintenance function, such as the pair site of pSClOl The selection cassettes within The plasmids are contained within the Spel-Xbal cassettes and can, for example, be used to encode the carbenicillin resistance (the gene) or the tetracycline resistance (the tetA gene, see Figure 1). The drug resistance cassette can be replaced with the ssb gene encoding the essential single-strand binding protein of Salmonella typhi, CVD908-htrA. The Xbal and EcoRI flanking sites between the selection cassette and Pomp are attempted for the insertion of additional maintenance functions, which include a PSK site, such as hok-sok (see Figures 1 and 2), or a function of additional division, such as the parA site of pR (see Figure 7). 6. 2 Modified ompC promoter Any promoter that controls the transcription of a heterologous gene is intended to be responsive to an environmental signal of biological relevance. For expression plasmids, described here, a cassette (Pompe) of the ompC promoter of E. coli is used, which is induced by increases in osmolarity. The construction of this cassette is based on the published sequence of Pompc / published by Norioka et al. (Norioka et al., 1986), and was carried out using synthetic sizing to create an EcoRI-Bg / lI cassette of 459 bp, where the natural RBS was removed. To confirm that this promoter was controlled osmotically with CVD 908-htrA, a derivative of pTETnirl5 was constructed in which the Pn? Ri5-toxC was replaced by a shell comprising Pompc that drives the expression of an aphA-2 shell without promoter, which It confers resistance to kanamycin. This plasmid, designated pKompC, was introduced into CVD 908-htrA, by electroporation, and the receptors were classified in kanamycin resistance in the medi LB. The osmotically regulated expression of aphA-2, s determined by inoculating CVD 908-htrA (pKompC) in 50 ml of supplemented cald nutrient (NB), containing increasing concentrations of kanamycin from 0 to 300 μg / ml; A conjugate palleelo of cultures was adjusted with identical intervals of aggregated kanamycin, but also containing 10% sucrose, to induce Pome- The cultures were incubated overnight at 37 ° C and the D.0.60o- was measured. are reported in Table 2, Experiment 1. Table 2 shows the osmolaride induction of the Pomcr promoter that controls the expression of kanamycin resistance, within the live vector of attenuated S. typhi, CVD 908-htrA. 1 A culture of CVD908-htrA (pKompC) was adjusted in LB broth supplemented with 0.0001 (weight / volume) of 2,3-dihydroxybenzoic acid (DHB) and 50 μg / ml kanamycin, and incubated for 16 hours at 37 ° C . This initial culture was then diluted to: 10 in a fresh medium and incubated at 37 ° C for two hours to provide a seed culture of bacteria that grow exponentially. 50 Rl of this culture were then inoculated with 50 ml of Nutrient Broth (NB) cultures supplemented with DHB as before, but with increasing concentrations of kanamycin; a parallel set of cultures was adjusted with identical intervals of added kanamycin, pro also containing 10% sucrose, to induce, as expected, the Pompc-incubated cultures overnight at 37 ° C and the DOβoo was measured - 2 A culture of CVD908-htr (pKompC) in supplemented caldoLB and kanamycin was incubated for 16 hours at 37 ° C, diluted 1:10 in a fresh medium, and incubated at 37 ° for two hours, to deliver a culture of sowing of bacteria that grows exponentially. Aliquots of 100 μl of this culture were then inoculated into 5 ml of NB broth cultures containing increasing concentrations of kanamycin from 200 to 800 μg / ml; a parallel set of cultures was adjusted, containing 300 m of NaCl, and all the cultures were incubated at 37 ° C for 16 hours and the D.O.600 was measured.

Regardless of the selective pressure used by kanamycin, the presence of 10% sucrose had an inhibitory effect on the growth of CVD 908-htrA (pKompC).

However, the results suggest that E. coli P, ompC s controlled osmotically when gene expression of AphA-2 was driven within CVD 908-htrA (pKompC). To confirm this, the CVD 908-htrA (pKompC) was inoculated into 50 m of supplemented NB broth, containing increasing concentrations of kanamycin from 200 to 800 μg / ml; a conjugate paléelo of cultures was again adjusted, which contains 300 mM of NaCl, to induce the Pompe. The cultures were incubated 37 ° C for 16 hours and the results are reported in Table 2, Experiment 2. It was confirmed that the Pome-driven expression of the aphA-2 gene within CVD 908-htr confers resistance to kanamycin at levels up to of 800 μg / ml, in an osmotically regulated manner.

The cassette of the aph gene was then replaced with a cassette of Bg / II - Nhel of 756 bp, which contains the allele gfpuv encoding GFPuv. During the visual classification of colonies of E. coli. sub-illuminated with ultraviolet light, one very bright fluorescent colony and another representative fluorescent coloni were chosen for further study, designated clone 1 and clone 3, respectively. In the purification of the plasmids involved, it was determined that clone 1 contained a plasmid that no longer carries a Bg / ll site that separates Pome and gfpuv, while clone 3 carried the expected Bg / ll site. We examined the induction of GFP expression when clones 1 and 3 grew in nutrient aga in the presence or absence of NaCl, and it was determined by visual inspection that clone 3 exhibited very little fluorescence when it grows on nutrient agar that does not contain NaCl, but it is brilliantly fluorescent when placed on nutrient agar containing 300 mM NaC (data not revealed) . However, clone 1 had a higher level of fluorescence when it was not induced, but fluoresced intensely when induced with 300 mM NaCl. Exclusion mutations within the gfpuv gene that can affect fluorescence, we replaced Pompe from clone i with pomp from clone 3, and confirmed the expected decrease in fluorescence as judged by underlining (data n shown). Therefore, we conclude that the observed differences in fluorescence are controlled by two genetically different versions of the Pome promoter, which we designate as Pompci (higher transcription levels with less osmotic control) and P0mPc3 (molded transcription levels with osmotic control similar to that observed for e Pome-aph cassette described above); we designate the plasmids containing these expression cassettes with pGFPPompCl and pGFPompC3, respectively. To quantify the differences in the induced and uninduced expression of gppuv controlled by Pompci and PomPc3 / the synthesis of GFPuv was monitored with both E. coli DH5 and S. Typhi CVD 908-htrA, using flow cytometry. This potent technique has the unique advantages of allowing rapid measurement of GFPuv expression within large numbers of individual bacteria, as well as accurately determining the average fluorescence intensity due to the synthesis of GFPuv within each bacterial population analyzed. To achieve this, pGFPompCl pGFPompC3 was introduced by electroporation, and the supplemented 1 x LB colonies, containing 100 μg / ml of carbenicillin, grown at 30 ° C for 48 hours were isolated. The isolated colonies then grew and the cultures frozen as a master stuff. Fresh colonies were then inoculated into any of the supplemented nutrient broth the supplemented nutrient broth containing 150 mM NaCl, and growing at 37 ° C / 250 rpm for 24 hours; the difference in D.0.6oo for any crop was never greater than 0.07. The induction of the expression of gfpuv, controlled by Pompc? omc3 were analyzed by flow cytometry and the results are presented in Table 3. Table 3 shows a comparison of induction of Pompci and Pompc3r that controls the expression of GFPuv, within the host strains of E. coli DH5a and CVD 908 -htrA1. 1 All strains were streaked with the master material frozen in 2X LB agar supplemented with DHB and 50 μg / ml carbenicillin, and incubated for 36 hours at 30 ° C. The isolated colonies were grouped in 300 μl of NB draft supplemented with DHB and carbenicillin, of which 25 μl was inoculated in 25 ml of supplemented NB broth, with and without 150 mM of NaCl, and incubated at 37 ° C, 250 rpm during 24 hours. Bacteria were formed into pellets, resuspended in 1 ml of PBS, pH 7.4 and then diluted 1: 1000 in PBS during flow cytometric analysis. 2 Defined as the ratio of mean fluorescent intensity, measured after induction with 150 mM NaCl, divided by the baseline level of fluorescent mean intensity, measured at low osmolarity. 3 NA = not applicable.

The basal level of expression for the Pompci-gfpuv cassette is 2.5 times higher than for the cassette of P0mpc3-gfpuv, when expressed in DH5a, and 2.1 times greater when expressed in CVD 908-htrA; however, the basal fluorescence level, detected by the GFPu synthesis, never exceeded an average fluorescent intensity of 5.37, independently of the host antecedent. If we define the induction relationship as the ratio of the mean fluorescence intensity, measured after induction, divided by the basal level of the mean fluorescence intensity, it is observed that when induced with 150 mM NaCl, Pompci and omPc3 exhibited within DH5a induction ratios of 1.7 and 2.4 respectively. Surprisingly, the induction ratio for Pompci when measured in CVD 908-htrA was 4.4 and produced a mean maximum fluorescence intensity of 2.4 for these experiments. Although the induction ratio for P0mpc2 within CVD-908-htrA was 6.7, the mean fluorescence intensity of 17.1 was less than that measured for P0mPc ?. Based on these data, it appears that Pompci is the strongest and still osmotically controlled promoter of the two ompC promoters. ompci / therefore, it was chosen for the widest possible synthesis of the antigen of the heterologous test to examine the effects of such synthesis on the stability of the plasmid. These data clearly show that when it drives the expression of gfpuv within the vecto strain alive CVD 908-htrA, Pompci and P? NoC3 can induce increasing osmolarity, although the basal level of transcription is still notable in both cases. The results observed under low osmolarity conditions further support our observations using the solid medium that Pompci drives the expression of heterologous antigen greater than Pompc3 • Since P0mc3 was noted to possess the attempted 31-terminal Bg / ll site, which was not detected for Pomci / s determined the nucleotide sequence of Pompc? to detect perhaps the point mutations that can explain the resistance of Pompc? • The only differences identified were placed in the 3 'terminal of the cassette. The sequence attempted within this region was 51-catataacAGATCTtaatcatccacAGGAGGatatctgATG-3 • (from left to right, the upper case denotes the Bg / II site, the ribosome binding site and the GFPuv start codon, respectively): the sequence real proved to be the 5 '-... catataacAGATCGATCTtaaAcatccacAGGAGGAtAtctgATG-3 (inserted or changed bases denoted with the upper case and bold and underlined). These changes detected within the ompCl promoter sequence are apparently responsible for increasing the observed resistance of Pompci by an unknown mechanism, since none of the basic ompC promoter sequence or the optimized ribosome binding site has been altered spontaneously. 6. 3 Origins of Response and Selection Cassettes The success of potentially expressing toxic or heterologous antigens otherwise problematic within CVD908-htrA in the copy number of plasmid d expression. In addition, the observed immune responses to a given heterologous antigen are affected by the copy number of the genes encoding the antigen, with antigens expressed by chromosomes, producing poorer immune responses, when compared to plasmid-based expression. An optimized immune response will depend on the plasmid-based expression of multiple copies of the heterologous plasmid antigens with the appropriate copy number. Since the appropriate number of copies for a given heterologous gene can not be known a priori, the present invention provides a set of expression plasmids containing the origin origins of response. (amplified from pAT153; number of copies -60), oril5 (amplified from pACYC184; number of copies -15) and orilO (amplified from pSClOl; number of copies -5). These self-contained response cassettes are all carried in the Bg / ll-BamHl fragments, and are illustrated by a set of expression plasmids resistant to tetracycline, shown in Figures 1A-1C. The expression of the gfpu expression cassette controlled by Pomci / contained in these expression plasmids d, was analyzed using flow cytometry. These experiments are designed to detect whether the differences in the level of fluorescence observed can be correlated with the expected copy number of a given expression plasmid. The CVD908-htrA strains carrying pGEN2, pGEN3 and pGEN4 were marked with strips on the rich medium SuperAgar supplemented with DHB and 20 μg / ml of tetracycline where appropriate. The SuperAgar was used because it is a very rich medium (3X LB agar). The plates were incubated at 30 ° C to reduce the toxicity of GFP synthesis and allow the bacteria to grow abundantly on the plates. The isolated colonies were then inoculated in 45 ml of SuperCaldo supplemented with DHB and 20 μg / ml of tetracycline, where appropriate, and incubated at 37 ° C for 16 hours. The bacteria were concentrated by centrifugation and resuspended in 1 ml of sterile PBS, pH = 7.4, and diluted 1: 100 in PBS, pH = 7.4 before FACS analysis. The bacteria were analyzed by flow cytometry, as described above, for two experiments that grow independently, and the results are shown in Table 4 at the end of this section. These data support the conclusion that the over expression of GFPuv within CVD908-htrA is toxic to bacteria. As the theoretical copy number increases for the plasmids pGEN4, pGEN3 and pGEN2, which express GFPuv under identical growth conditions from the identical Pomc promoter, the percentage of the growth population which fluoresces declines. It is expected that the "dim" bacteria are not viable bacteria and may no longer contain the plasmid d expression, since these cultures grow in the presence of 20 μg / ml of tetracycline. However, it is noted that when bands are marked on the solid medium and grow at 37 ° for 24 to 36 hours, the CVD908-htrA (pGEN2) grows poorly and fails to produce the isolated colonies, while the CVD908-htrA (pGEN3) and CVD908-htrA (pGEN4) grows very well and produces isolated fluorescent colonies. GFPuv is used here as representative of other heterologous antigens that will be of interest to be included in a live bacterial vector, such as the living vector based on S. typhi, however, it will be appreciated that GFPuv can be replaced by any metabolic n protein or peptide antigen. The above data show that, although the use of expression plasmids of medium copies containing the oriL replicons may be of use in the expression of some antigens, the expression of antigens d greater toxicity will be expressed more successful than the plasmid number. of minor copies, which employ replication origins that provide average copy numbers between 2 30, such as the origins of response orilOl or oril5A. 6. 4 The Elimination Site After the hok-sok antisense segregation Using the polymerase chain reaction, the PSK hok-sok genes were amplified using the plasmid-R pRl of multiple antibiotic resistance as the model for these reactions. All initial attempts to clone this site on plasmids of medium high copy number were unsuccessful. In order to directly select the hok-sok site during subcloning, a set of sizes was designated for use in overlapping the PCR reactions, so that the final product was a fragment containing a genetic fusion of the hok-so site. of pR1 and a non-promoter gene tetA of pBF322, which codes for resistance to tetracycline. This cassette was made by engineering so that the transcription of the hok gene will continue in tetA; the two sites within this cassette were separated by a Xbal restriction site for future manipulations. The construction of this cassette not only allowed the direct selection of the hok-sok site, but also allowed the confirmation that the PS function will operate in S. Typho CVD908-htrA. After electroporation of plasmids carrying the cassette into CVD908-htrA, transformants can be selected using tetracycline. The successful recovery of the isolated colonies indicates the successful synthesis of hok-tetA mRNA, and the successful synthesis of RNA or antisense to prevent the translation and synthesis of Hok, which would kill the bacteria. The recovery of the hok sok-tetA cassette then becomes direct, and is easily incorporated into our expression plasmids to create the selectable marker cassette of the plasmids pGEN2, pGEN and pGEN4, illustrated in Figures 1A-1C. Experiments are then initiated to determine the effect of the PSK function of hok-sok, on the stability of the expression plasmids, which contain oriEl and the bla resistance marker that encodes the β-lactamase which confers resistance to carbencillin. The hok-sok cassette inserted into the expression plasmid pTETnirl5, based on pAT153, in which the cassette of the heterologous antigen Pnirl5-Tox was replaced with our cassette Pompci-gfpuv, creating the plasmids pJN72 (without hok-sok) and pJN51 ( with Hok-sok). An additional set of plasmids was created by replacing P0mpc with the weaker promoter P0mpc3, creating pJNlO and pJN12; The structures of these four isogenic plasmids are represented in Figure 2. The CVD908-htrA strains carrying either pJN73, pJN51, pJNlO or pJN12, were stained in the rich medium of SuperAgar, supplemented with DHB 100 μg / ml of carbenicillin and the plates were incubated as before for pGEN plasmids at 30 ° C, to reduce the toxicity of GFPuv synthesis and allow the bacteria to grow abundantly on the plates. The isolated colonies were then incubated in 45 m of Super Caldo supplemented with DHB and 100 μg / ml of carbenicillin and grown at 37 ° C for 24 h, for analysis by fluorescence flow cytometry. A second independent experiment was carried out exactly like the first, except that the isolated colonies were suspended in 500 μl of Super Broth and 250 μl of each were inoculated in 45 ml of Supe Caldo cultures in pairs, with or without 300 mM of NaCl added to induce the cassettes of Pompc-gfpuv; the cultures were incubated at 37 ° C for 48 hours and again analyzed in the flow cytometry; and the results of both experiments are shown in Table 5. The fluorescence histograms for the non-induced and induced expression plasmids of experiment 2 are depicted in Figures 3A-3H.

These results of flow cytometry can be explained as follows: the expression of GFPuv (or other potentially harmful heterologous antigen) of a multiple copy expression plasmid, such as pJN72, increases the metabolic stress in the living vector CVD908-htrA ( pJN72) and increases the instability of plasmid in the absence of selection. Since the selectable marker 0 of the expression plasmid encodes the secreted enzyme, the β-lactamase, then conforms increases the time, the concentration of the carabicillin in the surrounding medium declines, the selective pressure decreases and the frequency of the plasmid loss increases; however, since plasmids of multiple copies are involved, relatively few bacteria managed to lose all the resident plasmids, but the average copy number of pJN72 per bacterium decreased. The quantification by flow cytometry of the production of GFPuv for an uninduced population of healthy growth CV 908-htr (pJN72) indicated that most bacteria express GFPuv and few non-fluorescent cells were detected (Figure 3A). However, the increased production of GFPuv by induction of the Pompci-gfpuv cassete increased the metabolic tension of CVD 908-htr (pJN72) and although the production of the double GFP, the percentages of the non-fluorescent bacteria increases as more plasmids are lost of the population (Figure 3B). In a similar growth population of CVD-908-htrA (pJN51), each bacterium carries multiple-copy plasmids that encode both GFPuv and PSK function. The frequency of plasmid loss for pJN51 remains the same as for pJN72, but, in this case, as the individual bacteria lose copies of the expression plasmid, the 1: 1 stoichiometry between the hok and sok mRNA levels is altered , and the production of Hok leads to cell death; therefore the only bacteria CVD 908-htrA (pJN51) that will grow rapidly will be those that retain all their expression plasmids. Therefore, it is not surprising that the quantification of the flow cytometry of GFPuv production for an induction population of CVD 908-htrA (pJN51) that grows healthy, now detects a population of fluorescent bacteria that exhibit fluorescence GFPuv levels equivalent that observed for CVD 908-htrA (pJN72) that grows under induction conditions (Figure 3C vs Figure 3B); however, the percentage of non-fluorescent bacteria was about half of the general population of organisms. The increased production of GFPuv in this population by induction of the Pompci-gfpuv cassette in CVD 908-htr (pJN51) again increases the metabolic tension in the living vector, but now the percentage of non-fluorescent bacteria reaches the few fluorescent bacteria, since many plasmids are presumably lost from the population and the bacteria are destroyed (Figure 2D). It would be expected that if a weaker promoter is used to control the expression of GFPuv, the general fluorescence of the population will decrease (compared to that observed for a similar population of organisms growing with a strong promoter expressing GFPuv under identical conditions), and the percentage of non-fluorescent bacteria will fall due to the general fall in the synthesis of GFPuv. However, as seen in Figures 3E-3H, the use of the weaker P0mPc3-gfpuv cassette does not significantly improve the viability of the induced bacteria carrying an elimination system, although the general expression of GFPuv is reduced. It is concluded that, in order to maximize the percentage of a population of live vectors that express the heterologous selection antigen, it is not sufficient to incorporate a PSK function in the given expression plasmid, it will be a marker of drug resistance, the asd system, an alternative ssb system, or the hok-sok elimination system. In addition to optimizing the number of copies and levels of expression, the segregation frequencies of these plasmids must also be improved to ensure that each daughter cell in the actively growing population will inherit at least one expression plasmid that will not be eliminated and removed from the population. Therefore, it is within the scope of the present invention to provide an expression plasmid which has a PSK function and which also has an optimized copy number and / or expression levels, coupled with incorporation of one or more SEG functions. 6. 5 Elimination system based on l Complementation It is also within the broad scope of the present invention to provide an expression plasmid comprising a deletion system based on l complementation, for example a system involving the deletion of the chromosomal ssb site of CVD908-htrA by homologous recombination and the trans-complementation of this lesion using the functional ssb that carries plasmids of multiple copies. To carry out such constructions the cloning of the relevant section of the S chromosome is required. Typhi encompasses the ssb gene and the flanking sequences, where specific deletions can be introduced for chromosomal mutagenesis. Since our original proposition, substantial progress has been made in the sequence of the Salmonella typhi chromosome at the Sanger Center in London. This Sanger Center is a genome research center, established in 1992 by the Wellcome Trust and the Medical Research Council, in order to increase our knowledge of genomes. Among other projects, the Sanger Center is studying the 4.5 Mb genome sequence of S. typhi, in collaboration with Gordon Douglas of the Department of Biochemistry, Imperial College, London. They study the sequence of strain CT18, a highly pathogenic, drug-resistant strain isolated from a typhoid patient at Cho Quan Hospital, Ho Chi Minh City, Vietnam. This cep is known to house the pVNIOO (a plasmid resistant multiple drugs, 130 kb) and a plasmid of 80 k cryptic. The genome is sequenced by a gun shot approach of the total genome using a 2 kb pU collection, generated locally from the chromosomal DNA provided by Prof. Dougan's lab. Each insert is always sequenced once from each end, the trigger phase is now complete, and termination is started. Currently there are 60 Contigs on 1 kb in the database, - a total of 5,106 Mb of the assembled sequence of 87,331 readings. Based on the updated results of October 4, 1999, we have identified Contig 343, which contains the S. typhi ssb site and the critical flanking sequences within a region of 205,199 bp. We have designated the sizes 1 and 4 (listed below) to be amplified by a PCR of a fragment of 3535 bp of S. typhi chromosome in which the ssb site is flanked by 1.5 kb of chromosomal sequence; This flank symmetry s requires for the optimal cross frequencies to introduce the sacB-neo cassette counter-selectable replace ssb. Using the previously presented methodology, we will use sizes 1 and 2 for the engineering of an Eco Rl - Xme I cassette of 1.5 kb, 5 '-proximal, upstream of ssb. The sizing 3 and 4 will be used to generate the Xma I - Eco Rl cassette, 1.5 kb, 3 'distal, current below ssb; both 1.5 kb cassettes will be ligated together, which form the 3 kb Eco Rl fragment, which contains the unique Xma site exactly in the cassette half. The sacB-neo cassette can now be easily inserted into the Xma I site, to complete the construction of the mutagenesis cassette to be inserted in the pCO (previously described in our first presentation). The required complementary ssb-1 cassette will be constructed using sizes 5 and 6 as a Nhe I cassette, for the replacement of drug-resistant markers within the Sba I-Spe I cassette of pGEN 211, pGEN 222, pGEN 206 or any other further version of the expression plasmids here detailed.

SIZING 1: 5"- gaattcGCGCGCTTCGGGATTCAGTCGCGTTCCTTCACA GCTGGCGCAGGGGCGATTACTGATGAA - 3 'SIZING 2: 5' - cccggGAGTCTCCTGAATACGTTTCATAAATAGTGTAA ACGCGTGAGTGTACCATTTCCACGTAGC - 3 'APRESTO 3: 5' - cccggGTAAAAAACTCAAAGCGTTATTTGCATTTTCGC TATAGTTCTCGTCTGCTGAAATGCCTGGTGT - 3 'APRESTO May 4' - gaattcCATTTCTATCAATAAATTACTATTAGTTTTGTCT TCTAACCAAGCCTCTATTTTATGAGTATCCTCTTCAG - 3 'APRESTO 5: 5" - gctagcATGGCCAGCAGAGGCGTAAACAAGGTGATTCT CGTTGGTAATCTGGGCCAGGACCCGGAAGTACGC - 3 'APRESTO 6: "" d' - gctagcTCAGAACGGAATGTCGTCGTCAAAATCCATTG GCGGTTCGTTAGACGGCGCTGGCGCG - 3 ' 6. 6 Stability of Expression Plasmids in l Absence of selection In order to develop a non-catalytic plasmid maintenance system, to increase the stability of multiple copy expression plasmids, which encode foreign antigens within CVD908-htrA, they initiated experiments to monitor the stabilization of plasmids, quantifying the expression of GFPuv by flow cytometry, when the strains are passed in the absence of antibiotic selection. These experiments were designed to address 3 fundamental questions: 1) What is the effect of the level of induction of ompCl on the stability of plasmids, which encode a heterologous antigen, such as GFPuv? 2) What is the effect of the number of copies on the stability of the plasmids that GFPuv expresses? 3) How do the maintenance functions of hok-sok, pair, and parA affect the retention of plasmids, both with individual components and synergistically? The initial experiments of flow cytometry were carried out where the replicons carried by CVD 908-htrA with any origin of response oriEl, oril5A or orilOl. It was easily determined that the replicone bearing the origins of May number of copies is very unstable, even when the strains grow in the presence of antibiotic selection. The results of flow cytometry indicated that even when cultured in the presence of carbenicillin, the percentage of bacterial populations no longer expresses detectable GFPuv ranging from approximately 50% of pGEN71 (which led to hok-sok) and pGEN84 (hok-sok + pair) at 62% for pGEN211 (hok-sok + pair + parA). Since replicons carrying oriin origin clearly do not allow for optimal synthesis of the heterologous GFPuv test antigen within the majority of a growing population of live vectore bacteria, this series of expression plasmids is not further examined.

The expression plasmids carrying CVD 908-htr with an origin of oril5A were then examined. Strains s inoculated in 25 ml cultures of IX LB + DHB (if antibiotic selection), containing 50 mM, 150 mM or 300 m NaCl. The cultures were incubated for 24 h at 37 ° C / 250 rpm, diluted 1: 1000 in a fresh medium of identical osmolarity and incubated for another 24 hours, samples from all the cultures were analyzed for the synthesis levels of GFPuv by cytometry flow. The results for the first passage in the absence of the selection are listed in Table 6, and the histograms representing these data are shown in Figure 8. Table 6 shows the stability within the 908-htrA CV of the oril5A replicons. , which contain the maintenance systems of increasingly complex plasmids, which grow without selection and in the presence of increasing osmolarity1. 1 These data are represented as histograms in Figure 8 2 All strains were marked with strips of frozen master material on 2x L agar supplemented with DHB and 50 μg / ml carbencillin, and incubated for 36 hours at 30 ° C. grouped in 300 μl of 1X LB broth supplemented with DHB, d which 25 μl was inoculated in 25 ml of 1X LB broth, containing DHB and 50 mM, 150 mM 300 mM NaCl; the cultures were incubated at 37 ° C, 250 rpm, for 24 hours. For the results presented in this table, the bacteria then formed globules, resuspended in 1 ml of PBS, pH 7.4, and then diluted 1: 1000 in PBS for flow cytometric analysis In general as the osmolarity increases and the induction of Pompci rises, the percentage of the population the average level of fluorescence intensity increased as expected. For example, in the presence of 50 mM d NaCl, 80.5% of a population of CVD 908-htrA (pGEN121) expresses GFPuv with an average fluorescence intensity of 53.3. As the concentration of NaCl increases to 300 mM, the percentage of GFPuv expressed by the population drops to 56.7%; however, the average intensity of fluorescence s rises to 105.3. NeverthelessIt is remarkable that strains that carry pGEN 222 with a complete plasmid maintenance system (ie hok-sok + pair + parA), the percentage d the population expressing the homologous antigen remains at approximately 95%, while the intensity Fluorescence mean increases from 52.1 (50 mM NaCl) to 89.2 (300 m NaCl). It is noted that in the further passage of these strains for an additional 24 hours, in the absence of selection of antibiotics, less than 5% of bacteria continue to express functional GFPuv. The bands of these cultures on the solid medium, before the analysis of the flow, indicate that the non-fluorescent bacteria remains viable, but is sensitive to the selection of antibiotics. When n fluorescent bacteria are sorted and plated, they confirm that they are sensitive to antibiotics and not fluorescent when irradiated with ultraviolet light, which indicates the loss of resident plasmids. A passage experiment involving expression plasmids carrying CVD 908-htrA with an orilO origin detected that there is no significant loss of GFPuv expression after passage of strains for 48 hours if selection, regardless of osmolarity. Therefore, the strains were passed in a separate experiment for 96 hors (ie 4 x 24 hours) in the presence of 50, 150 or 300 mM NaCl. The populations were analyzed by flow cytometry after 3 and 4 passages and the results are presented in Table 7. TABLE 7 shows the stability within CV 908-htrA of the orilOl replicons, which contain d plasmid maintenance systems. Increasing complexity, which grows without selection and in the presence of the growing osmolaride. 1 All strains were marked with strips of frozen master material on 2x L agar supplemented with DHB and 50 μg / ml carbenicillin, and incubated for 36 hours at 30 ° The isolated colonies were grouped in 300 μl of 1X LB broth, supplemented with DHB from which 25 μl were inoculated in 25 ml of 1X LB broth containing DHB and 50 mM, 15 mM or 300 mM NaCl; the cultures were incubated at 37 ° C, 250 rpm, for 24 hours (defined here as passage # 1). For passage # 2, 25 μl of passage # 1 was inoculated into 25 ml (ie 1: 1000 dilution) of an identical medium and incubated at 37 ° C, 250 rpm, for 24 hours without selection. Passes 3 and 4 were carried out identically, after the next passage was established and the remaining bacteria were formed and pellets were resuspended in 1 ml of PBS, pH 7.4 and then diluted 1: 1000 in PB for analysis by flow cytometry. ND = not done The live vectors that carry the replicons if stabilize orilOl, finally lost the ability to synthesize the heterologous antigen after 96 hours. For example, after growth of 96 hours in the presence of 50 mM NaCl, only 10.9% of CVD-htrA (pGEN132) expressed CFPuv and fluoresced. As the concentration of NaCl in the medium increased to 150 mM, fluorescence was detected in only 7.6% of the population; Interestingly, at 300 mM NaCl, the percentage recovered to 51.3% of fluorescent bacteria. Notably, the CVD 908-htrA carrying either d pGEN142 (hok.-sok) or pGEN206 (hok-sok + parA) siresi retained from GFPuv is greater than 95% of the population after 3 passages (72 hours), regardless of l osmolarity (see Table 7). The fluorescence percentage of CVD 908-htrA (pGEN142) remained almost this level after 4 passages (96 hours) while it decreased slightly for CVD 908-htrA (pGEN206). Taken together, these data show that the number of copies is reduced, the apparent stability of the resident plasmids and the benefit of a living vector pair synthesizing a heterologous antigen, such as GFPuv increases as the plasmid maintenance systems accumulate within a Given plasmid, the apparent stability and the synthesis of the antigen are also increased. Furthermore, since the induction of Pompci and the concomitant production of the heterologous antigen increases, the percentage of a population that remains capable of synthesizing the antigen can be drastically reduced. 6. 7 Bacterial Strains and Culture Conditions All constructs of plasmids recovered in strain DH5a or DH5aF'IQ (Gibco BRL) d Escherichia coli The construction of the hok-sok gene cassette used the pRl model of the DNA isolated from the strain of E. col J53 (pR1). a generous gift from James B. Kaper. The live vector of CVD 908-htrA from S. typhi is an auxotrophic derivative of the Ty2 wild-type strain with deletions in aroC, aroD and htrA (Taket et al., 1997b). All strains used for the examination of plasmid stability grew in a medium supplemented with 2,3-dihydroxybenzoic acid (DHB) as previously described (Hone et al., 1991 Galen et al, 1997). When grown in the solid medium, the strains carrying the plasmid of CVD 908-htrA formed a strip of frozen master material (-70 ° C) on the agar 2 Luria.- Bertani containing (per liter) 20 g of Bact tryptone, 10 g of Bact-yeast extract and 3 g of NaC (2X LB agar) plus carbecillin, at a concentration of 5 μg / ml. The plates were incubated at 30 ° C for 24-36 hours to obtain isolated colonies with a diameter of -2 mm; the strains were incubated at 30 ° C to minimize the toxicity of the expression of GFPuv in CVD 908-htrA. When grown in the liquid medium, the cultures were incubated at 37 ° C, 250 rpm, for 16 to 24 hours. To examine the osmotic induction of the ompC promoter (Pompe) within E. coli DH5 or CVD 908-htrA, the strains grew in the Bacto nutrient broth (Difco) containing DHB and NaCl sucrose; the cultures were supplemented either with 50 μg / ml of carbenicillin or with increasing concentrations of kanamycin, where the cassettes of Pompc -phA-2 were examined. For the quantification of GFPuv synthesis using flow cytometry, 6-8 colonies isolated from the master material in strips on 2X LB agar, as before, were inoculated into 25 ml of IX LB broth supplemented with 50 μg / ml of carbenicillin where desired and NaCl at increasing concentrations, to increase the induction of the ompC promoters. The cultures were incubated at 37 ° C, 250 rpm for 16 24 hours, prior to the formation of bacterial globules for flow cytometry, as described above. 6. 8 Molecular Genetic Techniques Standard techniques were used for the construction of the plasmids represented here (Sambrook et al., 1989). Unless otherwise noted, the native DNA polymerase (Gibco BRL) was used in the polymerase chain reactions (PCR). S. typhi was prepared by electroporation of the recombinant plasmids after harvesting from Miller Miller's LB (Gibco BRL) supplemented with DHB; After forming bacterial globules, the cells were washed three times with a sterilized distilled water culture volume and resuspended in sterilized distilled water to a final volume of 1/100 of the original culture volume. The electroporation of the strains was carried out in a Gene Pulser apparatus (Bio-Rad) adjusted to 2.5 kV, 200 and 25 μF. Following electroporation, the bacteria were repaired using the SOC medium and incubating at 37 ° C, 250 rpm, for 45 minutes; the bacteria were then plated onto the IX LB medium containing DHB plus 50 μg / ml of carbenicillin and incubated at 30 ° C for 24 hours. The isolated colonies were then swabbed in 2X L supplemented and incubated at 30 ° C for 16 hours. The frozen master material was prepared by collecting bacteria in the SOC medium without further supplementation and freezing 70 ° C. 6.9 Construction of Expression Vectors The expression vectors listed in the following Table 8 were prepared in the course of recent work. 6. 9.1 Construction of pJNl and pJN2 The expression plasmids, constructed from these studies, were composed of 3 basic cassettes, which encoded 1) the expression of a heterologous antigen, 2) an origin plasmid response, and 3) the selection and maintenance d functions. To achieve this, a basic replicon was built in which these cassettes were separated by unique restriction sites. The sizes used in the construction of the plasmid cassettes are indicated in the following Table 9: (Continuation) Relevant restriction sites are underlined and referenced in the text; ribosome binding sites and start codons are designated in cursive letters. 2 Refers to the sequence within the coding cord of a given gene, to which the sizing and homologue. 3 Refers to the sequence within the non-coding cord of a given gene, in which the sizing and homologue.

The pTETnir15 (see Table 8: Oxer et al., 1991) was redesigned so that the origin of the response and the gene are separated by a single Spel site. For this purpose, the oriEl cassette was synthesized by PCR using the Vent polymerase with sizes 1 and 2 and pCVD315 (Galen et al., 1990) as the model. The resulting 735 bp fragment designed the Spel and Bg / II 5 'sites-proximal to the transcript that controls the RNAII promoter and a 675-base Avrll designed site from these sites. A separate PCR reaction was carried out using the sizes 3 and 4 to create a blast cassette of 1234 bp, containing a site designed Xbal 5 '-proximal to the original EcoRI site. The products of these two PCR reactions were gel purified and used in an overlap PCR with the 1 4 sizes to deliver a final 1916 bp oriEl-bla fragment, which self-ligated to create pJN1. The Pniris toxC fragment from pTETnirl5 was cut as an Eco RI (partial digest). Aval fragment, in which the term Aval was polished, and inserted into the multiple cloning region of pSHlld (Brosius, 1989) split with Eco Rl and Stut; this cassette was then cut again as an Eco Rl (partial digestion) - Avrll fragment and was inserted in pJNl unfolded with Eco R - Avrl, creating pJN2 (see Table 8). 6. 9.2 Construction of pGFPo pC To facilitate the categorization of an osmotically regulated / d allele of Escherichia coli, an aphaA-2 cassette was constructed, which codes for resistance to the aminoglycosides of neomycin and kanamycin (Shaw et al., 1993). A polymerase chain reaction (PCR) was carried out using sizes 5 and 6 with model pIB279 (Blomfield et al., 1991) to generate a product of 1044 bp, of which a fragment of Bg / II-Nhel aphA-2 of 903 bp without promoter, was unfolded for the replacement of a cassette Bg / II-Nhel toxC, which codes for fragment C of tetanus toxin in pTETnirl5. The anaerobically regulated Pnirisr promoter was replaced with the 459 bp EcoRI-Bg / lI Pome allele, constructed using sizes 7 and 8 with the chromosomal DNA model of E. coli DH5a, to create pKompC. After confirming Pompe's osmotic induction by examining the increase in kanamycin resistance with increasing osmolarity, the aphA-2 cassette was then replaced with gfpuv encoding a codon-optimized, prokaryotic GFP allele (Clontech; Crameri et al., 1996). The gfpuv gene was recovered by PCR using the 9 and 10 primers with the model pGFPuv to generate a 751 bp / lI-Nhel fragment, which was inserted into pKompC, to generate the pGFPompC. The colonies were classified by the functional GFPuv. and the brightest colonies were then examined by fluorescence induction with increasing concentrations of NaCl. A Pompe-gfpuv cassette was unfolded from pGFPompCl as an EcoRI-Nhel fragment, and inserted into a derivative of pJN2 unfolded with EcoRI-Nhel to create pJJ4. 6. 9.3 Construction of pNRBl, pGEN2, pGEN3 and pGEN 4 Since it is intended that a number of copies n be influenced by the transcription originating from promoters outside the origin of response, it is necessary to ensure that each response cassette is flanked at both ends by the transcription terminators. Because these cassettes of origin and pJN2 antigen are separated by means of the trpA terminator, it is only necessary to insert an additional terminator between the cassettes of orig and bla. To facilitate the construction of additional plasmids afterwards, a tetA-TlT2 cassette was created. pYA292 (Galán et al., 1990) was first split with Hindi and Bg / ll the T1T2 terminator fragment was polished and inserted into the Smal site of the multiple cloning region KS (Stratagene) of pBluescript; when the appropriate orientation was identified, this cassette was resected as a BamHl -PstI fragment and inserted into pIB307 (Blomfield et al., 1991), unfolded with BarmHI-Pstl, which creates pJG14. It was then determined by the sequence analysis that the cassette has undergone a deletion of approximately 100 bp, removing half of the T2 terminator. Using pBR322 as a model, sizes 11 and 12 were used to synthesize a 1291 bp fragment, tetA Bg / II. This fragment of tetA Bg / ll was then inserted into the BamH1 site of pJG14, so that transcription of the tetA gene was terminated at the T1T2 terminator, creating pJG14tetA. Finally, this cassette tetA-TlT2 was unfolded from pJG14tetA as an EcoRI-PstI fragment in which the PstI site has been removed by polishing; the resulting fragment was inserted into pJJ4, unfolded with Spel, polished, and re-split with EcoRI to replace the bla casette and create pNRBl. The elimination function after the non-catalytic segregation to be incorporated in the plasmid maintenance systems of the expression plasmids described here was the hok-sok site of the R pRl factor of multidrug resistance. Initial attempts to recover the hok-sok site after PCR were not successful. Therefore it was necessary to use the overlap PCR to generate a cassette in which hok-sok were transcriptionally fused to a non-promoter gene tetA, such that the transcription originating from the hok promoter will continue in the tetA gene and will result in a transcript which encodes both Hok and resistance to tetracycline. Plasmid DNA pR1 was purified from E. coli J53 (pR1) in which pR1 encodes resistance to both carbenicillin and chloramphenicol. A 640 bp hok-sok fragment was synthesized using the sizes 13 and 14; a tetA fragment of 1245 bp, without promoter, was recovered in a separate PCR using the sizes 15 and 12 with pNRBl as the model. The products of these two PC reactions were then used in an overlap PCR, with the primers 12 and 13 supplying the final hok-sok-tetA fragment. This fragment was inserted as the EcoRI-Sphl fragment in pNRBl cleaved with EcoRI-Sphl, which regenerates the tetA gene and creates the pGENI. A set of 3 isogenic plasmids was then constructed, which differs only in the number of copies of which all additional expression plasmids will be derived. The origin Bg / ll -Avrll of response cassette of pGENl was replaced by a cassette Bg / II-Avrll oriEl of pJN2 to generate pGEN2. An oril5A response cassette was synthesized by PCR using primers 16 and 17 with pACYC184 as a model, to generate a 628 bp BamHl-Avrll fragment, which was inserted into pGEN2, unfolded with Bg / 11-AvrII to create pGEN3. Finally, an orilOl response cassette was synthesized by PCR using sizes 18 and 19 with the pSClOl model, which generates a 1949 bp BamHl-Avrll fragment, which was inserted into pGEN2, unfolded with Bg / II-Avrll to create the pGEN4. 6. 9.4 Construction of pJN5, pGEN51, pGEN91 and pGEN132 The main set of isogenic expression plasmids, to which the individual elements of a plasmid maintenance system are added in sequence, were composed of pGEN51 (containing oriEl), pGEN91 (containing oril5A), and pGEN132 (containing orilOl). The basic replicon from which these 3 plasmids were constructed was pJN5, which was assembled by unfolding the Pompc-gfpuv cartridge as the EcoRI-Nhel fragment of pGFPompC to replace the Pniris-toxC cassette of pJN2. The construction of pGEN51 was then achieved by removal of the response cassette of pGEN2 as a BamH1 fragment, and replacement of the response origin within pJN5 digested with Bg / II and BamH1, thus regenerating the gfpuv gene. The construction of pGEN91 and pGEN132 was constructed in an identical manner by cutting the source cassettes as BamH1 fragments of pGEN3 and pGEN4, respectively (see Figure 7, for the representation of isogenic expression plasmids based on pGEN91). 6. 9.5 Construction of pJN6, pGEN71, pGENlll and pGEN142 The hok-sok site was then inserted as a Xbal-SalI fragment in pJN5, unfolded with Xbal and Salí, regenerating the gfpv gene again, to create pJN6 (see Table 2) . The construction of pGEN71, pGENIII and pGEN142 was then carried out exactly as for pGEN51, pGEN91 pGEnl32, by the insertion in pJN6 of the cassettes of orige as BamHl fragments of pGEN2, pGEN3 and pGEN4 respectively. 6. 9. 6 Construction of pJN7, pGEN84 and pGEN121 The construction of plasmids of expressed oriEl and oril5A containing a plasmid maintenance system, composed of both the elimination system after segregation and at least one division d function, was first attempted using the pair function pSClOl. A BamHI-Bg / lI fragment of 377 bp was synthesized using the sizes 18 and 20 with the model DNA pSClOl; This fragment was inserted into pJN6 unfolded with Bg / II to create p N7. As in the previous constructions, the cassettes originating from pGEN2 and pGEN3 were then cut as BamH1 fragments and inserted into pJN7 digested with Bg / ll and BamH to create pGEN84 and pGEN121. 6. 9.7 Construction of pJN8, pGEN183, pGEN206, pGEN211 pGEN222 The final expression plasmids were constructed by introducing the active cleavage site for d pR1. As with hok-sok, initial attempts to recover the site for after the PCR were unsuccessful. It was necessary to use the overlap PCR to generate a cassette of aph. -parA, in which aph and parA were transcribed divergently separated by the Xbal and Xhol sites, to enable the subcloning of the parA site. A parA fragment of 1737 bp was synthesized using the sizes 21 and 22 with the model pR1; an aphA-2 fragment of 1076 bp was recovered in a separate PCR, using the 23 and 24 sizing with pIB279 as the model. The products of these two PCR reactions were then used in an overlap pCT with the sizes 22 and 23 to deliver the aphA2 fragment parA of 2743 bp. This fragment was inserted as the EcoRI-Spel fragment of 2703 in pJN6. The parA cassette was then cut again as an XhoI fragment and inserted again into pJN6 unfolded with XhoI, which regenerates the gppuv gene and creates p N8. The plasmids carrying a maintenance system composed of a hok-sok function of elimination after segregation and for, were constructed by cutting the oriEl and oril4A BamHl-Spel cassettes from pGEN51 and pGEN91, respectively, and inserting into pJN8 unfolded with BamHl and Spel to create pGEN183 and pGEN193, respectively. The plasmids containing the complete complement of hok-sok, pair and parA, as maintenance functions, were constructed by the insertion of cassettes of origin that contain pair as the BamHl-Spel cassettes of pGEN84, pGEN121 and pGEN132 in pJN8 unfolded with BamHl and Spel to create pGEN211, pGEN222 and pGEN208, respectively. 6. 10 Quantification of GFPuv and Maintenance of Plasmid The quantification of GFPuv and the maintenance of plasmid were analyzed by measuring the fluorescence of live vectors carrying plasmids, using a flow cytometry clarification system / Epics Elite ESP cell (Coulte), with the bacteria that excite the argon laser at 488 nm and the detected emissions at 525 nm, 25 ml of lXlb cultures that grow as described above and globules were formed, and the bacteria were resuspended in 1 ml of PBS. These cells were then diluted 1: 1000 in PBS before determination of viable counts and flow analysis. Forward versus lateral light disseminator, measured with logarithmic amplifiers, was used for entry into bacteria. A minimum of 50,000 events were acquired from each sample in the collection regime of approximately 3500 events per second. The mean fluorescence intensity for a given bacterial population was determined using the Epic Elite Software Analysis Package. Autofluorescence levels, determined using S. typhi strains without plasmids, CVD 908-htrA and E coli DH5a, were used to place markers that quantify the percentages of bacteria in a given population expressing GFPuv. 6. 11 Conclusions The general objective of the research presented in Sections 6.6 to 6.10 was to investigate the feasibility of developing a plasmid maintenance system for the stabilization of multiple-copy expression plasmids encoding foreign antigens in a live vector vaccine strain of S. typhi, without further modification of the chromosome. The maintenance of expression plasmids was increased by two independent levels. First, the dependence on the lethal-equilibrium maintenance systems involving the catalytic enzymes expressed from the plasmids of multiple copies was removed, this was accompanied by the incorporation in the expression plasmids of a system of elimination after segregation, based on the non-catalytic hok-sok plasmid addiction system of the pR1 factor of antibiotic resistance. At least one naturally occurring plasmid cleavage function was also introduced into these expression plasmids, to potentially eliminate the random segregation of such plasmids, thereby increasing their inheritance and stability.

Although these expression plasmids are ultimately intended to express immunogenic and protective antigens for delivery to the human immune system, GFPuv was selected as the test reportant antigen, because the quantification of mean fluorescence in a population of living vectors that grow it can be used as a measure of the stability of the resident plasmids within the living vector. All expression plasmids carry a cassette of identical antigen expression, with a Pompci allele, which controls transcription, and translation optimized by the incorporation of a consensus ribosome binding site. Because no catalytic activity is associated with the fluorescence of GFPuv, the level of fluorescence intensity as measured by flow cytometry within individual bacteria can be directly correlated with the gene dose and the number of copies. In addition, the use of the ompC promoter, osmotically regulated, allowed an evaluation of plasmid stability and the viability of the live vector, as the osmolarity induced by higher levels of GFPuv synthesis and presumably higher levels of metabolic stress increases. the vector alive. As seen in Table 2, we confirmed that the Pompcir allele designed for these studies was responsive to increased osmolarity, when the impulse expression of a resistance gene aph-2, resistance to less than 5 μg / ml kanamycin it was observed in the absence of the osmotic pressure, but the resistance increased to more than 800 μg / ml and the presence of 300 mM NaCl. It is surprising that, even though the Pompci allele was designed from the chromosomal site of E. coli, it appeared to function more efficiently in S. typhi. The non-induced level of GFPuv expression was the same for tant DH5a as CVD 908-htrA (mean fluorescence intensity d 4.45 vs. 5.37, respectively, Table 3). However, the synthesis of GFPuv increased 70% in DH5a after l induction, but increased more than 300% in CVD 908-htr (mean fluorescence intensity of 7.69 vs 23.4 respectively). This effect was not limited to the Pompc allele but was also remarkable when the Pompc3 was used (Table 3). These data are not in accordance with the recent observations of Martínez-Flores et al (1999) which reported that the genetic functions of coli ompC-lacZ constitutively expressed within S. typhi, and that this constitutive level of expression was comparable to levels induced within E. coli. Although we have identified a definite site of point mutations at the 3 'terminal of our E. coli Pompci allele, which may explain the osmotically controlled behavior, within S. typh CVD 908-htrA, such mutations were not identified within PomPc3 / who also respond to osmolariad within CV 908-htrA. It should be noted, however, that the genetic mergers studied by Martínez-Flores et al. Involved 1,150 bp of E. coli 5 '-ompC the control region upstream, while the Pompe alleles constructed here imply only 469 bp of the control region 5 '-proximal of ompC. Regardless of this discrepancy, it is supported that the highest levels of regulated heterologous gene expression were observed within the live vector vaccine strain of attenuated S. typhi.

The contributions of the various maintenance systems to the stability of the plasmids within the CVD 908-htrA, which grows in the absence of antibiotic selection, were then examined. No combination of maintenance functions can stabilize plasmids that contain oriEl response sources; in fact these constructions are difficult to propagate, even in the presence of an antibiotic. These observations raise doubts about the additional use of plasmids with higher copy numbers to optimize the expression of heterologous antigens within the cytoplasm of live vectors based on S. typhi, a strategy that, until now, has been followed by others groups that investigate Salmonella as living vectors (Covone et al., 1998).

The incorporation of plasmid maintenance systems in plasmids that carry an oril5A response source was more stimulated. When live vectors carrying such plasmids pass without selection for 24 hours at 37 ° C, the effects of various combinations of maintenance functions become apparent. In the absence of maintenance functions, the oril5A replicon pGEN91 was lost from more than 90% of the population, regardless of the level of induction of Pompc? (see Table 6 and Figure 8). With the incorporation of the elimination site after the hok-sok segregation in pGENIII, the percentage of bacteria expressing GFPuv tripled under all induction conditions, which make up the observations of others that the hok-sok site increases the stability of the oril5A replicons (Gerdes et al., 1985; Gerdes, 1988; Gerdes et al, 1997b). However, it was still noted that regardless of the induction conditions, more than 50% of the bacterial population is no longer fluorescent. Since it was confirmed that at least a portion of this population which is not fluorescent is still viable and lacks drug resistance, these data confirm the previous reports (Gerdes et al., 1986; Wu and Wood, 194; et al, 1997) that the presence of an elimination system after hok-sok segregation is insufficient by itself to ensure that viable bacteria without plasmids do not arise in a growing population.

A possible mechanism that allows the escape of the hok-sok influence involves spontaneous point mutations that arise within the open reading frame Hok lethal, which can inactivate Hok conformationally and thus allow the loss of plasmid to occur without being lethal. This point emphasizes the requirement of multiple mechanisms to increase the stability of resident plasmids within growing bacteria; when a maintenance function becomes inactive, the probability of other independent functions that become simultaneously inactivated are small, fading away. Indeed, such redundancy in maintenance functions are generally within the low copy number plasmids, which occur naturally (Nordastrom and Austin, 1989). For example, the sex F factor of Escherichia coli contains an active division function (sop) and two elimination systems (ccd and flm) (Loh et al., 1988; Golub and Panzer, 1988; Van Melderen et al. , 1994, Niki and Hiraga, 1997). Similarly, the pRl plasmid of drug resistance contains the function of active division (sop) and two elimination systems (ccd and flm) (Loh et al, 1988, Goulb and Panzer, 1988, Van Meideren et al., 1994, Niki and Hiraga, 1997).

Similarly, plasmid pRl drug resistance contains the active placement function parA, as does the elimination system after hok-sok segregation; in addition, it still has another kis-kud elimination system, recently defined (Bravo et al, 1987, Brava et al, 1998, Ruiz, -Echevarría et al, 1995). We demonstrate, in the work reported here, that the insertion in the oril5A replicons of multiple copies into a more complete maintenance system, that both the system after segregation, as well as the two division functions, drastically improve the stability of the expression plasmids in the absence of said selection, independently of the induction conditions for the expression of the heterologous antigen. However, after the passage without such selection for 48 hours, the plasmids were finally lost in the bacterial population, due to the escape of Hok lethality. This problem was recently addressed by Pecota et al (1997), who reported that the incorporation of double elimination systems significantly improves the stability of the plasmid when compared to the use of the hok-sok system alone.; no division functions are present in these plasmids. Perhaps the inclusion of the kis-kid elimination system, to more fully represent the complement of the stability functions of the pR1, may be required for the optimal stability of the expression plasmids of larger copies within the living vectors of S typhi since the phd-doc PSK cassettes have been recently constructed, we also examined the compatibility of this PSK function in our expression plasmids pGEN211 pGEN222 and pGEN206.

A comparison of the strains bearing pGEN12 (an oril5A replicon bearing hok-sok + pair; -15 copies by chromosomal equivalent) with plasmid pGEN142 with much smaller number of copies (an oril91 replicon carrying hok sok + pair, ~ 5 copies per chromosomal equivalent) shows that under the maximum induction conditions of Pompe, with 300 m NaCl, 57% of the population of CVD 908-htrA (pGEN121), passed for only 24 hours without selection, fluorescence with a average intensity of 105.3; for a population of CVD 908 htrA (pGEN142), it went through 96 hours without selection, under identical induction conditions, 94% of the bacteria analyzed by flow cytometry still maintain an average fluorescence intensity of 47.7. Based on such results with GFPuv as the test antigen, it can be speculated that an optimal level of the heterologous antigen presented by a live vector vaccine based on attenuated S. typhi, to the human immune system, can be achieved by decreasing the number of copies of Resident expression plasmids d to perhaps 5 copies per chromosomal equivalent. The efficiency of producing an immune response directed against a heterologous antigen will depend, in part, on the ability of the living vector to present such antigens to the immune system. The ability of a living vector to present antigens, in turn, will depend on the stability of the plasmids of expression of multiple copies that encode the heterologous antigens. Our results demonstrate that the inclusion of a plasmid maintenance system within multiple expression plasmids, without manipulation of the living vector, increases the stability of such expression plasmids. However, the presence of multiple-copy plasmids may also have an influence on the metabolic adaptation of the living vector. This is important because some foreign antigens of interest will exert a detrimental effect on the living vector.

While we are not trying to be united to this theory, we conclude that a significant metabolic load is placed in CVD 908-htrA, which carries a plasmid of expression of multiple copies; as the number of copies and / or the level of gene expression increases, the metabolic load also increases. Studies with E. coli have clearly established that bacteria carrying plasmids grow slower than non-plasmid bacteria (Boe et al., 1987; McDermott et al., 1993; Wu and Wood, 1994; Pectota et al., 1997; 1998). It has also been shown that as the number of copies increases, the growth rate of such strains decreases; similarly, as the induction of heterologous genes increases, the growth rate subsequently decreases (Wu and Woo, 1994, Pecota et al, 1997) Clearly, the loss of spontaneous plasmids will remove any metabolic load and allow bacteria without plasmids to grow more quickly that the population of bacteria that carry plasmids. In elegant studies, Wu and Wood (Wu and Wood, 1994), showed that strains of E. coli, which carry plasmids, plasmids maintained under conditions where the expression of the cloned gene was low for 100 hours when it happened in the absence of the selection; in contrast, under maximal induction conditions, the complete loss of plasmids occurred within 10 hors. Interestingly, when the hok-sok site is inserted into these expression plasmids, plasmids are maintained for 300 hours, under non-induced conditions, and 30 hours under induced conditions, such shifting in antigenic expression within a population of a bacterium of Live vector, would be expected to reduce the efficiency of the stimulus of any specific immune response to the foreign antigen. Our analyzes led us to conclude that the goal for a live vector vaccine based on S. effective multivalent typhi, is to optimize viability using smaller, stabilized copy number expression vectors capable of expressing high levels of heterologous antigen in response to the environmental signal, which will probably be found in vivo after the vaccine organisms have reached an appropriate ecological niche. We are currently testing this strategy, using an intranasal murine model to examine the immunogenicity of the C fragment of tetanus toxin expressed within CVD 908-htrA of our expression vectors pGEN211 (oriEl), pGEN222 (oril5A) and pGEN206 (orilOl) all which carry identical plasmid maintenance systems and differ only in the number of copies. The work presented here enables the development of simple oral doses of live vector vaccines based on S. typhi, capable of inducing protective immune responses against the multiple unrelated human pathogens.

List of sequences < 110 > Galßn, 'James < 120 > Plasmid maintenance system for the antigen < 130 > Request from James Galen < 140 > 09 / 204,117 < 141 > 1998-12-02 < 160 > 3 < 170 > Patßntln Ver. 2.0 < 210 > 1 < 211 > 4196 < 212 > DNA < 213 > . Artificial sequence < 220 > < 223 > Nucleotide sequence 1-4196 pGEN2 < 220 > < 223 > . Description of the artificial sequence: sequence 1-4196 nucledtide pGEN2 < 400 > i gaattctgtg gtagcacaga ataatgaaaa gtgtgtaaag aagggtaaaa aaaaccgaat 60 gcgaggcatc cggttgaaat aggggtaaac agacattcag aaatgaatga cggtaataaa 120 taaagttaat gatgatagcg ggagttattc tagttgcgag tgaaggtttt gttttgacat 180 tcagtgctgt caaatactta agaataagtt attgatttta accttgaatt attattgctt 240 gatgttaggt gcttacttcg ccattccgca ataatcttaa aaagttccct tgcatttaca 300 tctatagcga ttttgaaaca taaatgaaac atcttaaaag ttttagtatc atattcgtgt 360 tggattattc tgcatttttg gggagaatgg acttgccgac gggttaatca tgattaatga 420 gtatgcagtg gcataaaaaa gcaaataaag gcatataaca gatcgatctt aaacatccac 480 aggaggatat ctgatgagta aaggagaa? to acttttcact ggagttgtcc caattcttgt 540 tgaattagat ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga 600 tgcaacatac ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc 660 cttgtcacta atggccaaca tggtgttcaa ctttctctta gttatccgga tgcttttccc 720 tcatatgaaa cggcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaacg 780 cactatatct ttcaaagatg acgggaacta caagacgcgt 7gctgaagtca agtttgaagg 840 tgataccctt gttaatc tcgagttaaa gta aggtattga £ tttaaagaag atggaaacat 900 tctcggacac aaactcgagt acaactataa ctcacacaat gtatacatca cggcagacaa 950 acaaaagaat ggaatcaaag ctaacttcaa aattcgccac aacattgaag atggatccgt 1020 tcaactagca gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc 1080 agacaaccat tacctgtcga cacaatctgc cctttcgaaa gatcccaacg aaaagc? tga 1140 ccacatggtc cttcttgagt ttgtaactgc tgctgggatt acacatggca tggatgagct 1200 ctacaaataa tgagctagcc cgcctaatga gcgggctttt ttttctcggc ctagggccag 1260 ggaaccgcaa caaaaggcca aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 1320 atcacaaaaa cctgacgagc tcgacgctca ag cagaggt ggcgaaaccc gacaggacta 1380 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 1440 ccgcttaccg gatacctg c cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 1500 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 1560 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 1620 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 1680 aggtatgtag gcggtgctac ag agttcttg aagtggtggc ctaactacgg ctacactaga 1740 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 1800 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gt tttttgt ttgcaagcag 1860 gcagaaaaaa cagattacgc aggatctcaa gaagatcctt tgatcttttc tacggggtct 1920 gac ctc gt agatctaaaa cactaggccc aagagtttgt agaaacgcaa aaaggccarc 1980 cgtcaggatg gccttctgct taatttgatg cctggcagtt tatggcgggc gtcctgcccg 2040 ccaccctccg ggccgttgct tcgcaacgtt caaatccgct cccggcggat ttgtcctact 2100 caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc 2160 ctttcgtttt atttgatgcc tggcagttcc ctactc cgc atggggagac cccacactac 2220 catcggcgct acggcgtttc acttctgagt tcggcatggg gtcaggtggg accaccgcgc 2280 tactgccgcc aggcaaattc tgttttatca gaccgcttct gcgttctgat ttaatctgta 2340 tcaggctgaa aatcttctct catccgccaa aacagccaag ctggatcccc gatcttatca 2400 ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga caaggtatag 2460 ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg cataaatcgc 2520 cgtgacgatc agcgg-ccag tgatcgaa gt taggctggta agagccgcga gcgatccttg 2580 aagctg ccc tgatggtcgt catctacctg cctggacagc atggcctgca acgcgggcat 2640 cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc ctcgcgtcgc 2700 gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt 2760 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat 2820 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc 2880 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt 2940 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa 3000 ggctctcaag ggcatcggtc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc 3060 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat 3120 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct 3130 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc 3240 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcca 3300 gtggtcgcca caggacgggt tgatcgcgta gtcgatagtg gctccaacta gcgaagcgag 3360 cggcggccaa caggactggg agcggtcgga cagtgctccg agaacgggtg cgcatagaaa 3420 ttgcatcaac gcatatagcg ctagcagcac gccatagtga ctggcgatgc tgtcggaatg 3480 gacgatatcc cgcaagaggc ccggcagtac cggcataacc aagcctatgc czacagcatc 3540 cagggtgacg gtgccgagga tgacgatgag cgcattgtta gatttcattt ttttttcctc 3600 cttattttct agacaacatc agcaaggaga aaggggctac cggcgaacca gcagcccctt 3660 tataaaggcg cttcagtagt cagaccagca taagtcccga aaaggcgggc ctgcgcccgc 3720 ctccaggttg ctacttaccg gattcgtaag ccatgaaagc cgccacctcc ctgtgtccgt 3780 ctctgtaacg aatctcgcac agcgat TTTC gtgtcagata agtgaatatc aacagtgtga 3840 gacacacgat caacacacac cagacaaggg aacttcgtgg tagtf-catg gcctcttct 3900 agcgcggtaa ccttgcgcaa gaggctatcc tgatgtggac tagacatagg gatgcc? cgt 3960 ggrggttaat gaaaattaac ttactacggg gctatcttct ttctgccaca caacacggca 4020 acaaaccacc ttcacgtcat gaggcagaaa gcctcaa cg ccgggcacat catagcccat 4080 atacctgcac gctgaccaca ctcactttcc ctgaaaataa tccgctcatt cagaccgttc 4140 acgggaaatc cgtgtgattg ttgccgcatc acgctgcctc ccggagtttg tctcga 4196 <; 210 > 2 < 211 > 1197 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of . artificial sequence: Sequence 1201-3847 nucleotide pGEN4 (see figures 6a-e) < 400 > 2 ctacaaataa tgagctagcc cgcctaatga gcgggctttt ttttctcggc ctaggagata 60 cttaacaggg aagtgagagg gccgcggcaa agccgttttt ccataggctc cgcccccctg 120 cgaaatctga acaagcatca cgctcaaatc agtggtggcg aaacccgaca ggactataaa 180 gataccaggc gtttccccct ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt 240 ttaccggtgt cattccgctg ttatggccgc gtttgtctca ttccacgcct gacactcagt 300 tccgggtagg cagttcgctc caagctggac tgtatgcacg aaccccccgt tcagtccgac 360 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc tgcaaaagca cggaaagaca 420 ccactggcag cagccactgg taattgattt agaggagtta gtcttgaagt catgcgccgg 480 actgaaagga ttaaggctaa caagttttgg tgactgcgct cctccaagcc agttacctcg 540 gttcaaagag ttggtagctc agagaacctt cgaaaaaccg ccctgcaagg cggttttttc 600 gttttcagag caagagatta cgcgcagacc aaaacgatct caagaagatc atcttattaa 660 tcagataaaa tatttctagg atctaaaaca ctaggcccaa gagtttgtag aaacgcaaaa 720 aggccatccg tcaggatggc cttctgctta atttgatgcc tggcagttta tggcgggcgt 780 cctgcccgcc accctccggg ccgttgcttc gcaacgttca aatccgctcc cggcggattt B40 gtcctactca ggagagcg tt caccgacaaa caacagataa aacgaaaggc ccagtctttc 900 gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgcat ggggagaccc 960 cacactacca tcggcgctac ggcgt- cac ctcrgagttc ggcatggggt caggtgggac 1020 caccgcgcta ctgccgccag gcaaattctg ttttatcaga ccgcttctgc gttctgattt 1080 aggctgaaaa aatctgtatc tcttctctca tccgccaaaa cagccaagct ggatccccga 1140 tcttatcagg tcgaggrggc ccggctccat gcaccgcgac gcaacgcggg gaggcag 1197 < 210 > 3 < 211 > 2647 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: Sequence 1201-3847 (see figures 6a-e) < 400 > 3 ctacaaataa tgagctagcc cgcctaatga gcgggctttt ttttctcggc ctaggtttca 60 ccrgttctat taggtgttac atgctgttca tctgttacat tgtcgatctg ttcatggtga 120 atgcaccaaa acagctttaa aactcgtaaa agctct? Atg tatctatctt ttttacaccg 180 ttttcatctg tgcatatgga cagttttccc tttgatatct aacggtgaac agttgttcta 240 cttttgtttg ttagtcttga tgcttcactg atagatacaa gagccataag aacctcagat 300 ccttccgtat ttagccagta tgttctctag tgtggttcgt tgtttttgcg tgagccatga 360 gaacgaacca ttgagatcat gcttactttg catgtcáctc aaaaattttg cctcaaaact 420 ggtgagctga atttttgcag ttaaagcatc gtgtagtgtt tttcttagtc cgttacgtag 480 gtaggaatct gatgtaatgg ttgttggtat tttgtcacca ttcattttta tctggttgtt 540 ctcaagttcg gttacgagat ccatttgtct atctagttca acttggaaaa tcaacgtatc 600 agtcgggcgg cctcgcttat caaccaccaa tttcatattg ctgtaagtgt ttaaatcttt 660 acttattggt ttcaaaaccc attggt ag ccttttaaac tcatggtagt tattttcaag 720 cattaacatg aacttaaatt catcaaggct aatctctata tttgccttgt gagttttctt 780 ttgtgttagt tcttttaata aatcctcata accactcata gagtatttgt tttcaaaaga 840 cttaacatgt tccagat tat attttatgaa tttttttaac tggaaaagat aaggcaatat 900 ctcttcacta aaaactaatt ctaatttttc gcttgagaac ttggcatagt ttgtccactg 960 aagcctttaa gaaaatctca ccaaaggatt cctgatttcc acagttctcg tcatcagctc 1020 ttagctaata tctggttgct attttcccta caccataagc ctgatgttca tcatctgagc 1080 gtattggtta taagtgaacg ataccgtccg ttctttcctt gtagggtttt caatcgtggg 1140 gttgagtagt gccacacagc ataaaattag cttggtttca tgctccgtta agtcatagcg 1200 actaatcgct agttcatttg ctttgaaaac aactaattca gacatacatc tcaattggtc 1260 taggtgattt taatcactat accaattgag atgggctagt caatgataat tactagtcct 1320 gttgtgggta tttcctttga tc gtaaatt ctgctagacc tttgctggaa aacttgtaaa 1380 ccctctgtaa ttctgctaga attccgctag acctttgtgt gttttttttg tttatattca 1440 atttatagaa agtggttata taaagaaaga ataaaaaaag ataaaaagaa tagatcccag 1500 ccctgtgtat aactcactac tttagtcagt tccgcagtat tacaaaagga tgtcgcaaac 1560 cctctacaaa gctgtttgct acagacctta aaaccctaaa ggcttaagta gcaccctcgc 1620 aagctcgggc aaatcgctga atattccttt tgtctccgac catcaggcac ctgagtcgct 1680 gtctttttcg tgacattcag ttcg ctgcgc tcacggctct ggcagtgaat gggggtaaat 1740 ggcactacag gcgcctttta tggattcatg caaggaaact acccataata caagaaaagc 18C0 ccgtcacggg cttctcaggg cgttttatgg cgggtctgct atgtggtgct atctgacttt 1860 ttgctgttca gcagttcctg ccctctgatt ttccagtctg accacttcgg attatcccgt 1920 gacaggt at tcagactggc taatgcaccc agtaaggcag cggtatcatc aacaggctta 1980 cccgtcttac tgtcaaccgg atctaaaaca ctaggcccaa gagtttgtag aaacgcaaaa 2040 aggccatccg tcaggatggc cttctgctta atttgatgcc tggcagtttta tggcgggcgt 2100 cctgcccgcc accctccggg ccgttgcttc gcaacgttca aatccgctcc cggcggattt 2160 ggagagcgtt gtccractca cáccgacaaa caacagataa aacgaaaggc ccagtctttc 2220 gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgcat ggggagaccc 2280 cacactacca tcggcgctac ggcgtttcac ttctgagttc ggcatggggt caggtgggac 2340 caccgcgcta ctgccgccag gcaaattctg ttttatcaga ccgcttctgc gttctgattt 2400 aatctgtatc aggctgaaaa tcttctc tccgccaaaa ca 2460 cagccaagct ggatccccga tcttatcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca 2520 aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca 2580 taaatcgccg tgacgatcag cggtccagtg atcgaagtta ggctggtaag agccgcgagc gatcctt 2640 2647

Claims (155)

1. CLAIMS 1. An expression cassette, which functions independently, comprising a sequence of nucleotides encoding: (a) a response source; and (b) a nucleotide sequence, encoding a plasmid maintenance system, comprising: (i) at least one elimination function after segregation; and (ii) at least one division function.
2. 2. The expression cassette, which functions independently, of claim 1, wherein the source of response is selected from the group consisting of: oriEl, orilOl, oril5A and its derivatives.
3. 3. The expression cassette, which functions independently, of claim 1, wherein the elimination function, after segregation, is selected from the group consisting of asd, ssb, phd-doc and hok-sok.
4. 4. The expression cassette, which functions independently, of claim 1, wherein the elimination function, after segregation, is a substantial homolog of a deletion function, after segregation, which occurs naturally.
5. 5. The expression cassette, which functions independently, of claim 1, wherein the division function comprises an active division function.
6. 6. The expression cassette, which functions independently, of claim 1, wherein the division function comprises a passive partitioning function.
7. 7. The expression cassette, which functions independently, of claim 1, wherein the division function is the even site of pSClOl.
8. 8. The independently functioning expression cassette of claim 1, wherein the function. of division includes the parA.
9. 9. The independently functioning expression cassette of claim 1, wherein the division function is a substantial homologue of a division function, which occurs naturally.
10. 10. An amplifiable plasmid replicon, comprising the independently functioning expression cassette of claim 1.
11. 11. The amplifiable plasmid replicon of claim 10, further comprising an expression cassette, which functions independently, which includes a nucleotide sequence encoding an antigen of interest, transcriptionally controlled by a promoter.
12. 12. The amplifiable plasmid replicon of claim 11, which has a copy number that can be controlled to vary from 0 copies per cell to more than 75 copies per cell, and where the antigen of interest is a regulated test antigen for expression of a bacterium, so that the inducible promoter is placed to control the expression of the nucleotide sequence, so that when the induction of the promoter is increased, increase the expression of the test antigen, and the metabolic load of the bacterium is also increased.
13. 13. A bacterial cell, comprising the amplifiable plasmid replicon of claim 12.
14. 14. The amplifiable plasmid replicon of claim 11, wherein the promoter is derived from the ompC promoter.
15. 15. The amplifiable plasmid replicon of claim 11, wherein the promoter is the ompC promoter.
16. 16. The amplifiable plasmid replicon of claim 11, wherein the promoter is the ompC promoter fragment of E. coli, extending from nucleotides +70 to -389.
17. 17. The amplifiable plasmid replicon of claim 11, wherein the promoter is a modified ompC promoter, characterized in that this modified ompC promoter exhibits higher regimes of osmotically regulated expression, relative to a corresponding ompC promoter without said dot mutations.
18. 18. The amplifiable plasmid replicon of claim 17, wherein the modified ompC promoter comprises a modified Bg / ll site.
19. 19. The amplifiable plasmid replicon of claim 17, wherein the modified ompC promoter is without a Bg / ll site.
20. 20. The amplifiable plasmid replicon of claim 18, wherein the modified ompC promoter comprises the following 5 'to 3' sequence, which starts with the modified Bg / ll site and ends with the ATG start codon: AGATCX1x2TAAX3CATCCACAGGAGGATATCTGATG, where : (a) X1 is selected from the group consisting of G, C and A; (b) X2 is an insert having 1 to 5 nucleotides; (c) X3 is selected from the group consisting of A, T, G and C.
21. 21. The amplifiable plasmid replicon of claim 18, wherein X1 is G.
22. 22. The amplifiable plasmid replicon of claim 18, wherein X2 has from 1 to 4 nucleotides.
23. 23. The amplifiable plasmid replicon of claim 18, wherein X2 has 4 nucleotides.
24. 24. The amplifiable plasmid replicon of claim 18, wherein X2 has 4 nucleotides, independently selected from the group consisting of A, T, and C.
25. 25. The amplifiable plasmid replicon of claim 18, wherein X2 comprises a nucleotide or a nucleotide sequence, selected from the group consisting of ATCT; ATC; AT: TCT; CT; TC; TO; T; C; and T.
26. 26. The amplifiable plasmid replicon of claim 18, wherein X2 is selected from the group consisting of ATCT; ATC; AT; TCT; CT; TC; TO; T; and T.
27. 27. The amplifiable plasmid replicon of claim 18, wherein X2 is ATCT.
28. 28. The amplifiable plasmid replicon of claim 18, wherein X3 is A.
29. 29. The amplifiable plasmid replicon of claim 11, wherein the antigen of interest comprises the green fluorescent protein or a functional equivalent thereof.
30. 30. The amplifiable plasmid replicon of claim 11, wherein the antigen of interest comprises a detoxified Shiga toxin and / or a substantial homologue thereof.
31. 31. The amplifiable plasmid replicon of claim 11, wherein the antigen of interest comprises an antigen of Shiga toxin 2, selected from the group comprising: the pentamers of the toxin subunit 2B of Shiga and Shiga toxin 2, genetically detoxified (Stx 2).
32. 32. The amplifiable plasmid replicon of claim 30, wherein the gene encoding detoxified Shiga toxin 2 has modified segments, selected from the group consisting of: (797) ACÁ GCA GAC GCG TTA (811) (902) CTG AAC CTA GGG CGA (916) (1345) - GAA TTC GCG ACC AGT (1359); Y (1435) - GAA TCA GAT TCT GGA (1449)
33. 33. A bacterial cell, comprising the amplifiable plasmid replicon of claim 11.
34. 34. The amplifiable plasmid replicon of claim 10, further comprising an expression cassette, which functions independently, comprising a nucleotide sequence encoding a selectable marker.
35. 35. The amplifiable plasmid replicon of claim 34, wherein the selectable marker does not confer resistance to any antibiotic, which is ordinarily used in the medical treatment of humans.
36. 36. The amplifiable plasmid replicon of claim 34, wherein the selectable marker comprises the β-lactamase and / or a functional equivalent thereof.
37. 37. The amplifiable plasmid replicon of claim 34, wherein the nucleotide sequence encoding the resistance marker is selected from the group consisting of tetA, bla and functional equivalents thereof.
38. 38. A cell comprising the amplifiable plasmid replicon of claim 34.
39. 39. A bacterial cell, comprising the amplifiable plasmid replicon of claim 34.
40. 40. A vaccine of an attenuated bacterial vector, comprising a bacterial species containing a replicon, this replicon comprises: (a) a nucleotide sequence encoding an antigen of interest; and (b) a nucleotide sequence encoding a plasmid maintenance system
41. 41. The bacterial vector vaccine of claim 40, wherein the nucleotide sequence encoding the antigen of interest is contained in a genetic cassette, which functions independently.
42. 42. The bacterial vector vaccine of claim 50, wherein the sequence of nucleotides encoding the plasmid maintenance system is contained within genetic cassettes, which function independently.
43. 43. The attenuated bacterial live vector vaccine of claim 40, wherein the bacterial species is Salmonella typhi.
44. 44. The attenuated bacterial live vector vaccine of claim 40, wherein the replicon further comprises an ompC promoter, or a substantial homologue thereof.
45. 45. The attenuated bacterial live vector vaccine of claim 40, wherein the replicon further comprises a modified ompC promoter, which controls the expression of the antigen of interest, in which this modified ompC promoter exhibits higher osmotically controlled expression regimes, compared to a ompC promoter not modified.
46. 46. The attenuated bacterial live vector vaccine of claim 45, wherein the modified ompC promoter comprises a modified Bg / ll site.
47. 47. The attenuated bacterial live vector vaccine of claim 45, wherein the modified ompC promoter is without a complete Bg / ll site.
48. 48. The attenuated bacterial live vector vaccine of claim 46, wherein the modified ompC promoter comprises the following 5 'to 3' sequence, which starts with the modified Bg /? I site and ends at the ATG start codon: AGATCX1X2TAAX3CATCCACAGGAGGATATCTGATG, in which: (a) X1 is selected from the group consisting of G, C and A; (b) X2 is optionally present and is an insert having 1 to 5 nucleotides; (c) X3 is selected from the group consisting of A, T, G and C.
49. 49. The attenuated bacterial live vector vaccine of claim 48, wherein X1 is G.
50. 50. The attenuated bacterial live vector vaccine of claim 48, wherein X2 has from 1 to 4 nucleotides.
51. 51. The attenuated bacterial live vector vaccine of claim 48, wherein X2 has 4 nucleotides.
52. 52. The attenuated bacterial live vector vaccine of claim 48, wherein X2 has 4 nucleotides, independently selected from the group consisting of A, T, and C.
53. 53. The attenuated bacterial live vector vaccine of claim 48, wherein X2 comprises a nucleotide or a nucleotide sequence, selected from the group consisting of ATCT; ATC; AT: TCT; CT; TC; TO; T; C; and T.
54. 54. The attenuated bacterial live vector vaccine of claim 48, wherein X2 is selected from the group consisting of ATCT; ATC; AT; TCT; CT; TC; TO; T; and T.
55. 55. The attenuated bacterial live vector vaccine of claim 48, wherein X2 is ATCT.
56. 56. The attenuated bacterial live vector vaccine of claim 48, wherein X3 is A.
57. 57. The attenuated bacterial live vector vaccine of claim 40, wherein the plasmid maintenance system comprises: (a) at least one elimination function after segregation; and (b) at least one division function.
58. 58. The attenuated bacterial live vector vaccine of claim 57, wherein the elimination function, after segregation, is selected from the group consisting of balanced lethal functions, protein functions and antisense functions.
59. 59. The attenuated bacterial live vector vaccine of claim 57, wherein the elimination function, after segregation, is selected from the group consisting of asd, ssb, pha-doc, hok-sok and their substantial counterparts.
60. 60. The attenuated bacterial live vector vaccine of claim 57, wherein the division function comprises an active division function.
61. 61. The attenuated bacterial live vector vaccine of claim 57, wherein the division function comprises a passive division function.
62. 62. The attenuated bacterial live vector vaccine of claim 57, wherein the dividing function comprises the even site of pSClO1 and / or its substantial counterpart.
63. 63. The attenuated bacterial live vector vaccine of claim 57, wherein the dividing function comprises parA and / or its substantial counterpart.
64. 64. The attenuated bacterial live vector vaccine of claim 40, wherein the antigen of interest is a test antigen.
65. 65. The attenuated bacterial live vector vaccine of claim 64, wherein the test antigen is selected from the group consisting of the green fluorescent protein, functional equivalents thereof and the substantial homologs of this green fluorescent protein.
66. 66. The attenuated bacterial live vector vaccine of claim 40, wherein the antigen of interest is a detoxified Shiga toxin.
67. 67. The attenuated bacterial live vector vaccine of claim 40, wherein the antigen is one or more antigens of Shiga toxin 2, selected from the group comprising the pentamers of the subunit of Shiga toxin 2 and a detoxified Stx 2. genetically
68. 68. The attenuated bacterial live vector vaccine of claim 67, wherein the gene encoding detoxified Shiga toxin 2 has mutations selected from the group consisting of: (797) - ACÁ GCA GAC GCG TTA (811) (902) - CTG AAC CTA GGG CGA (916) (1345) GAA TTC GCG ACC AGT - (1359); Y (1435) - GAA TCA GAT TCT GGA (1449)
69. 69. The attenuated bacterial live vector vaccine of claim 40, wherein the replicon further comprises a nucleotide sequence encoding a resistance marker.
70. 70. The attenuated bacterial live vector vaccine of claim 69, wherein the nucleotide sequence, which encodes the resistance marker, is contained in a genetic cassette, which functions independently.
71. 71. The attenuated bacterial live vector vaccine of claim 69, wherein the selectable marker n confers resistance to some antibiotic, which is ordinarily used in the medical treatment of humans.
72. 72. The attenuated bacterial live vector vaccine of claim 69, wherein the nucleotide sequence, which encodes the resistance marker, comprises bla and / or its substantial homologue.
73. 73. The attenuated bacterial live vector vaccine of claim 69, wherein the nucleotide sequence encoding the resistance marker comprises the tet and / or its substantial counterpart.
74. 74. A conditionally unstable plasmid, to examine the changes in plasmid stability, which result from the incorporation of plasmid maintenance systems, this plasmid comprises a response source, which supplies an average copy number which falls within the range of approximately 2 to 75 copies and a promoter that promotes the expression of a protein or peptide, the over-expression of which imposes a metabolic load on a bacterium, which favors the loss of the plasmid.
75. 75. The unstable plasmid conditionally. of claim 74, wherein the average number of copies falls within the approximate range of 5 to 60 copies.
76. 76. The unstable plasmid conditionally. of claim 74, wherein the promoter comprises an ompC promoter or its substantial counterpart.
77. 77. The unstable plasmid conditionally. of claim 74, wherein the protein or peptide is selected from the group consisting of the green fluorescent protein, functional equivalents thereof and the substantial homologs of this green fluorescent protein.
78. 78. The unstable plasmid conditionally. of claim 74, wherein an average number of copies is selected from the group consisting of: about 5 copies per cell; approximately 15 copies per cell; and approximately 60 copies per cell.
79. 79. The unstable plasmid conditionally. of claim 74, wherein the response source is selected from the group consisting of the response origin of plasmid pSClOl, response origin of plasmid pACYC184, response origin of plasmid pAT153, and substantial homologs of any such response origins.
80. 80. The unstable plasmid conditionally. of claim 74, in which the response origin is d pSClOl, which confers a copy number of approximately 5 copies per genome equivalent.
81. 81. The unstable plasmid conditionally. of claim 74, wherein the response origin is d pACYC184, which confers a copy number of about 15 copies per genome equivalent.
82. 82. The unstable plasmid conditionally. of claim 74, wherein the response origin is d pAT153, which confers a copy number of approximately 60 copies per genome equivalent.
83. 83. A method for eliciting an immune response in a subject, this method comprises administering to the subject a live bacterial vector vaccine, including a bacterial cep, comprising an expression vector that includes: (a) a nucleotide sequence encoding an antigen; (b) a promoter that controls the expression of the antigen; and (c) a nucleotide sequence that encodes at least one plasmid maintenance system.
84. 84. The method of claim 83, wherein the live bacterial vector vaccine is administered in an immunizing effective amount.
85. 85. The method of claim 83, wherein the bacterial live vector is a species of attenuated Salmonella typh.
86. 86. The method of claim 83, wherein (a) and (b) are contained in a genetic cassette, which functions independently.
87. 87. The method of claim 83, wherein (C) is contained within genetic cassettes, which functions independently.
88. 88. The method of claim 83, wherein the promoter is an inducible promoter.
89. 89. The method of claim 83, wherein the promoter is an ompC promoter or a functional equivalent thereof.
90. 90. The method of claim 83, wherein the promoter is a modified ompC promoter, phenotypically characterized in that the promoter exhibits higher regimes of osmotically controlled expression, relative to the corresponding ompC promoter, without such dot mutations.
91. 91. The method of claim 90, wherein the modified ompC promoter comprises a modified Bg / II site or is without a complete Bg / II site.
92. 92. The method of claim 91, wherein the modified ompC promoter comprises the following d 5 'to 3' sequence, which starts with the modified Bg / ll site ends with the ATG start codon: AGATCX1X2TAAX3CATCCACAGGAGGATATCTGATG, in which: (d) X1 is selected from the group consisting of G, C and A; (e) X2 is an insert having from 1 to 5 nucleotides; (f) X3 is selected from the group consisting of A, T, G C.
93. The method of claim 92, wherein X1 is G.
94. 94. The method of claim 92, wherein X2 has from 1 to 4 nucleotides.
95. 95. The method of claim 92, wherein X2 has 4 nucleotides.
96. 96 The method of claim 92, wherein X2 has 4 nucleotides, independently selected from the group consisting of A, T, and C.
97. 97. The method of claim 92, wherein X2 comprises a nucleotide or a nucleotide sequence, selected from the group consisting of ATCT; ATC; AT: TCT; CT; TC; TO; T; C; and T.
98. The method of claim 92, wherein X2 is selected from the group consisting of ATCT; ATC; AT; TCT; CT; TC; TO; T; and T.
99. 99. The method of claim 92, wherein X2 is ATCT.
100. 100. The method of claim 92, wherein X3 is A.
101. 101. The method of claim 83, wherein the plasmid maintenance system comprises: (a) at least one elimination function after segregation; and (b) at least one division function.
102. 102. The method of claim 101, wherein the elimination function, after segregation, is contained in a genetic cassette, which functions independently.
103. 103. The method of claim 101, wherein the elimination function, after segregation, is selected from the group consisting of balanced letale functions, protein functions and antisense functions.
104. 104. The method of claim 101, wherein the elimination function, after segregation, is selected from the group consisting of asd, ssb, phd-doc, hok-sok and their substantial counterparts.
105. 105. The method of claim 101, wherein the dividing function is contained in a genetic cassette, which functions independently.
106. 106. The method of claim 101, wherein the division function comprises an active division function.
107. 107. The method of claim 101, wherein the division function comprises a passive division function.
108. 108. The method of claim 101, wherein the dividing function comprises the even site of pSClO1 and / or its substantial counterpart.
109. 109. The method of claim 101, wherein the dividing function comprises the parA and / or its substantial counterpart.
110. 110. The method of claim 101, wherein the antigens comprise at least one Shiga toxin, genetically detoxified.
111. 111. The method of claim 101, wherein the antigens include at least one toxin antigen Shiga (Stx2), selected from the group comprising the pentamers of the 2B toxin subunit of Shiga, and a genetically detoxified Stx.
112. 112. The method of claim 83, wherein the nucleotide sequence further comprises a sequence of nucleotides encoding a selectable marker, which confers no resistance to any antibiotic that is ordinarily used in the treatment of humans.
113. 113. The method of claim 112, wherein the nucleotide sequence, which encodes the selectable tag, is contained in a genetic cassette, which functions independently.
114. 114. The method of claim 83, wherein the subject is a human.
115. 115. The method of claim 83, wherein the subject is a bovine.
116. 116. A method for obtaining a stabilized bacterial live virus vaccine, comprising transforming a live bacterial vector with a replicon, which includes: (a) a plasmid maintenance system, which comprises: (i) at least one elimination function after segregation; e (ii) at least one division function; and (b) a nucleotide sequence, which encodes one or more antigens.
117. 117. The method of claim 116, wherein the elimination function after segregation is contained in a genetic cassette, which functions independently.
118. 118. The method of claim 116, wherein the dividing function is contained in a genetic cassette, which functions independently.
119. 119. The method of claim 116, wherein the elimination function, after segregation is selected from the group consisting of balanced lethal functions, protein functions and antisense functions.
120. 120. The method of claim 116, wherein the elimination function, after segregation, is selected from the group consisting of asd, ssb, phd-doc, hok sok and their substantial counterparts.
121. 121. The method of claim 116, wherein the division function is an active division function.
122. 122. The method of claim 116, wherein the division function is a passive division function.
123. 123. The method of claim 116, wherein the dividing function comprises the even site of pSClO1 and / or s substantial counterpart.
124. 124. The method of claim 116, wherein the dividing function comprises the parA and / or its substantial counterpart.
125. 125. The method of claim 116, wherein the antigen of interest is a detoxified Shiga toxin.
126. 126. The method of claim 116, wherein the antigen is one or more antigens of Shiga toxin 2, selected from the group comprising the pentamers of the subunit of Shiga toxin 2B and a detoxified Stx 2.
127. 127. The method of claim 125, wherein the gene encoding detoxified Shiga toxin 2 has mutations selected from the group consisting of: (797) - ACÁ GAC GAC GCG TTA (811) (902) - CTG AAC CTA GGG CGA (916) (1345) - GAA TTC GCG ACC AGT (1359); Y (1435) - GAA TCA GAT TCT GGA - (1449)
128. 128. The method of claim 116, wherein the promoter is a modified ompC promoter, phenotypically characterized in that said promoter exhibits regimes greater than the osmotically regulated expression, relative to a corresponding ompC promoter, without said d point mutations.
129. 129. The method of claim 116, wherein the modified ompC promoter is without the Bg / ll site.
130. 130. The method of claim 116, wherein the modified ompC promoter comprises a mutated Bg / ll site.
131. 131. The method of claim 116, wherein the modified ompC promoter comprises the following d 5 'to 3' sequence, which starts with the mutated Bg / ll site and terminates with the ATG start codon: AGATCX1X2TAAX3CATCCACAGGAGGATATCTGATG, in which: (a) X1 is selected from the group consisting of G, C and A; (b) X2 is an insert having 1 to 5 nucleotides; (c) X3 is selected from the group consisting of A, T, G C.
132. 132. A DNA comprising a modified omp promoter, phenotypically characterized in that the promoter exhibits higher regimes of osmotically regulated expression, relative to a corresponding non-mutated ompC promoter.
133. 133. The DNA of claim 132, wherein the modified ompC promoter comprises a mutated Bg / l site.
134. 134. The DNA of claim 133, wherein the modified ompC promoter comprises the following sequence from 5 'to 3', which starts with the mutated Bg / II site and terminates with the ATG start codon: AGATCX1X2TAAX3CATCCACAGGAGGATATCTGATG, in which: (a) X1 is selected from the group consisting of G, C and A; (b) X2 is an insert having 1 to 5 nucleotides; (c) X3 is selected from the group consisting of A, T, G C.
135. 135. The DNA of claim 134, wherein X1 and G.
136. 136. The DNA of claim 134, wherein X has from 1 to 4 nucleotides.
137. 137. The DNA of claim 134, wherein X has 4 nucleotides.
138. 138. The DNA of claim 134, wherein X has 4 nucleotides, independently selected from the group consisting of A, T, and C.
139. 139. The DNA of claim 134, wherein X comprises a nucleotide or nucleotide sequence selected from the group consisting of ATCT; ATC; AT: TCT; CT TC; TO; T; C; and T.
140. The method of claim 134, wherein X is selected from the group consisting of ATCT; ATC; AT; TCT CT; TC; TO; T; and T.
141. 141. The method of claim 134, wherein X is ATCT.
142. 142. The method of claim 134, wherein X is A.
143. 143. The method of claim 133, wherein the mutated Bg / II site of the ompC promoter comprises the AGATCG sequence.
144. 144. The DNA of claim 133, wherein the mutated Bg / ll site of the ompC promoter consists of the AGATCG sequence.
145. 145. The ADÑ of claim 133, wherein the modified ompC promoter comprises the following d 5 'to 3' sequence, between the mutated Bg / ll site and the ATG start codon: AGATCTTAAACATCCACAGGAGGATATCTGATG.
146. 146. The DNA of claim 132, further comprising a plasmid maintenance system, which includes: (a) at least one elimination function, after segregation; and (b) at least one division function.
147. 147. An expression plasmid, comprising the DNA of claim 146.
148. 148. The DNA of claim 132, further comprising a nucleotide sequence encoding a peptide or protein, the expression of which is controlled by a modified promoter.
149. 149. The DNA of claim 147, wherein the peptide or protein is selected from the group consisting of heterologous antigens and the green fluorescent protein.
150. 150. The DNA of claim 147, wherein the peptide or protein is selected from the group consisting of the detoxified Shiga toxin.
151. 151. The DNA of claim 147, wherein the peptide or protein is selected from the group consisting of the pentamers of the subunit of Shiga toxin 2b and a detoxified Stx.
152. 152. The DNA of claim 132, which also comprises a nucleotide sequence, which encodes or selectable marker, this marker does not confers resistance to any antibiotic that is ordinarily used in the treatment of humans.
153. 153. An expression plasmid comprising DNA of claim 152.
154. 154. The DNA of claim 132, further comprising a response origin and a transcription terminator sequence, at a 5 'position relative to the origin of response, so that the transcript of response origin is less disturbed relative to the alteration in the absence of such sequence of transcription terminator.
155. 155. An expression plasmid, comprising the DNA of any of claims 132 to 154.