FUSION POLYPEPTIDES

Abstract

The present invention relates to fusion polypeptides comprising (a) a 4-oxalocrotonate tautomerase (4-OT)-based polypeptide scaffold which is capable of forming multimers, and (b) a polypeptide antigen; and homo- and hetero-multimers thereof. The invention also provides nucleic acid molecules and vectors encoding the fusion polypeptides and multimers; and methods of using the fusion polypeptides, multimers, nucleic acid molecules and vectors to produce an immunogenic response against the polypeptide antigen.

Claims

1. A fusion polypeptide, wherein the fusion polypeptide comprises: (a) a 4-oxalocrotonate tautomerase (4-OT)-based polypeptide scaffold which is capable of forming multimers; and (b) a polypeptide antigen.

2. A fusion polypeptide as claimed in claim 1, wherein the 4-OT based polypeptide scaffold is from or derived from a bacterium of a genera selected from the group consisting of Acinetobacter, Bacillus, Bartonella, Bordetella, Burkholderia, Campylobacter, Citrobacter, Enterobacter, Escherichia, Haemophilus, Helicobacter, Leptospira, Mycobacterium, Neisseria, Pseudomonas, Rhodobacter, Salmonella, Staphylococcus, Streptococcus, Thermoanaerobacter, Vibrio and Yersinia.

3. A fusion polypeptide as claimed in claim 1 or claim 2, wherein 4-OT based polypeptide scaffold is from or derived from Staphylococcus aureus.

4. A fusion polypeptide as claimed in any one of claims 1 to 3, wherein the the 4-OT based polypeptide scaffold comprises or consists of an amino acid sequence as given in any one of SEQ ID NOs: 38-127, or a variant or derivative thereof having at least 50% sequence identity thereto, more preferably at least 60%, 70%, 80%, 90% or 95% sequence identity thereto, which retains the ability to multimerise and to present the polypeptide antigen.

5. A fusion polypeptide as claimed in any one of claims 1 to 3, wherein the 4-OT based polypeptide scaffold comprises or consists of an amino acid sequence as given in any one of SEQ ID NOs: 128-627, or a variant or derivative thereof having at least 80% sequence similarity thereto, more preferably at least 90% or 95% sequence similarity thereto, which retains the ability to multimerise and to present the polypeptide antigen

6. A fusion polypeptide as claimed in any one of claims 1 to 3, wherein the 4-OT based polypeptide scaffold comprises or consists of an amino acid sequence as given in any one of SEQ ID NOs: 128-627, or a variant or derivative thereof having at least 80% sequence identity thereto, more preferably at least 90% or 95% sequence identity thereto, which retains the ability to multimerise and to present the polypeptide antigen.

7. A fusion polypeptide as claimed in any one of claims 1 to 3, wherein the 4-OT based polypeptide scaffold comprises or consists of an amino acid sequence as given in any one of SEQ ID NOs: 1-29, or a variant or derivative thereof which retains the ability to multimerise and to present the polypeptide antigen.

8. A fusion polypeptide as claimed in claim 7, wherein the variant of the 4-OT based polypeptide scaffold comprises or consists of an amino acid sequence having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% amino acid sequence identity to any one of SEQ ID NOs: 1-29.

9. A fusion polypeptide as claimed in claim 7, wherein the derivative of the 4-OT based polypeptide scaffold comprises or consists of a fragment of any one of SEQ ID NOs: 1-29 which is at least 70%, 80%, 90%, 95% or 99% of the length of that SEQ ID NO.

10. A fusion polypeptide as claimed in any one of the preceding claims, wherein the polypeptide antigen is one which is presented on a microbe, parasite or neoplasm.

11. A fusion polypeptide as claimed in any one of the preceding claims, wherein the polypeptide antigen is a viral, bacterial, protozoan, animal, mammalian or human antigen.

12. A fusion polypeptide as claimed in any one of the preceding claims, wherein the polypeptide antigen is from a disease-causing bacterium, a disease-causing parasite or a disease-causing virus, preferably from a malaria-causing parasite or an influenza-causing virus.

13. A fusion polypeptide as claimed in any one of the preceding claims, wherein the polypeptide antigen is a bacterial or parasitic antigen which is from or derived from Staphylococcus, pathogenic Neisseria, Mycobacteria, Escherichia or from Apicomplexa (e.g. Plasmodium) or helminths, preferably from or derived from S. aureus, a pathogenic Neisseria species, M. tuberculosis, E. coli or P. falciparum.

14. A fusion polypeptide as claimed in any one of the preceding claims, wherein the polypeptide antigen is selected from the group consisting of ClfA, ClfB, FnBPA, FnBP, SdrC, SdrD, SdrE, SasA, SasB, SasC, SasD, SasX, SasF, SasG/AAp, MntC, IsdA, IsdB, IsdH, FhuD2, EsxA, EsxB, Spa, Coa, vWbp, HIa, HIgA, HIgB, HIgC, LukA, LukB, LukD, LukE, EpiP, Can, Csa1A, Csa1B, Csa1C, Csa1D, CsA2A, Csa3A, Csa3B, CsA3C, Csa3D, Csa3E, Csa3G, Csa3H, Csa31, Csa3J, Csa4A, Csa4B, Csa4c, scn, efb, efbc, or a variant or derivative thereof which maintains the immunogenic capacity of the antigen.

15. A fusion polypeptide as claimed in any one of claims 1 to 13, wherein the polypeptide antigen is S. aureus BitC; the extracellular domain of the S. aureus Clumping factor B precursor (ClfB); the S. aureus alpha toxin or a truncated form thereof; or the P. falciparum protein Pfs25.

16. A homo-multimer, wherein the homo-multimer comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16 or 20 fusion polypeptides as defined in any one of claims 1 to 15.

17. A hetero-multimer, wherein the hetero-multimer comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16 or 20 fusion polypeptides as defined in any one of claims 1 to 15.

18. A hetero-multimer as claimed in claim 17, wherein the hetero-multimer comprises 2, 3, 4, 5 or 6 different fusion polypeptides as defined in any one of claims 1 to 15.

19. A hetero-multimer as claimed in claim 17 or claim 18, wherein the hetero-multimer comprises the same 4-OT-based polypeptide scaffold but different polypeptide antigens.

20. A nucleic acid molecule encoding a fusion polypeptide as claimed in any one of claims 1 to 15, preferably wherein the nucleic acid molecule is DNA.

21. A vector or plasmid comprising a nucleic acid molecule as claimed in claim 20, preferably wherein the vector is an expression vector.

22. A vector as claimed in claim 21, wherein the vector is a viral vector, preferably an Adenoviral vector or a Modified Vaccinia Ankara (MVA) viral vector.

23. A host cell comprising a nucleic acid molecule as claimed in claim 20 or a vector or plasmid as claimed in claim 21 or claim 22, preferably wherein the host cell is a eukaryotic cell.

24. A pharmaceutical composition comprising a fusion polypeptide as claimed in any one of claims 1 to 15, a multimer as claimed in any one of claims 16-19, a nucleic acid molecule as claimed in claim 20, or a vector or plasmid as claimed in claim 21 or 22, optionally together with one or more pharmaceutically-acceptable carriers, excipients or diluents.

25. A vaccine composition comprising a fusion polypeptide as claimed in any one of claims 1 to 15, a multimer as claimed in any one of claims 16-19, a nucleic acid molecule as claimed in claim 20, or a vector or plasmid as claimed in claim 21 or 22, optionally together with an adjuvant.

26. A combined preparation comprising two or more components selected from the group consisting of fusion polypeptide as claimed in any one of claims 1 to 15, multimers as claimed in any one of claims 16-19, nucleic acid molecules as claimed in claim 20, and vectors or plasmids as claimed in claim 21 or 22, as a combined preparation in a form suitable for simultaneous, separate or sequential use, preferably for use in producing an immunogenic response to the polypeptide antigen in a subject.

27. A fusion polypeptide as claimed in any one of claims 1 to 15, a multimer as claimed in any one of claims 16-19, a nucleic acid molecule as claimed in claim 20, a vector or plasmid as claimed in claim 21 or 22, or a composition or preparation as claimed in any one of claims 24 to 26, for use in therapy or for use as a medicament.

28. A fusion polypeptide as claimed in any one of claims 1 to 15, a multimer as claimed in any one of claims 16-19, a nucleic acid molecule as claimed in claim 20, a vector or plasmid as claimed in claim 21 or 22, or a composition or preparation as claimed in any one of claims 24 to 26, for use in producing an immunogenic response to the polypeptide antigen in a subject, preferably for inducing a T-cell or a B-cell response to the polypeptide antigen in a subject.

29. Use of a fusion polypeptide as claimed in any one of claims 1 to 15, a multimer as claimed in any one of claims 16-19, a nucleic acid molecule as claimed in claim 20, a vector or plasmid as claimed in claim 21 or 22, or a composition or preparation as claimed in any one of claims 24 to 26, in the preparation of a medicament for producing an immunogenic response to the polypeptide antigen in a subject, preferably for inducing a T-cell or a B-cell response to the polypeptide antigen in a subject.

30. A method of inducing an immunogenic response to a polypeptide antigen in a subject, the method comprising administering an effective amount of a fusion polypeptide as claimed in any one of claims 1 to 15, a multimer as claimed in any one of claims 16-19, a nucleic acid molecule as claimed in claim 20, a vector or plasmid as claimed in claim 21 or 22, or a composition or preparation as claimed in any one of claims 24 to 26, to the subject.

31. A method of inducing an immunogenic response to a polypeptide antigen in a subject, method comprising administering to the subject: (i) an effective priming amount of a fusion polypeptide as claimed in any one of claims 1 to 15, a multimer as claimed in any one of claims 16-19, a nucleic acid molecule as claimed in claim 20, a vector or plasmid as claimed in claim 21 or 22, or a composition or preparation as claimed in any one of claims 24 to 26; and then (ii) an effective boosting amount of a fusion polypeptide as claimed in any one of claims 1 to 15, a multimer as claimed in any one of claims 16-19, a nucleic acid molecule as claimed in claim 20, a vector or plasmid as claimed in claim 21 or 22, or a composition or preparation as claimed in any one of claims 24 to 26.

32. A method as claimed in claim 31, the method comprising administering to the subject: (i) an effective priming amount of an adenovirus vector as claimed in claim 22 (preferably AdHu5); and then (ii) an effective boosting amount of a non-replicating poxvirus vector as claimed in claim 22 (preferably MVA).

33. A process for the production of one or more of fusion polypeptides as claimed in any one of claims 1 to 15, which process comprises expressing one or more nucleic acid molecules as claimed in claim 20 in a host cell, and recovering the fusion polypeptide product(s).

Description

BRIEF DESCRIPTION OF THE FIGURES

[0225] FIG. 1. Crystal structures of four multimerizing domains examined (PDB references are shown beneath each image pair).

[0226] FIG. 2. Antibody responses to BitC measured by LIPS after vaccination with different scaffolds fused to the c-terminus of BitC. Panel A) Design of pMono2 DNA vaccination vectors; CMV: CMV IE94 promoter, TPA: human tissue plasminogen activator signal sequence, Antigen: antigen being tested, V5: epitope tag and linker sequence, Scaffold: molecule under test, BGH pA: Bovine growth hormone polyadenylation sequence. Panel B) BitC-specific antibody response after prime-boost immunisations of groups of 4-5 BALB/c mice with 50 g BitC-scaffold DNA as measured by LIPS assay. Sera were collected 3 weeks post boost. Each symbol represents a single mouse. Dotted line: threshold for background response. *p<0.05.

[0227] FIG. 3. SAR1376 scaffold enhances immune responses to BitC and ClfB in BALB/c mice. Antigen specific antibody responses against BitC (panel A) and ClfB (panel C) as measured by LIPS assay, and number of IFN- producing T-cells induced by BitC (panel B) and ClfB (panel D) after vaccination with different scaffolds fused to the c-terminus of the antigens. Sera were collected 3 weeks post boost. Each symbol represents a single mouse. Dotted line: threshold for background response. **p<0.01; ***p<0.001.

[0228] FIG. 4. 4-OT family members. A) Family tree, 2780 discrete family members (modal length of 63 amino acids) were found across Eubacteria. B) Published crystal structures of 4-OT members from diverse genera, including SAR1376, whose positions are indicated in A. C) Cobalt alignment of twenty-four 4-OT like sequences from selected genera which include human pathogens.

[0229] FIG. 5. SAR1376 mutant P1A scaffold enhances immune responses to ClfB in BALB/c and CD1 mice. Antigen specific antibody responses against ClfB in BALB/c (panel A) and CD1 (panel C) mice as measured by LIPS assay, and number of IFN- producing T-cells induced by ClfB in BALB/c (panel B) and CD1 (panel D) mice after vaccination with mutant scaffolds fused to the c-terminus of the antigen. Sera were collected 3 weeks post boost. Each solid symbol represents a single mouse. Dotted line: threshold for background response. *p<0.05; **p<0.01.

[0230] FIG. 6. SAR1376 specific antibodies measured after vaccination with (mutant) scaffold fused to the c-terminus of the antigen. Panel A) antibody response against SAR1376 in BALB/c mice immunised with BitC-SAR1376 fusion protein; Panel B) anti-SAR1376 antibody levels after vaccination of BALB/c mice with ClfB fused to SAR1376, SAR1376-P1A or SAR1376-R35A, Panel C) anti-SAR1376 antibodies in CD1 mice immunised with ClfB-SAR1376 mutant PIA scaffold, as measured by LIPS assay. Each solid symbol represents a single mouse. ***p<0.001; ****p<0.0001.

[0231] FIG. 7. SAR1376 mutant PIA fused to truncated HIa increases (functional) antibody response when expressed in Viral Vectors. Panel A) Anti -hemolysin antibody levels, Panel B) anti SAR1376 antibody levels after vaccination of BALB/c mice with tHIa75-PIA, tHIa75-No scaffold, or empty vectors (controls) as assessed respectively by ELISA and LIPS assay. Sera were collected pre boost (day 56 post prime), and post boost (day 70 post prime). Panel C) -Toxin neutralizing activity of the antibodies was assessed in day 70 serum by Neutralisation Assay. Minimum and Maximum level as determined with mAb 8B7 as described in Methods. **p<0.01.

[0232] FIG. 8. Fusion of SAR3176-P1A to Pfs25 improves functional activity.

[0233] FIG. 8A) Plasmid construct for expression of pfs25-SAR1376 P1A in Pichia pastoris.

[0234] FIG. 8B) Antibody response after vaccination of BALB/c mice with Pfs2525 fused to SAR1376-P1A. Sera were collected at day 14, 28 and day 61 post prime and IgG responses measured using a standardised ELISA. Each solid symbol represents a single mouse.

[0235] FIG. 8C) Pooled-purified IgG mixed with malaria infected blood was fed to Anopheles stephensi mosquitos through a membrane. Midguts were dissected 9 days post-feed and the number of oocysts per gut were counted. Each solid symbol represents the count from one mosquito. AU=antibody units. *p<0.05.

EXAMPLES

[0236] The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1: Methods

[0237] Scaffolds Used and their Production

[0238] Chosen scaffold sequences were human codon optimised and DNA synthetized (GeneArt, Life Technologies Ltd) (SEQ ID NOs: 38-127). Scaffolds were fused to the C-terminus of the S. aureus antigens in a mammalian expression vector (pMono2) using a restriction enzyme based strategy. Antigens fused were S. aureus BitC, a S. aureus cell surface lipoprotein (accession NP_370379); the extracellular domain of the S. aureus Clumping factor B precursor [20] (ClfB, accession YP_001333563); S. aureus -hemolysin (accession YP_111574996, amino acids 1-75, designed tHIa75 here); and P. falciparum protein Pfs25 (accession AAN35500) [10]. Sequences for all these were synthesised by Life Technologies Ltd.

[0239] S. aureus tHIa75 was ligated into the pMono2 vector from which the construct was subcloned into shuttle vectors and transfected into replication-deficient adenovirus human serotype 5 (AdHu5) and Modified Vaccinia Ankara (MVA) as described elsewhere [29, 30].

[0240] The gene coding for Plasmodium falciparum transmission-blocking antigen, Pfs25, with a 6-his tag (His.sub.6-Pfs25) was fused 5 of the gene coding for SAR1376-PIA and ligated into the pPinka-HC plasmid (FIG. 8A) which puts protein expression under methanol-inducible control and allows secretion of the expression product from Pichia pastoris via the -mating factor secretion signal. Electrocompetent P. pastoris were transformed with the expression plasmid. Colonies were screened for optimal expression and the highest expressing clone selected for scale up. A one litre shake flask culture of the selected clone was grown under inducing conditions and the supernatant harvested. Supernatant containing the secreted expression product was harvested by centrifugation and the product purified by nickel-chelate affinity chromatography.

[0241] Vaccination Experiments

[0242] All mouse procedures were conducted in accordance to the Animal (Scientific Procedures) Act 1986 (Project licence 30/2825) and were approved by the University of Oxford Animal Care and Ethical Review Committee. Six to eight week old female BALB/c or CD1 mice from Harlan Laboratories UK were used.

[0243] In DNA vaccination experiments, groups of 4-12 mice (BALB/c or CD1) were immunised intramuscularly with 50 g vector DNA in 50 l PBS (25 l/hind leg). Immunisation was repeated 2 weeks later. On day 35, blood samples were taken from all animals under terminal anaesthesia (heart bleeds) for immune assays (IFN-gamma secreting T-cell numbers (ELISpot), and antibody levels by Luciferase ImmunoPrecipitation System (LIPS) assay) [31-32].

[0244] For viral vector immunization, groups of 6 mice were immunised intramuscularly with 10.sup.9 i.u. AdHu5 in 25 l PBS followed at least 8 weeks later by 10.sup.7 pfu MVA as prime-boost sequence, a regime we refer to as AM7. Venous blood samples were taken from the tail vein of all animals pre boost and 2 weeks post boost.

[0245] For immunization with recombinant proteins, groups of 6 BALB/c mice were immunised intramuscularly with 50 l aliquots (25 l/hind leg) of protein-in-Alhydrogel formulations, containing 2.5 g of either Pfs25-P1A or monomeric Pfs25 twice at 2 week intervals.

[0246] Blood was collected from the tail vein on day 14 (2 weeks post prime) and day 28 (2 weeks post boost).

[0247] Assessment of Immune Responses Against BitC and ClfB

[0248] A Luciferase ImmunoPrecipitation System (LIPS) assay was used to detect specific serum anti-S. aureus BitC and ClfB antibodies as described [32]. Briefly, recombinant BitC and ClfB fusion protein with Renilla luciferase were produced in 293 cells as described [32]. Serially diluted sera were incubated with Renilla luciferase-BitC or ClfB fusion proteins. The mix was added to filter plates loaded with A/G beads (Thermo Fisher). After incubation and subsequent washings, chemiluminescence was measured in a Luminometer (ClarioStar, BMG Labtech) after adding substrate (Renilla luciferase assay system, Promega UK Ltd.). Log transformation was applied to luminescence data prior to statistical analysis. Specific luminescence was generated by subtracting the assay background, which was considered to be the luminescence observed in the absence of any sera. The assay limit of detection was considered to be four standard deviations above the specific luminescence in the control groups.

[0249] Anti-Alpha Toxin Immune Responses

[0250] The anti-tHIa antibody levels and functional activity (neutralizing activity, NA) of the antibodies in serum were assessed respectively by ELISA and Toxin Neutralisation Assay (TNA) as described by Oscherwitz and Cease [33]. In brief, the ability of antibody to block recombinant alpha toxin (AT) cytotoxicity in vitro was assessed using the Jurkat T cell line (TIB-152, ATCC, Manassas, Va.). Mouse anti-Staphylococcal alpha hemolysin mAb (8B7) (IBT Bioservices #0210-001) was used as standard positive to obtain minimum and maximum levels for neutralisation of AT (H9395, Sigma Biologicals).

[0251] Anti-Pfs25 Immune Responses

[0252] Antibody levels in serum were assessed by standardised anti-Pfs25 ELISA, as described [10]. A serially-diluted standard reference serum with a known antibody titre was used to determine the antibody titre of individual samples. Total IgG was purified from the pooled serum of the mice immunised with Pfs25-SAR1376-P1A and assessed by functional assay by Standard Membrane Feeding Assay (SMFA). This assay involves feeding malaria infected blood mixed with purified IgG to Anopheles stephensi mosquitos through a membrane [34]. If the IgG has functional activity, it will block development of the malaria sexual stage in the mosquito midgut; and at 9 days post feed there will be a reduction in the number of oocysts observed in the gut compared to a non-functional IgG control.

[0253] Statistical Analysis

[0254] Data on antibody response and IFN-specific spots were statistically analysed for effect of added scaffold by means of an F-test after a log.sub.10 transformation and correction for background. Log(number of IFN- secreting cells) was used, because of the approximate log-normal distribution of ELISpot counts in the animals (not shown). Specific antibody levels from the LIPS assay were generated by subtracting the assay luminescence background, which was considered to be the luminescence observed in the absence of any sera, from the luminescence observed with serum dilutions added. The assay limit of detection was considered to be four standard deviations above the background. Post-hoc pairwise comparisons were performed using Dunnett's Multiple Comparison Test. Differences were considered significant when p<0.05. The statistical packages used were R 2.15 (http://www.cran.org), and GraphPad Prism version 5.04 (GraphPad Software, Inc.).

[0255] Bioinformatic Identification of 4-OT-Like Proteins.

[0256] The NCBI RefSeq database was queried using BLASTp and delta-BLAST [14] using default parameters with the S. aureus 4-OT enzyme (YP_040781.1) as a query. Further searches were performed using distant hits and results pooled, and then filtered using custom R scripts to include hits encoding proteins of 55 to 85 amino acids. Manual curation was performed, and the sequence start of predicted proteins was trimmed to begin with MP, as a proline is present in position 2 of all canonical family members [21], i.e. any amino acids purported to originate from upstream initiator codons were removed. A single sequence was selected per genus; using genus-specific sequences, an alignment was prepared using the NCBI Cobalt multiple-alignment engine [35] with default parameters. Additionally, a tree was constructed using PhyML [36] using default parameters, and visualised using Archeopterix [37] software.

[0257] Presentation of Crystal Structures

[0258] Crystal structures of proteins of interest were downloaded from the Protein data bank. A single hexameric structure was isolated from each set of crystal data using Pymol v.1.8.2 for Windows. For comparison of multiple 4-OT crystals, structures were aligned using CEAlign (Pymol) using default parameters. Pymol was also used to render images.

Example 2: Selection of Scaffolding Tags

[0259] Study of crystal structures within the Protein Data Bank revealed a number of bacterial proteins which form self-multimers of various orders. A subset was selected based on absence of intra- or inter-chain disulphide bonding, absence of transmembrane regions and absence of toxicity and oncogenic activity, with a view to increasing the probability of efficient expression.

[0260] In the first instance, a number of self-multimerising proteins were selected from S. aureus, a pathogenic microbe causing disease that is controlled by both T-cell and antibody-mediated mechanisms [17]. FIG. 1 shows the structures of the four proteins which were chosen: Dps-like Peroxide Resistance Protein Dpr, with a structure similar to that of ferritin [12]; QacR, a multidrug binding transcriptional repressor; SA138818, a protein of unknown function annotated as a homologue of E. coli ygbL aldolase class 2-like gene; and SAR1376, a 4-oxalocrotonate tautomerase (4-OT). The size of the crystallising unit varied from more than 20 nm (for QacR) to less than 5 nm (for SAR1376) (FIG. 1).

Example 3: Fusion of SAR1376 to S. aureus Antigens Enhances their Immunogenicity in Mice

[0261] Expression vectors producing fusions of the four proteins (see Sequences herein) with a series of S. aureus antigens were constructed. The expression cassette was composed of a human tissue plasminogen activator leader sequence, the antigen of interest, an epitope tag (V5) used to monitor protein expression, and the scaffolding domain via a GSG linker (FIG. 2A).

[0262] We studied scaffold fusions to two S. aureus antigens: BitC [19], a cell surface lipoprotein (accession NP_370379), and the extracellular domain of the Clumping factor B precursor [20] (ClfB, accession YP_001333563). As comparators, constructs expressing antigen without scaffold were constructed (FIG. 2A). Additionally, in some experiments, the multimerising tag IMX313 [2] fused to the antigen C-terminus was used as a positive control.

[0263] Groups of BALB/c mice were immunised intramuscularly with a mammalian expression DNA vector expressing BitC fused to one of the four test scaffolding domains. A priming and boosting immunisation was administered, separated by two weeks (FIG. 2). Measurement of the antibody response against BitC three weeks post-boost indicated that fusion of SAR1376 might increase immunogenicity of BitC relative to the no-scaffold comparator (p=0.16), while the mice vaccinated with the Dps scaffold responded poorly against BitC (FIG. 2B).

[0264] We further tested the SAR1376 and QacR scaffolds with both ClfB S. aureus antigen and with BitC, in the same 2-week prime-boost vaccination regimen. Humoral and cellular (IFN- ELISpot) responses were measured following the second immunisation. Comparator constructs containing the QacR domain were included in order to assess the specificity of the observed response. The immunogenicity results supported the previous experiment, in which a small increase was observed in antibody responses to BitC but not QacR (FIG. 3A; p=0.06 for BitC-SAR1376 when pooling data from experiments shown in FIGS. 2 and 3). A significantly higher antibody response to ClfB-SAR1376 was observed compared to ClfB without a scaffold (p=0.002, FIG. 3C). The SAR1376 scaffold did not enhance IFN- producing T-cell numbers with either of these two antigens (FIGS. 3B and 3D), although the IMX313 tag, used here as a positive control, did increase the T cell responses to BitC (FIG. 3B, p=0.01).

Example 4: The 4-OT Family

[0265] 4-OT-like enzymes are common in bacteria [21]. Using a protein-based search strategy, 2780 discrete family members (modal length of 63 amino acids) were found across Eubacteria, with examples in Archaea also noted (see Example 1). One example was chosen randomly from the 342 different genera identified. Extensive diversity is observed within the protein family, with only 20% identity in primary protein sequences between diverse members of the family (FIG. 4A). Examination of eleven bacterial crystal structures, however, showed a very similar crystal structures in family members despite huge evolutionary distance (FIG. 4B). Conserved motifs, including a highly conserved initial proline, do exist within the primary sequences from genera known to be pathogenic to man (FIG. 4C).

[0266] The crystal structure of SAR1376 reveals a hexamer forming an approximately spherical structure of about 5 nm diameter. It further suggests that the amino terminus of a short linker attached to the N-terminus of SAR1376 is surface accessible. Three such amino termini are present on each side of the sphere. This suggests a model in which fusion of antigens to the N-terminus of SAR1376 generates a small sphere with six antigens displayed outwards (FIG. 1).

Example 5: Inactivated SAR1376 Enzyme Retains the Adjuvant Activity

[0267] Since the mechanism of 4-OT catalysis has been heavily investigated, we mutated two critical residues involved in the active site: proline-1 (P1) and arginine-35 (R35), a site corresponding to R39 in other crystallised family members [21]. P1A mutations disrupt enzymatic activity, but leave the protein structure intact, whereas R39A or Q mutations disrupt catalysis and impair protein multimerisation [21]. The immunogenicity of ClfB fused to these variants was compared (FIG. 5). The SAR1376 mutant P1A enhanced both the antibody and T-cell responses to ClfB significantly compared to no scaffold (p=0.021) (FIGS. 5A and 5B). By contrast, the immune response to ClfB with the scaffold containing the R35A mutation [21] was not significantly different from ClfB without a scaffold (FIGS. 5A and 5B). The effect of SAR1376 mutant P1A on the ClfB antibody response was similar in both BALB/c (FIG. 5A) and outbred CD1 mice (FIG. 5C). Enhancement on IFN- producing T-cell numbers by P1A was observed in one mouse strain (FIGS. 5B and 5D). Taken together, this suggests that protein multimerisation contributes to the enhanced immunogenicity, but catalytic activity does not.

Example 6: SAR1376 and the PIA Variant are Immunogenic

[0268] We next investigated the capacity of SAR1376 fusion proteins to raise a humoral response against SAR1376 itself. Antibody responses against SAR1376 fused to either BitC (FIG. 6A) or ClfB (FIG. 6B) in immunised BALB/c mice were not significantly raised, as compared to the no-scaffold groups. By contrast, immunization with ClfB fusion constructs containing the SAR1376 mutant PIA scaffold raised a significant antibody response to SAR1376 in both BALB/c (FIG. 6B) and CD1 outbred (FIG. 6C) mice whereas the R35A mutation did not (FIG. 6B). In both strains, serological responses against SAR1376 in the absence of SAR1376 immunisation were indistinguishable from the assay background (FIG. 6), suggesting that SAR1376 homologues, if present in colonising murine microbiota, do not generate detectable cross reactivity with SAR1376.

[0269] In summary, fusion of two S. aureus antigens to SAR1376-PIA significantly increased antibody responses to the antigens, when expressed from DNA vaccine vectors. The enhancement was observed in two different mouse strains, and was abrogated by a mutation known to disrupt multimerisation of SAR1376. Baseline immune responses against SAR1376 were not detected in the two mouse strains, but SAR1376-P1A variant is itself immunogenic.

Example 7: Viral Vectored Vaccines Expressing SAR1376-PIA Fused to Truncated HIa Increase Immunogenicity

[0270] We investigated whether the pro-immunogenic effect of SAR1376 fusion was restricted to DNA vaccination. Because the effect of SAR1376 fusion appeared most marked on antibody induction, we elected to study the S. aureus alpha toxin (AT), a haemolytic multimeric -pore forming toxin which is a critical virulence factor in S. aureus [22] and is encoded by the H/a gene. AT can be neutralised by antibody [22]. We designed a truncated form of AT, designated tHIa75, comprising amino acids 1-75, the portion of the molecule reported to contain the receptor ADAM10 (A disintegrin and metalloproteinase 10) binding domain [23, 24]. Recombinant adenoviral (AdH5) and MVA vectors expressing SAR1376 fused to tHIa75 were constructed. BALB/c mice were vaccinated with AdH5-tHIa75 followed eight weeks later by MVA-tHIa, a prime-boost regime known to be highly immunogenic [2]. Analysis of the immune response against alpha toxin showed that SAR1376 fusion did not increase the immunogenicity of adenovirally expressed proteins (FIG. 7A), but two weeks post MVA boost, a significantly-enhanced antibody response was observed in the tHIa75-PIA group relative to the tHIa75-no scaffold group (FIG. 7A). However, the tHIa75 construct did not induce substantial functional (neutralising) antibody titres (FIG. 7C) in either configuration.

[0271] As expected, antibodies against SAR3176 were raised and boosted in tHIa75-PIA vaccinated group animals (FIG. 7B).

Example 8: Fusion of SAR1376-PIA to Pfs25 Recombinant Protein Improves Immunogenicity in BALB/c Mice

[0272] We investigated whether the pro-immunogenic effect of SAR1376 fusion extended to recombinant protein antigens by studying the effect SAR1376 fusion on immunogenicity of a P. falciparum protein, Pfs25. Pfs25 is a candidate antigen for a transmission blocking vaccine and antibodies against Pfs25 have been shown in several studies to interfere with sexual reproduction of the parasite in the mosquito vector [25]. Recently, we have reported that heptamerisation of Pfs25 by fusion to IMX313 increased its immunogenicity significantly in pre-clinical studies [10].

[0273] Recombinant monomeric Pfs25 and Pfs25-SAR1376-P1A proteins were produced in Pichia pastoris as secreted proteins and purified using a 6-Histidine tag (FIGS. 8A and 8B). Mice were immunised twice with either Pfs25-SAR1376-P1A or monomeric Pfs25 protein-in-Alhydrogel formulations at 2-week intervals.

[0274] Little response was seen in mice following vaccination with Pfs25. The antibody levels at all time points were significantly higher in the group that received Pfs25-SAR1376-P1A than the mice receiving monomeric Pfs25, demonstrating that fusion of Pfs25 to SAR1376-P1A significantly improved the immune response (FIG. 8C). The pooled and purified IgG from the Pfs25-SAR1376-P1A immunised mice (day 28) were able to completely block oocyst development in the mosquito midgut in the ex vivo Standard Membrane Feeding Assay (SMFA), demonstrating that antibodies produced by immunisation with this platform were functional (FIG. 8D).

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TABLE-US-00001 SEQUENCES SEQIDNO:1 Acinetobacter_baumannii_OIFC021gi444755286 MPFINIQTIEGLLDKDSKAELFKRITDLFVEIEGKGNPAFREHVWIRIDEYPPEQWQLGSLSPTKQMIELMTASK SEQIDNO:2 Bacillus_cereusgi487908138 MPYVTVKMLEGRTEEQKKALAEKVTAAVSETTGAPEENIIVFIEEMSKNHYAVGGKRLSDK SEQIDNO:3 Bartonella_elizabethae_Re6043vigi395417463 MPYVNIKITNEDVTSEQKRQLIEGATQLLVDVLNKNPRTTVVVIDEVDTDNWGIGGVPVTELRKL SEQIDNO:4 Bordetella_sp._FB-8gi518780346 MPYIRVEMLEGRSDEQKAKLAQAITNAMVEHAGAKPDSIFVVIEDVKKSNWATGGILMSQRK SEQIDNO:5 Burkholderia_sp._RPE67gi636800047 MPIVTIQVTREGNTPGANAVTADEKAQLIKGVSELLRDVLNKPLDSTFVVIQEVELENWGWGGLPVPEYRRQKQARD S SEQIDNO:6 Campylobacter_jejunigi657884310 MPFVNIRITKENGKPTTEQKQELIAGVTDLLAKVLNKNKSSTVVIIDEIDTDNYGLGGKSITQVRKEKS SEQIDNO:7 Citrobacter_freundiigi489932881 MPHVDIKCFPRDLNEEEKAALATDITEVIIRHLQSKESSVSVALNEVEPDAWQAVWDAEIAPQMTRLIKKPGYSM SEQIDNO:8 Enterobacter_cloacaegi654545569 MPHVDIKCFPRDLNDEQKTALAADIAEVIIRHFNSKDGSVSVALNQVNPEDWKAQVWDTEIGPKLDELIKKPGYSM SEQIDNO:9 Escherachia_coligi447042883 MPHIDIKCEPRELDEQQKAALAADITDVIIRHLNCKDSSISIALQQIQPESWQAIWDAEIAPQMEALIKKPGYSM SEQIDNO:10 Haemophilus_parainfluenzaegi503830159 MRYVNIKVTGGAEAPTAKQKAELIEGVTHLLQKVLNKNPETTVVVIDEVDMDNWGIGGKTVTSRRSMK SEQIDNO:11 Helicobacter_pylorigi639858229 MPFINIKLVPENGGSTNEQKQQLIEGVSDLMVKVLNKNKASIVVIIDEVDSNNYGLGGESVYHLRQKN SEQIDNO:12 Leptospira_interrogans_serovar_Djasiman_str._LT1649gi464164388 MPLVQIFVPAGSLSADMKNDMIQKVTDAVVETEGKPVVRRYTWVHINEVPDGGWGMSGKVVTIDAMKKSLEKTE SEQIDNO:13 Mycobacterium_intracellularegi497639745 MPLVEVTLVQGRAPHQLRTLISELTDAVETALGVSRSAIRVVLREVPDTHWAAGDVTIAERNKSS SEQIDNO:14 Neisseria_meningitidisgi488141741 MPYVNIKVTGGKEAPTAAQKAELIGGVTELLARVLGKNPETTVVVIDEVDTDNWGIGGKSVSERRKEGR SEQIDNO:15 Neisseria_gonorrhoeaegi636860961 MPYVNIKVTGGKEAPTAAQKAELIGGVIELLARVLGKNPETTVVVIDEVDTDNWDIGGKSVSERRKEGR SEQIDNO:16 Pseudomonas_aeruginosagi501023324 MPYVNIRVTREGVSADQKRQLIEGATDLLLKVLGKPPESTFVVIDEVDPDNWGVAGESVSALRRRQTPRTS SEQIDNO:17 Pseudomonas_putidagi639678172 MPIAQLYILEGRSDEQKESLIREISEAMSRSLDAPIERVRVIITEMPKNHFGIGGEPASKLNR SEQIDNO:18 Pseudomonas_aeruginosagi489251658 MPLVTVKGIEGVFSTEQKAEIISKITDAMVSVEGEKMRGVTWVVFEEVKSGDWGIGGEPITTEKVRALQNS SEQIDNO:19 Salmonella_entericagi555267960 MPHVDIKCFPRDLTDEAKLALAADITDVIIRHLQSKESSISVALNHVPPEEWQAVWDTEIAPQMETLIKKPGYTMSK SEQIDNO:20 Staphylococcus_aureusgi447046020 MPIVNVKLLEGRSDEQLKNLVSEVTDAVEKTTGANRQAIHVVIEEMKPNHYGVAGVRKSDQ SEQIDNO:21 Streptococcus_pneumoniaegi642960263 MPEVRIDLFEGRTLEQKKALAKEVTEAVVRNTGAPRSAVHVIINDMPEGTYFPQGEMRTK SEQIDNO:22 Streptococcus_agalactiaegi657924480 MPFVKIDLFEGRSQEQKNELAREVTEVVSRIAKAPKENIHVFINDMPEGTYYPQDELKKK SEQIDNO:23 Streptococcus_suisgi489024563 MPFVRIDLFEGRTEEQKIALAREVTEVVSRNTNAPKEAIHVFINDMPEGTYYPQGEMKRK SEQIDNO:24 Vibrio_fluvialisgi520908977 MPYINVKVTDDGVTKEQKQAIIKGCTQLMVDILNKNPEKTFVVIDEVNTDNWGVGFDQVTELRR SEQIDNO:25 Yersinia_pseudotuberculosisgi501054428 MPYVNIKITREGATAEQKKQLIAGVTQLLVDTLGKNPATTVVVIDEVDTDNWGIGGRSVTDLRQSS SEQIDNO:26 Yersinia_enterocoliticagi644933472 MPYVNIKITREGATAEQKKQLIAGVTQLLVDTLGKNPATTVVVIDEVDTDNWGIGGHSVTELRKSS SEQIDNO:27 SAR1376(aminoacidsequence) MMPIVNVKLLEGRSDEQLKNLVSEVTDAVEKTTGANRQAIHVVIEEMKPNHYGVAGVRKSDQ SEQIDNO:28 SAR1376P1A(aminoacidsequence,mutationunderlined) MMAIVNVKLLEGRSDEQLKNLVSEVTDAVEKTTGANKAIHVVIEEMKPNHYGVAGVRKSDQ SEQIDNO:29 SAR1376R35A(aminoacidsequence,mutationunderlined) MMPIVNVKLLEGRSDEQLKNLVSEVTDAVEKTTGANAQAIHVVIEEMKPNHYGVAGVRKSDQ SEQIDNO:30 SAR1376 ATGATGCCCATCGTGAACGTGAAGCTGCTGGAAGGCAGAAGCGACGAGCAGCTGAAGAACCTGGTGTCCGA AGTGACCGACGCCGTGGAAAAGACCACCGGCGCCAACAGACAGGCCATCCACGTCGTGATCGAGGAAATGAAGCCCA ACCACTACGGCGTGGCCGGCGTGCGGAAAAGCGATCAGTGATGA SEQIDNO:31 SAR1376P1A(mutationunderlined) ATGATGGCCATCGTGAACGTGAAGCTGCTGGAAGGCAGAAGCGACGAGCAGCTGAAGAACCTGGTGTCCGA AGTGACCGACGCCGTGGAAAAGACCACCGGCGCCAACAGACAGGCCATCCACGTCGTGATCGAGGAAATGAAGCCCA ACCACTACGGCGTGGCCGGCGTGCGGAAAAGCGATCAGTGATGA SEQIDNO:32 SAR1376R35A(mutationunderlined) ATGATGCCCATCGTGAACGTGAAGCTGCTGGAAGGCAGAAGCGACGAGCAGCTGAAGAACCTGGTGTCCGA AGTGACCGACGCCGTGGAAAAGACCACCGGCGCCAACGCCCAGGCCATCCACGTCGTGATCGAGGAAATGAAGCCCA ACCACTACGGCGTGGCCGGCGTGCGGAAAAGCGATCAGTGATGA SEQIDNO:33 SA1388 ATGAAGATCGCCGACCTGATGACCCTGCTGGACCACCACGTGCCCTTTAGCACAGCCGAGAGCTGGGACAA CGTGGGCCTGCTGATTGGCGACGGCGACGTGGAAGTGACCGGCGTGCTGACAGCCCTGGACTGCACACTGGAAGTCG TGAACGAGGCCATCGAGAAGGGCTACAACACCATCATCAGCCACCACCCCCTGATCTTCAAGGGCGTGACCAGCCTG AAGGCCAACGGCTACGGCCTGATCATCCGGAAGCTGATCCAGCACGACATCAACCTGATCGCCATGCACACCAACCT GGACGTGAACCCCTACGGCGTGAACATGATGCTGGCCAAGGCCATGGGCCTGAAGAACATCAGCATCATCAACAACC AGCAGGACGTGTACTACAAGGTGCAGACCTACATCCCCAAGGATAATGTGGGCCCCTTCAAGGACAAGCTGAGCGAG AATGGCCTGGCCCAGGAAGGCAACTACGAGTACTGCTTCTTCGAGAGCGAGGGCAGAGGCCAGTTCAAGCCTGTGGG CGAGGCCAACCCTACCATCGGCCAGATCGACAAGATCGAGGACGTGGACGAAGTGAAGATCGAGTTCATGATCGACG CCTACCAGAAGTCCAGAGCCGAGCAGCTGATCAAGCAGTACCACCCCTACGAGACACCCGTGTTCGACTTCATCGAG ATTAAGCAGACCTCCCTGTACGGCCTGGGCGTGATGGCCGAGGTGGACAACCAGATGACTCTGGAAGATTTCGCCGC CGACATCAAGAGCAAGCTGAACATCCCCTCCGTCAGATTCGTGGGCGAGAGCAACCAGAAGATCAAGCGGATCGCCA TCATCGGCGGCAGCGGCATCGGCTACGAGTATCAGGCTGTGCAGCAGGGCGCCGACGTGTTCGTGACAGGCGATATC AAGCACCACGACGCCCTGGACGCCAAGATCCATGGCGTGAACCTGATCGACATCAACCACTACAGCGAGTACGTGAT GAAGGAAGGCCTGAAAACCCTGCTGATGAACTGGTTCAATATCGAGAAGATTAACATTGATGTGGAAGCCAGCACCA TCAATACCGACCCCTTCCAGTACATCTGATGATGA SEQIDNO:34 Dps ATGAGCAACCAGCAGGACGTCGTGAAAGAACTGAATCAGCAGGTGGCCAACTGGACCGTGGCCTACACCAA GCTGCACAACTTCCATTGGTACGTGAAGGGCCCCAACTTCTTCAGCCTGCACGTGAAGTTCGAGGAACTGTACAACG AGGCCAGCCAGTACGTGGACGAGCTGGCCGAGAGAATCCTGGCCGTGGGCGGAAATCCTGTGGGCACCCTGACCGAG TGCCTGGAACAGAGCATTGTGAAAGAGGCCGCCAAGGGCTACAGCGCCGAGCAGATGGTGGAAGAACTGAGCCAGGA CTTCACCAACATCAGCAAGCAGCTGGAAAACGCCATCGAGATCGCCGGCAACGCTGGCGACGATGTGTCCGAGGACA TGTTCATCGGCATGCAGACCAGCGTGGACAAGCACAACTGGATGTTCAAGAGCTACCTGAGCTGATGATGA SEQIDNO:35 QacR ATGAACCTGAAGGACAAGATCCTGGGCGTGGCCAAAGAGCTGTTCATCAAGAACGGCTACAACGCCACCAC CACCGGCGAGATCGTGAAGCTGAGCGAGAGCAGCAAGGGCAACCTGTACTACCACTTCAAGACCAAAGAGAACCTGT TCCTGGAAATCCTGAACATCGAGGAATCCAAGTGGCAGGAACAGTGGAAGAAAGAACAGATCAAGTGCAAGACCAAC CGCGAGAAGTTCTACCTGTACAACGAGCTGAGCCTGACCACCGAGTACTACTACCCCCTGCAGAACGCCATCATCGA GTTCTGCACAGAGTACTACAAGACCAATAGCATCAACGAGAAGATGAACAAGCTGGAAAACAAGTACATCGACGCCT ACCACGTGATCTTCAAAGAGGGCAATCTGAACGGCGAGTGGTGCATCAATGACGTGAACGCCGTGTCCAAGATCGCC GCCAACGCCGTGAATGGCATCGTGACCTTCACCCACGAGCAGAACATCAATGAGCGGATCAAGCTGATGAACAAATT CAGCCAGATCTTCCTGAACGGCCTGAGCAAGTGATGA