Treatment and / or prevention of infectious diseases by monovalent / bivalent and polyvalent antigen particle-mediated immune responses

Abstract

The present invention relates to means and methods for targeted modulation of immune responses by contacting B cells with monovalent / bivalent and / or multivalent antigen particles. Targeted modulation of B cell immunity can be used in the therapy of infectious diseases. The present invention is based on the observation that the combination of multivalent and monovalent / bivalent antigen structures has the ability to enhance the immune response to antigens.

Description

[Technical field]

[0001] The present invention relates to means and methods for targeted modulation of immune responses by contacting B cells with monovalent / bivalent and / or multivalent antigen particles. Targeted modulation of B cell immunity can be used in the therapy of infectious diseases. The present invention is based on the observation that the combination of multivalent and monovalent / bivalent antigen structures has the ability to enhance the immune response to antigens.

[0002] Vaccines are one of the greatest inventions of modern medicine and the most economical and effective weapon for humans to fight against pathogens such as viruses and the diseases caused by viruses. Through the use of vaccines, humanity has succeeded in eradicating smallpox, virtually eradicating polio, and controlling most diseases that once plagued humanity, including tuberculosis, measles, diphtheria, and tetanus.

[0003] Currently, vaccine development relies on traditional models of B cell selection and development, which propose that central tolerance mechanisms remove the specificity of autoreactive B cells, resulting in a peripheral B cell repertoire that is devoid of autoreactive potential.

[0004] Since the outbreak of the global COVID-19 pandemic, laboratories around the world have intensified research into new ways to induce immune responses and improve vaccines. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there remains a need for novel and flexible approaches for the controllable modulation of immune responses to improve prevention and / or treatment against pathogens. [Means for solving the problem]

[0006] The above technical problems are solved by the embodiments disclosed herein and defined in the claims.

[0007] Thus, the present invention relates, inter alia, to the following embodiments: 1. A composition for use in the treatment and / or prevention of an infectious disease, comprising: (i) a monovalent / bivalent antigen particle comprising an antigen moiety comprising one or two antigen structures capable of inducing an antibody-mediated immune response against a target antigen, the target antigen being a pathogen-associated antigen; (ii) a multivalent antigen particle comprising an antigen moiety comprising three or more antigen structures capable of inducing an antibody-mediated immune response against a target antigen, wherein the three or more antigen structures are cross-linked and the target antigen is a pathogen-associated antigen. 2. The composition for use according to embodiment 1, wherein the three or more antigenic structures comprise a plurality of identical antigenic structures. 3. The composition for use according to embodiment 1 or 2, wherein the multivalent antigen particles further comprise a carrier moiety coupled to the antigen moiety and / or the monovalent / bivalent antigen particles further comprise a carrier moiety coupled to the antigen moiety. 4. The composition for use according to embodiment 3, wherein the carrier moiety comprises a structure selected from the group of a polypeptide, an immune CpG island, limpet hemocyanin (KLH), tetanus toxoid (TT), cholera toxin subunit B (CTB), a bacterium or bacterial ghost, a liposome, a chitosome, a virosome, a microsphere, a dendritic cell, a particle, a microparticle, a nanoparticle, or a bead. 5. The composition for use according to embodiments 1 to 4, wherein the crosslinks in the multivalent antigen particles are chemical crosslinks, such as bis-maleimide mediated crosslinks, or protein crosslinks, such as biotin-streptavidin mediated crosslinks. 6. The composition for use according to embodiments 1-5, wherein the multivalent antigen particle comprises a conjugate of the following formula ALA, where A is a moiety comprising the target antigen and L is a cross-linking linker, preferably L is a bismaleimide, and most preferably the conjugate is of the following structure (I), where R is a moiety comprising the target antigen.

[0008] [ka]

[0009] 7. The composition for use according to embodiments 1 to 5, wherein the multivalent antigen particles comprise a linker having a cross-linking reactive group for protein conjugation, preferably a linker having a cross-linking reactive group for stable protein conjugation. 8. The composition for use according to embodiment 7, wherein the crosslinking reactive groups are groups selected from carboxyl-amine reactive groups, amine reactive groups, sulfhydryl reactive groups, aldehyde reactive groups, and photoreactive groups. 9. The composition for use according to embodiment 7, wherein the crosslinking reactive group is a group selected from carbodiimides, NHS esters, imidoesters, pentafluorophenyl esters, hydroxymethylphosphines, maleimides, haloacetyls, hydrazides, alkoxyamines, diazirines, and arylazides. 10. The composition for use according to embodiments 1 to 9, wherein the multivalent antigen particles comprise at least two copies of the antigen structure in spatial proximity to each other, preferably within the range of 3 nm to 20 nm. 11. The composition for use according to any one of the preceding embodiments, wherein the pathogen-associated antigen comprises at least one substance selected from the group consisting of nucleic acids, carbohydrates, and peptides. 12. A composition for use according to embodiments 1 to 11, wherein the multivalent antigen particles are linked to the adjuvant, preferably the multivalent particles are covalently linked to the adjuvant. 13. The composition for use according to embodiment 12, wherein the adjuvant is IgG. 14. The composition for use according to embodiments 1 to 13, wherein the treatment and / or prevention comprises at least two administration times. 15. The composition for use according to embodiment 14, wherein the prevention comprises administering monovalent / bivalent antigen particles prior to multivalent antigen particles. 16. The composition for use according to embodiments 14-15, wherein the treatment and / or prevention comprises at least two administration times for monovalent / bivalent antigen particles and at least two administration times for multivalent antigen particles. 17. The composition for use according to any one of the preceding embodiments, wherein the antibody-mediated immune response is an IgG-mediated immune response and / or an IgM-mediated immune response. 18. The composition for use according to embodiments 1 to 17, wherein the pathogen is at least one pathogen selected from the group of parasites, bacteria, and viruses. 19. The composition for use according to embodiments 1 to 18, wherein the infectious disease is a viral infection. 20. The composition for use according to embodiment 19, wherein the viral infection is a coronavirus infection. 21. The composition for use according to embodiment 20, wherein the coronavirus infection is a SARS-CoV-2 infection. 22. The composition for use according to embodiment 21, wherein the pathogen-associated antigen comprises an amino acid sequence derived from a coronavirus spike protein, such as a receptor binding domain (RBD) sequence, preferably the complete RBD sequence, or a sequence comprising at least 80% sequence identity with the amino acid sequence of the SARS-CoV-2 RBD amino acid sequence (SEQ ID NO:1). 23. A method for producing an antibody that binds to a pathogen-associated antigen, comprising: (1) To a subject and / or cell capable of producing antibodies, (i) Monovalent / bivalent particles comprising an antigen moiety comprising one or two antigen structures capable of inducing an antibody-mediated immune response against a target antigen, the target antigen comprising a pathogen-associated antigen; and (ii) a multivalent antigen particle comprising an antigen moiety comprising three or more antigen structures capable of inducing an antibody-mediated immune response against a target antigen, wherein the three or more antigen structures are cross-linked, and the target antigen comprises a pathogen-associated antigen; and administering (2) isolating the antibody from the subject and / or cell, wherein the antibody binds to the target antigen. 24. A polynucleotide for use in the treatment and / or prevention of infectious diseases, comprising: (i) a monovalent / bivalent antigen particle comprising an antigen moiety comprising one or two antigen structures capable of inducing an antibody-mediated immune response against a target antigen, the target antigen comprising a pathogen-associated antigen; and (ii) a multivalent antigen particle comprising an antigen moiety comprising three or more antigen structures capable of inducing an antibody-mediated immune response against a target antigen, wherein the three or more antigen structures are cross-linked, and the target antigen comprises a pathogen-associated antigen; A polynucleotide encoding the 25. A vector comprising a polynucleotide for use according to embodiment 24.

[0010] Thus, the present invention relates to a composition for use in the treatment and / or prevention of infectious diseases, comprising: (i) a monovalent / bivalent antigen particle comprising an antigen moiety comprising one or two antigen structures capable of inducing an antibody-mediated immune response against a target antigen, wherein the target antigen comprises a pathogen-associated antigen; and (ii) a multivalent antigen particle comprising an antigen moiety comprising three or more antigen structures capable of inducing an antibody-mediated immune response against a target antigen, wherein the three or more antigen structures are cross-linked and the target antigen comprises a pathogen-associated antigen. [Brief description of the drawings]

[0011] [Figure 1] Antibody responses after immunization with SARS-CoV-2 derived RBD. Mice were pretreated as indicated 2 weeks prior to immunization. Mice were subsequently immunized on days 1 and 21. Serum was collected 28 days after immunization and used in ELISA to determine Ig concentrations. [Diagram 2]There is no antibody response after immunization with native RBD, while the complexed RBD induces a weak response. A. Schematic of the SARS-CoV-2 spike protein: the receptor-binding domain (RBD), which interacts with human angiotensin-converting enzyme 2 (ACE2) and thereby mediates the entry of viral particles into host cells, was described as a target for neutralizing antibodies. B. The native RBD (~27 kDa) was produced in HEK293-6E cells, biotinylated, and complexed by the addition of streptavidin (SAV), and samples were separated on a 10% Coomassie gel (here, under non-reducing conditions). The RBD forms self-aggregates that can be dissolved by reducing the disulfide bond with b-mercaptoethanol. C. Schematic of immunization procedure: WT mice were control immunized (CI), immunized intraperitoneally with 50 μg of native RBD (nRBD), RBD complexed to streptavidin (cRBD) in the presence of CpG-ODN #1826, or given repeated injections of native RBD (6 intraperitoneal doses of 50 μg each in the absence of adjuvant within 14 days). CI, nRBD-, and cRBD-immunized mice were boosted on day 21 with the same vaccination composition used for primary immunization. D. Blood was collected from immunized mice (described in C) at the indicated time points and RBD-specific IgM and IgG were measured by ELISA. Immunocomplexed RBD induces only weak antibody responses, detectable only after boosting. Repeated exposure to native RBD also induces antibody responses comparable to those induced by cRBD. [Diagram 3] Combining repeated nRBD treatment with cRBD immunization results in robust antibody responses. A. Schematic of the immunization procedure. B. Blood was collected from immunized mice (described in A) at the indicated time points. [Figure 4] The effect of repeated nRBD treatment can persist for extended periods of time. A. Schematic of the immunization procedure. B. Blood was collected from immunized mice (described in A) at the indicated time points. RBD-specific IgM and IgG were determined by ELISA. Pretreatment with native RBD primes for efficient antibody responses even when the primary immune response is delayed by 5 weeks. [Diagram 5]High antibody titers are required for virus neutralization in vitro. A. Concentrations of RBD-specific IgM (left), IgG (middle), and total Ig (right) determined by ELISA in samples used for neutralization assays. Serum was collected on day 28, one week after the boost. B-C. Neutralization potential was compared between sera collected after cRBD immunization in groups of PBS- (-PT) and nRBD-pretreated (+PT) mice. Neutralization capacity correlates with the concentration of total RBD-specific Ig. [Figure 6] Strong early antibody responses by IgD-deficient mice. [Figure 7] Mimicking immune complexes by random cross-linking of the RBD results in robust antibody responses. A. Native RBD (~27 kDa) was produced in HEK293-6E cells and chemically cross-linked by addition of maleimide (cRBD*MM). Samples were resolved on a 10% Coomassie gel (here under reducing conditions). B. Schematic of the immunization procedure. C. Blood was collected from immunized mice (described in B) at the indicated time points. RBD-specific IgM and IgG were measured by ELISA in both groups and compared to titers measured in CI mice. [Figure 8] Mimicking immune complexes by chemical cross-linking of the RBD results in robust antibody responses. A. Concentrations of RBD-specific IgM (left), IgG (middle), and total Ig (right) determined by ELISA in samples used for neutralization assays. B-C. Neutralization potential measured in sera from mice immunized with cRBD*MM. Results were compared to neutralization capacity determined in mice immunized with cRBD-SAV after RBD pretreatment. IgM is not exclusively required to achieve virus neutralization, which can also be achieved by samples containing mainly IgG. Higher concentrations of RBD-specific total Ig correlate with stronger neutralization capacity. cRBD MM: RBD conjugated with maleimide (MM). [Figure 9]Activating antigens form IgG complexes that boost immune responses. A. Schematic of SARS-CoV-2 spike protein with localization of the receptor binding domain (RBD). B. Reaction scheme of chemical cross-linking. At pH 6.5-7.5, the reactive groups of 1,2-phenylene-bis-maleimide (marked in red) oxidize with sulfhydryl groups on cysteine ​​residues of proteins, forming stable thioether linkages. C. Coomassie staining for RBD complexed by 1,2-phenylene-bis-maleimide (Bismale). RBD shows native RBD without cross-linking. D and E. Immunization with RBD. [Figure 10] Activating antigens form IgG complexes that boost immune responses. A. Schematic of SARS-CoV-2 spike protein with localization of the receptor binding domain (RBD). B. Reaction scheme of chemical cross-linking. At pH 6.5-7.5, the reactive groups of 1,2-phenylene-bis-maleimide (marked in red) oxidize with sulfhydryl groups on cysteine ​​residues of proteins to form stable thioether linkages. C. Analysis of 1,2-phenylene-bis-maleimide-complexed RBD under reducing conditions on a 10% SDS page by Coomassie staining. RBD indicates native RBD in the absence of 1,2-phenylene-bis-maleimide. RBD* indicates complexation with 20 μg of 1,2-phenylene-bis-maleimide per 100 μg of RBD, whereas RBD** indicates complexation with 100 μg of 1,2-phenylene-bis-maleimide per 100 μg of RBD. D. WT mice were immunized with either RBD* or RBD** produced as described in "C" using different amounts of cross-linker (maleimide). [Figure 11]Generation of antigen (Ag) complexes by biotinylation and subsequent incubation with streptavidin (SAV). A. Formation of biotin-SAV complexes requires additional steps involving biotinylation and SAV. B. Biochemical activation of antigens in the presence of IgG is simpler. MM, maleimide crosslinking. Antigen complexes with reactive maleimide groups form complexes with self-antigens, which boost the immune response. The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Figure 12] Activating antigens form IgG complexes that boost immune responses. A. Schematic of SARS-CoV-2 spike protein with localization of the receptor binding domain (RBD). B. Reaction scheme of chemical cross-linking. At pH 6.5-7.5, the reactive groups of 1,2-phenylene-bis-maleimide oxidize with sulfhydryl groups on cysteine ​​residues of proteins to form stable thioether linkages. C. Coomassie staining for RBD complexed by 1,2-phenylene-bis-maleimide (Bismale). RBD shows native RBD without cross-linking. D and E. Immunization with RBD.

[0012] The term "valent" as used within this application indicates the presence of a certain number of binding sites within an antibody or antigen molecule, respectively. Thus, the binding site of an antibody is a paratope, whereas the binding site within an antigen is generally referred to as an epitope. For example, a natural antibody or a full-length antibody according to the invention has two binding sites and is bivalent. Antigen proteins are monovalent / bivalent (when present as monomers), but when such antigen proteins are provided as multimers, they may contain two or more identical epitopes and are therefore multivalent, which may be bivalent, trivalent, tetravalent, etc. Thus, the term "trivalent" indicates the presence of three binding sites within an antibody molecule. Thus, the term "tetravalent" indicates the presence of four binding sites within an antibody molecule.

[0013] The term "monovalent / bivalent antigen particle" as described herein refers to a molecule or molecular complex, such as a protein or protein complex, that is antigenic and thus capable of stimulating an immune response in a vertebrate. Typically, a monovalent / bivalent antigen particle is composed of (i) one antigen moiety that includes no more than two of the antigen structures capable of inducing an antibody-mediated immune response against such antigen structure, or (ii) two antigen moieties that include no more than one of the antigen structures capable of inducing an antibody-mediated immune response against such antigen structure. As used herein, the term "monovalent / bivalent antigen particle" refers to a monovalent antigen particle, a bivalent particle, or a combination of a monovalent antigen particle and a bivalent antigen particle. In some embodiments, the term "monovalent / bivalent antigen particle" as described herein further includes a multivalent precursor that degrades into a monovalent / bivalent antigen particle in the subject's body before eliciting a substantial immune response (e.g., a prodrug that is activated upon contact with an enzyme in the body).

[0014] As used herein, the term "antigenic structure" refers to a fragment of an antigenic substance (e.g., a protein) that retains the ability to stimulate an antibody-mediated immune response. Such antigenic structures are understood to provide antigenic determinants or "epitopes," which refer to regions of a molecule that react specifically with an antibody, more specifically, with the paratope of an antibody. In a preferred embodiment of the present invention, the monovalent / bivalent antigen particles of the present invention contain no more than two copies of one specific epitope of the antigenic structure. Thus, preferably, only one / two antibody molecules of a certain antibody species with a particular paratope can bind to the monovalent / bivalent antigen particles according to the present invention.

[0015] The term "multivalent antigen particle" in the context of the invention disclosed herein refers to a molecule or molecular complex, such as a protein or protein complex, that is antigenic and thus capable of stimulating an immune response in a vertebrate. In the present invention, unlike monovalent / bivalent antigen particles, multivalent antigen particles are composed of antigen moieties that contain three or more antigen structures that can induce an antibody-mediated immune response. In some embodiments, the term "multivalent antigen particle" as described herein further includes low-valent (e.g., monovalent / bivalent) precursors that form the multivalent antigen particle in the subject's body before eliciting a substantial immune response (e.g., substances that are complexed and / or polymerized upon contact with enzymes in the body).

[0016] The term "treatment" (and grammatical variations thereof, such as "treat" or "treating"), as used herein, refers to a clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed for prophylaxis or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improving or ameliorating the disease state, and remission or improved prognosis.

[0017] The term "prevention", as used herein, relates to the ability to prevent, minimize or hinder the onset or development of a disorder, disease or condition before it develops.

[0018] In some embodiments, a disease or disorder as described herein refers to one or more symptoms and / or complications of a disease or disorder.

[0019] In a preferred embodiment of the invention, the multivalent antigen particles of the invention comprise three or more copies of one specific epitope of the antigen structure. In some embodiments, the multivalent antigen particles of the invention comprise three or more copies of one specific epitope of the antigen structure.

[0020] Thus, preferably, two or more antibody molecules of a certain antibody species having a specific paratope can be bound to a monovalent / bivalent antigen particle according to the present invention. Such a multivalent antigen particle may have a structure in which two or more antigen structures are covalently or non-covalently cross-linked to each other. Preferably, the two or more antigen structures contained in the antigen portion of the multivalent antigen particle comprise a plurality of identical antigen structures.

[0021] In the context of the present invention, the monovalent / bivalent antigen particles of the present invention are often referred to as "soluble" particles or antigens, whereas the multivalent antigen particles are referred to as "composite" particles or antigens.

[0022] The term "target antigen" as used herein refers to any molecule or structure that comprises an antigenic structure. The target antigen of the present invention may be a natural and / or synthetic immunogenic substance, such as a complete, fragmented or partial immunogenic substance, and the immunogenic substance may be selected from a nucleic acid, a carbohydrate, a peptide, or any combination thereof.

[0023] The term "crosslinking," as used herein, refers to a bond that links at least two antigen structures together, where the crosslinked complex has different physical properties than the isolated antigen structures. In some embodiments, the crosslinked complex is less soluble than the isolated antigen structures. In some embodiments, the crosslinks described herein comprise at least one covalent bond. In some embodiments, the crosslinks described herein comprise at least one ionic bond.

[0024] The term "pathogen," as used herein, refers to an agent that can cause disease, such as an infectious disease, in a subject. Pathogens include, for example, bacteria, viruses, prions, fungi, protozoa, helminths, nematodes, and any other pathogen that can make a subject ill or, if transmitted from a subject that would otherwise be disease-free, can cause disease in further subjects to which the pathogen is transmitted.

[0025] The term "pathogen-associated antigen" as used herein refers to any antigenic molecule, structure, or substance that can be found in a pathogen, preferably a molecule, structure, or substance that is specific to a pathogen (e.g., a pathogen-specific nucleic acid, carbohydrate, peptide, and / or protein). Thus, a pathogen-associated antigen is preferably a structure that is found in a pathogen but is substantially not found in a subject's body or has a higher biological relevance in a pathogen than in a subject's body. In some embodiments, a pathogen-associated antigen described herein is a carbohydrate and / or peptide that is found on the surface of a pathogen. In some embodiments, a pathogen-associated antigen described herein is a carbohydrate and / or peptide that is required for the pathogen's entry into a cell.

[0026] The term "infection," as used herein, refers to the invasion and proliferation of a pathogen within the body of a subject.

[0027] In the context of the present invention, a distinction is made between monovalent / bivalent antigen particles as opposed to multivalent antigen particles. Each particle is considered as a single molecular entity, which may contain covalently or non-covalently connected moieties. However, according to the present invention, each particle has immunogenic activity against a specific antigen. Thus, monovalent / bivalent antigen particles are understood to contain only one or two antigen structures capable of eliciting an immune response to the antigen, whereas multivalent antigen particles contain three or more, four or more copies of such antigen structures. In the context of the present invention, sometimes the term "soluble" antigen is also used for monovalent / bivalent antigen particles as opposed to the "complex" antigen of multivalent antigen particles. It should be understood that in most cases the antigen structure comprises or consists of an epitope that elicits an antibody immune response, while it is the binding site of the antibody produced on the basis of a cell-mediated immune response. In other words, the present invention distinguishes between the presentation of immune-eliciting epitopes as soluble single epitopes or the presentation of the same epitopes in a complex array.

[0028] The present invention is based, at least in part, on the surprising discovery that antigens can induce different immune responses depending on whether they are presented to immune cells as soluble antigens or as multivalent antigen particles. The combination of soluble antigens and composite multivalent antigen particles can increase immunity, reduce the need for adjuvants, and / or improve the effect of adjuvants and / or reduce the required dose (see, e.g., FIG. 1). Furthermore, the combinations described herein can suppress the production of protective IgM antibodies (FIGS. 2-8).

[0029] These findings establish a dynamic model of B cell activation in which immune responses are regulated by the relative amounts of antigen-forming B cells, thereby allowing the unlimited potential for adaptive immune responses.

[0030] Thus, the means and methods described herein provide a novel and versatile method for inducing and modifying immune responses. Antigens presented on antigen particles can be efficiently adapted to newly emerging pathogens, pathogen mutations and / or resistance mechanisms. The production of antigen particles can be standardized and does not have the biological variations of other immune response inducers, such as attenuated or inactivated virus vaccines. Furthermore, the distribution of antigen particles described herein can be controlled and predicted, unlike other immune response inducers, such as mRNA vaccines.

[0031] Thus, the present invention is based, at least in part, on the surprising discovery that a combination of monovalent / bivalent and multivalent antigen particles can be used to enhance and / or maintain antibody production.

[0032] In certain embodiments, the present invention relates to a composition for use according to the invention, wherein the three or more antigenic structures comprise a plurality of identical antigenic structures.

[0033] Thus, preferably, two or more antibody molecules of a certain antibody species having a specific paratope can be bound to the multivalent antigen particle according to the present invention. Such multivalent antigen particles can have a structure in which two or more of the antigen structures are covalently or non-covalently cross-linked to each other. Thus, in a preferred embodiment, the multivalent antigen particle comprises a complex comprising at least two identical epitopes that allows two antibodies to bind to the multivalent antigen particle simultaneously. Preferably, two or more of the antigen structures comprised in the antigen portion of the multivalent antigen particle comprise a plurality of identical antigen structures. Thus, in a preferred embodiment, the multivalent antigen particle comprises a complex comprising at least two, at least three or at least four identical epitopes that allows two antibodies to bind to the multivalent antigen particle simultaneously.

[0034] Compositions comprising such particles according to the present invention are capable of modulating immune responses (see, for example, Figures 1-8).

[0035] Thus, the present invention is based, at least in part, on the surprising discovery that multiple linked identical structures can modulate the immune response to a target antigen, as described herein.

[0036] In certain embodiments, the present invention relates to compositions for use according to the invention, wherein the multivalent antigen particles further comprise a carrier moiety coupled to the antigen moiety and / or the monovalent / bivalent antigen particles further comprise a carrier moiety coupled to the antigen moiety.

[0037] The term "carrier moiety" in the context of the invention disclosed herein preferably relates to a substance or structure which presents or comprises the antigenic structure of the particle of the invention.

[0038] In certain embodiments, the present invention relates to a composition for use according to the invention, wherein the carrier moiety comprises a structure selected from the group of a polypeptide, an immune CpG island, limpet hemocyanin (KLH), tetanus toxoid (TT), cholera toxin subunit B (CTB), a bacterium or bacterial ghost, a liposome, a chitosome, a virosome, a microsphere, a dendritic cell, a particle, a microparticle, a nanoparticle, or a bead.

[0039] In some embodiments of the invention, the multivalent antigen particle further comprises a carrier moiety attached to the antigen moiety, optionally via a linker, wherein the carrier, and optionally the linker, do not comprise another copy of the antigen structure, and the carrier moiety, and optionally the linker, are not capable of eliciting an antibody-mediated immune response against the target antigen. In another alternative or additional embodiment of the invention, the multivalent antigen particle further comprises a carrier moiety attached to the antigen moiety, optionally via a linker.

[0040] The term "linker", as described herein, refers to any molecule, peptide, or structure that can be used to covalently or non-covalently connect two moieties of the compounds of the invention to each other. In some embodiments, the linkers described herein are peptide linkers that can have any size and length suitable for a given application in the context of the present invention. The linker can have a length of 1 to 100 amino acids, preferably 2 to 50 amino acids. The linker can be a typical 4GS linker in 2, 3, 4, 5, 6, or more repeats.

[0041] In certain embodiments, the present invention relates to a composition for use according to the invention, wherein the crosslinks in the multivalent antigen particles are chemical crosslinks, such as bis-maleimide mediated crosslinks, or protein crosslinks, such as biotin-streptavidin mediated crosslinks.

[0042] In certain embodiments, the present invention relates to a multivalent particle for use according to the invention or a composition for use according to the invention, wherein the pathogen-associated antigen comprises at least one substance selected from the group of nucleic acids, carbohydrates, and peptides.

[0043] In certain embodiments, the invention relates to a composition for use according to the invention, wherein the multivalent antigen particle comprises a conjugate of the following formula ALA, where A is a moiety comprising a target antigen, L is a cross-linking linker, preferably L is a bismaleimide, and most preferably the conjugate is of the following structure (I), where R is a moiety comprising a target antigen.

[0044] [ka]

[0045] Preferably, neither the carrier moiety, and optionally the linker, is capable of eliciting an antibody-mediated immune response against the target antigen.

[0046] The carrier moiety can facilitate the presentation of the antigen to the immune system and improve the stability of the particle.

[0047] Thus, the present invention is based, at least in part, on the surprising discovery that carriers linked to antigen moieties can improve the antigenic, pharmacological and / or pharmacokinetic properties of multivalent antigen particles and thus affect the modulation of the immune response to the target antigens as described herein.

[0048] In certain embodiments, the present invention relates to a composition for use according to the invention, wherein the multivalent antigen particles comprise a linker having a cross-linking reactive group for protein conjugation.

[0049] The term "crosslinking reactive group for protein conjugation" as used herein refers to any chemical group or structure that allows for the formation of a bond between the antigen particles and a protein as described herein. Such crosslinking reactive groups and their preparation are well known to those skilled in the art (see, for example, Brinkley, M., 1992, Bioconjugate chemistry, 3(1), 2-13; Kluger, R., & Alagic, A, 2004, Bioorganic chemistry 32.6(2004):451-472.; Stephanopoulos, N.; Francis, MB, 2011, Nature Chemical Biology.7(12):876-884).

[0050] The inventors have found that linkers that are linked to antigen particles (e.g., multivalent antigen particles) described herein and that contain cross-linking reactive groups for binding to endogenous proteins in a subject can enhance the immune response (see, e.g., Figures 8-12, Examples 7, 9, 10).

[0051] In certain embodiments, the present invention relates to a composition for use according to the invention, wherein the multivalent antigen particles comprise a linker with cross-linking reactive groups for stable protein conjugation.

[0052] The term "stable protein conjugation" as used herein refers to a covalent protein conjugation that is not a SS bond. In some embodiments, the stable protein conjugation described herein is hydrolytically stable. In some embodiments, the stable protein conjugation described herein is an irreversible bond.

[0053] The present inventors have found that stable conjugation to endogenous proteins can enhance the immune response to the antigen particles described herein (Example 9).

[0054] In certain embodiments, the present invention relates to a composition for use according to the invention, wherein the cross-linking reactive group is attached to the protein at least one selected from the group consisting of a lysine amino acid residue, a cysteine ​​residue, a tyrosine residue, a tryptophan residue, the N-terminus and the C-terminus.

[0055] In certain embodiments, the present invention relates to compositions for use according to the invention, wherein the cross-linking reactive groups are groups selected from carboxyl-amine reactive groups, amine reactive groups, sulfhydryl reactive groups, aldehyde reactive groups, and photoreactive groups.

[0056] In certain embodiments, the present invention relates to a composition for use according to the invention, wherein the crosslinking reactive group is a group selected from carbodiimides, NHS esters, imidoesters, pentafluorophenyl esters, hydroxymethylphosphines, maleimides, haloacetyls, hydrazides, alkoxyamines, diazirines, and arylazides.

[0057] Thus, the present invention is based, at least in part, on enhancing the immune response by binding to endogenous proteins.

[0058] In certain embodiments, the present invention relates to a composition for use according to the invention, wherein the multivalent antigen particles comprise at least two copies of an antigen structure in spatial proximity to each other, preferably within the range of 3 nm to 20 nm.

[0059] The multivalent antigen particles of the present invention preferably comprise at least two copies of an antigen structure in spatial proximity to each other within a nanometer range selected from the ranges of about 1 nm to about 1000 nm, about 1 nm to about 500 nm, about 1 nm to about 100 nm, about 1 nm to about 50 nm, about 1 nm to about 20 nm, or about 3 nm to about 20 nm.

[0060] Methods for measuring spatial proximity are known to those of skill in the art (see, for example, F. Schueder et al., 2021, Angew. Chem. Int. Ed. 2021, 60, 716; Erickson, D. et al., 2008, Microfluidics and nanofluidics, 4(1-2), 33-52; Turkowyd, B., et al., 2016, Anal Bioanal Chem 408, 6885-6911).

[0061] The present inventors have found that multivalent particles of certain size ranges are particularly effective at eliciting certain immune responses.

[0062] Thus, the present invention is based, at least in part, on the surprising discovery that the size and / or spatial proximity of antigen particles can influence the modulation of the immune response to a target antigen, as described herein.

[0063] In certain embodiments, the present invention relates to a composition for use according to the invention, wherein the pathogen-associated antigen comprises at least one substance selected from the group of nucleic acids, carbohydrates, and peptides.

[0064] Nucleic acids, carbohydrates, and / or peptides are useful structures for copying or mimicking the antigenic patterns of pathogens, and they can be designed to induce specific immune responses without substantial side effects.

[0065] Thus, the present invention is based, at least in part, on the surprising discovery that certain antigen types can affect the modulation of immune responses to target antigens as described herein.

[0066] In certain embodiments, the invention relates to a composition for use according to the invention, wherein the multivalent antigen particles are linked to the adjuvant, preferably the multivalent particles are covalently linked to the adjuvant.

[0067] The term "adjuvant" as used herein refers to an agent that does not contain a target antigen and can enhance the immune response to the antigen particles described herein. In some embodiments, the adjuvant described herein comprises at least one adjuvant selected from the group of oils (e.g., paraffin oil, peanut oil), bacterial products, saponins, cytokines (e.g., IL-1, IL-2, IL-12), squalene, and IgG, and preferably, the adjuvant comprises a free SH group.

[0068] The inventors have found that the immune response, particularly that induced by multivalent antibodies, can be enhanced by linking the antigen particles described herein to an adjuvant (FIGS. 9D and 9E, 11, 12). This linking to an adjuvant substantially reduces the need to formulate the antigen particles described herein with more unlinked adjuvant. Furthermore, the adjuvant can increase the stability of the antigen particles described herein.

[0069] Thus, the present invention is based, at least in part, on the discovery that by linking the antigenic particles described herein to an adjuvant, the immune response elicited can be enhanced.

[0070] In certain embodiments, the present invention relates to a composition for use according to the invention, wherein the adjuvant is IgG.

[0071] The term "IgG" as used herein refers to a molecule consisting of or comprising a polypeptide of the immunoglobulin G class.

[0072] Traditional adjuvants are associated with side effects (see, e.g., Petrovsky, Nikolai. Drug safety 38.11 (2015): 1059-1074). The inventors have found that conjugating the antigen particles described herein with IgG is useful for enhancing immune responses and subsequently reducing the need to formulate the antigen particles described herein with traditional adjuvants (Figures 9D and 9E, Figures 11, 12).

[0073] Thus, the present invention is based, at least in part, on the discovery that by linking the antigenic particles described herein to IgG, the immune response elicited can be enhanced.

[0074] In certain embodiments, the present invention relates to a composition for use according to the invention, wherein the treatment and / or prevention comprises at least two administration times.

[0075] Thus, the components of the compositions of the present invention can be administered at different times to achieve a certain immune modulation, or can be administered repeatedly to boost and achieve an enhanced effect (see FIG. 1).

[0076] Thus, the present invention is based, at least in part, on the surprising discovery that priming and / or boosting modulate the altered immune response elicited by the means and methods of the present invention.

[0077] In certain embodiments, the invention relates to a composition for use according to the invention, wherein prophylaxis comprises administration of monovalent / bivalent antigen particles prior to multivalent antigen particles.

[0078] In certain embodiments, the present invention relates to a method for preventing and / or treating an infectious disease, comprising the steps of 1) priming by administration of monovalent / bivalent antigen particles and 2) boosting with multivalent antigen particles, wherein the monovalent / bivalent antigen particles and the multivalent antigen particles target the same antigen.

[0079] The means and methods of the various embodiments of the present invention in certain embodiments can be viewed as immunization methods for the generation of a particular desired antibody response, in this context, preferred embodiments of the methods of the present invention include a prime / boost immunization scheme for a subject.

[0080] The term "priming" an immune response to an antigen refers to the administration to a subject of an immunogenic composition that induces a higher level of immune response to the antigen upon subsequent administration of the same or a second composition than the immune response obtained by administration of a single immunogenic composition.

[0081] The term "boosting" an immune response to an antigen refers to administering a second, boosting immunogenic composition to a subject following administration of a priming immunogenic composition. In one embodiment, the boosting administration of the immunogenic composition occurs about 2-27 weeks, preferably 1-10 weeks, more preferably 1-5 weeks, and most preferably about 3 weeks after administration of the priming dose.

[0082] In some embodiments of the invention, the priming step is carried out with monovalent / bivalent antigen particles composed of antigen moieties comprising one or less of the antigen structures capable of inducing an antibody-mediated immune response against the target antigen, whereas the boosting step involves administration of multivalent antigen particles composed of antigen moieties comprising two or more of the antigen structures capable of inducing an antibody-mediated immune response against the target antigen, where two or more of the antigen structures are covalently or non-covalently cross-linked. In such prime / boost embodiments of the invention, the antigen structures used to induce the immune response in the priming and boosting steps are the same antigen structure.

[0083] In some embodiments of the invention, the boosting step may be performed with a composition of monovalent / bivalent and multivalent antigen particles.

[0084] Thus, the present invention is based, at least in part, on the surprising discovery that priming with monovalent / bivalent antigen particles increases the immune response to multivalent antigen particles.

[0085] In certain embodiments, the present invention relates to a composition for use according to the invention, wherein the treatment and / or prevention comprises at least two administration times for the monovalent / bivalent antigen particles and at least two administration times for the multivalent antigen particles.

[0086] In certain embodiments, the present invention relates to a composition for use according to the present invention, wherein the antibody-mediated immune response is an IgM-mediated immune response.

[0087] In certain embodiments, the present invention relates to a composition for use according to the present invention, wherein the antibody-mediated immune response is an IgG-mediated immune response.

[0088] In certain embodiments, the present invention relates to a composition for use according to the present invention, wherein the antibody-mediated immune response is an IgG-mediated immune response and an IgM-mediated immune response.

[0089] The present inventors have found that the composition of the present invention can selectively induce an IgG-mediated immune response and / or an IgM-mediated immune response (FIGS. 2 to 4, 7).

[0090] In certain embodiments, the present invention relates to a composition for use according to the invention, wherein the pathogen is at least one pathogen selected from the group of parasites, bacteria, and viruses.

[0091] Abiotrophia, Achromobacter, Acidaminococcus, Acidovorax, Acinetobacter, Actinobacillus. Actinobaculum、Actinomadura、Actinomyces、Aerococcus、Aeromonas、Aphipia、Agrobacterium、Alcaligenes、Alloiococcus AlteromonasAmycolata、Amycolatopsis、Anaerobospirillum、Anaerorhabdus、Anguillina」、Arachnia、Arcanobac terium, Arcobacter, Arthrobacter, Atopobium, Aureobacterium, Bacillus, Bacteroides, Balneatrix, Bartonella. Bergeyella, Bifidobacterium, Bilophila, Branhamella, Borrelia, Bordetella, Brachyspira, Brevibacillus, Bre vibacterium Brevundimonas Brucella Burkholderia Buttiauxella Butyrivibrio Calymmatobacterium Campyl obacter、Capnocytophaga、Cardiobacterium、Catonella、Cedecea、Cellulomonas、Centipeda、Chlamydia、Chlamydo phila, Chromobacterium, Chyseobacterium, Chryseomonas, Citrobacter, Clostridium, Collinsella, Comamonas, Co rynebacterium, Coxiella, Cryptobacterium, Delftia, Dermabacter, Dermatophilus, Desulfomonas, Desulfovibri o Dialister, Dichelobacter, Dolosicoccus, Dolosigranulum, Edwardsiella, Eggerthella, Ehrlichia, Eikenella.Empedobacter、Enterobacter、Enterococcus、Erwinia、Erysipelothrix、Escherichia、Eubacterium、Ewingella、Exiguobacterium、Facklamia、Filifactor、Flavimonas、Flavobacterium、Flexispira、Francisella、Fusobacterium、Gardnerella、Gemella Globicatella、Gordona、Haemophilus、Hafnia、Helicobacter、Helococcus、Holdemania、Ignavigranum、Johnsonella、Kingella、Klebsiella、Kocuria、Koserella、Kurthia、Kytococcus、Lactobacillus、Lactococcus、Lautropia、Leclercia、Legionella、Leminorella、Leptospira、Leptotrichia、Leuconostoc、Listeria、Listonella、Megasphaera、Methylobacterium、Microbacterium、Micrococcus、Mitsuokella、Mobiluncus、Moellerella、Moraxella、Morganella、Mycobacterium、Mycoplasma、Myroides、Neisseria、Nocardia、Nocardiopsis、Ochrobactrum、OeskoviaOligella、Orientia、Paenibacillus、Pantoea、Parachlamydia、Pasteurella、Pediococcus、Peptococcus、Peptostreptococcus、Photobacterium、Photorhabdus、Plesiomonas Porphyrimonas、Prevotella、Propionibacterium、Proteus、Providencia、Pseudomonas、Pseudonocardia、Pseudoramibacter、Psychrobacter、Rahnella、Ralstonia、Rhodococcus、Rickettsia, Rochalimaea, Roseomonas, Rothia, Ruminococcus, Salmonella, Selenomonas, Serpulina, Serratia, Shewenella, Shigella, Simkania, Slackia , Sphingobacterium, Sphingomonas, Spirillum, Staphylococcus, Stenotrophomonas, Stomatococcus, Streptobacillus, Streptococcus, Streptomyces, Su The present invention relates to a composition for use in which the bacterium is at least one bacterium from a genus selected from the group consisting of: Cincinivibrio, Sutterella, Suttonella, Tatumella, Tissierella, Trabulsiella, Treponema, Tropheryma, Tsukamurella, Turicella, Ureaplasma, Vagococcus, Veillonella, Vibrio, Weeksella, Wolinella, Xanthomonas, Xenorhabdus, Yersinia and Yokenella.

[0092] Bacteria Actimomyces europeus, Actimomyces georgiae, Actimomyces gerencseriae, Actimomyces graevenitzii, and Actimomyces israelii、Actimomyces meyeri、Actimomyces naeslundii、Actimomyces neuii neuii、Actimomyces neuii anitratus、Actimomyces odontolyticus、Actimomyces radingae、Actimomyces turicensis、Actimomyces viscosus、Arthrobacter creatinolyticus、Arthrobacter cumminsii、Arthrobacter woluwensis、Bacillus anthracis、Bacillus cereus、Bacillus circulans、Bacillus coagulans、Bacillus licheniformis、Bacillus megaterium、Bacillus myroides、Bacillus pumilus、Bacillus sphaericus、Bacillus subtilis、Bacillus thuringiensis、Borrelia afzelii、Borrelia andersonii、Borrelia bissettii、Borrelia burgdorferi、Borrelia garinii、Borrelia japonica、Borrelia lusitaniae、Borrelia tanukii、Borrelia turdi、Borrelia valaisiana Borrelia caucasica、Borrelia crocidurae、Borrelia recurrentis、Borrelia duttoni、Borrelia graingeri、Borrelia hermsii、Borrelia hispanica、Borrelia latyschewii、Borrelia mazzottii、Borrelia parking、Borreliapersica, Borrelia recurrentis, Borrelia turicatae, Borrelia venezuelensi, Bordetella bronchiseptica, Bordetella hinzii, Bordetella holmseii, Bordetella parapertussis, Bordetella pertussis, Bordetella trematum, Clostridium absonum, Clostridium argentinense, Clostridium baratii, Clostridium bifermentans, Clostridium beijerinckii, Clostridium butyricum, Clostridium cadaveris, Clostridium carnis, Clostridium celatum, Clostridium clostridioforme, Clostridium cochlearium, Clostridium cocleatum, Clostridium fallax, Clostridium ghonii, Clostridium glycolicum, Clostridium haemolyticum, Clostridium hastiforme, Clostridium histolyticum, Clostridium indolis, Clostridium innocuum, Clostridium irregulare, Clostridium leptum, Clostridium limosum, Clostridium malenominatum, Clostridium novyi, Clostridium oroticum, Clostridium paraputrificum, Clostridium piliforme, Clostridium putrefasciens, Clostridium ramosum, Clostridium septicum, Clostridium sordelii, Clostridium sphenoides, Clostridium sporogenes, Clostridium subterminale, Clostridiumsymbiosum, Clostridium tertium, Clostridium tetani, Escherichia coli, Escherichia fergusonii, Escherichia hermanii, Escherichia vulneris, Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus flavescens, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, Enterococcus raffinosus, Enterococcus solitarius, Haemophilus aegyptius, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus parainfluenzae, Haemophilus segnis, Haemophilus ducreyi, Haemophilus influenzae, Klebsiella ornitholytica, Klebsiella oxytoca, Klebsiella planticola, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella terrigena, Lysteria ivanovii, Lysteria monocytogenes, Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium alvei, Mycobacterium asiaticum, Mycobacterium aurum, Mycobacterium avium, Mycobacterium bohemicum, Mycobacterium bovis, Mycobacteriumbranderi, Mycobacterium brumae, Mycobacterium celatum, Mycobacterium chelonae, Mycobacterium chubense, Mycobacterium confluentis, Mycobacterium conspicuum, Mycobacterium cookii, Mycobacterium flavescens, Mycobacterium fortuitum, Mycobacterium gadium, Mycobacterium gastri, Mycobacterium genavense, Mycobacterium gordonae, Mycobacterium goodii, Mycobacterium haemophilum, Mycobacterium hassicum, Mycobacterium intracellulare, Mycobacterium interjectum, Mycobacterium heidelberense, Mycobacterium kansasii, Mycobacterium lentiflavum, Mycobacterium leprae, Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium microgenicum, Mycobacterium microti, Mycobacterium mucogenicum, Mycobacterium neoaurum, Mycobacterium nonchromogenicum, Mycobacterium peregrinum, Mycobacterium phlei, Mycobacterium scrofulaceum, Mycobacterium shimoidei, Mycobacterium simiae, Mycobacterium smegmatis, Mycobacterium szulgai, Mycobacterium terrae, Mycobacterium thermoresistabile, Mycobacterium triplex, Mycobacterium triviale, Mycobacteriumtuberculosis, Mycobacterium tusciae, Mycobacterium ulcerans, Mycobacterium vaccae, Mycobacterium wolinskyi, Mycobacterium xenopi, Mycoplasma buccale, Mycoplasma faucium, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma lipophilum, Mycoplasma orale, Mycoplasma penetrans, Mycoplasma pirum, Mycoplasma pneumoniae, Mycoplasma primatum, Mycoplasma salivarium, Mycoplasma spermatophilum, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas chlororaphis、Pseudomonas fluorescens、Pseudomonas luteola.Pseudomonas mendocina、Pseudomonas monteilii、Pseudomonas oryzihabitans、Pseudomonas pertocinogena、Pseudomonas pseudalcaligenes、Pseudomonas putida、Pseudomonas stutzeri、Rickettsia africae、Rickettsia akari、Rickettsia australis、Rickettsia conorii、Rickettsia felis、Rickettsia honei、Rickettsia japonica、Rickettsia mongolotimonae、Rickettsia prowazekii、Rickettsia rickettsiae、Rickettsia sibirica、Rickettsia slovaca、Rickettsia typhi、Salmonella choleraesuis choleraesuis、Salmonella choleraesuis arizonae、Salmonella choleraesuis bongori、Salmonella choleraesuis diarizonae、Salmonella choleraesuis houtenae、Salmonella choleraesuis indica、Salmonella choleraesuis salamae、Salmonella enteritidis、Salmonella typhi、Salmonella typhimurium、Shigella boydii、Shigella dysentaeriae、Shigella flexneri、Shigella sonnei、Staphylococcus aureus、Staphylococcus auricularis、Staphylococcus capitis capitis、Staphylococcus c.ureolyticus, Staphylococcus caprae, Staphylococcus aureus, Staphylococcus cohnii cohnii, Staphylococcus c.urealyticus, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus gallinarum, Staphylococcus haemolyticus, Staphylococcus hominis hominis, Staphylococcus h.novobiosepticius, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus lugdunensis, Staphylococcus pasteuri, Staphylococcus saccharolyticus, Staphylococcus saprophyticus, Staphylococcus schleiferi schleiferi, Staphylococcus s.coagulans, Staphylococcus sciuri, Staphylococcus simulans, Staphylococcus warneri, Staphylococcus xylosus, Streptococcus agalactiae, Streptococcus canis, Streptococcus dysgalactiae dysgalactiae, Streptococcus dysgalactiae equisimilis, Streptococcus equi equi, Streptococcus equi zooepidemicus, Streptococcus iniae, Streptococcus porcinus, Streptococcus pyogenes, Streptococcus anginosus, Streptococcus constellatus constellatus, Streptococcus constellatus pharyngidis, Streptococcus intermedius, Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, Streptococcus cristatus, Streptococcus gordonii, Streptococcus parasanguinis, Streptococcus salivarius, Streptococcus vestibularis, Streptococcus criceti, Streptococcus mutans, Streptococcus ratti, Streptococcus sobrinus, Streptococcus acidominimus, Streptococcus bovis, Streptococcus equinus, Streptococcus pneumoniae, Streptococcus suis, Vibrio alginolyticus, V. carchariae, Vibrio cholerae, C.The present invention relates to a composition for use according to the invention, wherein the at least one bacterium is selected from the group consisting of Vibrio cincinnatiensis, Vibrio damsela, Vibrio fluvialis, Vibrio furnissii, Vibrio hollisae, Vibrio metschnikovii, Vibrio mimicus, Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia pestis, Yersinia aldovae, Yersinia bercovieri, Yersinia enterocolitica, Yersinia frederiksenii, Yersinia intermedia, Yersinia kristensenii, Yersinia mollaretii, Yersinia pseudotuberculosis, and / or Yersinia rohdei.

[0093] In certain embodiments, the present invention relates to a composition for use according to the invention, wherein the pathogen is P. falciparum.

[0094] In certain embodiments, the present invention relates to a composition for use according to the present invention, wherein the pathogen is M. tuberculosis.

[0095] In certain embodiments, the present invention relates to a composition for use according to the invention, wherein the pathogen is selected from the group of multi-resistant bacteria (e.g., S. aureus).

[0096] In certain embodiments, the present invention relates to a multivalent particle for use according to the invention or a composition for use according to the invention, wherein the infectious disease is a viral infection. Preferably, in this embodiment, the pathogen is a virus.

[0097] In some embodiments, the viral infection described herein is an infection with a virus selected from the group consisting of adenoviridae, anelloviridae, arenaviridae, astroviridae, bunyaviridae, bunyavirus, caliciviridae, coronaviridae, filoviridae, flaviviridae, hepadnaviridae, herpesviridae, orthomyxoviridae, papillomaviridae, paramyxoviridae, parvoviridae, picornaviridae, pneumoviridae, polyomaviridae, poxviridae, reoviridae, retroviridae, rhabdoviridae, rhabdovirus, and togaviridae. In some embodiments, the viral infection described herein is an infection with an RNA virus. In some embodiments, the viral infection described herein is a viral infection of any of the following families: Amalgaviridae, Birnaviridae, Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae, Megabirnaviridae, Partitiviridae, Picobirnaviridae, Reoviridae, Totiviridae, Quadriviridae, Botybirnavirus, Unassigned dsRNA viruses, Arteriviridae, Coronaviridae (including among others the coronavirus, SARS-CoV), Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae, Marnaviridae, Picornaviridae, Secoviridae, Alphaflexivi ridae, Betaflexiviridae, Gammaflexiviridae, Tymoviridae, Alphatetraviridae, Alvernaviridae, Astroviridae, Barnaviridae, Benyviridae, Botourmiaviridae, Bromoviridae, Caliciviridae,Carmotetraviridae, Closteroviridae, Flaviviridae, Fusariviridae, Hepeviridae, Hypoviridae, Leviviridae, Luteoviridae, Polycipiviridae, Narnaviridae, Nodaviridae, Permutotetraviridae, Potyviridae, Sarthroviridae, Statovir us, Togaviridae, Tombusviridae, Virgaviridae, Unclassified Genus Plus Strand ssRNA Virus, Qinviridae, Aspiviridae, Chuviridae, Bornaviridae, Filoviridae, Mymonaviridae, Nyamiviridae, Paramyxoviridae, Pneumoviridae, Rhabdoviridae, Sunviridae, Anphev Infection with an RNA virus selected from the group consisting of irus, Arlivirus, Chengtivirus, Crustavirus, Wastrivirus, Yueviridae, Arenaviridae, Cruliviridae, Feraviridae, Fimoviridae, Hantaviridae, Jonviridae, Nairoviridae, Peribunyaviridae, Phasmaviridae, Phenuiviridae, Tospoviridae, Tilapineviridae, Amnoonviridae, Orthomyxoviridae, Satellite viruses (including, inter alia, Sarthroviridae, Albetovirus, Aumaivirus, Papanivirus, Virtovirus, chronic paralysis virus), Retroviridae, Metaviridae, and Pseudoviridae.

[0098] In some embodiments, the virus is selected from the group consisting of adeno-associated virus, Aichi virus, Australian bat lyssavirus, BK polyomavirus, Banna virus, Barmah forest virus, Bunyamwera virus, Bunyavirus La Crosse, Bunyavirus snowshoe hare, Cercopithecine herpesvirus, Chandipura virus, Chikungunya virus, Cosavirus A, Cowpox virus, Coxsackievirus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Dhori virus, Dugbe virus, Duvenhage virus, Eastern equine encephalitis virus, Ebola virus, Echovirus, Encephalomyocarditis virus, Epstein-Barr virus, European bat lyssavirus, GB Hepatitis C / G virus, Hantaan virus, Hendra virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Hepatitis Delta virus, Horsepox virus, Human adenovirus, Human astrovirus, Human coronavirus, Human cytomegalovirus, Human enterovirus 68, Human enterovirus 70, Human herpesvirus 1, Human herpesvirus 2, Human herpesvirus 6, Human herpesvirus 7, Human herpesvirus 8, Human immunodeficiency virus, Human papillomavirus 1, Human Papillomavirus 2, Human papillomavirus 16, Human papillomavirus 18, Human parainfluenza, Human parvovirus B19, Human respiratory syncytial virus, Human rhinovirus, Human SARS coronavirus, Human spumavirus, Human T-lymphotropic virus, Human torovirus, Influenza A virus, Influenza B virus, Influenza C virus, Isfahan virus, JC polyomavirus, Japanese encephalitis virus, Junin arenavirus, KI polyomavirus, Kunjin virus, Lagos bat virus, Lake Victoriamarburgvirus, Langat virus, Lassa virus, Lordsdale virus, Louping ill virus, Lymphocytic choriomeningitis virus, Machupo virus, Mayaro virus, MERS coronavirus, Measles virus, Mengo encephalomyocarditis virus, Merkel cell polyomavirus, Mokola virus, Molluscum contagiosum virus, Monkeypox virus, Mumps virus, Murray Valley encephalitis virus, New York virus, Nipah virus, Norwalk virus, O'nyong-nyong virus, Contagious pustular dermatitis virus, Oropouche virus, Pichinde virus, Poliovirus, Punta toro phlebovirus, Puumala virus, Rabies virus, Rift Valley fever virus, Rosavirus A, Ross river virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, Sagiyama virus, Salivirus A, Sandfly fever Sicily virus, Sapporo virus, SARS coronavirus 2, Semliki The virus selected from the group consisting of: Ardea forest virus, Seoul virus, Simian foamy virus, Simian virus 5, Sindbis virus, Southampton virus, St. louis encephalitis virus, Tick-borne powassan virus, Torque teno virus, Toscana virus, Uukuniemi virus, Vaccinia virus, Varicella-zoster virus, Variola virus, Venezuelan equine encephalitis virus, Vesicular stomatitis virus, Western equine encephalitis virus, WU polyoma virus, West Nile virus, Yaba monkey tumor virus, Yaba-like disease virus, Yellow fever virus, and / or Zika virus are described herein.

[0099] In certain embodiments, the present invention relates to a composition for use according to the present invention, wherein the pathogen is HHV-3.

[0100] In certain embodiments, the present invention relates to a composition for use according to the present invention, wherein the pathogen is HIV-1.

[0101] In some embodiments, the viruses described herein are variants having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% sequence identity to the viral genomic sequence of at least one virus described herein.

[0102] In certain embodiments, the present invention relates to a multivalent particle for use according to the invention or a composition for use according to the invention, wherein the viral infection is a coronavirus infection. Preferably, in this embodiment, the pathogen is a coronavirus.

[0103] In the present invention, the coronavirus may in particular be of the α-CoV, β-CoV, γ-CoV or δ-CoV genus. More specifically, the coronavirus may be selected from the group consisting of human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKU1), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63, New Haven coronavirus), Middle East respiratory syndrome-related coronavirus (MERS-CoV or "new coronavirus 2012"), severe acute respiratory syndrome coronavirus (SARS-CoV or "SARS-classic"), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or "new coronavirus 2019").

[0104] In a particular embodiment, the present invention relates to a multivalent particle for use according to the invention or a composition for use according to the invention, wherein the coronavirus infection is a SARS-CoV-2 infection. Preferably, in this embodiment, the pathogen is SARS-CoV-2.

[0105] In some embodiments, the SARS-CoV-2 described herein is a SARS-CoV-2 variant. In some embodiments, the SARS-CoV-2 variant described herein is a SARS-CoV-2 variant selected from the group of Lineage B.1.1.207, Lineage B.1.1.7, Cluster 5, 501.V2 variant, Lineage P.1, Lineage B.1.429 / CAL.20C, Lineage B.1.427, Lineage B.1.526, Lineage B.1.525, Lineage B.1.1.317, Lineage B.1.1.318, Lineage B.1.351, Lineage B.1.617, Lineage B.1.617.2, and Lineage P.3. In some embodiments, the SARS-CoV-2 variants described herein are SARS-CoV-2 variants described by a Nextstrain clade selected from groups 19A, 20A, 20C, 20G, 20H, 20B, 20D, 20F, 20I, and 20E.

[0106] In some embodiments, the SARS-CoV-2 variants described herein are SARS-CoV-2 variants selected from the group of alpha, delta, beta, gamma, eta, iota, kappa, lambda. In some embodiments, the SARS-CoV-2 viruses described herein are SARS-CoV-2 variants that include at least one mutation selected from the group of N440K, L452R, S477G / N, E484Q, E484K, N501Y, D614G, P681H, P681R, and A701V. In some embodiments, the SARS-CoV-2 variants described herein are SARS-CoV-2 variants or hybrids derived from the variants described herein.

[0107] The present invention provides means and methods for inducing an immune response against SARS-CoV-2, e.g., prior to exposure (Figure 1). Thus, the present invention provides a novel method for generating a vaccine against SARS-CoV-2. By replacing the presented antigen (i.e., in the examples, the RBD) with an antigen of another pathogen / virus / variant, the means and methods provided herein can be used for the prevention of any other pathogen.

[0108] The invention may also be used to boost an inadequate immune response during infection and may therefore be used therapeutically rather than prophylactically.

[0109] In certain embodiments, the invention relates to a composition for use according to the invention, wherein the pathogen-associated antigen comprises an amino acid sequence derived from a coronavirus spike protein, e.g., a receptor binding domain (RBD) sequence, preferably the complete RBD sequence, a fragment thereof, or a sequence that comprises at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of the SARS-CoV-2 RBD amino acid sequence (SEQ ID NO:1).

[0110] In certain embodiments, the present invention relates to a method of treatment, the method comprising: (i) administering to a subject a monovalent / bivalent antigen particle comprising one or two antigen structures capable of inducing an antibody-mediated immune response against a target antigen, the target antigen being a pathogen-associated antigen. and (ii) administering to the subject a multivalent antigen particle comprising an antigen moiety comprising three or more antigen structures capable of inducing an antibody-mediated immune response against a target antigen, wherein the three or more antigen structures are cross-linked, and the target antigen is a pathogen-associated antigen.

[0111] In certain embodiments, the present invention relates to a method of treatment comprising the steps of: (i) administering to a subject a multivalent antigen particle comprising an antigen moiety comprising three or more antigen structures capable of inducing an antibody-mediated immune response against a target antigen, wherein the three or more antigen structures are cross-linked and the target antigen is a pathogen-associated antigen; and (ii) administering to a subject a monovalent / bivalent antigen particle comprising an antigen moiety comprising one or two antigen structures capable of inducing an antibody-mediated immune response against the target antigen, wherein the target antigen is a pathogen-associated antigen.

[0112] In certain embodiments, the present invention relates to a method of inducing and / or modulating a humoral immune response and / or a B cell-mediated immune response against a pathogen-associated target antigen, the method comprising contacting one or more B cells with a combination comprising: (i) a monovalent / bivalent antigen particle comprising an antigen moiety comprising one or two antigen structures capable of inducing an antibody-mediated immune response against the target antigen, where the target antigen is a pathogen-associated antigen; and (ii) a multivalent antigen particle comprising an antigen moiety comprising three or more antigen structures capable of inducing an antibody-mediated immune response against an antigen associated with the target antigen, where the three or more antigen structures are crosslinked and the target antigen is a pathogen-associated antigen.

[0113] In certain embodiments, the present invention relates to a method for producing antibodies that bind to a pathogen-associated antigen, comprising the steps of: (1) administering to a subject and / or cells capable of producing antibodies (i) monovalent / bivalent and / or bivalent antigen particles comprising an antigen moiety comprising one or two antigen structures capable of inducing an antibody-mediated immune response against a target antigen, where the target antigen is a pathogen-associated antigen, and (ii) multivalent antigen particles and / or precursors thereof, where the multivalent antigen particles comprise an antigen moiety comprising three or more antigen structures capable of inducing an antibody-mediated immune response against the target antigen, where the three or more antigen structures are crosslinked, where the target antigen is a pathogen-associated antigen, and (2) isolating the antibodies from the subject and / or cells capable of producing the antibodies, where the antibodies bind to the target antigen.

[0114] The term "subject," as used herein, refers to animals such as mammals, including primates (human non-human primates, such as monkeys and chimpanzees), non-primates (such as cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses, and whales), or birds (e.g., ducks or geese). In some embodiments, the subjects described herein are non-human animals.

[0115] A "cell capable of producing an antibody" is preferably a b-cell, a hybridoma cell, a myeloma cell, and / or a cell genetically modified to produce an antibody. In some embodiments, a cell capable of producing an antibody described herein is a cell of a cell line.

[0116] Methods for isolating antibodies are known to those of skill in the art (see, for example, Huang J, Doria-Rose NA, et al., 2013, Nat Protoc. Oct; 8(10): 1907-15). Any method known to those of skill in the art can be used to isolate antibodies from a subject and / or cell. In some embodiments, isolating the antibodies described herein comprises at least one method selected from the group of physicochemical fractionation, class-specific affinity, and antigen-specific affinity.

[0117] In certain embodiments, the present invention relates to polynucleotides for use in the treatment and / or prevention of infectious diseases, encoding: (i) monovalent / bivalent antigen particles comprising an antigenic moiety comprising one or two antigenic structures capable of inducing an antibody-mediated immune response against a target antigen, wherein the target antigen is a pathogen-associated antigen; and (ii) multivalent antigen particles and / or precursors thereof, wherein the multivalent antigen particles comprise an antigenic moiety comprising three or more antigenic structures capable of inducing an antibody-mediated immune response against the target antigen, wherein the three or more antigenic structures are cross-linked, and the target antigen is a pathogen-associated antigen.

[0118] The term "polynucleotide" as used herein refers to a nucleic acid sequence. The nucleic acid sequence may be a DNA or RNA sequence, and preferably the nucleic acid sequence is a DNA sequence. The polynucleotides of the present invention are preferably provided either as isolated polynucleotides (i.e., isolated from their natural context) or in genetically modified form. The isolated polynucleotides referred to herein also encompass polynucleotides present in their natural cellular context, i.e., a cellular context other than a heterologous polynucleotide. The term polynucleotide encompasses single-stranded and double-stranded polynucleotides. Additionally, chemically modified polynucleotides, including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides, or artificially modified polynucleotides such as biotinylated polynucleotides, are also included.

[0119] In certain embodiments, the present invention relates to vectors comprising the polynucleotides for use in the present invention.

[0120] The term "vector" as used herein refers to a nucleic acid molecule that can transfer or transport another nucleic acid molecule. The transferred nucleic acid is generally linked to, i.e., inserted into, the vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication in a cell, or may contain sequences sufficient to allow integration into the host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.

[0121] The polynucleotides and / or vectors described herein may be used to produce the antigenic particles and / or portions thereof described herein.

[0122] The articles "a", "an" and "the" are used herein to refer to one or to more than one (ie to at least one or more) of the grammatical object of the article.

[0123] "Or" should be understood to mean either one, both, or any combination of the alternatives. "And / or" should be understood to mean either one, or both, of the alternatives.

[0124] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0125] The terms "include" and "comprise" are used synonymously. "Preferably" means one option within a set of options that does not exclude other options. "Eg" means one example that is not limited to the examples stated. "Consisting of" means including and limited to what follows the word "consisting of".

[0126] The terms "about" or "approximately" as used herein refer to "within 20%," more preferably "within 10%," and even more preferably "within 5%" of a given value or range.

[0127] References throughout this specification to "one embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," "a particular embodiment," or "a further embodiment," or combinations thereof, mean that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment of the invention. Thus, the appearances of such phrases or characteristics in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It is also understood that the positive recitation of a feature in an embodiment serves as a basis for excluding the feature in a particular embodiment.

[0128] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0129] Unless otherwise specified, the general methods and techniques described herein may be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990).

[0130] While aspects of the invention have been explained and described in detail in the drawings and the foregoing description, such explanations and descriptions are to be considered as illustrative or exemplary and not restrictive. Those skilled in the art will understand that changes and modifications may be made within the scope and spirit of the following claims. In particular, the invention encompasses further embodiments in any combination of features from the different embodiments described above and below. EXAMPLES

[0131] Example 1: Immunization Scheme Virus-derived peptides (Peptides & Elephants, Berlin) (SEQ ID NO:2, SEQ ID NO:3) were dissolved according to their water solubility in pure water or 1% dimethyl sulfoxide (DMSO). Virus-derived peptides (SEQ ID NO:2, SEQ ID NO:3) were conjugated to biotin or KLH, respectively. Quantities of 1 mg were purchased and dissolved in a volume of 1 ml. 10-50 μg of KLH-conjugated peptides were used for immunization of mice via intraperitoneal injection.

[0132] The impact of the immune concept of the present invention on vaccine design was tested using a pathogen-specific antigen derived from the SARS-CoV-2 coronavirus, which causes COVID-19. During infection, the SARS-CoV-2 coronavirus binds to angiotensin-converting enzyme 2 (ACE2) on the host cell surface via its receptor binding domain (RBD). Thus, eliciting an antibody response that blocks the RBD / ACE2 interaction is believed to be key to preventing coronavirus infection. Therefore, we used the RBD from SARS-CoV-2 to model the role of the antigen in the immune response during immunization.

[0133] Immunization with complexed RBD (cRBD) (a 4:1 ratio was used for complexation with streptavidin and biotinylated RBD; a minimum of 20 μg per 100 μg of RBD for complexation with 1,2-phenylene-bis-maleimide with 1,2-PBM) was found to induce a stronger IgG immune response compared to soluble RBD (sRBD). For production of RBD, an expression vector encoding a hexahistidine-tagged version of RBD was transiently transfected into HEK293-6E cells (Amanat, F., et al., 2020, Nature medicine, 26(7), 1033-1036). Soluble RBD was purified from the supernatant 5 days after transfection by nickel-based immobilized metal affinity chromatography (TaKaRa). However, the antibody concentration was not sufficient to allow virus neutralization using in vitro infection experiments (see Figures 1-8). Therefore, we tested whether pre-treating mice with sRBD prior to immunization would boost the immune response. Indeed, pre-treating mice with soluble RBD 2 weeks prior to immunization significantly enhanced the immune response (Figure 1). Importantly, sera from pre-treated mice showed a highly potent ability to prevent SARS-CoV-2 infection in vitro.

[0134] Furthermore, it was found that different ratios of sRBD to cRBD in the composition of the immunization cocktail resulted in different ratios of immunoglobulin isotypes (i.e., IgG to IgM) that allowed for precise control of the immune response following immunization (see Figures 2-8).

[0135] Example 2 Antibody responses after multiple injections of native or complexed RBD During infection, SARS-CoV-2 coronavirus binds to angiotensin-converting enzyme 2 (ACE2) on the host cell surface via its receptor-binding domain (RBD), and this binding appeared to be a critical step for viral infection. Thus, eliciting an antibody response that blocks the RBD / ACE2 interaction may be key to preventing coronavirus infection. Therefore, we generated recombinant RBD from SARS-CoV-2 and evaluated the role of antigenic form in the immune response during immunization.

[0136] We found that native RBD (nRBD) formed dimers under non-reducing conditions, and that higher molecular complexes of RBD (cRBD) could be formed after biotinylation (Figure 2A). While typical immunization by injecting nRBD on day 0 (primary immunization) and day 21 (secondary immunization or boost) failed to induce reliable antibody responses, cRBD was able to induce detectable antibody responses on day 28, one week after secondary immunization (Figures 2B and C). Interestingly, six repeated injections of nRBD over a two-week period were also able to induce detectable immune responses (Figure 2D).

[0137] Taken together, these data suggest that immunization with multivalent complex RBD or multiple injections of nRBD induces detectable RBD-specific antibody responses.

[0138] Example 3 Repeated nRBD treatment with cRBD results in robust antibody responses. Since the antibody responses described above should be increased to ensure immune protection, we tested whether combining six repeated injections of cRBD with nRBD could enhance the immune response. Therefore, we pretreated mice six times with nRBD prior to cRBD immunization (Figure 3A).

[0139] WT mice received repeated injections of either native RBD (6 intraperitoneal doses of 50 μg each in the absence of adjuvant within 14 days; +PT), whereas control animals were pretreated with PBS only (-PT). All animals were then immunized intraperitoneally with 50 μg of native RBD complexed to streptavidin (cRBD) in the presence of CpG-ODN #1826 and boosted 3 weeks later.

[0140] Indeed, pretreatment of mice with nRBD 2 weeks before immunization significantly enhanced the immune response ( Fig. 3B ).

[0141] RBD-specific IgM and IgG were measured by ELISA in both groups and compared to titers measured in CI mice. Mice repeatedly exposed to native RBD prior to immunization with cRBD mount a robust antibody response against RBD that can be detected already after the first application of cRBD.

[0142] Compared to cRBD without pretreatment (wo PT), pretreatment resulted in up to 40-fold higher concentrations of anti-RBD IgM at day 7 after cRBD immunization, and this IgM response was further increased at day 28, one week after the secondary immunization (Figure 3B). Anti-RBD IgG was also increased when nRBD pretreatment was combined with cRBD immunization, as measured by higher titers of anti-RBD IgG at days 14 and 28 (Figure 3B).

[0143] WT mice received repeated injections of either native RBD (6 intraperitoneal doses of 50 μg each in the absence of adjuvant within 14 days; +PT), whereas control animals were pretreated with PBS only (-PT). Animals were then immunized intraperitoneally with 50 μg of native RBD complexed to streptavidin (cRBD, day 0) in the presence of CpG-ODN #1826 and boosted 3 and 5 weeks later. A third group of mice was pretreated with RBD but received a primary immunization with cRBD 5 weeks later (Figure 4A).

[0144] RBD-specific IgM and IgG were analyzed by ELISA: The activity was determined by coating 96-well Maxisorp ELISA plates (Nunc) overnight with 50 μl / well of RBD at a concentration of 10 μg / ml.

[0145] After three washing steps with 200 μl ELISA washing buffer (PBS 0,1% Tween-20), non-specific binding sites were blocked with 100 μl / well ELISA blocking buffer (PBS 1% BSA) for 1 h at 37° C. After three additional washing steps with 200 μl / well ELISA washing buffer, 100 μl ELISA blocking buffer was added to each well. 150 μl of prediluted serum was applied in duplicate to the first column of the plate. Serial dilutions in a 1:3 ratio were prepared by transferring 50 μl from the first column to the second column and so on to the eighth column. Duplicate columns coated with either anti-mouse IgM (Southern Biotech, 1020-01) or IgG (Southern Biotech, 1030-01) at a concentration of 10 μg / ml and captured with mouse IgM (Southern Biotech, 0101-01) or IgG (Southern Biotech, 0107-01) served as standards. Two wells containing blocking buffer only served as blanks. Plates were incubated for an additional 2 hours at 37° C. for capture. Unbound antibodies were removed by washing three times with 200 μl / well of ELISA wash buffer, and 50 μl / well of secondary goat a-mouse IgM (Southern Biotech 1020-04, diluted 1:1,000 in ELISA blocking buffer) or IgG antibody conjugated to alkaline phosphatase (Southern Biotech 1030-04, diluted 1:2,000 in ELISA blocking buffer) solution was added and incubated for 1 hour at 37°C. The plate was then washed again three times with ELISA wash buffer to remove excess antibodies. Substrate solution containing 4-nitrophenyl phosphate (pNPP, Gennaxon) in diethanolamine buffer was added to each well. OD was measured at 405 nm using a Multiskan FC ELISA plate reader (Thermo Fisher Scientific) and antibody concentrations were determined using the SkanIt software provided with the machine.

[0146] Pretreatment with native RBD primes for efficient antibody responses even when the primary immune response is delayed by 5 weeks.

[0147] Interestingly, the effect of pretreatment with nRBD appears to be long-lasting, as immunization with cRBD induced robust antibody responses at day 35 after pretreatment similar to those induced at day 0 of immunization (Figure 4).

[0148] These data show that pretreatment with nRBD strongly enhances the immune response induced by cRBD, suggesting that repeated nRBD treatment may prime the immune system for efficient RBD-specific immune responses.

[0149] Example 4 High antibody titers are required for in vitro virus neutralization. To test whether the amount of antibodies induced by the combination treatment was sufficient for virus neutralization, an in vitro neutralization assay was performed using a pseudovirus preparation expressing the spike protein of Sars-CoV 2 (methods described in Hoffmann, M., et al., 2021, Cell, 184(9), 2384-2393).

[0150] The data show that the serum from pretreated mice exhibited obvious ability to prevent SARS-CoV-2 infection in vitro (Figure 5C). Furthermore, the data also show that the weak immune response induced by cRBD injection without pretreatment was not sufficient for virus neutralization (Figure 5).

[0151] Furthermore, it has been found that different ratios of sRBD to cRBD in the composition of the immunization cocktail result in different ratios of immunoglobulin isotypes (i.e., IgG to IgM) that allow for precise control of the immune response following immunization.

[0152] Thus, combining nRBD treatment with cRBD immunization induces robust antibody responses to neutralize Sars-CoV 2 infection.

[0153] Example 5 IgM BCR expression accelerates antibody responses. IgD-deficient mice were immunized in parallel with wild-type mice using a combination protocol of nRBD pretreatment and subsequent cRBD immunization. WT and IgD-KO mice were repeatedly exposed to native RBD (50 μg each intraperitoneally in the absence of adjuvant within 14 days; +PT). All animals were then immunized intraperitoneally with 50 μg of native RBD (cRBD) complexed to streptavidin in the presence of CpG-ODN #1826 and boosted 3 weeks later. Blood was collected from immunized mice at the indicated time points. RBD-specific IgM and IgG were measured in both groups by ELISA.

[0154] Consistent with the proposed role of IgD, primary (day 7) and secondary (day 28) IgM immune responses were greatly increased in IgD-deficient mice compared to wild-type controls (Figure 6). In contrast, secondary (day 28) IgG antibody responses were reduced in IgD-deficient mice (Figure 6). This indicates that B cell populations with reduced or absent IgD expression can elicit faster immune responses following immunization with cRBD. This suggests that individuals with significant production of newly produced B cells that have not reached the IgD-high stage are well protected from viral infections due to a more rapid primary response.

[0155] B cell responsiveness determines the strength and isotype of the antibody response. Newly generated B cells have much more IgM than IgD and are generated during lymphopoiesis, a process that declines with age. The difference between older and younger patients in surviving COVID-19 may be related to a weaker primary immune response in older patients.

[0156] Example 6. Robust antibody responses with RBD complexes generated by chemical cross-linking The above experiments suggest that RBD complexes are important for eliciting immune responses and that native RBD is required for efficient priming of immune responses. However, generation of immune complexes by biotinylating RBD and subsequent complex formation is unlikely to be practical for large-scale production of vaccines. Therefore, we tested whether chemical cross-linking could generate immunogenic cRBD. To this end, we used a compound typically used for irreversible cross-linking via sulfhydryl (SH) groups, 1,2-phenylene-bis-maleimide (hereafter referred to as bismaleimide). Different concentrations and incubation times were tested to generate different ratios of complex to native RBD (Figure 7A).

[0157] WT mice were immunized intraperitoneally with 50 μg of cRBD*MM in the presence of CpG-ODN #1826. The mice were boosted with the same compound 3 and 5 weeks later (FIG. 7B).

[0158] After dialysis, immunization experiments were performed by injecting similar amounts of chemically cross-linked RBD into wild-type mice at days 0 and 21. The experiments show that moderate amounts of IgM were detected at day 28, one week after secondary immunization, but IgG was strongly increased at this time point (Figure 7C). Mice immunized with cRBD*MM, which still contains monomeric RBD molecules, mount robust antibody responses with low RBD-specific IgM concentrations. These data indicate that chemical cross-linking produces a mixture of nRBD and cRBD with a very high capacity to induce antigen-specific immune responses.

[0159] Example 7 Antibodies induced by chemically cross-linked RBD have high neutralizing capacity Since recombinant RBD can be easily generated and used for primary and secondary immunization in a typical vaccination, chemical cross-linking of RBD may provide a practical method for the generation of SARS-CoV2 vaccines. Therefore, we tested whether the resulting antibodies could prevent viral infection (methods described in Hoffmann, M., et al., 2021, Cell, 184(9), 2384-2393). The results show that mice immunized with chemically cross-linked RBD have high competence in neutralization assays using pseudovirus preparations (Figure 8).

[0160] These data suggest that chemical cross-linking of the RBD may facilitate the design of efficient vaccines against SARS-CoV2.

[0161] Example 8 Activating antigens form IgG complexes that boost immune responses We noticed that chemical cross-linking with bismale slightly changed the behavior of monomeric RBD in Coomassie staining on SDS page (Figure 7A). We analyzed the sequence of the RBD and identified a single SH group that is not involved in an intramolecular disulfide bond. We proposed that bismale treatment of RBD or other proteins may result in saturation binding of bismale, such that additional proteins cannot be cross-linked by bismale molecules (Figure 9B, middle). However, it is possible that bismale treatment results in monomeric RBD conjugated by bismale, where a free maleimide group is still available (Figure 9B, bottom).

[0162] RBD* was complexed with 20 μg of Bismale per 100 μg of RBD, whereas RBD** shows complexation with 100 μg of Bismale per 100 μg of RBD (Figure 9C).

[0163] Immunizations were performed in WT C57BL6 / J mice with 50 μg of unconjugated native RBD (nRBD, n=3), 50 μg of RBD conjugated with 10 μg of Bismale (RBD*, n=3), or 50 μg of RBD conjugated with 10 μg of Bismale in the presence of 25 μg of polyclonal mouse IgG (RBD*IgG). 50 μg of CpG-ODN #1826 was used as adjuvant in all conditions. IgM or IgA isotypes were used instead of IgG for immunization with RBD*IgM and RBD*IgA. Mice were boosted with the same immunization mixture 21 days after the primary immunization. Serum was collected on day 28 for analysis. (Figure 9D).

[0164] WT mice were immunized with 50 μg of unconjugated native RBD+CpG-ODN (nRBD, n=3), 50 μg of RBD complexed with 10 μg of Bismare+CpG-ODN (RBD*, n=3), or 50 μg of RBD complexed with 10 μg of Bismare in the presence of 25 μg of mouse IgG but in the absence of CpG-ODN (RBD*IgG, n=2).

[0165] This results in an activated RBD that can undergo bioconjugation with other proteins in vitro or in vivo. Importantly, increasing amounts of bismale lead to a decrease in monomeric RBD, suggesting that more bismale leads to more protein complexes (Figure 9C). To test the possibility of forming heterocomplexes as well as to investigate the role of immunoglobulins in randomly formed complexes, IgM, IgA and IgG were included in the cross-linking reactions.

[0166] Interestingly, the results showed that while IgM and IgA failed to boost immune responses, cross-linking of RBD and IgG led to a dramatic increase in RBD-specific immune responses (Figure 9D). Importantly, addition of IgG after terminating Bismalle-mediated cross-linking did not boost immune responses suggesting that Bismalle-mediated cross-linking is important for IgG-mediated enhancement.

[0167] The enhancement observed by IgG led us to test whether IgG could serve as an adjuvant replacing traditional adjuvants such as alum or CpG. To this end, we compared the immune responses generated by complexed RBD injected in the presence of CpG or IgG as adjuvants. The results show that IgG containing immune complexes are able to induce robust antibody responses in the absence of traditional adjuvants such as CpG or alum that activate TLRs. In conclusion, the data suggest that IgG containing immune complexes can be generated by crosslinking IgG with specific antigens in vitro or by injecting the antigen after incubation in vitro with a bifunctional crosslinker containing two reactive groups. Such activating antigens represent a simple and efficient method for the development and generation of effective vaccines.

[0168] Example 9 Treatment of the bismaleimide-crosslinked immune complex with cysteine ​​in vitro quenches the still available reactive maleimide groups, restoring antigen activation and thereby reducing antibody production (Figure 10D).

[0169] 100 μg of activated RBD was quenched in at least 1 μl of freshly prepared 2 M L-cysteine ​​(Sigma-L-cysteine ​​BioUltra, ≥98.5% 30089-25G) solution and incubated overnight at room temperature. The next day, to remove unbound cysteine ​​and maleimide, the sample was rebuffered in a dialysis cassette (Thermo Fisher Scientific Slide-A-Lyzer™ 10K MWCO 66381) at 4°C under constant stirring with a magnetic stir bar. 1×PBS was replaced after overnight dialysis and the sample was dialyzed for an additional 4-6 hours at 4°C under constant stirring with a magnetic stir bar.

[0170] The data show that increasing the maleimide (RBD**) leads to an increased antibody response, and quenching the maleimide-treated antigen with cysteine ​​(RBD**C) dramatically reduces the antibody response, suggesting that maleimide treatment leads to the generation of activated antigens capable of forming complexes in vivo, and that this ability is important for the immune response.

[0171] Thus, antigen activation amplifies the immune response by making it reactive with SH groups on self-antigens. Inclusion of total IgG in antigen activation results in the generation of protein complexes that mimic immune complexes, thereby inducing an efficient antibody response.

[0172] Example 10 Antigen (Ag) complexes were generated by biotinylation and subsequent incubation with streptavidin (SAV). The complexed antigen induces an antibody response. Multivalency depends on the number of biotins per molecule. Multiple biotin groups allow multiple SAV binding and higher molecular complexes. Cross-linking with SAV results in higher molecular complexes and more efficient immune responses (Figures 11, 12).

Claims

1. A composition for use in the treatment and / or prevention of an infectious disease, comprising: (i) a monovalent / bivalent antigen particle comprising an antigen moiety comprising one or two antigen structures capable of inducing an antibody-mediated immune response against a target antigen, said target antigen being a pathogen-associated antigen; (ii) a multivalent antigen particle comprising an antigen moiety comprising three or more antigen structures capable of inducing an antibody-mediated immune response against said target antigen, wherein said three or more antigen structures are cross-linked, and said target antigen is a pathogen-associated antigen.

2. The composition for use according to claim 1 , wherein the three or more antigenic structures comprise a plurality of identical antigenic structures.

3. 3. The composition for use according to claim 1 or 2, wherein the multivalent antigen particles further comprise a carrier moiety bound to the antigen moiety and / or the monovalent / bivalent antigen particles further comprise a carrier moiety bound to the antigen moiety.

4. The composition for use according to claim 3, wherein the carrier moiety comprises a structure selected from the group of a polypeptide, an immune CpG island, limpet hemocyanin (KLH), tetanus toxoid (TT), cholera toxin subunit B (CTB), a bacterium or bacterial ghost, a liposome, a chitosome, a virosome, a microsphere, a dendritic cell, a particle, a microparticle, a nanoparticle, or a bead.

5. 5. The composition for use according to any one of claims 1 to 4, wherein the crosslinks in the multivalent antigen particles are chemical crosslinks, such as bis-maleimide mediated crosslinks, or protein crosslinks, such as biotin-streptavidin mediated crosslinks.

6. 6. The composition for use according to any one of claims 1 to 5, wherein the multivalent antigen particle comprises a conjugate of the following formula A-L-A, where A is a moiety comprising a target antigen and L is the linker of the cross-linking bond, preferably L is a bismaleimide, most preferably the conjugate is of the following structure (I), where R is a moiety comprising a target antigen. 【Chemistry 1】

7. 6. The composition for use according to any one of claims 1 to 5, wherein the multivalent antigen particles comprise a linker with cross-linking reactive groups for protein conjugation, preferably a linker with cross-linking reactive groups for stable protein conjugation.

8. The composition for use according to claim 7, wherein the crosslinking reactive groups are groups selected from carboxyl-amine reactive groups, amine reactive groups, sulfhydryl reactive groups, aldehyde reactive groups, and photoreactive groups.

9. 8. The composition for use according to claim 7, wherein the crosslinking reactive group is a group selected from carbodiimides, NHS esters, imidoesters, pentafluorophenyl esters, hydroxymethylphosphines, maleimides, haloacetyls, hydrazides, alkoxyamines, diazirines, and arylazides.

10. A composition for use according to any one of claims 1 to 9, wherein said multivalent antigen particles comprise at least two copies of said antigen structure in spatial proximity to each other, preferably within the range of 3 nm to 20 nm.

11. The composition for use according to any one of claims 1 to 10, wherein the pathogen associated antigen comprises at least one substance selected from the group of nucleic acids, carbohydrates and peptides.

12. The composition for use according to any one of claims 1 to 11, wherein said multivalent antigen particles are linked to an adjuvant, preferably said multivalent particles are covalently linked to an adjuvant.

13. The composition for use according to claim 12, wherein the adjuvant is IgG.

14. The composition for use according to any one of claims 1 to 13, wherein the treatment and / or prevention comprises at least two administration times.

15. 15. The composition for use according to claim 14, wherein the prevention comprises administering said monovalent / bivalent antigen particles prior to said multivalent antigen particles.

16. 16. The composition for use according to claim 14 or 15, wherein the treatment and / or prevention comprises at least two administration times for the monovalent / bivalent antigen particles and at least two administration times for the multivalent antigen particles.

17. The composition for use according to any one of claims 1 to 16, wherein the antibody-mediated immune response is an IgG-mediated immune response and / or an IgM-mediated immune response.

18. The composition for use according to any one of claims 1 to 17, wherein the pathogen is at least one pathogen selected from the group of parasites, bacteria and viruses.

19. The composition for use according to any one of claims 1 to 18, wherein the infectious disease is a viral infection.

20. 20. The composition for use according to claim 19, wherein the viral infection is a coronavirus infection.

21. The composition for use according to claim 20, wherein the coronavirus infection is a SARS-CoV-2 infection.

22. 22. The composition for use of claim 21 , wherein the pathogen associated antigen comprises an amino acid sequence derived from the coronavirus spike protein, e.g., a receptor binding domain (RBD) sequence, preferably the complete RBD sequence, or a sequence that comprises at least 80% sequence identity to the amino acid sequence of the SARS-CoV-2 RBD amino acid sequence (SEQ ID NO:1).

23. 1. A method for producing an antibody that binds to a pathogen-associated antigen, comprising: (1) to a subject and / or cell capable of producing antibodies; (i) a monovalent / bivalent particle comprising an antigen moiety comprising one or two antigen structures capable of inducing an antibody-mediated immune response against a target antigen, said target antigen comprising a pathogen-associated antigen; and (ii) a multivalent antigen particle comprising an antigen moiety comprising three or more antigen structures capable of inducing an antibody-mediated immune response against said target antigen, wherein said three or more antigen structures are cross-linked, and said target antigen comprises a pathogen-associated antigen; and administering (2) isolating an antibody from the subject and / or cell, wherein the antibody binds to the target antigen.

24. A composition comprising a polynucleotide for use in the treatment and / or prevention of an infectious disease, said polynucleotide comprising: (i) a monovalent / bivalent antigen particle comprising an antigen moiety comprising one or two antigen structures capable of inducing an antibody-mediated immune response against a target antigen, the target antigen comprising a pathogen-associated antigen; and (ii) a multivalent antigen particle comprising an antigen moiety comprising three or more antigen structures capable of inducing an antibody-mediated immune response against said target antigen, wherein said three or more antigen structures are cross-linked, and said target antigen comprises a pathogen-associated antigen; A composition encoding the 25. A composition comprising a vector for use in the treatment and / or prevention of an infectious disease, the vector comprising a polynucleotide described in claim 24.

26. A combination for use in the treatment and / or prevention of an infectious disease, comprising: (i) a monovalent / bivalent antigen particle comprising an antigen moiety comprising one or two antigen structures capable of inducing an antibody-mediated immune response against a target antigen, said target antigen being a pathogen-associated antigen; (ii) a multivalent antigen particle comprising an antigen moiety comprising three or more antigen structures capable of inducing an antibody-mediated immune response against said target antigen, wherein said three or more antigen structures are cross-linked, and said target antigen is a pathogen-associated antigen; and A combination comprising:

27. A combination for use in the treatment and / or prevention of an infectious disease, comprising: (i) a polynucleotide encoding a monovalent / bivalent antigen particle comprising an antigen moiety comprising one or two antigen structures capable of inducing an antibody-mediated immune response against a target antigen, the target antigen being a pathogen-associated antigen; and (ii) a polynucleotide encoding a multivalent antigen particle comprising an antigenic moiety comprising three or more antigenic structures capable of inducing an antibody-mediated immune response against said target antigen, wherein said three or more antigenic structures are cross-linked, and said target antigen is a pathogen-associated antigen; and A combination comprising:

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