Patent Application
20220332770
Sequence listings and related materials in the ASCII text file named “SEQ-URI2102 ST25.txt” and created on Apr. 11, 2022 with a size of about 78 kilobytes, is hereby incorporated by reference.
This invention was made with government support under grant number AI156510 awarded by the National Institutes of Health. The Government has certain rights in the invention.
The invention relates to virus-like particle (VLP)-based vaccines, vaccine delivery and vaccine adjuvants in general and particularly to a new vaccine platform that displays flagellin at high densities on virus-like particle (VLP) surface to greatly reduce any systemic adverse effects from flagellin-based vaccines while providing improved immunogenicity and efficacy.
Flagellin is the principal component of flagellar filament found in flagellated bacteria such as Salmonella, Escherichia coli, Vibrio vulnificus, Campylobacter coli, and P. aeruginosa. The flagellin protein typically has four structural domains (D0, D1, D2, and D3). D0 and D1 domains from the inner filament core and are composed of highly conserved N- and C-termini of flagellin in the form of α-helical coiled coils (
Extracellular flagellin can be recognized by toll-like receptor (TLR) 5 and intracellular flagellin can be recognized by NOD-like receptor (NLR) family, apoptosis inhibitory protein 5 (NAIP5) to initiate NLR family CARD domain containing 4 (NLRC4) inflammasome activation (see, F. Hayashi, et al., Nature 2001, 410(6832): 1099-103; Y. Zhao, et al., Nature 2011, 477(7366): 596-600). D1 domain contributes to TLR5 binding and filament formation and D0 domain contributes to NAIP5 recognition and NLRC4 inflammasome activation (see, e.g., W. S. Song, et al., Sci. Rep. 2017, 7: 40878). TLR5 binding leads to NFKB activation and release of pro-inflammatory cytokines, such as interleukin (IL)-6, while NLRC4 inflammasome activation leads to Caspase-1 activation (M. Vijay-Kumar, et al., Eur. J. Immunol. 2010, 40(12): 3528-34).
Flagellin has been explored as vaccine adjuvants and carriers as it appears to activate both the innate and adaptive arms of immunity and therefore holds great promise as highly immunogenic vaccine carriers when compared to other vaccine approaches (see, I.A. Hajam, et al., Exp. Mol. Med. 2017, 49(9): e373). FljB and FliC are two closely related but alternately expressed forms of flagellin in gram-negative Salmonella enterica serovar Typhimurium (S. Typhimurium) (see, H. R. Bonifield, et al., J. Bacteriol. 2003, 185(12): 3567-74), and indeed both have been explored for vaccine development.
Despite being highly immunogenic, two flagellin-based influenza vaccines (VAX125 and VAX102) induced significant systemic adverse reactions especially at high doses in clinical studies (J. J. Treanor, et al., Vaccine 2010, 28(52): 8268-74; C.B. Turley, et al., Vaccine 2011, 29(32): 5145-52). Specifically, both vaccines dose-dependently increased serum c-reactive protein (CRP) levels and, in some patients, systemic adverse reactions were accompanied with elevated serum IL-6 levels. Another flagellin-based influenza vaccine (VAX128) also dose-dependently increased serum CRP levels in the elderly and 4.8% subjects experienced at least 4-fold increase of serum IL-6 (D. N. Taylor, et al., Vaccine 2012, 30(39): 5761-9). Increase of serum CRP and IL-6 levels were also observed in a recent clinical trial of a different flagellin-based influenza vaccine (VAX2012Q) (L. Tussey, et al., Open Forum Infect. Dis. 2016, 3(1): ofw015). Considering TLR5 activation leads to IL-6 secretion that in turn activates CRP release, over activation of TLR5 on various types of lymphoid and non-lymphoid cells might explain, at least partly, systemic adverse reactions such as cytokine storm and fever that have been associated, without exception, with existing flagellin-based vaccine candidates (e.g., S. B. Mizel, et al., J. Immunol. 2010, 185(10): 5677-82).
Therefore, despite flagellin's great promise as a potential vaccine carrier, its high risk for inducing strong side effects has posed safety concerns that prevented it from surviving a clinical trial and making to the pharmaceutical market so far. Accordingly, there remains a strong need to develop safer flagellin-based yet highly immunogenic vaccine carriers.
Meanwhile, in pursuit of safer vaccination, researchers have turned to subunit vaccines more and more due to their significantly improved safety profiles as compared to whole pathogen-based vaccines. Yet, subunit vaccines have relatively low immunogenicity because of their weak uptake by antigen-presenting cells (APCs) and weak ability to stimulate APC activation. Efficient antigen uptake and stimulation of APC maturation are crucial to induce potent antigen-specific immune responses (R.M. Steinman, et al., Current Topics in Microbiology and Immunology 2006, 311: 17-58). Besides infectious disease vaccines, cancer vaccines based on tumor-associated antigens (TAAs) and neoantigens that are mainly presented on major histocompatibility complex (MHC) class II molecules (see, E. Blass, et al., Nat Rev Clin Oncol 2021, 18(4): 215-229). Failing to induce APC maturation and potent cytotoxic T lymphocyte (CTL) responses have presented significant challenges for the successful adoption of subunit vaccine technologies.
Vaccine delivery platforms facilitate antigen uptake by APCs and can be used to enhance subunit vaccine efficacy. Highly immunogenic proteins and virus-like particles (VLPs) are both popular vaccine delivery platforms (J. Wallis, et al., Clin Exp Immunol 2019, 196(2): 189-204). VLPs are self-assembled nanostructures, mimicking the size and shape of infectious viruses. VLPs can be presented on both MHC I and II molecules and elicit both humoral and cellular immune responses (see K. M. Frietze, et al., Curr Opin Virol 2016, 18: 44-9). Some VLPs can be used as vaccines alone, such as hepatitis b surface antigen (HBsAg), while others can be used to display vaccine antigens from other pathogens to serve as vaccine delivery platforms, such as Hepatitis B core (HBc) VLPs.
HBc self-assembles into VLPs and the c/el loop of HBc provides a site for high-density display of antigenic epitopes on VLP surface (e.g., U. Arora, et al., J. Nanobiotechnol. 2012, 10: 30). However, the c/e1 loop of HBc, localized at the tip of its spikes, allows the insertion of only short antigenic epitopes without imposing significant steric hindrance to negatively impact HBc VLP assembly (K. Roose, et al., Expert Review of Vaccines 2013, 12(2): 183-98). As of now, only two full-length proteins, green fluorescence protein (GFP, 238 aa) and outer surface protein C of the Lyme disease agent Borrelia burgdorferi (OspC, 189 aa) with closely juxtaposed N and C-termini, have been successfully displayed on HBc VLP surface via c/el loop insertion (P. A. Kratz, et al., Proc. Natl. Acad. Sci. U.S.A. 1999, 96(5): 1915-20; C. Skamel, et al., J. Biol Chem. 2006, 281(25): 17474-81). No success has been reported with recombinantly displaying a protein or fragments thereof with a size larger than 250 amino acids on HBc VLP surface. This shortcoming has greatly hampered the use of VLPs as a vaccine delivery platform.
The present invention, through unexpectedly successful display of flagellin or one or more portions thereof on a VLP surface, in a high-density fashion, provides a versatile, highly immunogenic, and surprisingly safe vaccine platform. The full-length flagellin protein is well over 450 amino acid residues and its incorporation would normally be expected to have hindered the proper folding and assembly of existing VLP platforms, and the expression of such a highly immunogenic molecule or even portions thereof would have also been expected to induce severe systemic adverse reactions in the body. However, due to unique structural geometries and clever engineering, the flagellin-displaying VLP platform of the invention turned out to be well tolerated and even more immunogenic than many existing vaccine carriers.
The present invention can be used to aid in vaccine development against emerging infectious diseases, such as COVID-19, or develop better, even universal, vaccines against known pathogens that mutate frequently such as the ever-evolving strains of influenza and other viruses. This is of particular public health importance since the current influenza vaccines vary from year to year in efficacy and induce only weak protection in the very young and the elderly. The platform based on the present invention can be used in many different ways: to make or deliver vaccines or immunogens, e.g., through recombinantly incorporating or chemically conjugating to an antigenic epitope or a full-length antigen (or a portion thereof); or to function as an adjuvant to boost the body's immune reaction when co-administered with an immunogenic agent, e.g., an existing vaccine.
In one aspect of the invention, the high-density display of flagellin proteins (or a portion thereof) on the VLP surface likely has successfully embedded the flagellin protein's D0 and D1 domains, or substantial portions thereof, in the interior of VLP and thus reduced flagellin's TLR5 activation ability and improved the systemic safety. At the same time, transition of flagellin from soluble to particulate form on VLPs may explain the increased immunogenicity that was recorded. In a further aspect of the invention, the D3 domain (and possibly the D2 domain as well) of flagellin which extends the furthest out on the VLP surface, allows recombinant insertion of anything ranging from small epitopes to large antigens without interfering or jeopardizing the VLP assembly. In yet another further aspect, part or all of the D3 domain and, optionally, part or all of the D2 domain of flagellin are replaced by a desired immunogen with little restrictions on the size and tertiary structure of such immunogen.
In one aspect, the invention relates to a fusion protein comprising parts as follows: (a) the amino acid sequence for the entire or substantial portions of two or more of the four domains of a flagellin protein, preferably the D0 and D1 domains or substantial portions thereof, and (b) the amino acid sequence for a polypeptide that can form a virus-like particle (VLP) (a “VLP-forming polypeptide”) in aggregate, wherein (a) and (b) are recombinantly linked to each other. The novel fusion protein preferably self-assembles with other such fusion proteins and forms a novel VLP in an aqueous environment (e.g., in a saline solution such as phosphate-buffered saline (PBS) or serum). A preferred embodiment of the VLP-forming polypeptide for constructing the fusion protein of the invention is a Hepatitis B core (HBc) protein or a substantial portion thereof. The fusion protein according to the present invention can further include: (c) an immunogenic amino acid sequence, e.g., an epitope or a full-length antigen, that serves as an immunogen such as an influenza epitope, a tumor-associated antigen (TAA) or a neoantigen. Alternately, the immunogen can be a molecule or compound chemically conjugated to the fusion protein having parts (a) and (b). Such immunogen can be a polysaccharide-based, hapten-based agent, e.g., a nicotine hapten molecule like a carboxymethlureido-nicotine molecule. The fusion protein of the invention, in the form of a self-assembled VLP, can be used as a vaccine, a vaccine carrier or a vaccine adjuvant.
In a particularly preferred embodiment, the present invention provides a flagellin-HBc fusion protein comprising the amino acid sequence of a full-length flagellin recombinantly inserted into any position within, or replacing part or all of, the amino acid sequence of the c/e1 loop (N75-L84) of a hepatitis b core (HBc) protein. The embodiment may further include at least one immunogenic sequence, such as a protein or a portion thereof found in an influenza virus, a sever acute respiratory syndrome (SARS) coronavirus, and so on. Other examples of such immunogenic sequences include an antigen expressed by a cancer or other diseased cell, e.g., tumor-associated antigens or neoantigens or a portion thereof. In an embodiment, the immunogenic sequence is recombinantly inserted into the amino acid sequence for the D2 or D3 domain of flagellin or recombinantly inserted by replacing part or entire D2, D3 or both D2 and D3 domains of flagellin. Exemplary embodiments include: (1) where D0 and D1 domains of FljB, flanked with optional short linker sequences, are inserted to replace the entire c/el loop of a truncated HBc (SEQ ID NO:28); and (2) where D0, D1, and D2 domains of FljB, flanked with optional short linker sequences, are inserted to replace the entire c/el loop of a truncated HBc (SEQ ID NO:29). Alternately, the invention provides a fusion protein with sequences for both flagellin and HBc (or substantial portions thereof) where the fusion protein is chemically conjugated to an immunogenic compound or biomolecule.
In one feature, the invention provides a fusion protein having amino acid sequences for (a) an antigen or immunogen, (b) a flagellin protein or a substantial portion thereof, (c) a virus-like particle (VLP)-forming polypeptide, where (a) (b) and (c) are recombinantly linked. In various embodiments, the fusion protein includes the amino acid sequence selected from the group of SEQ ID NOs: 23, 24, 27, 28 and 29, or a substantial portion thereof that is capable of self-assemble with the same or similar polypeptides into a virus-like particle (VLP) in an aqueous environment.
In one aspect, the present invention provides a virus-like particle (VLP) comprising an assembly of proteins, each being a fusion protein described herein.
In a further aspect, the invention provides genetic materials including polynucleotides that, when expressed, produce the fusion proteins of the invention. Such genetic materials include and are not limited to DNAs and RNAs including mRNA.
In another aspect, the invention provides a complex comprising a flagellin-containing VLP conjugated to an immunogen or antigenic agent such as a polysaccharide-based, hapten-based agent, e.g., a nicotine hapten molecule like a carboxymethlureido-nicotine molecule.
In another aspect, the invention features a virus-like particle (VLP) in an aqueous solution where the VLP displays at least 100, preferably at least 150, 180, or 240 full-length flagellin proteins or substantial portions thereof on an outer surface of the VLP.
Further aspects of the invention include a pharmaceutical composition, manufacture and use thereof where the composition comprises (a) a fusion protein, complex or VLP described herein, and (b) a pharmaceutically acceptable carrier, additive or excipient. Such compositions, when used as a vaccine, can optionally include at least an adjuvant such as cytosine phosphoguanine (CpG), a synthetic form of DNA that mimics bacterial and viral genetic material and aluminum-containing adjuvants (Alum). Other examples of adjuvants that can be used in conjunction with pharmaceutical compositions disclosed herein include squalene emulsion based MF59 and AS03 adjuvant, monophosphoryl lipid A (MPL) and so on. A method of treating a patient in need thereof by administering such a pharmaceutical composition is also part of the invention.
Preferred embodiments of the invention include vaccines against infectious pathogens such as an influenza vaccine that includes amino acid sequences for (a) an antigenic or immunogenic sequence against one or more influenza strains, (b) a flagellin protein or a substantial portion thereof, (c) a virus-like particle (VLP)-forming polypeptide or a substantial portion thereof, wherein (a) (b) and (c) are recombinantly linked. A preferred embodiment of the influenza vaccine is called a universal influenza vaccine that is effective against multiple influenza strains.
Another preferred embodiment of the invention is a cancer vaccine that includes amino acid sequences for: (a) an antigenic sequence expressed on a cancer cell, (b) a flagellin protein or a substantial portion thereof, (c) a virus-like particle (VLP)-forming polypeptide or a substantial portion thereof, where (a) (b) and (c) are recombinantly linked. Targeted cancers, in one embodiment, include: melanoma, breast cancer, and prostate cancer.
Another aspect of the invention relates to a method of making or using the VLPs of the invention: (a) using as an adjuvant with another antigenic or immunogenic agent, which can be any type of vaccine (e.g., recombinant, conjugated, VLP-based, toxoid-based, subunit-based, and so on); (b) making a vaccine by conjugating to an immunogenic agent; (c) making a vaccine by recombinantly inserting an immunogenic sequence into a fusion protein that forms VLP.
The foregoing and other objects, aspects, features, and advantages of the invention will become more apparent from the following description and from the claims.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
The objects and features of the invention can be better understood with reference to the drawings described below, and the claims. The drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the invention. In the drawings, like numerals are used to indicate like parts throughout the various views.
Other strains that are not presented here include and are not limited to: BA000037.2 Vibrio vulnificus major flagellin (FlaB), and so on.
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found, for example, in J. Krebs et al. (eds.), Lewin's Genes XII, published by Jones and Bartlett Learning, 2017 (ISBN 9781284104493); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Anmol Publications Pvt. Ltd, 2011 (ISBN 9788126531783); and other similar technical references.
As used in the specification and claims, the singular form “a”, “an”, or “the” includes plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells including mixtures thereof. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as support for the recitation in the claims of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitations, such as “wherein [a particular feature or element] is absent,” or “except for [a particular feature or element],” or “wherein [a particular feature or element] is not present (included, etc.) . . . ”.
When a dimensional measurement is given for a part herein, the value is, unless explicitly stated or clear from the context, meant to describe an average for a necessary portion of the part, i.e., an average for the portion of the part that is needed for the stated purpose. Any accessory or excessive portion is not meant to be included in the calculation of the value.
As used herein, the recitation of a numerical range for a variable is intended to convey that the invention may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the endpoints of the range. Similarly, for a variable which is inherently continuous, the variable can be equal to any real value within the numerical range, including the endpoints of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values >0 and <2 if the variable is inherently continuous.
As used herein, “about” means within plus or minus 10%. For example, “about 1” means “0.9 to 1.1”, “about 2%” means “1.8% to 2.2%”, “about 2% to 3%” means “1.8% to 3.3%”, and “about 3% to about 4%” means “2.7% to 4.4%.”
As used here, the term “adjuvant” refers to a substance or a combination of substances that enhances an immune response to an antigen. Typically, an adjuvant is formulated as part of a vaccine to enhance the vaccine's ability to induce protection in the body against infection by a pathogen, e.g., a virus or bacterium. Adjuvants are considered to enhance the immune response to an antigen in the vaccine in at least one of the following ways: prolong the presence of antigen in the body (blood and/or tissue); help antigen-presenting cells absorb antigen; activate antigen-presenting cells, macrophages, and lymphocytes; and support the production of cytokines. Adjuvants are also used in the production of antibodies from immunized animals. An “adjuvant effect” refers to enhancement in the immune response to a selected antigen in a host that receives the vaccine.
As used herein the terms “administration,” “administering,” or the like, when used in the context of providing a pharmaceutical composition to a subject generally refers to providing to the subject one or more pharmaceutical compositions comprising the agent, e.g., the novel pharmaceutical compositions of the invention, by any means such that the administered compound achieves one or more of the intended biological effects for which the compound was administered. By way of non-limiting example, a composition may be administered parenteral, subcutaneous, intravenous, intracoronary, rectal, intramuscular, intra-peritoneal, transdermal, or buccal routes of delivery.
The term “antigen” as used herein refers to substance, e.g., an amino acid sequence, that is intended or designed to elicit a specific immunological response in a host— however, not all antigens successfully do so; those that do can be called “immunogen.” An “antigen,” as used herein, includes a full-length sequence of a protein, analogs thereof, or immunogenic portions thereof. The term “immunogenic portion” refers to a portion or fragment of a protein that includes one or more epitopes and thus elicits an immunological response. Such portions can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris. Ed., Humana Press 1996, Totowa, N.J.). For example, linear epitopes may be determined by, e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al., Proc. Natl. Acad. Sci. USA 1984, 81:3998-4002; Geysen et al., Molec. Immunol. 1986, 23:709-715. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., X-ray crystallography and 2-dimensional nuclear magnetic resonance. Synthetic antigens are also included within the definition, for example, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens. See, e.g., Bergmann et al., Eur. J. Immunol. 1993, 23:2777-2781; Bergmann et al., J. Immunol. 1996, 157:3242-3249; Suhrbier, A., Immunol, and Cell Biol. 1997, 75:402-408. The term “antigenic” as used herein means associated with an antigen or having characteristics of an antigen.
The term “high density” as used herein refers to a VLP structure or assembly displaying a protein (e.g., flagellin) or a portion thereof at a concentration that is significantly higher than reported in prior art for any VLP. In various embodiments, a HBc-based VLP structure formed by fusion proteins of the invention displays the flagellin protein or a portion thereof at a density at about 80% (weight/weight) as opposed to only 1-8% (weight/weight) of the total protein displayed in prior art examples. Viewed in another way, an embodiment of a “high density” display of flagellin or a portion thereof means there is no fewer than 100, 150, 180, or even 240 units in a single VLP structure.
As used herein, the terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used interchangeably throughout and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. The nucleic acid molecule can be single-stranded or double-stranded. The double-stranded nucleic acid may have the two strands chemically linked and/or form at least one double-stranded region under suitable annealing conditions. The double-stranded region may contain at least one gap, nick, bulge, and/or bubble. In one embodiment, the nucleic acid molecules of the invention comprise a contiguous open reading frame encoding a fusion protein, an antibody, or a fragment, derivative, mutant, or variant thereof.
Nucleic acids encoding any of the various polypeptides disclosed herein may be synthesized chemically. Codon usage may be selected so as to improve expression in a cell. Such codon usage will depend on the cell type selected. Specialized codon usage patterns have been developed for E. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells and insect cells. See for example: Mayfield et al., Proc. Natl. Acad. Sci. USA. 2003 100(2):438-42; Sinclair et al. Protein Expr. Purif. 2002 (1):96-105; Connell N D. Curr. Opin. Biotechnol. 2001 12(5):446-9; Makrides et al. Microbiol. Rev. 1996 60(3):512-38; and Sharp et al. Yeast. 1991 7(7):657-78.
General techniques for nucleic acid manipulation are described for example in Sambrook et al., Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F. Ausubel et al., Current Protocols in Molecular Biology (Green Publishing and Wiley-Interscience: New York, 1987) and periodic updates, herein incorporated by reference. The DNA encoding the polypeptide is operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, viral, or insect genes. Such regulatory elements include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants is additionally incorporated.
The recombinant DNA can also include any type of protein tag sequence that may be useful for purifying the protein. Examples of protein tags include but are not limited to a histidine tag, a FLAG tag, a myc tag, an HA tag, or a GST tag. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts can be found in Cloning Vectors: A Laboratory Manual, (Elsevier, N.Y., 1985).
The expression construct is introduced into the host cell using a method appropriate to the host cell. A variety of methods for introducing nucleic acids into host cells are known in the art, including, but not limited to, electroporation; transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (where the vector is an infectious agent). Suitable host cells include prokaryotes, yeast, mammalian cells, bacterial cells, or plants.
Proteins disclosed herein can also be produced using cell-translation systems. For such purposes the nucleic acids encoding the polypeptide must be modified to allow in vitro transcription to produce mRNA and to allow cell-free translation of the mRNA in the particular cell-free system being utilized (eukaryotic such as a mammalian or yeast cell-free translation system or prokaryotic such as a bacterial cell-free translation system.
The expression “pharmaceutically acceptable carrier, additive or excipient” is intended to include a formulation, or substance used to stabilize, solubilize and otherwise be mixed with active ingredients to be administered to living animals, including humans. This includes any and all solvents, liquid or solid filler, diluent, excipient, encapsulating material, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Except insofar as any conventional media or agent is incompatible with the active compound, such use in the compositions is contemplated. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to a human subject. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate, magnesium stearate, and polyethylene oxide-polypropylene oxide copolymer as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
As used herein, the term “recombinantly linked,” when referring to amino acid sequences, peptides and polypeptides, means that the amino acid sequences, peptides and polypeptides are connected to each other as a result of original genetic materials (DNA or RNA) encoding them having been recombined often after being broken up first. For example, “recombinantly linked” amino acid sequence A and sequence B can be: “-A-B-,” “-A1-B-A2-,” or “-A1-B1-A2-B2-,” “-A1-B1-A2-N2-A3,” and so on with or without linker sequences anywhere in the chain, where A1, A2, A3, and so on are portions of the original sequence A, and B1, N2, N3, and so on are portions of the original sequence B. Amino acid sequences are recombinantly linked as long as they can be produced as part of a fusion protein that is encoded by genetic materials that have been manipulated using recombinant techniques. Recombinantly linked amino acid sequences can be produced using host cells, cell translation systems or otherwise chemically synthesized.
Similarly, as used herein, the term “recombinantly inserted,” when referring to two amino acid sequences, means that a first sequence is inserted into a second one as a result of the original genetic material for the first one being inserted into the original genetic material for the second one through recombinant techniques before both amino acid sequences are expressed as part of a fusion protein. Once the design of the new protein sequence is completed where the first amino sequence is “recombinantly inserted” into the second, the new sequence can be produced using host cells, cell translation systems, plants, or otherwise chemically synthesized.
As used herein, the term “subject” refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, canines, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
As used herein, the term “substantial portion” refers to part of an entity, short of its entirety, but with sufficient portions thereof such that it can function in a fashion that is equivalent to the full entity as far as this invention is concerned. For example, in the context of the flagellin domain(s), a substantial portion of a domain or domains can generate equivalent levels of immunity as the full domain or domains. In certain embodiments, such substantial portion refers to at least 90 or 95 percent of the full amino acid sequence of the full domain or domains. In the context of a VLP-forming polypeptide, a substantial portion thereof refers to a portion of the full-length polypeptide that, once expressed, can still form a VLP in an aqueous environment. For example, the first 149 aa of the full HBc sequence would qualify as a substantial portion of the HBc sequence in the context of being used as a VLP-forming polypeptide. Determination of exactly how much of the full sequence is needed in order to qualify as a substantial portion can be performed through routine investigation by one of ordinary skills in the proteomics or vaccine-research art using standard procedures such as point deletion or mutagenesis combined with functionality assessment.
The term “vaccine” as used herein refers to a biological preparation that induces immunity to a particular disease. A vaccine typically contains an antigen that resembles a disease-causing microorganism or compound and is often made from weakened or killed forms of the microbe, its subunit(s), epitope-containing portion(s), its toxins or one of its surface proteins, as well as genetic materials such as RNA that encodes parts of the microbe. Vaccines can be prophylactic (e.g., to prevent or ameliorate effects of a future infection by a pathogen such as the influenza virus), or therapeutic (e.g., vaccines against certain cancers that are being developed).
The flagellin protein is found in flagellated bacteria including but are not limited to Salmonella (e.g., Salmonella enterica and Salmonella bongori), Escherichia coli, Vibrio vulnificus, Campylobacter coli, Bacillus subtilis, Burkholderia pseudomallei and Pseudomonas aeruginosa. In Salmonella enterica serovar Typhimurium (S. Typhimurium), for instance, while there are two different forms of flagellin, FljB and FliC, only one is expressed at any given time in the cell (J. R. McQuiston et al., J. Clin. Microbiol. 2004, 42(5): 1923-32).
This homology is further confirmed by looking across at seven different variants of flagellin proteins across different bacterial species (
VLP-forming polypeptides can self-assemble into virus-like structures, but because these structures contain no viral genetic material, they are not infectious to the host. Typically, VLP-forming polypeptides are structural proteins found in a virion, such as the capsid. Polypeptides or structural proteins from many virus families have been found to be capable of forming VLPs, including Flaviviridae (e.g., Hepatitis B and C viruses), Retroviridae (e.g., HIV), Parvoviridae (e.g., adeno-associated virus), Paramyxoviridae (e.g., Nipha) and bacteriophages (see, A. Zeltins, Mol. Biotechnol. 2013, 53 (1): 92-107). VLPs, due to their small sizes and strong immunogenicity that results from high density display of viral surface proteins, may be useful as vaccines. An example of a VLP-forming polypeptide is the Hepatitis B core protein (HBc), with its crystal structure shown in
According to the present invention, a flagellin protein or a portion thereof is recombinantly expressed as operably linked to a VLP-forming polypeptide; the resultant fusion proteins then, in turn, self-assemble into their own VLP structure or assembly displaying, in high density (e.g., no fewer than 100, 150, 180, or even 240 units in a single VLP structure), the flagellin protein or a portion thereof. In various embodiments, about 180 or 240 molecules each having a flagellin protein or a portion thereof have been found in a single VLP of the invention, which provides it at much higher concentrations than any prior art has been able to. Further, the flagellin protein or a portion thereof displayed by the VLP is oriented in a way such that the D0 and D1 domains are not exposed on the surface of the VLP (or otherwise easily accessible from outside the VLP) whereas the D3 domain and possibly a part of the D2 domain are (
In prior art, flagellin has been chemically conjugated to HBc VLPs (Y. Lu, et al., Biotechnol. Bioeng. 2013, 110 (8): 2073-85; Y. Lu, et al., Sci. Rep. 2016, 6: 18379). In those studies, only a small fraction of HBc molecules were conjugated with flagellin, and low-density display of flagellin on HBc VLP surface was found not to significantly impact its TLRS activation ability (ditto). Besides HBc VLPs, flagellin was also displayed on influenza Matrix 1 (M1) VLP surface by co-expression of membrane-anchored flagellin and M1 in insect cells (B. Z. Wang, et al., J. Virol. 2008, 82 (23): 11813-23; E.J. Ko, et al., Vaccine 2019, 37 (26): 3426-3434). The content of flagellin in M1 VLPs was found to be about 1.3% or 8% of total protein (weight/weight). Low-density display of flagellin on M1 VLPs showed comparable TLRS activation ability to free flagellin (B.Z. Wang, et al., supra).
In sharp contrast to these prior strategies that resulted in low-density display of flagellin on VLP surface, self-assembly of the novel fusion protein where flagellin is expressed recombinantly linked to a VLP-forming polypeptide, led to high-density display of flagellin onto HBc VLP surface with —80% flagellin content in the VLP assembly. High-density display of flagellin on HBc VLP surface also showed at least 100-fold reduced TLR5 activation ability as compared to free flagellin. Besides significantly reduced TLR5 activation ability, the VLPs of the invention were also shown to have lost most of their NLRC4 inflammasome and Caspase 1 activation ability. D0/D1 domains of flagellin, crucial for TLR5 and NLRC4 inflammasome activation, are likely buried in the interior of the VLPs of the invention without access to TLR5 or NLRC4 inflammasome.
Despite significant reduction of TLR5 activation ability, surprisingly, VLPs of the invention showed similar adjuvant effects in boosting co-administered ovalbumin (OVA) immunization when compared to free flagellin. OVA is a standard reference protein for vaccination research. As the major protein constituent of chicken egg whites, ovalbumin is sufficiently large and complex to be mildly immunogenic. As a result, it is widely used as an antigen for immunization research and validation. Inventor's surprise finding means adjuvant effects of flagellin could be maintained even with a significant loss of TLR5 activation ability. Also, VLPs of the invention induced more than two-fold anti-flagellin antibody titer than flagellin itself, indicating improved immunogenicity. The high immunogenicity of the VLPs of the invention is supported by their increased uptake by APCs and induction of stronger dendritic cell (DC) maturation than free flagellin. The increased uptake of the VLPs of the invention may simply reflect more efficient uptake of particulate antigens by phagocytosis than soluble antigen uptake by non-receptor-mediated endocytosis (macropinocytosis), or the increased uptake of the VLPs of the invention may also have been mediated by cell surface receptors. The exact reasons for the surprise observation of the greater immunogenicity from the VLPs of the invention are to be determined by further investigation.
In a preferred embodiment of the invention, a full-length flagellin protein from Salmonella, FljB (506 aa, SEQ ID NO:1), or a portion thereof, is recombinantly inserted into the sequence of a VLP-forming polypeptide, the Hepatitis B core (HBc) protein or a substantial portion thereof, preferably anywhere within its c/el loop, or by replacing part or all of the c/el loop (
Inventors' work with VLPs made in accordance with principles of the invention, as exemplified by the FH VLPs, showed significantly reduced ability in activating TLR5 or inducing systemic IL-6 release than the flagellin protein itself. Flagellin but not immunization by VLPs of the invention was found to significantly increase rectal temperature of mice (a sign of fever), indicating improved safety profile with VLPs of the invention. Moreover, VLPs of the invention showed similar adjuvant effects to flagellin in boosting a co-administered antigen (e.g., OVA) immunization and VLPs of the invention induced more than two-fold higher anti-flagellin antibody titer than flagellin itself. Consistent with improved immunogenicity, VLPs of the invention could be more efficiently taken up by bone marrow-derived dendritic cells (BMDCs) and stimulate more potent dendritic cell (DC) maturation than flagellin. In addition, it was found that VLPs of the invention were a more immunogenic carrier than the original flagellin, VLP-forming polypeptides and the VLPs they assemble into, as well as other benchmarks used in the vaccine industry: (1) FH VLPs, an embodiment of the VLPs of the invention, performed better in all immunogenic tests than HBc VLPs, and the widely used keyhole limpet hemocyanin (KLH) for vaccine development, when FH VLPs were chemically conjugated to an nicotine hapten molecule; (2) VLPs formed by an exemplary fusion protein of the invention (FH) that is further recombinantly fused to an epitope (e.g., M2e in an influenza virus, or a CTL epitope of the ovalbumin protein, which are 146 and 48 aa including linkers) by displacing the highly variable D3 domain of FljB (
While not wishing to be bound by any particular theory, our data suggest that improvements in both safety profile and immunogenicity seen in the novel vaccine platform provided by the present invention in comparison to existing flagellin-based vaccine candidates may be contributed to the following: (a) the novel VLP assembly of the invention successfully hides the D0 and D1 domains or significant portions thereof inside the assembly--sequences essential for the recognition by TLR5 have been located in those two domains (e.g., ammino acid sequence segments 1-99 and 427-455 in S. Typhimurium FljB and segments 1-99 and 416-434 in S. Typhimurium FliC)--preventing access to them, thereby reducing unwanted TLR5 activation and associated systemic adverse reactions such as cytokine storm and fever; (b) immunogenicity improves due to flagellin or flagellin-associated immunogenic agent being transitioned from a soluble form to a particulate form; and (c) immunogenicity improves due to high-density display and concentration of flagellin or flagellin-associated immunogenic agent, as the flexibility of flagellin's D3 domain (and to a less extent, the D2 domain) allows the insertion of or substitution by not just one or more epitopes of small or moderate sizes but antigens of much larger sizes--the VLP-forming polypeptides, such as HBc, are not capable of such a feat without interfering or preventing the proper VLP assembly. In other words, the present invention has succeeded in combining features from both flagellin and prior art VLPs in an unexpected way that somehow remedies each other's shortcomings.
Application-wise, the fusion proteins of the invention and the resultant VLPs can be useful in the vaccine field in at least the following ways: (a) as adjuvant in a therapy co-administered with another antigenic agent, e.g., any type of vaccine including but not limited to: recombinant, conjugated, VLP-based, or toxoid-based vaccines; (b) conjugated to an immunogen, whether protein-based, polysaccharide-based or hapten-based; (c) further recombinantly fused to an immunogenic sequence, whether a single or multiple epitopes, a full-length antigen or a portion thereof (
First using nicotine vaccine as a model, the inventors compared relative immunogenicity of the VLPs of the invention to flagellin, HBc VLPs, and widely used KLH for purpose of nicotine vaccine development. VLPs of the invention (as exemplified by FH VLPs) were found to be a more immunogenic carrier than the widely used KLH, and KLH was more immunogenic than flagellin and HBc VLPs when tested for nicotine vaccine development. Immunization by VLPs of the invention chemically conjugated to nicotine induced about 4 times higher NicAb titer and more significantly inhibited nicotine entry into the brain than immunization by KLH-nicotine conjugates. It was further found that incorporation of Alum or CpG adjuvant into immunization by a vaccine based on VLPs of the invention that are conjugated to nicotine could further increase antibody production against nicotine and reduce nicotine entry into the brain after nicotine challenge.
In addition to high immunogenicity, VLPs of the invention showed a good systemic safety profile. Clinical adverse reactions include a significant increase of serum c-reactive protein (CRP) levels, significant increase of serum IL-6 levels, and increase of body temperature in some participants. Such adverse reactions were markedly absent from tested conducted here. For instance, VLPs of the invention induced more transient systemic IL-6 release than free flagellin in C57BL/6 mice and VLPs of the invention failed to significantly increase serum IL-6 levels in BALB/c mice. Furthermore, VLPs of the invention did not increase body temperature in mice even after repeated administrations at relatively high doses, while soluble flagellin at an equivalent dose consistently and significantly increased the body temperature of mice. No significant microscopic organ damage or body weight change relative to control group was observed either following immunization by VLPs of the invention. These results support the finding that VLPs of the invention have shown improved systemic safety when compared to flagellin, and can is indeed a good candidate for vaccines.
Besides developing vaccines using the presently disclosed VLPs in a “conjugate model,” the inventors then discovered through “recombinant models” (
This disclosure provides working examples where an infectious disease vaccine model and a cancer vaccine model are tested using recombinant fusion proteins of the invention. In most of the examples, the diverse antigens also replaced D3 domain of flagellin to prepare flagellin-based vaccines to compare its immunogenicity and safety with FH VLP-based vaccines; or inserted into c/el loop of HBc to explore its impact on HBc VLP assembly and prepare HBc or HBc VLP-based vaccines to compare its immunogenicity with FH VLP-based vaccines.
In a first recombinant model, a universal influenza vaccine was designed where ectodomains of influenza matrix protein 2 (M2e) from four different variants were recombinantly inserted to replace the D3 domain of the flagellin and expressed as part of a protein fused to a portion of HBc to assemble into a VLP structure (shorthanded as “FH-M2e” in this disclosure). With linkers, the antigenic sequence inserted into FH VLP was 146 amino acids residues in length. This fusion-protein-based vaccine embodiment was found to confer cross-protective immunity against various influenza A viral strains, offering strong potential as a universal influenza vaccine that can elicit anti-M2e antibody responses against seasonal, pandemic, and pre-pandemic viruses.
In a second recombinant model, the CTL epitope of ovalbumin (OVA) was also designed to replace the highly variable D3 domain of flagellin for display on the FH VLP surface (shorthanded as “FH-OVA” in this disclosure). Once expressed, our data suggest FH-OVA can be used to elicit OVA-specific CTL responses against OVA-expressing E.G7 lymphoma or Bl6F10 melanoma, therefore is a potential vaccine against multiple types of cancer.
FH VLP-based vaccines according to principles of the invention showed higher immunogenicity and protective efficacy as compared to FljB or HBc VLP-based vaccines. FH-M2e VLP mainly induces anti-M2e antibody responses to confer protection against viral challenges, while FH-OVA VLP mainly induces CTL responses to protect against OVA-expressing lymphoma or melanoma challenges. The ability of the FH VLP platform to elicit potent humoral and cellular immune responses against surface-displayed antigens supports its broad application in vaccine development against extracellular or intracellular pathogens or tumors. The high immunogenicity of FH VLP platform is expected to be due to the adjuvant effects of the particular form of FljB, which was found to potentiate Thl-biased antibody responses and vaccine-specific CTL responses. In comparison, soluble FljB was found to mainly potentiate Th2-biased antibody responses and only weakly stimulate vaccine-specific CTL responses.
Inventors further found that immunogenicity and protective efficacy of FH VLP-based vaccines could be substantially increased by incorporation of certain adjuvants, such as a clinical CpG 1018 adjuvant. To their surprise, a relatively low dose of CpG 1018 (2 μg) was found to significantly increase FH-M2e VLP-induced anti-M2e antibody titer by 6.5 folds and increase FH-M2e VLP-induced protection from 60% to 100%. Interestingly, 2 μg CpG 1018 was ineffective to enhance FH-OVA VLP-induced CTL responses and anti-tumor immunity (data not shown), which was achieved with increased CpG 1018 dose at 40 pg. This might reflect the differential CpG 1018 dose required to potentiate humoral and cellular immune responses elicited by FH VLP-based vaccines. Furthermore, inventors found incorporation of 2 μg CpG 1018 into FH-M2e VLP immunization didn't increase the risk of systemic adverse reactions in mice. Significantly increased immunogenicity and preserved safety strongly support low dose CpG 1018-adjuvanted FH-M2e VLP-based universal influenza vaccine approach in both human and animal models. As 2 μg CpG 1018 in mice translates to about 400 μg CpG 1018 in a regimen administered to human, in a preferred embodiment of low-dose administration, about or less than 400 μg per dose of CpG 1018 is administered as an adjuvant with a vaccine based on a VLP disclosed herein with. Incorporation of 40 μg CpG 1018 into FH-OVA VLP immunization induced a low level of IL-6 and TNFα release in mice, which gradually reduced after repeated immunizations, similar to that observed in FljB-OVA immunizations. Examples disclosed herein used OVA as a model to test whether compositions of the invention could effectively present tumor antigens, but it is within the contemplation of the present invention that tumor-associated antigens (TAAs) or neoantigens can be readily displayed on FH VLP surface by replacing D3 domain of FljB (or otherwise inserting into a flagellin sequence) to elicit potent Thl-biased antibody and CTL responses and incorporation of CpG 1018 can be further increase FH VLP-based tumor vaccine efficacy.
Similar to what was found with the conjugate model, tests conducted with the recombinant model also found that FH VLP-based vaccines showed significantly reduced ability to cause body temperature increases in mice or to induce systemic cytokine release, supporting the good safety profile of FH VLP platform. The good systemic safety of FH VLP or FH VLP-based vaccines is likely due to its significantly reduced TLR5 activation ability considering TLR5 activation leads to IL-6 synthesis, which further activates CRP release. The highly oriented surface display of FljB likely embeds its D1 domain, responsible for TLR5 activation, in interior of FH VLPs.
Accordingly, the present invention further provides a pharmaceutical composition, or a kit containing the fusion proteins of the invention preferably assembled as VLPs. The composition or kit can further include a pharmaceutically acceptable carrier, additive or excipient. The fusion proteins of the invention, as exemplified by the FljB-HBc fusion protein, can be conveniently expressed in bacterial systems such as E. Coli, which the pharmaceutical industry has lots of experience with, in large scale and refolded in vitro to form VLPs. Alternately, there are many other ways to produce the VLPs of the invention, for example, direct purification under native condition after expression in E. Coli. The fusion protein of the present invention can also be expressed by any other expression system known in the art, including systems that use yeast, other bacteria, insect cells, mammalian cells, cell-free expression systems, plants, or transgenic animals followed by purification under native or denatured conditions.
In a preferred embodiment, E. Coli cells are first used to express the fusion protein of the invention. After E. Coli cells are lysed, the harvested supernatant is purified under denatured condition. Subsequently, the fusion proteins are allowed to self-assembly into VLP after undergoing dialysis to remove denaturing agent (Urea). The process is well practiced and can find a good description in “Protein production and purification” (Nat. Methods 2008, 5(2): 135-146.), “Overview of the Purification of Recombinant Proteins” (Curr. Protoc. Protein Sci. 2015, 80: 6.1.1-6.1.35.), or “Protein Expression Handbook” (accessible online at thermofisher.com/content/dam/LifeTech/global/Forms/PDF/protein-expression-handbook.pdf).
The superior immunogenicity, good safety, and potential for large-scale production support the use of the VLPs of the invention as vaccine carriers, vaccines, or adjuvant.
In various embodiments, the fusion proteins and VLPs of the present invention can be utilized to make vaccines against all kinds of pathogens and diseases, including and are not limited to: influenza viruses, Hepatitis viruses, human papillomavirus (HPV), human immunodeficiency viruses (HIV), the Norwalk virus, Ebola, and Marburg viruses.
In one embodiment, a vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing COVID-19, is made based on principles of the present invention. Viral surface spike (S) protein of SARS-CoV-2 that mediates viral infection, or a portion thereof, is recombinantly expressed to be displayed on FH VLP surface to elicit neutralization antibodies in order to prevent viral infection. More conserved intracellular nucleocapsid (N) protein is also chosen to display on FH VLP surface to elicit cytotoxic T lymphocyte (CTL) responses with potentially broad cross-protective immunity to eliminate virus-infected cells and promote recovery. Thus, S and N co-displayed FH VLPs (SN/FH VLPs) may serve as a highly immunogenic and safe vaccine to tackle current and potential future COVID pandemics.
FljB gene of S. Typhimurium strain LT2 (SEQ ID NO:1) and partial HBc gene encoding 1-149 region of adw subtype (SEQ ID NO:16) were synthesized by Thermo Fisher Scientific. A forward primer containing an Nde I recognition site, his-tag sequence, and thrombin cleavage site and a reverse primer containing an Xho I recognition site and stop codon were used to amplify the FljB gene.
A forward primer containing an Nde I recognition site and a reverse primer containing an Xho I recognition site were used to amplify the HBc (1-149 aa) gene.
Polymerase chain reaction (PCR) products of FljB and HBc genes were then subjected to Xho I and Nde I digestion and subsequently ligated into pET-29a vector digested with the same enzymes.
Overlapping PCR was used to insert FljB gene into c/el loop (N75-L84 in the sequence) of HBc at A80-S81. In fact, insertion can be made at any other location within the c/el loop such as P79-A80, or a portion or even the entire c/el loop can be replaced with the sequence that is being inserted. A flexible linker, e.g., one with one or more glycines and serines, such as GGGGSGGGGS or (G4S)2 (SEQ ID NO:18) was inserted between HBc and FljB sequences to increase protein chain flexibility during refolding. Other well-known flexible linkers can also be inserted between HBc and FljB sequences to increase protein chain flexibility. PCR products were purified, digested with Xho I and Nde I, and ligated into pET-29a vector. Successful ligation was confirmed with sequencing after transformation of ligated pET-29a vector into competent DH5a cells.
As shown in
In Example 1, DNA was constructed to express full-length FljB between A80 and S81 of c/el loop of HBc and (G45)2 linker was inserted between FljB and HBc sequences to increase protein chain flexibility (
Next, FljB-HBc and HBc samples were subjected to dynamic light scattering (DLS) analysis in Zetasizer Nano ZS (Malvern). As shown in
In addition, immunogold labeling TEM was conducted to verify the presence of FljB on FH VLP surface. As shown in
Particle size of the different VLPs were further measured after immunogold labeling. As shown in
Flagellin activates both TLR5 and NLRC4 inflammasome. The ability of FH VLPs to activate TLR5 and NLRC4 inflammasome was explored and compared with FljB and FLA-ST. FLA-ST is ultrapure FliC purified from S. Typhimurium based on manufacturer's description. HEK293 cells co-transfected with murine TLR5 and SEAP reporter gene were incubated with FH VLP, FljB, and FLA-ST at different molar concentrations of respective proteins. As shown in
Next, the ability of FH VLPs to activate NLRC4 inflammasome was tested. NLRC4 inflammasome is a multi-protein complex, the activation of which cleaves pro-Caspase-1 to form p10/p20 heterodimer. Here Caspase-1 activation was directly analyzed following incubation of BMDCs with FH VLPs, FljB, and FLA-ST to reflect their NLRC4 inflammasome activation ability. As shown in
Considering that FljB does not require strong activation of innate immunity to exert its adjuvant effects, adjuvant effects from FH VLPs on boosting co-administered OVA immunization in BALB/c mice were tested. As shown in
To explore whether adjuvant effects of FH VLPs could be observed in another species of mice (C57BL/6 mice) and whether FH VLPs could dose-dependently increase OVA-induced antibody production, C57BL/6 mice were intradermally immunized with OVA alone or in the presence of increasing FH VLP doses (3, 10, and 30 μg). Results show that low dose but not medium or high dose of FH VLPs significantly increased anti-OVA antibody titer in C57BL/6 mice (
These mouse immunization studies indicate that FH VLPs possessed potent adjuvant effects across different species despite the lack of significant TLR5 and NLRC4 inflammasome activation abilities.
Example 4
Effective vaccine carriers need to be efficiently taken up by antigen presenting cells (APCs) in a mammalian subject. Accordingly, uptake of FH VLPs and FljB was tested in BMDCs. Soluble proteins are often endocytosed and sorted through early endosomes, late endosomes, and lysosomes (J. S. Blum, et al., Annu. Rev. Immunol. 2013, 31: 443-73). Particulate antigens are often phagocytosed and end up in phagolysosomes formed by fusion of phagosomes and lysosomes. Considering lysosomes and phagolysosomes have a pH of 4-4.5, LysoTracker capable of staining acidic organelles was used to stain both structures in this example.
BMDCs were incubated with AF555-conjugated FH VLPs, FljB, and HBc VLPs at equal molar concentrations of respective proteins. FH VLPs showed more significant uptake than FljB as evidenced by much stronger AF555 signals in FH VLP group than FljB group (Antigen,
BMDC uptake of FH VLPs, FljB, and HBc VLPs at equal molar concentrations was also examined through flow cytometry. As shown in
Similar results were observed once FH has been recombinantly engineered to carry an antigenic sequence (e.g., M2e) and allowed to fold and assemble into FH VLPs that also display the antigen in high density.
Besides efficient uptake, stimulation of DC maturation by vaccine carriers is also crucial to elicit potent vaccine-specific immune responses. Accordingly, the relative ability of FH VLPs to stimulate BMDC maturation is tested alongside FljB, FLA-ST, and HBc VLPs.
In brief, BMDCs were stimulated with FH VLPs, FljB, FLA-ST, and HBc VLPs at equal molar concentrations of respective proteins. Surface expression of costimulatory molecules CD40, CD80, and CD86 was tested 20 hours later. It was found that BMDCs could be divided into two populations based on CD40 expression: CD40high (CD40high) and CD40low (CD40lo), as shown in
FH VLPs and FLA-ST slightly increased MFI of CD80 and CD86 (
Next, percentages of CD40hi, CD80hi, and CD86hi DCs among the different groups were compared. The percentage of CD40hi, CD80hi, and CD86hi DCs was found to be significantly higher in the FLA-ST group than in the HBc VLP group, and the percentage of CD40hi DCs was significantly higher in the FH VLP group than in the HBc VLP group (
Further next, whether FH VLPs could also stimulate DC maturation in vivo was put to test. To explore this, C57BL/6 mice were intradermally injected with FH VLPs, FljB, and HBc VLPs. Skin was dissected 24 hours later for test of CD40, CD80, and CD86 expression on CD11c+ DCs. As shown in
More efficient uptake and stimulation of DC maturation indicate that FH VLPs might have improved immunogenicity as compared to FljB. To prove this, mice were immunized with FH VLPs and FljB (equal moles) and anti-FljB antibody responses were then measured and compared between groups.
Flagellin was reported to mainly induce Thl-biased IgG1 antibody responses with high dependence on MyD88. To test FH VLP-induced antibody isotype and the dependence on MyD88, MyD88 knock-out (KO) mice were also included in the above immunization studies. As shown in
FH VLP and FljB-induced isotype IgG1 and IgG2c antibody titers were also compared. As shown in
Due to the crucial role of IL-12 in skewing Thl -biased immune responses (C. Heufler, et al., Eur. J. Immunol. 1996, 26 (3): 659-68), IL-12 levels in BMDC stimulation studies were measured and it was found that FH VLPs but not FljB or FLA-ST stimulated significant IL-12 release (
The above studies strongly indicate that FH VLPs are more immunogenic than FljB in stimulating anti-FljB antibody production.
Significantly reduced TLR5 activation ability implicates that FH VLPs might have improved safety as compared to FljB. To prove this, serum IL-6 levels were first measured following FH VLP and FljB immunization of WT and MyD88 KO mice in the above studies (
As shown in
The relative ability of FH VLPs and FljB to induce systemic IL-6 release was also tested in BALB/c mice. FljB was found to significantly increase serum IL-6 levels at 6 hours, while FH VLPs failed to significantly increase serum IL-6 levels at the same time (
Besides induction of systemic IL-6 release, flagellin-based vaccines had also induced systemic adverse reactions, like fever, in clinical trials. Next, the abilities of FH VLPs and FljB to each induce fever-related systemic adverse reactions in murine models were compared. To test this, C57BL/6 mice were intradermally immunized with increased doses of FH VLPs and FljB (20 and 14.8 μg, respectively) weekly for 3 weeks and rectal temperature was measured right before and 24 hours after each immunization. Rectal temperature was measured with a mouse rectal temperature probe connected to PhysioSuite (Kent Scientific). As shown in
The above examples have indicated that FH VLPs might be a more immunogenic and safer carrier than FljB for vaccine development. Next, nicotine vaccines were used as a model to compare relative immunogenicity and safety of FH VLPs to FljB, HBc VLPs, or the widely used keyhole limpet hemocyanin (KLH) as each immunogen was conjugated to nicotine hapten and the resultant complex noted with “-nic”.
Specifically, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) was used to activate the carboxylic acid group of nicotine hapten 6-(carboxymethylureido)-(+/−)-nicotine (CMUNic) for conjugation to primary amine groups of protein immunogens. This procedure is well documented and reported in the art. In more detail, 1 mg CMUNic was activated with 5 mg EDC (E1769, Sigma) in 0.1M 2-(N-morpholino)ethanesulfonic acid (IVIES) buffer (pH4.5) at room temperature for 10 min. FH VLP or other immunogens (1 mg) dissolved 0.1M MES buffer (pH4.5) was added to the above mixture and kept at room temperature for 3 hours with continuous stirring. Nicotine conjugates were then dialyzed against PBS for 3 times to remove unreacted EDC or CMUNic.
Nicotine vaccines hold a great promise for anti-nicotine immunotherapy (T. Raupach, et al., Drugs 2012, 72 (4): el-16). Despite the unsatisfactory clinical trial outcomes for several nicotine vaccines (NicVAX, NicQb, Niccine), different strategies are under exploration to improve anti-nicotine immunotherapy that include making improvement in vaccine carriers (e.g., J.W. Lockner, et al., J. Med. Chem. 2015, 58 (2): 1005-11).
KLH was found to be a more immunogenic carrier than recombinant Pseudomonas Exoprotein A (rEPA) used in NicVAX for nicotine vaccine development (P.T. Bremer, et al., Pharmacol. Rev. 2017, 69(3): 298-315). Flagellin (FliC) was also explored as both vaccine carriers and adjuvants for nicotine vaccine development (N. T. Jacob, et al., J. Med. Chem. 2016, 59 (6): 2523-9). FliC-based nicotine vaccine was found to induce anti-nicotine antibodies with superior nicotine binding to Tetanus Toxoid (TT)-based nicotine vaccine. Here, relative immunogenicity of KLH was first compared to FljB and HBc VLPs for nicotine vaccine development, and tests showed that KLH-Nic elicited —2 times higher anti-nicotine antibody (NicAb) titer than FljB-Nic (data not shown). In another study, relative immunogenicity of KLH-Nic was compared to HBc-Nic, and it was found that KLH-Nic elicited —5 times higher NicAb titer than HBc-Nic (data not shown). These studies indicate that KLH was more immunogenic than FljB and HBc VLPs for nicotine vaccine development.
Next, the relative immunogenicity of FH-Nic was compared to KLH-Nic. Due to the reported synergy between two of the three adjuvants (FljB, CpG, and Alum) (see, e.g., J. W. Lockner, et al., Mol. Pharm. 2015, 12 (2): 653-62), the inventors also evaluated whether incorporation of CpG, Alum, or combinatorial CpG/Alum adjuvant could increase the immunogenicity of FH-Nic. As shown in
Antibody isotype analysis found FH-Nic mainly increased anti-nicotine IgG2a but not IgG1 antibody titer as compared to KLH-Nic (
Mice were further challenged with intravenous nicotine and brain and blood were collected 5 minutes later to quantify tissue nicotine levels. As compared to brain nicotine levels of non-immunized mice, KLH-Nic immunization reduced brain nicotine levels by 27% whereas FH-Nic immunization reduced brain nicotine levels by an impressive 63% (
Data of all groups were then pooled for linear correlation analysis. A negative correlation was identified between serum NicAb titer and brain nicotine levels (
Lastly, the inventors explored local and systemic safety of intradermal (ID) FH-Nic immunization and found that ID FH-Nic induced minimal local reactions as supported by the lack of visible changes of FH-Nic-injected skin. ID FH-Nic also failed to induce significant systemic reactions as supported by the baseline levels of serum IL-6 at 6 and 24 hours after immunization (data not shown).
The disclosure of information, methods and data contained in the inventors' publication Y. Zhao et al., Biomaterials, 2020, 249: 120030, is incorporated by reference in its entirety to the extent allowed by the law.
M2e (influenza matrix protein 2 ectodomain) is highly conserved region among influenza A viruses and has been a highly attractive target for universal influenza vaccine development. Yet, M2e has low immunogenicity and cannot mount potent immune responses. FljB as a potential vaccine carrier has been explored for M2e-based universal influenza vaccine development (FljB-M2e). Yet, FljB-M2e was found to induce systemic adverse reactions in humans due to its overt activation of TLR5 .
The VLPs of the invention (exemplied by FH VLPs) have shown significantly improved immunogenicity and safety as compared to FljB for nicotine vaccine development (Example 8 above). In this example, inventors set up to test the relative immunogenicity and safety of FH VLPs in comparison to FljB and HBc for M2e-based universal influenza vaccine development.
Tandem copies of human Hl/H3, swine H1, and avian H5/H7′s M2e sequences (SEQ ID NOS:19-22), as shown in
For comparison purposes, the same sequence of the four tandem copies of M2e variants (with linkers) was used to replace the D3 domain of FljB to generate a recombinant “FljB-M2e” vaccine, and also inserted into the c/el loop of HBc to see whether the resultant “HBc-M2e” fusion protein could self-assemble into VLPs. After recombinant plasmids were constructed, the different immunogens each with a foreign antigen inserted in its sequence (FH-M2e, FljB-M2e, HBc-M2e) were expressed in E. Coli, purified under denaturing conditions, and allowed to refold.
Protein expression was examined in SDS-PAGE (
Example 10: FH-M2e but not HBc-M2e self-assembles into VLPs
Transmission electron microscopy (TEM) was used to explore whether FH-M2e and HBc-M2e formed VLPs after refolding. As shown in
Dynamic light scattering (DLS) was then used to measure FH-M2e VLP size. As shown in
The uptake ability of the different M2e immunogens by bone marrow derived DCs (BMDCs) were compared. FH-M2e VLPs and HBc-M2e showed a strong uptake, while FljB-M2e and M2e peptide showed a weak uptake by BMDCs (data not shown). Furthermore, significant overlapping of antigen and lysosomal signals was found in FH-M2e VLP and HBc-M2e groups (data not shown), hinting efficient uptake of FH-M2e VLPs and HBc-M2e via endolysosomal pathways.
The ability of the different immunogens to stimulate BMDC maturation was also tested. As shown in
First, the inventors compared relative immunogenicity of FljB-M2e with HBc-M2e, and M2e peptide in the presence of Alum adjuvant (M2e/Alum). As shown in
Serum IL-6 and TNFα levels were also measured at before immunization, 3 hours after, and 18 hours after immunization since both are downstream of TLR5 and linked to systemic adverse reactions of FljB-M2e vaccine in humans. As shown in
Second, the inventors compared relative immunogenicity of FH-M2e VLPs and FljB-M2e. Furthermore, they also explored whether incorporation of CpG 1018 adjuvant could enhance immunogenicity of FH-M2e VLPs. CpG 1018 is a toll-like receptor (TLR) 9 agonist and has been a clinical adjuvant to boost hepatitis b vaccine efficacy. CpG 1018 is broadly effective in mice, non-human primates, and humans. As shown in
After PR8 viral challenges (8×LD50), mice in FH-M2e VLP groups showed significantly less body weight loss than that in FljB-M2e group (
These data strongly indicate the superiority of FH-M2e VLPs (a recombinant embodiment of VLPs of the invention) compared to FljB-M2e in eliciting immune protection against lethal PR8 viral challenges and that the incorporation of CpG could further enhance protective efficacy of FH-M2e VLPs.
The inventors further compared systemic cytokine release among immunization of the different vaccine candidates as a way to further study systemic safety issues. As shown in
Body temperature increase was not detected following FljB-M2e immunizations in the above studies. Based on the literature, intradermal immunization is likely to elicit more adverse reactions. To test this, mice were intradermally immunized with FljB-M2e and FH-M2e VLPs. Rectal temperatures of mice were measured before and 24 hours after intradermal immunization. As shown in
Example 13
In this example, any broad cross-protective abilities from FH-M2e VLP or FljB-M2e immunization were evaluated. Mice were prime/boost immunized and then challenged with three different influenza viruses. As shown in
In sum, our data support VLPs of the invention to be a more immunogenic, more versatile, and safer carrier for universal influenza vaccine development, at least by incorporating the M2e-immunogenic sequence into the platform, e.g., recombinantly as part of the fusion protein of the invention.
In a model for cancer vaccine development, the cytotoxic T-lymphocyte (CTL) epitope of ovalbumin (OVA) was recombinantly engineered to replace the highly variable D3 domain of flagellin for display on the FH VLP surface (referred to as “FH-OVA”, SEQ ID NO:24), as schematically shown in
For example, OVA247-274 (“DEVSGLEQLESIINFEKLTEWTSSNVME”) (SEQ ID NO: 25) encompassing the underlined CTL epitope of OVA257-264 (“SIINFEKL”) (SEQ ID NO: 26) in the middle region was used to elicit OVA-specific CTL responses against OVA-expressing E.G7 lymphoma or Bl6F10 melanoma (schematically illustrated in
After all the recombinant plasmids were constructed and OVA-epitope-inserted FljB-HBc, FljB and HBc polypeptides were expressed in E. Coli, purified, and refolded, protein expression was examined in SDS-PAGE and compared with theoretical molecular weight (
FH and HBc samples were further analyzed by TEM and DLS. Successful formation of FH VLP was found after replacement of the D3 domain of FljB with OVA247-274 (data not shown). Successful formation of HBc VLP was also found after insertion of OVA247-274 into its c/el loop (data not shown). DLS analysis found the OVA-displaying FH VLP to be similar in size as the M2e-displaying FH VLP reported in Example 10, which was bigger than OVA-displaying HBc VLP (data not shown).
Next, the relative potency of FH-OVA VLPs to induce OVA-specific CTL production was compared with HBc-OVA VLPs and FljB-OVA and also OVA247-274 peptide (shorthanded as “OVA”). Due to the low abundance of OVA-specific CD8+ T cells, the inventors adoptively transferred OVA-specific CD8+ T cells isolated from OT-I mice to native C57BL/6 mice followed by intradermal immunization with the different immunogens containing the same amount of OVA247-274 peptide. Draining lymph nodes were harvested 4 days later and CF SE+cells in CD8+ T cells were analyzed.
As shown in
Anti-tumor immunity was then compared following three repeated immunization with the different immunogens followed by challenging mice with OVA-expressing E.G7 lymphoma or Bl6F10 melanoma. Tumor growth and survival of tumor-bearing mice were monitored and compared among groups. FH-OVA VLP immunization most significantly retarded E.G7-OVA lymphoma growth with tumor volume significantly smaller than that in non-immunized group on day 13 and 16. In contrast, HBc-OVA VLP, FljB-OVA, and OVA peptide immunization failed to significantly reduce E.G7-OVA lymphoma growth with tumor volume at each time point showed no significant difference from that in non-immunized group (
Besides OVA-expression E.G7 lymphoma model, immunized mice were also challenged with OVA-expressing Bl6F10 melanoma in a different study. As shown in
FH-OVA VLP immunization also significantly extended survival of Bl6F10-OVA-bearing mice, while HBc-OVA VLP immunization failed to significantly extend survival of N16F10-OVA-bearing mice (
The above results indicate that FH VLPs provided the most immunogenic carrier to develop cancer vaccines to elicit the most potent CTL production and anti-tumor immunity, as compared to HBc VLPs and FljB. Although only a short CTL epitope was used as a model in the current example, longer antigenic epitopes and full-length neoantigens can be readily inserted for display on FH VLPs to elicit potent anti-tumor immunity as D3 domain of FljB allows insertion of diverse vaccine antigens with little restriction on length or 3D structure.
Although inventors mainly focused on exploring CTL and anti-tumor responses, anti-OVA247-274 antibody responses were also measured after the completion of the last immunization. In those studies, FljB-OVA and FH-OVA VLP but not HBc-OVA VLP induced significantly higher anti-OVA IgG titer as compared to OVA immunization alone (data not shown). Further, it was found that FljB-OVA induced significantly higher IgG1 titer, while FH-OVA VLP induced significantly higher IgG2c titer, as compared to OVA immunization alone (data not shown). This indicates that FH-OVA VLP induced mainly Thl-biased antibody responses, while FljB-OVA mainly induced Th2-biased antibody responses.
Furthermore, PBMCs were prepared 7 days after the last immunization and stimulated with synthetized OVA247-274 peptide followed by intracellular cytokine staining and flow cytometry analysis. HBc-OVA and FH-OVA VLP immunization induced the most significant increase of IFNy-secreting CD8+ T cells, while FH-OVA VLP but not HBc-OVA VLP immunization significantly increased IL4-secreting CD8+ T cells. These data support the high potency of FH-OVA VLPs to induce IFNy and IL4-secreting CTLs. Furthermore, FH-OVA VLP but not HBc-OVA VLP or other immunizations significantly increased IFNy and IL4-secreting CD4+ T cells (data not shown). Significant increase of CD4+ helper T cells was in line with the induction of highest anti-OVA antibody titer in FH-OVA VLP group.
Example 16: CpG 1018 boosts FH-OVA VLP immunization
In this example, inventors explored whether incorporation of CpG 1018, a Th1 adjuvant, could further enhance CTL production and anti-tumor immunity of FH-OVA VLPs.
Adoptive transfer and immunization studies were first conducted to evaluate OT-I cell expansion as well as Granzyme B, TNFa, and IFNy expression due to crucial roles of these cytokines in anti-tumor immunity. As shown in
Mice were then immunized with FH-OVA VLPs in the presence or absence of CpG 1018 or PBS to serve as control. Immunization was repeated three times and one week after the last immunization, PBMCs were collected, stimulated with CTL epitope of OVA, and cytokine-secreting OVA-specific CD8+ T cells were then analyzed. Incorporation of CpG 1018 into FH-OVA VLP immunization could significantly enhance OVA-specific single, dual, and triple cytokine-secreting CD8+ T cells, in line with the above adoptive transfer study (data not shown). Although inventors mainly focused on exploring CTL responses in this study, anti-OVA247-274 IgG, subtype IgG1 and IgG2c antibody responses were also measured. Results showed that CpG 1018 adjuvant significantly increased IgG2c but not IgG1 antibody production. The ratio of IgG2c to IgG1 was significantly increased by incorporation of CpG 1018 adjuvant (data not shown), hinting the induction of a Thl-biased antibody responses. CpG 1018 also increased total IgG level to a non-statistically significant level due to the large variations of IgG titer in both groups (data not shown).
Mice were then challenged with Bl6F10-OVA melanoma. As shown in
Example 17: Better systemic safety of FH VLP-based vaccines as compared to flagellin-based vaccines
Besides evaluation of the immunogenicity and protective efficacy, systemic safety of FH VLP-based vaccines was also tested and compared against flagellin-based vaccines. Since over-activation of TLR5 was likely contributed to the observed systemic safety of flagellin-based vaccines, inventors mainly measured serum IL-6 and TNFα levels as these two cytokines are downstream of TLR5 signaling pathways and linked to systemic adverse reactions of flagellin-based vaccines in humans.
In Example 12 above, inventors found FH-M2e VLPs did not significantly increase serum IL-6 levels at 3 hours in either prime or boost immunization and only slightly increased serum TNFα levels at 3 hours in boost but not prime immunization (
Besides M2e-based universal influenza vaccines, inventors also compared systemic cytokine release following three immunizations of OVA-based cancer vaccines in this example. FljB-OVA induced the most significant increase of serum IL-6 and TNFα levels at 3 hours following each immunization, while FH-OVA VLP immunization did not significantly increase serum IL-6 and TNFα levels (
The generation of FH VLP-bases vaccines involves construction of recombinant plasmid to express FljB-HBc fusion proteins, in which FljB is inserted into c/el loop of HBc and D3 domain of FljB is replaced with vaccine antigens, followed by E. Coli expression, purification, and self-assembly into VLPs (
While the present invention has been particularly shown and described with reference to the structure and methods disclosed herein and as illustrated in the drawings, it is not confined to the details set forth and this invention is intended to cover any modifications and changes as may come within the scope and spirit of the following claims. All publications and patent literature described herein are incorporated by reference in entirety to the extent permitted by applicable laws and regulations.
This application claims priority to and the benefit of co-pending U.S. provisional patent applications Ser. Nos. 63/173,994 and 63/277,011, filed Apr. 12, 2021 and Nov. 8, 2021, respectively, which applications are incorporated herein by reference in entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63173994 | Apr 2021 | US | |
| 63277011 | Nov 2021 | US |
