CN116528891A

CN116528891A - New antigens

Info

Publication number: CN116528891A
Application number: CN202180081227.1A
Authority: CN
Inventors: R·阿达莫; R·科齐; A·凡托尼; S·波加; R·罗西尼; M·斯卡谢利; N·瓦霍梅
Original assignee: Glaxosmithkline Biology Co ltd
Current assignee: Glaxosmithkline Biology Co ltd
Priority date: 2020-12-02
Filing date: 2021-11-30
Publication date: 2023-08-01

Abstract

The present invention relates to novel, modified FimH polypeptides, nucleic acids encoding them, and the use of polypeptides and nucleic acids in the treatment and/or prevention of diseases, particularly Urinary Tract Infections (UTI).

Description

New antigens

Sequence listing

The present application contains a sequence listing (VB 67013 FF Seq List_ST25.Txt; size: 356.838 bytes; date of creation: 2021, 10 months, 27 days) in the electronically submitted ASCII text file format, which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to novel, modified FimH polypeptides, nucleic acids encoding them, and the use of polypeptides and nucleic acids in the treatment and/or prevention of diseases, particularly Urinary Tract Infection (UTI).

Background

Pathogenic E.coli (UPEC) accounts for approximately 85% of all Urinary Tract Infections (UTI) (A.R. Ronald, urinary tract infection in adults: research priorities and strategies.Int.J. Antimicrob.Agents 17,343-348; 2001). Peak localization adhesin FimH of cilia type 1 allows UPEC to colonize the bladder epithelium during UTI by binding to mannosylated receptors on the surface of the urothelium (M.A. Mulvey, induction and evasion of host defences by type-piliated uropathogenic Escherichia coll.science 282,1494-1497; 1998).

FimH is phase-variable, environmental signals affect its expression, enabling bacteria to attach and avoid being excluded by faeces (effect. Immun.1998,66,3303). anti-FimH IgG is known to inhibit bacterial adhesion in the urinary bladder of mice and monkeys, and protection is associated with the presence of anti-FimH IgG in urine (Langermann S, et al science 1997Apr25;276 (5312): 607-11;Langermann S,et al.J Infect Dis.2000Feb;181 (2): 774-8). The exudation of serum functional IgG in the urogenital tract appears to be responsible for inhibiting bacterial adhesion.

FimH protein consists of N-terminal lectin domain (FimH) binding mannose through a pocket formed by three loop, 5-amino acid linker _L ) And a C-terminal pilin domain (FimH) attaching FimH to cilia _P ) Composition is prepared.

The crystal structure of FimH at various stages of cilia assembly shows that FimH _P Is composed of an incomplete immunoglobulin (Ig) -like fold that is stabilized by complementation with the partner FimC in the periplasm and with the donor strand of fimgs upon ciliated assembly. FimH _P Adopts a single conformation, but FimH _L At least two conformational states with different affinities for mannose can be presented- -a high affinity, i.e., relaxed (R) state, and a low affinity, i.e., strained (T) state (D.Choudhury, X-ray structure of the FimC-FimH clone-adhesin complex from uropathogenic Escherichia color. Science 285,1061-1066 (1999); C.- -S.Hung, structural basis of tropism of Escherichia coli to the bladder during urinary tract in section. Mol. Microbiol.44,903-915 (2002); I.le Trong, structural basis for mechanical force regulation of the adhesin FimH via finger trap-like beta sheet et rotation. Cell 141,645-655 (2010); G.Phan, crystal structure of the FimD usher bound to its cognate FimC-FimH subsystem. Nature 474,49-53 (2011); S.Geibel, structural and energetic basis of folded-protein transport by the FimD us. Nature 496,243-246 (2013)).

When FimH binds to FimC, fimH adopts an elongated conformation, wherein FimH _L And FimH _P Does not interact with each other, but FimH _L In a high affinity mannose binding state. When FimH binds to fimgs, fimH adopts a compact conformation in which FimH _L And FimH _P Closely interdynamic, fimH _L A low affinity mannose binding state is employed. FimH _P By reaction with FimH _L Base interactions of (2) reducing FimH ex situ _L Ability to bind mannose; mannose and FimH _L Binding of (C) induces FimH _L Conformations such that it does not interact with FimH _P Interaction.

It has been reported previously that FimH is directed against a low affinity conformation compared to monoclonal antibodies directed against the post-mannose binding form _L Can better inhibit adhesion to the bladder (Tchesnokova et al 2011,' Type 1Fimbrial Adhesin FimH Elicits an Immune Res)ponse That Enhances Cell Adhesion of Escherichia coli’Infect.Immun.79(10):3895-3904).

FimH with incomplete skin domains are unstable and tend to aggregate. Notably, fimH is typically used as an antigen complexed with the periplasmic protein FimC. The FimC component does not directly help to reduce bacterial colonization in mice, but rather helps to stabilize FimH, protecting it from degradation (Science 1997,276,607;FEMS Microbiol.Lett.2000,188,147). To produce stable FimH proteins, fimgs donor chain peptides (fimgs residues 1-14) have been added in vitro to replace the cilia assembly partner FimC on FimH. (Sauer MM, et al Nat Commun.2016Mar7; 7:10738.). FimH _L A low affinity conformation of (B) has also been obtained, incorporating a disulfide bridge, locking the mannose pocket (Kisiela DI, et al Proc Natl Acad Sci U S A2013 Nov19;110 (47): 19089-94).

The use of FimHC complexes involves a significant production burden-i.e. the production of two polypeptides which must then be reconstituted together-which presents an undesirable complication and significant storage problems, since the stability of the complex must be maintained during storage in order for the antigen to be effective. FimH with disulfide bridge due to the lower molecular weight of this moiety _L Is variable and has proved to be useful in FimH _L Full FimH with disulfide bridges in the domain is difficult to express.

Thus, there remains a need for an ExPEC antigen that is both immunologically effective and mass producible.

Detailed Description

Importantly, fimC stabilizes FimH in its extended post-binding form (nat. Commun.2016,7,10738). The inventors have surprisingly found that by means of a structure-directed design it is possible to stabilize the pre-bound form of FimH without FimC and/or to increase the ability of the generated anti-FimH antibodies to inhibit bacterial adhesion to urothelial cells.

Accordingly, in a first aspect the present invention provides a polypeptide, the amino acid sequence of which comprises or consists of:

(a) FimH; or variants, fragments and/or fusions of FimH

(b) The donor strand is complementary to the amino acid sequence,

wherein (b) is downstream of (a).

By "downstream" we mean that the amino acid sequence, or the amino acid sequence contained in the main amino acid sequence of the polypeptide, is located closer to the C-terminus of the polypeptide relative to the reference sequence.

Alternatively or additionally, the polypeptide of the invention comprises or consists of the amino acid sequence X- (a) -L- (b) -Y, wherein "(a)" is a FimH polypeptide; or variants, fragments and/or fusions of FimH; "L" is an optional first linker; "(b)" is the donor strand complementary amino acid sequence, "X" is the optional N-terminal amino acid sequence; "Y" is an optional C-terminal amino acid sequence, wherein "Y" is not derived from FimC or FimH or a fragment thereof.

By "donor strand complementary amino acid sequence" we mean an amino acid sequence capable of maintaining FimH in either (a) a high affinity conformation, a relaxed (R) state, or (b) a low affinity conformation, a strained (T) state. In a preferred embodiment, the donor strand complementary amino acid sequence is capable of maintaining FimH in a low affinity conformation, i.e., in a strained (T) state.

By 'high affinity conformation, the relaxed (R) state' we mean or include mannose binding affinity of FimH having a conformation closer to high affinity than the low affinity conformation (particularly when the polypeptide of the invention is derived or predominantly derived from FimH, especially complexed with FimC), e.g. at least 51%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of FimH in the high affinity conformation has mannose binding affinity, e.g. K _d <1.2. Mu.M, such as Kisiela DI, et al Proc Natl Acad Sci U S A.2013Nov19;110 (47) 19089-94.

By 'low affinity conformation, the stressed (T) state' we mean or include a mannose binding affinity of FimH having a higher affinity conformation (particularly where the polypeptide of the invention is derived or predominantly derived from FimH, especially when complexed with FimC) that is closer to the low affinity conformation than the high affinity conformation, e.g. less than 50%, 40%, 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% of the mannose binding affinity of FimH in the high affinity conformation, e.g. Kd ≡300 μm or more (i.e. no detectable mannose binding affinity), such as Kisiela DI, et al proc Natl Acad Sci U S a.2013nov19;110 (47) 19089-94. In one embodiment, the polypeptide of the invention is in a low affinity conformation, e.g., having a mannose binding affinity Kd of about 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm or 1mM, or no mannose binding affinity that is detectable.

Mannose binding may be determined using any suitable means known in the art, for example, surface Plasmon Resonance (SPR) may be used to verify binding, binding specificity and binding constants of FimH constructs to mannosylated bovine serum albumin (Man-BSA) and glucolated bovine serum albumin (Glc-BSA) (negative control), for example, rabani et al, 2018, 'Conformational switch of the bacterial adhesin FimH in the absence of the regulatory domain: engineering a minimalistic allosteric system' j.biol.chem.,293 (5): 1835-1849,and Bouckaert J,et al.Mol Microbiol.2005Jan;55 441-55, which are incorporated herein by reference.

The conformation of FimH can also be assessed by measuring binding of conformational antibodies using any suitable means known in the art, such as surface plasmon resonance, and as described in the examples. Exemplary antibodies are capable of recognizing epitopes that overlap differently with the mannose binding pocket of FimH, e.g., antibodies that bind to epitopes that overlap with the mannose binding pocket, e.g., epitopes that are limited to only one loop of the mannose binding pocket. Exemplary antibodies are those disclosed in WO2016/183501, or in Kisiela DI, et al Proc Natl Acad Sci U S A2013 Nov 19;110 (47) 19089-94,Kisiela DI,et al.PLoS Pathog.2015May 14;11 (5) antibodies disclosed in e1004857, and these are incorporated herein by reference. In one embodiment, the conformational antibody has a variable heavy chain (VH) sequence of SEQ ID NO. 125 and a variable light chain (VL) sequence of SEQ ID NO. 126. In one embodiment, the conformational antibody has the amino acid sequence of SEQ ID NO:127 and the variable heavy chain (VH) sequence of SEQ ID NO:128, and a variable light chain (VL) sequence.

QVQLQQSGAELATPGASVKMSCKASGYTSTNYWIHWVKQRPGQGLEWIGYINPTSGYTEYNQNFKDKATLTADKSSSTAYMQLTSLTSEDSAVYYCARGVIRDFWGQGTTLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSS

[ SEQ ID NO:125] -VH of mAb 926

DVLMTQTPLSLPVSLGDQASISCRSSQNIVHNNGNTYLEWYLQSPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIK [ SEQ ID NO:126] -VL (kappa) of mAb 926

QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPRDGDTNYNGKFMDKVTLTADKSSNTAYMQLSSLTSEDSAVYFCEVGRGFYGMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSS

[ SEQ ID NO:127] -VH of mAb475

DIVMTQSPKFMSTSVGDRVSVTCKASQNVSNVAWYQQKPGQSPKAMIYSASYRYSGVPGRFTGSGSGTDFTLTINNVQSEDLATYFCQQNSSFPFTFGGGTKLEIK [ SEQ ID NO:128] -VL (kappa) of mAb475

The term 'amino acid' as used herein includes standard 20 genetically encoded amino acids and their corresponding stereoisomeric 'D' forms (as compared to the natural 'L' form), omega amino acids and other naturally occurring amino acids, unconventional amino acids (e.g., α -disubstituted amino acids, N-alkyl amino acids, etc.) and chemically derivatized amino acids (see below).

Thus, when an amino acid is specifically recited, such as "alanine" or "Ala" or "a", the term refers to L-alanine and D-alanine unless explicitly stated otherwise. Other non-conventional amino acids may also be suitable components of the polypeptides of the invention, provided that the polypeptide retains the desired functional properties. For the polypeptides shown, each encoded amino acid residue is indicated by a single letter designation, where appropriate, corresponding to the trivial designation of a conventional amino acid.

By 'isolated' we mean that the features of the invention (e.g. polypeptides) are provided outside the environment in which they may naturally occur. Those skilled in the art will appreciate that 'isolated' means that 'hands-over' changes its natural state, that is, if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in an organism is not 'isolated' when in such an organism, but is an 'isolated' term as used in this disclosure when the same polynucleotide or polypeptide is isolated from coexisting materials in its natural state. In addition, a polynucleotide or polypeptide introduced into an organism by transformation, genetic manipulation, or any other recombinant means will be understood to be 'isolated' even though it is still present in the organism, which may be living or non-living, unless such transformation, genetic manipulation, or other recombinant means results in an organism that is not distinguishable from a naturally occurring organism.

By 'polypeptide' we mean or include polypeptides and proteins.

By "variant" of a polypeptide we mean an insertion, deletion and/or substitution, whether conservative or non-conservative. In particular, a variant polypeptide may be a non-naturally occurring variant (i.e., not present in nature, or not known to occur). Variants may have at least 50% sequence identity to a reference sequence, e.g., at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5%.

"sequence identity" or "identity" can be determined by the Smith Waterman homology search algorithm implemented in the MPSRCH program (Oxford Molecular), using biomimetic gap search with parameters gap open penalty=12 and gap extension penalty=1, or by the Needleman-Wunsch global alignment algorithm (see, e.g., rubin (2000) pediatric. Clin. North am. 47:269-285), using default parameters (e.g., using the EBLOSUM62 scoring matrix, gap open penalty=10.0, gap extension penalty=0.5). This algorithm is conveniently implemented in the needle tool of the EMBOSS software package. Unless otherwise indicated, where reference is made in application to sequence identity to a particular reference sequence, that identity is calculated over the entire length of that reference sequence. In addition, the percent identity may be determined by methods well known in the art, for example, using the LALIGN program (Huang and Miller, adv. (1991) 12:337-357, the disclosure of which is incorporated herein by reference) at the ExpASY facility website www.ch.embnet.org/software/LALIGN_form.html, using global alignment options, scoring matrix BLOSUM62, gap opening penalty-14, gap expansion penalty-4 as parameters. In addition, the percent sequence identity between two polypeptides can be determined using a suitable computer program, such as the alignX, vector NTI Advance (from Invitrogen corporation) or the GAP program (from the university of Wisconsin genetic computing group).

It will be appreciated that the calculation of percent identity is related to polymers (e.g., polypeptides or polynucleotides) to which sequences have been aligned.

Fragments and variants may be made using methods of protein engineering and site-directed mutagenesis well known in the art (see, e.g., molecular Cloning: a Laboratory Manual,3rd edition,Sambrook&Russell,2001,Cold Spring Harbor Laboratory Press, the disclosure of which is incorporated herein by reference).

It will be appreciated by those skilled in the art that the polypeptides of the invention, or fragments, variants or fusions thereof, may comprise one or more modified or derivatized amino acids.

Chemical derivatives of one or more amino acids can be achieved by reaction with a functional pendant group. Such derivatized molecules include, for example, those in which the free amino group has been derivatized to amine hydrochloride, p-toluenesulfonyl, carboxyphenoxy, butoxycarbonyl, chloroacetyl, or formyl. The free carboxyl groups may be derivatized to salts, methyl and ethyl esters or other types of esters and hydrazides. The free hydroxyl groups may be derivatized to O-acyl or O-alkyl derivatives. Also included as chemical derivatives are those polypeptides containing naturally occurring twenty standard amino acid derivatives. For example: 4-hydroxyproline can replace proline; 5-hydroxy lysine may replace lysine; 3-methylhistidine may replace histidine; homoserine may replace serine and ornithine may replace lysine. Derivatives also include peptides containing one or more additions or deletions, provided that the necessary activity is maintained. Other modifications include amidation, amino-terminal acylation (e.g., acetylating or thioglycolating), terminal carboxylation (e.g., with ammonia or methylamine), and similar terminal modifications.

Those skilled in the art will further appreciate that peptidomimetic compounds may also be useful. Thus, by 'polypeptide' we include peptidomimetic compounds that exhibit endo-lytic activity. The term 'peptidomimetic' refers to a compound that mimics the conformation and desirable characteristics of a particular polypeptide as a therapeutic agent.

For example, polypeptides described herein include not only molecules in which amino acid residues are linked by a peptide (-CO-NH-) but also molecules in which peptide bonds are reversed. Such inverted peptidomimetics can be made by methods known in the art, for example, as described in Meziere et al (1997) J.Immunol.159,3230-3237, the disclosure of which is incorporated herein by reference. Such reverse polypeptides containing NH-CO bonds rather than CO-NH peptide bonds are much more resistant to proteolytic degradation. Alternatively, the polypeptide of the invention may be a peptidomimetic compound in which one or more amino acid residues are linked by a-gamma (CH 2 NH) -linkage, in place of the traditional amide linkage.

It will be appreciated that the polypeptide may conveniently be blocked at its N-or C-terminus to help reduce sensitivity to external protective digestion, for example by amidation.

As discussed herein, various uncoded or modified amino acids, such as D-amino acids and N-methyl amino acids, may be used to modify the polypeptides of the invention. Furthermore, a putative bioactive conformation may be stabilized by covalent modification, such as cyclization or by addition of lactams, disulfides or other types of bridges. Methods for the synthesis of cyclic homologous and heterologous peptides, including disulfides, sulfides and alkylene bridges, have been disclosed in US 5,643,872. Examples of other cyclization processes are discussed and disclosed in US 6,008,058, the relevant disclosures of which are incorporated herein by reference. Another method of synthesizing a cyclic stable peptidomimetic compound is ring closure synthesis (RCM).

By 'fusion' of polypeptides we include polypeptides fused to any other polypeptide. For example, a polypeptide may comprise the insertion of one or more additional amino acids internally and/or at the N-and/or C-terminus of the amino acid sequence of a polypeptide of the invention.

Thus, as described herein, in one embodiment, the polypeptides of the first aspect of the invention include polypeptides of the invention to which are fused enzyme domains from different sources (e.g., from sources other than the polypeptides of the first aspect of the invention). Examples of suitable enzymatic domains include: L-alanyl-D-glutamate endopeptidase; D-glutamyl-m-DAP endopeptidase; a peptide-bridge specific endopeptidase; n-acetyl- β -D-glucosidase (=murine Li Xian hydrolase); n-acetyl- β -D-wall amidase (=lysozyme); cleavage of the transglutaminase. In addition, N-acetylurea-L-alanine amidase from other sources may also be utilized (see Loessner,2005,Current Opinion in Microbiology 8:480-487, the disclosure of which is incorporated herein by reference).

For example, the polypeptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A to facilitate purification of the polypeptide. Examples of such GST fusions are well known to those skilled in the art. Likewise, the polypeptide may be fused to an oligohistidine tag such as His6 or to an epitope recognized by an antibody such as the well-known Myc tag epitope. Fusion to any fragment, variant or derivative of said polypeptide is also included within the scope of the present invention. It will be appreciated that fusion (or variant or derivative thereof) retaining desirable properties, such as antigen activity, are preferred. It is also particularly preferred if the fusion is one which is suitable for use in the methods described herein.

For example, the fusion may comprise a further moiety that confers desirable properties on said polypeptide of the invention; for example, the moiety can be used to detect or isolate a polypeptide, to promote cellular uptake of a polypeptide, or to direct secretion of a protein from a cell. For example, the moiety may be a biotin group, a radioactive group, a fluorescent group, such as a small fluorescent group or a Green Fluorescent Protein (GFP) fluorescent group, as is well known to those skilled in the art. The moiety may be an immunological tag known to those skilled in the art, such as a Myc tag, or may be a lipophilic molecule or polypeptide structure known to those skilled in the art that is capable of promoting cellular uptake of the polypeptide.

Those of skill in the art will appreciate that the polypeptides of the invention also include pharmaceutically acceptable acid or base addition salts of the polypeptides described herein. The pharmaceutically acceptable acid addition salts of the above base compounds useful in the present invention are those which form non-toxic acid addition salts, i.e., salts containing pharmaceutically acceptable anions such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, succinate, maleate, fumarate, gluconate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [ i.e., 1' -methylenebis (2-hydroxy-3-naphthoate) ] salts, and the like.

Pharmaceutically acceptable base addition salts may also be used to produce pharmaceutically acceptable salt forms of the polypeptides. Chemical bases useful as reagents for preparing pharmaceutically acceptable base addition salts of the compounds of the invention are those which form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to, those derived from such pharmaceutically acceptable cations as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium), ammonium or water-soluble amine addition salts such as N-methyl glucamine- (meglumine), as well as lower alkanolamines and other base salts of pharmaceutically acceptable organic amines, and the like.

The polypeptide or fragment, variant, fusion or derivative thereof may also be stored lyophilized and reconstituted in a suitable carrier prior to use. Any suitable lyophilization process (e.g., spray drying, cake drying) and/or reconstitution techniques may be employed. Those skilled in the art will appreciate that lyophilization and reconstitution can result in varying degrees of activity loss, and that the level of use may have to be adjusted upward to compensate. Preferably, the lyophilized (freeze-dried) polypeptide loses no more than about 20%, or no more than about 25%, or no more than about 30%, or no more than about 35%, or no more than about 40%, or no more than about 45%, or no more than about 50% of its activity upon rehydration (prior to lyophilization).

The polypeptides of the invention are preferably provided in purified or substantially purified form, i.e., substantially free of other polypeptides (e.g., free of naturally occurring polypeptides), particularly free of other E.coli or host cell polypeptides, and generally at least about 50% pure (by weight), e.g., at least 70%, 80%, 90%, 95%, 96%, 97%, 98%99%, 99.5% or 100% pure (i.e., less than 50% of the composition is made up of other expressed polypeptides). Thus, the antigen in the composition is isolated from the whole organism expressing the antigenic molecule.

(a) The FimH of (a) may be any escherichia coli or klebsiella pneumoniae (or variant, fragment and/or fusion thereof), but alternatively or additionally (a) comprises or consists of:

(A) SEQ ID NO 1 (Genbank accession number: ELL41155.1 (fimH of E.coli J96)), SEQ ID NO 2, SEQ ID NO 100 (Genbank accession number: ABG72591.1 (fimH of UPEC 536)), SEQ ID NO 101,SEQ ID NO:102 (Genbank accession number: AAN83822.1 (fimH of CFT 073)), SEQ ID NO 103,SEQ ID NO:104 (Genbank accession number: AJE58925.1 (fimH of E.coli 789)), SEQ ID NO 105,SEQ ID NO:106 (Genbank accession number: AAC35864.1, corresponding to the amino acid sequence AF089840.1 (fimH of IHE 3034), or SEQ ID NO 107,

(B) With SEQ ID NO:1 (Genbank accession number: ELL41155.1 (FimH of E.coli J96)), SEQ ID NO:2, SEQ ID NO:100 (Genbank accession number: ABG72591.1 (FimH of UPEC 536)), SEQ ID NO:101,SEQ ID NO:102 (Genbank accession number: AAN83822.1 (FimH of CFT 073)), SEQ ID NO:103,SEQ ID NO:104 (Genbank accession number: AJE58925.1 (FimH of E.coli 789)), SEQ ID NO:105,SEQ ID NO:106 (Genbank accession number: AAC 35864.1), corresponding to the amino acid sequence AF089840.1 (FimH of IHE 3034), or SEQ ID NO:107 comprising an amino acid sequence of 1 to 10 single amino acid changes, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 single amino acid changes,

(C) With SEQ ID NO 1 (Genbank accession number: ELL41155.1 (FimH of E.coli J96)), SEQ ID NO 2, SEQ ID NO 100 (Genbank accession number: ABG72591.1 (FimH of UPEC 536)), SEQ ID NO 101,SEQ ID NO:102 (Genbank accession number: AAN83822.1 (FimH of CFT 073)), SEQ ID NO 103,SEQ ID NO:104 (Genbank accession number: AJE58925.1 (FimH of E.coli 789)), SEQ ID NO 105,SEQ ID NO:106 (Genbank accession number: AAC35864.1, corresponding to the amino acid sequence AF089840.1 (FimH of IHE 3034), or SEQ ID NO 107 having at least 70% sequence identity, and/or

(D) From SEQ ID NO 1 (Genbank accession number: ELL41155.1 (FimH of E.coli J96)), SEQ ID NO 2, SEQ ID NO 100 (Genbank accession number: ABG72591.1 (FimH of UPEC 536)), SEQ ID NO 101,SEQ ID NO:102 (Genbank accession number: AAN83822.1 (FimH of CFT 073)), SEQ ID NO 103,SEQ ID NO:104 (Genbank accession number: AJE58925.1 (FimH of E.coli 789)), SEQ ID NO 105,SEQ ID NO:106 (Genbank accession number: AAC35864.1, a fragment of at least 10 consecutive amino acids corresponding to the nucleic acid sequence AF089840.1 (IHE 3034), e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 275, 280, 290 or 300 consecutive amino acids of SEQ ID NO 107.

MKRVITLFAVLLMGWSVNAWSFACKTANGTAIPIGGGSANVYVNLAPVVNVGQNLVVDLSTQIFCHNDYPETITDYVTLQRGSAYGGVLSNFSGTVKYSGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNDVVVPTGGCDVSARDVTVTLPDYPGSVPIPLTVYCAKSQNLGYYLSGTTADAGNSIFTNTASFSPAQGVGVQLTRNGTIIPANNTVSLGAVGTSAVSLGLTANYARTGGQVTAGNVQSIIGVTFVYQ

[ SEQ ID NO:1] -GenBank:ELL41155.1 (underlined Signal peptide)

FACKTANGTAIPIGGGSANVYVNLAPVVNVGQNLVVDLSTQIFCHNDYPETITDYVTLQRGSAYGGVLSNFSGTVKYSGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNDVVVPTGGCDVSARDVTVTLPDYPGSVPIPLTVYCAKSQNLGYYLSGTTADAGNSIFTNTASFSPAQGVGVQLTRNGTIIPANNTVSLGAVGTSAVSLGLTANYARTGGQVTAGNVQSIIGVTFVYQ

[ SEQ ID NO. 2] -GenBank:ELL41155.1 minus 21aa signal peptide

MIVMKRVITLFAVLLMGWSVNAWSFACKTANGTAIPIGGGSANVYVNLAPAVNVGQNLVVDLSTQIFCHNDYPETITDYVTLQRGSAYGGVLSSFSGTVKYNGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNDVVVPTGGCDVSARDVTVTLPDYPGSVPIPLTVYCAKSQNLGYYLSGTTADAGNSIFTNTASFSPAQGVGVQLTRNGTIIPANNTVSLGAVGTSAVSLGLTANYARTGGQVTAGNVQSIIGVTFVYQ

[ SEQ ID NO:100] -Genbank accession number: ABG72591.1 (FimH of UPEC 536)) (underlined as signal peptide)

FACKTANGTAIPIGGGSANVYVNLAPAVNVGQNLVVDLSTQIFCHNDYPETITDYVTLQRGSAYGGVLSSFSGTVKYNGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNDVVVPTGGCDVSARDVTVTLPDYPGSVPIPLTVYCAKSQNLGYYLSGTTADAGNSIFTNTASFSPAQGVGVQLTRNGTIIPANNTVSLGAVGTSAVSLGLTANYARTGGQVTAGNVQSIIGVTFVYQ

[ SEQ ID NO:101] -Genbank accession number: ABG72591.1 (FimH of UPEC 536) minus signal peptide

MIVMKRVITLFAVLLMGWSVNAWSFACKTANGTAIPIGGGSANVYVNLAPAVNVGQNLVVDLSTQIFCHNDYPETITDYVTLQRGSAYGGVLSSFSGTVKYNGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNDVVVPTGGCDASARDVTVTLPDYPGSVPIPLTVYCAKSQNLGYYLSGTTADAGNSIFTNTASFSPAQGVGVQLTRNGTIIPANNTVSLGAVGTSAVSLGLTANYARTGGQVTAGNVQSIIGVTFVYQ

[ SEQ ID NO:102] -Genbank accession No. AAN83822.1 (FimH of CFT 073) (underlined is the Signal peptide)

FACKTANGTAIPIGGGSANVYVNLAPAVNVGQNLVVDLSTQIFCHNDYPETITDYVTLQRGSAYGGVLSSFSGTVKYNGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNDVVVPTGGCDASARDVTVTLPDYPGSVPIPLTVYCAKSQNLGYYLSGTTADAGNSIFTNTASFSPAQGVGVQLTRNGTIIPANNTVSLGAVGTSAVSLGLTANYARTGGQVTAGNVQSIIGVTFVYQ

[ SEQ ID NO:103] -Genbank accession number AAN83822.1 (FimH of CFT 073) minus signal peptide

MIVMKRVITLFAVLLMGWSVNAWSFACKTANGTAIPIGGGSANVYVNLAPVVNVGQNLVVDLSTQIFCHNDYPETITDYVTLQRGSAYGGVLSNFSGTVKYSGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNDVVVPTGGCDVSARDVTVTLPDYPGSVPIPLTVYCAKSQNLGYYLSGTTADAGNSIFTNTASFSPAQGVGVQLTRNGTIIPANNTVSLGAVGTSAVSLGLTANYARTGGQVTAGNVQSIIGVTFVYQ

[ SEQ ID NO:104] Genbank accession No. AJE58925.1 (FimH of E.coli 789) (underlined as signal peptide)

[ SEQ ID NO:105] Genbank accession number: AJE58925.1 (FimH of E.coli 789) minus signal peptide

MKRVITLFAVLLMGWSVNAWSFACKTANGTAIPIGGGSANVYVNLAPAVNVGQNLVVDLSTQIFCHNDYPETITDYVTLQRGAAYGGVLSSFSGTVKYNGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNDVVVPTGGCDVSARDVTVTLPDYPGSVPIPLTVYCAKSQNLGYYLSGTTADAGNSIFTNTASFSPAQGVGVQLTRNGTIIPANNTVSLGAVGTSAVSLGLTANYARTGGQVTAGNVQSIIGVTFVYQ

[ SEQ ID NO:106] Genbank accession No. AAC35864.1, (FimH of IHE 3034), (underlined is the signal peptide)

FACKTANGTAIPIGGGSANVYVNLAPAVNVGQNLVVDLSTQIFCHNDYPETITDYVTLQRGAAYGGVLSSFSGTVKYNGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNDVVVPTGGCDVSARDVTVTLPDYPGSVPIPLTVYCAKSQNLGYYLSGTTADAGNSIFTNTASFSPAQGVGVQLTRNGTIIPANNTVSLGAVGTSAVSLGLTANYARTGGQVTAGNVQSIIGVTFVYQ

[ SEQ ID NO:107] Genbank accession No. AAC35864.1, (FimH of IHE 3034), minus signal peptide

Alternatively or additionally, the polypeptide is a fragment, variant, fusion and/or derivative capable of inducing a specific immune response to a polypeptide selected from the group consisting of SEQ ID NO:1 (Genbank accession number: ELL41155.1 (FimH of E.coli J96)), SEQ ID NO:2, SEQ ID NO:100 (Genbank accession number: ABG72591.1 (FimH of UPEC 536)), SEQ ID NO:101,SEQ ID NO:102 (Genbank accession number: AAN83822.1 (FimH of CFT 073)), SEQ ID NO:103,SEQ ID NO:104 (Genbank accession number: AJE58925.1 (FimH of E.coli 789)), SEQ ID NO:105,SEQ ID NO:106 (Genbank accession number: AAC35864.1, corresponding to nucleic acid sequence AF089840.1 (FimH of IHE 3034), or SEQ ID NO: 107.

By "specific immune response" we mean or include the ability to induce (e.g., stimulate release of) an immune response in a subject that produces antibodies capable of binding to a specified amino acid sequence. Preferably, the antibody is capable of binding in vivo, i.e. under physiological conditions in which the amino acid sequence or polypeptide is present on or in the subject. Such binding specificity can be determined by methods well known in the art, such as ELISA, immunohistochemistry, immunoprecipitation, western blotting and flow cytometry using transfected cells expressing the polypeptides of the invention.

Alternatively, or in addition, the immune response is an immune activation response, e.g., a protective immune response. The polypeptide may elicit an in vitro protective immune response and/or an in vivo protective immune response when administered to a subject.

In the presence of a co-stimulatory signal, T cells differentiate into specific phenotype subtypes. Several of these subtypes are involved in inhibiting or terminating natural inflammatory signals. By "immune activation response" we mean and/or include the polypeptide inducing or being capable of inducing an immune response in a subject that does not result in the inhibition or termination of inflammation or an inflammatory signal, preferably in the activation or enhancement of inflammation or an inflammatory signal (e.g., a cytokine).

The protective immune response in vivo may be elicited in a mammal. Alternatively or additionally, the mammal is selected from armadillo (dasypus novemcinctus), baboon (papio and papio cynocephalus), camel (camelus bactrianus, camelus dromedarius, camel plus), cat (felis catus), dog (canis lupus familiaris), horse (equus ferus caballus), ferret (mustela putorius furo), goat (capra aegagrus hircus), guinea pig (cava pomelous), golden hamster (mesocricetus auratus), kangaroo (macropus rufus), camel horse (lama glama), mouse (mus musculus), pig (sus scrofa domesticus), rabbit (oryctolagus cuniculus), rat (rattus norvegicus), macaca mula, sheep (ovis aries), non-human primate, and human (Homo sapiens).

Alternatively or additionally, the connection FimH may be deleted _L And FimH _P To reduce FimH _L Reducing mannose binding. For example, relative to SEQ ID NO: glycine residues 196 and 197 of polypeptide part (a), relative to SEQ ID NO: glycine residues 180 and 181 of polypeptide part (a), relative to SEQ ID NO:100, glycine residues 183 and 184 of polypeptide part (a) relative to SEQ ID NO:102, glycine residues 183 and 184 of polypeptide part (a) relative to SEQ ID NO104, glycine residues 183 and 184 of polypeptide part (a), are:

(i) Presence; or (b)

(ii) And deleting.

Alternatively or additionally, one or more amino acids of a polypeptide known or predicted to be N-glycosylated or O-glycosylated are replaced by amino acids that are not or less easily glycosylated, such as serine (S), aspartic acid (D), alanine (A), or glutamine (Q). Alternatively or additionally, only polypeptide part (a) comprises amino acid substitutions to reduce or eliminate N-and/or O-glycosylation.

N-and/or O-glycosylation can be determined using any suitable means known in the art, for example, using NetNGlyc 1.0 and NetOGLlyc 4.0 servers (available at http:// www.cbs.dtu.dk/services/NetOGLyc/and http:// www.cbs.dtu.dk/services/NetOGLyc/obtained) using default settings.

Alternatively or additionally, polypeptide portion (a) comprises one or more of the following amino acid substitutions relative to SEQ ID No. 2: N28S, N91D, N249D, N256D, or one or more amino acid substitutions, e.g., one, two, three or four amino acid substitutions, at positions in SEQ ID NOS: 101, 103 and 105 corresponding to those positions in SEQ ID NO: 2.

Alternatively or additionally, the donor strand complementary amino acid sequence (b) comprises or consists of:

(i) 6-28 amino acids of SEQ ID NO. 3; or fragments and/or variants thereof, or

(ii) 8-36 amino acids of SEQ ID NO. 4; or fragments and/or variants thereof, asaatiqaADVTITVNGKVVAKPCTVSTT

[SEQ ID NO:3]Fimgdonor chain and flanking regions (donor chain underlined). PSMDKSKLTENTLQLAIIS RIKLYYRPAKLALPPDQ

[ SEQ ID NO:4] -FimC donor strand and flanking regions (donor strand underlined).

Alternatively or additionally, part (b) comprises or consists of 6-28 amino acids of SEQ ID No. 3 (or fragments and/or variants thereof) selected from the group consisting of:

(i) Amino acids 1-28 of SEQ ID NO. 3; or fragments and/or variants thereof,

(ii) Amino acids 2-27 of SEQ ID NO. 3; or fragments and/or variants thereof,

(iii) Amino acids 3-26 of SEQ ID NO. 3; or fragments and/or variants thereof,

(iv) Amino acids 4-25 of SEQ ID NO. 3; or fragments and/or variants thereof,

(v) Amino acids 5-24 of SEQ ID NO. 3; or fragments and/or variants thereof,

(vi) Amino acids 6-23 of SEQ ID NO. 3; or fragments and/or variants thereof,

(vii) Amino acids 7-22 of SEQ ID NO. 3; or fragments and/or variants thereof,

(viii) Amino acids 8-21 of SEQ ID NO. 3; or fragments and/or variants thereof,

(ix) Amino acids 9-20 of SEQ ID NO. 3; or fragments and/or variants thereof,

(x) Amino acids 10-19 of SEQ ID NO. 3; or fragments and/or variants thereof,

(xi) Amino acids 11-18 of SEQ ID NO. 3; or fragments and/or variants thereof, and

(xii) Amino acids 12-17 of SEQ ID NO. 3; or a fragment and/or variant thereof.

Alternatively or additionally, part (b) comprises or consists of 8-36 amino acids of SEQ ID NO. 4 (or fragments and/or variants thereof) selected from the group consisting of:

(i) Amino acids 1-36 of SEQ ID NO. 4; or fragments and/or variants thereof,

(ii) Amino acids 2-35 of SEQ ID NO. 4; or fragments and/or variants thereof,

(iii) Amino acids 3-34 of SEQ ID NO. 4; or fragments and/or variants thereof,

(iv) Amino acids 4-33 of SEQ ID NO. 4; or fragments and/or variants thereof,

(v) Amino acids 5-32 of SEQ ID NO. 4; or fragments and/or variants thereof,

(vi) Amino acids 6-31 of SEQ ID NO. 4; or fragments and/or variants thereof,

(vii) Amino acids 7-30 of SEQ ID NO. 4; or fragments and/or variants thereof,

(viii) Amino acids 8-29 of SEQ ID NO. 4; or fragments and/or variants thereof,

(ix) Amino acids 9-28 of SEQ ID NO. 4; or fragments and/or variants thereof,

(x) Amino acids 10-27 of SEQ ID NO. 4; or fragments and/or variants thereof,

(xi) Amino acids 11-26 of SEQ ID NO. 4; or fragments and/or variants thereof,

(xii) Amino acids 12-25 of SEQ ID NO. 4; or fragments and/or variants thereof,

(xiii) Amino acids 13-24 of SEQ ID NO. 4; or fragments and/or variants thereof,

(xiv) Amino acids 14-23 of SEQ ID NO. 4; or fragments and/or variants thereof,

(xv) Amino acids 15-24 of SEQ ID NO. 4; or fragments and/or variants thereof, and

(xvi) Amino acids 16-23 of SEQ ID NO. 4; or a fragment and/or variant thereof.

(A) The amino acid sequence of SEQ ID No. 5 or SEQ ID No. 6,

(B) An amino acid sequence comprising 1 to 10 single amino acid changes compared to SEQ ID NO. 5 or SEQ ID NO. 6, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 single amino acid changes,

(C) Fragments of at least 7 consecutive amino acids from SEQ ID NO. 5, for example at least 8, 9, 10, 11, 12 or 13 consecutive amino acids from SEQ ID NO. 5, and/or,

(D) Fragments of at least 7 consecutive amino acids from SEQ ID NO. 6, for example at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 consecutive amino acids from SEQ ID NO. 6.

ADVTITVNGKVVAK [ SEQ ID NO:5] -FimG donor chain

ENTLQLAIISRIKLYYRP [ SEQ ID NO:6] -FimC donor chain

In a preferred embodiment, the donor-strand complementary amino acid sequence (b) comprises or consists of the amino acid sequence of SEQ ID NO. 5. Alternatively or additionally, the donor strand complementary amino acid sequence (b) comprises or consists of the amino acid sequence of SEQ ID NO. 6.

Alternatively or additionally, the donor strand complementary amino acid sequence (b) is:

(i) Directly connected to the C-terminal of (a), or

(ii) Is connected to the C-terminal of (a) by a first linker.

Alternatively or additionally, the first linker (or "L") comprises or consists of 2-20 amino acids, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. Alternatively or additionally, the first linker starts with proline. In a preferred embodiment, the first linker starts with proline. Alternatively or additionally, the first linker comprises or consists of a polar amino acid, e.g. wherein the first linker is entirely made up of polar amino acids, or if the first linker starts with proline, the remaining amino acids are polar. Alternatively or additionally, the first linker comprises or consists of:

(i) PGDGN [ SEQ ID NO:7], or variants or fusions thereof, or

(ii) DNKQ [ SEQ ID NO:8], or a variant or fusion thereof.

In a preferred embodiment, the first linker (or "L") comprises or consists of SEQ ID NO. 7.

Alternatively or additionally, the polypeptide comprises a protein purification affinity tag, e.g. 6, 7, 8, 9 or 10 consecutive histidines, at the N-terminus, C-terminus and/or internally.

Alternatively or additionally, "X" comprises a cell secretion leader sequence. Alternatively or additionally, the polypeptide comprises a cell secretion leader sequence that:

(i) Upstream of (a), or

(ii) At the N-terminus of the polypeptide.

Alternatively or additionally, the cell secretion leader sequence is selected from the group consisting of:

(i) METDTLLLWVLLLWVPGSTGD [ SEQ ID NO:9], or variants or fusions thereof,

(ii) METDTLLLWVLLLWVPGSTGDAAQPARRARRTKLAL [ SEQ ID NO:10], or variants or fusions thereof,

(iii) MRLLAKIICLMLWAICVA [ SEQ ID NO:11], or variants or fusions thereof,

(iv) MGWSCIILFLVATATGVHS [ SEQ ID NO:12], or variants or fusions thereof,

(v) METPAELLFLLLLWLPDTTG [ SEQ ID NO:13], or variants or fusions thereof,

(vi) METDTLLLWVLLLWVPGSTG [ SEQ ID NO:108], or variants or fusions thereof, or

(vii) MEFGLSWVFLVAILEGVHC [ SEQ ID NO:14], or variants or fusions thereof.

Alternatively or additionally, "X" is a methionine (M) residue, particularly when the polypeptide is expressed in an E.coli host cell.

Alternatively or additionally, the polypeptide comprises a nanoparticle domain at the N-terminus or the C-terminus. Thus, in one embodiment, "X" comprises a nanoparticle domain or "Y" comprises a nanoparticle domain. By "nanoparticle domain" we mean or include amino acid sequences capable of self-assembly to form protein complexes, particularly globular protein complexes. By 'self-assembled', we mean or include assembly with the same type of nanoparticle domain (e.g., if the nanoparticle domain is a ferritin domain, it can be assembled with other ferritin domains to form a protein complex, such as a globular protein complex). In particular, the nanoparticle domains of the invention are capable of self-assembly when forming part of the a polypeptides of the invention.

Alternatively or additionally, the nanoparticle domain is selected from the following:

(a) Ferritin (e.g., [ SEQ ID NO:15] or [ SEQ ID NO:109] (helicobacter pylori), [ SEQ ID NO:16] (Escherichia coli)), or [ SEQ ID NO:149] - [ SEQ ID NO:152] (stabilized Escherichia coli), or variants and/or fragments thereof,

(b) iMX313 (e.g. [ SEQ ID NO:17 ]), or a variant and/or fragment thereof,

(c) mI3 (e.g. [ SEQ ID NO:18 ]), or a variant and/or fragment thereof,

(d) A packaging protein (e.g. [ SEQ ID NO:19 ]), or a variant and/or fragment thereof, or

(e) Self-assembled viral capsid proteins, such as the phage Acinetobacter AP205 capsid protein (NCBI reference sequence: NP-085472.1), hepatitis B virus core protein (HBc) [ SEQ ID NO:110], or phage qβ [ SEQ ID NO:111], or variants and/or fragments thereof. DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPV QLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEV LFKDILDKIELIGNENHGLYLADQYVKGIAKSRK [ SEQ ID NO:15] -H.pyri ferritin

DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS [ SEQ ID NO:109] -H.pyri ferritin (S-terminated)

LKPEMIEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINTVESPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYPTFNFLQWYVSEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDTQN [ SEQ ID NO:16] -E.coli ferritin

KKQGDADVCGEVAYIQSVVSDCHVPTAELRTLLEIRKLFLEIQKLKVELQGLSKEG[SEQ ID NO:17]iMX313

MKMEELFKKHKIVAVLRANSVEEAKKKALAVFLGGVHLIEITFTVPDADTVIKELSFLKEMGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPVEVAEKAKAFVEKIRGCTE[SEQ ID NO:18]mi3

MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLGEVEVLSDENEVVKWGLRKSLPLIELRATFTLDLWELDNLERGKPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKF [ SEQ ID NO:19] -parcel protein

MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLEDASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVV[SEQ ID NO:110]HBC

MAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQAYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY[SEQ ID NO:111]-Qbeta

LKPEMIEKLNEQMNLELYSSLLYQQMSAWCSYHGFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRIDTIPSPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYPTFNFLQWYVAEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDTQN [ SEQ ID NO:149] -1EUM_0_5-stabilized E.coli ferritin

LKPEMIEKLNEQMNLELYSSLLYQQMSAWCSYHGFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINTIPSPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYPTFNFLQWYVAEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDTQN [ SEQ ID NO:150] -1EUM_2-stabilized E.coli ferritin

LKPEMIEKLNEQMNLELYSSLLYQQMSAWCSYHGFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINTVPSPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYPTFNFLQWYVAEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDTQN [ SEQ ID NO:151] -1EUM_2_5-stabilized E.coli ferritin

LKPEMIEKLNEQMNLELYSSLLYQQMSAWCSYHGFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINTVESPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYPTFNFLQWYVSEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDTQN [ SEQ ID NO:152] -1EUM_6-stabilized E.coli ferritin

Alternatively or additionally, the nanoparticle domain is:

(i) Directly linked to a polypeptide, or

(ii) Is linked to the polypeptide by a second linker.

Alternatively or additionally, the second linker comprises or consists of 2-20 amino acids, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. Alternatively or additionally, the second linker comprises or consists of glycine (G) and/or serine (S), or comprises at least 50% glycine (G) and/or serine (S), e.g. at least 60%, 70%, 80%, 90% or 95% glycine (G) and/or serine (S).

Alternatively or additionally, the second linker is selected from the following:

(a) GSSGSGSGS [ SEQ ID NO:112] or variants and/or fusions thereof,

(b) GGSGS [ SEQ ID NO:113] or variants and/or fusions thereof,

(c) GGS or variants and/or fusions thereof,

(d) SGSHHHHHHHHGGS [ SEQ ID NO:114], or variants and/or fusions thereof,

(e) AKFVAAWTLKAAA [ SEQ ID NO:115] or variants and/or fusions thereof,

(f) GGGGSLVPRGSGGGGS [ SEQ ID NO:116], or variants and/or fusions thereof,

(g) EAAAKEAAAKEAAAKA [ SEQ ID NO:117], or variants and/or fusions thereof,

(h) SGSFVAAWTLKAAAGGS [ SEQ ID NO:118] or variants and/or fusions thereof, and

(i) SGSGSGGGGGGS [ SEQ ID NO:119] or variants and/or fusions thereof.

Linker AKFVAAWTLKAAA [ SEQ ID NO:115], also known as a pan HLA DR-binding epitope (PADRE), is a polypeptide capable of activating antigen-specific-CD4+ T cells, which is proposed as a carrier epitope suitable for the development of synthetic and recombinant vaccines, such as a "linear PADRE T helper epitope and carbohydrate B cell epitope conjugate, induces a specific high titer IgG antibody response 10.4049/jimmnol.164.3.1625, the disclosure of which is incorporated herein by reference. The linkers GGGGSLVPRGSGGGGS [ SEQ ID NO:116] and EAAAKEAAAKEAAAKA [ SEQ ID NO:117] are rigid linkers that cannot fold into an alpha helix.

Alternatively or additionally, the nanoparticle domain is:

(a) Upstream of (a),

(b) At the N-terminus of the polypeptide,

(c) Downstream of (b), or

(d) At the C-terminus of the polypeptide.

In another aspect, a designed and novel polypeptide monomer (and nucleic acid molecules encoding the same) capable of self-assembly into nanoparticles (i.e., protein nanoparticles) is provided. Host cells, vectors, or constructs, and methods of making or using such polypeptide monomers and protein nanoparticles are also provided. The invention also relates to Nanoparticles (NPs) whose surface structure comprises or consists of at least one such polypeptide monomer, and optionally carries one or more antigenic molecules.

The polypeptide monomers of the invention are mutated compared to their wild-type counterpart (i.e. E.coli bacterial ferritin [ SEQ ID NO:16 ]), and may thus have increased stability, e.g. improved heat stability or folding stability compared to their wild-type counterpart, which may thus form self-assembled nanoparticles, improved heat stability or folding stability compared to their wild-type counterpart.

By "increased stability" is meant that the molecule has a lower unfolding ratio, reduces misfolding, reduces protein domain movement, reduces protein domain rearrangement, increases half-life (in vitro or in vivo), increases shelf life, increases melting temperature (Tm) (meaning at least one increase in melting temperature if there are two or more molecules), lower folding free energy value (kcal/mol), lower binding free energy value (as in the case where one subunit binds to another subunit to form a macromolecule), or a combination thereof; under comparable or identical conditions, compared to a control molecule or its wild-type counterpart (e.g., temperature and/or pH). For clarity, a "control molecule" or its "wild-type counterpart" refers to a molecule that does not contain one or more stabilizing mutations if the stability of the molecule is increased by one or more mutations ("stabilizing mutations", such as one or more amino acid mutations). For purposes of the present invention, a monomer or nanoparticle may be described as having greater stability (e.g., increased thermostability and/or increased folding stability and/or increased binding stability) as compared to the wild-type counterpart molecule under comparable (or identical) conditions. As used herein, "conditions" include experimental and physiological conditions. See, for example, U.S. Pub. No.2011/0229507; clapp et al 20111 J.Pharm.Sci.100 (2): 388-401, discuss increasing stability by adjuvants and assessing antigen stability under altered pH, hydration and temperature conditions; and Rossi et al, 2016Infect. Immun.84 (6): 1735-1742. For clarity, "stability" may be designated as "thermal stability", which means the resistance of a molecule to folding at a particular temperature, typically expressed in the art by the melting temperature of the molecule, specifically by an increase in the melting temperature of the molecule (more than one melting temperature is possible for oligomeric proteins such as dimers or trimers), see Kumar et al 2000prot. Eng. Des. Sel. "Factors enhancing protein thermostability"13 (3): 179-191; and Miotto et al 2018bioRxiv doi 10.1101/354266"Insights on protein thermal stability:a graph representation of molecule interactions"). Depending on the context requirements, the thermal stability of two or more molecules (e.g. two or more modified molecules, each comprising one or more stabilizing mutations) may be compared, one of which may be said to be more thermally stable than the other (i.e. have an increased or increased thermal stability compared to the other). Stability, particularly thermostability, may be provided by a delta stability (dstabilityor dS) scoring method, which is the difference between the calculated relative thermostability of a determined on-line mutant protein and that of a control group or its wild-type counterpart (i.e., a non-stable mutant). Methods of determining dstatability are known (WO 2020/079586 (PCT/IB 2019/058777), MALITO, etc.), and may include the use of tools such as Molecular Operating Environment (MOE) software (REF: molecular Operating Environment (MOE) software; chemical Computing Group inc., available at WorldWideWeb (www). Chemcomp. dS is measured in kilocalories per mole. A lower dS value indicates higher protein stability, while a higher dS value indicates lower protein stability. It is clear that the mutant polypeptides of the invention have a higher relative thermostability (in kcal/mol) than the non-mutant polypeptides under the same or comparable experimental conditions. It can be further stated that the mutant polypeptides of the invention have lower dS values than non-mutant polypeptides under the same or comparable experimental conditions. It will be appreciated from the present invention that mutant polypeptides having lower dS values are more stable than non-mutant polypeptides under the same or comparable experimental conditions. The improvement in stability can be assessed by Differential Scanning Calorimetry (DSC), as discussed in Bruylants et al 2005Curr. Med. Chem.12:2011-2020and Calorimetry Sciences Corporation's"Characterizing Protein stability by DSC" (Life Sciences Application Note, doc. No.20211021306 Febrary 2006), or by Differential Scanning Fluorometry (DSF). An increase in (thermal) stability, as assessed by DSC or DSF, can be characterized by an increase in the thermal transition midpoint (Tm) of at least about 2 ℃. See, for example, thomas et al, 2013hum. Vaccine. Immunother.9 (4): 744-752. A "significant" increase or enhancement of thermal stability is defined as an increase in the calculated Tm of the complex of at least 5 ℃ (calculated for example by the protocol provided in example 4.7 of WO 2020/079586 (PCT/IB 2019/058777) MALITO et al). For clarity, "stability" herein may be designated as "folding stability," which refers to the free energy of folding of a molecule (reported in kilocalories per mole (kcal/mol)), which may be determined using a variety of known techniques (see, e.g., zhang et al 2012bioinformatics 28 (5): 664-671). Depending on the context requirements, the folding stability of two or more molecules can be compared, so that one molecule is more stable than the other because of its lower folding free energy value (units: kcal/mol). It is clearly pointed out that the monomers or nanoparticles of the invention have a higher/increased folding stability under the same or comparable conditions (experimental conditions) compared to the control molecule or its wild-type counterpart. A "significant" increase or enhancement of folding stability is defined as a folding free energy change value at least 100 kcal/mole less than the folding free energy change value (in kcal/mole) of a comparable or identical condition of the control molecule.

In one embodiment, the polypeptide monomers of the invention comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ id no:16, e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and have one or more mutations from: glycine (G) at a position aligned with residue 34 of SEQ ID No. 16 (T34G mutation), aspartic acid (D) at a position aligned with residue 70 of SEQ ID No. 16 (N70D mutation), isoleucine (I) at a position aligned with residue 72 of SEQ ID No. 16 (V72I mutation) and alanine (a) at a position aligned with residue 124 of SEQ ID No. 16 (S124A mutation);

in a preferred embodiment, the polypeptide monomer comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence SEQ ID NO:16, e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and has glycine (G) at a position aligned with residue 34 of SEQ ID NO:16 (T34G mutation), aspartic acid (D) at a position aligned with residue 70 of SEQ ID NO:16 (N70D mutation), isoleucine (I) at a position aligned with residue 72 of SEQ ID NO:16 (V72I mutation) and alanine (A) at a position aligned with residue 124 of SEQ ID NO:16 (S124A mutation). In some embodiments, the polypeptide monomer comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence SEQ ID NO:149, e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the polypeptide monomer comprises the amino acid sequence of SEQ ID NO: 149.

In one embodiment, the polypeptide monomer comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence SEQ ID NO. 16, e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and has glycine (G) at a position aligned with residue 34 of SEQ ID NO. 16 (T34G mutation), isoleucine (I) at a position aligned with residue 72 of SEQ ID NO. 16 (V72I mutation), and alanine (A) at a position aligned with residue 124 of SEQ ID NO. 16 (S124A mutation). In some embodiments, the polypeptide monomer comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence SEQ ID NO:150, e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the polypeptide monomer comprises the amino acid sequence of SEQ ID NO. 150.

In one embodiment, the polypeptide monomer comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence SEQ ID NO. 16, e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and has glycine (G) at a position aligned with residue 34 of SEQ ID NO. 16 (T34G mutation), and alanine (A) at a position aligned with residue 124 of SEQ ID NO. 16 (S124A mutation). In some embodiments, the polypeptide monomer comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence SEQ ID NO. 151, e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the polypeptide monomer comprises the amino acid sequence of SEQ ID NO. 151.

In one embodiment, the polypeptide monomer comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence SEQ ID NO. 16, e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and has glycine (G) at a position aligned with residue 34 of SEQ ID NO. 16 (T34G mutation). In some embodiments, the polypeptide monomer comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence SEQ ID NO 152, e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the polypeptide monomer comprises the amino acid sequence of SEQ ID NO. 152.

The design and novel polypeptide monomers of the present invention are capable of self-assembling into approximately spherical nanoparticles (e.g., having an outer surface structure with a diameter of about 5 nm to about 30 nm, preferably about 15 to about 20 nm). Thus, the polypeptide monomers of the invention can be used to provide self-assembled protein nanoparticles, and optionally, wherein the self-assembled protein nanoparticles carry (e.g., display) at least one antigen molecule, at least one immunostimulatory agent molecule, or at least one antigen molecule and at least one immunostimulatory agent molecule. In one embodiment, the nanoparticle of the invention (e.g., the approximately spherical nanoparticle of the invention) consists of 24 monomer subunits (e.g., wherein at least one monomer subunit is the polypeptide monomer of the invention) and has an octahedral symmetric basic geometry.

Nanoparticles (naturally occurring and recombinant nanoparticles, e.g., computationally engineered nanoparticles), methods of making them, and their use as scaffolds (or "carriers") for, e.g., one or more antigens or immunostimulants (i.e., "pharmaceutically acceptable nanoparticles") are known in the art.

As recognized in the art (see, e.g., ueda et al 2020ehife 9:e57659;Pan et al.2020Adv.Mater.32:2002940), the protein nanoparticles of the present invention may be used as a "scaffold" by which to carry (i.e., attach, join, fuse, bond, or connect to the outer surface structure of the nanoparticle) an antigen, an immunostimulant, multiple copies of the same antigen, multiple copies of the same immunostimulant, a mixture of two or more antigens (e.g., a mixture of two, three, four, or five antigens; i.e., an antigen bivalent, trivalent, tetravalent, or pentavalent), a mixture of two or more immunostimulants (e.g., two, three, four, or pentavalent; i.e., an immunostimulant bivalent, trivalent, tetravalent, or pentavalent), or a mixture of one or more antigens and one or more immunostimulants.

In certain embodiments, the self-assembly of the polypeptide monomers places their N-terminus on the exterior/outer surface of the nanoparticle and their C-terminus on the interior/core/inner surface of the nanoparticle. Thus, the antigen or immunostimulant attached to the N-terminus of the polypeptide monomer is displayed on the outer surface of the assembled nanoparticle. In other embodiments, the self-assembly of the polypeptide monomers places their C-terminus on the exterior/outer surface of the nanoparticle and their N-terminus on the interior/core/inner surface of the nanoparticle. Thus, the antigen or immunostimulant associated with the C-terminus of the polypeptide monomer is displayed on the outer surface of the assembled nanoparticle. In certain other embodiments, the antigen or immunostimulant is attached to the N-terminus of the polypeptide monomer and the antigen or immunostimulant is attached to the C-terminus of the polypeptide monomer (the same or different antigen and/or immunostimulant) such that the antigen or immunostimulant is displayed on the outer surface and carried on the inner surface of the assembled nanoparticle.

Thus, one embodiment of the invention provides a nanoparticle carrying one or more molecules (e.g., wherein the molecule is/is heterologous as compared to one or more (e.g., all) of the nanoparticle monomers), and optionally wherein the one or more molecules are/are displayed on the outer surface of the nanoparticle. When the one or more display molecules (e.g., antigen and/or immunostimulant) are proteins (e.g., all are proteins), they may be expressed as part of a polypeptide monomer (i.e., as a fusion protein monomer), such that self-assembly of the nanoparticle results in display of the protein on the nanoparticle outer surface. In addition, the protein display molecules may be attached to the assembled nanoparticle, for example, by chemical or biological conjugation as discussed herein and known in the art. In another embodiment of the invention, the display molecule is a polysaccharide or oligosaccharide (e.g., bacterial capsular polysaccharide); the saccharide may be linked to the nanoparticle to provide a "glycoconjugate". See Polonskaya et al 2017J.Clin. Invest.127 (4): 1492-1504; pan et al 2020adv. Mater.32:2002940.

In one embodiment, the antigen is a polypeptide having an amino acid sequence comprising or consisting of: (a) FimH; or a variant, fragment and/or fusion of FimH, and (b) a donor strand complementary amino acid sequence, wherein (b) is downstream of (a), or as described herein. In one embodiment, the antigen comprises or consists of the amino acid sequence: and the amino acid sequence SEQ ID NO:124 has at least 80% sequence identity, e.g., an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In one embodiment, the antigen comprises SEQ ID NO:124 or a sequence consisting of SEQ ID NO: 124.

In one embodiment, the amino acid sequence of the nanoparticle has at least 80% sequence identity to amino acid sequence SEQ ID NO 130 or 153, e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In one embodiment, the nanoparticle comprises or consists of the amino acid sequence of SEQ ID NO. 130 or 153.

Thus, certain embodiments of the invention provide polypeptides (i.e., polypeptide monomers) and nucleic acid molecules encoding such polypeptides that are capable of self-assembly into nanoparticles. The amino acid sequences herein may comprise or further comprise a tag (e.g., a purification tag such as a histidine (e.g., a 6xHis tag), an enterokinase tag, or a myc tag), as well as a linker between the polypeptide monomer and one or more molecules (e.g., antigens) carried by the nanoparticle. Furthermore, the nucleic acid sequences herein may encode amino acid sequences comprising tags and/or linkers.

Alternatively or additionally, the polypeptide comprises a phenylalanine (Phe, F) residue at the N-terminus of the FimH polypeptide. Alternatively or additionally, when the polypeptide comprises a nanostructure domain at the C-terminus or the N-terminus, the polypeptide comprises a phenylalanine (Phe, F) or aspartic acid (Asp, D) residue at the N-terminus of the mature polypeptide, i.e. after cleavage or removal of the leader sequence, if present. The presence of an aspartic acid (Asp, D) residue at the N-terminus of a mature polypeptide comprising a nanoparticle domain at the C-terminus or N-terminus is associated with improved secretion of the polypeptide when expressed by a mammalian host cell.

Alternatively or additionally, the polypeptide comprises or consists of an amino acid sequence corresponding to seq id no:

(a) SEQ ID NO. 20, or a variant and/or fragment thereof,

(b) SEQ ID NO. 21, or a variant and/or fragment thereof,

(c) SEQ ID NO. 22, or a variant and/or fragment thereof,

(d) SEQ ID NO. 23, or a variant and/or fragment thereof,

(e) 24, or a variant and/or fragment thereof,

(f) 25, or a variant and/or fragment thereof,

(g) 26 or a variant and/or fragment thereof,

(h) SEQ ID NO. 27, or a variant and/or fragment thereof,

(i) 28, or a variant and/or fragment thereof,

(j) 29, or a variant and/or fragment thereof,

(k) 30, or a variant and/or fragment thereof,

(l) 31 of SEQ ID NO, or a variant and/or fragment thereof,

(m) SEQ ID NO 32, or a variant and/or fragment thereof,

(n) SEQ ID NO. 33, or a variant and/or fragment thereof,

(o) SEQ ID NO 34, or a variant and/or fragment thereof,

(p) SEQ ID NO. 35, or variants and/or fragments thereof,

(q) SEQ ID NO. 36, or a variant and/or fragment thereof,

(r) SEQ ID NO. 37, or a variant and/or fragment thereof,

(s) SEQ ID NO. 38, or a variant and/or fragment thereof,

(t) SEQ ID NO 39, or a variant and/or fragment thereof,

(u) SEQ ID NO. 40, or a variant and/or fragment thereof,

(v) SEQ ID NO. 41, or a variant and/or fragment thereof,

(w) SEQ ID NO. 42, or a variant and/or fragment thereof,

(x) 43, or a variant and/or fragment thereof,

(y) SEQ ID NO 44, or a variant and/or fragment thereof,

(z) SEQ ID NO. 79, or variants and/or fragments thereof,

(aa) SEQ ID NO 80, or a variant and/or fragment thereof,

(bb) SEQ ID NO 81, or variants and/or fragments thereof,

(cc) SEQ ID NO. 82, or a variant and/or fragment thereof,

(dd) SEQ ID NO 83, or variants and/or fragments thereof,

(ee) SEQ ID NO. 84, or a variant and/or fragment thereof,

(ff) SEQ ID NO. 85, or a variant and/or fragment thereof,

(gg) SEQ ID NO 86, or a variant and/or fragment thereof,

(hh) SEQ ID NO 87, or a variant and/or fragment thereof,

(ii) 88, or a variant and/or fragment thereof,

(jj) SEQ ID NO 89, or a variant and/or fragment thereof,

(kk) any one of SEQ ID NOS 120-124, 129-143 and 153 or variants and/or fragments thereof.

In a preferred embodiment, the polypeptide comprises or consists of an amino acid sequence corresponding to SEQ ID NO. 123 or SEQ ID NO. 124. In one embodiment, the polypeptide comprises or consists of: an amino acid sequence having at least 70% sequence identity to SEQ ID NO. 123 or SEQ ID NO. 124, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 123 or SEQ ID NO. 124.

Alternatively or additionally, the mannose binding of the polypeptide is at least 20% lower, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% lower than the mannose binding of natural FimH complexed with natural FimHC (FimHC complex).

Mannose binding may be determined using any suitable means known in the art, for example, surface plasmon resonance may be used to verify binding, binding specificity and binding constants of FimH constructs to Man-BSA and Glc-BSA (negative controls), for example, see Rabani et al, 2018, 'Conformational switch of the bacterial adhesin FimH in the absence of the regulatory domain: engineering aminimalistic allosteric system' j.biol.chem.,293 (5): 1835-1849, incorporated herein by reference.

By 'native FimH' we mean or include wild-type FimH, particularly from which domain (a) of the polypeptide of the invention is derived (optionally, with removal of the native N-terminal secretion sequence). Alternatively or additionally, we refer to or include FimH of E.coli J96 (e.g., SEQ ID NO:1 or SEQ ID NO: 2), fimH of E.coli UPEC 536 (e.g., SEQ ID NO:100 or SEQ ID NO: 101), fimH of E.coli CFT073 (e.g., SEQ ID NO:102 or SEQ ID NO: 103), fimH of E.coli 789 (e.g., SEQ ID NO:104 or SEQ ID NO: 105), fimH of E.coli IHE3034 (e.g., SEQ ID NO:106 or SEQ ID NO: 107). In particular, we include FimH in a high affinity conformation, relaxed (R) state (see above).

By "native FimC" we mean or include wild-type FimC (optionally, with removal of the native N-terminal secretion sequence). In addition, we refer to or include FimC of e.coli J96, fimC of UPEC 536, fimC of e.coli CFT073, fimC of e.coli 789, fimC of e.coli IHE 3034.

By 'FimH complexed with natural FimHC' and 'FimHC complexes' we mean or include that, in the manner and/or conditions taught in this example section, in the manner and/or conditions taught by a) d.choudhury, X-ray structure of the FimC-FimH challenge-adhesin complex from uropathogenic Escherichia coll.science 285,1061-1066 (1999), (b) c. -s.hunt, structural basis of tropism of Escherichia coli to the bladder during urinary tract in effect.mol.44, 903-915 (2002), (c) i.le Trong, structural basis for mechanical force regulation of the adhesin FimH via finger trap-like β sheet et twisting.cell 141,645-655 (2010), (d) g.phan, crystal structure of the FimD usher bound to its cognate FimC-FimH substrate.nature 474,49-53 (2011), or (e) s.geibel, structural and energetic basis of folded-protein transport by the FimD us.nature 496,243-246 (2013), the binding of FimH to FimH is seen in the periplasm of bacteria naturally expressing FimH and FimH.

Alternatively or additionally, the polypeptide has at least 20% greater, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400% or 500% greater, immunogenicity than native FimH complexed with native FimC (in particular, we include FimH in a high affinity conformation, relaxed (R) state (see above)). Immunogenicity may be determined by any suitable means known in the art, for example, ELISA or Luminex (see examples section).

Alternatively or additionally, the autopolymerization induced by the polypeptide is at least 20% lower than native FimH, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% lower.

By 'polypeptide-induced autopolymerization is at least X%' (where 'X' is a number between 20 and 100) lower than that of native FimH, we mean or include polypeptides that when expressed by bacteria other than native FimH, induce bacterial autopolymerization at least X% lower than that of an equivalent bacteria expressing equivalent native FimH. By 'equivalent native FimH' we mean or include native FimH of the bacteria used in the assay, native FimH from which the polypeptides of the invention are derived and/or native FimH having the highest sequence identity to the polypeptides of the invention. The automated aggregation may be determined using any suitable method known in the art, but in one embodiment, the method used is schembi, christiansen and Klemm,2001, 'FimH-mediated autoaggregation of Escherichia coli' Molecular Microbiology,41 (6), 1419-1430, incorporated herein by reference; or Thomas et al, 2002, 'Bacterial adhesion to target cells enhanced by shear force' Cell,109 (7): 913-23, incorporated herein by reference; or Hartman et al 2012, 'Inhibition of bacterial adhesion to live human cells: activity and cytotoxicity of synthetic mannosides' FEBS Letters,586 (10): 1459-1465, incorporated herein by reference; or Falk et al, 1995, 'Chapter 9:Bacterial Adhesion and Colonization Assays'Meth.Cell,Biol, 45:165-192, which is incorporated herein by reference; or zanabani et al, 2016, 'a novel high-throughput assay to quantify the vaccine-induced inhibition of Bordetella pertussis adhesion to airway epithelia' BMC microbiol, 16:a215, which is incorporated herein by reference. Alternatively or additionally, bacterial adhesion (in short) was measured using the BAI assay, as follows: UPEC strains engineered to express mCherry fluorescent markers were incubated with SV-HUC-1 monolayers in 96-well plates for 30 min with specific serum or positive/negative controls for FimH derivatives. After adhesion, cells are extensively washed to remove unbound bacteria and fixed with formaldehyde. Finally, an automated high content screening microscope (Opera Phenix) was used to record specific fluorescent signals associated with adherent bacteria and quantified with Harmony software.

Alternatively or additionally, the polypeptide is capable of inhibiting bacterial adhesion by at least 20%, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

By 'inhibit bacterial adhesion' we mean or include an indicator of bacterial adhesion measurement by bacterial movement as described above or by a bacterial adhesion test (e.g. the BAI test) and/or in the examples section.

Alternatively or additionally, the polypeptide is capable of inhibiting hemagglutination of guinea pig erythrocytes by at least 2-fold, e.g., at least 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold.

By 'inhibit hemagglutination' we mean or include inhibition of hemagglutination as measured by Hulgren et al, effect Immun 1986,54,613-620and Jarvis C et al,ChemMedChem 2016,11,367-373 and/or the hemagglutination inhibition assay (HAI) described in the examples section.

Alternatively or additionally, the polypeptide is soluble, we mean or include at least 50% of the polypeptide w/w (e.g., present in the mixture and/or expressed by the cell) is in soluble form, e.g., at least 60%, 70%, 80%, 90%, 95% or 100% of the polypeptide is in soluble form.

In a second aspect the invention provides a nucleic acid, e.g. DNA or RNA, encoding a polypeptide according to the first aspect.

Alternatively or additionally, the nucleic acid has been codon optimized for expression in selected prokaryotic or eukaryotic cells, e.g., yeast cells (e.g., saccharomyces cerevisiae, pichia), insect cells (e.g., spodoptera frugiperda Sf21 cells, or Sf9 cells), or mammalian cells (Expi 293, expi293GNTI, chinese Hamster Ovary (CHO) cells, and human embryonic kidney 293 cells (HEK 293)). By "codon optimization" is meant modification in terms of codon usage to increase translation efficiency and/or half-life of the nucleic acid. Codon usage/optimization tables for many organisms are well known and publicly available (as provided by Athey et al 2017BMC Bioinf.18:391). The code optimization may be performed using any suitable means known in the art, for example, the method of the GeneArt operation.

Alternatively or additionally, the nucleic acid comprises or consists of a nucleic acid sequence corresponding to:

(1) SEQ ID NO. 45, or a variant and/or fragment thereof,

(2) SEQ ID NO. 46, or a variant and/or fragment thereof,

(3) SEQ ID NO. 47, or a variant and/or fragment thereof,

(4) SEQ ID NO. 48, or variants and/or fragments thereof,

(5) 49, or a variant and/or fragment thereof,

(6) SEQ ID NO. 50, or a variant and/or fragment thereof,

(7) SEQ ID NO. 51, or a variant and/or fragment thereof,

(8) SEQ ID NO. 52, or a variant and/or fragment thereof,

(9) 53, or variants and/or fragments thereof,

(10) 54, or a variant and/or fragment thereof,

(11) SEQ ID NO. 55, or a variant and/or fragment thereof,

(12) SEQ ID NO. 56, or a variant and/or fragment thereof,

(13) SEQ ID NO. 57, or a variant and/or fragment thereof,

(14) SEQ ID NO. 58, or a variant and/or fragment thereof,

(15) SEQ ID NO. 59, or a variant and/or fragment thereof,

(16) SEQ ID NO. 60, or a variant and/or fragment thereof,

(17) SEQ ID NO. 61, or a variant and/or fragment thereof,

(18) 62, or a variant and/or fragment thereof,

(19) 63, or a variant and/or fragment thereof,

(20) SEQ ID NO. 64, or a variant and/or fragment thereof,

(21) 65, or a variant and/or fragment thereof,

(22) 66 or a variant and/or fragment thereof,

(23) 67, or a variant and/or fragment thereof,

(24) 68 of SEQ ID NO, or a variant and/or fragment thereof,

(25) 69, or a variant and/or fragment thereof,

(26) SEQ ID NO. 70, or variants and/or fragments thereof,

(27) 71 or a variant and/or fragment thereof,

(28) SEQ ID NO. 72, or a variant and/or fragment thereof,

(29) 73, or a variant and/or fragment thereof,

(30) 74 of SEQ ID NO, or variants and/or fragments thereof,

(31) 75 of SEQ ID NO, or variants and/or fragments thereof,

(32) SEQ ID NO. 76, or a variant and/or fragment thereof,

(33) 77 or a variant and/or fragment thereof,

(34) 90, or a variant and/or fragment thereof,

(35) 91 of SEQ ID NO, or variants and/or fragments thereof,

(36) 92, or a variant and/or fragment thereof,

(37) 93, or a variant and/or fragment thereof,

(38) 94 of SEQ ID NO. 94, or variants and/or fragments thereof,

(39) 95 of SEQ ID NO, or variants and/or fragments thereof,

(40) 96, or variants and/or fragments thereof,

(41) SEQ ID NO. 97, or a variant and/or fragment thereof,

(42) 98, or variants and/or fragments thereof, and

(43) SEQ ID NO 99, or variants and/or fragments thereof.

The skilled artisan will immediately appreciate that when the nucleic acid of the invention is RNA, T is replaced with U in the nucleic acid sequence of the invention (e.g., SEQ ID NOs: 45-99).

In a third aspect the invention provides a vector comprising the nucleic acid of the second aspect. Alternatively or additionally, the vector is a plasmid, e.g., an expression plasmid. Alternatively or additionally, the plasmid is selected from the following: pCDNA3.1 (Life Technologies), pCDNA3.4 (Life Technologies), pFUSE, pBROAD, pSEC, pCMV, pDSG-IBA and pHEK293 Ultra. Alternatively or additionally, the plasmid is suitable for expression in a bacterial host cell and is selected from pACYCDuet-1, pTrcHis2A, pET, pET15TEV, pET22b+, pET303/CT-HIS, PET303/CT, pBAD/Myc-His A, pET303, pET24b (+), and the like.

Alternatively or additionally, the vector is a viral vector, e.g., an RNA viral vector. Alternatively or additionally, the viral vector is selected from the group consisting of an adenovirus vector and CHAD.

In a fourth aspect the invention provides a cell, such as a host cell, comprising a nucleic acid of the second aspect or a vector of the fourth aspect.

Suitable mammalian host cells are known in the art. Alternatively or additionally, the cell does not have N-acetylglucosamine transferase I (GnTI) activity. Alternatively or additionally, the cell is selected from the group consisting of an Expi293, an Expi293GNTI (Life Technologies), a Chinese Hamster Ovary (CHO) cell, a NIH-3T3 cell, a 293-T cell, a Vero cell, a HeLa cell, a perc.6 cell (ECACC storage 96022940), a Hep G2 cell, a MRC-5 (ATCC CCL-171), a WI-38 (ATCC CCL-75), a fetal rhesus lung cell (ATCC CL-160), a Madin-Darby bovine kidney ("MDBK") cell, a Madin-Darby canine kidney ("MDCK") cell (e.g., MDCK (NBL 2), ATCC CCL34, or MDCK 33016,DSM ACC 2219), a small hamster kidney (BHK) cell, such as BHK21-F, HKCC cell, human embryonic kidney 293 cell (HEK 293), and the like.

Suitable bacterial host cells are known in the art. Exemplary bacterial host cells include any of the following and derivatives thereof: coli strains BL21 (DE 3), HMS174 (DE 3), origami 2 (DE 3), BL21DE3T1r or T7shuffle expression.

In a fifth aspect the present invention provides a method of producing a polypeptide as defined in the first aspect by expressing a protein in a cell as defined in the fourth aspect.

In a sixth aspect the invention provides a vaccine comprising a polypeptide as defined in the first aspect, a nucleic acid as defined in the second aspect and/or a vector as defined in the third aspect. Alternatively or additionally, the vaccine comprises an adjuvant.

In one embodiment, the vaccine of the invention comprises a polypeptide as defined in the first aspect and an adjuvant comprising any one of the following: 3D-MPL, QS21 and liposomes, for example liposomes comprising cholesterol. In one embodiment, the vaccine of the invention comprises a polypeptide as defined in the first aspect and an adjuvant comprising 3D-MPL, QS21 and a liposome comprising cholesterol.

The inventors have surprisingly found that a vaccine comprising an adjuvant comprising 3D-MPL, QS21 and liposomes (containing cholesterol), such AS01 adjuvant, may elicit a better immune response. By "improved immune response" we mean or include that the serum and/or urine levels of immunoglobulin G (IgG) are higher in animals such as mice immunized with the vaccine than in animals such as mice immunized with a reference or vaccine. By "increase in IgG levels in serum and/or urine" we mean or include at least a 2-fold increase in cells, e.g., at least a 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, or 50-fold increase. The reference or control vaccine does not include adjuvants comprising 3D-MPL, QS21 and liposomes (containing cholesterol); for example, the reference or control vaccine comprises a PHAD adjuvant.

The inventors have also surprisingly found that a vaccine comprising a polypeptide AS defined in the first aspect and an adjuvant comprising 3D-MPL, QS21 and liposomes (containing cholesterol), such AS an AS01 adjuvant, is capable of eliciting a protective immune response after one or two doses.

An immune composition (e.g., vaccine) will be pharmaceutically acceptable. They typically include components other than antigen, for example, they typically include one or more pharmaceutical carriers, excipients, and/or adjuvants. For a thorough discussion of carriers and excipients, see Current Protocols in Molecular Biology (f.m. ausubel et al, eds., 1987) support 30, incorporated herein by reference. An exhaustive discussion of Vaccine adjuvants is found in Vaccine design: the Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press1995 (ISBN 0-306-44867-X); and Vaccine Adjuvants: preparation Methods and Research Protocols (Volume 42ofMethods in Molecular Medicine series), ISBN:1-59259-083-7.Ed.O' Hagan, incorporated herein by reference.

The compositions will generally be administered to the mammal in the form of an aqueous formulation. However, prior to application, the composition may be in a non-aqueous form. For example, while some vaccines are produced in aqueous form and then filled, dispensed and administered in aqueous form, other vaccines are lyophilized during production and reconstituted into aqueous form at the time of use. Thus, the compositions of the present invention may be dry, such as lyophilized, formulations. The composition may include a preservative such as thimerosal or 2-phenoxyethanol. Preferably, however, the vaccine should be substantially free (i.e. less than 5 μg/ml) of mercury species, such as free of thiomersal. Mercury-free vaccines are more preferred. Preservative-free vaccines are particularly preferred. To improve thermal stability, the composition may include a temperature protectant.

For controlling the tension, physiological salts, such as sodium salts, are preferably included. Sodium chloride (NaCl) is preferred, and may be present in an amount of between 1 and 20mg/ml, for example about 10.+ -. 2mg/ml NaCl. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride, and the like.

The osmolality of the composition is generally between 200mOsm/kg and 400mOsm/kg, preferably between 240 and 360mOsm/kg, more preferably in the range 290 to 310 mOsm/kg.

The composition may comprise one or more buffers. Typical buffers include: phosphate buffer; tris buffer; a borate buffer; succinate buffer; histidine buffer (especially containing aluminium hydroxide adjuvant); or citrate buffer. Buffers are typically in the range of 5-20 mM.

The pH of the composition is typically between 5.0 and 8.1, more typically between 6.0 and 8.0, for example between 6.5 and 7.5, or between 7.0 and 7.8.

The composition is preferably sterile. The composition is preferably non-pyrogenic, e.g. <1EU (endotoxin unit, a standard measure) per dose, preferably <0.1EU per dose. The composition is preferably gluten-free.

The composition may comprise materials for a single immunization, or may comprise materials for multiple immunizations (i.e., a "multi-dose" kit). Preservatives are preferably included in the multi-dose arrangement. As an alternative (or in addition) to adding a preservative to the multi-dose composition, the composition may be contained in a container with a sterile adapter to remove the material.

Human vaccines are typically administered at a dose of about 0.5 milliliters, although half doses (i.e., about 0.25 ml) may be used for children.

The immunogenic compositions of the invention may also comprise one or more immunomodulators. Preferably, the one or more immunomodulators comprise one or more adjuvants.

Adjuvant

The vaccines and immunogenic compositions of the present invention may comprise adjuvants in addition to the antigen. Adjuvants are used in vaccines to enhance and modulate the immune response to antigens. The adjuvants described herein may bind to any of the antigens described herein.

The adjuvant may be any adjuvant known to the skilled artisan, but the adjuvants include, but are not limited to, oil-in-water emulsions (e.g., MF59 or AS 03), liposomes, saponins, TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR6 agonists, TLR7 agonists, TLR8 agonists, TLR9 agonists, aluminum salts, nanoparticles, microparticles, immunostimulatory complexes (ISCOMS), calcium fluoride, and organic compound composites, or combinations thereof.

Oil-in-water emulsion

In one embodiment of the invention, there is provided a vaccine or immunogenic composition for use in the invention comprising an oil-in-water emulsion. The oil-in-water emulsion of the present invention comprises a metabolizable oil and an emulsifier. In order for any oil-in-water composition to be suitable for human use, the oil phase of the emulsion system must include a metabolizable oil. The meaning of the term metabolisable oil is well known in the art. Metabolizable is defined as "capable of being converted by metabolism" (Dorland's Illustrated Medical Dictionary, w.b. sanders Company,25th edition,1974). One particularly suitable metabolisable oil is squalene. Squalene (2, 6,10,15,19, 23-hexamethyl-2, 6,10,14,18, 22-tetracohexane) is an unsaturated oil, present in a major amount in shark liver oil, and less in olive oil, wheat germ oil, rice bran oil and yeast, is a particularly preferred oil for use in the oil-in-water emulsions of the present invention. Squalene is a metabolisable oil because it is an intermediate in cholesterol biosynthesis (Merck index,10th Edition,entry no.8619). In some embodiments, wherein the vaccine or immunogenic composition of the invention comprises an oil-in-water emulsion, the metabolisable oil is present in the vaccine or immunogenic composition in an amount of 0.5% to 10% (v/v) of the total volume of the composition. The oil-in-water emulsion further comprises an emulsifier. The emulsifier may suitably be polyoxyethylene sorbitol monooleate (POLYSORBATE 80). Furthermore, the emulsifier is suitably present in the vaccine or immune composition in an amount of 0.125 to 4% (v/v) of the total volume of the composition. The oil-in-water emulsion may optionally comprise tocopherol. Tocopherols are well known in the art and are described in EP 0382271B 1. Suitably, the tocopherol may be alpha-tocopherol or a derivative thereof, such as alpha-tocopherol succinate (also known as vitamin E succinate). The tocopherol is suitably present in the adjuvant composition in an amount of 0.25% to 10% (v/v) of the total volume of the immunogenic composition. The oil-in-water emulsion also optionally comprises sorbitol trioleate (SPAN 85).

Methods of producing oil-in-water emulsions are well known to those skilled in the art. Typically, the process comprises mixing the oil phase (optionally comprising a tocopherol) with a surfactant such as a PBS/TWEEN80 solution, and then homogenizing with a homogenizer; it will be clear to those skilled in the art that the method involving passing the mixture through the syringe needle twice is suitable for homogenizing small amounts of liquid. Likewise, the person skilled in the art can adjust the emulsification process in a microfluidic device (e.g. an M110S microfluidic device, with a maximum pressure input of 6 bar (output pressure of about 850 bar) up to 50 passes for 2 minutes) to produce smaller or larger volumes of emulsion. This adjustment can be achieved by routine experimentation, including measuring the emulsion produced until a formulation of oil droplets having the desired diameter is obtained.

In an oil-in-water emulsion, the oil and emulsifier should be in one aqueous carrier. The aqueous carrier may be, for example, phosphate buffered saline or citrate. In particular, the oil-in-water emulsion system used in the present invention has a small oil droplet size on the submicron scale. Suitably, the oil droplet size will be in the range 120 to 750 nm, in particular a size of 120 to 600 nm in diameter. Even more particularly, the oil-in-water emulsion comprises oil droplets wherein at least 70% of the intensity is less than 500 nanometers in diameter, more particularly at least 80% of the intensity is less than 300 nanometers in diameter, more particularly at least 90% of the intensity is in the range of 120 to 200 nanometers.

According to the invention the size of the oil droplets, i.e. the diameter, is given by the intensity. There are several methods by which the diameter of the oil droplet size can be determined by intensity. The intensity is measured by using a meter, suitably by dynamic light scattering, such as Malvern Zetasizer 4000 or preferably Malvern Zetasizer 3000HS. The first possibility is to determine the Z-average diameter ZAD by dynamic light scattering (PCS-photon correlation spectroscopy); this method also gives a polydispersity index (PDI), both ZAD and PDI can be calculated using a cumulative algorithm. These values do not require knowledge of the refractive index of the particles. The second method is to calculate the diameter of the oil droplets by determining the overall particle size distribution by another algorithm, either Contin, NNLS, or an automatic "Malvern" algorithm (the default algorithm provided by the applicator). In most cases, since the refractive index of the particles of the composite composition is unknown, only the intensity distribution and, if necessary, the average value of the intensity resulting from this distribution are considered.

ISCOMs

In some embodiments of the invention, vaccines comprising ISCOMs or immunogenic compositions of the invention are provided. ISCOMs are well known in the art (see Kersten & Crommelin,1995,Biochimica et Biophysica Acta 1241:117-138). ISCOMs comprise saponin, cholesterol and phospholipids forming an open cage structure, typically 40 nm in size. ISCOMs are produced by the interaction of saponin, cholesterol and further phospholipids. Typical reaction mixtures for preparing ISCOMs are 5 mg/ml saponin and 1 mg/ml cholesterol and phospholipids. Phospholipids suitable for use in ISCOMs include, but are not limited to, phosphorylcholine (didecyl-L-alpha-phosphatidylcholine [ DPC ], dilauroyl phosphatidylcholine-DPLC ], dimethyl phosphatidylcholine/DMPC ], dipalmitoyl phosphatidylcholine esterase [ DPPC ], di-tertiary acyl phosphatidylcholine [ DSPC ], dioleoyl phosphatidylcholine [ DOPC ], 1-palmitoyl, 2-oleoyl phosphatidylcholine esterase [ POPC ], di-Lai Duo phosphatidylcholine [ DEPC ]), phosphoglyceride (1, 2-dimercapto-sn-glycero-3-phosphoglyceride [ DMPG ], 1, 2-distearoyl-sn-glycero-3-phosphoglyceride [ DPPG ], 1, 2-distearoyl-sn-glycero-3-phosphoglyceride [ POPG ], 1-palmitoyl-2-oleoyl-sn-glyceric acid [ POPG ], phosphatide (1, 2-di-oleoyl-glycero-3-phosphatidate [ DMPA ], dipalmitoyl phosphatidylcholinesterase [ POPC ], di-2-oleoyl phosphatidylcholinesterase [ DPPE ], distearoyl-3-phosphoglyceride [ DSPA ], distearoyl-3-phosphoglyceride [ DSPE ], 2-stearoyl-3-phosphoglyceride [ DPPE ], 1, 2-distearoyl-3-phosphoglyceride-2-phosphoethanolamine [ DPPA ], phosphoethanolamine [ DPPE ], 1, 2-distearoyl-sn-3-phosphoglyceride-2-phosphoethanolamine [ DPPA (DPPA ] [ DPPA ] Polyglycerol phospholipids, functionalized phospholipids, terminally active phospholipids). In a particular embodiment, the ISCOMs include 1-palmitoyl-2-oleoyl-glycerol-3-phosphate ethanolamine. In a further specific embodiment, highly purified phosphatidylcholine is used, which may be selected from the following: phosphatidylcholine (from eggs), hydrogenated phosphatidylcholine (from eggs), phosphatidylcholine (from soybeans), hydrogenated phosphatidylcholine (from soybeans). In a further particular embodiment, the ISCOMs comprise phosphatidylethanolamine [ POPE ] or derivatives thereof. Some saponins are suitable for ISCOMs. The art has conducted extensive research on the adjuvant and hemolytic activity of individual saponins. For example, quil A (extracted from bark of the south America tree Quillaja Saponaria Molina) and fractions thereof are described in U.S. Pat. Nos. 5,057,540 and "Saponins as vaccine adjuvants", kensil, C.R., crit.Rev.Ther.Drug.Carrier Syst.,1996,12 (1-2): 1-55; and EP 0362279B 1. ISCOMs containing fractions of Quil A have been used to prepare vaccines (EP 0109942B 1). These structures are reported to have adjuvant activity (EP 0109942B 1; WO 96/11711). Fractions of QuilA, derivatives of QuilA and/or combinations thereof are suitable saponin formulations for ISCOMs. The haemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil a) have been described as effective adjuvants and their production methods are disclosed in US 5,057,540 and EP 0362279B 1. The use of QS7 (the non-hemolytic fraction of Quil-A) as an effective adjuvant for systemic vaccines is also described in these references. Kensil et al (1991.J.Immunology vol 146,431-437) further describe the use of QS 21. Combinations of QS21 and polysorbate or cyclodextrin are also known (WO 99/10008). Particulate adjuvant systems comprising QuilA fractions, such as QS21 and QS7, are described in WO 96/33739 and WO 96/11711, both of which are incorporated herein by reference. WO 96/01711 discloses other specific QuilA fractions in the form of ISCOMs, such as QH-A, QH-B, QH-C and mixtures of QH-A and QH-C, namely QH-703, which is incorporated herein by reference.

Microparticles

In some embodiments of the invention, there is provided a vaccine or immunogenic composition of the invention comprising microparticles. Microparticles, compositions comprising microparticles, and methods of producing microparticles are well known in the art (see Singh et al [2007Expert Rev.Vaccines 6 (5): 797-808] and WO 98/033487). The term "microparticles" as used herein refers to particles having a diameter or length of about 10 nanometers to 10,000 micrometers, derived from polymers having various molecular weights, and in the case of copolymers such as PLG, various lactams: the ratio of the hydantoin. In particular, the diameter of the microparticles should allow parenteral administration to a subject without clogging the administration device and/or capillaries of the subject. Microparticles are also referred to as microspheres. The size of the microparticles can be readily determined by techniques well known in the art, such as photon correlation spectroscopy, laser diffraction, and/or scanning electron microscopy. Microparticles for use herein will be formed from a sterilizable, non-toxic, and biodegradable material. Such materials include, but are not limited to: poly (a-hydroxy acid), polyhydroxy butyric acid, polycaprolactone, polyorthoester, and polyanhydride.

Liposome

In some embodiments of the invention, a vaccine or immunogenic composition of the invention comprising liposomes is provided. The term "liposome" generally refers to a lipid structure of a monolayer or multilamellar (in particular 2, 3, 4, 5, 6, 7, 8, 9 or 10 layers, depending on the number of lipid membranes formed) surrounding the interior of an aqueous solution. Liposomes and liposome formulations are well known in the art. Lipids capable of forming liposomes include all substances having fat or fat-like properties. The lipids capable of constituting the liposome may be selected from glycerides, glycerophospholipids, sulphur lipids, sphingolipids, phospholipids, isoprenaline, steroids, stearic acid, sterols, protolipids, synthetic cationic lipids and lipids containing carbohydrates. Liposomes can vary in size from 30 nanometers to several micrometers depending on the composition of the phospholipid and the method used for preparation. In certain embodiments of the invention, the liposomes will be between 50 nanometers and 500 nanometers in size, and in further embodiments, between 50 nanometers and 200 nanometers in size. Dynamic laser light scattering is a method for measuring liposome size and is well known to those skilled in the art. The liposome suitably comprises a neutral lipid, such as phosphatidylcholine, which is suitably amorphous at room temperature, such as egg yolk phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC) or dioleoyl phosphatidylcholine. In a particular embodiment, the liposomes of the invention contain DOPC. Liposomes can also contain a charged lipid that can increase the stability of the liposome-saponin structure composed of saturated lipids. In these cases, the amount of charged lipid is suitably 1 to 20% (weight ratio), preferably 5 to 10%. The ratio of sterols to phospholipids is 1 to 50% (mol/mol), suitably 20 to 25% (mol/mol).

Saponin

In some embodiments of the invention, the vaccine or immunogenic composition of the invention comprises a saponin. Particularly suitable saponins for use in the present invention are Quil a and its derivatives. Quil A is a saponin preparation isolated from the south America tree Quillaja Saponaria Molina and has been described for its first time by Dalsgaard et al in 1974 ("Saponin adjuvants", archiv. F U r die gesamte Virusforschung, vol.44, springer Verlag, berlin, p 243-254) as having adjuvant activity. Isolation of purified fragments of Quil a by HPLC retained adjuvant activity without Quil a-associated toxicity (EP 0362278), such as QS7 and QS21 (also known as QA7 and QA 21). QS-21 is a natural saponin extracted from Quillaja saponaria Molina bark, which induces CD8+ cytotoxic T Cell (CTL), th1 cell and main IgG2a antibody reaction, and is a special saponin in the present invention. The saponin adjuvant in the immunogenic compositions of the invention is especially an immunologically active component of Quil A, such as QS-7 or QS-21, suitably QS-21. In particular embodiments, the vaccine and/or immunogenic compositions of the invention contain an immunologically active saponin fraction in substantially pure form. In particular, the vaccine or immunogenic composition of the invention comprises substantially pure QS21, that is, QS21 is at least 75%, 80%, 85%, 90% pure, for example at least 95% pure, or at least 98% pure.

In a particular embodiment, QS21 is provided with exogenous sterols, such as cholesterol. Suitable sterols include beta-sitosterol, stigmasterol, ergosterol, and cholesterol. In another specific embodiment, the adjuvant composition includes cholesterol as a sterol. These sterols are well known in the art, for example, cholesterol is disclosed in Merck Index 11 th edition, page 341, a naturally occurring sterol found in animal fat.

In one embodiment, the liposomes of the invention comprising a saponin suitably comprise a neutral lipid, such as phosphatidylcholine, which is suitably amorphous at room temperature, such as egg yolk phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC) or dioleoyl phosphatidylcholine. Liposomes may also contain a charged lipid that increases the stability of the liposome-QS 21 structure composed of saturated lipids. In these cases, the amount of charged lipid is preferably 1 to 20% (w/w), particularly 5 to 10% (w/w). The ratio of sterols to phospholipids is 1 to 50% (mol/mol), suitably 20 to 25% (mol/mol).

When the active saponin moiety is QS21, the ratio of QS21 to sterol is generally from 1:100 to 1:1 (w/w), suitably between 1:10 and 1:1 (w/w), preferably from 1:5 to 1:1 (w/w). Suitably, excess sterols are present, the ratio of QS21 to sterols being at least 1:2 (w/w). In one embodiment, QS21: the ratio of sterols is 1:5 (w/w). Sterols are suitable cholesterol.

Other useful saponins are derived from plants Aesculus hippocastanum or Gyophilla struthium. Other saponins described in the literature include Escin, which is described in Merck Index (12 th edition: item 3737) as a saponin mixture occurring in the seeds of horse chestnut Lat: aesculus hippocastanum. Its isolation is described by chromatography and purification (Fiedler, arzneimitel-Forsch.4, 213 (1953)), and by ion exchange resins (Erbering et al, US 3,238,190). Fractions of epothilone have been purified and demonstrated to be biologically active (Yoshikawa et al, 1996,Chem Pharm Bull (Tokyo), 44 (8): 1454-1464). Sapoalbin from Gypsophilla struthium (R.Vochten et al 1968, J.Pharm.Belg.42:p213-226) is also described as an example in connection with ISCOM production.

Saponins, such as QS21, may be used in an amount of 1 to 100 μg per human dose of the adjuvant composition. QS21 may be used in an amount of about 50. Mu.g, for example between 40 and 60. Mu.g, suitably between 45 and 55. Mu.g or 49 and 51. Mu.g or 50. Mu.g. In another embodiment, the human dose of the adjuvant composition comprises QS21 in an amount of about 25 μg, for example between 20 and 30 μg, suitably between 21 and 29 μg or 22 to 28 μg or 28 to 27 μg or 24 to 26 μg, or 25 μg.

TLR4 agonists

In some embodiments, the vaccine or immunogenic composition of the invention comprises a TLR4 agonist. "TLR agonist" refers to a component capable of causing a signaling response through a TLR signaling pathway, and may be a direct ligand or may be an indirect ligand that causes a signaling response through the production of an endogenous or exogenous ligand (Sabroe et al,2003, JI p 1630-5). A TLR4 agonist is capable of eliciting a signaling response through the TLR-4 signaling pathway. One suitable example of a TLR-4 agonist is lipopolysaccharide, suitably a non-toxic derivative of lipid a, in particular mono-phospholipid a or more particularly 3-Deacylated mono-phospholipid a (3D-MPL).

3D-MPL is sold by the company Gelanin Smith under the name MPL, referred to as MPL or 3D-MPL throughout this document. See, for example, US 4,436,727; US 4,877,611; US 4,866,034 and US 4,912,094.3D-MPL primarily promotes CD4+ T cell responses with the IFN-gamma (Th 1) phenotype. 3D-MPL may be produced according to the methods disclosed in GB 2 220 211A. Chemically, it is a mixture of 3-deacylated Shan Linzhi A with 4, 5 or 6 acylated chains. In the compositions of the invention, small particle 3D-MPL may be used to prepare aqueous adjuvant compositions. The particle size of the small particle 3D-MPL may be sterile filtered through a 0.22 μm filter. Such formulations are described in WO 94/21292. Preferably, powdered 3D-MPL is used to prepare the aqueous adjuvant composition of the invention.

Other TLR-4 agonists that may be used are Alkyl Glucosamine Phosphates (AGPs), such as those disclosed in WO 98/50399 or US 6,303,347 (also disclosing the process for the preparation of AGPs), suitably pharmaceutically acceptable salts of AGPs as disclosed in RC527 or RC529 or US 6,764,840.

Other suitable TLR-4 agonists are described in WO 03/01223 and WO 03/099195, for example, compound I, compound II and compound III disclosed in WO 03/01223 on pages 4-5 or WO 03/099195 on pages 3-4, especially those disclosed in WO 03/01223, such as ER803022, ER803058, ER803732, ER804053, ER804057m ER804058, ER804059, ER804442, ER804680 and ER804764. For example, a suitable TLR-4 agonist is ER804057.

TLR-4 agonists, such as lipopolysaccharides, e.g. 3D-MPL, may be used in an amount of 1 to 100 μg per human dose of the adjuvant composition. 3D-MPL may be used at a dose of about 50 μg per person, for example 40 to 60 μg, suitably 45 to 55 μg or 49 to 51 μg or 50 μg. In another embodiment, the human dose of the adjuvant composition comprises about 25 μg, for example between 20 to 30 μg, suitably between 21 to 29 μg or 22 to 28 μg or 28 to 27 μg or 24 to 26 μg, or 25 μg 3D-MPL.

Synthetic derivatives of lipid a are known and considered TLR 4 agonists, including but not limited to:

OM174 (2-deoxy-6-o- [ 2-deoxy-2- [ (R) -3-dodecyloxy tetra-decanoylamino ] -4-O-phosphono-beta-D-glucopyranosyl ] -2- [ (R) -3-hydroxytetradecanoylamino ] -alpha-D-glucopyranosyl dihydrogen phosphate), (WO 95/14026)

OM294 DP (3S, 9R) -3- [ (R) -dodecyloxy-tetradecanoylamino ] -4-oxo-5-aza-9 (R) - [ (R) -3-hydroxytetradecanoylamino ] decane-1, 10-diol, 1, 10-bis (dihydrogen phosphate) (WO 99/64301 and WO 00/0462)

OM197 MP Ac-DP (3S-, 9R) -3- [ (R) -dodecyloxy-tetradecylamino ] -4-oxo-5-aza-9- [ (R) -3-hydroxytetradecylamino ] decane-1, 10-diol, 1-dihydro-10- (6-aminocaproic acid) (WO 01/46127).

PHAD (phosphorylated hexaacyl disaccharide).

Other suitable ligands for TLR-4 are capable of eliciting a signaling response through TLR-4 (Sabroe et al, JI 2003p 1630-5), e.g., lipopolysaccharides from gram negative bacteria and derivatives thereof, or fragments thereof, particularly non-toxic derivatives of LPS (e.g., 3D-MPL). Other suitable TLR agonists are: heat Shock Proteins (HSP) 10, 60, 65, 70, 75 or 90; surfactant protein a, hyaluronic acid oligosaccharides, heparin sulfate fragments, fibronectin fragments, fibrinogen peptides and b-defensin-2, muramyl Dipeptide (MDP) or Respiratory Syncytial Virus (RSV) F protein. In one embodiment, the TLR agonist is HSP 60, 70 or 90.

TLR agonists

In addition to TLR4 agonists, other natural or synthetic TLR molecular agonists may also be used in the vaccine or immunogenic composition of the invention. These include, but are not limited to, agonists of TLR2, TLR3, TLR5, TLR6, TLR7, TLR8 and TLR 9.

In one embodiment of the present invention, a TLR agonist capable of eliciting a signaling response through TLR-1 is used (Sabroe et al, JI 2003p 1630-5). Suitably, the TLR agonist capable of eliciting a signalling response through TLR-1 is selected from: a triacetylated Lipopeptide (LPs); phenol-soluble morelin; mycobacterium tuberculosis LP; s- (2, 3-bis (palmitoyloxy) - (2-RS) -propyl) -N-palmitoyl- (R) -Cys- (S) -Ser- (S) -Lys (4) -OH, tri-hydrochloride (Pam 3 Cys) LP, which mimics the acetylated amino terminus of bacterial lipoproteins and OspA LP from Serratia abalone.

In another embodiment, a TLR agonist capable of eliciting a signaling response through TLR-2 is used (Sabroe et al, JI 2003p 1630-5). Suitably, the TLR agonist capable of eliciting a signalling response through TLR-2 is one or more of a lipoprotein, peptidoglycan, bacterial lipopeptides from m.tubulosis, b.burgdorferi, t.pallidum, peptidoglycan from species including staphylococcus aureus, lipoalginic acid, mannitol, neisseria porin, bacterial pili, yersinia virulence factor, CMV virus, measles hemagglutinin and zymosan from yeast.

In another embodiment, a TLR agonist capable of eliciting a signaling response through TLR-3 is used (Sabroe et al, JI 2003p 1630-5). Suitably, the TLR agonist capable of eliciting a signalling response through TLR-3 is double stranded RNA (dsRNA), or polycreatine-polycytidylic acid (Poly IC), a molecular nucleic acid pattern associated with viral infection.

In another embodiment, a TLR agonist capable of eliciting a signaling response through TLR-5 is used (Sabroe et al, JI 2003p 1630-5). Suitably, the TLR agonist capable of eliciting a signalling response through TLR-5 is bacterial flagellin. The TLR-5 agonist may be a flagellin or may be a fragment of a flagellin that retains TLR-5 agonist activity. The flagellin may comprise a polypeptide selected from the group consisting of: h.pyri, s.tyrphimum, v.cholera, s.marcescens, s.flexneri, t.pallidium, l.pneumophilia, b.burgdorferi; difficile, r.meliloti, a.tumefaciens; lupine; claritdgeiae, p.mirabilis, b.subtitilis, l.mocytogenesis, p.aeroginosa and e.coli.

In a particular embodiment, the flagellin is selected from the group consisting of: s.tyrphinium flagellin B (Genbank Accession number AF 045151), fragments of s.tyrphinium flagellin B, escherichia coli flic (Genbank Accession number AB 028476); fragments of E.coli fliC; fragments of the S.tyrphinium flagellin FliC (ATCC 14028) and S.tyrphinium flagellin FliC.

In another specific embodiment, the TLR-5 agonist is a truncated flagellin, as described in WO 09/156405, i.e. wherein the hypervariable domain has been deleted. In one aspect of this embodiment, the TLR-5 agonist is selected from the group consisting of: fliC _Δ174-400 ；FliC _Δ161-405 And FliC _Δ138-405 。

In another specific embodiment, the TLR-5 agonist is a flagellin, as described in WO 09/128950. In another embodiment, a TLR agonist capable of eliciting a signaling response through TLR-6 is used (Sabroe et al, JI 2003p 1630-5). Suitably, TLR agonists capable of eliciting a signalling response through TLR-6 are mycolipoproteins, diacylated LP and phenol-soluble morelin. Further TLR6 agonists are described in WO 03/043572.

In another embodiment, a TLR agonist capable of eliciting a signaling response through TLR-7 is used (Sabroe et al, JI 2003p 1630-5). Suitably, the TLR agonist capable of eliciting a signalling response through TLR-7 is a single-stranded RNA (ssRNA), loxoribine, guanosine analogues or imidazoquinoline compounds at the N7 and C8 positions, or derivatives thereof. In a particular embodiment, the TLR agonist is imiquimod. Further TLR7 agonists are described in WO 02/085905.

In another embodiment, a TLR agonist capable of eliciting a signaling response through TLR-8 is used (Sabroe et al, JI 2003p 1630-5). Suitably, the TLR agonist capable of eliciting a signalling response through TLR-8 is a single-stranded RNA (ssRNA), an imidazoquinoline molecule with antiviral activity, such as resiquimod (R848); resiquimod is also recognized by TLR-7. Other TLR-8 agonists that may be used include those described in WO 04/071459.

In another embodiment, TLR agonists capable of eliciting a signaling response, such as agonists comprising CpG motifs, are used. The term "immunostimulatory oligonucleotide" as used herein refers to an oligonucleotide capable of activating a component of the immune system. In one embodiment, the immunostimulatory oligonucleotide comprises one or more unmethylated cytosine-guanosine (CpG) motifs. In another embodiment, the immunostimulatory oligonucleotide comprises one or more unmethylated thymosin-guanosine (TG) motifs or may be T-rich. By T-rich it is meant that the nucleotide composition of the oligonucleotide comprises greater than 50, 60, 70 or 80% thymidine. In one embodiment, the oligonucleotide is not an immunostimulatory oligonucleotide, nor does it contain unmethylated CpG motifs. In another embodiment, the immunostimulatory oligonucleotide is not enriched in T and/or does not comprise an unmethylated TG motif.

Oligonucleotides may be modified to improve stability in vitro and/or in vivo. For example, in one embodiment, the oligonucleotides are modified to include phosphorothioate backbones, i.e., internucleotide linkages. Other suitable modifications, including modifications of the biphosphinate, phosphoramide, and methylphosphonate, and alternative internucleotide linkages of the oligonucleotide are well known to those skilled in the art and are encompassed by the present invention.

In another embodiment, the vaccine or immune composition of the invention further comprises an immunostimulant selected from the group consisting of: a TLR-1 agonist, a TLR-2 agonist, a TLR-3 agonist, a TLR-4 agonist, a TLR-5 agonist, a TLR-6 agonist, a TLR-7 agonist, a TLR-8 agonist, a TLR-9 agonist, or a combination thereof.

Calcium composition

In some embodiments, the vaccine or immunogenic composition of the invention comprises a calcium fluoride composite comprising Ca, F, and Z. "Z" herein refers to an organic molecule. As used herein, "composite" refers to a material that exists in a solid form when dried and is insoluble or poorly soluble in pure water. In certain aspects, Z comprises a functional group that forms an anion upon ionization. Such functional groups include, but are not limited to, one or more functional groups selected from the group consisting of: hydroxyl, hydroxyl compound, hydroxyl, oxide, N-hydroxy acid salt, hydroxylamine, N-oxide, bicarbonate, carbonate, carboxylate, fatty acid, sulfate, organophosphate, dihydrogen phosphate, monohydrogen phosphate, monoester of phosphoric acid, diester of phosphoric acid, ester of phospholipids, phosphorothioate, sulfate, bisulfate, enoate, ascorbate phosphate, phenolate, and imidite.

In certain aspects, the calcium fluoride composites described herein include Z, wherein Z is an anionic organic molecule that possesses affinity for calcium and forms a water insoluble composite with calcium and fluorine. In a further aspect, the calcium fluoride composites described herein include Z, wherein Z can be categorized as including members of a chemical class selected from the group consisting of: hydroxy, oxide, N-hydroxy, N-oxide, bicarbonate, carbonate, carboxylates and dicarboxylates, salts of carboxylic acids, salts of QS21, extracts of Quillaja saponaria bark, extracts of immunologically active saponins, salts of saturated or unsaturated fatty acids, salts of oleic acid, salts of amino acids, thiolates, salts of thiols, salts of cysteine, salts of N-acetyl-cysteine, salts of L-2-oxo-4-thiazoline carboxylate, phosphates, dihydrophosphates, monohydrogenphosphates, salts of phosphoric acid, monoesters of phosphoric acid and salts thereof, diesters of phosphoric acid and salts thereof, esters of 3-O-deacetyl-4' -monophosphate A, esters of 3D-MLA, MPL, esters of phospholipids, DOPC, glycol phosphite derivatives, phosphates of CpG motifs, thiophosphates of the CpG family, sulphates, bisulphates, sulphates, enolates, ascorbates, phenolates, alpha-tocopherols, imine outsides, cytosines, methylcytosines, uracils, thymines, barbiturates, hypoxanthines, inosines, guanines, 8-oxo-adenines, xanthines, uric acids, pteroic acids, pteroylglutamic acids, folic acids, sinapins and flavins. In a further aspect, the calcium fluoride complexes described herein comprise Z, wherein Z is selected from the group consisting of: n-acetylcysteine; thiolactic acid; adipic acid; a carbonate salt; folic acid; glutathione; and uric acid. In some aspects, the calcium fluoride complexes herein comprise Z, wherein Z is selected from the group consisting of: n-acetylcysteine; adipic acid; a carbonate salt; and folic acid. In a further aspect, the calcium fluoride complex herein comprises Z, wherein Z is N-acetylcysteine, and the complex comprises between 51% Ca,48% F, no more than 1% N-acetylcysteine (w/w) and 37% Ca, 26% F, and 37% N-acetylcysteine (w/w). In a further aspect, the calcium fluoride complex herein comprises Z, wherein Z is thiolactic acid, and the complex comprises between 51% Ca,48% F, no more than 1% thiolactic acid (w/w) and 42% Ca, 30% F, 28% thiolactic acid (w/w). In a further aspect, the calcium fluoride complex herein comprises Z, wherein Z is adipic acid, the complex comprising 51% Ca,48% F, no more than 1% adipic acid (w/w) and 38% Ca,27% F,35% adipic acid (w/w). In a further aspect, the calcium fluoride complexes herein comprise Z, wherein Z is carbonate, the complexes comprise 51% Ca,48% F, no more than 1% carbonate (w/w) and 48% Ca,34% F,18% carbonate (w/w). In a further aspect, the calcium fluoride complex herein comprises Z, wherein Z is folic acid, and the complex comprises 51% Ca,48% F, no more than 1% folic acid (w/w) and 22% Ca, 16% F, 62% folic acid (w/w). In a further aspect, the calcium fluoride complex herein comprises Z, wherein Z is glutathione, the complex comprises 51% Ca,48% F, no more than 1% glutathione (w/w) and 28% Ca,20% F,52% glutathione (w/w). In a further aspect, the calcium fluoride complex herein comprises Z, wherein Z is uric acid, and the complex comprises 51% Ca,48% F, no more than 1% uric acid (w/w) and 36% Ca, 26% F, 38% uric acid (w/w).

Aluminium salt

In one embodiment, the vaccine or immunogenic composition of the invention comprises an aluminum salt. Suitable aluminum salt adjuvants are well known to the skilled artisan and include, but are not limited to, aluminum phosphate, aluminum hydroxide, or combinations thereof. Suitable aluminum salt adjuvants include, but are not limited to REHYDRAGEL HS, ALHYDROGEL 85, REHYDRAGEL PM, REHYDRAGEL AB, REHYDRAGEL HPA, REHYDRAGEL LV, ALHYDROGEL, or combinations thereof.

In particular, the protein adsorption capacity of the aluminium salt may be between 2.5 and 3.5, 2.6 and 3.4, 2.7 and 3.3 or 2.9 and 3.2, 2.5 and 3.7, 2.6 and 3.6, 2.7 and 3.5, or 2.8 and 3.4 proteins (BSA) per ml of aluminium salt. In a particular embodiment of the invention, the protein adsorption capacity of the aluminium salt is between 2.9 and 3.2 milligrams BSA/mg aluminium salt. The protein adsorption capacity of an aluminum salt can be measured by any means known to the skilled person. The protein adsorption capacity of aluminium salts can be measured by the method described in example 1 of WO 12/136823 (using BSA) or variations thereof.

The aluminum salts described herein (i.e., having the protein adsorption capacity described herein) can have a crystal size of between 2.8 and 5.7 nanometers, for example, 2.9 to 5.6 nanometers, 2.8 to 3.5 nanometers, 2.9 to 3.4 nanometers, or 3.4 to 5.6 nanometers, or 3.3 and 5.7 nanometers, as determined by X-ray diffraction. X-ray diffraction is well known to the skilled person. In a particular embodiment of the invention, the crystal size is measured using the method described in example 1 of WO 12/136823 or variations thereof.

The polypeptides and/or nucleic acids of the invention may be administered to a subject by any route of administration, e.g., orally, nasally, sublingually, intravenously, intramuscularly, intradermally (e.g., with microinjected skin patches) or transdermally (e.g., ointments or creams).

A seventh aspect of the invention provides a polypeptide as defined in the first aspect, a nucleic acid as defined in the second aspect, a vector as defined in the third aspect and/or a vaccine as defined in the sixth aspect for use in medicine.

An eighth aspect provides a polypeptide as defined in the first aspect, a nucleic acid as defined in the second aspect, a vector as defined in the third aspect and/or a vaccine as defined in the sixth aspect for use in generating an immune response in a mammal, e.g. for use in the treatment and/or prophylaxis of one or more diseases.

A ninth aspect provides a polypeptide as defined in the first aspect, a nucleic acid as defined in the second aspect, a vector as defined in the third aspect and/or a vaccine as defined in the sixth aspect for use in generating an immune response in a mammal, e.g. for use in the treatment and/or prophylaxis of one or more diseases.

A tenth aspect provides a polypeptide as defined in the first aspect, a nucleic acid as defined in the second aspect, a vector as defined in the third aspect and/or a vaccine as defined in the sixth aspect for use in the manufacture of a medicament for generating an immune response in a mammal, e.g. for use in the treatment and/or prophylaxis of one or more diseases.

An eleventh aspect provides a method of increasing an immune response in a mammal, the method comprising or consisting of: administering to a mammal an effective amount of a polypeptide as defined in the first aspect, a nucleic acid as defined in the second aspect, a vector as defined in the third aspect and/or a vaccine as defined in the sixth aspect.

The use or method of any of the seventh to eleventh aspects, wherein the one or more diseases is Urinary Tract Infection (UTI). Alternatively or additionally, UTI is caused by one or more bacteria selected from the group consisting of escherichia and klebsiella. Alternatively or additionally, the one or more bacteria are selected from the group consisting of escherichia coli and klebsiella pneumoniae. Alternatively or additionally, the E.coli is a urogenic E.coli (UPEC). Alternatively or additionally, the one or more bacteria are selected from the group consisting of E.coli J96, E.coli UPEC 536, E.coli CFT073, E.coli UMN026, E.coli CLONE Di14, E.coli CLONE Di2, E.coli CFT073; coli IA139, e.coli 536, e.coli NA114 and e.coli UTI89. Alternatively or additionally, the one or more bacteria are selected from the following k.pneumoniae strains: c3091,3824,3857,3858,3859,3860,3861,3928,3950,3951,4041,4121,4133, sp3, sp7, sp10, sp13, sp14, sp15, sp19, sp20, sp22, sp25, sp28, sp29, sp30, sp31, sp32, sp33, sp34, sp37, sp39, sp41, cas119, cas120, cas121, cas122, cas123, cas124, cas125, cas126, cas127, cas128, cas663, cas664, cas665, cas666, cas667, cas668, cas669, cas670, cas671, cas672, cas673, cas674, cas675, cas676, cas677, cas678, cas679, cas680, cas681, cas682, kp342 and MGH 7878.

Preferred, non-limiting examples embodying certain aspects of the present invention will now be described with reference to the following tables and accompanying drawings.

Schematic diagrams of fimh constructs in fig. 1A and 1b.

A) Fig. 1A. Structure of stable FimH (PDB: 4XO 9). Cartoon representation of FimH stabilized by FimGFimG donor chain (blue-indicated by arrow). FimH _L Domain yellow (top), fimH _P Red (bottom). The glycine natural linker between domains is represented by green rollers.

B) FIG. 1B Structure of FimH_DG_PGDGN_ferritin. Fimh_dg_pgdgn (light blue) fused to ferritin (red). A linker consisting of SGS-8H-GSG-links FimH to ferritin molecules. IgK leader sequences for expression and secretion into the medium in mammalian cells are yellow, followed by additional N-terminal charged residues. Three-dimensional structural model obtained with Rosetta general software. Cartoon representation of fimh_dg displayed on ferritin surface. 24 FimH subunits are present, the color being yellow/blue and ferritin being red.

FIGS. 2A and 2B. E.coli expression of FimH nanoparticles resulted in inclusion body formation:

a) FIG. 2A. FimH_DG_ (GSG 4) -ferritin, fimH _L cys-cys_QBeta、FimH _L cys-cys_mI3 and FimH _L SDS-PAGE analysis of E.coli cytoplasmic expression boiled and reduced samples of NOcys-MI 3. The construct was expressed, but only in the insoluble fraction (urea 8M, U8M) It was detected that it could not be detected in the soluble fraction (sol). Due to insolubility, no protein could be detected in the total lysate fraction (Tot); accumulation of insoluble material can be detected in the upper part of the gel. FimH _L Coli cytoplasmic expression of Nocys-MI3 anti-His western blot of boiled and reduced samples. FimH _L Mutations in the disulfide bridges within the domain do not improve solubility because only a weak band can be detected in the soluble portion.

B) FimH expressed around E.coli _L MI3 and cytoplasmic FimH _L SDS-PAGE analysis of ferritin. The detection of the corresponding FimH in the total lysate and insoluble fraction (U8M) _L -a band of fusion of MI3 and ferritin.

FIG. 3 prediction of N-glycosylated FimH sites using NetNGly prediction software.

Fig. 4. Stable FimH construct (fimh_Δgg_pgdgn_dg:930SI, fimH_DNKQ_DG:931SI, fimH_PGDGN_DG:932SI) and FimHC complex expression in mammalian cells.

FIG. 5 Western blot analysis of mammalian expression constructs containing N-terminal additional amino acids.

(A) Fig. 5A: after 3 and 6 days post-transfection, only the band of FIMH nanoparticles corresponding to FIMH_DG_PGDGN-ferritin (995 SI) was detected.

(B) Fig. 5B: cartoon representation of FimH from strain 536, three different residues compared to J96 are highlighted and represented in bars.

(C) Fig. 5C: PNG enzyme treatment of strain J96 fimh_dg_pgdgn_imx313 and fimh_dg_pgdgn_ferritin. After treatment, a transition of FIMH_DG_PGDGN_IMX313 at the correct MW was obtained, indicating that the protein is glycosylated in mammalian cells. FIMH_DG_PGDGN_ferritin from strain J96 was not detected in both untreated and treated PNG enzyme samples, indicating that the protein was degraded.

FIG. 6 MS-Spec peptide map.

FIG. 7 expression of candidate proteins without additional N-terminal amino acids was detected by Western blotting.

FIG. 8 Cryo-EM NS-EM with or without additional AA at the N-terminus (negative staining).

FIG. 9 Cryo-EM NS-EM without additional AA candidates at the N-terminus (negative staining).

A) Fig. 9A:1095SI FimH _L Negative staining microscopy image of ferritin (strain 536) without additional amino acids.

B) Fig. 9B: fimH _L Negative staining microscopy image of MI3 (strain J96) without additional amino acids.

C) Fig. 9C: negative staining microscopy images of 1184SI FIMH_DG_PGDGN_536-coat protein without additional amino acids.

Fig. 10.3D shows the presence of three "anchor" appendages on the tri-fold axis.

FIG. 11 measurement of IgG titres by ELISA assay. Mouse serum was tested 21 days after vaccination (I, green), 35 days (II, blue) and 45 days (III, red). FimH produced by E.coli _L Is used as a coating for ELISA plates.

FIG. 12 bacterial inhibition assay (BAI) on SV-HUC cells. Bacterial adhesion as measured by microscopic analysis (OPERA Phenix) and SV-HUC (ATCC) cells. The fluorescence volume or area (μm3 or 2) of the adherent bacteria was taken as a reading. Against recombinant protein FimHC, fimH _L Serum pools of cys (purified from E.coli) served as controls. Serum pools of recombinant proteins fimh_pgdgn_dg (932 SI), fimh_dnkq_dg (931 SI), fimh_dnkq_dg_degyc (951 SI) and fimh_pgdgn_dg-ferritin (995 SI) purified from the expgnti expression mammalian systems were used to measure their ability to inhibit bacterial binding to SV-HUC cells. Serum pools against AS01 were used AS negative controls.

FIG. 13 biochemical characterization of purified FimH_PGDGN_DG by SDS-PAGE, SE-UPLC and RP-UPLC.

FIG. 14 Biochemical characterization of purified FimH_DNKQ_DG by SDS-PAGE, SE-UPLC and RP-UPLC.

FIG. 15 biochemical characterization of purified FimH_DNKQ_DG_deglycosylated by SDS-PAGE, SE-UPLC and RP-UPLC.

FIG. 16. Biochemical features of purified FimH_DG_PGDGN_ferritin with additional AA (sequence from UPEC 536 strain) at the N-terminus.

Fig. 17: fimH specific total IgG (ELISA). FIG. 17A) anti-FimH IgG titer in mouse serum was correlated with MPL dose after 3. Figure 17B) anti-FimH IgG titers in mouse urine measured after 1 st, 2 nd and 3 rd vaccine doses. Preimmune serum served as a negative control. FimHC was immunized in combination with adjuvant with a protein content of 1.6 μg.

Fimh specific total IgG (ELISA): comparison of bacterial and mammalian expression systems in serum and urine. Fig. 18A) assumes that the antibody titers are a log-normal distribution and calculates the Geometric Mean Titer (GMT) and 95% ci on both sides thereof. To compare the groups, an analysis of variance model was performed on log10 titers, and the time points were repeatedly stated using the groups, time points and their interactions as fixed factors. Variance heterogeneity was considered between groups. From this model, the geometric mean ratios and their 95% cis were found. Antibody responses for each formulation were evaluated against FimHDG for ELISA plate coating. All statistical analyses were performed using SAS 9.4. Fig. 18B) FimH specific total urine IgG.

FIG. 19 FimH-specific total IgGs. ELISA results after dose I: assuming a log-normal distribution of antibody titers, geometric Mean Titers (GMTs) and their bilateral 95% cis were calculated. For comparison of the groups, log10 titers were subjected to an analysis of variance model, and the time points were repeatedly stated using the groups, time points and their interactions as fixed factors. The heterogeneity of variances is considered between groups. From this model, the geometric mean ratios and their 95% cis were found. Antibody responses for each formulation were evaluated against FimHDG for ELISA plate coating. All statistical analyses were performed using SAS 9.4.

FIG. 20 FimH-DG elicits a functional immune response. Bacterial inhibition assays of selected constructs compared to FimHC. The relative efficacy was calculated as reported in the examples.

FIG. 21 antibody ability of FimHDG antibodies to inhibit adhesion of ExPEC using bacterial inhibition assay (BAI). All candidates were formulated with AS 01.

FIG. 22 interaction of SPR analysis of FimH samples with mAb926 (Sensorgrams).

To investigate the interaction of FimH candidate and mAb926, SPR analysis was performed, yielding a sensor map representing the reaction (ordinal) versus time (abscissa), showing the progress of the interaction. The response is measured in Resonance Units (RU), proportional to the concentration of molecules on the surface of the sensor chip. Each sensor map consists of two parts, corresponding to the association and dissociation phases of the interaction. Binding is the first phase of biomolecular interactions, in which binding occurs when the analyte and ligand collide due to diffusion and the collision has the correct orientation and sufficient energy. Dissociation is the stage of ligand-analyte complex dissociation; the dissociated profile may provide information about the stability of the complex: the slower the dissociation, the higher the complex stability and vice versa.

FIG. 23A SDS page analysis of FimHDG-expressing unlabeled culture supernatants in mammalian cells. FimHDG unlabeled sds_page assay and SEC-UPLC assay purified from Expi293 cells and Expi cho cells. Fig. 23B: fimHDG purified from Expi293 and Expi cho cells had no nano DSF profile and melting temperature values of the tag compared to FimHDG containing a C-terminal His tag. Fig. 23C: mAbs 926 and 475 were analyzed for unlabeled SPR binding to FimHDG compared to FimHDG His. Mannose was analyzed for unlabeled SPR binding to FimHDG compared to FimHDG His. Fig. 23D: SDS-Page analysis was performed on supernatants of FimHDG-ferritin constructs containing different linkers and with or without the original Asp residues. Western blot analysis of the pellet of mammalian cells using anti-FimH specific mouse serum.

Fig. 24. Symmetrical monomers (relative to the other 23 strands) in octahedral e.coli nanoparticles (PDB 1 EUM) calculated based on PROSS to introduce stable mutations with increased affinity or stability (lower left of the graph).

Fig. 25. Fig. 25A: SDS page analysis of total (T) and soluble (S) extracts of WT E.coli ferritin and the different mutants. SEC profile for mutant 0.5 of fig. 25B. All constructs had a strong peak (arrow) in the dead volume, which was consistent with nanoparticle formation.

FIG. 26 NS-EM (negative staining) analysis of E.coli ferritin WT and different mutants (0.5,2,2.5,6).

FIG. 27 differential scanning fluorescence analysis and thermal profile of ferritin constructs. The left plot shows the derivative of fluorescence intensity with temperature. The circle on the right table represents the mutant with the highest Tm (0.5).

FIG. 28, left is Western blot analysis of supernatants of different nanoparticle constructs expressing FimH using anti-His antibodies. Star represents E.coli nanoparticle FimHDG-ferritin (mutant 0.5). On the right, TEM analysis shows the presence of correctly formed ferritin nanoparticles.

Examples

The present inventors devised stable non-complex (in the absence of FimC) variants of full-length FimH, wherein fimgs donor chain peptide [ SEQ ID NO:5]Through a 4 or 5 residue linker (DNKQ [ SEQ ID NO: 8)]Or PGDGN [ SEQ ID NO:7]) With FimH _P The C-terminal of (C-terminal) is genetically fused to obtain a 'FimH_DG' protein with FimH structure and functional characteristics on the assembled cilia. The linker was designed by selecting a highly polar charged residue (DNKQ) or inserting a proline residue (PGDGN linker) as the first residue of the linker, which was predicted to support the turn in the secondary structure and promote the correct protein structure. Furthermore, the construct is present in the ligation FimH _L And FimH _P Two glycine residues in the linker are deleted to further reduce FimH _L And reduced binding of mannose (fig. 1A).

In addition, nanoparticle designs of FimH can be used to expose multiple copies of stable FimH and further increase its immunogenicity as an adjunct to 1-2 doses of vaccine.

Virus-like particles (VLPs) and protein Nanoparticles (NPs) are display platforms for other antigens with the potential to induce potent B-cell and T-cell responses. They have the inherent ability to self-assemble into highly symmetrical stable and organized structures. Some chimeric VLPs/NPs are being investigated in preclinical and clinical studies worldwide. In particular, ferritin scaffolds have been genetically fused to viral hemagglutinin to obtain particles, with single doses being more immunogenic than seasonal influenza vaccines in the presence of an adjuvant (Nature 2013,49,104). The same approach has been used for preclinical studies of many other antigens (Chen Y, et al vaccine.2020jul 31;38 (35): 5647-5652). The challenge is not only to design a correctly assembled particle presenting the antigen of interest, but also to obtain its manufacturability and scalability. To explore the potential of self-assembled NPs and VLPs to display FimH candidates, different chimeras have been designed and tested by gene fusion.

Helicobacter pylori ferritin nanoparticles consist of 24 subunits, and in total, a trimer of 8 desired antigens can be shown in the highly symmetrical octahedral cage structure of ferritin nanoparticles (fig. 1B). Recently, protein i301 nanocages, a 60-mer NP based on Thermotoga maritima-keto-3-deoxy-phosphogluconate (KDGG) aldolase, has been designed by calculation (Hsia Y, et al Nature.2016Jul 7;535 (7610): 136-9.). The stability of i301 was further improved by mutating both cysteines (mI 3) and by fusing SpyCatcher to the N-terminus of the protein (Bruun TUJ, et al ACS Nano.2018Sep 25;12 (9): 8855-8866).

We constructed recombinant plasmids, which were used to stabilize ferritin, mI3 or encapulin with FimH_DG_PGDGN or FimH _L And FimH _L Gene fusion is carried out on Cys antigen. To separate the antigen and NP shown, a linker was added between the two sequences.

The tested linkers contain repeated Gly and Ser residues, but may also contain an internal 8xHis tag to facilitate protein purification. To increase protein expression and solubility in the E.coli cytoplasmic space of FimH NPs, internal S_S bridge mutant FimH was used _L Constructs (C24 SC 65S) were also fused to ferritin and mI3 and tested for expression and solubility.

Materials and methods

Cloning and E.coli expression

The FimH-NP bacterial construct was a DNA string synthesized by Geneart, and cloned directly into pET15-tev, pET21 or pET22 (see Table 1) using the Takara fusion cloning kit. Other constructs were purchased as synthetic genes from Geneart, and the protein of interest was cloned directly into an expression vector (pTRC-HIS 2A of Life Technologies). All synthetic genes were optimized for E.coli expression and contained N-terminal, C-terminal or internal HIS tags to allow affinity purification of the protein. Proteins were expressed in BL21DE3T1r (NEB) or T7 buffer at 20℃for 24 hours using HTMC medium and IPTG induction.

After particle recovery, the particles were resuspended in lysis buffer cell lytic express (Merk) or B-Per solution (Pierce) for 1 hour at 25 ℃. After centrifugation, visible Inclusion Body (IB) particles appeared and were dissolved in 8M urea (U8M). Protein expression and solubility were assessed by SDS-page of samples collected from soluble fraction (S) and insoluble fraction (IB).

Recombinant proteins are produced in mammalian cells.

FimH-NP mammalian constructs (see Table 2) were synthesized by the Geneart company as synthetic genes in the pCDNA3.1 or pCDNA3.4 (Life Technologies) vectors. All sequences are codon optimized for expression in mammalian cells and contain an N-terminal leader sequence for secretion into the cell culture medium. This sequence is IgK mouse leader METDTLLLWVLLLWVPGSTGD [ SEQ ID NO:9], or IgK mouse leader followed by 15 additional charged residues AAQPARRARRTKLAL [ SEQ ID NO:78]. (FIG. 1B)

To produce recombinant FimH-NPs, the expression vector was transfected into an Expi293GNTI cell according to the manufacturer's instructions (Life Technologies). The Expi293F GnTI-cell line was derived from engineered Expi293F cells that did not have the activity of N-acetylglucosaminyl transferase I (GnTI) and therefore lacked complex N-glycans, resulting in uniform glycosylation of the recombinant protein.

Briefly, 30 μg of pCDNA-FimH-NPs expression vector was transfected into 30ml of culture containing 75X106 Expi293F cells using the ExpiFectamine 293 reagent. Cells were incubated at 37℃and 120rpm at 8% CO2, after 24 hours, the transfection enhancers 1 and 2 were added to the cells. The cells were further incubated at 37℃for 144 hours. A batch of cultures was harvested every 24 hours and analyzed for NA expression by SDS-PAGE and Western Blot (WB). 72 hours and 144 hours after transfection, cell cultures were centrifuged at 1000rpm for 7 minutes, the supernatants were harvested, pooled, clarified by centrifugation, filtered with a 0.22 μm filter and stored at-20 ℃ until purification.

The glycosylation of mammalian expressed antigens was examined using PNGase F proteomics grade (P7367, sigma) according to manufacturer's instructions.

Using standard protocols, 1:1000 dilution with anti-his-HRP antibodies from sigma, or with FimH from bacteria _L anti-FimH produced in mice by cys purified proteins _L Western blots were performed with cys antibody and secondary anti-mouse HRP antibody.

NPs were purified from the culture supernatant by Ni2+ affinity chromatography. Fractions of interest were pooled and concentrated with a rotary concentrator (Millipore Amicon Ultra) cut off at 100 kDa; sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to check the purity of the protein. Recombinant FimH-NPs and FimH-DG antigens were purified by preparative Size Exclusion Chromatography (SEC) equilibrated with PBS buffer.

All fractions collected were checked for FimH-NPs or FimH-DG protein content by SDS-PAGE and fractions of interest were pooled, filtered at 22 microns, aliquoted and stored at-20 ℃.

To assess the size and purity of the proteins, analytical SEC-HPLC and reverse phase RP-UPLC were performed. In addition, fimH-NPs were analyzed by dynamic light scattering to further determine molecular weight and nanoparticle assembly, and sequence identity of proteins was assessed by LC-MS.

Immunization with

Each group of 12 CD1 mice (females) was immunized with 15 micrograms of the candidate expressed in the mammalian or bacterial system with ASO1 adjuvant. All mice were vaccinated by subcutaneous injection (SC) with 200 microliters (PBS diluted) of antigen mixture or adjuvant alone three times. Blood was collected via the tail vein 0 days post-inoculation (pre-immunization), 21 days (post-I), 35 days (post-II) and 45 or 49 days (post-III).

Analysis of FimH-specific antibodies

Serum FimH-specific lgG was determined by enzyme-linked immunosorbent assay (ELISA). Briefly, 100. Mu.l of antigen (1. Mu.g/ml) was coated onto 96-well microwell plates to each well of a 96-well Nunc Maxsorph plate, and incubated overnight at 4 ℃. Mu.l (PVP) saturated buffer was added to each well and incubated for 2 hours at 37 ℃. The tertiary wells were washed with PBT. Next, 100. Mu.l of diluted serum was added to each well, and the plate was incubated at 37℃for 2 hours. Wells were washed three times with PBT. Mu.l of alkaline phosphatase conjugated secondary antibody serum diluted 1:2000 in dilution buffer was added to each well and the plate incubated for 90 minutes at 37 ℃.

The tertiary wells were washed with PBT buffer. Mu.l of substrate p-nitrophenyl phosphate was added to each well and the plate was left at room temperature for 30 minutes. Mu.l of 4N NaOH was added to each well and the OD 405/620-630nm was followed. Antibody droplets were quantified as dilutions of serum using a multi-mode microplate reader to an absorbance of 0.4OD.

BAI detection

Bacteria (uti89 wt_mcherry clone 2) were cultured in static liquid culture for 3 passages: growth conditions that induce FimH expression. BAI assay was performed under selected conditions: the bacterial density was 0,012OD/ml and the incubation time was 30 minutes. Bacterial adhesion was measured by microscopic analysis (OPERA Phenix). SV-HUC (ATCC) cells were cultured in SV-HUC complete medium: f12K (Thermo Scientific) supplemented with 10% fbs and antibiotics. Pre-infection medium: complete medium, without antibiotics.

Test serum (heat inactivated):

SV-HUC cells (3X 106 cells/ml, 95% viability) from 3T 75 flasks were trypsinized (X5 min,37 ℃). Cells were seeded in 96-well plates, 60 wells/plate at 3.5x104 cells/well (vf=200 ul/well), 5% co at 37 °c ₂ And (5) incubating. Bacterial preparation static liquid culture comprising three passages: UTI89 strain was inoculated into 20ml LB (125 ml flask) and incubated at 37 ℃ under O/N, static conditions. Such dilution/incubationPassaging was repeated three times.

The medium of SV-HUC cells was exchanged with pre-infection medium without antibiotics (200 ul/well).

2x serum solutions were prepared in U-bottom 96-well plates with either f12k medium or f12k+10% fbs, as shown below, and further serially diluted.

1 ml of 3 rd generation bacterial culture UTI89 mcherry Clone2 was transferred to a single tube and centrifuged at 4500 Xg for 5 minutes at room temperature. Bacteria were washed with PBS and precipitated. Finally, bacterial pellet was resuspended in 0.5OD600/ml with infection medium.

The infection method is as follows: media was aspirated off on each plate, 50 ul/sample of 2x serum/mannose (20% d- (+) -mannose) solution or infection media (ctrl positive and negative) was added, followed by 50 ul/sample of 2x inoculum or infection media (ctrl negative). Plates were incubated for 30 minutes and 15% to 0.06% serum dilution was added. At 37 ℃,5% CO ₂ Incubate for 30 min under conditions, remove medium, wash wells three times with PBS. Bacteria were immobilized with 4% formaldehyde (200 ul/well) solution. After incubation for 20 minutes, the fixative was removed and the samples were washed 3 times with PBS (200 ul/well). DAPI (62248, thermo scientific) solution was diluted 1:5000 in PBS and 100ul was added per well. The samples were incubated at room temperature for 10 minutes (in the dark). The DAPI solution was removed and PBS (200 ul/well) was added to each well. Samples were stored in the dark at 4℃for 3 hours at RT before imaging with OPERA Phenix. The full aperture area was acquired with a 10x air objective under the Alexafluor488 setup. Each region was Z-stack (4 planes) acquired. The data were analyzed using Harmony software. The total fluorescence area of the bacteria (individual objects. Ltoreq.100.mu.m2) was calculated as the value of the adhesion.

Results

FimH stabilized monomeric antigen and FimH stabilized nanoparticles are secreted in a mammalian expression system as soluble proteins and can be easily purified by IMAC

As a first attempt, several FimH NPs constructs have been generated and tested under different conditions. The T7 and pTac promoters of pETvector and pTrcHIs2A vectors, respectively, were used to test candidates Solubility of the antigen in E.coli. In addition, cytoplasmic and periplasmic expression was tested, as well as different E.coli strains such as T7 Shuffle expression that optimized disulfide bridge formation into the cytoplasmic space. To increase protein expression and solubility in the E.coli cytoplasmic space of FimH NPs, internal S_S bridge mutant FimH was also used _L Constructs were fused to ferritin and ml 3 and tested for expression and solubility.

However, none of the constructs resulted in expression of soluble proteins, indicating that the expression system of e.coli may not be the best choice for obtaining FimH nanoparticles. FimH _L Mutations in disulfide bridges within the domain did not significantly improve solubility, as only a weak band in the soluble portion was found by Western blotting analysis.

Coli is a prokaryotic expression system, which is strongly recommended for its low cost fermentation and simple process. However, proteins produced with E.coli may result in recombinant proteins being expressed predominantly in the form of inclusion bodies, which are insoluble, inactive and may require a complex refolding process in vitro (FIG. 2).

To overcome the insolubility problem in E.coli, the inventors decided to adapt the mammalian EXPI293F expression system. First, fimH sequences were analyzed as N-and O-glycosylation sites that may be responsible for glycosylation. FIG. 3 reports the positions of putative N-glycosylation sites. No O-glycosylation sites were detected (data not shown).

In order to express bacterial proteins in mammalian cells, as much as possible to reduce the glycosylation that occurs in the system compared to the E.coli system, the present inventors used a genetically mutated EXPI293F cell line, called the Expi293F GnTI (thermo filter). The cell line was derived from engineered Expi293F cells, but it was devoid of the activity of N-acetylglucosamine transferase I (GnTI) and therefore lacks complex N-saccharides, resulting in uniform glycosylation of the recombinant protein.

With a secretory murine Ig-kappa chain leader sequence containing from E.coli strain 536 and/or J96 (plus FimH) _L Additional amino acids at the N-terminus of the domain (in some constructsThe method comprises the steps of carrying out a first treatment on the surface of the Table 1), fimH donor chain (FimH-DG) stabilized full length FimH proteins alone or fused to protein NPs (ferritin, mI3, IMX313, coat protein and HBc) were used to transfect EXPI293 GNTI cells. The accumulation of secreted recombinant proteins was described by measuring their expression in culture supernatants 72 hours and 144 hours post-transfection by WB and SDS-PAGE. Both analyses showed that several constructs could achieve high levels of FimH soluble expression, while others could not. Expressed and soluble FimH-DG stable proteins and FimH-NPs containing a C-terminal 6xHis tag or an internal 8xHis tag were purified from medium pooled at 72 hours and 144 hours using ionic metal fixation chromatography and preparative SEC chromatography. SDS-page analysis of proteins produced in mammalian expression systems found that they were of higher molecular weight than the corresponding bacterial proteins, indicating that they were glycosylated. Thus, two constructs lacking putative residues involved in N-glycosylation were mutated, resulting in FimH_DNKQ_DG_deglycol and FimH_PGDGN_DG_deglycol, containing the additional amino acid N-Term and the following mutations N28S, N91D, N249D, N D (Table 1).

Western blot analysis of supernatants of mammalian expression constructs containing N-terminal additional amino acids found expression bands corresponding to FimH_DNKQ_DG, fimH_PGDGN_DG and FimHC complexes. In contrast, fimH-. DELTA.GG-PGDGN-DG (connection FimH is missing) _L And FimH _P Gly residues of (C) were not detected 3 days and 6 days after transfection (FIG. 4). The protein characteristics of the purified products are reported in FIGS. 13-16.

The construct FimH_PGDGN_DG_ferritin (strain 536;995 SI) containing the N-terminal additional AA was successfully expressed and purified. In contrast, all FimH non-fimgs donor chain stable constructs (936 SI) -FimH-IMX313 j96; (935 SI) -fimh_mi3j 96; (929 SI) -FimH _L HIS-mI3 j96 (all containing N-terminal additional amino acids) was not detected in the culture supernatant.

Treatment of fimh_dg_pgdgn_imx313 and fimh_dg_pgdgn_ferritin of strain J96 with PNG enzyme found that fimh_dg_pgdgn_imx313 was converted at the correct MW in the treated samples, indicating that the protein was glycosylated in mammalian cells. FimH_DG_PGDGN_ferritin from the J96 strain was not detected in both untreated and treated PNG enzyme samples, indicating that the protein was degraded. Fimh_pgdgn_dg_ferritin (strain J96) (1000 SI) was not purified from the supernatant of 3 and 6 days of collection, even if the protein was detected immediately after sample collection, due to degradation and no construct was obtained (fig. 5A-C).

In addition, the predicted N-saccharides reported in FIG. 3 were mutated in serine or aspartic acid. The resulting fimh_dnkq_dgdeglycol candidates showed higher peptide map coverage compared to the WT sequence. This result suggests that possible glycosylation may occur at the correspondence of these specific mutated amino acids (fig. 6).

In addition, the representative constructs reported in FIG. 7 have the additional N-terminal amino acids (short leader sequence) removed upon expression. FimH-DG_PDGDN_ferritin (strain 536, additional N-terminal AA) was obtained by RP-UPLC in 88% purity. (998 SI) FimH_PGDGN_DG-HIS-IMX313 j96 was also expressed well and successfully purified.

All of these constructs were expressed in cell culture as secreted soluble proteins and further purified as described previously. anti-FimH prepared with bacterial stabilizing proteins _L Western blot analysis of supernatants by cys antibodies identified all mammalian expressed test NPs (FIG. 7).

To confirm proper assembly of FimH-Nps, purified proteins were checked by analytical SE-HPLC and DLS analysis. In SE-HPLC, they elute as a single large peak that is not sharp. The calculated MW of FimH-DG-PGDGN-ferritin Nps is consistent with Nps consisting of 24 subunits, as demonstrated in the DLS analysis, based on a comparison of the elution volume (Ev) of ferritin Nps with Ev of the Molecular Weight (MW) standard run under the same conditions.

The construct FimH-dg_pdgdn_ferritin SL (sequence from strain 536 or J96, lacking the additional N-terminal AA) resulted in high expression, with final purity estimated by RP-HPLC.

FimH _L The NPs construct was also successfully purified, and biochemical characterization confirmed the formation of NPs for (1095 SI) FimH _L -HIS-Fer 536, consisting of 24 subunits, for (1096 SI) FimH _L HIS-Mi 3J 96 consists of 60 subunits.

Visualization of FIMH-DG NPs produced

An additional confirmation was that the recombinant FimH-dg_pdgdn_ferritin extra AA (fig. 9A-B) fusion protein fimh_dg_pgdgn-HIS-ferritin 536 short leader sequence and fimh_pgdgn_dg_his-ferritin j96 were produced in mammalian expression systems, and that the formation of stable, correctly assembled NPs was obtained by visualizing the purified protein using negative staining electron microscope TEM. As shown in fig. 8B, (995 SI) fimh_pgdgn_dg_ferritin 536, samples containing N-terminal additional AA appear as a uniform population of octahedral particles in different directions, decorated by spikes. The bare ferritin particles showed a diameter of 13 nm, while the spiked ferritin showed a diameter of 30-32 nm. The difference in diameter (8.5 nm) corresponds to the length of FimH (calculated from FimH model). In addition, (1142 SI) fimh_dg_pgdgn-HIS-ferritin 536 was folded correctly and decorated with peaks of 8 FimH trimers. Bare ferritin particles were not present in the sample. The particles exhibit diameters of 30-32 nanometers. (1042 SI) FimH_PGDGN_DG_HIS-ferritin j96 samples present a mixed population of NPs with individual or aggregated proteins, correctly folded spike NPs present eight spikes, folded NPs with multiple spikes and incorrectly folded NPs. No naked ferritin particles were detected (fig. 8D).

(1095SI)FimH _L HIS-Fer 536 and (1096 SI) FimH _L (J96) The cryo-electron microscope NS-EM (negative staining) of mI3-his shows that NPs expressed in mammalian systems are fully assembled. FimH _L HIS-Mi 3J 96 (1096 SI) presents correctly folded nanoparticles, with icosahedral shape, highly symmetric 40nm, with spike decoration, with few aggregates (fig. 9A and 9B). In addition, 1185SI and 1184SI FIMH_DG_PGDGN_536-wrap proteins both have short leader sequences at the N-terminus and can be assembled correctly (FIGS. 9C and D). Constructs containing stabilized FimH fused to IMX313 were also successfully purified (1043 SI) FimH_DG_PGDGN_IMX313_HIS J96 and (998 SI) FimH_PGDGN_DG-HIS-IMX 313J 96, and biochemical characterization confirmed the formation of High Molecular Weight (HMW) species. However, TEM analysis of these constructs showed thatOnly aggregated protein was present (data not shown).

Three-dimensional reconstructed structural features of recombinant FimH-DG-ferritin NPs

To generate (995 SI) the three-dimensional structure of the assembled octahedral particles of fimh_pgdgn_dg_ferritin (FimH sequence from 536 strain), a single particle reconstruction method was applied to the TEM images. The FimH-dg_pdgdn_ferritin nanoparticles of a single box (box size 64x64 pixels) were first bandpass filtered to increase signal to noise ratio, then rotation and translation aligned, and finally centered before MSA classification. FIG. 10A shows the selection of the average number of the most abundant 2D classes of FimH-DG_PDGDN_ferritin, representing the different orientations of particles on carbon film support. The resulting 3D-EM structure of soluble FimH-dg_pdgdn_ferritin (fig. 10B) demonstrated that the structure consisted of a highly symmetrical octahedral cage structure with three "anchor" appendages on the 3 layers.

FIMH-DG stable protein and FIMH-DG_PGDGN-ferritin NPs are highly immunogenic in mice

To evaluate the immunogenicity of the candidates expressed in mammalian systems (fimh_pgdgn_dg, fimh_dnkq_dg, fimh_dnkq_dgdegyc and fimh_pgdgn_dg_ferritin), the FimH lectin domain expressed in e.coli (FimH _L ) As a coating, single plasma of immunized mice was analyzed by ELISA detection. Overall, all candidates elicited IgG responses. Fimh_pgdgn_dg and fimh_pgdgn_dg_ferritin showed similar IgG responses, whereas NP candidates showed more uniform and compact responses after II. This result indicates that NPs produce an earlier effective response than are candidates for recombinant protein expression. Immunization with two different doses (15 ug and 3 ug) of fimh_pgdgn_dg_ferritin showed comparable IgG response at the lower dose (3 ug) and the higher dose (15 ug), indicating that ferritin nanoparticles carrying recombinant fimh_dg_pgdgn protein were well immunogenic even at the lower test dose of 3 ug. Furthermore, the immune response caused by the ferritin form at the second dose was less diffuse than other candidates, including the corresponding FimH construct lacking the nanoparticle domain (fig. 11).

FimH-DG stable candidates (produced in mammalian systems) show a greater ability to inhibit bacterial adhesion than recombinant (bacterially produced) forms.

Serum against FimH stabilization candidates was tested for its ability to block bacteria from adhering to human bladder cells using an in vitro bacterial inhibition assay. Antibody ratio fimh_dnkq_dg or bacteria produced FimH against candidate vaccines fimh_pgdgn_dg and fimh_pgdgn_dg_ferritin _L The cys candidate vaccine is more effective in inhibiting bacterial adhesion to urethral cells. These results indicate that FimH-based stabilized candidate vaccines expressed in mammalian systems have great potential for further development of vaccines. Furthermore, the linker used to stabilize FimH plays a crucial role in the function of the produced antibodies (fig. 12), and constructs with PGDGN linkers are better at inhibiting bacterial adhesion.

Conclusion(s)

Our study investigated novel FimH candidates stabilized with donor chain strategy. Candidate vaccines are produced in the form of individual recombinant proteins, or assembled into nanoparticles carrying FimH subunits. Expression of soluble antigens has been achieved by transient transfection of EXPI293-GNTI cells using mammalian expression systems, as expression in E.coli results in insoluble products. To our knowledge, the use of such expression systems has never been used before for the production of bacterial proteins. In this case, mammalian expression systems increase protein solubility because FimH expressed in e.coli is insoluble under all test conditions. This expression system allows the production of stable fimh_dg antigen, which is soluble in both, as well as different FimH nanoparticles (FimH _L -MI3、FimH _L Ferritin and fimhh_dg_pgdgn-ferritin). In contrast, when unstabilized FimH was fused to NP, no expression was detected, indicating that stabilization by the fimgs complementary strand is necessary to produce full length isolated FimH protein in mammalian cells and to display antigen on ferritin NP. Deletion of two glycine residues, i.e. FimH _L And FimH _P The native linker between, resulting in the absence of FimH-. DELTA.GG-PGDGN-DG with an additional N-terminal AA, indicates that this deletion is detrimental to protein stability。

SDS-PAGE compares MW of bacterial insoluble proteins and the corresponding mammalian expressed proteins, showing that they have different molecular weights, indicating that mammalian proteins are glycosylated, as demonstrated by PNG enzyme treatment. All constructs with leader sequences (IgK murine leader sequence alone or with additional amino acids) successfully secreted FimH constructs into the expression medium. However, neither constructs with additional amino acids produced more uniform nanoparticles (fig. 7 and 9), nor naked ferritin NPs was observed.

Structural data confirm that all nanoparticles are correctly assembled, fimH peaks are detected at the surface of ferritin (24 peaks) and mI3 nanoparticles (60 peaks).

Our data indicate that FimH stabilizing candidates expressed into mammalian systems are immunogenic and that the resulting antibodies are able to inhibit bacterial adhesion to urothelial cells.

Effect of AS01 adjuvant: improvements to FimHC and FimH-DG

To assess the contribution of the PHAD and AS01 adjuvant systems to humoral responses, the FimHC protein complex was used AS a model antigen and was formulated in accordance with Langermann S, et al 1997Apr 25;276 (5312) expression was carried out as described in 607-11. IgG antibodies produced after vaccination were determined and relative titers were plotted AS a function of the amount of MPL contained in the PHAD and AS01 formulations. Overall, AS01 induced a higher total IgG response than PHAD in mouse serum (post-3) and urine (post-2 and-3). In addition, AS01B using 5 μg-MPL showed the same IgG levels AS compared to PHAD containing 12.5 μg-MPL (FIGS. 17A and 17B).

Improved antigen design and adjuvant formulation elicited a functional immune response after 2 doses (rather than 3)

To evaluate the FimHC complex and the stable His-tag form of FimH (FimHDG, i.e., fimH-PGDGN-DGN-DG, wherein DG represents the donor chain complementary peptide from FimG, fimHDG-ferritin, i.e., fimH-PGDGN-DGN-DG-linker (with His tag) -ferritin (from H.pylori)), different doses of antigen (0.55. Mu.g or 1.6. Mu.g) with PHAD or AS01 adjuvant were used for mouse immunization, the expression of FimHC complex AS Langermann S, et al, science.1997Apr 25;276 (5312): 607-11 describes the total IgG titer specific for FimH in bacteria or mammalian systems expressed in immune mouse serum and urine (determined by ELISA) after the second and third vaccine injections, the IgG titers (FIG. 18. Post-2 and post-3 antibodies were formulated) were used to elicit a significantly better response in the same dose of PHAD than the PHAD AS shown in the second and third vaccine injections than in the PHAD 01, and the PHAD (PHAD) with the same dose AS shown in the second vaccine system.

Finally, both stable FimH candidates purified in mammalian cells (FimHDG-HisTag mammal and FimHDG-HisTag ferritin) showed a higher response than FimHDG expressed in e. Both 1.6 and 0.55 micrograms of mammalian FimHDG constructs induced IgG levels that tended to stabilize after immunization 2 and 3. Furthermore, fimHDG elicited a higher response at the second administration (9.7 and 3 geometric mean ratios, respectively) at the two tested protein doses compared to 3 doses of FimHC-PHAD (fig. 18A). Antibody responses to FimHDG were assessed in urine collected with the immunization groups at higher protein doses following doses 1, 2 and 3. As observed in the test serum, the IgG titers measured were higher in mice vaccinated with the mammalian FimHDG formulation (fig. 18B).

For the selected immune group, the total IgG response of post-I was also determined. After dose I, fimHDG-ferritin nanoparticle induced GMT was twice as high as FimHDG without ferritin (at any Ag dose), although variability was higher than post-2 and post-3 responses (resulting in a 95% CI inclusion of 1).

The mammalian form with ASO1 adjuvant induced a higher IgG response after doses I and II (GMRs were observed to vary from 7.1 to 60.8, the lower limit of 95% ci was all above 1) compared to the bacterially derived antigen, while the response after the third dose was similar (GMRs were observed to be approximately 1.5 times) (fig. 19).

Comparison of different linkers (mammalian/bacterial) of constructs in terms of relative efficacy

To investigate the effect of different linkers, fimHDG candidates expressed in bacterial and mammalian systems were compared with FimHC in terms of inhibition of urothelial cell (BAI) adhesion by bacteria. FIG. 20 shows that all FimHDG constructs are more functional than FimHC, independent of the expression system (bacterial or mammalian) used for expression. Interestingly, fimHDG constructs carrying PGDGN linkers were more efficient than DNKQ constructs. These data indicate that analogs can stabilize FimH in different conformations, resulting in different functional antibody responses. The BAI assay is envisaged as a multi-dilution assay, with the tested samples being plated with a reference serum pool at different concentrations to estimate the dose-response curve. Before calculating the titer, the signal was normalized between 0% and 100%. Titers are expressed as the relative efficacy (RP) of the test samples against the reference cell, comparing the corresponding dose-response curves. Specifically, RP is calculated taking into account logarithmic dilution and fitting a 4-parameter logical (4 PL) constraint model (described in european doctor article chapter 5.3) in which the slope factors of the standard and test samples, the upper and lower asymptotes, are constrained to be equal. RP is calculated as reference value and sample EC ₅₀ The ratio between. EC (EC) ₅₀ Is calculated from the inflection point of the 4PL constraint and reverse transformed (inversely proportional). The model requires that the curves of the reference and sample have the same slope factor (parallelism) and the same maximum and minimum response levels (linearity) in the extreme parts. Suitability of parallelism and linearity assumptions is evaluated at each link, evaluating the P-value of the test parallelism deviation, the P-value of the test linearity deviation, and the slope ratio between the reference and sample.

FimH-DG induces a functional immune response in BAI, HAI and conformational mAb binding

In addition, the antibody ability of anti-FimHDG antibodies to inhibit adhesion of ExPEC was evaluated using bacterial inhibition assay (BAI). The data on antibody function show that serum produced against FimHDG constructs for bacteria and mammals shows a higher ability to inhibit bacterial adhesion than FimHC reference serum. Among the candidates tested, fimHDG-ferritin showed at least 10-fold function compared to the homologous FimHDG construct (fig. 21). Similar results were obtained by performing HAI tests. The FimHC complex is expressed as Langermann S, et al science 1997Apr 25;276 (5312) 607-11. "FimHDG" refers to FimH-PGDGN-DG, wherein DG represents the donor strand complementary peptide of FimG, and "FimHDG-ferritin" refers to FimH-PGDGN-DG-linker (His-tagged) -ferritin (from helicobacter pylori). The BAI assay and calculation of relative efficacy were performed as described in the examples above.

Binding of FimHDG to mAb962

To investigate the interaction of FimHDG (i.e., fimH-PGDGN-DG, where DG represents the donor strand complementary peptide of FimG) and mAb926 (Dagmara I. Kisiela et al (2015)), SPR analysis was performed. The monomeric form of FimHDG obtained from bacterial or mammalian systems (i.e., fimH-PGDGN-DG, wherein DG represents the donor strand complementary peptide to fimgs) shows similar binding to the mAb but is slightly different in the binding and dissociation curves. FimHDG-ferritin (FimH-PGDGN-DG-linker (His-tagged) -ferritin (from helicobacter pylori)) produced a more stable interaction compared to the monomeric form, probably due to the increased heat of multimeric effect. In contrast, mAb926 interacted less with FimHC than FimHDG, indicating that the latter stabilized in a pre-binding conformation as expected. In fact, mAb926 was generated against a FimH stabilized lectin domain whose mannose binding capacity (pre-binding conformation) was significantly reduced (Dagmara I. Kisiela et al, (2013), whereas FimC stabilized FimH in its extended post-binding-like form (Sauer et al, (2016), nature Communications volume 7,Article number:10738) (FIG. 22).

Evaluating new joints

Recombinant proteins are produced in mammalian cells.

To produce FimHDG (i.e., fimH-PGDGN-DG, wherein DG represents a donor strand complementary peptide from fimgs) and FimHDG-nanoparticles without internal or C-terminal repeating His residues, new constructs were designed with different linkers inserted between the fimh_dg gene and the Nanoparticle (NP) monomers. FimH-NP mammalian constructs were synthesized from Geneart or Twist as synthetic genes in pCDNA3.4 (Life technologies) vectors. All sequences are codon optimized for expression in mammalian cells and contain an N-terminal leader sequence for secretion into the cell medium. This sequence was the leader METDTLLLWVLLLWPGSTG of the IgK mice, or the leader of the IgK mice followed by aspartic acid residue METDTLLLWVLLLWPGSTGD to assess the contribution of this residue to efficient protein secretion. To produce recombinant FimH-NPs, expression vectors were transfected into Expi293 cells and Expi cho cells according to the manufacturer's instructions (Life Technologies), and culture supernatants were collected 5 days after transfection. Purification of the protein is achieved by ion exchange chromatography and preparative SEC purification steps.

Nano DSF analysis

To evaluate the fluorescence monitoring expansion of FimHDG constructs, we performed nano DSF analysis. Samples were manually loaded into nano-DSF standard capillaries in triplicate and transferred to a promethaus nt.48 nano-DSF device. For intrinsic tryptophan fluorescence measurements, the emission of tryptophan fluorescence was measured at 330 nm, 350 nm and their ratios (350 nm/330 nm) using an excitation wavelength of 280 nm. Data were analyzed using Prometheus PR. The control software (NanoTemper Technologies) analyzes the data and plots temperature using fluorescence comparison.

SPR analysis

FimHDG constructs were diluted with running buffer HBS-EP+ (0.01M HEPES,0.15MNaCl,0.003M EDTA and 0.05% v/v Surfactant P20) and captured on the surface of sensor chip NTA, which was previously activated by injection of 0.5mM Ni2+ ion solution and washed with 3mM EDTA. mAbs were captured at a concentration of 20ug/ml on the surface of the CM5 sensor coated with secondary anti-mouse IgG Fc. A fixed concentration of 50nM for each sample was injected onto the surface of the sensor chip for 180 seconds. The dissociation process lasted 600 seconds. Finally, the sensor chip was regenerated with 10mM Glycine-HCl pH 1.7. Experiments were performed using a Biacore T200 instrument (GE Healthcare) and analyzed using Biacore T200 evaluation software 3.0 (GE Healthcare).

Results:

full length FimH-DG stable unlabeled proteins containing a single secretory murine Ig-kappa chain leader sequence fused to the protein NPs (ferritin) were used to transfect EXPI293 and EXPI cho cells. Their expression in the culture supernatant 5 days after transfection was measured by SDS-PAGE to characterize the accumulation of secreted recombinant proteins. Analysis showed that high levels of FimHDG soluble expression could be obtained in the empty 293 cells and empty cho constructs (panel a). The protein was further purified from the culture supernatant and biochemically identified with previously purified His-tagged FimHDG and bacteria refolded FimHDG. In SDS-Page and SE-UPLC, the unlabeled FimHDG obtained from EXpi293 and ExpiCHO cells had good purity levels. Due to glycosylation occurring in mammalian cells, the molecular weight of the protein is higher in SDS-Page (about 42 kD) compared to bacterial cells, and is theoretically 31kD (FIG. 23A).

The folding of the unlabeled purified FimDG was analyzed by nano-DSF to obtain the melting temperature and compared with the temperature obtained for FimHDG-HIS. FimH-DG shows good thermal stability in nano-DSF with two thermal transitions relative to lectin (Tm 1) and Pi Lin (Tm 2) domains, whereas His-tagged FimHDF molecules show only one transition, possibly due to different folding. Furthermore, the unlabeled proteins show a higher stability of the Pi Lin structural transition (higher melting temperature value) than His-tagged molecules. Fig. 23B. This different folding of the His-tag construct compared to the non-tagged fimhdg may be due to the lack of the N-terminal aspartic acid residue and the C-terminal His-tag.

SPR analysis of the mammalian-produced unlabeled FimHDG construct (fig. 23C) showed that mAb 926 could bind to the construct differently than the His-tagged FimHDG protein. Furthermore, the unlabeled FimHDG protein showed weak interactions with mAb vh_475 and mannose, in contrast to His-tagged FimHDG, consistent with the different folding of the unlabeled construct with His-tagged proteins observed.

To produce unlabeled FimHDG-ferritin NPs, the His tag has been replaced with a different linker to isolate FimHDG molecules and nanoparticle monomer sequences. The designed and tested linker is composed of flexible residues such as glycine and serine, so that the linked protein domains can move freely relative to each other. We tested linkers of different lengths, longer linkers can ensure that two adjacent domains do not sterically interfere with each other, but can be more easily degraded. Linker akfvaaawtlkaaa, also known as pan HLA DR binding epitope (PADRE), is a polypeptide that activates antigen-specific-cd4+ T cells, which is proposed as a carrier epitope suitable for the development of synthetic and recombinant vaccines. The joints GGGGSLVPRGSGGGGS and EAAAAKEAAKAAKAAKA are rigid joints. The linker AEAAAKEAAAKEAAKA is stabilized by Glu-Lys salt bridges to form an alfa helix (Marquee & Baldwin, 1987). Since unlabeled FimHDG and His-tagged FimHDG also differ in the initial aspartic acid residues, some linkers were also tested without and with the N-terminal aspartic acid residues. Plasmids encoding the different constructs were used for the Expi293 transfection. 5 days after transfection, only constructs starting with the N-terminal aspartic acid residue (D) (no tag or his-tag) showed a SDS-page visible secretory protein band in the supernatant (FIG. 23D). Construct fimhdg_his_ferritin 1619SI and 1042SI have the same sequence except for the original aspartic acid residue, but only construct 1042SI can be secreted and present in the culture supernatant of EXPI, confirming the importance of this residue at the N-terminus of FimHDG for achieving efficient secretion of FimHDG-ferritin nanoparticles. Of the different linkers tested, only FimHDG-ferritin unlabeled 1623SI and 1627SI constructs from e.coli strains J96 and 536 had the original aspartic acid residue, resulting in secretion. To assess the expression of unlabeled FimHDG-ferritin 1433SI without the initial asparagine residue, we also performed Western blotting analysis to confirm that the protein was expressed in the pellet fraction, rather than only in the culture supernatant.

E. Silicon-based stability study of E.coli ferritin

Materials and methods

Sequence and structure based evolution constraint factor of E.coli ferritin design

The aim of this study was to make a symmetrical system design, such as self-assembled protein nanoparticles. This approach utilizes a combination of computational physics-based algorithms and evolutionary bioinformatics to introduce stable mutations. To achieve this, asymmetric units or monomers of E.coli ferritin (PDB: 1EUM WorldWideWeb (www). Rcsb. Org/structure/1 EUM) were motif designed, thermodynamic designs were made using Rosetta suite (Alford RF, et al J Chem Theory Comput.2017Jun13;13 (6): 3031-3048), and non-redundant evolutionary homologs (PSI-BLAST, altschul SF, et al nucleic Acids Res.1997Sep1;25 (17): 3389-402) were used to limit mutation space. The designed model is constrained within a symmetrical framework (DiMaio F, et al PLoS one.2011;6 (6): e 20450) to optimize the energy of the protein subunits at the geometric interface. A list of silicon stable sequences was then generated on the structural bioinformatics tool PROSS (Goldenzweig A, et al mol cell 2016, 7, 21; 63 (2): 337-346) (SEQ ID NOS: 149-152 and FIG. 24).

Expression and purification of proteins

The genes encoding the different mutants of E.coli-stable ferritin and wild-type ferritin were cloned into the pET15TEV vector containing an N-terminal 6XHIS tag and a TEV cleavage site. Plasmids encoding the different constructs were transformed into E.coli BL21DE3t1r competent cells. For protein expression, cells were grown in HTMC ON at 20℃and induced with 1mM IPTG at 20℃for 24 hours. Using CeLytic ^TM Express (Sigma Aldrich) the soluble proteins were extracted by chemical cleavage and purified by a nickel chelating column, followed by SuperdexPreparative size exclusion chromatography was performed on an Increase 10/300GL column (Cytiva) and purity was confirmed by SDS-PAGE (FIG. 25).

Transmission Electron Microscope (TEM) analysis

Negative staining: mu.l of the sample (diluted at 20 nanograms per microliter) was loaded onto a glow discharge copper 300 square grid for 30 seconds. After the excess was aspirated, the grid was negatively stained with Nano-W stain (Ted Pella, inc) for 30 seconds. Samples were analyzed with Tecnai G2 spirit and images were obtained with a Veleta CCD (FIG. 26).

ThermoFluor assay

The ThermoFluor assay is a rapid, temperature-based assay for assessing protein stability. In this method, each sample was diluted to a final concentration of 0.2mg/ml, and 4 μl of SYPRO Orange dye 1000X (Molecular Probes) was added, using buffer solution to a final volume of 40 μl. This mixture was transferred into wells of a 96-well thin-walled PCR plate (Bio-Rad) and water was added to the control sample. Each sample was analyzed in triplicate. The melting point (Tm) of each protein is determined by increasing the scan rate from 25℃to 100℃at 1℃per minute, and fluorescence measurements are made at each 1℃step. The unfolding curve and melting temperature were monitored by quantitative PCR thermal cycler (Stratagene). All DSF experiments were repeated three times. Derivatives of fluorescence intensity are plotted as a function of temperature, and reported Tm is the inflection point of a sigmoid curve determined using GraphPad Prism software (fig. 27).

Results

Recombinant production of silicon-stabilized E.coli ferritin nanoparticles

In order to obtain stable nanoparticles of specific antigen presenting E.coli stability (FimHDG, i.e. FimH-PGDGN-DG, where DG stands for the donor strand complementary peptide of FimG) from E.coli, a native ferritin scaffold was selected for repeated presentation of FimH and was computationally optimized. The Rosetta-based design approach maintains octahedral symmetry and focuses on the interface between the monomer and the other 23 chains in the symmetric system (fig. 24). This strategy of presenting E.coli specific antigens (FimH) from E.coli is a rational approach to show repeated preservation of species or genus specific designs of antigens by using natural scaffolds.

Coli WT ferritin and four mutants, representing all of the silicon stabilizing sequences produced by PROSS (SEQ ID NOS: 149-152), were highly expressed and soluble in E.coli cell lines as recombinant His-tagged proteins (FIG. 25). The construct was successfully purified by an affinity purification step followed by preparative size exclusion chromatography. Peaks corresponding to the high molecular weight fractions of all constructs were collected and further analyzed by electron microscopy to assess the correct formation of uniform and well-structured nanoparticles. From TEM analysis, all samples formed correctly folded ferritin nanoparticles, except mutant 2.5 which had a non-uniform morphology (fig. 26).

To determine the most stable E.coli ferritin nanoparticle, the thermostability of the recombinant ferritin constructs (WT, 0.5, 2, 6) was assessed by Differential Scanning Fluorometry (DSF) using Sypro Orange, which binds to hydrophobic residues and detects their exposure upon protein expansion. Ferritin shows very high thermal stability, as expected for protein nanocages, the first expansion transition is detected around 74-76 ℃. The DSF analysis showed that the e.coli mutant (0.5) protein exhibited the highest heat expansion transition, which, based on this increase in stability, resulted in its selection as the first construct for fusion with FimHDG antigen.

Mammalian production of E.coli-stabilized ferritin displaying FimHDG antigen

To test whether stable and native ferritin nanoparticles can act as scaffolds for displaying FimHDG antigen (i.e., fimH-PGDGN-DG, where DG stands for the donor strand complementary peptide from fimgs), and as a surrogate for helicobacter pylori ferritin, the sequence of FimHDG (containing the secretion sequence Igk) was genetically fused to the gene of stable ferritin (mutant 0.5). The two molecules are separated by a linker containing a repeating histidine sequence to affinity purify the recombinantly secreted nanoparticle in mammalian cell culture supernatant. This construct was used to transfect Expi293 Gnti cells and the accumulation of secreted recombinant protein was described by evaluating the expression levels in culture supernatants 5 days post-transfection by Western blot analysis using anti-His antibodies. The analysis showed that FimHDG-ferritin (mutant 0.5) nanoparticles were successfully secreted in the cell supernatant. The purified FimHDG-ferritin (mutant 0.5) nanoparticles were observed by transmission electron microscopy, confirming the correct morphology of ferritin stabilized nanoparticles and the surface display of FimHDG antigen, size of about 20nm (fig. 28).

This data suggests that stable escherichia coli ferritin nanoparticles displaying FimHDG can be successfully produced in mammalian cells, suggesting that it is possible to design nanoparticles with antigens and scaffolds that are both native to the pathogen of interest.

Table 1 (a): fimH-NP for bacterial testing

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Table 1 (B) FimH is expressed as a single recombinant protein in E.coli

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Table 2: mammalian-expressed FimH as a single recombinant protein and nanoparticle

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Table 3–construct nucleic acid sequences

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Claims

1. A polypeptide whose amino acid sequence comprises or consists of:

(a) FimH; or variants, fragments and/or fusions of FimH

(b) The donor strand is complementary to the amino acid sequence,

wherein (b) is downstream of (a).

2. A polypeptide comprising or consisting of the amino acid sequence X- (a) -L- (b) -Y, wherein "(a)" is a FimH polypeptide; or variants, fragments and/or fusions of FimH; "L" is an optional first linker; "(b)" is the donor strand complementary amino acid sequence, "X" is the optional N-terminal amino acid sequence; "Y" is an optional C-terminal amino acid sequence, wherein "Y" is not derived from FimC or FimH or a fragment thereof.

3. The polypeptide of claim 1 or 2, wherein (a) comprises or consists of:

(B) With SEQ ID NO:1 (Genbank accession number: ELL41155.1 (FimH of E.coli J96)), SEQ ID NO:2, SEQ ID NO:100 (Genbank accession number: ABG72591.1 (FimH of UPEC 536)), SEQ ID NO:101,SEQ ID NO:102 (Genbank accession number: AAN83822.1 (FimH of CFT 073)), SEQ ID NO:103,SEQ ID NO:104 (Genbank accession number: AJE58925.1 (FimH of E.coli 789)), SEQ ID NO:105,SEQ ID NO:106 (Genbank accession number: AAC 35864.1), corresponding to the nucleic acid sequence AF089840.1 (FimH of IHE 3034), or SEQ ID NO:107 comprising an amino acid sequence of 1 to 10 single amino acid changes, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 single amino acid changes,

4. The polypeptide of any one of the preceding claims, wherein one or more amino acids known or predicted to be N-glycosylated or O-glycosylated are substituted with serine (S), alanine (a), aspartic acid (D), or glutamine (Q).

5. The polypeptide of claim 4, wherein (a) comprises one or more of the following amino acid substitutions relative to SEQ ID No. 2: N28S, N91D, N249D, N256D, or one or more amino acid substitutions, e.g., one, two, three or four amino acid substitutions, at positions in SEQ ID NOS: 101, 103 and 105 corresponding to those positions in SEQ ID NO: 2.

6. The polypeptide of any one of the preceding claims, wherein (b) comprises or consists of:

(i) 6-28 amino acids of SEQ ID NO. 3; or fragments and/or variants thereof, or

(ii) 8-36 amino acids of SEQ ID NO. 4; or a fragment and/or variant thereof.

7. The polypeptide of claim 6, wherein the 6-28 amino acids of SEQ ID No. 3 correspond to:

(i) Amino acids 1-28 of SEQ ID NO. 3,

(ii) Amino acids 2-27 of SEQ ID NO. 3,

(iii) Amino acids 3-26 of SEQ ID NO. 3,

(iv) Amino acids 4-25 of SEQ ID NO. 3,

(v) Amino acids 5-24 of SEQ ID NO. 3,

(vi) Amino acids 6-23 of SEQ ID NO. 3,

(vii) Amino acids 7-22 of SEQ ID NO. 3,

(viii) Amino acids 8-21 of SEQ ID NO. 3,

(ix) Amino acids 9-20 of SEQ ID NO. 3,

(x) Amino acids 10-19 of SEQ ID NO. 3,

(xi) Amino acids 11-18 of SEQ ID NO. 3, and

(xii) Amino acids 12-17 of SEQ ID NO. 3.

8. The polypeptide of claim 6, wherein 8-36 amino acids of SEQ ID No. 4 correspond to:

(i) Amino acids 1-36 of SEQ ID NO. 4; or fragments and/or variants thereof,

(ii) Amino acids 2-35 of SEQ ID NO. 4; or fragments and/or variants thereof,

(iii) Amino acids 3-34 of SEQ ID NO. 4; or fragments and/or variants thereof,

(iv) Amino acids 4-33 of SEQ ID NO. 4; or fragments and/or variants thereof,

(v) Amino acids 5-32 of SEQ ID NO. 4; or fragments and/or variants thereof,

(vi) Amino acids 6-31 of SEQ ID NO. 4; or fragments and/or variants thereof,

(vii) Amino acids 7-30 of SEQ ID NO. 4; or fragments and/or variants thereof,

(viii) Amino acids 8-29 of SEQ ID NO. 4; or fragments and/or variants thereof,

(ix) Amino acids 9-28 of SEQ ID NO. 4; or fragments and/or variants thereof,

(x) Amino acids 10-27 of SEQ ID NO. 4; or fragments and/or variants thereof,

(xi) Amino acids 11-26 of SEQ ID NO. 4; or fragments and/or variants thereof,

(xii) Amino acids 12-25 of SEQ ID NO. 4; or fragments and/or variants thereof,

(xiii) Amino acids 13-24 of SEQ ID NO. 4; or fragments and/or variants thereof,

(xiv) Amino acids 14-23 of SEQ ID NO. 4; or fragments and/or variants thereof,

(xvi) Amino acids 16-23 of SEQ ID NO. 4; or a fragment and/or variant thereof.

9. The polypeptide of any one of the preceding claims, wherein (b) comprises or consists of:

(A) The amino acid sequence of SEQ ID No. 5 or SEQ ID No. 6,

(B) An amino acid sequence comprising 1 to 10 single amino acid changes compared to SEQ ID NO. 5 or SEQ ID NO. 6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 single amino acid changes,

(C) Fragments of at least 7 consecutive amino acids from SEQ ID NO. 5, for example at least 8, 9, 10, 11, 12 or 13 consecutive amino acids from SEQ ID NO. 5, or alternatively,

10. The polypeptide according to claim 1 or 2, wherein (b) comprises or consists of the amino acid sequence according to SEQ ID No. 5.

11. The polypeptide of any one of the preceding claims, wherein (b) is:

(i) Directly connected to the C-terminal of (a), or

(ii) Is connected to the C-terminal of (a) by a first connector.

12. The polypeptide of any one of claims 2 or 11, (ii) wherein the first linker or "L" comprises or consists of 2-20 amino acids, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

13. The polypeptide according to any one of claims 2 or 12, wherein the first linker starts with proline.

14. The polypeptide according to any one of claims 2, 11-13, wherein the first linker comprises or consists of a polar amino acid, e.g. wherein the first linker consists entirely of a polar amino acid, or, if the first linker starts with proline, the remaining amino acids are polar.

15. The polypeptide of any one of claims 2, 11-14, wherein the first linker comprises or consists of:

(i) PGDGN [ SEQ ID NO:7], or variants or fusions thereof, or

(ii) DNKQ [ SEQ ID NO:8], or a variant or fusion thereof.

16. The polypeptide according to any one of the preceding claims, wherein the polypeptide comprises a protein purification affinity tag, such as 6, 7, 8, 9 or 10 consecutive histidines, at the N-terminus, C-terminus and/or internally.

17. The polypeptide of any one of the preceding claims, wherein the polypeptide or "X" comprises a cell secretion leader sequence that:

(i) Upstream of (a), or

(ii) At the N-terminus of the polypeptide.

18. The polypeptide of claim 17, wherein the cell secretion leader sequence is selected from the group consisting of:

(i) METDTLLLWVLLLWVPGSTGD [ SEQ ID NO:9], or variants or fusions thereof,

(iii) MRLLAKIICLMLWAICVA [ SEQ ID NO:11], or variants or fusions thereof,

(iv) MGWSCIILFLVATATGVHS [ SEQ ID NO:12], or variants or fusions thereof,

(v) METPAELLFLLLLWLPDTTG [ SEQ ID NO:13], or variants or fusions thereof,

(vi) METDTLLLWVLLLWVPGSTG [ SEQ ID NO:108], or variants or fusions thereof, or

(vii) MEFGLSWVFLVAILEGVHC [ SEQ ID NO:14], or variants or fusions thereof.

19. The polypeptide of any one of the preceding claims, wherein the polypeptide comprises a nanoparticle domain at the N-terminus or C-terminus, optionally wherein "X" or "Y" comprises a nanoparticle domain.

20. The polypeptide of claim 19, wherein the nanoparticle domain is selected from the group consisting of:

(i) Ferritin (e.g., [ SEQ ID NO:15] or [ SEQ ID NO:109] (helicobacter pylori), [ SEQ ID NO:16] (Escherichia coli)), or [ SEQ ID NO:149] - [ SEQ ID NO:152] (stabilized Escherichia coli), or variants and/or fragments thereof,

(ii) iMX313 (e.g. [ SEQ ID NO:17 ]), or a variant and/or fragment thereof,

(iii) mI3 (e.g. [ SEQ ID NO:18 ]), or a variant and/or fragment thereof,

(iv) A packaging protein (e.g. [ SEQ ID NO:19 ]), or a variant and/or fragment thereof, or

(v) Self-assembled viral capsid proteins, such as the phage Acinetobacter AP205 capsid protein (NCBI reference sequence: NP-085472.1), hepatitis B virus core protein (HBc) [ SEQ ID NO:110], or phage qβ [ SEQ ID NO:111], or variants and/or fragments thereof.

21. The polypeptide of claim 19 or 20, wherein the nanoparticle domain:

(i) Directly linked to said polypeptide, or

(ii) Is linked to the polypeptide by a second linker.

22. The polypeptide of any one of claims 19-22, wherein the second linker comprises or consists of 2-20 amino acids, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

23. The polypeptide of any one of claims 19-22, wherein the second linker comprises or consists of glycine (G) and/or serine (S).

24. The polypeptide of any one of claims 19-23, wherein the second linker is selected from the group consisting of:

(i) GSSGSGSGS [ SEQ ID NO:112] or variants and/or fusions thereof,

(ii) GGSGS [ SEQ ID NO:113] or variants and/or fusions thereof,

(iii) GGS or variants and/or fusions thereof,

(iv) SGSHHHHHHHHGGS [ SEQ ID NO:114], or variants and/or fusions thereof,

(v) AKFVAAWTLKAAA [ SEQ ID NO:115] or variants and/or fusions thereof,

(vi) GGGGSLVPRGSGGGGS [ SEQ ID NO:116], or variants and/or fusions thereof,

(vii) EAAAKEAAAKEAAAKA [ SEQ ID NO:117], or variants and/or fusions thereof,

(viii) SGSFVAAWTLKAAAGGS [ SEQ ID NO:118] or variants and/or fusions thereof, and

(ix) SGSGSGGGGGGS [ SEQ ID NO:119] or variants and/or fusions thereof.

25. The polypeptide of any one of claims 19-24, wherein the nanoparticle domain is:

(i) Upstream of (a),

(ii) At the N-terminus of the polypeptide,

(iii) Downstream of (b), or

(iv) At the C-terminus of the polypeptide.

26. A polypeptide monomer comprising or consisting of the amino acid sequence:

(i) An amino acid sequence having at least 80% sequence identity to amino acid sequence SEQ ID NO. 16, for example at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and having one or more mutations from: glycine (G) at a position aligned with residue 34 of SEQ ID No. 16 (T34G mutation), aspartic acid (D) at a position aligned with residue 70 of SEQ ID No. 16 (N70D mutation), isoleucine (I) at a position aligned with residue 72 of SEQ ID No. 16 (V72I mutation) and alanine (a) at a position aligned with residue 124 of SEQ ID No. 16 (S124A mutation);

(ii) An amino acid sequence having at least 80% sequence identity to amino acid sequence SEQ ID No. 16, e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and having glycine (G) at a position aligned with residue 34 of SEQ ID No. 16 (T34G mutation), aspartic acid (D) at a position aligned with residue 70 of SEQ ID No. 16 (N70D mutation), isoleucine (I) at a position aligned with residue 72 of SEQ ID No. 16 (V72I mutation) and alanine (a) at a position aligned with residue 124 of SEQ ID No. 16 (S124A mutation), optionally wherein the polypeptide monomers comprise an amino acid sequence having at least 80% sequence identity to amino acid sequence SEQ ID No. 149, e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity;

(iii) An amino acid sequence having at least 80% sequence identity to amino acid sequence SEQ ID No. 16, e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and having glycine (G) at a position aligned with residue 34 of SEQ ID No. 16 (T34G mutation), isoleucine (I) at a position aligned with residue 72 of SEQ ID No. 16 (V72I mutation) and alanine (a) at a position aligned with residue 124 of SEQ ID No. 16 (S124A mutation), optionally wherein the polypeptide monomer comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence SEQ ID No. 150, e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity;

(iv) An amino acid sequence having at least 80% sequence identity to amino acid sequence SEQ ID No. 16, e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and having glycine (G) at a position aligned with residue 34 of SEQ ID No. 16 (T34G mutation), and alanine (a) at a position aligned with residue 124 of SEQ ID No. 16 (S124A mutation), optionally wherein the polypeptide monomer comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence SEQ ID No. 151, e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity;

(v) An amino acid sequence having at least 80% sequence identity to amino acid sequence SEQ ID NO. 16, e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and glycine (G) (a T34G mutation) at a position aligned with residue 34 of SEQ ID NO. 16, optionally wherein the polypeptide monomer comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence SEQ ID NO. 152, e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

27. The polypeptide monomer of claim 26, comprising an amino acid sequence selected from the group consisting of: 149,SEQ ID NO:150,SEQ ID NO:151, and 152.

28. A nanoparticle comprising the polypeptide monomer of claim 26 or 27.

29. The nanoparticle of claim 29, wherein the nanoparticle is a homomultimer.

30. The nanoparticle according to claim 28 or 29, wherein the outer surface structure or the inner surface structure of the nanoparticle carries one or more antigens and/or immunostimulants.

31. The nanoparticle according to claim 30, wherein the antigen comprises or consists of a polypeptide according to any one of claims 1 to 18.

32. The nanoparticle according to claim 31, comprising or consisting of an amino acid sequence having at least 80% sequence identity, such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, to the amino acid sequence SEQ ID NO 130 or 153.

33. The polypeptide according to any one of the preceding claims, wherein the polypeptide comprises or consists of an amino acid sequence corresponding to seq id no:

(i) SEQ ID NO. 20, or variants and/or fragments thereof,

(ii) SEQ ID NO. 21, or variants and/or fragments thereof,

(iii) SEQ ID NO. 22, or variants and/or fragments thereof,

(iv) SEQ ID NO. 23, or variants and/or fragments thereof,

(v) SEQ ID NO. 24, or variants and/or fragments thereof,

(vi) 25, or a variant and/or fragment thereof,

(vii) 26 or a variant and/or fragment thereof, (viii) 27 or a variant and/or fragment thereof, (ix) 28 or a variant and/or fragment thereof, (x) 29 or a variant and/or fragment thereof,

(xi) 30 or a variant and/or fragment thereof, (xii) 31 or a variant and/or fragment thereof, (xiii) 38 or a variant and/or fragment thereof, (xii) 40 or a variant and/or fragment thereof, (xxii) 41 or a variant and/or fragment thereof, (xxiii) 42 or a variant and/or fragment thereof, (xxiv) 43 or a variant and/or fragment thereof, (xxvii) 44 or a variant and/or fragment thereof, (xxiv) 82 or a variant and/or fragment thereof, (xxiv) 38 or a variant and/or fragment thereof, (xxiv) 39 or a variant and/or fragment thereof, (xxi) 40 or a variant and/or fragment thereof, (xxii) 41 or a variant and/or fragment thereof, (xxiii) 42 or a variant and/or fragment thereof, or a variant and/or fragment thereof, (xxxi) SEQ ID NO:84, or a variant and/or fragment thereof, (xxxii) SEQ ID NO:85, or a variant and/or fragment thereof, (xxxiii) SEQ ID NO:86, or a variant and/or fragment thereof, (xxxiv) SEQ ID NO:87, or a variant and/or fragment thereof, (xxxv) SEQ ID NO:88, or a variant and/or fragment thereof, (xxxvi) SEQ ID NO:89, or a variant and/or fragment thereof, and

(xxxvii) One of SEQ ID NOs 120-124,SEQ ID NO:129-143 and 153 or a variant and/or fragment thereof.

34. The polypeptide of any one of the preceding claims, wherein the polypeptide comprises or consists of: and SEQ ID NO:123 or SEQ ID NO:124, e.g., an amino acid sequence having at least 70% sequence identity to SEQ ID NO:123 or SEQ ID NO:124 have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

35. The polypeptide of any one of the preceding claims, wherein the polypeptide has a mannose binding at least 20% lower than native FimH (FimHC complex) complexed with native FimC, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% lower.

36. The polypeptide of any one of the preceding claims, wherein FimH is in a low affinity conformation, i.e., in a stressed (T) state.

37. The polypeptide of any one of the preceding claims, wherein the polypeptide induces auto-aggregation at least 20% lower than native FimH, such as at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% lower.

38. The polypeptide of any one of the preceding claims, wherein the polypeptide is capable of inhibiting bacterial adhesion by at least 20%, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

39. The polypeptide of any one of the preceding claims, wherein the polypeptide has an anti-FimH immunogenicity that is at least 20% higher, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400% or 500% higher than native FimH complexed with native FimC (in particular, we include FimH in a high affinity conformation, relaxed (R) state (see above)).

40. The polypeptide of any one of the preceding claims, wherein the polypeptide is capable of inhibiting hemagglutination of guinea pig erythrocytes by at least 2-fold, such as at least 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold.

41. Nucleic acid encoding a polypeptide according to any one of claims 1-40, e.g., consisting of SEQ ID NOs:45-77, 90-99 or variants and/or fragments thereof.

42. The nucleic acid of claim 42, wherein the nucleic acid has been codon optimized for expression in selected prokaryotic or eukaryotic cells, e.g., yeast cells (e.g., saccharomyces cerevisiae, pichia pastoris), insect cells (e.g., spodoptera frugiperda Sf21 cells, or Sf9 cells), or mammalian cells (Expi 293, expi293GNTI, chinese Hamster Ovary (CHO) cells, and human embryonic kidney 293 cells (HEK 293)).

43. A vector comprising the nucleic acid of claim 41 or 42.

44. The vector of claim 43, wherein the vector is a plasmid, e.g., an expression plasmid.

45. The vector of claim 43 or 44, wherein the plasmid is selected from the group consisting of pCDNA3.1 (Life Technologies), pCDNA3.4 (Life Technologies), pFUSE, pBROAD, pSEC, pCMV, pDSG-IBA and pHEK293 Ultra or from the group consisting of pACYCDuet-1, pTrcHis2A, pET, pET15TEV, pET22b+, pET303/CT-HIS, PET303/CT, pBAD/Myc-His A, pET303, pET24b (+).

46. The vector of claim 43, wherein the vector is a viral vector, e.g., an RNA viral vector.

47. A cell comprising the nucleic acid of claim 41 or 42 or the vector of any one of claims 43-46.

48. The cell of claim 46, wherein the cell does not have N-acetylglucosamine transferase I (GnTI) activity.

49. The cell of claim 47 or 48, wherein the host cell is selected from the group consisting of an Expi293, an Expi293GNTI (Life Technologies), a Chinese Hamster Ovary (CHO) cell, a NIH-3T3 cell, a 293-T cell, a Vero cell, a HeLa cell, a perc.6 cell (ECACC accession No. 96022940), a Hep G2 cell, a MRC-5 (ATCC CCL-171), a WI-38 (ATCC CCL-75), a fetal rhesus lung cell (ATCC CL-160), a Madin-Darby bovine kidney ("MDBK") cell, a Madin-Darby canine kidney ("MDCK") cell (e.g., MDCK (NBL 2), ATCC CCL34; or MDCK 33016, DSMACC 2219), a small hamster kidney (BHK) cell such as BHK21-F, HKCC cell, and a human embryonic kidney 293 cell (HEK 293).

50. The cell according to claim 47, wherein the host cell is selected from the group consisting of E.coli strains BL21 (DE 3), HMS174 (DE 3), origami 2 (DE 3), BL21DE3T1r or T7 buffer expression.

51. A cell culture comprising the cell of any one of claims 47-50.

52. A method of producing a polypeptide according to any one of claims 1-40 by expressing a protein in a cell according to any one of claims 47-50 under suitable conditions and in a suitable medium, thereby expressing the encoded polypeptide.

53. The method of claim 52, further comprising collecting the expressed polypeptide from the cultured cells and/or medium, and optionally purifying the collected polypeptide.

54. A pharmaceutical composition comprising the polypeptide of any one of claims 1-40, the nucleic acid of any one of claims 41 or 42, or the vector of any one of claims 43-46.

55. A vaccine comprising the polypeptide of any one of claims 1-40, the nucleic acid of claim 41 or 42, or the vector of any one of claims 43-46.

56. The vaccine of claim 55, further comprising an adjuvant.

57. The vaccine of claim 56, wherein the adjuvant comprises any one of: 3D-MPL, QS21 and liposomes, for example liposomes comprising cholesterol.

58. The vaccine of claim 57, wherein the adjuvant comprises 3D-MPL, QS21, and cholesterol-containing liposomes.

59. The vaccine of any one of claims 55-58, wherein the vaccine elicits a protective immune response after one or two doses.

60. A polypeptide according to any one of claims 1-40, a nucleic acid according to claim 41 or 42 or a vector according to any one of claims 43-46, or a vaccine according to claims 55-59 for use in medicine.

61. A polypeptide according to any one of claims 1-40, a nucleic acid according to claim 41 or 42 or a vector according to any one of claims 43-46 or a vaccine according to claims 55-59 for use in increasing an immune response in a mammal, e.g. for use in the treatment and/or prevention of one or more diseases.

62. A polypeptide according to any one of claims 1-40, a nucleic acid according to claim 41 or 42 or a vector according to any one of claims 43-46 or a vaccine according to claims 55-59 for use in increasing an immune response in a mammal, e.g. for use in the treatment and/or prevention of one or more diseases.

63. A polypeptide according to any one of claims 1-40, a nucleic acid according to claim 41 or 42 or a vector according to any one of claims 43-46 or a vaccine according to claims 55-59 for use in the manufacture of a medicament for increasing an immune response in a mammal, e.g. for the treatment and/or prevention of one or more diseases.

64. A method of increasing an immune response in a mammal, the method comprising or consisting of: administering to said mammal an effective amount of a polypeptide according to any one of claims 1-40, a nucleic acid according to claim 41 or 42, or a vector according to any one of claims 43-46, or a vaccine according to claims 55-59.