Nucleic acid chimera, chimeric flavivirus, its use, and immunogenic composition.

Abstract

The present invention relates to a chimeric flavivirus that includes non-coding regions, non-structural proteins, a capsid protein (c) and a portion of a premembrane signal sequence (prm) of an attenuated or wild-type dengue virus serotype 2 (DENV-2), and a portion of a prm signal sequence, a prm protein and at least one portion of an envelope protein (e) of a Zika virus (ZIKV) are described. Immunogenic compositions and methods for producing an immune response in an individual, such as an immune response directed against ZIKV, are also described.

Description

1 / 56 Nucleic acid chimera, chimeric flavivirus, its use, and immunogenic composition. CROSS-REFERENCE TO RELATED REQUESTS

[001] This application claims the benefit of the US Provisional Application. No. 62 / 359.812, filed on July 8, 2016, which is incorporated herein by reference in its entirety. FIELD

[002] This description refers to chimeric, attenuated flaviviruses containing structural proteins of an attenuated or wild-type dengue virus and at least one structural protein of a Zika virus. This description also refers to the use of chimeric flaviviruses in Zika virus vaccine compositions. BACKGROUND

[003] The Zika virus, a flavivirus classified within the Flaviviridae, along with other important mosquito-borne viruses, including yellow fever, dengue, West Nile virus, and Japanese encephalitis, has spread rapidly in a hemispheric epidemic since the virus was introduced to Brazil in 2015, reaching Central and North America, including territories of the United States, and now threatening the continental United States. Initially isolated in 1947 in Uganda, the virus was first linked to human disease in 1952 and has been sporadically recognized as a cause of mild, self-limiting febrile illness in Africa and Southeast Asia (Weaver et al., Antiviral Res 130: 69–80, 2016; Faria et al., Science 352 (6283): 345–349, 2016).However, in 2007, an outbreak appeared on Yap Island in the North Pacific, presumably transferred from Asia and subsequently spread from island to island across the Pacific, leading to an extensive outbreak in 2013-2014 in French Polynesia, with subsequent spread to New Caledonia, the Cook Islands, and finally Easter Island, far to the east. Petition 870260062389, dated 06 / 25 / 2026, p. 6 / 135 2 / 56 Asian lineage virus was subsequently transferred to the Western Hemisphere via routes that remain undetermined (Faria et al., Science 352 (6283): 345-349, 2016). The virus is anthroponically transmitted by Aedes aegypti, A. albopictus, and possibly A. hensilli and A. polynieseinsis (Weaver et al., Antiviral Res 130: 69-80, 2016).

[004] In late 2015, a significant increase in fetal anomalies (e.g., microcephaly) and Guillain-Barré syndrome (GBS) in areas of widespread Zika virus transmission raised concerns that the Zika virus was far more virulent than previously thought and prompted the World Health Organization (WHO) to declare a Public Health Emergency of International Concern (PHEIC) (Heymann et al., Lancet 387 (10020): 719–721, 2016). SUMMARY

[005] Chimeric flaviviruses are described here that include non-coding regions, non-structural proteins, a capsid protein (C), and a portion of a premembrane signal sequence (prM) from a dengue virus serotype 2 (DENV-2); and a portion of a prM signal sequence, a prM protein, and at least one portion of an envelope protein (E) from a Zika virus (ZIKV). Immunogenic compositions and methods for producing an immune response against ZIKV in an individual are also described.

[006] Nucleic acid chimeras are provided here comprising a first nucleic acid molecule comprising a 5' non-coding region, a nucleic acid encoding non-structural proteins and a protein C, and a 3' non-coding region, each from a DENV-2 strain genome, wherein the protein C comprises a portion of a prM signal sequence from the DENV-2 genome and a portion of a prM signal sequence from a ZIKV genome; and a second nucleic acid molecule operationally linked to the first. Petition 870260062389, dated 06 / 25 / 2026, p. 7 / 135 3 / 56 nucleic acid molecule, encoding a prM protein and at least a portion of an E protein from the ZIKV genome. In some embodiments, DENV-2 is an attenuated DENV-2 strain, such as the PDK-53 strain, or the attenuated strain includes one or more attenuating mutations present in the PDK-53 genome. In other embodiments, DENV-2 is a wild-type DENV-2, such as the 16681 strain.

[007] Chimeric flaviviruses are also provided, which include a nucleic acid chimera described herein. Immunogenic compositions that include a chimeric DENV-2 / ZIKV are additionally provided.

[008] In addition, methods are provided here for producing an immune response against ZIKV in an individual by administering to the individual a chimeric flavivirus described herein, or an immunogenic composition thereof.

[009] The objectives, features and advantages of the foregoing and others will become more apparent from the following detailed description, which continues with reference to the attached figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is a schematic of the genomic structure of chimeric viruses DENV-2 / Zika (D2 / ZKV). The C / prM and E / NS1 junction site sequences for DENV-2, ZIKV, and D2 / ZKV are also shown. For chimeric viruses, the ZIKV sequence is indicated by boxes. The C / prM junction sequences shown are from DENV-2 (nucleotides 388-447 of SEQ ID NO: 11; amino acids 98-117 of SEQ ID NO: 12), ZIKV (nucleotides 411-482 of SEQ ID NO: 7; amino acids 102-125 of SEQ ID NO: 8), D2 / ZKV Strategy 1 (nucleotides 388-459 of SEQ ID NO: 1 and amino acids 98-121 of SEQ ID NO: 2), and D2 / ZKV Strategy 2 (nucleotides 388-459 of SEQ ID NO: 3; amino acids 98-121 of SEQ ID NO: 4). Petition 870260062389, dated 06 / 25 / 2026, p. 8 / 135 4 / 56 E / NS1 junction sequences shown are from DENV-2 (nucleotides 2356-2430 of SEQ ID NO: 11; amino acids 754-778 of SEQ ID NO: 12), ZIKV (nucleotides 2424-2498 of SEQ ID NO: 7 and amino acids 773-797 of SEQ ID NO: 8) and D2 / ZKV (nucleotides 2401-2475 of SEQ ID NO: 1 and amino acids 769-793 of SEQ ID NO: 2). The NgoMIV introduced into the DENV-2 backbone at the E / NS1 junction site resulted in a Val to Ala substitution at amino acid 754 (DENV-2 residue E-482).

[0011] Figures 2A-2B are graphs showing the growth kinetics of D2 / ZK-P5 and D2 / ZK-V5. (Figure 2A) In Vero cells, both P5 and V5 chimeras reached peak titers greater than 1 x 107 pfu / ml, but V5 virus grew slightly more slowly than P5 virus. Both viruses replicated slightly less than wild-type ZIKV pRVABC59 in Vero cells. (Figure 2B) In C6 / 36 cells, P5 virus replicated significantly less than wild-type ZIKV and slightly less than its D2 16681 main-chain virus. However, P5 still replicated to more than 1 x 108 pfu / ml. V5 virus maintained the same incapacitated growth in C6 / 36 cells as the D2 PDK53 vaccine main-chain virus.

[0012] Figure 3 is a graph showing the neurovirulence of chimeric D2 / ZK and wild-type viruses in mice. Litters of newborn CD-1 mice (ICR) 0-2 days after birth were placed in groups of 10 according to age, and inoculated with 104 pfu / 30 pl of ZIKV, D2 / ZK-P5, D2 / ZK-V5, or D2 16681 via intracranial inoculation. All 29 mice (0, 1, or 2 days old) provoked with wild-type ZIKV became severely ill or died between 9 and 11 days post-inoculation. The 20 mice (0 or 1 day old) that received the wild-type D2 16681 virus also succumbed to virus infection between the days Petition 870260062389, dated 06 / 25 / 2026, p. 9 / 135 5 / 56 and 11. All 30 mice inoculated with D2 / ZK-P5 or D2 / ZK-V5 survived virus inoculation. SEQUENCE LISTING

[0013] The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter codes for amino acids, as defined in 37 CFR 1822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file, created on July 5, 2017, 562 KB, which is embedded by reference herein. In the accompanying sequence listing:

[0014] as SEQ ID NOs: 1 and 2 are nucleotide and amino acid sequences of D2 / ZK-V, respectively.

[0015] SEQ ID Nos: 3 and 4 are nucleotide and amino acid sequences of D2 / ZK-5V, respectively.

[0016] SEQ ID Nos: 5 and 6 are nucleotide and amino acid sequences of D2 / ZK-V2A, respectively.

[0017] SEQ ID Nos: 7 and 8 are nucleotide and amino acid sequences of the RIK3451 strain of ZIKV.

[0018] SEQ ID Nos: 9 and 10 are nucleotide and amino acid sequences of DENV-2 16681.

[0019] SEQ ID Nos: 11 and 12 are nucleotide and amino acid sequences of DENV-2 PDK-53.

[0020] SEQ IDs 13 and 14 are nucleotide and amino acid sequences of the ZIKV strain PRVABC59.

[0021] SEQ ID Nos: 15 and 16 are nucleotide and amino acid sequences of D2 / ZK-P-RFNN, respectively.

[0022] SEQ ID Nos: 17 and 18 are nucleotide and amino acid sequences of D2 / ZK-V-RFNN, respectively. Petition 870260062389, dated 06 / 25 / 2026, p. 10 / 135 6 / 56

[0023] SEQ ID Nos: 19 and 20 are nucleotide and amino acid sequences of D2 / ZK-P4, respectively.

[0024] SEQ ID Nos: 21 and 22 are nucleotide and amino acid sequences of D2 / ZK-V4, respectively.

[0025] SEQ ID Nos: 23 and 24 are nucleotide and amino acid sequences of D2 / ZK-P5, respectively.

[0026] SEQ ID Nos: 25 and 26 are nucleotide and amino acid sequences of D2 / ZK-V5, respectively. DETAILED DESCRIPTION I. Abbreviations ASD Average survival days C Capsid protein D2 / ZKV Chimeric dengue-2 / Zika virus DENV Dengue virus And envelope glycoprotein GBS Guillain-Barré Syndrome MOI Multiplicity of infection NS Non-structural pfu Plate forming unit pi Post-infection prM Pre-membrane protein PHEIC Public Health Emergency of International Concern WHO World Health Organization ZIKV Zika virus II. Terms and Methods

[0027] Unless otherwise indicated, technical terms are used in accordance with conventional usage. Definitions of terms common in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Petition 870260062389, dated 06 / 25 / 2026, p. 11 / 135 7 / 56 published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Table Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0028] To facilitate the review of the various description modalities, the following explanations of specific terms are provided:

[0029] Adjuvant: A substance or vehicle that non-specifically enhances the immune response to an antigen. Adjuvants may include a suspension of minerals (alum, aluminum hydroxide, or phosphate) in which the antigen is adsorbed; or a water-in-oil emulsion in which the antigen solution is emulsified in mineral oil (e.g., Freund's incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity. Immunostimulatory oligonucleotides (such as those including a CpG motif) may also be used as adjuvants (e.g., see U.S. Patents Nos. 6,194,388, 6,207,646, 6,214,806, 6,218,371, 6,239,116, 6,339,068, 6,406,705 and 6,429,199). Adjuvants also include biological molecules, such as co-stimulatory molecules. Examples of biological adjuvants include IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL.

[0030] To administer: As used herein, administering a composition (for example, an immunogenic composition, such as a chimeric virus) to an individual means providing, applying, or bringing the composition into contact with the individual. Administration may be carried out by any of several routes, such as topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal, and intramuscular.

[0031] Antibody: A protein (or protein complex) that includes one or more polypeptides substantially encoded by genes of Petition 870260062389, dated 06 / 25 / 2026, p. 12 / 135 8 / 56 Immunoglobulin or immunoglobulin gene fragments. Recognized immunoglobulin genes include the constant region genes kappa, lambda, alpha, gamma, delta, epsilon, and mu, as well as the myriad of immunoglobulin variable region genes. Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

[0032] The basic immunoglobulin (antibody) structural unit is generally a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having a light chain (approximately 25 kDa) and a heavy chain (approximately 50-70 kDa). The terminal end of each chain defines a variable region of approximately 100 to 110 or more amino acids, primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.

[0033] As used herein, the term antibodies includes intact immunoglobulins as well as various well-characterized fragments. For example, Fabs, Fvs, and single-chain Fvs (scFvs) that bind to the target protein (or epitope within a protein or fusion protein) would also be specific binding agents for that protein (or epitope). These antibody fragments are defined as follows: (1) Fab, the fragment containing a monovalent antigen-binding fragment of an antibody molecule produced by digestion of the whole antibody with the enzyme papain to produce an intact light chain and a portion of a heavy chain; (2) Fab', the fragment of an antibody molecule obtained by treatment of the whole antibody with pepsin, followed by reduction, to produce an intact light chain and a portion of the heavy chain; Petition 870260062389, dated 06 / 25 / 2026, p. 13 / 135 9 / 56 two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the antibody fragment obtained by treating the whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab')2, a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically modified fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single-chain antibody, a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single-chain molecule. The methods for manufacturing these fragments are routine (see, for example, Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999).

[0034] The antibodies for use in the methods and devices of this description may be monoclonal or polyclonal. By way of example only, monoclonal antibodies may be prepared from murine hybridomas according to the classic method of Kohler and Milstein (Nature 256: 495-97, 1975) or their derived methods. Detailed procedures for the production of monoclonal antibodies are described by Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999.

[0035] Antibody binding affinity: The strength of the bond between a single antibody binding site and a ligand (e.g., an antigen or epitope). The affinity of an antibody binding site X for a ligand Y is represented by the dissociation constant (Kd), which is the concentration of Y required to occupy half of the binding sites of X present in a solution. A smaller Kd indicates a stronger or greater affinity interaction between X and Y, and a lower concentration of ligand is required to occupy the sites. In general, antibody binding affinity can be affected by altering, Petition 870260062389, dated 06 / 25 / 2026, p. 14 / 135 10 / 56 modification and / or substitution of one or more amino acids in the epitope recognized by the antibody paratope. In one example, the antibody binding affinity is measured by final titration in an AgELISA assay.

[0036] Antigen: A compound, composition, or substance that can stimulate antibody production or a T-cell response in an animal, including compositions that are injected into or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogenes. In one embodiment, an antigen is a viral antigen, such as a flavivirus E protein.

[0037] Attenuated: In the context of a live virus, a virus is attenuated if its ability to infect a cell or individual and / or its ability to produce disease is reduced (e.g., eliminated) compared to a wild-type virus. Typically, an attenuated virus retains at least some ability to produce an immune response after administration to an immunocompetent individual. In some cases, an attenuated virus is able to produce a protective immune response without causing any signs or symptoms of infection. In some embodiments, the ability of an attenuated virus to cause disease in an individual is reduced by at least approximately 10%, at least approximately 25%, at least approximately 50%, at least approximately 75%, or at least approximately 90% relative to the wild-type virus. Consequently, an attenuating mutation is a mutation in the viral genome and / or an encoded polypeptide that results in an attenuated virus.

[0038] Biological sample: A sample obtained from an individual (such as a human or veterinary individual). Biological samples include, for example, fluid, cell, and / or tissue samples. In some embodiments, the biological sample is a sample Petition 870260062389, dated 06 / 25 / 2026, page 15 / 135 11 / 56 of fluid. The fluid sample includes, but is not limited to, serum, blood, plasma, urine, feces, saliva, cerebrospinal fluid (CSF), and bronchoalveolar lavage (BAL).

[0039] Capsid protein (C protein): A structural flavivirus protein that functions to package viral RNA into the nucleocapsid core during virus assembly. The C-terminal portion of the C protein includes an internal signal sequence (here called the C(ss) or prM signal sequence) for translocation of the prM protein to the endoplasmic reticulum, where cleavage of the C and prM proteins occurs. This signal sequence varies in length among different flaviviruses. For example, the C(ss) of both WNV and ZIKV is 18 amino acids, while the C(ss) of the DEN virus is 14 amino acids.

[0040] Chimera: A molecule (e.g., nucleic acid or protein) composed of parts from different origins (such as at least two nucleic acids or polypeptides) that, although not normally joined in their native state, are joined or linked to form a single continuous molecule. A chimera may include nucleic acids or polypeptides that are joined end-to-end (e.g., the amino terminus of one sequence is linked to the carboxyl terminus of a second sequence) or may include a sequence from one molecule that is embedded within another molecule (e.g., the amino terminus and the carboxyl terminus of the chimera are from one molecule, while an intervening sequence comes from another molecule).

[0041] A chimera may include a chimeric protein, for example, a protein that is composed of amino acids from more than one protein. A chimera may also include a chimeric nucleic acid composed of nucleic acid sequences from more than one source, such as a chimeric nucleic acid that codes for a chimeric protein. In other examples, a chimera may include a chimeric genome, such as a flavivirus genome, which is composed of Petition 870260062389, dated 06 / 25 / 2026, p. 16 / 135 12 / 56 sequences from two or more flaviviruses. For example, a chimeric flavivirus genome may comprise nucleic acid sequences from more than one flavivirus genome, such as a dengue virus and a Zika virus. In some examples, a chimeric flavivirus includes nucleic acids encoding one or more proteins from a first flavivirus and nucleic acids encoding one or more proteins from a second flavivirus. In particular examples, a chimeric flavivirus is composed of a nucleic acid encoding the non-structural proteins and a C protein or a portion thereof from a dengue virus genome linked to a nucleic acid encoding a prM protein and at least a portion of an E protein (and optionally a portion of a C protein) from a Zika virus genome.

[0042] Conservative substitution: A substitution of an amino acid residue in a protein sequence by a different amino acid residue having similar biochemical properties. Typically, conservative substitutions have little or no impact on the activity of a resulting polypeptide. For example, ideally, a flavivirus protein (such as a prM, E, or non-structural protein) including one or more conservative substitutions (e.g., 1-10, 2-5, or 5, 10, 20, 30, 40, or 50 substitutions) maintains the structure and function of the wild-type protein. A polypeptide can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence encoding that polypeptide using, for example, standard procedures such as site-directed mutagenesis or PCR.In one example, such variants can be readily selected for further testing by infecting cells with a virus containing a variant protein and determining its replication capacity, producing viruses containing a variant protein and determining their neurovirulence or neuroinvasion properties, and / or testing for antibody cross-reactivity. Petition 870260062389, dated 06 / 25 / 2026, page 17 / 135 13 / 56

[0043] Contact: Placement in direct physical association; includes both solid and liquid forms. Contact is often used synonymously with exposure. In some cases, contact includes transfection, such as the transfection of a nucleic acid molecule into a cell. In other examples, contact refers to incubating a molecule (such as an antibody) with a biological sample.

[0044] Control: A reference standard, for example, a positive control or a negative control. A positive control is known to give a positive test result. A negative control is known to give a negative result. However, the reference standard may be a theoretical or computed result, for example, a result obtained from a population.

[0045] Dengue virus (DENV): An RNA virus of the family Flaviviridae, genus Flavivirus. The dengue virus genome encodes the three structural proteins (C, prM, and E) that form the viral particle and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) that are only found in infected host cells but are necessary for virus replication. There are four serotypes of the dengue virus, designated DENV-1, DENV-2, DENV-3, and DENV-4. All four serotypes can cause the full spectrum of dengue disease. Infection with one serotype can produce lifelong immunity to that serotype. However, serious complications can occur after subsequent infection with a different serotype. The dengue virus is primarily transmitted by Aedes mosquitoes, particularly A. aegypti. Symptoms of dengue virus infection include fever, headache, muscle and joint pain, and a measles-like rash.In a small percentage of cases, the infection develops into a life-threatening dengue hemorrhagic fever, usually resulting in bleeding, low platelet levels, and leakage of blood plasma, or in the syndrome of... Petition 870260062389, dated 06 / 25 / 2026, page 18 / 135 14 / 56 Dengue shock characterized by dangerously low blood pressure.

[0046] Envelope glycoprotein (E protein): A structural protein of a flavivirus that mediates the binding of flavivirus virions to cellular receptors on host cells. The flavivirus E protein is required for membrane fusion and is the primary antigen that induces protective immunity to flavivirus infection. The flavivirus E protein affects host range, tissue tropism, and viral virulence. The flavivirus E protein contains three structural and functional domains, DI-DIII. In mature virus particles, the E protein forms head-to-tail homodimers that extend and form a dense network on the viral surface.

[0047] Flavivirus non-structural protein: There are seven non-structural (NS) proteins of a flavivirus, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, which are encoded by the portion of the flavivirus genome that is 3' to the structural proteins. NS1 has been implicated in RNA replication and has been shown to be secreted from infected mammalian cells (Post et al., Virus Res. 18: 291-302, 1991; Mackenzie et al., Virology 220: 232-240, 1996; Muylaert et al., Virology 222: 159-168, 1996). NS1 can produce strong humoral immune responses and is a potential vaccine candidate (Shlesinger et al., J. Virol. 60: 1153-1155, 1986; Qu et al., J. Gen. Virol. 74: 89-97, 1993). NS2 is cleaved into NS2A and NS2B. NS2A is involved in RNA replication and in the assembly and secretion of viral particles, and NS2B forms a complex with NS3 and functions as a cofactor for NS3 proteinase, which cleaves portions of the viral polyprotein.NS3 also functions as an RNA helicase and is used to unwind viral RNA during replication (Li et al., J. Virol. 73: 3108-3116, 1999). Although the exact functions of NS4A and NS4B remain to be elucidated, they are believed to be involved in RNA replication and trafficking. Petition 870260062389, dated 06 / 25 / 2026, p. 19 / 135 15 / 56 RNA (Lindenbach and Rice, In: Fields Virology, Knipe and Howley, eds., Lippincott, Williams and Wilkins, 991 to 1041, 2001). Finally, the NS5 protein is an RNA-dependent RNA polymerase involved in genome replication (Rice et al., Science 229: 726 to 733, 1985). NS5 also shows methyltransferase activity commonly found in RNA encapsulation enzymes (Koonin, J. Gen. Virol. 74: 733 to 740, 1993).

[0048] Flavivirus structural protein: The capsid (C), premembrane (prM), and envelope (E) proteins of a flavivirus are the viral structural proteins. Flavivirus genomes consist of positive-sense RNAs that are approximately 11 kb in length. The genome has a 5' cap but lacks a 3' polyadenylated tail (Wengler et al., Virology 89: 423 aa 437, 1978) and is translated into a polyprotein. The structural proteins (C, prM, and E) are at the amino-terminal end of the polyprotein, followed by the non-structural proteins (NS1-5). The polyprotein is cleaved by virus- and host-derived proteinases into individual proteins. Protein C forms the viral capsid, while proteins prM and E are embedded in the surrounding envelope (Russell et al., The Togaviruses: Biology, Structure, and Replication, Schlesinger, ed., Academic Press, 1980). Protein E functions in binding to host cell receptors, resulting in receptor-mediated endocytosis.At the low pH of the endosome, the E protein undergoes a conformational change causing fusion between the viral envelope and the endosomal membranes. It is believed that the prM protein stabilizes the E protein until the virus exits the infected cell, at which point prM is cleaved into the mature M protein (Reviewed by Lindenbach and Rice, In: Fields Virology, Knipe and Howley, eds., Lippincott, Williams and Wilkins, 991 to 1041, 2001).

[0049] Heterologous: Originating from different genetic sources or species. Petition 870260062389, dated 06 / 25 / 2026, page 20 / 135 16 / 56

[0050] Immune response: Response of a cell of the immune system, such as a B cell, T cell, macrophage, or polymorphonucleocyte, to a stimulus such as an antigen. An immune response may include any cell in the body involved in a host defense response, for example, an epithelial cell that secretes an interferon or a cytokine. An immune response includes, but is not limited to, an innate immune response or inflammation.

[0051] Immunize: To make an individual protected from an infectious disease, such as by vaccination.

[0052] Isolated: An isolated or purified biological component (such as a nucleic acid, peptide, protein, protein complex, or particle) has been substantially separated, produced separately, or purified from other components in a preparation or other components in the cell of the organism in which the component occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides, and proteins that have been isolated or purified thus include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins. The term isolated or purified does not require absolute purity; rather, it is intended as a relative term.Thus, for example, an isolated biological component is one in which the biological component is more enriched than the biological component that is in its natural environment within a cell, or other production container. Preferably, a preparation is purified in such a way that the biological component represents at least 50%, such as at least 70%, at least 90%, at least 95%, or more, of the total biological component content of the preparation. Petition 870260062389, dated 06 / 25 / 2026, p. 21 / 135 17 / 56

[0053] Nucleic acid molecule: A polymeric form of nucleotides, which may include sense and antisense strands of RNA, cDNA, and genomic DNA, and synthetic forms and mixed polymers of the foregoing. A nucleotide refers to a ribonucleotide, deoxynucleotide, or a modified form of any type of nucleotide. The term nucleic acid molecule, as used herein, is synonymous with nucleic acid and polynucleotide. A nucleic acid molecule is normally at least 10 bases long, unless otherwise specified. The term includes single and double-stranded forms of DNA. A polynucleotide may include wild-type or modified nucleotides linked together by wild-type and / or non-natural nucleotide bonds.

[0054] Operationally linked: A first nucleic acid is operationally linked to a second nucleic acid when the first nucleic acid is placed in a functional relationship with the second nucleic acid. Generally, operationally linked DNA sequences are contiguous and, when necessary, join two protein-coding regions in the same reading frame. Operationally linked nucleic acids include a first nucleic acid contiguous with the 5' or 3' end of a second nucleic acid. In other examples, a second nucleic acid is operationally linked to a first nucleic acid when it is incorporated into the first nucleic acid, for example, where the nucleic acid construct includes (in order) a portion of the first nucleic acid, the second nucleic acid, and the remainder of the first nucleic acid.

[0055] Pharmaceutically acceptable vehicle: The pharmaceutically acceptable vehicles (vehicles) useful in this description are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Petition 870260062389, dated 06 / 25 / 2026, page 22 / 135 18 / 56 Philadelphia, PA, 21st Edition (2005), describes compositions and formulations suitable for pharmaceutical administration of one or more therapeutic compositions, such as a chimeric virus, and additional pharmaceutical agents.

[0056] In general, the nature of the vehicle will depend on the particular mode of administration to be employed. For example, parenteral formulations generally comprise injectable fluids which include pharmaceutically and physiologically acceptable fluids such as water, physiological saline solution, balanced saline solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet or capsule forms), conventional non-toxic solid vehicles may include, for example, pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral vehicles, the pharmaceutical compositions to be administered may contain small amounts of non-toxic excipients such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate.

[0057] Pre-membrane protein (prM protein): Structural protein of flaviviruses. The prM protein is an approximately 25 kDa protein that is the intracellular precursor of the membrane protein (M). It is believed that prM stabilizes the E protein during the transport of the immature virion to the cell surface. When the virus exits the infected cell, the prM protein is cleaved to the mature M protein, which is part of the viral envelope (Reviewed by Lindenbach and Rice, in: Fields Virology, Knipe and Howley, eds., Lippincott, Williams, and Wilkins, 991 to 1041, 2001).

[0058] To prevent, treat or improve a disease: Preventing a disease refers to inhibiting the full development of a disease. Petition 870260062389, dated 06 / 25 / 2026, page 23 / 135 19 / 56 Treatment refers to a therapeutic intervention that improves a sign or symptom of a disease or pathological condition after it has begun to develop. Improvement refers to the reduction in the number or severity of one or more signs or symptoms of a disease.

[0059] Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified nucleic acid preparation is one in which the nucleic acid is more enriched than the nucleic acid in its natural environment (such as within a cell) or in a preparation or production container. In other examples, a purified virus preparation is one in which the virus is more enriched than in a cell or organism, a preparation, or a production container. A purified nucleic acid or virus also includes one that is substantially free of undesirable components, such as an inactivating agent. Preferably, a preparation is purified such that the nucleic acid or virus represents at least 50% of the total content of the preparation.In some embodiments, a purified preparation contains at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or more of the nucleic acid or virus.

[0060] Recombinant nucleic acid: A nucleic acid molecule (or protein or virus) that does not occur naturally or has a sequence that is made by an artificial combination of two otherwise separate sequence segments. This artificial combination is accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated nucleic acid segments, for example, by genetic engineering techniques such as those described by Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vols. 1–3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. The term recombinant includes nucleic acids and proteins that have been Petition 870260062389, dated 06 / 25 / 2026, p. 24 / 135 20 / 56 altered only by the addition, substitution, or deletion of a portion of a natural nucleic acid molecule or protein.

[0061] Sequence identity: The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise called sequence identity. Sequence identity is often measured in terms of percent identity (or similarity or homology); the higher the percent, the more similar the two sequences are.

[0062] Sequence alignment methods for comparison are well known in the art. Several alignment programs and algorithms are described by: Smith and Waterman (Adv. Appl. Math., 2: 482, 1981); Needleman and Wunsch (J. Mol. Biol., 48: 443, 1970); Pearson and Lipman (Proc. Natl. Acad. Sci., 85: 2444, 1988); Higgins and Sharp (Gene, 73: 237, 44, 1988); Higgins and Sharp (CABIOS, 5: 151-53, 1989); Corpet et al. (Nuc. Acids Res., 16: 10881-90, 1988); Huang et al. (Comp. Appls. Biosci. 8: 155, 65, 1992); and Pearson et al. (Meth. Mol. Biol., 24: 307-31, 1994). Altschul et al. (Nature Genet., 6: 119, 29, 1994) presents a detailed consideration of sequence alignment methods and homology calculations.

[0063] The ALIGN alignment tools (Myers and Miller, CABIOS 4: 11 to 17, 1989) or LFASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. 85: 2444 to 2448, 1988) can be used to perform sequence comparisons (Internet Program© 1996, WR Pearson and University of Virginia, fasta20u63 version 2.0u63, version date December 1996). ALIGN compares entire sequences with each other, while LFASTA compares regions of local similarity. These alignment tools and their respective tutorials are available on the Internet at the NCSA website. Alternatively, for comparisons of amino acid sequences with more than 30 Petition 870260062389, dated 06 / 25 / 2026, page 25 / 135 For 21 / 56 amino acids, the Blast 2 sequences function can be employed using the standard BLOSUM62 array for the standard parameters (existence cost of a gap of 11, and a residue gap cost of 1). When aligning short peptides (less than approximately 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 array set for the standard parameters (open gap penalty 9, extension gap penalty 1). The BLAST sequence comparison system is available, for example, on the NCBI network site; see also Altschul et al., J. Mol. Biol. 215: 40310, 1990; Gish and States, Nature Genet., 3: 266, 72, 1993; Madden et al., Meth. Enzymol. 266: 131-41, 1996; Altschul et al., Nucleic Acids Res., 25: 3389-402, 1997; and Zhang and Madden, Genome Res., 7: 649-56, 1997.

[0064] Serum: The fluid portion of blood that separates from clotted blood. Serum contains many proteins, including antibodies, but does not contain clotting factors.

[0065] Individual: Living multicellular vertebrate organisms, a category that includes human and non-human mammals (such as mice, rats, rabbits, sheep, horses, cows, and non-human primates).

[0066] Therapeutically effective amount: A quantity of a specified agent (such as a chimeric virus) sufficient to achieve a desired effect in an individual being treated with that agent. For example, this could be the amount of a virus vaccine useful for producing an immune response in an individual and / or for preventing infection by the virus. In the context of this description, a therapeutically effective amount of a Zika virus vaccine, for example, is an amount sufficient to increase resistance, prevent, improve, and / or treat infection caused by the Zika virus in an individual without causing a substantial cytotoxic effect in the individual. Petition 870260062389, dated 06 / 25 / 2026, page 26 / 135 22 / 56 The effective amount of a Zika virus vaccine (or Zika virus immunogenic composition) useful for increasing resistance, preventing, improving and / or treating infection in an individual will depend, for example, on the individual being treated, the method of administration of the therapeutic composition and other factors.

[0067] Transformed: A transformed cell is a cell into which a nucleic acid molecule (such as a heterologous nucleic acid) has been introduced by molecular biology techniques. The term encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

[0068] Vaccine: A preparation of immunogenic material capable of stimulating an immune response, administered for the prevention, inhibition, enhancement, or treatment of infectious diseases or other types of illnesses. The immunogenic material may include attenuated or inactivated (killed) microorganisms (such as bacteria or viruses), or antigenic proteins, peptides, or DNA derived from them. An attenuated virus is a virulent organism that has been modified to produce a less virulent form, but still retains the ability to produce antibodies and cell-mediated immunity against the virulent form. An inactivated (killed) virus is a previously virulent organism that has been inactivated with chemicals, heat, or other treatment, but produces antibodies against the organism. Vaccines can elicit prophylactic (preventive or protective) and therapeutic responses.Methods of administration vary depending on the vaccine, but may include inoculation, ingestion, inhalation, or other forms of administration. Vaccines may be administered with an adjuvant to enhance the immune response.

[0069] Vector: A vector is a nucleic acid molecule that Petition 870260062389, dated 06 / 25 / 2026, page 27 / 135 23 / 56 allows the insertion of foreign nucleic acid without interrupting the vector's ability to replicate and / or integrate into a host cell. A vector may include nucleic acid sequences that allow it to replicate in a host cell, such as an origin of replication. An insertion vector is capable of inserting itself into a host nucleic acid. A vector may also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the regulatory sequences necessary to allow the transcription and translation of inserted genes or genes.

[0070] Zika virus (ZIKV): A member of the Flaviviridae family of viruses and the Flavivirus genus. Other members of this genus include dengue virus, yellow fever virus, Japanese encephalitis virus (JEV), West Nile virus, and Spondweni virus. ZIKV is transmitted by the Aedes aegypti and A. albopictus mosquitoes, which are active during the day. This virus was first isolated from a Rhesus monkey from the Zika Forest in Uganda in 1947. Since the 1950s, ZIKV has been known to occur within a narrow equatorial belt from Africa to Asia. The virus spread eastward across the Pacific Ocean in 2013–2014, resulting in ZIKV outbreaks in Oceania to French Polynesia, New Caledonia, the Cook Islands, and Easter Island. In 2015, ZIKV spread through Mexico, Central America, the Caribbean, and South America, where it reached pandemic levels.ZIKV infection usually causes symptoms, which are mild, including mild headache, maculopapular rash, fever, malaise, conjunctivitis, and joint pain. ZIKV causes symptoms in approximately 20% of infected individuals, and no deaths from the virus have been reported so far. However, ZIKV infection has been associated with the birth of microcephalic babies following maternal infection, as well as an increase in GBS cases. (Reports follow.) Petition 870260062389, dated 06 / 25 / 2026, p. 28 / 135 Studies 24 / 56 also indicated that ZIKV has the potential for sexual transmission and transmission through human blood. ZIKV has also been found in human saliva and breast milk. Currently, there are no medical countermeasures available for the treatment or prevention of Zika virus infection (Malone et al., PLoS Negl Trop Dis 10 (3): e0004530, 2016).

[0071] Unless explicitly stated otherwise, all technical and scientific terms used herein have the same meaning as that commonly understood by one skilled in the art to which this description pertains. The singular terms a, an, and the include the plural referents unless the context clearly indicates otherwise. Comprising A or B means including A, or B, or A and B. It should be understood that all base sizes or amino acid sizes, and all molecular weights or molecular masses, given for nucleic acids or polypeptides are approximate and are provided for description. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present description, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, this specification, including explanations of terms, will prevail. Furthermore, the materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. III. Overview of Various Modalities

[0072] Chimeric flaviviruses that include non-coding regions, non-structural proteins, a capsid protein (C), and a portion of a premembrane signal sequence (prM) from a wild-type or attenuated dengue virus serotype 2 (DENV-2) are described herein; and a portion of a prM signal sequence, a prM protein, and at least one portion of an envelope protein (E) from a Zika virus (ZIKV). Petition 870260062389, dated 06 / 25 / 2026, p. 29 / 135 25 / 56 Tables 1 and 2 below provide start and stop positions of particular genes and proteins in an exemplary Zika virus (SPH2015) and an exemplary attenuated DENV-2 virus vaccine strain (PDK53). These sequences can serve as reference sequences and can be used to identify particular nucleotide or amino acid positions that correspond to referenced positions in the chimeric nucleic acids described herein, or proteins encoded by the chimeric nucleic acids described herein, for example, by producing an alignment of a chimera and one of the virus sequences provided herein. Table 1. Start and stop positions of non-coding regions (NCRs), structural proteins, and non-structural proteins in the strain. R103451 deZIKV Region Nucleotide start / stop position (SEQ ID NO: 7) Amino acid start / stop position (SEQ ID NO: 8) 5' NCR 1-107 — C 108-473 1-122 C(ss) 420-473 105-122 prM 474-977 123-290 M 753-977 216-290 E 978-2489 291-794 NS1 2490-3545 795-1146 NS2A 3546-4223 1147-1372 NS2B 4224-4613 1373-1502 NS3 4614-6464 1503-2119 NS4A 6465-6914 2120-2269 NS4B 6915-7667 2270-2520 NS5 7668-10376 2521-3423 Stop 10377-10379 — Petition 870260062389, dated 06 / 25 / 2026, page 30 / 135 26 / 56 3' NCR 10380-10807 — Table 2. Start and stop positions of NCRs, structural proteins, and non-structural proteins in the PDK-53 strain of the DENV-2 virus vaccine. Region Nucleotide start / stop position (SEQ ID NO: 11) Amino acid start / stop position (SEQ ID NO: 12) 5' NCR 1-96 — C 97-438 1-114 C(ss) 397-438 101-114 prM 439-936 115-280 M 712-936 206-280 E 937-2421 281-775 NS1 2422-3477 776-1127 NS2A 3478-4131 1128-1345 NS2B 4132-4521 1346-1475 NS3 4522-6375 1476-2093 NS4A 6376-6825 2094-2243 NS4B 6826-7569 2244-2491 NS5 7570-10269 2492-3391 3' NCR 10270-10723 —

[0073] In the described nucleic acid chimeras, the genome of ZIKV can be from any ZIKV strain, including an African genotype strain or an Asian genotype strain. In some embodiments, the ZIKV is an African genotype strain, such as MR766. In other embodiments, the ZIKV is an Asian genotype strain, such as SPH2015, PRVABC59, R103451, P6-740, or FSS 13025. In some embodiments, the ZIKV genome is from the R103451 strain (SEQ ID NO: 7 or deposited under GenBank Accession No. KX262887.1). The ZIKV genome can be a wild-type strain or an attenuated (or vaccine) strain. In some examples, the genome sequence of Petition 870260062389, dated 06 / 25 / 2026, p. 31 / 135 27 / 56 ZIKV is modified, for example, to introduce restriction sites for cloning purposes. These modifications can be silent mutations (e.g., changes in the nucleotide sequence that do not alter the amino acid sequence) or they can alter the amino acid sequence.

[0074] ZIKV sequences are publicly available. For example, GenBank Access numbers KX262887.1, KU321639.1, KU501215.1, KU955595.1, KU955594.1, KU955593.1, KU955592.1, KU955591.1, KU681082.3, KU681081.3, and KX247646.1, all of which are incorporated by reference as having been included in GenBank on June 14, 2016. In further examples, the ZIKV genome (or the ZIKV genome signal C, prM, and / or protein E sequence) is at least 90%, less than 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a publicly available ZIKV sequence.

[0075] In some embodiments, the genome of the DENV-2 strain is a genome of the attenuated DENV-2 strain. In some examples, the attenuated DENV-2 is the PDK-53 strain, whose genomic sequence is shown here as SEQ ID NO: 11.

[0076] In other embodiments, the DENV-2 strain genome is a wild-type DENV-2 strain genome. In some examples, the wild-type DENV-2 is strain 16681, whose genomic sequence is shown here as SEQ ID NO: 9.

[0077] In some examples, the described D2 / ZKV chimeras include one or more nucleic acid substitutions that result in an amino acid substitution that provides a desirable characteristic, for example, greater stability and / or replication in vaccine virus production cell culture (such as Vero cells), or decreased virus replication in mosquito cells (such as C6 / 36 cells) or live mosquitoes compared to non-virus. Petition 870260062389, dated 06 / 25 / 2026, p. 32 / 135 28 / 56 replaced or chimera.

[0078] Viruses containing the described nucleic acid chimeras can be readily produced by replication in cultured host cells. Methods of virus production are well known in the art (see, for example, Fields Virology, Knipe and Howley, eds., Lippincott, Williams and Wilkins, 2001; Flint et al., Principles of Virology, ASM Press, 2000). Host cell lines are generally selected to be easy to infect with viruses or transfect with viral genomic RNA, capable of stably maintaining foreign RNA with an un-rearranged sequence, and possessing the cellular components necessary for transcription, translation, post-translational modification, virus assembly, and secretion of the viral protein or particle. Furthermore, the cells are typically those with simple medium component requirements that can be adapted for growth in suspension culture.In some examples, the host cell line is a mammalian cell line that is adapted to growth in low-serum or serum-free media. Suitable exemplary host cell lines include Vero (monkey), C6 / 36 (mosquito), BHK21 (hamster), LLC-MK2 (monkey), SK6 (swine), L292 (mouse), HeLa (human), HEK (human), 2fTGH cells (human), HepG2 (human), and PDK (canine). Suitable cell lines can be obtained from the American Type Culture Collection (ATCC), Manassas, VA.

[0079] The description also provides D2 / ZKV chimeras having one or more nucleic acid or amino acid substitutions, insertions, deletions, or combinations thereof, such that the resulting chimera has enhanced characteristics, such as improved growth in Vero cells.

[0080] Manipulation of the nucleotide sequence of flaviviruses Petition 870260062389, dated 06 / 25 / 2026, page 33 / 135 29 / 56 chimeric variants described by standard procedures, including, for example, site-directed mutagenesis or PCR and M13 primer mutagenesis, can be used to produce variants with improved characteristics (such as virus titration or improved cell culture stability). Details of these techniques are well known. For example, protocols are provided by Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. The simplest modifications involve the substitution of one or more amino acids with amino acids with similar physicochemical and / or structural properties. These so-called conservative substitutions are likely to have minimal impact on the activity and / or structure of the resulting protein.Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of ​​substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) most of the side chain. Examples of conservative substitutions are shown below. Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln; Glu Petition 870260062389, dated 06 / 25 / 2026, p. 34 / 135 30 / 56 Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

[0081] The substitutions that are generally expected to produce the greatest changes in protein properties will not be conservative, for example, changes in which (a) a hydrophilic residue, for example, seryl or threonyl, is replaced by a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanine, valyl or alanyl (or vice versa); (b) a cysteine ​​or proline is replaced by any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl or histadyl, is replaced by an electronegative residue, for example, glutamyl or aspartyl (or vice versa); or (d) a residue having a bulky side chain, for example, phenylalanine, is replaced by one not having a side chain, for example, glycine (or vice versa).

[0082] In addition to the mutagenesis directed at producing variants of the described D2 / ZKV chimeras, mutations can occur after passage in cell culture that result in variants, some with desirable characteristics. The nucleic acid and amino acid substitutions, insertions, and / or deletions that accumulate in chimeric viruses during cell culture passages are readily determined by sequence analysis of amplified virus from isolated virus seed plates, and can be manipulated in infectious clones to generate D2 / ZKV chimera variants that have improved characteristics (such as replication to high titer). Consistent mutations identified from multiple seeds or Petition 870260062389, dated 06 / 25 / 2026, page 35 / 135 31 / 56 isolated plates are an indication of a desirable chimeric substitution in the cell type. Previous studies have successfully identified substitutions that occurred in cell culture and manipulated them in different chimeric virus constructs to produce chimeric viruses with improved characteristics (e.g., Huang et al., J. Virol. 77: 11436 to 11447, 2003; Huang et al., J. Virol. 12: 7300 to 7310, 2005; U.S. Patent No. 8,715,689 and WO 2015 / 196094).

[0083] Flavivirus nucleic acid chimeras are provided here. In some embodiments, the nucleic acid chimera includes a first nucleic acid molecule comprising a 5' non-coding region, a nucleic acid encoding non-structural proteins and a C protein, and a 3' non-coding region, each from a DENV-2 strain genome, wherein the C protein comprises a portion of a prM signal sequence from the DENV-2 genome and a portion of a prM signal sequence from a ZIKV genome; and a second nucleic acid molecule operationally linked to the first nucleic acid molecule, encoding a prM protein and at least a portion of an E protein from the ZIKV genome.

[0084] In some embodiments, the DENV-2 strain genome is an attenuated DENV-2 strain genome. In some examples, the attenuated DENV-2 genome includes a mutation in the 5' non-coding region at nucleotide position 57; a mutation at nucleotide position 2579 resulting in the presence of an aspartate at amino acid residue 53 of the NS1 protein; and / or a mutation at nucleotide position 5270 resulting in the presence of a valine at amino acid residue 250 of the NS3 protein. In particular examples, the attenuated DENV-2 is the PDK-53 strain. The attenuated DENV-2 may also include one or more mutations listed in Table 4.

[0085] In some embodiments, the genome of the DENV-2 strain is a genome of the wild-type DENV-2 strain. In some examples, the Petition 870260062389, dated 06 / 25 / 2026, p. 36 / 135 32 / 56 Wild type DENV-2 is strain 16681.

[0086] In some embodiments, the Zika virus is an African genotype virus, such as the MR-766 strain. In other embodiments, the Zika virus is an Asian genotype virus, such as the SPH2015, PRVABC59, R103451, P6-740 or FSS 13025 strains.

[0087] In some embodiments, the DENV-2 genome prM signal sequence portion includes the first three amino acids of the DENV-2 prM signal sequence and the ZIKV genome prM signal sequence portion includes the last 15 amino acids of the ZIKV prM signal sequence (see Strategy 1 in Figure 1). In some examples, the first three amino acids of the DENV-2 prM signal sequence include amino acids 101-103 of SEQ ID NO: 12 and / or the last 15 amino acids of the ZIKV prM signal sequence include amino acids 108-122 of SEQ ID NO: 8.

[0088] In other embodiments, the DENV-2 genome prM signal sequence portion includes the first five amino acids of the DENV-2 prM signal sequence and the ZIKV genome prM signal sequence portion includes the last 13 amino acids of the ZIKV prM signal sequence (see Strategy 2 in Figure 1). In some examples, the first five amino acids of the DENV-2 prM signal sequence include amino acids 101-105 of SEQ ID NO: 12 and / or the last 13 amino acids of the ZIKV prM signal sequence include amino acids 110-122 of SEQ ID NO: 8.

[0089] In other embodiments, the C / prM junction site includes a number of DENV-2 and ZIKV residues different from those listed above.

[0090] In some embodiments, a portion of protein E is from the DENV-2 genome. In some examples, the portion of protein E from the DENV-2 genome includes the last 14 amino acids of the modified DENV-2 protein E. In specific non-limiting examples, the last 14 amino acids of the DENV-2 protein E include the Petition 870260062389, dated 06 / 25 / 2026, page 37 / 135 33 / 56 amino acids 777-790 of SEQ ID NO: 2. In other examples, the E / NS1 junction site in the D2 / ZKV chimera may include an alternative number of DENV-2 residues, such as approximately 0, approximately 2, approximately 4, approximately 6, approximately 8, approximately 10, approximately 12, approximately 13, approximately 14, approximately 16, approximately 18, or approximately 20 DENV-2 residues.

[0091] In some embodiments, the nucleic acid chimera also includes at least one Vero cell adaptation mutation. In some examples, the Vero cell adaptation mutation results in a glutamine substitution for arginine at residue 465 of the D2 / ZKV E protein (also ZIKV E-465); an isoleucine substitution for threonine at residue 484 of the D2 / ZKV E protein (also ZIKV E-484); an isoleucine substitution for phenylalanine at residue 493 of the D2 / ZKV E protein (as DENV-2 E-484); a lysine substitution for asparagine at residue 99 of the NS2A protein (NS2A protein is DENV-2); and / or a substitution of aspartic acid for asparagine at residue 23 of the NS4A protein (the NS4A protein is from DENV-2) (Table 3).

[0092] In particular examples, nucleic acid chimeras include four adaptation mutations to Vero cells, where mutations result in a glutamine substitution for arginine at residue 465 of the D2 / ZKV E protein (also E-465 of ZIKV); an isoleucine substitution for phenylalanine at residue 493 of the D2 / ZKV E protein (also E-484 of DENV-2); a lysine substitution for asparagine at residue 99 of the NS2A protein (of DENV-2); and an aspartic acid substitution for asparagine at residue 23 of the NS4A protein (of DENV-2).

[0093] In other particular examples, nucleic acid chimeras include four adaptation mutations to Vero cells, in which Petition 870260062389, dated 06 / 25 / 2026, p. 38 / 135 34 / 56 mutations result in a glutamine substitution for arginine at residue 465 of the D2 / ZKV E protein (also E-465 of ZIKV); an isoleucine substitution for threonine at residue 484 of the D2 / ZKV E protein (also E-484 of ZIKV); an isoleucine substitution for phenylalanine at residue 493 of the D2 / ZKV E protein (also E484 of DENV-2); and a lysine substitution for asparagine at residue 99 of the NS2A protein (of DENV-2).

[0094] In other particular examples, nucleic acid chimeras include five adaptation mutations to Vero cells, wherein the mutations result in a glutamine substitution for arginine at residue 465 of the D2 / ZIKV E protein (also ZIKV E-465); an isoleucine substitution for threonine at residue 484 of the D2 / ZIKV E protein (also ZIKV E-484); an isoleucine substitution for phenylalanine at residue 493 of the D2 / ZIKV E protein (also DENV-2 E-484); a lysine substitution for asparagine at residue 99 of the NS2A protein (of DENV-2); and an aspartic acid substitution for asparagine at residue 23 of the NS4A protein (of DENV-2). Table 3. Potential amino acid mutations for fitness enhancement in Vero cells of DEN-2 / Zika chimeric viruses. NT position in chimeric DENV-2 / ZKV Protein AA position based on: AA substitution (WT-Mut) Chimeric DENV-2 / ZKV DENV-2 AA ZIKV AA 2347-2348 (CA to AG) E-465 NA E-465 Gln-Arg 2405 (T to C) E-484 NA E-484 Ile-Thr 2431 (A to T) E-493 E-484* NA Ile-Phe 3819 (G to T) NS2A-99 NS2A-99 NA Lys-Asn 6487 (G to A) NS4A-23 NS4A-23 NA Asp-Asn NA = not applicable; chimeric virus does not include the virus gene. * The E protein of DENV-2 is shorter than the E protein of ZIKV, Petition 870260062389, dated 06 / 25 / 2026, p. 39 / 135 35 / 56 resulting in different residue numbers between chimeric DENV-2 / ZKV and DENV-2.

[0095] In some embodiments, the nucleic acid chimera includes a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 25. In some examples, the nucleic acid chimera includes the nucleic acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 25.

[0096] In some embodiments, the nucleic acid chimera encodes an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26. In some examples, the nucleic acid chimera encodes the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26.

[0097] In some examples, the nucleic acid sequence is optimized by human codon.

[0098] Chimeric flaviviruses comprising a nucleic acid chimera described herein are also provided herein. Compositions, such as immunogenic compositions, which include chimeric flaviviruses are also provided by this description. In some embodiments, the immunogenic compositions further include a pharmaceutically acceptable carrier and / or one or more adjuvants.

[0099] In addition, methods are provided here for producing an immune response against ZIKV in an individual by administering to the individual a chimeric flavivirus or an immunogenic composition here Petition 870260062389, dated 06 / 25 / 2026, page 40 / 135 36 / 56 described. The immune response may include, for example, induction of specific antibodies to ZIKV (such as IgM and / or IgG antibodies) or induction of a virus-specific T cell response. In some instances, the immune response is a protective immune response.

[00100] In some embodiments, the method includes administering one to five doses (such as 1, 2, 3, 4, or 5 doses) of the immunogenic composition to the individual. In some examples, the first 1 or 2 doses are the described live attenuated chimeric DENV-2 / ZIKV immunogenic composition, and the subsequent dose(s) is / are inactivated or non-infectious ZIKV vaccine. In some examples, the method further includes administering a combination of the described live attenuated chimeric DENV-2 / ZIKV vaccine with an inactivated or non-infectious ZIKV vaccine (such as inactivated ZIKV or inactivated chimeric WN / ZIKV) simultaneously. In some examples, the method also includes administering one or more adjuvants to the individual.

[00101] A method is also provided for immunizing an individual against ZIKV by administering to the individual a chimeric flavivirus or immunogenic composition described herein.

[00102] In some modalities of the methods, the individual is a human. IV. Compositions and methods for producing an immune response

[00103] Methods are provided here for producing an immune response in an individual by administering to that individual a chimeric dengue serotype 2 / Zika virus (D2 / ZKV) described herein. In a particular example, the individual is human. The chimeric D2 / ZKV is used, for example, to produce an immune response that prevents or inhibits infection by a ZIKV.

[00104] In some examples, the method also includes selecting an individual who needs greater immunity to ZIKV. Individuals who need greater immunity to ZIKV include individuals who are in Petition 870260062389, dated 06 / 25 / 2026, page 41 / 135 37 / 56 risk of ZIKV infection, individuals who have been exposed to one or more ZIKV strains, and individuals who have already been vaccinated with ZIKV or other flavivirus vaccines. Residents or travelers to countries or regions where ZIKV is endemic are at risk of contracting ZIKV. Additional factors that contribute to the risk of ZIKV infection include the characteristics of the location, the presence of ZIKV in the area, exposure to mosquitoes, and lack of preventive measures (such as insect repellent).

[00105] One or more chimeric D2 / ZIKV are administered to an individual by any of the routes normally used to introduce a composition into an individual. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, parenteral, intravenous, subcutaneous, vaginal, rectal, intranasal, inhalation, or oral. Parenteral administration, like subcutaneous, intravenous, or intramuscular administration, is generally achieved by injection. Injectables may be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Solutions and suspensions for injection may be prepared from sterile powders, granules, and tablets of the type described above. Administration may be systemic or local.

[00106] Immunogenic compositions are administered in any suitable manner, such as with pharmaceutically acceptable vehicles. Pharmaceutically acceptable vehicles are determined in part by the particular composition to be administered as well as by the particular method used to administer the composition. See, for example, Remington: The Science and Practice of Pharmacy, University of the Sciences of Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st Edition (2005). Consequently, there is a wide variety of suitable formulations of compositions. Petition 870260062389, dated 06 / 25 / 2026, page 42 / 135 38 / 56 pharmaceuticals of the present description.

[00107] Immunogenic compositions can be conveniently presented in single-dose form and prepared using conventional pharmaceutical techniques. Such techniques include the step of combining the active ingredient and the pharmaceutical vehicle(s) or excipient(s). In general, formulations are prepared by uniformly and intimately combining the active ingredient with liquid vehicles. Formulations can be presented in single-dose or multi-dose containers, for example, sealed ampoules and vials, and can be stored in a lyophilized condition requiring only the addition of a sterile liquid vehicle, for example, water for injections, immediately before use. Solutions and suspensions for extemporaneous injection can be prepared from sterile powders, granules, and tablets commonly used by one skilled in the art.

[00108] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or fixed oils. Intravenous vehicles include fluid and nutrient replenishing agents, electrolyte replenishing agents (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases, and the like.

[00109] In some examples, the compositions described here include Petition 870260062389, dated 06 / 25 / 2026, p. 43 / 135 39 / 56 one or more adjuvants. In other examples, an adjuvant is not included in the composition but is administered separately to an individual (for example, in combination with a composition described herein) before, after, or substantially simultaneously with the administration of one or more of the compositions described herein. Adjuvants are agents that increase or enhance an immune response in an individual administered with an antigen, compared with administration of the antigen in the absence of an adjuvant. An example of an adjuvant is an aluminum salt, such as aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, or aluminum hydroxyphosphate.Other adjuvants include biological adjuvants such as cytokines (e.g., IL-2, IL-6, IL-12, RANTES, GM-CSF, TNF-α, or IFN-γ), growth factors (e.g., GMCSF or G-CSF), one or more molecules such as OX-40L or 4-1 BBL, immunostimulatory oligonucleotides (e.g., CpG oligonucleotides), Toll-like receptor agonists (e.g., TLR2, TLR4, TLR7 / 8, or TLR9 agonists), and bacterial lipopolysaccharides or their derivatives (such as 3D-MPL). Additional adjuvants include oil-in-water emulsions, squalene, or other agents. In one example, the adjuvant is a mixture of stabilizing detergents, micelle-forming agent, and oil available under the name PROVAX® (IDEC Pharmaceuticals, San Diego, CA). A person skilled in the art may select a suitable adjuvant or combination of adjuvants to be included in the compositions described herein or administered to an individual in combination with the compositions described herein.

[00110] Administration is performed in single or multiple doses. The dose administered to an individual in the context of this description should be sufficient to induce a beneficial therapeutic response in an individual over time, or to inhibit or prevent ZIKV infection. The required dose will vary from individual to individual. Petition 870260062389, dated 06 / 25 / 2026, page 44 / 135 40 / 56 depending on the species, age, weight and general condition of the individual, the severity of the infection to be treated, the specific immunogenic composition to be used and its mode of administration. An appropriate dose can be determined by one skilled in the art using only routine experimentation. In some examples, the dose of each chimeric virus (as in an immunogenic composition) administered to the individual is approximately 100 pfu to approximately 1,000,000 pfu. For example, a dose of the immunogenic composition may contain at least 100 pfu, at least 1,000 pfu, at least 5,000 pfu, at least 10,000 pfu, at least 50,000 pfu, at least 100,000 pfu, at least 500,000 pfu, or at least 1,000,000 pfu of the chimeric virus.

[00111] The volume of administration will vary depending on the route of administration. For example, intramuscular injections can range from approximately 0.1 ml to approximately 1.0 ml. Those skilled in the art will know appropriate volumes for different routes of administration.

[00112] Repeated immunizations may be necessary to produce an immune response in an individual. When administered in multiple doses, booster doses are given at various time intervals, such as weeks or months to years. In other examples, chimeric D2 / ZKV viruses are used as a booster after administration of one or more ZIKV vaccines. In one example, an individual is given a primary dose of a ZIKV vaccine followed by at least one booster dose of a chimeric D2 / ZKV virus described herein. In alternative examples, the chimeric D2 / ZKV virus is given first, followed by a booster dose of another ZIKV vaccine, such as an inactivated ZIKV vaccine. In some examples, the booster dose is given approximately 14, 30, 60, 90 or more days after administration of the primary dose. Petition 870260062389, dated 06 / 25 / 2026, p. 45 / 135 41 / 56 Additional boosters may be administered at subsequent time points if determined to be necessary or beneficial. Immunization protocols (such as immunogene quantity, number of doses, and timing of administration) may be determined experimentally, for example, using animal models (such as mice or non-human primates), followed by clinical trials in humans.

[00113] The following examples are provided to illustrate certain particular features and / or modalities. These examples should not be constructed to limit the description to the particular features or modalities described. EXAMPLES Example 1: Generation and characterization of chimeric flaviviruses

[00114] This example describes the construction of chimeric dengue-2 / Zika viruses that include the prM protein and at least one portion of the E protein of a ZIKV in an attenuated or wild-type DENV-2 backbone. Engineering and derivation of chimeric D2 / ZKVs

[00115] Using previously manipulated infectious clones of dengue virus serotype 2 (DENV-2) (Kinney et al., Virology 230: 300 to 308, 1997; Butrapet et al., Virol 74: 3011 to 3019, 2000), two chimeric DENV-2 / Zika viruses (D2 / ZKV-P and D2 / ZKV-V) were generated. D2 / ZKV-P and D2 / ZKV-V contain the prM and E genes of a Zika virus (ZIKV) in the genomic background of the parent virus (P) DENV-2 16681 and its vaccine strain (V) PDK-53, respectively. The DENV-2 PDK-53 vaccine strain was originally developed by serial passage of DENV-2 virus 16681 53 times in primary canine kidney cells. Table 4 presents a summary of the nucleotide and amino acid variation between the two strains (see also PCT Publication No. WO 01 / 060847, which is incorporated herein by reference). Petition 870260062389, dated 06 / 25 / 2026, p. 46 / 135 42 / 56 total). Table 4. Differences in Nucleotide and Amino Acid Sequences between DENV-2 16681 and its Vaccine-Derived Strain PDK-53 Genome Position Nucleotide 16681 Nucleotide PDK-53 Amino Acid 16681 Amino Acid PDK-53 Protein Position Polyprotein Position a 57 CT - - 524 AT Asp Val prM-29 2055 CT Phe Phe E-373 653 2579 GA Gly Asp NS1-53 828 4018 CT Leu Phe NS2A-181 1308 5270 AT Glu Val NS3-250 1725 5547 TC Arg Arg NS3-342 1817 6599 GC Gly Ala NS4A-75 2168 8571 CT Val Val NS5-334 2825

[00116] It has been previously determined that the three main determinants of DENV-2 PDK-53 attenuation reside in the 5' non-coding region (NCR), NS1 protein, and NS3 protein (Butrapet et al., Virol 74: 3011 to 3019, 2000), which are shown in bold in Table 4. Chimeric viruses containing the prM-E gene region of heterologous flaviviruses, within the genetic main chain of DENV-2 PDK-53, expressed the heterologous virus-specific E immunogenes and maintain the attenuated phenotype of the DENV-2 PDK-53 vaccine virus (Huang et al., J Virol 74: 3020 to 3028, 2000; Huang et al., Virol 77: 11436 to 11447, 2003; Kinney et al., Intervirology 44: 176 to 197, 2001; Huang et al., J. Virol 79: 7300 to 7310, 2005). The chimeric D2 / D1, D2, D2 / D3, D2 / D4, and D2 / WN viruses based on DENV-2 PDK53 were previously generated for a tetravalent live attenuated DENV vaccine and a WNV vaccine (US Patent Nos. 7,094,411; 7,641,909, 8,025,887 and 8,673).316, which are incorporated herein by reference in their entirety). The tetravalent DENV vaccine containing chimeric D2 / D1, D2, D2 / D3 and D2 / D4 has been manufactured and characterized for human clinical trials (Huang et al., PLoS Negl Trop Dis 7. Petition 870260062389, dated 06 / 25 / 2026, p. 47 / 135 43 / 56 (5): e2243, 2014), and is currently in phase 3 human clinical trials (George et al., J. Infect Dis 212 (7): 1032 to 1041, 2015; Osorio et al., Lancet Infect Dis 14: 830 to 838, 2014).

[00117] The chimeric constructs D2 / ZKV-P and D2 / ZKV-V were based on a similar engineering strategy used to generate chimeric D2 / WNV (Strategy 1 in Figure 1). The chimeric D2 / ZKV-V, which is based on the DENV-2 PDK-53 vaccine backbone, is used for the development of live attenuated ZIKV vaccines. The chimeric D2 / ZKV-P, which is based on the DENV-2 16681 parental strain, is used as a chimeric parent virus for virulence and attenuation comparisons with the chimeric D2 / ZKV-V vaccine, as well as for the development of ZIKV vaccines.

[00118] Viable chimeric D2 / ZKV-P virus was recovered from C6 / 36 cells transfected with chimeric viral RNA that was transcribed in vitro from modified chimeric cDNA. Because the DENV-2 PDK-53 vaccine virus does not replicate well in C6 / 36 cells (which is one of its characteristic attenuating phenotypes), initial efforts to recover chimeric D2 / ZKV-V from C6 / 36 cells did not produce detectable infectious virus. Furthermore, first-generation of the D2 / ZK-P and D2 / ZK-V constructs did not generate viable chimeric viruses from transfected Vero cells. The D2 / ZK-P seed virus recovered from C6 / 36 cells also did not efficiently infect Vero cells, suggesting mismatched chimeric genes of the virus for Vero cell infection. Since Vero cells are essential for the manufacture of live attenuated vaccine viruses, it is necessary to design a chimeric virus that replicates well and is stable in Vero cells.D2 / ZK-P obtained from C6 / 36 cells was adapted to grow in Vero cells by serial passage of the virus in Vero cells to a high multiplicity of infection (MOI).

[00119] After just one passage in Vero cells with high MOI, Petition 870260062389, dated 06 / 25 / 2026, page 48 / 135 44 / 56 The D2 / ZK-P virus successfully adapted to Vero cells was recovered and sequenced to identify the genetic mutations involved in Vero cell adaptation. Additional passages in Vero cells of the Vero-adapted D2 / ZK-P virus resulted in significantly higher titers of D2 / ZK-P virus seeds, which were also sequenced to identify further mutations that may improve the chimera's fitness in Vero cells. The necessary adapted mutations are incorporated into the D2 / ZK-V vaccine constructs. In previous studies, several chimeric viruses were modified with mutations for Vero cell adaptation to enhance and / or stabilize the chimeric viruses for Vero cell culture, and such modifications resulted in successful attenuated live dengue chimeric viruses and D2 / WN chimeric vaccine candidates using the same main chain as the DENV-2 PDK-53 vaccine. Chimeric D2 viruses / Zika

[00120] Below is a list of chimeric D2 / ZK viruses for generating live attenuated ZIKV vaccine candidates:

[00121] D2 / ZK-P (also called D2 / ZK-PS): This chimeric virus includes the parental (P) main strand of D2 16681 and the prM-E genes of the SPZ2015 (S) strain of Zika virus. The virus was constructed using strategy 1 illustrated in Figure 1. Viable virus was recovered from transfected C6 / 36 cells, but not from Vero cells, and could not be titrated on plates in Vero cells. Evidence of virus recovery was based on >90% of C6 / 36 cells IFA positive by 4G2, and a strong RT-PCR band from nucleic acid amplified from C6 / 36 culture fluid.

[00122] D2 / ZK-P Vero: A Vero cell-adapted version of D2 / ZK-P. To develop a virus adapted to Vero cells, Vero cells were infected with a high MOI of the D2 / ZK-P virus recovered from C6 / 36 cells. The first adaptation cycle resulted in Petition 870260062389, dated 06 / 25 / 2026, p. 49 / 135 45 / 56 7x10⁶ PFU / ml of chimeric virus replicated from Vero cells. The adapted virus was titrated on plates in Vero cells and exhibited clear plaques of varying sizes. Virus sequencing identified mutations in 7 amino acids (Table 5). Further passages in Vero cells are expected to increase the chimeric virus's adaptation to seed preparation. Plate purification of larger plaques of the chimera from infected Vero cells is also conducted to further determine the most useful mutation for Vero cell adaptation. Four of the identified mutations are manipulated in the chimeric constructs to improve D2 / ZKV growth in Vero cells (Table 3), and further confirmed as critical mutations for chimeric D2 / ZKV adaptation and stability in Vero cells (see Example 2). The other 3 mutations in E-191, NS4B24, and NS4B-245 proved to be unnecessary for adaptation or stability in Vero cells. Table 5. Mutations identified in viruses adapted to Vero cells. D2 / ZK-P NT position in chimeric DENV-2 / ZKV AA position of the AA-based substitution protein (WT-Mut) chimeric DENV-2 / ZKV DENV-2 AA position ZIKV AA position 1610 (A to T) E-191 NA E-191 His-Leu 2347-2348 (CA to AG) E-465 NA E-465 Gln-Arg 2405 (T to C) E-484 NA E-484 Ile-Thr 2431 (A to T) E-493 E-484 NA Ile-Phe 3819 (G to T) NS2A-99 NS2A-99 NA Lys-Asn 6941 (G to A) NS4B-24 NS4B-24 NA Glu-Gly 7603 (A to T) NS4B-245 NS4B-245 NA Asn-Leu

[00123] D2 / ZK-V (also called D2 / ZK-VS): This chimeric virus includes the vaccine (V) PDK-53 main chain and the prMPetição genes 870260062389, 25 / 06 / 2026, p. 50 / 135 46 / 56 And from the SPZ2015(S) strain of Zika virus. The nucleotide and amino acid sequences of this chimeric virus are presented here as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. No virus was recovered from C6 / 36 cells or Vero cells. The main chain of the vaccine strain is attenuated in C6 / 36 cells, therefore virus recovery was not expected. The chimera was also not viable in Vero cells (similar to D2 / ZK-P described above). The Vero cell-adapted mutations identified from Vero cell-adapted D2 / ZK-P virus are incorporated into the construct to make D2 / ZK-V viable as a candidate live attenuated virus vaccine.

[00124] D2 / ZK-P2A: This chimeric virus includes the parental main chain (P) of D2 16681, but with a mutation resulting in a methionine to valine substitution at residue 22 of the NS2A protein, and includes the prM-E gene from the Zika virus strain SPH2015. The M22V mutation was identified as a strong mutation from Vero cells to the earlier D2 / WNV chimera. Vero cells were transfected with D2 / ZK-P2A to assess virus growth. Transfected Vero cells were cultured at 37°C or 28°C to assess the temperature sensitivity of the chimeric construct. The results showed that the NS2A-22 mutation did not significantly improve virus stability in Vero cells.

[00125] D2 / ZK-V2A: This chimeric virus includes the vaccine (V) D2 PDK-53 main chain, but with a mutation resulting in a methionine to valine substitution at residue 22 of the NS2A protein and includes the prM-E gene from the Zika virus strain SPH2015. The nucleotide and amino acid sequences of this chimeric virus are shown here as SEQ ID NO: 5 and SEQ ID NO: 6, respectively. Vero cells were transfected with D2 / ZK-V2A to assess virus growth. Transfected Vero cells were cultured at 37°C or 28°C to Petition 870260062389, dated 06 / 25 / 2026, p. 51 / 135 47 / 56 evaluate the temperature sensitivity of the chimeric virus. On day 11 post-transfection, RT-PCR analysis of Vero cell culture at 28°C showed a strong positive result, but Vero cell culture at 37°C was negative. This result suggested that the NS2A-22 mutation alone is not sufficient for efficient adaptation of the chimeric virus to Vero cells.

[00126] D2 / ZK-5V: This chimeric virus is generated using the prM / E 2-joint strategy shown in Figure 1. The nucleotide and amino acid sequences of this chimeric virus are presented here as SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Differences in Zika prM-E genes in chimeric D2 / ZKV variants

[00127] The chimeric D2 / ZKV constructs were made using the ZIKV strain sequence SPH2015 obtained from GenBank (Accession No. KU321639.1), prior to the PRVABC59 and R103451 strains being isolated from travelers who acquired ZIKV infection during the 2015 outbreak at the CDC diagnostic laboratory. The R103451 and PRVABC59 strains are now available as wild-type (wt) ZIKV controls, but the SPH2015 strain is not. There is only a 1 amino acid (AA) sequence difference between SPH2015 and PRVABC59 (or R103451) within the prM-E gene region included in the chimeric D2 / ZIKV constructs. The difference lies in the 23rd amino acid position of protein E (E-23), with isoleucine (Ile) in the SPH2015 strain and valine (Val) in the PRVABC59 and R103451 strains. For the prM-E nucleotide sequences, there is one (silent) nucleotide difference between the chimeras and the R103451 strain, and six silent differences between the chimeras and the PRVABC59 strain. Attenuation characterization and vaccine development

[00128] The D2 / ZKV-V vaccine candidate is evaluated for previously established vaccine attenuation phenotypes. Petition 870260062389, dated 06 / 25 / 2026, page 52 / 135 48 / 56 DENV-2 PDK-53-based candidates, which include small plaques in Vero or LLC-MK2 cells, temperature sensitivity in Vero or LLC-MK2 cells, poor growth in C6 / 36 mosquito cells, neuroattenuation in mice, and decreased spread / transmission of mosquito gut / salivary gland infection (Huang et al., J. Virol. 77: 11436-11447, 2003; Huang et al., J. Virol 12: 7300-7310, 2005; Huang et al., PLoS Negl Trop Dis 7(5): e2243, 2014). The candidate vaccines are also tested in a small animal model (mouse) and non-human primate for immunogenicity and protective efficacy. Vaccine Applications

[00129] Based on previous success in developing DENV and WNV vaccines using the DENV-2 PDK-53-based chimeric virus platform, it is expected that this platform can be successfully used for the development of a live attenuated ZIKV vaccine. A D2 / ZKV chimeric can be used alone (univalent) or in combination with a tetravalent live attenuated DENV vaccine based on the same PDK-53 main chain (pentavalent vaccine). Combined vaccination strategies using both live attenuated and inactivated ZIKV candidate vaccines can be evaluated. Additional Chimeric Constructs

[00130] Additional chimeric D2 / ZKV cells are generated using alternative junction site strategies, incorporating adaptation mutations into Vero cells and / or introducing additional mutations to adjust the attenuation level or improve the genetic fitness / stability of the chimeric D2 / ZKV-V.

[00131] In one example, a chimeric D2 / ZKV is generated using Strategy 2 shown in Figure 1. In Strategy 2, the prM signal sequence includes the first five amino acids of DENV-2 PDK-53 and the Petition 870260062389, dated 06 / 25 / 2026, page 53 / 135 49 / 56 last 13 amino acids of a ZIKV (such as SPH2015).

[00132] In other examples, a chimeric D2 / ZIKV is generated by incorporating one or more Vero cell-adapting mutations identified from serial passages in Vero cells from D2 / ZK-P seeds. In one example, any of the mutations listed in Table 3 is incorporated into the D2 / ZK vaccine virus to improve adaptation to Vero cells. In other examples, any combinations of the mutations listed in Table 3 are incorporated into the D2 / ZKV vaccine to improve growth in Vero cells.

[00133] In another example, the chimeric D2 / ZKV includes an Ile-Val substitution at E23 to match the sequence of the ZIKV strains PRVABC59 and R103451. Example 2: Chimeric D2 / ZK viruses for vaccine development

[00134] This example describes the generation and characterization of 10 additional chimeric constructs of DENV-2 / ZKV (D2 / ZK) named D2 / ZKV-V2, P3.1, V3.1, V3.2, V3, P4, V4, P5, and V5 (see Table 6).

[00135] Based on the mutations identified from D2 / ZKVP adapted to Vero cells described in Example 1 (Table 5), some of the mutations were incorporated into the chimeric construct to determine mutations important for adaptation and stability to Vero cells. Among the 7 mutations, 4 of them (E-465, E-484, E-493, and NS22A-99) were identified for improving the fitness to Vero cells of the chimeric D2 / ZK viruses (Table 3). Interestingly, all three E mutations were within the transmembrane domain of the E protein, which would not affect the antigenic property of the E protein. Furthermore, two of them (E-484 and E-493) were close to the chimeric E / NS1 junction site, suggesting that the mutations could compensate for the defect caused by chimerization between the E proteins of ZIKV and DENV-2 (Figure 1 shows an example with E-484T and E-493F). Petition 870260062389, dated 06 / 25 / 2026, page 54 / 135 50 / 56 in D2 / ZKV). Table 3 also includes the NS4A-23 mutation that was identified during the study of the D2 / ZKV-V2 construct. Without any mutations adapted to Vero cells, both D2 / ZK-P and D2 / ZK-V are unable to replicate in Vero cells. It is possible to recover viable D2 / ZK-P viruses (in the D2 16681 main chain) from C6 / 36 cells, but not D2 / ZK-V viruses (in the D2 PDK-53 main chain).

[00136] Ten additional chimeric viruses that are viable in Vero cells were generated. However, each chimeric virus has a different plaque phenotype, growth efficiency, and genetic stability. A brief summary of each chimeric virus is provided below:

[00137] D2 / ZKV-V2: This chimeric virus construct contains E-465R and E-484T mutations, but is not yet stable in Vero cells; after 2 passages in Vero cells, it acquired multiple mutations. After purification in plates of five clonal V2 viruses, it was determined that three of them had acquired an NS4A-23N mutation, including one that also acquired an NS2A-99N mutation, and another clone acquired the E-493F mutation. Based on these results, the NS4A-23 mutations were included in subsequent constructs. The NS2A-99 and E493F mutations were previously identified in the original D2 / ZKV-P adapted to Vero cells, suggesting that these two mutations play important roles in adaptation to Vero cells.

[00138] D2 / ZKV-P3.1 and V3.1: Both of these chimeric viruses were engineered with 3 of the 5 mutations described in Table 3 above – E493, NS2A-99, and NS4A-23. Both viruses replicated well and produced clear, uniform plaques on Vero cells; however, after further sequencing analysis, it was determined that both recovered viruses had acquired the E-465R mutation. Although P3.1 and V3.1 viruses were not sufficiently stable for vaccine development, based on plaque phenotypes and consistency of Petition 870260062389, dated 06 / 25 / 2026, page 55 / 135 Given the 51 / 56 E-465 mutation in both chimeras, it was assumed that chimeras containing the E-465R, E-493F NS2A-99N, and NS4A-23N (RFNN) mutations would be stable for Vero cell amplification and would be potential live attenuated ZIKV vaccine candidates. The nucleotide and amino acid sequences of the PRFNN and V-RFNN chimeric viruses are presented here as SEQ ID NOs: 15-18.

[00139] D2 / ZKV-V3.2: This chimeric virus, which has the E-465, E-484, and NS4A-23 mutations, was not stable enough for growth in Vero cells. The virus grew weakly in Vero cells and produced diffuse pinpoint-sized plaques.

[00140] D2 / ZKV-V3: This virus contains an additional NS2A-99 mutation relative to the V3 virus. 2, but it is not yet stable enough for growth in Vero cells. After 2-3 passages in Vero cells, the chimera acquired an additional E-493 F mutation (as with V2 described above).

[00141] D2 / ZKV-P4 and V4: These chimeric viruses were constructed with E-465, E-484, E-493 and NS2A-99 mutations. They grew well and produced uniform plaques and are therefore candidates for the development of live attenuated virus vaccines. The nucleotide and amino acid sequences of the P4 and V4 chimeric viruses are presented here as SEQ ID NOs: 19-22.

[00142] D2 / ZKV-P5 and V5: These two chimeras, with either the 16681 (P5) or PDK-53 (V5) main strand, grew very well in Vero cells and produced plaques that were larger than all other constructs described above. Growth kinetics studies of these two viruses were performed in Vero cells, which confirmed that both viruses replicate efficiently in Vero cells. The V5 virus replicated slightly more slowly than the P5 virus, which is consistent with the slower growth of the D2 PDK-53 main strand virus than the D2 16681 virus. The plaque size of P5 and V5 was smaller than Petition 870260062389, dated 06 / 25 / 2026, page 56 / 135 52 / 56 of ZIKV, suggesting that both are replicated less efficiently than ZIKV in Vero cells. However, V5 virus plaques were slightly smaller than P5 virus plaques, which agrees with the previous observation that the PDK-53-based chimeric virus produces smaller plaques than chimeric viruses based on the D2 16681 main chain. V5 virus also maintained the attenuation phenotype as its D2 PDK-53 main chain in C6 / 36 cells. Furthermore, a mouse study was conducted to measure the neurovirulence levels of D2 / ZK-P5 and -V5 viruses (see below). The results indicated that both P5 and V5 are fully attenuated in newborn mice. Based on these results, these two viruses are good candidates for the development of live attenuated virus vaccines. The nucleotide and amino acid sequences of the chimeric viruses P5 and V5 are presented here as SEQ ID Nos: 23-26. Table 6. DEN-2 / Zika Chimeric Virus Virus names NT and AA position based on chimeric D2 / ZIKV genome Growth in Vero cells (D2 / ZIKV main strand)* Nt 2347-2348 (CA-AG) 2405 (TC) 2431 (AT) 3819 (GT) 6487 (GA) 16681 PDK-53 AA** E465 (QR) E484 (IT) E493 (IF) NS2A-99 (KN) NS4A-23 (DN) PV wt* QIIKD Not viable in Vero V2 P3.1 V3.1 RQTIIFKNDN Not stable, acquired multiple mutations Not stable Petition 870260062389, dated 06 / 25 / 2026, p. 57 / 135 53 / 56 Virus Names NT and AA Position Based on Chimeric D2 / ZIKV Genome Growth in Vero Cells (D2 / ZIKV main strand Nt 2347- 2405 2431 3819 6487)* 2348 (CA-AG) (TC) (AT) (GT) (GA) 16681 PDK-53 AA** E465 E484 E493 NS2A- NS4A- 99 23 (QR) (IT) (IF) (KN) (DN) acquired E465R P-RFNNV-RFNN RIFNN Evolved from 3.1 virus; potential candidates - V3. 2 RTIKN Weak growth, point plaques P3 V3 RTINN Not stable, evolved into P5 or V5 type virus P4 V4 RTFND Uniform plaques; potential candidates P5 V5 RTFNN Uniform plaques; potential candidates The underlined text indicates potential live attenuated ZIKB vaccine candidates * Chimeric viruses based on the parent virus D2 16681 called D2 / ZIKV-P#; Chimeras based on the vaccine virus D2 PDK-53 called D2 / ZIKV-V# ** Mutations in bold Petition 870260062389, dated 06 / 25 / 2026, p. 58 / 135 54 / 56 Attenuation Characterization

[00143] Vaccine candidate viruses have been characterized for previously established DENV-2 PDK-53 attenuation phenotypes. These include small plaques in Vero or LLC-MK2 cells, temperature sensitivity in Vero or LLC-MK2 cells, impaired growth in C6 / 36 mosquito cells, neuroattenuation in mice, and decreased transmission / spread of infection in the midgut / salivary gland of mosquitoes.

[00144] Plaque size in Vero cells: The four chimeras V4, V5, P4, and P5 exhibited smaller plaques than ZIKV. The plaques of the chimeras in the P main chain were slightly larger than the plaques produced from their V chimera counterparts. Furthermore, the plaques of P5 and V5 were larger than the P4 and V4 versions. Vero-adapted P-FRNN and V-RFNN (developed from P3.1 and V3.1) also showed similarly small plaques as the P4 and V4 viruses.

[00145] Growth kinetics in Vero cells: Viral growth of P5 and V5 viruses was tested in Vero cells. As shown in Figure 2A, both viruses replicated efficiently in Vero cells, but V5 was slightly slower than P5, and both were slower than ZIKV.

[00146] Growth kinetics in C6 / 36 cells: The growth kinetics of P5 and V5 viruses were evaluated in C6 / 36 cells. The results are shown in Figure 2B. Although P5 still grew efficiently in C6 / 36 cells, growth was significantly lower than wild-type ZIKV PRVABC59, and slightly lower than DENV-2 16681 main-chain virus. V5 virus maintained the attenuation phenotype like its DENV-2 PDK-53 vaccine main-chain virus in C6 / 36 cells. It is believed that most V viruses will maintain the attenuation phenotype from replication. Petition 870260062389, dated 06 / 25 / 2026, page 59 / 135 55 / 56 deficient in its DENV-2 PDK-53 vaccine, while most P viruses can still replicate well in C6 / 36 cells. Although P viruses may not have the in vitro attenuation phenotype in C6 / 36 cells, they can still be attenuated in live whole mosquitoes due to chimerization between DENV-2 and ZIKV.

[00147] Neurovirulence in mice: P5 and V5 viruses were compared with ZIKA and D2 16681 viruses in newborn ICR mice. An initial test in 7-day-old ICR (CD-1) mice showed that newborn mice at 7 days of age had already developed resistance to intracranial provocation with ZIKV and D2 16681. A neurovirulence provocation mouse model for D2 16681 virus using 1-day-old mice was previously established. Therefore, in the second experiment, newborn ICR mice aged 0-2 days were used. Litters of newborn mice (day 0-2 after birth) were grouped into 9-10 newborns / litter inoculated intracranially with 104 pfu of virus.All 29 baby mice (3 litters) inoculated with wild-type ZIKV died with a median survival time (ASD ± SD) of 8.4 ± 0.49 days, and all 20 mice (2 litters) inoculated with D2 16681 also died with an ASD of 9.05 ± 0.59 (Figure 3). Surprisingly, none of the 30 mice inoculated with P5 or V5 virus became ill, and there was no weight loss during the experiment. Immunogenicity and protective efficacy of candidate vaccines:

[00148] Studies are conducted to evaluate the immunogenicity and protective efficacy of candidate vaccines. Mice are administered single-dose or double-dose vaccines and are provoked with a lethal dose of wild-type ZIKV PRVABC59 one month after final immunization. Chimeric viruses are expected to induce strong Petition 870260062389, dated 06 / 25 / 2026, page 60 / 135 56 / 56 anti-ZIKV neutralizing antibodies protect mice from lethal provocation.

[00149] In view of the many possible embodiments to which the principles of the invention described may be applied, it should be recognized that the embodiments illustrated are only preferred examples of the invention and should not be considered as limiting the scope of the invention. On the contrary, the scope of the invention is defined by the following claims. Therefore, everything that is within the scope and spirit of these claims is claimed as such invention. Petition 870260062389, dated 06 / 25 / 2026, p. 61 / 135

Claims

1 / 5 CLAIMS 1. A nucleic acid chimera, characterized in that it comprises: a first nucleic acid molecule comprising a 5' non-coding region, a nucleic acid encoding non-structural proteins and a capsid protein (C) and a 3' non-coding region, each from a dengue virus serotype 2 (DENV-2) strain genome, wherein the C protein comprises a portion of a pre-membrane signal sequence (prM) from the DENV-2 genome and a portion of a prM signal sequence from a Zika virus (ZIKV) genome; and a second nucleic acid molecule operationally linked to the first nucleic acid molecule, encoding a prM protein and at least a portion of an envelope protein (E) from the ZIKV genome, wherein it further comprises: (i) four Vero cell adaptation mutations, wherein the mutations result in: a glutamine substitution for arginine at residue 465 of the E protein;(i) a substitution of isoleucine for threonine at residue 484 of protein E; a substitution of isoleucine for phenylalanine at residue 493 of protein E; and a substitution of lysine for asparagine at residue 99 of protein NS2A; or (ii) a substitution of glutamine for arginine at residue 465 of protein E; a substitution of isoleucine for threonine at residue 484 of protein E; a substitution of isoleucine for phenylalanine at residue 493 of protein E; a substitution of lysine for asparagine at residue 99 of protein NS2A; and a substitution of aspartic acid for asparagine at residue 23 of protein NS4A.

2. Nucleic acid chimera, according to claim 1, characterized in that the genome of the DENV-2 strain is an attenuated DENV-2 strain genome.

3. Nucleic acid chimera, according to claim 2, characterized in that the attenuated DENV-2 genome comprises a mutation in the 5' non-coding region at nucleotide position 57.

4. Nucleic acid chimera, according to claim 2 or 3, characterized in that the attenuated DENV-2 genome comprises a mutation at nucleotide position 2579 resulting in the presence of an aspartate at amino acid residue 53 of the NS1 protein.

5. Nucleic acid chimera, according to any one of claims 2 to 4, characterized in that the attenuated DENV-2 genome comprises a mutation at nucleotide position 5270 that results in the presence of a valine at amino acid residue 250 of the NS3 protein.

6. Nucleic acid chimera, according to any one of claims 2 to 5, characterized in that the attenuated DENV-2 is the PDK-53 strain.

7. Nucleic acid chimera, according to claim 1, characterized in that the genome of the DENV-2 strain is a wild-type DENV-2 strain genome.

8. Nucleic acid chimera, according to claim 7, characterized in that the wild-type DENV-2 is strain 16681.

9. Nucleic acid chimera, according to any one of claims 1 to 8, characterized in that ZIKV is the strain SPH2015, PRVABC59 or R103451.

10. Nucleic acid chimera, according to any one of claims 1 to 9, characterized in that the prM signal sequence portion of the DENV-2 genome comprises the first three amino acids of the DENV-2 prM signal sequence and the prM signal sequence portion of the ZIKV genome comprises the last 15 amino acids of the ZIKV prM signal sequence.

11. Nucleic acid chimera, according to claim 10, characterized in that the first three amino acids of the DENV-2 prM signal sequence comprise SAG (amino acids 101-103 of SEQ ID NO: 12).

12. Nucleic acid chimera, according to claim 10 or 11, characterized in that the last 15 amino acids of the ZIKV prM signal sequence comprise amino acids 108-122 of SEQ ID NO:

8.

13. Nucleic acid chimera, according to any one of claims 1 to 9, characterized in that the prM signal sequence portion of the DENV-2 genome comprises the first five amino acids of the DENV-2 prM signal sequence and the prM signal sequence portion of the ZIKV genome comprises the last 13 amino acids of the ZIKV prM signal sequence.

14. Nucleic acid chimera, according to claim 13, characterized in that the first five amino acids of the DENV-2 prM signal sequence comprise amino acids 101-105 of SEQ ID NO:

12.

15. Nucleic acid chimera, according to claim 13 or 14, characterized in that the last 13 amino acids of the ZIKV prM signal sequence comprise amino acids 110-122 of SEQ ID NO:

8.

16. Nucleic acid chimera, according to any one of claims 1 to 15, characterized in that a portion of the E protein is from the DENV-2 genome.

17. Nucleic acid chimera, according to claim 16, characterized in that the E protein portion of the DENV-2 genome comprises the last 14 amino acids of the DENV-2 E protein.

18. Nucleic acid chimera, according to claim 17, characterized in that the last 14 amino acids of the DENV-2 E protein comprise amino acids 777-790 of SEQ ID NO:

2.

19. Nucleic acid chimera, according to any one of claims 1 to 18, characterized in that it comprises the nucleic acid sequence SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO:

25.

20. Nucleic acid chimera, according to any one of claims 1 to 19, characterized in that the nucleic acid chimera encodes the amino acid sequence SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24 or SEQ ID NO:

26.

21. Chimeric Flavivirus, characterized in that it comprises the nucleic acid chimera as defined in any one of claims 1 to 20.

22. Immunogenic composition, characterized in that it comprises the chimeric flavivirus as defined in claim 21, and a pharmaceutically acceptable carrier.

23. Immunogenic composition, according to claim 22, characterized in that it further comprises one or more adjuvants.

24. Use of chimeric flavivirus, as defined in claim 21, characterized in that it is for the manufacture of a composition to produce an immune response against Zika Virus (ZIKV) in an individual.

25. Use, according to claim 24, characterized in that it comprises administering to the individual one to five doses of the chimeric flavivirus or immunogenic composition. Petition 870260062389, dated 06 / 25 / 2026, page 65 / 135 5 / 5 26. Use according to claim 24 or 25, characterized in that it further comprises administering one or more adjuvants to the individual.

27. Use, according to any one of claims 24 to 26, characterized in that it further comprises administering an inactivated Zika virus vaccine.

28. Use, according to any of claims 24 to 27, characterized by the fact that the individual is a human being. Petition 870260062389, dated 06 / 25 / 2026, p. 66 / 135

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