Patent Application
20110033501
The invention encompasses a recombinant bacterium and a vaccine comprising the recombinant bacterium. The recombinant bacterium may be used to elicit an immune response to one or more enteric pathogens.
Global disease burden data from UNICEF and WHO show that the top two major causes of infant and child mortality are respiratory and diarrheal diseases. While diarrheal diseases caused by enteric pathogens constitute one of the major causes of morbidity and mortality globally, obtaining accurate epidemiological information about the causative agents is essentially impossible. WHO estimates are just that and the apportionment of disease due to bacteria versus viruses versus parasites are often educated guesses at best and are further complicated due to infections with more than one causative pathogen. Enteric fever due to Salmonella Typhi and S. Paratyphi A & B is still a major worldwide problem affecting at least 20-30 million people with a significant mortality in medically underprivileged countries. Experts on Shigella infections estimate 165 million annual episodes of infection with 1.1 million deaths. If one collects all these data and the information of diarrheal diseases due to E. coli pathovars, C. perfringens type A, C. jejuni plus the enteric viruses and parasites, the total annual mortality due to diarrheal diseases approaches 5 million. Guerrant et al., Arch. Med. Res. 33:351-355 (2002) estimate that there are 3 million annual deaths from diarrheal diseases and thus might be closer to the truth. The WHO estimate that children in Africa experience an average of five diarrheal episodes per year with 800,000 deaths due to fluid loss and dehydration nevertheless indicates the magnitude of the problem. The results of a 4-year WHO study found that between 2000 and 2003, diarrheal diseases accounted for 18% of the 10.6 million annual deaths in children under 5 years of age.
The problem is exacerbated by cessation in use of subtherapeutic antibiotic additions to animal feed for growth promotion. This was first recognized in poultry and other livestock such as swine but C. perfringens also causes necrotizing enteritis in the small intestines of humans, which occurs sporadically in underdeveloped countries. Some factors that predispose to C. perfringens induced necrotic enteritis include protozoan and helminth infections. C. perfringens type associated diarrhea is one of the top 5 causes of food borne bacterial diarrheal disease ranked by CDC in the U.S. Alpha-toxin is particularly responsible for sublethal effects on enterocytes that could lead to malabsorption and stunting in children in developing countries. Studies also show the possible etiologic significance of early intestinal C. perfringens colonization and development of necrotizing enterocolitis in newborns. Yersinia enterocolitica and Y. pseudotuberculosis are other intestinal colonizers that may contribute to human diarrheal disease, but these enteropathogens have not been well studied as contributors to intestinal disease of humans in the developing world.
The problems of travelers in acquiring diarrheal diseases when abroad are legend and often ascribed to ETEC and EPEC strains but just as likely might be due to Norwalk and rotaviruses, Campylobacter, Listeria or Giardia, all predominantly waterborne or foodborne infections. Although enteric pathogens have a much larger detrimental effect on health in the developing world, these infections are not without major economic consequences in the U.S. and other developed countries. The USDA estimated that infections with Campylobacter, Salmonella (non-typhoidal), EHEC, STEC and Listeria had a 6.9 billion dollar negative economic impact. E. coli is the leading cause of both community-acquired and nosocomial urinary tract infections (UTI). As many as 50% of women have had at least one episode of UTI in their lifetime. E. coli also causes 12-50% of nosocomial infections. In regard to bacterial enteropathogens, a major problem, except for host-adapted Salmonella (i.e., S. Typhi and S. Paratyphi), Shigella sp. and some ETEC and EPEC strains, is the vast animal reservoir of Salmonella enterica serotypes (over 2000), Clostridium sp., Yersinia sp., APEC and other E. coli pathovars, Listeria and Campylobacter. Animals including companion animals, wildlife, and agriculturally important food animals are causes of water contamination or transmission of bacterial enteric pathogens through the food chain to humans. As such, a vaccine that reduces the probability of infection by necessitating higher infection doses or a vaccine that lessens the consequences of infection offers a definite public health benefit, especially in the developing world.
Approximately 1.4 million humans are infected with Salmonella enterica serotypes each year in the U.S. primarily causing gastroenteritis and lost time from work, but with a low incidence of more severe infections, sometimes leading to death in the very young, the elderly or in individuals with an immunocompromising condition, such as advanced HIV infection. In the U.S., Salmonella accounts for 31% of the fatalities due to foodborne pathogens whereas Listeria monocytogenes accounts for 28% and C. jejuni, which causes many more infections (2.5 million), accounts for 1% of the deaths. Twenty percent of all Salmonella cases or isolates are from children under 5 years of age.
Salmonella enterica has been subdivided into seven subspecies differentiated by biochemical and genetic tests, with subspecies I containing most of the serotypes that are implicated in warm-blooded animal and human infection. Although data collected from year to year and from country to country differ, it would appear that poultry (contaminated eggs and meat) constitute the major source of food-borne Salmonella infection in humans with contaminated pork, dairy products and vegetable/fruit crops accounting for the rest, but in decreasing frequency of causation. In a recent study, the Food Safety and Inspection Service (FSIS) determined Salmonella serotypes isolated from swine, ground turkey, ground beef and broilers in processing plants participating in the Hazard Analysis and Critical Control Point (HACCP) systems for pathogen reduction and found that 87% of the Salmonella isolates were from poultry sources. Using data from the Centers for Disease Control and Prevention collected in 2005, it is evident that some Salmonella serotypes that are most frequently isolated from humans are also very prevalent in poultry, with 8 of the Salmonella serotypes predominantly isolated from poultry being represented in the top 20 serotypes isolated from humans. Salmonella is a gram-negative bacterium, best known for causing enteric diseases. Within the Salmonella genus, there are two main species, S. bongori and S. enterica. However, within each species, there are over 2500 serovars. These numerous serovars are found in a wide variety of different environments and are associated with many different diseases. The vast majority of human isolates (>99.5%) are subspecies S. enterica. To simplify taxonomy, the Centers for Disease Control and Prevention recommend that Salmonella species be referred to only by their genus and serovar, e.g., Salmonella Typhi (or S. Typhi) instead of the more technically correct designation, Salmonella enterica subspecies enterica serovar Typhi.
One important use of genetically engineered microorganisms, such as Salmonella, is as a live vaccine for inducing immunity. The use of Salmonella for vaccine purposes requires that the Salmonella be attenuated such that the administration thereof does not induce disease symptoms associated with wild type Salmonella infection. In addition, the Salmonella vaccine also has to exhibit a high degree of immunogenicity. As such, the objective of much research on Salmonella-based vaccines is to construct a safe and efficacious Salmonella vector system that can be used repeatedly for multiple recombinant attenuated Salmonella vaccines (RASVs) and additionally, induce some level of cross-protective immunity to diarrheal diseases caused by the diverse S. enterica serotypes and other pathogenic enteric bacteria (e.g., Shigella sp. and E. coli pathovars).
In theory, the ideal attenuated Salmonella vaccine should exhibit wild-type abilities that are capable of withstanding all types of biological stress that is entailed with living in an individual. Examples of these types of biological stresses include: exposure to enzymes, acid, bile, osmotic pressures and ion stress. In addition, the ideal attenuated Salmonella vaccine should also be able to withstand host defenses encountered following administration (e.g., orally or intranasally). Further, the ideal attenuated Salmonella vaccine should also be able to colonize and invade host lymphoid tissues before displaying its attenuation and its inability to cause disease symptoms.
Another existing problem is that the recipient's immune system reacts to the Salmonella serotype-specific antigen. The combination of minimizing a recipient's immune response to the Salmonella serotype-specific antigen while maximizing the immune response against the undesired bacterial pathogens has not been effectively accomplished in the art. What is needed are compositions and/or vaccines of Salmonella that are capable of decreased or absent expression of Salmonella serotype-specific antigens that can elicit immune responses to antigens expressed by bacterial enteric pathogens.
All references, patents, and patent applications cited here are each incorporated by reference in their entirety for all purposes.
One aspect of the present invention encompasses a recombinant Salmonella bacterium. The bacterium is typically capable of the expression of at least one nucleic acid encoding at least two enteric antigens. When administered to a host, the bacterium is generally capable of eliciting an immune response against at least two enteric pathogens in addition to at least one Salmonella serotype.
Another aspect of the present invention encompasses a vaccine composition. The vaccine composition comprises a recombinant Salmonella bacterium. The bacterium is typically capable of the expression of at least one nucleic acid encoding at least two enteric antigens. When administered to a host, the bacterium is generally capable of eliciting an immune response against at least two enteric pathogens in addition to at least one Salmonella serotype.
Yet another aspect of the invention encompasses a method of inducing an immune response against an enteric pathogen. The method comprises administering a vaccine composition comprising a recombinant Salmonella bacterium. The bacterium is typically capable of the expression of at least one nucleic acid encoding at least two enteric antigens. When administered to a host, the bacterium is generally capable of eliciting an immune response against at least two enteric pathogens in addition to at least one Salmonella serotype.
Other aspects and iterations of the invention are described more thoroughly below.
The present invention provides a recombinant bacterium capable of eliciting an immune response against at least two enteric pathogens in addition to at least one Salmonella serotype. In some embodiments, the recombinant bacterium elicits an immune reponse against at least two Salmonella serotypes and at least two additional enteric pathogens. In an exemplary embodiment, the recombinant bacterium does not substantially induce an immune response to the serotype of the recombinant bacterium. The invention also encompasses a vaccine composition comprising the recombinant bacterium and methods for using the recombinant bacterium.
One aspect of the present invention encompasses a recombinant Salmonella bacterium. Generally speaking, the recombinant bacterium is capable of the expression of at least one nucleic acid encoding at least two enteric antigens. The bacterium, when administered to a host, typically elicits an immune response against at least two enteric pathogens and at least one Salmonella serotype. In exemplary embodiments, the recombinant bacterium does not substantially induce an immune response specific to the serotype of the recombinant bacterium.
In additional exemplary embodiments, a recombinant Salmonella bacterium of the invention is capable of colonizing a host to substantially the same extent as a wild-type bacterium of the same serotype. A bacterium of the invention, however, will preferably be substantially avirulent after colonization.
In some embodiments, the recombinant bacterium may be a Salmonella enterica serovar. In an exemplary embodiment, a bacterium of the invention may be derived from S. Typhimurium, S. Typhi, S. Paratyphi, S. Gallinarum, S. Enteritidis, S. Choleraesius, S. Arizonae, or S. Dublin. In an exemplary embodiment, a bacterium of the invention may be derived from S. Typhimurium, S. Paratyphi, or S. Typhi. In all cases, a recombinant bacterium of the invention generally does not comprise any drug resistance nucleic acid sequences or other sequence scars in the chromosomes of the recombinant strain.
Generally speaking, a recombinant bacterium of the invention is capable of the regulated expression of a nucleic acid encoding at least one serotype-specific antigen. As used herein, the phrase “serotype-specific antigen” refers to an antigen that elicits an immune response specific for the bacterial vector serotype. In some embodiments, the immune response to a serotype-specific antigen may also recognize closely related strains in the same serogroup, but in a different, but related, serotype. Non-limiting examples of serotype-specific antigens may include LPS O-antigen, one or more components of a flagellum, and Vi capsular antigen. In some embodiments, the expression of at least one, at least two, at least three, or at least four nucleic acid sequences encoding a serotype-specific antigen are regulated in a bacterium of the invention.
The phrase “regulated expression of a nucleic acid encoding at least one serotype-specific antigen” refers to expression of the nucleic acid encoding a serotype-antigen such that the bacterium does not substantially induce an immune response specific to the bacterial vector serotype. In one embodiment, the expression of the serotype-specific antigen is eliminated. In another embodiment, the expression is substantially reduced. In yet another embodiment, the expression of the serotype-specific antigen is reduced in a temporally controlled manner. For instance, the expression of the serotype-specific antigen may be reduced during growth of the bacterium in a host, but not during in vitro growth.
The expression of a nucleic acid encoding a Salmonella serotype-specific antigen may be measured using standard molecular biology and protein chemistry techniques known to one of skill in the art. As used herein, “substantial reduction” of the expression of a nucleic acid encoding a serotype-specific antigen refers to a reduction of at least about 1% to at least about 99.9% as compared to a Salmonella bacterium in which no attempts have been made to reduce serotype-specific antigen expression. In one embodiment, the expression of a nucleic acid encoding a serotype-specific antigen is reduced by 100% by using a deletion mutation. In other embodiments of the invention, the expression of a nucleic acid encoding a serotype-specific antigen is reduced by at least about 99.9%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90%. In yet other embodiments of the invention, the expression of a nucleic acid encoding a serotype-specific antigen is reduced by at least about 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80%. In still other embodiments of the invention, the expression of a nucleic acid encoding a serotype-specific antigen is reduced by at least about 75%, 70%, 65%, 60%, 55%, or 50%. In additional embodiments, the expression of a nucleic acid encoding a serotype-specific antigen is reduced by at least about 45%, 40%, 35%, 30%, 25%, or 20%. In yet additional embodiments, the expression of a nucleic acid encoding a serotype-specific antigen is reduced by at least about 15%, 10%, 5%, 4%, 3%, 2% or 1%.
Methods of regulating expression of a nucleic acid encoding at least one serotype-specific antigen are discussed in detail below, and in the examples.
i. Regulating the Expression of a Nucleic Acid Encoding LPS O-Antigen
In one embodiment, the expression of a nucleic acid encoding the serotype-specific antigen LPS O-antigen is regulated by mutating the pmi nucleic acid sequence, which encodes a phosphomannose isomerase needed for the bacterium to interconvert fructose-6-P and mannose-6-P. In some instances, the bacterium comprises a Δpmi mutation, such as a Δpmi-2426 mutation. (See
A bacterium of the invention that comprises a Δpmi mutation may also comprise other mutations that ensure that mannose available to the bacterium during in vitro growth is used for LPS O-antigen synthesis. For instance, a bacterium may comprise a Δ(gmd-fcl)-26 mutation. This mutation deletes two nucleic acid sequences that encode enzymes for conversion of GDP-mannose to GDP-fucose. This ensures that mannose available to the bacterium during in vitro growth is used for LPS O-antigen synthesis and not colanic acid production. Similarly, a bacterium may comprise the Δ(wcaM-wza)-8 mutation, which deletes all 19 nucleic acid sequences necessary for colanic acid production, and also precludes conversion of GDP-mannose to GDP-fucose.
In addition to regulating LPS O-antigen synthesis with mannose, the synthesis of LPS O-antigen may be regulated by arabinose, which is also absent in vivo. For instance, a bacterium may comprise the mutation ΔPrfc::TT araC PBAD rfc. (P stands for promoter and TT stands for transcription terminator.) The rfc nucleic acid sequence is necessary for the addition of O-antigen subunits, which typically comprise three or four sugars, in a repeat fashion. When the rfc nucleic acid sequence is absent, only one O-antigen repeat subunit is added to the LPS core polysaccharide. Normally, the serotype-specific O-antigen contains some 50 or so repeats of the O-antigen subunit, catalyzed by the enzyme encoded by the rfc nucleic acid sequence. In the case of a bacterium comprising the ΔPrfc::TT araC PBAD rfc deletion-insertion mutation, expression of the rfc nucleic acid sequence is dependant on the presence of arabinose that can be supplied during in vitro growth of the strain, but that is absent in vivo. Consequently, rfc expression ceases in vivo, resulting in the cessation of assembly of the O-antigen repeat structure. This reduces the bacterium's ability to induce an immune response against the serotype-specific O-antigen.
Another means to regulate LPS O-antigen expression is to eliminate the function of galE in a recombinant bacterium of the invention. The galE nucleic acid sequence encodes an enzyme for the synthesis of UDP-Gal, which is a substrate for LPS O-antigen, the outer LPS core and colanic acid. Growth of a bacterium comprising a suitable galE mutation in the presence of galactose leads to the synthesis of O-antigen and the LPS core. Non-phosphorylated galactose is unavailable in vivo, however, and in vivo synthesis of UDP-Gal ceases, as does synthesis of the O-antigen and the LPS outer core. One example of a suitable galE mutation is the Δ(galE-ybhC)-851 mutation.
In certain embodiments, a bacterium of the invention may comprise one or more of the Δpmi, ΔPrfc::TT araC PBAD rfc, and ΔgalE mutations, with or without a Δ(gmd-fcl)-26 or Δ(wcaM-wza)-8 mutation. Such a combination may yield a recombinant bacterium that synthesizes all components of the LPS core and O-antigen side chains when grown in vitro (i.e. in the presence of suitable concentrations of mannose, arabinose and galactose), but that ceases to synthesize the LPS outer core and O-antigen in vivo due to the unavailability of free unphosphorylated mannose, arabinose or galactose. Also, a recombinant bacterium with the inability to synthesize the LPS outer core and/or O-antigen is attenuated, as the bacterium is more susceptible to macrophages and/or complement-mediated cytotoxicity. Additionally, a bacterium with the inability to synthesize the LPS outer core and O-antigen in vivo, induces only a minimal immune response to the serotype-specific LPS O-antigen.
The regulated expression of one or more nucleic acids that enable synthesis of LPS O-antigen allows a recombinant bacterium of the invention to be supplied with required sugars such as mannose, arabinose and/or galactose during in vitro growth of the bacterium, ensuring complete synthesis of the LPS O-antigen. This is important, because the presence of the O-antigen on the recombinant bacterium cell surface is indispensable for the strain to invade and colonize lymphoid tissue, a necessary prerequisite for being immunogenic. In vivo, LPS O-antigen synthesis ceases due to the unavailability of the free unphosphorylated sugars. Consequently, the recombinant bacterium is attenuated, becoming more susceptible to complement-mediated cytotoxicity and macrophage phagocytosis. Also, when LPS O-antigen synthesis ceases, the LPS core is exposed. The core is a cross-reactive antigen with a similar structure in all Salmonella serotypes. In addition, when LPS O-antigen synthesis ceases, any cross-reactive outer membrane proteins expressed by the recombinant bacterium are exposed for surveillance by the host immune system.
ii. The Expression of a Nucleic Acid Encoding a Component of a Flagellum
In one embodiment, the expression of a nucleic acid encoding a serotype-specific component of a flagellum is regulated by mutating the nucleic acid that encodes FljB or FliC. For instance, a bacterium of the invention may comprise a ΔfljB217 mutation. Alternatively, a bacterium may comprise a ΔfliCI80 mutation. The ΔfljB217 mutation deletes the structural nucleic acid sequence that encodes the Phase II flagellar antigen whereas the ΔfliCI80 mutation deletes the 180 amino acids encoding the antigenically variable serotype-specific domain of the Phase I FliC flagellar antigen. The portion of the flagellar protein that interacts with TLR5 to recruit/stimulate innate immune responses represents the conserved N- and C-terminal regions of the flagellar proteins and this is retained and expressed by strains with the ΔfliCI80 mutation. In addition, the ΔfliCI80 mutation retains the CD4-dependent T-cell epitope. It should be noted, that expression of the Phase I flagellar antigen and not the Phase II flagellar antigen potentiates S. Typhimurium infection of mice. S. Typhimurium recombinant bacteria with the Δpmi-2426, AfljB2I7 and ΔfliC180 mutations, when grown in the absence of mannose, are not agglutinated with antisera specific for the B-group O-antigen or the S. Typhimurium specific anti-flagellar sera. These recombinant bacteria are also non-motile since the FliC180 protein that is synthesized at high levels is not efficiently incorporated into flagella. When these recombinant bacteria are evaluated using HEK293 cells specifically expressing TLR5, the level of NFκB production is about 50% higher than when using a ΔfliB217 F1iC+ strain that assembles flagellin into flagella and exhibits motility (there is no NFκB production by the control ΔfljB217 ΔfliC2426 strain with no flagella). Similarly, recombinant bacteria with the Δ(galE-ybhC)-851, ΔfljB217 and ΔfliC180 mutations, when grown in the absence of galactose, are not agglutinated with antisera specific for the B-group O-antigen or the S. Typhimurium specific anti-flagellar sera. These bacteria are also non-motile.
iii. The Expression of a Nucleic Acid Encoding the Vi Capsular Antigen
Certain Salmonella strains, such as S. Typhi and S. Dublin, express the Vi capsular antigen. This antigen is serotype-specific, inhibits invasion, and acts to suppress induction of a protective immune response. Consequently, when a recombinant bacterium of the invention is derived from a strain comprising the Vi capsular antigen, one or more nucleic acids encoding the Vi capsular antigen will be deleted such that the Vi capsular antigen is not synthesized.
Generally speaking, a recombinant bacterium of the invention is capable of the expression of at least one nucleic acid encoding at least two enteric antigens. As used herein, the phrase “enteric antigen” refers to an antigen that elicits an immune response against an enteric pathogen. Non-limiting examples of enteric antigens from Yersinia may include the V antigen and the psn nucleic acid product involved in iron acquisition. Non-limiting examples of enteric antigens from E. coli may include salmochelin, aerobactin, the sit operon antigens involved in iron and manganese uptake, the fimbriae encoded by the yagZ fimbrial operon, other fimbriae encoded by nucleic acids in various ETEC, EPEC, EHEC, APEC, UPEC and/or ExPEC strains, the tsh nucleic acid product, the iss nucleic acid product, and LTB, the non-toxic cell binding domain of the LT toxin. Non-limiting examples of enteric antigens from Shigella may include the IpaD and aerobactin antigens. Non-limiting examples of enteric antigens from C. jejuni may include PilA and CjaA antigen. Non-limiting examples of enteric antigens from C. perfringens may include the α-toxin and NetB antigens.
It is not necessary that the nucleic acid encoding an enteric antigen comprise the complete nucleic acid sequence of the antigen. It is only necessary that the enteric antigen sequence used be capable of eliciting an immune response. The antigen may be one that was not found in that exact form in the parent organism. For example, a sequence coding for an antigen comprising 100 amino acid residues may be transferred in part into a recombinant bacterium so that a peptide comprising only 75, 65, 55, 45, 35, 25, 15, or even 10, amino acid residues is produced by the recombinant bacterium. Alternatively, if the amino acid sequence of a particular enteric antigen or fragment thereof is known, it may be possible to chemically synthesize the nucleic acid fragment or analog thereof by means of automated nucleic acid sequence synthesizers, PCR, or the like and introduce said nucleic acid sequence into the appropriate copy number vector.
In certain embodiments, an enteric antigen of the invention may comprise a B cell epitope or a T cell epitope. Alternatively, an antigen to which an immune response is desired may be expressed as a fusion to a carrier protein that contains a strong promiscuous T cell epitope and/or serves as an adjuvant and/or facilitates presentation of the antigen to enhance, in all cases, the immune response to the antigen or its component part. This can be accomplished by methods known in the art. Fusion to tetnus toxin fragment C, CT-B, LT-B, hepatitis virus B core, and woodchuck hepatits virus core are particularly useful for these purposes, although other epitope presentation systems are well known in the art.
In certain embodiments, the expression of at least one nucleic acid encoding at least two enteric antigens may be regulated. In further embodiments, a nucleic acid sequence encoding an antigen of the invention may comprise a secretion signal. In other embodiments, an antigen of the invention may be toxic to the recombinant bacterium.
A recombinant bacterium may comprise a long sequence of nucleic acid encoding several nucleic acid sequence products, one or all of which may be enteric antigens. In some embodiments, the expression of at least one, at least two, at least three, at least four, at least five, at least six, or more nucleic acids encoding enteric antigens is regulated in a bacterium of the invention. These antigens may be encoded by two or more open reading frames operably linked to be expressed coordinately as an operon, wherein each antigen is synthesized independently. Alternatively, the two or more antigens may be encoded by a single open reading frame such that the antigens are synthesized as a fusion protein. In another alternative, the two or more antigens may be encoded by overlapping open reading frames.
In many cases, the high level expression of a nucleic acid sequence encoding an antigen in a bacterium reduces the bacterium's fitness, such that the bacterium grows slowly, is susceptible to stresses encountered in the host, and is generally less able to effectively colonize effector lymphoid tissues. High level expression of a nucleic acid sequence encoding an antigen, however, is highly desirable to maximize induction of an immune response against the antigen. Consequently, the phrase “regulated expression of at least one nucleic acid encoding at least two enteric antigens” refers to expression at least one nucleic acid encoding at least two enteric antigens in a bacterium such that the bacterium is capable of colonizing a host at levels similar to a wild-type bacterium, and yet is still capable of eliciting an immune response against an enteric pathogens when administered to the host. Methods of expressing, or regulating the expression of, at least one nucleic acid encoding at least two enteric antigens are discussed in detail below, and in the examples.
i. Chromosomally Integrated Nucleic Acid Sequence Encoding a Repressor
In one embodiment, the expression of a nucleic acid sequence encoding an enteric antigen is regulated by a chromosomally integrated nucleic acid sequence encoding a repressor and a vector. For instance, a recombinant bacterium of the invention that is capable of the regulated expression of a nucleic acid sequence encoding at least one enteric antigen may comprise, in part, at least one chromosomally integrated nucleic acid sequence encoding a repressor. Typically, the nucleic acid sequence encoding a repressor is operably linked to a regulatable promoter. The nucleic acid sequence encoding a repressor and/or the promoter may be modified from the wild-type nucleic acid sequence so as to optimize the expression level of the nucleic acid sequence encoding the repressor.
Methods of chromosomally integrating a nucleic acid sequence encoding a repressor operably-linked to a regulatable promoter are known in the art and detailed in the examples. Generally speaking, the nucleic acid sequence encoding a repressor should not be integrated into a locus that disrupts colonization of the host by the recombinant bacterium, or attenuates the bacterium. In one embodiment, the nucleic acid sequence encoding a repressor may be integrated into the relA nucleic acid sequence. In another embodiment, the nucleic acid sequence encoding a repressor may be integrated into the endA nucleic acid sequence.
In some embodiments, at least one nucleic acid sequence encoding a repressor is chromosomally integrated. In other embodiments, at least two, or at least three nucleic acid sequences encoding repressors may be chromosomally integrated into the recombinant bacterium. If there is more than one nucleic acid sequence encoding a repressor, each nucleic acid sequence encoding a repressor may be operably linked to a regulatable promoter, such that each promoter is regulated by the same compound or condition. Alternatively, each nucleic acid sequence encoding a repressor may be operably linked to a regulatable promoter, each of which is regulated by a different compound or condition.
As used herein, “repressor” refers to a biomolecule that represses transcription from one or more promoters. Generally speaking, a suitable repressor of the invention is synthesized in high enough quantities during the in vitro growth of the bacterial strain to repress the transcription of the nucleic acid encoding an antigen of interest on the vector, as detailed below, and not impede the in vitro growth of the strain. Additionally, a suitable repressor will generally be substantially stable, i.e. not subject to proteolytic breakdown. Furthermore, a suitable repressor will be diluted by about half at every cell division after expression of the repressor ceases, such as in a non-permissive environment (e.g. an animal or human host).
The choice of a repressor depends, in part, on the species of the recombinant bacterium used. For instance, the repressor is usually not derived from the same species of bacteria as the recombinant bacterium. For instance, the repressor may be derived from E. coli if the recombinant bacterium is from the genus Salmonella. Alternatively, the repressor may be from a bacteriophage.
Suitable repressors are known in the art, and may include, for instance, LacI of E. coli, C2 encoded by bacteriophage P22, or C1 encoded by bacteriophage λ. Other suitable repressors may be repressors known to regulate the expression of a regulatable nucleic acid sequence, such as nucleic acid sequences involved in the uptake and utilization of sugars. In one embodiment, the repressor is LacI. In another embodiment, the repressor is C2. In yet another embodiment, the repressor is C1.
The chromosomally integrated nucleic acid sequence encoding a repressor is operably linked to a regulatable promoter. The term “promoter”, as used herein, may mean a synthetic or naturally-derived molecule that is capable of conferring, activating or enhancing expression of a nucleic acid. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of a nucleic acid. The term “operably linked,” as used herein, means that expression of a nucleic acid is under the control of a promoter with which it is spatially connected. A promoter may be positioned 5′ (upstream) of the nucleic acid under its control. The distance between the promoter and a nucleic acid to be expressed may be approximately the same as the distance between that promoter and the native nucleic acid sequence it controls. As is known in the art, variation in this distance may be accommodated without loss of promoter function.
The regulated promoter used herein generally allows transcription of the nucleic acid sequence encoding a repressor while in a permissive environment (i.e. in vitro growth), but ceases transcription of the nucleic acid sequence encoding a repressor while in a non-permissive environment (i.e. during growth of the bacterium in an animal or human host). For instance, the promoter may be sensitive to a physical or chemical difference between the permissive and non-permissive environment. Suitable examples of such regulatable promoters are known in the art.
In some embodiments, the promoter may be responsive to the level of arabinose in the environment. Generally speaking, arabinose may be present during the in vitro growth of a bacterium, while typically absent from host tissue. In one embodiment, the promoter is derived from an araC-PBAD system. The araC-PBAD system is a tightly regulated expression system that has been shown to work as a strong promoter induced by the addition of low levels of arabinose. The araC-araBAD promoter is a bidirectional promoter controlling expression of the araBAD nucleic acid sequences in one direction, and the araC nucleic acid sequence in the other direction. For convenience, the portion of the araC-araBAD promoter that mediates expression of the araBAD nucleic acid sequences, and which is controlled by the araC nucleic acid sequence product, is referred to herein as PBAD. For use as described herein, a cassette with the araC nucleic acid sequence and the araC-araBAD promoter may be used. This cassette is referred to herein as araC-PBAD. The AraC protein is both a positive and negative regulator of PBAD. In the presence of arabinose, the AraC protein is a positive regulatory element that allows expression from PBAD. In the absence of arabinose, the AraC protein represses expression from PBAD. This can lead to a 1,200-fold difference in the level of expression from PBAD.
Other enteric bacteria contain arabinose regulatory systems homologous to the araC araBAD system from E. coli. For example, there is homology at the amino acid sequence level between the E. coli and the S. Typhimurium AraC proteins, and less homology at the DNA level. However, there is high specificity in the activity of the AraC proteins. For example, the E. coli AraC protein activates only E. coli PBAD (in the presence of arabinose) and not S. Typhimurium PBAD. Thus, an arabinose regulated promoter may be used in a recombinant bacterium that possesses a similar arabinose operon, without substantial interference between the two, if the promoter and the operon are derived from two different species of bacteria.
Generally speaking, the concentration of arabinose necessary to induce expression is typically less than about 2%. In some embodiments, the concentration is less than about 1.5%, 1%, 0.5%, 0.2%, 0.1%, or 0.05%. In other embodiments, the concentration is 0.05% or below, e.g. about 0.04%, 0.03%, 0.02%, or 0.01%. In an exemplary embodiment, the concentration is about 0.05%.
In other embodiments, the promoter may be responsive to the level of maltose in the environment. Generally speaking, maltose may be present during the in vitro growth of a bacterium, while typically absent from host tissue. The malT nucleic acid encodes MalT, a positive regulator of four maltose-responsive promoters (PPQ, PEFG, PKBM, and PS). The combination of malT and a mal promoter creates a tightly regulated expression system that has been shown to work as a strong promoter induced by the addition of maltose (6). Unlike the araC-PBAD system, malT is expressed from a promoter (PT) functionally unconnected to the other mal promoters. PT is not regulated by MalT. The malEFG-malKBM promoter is a bidirectional promoter controlling expression of the malKBM nucleic acid sequences in one direction, and the malEFG nucleic acid sequences in the other direction. For convenience, the portion of the malEFG-malKBM promoter that mediates expression of the malKBM nucleic acid sequence, and which is controlled by the malT nucleic acid sequence product, is referred to herein as PKBM, and the portion of the malEFG-malKBM promoter that mediates expression of the malEFG nucleic acid sequence, and that is controlled by the malT nucleic acid sequence product, is referred to herein as PEFG. Full induction of PKBM requires the presence of the MalT binding sites of PEFG. For use in the vectors and systems described herein, a cassette with the malT nucleic acid sequence and one of the mal promoters may be used. This cassette is referred to herein as ma/T-Pmal. In the presence of maltose, the MalT protein is a positive regulatory element that allows expression from Pmal.
In still other embodiments, the promoter may be sensitive to the level of rhamnose in the environment. Analogous to the araC-PBAD system described above, the rhaRS-PrhaB activator-promoter system is tightly regulated by rhamnose. Expression from the rhamnose promoter (Prha) is induced to high levels by the addition of rhamnose, which is common in bacteria but rarely found in host tissues. The nucleic acid sequences rhaBAD are organized in one operon that is controlled by the PrhaBAD promoter. This promoter is regulated by two activators, RhaS and RhaR, and the corresponding nucleic acid sequences belong to one transcription unit that is located in the opposite direction of the rhaBAD nucleic acid sequences. If L-rhamnose is available, RhaR binds to the PrhaRS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose in turn binds to the PrhaBAD and the PrhaT promoter and activates the transcription of the structural nucleic acid sequences. Full induction of rhaBAD transcription also requires binding of the Crp-cAMP complex, which is a key regulator of catabolite repression.
Although both L-arabinose and L-rhamnose act directly as inducers for expression of regulons for their catabolism, important differences exist in regard to the regulatory mechanisms. L-Arabinose acts as an inducer with the activator AraC in the positive control of the arabinose regulon. However, the L-rhamnose regulon is subject to a regulatory cascade; it is therefore subject to even tighter control than the araC PBAD system. L-Rhamnose acts as an inducer with the activator RhaR for synthesis of RhaS, which in turn acts as an activator in the positive control of the rhamnose regulon. In the present invention, rhamnose may be used to interact with the RhaR protein and then the RhaS protein may activate transcription of a nucleic acid sequence operably-linked to the PrhaBAD promoter.
In still other embodiments, the promoter may be sensitive to the level of xylose in the environment. The xylR-PxylA system is another well-established inducible activator-promoter system. Xylose induces xylose-specific operons (xylE, xylFGHR, and xylAB) regulated by XylR and the cyclic AMP-Crp system. The XylR protein serves as a positive regulator by binding to two distinct regions of the xyl nucleic acid sequence promoters. As with the araC-PBAD system described above, the xylR-PxylAB and/or xylR-PxylFGH regulatory systems may be used in the present invention. In these embodiments, xylR PxylAB xylose interacting with the XylR protein activates transcription of nucleic acid sequences operably-linked to either of the two Pxyl promoters.
The nucleic acid sequences of the promoters detailed herein are known in the art, and methods of operably-linking them to a chromosomally integrated nucleic acid sequence encoding a repressor are known in the art and detailed in the examples.
A nucleic acid sequence encoding a repressor and regulatable promoter detailed above, for use in the present invention, may be modified so as to optimize the expression level of the nucleic acid sequence encoding the repressor. The optimal level of expression of the nucleic acid sequence encoding the repressor may be estimated, or may be determined by experimentation. Such a determination should take into consideration whether the repressor acts as a monomer, dimer, trimer, tetramer, or higher multiple, and should also take into consideration the copy number of the vector encoding the antigen of interest, as detailed below. In an exemplary embodiment, the level of expression is optimized so that the repressor is synthesized while in the permissive environment (i.e. in vitro growth) at a level that substantially inhibits the expression of a nucleic acid sequence encoding an enteric antigen, and is substantially not synthesized in a non-permissive environment, thereby allowing expression of the nucleic acid encoding an enteric antigen.
As stated above, the level of expression may be optimized by modifying the nucleic acid sequence encoding the repressor and/or promoter. As used herein, “modify” refers to an alteration of the nucleic acid sequence of the repressor and/or promoter that results in a change in the level of transcription of the nucleic acid sequence encoding the repressor, or that results in a change in the level of synthesis of the repressor. For instance, in one embodiment, modify may refer to altering the start codon of the nucleic acid sequence encoding the repressor. Generally speaking, a GTG or TTG start codon, as opposed to an ATG start codon, may decrease translation efficiency ten-fold. In another embodiment, modify may refer to altering the Shine-Dalgarno (SD) sequence of the nucleic acid sequence encoding the repressor. The SD sequence is a ribosomal binding site generally located 6-7 nucleotides upstream of the start codon. The SD consensus sequence is AGGAGG, and variations of the consensus sequence may alter translation efficiency. In yet another embodiment, modify may refer to altering the distance between the SD sequence and the start codon. In still another embodiment, modify may refer to altering the −35 sequence for RNA polymerase recognition. In a similar embodiment, modify may refer to altering the −10 sequence for RNA polymerase binding. In an additional embodiment, modify may refer to altering the number of nucleotides between the −35 and −10 sequences. In an alternative embodiment, modify may refer to optimizing the codons of the nucleic acid sequence encoding the repressor to alter the level of translation of the mRNA encoding the repressor. For instance, non-A rich codons initially after the start codon of the nucleic acid sequence encoding the repressor may not maximize translation of the mRNA encoding the repressor. Similarly, the codons of the nucleic acid sequence encoding the repressor may be altered so as to mimic the codons from highly synthesized proteins of a particular organism. In a further embodiment, modify may refer to altering the GC content of the nucleic acid sequence encoding the repressor to change the level of translation of the mRNA encoding the repressor.
In some embodiments, more than one modification or type of modification may be performed to optimize the expression level of the nucleic acid sequence encoding the repressor. For instance, at least one, two, three, four, five, six, seven, eight, or nine modifications, or types of modifications, may be performed to optimize the expression level of the nucleic acid sequence encoding the repressor.
By way of non-limiting example, when the repressor is LacI, then the nucleic acid sequence of LacI and the promoter may be altered so as to increase the level of LacI synthesis. In one embodiment, the start codon of the LacI repressor may be altered from GTG to ATG. In another embodiment, the SD sequence may be altered from AGGG to AGGA. In yet another embodiment, the codons of lacI may be optimized according to the codon usage for highly synthesized proteins of Salmonella. In a further embodiment, the start codon of lacI may be altered, the SD sequence may be altered, and the codons of lacI may be optimized.
Methods of modifying the nucleic acid sequence encoding the repressor and/or the regulatable promoter are known in the art and detailed in the examples.
In some embodiments, the chromosomally integrated nucleic acid sequence encoding the repressor further comprises a transcription termination sequence. A transcription termination sequence may be included to prevent inappropriate expression of nucleic acid sequences adjacent to the chromosomally integrated nucleic acid sequence encoding the repressor and regulatable promoter.
A recombinant bacterium of the invention may also comprise a vector. For instance a bacterium that is capable of the regulated expression of at least one nucleic acid sequence encoding at least one enteric antigen may also comprise, in part, a vector. The vector comprises a nucleic acid sequence encoding at least one enteric antigen operably linked to a promoter. The promoter is preferably regulated by the chromosomally encoded repressor, such that the expression of the nucleic acid sequence encoding an antigen is repressed during in vitro growth of the bacterium, but the bacterium is capable of high level synthesis of the antigen in an animal or human host. In some cases, however, such regulated expression is not necessary, such as for expression of fimbrial adhesins encoded on a low copy number vector or where the synthesis of the enteric protective antigen does not compromise the growth and/or colonizing ability of the vaccine strain.
As used herein, “vector” refers to an autonomously replicating nucleic acid unit. The present invention can be practiced with any known type of vector, including viral, cosmid, phasmid, and plasmid vectors. The most preferred type of vector is a plasmid vector.
As is well known in the art, plasmids and other vectors may possess a wide array of promoters, multiple cloning sequences, transcription terminators, etc., and vectors may be selected so as to control the level of expression of the nucleic acid sequence encoding an antigen by controlling the relative copy number of the vector. In some instances in which the vector might encode a surface localized adhesin as the antigen, or an antigen capable of stimulating T-cell immunity, it may be preferable to use a vector with a low copy number such as at least two, three, four, five, six, seven, eight, nine, or ten copies per bacterial cell. A non-limiting example of a low copy number vector may be a vector comprising the pSC101 ori.
In other cases, an intermediate copy number vector might be optimal for inducing desired immune responses. For instance, an intermediate copy number vector may have at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 copies per bacterial cell. A non-limiting example of an intermediate copy number vector may be a vector comprising the p15A ori.
In still other cases, a high copy number vector might be optimal for the induction of maximal antibody responses or mucosal immune responses. A high copy number vector may have at least 31, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 copies per bacterial cell. In some embodiments, a high copy number vector may have at least 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 copies per bacterial cell. Non-limiting examples of high copy number vectors may include a vector comprising the pBR on or the pUC ori.
Additionally, vector copy number may be increased by selecting for mutations that increase plasmid copy number. These mutations may occur in the bacterial chromosome but are more likely to occur in the plasmid vector.
Preferably, vectors used herein do not comprise antibiotic resistance markers to select for maintenance of the vector.
A vector may comprise at least one nucleic acid sequence encoding at least two enteric antigens as detailed above.
The vector may comprise a nucleic acid sequence encoding at least one enteric antigen operably-linked to a promoter regulated by the repressor, encoded by a chromosomally integrated nucleic acid sequence. One of skill in the art would recognize, therefore, that the selection of a repressor dictates, in part, the selection of the promoter operably-linked to a nucleic acid sequence encoding an antigen of interest. For instance, if the repressor is LacI, then the promoter may be selected from the group consisting of LacI responsive promoters, such as Ptrc, Plac, PT7lac and Ptac. If the repressor is C2, then the promoter may be selected from the group consisting of C2 responsive promoters, such as P22 promoters PL and PR. If the repressor is C1, then the promoter may be selected from the group consisting of C1 responsive promoters, such as λ promoters PL and PR.
In each embodiment herein, the promoter regulates expression of a nucleic acid sequence encoding the antigen, such that expression of the nucleic acid sequence encoding an antigen is repressed when the repressor is synthesized (i.e. during in vitro growth of the bacterium), but expression of the nucleic acid sequence encoding an antigen is high when the repressor is not synthesized (i.e. in an animal or human host). Generally speaking, the concentration of the repressor will decrease with every cell division after expression of the nucleic acid sequence encoding the repressor ceases. In some embodiments, the concentration of the repressor decreases enough to allow high level expression of the nucleic acid sequence encoding an enteric antigen after about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 divisions of the bacterium. In an exemplary embodiment, the concentration of the repressor decreases enough to allow high level expression of the nucleic acid sequence encoding an enteric antigen after about 5 divisions of the bacterium in an animal or human host.
In certain embodiments, the promoter may comprise other regulatory elements. For instance, the promoter may comprise lacO if the repressor is LacI. This is the case with the lipoprotein promoter Plpp that is regulated by LacI since it possesses the LacI binding domain lacO.
In one embodiment, the repressor is a LacI repressor and the promoter is Ptrc.
As detailed above, generally speaking the expression of the nucleic acid sequence encoding the enteric antigen should be repressed when the repressor is synthesized. For instance, if the repressor is synthesized during in vitro growth of the bacterium, expression of the nucleic acid sequence encoding the enteric antigen should be repressed. Expression may be “repressed” or “partially repressed” when it is about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or even less than 1% of the expression under non-repressed conditions. Thus although the level of expression under conditions of “complete repression” might be exceeding low, it is likely to be detectable using very sensitive methods since repression can never by absolute.
Conversely, the expression of the nucleic acid sequence encoding the antigen should be high when the expression of the nucleic acid sequence encoding the repressor is repressed. For instance, if the nucleic acid sequence encoding the repressor is not expressed during growth of the recombinant bacterium in the host, the expression of the nucleic acid sequence encoding the antigen should be high. As used herein, “high level” expression refers to expression that is strong enough to elicit an immune response to the antigen. Consequently, the copy number correlating with high level expression can and will vary depending on the antigen and the type of immune response desired. Methods of determining whether an antigen elicits an immune response such as by measuring antibody levels or antigen-dependant T cell populations or antigen-dependant cytokine levels are known in the art, and methods of measuring levels of expression of antigen encoding sequences by measuring levels of mRNA transcribed or by quantitating the level of antigen synthesis are also known in the art. For more details, see the examples.
ii. Other Ways of Regulating the Expression of a Nucleic Acid Encoding at Least One Enteric Antigen
The invention also encompasses other means of regulating the expression of a nucleic acid sequence encoding at least one enteric antigen in a recombinant bacterium. For instance, in one embodiment, the enteric antigen of interest may be encoded on an extra-chromosomal vector. This can be used in the context of a balanced-lethal host-vector or balanced-attenuation host-vector system. Alternatively, the nucleotide sequence encoding the antigen of interest may be inserted into the chromosome but have its expression controlled by a regulatable system, e.g., LacI or C2, as with the regulated gene encoding the antigen of interest on an extra-chromosomal vector (e.g., a plasmid).
In each of the above embodiments, a recombinant bacterium of the invention may also be attenuated. “Attenuated” refers to the state of the bacterium wherein the bacterium has been weakened from its wild type fitness by some form of recombinant or physical manipulation. This includes altering the genotype of the bacterium to reduce its ability to cause disease. However, the bacterium's ability to colonize the gastrointestinal tract (in the case of Salmonella) and induce immune responses is, preferably, not substantially compromised. For instance, in one embodiment, regulated attenuation allows the recombinant bacterium to express one or more nucleic acids encoding products important for the bacterium to withstand stresses encountered in the host after immunization. This allows efficient invasion and colonization of lymphoid tissues before the recombinant bacterium is regulated to display the attenuated phenotype.
In one embodiment, a recombinant bacterium may be attenuated as described in section I(a)i above, i.e. regulating LPS O-antigen. In another embodiment, a recombinant bacterium may be attenuated as described in section (c)i below. In which case, both regulated attenuation and regulated expression of an enteric antigen encoding sequence may be dependent upon an arabinose regulatable system. Consequently, the concentration of arabinose needed for optimal expression of the regulated enteric antigen encoding sequence may not be the same as the concentration for optimal expression of attenuation. In an exemplary embodiment, the concentration of arabinose for the optimization of both regulated attenuation and regulated expression of sequences encoding antigen will be substantially the same.
Accordingly, the promoter and/or the nucleic acid sequence encoding an attenuation protein may be modified to optimize the system. Methods of modification are detailed above. Briefly, for example, the SD ribosome binding sequence may be altered, and/or the start codon may be altered from ATG to GTG for the nucleic acid sequences fur and phoPQ, so that the production levels of Fur and PhoPQ are optimal for both the regulated attenuation phenotype and the regulated expression when growing strains with a given concentration of arabinose. One of skill in the art will appreciate that other nucleic acid sequences, in addition to fur and phoPQ, may also be altered as described herein in combination with other well-known protocols. In addition, these attenuating nucleic acid sequences may be regulated by other systems using well-established protocols known to one of skill in the art. For example, they may be regulated using with promoters dependent on addition of maltose, rhamnose, or xylose rather than arabinose.
Other methods of attenuation are known in the art. For instance, attenuation may be accomplished by altering (e.g., deleting) native nucleic acid sequences found in the wild type bacterium. For instance, if the bacterium is Salmonella, non-limiting examples of nucleic acid sequences which may be used for attenuation include: a pab nucleic acid sequence, a pur nucleic acid sequence, an aro nucleic acid sequence, asd, a dap nucleic acid sequence, nadA, pncB, galE, pmi, fur, rpsL, ompR, htrA, hemA, cdt, cya, crp, dam, phoP, phoQ, rfc, poxA, galU, mviA, sodC, recA, ssrA, sirA, inv, hilA, rpoE, flgM, tonB, slyA, and any combination thereof. Exemplary attenuating mutations may be aroA, aroC, aroD, cdt, cya, crp, phoP, phoQ, ompR, galE, and htrA.
In certain embodiments, the above nucleic acid sequences may be placed under the control of a sugar regulated promoter wherein the sugar is present during in vitro growth of the recombinant bacterium, but substantially absent within an animal or human host. The cessation in transcription of the nucleic acid sequences listed above would then result in attenuation and the inability of the recombinant bacterium to induce disease symptoms.
The bacterium may also be modified to create a balanced-lethal host-vector system, although other types of systems may also be used (e.g., creating complementation heterozygotes). For the balanced-lethal host-vector system, the bacterium may be modified by manipulating its ability to synthesize various essential constituents needed for synthesis of the rigid peptidoglycan layer of its cell wall. In one example, the constituent is diaminopimelic acid (DAP). Various enzymes are involved in the eventual synthesis of DAP. In one example, the bacterium is modified by using a ΔasdA mutation to eliminate the bacterium's ability to produce β-aspartate semialdehyde dehydrogenase, an enzyme essential for the synthesis of DAP. One of skill in the art can also use the teachings of U.S. Pat. No. 6,872,547 for other types of mutations of nucleic acid sequences that result in the abolition of the synthesis of DAP. These nucleic acid sequences may include, but are not limited to, dapA, dapB, dapC, dapD, dapE, dapF, and asd. Other modifications that may be employed include modifications to a bacterium's ability to synthesize D-alanine or to synthesize D-glutamic acid (e.g., Δmurl mutations), which are both unique constituents of the peptidoglycan layer of the bacterial cell wall
Yet another balanced-lethal host-vector system comprises modifying the bacterium such that the synthesis of an essential constituent of the rigid layer of the bacterial cell wall is dependent on a nutrient (e.g., arabinose) that can be supplied during the growth of the microorganism. For example, a bacterium may comprise the ΔPmurA::TT araC PBAD murA deletion-insertion mutation. This type of mutation makes synthesis of muramic acid (another unique essential constituent of the peptidoglycan layer of the bacterial cell wall) dependent on the presence of arabinose that can be supplied during growth of the bacterium in vitro.
When arabinose is absent, however, as it is in an animal or human host, the essential constituent of the peptidoglycan layer of the cell wall is not synthesized. This mutation represents an arabinose dependant lethal mutation. In the absence of arabinose, synthesis of muramic acid ceases and lysis of the bacterium occurs because the peptidoglycan layer of the cell wall is not synthesized. It is not possible to generate AmurA mutations because they are lethal. The necessary nutrient, a phosphorylated muramic acid, cannot be exogenously supplied because enteric bacteria cannot take the nutrient up from the media. Recombinant bacteria with a ΔPmurA::TT araC PBAD murA deletion-insertion mutation grown in the presence of arabinose exhibit effective colonization of effector lymphoid tissues after oral vaccination prior to undergoing lysis due to the inability to synthesize muramic acid.
Similarly, various embodiments may comprise the araC PBAD c2 cassette inserted into the asd nucleic acid sequence that encodes aspartate semialdehyde dehydrogenase. Since the araC nucleic acid sequence is transcribed in a direction that could lead to interference in the expression of adjacent nucleic acid sequences and adversely affect vaccine strain performance, a transcription termination (TT) sequence is generally inserted 3′ to the araC nucleic acid sequence. The chromosomal asd nucleic acid sequence is typically inactivated to enable use of plasmid vectors encoding the wild-type asd nucleic acid sequence in the balanced lethal host-vector system. This allows stable maintenance of plasmids in vivo in the absence of any drug resistance attributes that are not permissible in live bacterial vaccines. In some of these embodiments, the wild-type asd nucleic acid sequence may be encoded by the vector described above.
In one embodiment, ΔasdA27::TT araC PBAD c2 has an improved SD sequence and a codon optimized c2 nucleic acid sequence. The C2 repressor synthesized in the presence of arabinose is used to repress nucleic acid sequence expression from P22 PR and PL promoters. In another embodiment, ΔasdA27::TT araC PBAD c2 has the 1104 base-pair asd nucleic acid sequence deleted (1 to 1104, but not including the TAG stop codon) and the 1989 base-pair fragment containing T4 ipIII TT araC PBAD c2 inserted. The c2 nucleic acid sequence in ΔasdA27::TT araC PBAD c2 has a SD sequence that was optimized to TAAGGAGGT. It also has an improved PBAD promoter such that the −10 sequence is improved from TACTGT to TATAAT. Furthermore, it has a codon optimized c2 nucleic acid sequence, in which the second codon was modified from AAT to AAA.
In further embodiments, the bacterium may be attenuated by regulating the murA nucleic acid sequence encoding the first enzyme in muramic acid synthesis and the asd nucleic acid sequence essential for DAP synthesis. These embodiments may comprise the chromosomal deletion-insertion mutations ΔasdA19::TT araC PBAD c2 or ΔasdA27::TT araC PBAD c2 and ΔPmurA7::TT araC PBAD murA or ΔPmurA12::TT araC PBAD murA or ΔPmurA25::TT araC PBAD murA. This host-vector grows in LB broth with 0.1% L-arabinose, but is unable to grow in or on media devoid of arabinose since it undergoes cell wall-less death by lysis. In some embodiments of the invention, the recombinant bacterium may comprise araBAD and araE mutations to preclude breakdown and leakage of internalized arabinose such that asd and murA nucleic acid sequence expression continues for a cell division or two after oral immunization into an environment that is devoid of external arabinose. (For example a strain with the ΔPmurA7::TT araC PBAD murA deletion-insertion mutation undergoes about two cell divisions and then commences to lyse in media made of mouse or chicken feed or chicken breast meat, unless they are supplemented with arabinose). Either GTG or TTG start codons for the murA and asd nucleic acid sequences are important to decrease translation efficiency on multi-copy plasmids. For instance plasmid vector pYA3681 (
i. Regulated Attenuation
The present invention also encompasses a recombinant bacterium capable of regulated attenuation. Generally speaking, the bacterium comprises a chromosomally integrated regulatable promoter. The promoter replaces the native promoter of, and is operably linked to, at least one nucleic acid sequence encoding an attenuation protein, such that the absence of the function of the protein renders the bacterium attenuated. In some embodiments, the promoter is modified to optimize the regulated attenuation.
In each of the above embodiments described herein, more than one method of attenuation may be used. For instance, a recombinant bacterium of the invention may comprise a regulatable promoter chromosomally integrated so as to replace the native promoter of, and be operably linked to, at least one nucleic acid sequence encoding an attenuation protein, such that the absence of the function of the protein renders the bacterium attenuated, and the bacterium may comprise another method of attenuation detailed in section I above.
Herein, “attenuation protein” is meant to be used in its broadest sense to encompass any protein the absence of which attenuates a bacterium. For instance, in some embodiments, an attenuation protein may be a protein that helps protect a bacterium from stresses encountered in the gastrointestinal tract or respiratory tract. Non-limiting examples may be the RpoS, PhoPQ, OmpR, Fur, and Crp proteins. In other embodiments, the protein may be necessary to synthesize a component of the cell wall of the bacterium, or may itself be a necessary component of the cell wall such as the protein encoded by murA. In still other embodiments, the protein may be listed in Section i above.
The native promoter of at least one, two, three, four, five, or more than five attenuation proteins may be replaced by a regulatable promoter as described herein. In one embodiment, the promoter of one of the proteins selected from the group comprising RpoS, PhoPQ, OmpR, Fur, and Crp may be replaced. In another embodiment, the promoter of two, three, four or five of the proteins selected from the group comprising RpoS, PhoPQ, OmpR, Fur, and Crp may be replaced.
If the promoter of more than one attenuation protein is replaced, each promoter may be replaced with a regulatable promoter, such that the expression of each attenuation protein encoding sequence is regulated by the same compound or condition. Alternatively, each promoter may be replaced with a different regulatable promoter, such that the expression of each attenuation protein encoding sequence is regulated by a different compound or condition such as by the sugars arabinose, maltose, rhamnose or xylose.
The native promoter of a nucleic acid encoding an attenuation protein is replaced with a regulatable promoter operably linked to the nucleic acid sequence encoding an attenuation protein. The term “operably linked,” is defined above.
The regulatable promoter used herein generally allows transcription of the nucleic acid sequence encoding the attenuation protein while in a permissive environment (i.e. in vitro growth), but cease transcription of the nucleic acid sequence encoding an attenuation protein while in a non-permissive environment (i.e. during growth of the bacterium in an animal or human host). For instance, the promoter may be responsive to a physical or chemical difference between the permissive and non-permissive environment. Suitable examples of such regulatable promoters are known in the art and detailed above.
In some embodiments, the promoter may be responsive to the level of arabinose in the environment, as described above. In other embodiments, the promoter may be responsive to the level of maltose, rhamnose, or xylose in the environment, as described above. The promoters detailed herein are known in the art, and methods of operably linking them to a nucleic acid sequence encoding an attenuation protein are known in the art.
In certain embodiments, a recombinant bacterium of the invention may comprise any of the following: ΔPfur::TT araC PBAD fur, ΔPcrp::TT araC PBAD crp, ΔPphoPQ::TT araC PBAD phoPQ, or a combination thereof. Growth of such strains in the presence of arabinose leads to transcription of the fur, phoPQ, and/or crp nucleic acid sequences, but nucleic acid sequence expression ceases in a host because there is no free arabinose. Attenuation develops as the products of the fur, phoPQ, and/or the crp nucleic acid sequences are diluted at each cell division. Strains with the ΔPfur and/or the ΔPphoPQ mutations are attenuated at oral doses of 109 CFU, even in three-week old mice at weaning. Generally speaking, the concentration of arabinose necessary to induce expression is typically less than about 2%. In some embodiments, the concentration is less than about 1.5%, 1%, 0.5%, 0.2%, 0.1%, or 0.05%. In certain embodiments, the concentration may be about 0.04%, 0.03%, 0.02%, or 0.01%. In an exemplary embodiment, the concentration is about 0.05%. Higher concentrations of arabinose or other sugars may lead to acid production during growth that may inhibit desirable cell densities. The inclusion of mutations such as ΔaraBAD or mutations that block the uptake and/or breakdown of maltose, rhamnose, or xylose, however, may prevent such acid production and enable use of higher sugar concentrations with no ill effects.
When the regulatable promoter is responsive to arabinose, the onset of attenuation may be delayed by including additional mutations, such as ΔaraBAD23, which prevents use of arabinose retained in the cell cytoplasm at the time of oral immunization, and/or ΔaraE25 that enhances retention of arabinose. Thus, inclusion of these mutations may be beneficial in at least two ways: first, enabling higher culture densities, and second enabling a further delay in the display of the attenuated phenotype that may result in higher densities in effector lymphoid tissues to further enhance immunogenicity.
Attenuation of the recombinant bacterium may be optimized by modifying the nucleic acid sequence encoding an attenuation protein and/or promoter. Methods of modifying a promoter and/or a nucleic acid sequence encoding an attenuation protein are the same as those detailed above with respect to repressors in Section (b).
In some embodiments, more than one modification may be performed to optimize the attenuation of the bacterium. For instance, at least one, two, three, four, five, six, seven, eight or nine modifications may be performed to optimize the attenuation of the bacterium.
In various exemplary embodiments of the invention, the SD sequences and/or the start codons for the fur and/or the phoPQ virulence nucleic acid sequences may be altered so that the production levels of these nucleic acid products are optimal for regulated attenuation.
A recombinant bacterium of the invention may be capable of eliciting an immune response against at least two Salmonella serotypes. This may be accomplished, for instance, by eliminating the serotype-specific LPS O-antigen side chains as discussed above. The remaining LPS core will elicit an immune response, inducing the production of antibodies against the LPS core. Since this LPS core is substantially identical in the several thousand Salmonella enterica serotypes, the antibodies potentially provide immunity against several diverse Salmonella enterica serotypes, such as Typhimurium, Heidelberg, Newport, Infantis, Dublin, Virchow, Typhi, Enteritidis, Berta, Seftenberg, Ohio, Agona, Braenderup, Hadar, Kentucky, Thompson, Montevideo, Mbandaka, Javiana, Oranienburg, Anatum, Paratyphi A, Schwarzengrund, Saintpaul, and Munchen.
In addition, the elimination of the LPS O-antigen provides the host immune system with better access to the outer membrane proteins of the recombinant bacterium, thereby enhancing induction of immune responses against these outer membrane proteins. In some embodiments, as described below, the outer membrane proteins may be upregulated to further enhance host immune responses to these proteins. Non-limiting examples of outer membrane proteins include proteins involved in iron and manganese uptake, as described below. Iron and manganese are essential nutrients for enteric pathogens and the induction of antibodies that inhibit iron and manganese uptake in effect starves the pathogens, conferring protective immunity on the host. Additionally, since these proteins are homologous among the enteric bacteria, such host immune responses provide immunity against multiple Salmonella enterica serotypes as well as to other enteric bacterial pathogens such as strains of Yersinia, Shigella and Escherichia. As evidence of this, the attenuated S. Typhimurium vaccine vector strain not expressing any Yersinia antigen is able to induce significant protective immunity to high doses of orally administered Y. pseudotuberculosis (see Example 9 and
The elicited immune response may include, but is not limited to, an innate immune response, a humoral immune response and a cell-mediated immune response. In one embodiment, Th2-dependent mucosal and systemic antibody responses to the enteric antigen(s) are observed. Immune responses may be measured by standard immunological assays known to one of skill in the art. In an exemplary embodiment, the immune response is protective.
Generally speaking, a recombinant bacterium of the invention is also capable of eliciting an immune response against at least two enteric pathogens in addition to Salmonella. This may be accomplished, for instance, by regulating the expression of an enteric antigen as described above. In an alternative embodiment, the enteric antigen may be an iron-regulated outer membrane protein (IROMP) or a manganese-regulated outer membrane protein (MnROMP). For instance, the mutation ΔPfur::TT araC PBAD fur in a Salmonella recombinant bacterium may cause up-regulation of IROMPS while the bacterium is growing in the host. This up-regulation may elicit a host immune response against the IROMPS of the Salmonella recombinant bacterium that cross-reacts with similar proteins from Shigella, E. coli, and Yersinia.
An “enteric pathogen,” as used herein, refers to a pathogen capable of invading and colonizing the intestines of a host and causing pathology. The pathology, however, is not limited to the gastrointestinal tract. Many enteric pathogens cause extra intestinal diseases such as typhoid fever, meningitis, septicemia, and urinary tract infections, and use the intestines as a route of entry or as a reservoir from which to spread to other sites of entry. Additionally, some enteric bacteria can invade and colonize via the respiratory tract. This is most common in birds, such as chickens and turkeys that scratch to aerosolize enteric bacteria that can invade the lungs and airsacs during breathing. These same enteric bacteria are also ingested by birds such that they are equally capable of residing in the intestinal tract. In either case, these enteric bacteria are still able to colonize internal tissues to cause disease independent of whether the invasion occurred via the gastrointestinal or respiratory tracks. Non-limiting examples of enteric pathogens may include E. coli serotypes O124, O86:B7, H37, O27, O124:B17, O6:H16, O25:H+, O27:H2O, O157:H7; Shigella Serogroup A or S. dysenteriae (12 serotypes), Serogroup B or S. flexneri (6 serotypes), Serogroup C or S. boydii (23 serotypes), Serogroup D or S. sonnei (1 serotype); Clostridium perfringens; Yersinia enterocolitica; Yersina pseudotuberculosis; and Campylobacter jejuni.
The elicited immune response may include, but is not limited to, an innate immune response, a humoral immune response and a cell-mediated immune response. In one embodiment, Th2-dependent mucosal and systemic antibody responses to the enteric antigen(s) are observed. Immune responses may be measured by standard immunological assays known to one of skill in the art. In an exemplary embodiment, the immune response is protective.
In some embodiments, a recombinant bacterium of the invention may also comprise a ΔPcrp::TT araC PBAD crp deletion-insertion mutation. Since the araC PBAD cassette is dependent both on the presence of arabinose and the binding of the catabolite repressor protein Crp, a ΔPcrp::TT araC PBAD crp deletion-insertion mutation may be included as an additional means to reduce expression of any nucleic acid sequence under the control of the PBAD promoter. This means that when the bacterium is grown in a non-permissive environment (i.e. no arabinose) both the repressor itself and the Crp protein cease to be synthesized, consequently eliminating both regulating signals for the araC PBAD regulated nucleic acid sequence. This double shut off of araC PBAD may constitute an additional safety feature ensuring the genetic stability of the desired phenotypes.
Generally speaking, the activity of the Crp protein requires interaction with cAMP, but the addition of glucose, which may inhibit synthesis of cAMP, decreases the ability of the Crp protein to regulate transcription from the araC PBAD promoter. Consequently, to avoid the effect of glucose on cAMP, glucose may be substantially excluded from the growth media, or variants of crp may be isolated that synthesize a Crp protein that is not dependent on cAMP to regulate transcription from PBAD. This strategy may also be used in other systems responsive to Crp, such as the systems responsive to rhamnose and xylose described above.
In some embodiments, a recombinant bacterium of the invention may be modified so as to reduce fluid secretion in the host. For instance, the bacterium may comprise the ΔsopB1925 mutation. Alternatively, the bacterium may comprise the ΔmsbB48 mutation. For more details, see the Examples.
Under certain embodiments, a live recombinant bacterium may possess the potential to survive and multiply if excreted from a host. This leads to the possibility that individuals not electing to be immunized may be exposed to the recombinant bacterium. Consequently, in certain embodiments, a recombinant bacterium of the invention may comprise one or more mutations that decrease, if not preclude, the ability of Salmonella vaccines to persist in the GI tract of animals.
In another embodiment, a recombinant bacterium of the invention may comprise one or more of the Δ(gmd fcl)-26 or Δ(wcaM-wza)-7, ΔagfBAC811 or Δ(PagfD agfG)-4, ΔbcsABZC2118 or ΔbcsEFG2319 and Δ(yshA-yihW)-157 mutations that block synthesis of colanic acid, thin aggregative fimbriae (i.e., curli), cellulose and extracellular polysaccharide, respectively, all of which contribute to biofilm formation. In addition, the mutation ΔyhiR36 that prevents use of DNA as a nutrient, Δ(shdA-ratB)-64, ΔmisL2 and ΔbigA3 that encode four proteins that enable Salmonella to adhere to host extracellular matrix proteins and ΔackA233 that blocks use of acetate, may be used as a means for biological containment. In exemplary embodiments, a recombinant bacterium comprising a biological containment mutation are not adversely effected in their virulence.
In some embodiments, the recombinant bacterium may comprise a method of regulated delayed lysis in vivo that prevents bacterial persistence in vivo and survival if excreted. These chromosomal mutations may include: Δ(gmd fcl)-26 or Δ(wcaM-wza)-8 that precludes synthesis of colanic acid that can protect cells undergoing cell wall-less death from lysing completely, ΔagfBAC811 that blocks synthesis of thin aggregative fimbriae (curli) that are critical for biofilm formation to enable persistent colonization on bile stones in the gall bladder, ΔasdA27::TT araC PBAD c2 insertion-deletion mutation to impose a requirement for the peptidoglycan constituent DAP and ΔPmurAI2::TTaraC PBAD murA insertion-deletion mutation as a conditional-lethal mutation blocking synthesis of the peptidoglycan constituent muramic acid. The latter two mutations are typically complemented by a regulated delayed lysis plasmid vector such as pYA3681 (
A recombinant bacterium of the invention may be particularly suited for use as a vaccine. Infection of a host with a Salmonella strain typically leads to colonization of the gut-associated lymphoid tissue (GALT) or Peyer's patches, which leads to the induction of a generalized mucosal immune response to the recombinant bacterium. Further penetration of the bacterium into the mesenteric lymph nodes, liver and spleen may augment the induction of systemic and cellular immune responses directed against the bacterium. Thus the use of recombinant Salmonella for oral immunization stimulates all three branches of the immune system, which is particularly important for immunizing against infectious disease agents that colonize on and/or invade through mucosal surfaces.
A recombinant bacterium of the invention may be administered to a host as a vaccine composition. As used herein, a vaccine composition is a composition designed to elicit an immune response to the recombinant bacterium, including any antigens that may be expressed by the bacterium. In an exemplary embodiment, the immune response is protective. As used herein, “protective” means that the immune response contributes to the lessening of any symptoms associated with infection of a host with the pathogen the antigen was derived from or designed to elicit a response against. For example, a protective antigen from a pathogen, such as Shigella, may induce an immune response that helps to ameliorate symptoms associated with Shigella infection or reduce the morbidity and mortality associated with infection with the pathogen. The use of the term “protective” in this invention does not necessarily require that the host is completely protected from the effects of the pathogen.
Immune responses to antigens are well studied and widely reported. A survey of immunology is given by Paul, W E, Stites D et al. and Ogra P L. et al. Mucosal immunity is also described by Ogra P L et al.
Vaccine compositions of the present invention may be administered to any host capable of mounting an immune response. Such hosts may include all vertebrates, for example, mammals, including domestic animals, agricultural animals, laboratory animals, and humans, and various species of birds, including domestic birds and birds of agricultural importance. Preferably, the host is a warm-blooded animal. The vaccine can be administered as a prophylactic or for treatment purposes.
In exemplary embodiments, the recombinant bacterium is alive when administered to a host in a vaccine composition of the invention. Suitable vaccine composition formulations and methods of administration are detailed below.
A vaccine composition comprising a recombinant bacterium of the invention may optionally comprise one or more possible additives, such as carriers, preservatives, stabilizers, adjuvants, and other substances.
In one embodiment, the vaccine comprises an adjuvant. Adjuvants, such as aluminum hydroxide or aluminum phosphate, are optionally added to increase the ability of the vaccine to trigger, enhance, or prolong an immune response. In exemplary embodiments, the use of a live attenuated recombinant bacterium may act as a natural adjuvant. The vaccine compositions may further comprise additional components known in the art to improve the immune response to a vaccine, such as T cell co-stimulatory molecules or antibodies, such as anti-CTLA4. Additional materials, such as cytokines, chemokines, and bacterial nucleic acid sequences naturally found in bacteria, like CpG, are also potential vaccine adjuvants.
In another embodiment, the vaccine may comprise a pharmaceutical carrier (or excipient). Such a carrier may be any solvent or solid material for encapsulation that is non-toxic to the inoculated host and compatible with the recombinant bacterium. A carrier may give form or consistency, or act as a diluent. Suitable pharmaceutical carriers may include liquid carriers, such as normal saline and other non-toxic salts at or near physiological concentrations, and solid carriers not used for humans, such as talc or sucrose, or animal feed. Carriers may also include stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers. Carriers and excipients as well as formulations for parenteral and nonparenteral drug delivery are set forth in Remington's Pharmaceutical Sciences 19th Ed. Mack Publishing (1995). When used for administering via the bronchial tubes, the vaccine is preferably presented in the form of an aerosol.
Care should be taken when using additives so that the live recombinant bacterium is not killed, or have its ability to effectively colonize lymphoid tissues such as the GALT, NALT and BALT compromised by the use of additives. Stabilizers, such as lactose or monosodium glutamate (MSG), may be added to stabilize the vaccine formulation against a variety of conditions, such as temperature variations or a freeze-drying process.
The dosages of a vaccine composition of the invention can and will vary depending on the recombinant bacterium, the regulated antigen, and the intended host, as will be appreciated by one of skill in the art. Generally speaking, the dosage need only be sufficient to elicit a protective immune response in a majority of hosts. Routine experimentation may readily establish the required dosage. Typical initial dosages of vaccine for oral administration could be about 1×107 to about 1×1010 CFU depending upon the age of the host to be immunized. In some embodiments, the initial oral dose may be about 1×105 to about 1×1010 CFU. Generally speaking, intranasal doses are lower than the dose for oral immunization. For instance, in some embodiments, the intranasal dose may be between about 80 and about 120 times lower than the oral dose. Similarly, parenteral doses are lower than the dose for intranasal immunization. For example, the parenteral dose may be between about 5 and 15 times lower than the intranasal dose. Administering multiple dosages may also be used as needed to provide the desired level of protective immunity.
In order to stimulate a preferred response of the GALT, NALT or BALT cells, administration of the vaccine composition directly into the gut, nasopharynx, or bronchus is preferred, such as by oral administration, intranasal administration, gastric intubation or in the form of aerosols, although other methods of administering the recombinant bacterium, such as intravenous, intramuscular, subcutaneous injection or intramammary, intrapenial, intrarectal, vaginal administration, or other parenteral routes, are possible.
In some embodiments, these compositions are formulated for administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Accordingly, these compositions are preferably combined with pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, and the like.
In an exemplary embodiment, the recombinant bacterium may be administered orally. Oral administration of a composition comprising a recombinant bacterium allows for greater ease in disseminating vaccine compositions for infectious agents to a large number of people in need thereof, for example, in Third World countries or during times of biological warfare. In addition, oral administration allows for attachment of the bacterium to, and invasion of, the gut-associated lymphoid tissues (GALT or Peyer's patches) and/or effective colonization of the mesenteric lymph nodes, liver, and spleen. This route of administration thus enhances the induction of mucosal immune responses as well as systemic and cellular immune responses.
The invention also encompasses kits comprising any one of the compositions above in a suitable aliquot for vaccinating a host in need thereof. In one embodiment, the kit further comprises instructions for use. In other embodiments, the composition is lyophilized such that addition of a hydrating agent (e.g., buffered saline) reconstitutes the composition to generate a vaccine composition ready to administer, preferably orally.
A further aspect of the invention encompasses methods of using a recombinant bacterium of the invention. For instance, in one embodiment the invention provides a method for modulating a host's immune system. The method comprises administering to the host an effective amount of a composition comprising a recombinant bacterium of the invention. One of skill in the art will appreciate that an effective amount of a composition is an amount that will generate the desired immune response (e.g., mucosal, humoral or cellular). Methods of monitoring a host's immune response are well-known to physicians, veternarians, and other skilled practitioners. For instance, assays such as ELISA, and ELISPOT may be used. Effectiveness may be determined by monitoring the amount of the antigen of interest remaining in the host, or by measuring a decrease in disease incidence caused by a given pathogen in a host. For certain pathogens, cultures or swabs taken as biological samples from a host may be used to monitor the existence or amount of pathogen in the individual.
In another embodiment, the invention provides a method for eliciting an immune response against an antigen in a host. The method comprises administering to the host an effective amount of a composition comprising a recombinant bacterium of the invention.
In still another embodiment, a recombinant bacterium of the invention may be used in a method for eliciting an immune response against an enteric pathogen in an individual in need thereof. In some embodiments, a recombinant bacterium of the invention may be used in a method for eliciting an immune response against at least 2, 3, 4, 5 or more than 5 enteric pathogens in an individual in need thereof. The method comprises administrating to the host an effective amount of a composition comprising a recombinant bacterium as described herein. In a further embodiment, a recombinant bacterium described herein may be used in a method for ameliorating one or more symptoms of an enteric disease in a host in need thereof. The method comprises administering an effective amount of a composition comprising a recombinant bacterium as described herein.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention. Those of skill in the art should, however, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention, therefore all matter set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.
The following examples illustrate various iterations of the invention.
Three developed means to permit a regulated delayed attenuation phenotype were used so that vaccine strains at the time of immunization exhibit nearly wild-type attributes for survival and colonization of lymphoid tissues and after five to ten cell divisions in vivo become avirulent. In all cases, we generated precise deletion and deletion-insertion mutations using allele replacement and P22HTint transduction methods that do not leave any drug resistance genes or other sequence scars in the chromosomes of mutated strains. The first strategy makes use of pmi mutants that lack phosphomannose isomerase needed to interconvert fructose-6-P and mannose-6-P. Strains with the Δpmi-2426 mutation (diagrams of all deletion mutations are depicted in
In addition to the Δpmi-2426 mutation to reduce serotype-specific immune responses to the S. Typhimurium B group O-antigen, we can include the ΔPrfc:: TTaraC PBAD rfc deletion-insertion mutation (
To provide further attenuation, we use two additional means for regulated delayed attenuation in vivo, one causes over expression of immunologically cross-reactive iron-regulated outer membrane proteins (IROMPs). These additional means to achieve regulated delayed attenuation rely on using a more tightly regulated araC PBAD activator-promoter than the original sequence from E. coli B/r. We deleted the promoter, including sequences for activator or repressor protein binding, for the fur gene encoding the Fur protein that represses all genes involved in iron acquisition. The strains constructed thus possess the ΔPfur::TT araC PBAD fur and ΔPcrp::TT araC PBAD crp deletion-insertion mutations. Without being bound by theory, the absence of Fur possibly attenuates Salmonella due to iron overload. The crp gene encoding cAMP receptor protein that is necessary for virulence of Salmonella and that is also needed for maximal transcription from the PBAD promoter is similarly regulated by an araC PBAD cassette causing crp synthesis to also be dependent on arabinose which is unavailable in vivo.
We then substituted the improved araC PBAD cassette to yield Salmonella strains with ΔPfur::TT araC PBAD fur and ΔPcrp::TT araC PBAD crp deletion-insertion mutations (P stands for promoter, TT for transcription terminator) (all deletion-insertion mutations are diagrammed in
Timing in phenotypic expression of attenuation in strains with araC PBAD regulated genes for virulence can be delayed for one or two cell doublings by including the ΔaraBAD23 and ΔaraE25 mutations (
To eliminate use of plasmid vectors with non-permitted drug resistance genes and to stabilize plasmid vectors in recombinant attenuated Salmonella vaccines (RASV) in vivo, we developed a balanced-lethal host-vector system using a vaccine host strain with deletion of the asd gene to impose an obligate requirement for diaminopimelic acid (DAP) and a plasmid vector with the wild-type asd gene. We now have two additional balanced-lethal vector-host systems based on genes for synthesis of D-alanine and muramic acid, two other unique essential constituents of the rigid layer of the bacterial cell wall. We have Asd+, DadB+ and MurA+ vectors with the pSC101 ori, pI5A ori, pBR ori and pUC ori to give multiple options for levels of antigen expression based on gene copy number. We can thus use all three Asd+, DadB+ and MurA+ vector systems in the same vaccine strain to encode multiple protective antigens. Such a vaccine host strain would have the ΔasdA33 (or ΔasdA27::TT araC PBAD c2, see below), Δalr-3, ΔdadB4 and ΔPmurAI2::TTaraC PBAD murA (ΔPmurA25::TTaraC PBAD murA) or mutations in addition to the ΔrecF126 chromosome mutation to prevent plasmid-plasmid recombination without altering virulence or colonizing ability. The chromosomal arabinose-dependant ΔPmurA12::TTaraC PBAD murA mutation is necessitated since ΔmurA mutations are lethal since the muramic acid must be phosphorylated and Salmonella is unable to incorporate such phosphorylated muramic acid.
The export of antigens to the periplasm of vaccine strains yields superior antibody titers than if the protective antigen was retained in the cytoplasm of the vaccine strain. We initially used the β-lactamase Type II secretion system (SS) but now have validated use of the phoA and ompA SSs with equally good results. In these various vectors, we use either Ptrc or PPR as promoters with expression levels controlled by the LacI and C2 repressors, respectively. Over expression of protective antigens, which will be necessary to enable ability to induce protective immunity to diverse enteric pathogens by RASV strains, can reduce colonizing ability and thus immunogenicity. In specifying expression of recombinant protective antigens by RASV strains, we have controlled expression by the LacI regulatable Ptrc and the C2 regulatable P22 PR or PL promoters. We therefore generated the optimized ΔrelA198::araC PBAD lacI TT insertion-deletion mutation, so that vaccine strains growing in the presence of arabinose synthesize the LacI repressor to repress transcription from Ptrc on Asd+ or MurA+ expression vectors until after vaccination when the vaccine strain has already colonized internal lymphoid tissues. This technology has been improved to increase expression of the lacI gene 40-fold by changing the SD sequence from AGGG to AGGA, the lacI start codon from GTG to ATG and by changing lacI codons to maximize translation efficiency in Salmonella. In other constructions such as with the DadB+ vectors, we use phage P22 PL and PR that are repressible by the C2 repressor. We have therefore constructed the ΔasdA27::TT araC PBAD c2 deletion-insertion mutation in which we have increased C2 production by improving the −10 promoter sequence, the SD sequence, changed codon 2 to A-rich to enhance translation and optimized c2 codons for high-level expression in Salmonella.
We more recently developed a balanced-attenuation vector-host system to provide additional plasmid vectors to enable delivery by our attenuated Salmonella vaccine strains of more protective antigens specified by cloned DNA from diverse enteric pathogens. We therefore have constructed plasmid vectors with the wild-type alleles for the aroA, aroC, aroD, ilvC and ilvE genes that render Salmonella and other pathogens attenuated. These AroA+, AroC+, AroD+, IlvC+ and InvE+ plasmid vectors possess the Ptrc promoter, a multiple cloning site, a transcription terminator and a pBR ori although other combinations of promoters and copy numbers are readily substituted to provide diversity of options for expression of antigen genes. The Salmonella vaccine stains thus have defined deletion mutations to result in strains with ΔaroA, ΔaroC, ΔaroD, ΔilvC and ΔilvE mutations to establish the balanced-attenuation vector-host systems.
Many of the vectors defined in this Example are listed in Table 8.
We have devised a host-vector system with regulated expression of the murA gene encoding the first enzyme in muramic acid synthesis and the asd gene essential for DAP synthesis. Both muramic acid and DAP are essential unique constituents of the rigid layer of the bacterial cell wall. The vector pYA3681 is diagramed in
In some instances, however, lysis in the intestinal tract may not be complete. This may be due to low levels of arabinose generated by normal flora while degrading consumed food. This may allows the survival of some vaccine cells with the regulated delayed lysis in vivo phenotype. We have therefore developed a means by which Salmonella rapidly catabolizes in vivo arabinose that could otherwise enable survival in the intestinal tract. We have constructed the mutation Δ(araC PBAD)-5::P22 PR araBAD (to replace the ΔaraBAD23 mutation) to use in a strain with the ΔasdA27::TT araC PBAD c2 mutation. Strains with this mutation exhibit constitutive transcription of the araBAD genes in vivo after several cell divisions to result in rapid degradation of any arabinose encountered.
We had shown that S. Typhimurium strains (see Table 7) with either the Δpmi-2426 or ΔPfur33::TTaraC PBAD fur mutation induced antibodies after a single oral immunization of female BALB/c mice that gave significant and similar titers by ELISA against outer membrane proteins isolated from diverse Salmonella serotypes and E. coli pathovars grown under conditions of iron limitation. We recently compared sera from mice orally immunized once with χ9088 (Δpmi-2426 Δ(gmd fcl)-26 ΔPfur33::TT araC PBAD fur ΔasdA33 with an Asd+ plasmid) and χ9241 (ΔpabA1516 ΔpabB232 ΔaraBAD23 ΔasdA16 ΔrelA198::araC PBAD lacI TT with an Asd+ plasmid) by western blot analyses of polyacrylamide gels of OMPs isolated from a diversity of Salmonella serotypes, various E. coli pathovars, Shigella flexneri strains, and three different Yersinia species all grown with or without iron limitation. In all cases, more antibody reactivity was observed with sera from χ9088 immunized mice than χ9241 immunized mice (compare
Acquisition of ion nutrients, in addition to iron, are undoubtedly essential for bacterial pathogen virulence. These abilities are likewise probably an essential means of host defense against these pathogens. We thus decided to genetically modify Salmonella vaccine strains to over express outer membrane proteins for acquisition of manganese and magnesium under the expectation that these will be cross-protective antigens. We therefore made constructions for regulated delayed constitutive expression of genes involved in these two transport functions. The repressor, MntR, regulates the expression of genes involved in manganese transport. Therefore, we have deleted the promoter, including sequences for activator or repressor protein binding, for the mntR gene and inserted the improved araC PBAD cassette to construct six different ΔPmntR::TT araC PBAD mntR insertion-deletion mutations (
Salmonella χ3761:
Salmonella χ3761:
Salmonella χ3761:
We introduced three of the ΔPmntR::TT araC PBAD mntR insertion-deletion mutations (expected to give high, medium and low levels of synthesis of the MntR repressor protein when strains are grown with arabinose) into attenuated S. Typhimurium strain χ9202 (ΔPcrp527::TT araC PBAD crp ΔaraBAD23). MntR synthesis in these constructs was regulated by arabinose availability as expected with the highest level of MntR synthesized by the strain with the ΔPmntR26 and the lowest by the strain with the ΔPmntR22 mutation (
We are also studying regulated delayed in vivo over expressing corA, mntH and mgtA genes on Asd+ vectors and yaeT and yaeT (bsa surface domain) genes on DadB+ vectors to determine whether over expression of these gene products induces immune responses that are improved relative to inducing cross protective immunity to bacterial enteric pathogens. Further modifications of these genetic constructions and evaluations of their contributions on induction of cross protective immunity are discussed below.
To reduce fluid secretion and potential mild gastroenteritis symptoms in human vaccinees, we include the ΔsopB1925 mutation, which we demonstrated to reduce fluid secretion and inflammation in rabbit ileal loop experiments. Others have demonstrated that sopB mutations also reduce induction of fluid secretion in calves. This mutation does not appreciably alter virulence or colonizing ability in mice. We have embarked on identification of mutations that render lipid A non toxic but retain ability to serve as a TLR4 agonist. One or more mutations conferring these attributes is included in a strain to be evaluated in humans. We had previously found that combination of the ΔsopB1925 and ΔmsbB48 mutations results in the least fluid secretion in rabbit ileal loops of any strain we tested. Strains with these two mutations are also well tolerated by newborn mice orally inoculated with 107 to 108 CFU on day of birth. An additional benefit of the ΔsopB1925 mutation is that it increases induced immune responses to expressed protective antigens.
A difficulty with live bacterial vaccines is the potential to survive and multiply if excreted. This leads to the possibility that individuals not electing to be immunized get immunized. We have developed a method of regulated delayed lysis in vivo that prevents vaccine persistence in vivo and survival if excreted as described in Example 3. This means of biological containment also serves both as a means or regulated delayed attenuation and as a means to release a bolus of expressed protective antigens upon lysis in vivo. However, we are developing other means that decrease, if not preclude, the ability of Salmonella vaccines to persist in the GI tract of animals. In this regard, the chicken cecum is highly desirable environment for the Salmonella. Consequently we use chickens as our animal to evaluate our successes or lack thereof in achieving biological containment without resorting to cell lysis.
We have constructed and evaluated strains with the Δ(gmd fcl)-26 or Δ(wcaL-wza)-7, ΔagfBAC811 or Δ(PagfDagfG)-4, ΔbcsABZC2118 or ΔbcsEFG2319 and Δ(yshA-yihW)-157 mutations that block synthesis of colanic acid, thin aggregative fimbriae (i.e., curli), cellulose and extracellular polysaccharide, respectively, all of which contribute to biofilm formation. Since the LPS O-antigen also enables biofilm formation, a strain with the Δpmi-2426, ΔPrfc174::TT araC PBAD rfc, and Δ(galE-ybhC)-851 mutations with or without a Δ(gmd-fcl)-26 or Δ(wcaM-wza)-8 mutation would be expected to survive less well in nature because of a dependency on the availability of three sugars simultaneously, an unlikely occurrence. Such a strain would thus exhibit a rough phenotype making it less able to survive in soil or even in the intestinal environment. We also have mutations such as ΔyhiR36 that prevents use of DNA as a nutrient, Δ(shdA-ratB)-64, ΔmisL2 and ΔbigA3 that encode four proteins that enable Salmonella to adhere to host extracellular matrix proteins and ΔackA233 that blocks use of acetate. Some of these mutations have been reported to reduce Salmonella persistence in the intestinal track of calves and in mice, but this is not so in the intestinal track of chickens. We have yet to combine these mutations abolishing the ability to synthesize biofilms, the ability to synthesize LPS O-antigen, the ability to bind to extracellular matrix proteins, and the inability to use DNA and acetate as nutrients in a single bacterium, but we surmise that such a strain would exhibit a high level of biological containment and be unable to persist in the intestinal tracks of birds or mammals. Additionally, such a strain would not survive in the environment if excreted. A further anticipated benefit of such a strain is the further removal of macromolecules that might mask immunological surveillance of surface localized LPS core and cross reactive outer membrane antigens. Thus we anticipate an enhancement in levels of induced immune responses to expressed antigens. Indeed, vaccine strains with the Δ(wcaM-wza)-8 mutation synthesize five to ten percent more protective antigen and induce similarly higher antibody titers to this antigen.
We have studied the avian pathogenic E. coli (APEC) strain χ7122 for some time and were first to describe an autotransporter (Type V secretion) protein (Tsh) in the Enterobacteriaceae, first to use genomic subtractive hybridization to define multiple genomic genetic islands in a pathogen, and first to use selective capture of transcribed sequences (SCOTS) to determine which genes are expressed in an animal. We have isolated and characterized three large plasmids from χ7122 and have sequenced the 103 kb pAPEC-1 plasmid that possesses the iroBCDN (salmochelin), tsh (hemoglobin protease, heme binding protein and hemagglutin), iucABCD iutA (aerobactin), sitABCD (iron and manganese uptake) and iss (serum resistance) loci known to contribute to virulence (and other loci that might also contribute). The iroBCDN and sitABCD previously identified as critical for ion acquisition and now the etsABC operon are ABC transport systems. Many of these genes involved in iron and/or manganese uptake are Fur and/or MntR regulated. Since pAPEC-1 is an IncF plasmid as is the S. Typhimurium virulence plasmid pSTV (184) (termed pSTUK100 for the UK-1 strain), we inserted the spvABCD operon that is responsible for the virulence attributes encoded on pSTUK100 into the cysG gene of the S. Typhimurium pSTUK100 cured strain χ9076 to yield χ9405 (ΔpSTUK100 ΔcysGI69::spvABCD). Oral infection of mice revealed that while the ΔpSTUK100 strain χ9076 was significantly attenuated compared to the pSTUK100 containing wild-type strain χ3761, strain χ9405 (ΔpSTUK100 ΔcysGI69::spvABCD) did not exhibit the expected virulence of χ3761 but was slightly attenuated. We therefore redesigned a stronger promoter for the inserted spvABCD operon and reconstructed the insertion into the cysG locus to result in the ΔcysG175::Pspv175 spvABCD deletion-insertion mutation in χ9876, which lacks the pSTUK100 virulence plasmid but now displays the same virulence as does the wild-type χ3761 strain. The nucleotide sequence for this improved spvABCD insertion is given in
pAPEC-1 can now be introduced into χ9876 or other multiply mutant ΔpSTUK100 strains using χ7122 as the conjugational donor. The pAPEC-1 plasmid is not self-conjugative due to a deletion of tra genes so once transferred to Salmonella it is stably maintained. The introduction of pAPEC-1 into a suitably designed Salmonella vaccine is expected to further enhance inducing protective immunity to several enteric pathogens. This is so since the aerobactin operon on pAPEC-1 is used by all Shigella and E. coli STEC/EIEC pathovars, the salmochelin operon is widely distributed in Salmonella serotypes and in UPEC strains and the sit operon is present in Salmonella, Shigella and UPEC strains. Most importantly, the proteins encoded by these operons possess considerable amino acid sequence homology and the proteins also have similar if not nearly identical conformations. These properties contribute to the induction of cross-protective immunity. Further enhancement by modifications of pAPEC-1 in inducing protection to Yersinia and Shigella pathogens in provided below in Example 21.
We have constructed various Asd+ plasmids encoding Y. pestis antigens for expression in S. Typhimurium χ8501 (Δcrp-28 ΔasdA16). Since the protective V antigen can interact with TLR2 to induce the non-inflammatory cytokine IL10 and this activity is encoded near the N-terminal end of the V antigen, we cloned a codon-optimized sequence encoding aa 131 to 327 as a β-lactamase SS fusion in the Asd+ vector pYA3620. Since this V antigen sequence is homologous to the V antigen sequence in the enteropathogens Y. enterocolitica and Y. pseudotuberculosis, we compared the ability of χ8501 with pYA3620 (vector control) versus χ8501 with the pYA3620-V antigen plasmid to induce protection. As indicated in
Since we now know the amino acid sequences that are necessary for interaction of the V antigen with TLR2 (Abramov et al, 2007, Sing et al, 2005), we engineered the truncated V antigen to express the N-terminal portion since it also contains protective epitopes. The sequence (979 bp) encoding AgV has been optimized for expression in Salmonella and cloned in pYA3620, generating the plasmid pYA4661. We have also introduced mutations into the sequence to modify sites of LCRV interaction with the receptor. (
The Yersinia V antigen, located at the tip of the Type III secretion needle, is essential for virulence and, if delivered in a modified form is a very protective antigen. The V antigen has structural homology to the IpaD protein of Shigella that is also located at the tip of the Type III secretion needle.
#Mice were challenged using an intra-lung model.
Jorge Giron at the University of Arizona has described the sequence of the yagZ fimbrial operon that encodes the ECP pili displayed by essentially all E. coli pathovars and Shigella but has gone undetected for many years since these fimbriae are only synthesized and assembled in response to a signal encountered in vivo (Proc. Natl. Acad. Sci. USA 104:10637-10642 (2007)). We have cloned this fimbrial operon on the low copy number Asd+ plasmid pYA3337 to yield pYA4428. This recombinant plasmid and the cloned pilus encoding DNA sequence are given in
Although the delivery of the B subunit of LT does not confer adequate or complete protection against ETEC strains, its delivery in a vaccine of the type we are constructing will be helpful. In addition, LT is delivered to the outer membrane surface of E. coli with the B subunits outward facing to enable interaction with the GM-1 ganglioside present on the surface of most host cells. The delivery of LT from bacteria to host cells is facilitated by formation of outer membrane vesicles (OMVs). At least three of the mutations so far included in the Salmonella strains described herein cause a 100-fold increase in OMV formation when grown under the conditions encountered in vivo (at least in regard to availability of sugars). Our vaccine strains should ultimately be superior in inducing antibody responses to LT-B and accordingly, we are testing this hypothesis. We cloned the human LT-B sequence under Ptrc control in the Asd+ vectors pYA3337, pYA3332, pYA3342 and pYA3341 with pSC101 ori, pI5A ori, pBR on and pUC ori, respectively, and introduced these plasmids into χ9241 (ΔpabA1516 ΔpabB232 ΔaraBAD23 ΔasdA16 ΔrelA198::araC PBAD lacI TT). Only the plasmids with the pSC101 ori and to a lesser extent the p15A ori caused synthesis and export of LT-B from the Salmonella cells. We have determined that S. Typhimurium strains with the Δpmi-2426 Δ(gmd-fcl)-26 or ΔfliC180 ΔfljB217 mutations generate high yields of outer membrane vesicles (OMVs). Since we already know that production of OMV enhances induction of high levels of immunity to other recombinant expressed antigens, we believe that these mutations will enhance immunogenicity of Salmonella vaccine strains expressing LT-B. We are validating that the LT-B is present in OMVs and will then proceed to determine what genetic alterations of Salmonella will optimize LT-B expression and surface localization to facilitate attachment and presumably invasion of recombinant Salmonella vaccine cells into host cells.
In this Example, we investigate whether a newly discovered pilus of critical importance for biofilm formation by and virulence and colonizing ability of C. jejuni can be used as an expressed antigen in our RASVs to induce immunity to C. jejuni colonization in chickens (and other target animals). The C. jejuni pilus gene was identified by isolating pilus proteins from cultures grown in a biofilm, separating them by SDS-PAGE, excising protein bands between 14.4-24 kDa that were subjected to trypsin digestion. The digests were subjected to liquid chromatography-mass spectrometer analysis and the sequence of each protein blasted against all known genomic sequences. The protein band at 18 kDa contained a sequence homologous to Ftp from Hemophilus spp and was then determined to be encoded by a gene designated pilA in the C. jejuni NCTC 11168 genome. The gene was disrupted by insertion of a chloramphenicol-resistance cassette. The C. jejuni pilA mutant was seeded onto polycarbonate membranes and cultured in Mueller-Hinton broth at 37° C. for 24 h. The pilA mutant produced sparse biofilms and SEM demonstrated the absence of pili in the mutant strain. Pili were not seen when the wild-type parent was grown under planktonic conditions. Fourteen day-old chicks were orally inoculated with 105 NCTC 11168 C. jejuni or the pilus minus NCTC 11168 strain. The chicks were euthanized ten days post-inoculation and a gram of cecal contents from each chick were 10-fold diluted, and plated on CEFEX plates. The results of three colonization evaluations are included in Table and demonstrate a 1000-fold reduction in colonization by the Pil strain. The pilA gene is highly conserved (98 to 100%) in all C. jejuni sequences available, has been cloned and has been cloned into the Asd+ expression vector pYA3493 and expressed as a β-lactamase SS fusion protein from pYA4495 (
RASV strain χ9088(pYA4495) was used to orally immunize chicks. The birds were challenged with 105 NCTC 11168 C. jejuni. A week later, cecal samples were collected and the number of CFU/g of cecal contents was determined. The results showed that immunization with χ9088(pYA4495), but not the control strain, resulted in a 4-log reduction C. jejuni recovered, indicating that the vaccine confers protection (Table 4).
C.
jejuni
C.
jejuni
C.
jejuni
The CjaA protein of C. jejuni is an outer membrane protein that is homologous to several prokaryotic solute-binding components of an ABC solute transporter system. Immunization of chickens with an attenuated Salmonella expressing CjaA has been shown to confer protection against C. jejuni challenge. We are constructing Salmonella vaccine strains that express both cjaA and pilA and are evaluating them in chickens.
The expression of C. jejuni genes in Salmonella can be problematic and lead to strain instability due to the low G+C content in the DNA of C. jejuni (31%) compared to the G+C content of Salmonella (50-53%). In addition, the codon usage in C. jejuni is different from Salmonella, which can lead to poor expression due to the presence of codons in the coding sequences that are rare in Salmonella. We have codon optimized the pilA and cjaA genes for expression in Salmonella (
All C. perfringens strains produce α-toxin, which is a potent phospholipase C enzyme with sphingomyelinase and hemolytic effects causing membrane disorganization (187). With MLD of <0.1 μg/mouse, it is the potent exotoxin among C. perfringens toxins that is explicitly implicated in gas-gangrene and as a major cause of necrotic enteritis. Chickens are considered 200 times more susceptible to α-toxin than β- or ε-toxin and develop severe necrotic enteritis or a widespread subclinical infection. In severe cases, necrotic enteritis causes significant destruction of the intestinal mucosa and mortality up to 30%. In sub clinical infections, α-toxin causes shortening of microvilli and thickens the intestinal mucosa to result in decreased nutrient uptake from digested food. To cause its lytic effect on cell membranes α-toxin has to bind to membranes by its C-terminal fragment and it has been shown that blockage of the C-terminus by specific antibody will neutralize α-toxin. Thus, we have cloned the C-terminal portion of the α-toxin gene onto the Asd+ pYA3493 vector as β-lactamase SS fusions and several slightly different constructs introduced into χ8133 (Δcrp-27 Δcya-27 ΔasdA16), χ8914 (ΔpabA1516 ΔpabB232 ΔasdA16) and χ9241 (ΔpabA1516 ΔpabB232 ΔasdA16 ΔaraBAD23, ΔrelA198::araC PBAD lacI TT).
Mice orally immunized with any of these RASV constructs were protected from C. perfringens-induced gas gangrene following C. perfringens intramuscular challenge and showed significant reduction in numbers of bacteria recovered after challenge. Immunization of chicks with the RASV induced toxin neutralizing antibodies evaluated by inhibition of RBC hemolysis and by neutralizing lecithinase activity. These chickens developed less severe lesions in experimental necrotic enteritis challenge. Most interestingly, sera from immunized chickens could reduce the titers of C. perfringens almost 1000-fold in 12-hour cultures (see Table 5). A similar result was observed in vivo with regard to C. perfringens titers following repetitive challenge (4 days in a row). It appears that α-toxin, is essential for the survival and replication of the bacteria in vivo (muscle or intestine), and antibodies to α-toxin bind to Tox+ C. perfringens cells (
C.
perfringens CFU/ml
A DNA sequence encoding the C-terminal 123 aa of plc, termed plcC (
A study was undertaken wherein we orally immunized birds at days 3 and 13 of life with χ8914 derivatives carrying these plasmids and expressing the plcC fusion proteins. The birds were challenged with C. perfringens over a four-day period, two weeks later. The next day, birds were examined for signs of necrotic enteritis. The results showed that immunization with any of the three vaccine strains expressing plcC reduced the number of macroscopic and microscopic lesions associated with necrotic enteritis and positively influenced weight gain after challenge (Table 6).
1Macroscopic lesions in the duodenum and jejunum.
2Frequency and severity of lesions were graded semi-quantitatively on scale of 0 to 5 (severe) in tissues collected one day after challenge.
3Body weight was measured before the C. perfringens challenge infection at day 35 of age and a week after the end of challenge infection, 44 days of age.
The netB gene encodes a protein that shows some homology with C. perfringens β-toxin. Among poultry isolates, it is found primarily in birds suffering from necrotic enteritis, but not from healthy animals and is required for the induction of necrotic enteritis symptoms in chickens. We have cloned the netB gene into a number of our Asd+ expression vectors and are performing immunological evaluations on Salmonella vaccine strains carrying these plasmids.
As mentioned above for C. jejuni, the expression of C. perfringens genes in Salmonella can be problematic and lead to strain instability due to the low G+C content in the DNA of C. perfringens (24-27%) compared to the G+C content of Salmonella (50-53%). In addition, the codon usage in C. perfringens is different from Salmonella, which can lead to poor expression due to the presence of codons in the coding sequences that are rare in Salmonella. We have optimized the sequences for plcC and netB for codon usage and G+C content (
We are investigating our RASV systems for induction of immunity to Giardia and this is in line with our recent success in inducing immunity to another intestinal parasite, Eimeria acervulina that causes coccidiosis in poultry. About 12 putative protective antigens have been identified by another party and our lab evaluated all the structures to determine how best to clone, express and deliver what parts of these antigens. The first four recombinants have been constructed. These are being evaluated in studies with mice.
We have constructed S. Paratyphi A vaccine strains in parallel while constructing and characterizing S. Typhimurium vaccine strains. χ9608 has the genotype ΔPfur8I::TT araC PBAD fur ΔPcrp527::TT araC PBAD crp Δpmi-2426 Δ(gmd fcl)-26 ΔrelA198::araC PBAD lacI TT ΔaraBAD23 ΔaraE25 ΔagfBAC811 ΔsopB1925 ΔasdA33. We can easily add any constellation of mutations described above or in the examples to enable use of multiple balanced-lethal or balanced-attenuation vectors to specify synthesis of described antigens of E. coli pathovars, Shigella, Yersinia, Campylobacter and Clostridium, mutations to up regulate expression of manganese and magnesium uptake antigens in vivo, mutations to confer biological containment, mutations to eliminate serotype specific flagellar antigens while retaining TLR5 interactions, and mutations to better expose common cross reactive surface antigens. We can also introduce the modified pAPEC-1 plasmid to specify cross-reactive protective antigens from E. coli, Salmonella, Shigella and Yersinia. It should be evident that similar vaccine constructs derived from S. Typhi strains are also possible.
Bacterial strains are listed in Table 7 and plasmids and plasmid vectors in Table 8. All strains for testing in mice and chickens are derived from the highly virulent S. Typhimurium strain UK-1 although some strains derived from SL1344 are also used. All bacterial strains that can be used are known to one of skill in the art including strains representing all Salmonella serotypes most frequently isolated from poultry, swine, cattle and humans. Defined deletion mutations with and without specific insertions that have been introduced into any of the strains constructed are depicted in
Salmonella
S.
Infantis NR29, group C1, O(6,7)
S.
Typhi
S.
Typhimurium UK-1
S.
Heidelber
S.
Gallinarum
S.
Paratyphi
S.
Enteritidis
S.
Typhimurium UK-1 χ3761 cured of
Escherichia
coli
Shigella
S.
flexneri 2457O, Hela invasion -;
S.
flexneri , Hela invasion +; Pcr+
S.
flexneri 2a, nonpigmented mutant
Yersinia
Y.
pestis , Subtype medievalis
Y.
pestis , Subtype orientalis
Y.
enterocolitica
Y.
pseudotuberculosis, Serotype IB
Clostridium
perfringens
C.
perfringens , Type A, Isolate from
C.
perfringens, wild type, Type A
C.
perfringens, wild type, Type A,
C.
perfringens Type A, Isolate from
In addition, we have collected nine additional S. enterica serotypes frequently associated with carriage in bovine, porcine and poultry species and/or transmitted, presumably through the food chain, to cause disease in humans. We have also discovered seventy other serotypes from our collection. Thus, there is a very extensive collection to use in our studies on induction of immune response to Salmonella of diverse serotypes. We have three new C. jejuni strains and eleven new Shigella strains representing diverse species and serotypes. Other strains from diverse locations are being collected.
As a new suicide vector donor strain, we constructed χ7378 with Δalr-9 and ΔdadX8 mutations in addition to the ΔasdA4 mutation to enable counter selection with omission of either D-alanine or DAP. This strain can also be used for conjugational delivery of recombinant DadB+ or Asd+ vectors while retaining a means for counter selection with a lethal marker. We also have a diversity of E. coli cloning hosts derived from χ6097 and χ6212 with the Δalr-9 and ΔdadX8 mutations in addition to the ΔasdA4 mutation.
Methods for DNA isolation, restriction enzyme digestion, DNA cloning and use of PCR for construction and verification of vectors are standard. DNA sequence analysis was performed at nominal charge in the DNA Sequence Laboratory in the School of Life Sciences at ASU. All oligonucleotide and/or gene segment syntheses were done commercially. Site-directed mutagenesis was used to optimize codons for translational efficiency in Salmonella. Stabilization of mRNA to prolong its half-life will involve site-directed mutagenesis to destroy RNase E cleavage sites and/or by fusing the ompA leader mRNA encoding sequence at the transcription start site for a gene encoding an antigen of interest. Phage P22HTint are used to transduce mutations of a selectable phenotype from one S. Typhimurium strain into other strains. Conjugational transfer of suicide vectors was performed by standard methods using the suicide vector donor strain χ7213 or its derivative with alr and dadB mutations χ7378. Plasmid constructs was evaluated by DNA sequencing, ability to complement various S. Typhimurium mutant strains and for ability to specify synthesis of proteins using gel electrophoresis and western blot analyses. His-tagged proteins was produced in χ7385, a new E. coli host, and used to obtain anti-protein rabbit antisera for western blot analyses.
Vaccine strains are fully characterized at each step in their construction and before use for immunization studies. These strains are compared with vector control strains for stability of plasmid maintenance, integrity and antigen synthesis ability when strains are grown in the presence of arabinose and/or DAP and/or D-alanine over a 50 generation period. Molecular genetic attributes are confirmed by use of PCR and/or Southern blot analyses with appropriate probes. Measurement of lipopolysaccharide or its absence is performed after electrophoresis using silver stained gels. This analysis is done after every step in any strain construction to eliminate rough variants if they arise. Motility tests and use of specific antisera for given flagellar antigens are used to reveal presence or absence of flagella. Presence of fimbrial adhesins are assayed using agglutination of yeast and red blood cells in the presence and absence of mannose as a function of growth conditions, Congo red binding assays and by transmission electron microscopy (TEM) using negative staining with phosphotungstic acid. Metabolic attributes of candidate vaccine strains are evaluated using API-20E tests.
The ability of various constructed Salmonella strains to attach to, invade into and survive in various murine and human macrophage cell lines are quantitated by well established methods used routinely. Similarly, ability to induce pyroptosis/apoptosis will use standard methods.
BALB/c and C57BL/6 female mice, six to eight weeks of age, are used for most experiments. Inbred mice with other MHC haplotypes and Swiss Webster outbred mice are also used in some studies. Mice are held in quarantine one-week before use in experiments. They are deprived of food and water 6 h before oral immunization. No bicarbonate is administered. Bacterial strains are grown under conditions to optimize expression of SPI-I encoded genes needed to facilitate invasion and colonization of lymphoid tissues. Food and water are returned 30 min after immunization. A second boosting immunization is given one, two or four weeks after the first dose, but after saphenous vein bleeding to collect serum and to collect feces and/or vaginal secretions for quantitation of SIgA. Candidate vaccine strains are quantitatively enumerated in various tissues as a function of time after inoculation. Generally, three mice are used per time point. The inoculation procedures are the same as in the immunization studies. All animals are housed in BL2 containment with filter bonnet covered cages. If high immunogenicity is observed in initial tests after primary immunization, subsequent studies are done to determine the lowest level of vaccine inocula to induce a significant immune response. Challenge studies involve many different bacterial pathogens all grown under optimal conditions for that pathogen and with the route of challenge also selected to reveal strengths and weaknesses of the immunizing strain or regime. We then calculate LD50 and mean day of death (MDD) values.
We have made purified antigens as His-tagged proteins from recombinant E. coli χ7385 for all antigens encoded by genes from various pathogens and specified by RASVs. χ7385 has been engineered to eliminate potential contamination of proteins with appendage proteins and LPS O-antigen. Salmonella LPS O-antigen is obtained commercially although purification of O-antigen and LPS core are in progress to facilitate determination of antibody titers. We have prepared an S. Typhimurium outer membrane protein (SOMP) fraction from χ9424 that has been engineered to be unable to produce flagella, all in vitro-expressed pilus antigens, and LPS O-antigen and a heat killed extract of the wild-type S. Typhimurium UK-1 strain χ3761. These antigens will be used as controls in western blots as well as for immunoassays as described below. We have OMP extracts from many Salmonella serotypes, E. coli, Shigella and Yersinia strains. Synthetic peptides for various T-cell epitopes to use in T-cell proliferation assays are prepared commercially.
Serum antibodies are measured in blood collected by saphenous vein bleeding and mucosal antibodies as extracted copro antibodies from feces or in vaginal secretions. In initial studies, sera and secretions are pooled from all mice in a group but in later studies with successful constructs, antibody titers are monitored in individual mice. We employ a doubling dilution method with the end point titer being the dilution giving an OD410 three times that for the reagent or unimmunized animal control. SIgA titers against the various antigens are monitored by ELISA in the same way. Since we are interested in distinguishing between a Th1 and Th2 response, the titers of IgG1 versus IgG2A are determined by standard methods known to one of skill in the art.
ELISPOT analysis is used to quantitate IgA secreting peripheral blood lymphocytes in evaluating RASVs for inducing mucosal immunity and for INF-γ produced by T cells from mice immunized with RASVs to stimulate T-cell immunity. INF-γ produced as a result of co-cultures of the lymphocytes and T cells are determined using a described ELISPOT assay.
These are performed on spleenocyte preparations obtained from groups of mice one, two and four weeks post-vaccination using standard methods. Lymphocytes are purified by Histopaque 1077 gradient centrifugation and examined for incorporation of [3H]-thymidine after stimulation with His-tagged antigens. Stimulation with specific peptides containing T-cell epitopes is also employed. Stimulation with S. Typhimurium outer membrane proteins and concanavalin A serve as controls.
CTL responses to T-cell epitopes are quantitated, if necessary, using the appropriate murine cell lines (P815 or EL-4) transfected with plasmids encoding the epitopes to serve as targets for assays of BALB/c or C57BL/6 immunized mice. Effector cells are obtained from spleens of non-immunized and immunized mice. Part of the effector cell population is used immediately in a Cytotox 96 nonradioactive CTL assay (Promega). The remaining cells are re-stimulated with the appropriate antigen for five to seven days prior to use in the CTL assay. The CTL assay measures lactate dehydrogenase released due to lysis of target cells. The values obtained are used to calculate the percentage of target cells lysed relative to the quantity of effector cells added. We have had very good success with this assay, but have also used the 51Cr release assay.
All results are analyzed using the most appropriate statistical test from the SAS program to evaluate the relative significance or lack thereof of results obtained.
To minimize induction of immune responses to serotype-specific antigens and maximize induction of cross protective immunity to common related antigens of S. enterica strains of diverse serotypes and to other bacterial enteric pathogens, especially E. coli pathovars, Shigella species and Yersinia species, further work was undertaken for genetic modification of S. Typhimurium.
We have one S. Typhimurium UK-1 starting strain χ9592 (Δpmi-2426 Δ(gmd-fcl)-26 ΔPfur8I::TT araC PBAD fur ΔPcrp527::TT araC PBAD crp ΔasdA27::TTaraC PBAD c2 ΔaraE25 ΔaraBAD23 ΔrelA198::araC PBAD ΔlacI TT ΔsopB1925 ΔagfBAC811Δf1iC180 ΔflijB217). We add mutations to this strain as their inclusion is validated by the experiments proposed in this and following sections. In addition, we then add the ΔrecF126 mutation and then derive three strains, one with the Δalr-3, ΔdadB4 mutations, one with the ΔPmurA12::TTaraC PBAD murA mutation and one with all three mutations. In another case, we will derive strains from S. Typhimurium χ9903 Δpmi-2426 Δ(wcaM-wza)-8 ΔPfur81::TT araC PBAD fur ΔPcrp527::TT araC PBAD crp ΔasdA27::TT araC PBAD c2 ΔaraE25 ΔaraBAD23 ΔrelA198::araC PBAD lacI TT ΔsopB1925 ΔagfBAC811 Δlrp-23.
We use our suicide vectors and P22 transduction methods to generate all the strain genotypes listed in the Introduction section above to generate stains lited in Table 7 and derivatives of χ9592 (Δpmi-2426 Δ(gmd-fcl)-26 ΔPfur81::TT araC PBAD fur ΔPcrp527::TT araC PBAD crp ΔasdA27::TT araC PBAD c2 ΔaraE25 ΔaraBAD23 ΔrelA198::araC PBAD lacI TT ΔsopB1925 ΔagfBAC811 ΔfljB217 ΔfliC180) and derivatives of χ9903 Δpmi-2426 Δ(wcaM-wza)-8 ΔPfur81::TT araC PBAD fur ΔPcrp527::TT araC PBAD crp ΔasdA27::TT araC PBAD c2 ΔaraE25 ΔaraBAD23 ΔrelA198::araC PBAD lacI TT ΔsopB1925 ΔagfBAC811 Δlrp-23. In making derivatives we will select from among the following mutations: ΔPrfc174::TT araC PBAD rfc, Δ(galE-ybhC)-851, ΔPmntR22::TT araC PBAD mntR, Δ(yshA-yihW)-207, ΔcysG175::Pspv175 spvABCD, ΔrecF126, Δalr-3 and ΔdadB4, ΔPmurA25::TTaraC PBAD murA, Δ(PagfD agfG)-4, ΔbcsABZC2118 or ΔbcsEFG2319, ΔyhiR36, Δ(shdA-ratB)-64, ΔmisL2, ΔbigA3, ΔackA233, ΔaroA21419, ΔaroC, ΔaroD, ΔilvE and ΔilvC.
At each step in the constructions we perform the strain characterizations described above. It should be noted that if improvements to the core genotype are developed prior to the completion of the construction plan above, one should include these improvements at the earliest step possible.
Studies were conducted to fully evaluate how best to maximize induction of cross-protective immunity, consequences of vaccination on normal flora, efficacy of mixtures of vaccines designed to protect against Salmonella and one or two other species versus a vaccine designed to protect against multiple bacterial enteric pathogens, and means to experimentally validate a vaccine designed to protect against multiple pathogens.
After making progress in constructing strains and vectors and in understanding how to best optimize vaccine efficacy, we are conducting a series of studies to examine optimal immunization regimens and doses, work out methods to evaluate induction of cross protective immunity without having to resort to hundreds of challenge studies, determine whether there are adverse consequences of vaccination on normal flora that impact the nutritional health of vaccinated individuals, determine whether antigen interference is observed such that vaccination with a mixture of vaccine strains will give better protection that achieved with a single genetically engineered vaccine, and devise means to validate vaccine-induced protection against multiple pathogens.
Initially we select two recombinant vaccines, one in which we expect protection against a lethal challenge (such as by EIEC or STEC) and the other in which we expect reduction in intestinal colonization (such as for C. perfringens or C. jejuni) in the mouse. We initially immunize orally with 107, 108 and 109 CFU of vaccine, monitor selected immune responses by ELISA and ELISPOT at two-week intervals for 8 to 10 weeks and then administer a boost immunization and follow antibody titers for another four weeks followed by challenge with the pathogen of choice at a reasonable dose. A second set of experiments is done with the best regimen followed by a range of challenge doses with the pathogens of choice. We then use two different vaccines to see if similar results were observed such as protection against S. Enteriditis or Y. enterocolitica or colonization by UPEC or EHEC pathovars.
The experiments described above are directed to defining an approach to this problem. In regard to cross protective immunity with Salmonella serotypes, the vast majority of strains are not very virulent in mice or chickens. We thus take four very virulent strains (for mice) of S. Typhimurium, S. Choleraesuis, S. Dublin and S. Enteriditis representative of serogroups B, C, D and D, respectively. If the vaccine prevents infection and severe disease with these strains, it can be inferred that the vaccine would induce protective immunity to strains of lower virulence. In other words, if we can identify strains of high virulence for any of the enteric pathogens and demonstrate protection to challenge either in terms of preventing disease symptoms or in eliminating persistent infection, we can infer protection and vaccine efficacy. During the course of these studies we can also determine whether antibody titers to a particular pathogen antigen increase as a consequence of challenge. In fact, we are examining this using cross-reactive antigens encoded by genes from enteric pathogens other than from the challenge strain. The issue is whether a challenge stimulates increased antibody titers to both homologous and some related cross-reactive antigens.
In other cases, we use strains of serotypes that do not cause lethal infection in mice or chickens, and determine whether immunization with a vaccine construct reduces colonization levels and/or persistence by these strains, especially in the gastrointestinal tract.
These studies are an extension of those described above in which we examine whether vaccination with a S. Typhimurium strain designed to maximize induction of cross protective immunity to enteric pathogens also engineered to express an ubiquitous E. coli pilus antigen does or does not have an adverse effect on normal E. coli flora. Thus, in addition to following titers of resident E. coli strains, we also measure weights, food consumption and general health over a period of some six months. If adverse consequences are observed, we examine whether administering probiotic microbial populations counters the adverse effect of vaccination.
Determining Whether Antigen Interference is Observed Such that Vaccination with a Mixture of Vaccine Strains Will Give Better Protection that Achieved with a Single Genetically Engineered Vaccine.
By careful analysis of data collected throughout these studies, we observe whether antibody titers to a particular antigen(s) decrease when a particular carrier vaccine strain is modified to deliver additional protective antigens. The rigorous test involves constructing a strain that expresses multiple antigens from the same vector or two or more vectors in the same strain. In this regard, we have fusions of two pneumococcal antigens on the same plasmid and the immune responses to each are independent of the order of antigens in the fusion. We thus engineer Asd+, DadB+ and MurA+ vectors to express the various antigens described in the studies described as above. In fact, we have a fourth balanced-lethal vector system using plasmid expression and chromosome deletion of the murl gene encoding glutamate racemase. The comparison with the multi-valiant vaccine would involve making a mixture of the individual vaccines generated among those proposed.
The basic idea to evaluate first is whether sera from immunized mice can passively protect naive unimmunized mice from any of the invasive pathogens capable of a lethal outcome. The studies described above likely identify strains of Salmonella, E. coli, Shigella (administered intranasally), Yersinia and C. perfringens suitable for these evaluations. We also investigate whether immunity could be transferred by transfer of periferal blood lymphocytes. Another assay would be to determine whether sera from immunized mice prevented invasion of any of the pathogens into cells in culture or protect against transcytosis using polarized cell monolayers. Our collective studies provide additional means by which a multi-valent vaccine protective against many enteropathogens could be evaluated for protective efficacy against the individual pathogens. Ultimate clinical evaluation would be by immunization of travelers and having them record bouts of diarrhea or other enteric disease during their subsequent travels.
Although not common, S. enterica strains of the non-typhoidal type can occasionally be associated with Reiter's syndrome or adjuvant arthritis. There is no present way to determine what Salmonella gene differences are responsible or whether our extensive genetic manipulation of the S. Typhimurium vaccine strain might decrease or even increase the possibility for such adverse sequela. For these reasons, we are developing S. Paratyphi A strains in parallel to the construction of the S. Typhimurium vaccine strains in case the risks of using a S. Typhimurium based vaccine in the developed world are judged to he unacceptably high.
We have constructed χ9608 (Δpmi-2426 Δ(gmd-fcl)-26 ΔPfur81::TT araC PBAD fur ΔPphoPQ107::TT araC PBAD phoPQ ΔPcrp527::TT araC PBAD crp ΔagfBAC811 ΔaraE25 ΔaraBAD23 ΔrelA198::araC PBAD lacI TT ΔsopB1925 ΔasdA33) which has the ΔPphoPQ107::TT araC PBAD phoPQ mutation not present in χ9558, the ΔasdA33 instead of the ΔasdA27::TT araC PBAD c2 mutation that are easily interchangeable. Safety of χ9608 has been demonstrated by oral inoculation of 108 CFU into day-of-birth mice. Since we have now shown that the presence of various ΔPphopQ::TT araC PBAD phoPQ constructions reduced induction of mucosal and serum antibody responses to protective antigens delivered by RASV strains, we have replaced the ΔPphoPQ107::TT araC PBAD phoPQ deletion-insertion mutation with the wild-type phoPQ alleles and now have several S. Paratyphi A strains all with the Δpmi-2426 Δ(gmd fcl)-26 ΔPfur81::TT araC PBAD fur ΔPcrp527::TT araC PBAD crp ΔaraE25 ΔrelA198::araCPBADlacITT ΔsopB1925 ΔagfBAC811 mutations (as present in χ9606) and derivatives with ΔaraBAD23, with ΔaraBAD23 and ΔasdA33 or with Δ(araC-PBAD)-5::P22PR araBAD mutation (χ9651, χ9762, χ9763). We have inserted additional mutations for use with multiple Asd+, DadB+ and MurA+ vectors, to confer biological containment and to add other desirable attributes based on our results with S. Typhimurium vaccine strains.
The difficulty in making a vaccine against extra-intestinal E. coli (ExPEC) in birds, animals and humans is related to the diversity of these strains. Targeting common genes among ExPEC strains will be the best strategy to have an efficient vaccine against ExPEC. pAPEC-1 encodes for different iron acquisition systems, common among ExPEC strains, mostly involved in their extra-intestinal life. Salmonella vaccine cured of pSTUK100 with the ΔcysG/75::Pspv175 spvABCD delection-insertion mutation and containing pAPEC-1 will be tested in chickens for its ability to protect against APEC infection, airsaculitis and septicemia, by challenging birds with APEC from worldwide serogroups O1, O2, and O78. The vaccine will be also tested in mice for its ability to protect against UPEC infections, by challenging mice with UPEC via urethral inoculation.
We are cloning the codon-optimized virulence genes psn from Yersinia and aerobactin operon (iutA iucABCD) from Shigella (
We recently constructed derivatives of χ9590 (Δpmi-2426 Δ(gmd-fcl)-26 ΔPfur81::TT araC PBAD fur ΔPcrp527::TT araC PBAD crp ΔasdA27:: TT araC PBAD c2 ΔaraE25 ΔaraBAD23 ΔrelA198::araC PBAD lacI TT ΔsopB1925 ΔagfBAC811 Δalr-3 ΔdadB4) (Table 7) with various types of rec mutations to investigate possible difficulties in the stable maintenance of two plasmids specifying two different protective anigens and with either the AsdA+ or DadB+ selective marker but with some DNA sequences in common due to using the same Ptrc promoter, termination sequence and pBR ori. Although we determined that recombination between plasmids as well as within plasmids was exceedingly rare (frequency of no more than 10−3 after full growth of a culture for some 30 generations), we found even greater stability in the χ9590 derivative strain χ9760 that has the ΔrecF126 allele in which recombination between plasmids was reduced another 10-fold. The inclusion of the ΔrecF126 deletion mutation in the wild-type S. Typhimurium UK-1 strain χ3761 has no effect on virulence having the same LD50. We will therefore include the ΔrecF126 mutation in strains to maintain multiple plasmids specifying synthesis and delivery of multiple protective antigens from diverse enteric pathogens. In addition to χ9760 that can stably maintain both AsdA+ and DadB+ plasmid vectors encoding two different protective antigens and in one case with one vector encoding a fusion to synthesize two different protective antigens to result in a strain deliverying three different protective antigens, we have constructed χ9822 (Δpmi-2426 Δ(gmd-fcl)-26 ΔPfur77::TT araC PBAD fur ΔPcrp527::TT araC PBAD crp ΔasdA27::TTaraCPBAD c2 ΔaraE25 ΔaraBAD23 Δrel1A98:: araC PBAD lacI TT ΔPmurA7::TT araC PBAD murA) to permit co-maintenance of AsdA+ and MurA+ plasmids each specifying one (or two as a fusion) different protective antigens.
It should be evident that inserts encoding protective antigens from enteric pathogens in AsdA+ vectors such as the Shigella IpaD antigen in pYA4418 (
Further enhancement in induction of cross-protective immunity against diverse enteric pathogens can be achieved as by introduction of the ΔPmnt22::TT araC PBAD mntR deletion-insertion mutation to cause an up-regulation of protein antigens for the uptake of manganese, an essential nutrient, in vaccine strains as has been done in χ9914 (Δpmi-2426 Δ(gmd-fcl)-26 ΔPfur81::TT araC PBAD fur ΔPcrp527::TT araC PBAD crp ΔasdA27:: TT araC PBAD c2 ΔaraE25 ΔaraBAD23 ΔrelA198::araC PBAD lacI TT ΔsopB1925 ΔagfBAC811 ΔaraBAD23 ΔPmnt22:TT araC PBAD mntR).
Further enhancement in inducing cross-protective immunity to diverse enteric pathogens is achieved by curing the S. Typhimurium virulence plasmid with insertion of the virulence plasmid-specified spvABCD operon as accomplished by the addition of the ΔcysG175::Pspv175 spvABCD deletion-insertion mutation in χ9876 (Example 8) followed by addition of pAPEC-1 that encodes many cross-reactive iron acquisition, manganese acquisition and serum resistance surface antigens that would be over expressed in vaccine strains with the ΔPfur81::TT araC PBAD fur ΔPmnt22::TT araC PBAD mntR ΔPcrp527::TT araC PBAD crp mutations. By introducing multiple additional mutations to enable use of diverse balanced-lethal and balanced-attenuation plasmid vectors one can cause synthesis of the many other protective antigens from diverse enteric pathogens as described in Examples 8, 9, 10, 11, 12, 13, 14, 15 20 and 21.
In design of a vaccine for humans, we would further modify a strain of S. Paratyphi A as described above in Examples 16 and 19. It should be noted that S. Paratyphi A does not have a virulence plasmid so it is immediately possible to introduce the modified pAPEC-1 plasmid with the ΔcvaAB::psn iutA iucABCD insertion described in Example 21 above. It is evident that all other modifications noted above could easily be introduced into such S. Paratyphi A derivative vaccines for humans.
This invention was made with government support under Grant No. 5U01 A1060557, Grant No. 5RO1A124533, and Grant No. 5ROI A1057885 awarded by The National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US08/78991 | 10/6/2008 | WO | 00 | 10/26/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60978084 | Oct 2007 | US |
