Patent Publication Number: US-2022213504-A1

Title: Zika Virus Constructs and Therapeutic Compositions Thereof

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of U.S. Patent Application No. 62/850,759, filed May 21, 2019, which is herein incorporated by reference in its entirety. 
    
    
     REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB 
     The content of the ASCII text file of the sequence listing named “20200520_034044_204WO1_ST25” which is 121 kb in size was created on May 19, 2020, and electronically submitted via EFS-Web herewith the application is incorporated herein by reference in its entirety. 
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention generally relates to Zika virus mutants and therapeutic compositions thereof, which may be used in the treatment of cancer. 
     2. Description of the Related Art 
     Gliomas are a type of brain tumor comprised of non-neuronal glial cells. Glioma accounts for 80% of all primary malignant brain tumors. Despite decades of research, patients with glioblastoma, the most common and deadly form of glioma, have a 5-year survival rate of 5.1%. Standard therapy for high-grade glioma requires a combination of surgical resection, radiation, and chemotherapy. Despite recent advances in malignant glioma research, only modest progress has been achieved in improving patient prognosis and quality of life. Glioblastoma (GB), the most common primary malignant brain tumor, is nearly universally fatal, with an average survival of 16 months following gross-total resection and adjuvant therapy. Glioblastoma is comprised of a heterogeneous cell population consisting of self-renewing cancer stem cells (CSCs) and varying degrees of differentiated tumor cells. Glioblastoma, also known as glioblastoma multiforme (GBM), likely arises from transformed glial populations of oligodendrocytes and the tumor propagating cells are a mix of oligodendrocyte progenitor cells (OPCs)/astrocytes/and undifferentiated glioblasts. 
     PCT publication WO 2018/035294 discloses the oncolytic effect of wild-type ZIKV strains on tumor cells, particularly gliomas and glioblastomas. Zika virus (ZIKV) is a mosquito-borne human pathogen of the Flaviviridae family. The ZIKV genome consists of a 10.8-kilobase single-stranded positive-sense RNA that codes for three structural proteins (capsid (C), membrane (prM/M), and envelope (E)) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). In addition, there are short UTRs on both the 5′ and 3′ ends of the genome. The mature ZIKV virion is roughly spherical and about 50 nm in diameter. It contains a nucleocapsid that is surrounded by an icosahedral shell consisting of 180 copies of both E glycoprotein and M protein anchored in a lipid bilayer. The flavivirus E protein, arranged as dimers on the surface of the mature virion, is the major viral protein involved in host-cell entry factor binding and fusion. Each E protein monomer consists of four domains—three ectodomains (DI, DII, and DIII) and a transmembrane domain (TM). The structurally central DI acts as a bridge between DII and DIII and contains one N-linked glycosylation site (N154). The N-linked glycosylation site is conserved across most flaviviruses and has been shown to be important for optimal infection of mosquito and mammalian cells. DII includes the dimerization interface and a fusion loop that interacts with the endosomal membrane after conformation change. The IgG-like DIII is a continuous polypeptide segment and is thought to be important for binding to entry factors. Several host entry factors, including DC-SIGN, AXL, and TYRO3, have been shown to be important for mediating ZIKV infection. 
     ZIKV infection is mostly asymptomatic, but it can cause influenza-like symptoms, such as fever, headache, joint pain, and maculopapular rash. Unfortunately, the recent outbreak of ZIKV in the Americas indicates that ZIKV can cause more serious disease, including microcephaly, other congenital malformations, and Guillain-Barré syndrome, and even death. 
     SUMMARY OF THE INVENTION 
     In some embodiments, the present invention provides a Zika Vector comprising a Zika virus genome, which lacks sequences that encode functional Zika virus capsid, matrix, and envelope proteins as described herein. In some embodiments, the present invention provides a ZIKV-E mutant, which comprises an E gene that encodes an envelope (E) protein having one or more amino acid mutations (i.e., substitutions, deletions, and/or insertions) as compared to the wildtype E protein (e.g., SEQ ID NO: 2), and (2) exhibits reduced E protein glycosylation as compared to a wildtype ZIKV strain (e.g., GenBank Accession Number KU501215, clinical isolate PRVABC59) having asparagine at amino acid position 154 (N154) in the envelope protein. The Zika Vector and the ZIKV-E mutant may be used to deliver one or more passenger sequences to a subject or a cell. In some embodiments the one or more passenger sequences are exogenous to the Zika virus genome of the given Zika Vector or ZIKV-E mutant. In some embodiments, the one or more passenger sequences is or encodes an immunomodulatory agent. In some embodiments, the immunomodulatory agent is IL2, IL15, IL18, CCL5 (RANTES), or TNFSF14 (LIGHT). In some embodiments, the one or more passenger sequences is or encodes a protein of interest, e.g., a therapeutic protein or an antigen or antigenic epitope thereof. In some embodiments, the Zika Vector is a ZIKV-Im mutant as described herein. In some embodiments, the ZIKV-E mutant is a ZIKV-Im mutant as described herein. In some embodiments, the present invention provides compositions which comprise one or more ZIKV constructs (i.e., one or more ZIKV-E mutants, one or more Zika Vectors, and/or one or moreZIKV-Im mutants) as described herein. 
     In some embodiments, the present invention provides a method of treating a cancer and/or aberrant neuroprogenitor cells in a subject, which comprises administering one or more ZIKV constructs or a pharmaceutical composition thereof to the subject. In some embodiments, the ZIKV construct is a ZIKV-E mutant as described herein. In some embodiments, the ZIKV construct is a ZIKV-Im mutant as described herein. In some embodiments, the ZIKV construct is both a ZIKV-E mutant and a ZIKV-Im mutant. In some embodiments, the ZIKV construct is a Zika Vector. In some embodiments, the one or more Zika Vectors and/or the one or more ZIKV-E mutants comprise one or more passenger sequences. In some embodiments the one or more passenger sequences are exogenous to the Zika virus genome of the given Zika Vector or ZIKV-E mutant. In some embodiments, the passenger sequence is or encodes an immunomodulatory agent. In some embodiments, the immunomodulatory agent is IL2, IL15, IL18, CCL5 (RANTES), or TNFSF14 (LIGHT). In some embodiments, the passenger sequences is or encodes a therapeutic protein such as CT20p-NP, Poropeptide-Bax, R8-Bax, RRM-IL12, RRM-MV, or TAT-Bim, BAC1-ELP-H1, or RGD-PEG-Suc-PD0325901. In some embodiments, a therapeutically effective amount of the one or more ZIKV constructs is administered to the subject by, for example, by subcutaneous delivery, intravenous delivery, intratumoral delivery, intracerebral delivery, and/or intracarotid delivery. In some embodiments, the one or more ZIKV constructs are administered to the subject a ratio of about 1:1 to about 10:1 of the cells to be treated or infected by the given construct. In some embodiments, the method further comprises administering one or more AKT inhibitors, e.g., MK-2206, to the subject. In some embodiments, the cancer is a ZIKV-treatable cancer such as a glioma, a glioblastoma, a neuroblastoma, or a retinoblastoma. In some embodiments, subject is mammalian. In some embodiments, subject is human. 
     In some embodiments, the present invention provides a method of delivering one or more passenger sequences to a subject or a cell, which comprises administering one or more Zika Vectors comprising the one or more passenger sequences to the subject or cell. In some embodiments the one or more passenger sequences are exogenous to the Zika virus genome of the one or more Zika Vectors. In some embodiments, the one or more passenger sequences are or encode an immunomodulatory agent. In some embodiments, the immunomodulatory agent is IL2, IL15, IL18, CCL5 (RANTES), or TNFSF14 (LIGHT). In some embodiments, the one or more passenger sequences encode a protein of interest, e.g., a therapeutic protein or an antigen or antigenic epitope thereof. 
     In some embodiments, the present invention provides a method of inducing an immune response in a subject, which comprises administering to the subject one or more ZIKV constructs as described herein. In some embodiments, the ZIKV construct is a ZIKV-E mutant as described herein. In some embodiments, the ZIKV construct is a ZIKV-Im mutant as described herein. In some embodiments, the ZIKV construct is both a ZIKV-E mutant and a ZIKV-Im mutant. In some embodiments, the ZIKV construct is a Zika Vector. In some embodiments, the one or more Zika Vectors and/or the one or more ZIKV-E mutants comprise one or more passenger sequences. In some embodiments the one or more passenger sequences are exogenous to the Zika virus genome of the given Zika Vector or ZIKV-E mutant. In some embodiments, the one or more passenger sequences are or encode an immunomodulatory agent. In some embodiments, the immunomodulatory agent is IL2, IL15, IL18, CCL5 (RANTES), or TNFSF14 (LIGHT). In some embodiments, the one or more passenger sequences encode an antigen or an antigenic epitope thereof. In some embodiments, the antigen or the antigenic epitope is of a pathogen. In some embodiments, the one or more passenger sequences encode a nucleocapsid protein, an envelope protein, a spike protein, or one or more fragments thereof of a given virus. In some embodiments, an immunogenic amount of the one or more ZIKV constructs is administered to the subject. 
     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. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
       This invention is further understood by reference to the drawings wherein: 
         FIG. 1  schematically shows some of the ZIKV constructs exemplified herein: a wild-type ZIKV strain (ZIKV-WT), a GFP-labeled ZIKV-E mutant (GFP-ZIKV-E), a ZIKV-E mutant comprising a passenger sequence, Gene X (PS-ZIKV-E), a GFP-labeled Zika Vector, which lacks the structural genes encoding capsid, prM, and E proteins (GFP-Zika Vector), and a Zika Vector comprising a passenger sequence, Gene X (PS-Zika Vector). GFP=green fluorescent protein, P2A=F2A linker sequence, Gene X=gene encoding a passenger sequence such as an immunomodulatory agent (e.g., a cytokine such as CCL5, IL18, IL2, and TNFSF14). 
         FIG. 2 - FIG. 5 : Safety profile of ZIKV-E mutants.  FIG. 2  is a graph showing the induction of apoptotic cell death by a ZIKV-E mutant (VAX2B) in glioblastoma cancer cells at 24 hours post infection. The first bar on the left is Mock followed by two sets of bars in which the first bar of each set is ZIKV wild-type (PRVABC59), and the second bar of each set is VAX2B.  FIG. 3  is a Kaplan-Meier survival plot showing the percent survival of Ifnar1−/− mice following infection with wild-type ZIKV (PRVABC59 and MR766) and ZIKV-E mutants (VAX2A and VAX2B). Negative control mice (Mock) and mice infected with the ZIKV-E mutants exhibited 100% survival over the entire study period.  FIG. 4  shows the serum viral load of VAX2B at 7 days post infection (dpi).  FIG. 5  shows the mean percent body weight change of mice post-subcutaneous inoculation with Mock, wild-type PRVABC59, wild-type MR766 and two different ZIKV-E mutants, VAX2A and VAX2B. Furthermore, body weight of individual mouse for each group is given for mock, wild-type PRVABC59, wild-type MR766, VAX2A, and VAX2B. Error bars represent the standard deviation (SD). Students t-test, p value &lt;0.05(*), and p&lt;0.0001(***). 
         FIG. 6 - FIG. 10 : Glioblastoma Multiforme (GBM) cell line screening assays. Multiple GBM cell lines were infected with a ZIKV-E mutant (VAX2B) at viral doses MOI 1 and MOI 10 in 96-well plate format in triplicate and 2 independent platings. Tumoroid spheres attached to the plate. Every other day, fresh neuronal supplement (NS) media was added.  FIG. 6  shows exemplary bright field images taken 7 days post-infection (dpi). Cell lines that were susceptible to the ZIKV-E mutant are categorized as “Responders” and cell lines that were less susceptible are categorized as “Non-Responders”.  FIG. 7  is a graph showing cell viability of the indicated cell lines at 7 dpi.  FIG. 8  is a graph summarizing the cleaved caspase 3/7 activity (apoptosis) of the indicated cell lines at 7 dpi.  FIG. 9  and  FIG. 10 : Graphs showing that the indicated Non-Responder ( FIG. 9 ) and Responder ( FIG. 10 ) GBM cell lines support high levels of ZIKV-E mutant viral production. 
         FIG. 11  is a graph showing that the combination of an AKT pathway inhibitor, MK-2206, and a ZIKV-E mutant (VAX2B) synergistically increases the percent apoptosis of non-responder GBM primary cells (GS054). 
         FIG. 12 - FIG. 15 : ZIKV-E mutants are safe and effective oncolytic therapeutics.  FIG. 12  schematically shows the experimental design of in vivo experiments using 8-10 week old Female NSG mice. Subjects were divided into three groups: Control (PBS), ZIKV-E, and ZIKV-E Pre-Treatment. Subjects of the ZIKV-E Pre-Treatment Group were treated with the ZIKV-E mutant 4 hours before xenograft implantation. Xenograft implantation: 2×10 6  U-87 MG cells were implanted SQ in the left hind flank of each subject. On Day 17 and Day 24, Controls and ZIKV-E Pre-Treatment subjects were administered PBS while the ZIKV-E subjects were treated with the ZIKV-E mutant. The dose of the ZIKV-E mutant (VAX2B) administered was MOI 1 (2×10 6  PFU/mouse).  FIG. 13  is a graph showing that subjects pretreated with the ZIKV-E mutant do not develop tumors and treatment with the ZIKV-E mutant effectively reduced tumor volume (mm 3 ) by Day 34. By Day 43, only a small connective tissue tumor remnant in some ZIKV-E subjects.  FIG. 14  is a Kaplan-Meier Survival curve (n=8 mice/group) showing that untreated positive controls (Group A, PBS +tumor xenograft) died by Day 40, whereas subjects of the negative control, ZIKV-E, and ZIKV-E Pre-Treatment Groups exhibit 100% survival past Day 40.  FIG. 15  is a graph summarizing the ZIKV-E mutant viral titers in subjects of the positive control group (taken Days 30-38) and the ZIKV-E group (taken Day 43). Error bars represent the standard deviation (SD). Students t-test indicated statistically significant (p&lt;0.0001) differences between Group A and ZIKV-E group. 
         FIG. 16 - FIG. 18 : ZIKV-E mutants are effective oncolytic therapeutics against cancer cells that tend to be resistant to epidermal growth factor receptor (EGFR) inhibitors. 8-10 week old Female NSG mice were implanted with 2×10 6  GBM GS025 cells SQ in the left hind flank. On Day 41 and Day 48 the control group (n=8) received PBS and the treatment group (n=8) received VAX2B (2×10 6  pfu/mouse) intratumorally.  FIG. 16  is a graph showing that the ZIKV-E treated subjects maintained their body weight.  FIG. 17  is a Kaplan-Meier survival plot showing 100% survival of ZIKV-E subjects.  FIG. 18  is a graph showing that the ZIKV-E mutant, at least, inhibited the growth of the GS025 tumor cells. Error bars represent the standard deviation (SD). Students t-test, p&lt;0.05(*), p&lt;0.001(**), and p&lt;0.0001(***). 
         FIG. 19  schematically shows the design of a Zika Vector as described herein. The structural genes of a ZIKV virus are removed or can be replaced with a given passenger sequence, e.g., a nucleic acid sequence that encodes a desired therapeutic protein such as an immunomodulatory agent. 
         FIG. 20  schematically shows a Zika Vector as exemplified herein. The Zika Vector has a restriction enzyme linker sequence (BsiWI linker sequence) in which one or more passenger sequences may be inserted. 
         FIG. 21  schematically shows the VAXR1 construct as exemplified herein. The VAXR1 construct is the Zika Vector of  FIG. 20  comprising a sequence that encodes eGFP inserted in the BsiWI linker. The VAXR1 construct also comprises an F2A insert and a Not1 linker sequence. 
         FIG. 22  is a DNA gel electrophoresis image of digestion of ZIKV-Im mutant constructs. Genes encoding GFP, CCL5, IL18, IL2, and TNFSF14 were cloned into Zika Vector of  FIG. 20 . Gel image shows two independent clones per construct. M: 1 kb plus DNA ladder. 
         FIG. 23 : Vaccine production and validation. VAXR1 was used as being representative of the production of Zika Vectors comprising passenger sequences. Graph shows production of vaccine particles by 293T cells between days 5 to 11. Flow cytometry assays indicated vaccine particle production efficiencies of 43.22% (Day 5), 58.72% (Day 6), 66.69% (Day 7), 69.44% (Day 8), 65.03% (Day 9), 49.60% (Day 10) and 43.71% (Day 11). 
         FIG. 24  and  FIG. 25 : Safety profile of Zika Vectors in neonatal mice. VAXR1 was used to show that Zika Vectors are safe as compared to wildtype ZIKV (PRV-WT) in subjects.  FIG. 24 : Kaplan-Meier survival graph shows 100% mortality in wildtype ZIKV infected pups and complete protection in VAXR1 inoculated pups. 
         FIG. 25 : VAXR1 inoculated mice did not have any replicating virus, which had below detectable level of virus compared to wild-type virus infected pups. *** p&lt;0.0001 
         FIG. 26 - FIG. 29 : Show that Zika Vectors protect subjects from infection by ZIKV. Particularly, VAXR1 immunization protects breeding females and fetuses from Zika viral disease.  FIG. 26 : Schematic diagram of immunization and key timepoints.  FIG. 27 : Percent body weight change in PBS control (Un-Vac) and VAXR1 (Vac) immunized mice.  FIG. 28 : Percent body weight change of pregnant animals.  FIG. 29 : Body weight of E20.5 fetuses of vaccinated (Vac) and un-vaccinated (Un-Vac) pregnant mice. Growth retardation was observed in fetuses of un-vaccinated mothers, but not in fetuses of vaccinated mothers. 
         FIG. 30  and  FIG. 31 : VAXR1 immunization leads to protective immune responses against subcutaneous ZIKV challenge and T cell memory populations.  FIG. 30 : The percentage of splenic monocytes, neutrophils, dendritic cells, and B-cells were determined for PBS, and vaccinated and non-vaccinated mice at 8 days after subcutaneous ZIKV challenge.  FIG. 31 : Percentage of splenic CD4+ T-cells, central memory CD4+ T-cells (CD4+CD44+CD62L+), effector memory CD4+ T-cells (CD4+CD44+CD62L−), CD8a+ T-cells, central memory CD8+ T-cells (CD8+CD44+CD62L+), and effector memory CD8+ T-cells (CD8+CD44+CD62L−) at 8 days after subcutaneous ZIKV challenge. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       FIG. 1  schematically shows a wild-type Zika virus (ZIKV-WT) and ZIKV constructs: ZIKV-E mutants (GFP-ZIKV-E and PS-ZIKV-E) and Zika Vectors (GFP-Zika Vector and PS-Zika Vector). As provided herein, ZIKV-E mutants exhibit oncolytic activity and better in vivo safety profiles compared to wildtype ZIKV strains (e.g., PRVABC59, and MR766) and Zika Vectors are safe delivery vectors for passenger sequences. That is, subjects infected with wildtype ZIKV strains had significantly more weight loss, viral replication, and mortality compared with subjects infected with the recombinant ZIKV-E mutants and Zika Vectors as described herein. 
     As shown in  FIG. 2 , glioblastoma (GB) cells infected with a ZIKV-E mutant (with an envelope (E) protein having SEQ ID NO: 1, wherein the amino acid residue at position 154 is Thr) as compared to a wildtype E protein (SEQ ID NO: 2) had poor viability and underwent caspase 3/7-mediated apoptotic cell death. The safety profile of the ZIKV-E mutant was evaluated in a pre-clinical mice model. 8-12 weeks old ifnral-1-were infected with a dose of 1×10 6  pfu/mouse through subcutaneous route and the mice were followed up for 21 days. As shown in  FIG. 5 , mice infected with the ZIKV-E mutants remained healthy without having weight loss. As shown in  FIG. 4 , at Day 7 post-infection, mice infected with the ZIKV-E mutants had an order of magnitude lower blood viral load than those infected with wildtype ZIKV strains. As shown in  FIG. 3 , mice infected with the ZIKV-E mutants exhibited 100% survival, whereas mice infected with wildtype ZIKV strains exhibited at least 60% mortality before 9 days post infection. Therefore, compared to wildtype ZIKV strains (e.g., PRVABC59 and MR766), ZIKV-E mutants provide a superior in vivo safety profile. 
     Also disclosed herein are ZIKV-Im mutants, which are Zika Vectors that express an immunomodulatory agent, e.g., an immunoadjuvant. Specifically, sequences encoding human cytokines CCL5 (SEQ ID NO: 3), IL18 (SEQ ID NO: 4), IL2 (SEQ ID NO: 5), and TNFSF14 (LIGHT) (SEQ ID NO: 6) were cloned into a Zika Vector lacking structural genes (Capsid, prM, and envelope), e.g., SEQ ID NO: 7, to result in ZV-CCL5 (SEQ ID NO: 8), ZV-IL18 (SEQ ID NO: 9), ZV-IL2 (SEQ ID NO: 10), and ZV-TNFSF14 (SEQ ID NO: 11), respectively. For constructing these recombinant viruses, cloning strategies, and primers for PCR amplifications of the target genes were designed and developed to create two overlapping segments. These two segments were then PCR stitched into one segment and inserted into a specific region in the 5′ end of the envelope region. The final PCR product was cloned into the plasmid encoding the parental wild-type ZIKV strain (PRVABC59) and the insert (i.e., nucleic acid molecule encoding the immunomodulatory agent) was confirmed by restriction analysis using EcoRI-KpnI enzymes and sequenced for verification. See  FIG. 22 . 
     While generating the ZV-CCL5, ZV-IL18, ZV-IL2, and ZV-TNFSF14 constructs, the structural genes, i.e., Capsid, prM, and envelope (E), were removed to make these constructs into one-cycle replication competent vectors. For production of the viral particles, the structural genes and the constructs containing the gene encoding the immunomodulatory agent can be packaged using 293T cells. The resulting viral particles can be used as therapeutic agents alone or in combination with replication competent ZIKV-E mutants and/or other ZIKV constructs. 
     ZIKV-E Mutants, Zika Vectors, and ZIKV-Im Mutants 
     As used herein, “ZIKV constructs” include ZIKV-E mutants, Zika Vectors, and/or ZIKV-Im mutants as described herein. 
     In some embodiments, ZIKV-E mutants are contemplated herein. As used herein, a “ZIKV-E mutant” refers to a recombinant Zika virus (ZIKV) that (1) has an E gene that encodes an envelope (E) protein having one or more amino acid mutations (i.e., substitutions, deletions, and/or insertions) as compared to the wildtype E protein (SEQ ID NO: 2), and (2) exhibits reduced E protein glycosylation as compared to a wildtype ZIKV strain (e.g., GenBank Accession Number KU501215, clinical isolate PRVABC59) having asparagine at amino acid position 154 (N154) in the envelope protein. In some embodiments, the one or more amino acid mutations of the ZIKV-E mutants comprise an N154X substitution, wherein X is any amino acid other than asparagine. In some embodiments, the N154X substitution is N154T, N154H, N154D, N154Y, N154T, N154S, N154I, N154K, or N154K. In some embodiments, the N154X substitution is N154T, N154D, N154E, N154S, N154A, N154V, or N154Q. In some embodiments, the N154X substitution is N154T, N154D, N154E, N154S, or N154A. In some embodiments, the N154X substitution is N154T or N154D. In some embodiments, the ZIKV-E mutants have an E protein having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, and an amino acid residue other than Asn at amino acid position 154, and preferably the amino acid at position 154 is Thr, Asp, Glu, Ser, Ala, Val, or Gln, more preferably the amino acid at position 154 is Thr, Asp, Glu, Ser, Ala, and most preferably the amino acid at position 154 is Thr or Asp. In some embodiments, the ZIKV-E mutants are also ZIKV-Im mutants as described in the paragraph below. In some embodiments, the ZIKV-E mutants lack one or more structural proteins (e.g., genes encoding Capsid, prM, and/or Envelope proteins). 
     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, including those set forth in WO 2018035294. In some embodiments, the Zika virus genome has 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to Accession No. NC_012532.1. In some embodiments, the Zika virus genome comprises the sequence set forth in Accession No. NC 012532.1. In some embodiments, the Zika virus genome comprises a sequence as set forth in 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 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 comprises a CMV promoter. In some embodiments, the Zika Vector comprises a BsiWI linker sequence. In some embodiments, the Zika Vector comprises a hepatitis delta virus ribozyme (HDVR) sequence. In some embodiments, the Zika Vector comprises an SV40 PolyA sequence. In some embodiments, the Zika Vector comprises a pBR322 cloning vector sequence. 
     As used herein, a “passenger sequence” refers to any nucleic acid molecule of interest that are exogenous to the ZIKV genome. Non-limiting examples of passenger sequences include nucleic acid sequences that encode immunomodulatory agents such as cytokines, antigens and antigenic epitopes, and therapeutic proteins. Exemplary therapeutic proteins include Bac-7-ELP-p21, BAC1-ELP-H1, BR2, Buforin IIb, Cardiac natriuretic peptides, CT20p-NP, Dox-TAT, F8A, FWCS, Int-H1-S6A, LPD-PEG-NGR, Magainin II, NRC-3, NRC-7, p16, Pen-ELP-H1, Pen-ELP-p21, PNC-2, PNC-21, PNC-27, PNC-28, PNC-7, Poropeptide-Bax, R8-Bax, RGD-PEG-Suc-PD0325901, RGD-SSL-Dox, RRM-IL12, RRM-MV, TAT-Bim, Tat-aHDM2, VWCS, Neo-2/15, and the like. In some embodiments, the therapeutic protein is CT20p-NP, Poropeptide-Bax, R8-Bax, RRM-IL12, RRM-MV, or TAT-Bim. In some embodiments, the therapeutic protein is BAC1-ELP-H1 or RGD-PEG-Suc-PD0325901. 
     In some embodiments, ZIKV-Im mutants are contemplated herein. As used herein, a “ZIKV-Im mutant” refers to a Zika virus (ZIKV) (including Zika Vectors) that has been modified to express an exogenous immunomodulatory agent, such as a cytokine. In some embodiments, the cytokine is a human cytokine. In some embodiments, the cytokine is IL2, IL15, IL18, CCL5 (RANTES), or TNFSF14 (LIGHT). In some embodiments, the ZIKV-Im mutants are also ZIKV-E mutants as described in the paragraph above. In some embodiments, the ZIKV-Im mutants lack one or more structural proteins genes (e.g., genes encoding Capsid, prM, and/or Envelope proteins). 
     Compositions 
     Compositions, including pharmaceutical compositions and vaccines, comprising, consisting essentially of, or consisting of one or more ZIKV constructs are contemplated herein. As used herein, the phrase “consists essentially of” in the context of “a composition consisting essentially of [a given] ZIKV construct” means that the composition may comprise additional ingredients, including active pharmaceutical ingredients, except for other ZIKV constructs other than the given ZIKV construct. The term “pharmaceutical composition” refers to a composition suitable for pharmaceutical use in a subject. A pharmaceutical composition generally comprises a therapeutically effective amount of an active agent, e.g., one or more ZIKV constructs as contemplated herein, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions comprise one or more ZIKV constructs and a pharmaceutically acceptable carrier that is suitable for administration to a subject. 
     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 &amp; Wilkins, Baltimore, Md. In some embodiments, the pharmaceutically acceptable vehicle is one that is suitable for intravenous, subcutaneous, intranasal, or intratumoral 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 ZIKV 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 sufficient to produce a desired therapeutic result as compared to a negative control. The desired therapeutic result may comprise an objective or subjective improvement in, e.g., long-term survival, effective prevention of a disease state, the level or concentration of a given biomarker, and the like. Therapeutically effective amount includes amounts that treat, prevent, or inhibit a given disease or condition, such as ZIKV-treatable cancer, in a subject as compared to a control, such as a placebo. The skilled artisan will appreciate that certain factors may influence the amount required to effectively treat a subject, including the degree of the given disease or affliction, previous treatments, the general health and age of the subject, and the like. Nevertheless, therapeutically effective amounts may be readily determined by methods in the art. In some embodiments, a therapeutically effective amount of a given ZIKV 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. The therapeutically effective amount may depend the extent of the given disease or amount of tissue to be treated. For example, tumor volumes of about 1 cm 3  can be treated with about 10e3 to 10e9 viral particles and tumor volumes of about 100 cm 3  can be treated with about 10e6 to 10e11 viral particles. In some embodiments, the therapeutically effective amount is one that provides a multiplicity of infection (MOI) of at least 1, an MOI of 1-10, or an MOI of 1-5. In some embodiments, the therapeutically effective amount is one that provides an MOI of 1, 2, 3, 4, or 5. In some embodiments, the therapeutically effective amount is at least about the amount of cells to be treated (i.e., intended to be infected by the viral particles). In some embodiments, the ratio of viral particles to cells to be treated ranges from about 1:1 to about 10:1. In some embodiments, the ratio of viral particles to cells to be treated ranges from about 1:1 to about 5:1. In some embodiments, the ratio of viral particles to cells to be treated is about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1. 
     In addition to the one or more ZIKV constructs, pharmaceutical compositions may include one or more supplementary agents. Examples of suitable supplementary agents include chemotherapeutics such as taxanes; epothilones; histone deacetylase inhibitors; inhibitors of topoisomerase I; inhibitors of topoisomerase II; kinase inhibitors; retinoids, vinca alkaloids, and the like. In some embodiments, the one or more supplementary agents are temozolomide, procarbazine, carmustine, lomustine, vincristine, and the like. In some embodiments, the pharmaceutical compositions comprising one or more ZIKV constructs further include on or more AKT inhibitors such as MK-2206 (8-[4-(1-Aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3-one), Perifosine (KRX-0401), miltefosine, Capivasertib (AZD5363), Ipatasertib (RG7440), Pictilisib (GDC-0941), Wortamannin, and the like. 
     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 ZIKV 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 ZIKV construct, the degree of the given infection, the manner of administration, the general state of health of the subject, and the like. The typical immunogenic amounts of a given ZIKV 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 ZIKV 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 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 immunization 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. 
     In addition to the one or more ZIKV constructs, the pharmaceutical compositions may include an adjuvant and/or stabilizers in the art, e.g., MgCl 2 , MgSO 4 , 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 ZIKV construct as disclosed herein, aids the pharmaceutically active agent in its mechanism of action. Thus, an adjuvant as contemplated herein is a substance that aids the ability of the given ZIKV construct to cause oncolysis and/or elicit an immune response against cancer cells. Suitable adjuvants include incomplete Freund&#39;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. In some embodiments, the adjuvant is an immunoadjuvant, such as a cytokine. The effectiveness of an adjuvant may be determined by methods in the art. In some embodiments, the immunoadjuvant is provided as a recombinant expression product, i.e., an immunoadjuvant which the given ZIKV construct has been recombinantly modified to express. 
     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, intratumoral, 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 ZIKV 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 ZIKV constructs 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 ZIKV 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. 
     Therapeutic Methods 
     Because ZIKV strains activate antitumor immunity in vivo (Chen, et al. (2018) mBio 9(5): e01683-18) and, as provided herein, ZIKV constructs exhibit oncolytic activity, methods of treating subjects for a cancer, which comprise administering to the subjects one or more ZIKV constructs (preferably one or more ZIKV-E mutants and/or one or more ZIKV-Im mutants), are contemplated herein. In some embodiments, the cancer is a ZIKV-treatable cancer. 
     As used herein, a “ZIKV-treatable cancer” includes craniopharyngiomas, ependymomas (e.g., myxopapillary ependymoma papillary ependymomas, subependymomas, anaplastic ependymomas), glioblastoma multiforme (GBM), glioblastomas, gliomas, low, mid, and high grade astrocytomas, medulloblastomas, meningiomas, neuroblastomas, oligodendrogliomas, oligodendrogliomas, pituitary adenomas, primitive neuroectodermal tumors, retinoblastomas, and cancers in which the cancer cells express one or more flavivirus receptors involved in flavivirus cell entry. See, e.g., Laureti, et al. (2018). Frontiers in Immunology 9: 2180. In some embodiments, the one or more flavivirus receptors is GRP78, SDC2, HSP90AB1, TYRO2, AXL, and/or MERTK. In some embodiments, the one or more flavivirus receptors is AXL. In some embodiments, the ZIKV-treatable cancer is a glioblastoma, a glioma, a neuroblastoma, or a retinoblastoma. Examples of cancers in which the cancer cells express a flavivirus receptor involved in flavivirus cell entry include skin cancers (e.g., basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, atypical fibroxanthoma, cutaneous lymphoma, and dermatofibrosarcoma), lung cancers (e.g., small cell lung cancers (SCLC) and non-small cell lung cancers (NSCLC)) and gastrointestinal cancers (e.g., esophageal cancer (carcinoma), stomach cancer (gastric cancer), liver cancer (hepatocellular carcinoma), pancreatic cancer, and colorectal cancers). 
     In some embodiments, the ZIKV-treatable cancer is glioblastoma multiforme (GBM). In some embodiments, the ZIKV-treatable cancer is glioblastoma multiforme (GBM) and treatment with a therapeutically effective amount of one or more ZIKV constructs (preferably one or more ZIKV-E mutants and/or one or more ZIKV-Im mutants) increases the overall survival of the subject by about 2 to about 6 months as compared to negative control subjects. In some embodiments, the ZIKV-treatable cancer is glioblastoma multiforme (GBM) and treatment with a therapeutically effective amount of one or more ZIKV-E mutants and/or one or more ZIKV-Im mutants results in about 30% or more reduction in tumor growth and/or tumor size. In some embodiments, the ZIKV-treatable cancer is glioblastoma multiforme (GBM) and treatment with a therapeutically effective amount of one or more ZIKV-E mutants and/or one or more ZIKV-Im mutants results in about 70% or more reduction in tumor growth and/or tumor size. 
     Because ZIKV preferentially infects and/or replicates in neuroprogenitor cells over adult brain cells, methods of treating subjects for diseases and disorders caused by or associated with aberrant neuroprogenitor cells, which comprise administering to the subject one or more ZIKV constructs (preferably one or more ZIKV-E mutants and/or one or more ZIKV-Im mutants), are contemplated herein. Diseases and disorders caused by or associated with aberrant neuroprogenitor cells include epilepsy, schizophrenia, autism spectrum disorders (ASDs), neurodegenerative diseases (e.g., Parkinson&#39;s disease (PD) and Alzheimer&#39;s disease (AD)), and the like. See, e.g., Ladran, et al. (2013) Neural stem and progenitor cells in health and disease. Wiley interdisciplinary reviews. Systems biology and medicine, 5(6): 701-715. 
     In some embodiments, the methods comprise administering a therapeutically effective amount of one or more ZIKV constructs to a subject. In some embodiments, the therapeutically effective amount 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. The therapeutically effective amount may depend the extent of the given disease or amount of tissue to be treated. For example, tumor volumes of about 1 cm 3  can be treated with about 10e3 to 10e9 viral particles and tumor volumes of about 100 cm 3  can be treated with about 10e6 to 10e11 viral particles. In some embodiments, the therapeutically effective amount is one that provides a multiplicity of infection (MOI) of at least 1, an MOI of 1-10, or an MOI of 1-5. In some embodiments, the therapeutically effective amount is one that provides an MOI of 1, 2, 3, 4, or 5. In some embodiments, the therapeutically effective amount is at least about the amount of cells to be treated (i.e., intended to be infected by the viral particles). In some embodiments, the ratio of viral particles to cells to be treated ranges from about 1:1 to about 10:1. In some embodiments, the ratio of viral particles to cells to be treated ranges from about 1:1 to about 5:1. In some embodiments, the ratio of viral particles to cells to be treated is about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1. 
     In some embodiments, one or more ZIKV constructs may be administered to a subject to induce in a subject an immune response against a given target. In some embodiments, the given target is a ZIKV protein or antigenic epitope thereof. In some embodiments, the given target is a virus and the one or more ZIKV constructs is a Zika Vector that comprises one or more passenger sequences that encode a nucleocapsid protein, an envelope protein, a spike protein, and/or one or more antigenic fragments thereof of the virus. In some embodiments, the immunogenic amount administered to the subject for the initial vaccination and boosting immunization, if any, 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, the immunogenic amount administered to a human subject for the initial vaccination and boosting immunization, if any, is 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, the immunogenic amount administered to a human subject for the initial vaccination and boosting immunization, if any, is about 1-25 μg, about 5-20 μg, or about 7.5-16 μg. In some embodiments, the subject is administered a boosting 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. 
     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 about 1×10 4  to about 1×10 6  pfu of the one or more ZIKV constructs is administered to the subject. In some embodiments, a dose of the one or more ZIKV constructs, which are replication competent, results in about 10 3  to about 10 5  copies of the in the blood of the subject. 
     In some embodiments, the one or more ZIKV constructs are administered in the form of a pharmaceutical composition, such as those contemplated herein, including those described in the “Compositions” section above. In some embodiments, the one or more ZIKV constructs are intravenously administered. In some embodiments, the one or more ZIKV constructs are subcutaneously administered. In some embodiments, the one or more ZIKV constructs are intratumorally administered. 
     In some embodiments, the subject to be treated is mammalian. In some embodiments, the subject is an animal such as a rodent or a non-human primate. In some embodiments, the subject is human. In some embodiments, the subject the subject is “in need thereof”. As used herein, a subject “in need thereof” is one who has been diagnosed as having a cancer, such as a ZIKV-treatable cancer, aberrant neuroprogenitor cells (in the case of treating cancer or aberrant neuroprogenitor cells), or one who is at risk of infection by a given pathogen (in the case of inducing an immune response in a subject against the given pathogen). 
     Toxicity and therapeutic efficacy of ZIKV 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, LC 50  (the dose expressed as concentration x exposure time that is lethal to 50% of the population) or the LD 50  (the dose lethal to 50% of the population), and the ED 50  (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 LD 50 /ED 50 . ZIKV constructs s which exhibit large therapeutic indices are preferred. While ZIKV 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 ED 50  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 a given ZIKV construct 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 IC 50  (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 
     Kits comprising one or more ZIKV 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 ZIKV constructs or compositions thereof are packaged together with one or more reagents or drug delivery devices for administering the ZIKV constructs or compositions thereof to a subject. In some embodiments, the kits comprise the one or more ZIKV 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. 
     EXAMPLES 
     Cell Lines and Subjects 
     U-87 MG cell line (human glioblastoma; astrocytoma; classified as grade IV) was purchased from the American Type Culture Collection (ATCC, USA) and other GBM cell lines were maintained using complete Eagle&#39;s Minimum Essential Medium (EMEM) (Fisher Scientific). Complete EMEM was supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml), and streptomycin (100 mg/ml) (Life Technologies). The cells were maintained at 37° C. with 5% CO2 and were passage every third day using 1× trypsin with EDTA. 
     NGS (NOD scid gamma) mice. Immunodeficient mice obtained from the Jackson Laboratory (Mount Desert Island, Me.). Because NGS mice are immunodeficient, the results of the experiments herein indicate is likely linked to direct cell killing and phagocytic removal of debris by ZIKV-E mutants. 
     ZIKV Wild-Type Strains and Parental Construct 
     An Asian genotype, clinical isolate PRVABC59 (GenBank accession number KU501215) and MR766 (GenBank accession number LC002520) were used as wild-type ZIKV strains. The original stocks (P=3) were subjected to additional passages in Vero cells to generate working viral stocks. Viral titers were measured by plaque assay as described previously. 
     PRVABC59 was amplified in C6/36 cells, and total RNA of infected cells was extracted with Purelink RNA Mini Kit (Ambion). ZIKV cDNA covering the complete genome was synthesized using SuperScript III reverse transcriptase (Thermo Fisher) with random hexamer (Fragments 1 to 5) or ZIKV specific primer against the last 20 nt of ZIKV 3′-end (Fragment 6). Then, six ZIKV sub-genomic fragments covering the whole genome were amplified using KOD polymerase (Millipore). Six fragments were assembled into EcoR I linearized pBR322 plasmid to generate pZ-PR plasmid using HiFi DNA Assembly Cloning Kit (NEB), and further transformed into DH10B  E. coli . The whole sequence of pZ-PR plasmid (Accession No. KY583506) was verified by Sanger DNA sequencing. 
     Recombinant ZIKV was generated from plasmid pZ-PR, containing the full-length cDNA of ZIKV Puerto Rico strain, using the previously describe method (Shan et al., 2016) with some modifications. In brief, pZ-PR plasmid was amplified in DH10B  E. coli  and then purified using PureLink HiPure Plasmid Midiprep Kit (Thermo Fisher). To generate DNA template for RNA in vitro transcription, 100 μg of pZ-PR was linearized with BstBI, followed by end blunting with Mung Bean Nuclease (NEB). Then, the end-blunted template DNA was purified with phenol-chloroform, precipitated with ethanol, and resuspended in 20 μl RNase-free water. 5′-capped RNA was in vitro transcribed using mMESSAGE mMACHINE T7 kit (Ambion) with 1 μg template DNA and an additional 1 μl of 30 mM GTP solution. In vitro transcribed RNA was further purified with phenolchloroform, precipitated with ethanol, resuspended in 100 μl RNase-free water, aliquoted, and stored in −80° C. freezer. 
     For cell transfection, 12 μg of RNA was mixed with 5×10 6  BHK21 cells in 200 Electroporation Solution (Bioland), and electroporated in 4-mm cuvette with the GenePulser apparatus (Bio-Rad) at the setting of 240 V and 950 μF, pulsing once. After a 10-minute recovery at room temperature, transfected cells were resuspended in 10 ml warm culturing medium, incubated in cell incubator overnight, and then washed once and replenished with fresh medium. Three to four days post electroporation when about 40% of cells showing CPE, supernatants were collected, span at 8000 g for 10 minutes at 4° C. to remove cellular debris, aliquoted, and stored at −80° C. 
     Zika Viral Infection 
     Glioblastoma cells were seeded at a density of 3×10 4  cells per well in a 48-well plate (1.5×10 4  cells per well of 96-well plate). The next day, a viral inoculum was prepared in a serum-free base media at a Multiplicity of Infection (MOI) of 1 or 10. 200 (100 μL per well of 96-well plate) of viral inoculum was added to each well and the inoculated plates were incubated in 37° C. with 5% CO2 for 2-4 hours. At the end of incubation, the inoculum was replaced with 10% FBS supplemented media. For the mock infection control, the cells received only basic media that was used for preparing the viral inoculum. This mock or uninfected control cells were included for each time point for comparison. At the indicated time points, samples were collected for various assays, including immunofluorescence assay, cell viability assay, caspase 3/7 assay and reverse transcription quantitative PCR (RT-qPCR). 
     Caspase 3/7 Activity Assay for Apoptosis Measurement 
     Caspase-Glo 3/7 Assay (Promega, USA) was performed as per the manufacturer&#39;s protocol. At indicated time points, Zika virus infected and control mock infected cells in the 96-well plate were incubated with the proluminescent caspase-3/7 substrate for 1 hour at room temperature. Subsequently, 100 μL of lysate was transferred to a white 96 well microtiter plate for reading the luminescence signal using a luminometer (Glomax Microplate Luminometer, Promega). 
     Cell Viability Assay 
     CellTiter-Glo Luminescent assay (Promega, USA) to measure intracellular ATP content is performed as per the manufacturer&#39;s protocol. At indicated time points, Zika virus infected and control mock infected cells in the 96-well plate are incubated with the substrate for 1 hour at room temperature. Subsequently, 100 μL of lysate is transferred to a white 96 well microtiter plate for reading the luminescence signal using a luminometer (Glomax Microplate Luminometer, Promega). 
     Reverse Transcription Quantitative PCR Analysis 
     Total RNA is extracted from mock and infected glioma cells at the designated time points using an RNeasy Mini Kit (QIAGEN). After treatment with RNase-free DNase, 1 μg of RNA is reverse-transcribed into cDNA using random hexamer primer and the SuperScript III Reverse Transcriptase Kit (Life Technologies) as recommended by the manufacturer. The following conditions are used for cDNA amplification: 65° C. for 5 minutes; 4° C. for 1 minute followed by 55° C. for 60 minutes and 72° C. for 15 minutes. Quantitative real-time PCR is carried out using Platinum SYBR Green qPCR SuperMix-UDG with ROX Kit (Life Technologies) by the QuantStudioTM 12K Flex Real-Time PCR System (Life Technologies). Known copy numbers (from 10e0 to 10e10) of ZIKV gene template are included as a standard. The relative concentration of each transcript is calculated using 2 −ΔCT  method using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) threshold cycle (C T ) values for normalization. The normalized C T  values are used for calculating copy numbers. The following conditions are used for transcript amplification: 50° C. for 2 minutes; 95° C. for 2 minutes followed by 40 cycles of 95° C. for 15 seconds and 60° C. for 1 minute, in conjunction with the appropriate primer pairs. 
     Immunofluorescence Assay 
     Immunofluorescence assay is performed on both the mock and infected Vero cells fixed with methanol. The fixed cells are incubated at −20° C. for 30 minutes, then washed three times with 1×PBS. Following three PBS washes, the cells are permeabilized and blocked with 10% fetal bovine serum, 3% BSA, 0.1% Triton-x 100 in PBS. Subsequently, the fixed and permeabilized cells are incubated with mouse monoclonal antibody for Flavivirus group antigen (D1-4G2-4-15 (4G2)) (Absolute Antibody Ltd.) at a 1:200 dilution for up to 6 hours or overnight incubation at 4° C. The secondary antibodies, goat anti-mouse polyclonal antibody (Alexa Fluor® 488) (Life Technologies, USA) are added at 1:1000 dilutions and incubated for 1 hour at room temperature. Between antibody changes the cells are washed five times with PBS. The nuclei were stained with the addition of 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies, USA). 
     Example 1. Zikv-E Mutants 
     ZIKV-E mutants were prepared as described in Gong, et al. (2018) iScience 1: 97-111. Briefly, the Asian genotype Zika virus (GenBank accession number KU501215, clinical isolate PRVABC59) was modified using recombinant techniques in the art to result in N154X substitutions in the envelope protein. “VAX2A” refers to a ZIKV-E mutant that has an N154D substitution in the envelope protein. “VAX2B” refers to a ZIKV-E mutant that has an N154T substitution in the envelope protein. 
     D. Construction of ZIKV-E Mutants 
     To generate the plasmid construct for ZIKV-E mutants, PCR primers were designed with desired mutation in envelope amino acid position 154. To generate full length fragments for cloning, the corresponding left and right fragments flanking the desired mutation site were amplified using high fidelity KOD DNA polymerase (Millipore) for 18 cycles. Each corresponding left and right fragments were combined, and the resulting full-length DNA inserts were generated by amplification with KOD polymerase for 20 cycles. Then, the full-length inserts were digested with KpnI and EcoRI, while the vector pZ-PR plasmid was digested with KpnI and EcoRI followed by treatment with Shrimp Alkaline Phosphatase (NEB). Ligation was performed for each of the three sub libraries with T4 DNA ligase (NEB) with a vector: insert ratio of 1:5. Ligated products were purified with phenol-chloroform, precipitated with ethanol, resuspended in 10 μl sterilized water, and electroporated into DH10B  E. coli  competent cells. For each of the three sub libraries, about 40,000 colonies were collected from LB plates, and directly subjected to plasmid DNA purification. DNA plasmids were stored at −80° C. in aliquot. 
     To generate ZIKV-E mutants, BHK21 cells were electroporated with in vitro transcribed RNA, and viral supernatant were collected at 4 days post transfection. ZIKV-E mutants were further amplified in C6/36 cells, then supernatants were collected, clarified of debris, and stored at −80° C. in aliquot. Virion RNA was also extracted, and reverse transcribed to cDNA. E protein coding sequence of all ZIKV-E mutants were PCR amplified individually, followed by Sanger DNA sequencing to verify the sequence. SEQ ID NO: 12 is the construct of VAX2B.  FIG. 2  shows that the VAX2B retained the anti-tumoral activity by inducing apoptotic death of infected U87 GBM cancer cells. 
     E. Preclinical Safety Study of ZIKV-E Mutants 
     To assess the safety profile of ZIKV-E mutants, 4-6 week old Ifnra1 −/−  mice were inoculated with VAX2B (1×10 6  pfu per mouse; subcutaneous route). As a positive control, mice were infected with unmodified wild-type Zika viruses (PRVABC59 and MR766 strains). Infected animals were monitored twice daily for three weeks. The wild-type virus infected animals had significant mortality and exhibited signs of neurological disease (paralysis). Negative control mice (Mock) and mice infected with VAX2B exhibited 100% survival over the entire study period  FIG. 3 ). Moreover, the serum viral load of VAX2B was 10-100 fold lower than wild-type virus at 7 days post infection (dpi) ( FIG. 4 ) indicating viral attenuation. Furthermore, VAX2B infected mice stayed healthy without any weight loss throughout the study ( FIG. 5 ) suggesting loss of virulence. 
     D. Assessing Susceptibility of Various Primary Human GBM Lines to ZIKV-E Mutants 
     Primary human GBM lines were infected with VAX2B at an MOI of 1 and 10 to determine susceptibility to infection. After infection, based on the level of apoptotic cell death the primary GBM lines were categorized into two different groups, responders and non-responders ( FIG. 6 ). The responder group demonstrated decreased in cell viability and increase cell apoptosis, whereas non-responders exhibited little decrease in cell viability ( FIG. 7 ) and minimal apoptotic activity ( FIG. 8 ). Both responder and non-responder lines exhibited high viral titers and active viral replication of VAX2B ( FIG. 9  and  FIG. 10 ). 
     F. Combination Therapy 
     Active replication of virus in non-responder lines with additional treatment can potentially create a synergistic effect to provide a strong kill response in an otherwise non-responding line. Detailed analysis of the exomes revealed that the many of the non-responder cell lines had phosphatase and tensin homolog (PTEN) loss of function mutations. Hypothesizing that PTEN loss can lead to un-inhibited activation of PI3K-AKT pathway and contribute to resistance to apoptosis induced by ZIKV-E mutants, the effects of VAX2B (at an MOI of 1 or 10) combined with different concentrations of an AKT inhibitor, MK-2206, on the non-responder cell line GS054 were evaluated.  FIG. 11  shows that the combination of a ZIKV-E mutant and AKT inhibitors may be synergistic against cancer cells having PTEN loss of function nutations. Western blot analysis confirmed the inhibition of AKT phosphorylation in the treated cells. AKT inhibitors did not significantly impact viral titers. 
     G. In Vivo Efficacy Studies in Mouse Xenograft Model 
     To evaluate the anti-tumor therapeutic properties of VAX2B, NSG mice were xenografted with U87 cells in the left flank region on Day 0. A schematic of study outline is provided in  FIG. 12 . Except for the ZIKV-E Pretreatment Group, subjects developed visible and palpable tumor mass in the inoculated site at Day 17. Subsequently, the tumor containing mice (ZIKV-E Group) received VAX2B (2×10 6  pfu/mouse) intratumorally. The tumor mass in the untreated (PBS Group) gradually increased in size ( FIG. 13 ) reaching euthanasia endpoint. By Day 40, all the untreated subjects succumbed to cancer burden, whereas VAX2B treated subjects underwent tumor remission ( FIG. 14 ). Viral titers from subjects indicate that that the anti-tumor activity is due to direct oncolytic effect of the ZIKV-E mutant ( FIG. 15 ). Histopathology and immunohistochemical analysis revealed viral replication in tumor cells and infiltration of inflammatory cells and apoptotic cell death. 
     In a subsequent study, the in vivo efficacy of VAX2B against tumors resistant to EGFR inhibitors was evaluated. NSG mice were xenografted with human GBM derived GS025 cells in the left flank region on Day 0 GS025 cells have amplified EGF receptors and cells develop resistance to EGFR inhibitors. On Day 41 and Day 48 the control group received PBS and the treatment group received VAX2B (2×10 6  pfu/mouse) intratumorally. Subject treated with VAX2B maintained their body weight ( FIG. 16 ) and 100% survived ( FIG. 17 ). Unlike treated subjects, the tumor mass in untreated subjects gradually increased in size ( FIG. 18 ). Viral titers in subjects indicate that that the anti-tumor activity is due to direct oncolytic effect of the ZIKV-E mutant. 
     These studies show that ZIKV-E mutants, alone or in combination, with other agents, e.g., AKT inhibitors, are safe and effective in treating cancers such as glioblastomas. 
     Example 2. Zika Vector and Zikv-Im Mutants 
     A replication deficient Zika Vector was made by splitting a Zika virus (ZIKV) genome into structural and non-structural (NS) regions ( FIG. 19 ) and deleting the nucleic acid sequences that encode the structural genes: Core (C), PrM, and Envelope (E) (which based on Zika virus strain PRVABC59 (Accession No. KU501215.1), a total of 2223 nucleotides (i.e., nucleotides positions 183 to 2405) were deleted to give a ZIKV sequence. The Zika Vector exemplified herein is flanked by a CMV promoter at the beginning and hepatitis delta virus (HDV) ribozyme/SV40 poly A sequences at the end. The Zika Vector is schematically shown in  FIG. 20  (SEQ ID NO: 7, wherein nucleotides: 7-741 is a CMV promoter; 742-9337 is the ZIKV sequence; 924-935 is a BsiWI linker sequence (SEQ ID NO: 13); 9338-9421 is a hepatitis delta virus ribozyme (HDVR) sequence; 9422-9460 is a linker sequence; 9461-9682 is an SV40 Poly A sequence; and 9683-14020 is a pBR322 cloning vector sequence. 
     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: 16) total size 2382 nucleotides; 794 amino acids (SEQ ID NO: 17)) cloned into a mammalian expression vector driven by CMV promoter and at the end SV40 poly A sequence. 
       FIG. 21  schematically shows the VAXR1 construct (SEQ ID NO: 14), which comprises a sequence (SEQ ID NO: 15) that encodes eGFP inserted in the BsiWI linker sequence of the Zika Vector. The VAXR1 construct also comprises an F2A insert (SEQ ID NO: 18) and a Not1 linker sequence after the BsiWI linker sequence. 
     The Zika Vector and packaging plasmid described in Example 2 and methods in the art were used to generate the following ZIKV-Im mutants: ZV-CCL5, ZV-IL18, ZV-IL2, and ZV-TNFSF14. 
     A. Vaccine Production 
     293T cells were cultured in IMDM containing 10% FCS and antibiotics in an incubator (37° C. and 5% CO 2 ). One day before transfection, 293T cells (1.4×10 7  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 ( FIG. 23 ). 
     B. Safety and Efficacy 
     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 1×10 3  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 ( FIG. 24 ). This data suggests that the replication deficient Zika Vector is safe and non-lethal. 
     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 ( FIG. 25 ). However, wild-type ZIKV exposed mice had a mean viral load of 10 million pfu per ml of blood. These findings suggest that the replication deficient Zika Vector is a safe construct. 
     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  FIG. 26 . Female mice (n=10) were immunized with VAXR1 via subcutaneous route. The un-vaccinated mice (n=18) received PBS. Vaccinated mice were boosted on Day 14. Mice received VAXR1 stayed healthy and active suggesting that the Zika Vector is well tolerated and safe in adult mice. These animals maintained bodyweight similar to that of PBS mice ( FIG. 27 ). 
     Both the vaccinated and un-vaccinated mice were subjected to mating on Day 21. Pregnant mice were challenged with wild type ZIKV (1×10 6  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 ( FIG. 28 ). 100% of the vaccinated mice were protected. Importantly, the fetuses of vaccinated mothers were healthy and maintained normal body weight ( FIG. 29 ). The fetuses of un-vaccinated mothers had reduced body weight and several of the fetuses were reabsorbed in uterus. Also, dead and partially decomposed fetuses were found in unvaccinated animal&#39;s uterus. There were 18 non-viable pubs born in the un-vaccinated group. All the pups born in the vaccinated group were healthy and similar to the pups of uninfected mothers. 
     C. Immune Responses 
     Mass cytometry was performed to evaluate the various sub-sets of immune cell populations ( FIG. 29 ). Vaccination of breeding mice resulted in increased immune response ( FIG. 30 ) and no inflammatory response as determined by normal level of macrophages and monocytes in the spleen was observed. 
     REFERENCES 
     The following references are herein incorporated by reference in their entirety with the exception that, should the scope and meaning of a term conflict with a definition explicitly set forth herein, the definition explicitly set forth herein controls:
     WO 2018/035294   Chen, et al. (2018) Treatment of Human Glioblastoma with a Live Attenuated Zika Virus Vaccine Candidate. mBio 9(5): e01683-18.   Gong, et al. (2018) High-Throughput Fitness Profiling of Zika Virus E Protein Reveals Different Roles for Glycosylation during Infection of Mammalian and Mosquito Cells. iScience 1: 97-111, and Supplemental Information.   Al-Mawsawi, et al. (2014) High-throughput profiling of point mutations across the HIV-1 genome. Retrovirology, 11, 124.   Crooks, et al. (2004) WebLogo: a sequence logo generator. Genome Res, 14, 1188-90.   Gong, et al. (2016) A Herpesvirus Protein Selectively Inhibits Cellular mRNA Nuclear Export. Cell Host Microbe, 20, 642-653.   Qi, et al. (2015) Murine Gammaherpesvirus 68 ORF48 Is an RTA-Responsive Gene Product and Functions in both Viral Lytic Replication and Latency during In Vivo Infection. J Virol, 89, 5788-800.   Shan, et al. (2016) An Infectious cDNA Clone of Zika Virus to Study Viral Virulence, Mosquito Transmission, and Antiviral Inhibitors. Cell Host Microbe, 19, 891-900.   Wu, et al. (2015) Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality. PLoS Genet, 11, e1005310.   Marqus, et al. (2017) Evaluation of the use of therapeutic peptides for cancer treatment. J Biomed Sci. 24(1):21.   

     All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. 
     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 &amp; Waterman, Adv Appl Math 2:482 (1981), by the homology alignment algorithm of Needleman &amp; Wunsch, J Mol Biol 48:443 (1970), by the search for similarity method of Pearson &amp; 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, Wis.), 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. 
     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.