Patent Application
20230090746
This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “10457-465PC0_ST25” created on Feb. 8, 2021 is 92 KB in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
H. pylori is a Gram-negative bacterium infecting the stomach of more than half of the Earth's human population and is the main cause of gastric pathologies including peptic ulcers, dyspepsia and gastric cancer [1, 2]. Prevalence differs from 10 to >70% [3]. H. pylori has been confirmed as a class I carcinogen by the World Health Organization [4]. Gastric cancer is still the second leading cause of death by cancer worldwide [5]. Eradication of H. pylori has been frequently considered as an efficient approach to cure peptic ulcer disease in addition to gastric cancer [6]. No doubt, antibiotics are the first choice in treatment. However, H. pylori infections present numerous challenges to successful antimicrobial therapy, some of which are limited to H. pylori and others are experienced in the treatment of other infections. Challenges arise from the fact that H. pylori colonize the stomach where they are protected by a dense mucus layer and an acidic environment. Additionally, the stomach is continuously secreting acid and discharging its contents such that typical therapy would be diluted and washed out [7]. The efficacy of several antimicrobials is greatly reduced at acidic pH and proper pH is needed for them to be effective. H. pylori can obtain resistant genotypes and become multi-drug resistant (MDR). Importantly, a recent study has shown that eradication of toxigenic H. pylori expressing VacA is not achievable using only antibiotics [8]. In clinical practice, the quick emergence of resistance raised concerns about the correct management of this bacterial infection [9, 10]. Thus, protective and therapeutic vaccines could be an alternative method for antibiotic treatment against H. pylori infection. The importance of CD4+ T cells in protective immunity against H. pylori has been broadly accepted [11]. Oral administration of recombinant Salmonella vectored vaccines could provoke classical Th1-type responses and also induce a significant mucosal SIgA response [12, 13] through transcytosis by micro-fold cells [14] or by direct antigen presentation by interstitial dendritic cells [15]. It was reported that a large number of foreign antigens, synthesized and delivered by live attenuated Salmonella, protected animals against a diversity of pathogens including viruses, bacteria and parasites [16]. Oral administration of a live attenuated Salmonella vector vaccine synthesizing and delivering H. pylori protective antigens could, therefore, be a promising approach to eradicate H. pylori infections.
SEQ ID NOs: 1-4 show the natural nucleic acid sequence, and optimized nucleic acid and amino acid sequences of HopM antigen, and signal peptide, respectively.
SEQ ID NOs: 5-8 show the natural nucleic acid sequence, and optimized nucleic acid and amino acid sequences of of HpaA antigen and signal peptide, respectively.
SEQ ID NOs: 9-11 show the natural nucleic acid sequence, and optimized nucleic acid and amino acid sequences of of UreA antigen, respectively.
SEQ ID NOs: 12-14 show the natural nucleic acid sequence, and optimized nucleic acid and amino acid sequences of of CagA antigen, respectively.
SEQ ID NOs: 15-20 show the nucleic acid and amino acid sequences of VacA, BabA, and NapA, respectively.
SEQ ID NOs: 21 and 22 show the nucleic acid and amino acid sequences of a chimeric gene and protein, respectively, based on fliD, vacA, ureB, and cagA.
SEQ ID NOs: 23-25 show the natural nucleic acid sequence, and optimized nucleic acid and amino acid sequences of UreB antigen, respectively
As disclosed herein, PIESVs have been developed that specifically synthesize heterologous antigens and enhance the induction of immune responses and protection against viruses and bacteria [17, 18]. Such mucosal PIESV delivery induces strong mucosal, systemic and cellular immunities against the targeted pathogen [19-24]. Since H. pylori colonize and move through a mucosal body surface, the induction of mucosal immunity dependent on the production of secretory antibodies (SIgA/SIgM) as well as cellular immunity offers the first line of defense against infection with such pathogens [25-30]. Injectable subunit recombinant protein vaccines are unable to induce such mucosal immunity in contrast to PIESVs that induce mucosal, systemic and cellular immunities that can block infections and preclude disease [18, 31-38]. Here, disclosed is PIESVs synthesizing H. pylori protective antigens against H. pylori SS1 infection.
As used herein the specification, “a” or “an” may mean one or more, unless clearly indicated otherwise. As used herein in the claims, when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.
The terms “attenuated” or “attenuation” as used herein refer to the process of rendering certain pathogen virulence attributes needed to cause diseases less able to cause such disease symptoms. In one example, attenuation involves imparting an attenuation mutation in the pathogen.
The term “attenuating mutation” refers to a mutation imparted into a pathogen that reduces infectivity, virulence, toxicity, induction of disease symptoms, and/or impairment of a subject upon administration of the pathogen (e.g. PIESV strain). Examples of attenuating mutations include those mutations that facilitate lysis in vivo (e.g. impairing synthesis of essential constituents of peptidoglycan layer), reduce or impair synthesis of LPS or other cell-surface components, and one or more mutations that provide auxotrophy (e.g. dependence on an amino acid, purine, pyrimidine, or vitamin for growth).
The term “attenuated derivative of an invasive pathogenic microorganism” refers to a microorganism that is derived from an invasive pathogenic microorganism but which has been genetically modified to be attenuated.
As used herein, “codon” means, interchangeably, (i) a triplet of ribonucleotides in an mRNA which is translated into an amino acid in a polypeptide or a code for initiation or termination of translation, or (ii) a triplet of deoxyribonucleotides in a gene whose complementary triplet is transcribed into a triplet of ribonucleotides in an mRNA which, in turn, is translated into an amino acid in a polypeptide or a code for initiation or termination of translation. Thus, for example, 5′-TCC-3′ and 5′-UCC-3′ are both “codons” for serine, as the term “codon” is used herein.
The term “codon optimized” or “codon optimization” as used herein refers to enhancing the ability of the antigen encoding sequence to be expressed in the Salmonella vaccine strain by selecting codons that are used for highly expressed genes in Salmonella.
Such codon optimization also includes changing the GC content of the antigen encoding sequence to be similar to that used for Salmonella (i.e., ˜52% GC). In addition, the codon optimization can also be used to enhance the stability of the mRNA encoded by the antigen encoding sequence so as to be less likely to be degraded by RNases.
The term “delayed attenuation” as used herein refers to a means of gene regulation such that the attenuation attribute is not expressed during growth of the vaccine strain or during its administration to an animal host but is not expressed after the vaccine enters the animal host and is manifest as a consequence of vaccine cell division in vivo with gradual dilution of the virulence gene product by at least half at each cell division in vivo.
The term “high level synthesis” refers to a means of synthesizing a protein antigen at a level that exceeds that level of synthesis that would be synthesized by a chromosomal gene encoding that antigen and can be achieved by encoding the antigen gene on a multi-copy plasmid and/or by placing the antigen encoding sequence under the control of a promoter known to cause gene product synthesis at an elevated level greater than would be caused by using the native promoter for that antigen encoding gene.
The term “balanced-lethal plasmid-host” is the design of the plasmid-host composition such that survival of the host is dependent on the maintenance of the plasmid such that loss of the plasmid results in death of the host. (See Nakayama, K., Kelly, S. & Curtiss, R. Construction of an ASD+ Expression-Cloning Vector: Stable Maintenance and High Level Expression of Cloned Genes in a Salmonella Vaccine Strain. Nat Biotechnol 6, 693-697 (1988) or Galán J E, Nakayama K, Curtiss R 3rd. Cloning and characterization of the asd gene of Salmonella typhimurium: use in stable maintenance of recombinant plasmids in Salmonella vaccine strains. Gene. 1990 Sep. 28; 94(1):29-35, whose teachings are incorporated by reference).
The term “regulated delayed lysis” refers to a construction in which the expression of one or more genes specifying synthesis of peptidoglycan precursors such as but not limited to diaminopimelic acid and muramic acid are regulated by a sugar-dependent process such that the genes are expressed in the presence of a sugar such as but not limited to arabinose supplied during cultivation of the strain and cease to be expressed in vivo since the sugar is absent to result in lysis as a consequence of cell division of the PIESV strain in vivo. The genes conferring the regulated delayed lysis phenotype may be either chromosomal and/or plasmid encoded.
The term “regulated delayed lysis plasmid” refers to a construction in which the expression of one or more genes specifying synthesis of peptidoglycan precursors such as but not limited to diaminopimelic acid and muramic acid that are regulated by a sugar-dependent process are located on a plasmid vector encoding synthesis of one or more protective antigens.
The terms “animal host”, “host” or “subject” are used interchangeably and refers to a human or nonhuman mammal into which an attenuated derivative of an invasive pathogenic microorganism has been administered. In a specific embodiment, the animal host is a human.
The term “operably linked” as used herein means that one nucleic acid sequence is linked to another nucleic acid sequence, and therefore the function or expression thereof is influenced by the linked nucleic acid sequence.
The term “administering” or “administration” of an agent as used herein means providing the agent to a subject using any of the various methods or delivery systems for administering agents or pharmaceutical compositions known to those skilled in the art. Agents described herein may be administered by oral, intradermal, intravenous, intramuscular, intraocular, intranasal, intrapulmonary, epidermal, subcutaneous, mucosal, or transcutaneous administration.
A used herein, the term “immune response” includes a response by a subject's immune system to a vaccine Immune responses include both cell-mediated immune responses (responses mediated by antigen-specific T cells and non-specific cells of the immune system) and humoral immune responses (responses mediated by antibodies present in the plasma lymph, and tissue fluids). The term “immune response” encompasses both the initial responses to an immunogen as well as memory responses that are a result of “acquired immunity.”
The term “Protective Immunity Enhanced Salmonella Vaccine” or “(PIESV)” refers to a vector strain that has been engineered to synthesize and deliver an immunogen (e.g. Helicobacter protein or other type of antigen).
The term “Protective Immunity Enhanced Salmonella Vaccine vector strain” or “PIESV vector strain” refers to a strain of Salmonella that has one or more attenuating mutations and is capable of being engineered to express an immunogen insertion.
The term “protective immunity” as used herein refers to induction of an immune response upon administration of a vaccine sufficient to confer protection against a pathogen.
As used herein, the term “vaccine” refers to an immunogen or a composition comprising an immunogen that elicits an endogenous immune response in a subject (e.g., a human or animal) The endogenous immune response may result in, for example, the switching of a Th1 biased immune response to a Th2 biased immune response, the activation or enhancement of T effector cell responses and/or the reduction of T regulatory cell response, the activation of antigen-specific naive lymphocytes that may then give rise to antibody-secreting B cells or antigen-specific effector and memory T cells or both, and/or the direct activation of antibody-secreting B cells. Typically, a vaccine provides for protective immunity against a pathogen.
The term “biologically active fragment” or “biologically active variant” refers to a fragment or variant of a sequence that maintains its biological activity. In the context of H. pylori antigen sequences, a biologically active fragment or biologically active variant is a fragment or variant of an antigen amino acid sequence that elicits an immune response in a host.
The term “variant” as used herein refers to a nucleic acid sequence or amino acid sequence that possesses at least about 85, 90, 95, 96, 97, 98 or 99 percent sequence identity to another nucleic acid sequence or amino acid sequence, respectively.
The term “sequence identity” or “identity,” as used herein in the context of two polynucleotides or polypeptides, refers to the residues in the sequences of the two molecules that are the same when aligned for maximum correspondence over a specified comparison window.
As used herein, the term “percentage of sequence identity” or “percent sequence identity” may refer to the value determined by comparing two optimally aligned sequences (e.g., nucleic acid sequences or polypeptide sequences) of a molecule over a comparison window, wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleotide or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. A sequence that is identical at every position in comparison to a reference sequence is said to be 100% identical to the reference sequence, and vice-versa. The term “about” with respect to a numerical value of a sequence length means the stated value with a +/− variance of up to 1-5 percent. For example, about 30 contiguous nucleotides means a range of 27-33 contiguous nucleotides, or any range in between. The term “about” with respect to a numerical value of percentage of sequence identity means the stated percentage value with a +/− variance of up to 1-3 percent rounded to the nearest integer. For example, about 90% sequence identity means a range of 87-93%. However, the percentage of sequence identity cannot exceed 100 percent. Thus, about 98% sequence identity means a range of 95-100%.
Salmonella has been extensively investigated among bacterial genera for delivering recombinant protective antigens and DNA vaccine vectors due to its capability to be delivered mucosally, thereby stopping the use of needles for immunization [18]. Live attenuated bacterial vaccines preferably comprise strains that possess two or more stable attenuating mutations dispersed in the bacteria chromosome and plasmids (without antibiotic-resistant markers) into which genes encoding heterologous antigens from microbial pathogens can be inserted. The attenuating mutations should be in genes encoding essential components of bacterial cell structures or biosynthetic pathways for crucial nutrients that are not freely available in any environment that the PIESV may reside outside of the laboratory [18].
In this study, PIESVs were employed to deliver and synthesize nine recombinant protective antigens of H. pylori. Mice were immunized with each construct alone or a combination of them. After that, Based on the results obtained, a series of plasmids were constructed encoding for synthesis of two antigens for delivery by the PIESV construct. Immunization of mice with mixtures of these constructs revealed the best antigen combinations. It was found that immunization of mice with the combination of two plasmids each carrying genetic sequences encoding two different antigens (totally four antigens including HpaA, NapA (also termed Hp-NAP), UreA and UreB) provided statistically significant protection against H. pylori SS 1 challenge infection. These results not only emphasize the efficacy of the cocktail vaccine to provide sterile protection but also show the strong applicability of PIESVs, especially the improved plasmids used in this system. The induction of protective immunity by a PIESV delivering multiple antigens in the hostile environment of the stomach was a new and surprising result. To unravel the mechanism of protection provided by the cocktail vaccine, the humoral and cellular immune responses induced by immunization were measured and compared to mice immunized with a combination of the two PIESV-empty vector strains or PBS.
Although the detailed mechanism by which immunization protects against H. pylori remains to be clarified, the vital role of cell-mediated immunity has been well-known by showing that protection against H. pylori infection can be obtained even in the lack of B cells [77]. In this regard, it has been shown that immunological eradication of H. pylori usually is contingent on Th1-type responses and IFN-γ, the prototype Th1 cytokine, playing a main role in the control of H. pylori infection by activating macrophages and changing antibody responses toward protective IgG2c in C57BL/6 mice [78]. The data herein shows very high levels of Th1 (IL-12 and IFN-γ) and Th17 (IL-17) cytokines during recall responses of T cells from mice immunized with the cocktail mixture of PIESV strains each delivering two antigens each.
Consistent with the proliferative response, T cells from mice immunized with the vaccine, produced higher levels of IFN-γ in response to each component of the cocktail than mice that received PBS or the two empty-vector PIESV strains alone. In addition, T cells of immunized mice produced high levels of IL-6 when re-exposed to the immunizing antigens. IL-6 is deemed one of the mediators directing the transition between innate and adaptive immunity. Its production is mostly targeted by the PAMP-mediated TLR signaling cascade and as a result, IL-6 gene-deficient mice display impaired defense against some bacterial infections [79]. Thus, it is possible that this cytokine plays a key role in providing immunity against H. pylori. Besides, Th17 cells and their effector cytokines seem to mediate immunity against numerous infections, mainly those caused by extracellular pathogens like H. pylori [80]. Th17 immune responses include neutrophil recruitment, secretion of antimicrobial peptides, and Th1-mediated protection driven by IL-17 [81,82]. In the study described in the Examples herein, a significant amount of IL-17 was measured after the re-stimulation of T cells with each recombinant antigen in the cocktail vaccine.
Consistent with the proliferative response, T cells from mice immunized with the cocktail mixture PIESV vaccine delivering four protective antigens, produced higher levels of IFN-γ in response to each component of the cocktail than mice that received PBS or a mixture of the empty vector PIESV strains. In addition, T cells of immunized mice produced high levels of IL-6 when re-exposed to the immunizing antigens. IL-6 is deemed one of the mediators directing the transition between innate and adaptive immunity. Its production is mostly targeted by the PAMP-mediated TLR signaling cascade and as a result, IL-6 gene-deficient mice display impaired defense against some bacterial infections (79). Thus, it is possible that this cytokine plays a key role in providing immunity against H. pylori infection. Besides, Th17 cells and their effector cytokines seem to mediate immunity against numerous infections, mainly those caused by extracellular pathogens like H. pylori (80). Th17 immune responses include neutrophil recruitment, secretion of antimicrobial peptides, and Th1-mediated protection driven by IL-17 (81, 82). Thus, a significant amount of IL-17 was measured after the re-stimulation of T cells with each recombinant antigen in the cocktail vaccine. The level of circulating IL-22 cytokine also spiked after immunization with the PIESV-Hp mixture and the PIESV-empty vector mixture compared to the non-immunized control group only receiving PBS. It seems that Salmonella vaccines are able to induce the production of circulating of IL-22 that has been shown to play an important role in protecting the gut tissue integrity and enhance disease lessening through chronic Salmonella Infection (83).
To gain insight further into the underlying mechanism, the kinetics of antibody production against each component of the vaccine were observed and it was found that both antigen-specific IgG1 and IgG2c antibodies were produced. Notably, the IgG1/IgG2c ratio also showed that the response elicited in immunized mice had a strong Th1 bias. Since IFN-γ and IgG2c are markers of the Th1 type of immune response in C57BL/6 mice, and synthesis of IgG1 can be controlled by Th2 clones, seeing the results obtained from cytokine assays and these findings, one might conclude that the immune responses elicited by the vaccine was of a mixed Th1/Th17 type [78].
The mucosal immune responses produced by immunization were measured. With that goal, the gastric IgA secretion into the gastric mucosa and immune-mediated inflammation were measured in immunized mice. Compared to control immunized mice, immunized mice elicited significant levels of each antigen-specific gastric IgA. These surprising findings indicate that immunization with PIESV strains delivering heterologous antigens efficiently stimulated gastric mucosal immune responses.
The responses of other components of the immune system in immunized mice against infection by H. pylori were also investigated. Thus, synthesis of several antimicrobial peptides and chemokines were assessed. The results only showed that production of CXCL2 was significantly upregulated in the stomach of immunized mice. CXCL2 is also a mighty neutrophil chemoattractant and is involved in numerous immune responses comprising wound healing, cancer metastasis, and angiogenesis [84]. This result shows that the composite vaccine is capable to induce recall innate as well as acquired immunity. Systemic immune responses were also studied in immunized mice. Although in proliferation assays, CD8+ T cells were propagated more than CD4+ T cells in response to re-stimulation, the number of CD4+ T cells but not CD8+ T cells expressing IFN-γ increased after infection with H. pylori SS1 in the spleen of immunized mice. This supports the IgG titer data that indicate that systemic immunity was skewed towards a Th1 response after immunization. However, local immunity in the stomach tissues may be more dependent on CD8+ T cells. Further investigation is therefore needed to clarify the contribution of CD4 versus CD8 T cells in the stomach tissue.
The result of histopathology measurements showed an increased number of gram-positive rod-shaped bacteria in the stomach of control groups compared to the immunized group. Although the species of this population was not verified, this finding shows the role of H. pylori infection in increasing the number of other gram-positive bacilli which might have a role in worsening the stomach complications caused by H. pylori infection. In this regard, it has been indicated that gastric colonization by non-H. pylori bacteria, such as Bacteroides, Actinobacteria, Fusobacteria, Firmicutes, and Proteobacteria, could influence the risk for gastric cancer [85-89]. Notably, the vaccine embodiments disclosed herein not only provided sterile protection in seven out of 10 of the immunized mice but it also caused a significant reduction in the titer of gram-positive rod-shaped bacteria.
In sum, a S. Typhimurium-based live vaccine including the delivery of four protective antigens of H. pylori confers significantly high protection against H. pylori infection in mice. Additionally, immunological studies showed the induction of both specific acquired immune responses against each antigen and the innate immunity responses. The findings herein also showed the increased number of gram-positive rod-shaped bacteria in the presence of H. pylori in control groups compared to the immunized mice.
An attenuated microorganism as described herein that is capable of the regulated expression of at least one nucleic acid sequence encoding a Helicobacter antigen may also comprise, in part, a plasmid vector. The plasmid vector comprises a nucleic acid sequence encoding at least one Helicobacter antigen operably linked to a promoter. The promoter is 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 microorganism, but the microorganism is capable of high-level synthesis of the antigen in an animal or human host.
As used herein, “plasmid vector” refers to an autonomously replicating nucleic acid unit. The term plasmid vector is used in its broadest sense and includes many types of vectors. Examples include bacteriophage viral vectors, cosmid, phasmid, in addition to conventional plasmid vectors. It is noted that bacteriophage viral vectors include integrated prophages or plasmid derivatives of phages (e.g. lambda dv is a plasmid derivative of lambda phage that can be used as a vector). Also, it is noted that some bacteriophages infect bacteria as prophages but exist as in plasmid form upon infection into the bacterium (e.g. P1 bacteriophage of Shigella and E. coli.).
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 ori 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 one or more than one nucleic acid sequence encoding a Helicobacter antigen, whether regulated or not, as detailed above (SEQ ID NOs: 1-23).
In each of the above embodiments, the microorganism capable of regulated expression of Helicobacter antigens also is attenuated. “Attenuated” refers to the state of the microorganism (e.g., bacterium) wherein the microorganism has been weakened from its wild-type fitness by some form of recombinant or physical manipulation. This may include altering the genotype of the microorganism to reduce its ability to cause disease. However, the bacterium's ability to colonize the gut (in the case of Salmonella) and internal effector lymphoid tissues to induce immune responses is, preferably, not substantially compromised. For instance, in one embodiment, regulated attenuation allows the recombinant microorganism to express one or more nucleic acids encoding products important for the microorganism to withstand stresses encountered in the host after immunization. This allows efficient invasion and colonization of lymphoid tissues before the recombinant microorganism is regulated to display the attenuated phenotype.
In one embodiment, a microorganism may be attenuated by regulating LPS O-antigen synthesis. In another embodiment, a recombinant microorganism may be attenuated as described below. In which case, both regulated attenuation and regulated expression of a Helicobacter 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. In other embodiments, such regulation of attenuation or regulation of gene expression may be dependent on other sugars such as mannose or rhamnose, which are unavailable in a non-phosphorylated form in vivo.
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 encoding the virulence protein, so that the production levels of the virulence protein are optimal for both the regulated attenuation phenotype and the regulated expression when growing strains with a given concentration of arabinose. 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 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 microorganism 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, rfaH, waaL, poxA, galU, mviA, sodC, recA, ssrA, sirA, sifA, 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 microorganism to induce disease symptoms.
The microorganism 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 microorganism 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 microorganism 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. In any of these cases, plasmid vectors can possess the wild-type gene to complement the deleted chromosomal gene to establish the balanced-lethal vector-host system. This system is stably maintained in vivo due to the absence of the required nutrient imposed by the chromosomal mutation with loss of the plasmid resulting in death of the vaccine cell.
Yet another balanced-lethal host-vector system comprises modifying the microorganism 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 microorganism may comprise the ΔPmurA::TT araC ParaBAD 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 microorganism 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 dependent lethal mutation. In the absence of arabinose, synthesis of muramic acid ceases and lysis of the microorganism occurs because the peptidoglycan layer of the cell wall is not synthesized. It is not possible to generate ΔmurA 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 ParaBAD 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 ParaBAD 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 ParaBAD 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 ParaBAD 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 ParaBAD c2 inserted. The c2 nucleic acid sequence in ΔasdA27::TT araC ParaBAD c2 has a SD sequence that was optimized to TAAGGAGGT. It also has an improved ParaBAD 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 microorganism 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 ΔasdA27::TT araC ParaBAD c2 and ΔPmurA25::TT araC ParaBAD 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. 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 pG8R114 contains the murA nucleic acid sequence (with altered start codon sequences to decrease translation efficiency) under the control of an araC ParaBAD promoter. Also, the second nucleic acid sequence under the direction of this promoter is the asd nucleic acid sequence (with altered start codon sequences to decrease translation efficiency). The P22 PR promoter is in the anti-sense direction of both the asd nucleic acid sequence and the murA nucleic acid sequence. The P22 PR is repressed by the C2 repressor made during growth of the strain in media with arabinose (due to the ΔasdA27::TT araC ParaBAD c2 deletion-insertion). However, C2 concentration decreases due to cell division in vivo to cause PR directed synthesis of anti-sense mRNA to further block translation of asd and murA mRNA. The araC ParaBAD sequence is also not from E. coli B/r as originally described but represents a sequence derived from E. coli K-12 strain χ289 with tighter control and less leakiness in the absence of arabinose. In the preferred embodiment, transcription terminators (TT) flank all of the domains for controlled lysis, replication, and expression so that expression in one domain does not affect the activities of another domain. As a safety feature, the plasmid asd nucleic acid sequence does not replace the chromosomal asd mutation since they have a deleted sequence in common, consequently, the E. coli murA nucleic acid sequence was used in the plasmid instead of using the Salmonella murA nucleic acid sequence. The recombinant microorganism of this embodiment is avirulent at oral doses in excess of 109 CFU to BALB/c mice. In addition to being fully attenuated, this construction exhibits complete biological containment with no in vivo recombinant bacteria survivors detectable after 21 days and no recombinant bacteria survivors during or after excretion. This property enhances vaccine safety and minimizes potential for immunization of those not intended to be immunized or in humans not elected to be immunized.
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 microorganism 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.
Various Helicobacter antigens and sequences may be implemented in the vaccine system and microorganism disclosed herein. Examples of Helicobacter antigens that may be implemented include but are not limited to (1) Hp-NAP (also termed NapA) (e.g. SEQ ID No. 20), (2) HpaA (e.g. SEQ ID NO. 7), (3) UreA (e.g. SEQ ID NO. 11), (4) UreB (e.g. SEQ ID NO. 25), (5) HopM (e.g. SEQ ID NO. 3), (6) BabA (e.g. SEQ ID NO. 18), (7) Chimeric Protein (e.g. SEQ ID NO. 22), (8) CagA (e.g. SEQ ID NO. 14), (9) VacA (e.g. SEQ ID NO. 16) antigens. Nucleic acid and amino acid sequences relating to the aforementioned 1-9 antigens are provided in SEQ ID NOs 1-25. The sequences of these antigens are known and provided in the cited literature. In preparation for vector construction, we do a complete bioinformatic analysis of all structural properties of each protein and the structure of the transcribed mRNA analyzed to then modify DNA codons to enhance stability of mRNA and efficiency of translation in Salmonella. Sometimes protein engineering may be conducted to delete sequences that interfere with stability or synthesis by the PIESV vector strain that impair growth and that are not likely important for immunogenicity. In certain embodiments, nucleic acid sequences encoding the aforementioned nine Helicobacter antigens may include the native sequences of the foregoing 1-9 antigens or codon optimized versions of these 1-9 antigens. In specific examples, embodiments relate to the Helicobacter antigens (1-9 above), or biologically active fragments or biologically active variants of these antigens, or nucleic acid acids encoding the same. Thus, nucleic acid sequences encoding a given antigen may be incorporated into engineered vectors as an expression construct for expression in a host.
For Helicobacter antigens without native signal sequences, the commercially synthesized DNA sequences were most often inserted into pG8R111 (pBR ori), or optionally, pYA4589 (p15A ori). For those proteins with a native signal sequence, the native signal sequence is removed, and the codon-optimized sequence lacking the native SS is inserted into the pG8R114 (pBR ori) and pG8R113 (p15A ori) vectors with the improved bla SS (43) to maximize antigen synthesis levels and secretion to enhance production of immunogenic outer membrane vesicles. We also insert sequences without their native signal peptides into the T3SS vector pG8R110 (p15A ori).
As taught herein, secretory signal sequences may be included in the plasmid vectors to direct delivery of the expressed sequence out of the cell. These include secretory signal sequences for the type 2 and type 3 secretory systems. The improved bla SS used in pG8R114 and other plasmid vectors is described in Jiang et al. [43] and the sequence for the T3SS with fusion to the N-terminal 80 amino acids of the SopE protein as used in pG8R110 is given in Juarez et al. [64].
Under certain embodiments, a live attenuated microorganism 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 attenuated microorganism. It is also possible that attenuated microorganisms that survive and persist in an animal host destined for food consumption might contaminate the meat during slaughter and be transmitted through the food chain to human consumers. Consequently, in certain embodiments, an attenuated microorganism 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 some embodiments, the attenuated microorganism may comprise a method of regulated delayed lysis in vivo that prevents bacterial persistence in vivo and survival if excreted. These mutations include: Δ(wza-wcaM)-8 that prevents synthesis of colanic acid and other polysaccharide capsules that protect lysing cells from display of complete lysis and thus enhances the level of biological containment afforded by using the regulated delayed lysis in vivo attribute. ΔasdA27::TT araC ParaBAD c2 insertion-deletion mutation to impose a requirement for the peptidoglycan constituent DAP and ←PmurA25::TT araC ParaBAD 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 pG8R110, pG8R111 and pG8R114 (
An attenuated microorganism of the invention has been modified to enhance its ability to synthesize and deliver antigens that would induce protective immunity to infections caused by other pathogens, in this case from Helicobacter. As such this recombinant attenuated microorganism may be particularly suited for use as a vaccine. Infection of an animal 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 attenuated microorganism. Further penetration of the bacterium into the mesenteric lymph nodes, liver and spleen augments 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.
An attenuated microorganism 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 attenuated microorganism, 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 Helicobacter spp, may induce an immune response that helps to ameliorate symptoms associated with Helicobacter 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.
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, human or nonhuman mammals. In a specific embodiment, the mammal is a ruminant such as cow, horse, pig, goat, or sheep. The vaccine can be administered as a prophylactic or for treatment purposes. In an alternative embodiment, the animal host is a bird, such as turkey, geese or chickens.
In exemplary embodiments, the attenuated microorganism is alive when administered to a host in a vaccine composition. Suitable vaccine composition formulations and methods of administration are detailed below.
Vaccine Composition
The Salmonella vaccines discussed herein are typically lyophilized after production and may be reconstituted in a pharmaceutically acceptable carrier prior to administration. Such a carrier may be any solvent or solid material for encapsulation that is non-toxic to the inoculated host and compatible with the attenuated microorganism. 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 respiratory tract, the vaccine is preferably presented in the form of an aerosol.
The dosages of a vaccine or vaccine composition disclosed herein can and will vary depending on the attenuated microorganism, 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 1×1010 CPU depending upon the age of the host to be immunized. Administering multiple dosages may also be used as needed to provide the desired level of protective immunity.
Methods of Administration
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 attenuated microorganism, such as intravenous, intramuscular, subcutaneous injection or intramammary, intrapenile, 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, attenuated microorganisms may be administered orally. Oral administration of a composition comprising an attenuated microorganism 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.
In another embodiment, attenuated microorganisms may be administered by coarse spray. The vaccines are administered by this whole-body spray route in an amount that is effective in eliciting an immune response, i.e. antibody and/or cellular immunity. Whole-body spray administration is surprisingly effective for vaccines comprising a live avirulent derivative of an enteropathogenic bacteria such as attenuated Salmonella. The effective doses, which elicit an immune response, are roughly comparable to doses that are effective by the oral route of administration, such as administration in the drinking water.
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 an attenuated microorganism 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 an attenuated microorganism 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, veterinarians, 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 a Helicobacter antigen in a host. The method comprises administering to the host an effective amount of a composition comprising an attenuated microorganism of the invention.
In still another embodiment, an attenuated microorganism of the invention may be used in a method for eliciting an immune response against Helicobacter in a host in need thereof. The method comprises administrating to the host an effective amount of a composition comprising an attenuated microorganism as described herein. In a further embodiment, an attenuated microorganism described herein may be used in a method for ameliorating one or more symptoms of Helicobacter infection in a host in need thereof. The method comprises administering an effective amount of a composition comprising an attenuated microorganism 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 ΔPmurA::TT araC ParaBAD murA and ΔasdA::TT araC ParaBAD c2 mutations regulate synthesis of diaminopimelic acid acid and muramic acid, two essential constituents of peptidoglycan, which enable PIESV lysis in the absence of DAP and inability to synthesize the MurA enzyme without arabinose in vivo (40). The Δpmi ΔwaaL ΔpagL::TT rhaRS PrhaBAD waaL mutations collectively provide regulated delayed attenuation and cause cessation in the synthesis of LPS O-antigen in the absence of the sugars mannose and rhamnose in vivo (41, 61, 62). The deletion Δ(wza-wcaM) enhances complete lysis, blocks the synthesis of cell surface polymers to prevent biofilm formation and enhances induced immunities (63). The ΔrelA::araC ParaBAD lacI TT mutation confers the regulated delayed antigen synthesis phenotype that enhances PIESV colonization and induction of immune responses by the arabinose-dependent regulated LacI synthesis so that Ptrc-regulated genes on plasmid vectors are gradually expressed as LacI is diluted at each cell division during in vivo growth (64-66). The ΔrecF mutation decreases inter- and intra-plasmidic recombination to stabilize PIESVs (67). The ΔsifA mutation enables PIESV escape from the Salmonella-containing vesicles (SCVs) to cause PIESV cells to lyse in the cytosol (68, 69). This enables delivery of protective antigens to the proteasome for class I presentation to generate CD8−, CD17− and NKT-dependent immunities.
Codon-optimized sequences encoding all nine H. pylori antigens were successfully inserted into the plasmid vectors pG8R111 or pG8R114 (
To determine whether immunization with vaccine candidates lowers the bacterial load in the stomachs of infected mice, we ascertained CFUs of H. pylori using quantitative bacterial culture procedures. Immunized mice were infected with H. pylori SS1 two weeks after the last immunization. Then, four weeks after challenge, the stomachs of euthanized mice were removed, minced, homogenized, serially diluted and then cultured on the selective agar medium. As shown in Table 2 and
To study the humoral response against antigens in mice immunized with PIESV vector strains delivering four antigens, sera were obtained at several time points over three months following initial immunization Immunization of mice induced strong and specific immunoglobulin G (IgG) responses against each component of the cocktail vaccine including pG8R230 (UreA+UreB) and pG8R262 (NapA+HpaA), in which IgG2c (Th1-related isotypes) titers were usually slightly above those of the IgG1 subtype (
To find out the dominant subset of T cells in the spleens of immunized mice and assess whether such T cells could propagate after re-stimulation with related antigens in vitro, T cells were co-cultured with dendritic cells pulsed with each component of the cocktail vaccine. The percentage of CD4+ and CD8+ cells in the immunized group were significantly increased in comparison to PBS or PIESV-empty vector immunized mice (
Since cytokines produced by activated T cells are indicators of the type of Th responses, we also measured the amounts of ten different cytokines in supernatants obtained in T-cell propagation experiments. Cytokine secretion by the re-stimulated T cells was evaluated by Multiplex ELISA. In comparison to the controls, T cells of mice immunized with the combination of pG8R230 (UreA+UreB) and pG8R262 (NapA+HpaA) synthesized and delivered by two PIESV vector strains secreted significantly higher amounts of IFN-γ, IL-5, IL-6, IL-22, IL-17A and TNF-α in response to each recombinant protein used for immunization (
Immunized Mice Increase IFN-γ+ CD4 T and CXCL2 After Infection With H. pylon SS1
To obtain a deeper understanding of the roles of antimicrobial peptides and immunological markers in the protection provided in immunized mice, ten days after infecting the tested mice with H. pylori SS1, mouse stomachs and spleens were obtained. Reg3a, Reg3b, CXCL1, CXCL2, and CXCL5 in stomach tissues were investigated by qPCR. Spleen samples were further analyzed for T-cell markers and cytokines. In the stomachs of immunized mice after infection with H. pylori SS1, the expression of CXCL2 increased two-fold while the expression of the remaining genes did not change (
In response to H. pylori SS1 infection, IFN-γ+ CD4 T cells increased in the spleens of immunized mice compared to the controls. Additionally, no significant changes in TNF+ or IL-17A+ CD4 T cells in the spleen for any test groups were seen. There also were no significant changes in IFN-γ+, TNF+ or GranzymeB+ CD8 T cells in the spleen for any test group (
The stomach tissues were evaluated and scored based on severity 0 to 3 for criteria including mucosal inflammation and type, submucosal inflammation and type, presence of non-H. pylori bacteria, mucosal ulceration, and hyperkeratosis of squamous portions of the stomach. The scale 0-3 refers to 0=normal, 1=mild, 2=moderate, 3=severe. The inflammation ranged from nominal to severe both in the mucosa and submucosa of both the glandular and squamous portions of the stomach and consisted of lymphocytes, plasma cells with or without smaller numbers of neutrophils. Mice in control group had large amounts of hyperkeratosis in the squamous portions of the stomach which are often associated with moderate to large numbers of large, Gram-positive, rod-shaped bacteria on the surface or within the laminated keratin. The hyperkeratosis and associated bacteria are suggestive of hyporexia or anorexia for a relatively prolonged period of at least 2 days. In anorectic rodents, the build-up of excess keratin is presumably caused by reduced mechanical removal by the passage of food (70). The hyperkeratosis was not seen in a single area as suggested at the limiting ridge, but throughout all of the squamous lined areas of the forestomach.
However, significant weight loss was not seen in any group of mice. Overall, the mice receiving the PIESV-empty vector had relatively higher inflammation and numbers of non-H. pylori bacteria (gram-positive rod-shaped bacilli) compared to immunized mice. These findings might suggest the role of other gram-positive bacteria as well as H. pylori infection in the development of stomach complications. Additionally, these findings demonstrated that the vaccine candidate not only protects against H. pylori SS1 but also against other non-H. pylori flora, which may have a role in stomach complications (
Bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli and S. Typhimurium UK-1 derivative strains were routinely cultured at 37° C. in LB broth [19] or on LB agar. S. Typhimurium UK-1 mutant strains were supplemented with 50 μg/ml of diaminopimelic acid (DAP), 0.05% arabinose, 0.1% mannose and 0.1% rhamnose when necessary for bacterial growth as described in previous work (39-41). For animal experiments, S. Typhimurium χ12341 was cultured in LB broth with appropriate supplements. Overnight cultures were diluted 1:100 and grown with aeration (200 rpm) to an optical density at 600 nm of ˜0.85. Bacteria were then centrifuged at 5,000×g for 15 min at room temperature and resuspended in buffered saline with 0.01% gelatin (BSG) (42). The H. pylori strain SS1 (a kind gift from Prof. James G. Fox, Massachusetts Institute of Technology) was grown on Brucella agar supplemented with 5% sheep blood, 25 μg/ml trimethoprim, 3.3 μg/ml polymyxin B, 100 μg/ml vancomycin, 50 μg/ml amphotericin B, 200 μg/ml bacitracinin and 10 μg/ml nalidixic acid in an anaerobic jar with a microaerophilic gas generating kit (BD, USA) for 5 days at 37° C.
The regulated lysis vector pG8R111 (
Determination of Plasmid Stability
Measurement of plasmid stability is described previously (42). Briefly, vaccine strains grown overnight (G0) were diluted 1:1000 into pre-warmed fully supplemented LB grown (permissive growth conditions) with aeration for 12 h at 37° C. This process was repeated for approximately 50 generations (the last subculture is called G5) and the proportions of cells holding the Asd+ plasmids were determined for each culture. The percentage of clones holding the plasmids from each culture was determined by counting the colonies grown on LB agar with and without DAP. The continued ability of these clones to synthesize H. pylori antigens was also checked after 50 generations using western blotting.
S. Typhimurium strain χ12341 carrying recombinant plasmids (PIESV-Hp) or empty vectors (PIESV-empty vector) were cultured in LB containing 0.05% arabinose, 0.1% mannose and 0.1% rhamnose at 37° C. LB containing kanamycin 50 μg/ml was used to culture E. coli BL21 carrying recombinant plasmids as well. When the bacteria reached an OD600 of 0.6, 1 mM IPTG was added to the cultures to induce heterologous H. pylori protein syntheses. Protein samples from bacterial cultures were separated and analyzed by SDS-polyacrylamide gel electrophoresis with transfer to nitrocellulose membranes as previously described (55). Recombinant proteins were detected by anti-6xHis peroxidase (Roche, Basel, Switzerland) (1/40,000) for 2 h. E. coli BL21 carrying pG8R289 (ureA-6xHis), pG8R290 (ureB-6xHis), pG8R291 (hpaA-6xHis) or pG8R292 (napA-6xHis) were grown overnight at 37° C. in LB broth supplemented with 50 μg/ml kanamycin. The procedures for protein synthesis and purification have been described in our previous study (4). Prestained Protein Ladder, 10 to 180 kDa (Thermofisher, USA) was used as the protein marker.
All animal experiments were approved by the University of Florida Institutional Animal Care and Use Committee. Six-to eight-week-old SPF female C57BL/6 mice (n=10/group), were purchased from Charles River Laboratories (Wilmington, Mass.). Mice were acclimated for one week after arrival before starting the experiments.
Immunization procedures followed the previous description (4, 55, 56). Briefly, food and water were not given to mice for 6 h prior to the immunization and re-supplied 30 min later. Mice were orally immunized with 20 μl BSG containing 109 CFU of each strain, combination of strains, or with 20 μl BSG alone as the negative control on day 0 and boosted on days 14 and 28. Blood samples were collected individually on days 0, 14, 28, 42 and 72 and serum collected individually after centrifugation for individual analysis of antibody responses. Mice (10 mice) were immunized with 109 CFU of each PIESV synthesizing and delivering different antigens. A mixture of PIESV strains delivering pG8R111 and pG8R114 (PIESV-empty vectors) were used as controls.
To assess whether the vaccination of mice with Salmonella delivering H. pylori antigens was able to reduce bacterial load of H. pylori in the stomachs of infected mice, mice were infected with 5×108 H. pylori SS1 thrice at one day intervals two weeks after the last immunization. Four weeks post infection, mice were euthanized and their stomachs removed and H. pylori CFUs quantified. For this purpose, the quantitative bacterial culture of mouse stomach was used. Briefly, a half section of the stomach from each euthanized mouse was completely homogenized, serially diluted, and then cultured on selective medium as described above.
To specifically evaluate serum total IgG, IgG1 and IgG2c titers in immunized mice, an enzyme-linked immunosorbent assay (ELISA) was used. 96-well polystyrene plates (Nalge Nunc. Rochester, N.Y., USA) were coated with purified recombinant HpaA, UreA, UreB and Hp-NAP (5 mg/ml). Upon over-night incubation, the plates were washed three times with TBST buffer (Tris-buffered saline, pH 7.4, containing 0.05% Tween 20) followed by blocking with 300 μl PBS containing 10% FBS for 2 h at 37° C. After adding serial dilutions of mouse sera to the plates, they were incubated for 2 h at room temperature and then washed. HRP-conjugated goat-anti-mouse IgG1, IgG2c or total IgG (Southernbiotech, Birmingham, Ala., USA) were added to the wells and incubated for another 90 min at 37° C. After the last washing step, specific reactivity was calculated by the addition of 50 μl/well of the enzyme-substrate TMB (Thermofisher, USA). The reaction was stopped by adding 15 μl of 2 M H2SO4. Next, the absorbance at 450 nm was measured. To define a cut-off value for the test, the mean specific OD plus three-fold SD of sera from mice immunized with BSG was calculated at a 1:100 dilution. Serum titers were calculated as the reciprocal of the last serum dilution providing an OD higher than the cut-off (57). To assess stomach mucosal IgA production, the secretory IgA was extracted by incubation of mouse stomachs in PBS containing 5% non-fat dry milk, 4-(2-aminoethyl)-benzenesulphonyl fluorid (Calbiochem), 1 mg/ml 3.25 mM aprotinin, 10 mM leupeptin (Sigma), and bestatin (Boehringer-Mannheim Biochemicals). After extensive vortexing and centrifugation (16,000 g for 10 min), the supernatants were used for the determination of antibody titers. An ELISA was used as described above.
C57BL/6 mice were euthanized, femurs were removed under sterile conditions, and then soaked in 70% alcohol for a minute. Both ends of the bone were cut off with scissors, and the needle of a 1-mL syringe was inserted into the bone cavity to rinse the BM out of the cavity into a sterile culture dish with RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal bovine serum. The cell suspension in the dish was collected and counted. The cells suspended were distributed in plates at a density of 2×106 cells/per plate. Subsequently, GM-CSF was added into the medium to a final concentration of 20 ng/mL. The cells were cultured at 37° C. in an incubator containing 5% CO2. The culture medium was replaced 72 h later to remove the unattached cells and cell debris, then the fresh medium was supplemented with GM-CSF. On day 10, the semi-suspended cells and loosely attached cells were collected by gently pipetting the medium against the plate. The cells were pulsed with recombinant proteins (HpaA, Hp-NAP, UreA and UreB) and incubated in complete medium overnight before co-culturing with T cells.
Mouse spleens were obtained from mice immunized with PIESV-Hp (hpaA, napA, ureA, ureB), PIESV-empty vector or PBS, at 45 days post the first immunization. T cells were isolated and counted using EasySep™ Mouse T Cell Isolation Kit (STEMCELL, Cambridge, Mass. USA). T cells were then stained with CellTrace (Thermofisher, USA) to trace the propagation of T cells after re-stimulation by flow cytometry. After that, T cells and DCs already pulsed with recombinant proteins (HpaA, UreA, UreB and Hp-NAP) were co-cultured 10 to 1, (T cells to DCs, respectively) and incubated for 7 days. Supernatants were collected and cells were stained by antibodies against CD3, CD4, and CD8. Using Flow cytometer, T-cell propagation against each antigen in addition to different types of T cells were investigated.
The concentration of ten different cytokines including IFN-γ, TNF-alpha, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17A, IL-22, and IL-23 were measured in both sera and supernatants described in the previous section. We used Multiplex Assays Using Luminex (Millipore-Sigma, USA) to evaluate the concentration of cytokines in the samples. In this method, the sample is added to a mixture of color-coded beads, pre-coated with different cytokine-specific capture antibodies. The antibodies finally bind to the interested cytokines. Specific detection antibodies of cytokines of interest conjugated with biotin are added and form an antibody-antigen sandwich. PE-conjugated streptavidin is then added. It attaches to the biotinylated detection antibodies. Polystyrene beads are read on a dual-laser flow-based detection set, using Luminex 200™. One laser categorizes the bead and defines the cytokine that is being detected. The second laser defines the greatness of the PE-derived signal, which is in direct quantity to the amount of cytokine bound.
Immunological Responses in Stomach of Immunized Mice After Infection With H. pylori
To obtain further information about how the immune system responded in the immunized mice after infection with H. pylori, ten days after the challenge (day 55) mouse spleens were collected. Single-cell suspensions from spleens were prepared by pressing spleens through 100 μm cell strainers. Cell suspensions were washed in PBS and resuspended in red blood cell lysis buffer [155 mM NH4Cl; 10 mM KHCO3; 0.1 mM EDTA] for 8 min on ice. Cell suspensions were washed again with PBS before staining. Antibodies were purchased from eBioscience (CD8a, IFNγ, IL-17A, CD16/32, and TNFα), BioLegend (GranzymeB, Aqua Live/Dead), or TONBO (CD3 and CD4). CD16/32 antibody was used to block nonspecific binding to Fc receptors before all surface staining. For cytokine staining, cells were stimulated with 50 ng/mL phorbol-12-myristate-13-acetate (PMA) and 500 ng/mL ionomycin for 4 h, and Brefeldin A (2 mg/mL) was added 2 h before cell harvest. Dead cells were discriminated by LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen). Sample acquisition was performed on FACSCantoII and analyzed with FlowJo (version 10.2, Tree Star).
The expression of CXCL1, CXCL2, CXCL5, Reg3a, Reg3b, Reg3d and Reg3g were investigated using qPCR (58, 59). Briefly, extracted RNA from mouse stomach biopsy specimens obtained ten days after infection were reverse-transcribed to cDNA by PrimeScript™ RT reagent Kit (ThermoFisher). Real-time PCR was performed on the IQ5 (Bio-Rad) with PowerUp™ SYBER™ Green Master Mix (appliedbiosystems, USA) according to the manufacturer's specifications. β-actin was used for normalizing the expression. The relative gene expression was expressed as fold change calculated by the ΔΔCt method (60).
Mouse stomachs were obtained 75 days post the first immunization. Stomach tissues were fixed in 10% neutral buffered formalin for at least 24 h. The trimmed tissues were routinely processed, paraffin-embedded, sectioned at 5 μm, and stained with hematoxylin and eosin. Selected sections of stomach tissues were also evaluated with Warthin-Starry and Gram stains to identify bacteria. The pathologist was blinded as to the experimental groups and treatments of the study. Histologic samples of both glandular and squamous portions of the stomach were examined and evaluated and scored based on intensity 0 to 3 for criteria including mucosal inflammation and type, submucosal inflammation and type, non-H. pylori bacteria, mucosal ulceration, and hyperkeratosis of squamous stomach.
Statistical analysis was done using SPSS computer software, version 17.0. The statistical differences between two groups were studied by t-test and results among several groups were obtained by one factor analysis of variance (ANOVA) and Tukey's post hoc test. The statistical border for accepting significance was p<0.05. Data were envisaged using the GraphPad Prism 7 program.
E. coli
Salmonella
H. pylori lysate
Groups refer to immunization of groups of ten mice with χ12341 containing the plasmids listed in Table 1 encoding the H. pylori antigens specified.
As described in Example 1, the S. Typhimurium PIESV vector strain χ12341was used to synthesize and deliver the H. pylori protein antigens to vaccinated mice to generate the results described in Examples 2, 3 and 4. As described in the Materials and Methods for Examples 1-4, in the section on Immunization of mice, the mice were immunized on day 0 and boosted with the same dose of vaccine on days 14 and 28. This vaccination regimen was determined to be optimal to achieve the levels of protection observed. However, it would be preferable to enable induction or protective immunity after a single vaccination. This would reduce the expense associated with having to give multiple vaccinations and the problems in ensuring that vaccinated individuals return for their second and third vaccine doses. In other studies, it has been determined that χ12341, although excellent for delivering antigens to induce protective immunity to broiler chickens against bacterial pathogens, does not persist long enough in mice after a single vaccination to induce a high level of protective immunity.
χ12341 with the plasmid vectors pG8R111 and pG8R114 (
Based on these discoveries described in co-pending U.S. Provisional Application No. 63/120,940 filed Dec. 3, 2020, we described the construction of S. Typhimurium χ12615 with the genotype ΔPmurA25::TT araC ParaBAD murA ΔasdA27::TT araC ParaBAD c2 ΔwaaL46 ΔpagL38::TT rhaRS PrhaBAD2 waaL2 Δ(wza-wcaM)-8 ΔrelA1123 ΔrecF126 ΔsifA26 ΔaraBAD65::TT ΔrhaBADSR515 ΔpagP8 ΔlpxR9 (pSTUK201 Δ(traM-traX)-36::araC ParaBAD lacI TT. Many of the mutations in χ12615 are also present in χ12341 from which χ12615 was derived. These beneficial mutations present in χ12341 have thus been retained. These include Δ(wza-wcaM)-8 that eliminates some 20 genes that encode enzymes that Salmonella can use to synthesize extracellular polysaccharides such as colanic acid (2) that is made under stress and reduces completeness of regulated cell lysis (3-7) and LPS O-antigen capsule (8-10) that both act to suppress induction of immune responses. In addition, the Δ(wza-wcaM)-8 mutation enhances early onset induced antibody responses (2). The ΔrecF126 mutation reduces inter- and intra-plasmidic recombination and thus stabilizes maintenance of the genetic integrity of regulated lysis plasmid vectors (11). The ΔsifA26 mutation enables PIESV constructs to escape from the Salmonella Containing Vacuole (SCV, an endosome compartment) so that lysis of some vaccine cells occurs in the cytosol so that protective antigens released during lysis can be presented to the proteasome for Class I presentation to facilitate induction of CD8, CD17 and NKT cellular immune responses (12-15).
It is well recognized that animal sensitivity to the endotoxin of gram-negative bacteria composed of the LPS lipid A moiety varies over a 1000-fold range with birds such as chickens being very tolerant/resistant while humans and especially horses are extremely sensitive (16-21). Since deletion of genes for synthesis of lipid A is lethal due to the importance of lipid A in constituting the lipid in the outer layer of the outer membrane in gram negative bacteria, it is critical to determine the components of lipid A leading to toxicity to determine how they might be modified or eliminated to reduce toxicity. However, lipid A constitutes a Pathogen-Associated Molecular Pattern (PAMP) now generally referred to as a Microbe-Associated Molecular Pattern (MAMP) since they are present on all gram-negative bacteria) that interacts with TLR4 to recruit innate immunity. This interaction is therefore of critical importance in enhancing the immunogenicity of bacterial vectored vaccines. We have therefore included the ΔpagP8 and ΔlpxR9 mutations in χ12615 to lessen lipid A toxicity and can then easily replace the ΔpagP8 mutation with the ΔpagP81::Plpp lpxE deletion-insertion mutation that synthesizes the Francisella tularensis LpxE enzyme that eliminates the 1′ phosphate from lipid A to render lipid A totally nontoxic while retaining ability to bind to TLR4 (22).
The inclusion of the ΔaraBAD65::TT and ΔrhaBADSR515 mutations to block catabolism of arabinose or rhamnose present in vaccine cells at the time of harvesting from the fermenter delays by one to three in vivo cell divisions the cessation in synthesis of arabinose and rhamnose regulated genes. This then delays the time in which vaccine cells commence to synthesize protective antigens expressed under the control of the LacI repressible Ptrc promoter, the time for cessation of LPS O-antigen and the time for commencement of cell lysis.
Since the Δpmi-2426 mutation in χ12341 leaves two sugars still attached to the LPS core, it was removed in the construction of χ12615. Since the elimination of the WaaL enzyme which is the ligase that attaches LPS O-antigen units to the LPS core, the elimination of the Δpmi-2426 mutation increases the sensitivity of vaccine cells to C′-mediated cytotoxicity. While this is an added safety benefit, dead cells cannot synthesize protective antigens or undergo programmed continued cell lysis. We thus replaced the ΔpagL64::TT rhaRS PrhaBAD1 waaL1 mutation in χ12341 with the ΔpagL38::TT rhaRS PrhaBAD1 waaL2 sequence in χ12615 to cause a higher level of WaaL synthesis that would require more in vivo cell divisions for the amount of WaaL to be diluted out. This in turn increases the number of in vivo cell divisions for complete loss of LPS O-antigen and display of complete sensitivity to C′-mediated cytotoxicity. To achieve this the PrhaBAD-10 sequence in the ΔpagL64::TT rhaRS PrhaBAD1 waaL1 deletion-insertion mutation was changed from TAGACT to TATAAT, the SD sequence changed from AAGG to AGGA and two Lys AAA codons inserted after the ATG start codon in the ΔpagL38::TT rhaRS waaL2. These three changes increased RNA polymerase binding to the promoter, ribosome loading at the ribosome-binding site and efficiency of translation initiation.
The last significant improvement in χ12615 was to replace the chromosomal ΔrelA::araC ParaBAD lacI TT mutation with the Δ(traM-traX)-36::araC ParaBAD lacI TT) mutation that is present in the S. Typhimurium virulence plasmid now designated pSTUK201. This mutation deletes a large segment of the virulence plasmid sequence encoding genes that permitted the virulence plasmid to be conjugationally transferred to other bacterial cells. Thus, this substitution enhances the safety of the vaccine vector while retaining the regulated delayed antigen synthesis attribute. χ12615 also has the chromosomal ΔrelA1123 mutation which uncouples a dependance on protein synthesis for continued cell growth that is required to achieve complete lysis of the vaccine construct in vivo to release a bolus of protective antigens.
The detailed methods with description of suicide vectors used in strain modification to construct χ12615 and the nucleotide sequences of the genetic modifications are provided in co-pending U.S. Provisional Application No. 63/120,940 filed Dec. 3, 2020.
We have therefore constructed derivatives of χ12615 with the regulated delayed lysis plasmids pG8R111 and pG8R114 (Table 1) to serve as empty-vector control constructs and the plasmid derivatives encoding H. pylori protective antigens pG8R230 and pG8R262 (
In ongoing studies as disclosed in co-pending U.S. Provisional Application No. 63/120,940 filed Dec. 3, 2020, we are investigating other improvements to further enhance the safety and immunogenicity of χ12615.
S. Typhimurium has some 12 operons encoding fimbrial appendages. Some of these fimbriae contribute to intestinal colonization because of adherent components on the fimbriae. However, some of these fimbriae fail to be synthesized under any in vitro condition and are not synthesized in the GI tract either. However, the Sta and Saf fimbriae that are not synthesized under any laboratory experimental condition are synthesized and assembled in vivo in spleens (23). This in vivo up-regulation in synthesis and assembly was an important observation since the spleen is possibly the most important internal effector lymphoid tissue and is responsible for generating long-lasting protective immunity. Constitutive in vivo synthesis of either the Saf fimbriae or the Stc fimbriae in PIESV strains can be achieved by the addition of either the ΔPsaf5::PmurA safA or ΔPstc::PmurA stcA deletion-insertion mutation, in which synthesis and assembly is specified by the constitutive promoter of the mur operon, essential for synthesis of the rigid peptidoglycan layer of the cell wall. Such PIESV vaccine vector strains delivering the protective Streptococcus pneumoniae PspA protective antigen enhanced both the anti-PspA antibody responses and increased the levels of protective immunity to challenge of vaccinated animals with a wild-type virulent S. pneumoniae strain (23). Representative results demonstrating that constitutive expression of the operons encoding the Saf and Stc fimbriae in antigen delivery vaccine vector strains are better at conferring protective immunity to pathogen challenge are presented in Table 3.
Seven-week-old BALB/c mice were immunized orally with ˜1×108 CFU of the indicated S. Typhimurium vaccine strains and boosted with the same dose 6 weeks later. All mice were challenged by intraperitoneal inoculation 4 weeks after the booster dose with ˜1×104 CFU of virulent S. pneumoniae strain WU2. Deaths were recorded until 3 weeks post-infection.
It is also likely that the Saf and Stc fimbriae that represent MAMPs recruit innate immunity by interaction with some TLR, which has yet to be identified. Based on these observations, the addition of either or both the ΔPsaf5::PmurA safA and APstc::PmurA stcA deletion-insertion mutation to the PIESV vector strain χ12615 should enhance colonization of the spleen to enhance the level of induced protective immunity of delivered H. pylori protective antigens.
In previous work we have modified Salmonella strains to undergo rapid in vivo lysis to recruit rapid induction of innate immune responses to enhance induction of protective immunity by co-administered subunit, live attenuated and live vectored vaccines. The construction and properties of these SDAAS strains are described in U.S. Ser. No. 63/017,866 (Live self-destructing bacterial adjuvants to enhance induction of immunity). Enhancement in the levels of protective immunity induced by the PIESV constructs synthesizing and delivering H. pylori protective antigens can thus be achieved by co-administration of SDAAS strains in either the Family A or Family B lineages (that differ in their rates of lysis in vivo).
Thus, the Family A SDAAS strain χ12661 (Δalr-3 ΔdadB4 ΔasdA33 ΔfliC180 Δ(hin-fljBA)-219 ΔpagP8 ΔlpxR9 ΔpagL7 ΔeptA4 ΔarnT6 ΔsifA26 ΔrecA62) can be administered either mucosally (intranasal) or parenterally (subcutaneous, intramuscular, intravenous) at the same time as the PIESV strains delivering H. pylori antigens are orally administered. In this case, χ12615 (pG8R230) plus χ12615 (pG8R262) or improved derivatives would be used to maximize the induction of protective immunity against the consequences of H. pylori infections. χ12661 will efficiently recruit innate immunity via interaction with TLR4 (due to the ΔpagP8 ΔlpxR9 ΔpagL7 ΔeptA4 ΔarnT6 mutations) and TLR5 (due to the ΔfliC180 and Δ(hin-fljBA)-219 mutations) that are on the surface of many cell types in the vaccinated animal host. After invasion into host cells and escape from the SCV (due to the ΔsifA26 mutation), χ12661 will rapidly lyse (due to the Δalr-3 ΔdadB4 ΔasdA33 mutations) to liberate peptidoglycan-derived components to interact with internal Nod1 and Nod2 and DNA partially degraded due to the ΔrecA62 mutation to generate CpG motifs that will interact with TLR9 (also on the internal membrane surfaces of host cells). Thus, the administration of χ12661 will stimulate induction of innate immunity by multiple means.
Alternatively, if the Family A SDAAS strain lyses too rapidly to minimize the impact of stimulating innate immunity via Nod1, Nod2 and TLR9 that requires entry into host cells prior to lysis, we can use a Family B SDAAS strain as the co-administered adjuvant Family B SDAAS strains possess sugar-regulated expression of genes for peptidoglycan precursors such that lysis occurs after several cell divisions in vivo. This provides more time for invasion of the SDAAS strain into host cells prior to lysis. In such a case we could use the Family B strain χ12626 (ΔPasdA55::TT araC ParaBAD asd Δalr-3 ΔPdadB66::TT araC ParaBAD dadB ΔfliC180 ΔpagP81::PlpplpxE ΔpagL7 ΔlpxR9 Δ(hin-fljBA)-219 ΔarnT6 ΔeptA4 ΔsifA26 ΔwbaP45). Since the Family B SDAAS strain increases in number due to several cell divisions prior to lysis, there is an enhanced probability of toxicity due to released lipid A (even though modified by the ΔpagL7 ΔlpxR9 ΔarnT6 ΔeptA4 mutations). We therefore included the ΔpagP81::PlpplpxE mutation that eliminated the 1′ phosphate on lipid A responsible for most of the toxicity of lipid A [22]. Other mutations and associated phenotypes are similar in the Family A and B strains or they could be identical. For example, the ΔwbaP45 mutation in χ12626, which eliminates the LPS O-antigen to expose the LPS core, enhances recruitment via interaction with TLR4.
Helicobacter species are prevalently associated with chronic stomach ailments and disease in various animal species. H. suis infects pigs and causes disease and economic losses in the pork industry [24]. H. suis is also reasonably prevalent in humans and contributes to gastric stress and disease [24]. Since the antigen genes in H. pylori for the four identified protective antigens UreA, UreB, NapA and HapA are all present in S. suis and have sizes and amino acid sequences that are very similar (see Table 4), it is possible that the vaccine constructs χ12615 (pG8R230) and χ12615 (pG8R262) would induce some level of protective immunity to vaccinated pigs against infection and disease by H. suis. Although S. Typhimurium vectored vaccines work reasonably well in swine, it might be preferable to use S. Choleraesuis vaccine vector with the regulated delayed lysis phenotype since S. Choleraesuis is host-adapted to swine as a very invasive S. enterica serotype.
The teachings of the references provided herein are incorporated in their entirety to the extent not inconsistent with the teachings herein.
This disclosure is written to describe the invention to a person having ordinary skill in the art, who will understand that this disclosure is not limited to the specific examples or embodiments described. The examples and embodiments are single instances of the invention which will make a much larger scope apparent to the person having ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by the person having ordinary skill in the art. It is also to be understood that the terminology used herein is for the purpose of describing examples and embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
All the features disclosed in this specification (including any accompanying claims, abstract, and drawings) may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. The examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to the person having ordinary skill in the art and are to be included within the spirit and purview of this application. Many variations and modifications may be made to the embodiments of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure. For example, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.
This invention was made with government support under AI056289, AI1216172 and TR1000427 awarded by The National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/017083 | 2/8/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62971365 | Feb 2020 | US |
