Patent Application
20240252619
The content of the ASCII text file of the sequence listing named “20220520_034044_219W01_ST25” which is 293,529 bytes in size was created on May 20, 2022, and electronically submitted via EFS-Web herewith the application is incorporated herein by reference in its entirety.
The field generally relates to compositions and methods for inducing an immune response against coronavirus antigens.
The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak precipitated a major global health crisis. To date, there is yet to be a safe and effective vaccine against SARS-CoV-2.
In some embodiments, the present invention is a nucleic acid construct comprising, consisting essentially of, or consisting of a Zika Vector having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 44 or SEQ ID NO: 43 and lacking sequences encoding functional Zika virus capsid, matrix, and envelope proteins. In some embodiments, the nucleic acid construct further comprises one or more coronavirus sequences. In some embodiments, the Zika Vector has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 43 or SEQ ID NO: 44. In some embodiments, the nucleic acid construct further comprises a CMV promoter or a T7 promoter. In some embodiments, the nucleic acid construct further comprises a sequence that encodes Bacteriophage T4 Fibritin foldon and/or a sequence that encodes an IgK signal peptide. In some embodiments, the nucleic acid construct further comprises a SV40 PolyA sequence. In some embodiments, the nucleic acid construct further comprises a Hepatitis delta virus ribozyme (HDVR) sequence. In some embodiments, the nucleic acid construct further comprises a pBR322 cloning vector sequence. In some embodiments, the nucleic acid construct further comprises a sequence encoding a 2A self-cleaving peptide, e.g., F2A (SEQ ID NO: 32). In some embodiments, the nucleic acid construct further comprises one or more linker sequences selected from SEQ ID NO: 2, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 33, and SEQ ID NO: 35. In some embodiments, the one or more coronavirus sequences encodes a peptide sequence having (a) 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 75, at least 100, at least 125, at least 150, or at least 200 consecutive amino acid residues of a known coronavirus protein; (b) 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 75, at least 100, at least 125, at least 150, or at least 200 consecutive amino acid residues of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, or SEQ ID NO: 42; or (c) SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, or SEQ ID NO: 42. In some embodiments, the nucleic acid construct has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40.
In some embodiments, the present invention is a nucleic acid construct comprising, consisting essentially of, or consisting of a Zika Vector and one or more coronavirus sequences. In some embodiments, the Zika Vector is (a) a replication defective viral vector that comprises a Zika virus genome except for the sequences that encode functional Zika virus capsid, matrix, and envelope proteins; or (b) a replication defective viral vector that comprises a Zika virus genome and lacks the sequences that encode Zika virus capsid, matrix, and envelope proteins. In some embodiments, the Zika virus genome encodes the proteins encoded by SEQ ID NO: 45 (Accession No. NC_012532.1). In some embodiments, the Zika virus genome has 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 45 (Accession No. NC_012532.1). In some embodiments, the Zika Vector has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 43 or SEQ ID NO: 44. In some embodiments, the nucleic acid construct further comprises a CMV promoter or a T7 promoter. In some embodiments, the nucleic acid construct further comprises a sequence that encodes Bacteriophage T4 Fibritin foldon and/or a sequence that encodes an IgK signal peptide. In some embodiments, the nucleic acid construct further comprises a SV40 PolyA sequence. In some embodiments, the nucleic acid construct further comprises a Hepatitis delta virus ribozyme (HDVR) sequence. In some embodiments, the nucleic acid construct further comprises a pBR322 cloning vector sequence. In some embodiments, the nucleic acid construct further comprises a sequence encoding a 2A self-cleaving peptide, e.g., F2A (SEQ ID NO: 32). In some embodiments, the nucleic acid construct further comprises one or more linker sequences selected from SEQ ID NO: 2, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 33, and SEQ ID NO: 35. In some embodiments, the one or more coronavirus sequences encodes a peptide sequence having (a) 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 75, at least 100, at least 125, at least 150, or at least 200 consecutive amino acid residues of a known coronavirus protein; (b) 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 75, at least 100, at least 125, at least 150, or at least 200 consecutive amino acid residues of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, or SEQ ID NO: 42; or (c) SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, or SEQ ID NO: 42. In some embodiments, the nucleic acid construct has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40.
In some embodiments, the present invention is a composition comprising, consisting essentially of, or consisting of one or more nucleic acid constructs as described herein and a pharmaceutically acceptable carrier and/or an adjuvant.
In some embodiments, the present invention is a method of inducing an immune response in a subject, which comprises, consists essentially of, or consists of administering to the subject an immunogenic amount of one or more nucleic acid constructs or a composition thereof as described herein.
In some embodiments, the present invention is a method of treating or inhibiting an infection by a coronavirus in a subject, which comprises, consists essentially of, or consists of administering to the subject a therapeutically effective amount or an immunogenic amount of one or more nucleic acid constructs or a composition according thereof as described herein.
In some embodiments, the present invention is a method of treating a subject for a COVID disease, which comprises, consists essentially of, or consists of administering to the subject a therapeutically effective amount of one or more nucleic acid constructs or a composition thereof as described herein.
Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention, and together with the description explain the principles of the invention.
This invention is further understood by reference to the drawings wherein:
The schematic representations of the constructs were modified to improve reproducibility of the drawings. While some of the detail, e.g., restriction enzyme sites, appear slightly different from those presented in U.S. Application Nos. 63/191,829 and 63/197,031, the nucleic acid sequences are the same such that the lack of any detail that was previously presented is in no way a disclaimer of subject matter.
Disclosed herein are “coronavirus constructs” which comprise one or more coronavirus sequences inserted in a Zika Vector as schematically shown in
The coronavirus constructs may be targeted or delivered to antigen presenting cells such as dendritic cells and macrophages in a subject After administering to a subject, the coronavirus constructs induce a humoral and T cell immune response against the coronavirus sequences.
The coronavirus constructs as described herein may be used to induce an immune response in a subject, treat or inhibit an infection by a coronavirus in a subject, or treat a subject for a COVID disease. Therefore, in some embodiments, the present invention provides methods for inducing an immune response in a subject, which comprise administering to the subject an immunogenic amount of one or more coronavirus constructs or a composition thereof. In some embodiments, the present invention provides methods for treating or inhibiting an infection by a coronavirus in a subject, which comprise administering to the subject a therapeutically effective amount or an immunogenic amount of one or more coronavirus constructs or a composition thereof. In some embodiments, the present invention provides methods for treating a subject for a COVID disease, which comprise administering to the subject a therapeutically effective amount of one or more of one or more coronavirus constructs or a composition thereof.
As used herein, a “COVID disease” refers to a disease, such as COVID-19, caused by an infection by a coronavirus.
Unless explicitly indicated otherwise, the term “coronavirus” is used to refer to any virus classified as belonging to Coronaviridae. In some embodiments, the coronavirus is an alphacoronavirus. In some embodiments, the coronavirus is a betacoronavirus. In some embodiments, the coronavirus is a gammacoronavirus. In some embodiments, the coronavirus is a deltacoronavirus. In some embodiments, the coronavirus is a Severe Acute Respiratory Syndrome-Related Coronavirus strain (e.g., SARS-CoV, SARS-CoV-2). In some embodiments, the coronavirus is a Middle East Respiratory Syndrome-Related Coronavirus strain (e.g., MERS-CoV). In some embodiments, the coronavirus has a genome that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to Accession No. NC_004718.3 (SARS-CoV), Accession No. NC_045512.2 (SARS-CoV-2), or Accession No. NC_019843.3 (MERS-CoV). In some embodiments, the coronavirus has a genome that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to polyprotein encoded by Accession No. NC_004718.3 (SARS-CoV), Accession No. NC_045512.2 (SARS-CoV-2), or Accession No. NC_019843.3 (MERS-CoV).
As used herein, “coronavirus sequences” refer to sequences that encode peptide sequences of proteins (e.g., spike (S) proteins, nucleocapsid (N) proteins, matrix/membrane (M) proteins, envelope (E) proteins, RNA dependent RNA polymerases (RdRp)), and fragments thereof of a coronavirus. In some embodiments, the coronavirus sequence encodes a peptide sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Accession Nos.: YP_009724389.1, YP_009725295.1, YP_009724390.1, YP_009724391.1, YP_009724392.1, YP_009724393.1, YP_009724395.1, YP_009724397.2, NP_828849.2, NP 828850.1, NP_828851.1, NP_828852.2, NP_828853.1, NP_828855.1, NP_828857.1, NP_828858.1, YP_009047202.1, YP_009047203.1, YP_009047204.1, YP_009047205.1, YP_009047206.1, YP_009047207.1, YP_009047208.1, YP_009047209.1, YP_009047210.1, YP_009047211.1, YP_009047212.1, and fragments thereof. In some embodiments, the coronavirus sequence encodes a peptide sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and fragments thereof. In some embodiments, the peptide sequences are at least 75, at least 100, at least 125, at least 150, or at least 200 amino acid residues in length. In some embodiments, the coronavirus sequence encodes a peptide sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 75, at least 100, at least 125, at least 150, or at least 200 consecutive amino acid residues of a known coronavirus protein. In some embodiments, the coronavirus sequence encodes a peptide sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 75, at least 100, at least 125, at least 150, or at least 200 consecutive amino acid residues of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, or SEQ ID NO: 19.
In some embodiments, the present invention is directed to a Zika Vector and compositions comprising one or more Zika Vectors. As used herein, a “Zika Vector” or “Zika Vaccine Vector” refers to a replication defective viral vector that comprises a Zika virus genome except for sequences encoding functional Zika virus capsid, matrix, and envelope proteins. In some embodiments, the Zika Vectors lack sequences that encode Zika virus capsid, matrix, and envelope proteins. The Zika virus genome may be of any Zika virus known in the art. In some embodiments, the Zika virus genome has 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 45 (Accession No. NC_012532.1). In some embodiments, the Zika virus genome comprises the sequence set forth in SEQ ID NO: 45 (Accession No. NC_012532.1). In some embodiments, the Zika virus genome comprises a sequence as set forth in SEQ ID NO: 45 (Accession No. NC_012532.1), with one or more silent mutations thereof. In some embodiments, the Zika Vector comprises a sequence that has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 43. In some embodiments, the Zika Vector comprises a sequence that has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 44. In some embodiments, the Zika Vector comprises a sequence that has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to nucleotides 742-9337 of SEQ ID NO: 1. In some embodiments, the Zika Vector comprises nucleotides 742-9337 of SEQ ID NO: 1, with one or more silent mutations. In some embodiments, the Zika Vector is codon-optimized for expression in primates, preferably humans. In some embodiments, the Zika Vector comprises a sequence that has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 22, with one or more silent mutations. In some embodiments, the Zika Vector comprises a CMV promoter, e.g., SEQ ID NO: 23. In some embodiments, the Zika Vector comprises a T7 promoter, e.g., SEQ ID NO: 24. In some embodiments, the Zika Vector comprises a BsiWI restriction site, e.g., SEQ ID NO: 1 or SEQ ID NO: 25. In some embodiments, the Zika Vector comprises a hepatitis delta virus ribozyme (HDVR) sequence, e.g., SEQ ID NO: 34. In some embodiments, the Zika Vector comprises an SV40 PolyA sequence, e.g., SEQ ID NO: 36. In some embodiments, the Zika Vector comprises a pBR322 cloning vector sequence, e.g., SEQ ID NO: 37. In some embodiments, the Zika Vector comprises a sequence that encodes an IgK signal peptide (e.g., SEQ ID NO: 27), a bacteriophage T4 Fibritin Foldon protein (e.g., SEQ ID NO: 30), and/or an 2A self-cleaving peptide such as F2A (SEQ ID NO: 32).
As used herein, a given percentage of “sequence identity” refers to the percentage of nucleotides or amino acid residues that are the same between sequences, when compared and optimally aligned for maximum correspondence over a given comparison window, as measured by visual inspection or by a sequence comparison algorithm in the art, such as the BLAST algorithm, which is described in Altschul et al., (1990) J Mol Biol 215:403-410. Software for performing BLAST (e.g., BLASTP and BLASTN) analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The comparison window can exist over a given portion, e.g., a functional domain, or an arbitrarily selection a given number of contiguous nucleotides or amino acid residues of one or both sequences. Alternatively, the comparison window can exist over the full length of the sequences being compared. For purposes herein, where a given comparison window length and/or position is not provided, the recited sequence identity is over 100% of the reference sequence. Additionally, for the percentages of sequence identity of the proteins provided herein, the percentages are determined using BLASTP 2.8.0+, scoring matrix BLOSUM62, and the default parameters available at blast.ncbi.nlm.nih.gov/Blast.cgi. See also Altschul, et al., (1997) Nucleic Acids Res 25:3389-3402; and Altschul, et al., (2005) FEBS J 272:5101-5109.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv Appl Math 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J Mol Biol 48:443 (1970), by the search for similarity method of Pearson & Lipman, PNAS USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection.
As used herein, the terms “protein”, “polypeptide” and “peptide” are used interchangeably to refer to two or more amino acids linked together. Groups or strings of amino acid abbreviations are used to represent peptides. Except when specifically indicated, peptides are indicated with the N-terminus on the left and the sequence is written from the N-terminus to the C-terminus.
In some embodiments, the present invention provides methods of making antibodies and/or a T cell mediated immune response against one or more coronavirus sequences, which comprises administering one or more coronavirus constructs as described herein to a subject. In some embodiments, the present invention provides methods of making antibodies against one or more coronavirus sequences, which comprises synthetically engineering the antibodies based on the sequences of those produced in a subject who has been administered one or more coronavirus constructs as described herein. As used herein, “antibody” refers to naturally occurring and synthetic immunoglobulin molecules and immunologically active portions thereof (i.e., molecules that contain an antigen binding site that specifically bind the molecule to which antibody is directed against). As such, the term antibody encompasses not only whole antibody molecules, but also antibody multimers and antibody fragments as well as variants (including derivatives) of antibodies, antibody multimers and antibody fragments. Examples of molecules which are described by the term “antibody” herein include: single chain Fvs (scFvs), Fab fragments, Fab′ fragments, F(ab′)2, disulfide linked Fvs (sdFvs), Fvs, and fragments comprising or alternatively consisting of, either a VL or a VH domain.
Compositions, including pharmaceutical compositions and vaccines, comprising, consisting essentially of, or consisting of one or more coronavirus constructs (e.g., COVID-VAX1, COVID-VAX2, COVID-VAX3, COVID-VAX4, and/or COVID-VAX5) are contemplated herein. As used herein, the phrase “consists essentially of” in the context of “a composition consisting essentially of [a given] coronavirus construct” means that the composition may comprise additional ingredients, including active pharmaceutical ingredients, except for coronavirus constructs other than the given coronavirus construct. The term “pharmaceutical composition” refers to a composition suitable for pharmaceutical use in a subject. A composition generally comprises an effective amount of an active agent and a diluent and/or carrier. A pharmaceutical composition generally comprises a therapeutically effective amount or an immunogenic amount, of an active agent and a pharmaceutically acceptable carrier. In addition to the one or more coronavirus constructs, pharmaceutical compositions may include one or more supplementary agents. Examples of suitable supplementary agents include vaccines and antivirals known in the art.
As used herein, a “pharmaceutically acceptable vehicle” or “pharmaceutically acceptable carrier” are used interchangeably and refer to solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration and comply with the applicable standards and regulations, e.g., the pharmacopeial standards set forth in the United States Pharmacopeia and the National Formulary (USP-NF) book, for pharmaceutical administration. Thus, for example, unsterile water is excluded as a pharmaceutically acceptable carrier for, at least, intravenous administration. Pharmaceutically acceptable vehicles include those known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th ed (2000) Lippincott Williams & Wilkins, Baltimore, MID. In some embodiments, the pharmaceutically acceptable vehicle is one that is suitable for intravenous, subcutaneous, or intranasal administration to a subject. In some embodiments, the pharmaceutically acceptable carrier is sterile. In some embodiments, the pharmaceutically acceptable carrier is sterile saline, which may be buffered. Preferred pharmaceutical compositions are those comprising, consisting essentially of, or consisting of one or more coronavirus constructs in a therapeutically effective amount or an immunogenic amount, and a pharmaceutically acceptable vehicle.
As used herein, an “effective amount” refers to a dosage or amount sufficient to produce a desired result. The desired result may comprise an objective or subjective change as compared to a control in, for example, in vitro assays, and other laboratory experiments. As used herein, a “therapeutically effective amount” refers to an amount that may be used to treat, prevent, or inhibit a given disease or condition in a subject as compared to a control, such as a placebo. Again, the skilled artisan will appreciate that certain factors may influence the amount required to effectively treat a subject, including the degree of the condition or symptom to be treated, previous treatments, the general health and age of the subject, and the like. Nevertheless, effective amounts and therapeutically effective amounts may be readily determined by methods in the art.
The one or more coronavirus constructs may be administered, preferably in the form of pharmaceutical compositions, to a subject. Preferably the subject is mammalian, more preferably, the subject is human. Preferred pharmaceutical compositions are those comprising at least one coronavirus construct in a therapeutically effective amount or an immunogenic amount, and a pharmaceutically acceptable vehicle. In some embodiments, a therapeutically effective amount of a coronavirus construct ranges from about 0.01-0.5 μg/kg, about 0.1-0.5 μg/kg, or about 0.35-0.5 μg/kg body weight of a subject. In some embodiments, a therapeutically effective amount of a given coronavirus construct comprises about 10e3 to 10e11 (log scale) viral particles (VP). In some embodiments, the therapeutically effective amount comprises about 10e4 to 10e11 (log scale) viral particles (VP). In some embodiments, the therapeutically effective amount comprises about 10e3, 10e4, 10e5, 10e6, 10e7, 10e8, 10e9, 10e10, or 10e11 of VP. It should be noted that treatment of a subject with a therapeutically effective amount or an immunogenic amount may be administered as a single dose or as a series of several doses. The dosages used for treatment may increase or decrease over the course of a given treatment. Optimal dosages for a given set of conditions may be ascertained by those skilled in the art using dosage-determination tests and/or diagnostic assays in the art. Dosage-determination tests and/or diagnostic assays may be used to monitor and adjust dosages during the course of treatment. In some embodiments a single dose of a coronavirus construct for a human subject ranges from about 1-50 μg, about 1-40 μg, about 1-35 μg, about 1-30 μg, about 1-25 μg, about 1-20 μg, or about 1-15 μg. In some embodiments a single dose of a coronavirus construct for a human subject ranges from about 1-25 μg, about 5-20 μg, or about 7.5-16 μg.
Vaccines provide a protective immune response when administered to a subject. In some embodiments, a “vaccine”, is a pharmaceutical composition that comprises an immunogenic amount of at least one coronavirus construct and provides a protective immune response when administered to a subject. The protective immune response may be complete or partial, e.g., a reduction in symptoms as compared with an unvaccinated subject. As used herein, an “immunogenic amount” is an amount that is sufficient to elicit an immune response in a subject and depends on a variety of factors such as the immunogenicity of the given coronavirus construct, the degree of the given COVID disease, the manner of administration, the general state of health of the subject, and the like. The typical immunogenic amounts of a given coronavirus construct for initial and boosting immunizations range from about 0.01-0.5 μg/kg, about 0.1-0.5 μg/kg, or about 0.35-0.5 μg/kg body weight of a subject. For example, the typical immunogenic amount for initial and boosting immunization for therapeutic or prophylactic administration for a human subject ranges from about 1-50 μg, about 1-40 μg, about 1-35 μg, about 1-30 μg, about 1-25 μg, about 1-20 μg, or about 1-15 μg. In some embodiments a single dose of a coronavirus construct for a human subject ranges from about 1-25 μg, about 5-20 μg, or about 7.5-16 μg. Examples of suitable immunization protocols include an initial immunization vaccination (time 0), followed by one or more booster immunization at 1, 2, 3, and/or 4 weeks, or 1, 2, 3, 4, 5, and/or 6 months, or 1 or 2 years which these initial vaccination may be followed by further booster immunization if needed or desired. For example, an exemplary two dose schedule is a booster immunization 6 to 12 months after the initial vaccination and an exemplary three dose schedule is a first booster immunization at 2 months and a second booster immunization at 6 months after the initial vaccination.
The compositions contemplated herein may include an adjuvant and/or stabilizers in the art, e.g., MgCl2, MgSO4, lactose-sorbitol, and sorbitol-gelatine. As used herein, an “adjuvant” refers to any substance which, when administered in conjunction with (e.g., before, during, or after) a pharmaceutically active agent, such as a coronavirus construct as disclosed herein, aids the pharmaceutically active agent in its mechanism of action. Thus, an adjuvant in a vaccine is a substance that aids the at least one coronavirus construct in eliciting an immune response. Suitable adjuvants include incomplete Freund's adjuvant, alum, aluminum phosphate, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, nor-MDP), N-acetylmuramyl-Lalanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipa-lmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, MTP-PE), and RIBI, which comprise three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (NPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. The effectiveness of an adjuvant may be determined by methods in the art.
Pharmaceutical compositions may be formulated for the intended route of delivery, including intravenous, intramuscular, intra peritoneal, subcutaneous, intraocular, intrathecal, intraarticular, intrasynovial, cisternal, intrahepatic, intralesional injection, intracranial injection, infusion, and/or inhaled routes of administration using methods known in the art. Pharmaceutical compositions may include one or more of the following: pH buffered solutions, adjuvants (e.g., preservatives, wetting agents, emulsifying agents, and dispersing agents), liposomal formulations, nanoparticles, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions. The compositions and formulations may be optimized for increased stability and efficacy using methods in the art. See, e.g., Carra et al., (2007) Vaccine 25:4149-4158.
The compositions may be administered to a subject by any suitable route including oral, transdermal, subcutaneous, intranasal, inhalation, intramuscular, and intravascular administration. It will be appreciated that the preferred route of administration and pharmaceutical formulation will vary with the condition and age of the subject, the nature of the condition to be treated, the therapeutic effect desired, and the particular coronavirus construct used.
The pharmaceutical compositions may be provided in dosage unit forms. As used herein, a “dosage unit form” refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of the one or more coronavirus construct calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the given coronavirus construct and desired therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
Toxicity and therapeutic efficacy of coronavirus constructs according to the instant invention and compositions thereof can be determined using cell cultures and/or experimental animals and pharmaceutical procedures in the art. For example, one may determine the lethal dose, LC50 (the dose expressed as concentration×exposure time that is lethal to 50% of the population) or the LD50 (the dose lethal to 50% of the population), and the ED50 (the dose therapeutically effective in 50% of the population) by methods in the art. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Coronavirus constructs s which exhibit large therapeutic indices are preferred. While coronavirus constructs that result in toxic side-effects may be used, care should be taken to design a delivery system that targets such compounds to the site of treatment to minimize potential damage to uninfected cells and, thereby, reduce side-effects.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. Preferred dosages provide a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary depending upon the dosage form employed and the route of administration utilized. Therapeutically effective amounts and dosages of one or more coronavirus constructs can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. Additionally, a dosage suitable for a given subject can be determined by an attending physician or qualified medical practitioner, based on various clinical factors.
Kits comprising one or more coronavirus constructs as described herein and/or compositions thereof, optionally in combination with one or more supplementary agents, are contemplated herein. In some embodiments, the one or more coronavirus constructs or compositions thereof are packaged together with one or more reagents or drug delivery devices for administering the coronavirus constructs or compositions thereof to a subject. In some embodiments, the kits comprise the one or more coronavirus constructs, optionally in one or more unit dosage forms, packaged together as a pack and/or in drug delivery device, e.g., a pre-filled syringe.
In some embodiments, the kits include a carrier, package, or container that may be compartmentalized to receive one or more containers, such as vials, tubes, and the like. In some embodiments, the kits optionally include an identifying description or label or instructions relating to its use. In some embodiments, the kits include information prescribed by a governmental agency that regulates the manufacture, use, or sale of compounds and compositions as contemplated herein.
The following examples are intended to illustrate but not to limit the invention.
The replication deficient Zika Vector was made by splitting a Zika virus (ZIKV) genome into structural and non-structural (NS) regions (
A structural gene sequence based on the same ZIKV strain was used to package the Zika Vector. The packaging plasmid comprised nucleotides 108 to 2489 of PRVABC59 (SEQ ID NO: 20) total size 2382 nucleotides; 794 amino acids (SEQ ID NO: 21)) cloned into a mammalian expression vector driven by CMV promoter and at the end SV40 poly A sequence.
Methods in the art were used to make the control and coronavirus constructs exemplified herein.
Vaccine production: 293T cells were cultured in IMDM containing 10% FCS and antibiotics in an incubator (37° C. and 5% CO2). One day before transfection, 293T cells (1.4×107 cells) were seeded in T175 flask coated with 250 μg/ml rat collagen 1. The cells were transfected with 37.5 μg packaging plasmid and 12.5 μg of control (VAXR1) or a coronavirus construct. One day after transfection, the transfected cells were cultured at 30° C. Three days after transfection, the culture medium was changed to AIM-V (ThermoFisher Scientific) supplemented with antibiotics. The supernatant was collected 5, 6, 7, 8, 9, 10, and 11 days after transfection. After harvesting the supernatant, the same amount of fresh medium was replenished. The cells and debris in the harvested supernatant were removed by filtration by filters (0.22 or 0.45 μm pore size) and/or centrifugation (2000×g, 10 mins, 4° C.) and frozen at −80° C. For measuring the vaccine particle production, Vero cells or 293T-TIM-1 cells were inoculated with 100 μl of diluted or 1 in 10 diluted vaccine particles and 48 hours later flow cytometry was performed. Optimum vaccine particle production was observed between Days 7-9 (
For assessing safety, the VAXR1 construct was tested in neonatal Ifnar1−/− mice. The wild-type PRVABC59 ZIKV was used as a positive control. The pups received 1×103 PFU/mouse of PRVABC59 virus and VAXR1 and were followed up for 14 days. The wild-type virus infected pups had 100% mortality, whereas VAXR1 inoculated pups were 100% viable (
Subsequently, the virus load in wild-type virus and vaccine exposed pups was investigated. The data shows that VAXR1 did not replicate, thus no infection detected (
Based on safety study in neonatal mice, breeding female mice were immunized with the VAXR1 construct. The timeline of various key steps in this experiment is provided in
Both the vaccinated and un-vaccinated mice were subjected to mating on Day 21. Pregnant mice were challenged with wild type ZIKV (1×106 pfu/mouse). The vaccinated and mock infected (healthy) pregnant animals gained weight, whereas un-vaccinated pregnant mice continue to lose weight and by Day 8, these animals reached the endpoint (
Mass cytometry was performed to evaluate the various sub-sets of immune cell populations (
Adult Ifnar1−/− mice (mixed sex; 14-18 week old; n=8-10 animals/group) were immunized with a given coronavirus construct (dose 1×104 foci forming unit/mouse, subcutaneous route). The subjects were followed up for two weeks. Body weight and animal death (if any) were the parameters monitored. All subjects survived (
Because the coronavirus constructs result in expression of the given coronavirus antigen encoded by the given one or more coronavirus sequences and a ZIKV antigen present in the Zika Vector in equal amounts, immune responses against ZIKV challenge are indirectly representative of the likely immune responses against coronaviruses that express the given coronavirus antigen. Subjects were administered booster vaccinations two weeks post the primary dose. Then, at 4 weeks post-immunization, the subjects were challenged with wild-type ZIKV. Subjects vaccinated with coronavirus constructs were protected from lethal Zika challenge (
These results indicate that the coronavirus constructs are safe and likely effective in inducing an immune response against SARS-CoV-2.
Methods in the art were used to make ZIKV codon-optimized coronavirus constructs. The codon-optimized ZIKV vaccine vector has the nucleic acid sequence set forth in SEQ ID NO: 22. The codon-optimized ZIKV vaccine vector comprises a BsiWI restriction site (SEQ ID NO: 25), an SG linker sequence (SEQ ID NO: 28), and a NotI linker sequence (SEQ ID NO: 33) and sequences encoding an IgK signal peptide (SEQ ID NO: 27), Bacteriophage T4 Fibritin Foldon (SEQ ID NO: 30), and F2A (SEQ ID NO: 32).
Table 4 shows the features that are shared among the exemplified COVID vaccines.
All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified.
Except when specifically indicated, peptides are indicated with the N-terminus on the left and the sequences are written from the N-terminus to the C-terminus. Similarly, except when specifically indicated, nucleic acid sequences are indicated with the 5′ end on the left and the sequences are written from 5′ to 3′.
As used herein, the terms “subject”, “patient”, and “individual” are used interchangeably to refer to humans and non-human animals. The terms “non-human animal” and “animal” refer to all non-human vertebrates, e.g., non-human mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects and test animals. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.
As used herein, the term “diagnosing” refers to the physical and active step of informing, i.e., communicating verbally or by writing (on, e.g., paper or electronic media), another party, e.g., a patient, of the diagnosis. Similarly, “providing a prognosis” refers to the physical and active step of informing, i.e., communicating verbally or by writing (on, e.g., paper or electronic media), another party, e.g., a patient, of the prognosis.
The use of the singular can include the plural unless specifically stated otherwise. As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” can include plural referents unless the context clearly dictates otherwise.
As used herein, “and/or” means “and” or “or”. For example, “A and/or B” means “A, B, or both A and B” and “A, B, C, and/or D” means “A, B, C, D, or a combination thereof” and said “A, B, C, D, or a combination thereof” means any subset of A, B, C, and D, for example, a single member subset (e.g., A or B or C or D), a two-member subset (e.g., A and B; A and C; etc.), or a three-member subset (e.g., A, B, and C; or A, B, and D; etc.), or all four members (e.g., A, B, C, and D).
As used herein, the phrase “one or more of”, e.g., “one or more of A, B, and/or C” means “one or more of A”, “one or more of B”, “one or more of C”, “one or more of A and one or more of B”, “one or more of B and one or more of C”, “one or more of A and one or more of C” and “one or more of A, one or more of B, and one or more of C”.
The phrase “comprises, consists essentially of, or consists of A” is used as a tool to avoid excess page and translation fees and means that in some embodiments the given thing at issue: comprises A, consists essentially of A, or consists of A. For example, the sentence “In some embodiments, the composition comprises, consists essentially of, or consists of A” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises A. In some embodiments, the composition consists essentially of A. In some embodiments, the composition consists of A.”
Similarly, a sentence reciting a string of alternates is to be interpreted as if a string of sentences were provided such that each given alternate was provided in a sentence by itself. For example, the sentence “In some embodiments, the composition comprises A, B, or C” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises A. In some embodiments, the composition comprises B. In some embodiments, the composition comprises C.” As another example, the sentence “In some embodiments, the composition comprises at least A, B, or C” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises at least A. In some embodiments, the composition comprises at least B. In some embodiments, the composition comprises at least C.”
To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated.
Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein, but is only limited by the following claims.
This application claims the benefit of U.S. Patent Application No. 63/191,829, filed May 21, 2021, and U.S. Patent Application No. 63/197,031, filed Jun. 4, 2021, both of which are herein incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/030282 | 5/20/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63191829 | May 2021 | US | |
| 63197031 | Jun 2021 | US |
