Patent Grant
10806778
This application claims priority to European Application No. 19154550.8, filed Jan. 30, 2019, the disclosure of which is incorporated herein by reference in its entirety.
The present invention relates to the modification of an attenuated strain of Salmonella enterica serovar Typhi, such that its natural surface-exposed polysaccharide and flagellin antigens are converted to, or augmented by, those from other strains of Salmonella, including S. enterica serovars Paratyphi, Typhimurium and Enteritidis. Such a modification utilises the long history of safe use of strains of S. Typhi in humans as a typhoid vaccine, to deliver homologous antigens from other members of the genus Salmonella as components of vaccines for enteric fever and Salmonellosis.
Accompanying this filing is a Sequence Listing entitled “Sequence-Listing_ST25.txt”, created on Feb. 25, 2020 and having 72,899 bytes of data, machine formatted on IBM-PC, MS-Windows operating system. The sequence listing is hereby incorporated herein by reference in its entirety for all purposes.
Enteric fever is exclusive to humans and is caused by two serovars of Salmonella enterica: Typhi and Paratyphi, the latter comprising serovars A, B and C. Typhoid fever is estimated to have caused 21.7 million illnesses leading to 217,000 deaths in 2000, with 5.4 million cases of paratyphoid fever annually (Crump et al. 2004, Bull. World Health Organ. 82: 346-353). Typhoid and paratyphoid fevers are very similar infections of the reticuloendothelial system, intestinal lymphoid tissue and gallbladder, leading to acute febrile illnesses. Blood culture or serological tests are required to differentiate them. Outbreaks of typhoid fever are frequent in sub-Saharan Africa and Asia, with S. Paratyphi A responsible for up to 50% of enteric fever cases in Asia; enteric fever is also endemic in Latin America (Crump & Mintz 2010, Clin. Infectious Dis. 50: 241-246). S. Paratyphi A is the most abundant strain causing paratyphoid fever globally, with several reports showing it causing an increasing number of the total enteric fever cases (Fangtham & Wilde 2008, Int. J. Travel Med. 15: 344-350).
All licensed injected typhoid vaccines use the Vi capsular polysaccharide antigen purified from S. Typhi, and are single-dose with boosting recommended every 2-3 years (Martin 2012, Curr. Opin. Infect. Dis. 25: 489-499). The main adverse event is pain at the injection site. The only live attenuated typhoid vaccine is S. Typhi Ty21a (Vivotif®), developed by chemical mutagenesis of S. Typhi Ty2 and administered orally in 3-4 doses, with boosting required after 5-7 years (Martin 2012, Curr. Opin. Infect. Dis. 25: 489-499). Ty21a is very safe and well tolerated. In a comparative clinical study of injected vaccine Typherix® versus Vivotif®, only the latter was found to generate immune responses that mimic the natural infection (Kantele et al. 2013, Plos One 8: e60583).
The Vi antigen is not present in S. Paratyphi A or B (but is expressed by S. Paratyphi C), so injected Vi vaccines are ineffective against the two most prevalent S. Paratyphi strains. Ty21a has been proven to confer cross-protection against S. Paratyphi B in field studies (Levine et al. 2007, Clin. Infectious Dis. 45: S24-S28). However, field studies using Ty21a showed little or no cross-protection against S. Paratyphi A, despite the generation of cross-reactive antibody responses (Wahid et al. 2012, Clin. & Vaccine Immunol. 19: 825-834).
To try to address the short duration of protection and lack of memory response of Vi vaccines, Vi polysaccharide has been conjugated to carrier proteins in a new generation of Vi glycoconjugate vaccines. Carrier proteins include Pseudomonas aeruginosa exotoxin, tetanus and diphtheria toxoids (Martin 2012, Curr. Opin. Infect. Dis. 25: 489-499). Injectable conjugates of O-antigens purified from S. Paratyphi A have also been developed, primarily O2 conjugated to tetanus toxoid (O2-TT), to diphtheria toxoid (O2-DT) and to a detoxified mutant of the diphtheria toxin (O2-CRM197), co-administered with Vi conjugated to the same carrier protein as enteric fever vaccines targeting S. Typhi and S. Paratyphi A (Martin et al. 2016, Vaccine 34: 2900-2902).
The live attenuated approach to enteric fever vaccine development has significant advantages over injectable Vi vaccines: longer duration of protection, generation of immunological memory, closer immunological profile to the natural infection and elimination of needles. In addition to the licensed chemically mutagenised typhoid vaccine strain Ty21a, other specifically mutated live vaccine strains of S. Typhi have been evaluated in clinical trials: CVD 906 and CVD 908 (AaroC, AaroD); CVD 906-htrA and CVD 908-htrA (ΔaroC, ΔaroD, ΔhtrA); CVD 909 (ΔaroC, ΔaroD, ΔhtrA and constitutive expression of Vi); M01ZH09 (ΔaroC, ΔssaV); Ty800 (ΔphoP, ΔphoQ); χ3927 (Δcya, Δcrp) (Tennant & Levine 2015, Vaccine 33: C36-C41) and χ4073 (Δcya, Δcrp, Δcdt) (Paterson & Maskell 2010, Hum. Vaccines 6: 379-384). It is reasonable to expect a degree of cross-protection from these specifically mutated S. Typhi strains to S. Paratyphi B as is the case for Ty21a.
Attenuated strains of S. Paratyphi A have also been produced, including ΔphoPQ mutants tested pre-clinically (Roland et al. 2010, Vaccine 28: 3679-3687), and CVD 1902 (ΔguaBA, ΔcIpX) which has been evaluated in a clinical trial (Tennant & Levine 2015, Vaccine 33: C36-C41). A combination of CVD 909 and CVD 1902 is in clinical development as a vaccine targeting S. Typhi and S. Paratyphi A (Martin et al. 2016, Vaccine 34: 2900-2902). However, this strategy requires the clinical evaluation of S. Paratyphi A, which does not have the long history of safe use of S. Typhi.
Non-typhoidal Salmonella (NTS) cause gastroenteritis, with symptoms including diarrhoea and fever. The increase in cases of an invasive form of non-typhoidal Salmonella (iNTS), predominantly in Africa, is an important public health issue. The strains responsible for the vast majority of iNTS cases are S. enterica serovars Typhimurium and Enteritidis, and multidrug resistant isolates are of particular concern (MacLennan & Levine 2013, Expert Rev. Anti Infect. Ther. 11:443-446). iNTS strains cause a significantly more severe form of the disease, with prolonged symptoms and shedding of bacteria lasting for several weeks. There are currently no vaccines for NTS approved for human use.
The benefits of live attenuated vaccines include the induction of mucosal and cell-mediated immune responses, in addition to systemic antibody responses, and the duration of these responses can be longer than those from injected subunit vaccines as descried above for typhoid. Attenuated S. Typhi strains have been administered to millions of people as experimental and licensed vaccines with an excellent record of safety and immunogenicity. This serovar also lacks the ability to persist in environmental reservoirs due to its exclusivity to humans, thus increasing its biosafety. Therefore, there are several reasons why it is advantageous to use live attenuated S. Typhi as a vector for delivery of homologous antigens from other serovars of S. enterica, rather than attenuating the wild-type strains where the effect of the attenuating mutations may not be predictable. For example, the S. Typhi vaccine candidate ZH9 carrying mutations in the genes aroC and ssaV has been shown to be safe and well tolerated in multiple clinical trials (Lyon et al. 2010, Vaccine 28: 3602-3608), whereas the same mutations introduced into S. Typhimurium resulted in prolonged shedding in stools (Hindle et al. 2002, Infect. Immun. 70: 3457-3467).
The three most important surface antigens of the S. enterica serovars for the induction of protective immunity are lipopolysaccharide O-antigens, flagella (H-antigens) and Vi. The table below summarises the antigenic compositions of the principle enteric fever and iNTS strains following the Kauffmann-White-Le Minor scheme classification scheme (Grimont & Weill 2007, Antigenic formulae of the Salmonella serovars, 9th Edition).
Typhi
Paratyphi A
Paratyphi B
Paratyphi C
Typhimurium
Enteritidis
[ ] indicates antigens exceptionally found in wild-type strains.
Salmonella lipopolysaccharides consists of lipid A linked to the KDO (3-deoxy-D-manno-octulosonic acid) terminus of a conserved core region, which is then linked to a variable, repeated O-antigen trisaccharide. In S. Typhi, S. Paratyphi A, S. Paratyphi B, S. Typhimurium and S. Enteritidis this repeated O-antigen is O12, a triglyceride of mannose (Man), rhamnose (Rha) and galactose (Gal). In S. Paratyphi A, a branch of paratose (Par; 3,6-dideoxy-D-ribo-hexose) from the C-3 of Man confers serogroup specificity: O2 (
Except for the flagella produced by S. Typhi Ty21a, flagellin is not a component of any current licensed vaccine for an S. enterica infection. Flagellin is an important pathogen-associated molecular pattern (PAMP) that is recognised by toll-like receptor 5 (TLR5) and is highly immunogenic, making it an important component of a live vaccine for S. enterica. The flagella filament of S. enterica is composed of approximately 20,000 flagellin (FliC or FljB) proteins with a terminal cap encoded by fliD (Haiko & Westerlund-VVikstrOm 2013, Biology 2: 1242-1267). S. Typhi and S. Paratyphi A are generally monotypic for flagellin, expressing only FliC.
There is a particular need in the art for improved vaccines directed toward S. enterica serovars Paratyphi A, B, C, Typhimurium and Enteritidis.
In a first aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is modified to express the lipopolysaccharide O2 O-antigens and the flagella proteins of Salmonella enterica serovar Paratyphi A.
In a second aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is modified to express the lipopolysaccharide O4 O-antigens and the flagella proteins of Salmonella enterica serovar Paratyphi B and Salmonella enterica serovar Typhimurium.
In a third aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is further modified to contain a functional fepE gene, such that long O-antigen chains are generated, preferably wherein the O-antigen chains are 100 repeated units of the trisaccharide backbone in length.
In a fourth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is modified to either constitutively express the gtrC gene (encoding rhamnose acetyltransferase), or alternatively, wherein said strain is modified to express the gtrC gene in trans.
In a fifth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain has its native fliC gene (SEQ ID NO: 1) substituted with the fliC gene of Salmonella enterica serovar Paratyphi A (SEQ ID NO: 2), Salmonella enterica serovar Paratyphi B (SEQ ID NO: 3), Salmonella enterica serovar Paratyphi C (SEQ ID NO: 5), Salmonella enterica serovar Typhimurium (SEQ ID NO: 7) and Salmonella enterica serovar Enteritidis (SEQ ID NO: 9), such that the conferred serotype is altered from an Hd serotype to a Ha, Hb, Hc, Hi and Hg,m serotype respectively.
In a sixth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein the fljBA locus, controlling expression of the fljB gene of Salmonella enterica serovar Paratyphi B (SEQ ID NO: 4), Salmonella enterica serovar Paratyphi C (SEQ ID NO: 6) and Salmonella enterica serovar Typhimurium (SEQ ID NO: 8) are inserted into the chromosome of Salmonella enterica serovar Typhi or expressed in trans.
In a seventh aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain expresses the atypical variants of flagellin of Salmonella enterica serovar Paratyphi A, Salmonella enterica serovar Paratyphi B, Salmonella enterica serovar Paratyphi C, Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis.
In an eighth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain has inserted a second copy of the tviA gene (SEQ ID NO: 10).
The present invention further includes a vaccine comprising one or more said modified strains for use in enhancing immunogenicity against Salmonella enterica serovar Paratyphi A, Paratyphi B, Paratyphi C, Typhimurium and Enteritidis.
The following description is presented to enable any person skilled in the art to make and use the present invention. Various modifications to the disclosed embodiments will be readily apparent to those skilled in the art.
In a first aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain may be modified to express the lipopolysaccharide O2 O-antigens and the flagella proteins of Salmonella enterica serovar Paratyphi A.
The term ‘live attenuated strain’ in the context of the present invention refers to the alteration of said strain to reduce its pathogenicity, rendering it harmless to the host, whilst maintaining its viability. This method is commonly used in the development of vaccines due to its ability to elicit a highly specific immune response whilst maintaining an acceptable safety profile. Development of attenuated live bacterial vaccines may involve a number of methods, examples include, but are not limited to; passing the pathogens under in vitro conditions until virulence is lost, chemical mutagenesis and genetic engineering techniques.
It is envisaged that the lipopolysaccharide O9 O-antigens of Salmonella enterica serovar Typhi may be replaced with the O2 O-antigens of Salmonella enterica serovar Paratyphi A.
The S. Paratyphi A O-antigen biosynthetic pathway involves the precursor CDP-4-keto-3,6-dideoxy-D-glucose being converted to CDP-Par by the CDP-paratose synthase, RfbS. In addition to rfbS (previously called prt), S. Typhi has a functional fbE gene (previously called tyv) encoding CDP-paratose 2-epimerase, which converts CDP-Par to CDP-Tyv (
The present invention involves the inactivation of the chromosomal rfbE in Salmonella enterica serovar Typhi (
The rfbE inactivation prevents Tyv from being synthesised, resulting in Par being attached to Man. This alteration in the biochemical pathway introduces the Salmonella enterica serovar Paratyphi A O2 O-antigen (
In a preferred embodiment, the modification to inactivate the rfbE gene retains non-coding DNA without disrupting the expression of downstream (non-rfbE) coding sequences.
In a preferred embodiment, the deletion of the fbE cistron is accompanied by the insertion of a non-coding spacer region intended to maintain the correct reading frame.
The spacer region may be any suitable non-coding DNA sequence which retains the correct reading frame when inserted. Preferably, the spacer region of DNA is the cistron of the Escherichia coli gene wbdR which results in the production of long LPS.
It is a surprising finding that the modifications disclosed herein result in a long LPS with the S. Paratyphi A O2 O-antigen characteristic. This has benefits in vaccine production, allowing live attenuated strains of S. Typhi, and/or derivatives thereof, to be produced, offering additional protection against S. Paratyphi A. The vaccine therefore has benefits over conventional vaccines which protect only against S. Typhi.
The term ‘spacer region of DNA’ in the context of the present invention refers to a region of non-coding DNA located between genes. The term ‘cistron’ refers to a section of DNA which encodes for a specific polypeptide in protein synthesis. The insertion of a spacer region of DNA may involve the transformation of an electrocompetent plasmid with a replacement cassette. See Example 1 for further details.
Where the methods herein described involve the use of a plasmid, said plasmid will ideally have an origin of replication selected from pMB1, ColEl, p15A, pSC101 and RK2. The plasmid may contain an antibiotic resistance gene selected from β-lactamase (bla), kanamycin phosphotransferase (kan), tetracycline efflux protein (tetA) or chloramphenicol acetyltransferase (cat). Ideally the antibiotic resistance gene will be excised prior to or shortly after transformation into the live bacterial vector strain, for example by a mechanism such as ‘X-mark’ (Cranenburgh & Leckenby 2012, WO2012/001352). A plasmid maintenance system may be required to prevent plasmid loss. These may include mechanisms to place a native chromosomal gene under a heterologous promoter such as the ‘Operator-Repressor Titration for Vaccines’ (ORT-VAC; Garmory et al. 2005, Infect. Immun. 73: 2005-2011) or ‘oriSELECT’ (Cranenburgh 2005, WO 2005/052167) systems, neither of which require an additional selectable marker gene to be present on the plasmid. Alternatively, a selectable marker gene will be used that is not an antibiotic resistance gene, such as a gene to complement a host cell mutation (Degryse 1991, Mol. Gen. Genet. 227: 49-51).
Preferably, the spacer region of DNA is the cistron of the Escherichia coli gene wbdR. Other non-functional genes of Salmonella enterica serovar Typhi of approximately the same length as the rfbE cistron may also be used for this purpose. It is preferable that the chosen spacer DNA used for this purpose will be approximately 50-2000 base pairs in length as well as lacking a terminator sequence. The use of this spacer region results in the inactivation of rfbE without causing any downstream effects (SEQ ID NO: 20) and effectively changing Salmonella enterica serovar Typhi LPS to Salmonella enterica serovar Paratyphi A.
The inventors have shown that deletion of rfbE whilst maintaining the original reading frame (via the use of a spacer region of DNA) is a crucial requirement of the above process.
Preferably, the resulting lipopolysaccharide O2 O-antigens of Salmonella enterica serovar Paratyphi A are at least equivalent in length to the lipopolysaccharide O9 O-antigens of Salmonella enterica serovar Typhi. It is preferable that the resulting lipopolysaccharide will be 16-35 O-antigen repeat units in length, a range which constitutes a ‘long’ lipopolysaccharide species. A person skilled in the art will understand the desirability of the presence of O-antigen repeat units in triggering an immunogenic reaction.
It is envisaged that the present invention may also include the live attenuated strain, according to above, wherein said strain may have its native fliC gene replaced with the fliC gene of Salmonella enterica serovar Paratyphi A, such that the conferred serotype is altered from an Hd serotype to a Ha serotype, where ‘serotype’ refers to a distinct variation within the bacterial species.
The Phase 1 flagellum of S. Typhi is essential for motility and invasion, and confers the serotype Hd. The filament consists of the flagellum protein FIiC, with a FliD cap. The inventors have discovered that replacing the fliC on the S. Typhi chromosome with that of S. Paratyphi A results in the conversion from the Hd to the Ha serotype of functional flagella.
Chromosomal replacement may be used to achieve the above substitution. The substitution may be a full or partial replacement. In the context of a partial replacement, it is preferable that the replacement of the amino acids in positions 176-414 is carried out. The latter may involve the transformation of an electrocompetent plasmid with a replacement cassette. See Example 2 for further details. Alternatively, the substituted fliC gene may be expressed in trans from a plasmid or additional chromosomal location.
An additional embodiment of the present invention is the live attenuated strain described above wherein the strain may be further modified to contain a functional fepE gene, such that long O-antigen chains are generated, preferably wherein the O-antigen chains are 100 repeated units of the trisaccharide backbone in length.
The fepE gene encodes the length regulator of very long O-antigen chains, wherein ‘very long’ is taken to mean more than 100 repeated units of the trisaccharide backbone. S. Typhi does not possess these long O-antigen chains due to a mutation introducing a stop codon into the gene (SEQ ID NO: 21). S. Typhi may be manipulated into expressing these long O-antigen chains via a number of methods; the natural promoter of fepE may be replaced with an alternative promoter, for example P araBAD, the chromosomal mutation of fepE in S. Typhi may be repaired or a functional copy of fepE (SEQ ID NO: 11) may be inserted elsewhere in the S. Typhi chromosome. For vaccine applications, an in vivo-induced promoter or a constitutive promoter may be utilised, examples of such promoters include PpagC, PnirB, PssaG, PsifA, PsifB, PsseA, PsseG Pssej, Plac, Ptac, Ptrc and lambda PL/PR.
A ‘promoter’ refers to a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3′ direction) coding or non-coding sequence. A promoter may also be a regulatory DNA sequence that affects the binding of RNA polymerase at the transcription initiation site. For the purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence may be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase.
Promoters can be constitutively active (wherein ‘active’ means transcription is ‘on’), spatially restricted or inducible. As used herein ‘spatially restricted’ refers to a promoter that is only active in a specific subset of cells or cellular compartment of a multicellular organism. A spatially restricted promoter can thus be used to activate the expression of a nucleic acid in a particular tissue or cell type of a multicellular organism.
As used herein an ‘inducible promoter’ refers to a promoter that enables the temporal and/or spatial activation of transcription in response to external physical or environmental stimuli. Inducible promoters include those activated by the presence of specific small molecules that alleviate transcriptional repression. For example, transcription from such an inducible promoter may be regulated by a ‘repressor protein’. As used herein, ‘repressor protein’ refers to a polypeptide that binds to and occupies the inducible promoter to prevent transcription initiation. When bound to the promoter, said repressor protein can prevent binding or recruitment of RNA polymerase or associated co-factors to the transcription initiation site to prevent the activation of transcription. However, upon binding its relevant small molecule, or encountering its relevant physical or environmental stimulus, the repressor protein can no longer bind to the promoter sequence, and thus transcriptional repression is relieved. Where the above examples include in vivo-induced promoters for expression of cistrons encoding enzymes involved in O-antigen biosynthesis, or for expression of alternative fliC cistrons or TviA, such promoters include but are not limited to: PpagC, PnirB PssaG, PsifA PsifB, PsseA, PsseG and PsseJ (Dunstan et al. 1999, Infect. Immun. 67: 5133-5141; Xu et al. 2010, Infect. Immun. 78: 4828-4838; Kroger et al. 2013, Cell Host & Microbe 14: 683-695). Other promoters of use include lambda a and PR the temperature-induced lambda repressor cl including its thermo-labile mutant repressor cl857 (Love et al. 1996, Gene 176:49-53; SEQ ID NO: 24 & 25) and promoters that are constitutive in Salmonella in the absence of the LacI repressor such as Plac, Ptac and Ptrc (Terpe 2006, Appl. Microbiol. Biotechnol. 72: 211-222). In some embodiments, the functional variants include those having similar or modified sequences to PpagC, PnirB, PssaG, PsifA, PsifB, PsseA, PsseG Pssej and lambda PL/PR, and similar or substantially identical promoter activity as the wild-type promoter from which the variant is derived, particularly with respect to its ability to induce expression in vivo. Similar modified sequences may include having at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the wild-type sequence of any of PpagC, PnirB, PssaG, PsifA, PsifB, PsseA, PsseG, PsseJ and lambda PL/PR.
Preferably, the introduction of these long O-antigen chains may be beneficial in inducing an LPS-specific immune response. There may be an additional benefit where the LPS is naturally very long such as from expression of fepE.
It is further envisaged that the live attenuated strain described above may be modified to constitutively express gtrC or to express gtrC in trans.
Particular S. enterica serovars are acetylated on the rhamnose on the O-antigen, a feature which has been demonstrated as important for O2 O-antigen specificity in S. Paratyphi A. The family 2 gtr operon (SEQ ID NO: 22) encodes the rhamnose acetyltransferase GtrC in S. Typhi and S. Paratyphi A. To achieve a greater and more consistent level of rhamnose acetylation it may be desirable to make gtrC constitutively expressed, for example, either on a plasmid or from an additional chromosomal locus. Alternatively, the native family 2 gtr operon promoter responsible for phase variation can be replaced with a constitutive promoter or one that is conditionally expressed in vivo.
It is further envisaged that the live attenuated strain described above may be further modified to contain an additional copy of the tviA gene under the control of a phagosomally induced promoter.
The Vi capsular polysaccharide antigen contributes to the virulence of S. Typhi but is naturally down-regulated upon invasion of the liver and spleen (Janis et al. 2011, Infect. Immun. 79: 2481-2488). Regulation of Vi expression is carried out by the positive transcriptional regulator TviA.
The insertion of a second copy of the tviA gene into S. Typhi may induce immune responses against Vi and enhance the anti-flagellin response. The second copy may be inserted into the S. Typhi in trans, either on a plasmid (
A further embodiment of the present invention may be a vaccine comprising the live attenuated strains herein disclosed, for use in enhancing immunogenicity against S. Paratyphi A and for use in the treatment or prevention of enteric fever and salmonellosis. The vaccine may contain a single live attenuated strain or combine more than one live attenuated strain, for example, the vaccine may contain ZH9 and/or one of its derivative strains; ZH9PA, ZH9PL2, ZH9W or ZH9PF. For example, combinations may include ZH9+ZH9PL2, ZH9+ZH9W, ZH9+ZH9PF, preferably the combination is ZH9+ZH9PA.
The term ‘immunogenicity’ refers to the ability of a particular substance to provoke an immune response.
The term ‘vaccine’ may be taken to comprise a number of additional elements in addition to the attenuated live strain herein disclosed. The attenuated live strain may be present in a composition together with any other suitable adjuvant, diluent or excipient. Examples of suitable adjuvants, diluents or excipients include, but are not limited to; disodium hydrogen phosphate, soya peptone, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, magnesium sulphate, calcium chloride, sucrose, sterile saline and sterile water.
The vaccine may be administered by any appropriate route, preferably orally or intranasal routes; however the former is the preferred route of administration. The vaccine strain or strains will preferentially be lyophilised by a process such as freeze-drying and will be stored in sachets for later rehydration and oral administration to young children. Alternatively, they may be dispensed into enterically coated capsules for oral administration to older children and adults. For the encapsulated formulation, the lyophilised Salmonella will ideally be mixed with a bile-adsorbing resin such as cholestyramine to enhance survival when released from the capsule into the small intestine (Edwards and Slater 2009, Vaccine 27: 3897-3903).
The skilled person will appreciated that the vaccine may contain the aforementioned live attenuated strains (e.g. ZH9 and ZH9PA) of Salmonella entertica serovar Typhi at a density of 108, 109, 1010, 1011 or 1012 colony-forming units per dose. The dosing regime may involve a single dose or multiple doses, ideally the vaccine will be administered in 1-3 doses.
In a second aspect, the present invention provides for a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is modified to express the lipopolysaccharide O4 O-antigens and the flagella proteins of Salmonella enterica serovar Paratyphi B and Salmonella enterica serovar Typhimurium.
To achieve the above modification, the gene rfbJ (previously called abe), encoding CDP-abequose synthase from S. Paratyphi B or S. Typhimurium, can be inserted to replace rfbS with or without the simultaneous replacement of rfbE (
Alternatively to the replacement method described above, and rfbJ may be expressed in trans, either from a plasmid or alternative chromosomal locus, leading to a mixture of 04 and O9 O-antigens, designed to induce antibody responses to S. Typhi, S. Paratyphi B and S. Typhimurium.
The invention further intends the live attenuated strain, according to the second aspect of the present invention, may have its native fliC gene replaced with the fliC gene of Salmonella enterica serovar Paratyphi B and/or Salmonella enterica serovar Typhimurium, such that the conferred serotype is altered from an Hd serotype to a Hb and Hi serotype respectively.
It is further envisaged that the live attenuated strain, according to the second aspect of the present invention, may have the fljBA locus of Salmonella enterica serovar Paratyphi B and Salmonella enterica serovar Typhimurium inserted into the chromosome of Salmonella enterica serovar Typhi or expressed in trans.
Several serovars of S. enterica (including S. Paratyphi B and C, and S. Typhimurium) have an additional antigenically distinct flagellin gene fljB, which is subject to phase variation such that flagella composed of either FliC or FljB is produced (
It is envisaged that the fljBA locus of S. Paratyphi B, S. Paratyphi C or S. Typhimurium may be inserted into the chromosome of S. Typhi or expressed in trans from a plasmid, thus introducing the phase-variable flagella phenotype of the desired serovar. Alternatively, one or both of the hix sites flanking the native promoter of the fljBA operon, or the hin recombinase gene, may be mutated to prevent DNA inversion, leading to constitutive expression such that flagella filaments are comprised of only FIjB. The latter approach may be coupled with the pre-described modification of the S. Typhi fliC.
It is further envisaged that the live attenuated strain, according to the second aspect of the present invention, may be further modified to include the additional modifications previously described regarding fepE, gtrC and tviA expression.
A further intended application of the present invention is a vaccine comprising the live attenuated strain, according to the second aspect of the present invention, for use in enhancing immunogenicity against Salmonella enterica serovar Paratyphi B and/or Salmonella enterica serovar Typhimurium.
In a third aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is further modified to contain a functional fepE gene, such that long O-antigen chains are generated, preferably wherein the O-antigen chains are 100 repeated units of the trisaccharide backbone in length. The method by which this effect may be achieved, and further details regarding this aspect of the invention, have been previously outlined on pages 12 and 13 of the present application.
In a fourth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is modified to either constitutively express the gtrC gene, or alternatively, wherein said strain is modified to express the gtrC gene in trans. The method by which this effect may be achieved, and further details regarding this aspect of the invention, have been previously outlined on page 14 and 15 of the present application.
In a fifth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain has its native fliC gene substituted with the fliC gene of Salmonella enterica serovar Paratyphi A, Salmonella enterica serovar Paratyphi B, Salmonella enterica serovar Paratyphi C, Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis, such that the conferred serotype is altered from an Hd serotype to a Ha, Hb, Hc, Hi and Hg,m serotype respectively. The method by which this effect may be achieved, and further details regarding this aspect of the invention, have been previously outlined on page 12 and 17 of the present application.
In a sixth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein the fljBA locus of Salmonella enterica serovar Paratyphi B, Salmonella enterica serovar Paratyphi C and Salmonella enterica serovar Typhimurium are inserted into the chromosome of Salmonella enterica serovar Typhi or expressed in trans. The method by which this effect may be achieved, and further details regarding this aspect of the invention, have been previously outlined on pages 17 and 18 of the present application.
In a seventh aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain expresses the atypical variants of flagellin of Salmonella enterica serovar Paratyphi A, Salmonella enterica serovar Paratyphi B, Salmonella enterica serovar Paratyphi C, Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis.
S. Typhi isolates are subject to phase variation: one such isolate expresses a variant of FljB called Hz66 from a linear plasmid called pBSSB1 (Baker et al. 2007, Plos Pathogens 3: e59), another flagella variant is Hj which has a 261 bp in-frame deletion of the central region of the Hd fliC gene (Frankel et al. 1989, EMBO J. 8: 3149-3152).
For Hz66 this can be achieved by the inclusion of the pBSSB1 plasmid in the S. Typhi-derived vaccine strain. Alternatively, FljBz66 or Hj may be expressed from a chromosomal location which may be the location of the deleted fljB gene, the native chromosomal location of fliC (thereby replacing it with a variant), or expressed on a plasmid from its native promoter or from a phagosomally induced promoter such as PssaG The amino acid sequences of the Hz66 and Hj are described in Schreiber et al. 2015, Nature 5: 7947.
In an eighth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain has inserted a second copy of the tviA gene. The method by which this effect may be achieved, and further details regarding this aspect of the invention, have been previously outlined on page 15 of the present application.
It is envisaged that the live attenuated strains herein disclosed may be administered in isolation or in combination (e.g. ZH9 and ZH9PA), in the form of a vaccine, to give the subject a broad protection against a variety of S. enterica serovars, specifically, Salmonella enterica serovar Paratyphi A and B, and Salmonella enterica serovar Typhimurium.
The table lists S. Typhi ZH9 and its derivative strains altered for LPS and flagellin:
The invention will now be illustrated in the following examples with reference to the accompanying drawings.
To construct S. Typhi ZH9 expressing S. Paratyphi A LPS, the rfbE gene was deleted in two different ways. In one method of deletion, a spacer cistron wbdR was synthesised flanked with 700 bp of DNA homologous to rfbE upstream gene rfbS and downstream gene rfbX to create a deletion cassette. A NotI restriction site at the 3′ end of the spacer cistron was included to clone a dif-flanked antibiotic resistance marker gene cat gene amplified with primers designed with NotI restriction site. The cat gene was amplified from pBRT1N plasmid synthetically generated with 5NotIdifcat and 3NotIdifcat primers designed with dif sequences. E. coli TOP10 cells were used for cloning operations to generate the pUCpW_difCAT plasmid. Chromosomal replacement of the rfbE gene with the spacer gene was carried out as described in Bloor and Cranenburgh 2006 (Appl. Environ. Microbiol. 72: 2520-2525). Briefly, S. Typhi ZH9 was first transformed with a pLGBEK plasmid coding for λ Red gene functions for integration of linear DNA. Electrocompetent ZH9(pLGBEK) was then transformed with the deletion cassette linearised using SalI and SacI restriction enzymes (SEQ ID NO: 26), and transformants were selected on LB-aro (LB medium containing aromatic amino acids and precursors of aromatic amino acid biosynthesis) agar plates supplemented with 20 μg/ml chloramphenicol. Single colonies were isolated and cultured overnight in LB-aro broth in the absence of antibiotics. Xer recombination deleted the cat gene to generate chloramphenicol-sensitive colonies of ZH9W.
In the other method of deletion, the dif-flanked antibiotic resistance gene cat was amplified with the primers rfbEdelF and rfbEdelR designed with homologous sequences to rfbS and rfbX genes respectively. The difCAT cassette was amplified from the synthetic pMKtetORTDAP plasmid to generate the PL deletion cassette (SEQ ID NO: 27). Electrocompetent ZH9(pLGBEK) were then transformed with the amplified DNA sequence and transformants were selected on LB-aro agar plates supplemented with 20 μg/ml chloramphenicol. Single colonies were isolated and cultured overnight in LB-aro broth in the absence of antibiotics. Xer recombination deleted the cat gene to generate chloramphenicol-sensitive colonies of ZH9PL2.
To construct S. Typhi ZH9 expressing S. Paratyphi A flagellin, the native fliC gene was replaced with the S. Paratyphi A fliC. The replacement cassette was synthesised with S. Paratyphi A fliC flanked with 700 bp of DNA homologous to the gene fliD upstream of fliC, and a pseudogene downstream of fliC. A NotI restriction site at the 3′ end of S. Paratyphi A fliC was included to clone a dif-flanked antibiotic resistance marker gene cat gene amplified with primers designed with NotI restriction site. The cat gene was amplified from pBRT1N plasmid synthetically generated with 5NotIdifcat and 3NotIdifcat primers designed with dif sequences. TOP10 E. coli cells were used for cloning operations to generate pUCpF_difCAT plasmid. Chromosomal replacement of S. Typhi FliC gene with S. Paratyphi A FliC was carried out as described in Example 1: electrocompetent ZH9(pLGBEK) was transformed with the pUCpF_difCAT replacement cassette excised using SalI and SacI restriction enzymes (SEQ ID NO: 28), and transformants were selected on LB-aro agar plates supplemented with 20 μg/ml chloramphenicol. Single colonies were isolated and cultured overnight in LB-aro mix broth in the absence of antibiotic. Xer recombination resulted in the deletion of the cat gene to generate chloramphenicol-sensitive colonies of ZH9PF.
To construct S. Typhi ZH9 expressing both S. Paratyphi A LPS and Flagellin, electrocompetent ZH9PF (pLGBEK) was then transformed with the replacement cassette generated from the pUCpW_difCAT cassette excised using SalI and SacI restriction enzymes, and transformants were selected on LB-aro agar plates supplemented with 20 μg/ml chloramphenicol. Single colonies were isolated and cultured overnight in LB-aro broth in the absence of antibiotic. Xer recombination resulted in the deletion of the cat gene to generate chloramphenicol-sensitive colonies of ZH9PA.
To construct the medium copy-number expression plasmid pBRT4tviA (
To construct the medium copy number expression plasmid pBAD2fepE (SEQ ID NO: 30), primers fepE5_pBAD and fepE3_pBAD_pSC were used to amplify the fepE gene of S. Typhimurium WT05 (aroC, ssaV) from chromosomal DNA. The PCR product and expression plasmid pBAD2 were digested using NdeI and SalI and ligated to generate the plasmid pBAD2fepE. pBAD2fepE has fepE under the control of arabinose promoter, which is not active in nutrient broths such as LB or TB and require the addition of arabinose at 0.02% to induce the expression of the length regulator of very long (VL) O antigen chains. E. coli NEB5-alpha was used for cloning operations. The pBAD2fepE plasmid was transformed into S. Typhi ZH9, and transformants were selected on LB-aro agar plates supplemented with 50 μg/ml kanamycin. Single colonies of ZH9 (pBAD2fepE) were isolated and cultured overnight in LB-aro broth supplemented with 50 μg/ml kanamycin and 1:1000 of 20% arabinose to induce expression of LPS with very long (VL) O-antigen chains.
For immunofluorescence microscopy, S. Typhi ZH9 and its derivative strains ZH9W, ZH9PL, ZH9PF and ZH9PA were cultured for 18 hours in LB-aro broth at 37° C. and 200 r.p.m. A volume of each culture equivalent to an optical density of A600=1 was collected and washed in PBS. Pellets were resuspended in 10 μl of PBS with 1 μl of primary antibody and incubated for 10 minutes at ambient temperature. LPS analysis was carried out by staining ZH9, ZH9W, ZH9PL and ZH9PA with one of the following primary antibodies: anti-S. Typhi LPS monoclonal antibody B348M (Genetex), anti-S. Paratyphi A LPS monoclonal antibody (Bio-rad), 0:9 antiserum (SSI) and 0:2 antiserum (SSI). Flagellin analysis was carried out by staining ZH9, ZH9PF and ZH9PA with the following primary antibodies: H:d antiserum (SSI) and H:a antiserum (SSI). Bacterial cells primary stained were then washed in PBS and pellets were resuspended in 10 μl of PBS with 1 μl of secondary antibody conjugated to Dylight 488 fluorochrome. After 10 minutes incubation at room temperature, cells were washed in PBS and a small volume were applied on a microscope slide to be visualised using a fluorescent microscope (Zeiss Axiophot) with attached Zeiss Axiocam camera. Fluorescence imaging demonstrated the conversion of the 09 to the 02 serotype of LPS in ZH9W and ZH9PL (
To analyse LPS O-antigen length, ZH9 and ZH9(pBAD2fepE) pre-cultures were used to inoculate LB-aro broth and grown at 37° C. and 200 r.p.m., with ZH9(pBAD2fepE) supplemented with 50 μg/ml kanamycin and induced by adding 0.02% arabinose. When exponential phase was reached, 4 mL of each culture was lysed to prepare LPS using an LPS extraction kit (Intron Biotechnology). LPS samples were run on an SDS-PAGE gel and silver-stained.
To assess the immunogenicity of the ZH9PA strain and to confirm retained immunogenicity of both ZH9 and ZH9PA strains when co-administered, an immunogenicity study was conducted in mice. Balb/c animals were immunized via a single subcutaneous immunization with ZH9 alone (1×108 cfu/mouse), ZH9PA alone (1×108cfu/mouse) or combination of the two ZH9+ZH9PA (0.5×108 cfu ZH9+0.5×108 cfu ZH9PA/mouse). Pre-immune serum samples were collected prior to immunization and terminal serum samples were collected 35 days after immunization. All samples were centrifuged for serum isolation and serum stored at −80° C.
The sera were used to run in house standardized ELISA assays aimed at assessing the titers specific against Salmonella Typhi LPS (O:9) and flagellin (H:d), S. Paratyphi A LPS (O:2) and flagellin (H:a). Briefly, half-area ELISA plates (Corning) were coated with the following specific antigens (all provided by The Native Antigen Company), diluted in 50 mM carbonate/bicarbonate buffer (pH 9.6):
Plates were incubated at 4° C. overnight (˜16 h). The day after, plates were washed with PBS+0.05% Tween-20 before blocking with Pierce™ Protein-Free (PBS) blocking buffer for 1 hour at 37° C. After washing plates as before, mouse sera from the oral immunogenicity study were added for 1 additional hour at 37° C. A standard curve was also generated for each antigen by using serovar-specific reagents as follows:
Reacting sera and standard curve for S. Typhi and Paratyphi A LPS were detected using a secondary goat anti-mouse antibody conjugated directly to horse radish peroxidase (HRP), whilst standard curve for S. Typhi and Paratyphi A flagellin with HRP-conjugated goat anti-rabbit antibody. After washing plates as above, secondary antibodies were added at 1:2000 dilution in Pierce™ Protein-Free (PBS) blocking buffer and incubated at room temperature for 1 hour. Positive sera were revealed using 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate and measured using a spectrophotometer at 450 nM. Absorbance (OD) was plotted using a 4 parametric logistic curve of the positive control; then end point titers were determined as logarithm of the dilution value at which the serial dilution curve of each mouse serum met OD equal 1.
Sequences Used Throughout the Specification and Forming Part of the Description:
SEQ ID NO: 1 (S. typhi fliC Cistron)
SEQ ID NO: 2 (S. paratyphi A fliC Cistron)
SEQ ID NO: 3 (S. paratyphi B fliC Cistron)
SEQ ID NO: 4 (S. paratyphi B fljB Cistron)
SEQ ID NO: 5 (S. paratyphi C fliC Cistron)
SEQ ID NO: 6 (S. paratyphi C fliC Cistron)
SEQ ID NO: 7 (S. typhimurium fliC Cistron)
SEQ ID NO: 8 (S. typhimurium fljB Cistron)
SEQ ID NO: 9 (S. Enteritidis fliC Cistron)
SEQ ID NO: 10 (S. Typhi tviA Cistron)
SEQ ID NO: 11 (S. Typhimurium fepE)
SEQ ID NO: 12 (S. Typhi rfbE Cistron)
SEQ ID NO: 13 (E. coli O157:H7 wbdR Cistron)
SEQ ID NO: 14 (S. Typhi rfbS Cistron)
SEQ ID NO: 15 (S. Typhimurium rfbJ Cistron)
SEQ ID NO: 16 (S. Typhimurium PssaG Promoter Region)
SEQ ID NO: 17 (E. coli araC Repressor and ParaBAD Promoter)
SEQ ID NO: 18 (S. Paratyphi A rfbE Pseudogene)
SEQ ID NO: 19 (S. typhi rfbE Locus with a Partial Deletion of rfbE)
SEQ ID NO: 20 (S. Typhi rfbE Locus with rfbE Disrupted by wbdR)
SEQ ID NO: 21 (S. Typhi fepE Pseudogene)
SEQ ID NO: 22 (S. Typhi Family 2 gtr Operon)
SEQ ID NO: 23 (S. Typhimurium hin-fljBA Locus)
SEQ ID NO: 24 (Bacteriophage Lambda Tandem PR and PL Promoters)
SEQ ID NO: 25 (Bacteriophage Lambda Thermo-Labile Repressor cl857 Cistron)
SEQ ID NO: 26 (pUCpW_difCAT rfbE Deletion Cassette)
SEQ ID NO: 27 (PL rfbE Deletion Cassette)
SEQ ID NO: 28 (pUCpF_difCAT fliC Replacement Cassette)
SEQ ID NO: 29 (tviA Expression Plasmid pBRT4tviA)
SEQ ID NO: 30 (fepE Expression Plasmid pBAD2fepE)
| Number | Date | Country | Kind |
|---|---|---|---|
| 19154550 | Jan 2019 | EP | regional |
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|---|---|---|---|
| 6130082 | Majarian et al. | Oct 2000 | A |
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| 9714782 | Apr 1997 | WO |
| 9826799 | Jun 1998 | WO |
| 2005052167 | Jun 2005 | WO |
| 2007053489 | May 2007 | WO |
| 2012001352 | Jan 2012 | WO |
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| Number | Date | Country | |
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| 20200237887 A1 | Jul 2020 | US |
