WO2023201222A1

WO2023201222A1 - Ferritin nanoparticles and methods of use thereof

Info

Publication number: WO2023201222A1
Application number: PCT/US2023/065620
Authority: WO
Inventors: Matthew J. VUKOVICH; Ivelin Stefanov Georgiev
Original assignee: Vanderbilt University
Priority date: 2022-04-11
Filing date: 2023-04-11
Publication date: 2023-10-19

Abstract

The present disclosure provides novel ferritin nanoparticles and uses thereof for treatment of diseases and infections and production of broadly neutralizing antibodies.

Description

FERRITIN NANOPARTICLES AND METHODS OF USE THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 63/329,751, filed April 11, 2022, the disclosure of which is expressly incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbers AU47768 awarded by National Institutes of Health. The government has certain rights in the invention.

FIELD

The invention is in the fields of ferritin nanoparticles and methods of use. The present disclosure more specifically relates to ferritin nanoparticles comprising multivalent antigens.

BACKGROUND

The diversity of the HIV-1 envelope glycoprotein (Env) between strains makes creation of a protective vaccine especially challenging. Attempts at incorporating Env diversity in vaccination strategies have had limited success. What is needed are compositions for eliciting broadly neutralizing antibody responses.

SUMMARY

Disclosed herein is a nanoparticle comprising at least one light chain of a ferritin protein and/or at least one heavy chain of a ferritin protein, wherein the at least one light chain and the at least one heavy chain each has at least one amino acid mutation to cysteine.

In some embodiments, the at least one amino acid mutation is at an interface where a heavy chain and a light chain interact. In some embodiments, the light chain comprises a A26C mutation relative to SEQ ID NO: 2. In some embodiments, the light chain comprises a A26C mutation relative to SEQ ID NO: 1. In some embodiments, the ferritin protein is an insect protein. In some embodiments, the ferritin protein is encoded by an insect gene (e.g., a Trichoplusia ni ferritin gene). In some embodiments, the ferritin protein is a Trichoplusia ni ferritin protein.

In some embodiments, the light chain and/or the heavy chain is truncated. In some embodiments, the truncated light chain comprises a deletion of amino acid residues 1-28 or 1-24 relative to SEQ ID NO: 2. In some embodiments, the truncated light chain comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the truncated heavy chain comprises a deletion of amino acid residues 1-25 relative to SEQ ID NO: 1. In some embodiments, the truncated heavy chain comprises the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the at least one light and/or the at least one heavy chain is further linked to one or more polypeptides. In some embodiments, the one or more polypeptides are one or more antigens. In some embodiments, the one or more polypeptides are one or more viral antigens (e.g., HIV-1 proteins, influenza proteins, or RSV proteins). In some embodiments, the HIV-1 proteins are HIV-1 Env proteins. In some embodiments, the HIV-1 Env proteins are from one or more subtypes of HIV- 1.

In some aspects, disclosed herein is a vaccine comprising the nanoparticle of any preceding aspect.

In some aspects, disclosed herein is a method for treating or preventing a viral infection in a subject, comprising administrating to the subject a therapeutically effective amount of the vaccine of any preceding aspect. In some embodiments, the viral infection is an HIV-1 infection, RSV infection, or influenza infection.

In some aspects, disclosed herein is a method for producing broadly neutralizing antibodies, comprising administering to a subject an effective amount of the nanoparticle of any one of any preceding aspect; obtaining a biological sample from the subject; and isolating the broadly neutralizing antibodies from the biological sample.

In some embodiments, the broadly neutralizing antibodies are specific for two or more subtypes of a virus. In some embodiments, the broadly neutralizing antibodies are specific for two or more subtypes of HIV- 1. BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

FIG. 1 shows the output strains from a strain selection algorithm used to identify HIV-1 Env strains that can provide broad protection.

FIG. 2 shows the structure of insect ferritin nanoparticles.

FIG. 3 shows Stabilizing Insect Ferritin (Stifer) white lines are disulfide bonds. Stifer mutant introduces extra disulfide bonds (black lines) between monomers.

FIG. 4 shows the result of SDS-PAGE. Wild type is referred as WT. Stifer mutant is referred to as (LC26 HC49) in this gel. Gel shows evidence of an extra disulfide bond introduced by mutations. Non-Reduced conditions show higher molecular weight for the mutant compared to WT. Reducing conditions show all monomers run at the same molecular weights. Extra disulfide bond is introduced between monomers that did not have a disulfide bond in the WT.

FIG. 5 shows negative stain electron microscopy of insect ferritin nanoparticles that contain the Stifer mutation set. This data confirmed that the stiffer mutations do not disrupt the spherical structure of the nanoparticles.

FIG. 6 shows evidence of nanoparticle formation using negative stain EM with the Stifer mutant.

DETAILED DESCRIPTION

Disclosed herein are polypeptides, nanoparticle compositions, and vaccines and uses thereof for producing immune responses and treating diseases.

Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed.

The following definitions are provided for the full understanding of terms used in this specification.

Terminology

The single letter designation for amino acids is used predominately herein. As is well known by one of skill in the art, such single letter designations are as follows: A is alanine; C is cysteine; D is aspartic acid; E is glutamic acid; F is phenylalanine; G is glycine; H is histidine; I is isoleucine; K is lysine; L is leucine; M is methionine; N is asparagine; P is proline; Q is glutamine; R is arginine; S is serine; T is threonine; V is valine; W is tryptophan; and Y is tyrosine.

As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to a “polypeptide” means one or more polypeptides.

The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, or ±1% from the measurable value.

“Administration” to a subject includes any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, for example, any ocular route. In some embodiments, administration is carried out by intraocular route. Administration includes self-administration and the administration by another. The phrases "concurrent administration", "administration in combination", "simultaneous administration" or "administered simultaneously" as used herein, means that the compounds are administered at the same point in time or immediately following one another.

As used herein, the terms “antigen” or “immunogen” are used interchangeably to refer to a substance, typically a protein, a nucleic acid, a polysaccharide, a toxin, or a lipid, which is capable of inducing an immune response in a subject. The term also refers to proteins that are immunologically active in the sense that once administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector that encodes the protein) is able to evoke an immune response of the humoral and/or cellular type directed against that protein. The term “biocompatible" generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject.

The term “biological sample” as used herein means a sample of biological tissue or fluid. Such samples include, but are not limited to, tissue isolated from animals. Biological samples can also include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histologic purposes, blood, plasma, serum, sputum, stool, tears, mucus, hair, and skin. Biological samples also include explants and primary and/or transformed cell cultures derived from patient tissues. A biological sample can be provided by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods as disclosed herein in vivo. Archival tissues, such as those having treatment or outcome history can also be used.

“Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc. In some aspects, the composition disclosed herein comprises the ferritin polypeptides disclosed herein.

By the term “effective amount” of a therapeutic agent is meant a nontoxic but sufficient amount of a beneficial agent to provide the desired effect. The amount of beneficial agent that is “effective” will vary from subject to subject, depending on the age and general condition of the subject, the particular beneficial agent or agents, and the like. Thus, it is not always possible to specify an exact “effective amount.” However, an appropriate “effective” amount in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of a beneficial can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts.

The term “isolating” as used herein refers to isolation from a biological sample, i.e., blood, plasma, tissues, exosomes, or cells. As used herein the term “isolated,” when used in the context of, e.g., an antibody, refers to an antibody of interest that is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, and even at least 99% free from other compounds, materials, matter, mass and/or substances with which the cell is associated with prior to purification.

As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

The term "nucleic acid" as used herein means a polymer composed of nucleotides, e.g. deoxyribonucleotides (DNA) or ribonucleotides (RNA). The terms "ribonucleic acid" and "RNA" as used herein mean a polymer composed of ribonucleotides. The terms "deoxyribonucleic acid" and "DNA" as used herein mean a polymer composed of deoxyribonucleotides.

The term "polypeptide" refers to a compound made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds.

The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.

As used herein, "operatively linked" can indicate that the regulatory sequences useful for expression of the coding sequences of a nucleic acid are placed in the nucleic acid molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and/or transcription control elements (e.g., promoters, enhancers, and termination elements), and/or selectable markers in an expression vector. The term "operatively linked" can also refer to the arrangement of polypeptide segments within a single polypeptide chain, where the individual polypeptide segments can be, without limitation, a protein, fragments thereof, linking peptides, and/or signal peptides. The term operatively linked can refer to direct fusion of different individual polypeptides within the single polypeptides or fragments thereof where there are no intervening amino acids between the different segments as well as when the individual polypeptides are connected to one another via one or more intervening amino acids.

The “fragments,” whether attached to other sequences or not, can include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified peptide or protein. These modifications can provide for some additional property, such as to remove or add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the fragment possesses a bioactive property (for example, forming a ferritin nanoparticle).

A “variant” refers to a molecule substantially similar in structure and immunoreactivity. Thus, provided that two molecules possess a common immunoactivity and can substitute for each other, they are considered “variants” as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the amino acid or nucleotide sequence is not identical. Thus, in one embodiment, a variant refers to a protein whose amino acid sequence is similar to a reference amino acid sequence, but does not have 100% identity with the respective reference sequence. The variant protein has an altered sequence in which one or more of the amino acids in the reference sequence is deleted or substituted, or one or more amino acids are inserted into the sequence of the reference amino acid sequence. As a result of the alterations, the variant protein has an amino acid sequence which is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the reference sequence. For example, variant sequences which are at least 95% identical have no more than 5 alterations, i.e. any combination of deletions, insertions or substitutions, per 100 amino acids of the reference sequence. Percent identity is determined by comparing the amino acid sequence of the variant with the reference sequence using any available sequence alignment program. An example includes the MEGALIGN project in the DNA STAR program. Sequences are aligned for identity calculations using the method of the software basic local alignment search tool in the BLAST network service (the National Center for Biotechnology Information, Bethesda, Md.) which employs the method of Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410. Identities are calculated by the Align program (DNAstar, Inc.) In all cases, internal gaps and amino acid insertions in the candidate sequence as aligned are not ignored when making the identity calculation.

The term “subject” refers to a human in need of treatment for any purpose, and more preferably a human in need of treatment to treat infection. The term “subject” can also refer to non-human animals, such as dogs, cats, horses, cows, pigs, sheep and non-human primates, among others.

"Pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

"Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents.

As used herein, the term “carrier” encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2005. Examples of physiologically acceptable carriers include saline, glycerol, DMSO, buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™ (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, NJ). To provide for the administration of such dosages for the desired therapeutic treatment, compositions disclosed herein can advantageously comprise between about 0.1% and 99% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.

As used herein, the terms “treating” or “treatment” of a subject includes the administration of a drug to a subject with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting a disease or disorder, or a symptom of a disease or disorder. The terms “treating” and “treatment” can also refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, and improvement or remediation of damage.

As used herein, the terms "prevent", "preventing" and "prevention" refer to the prevention of the recurrence or the onset of one or more symptoms of a disorder or disease, especially in individuals which have been analyzed to be susceptible or likely to develop the disease.

“Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

“Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g., a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the treatment or prevention of infection or disease. In some embodiments, a desired therapeutic result is a reduction of viral levels in a subject. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as coughing relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.

The term “increased” or “increase” as used herein generally means an increase by a statically significant amount; for the avoidance of any doubt, “increased” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level so long as the increase is statistically significant.

The term “reduced”, “reduce”, “reduction”, or “decrease” as used herein generally means a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10- 100% as compared to a reference level so long as the decrease is statistically significant.

The term “nanoparticle” as used herein refers to a particle or structure which is biocompatible with and sufficiently resistant to chemical and/or physical destruction by the environment of such use so that a sufficient number of the nanoparticles remain substantially intact after delivery to the site of application or treatment and whose size is in the nanometer range. For the purposes of the present invention, a nanoparticle typically ranges from about 1 nm to about 1000 nm, preferably from about 5 nm to about 50 nm, more preferably from about 10 nm to about 15 nm.

Compositions

In some aspects, disclosed herein is a nanoparticle comprising at least one ferritin polypeptide, wherein the at least one ferritin polypeptide comprises at least one amino acid mutation to cysteine. In some embodiments, the at least one ferritin polypeptide comprises a light chain of a ferritin protein or a heavy chain of a ferritin protein, or a combination thereof.

In some aspects, disclosed herein is a nanoparticle comprising at least one light chain of a ferritin protein and at least one heavy chain of a ferritin protein, wherein the at least one light chain and the at least one heavy chain each has at least one amino acid mutation to cysteine. In some embodiments, the ferritin protein is an insect ferritin protein or a mammalian ferritin protein. In some embodiments, the nanoparticle comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, seventeen, eighteen, nineteen, twenty, twenty one, or twenty five light chains and at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, seventeen, eighteen, nineteen, twenty, twenty one, or twenty five heavy chains. In some embodiments, the nanoparticle comprises about 4 to 25, 4 to 10, 6 to 12, 8 to 14, 10 to 16, 12 to 18, 14 to 20, or 16 to 24 light chains and about 4 to 25, 4 to 10, 6 to 12, 8 to 14, 10 to 16, 12 to 18, 14 to 20, or 16 to 24 heavy chains. In some embodiments, the nanoparticle comprises about 12 light chains and 12 heavy chains.

In some embodiments, the ferritin protein is an insect protein. In some embodiments, the ferritin protein is encoded by an insect gene (e.g., a Trichoplusia ni ferritin gene). In some embodiments, the ferritin protein is a Trichoplusia ni ferritin protein.

The nanoparticle structure of wild type ferritin is unstable. The amino acid mutations to cysteines disclosed herein can form disulfide bonds, for example, between a light chain and a light chain of a ferritin protein, between a light chain and a heavy chain of a ferritin protein, or between a heavy chain and a heavy chain of a ferritin protein. These additional disulfide bonds form between the additional cysteine residues can stabilize a ferritin nanoparticle structure. Accordingly, in some aspects, disclosed herein is a nanoparticle comprising at least one light chain of a ferritin protein and at least one heavy chain of a ferritin protein, wherein the at least one light chain and the at least one heavy chain each has at least one amino acid mutation to cysteine, wherein the at least one amino acid mutation is at an interface where a heavy chain and a light chain interact.

In some embodiments, the light chain comprises one amino acid mutation to cysteine. In some embodiments, the light chain comprises two amino acid mutations to cysteines. In some embodiments, the light chain comprises three amino acid mutations to cysteines. In some embodiments, the light chain comprises four amino acid mutations to cysteines. In some embodiments, the light chain comprises five amino acid mutations to cysteines. In some embodiments, the light chain comprises a polypeptide sequence having at or greater than about

80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 2, or a polypeptide comprising a portion of SEQ ID NO: 2. In some embodiments, the light chain comprises a A26C mutation relative to SEQ ID NO: 2. In some embodiments, the light chain comprises the polypeptide sequence of SEQ ID NO: 2 or a fragment thereof.

In some embodiments, the heavy chain comprises one amino acid mutation to cysteine. In some embodiments, the heavy chain comprises two amino acid mutations to cysteines. In some embodiments, the heavy chain comprises three amino acid mutations to cysteines. In some embodiments, the heavy chain comprises four amino acid mutations to cysteines. In some embodiments, the heavy chain comprises five amino acid mutations to cysteines. In some embodiments, the heavy chain comprises a polypeptide sequence having at or greater than about

80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 1, or a polypeptide comprising a portion of SEQ ID NO: 1. In some embodiments, the heavy chain comprises a D49C mutation relative to SEQ ID NO: 1. In some embodiments, the heavy chain comprises the polypeptide sequence of SEQ ID NO: 1 or a fragment thereof.

In some embodiments, disclosed herein is a nanoparticle comprising at least one light chain of a ferritin protein and at least one heavy chain of a ferritin protein, wherein the light chain comprises the polypeptide sequence of SEQ ID NO: 2 or a fragment thereof, and wherein the heavy chain comprises the polypeptide sequence of SEQ ID NO: 1 or a fragment thereof.

In some embodiments, the light chain and/or the heavy chain is truncated. In some embodiments, the truncated light chain comprises a deletion of an N-terminal portion or a C- terminal portion of the light chain. In some embodiments, the truncated light chain comprises a deletion of amino acid residues 1-28 or 1-24 relative to SEQ ID NO: 2. In some embodiments, the truncated light chain comprises the amino acid sequence of SEQ ID NO: 6 or 8 or a fragment thereof.

In some embodiments, the truncated heavy chain comprises a deletion of an N-terminal portion or a C-terminal portion of the heavy chain. In some embodiments, the truncated heavy chain comprises a deletion of amino acid residues 1-25 relative to SEQ ID NO: 1. In some embodiments, the truncated heavy chain comprises the amino acid sequence of SEQ ID NO: 5 or 7 or a fragment thereof.

In some embodiments, the at least one light chain is further linked to one or more polypeptides. In some embodiments, the polypeptide is linked to the N-terminus or C-terminus of the light chain. In some embodiments, the at least one heavy chain is further linked to one or more polypeptides. In some embodiments, the polypeptide is linked to the N-terminus or C-terminus of the heavy chain. In some embodiments, the polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 13, or a polypeptide comprising a portion of SEQ ID NO: 13. In some embodiment, the light chain linked to the polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 10, or a polypeptide comprising a portion of SEQ ID NO: 10. In some embodiment, the heavy chain linked to the polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 9, or a polypeptide comprising a portion of SEQ ID NO: 9.

In some embodiments, the one or more polypeptides are one or more antigens (e.g., antigen of a pathogen or oncogene-encoded proteins). Accordingly, also disclosed herein is a nanoparticle comprising at least one light chain of a ferritin protein and at least one heavy chain of a ferritin protein, wherein the at least one light chain and the at least one heavy chain each has at least one amino acid mutation to cysteine, wherein the at least one light chain and/or the at least one heavy chain is further linked to an antigen.

In some embodiments, the at least one light chain and/or the at least one heavy chain is further linked to one or more bacterial antigens, viral antigens, or parasitic antigens. In some embodiments, the viral antigen is a protein of Herpes Simplex virus- 1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus, Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papillomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Zika virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, or Human Immunodeficiency virus type-2. In some embodiments, the one or more viral antigens are HIV-1 proteins, influenza proteins, or RSV proteins. In some embodiments, the antigen forms a trimeric structure on the surface of the nanoparticle.

In some embodiments, the one or more polypeptides are selected from the group consisting of an HIV-1 Gag peptide, an HIV-1 Pol peptide, an HIV-1 Env peptide, an HIV-1 Vif peptide, an HIV-1 Vpr peptide, an HIV-1 Tat peptide, an HIV-1 Vpu peptide, and an HIV-1 Nef peptide. In some embodiments, the HIV-1 Env proteins are from one or more subtypes of HIV- 1, for example, clades A, B, and/or C or strains DU172.17, 286.36, KNH1209.18, HT593.1, 5768.04, and/or MB539.2B7.

In some embodiments, the nanoparticle has a diameter from about 1 nm to about 1000 nm. In some embodiments, the nanoparticle has a diameter less than, for example, about 1000 nm, about 950 nm, about 900 nm, about 850 nm, about 800 nm, about 750 nm, about 700 nm, about 650 nm, about 600 nm, about 550 nm, about 500 nm, about 450 nm, about 400 nm, about 350 nm, about 300 nm, about 290 nm, about 280 nm, about 270 nm, about 260 nm , about 250 nm, about 240 nm, about 230 nm, about 220 nm, about 210 nm, about 200 nm, about 190 nm, about 180 nm, about 170 nm, about 160 nm, about 150 nm, about 140 nm, about 130 nm, about 120 nm, about 110 nm, about 100 nm, about 90 nm, about 80 nm, about 70 nm, about 60 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, or about 10 nm. In some embodiments, the nanoparticle has a diameter, for example, from about 5 nm to about 100 nm, from about 5 nm to about 50 nm, from about 5 nm to about 30 nm, from about 5 nm to about 20 nm, from about 10 nm to about 20 nm, or from about 10 nm to about 15 nm. In some embodiments, the nanoparticle has a diameter from about 10 nm to about 15 nm. In some aspects, disclosed herein is a vaccine comprising the nanoparticle of disclosed herein.

In some aspects, disclosed herein is a polynucleotide encoding the polypeptide disclosed herein.

Methods

In some aspects, disclosed herein is a method for treating or preventing an infection (e.g., a bacterial infection, a viral infection, or a parasitic infection) in a subject, comprising administrating to the subject a therapeutically effective amount of the vaccine or nanoparticle disclosed herein. In some embodiments, the viral infection is an HIV-1 infection, RSV infection, or influenza infection.

In some aspects, disclosed herein is a method for treating or preventing a disease in a subject, comprising administrating to the subject a therapeutically effective amount of the vaccine or nanoparticle disclosed herein.

In some embodiments, the vaccine comprises a nanoparticle comprising at least one light chain of a ferritin protein and/or at least one heavy chain of a ferritin protein, wherein the at least one light chain and the at least one heavy chain each has at least one amino acid mutation to cysteine.

In some embodiments, the at least one amino acid mutation is at an interface where a heavy chain and a light chain interact. In some embodiments, the light chain comprises a A26C mutation relative to SEQ ID NO: 2. In some embodiments, the light chain comprises a A26C mutation relative to SEQ ID NO: 1.

Also disclosed herein is a system for use to produce broad broadly neutralizing antibodies, wherein the system comprises a nanoparticle comprising at least one light chain of a ferritin protein and at least one heavy chain of a ferritin protein, wherein the at least one light chain and the at least one heavy chain each has at least one amino acid mutation to cysteine, wherein the at least one light chain and/or the at least one heavy chain is further linked to one or more antigens.

In some embodiments, the broadly neutralizing antibodies are specific for two or more subtypes of a virus. In some embodiments, the broadly neutralizing antibodies are specific for two or more subtypes of HIV-1, for example, clades A, B, and/or C or strains DU172.17, 286.36, KNH1209.18, HT593.1, 5768.04, and/or MB539.2B7.

In some embodiments, the at least one light chain is further linked to one or more polypeptides. In some embodiments, the polypeptide is linked to the N-terminus or C-terminus of the light chain. In some embodiments, the at least one heavy chain is further linked to one or more polypeptides. In some embodiments, the polypeptide is linked to the N-terminus or C-terminus of the heavy chain. In some embodiments, the polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 13, or a polypeptide comprising a portion of SEQ ID NO: 13. In some embodiment, the light chain linked to the polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 10, or a polypeptide comprising a portion of SEQ ID NO: 10. In some embodiment, the heavy chain linked to the polypeptide comprises a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 9, or a polypeptide comprising a portion of SEQ ID NO: 9. The present invention may be better understood with reference to the accompanying examples that are intended for purposes of illustration only and should not be construed to limit the scope of the invention, as defined by the claims appended hereto.

EXAMPLES

The following examples are set forth below to illustrate the polypeptides, compositions, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

Example 1. Stifer Mutations in Insect Ferritin: Multivalent Immunogens for the Elicitation of HIV-1 Env Broadly Neutralizing Antibodies

The incredible diversity of the HIV-1 envelope glycoprotein (Env) between strains makes creation of a protective vaccine especially challenging. Attempts at incorporating Env diversity in vaccination strategies have had limited success, due to relatively arbitrary strain selection. This study developed a multi-objective optimization algorithm that simultaneously optimized Env strain selection based on factors that favor the elicitation of broadly neutralizing antibodies (bNAbs) including glycan shield coverage, availability of bNAb epitopes, and the level of sequence diversity between strains. Immunizations of guinea pigs with this optimized strain set will elicit antibodies with a wider breadth of Env neutralization compared to previous immunization studies. Furthermore, this study compared different immunization strategies with the strain set disclosed herein including sequential, cocktail, and multivalent display on nanoparticles. Nanoparticles that display multiple different Env strains on one particle can more strongly engage low affinity bNAb precursor B cell receptors due to increased avidity. We are using a modified form of insect ferritin that has been truncated and fused to Env to construct nanoparticles that each display two different Env strains from our optimized set. To increase nanoparticle stability, a mutation was added tointroduce a disulfide bond between monomers. Thus, disclosed herein are innovative strategies for immunogen design and through a systematic evaluation of critical variables, in order to develop a vaccine that can elicit bNAb responses. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference. Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.

SEQUENCES

Native Insect Ferritin Heavy Chain (SEQ ID NO: 1)

TQCNVNPVQIPKDWITMHRSCRNSMRQQIQMEVGASLQYLAMGAHFSKDWNRPGFA QLFFD AASEEREHAMKLIEYLLMRGELTND VS SLLQVRPPTRS S WKGGVEALEHALSME SDVTKSIRNVIKACEDDSEFNDYHLVDYLTGDFLEEQYKGQRDLAGKASTLKKLMDRH EALGEFIFDKKLLGIDV

Native Insect Ferritin Light Chain (SEQ ID NO: 2)

ADTCYNDVALDCGITSNSLALPRCNAVYGEYGSHGNVATELQAYAKLHLERSYDYLLS AAYFNNYQTNRAGFSKLFKKLSDEAWSKTIDIIKHVTKRGDKMNFDQHSTMKTERKNY TAENHELEALAKALDTQKELAERAFYIHREATRNSQHLHDPEIAQYLEEEFIEDHAEKIR TLAGHTSDLKKFITANNGHDLSLALYVFDEYLQKTV

Stifer Mutant Insect Ferritin Heavy Chain (SEQ ID NO: 3)

TQCNVNPVQIPKDWITMHRSCRNSMRQQIQMEVGASLQYLAMGAHFSKCWNRPGFA QLFFD AASEEREHAMKLIEYLLMRGELTND VS SLLQVRPPTRS S WKGGVEALEHALSME SDVTKSIRNVIKACEDDSEFNDYHLVDYLTGDFLEEQYKGQRDLAGKASTLKKLMDRH EALGEFIFDKKLLGIDV

Stifer Mutant Insect Ferritin Light Chain (SEQ ID NO: 4)

ADTCYNDVALDCGITSNSLALPRCNCVYGEYGSHGNVATELQAYAKLHLERSYDYLLS AAYFNNYQTNRAGFSKLFKKLSDEAWSKTIDIIKHVTKRGDKMNFDQHSTMKTERKNY TAENHELEALAKALDTQKELAERAFYIHREATRNSQHLHDPEIAQYLEEEFIEDHAEKIR TLAGHTSDLKKFITANNGHDLSLALYVFDEYLQKTV

Truncated Trimer-Optimized Insect Ferritin Heavy Chain (SEQ ID NO: 5)

RSCRNSMRQQIQMEVGASLQYLAMGAHFSKDWNRPGFAQLFFDAASEEREHAMKLIE YLLMRGELTNDVSSLLQVRPPTRSSWKGGVEALEHALSMESDVTKSIRNVIKACEDDSE FNDYHLVDYLTGDFLEEQYKGQRDLAGKASTLKKLMDRHEALGEFIFDKKLLGIDV

Truncated Trimer-Optimized Insect Ferritin Light Chain (SEQ ID NO: 6)

GEYGSHGNVATELQAYAKLHLERSYDYLLSAAYFNNYQTNRAGFSKLFKKLSDEAWS KTIDIIKHVTKRGDKMNFDQHSTMKTERKNYTAENHELEALAKALDTQKELAERAFYIH REATRNSQHLHDPEIAQYLEEEFIEDHAEKIRTLAGHTSDLKKFITANNGHDLSLALYVF DEYLQKTV Stifer Mutant Trimer-Optimized Insect Ferritin Heavy Chain (SEQ ID NO: 7)

RSCRNSMRQQIQMEVGASLQYLAMGAHFSKCWNRPGFAQLFFDAASEEREHAMKLIE YLLMRGELTNDVSSLLQVRPPTRSSWKGGVEALEHALSMESDVTKSIRNVIKACEDDSE FNDYHLVDYLTGDFLEEQYKGQRDLAGKASTLKKLMDRHEALGEFIFDKKLLGIDV

Stifer Mutant Truncated Trimer-Optimized Insect Ferritin Light Chain (SEQ ID NO: 8)

NCVYGEYGSHGNVATELQAYAKLHLERSYDYLLSAAYFNNYQTNRAGFSKLFKKLSDE AWSKTIDIIKHVTKRGDKMNFDQHSTMKTERKNYTAENHELEALAKALDTQKELAERA FYIHREATRNSQHLHDPEIAQYLEEEFIEDHAEKIRTLAGHTSDLKKFITANNGHDLSLAL YVFDEYLQKTV

SOSIP-Fused Stifer Mutant Trimer-Optimized Insect Ferritin Heavy Chain (SEQ ID NO:

9)

MPMGSLQPLATLYLLGMLVASVLATEKLWVTVYYGVPVWKEATTTLFCASDAKA

YETEVHNVWATHACVPTDPNPQEVLLENVTENFNMWKNNMVEQMQEDIISLWDQ SLKPCVKLTPLCVTLECHDVNVNGTANNGTTNVTESGVNSSDVTSNNVTNSNWGT

MEKGEIKNCSFNITTNIRDKMQKETAQFYKLDIVPIEDQNKTNNTLYRLINCNTSVC

TQACPKVSFEPIPIHYCTPAGFAILKCNDRNFNGTGPCKNVSTVQCTHGIKPWSTQ

LLLNGSLAEAEWIRSENFTNNAKTIIIQLNETVEINCTRPNNNTSKRISIGPGRAFRA

TKHGNIRQAHCNISRATWNSTLKKIVAKLREQFGNKTIVFQPSSGGDPEIVMHSFN

CGGEFFYCNTTQLFNSTWNSTEESNSTEEGTITLPCRIKQHNMWQEVGKCMYAPPI

EGQIRCSSNITGLLLTRDGGNNNKTNGTEIFRPGGGDMRDNWRSELYKYKWKIEP

LGVAPTKCKRRWQGGSGGGGSGGGGSGGAVGIVGAMFLGFLGAAGSTMGAAS MTLTVQARLLLSGIVQQQNNLLRAPEAQQHLLQLTVWGIKQLQARVLAVERYLK

DQQLLGIWGCSGKLICCTTVPWNTSWSNKSLSEIWDNMTWMQWEREIDNYTSLIY

TLIEESQNQQEKNEQELLELDGGSGGRSCRNSMRQQIQMEVGASLQYLAMGAHFSKC WNRPGF AQLFFD AASEEREH AMKLIEYLLMRGELTND VS SLLQ VRPPTRS S WKGGVE ALEHALSMESDVTKSIRNVIKACEDDSEFNDYHLVDYLTGDFLEEQYKGQRDLAGKAST LKKLMDRHEALGEFIFDKKLLGIDV

SOSIP-FUSED Stifer Mutant Truncated Trimer-Optimized Insect Ferritin Light Chain (SEQ ID NO: 10)

MPMGSLQPLATLYLLGMLVASVLATEKLWVTVYYGVPVWKEATTTLFCASDAKA YETEVHNVWATHACVPTDPNPQEVLLENVTENFNMWKNNMVEQMQEDIISLWDQ SLKPCVKLTPLCVTLECHDVNVNGTANNGTTNVTESGVNSSDVTSNNVTNSNWGT

MEKGEIKNCSFNITTNIRDKMQKETAQFYKLDIVPIEDQNKTNNTLYRLINCNTSVC

TQACPKVSFEPIPIHYCTPAGFAILKCNDRNFNGTGPCKNVSTVQCTHGIKPWSTQ LLLNGSLAEAEWIRSENFTNNAKTIIIQLNETVEINCTRPNNNTSKRISIGPGRAFRA TKHGNIRQAHCNISRATWNSTLKKIVAKLREQFGNKTIVFQPSSGGDPEIVMHSFN CGGEFFYCNTTQLFNSTWNSTEESNSTEEGTITLPCRIKQIINMWQEVGKCMYAPPI EGQIRCSSNITGLLLTRDGGNNNKTNGTEIFRPGGGDMRDNWRSELYKYKWKIEP LGVAPTKCKRRWQGGSGGGGSGGGGSGGAVGIVGAMFLGFLGAAGSTMGAAS MTLTVQARLLLSGIVQQQNNLLRAPEAQQHLLQLTVWGIKQLQARVLAVERYLK DQQLLGIWGCSGKLICCTTVPWNTSWSNKSLSEIWDNMTWMQWEREIDNYTSLIY

TLIEESONOOEKNEOELLELDGGSGGSGGNCVYGEYGSHGNVATELQAYAKLHLERS YDYLLSAAYFNNYQTNRAGFSKLFKKLSDEAWSKTIDIIKHVTKRGDKMNFDQHSTMK TERKNYTAENHELEALAKALDTQKELAERAFYIHREATRNSQHLHDPEIAQYLEEEFIED HAEKIRTLAGHTSDLKKFITANNGHDLSLALYVFDEYLQKTV

Linker 1 (SEQ ID NO: 11)

GGSGG

Linker 2 (SEQ ID NO: 12)

GGSGGSGG

SOSIP sequence (SEQ ID NO: 13)

MPMGSLQPLATLYLLGMLVASVLATEKLWVTVYYGVPVWKEATTTLFCASDAKAYET EVHNVWATHACVPTDPNPQEVLLENVTENFNMWKNNMVEQMQEDIISLWDQSLKPCV KLTPLCVTLECHDVNVNGTANNGTTNVTESGVNSSDVTSNNVTNSNWGTMEKGEIKNC SFNITTNIRDKMQKETAQFYKLDIVPIEDQNKTNNTLYRLINCNTSVCTQACPKVSFEPIPI HYCTPAGFAILKCNDRNFNGTGPCKNVSTVQCTHGIKPVVSTQLLLNGSLAEAEVVIRSE NFTNNAKTIIIQLNETVEINCTRPNNNTSKRISIGPGRAFRATKIIGNIRQAHCNISRATWNS TLKKIVAKLREQFGNKTIVFQPSSGGDPEIVMHSFNCGGEFFYCNTTQLFNSTWNSTEES NSTEEGTITLPCRIKQIINMWQEVGKCMYAPPIEGQIRCSSNITGLLLTRDGGNNNKTNGT EIFRPGGGDMRDNWRSELYKYKWKIEPLGVAPTKCKRRWQGGSGGGGSGGGGSGG AVGIVGAMFLGFLGAAGSTMGAASMTLTVQARLLLSGIVQQQNNLLRAPEAQQHLLQL

TVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICCTTVPWNTSWSNKSLSEIWDNM TWMQWEREIDNYTSLIYTLIEESQNQQEKNEQELLELD

Claims

CLAIMS We claim:

1. A nanoparticle comprising at least one light chain of a ferritin protein and at least one heavy chain of a ferritin protein, wherein the at least one light chain and the at least one heavy chain each has at least one amino acid mutation to cysteine.

2. The nanoparticle of claim 1, wherein the at least one amino acid mutation is at an interface where a heavy chain and a light chain interact.

3. The nanoparticle of claim 1 or 2, wherein the light chain comprises a A26C mutation relative to SEQ ID NO: 2.

4. The nanoparticle of claim 1 or 2, wherein the heavy chain comprises a D49C mutation relative to SEQ ID NO: 1.

5. The nanoparticle of any one of claims 1-4, wherein the light chain and/or the heavy chain is truncated.

6. The nanoparticle of claim 5, wherein the truncated light chain comprises a deletion of amino acid residues 1-28 or 1-24 relative to SEQ ID NO: 2.

7. The nanoparticle of claim 6, wherein the truncated light chain comprises the amino acid sequence of SEQ ID NO: 8.

8. The nanoparticle of claim 5, wherein the truncated heavy chain comprises a deletion of amino acid residues 1-25 relative to SEQ ID NO: 1.

9. The nanoparticle of claim 8, wherein the truncated heavy chain comprises the amino acid sequence of SEQ ID NO: 7. The nanoparticle of any one of claims 1-9, wherein the at least one light chain and/or the at least one heavy chain is further linked to one or more polypeptides. The nanoparticle of claim 10, wherein the one or more polypeptides are one or more antigens. The nanoparticle of claim 10 or 11, wherein the one or more polypeptides are one or more viral antigens. The nanoparticle of claim 12, wherein the one or more viral antigens are HIV-1 proteins, influenza proteins, or RSV proteins. The nanoparticle of claim 13, wherein the HIV-1 proteins are HIV-1 Env proteins. The nanoparticle of claim 14, wherein the HIV-1 Env proteins are from one or more subtypes of HIV - 1. A vaccine comprising the nanoparticle of any one of claims 1-15. A method for treating or preventing a viral infection in a subject, comprising administrating to the subject a therapeutically effective amount of the vaccine of claim 16. The method of claim 17, wherein the viral infection is an HIV-1 infection, RSV infection, or influenza infection. A method for producing broadly neutralizing antibodies, comprising administering to a subject an effective amount of the nanoparticle of any one of claims 1-15; obtaining a biological sample from the subject; and isolating the broadly neutralizing antibodies from the biological sample. The method of claim 19, wherein the broadly neutralizing antibodies are specific for two or more subtypes of a virus. The method of claim 19 or 20, wherein the broadly neutralizing antibodies are specific for two or more subtypes of HIV- 1.