WO2020257315A1 - Multiple antigen protein displayed adjuvant systems - Google Patents

Abstract

The present invention provides novel 'Multiple Antigen Protein Displayed Adjuvant' [MAPDAdjuvant] systems based on adjuvanted nanoparticles'. These nanoparticles are designed and produced in a manner comprising assemblies of peptides that present multiple copies of protein antigens and their critical immunogenic epitopes in ordered arrays displayed on the adjuvants. In one aspect, an objective of the MAPDAdjuvants is the proper orientation and presentation of immunogens having high density (high copy number of antigens) to support multiple binding events to occur simultaneously between the nanoparticle and the host cell B Cell Receptors (BCRs). These multiple binding events, generated by the adjuvanted nanoparticles, eventually provide stronger antigen-antibody interactions compared to the weak and low affinity interactions provided by the monovalent binding generated by single soluble protein immunogens.

Description

MULTIPLE ANTIGEN PROTEIN DISPLAYED ADJUVANT SYSTEMS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority to United States Provisional Application No. 62/862,913, filed June 18, 2019, the contents of which are incorporated herein by reference.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on June 17, 2020, is named 12063-001W01-SEQ_LIST_ST25.txt and is 22 kilobytes in size.

BACKGROUND

[0003] Stimulation of protective immune responses by antigens is the basis for effective prophylactic vaccines against pathogenic microorganisms. Not all antigens induce an appropriate immune response, however. Development of new and improved systems for stimulating protective immune responses is an active area.

SUMMARY

[0004] The invention relates to novel Multiple Antigen Protein Displayed Adjuvant (MAPD Adjuvant) systems for immunogenicity enhancement and/or stability

enhancement. An ingredient of the MAPD Adjuvant system is a recombinantly expressed Multifunctional Chimeric Fusion Protein (MCFP) which in turn is composed of (i) protein/glycoprotein antigen; (ii) linker peptide, optionally fused to heterologous T-cell epitopes; and (iii) Dual Function Peptide (DFP) which can act as a purification aid as being capable of binding to the adjuvant via non-covalent affinity interactions.

Specifically, in some examples, the present invention relates to the production of adjuvanted nanoparticles, and more specifically, to the design and production of adjuvanted nanoparticles to minimize non-specific interactions between critical immunogenic epitopes of the antigens and the adjuvants. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] In the accompanying drawings, which are incorporated in and constitute a part of the specification, embodiments of the disclosed inventions are illustrated. It will be appreciated that the embodiments illustrated in the drawings are shown for purposes of illustration and not for limitation. It will be appreciated that changes, modifications and deviations from the embodiments illustrated in the drawings may be made without departing from the spirit and scope of the invention, as disclosed below.

[0006] Fig. 1 depicts the current classical vaccine formulation process of the mixing of the antigen (drug substance) with the adjuvant to produce the“drug product”. In some examples, component (A) in the figure may be called a“critical immunogenic epitope.”

[0007] Fig. 2 depicts example components and steps involved in the production of an example MAPD Adjuvant system. In the first step, Multifunctional Chimeric Fusion Protein (MCFP) is produced by recombinant expression. MCFP drug substance is composed of (A) critical immunogenic epitope; (B) protein/glycoprotein antigen; (C) linker peptide, optionally fused to (D) heterologous T-cell epitope; and (E) Dual Function Peptides (DFP) which can act as a purification aid as well having the non-covalent affinity to bind to an adjuvant. In the second formulation step, the MCFP is mixed with an adjuvant to produce the MAPD Adjuvant (Drug Product).

[0008] Fig. 3 depicts example components and steps involved in the production of an example MAPD Adjuvant system. In the first step Multifunctional Chimeric Fusion Protein (MCFP) is produced by recombinant expression. MCFP drug substance is composed of (A) critical immunogenic epitope; (B) protein/glycoprotein antigen; (C) linker peptide, optionally fused to (D) heterologous T-cell epitope; and (E) Dual Function Peptide (DFP) which can act as a purification aid as well having the non-covalent affinity to bind to an adjuvant. In the second formulation step, the MCFP is mixed with (G) a core adjuvant, precoated with (F) polypeptide or peptide adjuvant (may be ionic), to produce the MAPD Adjuvant (Drug Product).

[0009] Fig. 4 is an expression and solubility assessment of CTRNV6 (containing

Staphylococcus aureus Clf A protein antigen). Samples were collected prior to induction with IPTG (denoted with“0”) and at three hours post-induction with IPTG (denoted with “3”). Protein expression was assessed in whole cell protein samples (denoted with “WC”), and solubility of the recombinant protein was assessed in post-induction samples, where“S” indicates the soluble protein fraction and“I” indicates the insoluble protein fraction. Sizes of relevant molecular weight standards are shown at the left of the figure, and the CTRNV6 protein is indicated with a solid arrow at the right of the figure.

[0010] Fig. 5A and 5B includes data showing purification of CTRNV6 (containing S. aureus ClfA protein antigen). CTRNV6 was purified from the soluble protein fraction by Immobilized Metal Affinity Chromatography (IMAC) in batch mode. The progression of purification was followed throughout the procedure by SDS-PAGE. Fig. 5A. M: Protein molecular weight standard; 1: Whole cell protein from cells prior to induction with IPTG; 2: Whole cell protein from cells post- induction with IPTG; 3: Proteins not bound during incubation with IMAC resin; 4: Proteins removed by first wash in buffer containing 5 mM imidazole; 5: Proteins removed by second wash in buffer containing 5 mM imidazole; 6: Proteins removed by first wash in buffer containing 60 mM imidazole; 7: Proteins removed by second wash in buffer containing 60 mM imidazole; 8: Proteins removed by third wash in buffer containing 60 mM imidazole. Fig. 5B. M: Protein molecular weight standard; 1: Whole cell protein from cells prior to induction with IPTG; 2: Whole cell protein from cells post- induction with IPTG; 3: Proteins eluted from the resin from the first wash in buffer containing 1 M imidazole; 4: Proteins eluted from the resin from the second wash in buffer containing 1 M imidazole; 5: Proteins eluted from the resin from the third wash in buffer containing 1 M imidazole; 6: Proteins eluted from the resin from the fourth wash in buffer containing 1 M imidazole; 7:

Proteins eluted from the resin from the fifth wash in buffer containing 1 M imidazole. Sizes of relevant molecular weight standards are shown at the left of each panel.

CTRNV6 protein is indicated with a solid arrow at the right of the Panel B.

DETAILED DESCRIPTION

Definitions

[0011] As used herein, the singular forms "a," "an," and "the," refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term "an antigen" or“immunogen” includes single or plural antigens or immunogens and can be considered equivalent to the phrase "at least one antigen" or“at least one immunogen”. [0012] The term "adjuvant" refers to a substance capable of enhancing, accelerating, or prolonging the body's immune response to an immunogen or immunogenic composition, such as a vaccine (although it is not immunogenic by itself). An adjuvant may be included in the immunogenic composition, such as a vaccine.

[0013] The term "administration" refers to the introduction of a substance or composition into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intramuscular, the composition (such as a composition including a disclosed immunogen) is administered by introducing the composition into a muscle of the subject.

[0014] The term "antigen" refers to a molecule that can be recognized by an antibody. Examples of antigens include polypeptides, peptides, lipids, polysaccharides, and nucleic acids containing antigenic determinants, such as those recognized by an immune cell.

[0015] The term“bound to” refers to the association of two different molecules via covalent or non-covalent interactions.

[0016] The term“dual function peptide” (DFP) refers to a peptide domain that aids in protein purification as well as having the affinity to bind to adjuvants, in some examples, via non-covalent interactions.

[0017] The term "effective amount" refers to an amount of agent that is sufficient to generate a desired response. For instance, this can be the amount necessary to inhibit viral replication or to measurably alter outward symptoms of the viral infection.

[0018] The term "epitope" (or "antigenic determinant" or "antigenic site") refers to the region of an antigen to which an antibody, B cell receptor, or T cell receptor binds or responds. Epitopes can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by secondary, tertiary, or quaternary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by higher order folding are typically lost on treatment with denaturing solvents.

[0019]“Critical immunogenic epitopes” are designed to trigger the immune responses of immune cells to generate an optimal immune response to produce‘functional antibody’ molecules, and are ideal for vaccine design. Not all epitopes can generate the immune response required to produce functional antibody molecules. Peptide antigens are cleaved into short peptides, and some peptides are transported into the endoplasmic reticulum (ER) by the antigen presenting proteins. Only some epitopes will bind to major histocompatibility complex (MHC) molecules and form the MHC-peptide complexes. This binding is governed by a number factors including the length, sequence, type of amino acids and the shape of the epitope which determines the‘antigen- antibody complex’ interactions. Finally, the complexes are presented on the cell surface to induce the immune response. T-cell epitopes are defined as the antigen segments that bind to major histocompatibility molecules. The major histocompatibility complex (MHC) is the cell surface molecules in vertebrates that are encoded by a specified gene family. The MHC molecules are of two categories: MHC-I and MHC-II. MHC-I molecules usually present epitopes of 9 amino acids, whereas epitopes binding to MHC-II may consist of 12-25 amino acids.

[0020] The term "glycoprotein" refers to a protein that contains oligosaccharide chains (glycans) covalently attached to polypeptide side-chains.

[0021] The term "glycosylation site" refers to an amino acid sequence on the surface of a polypeptide, such as a protein, which accommodates the attachment of a glycan.

[0022] The term“heterologously derived” refers to incorporation of a sequence, either nucleotide or amino acid, that is not naturally present in a sequence of interest. Incorporation of heterologous sequences can be accomplished, for example, by recombinant DNA technology.

[0023] The term "host cells" refers to cells in which a vector can be propagated and its DNA or RNA expressed. The cell may be prokaryotic or eukaryotic.

[0024] In another aspect, the present invention provides nucleic acid molecules that encode peptide-linked protein immunogen described herein above. These nucleic acid molecules include DNA, cDNA, and RNA sequences. The nucleic acid molecule can be incorporated into a vector, such as an expression vector.

[0025] The term "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence. Methods of alignment of sequences for comparison are well known in the art. Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a peptide sequence that has 150 matches when aligned with a test sequence having 300 amino acids is 50.0 percent identical to the test sequence (150/300*100 = 50.0).

[0026] In some examples, the sequences disclosed and/or claimed herein may be 60, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the actual sequences in any of the SEQ ID NOs that are part of this application.

[0027] In some examples, the sequences disclosed and/or claimed herein may be at least 60, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the actual sequences in any of the SEQ ID NOs that are part of this application.

[0028] In some examples, the sequences disclosed and/or claimed herein may be less than 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the actual sequences in any of the SEQ ID NOs that are part of this application.

[0029] In some examples, the sequences disclosed herein may be or may be 60, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical or at least 60, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical, and less than 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical, as appropriate, to the actual sequences in any of the SEQ ID NOs that are part of this application.

[0030] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman and Wunsch, Mol. Biol. 48:443, 1970, by the search for similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th ed, Cold Spring Harbor, N.Y., 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley and Sons, New York, through supplement 104, 2013).

[0031] The term "immunogen" refers to a compound, composition, or substance that is immunogenic as defined herein below.

[0032] The term "immunogenic" refers to the ability of a substance to cause, elicit, stimulate, or induce an immune response against a particular antigen, in a subject, whether in the presence or absence of an adjuvant.

[0033] The term“immunogenicity”refers to the ability of a foreign substance, such as an antigen, to provoke an immune response in the body of a human or other animal, whether in the presence or absence of an adjuvant.

[0034] The term "immune response" refers to any detectable response of a cell or cells of the immune system of a host mammal to a stimulus (such as an immunogen), including, but not limited to, innate immune responses (e.g., activation of Toll receptor signaling cascade), cell-mediated immune responses (e.g., responses mediated by T cells, such as antigen- specific T cells, and non-specific cells of the immune system), and humoral immune responses (e.g., responses mediated by B cells, such as generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids). Examples of immune responses include an alteration (e.g., increase) in Toll-like receptor activation, lymphokine (e.g., cytokine (e.g., Thl, Th2 or Thl7 type cytokines) or chemokine) expression or secretion, macrophage activation, dendritic cell activation, T cell (e.g., CD4+ or CD8+ T cell) activation, NK cell activation, B cell activation (e.g., antibody generation and/or secretion), binding of an immunogen (e.g., antigen (e.g., immunogenic polypeptide)) to an MHC molecule, induction of a cytotoxic T lymphocyte ("CTL") response, induction of a B cell response (e.g., antibody production), and, expansion (e.g., growth of a population of cells) of cells of the immune system (e.g., T cells and B cells), and increased processing and presentation of antigen by antigen presenting cells. The term "immune response" also encompasses any detectable response to a particular substance (such as an antigen or immunogen) by one or more components of the immune system of a vertebrate animal in vitro.

[0035] The term“immunogenic composition" refers to a composition comprising an immunogen. [0036] The term“immunogenicity enhancement” refers to a significant increase in the level of immunogenic response resulting in the increased production of antibody molecules specific to the antigen

[0037] The term“ionic polypeptide” refers to polypeptide that bears overall anionic (negative) or cationic (positive) charge

[0038] The term“linker peptide” refers to small peptides that connect protein and polypeptide subunits, and also provide many other functions, such as maintaining cooperative inter-domain interactions or preserving biological activity. Peptide linkers in multi-domain proteins are helpful for the rational design of recombinant fusion proteins. Similar to recombinant fusion proteins, several naturally-occurring multi- domain proteins are composed of two or more functional domains joined by linker peptides.

[0039] The term Multiple Antigen Protein Displayed Adjuvant (MAPD Adjuvant) is defined as a Chimeric Fusion Protein (CFP) that can bind to an adjuvant at multiple binding locations preferably via the adjuvant binding peptide region of the CFP and displaying the antigens away from the adjuvant The primary goal of the MAPD Adjuvant is to minimize the direct interactions between the critical immunogenic epitopes of the antigen and the adjuvant. The adjuvant binding peptides are covalently linked to the antigen with or without a linker peptide between the antigen and the adjuvant binding peptide.

[0040] The term Multifunctional Chimeric Fusion Protein (MCFP) is defined as a Chimeric Fusion Protein (CFP) that is recombinantly produced minimally comprising (i) an antigen and (ii) dual function peptide. The purpose of the dual function is to act as (i) a purification aid for the CFP as well as (ii) possessing the capability to bind to an adjuvant, preferably via non-covalent interactions.

[0041] The term "native" or "wild-type" protein, sequence, or polypeptide refers to a naturally existing protein, sequence, or polypeptide that has not been artificially modified by selective mutations.

[0042] The term "pharmaceutically acceptable carriers" refers to a material or composition which, when combined with an active ingredient, is compatible with the active ingredient and does not cause toxic or otherwise unwanted reactions when administered to a subject, particularly a mammal. Examples of pharmaceutically acceptable carriers include solvents, surfactants, suspending agents, buffering agents, lubricating agents, emulsifiers, absorbants, dispersion media, coatings, and stabilizers.

[0043] The term“polyhistidine tags” refers to an amino acid motif in proteins that typically consists of at least six histidine (His) residues, often at the N- or C-terminus of the protein.

[0044] The term "soluble protein" refers to a protein capable of dissolving in aqueous liquid and remaining dissolved. The solubility of a protein may change depending on the concentration of the protein in the water-based liquid, the buffering condition of the liquid, the concentration of other solutes in the liquid, for example salt and protein concentrations, and the temperature of the liquid.

[0045] The term“specific binding” refers to the defined interactions between two different molecules or fragments of molecules such as an interaction between an antigen and an antigen presenting cell or an antibody molecule to form an immune complex. An immune complex, typically, acts as a unitary object, effectively an antigen of its own with a specific epitope. Any interactions that interfere with the formation of an immune complex are considered non-specific if the complexes do not result in the formation of unitary objects.

[0046] The term“stability enhancement” refers to an increase in shelf-life of a molecule due to the decreased chances for disassociation or degradation of a molecule

[0047] The term "subject" refers to either a human or a non-human mammal. The term "mammal" refers to any animal species of the Mammalia class. Examples of mammals include: humans; non-human primates such as monkeys; laboratory animals such as rats, mice, guinea pigs; domestic animals such as cats, dogs, rabbits, cattle, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, elephants, and the like.

[0048] The term "vaccine" refers to a pharmaceutical composition comprising an immunogen that is capable of eliciting a prophylactic or therapeutic immune response in a subject. Typically, a vaccine elicits an antigen- specific immune response to an antigen of a pathogen, for example a viral pathogen. Vaccine Systems

[0049] Even though pathogenic microorganisms contain a number of antigenic components such as proteins, glycoproteins, capsular polysaccharides,

lipopolysaccharides, etc., only a selected few of these will induce the appropriate immune response. Therefore, the selection of the immunogen, multi-faceted design criteria, stability and viable manufacturing process criteria along with the delivery strategy in the form of an optimal vaccine construct are essential considerations for the development of an effective vaccine product [Newman et al., 1995; Prasad, 2018].

[0050] Many of the currently commercially licensed vaccines typically comprise an antigen (immunogen) and an adjuvant. Antigens are usually macromolecules, such as peptides, carbohydrates, proteins etc., having epitopes (specific antigenic sites) that recognize, interact and bind with various components of the immune systems, such as B lymphocytes. Typically, antigens are perceived by immune systems of living organisms as being foreign, toxic or dangerous and produce antibody molecules to combat these “foreign” antigens. Some antigens such as short peptides, small molecule haptens, some proteins and carbohydrates elicit a poor immunogenic response. One approach to address the poor antibody response of these antigens is through the conjugation of the antigens to carrier proteins to elicit a more robust immunogenic response. In this type of immune response, elicited by the antigens conjugated to carrier proteins, antibodies are produced and secreted by the B-lymphocytes in conjunction with the T-helper (Th) cells. Using a synergistic approach, the B and T cells synchronize to induce an antigen specific antibody response that is robust and long-lasting memory.

[0051] Peptide antigens used to generate site-specific antibodies to proteins have been described in the art towards the development of vaccines. The poor immunogenic response of a short peptide could be amplified by Multiple Antigen Peptide (MAP) based systems [Tam et al., 1988; Posnett, et al., 1988; Posnett, et al.,1989] to overcome the need for chemical conjugation to the carrier protein, forming nanoparticles. In this MAP system, multiple copies of antigenic peptides are simultaneously bound to the a- and e- amino groups of a non-immunogenic Lys-based dendritic scaffold.

[0052] The MAP system allows the formation of arrays of a wide range of peptidic nanoparticles in a controlled fashion. The orientation and the subsequent presentation of the immunogen is a critical quality attribute for the generation of functional antibodies specific to the antigen. These multimeric MAPs have been demonstrated to be highly immunogenic, allowing production of polyclonal and monoclonal antibodies. The majority of these antibodies react with the peptide in its monomeric form as well as its multimeric form. The antigenic determinants of the peptide that are typically recognized by these antibodies include continuous type as well as conformational type of

determinants.

[0053] Several Multiple antigen-presenting peptide vaccine systems have been developed and described in the art [Fujita et al., 2011; Moyer et al., 2016]. These include: (1) the addition of functional components, e.g., T-cell epitopes, cell-penetrating peptides, and lipophilic moieties; and (2) synthetic approaches using size-defined nanomaterials, e.g., self-assembling peptides, non-peptidic dendrimers, and gold nanoparticles, as antigen-displaying platforms; (3) chemical conjugation of adjuvant compounds to protein antigens [Wille-Reece et al., 2005]; (4) the incorporation of antigen or adjuvant into particulate vehicles by conjugation to the surface of nanoparticles [de Titta A, et al., 2013]; (5) the incorporation of antigen or adjuvant into particulate vehicles by the entrapment within lipid vesicles or capsules [Moon et al., 2011]; (6) the encapsulation within polymer particles [Ilyinskii, et al., 2014]; and (7) Virus Like Particles (VLPs) [Cimica et al., 2016; Donaldson et al., 2018]. Liposomes and poly(lactide-co-glycolide) (PLGA) have been described in the art as vaccine vehicles [Silva et al., 2016].

[0054] The MAP vaccines, described in the art, can carry several copies of peptide antigens on a carrier or nanoparticle and can elicit higher antibody titers than single peptide monomers and carrier protein-peptide conjugates. However, the main limitation in the MAP systems is the need for additional components, such as an adjuvant, were required in many cases to elicit robust immunogenicity. Therefore, subsequent research efforts have been directed towards improvement of these MAP vaccines by the incorporation of multiple functions into a single vaccine product using helper T-cell epitopes, immune- stimulant lipid moieties, or cell-penetrating peptides, etc.

[0055] Another approach to boost the immunogenicity of various protein antigens is through the use of adjuvants such as aluminum- and calcium-based compounds, saponin- based compounds such as QS-21, squalene-based compounds such as MF-59,

Monophosphoryl Lipid A, etc. [Reed at al., 2013]. The current“antigen plus adjuvant” vaccine products are comprised of two key components“antigen” (drug substance) and “adjuvant” followed by a formulation process step which may contain additional inert ingredients such as“stabilizers” and“excipients” to form the drug product. In this context, it is important that the biodistribution and pharmacokinetics of the vaccine are optimized and controlled for robust immunogenicity (most potent and specific immune response towards the antigen in question), stability and safety.

[0056] There are several outstanding challenges associated with the production of optimal vaccine constructs against infectious disease and tumors [Moyer et al, 2016]. For example, several soluble protein vaccines containing mono-antigens often fail to generate a robust immunogenic response resulting in suboptimal efficacy. In the case of prophylactic vaccines, one of the key requirements to elicit a robust immunogenic response that results in an optimal efficacy is the production of sufficient number of long- lasting antibody -producing cells and memory T and B cell populations. For

immunotherapeutic vaccines against cancer strong CD8+T cell responses are required.

[0057] Antigen architecture in a vaccine construct, which defines epitope density features such as spacing, density and the rigidity/flexibility may significantly influence B cell responses, based on data from animal studies. There are several methods described in the art that demonstrate high density protein antigens, increased valency through multimerization or conjugation to polymers/carrier proteins, or multiple antigen display from nanoparticles result in the increase of B cell triggering, antigen internalization and presentation to T-helper cells in animal models. It has been described in the art that B cells optimally recognize viruses and bacteria that typically express dense, arrayed repetitive copies of proteins at their surfaces.

[0058] It has been described in that art that peptides with specific amino acid sequences, having certain helical or b-hairpin/sheet secondary structures, can assemble themselves to form nanoparticles via non-covalent interactions such as van der Waals bonds, electrostatic interactions, hydrogen bonds or stacking interactions, etc. These nanomaterials have found applications in several fields such as tissue engineering, drug delivery, vaccine development, etc. [Hartgerink et ah, 1996; Rajagopal et ah, 2004].

[0059] It is well known in the art that assemblies of polypeptides that present antigens with optimal density in defined orientations can potentially mimic the repetitiveness, geometry, size, and shape of the natural host-pathogen surface interactions [Lopez-Sagaseta et al., 2016]. Such nanoparticles offer a combined strength of multiple antigen binding sites (avidity) to provide enhanced stability and robust immunogenicity. The self-assembling properties could be leveraged for the display of various immunogens in order to mimic the repetitive display architecture of a natural microbe, e.g. a virus capsid.

[0060] Several exogenous multimerization domains that promote formation of stable multimers of soluble proteins are known in the art. Examples of such multimerization domains that can be linked to an immunogen provided by the present disclosure include: (1) the GCN4 leucine zipper [Harbury et al. 1993]; (2) the trimerization motif from the lung surfactant protein [Hoppe et al. 1994]; (3) collagen [McAlinden et al. 2003] and (4) the phage T4 fibritin foldon [Miroshnikov et al. 1998].

[0061] It has been described in the art that coiled-coil sequences in proteins which consist of heptad repeats containing two characteristic hydrophobic positions have been exploited for self-assembly for the production of nanoparticles [Miroshnikov et al., 1998; Todd et al., 2002; McAlinden et al.,2003]. The role of these buried hydrophobic residues in determining the structures of coiled coils was investigated by studying mutants of the GCN4 leucine zipper [Harbury, 1993]. Short stretches of amino acids can form structural motifs responsible for the tight parallel association and trimerization of the three identical polypeptide chains of lung surfactant protein D, which contains both collagen regions and C-type lectin domains [Hoppe 1994].

[0062] It has been described in the art that several Self-assembling Peptidic

Nanoparticles (SANPs) composed of a pentameric coiled-coil oligomerization domain derived from cartilage protein [Jung et al., 2009] and a trimeric coiled-coil

oligomerization domain [Rudra et al., 2010] have been exploited to produce as multiple antigen-display platforms [Burkhard et al., 2001; Pimantel et al., 2009; Kaba et al., 2009]. The above described nanoparticles play a wide variety of physiological roles and this could be exploited more widely in the arena of vaccine design and development. It is also well known in the art that in silico molecular modeling approaches could be used to design such self-assembling nanoparticles for vaccine design [Lapelosa et al., 2009].

[0063] It is known in the art that the crystal structure of peptide epitopes in complex with the corresponding broadly neutralizing human monoclonal antibody have the potential to provide targets for structure-based vaccine design strategies aimed at identifying optimal antigenic activity [McLellan et al., 2013].

[0064] A number of methods are known in the art that describe the utility of natural or engineered protein nanoparticle scaffolds to add heterologous epitopes or antigens onto the‘nanoparticle, to produce‘chimeric’ nanoparticle antigens. These chimeric nanoparticles can be generated by self-assembly, or by covalent chemical conjugation of an antigen/immunogen to a nanoparticle.

[0065] Examples of chimeric nanoparticles include virus-like particles (VLPs) composed of single or multiple viral antigens, in some instances anchored in a lipid bilayer. A number of methods have been described in the art that involve usage of proteins from various microorganisms as templates for the production of such

nanoparticles and for the presentation of immunogenic epitopes. Phage particles have been described in the art as an attractive antigen delivery system to design new vaccines [Prisco et ah, 2012]. For example, filamentous phage fd has been identified as an antigen delivery platform for peptide vaccines for immunotherapeutic targets. Peptides displayed on the surface of filamentous bacteriophage fd were shown to induce humoral as well as cell-mediated immune responses. The immune response induced by phage-displayed peptides can be enhanced by targeting phage particles to the antigen presenting cells, utilizing a single-chain antibody fragment that binds a dendritic cell receptor. Other examples include the protein pill of the filamentous phage fl, the Ty component from Saccharomyces cerevisiae, antigens of the hepatitis B vims, surface or coat proteins of human parvovirus B 19, Sindbis vims, and papillomavims.

[0066] Examples of the vaccines based on the use of self-assembled VLPs that exploit the design principles described above include the licensed human papilloma vims vaccines, hepatitis B vaccine, etc. A malaria vaccine candidate RTS,S was developed using the above design principles. This vaccine has been recommended for licensure by EMEA, after undergoing large scale phase 3 evaluation [Mahmoudi et ah, 2017] and it was introduced in Ghana in April 2019. This vaccine is based on the hepatitis B surface antigen VLP platform, which includes the carboxy terminus (amino acids 207-395) of the Plasmodium falciparum circumsporozoite (CS) antigen along with the GSL AS01 adjuvant, a mixture of liposomes, MPL and QS-21. The vaccine formulation with the mixture of adjuvants was demonstrated to induce humoral and cellular immune responses to the antigen.

[0067] A number of new vaccine design approaches have been described in the art, aided by the identification of human monoclonal antibodies with high specificity for critical immunogenic epitopes [Melero et ah, 2016]. In conjunction with the capability of modem molecular modeling tools to obtain atomic-level structural information for protein antigens and precision engineering to produce self-assembling nanoparticles, clinical proof-of-concept for structure-based vaccine design may first be achieved for respiratory syncytial vims (RSV). These strategies address the longstanding challenges in the vaccine development with respect to the conformation-dependent access to neutralization- sensitive epitopes of protein immunogens which could determine the capacity to induce potent neutralizing activity [Graham et al., 2019]. Success with RSV has motivated structure-based vaccine design and stabilization of other protein antigens for use as immunogens.

[0068] Helicobacter pylori ferritin has been employed to develop a vaccine candidate which elicited broadly neutralizing H1N1 antibodies, in animal studies

[Kanekiyo et al. 2013]. Ferritin is a natural protein that can be found in cells from all living species. Ferritin is useful as a vaccine platform since it provides particles that can display multiple antigens on its surface, mimicking the natural organization.

Hemagglutinin (HA) of influenza virus was inserted at the interface of adjacent subunits to generate eight trimeric viral spikes on the surface of ferritin nanoparticle via self- assembly [Darricarrere et al., 2018]. A candidate vaccine using this influenza-ferritin self- assembled nanoparticle vaccine entered Phase 1 clinical trials in 2019 [NIH News, 2019]. A prototype universal influenza vaccine which displays part of HA (stem region only) on the surface of a nanoparticle made of nonhuman ferritin was developed by NIH scientists. This H1N1 candidate vaccine protected animals from infection of H5N1 a different influenza subtype, indicating that the antibodies induced by the vaccine can protect against other influenza subtypes within“group 1,” which includes both HI and H5.

[0069] The strategies discussed above, however, address only the key features required for the‘optimal drug substance’ and not necessarily an‘optimal drug product’. However, it is important to note that for many vaccine candidates the role of adjuvants provides an additional layer of assurance for the development of an optimal vaccine construct towards the generation of a durable and robust immunogenic response.

[0070] The design features of the optimal vaccine construct, therefore, should incorporate the particle shape to mimic the microbial structure such as the multiple antigen display architecture on the particle surface and repetition pattern (antigen density /copy number). In addition, delivery of the antigen and adjuvant as well as the stability are important, as part of a comprehensive vaccine design and development strategy [Moyer, 2016; Prasad, 2018].

[0071] The formulation process, currently used for the manufacture of various vaccines to produce the final Drug Product (DP) with various adjuvants such as aluminum phosphate, Aluminum Hydroxide, Calcium Phosphate etc., is typically not a fully controlled process step. These adjuvants have been used for several decades to enhance the immune response to vaccines. The control of the formulation process parameters is vital for manufacturing consistency since they have a direct impact on the physical, chemical, and biological properties of these adjuvants [HogenEsch 2018]. The optimization of the final construct of the vaccine could ultimately determine vaccine performance. The non-specific interactions between adjuvants and antigens, therefore, may have a direct impact on the potency of vaccines.

[0072] Another approach to boost the immunogenicity of various protein antigens is through the use of mucoadhesive adjuvants such as the derivatives of polyglutamic acids (PGA), chitosan, etc. Several PGA derivatives have been described in the art as potential mucoadhesive adjuvants. For example, a PGA-based complex has been described as an efficient mucosal adjuvant system for an influenza vaccine based on the recombinant fusion protein sM2HA2, which contains the consensus matrix protein 2 (sM2) and the stalk domain of HA (HA2) [Noh et al., 2019] . The g-PGA synthesized naturally by microbial species (e.g. Bacillus subtilis and Bacillus licheniformis) is a highly anionic polymer that is used in a variety of applications (e.g., food products, cosmetics and medicines) and has been shown to have excellent biocompatibility and noncytotoxicity [Buescher et al., 2007, Kim et al., 2007]. It can act as a mucoadhesive delivery vehicle for recombinant protein antigens and also provide an easy and robust strategy for the incorporation of hydrophobic immuno stimulatory compounds such monophosphoryl lipid A (MPL) dervatives, QS21 and intracellular stimulator of interferon genes (STING) agonist adjuvants.

[0073] Vaccine products encounter various types of interfacial stress during development, manufacturing, and clinical administration [Li et al., 2019]. Protein antigens come in contact with various surfaces during various steps of formulation, with adjuvants, excipients and stabilizers. These additional interfaces can negatively impact the final vaccine drug product quality attributes. During the various processing steps of the vaccine drug substance including final formulation, additional chances for the formation of undesirable modifications and side products could arise. These undesirable modifications include formation of visible particles, subvisible particles, or soluble aggregates and/or changes in target protein concentration due to the potential adsorption of the molecule to various interfaces. Protein aggregation at interfaces is often

accompanied by changes in conformation, in response to interfacial stresses such as hydrophobicity, charge, and mechanical stress. Formation of aggregates due to these non specific interactions may bury critical immunogenic epitopes from optimal interactions leading to suboptimal immunogenicity. Therefore, it is important to minimize

opportunities for aggregation throughout the product design and development cycle and to develop appropriate mitigation strategies.

[0074] The current classical formulation process, widely used in the manufacture of licensed vaccines as well as most clinical candidates, typically involves mixing of various vaccine antigens (drug substances) with a given adjuvant such as an aluminum salt, using a few control parameters such as excipients, salts, pH, adjuvant concentration, etc., to produce the final drug product (Figure 1). However, the control of the formulation process using a few selected process parameters results only in the partial control of the formulation process. The partial control of formulation process parameters, without addressing the key aspect of the optimal presentation of the critical immunogenic epitopes, may result in the random burial of the critical immunogenic epitopes, in the final formulated drug product, preventing their ability to interact with the antigen presenting cells (APCs).

[0075] It is, therefore, important that this key aspect of optimal antigen presentation in the final formulated drug product is addressed in an adequate manner to elicit a robust immunogenic response. In addition to the optimal presentation of the critical immunogenic epitopes, it is also important to control the antigen number (antigen density) in a consistent manner. The formulation processing, using adjuvants, without proper optimization due to incomplete understanding of the orientation and presentation critical immunogenic epitopes may also result in the choice of an inappropriate adjuvant. In addition to the presentation, adsorption of the antigen results in the conversion of the soluble antigens to particulate form, which enhances uptake through phagocytosis by dendritic cells. The proper control of the process of adsorption during the formulation step also results in the control of key attributes of the particulates, such as molecular size. The process of adsorption to the adjuvant also results in the retention of the antigen at the injection site, allowing time for recruitment of APCs through release of cytokines and the induction of a local inflammatory reaction. The proper control of the process of adsorption during the formulation step, therefore, results also in the control of key attributes that define the retention of the antigen at the injection site.

[0076] An optimal vaccine formulation, therefore, requires that three key criteria are met in order to elicit a robust immunogenic response resulting in efficacy (i)

minimization of non-specific interactions between the antigen and the adjuvant; (ii) transport of vaccines to lymphoid tissues, (iii) trigger antigen induced signals to immune cells, and (iv) control of the kinetics of antigen presentation to immune cells. A well- designed vaccine strategy for the delivery of the antigen and the adjuvant in a concerted manner to the lymphoid cells helps to address the four key criteria outlined above. These design features and the formulation strategy ultimately are the drivers that help shape the elicitation of an optimal and robust immunogenic response as well as the stability of the vaccine construct.

[0077] An optimal vaccine formulation may further contain additional

“pharmaceutically acceptable carriers" in combination with an immunogen, is compatible with the immunogens and does not cause toxic or otherwise unwanted reactions when administered to a subject, particularly a mammal. Examples of pharmaceutically acceptable carriers include solvents, surfactants, suspending agents, buffering agents, lubricating agents, emulsifiers, absorbants, dispersion media, coatings, and stabilizers.

[0078] In light of the deficiencies associated with the current‘partially controlled’ formulation process employed for many licensed vaccines and many clinical candidates, there is a critical need for process control by directing the critical immunogenic epitopes for optimal presentation of the antigen in the drug product.

[0079] It is the object of the current invention to design and develop vaccine constructs for optimal presentation of the antigen in the drug product with better process control.

Multiple Antigen Protein Displayed Adjuvant Systems (MAPD Adjuvant)

[0080] In contrast to the classical formulation approaches described above, it is the objective of the invention to design novel‘Multiple Antigen Protein Displayed

Adjuvants’ [MAPD Adjuvant]. These adjuvanted nanoparticles are designed and produced in a manner, that present multiple copies of protein antigens and their critical immunogenic epitopes in arrays displayed on the adjuvants. It is an additional objective of the invention to design adjuvanted nanoparticles which provide the following vaccine design advantages (a) optimal immunogen density due to multiple binding sites (avidity); (b) optimal display (orientation) of critical immunogenic epitopes; (c) reduce non-specific interactions between the critical immunogenic epitopes and the adjuvant; (d) enhanced stability in order to provide improved antigen shelf-life (storage) and robust

immunogenicity .

[0081] The ultimate objective of the MAPD Adjuvants is the proper orientation and presentation of immunogens having high density (high copy number of antigens) to support multiple binding events to occur simultaneously between the nanoparticle and the host cell B Cell Receptors (BCRs). These multiple binding events, generated by the adjuvanted nanoparticles, eventually provide stronger antigen- antibody interactions compared to the low affinity interactions provided by the monovalent binding generated by single soluble protein immunogens.

[0082] It is the object of the current invention to incorporate the above described design features and the three key criteria to produce MAPD Adjuvant for immunogenicity enhancement as well as enhanced stability. Specifically, the current invention is directed towards incorporating these design features to produce adjuvanted nanoparticles. It is the object of this invention to produce nanoparticles that incorporate the particle shape to mimic the microbial structure such as the multiple antigen display architecture on the particle surface and repetition pattern (antigen density /copy number). In addition, these adjuvanted nanoparticles systems incorporate the design features for the delivery of the antigen and adjuvant as well as the stability, as part of a comprehensive vaccine design and development strategy. The following examples describe the method by which these MAPD Adjuvant could be produced for immunogenicity and stability enhancement.

[0083] The key ingredient, of the current invention, the MAPD Adjuvant system is first recombinantly expressed involving Multifunctional Chimeric Fusion Protein (MCFP) which in turn is composed of (i) protein/glycoprotein antigen; (ii) linker peptide, optionally fused to heterologous T-cell epitope; and (iii) Dual Function Peptide (DFP), which can act as a purification aid as well as being capable of binding to the adjuvant via non-covalent interactions.

[0084] There are four main types of noncovalent bonds in biological systems:

hydrogen bonds, ionic bonds, van der Waals interactions, and hydrophobic bonds. The bond energies for these interactions range from about 1 to 5 kcal/mol.

[0085] The current invention provides the design principles involved in the production of the MAPD Adjuvant system (Figure 2). In the first step, MCFP is produced by recombinant expression. MCFP (the drug substance) is composed of

protein/glycoprotein antigen; linker peptides, optionally fused to a heterologous T-cell epitope; and Dual Function Peptide (DFP) which can act as a purification aid as well as being capable of binding to the adjuvant via non-covalent interactions. In the second formulation step, the MCFP is mixed with an adjuvant to produce the MAPD Adjuvant (Drug Product).

[0086] The current invention also provides the design principles involved in the production of the MAPD Adjuvant system, by an alternate process. Figure 3 illustrates the key components and steps involved in the production of the MAPD Adjuvant system. In the first step the MCFP is produced by recombinant expression. MCFP drug substance is composed of (B) protein/glycoprotein antigen; (C) linker peptides, optionally fused to (D) heterologous T-cell epitope; and (E) Dual Function Peptide (DFP) which can act as a purification aid as well as capable of binding to the adjuvant via non-covalent

interactions. In the second formulation step, the MCFP is mixed with an adjuvant- 1 (G), precoated earlier, separately, with (F) polypeptide adjuvant-2, to produce the

MAPD Adjuvant (Drug Product). For example, this alternate strategy, of using two adjuvants, is particularly useful for the DFP having polypeptide tags such as a

polyhistidine (His6-Hisl0) tag which can bind to negatively charged PGA.

[0087] Anionic polypeptides, such as PGA could be used as pre-coating adjuvant-2, layered on positively charged adjuvant- 1 such as aluminum and calcium salts

(phosphates), which also can bind involving non-covalent ionic interactions.

[0088] The present invention is directed to the methods for design of Multiple Antigen Protein Displayed Adjuvant Systems [MAPD Adjuvant] for immunogenicity and stability enhancement, applicable for the development of optimal nanoparticle vaccine constructs.

[0089] Furthermore, the invention also relates to such multiple antigen protein displayed adjuvanted immunogenic products and immunogenic compositions containing such immunogenic products and immunogenic compositions containing such

immunogenic systems and immunogenic products made by such methods. The invention provides Multiple Antigen Protein Displayed Adjuvant Systems (MAPD Adjuvant) to be used as efficient vaccines comprising‘functionalized protein nanoparticles containing both an antigen and an adjuvant’, and a method of vaccinating humans or non-human animals using such functionalized nanoparticles containing adjuvants. The invention also provides processes for making adjuvanted protein nanoparticles and functionalized adjuvanted protein nanoparticles.

[0090] The present invention combines and integrates the above described key features into a single process that can enhance the immunogenic response, namely the use of the adjuvant in two steps (Figure 2).

[0091] In one embodiment, the present invention is directed to the methods for design of novel Multiple Antigen Protein Displayed Adjuvant Systems [MAPD Adjuvant] for immunogenicity and stability enhancement, applicable for the development of optimal nanoparticle vaccine constructs. In other embodiment, these novel MAPD Adjuvant systems are constructed more specifically using two inventive steps comprising: (i) the recombinant production of MCFP vaccine antigens comprising DFPs, acting as purification aids as well having the capability to bind with adjuvants; and (ii)

multimerization of the vaccine antigens to form adjuvanted nanoparticles, via binding of the DFPs with the adjuvant involving non-covalent interactions. These two directed steps help minimize non-specific interactions between the critical immunogenic epitopes of the vaccine antigen and the adjuvant in order to optimize the chances for antigen- antibody interactions for immunogenicity and/or stability enhancement.

[0092] In another embodiment, the invention provides MAPD Adjuvant to be used as efficient vaccines comprising‘ functionalized protein nanoparticles containing both an antigen and an adjuvant’, and a method of vaccinating humans or non-human animals using such functionalized protein antigen nanoparticles containing adjuvants. The invention also provides processes for making protein nanoparticles and functionalized protein nanoparticles.

[0093] As outlined in Figures 2 and 3, the present invention is specifically directed to address the key challenge of the proper orientation and the preservation, protection and presentation of the critical immunogenic epitopes in a stepwise manner.

[0094] In another embodiment, the invention also relates to such multiple antigen protein displayed adjuvanted immunogenic products and immunogenic compositions containing such immunogenic products and immunogenic compositions containing such immunogenic systems and immunogenic products made by such methods.

[0095] The current invention exemplifies the design and development of the MCFP based on a vaccine protein antigen, clumping factor A (ClfA) derived from the bacterial pathogen, Staphylococcus aureus to produce the MAPD Adjuvant nanoparticle.

[0096] Infections caused by S. aureus pose a significant medical challenge worldwide, with disease manifestations ranging from mild skin infections to

overwhelming sepsis and death. The fibrinogen (Fg)-binding microbial surface component clumping factor A (ClfA) is an important virulence factor of S. aureus. ClfA promotes the attachment of the bacteria to a number of biomaterials promoting them to colonize and form thick biofilms. ClfA protein antigen has been included as a component in a number of multivalent S. aureus vaccines clinical trials, due to its protective role against infection [Frenck et al., 2017]. The N-terminal A region of ClfA is composed of three subdomains: Nl, N2, and N3. The C-terminus of the g-chain of Fg docks in a ligand-binding trench located between subdomains N2 and N3. The mechanism involves conformational changes of the adhesin that result in a greatly stabilized adhesin-ligand complex. ClfA functions as a force- sensitive molecular switch that regulates the strength of adhesion of S. aureus to protein-conditioned biomaterials, thus emphasizing the role that physical forces, such as shear rate of adhesion, could play in activating the function of bacterial adhesins [Herman-Bausiera, 2017].

[0097] Due to the important role played by the conformation of the ClfA protein in the adhesion properties, it is important to ensure that all the epitopes that play a critical role in vaccine function need to be shielded away from any non-specific interactions with the adjuvant during the formulation step. The MAPAdjuvant nanoparticles can play a crucial role towards realizing this‘controlled formulation approach’ to minimize the interactions between the critical epitopes of protein antigens, such as ClfA, and the adjuvant.

[0098] In certain embodiments the MAPD Adjuvant nanoparticle vaccine

compositions hereof comprise aluminum as an adjuvant, e.g., in the form of aluminum hydroxide, aluminum phosphate, aluminum potassium phosphate, calcium phosphate or combinations thereof, in concentrations of 0.05-5 mg, e.g., from 0.075-1.0 mg, of aluminum content per dose.

[0099] In other aspects, the present disclosure provides a method of eliciting an immune response against the immunogen that is part of the MAPD Adjuvant nanoparticle composition in a subject, such as a human, comprising administering to the subject an effective amount of the immunogen that is part of the MAPD Adjuvant nanoparticle composition; a nucleic acid molecule encoding the immunogen that is part of the

MAPD Adjuvant nanoparticle composition; or a composition comprising the immunogen that is part of the MAPD Adjuvant nanoparticle composition or nucleic acid molecule.

The present disclosure also provides a method of preventing infection in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition, such as a vaccine, comprising a the immunogen that is part of the

MAPD Adjuvant nanoparticle composition, a nucleic acid encoding the immunogen that is part of the MAPD Adjuvant nanoparticle composition. In some particular embodiments, the pharmaceutical composition comprises the immunogen that is part of the

MAPD Adjuvant nanoparticle composition. In some embodiments of the methods provided herein above, the subject is a human. In some particular embodiments, the human is a child, such as an infant. In some other particular embodiments, the human is a woman, particularly a pregnant woman. [00100] The effective amount administered to the subject is an amount that is sufficient to elicit an immune response against an antigen defined by the immunogen that is part of the MAPD Adjuvant nanoparticle composition in the subject.

[00101] In addition to the immunogenic component, the MAPD Adjuvant

nanoparticle vaccine compositions further comprise an immunomodulatory agent, such as an adjuvant. Examples of suitable adjuvants include aluminum salts such as aluminum hydroxide, aluminum phosphate and/or calcium phosphate; derivatives of polyglutamic acid (PGA) CpG-motif containing oligonucleotides, ADP-ribosylating bacterial toxins or mutants thereof, such as E. coli heat labile enterotoxin LT, cholera toxin CT, and the like.

[00102] The term "glycoprotein" refers to a protein that contains oligosaccharide chains (glycans) covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification known as glycosylation. The term "glycosylation site" refers to an amino acid sequence on the surface of a polypeptide, such as a protein, which accommodates the attachment of a glycan. An N-linked glycosylation site is triplet sequence of NX(S/T) in which N is asparagine, X is any residue except proline, and (S/T) is a serine or threonine residue. A glycan is a polysaccharide or oligosaccharide. Glycan may also be used to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan.

[00103] The MCFPs provided by the present disclosure can be prepared by routine methods known in the art, such as by expression in a recombinant host system using a suitable vector. Suitable recombinant host cells include, for example, insect cells, mammalian cells, avian cells, bacteria, and yeast cells. Examples of suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (a clonal isolate derived from the parental Trichoplusia ni BTI-TN-5B 1-4 cell line (Invitrogen)). Examples of suitable mammalian cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 or Expi 293 cells, typically transformed by sheared adenovirus type 5 DNA), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells. Suitable avian cells include, for example, chicken embryonic stem cells (e.g., EBx.RTM. cells), chicken embryonic fibroblasts, chicken embryonic germ cells, quail fibroblasts (e.g. ELL-O), and duck cells. Suitable insect cell expression systems, such as baculovirus-vectored systems, are known to those of skill in the art and described in, e.g., Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovims/insect cell expression systems are commercially available in kit form from Invitrogen, San Diego Calif. Avian cell expression systems are also known to those of skill in the art and described in, e.g., U.S. Pat. Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168; and 6,500,668. Similarly, bacterial and mammalian cell expression systems are also known in the art and described in, e.g., Yeast Genetic Engineering (Barr et ah, eds., 1989) Butterworths, London.

[00104] A number of suitable vectors for expression of recombinant proteins in insect or mammalian cells are well-known and conventional in the art. Suitable vectors can contain a number of components, including, but not limited to one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as a transcriptional control element (e.g., a promoter, an enhancer, a terminator), and/or one or more translation signals; and a signal sequence or leader sequence for targeting to the secretory pathway in a selected host cell (e.g., of mammalian origin or from a heterologous mammalian or non-mammalian species). For example, for expression in insect cells a suitable baculovirus expression vector, such as pFastBac (Invitrogen), is used to produce recombinant baculovirus particles. The baculovirus particles are amplified and used to infect insect cells to express recombinant protein. For expression in mammalian cells, a vector that will drive expression of the construct in the desired mammalian host cell (e.g., Chinese hamster ovary cells) is used.

[00105] The peptide-linked immunogens used in the MAPD Adjuvant nanoparticles can be purified using any suitable methods. For example, methods typically used for protein antigens such as immunoaffinity chromatography are known in the art. Suitable methods for purifying desired peptide-linked protein immunogens include precipitation and various types of chromatography, such as hydrophobic interaction, ion exchange, affinity, chelating and size exclusion are well-known in the art. Suitable purification schemes can be created using two or more of these or other suitable methods. If desired, the peptide-linked protein immunogens can include a "tag" that facilitates purification, such as an epitope tag or a histidine (His) tag. Such tagged polypeptides can conveniently be purified, for example from conditioned media, by chelating chromatography or affinity chromatography .

Examples [00106] The invention is further described by the following illustrative examples.

The examples do not limit the invention in any way. They merely serve to clarify the invention.

Example 1. DNA manipulations and molecular cloning of MCFP comprising ClfA protein antigen (CTRNV6)

[00107] The mature sequence of Staphylococcus aureus strain Newman (SEQ ID NO:l) was selected for cloning and expression. For non-native sequences, codon- optimization was performed (Codon Optimization Tool found at

https://www.idtdna.com/CodonOpt) for expression in Escherichia coli. Gene synthesis was performed by IDT (Coralville, IA). All oligonucleotides were purchased from Sigma-Aldrich (St. Louis, MO), and restriction endonucleases were purchased from New England Biolabs (Ipswich, MA). DNA assembly reactions were performed with

NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs). pET28a

(MilliporeSigma, Burlington, MA) expression vector was digested with /Vco/-HF and BamHI- HF, and overlapping sequences to facilitate insertion of the two synthetic DNA fragments (SEQ ID NO:6 and SEQ ID NO: 7) into the vector were added during DNA synthesis. Additional overlapping sequences were included to facilitate splicing of the two synthetic gene fragments together. Cloning reactions were transformed into E. coli DH5a cells, and selection was performed on LB plates containing 30 pg/mL kanamycin. Plasmid purifications were performed with the Wizard Plus SV Minipreps DNA

Purification System (Promega, Madison, WI). The presence of the intended nucleotide sequence (SEQ ID NO: 4) was confirmed by DNA sequencing (Eurofins, Louisville,

KY).

Example 2. Recombinant MCFP comprising ClfA protein antigen (CTRNV6) expression

[00108] E. coli BL21 (DE3) was used as the expression host. Cells harboring expression plasmids were cultured overnight in LB medium containing 50 pg/mL kanamycin. The following day, cells were inoculated into 2X YT medium to a starting optical density at 600 nm (OD600) between 0.005 and 0.02. The cultures were incubated at 37°C with shaking at 250 rpm. When the cultures reached OD600 values around 0.6 to 0.8, a pre-induction sample (t = 0) was collected and frozen at -20°C, and IPTG was added to the remaining culture at a final concentration of 1 mM. The cultures were incubated for an additional 3 hours at 37 °C with shaking at 250 rpm. Following this incubation, cell samples were harvested (t = 3) and frozen at -20°C.

Example 3. Recombinant MCFP comprising ClfA protein (CTRNV6) purification (SEQ ID NO: 51

[00109] The recombinant MCFP comprising ClfA protein CTRNV6 (SEQ ID NO:

5), was purified by IMAC in batch mode. Frozen cell pellets were resuspended in IX BugBuster Protein Extraction Reagent (MilliporeSigma) containing 100 mM Tris, pH 7.9, 100 mM NaCl, 5 mM imidazole, Benzonase (MilliporeSigma), and cOmplete EDTA-free Protease Inhibitor (Roche, Indianapolis, IN). Cells were lysed at room temperature for 20 min, and insoluble material was removed by centrifugation. The soluble protein fraction was incubated with equilibrated HisPur Ni-NTA Resin (Thermo Fisher Scientific, Pittsburgh, PA), and the resin was washed with buffers containing increasing

concentrations of imidazole. Following the washes, the recombinant ClfA protein was eluted in buffer containing 20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 1 M imidazole.

Example 4. Formulation

[00110] The recombinant MCFP comprising ClfA protein (drug substance) is formulated by mixing with Polyglutamic acid (PGA) adjuvant to form the drug product after adjusting the pH to 5.5 - 7.5.

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Claims

1. A composition, comprising a plurality of components bound to an adjuvant core, the components comprising: a recombinantly expressed Multifunctional Chimeric Fusion Protein (MCFP) and/or glycoprotein vaccine antigen that is part of the MCFP; a linker peptide covalently linked with the MCFP and/or glycoprotein vaccine antigen; a heterologously derived T-cell epitope peptide, optionally, covalently linked to the MCFP/glycoprotein vaccine antigen; and a dual function peptide, that is part of the MCFP, capable of acting as a purification aid as well as binding to the adjuvant and blocking core adjuvant surface space from non-specific binding, thereby controlling the density of the vaccine antigen capable of forming adjuvanted nanoparticles.

2. A Multiple Antigen Protein Displayed Adjuvant (MAPD Adjuvant) system for immunogenicity enhancement and/or stability enhancement, having a recombinantly expressed Multifunctional Chimeric Fusion Protein (MCFP), and comprising a plurality of components that are bound to an adjuvant core; wherein:

(i) a first component of said plurality comprises a vaccine protein/glycoprotein antigen;

(ii) a second component of said plurality comprises a linker peptide covalently linked with the protein/glycoprotein antigen

(iii) a third, optional component, of said plurality comprises a heterologously derived T-cell epitope peptide covalently linked to the protein/glycoprotein antigen; and

(iv) a fourth component of said plurality, that is part of MCFP, is a dual function peptide capable of binding to the adjuvant core and blocking core adjuvant surface space from non-specific binding, thereby controlling the density of the protein/glycoprotein antigen capable of forming adjuvanted nanoparticles.

3. A Multiple Antigen Protein Displayed Adjuvant (MAPD Adjuvant) system for immunogenicity enhancement and/or stability enhancement, having a recombinantly expressed Multifunctional Chimeric Fusion Protein (MCFP), and comprising a plurality of components that are bound to a core adjuvant, wherein:

(i) a first component of said plurality comprises a polypeptide adjuvant coated onto the core adjuvant;

(ii) a second component of said plurality comprises a vaccine protein/glycoprotein antigen;

(iii) a third component of said plurality comprises a linker peptide covalently linked with the protein/glycoprotein antigen;

(iv) a fourth, optional component of said plurality comprises a heterologously derived T-cell epitope peptide covalently linked to the protein/glycoprotein antigen; and

(v) a fifth component of said plurality, that is part of MCFP, comprises a dual function peptide capable of binding to the polypeptide adjuvant and blocking core adjuvant surface space from non-specific binding, thereby controlling the density of the vaccine capable of forming a nanoparticle.

4. The MAPDAdjuvant of one of claims 2 or 3, wherein at least one of the plurality of components is a Multifunctional Chimeric Fusion Protein produced by recombinant protein expression in Escheria coli.

5. The MAPDAdjuvant of any one of claims 2-4, wherein at least one of the plurality of components acts as a purification aid of the recombinantly produced MCFP

6. The MAPDAdjuvant of claim 5, wherein at least one of the plurality of components is bound to the core adjuvant via the formation of adjuvanted nanoparticles.

7. The MAPDAdjuvant of any one of claims 2-6, comprising a recombinantly expressed Clumping factor A (ClfA)(SEQ ID NO: 5) derived from S. aureus.

8. The MAPDAdjuvant of any one of claims 2-7, comprising a recombinantly expressed ClfA (SEQ ID NO: 5) linked to dual function peptides capable of performing protein purification as well as having the affinity to non-covalently bind to adjuvants.

9. The MAPD Adjuvant of any one of claims 2-8, wherein the dual function peptide comprises a polyhistidine tag having 6-10 contiguous histidine residues.

10. The MAPD Adjuvant of any one of claims 2-9, comprising a protein/peptide vaccine antigen.

11. The MAPD Adjuvant of any one of claims 2-10 , comprising a protein vaccine antigen derived from a bacterial pathogen that is SEQ ID NO: 5.

12. The MAPD Adjuvant of any one of claims 2-6, wherein the protein/glycoprotein antigen is derived from a viral pathogen.

13. The MAPD Adjuvant of any one of claims 2-12, comprising heterologously sourced T-cell peptides.

14. The MAPD Adjuvant of claim 6, wherein the adjuvanted nanoparticles stimulate an innate immune response and/or improve stability of the antigen.

15. The MAPDAdjuvant of claim 14, wherein the nanoparticle has a mean diameter between 10 and 100 nm.

16. The MAPDAdjuvant of any one of claims 2-15, comprising a recombinantly expressed MCFP, in a multicomponent vaccine.

17. The MAPDAdjuvant of any one of claims 2-16, comprising a DFP in a recombinantly expressed MCFP, in a multicomponent vaccine.

18. The MAPDAdjuvant of one of claims 7 or 8, comprising a recombinantly expressed Clumping factor A (ClfA) derived from S. aureus in a multicomponent vaccine against S. aureus (SEQ ID No. 5).