Modified nanoparticle subunits and use thereof

Abstract

The present invention relates to the field of modified nanoparticle subunits, immunogenic compositions and vaccines comprising the modified nanoparticles, their manufacture and the use of such compositions in medicine.

Description

[0001] MODIFIED NANOPARTICLE SUBUNITS AND USE THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of improved dodecin nanoparticle subunits, immunogenic compositions and vaccines comprising the improved dodecin nanoparticle subunits that display polypeptide antigens, their manufacture and method of making and the use of such compositions in medicine. More particularly, it relates to a modified nanoparticle (NP) subunit of dodecin, which can be used to display polypeptide antigens and methods for preparing an assembled modified dodecin nanoparticle in a host cell. It also relates to methods for preparing an assembled modified dodecin nanoparticle in a host cell (e.g., a bacterial host cell), wherein the host cell expresses a modified dodecin nanoparticle subunit. In aspects, the modified dodecin nanoparticle subunit protein is expressed in the cytoplasm, in the periplasm of the host cell, or is secreted by the host cell.

[0004] BACKGROUND

[0005] Protein nanoparticles (NPs) have the ability to self-assemble into highly ordered symmetric and stable structures. NPs can be used for displaying target antigens in a defined pattern at high density on their surface, which resembles pathogen-associated molecular patterns (PAMPs) in nature, inducing both innate and adaptive immune responses. For instance, protein NPs may act as scaffolds to present antigens in highly ordered repetitive antigen arrays (see e.g., W002 / 056905). Accordingly, self-assembling protein nanoparticles have demonstrated the ability to evoke an immune response and may be used in the development and generation of new types of vaccine technologies.

[0006] In the past some efforts were put in displaying polypeptide antigens onto these NP, e.g., by providing a fusion protein consisting in the amino acid sequence of the NP subunit being covalently linked with the amino acid sequence of the polypeptide antigen.

[0007] Examples of fusion with dodecin have been reported e.g., in WO2019 / 086548.

[0008] However, stability and misfolding can arise, which result in a loss of either NP assembly and / or a loss of immunogenicity of the polypeptide antigen moiety. There exists a need for improved vaccine technologies. In particular, there exists a need for an adaptable nanoparticle platform, capable of producing a nanoparticle subunit that selfassembles while exposing at its surface a polypeptide antigen. Such a platform would be capable of producing a stable antigen surface-exposed nanoparticle in a single step. Suitably, such a platform would be suitable for preparation of immunogenic compositions and / or vaccines for different nanoparticles and different polypeptide antigens.

[0009] SUMMARY

[0010] In one aspect, a modified, in particular stabilized, dodecin nanoparticle (NP) subunit is provided, having an amino acid sequence at least 80% identical to amino acid residues 2-70 of SEQ ID NO: 1 and comprising at least one substitution at position A53, with respect to SEQ ID NO: 1.

[0011] In a further aspect, the invention pertains to a chimeric antigenic polypeptide comprising a) the modified subunit of the instant invention, and b) a polypeptide antigen.

[0012] In another aspect, is provided a method for preparing an assembled antigenic nanoparticle (NP) comprising the steps of: i) providing a host cell; ii) expressing the chimeric antigenic polypeptide according to the invention in said host cell, so as to produce an assembled antigenic nanoparticle; iii) recovering / obtaining the assembled antigenic nanoparticle.

[0013] Another aspect of the invention pertains to an isolated nucleic acid molecule, comprising a nucleotide sequence encoding the modified, in particular stabilized, nanoparticle subunit or the chimeric antigenic polypeptide according to the instant invention.

[0014] In one aspect, is provided a vector, comprising the isolated nucleic acid molecule according to the invention. In one further aspect, the invention concerns a host cell, comprising the isolated nucleic acid molecule or the vector of the instant invention.

[0015] Another aspect of the invention relates to an assembled antigenic nanoparticle comprising the chimeric antigenic polypeptide as disclosed herein.

[0016] In a further aspect, is also provided an immunogenic composition comprising i) the chimeric antigenic polypeptide and / or the assembled antigenic nanoparticle according to the instant invention, and ii) a pharmaceutically acceptable vehicle.

[0017] Another further aspect of the invention concerns a vaccine comprising the immunogenic composition as disclosed herein.

[0018] In one aspect, the invention provides a method of inducing an immune response in a human subject, comprising administering to the subject an immunologically effective amount of the assembled nanoparticle, the immunogenic composition, or the vaccine according to the instant invention.

[0019] DESCRIPTION OF DRAWINGS / FIGURES

[0020] FIG. 1. Graph showing the binding energy (expressed as kcal / mol) plotted against the folding stability (also expressed as kcal / mol) for the modified dodecin NP subunits generated by computational design. Minimization and residue mutant scanning was performed in silico on the wild type dodecin subunit sequence (SEQ ID NO: 1); the starting structure for the nanoparticles was refined and designed using an updated version of the PROSS algorithm (with symmetry restraints and the beta energy scoring function). The Rosetta FilterScan mover was used to perform single point mutagenesis of all the residues to the preferred PSSM mutations. The mutation scan was binned within twelve different energy thresholds (-0.5, -1, -1.5, -2, -2.5, -3, -3.5, -4, -4.5, -5, -5.5, -6 kcal / mol) to increase mutation sequence diversity. For example, a combination of -6 kcal / mol single point mutations would result in fewer mutations due to a higher energetic barrier for introducing new mutations.

[0021] FIG. 2. Graph showing a SDS-PAGE analysis on pooled fraction after purification by IMAC and SEC. The upper arrow refers to not completely folded protein, whereas the lower arrow refers to the monomeric dodecin subunits.

[0022] FIG. 3A, FIG. 3B and FIG. 3C. TEM analysis showing the wild type (FIG. 3A), the “0.5” construct of SEQ ID NO: 8 (FIG. 3B) and the “6” construct of SEQ ID NO: 2 (FIG. 3C). Black circles represent correctly assembled NPs, whereas white circles represent NPs that adhere to each other.

[0023] FIG. 4A and FIG. 4B. FIG. 4A: Graph showing a SDS-PAGE analysis on pooled fractions after IMAC and after SEC. The arrow indicates the single nanoparticle subunit fused to MrkA. High molecular aggregates are however visible due to the high stability of the selected NP. FIG. 4B: Western blot analysis on the purified MrkA-NP using a rabbit polyclonal anti-MrkA antibody.

[0024] FIG. 5A and FIG. 5B. FIG. 5 A: DLS measurement of purified MrkA-s / dodecin NP compared to the NP not displaying MrkA. FIG. 5B: TEM analysis showing a homogenous layer of MrkA- s / dodecin NP. (As used herein, the term “s / dodecin” refers to a stabilized dodecin subunit with one or more stabilizing mutation with respect to the wild type dodecin subunit).

[0025] FIG. 6. SDS PAGE analysis of recombinant MrkA monomer after IMAC purification.

[0026] FIG. 7A and FIG. 7B. Graphs showing the anti-MrkA total IgG ELISA, showing the geometric mean and individual EU / mL of all groups at day 27 (FIG. 7A) and day 42 (FIG. 7B). LD is for low dose (2 mg) as compared to a reference dose (10 mg); group 6 received 1 dose in lieu of the reference immunization schedule of 2 doses (see example 2).

[0027] FIG. 8. Graphs showing the anti-MrkA IgG response induced by recombinant MrkA monomer and MrkA-s / dodecin nanoparticle tested with Alhydrogel or AS01, at day 27 and day 42, using the Mann-Whitney unpaired two-tailed t test (groups 1-2 and groups 3-4). For day 27, *** p=0.0001; **** p<o 0001. For day 42, ** p=0.0027; *** p=0.0009.

[0028] FIG. 9. Graphs showing the anti-MrkA IgG response induced by MrkA-s / dodecin NP tested without adjuvants or with Alhydrogel or AS01, using the non-parametric Kruskall-Wallis test with Dunn's multiple comparison test (groups 1, 3, 5). The variation of the median values were not statistically significant (none of the p values were inferior to 0.05). FIG. 10. Graphs showing anti-MrkA IgG response induced by MrkA-s / dodecin NP tested with a one-dose or a two-doses schedule, using the Mann-Whitney unpaired two-tailed t test (groups 1, 6; day 42 only). *** p=0.0003.

[0029] FIG. 11. Graphs showing anti-MrkA IgG response induced by MrkA-s / dodecin NP was also assessed at 2 pg / dose and at 10 pg / dose both with AS01 as adjuvant, using the Mann- Whitney unpaired two-tailed t test (groups 2, 7). ** p=0.0014; **** p< 0.0001).

[0030] FIG. 12. TEM analysis showing the assembly of NPs from s / dodecin subunits fused to the 0-barrel of the fHbp protein antigen from N. meningitis.

[0031] FIG. 13. TEM analysis showing the assembly of NPs from s / dodecin subunits fused at the N- terminus to the TAG moiety of the Galack DOCK / TAG protein ligation system.

[0032] FIG. 14. TEM analysis showing the assembly of NPs from s / dodecin subunits fused at the N- terminus to the DOCK moiety of the Galack DOCK / TAG protein ligation system.

[0033] FIG. 15A and FIG. 15B. Graphs showing the result of the purification by size exclusion chromatography (SEC) using a Superdex 200 10 / 300 column equilibrated in PBS buffer, with a flow rate of 0.5 mL / min, of M0MPD-L1 -s / dodecin nanoparticles (loop 1 of MOMP serovar D, FIG. 15A) and MOMPD-Ll-L4-s / dodecin nanoparticles (loops 1 and 4 of MOMP serovar D, FIG. 15B)

[0034] FIG. 16. TEM analysis showing the assembly of NPs from s / dodecin subunits fused to loop 1 of the MOMP antigen of serovar D.

[0035] FIG. 17. Graphs showing anti-MOMP serovar D IgG response induced by M0MPD-L1 -s / dodecin NP (loop 1 of MOMP serovar D) or MOMPD-Ll-L4-s / dodecin NP (loops 1 and 4 of MOMP serovar D) was assessed at respectively 4.5 pg / dose and at 6.5 pg / dose both with ASO l as adjuvant. P2 corresponds to day 27 after immunization; P3 corresponds to day 42 after immunization of the mice

[0036] DETAILED DESCRIPTION

[0037] Definitions

[0038] As used herein, the term “naturally occurring amino acid residues” refers to amino acids that are naturally incorporated into polypeptides. In particular, the 20 amino acids encoded by the universal genetic code: alanine (ala, A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gin, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0039] “Modified” refers to a modification made to a nucleic acid sequence of a dodecin nanoparticle monomer subunit as compared to a wild type (wt) sequence or within the amino acid sequence of the dodecin nanoparticle monomer subunit itself; the term “modified” may or can also refer to mutations made to an amino acid sequence to improve the stability of the polypeptide it encodes.

[0040] “Stabilized” refers to introducing one or more mutations into an amino acid sequence of a dodecin nanoparticle monomer subunit, in order to improve the stability of the nanoparticle (e.g., with regards to self-assembly). In other words, the stabilized dodecin nanoparticle monomer subunits self-assemble in an improved manner, for example, as measured by a particle size distribution pattern, meaning a pattern for which aggregation of nanoparticles is less frequently observed, detected and / or measured. In some embodiments, the particle size distribution pattern does not show any substantial aggregation of the nanoparticles.

[0041] “Nanoparticle” or “NP” refers to a three-dimensional structure that self assembles from a plurality of nanoparticle monomer subunits. A “nanoparticle monomer” or “nanoparticle subunit” is intended to refer to one subunit of a nanoparticle. A “self-assembling fragment” of a nanoparticle monomer subunit is intended to refer to a fragment that assembles into a nanoparticle and displays target polypeptide antigens to generate an immune response against the target polypeptide antigen. In some instances, the term “dodecin nanoparticle subunit”, “dodecin nanoparticle polypeptide” and “dodecin nanoparticle subunit protein” are meant to refer to the same molecule.

[0042] In aspects, a nanoparticle may refer to one nanoparticle or a plurality of nanoparticles. In aspects, a nanoparticle may refer to a plurality of assembled monomer subunits, e.g., wherein each monomer subunit comprises a protein antigen fused to a single nanoparticle monomer subunit. As used herein, the term "immunogenic fragment" means a portion of a polypeptide antigen smaller than the whole, that is capable of eliciting a humoral and / or cellular immune response in a host animal, e.g., human, specific for that fragment. Fragments of a polypeptide or a protein can be produced using techniques known in the art, e.g., recombinantly, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide. Typically, fragments comprise at least 10, 20, 30, 40 or 50 contiguous amino acids of the full-length sequence. Fragments may be readily modified by adding or removing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 amino acids from either or both of the N-terminus and C-terminus.

[0043] As used herein, the term “conservative amino acid substitution” involves substitution of a native amino acid residue with a non-native residue such that there is little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position, and without resulting in decreased immunogenicity. For example, these may be substitutions within the following groups: (1) valine, glycine; (2) glycine, alanine; (3) valine, isoleucine, leucine; (4) aspartic acid, glutamic acid; (5) asparagine, glutamine; (6) serine, threonine; (7) lysine, arginine; and (8) phenylalanine, tyrosine. Conservative amino acid modifications to the sequence of a polypeptide (and the corresponding modifications to the encoding nucleotides) may produce polypeptides having functional and chemical characteristics similar to those of a parental polypeptide.

[0044] As used herein, the term “deletion” refers to the removal of one or more amino acid residues from the protein molecule sequence. Typically, no more than about from 1 to 10 (e.g. 1 to 7 residues, 1 to 6 residues or 1 to 4 residues) are deleted at any one site within the protein molecule sequence.

[0045] As used herein, the terms “insertion” or “addition” (including other tenses thereof such as “inserted”) means the addition of one or more non-native amino acid residues in the protein molecule sequence or, as the context requires, addition of one or more non-native nucleotides in the polynucleotide sequence. Typically, no more than about from 1 to 10 residues, (e.g., 1 to 7 residues, 1 to 6 residues, or 1 to 4 residues) are inserted at any one site within the protein molecule sequence. As used herein, the term “added next to” refers to the addition of one or more non-native amino acid residues in the protein sequence at a position adjacent to the referenced amino acid or amino acid region. For example, “added next to one or more amino acids between amino acid residues 198-218” means the addition at a position adjacent to any one of amino acid residues 198- 218 (including adjacent to amino acid residues 198 or 218).

[0046] Unless specifically stated otherwise, providing a numeric range (e.g., “25-30”) is inclusive of endpoints (i.e. includes the values 25 and 30). For example, “between amino acids 198 to 218...of SEQ ID NO: X” refers to a position in the amino acid sequence between amino acid 198 and amino acid 218 of SEQ ID NO: X including both amino acids 198 and 218, as well as amino acids 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216 and 217.

[0047] The terms “identical” or percent “identity” refer to nucleotide sequences or amino acid sequences that are the same or have a specified percentage of nucleotide residues or amino acid residues that are the same (e.g. 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity over a specified region), when compared and aligned for maximum correspondence using, for example, sequence comparison algorithms or by manual alignment and visual inspection. Identity between polypeptides may be calculated by various algorithms. In general, when calculating percentage identity the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment. For example, the Needleman Wunsch algorithm (Needleman and Wunsch 1970, J. Mol. Biol. 48: 443-453) for global alignment, or the Smith Waterman algorithm (Smith and Waterman 1981, J. Mol. Biol. 147: 195- 197) for local alignment may be used, e.g., using the default parameters (Smith Waterman uses BLOSUM 62 scoring matrix with a Gap opening penalty of 10 and a Gap extension penalty of 1). A preferred algorithm is described by Dufresne et al. in Nature Biotechnology in 2002 (vol. 20, pp. 1269-71) and is used in the software GenePAST (Genome Quest Life Sciences, Inc. Boston, MA). The GenePAST “percent identity” algorithm finds the best fit between the query sequence and the subject sequence and expresses the alignment as an exact percentage. GenePAST makes no alignment scoring adjustments based on considerations of biological relevance between query and subject sequences. Identity between two sequences is calculated across the entire length of both sequences and is expressed as a percentage of the reference sequence.

[0048] As used herein the term “recombinant” means artificial or synthetic. In certain embodiments, a “recombinant polypeptide” refers to a protein that has been made using recombinant nucleotide sequences (nucleotide sequences introduced into a host cell). In certain embodiments, the nucleotide sequence that encodes a “recombinant polypeptide” is heterologous to the host cell.

[0049] As used herein the term “chimeric” means artificial or synthetic fusion of two or more amino acid sequences or alternatively two or more nucleic acid sequences from distinct origins. In certain embodiments, a “chimeric polypeptide” refers to a polypeptide that has been made using recombinant nucleotide sequences (nucleotide sequences introduced into a host cell). In certain embodiments, the nucleotide sequence that encodes a “chimeric polypeptide” is heterologous to the host cell. In some embodiments, the chimeric polypeptide is not glycosylated, i.e. does not comprise any saccharide moiety, including oligosaccharide and polysaccharide moieties.

[0050] As used herein the terms “isolated” or “purified” mean a polypeptide, protein, polynucleotide, or vector in a form not found in nature, as such. This includes, for example, a polypeptide, protein, polynucleotide, or vector having been separated from host cell or organism (including crude extracts) or otherwise removed from its natural environment. In certain embodiments, an isolated or purified polypeptide is a polypeptide essentially free from all other polypeptides with which the protein is innately associated (or innately in contact with).

[0051] As used herein, the term “subject” refers to an animal, in particular a mammal such as a primate (e.g., human).

[0052] As used herein, the term “effective amount,” in the context of administering a therapy (e.g., an immunogenic composition or vaccine of present embodiments) to a subject refers to the amount of a therapy which has a prophylactic and / or therapeutic effect(s). In certain embodiments, an “effective amount” refers to the amount of a therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of a microbial, including yeast, bacterial or viral, infection or symptom associated therewith; (ii) reduce the duration of a microbial, including yeast, bacterial or viral, infection or symptom associated therewith; (iii) prevent the progression of a microbial, including yeast, bacterial or viral, infection or symptom associated therewith; (iv) cause regression of a microbial, including yeast, bacterial or viral, infection or symptom associated therewith; (v) prevent the development or onset of a microbial, including yeast, bacterial or viral, infection, or symptom associated therewith; (vi) prevent the recurrence of a microbial, including yeast, bacterial or viral, infection or symptom associated therewith; (vii) reduce organ failure associated with a microbial, including yeast, bacterial or viral, infection; (viii) reduce hospitalization of a subject having a microbial, including yeast, bacterial or viral, infection; (ix) reduce hospitalization length of a subject having a microbial, including yeast, bacterial or viral, infection; (x) increase the survival of a subject with a microbial, including yeast, bacterial or viral, infection; (xi) eliminate a microbial, including yeast, bacterial or viral, infection in a subject; (xii) inhibit or reduce a microbial, including yeast, bacterial or viral, replication in a subject; and / or (xiii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

[0053] The term “comprises” is open-ended and means “includes.” Thus, unless the context requires otherwise, the word “comprises” or “has”, and variations thereof (including “comprise” and “comprising” or “have” and “having”, respectively), will be understood to imply the inclusion of a stated compound(s), molecule(s), composition(s), or steps, but not to the exclusion of any other compound(s), molecule(s), composition(s), or steps or group of compounds or steps. The terms “comprising” and “having” when used as a transition phrase herein are open-ended whereas the term “consisting of’ when used as a transition phrase herein is closed (i.e., limited to that which is listed and nothing more). The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

[0054] “Nanoparticle subunit” is intended to refer to a nanoparticle monomer that self-assembles with other nanoparticle monomers to form a nanoparticle. The nanoparticle subunit may be genetically fused to an antigen, in particular a polypeptide antigen. In some embodiments, the polypeptide antigen is not glycosylated, i.e. does not comprise any saccharide, including oligosaccharide and polysaccharide, moieties.

[0055] A “modified nanoparticle subunit” refers to a nanoparticle subunit that can be modified for stability (e.g., one or more mutation(s)). In embodiments, “modified nanoparticle subunit” and “stabilized nanoparticle subunit” are meant to refer to the same subject matter and may substitute one another. The modified nanoparticle subunit self-assembles with other subunits to form an assembled nanoparticle (e.g., composed of multiple subunits). A modified nanoparticle subunit that is directly or indirectly fused to one or more polypeptide antigen(s) may be part of a pharmaceutical composition, an immune composition, or a vaccine composition, designed to elicit an immune response against the one or more polypeptide antigen molecules exposed at the surface of the nanoparticle subunit. In aspects, nanoparticles may be used to display polypeptide antigens to induce a host response such as an effective B cell response and / or a T cell response. In aspects, the modified nanoparticle subunit includes one or more stabilizing mutation(s).

[0056] Assembled nanoparticles may display multiple molecules (one or more polypeptide antigens) in an ordered array. With respect to a nanoparticle displaying one or more polypeptide antigen(s), it is thought that an ordered multiplicity of polypeptide antigens presented on a nanoparticle exterior surface allows multiple binding events to occur simultaneously between the nanoparticle and host cell, which favors the induction of a potent host immune response (see e.g. Lopez-Sagaseta et al., Comput Struct Biotechnol J, 14:58-68 (2016); see also Perotti and Perez 2020 Viruses 12(35): doi: 10.3390 / vl2010035 (17 total pages); Ueda et al. 2020 eLife 9: e57659 (30 total pages)) along with conjugation to a polysaccharide (see Polonskaya et al. 2017 J. Clin. Invest. 127(4): 1492-1504; Pan et al. 2020 Adv. Mater. 32:2002940)). Nanoparticles may undergo self-assembly into highly symmetric stable and organized structures and may be modified to display polypeptide antigen(s) in order to mimic the surface of a microbe, including a yeast, a bacterium or a virus.

[0057] As used herein, the terms “modified polypeptide” “modified protein” mean a polypeptide or a protein that is altered (in one or more ways) as compared to wild type, as found in nature (e.g., a “modified protein” excludes a wild type protein).

[0058] Certain embodiments provide a nucleic acid molecule comprising a polynucleotide that encodes such modified, in particular stabilized, nanoparticle subunit (comprising one or more stabilization mutation(s)) or chimeric antigenic polypeptide molecules, comprising a stabilized dodecin nanoparticle subunit fused to a target polypeptide antigen.

[0059] Certain embodiments provide a pharmaceutical composition comprising one or more nanoparticle monomer(s) or one or more chimeric antigenic polypeptide molecule(s). Certain other embodiments provide a composition comprising a nanoparticle subunit fused or linked to one or more polypeptide antigen molecule(s). The nanoparticle subunits may assemble into nanoparticles.

[0060] Computational and structure-based design have been applied to design suitable nanoparticles and to identify positions within nanoparticle subunits for stabilization. For example, Rosetta and MOE may be used to identify suitable sites for modification (e.g., modifications / mutations to improve NP stability). Once a lead design is identified, these leads may be cloned and expressed to evaluate their expression by the host cell, either in the cytoplasm or in the periplasm, as well as suitability for obtaining a modified, in particular stabilized, NP, or a modified, in particular stabilized, NP decorated with polypeptide antigens at its surface.

[0061] Illustratively, competent Gram-negative E. coli strains transformed with different plasmids carrying different nanoparticle genes and leader sequences may be screened for efficient nanoparticle expression and assembly in the periplasm. Such experiments revealed that it is technically feasible to obtain stable nanoparticles that are correctly assembled in the bacterial periplasm to generate antigenic nanoparticles for use as vaccines against microbial infection.

[0062] Stabilized dodecin nanoparticle subunit

[0063] It is an object of present embodiments to provide a nanoparticle (NP) platform for producing stabilized nanoparticles. It should be understood that the modified, in particular stabilized, nanoparticle subunits according to the instant disclosure are produced in a host cell. The modified, in particular stabilized, nanoparticle subunits according to the instant disclosure are alternatively produced in the cytoplasm of the host cell; in the periplasm of the host cell, when the host cell is a Gram-negative bacterium; or secreted by the host cell.

[0064] In one embodiment, the modified, in particular stabilized, nanoparticle subunit is translocated to or expressed in the periplasm of a host cell, in particular a Gram-negative bacterial host cell (e.g. a bacterial host cell such as E. coli).

[0065] The present inventors have surprisingly found that some mutation(s) of the dodecin subunit amino acid sequence achieve a modified, in particular stabilized, dodecin NP that is less prone to self-aggregate and display a more homogeneous particle size distribution. As it would be apparent from the example section below, the stabilized, dodecin subunit of amino acid sequence SEQ ID NO: 8 was successfully synthesized in E. coli, and when the resulting assembled nanoparticles were analyzed by transmission electron microscopy or TEM, they significantly showed reduced aggregation when compared to nanoparticles produced with the wild type dodecin subunits (of amino acid sequence SEQ ID NO: 1). In addition, the stabilized dodecin nanoparticle subunit of sequence SEQ ID NO: 8 was successfully fused to distinct polypeptide antigens and the resulting chimeric antigenic polypeptides retained immunogenic properties, when assessed in in vivo animal models, in some cases after one unique dose.

[0066] Remarkably, the stabilized dodecin nanoparticle subunit was successfully fused to the poor immunogenic MrkA protein of Klebsiella pneumoniae, and the resulting assembled nanoparticles achieved improved immunogenicity as compared to unfused MrkA.

[0067] Similarly, the stabilized dodecin nanoparticle subunit was successfully fused to loop 1 and / or loop 4 of the highly insoluble MOMP protein of Chlamydia trachomatis, which is considered as one major antigen. Therefore, the invention offers means to improve the feasibility to design vaccine against this pathogenic bacterium.

[0068] Thus, in an aspect, present embodiments relate to self-assembling dodecin nanoparticles produced by the host cell that display antigenic polypeptide molecules on the external surface of the nanoparticle, to compositions comprising such nanoparticles, and to methods of making and using such nanoparticles and compositions.

[0069] In an embodiment, the modified, in particular stabilized, dodecin nanoparticle subunit comprises a signal sequence which is capable of directing the nanoparticle subunit to the periplasm of a host cell, in particular a Gram-negative bacterial host cell (e.g., an E. coli bacterium). Signal sequences, including periplasmic signal sequences, are usually removed during translocation of the protein into, for example, the periplasm by signal peptidases (i.e., a mature protein is a protein from which at least the signal sequence has been removed). The signal sequence may be from E. coli DsbA [MKKIWLALAGLVLAFSASA (SEQ ID NO: 14)], E. coli TolB [MKQALRVAFGFLILWASVLHA (SEQ ID NO: 15)], E. coli flagellm (Figi) [MIKFLSALILLLVTTAAQA (SEQ ID NO: 16)], E. coli outer membrane porin A (OmpA) [MKKTAIAIAVALAGFATVAQA (SEQ ID NO: 17)], E. coli maltose binding protein (MalE) [MKIKTGARILALSALTTMMFSASALA (SEQ ID NO: 18)], Erwinia carotovorans pectate lyase (PelB) [MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 19)], heat labile E. coli enterotoxin LTIIb [MSFKKIIKAFVIMAALVSVQAHA (SEQ ID NO: 20)], Bacillus subtilis endoxylanase XynA [MFKFKKKFLVGLTAAFMSISMFSATASA (SEQ ID NO: 21], or SipA [MKMNKKVLLTSTMAASLLSVASVQAS (SEQ ID NO: 22], In a specific embodiment, the signal sequence is from E. coli D sb A [MKKIWLALAGLVLAFSASA (SEQ ID NO: 14)].

[0070] Thus, present embodiments provide a modified, in particular stabilized, dodecin nanoparticle subunit, wherein the amino acid sequence further comprises a signal sequence which is capable of directing the dodecin nanoparticle subunit to the periplasm of a host cell (e.g., bacterium), said signal sequence being any suitable sequence. Techniques for fusing signal peptide to a nanoparticle subunit are known in the art (e.g., a signal peptide of the protein DsbA from E. coli can be genetically fused to the N-terminus of the mature nanoparticle subunit amino acid sequence) (see, Schulz, H., Hennecke, H., and Thony-Meyer, L., Science, 281, 1197-1200, 1998).

[0071] It will be understood by a person skilled in the art, that reference to “between amino acids ...” (for example “between amino acids 45-55”) is referring to the amino acid number counting consecutively from the N-terminus of the amino acid sequence. For example, “between amino acids 49-55 of SEQ ID NO: X” refers to position in the amino acid sequence between amino acid 49 and amino acid 55 of SEQ ID NO: X including both amino acids 49 and 55, as well as amino acid at position 50, 51, 52, 53 and 54.

[0072] The amino acid numbers referred to herein correspond to the amino acids in SEQ ID NO: X and as described above, a person skilled in the art can determine equivalent amino acid positions in an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: X by alignment. The addition or deletion of amino acids from the variant and / or fragment of SEQ ID NO: X could lead to a difference in the actual amino acid position of the consensus sequence in the mutated sequence, however, by lining the mutated sequence up with the reference sequence, the amino acid in an equivalent position to the corresponding amino acid in the reference sequence can be identified and hence the appropriate position for addition or substitution of the consensus sequence can be established. In aspects, mutation positions listed with respect to a sequence may refer to a first sequence (e.g., having mutations) aligned with a reference sequence (e.g. wt sequence). Specific examples follow.

[0073] Nanoparticles made of non-viral protein subunits may display antigenic molecules on the exterior surface, in particular polypeptide antigens. Such nanoparticles include those made of bacterial, insect, and mammalian proteins that naturally self-assemble into nanoparticles. Herein, host cells were engineered to produce modified, in particular stabilized, dodecin nanoparticles, as well as modified, in particular stabilized, dodecin nanoparticles that display one or more polypeptide antigen(s). In one aspect, the modified, in particular stabilized, dodecin nanoparticle subunit, in the form of a monomer, is expressed in the cytoplasm of the host cell, wherein it undergoes assembly to form an assembled nanoparticle. In another aspect, the modified, in particular stabilized, dodecin nanoparticle subunit, in the form of a monomer, is translocated to or expressed in the periplasm of the host cell, in particular the periplasm of a Gram-negative bacterial host cell, wherein it undergoes assembly to form an assembled nanoparticle. In aspects, a leader sequence is present. In one further aspect, the modified, in particular stabilized, dodecin nanoparticle subunit, in the form of a monomer, is secreted by the host cell, wherein it undergoes assembly to form an assembled nanoparticle. In aspects, the secreted assembled nanoparticle may be found at the external surface of the host cell, and / or found in the culture medium. As it is understood herein, the modified, in particular stabilized, dodecin nanoparticle subunit may include one, two or three mutations for stability, so as to decrease aggregation of nanoparticles subunits and the assembly of nanoparticles with an improved size particle distribution.

[0074] Dodecin Subunit Protein

[0075] Dodecin subunit protein (also known as mtDod) is a soluble secretory protein of about 8 kDa with a P«PP topology (Bourdeaux et al., Nature Scientific Reports, 2020, 10: 13297), encoded by the gene Rvl498a of Mycobacterium tuberculosis (Liu et al. Journal of Structural Biology, 2011, 175:31-18), which self-assembles into a dodecamer of spherical shape. In aspects, N- and C-termini are exposed at the protein surface (Bourdeaux et al., Nature Scientific Reports, 2020, 10: 13297), facilitating its use as a carrier molecule. A dodecin nanoparticle may assemble from 12 subunits to form a spherical shape with about a 6 nm diameter. A dodecin subunit polypeptide of present embodiments can be produced by methods provided herein. The amino acid sequence of wildtype dodecin is shown in SEQ ID NO: 1 (Uniprot: Q8VK10). Modifications to dodecin were made to improve stability, with said modifications at one, two or three of positions A53, G25 and V50 (e.g., A53T, G25N and V50T), wherein the positions are with respect to the wild type amino acid sequence, as found in nature, - (i.e. the amino acid sequence SEQ ID NO: 1).

[0076] In some aspects, the invention relates to a modified, in particular stabilized, dodecin NP subunit having an amino acid sequence at least 80% identical to amino acid residues 2-70 of SEQ ID NO: 1 and comprising at least one substitution at position A53, with respect to SEQ ID NO: 1.

[0077] In aspects, dodecin may optionally be linked to a signal sequence at the N- terminus and / or a histidine tag for purification (e.g., at the N- or C- terminus). The modified, in particular stabilized, dodecin subunit protein further undergoes self-assembly in the periplasm to form an assembled nanoparticle.

[0078] In some embodiments, the modified, in particular stabilized, dodecin subunit protein further comprises one or more substitution(s) at position(s) G25 and / or V50, with respect to SEQ ID NO: 1.

[0079] The modified, in particular stabilized, dodecin subunit protein may comprise stabilizing mutations at one or more of alanine to threonine (A53T), glycine to asparagine (G25N) and valine to threonine (V50T), with reference to the amino acid sequence of SEQ ID NO: 1 (or an equivalent position in an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1). In some embodiments, the substitutions are selected from the group consisting of A53T, G25N and V50T, with respect to SEQ ID NO: 1.

[0080] The modified, in particular stabilized, dodecin subunit protein of the invention may be the amino acid sequence of SEQ ID NO: 1 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1) and comprising substitution of alanine to threonine (A53T). The modified, in particular stabilized, dodecin subunit protein may be the amino acid sequence of SEQ ID NO: 1 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1) and comprising substitution of glycine to asparagine (G25N). The modified, in particular stabilized, dodecin subunit protein of present embodiments may be the amino acid sequence of SEQ ID NO: 1 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1) and comprising substitution of valine to threonine (V50T).

[0081] Thus, in aspects, stability mutations may include (i) A53T, (ii) G25N and A53T, (ii) V50T and A53T, or (iv) G25N, V50T, and A53T. In a preferred embodiment, the specific mutations may include G25N, V50T, and A53T.

[0082] In some embodiments, the one or more substitutions are selected from the group consisting of A53T, G25N and V50T, with respect to SEQ ID NO: 1.

[0083] In still other aspects, a signal sequence may replace one or more N-terminal residues or be inserted / appended at the N terminus. Any suitable signal sequence (e.g. DsbA (SEQ ID NO: 14), TolB (SEQ ID NO: 15), PelB (SEQ ID NO: 16), Figi (SEQ ID NO: 16), LtllB (SEQ ID NO: 20)) may be used. In aspects, a signal sequence may be appended at the N terminus of the amino acid sequence of SEQ ID NO: 4 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 4, provided the amino acid comprises A53T substitution, the position being with respect to SEQ ID NO: 1)).

[0084] In aspects, the modified sequence optionally may include a Poly His tag (e.g., a string of 5 to 7 histidine residues, or 6 histidine resides) at the N- or C-terminus for ease of purification of the amino acid sequences of SEQ ID NOs: 4, 6, 7 or 8 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 4, 6, 7 or 8).

[0085] A person skilled in the art will understand that for variant dodecin amino acid sequences with respect to SEQ ID NO: 1 and / or fragments of an amino acid sequence of SEQ ID NO: 1, such an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, and with reference to “between amino acids ...” refers to the position that would be equivalent to the defined position, if this sequence was lined up with an amino acid sequence of SEQ ID NO: 1 in order to maximise the sequence identity between the two sequences (Sequence alignment tools are not limited to Clustal Omega (www(.)ebi(.)ac(.)ac(.)uk) MUSCLE (www(.)ebi(.)ac(.)uk), or T-coffee (www(.)tcoffee(.)org). In one aspect, the sequence alignment tool used is Clustal Omega (www(.)ebi(.)ac(.)ac(.)uk).

[0086] In aspects, the modified sequence optionally may include a Poly His tail (e.g. a string of 5 to 7 histidine residues, or 6 histidine resides) inserted / appended to the N-terminus or C-terminus for purification of the corresponding polypeptides. In some embodiments, the modified, in particular stabilized, dodecin subunit protein of the invention is not glycosylated. In these embodiments, the modified, in particular stabilized, dodecin subunit protein does not comprise any saccharide, including oligosaccharide and polysaccharide, moieties.

[0087] Chimeric

[0088] In other aspects, modified, in particular stabilized, dodecin nanoparticle subunits may be fused to a polypeptide antigen, according to techniques known in the art, in particular by genetic fusion of the corresponding coding nucleic acid sequences. The chimeric antigenic polypeptides may undergo expression in the host cell and may self-assemble in the periplasm.

[0089] In aspects, the modified, in particular stabilized, dodecin nanoparticle subunit polypeptide may further comprise a “peptide tag” or “tag”, i.e. a sequence of amino acids that allows for the isolation and / or identification of the modified, in particular stabilized, subunit protein. For example, adding a tag to a modified, in particular stabilized, dodecin nanoparticle subunit polypeptide can be useful in the purification of that protein and, hence, the purification of product vaccines comprising the tagged modified, in particular stabilized, nanoparticle subunit polypeptide. Exemplary tags that can be used herein include, without limitation, histidine (HIS) tags (e.g. hexa-histidine tag, or 6Xhis-Tag), FLAG-TAG, and HA tags. In one embodiment, the tag is a hexa-histidine tag. The tags used herein are removable, e.g. removal by chemical agents or by enzymatic means, once they are no longer needed, e.g. after the protein has been purified. Thus, the modified, in particular stabilized, nanoparticle subunit polypeptide may further comprise a peptide tag. Optionally the peptide tag is located either at the N-terminus or at the C-terminus of the amino acid sequence of the target polypeptide. Optionally the tag comprises six histidine residues at the N- terminus or at the C-terminus of the amino acid sequence. In one aspect, the modified, in particular stabilized, nanoparticle subunit polypeptide comprises (or consists of) an amino acid sequence which is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to any one of the nanoparticle subunit sequences provided herein and a peptide tag (e.g. six histidine residues at the N- or C-terminus of the amino acid sequence).

[0090] In some embodiments, the chimeric antigenic polypeptide may be synthesized as a polypeptide having an amino acid sequence of formula NH2 — A-[L]-B — COOH, wherein each A and B represent the modified, in particular stabilized, dodecin nanoparticle subunit and the polypeptide antigen amino acid sequences (irrespective of the order), and L represents an optional linker amino acid sequence.

[0091] In some embodiment, the linker amino acid sequence “L” is a glycine polymer or glycineserine polymer linker. Exemplary linkers include, but are not limited to, “GSG”, “GSS”, “GGSG”, “GGSGG”, “GSGSG”, “GSGGG”, “GGGSG”, “GSSSG” and “GSGGGG”. Other suitable glycine or glycine-serine polymer linkers will be apparent to the skilled person.

[0092] In some embodiments, the chimeric polypeptide, including the modified dodecin nanoparticle subunit and / or the polypeptide antigen, is not glycosylated. In these embodiments, one understands that the chimeric polypeptide does not comprise any saccharide, including oligosaccharide and polysaccharide, moieties.

[0093] In certain embodiments, the chimeric antigenic polypeptide comprises a polypeptide antigen selected from Chlamydia trachomatis serovar D MOMP (major outer membrane protein), Chlamydia trachomatis serovar E MOMP, and one or more immunogenic fragment(s) thereof. As used herein, an immunogenic fragment is intended to refer to a portion of the polypeptide antigen, hence excluding the full-length polypeptide antigen, which is capable of eliciting an immune response.

[0094] In some embodiments, the chimeric antigenic polypeptide comprises a polypeptide antigen selected from Chlamydia trachomatis serovar D MOMP (major outer membrane protein) or one or more immunogenic fragment(s) thereof and a polypeptide antigen selected from Chlamydia trachomatis serovar E MOMP or one or more immunogenic fragment(s) thereof. As used herein, an immunogenic fragment is intended to refer to a portion of the polypeptide antigen, hence excluding the full-length polypeptide antigen, which is capable of eliciting an immune response. In some embodiments, the one or more immunogenic fragment(s) of MOMP comprise(s) loop 1 , loop 2, loop 3, loop 4, and any combination thereof. In particular, in certain embodiments, the Chlamydia trachomatis MOMP polypeptide antigen comprises loop 1 and / or loop 4 of MOMP of Chlamydia trachomatis serovar D.

[0095] In certain embodiments, the chimeric antigenic polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11. In other embodiments, the chimeric antigenic polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12.

[0096] In certain embodiments, the chimeric antigenic polypeptide comprises a polypeptide antigen consisting of Klebsiella pneumoniae MrkA. In said embodiments, the chimeric antigenic polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 23.

[0097] In some embodiments, the chimeric antigenic polypeptide comprises a polypeptide antigen consisting of Neisseria meningitidis fHbp. In said embodiments, the chimeric antigenic polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 24 or SEQ ID NO: 25.

[0098] In some embodiments, the antigen polypeptides may be genetically fused to the modified, in particular stabilized, dodecin nanoparticle subunit at its N-terminus, at its C-terminus, or alternatively at a position distinct from the N-terminus and the C-terminus, with the proviso that the nanoparticle subunit, within the chimeric antigenic polypeptide, retains the ability to produce assembled nanoparticles of the appropriate size and conformation. As a non-limiting example, the MRA amino acids in position 34-36 of SEQ ID NO: 8 may be substituted with the amino acid of an antigen polypeptide of interest.

[0099] An embodiment comprises a modified, in particular stabilized, dodecin nanoparticle subunit displaying one or more polypeptide antigen(s) on the exterior surface once the stabilized nanoparticle is assembled. In one aspect of the present invention, the polypeptide antigen(s) may be displayed at alternative locations on the nanoparticle subunit, including on its N-terminus or its C-terminus. In other embodiments of the present invention, the polypeptide antigens may be displayed on the nanoparticle subunit on its N-terminus, its C-terminus and both its N-terminus and C-terminus. Without being limited by theory, these embodiments are facilitated by the fact that both the N-terminus and the C-terminus of the dodecin NP subunit are on the same side of the subunit and exposed at the surface of the assembled NP.

[0100] The polypeptide antigen displayed by the nanoparticle may be a bacterial polypeptide, a yeast polypeptide or a viral polypeptide. In an embodiment, the polypeptide antigen is a bacterial polypeptide from a Gram-negative bacterium, or a polypeptide from a Gram-positive bacterium.

[0101] In a further embodiment, the polypeptide antigen is selected from the group consisting of Chlamydia species, Escherichia species, Shigella species, Klebsiella species, Salmonella species, Yersinia species, Helicobacter species, Proteus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Enterococcus species, Staphylococcus species, Bacillus species, Clostridium species, Listeria species, Campylobacter species, Neisseria species and Candida polypeptide antigens.

[0102] In some embodiments, the polypeptide antigen displayed on the modified, in particular stabilized, nanoparticle surface is selected from the group consisting of a polypeptide antigen from Chlamydia species, Escherichia species, Shigella species, Klebsiella species, Salmonella species, Yersinia species, Helicobacter species, Proteus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Enterococcus species, Staphylococcus species, Bacillus species, Clostridium species, Listeria species, Campylobacter species, Neisseria species and Candida polypeptide antigens.

[0103] Examples include but are not limited to Chlamydia trachomatis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, or Staphylococcus aureus. In an embodiment, the polypeptide antigen of present embodiments is a bacterial polypeptide selected from Chlamydia trachomatis, Shigella flexneri, Klebsiella pneumoniae and Streptococcus pneumoniae polypeptides.

[0104] In some embodiments, the polypeptide antigen is selected from the group consisting of Chlamydia species polypeptide antigens, Klebsiella species polypeptide antigens and Neisseria species polypeptide antigens.

[0105] In some embodiments, the polypeptide antigen is selected from the group consisting of Chlamydia trachomatis polypeptide antigens, Klebsiella pneumoniae polypeptide antigens and Neisseria meningitidis polypeptide antigens.

[0106] In certain embodiments, the polypeptide antigen comprises MOMP (Major Outer Membrane Protein) from Chlamydia trachomatis serovar D. In some embodiments, the polypeptide antigen comprises one or more loops selected in the group of loop 1 , loop 2, loop 3 and loop 4 of MOMP from Chlamydia trachomatis serovar D. In some embodiments, the polypeptide antigen comprises at least loop 1 and / or loop 4 of MOMP from Chlamydia trachomatis serovar D.

[0107] In certain embodiments, the polypeptide antigen comprises MOMP from Chlamydia trachomatis serovar E. In some embodiments, the polypeptide antigen comprises one or more loops selected in the group of loop 1, loop 2, loop 3 and loop 4 of MOMP from Chlamydia trachomatis serovar E. In some embodiments, the polypeptide antigen comprises at least loop 1 and / or loop 4 of MOMP from Chlamydia trachomatis serovar E.

[0108] In certain embodiments, the polypeptide antigen comprises MrkA from Klebsiella pneumoniae.

[0109] In certain embodiments, the polypeptide antigen comprises a fHbp from Neisseria meningitidis. In certain embodiments, the polypeptide antigen is from a Gram-negative bacterium (e.g. Salmonella species, Shigella species, Pseudomonas species or Klebsiella species such as Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, or Klebsiella pneumoniae.

[0110] In certain embodiments, the polypeptide antigen is from a Gram-negative or Gram-positive bacteria such as Neisseria meningitidis serogroup A, Neisseria meningitidis serogroup B, N. meningitidis serogroup C, N. meningitidis serogroup Y, N. meningitidis serogroup W, H. influenzae, Group B Streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus. In certain embodiments, the polypeptide antigen is a polypeptide from Streptococcus species or Staphylococcus species (e.g. Streptococcus pneumoniae or Staphylococcus aureus). In an embodiment, the polypeptide antigen is a polypeptide from Staphylococcus aureus. For example, the polypeptide antigen may be a polypeptide from Staphylococcus aureus type 5 and 8. In an embodiment, the polypeptide antigen is a polypeptide from Streptococcus pneumoniae (e.g. any of the 90+ serotypes (see, Shoji et al., Infection and Drug Resistance (2018) vol. 11, pp 1387- 1400)).

[0111] Antigenic display

[0112] Molecules, including antigenic polypeptide molecules, directly or indirectly attached to the exterior surface of a modified, in particular stabilized, dodecin nanoparticle subunit may be referred to herein as “display” or “displayed” molecules. Antigen-displaying modified, in particular stabilized, nanoparticles preferably display multiple copies of antigenic polypeptide molecules in an ordered array, such an ordered array presented on a nanoparticle surface, to allow multiple binding events to occur simultaneously between the nanoparticle and host cell, which may favor the induction of a potent host immune response (see e.g. Lopez-Sagaseta etal., (2016)). Presentation of polypeptide antigens on nanoparticles has been exploited to improve the immunogenicity of subunit protein antigens (see, Jardine et al, (2013); Correira et al, (2014)).

[0113] In a non-limitative example, multiple antigens display may be achieved by fusing a modified, in particular stabilized, dodecin nanoparticle subunit with distinct antigen polypeptide at N-terminus and C-terminus, or mixing two or more distinct chimeric antigenic polypeptides.

[0114] Nucleic acids and vectors

[0115] In one aspect, the invention pertains to an isolated nucleic acid molecule, comprising a nucleotide sequence encoding the modified, in particular stabilized, nanoparticle subunit or the chimeric antigenic polypeptide according to the instant invention.

[0116] In a further aspect, is provided a vector, comprising the isolated nucleic acid molecule according to the invention herein.

[0117] A vector comprising such a polynucleotide is a further aspect of present embodiments. In specific embodiments, heterologous nucleotide sequences are introduced into the host cells using a plasmid, e.g. the heterologous nucleotide sequences are expressed in the host cells by a plasmid (e.g. an expression vector). It is to be understood that the vector may be a DNA-based vector or a RNA-based vector. When the vector is a RNA-based vector, it may be in particular a mRNA-based vector.

[0118] Host cell

[0119] Host cells that can be used to produce the chimeric antigenic polypeptide and / or the assembled antigenic nanoparticles of present embodiments, include archea, prokaryotic host cells, and eukaryotic host cells. In certain embodiments, the host cell is a non-human host cell. Exemplary prokaryotic host cells for use in production of the modified, in particular stabilized, dodecin nanoparticle subunit, the chimeric antigenic polypeptide or the assembled nanoparticle according to present embodiments include Escherichia species, Chlamydia species, Neisseria species, Shigella species, Klebsiella species, Xanthomonas species, Salmonella species, Yersinia species, Lactococcus species, Lactobacillus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Staphylococcus species, Bacillus species, and Clostridium species. In aspects, the host cell is E. coli.

[0120] Host cells may be modified to delete or modify genes in the host cell genetic background (genome) that compete or interfere with the synthesis of the polypeptide of interest. These genes can be deleted or modified in the host cell background (genome) in a manner that makes them inactive / dysfunctional (i.e. the host cell nucleotide sequences that are deleted / modified do not encode a functional protein or do not encode a protein whatsoever). In an embodiment, when nucleotide sequences are deleted from the genome of the host cells of present embodiments, they are replaced by a desirable sequence, e.g. a sequence that is useful for polypeptide production.

[0121] In some embodiments, the host cell of present embodiments may be E. coli.

[0122] The host cells of the present invention are engineered to comprise heterologous nucleotide sequences. The host cells of the present invention are engineered to comprise a nucleotide sequence that encodes a modified, in particular stabilized, dodecin nanoparticle subunit protein, optionally within a plasmid. Thus, host cells of present embodiments can produce a chimeric antigenic polypeptide comprising an antigen, for example a microbial antigenic polypeptide (e.g. a bacterial, yeast or mammalian polypeptide antigen) which is fused to a modified, in particular stabilized, dodecin nanoparticle subunit molecule.

[0123] Methods assembled

[0124] The nanoparticle subunit polypeptide or chimeric antigenic polypeptide of present embodiments may be produced by any suitable means, including by recombinant expression, and purified (if necessary) using any suitable method known in the art. The nanoparticle products may be analyzed using methods known in the art, e.g. by crystallography, Dynamic Light Scattering (DLS), Nano-Differential Scanning Fluorimetry (Nano-DSF), and Electron Microscopy, to confirm production of suitable nanoparticles.

[0125] Methods of recombinant expression suitable for the production of the nanoparticle subunit polypeptides or chimeric antigenic polypeptides are known in the art. The expressed polypeptide may include a purification tag and / or a protease site. Various expression systems are known in the art, including those using human (e.g. HeLa) host cells, mammalian (e.g. Chinese Hamster Ovary (CHO)) host cells, prokaryotic host cells (e.g. E. coli), or insect host cells. The host cell is typically transformed with the recombinant nucleic acid sequence encoding the desired polypeptide molecule, cultured under conditions suitable for expression of the molecule, and the molecule purified from the cell or culture medium. Cell culture conditions are particular to the cell type and expression vector, as is known in the art.

[0126] Host cells can be cultured in conventional nutrient media modified as appropriate and as will be apparent to those skilled in the art (e.g. for activating promoters). Culture conditions, such as temperature, pH and the like, may be determined using knowledge in the art, see e.g. Freshney (1994) and the references cited therein. In bacterial host cell systems, a number of expression vectors is available including, but not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene) or pET vectors (Novagen, Madison WI). In mammalian host cell systems, a number of expression systems, including both plasmids and viralbased systems, is available commercially.

[0127] Eukaryotic or microbial host cells expressing nanoparticle subunit polypeptides or chimeric antigenic polypeptides can be disrupted by any convenient method (including freezethaw cycling, sonication, mechanical disruption), and polypeptides and / or self-assembled nanoparticles can be recovered and purified from recombinant cell culture by any suitable method known in the art (including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography (e.g. using any of the tagging systems noted herein), hydroxyapatite chromatography, and lectin chromatography)). High performance liquid chromatography (HPLC) can be employed in the final purification steps.

[0128] In general, and using methods as are known in the art, expression of a recombinantly encoded nanoparticle subunit polypeptide or chimeric antigenic polypeptide involves preparation of an expression vector comprising a recombinant polynucleotide under the control of one or more promoters, such that the promoter stimulates transcription of the polynucleotide and promotes expression of the encoded polypeptide. “Recombinant Expression” as used herein refers to such a method.

[0129] “Recombinant expression vectors” comprise a recombinant nucleic acid sequence operatively linked to control sequences capable of effecting expression of the gene product. “Control sequences” are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules and need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. “Recombinant host cells” comprise such recombinant expression vectors.

[0130] A further embodiment is a method of producing a nanoparticle comprising microbial, including yeast, bacterial or viral, polypeptide antigens on the exterior surface of the nanoparticle. The method comprises the steps of (a) culturing a recombinant host cell under conditions conducive to (1) expressing or translocating the modified, in particular stabilized, nanoparticle subunit polypeptide(s) in the periplasm of the host cell, and under conditions conducive to selfassembly of the nanoparticle; (b) recovering or purifying assembled antigenic nanoparticles from the host cell or the culture medium in which the host cell is grown, as is suitable.

[0131] There is provided in one aspect, a method for preparing an assembled modified nanoparticle (NP) comprising the steps of providing a host cell; expressing a nanoparticle subunit in the periplasm of the host cell; producing an assembled antigenic nanoparticle from the nanoparticle subunits; and optionally purifying the assembled antigenic nanoparticle.

[0132] A further embodiment is a method of producing a nanoparticle comprising one or more microbial, including yeast, bacterial or viral, polypeptide antigen(s) on the exterior surface of the nanoparticle. In one aspect, the method comprises the steps of (a) culturing a recombinant host cell under conditions conducive to (1) expressing the modified, in particular stabilized, dodecin nanoparticle subunit polypeptide(s) or the chimeric antigen polypeptide in the host cell, under conditions conducive to self-assembly of the nanoparticle; (b) recovering or purifying assembled dodecin nanoparticles from the cytoplasm of the host cell. Alternatively, in some aspects, thee method comprises the steps of (a) culturing a recombinant host cell under conditions conducive to (1) expressing the modified, in particular stabilized, dodecin nanoparticle subunit polypeptide(s) or the chimeric antigen polypeptide in the host cell, under conditions conducive to self-assembly of the nanoparticle; (b) recovering or purifying assembled dodecin nanoparticles from the periplasm of the host cell or the culture medium in which the host cell is grown, as is suitable.

[0133] There is also provided in one aspect, a method for preparing an assembled modified, in particular stabilized, dodecin nanoparticle (NP) comprising the steps of providing a host cell; expressing a modified, in particular stabilized, dodecin nanoparticle subunit or chimeric antigen polypeptide in the host cell; and producing an assembled dodecin nanoparticle from the modified, in particular stabilized, dodecin nanoparticle subunit or chimeric antigen polypeptide in the cytoplasm of the host cell.

[0134] There is further provided in one aspect, a method for preparing an assembled modified, in particular stabilized, dodecin nanoparticle (NP) comprising the steps of providing a host cell; expressing a modified, in particular stabilized, dodecin nanoparticle subunit or chimeric antigen polypeptide in the host cell; and producing an assembled dodecin nanoparticle from the modified, in particular stabilized, dodecin nanoparticle subunit or chimeric antigen polypeptide in the periplasm of the host cell, in particular the periplasm of a Gram-negative bacterial host cell.

[0135] Purification

[0136] The term “purification” as used herein refers to the separation or isolation of a defined product (e.g. a recombinantly expressed nanoparticle or a recombinantly expressed chimeric antigen polypeptide from a composition containing other components (e.g. a host cell or host cell medium).

[0137] It is to be understood that the chimeric antigenic polypeptides or the assembled modified nanoparticles according to the instant disclosure may be purified from the cytoplasm of the host cell, the periplasm of a Gram-negative bacterial host cell, or the extracellular culture medium in which the host cell has been grown.

[0138] A composition that has been fractionated to remove undesired components, and which composition retains its biological activity, is considered purified. A purified assembled nanoparticle retains its biological activity. The term “purified” is intended to mean “removed from its natural environment and substantially free of impurities from that natural environment” (such as other chromosomal and extra-chromosomal DNA and RNA, organelles, and proteins (including other proteins, lipids, or polysaccharides which are also secreted into culture medium or result from lysis of host cells)). For clarity and as would be well understood by a person in the field, herein a molecule (such as an antigen or immunostimulant) within, or suitable for use within, a pharmaceutical, immunogenic, vaccine, or adjuvant composition is a purified molecule (such as a purified antigen or purified immunostimulant) whether or not the word “purified” is recited. It is understood in the field that for a molecule (such as an antigen, agent, immunostimulant, additive, vector, or other compound) or composition to be suitable (i.e., safe) for pharmaceutical or vaccine use (e.g. administration) with a human or non-human mammal (i.e., for the molecule to be pharmaceutically acceptable), it is at least purified (i.e., not crude).

[0139] Purified is a relative term and that absolute (100%) purity is not required for, e.g. pharmaceutical or vaccine use. A molecule may be at a purity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95% of a composition’s total proteinaceous content or mass (determined by, e.g. gel electrophoresis such as assessing the number of polypeptides within a fraction by SDS / PAGE analysis). Stated another way, “purification” or “purifying” refers to the process of removing undesired components from a composition or host cell or culture. Various methods for use in purifying polypeptides and NPs are known in the art and include, e.g. various types of chromatography such as High Performance Liquid Chromatography (HPLC), ionexchange chromatography, and size-exclusion chromatography (SEC), hydrophobic interaction, affinity, chelating; electrophoresis such as gel electrophoresis; centrifugation such as density gradient centrifugation; dialysis; filtration; precipitation; antibody capture; solvent extraction, affinity purification, and combinations thereof. Polypeptides NPs may be expressed with a tag operable for affinity purification, such as a 6xHistidine tag as is known in the art. A His-tagged polypeptide may be purified using Immobilized Metal Ion Affinity Chromatography (IMAC), for example, Ni-NTA column chromatography, by or using anti-6xHis antibody fused to a solid support.

[0140] A “substantially pure” preparation of polypeptides (or nanoparticles, or chimeric antigen polypeptide) or nucleic acid molecules is one in which the desired component represents at least 50% of the total polypeptide or nucleic acid content of the preparation, respectively. In certain embodiments, a substantially pure preparation will contain at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more of the total polypeptide or nucleic acid content of the preparation. Methods for quantifying the degree of purification of expressed polypeptides are known in the art and include, for example, determining the specific activity of an active fraction, or assessing the number of polypeptides within a fraction by SDS / PAGE analysis. The term “purification” or “purifying” herein refers to the process of removing components from a composition or host cell or culture, the presence of which is not desired. Molecules which have not been subjected to any purification steps (i.e., the molecule as it is found in nature or a “crude” molecule) are not suitable for pharmaceutical use (i.e., not suitable for administration to a subject).

[0141] In some instances, the terms “purified” and “isolated” are meant to be equivalent.

[0142] In some embodiments, the purified assembled nanoparticles may further be analysed, for determining their particle size distribution. In said embodiments, any suitable technique know from the state of the art may be appropriate. Non limiting examples of such techniques include laser diffraction, dynamic light scattering, transmission electron microscopy (TEM) and scanning electron microscopy (SEM).

[0143] A further embodiment concerns immunogenic compositions or pharmaceutical compositions, such as vaccines, which comprise nanoparticles displaying microbial, including bacterial and viral, polypeptide antigens, and a pharmaceutically acceptable diluent, or excipient. In certain instances, immunogenic compositions are administered to subjects to elicit an immune response that protects the subject against infection by a microbial pathogen, or decreases symptoms or conditions induced by a microbial pathogen. In the context of this disclosure, the term immunogenic composition will be understood to encompass compositions that are intended for administration to a subject or population of subjects for the purpose of eliciting a protective or palliative immune response against said microbes, including bacteria and viruses.

[0144] An “immunogenic composition” is a composition of matter suitable for administration to a human or non-human mammalian subject and which, upon administration of an immunologically effective amount, elicits a specific immune response, e.g. against a polypeptide antigen displayed on the modified, in particular stabilized, dodecin nanoparticle. An immunogenic composition of the present invention can include one or more additional components, such as an excipient, and / or adjuvant. While administration of a polypeptide antigen displayed on nanoparticles may enhance a subject’s immune response to the polypeptide antigen (as compared to administration of the polypeptide antigen in the absence of the nanoparticle), as used herein, the nanoparticles, used as scaffolds, are not defined as an adjuvant.

[0145] Numerous pharmaceutically acceptable diluents and / or pharmaceutically acceptable excipients or vehicles are known in the art and are described, e.g. in Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15thEdition (1975). The adjective “pharmaceutically acceptable” indicates that the diluent or excipient is suitable for administration to a subject (e.g. a human or non-human mammalian subject). In general, the nature of the diluent and / or excipient and / or vehicle will depend on the particular mode of administration being employed. For instance, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In certain formulations (for example, solid compositions, such as powder forms), a liquid diluent is not employed. In such formulations, non-toxic solid components can be used, including for example, pharmaceutical grades of trehalose, mannitol, lactose, starch or magnesium stearate. Suitable solid components are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles.

[0146] Accordingly, suitable excipients can be selected by those of skill in the art to produce a formulation suitable for delivery to a subject by a selected route of administration.

[0147] In one embodiment, the immunogenic or pharmaceutical compositions comprising nanoparticles do not further comprise an adjuvant. In another embodiment, the immunogenic or pharmaceutical compositions comprising nanoparticles do further comprise an adjuvant.

[0148] Preparation of immunogenic compositions, such as vaccines, including those for administration to human subjects, is generally described in Pharmaceutical Biotechnology, vol.61 Vaccine Design - the subunit and adjuvant approach, edited by Powell and Newman, Plenum Press, 1995 (see also, New Trends and Developments in Vaccines, edited by Voller el al., University Park Press, Baltimore, Maryland, U.S.A. 1978).

[0149] The chimeric antigenic polypeptide and / or assembled antigenic nanoparticles of present embodiments are particularly suited for inclusion in immunogenic compositions and vaccines.

[0150] The present embodiments provide an immunogenic composition comprising chimeric antigenic polypeptide and / or assembled antigenic nanoparticles, and optionally a pharmaceutically acceptable excipient and / or carrier.

[0151] Immunogenic compositions comprise an immunologically effective amount of the chimeric antigenic polypeptide and / or assembled antigenic nanoparticles, as well as any other components. By “immunologically effective amount,” it is meant that the administration of that amount to an individual, either as a single dose or as part of a series is effective for treatment or prevention. This amount varies depending on the health and physical condition of the individual to be treated, age, the degree of protection desired, the formulation of the vaccine and other relevant factors.

[0152] Pharmaceutically acceptable excipients and carriers are described, for example, in Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co. Easton, PA, 5th Edition (1975). Pharmaceutically acceptable excipients can include a buffer, such as a phosphate buffer (e.g. sodium phosphate). Pharmaceutically acceptable excipients can include a salt, for example, sodium chloride. Pharmaceutically acceptable excipients can include a solubilizing / stabilizing agent, for example, polysorbate (e.g. TWEEN 80). Pharmaceutically acceptable excipients can include a preservative, for example 2-phenoxyethanol or thiomersal. Pharmaceutically acceptable excipients can include a carrier such as water or saline.

[0153] Compositions comprising chimeric antigenic polypeptides and / or assembled antigenic nanoparticles may further comprise one or more pharmaceutically acceptable additives such as buffers, carriers, excipients, tonicity agents, wetting or emulsifying agents, detergents, antimicrobials, and diluents. Pharmaceutically acceptable additives are known in the field (e.g. in Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975)). Accordingly, suitable composition components can be selected by an artisan to produce a suitable formulation (e.g. suitable for pharmaceutical use).

[0154] The additive may comprise a pharmaceutically acceptable diluent (e.g. sterile water), saline, glycerol, etc. Additionally, a pharmaceutically acceptable additive may comprise auxiliary substances, such as wetting or emulsifying agents, or pH buffering substances.

[0155] The additive may comprise a pharmaceutically acceptable excipient. Such excipients include, without limitation: glycerol, polyethylene glycol (PEG), glass forming polyols (such as, sorbitol, trehalose) N-lauroylsarcosine (e.g. sodium salt), L-proline, non-detergent sulfobetaine, guanidine hydrochloride, urea, trimethylamine oxide, KC1, Ca2+, Mg2+ , Mn2+ , Zn2+ (and other divalent cation related salts), dithiothreitol (DTT), dithioerytrol, B-mercaptoethanol, detergents (including, e.g. Tween 80, Tween 20, Triton X-l 00, NP-40, Empigen BB, Octylglucoside, Lauroyl maltoside, Zwittergent 3-08, Zwittergent 3-10, Zwittergent 3-12, Zwittergent 3-14, Zwittergent 3- 16, CHAPS, sodium deoxy cholate, sodium dodecyl sulphate, and cetyltrimethylammonium bromide).

[0156] A pharmaceutically acceptable additive for use herein may be a detergent, e.g. a TWEEN (polysorbate), such as TWEEN 80. Detergents are generally present at low levels e.g. <0.01 %.

[0157] The pH of a composition comprising a protein nanoparticle may be between 6 and 8, for example between 6.5 and 7.5 (e.g. about 7). Stable pH may be maintained by the use of a buffer (e.g. an acetate buffer, citrate buffer, histidine buffer, maleate buffer, phosphate buffer, succinate buffer, tartrate buffer, or Tris buffer). Thus, a composition will generally include a buffer. A composition may be sterile and / or pyrogen-free. Compositions may be isotonic with respect to humans.

[0158] Also provided is a method of making the immunogenic composition comprising the step of mixing the chimeric antigenic polypeptide and / or assembled antigenic nanoparticles with a pharmaceutically acceptable excipient and / or carrier.

[0159] Also provided is an immunogenic composition (e.g. a vaccine composition) optionally comprising an adjuvant.

[0160] The term “adjuvant” refers to a compound that when administered in conjunction with or as part of an immunogenic composition of the vaccine of present embodiments augments, enhances and / or boosts the immune response to chimeric antigenic polypeptide and / or assembled antigenic nanoparticles, but when the compound is administered alone does not generate an immune response to the chimeric antigenic polypeptide and / or assembled antigenic nanoparticles. Adjuvants can enhance an immune response by several mechanisms including, e.g. lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages. Specific examples of adjuvants include, but are not limited to, aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3 De-O-acylated monophosphoryl lipid A (MPL) (see United Kingdom Patent GB2220211), MF59 (Novartis), AS01 (GlaxoSmithKline), AS03 (GlaxoSmithKline) and saponins, such as QS21 (see Kensil et al. in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); U.S. Pat. No. 5,057,540). In some embodiments, the adjuvant is Freund’s adjuvant (complete or incomplete). Other adjuvants are oil in water emulsions (such as squalene or peanut oil), optionally in combination with immune stimulants, such as monophosphoryl lipid A (see Stoute et al. N. Engl. J. Med. 336, 86-91 (1997)).

[0161] Immunogenic compositions of the present invention may additionally include one or more adjuvants. As used herein, an “adjuvant” is an agent that enhances the production of an immune response in a non-specific manner. Common adjuvants include suspensions of minerals (e.g. alum, aluminum hydroxide, aluminum phosphate); saponins such as QS21; emulsions, including water- in-oil, and oil-in-water (and variants thereof, including double emulsions and reversible emulsions), liposaccharides, lipopolysaccharides, immunostimulatory nucleic acid molecules (such as CpG oligonucleotides), liposomes, Toll Receptor agonists, Toll-like Receptor agonists (particularly, TLR2, TLR4, TLR7 / 8 and TLR9 agonists), and various combinations of such components. For the purposes of the present embodiments, the assembled nanoparticle is not considered an adjuvant.

[0162] In some embodiments, alum is not used as adjuvant to enhance the immune response of the vaccine, but rather as adsorbent to further improve the safety profile.

[0163] Also provided is a method of making the immunogenic composition comprising the step of mixing the chimeric antigenic polypeptide and / or assembled antigenic nanoparticles with a pharmaceutically acceptable excipient and / or carrier and an adjuvant. Vaccine preparation is generally described in Vaccine Design (“The subunit and adjuvant approach” (eds Powell M.F. & Newman M.J.) (1995) Plenum Press New York).

[0164] The immunogenic compositions of present embodiments can be included in a container, pack, or dispenser together with instructions for administration.

[0165] The immunogenic compositions or vaccines of thereof can be stored before use, e.g. the compositions can be stored frozen (e.g. at about -20°C or at about -70°C); stored in refrigerated conditions (e.g. at about 4°C); or stored at room temperature. The immunogenic compositions or vaccines of present embodiments may be stored in solution or lyophilized. In an embodiment, the solution is lyophilized in the presence of a sugar such as sucrose, trehalose or lactose. In another embodiment, the vaccines of present embodiments are lyophilized and extemporaneously reconstituted prior to use. Administration and

[0166] Immunogenic compositions or vaccines of present embodiments may be used to protect or treat a subject (e.g. mammal), by means of administering said immunogenic composition or vaccine via systemic or mucosal route. These administrations may include injection via the intramuscular (IM), intraperitoneal, intradermal (ID) or subcutaneous (SC) routes; or via mucosal administration to the oral / alimentary, respiratory, genitourinary tracts.

[0167] In one aspect, the immunogenic composition or vaccine of present embodiments is administered by the intramuscular delivery route. Intramuscular administration may be to the thigh or the upper arm. Injection is typically via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used. A typical intramuscular dose is from about 0.1 to about 5 ml, in particular from about 0.1 to about 1 ml, in particular about 0.5 ml.

[0168] In another aspect, the immunogenic composition or vaccine of present embodiments is administered by the intradermal administration. Human skin comprises an outer "horny" cuticle, called the stratum corneum, which overlays the epidermis. Underneath this epidermis is a layer called the dermis, which in turn overlays the subcutaneous tissue. The conventional technique of intradermal injection, the "mantoux procedure," comprises steps of cleaning the skin, and then stretching with one hand, and with the bevel of a narrow-gauge needle (26 to 31 gauge) facing upwards the needle is inserted at an angle of between 10 to 15°. Once the bevel of the needle is inserted, the barrel of the needle is lowered and further advanced whilst providing a slight pressure to elevate it under the skin. The liquid is then injected very slowly thereby forming a bleb or bump on the skin surface, followed by slow withdrawal of the needle.

[0169] In another aspect, the immunogenic composition or vaccine of present embodiments is administered by the intranasal administration. Typically, the immunogenic composition or vaccine is administered locally to the nasopharyngeal area, e.g. without being inhaled into the lungs. It is desirable to use an intranasal delivery device which delivers the immunogenic composition or vaccine formulation to the nasopharyngeal area, without or substantially without it entering the lungs. Suitable devices for intranasal administration of the vaccines according to present embodiments are spray devices. Suitable commercially available nasal spray devices include ACCUSPRAY™ (Becton Dickinson). The amount of the chimeric antigenic polypeptide and / or assembled antigenic nanoparticles in each immunogenic composition or vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and how it is presented. In some embodiment, the content of the chimeric antigenic polypeptide and / or assembled antigenic nanoparticles will typically be in the range from about 1 to aboutl,000 pg, suitably from about 5 to about 500 pg, in particular from about 10 to aboutlOO pg.

[0170] In some embodiment, the content of the chimeric antigenic polypeptide and / or assembled antigenic nanoparticles will typically be in the range of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 to 100 pg.

[0171] In certain embodiment, the content of the chimeric antigenic polypeptide and / or assembled antigenic nanoparticles will typically be in the range of 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,

[0172] 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,

[0173] 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740,

[0174] 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930,

[0175] 940, 950, 960, 970, 980, 990 to 1,000 pg.

[0176] ProDhylactic and Therapeutic Uses

[0177] The present invention also provides an immunogenic composition of present embodiments, or the vaccine of present embodiments, for use in medicine. The present invention also provides an immunogenic composition of present embodiments, or the vaccine of present embodiments, for use in the treatment and / or the prevention of a microbial infection.

[0178] The present invention further provides the use of an immunogenic composition of present embodiments, or the vaccine of present embodiments, for the preparation or the manufacture of a medicament. The present invention further provides the use of an immunogenic composition of present embodiments, or the vaccine of present embodiments, for the treatment and / or the prevention of a microbial infection.

[0179] A method is provided of inducing an immune response in a subject (e.g. human), the method comprising administering a therapeutically or prophylactically effective amount of the chimeric antigenic polypeptide and / or assembled antigenic nanoparticles, an immunogenic composition thereof, or a vaccine thereof, to a subject (e.g. human) in need thereof. Also provided is a chimeric antigenic polypeptide and / or assembled antigenic nanoparticle, the immunogenic composition thereof, or the vaccine thereof for use in the manufacture of a medicament for inducing an immune response in a subject (e.g. human).

[0180] A further aspect is a method of inducing an immune response for the purpose of treating and / or preventing a microbial, including yeast, bacterial and viral, infection of a subject, comprising administering to the subject an immunologically effective amount of the microbial, including bacterial and viral, antigenic polypeptide molecule displayed on the surface of nanoparticles to which an immune response is desired, where said polypeptide antigens can induce a protective or therapeutic immune response. Such microbial, including bacterial and viral, antigenic polypeptide molecule displayed on the surface of nanoparticles may be within an immunogenic or pharmaceutical composition as described herein. In one embodiment of present embodiments, a single dose is administered to the subject. The dose may be adjuvant-free, or it may further comprise an adjuvant.

[0181] Also provided herein are methods of inducing an immune response in a subject against a microbe, including a yeast, bacterium and a virus, comprising administering to the subject chimeric antigenic polypeptide and / or assembled antigenic nanoparticles of present embodiments, an immunogenic composition thereof or a vaccine thereof. The chimeric antigenic polypeptide and / or assembled antigenic nanoparticles of present embodiments is an immunogenic composition or a vaccine that can be used to induce an immune response against a bacterium, e.g. Chlamydia species, Shigella species, Pseudomonas aeruginosa, Klebsiella pneumoniae, N. meningitidis, H. influenzae type b (Hib), Group B Streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus.

[0182] In some instances, the immunogenic composition or the vaccine according to the invention may induce an immune response against a bacterium selected from the group consisting of Chlamydia species, Escherichia species, Shigella species, Klebsiella species, Salmonella species, Yersinia species, Helicobacter species, Proteus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Enterococcus species, Staphylococcus species, Bacillus species, Clostridium species, Listeria species, Campylobacter species, Neisseria species and Candida species.

[0183] In an embodiment, the chimeric antigenic polypeptide and / or assembled antigenic nanoparticles of present embodiments is an immunogenic composition or a vaccine that can be used to induce an immune response against a bacterium, e.g. Streptococcus species, Shigella species, Pseudomonas species, Klebsiella species, or Staphylococcus species, (e.g. Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, or Staphylococcus aureus). In one embodiment, said subject has a bacterial infection at the time of administration. In another embodiment, said subject does not have a bacterial infection at the time of administration.

[0184] Also provided are methods of treating and / or preventing a microbial, including yeast, bacterial and viral, infection in a subject comprising administering to the subject a chimeric antigenic polypeptide and / or assembled antigenic nanoparticle. The chimeric antigenic polypeptide and / or assembled antigenic nanoparticles may be in the form of an immunogenic composition or vaccine. Thus, a method of treating and / or preventing a microbial, including yeast, bacterial and viral, infection in a subject (e.g. human) is provided, the method comprising administering a therapeutically or prophylactically effective amount of a chimeric antigenic polypeptide and / or assembled antigenic nanoparticles, an immunogenic composition thereof or a vaccine thereof, to a subject (e.g. human) in need thereof. A chimeric antigenic polypeptide and / or assembled antigenic nanoparticles, an immunogenic composition thereof or a vaccine thereof, is provided for use in treating and / or preventing a microbial, including yeast, bacterial or viral, infection in a subject (e.g. human). Also provided is a chimeric antigenic polypeptide and / or assembled antigenic nanoparticles, the immunogenic composition thereof or the vaccine thereof for use in the manufacture of a medicament for treating and / or preventing microbial, including yeast, bacterial or viral, infections in a subject (e.g. human).

[0185] In a specific embodiment, the immunogenic composition or vaccine provided herein is used in the prevention of infection of a subject by a bacterium. Bacterial infections that can be treated and / or prevented using the chimeric antigenic polypeptide and / or assembled antigenic nanoparticles provided herein including those caused by Chlamydia species, N. meningitidis, H. influenzae type b (Hib), Streptococcus species, Shigella species, Pseudomonas species, Klebsiella species, or Staphylococcus species, (e.g. Chlamydia trachomatis, Shigella dysenteriae, Shigella jlexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae or Staphylococcus aureus).

[0186] A further aspect is a method of inducing an immune response in a mammalian subject, such as a human subject, wherein said immune response is specific for a microbial, including yeast, bacterial or viral, antigenic polypeptide molecule displayed on the surface of nanoparticles provided herein. The method comprises administering to a subject an immunologically effective amount of the microbial, including yeast, bacterial or viral, antigenic polypeptide molecule displayed on the surface of nanoparticles to which an immune response is desired. The subject may have a microbial, including yeast, bacterial or viral, infection at the time of administration, or the administration may be given prophylactically to a subject who does not have a microbial, including yeast, bacterial or viral, infection at the time of administration. In one embodiment, the nanoparticles administered display polypeptide antigens from at least two disease-causing serotypes. This may be achieved by administering a mixture of nanoparticles where each nanoparticle displays a single serotype polypeptide antigen, or by administering nanoparticles that display multiple serotype polypeptide antigens. The antigens may be microbial polypeptides or immunogenic fragments thereof, or a mixture thereof.

[0187] Also provided herein are methods of inducing the production of opsonophagocytic antibodies in a subject against a bacterium, comprising administering to the subject a chimeric antigenic polypeptide and / or assembled antigenic nanoparticles, an immunogenic composition thereof, or a vaccine thereof. The chimeric antigenic polypeptide and / or assembled antigenic nanoparticle is an immunogenic composition or a vaccine that can be used to induce the production of opsonophagocytic antibodies in a subject against a microbe, including a bacterium, e.g. Chlamydia species, Shigella species, Pseudomonas aeruginosa, Klebsiella pneumoniae, N. meningitidis, H. influenzae type b (Hib), Group B Streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus. In an embodiment, the chimeric antigenic polypeptide and / or assembled antigenic nanoparticle which is an immunogenic composition or a vaccine can be used to induce the production of opsonophagocytic antibodies in a subject against a microbe, including a bacterium, e.g. Chlamydia species, Streptococcus species, Shigella species, Pseudomonas species, Klebsiella species, or Staphylococcus species, (e.g. Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae or Staphylococcus aureus). In some instances, the immunogenic composition or the vaccine according to the invention may induce the production of opsonophagocytic antibodies in a subject against a bacterium selected from the group consisting of Chlamydia species, Escherichia species, Shigella species, Klebsiella species, Salmonella species, Yersinia species, Helicobacter species, Proteus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Enterococcus species, Staphylococcus species, Bacillus species, Clostridium species, Listeria species, Campylobacter species, Neisseria species and Candida species.

[0188] Another embodiment is a method of immunizing a human subject in order to decrease the risk of disease, wherein the subject receives both a priming dose and a boosting dose of a composition, and where the priming and the boosting dose each elicit in the subject IgG antibodies specific for the same disease-causing serotype(s). In one embodiment, the boosting dose is administered more than 14 days, 21 days, 30 days, 2 months, 3 months, 6 months, 1 year or more after the priming dose. The priming and / or boosting dose may be adjuvant-free, or either or both may further comprise an adjuvant.

[0189] In one embodiment, the chimeric antigenic polypeptide and / or assembled antigenic nanoparticle and compositions thereof are utilized in methods of immunizing a subject to achieve a protective (prophylactic) immune response in both the subject and (via transplacental transfer of maternal antibodies) to an infant born to the subject.

[0190] The immunogenic compositions provided herein are conventionally administered parenterally, e.g. by injection, either subcutaneously, intraperitoneally, transdermally, or intramuscularly. Dosage treatment may be a single dose schedule or a multiple dose schedule.

[0191] The various features which are referred to in individual sections above apply, as appropriate, to other sections. Consequently, features specified in one section may be combined with features specified in other sections, as appropriate. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments (or aspects of the disclosure) described herein.

[0192] Embodiments Embodiments are further described in the subsequent numbered paragraphs; embodiments are meant to be combined when applicable:

[0193] 1. A modified, in particular stabilized, dodecin nanoparticle (NP) subunit having an amino acid sequence at least 80% identical to amino acid residues 2-70 of SEQ ID NO: 1 and comprising at least one substitution at position A53, with respect to SEQ ID NO: 1.

[0194] 2. The modified, in particular stabilized, subunit of embodiment 1 , wherein the amino acid sequence is at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 2-70 of SEQ ID NO: 1 and comprising at least one substitution at position A53, with respect to SEQ ID NO: 1.

[0195] 3. The modified, in particular stabilized, subunit of any preceding embodiment, wherein the amino acid sequence is identical to amino acid residues 2-70 of SEQ ID NO: 1 and comprising at least one substitution at position A53, with respect to SEQ ID NO: 1.

[0196] 4. The modified, in particular stabilized, subunit of any preceding embodiment, wherein it further comprises one or more substitution(s) at position(s) G52 and / or V50, with respect to SEQ ID NO: 1.

[0197] 5. The modified, in particular stabilized, subunit of any preceding embodiment, wherein it further comprises one substitution at position G52, with respect to SEQ ID NO: 1.

[0198] 6. The modified, in particular stabilized, subunit of any preceding embodiment, wherein it further comprises one substitution at position V50, with respect to SEQ ID NO: 1.

[0199] 7. The modified, in particular stabilized, subunit of any preceding embodiment, wherein the substitutions selected from the group consisting of A53T, G25N, and V50T, with respect to SEQ ID NO: 1. 8. The modified, in particular stabilized, subunit of any preceding embodiment, comprising the substitution is A53T, with respect to SEQ ID NO: 1.

[0200] 9. The modified, in particular stabilized, subunit of any preceding embodiment, comprising the substitution is G25N, with respect to SEQ ID NO: 1.

[0201] 10. The modified, in particular stabilized, subunit of any preceding embodiment, comprising the substitution is V50T, with respect to SEQ ID NO: 1.

[0202] 11. The modified, in particular stabilized, subunit of any preceding embodiment, comprising the substitutions are A53T and G25N, with respect to SEQ ID NO: 1.

[0203] 12. The modified, in particular stabilized, subunit of any preceding embodiment, comprising an amino acid sequence at least 80% identical to amino acid residues 2-70 of SEQ ID NO: 6.

[0204] 13. The modified, in particular stabilized, subunit of any preceding embodiment, comprising an amino acid sequence at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 2-70 of SEQ ID NO: 6.

[0205] 14. The modified, in particular stabilized, subunit of any preceding embodiment, comprising an amino acid sequence identical to amino acid residues 2-70 of SEQ ID NO: 6.

[0206] 15. The modified, in particular stabilized, subunit of any preceding embodiment, comprising the substitutions are A53T and V50T, with respect to SEQ ID NO: 1.

[0207] 16. The modified, in particular stabilized, subunit of any preceding embodiment, comprising an amino acid sequence at least 80% identical to amino acid residues 2-70 of SEQ ID NO: 7.

[0208] 17. The modified, in particular stabilized, subunit of any preceding embodiment, comprising an amino acid sequence at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 2-70 of SEQ ID NO: 7. 18. The modified, in particular stabilized, subunit of any preceding embodiment, comprising an amino acid sequence identical to amino acid residues 2-70 of SEQ ID NO: 7.

[0209] 19. The modified, in particular stabilized, subunit of any preceding embodiment, comprising each substitution A53T, G25N, and V50T, with respect to SEQ ID NO: 1.

[0210] 20. The modified, in particular stabilized, subunit of any preceding embodiment, comprising an amino acid sequence at least 80% identical to amino acid residues 2-70 of SEQ ID NO: 8.

[0211] 21. The modified, in particular stabilized, subunit of any preceding embodiment, comprising an amino acid sequence at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 2-70 of SEQ ID NO: 8.

[0212] 22. The modified, in particular stabilized, subunit of any preceding embodiment, comprising an amino acid sequence identical to amino acid residues 2-70 of SEQ ID NO: 8.

[0213] 23. The modified, in particular stabilized, subunit of any preceding embodiment, comprising an amino acid sequence at least 80% identical to amino acid sequence of SEQ ID NO: 8.

[0214] 24. The modified, in particular stabilized, subunit of any preceding embodiment, comprising an amino acid sequence at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid sequence of SEQ ID NO: 8.

[0215] 25. The modified, in particular stabilized, subunit of any preceding embodiment, comprising an amino acid sequence of SEQ ID NO: 8.

[0216] 26. A chimeric antigenic polypeptide comprising a) the modified, in particular stabilized, subunit of any preceding embodiment, and b) a polypeptide antigen. 27. The chimeric antigenic polypeptide of the preceding embodiment, which is intended to generate a specific immune response.

[0217] 28. The chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from the group consisting of Chlamydia species, Escherichia species, Shigella species, Klebsiella species, Salmonella species, Yersinia species, Helicobacter species, Proteus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Enterococcus species, Staphylococcus species, Bacillus species, Clostridium species, Listeria species, Campylobacter species, Neisseria species and Candida polypeptide antigens.

[0218] 29. The chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Chlamydia species polypeptide antigens.

[0219] 30. The chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Chlamydia trachomatis polypeptide antigens.

[0220] 31. The chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is Chlamydia trachomatis MOMP (major outer membrane protein).

[0221] 32. The chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Chlamydia trachomatis serovar D MOMP ( ^Chlamydia trachomatis serovar E MOMP, and one or more immunogenic fragment(s) thereof.

[0222] 33. The chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Chlamydia trachomatis serovar D MOMP (major outer membrane protein) or one or more immunogenic fragment(s) thereof and a polypeptide antigen selected from Chlamydia trachomatis serovar E MOMP or one or more immunogenic fragment(s) thereof. 34. The chimeric antigenic polypeptide of the preceding embodiment, wherein the one or more immunogenic fragment(s)of MOMP comprise(s) loop 1, loop 2, loop 3, loop 4, or any combination thereof.

[0223] 35. The chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen comprises loop 1 and loop 4 of MOMP.

[0224] 36. The chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11.

[0225] 37. The chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12.

[0226] 38. The chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Klebsiella species polypeptide antigens.

[0227] 39. The chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Klebsiella pneumoniae polypeptide antigens.

[0228] 40. The chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is Klebsiella pneumoniae MrkA.

[0229] 41. The chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 23. 42. The chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Neisseria Species polypeptide antigens.

[0230] 43. The chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Neisseria meningitidis polypeptide antigens.

[0231] 44. The chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is Neisseria meningitidis fHbp.

[0232] 45. The chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 24 or SEQ ID NO: 25.

[0233] 46. The chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Pseudomonas species polypeptide antigens.

[0234] 47. The chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Pseudomonas aeruginosa polypeptide antigens.

[0235] 48. Use of a chimeric antigenic polypeptide comprising a) the modified subunit of any preceding embodiment of the same category, and b) a polypeptide antigen, to generate a specific immune response.

[0236] 49. The use of the chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from the group consisting of Chlamydia species, Escherichia species, Shigella species, Klebsiella species, Salmonella species, Yersinia species, Helicobacter species, Proteus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Enterococcus species, Staphylococcus species, Bacillus species, Clostridium species, Listeria species, Campylobacter species, Neisseria species and Candida polypeptide antigens. 50. The use of the chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Chlamydia species polypeptide antigens.

[0237] 51. The use of the chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Chlamydia trachomatis polypeptide antigens.

[0238] 52. The use of the chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is Chlamydia trachomatis major outer membrane protein (MOMP).

[0239] 53. The use of the chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Chlamydia trachomatis serovar D MOMP, Chlamydia trachomatis serovar E MOMP, and one or more immunogenic fragment(s) thereof.

[0240] 54. The use of the chimeric antigenic polypeptide of the preceding embodiment, wherein the polypeptide antigen is selected from Chlamydia trachomatis serovar D MOMP or one or more immunogenic fragment(s) thereof and a polypeptide antigen selected from Chlamydia trachomatis serovar E MOMP (major outer membrane protein) or one or more immunogenic fragment(s) thereof.

[0241] 55. The use of the chimeric antigenic polypeptide of the preceding embodiment, wherein the one or more immunogenic fragment(s)of MOMP comprise(s) loop 1, loop 2, loop 3, loop 4, or any combination thereof.

[0242] 56. The use of the chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen comprises loop 1 and / or loop 4 of MOMP. 57. The use of the chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11.

[0243] 58. The use of the chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12.

[0244] 59. The use of the chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Klebsiella species polypeptide antigens.

[0245] 60. The use of the chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Klebsiella pneumoniae polypeptide antigens.

[0246] 61. The use of the chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is Klebsiella pneumoniae MrkA.

[0247] 62. The use of the chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 23.

[0248] 63. The use of the chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Neisseria Species polypeptide antigens.

[0249] 64. The use of the chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Neisseria meningitidis polypeptide antigens. 65. The use of the chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is Neisseria meningitidis fflbp.

[0250] 66. The use of the chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 24 or SEQ ID NO: 25.

[0251] 67. The use of the chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Pseudomonas species polypeptide antigens.

[0252] 68. The use of the chimeric antigenic polypeptide of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Pseudomonas aeruginosa polypeptide antigens.

[0253] 69. A chimeric antigenic polypeptide comprising a) the modified subunit of any preceding embodiment of the same category, and b) a polypeptide antigen, for use to prevent a bacterial infection.

[0254] 70. The chimeric antigenic polypeptide for use as of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from the group consisting of Chlamydia species, Escherichia species, Shigella species, Klebsiella species, Salmonella species, Yersinia species, Helicobacter species, Proteus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Enterococcus species, Staphylococcus species, Bacillus species, Clostridium species, Listeria species, Campylobacter species, Neisseria species and Candida polypeptide antigens.

[0255] 71. The chimeric antigenic polypeptide for use as of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Chlamydia species polypeptide antigens. 72. The chimeric antigenic polypeptide for use as of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Chlamydia trachomatis polypeptide antigens.

[0256] 73. The chimeric antigenic polypeptide for use as of any preceding embodiment of the same category, wherein the polypeptide antigen is Chlamydia trachomatis MOMP (major outer membrane protein).

[0257] 74. The chimeric antigenic polypeptide for use as of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Chlamydia trachomatis serovar D MOMP, Chlamydia trachomatis serovar E MOMP, and one or more immunogenic fragment(s) thereof.

[0258] 75. The chimeric antigenic polypeptide for use as of the preceding embodiment, wherein the polypeptide antigen is selected from Chlamydia trachomatis serovar D MOMP or one or more immunogenic fragment(s) thereof and a polypeptide antigen selected from Chlamydia trachomatis serovar E MOMP or one or more immunogenic fragment(s) thereof.

[0259] 76. The chimeric antigenic polypeptide for use as of the preceding embodiment, wherein the one or more immunogenic fragment(s)of MOMP comprise(s) loop 1, loop 2, loop 3, loop 4, or any combination thereof.

[0260] 77. The chimeric antigenic polypeptide for use as of any preceding embodiment of the same category, wherein the polypeptide antigen comprises loop 1 and / or loop 4 of MOMP.

[0261] 78. The chimeric antigenic polypeptide for use as of any preceding embodiment of the same category, wherein the polypeptide antigen comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11.

[0262] 79. The chimeric antigenic polypeptide for use as of any preceding embodiment of the same category, wherein the polypeptide antigen comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12.

[0263] 80. The chimeric antigenic polypeptide for use as of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Klebsiella species polypeptide antigens.

[0264] 81. The chimeric antigenic polypeptide for use as of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Klebsiella pneumoniae polypeptide antigens.

[0265] 82. The chimeric antigenic polypeptide for use as of any preceding embodiment of the same category, wherein the polypeptide antigen is Klebsiella pneumoniae MrkA.

[0266] 83. The chimeric antigenic polypeptide for use as of any preceding embodiment of the same category, wherein the polypeptide antigen comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 23.

[0267] 84. The chimeric antigenic polypeptide for use as of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Neisseria Species polypeptide antigens.

[0268] 85. The chimeric antigenic polypeptide for use as of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Neisseria meningitidis polypeptide antigens.

[0269] 86. The chimeric antigenic polypeptide for use as of any preceding embodiment of the same category, wherein the polypeptide antigen is Neisseria meningitidis fHbp.

[0270] 87. The chimeric antigenic polypeptide for use as of any preceding embodiment of the same category, wherein the polypeptide antigen comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 24 or SEQ ID NO: 25. 88. The chimeric antigenic polypeptide for use as of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Pseudomonas species polypeptide antigens.

[0271] 89. The chimeric antigenic polypeptide for use as of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Pseudomonas aeruginosa polypeptide antigens.

[0272] 90. A method for preparing an assembled antigenic nanoparticle (NP) comprising the steps of: i) providing a host cell; ii) expressing the chimeric antigenic polypeptide of any preceding embodiment in said host cell, so as to produce an assembled antigenic nanoparticle; iii) recovering / obtaining the assembled antigenic nanoparticle.

[0273] 91. The method of the preceding embodiment, wherein the host cell consists in a bacterial cell.

[0274] 92. The method of any preceding embodiment of the same category, wherein the host cell consists in a Gram-negative bacterial cell.

[0275] 93. The method of any preceding embodiment of the same category, wherein the host cell consists in an E. coli bacterial cell.

[0276] 94. The method of any preceding embodiment of the same category, wherein the chimeric antigenic polypeptide comprises a polypeptide antigen selected from the group consisting of Chlamydia species, Escherichia species, Shigella species, Klebsiella species, Salmonella species, Yersinia species, Helicobacter species, Proteus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Enterococcus species, Staphylococcus species, Bacillus species, Clostridium species, Listeria species, Campylobacter species, Neisseria species and Candida polypeptide antigens. 95. The method of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Chlamydia species polypeptide antigens.

[0277] 96. The method of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Chlamydia trachomatis polypeptide antigens.

[0278] 97. The method of any preceding embodiment of the same category, wherein the polypeptide antigen is Chlamydia trachomatis MOMP.

[0279] 98. The method of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Chlamydia trachomatis serovar D MOMP, Chlamydia trachomatis serovar E MOMP, and one or more immunogenic fragment(s) thereof.

[0280] 99. The method of the preceding embodiment, wherein the one or more immunogenic fragment(s) of MOMP comprise(s) loop 1, loop 2, loop 3, loop 4, or any combination thereof.

[0281] 100. The method of any preceding embodiment of the same category, wherein the polypeptide antigen comprises loop 1 and / or loop 4 of MOMP.

[0282] 101. The method of any preceding embodiment of the same category, wherein the polypeptide antigen comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11.

[0283] 102. The method of any preceding embodiment of the same category, wherein the polypeptide antigen comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12.

[0284] 103. The method of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Klebsiella species polypeptide antigens.

[0285] 104. The method of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Klebsiella pneumoniae polypeptide antigens.

[0286] 105. The method of any preceding embodiment of the same category, wherein the polypeptide antigen is Klebsiella pneumoniae MrkA.

[0287] 106. The method of any preceding embodiment of the same category, wherein the polypeptide antigen comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 23.

[0288] 107. The method of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Neisseria Species polypeptide antigens.

[0289] 108. The method of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Neisseria meningitidis polypeptide antigens.

[0290] 109. The method of any preceding embodiment of the same category, wherein the polypeptide antigen is Neisseria meningitidis fflbp.

[0291] 110. The method of any preceding embodiment of the same category, wherein the polypeptide antigen comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 24 or SEQ ID NO: 25.

[0292] 111. The method of any preceding embodiment of the same category, wherein the polypeptide antigen is selected from Pseudomonas species polypeptide antigens.

[0293] 112. Assembled nanoparticle obtained by the method of any preceding embodiment of the same category.

[0294] 113. The assembled nanoparticles of the preceding embodiment having a size ranging from about 5 nm to 100 nm.

[0295] 114. The assembled nanoparticles of the preceding embodiment having a size ranging from about 5 nm to about 75 nm.

[0296] 115. The assembled nanoparticles of the preceding embodiment having a size ranging from about 6 nm to about 75 nm.

[0297] 116. The assembled nanoparticles of the preceding embodiment having a size ranging from about 6 nm to about 50 nm.

[0298] 117. The assembled nanoparticles of the preceding embodiment having a size ranging from about 6 nm to about 25 nm.

[0299] 118. The assembled nanoparticles of the preceding embodiment having a size ranging from about 6 nm to about 24 nm.

[0300] 119. An isolated nucleic acid molecule, comprising a nucleotide sequence encoding the modified nanoparticle subunit or the chimeric antigenic polypeptide of any preceding embodiment of the same category.

[0301] 120. A vector, comprising the isolated nucleic acid molecule of any preceding embodiment of the same category.

[0302] 121. The vector of the preceding embodiment selected in from the group consisting of DNA vector and RNA vector. 122. The vector of any preceding embodiment of the same category consisting of a DNA vector.

[0303] 123. The vector of any preceding embodiment of the same category consisting of a RNA vector.

[0304] 124. The vector of any preceding embodiment of the same category consisting of a mRNA vector.

[0305] 125. A host cell, comprising the isolated nucleic acid molecule of any preceding embodiment or the vector of any preceding embodiment.

[0306] 126. The host cell of the preceding embodiment consisting in a bacterial cell.

[0307] 127. The host cell of the preceding embodiment consisting in a Gram-negative bacterial cell.

[0308] 128. The host cell of the preceding embodiment consisting in an E. coli bacterial cell.

[0309] 129. An assembled antigenic nanoparticle comprising the chimeric antigenic polypeptide of any preceding embodiment of the same category.

[0310] 130. The assembled nanoparticles of the preceding embodiment of the same category having a size ranging from about 5 nm to 100 nm.

[0311] 131. The assembled nanoparticles of the preceding embodiment having a size ranging from about 5 nm to about 75 nm.

[0312] 132. The assembled nanoparticles of the preceding embodiment having a size ranging from about 6 nm to about 75 nm. 133. The assembled nanoparticles of the preceding embodiment having a size ranging from about 6 nm to about 50 nm.

[0313] 134. The assembled nanoparticles of the preceding embodiment having a size ranging from about 6 nm to about 25 nm.

[0314] 135. The assembled nanoparticles of the preceding embodiment having a size ranging from about 6 nm to about 24 nm.

[0315] 136. An immunogenic composition comprising i) the chimeric antigenic polypeptide and / or the assembled antigenic nanoparticle of any preceding embodiment of the same category, and ii) a pharmaceutically acceptable vehicle.

[0316] 137. A vaccine comprising the immunogenic composition of any preceding embodiment of the same category.

[0317] 138. The immunogenic composition or the vaccine of any preceding embodiment of the same category, further comprising an adjuvant selected from the group consisting of alum, aluminium hydroxide, aluminium phosphate, a saponin, a water-in-oil emulsion, an oil-in-water emulsion, a liposaccharide, a lipopolysaccharide, an immunostimulatory nucleic acid molecule, a liposome, and a Toll Receptor or Toll-Like Receptor agonist.

[0318] 139. A method of inducing an immune response in a human subject, comprising administering to the subject an immunologically effective amount of the assembled nanoparticle, the immunogenic composition, or the vaccine of any preceding embodiment of the same category.

[0319] 140. Assembled nanoparticle, the immunogenic composition, or the vaccine of any preceding embodiment of the same category for use as a medicament. 141. Assembled nanoparticle, the immunogenic composition, or the vaccine of any preceding embodiment for use in preventing a microbial infection.

[0320] 142. Use of an assembled nanoparticle, the immunogenic composition, or the vaccine of any preceding embodiment of the same category as a medicament.

[0321] 143. Use of an assembled nanoparticle, the immunogenic composition, or the vaccine of any preceding embodiment of the same category for the preparation or manufacture of a medicament.

[0322] 144. Use of an assembled nanoparticle, the immunogenic composition, or the vaccine of any preceding embodiment of the same category for preventing a microbial infection.

[0323] 145. Use of an assembled nanoparticle of any preceding embodiment of the same category, for the display of one or more microbial polypeptide antigen(s).

[0324] 146. The use of an assembled nanoparticle of the preceding embodiment, for the display of one or more yeast polypeptide antigen(s).

[0325] 147. The use of an assembled nanoparticle of the preceding embodiment of the same category, for the display of one or more bacterial polypeptide antigens(s).

[0326] 148. The use of an assembled nanoparticle of the preceding embodiment of the same category, for the display of one or more or viral polypeptide antigen(s).

[0327] Miscellaneous

[0328] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The term “plurality” refers to two or more. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.

[0329] Additionally, numerical limitations given with respect to concentrations or levels of a substance, such as an antigen, are intended to be approximate. Thus, where a concentration is indicated to be at least (for example) 200 pg, it is intended that the concentration be understood to be at least approximately (or “about” or “~”) 200 pg.

[0330] It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acid molecules or polypeptides are approximate and are provided for description. Additionally, numerical limitations given with respect to concentrations or levels of a substance, such as an antigen, are intended to be approximate. Thus, where a concentration is indicated to be at least (for example) 200 pg, it is intended that the concentration be understood to be at least approximately (or “about” or “~”) 200 pg.

[0331] The term "and / or" as used in a phrase such as "A and / or B" is intended to include “A and B," "A or B," "A," and "B." Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0332] Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus, components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc. Similarly, while steps of a method may be numbered (such as (1), (2), (3), etc. or (i), (ii), (iii)), the numbering of the steps does not mean that the steps must be performed in that order (i.e., step 1 then step 2 then step 3, etc.). The word “then” or “followed by” may be used to specify the order of a method’s steps.

[0333] The present invention is not limited to particular embodiments described herein. It is appreciated that certain features of the invention which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0334] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

[0335] The entire disclosure of published references, patents, and published patent applications cited herein are incorporated herein by reference in their entirety.

[0336] The following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the present embodiments in any manner.

[0337] EXAMPLES

[0338] EXAMPLE 1. MODIFIED DODECIN SUBUNITS

[0339] 1. Materials and Methods a) Strains and transformation

[0340] Recombinant plasmids have been provided by Twist Bioscience cloned in pEt24b+ plasmid with the sequence optimized for the expression in E. coli. DNA has been transformed in chemically competent E. coli BL21(DE3) T1R (fhuA2 [Ion] ompT gal (A. DE3) [dem] AhsdS. A. DE3= A. sBamHlo AecoRI-B int::(lad::PlacUV5::T7 genel) i21 Xnin5) cells provided by New England Biolabs (NEB) through heat shock procedure. b) Pre-culture and glycerol stock preservation

[0341] The recombinant colonies grown on selective plates were picked and incubated with 5-10 mL of LB (Tryptone lOg / L, yeast extract 5g / L, NaCl lOg / L) medium at 37°C and 180 rpm overnight (about 16h). Pre-culture was used to set up batch or small-scale cultivation and 500 pL were mixed with 200 pL of 80% sterile glycerol and preserved in cryo vials at -80°C. c) Small scale protein expression and purification with IMAC Pre-culture has been diluted 1 :100 in ImL HTMC medium (glycerol 15 g / L, yeast extract 30 g / L, MgSO4X7H2O 0.5 g / L, KH2PO4 5 g / L, K2HPO4 20 g / L, KOH IM at pH final 7.35±0.1) + kanamycin [50 pg / mL] in 96 well plate and incubated for 16h at 20°C and 160 rpm. The production of interest protein was induced with ImM sterile IPTG and protein expression was followed for 24h at 20°C and 160 rpm. The plate has been centrifuged at 3,600 rpm for 20 min, the supernatant discarded and the pellet lysed with 600 uL of Cell-lytic express lysis buffer.

[0342] The proteins have been then purified through affinity chromatography with King Fisher and magnetic beads (Dynabeads TALON). The protein of interest has been eluted in 100 uL of elution buffer (150 mM Imidazole, 50 mM NaP pH 8.0, 300 mM NaCl, 0.01% Tween 20).

[0343] 9 pL of elution were mixed with 3 pL of 4X reducing mix (LSD and DDT) and boiled at 98°C for

[0344] 10 min. 12 pL of samples and pre-stained protein molecular marker (Thermo Fisher Scientific) were loaded on precast polyacrylamide gels (Thermo Fisher Scientific). The run was performed in IX 2-(N-morpholino) ethane sulfonic acid (MES), at 150 V 1,000 mA and 180 W for 35 min. Gels were stained with Coomassie ProBlue Safe stain (Giotto) for protein visualization and images were acquired with GelDoc (Bio-Rad). d) Size Exclusion-Ultra high Performance Liquid Chromatography (SE-UPLC)

[0345] 25 uL of purified protein produced in small scale have been analyzed in SE-UPLC in order to assess the presence of NP. The run as been performed in PBS with Superdex 200 increase (Cytiva) column and signals in Fluorescence have been recovered.

[0346] The cultivation was set up in a 250 mL shaker flask with 75 mL of sterile HTMC medium (glycerol 15 g / L, yeast extract 30 g / L, MgSCU x7H2O 0.5 g / L, KH2PO4 5 g / L, K2HPO4 20 g / L, KOH IM at pH final 7.35±0.1), kanamycin (50 pg / mL) and preculture diluted 1: 100. The flask was incubated for 16h at 20°C and 160 rpm. 500 pL of not-induced sample were collected in 1,5 mL Eppendorf tube and stored at 4°C. The production of interest protein was induced with ImM sterile IPTG and protein expression was followed for 24h at 20°C and 160 rpm. 500 pL of induced sample were collected in 1,5 mL Eppendorf tube and stored at 4°C.

[0347] The whole cultivation was transferred in 50 mL falcon and centrifuged for 8,000 rpm at 4 °C for 10 min. Medium was discarded and pellet, after Ih at -20°C, was lysed with CelLytic Express lysis buffer. Each g of pellet was resuspended with 10 mL of lysis buffer and incubated at RT for Ih under gently shaking. After lysis the samples were diluted 1:2 with PBS and centrifuged at 8,000 rpm at 4°C for 10 min to collect soluble fraction. f) Immobilized metal affinity chromatography (IMAC)

[0348] Empty PD 10 column were prepared with the appropriate filter and filled with 2 mL of homogenous suspension of Ni-NTA Agarose resin (Thermo Fisher Scientific). When the matrix was settled, the resin was decanted and the leftover liquid was discarded, leaving 1 cm above the column head to prevent drying out. The column was washed with five column volumes (CV) of distilled water and then equilibrated with ten CV of equilibration buffer (PBS or 20 mM Tris, 150mM NaCl pH 8). Crude cell lysate was applied on the column, incubated for 10 min, and the flow through was collected. The column was washed with five CV of equilibration buffer supplied with 20 mM imidazole. Target protein was eluted with about 5-10 mL of elution buffer containing 350 mM imidazole. The column was washed with two CV of equilibration buffer, ten CV of distilled water and it was preserved in one CV of 20% ethanol. All fractions collected during IMAC purification were analysed with SDS-PAGE as described previously. The elution fractions containing interest proteins were transferred into ultrafiltration device and centrifuged at 4,500 rpm until to reach desired final volume. g) Size exclusion chromatography (SEC)

[0349] Recombinant chimeric nanoparticles were purified with size exclusion chromatography in order to separate correctly assembled molecules from their monomers. 0.5, 1 or 5 mL of samples were loaded on preparative Superdex 200 10 / 300-26 / 600 or in Superose 6 columns equilibrated with IX PBS. All the collected fractions were checked for the content of target protein by SDS-PAGE analysis. Fractions of interest were pooled, filtered at 22 pm, concentred and stored at -20°C. h) Protein content evaluation

[0350] Protein concentration was determined with Nanodrop measuring the absorbance at 280 nm and normalizing with the calculated protein extinction coefficient. The first measurement was the blank solution represented by the buffer alone. Then the concentration of each sample was measured, and the value obtained was expressed as mg / mL. i) Transmission electron microscopy

[0351] The electron microscopy analysis was performed loading 5 pl of sample concentrated 20 ng / pL onto a glow discharged copper 300-square mesh grid for 30 sec. Blotted the excess, the grid was negatively stained using NanoW for 30 sec. The samples were analysed using a Tecnai G2 spirit and the images were acquired using a Tvips TemCam-F216 (EM-Menu software). j) Differential scanning calorimetry (DSC)

[0352] Using a MicroCai VP-Capillary DSC instrument (GE Healthcare), protein samples (10 pM) were subj ected to a temperature scan range from 10-110°C with a rate of 180°C / h, and a 4-s filter period. During the analysis the heat capacity of denaturation (Cp) was constantly measured as Kcal / mole / °C. The results, analysed with Origin 7 software, were reported as Cp in function of increasing temperature with a resulting Gaussian curve. The temperature corresponding to the maximum of the curve represents the Tm of the protein. k) Dynamic light scattering (PLS)

[0353] The hydrodynamic diameter of NP in solution was measured with dynamic light scattering. The measurement was performed using Malvern Zetasizer Nano ZS equipped with a 633 nm He-Ne laser and using an angle of 173°. Scattering light detected was automatically adjusted by laser attenuation filters. For data analysis, the refractive index (RI) and viscosity of PBS were used at 25°C. To analyse the data the Zetasizer software version 7.11 was used. Temperature was set at 25°C. 80 pL of the sample were transferred into a single-use polystyrene microcuvette (ZEN0040, Alfatest) with a path length of 10 mm. The hydrodynamic diameter of NPs was expressed by a Z- average providing also the poly disperse index.

[0354] 2. Results

[0355] The starting structure for the Rvl498A protein from Mycobacterium tuberculosis (hereunder referred to as wt dodecin, having an amino acid sequence corresponding to sequence of SEQ ID NO: 1) was refined and designed using an updated version of the PROSS algorithm (with symmetry restraints and the beta energy scoring function). The Rosetta FilterScan mover was used to perform single point mutagenesis of all the residues to the preferred PSSM mutations. The mutation scan was binned within twelve different energy thresholds (-0.5, -1, -1.5, -2, -2.5, -3, - 3.5, -4, -4.5, -5, -5.5, -6 kcal / mol) to increase mutation sequence diversity (FIG. 1). For example, a combination of -6 kcal / mol single point mutations would result in fewer mutations due to a higher energetic barrier for introducing new mutations.

[0356] Modified dodecin subunits with the energy threshold of -6 kcal / mol (bearing a G25N mutation with respect to SEQ ID NO: 1 and corresponding to SEQ ID NO: 2) and -0.5 kcal / mol (bearing a G25N, V50T and A53T triple mutation with respect to SEQ ID NO: 1 and corresponding to SEQ ID NO: 8) were expressed and analyzed on SDS-PAGE electrophoresis. As seen in FIG. 2, both modified dodecin subunits were expressed and visible under a completely unfolded conformation (lower gel bands) and under a not completely unfolded conformation (upper gel bands).

[0357] When further analyzed under TEM method, it appeared that the wt dodecin (of sequence SEQ ID NO: 1) displayed aggregated nanoparticles (FIG. 3A; e.g., white circle) among the non-aggregated nanoparticles (FIG. 3 A; black circles). In contrast, the -0.5 kcal / mol modified dodecin (of sequence SEQ ID NO: 8) displayed significantly less aggregated nanoparticles (FIG. 3B; e.g., black circles). Finally, similarly to the observations made with the wt dodecin subunit, the -6 kcal / mol modified dodecin (of sequence SEQ ID NO: 2) also displayed aggregated nanoparticles (FIG. 3C; e.g., white circle).

[0358] Therefore, this study achieved the identification of a modified dodecin of SEQ ID NO: 8 with improved stability, as it is less prone to display aggregated nanoparticles. The modified dodecin of sequence SEQ ID NO: 8 varies from the wt dodecin subunit in that is bears 3 mutations, namely G25N, V50T and A53T, and is referred further as to s / dodecin (for stabilized dodecin).

[0359] EXAMPLE 2. STABILIZED DODECIN SUBUNIT FOR THE DISPLAY OF THE MRKA ANTIGEN OF KLEBSIELLA PNEUMONIAE

[0360] Klebsiella pneumoniae (Kp) is a Gram-negative bacterium belonging to Enterobacteriaceae family, in which type 3 fimbriae appear to be highly conserved antigens.

[0361] Within the type 3 fimbrial complex, MrkA, a 20 KDa protein, represents the major structural component of this complex. It is involved in biofilm formation and establishment of infection. Its amino acid sequence is highly conserved among the majority of Enter obacteriaceae strains analyzed so far. However, while exploring MrkA immunogenicity, it was observed that anti-MrkA IgG response is relatively low after two immunizations with recombinant MrkA (rMrkA), even in the presence of an adjuvant.

[0362] In order to improve MrkA immunogenicity, we have explored the possibility to use protein NPs as delivery systems for MrkA protein antigen, and notably the stabilized version of dodecin NP subunit of example 1 (dodecin bearing the 3 mutations G25N, V50T and A53T as compared to the wt dodecin of SEQ ID NO: 8; further referred to as s / dodecin). The MrkA coding sequence was genetically fused to the N-term of the NP subunit, spaced by a glycine-serine linker. A 6x Histidine tag (His-Tag) was also added in the coding sequence of MrkA to facilitate chimeric NP purification by affinity chromatography.

[0363] MrkA chimeric fusion protein with stabilized dodecin subunit has an amino acid sequence of SEQ ID NO: 23.

[0364] 1. Materials and methods a) Production of the MrkA chimeric fusion protein with stabilized dodecin subunit MrkA-s / dodecin fusion construct was cloned into the expression vector pET29b under the control of the IPTG-inducible T7 promoter and transformed into E. coli BL21 (DE3). The cells were grown in 250 mL of HTMC medium, under shaking (180 rpm) at 37° C until reaching an optical density OD600 of 0.8 followed by induction with ImM IPTG for 20 h at 18°C. Soluble expression was checked by performing a western blot with an anti-His-tag antibody. Bacteria cells were harvested by centrifugation at 4,000 rpm for 30 minutes. Then pelleted cells were resuspended in 50 mM Tris, 500 mM NaCl and 30 mM of imidazole at pH 8 (10 ml for each gram of bacteria pelleted) and lysed by sonication for 35 minutes alternating 35 cycles of 30s pulse and 30s stop (1 cycle for 1 ml). The protein of interest was purified from soluble fractions with a two-step purification procedure.

[0365] The first purification step was performed by immobilized metal affinity chromatography (IMAC) and an elution buffer containing 50 mM Tris, 500 mM NaCl and 500 mM of imidazole (pH 8). Fractions containing the target protein were collected and further purified by size exclusion chromatography (SEC) using a Superdex 200 10 / 300 column equilibrated in PBS buffer, with a flow rate of 0.5 mL / min. SDS-PAGE analysis was performed to check protein purity (FIG. 4A and 4B) and the concentration was determined by colorimetric protein assay micro BCA.

[0366] Dynamic light scattering (DLS; FIG. 5 A) and Transmission electron microscopy (TEM; FIG. 5B) confirmed the assembly of a well-structured NP with a size of 17 nm, confirming the theoretical size of the model monomeric MrkA-s / dodecin (FIG. 5A and 5B).

[0367] Recombinant MrkA was produced as a soluble monomer in / / . coli BL21 (DE3) cells and purified via affinity chromatography using the His-tag present at the N-terminus of the protein sequence. Its purity was verified by High Performance Liquid Chromatography (HLPC-SEC) and SDS- PAGE analysis, as shown in FIG. 6. b) Animal immunization studies

[0368] 7 groups of 10 female CD1 mice, 4-6 weeks old, were vaccinated subcutaneous (SC) with 200 pL of vaccine at day 0 (all groups 1-7) and day 28 (Groups 1-5, 7). Sera were collected at day 27 (sera were maintained single). Final bleed was performed at day 42 (sera were maintained single) and mice were euthanized.

[0369] Sera collected from mice at day 27 and 42 (all groups) were analyzed by ELISA.

[0370] Table 1 : Protocol design

[0371] 1AS01 was used at a final concentration of 100 pg / mL.2Alhydrogel was used a final concentration of 10.3 mg / mL in Al3+.3Low dose (LD). c) Assessment of the binding of sera to wildtype MrkA fimbriae

[0372] Sera from group 1, 3, 4 and 5 were pooled and tested against K61:O3_long at dilution 500. The FACSanalysis was performed in a 96 wells plate, 0.05 OD of bacteria were fixed in each well with cytofix 4% and incubated with pooled sera. After washing and centrifugation, a second incubation with Alexa Fluor 488 goat anti-mouse IgG secondary antibody was followed. Samples were measured and analyzed using FlowJo.

[0373] 2. Results

[0374] Anti-MrkA total IgG ELISA graphs, showing the geometric mean and individual EU / mL of all groups at days 27 and 42 for all groups of mice are shown in FIG. 7A and 7B, respectively. The anti -MrkA IgG response induced by recombinant MrkA monomer and MrkA-s / dodecin nanoparticle was assessed with Alhydrogel or AS01, using the Mann-Whitney unpaired two-tailed t test (groups 1-2 and groups 3-4).

[0375] As shown in FIG. 8, MrkA-s / dodecin NP adjuvanted with AS01 induces significantly higher anti- MrkA IgG titers in comparison to recombinant MrkA monomer with AS01 both at day 27 and day 42 (P value < 0.0001); whereas MrkA-s / dodecin NP adjuvanted with alhydrogel induces significantly higher anti-MrkA IgG titers in comparison to recombinant MrkA monomer with Alhydrogel both at day 27 and day 42 (P value = 0.0009; 0.0027). The anti-MrkA IgG response induced by MrkA-s / dodecin NP was further assessed without adjuvants or with Alhydrogel or AS01, using the non-parametric Kruskall-Wallis test with Dunn's multiple comparison test (groups 1, 3, 5).

[0376] As shown in FIG. 9, MrkA-s / dodecin NP induces comparable anti-MrkA IgG titers when adjuvanted with AS01 or Alhydrogel or when unadjuvanted. Although, the total geomean of group immunized with MrkA-s / dodecin NP adjuvanted with Alhyrdogel is a logarithm superior than the unadj uvanted.

[0377] The anti-MrkA IgG response induced by MrkA-s / dodecin NP was also assessed with a one-dose or a two-doses schedule (reference schedule), using the Mann- Whitney unpaired two-tailed t test (groups 1, 6; day 42 only).

[0378] As shown in FIG. 10, MrkA-s / dodecin NP administrated only once induces lower anti-MrkA IgG at day 42 in comparison to two administrations at days 0 and 28 (P value= 0.0003). However, one dose of MrkA-s / dodecin NP elicits a significantly higher immune response compared to two doses of MrkA monomer at day 42: (P value=0.0003). The anti-MrkA IgG response induced by MrkA- s / dodecin NP was also assessed at 2 pg / dose (low dose) and at 10 pg / dose (reference dose) both with AS01 as adjuvant, using the Mann- Whitney unpaired two-tailed t test (groups 2, 7).

[0379] As shown in FIG. 11, 2 pg of MrkA-s / dodecin NP induce lower anti-MrkA IgG compared to anti- MrkA IgG elicited by 10 pg of MrkA-s / dodecin NP (P value at day 27 = 0.0014; P value at day 42 < 0.0001). Finally, at dilution 500, pooled sera from group 1, 3 and 5 were perfectly able to detect MrkA; group 4, instead, revealed to be negative and it was probably due to its low anti-MrkA total IgG.

[0380] Finally, immunologic data (anti-MrkA IgGs levels) were obtained with the MrkA monomers and the s / dodecin-MrkA fusion protein in a rabbit model, formulated in alhydrogel. These data confirmed the superiority of the immune response with the fusion protein as compared to the monomer (data not shown).

[0381] 3- Conclusions

[0382] In this study, s / dodecin self-assembling NP has been decorated with MrkA (MrkA-s / dodecin) through genetic fusion and its immunogenicity has been tested in mice.

[0383] The poor immunogenicity of recombinant MrkA monomer was confirmed in the present study. Indeed, the anti-MrkA IgG response elicited by MrkA monomer adjuvanted both with AS01 or Alhydrogel at day 27 and day 42 was very low. The use of a larger scaffold as s / dodecin combined with the multidisplay of the MrkA antigen allowed to significantly increase the anti-MrkA IgG response. Indeed, comparing groups immunized with MrkA-s / dodecin NP and MrkA monomer formulated both with AS01 or Alhydrogel, anti-MrkA IgG were significantly higher for MrkA-NP system (day 27 p< 0.0001; day 42 p=0.0009; 0.0027 respectively), regardless of the adjuvant system used. Moreover, differently from what observed with recombinant MrkA monomer which requires the use of an adjuvant, MrkA-s / dodecin NP induced comparable anti-MrkA IgG when adjuvanted with AS01 or Alhydrogel or unadjuvanted (day 27 p=0.0942; day 42 p=0.4037), suggesting that the NP system per se was able to increase the response.

[0384] Both administration schedule with one or two injections of MrkA-s / dodecin NP induced at day 27 and day 42 significant high anti-MrkA IgG levels.

[0385] In conclusion, this study demonstrates the capacity of the self-assembling NP s / dodecin to increase the immune response of a poor immunogenic antigen, as MrkA of Klebsiella pneumoniae, even after 1 single dose. Finally, it was able to verify the binding of sera obtained from mice immunize with the NP MrkA-s / dodecin constructs to wildtype MrkA fimbriae.

[0386] EXAMPLE 3. STABILIZED DODECIN SUBUNIT FOR THE DISPLAY OF A B- BARREL CONTAINING ANTIGEN

[0387] The factor H binding protein (fHbp) of N. meningitis serogroup B has been previously identified as antigenic and is one antigen present in the BEXSERO product (BEXSERO is a Trademark owned by or licensed to the GSK group of companies), which is used against the N. meningitis serogroup B infection.

[0388] The coding sequence of fHbp was fused to s / dodecin (of amino acid sequence SEQ ID NO: 8) at its N-terminus (resulting in a chimeric antigenic polypeptide of sequence SEQ ID NO: 24) or its C-terminus (resulting in a chimeric antigenic polypeptide of sequence SEQ ID NO: 25), and the assembled nanoparticles were purified and analyzed by TEM method.

[0389] As shown in FIG. 12, s / dodecin was successful in displaying the fHbp antigen of N. meningitis serogroup B at its N-terminus, as the chimeric fusion retained the ability to form nanoparticles. The same observation was made when the fHbp antigen when fused to s / dodecin at its C-terminus (not shown).

[0390] EXAMPLE 4. STABILIZED DODECIN SUBUNIT FOR THE DISPLAY OF GALACK DOC / TAG PROTEIN LIGATION SYSTEM

[0391] Similarly to the examples above, the feasibility to fuse the s / dodecin subunits (dodecin of amino acid sequence of SEQ ID NO: 8) to additional polypeptide while maintaining the ability of the nanoparticle subunit moiety to correctly assemble as nanoparticles of the appropriate size and shape was further assessed with the DOCK and TAG proteins of the GALACK ligation system.

[0392] As depicted in FIG. 13 and FIG. 14, the successful assembly of NPs from s / dodecin subunits fused at the N-terminus to either TAG (SEQ ID NO: 26) or the DOCK (SEQ ID NO: 27) moiety of the Galack DOCK protein ligation system, respectively, was demonstrated when analyzed by TEM method.

[0393] EXAMPLE 5. STABILIZED DODECIN SUBUNIT FOR THE DISPLAY OF MOMP

[0394] ANTIGENS OF CHLAMYDIA

[0395] 1. Materials and methods

[0396] Loopl (LI) and Loop 4 (L4) of MOMP serovar D-s / dodecin (s / dodecin of an animo acid sequence SEQ ID NO: 8) fusion constructs were cloned into the expression vector pET29b under the control of the IPTG-inducible T7 promoter and transformed into E. coli BL21 (DE3). The cells were grown in 75 mL of HTMC medium, under shaking (160 rpm) at 20°C for 16 h followed by induction with ImM IPTG for 24 h at 20°C. Soluble expression was checked by performing a SDS-PAGE analysis. Bacteria cells were harvested by centrifugation at 8,000 rpm for 10 minutes. Then pelleted cells were resuspended in Cell-lytic express lysis buffer (Sigma- Aldrich) (10 ml for each gram of bacteria pelleted) and incubated for Ih at RT under shaking.

[0397] The protein of interest was purified from soluble fractions with a two-step purification procedure. The first purification step was performed by immobilized metal affinity chromatography (IMAC) and an elution buffer containing IX PBS + 350 mM of imidazole (pH 8). Fractions containing the target protein were collected and further purified by size exclusion chromatography (SEC) using a Superdex 200 10 / 300 column equilibrated in PBS buffer, with a flow rate of 0.5 mL / min (see FIG. 15 A and 15B). Dynamic light scattering (DLS) and Transmission electron microscopy (TEM; FIG. 16) confirmed the assembly of a well-structured NP with a size of 17 nm, confirming the theoretical size of the model MOMP -LI or MOMP-Ll / MOMP-L4-s / dodecin.

[0398] Table 2: Sample specifications (storage in PBS IX at -20°C)

[0399] 4 groups of 12 female C57 / B16 mice, 4-6 weeks old, were vaccinated intramuscularly (IM) with 50 pL of NPs (s / dodecin, s / dodecin M0MPD-L1, s / dodecin M0MPD-L1-L4) formulated with AS01 adjuvant or adjuvant alone at day 0 (all groups 1-7) day 20 and day 41. Sera were collected at day 42 (sera were maintained single). Final bleed was performed at day 57 (sera were maintained single) and mice were euthanized. Sera collected from mice at day 42 (P2) and 57 (P3) were analyzed by ELISA.

[0400] Briefly Nunc Maxisorp microtiter plates were coated with MOMPD. After over-night incubation, plates were washed and 100 pL of diluted (1 :400) mice sera were added to each well and the plates incubated for 2 hours at 37°C. After primary antibody incubation, plates were washed and the secondary Alkaline phosphatase-conjugated antibodies diluted 1:2,000 in dilution buffer (1% BSA, 0.05% Tween-20) were dispensed and incubated for 90 minutes at 37°C. Plates were then washed and a chromogenic substrate solution of p-nitrophenyl phosphate was added to each well and the plates incubated at room temperature for 30 minutes. After this step 100 pL 4N NaOH were added to each well to stop the enzymatic reaction. Plates were finally read at 405-620 nm and the antibody titers were quantified as the dilution of serum that gives an absorbance of Optical Density (OD) value equal to 0.4. 2. Results

[0401] As shown in FIG. 17, MOMP-s / dodecin NP adjuvanted with AS01 induces specific anti-MOMP titers both at day 27 (P2) and day 42 (P3). ELISA data showed that both s / dodecin MOMP constructs elicited significant higher IgG MOMP D titers at both timepoints compared to s / dodecin or adjuvant alone.

[0402] Consequently, the stabilized dodecin (bearing the triple mutation G25N, V50T and A53T) is capable of displaying antigenic loops of the MOMP antigen from serovar D, retaining the ability to form nanoparticles and eliciting an immune response in a mouse model.

[0403] SEQUENCE LISTING

[0404] SEO ID NO: 1

[0405] Dodecin wild type amino acid sequence (Q8VK10)

[0406] MSNHTYRVIEIVGTSPDGVDAAIQGGLARAAQTMRALDWFEVQSIRGHLVDGAVAHFQ VTMKVGFRLEDS

[0407] SEO ID NO: 2

[0408] Dodecin modified amino acid sequence (G25N)

[0409] MSNHTYRVIEIVGTSPDGVDAAIQNGLARAAQTMRALDWFEVQSIRGHLVDGAVAHFQ VTMKVGFRLEDS

[0410] SEO ID NO: 3

[0411] Dodecin modified amino acid sequence (V50T)

[0412] MSNHTYRVIEIVGTSPDGVDAAIQGGLARAAQTMRALDWFEVQSIRGHLTDGAVAHFQ

[0413] VTMKVGFRLEDS

[0414] SEO ID NO: 4

[0415] Dodecin modified amino acid sequence (A53T)

[0416] MSNHTYRVIEIVGTSPDGVDAAIQGGLARAAQTMRALDWFEVQSIRGHLVDGTVAHFQ

[0417] VTMKVGFRLEDS SEQ ID NO: 5

[0418] Dodecin modified amino acid sequence (G25N, V50T)

[0419] MSNHTYRVIEIVGTSPDGVDAAIQNGLARAAQTMRALDWFEVQSIRGHLTDGAVAHFQ

[0420] VTMKVGFRLEDS

[0421] SEO ID NO: 6

[0422] Dodecin modified amino acid sequence (G25N, A53T)

[0423] MSNHTYRVFEIVGTSPDGVDAAIQNGLARAAQTMRALDWFEVQSIRGHLVDGTVAHFQ

[0424] VTMKVGFRLEDS

[0425] SEQ ID NO: 7

[0426] Dodecin modified amino acid sequence (V50T, A53T)

[0427] MSNHTYRVFEIVGTSPDGVDAAIQGGLARAAQTMRALDWFEVQSIRGHLTDGTVAHFQ

[0428] VTMKVGFRLEDS

[0429] SEQ ID NO: 8

[0430] Dodecin modified amino acid sequence (G25N, V50T, A53T; referred as to s / dodecin)

[0431] MSNHTYRVIEIVGTSPDGVDAAIQNGLARAAQTMRALDWFEVQSIRGHLTDGTVAHFQ

[0432] VTMKVGFRLEDS

[0433] SEQ ID NO: 9

[0434] Dodecin modified amino acid sequence (G25N, V50T, A53T) and His tag

[0435] MSNHTYRVIEIVGTSPDGVDAAIQNGLARAAQTMRALDWFEVQSIRGHLTDGTVAHFQ

[0436] VTMKVGFRLEDS GSGHHHHHH

[0437] SEO ID NO: 10

[0438] Dodecin modified nucleic acid sequence

[0439] ATGTCTAACCATACTTACCGTGTTATCGAAATCGTGGGAACTTCGCCGGACGGAGTG

[0440] GACGCCGCGATTCAGAACGGCTTAGCCCGTGCTGCCCAAACCATGCGTGCGCTTGAT TGGTTTGAGGTGCAGTCAATTCGCGGACATCTGACAGATGGCACGGTCGCTCACTTC

[0441] CAAGTAACCATGAAAGTGGGGTTTCGCCTGGAGGACTCTGGCAGTGGCCACCACCA

[0442] CCACCATCACTAACGTTGCGCGGAGTTTCTGCGTATGCACAACCTGGAAGACAGCTG

[0443] CTTCAGCTTTCTGTAA

[0444] SEQ ID NO: 11 s / dodecin_MOMP-Ll (modified dodecin fused to loop 1 of MOMP serovar D of Chlamydia trachomatis)

[0445] MSNHTYRVIEIVGTSPDGVDAAIQNGLARAAQTDRVLKTDVNKEFQMGAKPTTDTGNS

[0446] AAPSTLTARENPAYGRHMQDAEMFTNLDWFEVQSIRGHLTDGTVAHFQVTMKVGFRL

[0447] EDSGSGHHHHHH

[0448] SEO ID NO: 12

[0449] MOMP-L4_s / dodecin_MOMP-Ll (modified dodecin fused to both loops 1 and 4 of MOMP serovar D of Chlamydia trachomatis)

[0450] MDADHRIAQPKSATAIFDTTTLNPTIAGAGDVKTGAEGQLGDTMQIVSLQLNKMKSSN

[0451] HTYRVIEIVGTSPDGVDAAIQNGLARAAQTDRVLKTDVNKEFQMGAKPTTDTGNSAAP

[0452] STLTARENPAYGRHMQDAEMFTNLDWFEVQSIRGHLTDGTVAHFQVTMKVGFRLEDS

[0453] GSGHHHHHH

[0454] Histidine tag

[0455] MGSSHHHHHHS

[0456] SEO ID NO: 14

[0457] Signal Sequence DsbA

[0458] MKKIWLALAGLVLAFSASA

[0459] SEQ ID NO: 15

[0460] Signal sequence (TolB) MKQALRVAFGFLILWASVLHA

[0461] Signal sequence (Figi)

[0462] MIKFLSALILLLVTTAAQA

[0463] SEO ID NO: 17

[0464] Signal sequence (OmpA)

[0465] MKKTAIAIAVALAGFATVAQA

[0466] SEO ID NO: 18

[0467] Signal sequence (MalE)

[0468] MKIKTGARILALSALTTMMFSASALA

[0469] SEO ID NO: 19

[0470] Signal sequence (PelB)

[0471] MKYLLPTAAAGLLLLAAQPAMA

[0472] SEO ID NO: 20

[0473] Signal sequence (LTIIb)

[0474] MSFI<I<III<AFVIMAALVSVQAHA

[0475] SEO ID NO: 21

[0476] Signal sequence (XynA)

[0477] MFKFKKKFLVGLTAAFMSISMF S ATAS A

[0478] Signal sequence (SipA)

[0479] MKMNKKVLLTSTMAASLLSVASVQAS SEQ ID NO: 23 s / dodecin_MrkA ((modified dodecin fused to MrkA of Klebsiella pneumoniae)

[0480] CTVSVNGQGSDANVYLSPVTLTEVKAAAADTYLKPKSFTIDVSNCQAADGTKQDDVSK

[0481] LGVNWTGGNLLAGATSKQQGYLANTEASGAQNIQLVLSTDNATALTNKIIPGDSTQPKA

[0482] KGDASAVADGARFTYYVGYATSAPTTVTTGWNSYATYEITYQGGGGGGADTTVGGG

[0483] QVNFFGKVTDVSGGSGGSGGSGGSSNHTYRVIEIVGTSPDGVDAAIQNGLARAAQTMR

[0484] ALDWFEVQSIRGHLTDGTVAHFQVTMKVGFRLEDSGSHHHHHHHHHHGS

[0485] SEO ID NO: 24 s / dodecin_fHbp (modified dodecin fused to fHbp of N. meningitis, at N-terminus)

[0486] MGSSHHHHHHENLYFQGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTIDFA

[0487] AKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQ

[0488] EVAGSAEVKTVNGIRHIGLAAKQGGSGGSGGSGGSSNHTYRVIEIVGTSPDGVDAAIQN

[0489] GLARAAQTMRALDWFEVQSIRGHLTDGTVAHFQVTMKVGFRLEDS

[0490] SEQ ID NO: 25 s / dodecin fHbp (modified dodecin fused to fHbp of N. meningitis, at C-terminus)

[0491] MSNHTYRVIEIVGTSPDGVDAAIQNGLARAAQTMRALDWFEVQSIRGHLTDGTVAHFQ

[0492] VTMKVGFRLEDSGGSGGSGGSGGSGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGK

[0493] LTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSL

[0494] GIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQGSGHHHHHH

[0495] SEQ ID NO: 26 s / dodecin TAG (modified dodecin fused to TAG of the GALACK system at N-terminus)

[0496] MASYSKGSETDIEYTQGSKVNDAQQVINKKVTIGGSGGSMSNHTYRVIEIVGTSPDGVD

[0497] AAIQNGLARAAQTMRALDWFEVQSIRGHLTDGTVAHFQVTMKVGFRLEDSGSGHHHH

[0498] HH

[0499] SEO ID NO: 27 s / dodecin_DOCK (modified dodecin fused to DOCK of the GALACK system at N-terminus)

[0500] TYGRKFVKTNKDGKIRLAGAVFLVKKDGKYLARKTGDASEEQKAAVDEAKKELDKAI

[0501] KKYNDLTKEQQEGEDGKKALATVTEKQKEYNDAFKKANYRYEWVDDKNAENWHLI

[0502] SNDKGQFEITGLTEGTYSLEETQAPTGYAPLSGDVSFNVNGGSGGSMSNHTYRVIEIVGT SPDGVDAAIQNGLARAAQTMRALDWFEVQSIRGHLTDGTVAHFQVTMKVGFRLEDSG

[0503] SGHHHHHH

Claims

CLAIMS1. A modified dodecin nanoparticle (NP) subunit having an amino acid sequence at least 80% identical to amino acid residues 2-70 of SEQ ID NO: 1 and comprising at least one substitution at position A53, with respect to SEQ ID NO: 1.

2. The modified subunit of claim 1 , further comprising one or more substitution(s) at position(s) G52 and / or V50, with respect to SEQ ID NO: 1.

3. The modified subunit of claim 1 or 2, wherein the substitutions are selected from the group consisting of A53T, G25N and V50T, with respect to SEQ ID NO: 1.

4. The modified subunit of claim 1, comprising each substitution A53T, G25N and V50T, with respect to SEQ ID NO: 1.

5. The modified subunit of claim 1, comprising an amino acid sequence identical to amino acid residues 2-70 of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.

6. A chimeric antigenic polypeptide comprising a) the modified subunit of claim 1 to 5, and b) a polypeptide antigen.

7. The chimeric antigenic polypeptide of claim 6, which is intended to generate a specific immune response.

8. The chimeric antigenic polypeptide of claim 6 or 7, wherein the polypeptide antigen is selected from the group consisting of Chlamydia species, Escherichia species, Shigella species, Klebsiella species, Salmonella species, Yersinia species, Helicobacter species, Proteus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Enterococcus species, Staphylococcus species, Bacillus species, Clostridium species, Listeria species, Campylobacter species, Neisseria species andCandida polypeptide antigens.

9. The chimeric antigenic polypeptide of claim 8, wherein the polypeptide antigen is selected from the group consisting of Chlamydia species polypeptide antigens, Klebsiella species polypeptide antigens and Neisseria species polypeptide antigens.

10. A method for preparing an assembled antigenic nanoparticle (NP) comprising the steps of: i) providing a host cell; ii) expressing the chimeric antigenic polypeptide of claim 6 to 9 in said host cell, so as to produce an assembled antigenic nanoparticle; iii) recovering / obtaining the assembled antigenic nanoparticle.

11. An isolated nucleic acid molecule, comprising a nucleotide sequence encoding the modified nanoparticle subunit of claim 1 to 5 or the chimeric antigenic polypeptide of claim 6 to 9.

12. A vector, comprising the isolated nucleic acid molecule of claim 11.

13. A host cell, comprising the isolated nucleic acid molecule of claim 1 lor the vector of claim 12.

14. An assembled antigenic nanoparticle comprising the chimeric antigenic polypeptide of claim 6 to 9.

15. An immunogenic composition comprising i) the chimeric antigenic polypeptide of claim 6 to 9 and / or the assembled antigenic nanoparticle of claim 14, and ii) a pharmaceutically acceptable vehicle.

16. A vaccine comprising the immunogenic composition of claim 15, and optionally an adjuvant.

17. The immunogenic composition of claim 15 or the vaccine of claim 16, further comprising an adjuvant selected from the group consisting of alum, aluminium hydroxide, aluminium phosphate, a saponin, a water-in-oil emulsion, an oil-in-water emulsion, a liposaccharide, a lipopolysaccharide, an immunostimulatory nucleic acid molecule, a liposome, and a Toll Receptor or Toll-Like Receptor agonist.

18. A method of inducing an immune response in a human subject, comprising administering to the subject an immunologically effective amount of the assembled nanoparticle of claim 14, the immunogenic composition of claim 15, or the vaccine of claim 16.

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