Compositions and methods for preventing and treating Zika virus infection
10611801 ยท 2020-04-07
Assignee
Inventors
Cpc classification
C12N2770/24122
CHEMISTRY; METALLURGY
C12N2770/24134
CHEMISTRY; METALLURGY
A61K2039/55
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
C12N2710/00043
CHEMISTRY; METALLURGY
International classification
A61K39/00
HUMAN NECESSITIES
Abstract
The invention relates to immunogenic compositions and vaccines containing a ZIKV protein or a polynucleotide encoding a Zika virus (ZIKV) protein and uses thereof. The invention also provides methods of treating and/or preventing a ZiKV infection by administering an immunogenic composition or vaccine of the invention to a subject (e.g., a human).
Claims
1. An isolated nucleic acid molecule comprising: (i) a nucleotide sequence having at least 85% sequence identity to the sequence of any one of SEQ ID NOs: 1, 3, 5, 7, 9, and 11, or a complementary sequence thereof; and/or (ii) a nucleotide sequence that encodes a polypeptide having at least 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, and 12.
2. An isolated polypeptide comprising an amino acid sequence having at least 85% sequence identity to the sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, or 12.
3. A vector comprising one or more of the nucleic acid molecules of claim 1.
4. The vector of claim 3, wherein the vector is a mammalian, bacterial, or viral derived expression vector.
5. The vector of claim 4, wherein the vector is: (i) a viral vector derived from a virus selected from the group consisting of a retrovirus, adenovirus, adeno-associated virus, parvovirus, coronavirus, negative strand RNA viruses, orthomyxovirus, rhabdovirus, paramyxovirus, positive strand RNA viruses, picornavirus, alphavirus, double stranded DNA viruses, herpesvirus, Epstein-Barr virus, cytomegalovirus, fowlpox, and canarypox; (ii) an adenoviral vector derived from an adenovirus selected from the group consisting of Ad2, Ad5, Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52, and Pan9; or (iii) an adenoviral vector derived from a human, chimpanzee, or rhesus adenovirus.
6. A composition comprising the nucleic acid molecule of claim 1, a polypeptide encoded by the nucleic acid molecule, or a vector comprising the nucleic acid molecule, wherein, optionally, the composition further comprises: (i) a pharmaceutically acceptable carrier, excipient, or diluent; and/or (ii) an adjuvant or an immunostimulatory agent.
7. An immunogenic composition comprising the composition of claim 6, wherein said immunogenic composition: (i) is capable of treating or reducing the risk of a ZIKV infection in a subject in need thereof; and/or (ii) elicits production of neutralizing anti-ZIKV antisera after administration to said subject.
8. An isolated antibody or an antigen-binding fragment thereof that specifically binds to the polypeptide of claim 2.
9. The antibody of claim 8, wherein the antibody or antigen-binding fragment thereof is generated by immunizing a mammal with the nucleic acid molecule of claim 1, a polypeptide encoded by the nucleic acid molecule, a vector comprising the nucleic acid molecule, a composition comprising the nucleic acid molecule, polypeptide, or vector, and/or an immunogenic composition comprising the composition.
10. A method of producing anti-ZIKV antibodies, comprising administering to a subject an amount of at least one of the nucleic acid molecule of claim 1, a polypeptide encoded by the nucleic acid molecule, a vector comprising the nucleic acid molecule, a composition comprising the nucleic acid molecule, polypeptide or vector, and/or an immunogenic composition comprising the composition, wherein the amount is sufficient to elicit production of neutralizing anti-ZIKV antisera after administration to said subject.
11. An isolated anti-ZIKV antibody produced by the method of claim 10, wherein the isolated anti-ZIKV antibody binds to an epitope within any one of SEQ ID NOs: 2, 4, 6, 8, 10, or 12.
12. A method of treating or reducing the risk of a ZIKV infection in a subject in need thereof and/or reducing a ZIKV-mediated activity in a subject infected with a ZIKV, wherein said ZIKV-mediated activity is viral titer, viral spread, infection, or cell fusion, the method comprising administering to the subject a therapeutically effective amount of at least one of the nucleic acid molecule of claim 1, a polypeptide encoded by the nucleic acid molecule, a vector comprising the nucleic acid molecule, a composition comprising the nucleic acid molecule, polypeptide or vector, an immunogenic composition comprising the composition, an antibody specifically binding to the polypeptide, and/or a neutralizing anti-ZIKV antisera produced by administering to a subject the nucleic acid molecule, polypeptide, vector, composition, or immunogenic composition.
13. The method of claim 12, wherein: (i) the therapeutically effective amount of the nucleic acid molecule, the polypeptide, the vector, the composition, the immunogenic composition, the antibody, and/or the neutralizing anti-ZIKV antisera is sufficient to produce a log serum anti-Env antibody titer greater than 2 in the subject, as measured by an ELISA assay; (ii) the therapeutically effective amount is between 15 g and 300 g of the nucleic acid molecule, the polypeptide, the vector, the composition, the immunogenic composition, the antibody, and/or the neutralizing anti-ZIKV antisera; (iii) the ZIKV titer is decreased after administration of the nucleic acid molecule, the polypeptide, the vector, the composition, the immunogenic composition, the antibody, and/or the neutralizing anti-ZIKV antisera; and/or (iv) the ZIKV is undetectable after administration of the nucleic acid molecule, the polypeptide, the vector, the composition, the immunogenic composition, the antibody, and/or the neutralizing anti-ZIKV antisera.
14. The method of claim 12, wherein said administering occurs: (i) prior to exposure to a ZIKV; (ii) at least 1 hour prior to exposure to said ZIKV; (iii) post-exposure to the ZIKV; or (iv) at least 15 minutes post-exposure to said ZIKV.
15. The method of claim 12, wherein: (i) the nucleic acid molecule, the polypeptide, the vector, the composition, the immunogenic composition, the antibody, and/or the neutralizing anti-ZIKV antisera is administered to said subject in at least one dose, in at least two doses, as a prime, as a boost, or as a prime-boost; and/or (ii) the nucleic acid molecule, the polypeptide, the vector, the composition, the immunogenic composition, the antibody, and/or the neutralizing anti-ZIKV antisera is administered to said subject intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivelly, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, by gavage, in creams, or in lipid compositions.
16. The method of claim 12, wherein: (i) the subject is a mammal; (ii) the subject is a human; (iii) the method promotes an immune response in said subject; and/or (iv) the method promotes a humoral immune response in said subject.
17. A method of manufacturing an immunogenic composition for treating or reducing the risk of a ZIKV infection in a subject in need thereof, said method comprising the steps of: (a) forming the immunogenic composition by admixing at least one of the nucleic acid molecule of claim 1, a polypeptide encoded by the nucleic acid molecule, a vector comprising the nucleic acid molecule, a composition comprising the nucleic acid molecule, polypeptide or vector, an antibody specifically binding to the polypeptide, and/or a neutralizing anti-ZIKV antisera produced by administering to a subject the nucleic acid molecule, the polypeptide, the vector, or the composition with a pharmaceutically acceptable carrier, excipient, or diluent; and (b) placing the immunogenic composition in a container.
18. A kit comprising: (a) a first container comprising at least one of the nucleic acid molecule of claim 1, a polypeptide encoded by the nucleic acid molecule, a vector comprising the nucleic acid molecule, a composition comprising the nucleic acid molecule, polypeptide or vector, an immunogenic composition comprising the composition, an antibody specifically binding to the polypeptide, and/or a neutralizing anti-ZIKV antisera produced by administering to a subject the nucleic acid molecule, the polypeptide, the vector, the composition or the immunogenic composition; (b) instructions for use thereof; and optionally (c) a second container comprising a pharmaceutically acceptable carrier, excipient, or diluent.
19. The kit of claim 18, wherein: (i) the first container further comprises a pharmaceutically acceptable carrier, excipient, or diluent; and/or (ii) the kit optionally includes an adjuvant and/or an immunostimulatory agent.
20. A composition comprising the polypeptide of claim 2.
21. A composition comprising the vector of claim 3.
22. An immunogenic composition comprising the composition of claim 20.
23. An immunogenic composition comprising the composition of claim 21.
24. The isolated antibody or antigen-binding fragment thereof of claim 8, wherein the isolated antibody or antigen-binding fragment thereof is an isolated humanized antibody or an antigen-binding fragment thereof, an isolated IgG antibody or an antigen-binding fragment thereof, or a bis-Fab, Fv, Fab, Fab-SH, F(ab).sub.2, a diabody, a linear antibody, or a scFV.
25. The method of claim 12, wherein: (i) the ZIKV is a ZIKV strain from an Asian or African lineage; or (ii) the ZIKV is a ZIKV strain from Brazil or Puerto Rico, wherein optionally the ZIKV is Brazil-ZKV2015 or PRVABC59.
26. The method of claim 12, wherein the antibody is an isolated humanized antibody or an antigen-binding fragment thereof, an isolated IgG antibody or an antigen-binding fragment thereof, or a bis-Fab, Fv, Fab, Fab-SH, F(ab).sub.2, a diabody, a linear antibody, or a scFV.
27. The method of claim 17, wherein the antibody is an isolated humanized antibody or an antigen-binding fragment thereof, an isolated IgG antibody or an antigen-binding fragment thereof, or a bis-Fab, Fv, Fab, Fab-SH, F(ab).sub.2, a diabody, a linear antibody, or a scFV.
28. The kit of claim 18, wherein the antibody is an isolated humanized antibody or an antigen-binding fragment thereof, an isolated IgG antibody or an antigen-binding fragment thereof, or a bis-Fab, Fv, Fab, Fab-SH, F(ab).sub.2, a diabody, a linear antibody, or a scFV.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
(47) We have discovered that Zika virus (ZIKV) polypeptides can be used to elicit protective and therapeutic immune responses against a ZIKV infection when administered to a subject (e.g., a human subject) infected with or likely to be exposed to a ZIKV. The compositions that can be prepared for administration to a subject include a ZIKV protein (e.g., a prM-Env, prM-Env.dTM, prM-Env.dStem, Env, Env.dTM, and/or Env.dStem or a portion thereof) or a vector containing a nucleic acid sequence that encodes the ZIKV protein (e.g., an expression vector, such as a plasmid, or a viral vector, such as an adenovirus, poxvirus, adeno-associated virus, retroviral, or other viral vector, or naked or encapsulated DNA.
(48) In particular, we describe the generation of DNA vaccines expressing a truncated ZIKV pre-membrane and envelope (prM-Env) region, the envelope region alone (Env), and deletion mutants that remove either the transmembrane (TM) or stem (Stem) polyproteins (Table 1) that provide protection from ZIKV infection. The ZIKV DNA vaccines of the invention were generated by incorporating a polynucleotide of Table 1 into the mammalian expression vector pcDNA3.1+ (Invitrogen, CA, USA) to generate the prM-Env vaccine (DNA-prM-Env), prM-Env.dTM DNA vaccine (DNA-prM-Env.dTM), the prM-Env.dStem DNA vaccine (DNA-prM-Env.dStem), the Env vaccine (DNA-Env), the Env.dTM vaccine (DNA-Env.dTM), and the Env.dStem vaccine (DNA-Env.dStem).
(49) We demonstrate that the DNA vaccines of the invention provide protection against ZIKV challenge, and that protective efficacy is correlated with Env-specific antibody titers. Additionally, we show that adoptive transfer of purified IgG from a vaccinated subject confers passive protection from ZIKV infection.
(50) The nucleic acid molecules, polypeptides, vectors, vaccines, compositions, antibodies, and methods treating and preventing a ZIKV infection of the invention are described herein.
(51) TABLE-US-00001 TABLE 1 ZIKV derived polynucleotide and polypeptide molecules SEQ ID NO. Region of ZIKV polynucleotide polypeptide prM-Env 1 2 (prM-Env (216-794) or M-Env or prM-Env (pr deleted)) prM-Env.dTM 3 4 prM-Env.dStem 5 6 Env 7 8 Env.dTM 9 10 Env.dStem 11 12 prM-Env (full length) 24 25 prM-Env with JEV Stem/TM 26 27
I. COMPOSITIONS AND METHODS
(52) Nucleic Acid Molecules of the Invention
(53) The nucleic acid molecules of the invention (Table 1) were designed based on the Zika virus (ZIKV) strain BeH815744 (accession number KU365780 (SEQ ID NOs: 15-16)). The nucleic acid molecules of the invention encode regions of the Zika virus (ZIKV) polyprotein, for example, the pre-membrane and envelope (prM-Env) region, the Env region alone, or deletion mutants of the prM-Env or Env regions in which the transmembrane (TM) or Stem region have been removed. The nucleic acid molecules of the invention prM-Env (SEQ ID NO: 1), prM-Env.dTM (SEQ ID NO: 3), prM-Env.dStem (SEQ ID NO: 5), Env (SEQ ID NO: 7), Env.dTM (SEQ ID NO: 9), and Env.dStem (SEQ ID NO: 11) have been optimized relative to the wild-type BeH815744 nucleotide sequences for improved expression in host cells (e.g., mammalian (e.g., human) host cells) and particle formation, and encode the polypeptides set forth in SEQ ID NOs: 2, 4, 6, 8, 10, or 12, respectively (Table 1). Optimization can included the addition of a leader sequence, such as a Japanese encephalitis virus (JEV) leader sequence (e.g., SEQ ID NO: 13), restriction site (e.g., SEQ ID NOs: 21-22), and/or a Kozak sequence (e.g., SEQ ID NO: 23).
(54) The prM-Env (full length) (e.g., SEQ ID NOs: 24-25) contains the full-length sequence of the prM-Env region, while prM-Env with JEV Stem/TM (e.g., SEQ ID NOs: 26-27) includes the ZIKV prM signal region of Japanese encephalitis virus (JEV) with the final 98 amino acids comprising the stem and transmembrane regions exchanged with corresponding JEV sequences.
(55) The nucleic acid molecules have a nucleotide sequence with at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to, all or a portion of any one of SEQ ID NOs: 1, 3, 5, 7, 9, or 11, or a complementary sequence thereof. Alternatively, an isolated nucleic acid molecule has a nucleotide sequence that encodes a ZIKV polypeptide with at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, or 12.
(56) The nucleic acid molecules of the invention may be further optimized, such as by codon optimization, for expression in a targeted mammalian subject (e.g., human).
(57) The nucleic acid molecules may also be inserted into expression vectors, such as a plasmid, or a viral vector, such as an adenovirus, poxvirus, adeno-associated virus, retroviral, or other viral vector, or prepared as naked or encapsulated DNA and incorporated into compositions of the invention.
(58) Polypeptides of the Invention
(59) The polypeptides of the invention are ZIKV polypeptides corresponding to, for example, the pre-membrane and envelope (prM-Env) region, the Env region alone, or deletion mutants of the prM-Env or Env regions in which the transmembrane (TM) or Stem region has been removed. Polypeptides of the invention include prM-Env (SEQ ID NO: 2), prM-Env.dTM (SEQ ID NO: 4), prM-Env.dStem (SEQ ID NO: 6), Env (SEQ ID NO: 8), Env.dTM (SEQ ID NO: 10), and Env.dStem (SEQ ID NO: 12) and variants having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to, all or a portion of any one of SEQ ID NOs: 2, 4, 6, 8, 10, or 12. The polypeptides of the invention may include at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or 1800 or more continuous or non-continuous amino acids of any one of SEQ ID NOs: 2, 4, 6, 8, 10, or 12. Polypeptides of the invention may also include a leader sequence, such as a Japanese encephalitis virus (JEV) leader sequence (SEQ ID NO: 14). The polypeptides may also be isolated from other components (e.g., components with which the polypeptides are natively associated) and incorporated into compositions of the invention.
(60) Vectors of the Invention
(61) The invention also features recombinant vectors including any one or more of the polynucleotides described above. The vectors of the invention can be used to deliver an nucleic acid expressing an immunogen of the invention (e.g., one of more of SEQ ID NOs: 2, 4, 6, 8, 10, or 12 or variants thereof, having at least 85-99% sequence identity thereto, for example at least greater than 90% sequence identity thereto), and in include mammalian, viral, and bacterial expression vectors. The mammalian, viral, and bacterial vectors of the invention can be genetically modified to contain one or more nucleic acid sequences set forth in SEQ ID NOs: 1, 3, 5, 7, 9, or 11 or variants thereof, having at least 85-99% sequence identity thereto, for example at least greater than 90% sequence identity thereto, and complements thereof.
(62) The vectors may be, for example, plasmids, artificial chromosomes (e.g. BAG, PAC, YAC), and virus or phage vectors, and may optionally include a promoter, enhancer, or regulator for the expression of the polynucleotide. The vectors may also contain one or more selectable marker genes, for example an ampicillin, neomycin, and/or kanamycin resistance gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example, for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell, e.g., for the production of protein encoded by the vector. The vectors may also be adapted to be used in vivo, for example in a method of DNA vaccination or of gene therapy.
(63) Promoters and other expression regulation signals may be selected to be compatible with the host cell for which expression is designed. For example, mammalian promoters include the metallothionein promoter, which can be induced in response to heavy metals, such as cadmium, and the -actin promoter. A viral promoter, which can be obtained from the genome of a virus, such as, for example, polyoma virus, fowlpox virus, adenovirus (A), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus, hepatitis-B virus, and Simian Virus 40 (SV40), and human papillomavirus (HPV), may also be used. These promoters are well known and readily available in the art.
(64) A preferred promoter element is the CMV immediate early promoter. In some embodiments, the expression plasmid is pcDNA3.1+ (Invitrogen, CA, USA). In some embodiments, the expression vector is a viral vector, such as a vector derived from adenovirus or poxvirus.
(65) Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into the genome of a cell (e.g., a eukaryotic or prokaryotic cell). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Examples of viral vectors that can be used to deliver a nucleic acid expressing an immunogen of the invention (e.g., one of more of SEQ ID NOs: 2, 4, 6, 8, 10, or 12 or variants thereof having at least 85-99% sequence identity thereto, for example at least greater than 90% sequence identity thereto) include a retrovirus, adenovirus (e.g., Ad2, Ad5, Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52 (e.g., RhAd52), and Pan9 (also known as AdC68)), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picomavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses useful for delivering polynucleotides encoding immunogens (e.g., polypeptides) of the invention include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin. J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). These adenovirus vectors can be derived from, for example, human, chimpanzee, or rhesus adenoviruses. Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in McVey et al., (U.S. Pat. No. 5,801,030); incorporated herein in its entirety by reference. The nucleic acid material (e.g., including a nucleic acid molecule of the invention) of the viral vector may be encapsulated, e.g., in a lipid membrane or by structural proteins (e.g., capsid proteins), that may include one or more viral polypeptides (e.g., a glycoprotein). The viral vector can be used to infect cells of a subject, which, in turn, promotes the translation of the heterologous gene(s) of the viral vector into the immunogens of the invention. For example, a viral vector of the invention can be genetically modified to contain one or more nucleic acid sequences set forth in SEQ ID NOs: 1, 3, 5, 7, 9, or 11 or variants thereof having at least 85-99% sequence identity thereto, for example at least greater than 90% sequence identity thereto, and complements thereof.
(66) Adenoviral vectors disclosed in International Patent Application Publications WO 2006/040330 and WO 2007/104792, each incorporated by reference herein, are particularly useful as vectors of the invention. These adenoviral vectors can encode and/or deliver one or more of the immunogens of the invention (e.g., ZIKV polypeptides) to treat a subject having a pathological condition associated with a viral infection (e.g., a ZIKV infection). In some embodiments, one or more recombinant adenovirus vectors can be administered to the subject in order to express more than one type of immunogen (e.g., ZIKV polypeptide) of the invention. Besides adenoviral vectors, other viral vectors and techniques are known in the art that can be used to facilitate delivery and/or expression of one or more of the immunogens of the invention in a subject (e.g., a human). These viruses include poxviruses (e.g., vaccinia virus and modified vaccinia virus Ankara (MVA); see, e.g., U.S. Pat. Nos. 4,603,112 and 5,762,938, each incorporated by reference herein), herpesviruses, togaviruses (e.g., Venezuelan Equine Encephalitis virus; see, e.g., U.S. Pat. No. 5,643,576, incorporated by reference herein), picomaviruses (e.g., poliovirus; see, e.g., U.S. Pat. No. 5,639,649, incorporated by reference herein), baculoviruses, and others described by Wattanapitayakul and Bauer (Biomed. Pharmacother. 54:487 (2000), incorporated by reference herein).
(67) Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide of the invention into the host genome, although such recombination is not preferred. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression.
(68) Vectors capable of driving expression in insect cells (for example baculovirus vectors), in human cells, in yeast or in bacteria may be employed in order to produce quantities of the ZIKV protein encoded by the polynucleotides of the present invention, for example, for use as subunit vaccines or in immunoassays.
(69) Antibodies of the Invention
(70) Anti-ZIKV antibodies of the invention are capable of specifically binding to a ZIKV polypeptide and are capable of inhibiting a ZIKV-mediated activity (e.g., viral spread, infection, and or cell fusion) in a subject (e.g., a human). The result of such binding may be, for example, a reduction in viral titer (e.g., viral load), by about 1% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or more, after administration of an antibody of the invention to a subject infected with ZIKV. The anti-ZIKV antibodies of the invention may selectively bind to an epitope comprising all, or a portion of, the Env region of the ZIKV polyprotein. In particular, the anti-ZIKV antibodies of the invention may selectively bind to an epitope comprising all, or a portion of, any one of SEQ ID NOs: 2, 4, 6, 8, 10, or 12. The antibodies of the invention can therefore be used to prevent or treat an ZIKV infection.
(71) The specific binding of an antibody or antibody fragment of the invention to a ZIKV polyprotein can be determined by any of a variety of established methods. The affinity can be represented quantitatively by various measurements, including the concentration of antibody needed to achieve half-maximal inhibition of viral spread (e.g., viral titer) in vitro (IC.sub.50) and the equilibrium constant (K.sub.D) of the antibody-ZIKV polyprotein complex dissociation. The equilibrium constant, K.sub.D, that describes the interaction of ZIKV polyprotein with an antibody of the invention is the chemical equilibrium constant for the dissociation reaction of a ZIKV polyprotein-antibody complex into solvent-separated ZIKV polyprotein and antibody molecules that do not interact with one another.
(72) Antibodies of the invention are those that specifically bind to a ZIKV polyprotein (e.g., the Env region of ZIKV) with a K.sub.D value of less than 1 M (e.g., 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM). In certain cases, antibodies of the invention are those that specifically bind to a ZIKV polyprotein with a K.sub.D value of less than 1 nM (e.g., 990 pM, 980 pM, 970 pM, 960 pM, 950 pM, 940 pM, 930 pM, 920 pM, 910 pM, 900 pM, 890 pM, 880 pM, 870 pM, 860 pM, 850 pM, 840 pM, 830 pM, 820 pM, 810 pM, 800 pM, 790 pM, 780 pM, 770 pM, 760 pM, 750 pM, 740 pM, 730 pM, 720 pM, 710 pM, 700 pM, 690 pM, 680 pM, 670 pM, 660 pM, 650 pM, 640 pM, 630 pM, 620 pM, 610 pM, 600 pM, 590 pM, 580 pM, 570 pM, 560 pM, 550 pM, 540 pM, 530 pM, 520 pM, 510 pM, 500 pM, 490 pM, 480 pM, 470 pM, 460 pM, 450 pM, 440 pM, 430 pM, 420 pM, 410 pM, 400 pM, 390 pM, 380 pM, 370 pM, 360 pM, 350 pM, 340 pM, 330 pM, 320 pM, 310 pM, 300 pM, 290 pM, 280 pM, 270 pM, 260 pM, 250 pM, 240 pM, 230 pM, 220 pM, 210 pM, 200 pM, 190 pM, 180 pM, 170 pM, 160 pM, 150 pM, 140 pM, 130 pM, 120 pM, 110 pM, 100 pM, 90 pM, 80 pM, 70 pM, 80 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM).
(73) Antibodies of the invention can also be characterized by a variety of in vitro binding assays. Examples of experiments that can be used to determine the K.sub.D or IC.sub.50 of a ZIKV antibody include, e.g., surface plasmon resonance, isothermal titration calorimetry, fluorescence anisotropy, and ELISA-based assays, among others. ELISA represents a particularly useful method for analyzing antibody activity, as such assays typically require minimal concentrations of antibodies. A common signal that is analyzed in a typical ELISA assay is luminescence, which is typically the result of the activity of a peroxidase conjugated to a secondary antibody that specifically binds a primary antibody (e.g., a ZIKV antibody of the invention). Antibodies of the invention are capable of binding ZIKV and epitopes derived thereof, such as epitopes containing one or more of residues of any one of SEQ ID NOs: 2, 4, 6, 8, 10, or 12, as well as isolated peptides derived from ZIKV that structurally pre-organize various residues in a manner that may simulate the conformation of these amino acids in the native protein. For instance, antibodies of the invention may bind peptides containing the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, or 12, or a peptide containing between about 10 and about 30 continuous or discontinuous amino acids of any one of SEQ ID NOs: 2, 4, 6, 8, 10, or 12. In a direct ELISA experiment, this binding can be quantified, e.g., by analyzing the luminescence that occurs upon incubation of an HRP substrate (e.g., 2,2-azino-di-3-ethylbenzthiazoline sulfonate) with an antigen-antibody complex bound to a HRP-conjugated secondary antibody.
(74) Antibodies of the invention include those that are generated by immunizing a host (e.g., a mammalian host, such as a human) with the polypeptides of SEQ ID NOs: SEQ ID NOs: 2, 4, 6, 8, 10, or 12. The antibodies can be prepared recombinantly and, if necessary, humanized, for subsequent administration to a human recipient if the host in which the anti-ZIKV antibodies are generated is not a human.
(75) Compositions of the Invention
(76) Compositions of the invention include DNA vectors containing a heterologous nucleic acid molecule encoding an antigenic or therapeutic gene product, or fragment thereof, from a ZIKV (e.g., all or a portion of the nucleic acid molecule of SEQ ID NO: 1, 3, 5, 7, 9, or 11, or a variant thereof having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 1, 3, 5, 7, 9, or 11, and complements thereof). Additional compositions of the invention include an immunogenic polypeptide, or fragment thereof, from a ZIKV polyprotein (e.g., all or a portion of the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, or 12, or a variant thereof having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 2, 4, 6, 8, 10, or 12). The compositions of the invention may also include a ZIKV antibody (e.g., an anti-Env antibody) capable of binding ZIKV and epitopes derived thereof, such as epitopes containing one or more of residues of any one of SEQ ID NOs: 2, 4, 6, 8, 10, or 12. The antibody may be generated by immunization of a host with a polypeptide of any one of SEQ ID NOs: 2, 4, 6, 8, 10, or 12.
(77) Optionally, the compositions can be formulated, for example, for administration via a viral vector (e.g., an adenovirus vector or a poxvirus vector). Recombinant adenoviruses offer several significant advantages for use as vectors for the expression of, for example, one or more of the immunogens of the invention (e.g., ZIKV polypeptides). The viruses can be prepared to high titer, can infect non-replicating cells, and can confer high-efficiency transduction of target cells ex vivo following contact with a target cell population. Furthermore, adenoviruses do not integrate their DNA into the host genome. Thus, their use as expression vectors has a reduced risk of inducing spontaneous proliferative disorders. In animal models, adenoviral vectors have generally been found to mediate high-level expression for approximately one week. The duration of transgene expression (expression of a nucleic acid molecule of the invention) can be prolonged by using cell or tissue-specific promoters. Other improvements in the molecular engineering of the adenovirus vector itself have produced more sustained transgene expression and less inflammation. This is seen with so-called second generation vectors harboring specific mutations in additional early adenoviral genes and gutless vectors in which virtually all the viral genes are deleted utilizing a Cre-Lox strategy (Engelhardt et al., Proc. Natl. Aced. Sci. USA 91:6196 (1994) and Kochanek et al., Proc. Natl. Acad. Sci. USA 93:5731 (1996), each herein incorporated by reference).
(78) Therapeutic formulations of the compositions of the invention are prepared for administration to a subject (e.g., a human) using standard methods known in the art by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (20.sup.th edition), ed. A. Gennaro, 2000. Lippincott, Williams & Wilkins, Philadelphia, Pa.). Therapeutic formulations of the compositions of the invention are prepared using standard methods known in the art by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (20.sup.th edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.). Acceptable carriers, include saline, or buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagines, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN, PLURONICS, or PEG.
(79) Optionally, but preferably, the formulation contains a pharmaceutically acceptable salt, preferably sodium chloride, and preferably at about physiological concentrations. Optionally, the formulations of the invention can contain a pharmaceutically acceptable preservative. The preservative concentration may range from about 0.1 to about 2.0%, typically v/v. Suitable preservatives include those known in the pharmaceutical arts, such as benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben. Optionally, the formulations of the invention can include a pharmaceutically acceptable surfactant at a concentration of about 0.005 to about 0.02%.
(80) Optionally, the compositions of the invention may be formulated to include for co-administration, or sequential administration with, an adjuvant and/or an immunostimulatory agent, (e.g., a protein), such as receptor molecules, nucleic acids, immunogenic proteins, pharmaceuticals, chemotherapy agents, and accessory cytokines. For example, interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), lipid A, phospholipase A2, endotoxins, staphylococcal enterotoxin B, Type I interferon, Type II interferon, transforming growth factor- (TGF-), lymphotoxin migration inhibition factor, granulocyte-macrophage colony-stimulating factor (CSF), monocyte-macrophage CSF, granulocyte CSF, vascular epithelial growth factor (VEGF), angiogenin, transforming growth factor (TGF-), heat shock proteins (HSPs), carbohydrate moieties of blood groups, Rh factors, fibroblast growth factors, nucleotides, DNA, RNA, mRNA, MART, MAGE, BAGE, mutant p53, tyrosinase, AZT, angiostatin, endostatin, or a combination thereof, may be included in formulations of, or for co-administration with, the compositions of the invention.
(81) The pharmaceutical compositions of the invention can be administered in a therapeutically effective amount that provides an immunogenic and/or protective effect against an infective agent (e.g., a ZIKV. In some embodiments, a composition comprising a nucleic acid molecule, polypeptide, vector, and/or antibodies of the invention may be formulated for administration at a dose of at least 1-1,000 g (e.g., at least 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, or 300 g or more). In some embodiments, a composition comprising a nucleic acid molecule, vector, and/or vaccine of the invention of the invention is administered at a dose of 50 g.
(82) The compositions utilized in the methods described herein can be formulated, for example, for administration intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, by gavage, in cremes, or in lipid compositions.
(83) Pharmaceutical compositions according to the invention described herein may be formulated to release the composition immediately upon administration (e.g., targeted delivery) or at any predetermined time period after administration using controlled or extended release formulations. Administration of the pharmaceutical composition in controlled or extended release formulations is useful where the composition, either alone or in combination, has (i) a narrow therapeutic index (e.g., the difference between the plasma concentration leading to harmful side effects or toxic reactions and the plasma concentration leading to a therapeutic effect is small; generally, the therapeutic index, TI, is defined as the ratio of median lethal dose (LD.sub.50) to median effective dose (ED.sub.50)); (ii) a narrow absorption window at the site of release (e.g., the gastro-intestinal tract); or (iii) a short biological half-life, so that frequent dosing during a day is required in order to sustain a therapeutic level.
(84) Many strategies can be pursued to obtain controlled or extended release in which the rate of release outweighs the rate of metabolism of the pharmaceutical composition. For example, controlled release can be obtained by the appropriate selection of formulation parameters and ingredients, including, e.g., appropriate controlled release compositions and coatings. Suitable formulations are known to those of skill in the art. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes.
(85) The compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation may be administered in powder form or combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 8 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of an immunogenic composition (e.g., a vaccine or an anti-ZIKV antibody) of the invention and, if desired, one or more immunomodulatory agents, such as in a sealed package of tablets or capsules, or in a suitable dry powder inhaler (DPI) capable of administering one or more doses.
(86) Methods of Treatment Using Compositions of the Invention
(87) The pharmaceutical compositions (e.g., immunogenic compositions and anti-ZIKV antibodies) of the invention can be used to treat a subject (e.g., a human) at risk of exposure (e.g., due to travel to a region were Zika virus (ZIKV) infection is prevalent) to a ZIKV or to treat a subject having a ZIKV infection. In particular, the compositions of the invention can be used to treat (pre- or post-exposure) infection by a ZIKV. In some embodiments, treatment with a composition of the invention may reduce a ZIKV-mediated activity in a subject, such as viral titer, viral spread, infection, and or cell fusion. In some embodiments, ZIKV titer in a treated subject infected with ZIKV is decreased by about 1% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or more after administration of a composition (e.g., vaccine) of the invention to the subject. The ZIKV infection and/or exposure may be to a strain of the Asian Lineage (
(88) The vectors (e.g., mammalian, bacterial, or viral derived expression vectors) of the invention can be used to deliver a nucleic acid expressing an immunogen of the invention (e.g., one of more of SEQ ID NOs: 2, 4, 6, 8, 10, or 12 or variants thereof, having at least 85-99% sequence identity thereto, for example at least greater than 90% sequence identity thereto) to a subject in a method of preventing and/or treating a ZIKV infection. The vectors (e.g., mammalian, bacterial, or viral derived expression vectors) of the invention can be genetically modified to contain one or more nucleic acid sequences set forth in SEQ ID NOs: 1, 3, 5, 7, 9, or 11 or variants thereof having at least 85-99% sequence identity thereto, for example at least greater than 90% sequence identity thereto, and complements thereof. In particular, adenoviral vectors (e.g., vectors derived from Ad2, Ad5, Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52, and Pan9 (also known as AdC68)) disclosed in International Patent Application Publications WO 2006/040330 and WO 2007/104792, each incorporated by reference herein, are particularly useful as vectors of the invention in methods of delivering an immunogen of the invention to a subject. Other examples of vectors are described, for example, in McVey et al., (U.S. Pat. No. 5,801,030); incorporated herein, in its entirety, by reference.
(89) Useful gene therapy methods for the delivery of immunogens of the invention to a subject in need thereof include those described in PCT publication no. WO 2006/060641, U.S. Pat. No. 7,179,903, and PCT publication no. WO 2001/036620, which described the use of, for example, an adenovirus vector (e.g., vectors derived from Ad2, Ad5, Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52, and Pan9 (also known as AdC68)) for therapeutic protein delivery.
(90) Administration
(91) The pharmaceutical compositions of the invention can be administered to a subject (e.g., a human) pre- or post-exposure to an infective agent (e.g., a ZIKV) to treat, prevent, ameliorate, inhibit the progression of, or reduce the severity of one or more symptoms of virus infection (e.g., ZIKV infection). For example, the compositions of the invention can be administered to a subject having a ZIKV infection. Examples of symptoms of diseases caused by a viral infection, such as ZIKV, that can be treated using the compositions of the invention include, for example, fever, joint pain, rash, conjunctivitis, muscle pain, headache, retro-orbital pain, edema, lymphadenopathy, malaise, asthenia, sore throat, cough, nausea, vomiting, diarrhea, and hematospermia. These symptoms, and their resolution during treatment, may be measured by, for example, a physician during a physical examination or by other tests and methods known in the art.
(92) The preferred method of administration can vary depending on various factors (e.g., the components of the composition being administered and the severity of the condition being treated). Formulations suitable for oral or nasal administration may consist of liquid solutions, such as an effective amount of the composition dissolved in a diluent (e.g., water, saline, or PEG-400), capsules, sachets, tablets, or gels, each containing a predetermined amount of the chimeric Ad5 vector composition of the invention. The pharmaceutical composition may also be an aerosol formulation for inhalation, for example, to the bronchial passageways. Aerosol formulations may be mixed with pressurized, pharmaceutically acceptable propellants (e.g., dichlorodifluoromethane, propane, or nitrogen). In particular, administration by inhalation can be accomplished by using, for example, an aerosol containing sorbitan trioleate or oleic acid, for example, together with trichlorofluoromethane, dichlorofluoromethane, dichlorotetrafluoroethane, or any other biologically compatible propellant gas.
(93) Immunogenicity of the composition of the invention may be significantly improved if it is co-administered with an immunostimulatory agent and/or adjuvant. Suitable adjuvants well-known to those skilled in the art include, for example, aluminum phosphate, aluminum hydroxide, QS21, Quil A (and derivatives and components thereof), calcium phosphate, calcium hydroxide, zinc hydroxide, glycolipid analogs, octodecyl esters of an amino acid, muramyl dipeptides, polyphosphazene, lipoproteins, ISCOM matrix, DC-Chol, DDA, cytokines, and other adjuvants and derivatives thereof.
(94) The compositions of the invention may be administered to provide pre-exposure prophylaxis or after a subject has been diagnosed as having a viral infection (e.g., ZIKV infection) or a subject exposed to an infective agent, such as a virus (e.g., a ZIKV). The composition may be administered, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 35, 40, 45, 50, 55, or 60 minutes, 2, 4, 6, 10, 15, or 24 hours, 2, 3, 5, or 7 days, 2, 4, 6 or 8 weeks, or even 3, 4, or 6 months pre-exposure to a ZIKV, or may be administered to the subject 15-30 minutes or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 20, 24, 48, or 72 hours, 2, 3, 5, or 7 days, 2, 4, 6 or 8 weeks, 3, 4, 6, or 9 months, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 years or post-exposure to a ZIKV.
(95) When treating viral infection (e.g., a ZIKV infection), the compositions of the invention may be administered to the subject either before the occurrence of symptoms or a definitive diagnosis or after diagnosis or symptoms become evident. For example, the composition may be administered, for example, immediately after diagnosis or the clinical recognition of symptoms or 2, 4, 6, 10, 15, or 24 hours, 2, 3, 5, or 7 days after diagnosis or detection of symptoms.
(96) One or more doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses) of an immunogenic composition or anti-ZIKV antibody-containing composition of the invention may be administered to a subject in need thereof. In some embodiments, a subject is administered at least one dose. In some embodiments, a subject is administered at least two doses. In some embodiments, an immunogenic composition of the invention is administered to a subject in need thereof as a prime, a boost, or as a prime-boost.
(97) Dosages
(98) The dose of the compositions of the invention or the number of treatments using the compositions of the invention may be increased or decreased based on the severity of, occurrence of, or progression of, the disease in the subject (e.g., based on the severity of one or more symptoms of, e.g., viral infection).
(99) The pharmaceutical compositions of the invention can be administered in a therapeutically effective amount that provides an immunogenic and/or protective effect against an infective agent (e.g., a ZIKV). In some embodiments, a composition comprising a nucleic acid molecule, polypeptide, vector, and/or antibodies of the invention may be administered in a dose of at least 1 g to 10 mg (e.g., at least 10 g, 20 g, 30 g, 40 g, 50 g, 60 g, 70 g, 80 g, 90 g, 100 g, 125 g, 150 g, 175 g, 200 g, 225 g, 250 g, 275 g, 300 g, 325 g, 350 g, 375 g, 400 g, 425 g, 450 g, 475 g, 500 g, 525 g, 550 g, 575 g, 600 g, 625 g, 650 g, 875 g, 700 g, 725 g, 750 g, 775 g, 800 g, 825 g, 850 g, 875 g, 900 g, 925 g, 950 g, 975 g, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, or 9 mg or more). In some embodiments, a composition comprising a nucleic acid molecule, vector, and/or antibody of the invention of the invention is administered at a dose of about 50 g (e.g., a dose between about 25 g and about 75 g). In some embodiments, a composition comprising a nucleic acid molecule, vector, and/or antibody of the invention of the invention is administered at a dose of about 5 mg (e.g., a dose of about 1 mg to about 10 mg).
(100) In some instances, administration of an effective amount of a composition of the invention (e.g., an immunogen of the invention, such as SEQ ID NO: 1) reduces ZIKV serum viral loads determined from a subject having a ZIKV infection by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to viral loads determined from the patient prior to administration of an effective amount of a composition of the invention. In some instances, administration of an effective amount of a composition of the invention reduces serum viral loads to an undetectable level compared to viral loads determined from the patient prior to administration of an effective amount of a composition of the invention. In some instances, administration of an effective amount of a composition of the invention results in a reduced and/or undetectable serum viral load that may be maintained for at least about 1, 2, 3, 4, 5, 6, 7 days; 1, 2, 3, 4, weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months; or 1 year or more.
(101) The dosage administered depends on the subject to be treated (e.g., the age, body weight, capacity of the immune system, and general health of the subject being treated), the form of administration (e.g., as a solid or liquid), the manner of administration (e.g., by injection, inhalation, or dry powder propellant), and the cells targeted (e.g., epithelial cells, such as blood vessel epithelial cells, nasal epithelial cells, or pulmonary epithelial cells). The composition is preferably administered in an amount that provides a sufficient level of the antigenic or therapeutic gene product, or fragment thereof (e.g., a level of an antigenic gene product that elicits an immune response without undue adverse physiological effects in the host caused by the antigenic gene product).
(102) The method of delivery, for example of a DNA vaccine, may also determine the dose amount. In some cases, dosage administered by injections by intravenous (i.v.) or intramuscular (i.m.) route may require variable amounts of a DNA vaccine, for example from 10 g-1 mg. However, administration using a gene gun may require a dose of a DNA vaccine between 0.2 g and 20 g (e.g., 0.2, 0.1, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 g). In some instances, the use of a gene gun to deliver a dose of a DNA vaccine may require only ng quantities of DNA, for example between 10 ng and 200 ng (e.g., 10, 12, 13, 14, 15, 16, 17, 18, 19, 20.30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 ng).
(103) In other embodiments wherein the delivery vector is a virus, the subject can be administered at least about 110.sup.3 viral particles (VP)/dose or between 110.sup.1 and 110.sup.20 VP/dose (e.g., 110.sup.1, 110.sup.2, 110.sup.3, 110.sup.4. 110.sup.5. 110.sup.6, 110.sup.7, 110.sup.8, 110.sup.9, 110.sup.10, 110.sup.11, 110.sup.12, 110.sup.13, 110.sup.14, 110.sup.15. 110.sup.16, 110.sup.17, 110.sup.18, 110.sup.19, and 110.sup.20 VP/dose).
(104) In addition, single or multiple administrations of the compositions of the present invention may be given (pre- or post-exposure and/or pre- or post-diagnosis) to a subject (e.g., one administration or administration two or more times). For example, subjects who are particularly susceptible to, for example, viral infection (e.g., a ZIKV infection) may require multiple treatments to establish and/or maintain protection against the virus. Levels of induced immunity provided by the pharmaceutical compositions described herein can be monitored by, for example, measuring amounts of neutralizing secretory and serum antibodies. The dosages may then be adjusted or repeated as necessary to trigger the desired level of immune response. For example, the immune response triggered by a single administration (prime) of a composition of the invention may not sufficiently potent and/or persistent to provide effective protection. Accordingly, in some embodiments, repeated administration (boost), such that a prime boost regimen is established, can significantly enhance humoral and cellular responses to the antigen of the composition.
(105) Alternatively, the efficacy of treatment can be determined by monitoring the level of the antigenic or therapeutic gene product, or fragment thereof, expressed in a subject (e.g., a human) following administration of the compositions of the invention. For example, the blood or lymph of a subject can be tested for antigenic or therapeutic gene product, or fragment thereof, using, for example, standard assays known in the art.
(106) In some instances, efficacy of treatment can be determined by monitoring a change in the serum viral load from a sample from the subject obtained prior to and after administration of an effective amount of a composition of the invention (e.g., an immunogen of the invention, such as SEQ ID NO: 1). A reduction in serum viral load of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to viral load determined from the subject prior to administration of an effective amount of a composition of the invention may indicate that the subject is receiving benefit from the treatment. If a viral load does not decrease by at least about 10%, 20%, 30%, or more after administration of a composition of the invention, the dosage of the composition to be administered may be increased. For example, by increasing the g or mg amount of a DNA vaccine (e.g., a DNA vaccine containing SEQ ID No: 1) administered to the subject or by increasing the number of viral particles (VP) of an adenovirus vector-based vaccine (e.g., an adenovirus vector-based vaccine containing SEQ ID NO: 1).
(107) A single dose of a composition of the invention may achieve protection, pre-exposure or pre-diagnosis. In addition, a single dose administered post-exposure or post-diagnosis can function as a treatment according to the present invention.
(108) A single dose of a composition of the invention can also be used to achieve therapy in subjects being treated for an infection (e.g., a ZIKV infection). Multiple doses (e.g., 2, 3, 4, 5, or more doses) can also be administered, in necessary, to these subjects.
II. EXAMPLES
(109) The following examples are to illustrate the invention. They are not meant to limit the invention in any way.
Example 1. Development and Characterization of ZIKV DNA Vaccines
(110) Introduction
(111) Zika virus (ZIKV) is believed to cause neuropathology in developing fetuses by crossing the placenta and targeting cortical neural progenitor cells, leading to impaired neurogenesis and resulting in microcephaly and other congenital malformations. ZIKV also has been associated with neurologic conditions such as Guillain-Barre syndrome. Vaccines have been developed for other flaviviruses, including yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, and dengue viruses, but no vaccine currently exists for ZIKV.
(112) Generation of Zika Virus Challenge Stocks
(113) Zika virus (ZIKV) stocks were provided by the University of So Paulo, Brazil (Brazil ZKV2015, accession number KU497555.1 (SEQ ID NOs: 17-18); ZIKV-BR) (Cugola et al., Nature 2016) and the U.S. Centers for Disease Control and Prevention, USA (Puerto Rico PRVABC59, accession number KU501215.1 (SEQ ID NOs: 19-20); ZIKV-PR) (
(114) Both the ZIKV-BR and ZIKV-PR strains were passage number three. To generate challenge stocks low passage number Vero E6 cells were infected at a multiplicity of infection (MOI) of 0.01 plaque-forming units (PFU)/cell. Supernatants were screened daily for viral titers and harvested at peak growth. Culture supernatants were clarified by centrifugation, and fetal bovine serum was added to 20% final concentration (v/v) and stored at 80 C.
(115) The concentration and infectivity of the stocks were determined by RT-PCR and PFU assays. The PFU assay was conducted as follows. Vero WHO cells were seeded in a MVV6 plate to reach confluency at day three. Cells were infected with log dilutions of ZIKV for one hour and overlaid with agar. Cells were stained after six days of infection by neutral red staining. Plaques were counted, and titers were calculated by multiplying the number of plaques by the dilution and divided by the infection volume.
(116) The RT-PCR assay was conducted as follows. Cap genes of available ZIKV genomes were aligned using Megalign (DNAstar, WI, USA), and primers and probes to a highly conserved region were designed using primer express v3.0 (Applied Biosystems, CA, USA). Primers were synthesized by Integrated DNA Technologies (Coralville, Iowa, USA) and probes by Biosearch Technologies (Petaluma, Calif., USA). To assess viral loads, RNA was extracted from serum with a QIAcube HT (Qiagen, Germany). Reverse transcription and RT-PCR were performed as previously described (Larocca et al., Science. 353(6304):1129-1132, 2016). The wildtype ZIKV BeH815744 Cap gene was utilized as a standard and was cloned into pcDNA3.1+, and the AmpliCap-Max T7 High Yield Message Maker Kit was used to transcribe RNA (Cellscript, WI, USA). RNA was purified using the RNA clean and concentrator kit (Zymo Research, CA, USA), and RNA quality and concentration was assessed by the BIDMC Molecular Core Facility. Log dilutions of the RNA standard were reverse transcribed and included with each RT-PCR assay.
(117) Viral loads were calculated as virus particles (VP) per ml. The viral particle (VP) to plaque-forming unit (PFU) ratio of both stocks was approximately 1,000.
(118) Design of ZIKV Immunogens and ZIKV DNA Vaccines
(119) Zika virus (ZIKV) strain BeH815744 (accession number KU365780 (SEQ ID NOs: 15-16)) (
(120) To assess transgene expression (e.g., polypeptide expression (e.g., immunogen expression)) from DNA vaccines, cell lysates obtained 48 hour following lipofectamine 2000 (Invitrogen, CA, USA) transient transfection of 293T cells were mixed with reducing sample buffer, heated for five min at 100 C., cooled on ice, and run on a precast 4-15% SDS-PAGE gel (Biorad, CA, USA). Protein was transferred to PVDF membranes using the iBlot dry blotting system (Invitrogen, CA, USA), and the membranes were blocked overnight at 4 C. in PBS-T (Dulbeco's Phosphate Buffered Saline+0.2% V/V Tween 20+5% W/V non-fat milk powder). Following overnight blocking, the membranes were incubated for one hour with PBS-T containing a 1:5000 dilution of mouse anti-ZIKV Env mAb (BioFront Technologies, FL, USA). Membranes were then washed three times with PBS-T and incubated for one hour with PBS-T containing a 1:1000 dilution of rabbit anti-mouse HRP (Jackson ImmunoResearch, PA, USA). Membranes were then washed three times with PBS-T and developed using the Amersham ECL plus Western blotting detection system (GE Healthcare, Chicago, USA). Transgene expression was verified by Western blot (
(121) In Vivo Assessment of Immunologic Response to ZIKV Immunogens
(122) To assess the immunogenicity of the DNA vaccines prM-Env (DNA-prM-Env, comprising SEQ ID NO: 1), prM-Env.dTM (DNA-prM-Env.dTM, comprising SEQ ID NO: 3), prM-Env.dStem (DNA-prM-Env.dStem, comprising SEQ ID NO: 5), Env (DNA-Env, comprising SEQ ID NO: 7), Env.dTM (DNA-Env.dTM, comprising SEQ ID NO: 9), and Env.dStem (DNA-Env.dStem, comprising SEQ ID NO: 11), groups of Balb/c mice (N=5-10/group) received a single immunization of 50 g of DNA vaccine by the intramuscular (i.m.) route at week zero. Env-specific antibody responses were evaluated at week three by ELISA. Mouse ZIKV Env ELISA kits (Alpha Diagnostic International, TX, USA) were used to determine endpoint antibody titers using a modified protocol. 96-well plates were first equilibrated at room temperature with 300 l of kit working wash buffer for five min. 6 l of mouse serum was added to the top row, and 3-fold serial dilutions were tested in the remaining rows. Samples were incubated at room temperature for one hour, and plates were washed four times. 100 l of anti-mouse IgG HRP-conjugate working solution was then added to each well and incubated for 30 min at room temperature. Plates were then washed five times, developed for 15 min at room temperature with 100 l of TMB substrate, and stopped by the addition of 100 l of stop solution. Plates were analyzed at 450 nm/550 nm on a VersaMax microplate reader using Softmax Pro 6.0 software (Molecular Devices, CA, USA). ELISA endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance >2-fold over background values. The full-length prM-Env DNA vaccine elicited higher Env-specific antibody titers than did the Env DNA vaccine and all the dTM and dStem deletion mutants (
(123) ZIKV-specific cellular immune responses were assessed by interferon- (IFN-) ELISPOT assays using a pool of overlapping 15-amino-acid peptides covering the prM or Env proteins (JPT, Berlin, Germany). The assay was performed as follows. 96-well multiscreen plates (Millipore, MA, USA) were coated overnight with 100 l/well of 10 g/ml anti-mouse IFN- (BD Biosciences, CA, USA) in endotoxin-free Dulbecco's PBS (D-PBS). The plates were then washed three times with D-PBS containing 0.25% Tween 20 (D-PBS-Tween), blocked for two hour with D-PBS containing 5% FBS at 37 C., washed three times with D-PBS-Tween, rinsed with RPMI 1640 containing 10% FBS to remove the Tween 20, and incubated with 2 g/ml of each peptide and 510 murine splenocytes in triplicate in 100 l reaction mixture volumes. Following an 18 hour incubation at 37 C., the plates were washed nine times with PBS-Tween and once with distilled water. The plates were then incubated with 2 g/ml biotinylated anti-mouse IFN- (BD Biosciences, CA, USA) for two hour at room temperature, washed six times with PBS-Tween, and incubated for two hour with a 1:500 dilution of streptavidin-alkaline phosphatase (Southern Biotechnology Associates, AL, USA). Following five washes with PBS-Tween and one with PBS, the plates were developed with nitroblue tetrazolium-5-bromo-4-chloro-3-indolyl-phosphate chromogen (Pierce, Ill., USA), stopped by washing with tap water, air dried, and read using an ELISPOT reader (Cellular Technology Ltd., OH, USA). The numbers of spot-forming cells (SFC) per 10.sup.6 cells were calculated. The medium background levels were typically <15 SFC per 10.sup.6 cells.
(124) ZIKV-specific CD4.sup.+ and CD8.sup.+ T lymphocyte responses were assessed using splenocytes and analyzed by flow cytometry (
(125) In Vivo Assessment of the Protective Efficacy of ZIKV DNA Vaccines Against ZIKV Challenge
(126) To assess the protective efficacy of these DNA vaccines against ZIKV challenge, vaccinated or sham control Balb/c mice were challenged at week four post immunization by the intravenous (i.v.) route with 10.sup.5 viral particles (VP) [10.sup.2 plaque-forming units (PFU)] of ZIKV-BR or ZIKV-PR. Viral loads following ZIKV challenge were determined by RT-PCR (Larocca et al., Science. 353(6304):1129-1132, 2016), as generally described herein. Sham vaccinated mice inoculated with ZIKV-BR developed approximately 6 days of detectable viremia with a mean peak viral load of 5.42 log copies/ml (range 4.55-6.57 log copies/ml; N=10) on day three following challenge (
(127) The prM-Env DNA vaccine also provided complete protection against ZIKV-BR challenge in SJL mice (
(128) Protective Efficacy of Antibodies Produced from DNA-prM-Env Immunization
(129) To investigate the immunologic mechanism of protection against ZIKV-BR challenge, serum was collected from prM-Env DNA vaccinated mice or nave mice, and polyclonal IgG was purified using protein G purification kits (Thermo Fisher Scientific, MA, USA). Varying amounts of purified IgG was infused by the intravenous (i.v.) route into nave recipient mice prior to ZIKV challenge. Passive infusion of varying quantities of purified IgG (e.g., 100 uL at varying titers between 25-2025) by the i.v. route resulted in median Env-specific log serum antibody titers of 2.82 (high), 2.35 (mid), and 1.87 (low) in recipient mice following adoptive transfer (
(130) Depletion of T Lymphocytes We next depleted CD4.sup.+ and/or CD8.sup.+ T lymphocytes (>99.9% efficiency) in prM-Env vaccinated mice on day 2 and day 1 prior to challenge (
Conclusion
(131) The data presented here demonstrates that a single immunization with a DNA vaccine provided complete protection against parenteral ZIKV challenges in mice. The prM-Env DNA vaccine afforded protection in three strains of mice, and against ZIKV isolates from both Brazil and Puerto Rico, suggesting the generalizability of these observations. Moreover, the vaccine immunogens were designed to be heterologous sequences compared with the challenge viruses (
(132) The robust protection observed in the present studies and the clear immune correlate of protection confirm the applicability of ZIKV vaccine development for use in humans. Moreover, the ZIKV-BR challenge isolate used in the present study has been shown in wildtype SJL mice to recapitulate certain key clinical findings of ZIKV infection in humans, including fetal microcephaly and intrauterine growth retardation. In addition, ZIKV-BR induced comparable magnitude and duration of viremia in Balb/c and SJL mice in our studies as compared with humans, suggesting the potential relevance of this model. It is notable that ZIKV-BR replicated efficiently in Balb/c and SJL mice (
(133) The explosive epidemiology of the current ZIKV outbreak and the devastating clinical consequences for fetuses in pregnant women who become infected confirm the need for a ZIKV vaccine, such as those described herein. Our data demonstrate that complete protection against ZIKV challenge was reliably and robustly achieved with DNA vaccines and purified inactivated virus vaccines in susceptible mice. The compositions described herein offer safety advantages over live attenuated and replicating flavivirus vaccines, particularly for pregnant women. Moreover, the magnitude of Env-specific antibody titers that provide complete protection against ZIKV challenge in mice can be expected in humans as well, using DNA vaccines.
Example 2. Administration of a DNA Vaccine to a Human Subject
(134) Compositions of the invention may be administered to human subjects, pre- or post-exposure to a ZIKV, according to the methods of the invention. The human subject may be one identified as being at high risk for infection, such as an individual who has or will be traveling to a region where ZIKV infection is prevalent.
(135) For example, a pregnant woman or a women of child-bearing age identified as having a risk of ZIKV infection may be administered a DNA vaccine containing a nucleic acid molecule encoding a ZIKV nucleic acid of the invention (e.g., prM-Env (DNA-prM-Env, SEQ ID NO: 1)), e.g., in an adenoviral vector at a dose of between 10 g and 10 mg. The patient is then monitored for presentation of symptoms of ZIKV infection or the resolution of symptoms. If necessary, a second dose or additional doses of the DNA vaccine can be administered.
Example 3. Administration of an Immunogenic ZIKV Polypeptide to a Human Subject
(136) A human subject identified as having a risk of ZIKV infection may be administered a ZIKV immunogen of the invention (e.g., prM-Env polypeptide (SEQ ID NO: 2)) or a nucleic acid molecule encoding a ZIKV polypeptide (e.g., SEQ ID NO: 1), e.g., in an adenoviral vector at a dose of between 10 g and 10 mg. The patient is then monitored for presentation of symptoms of ZIKV infection or the resolution of symptoms. If necessary, a second dose of the DNA vaccine can be administered.
Example 4. Administration of Anti-ZIKV Antibodies to a Human Subject at Risk of ZIKV Infection
(137) A human subject identified as having a risk of ZIKV infection (e.g., due to travel to a region where ZIKV infection is prevalent, or the subject being a pregnant woman or a woman of childbearing age) may be administered an anti-ZIKV antibody that binds to an epitope within the prM-Env (SEQ ID NO: 2) polypeptide (e.g., the antibody may have been generated against the prM-Env polypeptide of SEQ ID NO: 2) at a dose of between 1-1.000 mg as a prophylactic therapy. The subject may be administered the anti-ZIKV antibody as a prophylactic therapy prior to or post-exposure to a ZIKV. The patient can then be monitored for presentation of symptoms of ZIKV infection or the resolution of symptoms. If necessary, a second dose or additional doses of the anti-ZIKV antibody can be administered.
Example 5. Administration of Anti-ZIKV Antibodies to a Human Subject Presenting Symptoms of ZIKV Infection
(138) A human subject identified as presenting symptoms of ZIKV may be administered an anti-ZIKV antibody that binds to an epitope within the prM-Env (SEQ ID NO: 2) polypeptide (e.g., the antibody may have been generated against the prM-Env polypeptide of SEQ ID NO: 2) at a dose of between 1-1,000 mg. The subject (e.g., a male or female subject, such as a pregnant woman or a woman of childbearing age) may have recently traveled to a region where ZIKV infection is prevalent. After diagnosis of ZIKV infection by a medical practitioner, the subject can be administered a dose of the anti-ZIKV antibody. The patient can then be monitored for resolution of symptoms. If necessary, a second dose or additional doses of the anti-ZIKV antibody can be administered.
Example 6. Development and Characterization of ZIKV Adenovirus Vector-Based Vaccines
(139) Design of ZIKV Adenovirus Vaccines
(140) Adenovirus vaccines were generated by incorporating a nucleic acid molecule of
(141) In Vivo Assessment of the Protective Efficacy of ZIKV Adenovirus Vector-Based Vaccines Against ZIKV Challenge
(142) To assess the protective efficacy of these adenovirus vector-based vaccines against ZIKV challenge, vaccinated or sham vaccinated (i.e., unvaccinated) control Balb/c mice were challenged at week four post immunization by the intramuscular (i.m.) route with 10.sup.5 viral particles (VP) [10.sup.2 plaque-forming units (PFU)] of ZIKV-BR. Viral loads following ZIKV challenge were determined by RT-PCR (Larocca et al. Science. 353(6304):1129-1132, 2016), as generally described herein. Sham vaccinated mice inoculated with ZIKV-BR developed approximately 6 days of detectable viremia following challenge (
Example 7. Optimization of ZIKV prM-Env Immunogens and ZIKV prM-Env DNA Vaccines
(143) Following the methodology described in Example 1, additional DNA vaccines were generated by incorporating a nucleic acid molecule of
(144) Immunogenicity of the DNA vaccines prM-Env or M-Env (DNA-prM-Env (M-Env), comprising SEQ ID NO: 1), prM-Env (full-length) (DNA-prM-Env (full-length), comprising SEQ ID No: 24), and prM-Env with JEV Stem/TM vaccine (DNA-prM-Env (JEV Stem), comprising SEQ ID No: 26) was compared using the methods described in Example 1. The prM-Env or M-Env vaccine, prM-Env (full-length) vaccine, and prM-Env with JEV Stem/TM vaccine were found elicited approximately equivalent median Env-specific antibody titers (
(145) These data suggest that the prM-Env or M-Env vaccine, comprising SEQ ID NO: 1, provides increased antigen expression, immunogenicity, and improved protective efficacy in mice over the prM-Env (full-length) vaccine and the prM-Env with JEV Stem/TM vaccine. Additionally, the addition of the JEV stem was found to impair protective efficacy.
Example 8. Evaluation of ZIKV DNA and Adenovirus Vector-Based Vaccines in Rhesus Monkeys
(146) To assess the immunogenicity of the DNA vaccine prM-Env or M-Env (DNA-prM-Env (M-Env). comprising SEQ ID NO: 1) and the adenovirus vector-based vaccine RhAd52-prM-Env (RhAd52-prM-Env, comprising SEQ ID No: 1) groups of rhesus monkeys (N=4/group) received immunization with 5 mg of DNA vaccine by the intramuscular (i.m.) route at week zero and week four, or a single immunization with 10.sup.10 virus particles (VP) of RhAd52-prM-Env vaccine at week zero. Cellular immune responses were measured using IFN- ELISPOT assays to prM, Env, Cap, and NS1 at week 6 for the DNA vaccine or at week 2 for the RhAd52-prM-Env vaccine. The DNA-prM-Env vaccine induced ZIKV-specific neutralizing antibody titers in all animals after the week 4 boost immunization, although only minimal 50% microneutralization (MN50) titers were detected after the initial priming immunization (
(147) Additionally, the durability of the protective efficacy of immunization with DNA-prM-Env or RhAd52-prM-Env, as described above, was assessed one year after immunization. One year post immunization monkeys were challenged with 10.sup.8 VP (10.sup.3 PFU) of ZIKV-BR, generally as described herein. Detectable Env-specific antibody responses were observed 2, 4, 6, 8, 10, 14, 18, 23, and 34 weeks post immunization (
Example 9: Evaluating the Durability of the Protective Effect of ZIKV DNA and Adenovirus Vector-Based Vaccines in Balb/c Mice
(148) To access the durability of the protective efficacy of the ZIKV DNA and adenovirus vector-based vaccines of the invention vaccinated or naive control Balb/c mice were challenged at week 20 post immunization by the intramuscular (i.m.) route with 10.sup.2 plaque-forming units (PFU) of ZIKV-BR (
(149) Generally, an adenovirus vector-based vaccine containing the prM-Env immunogen (SEQ ID NO: 1) was found to offer robust protection to both mice and monkeys, when administered as a single shot vaccine. Adenovirus vector-based vaccine containing the prM-Env Immunogen (SEQ ID NO: 1) were also found to be more potent than DNA vaccines containing the prM-Env immunogen (SEQ ID NO: 1) in both mice and monkeys.
Example 10: Evaluating the Protective Effect of ZIKV DNA and Adenovirus Vector-Based Vaccines in Balb/c Mice Having a Baseline Flavivirus Immunity
(150) The protective efficacy of the ZIKV DNA and adenovirus vector-based vaccines of the invention was evaluated in Balb/c mice having a baseline immunity to a Flavivirus or naive controls (
Other Embodiments
(151) All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.
(152) While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.
(153) Other embodiments are within the claims.