A new vaccine against the Zika virus

Abstract

An aspect of the present invention relates to nucleic acid structures capable of expressing a Zika antigen that elicits an immune response against the Zika virus in mammals, and methods for using the same. Additionally, a DNA plasmid vaccine capable of eliciting an immune response against the Zika virus in mammals is presented, comprising a DNA plasmid and a pharmaceutically acceptable excipient, and methods for using the same. The DNA plasmid can express the Zika antigen in mammalian cells in an amount effective for eliciting a cross-reactive immune response against all Zika strains in mammals.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] The present application claims priority to US provisional application No. 62 / 300,030 filed February 25, 2016, US provisional application No. 62 / 305,183 filed March 8, 2016, US provisional application No. 62 / 396,742 filed September 19, 2016, US provisional application No. 62 / 417,100 filed November 3, 2016, and US provisional application No. 62 / 462,249 filed February 22, 2017, each incorporated herein by reference in their entirety.

[0003] Field of invention

[0004] The present invention relates to a Zika vaccine, an improved method for inducing an immune response, and an improved method for prophylactically and / or therapeutically immunizing an individual against the Zika virus. Background Technology

[0005] background

[0006] The Zika virus (ZIKAV) is a small, enveloped, positive-stranded RNA virus belonging to the genus Flavivirus of the family Flaviviridae. The virus is known to be transmitted by diurnal Aedes mosquitoes, such as *Aedes aegypti* and *Aedes albopictus*. Its name originates from the Zika forests of Uganda, where the virus was first isolated in 1947.

[0007] The infection known as Zika fever, similar to a mild form of dengue fever, often causes no symptoms or only mild symptoms. Since the 1950s, it has been known to occur in a narrow equatorial belt from Africa to Asia. Between 2013 and 2014, the virus spread across the Pacific to French Polynesia, New Caledonia, the Cook Islands, and Easter Island, and in 2015, it spread eastward to Mexico, Central America, the Caribbean, and South America, where Zika outbreaks reached pandemic levels. Since 2016, the disease cannot be prevented by drugs or vaccines. Since February 2016, it has been evidenced that Zika fever in pregnant women can cause abnormal brain development in their fetuses through maternal genetic transmission, which may lead to miscarriage or microcephaly.

[0008] Due to the combination of the increasing global spread of the virus and the absence of any treatment or vaccine against it, the Zika virus is causing global health concern.

[0009] For this reason, there is still a need to develop an economical and effective vaccine that provides broad immunity against the Zika virus, and preferably cross-replicates all serotypes. Furthermore, there is still a need for effective methods to administer vaccines, such as DNA vaccines or DNA plasmid vaccines, to mammals to provide prophylactic or therapeutic immunization against the Zika virus. The problem to be solved

[0010] Summary of the Invention

[0011] One aspect of the present invention provides a nucleic acid construct capable of expressing a polypeptide that elicits an immune response in mammals against the Zika virus. These nucleic acid constructs consist of an encoding nucleotide sequence and a promoter operably linked to said encoding nucleotide sequence. said encoding nucleotide sequence expresses a polypeptide, wherein said polypeptide comprises a common Zika antigen comprising a pre-membrane-envelope (prM+Env or prME). said promoter regulates the expression of said polypeptide in mammals.

[0012] Another aspect of the present invention provides a DNA plasmid vaccine capable of producing an immune response to the Zika virus in mammals. The DNA plasmid vaccine comprises a DNA plasmid capable of expressing a common Zika antigen in mammals and a pharmaceutically acceptable excipient. The DNA plasmid comprises a promoter operably linked to a coding sequence encoding the common Zika antigen. The common Zika antigen comprises a common prME.

[0013] Another aspect of the present invention provides a method for inducing an immune response against the Zika virus in mammals, the method comprising delivering a DNA plasmid vaccine to a mammalian tissue, wherein the DNA plasmid vaccine comprises a DNA plasmid capable of expressing a common antigen of the Zika virus in mammalian cells to induce an immune response in the mammal, and electroporating the cells of the tissue to allow the entry of the DNA plasmid into the cells. Brief explanation of the drawing

[0014] Brief explanation of the drawing Figure 1 illustrates examples of Zika virus particles, Zika RNA genomes, and their translated genes. Figure 2 illustrates a plasmid map for a Zika vaccine showing the locations of the insert (expression cassette) encoding the Zika antigen. Figure 3 illustrates a diagram showing the linear structure of various Zika antigen designs. Figure 4 illustrates the annotated amino acid sequence for the Zika antigen - leader sequence + prME. Figures 5 and 6 illustrate the genetic relationships between various Zika virus strains: Figure 5 shows the genetic distance between isolates, and Figure 6 illustrates the genetic lineage. Figure 7 illustrates a plasmid map for the Zika vaccine showing the location of the insert (expression cassette) encoding Zika-prM+Env. Figure 8 illustrates a gel electrophoresis image showing the presence of an expression cassette. Figures 9a and 9b illustrate Western blot gels showing Zika envelope proteins: Figure 9a shows non-specific binding to antiserum in cell lysates; Figure 9b shows specific binding to anti-pan-flavivirus in cell lysates. Figure 10a illustrates an SDS-PAGE gel showing the purification of Zika-coat protein. Figure 10b illustrates a Western blot gel showing the purification of Zika-coat protein. Figures 11 and 12 illustrate bar graphs showing spike-specific CD8 T-lymphocyte responses assessed by the IFN-gamma ELISpot assay against a peptide pool covering the pre-M+ envelope antigen. Figure 11. Individual mouse mean. Figure 12. Group mean. Mean response in each group 1 week after third immunization. Figures 13a and 13b illustrate graphs representing the binding ELISA of samples, showing that Zika prM+Env vaccination of mice elicits a positive antibody response reacting with Zika-skin antigen. Figures 14a and 14b illustrate graphs showing that the ZV-prME immunogen elicits a significant antibody response specifically reacting with the Zika-env antigen. The cross-reactivity of ZpME serum to Dengue 1, 2, 3, and 4 antigens Env was negative, whereas its cross-reactivity to Zika Env showed strong binding. Figures 15a–15e illustrate analyses indicating that ZV-prME vaccine-induced serum does not cross-react with dengue 1-4 recombinant Env. The analyses also support that anti-CHIKV vaccine-induced serum does not bind to Zika Env. Figure 16, including Figures 16a through 16e, depicts experimental results showing the construction of the ZV-prME common DNA vaccine. Figure 16a depicts a phylogenetic tree at the amino acid level of the ZIKV envelope sequence between ZIKV isolates and envelope strains. A common design strategy was adopted for the ZIKV-prME common sequence. The ruler indicates the amino acid distance between regions. The analysis was performed using MEGA version 5 software. Red stars indicate Zika-prME common. Figure 16b depicts a schematic representation of the ZIKV-prME DNA vaccine directing the cloning of prME (prM+Env) into the pVax1 mammalian expression vector, pGX0001. The codon-optimized synthetic gene of the prME construct contained an IgE leader sequence. The entire gene construct was inserted into the BamH1 and Xho1 regions of the pVax1 vector under the control of the CMV promoter. Figure 16c depicts the agarose gel electrophoresis analysis of the ZIKV-prME DNA vaccine. Lane 1 shows the uncleaved vaccine construct; Lane 2, restriction cleavage of the plasmid with BamH1 / Xho1; Lane 3, DNA molecular size marker (kDa). Figure 16d depicts the expression analysis of ZIKV-prME protein expression in 293T cells by SDS-PAGE using Western blot evaluation and IFA detection. 293T cells were transfected with the ZIKV-prME plasmid, and cell lysates and supernatants were analyzed for expression. Lane 1 contains the protein molecular weight marker (kDa); Lane 2, pVax1 control cell lysate; Lane 3, cell lysate from ZIKV-prME transfected cells; Lane 4, supernatant from ZIKV-prME transfected cells; Lane 5, recombinant prME positive control. Figure 16e depicts the immunofluorescence assay (IFA) assay for ZIKV-prME protein expression in 293T cells. 293T cells were transfected with 5 μg of ZIKV-prME plasmid.Immunofluorescence labeling was performed 24 hours after transfection with serum (1:100) from immunized mice and anti-mouse IgG FITC. Staining with serum from mice immunized with ZIKV-prME and pVax1 is shown. Figure 17, including Figures 17a through 17c, depicts experimental results demonstrating the characterization of cellular immune responses in mice following inoculation with the ZIKV-prME DNA vaccine. Figure 17a depicts the ELISpot assay measuring IFN-γ secretion in splenocytes. C57 / BL6 mice (n = 5 / group) were intramuscularly immunized three times with 25 μg of either pVax1 or ZIKV-prME DNA vaccine, followed by in vivo EP. IFN-γ production was measured by the IFN-γ ELISpot assay as an indicator of the induction of a cellular immune response. Splenocytes harvested 7 days after the third immunization were cultured in the presence of one of six peptide pools covering the entire prM and envelope proteins. The results are plotted in a stacked bar graph. The data represent the average number of SFU (Spot-forming Units) per million splenocytes as values ​​representing the mean response ± SEM for each group (n = 4). Figure 17b depicts the epitope composition of the ZIKV-prME-specific IFN-γ response as determined by stimulation with the matrix peptide pool one week after the third immunization. Values ​​represent the mean response ± SEM for each group (n = 4). Experiments were performed independently at least three times, and the results were similar. Figure 17c illustrates that immunization with ZIKV-prME induces a higher number of IFN-γ and TNF-α secreting cells when stimulated by the ZIKV peptide. One week after the final immunization with the ZIKV-prME vaccine, splenocytes were cultured in the presence of collected ZIKV peptide (5 μM) or tissue culture medium alone. The frequency of ZIKV peptide-specific IFN-γ and TNF-α secreting cells was measured by fluorescence-activated cell sorting (FACS). A single-function gate was set based on a negative control (unstimulated) sample and placed consistently across the entire sample. The percentage of total CD8+ T cell responses is plotted.These data represent two independent immunization experiments. Figure 18, including Figures 18a through 18d, depicts the profiles of IFN-γ production and antibody levels by splenocytes in serum collected from pZIKV-prME (MR766) and pZIKV-prME (Brazil)-immunized mice. Six-week-old C57 / BL6 mice were immunized as described in Materials and Methods. Serum and splenocytes were collected one week after the third immunization and cultured with ZIKV-specific prME peptides, and the number of IFN-γ SFUs per million cells was determined by ELISPOTS. Figure 18a depicts the ELISPOTS analysis of serum collected from MR766-immunized mice. Figure 18b depicts the ELISPOTS analysis of serum collected from Brazil-immunized mice. Anti-ZIKV Env antibody levels in serum were measured by ELISA (C&D). Figure 18c depicts the levels of anti-ZIKV Env antibodies in serum measured by ELISA in MR766-immunized mice. Figure 18d depicts the levels of anti-ZIKV Env antibodies in serum measured by ELISA in Brazil-immunized mice. Figure 19, including Figures 19a through 19e, depicts experimental results demonstrating that an anti-ZIKV antibody response is induced by ZIKV-prME plasmid inoculation. C57BL / 6 mice were intramuscularly immunized three times at 2-week intervals with 25 μg of ZIKV-prME plasmid or pVax1. Binding to the envelope antigen was analyzed in serum from animals at different time points after immunization at various dilutions. ELISA plates were coated with vaccine-conformed recombinant ZIKV-envelope protein. Figure 19a depicts that results are presented from one of two independent experiments. Similar results were obtained in the second experiment. Figure 19b depicts that differences in anti-ZIKV endpoint titers produced in response to the ZIKV-prME immunogen were analyzed in serum from immunized animals after each additional inoculation. Figure 19c depicts the Western blot analysis of ZIKV-env antigen expression. Recombinant ZIKV-Env proteins at various concentrations were electrophoresed on a 12.5% ​​SDS polyacrylamide gel and analyzed by Western blot analysis with serum from mice immunized with pVax1 or ZIKV-prME as indicated. Expression of ZIKV-Env proteins is indicated by arrowheads. Figure 20d depicts the immunofluorescence analysis of Vero cells infected with or mock-infected with ZIKV-MR766 after incubation with serum from mice immunized with ZIKV-prME or pVax1. Serum samples from mice immunized with pZIKV-prME were tested for their ability to neutralize ZIKV infectivity in vitro by the Plaque-Reduction Neutralization (PRNT) assay. PRNT50 was defined as a serum dilution factor capable of inhibiting 50% of the input virus. The value in parentheses indicates PRNT50. The control plasmid pZIKV-capsid and pVax1 serum were used as negative controls. Figure 20, including Figures 20a through 20e, depicts experimental results demonstrating the induction of a ZIKV-specific cellular immune response following ZIKV=prME DNA inoculation in NHPs. Figure 20a depicts Himalayan monkeys being intradermally (ID) immunized with 2 mg of ZIKV-prME plasmid administered as 1 mg at each of the two sites at weeks 0 and 4, followed immediately by intradermal EP. PBMCs were isolated prior to immunization and at week 6 and used in the ELISPOT assay to detect IFN-γ-secreting cells in response to stimulation with ZIKV-prME peptides. The number of IFN-γ-producing cells acquired per million PBMCs against six peptide pools covering the entire prME protein is indicated on the y-axis for the inoculated group. Values ​​represent the mean response ± SEM for each group (n = 5). Figure 20b depicts the detection of ZIKV-prME-specific antibody responses following DNA inoculation. Anti-ZIKV IgG antibodies were measured by ELISA before immunization and at week 6. Figure 20c depicts endpoint ELISA titers for anti-ZIKV envelope antibodies plotted after the first and second immunizations. Figure 20d depicts Western blot analysis using week 6 collected monkey serum demonstrating binding to the recombinant envelope protein. Figure 20e depicts immunofluorescence analysis of Vero cells infected with ZIKV MR766 at 10 PFU. Cells were probed 24 hours after infection with week 6 collected monkey serum at a 1:100 ratio and subsequently detected as secondary anti-human IgG-AF488. Figure 21, including Figures 21a through 21c, depicts experimental results demonstrating the plaque-reduction neutralizing activity of serum from Himalayan monkeys immunized with ZIKV-prME. Himalayan monkeys were immunized as described in Materials and Methods. Figure 21a depicts that pre-immunization and 6-week immunized serums from individual monkeys were tested by the Plaque Reduction Neutralization (PRNT) assay for their ability to neutralize ZIKV infectivity in vitro. PRNT50 was defined as the serum dilution factor capable of inhibiting 50% of the input virus. The calculated IC50 values ​​for each monkey are listed. Figures 21b and 21c depict the cytopathic effects of ZIKV MR766 and PR209 on Vero, SK-N-SH, and U87MG cells. Figure 21b depicts Vero cells that are mock-infected or infected with the MR766 or PR209 viruses. Figure 21c depicts SK-N-SH and U87MG cells being mock-infected or infected with MR766 at an MOI of 0.001 PFU / cell in the presence of collected NHP serum immunized with the ZIKV-prME vaccine (Week 6). Syncoplasmic reticulum formation (CPE) and the induction of prME protein expression were analyzed by indirect immunofluorescence assay (IFA) using immunized NHP serum at 48 hours post-infection. Photographs were taken at 4x magnification. Figure 22, including Figures 22a through 22c, depicts experimental results demonstrating the profile of IFN-γ and antibody production by splenocytes isolated from pZIKV prME in mice lacking type I interferon α, β receptors. Figure 22a depicts that IFN α, β receptor knockout mice (4 to 6) were intramuscularly immunized with 25 μg of pZIKV-prME or pVax1 plasmid three times at 2-week intervals. Splenocytes were collected 2 weeks after the last immunization and cultured with the prME peptide, and the number of IFN-γ-producing cells was counted by ELISA. Figure 22b depicts that serum antibodies specific to the ZIKV Env protein in the immunized animals were counted by ELISA at various dates after immunization. Figure 22c depicts endpoint titers at 0, 1, 2, 3, 4, and 5 weeks after immunization. Figure 23, including Figures 23a through 23f, depicts experimental results showing survival data for immunized mice lacking type I interferon α, β receptors after Zika virus infection. Survival of IFN-α / β receptor knockout mice after Zika infection. Figure 23a depicts mice being immunized once and then challenged with 106 PFU of ZIKV-PR209 two weeks later. Figure 23b depicts mice being immunized twice at a two-week interval and then challenged with 106 PFU of ZIKV-PR209 seven days after the second immunization. Figure 23c depicts mice being immunized twice at a two-week interval and then challenged with 2 x 106 PFU of ZIKV-PR209 seven days after the second immunization. Survival curves were constructed using data from two separate experiments. Figure 23d depicts changes in body weight for 2x immunized animals; the data reflect results from two independent experiments with 10 to 15 mice per group. Figure 23e depicts the clinical scores for the animals in Figure 23b. Figure 23f depicts the clinical scores for the animals in Figure 23c. The designations for the clinical scores are as follows: 1-no disease, 2-reduced mobility; 3-crouching posture and reduced mobility; 4-hind knuckle gait (partial paralysis), 5-paralysis of one hind leg, and 6-paralysis of both hind legs. Figure 24, including Figures 24a through 24e, depicts experimental results showing the construction of the ZIKV-prME common DNA vaccine. Figure 24a depicts a schematic representation of the ZIKV-prME DNA vaccine directing the cloning of rME into the pVax1 mammalian expression vector. A common design strategy for the ZIKV-prME common sequence was adopted. The codon-optimized synthetic gene of the prME construct contained a synthetic IgE leader sequence. The optimized gene construct was inserted into the BamH1 and Xho1 regions of the modified pVax1 vector under the control of the CMV promoter. Figure 24b depicts that the model construction of the ZIKV-E protein shows overlap with potentially relevant epitope regions of the vaccine target. Several modifications made for vaccine design purposes are located in domains II and III (located within the dashed line of the inset, middle left). Vaccine-specific residue changes in these regions are illustrated in purple CPK format in the depiction of the ribbon core of the E (envelope) protein dimer (each chain in light green and dark green, respectively). Regions corresponding to the defined EDE are indicated in cyan, and fusion loops are indicated in blue. Residue Ile156 (T156I) of the vaccine E protein, modeled as exposed on the surface of 150 loops, is part of the N-linked glycosylation motif NXS / T in multiple dengue virus lineages as well as several different ZIKV strains. Figure 24c depicts the expression analysis of ZIKV-prME protein expression by SDS-PAGE in 293T cells using Western blot analysis. 293T cells were transfected with the ZIKV-prME plasmid, and cell lysates and supernatants were analyzed for the expression of the vaccine construct in pan-flavivirus immunized serum. Protein molecular weight marker (kDa); Cell lysates and supernatants from ZIKV-prME transfected cells and rZIKV-E positive controls were loaded as indicated.Figure 24d depicts the expression analysis of ZIKV-prME protein expression in 293T cells by SDS-PAGE using Western blot analysis. 293T cells were transfected with the ZIKV-prME plasmid, and cell lysates and supernatants were analyzed for the expression of the vaccine construct in ZIKV-prME immunized serum. Protein molecular weight marker (kDa); cell lysates and supernatants from ZIKV-prME transfected cells and rZIKV-E positive controls were loaded as indicated. Figure 24e depicts the immunofluorescence assay (IFA) analysis for ZIKV-prME protein expression in 293T cells. These cells were transfected with 5 μg of the ZIKVprME plasmid. 24 hours after transfection, immunofluorescence labeling was performed with the addition of serum (1:100) from ZIKV-prME immunized mice, followed by the addition of a second anti-mouse IgG-AF488 antibody for detection. Staining with serum from ZIKV-prME and pVax1 immunized mice is shown. The DAPI panel shows the counterstaining of cell nuclei. The overlay panel is a combination of anti-mouse IgG-AF488 and DAPI staining patterns. DAPI, 4',6-diamidino-2-phenylindole; ZIKV-prME, Zika virus precursor membrane and envelope. Figure 25, including Figures 25a through 25d, depicts experimental results demonstrating the characterization of cellular immune responses in mice following inoculation with the ZIKV-prME DNA vaccine. Figure 25a depicts the timeline of vaccine immunization and immunoassay used in the study. Figure 25b depicts the ELISpot assay measuring IFN-γ secretion from splenocytes in response to ZIKV-prME immunization. C57BL / 6 mice (n=4 / group) were immunized three times with 25 μg of either pVax1 or ZIKV-prME DNA vaccine, followed by electroporation. IFN-γ production, as an indicator of the induction of a cellular immune response, was measured by the IFN-γ ELISpot assay. Splenocytes harvested one week after the third immunization were cultured in the presence of one of six peptide pools covering the entire prM and envelope proteins. The results are plotted in a stacked bar graph. The above data represent the average number of SFU (spot-forming units) per million splenocytes as values ​​representing the mean response ± SEM for each group. Figure 25c depicts the epitope composition of ZIKVprME-specific IFN-γ responses determined by stimulation with the matrix peptide pool one week after the third immunization. These values ​​represent the mean response ± SEM for each group. Experiments were performed independently at least three times, and the results were similar. Figure 25d depicts the flow cytometry analysis of T-cell responses. Immunization with ZIKV-prME induces higher numbers of IFN-γ and TNF-α secreting cells when stimulated by the ZIKV peptide. One week after the final immunization with the ZIKV-prME vaccine, splenocytes were cultured in the presence of the collected ZIKV peptide (5 μM) or R10 alone. The frequency of ZIKV peptide-specific IFN-γ and TNF-α secreting cells was measured by flow cytometry. Single-function gates were set up based on negative control (unstimulated) samples and consistently placed across the entire sample.All CD8. + The percentage of T-cell response is plotted. These data represent two independent immunization experiments. IFN, interferon; TNF, tumor necrosis factor; ZIKV-prME, Zika virus precursor membrane and envelope. Figure 26, including Figures 26a through 26e, depicts experimental results demonstrating that an anti-ZIKV antibody response is induced by ZIKV-prME inoculation. Figure 26a depicts an ELISA assay measuring binding antibody production in immunized mice (measured by the OD450 value). C57BL / 6 mice (n = 4) were immunized three times at 2-week intervals with 25 μg of ZIKV-prME plasmid or pVax1. Binding to rZIKV-E was analyzed in serum from animals at different time points (days 21, 35, and 50) after immunization at various dilutions. The data shown represent at least three distinct experiments. Figure 26b depicts the endpoint binding titer assay. Differences in anti-ZIKV endpoint titers produced in response to the ZIKV-prME immunogen were analyzed in serum from immunized animals after each additional inoculation. Figure 26c depicts the Western blot analysis of rZIKV-E specific antibodies induced by ZIKV-prME immunization. rZIKV-E proteins were electrophoresed on a 12.5% ​​SDS polyacrylamide gel and analyzed by Western blot analysis with mixed serum from ZIKV-prME immunized mice (Day 35). Binding to rZIKV-E is indicated by arrowheads. Figure 26d depicts the immunofluorescence analysis of ZIKV-specific antibodies induced by ZIKV-prME immunization. Vero cells infected with or mock-infected with ZIKV-MR766 were stained with mixed serum from ZIKV-prME immunized mice (Day 35), followed by the anti-mouse AF488 secondary antibody for detection. Figure 26e illustrates the plaque-reduction neutralization (PRNT) assay of neutralizing antibodies induced by ZIKV-prME immunization. Serum samples from ZIKV-prME immunized mice were tested for their ability to neutralize ZIKV infectivity in vitro. PRNT50 was defined as a serum dilution factor capable of inhibiting 50% of the input virus.The value in parentheses indicates PRNT50. Control ZIKV-Cap (DNA vaccine expressing ZIKV capsid protein) and pVax1 serotypes were used as negative controls. ZIKV-prME, Zika virus precursor membrane and envelope. Figure 27, including Figures 27a through 27e, depicts experimental results demonstrating the induction of a ZIKV-specific cellular immune response following ZIKV-prME inoculation in non-human primates (NHPs). Figure 27a depicts an ELISPOTT assay measuring IFN-γ secretion from peripheral blood mononuclear cells (PBMCs) in response to ZIKV-prME immunization. Himalayan monkeys were intradermally immunized with 2 mg of ZIKV-prME plasmid administered as 1 mg at each of the two sites at weeks 0 and 4, followed immediately by intradermal electroporation. PBMCs were isolated prior to immunization and at week 6 and used for an ELISPOTT assay to detect IFN-γ-secreting cells in response to stimulation with the ZIKV-prME peptide, as described in the 'Materials and Methods' section. The number of IFN-γ-producing cells obtained per million PBMCs against six peptide pools covering the entire prME protein is plotted. These values ​​represent the mean response ± SEM for each group (n=5). Figure 27b depicts the detection of ZIKV-prME-specific antibody responses after DNA inoculation. Anti-ZIKV IgG antibodies were measured by ELISA before immunization and at week 6. Figure 27c depicts the endpoint ELISA titers for anti-ZIKV envelope antibodies plotted after the first and second immunizations. Figure 27d depicts that Western blot analysis using RM immunized serum at week 6 demonstrated binding to the recombinant envelope protein. Figure 27e depicts the PRNT activity of serum from RM immunized with ZIKV-prME. Pre-immunization and 6-week immunization serums from individual monkeys were tested for their ability to neutralize ZIKV infectivity in vitro using the Plaque-Reduction Neutralization (PRNT) assay. PRNT50 was defined as a serum dilution factor capable of inhibiting 50% of the input virus. The calculated (PRNT50) values ​​for each monkey are listed.IFN, interferon; ZIKV-prME, Zika virus precursor membrane and envelope. Figure 28, including Figures 28a through 28f, depicts experimental results showing survival data for immunized mice lacking type I interferon α, β receptors after ZIKV infection. Figure 28a shows IFNAR after ZIKV infection. - / - Describes the survival of mice. Mice were immunized with 25 μg of ZIKV-prME DNA vaccine twice at a 2-week interval, and 1 x 10 6 One week after the second immunization with plaque-forming units, they were challenged with the ZIKV-PR209 virus. Figure 28b shows IFNAR after ZIKV infection. - / - Describes the survival of mice. Mice were immunized with 25 μg of ZIKV-prME DNA vaccine twice at a 2-week interval, and 2 x 10 6 Plaque-forming units were challenged with ZIKV-PR209 virus one week after the second immunization. Figure 28c shows 1 x 10 6 Depicts changes in body weight of animals immunized with plaque-forming units. Drawing 28d is 2 x 10 6 Depicts changes in body weight of animals immunized with plaque-forming units. Figure 28e is 1 x 10 6 Depicts the clinical score of animals immunized with plaque-forming units. Figure 28f is 2 x 10 6 Describes the clinical scores of animals immunized with plaque-forming units. The designations for clinical scores are as follows: 1: No disease, 2: Reduced mobility; 3: Crouching posture and reduced mobility; 4: Hindlimb knuckle gait (partial paralysis), 5: Paralysis of one hindlimb; and 6: Paralysis of both hindlimbs. The above data reflect results from two independent experiments with 10 mice per group. ZIKV-prME, the precursor membrane and envelope of the Zika virus. Figure 29, comprising Figures 29a through 29d, depicts experimental results demonstrating that monoimmunization with the ZIKV-prME vaccine provides protection against ZIKV challenge in mice lacking type I interferon α, β receptors. Mice were immunized once, and 2 x 10 at 2 weeks after monoimmunization 6 The plaque-forming unit was challenged with ZIKV-PR209. The survival curve depicts 10 mice per group per experiment. Figure 29a demonstrates that the ZIKV-prME vaccine prevents Zika-induced neurological abnormalities in mouse brains. Figure 29b depicts brain sections collected from pVax1 and ZIKV-prME vaccinated groups 7–8 days after challenge and stained with H&E (hematoxylin and eosin) for histology. Representative sections taken from unprotected pVax1 control animals show pathology: (i): nuclear fragment within the neural network of the cerebral cortex (inset shows higher magnification, and arrows highlight the nuclear fragment); (ii): perivascular infiltration, lymphocyte infiltration, and degenerating cells within the cortex; (iii): perivascular infiltration, cytodegeneration, and nuclear fragment within the cerebral cortex; and (iv): degenerating neurons within the hippocampus (arrows). Examples of normal tissue from mice vaccinated with ZIKV-prME appeared to be within normal limits (v and vi). Figure 29c depicts the levels of ZIKV RNA in plasma specimens from mice after vaccination and viral challenge on specified dates post-infection. Results are expressed as genomic equivalents per milliliter of plasma. Figure 29d depicts the levels of ZIKV-RNA in brain tissue analyzed at 28 days post-infection. Results are expressed as genomic equivalents per gram of tissue. ZIKV-prME, Zika virus precursor membrane and envelope. Figure 29, including Figures 30a and 30b, depicts experimental results demonstrating protection of mice lacking type I interferon α, β receptors following passive delivery of anti-ZIKV immune serum after ZIKV challenge. Collected NHP anti-ZIKV immune serum, titrated against anti-Zika virus IgG, was administered to Zika virus (10 mice per mouse). 6 Plaque-forming units were administered ip to mice on day 1 after sc challenge (150 μl / mouse). As controls, normal monkey serum and phosphate-buffered saline (PBS) were administered to age-matched mice (150 μl / mouse). Figure 30a depicts changes in mouse body weight during the course of infection and treatment. Each point represents the mean and standard error of the calculated percentage pre-challenge (day 0) body weight for each mouse. Figure 30b depicts the survival of mice after administration of NHP immune serum. ZIKV-prME, Zika virus precursor membrane and envelope. Figure 31, including Figures 31a through 31d, depicts experimental results demonstrating the characterization of the immune response to the ZIKV-prME-MR766 or ZIKV-prME-Brazil vaccines in C57BL / 6 mice. Figure 31a depicts ELISpot and ELISA assays measuring cellular and antibody responses after inoculation with the ZIKV-prME-MR766 and ZIKV-prME-Brazil DNA vaccines. C57BL / 6 mice (n = 4 / group) were intramuscularly immunized three times with 25 μg of ZIKV-prME-MR766, followed by in vivo EP. IFN-γ production was measured by an IFN-γ ELISpot as an indicator of the induction of a cellular immune response. Splenocytes harvested one week after the third immunization were cultured in the presence of one of six peptide pools covering the entire prM and E proteins. The results are plotted in a stacked bar graph. The data represent the average number of SFU (spot-forming units) per million splenocytes, with values ​​representing the mean response ± SEM for each. Figure 31b depicts ELISpot and ELISA assays measuring cellular and antibody responses following immunization with the ZIKV-prME-MR766 and ZIKV-prME-Brazil DNA vaccines. C57BL / 6 mice (n = 4 / group) were intramuscularly immunized three times with 25 μg of ZIKV-prME-Brazil, followed by in vivo EP. IFN-γ yield was measured by the IFN-γ ELISpot as an indicator of the induction of a cellular immune response. Splenocytes harvested one week after the third immunization were cultured in the presence of one of six peptide pools covering the entire prM and E proteins. The results are plotted in a stacked bar graph. The above data represent the average number of SFU (spot-forming units) per million splenocytes, with values ​​representing the mean response ± SEM in each case. Figure 31c depicts an ELISA assay measuring binding antibody production in immunized C57BL / 6 mice.Binding to rZIKV-E was analyzed in serum from mice 35 days after immunization at various dilutions. Figure 31d depicts an ELISA assay measuring binding antibody production in immunized C57BL / 6 mice. Binding to rZIKV-E was analyzed in serum from mice 35 days after immunization at various dilutions. Figure 32, including Figures 32a through 32d, depicts experimental results demonstrating the expression, purification, and characterization of ZIKV-envelope proteins. Figure 32a depicts the cloning plasmid for rZIKV-E expression. Figure 32b depicts the characterization of recombinant ZIKV-E (rZIKV-E) proteins by SDS-PAGE and Western blot analysis. Lane 1 - BSA control; Lane 2 - Lysates from E. coli cultures transformed with the pET-28a vector plasmid were purified by a nickel metal affinity resin column and separated by SDS-PAGE after IPTG induction. Lanes 3, 37 - Purified recombinant ZV-E proteins were analyzed by Western blot with an anti-His tag antibody. Lane M - Protein molecular weight marker. Figure 32c depicts that the purified rZIKV-E proteins were evaluated for antigenicity. ELISA plates were coated with rZIKV-E and subsequently incubated with various dilutions of immune serum from mice immunized with the ZIKV-prME vaccine or pan-flavivirus antibodies as a positive control. Conjugated IgG was detected by the addition of a peroxidase-conjugated mouse antibody, followed by a tetramethylbenzidine substrate, as described in the experimental examples. Figure 32d depicts the Western blot detection of purified rZIKV-E protein with immune serum from mice immunized with ZIKV prME. Various concentrations of purified rZIKV-E protein were loaded onto an SDS-PAGE gel as described. Dilutions of 1:100 immune serum and 1:15,000 goat mice were used at room temperature for 1 hour. After washing, the membranes were imaged using an Odyssey infrared imaging device. Odyssey protein molecular weight standards were used. Arrows indicate the location of the rZIKV-E protein. Figure 33, including Figures 33a through 33c, is IFNAR - / -We describe experimental results demonstrating the characterization of the immune response ZIKA-prME in mice. IFNAR - / - ELISpot and ELISA assays measuring cellular and antibody responses to ZIKV-prME in mice. Mice (n = 4 / group) were intramuscularly immunized three times with 25 μg of ZIKV-prME, followed by in vivo EP. Figure 33a depicts IFN-γ output being measured by IFN-γ ELISpot as an indicator of cellular immune response induction. Figure 33b shows immunized IFNAR - / - Describes an ELISA assay measuring the production of binding antibodies in mice. Binding to rZIKV-E was analyzed in serum from mice at various time points after immunization. Figure 33c shows immunized IFNAR - / - Describes the analysis of endpoint titers of anti-ZIKV antibodies produced in mice. Figure 34, including Figures 34a through 34d, depicts experimental results demonstrating the neutralizing activity of immune serum from Himalayan monkeys immunized against ZIKV-prME. SK-N-SH and U87MG cells were either mock-infected or infected with MR766 at an MOI of 0.01 PFU / cell in the presence of collected NHP serum immunized with the ZIKV-prME vaccine (Week 6). Zika virus infectivity was analyzed by indirect immunofluorescence assay (IFA) using serum from ZIKV-prME-vaccinated NHPs 4 days after infection. Figure 34a depicts a photograph of stained tissue specimen slices taken at 20x magnification demonstrating inhibition of infection by ZIKV viruses MR766 and PR209 in Vero, SK-N-SH, and U87MG. Figure 34b depicts a photograph of a stained tissue specimen slice taken at 20x magnification demonstrating inhibition of infection by ZIKV viruses SK-N-SH and U87MG in Vero, SK-N-SH and U87MG. Figure 34c depicts a bar graph showing the percentage of infected cells (GFP-positive cells) demonstrating inhibition of infection by ZIKV viruses MR766 and PR209 in Vero, SK-N-SH and U87MG. Figure 34d depicts a bar graph showing the percentage of infected cells (GFP-positive cells) demonstrating inhibition of infection by ZIKV viruses SK-N-SH and U87MG in Vero, SK-N-SH and U87MG. Figure 35, including Figures 35a to 35d, is ZIKV's IFNAR - / - It describes experimental results proving toxicity in mice. These data show that ZIKV is IFNAR - / - It is confirmed that it is toxic and causes morbidity and death. Figure 35a is 10 6 Intracranial IFNAR inoculated with pfu ZIKV-PR209 virus - / -Depicts the Kaplan-Meier survival curve of a mouse. Figure 35b is 10 6 IFNAR administered intravenously with pfu ZIKV-PR209 virus - / - Depicts the Kaplan-Meier survival curve of a mouse. Figure 35c is 10 6 IFNAR inoculated intraperitoneally with pfu ZIKV-PR209 virus - / - Depicts the Kaplan-Meier survival curve of a mouse. Figure 35d is 10 6 IFNAR inoculated subcutaneously with pfu ZIKV-PR209 virus - / - Depicts the Kaplan-Meier survival curve of mice. Figure 35a depicts the change in mouse body weight during the course of infection for all roots. Figure 36, including Figures 36a through 36c, depicts experimental results demonstrating the induction of a ZIKV-specific cellular immune response following ZIKV-prME inoculation in non-human primates (NHPs). Figure 36a is a schematic representation of the NHP immunization study. Figure 36b depicts the results after a single immunization. Figure 36c depicts the results after two immunizations. ELISpot assay measuring IFN-g secretion in PBMCs in response to ZIKV-prME immunization. Himalayan monkeys were intradermal (id) immunized with 2 mg of ZIKV-prME plasmid administered as 1 mg at each of the two sites at weeks 0 and 4, followed immediately after immunization. PBMCs were isolated before immunization and at week 6, and used in the ELISPOT assay to detect IFN-γ-secreting cells in response to stimulation with ZIKV-prME peptides as described in Materials and Methods. The number of IFN-γ-producing cells obtained per million PBMCs against six peptide pools covering the entire prME protein is plotted. Values ​​represent the mean response ± SEM for each group (n = 5). Figure 37, including Figures 37a and 37b, depicts experimental results demonstrating that an anti-ZIKV antibody response is induced by ZIKV-prME inoculation in non-human primates (NHP). Figure 37a depicts the detection of a ZIKV-prME-specific antibody response after a single DNA inoculation. Anti-ZIKV IgG antibodies were measured by ELISA before immunization and at week 6. Figure 37b depicts the detection of a ZIKV-prME-specific antibody response after two DNA inoculations. Anti-ZIKV IgG antibodies were measured by ELISA before immunization and at week 6. Figure 38, including Figures 38a through 38d, depicts experimental results demonstrating that Zika-prME immunization confers protection against Zika challenge. Figure 38a is a schematic representation of the NHP Zika challenge study. Himalayan monkeys were vaccinated twice with pZV-prME DNA via the ID route using EP at weeks 0 and 4. At week 8, these animals were subcutaneously challenged with a Zika-PR209 virus strain. As a control, five inexperienced animals were infected with the ZV-PR209 virus. Figure 38b depicts the determination of sequential viral load for individual animals in inexperienced NHPs. Figure 38c depicts the determination of sequential viral load for individual animals in NHPs vaccinated once. Figure 38d depicts the determination of sequential viral load for individual animals in NHPs vaccinated twice. The panel shows that the peak viral load for each animal is plotted with standard error bars for these three groups (log of viral RNA copies / mL plasma). Figure 39, including Figures 39a and 39b, depicts experimental results from a Phase 1 Zika DNA vaccine study. Figure 39a depicts experimental results from a binding ELISA study. Figure 39b depicts experimental results demonstrating passive delivery and protection. Figure 40, including Figures 40a and 40b, depicts experimental results from immunofluorescence analysis. Figure 41, including Figures 41a and 41b, depicts experimental results demonstrating the characterization of the percentage of combined reactants. Figure 42 depicts experimental results demonstrating neutralization after two doses. Specific details for implementing the invention

[0015] Detailed description of a desirable embodiment

[0016] The following abbreviated or shortened definitions are provided to aid in understanding preferred embodiments of the present invention. In this specification, these abbreviated definitions are by no means exhaustive and do not contradict definitions understood in the field or in the dictionary sense. These abbreviated definitions are provided to supplement or to define definitions known in the field more clearly.

[0017] definition

[0018] Sequence homology for nucleotides and amino acids as used herein is derived from FASTA, BLAST, and Gapp BLAST (Altschul et al., Nuc. Acids Res., 1997, 25, 3389, which is incorporated herein by reference in its entirety), and PAUP *This can be determined using 4.0b10 software (DL Swofford, Sinauer Associates, Massachusetts). Simply put, the BLAST algorithm, which refers to a basic local alignment search tool, is suitable for determining sequence similarity (Altschul et al., J. Mol. Biol., 1990, 215, 403-410, incorporated herein by reference in its entirety). Software for performing BLAST analysis is available through the National Center for Biotechnology Information. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an estimate of the probability that a match will occur by chance between two nucleotide sequences. For example, a nucleic acid is considered similar to another if, in a comparison of the test nucleic acid to another nucleic acid, the smallest sum probability is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. "Percentage of similarity" is PAUP * It can be calculated using 4.0b10 software (DL Swofford, Sinauer Associates, Massachusetts). The average similarity of common sequences is calculated by comparing them with all sequences within the phylogenetic tree.

[0019] As used herein, the term “nucleic acid construct” refers to a DNA or RNA molecule comprising a nucleotide sequence encoding a protein. The coding sequence or “encoding nucleic acid sequence” may include initiation and termination signals operably linked to a regulatory element, including a promoter and a polyadenylation signal, which can drive expression in the cells of an individual to which the nucleic acid molecule is administered.

[0020] As used herein, the term “expressible form” refers to a nucleic acid construct containing a necessary regulatory element operably linked to a coding sequence encoding a protein so that the coding sequence is expressed when present within a cell of an organism.

[0021] In this specification, the term “constant current” is used herein to define the current received or experienced by a tissue or a cell defining said tissue during the duration of an electrical pulse delivered to said tissue. The electrical pulse is delivered from the electroporation device described herein. This current remains at a constant amperage within said tissue during the lifetime of the electrical pulse because the electroporation device presented herein preferably possesses a feedback element that provides immediate feedback. This feedback element measures the resistance of the tissue (or cell) during the duration of the pulse and may induce the electroporation device to change its electrical energy output (e.g., increase the voltage) so that the current within said tissue remains constant throughout the electrical pulse (approximately microseconds) and between pulses. In some embodiments, the feedback element includes a control device.

[0022] The terms "feedback" or "current feedback" are used synonymously and refer to the active response of the presented electroporation device, which involves measuring the current within the tissue between electrodes and adjusting the energy output delivered by the EP device accordingly to maintain the current at a constant level. This constant level is preset by the user prior to the initiation of a pulse sequence or electrotreatment. Preferably, feedback is achieved by the electroporation component, for example, the control unit of the electroporation device, because the electrical circuit included herein can continuously monitor the current within the tissue between electrodes, compare the monitored current (or current within the tissue) to a preset current, and continuously perform energy-output adjustments to maintain the monitored current at a preset level. In some embodiments, the feedback loop is immediate because it is analog closed-loop feedback.

[0023] The terms “electroporation,” “electropermeability,” or “kinetic enhancement” (“EP”) used interchangeably herein refer to the use of membrane-crossing electric field pulses to induce microscopic pathways (holes) in a biological membrane; the presence of these allows biomolecules, e.g., plasmids, oligonucleotides, siRNA, drugs, ions and / or water, to move from one side of the cell membrane to the other.

[0024] The term "decentralized current" is used herein to define the pattern of current delivered from various needle electrode arrays of the electroporation device described herein, wherein these patterns minimize or, preferably, eliminate the occurrence of electroporation-related heat stress at any part of the electroporated tissue.

[0025] As used herein, the term "feedback mechanism" refers to a process performed by software or hardware (or firmware) that receives and compares the impedance of a desired tissue having a preset value, preferably a current (before, during, and / or after the delivery of an energy pulse), and adjusts the delivered energy pulse to achieve the preset value. As used herein, the term "impedance" is used when discussing the feedback mechanism and can be converted into a current value according to Ohm's law, thus enabling comparison with a preset current. In a preferred embodiment, the "feedback mechanism" is performed by an analog closed-loop circuit.

[0026] The term “immune response” is used herein to refer to the activation of the host’s immune system, e.g., the mammalian immune system, in response to the introduction of a Zika antigen, e.g., universal Zika antigen, via a provided DNA plasmid vaccine. The immune response may be in the form of a cellular or humoral response, or both.

[0027] The terms “common” or “common sequence” are used herein to refer to a synthetic nucleic acid sequence or a corresponding polypeptide sequence constructed based on the analysis of the alignment of multiple lineages of the Zika gene. A common universal Zika can be used to induce broad immunity against multiple subtypes or serotypes of the Zika virus.

[0028] The term "adjuvant" is used herein to mean any molecule added to the DNA plasmid vaccine described herein to enhance the antigenicity of the Zika antigen encoded by the DNA plasmid and encoding nucleic acid sequence described below.

[0029] The terms “subtype” or “serotype” are interchangeably used herein with respect to a virus, e.g., Zika virus, and refer to a genetic variant of a viral antigen that allows one subtype to be recognized by the immune system separately from other subtypes. For example, Zika virus subtype 1 is immunologically distinguishable from Zika virus subtype 2.

[0030] One aspect of the present invention provides a nucleic acid construct capable of expressing a polypeptide that elicits an immune response in mammals against the Zika virus. These nucleic acid constructs consist of an encoding nucleotide sequence and a promoter operably linked to said encoding nucleotide sequence. said encoding nucleotide sequence expresses a polypeptide, wherein said polypeptide comprises a common Zika antigen comprising prME. said promoter regulates the expression of said polypeptide in mammals.

[0031] In some embodiments, the nucleic acid construct may further comprise an IgE reader sequence operably linked to the N-terminal end of the coding sequence and operably linked to a promoter. Preferably, the IgE reader has the sequence of SEQ ID NO: 12. The nucleic acid construct may also comprise a polyadenylation sequence attached to the C-terminal end of the coding sequence. Preferably, the nucleic acid construct is codon-optimized.

[0032] In a preferred embodiment, the nucleic acid sequence and amino acid sequence may be selected from the following:

[0033] Sequence Number Description

[0034] 1 Common Zika IgE Leader-prME Protein

[0035] 2 Common Zika IgE Reader-prME (Construct 1) DNA

[0036] 3 Common Zika IgE Leader-prME (Construct 1) Protein

[0037] 4 Common Zika IgE Leader-NS1 DNA

[0038] 5 Common Zika IgE Leader-NS1 Proteins

[0039] 6 Common Zika IgE Leader-Capsid DNA

[0040] 7 Common Zika IgE Leader-Capsid Proteins

[0041] 8 Zika IgE Leader-prME MR766 DNA

[0042] 9 Zika IgE Leader-prME MR766 Protein

[0043] 10 Zika IgE Leader-prME Brazil DNA

[0044] 11 Zika IgE Leader-prME Brazilian Protein

[0045] 12 IgE leaders

[0046] 13 Common Zika IgE Leader-NS1 DNA (pGX7211)

[0047] 14 Common Zika IgE Leader-Capsid DNA (pGX7212)

[0048] 15 Zika IgE Leader-prME Brazilian DNA (pGX7213)

[0049] 16 Zika IgE Leader-prME MR766 DNA (pGX7214)

[0050] 17 Zika PreEnv (MR766) w / out capsid DNA (pGX7210)

[0051] 18 Zika PreEnv (MR766) w / out capsid protein (pGX7210)

[0052] In some embodiments, the DNA sequence herein may have the IgE leader sequence (nucleotide sequence encoding sequence number: 12) removed from the 5' end, and the protein sequence herein may have the IgE leader sequence of sequence number: 12 removed from the N end.

[0053] Another aspect of the present invention provides a DNA plasmid vaccine capable of producing an immune response to the Zika virus in mammals. The DNA plasmid vaccine comprises a DNA plasmid capable of expressing a common Zika antigen in mammals and a pharmaceutically acceptable excipient. The DNA plasmid comprises a promoter operably linked to a coding sequence encoding the common Zika antigen. The common Zika antigen consists of a common prME, NS1, a capsid, or a fusion of one or more of the aforementioned antigens. In one embodiment, the DNA plasmid encodes the common Zika antigen. In one embodiment, the DNA plasmid encodes the common Zika antigen having the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:18.

[0054] In one embodiment, the DNA plasmid comprises a sequence including, but not limited to, sequence number: 2, sequence number: 4, sequence number: 6, sequence number: 8, sequence number: 10, sequence number: 13, sequence number: 14, sequence number: 15, sequence number: 16 and sequence number: 17.

[0055] In some embodiments, the DNA plasmid comprises an encoding sequence encoding a Zika antigen, wherein an IgE leader sequence is excluded from the N-terminal end of the coding sequence. In some embodiments, the DNA plasmid further comprises an IgE leader sequence attached to the N-terminal end of the coding sequence and operably linked to a promoter. Preferably, the IgE leader has the sequence of SEQ ID NO:12.

[0056] The DNA plasmid may further include a polyadenylation sequence attached to the C-terminal end of the coding sequence. Preferably, the DNA plasmid is codon-optimized.

[0057] In some embodiments, the pharmaceutically acceptable excipient is an adjuvant. Preferably, the adjuvant is selected from the group consisting of: IL-12 and IL-15. In some embodiments, the pharmaceutically acceptable excipient is a transfectivity enabling agent. Preferably, the transfectivity enabling agent is a polyvalent anion, a polyvalent cation, or a lipid, and more preferably, poly-L-glutamate. Preferably, poly-L-glutamate is present at a concentration less than 6 mg / ml. Preferably, the DNA plasmid vaccine has a total DNA plasmid concentration of 1 mg / ml or more.

[0058] In some embodiments, the DNA plasmid comprises a plurality of distinct DNA plasmids, wherein each of the plurality of distinct DNA plasmids encodes a polypeptide comprising a common prME protein, a common prME (structure 1), a common NS1 DNA, or a common capsid protein.

[0059] A DNA plasmid vaccine may include a DNA plasmid encoding an amino acid sequence including, but not limited to, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:18.

[0060] In one embodiment, the DNA plasmid vaccine may comprise a DNA plasmid comprising a sequence including, but not limited to, sequence number: 2, sequence number: 4, sequence number: 6, sequence number: 8, sequence number: 10, sequence number: 13, sequence number: 14, sequence number: 15, sequence number: 16, and sequence number: 17.

[0061] In some embodiments, the mammal that produces an immune response to the DNA plasmid vaccine is a primate. Preferably, the mammal is a primate. The immune response may be a humoral response or a cellular response, and preferably both.

[0062] Another aspect of the present invention provides a method for inducing an immune response against the Zika virus in mammals, the method comprising delivering a DNA plasmid vaccine to a mammalian tissue, wherein the DNA plasmid vaccine comprises a DNA plasmid capable of expressing a common antigen of the Zika virus in mammalian cells to induce an immune response in the mammal, and electroporating the cells of the tissue to allow the entry of the DNA plasmid into the cells.

[0063] In some embodiments, the method for inducing an immune response includes a delivery step comprising injecting a DNA plasmid vaccine into the dermis, subcutaneous tissue, or muscle tissue.

[0064] In some embodiments, the method for inducing an immune response may further include pre-setting a current desired to be delivered to the tissue; and electroporating the cells of the tissue with energy pulses at a constant current equivalent to the pre-set current.

[0065] In some embodiments, the method for eliciting an immune response further comprises measuring the impedance in an electroporated cell; and adjusting the energy level of an energy pulse relative to the measured impedance to maintain a constant current in the electroporated cell. The measurement step and the adjustment step preferably occur within the lifetime of the energy pulse.

[0066] In some embodiments, the electroporation step includes delivering energy pulses to a plurality of electrodes according to a pulse chain pattern that delivers energy pulses in a dispersion pattern.

[0067] In some embodiments of the present invention, the DNA plasmid vaccine may further comprise an adjuvant. In some embodiments, the adjuvant is selected from the group consisting of alpha-interferon, gamma-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), skin T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosal layer-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, and CD86 including IL-15, in which the signal sequence is deleted and a signal peptide from IgE is optionally included. Other genes that may be useful adjuvants include those encoding the following: MCP-1, MIP-1-alpha, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, mutant forms of IL-4 and IL-18, CD40, CD40L, angiogenesis factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon response gene, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof. In some preferred embodiments, the adjuvant is selected from IL-12, IL-15, CTACK, TECK, or MEC.

[0068] In some embodiments, pharmaceutically acceptable excipients are transfectivity enabling agents, which may include: surfactants, e.g., immunostimulatory complexes (ISCOMS), Freund incomplete adjuvants, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles, e.g., squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyvalent anions, polyvalent cations, or nanoparticles, or other known transfectivity enabling agents. Preferably, the transfectivity enabling agent is a polyvalent anion, a polyvalent cation including poly-L-glutamate (LGS), or a lipid. Preferably, the transfectivity enabling agent is poly-L-glutamate, and more preferably, poly-L-glutamate is present in the DNA plasmid vaccine at a concentration less than 6 mg / ml. In some embodiments, the concentration of poly-L-glutamate in the DNA plasmid vaccine is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml, or less than 0.010 mg / ml.

[0069] In some embodiments, the DNA plasmid vaccine may be delivered to a mammal to induce an immune response; preferably, the mammal is a primate including human and non-human primates, cattle, pigs, chickens, dogs, or sables. More preferably, the mammal is a human primate.

[0070] One aspect of the present invention relates to a method for inducing an immune response to the Zika virus in mammals. The method comprises delivering a DNA plasmid vaccine to mammalian tissues and electroporating cells of said tissues with energy pulses at a constant current effective for allowing the entry of said DNA plasmid into said cells. The DNA plasmid vaccine comprises a DNA plasmid capable of expressing a Zika antigen, preferably a common antigen, in mammalian cells to induce an immune response in mammals. The method for inducing an immune response comprises electroporating cells of said tissues with energy pulses at a constant current effective for allowing the entry of said DNA plasmid into said cells.

[0071] In some embodiments, the method of the present invention comprises a delivery step comprising injecting a DNA plasmid vaccine into dermal, subcutaneous, or muscle tissue. Preferably, these methods utilize an in vivo electroporation device for presetting a current desired to be delivered to the tissue; and electroporate cells of said tissue with energy pulses at a constant current equivalent to the preset current. In some embodiments, the electroporation step further comprises measuring the impedance in the electroporated cells; and adjusting the energy level of the energy pulse relative to the measured impedance to maintain a constant current in the electroporated cells; wherein the measurement step and the adjustment step occur within the lifetime of the energy pulse.

[0072] In some embodiments, the electroporation step includes delivering energy pulses to a plurality of electrodes according to a pulse chain pattern that delivers energy pulses in a dispersion pattern.

[0073] The present invention also comprises DNA fragments encoding polypeptides capable of eliciting an immune response substantially similar to a non-fragment immune response to Zika antigen in mammals. These DNA fragments are fragments selected from at least one of the various encoding nucleotide sequences of the present invention, including a nucleotide sequence encoding SEQ ID NO:1, a nucleotide sequence encoding SEQ ID NO:2, a nucleotide sequence encoding SEQ ID NO:3, a nucleotide sequence encoding SEQ ID NO:4, a nucleotide sequence encoding SEQ ID NO:5, a nucleotide sequence encoding SEQ ID NO:6, a nucleotide sequence encoding SEQ ID NO:7, a nucleotide sequence encoding SEQ ID NO:8, a nucleotide sequence encoding SEQ ID NO:9, a nucleotide sequence encoding SEQ ID NO:10, a nucleotide sequence encoding SEQ ID NO:11, a nucleotide sequence encoding SEQ ID NO:17, a nucleotide sequence encoding SEQ ID NO:18, and SEQ ID NO:14-16, and may be one of the DNA fragments described below when applied to a specific encoding nucleic acid sequence provided herein. In some embodiments, the DNA fragment may comprise 30 or more, 45 or more, 60 or more, 75 or more, 90 or more, 120 or more, 150 or more, 180 or more, 210 or more, 240 or more, 270 or more, 300 or more, 320 or more, 340 or more, or 360 or more nucleotides. In some embodiments, the DNA fragment may comprise a coding sequence for an immunoglobulin E (IgE) leader sequence. In some embodiments, the DNA fragment may contain fewer than 60, fewer than 75, fewer than 90, fewer than 120, fewer than 150, fewer than 180, fewer than 210, fewer than 240, fewer than 270, fewer than 300, fewer than 320, fewer than 340, or fewer than 360 nucleotides.

[0074] The present invention comprises polypeptides encoded by these encoding nucleotide sequences and may comprise polypeptides having the amino acid sequences of SEQ NOs: 1, 3, 5, 7, 9, 11, 18. The present invention also comprises polypeptide fragments capable of eliciting an immune response substantially similar to the immune response of non-fragment to Zika antigen in mammals. These polypeptide fragments are selected from at least one of the various polypeptide sequences of the present invention, including SEQ NOs: 1, 3, 5, 7, 9, 11, 18, and may be one of the polypeptide fragments described below when applied to a specific polypeptide sequence provided herein. In some embodiments, the polypeptide fragment may comprise 15 or more, 30 or more, 45 or more, 60 or more, 75 or more, 90 or more, 100 or more, 110 or more, or 120 or more amino acids. In some embodiments, the polypeptide fragment may comprise fewer than 30, fewer than 45, fewer than 60, fewer than 75, fewer than 90, fewer than 100, fewer than 110, or fewer than 120 amino acids.

[0075] The determination of a functional fragment that elicits an immune response substantially similar to that of a non-fragment immune response to the Zika antigen in mammals can be easily made by a person skilled in the art. The fragment can be analyzed to contain at least one, preferably one or more, antigenic epitopes as presented in publicly available databases, for example, the National Center for Biotechnology Information (NCBI). In addition, immune response studies can be performed in mice using antibody titer and ELISpots analysis, for example, as described in the examples below.

[0076] vaccine

[0077] In some embodiments, the present invention provides an enhanced vaccine by providing a protein having an epitope that makes it particularly effective as an immunogen capable of inducing an immune response, and a gene construct encoding these proteins. Thus, a vaccine capable of inducing a therapeutic or prophylactic immune response may be provided.

[0078] According to some embodiments of the present invention, a vaccine according to the present invention is delivered to an individual to modulate the activity of the individual's immune system and, therefore, to enhance the immune response. When a nucleic acid molecule encoding a protein is absorbed by a cell of the individual, said nucleotide sequence is expressed in the cell and, therefore, said protein is delivered to the individual. An aspect of the present invention provides a method for delivering a coding sequence of a protein to a nucleic acid molecule, for example, a plasmid.

[0079] According to some aspects of the present invention, a composition and a method for prophylactically and / or therapeutically immunizing an individual are provided.

[0080] When absorbed by the cell, the DNA plasmid may remain within the cell as separate genetic material. Alternatively, RNA may be administered to the cell. It is also anticipated that the gene construct be provided as a linear chromosome containing a centromere, telomere, and replication origin. The gene construct contains regulatory elements necessary for the gene expression of nucleic acid molecules. These elements include: a promoter, a start codon, a stop codon, and a polyadenylation signal. In addition, enhancers are often required for the gene expression of sequences encoding target proteins or immune control proteins. These elements must be operablely linked to the sequence encoding the desired protein, and these regulatory elements must be operable in the individual to which they are administered.

[0081] Start and stop codons are generally considered to be part of the nucleotide sequence encoding the desired protein. However, these elements must be functional in the mammal to which the nucleic acid construct is administered. The start and stop codons must be inframe with the coding sequence.

[0082] The promoter and polyadenylation signal used must be functional within the organism's cells.

[0083] Examples of promoters useful for carrying out the present invention, particularly useful in the production of human gene vaccines, include but are not limited to promoters from primate virus 40 (SV40), mouse mammary tumor virus (MMTV) promoters, human immunodeficiency virus (HIV), e.g., bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoters, Molony virus, avian leukemia virus (ALV), cytomegalovirus (CMV), e.g., CMV ultra-early promoters, Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), as well as promoters from human genes, e.g., human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein; in other embodiments, the promoter may be a tissue-specific promoter, e.g., a muscle or skin-specific promoter (natural or synthetic). An example of such a promoter is described in US patent application publication number US20040175727, which is incorporated herein in its entirety.

[0084] Examples of polyadenylation signals useful for carrying out the present invention, particularly useful in the production of human gene vaccines, include, but are not limited to, SV40 polyadenylation signals, LTR polyadenylation signals, bovine growth hormone (bGH) polyadenylation signals, human growth hormone (hGH) polyadenylation signals, and human β-globin polyadenylation signals. In particular, the SV40 polyadenylation signal within the pCEP4 plasmid (Invitrogen, San Diego, CA), referred to as the SV40 polyadenylation signal, may be used.

[0085] In addition to the regulatory elements required for DNA expression, other elements may also be included within the DNA molecule. These additional elements include enhancers. Enhancers may be selected from a group including, but not limited to, human actin, human myosin, human hemoglobin, human muscle creatine, and viral enhancers, such as those from CMV, RSV, and EBV.

[0086] A genetic construct may be provided with a mammalian replication origin to maintain the construct extrachromosomally and produce multiple copies of the construct within the cell. Plasmids pVAX1, pCEP4, and pREP4 from Invitrogen (San Diego, CA) contain an Epstein-Barr virus replication origin and a nuclear antigen EBNA-1 coding region that yield high-copy episomal replication without integration.

[0087] To maximize protein production, a regulatory sequence sufficiently suitable for gene expression in the cell to which the construct is administered can be selected. Furthermore, a codon encoding said protein that is transcribed most efficiently in the host cell can be selected. A person skilled in the art can produce a functional DNA construct in a cell.

[0088] In some embodiments, a nucleic acid construct may be provided in which the coding sequence for the protein described herein is linked to an IgE leader peptide, or such an IgE leader is removed. In some embodiments, the protein described herein is linked to an IgE signal peptide, or such an IgE leader is removed.

[0089] In some embodiments where proteins are used, for example, a person skilled in the art may produce the proteins of the present invention using widely known techniques and isolate the proteins using widely known techniques. In some embodiments where proteins are used, for example, a person skilled in the art may insert a DNA molecule encoding the proteins of the present invention into a commercially available expression vector for use in a widely known expression system using widely known techniques. For example, the commercially available plasmid pSE420 (Invitrogen, San Diego, Calif.) may be used for the production of proteins in Escherichia coli (E. coli). The commercially available plasmid pYES2 (Invitrogen, San Diego, Calif.) may be used for production, for example, in a Saccharomyces cerevisiae yeast strain. The commercially available MAXBAC™ complete baculovirus expression system (Invitrogen, San Diego, Calif.) can be used for production in insect cells, for example. The commercially available plasmid pcDNA or pcDNA3 (Invitrogen, San Diego, Calif.) can be used for production in mammalian cells, for example, Chinese hamster ovary (CHO) cells. Those skilled in the art can use these commercial expression vectors and systems or others to produce proteins using routine techniques and readily available starting materials (see, for example, Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989)). Thus, the desired protein can be produced in both prokaryotic and eukaryotic systems, and the spectrum of the processed form of said protein can be obtained.

[0090] Those skilled in the art may produce vectors using other commercially available expression vectors and systems, or by using widely known methods and readily available starting materials. Expression systems containing essential control sequences, e.g., promoters and polyadenylation signals, and preferably enhancers, are readily available and known in the art for various hosts. See, for example, Sambrook et al., *Molecular Cloning a Laboratory Manual*, Second Ed., Cold Spring Harbor Press (1989). Gene constructs comprise protein-coding sequences operably linked to functional promoters in the cell lines or cells of the targeted tissue to which these constructs are transfected. Examples of structural promoters include promoters from cytomegalovirus (CMV) or SV40. Examples of inducible promoters include mouse mammary leukemia virus or metallothionein promoters. Those skilled in the art may readily produce gene constructs useful for transfecting cells with DNA encoding the protein of the present invention from readily available starting materials. An expression vector containing DNA encoding the above protein is used to transform a compatible host, and the host is subsequently cultured and maintained under conditions in which the expression of foreign DNA occurs.

[0091] The produced protein is recovered from the culture medium, or, if feasible, from the culture medium by lysing the cells, as is known to those skilled in the art. Those skilled in the art may isolate the protein produced using such an expression system by utilizing widely known techniques. As previously described, the method of purifying proteins from natural sources using antibodies that specifically bind to a particular protein can be equally applied to purifying proteins produced by recombinant DNA methodologies.

[0092] In addition to producing proteins by recombinant technology, automated peptide synthesis devices may also be used to produce isolated and essentially pure proteins. Such technology is widely known to those skilled in the art and is useful in cases where derivatives with substitutions are not provided in the production of DNA-encoded proteins.

[0093] Nucleic acid molecules may be delivered using one of several widely known techniques, including DNA injection with and without in vivo electroporation (also referred to as DNA vaccination), liposome-mediated technology, nanoparticle-assisted technology, and recombinant vectors, e.g., recombinant adenoviruses, recombinant adenovirus-associated viruses, and recombinant cowpox. Preferably, the nucleic acid molecules described herein, e.g., DNA plasmids, are delivered via DNA injection and with in vivo electroporation.

[0094] Routes of administration include, but are not limited to, intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intra-arterial, intraocular, oral, and local, transdermal, inhalation or suppository, or administration into mucosal tissues, e.g., vaginal, rectal, urethral, ​​oral mucosa and sublingual tissues by lavage. Preferred routes of administration include intramuscular, intraperitoneal, intradermal, and subcutaneous injection. Gene constructs may be administered by means including, but not limited to, traditional syringes, needleless injection devices, "microprojectile bombardment gene guns," or other physical methods, e.g., electroporation ("EP"), "hydrodynamic methods," or ultrasound.

[0095] Examples of preferred electroporation devices and electroporation methods for facilitating the delivery of the DNA vaccine of the present invention include those described in US Patent No. 7,245,963 (Draghia-Akli, et al.) and US Patent Publication No. 2005 / 0052630 (filed by Smith, et al.), and these documents are incorporated into the present invention purely by reference. In addition, to facilitate the delivery of these DNA vaccines, the electroporation apparatus and electroporation method described in US provisional application No. 60 / 852,149 filed on October 17, 2006, under 35 USC 119(e), and concurrently pending joint US patent application No. 11 / 874072 filed on October 17, 2007, claiming priority to US provisional application No. 60 / 978,982 filed on October 10, 2007, are also preferred, and all of these documents are incorporated into the present invention purely by reference.

[0096] US Patent No. 7,245,963 (Draghia-Akli, et al.) describes a modular electrode system for facilitating the introduction of biomolecules into cells of selected tissues from the body or plants and the use thereof. The modular electrode system comprises a plurality of needle electrodes; a subcutaneous injection needle; an electrical connector providing a conductive link from a programmable constant current pulse controller to the plurality of needle electrodes; and a power source. An operator can grasp the plurality of needle electrodes mounted on a support structure and firmly insert them into tissues selected from the body or plants. Biomolecules are then delivered into the selected tissue via the subcutaneous injection needle. The programmable constant current pulse controller is activated, and a constant current electrical pulse is applied to the plurality of needle electrodes. The applied constant current electrical pulse facilitates the introduction of biomolecules into cells between the plurality of electrodes. The entire contents of US Patent No. 7,245,963 are incorporated herein by reference.

[0097] US Patent Publication 2005 / 0052630 (filed by Smith, et al.) describes an electroporation device that can be used to effectively facilitate the introduction of biomolecules into cells of selected tissues from the body or plants. The electroporation device comprises a kinetic device ("EKD device") whose operation is specified by software or firmware. The EKD device produces a series of programmable constant current pulse patterns between electrodes within an array based on user control and input of pulse parameters, and allows for the storage and acquisition of current waveform data. The electroporation device also comprises a replaceable electrode disk having an array of needle electrodes, a central injection passage for an injection needle, and a removable guide disk. The entire contents of US Patent Publication 2005 / 0052630 are incorporated herein by reference.

[0098] The electrode array and method described in US Patent No. 7,245,963 and US Patent Publication 2005 / 0052630 are adapted for deep penetration into tissues, e.g., muscles, as well as other tissues or organs. Due to the shape of the electrode array, an injection needle (for delivering a selected biomolecule) is also fully inserted into the target organ, and the injection is administered perpendicularly to the target tissue within the zone pre-described by the electrodes. The electrodes described in US Patent No. 7,245,963 and US Patent Publication 2005 / 005263 are preferably 20 mm long and 21 gauge.

[0099] The following is an example of the method of the present invention, and is discussed in more detail in the aforementioned patent references: An electroporation device may be configured by a user to deliver energy pulses that produce a constant current similar to a preset current input to a desired tissue of a mammal. The electroporation device comprises an electroporation component and an electrode assembly or a handle assembly. The electroporation component may include and integrate one or more of various elements of the electroporation device, including a controller, a current waveform generator, an impedance tester, a waveform logger, an input element, a status reporting element, a communication port, a memory component, a power source, and a power switch. The electroporation component may function as one element of the electroporation device, and other elements are separate elements (or components) that communicate with said electroporation component. In some embodiments, the electroporation component may function as one or more elements of the electroporation device and may still communicate with other elements of the electroporation device that are distinct from the electroporation component. The invention is not limited to elements of the electroporation device existing as a single electromechanical device or as part of a mechanical device, because these elements may function as a single device or as distinct elements communicating with each other. The electroporation component may deliver energy pulses that produce a constant current in the desired tissue and includes a feedback mechanism.The electrode assembly comprises an electrode array having multiple electrodes in a spatial arrangement, wherein the electrode assembly receives an energy pulse from an electroporation component and transmits the energy pulse to a desired tissue through the electrodes. At least one of the multiple electrodes is neutral during the transmission of the energy pulse, measures the impedance in the desired tissue, and transmits the impedance to the electroporation component. A feedback mechanism can receive the measured impedance and adjust the energy pulse transmitted by the electroporation component to maintain a constant current.

[0100] In some embodiments, a plurality of electrodes can transmit energy pulses in a dispersion pattern. In some embodiments, a plurality of electrodes can transmit energy pulses in a dispersion pattern through the control of the electrodes under a programmed sequence, and said programmed sequence is input into an electroporation component by a user. In some embodiments, the programmed sequence includes a plurality of pulses transmitted in sequence, wherein each pulse of the plurality of pulses is transmitted by at least two active electrodes together with one neutral electrode that measures impedance, and a subsequent pulse among the plurality of pulses is transmitted by a different one of at least two active electrodes together with one neutral electrode that measures impedance.

[0101] In some embodiments, the feedback mechanism is performed by hardware or software. Preferably, the feedback mechanism is performed by an analog closed-loop circuit. Preferably, such feedback occurs every 50 μs, 20 μs, 10 μs, or 1 μs, and more preferably is real-time feedback or immediate (i.e., substantially immediate when determined by available techniques for determining the response time). In some embodiments, a neutral electrode measures the impedance in the desired tissue and transmits the impedance to the feedback mechanism, and the feedback mechanism responds to the impedance and adjusts the energy pulse so that the constant current is maintained at a value similar to a preset current. In some embodiments, the feedback mechanism maintains the constant current continuously and immediately during the transmission of the energy pulse.

[0102] Pharmaceutically acceptable excipients may include functional molecules known and readily available in the art, e.g., carriers, adjuvants, carriers, or diluents. Preferably, the pharmaceutically acceptable excipient is an adjuvant or a transfection facilitating agent. In some embodiments, a nucleic acid molecule, or a DNA plasmid, is delivered to a cell in combination with the administration of a polynucleotide function enhancer or a gene vaccine promoter (or transfection facilitating agent). Polynucleotide function enhancers are described in US Serial Nos. 5,593,972, 5,962,428 and International Application Serial No. PCT / US94 / 00899 filed January 26, 1994, each of which is incorporated by reference into the present invention. Gene vaccine promoters are described in US Serial No. 021,579 filed April 1, 1994, which is incorporated by reference into the present invention. The transfection-enabled agent may be administered as a mixture with a nucleic acid molecule in combination with said nucleic acid molecule, or administered separately and simultaneously before and after the administration of the nucleic acid molecule. Examples of transfection-enabled agents include surface active agents, e.g., immune-stimulating complexes (ISCOMS), Freund incomplete adjuvants, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, and vesicles, e.g., squalene and squalene, and hyaluronic acid may also be used for co-administration with the gene construct. In some embodiments, the DNA plasmid vaccine may also comprise a transfection-enabler, for example, lipids, liposomes including lecithin liposomes or other liposomes known in the art, DNA-liposome mixtures (see e.g., W09324640), calcium ions, viral proteins, polyvalent anions, polyvalent cations, or nanoparticles, or other known transfection-enabler.Preferably, the transfection-enabled agent is a polyvalent anion, a polyvalent cation including poly-L-glutamate (LGS), or a lipid.

[0103] In some preferred embodiments, the DNA plasmid is delivered along with an adjuvant, which is a gene for a protein that further enhances the immune response to these target proteins. Examples of such genes include other cytokines and lymphokines, such as alpha-interferon, gamma-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, MHC, CD80, CD86, and those encoding IL-15, including IL-15, in which the signal sequence is deleted and a signal peptide from IgE is selectively included. Other genes that may be useful include those encoding the following: MCP-1, MIP-1α, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, mutant forms of IL-4 and IL-18, CD40, CD40L, angiogenesis factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon response gene, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and their functional fragments.

[0104] The DNA plasmid vaccine according to the present invention comprises an amount of DNA of about 1 nanogram to 10 milligrams; about 1 microgram to about 10 milligrams; or preferably about 0.1 microgram to about 10 milligrams; or more preferably about 100 micrograms to about 1 milligram. In some preferred embodiments, the DNA plasmid vaccine according to the present invention comprises about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the DNA plasmid vaccine contains about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the DNA plasmid vaccine contains about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the DNA plasmid vaccine contains about 1 to about 350 micrograms of DNA. In some preferred embodiments, the DNA plasmid vaccine contains about 25 to about 250 micrograms of DNA. In some preferred embodiments, the DNA plasmid vaccine contains about 100 micrograms to about 1 milligram of DNA.

[0105] The DNA plasmid vaccine according to the present invention is prepared according to the method of administration used. When the DNA plasmid vaccine is an injectable composition, it is sterile and / or free of pyrogens and / or particulates. Preferably, an isotonic formulation is used. Generally, additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, an isotonic solution, for example, phosphate-buffered saline, is preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation. In some embodiments, a stabilizer, for example, LGS or other polyvalent cations or polyvalent anions, is added to the formulation to enable the formulation to remain stable at room temperature or ambient temperature for an extended period.

[0106] In some embodiments, a method for inducing an immune response to a common Zika antigen in mammals includes a method for inducing a mucosal immune response. Such a method comprises administering to a mammal one or more of the CTACK protein, TECK protein, MEC protein, and their functional fragments or their expressible coding sequences in combination with the DNA plasmid containing the common Zika antigen described above. One or more of the CTACK protein, TECK protein, MEC protein, and their functional fragments may be administered prior to, simultaneously with, or after administration of the DNA plasmid Zika vaccine presented herein. In some embodiments, an isolated nucleic acid molecule encoding one or more proteins selected from the group consisting of CTACK, TECK, MEC, and their functional fragments is administered to a mammal.

[0107] Examples

[0108] The present invention is further illustrated in the following examples. While these examples represent preferred embodiments of the invention, they are provided for illustrative purposes only. From the above detailed description and these examples, those skilled in the art can identify the essential features of the invention and make various modifications of the invention suitable for various uses and conditions without departing from the technical spirit and scope of the invention. Accordingly, various modifications of the invention other than those shown and described herein will be apparent to those skilled in the art from the above detailed description. Such modifications are also intended to fall within the scope of the appended claims.

[0109] Preferably, the DNA preparation used with the muscle or skin EP device described herein has a high DNA concentration, preferably a small volume optimal for skin delivery, preferably a small injection volume, ideally 25 to 200 microliters (μL), containing an amount of micrograms to tens of milligrams, and more preferably a milligram amount of DNA. In some embodiments, the DNA preparation has a high DNA concentration, for example, 1 mg / mL or more (mg DNA / volume of preparation). More preferably, the DNA preparation has a DNA concentration providing a gram amount of DNA in a 200 μL formula, and more preferably a gram amount of DNA in a 100 μL formula.

[0110] DNA plasmids used with the EP device of the present invention may be prepared or manufactured using a combination of known devices and techniques, but preferably, they are manufactured using the optimized plasmid manufacturing technique described in US Application No. 12 / 126611 disclosed on January 1, 2009, as US Publication No. 20090004716. In some examples, the DNA plasmids used in these studies may be prepared at a concentration of 10 mg / mL or higher. The manufacturing technique also includes or incorporates various devices and protocols widely known to those skilled in the art, in addition to those described in US Publication No. 20090004716 and those described in US Patent No. 7,238,522 granted on July 3, 2007. High concentration plasmids used with the skin EP device and delivery technique described herein enable administration of the plasmid into the ID / SC space at a reasonably low volume and assist in enhancing expression and immunization effects. Publications, US Publication No. 20090004716 and US Patent No. 7,238,522 are incorporated herein in their entirety.

[0111] Example 1: Zika prME vaccine

[0112] Zika vaccine approach

[0113] As shown in Figure 2, a Zika antigen expression construct was produced with the backbone depicted therein. The expression cassette was inserted after the CMV promoter and with a trailing polyadenylated tail. The cassette may include the encoding sequence for the antigen depicted in Figure 3, including prME, NS1, and a capsid.

[0114] Phylogenetic Analysis of Zika prME and Vaccine Design

[0115] Phylogenetic analysis was performed as shown in Figures 5 and 6. Stars indicate the location of the common prME sequence number:3. This common prME is shown inserted into the cloning site within the expression vector in Figure 7.

[0116] The expressed proteins were characterized by Western blot analysis as shown in Figures 9a and 9b, which show specific binding to anti-flavivirus antibodies.

[0117] The above protein was subsequently purified as shown in Figures 10a and 10b.

[0118] Mouse Immunization

[0119] Animals - Balb / C mice (8 groups)

[0120] Plasmid - Zika-prME (encoding sequence containing sequence number: 2)

[0121] Device - 3P Electroporation Device (Inovio Pharmaceuticals, Plymouth Meeting, PA)

[0122] Immunization Schedule:

[0123] Mice were immunized with DNA a total of three times: once on day 0 (primary), and boosters on days 14 and 28. Immunological assays were performed one week after the third DNA immunization.

[0124] Injection method - Intramuscular

[0125] Blood collection schedule - Before blood collection and on the 14th, 28th & 35th days

[0126] Blood collection method - behind the eye

[0127] Groups & Animals - 10 Animals / Group X 3 Groups = 30

[0128] 1) pVax1

[0129] 2) pVax-1 Zika preme (Sequence No.: 2)

[0130] Cellular immune response induced by the Zika prME vaccine

[0131] Spike-specific CD8 T-lymphocyte responses were assessed by the IFN-g ELISpot assay for the peptide pool covering the prME antigen. Refer to Figures 11 and 12. The mean response in each group was 1 week after the third immunization. Error bars indicate the standard error. Responses to the pVax control are shown.

[0132] Induction of antibodies in mice

[0133] Zika prME vaccination of mice induced a positive antibody response that reacted with Zika-skin antigen. Refer to Figures 13 and 14.

[0134] The Zika prME vaccine was found to be immunogenic in mice based on binding to the recombinant protein E antigen. Seroconversion was observed in immunized animals by Western blot analysis and ELISA.

[0135] Example 2: A novel DNA vaccine against Zika virus prME induces protective immunity in vivo.

[0136] A novel synthetic DNA co-based vaccine targeting the Zika virus membrane + envelope proteins is described herein. Following confirmation of construct expression, mice and non-human primates were immunized via electroporation, demonstrating the induction of both cellular and humoral immunity with neutralizing activity in vaccinated animals. IFN-α / β R - / -In mice, single or double-dose immunization provided 100% protection against weight loss or death in these lethal challenge models. This represents the first Zika virus vaccine approved for human trials.

[0137] The materials and methods will be explained from now on.

[0138] Cells, viruses, and animals

[0139] Human embryonic kidney (HEK) 293T (American Type Culture Collection (ATCC) #CRL-N268, Manassas, VA) and Vero CCL-81 (ATCC #CCL-81) cells were maintained in Dulbecco's modified Eagle medium (DMEM; Gibco-Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin, and were passaged at confluence. Neuronal tumor cell lines SK-N-SH (ATCC HTB-11) and U87MG (ATCC HTB-14) were maintained in Eagle minimal essential medium (MEM; Corning-Cellgro) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin, and were passaged at confluence. Both Zika virus strains MR766 (a goodwill gift from Dr. Susan Weiss) and PR209 (Bioqual, MD) were amplified in Vero cells, and the stocks were titrated in Vero cells by a standard plaque assay.

[0140] C57 / BL6 and IFNAR - / - Mice and Himalayan monkey procedures were performed under ketamine anesthesia. These animals were housed in adjacent individual primate cages allowing for social interaction under controlled conditions of humidity, temperature, and lighting (12-hour light / 12-hour dark cycle). Food and water were available in unlimited quantities. These animals were monitored twice daily, and commercial monkey food, treats, and fruit were fed twice daily.

[0141] DNA vaccine constructs and synthesis

[0142] The Zika-prM+Env plasmid DNA construct encodes the full-length precursors of the membrane (prM) protein and the envelope (E) protein. A common strategy was utilized, and the common sequence was determined by the alignment of the current Zika prM+E protein sequences. The vaccine insert was genetically optimized for enhanced expression in humans (i.e., codon and RNA optimization), and an IgE leader sequence was added to facilitate expression. This construct was commercially synthesized (Genscript, NJ) and subsequently subcloned into a modified pVax1 expression vector under the control of a cytomegalovirus ultra-early promoter, as described in the existing literature (Muthumani et al., 2015, Sci Trans Med 7:301ra132). The final construct was named the ZIKV-prME vaccine, and the control plasmid backbone is pVax1. In addition, several other aligned DNA constructs encoding the prM and Env genes from the MR766 and 2016 Brazilian strains were also designed for further evaluation. Large-scale amplification of the DNA constructs was performed by Inovio (Plymouth Meeting, PA), and the purified plasmid DNA was prepared in water for immunization. The size of the DNA inserts was confirmed by agarose gel electrophoresis. Phylogenetic analysis was performed by multiple alignment with ClustalW using MEGA version 5 software (Muthumani et al., 2015, Sci Trans Med 7:301ra132).

[0143] DNA Immunization and Electroporation

[0144] Mouse Immunogenicity Study: Female C57BL / 6 Mice (6 to 8 weeks old) and IFNAR - / -Mice (5 to 7 weeks of age) were immunized with 25 μg of DNA in a total volume of 20 or 30 μl of water delivered into the tibialis anterior muscle via in vivo EP delivery (n=4). In vivo EP was delivered to the same site immediately after immunization using a CELLECTRA adaptive constant current EP device (Inovio Pharmaceuticals, PA). The 3-pronged CELLECTRA minimally invasive device was inserted approximately 2 mm into the muscle. Square wave pulses were delivered through a triangular 3-electrode array consisting of 26-gauge solid stainless steel electrodes, and two constant current pulses of 0.1 Amp were delivered for 52 microseconds / pulse separated by a 1-second delay. Additional protocols for the use of EP were described in detail previously. Mice were immunized three times at 2-week intervals and sacrificed 1 week after final immunization. Blood was collected after each immunization for analysis of cellular and humoral immune responses (Muthumani et al., 2015, Sci Trans Med 7:301ra132). Himalayan monkey immunogenicity study: Five Himalayan monkeys were ID-immunized with 2 mg ZIKV-prME vaccine at two sites at 4-week intervals. EP was delivered immediately using the same device described for mouse immunization.

[0145] IFNAR - / - Challenge inoculation study in mice

[0146] IFNAR - / - The mice were divided into three groups. The first group of mice was immunized once, and 10 at 2 weeks after immunization 6 They were challenged with PFU ZIKV-PR209. The second group of mice was immunized twice at a 2-week interval and challenged with 106 PFU ZIKV-PR209 one week after the second immunization. The third group of mice was immunized twice at a 2-week interval and challenged with 2 x 10⁶ at one week after the second immunization. 6The animals were challenged with PFU ZIKV PR209. After challenge, the animals were weighed, and their body temperature was measured daily using a subcutaneous temperature chip. In addition, clinical signs of the disease (decreased mobility; crouching posture; hind leg knuckle gait (partial paralysis), paralysis of one or both hind legs) were observed twice daily. Criteria for euthanasia on a welfare basis consisted of a 20% weight loss or the observation of any abnormal clinical signs.

[0147] Western blot and immunofluorescence assay

[0148] For in vitro expression studies, transfection was performed using GeneJammer reagents according to the manufacturer's protocol (Agilent). Briefly, cells were grown to 50% confluence in 35-mm dishes and transfected with 1 µg of Zika preM+Env vaccine. These cells were harvested 2 days after transfection, washed twice with phosphate-buffered saline (PBS), and lysed with cell lysis buffer (Cell Signaling Technology). Western blots were used to demonstrate the expression of Zika preM+Env proteins from the harvested cell lysates, as described in the literature (Muthumani et al., 2015, Sci Trans Med 7:301ra132).

[0149] The specificity of mice and RM immune serum was confirmed using Western blot analysis. 3-12% Bis-Tris NuPAGE gels (Life Technologies) were dropwise loaded with 5 μg or 1 μg of ZIKV Env recombinant protein and Odyssey protein molecular weight marker (Product # 928-40000). The gels were transferred in MOPS buffer at 200 V for 50 minutes. These proteins were transferred onto nitrocellulose membranes using an iBlot 2 Gel delivery device (Life Technologies). These membranes were blocked in PBS Odyssey blocking buffer (LI-COR Biosciences) for 1 hour at room temperature. Anti-Flavivirus herd antigen (MAB10216-clone D1-4G2-4-15) antibodies were diluted 1:500 to detect vaccine expression, and immune serum from mice and RMs was diluted 1:50 in Odyssey blocking buffer containing 0.2% Tween 20 (Bio-Rad) and incubated with these membranes overnight at 4°C. These membranes were washed with PBST and subsequently incubated with appropriate secondary antibodies [goat anti-mouse IRDye680CW (LICOR) for mouse serum and flavivirus antibodies; and goat anti-human IRDye800CW (LICOR) for RM serum] at a dilution of 1:15,000 for mouse serum for 1 hour at room temperature. After washing, these membranes were imaged on an Odyssey infrared imaging device (LI-COR).

[0150] For immunofluorescence assays, HeLa or Vero cells were grown on coverslips and transfected with 5 μg of Zika preM+Env vaccine. Two days after transfection, these cells were fixed with 4% PFA for 15 minutes. Non-specific binding was subsequently blocked with normal goat serum diluted in PBS for 1 hour at room temperature. The slides were then washed in PBS for 5 minutes and subsequently incubated with serum from immunized mice or RMs at a 1:100 dilution overnight at 4°C. These slides were washed as previously described and incubated with appropriate secondary antibodies [goat anti-mouse IgG-AF488 (Sigma) for mouse serum and goat anti-human IgG-AF488 for RM serum] at a 1:200 dilution for 1 hour at room temperature. After washing, Flouroshield fixation medium containing DAPI (Abcam) was added to stain the nuclei of all cells. Subsequently, coverslips were loaded, and slides were observed under a microscope (EVOS Cell Imaging Systems; Life Technologies) (Muthumani et al., 2015, Sci Trans Med 7:301ra132). Additionally, Vero, SK-N-SH, or U87-MB cells were grown on 4-chamber tissue culture-treated glass slides (Falcon cat#354114), infected with MR766 ZV at an MOI of 0.01 for 4–6 days, and subsequently stained as described.

[0151] Isolation of splenocytes and PBMCs

[0152] Single-cell suspensions of splenocytes were prepared from all mice. Briefly, splenocytes from mice were individually collected in 5 ml of RPMI 1640 supplemented with 10% FBS (R10) and subsequently processed in a Stomacher 80 paddle blender (AJ Seward and Co. Ltd.) at high speed for 30 seconds. The processed splenocyte samples were filtered through a 45-mm nylon filter and subsequently centrifuged at 1500 rpm for 10 minutes at 4°C. The cell pellet was resuspended in 5 ml of ACK (Ammonium Chloride-Potassium) lysis buffer (Life Technologies) for 5 minutes at room temperature, after which PBS was added to stop the reaction. The samples were centrifuged once again at 1500 rpm for 10 minutes at 4°C. The cell pellet was resuspended in R10 at a concentration of 1 x 10⁷ cells / ml and subsequently passed through a 45-mm nylon filter before use in the ELISpot assay and flow cytometry analysis (Muthumani et al., 2015, Sci Trans Med 7:301ra132). For RM, blood (20 ml at each time point) was collected in EDTA tubes, and peripheral blood mononuclear cells (PBMCs) were isolated into Accuspin tubes (Sigma-Aldrich, St. Louis, MO) using the standard Ficoll-Hypaque procedure.

[0153] ELISpot test

[0154] Briefly, 96-well ELISpot plates (Millipore) were coated with antimicrobial mouse IFN-γ capture antibodies (R&D Systems) and incubated overnight at 4°C. The next day, the plates were washed with PBS and blocked with PBST + 1% BSA for 2 hours. Two hundred thousand splenocytes from pZV-prM+Env-immunized mice were added to each well and incubated overnight at 37°C in 5% CO2 in the presence of medium alone (negative control), medium containing PMA / ionomycin (positive control), or medium containing a peptide pool (1 μg / ml) consisting of 15-mers (Genscript) that overlap by 9 amino acids and span the length of the Zika coat protein. After 24 hours, these cells were washed and subsequently incubated overnight at 4°C with biotinylated antimicrobial mouse IFN-γ antibodies (R&D Systems). Streptavidin-alkaline phosphatase (R&D Systems) was added to each well after washing and subsequently incubated at room temperature for 2 hours. The plates were washed and subsequently 5-bromo-4-chloro-3'-indolylphosphate p-toluidine salt and nitro blue tetrazolium chloride (dye source color reagent; R&D Systems) were added. Finally, the plates were rinsed with distilled water, dried at room temperature, and quantified by an automated ELISpot reader (CTL Limited), and the raw values ​​were normalized to SFU per million splenocytes. In the case of RM specimens, ELISPOTPRO (MABTECH) for the monkey IFN-γ kit was used as described by the manufacturer, 200,000 PBMCs were stimulated with a peptide pool, and the plates were washed and the spots were examined according to existing literature (Muthumani et al., 2015, Sci Trans Med 7:301ra132; Mallilankaraman et al.It was colored and counted as described in , 12011, PLoS Negl Trop Dis 5:e928).

[0155] Humoral Immune Response: Antibody-Binding ELISA

[0156] Enzyme-linked immunosorbent assay (ELISA) was used to determine the titers of mouse and RM serum, as described in the existing literature (Muthumani et al., 2015, Sci Trans Med 7:301ra132). Briefly, 1 μg / ml of purified Zika coat protein was used to coat 96-well micro-titer plates (Nalgene Nunc International, Naperville, IL) overnight at 4°C. After blocking with 10% FBS in PBS for at least 1 hour, the plates were washed four times with 0.05% PBST (Tween20 in PBS). Serum samples from immunized mice and RM were serially diluted in 1% FBS and 0.2% PBST, added to the plates, and subsequently incubated at room temperature for 1 hour. The plates were washed four times again with 0.05% PBST and subsequently incubated with HRP-conjugated antimicrobial mouse IgG (Sigma) at a dilution of 1:35,000 for mouse serum for 1 hour at room temperature. For RM serum, anti-monkey IgG HRP (Southern Biotech) was used at a dilution of 1:5,000 for 1 hour at room temperature. The conjugated enzyme was detected by adding SIGMAFAST™ OPD (o-phenylenediamine bihydrochloride) tablets according to the manufacturer's instructions (Sigma Aldrich). The reaction was stopped after 15 minutes with the addition of 1N H2SO4. The plates were then read at an absorbance of 450 nm. All mouse serum and RM serum samples were double-checked. The endpoint potency was determined using the method described by Frey et al. (Frey et al., 1998, J Immunol Methods 221:35-41).

[0157] Junghwa (PRNT) 50 ) black

[0158] Plaque-reduction neutralization tests (PRNT) involving MR766 and Vero cells were described in existing literature (Sun et al., 2006, J Infect Dis 193:1658-65). Briefly, mouse or RM serum was serially diluted in serum-free DMEM (from 1:10 to 1:1280) and incubated with an equivalent volume of MR766 Zika virus (100 pfu) at 37°C for 2 hours. The mixture was added to the confluent layer of Vero cells and left at 37°C for 2 hours for adsorption. A 2XDMEM medium:soft agar (1:1) overlay was added over the cells, and the plates were incubated at 37°C for 5 days. The agar overlay was removed from the wells, and the cells were fixed with 4% paraformaldehyde, washed with 1X PBS, stained with crystal violet solution, washed with 1X PBS, and the plates were left to dry. In the assay performed on 24-well plates, plaques were manually counted. In the assay performed on 96-well plates, plaques were scanned with an automated immunospot reader (CTL Limited), and plaques in the sample wells, as well as in the negative control (DMEM alone) and positive control (100 pfu MR766 Zika virus alone), were counted using automated software equipped with an ELISpot reader. The percentage of plaque reduction was calculated as follows: % reduction = 100 X [1 - (average number of plaques per dilution in the positive control well / average number of plaques per average number)]. GraphPad Prism software was used to perform a non-linear regression analysis of plaque reduction % against the log transformation of each individual serum dilution to facilitate linear interpolation of the actual 50% PRNT titer at the peak response after vaccination. The median and interquartile ranges at 50% neutralization were calculated for each neutralization target, both globally and by vaccine treatment group; the geometric mean titer was also calculated.The titer is the reciprocal of the maximum dilution that induces a 50% reduction in the total number of plaques.

[0159] Flow cytometry and intracellular cytokine staining (ICS) assay

[0160] Splenocytes were added to 96-well plates (2 x 10⁶ / well) and stimulated with ZikapreM and enveloped peptides for 5 hours at 37°C / 5% CO₂ in the presence of a protein transport inhibitor cocktail (Brefeldin A and Monensin) (eBioscience). A cell stimulation cocktail (plus protein transport inhibitors) (Forbol 12-myristate 13-acetate (PMA), ionomycin, Brefeldin A, and Monensin) (eBioscience) was used as a positive control, and R10 medium was used as a negative control. All cells were subsequently stained for surface and intracellular proteins as described in the manufacturer's instructions (BD, San Diego, CA). Briefly, these cells were washed in FACS buffer (PBS containing 0.1% sodium azide and 1% FCS) prior to surface staining with fluorochrome-conjugated antibodies. Cells were washed with FACS buffer, fixed, and permeated using BD Cytofix / Ctyoperm™ (BD, San Diego, CA, USA) according to the manufacturer's protocol, followed by intracellular staining. The following antibodies were used for surface staining: LIVE / DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen), CD19 (V450; clone 1D3; BD Biosciences), CD4 (FITC; clone RM4-5; ebioscience), CD8 (APC-Cy7; clone 53-6.7; BD Biosciences); CD44 (BV711; clone IM7; Biolegend). For intracellular staining, the following antibodies were used: IFN-γ (APC; clone XMG1.2; Biolegend), TNF-α (PE; clone MP6-XT22; ebioscience), CD3 (PerCP / Cy5.5; clone 145-2C11; Biolegend); IL-2 (PeCy7; clone JES6-SH4; ebioscience).All data were collected using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR).

[0161] Statistical analysis

[0162] Graphpad, Prism 4 (Graphpad software, Inc. San Diego, CA) was used for statistical analysis. A log-10 transformation was applied to endpoint-bound ELISA titers and total viral PRNT50 titers.

[0163] The results of these experiments will now be explained.

[0164] Preparation of ZIKV-prME common DNA vaccine

[0165] The common sequence of Zika prM (progenitor membrane) and E (envelope) genes (ZIKV-prME) was derived using prM and E sequences from various Zikas isolated between 1952 and 2015 that caused human infection (Fig. 16a). The Zika-prME common sequence was cloned into the pVax1 vector after further modification and optimized to enhance its in vivo expression, including the addition of a highly efficient immunoglobulin E (IgE) leader peptide sequence (Fig. 16b). Endonuclease restriction cleavage and gene sequencing were used to validate the final vaccine plasmid (Fig. 16c). Expression of the Zika-prME protein from the plasmid was confirmed by performing Western spectroscopy and indirect immunofluorescence assays on vaccine-transfected 293T cells 48 hours after 84 transfections (Figs. 16d and 16e).

[0166] The Zika-pME DNA vaccine induces antigen-specific T cell or functional humoral responses in mice.

[0167] The ability of the Zika-prME plasmid vaccine to induce a cellular immune response was evaluated. A group of five C57 / BL6 mice was immunized with either the control plasmid core (pVax1) or the Zika-prME plasmid vaccine by three intramuscular injections at 2-week intervals, followed by electroporation (EP) at 92 delivery sites as described in the literature (Muthumani et al., 2015, Sci Trans Med 7:301ra132). Animals were sacrificed one week after their third injection, and bulk splenocytes harvested from each animal were evaluated for their ability to secrete interferon-γ after debody exposure to a peptide pool containing Zika-Env using a standard enzyme-linked immunospot assay. The results of the assay showed that splenocytes from Zika-prME-immunized mice produced a distinct cellular immune response after stimulation with multiple Zika-Env peptide pools (Fig. 17a). The regions of Zika-Env that induced the strongest cellular response(s) were evaluated by mapping the analysis ELISpots in a matrix format using 22 peptide pools consisting of 15-mers (overlapping by 11 amino acids) spanning the entire Zika-prME protein. As observed in Figure 17b, while several pools induce elevated T cell responses, peptide pool 15 is 10 6 The highest SFU was induced for each reaction. Mapping data revealed a single dominant prME epitope 'IRCIGVSNRDFVEGM (SEQ No.: 18)' for the sequence. The listed dominant peptide was confirmed to contain a single H2-Db restricted epitope by using the IDEP Common Tool, an immunoepitope database analysis resource, which implies effective processing of this antigen.

[0168] Further evaluation of the cellular immunogenicity of the Zika-prME vaccine involved determining the pluripotent nature of CD8+ T cells collected 1 week after final immunization. These results show that the Zika-prME vaccine increased the proportion of pluripotent vaccine-specific T cells expressing tumor necrosis factor-α (TNF-α) and IFN-γ (Fig. 17c). Importantly, Zika-prME vaccination exhibited a strong ability to amplify T-cell functionality. For comparative studies, additional vaccine research was performed using Plasmid 115 encoding the prME sequence of the recently identified Brazilian Zika strain or the original MR766 Zika strain. Induction of the cellular immune response in mice immunized with either plasmid was measured by IFN-γ ELISpot 1 week after the third injection, following stimulation of splenocytes with the same Zika-preME peptide pool as used in Fig. 17a. The results show that T cell and antibody responses induced by the novel common Zika-prME DNA vaccine construct were at least twice as high as those produced by either of these two non-common plasmid vaccines (Figures 18a and 18b). The aforementioned mapping analysis of cellular responses induced by the Brazil or MR766 prME vaccines revealed that both also induced their most significant cellular responses to the dominant Env-specific CTL epitope identified in Figure 17b for the common Zika-prME plasmid (data not presented). Overall, these common immunogens appeared to be consistently more robust in these assays and were further investigated.

[0169] The ability of the common Zika-prME vaccine to induce a humoral immune response in mice was evaluated. A group of C57 / BL6 mice was immunized three times at 2-week intervals with either an immature injection, followed by immunization with 25 μg of either an empty control plasmid or a common Zika vaccine plasmid via EP. Serum was obtained from each injected mouse on Day 0 (prior to the first immunization), Day 14 (2 weeks after the first immunization), Day 21 (1 week after the second immunization), and Day 35 (1 week after the third immunization). Each collected serum was tested by ELISA for Zika-specific IgG responses using fixed rZIKA-Env as the capture antigen. A significant increase in anti-Zika-specific IgG was observed on Day 21, and a further increase in serum IgG levels was observed in the Day 35 serum (Figure 19a). Sixty serum samples from animals vaccinated at day 138 demonstrate that the high antibody response observed in the day 35 serum was maintained for a long period after the final booster dose. Most importantly, the serum from the vaccinated mice contained very high levels of antibodies, as indicated by the endpoint titer (Fig. 19b). Further assessment of the specificity of the vaccine-induced antibody was performed by screening the day 35 mixed serum for its ability to detect rZIKA-E by Western spectroscopy (Fig. 19c) and to stain Zika-infected cells by immunofluorescence assay (Fig. 19d). From both of these analyses, the results confirmed the specificity.

[0170] In addition, Zika-specific binding antibody responses were also assessed in mice immunized with plasmids encoding prME sequences from the previously described Brazilian and MR766 strains. Day 35 serum from sham- or vaccine-immunized mice was analyzed in ELISA for binding to rZIKA-E. These analyses indicate that both plasmids induce significant antibody binding (Figures 18c and 18d), and that immunization with the common Zika-prME DNA vaccine yields an excellent humoral response with increased affinity for the heterologous Zika coat.

[0171] Plaque Reduction Neutralization Test (PRNT) assays were performed on 35-day mixed serum from mice immunized three times with empty pVax1, common Zika-prME plasmid vaccine, or common Zika-capsid plasmid vaccine using a method adapted from the previously described technique for analyzing DV, WNV, and other flaviviruses. As shown in Figure 19e, the anti-Zika mutual PRNT50 titer after the third vaccination was significantly higher in mice administered the Zika-prME vaccine than in mice administered the Zika-capsid DNA vaccine or control DNA pVax1. The neutralizing antibodies induced by the Zika-prME vaccine used in these experiments had a PRNT50 titer of 456. Representative photographs of viral plaques are shown below for a 1:100 dilution of serum.

[0172] Cellular and humoral responses induced by the Zika-prME DNA vaccine in non-human primates

[0173] NHPs were immunized intradermally (ID) followed by electroporation, based on previous research results showing that this method can enhance antigen-specific humoral immune responses induced by DNA vaccines. Himalayan monkeys (RM; n=5 / group) were ID-administered with 2.0 mg of the vaccine plasmid as EP, and serum and PBMCs were collected from RMs at Day 0 (pre-immunization prior to the first immunization), Week 2 (2 weeks after the first immunization), and Week 6 (2 weeks after the second immunization). To measure vaccine-induced cellular immune responses, ELISpot analysis was performed on Week 6 PBMC exoskeletons stimulated with the Zika-E peptide pool used in Figure 17a. These results show that Zika prME immunization boosted anti-Zika T-cell responses in all RMs and broadened their antigen recognition compared to responses in pre-immunization serum (Figure 20a).

[0174] Specific anti-Zika virus antibody responses in serum 181 from ID+EP-vaccinated RMs were assessed by ELISA. After the primary vaccination, Zika-Env-specific binding antibodies were detectable in RMs 2 weeks after the first immunization and were further boosted by subsequent immunizations (Fig. 20b). From the same time point after immunization, serum from vaccinated RMs was diluted to the endpoint titer and tested once again for rZIKA-Env (Fig. 20c). The ELISA results were confirmed by Western spectroscopy using RM serum collected from the vaccinated group (Fig. 20d). Furthermore, serum from immunized RMs was also able to recognize Zika-MR766-infected Vero cells in an immunofluorescence assay (Fig. 20e). Subsequently, attempts were made to detect neutralizing antibody (nAb) responses in serum from Zika-immunized RMs. PRNT50 (the inverse of the serum dilution at which 50% of the control Zika infection was inhibited) was used to test NAb activity and was performed on each individual immunized animal. Specimens with antibody titers < 10, which is the detection limit of the assay, were assigned to each group of animals. Interestingly, Zika-prME immunized monkeys had titer ranges from 161 to 1380 (mean 501±224) (Figure 21a).

[0175] Ability of NHP immune serum to block infection in important Zika-infected neuroblastoma cells (SK-N-SH cells) and neurostria cells (U-87MG). Cell lines with MR766 or PR209 in control or vaccine serum, and analyzed for infection at 24 hours. Serum from vaccinated RM inhibited either virus in both cell lines after infection (infection multiplicity of 1.0) (Figures 21b and 21c). These data support the utility of serum from Zika-prME DNA-vaccinated RM in inhibiting Zika infection.

[0176] Zika-specific functional immune response and protection against Zika virus in mice lacking the receptor for type I interferon (IFNAR) immunized with the Zika DNA vaccine

[0177] The mechanisms of Zika-induced disease and immunity are ill-defined, and the protective versus hypothetical pathogenic nature of the immune response to Zika infection is not yet clear. While most mouse strains are resistant to Zika infection, mice lacking the IFN-α / β receptor (IFNAR) are susceptible to infection and disease, with most found to collapse within 6–7 days of challenge. The ability of the common Zika-prME plasmid vaccine to induce cellular and humoral immune responses in these mouse strains was investigated. The IFNAR mouse population was immunized three times at 2-week intervals with either an EP-empty control plasmid or the common Zika-prME plasmid. Serum was collected from immunized mice on days 0, 14, 21, and 35, and splenocytes were harvested from mice one week after final immunization. Splenocytes from vaccine-immunized IFNAR mice produced a clear cellular immune response, as indicated by the level of SFU per 106 cells in the ELISpot assay (Fig. 22a). Results from ELISAs using rZIKA-Env as the capture antigen show that the animals had detectable anti-Zika serum IgG by day 14, and that these levels were boosted at subsequent collection points (Fig. 22b). Serum from the vaccinated mice contained significant levels of antibodies, as indicated by the endpoint titer (Fig. 22b). These results indicate that IFNAR mice immunized with the common Zika-prME vaccine can induce anti-Zika cellular and humoral immune responses, supporting further studies on vaccine protection in these potential challenge models.

[0178] In an exploratory study, IFNAR mice were challenged with 1 x 10⁶ PFU of PR209 isolates administered subcutaneously (sc); intraperitoneally (iv); intracranially (ic); and intravenously (iv). After challenge, all animals were monitored for transient body weight and body temperature measurements, as well as other clinical signs of necrosis, such as hindlimb weakness and paralysis. No changes in the mice's overall appearance were observed during the first two days after inoculation. However, after the third day, all four infection routes exhibited reduced activity, decreased mobility, and a crouching posture; accompanied by hindlimb weakness, water intake, and apparent body weight loss. Regardless of the challenge site, the animals collapsed with infection between days 6 and 8, and these challenge doses were utilized in subsequent studies.

[0179] Two groups of vaccinated animals (10 animals per group) or two sets of pVax1-immunized controls were vaccinated 1x on day 0 and 1x on day 14, and 1x on day 21. 6 PFU or 2x10 6 They were challenged with PFU's PR209 (Figures 23b and 23c). 100% of the vaccinated animals survived, whereas 1x10 6 30% or 2 x 10 of the PFU-challenged controls 6Only 10% of the PFU challenged controls survived. Next, the group of animals was immunized once and challenged 14 days after immunization. 100% of these animals survived, whereas only 10% of the control animals survived. All mice that were vaccinated once with Zika-prME and subsequently challenged with the Zika virus were protected from lethal challenge (Figure 23a). In all challenges, the vaccinated animals also did not exhibit symptoms of the disease and were protected from body weight loss (Figure 23d). Infection of control mice with the Zika virus resulted in a significant decrease in body weight, often combined with reduced mobility, a crouching posture, hind leg knuckle gait, and / or bilateral hind leg paralysis, along with a significant mortality rate (Figures 23e and 23f). Taken together, these data exemplify that the ZV-prME DNA vaccine mediated an immune response that protected mice against Zika challenge.

[0180] In this study, humoral and cellular responses utilizing prME derived from electroporation as an antigen, plus a DNA-based vaccine, were documented in rodents and non-human primates. The optimized enhanced DNA vaccine technology via the EP delivery approach was effective in stimulating robust and broad immune responses and monoimmunization-induced immunity that protect against disease and death in IFNAR mice. This study supports the concept that protective immunity can be induced against these serious emerging viral infections using flexible and rapidly clinically viable DNA vaccination strategies.

[0181] Example 3: In vivo protection against ZIKV infection and onset via passive antibody transfer and active immunization with prMEnv DNA vaccine

[0182] In this study, a novel synthetic DNA vaccine targeting the ZIKV pre-membrane + envelope protein (prMEnv) was produced and evaluated for in vivo efficacy. Following the initial in vitro development and evaluation studies of the aforementioned plasmid construct, mice and non-human primates were immunized with this prMEnv DNA-based immunogen via electroporation-mediated enhanced DNA delivery. Vaccinated animals were found to produce antigen-specific cellular and humoral immunity and neutralizing activity. Mice lacking receptors for interferon (IFN)-α / β (IFNAR - / - Immunization with these DNA vaccines (designated as) induced 100% protection against infection-associated weight loss or mortality, in addition to preventing viral pathology in brain tissue following in vivo viral challenge. Furthermore, passive delivery of non-human primate anti-ZIKV immune serotypes provided IFNAR protection against subsequent viral challenge. - / - This initial study of the ZIKV vaccine in a pathogenic mouse model supports the importance of immune responses targeting prME in ZIKV infection and suggests that further research on this vaccine approach may be relevant for ZIKV control in humans.

[0183] The materials and methods will be explained from now on.

[0184] Cells, viruses, and animals

[0185] Human embryonic kidney 293T (American Type Culture Collection (ATCC) #CRL-N268, Manassas, VA, USA) and Vero CCL-81 (ATCC #CCL-81) cells were maintained in DMEM (Dulbecco's Modified Eagle Medium; Gibco-Q3 Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin, and passaged at confluence. Both ZIKV virus strains MR766 (goodwill gift from Dr. Susan Weiss) and PR209 (Bioqual, MD) were amplified in Vero cells, and stocks were titrated in Vero cells by the standard plaque assay. 5- to 6-week-old female C57BL / 6 (The Jackson Laboratory) and IFNAR - / - (MMRRC Repository - The Jackson Laboratory) Mice were housed, treated, or vaccinated in temperature-controlled, light-cycle facilities in accordance with the guidelines of the National Institutes of Health (NIH), Wistar, and the Public Health Agency of Canada's IACUC (Institutional Animal Care and Use Committee).

[0186] RMs were received, treated, or vaccinated at Bioqual, MD, USA. This study was conducted in strict compliance with the recommendations described in the Guide for the Care and Use of Laboratory Animals from the NIH, the Office of Animal Welfare, and the U.S. Department of Agriculture. All animal immunization procedures were approved by the Bioqual Animal Care and Use Committee (IACUC). Bioqual is accredited by the American Association for Accreditation of Laboratory Animal Care. All procedures were performed under ketamine anesthesia by skilled personnel under the supervision of veterinary staff, and every effort was made to protect animal welfare and minimize animal suffering in accordance with the recommendations of the Weatherall report for the use of non-human primates. These animals were housed in adjacent individual primate cages that allowed for social interaction under controlled conditions of humidity, temperature, and lighting (12-hour light / 12-hour dark cycle). Food and water were available in unlimited quantities. These animals were monitored twice a day, and commercial monkey food, treats, and fruit were fed twice a day by skilled personnel.

[0187] Preparation of ZIKV-prME DNA vaccine

[0188] A ZIKV-prME plasmid DNA construct encoding the full-length precursors of the membrane (prM), envelope (E), and capsid proteins was synthesized. A common strategy was utilized, and the common sequence was determined by the alignment of the current ZIKV prME protein sequence. The vaccine insert was genetically optimized for enhanced expression in humans (i.e., codon and RNA optimization), and an IgE leader sequence was added to facilitate expression. This construct was commercially synthesized (Genscript, NJ, USA) and subsequently subcloned into a modified pVax1 expression vector under the control of a cytomegalovirus ultra-early promoter, as described in the existing literature (Muthumani et al., 2016, Sci Transl Med 7:301ra132). The final construct was named the ZIKV-prME vaccine, and the control plasmid backbone is pVax1. In addition, several other aligned DNA constructs encoding the prM and E genes from the MR766 (DQ859059.1) and 2016 Brazil (AMA12084.1) strains were also designed for further evaluation. Large-scale amplification of the DNA constructs was performed by Inovio Pharmaceuticals Inc. (Plymouth Meeting, PA, USA), and the purified plasmid DNA was prepared in water for immunization. The size of the DNA inserts was confirmed by agarose gel electrophoresis. Phylogenetic analysis was performed by multiple alignment with ClustalW using MEGA version 5 software (Muthumani et al., 2016, Sci Transl Med 7:301ra132).

[0189] DNA Immunization and Electroporation-Mediated Delivery Enhancement

[0190] Female C57BL / 6 mice (6-8 weeks old) and IFNAR - / -Mice (5–6 weeks old) were immunized with 25 μg of DNA in a total volume of 20 or 30 μl of water delivered into the tibialis anterior muscle via in vivo electroporation. In vivo electroporation was performed at the same site immediately after DNA injection using a CELLECTRA adaptive constant current EP device (Inovio Pharmaceuticals). The 3-pronged CELLECTRA minimally invasive device was inserted approximately 2 mm into the muscle. Square wave pulses were delivered through a triangular 3-electrode array consisting of 26-gauge solid stainless steel electrodes, and two constant current pulses of 0.1 Amp were delivered for 52 μs / pulse separated by a 1-second delay. Additional protocols for the use of electroporation are described in detail in the existing literature (Flingai et al., 2015, Sci Rep 5:12616). These mice were immunized three times at 2-week intervals and sacrificed 1 week after final immunization. Blood was collected after each immunization for analysis of cellular and humoral immune responses (Muthumani et al., 2016, Sci Transl Med 7:301ra132). Himalayan monkey immunogenicity study: Five Himalayan monkeys were intradermally immunized with 2 mg ZIKV-prME vaccine at two sites at 5-week intervals. Electroporation was performed immediately using the same device described for mouse immunization.

[0191] Western blot analysis

[0192] For in vitro expression studies, transfection was performed using GeneJammer reagents according to the manufacturer's protocol (Agilent). Briefly, cells were grown to 50% confluence in 35 mm dishes and transfected with 1 μg of ZIKV-prME vaccine. These cells were collected 2 days after transfection, washed twice with PBS, and lysed with cell lysis buffer (Cell Signaling Technology). Western blot was used to demonstrate the expression of ZIKV-prME protein from harvested cell lysates and the immunospecificity of mice and RM serum through the use of anti-flavivirus (Flavivirus) or immune serum from ZIKV-prME vaccinated mice, as described in the literature (Muthumani et al., 2016, Sci Transl Med 7:301ra132). Briefly, 3-12% Bis-Tris NuPAGE gels (Life Technologies) were dropwise loaded with 5 μg or 1 μg of ZIKV envelope recombinant protein (rZIKV-E); transfected cell lysates or supernatants; and Odyssey protein molecular weight markers (Product # 928-40000). The gels were moved in MOPS buffer at 200 V for 50 minutes. These proteins were transferred onto nitrocellulose membranes using an iBlot 2 Gel delivery device (Life Technologies). These membranes were blocked in PBS Odyssey blocking buffer (LI-COR Biosciences) for 1 hour at room temperature. To detect vaccine expression, the anti-Flavivirus (Flavivirus) herd antigen (MAB10216-clone D1-4G2-4-15) antibody was diluted 1:500, and immune serum from mice and RMs was diluted 1:50 in Odyssey blocking buffer containing 0.2% Tween 20 (Bio-Rad) and incubated with these membranes overnight at 4°C.These membranes were washed with PBST and subsequently incubated with appropriate secondary antibodies (goat anti-mouse IRDye680CW for mouse serum and flavivirus antibody; LI-COR Biosciences; and goat anti-human IRDye800CW for RM serum (LI-COR Biosciences)) at a dilution of 1:15,000 against mouse serum for 1 hour at room temperature. After washing, these membranes were imaged on an Odyssey infrared imaging device (LI-COR Biosciences).

[0193] Immunofluorescence assay

[0194] For immunofluorescence assays, cells were grown on coverslips and transfected with 5 μg of ZIKV-prME vaccine. Two days after transfection, these cells were fixed with 4% paraformaldehyde for 15 minutes. Non-specific binding was subsequently blocked with normal goat serum diluted in PBS for 1 hour at room temperature. The slides were then washed in PBS for 5 minutes and subsequently incubated with serum from immunized mice or RM at a 1:100 dilution overnight at 4°C. These slides were washed as previously described and incubated with appropriate secondary antibodies (goat anti-mouse IgG-AF488 for mouse serum and goat anti-human IgG-AF488 for RM serum; Sigma) at a 1:200 dilution for 1 hour at room temperature. After washing, Flouroshield fixation medium containing DAPI (Abcam) was added to stain the nuclei of all cells. Subsequently, coverslips were loaded, and slides were observed under a microscope (EVOS Cell Imaging Systems; Life Technologies) (Muthumani et al., 2016, Sci Transl Med 7:301ra132). In addition, Vero, SK-N-SH, or U87-MB cells were grown on glass slides treated with 4-chamber tissue culture medium, infected with ZIKV-MR766 or PR209 pre-cultured with / without RM immunoseroles (1:200) at an MOI of 0.01, and stained 4 days after ZIKV infection using pan-flavivirus antibodies as described in the literature (Rossi et al., 2016, J Rop Med Hyg 94:1362-9).

[0195] Histopathology analysis

[0196] For histopathology, formalin-fixed, paraffin-embedded brain tissue was divided into 5 μm thick sagittal sections, placed on Superfrost microscope slides (Fisher Scientific), and supported overnight at 37°C. These sections were deparaffinized using two changes of xylene and rehydrated by immersion in 100%, 90%, and subsequently 70% ethanol. These sections were stained for nuclear structures using Harris hematoxylin (Surgipath) for 2 minutes, followed by differentiation in 1% acidic alcohol (Surgipath) and treatment with Scott tap water for 2 minutes. Subsequently, these sections were counterstained for cytoplasmic structures using eosin (Surgipath) for 2 minutes. Slides were dehydrated with 70%, 90%, and 100% ethanol, cleaned in xylene, and loaded using Permount (Fisher Scientific).

[0197] Isolation of splenocytes and PBMCs

[0198] Single-cell suspensions of splenocytes were prepared from all mice. Briefly, splenocytes from mice were individually collected in 5 ml of RPMI 1640 supplemented with 10% FBS (R10) and subsequently processed in a Stomacher 80 paddle blender (AJ Seward and Co. Ltd.) at high speed for 30 seconds. The processed splenocyte specimens were filtered through a 45 mm nylon filter and subsequently centrifuged at 1,500 g for 10 minutes at 4°C. The cell pellets were resuspended in 5 ml of ACK (Ammonium Chloride-Potassium) lysis buffer (Life Technologies) for 5 minutes at room temperature, after which PBS was added to stop the reaction. These specimens were once again centrifuged at 1,500 g for 10 minutes at 4°C. The cell pellet was resuspended in R10 and subsequently passed through a 45 mm nylon filter before use in the ELISpot assay and flow cytometry analysis (Muthumani et al., 2016, Sci Transl Med 7:301ra132). For RM, blood (20 ml at each time point) was collected in EDTA tubes, and PBMCs were isolated into Accuspin tubes (Sigma-Aldrich, St. Louis, MO, USA) using the standard Ficoll-hypaque procedure. 5 ml of blood was also collected into serum tubes at each time point for serum isolation.

[0199] Flow cytometry and intracellular cytokine staining

[0200] Splenocytes in a 96-well plate (2x10 6Cells were added to the wells and stimulated with ZIKV-prME collected peptides for 5 hours at 37°C / 5% CO2 in the presence of a protein transport inhibitor cocktail (Brefeldin A and Monensin; eBioscience). A cell stimulation cocktail (plus protein transport inhibitors; PMA (Forbol 12-Myristate 13-Acetate), Ionomycin, Brefeldin A, and Monensin; eBioscience) was used as a positive control, and R10 medium was used as a negative control. All cells were subsequently stained for surface and intracellular proteins as described in the manufacturer's instructions (BD Biosciences, San Diego, CA, USA). Briefly, these cells were washed in FACS buffer (PBS containing 0.1% sodium azide and 1% FBS) prior to surface staining with fluorochrome-conjugated antibodies. These cells were washed with FACS buffer, fixed, and permeated using BD Cytofix / Ctyoperm™ (BD Biosciences) according to the manufacturer's protocol, followed by intracellular staining. The following antibodies were used for surface staining: LIVE / DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen), CD19 (V450; clone 1D3; BD Biosciences), CD4 (FITC; clone RM4-5; eBioscience), CD8 (APC-Cy7; clone 53-6.7; BD Biosciences); CD44 (BV711; clone IM7; BioLegend). For intracellular staining, the following antibodies were used: IFN-γ (APC; clone XMG1.2; BioLegend), TNF-α (PE; clone MP6-XT22; eBioscience), CD3 (PerCP / Cy5.5; clone 145-2C11; BioLegend); IL-2 (PeCy7; clone JES6-SH4; eBioscience).All data were collected using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

[0201] ELISpot test

[0202] Briefly, 96-well ELISpot plates (Millipore) were coated with antimicrobial mouse IFN-γ capture antibodies (R&D Systems) and incubated overnight at 4°C. The next day, the plates were washed with PBS and blocked with PBST + 1% BSA for 2 hours. Two hundred thousand splenocytes from immunized mice were added to each well and incubated overnight at 37°C in 5% CO2 in the presence of medium alone (negative control), medium containing PMA / ionomycin (positive control), or medium containing a peptide pool (1 μg / ml) consisting of a 15-mer (Genscript) that overlaps by 9 amino acids and spans the length of the ZIKV prME protein. After 24 hours, these cells were washed and subsequently incubated overnight at 4°C with biotinylated antimicrobial mouse IFN-γ antibodies (R&D Systems). Streptavidin-alkaline phosphatase (R&D Systems) was added to each well after washing and subsequently incubated at room temperature for 2 hours. The plates were washed and subsequently, 5-bromo-4-chloro-3'-indolylphosphate p-toluidine salt and nitro blue tetrazolium chloride (dye source color reagent; R&D Systems) were added. Finally, the plates were rinsed with distilled water, dried at room temperature, and SFU was quantified by an automated ELISpot reader (CTL Limited), and raw values ​​were normalized to SFU per million splenocytes. For RM specimens, ELISpot for the Monkey IFN-γ Kit. PRO(MABTECH) was used as described by the manufacturer, 200,000 PBMCs were stimulated with a peptide pool, and the plates were washed and spots were colored and counted as described in existing literature (Muthumani et al., 2016, Sci Transl Med 7:301ra132).

[0203] Humoral Immune Response: Antibody-Binding ELISA

[0204] ELISA was used to determine the titers of mouse and RM serum as described in the existing literature (Muthumani et al., 2016, Sci Transl Med 7:301ra132). Briefly, 1 μg of purified rZIKV-E protein was used to coat 96-well micro-titer plates (Nalgene Nunc International, Naperville, IL, USA) overnight at 4°C. After blocking with 10% FBS in PBS for at least 1 hour, the plates were washed four times with 0.05% PBST (Tween20 in PBS). Serum samples from immunized mice and RM were serially diluted in 1% FBS, added to the plates, and subsequently incubated at room temperature for 1 hour. These plates were washed four times again with 0.05% PBST and subsequently incubated with HRP-conjugated antimicrobial mouse IgG (Sigma) at a dilution of 1:35,000 for mouse serum for 1 hour at room temperature. For RM serum, anti-monkey IgG HRP (Southern Biotech) was used at a dilution of 1:5,000 for 1 hour at room temperature. The conjugated enzyme was detected by adding SIGMAFAST OPD (o-phenylenediamine bihydrochloride) substrate solution according to the manufacturer's instructions (Sigma-Aldrich). The reaction was stopped after 15 minutes with the addition of 1N H2SO4. Absorbance at 450 nm was read on a Synergy plate reader. All mouse and RM serum specimens were double-checked. The endpoint potency was determined using the method described in the existing literature (Frey et al., 1998, J Immunol Methods 21:35-41).

[0205] Junghwa (PRNT) 50 ) black

[0206] PRNT involving MR766 and Vero cells has been described in existing literature (Sun et al., 2006, J Infect Dis 193:1658-65). Briefly, heat-inactivated mouse or RM serum was serially diluted (from 1:10 to 1:1280) in serum-free DMEM and incubated with an equivalent volume of ZIKV MR766 (100 pfu) for 2 hours at 37°C. The mixture was added to the confluent layer of Vero cells and left at 37°C for 2 hours for adsorption. A 2 x DMEM medium:soft agar (1:1) overlay was added over the cells, and the plates were incubated at 37°C for 5 days. The agar overlay was removed, and the cells were fixed with 4% paraformaldehyde, washed with 1x PBS, stained with crystal violet solution, washed with 1x PBS, and the plates were left to dry. In the assay performed on 24-well plates, plaques were scanned using an automated immunospot reader (CTL Limited), and plaques in the sample wells, as well as in the negative control (DMEM alone) and positive control (100 pfu MR766 ZIKV virus alone) wells, were counted using automated software equipped with an ELISpot reader. The percentage of plaque reduction was calculated as follows: % reduction = 100 x {1 - (each dilution of plaques in the positive control wells / average number of plaques for each average number)}. GraphPad Prism software was used to perform a non-linear regression analysis of the % plaque reduction against the log-transform of each individual serum dilution to facilitate linear interpolation of the true 50% PRNT titer in the post-vaccination peak response. The median and interquartile ranges at 50% neutralization were calculated for each neutralization target, both overall and by vaccine treatment group; geometric mean titers were also calculated. These titers are the reciprocals of the maximum dilution that induces a 50% reduction in the total number of plaques.

[0207] IFNAR - / - ZIKV challenge study in mice

[0208] For the Zika challenge study, IFNAR - / - Mice (n = 10 / group) were immunized once or twice with the Zika-prME vaccine or pVax1. These mice were vaccinated at 1 x 10⁶ on day 15 (single-immunization group) or on day 21 (double-immunization group), one week after the second immunization. 6 PFU or 2 x 10 6 Immunized with PFU ZIKV-PR209 virus. Also, IFNAR - / - An additional group of mice (n = 10 / group) was immunized once, and 2x10 on day 15 6 The animals were challenged with the PFU ZIKV-PR209 virus. After challenge, the animals were weighed, and their body temperature was measured daily using a subcutaneous temperature chip. In addition, they were observed twice daily for clinical signs of the disease (reduced mobility; crouching posture; hind leg knuckle gait (partial paralysis), paralysis of one or both hind legs), and blood was drawn to determine the viral load. Criteria for euthanasia on a welfare basis consisted of a 20% reduction in body weight or paralysis of one or both hind legs.

[0209] Real-time RT-PCR assay for ZIKV load measurement

[0210] Brains from treated mice were placed in RNAlater (Ambion) at 4°C for 1 week and subsequently stored at -80°C. The brain tissue was then quantified and homogenized in 600 μl RLT buffer in a 2 ml cryo-vial using a tissue pulverizer (Qiagen) containing stainless steel beads at 30 cycles / s for 6 minutes. Viral RNA was also isolated from blood using the RNeasy Plus Mini Kit (Qiagen). A ZIKV-specific real-time RT-PCR assay was utilized for the detection of viral RNA from individual animals. RNA was reverse transcribed and amplified using primers ZIKV 835 and ZIKV 911c and probe ZIKV 860FAM in conjunction with the TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems). Standard curves were calculated in parallel for each plate and used to quantify the viral genome copy number. StepOnePlus Real-time PCR System (ABI) software version 2.3 was used to calculate the cycle threshold (Ct) value, and a Ct value ≤38 for at least one replicate was considered positive as described in existing literature (Lanciotti et al., 2008, Emerg Infect Dis 14:1232-9). Whole blood (pre-bleed) was negative in this test.

[0211] Statistical analysis

[0212] Differences in the fold increase of antibody titers were compared using Mann-Whitney analysis. Statistical analysis was performed using Graphpad, Prism 4 (Graphpad software, Inc. San Diego, CA, USA). For all analyses, P<0.05 was considered significant. Log 10 Transformation endpoints: ELISA titer and whole virus PRNT 50 It was applied to the potency.

[0213] The results of these experiments will now be explained.

[0214] Preparation of ZIKV-prME common DNA vaccine

[0215] The common sequence of ZIKV prM (precursor membrane) and Env (envelope) genes (ZIKV-prME) was derived using prM and Env sequences from various ZIKV strains isolated between 1952 and 2015 that caused human infection. The ZIKV-prME common sequence was cloned into the pVax1 vector after further modification and optimized to enhance in vivo expression, including the addition of a highly efficient immunoglobulin E (IgE) leader peptide sequence (Fig. 24a). Optimal alignment of the ZIKV-envelope sequence was performed using homology models and visualization in Discovery Studio 4.5. Reference models included PDB 5JHM and PDB 5IZ7. Aligned residues corresponding to specific regions on the prME antigen were labeled in these models for visualization purposes (Fig. 24b). Optimized common vaccine screening is generally conserved or semi-conserved compared to the multiple ZIKV lineages analyzed in this study. Structural studies of EDE-specific neutralizing antibodies revealed that these recognition determinants can be found in serotype-invariant sites at the envelope-dimer interface, which include the exposed major chain of the fusion loop and two conserved glycan chains (N67- and N153-linked glycans) (Rouvinski et al., 2015, Nature 520:109-13). These two glycosylation sites are not highly conserved in other flaviviruses. Furthermore, ZIKV does not possess an N67-linked glycosylation site, and an N154-linked glycosylation site (equivalent to the N153-linked glycosylation site in dengue) is absent in some of the isolated ZIKV lineages. For this reason, as part of a common design, a construct excluding these glycosylation sites was designed. The absence of glycosylation at these sites was correlated with enhanced binding of EDE1-type broad-spectrum neutralizing antibodies (bnAbs) to the ZIKV-envelope protein (Rouvinski et al., 2015, Nature 520:109-13).

[0216] Following synthesis, the expression of ZIKV-prME protein from the plasmid was confirmed by Western blot analysis and indirect immunofluorescence assay. Protein extracts prepared from cells transiently transfected with ZIKV-prME were analyzed for expression by Western blot using panflavivirus antibodies (Fig. 24c) and serum collected from ZIKV-prME immunized mice (Fig. 24d). ZIKV-prME expression was further detected by IFA by staining 293T cells transfected with the ZIKV-prME plasmid with an anti-ZIKV-prME specific antibody 48 hours after transfection (Fig. 24e).

[0217] The ZIKV-prMEnv DNA vaccine induces antigen-specific T cells in C57BL / 6 mice.

[0218] The ability of the ZIKV-prMEnv plasmid vaccine to induce a cellular immune response was evaluated. A group of four female C57BL / 6 mice were immunized with either the control plasmid core (pVax1) or the ZIKV-prME plasmid vaccine three times at 2-week intervals, followed by intramuscular (im) injection and subsequent electroporation at the delivery site (Fig. 25a). These animals were euthanized one week after their third injection, and bulk splenocytes harvested from each animal were evaluated in the ELISpot assay for their ability to secrete interferon-γ (IFN-γ) after desalination and exposure to a peptide pool containing ZIKV-prME. The assay results showed that splenocytes from ZIKV-prME immunized mice produced a cellular immune response after stimulation with multiple ZIKV-E peptide pools (Fig. 25b). The regions of ZIKVEnv that induced the strongest cellular response(s) were evaluated by ELISpot assay in a matrix format using a pool of 22 peptides consisting of 15-mers (overlapping by 11 amino acids) spanning the entire ZIKV-prME protein. Several pools showed elevated T-cell responses, with peptide pool 15 exhibiting the highest number of spot-forming units (SFU) (Fig. 25c). This matrix mapping analysis revealed the dominant prME epitope, 'IRCIGVSNRDFVEGM (SEQ No.: 17)' (aa167-181). This peptide was confirmed to contain an H2-Db limited epitope through analysis utilizing an immunoepitope database analysis resource tool, which supports that the antigen is effectively processed in this haplotype.

[0219] Further evaluation of the cellular immunogenicity of the ZIKV-prMEnv vaccine was performed using CD8 collected 1 week after final immunization. +It involved the determination of the pluripotent properties of T cells. The results show that ZIKV-prMEnv vaccination increased the proportion of dual-function vaccine-specific T cells expressing TNF-α (tumor necrosis factor-α) and IFN-γ. Importantly, ZIKV-prMEnv vaccination exhibited a strong ability to amplify T-cell functionality (Fig. 25d).

[0220] In addition, comparative immunological studies were performed using optimized plasmids encoding the prMEnv sequences of the recently identified Brazilian ZIKV lineage or the original MR766 ZIKV lineage. The induction of cellular immune responses in mice immunized with either plasmid was measured via the IFN-γ ELISpot assay one week after the third vaccination, following the stimulation of splenocytes with the ZIKV-prMEnv peptide pool. The results showed that T cell responses induced by the common ZIKVprME DNA vaccine construct were consistently higher than those produced by either of these two non-common plasmid vaccines (Figures 31a and 31b). The above-described mapping analysis of cellular responses induced by the Brazil or MR766 prME vaccines revealed that both vaccines induced significant cellular responses to the dominant Env-specific CTL epitopes identified in Figures 25b and 25c for the common ZIKV-prMEnv plasmid (data not presented). The common immunogen consistently induced more robust responses in these T-cell assays at the same dose and was further evaluated in additional assays.

[0221] Production of ZIKV recombinant coat protein

[0222] At the start of these studies, there were no commercially available reagents to evaluate specific anti-ZIKV immune responses. For this reason, recombinant ZIKV-envelope protein (rZIKV-E) was produced out of necessity to support the assays performed in this study. To produce this reagent, a common ZIKV-envelope sequence based on the ZIKV-prME vaccine common antigen was cloned into a pET30a Escherichia coli expression vector (Fig. 32a). The rZIKV-E antigen was produced from E. coli cultures, purified using nickel column chromatography, and analyzed using SDS-PAGE, which revealed an overexpressed protein of predicted size in the lysate from rZIKV-E transfected bacteria that could be detected by Western chromatography using an anti-His tag antibody (Fig. 32b). Serum from mice immunized with the ZIKV-prME vaccine bound to rZIKV-Env, which is used as a capture antigen in an ELISA (enzyme-linked immunosorbent assay; Fig. 32c). A commercial antibody that reacts to the envelope protein of multiple flaviviruses (designated as pan-flavivirus) also bound to rZIKV-E. Western analysis demonstrated that the immune serum from mice immunized with ZIKV-prMEnv specifically recognizes rZIKV-E (Fig. 32d). These data indicate that the produced rZIKV-E specifically reacted with the immune serum from mice vaccinated with ZIKV-prMEnv, and thus these recombinant proteins were used for further immunogenicity studies.

[0223] Induction of functional humoral response in C57BL / 6 mice by ZIKV-prME DNA vaccine

[0224] The ability of the common ZIKV-prMEnv vaccine to induce a humoral immune response in mice was evaluated. A group of four C57BL / 6 mice was intramuscularly immunized with 25 μg of either empty control pVax1 or the common ZIKV-prMEnv vaccine plasmid via three electroporation-mediated deliveries at 2-week intervals (im). Serum was obtained from each immunized mouse and tested for ZIKV-specific IgG responses by ELISA using immobilized rZIKV-E as the capture antigen. A significant increase in anti-ZIKV-specific IgG was observed at day 21, and a further increase in serum IgG levels was observed at day 35 (Figure 26a). At day 60, serum from the vaccinated animals showed that the elevated ZIKV-specific antibody response was maintained for an extended period following the final booster dose. Most importantly, serum from vaccinated mice contained very high levels of rZIKV-E-specific antibodies, as indicated by the endpoint titer (Fig. 26b). Further assessment of the specificity of the vaccine-induced antibodies was performed by screening mixed serum from mice inoculated with the ZIKVprMEnv plasmid for their ability to detect rZIKV-E (envelope) by Western spectroscopy (Fig. 26c) and stain ZIKV (MR766 lineage)-infected cells by immunofluorescence assay (Fig. 26d). From both of these analyses, the results confirmed the specificity of the vaccine-induced humoral response.

[0225] Furthermore, ZIKV-specific binding antibody responses were also assessed in mice immunized with plasmids encoding the prMEnv sequence from the previously described Brazilian and MR766 strains. At day 35 (1 week after the third immunization), serum from pVax1- and both non-common vaccine-immunized mice was analyzed by ELISA for binding to rZIKV-E. These analyses indicate that both the MR766 and Brazilian vaccine plasmids induce significant antibody binding, and that immunization with the common ZIKV-prME DNA vaccine yields an effective humoral response to rZIKV-E (Figures 31c and 31d).

[0226] Plaque Reduction Neutralization Test (PRNT) assays were performed on 35-day serum collected from (3 x) mice immunized with a control pVax1 plasmid, the common ZIKV-prMEnv plasmid vaccine, or the common ZIKV-C (capsid) plasmid vaccine. The PRNT assay used was a method adapted from a technique described in existing literature (Davis et al., 2001, J Virol 75:4040-7) for analyzing dengue virus, West Nile virus, and other flaviviruses. As shown in Figure 26e, ZIKV-prME immunization resulted in an anti-ZIKV reciprocal PRNT of 456 ± 5. 50 A significant neutralization response was produced at the dilution titer (the inverse of the serum dilution level at which 50% of the control ZIKV infection was inhibited), whereas mice vaccinated with the ZIKV-Cap DNA vaccine showed only a titer (33 ± 6) that was minimally higher than that of animals vaccinated with the pVax1 control plasmid (titer = 15 ± 2).

[0227] Mice lacking type I interferon receptors after immunization with ZIKV-prME DNA vaccine (IFNAR - / - Immune response and protection against ZIKV in )

[0228] The mechanisms of ZIKV-induced disease and immunity are ill-defined, and the protective versus hypothetical pathogenic nature of the immune response to ZIKV infection is not yet clear (Rossi et al., 2016, J Rop Med Hyg 94:1362-9). While most mouse strains are resistant to ZIKV infection, mice lacking IFN-α / β receptors (IFNAR - / - It was found that ) is susceptible to infection and disease, and most collapse within 6–7 days after challenge (Lazear et al., 2016, Cell Host Microbe 19:720–30). The ability of the common ZIKV-prME plasmid vaccine to induce cellular and humoral immune responses in these mouse strains was investigated. 5 to 6-week-old female IFNAR - / - Mice (n = 4) were im-immunized with either the control pVax1 plasmid or the ZIKV prME vaccine plasmid via three electroporation-mediated deliveries at 2-week intervals. Serum was collected from immunized mice on days 0, 14, 21, and 35, and splenocytes were harvested from mice 1 week (day 35) after final immunization. Splenocytes from vaccine-immunized mice were 10 in the ELISpot assay 6 Each cell produced a distinct cellular immune response as indicated by the SFU levels (Figure 33a). From the ELISA assay using rZIKV-E as the capture antigen, results showed detectable anti-ZIKV serum IgG (titers of ~1:1,000) up to day 14, and these levels were boosted by subsequent vaccination, with binding antibody titers reaching at least 1:100,000 (Figures 33b and 33c). In contrast, PRNT for samples 35 days post-immunization 50 The titer was 1:60. These results are for IFNAR immunized with the common ZIKV-prMEnv vaccine - / -This indicates that mice can produce anti-ZIKV cellular and humoral immune responses that support further studies in this model of presumed vaccine efficacy upon pathogenic challenge.

[0229] ZIKV-specific functional cellular and humoral responses induced by the ZIKV-prMEnv DNA vaccine in non-human primates

[0230] Based on recent studies showing a potent immune response in a lower voltage intradermal format, NHP was immunized by intradermal immunization using intradermal electroporation (Hutnick et al., 2012, Hum gene Ther 23:943-50; Broderick et al., Mol Ther Nucleic Acids 1:e11). Himalayan monkeys (RM; n = 5 / group) were administered 2.0 mg of vaccine plasmid intradermally via electroporation, with each animal vaccinated twice at a 4-week interval. Serum and peripheral blood mononuclear cells (PBMCs) were collected on day 0 (before immunization) and week 6 (2 weeks after the second immunization). ELISpot analysis of 6-week-old PBMCs pre-immunization and debody-stimulated with the ZIKV-prMEnv peptide pool showed that ZIKV-prMEnv immunization induces a robust anti-ZIKV T cell response in RM (Figure 27a).

[0231] Specific anti-ZIKV antibody responses in serum from vaccinated RMs were assessed by ELISA. At week 6, rZIKV-Env-specific binding antibodies were detectable in animals vaccinated with ZIKV-prMEnv (Figure 27b). Endpoint titers were determined for each animal at week 2 (after one immunization) and week 6 (after two immunizations; Figure 27c). The ELISA results were confirmed by Western blot analysis using RM serum from individual vaccinated animals (Figure 27d). The neutralizing activity of antibodies produced from RMs at week 6 was PRNT 50 It was evaluated by assay. All vaccinated monkeys showed anti-ZIKV reciprocal PRNT varying in the range of 161 to 1380.50 It had significant neutralizing activity at dilution titers (mean 501 ± 224 standard error of the mean above; Figure 27e). PRNT titers were not directly correlated with ELISA titers (data not presented).

[0232] The ability of NHP vaccine immunized serum to block ZIKV infection in Vero cells, neuroblastoma (SK-N-SH), or neurostriatal (U-87MG) cells in vitro was investigated by IFA. ZIKV Q2 lineages (MR766 or PR209) were pre-cultured in serum or dilutions of NHP-immunized serum and added to monolayers of each cell type. Four days post-infection, ZIKV-positive cells were identified by IFA using panflavivirus antibodies (Figures 34a-34c), and ZIKV-positive cells were quantified (Figures 34b-34d). Serum from Zika-prME-vaccinated RMs inhibited ZIKV infection in each cell type.

[0233] IFNAR after ZIKV-prME immunization - / - Protection against ZIKV infection and disease in mice

[0234] In an exploratory study, 5-6 week old IFNAR (- / - ) Mice (n = 10) were administered 1 x 10⁶ by subcutaneous (sc); intraperitoneal (ip); intracranial; or intravenous (iv) route. 6The animals were challenged with ZIKV-PR209 isolates containing plaque-forming units (PFU). After challenge, all animals were monitored for clinical signs of infection, including transient body weight measurements as well as other signs of necrosis, such as hindlimb weakness and paralysis. No changes in the overall appearance of the mice were observed during the first four days after inoculation. However, after the fourth day, mice in each group exhibited reduced overall activity, decreased mobility, and a crouching posture, often accompanied by hindlimb weakness, reduced water intake, and apparent body weight loss. These animals collapsed with infection between days 6 and 8, regardless of the viral challenge route (Figures 35a-35e). Based on these data, subsequent studies to evaluate ZIKV-prME-mediated protection in these models utilized the sc route for challenge.

[0235] The protective effect of the ZIKV-prMEnv vaccine is then, these IFNARs - / - It was evaluated in mouse models. Two groups of mice (n = 10) were immunized with the ZIKV-prME vaccine via the im route through electroporation-mediated delivery (25 μg of vaccine). Additionally, two groups of 10 mice were immunized with the control pVax1 vector via the im route through electroporation-mediated delivery. Immunization was performed twice at a 2-week interval, and all animals were challenged on day 21 (1 week after the second immunization). One set of control and immunized mice was immunized with 1 x 10⁶ via the sc route. 6 We received ZIKV-PR209 from the PFU, and different sets for each group were provided via the sc route, totaling 2 x 10 6They were challenged with PFU ZIKV-PR209. At 3 weeks post-challenge, 100% of all ZIKV-prME-inoculated animals survived, whereas only 30% of single-challenged controls or 10% of double-dose challenged controls survived (Figures 28a and 28b). In all challenges, the vaccinated animals showed no signs of disease, including no evidence of body weight loss (Figures 28c and 28d). Infection of control mice with the ZIKV-PR209 virus resulted in a significant decrease in body weight, accompanied by reduced mobility, a crouching posture, hind leg knuckle gait, and / or paralysis of one or both hind legs (Figures 28e and 28f).

[0236] IFNAR from ZIKV challenge inoculation - / - The potential capability of monoimmunization with the ZIKVprME DNA vaccine protecting mice was evaluated. A group of 10 mice was im-immunized by electroporation with either a control plasmid or the ZIKV-prME vaccine, and after 2 weeks, 2 x 10 6The animals were challenged with a double full dose of PFU ZIKV-PR209. At 3 weeks post-challenge, 100% of the animals vaccinated with ZIKV-prME survived, whereas only 10% of the control animals survived (Figure 29a). To determine macroscopic histopathological changes, brain tissues were divided into 5 μm-thick sagittal sections, stained for nuclear structures, and counterstained for cytoplasmic structures using eosin (Figure 29b). Mice were euthanized on days 7 or 8 post-challenge for histological and viral load analysis. ZIKV infection induced severe brain pathology in these mice. Brain sections of unvaccinated control (pVax1) mice show nuclear fragments within neutrophils (Figure 29b); It showed perivascular infiltration of blood vessels within the cortex, lymphocytic infiltration and degenerating cells in the cerebral cortex (Fig. 29b), and degenerating neurons within the hippocampus (Fig. 29b). However, in contrast, animals vaccinated with ZIKV prME exhibited normal histopathology in brain tissue (Fig. 29b), which supports the idea that protective antibodies induced by immunization with the synthetic Zika-prME vaccine were able to limit virus-induced disease in the brain. These observations demonstrate the potential of brain-protective vaccination in this model. Following ZIKV challenge, high (2 x 10⁶) values ​​were consistently observed in vaccinated mice, along with improvements in body weight loss and mobility impairment. 6 In the group challenged with the PFU dose, significantly lower viral loads were observed in the blood (Fig. 29c) and brain (Fig. 29d) of animals vaccinated with ZIKV-prME compared to animals vaccinated with pVax1 challenged with the virus. Taken together, these data demonstrate that the ZIKV-prME DNA vaccine-mediated immune response can protect mice against ZIKV challenge.

[0237] Passive delivery of anti-ZIKV immune serum protects mice against ZIKV infection.

[0238] Next, the transfer of immune serum from ZIKV-prMEnv-vaccinated RM is IFNAR - / - Whether it prevents ZIKV-mediated disease development in mice was tested. To this end, 150 μg equivalents of IgG (PRNT) from 6-week RM were used. 50 1 / 160) IFNAR on day 1 after ZIKV virus challenge inoculation - / - It was adopted into mice. Two groups of control mice were included: one group received pre-immunization serum from RM, and the other group received phosphate-buffered saline (PBS). Mice receiving PBS or control serum lost 15 to 25% of their original body weight during the course of infection, and all of them died 6 to 8 days after infection. When vaccine-immunization serum from RM was transferred to infection-susceptible mice, these animals lost body weight on days 3 and 4, but subsequently recovered their body weight starting from day 5, and 80% ultimately survived infectious challenge (Fig. 30a), which demonstrates the ability of NHP serotransfer to confer protection against the clinical manifestations of ZIKV infection after viral challenge (Fig. 30b). In repeated experiments performed to evaluate the efficacy of immune serotransmission in protection against ZIKV challenge, survival among ZIKV-prME immune serorecipients ranged from 80% to 100%. These studies demonstrate that anti-ZIKV vaccine immune serotransmission has the ability to confer significant protection against ZIKV infection in the absence of an acquired adaptive anti-ZIKV immune response.

[0239] Vaccination with ZIKV-prME common structure

[0240] The recent spread of ZIKV and its associated outbreaks in humans have raised serious concerns. Currently, there are no licensed vaccines or treatments for this emerging pathogen. Very recently, a collection of experimental ZIKV vaccines was found to lower viral load after challenge in non-pathogenic animal infection models (Larocca et al., 2016, Nature 536:474-8; Abbink et al., 2016, Science 353:1192-32). These data are encouraging. In this regard, it is important to investigate additional novel vaccine approaches targeting Zika in additional models. Here, synthetic DNA vaccines designed to express novel common ZIKV-prM and E antigens were evaluated for immunogenicity following electroporation-enhanced immunization in mice and non-human primates. ZIKV-prM DNA inoculation was observed to be immunogenic and to yield antigen-specific T cells and binding and neutralizing antibodies in both mice and NHPs. Uniquely, these NHPs were immunized with ZIKV-prME via intradermal electroporation, which utilizes lower voltage and a smaller transfection zone than im electroporation as recently described (Trimble et al., 2016, Lancet 386:2078-88). Further research on this approach could provide benefits in a clinical setting.

[0241] The ZIKV-prME common construct includes engineered modifications of the potential NXS / T motif that remove putative glycosylation sites. The deletion of glycosylation at these sites was correlated with enhanced binding of EDE1-type bnAbs (broad-spectrum neutralizing antibodies) to ZIKV-E proteins (Muthumani et al., 2016, Sci Transl Med 7:301ra132). Antibody responses induced by the common ZIKV-prME appear to be robust in size or, in some cases, superior compared to those induced by the similarly developed ZIKV-prME-MR766 and ZIKV-prME-Brazil vaccines. These constructs were sequences matched to the original ZIKV-MR766 isolate or the recently circulating ZIKV strain from Brazil, respectively. Although this is merely supporting, further studies will provide more insights into the effects of these integrated engineered modifications on the induced immune response.

[0242] Because there are very few pathogenic challenge models against ZIKV, C57BL / 6 and IFNAR - / - The putative protective nature of the immune response to the ZIKV-prME vaccine in mice was compared. Both mouse strains responded with a robust humoral immune response when immunized with ZIKV-prME. Although a T-cell response was also induced, IFNAR - / - Compared to those derived from animals, wild-type C57BL / 6 appears more robust, which supports a partial defect in the transition from innate to adaptive immunity as expected due to the knockout phenotype in the aforementioned mice. However, based on the induction of antigen-specific immunity, the model was useful for evaluating the impact of the vaccine on both infection and disease development. IFNAR - / -Monovaccination with ZIKV-prME in mice was protective against disease and mortality in these models, including protection against neuropathy. Flavivirus-neutralizing antibodies directed toward the Env antigen are thought to play a key role in protection against disease; this concept is supported directly by passive antibody transduction experiments in animal models and indirectly by epidemiological data from prospective studies in geographical regions vulnerable to mosquito-borne viral infections (Weaver et al., 2016, Antiviral Res 130:69-80; Roa et al., 2016, Lancet 387:843; Samarasekera et al., 2016, Lancet 387:521-4). Although IFNAR with the ZIKV-prME DNA vaccine - / - Although mouse immunization as well as serum transfer from immunized NHPs is protective in these murine models, in contrast to sero-transferred mice, vaccinated IFNAR - / - It exhibited improved control of weight loss as an indication of control of the onset. Although further research is needed, these results potentially suggest a role for T-cell responses in this aspect of protection in this model. In addition, control IFNARs that recovered from challenge - / - Mice were observed to remain virus-positive by PCR for at least several weeks, suggesting additional benefits of vaccination. This study supports the potential of vaccination to impact disease prevention in susceptible hosts, and in this case, of such synthetic DNA vaccination.

[0243] Example 4: DNA vaccine induces protective immunity against Zika virus prME in non-human primates

[0244] Himalayan monkeys were dermal (id) immunized with 2 mg of ZIKV-prME plasmid administered as 1 mg at each of the two sites at weeks 0 and 4, followed immediately by dermal electroporation (EP). PBMCs were isolated before immunization and at week 6 and used in the ELISPOT assay to detect IFN-γ-secreting cells in response to stimulation with ZIKV-prME peptides (Figure 36a). NHPs receiving one immunization and NHPs receiving two immunizations showed an increase in IFN-γ-producing cells acquired per million PBMCs for six peptide pools covering the entire prME protein (Figures 36b and 36c), which demonstrates the induction of a ZIKV-specific cellular immune response following ZIKV-prME inoculation. As shown in Figure 37, an anti-ZIKV antibody response is induced by ZIKV-prME vaccination of NHP.

[0245] Himalayan monkeys were vaccinated twice with pZV-prME DNA via the ID route using EP at weeks 0 and 4. At week 8, these animals were subcutaneously challenged with a Zika-PR209 virus strain. As a control, five inexperienced animals were infected with the ZV-PR209 virus (Figure 38a). Inexperienced NHPs infected with ZV-PR209 each exhibited a significant viral load (Figure 38b). NHPs immunized once or twice with pZV-prME DNA did not have a detectable viral load (Figures 37c and 38d). These studies demonstrate that Zika-prME immunization confers protection against Zika challenge.

[0246] Example 5: Phase 1 Zika DNA Vaccine Study ID-EP Interim Analysis

[0248] Zika-001 Clinical Protocol

[0249] The first Phase I study was an open-label, dose-range study evaluating the safety, tolerability, and immunogenicity of GLS-5700 administered ID, followed by EP, in dengue virus-naive adults, and was conducted at three locations in the US and Canada.

[0250] The primary objective of the above study was to evaluate the safety and tolerability of GLS-5700 when administered via ID injection and subsequently via EP in healthy adult subjects who were inexperienced with the dengue virus up to 14 days from the final vaccine administration.

[0251] In this study, the primary safety endpoints include: (1) the occurrence of adverse events classified by the System of Occlusion (SOC), priority term (PT) severity, and their relationship to the study treatment and schedule up to 14 days after vaccination; (2) administration (injection) site reactions (described by frequency and severity grades) and administration site pain up to 14 days after the final vaccination; and (3) changes in safety laboratory parameters (e.g., liver panel tests, vital signs) described by frequency and severity grades.

[0252] Secondary objectives include: (1) to evaluate the safety of GLS-5700 up to 1 year after vaccination in dengue-virus-naive adults; and (2) to evaluate the cellular and humoral responses of GLS-5700 when delivered to ID and thereafter, EP in dengue-virus-naive adults.

[0253] Secondary immunological endpoints include: (1) titers of binding antibodies against Zika envelope (E) protein when measured by ELISA; (2) titers of neutralizing antibodies against Zika virus when measured in a virus neutralization assay; and (3) antigen-specific cellular immune response against Zika virus when determined by interferon-gamma (IFN-γ) ELISA spot and / or intracellular staining (ICS) assay.

[0254] This Phase I clinical trial evaluates whether GLS-5700, administered via ID injection and subsequently via electroporation (EP), is safe, tolerable, and capable of producing an immune response against Zika virus in dengue virus-naive participants, and whether the immunoresponsiveness is dose-dependent. The injection is administered via the deltoid muscle, immediately followed by CELLECTRA ® EP will continue with the -3P device.

[0255] GLS-5700 contains plasmid pGX7201, which encodes the common sequence of the Zika virus's pre-membrane (prM) and envelope (E) proteins.

[0256] Currently, there are no approved treatments or preventive vaccines for the Zika virus. Furthermore, no vaccine candidate for the Zika virus has advanced to clinical trials.

[0257] Evaluation of GLS-5700 ID administration :

[0258] There are two potent parts for Zika-001 (Table 1). Participants (n=20 per group) will be administered GLS-5700 at one of two dose levels: 1 mg or 2 mg DNA / dose. The vaccine is administered as a 0.1 ml ID injection, followed by CELLECTRA ® EP will be administered via a -3P device. Participants will receive one or two injections into the deltoid region during the vaccination (three-dose vaccination series) at weeks 0, 4, and 12.

[0259] Table 1. Potentially Involved Parts for Medication and Dietary Management

[0260] army vaccine schedule n Root Injection with every medication dose Dosage (mg) 1 GLS-5700 0-4-12 weeks 20 ID 1 1 2 GLS-5700 0-4-12 weeks 20 ID 2 2 Total 40

[0261] To assess safety, participants are monitored for adverse events using the "Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials" (Appendix B) at labs assessed according to site normal values. Pain is assessed immediately after EP and 30 minutes after EP. Laboratory safety assessments will be obtained at screening, one week after the first vaccination, and two weeks after the second and third vaccinations. Adverse events, including the assessment of injection site reactions, are monitored up to 12 months after the final vaccination.

[0262] The inclusion criteria for the study population are as follows:

[0263] a. Age 18-65;

[0264] b. May provide a consent form for participation and sign the Informed Consent Form (ICF);

[0265] c. Able to and willing to comply with all research procedures;

[0266] d. The woman of potential pregnancy agrees to use medically effective contraception (oral contraception, barrier methods, spermicides, etc.), or her partner is infertile for 3 months from registration until the last injection, or her partner cannot medically induce pregnancy.

[0267] e. Sexually active men considered to be sexually fertile must agree to use a barrier contraceptive method during the study and to continue using it for at least 3 months after the final injection, or their partner is permanently infertile or medically unable to conceive;

[0268] f. Normal screening ECG or screening ECG with no clinically significant findings;

[0269] g. The screening laboratory must be within normal limits or have only Grade 0-1 investigation results;

[0270] h. No history of clinically significant immunosuppressive or autoimmune diseases.

[0271] i. No history of dengue virus vaccination or disease; no history of yellow fever vaccination.

[0272] j. Dengue seronegative at baseline by screening laboratory evaluation

[0273] k. Not taking immunosuppressants currently or within the previous 4 weeks (excluding inhaled, topical skin and / or eye drops—intravenous corticosteroids, low-dose methotrexate, or prednisone at a dose less than 10 mg / day or steroid dose-equivalents).

[0274] The exclusion criteria for the study population are as follows:

[0275] a. Administration of the investigated compound within 30 days of the current or first dose;

[0276] b. Prior receipt of the investigational product for the treatment or prevention of Zika virus, except where it is demonstrated that the participant received a placebo;

[0277] c. Administration of any vaccine within 4 weeks of the first dose;

[0278] d. Administration of any monoclonal or polyclonal antibody product within 4 weeks of the first dose

[0279] e. Administration of any blood product within 3 months of the first dose;

[0280] f. Pregnancy or breastfeeding nutrition or pregnancy planning during the course of the study;

[0281] g. A positive serotype for dengue virus (any serotype), or a history of receiving dengue virus or yellow fever virus vaccination at any point in the past;

[0282] h. Positive serological test for HIV, hepatitis B surface antigen (HBsAg); or any potential infectious disease as determined by a primary investigator or medical monitor;

[0283] i. Positive serological test for hepatitis C (Exclusion: successful treatment with confirmation of a sustained viral response)

[0284] j. Baseline evidence of kidney disease (CKD stage II or higher) as measured by creatinine greater than 1.5;

[0285] k. Baseline screening lab(s) having Grade 2 or higher abnormalities, excluding Grade 2 creatinine;

[0286] l. Chronic liver disease or cirrhosis;

[0287] m. History of immunosuppressive diseases, including blood cancer, or parenchymal or bone marrow transplantation;

[0288] n. Current or anticipated concomitant immunosuppressive therapy (excluding inhaled, topical skin and / or ophthalmic-infused corticosteroids, low-dose methotrexate, or prednisone at doses greater than 10 mg / day or steroid dose-equivalents);

[0289] o. Current or anticipated treatment with TNF-α inhibitors, e.g., infliximab, adalimumab, etanercept;

[0290] p. Previous major surgery, or random radiation therapy within 4 weeks of group assignment;

[0291] q. Any early excitation syndrome, e.g., Wolff-Parkinson-White syndrome;

[0292] r. Presence of a pacemaker or an automated implantable cardioverter-defibrillator (AICD)

[0293] s. A metal implant within 20 cm of the planned injection site(s);

[0294] t. Presence of keloid scar formation or hypertrophic scar as a clinically significant medical condition at the planned injection site(s).

[0295] u. A prisoner or participant forcibly detained (involuntary confinement) for the treatment of a physical or mental illness;

[0296] v. Active drugs, or alcohol use or dependence that, in the investigator's opinion, interfere with adherence to study requirements or assessment of immunological endpoints; or

[0297] w. No willingness to allow the storage and future use of specimens for research related to the Zika virus

[0298] x. Any disease or condition that, in the investigator's opinion, may affect the safety of the participant or the evaluation of any study endpoint.

[0299] Zika-002 Clinical Protocol

[0300] The second Phase I study was a placebo-controlled, double-blind study to evaluate the safety, tolerability, and immunogenicity of GLS-5700 administered by electroporation at ID and thereafter in dengue-seropositive adults in Puerto Rico. The primary objective of the study was to evaluate the safety and tolerability of GLS-5700 when administered by ID injection and then by EP in healthy dengue-seropositive adult individuals up to 12 weeks from the final vaccination.

[0301] The primary safety endpoints in this study include: (1) the occurrence of adverse events classified by the System of Organ Classification (SOC), priority term (PT) severity, and their relationship to the study treatment and schedule up to 12 weeks after final vaccination; (2) administration (injection) site reactions (described by frequency and severity grades) and administration site pain up to 12 weeks after final vaccination; and (3) changes in safety laboratory parameters (e.g., liver panel test, vital signs) described by frequency and severity grades. The secondary safety endpoints in this study include: (1) evaluating the safety of GLS-5700 up to 1 year after final vaccination in dengue-virus seropositive adults; and (2) evaluating the cellular and humoral responses to EP-delivered GLS-5700 in dengue-virus seropositive adults at ID and thereafter.

[0302] Secondary immunological endpoints include: (1) binding antibody titers against Zika envelope (E) measured by ELISA; (2) neutralizing antibody against Zika virus when measured in a neutralization assay; and (3) antigen-specific cellular immune response against Zika virus when determined by interferon-gamma (IFN-γ) ELISA spot and / or intracellular staining (ICS) assay.

[0303] This Phase I clinical trial evaluates whether GLS-5700, administered via ID injection and subsequently via electroporation (EP), is safe, tolerable, and capable of producing an immune response against the Zika virus in dengue seropositive adults. The injection is administered intradermally in the deltoid region, immediately followed by CELLECTRA ® EP continues through the -3P device.

[0304] GLS-5700 contains plasmid pGX7201, which encodes the common sequence of the Zika virus's pre-membrane (prM) and envelope (E) proteins.

[0305] Currently, there are no approved treatments or preventive vaccines for the Zika virus. Furthermore, no vaccine candidate for the Zika virus has advanced to clinical trials.

[0306] Evaluation of GLS-5700 ID administration :

[0307] Subjects (n=80 per group) will be randomized to receive GLS-5700 at a dose of 2 mg DNA (GLS-5700 is prepared at SSC) or a placebo (SSC, compositional buffer for GLS-5700). The vaccine or placebo is administered as two 0.1 mL ID injections, followed by CELLECTRA ® EP will follow with a -3P device. Subjects will be administered vaccinations within the deltoid region at 0, 4, and 12 weeks (3-dose vaccination series; Table 2).

[0308] Table 2. Potentially Involved Parts for Medication and Dietary Management

[0309] army vaccine schedule n Root Dosage (mg) 1 Penalty 0-4-12 weeks 80 ID 0 2 GLS-5700 0-4-12 weeks 80 ID 2 Total 160

[0310] To assess safety, subjects are monitored for adverse events using the "Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials" (Appendix B) in labs assessed according to site normal values. Pain is assessed 30 minutes after EP. Laboratory safety assessments will be obtained, if applicable, at screening, 1 week after the first vaccination, and 2 weeks after the second and third vaccinations. Adverse events, including assessment of injection site reactions, will be monitored up to 12 months after the final vaccination.

[0311] The inclusion criteria for the study population are as follows:

[0312] a. Age 18-65;

[0313] b. May provide a consent form for participation and sign the Informed Consent Form (ICF);

[0314] c. Able to and willing to comply with all research procedures;

[0315] d. The woman of potential pregnancy agrees to use medically effective contraception (oral contraception, barrier methods, spermicides, etc.), or her partner is infertile for 3 months from registration until the last injection, or her partner cannot medically induce pregnancy.

[0316] e. Sexually active men considered to be sexually fertile must agree to use a barrier contraceptive method during the study and to continue using it for at least 3 months after the final injection, or their partner is permanently infertile or medically unable to conceive;

[0317] f. Positive dengue virus seroreaction at screening;

[0318] g. Normal screening ECG or screening ECG with no clinically significant findings;

[0319] h. The screening laboratory must be within normal limits or have only Grade 0-1 investigation results;

[0320] i. No history of clinically significant immunosuppressive or autoimmune diseases.

[0321] j. No history of dengue virus vaccination; no history of yellow fever vaccination.

[0322] k. Not taking immunosuppressants currently or within the past 4 weeks (excluding inhaled, topical skin and / or eye drops—intravenous corticosteroids, low-dose methotrexate, or prednisone at a dose less than 10 mg / day or steroid equivalents).

[0323] The exclusion criteria for the study population are as follows:

[0324] a. Administration of the investigated compound within 30 days of the current or first dose;

[0325] b. Prior receipt of the investigation product for the treatment or prevention of Zika virus, except where it is proven that the subject received a placebo;

[0326] c. Administration of any vaccine within 4 weeks of the first dose;

[0327] d. Administration of any monoclonal or polyclonal antibody product within 4 weeks of the first dose

[0328] e. Administration of any blood product within 3 months of the first dose;

[0329] f. Pregnancy or breastfeeding nutrition or pregnancy planning during the course of the study;

[0330] g. Negative serological result for dengue virus

[0331] h. History of receiving dengue virus or yellow fever virus vaccination at any point in the past;

[0332] i. Positive serological test for HIV, hepatitis B surface antigen (HBsAg); or history of any potential infectious disease as determined by a primary investigator or medical monitor;

[0333] j. Positive serological test for hepatitis C (Exclusion: successful treatment with confirmation of a sustained viral response)

[0334] k. Baseline evidence of kidney disease (CKD stage II or higher) as measured by creatinine greater than 1.5;

[0335] l. Baseline screening lab(s) having Grade 2 or higher abnormalities, excluding Grade 2 creatinine;

[0336] m. Chronic liver disease or cirrhosis;

[0337] n. History of immunosuppressive diseases, including blood cancer, or parenchymal or bone marrow transplantation;

[0338] o. Current or anticipated concomitant immunosuppressive therapy (excluding inhaled, topical skin and / or ophthalmic-infused corticosteroids, low-dose methotrexate, or prednisone at a dose or steroid equivalent of 10 mg / day or greater than this);

[0339] p. Current or anticipated treatment with TNF-α inhibitors, e.g., infliximab, adalimumab, etanercept;

[0340] q. Previous major surgery, or random radiation therapy within 4 weeks of military assignment;

[0341] r. Any early excitation syndrome, e.g., Wolff-Parkinson-White syndrome;

[0342] s. Presence of a pacemaker or an automated cardiodactyl defibrillator (AICD)

[0343] t. Metal implant within 20 cm of the planned injection site(s);

[0344] u. Presence of keloid scar formation or hypertrophic scar as a clinically significant medical condition at the planned injection site(s).

[0345] v. A prisoner or individual forcibly detained (involuntary confinement) for the treatment of physical or mental illness;

[0346] w. Active drugs, or alcohol use or dependence that, in the investigator's opinion, interfere with adherence to study requirements or assessment of immunological endpoints; or

[0347] x. No willingness to allow the storage and future use of specimens for research related to the Zika virus

[0348] y. Any disease or condition that, in the investigator's opinion, may affect the safety of the individual or the evaluation of any study endpoint.

[0349] Clinical results

[0350] Patients were immunized with pZV-prME DNA on day 0 and week 4 (Figures 39-42). To determine the percentage of bound responders, serum was incubated at the indicated dilutions on plates coated with Zika Env protein, and a secondary antibody detected the total IgG response.

[0351] Vero cells were infected with the Zika virus, and after 3 days, the cells were fixed and subsequently cultured with a 1:100 dilution of serum from day 0 and week 6.

[0352] Neutralization studies of patient serum IC 50 It was carried out by determining (the serum dilution that neutralizes 50% of ZIKV PR209 infection in Vero cells).

[0353] Figure 39a provides the results of the binding ELISA assay. Figure 39b provides experimental results demonstrating passive delivery and protection.

[0354] Figure 40 provides exemplary immunofluorescence data demonstrating an increase in anti-human IgG-AF488 staining after two doses.

[0355] Figure 41 provides data demonstrating the characterization of the combined reactant.

[0356] Figure 42 provides data proving that there was an increase in neutralization after two doses.

Claims

Claim 1 An isolated nucleic acid molecule, wherein the nucleic acid molecule encodes a common Zika antigen, and the common Zika antigen comprises at least one amino acid sequence selected from the group composed as follows: Sequence No. 1, an amino acid sequence that is at least 95% identical to Sequence No. 1, a fragment of Sequence No. 1 excluding the IgE signal peptide sequence, and an amino acid sequence that is at least 95% identical to the fragment of Sequence No. 1 excluding the IgE signal peptide sequence; Sequence No. 3, an amino acid sequence that is at least 95% identical to Sequence No. 3, a fragment of Sequence No. 3 excluding the IgE signal peptide sequence, and an amino acid sequence that is at least 95% identical to the fragment of Sequence No. 3 excluding the IgE signal peptide sequence; Sequence No. 5, an amino acid sequence that is at least 95% identical to Sequence No. 5, a fragment of Sequence No. 5 excluding the IgE signal peptide sequence, and an amino acid sequence that is at least 95% identical to the fragment of Sequence No. 5 excluding the IgE signal peptide sequence; Sequence No. 7, an amino acid sequence that is at least 95% identical to Sequence No. 7, The fragment of Sequence No. 7 with the IgE signal peptide sequence excluded, and an amino acid sequence that is at least 95% identical to the fragment of Sequence No. 7 with the IgE signal peptide sequence excluded. Claim 2 An isolated nucleic acid molecule according to claim 1, wherein the isolated nucleic acid comprises a sequence selected from the group consisting of sequence number:2, a sequence that is at least 95% identical to sequence number:2, sequence number:2 from which a nucleotide sequence encoding an IgE signal peptide is excluded, a fragment of sequence number:2 from which a nucleotide sequence encoding an IgE signal peptide is excluded, and a nucleotide sequence encoding an amino acid sequence that is at least 95% identical to sequence number:2 from which a nucleotide sequence encoding an IgE signal peptide is excluded. Claim 3 An isolated nucleic acid molecule according to claim 1, wherein the isolated nucleic acid comprises a sequence selected from the group consisting of sequence number:4, a sequence that is at least 95% identical to sequence number:4, sequence number:4 from which a nucleotide sequence encoding an IgE signal peptide has been excluded, a fragment of sequence number:4 from which a nucleotide sequence encoding an IgE signal peptide has been excluded, and a nucleotide sequence encoding an amino acid sequence that is at least 95% identical to sequence number:4 from which a nucleotide sequence encoding an IgE signal peptide has been excluded. Claim 4 An isolated nucleic acid molecule according to claim 1, wherein the isolated nucleic acid comprises a sequence selected from the group consisting of sequence number:6, a sequence that is at least 95% identical to sequence number:6, sequence number:6 from which a nucleotide sequence encoding an IgE signal peptide is excluded, a fragment of sequence number:6 from which a nucleotide sequence encoding an IgE signal peptide is excluded, and a nucleotide sequence encoding an amino acid sequence that is at least 95% identical to sequence number:6 from which a nucleotide sequence encoding an IgE signal peptide is excluded. Claim 5 An isolated nucleic acid molecule according to claim 1, characterized in that the isolated nucleic acid molecule is a plasmid. Claim 6 A composition for inducing an immune response against Zika virus comprising a nucleic acid molecule, wherein the nucleic acid molecule encodes a common Zika antigen, and the common Zika antigen comprises at least one amino acid sequence selected from the group consisting of: Sequence No. 1, an amino acid sequence at least 95% identical to Sequence No. 1, a fragment of Sequence No. 1 excluding an IgE signal peptide sequence, and an amino acid sequence at least 95% identical to the fragment of Sequence No. 1 excluding an IgE signal peptide sequence; Sequence No. 3, an amino acid sequence at least 95% identical to Sequence No. 3 excluding an IgE signal peptide sequence, a fragment of Sequence No. 3 excluding an IgE signal peptide sequence, and an amino acid sequence at least 95% identical to the fragment of Sequence No. 3 excluding an IgE signal peptide sequence; Sequence No. 5, an amino acid sequence at least 95% identical to Sequence No. 5 excluding an IgE signal peptide sequence, a fragment of Sequence No. 5 excluding an IgE signal peptide sequence, and an amino acid sequence at least 95% identical to the fragment of Sequence No. 5 excluding an IgE signal peptide sequence; Sequence No. 7, sequence An amino acid sequence that is at least 95% identical to No.:7, a fragment of Sequence No.:7 with the IgE signal peptide sequence excluded, and an amino acid sequence that is at least 95% identical to the fragment of Sequence No.:7 with the IgE signal peptide sequence excluded. Claim 7 A composition according to claim 6, characterized in that it is prepared for delivery to an individual using electroporation. Claim 8 A composition according to claim 6, further comprising a nucleic acid sequence encoding one or more proteins selected from the group consisting of IL-12, IL-15, and IL-28. Claim 9 A method for inducing an immune response to the Zika virus, characterized by comprising administering a nucleic acid molecule of any one of claims 1 to 5 to an individual other than a human in an amount effective for inducing an immune response in said individual other than a human. Claim 10 A method for treating an individual diagnosed with Zika virus other than a human, characterized by including administering a therapeutically effective amount of a nucleic acid molecule according to any one of claims 1 to 5 to the individual other than a human. Claim 11 A method for preventing Zika virus infection in individuals other than humans, characterized by including administering a preventive effective amount of a nucleic acid molecule according to any one of claims 1 to 5 to individuals other than humans. Claim 12 A composition for the prevention or treatment of Zika virus infection comprising a nucleic acid molecule, wherein the nucleic acid molecule encodes a common Zika antigen, and the common Zika antigen comprises at least one amino acid sequence selected from the group consisting of: Sequence No. 1, an amino acid sequence identical to Sequence No. 1 by at least 95%, a fragment of Sequence No. 1 excluding the IgE signal peptide sequence, and an amino acid sequence identical to the fragment of Sequence No. 1 excluding the IgE signal peptide sequence by at least 95%; Sequence No. 3, an amino acid sequence identical to Sequence No. 3 by at least 95%, a fragment of Sequence No. 3 excluding the IgE signal peptide sequence, and an amino acid sequence identical to the fragment of Sequence No. 3 excluding the IgE signal peptide sequence by at least 95%; Sequence No. 5, an amino acid sequence identical to Sequence No. 5 by at least 95%, a fragment of Sequence No. 5 excluding the IgE signal peptide sequence, and an amino acid sequence identical to the fragment of Sequence No. 5 excluding the IgE signal peptide sequence by at least 95%; Sequence No. 7, sequence An amino acid sequence that is at least 95% identical to No.:7, a fragment of Sequence No.:7 with the IgE signal peptide sequence excluded, and an amino acid sequence that is at least 95% identical to the fragment of Sequence No.:7 with the IgE signal peptide sequence excluded. Claim 13 A composition according to claim 12, characterized in that it is prepared for delivery to an individual using electroporation. Claim 14 A composition according to claim 12, further comprising a nucleic acid sequence encoding one or more proteins selected from the group consisting of IL-12, IL-15, and IL-28. Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete

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