VACCINE COMPOSITIONS COMPRISING ENDOGENOUS GAG POLYPEPTIDES
Described herein is a composition comprising: 1) an ARC polypeptide or an endogenous gag (endo-gag) polypeptide; 2) a pathogen-associated antigen; and 3) an adjuvant. Also described herein are vaccines and methods of vaccination using compositions comprising: 1) an ARC polypeptide or an endogenous gag (endo-gag) polypeptide; 2) a pathogen-associated antigen; and 3) an adjuvant.
This application claims the benefit of U.S. Pat. Application No. 16/876,731, filed May 18, 2020, which is incorporated herein by reference in its entirety.
SEQUENCE LISTINGThe instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 13, 2021, is named 54838-708_601_SL.txt, and is 76,601 bytes in size.
SUMMARYDescribed herein are vaccine compositions, methods of vaccinating using the compositions, and methods of making the compositions. Such compositions are useful for the vaccination of individuals against pathogenic diseases caused by bacteria, viruses, fungi, or parasites.
The compositions described herein are virus-like particles (VLPs) that display antigens and/or adjuvants. These VLPs comprise endogenous Gag-like polypeptides such as ARC polypeptides and are sometimes referred to as endogenous virus-like particles (endo-VLPs). When a human individual is the one vaccinated, the ARC polypeptides may comprise human ARC or other human endogenous Gag (endo-Gag) polypeptides. The endo-VLPs described herein provide advantages compared to known methods of vaccination.
Previous methods of vaccination, including the use of live attenuated viruses, inactivated viruses, and recombinant subunit-based vaccines, all suffer from drawbacks. One is that engineering and attenuating live virus-based vaccines are labor and time-intensive processes. While addressing this limitation, recombinant subunit vaccines may lack potency due to the lack of viral, bacterial, or other contexts that leads to adequate immune priming or boosting. Nanoparticle vaccines offer a potential solution to this limitation when using recombinant proteins for vaccine production. By introducing antigens, agonists for innate immune receptors, and/or adjuvants together on a single nanoparticle, one can leverage the benefits of a recombinant vaccine in a package that appears to the immune system to be a virus. When antigen and adjuvant molecules are mere nanometers apart on a single nanoparticle, the likelihood of immune cell recognition of both is greatly increased. This combination of benefits makes recombinant antigen delivery using endoVLP delivery systems a rapid approach to developing effective vaccines targeting emerging threats to human health. Further, the use of endoVLPs based upon human ARC and other human Gag-like proteins allows for a platform amenable to repeated administration since an immune response is not generated against the endoVLPs.
In one embodiment, a composition comprises: 1) an ARC polypeptide or an endogenous gag (endo-gag) polypeptide; 2) a pathogen-associated antigen; and 3) an adjuvant.
In some embodiments, the composition comprises an ARC polypeptide comprising an amino acid sequence that is SEQ ID NO: 1 or an amino acid sequence that is at least 90% identical to SEQ ID NO: 1. In some embodiments, the endo-gag polypeptide comprises an amino acid that is SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. In other embodiments, the endo-gag polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. In some instances, the endo-gag polypeptide comprises any combination of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28.
In some embodiments, the pathogen-associated antigen and the adjuvant are different compounds or polypeptides. In one embodiment, the pathogen-associated antigen comprises a polypeptide. In some embodiments, the pathogen-associated antigen is a bacterial antigen, a fungal antigen, a parasitic antigen, or a viral antigen. In one embodiment, the pathogen-associated antigen is a bacterial antigen. In another embodiment, the pathogen-associated antigen is a fungal antigen. In one instance, the pathogen-associated antigen is a parasitic antigen. In another instance, the pathogen-associated antigen is a viral antigen. In one embodiment, the viral antigen is a respiratory virus antigen. In another embodiment, the viral antigen is a Coronaviridae antigen. In one instance, Coronaviridae exhibits human tropism. In some embodiments, Coronaviridae is selected from the list consisting of SARS Coronavirus (SARS-CoV-1), COVID-19 (SARS-CoV-2), MERS-coronavirus (MERS-CoV), or any combination thereof.
In some embodiments, the viral antigen comprises a spike protein, an envelope protein, a nucleocapsid protein, a membrane protein, a membrane glycoprotein, or a non-structural protein. In other embodiments, the viral antigen comprises a SARS Coronavirus (SARS-CoV-1) antigen, a COVID-19 (SARS-CoV-2) antigen, a MERS-coronavirus (MERS-CoV) antigen, or any combination thereof. In some instances, the viral antigen comprises a spike protein, an envelope small membrane protein, a membrane protein, a non-structural protein 6 (NSP6), a nucleoprotein, an ORF10 protein, Protein 3a, Protein7a, Protein 9b, structural protein 8, uncharacterized protein 4, or any combination thereof. In some embodiments, the viral antigen comprises an amino acid residue sequence as set forth in any one of SEQ ID NOs: 30 to 42, and combinations thereof. In other embodiments, the viral antigen consists of an amino acid residue sequence as set forth in any one of SEQ ID NOs: 30 to 42, and combinations thereof. In some embodiments, the viral antigen is an Influenza antigen. In some embodiments, the Influenza antigen is an M1 matrix protein. In some embodiments, the M1 matrix protein is derived from influenza H5N1 and/or comprises SEQ ID NO: 45 or a fragment thereof.
In some embodiments, the ARC polypeptide or the endo-gag-polypeptide is coupled to the pathogen-associated antigen. In one embodiment, the ARC polypeptide or the endo-gag-polypeptide is coupled to the pathogen-associated antigen by a peptide bond. In another embodiment, the pathogen-associated antigen is N-terminal to the ARC polypeptide or the endo-gag-polypeptide. In one instance, the pathogen-associated antigen is C-terminal to the ARC polypeptide or the endo-gag-polypeptide. In another instance, a flexible peptide linker separates the pathogen-associated antigen and the ARC polypeptide or the endo-gag-polypeptide. In one embodiment, the ARC polypeptide or the endo-gag-polypeptide is coupled to the pathogen-associated antigen by a bond formed from the reaction of an NHS-ester and a primary amine of the ARC polypeptide.
In some embodiments, the adjuvant comprises an immune stimulatory compound. In other embodiments, the immune stimulatory compound comprises a lipid, a nucleic acid, an aluminum compound, an water-in-oil emulsion, a polypeptide, or any combination thereof. In one embodiment, the immune stimulatory compound comprises a lipid. In other embodiments, the immune stimulatory compound comprises a nucleic acid. In one embodiment, the nucleic acid is a DNA. In some embodiments, the DNA comprises CPG-1018, CPG-1826, CPG-2007, CPG-2006, or any combination thereof. In another embodiment, the nucleic acid is an RNA. In some embodiments, the RNA comprises CV1802, Poly(U), Poly(I:C), ssRNA40, GFP RNA, RNA41, RNA42, RNA33, RNA35, 5′-phosphorylated blunt ended viral genomic dsRNA <300bp, long dsRNA >1000bp, genomic ssRNA, ssRNA40, or any combination thereof. In other embodiments, the immune stimulatory compound comprises an aluminum compound. In some instances, the aluminum compound comprises alum. In some embodiments, the immune stimulatory compound comprises an water-in-oil emulsion. In other embodiments, the immune stimulatory compound comprises an agonist for a toll-like receptor, a NOD-like receptor, a RIG-1 or MDA-5 receptor, a C-type lectin receptor, a costimulatory molecule, a cytokine receptor, a STING pathway, or any combination thereof. In some instances, the toll-like receptor agonist is selected from the list consisting of CpG oligonucleotide, SD-101, LFX453, imiquimod, Bacillus Calmette-Guerin (BCG), monophosphoryl lipid A, Poly ICLC, GSK1795091, or any combination thereof. In some embodiments, the NOD-like receptor agonist is selected from the list consisting of bacterial peptidoglycan, an acylated derivative of iE-DAP (C12-iE-DAP), D-gamma-Glu-mDAP (iE-DAP), L-Ala-gamma-D-Glu-mDAP (Tri-DAP), muramyl dipeptide (MDP), muramyl tripeptide, L18-MDP, M-TriDAP, murabutide, PGN-ECndi, PGN-ECndss, PGN-SAndi, N-glycosylated muramyl dipeptide, murabutide, or any combination thereof. In other embodiments, the RIG-1 or MDA-5 receptor agonist is selected from the list consisting of poly(I:C), Poly(dA:dT), Poly(dG:dC), 3p-hpRNA, 5′ppp-dsRNA, or any combination thereof. In some instances, the C-type lectin receptor agonist is selected from the list consisting of Beta-1,3-glucan, zymosan, Heat-killed C. albicans, cord factor, and Trehalose-6,6-dibehenate, or any combination thereof. In one embodiment, the immune stimulatory compound comprises a polypeptide. In some embodiments, the polypeptide is a polypeptide from Brucella abortus, Bordetella pertussis, Chlamydia trachomatis, Fusobacterium nucleatum, Mycobacterium tuberculosis, Neisseria meningitidis, Staphylococcus aureus, Shigella dysenteriae, Shigella flexneri, Streptococcus pneumoniae, Vibrio cholerae, Brucella abortus, Mycobacterium paratuberculosis, Neisseria meningitidis, Streptococcus pneumoniae, or any combination thereof. In other embodiments, the polypeptide is from BCSP31, FHA, MOMP, PorB, PVL, Porin, OmpA, 34 kDa MOMP, PepO, OmpU, Lumazine synthase, Omp16, Omp19, BCSP31, CobT, RpfE, Rv0652, HBHA, NhhA, DnaJ, Pneumolysin, ΔA146 Pneumolysin, or any combination thereof. In another embodiment, the polypeptide is a polypeptide from human heat shock protein 70.
In one embodiment, the adjuvant is coupled to the ARC polypeptide or the endo-gag-polypeptide. In another embodiment, the adjuvant is coupled to the ARC polypeptide or the endo-gag-polypeptide by a bond formed from the reaction of an NHS-ester and a primary amine of the ARC polypeptide. In one instance, the adjuvant is coupled to the ARC polypeptide or the endo-gag-polypeptide by a peptide bond. In another instance, the adjuvant is N-terminal to the ARC polypeptide or the endo-gag-polypeptide. In one embodiment, the adjuvant is C-terminal to the ARC polypeptide or the endo-gag-polypeptide.
In some embodiments, the composition comprises a flexible peptide linker separating the adjuvant and the ARC polypeptide or the endo-gag-polypeptide. In other embodiments, the composition comprises a virus-like particle. In some instances, the virus-like particle comprises a mixture of the ARC or the endo-gag-polypeptide polypeptide coupled to the pathogen-associated antigen and the ARC polypeptide or the endo-gag-polypeptide coupled to the adjuvant. In some embodiments, the virus-like particle comprises ARC polypeptide or the endo-gag-polypeptide not coupled to adjuvant or pathogen-associated antigen. In other embodiments, the ARC polypeptide or the endo-gag-polypeptide coupled to the adjuvant comprises from about 5% to about 20% of the virus-like particle. In some instances, the ARC polypeptide or the endo-gag-polypeptide coupled to the adjuvant comprises from about 5% to about 15% of the virus-like particle. In some embodiments, the ARC polypeptide or the endo-gag-polypeptide coupled to the adjuvant comprises about 10% of the virus-like particle. In other embodiments, the ARC polypeptide or the endo-gag-polypeptide coupled to the pathogen-associated antigen comprises from about 5% to about 20% of the virus-like particle. In some instances, the ARC polypeptide or the endo-gag-polypeptide coupled to the pathogen-associated antigen comprises from about 5% to about 15% of the virus-like particle. In some embodiments, the ARC polypeptide or the endo-gag-polypeptide coupled to the pathogen-associated antigen comprises about 10% of the virus-like particle. In other embodiments, the ARC polypeptide or the endo-gag-polypeptide not coupled to adjuvant or pathogen-associated antigen comprises from about 90% to about 60% of the virus-like particle. In some instances, the ARC polypeptide or the endo-gag-polypeptide not coupled to adjuvant or pathogen-associated antigen comprises from about 90% to about 70% of the virus-like particle. In some embodiments, the ARC polypeptide or the endo-gag-polypeptide not coupled to adjuvant or pathogen-associated antigen comprises about 80% of the virus-like particle.
In some embodiments, the composition of any one of the previous embodiments comprises a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition of any one of the previous embodiments is used as a vaccine. In some embodiments, the composition of any one of the previous embodiments is used in priming or boosting an adaptive immune response to the pathogen-associated antigen. In one embodiment, the adaptive immune response is an antibody response to the pathogen-associated antigen. In another embodiment, the antibody response produces IgG antibodies that specifically bind the pathogen-associated antigen. In one instance, the adaptive immune response is a cellular immune response to the pathogen-associated antigen.
In some embodiments, a method of priming an adaptive immune response to a pathogen-associated antigen in an individual comprises administering the composition of any one the previous embodiments to the individual, thereby priming an adaptive immune response to the pathogen-associated antigen. In one embodiment, the individual is a human individual. In another embodiment, the adaptive immune response is an antibody response to the pathogen-associated antigen. In one instance, the antibody response produces IgG antibodies that specifically bind the pathogen-associated antigen. In one embodiment, the adaptive immune response is a cellular immune response to the pathogen-associated antigen.
In some embodiments, a method of vaccinating an individual comprises administering the composition of any one of the previous embodiments to the individual, thereby vaccinating the individual. In one embodiment, the method of vaccinating protects from Coronaviridae infection or symptoms. In some embodiments, Coronaviridae comprises SARS Coronavirus (SARS-CoV-1), COVID-19 (SARS-CoV-2), MERS-coronavirus (MERS-CoV), or any combination thereof.
In some embodiments, a nucleic acid encodes the ARC polypeptide or the endo-gag-polypeptide, the ARC polypeptide or the endo-gag-polypeptide that is coupled to the pathogen-associated antigen by a peptide bond, the pathogen-associated antigen that is N-terminal to the ARC polypeptide or the endo-gag-polypeptide, the pathogen-associated antigen that is C-terminal to the ARC polypeptide or the endo-gag-polypeptide, the pathogen-associated antigen and the ARC polypeptide or the endo-gag-polypeptide separated by a flexible peptide linker, the adjuvant coupled to the ARC polypeptide or the endo-gag-polypeptide by a peptide bond, the adjuvant that is N-terminal to the ARC polypeptide or the endo-gag-polypeptide, the adjuvant that is C-terminal to the ARC polypeptide or the endo-gag-polypeptide, or the adjuvant and the ARC polypeptide or the endo-gag-polypeptide separated by a flexible peptide linker.
In some embodiments, a vector comprises the nucleic acid encoding the ARC polypeptide or the endo-gag-polypeptide, the ARC polypeptide or the endo-gag-polypeptide that is coupled to the pathogen-associated antigen by a peptide bond, the pathogen-associated antigen that is N-terminal to the ARC polypeptide or the endo-gag-polypeptide, the pathogen-associated antigen that is C-terminal to the ARC polypeptide or the endo-gag-polypeptide, the pathogen-associated antigen and the ARC polypeptide or the endo-gag-polypeptide separated by a flexible peptide linker, the adjuvant coupled to the ARC polypeptide or the endo-gag-polypeptide by a peptide bond, the adjuvant that is N-terminal to the ARC polypeptide or the endo-gag-polypeptide, the adjuvant that is C-terminal to the ARC polypeptide or the endo-gag-polypeptide, or the adjuvant and the ARC polypeptide or the endo-gag-polypeptide separated by a flexible peptide linker. In other embodiments, the vector comprises a promoter operatively coupled to the nucleic acid encoding the ARC polypeptide. In some instances, a host cell comprises the vector of the previous embodiments. In some embodiments, a method of manufacturing a vaccine comprises isolating the ARC polypeptide or the endo-gag polypeptide from the host cell of any one of the previous embodiments and contacting the polypeptide to a solution comprising a salt concentration of 100 mM to 1000 mM. In other embodiments, the salt is NaCl or NaPO4.
The novel features described herein are set forth with particularity in the appended claims. A better understanding of the features and advantages of the features described herein will be obtained by reference to the following detailed description that sets forth illustrative examples, in which the principles of the features described herein are utilized, and the accompanying drawings of which:
In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the embodiments provided may be practiced without these details. Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.
As used herein, the term “about” refers to an amount that is near the stated amount by 10% or less.
As used herein, the term “individual,” “patient,” or “subject” refers to individuals diagnosed with, suspected of being afflicted with, or at-risk of developing at least one disease for which the described compositions and method are useful for treating. In certain embodiments, the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. In certain embodiments, the individual is a human.
The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains and other peptides, e.g., linkers and binding peptides, may include natural and/or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., South San Francisco, Calif., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program’s alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.
The polypeptides described herein can be encoded by a nucleic acid. A nucleic acid is a type of polynucleotide comprising two or more nucleotide bases. In certain embodiments, the nucleic acid is a component of a vector that can be used to transfer the polypeptide-encoding polynucleotide into a cell. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a genomic integrated vector, or “integrated vector,” which can become integrated into the chromosomal DNA of the host cell. Another type of vector is an “episomal” vector, e.g., a nucleic acid capable of extra-chromosomal replication. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.” Suitable vectors comprise plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. In the expression vectors, regulatory elements such as promoters, enhancers, polyadenylation signals for use in controlling transcription can be derived from mammalian, microbial, viral, or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated. Vectors derived from viruses, such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, and the like, may be employed. Plasmid vectors can be linearized for integration into a chromosomal location. Vectors can comprise sequences that direct site-specific integration into a defined location or restricted set of sites in the genome (e.g., AttP-AttB recombination). Additionally, vectors can comprise sequences derived from transposable elements.
As used herein, the terms “homologous,” “homology,” or “percent homology” when used herein to describe an amino acid sequence or a nucleic acid sequence, relative to a reference sequence, can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such a formula is incorporated into the basic local alignment search tool (BLAST) programs of Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Percent homology of sequences can be determined using the most recent version of BLAST, as of the filing date of this application.
The nucleic acids encoding the polypeptides described herein can be used to infect, transfect, transform, or otherwise render a suitable cell transgenic for the nucleic acid, thus enabling the production of polypeptides for commercial or therapeutic uses. Standard cell lines and methods for the production of antibodies from a large-scale cell culture are known in the art. See e.g., Li et al., “Cell culture processes for monoclonal antibody production.” Mabs. 2010 Sep-Oct; 2(5): 466-477. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell. In certain embodiments, the mammalian cell line useful for producing antibodies is a Chines Hamster Ovary (CHO) cell, an NS0 murine myeloma cell, or a PER.C6® cell. In certain embodiments, the nucleic acid encoding the antibody is integrated into a genomic locus of a cell useful for producing antibodies. In certain embodiments, described herein is a method of making an antibody comprising culturing a cell comprising a nucleic acid encoding an antibody under conditions in vitro sufficient to allow production and secretion of said antibody.
In certain embodiments, described herein, is a master cell bank comprising: (a) a mammalian cell line comprising a nucleic acid encoding a polypeptide described herein integrated at a genomic location; and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol or DMSO. In certain embodiments, the master cell bank is contained in a suitable vial or container able to withstand freezing by liquid nitrogen.
Also described herein are methods of making a polypeptide described herein. Such methods comprise incubating a cell or cell-line comprising a nucleic acid encoding the polypeptide in a cell culture medium under conditions sufficient to allow for expression and secretion of the polypeptide, and further harvesting the polypeptide from the cell culture medium. The harvesting can further comprise one or more purification steps to remove live cells, cellular debris, nonpolypeptide of interest proteins, undesired salts, buffers, and medium components. In certain embodiments, the additional purification step(s) include centrifugation, ultracentrifugation, protein A, protein G, protein A/G, or protein L purification, and/or ion exchange chromatography.
As used herein, the term “prime” or “priming” in reference to an immune response refers to introducing an antigen to the immune system of an individual, wherein the individual is naive to the antigen introduced. The term “boost” or “boosting” in reference to an immune response refers to introducing an antigen to the immune system of an individual, wherein the immune system of the individual has experienced the antigen before, either through a previous vaccine administration or through natural infection.
As used herein, a “vaccine” refers to a composition of matter that is intended to prime or boost an immune response in an individual. The term “vaccinating” refers to the act of administering a vaccine to an individual. Vaccines may be administered prophylactically to prevent any disease or severe disease, or one or more unwanted symptoms such as fever, cough, sore throat, rhinorrhea, nasopharyngeal or chest congestion, respiratory distress, diarrhea, or vomiting. Vaccines may also be administered post-recovery from disease to prevent reinfection or preserve immunity gained from natural infection.
The term “adaptive immune response” as used herein refers to the components of the immune response that respond in an antigen-restricted way and encompasses cellular immune responses attributable to T lymphocytes and humoral or antibody response attributable to B cells and plasma cells. A “cellular immune response” is indicated by any one or more of the following: cytokine/chemokine release by T cells; T-cell homing to secondary lymphoid organs; T-cell proliferation; and cytotoxic T-cell responses. Several methods can be used to verify an antigen-specific cellular immune response, including ex vivo antigen stimulation assays of T lymphocytes and in vivo assays, such as tetramer staining of T lymphocytes. An “antibody response” is indicated by any one or more of the following: B cell proliferation, B-cell cytokine/chemokine release, B-cell homing to secondary lymphoid organs, antibody secretion, isotype switching to IgG type antibodies, or plasma cell differentiation. An antibody response can be verified by several methods, but a predominant method is the detection of antigen-specific antibodies in the serum or plasma of a vaccinated individual.
An “adjuvant” as described herein refers to a substance that in combination with an antigen promotes an adaptive immune response to the antigen. An “immune stimulatory compound” refers to a substance that specifically interacts with the innate immune system to initiate a “danger signal” that ultimately leads to the development of the adaptive components of the immune response (e.g., B cell, T cells). Immune stimulatory compounds include pathogen-associated molecular patterns (PAMPs) such as dsRNA, lipopolysaccharide, and CpG DNA, either naturally occurring or synthetic. Immune stimulatory compounds are agonists of various innate immune receptors including Toll-like receptors (TLRs), NOD-like receptors, RIG-1 or MDA-5 receptors, C-type lectin receptors, or the STING pathway.
A “pathogen-associated antigen” as described herein includes antigenic determinants derived from pathogenic organisms, including viruses, bacteria, fungi, or parasites capable of causing disease in an individual. Generally, such antigens will comprise polypeptides that may elicit an adaptive immune response specific for said antigen.
Coronaviridae refers to a family of enveloped, positive-sense, single-stranded RNA viruses. Coronaviridae encompass alpha-, beta-, gamma-, and delta-coronaviruses. Several coronaviruses cause disease in humans, including SARS Coronavirus (SARS-CoV-1), COVID-19 (SARS-CoV-2), and MERS-coronavirus (MERS-CoV).
Virus-like particles (VLPs) are molecules that closely resemble viruses but are noninfectious because they contain no viral genetic material. VLPs generally comprise one or more viral structural proteins (e.g., envelop, capsid, or membrane proteins) and can comprise structural proteins from different viruses. Generally, VLPs form a hollow structure that can be used to carry therapeutic payloads, such as small molecule inhibitors or non-viral nucleic acids.
ARC Based VaccinesArc (activity-regulated cytoskeleton-associated protein) regulates the endocytic trafficking of a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMP A) type glutamate receptors. Arc activities have been linked to synaptic strength and neuronal plasticity. Phenotypes of loss of Arc in experimental murine models include defective formation of long-term memory and reduced neuronal activity and plasticity. Arc exhibits similar molecular properties to retroviral Gag proteins. The Arc gene may have originated from the Ty3 /gypsy retrotransposon. An endogenous Gag (endo-Gag) protein is any protein endogenous to a eukaryotic organism, including Arc, that has predicted and annotated similarity to viral Gag proteins. Exemplary endo-Gag proteins are disclosed in Campillos M, Doerks T, Shah PK, and Bork P. “Computational characterization of multiple Gag-like human proteins,” Trends Genet. 2006 Nov;22(11):585-9.
The compositions of ARC/endo-gag proteins and endoVLPs described herein are useful to vaccinate or otherwise prime and/or boost an adaptive immune response directed against a pathogen-associated antigen. Such immune responses can prevent natural infection against a pathogen from which the antigen is derived, reduce the severity of symptoms of or the mortality associated with the pathogen, or reduce an individual’s ability to act as a carrier for the pathogen.
Referring to
The ARC/endo-gag -VLP polypeptide vaccines described herein, in certain embodiments, comprise an adjuvant coupled to an ARC polypeptide. The adjuvant may be a nonpolypeptide compound such as a CpG oligonucleotide, double-stranded RNA, lipopolysaccharide, a TLR agonist, a RIG agonist, a STING agonist, or a C-type lectin receptor agonist. In such instances wherein the adjuvant is not a polypeptide, the adjuvant can be crosslinked to an ARC/endo-gag polypeptide using any suitable chemistry. In certain embodiments, the adjuvant is coupled by a crosslinking reaction after VLP formation. In certain embodiments, the adjuvant is coupled by the reaction of an NHS ester and a primary amine on the ARC/endo-gag polypeptide. In certain embodiments, the adjuvant is coupled by a peptide linkage as a fusion protein. In certain embodiments, the adjuvant is coupled by a peptide linkage N-terminal to the ARC/endo-gag polypeptide. In certain embodiments, the adjuvant is coupled by a peptide linkage C-terminal to the ARC/endo-gag polypeptide. In certain embodiments, the adjuvant and the ARC/endo-gag polypeptide are connected via a flexible polypeptide linker. In certain embodiments, the adjuvant and the ARC/endo-gag polypeptide are connected via a flexible polypeptide linker, wherein the adjuvant polypeptide is N-terminal to the ARC/endo-gag polypeptide. In certain embodiments, the adjuvant and the ARC/endo-gag polypeptide are connected via a flexible polypeptide linker, wherein the adjuvant polypeptide is C-terminal to the ARC/endo-gag polypeptide.
The ARC/endo-gag-VLP polypeptide vaccines described herein, in certain embodiments, comprise a pathogen-associated antigen coupled to an ARC/endo-gag polypeptide. In certain embodiments, the pathogen-associated antigen comprises a short polypeptide comprising 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 34, 25, 26, 27, 28, 29, 30, or more amino acids in length. In certain embodiments, the pathogen-associated antigen comprises a polypeptide comprising all or substantially all of an antigenic protein. In certain embodiments, the pathogen-associated antigen comprises a polypeptide comprising an antigenic domain of a protein. In certain embodiments, the pathogen-associated antigen comprises a polypeptide comprising an antigenic region of a protein. In certain embodiments, the pathogen-associated antigen comprises a polypeptide comprising two or more antigenic domains or regions of a protein, or combinations thereof. In certain embodiments, the pathogen-associated antigen comprises a polypeptide at least about 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, or more amino acids in length. In certain embodiments, the pathogen-associated antigen comprises a polypeptide less than about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000 amino acids in length.
In such instances wherein the pathogen-associated antigen is not a polypeptide, the pathogen-associated antigen can be crosslinked to an ARC/endo-gag polypeptide using any suitable chemistry. In certain embodiments, the pathogen-associated antigen is coupled by a crosslinking reaction after VLP formation. The cross-linking reaction can be any suitable reaction, including but not limited to, an amine-to-amine, sulfhydryl-to-sulfhydryl, amine-to-sulfhydryl, carboxyl-to-amine, or sulfhydryl-to-carboxyl. In certain embodiments, the pathogen-associated antigen is coupled by the reaction of an NHS ester and a primary amine on the ARC/endo-gag polypeptide (e.g., a lysine residue). In certain embodiments, the pathogen-associated antigen is coupled by the reaction of an imidoester and a primary amine on the ARC/endo-gag polypeptide. In certain embodiments, the pathogen-associated antigen is coupled by the reaction of a maleimide group and a sulfhydryl group (e.g., cysteine residue) on the ARC/endo-gag polypeptide. In certain embodiments, the pathogen-associated antigen is coupled by the reaction of a maleimide group and a primary amine group on the ARC/endo-gag polypeptide. In certain embodiments, the pathogen-associated antigen is coupled by the reaction of a haloacetyl group and a sulfhydryl group on the ARC/endo-gag polypeptide. In certain embodiments, the pathogen-associated antigen is coupled by the reaction of a haloacetyl group and a primary amine group on the ARC/endo-gag polypeptide. In certain embodiments, the pathogen-associated antigen is coupled by the reaction of a pyridyldithiol and a sulfhydryl group on the ARC/endo-gag polypeptide. In certain embodiments, the pathogen-associated antigen is coupled by the reaction of a pyridyldithiol and a primary amine group on the ARC/endo-gag polypeptide. In certain embodiments, the pathogen-associated antigen is coupled by the reaction of a carbodiimide and a primary amine group on the ARC/endo-gag polypeptide. In certain embodiments, is coupled by a heterobifunctional linker. In certain embodiments, the pathogen-associated antigen is coupled by the reaction of a maleimide/hydrazide and a sulfhydryl group on the ARC/endo-gag polypeptide. In certain embodiments, the pathogen-associated antigen is coupled by the reaction of a pyridyldithiol /hydrazide and a sulfhydryl group on the ARC/endo-gag polypeptide. In certain embodiments, the crosslinker is a photoreactive crosslinker. In certain embodiments, the pathogen-associated antigen is coupled by a peptide linkage as a fusion protein. In certain embodiments, the pathogen-associated antigen is coupled by a peptide linkage N-terminal to the ARC/endo-gag polypeptide. In certain embodiments, the pathogen-associated antigen is coupled by a peptide linkage C-terminal to the ARC/endo-gag polypeptide. In certain embodiments, the pathogen-associated antigen and the ARC/endo-gag polypeptide are connected via a flexible polypeptide linker. In certain embodiments, the pathogen-associated antigen and the ARC/endo-gag polypeptide are connected via a flexible polypeptide linker, wherein the pathogen-associated antigen polypeptide is N-terminal to the ARC/endo-gag polypeptide. In certain embodiments, the pathogen-associated antigen and the ARC/endo-gag polypeptide are connected via a flexible polypeptide linker, wherein the pathogen-associated antigen polypeptide is C-terminal to the ARC/endo-gag polypeptide.
The ARC/endo-gag-VLP compositions comprise unlabeled or uncoupled ARC/endo-gag polypeptides included with adjuvant coupled ARC/endo-gag polypeptides and pathogen-associated antigen coupled ARC/endo-gag polypeptides as specific percentages. Percentages described herein are based upon a percentage of the ARC/endo-gag protein with the indicated coupling.
In certain embodiments, adjuvant coupled ARC/endo-gag is present at about 1% to about 10% in the ARC/endo-gag-VLP vaccine compositions. In certain embodiments, adjuvant coupled ARC/endo-gag is present at about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 1% to about 6%, about 1% to about 7%, about 1% to about 8%, about 1% to about 9%, about 1% to about 10%, about 2% to about 3%, about 2% to about 4%, about 2% to about 5%, about 2% to about 6%, about 2% to about 7%, about 2% to about 8%, about 2% to about 9%, about 2% to about 10 %, about 3% to about 4%, about 3% to about 5%, about 3% to about 6%, about 3% to about 7%, about 3% to about 8%, about 3% to about 9%, about 3% to about 10%, about 4% to about 5%, about 4% to about 6%, about 4% to about 7%, about 4% to about 8%, about 4% to about 9%, about 4% to about 10%, about 5% to about 6%, about 5% to about 7%, about 5% to about 8%, about 5% to about 9%, about 5% to about 10 %, about 6% to about 7%, about 6% to about 8%, about 6% to about 9%, about 6% to about 10%, about 7% to about 8%, about 7% to about 9%, about 7% to about 10%, about 8% to about 9%, about 8% to about 10%, or about 9% to about 10%. In certain embodiments, adjuvant coupled ARC/endo-gag is present at about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In certain embodiments, adjuvant coupled ARC/endo-gag is present at, at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, or about 9%. In certain embodiments, adjuvant coupled ARC/endo-gag is present at, at most about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.
In certain embodiments, adjuvant coupled ARC/endo-gag is present at about 5% to about 40% in the ARC/endo-gag-VLP vaccine compositions. In certain embodiments, adjuvant coupled ARC/endo-gag is present at about 5% to about 10%, about 5% to about 15%, about 5% to about 20%, about 5% to about 25%, about 5% to about 30%, about 5% to about 35%, about 5% to about 40%, about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 30%, about 10% to about 35%, about 10% to about 40 %, about 15% to about 20%, about 15% to about 25%, about 15% to about 30 %, about 15% to about 35%, about 15% to about 40%, about 20% to about 25%, about 20% to about 30 %, about 20% to about 35%, about 20% to about 40%, about 25% to about 30%, about 25% to about 35%, about 25% to about 40%, about 30% to about 35%, about 30% to about 40%, or about 35% to about 40%. In certain embodiments, adjuvant coupled ARC/endo-gag is present at about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40%. In certain embodiments, adjuvant coupled ARC/endo-gag is present at, at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%. In certain embodiments, adjuvant coupled ARC/endo-gag is present at, at most about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40%.
In certain embodiments, adjuvant coupled ARC/endo-gag is present at less than 2% in the ARC/endo-gag-VLP vaccine compositions. In certain embodiments the adjuvant coupled ARC/endo-gag is present at about 0.01% to about 2%, about 0.01% to about 1.5%, about 0.1% to about 1.5%, about 0.2% to about 1%, about 0.2% to about 0.5%, about 0.01 % to about 1.0%, about 0.01% to about 0.5%, about 0.01% to about 0.25%, about 0.01% to about 0.1%. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, about 15, about 20, about 25 or about 30 copies of an adjuvant coupled ARC/endo-gag polypeptide are present in an ARC/endo-gag-VLP vaccine particle. In certain embodiments, the ARC/endo-gag-VLP vaccine compositions contain no adjuvant coupled ARC/endo-gag.
In certain embodiments, pathogen-associated antigen coupled ARC/endo-gag is present at about 1% to about 10% in the ARC/endo-gag-VLP vaccine compositions. In certain embodiments, pathogen-associated antigen coupled ARC/endo-gag is present at about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 1% to about 6%, about 1% to about 7%, about 1% to about 8%, about 1% to about 9%, about 1% to about 10%, about 2% to about 3%, about 2% to about 4%, about 2% to about 5%, about 2% to about 6%, about 2% to about 7%, about 2% to about 8%, about 2% to about 9%, about 2% to about 10 %, about 3% to about 4%, about 3% to about 5%, about 3% to about 6%, about 3% to about 7%, about 3% to about 8%, about 3% to about 9%, about 3% to about 10%, about 4% to about 5%, about 4% to about 6%, about 4% to about 7%, about 4% to about 8%, about 4% to about 9%, about 4% to about 10%, about 5% to about 6%, about 5% to about 7%, about 5% to about 8%, about 5% to about 9%, about 5% to about 10 %, about 6% to about 7%, about 6% to about 8%, about 6% to about 9%, about 6% to about 10%, about 7% to about 8%, about 7% to about 9%, about 7% to about 10%, about 8% to about 9%, about 8% to about 10%, or about 9% to about 10%. In certain embodiments, pathogen-associated antigen coupled ARC/endo-gag is present at about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In certain embodiments, pathogen-associated antigen coupled ARC/endo-gag is present at, at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, or about 9%. In certain embodiments, pathogen-associated antigen coupled ARC/endo-gag is present at, at most about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.
In certain embodiments, pathogen-associated antigen coupled ARC/endo-gag is present at about 5% to about 40% in the ARC/endo-gag-VLP vaccine compositions. In certain embodiments, pathogen-associated antigen coupled ARC/endo-gag is present at about 5% to about 10%, about 5% to about 15%, about 5% to about 20%, about 5% to about 25%, about 5% to about 30%, about 5% to about 35%, about 5% to about 40%, about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 30%, about 10% to about 35%, about 10% to about 40 %, about 15% to about 20%, about 15% to about 25%, about 15% to about 30 %, about 15% to about 35%, about 15% to about 40%, about 20% to about 25%, about 20% to about 30 %, about 20% to about 35%, about 20% to about 40%, about 25% to about 30%, about 25% to about 35%, about 25% to about 40%, about 30% to about 35%, about 30% to about 40%, or about 35% to about 40%. In certain embodiments, pathogen-associated antigen coupled ARC/endo-gag is present at about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40%. In certain embodiments, pathogen-associated antigen coupled ARC/endo-gag is present at, at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%. In certain embodiments, pathogen-associated antigen coupled ARC/endo-gag is present at, at most about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40%.
In certain embodiments, pathogen-associated antigen coupled ARC/endo-gag is present at about 30% to 100% in the ARC/endo-gag-VLP vaccine compositions. In certain embodiments, pathogen-associated antigen coupled ARC/endo-gag is present at about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95%, about 95% to 100%. In certain embodiments, pathogen-associated antigen coupled ARC/endo-gag is present at about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In certain embodiments, pathogen-associated antigen coupled ARC/endo-gag is present at, at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 98%. In certain embodiments, pathogen-associated antigen coupled ARC/endo-gag is present at, at most about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 98%.
In certain embodiments, pathogen-associated antigen coupled ARC/endo-gag is present at less than 2% in the ARC/endo-gag-VLP vaccine compositions. In certain embodiments the pathogen-associated antigen coupled ARC/endo-gag is present at about 0.01% to about 2%, about 0.01% to about 1.5%, about 0.1% to about 1.5%, about 0.2% to about 1%, about 0.2% to about 0.5%, about 0.01% to about 1.0%, about 0.01% to about 0.5%, about 0.01% to about 0.25%, about 0.01% to about 0.1%. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, about 15, about 20, about 25 or about 30 copies of a pathogen-associated antigen coupled ARC/endo-gag polypeptide are present in an ARC/endo-gag-VLP vaccine particle. In certain embodiments, the ARC/endo-gag-VLP vaccine compositions contain no pathogen-associated antigen coupled ARC/endo-gag.
In certain embodiments, uncoupled ARC/endo-gag is present at about 90% to about 99% in the ARC/endo-gag-VLP vaccine compositions. In certain embodiments, uncoupled ARC/endo-gag is present at about 90% to about 91%, about 90% to about 92%, about 90% to about 93%, about 90% to about 94%, about 90% to about 95%, about 90% to about 96%, about 90% to about 97%, about 90% to about 98%, about 90% to about 99%, about 91% to about 92%, about 91% to about 93%, about 91% to about 94%, about 91% to about 95%, about 91% to about 96%, about 91% to about 97%, about 91% to about 98%, about 91% to about 99%, about 92% to about 93%, about 92% to about 94%, about 92% to about 95%, about 92% to about 96%, about 92% to about 97%, about 92% to about 98%, about 92% to about 99%, about 93% to about 94%, about 93% to about 95%, about 93% to about 96%, about 93% to about 97%, about 93% to about 98%, about 93% to about 99%, about 94% to about 95%, about 94% to about 96%, about 94% to about 97%, about 94% to about 98%, about 94% to about 99%, about 95% to about 96%, about 95% to about 97%, about 95% to about 98%, about 95% to about 99%, about 96% to about 97%, about 96% to about 98%, about 96% to about 99%, about 97% to about 98%, about 97% to about 99%, or about 98% to about 99%. In certain embodiments, uncoupled ARC/endo-gag is present at about 90 %, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%. In certain embodiments, uncoupled ARC/endo-gag is present at, at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, or about 98%. In certain embodiments, uncoupled ARC/endo-gag is present at, at most about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.
In certain embodiments, uncoupled ARC/endo-gag is present at about 60% to about 90% in the ARC/endo-gag-VLP vaccine compositions. In certain embodiments, uncoupled ARC/endo-gag is present at about 60% to about 65%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 65% to about 70%, about 65% to about 75%, about 65% to about 80%, about 65% to about 85%, about 65% to about 90%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 80% to about 85%, about 80% to about 90%, or about 85% to about 90%. In certain embodiments, uncoupled ARC/endo-gag is present at about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90%. In certain embodiments, uncoupled ARC/endo-gag is present at, at least about 60%, about 65%, about 70%, about 75%, about 80%, or about 85%. In certain embodiments, uncoupled ARC/endo-gag is present at, at most about 65%, about 70%, about 75%, about 80%, about 85%, or about 90%.
In certain embodiments, an ARC/endo-gag-VLP vaccine comprises about 80% uncoupled ARC/endo-gag, about 10% ARC/endo-gag coupled to adjuvant, and about 10% ARC/endo-gag coupled to a pathogen-associated antigen. In certain embodiments, an ARC/endo-gag-VLP vaccine comprises about 80% uncoupled ARC/endo-gag, about 15% ARC/endo-gag coupled to adjuvant, and about 5% ARC/endo-gag coupled to a pathogen-associated antigen. In certain embodiments, an ARC/endo-gag-VLP vaccine comprises about 80% uncoupled ARC/endo-gag, about 5% ARC/endo-gag coupled to adjuvant, and about 15% ARC/endo-gag coupled to a pathogen-associated antigen. In certain embodiments, an ARC/endo-gag-VLP vaccine comprises about 80% uncoupled ARC/endo-gag, about 1% ARC/endo-gag coupled to adjuvant, and about 19% ARC/endo-gag coupled to a pathogen-associated antigen. In certain embodiments, an ARC/endo-gag-VLP vaccine comprises about 80% uncoupled ARC/endo-gag, about 2% ARC/endo-gag coupled to adjuvant, and about 18% ARC/endo-gag coupled to a pathogen-associated antigen. In certain embodiments, an ARC/endo-gag-VLP vaccine comprises about 80% uncoupled ARC/endo-gag, about 3% ARC/endo-gag coupled to adjuvant, and about 17% ARC/endo-gag coupled to a pathogen-associated antigen. In certain embodiments, an ARC/endo-gag-VLP vaccine comprises about 80% uncoupled ARC/endo-gag, about 4% ARC/endo-gag coupled to adjuvant, and about 16% ARC/endo-gag coupled to a pathogen-associated antigen. In certain embodiments, an ARC/endo-gag-VLP vaccine comprises about 90% uncoupled ARC/endo-gag, about 5% ARC/endo-gag coupled to adjuvant, and about 5% ARC/endo-gag coupled to a pathogen-associated antigen. In certain embodiments, an ARC/endo-gag-VLP vaccine comprises about 90% uncoupled ARC/endo-gag, about 1% ARC/endo-gag coupled to adjuvant, and about 9% ARC/endo-gag coupled to a pathogen-associated antigen. In certain embodiments, an ARC/endo-gag-VLP vaccine comprises about 90% uncoupled ARC/endo-gag, about 2% ARC/endo-gag coupled to adjuvant, and about 8% ARC/endo-gag coupled to a pathogen-associated antigen. In certain embodiments, an ARC/endo-gag-VLP vaccine comprises about 90% uncoupled ARC/endo-gag, about 3% ARC/endo-gag coupled to adjuvant, and about 7% ARC/endo-gag coupled to a pathogen-associated antigen. In certain embodiments, an ARC/endo-gag-VLP vaccine comprises about 90% uncoupled ARC/endo-gag, about 4% ARC/endo-gag coupled to adjuvant, and about 6% ARC/endo-gag coupled to a pathogen-associated antigen.
The ARC polypeptides of the VLPs described herein in certain embodiments, comprise a human ARC polypeptide. In certain embodiments, the ARC polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% identical to SEQ ID NO: 1. In certain embodiments, the ARC polypeptide comprises an amino acid sequence identical to SEQ ID NO: 1. In certain embodiments, the ARC polypeptide comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids from the N- or C-terminus of the ARC polypeptide.
In certain embodiments, the polypeptide of the VLP is an endo-gag. The endo-gag polypeptide may be any endo-gag polypeptide encoded by the genome of a cell, whether expressed or non-expressed. In some embodiments, the endo-gag has a deletion of the N-terminal methionine. In some embodiments, the endo-gag has an insertion of one or more amino acid residues at the N-terminus, for example, insertion of a glycine. In some embodiments, the N-terminal methionine of the endo-gag is replaced with another amino acid residue, for example, glycine.
In certain embodiments, the polypeptide of the VLP is an endo-gag. In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% identical to SEQ ID NO: 16. In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence identical to SEQ ID NO: 16. In certain embodiments, the endo-gag comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids from the N- or C-terminus of the endo-gag.
In certain embodiments, the polypeptide of the VLP is an Endogenous Gag (Endo-gag). In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% identical to SEQ ID NO: 17. In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence identical to SEQ ID NO: 17. In certain embodiments, the endo-gag comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids from the N- or C-terminus of the endo-gag.
In certain embodiments, the polypeptide of the VLP is an Endogenous Gag (Endo-gag). In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% identical to SEQ ID NO: 18. In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence identical to SEQ ID NO: 18. In certain embodiments, the endo-gag comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids from the N- or C-terminus of the endo-gag.
In certain embodiments, the polypeptide of the VLP is an Endogenous Gag (Endo-gag). In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% identical to SEQ ID NO: 19. In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence identical to SEQ ID NO: 19. In certain embodiments, the endo-gag comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids from the N- or C-terminus of the endo-gag.
In certain embodiments, the polypeptide of the VLP is an Endogenous Gag (Endo-gag). In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% identical to SEQ ID NO: 20. In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence identical to SEQ ID NO: 20. In certain embodiments, the endo-gag comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids from the N- or C-terminus of the endo-gag
In certain embodiments, the polypeptide of the VLP is an Endogenous Gag (Endo-gag). In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% identical to SEQ ID NO: 21. In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence identical to SEQ ID NO: 21. In certain embodiments, the endo-gag comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids from the N- or C-terminus of the endo-gag.
In certain embodiments, the polypeptide of the VLP is an Endogenous Gag (Endo-gag). In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% identical to SEQ ID NO: 22. In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence identical to SEQ ID NO: 22. In certain embodiments, the endo-gag comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids from the N- or C-terminus of the endo-gag.
In certain embodiments, the polypeptide of the VLP is an Endogenous Gag (Endo-gag). In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% identical to SEQ ID NO: 23. In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence identical to SEQ ID NO: 23. In certain embodiments, the endo-gag comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids from the N- or C-terminus of the endo-gag.
In certain embodiments, the polypeptide of the VLP is an Endogenous Gag (Endo-gag). In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% identical to SEQ ID NO: 24. In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence identical to SEQ ID NO: 24. In certain embodiments, the endo-gag comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids from the N- or C-terminus of the endo-gag.
In certain embodiments, the polypeptide of the VLP is an Endogenous Gag (Endo-gag). In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% identical to SEQ ID NO: 25. In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence identical to SEQ ID NO: 25. In certain embodiments, the endo-gag comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids from the N- or C-terminus of the endo-gag.
In certain embodiments, the polypeptide of the VLP is an Endogenous Gag (Endo-gag). In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% identical to SEQ ID NO: 26. In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence identical to SEQ ID NO: 26. In certain embodiments, the endo-gag comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids from the N- or C-terminus of the endo-gag.
In certain embodiments, the polypeptide of the VLP is an Endogenous Gag (Endo-gag). In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% identical to SEQ ID NO: 27. In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence identical to SEQ ID NO: 27. In certain embodiments, the endo-gag comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids from the N- or C-terminus of the endo-gag.
In certain embodiments, the polypeptide of the VLP is an Endogenous Gag (Endo-gag). In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% identical to SEQ ID NO: 28. In certain embodiments, the endo-gag polypeptide comprises an amino acid sequence identical to SEQ ID NO: 28. In certain embodiments, the endo-gag comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids from the N- or C-terminus of the endo-gag.
The VLPs may comprise a single type of ARC or endo-gag polypeptide or a mixture of any 1, 2, 3, 4, 5, or more ARC/endo-gag polypeptides selected from any of SEQ ID NOs: 1, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28.
AdjuvantsThe ARC/endo-gag compositions described herein further comprise an adjuvant. Adjuvants allow for activation of the immune system and can act specifically through specific innate immune signaling molecules (Toll-like receptors, RIG receptors, NOD receptors, etc.) or non-specifically (e.g., water-in-oil emulsions, aluminum compounds). The adjuvants used in the compositions are, in certain embodiments, not conjugated to an ARC/endo-gag polypeptide, but mixed with the ARC/end-gag VLPs in an injectable form. In other embodiments, the adjuvants are coupled to a certain amount or percentage of VLP ARC/endo-gag polypeptides. Such coupling can be covalent or non-covalent. When the coupling is covalent, the coupling can be through a peptide bond (e.g., a fusion protein) or a non-peptide bond conjugation.
The adjuvant polypeptides described herein, are in certain embodiments, coupled to ARC/endo-gag peptides as a fusion protein. In certain embodiments, the adjuvant polypeptide is fused to the N-terminus of an ARC/end-gag polypeptide; the fusion optionally comprises a flexible polypeptide linker between the adjuvant polypeptide and the ARC/endo-gag polypeptide. In certain embodiments, the adjuvant polypeptide is fused to the C-terminus of an ARC/end-gag polypeptide; the fusion optionally comprises a flexible polypeptide linker between the adjuvant polypeptide and the ARC/endo-gag polypeptide.
Adjuvant polypeptides or adjuvant non-polypeptides (e.g., nucleic acids, bacterial compounds) are, in certain embodiments, covalently coupled or cross-linked by non-peptide linkages. In certain embodiments, the adjuvant polypeptide is coupled to an ARC/endo-gag polypeptide by a primary amine (e.g., lysine or N-terminus of the polypeptide chain), a sulfhydryl (e.g., cysteine), a carboxyl (e.g., aspartic acid, glutamic acid or C-terminus of the polypeptide chain). The cross-linking reaction can be any suitable reaction, including but not limited to, an amine-to-amine, sulfhydryl-to-sulfhydryl, amine-to-sulfhydryl, carboxyl-to-amine, or sulfhydryl-to-carboxyl. In certain embodiments, the adjuvant is coupled by the reaction of an NHS ester and a primary amine on the ARC/endo-gag polypeptide (e.g., a lysine residue). In certain embodiments, the adjuvant is coupled by the reaction of an imidoester and a primary amine on the ARC/endo-gag polypeptide. In certain embodiments, the adjuvant is coupled by the reaction of a maleimide group and a sulfhydryl group (e.g., cysteine residue) on the ARC/endo-gag polypeptide. In certain embodiments, the adjuvant is coupled by the reaction of a maleimide group and a primary amine group on the ARC/endo-gag polypeptide. In certain embodiments, the adjuvant is coupled by the reaction of a haloacetyl group and a sulfhydryl group on the ARC/endo-gag polypeptide. In certain embodiments, the adjuvant is coupled by the reaction of a haloacetyl group and a primary amine group on the ARC/endo-gag polypeptide. In certain embodiments, the adjuvant is coupled by the reaction of a pyridyldithiol and a sulfhydryl group on the ARC/endo-gag polypeptide. In certain embodiments, the adjuvant is coupled by the reaction of a pyridyldithiol and a primary amine group on the ARC/endo-gag polypeptide. In certain embodiments, the adjuvant is coupled by the reaction of a carbodiimide and a primary amine group on the ARC/endo-gag polypeptide. In certain embodiments, the adjuvant is coupled by a heterobifunctional linker. In certain embodiments, the adjuvant is coupled by the reaction of a maleimide/hydrazide and a sulfhydryl group on the ARC/endo-gag polypeptide. In certain embodiments, the adjuvant is coupled by the reaction of a pyridyldithiol/hydrazide and a sulfhydryl group on the ARC/endo-gag polypeptide. In certain embodiments, the crosslinker is a photoreactive crosslinker. For such conjugations, VLPs may be formed beforehand, and appropriate coupling reactions are carried out after VLP formation. Alternatively, the ARC/endo-gag polypeptides may be coupled with adjuvant before formation of VLPs.
An adjuvant according to the methods of this disclosure can be a pathogen-associated molecular pattern (PAMP) or a synthetic version thereof. PAMPs are small molecules conserved within a class of microbes and include, without limitation, glycans, glycol-conjugations, bacterial flagellin, lipoteichoic acid, peptidoglycan, CpG oligonucleotides, and double-stranded RNA. PAMPs activate a variety of innate immune receptors, known as pattern recognition receptors, expressed in antigen-presenting cells and initiate adaptive immune response attributable to B and T cells. Dendritic cells and other antigen-presenting cells express a variety of pattern recognition receptors and are activated in response to their binding to PAMPs. Pattern recognition receptors include, without limitation, Toll-like receptors, NOD-like receptors, RIG-1 receptors, MDA-5 receptors, and the STING pathway. In one embodiment, the dendritic cell-activating molecule activates dendritic cell sthrough a Toll-like receptor, a NOD-like receptor, a RIG-1 or MDA-5 receptor, a C-type lectin receptor, or the STING pathway.
Toll-like receptors are a class of receptors that are involved in the innate immune system. In a certain embodiment, the adjuvant activates a toll-like receptor. In another embodiment, the dendritic cell-activating molecule is a toll-like receptor agonist from the list consisting of a CpG oligonucleotide, SD-101, LFX453, imiquimod, Bacillus Calmette-Guerin (BCG), Poly ICLC, GSK1795091, and combinations thereof.
NOD-like receptors are a class of pattern recognition receptors found intracellularly in antigen-presenting cells that bind PAMPs and play a role in the innate immune system. In certain embodiments, the adjuvant activates a NOD-like receptor. In another embodiment, the dendritic cell activating-molecule is a NOD-like receptor agonist selected from the list consisting of bacterial peptidoglycan, an acylated derivative of iE-DAP (C12-iE-DAP), D-gamma-Glu-mDAP (iE-DAP), L-Ala-gamma-D-Glu-mDAP (Tri-DAP), muramyl dipeptide (MDP), muramyl tripeptide, L18-MDP, M-TriDAP, murabutide, PGN-ECndi, PGN-ECndss, PGN-SAndi, N-glycolylated muramyl dipeptide, murabutide, and combinations thereof.
RIG-1 and MDA-5 receptors also recognize PAMPs. Specifically, both RIG-1 receptors and MDA-5 receptors are involved in the recognition of viruses by the innate immune system. RIG-1 receptors generally bind to single- or double-stranded RNA strands of less than 2000 base pairs, while MDA-5 receptors generally bind to virally-derived single or double RNA strands greater than 2000 base pairs. When activated, these receptors promote interferon signaling and other responses of the innate immune system. In certain embodiments, the adjuvant activates a RIG-1 or MDA5 receptor. In another embodiment, the dendritic cell-activating molecule is a RIG-1 or MDA-5 receptor agonist selected from the list consisting of poly(I:C), Poly(dA:dT), Poly(dG:dC), 3p-hpRNA, 5′ppp-dsRNA, and combinations thereof.
C-type lectin receptors are involved in recognition of PAMPs, particularly those derived from fungi and mycobacteria. When a PAMP binds to a C-type lectin receptor, the innate immune system is activated. In certain embodiments, the adjuvant activates a C-type lectin receptor. In another embodiment, the adjuvant is a C-type lectin receptor agonist selected from the list consisting of Beta-1,3-glucan, zymosan, heat-killed C. albicans, cord factor, Trehalose-6,6-dibehenate, and combinations thereof.
The STING pathway is involved in innate immunity and the detection of PAMPs. Activation of the STING pathway results in expression of type I interferon. In certain embodiments, the adjuvant activates the STING pathway. In certain embodiments, the adjuvant is a STING agonist selected from the list consisting of 2′,3′-cGAMP (CAS Number, 1441190-66-4), 4-[(2-Chloro-6-fluorophenyl)methyl]-N-(furan-2-ylmethyl)-3-oxo-1,4-benzothiazine-6-carboxamide, MK-1454, ADU-S100/MIW815, SRCB-0074, SYNB1891, E-7766, or SB11285, and combinations thereof.
CD40 is a TNF-family receptor expressed on dendritic cells and other antigen-presenting cells. CD40 signaling results in the expression of costimulatory ligands, cytokines, enhanced antigen presentation, and trafficking to the draining lymph node. In certain embodiments, the adjuvant comprises a CD40 agonist. In certain embodiments, the CD40 agonist is a CD40 agonistic antibody. Examples of CD40 agonist antibodies include, but are not limited to, dacetuzumab (also known as SGN-40, Seattle Genetics), CP-870,893 (University of Pennsylvania/Hoffmann-LaRoche), ADC-1013 (Alligator Bioscience AB), 2141-v11 (Rockefeller University), APX005M (Apexigen, Inc), Chi Lob 7/4 (Cancer research UKK), BG9588 (NIAMS), CFZ533 (Novartis), PG10 (PanGenetics UK Limited), BMS-986004 (Bristol-Myer Squibbs), lucatumumab (also known as HCD122, Novartis), HCD122 (Novartis), JNJ-64457107 (Janssen Research & Development), selicrelumab (also known as RO7009789, Hoffman-La Roche), ASKP1240 (Astellas Pharma Global Development), and SEA-CD40 (Seattle Genetics).
Immunity can be primed by cytokines and chemokines. Cytokines can control the maturation of immature dendritic cells and activate dendritic cells. In certain embodiments, the adjuvant promotes signaling or is an agonist of a cytokine receptor. In other embodiments, the adjuvant comprises a cytokine selected from the list consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-15 (IL-15), tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), and combinations thereof.
In certain embodiments, the adjuvant comprises a water-in-oil emulsion, such as the Montanide series of adjuvants, including but not limited to, ISA-25™, ISA-50™, ISA-51™, ISA-206™, ISA-720™, or SEPPIC™. Other water-in-oil emulsion adjuvants comprise complete Freund’s adjuvant, incomplete Freund’s adjuvant, MF59, squalene-water emulsions, OW-14, and combinations thereof.
Certain nucleic acids can act as adjuvants. In certain embodiments, the adjuvant comprises a nucleic acid. In certain embodiments, the nucleic acid is a DNA. In certain embodiments, the DNA comprises CPG-1018, CPG-1826, CPG-2007, CPG-2006, or any combination thereof. In certain embodiments, the nucleic acid is an RNA. In certain embodiments, the RNA comprises CV1802, Poly(U), Poly(I:C), ssRNA40, GFP RNA, RNA41, RNA42, RNA33, RNA35, 5′-phosphorylated blunt-ended viral genomic dsRNA <300bp, long dsRNA >1000bp, Genomic ssRNA, ssRNA40, or any combination thereof.
Aluminum compounds have been included in vaccine formulations as adjuvants. In certain embodiments, the adjuvant comprises an aluminum compound. In certain embodiments, the aluminum compound comprises alum.
Many polypeptides from a wide range of pathogens have been noted for their adjuvant effects. Therefore, in certain embodiments, the adjuvant comprises a polypeptide from a pathogen. In certain embodiments, the polypeptide is a polypeptide from Brucella abortus, Bordetella pertussis, Chlamydia trachomatis, Fusobacterium nucleatum, Mycobacterium tuberculosis, Neisseria meningitidis, Staphylococcus aureus, Shigella dysenteriae, Shigella flexneri, Streptococcus pneumoniae, Vibrio cholerae, Brucella abortus, Mycobacterium paratuberculosis, Neisseria meningitidis, Streptococcus pneumoniae, or any combination thereof.
In certain embodiments, the adjuvant polypeptide is from a protein selected from the list consisting of: BCSP31, FHA, MOMP, PorB, PVL, Porin, OmpA, 34 kDa MOMP, PepO, OmpU, Lumazine synthase, Omp16, Omp19, BCSP31, CobT, RpfE, Rv0652, HBHA, NhhA, DnaJ, Pneumolysin, ΔA146 Pneumolysin, or any combination thereof.
In certain embodiments, the adjuvant polypeptide is from a bacterial species and protein selected from the list consisting of: Brucella abortus (BCSP31), Bordetella pertussis (FHA), Chlamydia trachomatis (MOMP), Neisseria meningitidis (PorB), Staphylococcus aureus (PVL), Shigella dysenteriae (Porin), Shigella flexneri (OmpA, 34 kDa MOMP), Streptococcus pneumoniae (PepO), Vibrio cholerae (OmpU), Brucella abortus (Lumazine synthase, Omp16, Omp19, BCSP31), Mycobacterium paratuberculosis (CobT, RpfE, Rv0652, HBHA), Neisseria meningitidis (NhhA), Streptococcus pneumoniae (DnaJ, Pneumolysin, ΔA146 Pneumolysin), or any combination thereof.
In certain embodiments, the adjuvant polypeptide is from bacterial flagellin.
In certain embodiments, the adjuvant polypeptide is from human heat shock protein 70.
The VLPs described herein can comprise a single type of adjuvant or a mixture of different adjuvants. In certain embodiments, the ARC/endo-gag VLPs comprise a single type of adjuvant. In certain embodiments, the ARC/endo-gag VLPs comprise a plurality of different types of adjuvant. In certain embodiments, the ARC/endo-gag VLPs comprise two or more types of adjuvant. In certain embodiments, the ARC/endo-gag VLPs comprise three or more types of adjuvant. In certain embodiments, the ARC/endo-gag VLPs comprise four or more types of adjuvant. In certain embodiments, the ARC/endo-gag VLPs comprise five or more types of adjuvant. In certain embodiments, the ARC/endo-gag VLPs comprise two, three, four, five, six, or seven different types of adjuvant.
Pathogen-Associated AntigensThe ARC/endo-gag compositions described herein further comprise a pathogen-associated antigen (PAA). Immune responses directed against the pathogen-associated antigen allow for the generation of pathogen-specific adaptive immune responses that can serve to protect an individual from infection, symptoms, severe diseases, or mortality associated with the pathogen. In certain embodiments, the pathogen-associated antigens are coupled to a certain amount or percentage of the ARC/endo-gag polypeptides of a VLP. Such coupling can be covalent or non-covalent. When the coupling is covalent, the coupling can be through a peptide bond (e.g., fusion protein) or a non-peptide bond conjugation.
The pathogen-associated antigens described herein are, in certain embodiments, coupled to ARC/endo-gag peptides as a fusion protein. In certain embodiments, the pathogen-associated antigen is fused to the N-terminus of an ARC/end-gag polypeptide, and the fusion optionally comprises a flexible polypeptide linker between the adjuvant polypeptide and the ARC/endo-gag polypeptide. In certain embodiments, the pathogen-associated antigen is fused to the C-terminus of an ARC/end-gag polypeptide, and the fusion optionally comprises a flexible polypeptide linker between the pathogen-associated antigen and the ARC/endo-gag polypeptide.
Pathogen-associated antigens are, in certain embodiments, covalently coupled by non-peptide linkages. In certain embodiments, the pathogen-associated antigen is coupled to an ARC/endo-gag polypeptide by a primary amine (e.g., lysine or N-terminus of the polypeptide chain), a sulfhydryl (e.g., cysteine), a carboxyl (e.g., aspartic acid, glutamic acid or C-terminus of the polypeptide chain). For such conjugations, VLPs may be formed beforehand, and appropriate coupling reactions are carried out after VLP formation. Alternatively, the ARC/endo-gag polypeptides may be coupled with pathogen-associated antigen before the formation of VLPs.
The pathogen-associated antigens can comprise full proteins derived from a pathogen or fragments of polypeptides derived from a pathogen. Full-proteins or large polypeptide fragments of proteins (> 40 amino acids in length) can be used in the ARC/endo-gag polypeptides. Without being bound by theory, large polypeptides and shorter polypeptides may be phagocytosed or otherwise internalized and processed to be presented to T cells by the MHC molecules on antigen-presenting cells such as macrophages, dendritic cells, or B cells, leading to initiation of an immune response. Such an immune response results in an adaptive immune response comprising an antibody response, a cellular immune response, or a combination thereof. In certain embodiments, the ARC/endo-gag VLPs generate an antibody response, as indicated by pathogen-associated antigen-specific antibodies. In certain embodiments, the antibodies comprise IgG isotype antibodies. IgG antibodies are indicative of the establishment of immunological memory and are generally higher affinity than IgM antibodies. In certain embodiments, the ARC/endo-gag VLPs generate a cellular immune response represented by the generation of pathogen-associated antigen-specific helper T cells or pathogen-associated antigen-specific cytotoxic T cells.
In certain embodiments, the pathogen-associated antigen is configured to bind an MHC class I protein. In certain embodiments, the MHC class I is a human MHC class I. In certain embodiments, the pathogen-associated antigen is a polypeptide at least 8 amino acids in length. In certain embodiments, the pathogen-associated antigen is a polypeptide that is between 8 and 11 amino acids in length. In certain embodiments, the pathogen-associated antigen is a polypeptide that is 8, 9, 10, or 11 amino acids in length.
In certain embodiments, the pathogen-associated antigen is configured to bind an MHC class II protein. In certain embodiments, the MHC class II is a human MHC class II. In certain embodiments, the pathogen-associated antigen is a polypeptide at least 10 amino acids in length. In certain embodiments, the pathogen-associated antigen is a polypeptide that is between 10 and 40 amino acids in length. In certain embodiments, the pathogen-associated antigen is a polypeptide that is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length.
In certain embodiments, the pathogen-associated antigen is large polypeptide greater than about 40 amino acids in length, which may contain a plurality of antigens capable of binding MHC class I or MHC class II. Such polypeptides can be from pathogen-derived antigens or polypeptides known to be immunogenic.
In certain embodiments, the pathogen-associated antigen comprises or consists of about 40 amino acids to about 1,000 amino acids. In certain embodiments, the pathogen-associated antigen comprises or consists of about 40 amino acids to about 50 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 300 amino acids, about 40 amino acids to about 400 amino acids, about 40 amino acids to about 500 amino acids, about 40 amino acids to about 750 amino acids, about 40 amino acids to about 1,000 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 750 amino acids, about 50 amino acids to about 1,000 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 1,000 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 1,000 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 750 amino acids, about 300 amino acids to about 1,000 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about 1,000 amino acids, about 500 amino acids to about 750 amino acids, about 500 amino acids to about 1,000 amino acids, or about 750 amino acids to about 1,000 amino acids. In certain embodiments, the pathogen-associated antigen comprises or consists of about 40 amino acids, about 50 amino acids, about 100 amino acids, about 200 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, about 750 amino acids, or about 1,000 amino acids. In certain embodiments, the pathogen-associated antigen comprises or consists of at least about 40 amino acids, about 50 amino acids, about 100 amino acids, about 200 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, or about 750 amino acids. In certain embodiments, the pathogen-associated antigen comprises or consists of at most about 50 amino acids, about 100 amino acids, about 200 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, about 750 amino acids, or about 1,000 amino acids.
In certain embodiments, the pathogen-associated antigen is from a virus, a bacterium, a fungus, or a parasite.
In certain embodiments, the pathogen-associated antigen is from a bacterium, including without limitation a Streptococcus, a Pseudomonas, a Shigella, a Campylobacter, a Salmonella, a Clostridium, an Escherichia, or Bacillus Anthracis.
In certain embodiments, the pathogen-associated antigen is from a pathogen that causes human disease which include but are not limited to, Bacillus anthracis (anthrax), Clostridium botulinum toxin (botulism), Yersinia pestis (plague). Variola major (smallpox) and other related pox viruses, Francisella tularensis (tularemia), Viral hemorrhagic fevers, Arenaviruses, (e.g., Junin, Machupo, Guanarito, Chapare, Lassa, and/or Lujo), Bunyaviruses (e.g., Hantaviruses causing Hanta Pulmonary syndrome, Rift Valley Fever, and/or Crimean Congo Hemorrhagic Fever), Flaviviruses, Dengue, Filoviruses (e.g., Ebola and Marburg viruses), Burkholderia pseudomallei (melioidosis), Coxiella burnetii (Q fever), Brucella species (brucellosis), Burkholderia mallei (glanders), Chlamydia psittaci (Psittacosis), Ricin toxin (Ricinus communis), Epsilon toxin (Clostridium perfringens), Staphylococcus enterotoxin B (SEB), Typhus fever (Rickettsia prowazekii), Food- and waterborne pathogens, Diarrheagenic E.coli , Pathogenic Vibrios, Shigella species, Salmonella, Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica, Caliciviruses, Hepatitis A, Cryptosporidium parvum, Cyclospora cayatanensis, Giardia lamblia,Entamoeba histolytica, Toxoplasma gondii, Naegleria fowleri, Balamuthia mandrillaris, Fungi, Microsporidia, Mosquito-borne viruses (e.g., West Nile virus (WNV), LaCrosse encephalitis (LACV), California encephalitis, Venezuelan equine encephalitis (VEE), Eastern equine encephalitis (EEE), Western equine encephalitis (WEE), Japanese encephalitis virus (JE), St. Louis encephalitis virus (SLEV), Yellow fever virus (YFV), Chikungunya virus, Zika virus, Nipah and Hendra viruses, Additional hantaviruses, Tickborne hemorrhagic fever viruses, Bunyaviruses, Severe Fever with Thrombocytopenia Syndrome virus (SFTSV), Heartland virus, Flaviviruses (e.g., Omsk Hemorrhagic Fever virus, Alkhurma virus, Kyasanur Forest virus), Tickborne encephalitis complex flaviviruses, Tickborne encephalitis viruses, Powassan/Deer Tick virus, Tuberculosis, including drug-resistant Tuberculosis, Influenza virus, Prions, Streptococcus, Pseudomonas, Shigella, Campylobacter, Salmonella, Clostridium, Escherichia, Hepatitis C, papillomavirus, Epstein-Barr virus, varicella, variola, Orthomyxovirus, Severe acute respiratory syndrome associated coronavirus (SARS-CoV), SARS-CoV-2 (COVID-19), MERS-CoV, other highly pathogenic human coronaviruses, or any combination thereof.
In certain embodiments, the virus is a respiratory virus that primarily results in respiratory symptoms including, without limitation, coronaviruses, influenza viruses, adenoviruses, rhinoviruses, coxsackieviruses, and metapneumoviruseses. In certain embodiments, the virus is an enteric virus that primarily results in digestive symptoms including, without limitation, enteroviruses, noroviruses, heptoviruses, reoviruses, rotaviruses, parvoviruses, toroviruses, and mastadenovirus. In certain embodiments, the virus is a hemorrhagic fever virus including, without limitation, Ebola virus, Marburg virus, dengue fever virus, yellow fever virus, Rift valley fever virus, hanta virus, and Lassa fever virus.
In certain embodiments, the pathogen-associated antigen is from an influenza virus. In certain embodiments, the pathogen-associated antigen is from an influenza A virus, such as the H5N1 strain. In certain embodiments, the pathogen-associated antigen is from an influenza B virus. In certain embodiments, the pathogen-associated antigen is an influenza matrix M1 protein or a fragment thereof. In certain embodiments, the pathogen-associated antigen is an influenza neuraminidase or a fragment thereof. In certain embodiments, the pathogen-associated antigen is an influenza hemagglutinin or a fragment thereof. For example, the pathogen-associated antigen may comprise an entire hemagglutinin, an HA1 domain, an HA2 domain or any antigenic portion thereof.
In certain embodiments, the pathogen-associated antigen is a Coronaviridae antigen. In certain embodiments, the Coronaviridae exhibits human tropism. In certain embodiments, the Coronaviridae is selected from the list consisting of SARS Coronavirus (SARS-CoV-1), COVID-19 (SARS-CoV-2), MERS-coronavirus (MERS-CoV), or any combination thereof. In certain embodiments, the Coronaviridae comprises SARS Coronavirus (SARS-CoV-1). In certain embodiments, the Coronaviridae comprises COVID-19 (SARS-CoV-2). In certain embodiments, the Coronaviridae comprises MERS-coronavirus (MERS-CoV). In certain embodiments, the Coronaviridae antigen comprises a spike protein, an envelope protein, a nucleocapsid protein, a membrane protein, a membrane glycoprotein, or a non-structural protein. In certain embodiments, the Coronaviridae antigen comprises a spike protein, an envelope small membrane protein, a membrane protein, a non-structural protein 6 (NSP6), a nucleoprotein, an ORF 10 protein, Protein 3a, Protein7a, Protein 9b, structural protein 8, uncharacterized protein 4, or any combination thereof. In certain embodiments, the Coronaviridae antigen comprises an amino acid residue sequence as set forth in any one of SEQ ID NOs: 30 to 42, and combinations thereof. In certain embodiments, the Coronaviridae antigen consists of an amino acid residue sequence as set forth in any one of SEQ ID NOs: 30 to 42, and combinations thereof. In certain embodiments, the Coronaviridae antigen comprises a spike protein comprising SEQ ID NO: 43 or SEQ ID NO: 44, or a fragment thereof
The VLPs described herein can comprise a single type of pathogen-associated antigen or a mixture of different pathogen-associated antigens. In certain embodiments, the ARC/endo-gag VLPs comprise a single pathogen-associated antigen. In certain embodiments, the ARC/endo-gag VLPs comprise a plurality of different pathogen-associated antigens. In certain embodiments, the ARC/endo-gag VLPs comprise two or more pathogen-associated antigens, wherein the two or more pathogen-associated antigens are from the same pathogen. In certain embodiments, the ARC/endo-gag VLPs comprise two or more pathogen-associated antigens, wherein the two or more pathogen-associated antigens are from a different pathogen. In certain embodiments, the ARC/endo-gag VLPs comprise three or more pathogen-associated antigens, wherein the three or more pathogen-associated antigens are from the same pathogen. In certain embodiments, the ARC/endo-gag VLPs comprise four or more pathogen-associated antigens, wherein the four or more pathogen-associated antigens are from the same pathogen. In certain embodiments, the ARC/endo-gag VLPs comprise five or more pathogen-associated antigens, wherein the five or more pathogen-associated antigens are from the same pathogen. In certain embodiments, the ARC/endo-gag VLPs comprise two, three, four, five, six, or seven different pathogen-associated antigens, wherein the pathogen-associated antigens are from the same pathogen.
Methods of MakingThe VLP vaccine compositions comprising ARC polypeptides, endo-gag polypeptides, or a mixture thereof are assembled from ARC or endo-gag polypeptides that are encoded by nucleic acid vectors that have been transferred into a suitable host cell.
In certain embodiments, the Arc polypeptides, endo-Gag polypeptides, engineered Arc polypeptides, and engineered endo-Gag polypeptides described herein are encoded by plasmid vectors. In some embodiments, vectors include any suitable vectors derived from either a eukaryotic or prokaryotic source. In some cases, vectors are obtained from bacteria (e.g., E. coli), insects, yeast (e.g., Pichia pastoris), algae, or mammalian sources. Exemplary bacterial vectors include pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA3 1-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.Exemplary insect vectors include pFastBacl, pFastBac DETAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, FLAG vectors such as pPolh-FLAGl or pPolh-MAT 2, or MAT vectors such as pPolh-MATl, or pPolh-MAT2. In some cases, yeast vectors include Gateway® pDEST™ 14 vector, Gateway® pDEST™ 15 vector, Gateway® pDEST™ 17 vector, Gateway® pDEST™ 24 vector, Gateway® pYES-DEST52 vector, pBAD-DEST49 Gateway® destination vector, pA0815 Pichia vector, pFLDl Pichi pastoris vector, pGAPZA,B, & C Pichia pastoris vector, pPIC3.5K Pichia vector, pPIC6 A, B, & C Pichia vector, pPIC9K Pichia vector, pTEFl/Zeo, pYES2 yeast vector, pYES2/CT yeast vector, pYES2/NT A, B, & C yeast vector, or pYES3/CT yeast vector. Exemplary algae vectors include pChlamy-4 vector or MCS vector. Examples of mammalian vectors include transient expression vectors or stable expression vectors. Mammalian transient expression vectors include p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1, pFLAG-CMV 5a,b,c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. Mammalian stable expression vector include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.
In some embodiments, a host cell includes any suitable cell, such as a naturally derived cell or a genetically modified cell. In some instances, a host cell is a production host cell. In some instances, a host cell is a eukaryotic cell. In other instances, a host cell is a prokaryotic cell. In some cases, a eukaryotic cell includes fungi (e.g., a yeast cell), an animal cell, or a plant cell. In some cases, a prokaryotic cell is a bacterial cell. Examples of a bacterial cell include gram-positive bacteria or gram-negative bacteria. In some embodiments, the gram-negative bacterium is anaerobic, rod-shaped, or both. In some instances, gram-positive bacteria include Actinobacteria, Firmicutes or Tenericutes. In some cases, gram-negative bacteria include Aquifwae, Deinococcus-Thermus, Fibrobacteres ChlorobilBacteroidetes (FCB group), Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes Verrucomicrobial Chlamydiae (PVC group), Proteobacteria, Spirochaetes or Synergistetes. In some embodiments, the bacterium is Acidobacteria, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Dictyoglomi, Thermodesulfobacteria, or Thermotogae. In some embodiments, a bacterial cell is Escherichia coli, Clostridium botulinum, or Coli bacilli. Exemplary prokaryotic host cells include, but are not limited to, BL21, Maehl™, DH10B™, TOP10, DH5a, DHlOBac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F’, INVaF, TOP10/P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™. In some instances, animal cells include a cell from a vertebrate or from an invertebrate. In some cases, an animal cell includes a cell from a marine invertebrate, fish, insect, amphibian, reptile, mammal, or human. In some cases, a fungus cell includes a yeast cell, such as brewer’s yeast, baker’s yeast, or wine yeast. Fungi include ascomycetes such as yeast, mold, filamentous fungi, basidiomycetes, or zygomycetes. In some instances, yeast includes Ascomycota or Basidiomycota. In some cases, Ascomycota includes Saccharomycotina (true yeasts, e.g., Saccharomyces cerevisiae (baker’s yeast)) or Taphrinomycotina (e.g., Schizosaccharomycetes (fission yeasts)). In some cases, Basidiomycota includes Agaricomycotina (e.g., Tremellomycetes) or Pucciniomycotina (e.g., Microbotryomycetes). Exemplary yeast or filamentous fungi include, for example, the genus: Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, Kluyveromyces, Zygosaccharomyces,Yarrowia, Trichosporon, Rhodosporidi, Aspergillus, Fusarium, or Trichoderma. Exemplary yeast or filamentous fungi include, for example, the species: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Candida boidini, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida viswanathii, Candida lusitaniae, Rhodotorula mucilaginosa, Pichia metanolica, Pichia angusta, Pichia pastoris, Pichia anomala, Hansenula polymorpha,Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowia lipolytica, Trichosporon pullulans, Rhodosporidium toru-Aspergillus niger, Aspergillus nidulans, Aspergillus awamori, Aspergillus oryzae, Trichoderma reesei, Yarrowia lipolytica, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii, Zygosaccharomyces bailii, Cryptococcus neoformans, Cryptococcus gattii, or Saccharomyces boulardii. Exemplary yeast host cells include, but are not limited to, Pichia pastoris yeast strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33; and Saccharomyces cerevisiae yeast strain such as INVScl. In some instances, additional animal cells include cells obtained from a mollusk, arthropod, annelid or sponge. In some cases, an additional animal cell is a mammalian cell, e.g., from a human, primate, ape, equine, bovine, porcine, canine, feline or rodent. In some cases, a rodent includes mouse, rat, hamster, gerbil, hamster, chinchilla, fancy rat, or guinea pig. Exemplary mammalian host cells include, but are not limited to, HEK 293 cells, 293 A cell line, 293FT cell line, 293F cells, 293 H cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, Expi293F™ cells, Flp-In™ T-REx™ 293 cell line, Flp-In™-293 cell line, Flp-In™-3T3 cell line, Flp-In™-BHK cell line, Flp-In™-CHO cell line, Flp-In™-CV-1 cell line, Flp-In™-Jurkatcell line, FreeStyle™ 293 -F cells, FreeStyle™ CHO-S cells, GripTite™ 293 MSR cell line, GS-CHO cell line, HepaRG™ cells, T-REx™ Jurkat cell line, Per.C6 cells, T-REx™-293 cell line, T-REx™-CHO cell line, and T-REx™-HeLa cell line. In some instances, a mammalian host cell is a primary cell. In some instances, a mammalian host cell is a stable cell line or a cell line that has incorporated a genetic material of interest into its own genome and has the capability to express the product of the genetic material after many generations of cell division. In some cases, a mammalian host cell is a transient cell line or a cell line that has not incorporated a genetic material of interest into its own genome and does not have the capability to express the product of the genetic material after many generations of cell division. Exemplary insect host cells include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf2l cells, High Five™ cells, and expresSF+® cells. In some instances, plant cells include a cell from algae. Exemplary plant cell lines include, but are not limited to, strains from Chlamydomonas reinhardtii 137c, orSynechococcus elongatus PPC 7942.
Production of the VLPs described herein comprises recovering the ARC/end-gag polypeptides from a cell culture supernatant of a cell expressing the ARC/end-gag polypeptides and incubating the recovered polypeptides at conditions to form the VLPs. VLPs can self-assemble at salt concentrations ranging from 100 mM - 1000 mM, 200 mM - 800 mM, 300 mM - 600 mM, or about 500 mM. The salt may comprise NaCl, KCl, CaSO4, Na2CO3, NaHC03, MgSO4, sodium acetate, sodium bicarbonate, sodium borate, sodium citrate, sodium phosphate, sodium sulfate, sodium sulfide, sodium sulfite, sodium thiosulfate, ammonium acetate, ammonium chloride, ammonium sulfate, magnesium chloride, potassium acetate, potassium carbonate, or potassium phosphate. In certain embodiments, ARC/endo-gag polypeptides recovered from supernatant can be subjected to one or more of centrifugation, ultracentrifugation, dialysis, filtration, ultrafiltration, buffer exchange, column chromatography, anion exchange chromatography or nickel affinity chromatography.
Therapeutic MethodsThe ARC/endo-gag polypeptides described herein can be included with one or more pharmaceutically acceptable excipients, carriers, or diluents in a vaccine composition. The vaccine compositions described herein can be used in a method of priming an adaptive immune response in an individual against the pathogen from which the pathogen-associated antigen is derived. The vaccine compositions described herein can be used in a method of boosting an adaptive immune response in an individual against the pathogen from which the pathogen-associated antigen is derived. In certain embodiments, the adaptive immune response primed or boosted reduces the probability that the individual will be infected with the pathogen from which the pathogen-associated antigen is derived. In certain embodiments, the adaptive immune response primed or boosted reduces the probability that the individual will spread the pathogen from which the pathogen-associated antigen is derived.
Also described herein is a method of vaccinating an individual against a pathogen comprising administering to the individual a composition comprising: 1) an ARC polypeptide or an endogenous gag (endo-gag) polypeptide; 2) a pathogen-associated antigen derived from the pathogen; and 3) an adjuvant.
Also described herein is a method of inducing an antibody response to a pathogen in an individual comprising administering to the individual a composition comprising: 1) an ARC polypeptide or an endogenous gag (endo-gag) polypeptide; 2) a pathogen-associated antigen derived from the pathogen; and 3) an adjuvant. In certain embodiments, the antibody response is an IgG response.
Also described herein is a method of inducing a cellular immune response to a pathogen in an individual comprising administering to the individual a composition comprising: 1) an ARC polypeptide or an endogenous gag (endo-gag) polypeptide; 2) a pathogen-associated antigen derived from the pathogen; and 3) an adjuvant. In certain embodiments, the cellular immune response is a CD4+ helper T-cell response. In certain embodiments, the cellular immune response is a CD8+ cytotoxic T-cell response.
Pharmaceutically Acceptable Excipients, Carriers, and DiluentsIn certain embodiments, the polypeptides or virus-like particles of the current disclosure are included in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. Various pharmaceutically acceptable ingredients are described in Remington: The Science and Practice of Pharmacy, Joseph P Remington and Loyd V. Allen, 22nd edition, Pharmaceutical Press, 2013.
The compositions of the present invention are in a biologically compatible form suitable for administration in vivo for subjects. The pharmaceutical compositions further comprise a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the VLP is administered.
Also described herein are kits comprising one or more of the polypeptides or virus-like particles described herein in a suitable container and one or more additional components selected from: instructions for use; a diluent, an excipient, a carrier, and a device for administration.
In certain embodiments, described herein is a method of preparing a vaccine comprising admixing one or more pharmaceutically acceptable excipients, carriers, or diluents and one or more Arc or endo-Gag polypeptides of the current disclosure. In certain embodiments, described herein is a method of preparing a vaccine for storage or shipping comprising lyophilizing one or more Arc or endo-Gag polypeptide compositions of the current disclosure.
EXAMPLESThe following illustrative examples are representative of embodiments of compositions and methods described herein and are not meant to be limiting in any way.
Example 1 – Assembly of endoVLPsA robust recombinant system was established to purify monomeric Arc or endo-Gag proteins and form highly pure and concentrated endoVLPs resembling viral capsids (
A certain protocol for expression of recombinant ARC polypeptide and assembly into endVLPs is detailed below.
Expression vectors constructs comprising Arc and endo-Gag open reading frames were transformed into the Rosetta 2 (DE3)pLysS E. coli strain (Millipore Sigma, Cat# 71403). Arc was induced with 0.1 mM IPTG followed by a 16-hour incubation at 16° C. Cell pellets were lysed by sonication in 20 mM sodium phosphate pH 7.4, 0.1 M NaCl, 40 mM imidazole, 1 mM DTT, and 10% glycerol. The lysate was treated with excess TETRBO DNase (Thermo Fisher Scientific, Cat# AM2238), RNase Cocktail (Thermo Fisher Scientific, Cat# AM2286), and Benzonase Nuclease (Millipore Sigma, Cat# 71205) to eliminate nucleic acids. NaCl was added to lysate in order to adjust the NaCl concentration to 0.5 M followed by centrifugation and filtration to remove cellular debris. 6XHIS-tagged (SEQ ID NO: 47) recombinant protein was loaded onto a HisTrap HP column (GE Healthcare, Cat# 17-5247-01), washed with buffer A (20 mM sodium phosphate pH 7.4, 0.5 M NaCl, 40 mM imidazole, and 10% glycerol), and eluted with a linear gradient of buffer B (20 mM sodium phosphate pH 7.4, 0.5 M NaCl, 500 mM imidazole, and 10% glycerol). Collection tubes were supplemented in advance with 10 mL of 0.5 M EDTA pH 8.0 per 1 ml eluate. The resulting Arc or protein is generally more than 95% pure, with a yield of up to 50 mg per 1 L of bacterial culture.
Residual nucleic acid was removed by anion exchange chromatography on a Mono Q 5/50 GL column (GE Healthcare, Cat# 17516601). Before loading to the column, recombinant protein was buffer exchanged to buffer C (20 mM Tris-HCl pH 8.0, 100 mM NaCl, and 10% glycerol) using Pierce Protein Concentrator PES, 10 K MWCO, 5-20 ml” (Thermo Scientific, Cat# 88528) according to the manufacturer’s protocol. After loading, the mono Q resin was washed with 2 ml of buffer C. Arc and endo-Gag proteins were eluted using a linear gradient of buffer D (20 mM Tris-HCl pH 8.0, 500 mM NaCl, and 10% glycerol). RNA efficiently separated from Arc and eluted at 600 mM NaCl.
The N-terminal 6XHIS tag (SEQ ID NO: 47) and spacer were removed from concentrating peak fractions of the mono Q purified Arc using a 10 kDa MWCO PES concentrator and then treating with 10% v/v of AcTEV™ Protease (Invitrogen™ #12575023). The cleavage efficiency is above 99%, as revealed by an SDS-PAGE assay. The protein is then diluted into HisTrap Buffer A and cleaned with HisTrap HP resin. The resulting purified Arc has an N-terminal Glycine residue and does not contain the initial methionine.
Cleaved Arc protein (1 mg/mL) was loaded into a 20 kDa MWCO dialysis cassette and dialyzed overnight in 1 M sodium phosphate (pH 7.5) at room temperature. The following day, the solution was removed from the cassette, transferred to microcentrifuge tubes, and spun at max speed for 5 minutes in a tabletop centrifuge. The supernatant was transferred to a 100 kDa MWCO Regenerated Cellulose Amicon ETltrafiltration Centrifugal concentrator. The buffer was exchanged to PBS pH 7.5, and the volume was reduced 20-fold.
Example 3 – Assembly of Endo VLPs From ARC Fusion ProteinsIn order to utilize a recombinant Arc endoVLP delivery system for vaccine development, the Arc protein must be amenable to stable tethering of candidate antigens and adjuvant molecules while retaining the ability to assemble into VLP capsids. Various Arc protein fusions were generated, including fusion proteins with N- and C-terminal Cre domains separated from Arc by a 5XGS flexible protein linker(SEQ ID NO: 46) (
As an alternative to direct tethering, proteins will be conjugated directly to fully formed Arc endoVLPs. After confirming the assembly of highly pure recombinant Arc endoVLP preparations by TEM, endoVLP structures were fluorescently labeled through chemical crosslinking using Alexa Fluor (Thermo) NHS-ester (
A mouse model system can provide in vivo proof-of-principle data for endoVLP vaccines comprising Arc or endo-Gag polypeptides. The mouse Arc protein (SEQ ID NO: 29) is used for these experiments. Groups of ten, split gender, eight-week-old BALB/C mice will be administered either naked endoVLPs (control) or an endoVLP SARS-CoV- 2 vaccine (with spike protein as the antigen), with a second administration after two weeks. Three weeks after booster administration, serum and plasma will be collected, with an additional assessment of an antibody response against the viral antigen using a commercially available ELISA for SARS-CoV-2 spike protein. In vivo studies can also examine immune response in both B and T-cells. To determine the humoral immune responses by B-cells, a neutralization assay based on heat-inactivated animal sera would be employed. To assess cell-mediated immunity, the release of various cytokines in response to SARS-CoV-2 antigen-presenting target cells can be used.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.
All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.
Claims
1. A composition comprising: 1) an ARC polypeptide or an endogenous gag (endo-gag) polypeptide; 2) a pathogen-associated antigen; and 3) an adjuvant.
2. The composition of claim 1, wherein the arc polypeptide comprises:
- a) an amino acid sequence that is SEQ ID NO: 1 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 1;.
3. The composition of claim 1 or 2, wherein the endo-gag polypeptide comprises:
- a) an amino acid sequence that is SEQ ID NO: 16 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 16;
- b) an amino acid sequence that is SEQ ID NO: 17 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 17;
- c) an amino acid sequence that is SEQ ID NO: 18 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 18;
- d) an amino acid sequence that is SEQ ID NO: 19 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 19;
- e) an amino acid sequence that is SEQ ID NO: 20 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 20;
- f) an amino acid sequence that is SEQ ID NO: 21 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 21;
- g) an amino acid sequence that is SEQ ID NO: 22 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 22; or
- h) an amino acid sequence that is SEQ ID NO: 23 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 23; or
- i) an amino acid sequence that is SEQ ID NO: 24 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 24; or
- j an amino acid sequence that is SEQ ID NO: 25 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 25; or
- k) an amino acid sequence that is SEQ ID NO: 26 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 26; or
- 1) an amino acid sequence that is SEQ ID NO: 27 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 27; or
- m) an amino acid sequence that is SEQ ID NO: 28 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 28;
- n) or any combination thereof.
4. The composition of claim 1 or 3, wherein the pathogen-associated antigen and the adjuvant are different compounds or polypeptides.
5. The composition of any one of claims 1 to 4, wherein the pathogen-associated antigen comprises a polypeptide.
6. The composition of any one of claims 1 to 5, wherein the pathogen-associated antigen is a bacterial antigen, a fungal antigen, a parasitic antigen or a viral antigen.
7. The composition of any one of claims 1 to 5, wherein the pathogen-associated antigen is a bacterial antigen.
8. The composition of any one of claims 1 to 5, wherein the pathogen-associated antigen is a fungal antigen.
9. The composition of any one of claims 1 to 5, wherein the pathogen-associated antigen is a parasitic antigen.
10. The composition of any one of claims 1 to 5, wherein the pathogen-associated antigen is a viral antigen.
11. The composition of claim 10, wherein the viral antigen is a respiratory virus antigen.
12. The composition of claim 10, wherein the viral antigen is a Coronaviridae antigen.
13. The composition of claim 12, wherein the Coronaviridae exhibits human tropism.
14. The composition of claim 12 or 13, wherein the Coronaviridae is selected from a list consisting of SARS Coronavirus (SARS-CoV-1), COVID-19 (SARS-CoV-2), MERS-coronavirus (MERS-CoV), or any combination thereof.
15. The composition of any one of claims 10 to 14, wherein the viral antigen comprises a spike protein, an envelope protein, a nucleocapsid protein, a membrane protein, a membrane glycoprotein, or a non-structural protein.
16. The composition of claim 15, wherein the viral antigen comprises a SARS Coronavirus (SARS-CoV-1) antigen, a COVID-19 (SARS-CoV-2) antigen, a MERS-coronavirus (MERS-CoV) antigen, or any combination thereof.
17. The composition of claim 16, wherein the viral antigen comprises a spike protein, an envelope small membrane protein, a membrane protein, a non-structural protein 6 (NSP6), a nucleoprotein, an ORF10 protein, Protein 3a, Protein7a, Protein 9b, structural protein 8, uncharacterized protein 4, or any combination thereof.
18. The composition of claim 16, wherein the viral antigen comprises an amino acid residue sequence as set forth in any one of SEQ ID NOs: 30 to 42, and combinations thereof.
19. The composition of claim 16, wherein the viral antigen consists of an amino acid residue sequence as set forth in any one of SEQ ID NOs: 30 to 42, and combinations thereof.
20. The composition of claim 10, wherein the viral antigen is an Influenza antigen.
21. The composition of claim 20, wherein the Influenza antigen is an M1 matrix protein or a fragment thereof.
22. The composition of any one or claims 1 to 21, wherein the ARC polypeptide or the endo-gag-polypeptide is coupled to the pathogen-associated antigen.
23. The composition of claim 22, wherein the ARC polypeptide or the endo-gag-polypeptide is coupled to the pathogen-associated antigen by a peptide bond.
24. The composition of claim 23, wherein the pathogen-associated antigen is N-terminal to the ARC polypeptide or the endo-gag-polypeptide.
25. The composition of claim 23, wherein the pathogen-associated antigen is C-terminal to the ARC polypeptide or the endo-gag-polypeptide.
26. The composition of any one of claims 23 to 25, comprising a flexible peptide linker separating the pathogen-associated antigen and the ARC polypeptide or the endo-gag-polypeptide.
27. The composition of claim 22, wherein the ARC polypeptide or the endo-gag-polypeptide is coupled to the pathogen by a bond formed from the reaction of an NHS-ester and a primary amine of the ARC polypeptide.
28. The composition of any one of claims 1 to 27, wherein the adjuvant comprises an immune stimulatory compound.
29. The composition of claim 27, wherein the immune stimulatory compound comprises a lipid, a nucleic acid, an aluminum compound, an water-in-oil emulsion, a polypeptide, or any combination thereof.
30. The composition of claim 29, wherein the immune stimulatory compound comprises a lipid.
31. The composition of claim 29, wherein the immune stimulatory compound comprises a nucleic acid.
32. The composition of claim 31, wherein the nucleic acid is a DNA.
33. The composition of claim 32, wherein the DNA comprises CPG-1018, CPG-1826, CPG-2007, CPG-2006, or any combination thereof.
34. The composition of claim 31, wherein the nucleic acid is an RNA.
35. The composition of claim 32, wherein the RNA comprises CV1802, Poly(U), Poly(I:C), ssRNA40, GFP RNA, RNA41, RNA42, RNA33, RNA35, 5′-phosphorylated blunt-ended viral genomic dsRNA <300bp, long dsRNA >1000bp, genomic ssRNA, ssRNA40, or any combination thereof.
36. The composition of claim 29, wherein the immune stimulatory compound comprises an aluminum compound.
37. The composition of claim 36, wherein the aluminum compound comprises alum.
38. The composition of claim 29, wherein the wherein the immune stimulatory compound comprises an water-in-oil emulsion.
39. The composition of claim 29, wherein the immune stimulatory compound comprises an agonist for a toll-like receptor, a NOD-like receptor, a RIG-1 or MDA-5 receptor, a C-type lectin receptor, a costimulatory molecule, a cytokine receptor, a STING pathway, or any combination thereof.
40. The composition of claim 39, wherein the toll-like receptor agonist is selected from a list consisting of CpG oligonucleotide, SD-101, LFX453, imiquimod, Bacillus Calmette-Guerin (BCG), monophosphoryl lipid A, Poly ICLC, GSK1795091, or any combination thereof.
41. The composition of claim 39, wherein the NOD-like receptor agonist is selected from a list consisting of bacterial peptidoglycan, an acylated derivative of iE-DAP (C12-iE-DAP), D-gamma-Glu-mDAP (iE-DAP), L-Ala-gamma-D-Glu-mDAP (Tri-DAP), muramyl dipeptide (MDP), muramyl tripeptide, L18-MDP, M-TriDAP, murabutide, PGN-ECndi, PGN-ECndss, PGN-SAndi, N-glycolylated muramyl dipeptide, murabutide, or any combination thereof.
42. The composition of claim 39, wherein the RIG-1 or MDA-5 receptor agonist is selected from a list consisting of poly(I:C), Poly(dA:dT), Poly(dG:dC), 3p-hpRNA, 5′ppp-dsRNA, or any combination thereof.
43. The composition of claim 39, wherein the C-type lectin receptor agonist is selected from a list consisting of Beta-1,3-glucan, zymosan, Heat-killed C. albicans, cord factor, and Trehalose-6,6-dibehenate, or any combination thereof.
44. The composition of claim 29, wherein the immune stimulatory compound comprises a polypeptide.
45. The composition of claim 44, wherein the polypeptide is a polypeptide from Brucella abortus, Bordetella pertussis, Chlamydia trachomatis, Fusobacterium nucleatum, Mycobacterium tuberculosis, Neisseria meningitidis, Staphylococcus aureus, Shigella dysenteriae, Shigella flexneri, Streptococcus pneumoniae, Vibrio cholerae, Brucella abortus, Mycobacterium paratuberculosis, Neisseria meningitidis, Streptococcus pneumoniae, or any combination thereof.
46. The composition of claim 45, wherein the polypeptide is from BCSP31, FHA, MOMP, PorB, PVL, Porin, OmpA, 34 kDa MOMP, PepO, OmpU, Lumazine synthase, Omp16, Omp19, BCSP31, CobT, RpfE, Rv0652, HBHA, NhhA, DnaJ, Pneumolysin, ΔA146 Pneumolysin, or any combination thereof.
47. The composition of claim 44, wherein the polypeptide is polypeptide from bacterial flagellin.
48. The composition of claim 44, wherein the polypeptide is polypeptide from human heat shock protein 70.
49. The composition of any one or claims 1 to 48, wherein the adjuvant is coupled to the ARC polypeptide or the endo-gag-polypeptide.
50. The composition of claim 49, wherein the adjuvant is coupled to the ARC polypeptide or the endo-gag-polypeptide by a bond formed from the reaction of an NHS-ester and a primary amine of the ARC polypeptide.
51. The composition of claim 49, wherein the adjuvant is coupled to the ARC polypeptide or the endo-gag-polypeptide by a peptide bond.
52. The composition of claim 51, wherein the adjuvant is N-terminal to the ARC polypeptide or the endo-gag-polypeptide.
53. The composition of claim 51, wherein the adjuvant is C-terminal to the ARC polypeptide or the endo-gag-polypeptide.
54. The composition of any one of claims 51 to 53, comprising a flexible peptide linker separating the adjuvant and the ARC polypeptide or the endo-gag-polypeptide.
55. The composition of any one of claims 1 to 54, wherein the composition comprises a virus-like particle.
56. The composition of claim 55, wherein the virus-like particle comprises a mixture of the ARC or the endo-gag-polypeptide polypeptide coupled to the pathogen-associated antigen and the ARC polypeptide or the endo-gag-polypeptide coupled to the adjuvant.
57. The composition of claim 55 or 56, wherein the virus-like particle comprises ARC polypeptide or the endo-gag-polypeptide not coupled to adjuvant or pathogen-associated antigen.
58. The composition of any one of claims 55 to 56, wherein the ARC polypeptide or the endo-gag-polypeptide coupled to the adjuvant comprises from about 5% to about 20% of the virus-like particle.
59. The composition of any one of claims 55 to 56, wherein the ARC polypeptide or the endo-gag-polypeptide coupled to the adjuvant comprises from about 5% to about 15% of the virus-like particle.
60. The composition of any one of claims 55 to 56, wherein the ARC polypeptide or the endo-gag-polypeptide coupled to the adjuvant comprises about 10% of the virus-like particle.
61. The composition of any one of claims 55 to 59, wherein the ARC polypeptide or the endo-gag-polypeptide coupled to the pathogen-associated antigen comprises from about 5% to about 20% of the virus-like particle.
62. The composition of any one of claims 55 to 59, wherein the ARC polypeptide or the endo-gag-polypeptide coupled to the pathogen-associated antigen comprises from about 5% to about 15% of the virus-like particle.
63. The composition of any one of claims 54 to 59, wherein the ARC polypeptide or the endo-gag-polypeptide coupled to the pathogen-associated antigen comprises about 10% of the virus-like particle.
64. The composition of any one of claims 54 to 63, wherein the ARC polypeptide or the endo-gag-polypeptide not coupled to adjuvant or pathogen-associated antigen comprises from about 90% to about 60% of the virus-like particle.
65. The composition of any one of claims 54 to 63, wherein the ARC polypeptide or the endo-gag-polypeptide not coupled to adjuvant or pathogen-associated antigen comprises from about 90% to about 70% of the virus-like particle.
66. The composition of any one of claims 54 to 63, wherein the ARC polypeptide or the endo-gag-polypeptide not coupled to adjuvant or pathogen-associated antigen comprises about 80% of the virus-like particle.
67. The composition of any one of claims 1 to 65, comprising a pharmaceutically acceptable excipient, carrier, or diluent.
68. The composition of any one of claims 1 to 67, for use in priming or boosting an adaptive immune response to the pathogen-associated antigen.
69. The use of claim 68, wherein the adaptive immune response is an antibody response to the pathogen-associated antigen.
70. The use of claim 69, wherein the antibody response produces IgG antibodies that specifically bind the pathogen-associated antigen.
71. The use of claim 68, wherein the adaptive immune response is a cellular immune response to the pathogen-associated antigen.
72. The composition of any one of claims 1 to 67, for use as a vaccine.
73. A method of priming an adaptive immune response to a pathogen-associated antigen in an individual comprising administering the composition of any one of claims 1 to 67 to the individual thereby priming an adaptive immune response to the pathogen-associated antigen.
74. The method of claim 73, wherein the individual is a human individual.
75. The method of claim 73, wherein the adaptive immune response is an antibody response to the pathogen-associated antigen.
76. The method of claim 73, wherein the antibody response produces IgG antibodies that specifically bind the pathogen-associated antigen.
77. The method of claim 73, wherein the adaptive immune response is a cellular immune response to the pathogen-associated antigen.
78. A method of vaccinating an individual comprising administering the composition of any one of claims 1 to 68 to the individual thereby vaccinating the individual.
79. The method of claim 78, wherein the method of vaccinating protects from Coronaviridae infection or symptoms.
80. The method of claim 79, wherein the Coronaviridae comprises SARS Coronavirus (SARS-CoV-1), COVID-19 (SARS-CoV-2), MERS-coronavirus (MERS-CoV), or any combination thereof.
81. A nucleic acid encoding the ARC polypeptide or the endo-gag-polypeptide of any one of claims 21 to 24 or 49 to 53.
82. A vector comprising the nucleic acid of claim 81.
83. The vector of claim 82 comprising a promoter operatively coupled to the nucleic acid encoding the ARC polypeptide.
84. A host cell comprising the vector of claim 82 or 83.
85. A method of manufacturing a vaccine comprising isolating the ARC polypeptide or the endo-gag polypeptide from the host cell of claim 84 and contacting the polypeptide to a solution comprising a salt concentration of 100 mM to 1000 mM.
86. The method of claim 85, wherein the salt is NaCl or NaPO4.
Type: Application
Filed: May 17, 2021
Publication Date: Jun 29, 2023
Inventors: Zachary GILBERT (Brooklyn, NY), Colin MALONE (Brooklyn, NY)
Application Number: 17/998,856
