Ferritin protein

Abstract

Disclosed are ferritin proteins comprising mutations replacing surface-exposed amino acids with cysteines, N- or C-terminal linkers comprising cysteines, and / or one or more immunostimulatory moieties linked to the ferritin protein via surface-exposed amino acids, which may further comprise non-ferritin polypeptides and be antigenic, e.g., for use in eliciting antibodies against the non-ferritin polypeptides. [Selected Figure] Figure 1D

Description

[Technical Field]

[0001] This application claims the interests of U.S. Provisional Patent Application No. 62 / 652,217, No. 62 / 652,199, No. 62 / 652,201, No. 62 / 652,201, No. 62 / 652,210, and No. 62 / 652,204, all filed on April 3, 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] This application includes sequence listings, which have been electronically submitted in ASCII format and are incorporated herein by reference in their entirety. The ASCII copy was created on March 27, 2019, named 2019-03-27_01121-0035-00PCT_SL_ST25.txt, and has a size of 1,115,992 bytes. [Background technology]

[0003] Despite numerous successes in the field of vaccinology, new breakthroughs are needed to protect humans from many life-threatening infectious diseases. Many currently approved vaccines rely on decades-old technology to produce live attenuated or inactivated dead bacterial vaccines, which have inherent safety concerns and often only stimulate a very short-lived, weak immune response, requiring multiple doses. Advances in genetic and biochemical engineering have made it possible to develop therapeutics for challenging disease targets, but their applications in the field of vaccinology have not yet been fully realized.

[0004] Recombinant protein technology now enables the design of improved antigenic polypeptides. In addition, nanoparticles are increasingly demonstrating their potential for effective antigen presentation and targeted drug delivery. Ferritin particles have been shown to possess increased binding affinity, given by the multivalent presentation of their molecular cargo, and the ability to more efficiently pass through biological barriers due to their microscopic size. Helicobacter pylori (H. pylori) ferritin particles fused to the influenza virus hemagglutinin (HA) protein enabled improved antigen stability and increased immunogenicity in a mouse influenza model (see Non-Patent Literature 1). This fusion protein self-assembles into octahedral symmetric nanoparticles, presenting eight trimer HA spike structures, which, when used with adjuvants, elicit a potent immune response in various preclinical models. However, it was unclear whether these particles could be used as a suitable platform for non-HA influenza polypeptides that are not self-adjuvants and have lower immunogenicity than HA.

[0005] This specification presents a set of novel polypeptides, nanoparticles, compositions, methods, and uses involving ferritin. This specification describes a self-adjuvant platform in which an immunostimulatory moiety, such as an adjuvant, is conjugated to ferritin via surface-exposed amino acids or via a linker between ferritin and a non-ferritin polypeptide. Antigenic ferritin polypeptides were generated by combining a non-ferritin polypeptide with ferritin. Conjugating the immunostimulatory moiety with ferritin combined with a non-ferritin polypeptide allows for targeted and simultaneous delivery of the immunostimulatory moiety and the non-ferritin polypeptide as a single macromolecular entity, thereby significantly reducing the potential for systemic toxicity associated with conventional vaccines containing immunostimulatory moieties, such as antigens and adjuvants, as separate molecules. Simultaneous delivery of the immunostimulatory moiety together with the non-ferritin polypeptide as a macromolecular entity, and its polyvalent presentation on ferritin particles, also reduces the overall required vaccine dose, thereby lowering manufacturing burden and cost. Similarly, antigenic ferritin polypeptides, nanoparticles, and compositions for use in immunizing against respiratory syncytial virus (RSV), Epstein-Barr virus (EBV), influenza infection, and Lyme disease are also disclosed herein.

[0006] Furthermore, the polypeptides, nanoparticles, compositions, and uses disclosed herein enable the co-delivery of pathogen-derived non-ferritin polypeptides and targeted immune signals capable of inducing specific types of immune responses, in accordance with desired immunological outcomes against specific pathogens. One example is the induction of a Th1-type response by a TLR7 / 8 agonist conjugated to hemagglutinin (HA), which leads to the production of an IgG2a class-switched antibody known to be more effective in associating with FcγR and eliminating virus-infected cells via the ADCC mechanism (see Non-Patent Literature 2). Moreover, by co-delivering the ferritin-conjugated immunostimulatory moiety and the non-ferritin polypeptide as a single molecular entity, it is possible to ensure that the stimulation of immune cells occurs reliably in the presence of the non-ferritin polypeptide. In contrast, mixtures of the same immunostimulatory molecules without conjugation result in systemic distribution, generally require higher doses, and similarly carry the risk of undesirable effects due to indiscriminate immunostimulation in cells that have not come into contact with the antigen.

[0007] This specification also describes a platform for incorporating multiple polypeptides into ferritin particles by providing, for example, ferritin heavy and light chains containing first and second non-ferritin polypeptides. This platform is divalent and can provide a single polymer entity having other advantages related to ferritin therapeutics, such as the conjugation of an immunostimulant moiety described herein. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Kanekiyo et al., Nature 499: pp. 102-106 (2013) [Non-Patent Document 2] DiLillo et al., Nature Medicine 20: pp. 143-151 (2014) [Overview of the project] [Problems that the invention aims to solve]

[0009] The purpose of this disclosure is to provide compositions, kits, methods, and uses that can provide one or more of the above-described advantages, or at least provide the public with a useful selection. [Means for solving the problem]

[0010] Embodiment 1 is a ferritin protein that includes a mutation in which an amino acid exposed on the surface is replaced with cysteine.

[0011] Embodiment 2 is a ferritin protein containing a cysteine-containing N- or C-terminal linker.

[0012] Embodiment 3 is a ferritin protein comprising one or more immunostimulatory moieties linked to the ferritin protein via surface-exposed amino acids.

[0013] Embodiment 4 is a ferritin protein according to any one of Embodiments 1 to 3, which is an antigenic ferritin protein further comprising a non-ferritin polypeptide.

[0014] Embodiment 5 is an antigenic ferritin protein comprising (i) a mutation that replaces an amino acid exposed on the surface with cysteine ​​and an immunostimulatory moiety linked to cysteine; and (ii) a non-ferritin polypeptide.

[0015] Embodiment 6 is an antigenic ferritin protein comprising (i) cysteine ​​exposed on its surface, (ii) a peptide linker at the N-terminus of the ferritin protein, and (iii) a non-ferritin polypeptide at the N-terminus of the peptide linker.

[0016] Embodiment 7 is the ferritin protein according to any one of Embodiments 1 to 6, further including a mutation that replaces asparagine exposed on the surface with a non-asparagine amino acid.

[0017] Embodiment 8 is the ferritin protein according to any one of Embodiments 1 to 7, further including a mutation that replaces internal cysteine with a non-cysteine amino acid.

[0018] Embodiment 9 is the ferritin protein according to Embodiment 8, wherein the internal cysteine is at position 31 of H. pylori ferritin or at a position corresponding to position 31 of H. pylori ferritin determined by pairwise or structural alignment.

[0019] Embodiment 10 is an antigenic ferritin protein, comprising: a. A mutation that replaces an amino acid exposed on the surface with cysteine and an immunostimulatory moiety linked to cysteine; b. A mutation that replaces the internal cysteine at position 31 of H. pylori ferritin with a non-cysteine amino acid, or a mutation that replaces the internal cysteine at a position similar to position 31 of non-H. pylori ferritin determined by pairwise or structural alignment with a non-cysteine amino acid; c. A mutation that replaces asparagine exposed on the surface with a non-asparagine amino acid; and d. A non-ferritin polypeptide and is an antigenic ferritin protein.

[0020] Embodiment 11 is the ferritin protein according to any one of Embodiments 8 to 10, wherein the non-cysteine amino acid is serine.

[0021] Embodiment 12 is the ferritin protein according to any one of Embodiments 7 to 11, wherein asparagine is at position 19 of H. pylori ferritin or at a similar position of non-H. pylori ferritin determined by pairwise or structural alignment.

[0022] Embodiment 13 is a ferritin protein according to any one of Embodiments 1 to 12, wherein the ferritin comprises one or more E12C, S26C, S72C, A75C, K79C, S100C, and S111C mutations in H. pylori ferritin, or one or more corresponding mutations in non-H. pylori ferritin determined by pairwise or structural alignment.

[0023] Embodiment 13a is the ferritin protein according to Embodiment 13, wherein the ferritin includes the E12C mutation of H. pylori ferritin, or the corresponding mutation in non-H. pylori ferritin, determined by pairwise or structural alignment.

[0024] Embodiment 13b is the ferritin protein according to Embodiment 13, wherein the ferritin includes the S26C mutation in H. pylori ferritin, or the corresponding mutation in non-H. pylori ferritin, determined by pairwise or structural alignment.

[0025] Embodiment 13c is the ferritin protein according to Embodiment 13, wherein the ferritin includes the S72C mutation of H. pylori ferritin, or the corresponding mutation in non-H. pylori ferritin, determined by pairwise or structural alignment.

[0026] Embodiment 13d is the ferritin protein according to Embodiment 13, wherein the ferritin includes the A75C mutation in H. pylori ferritin, or the corresponding mutation in non-H. pylori ferritin, determined by pairwise or structural alignment.

[0027] Embodiment 13e is the ferritin protein according to Embodiment 13, wherein the ferritin includes the K79C mutation in H. pylori ferritin, or the corresponding mutation in non-H. pylori ferritin, determined by pairwise or structural alignment.

[0028] Embodiment 13f is the ferritin protein according to Embodiment 13, wherein the ferritin includes the S100C mutation in H. pylori ferritin, or the corresponding mutation in non-H. pylori ferritin, determined by pairwise or structural alignment.

[0029] Embodiment 13g is the ferritin protein according to Embodiment 13, wherein the ferritin contains the S111C mutation of H. pylori ferritin, or the corresponding mutation in non-H. pylori ferritin, determined by pairwise or structural alignment.

[0030] Embodiment 14 is a ferritin protein according to any one of Embodiments 4 to 13 g, wherein the non-ferritin polypeptide is a polypeptide derived from influenza, Epstein-Barr virus, respiratory syncytial virus (RSV), or Borrelia.

[0031] Embodiment 14a is a ferritin protein according to any one of Embodiments 4 to 13 g, wherein the non-ferritin polypeptide comprises an influenza-derived polypeptide, and optionally the polypeptide comprises a hemagglutinin polypeptide.

[0032] Embodiment 14b is a ferritin protein according to any one of Embodiments 4 to 13 g, wherein the non-ferritin polypeptide comprises a polypeptide derived from Epstein-Barr virus, and optionally the polypeptide comprises one or more of the following polypeptides: gL, gH, gL / gH, gp220, or gp42.

[0033] Embodiment 14c is a ferritin protein according to any one of Embodiments 4 to 13g, wherein the non-ferritin polypeptide comprises a polypeptide derived from respiratory syncytial virus, and optionally the polypeptide comprises an RSV F or RSV G polypeptide.

[0034] Embodiment 14d is a ferritin protein according to any one of Embodiments 4 to 13g, wherein the non-ferritin polypeptide comprises a polypeptide derived from Borrelia, and optionally the polypeptide comprises an OspA polypeptide.

[0035] Embodiment 15 is a ferritin protein according to Embodiment 14 or 14c, wherein the non-ferritin polypeptide comprises an RSV G polypeptide, and optionally the RSV G polypeptide comprises a G polypeptide central conservation region.

[0036] Embodiment 15a is the ferritin protein according to Embodiment 15, wherein the RSV G polypeptide is not glycosylated.

[0037] Embodiment 15b is a ferritin protein according to Embodiment 15 or 15a, wherein an RSV G polypeptide is chemically conjugated to the ferritin protein.

[0038] Embodiment 16 is a ferritin protein according to any one of Embodiments 4-5 or 7-15b, further comprising a peptide linker between ferritin and a non-ferritin polypeptide.

[0039] Embodiment 17 is a ferritin protein according to any one of Embodiments 1 to 16, comprising an immunostimulatory moiety that is linked to cysteine ​​and is capable of hydrogen bonding or ionic bonding.

[0040] Embodiment 18 is a ferritin protein according to any one of Embodiments 1 to 17, comprising an immunostimulatory moiety that is an agonist of TLR2, TLR7 / 8, TLR9, or STING.

[0041] Embodiment 18a is a ferritin protein according to any one of Embodiments 1 to 17, comprising an immunostimulatory moiety that is an agonist of TLR2, wherein the agonist is optionally PAM2CSK4, FSL-1, or PAM3CSK4.

[0042] Embodiment 18b is a ferritin protein according to any one of Embodiments 1 to 18a, comprising an immunostimulatory moiety that is an agonist of TLR7 / 8, wherein the agonist is optionally single-stranded RNA, imidazoquinoline, a nucleoside analog, 3M-012, or SM7 / 8a.

[0043] Embodiment 18c is a ferritin protein according to any one of Embodiments 1 to 18b, comprising an immunostimulatory moiety that is an agonist of TLR9, wherein the agonist is optionally a CpH oligodeoxynucleotide (ODN), an ODN containing one or more hexameric CpG motifs including 5'purine (Pu)-pyrimidine (Py)-CG-Py-Pu3', an ODN containing the sequence of SEQ ID NO: 210, or ISS-1018.

[0044] Embodiment 18d is the ferritin protein described in Embodiment 18c, wherein the TLR9 agonist comprises a skeleton including a phosphorothioate bond.

[0045] Embodiment 18e is a ferritin protein according to any one of Embodiments 1 to 18d, comprising an immunostimulatory moiety that is an agonist of STING, wherein the agonist is optionally a cyclic dinucleotide (CDN), cdA, cdG, cAMP-cGMP, and 2'-5',3'-5'cGAMP, or DMXAA.

[0046] Embodiment 19 is one of sequence numbers 201-207 or 211-215 and 9 The ferritin protein according to any one of Embodiments 1 to 18e comprises an amino acid sequence having 0%, 95%, 98%, or 99% identity.

[0047] Embodiment 19a is represented by Sequence ID No. 201 and 9 The ferritin protein according to any one of Embodiments 1 to 19 comprises an amino acid sequence having 0%, 95%, 98%, or 99% identity.

[0048] Embodiment 19b is shown in Sequence ID No. 202 and 9 The ferritin protein according to any one of Embodiments 19a comprises an amino acid sequence having 0%, 95%, 98%, or 99% identity.

[0049] Embodiment 19c is represented by Sequence ID No. 203 and 9 A ferritinta according to any one of Embodiments 1 to 19b, comprising an amino acid sequence having 0%, 95%, 98%, or 99% identity. It is protein.

[0050] Embodiment 19d corresponds to sequence numbers 201-207 or 211-215 and 9 The ferritin protein according to any one of Embodiments 1 to 19c comprises an amino acid sequence having 0%, 95%, 98%, or 99% identity.

[0051] Embodiment 19e is represented by Sequence ID No. 204 and 9 The ferritin protein according to any one of Embodiments 1 to 19d comprises an amino acid sequence having 0%, 95%, 98%, or 99% identity.

[0052] Embodiment 19f is sequence number 205 and 9 The ferritin protein according to any one of Embodiments 1 to 19e comprises an amino acid sequence having 0%, 95%, 98%, or 99% identity.

[0053] Embodiment 19g is represented by Sequence ID No. 206 and 9 The ferritin protein according to any one of Embodiments 1 to 19f comprises an amino acid sequence having 0%, 95%, 98%, or 99% identity.

[0054] Embodiment 19h is represented by Sequence ID No. 207 and 9 The ferritin protein is described in any one of Embodiments 1 to 19 g, and contains an amino acid sequence having 0%, 95%, 98%, or 99% identity.

[0055] Embodiment 19i is represented by Sequence ID No. 211 and 9 The ferritin protein according to any one of Embodiments 1 to 19h comprises an amino acid sequence having 0%, 95%, 98%, or 99% identity.

[0056] Embodiment 19j is represented by Sequence ID No. 212 and 9 The ferritin protein according to any one of Embodiments 1 to 19i comprises an amino acid sequence having 0%, 95%, 98%, or 99% identity.

[0057] Embodiment 19k is represented by Sequence ID No. 213 and 9 The ferritin protein according to any one of Embodiments 1 to 19j comprises an amino acid sequence having 0%, 95%, 98%, or 99% identity.

[0058] Embodiment 19l is represented by Sequence ID No. 214 and 9 The ferritin protein according to any one of Embodiments 1 to 19k comprises an amino acid sequence having 0%, 95%, 98%, or 99% identity.

[0059] Embodiment 19m is represented by Sequence ID No. 215 and 9 The ferritin protein according to any one of Embodiments 1 to 19l comprises an amino acid sequence having 0%, 95%, 98%, or 99% identity.

[0060] Embodiment 20 is a ferritin particle containing the ferritin protein described in any one of Embodiments 1 to 19m.

[0061] Embodiment 21 is a composition comprising a ferritin protein or ferritin particles as described in any one of Embodiments 1 to 20, and a pharmaceutically acceptable carrier.

[0062] Embodiment 22 is a composition comprising a first ferritin protein and a second ferritin protein, wherein the first ferritin protein comprises a ferritin heavy chain and a first non-ferritin polypeptide, the second ferritin protein comprises a ferritin light chain and a second non-ferritin polypeptide, and the first and second non-ferritin polypeptides are different, and optionally the ferritin particles comprise the first ferritin protein and the second ferritin protein.

[0063] Embodiment 23 is the composition according to Embodiment 21 or 22, further comprising an adjuvant.

[0064] Embodiment 24 is a ferritin protein, ferritin particles, or composition according to any one of Embodiments 4 to 23, for use in the target vaccine.

[0065] Embodiment 25 is a method of vaccinating a subject, comprising administering to the subject a ferritin protein, ferritin particles, or composition described in any one of Embodiments 4 to 23.

[0066] Embodiment 26 is a ferritin protein, ferritin particles, or composition described in Embodiment 24, or a method described in Embodiment 25, wherein the subject is a human.

[0067] Embodiment 26a is a ferritin protein, ferritin particles, or composition described in Embodiment 24, or the method described in Embodiment 25, wherein the subject is a mammal, and the mammal may be a primate or a domestic mammal, and the primate may be a non-human primate, monkey, macaque, rhesus macaque, crab-eating macaque, or ape, or the domestic mammal may be a dog, rabbit, cat, horse, sheep, cow, goat, camel, or donkey.

[0068] Embodiment 27 is a nucleic acid encoding a ferritin protein according to any one of Embodiments 1 to 26a, wherein the nucleic acid is optionally mRNA.

[0069] Embodiment F1 is an antigenic influenza-ferritin polypeptide comprising (i) a ferritin protein having a mutation that replaces an amino acid exposed on the surface with cysteine, and (ii) an influenza polypeptide.

[0070] Embodiment F2 is an antigenic influenza-ferritin polypeptide comprising (i) a ferritin protein having a mutation that replaces surface-exposed amino acids with cysteine ​​and an immunostimulatory moiety conjugated to cysteine; and (ii) an influenza polypeptide.

[0071] Embodiment F3 is the antigenic influenza-ferritin polypeptide according to Embodiment F1, further comprising an immunostimulatory moiety conjugated to a ferritin protein via cysteine.

[0072] Embodiment F4 is an antigenic influenza-ferritin polypeptide according to any one of Embodiments F1 to F3, wherein the influenza polypeptide comprises a hemagglutinin (HA) or neuraminidase (NA) polypeptide.

[0073] Embodiment F5 is the antigenic influenza-ferritin polypeptide described in Embodiment F4, wherein the HA polypeptide includes a conserved region.

[0074] Embodiment F6 is the antigenic influenza-ferritin polypeptide according to Embodiment F5, wherein the conserved region comprises all or part of the stem region of HA.

[0075] Embodiment F7 is an antigenic influenza-ferritin polypeptide according to any one of Embodiments F1 to F6, wherein the influenza antigen comprises an HA antigen containing the Y98F mutation.

[0076] Embodiment F8 is an antigenic influenza-ferritin polypeptide according to any one of Embodiments F1 to F7, further comprising a mutation that replaces an internal cysteine ​​with a non-cysteine ​​amino acid.

[0077] Embodiment F9 is an antigenic influenza-ferritin polypeptide according to Embodiment F8, wherein the internal cysteine ​​is located at position 31 of H. pylori ferritin, or at a position corresponding to position 31 of H. pylori ferritin, as determined by pairwise or structural alignment, and the internal cysteine ​​is optionally mutated to serine.

[0078] Embodiment F10 is an antigenic influenza-ferritin polypeptide according to any one of Embodiments F1 to F9, further comprising a mutation that replaces surface-exposed asparagine with a non-asparagine amino acid, wherein the non-asparagine amino acid is glutamine.

[0079] Embodiment F11 is an antigenic influenza-ferritin polypeptide according to any one of Embodiments F1 to F10, wherein the amino acids exposed on the surface are mutations of E12, S26, S72, A75, K79, S100, or S111 in H. pylori ferritin, or similar amino acids in non-H. pylori ferritin.

[0080] Embodiment F12 is an antigenic influenza-ferritin polypeptide according to Embodiment F11, wherein the amino acid mutations exposed on the surface are E12C, S26C, S72C, A75C, K79C, S100C, or S111C of H. pylori ferritin, or similar amino acids in non-H. pylori ferritin, determined by pairwise or structural alignment.

[0081] Embodiment F13 is an antigenic influenza-ferritin polypeptide according to any one of Embodiments F1 to F12, wherein the immunostimulatory portion is an agonist of TLR7 or TLR8.

[0082] Embodiment F14 is an antigenic influenza-ferritin polypeptide according to any one of Embodiments F1 to F13, wherein the immunostimulatory portion is a TLR9 agonist.

[0083] Embodiment F15 is an antigenic influenza-ferritin polypeptide according to any one of Embodiments F1 or F3-F14, further comprising a linker between the immunostimulatory portion and the ferritin protein.

[0084] Embodiment F16 is the antigenic influenza-ferritin polypeptide described in Embodiment F15, wherein the linker comprises one, two, or three maleimide moieties, polyethylene glycol (PEG) moieties, and dibenzocyclooctin (DBCO) moieties.

[0085] Embodiment F17 is an antigenic influenza-ferritin polypeptide according to any one of Embodiments F1 to F16, further comprising a peptide linker between the ferritin protein and the influenza polypeptide.

[0086] Embodiment F18 is a ferritin particle containing the antigenic influenza-ferritin polypeptide described in any one of Embodiments F1 to F17.

[0087] Embodiment F19 is a composition comprising an antigenic influenza-ferritin polypeptide or ferritin particles described in any one of Embodiments F1 to F18, and a pharmaceutically acceptable carrier.

[0088] Embodiment F20 is the composition of Embodiment F19, further comprising a ferritin protein and a second antigenic influenza-ferritin polypeptide containing a different influenza polypeptide.

[0089] Embodiment F21 is the composition according to Embodiment F20, wherein the influenza polypeptide is derived from influenza A, the influenza polypeptide of the second antigenic influenza-ferritin polypeptide is derived from influenza B, or the influenza polypeptide and the influenza polypeptide of the second influenza-ferritin polypeptide are derived from subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, or H18, or one or both of the influenza polypeptides are an engineered stabilized stem antigen derived from subtypes H1, H3, H7, or H10.

[0090] Embodiment F22 is an antigenic influenza-ferritin polypeptide, ferritin particles, or composition according to any one of Embodiments F1 to F21 for use in a method of inducing an immune response to influenza or in protecting an influenza-infected subject.

[0091] Embodiment F23 is a method for inducing an immune response to influenza or protecting a subject from influenza infection, comprising the step of administering one or more antigenic influenza-ferritin polypeptides, ferritin particles, or compositions described in any one of Embodiments F1 to F22 to the subject.

[0092] Embodiment F24 is an antigenic influenza-ferritin polypeptide, ferritin particles, composition, or method according to any one of Embodiments F1 to F23, wherein the subject is a human.

[0093] Embodiment F25 is a nucleic acid encoding an antigenic influenza-ferritin polypeptide according to any one of Embodiments F1 to F17, wherein the nucleic acid is optionally mRNA.

[0094] Additional objectives and benefits are partially described in the following description, partially evident from the description, or can be learned through practice. These objectives and benefits are realized and achieved by the elements and combinations specifically indicated in the attached claims.

[0095] The general description above and the detailed description below are for illustrative purposes only and do not limit the scope of the claims.

[0096] The following drawings, incorporated herein and constituting part of this specification, illustrate several embodiments and are useful in illustrating the principles described herein together with the description. [Brief explanation of the drawing]

[0097] [Figure 1A] This figure shows an exemplary design of OspA-ferritin nanoparticles. Figure 1A. OspA genetically fused to ferritin forms a fusion protein. The OspA and ferritin sequences are separated by a glycine-serine linker (-GS-). [Figure 1B] This figure shows an exemplary design of OspA-ferritin nanoparticles. Figure 1B shows the structure of the ectodomain of OspA. The C-terminus of OspA bound to ferritin is indicated by an asterisk. [Figure 1C] This figure shows an exemplary design of OspA-ferritin nanoparticles. Figure 1: Exemplary ferritin nanoparticles composed of 24 monomers of C.H. pylori ferritin. [Figure 1D] This figure shows an exemplary design of OspA-ferritin nanoparticles. Figure 1D. Exemplary OspA-ferritin fusion protein nanoparticles. Ferritin (light gray), glycine-serine linker (GS) position, and OspA (dark gray and black) are shown (n: number of subunits). [Figure 2-1]Figures 2A-2D illustrate the expression and purification of exemplary OspA-ferritin. Figure 2A. Size exclusion chromatography (SEC) profile of exemplary OspA-ferritin nanoparticles purified on a Superose 6 column. Figure 2B. SDS-PAGE gel of purified exemplary OspA-ferritin from Expi293 cells. Figure 2C. Dynamic light scattering (DLS) profile of exemplary OspA-ferritin nanoparticles. The radius is 13 nm, the Pd% (standardized polydispersity measure) is 7.4, and the mass is 100%. Figure 2D. Composite image of exemplary OspA-ferritin composed of class mean transmission electron micrographs of 318 particles at 67,000x magnification. Ferritin nanoparticles appear on the transmission electron microscope as a strong circular density with a hollow center. Each nanoparticle is surrounded by numerous short shapes corresponding to OspA, which appear annular or slightly oval. [Figure 2-2] Continuation of Figure 2-1. [Figure 2-3] Continuation of Figure 2-2. [Figure 3A] This figure shows the generation of alternative serotype OspA nanoparticles in Escherichia coli. Figure 3A. Biochemical analysis of OspA-ferritin serotypes 1-5 and 7 purified by size exclusion chromatography by SDS-PAGE. [Figure 3B] This figure shows the generation of surrogate serotype OspA nanoparticles in Escherichia coli. Figure 3B. Transmission electron microscopy of OspA-ferritin serotypes 1-5 and 7 (98,000x magnification). [Figure 4]This figure compares the immunogenicity and duration of exemplary serotype 1 OspA-ferritin nanoparticles with RECOMBITEK® Lime (a liquid suspension of purified outer surface protein A (OspA) from Borrelia burgdorferi). C3H mice (n=5) were intramuscularly immunized with 1 μg of OspA-ferritin + Ribi adjuvant (Sigma adjuvant system, catalog no. S6322-1vl) or RECOMBITEK® Lime at weeks 0 and 4. Antibody responses were evaluated by measuring endpoint titers via ELISA 2 weeks after the second immunization (week 6) and 21 weeks after the second immunization (week 25) using each composition. [Figure 5-1]Figures 5A–5C are diagrams providing information on exemplary OspA-ferritin. The OspA polypeptide is modified with an OspA serotype 1 epitope that has homology to a fragment of the human leukocyte function-associated antigen-1 (hLFA-1) sequence. Figure 5A. Structure showing the location of the LFA-1 homologous site (amino acids 165–173 of SEQ ID NO: 83) within the OspA ectodomain. Figure 5B. Dendrodia showing the relationship between OspA amino acids 165–173 of SEQ ID NO: 83 (OspA of B. brudolferi serotype 1) and the corresponding sequences of hLFA-1 and other Borrelia species and serotypes. Figure 5C compares the 9-amino acid segment (nonapeptide) at amino acids 165-173 of SEQ ID NO: 83 (labeled "OspA") with the corresponding nonapeptides from serotype 2 and serotype 3 OspA ("S2" (SEQ ID NO: 79) and "S3" (SEQ ID NO: 80), respectively), a rationally designed substituted nonapeptide ("RD2" (SEQ ID NO: 81)), and the corresponding nonapeptide from hLFA-1 (SEQ ID NO: 78). Figure 5C discloses SEQ ID NOs: 77, 79-81, and 78, respectively, in order of appearance. Figure 5D shows C3H mice (n=5) immunized intramuscularly (IM) at weeks 0 and 4 with 1 μg dose of OspA serotype 1-ferritin nanoparticles using AddaVax® adjuvant (squalene-based oil-in-water nanoemulsion, InvivoGen, available from catalog no. vac-adx-10). The OspA sequences contained either a wild-type hLFA-1 homologous site (i.e., amino acids 165–173 of SEQ ID NO: 83, "Sero1") or substitution sequences such as SEQ ID NO: 81 ("RD"), SEQ ID NO: 80 ("Sero 3 substitution"), and SEQ ID NO: 79 ("Sero 2 substitution"). Antibody response was assessed via endpoint titers measured by ELISA two weeks after a second immunization with the indicated construct. [Figure 5-2] Continuation of Figure 5-1. [Figure 5-3] Continuation of Figure 5-2. [Figure 6A]Figure 6A presents information on exemplary immunostimulatory moieties: exemplary OspA-ferritin nanoparticles conjugated to a TLR7 / 8 agonist (3M-012). A two-step click chemistry strategy was used to conjugate 3M-012 to ferritin. A DBCO-PEG4-maleimide linker was first attached to surface-exposed cysteine ​​on ferritin. After removing excess linker, azide-3M-012 was added. [Figure 6B] Exemplary immunostimulatory portion: This figure presents information on exemplary OspA-ferritin nanoparticles conjugated to the TLR7 / 8 agonist (3M-012). Figure 6B. C3H mice (n=5) were immunized intramuscularly with 1 μg of the composition shown at weeks 0 and 4 and analyzed after 2 weeks. "Conjugate" indicates OspA-ferritin-3M-012 conjugated nanoparticles. "Mixed" indicates unconjugated mixtures of the same OspA-ferritin administered with 29 ng or 20 μg of 3M-012 or alum. The 29 ng "mixed" mixture of OspA-ferritin and 3M-012 represents the molar equivalent of 3M-012 on the conjugated nanoparticles. [Figure 7-1]Figures 7A–7C provide information on exemplary OspA-ferritin nanoparticles conjugated to an exemplary immunostimulatory moiety:ISS-1018 CpG (SEQ ID NO: 210). Figure 7A. CPG was conjugated to ferritin using a two-step click chemistry strategy. A DBCO-PEG4-maleimide linker was initially attached to surface-exposed cysteine ​​on ferritin. After removing excess linker, azide-CpG was added. Figure 7A discloses SEQ ID NO: 534. Figure 7B. Biochemical analysis of CpG conjugation by SDS-PAGE gel reveals a molecular weight shift after conjugation to CpG, with 92% of OspA-ferritin conjugated to CpG being present. Figure 7C. C3H mice (n=5) were intramuscularly immunized with 1 μg of the indicated composition at weeks 0 and 4 and analyzed at week 2. "Conjugate" refers to OspA-ferritin-CPG conjugate nanoparticles. "Mixed" refers to a non-conjugate mixture of the same OspA-ferritin administered with 339 ng or 50 μg of CpG or alum. The 339 ng "mixed" mixture of OspA-ferritin and CPG represents the molar equivalent of CpG on the conjugate nanoparticles. [Figure 7-2] Continuation of Figure 7-1. [Figure 8-1] Figures 8A-8F compare the antibody responses to serotype 1 (Figure 8A), serotype 2 (Figure 8B), serotype 3 (Figure 8C), serotype 4 (Figure 8D), serotype 5 (Figure 8E), and serotype 7 (Figure 8F) after administration of a hexavalent composition containing monovalent serotype-matched OspA-ferritin (1 μg / dose) with alum adjuvant ("monovalent"), or serotype 1, serotype 2, serotype 3, serotype 4, serotype 5, and serotype 7 OspA-ferritin (1 μg / dose each) together with alum adjuvant ("hexavalent"). Antibody response was assessed via endpoint titers measured by ELISA after intramuscular immunization of C3H mice (n=5) at week 0 and week 4, and 2 weeks later. ELISA plates were coated with specific serotypes of OspA. [Figure 8-2] Continuation of Figure 8-1. [Figure 8-3] Continuation of Figure 8-2. [Figure 9-1] Figures 9A-9G show the antibody responses in mice to serotype 1 (Figure 9A), serotype 2 (Figure 9B), serotype 3 (Figure 9C), serotype 4 (Figure 9D), serotype 5 (Figure 9E), serotype 6 (Figure 9F), and serotype 7 (Figure 9G) observed after administration of conjugated and unconjugated hexavalent OspA-ferritin nanoparticle compositions. Except for "Hexavalent-CPG" and "Hexavalent-3M-012" indicating that nanoparticles are chemically conjugated to CPG and 3M-012, the hexavalent composition contains OspA-ferritin of serotype 1, serotype 2, serotype 3, serotype 4, serotype 5, and serotype 7, respectively, as shown in Figures 8A–F (see Figures 7A and 6A, and the attached description). Antibody response was evaluated via endpoint titers measured by ELISA after 2 weeks. ELISA plates were coated with specific serotypes of OspA. [Figure 9-2] Continuation of Figure 9-1. [Figure 9-3] Continuation of Figure 9-2. [Figure 9-4] Continuation of Figure 9-3. [Figure 10-1]Figures 10A-10G show the antibody response to hexavalent OspA-ferritin nanoparticle composition against serotypes 1-7 in rhesus monkeys (n=3 monkeys / group), respectively. This is the same as Figures 9A-G, except that the dose was 60 μg total (10 μg for each serotype) and contained an unconjugated AF03 adjuvant. Monkeys were immunized intramuscularly at weeks 0 and 6. Antibody response was analyzed via endpoint titers measured by ELISA at 2 weeks post-immunization. RECOMBITEK® lime was used as a control at a dose of 10 μg. For all experiments, ELISA plates were coated with the OspA serotypes shown in each panel. Figures 10H-10N show the antibody response to hexavalent OspA-ferritin nanoparticle composition against serotypes 1-7 in rhesus monkeys (n=3 monkeys / group), respectively. This was the same as described in Figures 10A–10G, except that the AF03 adjuvant was not used, and instead nanoparticles were conjugated to 3M-012 or CpG (see Figures 6A and 7A, and the attached notes). The dose was 60 μg total (10 μg for each serotype). Monkeys were immunized intramuscularly at weeks 0 and 6. Antibody response was analyzed at 2 weeks post-immunization via endpoint titers measured by ELISA. For all experiments, ELISA plates were coated with the OspA serotypes shown in each panel. [Figure 10-2] Continuation of Figure 10-1. [Figure 10-3] Continuation of Figure 10-2. [Figure 10-4] Continuation of Figure 10-3. [Figure 10-5] Continuation of Figure 10-4. [Figure 11]This figure shows the results of a tick loading test of the 3M-012-conjugated OspA-ferritin composition. Mice were immunized with a 1 μg dose of the composition shown at weeks 0 and 4. The monovalent composition contained 1 μg of OspA-ferritin serotype 1 conjugated to 3M-012. The "hexavalent-3M-012" composition was as described in Figures 9A-G. Control particles lacked the OspA polypeptide. Mice were loaded with 5-6 ticks infected with Borrelia burgdorferi N40 strain (serotype 1) for 5 days at 2 weeks after the second immunization, and sacrificed at 2 weeks. Tissue samples from the heart, ankle, and ear were cultured for 6 weeks in BSK medium supplemented with antibiotics against B. burgdorferi. Negative samples were tested for the presence of B. burgdorferi by PCR. Positive samples were positive by either culture or PCR. [Figure 12] This figure shows confirmation of the conjugation of OspA-ferritin nanoparticles to the TLR7 / 8 agonist 3M-012 by mass spectrometry. The upper panel shows the unconjugated construct, and the lower panel shows the conjugated construct. The data showed a mass shift of 586.69 daltons, consistent with the addition of 3M-012. [Figure 13] This figure shows the antibody response in mice to the non-glycosylated mutant OspA-ferritin (NG-RD) compared to the glycosylated counterpart (RD) as measured by ELISA across the indicated dilution series. RD = SEQ ID NO: 52. NG-RD = SEQ ID NO: 53. Mice were vaccinated with a 1 μg dose at weeks 0 and 4. [Figure 14] This figure shows the antibody response in mice to OspA-ferritin (SEQ ID NO: 52). The OspA-ferritin glycosylation mutant N>Q (SEQ ID NO: 53) and the glycosylation mutant S / T>A (SEQ ID NO: 63) were compared to RECOMBITEK® lime controls and negative (pre-immuno) controls, as measured by ELISA across the indicated dilution series. [Figure 15-1]Figures 15A–15E show the purification and characterization of OspA constructs containing different linkers (GS, Gly-Ser linker, GS1, Gly-Gly-Gly-Ser linker (SEQ ID NO: 443), GS2, SEQ ID NO: 91 linker, GS5, SEQ ID NO: 92 linker, construct sequences are SEQ ID NOs. 53 and 60–62, respectively). Figure 15A. Coomassy staining of purified OspA constructs containing the indicated linkers. Figure 15A discloses SEQ ID NOs. 443–445, respectively, in order of appearance. Figure 15B. Dynamic light scattering (DLS) of OspA-ferritin nanoparticles containing GS1 (SEQ ID NO: 60). Figure 15C. DLS of OspA-ferritin nanoparticles containing GS2 (SEQ ID NO: 61). Figure 15D. Electron micrograph (EM) of OspA-ferritin nanoparticles containing GS5 (SEQ ID NO: 62). Figure 15E. DLS of OspA-ferritin nanoparticles containing GS5 (SEQ ID NO: 62). [Figure 15-2] Continuation of Figure 15-1. [Figure 15-3] Continuation of Figure 15-2. [Figure 16] This figure shows the antibody response of mice to OspA-ferritin constructs containing different linkers (linker 1×GGGS construct, SEQ ID NO: 60 (disclosed as SEQ ID NO: 443, "1×GGGS"), linker 2×GGGS construct, SEQ ID NO: 61 (disclosed as SEQ ID NO: 444, "2×GGGS"), linker 5×GGGS construct, SEQ ID NO: 62 (disclosed as SEQ ID NO: 445, "5×GGGS")) compared to RECOMBITEK® lime and negative (pre-immuno) controls, measured by ELISA across the indicated dilution series. [Figure 17-1] Figures 17A-17C illustrate the characterization of the lumazine synthase OspA serotype 4 construct (SEQ ID NO: 18). Figure 17A: DLS data. Figure 17B: Coomasiegels of fractions 22-64 showing size exclusion chromatography (SEC) traces. Figure 17C: EM data. [Figure 17-2] Continuation of Figure 17-1. [Figure 18]This figure shows the antibody response in mice to the OspA serotype 4-ferritin construct (SEQ ID NO: 4) and the OspA serotype 4-lumazine synthase construct (SEQ ID NO: 18), with or without alum. [Figure 19-1] Figures 19A-19C illustrate the characterization of the OspA serotype 1-lumazine synthase construct (SEQ ID NO: 12). Figure 19A: EM data. Figure 19B: Kouma Siegels of fractions 20-40 showing SEC traces. Figure 19C: DLS data. [Figure 19-2] Continuation of Figure 19-1. [Figure 20-1] Figures 20A-20C show the characterization of the OspA serotype 2-lumazine synthase construct (SEQ ID NO: 16). Figure 20A: EM data. Figure 20B: Coomasiegels of fractions 27-56 showing SEC traces. Figure 20C: DLS data. [Figure 20-2] Continuation of Figure 20-1. [Figure 21A] This figure shows the characterization of the OspA serotype 3-lumazine synthase construct (SEQ ID NO: 17). Figure 21A. Coomasiegels of fractions 23-39 showing SEC traces. [Figure 21B] This figure shows the characterization of the OspA serotype 3-lumazine synthase construct (SEQ ID NO: 17). Figure 21B. DLS data. [Figure 22A] This figure shows the characterization of the OspA serotype 5-lumazine synthase construct (SEQ ID NO: 19). Figure 22A. EM data. [Figure 22B] This figure shows the characterization of the OspA serotype 5-lumazine synthase construct (SEQ ID NO: 19). Figure 22B. Coomasiegels of fractions 22-38 showing SEC traces. [Figure 22C] This figure shows the characterization of the OspA serotype 5-lumazine synthase construct (SEQ ID NO: 19). Figure 22C. DLS data. [Figure 23A] Characterization of the OspA serotype 7-lumazine synthase construct (SEQ ID NO: 21) is shown. Figure 23A. EM data. [Figure 23B]Characterization of the OspA serotype 7-lumazine synthase construct (SEQ ID NO: 21) is shown. Figure 23B. Coomasiegels of fractions 20-38 showing SEC traces. [Figure 23C] Characterization of the OspA serotype 7-lumazine synthase construct (SEQ ID NO: 21) is shown. Figure 23C. DLS data. [Figure 24-1] Figures 24A-24G show the antibody responses to serotypes 1-7 in C3H mice (n=5 mice / group) to a heptavalent OspA-ferritin nanoparticle composition of 1 μg each of OspA-ferritin nanoparticles corresponding to OspA serotypes 1-7 (7 μg in total), adjuvanted with either alum or AF03, or to RECOMBITEK® lime, respectively. In all experiments, ELISA plates were coated with the OspA serotype indicated as "SX" (where X is the serotype number) on each panel. [Figure 24-2] Continuation of Figure 24-1. [Figure 24-3] Continuation of Figure 24-2. [Figure 24-4] Continuation of Figure 24-3. [Figure 25] This figure shows the time course of endpoint antibody titers in rhesus monkeys. At weeks 0 and 6, monkeys were intramuscularly inoculated with a hexavalent OspA-ferritin vaccine (containing OspA serotypes 1, 2, 3, 4, 5, and 7 in separate nanoparticles) using AF03 adjuvant or RECOMBITEK®. ELISA plates were coated with OspA serotype 1. [Figure 26-1]Figures 26A-26B show sequence comparisons and homologies of representative H1N1 influenza virus strains. (Figure 26A) Sequence alignment of candidate antigenic HA polypeptides using vector NTI AlignX software. Black on white: Consensus residues derived from residues that are completely conserved at the given position. White on black: Consensus residues derived from the occurrence of one residue more than 50% at the given position. Black underlined and bold on white: Residues that are weakly similar to the consensus residue at the given position. Double underlined and italicized black on white: Consensus residues derived from blocks of similar residues at the given position. Bold on white: Dissimilar residues. The sequences shown are the HA portions + serine linkers at the C-terminus of the following sequences: CA09 HA-Np = residues 1-519 of SEQ ID NO: 315. COBRA P1 HA-Np = residues 1-519 of SEQ ID NO: 327. COBRA X6 HA-Np = residues 1-518 of SEQ ID NO: 329. NC99 HA-Np = residues 1-518 of SEQ ID NO: 301. HK77 HA-Np = residues 1-519 of SEQ ID NO: 318. FM47 HA-Np = residues 1-519 of SEQ ID NO: 317. DV57 HA-Np = residues 1-518 of SEQ ID NO: 321. MAL54 HA-Np = residues 1-519 of SEQ ID NO: 316. (Figure 26B) Dendromes constructed using the neighbor-joining method (Vector NTI) for the listed HA protein sequences from influenza strains. Arrows indicate strains selected as candidates for evaluation by generating HA-ferritin nanoparticles from these sequences and testing their immunogenicity in mice. [Figure 26-2] Continuation of Figure 26-1. [Figure 26-3] Continuation of Figure 26-2. [Figure 27] This figure shows manipulated surface-exposed cysteine ​​(Cys) on ferritin. The positions of the cysteine ​​resulting from mutations that replace surface-exposed amino acids are shown in relation to ferritin nanoparticles. [Figure 28A]This figure shows the conjugation of Toll-like receptor (TLR) agonists to ferritin. (Figure 28A) The results of conjugation of SM7 / 8a small molecules to cysteine, resulting from mutations that replace surface-exposed amino acids of ferritin via a PEG4 linker with a maleimide-reactive group, are shown in association with ferritin nanoparticles. [Figure 28B] This figure shows the conjugation of Toll-like receptor (TLR) agonists to ferritin. (Figure 28B) The results of conjugation of CpG (SEQ ID NO: 535) to cysteine ​​resulting from mutations that replace surface-exposed amino acids of ferritin are shown in association with ferritin nanoparticles using a maleimide-DBCO bifunctional linker and azide-functionalized CpG reagents, using two-step click chemistry. [Figure 29A] This figure shows the conjugation of Toll-like receptor (TLR) agonists to ferritin. (Figure 29A) A model of ferritin nanoparticles containing HA polypeptide and 3M-012 conjugated to surface-exposed cysteine ​​of ferritin using a two-step click chemistry reaction via a maleimide-DBCO bifunctional linker and an azide-functionalized 3M-012 reagent. [Figure 29B] This figure shows the conjugation of a Toll-like receptor (TLR) agonist to ferritin. (Figure 29B) A model of ferritin nanoparticles containing HA polypeptide and CpG conjugated to surface-exposed cysteine ​​of ferritin using a two-step click chemistry reaction with a maleimide-DBCO bifunctional linker and an azide-functionalized CpG reagent. Figure 29B discloses Sequence ID No. 534. [Figure 30]This figure shows the trypsin cleavage sites within the ferritin moiety of a specific ferritin nanoparticle construct. Some ferritin nanoparticles contain trypsin cleavage sites in addition to unpaired surface-exposed cysteine ​​for adjuvant conjugation. The amino acid sequence shown in Figure 30 is a "generalized" sequence that shows sequences common to multiple constructs. For example, residues 519-694 of SEQ ID NO: 314 contain the "generalized" sequence shown in Figure 30. The "XXX" sequence represents the influenza polypeptide. The location of the ferritin S111C mutation present in this sequence is also shown. [Figure 31] Figures 31A and 31B show the gel shift and mass spectrometry (MS) results with and without conjugation. (Figure 31A) Gel shift and mass spectrometry results of H1 / Stem-Np (SEQ ID NO: 343) with or without conjugation to maleimide-PEG4-SM7 / 8a. (Figure 31B) Gel shift and mass spectrometry results of H5 / hCobra2-Np (SEQ ID NO: 332) with or without conjugation to maleimide-PEG4-SM7 / 8a. [Figure 32A] This figure shows the results of further gel shifts with and without conjugation. (Figure 32A) Gel shift results of H1 / Stem-Np after peptide N-glycosidase (PNGase) treatment with or without conjugation to maleimide-PEG4-SM7 / 8a, or results from two-step click chemistry using maleimide-PEG4-DBCO and azide-CpG. [Figure 32B] This figure shows the results of further gel shifts with and without conjugation. (Figure 32B) Gel shift results for H1 / Stem-Np after trypsin treatment, with and without conjugation to either maleimide-PEG4-SM7 / 8a or maleimide-PEG4-DBCO and azide-CpG, by two-step click chemistry. [Figure 33A]This figure shows the mass spectra of maleimide-PEG4-SM7 / 8a with and without conjugation to H1 / Stem-Np. (Figure 33A) The MS data after PNGase treatment shows the mass of H1 / Stem-Np before and after conjugation to maleimide-PEG4-SM7 / 8a. [Figure 33B] This figure shows the mass spectra of maleimide-PEG4-SM7 / 8a with and without conjugation to H1 / Stem-Np. (Figure 33B) The MS data after treatment of H1 / Stem-Np with trypsin shows the mass of cleaved ferritin before and after conjugation to maleimide-PEG4-SM7 / 8a. [Figure 34] This figure shows the mass spectra of the maleimide-PEG4-DBCO linker with and without conjugation to H1 / Stem-Np. The addition of the maleimide-PEG4-DBCO linker to H1 / Stem-Np was confirmed by the mass change measured by MS after linker addition. [Figure 35] This figure shows the SDS-PAGE of H1 / Stem-Np before and at various stages of CpG conjugation via two-step click chemistry. "After azide-CpG conjugation" refers to the final product of the two-step click reaction. [Figure 36] This figure characterizes the conjugation of linkers containing 3M-012 to the NC99 HA-TEV-Np construct. The constructs in the WT and +TEV lanes have the sequence of SEQ ID NO: 313, S26C refers to SEQ ID NO: 310, S72C refers to SEQ ID NO: 311, A75C refers to SEQ ID NO: 312, and S111C refers to SEQ ID NO: 309. All samples except WT were treated with tobacco etch-second viral protease (TEV). [Figure 37-1]Figures 37A-37E show the mass spectra of various constructs with and without reduction or conjugation. (Figure 37A) H1 / Stem-Np containing S111C before reduction. (Figure 37B) H1 / Stem-Np containing S111C after reduction. The decrease in mass (115Da) observed after reduction is consistent with the removal of post-translational modifications that inhibit the reactivity of cysteine. (Figure 37C) Mass spectra of NC99 HA-TEV-Np-S26C nanoparticles (SEQ ID NO: 310) with and without conjugation with 3M012. (Figure 37D) Mass spectra of NC99 HA-TEV-Np-A75C nanoparticles (SEQ ID NO: 312) with and without conjugation to 3M012. (Figure 37E) Mass spectra of NC99 HA-TEV-Np-S111C nanoparticles (SEQ ID NO: 309) with and without conjugation to 3M012. [Figure 37-2] Continuation of Figure 37-1. [Figure 37-3] Continuation of Figure 37-2. [Figure 37-4] Continuation of Figure 37-3. [Figure 38-1] Figures 38A-38F show negative stained electron microscope (EM) images of H1 / Stem-Np or NC99 HA-Np nanoparticles with or without conjugation to SM7 / 8a, 3M012, or CpG. (Figure 38A) Unconjugated H1 / Stem-Np. (Figure 38B) H1 / Stem-Np-SM7 / 8a conjugate. (Figure 38C) H1 / Stem-Np-CpG conjugate. (Figure 38D) NC99 HA-Np (SEQ ID NO: 309). (Figure 38E) NC99 HA-Np-3M012 conjugate. (Figure 38F) NC99 HA-Np-CpG conjugate. [Figure 38-2] Continuation of Figure 38-1. [Figure 39-1] Figures 39A-39C show the dynamic light scattering (DLS) analysis of H1 / Stem-Np-SM7 / 8a conjugate (Figure 39A), H1 / Stem-Np-CpG conjugate (Figure 39B), and unconjugated H1 / Stem-Np (Figure 39C). [Figure 39-2]Continuation of Figure 39-1. [Figure 39-3] Continuation of Figure 39-2. [Figure 40A] This figure shows the antibody response to H1 / Stem-Np formulated with the shown mixed adjuvant, or conjugated to a TLR agonist such as SM7 / 8a or CpG via a PEG4 linker, as shown in Figures 28A-B. Serum was collected from mice (n=5) 5 weeks post-immunization (weeks 0 and 3), and antibody titers were measured by enzyme-linked immunosorbent assay (ELISA). This data demonstrates the autoadjuvant properties of the H1 / Stem-Np-TLR-agonist conjugate. Figure 40A shows the ELISA against the H1 / New Caledonia / 20 / 1999 HA trimer. PAA = polyacrylic acid, mixed equimolar = 83.3 ng of SM7 / 8a (equivalent to the dose administered using ferritin nanoparticles conjugated with SM7 / 8), high dose = 21.84 μg (higher than the dose administered using ferritin nanoparticles conjugated with SM7 / 8). [Figure 40B] This figure shows the antibody response to H1 / Stem-Np formulated with the shown mixed adjuvant, or conjugated to a TLR agonist such as SM7 / 8a or CpG via a PEG4 linker, as shown in Figures 28A-B. Serum was collected from mice (n=5) 5 weeks post-immunization (weeks 0 and 3), and antibody titers were measured by enzyme-linked immunosorbent assay (ELISA). This data demonstrates the autoadjuvant properties of the H1 / Stem-Np-TLR-agonist conjugate. Figure 40B shows the ELISA against the H1 / Stem trimer. Mixed equimolar = 850 ng of CpG (equivalent to the dose administered using ferritin nanoparticles conjugated with CpG), high dose = 20 μg of CpG. [Figure 41-1]Figures 41A-41C show a comparison of titers obtained using NC99 HA-Np with and without conjugated or isolated 3M-012 (SEQ ID NO: 309). Antibody responses to nanoparticles (0.22 μg / dose) conjugated with NC99 HA-Np-3M012 were tested in mice. Mixed controls included a mixture of HA-Np (0.22 μg / dose) and 10 μg of 3M012 (typical literature dose), and a mixture of HA-Np (0.22 μg / dose) and 1.7 ng of 3M012 (equimolar match with the conjugated). Additional controls included unconjugated HA-Np and IIV administered at matched HA content (0.17 μg HA / dose). Based on ELISA endpoint titers (Figure 41A), pseudovirus (PsV) neutralizing IC50 titers, and (Figure 41B) hemagglutination inhibition (HAI) titers (Figure 41C), the HA-Np-3M012 conjugate induced a significantly stronger antibody response than the equimolar mixed control. Conjugated nanoparticles also induced a stronger response (ELISA) and a stronger neutralizing antibody response (PsV) than unconjugated nanoparticles and standard IIV treatment, although this result was not statistically significant in the HAI assay. Assays were performed on serum from 2 weeks after boost. All samples were triple-duplicated for the experiment. Median ± SEM is graphed. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. [Figure 41-2] Continuation of Figure 41-1. [Figure 42-1]Figures 42A-42C show a comparison of titers obtained using NC99 HA-Np with and without conjugated or separated CpG (SEQ ID NO: 309). Antibody responses to HA-Np-CpG conjugated nanoparticles (0.22 μg / dose) were tested in mice. Mixed controls included a mixture of HA-Np (0.22 μg / dose) and 20 μg of CpG (typical therapeutic dose), and a mixture of HA-Np (0.22 μg / dose) and 21 ng of CpG (equimolar match with the conjugated). Additional controls included unconjugated HA-Np and IIV administered at matched HA content (0.17 μg HA / dose). Based on ELISA endpoint titers (Figure 42A) and pseudoviral neutralization IC50 titers (Figure 42B), the conjugate induced stronger binding and neutralizing antibody responses than the matched mixture, unconjugated particles, or IIV. Furthermore, the HA-Np-CpG conjugate induced significantly stronger HAI titers (Figure 42C) than the equimolar mixed controls. Additionally, HAI titers were 2.6-fold and 3.0-fold higher than those of unconjugated HA-Ferr and IIV, respectively, although these results were not statistically significant. ELISA and PsV assays were performed on serum 2 weeks after boost, and HAI assays were performed on serum 5 weeks after boost. All samples were triple-duplicated for the experiment, and median ± SEM values ​​were graphed. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. [Figure 42-2] Continuation of Figure 42-1. [Figure 43-1]Figures 43A–43C illustrate the characterization of self-assembled HA-ferritin nanoparticles derived from six evolutionarily branched H1 hemagglutinin (HA) antigens and two computationally generated (COBRA) antigens. (Figure 43A) Nanoparticle size and polydispersity ("dispersibility") were measured by dynamic light scattering (DLS). (Figure 43B) The purity of HA-nanoparticles was evaluated by SDS-PAGE Coomersie staining. (Figure 43C) The integrity of nanoparticles was visualized by 80,000x negative staining electron microscopy. FM47=A / Fort Monmouth / 1-JY2 / 1947, MAL54=A / Malaysia / 302 / 1954, DV57=A / Denver / 1957 (DV57), HK77=A / Hong Kong / 117 / 1977, NC99=A / New Caledonia / 20 / 99, CA09=A / California / 4 / 2009. COBRA P1 and COBRA X6 are consensus sequences generated computationally from multiple sequences recently described by Carter DM et al., J Virol 90:4720-4734 (2016). For sequence numbers, please refer to the legend in Figures 26A-26B. [Figure 43-2] Continuation of Figure 43-1. [Figure 44-1] Figures 44A-44H show the efficacy and range of immune responses induced by various HA-ferritin nanoparticles (see the legend in Figures 26A-26B for sequence numbers). Hemagglutination inhibition (HAI) titers (log2) of mouse serum 6 weeks after immunization with the indicated HA-Np vaccine were assayed against a diverse panel of H1N1 influenza viruses. Mice (n=5) were immunized with hemagglutinin nanoparticles (HA-Nps) at weeks 0 and 3. The dashed line indicates the detection limit of the assay. (Figures 44A-44E) Data using Ribi adjuvant. (Figures 44F-44H) Data using AF03 adjuvant. The x-axis shows a panel of H1N1 influenza strains for the baseline years (1934-2013). Table 2 shows the full names of the strains corresponding to each year. An asterisk indicates a matching strain. [Figure 44-2] Continuation of Figure 44-1. [Figure 44-3] Continuation of Figure 44-2. [Figure 44-4] Continuation of Figure 44-3. [Figure 45-1] Figures 45A–45F show the HA antibody response induced by HA-ferritin nanoparticles. (Figure 45A) Mice [n=5] were immunized with specific nanoparticles or combinations of nanoparticles using the Sigma adjuvant system (catalog no. S6322). IIV refers to the inactivated influenza vaccine. (Figures 45B–F) Antibody responses were measured by determining ELISA titers against HA trimers from A / Fort Monmouth / 1 / 1947 (Figure 45B), A / Malaysia / 302 / 1954 (Figure 45C), A / Hong Kong / 117 / 1977 (Figure 45D), A / New Caledonia / 20 / 99 (Figure 45E), or A / California / 4 / 2009 (Figure 45F). ELISA titers were measured 5 weeks after the initial immunization. The lowest serum dilution tested sets the assay limit of detection, as indicated by the dotted line. The white circles indicate the agreement between the strain from which the nanoparticles administered to the mice originated and the strain assayed by ELISA. [Figure 45-2] Continuation of Figure 45-1. [Figure 45-3] Continuation of Figure 45-2. [Figure 46-1]Figures 46A–46H show the antibody response to HA mixtures of multiple ferritin nanoparticles administered to mice, as measured by HAI titer assays. (Figures 46A–46B) Bivalent combinations. (Figures 46C–46E) Trivalent combinations. (Figures 46F–46H) Tetravalent combinations. HAI titers (log2) of a series of branched H1N1 influenza viruses were assayed. Mice (n=5) were immunized with the shown HA-nanoparticle combinations using adjuvants at weeks 0 and 3. The bivalent combination COBRA X6+COBRA P1 HA-Nps was tested with the AF03 adjuvant to match the evaluation of monovalent COBRA HA-Nps. Similarly, individual strain HA-ferritin nanoparticle combinations were tested with the Ribi adjuvant to match the evaluation of individual monovalent strain HA-Nps. Asterisks indicate agreement between the strains from which the nanoparticles administered to mice originated and the strains assayed by ELISA. The x-axis shows a panel of H1N1 influenza strains for the base years (1934–2013). Table 2 shows the full names of the strains for each year. The dashed lines indicate the detection limits of the assay. [Figure 46-2] Continuation of Figure 46-1. [Figure 46-3] Continuation of Figure 46-2. [Figure 46-4] Continuation of Figure 46-3. [Figure 47-1]Figures 47A–47F show the antibody response to compositions containing Ribi or AF03 adjuvant, as measured by HAI titer assays. Compared to the results obtained with AF03 adjuvant (Figures 47B, 47D, and 47F), the results obtained with Ribi adjuvant (Figures 47A, 47C, and 47E) are similar for the bivalent combination of NC99 and CA09 HA-Nps (Figures 47A–47B), the trivalent combination of NC99, CA09 and HK77 HA-Nps (Figures 47C–47D), and the trivalent combination of NC99, CA09 and FM47 HA-Nps (Figures 47E–47F). HAI titers were measured in mouse serum 6 weeks after prime administration and boosted 3 weeks later. Mice [n=5] were immunized with either Ribi or AF03 adjuvant using the HA-Nps combinations, as shown. Asterisks indicate agreement between the strains from which the nanoparticles administered to mice originated and the strains assayed by ELISA. The x-axis shows a panel of H1N1 influenza strains for the baseline years (1934–2013). Table 2 shows the full names of the strains for each year. Dashed lines indicate the detection limits of the assay. [Figure 47-2] Continuation of Figure 47-1. [Figure 47-3] Continuation of Figure 47-2. [Figure 48-1]Figures 48A-48C show comparative antibody responses induced by egg-produced NC99 and CA09 inactivated influenza vaccines (IIV), as measured by HAI titer assays. (Figure 48A) NC99 IIIV. (Figure 48B) CA09 IIV. (Figure 48C) NC99 + CA09 IIV. Hemagglutination inhibition (HAI) titers (log2) of mouse-derived serum 6 weeks after immunization with the indicated IIV vaccines, either as single-component or co-administered, against a diverse panel of H1N1 influenza viruses. Mice (n=5) were immunized with 170 ng (HA content) of each vaccine at weeks 0 and 3 using Ribi adjuvant. The x-axis shows a panel of H1N1 influenza strains tested in the baseline years from 1934 to 2013 (see also Table 2). The x-axis shows a panel of H1N1 influenza strains tested in the baseline years (1934-2013). Table 2 shows the complete names of the strains for each year. The dashed lines indicate the detection limits of the assay. [Figure 48-2] Continuation of Figure 48-1. [Figure 49-1]Figures 49A–49D show the antibody response to various influenza viruses after immunization of ferrets with HA-ferritin nanoparticle composition or IIV, as measured by HAI titer assays. HAI titers (log2) of serum from ferrets (n=12 / group) after two immunizations mixed with AF03 adjuvant and the following: Figure 49A: PBS alone, Figure 49B: A / California / 2009 IIV, Figure 49C: NC99+CA09+HK77 HA-Nps, and Figure 49D: COBRA-X6+P1+HK77 HA-Nps. Figure 49E shows heterologous loading of ferrets immunized with HA-ferritin nanoparticle composition, IIV, or PBS. Ferrets were immunized as described in Figures 49A-D and loaded with 1 mL of A / Fortmonmouth / 1 / 1947 virus intranasally at 104.65 times the 50% tissue culture infectious dose (TCID50) four weeks after final immunization. Viral titers were quantified in nasal lavage over seven days after loading. The dashed line indicates the detection limit of the assay. Viral titers were significantly reduced five days after loading in ferrets immunized with the HA-Nps combination compared to vehicle (PBS) controls, as measured by one-tail unpaired t-test and one-way ANOVA [F(3,44)=5.18, p=0.00375], but not in CA09 IV. ***=P≦0.001, Student's t-test. [Figure 49-2] Continuation of Figure 49-1. [Figure 49-3] Continuation of Figure 49-2. [Figure 50-1]Figures 50A–C show the antibody response of cynomolgus monkeys (Macaca fascicularis) after immunization with 50 μg of H1 / Stem-Np formulated with a mixed AF03 adjuvant, or 200 μg of H1 / Stem-Np (without adjuvant control), or 200 μg of H1 / Stem-Np-SM7 / 8a conjugate (shown in Figure 28A), or 200 μg of H1 / Stem-Np-CpG conjugate (shown in Figure 28B). Figure 50A shows the HA antibody titers measured by enzyme-linked immunosorbent assay (ELISA) on plates coated with H1 / New Caledonia / 20 / 1999 HA trimers at indicated time points, immunized at weeks 0, 4, and 10. Figure 50B shows the neutralizing IC50 of lentiviruses pseudotyped using H1 / New Caledonia / 20 / 1999 HA and NA. Figure 50C shows the neutralizing IC50 of lentiviruses pseudotyped using H5 / Vietnam / 1203 / 2004 HA and NA. This data demonstrates the self-adjuvant properties of the H1 / Stem-Np-TLR-agonist conjugate in a primate model. [Figure 50-2] Continuation of Figure 50-1. [Figure 51-1]Figures 51A-51B show a comparison of titers obtained with and without conjugated or isolated 3M-012 (SEQ ID NO: 309). Antibody responses to nanoparticles conjugated with NC99 HA-Np-3M012 were at four doses: 0.1 μg, 0.5 μg, 2.5 μg, and 12.5 μg. Mixed controls and unconjugated nanoparticles were included for comparison. The amount of TLR agonist used in the “equomolar” and “high-dose” mixed controls was calculated to maintain the antigen-to-adjuvant ratio used in the initial conjugation test (0.22 μg of HA-NP with either 1.7 ng of 3M012 for “equomolar” or 10 μg for “high-dose”). Serum neutralization was measured in the NC99 lentiviral reporter assay (left, Figure 51A) and the NC99 HAI assay (right, Figure 51B). The arrows highlight the 2.5 μg dose, showing that the HA-NP-3M012 conjugate induced similar antibody titers to HA-NP+3M012 despite containing less than 5000 times the 3M012 adjuvant. The mean ± standard error is graphed. [Figure 51-2] Continuation of Figure 51-1. [Figure 52-1] Figures 52A and 52B show single-stranded gL and gH monomers (Figure 52A) (SEQ ID NO: 406) and trimers (Figure 52B) (SEQ ID NO: 411), with and without removal of the His-tag by Coomassi and Western blot analysis. Figure 52B also shows UV absorbance traces of fractions purified by size exclusion column (Superose® 6). [Figure 52-2] Continuation of Figure 52-1. [Figure 53-1]Figures 53A–53E show the purification and characterization of single-chain gL / gH-ferritin nanoparticles (SEQ ID NO: 414). UV absorbance traces of the Superose® 6 purified fraction (Figure 53A), and Coomassie (Figure 53B) and Western blot (Figure 53C) analyses of fractions selected from the purification (L represents the molecular weight ladder, with the 150 and 250 kDa bands labeled at the 150 and 250 kDa band positions, shown on the right in Figure 53B). Dynamic light scattering (Figure 53D) and electron microscopy (Figure 53E) analyses of the single-chain nanoparticles are also presented. [Figure 53-2] Continuation of Figure 53-1. [Figure 53-3] Continuation of Figure 53-2. [Figure 53-4] Continuation of Figure 53-3. [Figure 54] This figure shows different representative single-stranded gL / gH-ferritin constructs. [Figure 55] This figure shows antibody titers after immunizing mice with single-chain gL / gH trimers or nanoparticles (NPs) mixed with AF03 adjuvant, a squalene emulsion-based adjuvant. *p-value = <0.05 when comparing NP constructs to their corresponding trimer constructs. From left to right, the constructs were SEQ ID NOs: 416, 410, 411, 413, 412, and 414. [Figure 56] Figures 56A-56B show the mouse anti-gL / gH antibody response to a bivalent composition containing both gp220 nanoparticles (SEQ ID NO: 401) and single-stranded gL / gH nanoparticles ("gL_gH_C5 NP", SEQ ID NO: 419) compared to single-stranded gL / gH nanoparticles and a negative control of naked ferritin (i.e., ferritin not bound to any non-ferritin polypeptide or immunostimulatory moiety). The results indicate that the use of the bivalent composition does not result in interference with the anti-gL / gH antibody response compared to the results for single-stranded gL / gH with the negative control of naked ferritin. Both compositions contained the AF03 adjuvant. ELISA results for individual dilutions (Figure 56A) and binding titers (Figure 56B) are shown. [Figure 57]Figures 57A-57B show the anti-gp220 antibody response to a bivalent composition containing both gp220 nanoparticles and single-stranded gL / gH nanoparticles, as described in Figures 57A-57B. The results show that using the bivalent composition does not result in interference with the anti-gp220 antibody response compared to the results for gp220 nanoparticles using a negative control of naked ferritin. Both compositions contained the AF03 adjuvant. ELISA results for individual dilutions (Figure 56A) and binding titers (Figure 56B) are shown. [Figure 58] Figure 58A shows a design of nanoparticles containing EBV polypeptide and ferritin, including a mutation that replaces surface-exposed amino acids with cysteine ​​for conjugation to immunostimulatory moieties such as Toll-like receptor (TLR) agonists. See Sequence ID No. 414 for an exemplary sequence corresponding to this design. Here, the single-stranded gL / gH antigen is bound to ferritin by a flexible 46-amino acid linker. Figure 58B shows a representative Toll-like receptor agonist (SM7 / 8a with a PEG4-maleimide linker) suitable for conjugation to the construct according to Figure 58A. Figure 58C shows an electron micrograph (EM) image of gL / gH nanoparticles having SM7 / 8a conjugated via cysteine ​​on the ferritin surface and the PEG4-maleimide linker. [Figure 59A] Figure 59A shows a partial structure of ferritin, including a mutation in which a surface-exposed amino acid is replaced with cysteine, and the position of cysteine ​​is indicated. [Figure 59B] Figure 59B shows the conjugation of a CpG adjuvant (SEQ ID NO: 535) to ferritin by aligning ferritin, a linker, and the CpG adjuvant, which are oriented to show portions of each that will bind in close proximity to one another. [Figure 60A]This figure shows the mass spectrometry (MS) spectra of the form without gL / gH-ferritin conjugate (Figure 60A) and the form with SM7 / 8a conjugate (Figure 60B). The difference in mass of the main peak is 711 Da, which is approximately the difference predicted from the conjugate of SM7 / 8a and the linker. [Figure 60B] This figure shows the mass spectrometry (MS) spectra of the form without gL / gH-ferritin conjugate (Figure 60A) and the form with SM7 / 8a conjugate (Figure 60B). The difference in mass of the main peak is 711 Da, which is approximately the difference predicted from the conjugate of SM7 / 8a and the linker. [Figure 61A] This figure shows the mass spectrometry (MS) spectra of gp220-ferritin in its unconjugated form (Figure 61A) and in its conjugated form with SM7 / 8a (Figure 61B). The difference in mass of the main peak is 714.7 Da, which is approximately the difference predicted from the conjugation of SM7 / 8a with the linker. [Figure 61B] This figure shows the mass spectrometry (MS) spectra of gp220-ferritin in its unconjugated form (Figure 61A) and in its conjugated form with SM7 / 8a (Figure 61B). The difference in mass of the main peak is 714.7 Da, which is approximately the difference predicted from the conjugation of SM7 / 8a with the linker. [Figure 62] Figures 62A–62D show electron microscope (EM) images of unconjugated (Figures 62A, C) and conjugated (Figures 62B, D) single-chain gL / gH (Figures 62A, B) and gp220 (Figures 62C, D) ferritin nanoparticles, demonstrating that conjugation of SM7 / 8a to these nanoparticles did not disrupt the nanoparticle structure. [Figure 63]Figures 63A-63B show the antibody response in mice after treatment with ferritin nanoparticles containing single-stranded gL / gH, AF03 adjuvant as a separate molecule, or conjugated SM7 / 8a. ELISA results are shown as individual dilutions (Figure 63A) and binding titers (Figure 63B). [Figure 64] Figures 64A-64B show the antibody response in mice after treatment with nanoparticles containing either AF03 adjuvant alone, as a distinct molecule, or conjugated SM7 / 8a. ELISA results are shown as individual dilutions (Figure 64A) and binding titers (Figure 64B). [Figure 65] Figures 65A-65B show the anti-gL / gH antibody response in mice treated with gp220 nanoparticles conjugated to SM7 / 8a and single-stranded gL / gH ferritin nanoparticles conjugated to SM7 / 8a, compared to treatment with single-stranded gL / gH ferritin nanoparticles conjugated to SM7 / 8a and unconjugated ferritin, as measured by ELISA. Results are shown for experiments without mixed AF03 (Figure 65A) or with mixed AF03 (Figure 65B). [Figure 66] Figures 66A-66B show the anti-gp220 antibody response in mice after treatment with SM7 / 8a-conjugated gp220 nanoparticles and SM7 / 8a-conjugated single-stranded gL / gH nanoparticles, compared to treatment with SM7 / 8a-conjugated gp220 nanoparticles and unconjugated ferritin, as measured by ELISA. Results are shown for experiments without mixed AF03 (Figure 66A) or with mixed AF03 (Figure 66B). [Figure 67]This figure shows the anti-gL / gH antibody response in mice treated with single-stranded gL / gH nanoparticles (gL / gH_C5, SEQ ID NO: 419) and naked ferritin, with or without a mixed AF03 adjuvant and / or SM7 / 8a conjugated to single-stranded gL / gH nanoparticles, as measured by ELISA endpoint titer. [Figure 68] This figure shows the anti-gp220 antibody response in mice after treatment with gp220 nanoparticles and bare ferritin, with or without a mixed AF03 adjuvant and / or SM7 / 8a conjugated to gp220 nanoparticles. [Figure 69] This figure shows the anti-gL / gH antibody response in mice after treatment with a divalent composition containing gp220 nanoparticles and single-stranded gL / gH nanoparticles. As shown in the legend, some nanoparticles were conjugated with SM7 / 8a, and some were mixed with AF03. The key top-to-bottom order of symbols corresponds to the left-to-right order of symbols in the graph. [Figure 70] This shows the anti-gp220 antibody response in mice after treatment with a bivalent composition containing single-chain gL / gH nanoparticles, gp220 nanoparticles, and / or naked ferritin. As shown in the legend, some nanoparticles were conjugated with SM7 / 8a, and some were mixed with AF03. The order of symbols from top to bottom in the key corresponds to the order of symbols from left to right in the graph. [Figure 71-1]Figures 71A–71D show the antibody response in mice treated with gL_gH_C7 nanoparticles (SEQ ID NO: 420) with or without conjugation to mixed AF03 and / or SM7 / 8a (Figures 71A, 71C, or 71D) or CpG oligodeoxynucleotide (Figure 71B). Shown are endpoint titers from prime blood collection measured by ELISA (Figure 71A), ELISA results for individual dilutions from booster blood collection (Figure 71B), and endpoint titers measured by ELISA from booster blood collection (Figure 71C) and final blood collection (Figure 71D). [Figure 71-2] Continuation of Figure 71-1. [Figure 72] This figure shows the antibody response in mice treated with gL_gH_C5 nanoparticles (SEQ ID NO: 419), with or without conjugation to a mixed AF03 adjuvant and / or SM7 / 8a, as endpoint titers measured by ELISA from prime, boost, and final blood collections. [Figure 73] This figure shows the antibody response in mice treated with gp220 nanoparticles with or without conjugation to a mixed AF03 adjuvant and / or SM7 / 8a, as endpoint titers measured by ELISA from prime, boost, and final blood collections. [Figure 74A] Figure 74A shows the light and heavy chains of T.ni ferritin, visualized by Coomersie staining, with and without fusion to either gp220 or gL / gH. Markers at 20, 25, 75, 100, and 150 kDa are labeled in the rightmost lane of Figure 74A. [Figure 74B] Figure 74B provides examples of constructs containing the light and heavy chains of T.ni ferritin, with or without fusion with either gp220 or gL / gH. [Figure 75-1]Figures 75A–75D illustrate the ion-exchange (Q column) chromatography and size exclusion chromatography (SEC) purification of gp220-T.ni ferritin. Absorbance traces from the Q column (Figure 75A) and SEC Superose® 6,16 / 600 (Figure 75B) at pH 7 are shown, Coomassi staining of the fraction from the Q column at pH 7 (Figure 75C) (lanes from left are input ("In"), flow-through ("FT"), molecular weight ladder (size shown in kD on the left), and selected fraction), and Coomassi staining of the fraction from SEC Superose® 6,16 / 600 (Figure 75D) (lanes from left are molecular weight ladder (size shown in kD on the left) and selected fraction). Figure 75E shows an example of a construct. [Figure 75-2] Continuation of Figure 75-1. [Figure 75-3] Continuation of Figure 75-2. [Figure 76-1] Figures 76A–76D illustrate the ion exchange (Q column) chromatography and size exclusion chromatography (SEC) purification of gL / gH(light) / gp220(heavy)-T.ni ferritin. Absorbance traces from the Q column (Figure 76A) and SEC Superose® 6,16 / 600 (Figure 76B) at pH 7 are shown, Coomassi staining of the fraction from the Q column at pH 7 (Figure 76C) (lanes from left are input ("In"), flow-through ("FT"), molecular weight ladder (size shown in kD on the left), and selected fraction), and Coomassi staining of the fraction from SEC Superose® 6,16 / 600 (Figure 76D) (lanes from left are molecular weight ladder (size shown in kD on the left) and selected fraction). Figure 76E shows an example of a construct. [Figure 76-2] Continuation of Figure 76-1. [Figure 76-3] Continuation of Figure 76-2. [Figure 77-1]Figures 77A–77H show gp220-T.ni ferritin or gL / gH(light chain) / gp220(heavy chain)-T.ni ferritin constructs visualized by Coomersie staining (Figures 77A and 77E), schematically illustrated (Figures 77B and 77F), characterized by dynamic light scattering (DLS) (Figures 77D and 77H), and visualized by electron micrographs (Figures 77C and 77G). Figures 77A–77D show data using gp220 fused to both the light and heavy chains (Figure 77B). Figures 77E–77H show data using gp220 fused to the heavy chain and gL / gH fused to the light chain (Figure 77F). [Figure 77-2] Continuation of Figure 77-1. [Figure 77-3] Continuation of Figure 77-2. [Figure 77-4] Continuation of Figure 77-3. [Figure 78-1] Figures 78A–78C show naked T.ni ferritin particles (i.e., not fused to non-ferritin polypeptides) visualized by Coomassie staining (Figure 78A), visualized by electron microscopy (Figure 78B), and characterized by DLS (Figure 78C). [Figure 78-2] Continuation of Figure 78-1. [Figure 79A] Figure 79A shows the SDS-modified Coomassie-stained gel (left) and (right) the size exclusion chromatography (SEC) peaks of the gH / gL / gp42 NP construct (SEQ ID NO: 227) expressed in 293 expi cells. The horizontal axis of the SEC chromatogram is in mL. [Figure 79B] Figure 79B shows gH / gL / gp42 NPs purified from the CHO pool to have a dynamic light scattering radius of approximately 26.2 nm. [Figure 80] Figures 80A and 80B show the evaluation of immune responses induced by monovalent gH / gL / gp42 nanoparticle compositions combined with bare ferritin nanoparticles, or by a divalent composition (gH / gL / gp42 nanoparticles combined with gp220). Figure 80A shows B cell neutralization. Figure 80B shows epithelial cell neutralization. [Figure 81-1] Figures 81A-E show the endpoint binding titers to the indicated antigens. Figures 81F-G show the EBV virus neutralization assays of serum from vaccinated ferrets as shown. Prime = Inj.1, Boost = Inj.2. [Figure 81-2] Continuation of Figure 81-1. [Figure 81-3] Continuation of Figure 81-2. [Figure 81-4] Continuation of Figure 81-3. [Figure 82A] Figure 82A shows the purification of gH / gL / gp42_NP_C12 (SEQ ID NO: 228) using Superose 6-size exclusion chromatography. The arrows indicate fractions recovered from peaks by denatured Kuuma-Siegel analysis and Western blotting using anti-ferritin antibodies. [Figure 82B] Figure 82B shows the dynamic light scattering analysis of the sample in Figure 31A, which exhibits a particle size radius of 20.6 nm. [Figure 83A] Figure 83A shows the purification of gH / gL / gp42_NP_C13 (SEQ ID NO: 229) using Superose 6-size exclusion chromatography. The arrows indicate fractions recovered from peaks by denatured Kumasiegel analysis and Western blotting using anti-ferritin antibodies. [Figure 83B] Figure 83B shows the dynamic light scattering analysis of the sample in Figure 32A, which has a particle size radius of 17.1 nm. [Figure 84A] Figure 84A shows the purification of gH / gL / gp42_NP_C14 (SEQ ID NO: 230) using Superose 6-size exclusion chromatography. The arrows indicate fractions recovered from peaks by denatured Kumasiegel analysis and Western blotting using anti-ferritin antibodies. [Figure 84B] Figure 84B shows the dynamic light scattering analysis of the sample in Figure 84A, which has a particle size radius of 16.9 nm. [Figure 85]The SDS-reduced Kooma-Siegel on the left shows the purified single-stranded gH / gL / gp42-His product (SEQ ID NO: 226). The protein was purified using nickel affinity chromatography. On the right is the 2.9 angstrom crystal structure of the single-stranded gH / gL / gp42-His product (SEQ ID NO: 226). Gp42 (dark gray, indicated by the arrow) interacts with the gH / gL heterodimer. [Figure 86] Figures 86A–E show a diagram of the single-stranded constructs of gH / gL / gp42 fused to ferritin (as in each of Sequence IDs 227–231), with Figure 86A being the diagram. Fusion between each protein is via a flexible amino acid linker or a rigid amino acid linker as specified above. The single-stranded gH / gL / gp42 molecule confirms a 1:1:1 ratio for heterotrimer formation on nanoparticles. The crystalline structure of this heterotrimer has been elucidated to show that single-stranded gH / gL / gp42 can employ heterotrimer formation similar to that of naturally occurring wild-type gH, gL, and gp42 proteins (see also Figures 86B and 85). Figure 86C is a model of how this single-stranded gH / gL / gp42 heterotrimer is presented on nanoparticles via fusion with ferritin. There are 24 copies of single-stranded gH / gL / gp42 presented on a single nanoparticle. Figure 86D shows the purification after expression of SEQ ID NO: 227 in 293Expi cells. Modified SDS-Kumasiegel shows that gH / gL / gp42 fused to ferritin exceeds 150 kD due to glycosylation. Figure 35E shows negative staining electron microscopy analysis of the purified product, showing that single-stranded gH / gL / gp42 fused to ferritin can successfully form nanoparticles that present the gH / gL / gp42 antigen on their surface. [Figure 87A]This figure shows an exemplary RSV Pre-F-NP polypeptide structure (Figure 87A). A linear diagram listing the residue numbers corresponding to the N-terminus of each segment. Numbering is according to Sequence ID No. 526. Domains 1-3 are denoted as DI, DII, and DIII, respectively, and the 7-residue (heptad) repeat region A (HRA) and 7-residue repeat region B (HRB) are also labeled. C-terminal ferritin is labeled (ferritin nanoparticles). The F1 and F2 fragments of the RSV F portion are shown below the drawing. The region between the F1 and F2 fragments, formed by deleting the peptide 27 fragment (p27) fusion peptide (FP) and the furin cleavage site (furin site) and replacing it with a flexible linker to form a single-stranded F construct, is shown as a line above the drawing. The asterisks above the figure indicate the approximate locations of the manipulated glycosylation sites E328N, S348N, and R507N. [Figure 87B] This figure shows an exemplary RSV Pre-F-NP polypeptide structure (Figure 87B). It is a structural model of the pre-fusion RSV F portion, showing key neutralizing (Nab) epitopes for D25, AM14, 101F, and palivizumab antibodies. Approximate regions of shared pre-fusion and post-fusion structural epitopes are indicated by white triangles. The positions of exemplary manipulated glycosylation sites E328N, S348N, and R507N are labeled. The manipulated glycosylation sites are structurally shared between the pre-fusion and post-fusion conformations and map to regions distant from key neutralizing epitopes recognized by antibodies such as D25, AM14, 101F, and palivizumab. Thus, constructs containing these manipulated glycan sites still bind to the aforementioned neutralizing antibodies (data not shown). [Figure 87C] This figure shows an exemplary RSV Pre-F-NP polypeptide structure (Figure 87C). The structural model of the RSV pre-F protein nanoparticle (Pre-F-NP) with HRA and HRB regions is shown in darker shades. The resulting folded Pre-F-NP construct can form a 24-mer presenting the key epitopes listed in Figure 87B. [Figure 87D]This figure shows an exemplary RSV Pre-F-NP polypeptide structure. (Figure 87D) The 2D class mean of electron micrographs of RSV Pre-F-NP construct RF8085 (SEQ ID NO: 501) shows the symmetry of the RSV F trimer portion on 24-mer ferritin nanoparticles. [Figure 88] This figure shows the small-scale expression of several Pre-F-NP constructs expressed in 293-cell conditioned medium, as measured by D25 antibody Western blotting. RF8090 is SEQ ID NO: 502, which is a cloning variant used in CHO expression having the same sequence as RF8085, i.e., SEQ ID NO: 501. RF8085 and RF8090 are Pre-F-NP constructs with disulfide and cavity-filling mutations of DS-CAV with deletions and single-strand linkers as described in Figure 87A, fused to ferritin at the N-terminus. RF8100-RF8105 and RF8108-RF8112 have sequences SEQ ID NOs: 503-508 and 511-514, respectively. scF-pFerr=RSV is a fusion protein of F polypeptide and ferritin. Mutations that appear to improve construct expression against the RF8090 benchmark are shown below the Western blot. Notable mutations include the addition of glycan sites via the E328N, S348N, and R507N mutations, as well as the central helix-capping mutation I327P, which increased the expression and secretion of RSV F nanoparticles into conditioned medium as measured by Western blotting. [Figure 89] This figure shows the expression of RF8085 (SEQ ID NO: 501, control construct) and RF8106 (SEQ ID NO: 509, containing the I217P mutation in RF8108 and the I217P mutation that deletes the disulfide (DS) mutation of DS-CAV1), as measured by Western blot analysis of conditioned medium from 293 expression. Replacing DS with the central helix-capping mutation I217P significantly increased expression. Replacing DS with the central helix-capping mutation did not affect the binding of the construct to pre-fusion specific antibodies D25 and AM14. [Figure 90]The results of size exclusion chromatography purification of the RF8106 construct (SEQ ID NO: 509) are shown. The retention volume of approximately 65 ml of RF8106 nanoparticles on a Superose 6 prepared SEC column was consistent with that of folded 24-mer nanoparticles, suggesting that the mutation in RF8106 did not hinder nanoparticle formation. [Figure 91] Figures 91A-91B show the dynamic light scattering (DLS) analyses of non-reducible (79A) and reduced (79B) RF8106. Similar to the SEC analysis, DLS demonstrated that RSV Pre-F-NPs form the expected folded nanoparticles. Furthermore, the reduction data indicates that the particles were not destroyed by reduction, which occurred before adjuvant conjugation to surface-exposed cysteine ​​introduced on ferritin by mutation (see Figure 92). [Figure 92] This figure shows the Coomassi-stained SDS-PAGE gel analysis of RF8106 with and without conjugation to the TLR9 agonist CpG. The increased gel shift of the CpG-treated nanoparticles demonstrated that the CpG adjuvant can be added to RSV F nanoparticles up to approximately 40–50% completion. Conjugation of CpG, or other immunostimulant moieties such as the TLR7 / 8 agonist SM7 / 8, did not inhibit the ability of the particles to bind to pre-fusion specific antibodies D25 and AM14. [Figure 93]This figure shows Western blots of nanoparticles containing RSV F with (RF8117, SEQ ID NO: 517) and RSV F without additional glycans (RF8085, SEQ ID NO: 501, and RF8113, SEQ ID NO: 516). RF8113 is similar to RF8106, but the S111C surface-exposed cysteine ​​from RF8106 (using ferritin residue numbering, i.e., corresponding to its position in the ferritin sequence of SEQ ID NO: 208) is replaced with a K79C surface-exposed cysteine ​​(also using ferritin residue numbering) to further separate the conjugation site from the Pre-F moiety. Similar to RF8106, RF8113 retains improved expression compared to the benchmark molecule RF8085. RF8117 is similar to RF8113 but further includes three glycosylation mutations identified in Figure 88, namely E328N, S348N, and R507N, further improving expression and blocking a non-neutralizing epitope shared between pre-fusion and post-fusion F conformations, as described in Figure 87B. [Figure 94] This figure shows the expression of RSV F constructs with different substitutions at the potential trypsin-like protease cleavage site. In CHO cell line expression of RF8090 (same protein sequence as RF8085, but with a different DNA sequence adapted to the CHO expression vector), it was observed that the polypeptide was cleaved between the F and ferritin moieties, resulting in decreased expression. Based on the resulting mass of the F moiety, it was hypothesized that proteolysis might occur near the HRB, the bull-frog linker region of the Pre-F-NP construct. Mutations in lysine and arginine residues (approximately residues 450-550) within this region were explored to eliminate potential trypsin-like proteolysis of the construct. Mutations of RF8122 (SEQ ID NO: 518) to RF8117 (K498L and K508Q) resulted in improved expression in 293 cells and may reduce or eliminate proteolysis in CHO cells. Selective mutations limited expression. [Figure 95-1]Figures 95A-B show the expression of RF8090, RF8117, and RF8140 in stably transfected CHO cells. The expression yield of RF8090 (SEQ ID NO: 501) was observed at a low level. RF8117 (SEQ ID NO: 517) and RF8140 (SEQ ID NO: 523) were constructed by introducing mutations replacing the disulfide of DS-CAV1 and mutations to the linker between the F and ferritin portions to remove a potential typsin cleavage site, as described above, and cloned into stably expressing CHO cells. (Figure 95A) The expression of RF8117 and RF8140, respectively, from three and four pools of CHO cells in CHO-conditioned medium were compared to the yield of RF8090 in CHO-conditioned medium by D25-Western blot analysis. All three CHO pools of RF8117 and all four CHO pools of RF8140 expressed RF8117 in higher yields than RF8090. (Figure 95B) Expression of RF8117 in CHO-conditioned medium as measured by Octet using a D25-pre-fusion F-specific antibody. The left panel shows the response of purified RF8140 from known concentrations of 293 medium plotted against the response to binding to D25 on a protein A chip providing a standard curve. Individual dots represent the response to D25 binding from RF8117 CHO-conditioned medium. The right panel shows the calculated yield of RF8117 or RF8140 in CHO-conditioned medium based on the D25 binding response. Both RF8117 and RF8140 were expressed in the medium as measured by binding to D25 and AM14, demonstrating that CHO cells, like 293 cells, can express Pre-F-NPs in a folded manner that retains the pre-fusion F trimer structure. [Figure 95-2] Continuation of Figure 95-1. [Figure 96]Figures 96A-B show the neutralizing antibody response to Pre-F-NP RF8117. (Figure 96A) RSV neutralizing titers induced by high-dose (1 μg) and low-dose (0.1 μg) immunization of DS-CAV1 (Pre-F trimer, SEQ ID NO: 525), post-F trimer (Post-F trimer, SEQ ID NO: 524), or Pre-F-NP with manipulated glycosylation (Pre-F-NP, RF8117, SEQ ID NO: 517) were compared by VERO cell assay. All RSV polypeptides were administered with the adjuvant AF03 as described herein. Throughout, unless otherwise noted, AF03 was administered with RSV polypeptides or nanoparticles but not conjugated to them. RSV polypeptides and doses are shown below the x-axis. Statistical analysis of the high-dose response compared to Pre-F-NP immunization is shown. (Figure 96B) RSV neutralizing titers induced by high-dose (1 μg) and low-dose (0.1 μg) immunization using DS-CAV1 (Pre-F trimer), manipulated unglycosylated Pre-F-NP (RF8113, SEQ ID NO: 516), or manipulated glycosylated Pre-F-NP were compared by VERO cell assay. All RSV polypeptides were administered with adjuvant AF03 (unconjugated to any polypeptide or nanoparticle) as described herein. RSV polypeptides and doses are shown below the x-axis. [Figure 97-1]Figures 97A-D show a comparison of pre-fusion F trimer (DS-CAV1) bound antibodies and RSV neutralizing antibodies induced by immunization with post-fusion F trimer (SEQ ID NO: 524) or Pre-F-NP (RF8140, SEQ ID NO: 523) in mouse or non-human primate models. (Figure 97A) Compares the pre-fusion F trimer bound antibody response induced in mice from immunization with post-fusion F and Pre-F-NP (RF8140, SEQ ID NO: 523). (Figure 97B) Shows the neutralizing antibody response induced in mice from immunization with post-fusion F and Pre-F-NP (RF8140, SEQ ID NO: 523). (Figure 97C) Compares the pre-fusion F trimer bound antibody response (AF03, shown in parentheses below) induced by Pre-F-NP in non-human primates with and without adjuvants. (Figure 97D) RSV neutralizing titers induced by immunization with Pre-F-NP (RF8140, SEQ ID NO: 523) are compared with and without the AF03 adjuvant. In mice, Pre-F-NP induces a higher pre-fusion F-binding response and RSV neutralization reaction compared to the post-fusion trimer. In non-human primates, Pre-F-NP induces a potent neutralization reaction. [Figure 97-2] Continuation of Figure 97-1. [Figure 98]Figures 98A-98B illustrate how the manipulated glycosylation site blocks the post-fusion epitope. (Figure 98A) Shows the antibody response to pre-fusion F (DS-CAV1) induced by immunization with manipulated glycosylation-free Pre-F-NP (RF8113) or manipulated glycosylation-containing Pre-F-NP (manipulated Gly particles), measured by Octet. (Figure 98B) Shows the antibody response to the post-fusion trimer induced by immunization with manipulated glycosylation-free Pre-F-NP (RF8113) or manipulated glycosylation-containing Pre-F-NP (RF8117), measured by Octet. As described above, all RSV polypeptides were mixed with AF03 during immunization. Both RF8113 and RF8117 induce a robust antibody response to pre-fusion F, but the post-fusion F antibody response induced by RF8117 is significantly reduced. This is due to manipulated glycan mapping to shared pre-fusion and post-fusion epitopes (Figure 88B). [Figure 99-1] Figures 99A-C illustrate the blocking of non-neutralizing epitopes by manipulated glycosylation sites. (FIG99A) Comparison of RSV neutralizing titers induced by immunization with Pre-F NPs having wild-type glycosylation sites ("Wt glycan particles", RF8113, SEQ ID NO: 516) versus Pre-F NPs having additional manipulated glycosylation sites ("+ glycan particles", RF8117, SEQ ID NO: 517) at a 0.1 μg dose in a mouse study. (Figure 99B) Comparison of post-RSV fusion F trimer-bound antibody responses induced by immunization with Wt glycan particles (RF8113, SEQ ID NO: 516) versus + glycan particles (RF8117, SEQ ID NO: 517) at a 0.1 μg dose in a mouse study. (Figure 99C) The ratio of the measured neutralization titer to the binding titer from panels A and B demonstrates that the manipulated glycan did not reduce the functional neutralizing antibody response, but reduced the non-neutralizing antibody induced on the shared pre- and post-fusion epitopes (Figure 87B), thereby improving the neutralization / binding antibody ratio. [Figure 99-2] Continuation of Figure 99-1. [Figure 100-1] Figures 100A-D illustrate the characterization of the RSV G central domain peptide (Gcc) conjugated to ferritin nanoparticles. (Figure 100A) Coomassie-stained SDS-PAGE gel showing click conjugation of the RSV G central domain (SEQ ID NO: 529) to ferritin nanoparticles, forming the Gcc-NP antigen. (Figure 100B) Structural model of Gcc-NP. (Figure 100C) Comparison of Gcc-binding antibody responses induced by immunization with Gcc peptide alone (Gcc peptide, SEQ ID NO: 529) versus Gcc peptide conjugated to nanoparticles (Gcc-NP) in a mouse study. Representative responses from untreated serum are shown in white boxes, responses after the second immunization are shown in light gray boxes, and responses after the third immunization are shown in dark gray boxes. (Figure 100D) Comparison of RSV neutralizing titers induced by immunization with Gcc peptide (SEQ ID NO: 529) versus Gcc-NP in a mouse study after the third injection, as measured by HAE cell assay. Serum from untreated animals and serum from animals immunized with Gcc peptide were pooled, and their titers are shown as bars. [Figure 100-2] Continuation of Figure 100-1. [Figure 101-1]Figures 101A-C show that simultaneous administration of RSV Pre-F-NP (RF8140) and Gcc-NP induces a neutralization reaction. Mice were immunized with a dose of 1 μg per antigen with Pre-F-NP (RF8140) alone, Gcc-NP alone, or a combination of Pre-F-NP and Gcc-NP. As described above, all immunizations were adjuvated with AF03. (Figure 101A) Immunization of mice with RF8140 alone (Pre-F-NP) or RF8140 and Gcc-NP (Pre-F-NP+Gcc-NP) induced antibodies that bind to the pre-fusion F trimer. (Figure 101B) Immunization of mice with Gcc-NP alone (Gcc-NP) or RF8140 and Gcc-NP (Pre-F-NO+Gcc-NP) induced antibodies that bind to the Gcc peptide. (Figure 101C) Animals immunized with Pre-F-NP alone, Gcc-NP alone, or Pre-F-NP and Gcc-NP simultaneously induced neutralization after the second and third immunizations, as measured by the HAE neutralization assay. Co-administration of Pre-F-NP and Gcc-NP induced a better neutralization response than immunization with Pre-F-NP alone. [Figure 101-2] Continuation of Figure 101-1. [Figure 102]Figures 102A-B show that co-administration of Pre-F-NP and Gcc-NP does not interfere with the induction of antibodies binding to Pre-F-fused F trimers or Gcc-nanoparticles. Neutralizing titers measured by F-sensitive VERO cell assays are shown on the left side of Figure 102A, and neutralizing titers measured by F and G-sensitive HAE assays are shown on the right side of Figure 102B. Immunization of animals was similar to that in Figure 101. The RSV polypeptide used for immunization is shown below the horizontal axis. Black bars represent serum pooled from the immunization groups described in Figure 101 and are similarly labeled. Serum from untreated animals is also shown as a black bar and is displayed for comparison. Serum depleted with pre-F-fused F trimers is shown in white and is to the right of the corresponding black bar. Serum depleted with the G-ectodomain is shown as a diagonally striped bar just to the right of the corresponding black bar. Serum depleted with pre-F-fused F trimers and subsequently with the G-ectodomain is shown as a vertically striped bar. (Figure 102A) Neutralizing titer was observed in VERO cell assays for serum from RF8140 immunization and RF8140 + Gcc-NP co-administration, but not in untreated serum or serum from Gcc-NP immunization alone. Depletion of serum from RF8140 or RF8140 + Gcc-NP groups with pre-fusion F trimers resulted in a decrease in measurable neutralizing titer. (Figure 102B) Neutralizing titer was observed in HAE cell assays for serum from animals immunized with RF8140, Gcc-NP, or RF8140 co-administered with Gcc-NP. Serum from untreated animals did not show a neutralization reaction. Serum from animals immunized with RF8140 depleted with pre-fusion F trimers showed a decrease in measurable neutralizing titer. Serum from animals immunized with Gcc-NP depleted with the G ectodomain also showed a decrease in measurable neutralizing titer. Serum from animals immunized with simultaneous administration of RF8140 and Gcc-NP did not show a measurable decrease in neutralizing titer when the pre-F trimer alone was depleted, but it did show a measurable decrease in neutralizing titer when both the pre-F trimer and the G ectodomain were depleted. In summary, these data suggest that simultaneous administration of Pre-F-NP and Gcc-NP does not interfere with the respective ability of the antigens to induce neutralizing antibodies against pre-F or G. [Figure 103] Figures 103A-B show that adjuvantization of RF8117 or RF8140 with AF03, SPA09, or alum induces superior neutralization in mice compared to unadjuvanted RF8117. (Figure 103A) Serum neutralization titers from mice immunized with unadjuvanted (No Adj), alum-adjuvanted, or AF03-adjuvanted RF8117 are shown as measured by VERO cell assay. (Figure 103B) Serum neutralization titers from mice immunized with unadjuvanted (No Adj) RF8117, SPA09-adjuvanted RF8117, or AF03-adjuvanted RF8140 are shown as measured by VERO cell assay. In all cases for either RF8117 or RF8140, the adjuvanted group of untreated mice induced a higher neutralizing titer than the unadjuvanted group. [Figure 104-1] Figures 104A-B show that adjuvantization of RF8140 with AF03 or SPA09 induces a superior neutralization response in non-human primates (NHPs) compared to unadjuvanted RF8140 immunization. (Figure 104A) Pre-fusion F trimer binding response measured in NHP serum after immunization with either unadjuvanted (no adjuvant), adjuvanted with AF03, or adjuvanted with SPA09 (using two doses of SPA09 as shown below), as measured by ELISA. At all time points, adjuvantization with AF03 or SPA09 induces a superior neutralization response. (Figure 104B) Serum neutralizing titers from NHP immunized with RF8140 were measured by VERO cell assay, either unadjuvanted (No Adj), adjuvanted with AF03, or adjuvanted with SPA09 (using two doses of SPA09 as shown below). In all cases, immunization with RF8140 with adjuvants induced higher neutralizing titers than the adjuvanted and unadjuvanted groups at all time points. [Figure 104-2] Continuation of Figure 104-1. [Figure 105]Figures 105A-B show that conjugation of RF8140 to the TLR7 / 8 agonist SM7 / 8 or the TLR9 agonist CpG induces superior pre-fusion fluorine binding titer compared to unadjuvanted RF8140 alone. (Figure 105A) Pre-fusion fluorine trimer binding response measured in serum from untreated mice, mice immunized with unadjuvanted RF8140, mice immunized with RF8140 conjugated with SM7 / 8 adjuvant, RF8140 adjuvanted with 130 ng of SM7 / 8, or RF8140 adjuvanted with 20 μg of SM7 / 8. RF8140 conjugated with SM7 / 8 induces a higher pre-fusion fluorine trimer binding titer than the unadjuvanted or SM7 / 8 adjuvanted groups. (Figure 105B) Prefusion F trimer binding response measured in serum from untreated mice, mice immunized with unadjuvanted RF8140, RF8140 conjugated with a CpG adjuvant, RF8140 adjuvanted with 680 ng of CpG, or RF8140 adjuvanted with 20 μg of SM7 / 8. RF8140 conjugated with SM7 / 8 induces a higher prefusion F trimer binding titer than the unadjuvanted or SM7 / 8 adjuvanted groups. [Figure 106-1]Figures 106A-G show that F subunit vaccine candidates induce Pre-F-targeted neutralizing antibody and Th1 CD4+ T cell responses in the MIMIC system. (Figure 106A) Anti-pre-F titers in the MIMIC system were measured by AF after priming each Ag with molar equivalent concentrations of F in 10 ng / ml Pre-F NPs (n=48-49 donors / group). (Figure 106B) Microneutralizing titers were measured and expressed in international units / ml (IU / ml). (Figure 106C) A ratio of anti-pre-F to post-F > 1 indicates a higher level of pre-F binding antibody compared to post-F binding antibody, while a ratio < 1 indicates a greater antibody response to post-F. (Figure 106D) TNFα production in restimulated activated CD154+ / CD4+ T cells with F protein-loaded target cells was measured using flow cytometry (n=48). Statistical significance was determined by Tukey-Kramer-HSD multiple comparisons (Figure 106E). Pre-existing antibody titers (serum status) in human subjects strongly correlated with the magnitude of the RSV immune response in the MIMIC system. Linear regression plots showing anti-Pre-F IgG in serum from each donor versus the total anti-Pre-F IgG response were generated by software or algorithm, and p-values ​​for the common slope were analyzed statistically (n=50). The Y-axis represents the anti-Pre-F IgG level obtained after priming with RSV (Figure 106F). Similar to Figure 106E, linear regression plots showing anti-Pre-F IgG in serum from each donor versus the total anti-Pre-F IgG after priming with the F subunit vaccine candidate are shown (squares for post-F, circles for Pre-F-NP, and diamonds for DC-Cav1). Pre-existing circulating titers of anti-Pre-IgF IgG ranged from 199,800 to 3,037,600,000. Each donor represents the IgG value of that donor. (Figure 106G) Comparison of Gcc-binding antibody responses induced by treatment with Gcc peptide alone (Gcc peptide) versus Gcc peptide conjugated to nanoparticles (Gcc-NP) in human B cells. As mentioned above, the untreated group is shown for comparison. [Figure 106-2] Continuation of Figure 106-1. [Figure 106-3] Continuation of Figure 106-2. [Figure 106-4] Continuation of Figure 106-3. [Figure 107] Figures 107A-C show the neutralizing antibody titers induced by a low dose (0.5 μg) of RSV Gcc-ferritin nanoparticles ("Gcc-NP"). Using untreated and highly immunized sera as negative and positive controls, the RSV A strain HAE neutralizing titers induced by immunization with RSV Gcc-NP containing the RSV A2 Gcc sequence (formulated in AF03) are shown from serum collected 2 weeks after the second immunization (2wp2) (Figure 107A) or 2 weeks after the third immunization (2wp3) (Figure 107B). The RSV B strain HAE neutralizing titers induced by immunization with RSV Gcc-NP containing the RSV A2 Gcc sequence (formulated in AF03) are also shown from serum collected 2 weeks after the third immunization (2wp3) (Figure 107C). [Figure 108] Figures 108A-B show the RSV A2 strain antigen-binding antibody response induced by RSV Gcc-NP. (Figure 108A) Gcc-binding antibody response induced against the Gcc A2 strain, measured 2 weeks after the second injection (light gray box) and 2 weeks after the third injection (dark gray box), induced by a high dose (5 μg) of RSV Gcc-NP. Serological responses from untreated mice are shown as a negative control. (Figure 108B) Gcc-binding antibody response induced against the Gcc A2 strain, measured 2 weeks after the second injection (light gray box) and 2 weeks after the third injection (dark gray box), induced by a low dose (0.5 μg) of RSV Gcc-NP. [Figure 109]Figures 109A-B show the RSV B1 strain antigen-binding antibody response induced by RSV Gcc-NP. (Figure 109A) Gcc-binding antibody response induced against the Gcc B1 strain, measured 2 weeks after the second injection (light gray box) and 2 weeks after the third injection (dark gray box), induced by a high dose (5 μg) of RSV Gcc-NP. Serum responses from untreated mice are shown as a negative control. (Figure 109B) Gcc-binding antibody response induced against the Gcc B1 strain, measured 2 weeks after the second injection (light gray box) and 2 weeks after the third injection (dark gray box), induced by a low dose (0.5 μg) of RSV Gcc-NP. [Modes for carrying out the invention]

[0098] This specification provides novel ferritin platform polypeptides and nanoparticles for use in immunotherapy. These ferritins may include mutations that convert non-cysteine ​​amino acids to cysteine ​​in surface-exposed amino acids, allowing the immunostimulatory moiety to be directly conjugated to cysteine ​​exposed on the manipulated surface. The ferritin polypeptides provided herein may further include non-ferritin polypeptide components and may be antigenic when administered alone with adjuvants as separate molecules and / or as part of nanoparticles, and may be autoadjuvant. The design of the ferritin platform proteins and nanoparticles may increase immunogenicity and / or eliminate or reduce the need to administer adjuvants separately, and similarly may reduce the amount of adjuvant / immunostimulatory moiety required to induce an immune response to ferritin-associated (e.g., fused) non-ferritin polypeptides. Nucleic acids encoding the polypeptides described herein are also provided.

[0099] I. Definition When used herein, "ferritin" or "ferritin protein" refers to H. pylori ferritin (SEQ ID NO: 208 or 209) or P. frusus (P. fur). This refers to proteins that have detectable sequence identity with other ferritins considered herein, such as *Iosus* ferritin, *Trichoplusia ni* ferritin, or human ferritin, and that play a role in storing iron, for example, within cells or tissues, or transporting iron into the bloodstream. Such exemplary ferritins, including ferritins that exist as two polypeptide chains known as a heavy chain and a light chain (e.g., *Trichoplusia ni* and human ferritin), are discussed in detail below. In some embodiments, the ferritin is described herein as having at least the ferritin sequence disclosed, for example, in Table 1 (Sequence Listing). 9 Includes sequences with 0%, 95%, 97%, 98%, 99%, or 99.5% identity. 。「 When used herein, "wild-type ferritin" refers to ferritin whose sequence consists of a naturally occurring sequence. 。

[0100] As used herein, “ferritin monomer” refers to a single ferritin molecule that is not aggregated with other ferritin molecules (e.g., a single ferritin heavy or light chain, where applicable). “Ferritin polymer” includes multiple associated ferritin monomers. “Ferritin protein” includes monomeric ferritin and polymeric ferritin.

[0101] As used herein, “ferritin particles” refers to ferritin that has self-assembled into a spherical shape. Ferritin particles are sometimes referred to as “ferritin nanoparticles” or simply “nanoparticles.” In some embodiments, ferritin particles comprise 24 ferritin monomers (or, where applicable, 24 heavy and light chains in total).

[0102] As used herein, “hybrid ferritin” refers to ferritin containing H. pylori ferritin having the amino-terminal extension of bullfrog ferritin. An exemplary sequence used as the amino-terminal extension of bullfrog ferritin is shown as Sequence ID No. 217. In hybrid ferritin, the amino-terminal extension of bullfrog ferritin can be fused to H. pylori ferritin so that the immunostimulatory moiety binding sites are uniformly distributed on the surface of the ferritin particles. As used herein, “bullfrog linker” refers to a linker containing the sequence of Sequence ID No. 217. Hybrid ferritin is also sometimes referred to as “bfpFerr” or “bfp ferritin.” Any construct containing the bullfrog sequence can be provided without the bullfrog sequence, for example, without a linker or alternative linker. Exemplary bullfrog linker sequences are provided in Table 1. Table 1 shows bullfrog linkers, but the same constructs can be prepared without a linker or alternative linker.

[0103] As used herein, "N-glycan" refers to a carbohydrate chain attached to a protein at the amide nitrogen of an N (asparagine) residue. Therefore, N-glycans are formed by the process of N-glycosylation. This glycan may be a polysaccharide.

[0104] As used herein, "glycosylation" refers to the addition of carbohydrate units to a protein.

[0105] As used herein, “immune response” refers to the response of cells of the immune system, such as B cells, T cells, dendritic cells, macrophages, or polymorphonuclear cells, to a stimulus such as an antigen or vaccine. An immune response may include any cells of the body involved in the host defense response, including, for example, epithelial cells that secrete interferon or cytokines. An immune response includes, but is not limited to, innate and / or adaptive immune responses. As used herein, “protective immune response” refers to an immune response that protects a subject from infection (e.g., preventing infection or the development of an infection-related disease). Methods for measuring immune responses are well known in the art and include, for example, measuring the proliferation and / or activity of lymphocytes (e.g., B or T cells), the secretion of cytokines or chemokines, inflammation, antibody production, etc. “Antibody response” is an immune response in which antibodies are produced.

[0106] As used herein, “antigen” refers to an activator that, upon exposure to or administration to an organism, elicits an immune response, and / or an activator to which a T cell receptor (e.g., when presented by an MHC molecule) or an antibody (e.g., when produced by a B cell) binds. In some embodiments, an antigen elicits a humoral response in an organism (e.g., including the production of antigen-specific antibodies). Or, in addition, in some embodiments, an antigen elicits a cellular response in an organism (e.g., including T cells whose receptors specifically interact with the antigen). A particular antigen may elicit an immune response in one or more members of a target organism (e.g., mouse, rabbit, primate, human), but not in all members of the target species. In some embodiments, the antigen elicits an immune response in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of members of the target species. In some embodiments, the antigen binds to antibodies and / or T cell receptors, but may or may not induce a specific physiological response in the organism. In some embodiments, for example, the antigen may bind to antibodies and / or T cell receptors in vitro, regardless of whether such an interaction occurs in vivo. In some embodiments, the antigen reacts with products of specific humoral or cellular immunity, including products induced by heterologous immunogens. The antigen comprises antigenic ferritin proteins, including ferritin (e.g., including one or more mutations) and non-ferritin polypeptides as described herein.

[0107] When used herein, “immunostimulatory moiety” refers to a moiety that can covalently bind to ferritin or an antigenic ferritin polypeptide and activate components of the immune system (either alone or when bound to ferritin or an antigenic ferritin polypeptide). Exemplary immunostimulatory moieties include agonists of toll-like receptors (TLRs), such as TLR4, 7, 8, or 9. In some embodiments, the immunostimulatory moiety is an adjuvant.

[0108] As used herein, "adjuvant" refers to a substance or vehicle that nonspecifically enhances the immune response to an antigen. Adjuvants may include, but are not limited to, suspensions of inorganic substances (e.g., alum, aluminum hydroxide, or phosphate) to which an antigen has been adsorbed, or water-in-oil or oil-in-water emulsions (e.g., Freund's incomplete adjuvant) obtained by emulsifying an antigen solution in mineral oil or water. Sometimes, dead mycobacteria (e.g., Freund's complete adjuvant) are included to further enhance antigenicity. Immunostimulatory oligonucleotides (e.g., CpG motifs) can also be used as adjuvants (see, for example, U.S. Patents 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants may also include biological molecules such as Toll-Like receptor (TLR) agonists and co-stimulatory molecules. Adjuvants may be administered as individual molecules in the composition or covalently (conjugated) to ferritin or antigenic ferritin polypeptides.

[0109] "Antigenic ferritin polypeptide" and "antigenic ferritin protein" are used interchangeably herein and refer to polypeptides comprising ferritin and a non-ferritin polypeptide of sufficient length to be antigenic with respect to a non-ferritin polypeptide. Antigenic ferritin polypeptides may further include an immunostimulatory moiety. Antigenicity may be a feature of the non-ferritin sequence as part of a larger construct. That is, the construct is sufficient if it can act as an antigen to the non-ferritin polypeptide, regardless of whether the non-ferritin polypeptide (and, where applicable, the immunostimulatory moiety) can do so. In the context of antigenic ferritin polypeptides, a non-ferritin polypeptide may be a molecule obtained from, derived from, or similar to a polypeptide of a pathogen, e.g., an entire molecule or a fragment of a molecule derived from a pathogen, which can produce a protective immune response against the pathogen in its host. Non-ferritin polypeptides may include naturally occurring sequences or may be artificially designed or modified so that their structure is non-identical to naturally occurring molecules. For example, a polypeptide may differ from its naturally occurring form so that it has greater immunogenicity or a reduced risk of mediating an inappropriate response (e.g., an autoimmune response) in a subject. In some embodiments, the non-ferritin polypeptide is an RSV, influenza, EBV, or OspA polypeptide, in which case the antigenic ferritin polypeptide is also an "antigenic X polypeptide," where X is RSV, influenza, EBV, or OspA. However, for clarity, the antigenic RSV, influenza, EBV, or OspA polypeptide does not need to contain ferritin. "Antigenic polypeptide," as used herein, refers to a polypeptide that is either or both an antigenic ferritin polypeptide and an antigenic RSV, EBV, or OspA polypeptide.

[0110] When used herein, "antigenic EBV polypeptide" refers to a polypeptide comprising all or part of an EBV amino acid sequence of sufficient length for the molecule to be antigenic with respect to EBV. Antigenicity may be a feature of the EBV sequence as part of a construct further comprising heterologous sequences, such as ferritin or rumazine synthase protein, and / or immunostimulatory moieties. That is, if the EBV sequence is part of a construct further comprising heterologous sequences, it is sufficient that the construct can serve as an antigen to produce anti-EBV antibodies, regardless of whether the EBV sequence without heterologous sequences can do so.

[0111] As used herein, "antigenic RSV polypeptide" refers to a polypeptide comprising all or part of an RSV amino acid sequence of sufficient length for the molecule to be antigenic with respect to RSV. Antigenicity may be a feature of the RSV sequence as part of a construct further comprising heterologous sequences, such as ferritin, and / or immunostimulatory moieties. That is, if the RSV sequence is part of a construct further comprising heterologous sequences, it is sufficient that the construct can serve as an antigen to produce anti-RSV antibodies, regardless of whether an RSV sequence without heterologous sequences can do so.

[0112] "Antigenic influenza-ferritin polypeptide," as used herein, refers to a molecule comprising ferritin and an influenza polypeptide, the molecule being antigenic with respect to the influenza polypeptide. Antigenicity may be a feature of the influenza polypeptide as part of a larger construct. That is, the construct only needs to be able to act as an antigen that produces antibodies against the influenza polypeptide, regardless of whether an influenza polypeptide without ferritin can do so. In some embodiments, the influenza polypeptide and ferritin are genetically fused as a fusion protein. In some embodiments, the influenza polypeptide and ferritin are non-genetically linked, for example, by chemical conjugation.

[0113] As used herein, "antigenic OspA polypeptide" refers to a polypeptide containing all or part of an OspA of sufficient length to be antigenic with respect to OspA. A full-length OspA includes a transmembrane domain and an ectodomain, as defined below. Antigenicity may be a feature of an OspA sequence as part of a construct further containing heterologous sequences, such as ferritin or rumazine synthase protein. That is, if OspA is part of a construct further containing heterologous sequences, it is sufficient that the construct can serve as an antigen to produce an anti-OspA antibody, regardless of whether an OspA sequence without heterologous sequences can do so.

[0114] As used herein, "auto-adjuvant" refers to a composition or polypeptide comprising ferritin and an immunostimulant moiety directly conjugated to ferritin, such that ferritin and the immunostimulant moiety reside in the same molecular entity. An antigenic ferritin polypeptide, including a non-ferritin polypeptide, may be conjugated to an immunostimulant moiety to produce an auto-adjuvant polypeptide.

[0115] As used herein, "surface-exposed" amino acids, where applicable, refer to amino acid residues in proteins (e.g., ferritin) that have side chains to which solvent molecules can come into contact when the protein is in its original three-dimensional conformation after multimerization. Thus, in the case of ferritin, for example, which forms a 24-mer, surface-exposed amino acid residues are residues whose side chains can come into contact with the solvent when ferritin assembles as a 24-mer, for example, as a ferritin multimer or ferritin particles.

[0116] As used herein, “Subject” refers to any member of the animal kingdom. In some embodiments, “Subject” refers to a human. In some embodiments, “Subject” refers to a non-human animal. In some embodiments, “Subject” includes, but is not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In certain embodiments, a non-human subject is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, a subject may be a transgenic animal, a genetically modified animal, and / or a clone. In certain embodiments of the present invention, a subject may be an adult, adolescent, or juvenile. In some embodiments, the terms “individual” or “patient” are used interchangeably with “subject” and are intended to be interchangeable.

[0117] As used herein, the terms “vaccination” or “to vaccinate” refer to the administration of a composition intended to produce an immune response to, for example, a pathogen. Vaccination may be administered before, during, and / or after exposure to a pathogen and / or the onset of one or more symptoms, and in some embodiments, it may be administered before, during, and / or immediately after exposure to a pathogen. In some embodiments, vaccination may involve multiple doses of a vaccine composition at appropriate intervals.

[0118] As used herein, “EBV polypeptide” refers to a polypeptide containing all or part of the amino acid sequence encoded by EBV. Similarly, gL, gH, gp42, and gp220 polypeptides refer to polypeptides containing all or part of the gL, gH, gp42, or gp220 amino acid sequence encoded by EBV, respectively. For example, a polypeptide having at least 80% identity with the EBV-coded polypeptide necessarily contains part of the EBV-coded polypeptide. The terms “gL polypeptide,” “gH polypeptide,” “gp42 polypeptide,” and “gp220 polypeptide” are used interchangeably with “EBV gL polypeptide,” “EBV gH polypeptide,” “EBV gp42 polypeptide,” and “EBV gp220 polypeptide,” respectively. Immunity with EBV polypeptide as part or all of the antigenic polypeptide may confer protection against EBV infection. Unless otherwise stated herein, any polypeptides disclosed herein, including EBV polypeptides, may include all or part of a plurality of sequences encoded by EBV (e.g., all or part of EBV's gL and gH, or all or part of EBV's gL, gH, and gp42).

[0119] As used herein, “monomer” or “monomer construct” in the context of EBV polypeptides refers to a construct expressed as a single-stranded protein. A monomer may include single-stranded gL and gH of EBV, or single-stranded gL, gH, and gp42 of EBV.

[0120] As used herein, “trimer” or “trimer construct” in the context of EBV polypeptide refers to a construct comprising gL and / or gH of EBV along with a trimer-forming domain, such as the Foldon trimer-forming domain derived from T4 phage fibrin. Other trimer-forming domains, such as the human collagen XVIII trimer-forming domain (see, e.g., Alvarez-Cienfuegos et al., Scientific Reports 2016;6:28643) and the L1ORF1p trimer-forming domain (see, e.g., Khazina et al., Proc Natl Acad Sci USA 2009 Jan 12;106(3):731-36), are also known in the art and can be used in trimer constructs.

[0121] When used herein, "antigenic site 0" or "site 0 epitope" refers to the apex site of the pre-fusion RSV F trimer, including amino acid residues 62-69 and 196-209 of wild-type RSV (SEQ ID NO: 526). The site 0 epitope is a binding site for antibodies specific to pre-fusion RSV F, such as D25 and AM14, and antibody binding to the site 0 epitope blocks RSV cell surface binding (see McLellan et al., Science 340(6136):1113-1117(2013)).

[0122] As used herein, “antigen stability” refers to the stability of an antigen over time or in solution.

[0123] As used herein, "cavity-filling substitution" refers to an manipulated hydrophobic substitution to fill cavities present in the RSV F trimer before fusion.

[0124] "F protein" or "RSV F protein" refers to the RSV protein involved in promoting the fusion of the viral envelope with the host cell membrane during viral entry.

[0125] "RSV F polypeptide" or "F polypeptide" refers to a polypeptide that contains at least one epitope of the F protein.

[0126] "Glycanization," as used herein, refers to the addition of a mutation that introduces a glycosylation site not present in the wild-type sequence (e.g., wild-type RSV F), which can be manipulated to increase construct expression, increase construct stability, or block epitopes shared between pre- and post-fusion conformations. Modified proteins involving glycanization will have more glycosylation and therefore a higher molecular weight. Glycanization can reduce the degree to which the RSV F polypeptide evokes antibodies against the post-fusion conformation of RSV F.

[0127] When used herein, “G protein” or “RSV G protein” refers to a binding protein involved in the association of RSV with human airway epithelial cells. An exemplary wild-type RSV G amino acid sequence is provided as SEQ ID NO: 527. The RSV G protein contains an extracellular ectodomain (approximately amino acids 66–297 of RSV G (SEQ ID NO: 527)). Within the ectodomain of RSV G is a centrally conserved region (Gcc or CCR, approximately amino acids 151–193 of SEQ ID NO: 527). The CCR of RSV G contains a CX3C motif. The CX3C motif mediates the binding of the G protein to the CX3CR1 receptor.

[0128] When used herein, "Helix PRO capping" or "Helix Proline capping" refers to the ability to stabilize helix formation when the helix cap contains proline.

[0129] When used herein, "promoter-stabilizing substitution" describes an amino acid substitution in RSV F that stabilizes the pre-fusion conformation by stabilizing the interactions within the promoter of the RSV F trimer.

[0130] When used herein, "promoter-stabilizing substitution" describes an amino acid substitution in RSV F that stabilizes the pre-fusion conformation by stabilizing the interaction between the promoters of the RSV F trimer and each other.

[0131] As used herein, "protease cleavage" refers to the proteolytic degradation (sometimes also referred to as "clipping" in the art) of a sensitive residue (e.g., lysine or arginine) in a polypeptide sequence.

[0132] When used herein in relation to RSV F, "post-fusion" refers to the stable conformation of RSV F that occurs after the fusion of the virus with the cell membrane.

[0133] When used herein in relation to RSV F, "pre-fusion" refers to the conformation of RSV F that is incorporated prior to the virus-cell interaction.

[0134] As used herein, "protomer" refers to the structural unit of an oligomeric protein. In the case of RSV F, each individual unit of the RSV F trimer is a protomer.

[0135] "Hemagglutinin" or "HA," as used herein, refers to any influenza virus glycoprotein involved in binding to sialic acid on host cell membranes (an exemplary hemagglutinin is UniProt accession number: P03451). HA encompasses synthetic polypeptides that are recognized by or can induce anti-HA antibodies, such as COBRA P1, COBRA X6, and COBRA X3, described below.

[0136] As used herein, "HA stem" refers to an engineered influenza polypeptide designed from a conserved region of HA within the HA ectodomain. "Conserved" means that the region maintains significantly higher sequence identity among HAs from different strains of influenza with different HA subtypes than the sequence identity of HA as a whole. HA stem antigens are discussed in detail, for example, Impagliazzo et al., Science 2015 Sep 18, 349(6254): pp. 1301-1306; Valkenburg et al., Sci Rep. 2016 Mar 7, 6: pp. 22666; and Mallajosyula et al., Front Immunol. 2015, 6: pp. 329.

[0137] "Neuraminidase" or "NA," as used herein, refers to any influenza virus glycoprotein involved in catalyzing the removal of terminal sialic acid residues from viral and cellular glycoconjugates (an exemplary neuraminidase is UniProt accession number: P03472).

[0138] As used herein, the "Y98F mutation" refers to the substitution of tyrosine with phenylalanine in the wild-type HA sequence that is in direct contact with sialic acid. The location of the phenylalanine resulting from this mutation is shown in Figure 26A. The exact location may differ in some HA subtypes, but can be identified by sequence alignment or structural analysis. The presence of the Y98F mutation in the HA sequence implies that the corresponding wild-type HA is a subtype containing tyrosine in direct contact with sialic acid.

[0139] When used herein, “immunostimulatory moiety” refers to a moiety covalently bound to ferritin or an antigenic ferritin polypeptide that can activate components of the immune system (either alone or when bound to ferritin or an antigenic ferritin polypeptide). Exemplary immunostimulatory moieties include agonists of toll-like receptors (TLRs), such as TLR4, 7, 8, or 9. In some embodiments, the immunostimulatory moiety is an adjuvant.

[0140] As used herein, the term "kit" refers to a packaged set of related components, such as one or more compounds or compositions, and one or more related materials, such as solvents, solutions, buffers, instructions, or desiccants.

[0141] As used herein, "N-glycan" refers to a carbohydrate chain that binds to a protein at the amide nitrogen of the N-(asparagine) residue of the protein. Thus, N-glycans are formed by the N-glycosylation process. This glycan can be a polysaccharide.

[0142] As used herein, "OspA ectodomain" refers to amino acid residues approximately 27 - 273 of B. burgdorferi OspA (UniProt accession number P0CL66), or the corresponding positions of its homologs identified by pairwise or structural alignment. Further examples of the OspA ectodomain include positions 27 - X of any of SEQ ID NOs: 83 - 89, where X is the C-terminal position of the relevant sequence, and optionally the C-terminal Lys is not included. In some embodiments, the ectodomain further includes, at its N-terminus, the 26th residue or the 25th and 26th residues of the corresponding full-length wild-type sequence; in SEQ ID NOs: 83 - 89, the 25th and 26th residues are Asp and Glu. Still further examples of the OspA ectodomain include any of SEQ ID NOs: 94 - 102, optionally without the 1, 2, or 3 N-terminal residues (Met - Asp - Glu), and further optionally without the C-terminal Lys.

[0143] As used herein, "OspA transmembrane domain" refers to amino acid residues approximately 2 - 24 of B. burgdorferi OspA (UniProt accession number P0CL66), or the corresponding positions of its homologs identified by pairwise or structural alignment.

[0144] The present disclosure describes nucleic acid sequences and amino acid sequences that each have a certain degree of identity to a predetermined nucleic acid sequence or amino acid sequence (reference sequence).

[0145] "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides identified between the sequences. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences.

[0146] The terms "percent identical", "percent identity", or similar terms are intended to refer to the percentage of nucleotides or amino acids that are identical in the optimal alignment between the sequences being compared. The percentage is purely statistical, and the differences between two sequences may be distributed across the full length of the sequences being compared, but need not be distributed randomly. Comparison of two sequences is typically performed by comparing the sequences over segments or "comparison windows" after optimal alignment to identify local regions of the corresponding sequences. Optimal alignment for comparison may be performed manually or with the aid of a computer program using the algorithms of Smith and Waterman, 1981, Adv. Appl. Math. 2, 482; Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443; Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444; or GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0147] Percentage identity is obtained by determining the number of corresponding identical positions in the arrays being compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference array), and multiplying the result by 100.

[0148] In some embodiments, the degree of identity is given with respect to a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the total length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given with respect to at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, or in some embodiments, with respect to consecutive nucleotides. In some embodiments, the degree of identity is given over the entire length of the reference sequence.

[0149] A nucleic acid sequence or amino acid sequence having a specific degree of identity with respect to a given nucleic acid sequence or amino acid sequence can have at least one functional property of the given sequence, and in some cases, is functionally equivalent to the given sequence. One important property is, in particular, the ability to act as a cytokine when administered to a subject. In some embodiments, a nucleic acid sequence or amino acid sequence having a specific degree of identity with respect to a given nucleic acid sequence or amino acid sequence is functionally equivalent to the given sequence.

[0150] As used herein, the term “kit” refers to a packaged set of related components, such as one or more compounds or compositions, and one or more related materials, such as solvents, solutions, buffers, instructions, or desiccants.

[0151] II. Exemplary ferritins, antigenic ferritin polypeptides, conjugates, compositions, methods, and uses Ferritin proteins self-assemble into spherical protein complexes containing multiple individual monomers. These self-assembled ferritin complexes can be called ferritin particles or nanoparticles.

[0152] Ferritin genes are found in many species and generally exhibit a highly conserved alpha-helix structure, although their sequences vary. Therefore, any ferritin, including bacterial, insect, and human ferritin, can be used in this invention, despite its sequence identity with any particularly described ferritin.

[0153] In some embodiments, ferritin is a bacterium, insect, fungus, bird, or mammal. In some embodiments, ferritin is human. In some embodiments, ferritin is a bacterium. In some embodiments, ferritin is H. pylori ferritin.

[0154] In some embodiments, ferritin is light-chain and / or heavy-chain ferritin. In some embodiments, ferritin is human heavy-chain ferritin (FTH1, GENE ID number: 2495) or human light-chain ferritin (FTL, GENE ID number: 2512), optionally having one or more modifications described herein. In some embodiments, ferritin is nettle-type heavy-chain ferritin (GenBank: AY970291.1) or nettle-type light-chain ferritin (AY970292.1), optionally having one or more mutations described herein. In some embodiments, ferritin nanoparticles contain 24 subunits in total from heavy-chain and light-chain ferritin, for example, 12 heavy-chain subunits and 12 light-chain subunits. In some embodiments, ferritin includes mutations that replace surface-exposed amino acids with cysteine.

[0155] In some embodiments, antigenic ferritin polypeptides are provided, comprising ferritin and a non-ferritin polypeptide of sufficient length for the molecule to be antigenic with respect to a non-ferritin polypeptide.

[0156] In some embodiments, the antigenic ferritin polypeptide comprises heavy-chain ferritin or non-ferritin polypeptide, as well as light-chain ferritin and non-ferritin polypeptides. Such polypeptides can be combined to express the same or different two non-ferritin polypeptides on a single ferritin polymer or particle. In some embodiments, the two different non-ferritin polypeptides are encoded by a single infectious agent. In some embodiments, the two different non-ferritin polypeptides are encoded by two different infectious agents. In some embodiments, the infectious agent is a virus or a bacterium. In some embodiments, the two different non-ferritin polypeptides are encoded by two different infectious agents, such as different pathogens like influenza, Borrelia, RSV, or EBV, or different strains or types of pathogens like influenza, Borrelia, RSV, or EBV, and bind to heavy-chain and light-chain ferritin to assemble into nanoparticles.

[0157] In some embodiments, the antigenic ferritin polypeptide comprises heavy chain ferritin and non-ferritin polypeptides, which assemble with light chain ferritin and non-ferritin polypeptides to produce a bivalent composition. In some embodiments, the ferritin is H. pylori ferritin having one or more of the mutations described herein (see SEQ ID NOs. 208 or 209 for exemplary H. pylori ferritin sequences). In some embodiments, the lower sequence homology between H. pylori ferritin (or other bacterial ferritin) and human ferritin may reduce the potential for autoimmunity when used as a vaccine platform (see Kanekiyo et al., Cell 162, pp. 1090-1100 (2015)).

[0158] In some embodiments, ferritin is Pyrococcus furiosus ferritin (NCBI seq WP_011011871.1) having one or more of the mutations described herein.

[0159] In some embodiments, ferritin is wild-type ferritin and 9 Includes sequences with identity greater than 0%, greater than 95%, greater than 97%, greater than 98%, or greater than 99%.

[0160] In some embodiments, a different protein capable of forming nanoparticles is used instead of ferritin. In some embodiments, this protein is lumazine synthase (see Ra et al., Clin Exp Vaccine Res 3: pp. 227-234 (2014)). In some embodiments, this protein is lumazine synthase serotype 1, 2, 3, 4, 5, 6, or 7. Exemplary lumazine synthase sequences are provided as SEQ ID NOs. 216 and 219. In some embodiments, the lumazine synthase contains a sequence having 80%, 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NOs. 216 or 219.

[0161] A. Ferritin mutation This specification discloses ferritins containing one or more mutations. In some embodiments, one or more mutations include, for example, changes in the amino acid sequence of wild-type ferritin and / or insertions at the N or C terminus. In some embodiments, one, two, three, four, five, or more different amino acids are mutated in the ferritin compared to wild-type ferritin (in some embodiments, in addition to any N-terminal insertions). One or more mutations can alter the functional properties of ferritin, as will be discussed in detail below, for example. Generally, a mutation simply refers to a difference in sequence (such as substituted, added, or deleted amino acid residues or multiple residues) compared to the corresponding wild-type ferritin.

[0162] 1. Cysteine for conjugation In some embodiments, ferritin is mutated to provide a chemical handle for conjugation of an immunostimulatory moiety and / or a non-ferritin polypeptide. This can be achieved by mutations that replace non-cysteine surface-exposed amino acids with cysteine. To avoid misunderstanding, expressions such as "replacing a surface-exposed amino acid with cysteine" necessarily imply that the surface-exposed amino acid in the wild-type or pre-mutation sequence is not cysteine. Another approach to providing a chemical handle for conjugation of an immunostimulatory moiety or a non-ferritin polypeptide is to include a segment of amino acids, such as a linker, at the N or C terminus of ferritin, and the segment of amino acids includes cysteine. In some embodiments, this cysteine (which replaces a surface-exposed amino acid or is in the N- or C-terminal linker) is unpaired, meaning that it does not have an appropriate partner cysteine to form a disulfide bond. In some embodiments, this cysteine does not change the secondary structure of ferritin. In some embodiments, this cysteine does not change the tertiary structure of ferritin.

[0163] In some embodiments, this cysteine can be used to conjugate an agent, such as an immunostimulatory moiety, to ferritin. In some embodiments, this cysteine provides a free thiol group that is reactive. In some embodiments, the agent conjugated to this cysteine on ferritin is exposed on the surface of the assembled ferritin particles. In some embodiments, this cysteine can interact with molecules and cells of a subject while the ferritin particles are assembling after administration.

[0164] In some embodiments, the presence of this cysteine enables conjugation of one or more immunostimulatory moieties, such as adjuvants. In some embodiments, conjugation of the immunostimulatory moiety does not occur in the absence of this cysteine.

[0165] In some embodiments, the non-cysteine ​​amino acids substituted for cysteine ​​are selected from E12, S72, A75, K79, S100, and S111 of H. pyloriferritin. Thus, in some embodiments, the surface-exposed amino acids substituted for cysteine ​​are amino acid residues corresponding to E12, S26, S72, A75, K79, S100, or S111 of H. pyloriferritin. Similar amino acids can be found in non-H. pyloriferritin by pairwise or structural alignment. In some embodiments, the non-cysteine ​​amino acids substituted for cysteine ​​can be selected from amino acids corresponding to S3, S19, S33, I82, A86, A102, and A120 of human light chain ferritin. In some embodiments, the surface-exposed amino acid that is replaced with cysteine ​​is selected based on the understanding that, when the original amino acid is replaced with cysteine, this is reactive in the aggregated ferritin polymer or particle, and / or this cysteine ​​does not interfere with the stability of the ferritin polymer or particle, and / or this cysteine ​​does not result in a reduction in ferritin expression levels.

[0166] In some embodiments, ferritin contains an E12C mutation. In some embodiments, the E12C residue can be used to conjugate an active agent (e.g., an immunostimulatory moiety and / or a non-ferritin polypeptide) to ferritin. In some embodiments, the E12C residue provides a reactive free thiol group. In some embodiments, the active agent conjugated to the E12C residue on the ferritin monomer is expressed on the surface of the assembled ferritin multimer or particle. In some embodiments, 24 E12C residues (one from each monomer) are present on the surface of the ferritin multimer or particle.

[0167] In some embodiments, ferritin contains an S26C mutation. In some embodiments, the S26C residue can be used to conjugate an active agent (e.g., an immunostimulatory moiety and / or a non-ferritin polypeptide) to ferritin. In some embodiments, the S26C residue provides a reactive free thiol group. In some embodiments, the active agent conjugated to the S26C residue on the ferritin monomer is expressed on the surface of an assembled ferritin multimer or particle. In some embodiments, 24 S26C residues (one from each monomer) are present on the surface of the ferritin multimer or particle.

[0168] In some embodiments, ferritin contains the S72C mutation. In some embodiments, the S72C residue can be used to conjugate an active agent (e.g., an immunostimulatory moiety and / or a non-ferritin polypeptide) to ferritin. In some embodiments, the S72C residue provides a reactive free thiol group. In some embodiments, the active agent conjugated to the S72C residue on the ferritin monomer is expressed on the surface of the assembled ferritin multimer or particle. In some embodiments, 24 S72C residues (one from each monomer) are present on the surface of the ferritin multimer or particle.

[0169] In some embodiments, ferritin contains the A75C mutation. In some embodiments, the A75C residue can be used to conjugate an active agent (e.g., an immunostimulatory moiety and / or a non-ferritin polypeptide) to ferritin. In some embodiments, the A75C residue provides a reactive free thiol group. In some embodiments, the active agent conjugated to the A75C residue on the ferritin monomer is expressed on the surface of the assembled ferritin multimer or particle. In some embodiments, 24 A75C residues (one from each monomer) are present on the surface of the ferritin multimer or particle.

[0170] In some embodiments, ferritin contains the K79C mutation. In some embodiments, the K79C residue can be used to conjugate an active agent (e.g., an immunostimulatory moiety and / or a non-ferritin polypeptide) to ferritin. In some embodiments, the K79C residue provides a reactive free thiol group. In some embodiments, the active agent conjugated to the K79C residue on the ferritin monomer is expressed on the surface of the assembled ferritin multimer or particle. In some embodiments, 24 K79C residues (one from each monomer) are present on the surface of the ferritin multimer or particle.

[0171] In some embodiments, ferritin contains the S100C mutation. In some embodiments, the S100C residue can be used to conjugate an active agent (e.g., an immunostimulatory moiety and / or a non-ferritin polypeptide) to ferritin. In some embodiments, the S100C residue provides a reactive free thiol group. In some embodiments, the active agent conjugated to the S100C residue on the ferritin monomer is expressed on the surface of an assembled ferritin multimer or particle. In some embodiments, 24 S100C residues (one from each monomer) are present on the surface of the ferritin multimer or particle.

[0172] In some embodiments, ferritin contains the S111C mutation. In some embodiments, the S111C residue can be used to conjugate an active agent (e.g., an immunostimulatory moiety and / or a non-ferritin polypeptide) to ferritin. In some embodiments, the S111C residue provides a reactive free thiol group. In some embodiments, the active agent conjugated to the S111C residue on the ferritin monomer is expressed on the surface of an assembled ferritin multimer or particle. In some embodiments, 24 S111C residues (one from each monomer) are present on the surface of the ferritin multimer or particle.

[0173] 2. Removal of internal cysteine In some embodiments, ferritin includes mutations that replace an internal cysteine ​​with a non-cysteine ​​amino acid. By removing the original internal cysteine ​​residue, it is possible to ensure that there is only one unpaired cysteine ​​per ferritin monomer, thereby avoiding undesirable reactions such as disulfide formation and potentially leading to more stable and efficient results (e.g., adjuvant presentation). In some embodiments, C31 of H. pyloriferritin is replaced with a non-cysteine ​​amino acid. In some embodiments, C31 of H. pyloriferritin is replaced with serine (C31S), but any non-cysteine ​​residue, such as alanine, glycine, threonine, or asparagine, may be used. Similar amino acids can be found in non-H. pyloriferritin by pairwise or structural alignment. Thus, in some embodiments, the internal cysteine ​​replaced for non-cysteine ​​is an amino acid residue that aligns with C31 of H. pyloriferritin. Exemplary ferritin sequences exhibiting the C31S mutation are shown in SEQ ID NOs. 201–207. In some embodiments, if more than one internal cysteine ​​is present in ferritin, two or more (e.g., each) internal cysteine ​​are replaced by a non-cysteine ​​amino acid such as serine, or an amino acid selected from serine, alanine, glycine, threonine, or asparagine.

[0174] 3. Glycosylation Human-compatible glycosylation may be relevant to the safety and efficacy of recombinant drug products. Regulatory approval may be conditional on demonstrating appropriate glycosylation as a critical quality attribute (see Zhang et al., Drug Discovery Today 21(5):740-765 (2016)). N-glycans can result from glycosylation of the asparagine side chain and may differ in structure between humans and other organisms such as bacteria and yeast. Thus, it would be desirable to reduce or eliminate non-human glycosylation and / or N-glycan formation in ferritin according to this disclosure. In some embodiments, controlling the glycosylation of ferritin improves the efficacy and / or safety of the composition, particularly when used for human vaccine administration.

[0175] In some embodiments, ferritin is mutated to inhibit N-glycan formation. In some embodiments, the mutated ferritin has reduced glycosylation compared to its corresponding wild-type ferritin.

[0176] In some embodiments, ferritin includes a mutation that replaces surface-exposed asparagine with a non-asparagine amino acid. In some embodiments, surface-exposed asparagine is at N19 of H. pylori ferritin, or at the position corresponding to position 31 of H. pylori ferritin, as determined by pairwise or structural alignment. In some embodiments, mutating such asparagine, e.g., N19 of H. pylori ferritin, reduces the glycosylation of ferritin. In some embodiments, the mutation replaces asparagine with glutamine. In some embodiments, ferritin is H. pylori ferritin containing the N19Q mutation. Sequence IDs 201–207 are exemplary ferritin sequences containing the N19Q mutation.

[0177] Mammals exposed to glycosylated proteins produced in bacteria or yeast may develop an immune response to the glycosylated protein because the glycosylation pattern of a given protein in bacteria or yeast may differ from the glycosylation pattern of the same protein in mammals. Thus, some glycosylated therapeutic proteins may not be suitable for production in bacteria or yeast.

[0178] In some embodiments, reduced ferritin glycosylation due to amino acid mutations promotes protein production in bacteria or yeast. In some embodiments, reduced ferritin glycosylation reduces the potential for adverse effects in mammals when mutant ferritin expressed in bacteria or yeast is administered. In some embodiments, the reactiongenicity of mutant ferritin produced in bacteria or yeast in human subjects is low due to reduced glycosylation. In some embodiments, the incidence of hypersensitivity responses in human subjects is lower after treatment with mutant ferritin having reduced glycosylation compared to wild-type ferritin.

[0179] In some embodiments, the degradation of a composition containing mutant ferritin with reduced glycosylation in a subject is slower compared to a composition containing wild-type ferritin or a composition containing the corresponding ferritin with wild-type glycosylation. In some embodiments, a composition containing mutant ferritin with reduced glycosylation has reduced clearance in a subject compared to a composition containing wild-type ferritin or a corresponding ferritin with wild-type glycosylation. In some embodiments, a composition containing mutant ferritin with reduced glycosylation has a longer serum half-life compared to a composition containing wild-type ferritin or a corresponding ferritin with wild-type glycosylation.

[0180] 4. Combinations of mutations In some embodiments, ferritin comprises more than one of the mutation types described herein. In some embodiments, ferritin comprises one or more mutations independently selected from mutations that reduce glycosylation, mutations that remove internal cysteine, and mutations that produce surface-exposed cysteine. In some embodiments, ferritin comprises mutations that reduce glycosylation, mutations that remove internal cysteine, and mutations that produce surface-exposed cysteine.

[0181] In some embodiments, ferritin includes the N19Q mutation, the C31S mutation, and a mutation that produces surface-exposed cysteine. In some embodiments, ferritin includes the N19Q mutation, the C31S mutation, and the E12C mutation. In some embodiments, ferritin includes the N19Q mutation, the C31S mutation, and the S72C mutation. In some embodiments, ferritin includes the N19Q mutation, the C31S mutation, and the A75C mutation. In some embodiments, ferritin includes the N19Q mutation, the C31S mutation, and the K79C mutation. In some embodiments, ferritin includes the N19Q mutation, the C31S mutation, and the S100C mutation. In some embodiments, ferritin includes the N19Q mutation, the C31S mutation, and the S111C mutation. In some embodiments, ferritin contains mutations corresponding to any of the aforementioned sets of mutations, the corresponding mutations changing N to Q, C to S, and amino acids exposed on the non-cysteine ​​surface to cysteine ​​at positions determined by pairwise alignment of the ferritin amino acid sequence with the H. pylori ferritin amino acid sequence (SEQ ID NO: 208 or 209).

[0182] Exemplary ferritins containing more than one type of mutation are provided in SEQ ID NOs. 201–207.

[0183] 5. Structural Alignment As discussed above, the mutational locations corresponding to the mutations described for H. pylori ferritin can be identified by pairwise or structural alignment. Structural alignment is suitable for large protein families like ferritins, where proteins share similar structures despite considerable sequence variation, and many members of the family are structurally characterized. This can also be used to identify corresponding locations in different forms of other polypeptides described herein, such as influenza (e.g., hemagglutinins), Borrelia (e.g., OspA), RSV (e.g., RSV F or G), and EBV (e.g., gL, gH, gp220, or gp42). The Protein Databank (PDB) contains 3D structures for many ferritins, including the ferritins listed below along with their accessions.

[0184] 2jd6,2jd7-PfFR-Purococcus phryosus. 2jd8-PfFR+Zn. 3a68-soFR-soybean derived from gene SferH4. 3a9q-soFR derived from gene SferH4 (mutant). 3egm,3bvf,3bvi,3bvk,3bvl-HpFR-Helicobacter pylori. 5c6f-HpFR(mutant)+Fe. 1z4a,1vlg-FR-Thermotoga maritime. 1s3q,1sq3,3kx9-FR-Archaeoglubus fulgidus,1krq-FR-Campylobacter jejuni. 1eum-EcFR-Escherichia coli. 4reu-EcFR+Fe. 4xgs-EcFR(mutant)+Fe2O2. 4ztt-EcFR(mutant)+Fe2O+Fe2+Fe+O2. 1qgh-LiFR-Listeria innocua. 3qz3-VcFR-Vibrio cholerae. 3vnx-FR-Ulva pertusa. 4ism,4isp,4itt,4itw,4iwj,4iwk,4ixk,3e6s-PnmFR-Pseudo-nitschia multiseries. 4zkh,4zkw,4zkx,4zl5,4zl6,4zlw,4zmc-PnmFR(mutant)+Fe. 1z6o-FR-Nettle moth. 4cmy-FR+Fe-Green sulfur bacteria (Chlorobaculum tepidum). Ferritin light chain (FTL). 1lb3,1h96-mFTL-mouse. 1rcc,1rcd,1rci-bFTL+Tartrate+Mg. 1rce,1rcg-bFTL+Tartrate+Mn. 3noz,3np0,3np2,3o7r-hoFTL(mutant)-Horse. 3o7s,3u90-hoFTL. 4v1w-hoFTL-cryoEM. 3rav,3rd0-hoFTL+Barbiturate. Ferritin light chain + heavy chain:5gn8-hFTH+Ca.

[0185] Structural alignment involves identifying corresponding residues across two (or more) polypeptide sequences by (i) modeling the structure of the first sequence using a known structure of the second sequence, or (ii) comparing the structures of the first and second sequences, both of which are known, and identifying the residue in the first sequence that is most similar to the residue of interest in the second sequence. The corresponding residues are identified in some algorithms based on minimizing the alpha-carbon distances in the superimposed structures (e.g., which pair of alpha-carbon pairs provides the least mean squared deviation for alignment). When identifying the positions in non-H. pyloriferritin that correspond to positions described for H. pyloriferritin, H. pyloriferritin may be the “second” sequence. If the target non-H. pylori ferritin does not have a known structure available, but is more closely related to another non-H. pylori ferritin with a known structure than to H. pylori ferritin, it may be most effective to model the target non-H. pylori ferritin using the known structure of the closely related non-H. pylori ferritin, and then compare that model to the H. pylori ferritin structure to identify the desired corresponding residues in the target ferritin. Extensive literature exists on structural modeling and alignment, with representative disclosures including U.S. Patent No. 6,859,736; U.S. Patent No. 8,738,343; and the disclosure cited in Aslam et al., Electronic Journal of Biotechnology 20 (2016), pp. 9-13. For considerations of structural modeling based on known related structures or multiple structures, see, for example, Bordoli et al., Nature Protocols 4 (2009), pp. 1-13, and the references cited therein.

[0186] B. Immunostimulatory moiety; adjuvant; conjugated non-ferritin polypeptide In some embodiments, an immunostimulatory moiety, such as a non-ferritin polypeptide and / or adjuvant, binds to a surface-exposed amino acid. In some embodiments, the surface-exposed amino acid is, for example, cysteine ​​resulting from the mutations discussed above. In some embodiments, the surface-exposed amino acid is lysine, aspartate, or glutamate. Conjugation procedures using glutaraldehyde (for conjugating lysine to an amino-supported linker or portion) or carbodiimide (e.g., 1-cyclohexyl-3-(2-morpholin-4-yl-ethyl)carbodiimide or 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC; EDAC) for conjugating aspartate or glutamate to an amino-supported linker or portion, or for conjugating lysine to a carboxyl-supported linker or portion) are described in Chapter 4 of Holtzhauer, M., Basic Methods for the Biochemical Lab, Springer 2006, ISBN 978-3-540-32785-1, available, for example, from www.springer.com.

[0187] In some embodiments, an immunostimulatory moiety, such as an adjuvant, binds to an amino acid exposed on the surface of ferritin. In some embodiments, more than one immunostimulatory moiety, such as an adjuvant, binds to an amino acid exposed on the surface of ferritin. In some embodiments, 24 immunostimulatory moieties bind to a ferritin polymer or particle (e.g., one portion of each monomer in H. pylori ferritin particles). In some embodiments where multiple immunostimulatory moieties are bound to ferritin nanoparticles, all immunostimulatory moieties are identical. In some embodiments where multiple immunostimulatory moieties are bound to ferritin nanoparticles, the immunostimulatory moieties are not all identical.

[0188] 1. Type of immunostimulatory component; adjuvant An immunostimulatory moiety capable of binding to surface-exposed amino acids (e.g., cysteine) can be used in ferritin in accordance with this disclosure. In some embodiments, the immunostimulatory moiety is a B cell agonist.

[0189] In some embodiments, the immunostimulatory moiety is not hydrophobic. In some embodiments, the immunostimulatory moiety is hydrophilic. In some embodiments, the immunostimulatory moiety is polar. In some embodiments, the immunostimulatory moiety is capable of hydrogen bonding or ionic bonding, and includes, for example, hydrogen bond donors, hydrogen bond acceptors, cationic moieties, or anionic moieties. A moiety is considered cationic or anionic if it is ionized in aqueous solution at physiologically relevant pH values ​​such as pH 6, 7, 7.4, or 8.

[0190] In some embodiments, the immunostimulatory portion is an adjuvant. In some embodiments, the adjuvant includes a pathogen-associated molecular pattern (PAMP). In some embodiments, the adjuvant is a stimulant of a toll-like receptor (TLR) agonist or an interferon gene (STING) agonist. In some embodiments, the adjuvant activates TLR signaling in B and / or T cells. In some embodiments, the adjuvant modulates an adaptive immune response.

[0191] a) TLR2 agonist In some embodiments, the immunostimulatory moiety is a TLR2 agonist. In some embodiments, the immunostimulatory moiety stimulates TLR2 signaling. In some embodiments, the immunostimulatory moiety is a synthetic small molecule ligand for TLR2. In some embodiments, the immunostimulatory moiety is a synthetic small molecule agonist for TLR2 signaling.

[0192] In some embodiments, the TLR2 agonist is PAM2CSK4, FSL-1, or PAM3CSK4.

[0193] b) TLR7 / 8 Agonist In some embodiments, the immunostimulatory moiety is a TLR7 and / or TLR8 agonist (i.e., at least one agonist of TLR7 and TLR8). In some embodiments, the immunostimulatory moiety stimulates TLR7 and / or TLR8 signaling. In some embodiments, the immunostimulatory moiety is a synthetic small molecule ligand of TLR7 and / or TLR8. In some embodiments, the immunostimulatory moiety is a synthetic small molecule agonist of TLR7 and / or TLR8 signaling.

[0194] In some embodiments, the TLR7 and / or TLR8 agonist is single-stranded (ssRNA). In some embodiments, the TLR7 and / or TLR8 agonist is imidazoquinoline. In some embodiments, the TLR7 and / or TLR8 agonist is a nucleoside analog.

[0195] In some embodiments, the TLR7 and / or TLR8 agonist is an imidazoquinolineamine Toll-like receptor (TLR) agonist such as 3M-012 (3M Pharmaceuticals). The structure of free 3M-012 is: [ka] It is understood that an immunostimulant moiety, such as 3M-012 or any moiety considered herein, can be conjugated to ferritin by substituting a suitable terminal atom (e.g., hydrogen) of the moiety for a bond with ferritin as described herein, for example, with the sulfur of cysteine ​​exposed on the surface, or by a linker that binds to such sulfur. Thus, when conjugated to ferritin, the structure of the immunostimulant moiety differs slightly from the structure of the free molecule.

[0196] In some embodiments, the TLR7 and / or TLR8 agonist is SM7 / 8a. The structure of free SM7 / 8a is: [ka] That is the case.

[0197] For example, see Nat Biotechnol.2015 Nov;33(11):1201~10pp.doi:10.1038 / nbt.3371.

[0198] c) TLR9 Agonist In some embodiments, the immunostimulatory moiety is a TLR9 agonist. In some embodiments, the immunostimulatory moiety stimulates TLR9 signaling. In some embodiments, the immunostimulatory moiety is a synthetic small molecule ligand for TLR9. In some embodiments, the immunostimulatory moiety is a synthetic small molecule agonist for TLR9 signaling.

[0199] In some embodiments, the TLR9 agonist is a CpG oligodeoxynucleotide (ODN). In some embodiments, the TLR9 agonist is an unmethylated CpG ODN. In some embodiments, the CpG ODN contains a partial or complete phosphorothioate (PS) skeleton instead of the native phosphodiester (PO) skeleton found in normal DNA.

[0200] In some embodiments, the CpG ODN is a class B ODN, which comprises one or more hexameric CpG motifs including 5'purine (Pu)-pyrimidine (Py)-CG-Py-Pu3'; has a well-phosphorothioate-modified (i.e., PS-modified) skeleton; and has a length of 18 to 28 nucleotides. In some embodiments, the CpG ODN comprises the sequence of SEQ ID NO: 210, and optionally includes a phosphorothioate bond in the skeleton.

[0201] In some embodiments, the TLR9 agonist includes an immunostimulatory sequence (ISS). In some embodiments, the TLR9 agonist is ISS-1018 (Dynavax) (SEQ ID NO: 210).

[0202] d) STING Agonist In some embodiments, the immunostimulatory moiety is a STING (interferon gene protein stimulator, also known as endoplasmic reticulum IFN stimulator) agonist. In some embodiments, the immunostimulatory moiety stimulates STING signaling. In some embodiments, the immunostimulatory moiety is a synthetic small molecule ligand for STING. In some embodiments, the immunostimulatory moiety is a synthetic small molecule agonist of STING signaling.

[0203] In some embodiments, the STING agonist is a cyclic dinucleotide (CDN). See, for example, Danilchanka et al., Cell 154: pp. 962-970 (2013). Exemplary CDNs include cdA, cdG, cAMP-cGMP, and 2'-5',3'-5'cGAMP (see Danilchanka et al. for structures). STING agonists also include synthetic agonists such as DMXAA. [ka]

[0204] 2. Conjugated non-ferritin polypeptides In some embodiments, the non-ferritin polypeptide is conjugated to amino acids exposed on the surface of ferritin. In some embodiments, the non-ferritin polypeptide is a polypeptide derived from a pathogen that makes the ferritin protein antigenic. In some embodiments, the non-ferritin polypeptide is antigenic on its own, but in some embodiments, the non-ferritin polypeptide is antigenic due to its association with ferritin. In some embodiments, the non-ferritin polypeptide is any one of the non-ferritin polypeptides described herein.

[0205] 3. Conjugation In some embodiments, a surface-exposed cysteine ​​(e.g., resulting from a mutation described herein) or cysteine ​​in a peptide linker bound to ferritin (e.g., the N-terminus of ferritin) is used to conjugate an immunostimulatory moiety, such as an adjuvant, or a non-ferritin polypeptide to ferritin. In some embodiments, the linker is conjugated to such cysteine, and the linker can then be conjugated to an immunostimulatory moiety, such as an adjuvant, or a non-ferritin polypeptide. In some embodiments, such cysteine ​​provides a chemical handle for the conjugation reaction that links the adjuvant, linker, or non-ferritin polypeptide. In some embodiments, a bioconjugate is produced, and the immunostimulatory moiety, such as an adjuvant, or a non-ferritin polypeptide is linked to ferritin after reduction of such cysteine. In some embodiments, the cysteine ​​is a surface-exposed unpaired cysteine, i.e., cysteine ​​lacking a partner cysteine ​​at the appropriate position for forming a disulfide bond. In some embodiments, the cysteine ​​is an unpaired cysteine ​​containing a free thiol side chain.

[0206] a) Types of conjugation chemistry Using any type of chemistry, an immunostimulatory moiety or non-ferritin polypeptide, such as an adjuvant, can be conjugated to ferritin via the reaction of surface-exposed amino acids, such as cysteine ​​or another amino acid like Lys, Glu, or Asp.

[0207] In some embodiments, conjugation is carried out using click chemistry. As used herein, “click chemistry” refers to a reaction between a pair of functional groups that react rapidly and selectively with each other (i.e., “click”). In some embodiments, click chemistry can be carried out under mild aqueous conditions. In some embodiments, the click chemistry reaction utilizes cysteine ​​on the surface of ferritin, such as cysteine ​​resulting from amino acid mutations exposed on the surface, and carries out click chemistry using a functional group that can react with cysteine.

[0208] A variety of reactions that meet the criteria of click chemistry are known in the art, and those skilled in the art can use any of the published methodologies (see, for example, Hein et al., Pharm Res 25(10):2216-2230 (2008)). A wide range of commercially available reagents, such as those from Sigma Aldrich, Jena Bioscience, or Lumiprobe, can be used for click chemistry. In some embodiments, conjugation is carried out using click chemistry as described in the following examples.

[0209] In some embodiments, click chemistry occurs after the reduction of ferritin.

[0210] In some embodiments, click chemistry can be a one-step click reaction. In some embodiments, click chemistry can be a two-step click reaction.

[0211] In some embodiments, the reaction involves metal-free click chemistry. In some embodiments, the reaction involves thiol-maleimide and / or disulfide exchange.

[0212] Metal-free click chemistry Metal-free click chemistry can be used in conjugation reactions to avoid potential protein oxidation. Metal-free click chemistry has been used to form antibody conjugates (see van Geel et al., Bioconjugate Chem. 2015, 26, pp. 2233-2242).

[0213] In some embodiments, metal-free click chemistry is used in the reaction to conjugate the adjuvant to ferritin. In some embodiments, copper-free conjugation is used in the reaction to conjugate the adjuvant to ferritin. In some embodiments, the metal-free click chemistry is bicyclo[6.1.0]nonine (BCN). In some embodiments, the metal-free click chemistry is dibenzoazacyclooctin (DBCO). In some embodiments, BCN or DBCO reacts with an azide group.

[0214] DBCO exhibits high specificity for azide groups via a strain-enhanced click reaction in the absence of a catalyst, yielding stable triazoles in high yield. In some embodiments, DBCO reacts with azides in the absence of a copper catalyst.

[0215] In some embodiments, metal-free click chemistry is used in one-step click reactions. In some embodiments, metal-free click chemistry is used in two-step click reactions.

[0216] Thiol-maleimide and disulfide exchange The ferritin used herein may include cysteine ​​containing a thiol, also known as sulfhydryl, which is available for reaction with (or may become available through reduction with) sulfhydryl reactive chemical groups. Thus, cysteine ​​allows for chemoselective modification to add immunostimulatory moieties, such as adjuvants, to ferritin. Under basic conditions, cysteine ​​is deprotonated to produce a thiolate nucleophile, which can react with weak electrophiles such as maleimide and iodoacetamide. The reaction of cysteine ​​with maleimide or iodoacetamide results in a carbon-sulfur bond.

[0217] In some embodiments, the sulfhydryl reactive chemical group reacts with cysteine ​​in the linker of cysteine ​​or ferritin exposed on the surface. In some embodiments, the sulfhydryl reactive chemical group is haloacetyl, maleimide, aziridine, acryloyl, arylating agent, vinyl sulfone, pyridyl disulfide, or TNB-thiol.

[0218] In some embodiments, the sulfhydryl reactive chemical group is conjugated to the sulfhydryl of cysteine ​​by alkylation (i.e., formation of a thioether bond). In some embodiments, the sulfhydryl reactive chemical group is conjugated to the sulfhydryl of cysteine ​​by disulfide exchange (i.e., formation of a disulfide bond).

[0219] In some embodiments, the reaction to conjugate an immunostimulatory moiety, such as an adjuvant, to ferritin is a thiol-maleimide reaction.

[0220] In some embodiments, the sulfhydryl reactive chemical group is maleimide. In some embodiments, the reaction of maleimide with cysteine ​​results in the formation of a stable, non-reversible thioester bond, for example. In some embodiments, maleimide does not react with tyrosine, histidine, or methionine in ferritin. In some embodiments, the unreacted maleimide is quenched at the end of the reaction by adding, for example, an excess of free thiols.

[0221] In some embodiments, the reaction to conjugate an immunostimulatory moiety, such as an adjuvant, to ferritin is a thiol-disulfide exchange, also known as disulfide interconversion. In some embodiments, the reaction involves the formation of a mixed disulfide containing a portion of the original disulfide. In some embodiments, the original disulfide is a cysteine ​​introduced into ferritin by a mutation of an amino acid exposed on the surface or the addition of an N-terminal linker.

[0222] In some embodiments, the sulfhydryl reactive chemical group is a pyridyldithiol. In some embodiments, the sulfhydryl reactive chemical group is a TNB-thiol group.

[0223] b) Linker for conjugation In some embodiments, an immunostimulatory moiety, such as an adjuvant, or a non-ferritin polypeptide, is bound to ferritin via a linker covalently bonded to a surface-exposed amino acid, such as cysteine. In some embodiments, the linker includes polyethylene glycol, e.g., a PEG linker. In some embodiments, the polyethylene glycol (e.g., PEG) linker increases the water solubility and ligation efficiency of ferritin linked to the immunostimulatory moiety, such as an adjuvant. The PEG linker is between 2 and 18 PEG lengths, e.g., PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14, PEG15, PEG16, PEG17, and PEG18.

[0224] In some embodiments, the linker comprises maleimide. In some embodiments, the linker comprises an immunostimulatory moiety (ISM)-linker-maleimide component. In some embodiments, the ISM-linker-maleimide is conjugated to ferritin in a one-step click chemistry by the reaction of maleimide with cysteine ​​of ferritin. In some embodiments, the ISM of the adjuvant-linker-maleimide is SM7 / 8a. In some embodiments, the linker of the ISM-linker-maleimide is PEG4. In some embodiments, the ISM-linker-maleimide is SM7 / 8a-PEG4-maleimide.

[0225] In some embodiments, a two-step click chemistry protocol is used with a linker containing a sulfhydryl reactive chemical group at one end and an amine reactive group at the other end. In such a two-step click chemistry protocol, the sulfhydryl reactive chemical group reacts with the cysteine ​​of ferritin, while the amine reactive group reacts with a reagent conjugated to the ISM. In this way, the ISM is conjugated to ferritin via a set of two-step chemistry reagents.

[0226] In some embodiments of the two-step click chemistry protocol, the sulfhydryl reactive chemical group is maleimide. In some embodiments of the two-step click chemistry protocol, maleimide reacts with cysteine ​​introduced into ferritin by mutation of surface-exposed amino acids or addition of an N-terminal linker.

[0227] In some embodiments of the two-step click chemistry protocol, the amine reactant is DBCO. In some embodiments of the two-step click chemistry protocol, DBCO reacts with an azide group bonded to the ISM.

[0228] In some embodiments, maleimide-linker-DBCO is used. In some embodiments, maleimide-linker-DBCO is conjugated to ferritin after reduction of ferritin. In some embodiments, maleimide-linker-reagent is conjugated to ferritin by the reaction of maleimide with the cysteine ​​of ferritin in the first step. In some embodiments, DBCO is used to couple to an azide-coupled ISM. In some embodiments, the azide-coupled ISM is ISS-1018. In some embodiments, the adjuvant coupled to the azide is 3M-012 or CpG.

[0229] In some embodiments, a linker having a reactive group is added to the ISM. In some embodiments, the linker is a PEG4-azid linker or a PEG4-maleimide linker.

[0230] In some embodiments, the PEG4 azid linker is conjugated to 3M-012. An exemplary structure of 3M-012 conjugated to the PEG4 azid linker is: [ka] That is the case.

[0231] In some embodiments, the PEG4-azid linker is conjugated to SM7 / 8a. An exemplary structure of SM7 / 8a conjugated to the PEG4-azid linker is: [ka] That is the case.

[0232] In some embodiments, the PEG4-maleimide linker is conjugated to SM7 / 8a. An exemplary structure of SM7 / 8a conjugated to the PEG4-maleimide linker is: [ka] That is the case.

[0233] In some embodiments, the azide group is conjugated to ISS-1018. An exemplary structure of ISS-1018 conjugated to an NHS ester-azide linker is: [ka] That is the case.

[0234] C. Antigenic ferritin polypeptide In some embodiments, the ferritin described herein is part of an antigenic ferritin polypeptide further comprising a non-ferritin polypeptide. In some embodiments, the antigenic ferritin polypeptide is a fusion protein comprising ferritin coupled to a non-ferritin polypeptide. In some embodiments, the non-ferritin polypeptide is fused to the N-terminus of ferritin. In some embodiments, the non-ferritin polypeptide is fused to the C-terminus of ferritin. The non-ferritin polypeptide can also be conjugated to the ferritin discussed above, for example, via cysteine ​​resulting from surface-exposed amino acid mutations or via cysteine ​​introduced into the N- or C-terminal linker.

[0235] 1. Linker In some embodiments, the linker separates the amino acid sequence of the non-ferritin polypeptide from the amino acid sequence of ferritin. Any linker may be used. In some embodiments, the linker is a peptide linker that can facilitate the expression of the antigenic ferritin polypeptide as a fusion protein (e.g., derived from a single open reading frame). In some embodiments, the linker is a glycine-serine linker. In some embodiments, the glycine-serine linker is GS, GGGS (SEQ ID NO: 443), 2XGGGS (i.e., GGGSGGGS) (SEQ ID NO: 444), or 5XGGGS (SEQ ID NO: 445). The linker is N-terminus or C-terminus relative to ferritin.

[0236] In some embodiments, the linker is 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid lengths. In some embodiments, the linker is about 2–4, 2–6, 2–8, 2–10, 2–12, or 2–14 amino acid lengths. In some embodiments, the linker is at least 15 amino acid lengths. In some embodiments, the linker is at least 25 amino acid lengths. In some embodiments, the linker is at least 30 amino acid lengths. In some embodiments, the linker is at least 35 amino acid lengths. In some embodiments, the linker is at least 40 amino acid lengths. In some embodiments, the linker is less than or equal to 60 amino acid lengths. In some embodiments, the linker is less than or equal to 50 amino acid lengths. In some embodiments, the linker is about 16, 28, 40, 46, or 47 amino acid lengths. In some embodiments, the linker is flexible. In some embodiments, the linker includes cysteine ​​for use as a conjugation site for, for example, an immunostimulatory moiety (e.g., an adjuvant); an exemplary linker including cysteine ​​is provided as SEQ ID NO: 225. In some embodiments, the linker includes a sequence having at least 75%, 80%, 85%, 90%, or 95% identity with SEQ ID NO: 225, and further includes cysteine ​​corresponding to the cysteine ​​in SEQ ID NO: 225. In some embodiments, the linker includes at least 25 amino acids (e.g., 25 to 60 amino acids), and the cysteine ​​is located in the range from the 8th amino acid from the N-terminus to the 8th amino acid from the C-terminus, or within 10 amino acids of the central residue, or at the linker's binding site.

[0237] In some embodiments, the linker comprises the amino acid glycine (G) and / or serine (S). In some embodiments, the linker comprises or consists of the amino acids glycine (G), serine (S), asparagine (N), and / or alanine (A), and optionally cysteine ​​as discussed above. In some embodiments, the linker comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 222. In some embodiments, the linker comprises GGGSGGGGSGGGGSG (SEQ ID NO: 220), GGSGSGSNSSASSGASSGGASGGSGGSG (SEQ ID NO: 221), GGSGSASSGASASGSSNGSGSGSGSNSSASSGASSGGASGGSGGSG (SEQ ID NO: 222), or GS. In some embodiments, the linker comprises FR1 (SEQ ID NO: 223) or FR2 (SEQ ID NO: 224). In some embodiments, the linker includes sequence numbers 233-238.

[0238] In some embodiments, ferritin comprises H. pylori ferritin (referred to as hybrid ferritin) having an amino-terminal extension of bullfrog ferritin. In some embodiments, this hybrid ferritin forms a polymer having non-ferritin polypeptide binding sites uniformly distributed on the surface (see Kanekiyo 2015). In some embodiments, an N-terminal fusion protein with hybrid ferritin enables the presentation of non-ferritin polypeptides on the surface of ferritin nanoparticles. In some embodiments, the non-ferritin polypeptide is a viral or bacterial polypeptide. In some embodiments, ferritin contains glutamate at a position corresponding to position 13 of SEQ ID NO: 208 (hybrid ferritin containing this glutamate) or position 6 of SEQ ID NO: 209 (wild-type H. pylori ferritin with isoleucine at position 6). When combined with the bullfrog linker, this glutamate is thought to maintain the conserved salt crosslinks found in human and bullfrog ferritin (6R and 14E in both human light chain and bullfrog lower subunit ferritin). See Kanekiyo et al., Cell 162, pp. 1090-1100 (2015).

[0239] In some embodiments, a non-ferritin polypeptide is linked to ferritin via a cysteine-thrombin-histidine linker. In some embodiments, this linker is used to directly conjugate a moiety (e.g., an immunostimulant moiety or a non-ferritin polypeptide) to ferritin via click chemistry. An exemplary sequence containing a cysteine-thrombin-histidine linker is Sequence ID No. 218. Suitable click chemistry for conjugation reactions involving a cysteine-thrombin-histidine linker is discussed above.

[0240] In some embodiments, a linker containing cysteine ​​as a conjugation site for an immunostimulatory moiety, such as an adjuvant, is used in a construct containing a ferritin molecule lacking surface-exposed unpaired cysteine, or in a construct containing a ferritin molecule containing surface-exposed unpaired cysteine.

[0241] In some embodiments, the structure does not include a linker. In some embodiments, the structure includes one linker. In some embodiments, the structure includes two or more linkers.

[0242] Representative pathogens (viruses and bacteria) that can be used as sources of non-ferritin polypeptides for incorporation into antigenic ferritin polypeptides include Epstein-Barr virus (EBV), influenza, Borrelia (e.g., Borrelia species that cause Lyme disease, such as B. burgdorferi), and respiratory syncytial virus (RSV). In some embodiments, the pathogen-derived non-ferritin polypeptide includes a peptide sequence derived from a protein expressed or encoded by the pathogen. In some embodiments, the amino acids of the non-ferritin polypeptide are linked to the amino acid sequence of hybrid ferritin, and the non-ferritin polypeptide sequence is located before the N-terminal elongation of bullfrog ferritin.

[0243] In some embodiments, the amino acids of a non-ferritin polypeptide bind to the amino acid sequence of hybrid ferritin to generate a fusion protein. In some embodiments, this fusion protein contains hybrid ferritin together with the non-ferritin polypeptide, such that the non-ferritin polypeptide is present in each monomer of ferritin. In some embodiments, these monomers self-assemble into ferritin nanoparticles. In some embodiments, ferritin nanoparticles containing hybrid ferritin monomers contain multiple copies of the non-ferritin polypeptide on the nanoparticle surface. In some embodiments, an aggregate of 24 hybrid ferritin monomers forms ferritin nanoparticles together with 24 non-ferritin polypeptides on the nanoparticle surface.

[0244] In some embodiments, the antigenic ferritin polypeptide comprises one of the sequences SEQ ID NOs: 1-76, 301-343, 401-403, 410, 413-414, 417-427, or 501-523. In some embodiments, the fusion protein comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with any of the aforementioned sequences, and the ferritin sequence comprises mutations that replace surface-exposed amino acids with cysteine. Such ferritin sequences may further comprise an Asn-to-Glu mutation at a position corresponding to position 19 of free H. pylori ferritin (SEQ ID NO: 208), and / or a Cys-to-Ser mutation at a position corresponding to position 31 of free H. pylori ferritin (SEQ ID NO: 208). In some embodiments, cysteine ​​resulting from surface-exposed amino acid mutations corresponds to any cysteine ​​position disclosed herein, for example, positions 12, 26, 72, 75, 79, 100, or 111 of H. pyrroferritin.

[0245] 2. EBV polypeptide as a non-ferritin polypeptide In some embodiments, the ferritin polypeptide described herein further comprises an EBV polypeptide. In some embodiments, the non-ferritin polypeptide of the antigenic ferritin polypeptide described herein is an EBV polypeptide. In some embodiments, the antigenic ferritin polypeptide described herein is also an antigenic EBV polypeptide.

[0246] a) EBV polypeptides containing gL and gH polypeptides EBV possesses three glycoproteins, namely glycoproteins B (gB), gH, and gL, which form a core membrane fusion mechanism that allows the virus to penetrate into cells. gL and gH have been previously described, for example, by Matsuura et al., Proc Natl Acad Sci US A. 2010 Dec 28;107(52):226 pp. 41-6. Monomers and trimers of gL and gH for use as vaccines have been described, for example, by Cui et al., Vaccine. 2016 Jul 25;34(34):40 pp. 50-5. The gH and gL proteins associate to form a heterodimer complex that is thought to be necessary for efficient membrane fusion and binding to epithelial cell receptors, which are required for viral entry.

[0247] In some embodiments, the EBV polypeptide comprises EBV gL and EBV gH. In some embodiments, the polypeptide exists as a single chain. In some embodiments, the polypeptide forms nanoparticles (e.g., ferritin or rumazine synthase particles) through, for example, ferritin multimerization or rumazine synthase. In some embodiments, the antigenic EBV polypeptide according to this disclosure comprises the EBV gL polypeptide and the EBV gH polypeptide, as well as a linker having at least 15 amino acids in length separating the EBV gL polypeptide and the EBV gH polypeptide. Relatively long linkers have been found to provide benefits such as improved expression and / or immunogenicity.

[0248] In some embodiments, the EBV gH and / or gL polypeptide comprises the full-length gH and / or gL (see GenBank accession numbers CEQ35765.1 and YP_001129472.1, respectively, for exemplary full-length sequences). In some embodiments, the EBV gH and / or gL polypeptide is a fragment of gH and / or gL. In some embodiments, the gL polypeptide is a gL(D7) construct having a 7-amino acid deletion at the C-terminal end of gL. In some embodiments, the gH polypeptide contains a mutation at C137, such as the C137A mutation. In some embodiments, the C137 mutation removes the original unpaired cysteine ​​to avoid nonspecific conjugation. In some embodiments, the gH polypeptide contains a mutation that removes the position corresponding to cysteine ​​137 of SEQ ID NO: 437, such as the C137A mutation. In some embodiments, the C137 mutation removes the original unpaired cysteine ​​to avoid nonspecific conjugation.

[0249] In some embodiments, the EBV gL polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 436. In some embodiments, the EBV gH polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 437.

[0250] In some embodiments, the mammalian leader sequence (also known as the signal sequence) is added to the N-terminus of an EBV polypeptide, such as a gH or gL polypeptide, for example, at the N-terminus of the polypeptide. In some embodiments, the mammalian leader sequence, when expressed in mammalian cells, results in protein secretion.

[0251] The original EBV gH and / or gL sequences are shown in GenBank accession number NC_009334.1 (Human Herpesvirus 4, Complete Genome, dated 26-Mar-2010). Full-length or fragmented original EBV gH and / or gL may be used as non-ferritin polypeptides. In some of the constructs disclosed herein, amino acids 23-137 of the gL amino acid sequence in NC_009334.1 are used as the gL polypeptide, and the original signal peptide (amino acids 1-22 of the NCBI sequence) is replaced with an IgGκ reader sequence. In some constructs, amino acids 19-678 of the gH amino acid sequence in NC_009334.1 are used as the gH polypeptide. In some embodiments, gL and gH are linked via a linker as shown in the sequence listing described herein.

[0252] In some embodiments, the gL and gH polypeptides are expressed as single-stranded monomers. In some embodiments, the monomeric composition includes or consists of sequences shown in the sequence listing and described as “monomers.” Single-stranded monomers containing the gL and gH polypeptides may be referred to as “gL / gH,” and these can be used interchangeably with “gH_gL,” “gL_gH,” or “gL / gH.”

[0253] The gL / gH polypeptide can be combined with either ferritin or lumazine synthase as discussed herein. For example, in some embodiments, the antigenic EBV polypeptide comprises monomeric or trimer gL / gH polypeptide (+ / -gp42 and / or gp220), and ferritin, i) heavy-chain or light-chain ferritin (e.g., nettle chain heavy-chain or light-chain ferritin); or ii) optionally containing surface-exposed cysteine.

[0254] In addition, in some embodiments, any antigenic EBV polypeptide, including EBV gL / gH polypeptide and ferritin, may be present in a composition containing ferritin, as well as other polypeptides disclosed herein, such as other antigenic EBV polypeptides other than gL / gH, for example, gp220 and / or gp42.

[0255] b) EBV polypeptide containing gp220 polypeptide In some embodiments, the EBV polypeptide comprises the gp220 polypeptide. The gp220-hybrid bullfrog / H. pylori ferritin nanoparticles have already been described in Kanekiyo Cell. 2015 Aug 27;162(5):1090-100. These nanoparticles do not contain mutations that provide surface-exposed cysteine ​​or cysteine-containing linkers, among other differences from certain ferritins described herein.

[0256] In some embodiments, the gp220 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 438.

[0257] In some embodiments, a mammalian leader sequence (also known as a signal sequence) is added to the N-terminus of the gp220 polypeptide. In some embodiments, the mammalian leader sequence, when expressed in mammalian cells, results in protein secretion.

[0258] The gp220 polypeptide can be combined with any ferritin or lumazine synthase considered herein. For example, in some embodiments, the antigenic EBV polypeptide comprises the gp220 polypeptide (+ / -gL / gH and / or gp42), and ferritin comprising i) heavy-chain or light-chain ferritin (e.g., nettle-leaved ferritin heavy-chain or light-chain ferritin); or ii) optionally surface-exposed cysteine ​​as described herein.

[0259] In addition, in some embodiments, any antigenic EBV polypeptide, including the gp220 polypeptide and ferritin, may be present in a composition containing ferritin, as well as other polypeptides disclosed herein, such as other antigenic EBV polypeptides other than gp220, for example, gL / gH and / or gp42.

[0260] c) EBV polypeptide containing gp42 polypeptide In some embodiments, the EBV polypeptide comprises the gp42 polypeptide. An exemplary gp42 sequence is provided as SEQ ID NO: 434. A further exemplary gp42 sequence suitable for inclusion in a fusion with gL and gH polypeptides, for example, is provided as SEQ ID NO: 239. A further exemplary gp42 sequence suitable for inclusion in a fusion with gL and gH polypeptides is provided as SEQ ID NO: 240.

[0261] In some embodiments, the gp42 polypeptide includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 434. In some embodiments, the gp42 polypeptide includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 239. In some embodiments, the gp42 polypeptide includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 240.

[0262] In some embodiments, a mammalian leader sequence (also known as a signal sequence) is added to the N-terminus of the gp42 polypeptide. In some embodiments, the mammalian leader sequence, when expressed in mammalian cells, results in protein secretion. An exemplary leader sequence is amino acids 1-22 of SEQ ID NO: 226.

[0263] In some embodiments, an antigenic EBV polypeptide comprising gH and / or gL polypeptides further comprises a gp42 polypeptide. Any EBV polypeptide comprising the above gH and / or gL polypeptides may further comprise a gp42 polypeptide. In some embodiments, the gp42 polypeptide is located at the C-terminus of the gH and / or gL polypeptide, as illustrated in SEQ ID NOs. 421 and 226-231. In some embodiments, the gp42 polypeptide is located at the N-terminus of ferritin, as illustrated in SEQ ID NOs. 421 and 227-231. Thus, for example, an antigenic EBV polypeptide comprises, from N to C-terminus, a gL polypeptide, a gH polypeptide, a gp42 polypeptide, and optionally ferritin. A linker, such as the linker described herein, separates the gp42 polypeptide from the EBV polypeptide, and / or the ferritin located at its N-terminus and / or C-terminus. In some embodiments, the linker separates each EBV polypeptide (e.g., gL polypeptide, gH polypeptide, and gp42 polypeptide) in the antigenic ferritin polypeptide, and if present, a further linker may be present between ferritin and the EBV polypeptide proximal to it (e.g., gp42 polypeptide).

[0264] In some embodiments, linkers having at least 15 amino acid lengths separate the EBV gH polypeptide and the EBV gp42 polypeptide. Such linkers may have lengths of 15–60 amino acids, 20–60 amino acids, 30–60 amino acids, 40–60 amino acids, 30–50 amino acids, or 40–50 amino acids. In some embodiments, the linker contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 234.

[0265] In some embodiments, if gp42 and ferritin are present in the polypeptide, the linker separates the EBV gp42 polypeptide and ferritin. Such a linker may have a length of at least 15 amino acids, or have lengths of 15-60 amino acids, 20-60 amino acids, 30-60 amino acids, 40-60 amino acids, 30-50 amino acids, or 40-50 amino acids. In some embodiments, such a linker contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NOs: 233, 234, 235, 236, 237, or 238.

[0266] The gp42 polypeptide can be combined with either ferritin or lumazine synthase as discussed herein. For example, in some embodiments, the polypeptide comprises the gp42 polypeptide (+ / -gL / gH and / or gp220) and heavy-chain or light-chain ferritin (e.g., nettle heavy-chain or light-chain ferritin); or ii) ferritin comprising surface-exposed cysteine ​​as described herein.

[0267] In some embodiments, the antigenic EBV polypeptide includes a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with amino acids 23-1078 of SEQ ID NO: 226. In some embodiments, the antigenic EBV polypeptide includes a sequence having 80%, 85%, 90%, 95%, 98%, or 99% identity with amino acids 1-1078 of SEQ ID NO: 226. In some embodiments, the antigenic EBV polypeptide includes a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with any one of SEQ ID NOs: 226, 227, 228, 229, 230, or 231, and may be missing a leader sequence (for example, missing any or all of amino acids 1-22 of these sequences).

[0268] In addition, in some embodiments, any antigenic EBV polypeptide, including the gp42 polypeptide and ferritin, may be present in a composition containing ferritin and other polypeptides disclosed herein, such as other antigenic EBV polypeptides other than gp442, for example, gL / gH and / or gp220.

[0269] d) Mutations in the gL, gH, gp42, linker, and / or ferritin sequences to remove possible oxidation, deamide, or isoaspartate formation sites. In some embodiments, the antigenic EBV polypeptide includes one or more mutations to remove possible oxidation, deamide, or isoaspartate formation sites, such as the exemplary mutations listed in Table 1 below.

[0270] For example, in some embodiments, the gL sequence may include one or more mutations to remove possible succinimide / isoaspartate or deamide sites. For instance, the gL sequence may include a G-to-A mutation at the position corresponding to position 36 of SEQ ID NO: 227; an N-to-Q mutation at the position corresponding to position 47 of SEQ ID NO: 227; or an N-to-Q mutation at the position corresponding to position 105 of SEQ ID NO: 227. When aligning these positions using default parameters according to a standard sequence alignment algorithm such as the Smith-Waterman algorithm, a given position in the amino acid sequence "corresponds" to a given position in SEQ ID NO: 227.

[0271] In some embodiments, the linker includes one or more mutations to remove possible deamide sites. For example, the linker sequence may include an N-to-G mutation at the position corresponding to position 132 or 141 of sequence number 227.

[0272] In some embodiments, the gH sequence includes one or more mutations to remove possible succinimide / isoaspartate or oxidation sites. For example, the gH sequence may include an M-to-L mutation at positions 189, 401, or 729 of SEQ ID NO: 227; a D-to-E mutation at position 368 of SEQ ID NO: 227; an M-to-I mutation at positions 499 or 639 of SEQ ID NO: 227; or an N-to-Q mutation at position 653 of SEQ ID NO: 227.

[0273] In some embodiments, the gp42 sequence may include one or more mutations to remove possible deamide sites. For example, the gp42 sequence may include an N-to-Q mutation at the position corresponding to position 959 or 990 of SEQ ID NO: 227; or an N-to-S mutation at the position corresponding to position 988 of SEQ ID NO: 227.

[0274] In some embodiments, the ferritin sequence includes one or more mutations to eliminate possible deamide, oxidation, or isoaspartate formation sites. For example, the ferritin sequence may include a Q-to-S mutation at the position corresponding to position 1150 of SEQ ID NO: 227; an M-to-I mutation at the position corresponding to position 1168 of SEQ ID NO: 227; an M-to-L mutation at the position corresponding to position 1177 of SEQ ID NO: 227; a G-to-A mutation at the position corresponding to position 1188 of SEQ ID NO: 227; or an N-to-Q mutation at the position corresponding to position 1253 or 1296 of SEQ ID NO: 227.

[0275] Exemplary mutations are shown in Table 6 below. The position numbering corresponds to sequence number 227.

[0276] [Table 1]

[0277] 3. Influenza polypeptide as a non-ferritin polypeptide In some embodiments, the ferritin polypeptide described herein further comprises an influenza polypeptide. In some embodiments, the non-ferritin polypeptide of the antigenic ferritin polypeptide described herein is an influenza polypeptide. In some embodiments, the antigenic ferritin polypeptide described herein is an antigenic influenza-ferritin polypeptide.

[0278] a) HA and NA polypeptides In some embodiments, the influenza polypeptide is an HA or NA polypeptide containing full-length or partial-length HA or NA. Any HA or NA polypeptide may be used. The HA or NA polypeptide may be naturally occurring or may be derived from nature. In some embodiments, HA is derived from one of H1 to H18. In some embodiments, NA is derived from one of N1 to N11.

[0279] In some embodiments, the HA polypeptide includes an HA ectodomain. The HA ectodomain may be derived from any subtype of influenza, including H1 to H18.

[0280] In some embodiments, the HA polypeptide includes the stem region of HA. The stem region of HA may be derived from any subtype of influenza, including H1 to H18.

[0281] In some embodiments, the HA polypeptide is derived from influenza A virus. Influenza A viruses include A / Puerto Rico / 1934, A / Weiss / 1 / 1943, A / Fort Monmouth / 1 / 1947 (FM47), A / Malaysia / 302 / 54 (MAL54), A / Denver / 1 / 1957 (DV57), A / New Jersey / 8 / 1976, A / USSR / 90 / 1977, A / Hong Kong / 117 / 1977 (HK77), A / Brazil / 11 / 1978, and A Possible locations include Chile (1 / 1983), Taiwan (1 / 1986), Texas (36 / 1991), Beijing (262 / 1995), New Caledonia (20 / 1999, NC99), Solomon Islands (6 / 2006), Brisbane (59 / 2007), California (07 / 2009, CA09), Bangladesh (2021 / 2012), or Vietnam (3050 / 2013).

[0282] In some embodiments, the HA polypeptide is derived from the H1 influenza virus. In some embodiments, the H1 virus is A / South Carolina / 1 / 18.

[0283] In some embodiments, the HA polypeptide is derived from the H2 influenza virus. In some embodiments, the H2 virus is the H2N2 influenza A virus that caused the 1957 pandemic.

[0284] In some embodiments, the HA polypeptide is derived from an H3 influenza virus. In some embodiments, the H3 influenza virus is an H3N8 virus. In some embodiments, the H3N8 virus is Uma-Ohio 2003. In some embodiments, the H3N8 virus is Uma-Bari 2005. In some embodiments, the H3N8 virus is Uma-Aboin 2003. In some embodiments, the H3 influenza virus is an H3N2 virus. In some embodiments, the H3N2 virus is Perth 2009. In some embodiments, the H3N2 virus is Victoria 2011.

[0285] In some embodiments, the HA polypeptide is derived from the H5 influenza virus. In some embodiments, the H5 influenza virus is the H5 / N1 virus. In some embodiments, the H5 / N1 virus is Indonesia 2005. In some embodiments, the H5 / N1 virus is Indian goose 2005. In some embodiments, the H5 / N1 virus is Whooper swan 2005. In some embodiments, the H5 / N1 virus is Mallard / Hua Dong 2003.

[0286] In some embodiments, the HA polypeptide is derived from influenza B virus. In some embodiments, the B virus is Wisconsin 2010. In some embodiments, the B virus is Massachusetts 2012. In some embodiments, the B virus is Phuket 2013. In some embodiments, the B virus is Brisbane 2008. In some embodiments, the Brisbane 2008 sequence contains the D197N mutation. This mutation has been found to improve the expression of this nanoparticle, and it is a naturally occurring mutation in other strains such as B / Brisbane / 2009 and B / Phuket / 2013. This amino acid may be involved in sialic acid receptor contact.

[0287] In some embodiments, the HA polypeptide includes a computationally optimized broadly reactive antigen (COBRA) generated according to the examples in Giles BM and Ross TM, Vaccine 29(16):3043-54 (2011) or Carter DM et al., J Virol 90:4720-4734 (2016).

[0288] In some embodiments, the COBRA sequence is generated from human H1N1 influenza sequences. In some embodiments, the COBRA sequence is generated from human H1N1 influenza sequences from the period 1999–2012. An example COBRA sequence generated from human H1N1 influenza sequences from the period 1999–2012 is COBRA X6, which is included in SEQ ID NO: 329. In some embodiments, the COBRA sequence is generated from human H1N1 strains from the periods 1933–1957 and 2009–2011, in addition to swine H1N1 influenza strains from the period 1931–1998. An example COBRA sequence generated from human H1N1 strains from the periods 1933–1957 and 2009–2011, in addition to swine H1N1 influenza strains from the period 1931–1998 is COBRA P1, which is included in SEQ ID NO: 327.

[0289] In some embodiments, the COBRA sequence is X3. In some embodiments, the COBRA sequence is hCOBRA-2, which is generated from H5N1.

[0290] A mutation (Y98F) that removes the HA receptor binding site was described by Whittle et al., Journal of Virology 11(8): pp. 4047-4057 (2014). In some embodiments, the HA polypeptide contains the Y98F mutation. Any of the above HAs can be modified to contain the Y98F mutation. In some embodiments, the HA is derived from the H1 / New Caledonia / 1999 (NC99) virus and contains the Y98F mutation.

[0291] 4. Borrelia and OspA polypeptides as non-ferritin polypeptides In some embodiments, the ferritin polypeptide described herein further comprises a borrelia polypeptide. In some embodiments, the non-ferritin polypeptide of the antigenic ferritin polypeptide is a borrelia polypeptide. In some embodiments, the borrelia polypeptide is derived from B. burgdorferi. In some embodiments, the borrelia polypeptide is derived from a borrelia species corresponding to serotype 1, 2, 3, 4, 5, 6, or 7. In some embodiments, the borrelia can be carried by ticks of the genus Ixodes.

[0292] In some embodiments, the Borrelia polypeptide is an OspA polypeptide. In some embodiments, the antigenic ferritin polypeptide described herein is also an antigenic OspA polypeptide.

[0293] In some embodiments, the OspA polypeptide comprises Borrelia's modified outer surface protein A (OspA). OspA exists in multiple serotypes, as defined by its reactivity with monoclonal antibodies against different species of OspA (see Wilske et al., J Clin Microbio 31(2): pp. 340-350 (1993)). These serotypes correlate with different genetic species of Borrelia bacteria. In some embodiments, OspA is one of serotypes 1-7. In some embodiments, OspA is derived from Borrelia burgdorferi, Borrelia mayonii, Borrelia afzelii, Borrelia garinii, or Borrelia bavariensis. In some embodiments, OspA is Borrelia burgdorferi OspA. In some embodiments, Borrelia can be carried by ticks of the genus Ixodes. In some embodiments, Borrelia is Borrelia burgdorferi, Borrelia mayonii, Borrelia afzeri, Borrelia gallini, or Borrelia bavaliensis.

[0294] In some embodiments, the OspA polypeptide is an OspA serotype 1 polypeptide, such as the OspA serotype 1 ectodomain. Literature has reported that the OspA serotype 1 epitope at amino acids 165-173 of SEQ ID NO: 83 has homology to human leukocyte function-associated antigen-1 (hLFA-1), i.e., a fragment of the sequence of SEQ ID NO: 78 (see Gross, DM et al., Science 281(5377): pp. 703-706 (1998)). Amino acids 165-173 of SEQ ID NO: 83 are shown as an isolated nonapeptide in SEQ ID NO: 77 and are referred to as the hLFA-1 homology site. SEQ ID NO: 83 is an exemplary wild-type serotype 1 OspA sequence used herein as a reference sequence for consideration of amino acid positions in OspA. This homology site may play a role in the development of Lyme arthritis, including antibiotic-resistant Lyme arthritis. In some embodiments, the OspA polypeptide comprises a modified OspA serotype 1 polypeptide of Borrelia, where the modified OspA does not contain the sequence of SEQ ID NO: 77. Such polypeptides may have improved safety, for example, a reduced risk of inducing an autoimmune response, when used to induce antibodies. In some embodiments, the OspA serotype 1 polypeptide has one or more modifications that reduce its identity with hLFA-1. Any modifications that reduce homology with SEQ ID NO: 78, reduce identity with SEQ ID NO: 78, or introduce one or more non-conservative substitutions compared to SEQ ID NO: 78 are included.

[0295] In some embodiments, the OspA polypeptide comprises a Borrelia OspA serotype 1 polypeptide, and the OspA polypeptide does not contain the sequence of SEQ ID NO: 77. In some embodiments, the OspA polypeptide comprises an ectodomain of OspA serotype 1, and the ectodomain does not contain the sequence of SEQ ID NO: 77. In some embodiments, the OspA serotype 1 polypeptide comprises a sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% identity with any one of the sequences of SEQ ID NOs: 94-102.

[0296] "Reducing homology" encompasses reducing sequence identity and / or sequence similarity, where each member of a set of amino acids listed as a conserved substitution in Table 4 below is considered similar to the original residue and the other members of the set; for example, column 1 of the table shows that alanine, valine, leucine, and isoleucine are similar to each other, and column 8 shows that alanine and glycine are similar to each other. Similarity is not transitive, and therefore, for example, isoleucine and glycine are not considered similar. In some embodiments, the OspA polypeptide comprises an OspA serotype 1 protein having reduced homology to hLFA-1 compared to the wild-type OspA serotype. In some embodiments, the modified OspA comprises an OspA serotype 1 that includes modifications to any one or more amino acids of SEQ ID NO: 77. In some embodiments, the modifications to SEQ ID NO: 77 are non-conserved amino acid substitutions. Non-conserved substitutions are substitutions that are different from the conserved substitutions shown in the table below.

[0297] [Table 2]

[0298] In some embodiments, the OspA polypeptide comprises an OspA serotype 1 protein in which one or more amino acids in SEQ ID NO: 77 are replaced with the corresponding amino acids of a non-serotype 1 OspA, such as OspA of serotype 2, 3, 4, 5, 6, or 7. In some embodiments, each of the amino acids in SEQ ID NO: 77 is replaced with the corresponding amino acids of OspA of serotype 2, 3, 4, 5, 6, or 7. In some embodiments, the amino acids in SEQ ID NO: 77 are replaced with the corresponding amino acids of serotype 2 (S2, SEQ ID NO: 79) or serotype 3 (S3, SEQ ID NO: 80).

[0299] In some embodiments, the OspA polypeptide includes SEQ ID NO: 81. In some embodiments, the OspA polypeptide includes SEQ ID NO: 82. SEQ ID NOs: 81 and 82 are intended to replace SEQ ID NO: 77, thereby reducing homology to SEQ ID NO: 78.

[0300] In some embodiments, the OspA polypeptide is a full-length OspA (including, for example, a transmembrane domain and an ectodomain that may or may not include modifications to reduce homology to hLFA-1 as described herein).

[0301] In some embodiments, the OspA polypeptide lacks a transmembrane domain. In some embodiments, the polypeptide lacks a portion of the transmembrane domain, for example, amino acids 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 at the N-terminus of the wild-type OspA sequence. In some embodiments, the OspA polypeptide lacks amino acid 17 of OspA serotype 1, or a segment containing the corresponding position in its homolog, as identified by pairwise or structural alignment. In some embodiments, the OspA polypeptide lacks at least amino acids 1–17 of OspA, such as OspA serotype 1, or the corresponding amino acids in its homolog, as identified by pairwise or structural alignment. In some embodiments, the OspA polypeptide lacks at least the N-terminal 18, 19, 20, 21, 22, 23, or 24 amino acids of OspA, such as OspA serotype 1, or the corresponding amino acids in its homolog, as identified by pairwise or structural alignment. In some embodiments, the OspA polypeptide lacks amino acids 1-25 of OspA, such as OspA serotype 1, or the corresponding amino acids in its homolog, as identified by pairwise or structural alignment. In some embodiments, the OspA polypeptide lacks amino acids 1-26 of OspA serotype 1, or the corresponding amino acids in its homolog, as identified by pairwise or structural alignment. To avoid misunderstanding, the absence of a transmembrane domain does not require the polypeptide to lack an N-terminal methionine; for example, a polypeptide in which the first residue is methionine, the second residue corresponds to residue 26 of wild-type OspA, followed by residues corresponding to wild-type OspA residues such as the 27th, 28th, etc., is considered to lack a transmembrane domain. In some embodiments, polypeptides containing OspA polypeptides lack lipid attachment sites, such as the lipid attachment site contained within the transmembrane domain of wild-type OspA serotype 1. In some embodiments, OspA polypeptides lack cysteine ​​17 of OspA serotype 1.In some embodiments, the OspA polypeptide does not contain cysteine ​​corresponding to any of the positions 1-25 of wild-type OspA, for example, SEQ ID NOs. 83-89. In some embodiments, the polypeptide lacks cysteine ​​17 of OspA serotype 1 or has a substitution with cysteine ​​17. In some embodiments, the OspA polypeptide lacks at least a portion of the wild-type OspA transmembrane domain to lack a lipid attachment site. In some embodiments, the OspA polypeptide lacks amino acids that align with amino acids 1-17 of OspA serotype 1.

[0302] In some embodiments, the OspA polypeptide does not contain a palmitoyl group. In some embodiments, the OspA polypeptide does not contain a diacylglycerol group. In some embodiments, the OspA polypeptide is not lipid-added. In some embodiments, the OspA polypeptide lacks a lipid-adding site. In some embodiments, this lipid-adding site is contained within a transmembrane domain. In some embodiments, the lipid-adding site removed is cysteine ​​17 of OspA serotype 1. In some embodiments, the OspA polypeptide either lacks cysteine ​​17 of OspA serotype 1 or has a substitution with cysteine ​​17.

[0303] In some embodiments, the removal of the OspA lipid attachment site and / or the transmembrane domain or part thereof, and / or the absence of palmitoyl and / or diacylglycerol groups, allows for easier protein purification, for example, by improving protein solubility and / or by making the protein more susceptible to purification by techniques such as ion exchange and other forms of chromatography.

[0304] In some embodiments, the OspA polypeptide includes a mammalian leader sequence (also known as a signal sequence). In some embodiments, the mammalian leader sequence, when expressed in mammalian cells, leads to polypeptide secretion.

[0305] In some embodiments, the OspA polypeptide lacks a glycosylation site. Modifications that remove the glycosylation site are described in detail herein. OspA polypeptides according to this disclosure may include any such modifications that can be combined with any other modifications described herein, including modifications to the hLFA-1 homology site and / or deletion of part or all of the transmembrane domain. In some embodiments, the polypeptide does not contain SEQ ID NO: 77 (e.g., having reduced homology to hLFA-1a), has a mutation that reduces glycosylation, and / or lacks a transmembrane domain.

[0306] a) Modification of glycosylation N-linked glycosylation is the binding of a glycan to the amide nitrogen of an asparagine (Asn;N) residue in a protein. The binding process results in a glycosylated protein. Glycosylation can occur at any asparagine residue in a recognized protein that is accessible by glycosylation enzymes after translation, most commonly at accessible asparagine that is part of the NXS / TX site, where the second amino acid residue following asparagine is serine or threonine. Non-human glycosylation patterns (e.g., resulting from the expression of polypeptides containing glycosylation sites in certain non-human cell types) can make polypeptides undesirably reactive when used to induce antibodies. In addition, glycosylation of polypeptides that are not normally glycosylated can alter their immunogenicity. For example, glycosylation can mask important immunogenic epitopes within a protein. Thus, to reduce or eliminate glycosylation, either the asparagine residue or the serine / threonine residue can be modified, for example, by substitution with another amino acid.

[0307] In some embodiments, polypeptides containing the OspA polypeptide are modified to reduce or eliminate glycosylation. In some embodiments, one or more N-glycosylation sites in OspA are removed. In some embodiments, removal of the N-glycosylation site reduces the glycosylation of OspA. In some embodiments, the polypeptide has reduced glycosylation compared to wild-type OspA, such as wild-type serotype 1OspA. In some embodiments, removal of the N-glycosylation site eliminates the glycosylation of OspA.

[0308] In some embodiments, one or more asparagines in OspA are replaced with non-asparagine amino acids. In some embodiments, each asparagine in OspA is replaced with a non-asparagine amino acid. Any native or non-native amino acid found in the protein, such as glutamine, may be used to replace the asparagine. In some embodiments, modifications to reduce or remove glycosylation modify the NXS / TX glycosylation site (the second residue after N is S or T). In some embodiments, the first X and / or the second X in the NXS / TX site are not proline. In some embodiments, modifications to reduce or remove glycosylation are substitutions of N to Q. In some embodiments, modifications to reduce or remove glycosylation are substitutions of S / T to A.

[0309] A detailed discussion of positions that can be modified to reduce or eliminate glycosylation is given below. Position numbers refer to their locations in the full-length OspA sequences provided as SEQ ID NOs. 83–89. It is understood that position numbers should be appropriately adjusted with respect to partial and modified OspA sequences (for example, if an N-terminal deletion results in a true shortening of 25 amino acid residues, the position number should be decreased by 25).

[0310] In some embodiments, modifications to reduce or eliminate glycosylation include substitution of any one or more of N20, N71, N190, N202, and N251 in OspA serotype 1 (SEQ ID NO: 83). In some embodiments, modifications include modifications in each of N71, N190, N202, and N251 in OspA serotype 1. In some embodiments, modifications to reduce or eliminate glycosylation include one or more of N20Q, N71Q, N190Q, N202Q, or N251Q in OspA serotype 1. The corresponding amino acids can be found in OspA of different serotypes by pairwise alignment. Thus, in some embodiments, the asparagine residues substituted in OspA of serotypes 2-7 are amino acid residues that align with N20, N71, N190, N202, and N251 in OspA serotype 1. In some embodiments, modifications to reduce or eliminate glycosylation include the substitution of any one or more Ser or Thr residues at positions 22, 73, 192, 204, and 253 of OspA serotype 1. In some embodiments, modifications include the substitution of one or more Ser or Thr residues with alanine at positions 22, 73, 192, 204, and 253 of OspA serotype 1.

[0311] In some embodiments, modifications to reduce or eliminate glycosylation include substitution of any one or more of N20, N71, N141, N164, N202, and N205 in OspA serotype 2 (SEQ ID NO: 84). In some embodiments, modifications include modifications in each of N20, N71, N141, N164, N202, and N205 in OspA serotype 2. In some embodiments, modifications to reduce or eliminate glycosylation include one or more of N20Q, N71Q, N141Q, N164Q, N202Q, or N205Q in OspA serotype 2. Similar amino acids can be found in different serotypes of OspA by pairwise alignment. Thus, in some embodiments, the asparagine residues substituted in OspA of serotypes 1 or 3-7 are amino acid residues that align with N20, N71, N141, N164, N202, and N205 of OspA serotype 2. In some embodiments, modifications to reduce or eliminate glycosylation include the substitution of any one or more Ser or Thr residues at positions 22, 73, 143, 166, 204, and 207 of OspA serotype 2. In some embodiments, modifications include the substitution of one or more Ser or Thr residues with alanine at positions 22, 73, 143, 166, 204, and 207 of OspA serotype 2.

[0312] In some embodiments, modifications to reduce or eliminate glycosylation include substitution of any one or more of N20, N71, N95, N141, N191, and N203 in OspA serotype 3 (SEQ ID NO: 85). In some embodiments, modifications include modifications in each of N20, N20, N71, N95, N141, N191, and N203 in OspA serotype 3. In some embodiments, modifications to reduce or eliminate glycosylation include one or more of N20Q, N71Q, N95Q, N141Q, N191Q, or N203Q in OspA serotype 3. Similar amino acids can be found in different serotypes of OspA by pairwise alignment. Thus, in some embodiments, the asparagine residues substituted in OspA of serotypes 1-2 or 4-7 are amino acid residues that align with N20, N20, N71, N95, N141, N191, and N203 of OspA serotype 3. In some embodiments, modifications to reduce or eliminate glycosylation include the substitution of any one or more Ser or Thr residues at positions 22, 73, 97, 143, 193, and 205 of OspA serotype 3. In some embodiments, modifications include the substitution of one or more Ser or Thr residues with alanine at positions 22, 73, 97, 143, 193, and 205 of OspA serotype 3.

[0313] In some embodiments, modifications to reduce or eliminate glycosylation include substitution of any one or more of N20, N71, N141, N202, N205, and N219 in OspA serotype 4 (SEQ ID NO: 86). In some embodiments, modifications include modifications in each of N20, N71, N141, N202, N205, and N219 in OspA serotype 4. In some embodiments, modifications to reduce or eliminate glycosylation include one or more of N20Q, N71Q, N141Q, N202Q, N205Q, or N219Q in OspA serotype 4. Similar amino acids can be found in different serotypes of OspA by pairwise alignment. Thus, in some embodiments, the asparagine residues substituted in OspA of serotypes 1-3 or 5-7 are amino acid residues that align with N20, N71, N141, N202, N205, and N219 of OspA serotype 4. In some embodiments, modifications to reduce or eliminate glycosylation include the substitution of any one or more Ser or Thr residues at positions 22, 73, 143, 204, 207, and 221 of OspA serotype 4. In some embodiments, modifications include the substitution of one or more Ser or Thr residues with alanine at positions 22, 73, 143, 204, 207, and 221 of OspA serotype 4.

[0314] In some embodiments, modifications to reduce or eliminate glycosylation include substitution of any one or more of N20, N71, and N141 in OspA serotype 5 (certain serotypes, including serotypes 5-7, contain fewer glycosylation sites than certain other OspA sequences, such as serotype 1). In some embodiments, modifications include modifications at each of N20, N71, and N141 in OspA serotype 5 (SEQ ID NO: 87). In some embodiments, modifications to reduce or eliminate glycosylation include one or more of N20Q, N71Q, or N141Q in OspA serotype 5. Similar amino acids can be found in OspA of different serotypes by pairwise alignment. Thus, in some embodiments, the asparagine residues substituted in OspA of serotypes 1-4, or 6-7, are amino acid residues that align with N20, N71, and N141 in OspA serotype 5. In some embodiments, modifications to reduce or eliminate glycosylation include the substitution of any one or more Ser or Thr residues at positions 22, 73, and 143 of OspA serotype 5. In some embodiments, modifications include the substitution of one or more Ser or Thr residues with alanine at positions 22, 73, and 143 of OspA serotype 5.

[0315] In some embodiments, modifications to reduce or eliminate glycosylation may include substitution of any one or more of N20, N71, and N141 in OspA serotype 6 (SEQ ID NO: 88). In some embodiments, the modification includes modifications in each of N20, N71, and N141 in OspA serotype 6. In some embodiments, the modification to reduce or eliminate glycosylation includes one or more of N20Q, N71Q, or N141Q in OspA serotype 6. Similar amino acids can be found in OspA of different serotypes by pairwise alignment. Thus, in some embodiments, the asparagine residues substituted in OspA of serotypes 1-5, or 7 are amino acid residues that align with N20, N71, and N141 in OspA serotype 6. In some embodiments, modifications to reduce or eliminate glycosylation include substitution of any one or more Ser or Thr residues at positions 22, 73, and 143 of OspA serotype 6. In some embodiments, modifications include substitution of one or more Ser or Thr residues with alanine at positions 22, 73, and 143 of OspA serotype 6.

[0316] In some embodiments, modifications to reduce or eliminate glycosylation include substitution of any one or more of N20, N71, N141, and N191 in OspA serotype 7 (SEQ ID NO: 89). In some embodiments, modifications include modifications in each of N20, N71, N141, and N191 in OspA serotype 7. In some embodiments, modifications to reduce or eliminate glycosylation include one or more of N20Q, N71Q, N141Q, or N191Q in OspA serotype 7. Similar amino acids can be found in OspA of different serotypes by pairwise alignment. Thus, in some embodiments, the asparagine residues substituted in OspA of serotypes 1-6 are amino acid residues that align with N20, N71, N141, and N191 in OspA serotype 7. In some embodiments, modifications to reduce or eliminate glycosylation include the substitution of any one or more Ser or Thr residues at positions 22, 73, 143, and 193 of OspA serotype 7. In some embodiments, modifications include the substitution of one or more Ser or Thr residues with alanine at positions 22, 73, 143, and 193 of OspA serotype 7.

[0317] 5. RSV polypeptide as a non-ferritin polypeptide In some embodiments, the ferritin polypeptide described herein further comprises an RSV polypeptide. In some embodiments, the non-ferritin polypeptide of the antigenic ferritin polypeptide described herein is an RSV polypeptide. In some embodiments, the antigenic ferritin polypeptide described herein is an antigenic RSV polypeptide. The RSV polypeptide may be an RSV F polypeptide, such as any RSV F polypeptide described herein. The RSV F polypeptide may comprise the entire sequence of RSV F or a portion of RSV F. The RSV F polypeptide may comprise one or more modifications (e.g., amino acid substitutions) compared to the wild-type sequence. The RSV polypeptide may be an RSV G polypeptide, such as any RSV G polypeptide described herein.

[0318] a) RSV F polypeptide In some embodiments, the RSV F polypeptide is a full-length or fragmentary wild-type RSV F polypeptide. In some embodiments, the epitope of the RSV polypeptide shared between pre-fusion RSV F and post-fusion RSV F is blocked. Blocking the epitope reduces or eliminates the production of antibodies against the epitope when the antigenic RSV polypeptide is administered to a target. This can increase the proportion of antibodies that target the epitope specifically for a particular conformation of F, such as the pre-fusion conformation. Since F has a pre-fusion conformation of the virus that has not yet entered a cell, an increase in the proportion of antibodies targeting pre-fusion F can provide a greater degree of neutralization (expressed, for example, as a neutralization-to-binding ratio as described herein). Blocking can be achieved by manipulating a bulky portion, such as an N-glycan, proximal to the shared epitope. For example, an N-glycosylation site not present in wild-type F can be added, for example, by mutating an appropriate residue to asparagine. In some embodiments, the blocked epitope is the epitope of antigenic site 1 of RSV F. In some embodiments, two or more epitopes shared between pre-fusion and post-fusion RSV F are blocked. In some embodiments, two or more epitopes of antigenic site 1 of RSV F are blocked. In some embodiments, one or more, or all, epitopes that topographically overlap with the blocked epitope are similarly blocked, and the blocked epitope is optionally the epitope of antigenic site 1 of RSV F.

[0319] In some embodiments, the RSV F polypeptide contains asparagine corresponding to positions 328, 348, or 507 of SEQ ID NO: 526. In some embodiments, the polypeptide contains asparagine corresponding to at least two of positions 328, 348, or 507 of SEQ ID NO: 526. In some embodiments, the polypeptide contains asparagine corresponding to positions 328, 348, or 507 of SEQ ID NO: 526. As described in the examples, such asparagine has been found to function as a glycosylation site. Furthermore, although we do not wish to be bound by any particular theory, glycans at these sites may inhibit the development of antibodies against nearby epitopes, including epitopes common to pre- and post-fusion RSV F proteins, when the polypeptide is administered to a target. In some embodiments, glucosylation of asparagine corresponding to positions 328, 348, or 507 of SEQ ID NO: 26 blocks at least one epitope shared between pre- and post-fusion RSV F, such as the epitope at antigenic site 1. Inhibiting the generation of antibodies against epitopes common to pre- and post-fusion RSV proteins may be beneficial because it can direct the generation of antibodies against epitopes specific to the pre-fusion RSV F protein, such as the site 0 epitope, which may have more effective neutralizing activity than antibodies against other RSV F epitopes. The site 0 epitope is associated with amino acid residues 62-69 and 196-209 of SEQ ID NO: 526. Therefore, in some embodiments, the RSV F polypeptide contains amino acid residues 62-69 and 196-209 of SEQ ID NO: 526.

[0320] It should be noted that the constructs described herein may have deletions or substitutions of different lengths compared to the wild-type RSV F. For example, in SEQ ID NO: 523 and other constructs, replacing positions 98–144 of the wild-type sequence (SEQ ID NO: 526) with GSGNVGL (positions 98–104 of SEQ ID NO: 523; similarly, SEQ ID NO: 531) results in a true removal of 40 amino acids, thereby causing positions 328, 348, or 507 of SEQ ID NO: 526 to correspond to positions 288, 308, and 467 of SEQ ID NO: 523. In general, the positions in the constructs described herein can be mapped onto the wild-type sequence of SEQ ID NO: 526 by pairwise alignment, for example, using the Needleman-Wunsch algorithm with standard parameters (EBLOSUM62 matrix, gap penalty 10, gap elongation penalty 0.5). Similarly, see also the consideration of structural alignment provided herein as an alternative approach to identifying corresponding positions.

[0321] In some embodiments, the RSV F polypeptide includes mutations that add glycans to block epitopes on the pre-fusion antigen that are structurally similar to epitopes on the surface of the pre-fusion RSV F. In some embodiments, the glycans are added to specifically block epitopes that may be present in the pre-fusion conformation of RSV F. In some embodiments, the glycans are added to block epitopes that may be present in the pre-fusion conformation of RSV F, but do not affect one or more epitopes present in the pre-fusion conformation of RSV F, such as the site 0 epitope.

[0322] In some embodiments, glycans added at one or more of the above glycosylation sites increase secretion in expression systems such as mammalian cells compared to other constructs.

[0323] In some embodiments, the RSV F polypeptide contains a sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identity with amino acids 1-478 of SEQ ID NO: 517. In some embodiments, the RSV F polypeptide contains a sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identity with the sequence of SEQ ID NO: 517. In some embodiments, the RSV F polypeptide contains amino acids 1-478 of SEQ ID NO: 517. In some embodiments, the RSV F polypeptide contains the sequence of SEQ ID NO: 517.

[0324] In some embodiments, the RSV F polypeptide contains a sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identity with amino acids 1-478 of SEQ ID NO: 523. In some embodiments, the RSV F polypeptide contains a sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identity with the sequence of SEQ ID NO: 523. In some embodiments, the RSV F polypeptide contains amino acids 1-478 of SEQ ID NO: 523. In some embodiments, the RSV F polypeptide contains the sequence of SEQ ID NO: 523.

[0325] In some embodiments, the RSV F polypeptide includes a DS-CAV1 sequence (e.g., described in McLellan, JS et al., Science 342(6158): pp. 592-598 (2013)) (SEQ ID NO: 525) from which further modifications can be made to include at least one, two, or three of the above-mentioned asparagines.

[0326] In some embodiments, the polypeptide further comprises a ferritin protein. The ferritin protein may further comprise any or a combination of the features described below in the chapter on ferritin.

[0327] The RSV F polypeptide may, or in addition, include any additional features described in the following discussion, or any feasible combination of such features.

[0328] single stranded construct In some embodiments, the RSV F polypeptide is a single-stranded construct, such as an RSV F polypeptide lacking a furin cleavage site. In some embodiments, RSV F contains one or more furin cleavage sites. The furin cleavage site-lacking construct is expressed as a single polypeptide that is not cleaved into the biological F1 / F2 fragments of the original F protein.

[0329] Amino acid substitutions In some embodiments, RSV F contains a single amino acid substitution compared to the wild-type sequence. In some embodiments, RSV F contains one more single amino acid substitution compared to the wild-type sequence, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 substitutions. An exemplary wild-type sequence is SEQ ID NO: 526.

[0330] In some embodiments, amino acid substitutions or pairs of amino acid substitutions are protomer-stabilizing substitutions. Exemplary substitutions that can be protomer-stabilizing are V207L;N228F;I217V and E218F;I221L and E222M; or Q224A and Q225L, using the positional numbering in Sequence ID No. 526.

[0331] In some embodiments, amino acid substitutions or pairs of amino acid substitutions constitute interprotomer stabilization. Exemplary substitutions that can constitute interprotomer stabilization are V220I, as well as A74L and Q81L, using the positional numbering in SEQ ID NO: 526.

[0332] In some embodiments, amino acid substitutions are expected to stabilize the helix, i.e., the helix domain of RSV F. Stabilization of the helix domain may contribute to the stability of the site 0 epitope and the stability of the pre-fusion conformation of RSV F. Exemplary substitutions that may cause helix stabilization are N216P or I217P, using the positional numbering in SEQ ID NO: 526.

[0333] In some embodiments, the amino acid substitution is helix capping. In some embodiments, the amino acid substitution is helix PRO capping. Helix capping is based on the biophysical observation that a proline residue mutation site in the alpha-helix may interfere with helix formation, but proline at the N-terminus of the helix region may help induce helix formation by stabilizing the PHI / PSI bond angle. Exemplary substitutions that can be helix capping are N216P or I217P, using the positional numbering in SEQ ID NO: 526.

[0334] In some embodiments, amino acid substitutions replace disulfide mutations in DS-CAV1. In some embodiments, the manipulated disulfide of DS-CAV1 is reverted to the wild type (C69S and / or C212S mutations in DS-CAV1, using the positional numbering of SEQ ID NO: 526). In some embodiments, one or more C residues in DS-CAV1 are replaced with S residues to remove the disulfide bond. In some embodiments, C69S or C212S substitutions using the positional numbering of SEQ ID NO: 526 remove the disulfide bond. In some embodiments, the RSV F polypeptide contains both C69S and C212S using the positional numbering of SEQ ID NO: 526. In some embodiments, such cysteine ​​substitutions and the resulting removal of the disulfide bond block the reduction of the RSV F polypeptide (i.e., the acceptance of electrons from the reducing agent). In some embodiments, the I217P substitution using the positional numbering of SEQ ID NO: 526 is included in the antigen instead of substitutions at C69 and / or C212. Position 217 in sequence number 526 corresponds to position 177 in sequence number 523.

[0335] In some embodiments, amino acid substitutions prevent proteolysis by trypsin or trypsin-like proteases. In some embodiments, the amino acid substitutions that prevent such proteolysis are located in the 7-residue repeat region B (HRB) of RSV F. The appearance of fragments consistent with proteolysis of RSV F-ferritin constructs containing the wild-type HRB region suggests that lysine or arginine in this region is a target for proteolysis. Amino acid substitutions that remove a K or R residue can be called knockouts (KO). In some embodiments, K or R is used instead of L or Q. In some embodiments, K is used instead of L or Q. In some embodiments, the RSV F polypeptide contains K498L and / or K508Q using the positional numbering in SEQ ID NO: 526. The corresponding positions in SEQ ID NO: 523 are 458 and 468, respectively. In some embodiments, the RSV F polypeptide contains both K498L and K508Q.

[0336] In some embodiments, amino acid substitutions add glycans. In some embodiments, amino acid substitutions increase glycosylation by adding glycans to the RSV F polypeptide. Substitutions that add glycans can also be called manipulated glycosylation compared to intrinsic glycosylation (which does not have additional glycans).

[0337] In some embodiments, the amino acid substitution that adds glycan was a substitution to N. In some embodiments, the amino acid substitution to N enables N-linked glycosylation. In some embodiments, the substitution to N is accompanied by a substitution to T or S at a second amino acid position C-terminal to N, which forms an NxT / S glycosylation motif. In some embodiments, N is exposed on the surface. Mutations that increase glycosylation can provide increased expression of polypeptides including RSV F polypeptide, as shown in the following examples.

[0338] Changes in the properties of the RSV F polypeptide based on modification Modifications to the amino acid sequence of RSV F can alter the properties of the RSV F polypeptide. These properties may include any structural or functional characteristics of the RSV F polypeptide.

[0339] In some embodiments, a single modification to the amino acid sequence alters several properties of the RSV F polypeptide. In some embodiments, the RSV F polypeptide may include multiple modifications that alter different properties of the RSV F polypeptide. In some embodiments, multiple modifications result in a greater change in the properties of the RSV F polypeptide.

[0340] In some embodiments, multiple modifications may have an additive effect on specific properties. For example, two amino acid substitutions that add glycans can result in a greater increase in glycosylation of the RSV F polypeptide compared to a single amino acid substitution.

[0341] In some embodiments, multiple modifications affect different properties of the RSV F polypeptide. For example, one or more amino acid substitutions that increase glycosylation can be fabricated together with one or more amino acid substitutions that block reduction.

[0342] In some embodiments, modifications to the RSV F polypeptide stabilize the pre-fusion conformation.

[0343] In some embodiments, the modification stabilizes the site 0 epitope (also known as antigenic site 0) of pre-fusion RSV F, as described, for example, by McLellan et al., Science 340(6136): pp. 1113-1117 (2013). In some embodiments, the modification that stabilizes the site 0 epitope is protomer stabilization. In some embodiments, the modification that stabilizes the site 0 epitope stabilizes pre-fusion F when measured by site 0 and site V binding, respectively, by binding to antibody D25 or AM14.

[0344] In some embodiments, the modification increases the expression of RSV F in the expression system. In some embodiments, the modification increases the secretion of RSV F in the expression system. In some embodiments, the modification increases the stability of recombinant RSV F after expression. This change may be present in any type of expression system, such as bacteria, fungi, insects, or mammals.

[0345] In some embodiments, amino acid substitutions introducing proline increase expression compared to other constructs. In some embodiments, amino acid substitutions adding glycans increase expression compared to other constructs. In some embodiments, amino acid substitutions replacing K or R with other amino acids increase expression compared to other constructs. Observable increases in expression may be due to any mechanism that increases the yield of fermentation or other production processes, including relative inhibition of protease cleavage or degradation in the host cell or extracellular environment and / or increased stability. In some embodiments, amino acid substitutions replacing one or more K residues in the HRB region of RSV F with other amino acids increase expression compared to other constructs.

[0346] In some embodiments, amino acid substitutions, where K is replaced with another amino acid, increase the stability of the RSV F polypeptide. In some embodiments, amino acid substitutions, where one or more K residues in the HRB region of RSV F are replaced with other amino acids, increase the stability of the RSV F polypeptide. In some embodiments, this increase in stability is due to a reduction in protease cleavage.

[0347] In some embodiments, RSV F includes mutations that remove a disulfide, for example, to prevent conjugation after reduction. In some embodiments, the I217P substitution blocks the reduction of the RSV F polypeptide. In some embodiments, amino acid substitutions that replace K with another amino acid block the reduction of the RSV F polypeptide in the presence of a reducing agent.

[0348] In some embodiments, single-stranded constructs increase expression compared to other constructs.

[0349] In some embodiments, the RSV F polypeptide comprises the DS-CAV1 sequence (SEQ ID NO: 525) (for example, as described in McLellan, JS et al., Science 342(6158): pp. 592-598 (2013)). In some embodiments, the RSV F polypeptide comprises the DS-CAV1 sequence from which further modifications are made, including at least one, two, or three of the above-mentioned asparagines.

[0350] b) RSV G polypeptide As used herein, the RSV G polypeptide may comprise the entire RSV G sequence or a portion of the RSV G sequence. The RSV G polypeptide may include modifications compared to the wild-type sequence. In some embodiments, the RSV G polypeptide is a modified RSV G compared to the wild-type RSV G (SEQ ID NO: 527). In some embodiments, these modifications are changes in the amino acids of the RSV G polypeptide compared to the wild-type RSV G.

[0351] In some embodiments, the RSV G polypeptide comprises all or part of the ectodomain of RSV G (SEQ ID NO: 528 or the corresponding position). In some embodiments, the RSV G polypeptide comprises all or part of the Gcc region (amino acids 151-193 of RSV G (SEQ ID NO: 527)). In some embodiments, the RSV G polypeptide comprises a CX3C motif. In some embodiments, the RSV G polypeptide binds to the CX3CR1 receptor. The Gcc region is conserved and immunogenic and can thus be used to induce antibodies with broad activity against RSV strains. In some embodiments, RSV Gcc strain A, shown in SEQ ID NO: 536, is provided. In some embodiments, RSV Gcc strain B, shown in SEQ ID NO: 537, is provided.

[0352] In some embodiments, the RSV G polypeptide is not glycosylated. For example, the RSV G polypeptide may lack the NXS / TX glycosylation site due to cleavage or mutation of an N or S / T residue (e.g., to Q or A, respectively) or a combination thereof.

[0353] In some embodiments, the RSV G polypeptide can be conjugated to ferritin as described herein, for example, via cysteine ​​exposed on the surface of ferritin. In some embodiments, the ferritin nanoparticles are a fusion protein also comprising the RSV F polypeptide and the ferritin protein described above, such as any polypeptide containing the RSV F polypeptide.

[0354] D. Exemplary compositions, kits, nucleic acids, uses, and methods In some embodiments, the present invention provides a method for immunizing a subject against infection by a pathogen. The present invention further provides a method for inducing an immune response to a pathogen in a subject. In some embodiments, the method of the present invention includes the step of administering an effective amount of a pharmaceutical composition described herein to a subject. In some embodiments, the method of the present invention includes the step of administering an effective amount of an antigenic ferritin polypeptide or nanoparticles described herein to a subject.

[0355] In some embodiments, the compositions described herein are administered to a subject, including a human, to immunize the subject against infection by a pathogen. In some embodiments, the compositions described herein are for use in immunizing a subject such as a human. In some embodiments, the administered composition comprises a polypeptide containing one of the sequences SEQ ID NOs: 1-76, 301-343, 401-403, 410, 413-414, 417-427, or 501-523. In some embodiments, the administration immunizes against influenza, EBV, RSV, or Borrelia.

[0356] Similarly, the composition may be administered to induce a protective immune response against future infections. In some embodiments, the future infections are influenza, EBV, RSV, or Borrelia infections.

[0357] In some embodiments, the protective immune response reduces the incidence of hospitalization. In some embodiments, if the composition contains an influenza polypeptide, the protective immune response reduces the incidence of laboratory-confirmed influenza infection. In some embodiments, if the composition contains an EBV polypeptide, the protective immune response reduces the incidence of EBV infection, mononucleosis, complications caused by mononucleosis (e.g., hepatitis, encephalitis, severe hemolytic anemia, or splenomegaly), nasopharyngeal cancer, gastric cancer, or B lymphoma (including Burkitt or Hodgkin lymphoma). In some embodiments, if the composition contains an RSV polypeptide, the protective immune response reduces the incidence of RSV infection, pneumonia, bronchitis, or asthma. In some embodiments, if the composition contains a Borrelia polypeptide, the protective immune response reduces the incidence of acute or chronic Lyme disease, including joint inflammation, neurological symptoms, cognitive impairment, or irregular heart rhythm.

[0358] 1. Target In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0359] In some embodiments, the subjects are adults (aged 18 or older or equal to 18). In some embodiments, the subjects are children or adolescents (under 18). In some embodiments, the subjects are elderly (aged 60 or older). In some embodiments, the subjects are non-elderly adults (aged 18 or older or equal to 18, and under 60 or equal to 60).

[0360] In some embodiments, the composition is administered to the target group in more than one dose. In some embodiments, booster doses improve the immune response.

[0361] In some embodiments, one or more of the antigenic polypeptides or compositions described herein are intended for use in mammals such as primates (e.g., monkeys (e.g., macaques such as rhesus macaques or crab-eating macaques) or non-human primates such as apes), rodents (e.g., mice or rats), or domestic mammals (e.g., dogs, rabbits, cats, horses, sheep, cattle, goats, camels, or donkeys). In some embodiments, one or more of the antigenic polypeptides or compositions described herein are intended for use in birds such as poultry (e.g., chickens, turkeys, ducks, geese, guinea fowl, or swans).

[0362] 2. Adjuvant When used herein, adjuvants may be conjugated to ferritin via surface-exposed amino acids, such as cysteine. Unconjugated adjuvants may also be administered to subjects together with the antigenic ferritin polypeptide described herein. In some embodiments, administration of an adjuvant together with the antigenic ferritin polypeptide produces higher titer antibodies against the non-ferritin polypeptide in subjects compared to administration of the non-ferritin polypeptide alone or the antigenic ferritin polypeptide alone without an adjuvant. Adjuvants may promote an earlier, stronger, or more sustained immune response to the antigenic polypeptide.

[0363] In some embodiments, the composition contains one adjuvant. In some embodiments, the composition contains more than one adjuvant. In some embodiments, the composition does not contain any adjuvants.

[0364] In some embodiments, the adjuvant contains aluminum. In some embodiments, the adjuvant is aluminum phosphate. In some embodiments, the adjuvant is alum (Alyhydrogel '85 2%; Brenntag - catalog number 21645-51-2).

[0365] In some embodiments, the adjuvant is an organic adjuvant. In some embodiments, the adjuvant is an oil-based adjuvant. In some embodiments, the adjuvant includes an oil-in-water nanoemulsifier.

[0366] In some embodiments, the adjuvant contains squalene. In some embodiments, the squalene-containing adjuvant is Ribi (Sigma adjuvant system catalog number S6322-1vl), Addavax® MF59, AS03, or AF03 (see U.S. Patent No. 9,703,095). In some embodiments, the squalene-containing adjuvant is a nanoemulsion.

[0367] In some embodiments, the adjuvant comprises a polyacrylic acid polymer (PAA). In some embodiments, the adjuvant comprising PAA is SPA09 (see WO2017218819).

[0368] In some embodiments, the adjuvant includes a non-metabolizable oil. In some embodiments, the adjuvant includes a Freund's incomplete adjuvant (IFA).

[0369] In some embodiments, the adjuvant comprises a non-metabolized oil and dead Mycobacterium tuberculosis. In some embodiments, the adjuvant is Freund's complete adjuvant (CFA).

[0370] In some embodiments, the adjuvant is a lipopolysaccharide. In some embodiments, the adjuvant is monophosphoryl A (MPL or MPLA).

[0371] 3. Pharmaceutical Compositions Various embodiments provide pharmaceutical compositions comprising antigenic ferritin polypeptides and / or related entities as described herein. In some embodiments, the pharmaceutical composition is an immunogenic composition (e.g., a vaccine) capable of inducing an immune response, such as a protective immune response against a pathogen.

[0372] For example, in some embodiments, the pharmaceutical composition may comprise one or more of the following: (1) an antigenic ferritin protein comprising (i) a mutation that replaces a surface-exposed amino acid with cysteine, and (ii) a non-ferritin polypeptide; (2) an antigenic ferritin protein comprising (i) a mutation that replaces a surface-exposed amino acid with cysteine, and an immunostimulatory moiety linked to the cysteine; and (ii) a non-ferritin polypeptide; (3) an antigenic ferritin protein comprising (i) a surface-exposed cysteine, (ii) an N-terminal peptide linker to the ferritin protein, and (iii) an N-terminal non-ferritin polypeptide to the peptide linker (4)(i) mutations that replace surface-exposed amino acids with cysteine ​​and immunostimulatory moieties linked to cysteine, (ii) mutations that replace the internal cysteine ​​at position 31 of H. pylori ferritin, or the internal cysteine ​​at a position similar to position 31 of non-H. pylori ferritin, as identified by pairwise or structural alignment, with a non-cysteine ​​amino acid, (iii) mutations that replace surface-exposed asparagine with a non-asparagine amino acid, and (iv) antigenic ferritin proteins containing non-ferritin polypeptides; or (5) ferritin particles containing the aforementioned ferritin proteins.

[0373] In some embodiments, the present invention provides a pharmaceutical composition comprising an antibody or other active substance related to the antigenic polypeptide described herein. In one embodiment, the pharmaceutical composition comprises an antibody that binds to and / or competes with the antigenic polypeptide described herein. Alternatively, the antibody may recognize viral particles or bacteria containing the non-ferritin polypeptide component of the antigenic polypeptide described herein.

[0374] In some embodiments, the pharmaceutical compositions described herein are administered alone or in combination with one or more active ingredients for enhancing the immune response, such as adjuvants described herein. In some embodiments, the pharmaceutical compositions further comprise the adjuvants described herein.

[0375] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. As used herein, the term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. In exemplary embodiments, the carrier may include sterile liquids, such as water, and oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc., of petroleum, animal, plant, or synthetic origin. In some embodiments, the carrier is one or more solid components or comprises them. Pharmaceutically acceptable carriers may also include, but are not limited to, saline, buffered saline, dextrose, glycerol, ethanol, and combinations thereof. As used herein, an excipient is any non-therapeutic agent that can be included in the pharmaceutical composition to provide or contribute to, for example, a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, but are not limited to, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. In various embodiments, the pharmaceutical composition is sterile.

[0376] In some embodiments, the pharmaceutical composition contains trace amounts of wetting agents or emulsifiers, or pH buffers. In some embodiments, the pharmaceutical composition may contain any variety of additives such as stabilizers, buffers, or preservatives. In addition, it may contain auxiliaries, stabilizers, thickeners, lubricants, and colorants.

[0377] In various embodiments, pharmaceutical compositions can be formulated to suit any desired mode of administration. For example, pharmaceutical compositions can take the form of liquids, suspensions, emulsions, drops, tablets, pills, pellets, capsules, capsules containing liquid, gelatin capsules, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, lyophilized powders, freeze suspensions, dry powders, or any other form suitable for use. General studies on the formulation and manufacture of pharmaceuticals are incorporated herein by reference, for example, Remington's Pharmaceutical Sciences, 19 th This can be found in ed., Mack Publishing Co., Easton, PA, 1995.

[0378] The pharmaceutical composition may be administered via any route of administration. These routes include, for example, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, mucosal, epidural, sublingual, intranasal, intracerebral, vaginal, transdermal, rectal, intratracheal infusion, bronchial infusion, inhalation, or topical administration. Administration may be topical or systemic. In some embodiments, administration is carried out orally. In other embodiments, administration is by parenteral injection. In some examples, administration results in the release of the antigenic ferritin polypeptide described herein into the bloodstream. The mode of administration may be left to the discretion of the physician.

[0379] In some embodiments, the pharmaceutical composition is suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, and subcutaneous). Such compositions can be formulated, for example, as solutions, suspensions, dispersants, emulsions, etc. They may also be manufactured in the form of sterile solid compositions (e.g., lyophilized compositions) that can be dissolved or suspended in a sterile injectable medium immediately before use. For example, parenteral administration can be achieved by injection. In such embodiments, the injectable preparation is prepared in its usual form, i.e., a liquid solution or suspension, a solid form suitable for a solution or suspension in a liquid before injection, or an emulsion. In some embodiments, the injectable solutions and suspensions are prepared from sterile powders, lyophilized powders, or granules.

[0380] In further embodiments, the pharmaceutical composition is formulated for delivery by inhalation (e.g., direct delivery to the lungs and respiratory tract). For example, the composition may take the form of a nasal spray or any other known aerosol formulation. In some embodiments, the preparation for inhalation or aerosol delivery comprises a plurality of particles. In some embodiments, such preparation may have an average particle size of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or about 13 microns. In some embodiments, the preparation for inhalation or aerosol delivery is formulated as a dry powder. In some embodiments, the preparation for inhalation or aerosol delivery is formulated as a wet powder, for example, by including a wetting agent. In some embodiments, the wetting agent is selected from the group consisting of water, saline solution, or other liquids with a physiological pH.

[0381] In some embodiments, the pharmaceutical composition according to the present invention is administered as drops into the nose or mouth. In some embodiments, the dose may consist of multiple drops (e.g., 1 to 100, 1 to 50, 1 to 20, 1 to 10, 1 to 5 drops, etc.).

[0382] The pharmaceutical compositions of the present invention can be administered in any dose appropriate to achieve a desired outcome. In some embodiments, the desired outcome is the induction of a long-lasting adaptive immune response to a pathogen, such as the origin of non-ferritin polypeptides present in the antigenic ferritin polypeptides in the composition. In some embodiments, the desired outcome is a reduction in the intensity, severity, frequency, and / or delay of the onset of one or more symptoms of an infection. In some embodiments, the desired outcome is the inhibition or prevention of an infection. The required dose varies from subject to subject depending on the species, age, weight, and general condition of the subject, the severity of the infection to be prevented or treated, the specific composition used, and its mode of administration.

[0383] In some embodiments, the pharmaceutical composition according to the present invention is administered in single or multiple doses. In some embodiments, the pharmaceutical composition is administered in multiple doses administered on different days (e.g., a prime-boost vaccination strategy). In some embodiments, the pharmaceutical composition is administered as part of a booster regimen.

[0384] In various embodiments, the pharmaceutical composition is administered concurrently with one or more additional therapeutic agents. Concurrent administration is not necessary if the timing of their administration overlaps in terms of the pharmacological activity of the additional therapeutic agents and the active ingredients in the pharmaceutical composition, thereby producing a combined therapeutic effect. Generally, each active substance is administered according to a dose and time schedule determined for that active substance.

[0385] 4.Nucleic acid / mRNA Similarly, nucleic acids encoding antigenic polypeptides described herein are also provided. In some embodiments, the nucleic acid is mRNA. Any nucleic acid capable of translation resulting in a polypeptide is considered to be mRNA for the purposes of this disclosure.

[0386] 5. Kit Similarly, this specification also provides kits comprising one or more antigenic polypeptides, nucleic acids, antigenic ferritin particles, antigenic lumazine synthase particles, compositions, or pharmaceutical compositions described herein. In some embodiments, the kit further comprises one or more solvents, solutions, buffers, instructions, or desiccants.

[0387] This description and exemplary embodiments should not be construed as limiting. With respect to the subject matter of this specification and the appended claims, unless otherwise specifically indicated, all numerical values ​​expressing quantities, percentages, or proportions, as well as other numerical values ​​used herein and in the claims, should be understood in all examples to be modified by the term “about” to the extent not already so. “About” indicates a degree of variation that does not substantially affect the characteristics of the subject matter described, e.g., variation of 10%, 5%, 2%, or 1% or less. Therefore, unless otherwise indicated, the numerical parameters described in the following specification and the appended claims are approximations that may vary depending on the desired characteristics to be obtained. Each numerical parameter should be interpreted, at least by considering the reported significant figures and applying common rounding techniques, and not as an attempt to limit the application of the claims and the doctrine of equivalents.

[0388] When used herein and in the appended claims, the singular forms “a,” “an,” and “it,” as well as any singular form of any other term, should be noted to include multiple referents unless explicitly and explicitly limited to one. When used herein, the term “includes” and its grammatical variations are intended to be non-restrictive, and the citation of an item in a list does not exclude other similar items that may be substituted for or added to the listed item. The term “or” is used in an inclusive sense and is equivalent to “and / or” unless the text indicates otherwise.

[0389] [Table 3] [Table 4] [Table 5] [Table 6] Table 7 Table 8 Table 9 Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16 Table 17 Table 18 Table 19 Table 20 Table 21 Table 22 Table 23 Table 24 Table 25 Table 26 Table 27 Table 28 Table 29 Table 30 Table 31 Table 32 Table 33 Table 34 Table 35 Table 36 Table 37 Table 38 Table 39 Table 40 Table 41 Table 42 Table 43 Table 44 Table 45 Table 46 Table 47 Table 48 Table 49 Table 50 Table 51 Table 52 Table 53 Table 54 Table 55 Table 56 Table 57 Table 58 Table 59 [Table 60] [Table 61]

[0390] Examples The following embodiments are provided to illustrate the specific embodiments disclosed and should not be construed as limiting the scope of this disclosure. [Examples]

[0391] Preparation of OspA-ferritin antigenic polypeptide Antigenic polypeptides containing OspA and ferritin were produced.

[0392] OspA is the following sequence: Borrelia burgdorferi strain B31 (serotype 1) NBCI sequence ID WP_010890378.1, Borrelia afzelii strain PKO (serotype 2) NCBI sequence: WP_011703777.1, Borrelia garinii strain PBr (serotype 3) GenBank: CAA56549.1, Borrelia bavariensis (serotype 4) NCBI sequence WP_011187157.1, Borrelia garinii (serotype 5) GenBank CAA59727.1, Borrelia garinii (Borrelia The ferritin was synthesized by Genescript from *Borrelia garinii* (serotype 6) GenBank:CAA45010.1 and *Borrelia garinii* (serotype 7) GenBank CAA56547.1. *H. pylori* ferritin with an inserted N-terminal bullfrog ferritin sequence was synthesized by Genescript. The bullfrog ferritin sequence used was the same as that used in previous studies (see Kanekiyo, M. et al., Cell 162(5): pp. 1090-100 (2015)). The pet21a vector was used to express His-tagged OspA and OspA-ferritin nanoparticles in *E. coli*. A mammalian expression vector similar to that used previously was used for expression in Expi293 cells (see Xu, L. et al., Science 358(6359): pp. 85-90 (2017)).

[0393] OspA-ferritin nanoparticles were created by genetically fusing the ectodomain of OspA to the amino terminus of ferritin to produce an antigenic polypeptide (Figure 1A). OspA is a 31 kDa lipoprotein with an extended β-sheet structure, made up of 21 consecutive antiparallel β-strands, each having a single carboxy-terminal α-helix (Figure 1B) (see Kitahara, R. et al., Biophys J 102(4):916-926 (2012)). The carboxy-terminus of OspA also has a remarkably large cavity (approximately 200 Å), which corresponds to a site suitable for linking to ferritin using a glycine-serine sequence (Figure 1B). The 24 subunits of ferritin spontaneously assemble to form hollow spherical nanoparticles (Figure 1C). The ferritin used in this study contains the amino-terminal sequence of bullfrog ferritin fused to Helicobacter pylori ferritin, creating a chimera with minimal association with human ferritin (see Kanekiyo, 2015). The amino-terminal bullfrog ferritin sequence protrudes radially from the core of the nanoparticle (see Trikha, J. et al., J Mol Biol 248(5): pp. 949-67 (1995)), facilitating the uniform presentation of OspA on the surface of the nanoparticle.

[0394] To improve the functionality of ferritin, three additional structural modifications were made to ferritin: N19Q, C31S, and S111C. The substitution of N19Q removed a site potentially capable of glycosylation at the amino terminus. The substitution of S111C introduced a cysteine ​​exposed on the surface of ferritin, which can be used, for example, to conjugate an adjuvant by click chemistry. Finally, cysteine ​​31 was modified to serine so that only one cysteine ​​is modified by conjugation. The presentation of OspA on the surface of the nanoparticles provides 24-mer antigenic nanoparticles (Figure 1D).

[0395] For purification from E. coli, BL21 Star (DE3) (Invitrogen, Cat#C601003) was used. Protein induction was performed with 100 μM IPTG overnight at 16°C. The cell pellet was lysed by sonication in Tris buffer, pH 8, 50 mM NaCl. The filtered supernatant was purified by collecting OspA-ferritin from the flow-through using an anion exchange column (HiTrap Q HP, GE). Endotoxin was then removed by repeating 1% Triton X114 extraction six times. The aqueous phase was then concentrated using an Amicon 100MW cutoff filter (Millipore, Cat#UFC910096), and the nanoparticles were further purified at 4°C using a 120 ml SEC column for Superose 6 preparation. For purification from mammalian cell culture, plasmid DNA was transfected into Expi293 cells using FectoPRO transfection reagent (Polyplus, Cat#116-100) according to the manufacturer's instructions. The transfected cells were cultured on day 5, and the supernatant was collected and filtered. Endotoxin-free reagents and glassware were used, and an endotoxin-free protocol was followed. Q Sepharose fast-flow beads (GE, Cat#17-0510-01) were prepared using 50 mM Tris, pH 7, and 50 mM NaCl, and added to the filtered supernatant by gravity. The flow-through was collected and concentrated to 4 ml using an Amicon 100 MW cutoff filter. The nanoparticles were then further purified at room temperature using a 120 ml SEC column for Superose 6 preparation.

[0396] For the purification of His6-tagged (SEQ ID NO: 442) OspA, OspA from serotypes 1, 4, 5, and 7 was purified from E. coli BL21 (DE3) (Invitrogen, Cat#C600003), and OspA from serotypes 2 and 3 was purified from Expi293 cells. These constructs lacked the transmembrane domain, contained a C-terminal His6 tag (SEQ ID NO: 442), and were otherwise wild-type. For E. coli purification, proteins were induced in 500 μM IPTG for 5 hours, and the cells were pelleted and frozen at -20°C. The pellets were resuspended in 1% Triton in TBS buffer containing Complete Protease Inhibitor (Sigma-Aldrich, Cat#11697498001), and the cells were lysed by sonication. The supernatant was filtered and sterilized. For mammalian cell culture, the supernatant was collected 5 days after transfection and filtered and sterilized. The supernatant was passed through a GE HiTrap HP 5ml column (Cat#17-5248-02) attached to an AKTA Pure FPLC. The column was washed with 20 mM imidazole in TBS, loaded, and washed again. The final protein was eluted with 250 mM imidazole in TBS.

[0397] OspA serotype 1 was expressed from B. burgdolferri strain B31 fused to ferritin in transformed human renal epithelial cell line Expi293 (Figures 2A-2D, SEQ ID NO: 52). Nanoparticle formation and protein purity were confirmed by size exclusion column chromatography (SEC) and SDS-PAGE (Figures 2A and 2B, respectively). SEC analysis revealed a single, symmetrical peak at the expected retention time (Figure 2A).

[0398] Dynamic light scattering (DLS) analysis was also performed. Purified nanoparticles were loaded at a concentration of approximately 0.4 μg / ml onto a black 384-well plate with a transparent bottom (Corning, Cat#3540). The samples were read using a DynaPro plate reader II (Wyatt) at a controlled temperature of 25°C. DLS recorded pure, aggregate-free nanoparticles with low polydispersity (7.4%) and a particle size of 13 nm (Figure 2C). The ease of purification was improved by removing the transmembrane domain of OspA (amino acids 1-25) containing the lipidization site. The OspA sequence contains four sites that are capable of amino-linked glycosylation, and OspA-ferritin purified from mammalian cells migrated at a high molecular weight consistent with glycan addition (Figure 2B).

[0399] Transmission electron microscopy negative staining imaging and 2D class-mean analysis were performed on OspA-ferritin nanoparticles (Figure 2D). OspA-ferritin nanoparticle samples were diluted 300-fold with 1×TBS and imaged on a continuous carbon layer supported by nitrocellulose on a 400-mesh copper grid. The grid was prepared by spreading 3 μl of the sample suspension onto a clean grid, absorbing it with filter paper, and immediately staining it with uranyl formate. Electron microscopy observations were performed using an FEI Tecnai T12 electron microscope equipped with an FEI Eagle 4k×4K CCD camera. High-magnification images were acquired at 67,000x magnification (0.16 mm / pixel). Images showed nominal underfocus of -1.9 μm to -0.8 μm and approximately 30 e - / Å 2 The images were acquired with a specific electron content. Individual particles were selected from 67,000x high-magnification images using an automated extraction protocol (see Lander, GC et al., J Struct Biol, 166(1): pp. 95-102 (2009)). A referenceless alignment strategy based on the XMIPP processing package was used (see Sorzano, CO et al., J Struct Biol 148(2): pp. 194-204 (2004)). The algorithm in this package aligns the selected particles and classifies them into self-similar groups or classes.

[0400] Ferritin nanoparticles appeared as circular, high-density structures with a hollow center (Figure 2D). Each nanoparticle was surrounded by numerous short, uniform spikes of OspA, which appeared somewhat elliptical. The particles had an overall diameter ranging from approximately 194 to 220 Å, with a ferritin core diameter of 125 Å. The spikes extended from the particle surface to a length of 45 Å and were uniform in size, shape, and orientation. The OspA spikes were approximately 30 Å wide and tapered to their lowest density in the glycine-serine linker of ferritin.

[0401] When the LYMErix™ vaccine was discontinued in 2002, concerns arose that this vaccine contained an epitope (amino acids 165-173 of SEQ ID NO: 83) homologous to the nonapeptide segment (SEQ ID NO: 78) of human leukocyte function-associated antigen-1 (hLFA-1, see Gross, DM et al., Science 281(5377): pp. 703-706 (1998)) (Figure 5A). Amino acids 165-173 of SEQ ID NO: 83 are referred to as the hLFA-1 homology site. OspA serotype 1 is the only serotype that contains this sequence homology (Figure 5B). To avoid any potential concerns regarding this sequence, the hLFA-1 homology site was replaced with the corresponding OspA serotype 2 (SEQ ID NO: 79) or serotype 3 (SEQ ID NO: 80) nonapeptide sequence, or point substitutions were introduced to reduce similarity to hLFA-1 and prevent the production of antibodies that bind to hLFA-1 (RD2, SEQ ID NO: 81) (Figure 5C). For point substitutions, amino acids exposed on the surface were substituted to reduce similarity to hLFA-1 while avoiding or minimizing destabilization of the β-sheet structure.

[0402] The immunogenicity of hLFA-1 nanoparticles with modified hLFA-1 homology sites was tested in mice, and the immune response was compared to that of unmodified OspA-ferritin nanoparticles (Figure 5D). The antibody titers induced by the nanoparticles with modified hLFA-1 homology sites were potent, and there was no significant difference compared to nanoparticles with unmodified hLFA-1 homology sites. [Examples]

[0403] Characterization of the immunogenicity of OspA-ferritin nanoparticles To evaluate the immunogenicity of OspA-ferritin nanoparticles, C3H mice were immunized twice with either serotype 1 OspA-ferritin nanoparticles in the presence of Ribi adjuvant, or with RECOMBITEK® Lyme (a lipid suspension of purified outer surface protein A (OspA) from B. burgdorferi), a canine vaccine containing serotype 1 lipid-modified full-length recombinant OspA (Figure 4). C3H / HeN mice were intramuscularly vaccinated at 0 and 4 weeks. Serum was ELISA performed starting 2 weeks after the second dose. Ribi (Sigma adjuvant system Cat#S6322-1vl) was resuspended in 1 ml of PBS, vortexed for 1 minute, and added to the antigen in an equal volume prior to immunization.

[0404] Antibody response was determined using enzyme-coupled immunosorbent assay (ELISA) against recombinant OspA. Briefly, a 96-well plate was coated with 1 μg / ml OspA-His diluted in PBS and incubated overnight at 4°C. After removing the OspA-His, the plate was blocked with 5% skim milk dissolved in PBST. After removing the blocking reagent, primary serum samples were added sequentially diluted in PBST. Equivolutes of primary samples were added to the blocking solution to a final blocking solution concentration of 50%. After incubation for 1 hour, the plate was washed with PBST and incubated with HRP-coupled goat anti-mouse IgG secondary antibody (5,000-fold dilution in blocking solution) at room temperature for 1 hour. The secondary antibody was aspirated and removed, the plate was washed, and incubated with Sure Blue TMB peroxidase substrate (KPL, Gaithersburg, MD), followed by the addition of an equivolute stop solution (0.5N sulfuric acid). Absorbance was measured at 450 nm.

[0405] Immunization with OspA-ferritin induced an endpoint titer 4.4 times higher at 6 weeks than RECOMBITEK® Lyme (p<0.001). Antibody titers at 25 weeks were also significantly higher than those with RECOMBITEK® Lyme (p<0.005) (Figure 4). [Examples]

[0406] Glycosylated mutants; evaluation of efficacy To evaluate the protective efficacy of OspA-ferritin, a challenge model was used in which immunized mice or control mice were infected with mites carrying B. burgdolferi (see Rosa, PA et al., Nat Rev Microbiol 3(2): pp. 129-43 (2005)).

[0407] C3H / HeN mice were intramuscularly vaccinated with either 1 μg of OspA-ferritin nanoparticles mixed 1:1 with AddaVax® or 1 μg of ferritin nanoparticles. Mice were vaccinated at 0 and 4 weeks of age. To immunize the mice, serotype 1OspA-ferritin nanoparticles (SEQ ID NO: 53) were used, which had been modified with a rationally designed modification of the hLFA-1 homology site and a modification to remove all potential N-glycosyl sites. This is because OspA naturally expressed in bacteria is not glycosylated at these sites. To prevent glycosylation, the sequence included the following N>Q substitutions: N71Q, N190Q, N202Q, and N251Q. Its immunogenicity was similar to that of glycosylated nanoparticles (Figure 13).

[0408] When the serine / threonine glycosylation site was mutated to alanine (SEQ ID NO: 63), glycosylated variants of OspA-ferritin were also tested. Both this construct and the N>Q construct discussed above produced a stronger immune response compared to OspA-ferritin with the wild-type glycosylation site (SEQ ID NO: 52) and were superior to the RECOMBITEK® Lyme control (Figure 14).

[0409] In further experiments evaluating the protective efficacy of OspA-ferritin nanoparticles in a mite challenge model, Ixodes scapularis mite larvae were obtained from the National Tick Research and Education Center, Oklahoma State University (Stillwater, OK). Infected nymphs with B. burgdorferi were produced by allowing uninfected larvae to feed until gorged on SCID mice infected with B. burgdorferi strain N40. The congested larvae were collected and molted for 4–6 weeks at room temperature and high relative humidity to develop into nymphs. The B. burgdorferi infection rate in the blood-feeding larvae was determined by culturing a portion of the mites recovered from each batch.

[0410] Mice were immunized twice at weeks 0 and 4 with 1 μg doses of OspA-ferritin (SEQ ID NO: 53) and AddaVax® adjuvant, or control ferritin, while a control group was immunized in parallel with RECOMBITEK® Lyme. At week 6 (i.e., 2 weeks after the second vaccination), mice were challenged by allowing 5-6 B. burgdorferi-infected mite nymphs to feed until saturated. B. burgdorferi infection was assayed by collecting the fed nymphs and culturing them in BSK medium. Mice were sacrificed 2 weeks after the challenge, and B. burgdorferi infection was assayed by culturing the ears, ankles, and hearts. The presence of B. burgdorferi was determined by observing the cultures under a dark-field microscope. Mice were defined as infected with B. burgdorferi if one or more organ cultures were positive under a dark-field microscope. Negative cultures were also tested by PCR specific to B. burgdorferi.

[0411] Mice were sacrificed after two weeks. Tissue samples from the heart, ankle, and ear were cultured in BSK medium for six weeks in the presence of an antibiotic against B. burgdorferi. Negative samples were tested for the presence of B. burgdorferi by PCR. All negative cultures were PCR-negative. Protection was calculated as the percentage of mice that did not become infected.

[0412] Compositions containing OspA-ferritin and AddaVax® adjuvant showed no infection (0 / 4), in contrast to negative control ferritin, in which 4 out of 5 animals were infected (Table 5, p<0.01).

[0413] [Table 62] [Examples]

[0414] Evaluation of the efficacy of OspA-ferritin conjugated to the immunostimulatory portion. Autoadjuvant constructs were produced by manipulating cysteine ​​on the surface of ferritin nanoparticles (S111C), enabling direct conjugation of immunostimulatory moieties such as TLR agonists (Figure 6A) or CpG (SEQ ID NO: 210; ISS-1018, Figure 7A) via click chemistry. The direct conjugation procedure was as follows: Materials derived from mammals were reduced to 10 mM TCEP (Amresco, K831-10G) in 50 mM Tris, pH 8.5 for 1 hour to remove cysteine. The protein was then dialyzed in 100 mM Tris, pH 8, 50 mM NaCl to remove TCEP. Materials derived from E. coli did not require reduction. DBCO-PEG4-maleimide linker (Sigma-Aldrich, cat#760676, 5 mg) was resuspended in DMSO at a concentration of 5 mg / ml. 2.5 mg of linker was added to 3 mg of protein in 10 ml volume (final DMSO concentration was 5%). The linker was incubated with the reduced protein at room temperature for 30 minutes. Excess linker was removed by buffer exchange using an Ambicon 100 MW cutoff filter concentrator (Millipore Cat#UFC910096). Azide-PEG4-3M-012 (synthesized in-house) and azide-CPG (ISS-1018, custom synthesized by IDT) were used in the final click chemistry step. As the final conjugation step, 0.5 mg of adjuvant was added to 0.5 mg of protein and incubated at 37°C for 6 hours, followed by incubation at 4°C overnight. Excess adjuvant was removed by buffer exchange using an Ambicon 100 MW cutoff filter concentrator. The efficiency of conjugation was confirmed by mass spectrometry for 3M-012 and by SDS-PAGE analysis for CPG.

[0415] We used 3M-012, a TLR7 / 8 agonist previously shown to enhance the antibody response when directly conjugated to the HIV Gag protein (see Wille-Reece, U. et al., Proc Natl Acad Sci USA 102(42); pp. 15190-1514 (2005)). 3M-012 was conjugated to the nanoparticle of SEQ ID NO: 53 using a two-step click chemistry approach. First, a DBCO-PEG4-maleimide linker was linked to cysteine, and then the modified 3M-012 was added along with a PEG4-azide linker by copper-free azide-alkyne cycloaddition (Figure 6A). Mass spectrometry confirmed a conjugation efficiency of over 99%, with a mass shift of 587 Daltons (Figure 12). In addition to azide-3M-012, we also succeeded in adding azide-CPG (Figure 7A). Conjugation was confirmed by gel shift (Figure 7B).

[0416] Nearly complete conjugation of ferritin was observed, suggesting that most nanoparticles possessed 24 agonist molecules. The immunogenicity of the conjugated OspA-ferritin nanoparticles was then evaluated in mice. C3H / HeN mice were intramuscularly vaccinated at 0 and 4 weeks. ELISA was performed on serum starting 2 weeks after the second dose. Prior to immunization, Alam (Alyhydrogel '85 2%, Brenntag, Cat#21645-51-2) was added to the antigen in an equal volume. Ribi (Sigma adjuvant system, Cat#S6322-1vl) was resuspended in 1 ml of PBS, vortexed for 1 minute, and added to the antigen in an equal volume prior to immunization.

[0417] Mice immunized with 3M-012 conjugated particles produced a 4.5-fold higher OspA antibody response compared to unconjugated material (Figure 6B, p<0.05). The antibody response induced by 3M-012 conjugated particles was greater than that of particles mixed with equimolar amounts of 3M-012 (29 ng) and comparable to that of particles mixed with 1000-fold higher doses (20 μg) of 3M-012 or standard alum (Alyhydrogel '85, 2%, Brenntag - Cat#21645-51-2) adjuvant.

[0418] Similar enhancement of antibody production was observed with CPG-conjugated OspA-ferritin nanoparticles (SEQ ID NO: 53), resulting in a 6.3-fold increase in immune response compared to unconjugated particles and a 4.7-fold increase compared to an equal amount of unconjugated CPG mixed with the nanoparticles (Figure 7C).

[0419] Therefore, targeted delivery of adjuvants conjugated to OspA-ferritin nanoparticles makes it possible to stimulate an effective and specific antibody response while substantially reducing the amount of adjuvant required. [Examples]

[0420] Evaluation of immunogenicity of OspA-ferritin nanoparticles containing various serotypes Serotype 1 OspA strain B. burgdolferi causes disease in the United States, while B. afzeri (serotype 2), B. gallini (serotypes 3, 5, 6, 7), and B. bavaliensis (serotype 4) cause disease in Europe, Asia, and other regions. To produce a composition that provides broad cross-protection, OspA-ferritin nanoparticles were designed for serotypes 1, 2, 3, 4, 5, and 7 (SEQ ID NOs: 1, 5, 6, 7, 8, and 10). These particles were expressed and purified from E. coli using anion exchange and size exclusion chromatography (Figure 3A). All OspA-ferritin antigenic polypeptides had the expected molecular weight of 47 kDa, and the formation of all six OspA nanoparticles was confirmed by DLS analysis and transmission electron microscopy (Figure 3B).

[0421] A six-component composition was produced by combining OspA-ferritin of serotypes 1-5 and 7 in equimolar proportions.

[0422] The immunogenicity of this six-component composition (i.e., hexavalent) with added arum was compared in mice with single-serotype particles (i.e., monovalent) with the same adjuvant (Figures 8A-8F). For each serotype, the monovalent composition was administered at a dose of 1 μg, and the hexavalent composition at a dose of 1 μg (total dose of 6 μg). Prior to immunization, arum (Alyhydrogel '85, 2%, Brenntag - Cat#21645-51-2) was added to the antigen in equivolute. The six-component composition induced a potent antibody response against all six OspA serotypes 1-5 and 7. Furthermore, the response against single-serotype controls was similar to that of the mixture, indicating no interference among the six serotype combinations. Against serotype 4, an improved immune response was observed with the hexavalent composition compared to the single-component composition (see Figure 8D, serotype 4).

[0423] Since it was established that hexavalent compositions are immunogenic and, in some cases, superior to monovalent compositions, conjugates were prepared of each of the six OspA-ferritin nanoparticles with 3M-012 and CpG. Compositions conjugated with two types of six components were created by combining the six OspA-ferritin nanoparticles conjugated with 3M-012 and separately combining the six OspA-ferritin nanoparticles conjugated with CpG. The CpG-conjugated hexavalent composition and the 3M-012-conjugated hexavalent composition showed a significant increase in antibody response in mice compared to the unconjugated hexavalent composition for all seven globally known OspA serotypes (Figures 9A-9G), demonstrating that the hexavalent formulation also provides protection against serotype 6, even though OspA serotype 6 polypeptide is not present in the composition.

[0424] In non-human primates (NHP, rhesus macaques), the hexavalent nanoparticle composition with AF03 adjuvant (unconjugated) performed better than the RECOMBITEK® Lyme control, showing 11 to 200 times higher Ab titers against all seven circulating Borrelia serotypes (Figures 10A to 10G). Similar to mice, the hexavalent composition induced a high-titer antibody response in the presence of the adjuvant. Compositions conjugated with 3M-012 and CpG induced a similar response in NHP to the RECOMBITEK® Lyme control (Figures 10A to 10G compared to Figures 10H to 10N, respectively). The antibody titers of the hexavalent vaccine in NHP remained potent even 19 weeks after boost administration, maintaining its superiority over the RECOMBITEK® Lyme control (Figure 25).

[0425] The conjugated compositions were also tested in a tick challenge model. Mice were vaccinated with 1 μg of antigen at weeks 0 and 4. The monovalent composition contained 1 μg of OspA-ferritin serotype 1 conjugated to 3M-012. The hexavalent compositions contained 1 μg of OspA serotypes 1, 2, 3, 4, 5, and 7 conjugated to 3M-012, respectively. Two weeks after the second immunization, mice were challenged for 5 days with 5-6 ticks infected with Borrelia burgdorferi strain N40 (serotype 1), and sacrificed two weeks later. Tissue samples from the heart, ankle, and ear were cultured for 6 weeks in BSK medium supplemented with antibiotics against B. burgdorferi. Negative samples were tested for the presence of B. burgdorferi by PCR. Positive samples were positive by either culture or PCR (Figure 11).

[0426] Furthermore, a heptavalent vaccine containing all seven serotypes was tested in mice. Mice were immunized intramuscularly at weeks 0 and 4 with a heptavalent OspA-ferritin nanoparticle composition containing 1 μg each (7 μg total) of OspA-ferritin nanoparticles corresponding to OspA serotypes 1-7, with Aram or AF03 added as an adjuvant, or with RECOMBITEK® Lyme (1 μg dose). Antibody response was analyzed by endpoint titer measured by ELISA two weeks after immunization. A more potent immune response was demonstrated compared to RECOMBITEK® (Figures 24A-24G).

[0427] Thus, OspA-ferritin nanoparticles induced high-titer antibody responses against seven major serotypes. Furthermore, the seven-component Lyme vaccine candidate suggests potential for controlling the global spread of Lyme disease. [Examples]

[0428] Characterization of OspA-ferritin constructs with various flexible linkers Several different linkers were tested to provide flexibility between OspA and ferritin. The constructs ranged from one to five -GGGS-(SEQ ID NO: 443) sequences. Various linker constructs were purified to form nanoparticles of uniform size.

[0429] All OspA-linker-ferritin constructs containing the GS1(GGGS)(SEQ ID NO: 443), GS2(SEQ ID NO: 91), or GS5(SEQ ID NO: 92) linkers could be expressed (Figure 15A) and showed consistent DLS (Figures 15B, 15C, and 15E) and EM (Figure 15D) profiles.

[0430] Furthermore, various GGGS-linker constructs (disclosed as "GGGS" in Sequence ID No. 443) (linker 1×GGGS (disclosed as "1×GGGS" in Sequence ID No. 443) [Sequence ID No. 60], linker 2×GGGS (disclosed as "2×GGGS" in Sequence ID No. 444) [Sequence ID No. 61], and linker 5×GGGS (disclosed as "5×GGGS" in Sequence ID No. 445) [Sequence ID No. 62]) all showed a strong immune response in C3H mice (Figure 16). [Examples]

[0431] Characterization of Lumazine Synthase OspA Nanoparticles We investigated the OspA antigen presentation of lumazine synthase derived from another nanoparticle, Aquifex aeolicus. OspA-lumazine synthase particles containing various serotypes were readily purified from E. coli cells by anion exchange chromatography and size exclusion chromatography. Constructs consisting of OspA serotype 1 (SEQ ID NO: 12, Figures 19A-19C), OspA serotype 2 (SEQ ID NO: 16, Figures 20A-20C), OspA serotype 3 (SEQ ID NO: 17, Figures 21A-21B), OspA serotype 4 (SEQ ID NO: 18, Figures 17A-17C), OspA serotype 5 (SEQ ID NO: 19, Figures 22A-22C), and OspA serotype 7 (SEQ ID NO: 21, Figures 23A-23C) were produced and characterized. OspA-lumazine synthase particles formed 15.8 nm particles in EM and were uniform in size in DLS.

[0432] OspA serotype 4 lumazine synthase particles (SEQ ID NO: 18) were tested for immunogenicity in mice (Figure 18). OspA lumazine synthase particles with or without alum produced a strong immune response that appeared to be at least as potent as the immune response of similar OspA serotype 4 ferritin nanoparticles (SEQ ID NO: 7).

[0433] Therefore, antigenic polypeptides, including lumazine synthase and OspA polypeptide, can also be used to induce an anti-OspA antibody response. [Examples]

[0434] Design, purification, and characterization of HA-ferritin nanoparticles HA nanoparticles (HA-Np) were produced by fusing the ectodomain sequence of HA (with 48 C-terminal transmembrane residues deleted) to the N-terminus of ferritin, generating self-assembling nanoparticles in mammalian cells. The ferritin contained mutations that replaced surface-exposed amino acids with cysteine ​​(resulting in S26C, A75C, or S111C mutations in the ferritin sequence of SEQ ID NO: 208), enabling conjugation of adjuvants to HA-Np. Such cysteine ​​representations are shown in Figure 27.

[0435] Cysteine ​​resulting from the above mutations can be used to conjugate the immunostimulatory moiety. Figure 28A illustrates the results of an exemplary one-step click chemistry reaction in which a TLR agonist is conjugated to ferritin using a maleimide-PEG4-SM7 / 8a click reagent. In this reaction, maleimide reacts with an unpaired cysteine, and via a linker interposed at the surface-exposed cysteine, the TLR7 / 8 agonist molecule is covalently conjugated to ferritin, producing a covalent TLR7 / 8 agonist-ferritin conjugate. In this example, one cysteine ​​on the surface of one monomer is shown. Ferritin nanoparticles are polymers, consisting of, for example, 24 monomers. Therefore, ferritin nanoparticles can contain an equal number of surface-exposed cysteine ​​as the number of monomers, and each of the surface-exposed cysteine ​​can be conjugated.

[0436] Figures 28B and 29A-29B illustrate an exemplary two-step click chemistry that can be used to conjugate an adjuvant to ferritin nanoparticles, and how CpG and 3M-012 (sometimes referred to as 3M012) can be conjugated to ferritin by a two-step click chemistry reaction via an intermediate bifunctional linker. The exemplary linker is Sigma's PEG linker (catalog #760676) containing maleimide and DBCO reactive groups. In this example, the TLR agonist is functionalized with a reactive azide group.

[0437] In the experiments described below, mass spectrometry (MS) was used to characterize influenza-ferritin with and without the conjugated immunostimulatory moiety. In some cases, the analyte subjected to MS was the trypsin digest of influenza-ferritin. Trypsin generally cleaves after lysine and arginine residues, except when followed by proline. However, the resulting nanoparticles (the C-terminus of the indicated trypsin site) are resistant to proteolysis. Figure 30 shows the distal (C-terminus) trypsin site cleaved by trypsin in bullfrog-H. pylori ferritin constructs such as SEQ ID NO: 314 under natural conditions. Thus, trypsin digestion releases proteolysis-resistant ferritin particles suitable for MS analysis.

[0438] Therefore, assuming that the linker sequence is more accessible by trypsin, conjugation to nanoparticles can be evaluated using trypsin digestion and subsequent simplified MS analysis, regardless of the presence of the N-terminal antigen in the uncleaved polypeptide. This method was devised to overcome the complexities that glycoprotein antigens present to MS analysis. For example, H1 / Stem-Np can only be analyzed by MS after PNGase treatment (Figure 33A).

[0439] HA nanoparticles were constructed by genetically fusing HA sequences from the following influenza strains to the N-terminus of Helicobacter pylori-bulb frog hybrid ferritin. A / Fort Mammoth / 1-JY2 / 1947 (GenBank CY147342, Amino Acids 1-518, Y108F); A / Malaysia / 302 / 1954 (GenBank CY009340.1, Amino Acids 1-518, Y108F); A / Denver / 1957 (GenBank CY008988, Amino Acids 1-517, Y108F); A / Hong Kong / 117 / 1977 (GenBank CY009292, Amino Acids 1-518, Y108F); A / New Caledonia / 20 / 99 (GenBank AHJ09883.1, Amino Acids 1-518); A / California / 4 / 2009 (GenBank AHJ09884.1, Amino Acids 1-518); COBRA P1 (Sequence ID 302, amino acids 1-518, Y108F, U.S. Patent Application Publication No. 20150017196A1) and COBRA X6 (U.S. Patent Application Publication No. 20140127248A1, amino acids 1-517, Y108F). COBRA P1 and COBRA X6 were produced by a computerized method of hierarchical sequence averaging (Carter DM et al., J Virol 90: pp. 4720-4734 (2016)). COBRA X6 was produced from human H1N1 influenza sequences from 1999 to 2012, and COBRA P1 was produced from human H1N1 strains from 1933 to 1957 and 2009 to 2011, as well as from porcine H1N1 influenza strains from 1931 to 1998 (Carter, 2016). The HA-ferritin gene was cloned into the XbaI / BamHI region of the SIB002 vector, along with the gccacc kozak sequence preceding the ATG start codon, for expression in mammals. All sequences were codon-optimized for expression in human cell lines.

[0440] The HA-Np plasmid was purified using the Powerprep kit (Origene catalog #NP100009) and used to transfect Expi293 cells (ThermoFisher catalog #A14635). FectoPRO DNA transfection reagent (Polyplus #116-100) was used under standard conditions (DNA 0.5 μg / mL, FectoPRO reagent 0.75 μl / mL, enhancer 0.45 μl / mL). Nanoparticles were collected from the supernatant of Expi293 cells 4-6 days after transfection by centrifugation at 3,488 g for 15 minutes at 4°C and filtered through a 0.45 μm vacuum-driven filter unit (Thermo Scientific catalog #167-0045). HA-Np was passed through a Q-Sepharose Fast Flow column (GE catalog #17051001) by gravity flow, the flow-through was collected, diluted 3-fold with water, and the pH was adjusted to a final concentration of 50 mM by adding Tris buffer pH 8.5. Alternatively, instead of using the initial Q-Sepharose column, the supernatant was diluted 5-fold with buffer A (50 mM Tris, pH 8.5, 5 mM NaCl). The sample was then loaded onto a Q-Sepharose column (HiTrap Q HP, GE catalog #17115401), and the protein was eluted with a NaCl gradient of a mixture of buffer A (50 mM Tris, pH 8.5, 5 mM NaCl) and buffer B (50 mM Tris, pH 8.5, 1 M NaCl) at a volume 30 times the column volume, ranging from 0 to 60%. The HA-nanoparticle protein fraction was collected, concentrated using an Amicon Ultra-15 centrifugal filter unit (Millipore catalog #UFC910024), and further purified by size exclusion chromatography using a Superose 6 column PG XK 16 / 70, 60-65 cm (catalog #90100042) in phosphate-buffered saline. The final fraction was concentrated and filtered through a 0.22 μm filter (Millipore SLGV004SL). The final protein was tested using an endotoxin-free solution with a Charles Rivers Endosafe PTS instrument equipped with a LAL cartridge with a detection limit of 0.05 EU / ml.

[0441] The trypsin digestion of H1 / Stem-Np (SEQ ID NO: 343, Figure 31A) and H5 / COBRA-Np (SEQ ID NO: 332, Figure 31B) is shown. In both examples, the molecular weight shift due to trypsin digestion (indicated by "+") corresponds to the molecular weight of SM7 / 8a (711 daltons). Therefore, these data indicate that the conjugation of SM7 / 8a to ferritin nanoparticles containing surface-exposed cysteine ​​was successful.

[0442] Determining the mass change of ferritin nanoparticles after conjugation with low molecular weight molecules such as maleimide-PEG4-SM7 / 8a is not clear in gel shift experiments using PNGase to remove glycosylation of H1 / Stem-Np without trypsin treatment (Figure 32A). As shown in Figure 32B, trypsin treatment makes it possible to confirm the conjugation of small molecules such as SM7 / 8a to H1 / Stem-Np by gel shift assay. Conjugation of higher molecular weight molecules such as CpG can be observed by gel shift assay of deglycosylated H1 / Stem-Np (Figure 32A) and by gel shift assay of ferritin nanoparticles released from H1 / Stem-Np by trypsin digestion under natural conditions. Therefore, by utilizing trypsin digestion of ferritin nanoparticles under natural conditions, a method for characterizing core fragments conjugated by TLR agonists is provided as a platform technology for analyzing given antigens with properties that may interfere with these analytical assays, otherwise difficult to analyze by gel shift or mass spectrometry (MS) experiments.

[0443] SM7 / 8a-PEG4-maleimide was conjugated to H1 / Stem-Np, and after PNGase treatment solely for analytical purposes, the conjugation product was found to have a mass of 42930 Da on MS. This was approximately 715 kDa heavier than the unconjugated H1 / Stem-Np (Figure 33A). After trypsin digestion, the reduced ferritin nanoparticles had a mass of 19510 Da (based on cleavage of the H1 / Stem portion of the nanoparticles) (Figure 33B). After conjugation, the mass of the trypsin-digested conjugated nanoparticles was also approximately 715 kDa heavier. Therefore, both Figures 33A and 33B support the conjugation efficiency of the adjuvant-containing linker to ferritin nanoparticles being approximately 100%.

[0444] The maleimide-PEG4-DBCO linker was conjugated to H1-stem-Np via a two-step click chemistry reaction. In this reaction, the maleimide reacted with cysteine ​​exposed on the surface of the ferritin nanoparticles, and MS analysis after trypsin digestion showed a mass increase that was approximately consistent with the molecular weight of linker addition, which is 675 Da (Figure 34).

[0445] Next, azide-CpG was added to the H1 / Stem-Np-PEG4-DBCO intermediate (labeled "after Mal-PEG4-DBCO" in Figure 35). This was confirmed by a gel shift assay. Azide-CpG was ordered from IDT (SEQ ID NO: 345). The conjugate and control were treated with PNGase before SDS-PAGE. The CpG conjugation resulted in an upward gel shift compared to the linker conjugation due to the added mass (approximately 7.5 kDa). Figure 36 shows an example of a 50% conjugation efficiency quantified by densitometry of two bands labeled "Stem-Np-CpG" and "Stem-Np". Stem-Np is H1 / Stem-Np (SEQ ID NO: 343). Untreated represents H1 / Stem-Np before reduction. TCEP reduction refers to H1 / Stem-Np treated with 3 mM TCEP at room temperature for 1 hour, followed by overnight dialyzing against PBS at 4°C.

[0446] To evaluate conjugation, the tobacco etch virus (TEV) protease cleavage site (SEQ ID NO: 344) was also used. 3M-012 was conjugated to various NC99 HA-TEV-Np constructs (SEQ ID NOs: 309-312) ...

Claims

1. Antigenic ferritin protein, (i) a ferritin protein comprising a mutation that replaces an amino acid exposed on the surface with cysteine, and an immunostimulatory moiety linked to the cysteine; (ii) A peptide linker at the N-terminal end of the ferritin protein, comprising a sequence having at least 80% sequence identity with SEQ ID NO: 217; and, (iii) The non-ferritin polypeptide at the N-terminal end of the peptide linker. The antigenic ferritin protein, including the antigenic ferritin protein.

2. Antigenic ferritin protein, (i) a ferritin protein containing cysteine ​​exposed on its surface, and an immunostimulatory moiety linked to the cysteine; (ii) The N-terminal peptide linker of the ferritin protein, wherein the sequence of the peptide linker comprises a sequence having at least 80% sequence identity with SEQ ID NO: 217; and (iii) The non-ferritin polypeptide at the N-terminal end of the peptide linker. The antigenic ferritin protein, including the antigenic ferritin protein.

3. The antigenic ferritin protein according to claim 1 or 2, further comprising a mutation in the ferritin protein portion that replaces an internal cysteine ​​with a non-cysteine ​​amino acid.

4. The antigenic ferritin protein according to claim 3, wherein the internal cysteine ​​is located at position 31 of H. pylori ferritin, or at a position corresponding to position 31 of H. pylori ferritin, as determined by pairwise or structural alignment.

5. The antigenic ferritin protein according to any one of claims 1 to 4, further comprising a mutation in the ferritin protein portion that replaces surface-exposed asparagine with a non-asparagine amino acid.

6. Antigenic ferritin protein: a. The following mutations: (i) Mutations that replace surface-exposed amino acids with cysteine; (ii) Mutations that replace the internal cysteine ​​at position 31 of H. pyloriferritin, or an internal cysteine ​​at a position similar to that at position 31 of non-H. pyloriferritin, as determined by pairwise or structural alignment, with a non-cysteine ​​amino acid; and (iii) Mutation that replaces surface-exposed asparagine with non-asparagine amino acids ferritin protein, including; b. The immunostimulatory moiety linked to cysteine ​​in the mutation of a(i) above; c. A peptide linker at the N-terminal end of the ferritin protein, comprising a sequence having at least 80% sequence identity with SEQ ID NO: 217; and d. The non-ferritin polypeptide at the N-terminal end of the peptide linker. The antigenic ferritin protein, including the antigenic ferritin protein.

7. The antigenic ferritin protein according to any one of claims 3 to 6, wherein the non-cysteine ​​amino acid is serine.

8. The antigenic ferritin protein according to claim 5 or 6, wherein the asparagine exposed on the surface is at position 19 of H. pyloriferritin, or at an equivalent position of non-H. pyloriferritin, as determined by pairwise or structural alignment.

9. The antigenic ferritin protein according to any one of claims 1 to 8, wherein the ferritin protein comprises one or more E12C, S26C, S72C, A75C, K79C, S100C, and S111C mutations of H. pylori ferritin, or one or more corresponding mutations in non-H. pylori ferritin determined by pairwise or structural alignment.

10. The antigenic ferritin protein according to any one of claims 1 to 9, wherein the non-ferritin polypeptide is a polypeptide derived from influenza, Epstein-Barr virus, respiratory syncytial virus, or Borrelia.

11. The non-ferritin polypeptide comprises an RSV G polypeptide, and optionally the RSV The antigenic ferritin protein according to claim 10, wherein the G polypeptide includes a central conserved region of the G polypeptide.

12. The antigenic ferritin protein according to any one of claims 1 to 11, wherein the immunostimulatory portion includes a portion capable of hydrogen bonding or ionic bonding.

13. The antigenic ferritin protein according to any one of claims 1 to 12, wherein the immunostimulatory portion is an agonist of TLR2, TLR7 / 8, TLR9, or STING.

14. The antigenic ferritin protein according to any one of claims 1 to 13, wherein the ferritin protein comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity with any one of SEQ ID NOs. 201 to 207 or 211 to 215.

15. Ferritin particles comprising the antigenic ferritin protein according to any one of claims 1 to 14.

16. A composition comprising an antigenic ferritin protein or ferritin particles according to any one of claims 1 to 15, and a pharmaceutically acceptable carrier.

17. The composition according to claim 16, further comprising an adjuvant.

18. An antigenic ferritin protein, ferritin particles, or composition according to any one of claims 1 to 17, for use in the target vaccine.

19. A pharmaceutical composition comprising the composition according to any one of claims 16 to 18 for use in a method of vaccinating a target, wherein the method comprises the step of administering the pharmaceutical composition to a target.

20. The pharmaceutical composition according to claim 19, wherein the subject is a human.

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