A Zika / dengue vaccine and its application

Abstract

The present invention relates to the field of biotechnology, and particularly to a Zika / dengue vaccine and its application. Based on crystal structure analysis and other structural and functional analyses, the present invention has obtained the epitope information of the main antibody causing the ADE effect. The antigen provided in the present invention introduces mutations in the FL fusion region of the E protein of Zika virus or dengue virus, and the antigens with such mutations cannot bind to the antibody causing ADE (FLE antibody); the vaccine provided by the present invention, after immunization, can avoid the production of antibodies induced by the FL epitope, thereby achieving the reduction or elimination of the ADE effect.

Description

[0001] This application is a divisional application of the invention patent application with the application date of November 9, 2020, the application number of 202011243025.6, and the invention name of "A Zika / Dengue Vaccine and Its Application". Technical Field

[0002] The present invention relates to the field of biotechnology, and specifically relates to a Zika / Dengue vaccine and its application. Background Art

[0003] Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus of the family Flaviviridae. The ZIKV epidemic that broke out in the Americas in 2015-2016 spread to 84 countries around the world, including China. However, there are still no vaccines and drugs available so far. Although the global incidence of ZIKV infection has weakened now, ZIKV still poses a threat to people living in endemic areas. Therefore, the development of a ZIKV vaccine is urgent.

[0004] DENV virus (dengue virus, DENV) has 4 serotypes and is also a mosquito-borne virus of the genus Flavivirus of the family Flaviviridae.

[0005] The structures of ZIKV virus and DENV virus are relatively similar, both presenting an icosahedral spherical structure, having an envelope, and the envelope surface contains envelope (E) protein. The internal viral genome is single-stranded positive-strand RNA, about 11 kb in length, containing only one open reading frame. The polyprotein translated therefrom can be cleaved into 3 structural proteins (C, prM, E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5).

[0006] The E protein is about 53 kD in size and is the main protein on the surface of ZIKV and DENV. It mediates virus entry into cells and membrane fusion. Therefore, it is an important target for activating neutralizing antibodies and is also an important target protein in vaccine design. The E protein has 504 amino acids and exists in the form of a dimer. Each monomer has three domains, namely DI, DII, and DIII. The head of DII (amino acids 98-109) contains a highly conserved fusion loop (FL). Among them, the FL region sequences of ZIKV virus and DENV virus are exactly the same, both being D98-R99-G100-W101-G102-N103-G104-C105-G106-L107-F108-G109. The FL region plays a key role in the membrane fusion process of virus invasion. During the virus infection process, immune cells will produce a large number of antibodies against FL.

[0007] The prM protein is approximately 26 kD in size and assists in the correct folding of the E protein. The transmembrane region at the 3' end of prM / E serves as an endoplasmic reticulum retention signal to assist in the formation of a heterodimer between prM and E. A major function of the prM protein is to maintain the stability of the E protein. In immature virus particles, the pr polypeptide is located at the tip of the E protein, forming a pr-E spike that hides the fusion peptide of the E protein. At this time, due to steric hindrance, prM is not easily accessible to furin for cleavage. Subsequently, the acidic environment in the Golgi apparatus induces a rearrangement reaction, exposing the furin cleavage site, and the prM protein is cleaved by furin to become the M protein. At this point, the cleaved pr polypeptide does not immediately dissociate from the virus particle but needs to be exposed to a neutral pH cellular environment for the pr polypeptide to be released, resulting in the appearance of mature virus particles.

[0008] Since the genetic composition and antigenic properties of ZIKV are very similar to those of the four serotypes of dengue virus (DENV), with an amino acid similarity of approximately 56%, the antibodies induced by ZIKV infection can have a strong cross-reaction with DENV, which is also a factor to be considered in vaccine safety issues. A large amount of evidence indicates that pre-existing antibodies after ZIKV infection can enhance subsequent DENV infection due to cross-reaction with DENV (Fowler et al., 2018; George et al., 2017; Li et al., 2017; Richner et al., 2017; Stettler et al., 2016; Valiant et al., 2018). This phenomenon is called antibody-dependent enhancement (ADE), which refers to the enhancement of virus infection when antibodies are insufficient to neutralize the virus or are at sub-neutralizing concentrations (Beltramello et al., 2010; Dejnirattisai et al., 2010). Although epidemiological investigations are still lacking, pre-existing ZIKV antibodies from humans, monkeys, and mice have been shown to enhance DENV infection in cell experiments (George et al., 2017; Richner et al., 2017; Stettler et al., 2016; Valiant et al., 2018). In addition, antibodies obtained by ZIKV infection, vaccine immunization, or from the fetus to the mother in monkey and mouse models have also been confirmed to exacerbate DENV infection symptoms (Fowler et al., 2018; George et al., 2017; Richner et al., 2017; Stettler et al., 2016). Therefore, the ADE effect on future DENV infection should be considered in the design of ZIKV vaccines.

[0009] Antibodies that cause ADE reactions are mainly induced by the FL fusion region of the virus (Beltramello et al., 2010; Dejnirattisai et al., 2010). In flavivirus infections, such antibodies account for a relatively large proportion of the total induced antibodies. Due to the high conservation of the epitopes, these antibodies usually cross-react between different serotypes. They mostly have low neutralizing activity and are prone to cause ADE reactions, while most antibodies with high neutralizing activity bind to other epitopes on the E protein. A series of ZIKV neutralizing monoclonal antibodies have been identified, which target Domain I (DI), Domain II (DII), and Domain III (DIII) of the E protein or quaternary structure epitopes (Barba-Spaeth et al., 2016; Stettler et al., 2016; Wang et al., 2017; Wang et al., 2016; Zhao et al., 2016). Therefore, an ideal ZIKV vaccine design strategy is to shift the hot epitopes of the immune response from the FL region to other neutralizing epitopes.

[0010] Antibodies bind to virus particles and are then taken up by cells through binding to Fcγ receptor proteins on the surface of myeloid cells, subsequently promoting virus infection. Since there are 4 serotypes of DENV, when a person is reinfected with DENV of a different serotype for the second time, ADE is very likely to occur, which explains the more severe disease phenomena in humans infected with DENV (Katzelnick et al., 2017). ADE has been used to explain the usage limitations of the currently only approved DENV vaccine and is recommended to be used only for DENV seropositive individuals. For seronegative individuals, vaccination may instead exacerbate dengue infection (Rey et al., 2018; Slon-Campos et al., 2019). Therefore, how to avoid the occurrence of ADE is also a challenge in the development of DENV vaccines.

[0011] The information disclosed in this background art section is only intended to enhance the overall understanding of the present invention and should not be regarded as an admission or any form of suggestion that this information constitutes prior art already known to those of ordinary skill in the art. Summary of the Invention

[0012] Object of the Invention

[0013] The object of the present invention is to provide a Zika / dengue vaccine that avoids ADE reaction and its application. Based on crystal structure analysis and other structural and functional analyses, the present invention obtains the epitope information of the main antibody causing the ADE effect. The antigen provided in the present invention introduces mutations in the FL fusion region of the E protein of Zika virus or dengue virus, and the antigen with such mutations cannot bind to the antibody (FLE antibody) causing ADE; the vaccine provided based on the antigen can avoid the production of antibodies induced by the FL epitope after immunization, thereby reducing or eliminating the ADE effect.

[0014] Solution

[0015] To achieve the object of the present invention, the present invention provides an antigen, the antigen has the FL fusion region of the E protein of Zika virus or dengue virus, and the FL fusion region of the E protein has one of the following mutations:

[0016] (1) One or both of the mutations at the D98 site and the N103 site in combination with the mutations at the three sites of G106, L107, and F108;

[0017] (2) One or a combination of the mutations at the G106 site, the L107 site, and the F108 site;

[0018] (3) Mutation at the W101 single site.

[0019] (1) One or both of the mutations at the D98 site and the N103 site in combination with the mutations at the three sites of G106, L107, and F108 are any one of the following: mutations at the five sites of D98, N103, G106, L107, and F108; mutations at the four sites of D98, G106, L107, and F108; mutations at the four sites of N103, G106, L107, and F108.

[0020] (2) One or a combination of the mutations at the G106 site, the L107 site, and the F108 site are any one of the following: single-site mutations selected from the mutations at the G106 site, the L107 site, and the F108 site; double-site mutations selected from the mutations at the G106 site, the L107 site, and the F108 site; mutations at the three sites of G106, L107, and F108.

[0021] The D98 site mutation refers to the substitution of aspartic acid (D) at position 98 of the E protein with any amino acid other than aspartic acid; the N103 site mutation refers to the substitution of asparagine (N) at position 103 of the E protein with any amino acid other than asparagine; the G106 site mutation refers to the substitution of glycine (G) at position 106 of the E protein with any amino acid other than glycine; the L107 site mutation refers to the substitution of leucine (L) at position 107 of the E protein with any amino acid other than leucine; the F108 site mutation refers to the substitution of phenylalanine (F) at position 108 of the E protein with any amino acid other than phenylalanine; the W101 site mutation refers to the substitution of tryptophan (W) at position 101 of the E protein with any amino acid other than tryptophan.

[0022] The D98 site, W101 site, N103 site, G106 site, L107 site or F108 site is located in the FL fusion region of the E protein. The FL (fusion region) sequence is highly conserved among flavivirus viruses. Among them, the FL sequences of Zika virus (ZIKV) and Dengue virus (DENV) are exactly the same, both being D98-R99-G100-W101-G102-N103-G104-C105-G106-L107-F108-G109. In the present invention, the numbering of the D98, W101, N103, G106, L107, and F108 sites refers to the positions in the E protein sequences of Zika virus and Dengue virus. Specifically, it can refer to the 98th, 101st, 103rd, 106th, 107th, and 108th sites shown in SEQ ID NO.1 of the E protein of Zika virus (taking the ZIKV FSS13025 strain as an example, GenBank: JN860885.1).

[0023] In one possible implementation of the above antigen, the mutations in the FL fusion region of the E protein are selected from: the five-site mutations of D98, N103, G106, L107, and F108;

[0024] or, the mutations in the FL fusion region of the E protein are selected from: the three-site mutations of G106, L107, and F108;

[0025] or, the mutations in the FL fusion region of the E protein are selected from: the two-site mutations of G106 and L107;

[0026] or, the mutations in the FL fusion region of the E protein are selected from: the two-site mutations of G106 and F108;

[0027] or, the mutations in the FL fusion region of the E protein are selected from: the two-site mutations of L107 and F108;

[0028] or, the mutations in the FL fusion region of the E protein are selected from: the single-site mutation of G106;

[0029] Alternatively, the mutation in the E protein FL fusion region is selected from: single-site mutation of L107;

[0030] Alternatively, the mutation in the E protein FL fusion region is selected from: single-site mutation of F108;

[0031] Alternatively, the mutation in the E protein FL fusion region is selected from: single-site mutation of W101.

[0032] In a possible implementation of the above antigen, the mutation in the E protein FL fusion region is selected from the following mutations or combinations of mutations:

[0033]

[0034]

[0035]

[0036] In the above table, the D98N mutation means that aspartic acid (D) at position 98 of the E protein is replaced by asparagine (N);

[0037] The N103T mutation means that asparagine (N) at position 103 of the E protein is replaced by threonine (T);

[0038] The G106F mutation means that glycine (G) at position 106 of the E protein is replaced by phenylalanine (F); the G106L mutation means that glycine (G) at position 106 of the E protein is replaced by leucine (L);

[0039] The L107E mutation means that leucine (L) at position 107 of the E protein is replaced by glutamic acid (E); the L107K mutation means that leucine (L) at position 107 of the E protein is replaced by lysine (K);

[0040] The F108W mutation means that phenylalanine (F) at position 108 of the E protein is replaced by tryptophan (W);

[0041] The mutations of amino acids are extrapolated in turn, and the types of amino acids represented by single letters are the conventional understandings of those skilled in the art.

[0042] In a possible implementation of the above antigen, when the antigen has the E protein FL fusion region of Zika virus, the antigen further includes the full-length or partial M protein sequence of Zika virus; optionally, the antigen further includes the full-length sequence of Zika virus M protein;

[0043] When the antigen has the E protein FL fusion region of dengue virus, the antigen further includes the full-length or partial M protein sequence of dengue virus; optionally, the antigen further includes the full-length sequence of dengue virus M protein.

[0044] The M protein is formed after the prM structural protein is cleaved by furin protease. The full-length or partial M protein sequence refers to 0.5%-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100% of the M protein sequence. The sequence can be either a continuously selected sequence from the M protein or a combination of fragments separately selected from the M protein sequence.

[0045] In one possible implementation of the above antigen, when the antigen has the E protein FL fusion region of Zika virus, the antigen further includes the full-length or partial prM protein of Zika virus; optionally, the antigen further includes the full-length sequence of the Zika virus prM protein;

[0046] When the antigen has the E protein FL fusion region of dengue virus, the antigen further includes the full-length or partial prM protein of dengue virus; optionally, the antigen further includes the full-length sequence of the dengue virus prM protein.

[0047] The prM protein is a structural protein of Zika virus or dengue virus, with a size of approximately 26 kD, and is used to assist the correct folding of the E protein. The full-length or partial prM protein sequence refers to 0.5%-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100% of the prM protein sequence. The sequence can be either a continuously selected sequence from the prM protein or a combination of fragments separately selected from the prM protein sequence.

[0048] In one possible implementation of the above antigen, when the antigen has the E protein FL fusion region of Zika virus, the antigen has the full-length or partial E protein of Zika virus; optionally, the antigen includes the full-length sequence of the E protein of Zika virus;

[0049] When the antigen has the E protein FL fusion region of dengue virus, the antigen has the full-length or partial E protein of dengue virus; optionally, the antigen includes the full-length sequence of the E protein of dengue virus.

[0050] Including the full-length or partial E protein sequence refers to 0.5%-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100% of the E protein sequence. The sequence can be either a continuously selected sequence from the E protein or a combination of fragments separately selected from the E protein sequence.

[0051] The sequences of the E protein, prM protein, and M protein of the Zika virus can be obtained according to the full sequences of various Zika virus strains publicly available in NCBI and reports in the prior art. The sequences of the E protein, prM protein, and M protein of the dengue virus can be obtained according to the full sequences of various virus strains of the four serotypes of the dengue virus publicly available in NCBI and the prior art.

[0052] In one possible implementation of the above antigen, the Zika virus includes all Zika virus strains, such as the ZIKVFSS13025 strain (GenBank: JN860885.1), the ZIKK SMGC-1 strain.

[0053] In one possible implementation of the above antigen, the dengue virus includes various dengue virus strains of the four serotypes of the dengue virus, such as DENV1 (Hawaii strain, GenBank: KM204119), DENV2 (New Guinea C strain, GenBank: KM204118.1), DENV3 (YN02 strain, GenBank: KF824903), and DENV4 (Guangzhou B5 strain in China, GenBank: AF289029).

[0054] The present invention also provides an antigenic epitope of the FL fusion region of the Zika virus E protein. The FL fusion region of the Zika virus E protein has one of the following mutations based on the amino acid sequence D98-R99-G100-W101-G102-N103-G104-C105-G106-L107-F108-G109:

[0055] (1) One or both of the mutations at the D98 site and the N103 site combined with the mutations at the three sites of G106, L107, and F108;

[0056] (2) One or a combination of the mutations at the G106 site, the L107 site, and the F108 site;

[0057] (3) Mutation at the W101 site.

[0058] The present invention also provides a Zika virus antigen containing the above antigenic epitope.

[0059] In one possible implementation of the above Zika virus antigen, it further includes one or more of the following sequences:

[0060] The full-length or partial E protein sequence of the Zika virus;

[0061] The full-length or partial M protein sequence of the Zika virus;

[0062] The full-length or partial prM protein of the Zika virus.

[0063] An epitope of the dengue virus E protein FL fusion region, wherein the dengue virus E protein FL fusion region has one of the following mutations based on the amino acid sequence D98-R99-G100-W101-G102-N103-G104-C105-G106-L107-F108-G109:

[0064] (1) One or both of the mutations at the D98 site and the N103 site in combination with the mutations at the three sites of G106, L107, and F108;

[0065] (2) One or a combination of the mutations at the G106 site, the L107 site, and the F108 site;

[0066] (3) Mutation at the W101 single site.

[0067] The present invention also provides a dengue virus antigen containing the above epitope.

[0068] In a possible implementation manner, the above dengue virus antigen further includes one or more of the following sequences:

[0069] The full-length or partial E protein sequence of the dengue virus;

[0070] The full-length or partial M protein sequence of the dengue virus;

[0071] The full-length or partial prM protein of the dengue virus.

[0072] The present invention also provides antibodies obtained from the above antigen, Zika virus antigen, and dengue virus antigen.

[0073] The present invention also provides a polynucleotide encoding the above antigen, epitope, Zika virus antigen, and dengue virus antigen.

[0074] The present invention also provides an expression cassette, recombinant vector, transgenic cell line, recombinant bacterium, adenovirus, lentivirus, or virus particle containing the above polynucleotide.

[0075] The present invention also provides an mRNA encoding the above antigen, epitope, Zika virus antigen, and dengue virus antigen.

[0076] The present invention also provides a vaccine, which includes the above antigen, the above Zika virus antigen, the above dengue virus antigen, the above polynucleotide, the above expression cassette, recombinant vector, transgenic cell line, recombinant bacterium, adenovirus, lentivirus, or virus particle, or the above mRNA as an active ingredient.

[0077] In one possible implementation of the above-mentioned vaccine, the vaccine includes one or more of inactivated vaccines, attenuated vaccines, DNA vaccines, mRNA vaccines, adenovirus vaccines, other viral vector vaccines, subunit vaccines, or virus-like particles.

[0078] In one possible implementation of the above-mentioned vaccine, the vaccine is an adenovirus vaccine.

[0079] In one possible implementation of the above-mentioned vaccine, the vaccine further includes a pharmaceutically or veterinarily acceptable vehicle, diluent, adjuvant, or excipient.

[0080] The present invention also provides the use of the above-mentioned antigen, the above-mentioned antigenic epitope, the above-mentioned antibody, the above-mentioned polynucleotide, the above-mentioned expression cassette, recombinant vector, transgenic cell line, recombinant bacterium, adenovirus, lentivirus, or virus-like particle, or the above-mentioned mRNA in the preparation of a vaccine for preventing and / or treating flavivirus infection.

[0081] The present invention also provides the use of the above-mentioned antigen, the above-mentioned antigenic epitope, the above-mentioned antibody, the above-mentioned polynucleotide, the above-mentioned expression cassette, recombinant vector, transgenic cell line, recombinant bacterium, adenovirus, lentivirus, or virus-like particle, or the above-mentioned mRNA in the preparation of a detection reagent or kit for detecting flavivirus infection.

[0082] Advantageous Effects

[0083] (1) Based on crystal structure analysis and other structural and functional analyses, the present invention obtained the epitope information of the main antibody causing the ADE effect. The antigen provided in the present invention introduces one of the following mutations in the FL fusion region of the E protein of Zika virus or Dengue virus: i. a combination of one or two of the mutations at the D98 site and the N103 site and the mutations at the three sites of G106, L107, and F108; ii. one or a combination of the mutations at the G106 site, the L107 site, and the F108 site; iii. a single-site mutation at the W101 site. The antigen with this mutation cannot bind to the antibody (FLE antibody) causing ADE. The vaccine obtained based on the antigen provided by the present invention can avoid the production of antibodies induced by the FL epitope after immunization, thereby reducing or eliminating the ADE effect.

[0084] (2) The antigen provided in the present invention cannot bind to the antibody (FLE antibody) that causes ADE, but the binding ability to antibodies of other epitopes is not affected. Taking several adenovirus vaccines of Zika virus obtained based on the antigen as an example, the present invention proves that the obtained recombinant adenovirus vaccine does not reduce the immunogenicity of the antigen, and can still activate the production of neutralizing antibodies well; and plays a complete protective role in mouse challenge; can well protect mice against viremia and tissue and organ infections; and can reduce or even eliminate the ADE effect on 4 serotype DENV viruses after immunization.

[0085] It is also proved that the mutated E protein can still maintain the dimer form, and only the amino acid side chains where FL is mutated have changed, without affecting other neutralizing antibody epitopes. Single-cell sequencing analysis of germinal center (GC) B cells on the antibody response induced by the recombinant adenovirus vaccine in mice proves that the recombinant adenovirus vaccine with the FL region mutated significantly reduces the antibodies induced by the FL epitope compared with the wild-type vaccine in the FL region, and the dominant epitope of the antigen has shifted. These explain the mechanism of action by which the vaccine provided by the present invention based on the antigen can eliminate the ADE effect.

[0086] (3) Taking several adenovirus expression plasmids of dengue virus obtained based on the antigen as an example, the present invention proves that the obtained antigens cannot bind to the antibody (FLE antibody) that causes ADE; this shows that after immunization with the dengue virus vaccine obtained based on the antigen of the present invention, the production of antibodies induced by the FL epitope can be avoided, thereby reducing or eliminating the ADE effect caused by subsequent DENV virus infection after immunization.

[0087] (4) The vaccine provided by the present invention obtained based on the antigen can include various forms, such as nucleic acid vaccines, mRNA vaccines, adenovirus vector vaccines, other virus vector vaccines, virus-like particles, virus attenuated or inactivated vaccines based on the antigen sequence, chimeric vaccines of other backbones, etc., and can be used to prepare Zika and tetravalent dengue vaccines that eliminate the ADE effect. Description of the Drawings

[0088] One or more embodiments are exemplarily illustrated by the pictures in the corresponding drawings, and these exemplary illustrations do not constitute a limitation on the embodiments. The special word "exemplary" here means "serving as an example, an embodiment or an illustration". Any embodiment illustrated here as "exemplary" does not have to be construed as superior to or better than other embodiments.

[0089] Figure 1 It is the neutralization experiment results of immunizing mice with wild-type recombinant adenovirus vaccines AdC7-M / E-WT and AdC7-prM / E-WT.

[0090] Figure 2These are the experimental results of sera from mice immunized with wild-type recombinant adenovirus vaccines AdC7-M / E-WT and AdC7-prM / E-WT enhancing DENV-infected cells.

[0091] Figure 3 Z6 antibody has a certain degree of ADE against DENV-infected cells of 4 serotypes.

[0092] Figure 4 This is the complex structure of the Fab fragment of Z6 antibody and ZIKV E protein.

[0093] Figure 5 These are the complex structures of different antibodies and flavivirus E proteins. Among them, A in the figure is the complex structure of Z6 antibody and ZIKV E protein, B is the complex structure of 2A10G6 antibody and ZIKV E protein, C is the complex structure of E53 antibody and WNV E protein, and D is the overlapping map of the structures of the first three antibodies and ZIKV E protein.

[0094] Figure 6 This is the amino acid site analysis of the binding of ZIKV E protein to Z6 antibody, ZIKV E protein to 2A10G6 antibody, and WNV E protein to E53 antibody.

[0095] Figure 7 This is the phylogenetic tree of flaviviruses.

[0096] Figure 8 These are the mutation sites and sequences of ZIKV M / E MutA / B / C mutants.

[0097] Figure 9 These are the results of flow cytometry detecting the antigen activity of M / E-MutA / B / C.

[0098] Figure 10 This is the construction process of pAdC7-M / E-MutB / MutC recombinant plasmid.

[0099] Figure 11 These are the serum neutralizing antibody titers after immunizing BALB / c mice with ZIKV recombinant adenovirus vaccine.

[0100] Figure 12 These are the serum neutralizing antibody titers after immunizing Ifnar1 - / - mice with ZIKV recombinant adenovirus vaccine.

[0101] Figure 13 These are the serum neutralizing antibody titers after immunizing Ifnar1 - / - mice with ZIKV recombinant adenovirus vaccine and then challenging them with the virus, including the mortality rate and body weight change results.

[0102] Figure 14It is the experimental result of the ZIKV recombinant adenovirus vaccine protecting Ifnar1 - / - mice against viremia caused by ZIKV infection.

[0103] Figure 15 It is the experimental result of the ZIKV recombinant adenovirus vaccine protecting Ifnar1 - / - mice against tissue and organ infection after ZIKV challenge.

[0104] Figure 16 It is the experimental result of the change in neutralizing antibody titer in the serum of Ifnar1 - / - mice before and after ZIKV injection after immunization with the ZIKV recombinant adenovirus vaccine.

[0105] Figure 17 It is the experimental result of the cross - reaction of the serum of BALB / c mice immunized with the ZIKV recombinant adenovirus vaccine against 4 serotypes of DENV.

[0106] Figure 18 It is the experimental result of the ADE of the serum of BALB / c mice immunized with the ZIKV recombinant adenovirus vaccine against 4 serotypes of DENV - infected cells.

[0107] Figure 19 It is the result of in - vivo experiments detecting the effect of the ZIKV recombinant adenovirus vaccine on the pathogenesis of DENV2 - enhanced infection of Ifnα / βr - / - Ifnγr - / - in mice.

[0108] Figure 20 It is the condition for sorting GC B cells binding to the ZIKV E protein by flow cytometry from the lymph node cells of BALB / c mice immunized with the ZIKV recombinant adenovirus vaccine

[0109] Figure 21 It is the analysis result of the antibody spectrum of GC B cells binding to the ZIKV E protein in BALB / c mice immunized with the ZIKV recombinant adenovirus vaccine.

[0110] Figure 22 It is the result of paired HV and LV in the antibody spectrum induced by immunizing BALB / c mice with the AdC7 - M / E - WT vaccine.

[0111] Figure 23 It is the result of paired HV and LV in the antibody spectrum induced by immunizing BALB / c mice with the AdC7 - M / E - MutB vaccine.

[0112] Figure 24-1 It is the result of paired HV and LV in the antibody spectrum induced by immunizing BALB / c mice with the AdC7 - M / E - MutC vaccine.

[0113] Figure 24-2 These are the paired HV and LV results in the antibody repertoire induced by immunizing BALB / c mice with the AdC7-M / E-MutC vaccine.

[0114] Figure 25 This is to identify the binding ability of antibodies induced by immunizing BALB / c mice with the ZIKV recombinant adenovirus vaccine to different ZIKV E proteins and DENV E proteins.

[0115] Figure 26 These are the experimental results of detecting the ADE effect of antibodies induced by immunizing mice with the representative AdC7-M / E-WT vaccine to promote DENV-infected cells.

[0116] Figure 27 This is a comparison of the V gene loci used by monoclonal antibodies induced by immunizing mice with the AdC7-M / E-WT vaccine with the reported FLE monoclonal antibodies.

[0117] Figure 28-1 This is a comparison of the sequence similarity between monoclonal antibodies induced by immunizing mice with the AdC7-M / E-WT vaccine and the reported FLE monoclonal antibodies.

[0118] Figure 28-2 This is a comparison of the sequence similarity between monoclonal antibodies induced by immunizing mice with the AdC7-M / E-WT vaccine and the reported FLE monoclonal antibodies.

[0119] Figure 29 These are the results of the affinity of the ZIKV sE-WT protein and the ZIKV sE-MutC protein to the FLE antibody detected by BIAcore.

[0120] Figure 30 These are the results of the affinity of the ZIKV sE-WT protein and the ZIKV sE-MutC protein to non-FLE neutralizing antibodies detected by BIAcore.

[0121] Figure 31 This is the complex protein structure of the ZIKV sE-MutC and the Z3L1 single-chain variable region fragment (scFv).

[0122] Figure 32 This is the dimer structure of the ZIKV sE-MutC protein.

[0123] Figure 33 This is the comparative analysis after the superposition of the complex structures of Z3L1 / ZIKV sE MutC and Z3L1 / ZIKV sE WT (PDB: 5GZN).

[0124] Figure 34It is a comparative analysis after the FL region of ZIKV sE-MutC overlaps with the FL region of ZIKV sE-WT (PDB: 5JHM).

[0125] Figure 35 It is a comparative analysis of the complex structure of the FLE antibody and the E protein in the flavivirus genus after the FL of ZIKV sE MutC is overlapped. In the figure, A, B, and C use the Z6 antibody and the ZIKV sE protein, the 2A10G6 antibody and the ZIKV sE protein (PDB: 5JHL), and the E53 antibody and the WNV sE protein (PDB: 3I50) respectively.

[0126] Figure 34 It is a comparative analysis after the FL region of ZIKV sE-MutC overlaps with the FL region of ZIKV sE-WT (PDB: 5JHM).

[0127] Figure 35 It is a comparative analysis of the complex structure of the FLE antibody and the E protein in the flavivirus genus after the FL of ZIKV sE MutC is overlapped. A, B, and C use the Z6 antibody and the ZIKV sE protein, the 2A10G6 antibody and the ZIKV sE protein (PDB: 5JHL), and the E53 antibody and the WNV sE protein (PDB: 3I50) respectively.

[0128] Figure 36 It is the result of detecting the antigen activity of DENV2 wild-type and mutant E proteins by flow cytometry.

[0129] Figure 37 It is the result of detecting the antigen activity of the 101st tryptophan of the ZIKV E protein mutated into other 19 amino acids by flow cytometry. Detailed implementation manners

[0130] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention. Unless otherwise clearly stated, in the whole specification and claims, the term "comprise" or its variations such as "comprises" or "including" etc. will be understood to include the stated elements or components, without excluding other elements or other components.

[0131] In addition, for a better illustration of the present invention, numerous specific details are provided in the following specific embodiments. Those skilled in the art should understand that the present invention can be implemented without certain specific details. In some embodiments, raw materials, components, methods, means, etc. well-known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.

[0132] Example 1 Detection of Humoral Immune Response in Mice Induced by Recombinant Chimpanzee Adenovirus Vaccines Constructed with ZIKV Wild-Type M / E and prM / E Antigens

[0133] The M / E antigen of the ZIKV FSS13025 virus strain (GenBank: JN860885.1) was constructed into a chimpanzee adenovirus serotype 7 vector. After adenovirus packaging, culture, and purification, the recombinant adenovirus vaccine AdC7-M / E-WT was obtained (for the construction of the recombinant adenovirus vaccine AdC7-M / E-WT, refer to: Xu et al. (2018) Journal of virology. vol.92, 6e01722-17.26 Feb.). Experiments have proven that it has good protective effects on mice. There are literature reports that adenovirus vaccines constructed with ZIKV prM / E antigens also have protective effects. Therefore, the prM / E antigen of the ZIKV-SMGC-1 virus strain was constructed into a chimpanzee adenovirus serotype 7 vector, and the recombinant adenovirus AdC7-prM / E-WT was packaged (for the construction of the recombinant adenovirus AdC7-prM / E-WT, refer to Hassan, Ahmed O et al. (2019) Cell reports, vol.28, 10:2634-2646.e4.), which was used as a control for subsequent experiments.

[0134] The humoral immune responses induced by the recombinant adenovirus vaccines AdC7-M / E-WT and AdC7-prM / E-WT in mice were evaluated as follows:

[0135] Fifteen BALB / c mice were randomly divided into 3 groups and immunized with 1.6x 10 11 vp (virus particles) of the AdC7-M / E-WT adenovirus vaccine, the AdC7-prM / E-WT adenovirus vaccine, and PBS. Four weeks later, blood was collected, and the serum was separated by centrifugation. The serum was inactivated by heating at 56 °C for 30 minutes, and the neutralizing antibody titers in the serum were detected using a micro-neutralization assay.

[0136] The process of the micro-neutralization assay is as follows: VERO cells are seeded in 96-well plates one day in advance. On the second day, the serum is serially diluted in DMEM medium (Invitrogen, C11995500BT) containing 1% FBS (Gibco, 10270-106) using a 96-well plate, and the virus is also diluted in DMEM medium with 1% FBS. The serum and virus solution are mixed well, and 100 FFU of ZIKV-SMGC-1 is added to each well. The plate is incubated at 37 °C for 2 hours. The supernatant medium in the VERO cell plate is removed, and the mixture of serum and virus is added. After culturing for 2 hours, DMEM containing 10% FBS is supplemented. The cells are cultured in a 37 °C incubator for 4 days. After 4 days, the cell culture plate is taken out, all the supernatant is discarded, the cells are washed once with PBS, and then fixed with 150 μl of methanol and placed in a -20 °C refrigerator for 15-20 minutes. Then the cells are washed twice with PBS. Blocking is performed using 2% skim milk (blocking solution) prepared with PBS and incubated at room temperature for 30 minutes. Then the primary antibody is added. The primary antibody is the Z6 antibody that binds to the ZIKV E protein and is diluted to a working concentration of 5 μg / mL using the blocking solution and incubated at room temperature for 2 hours. Then the cells are washed three times with PBST, and the secondary antibody is added. The secondary antibody is a goat anti-human antibody conjugated with HRP (Proteintech, SA00001-17), which is diluted 1500-fold with the blocking solution and incubated at room temperature for 2 hours. Then the cells are washed four times with PBST. 50 μl of TMB chromogenic solution (Beyotime, P0209) is added and incubated at room temperature. The reaction time is about 20 minutes. The color change is observed, and 50 μl of 2 M hydrochloric acid is added to terminate the reaction. The OD450 absorbance value is read on an ELISA reader. The data is non-linearly fitted using GraphPad Prism software to calculate the serum dilution factor corresponding to neutralizing 50% of cell infections, which is the neutralization titer value (MN 50 ). When the serum at the lowest dilution factor still cannot neutralize 50% of cell infections, the MN of this sample is defined as 50 half of the lowest dilution factor.

[0137] The results of the neutralization assay are shown as Figure 1 . The group immunized with PBS is the negative control Sham group (denoted as Sham in the figure), and no neutralizing antibodies can be detected in the serum of this group; the Log MN of neutralizing antibodies can be detected in the mice of the AdC7-M / E-WT group (denoted as M / E in the figure) and the AdC7-prM / E-WT group (denoted as prM / E in the figure). 50 The average value is between 2-2.5. Statistical analysis shows that there is no significant difference between these two groups, indicating that both the recombinant adenovirus vaccines AdC7-M / E-WT and AdC7-prM / E-WT induce high levels of neutralizing antibodies in mice.

[0138] Example 2. In vitro experiment to detect antibody-dependent enhancement (ADE) of DENV by sera from mice immunized with wild-type recombinant adenovirus vaccines AdC7-M / E-WT and AdC7-prM / E-WT

[0139] Since the fusion loop (FL) sequence of the envelop (E) protein of flavivirus is highly conserved, infection with Zika virus (ZIKV) or immunization with a vaccine expressing the ZIKV E protein can induce antibodies that cross-react with dengue virus (DENV), leading to antibody-dependent enhancement (ADE) of DENV (Stettler, K., et al. (2016) Science: science.aaf8505.). Therefore, it was detected whether the sera from mice immunized with the recombinant adenovirus vaccines AdC7-M / E-WT and AdC7-prM / E-WT constructed above could cause ADE of DENV.

[0140] The sera (from the mice immunized with the recombinant adenovirus vaccine in Example 1) were serially diluted with RPMI-1640 medium (Invitrogen, C11875500BT) containing 1% FBS, and the diluted samples were added to a 96-well plate, 10 μl per well. Subsequently, the corresponding DENV (DENV2, GenBank: KM204118.1; DENV3, GenBank: KF824903; DENV4, GenBank: AF289029; DENV1 was a virus strain isolated from an infected patient sample at the Third People's Hospital of Shenzhen) was added, and the plate was placed in a 37 °C cell culture incubator for 1 hour.

[0141] The K562 cells expressing the FcγRIIA receptor on the cell surface were centrifuged at 800g for 5 minutes, resuspended with RPMI-1640 medium containing 1% FBS, and the cells were counted. Then the cell density was adjusted to 3 x 10 6Per ml, and then added to the mixture of virus and serum, 10 μl per well, and incubated in a 37 °C cell culture incubator for 2 hours. Supplement with RPMI-1640 medium containing 2% FBS, 100 μl per well, and continue to culture in a 37 °C cell culture incubator for 4 days. After 4 days, transfer the cells to a 96-well plate, centrifuge at 800 g for 5 minutes, remove the supernatant, then wash once with PBS and centrifuge to collect the cells. Add Fixation and Permeabilization solution (BD, 554722) 100 μl per well to the 96-well plate, and place it in a 4 °C refrigerator for 20 minutes. Then centrifuge at 800 g for 5 minutes to collect the cells and wash twice with 1×Perm / Wash buffer (BD, 554723). Add FITC-labeled Z6 antibody (Z6-FITC), 50 μl per well, and place it in a 4 °C refrigerator for 1 hour. Centrifuge to collect the cells and wash twice with 1×Perm / Wash buffer. Resuspend the cells with PBS (200 μl per well), and use a flow cytometer to detect the proportion of positive cells infected with the virus.

[0142] The results are as Figure 2 shown. K562 cells expressing the FcγRIIA receptor on the cell surface were used as a cell infection model in the experiment. The sera of mice immunized with the recombinant adenovirus vaccine AdC7-M / E-WT group (denoted as M / E in the figure) and AdC7-prM / E-WT group (denoted as prM / E in the figure) enhanced the infection of K562 cells with 4 serotypes of DENV. The sera of the control group Sham group (denoted as Sham in the figure) did not have ADE. It is speculated that the sera of mice immunized with the recombinant adenovirus vaccines AdC7-M / E-WT and AdC7-prM / E-WT have cross-reactivity with ZIKV and DENV, and the antibodies in some sera can bind to DENV, thus inducing an ADE reaction.

[0143] Example 3 Detection of ADE of Monoclonal Antibodies against DENV1-4 in vitro

[0144] The Z6 antibody is a monoclonal antibody that binds to the ZIKV E protein and is recombinantly expressed and purified after sequencing the antibody sequence from B cells isolated from the blood of a ZIKV-infected patient. Previous experiments have shown that it mainly binds to the FL epitope of the ZIKV E protein (Wang, Qihui, et al. Science translational medicine 8.369(2016):369ra179.). Since the FL sequences of ZIKV and DENV are highly conserved, and the Z6 antibody has low neutralizing activity (Wang, Qihui, et al. Science translational medicine 8.369(2016):369ra179.), it is speculated that the Z6 antibody is likely to cause ADE to DENV. The recombinant chimpanzee adenovirus vaccines AdC7-M / E-WT and AdC7-prM / E-WT constructed using ZIKV wild-type M / E and prM / E antigens are likely to activate the production of antibodies targeting the FL epitope, thus leading to ADE.

[0145] The ADE effect of the Z6 antibody was detected, and the results are shown in Figure 3 . Figure 3 The results showed that the Z6 antibody had a certain degree of ADE against all four serotypes of DENV. The Z6 antibody with low neutralizing activity cross-reacted with DENV, and it is speculated that it is likely to cause ADE by binding to the FL epitope of the DENV E protein, and further research was carried out on this.

[0146] Example 4 Structural analysis of the complex of ZIKV E protein and Z6 antibody

[0147] Two structures of complexes of antibodies and antigens at the FL epitope in the Flavivirus genus have been reported in the literature, namely the structure of the 2A10G6 antibody and the ZIKV soluble E protein (sE) (Dai, Lianpan, et al. Cell Host & Microbe (2016): S1931312816301494.), and the structure of the E53 antibody and the WNV E protein (Cherrier, Mickael V., et al. The EMBO Journal 28.20(2009):3269-3276.). 2A10G6 is a flavivirus broad-spectrum neutralizing antibody that can neutralize DENV1-4, WNV, YFV, and ZIKV, and it binds to the FL and bc loop of the ZIKV E protein (Dai, Lianpan, et al. Cell Host & Microbe (2016): S1931 312 816 301 494.).

[0148] To further analyze the antibody-binding mechanism of ZIKV FL epitopes, we obtained the complex structure of the Fab fragment of antibody Z6 and ZIKV E protein at a resolution of by combining protein crystallization and X-ray diffraction methods with data analysis. The results are shown in Figure 4 . The data collection and parameter refinement of the complex structure are shown in Table 1.

[0149] As can be seen from Figure 4 , similar to antibody 2A10G6, antibody Z6 binds to the top of DII of E protein at an almost perpendicular angle and interacts with the FL and bc loops of E protein. However, the difference is that both the heavy chain and light chain of Z6 interact with ZIKV E protein, while 2A10G6 focuses more on the binding of the heavy chain. The results are shown in Figure 5 . By analyzing the amino acid sites of the binding, as shown in Figure 6 , it was found that antibody Z6 has more interactions with W101, G106, L107, and F108 of ZIKV E protein, and these 4 amino acid sites also play key roles in binding antibody 2A10G6; the interaction of E53 with E protein does not depend on W101, but it also has more contacts with G106 and L107.

[0150] Therefore, it is predicted that modifying the immunogen at these 4 amino acid sites can avoid the production of antibodies induced by FL epitopes, thereby achieving the purpose of reducing or eliminating ADE to DENV.

[0151] There have been literature reports that ZIKV vaccines based on M / E antigens have good protective effects (Abbink, Peter, et al. Science Translational Medicine 9.420 (2017).). Therefore, we selected the M / E antigen for subsequent mutagenesis design.

[0152] In addition, there have been literature reports that ZIKV vaccines based on prM / E antigens also have good protective effects (Dowd et al, Science, 2016, Vol 354, Issue 6309), and our previous data also demonstrated that the mutagenesis design based on M / E antigens has similar effects on prM / E antigens. Therefore, the experimental results of the mutagenesis design based on prM / E antigens in the embodiments of the present invention will not be elaborated. The mutagenesis design of M / E antigens in the embodiments of the present invention is equally applicable to prM / E antigens.

[0153] Table 1. Data collection and refinement parameters of ZIKV sE-Z6 complex

[0154]

[0155]

[0156] Example 5 Construction of chimeric virus antigen protein using Flavivirus FL with a relatively distant evolutionary distance

[0157] By aligning the amino acid sequences of the FL epitopes of the E proteins of Flavivirus, we found that the FL sequences of most viruses are very conserved, but there are still some FL sequences of viruses with relatively distant evolutionary distances that are different from the FL sequence of ZIKV. In order not to affect the normal protein folding and the display of other neutralizing epitopes while disrupting the ZIKV FL epitope, we used the amino acid sequences of FL of viruses with a relatively distant evolutionary distance from ZIKV in Flavivirus (the Flavivirus phylogenetic tree is as Figure 7 shown) to construct chimeric virus antigen proteins, speculating that it can better maintain the overall conformation of the antigen and reduce the impact on epitopes other than the mutant sites.

[0158] We designed mutants of the ZIKV M / E antigen with the FL sequences of these viruses as a reference. The sites and sequences of mutations in the FL fusion region of the full-length M and E are as Figure 8 shown. Among them, MutA is derived from AEFV (Aedes flavivirus, GenBank: KC181923.1), and the mutation sites are D98N, N103T, G106F, L107E, and F108W;

[0159] MutB is derived from CFAV (Cell fusing agent virus, GenBank: NC_001564.2), and the mutation sites are D98N, N103T, G106F, L107K, and F108W;

[0160] MutC is derived from NAKV (Nakiwogo virus, GenBank: NC_030400.1), and the mutation sites are D98N, N103T, G106L, L107E, and F108W.

[0161] The primers used in constructing the pCAGGS-M / E-MutA / B / C plasmid are shown in Table 2. Taking the construction of MutA as an example, the plasmid pCAGGS-M / E-WT was used as a template, and the products mutA-1 were obtained by PCR with WT-F and mutA-R as primers. The plasmid pCAGGS-M / E-WT was used as a template, and the products mutA-2 were obtained by PCR with WT-R and mutA-F as primers. Then, mutA-1 and mutA-2 were mixed in a molar ratio of 1:1 as a template, and WT-F and WT-R were used as primers for PCR to obtain the PCR product mutA. The pCAGGS plasmid was digested with XhoI and EcoRI to obtain a linear plasmid with sticky ends. The digested linear plasmid was mixed with the PCR product mutA in a molar ratio of 1:5, and recombinant was carried out using the In-Fusion kit. The recombinant product was transformed into DH5α competent cells, spread on an ampicillin-resistant plate and cultured at 37 °C. Then, clones were picked for PCR identification and sequencing identification, and then the plasmid (pCAGGS-M / E-MutA) was extracted for subsequent experiments.

[0162] Table 2 Primers used in constructing pCAGGS-M / E-MutA / B / C

[0163]

[0164]

[0165] Example 6 Detection of the activity of M / E-MutA / B / C antigens

[0166] The wild-type plasmid pCAGGS-M / E-WT and the mutant plasmids pCAGGS-M / E-MutA, pCAGGS-M / E-MutB and pCAGGS-M / E-MutC were transfected into 293T cells respectively. After 48 hours, the cells were collected, digested into single cells, fixed and permeabilized, incubated with an antibody binding to ZIKV E, incubated with a goat anti-human FITC secondary antibody, and finally the positive ratio of the samples was detected by flow cytometry. The results are as Figure 9 shown.

[0167] Z3L1, Z20 and Z23 are all ZIKV-specific antibodies with high neutralizing activity, which bind to DI, DII and DIII of the ZIKV E protein respectively (Wang, Qihui, et al. (2016) Science translational medicine 8.369:369ra179.).

[0168] By Figure 9It can be seen that the antibodies (Z6 and 2A10G6) that bind to the FL epitope of the ZIKV E protein can bind to cells expressing the wild-type M / E-WT antigen, but neither binds to cells expressing the three mutant antigens. The Z3L1, Z23, and Z20 antibodies with high neutralizing activity that bind to non-FL epitopes bind to both cells expressing the wild-type M / E-WT antigen and cells expressing the three mutant antigens. This indicates that the FL epitopes on the M / E-MutA, M / E-MutB, and M / E-MutC antigens are disrupted, resulting in the inability of the corresponding antibodies to bind, but there is no change in the epitopes bound by other ZIKV potent neutralizing antibodies, and the corresponding antibodies can still bind.

[0169] That is, the M / E-MutA, M / E-MutB, and M / E-MutC antigens can induce fewer or no antibodies that bind to DENV FL, thereby reducing the ADE to DENV; at the same time, the M / E-MutA, M / E-MutB, and M / E-MutC antigens do not affect other antibody epitopes.

[0170] Example 7 uses the M / E-MutB and M / E-MutC antigens of ZIKV to construct recombinant chimpanzee adenovirus vaccines AdC7-M / E-MutB and AdC7-M / E-MutC

[0171] From Figure 9 It can be seen that the intensity of cell positivity of MutB and MutC is slightly higher than that of MutA. Therefore, MutB and MutC are mainly used in subsequent experiments.

[0172] First, clone the M / E-MutB and M / E-MutC antigens into the pshuttle vector. Using the plasmid pCAGGS-M / E-MutB or pCAGGS-M / E-MutC as a template, perform a PCR reaction with to_pshuttle-F and to_pshuttle-R as primers to obtain the PCR products to_pshuttle-mutB and to_pshuttle-mutC. Digest the pshuttle plasmid with XbaI (Thermo, FD0684) and KpnI (Thermo, FD0524) to obtain a linear plasmid with sticky ends. Mix the digested linear plasmid with the PCR product to_pshuttle-mutB or to_pshuttle-mutC at a molar ratio of 1:5, perform recombination using the In-Fusion kit, transform the recombinant product into DH5α competent cells, spread them on a kanamycin-resistant plate and culture at 37°C. Then pick colonies for PCR identification and sequencing identification, and then extract the plasmid. After that, construct the cassettes expressing M / E-MutB and M / E-MutC on the pshuttle plasmid into the AdC7 vector. See the above construction steps in: Xu, Kun et al. (2018) Journal of virology. vol.92, 6e01722-17. 26 Feb.

[0173] Using the plasmid pshuttle-M / E-MutB or MutC as a template, perform a PCR reaction with to_AdC7-F and to_AdC7-R as primers to obtain the PCR products to_AdC7-MutB and to_AdC7-MutC. Digest the AdC7 plasmid with PI-SceI (NEB, R0696S) and I-CeuI (NEB, R0699S) to obtain a linear plasmid with sticky ends. Mix the digested linear plasmid with the PCR product to_AdC7-MutB or to_AdC7-MutC at a molar ratio of 1:5, perform recombination using the In-Fusion kit, transform the recombinant product into stbl2 competent cells, spread them on an ampicillin-resistant plate and culture at 30°C. Then pick colonies for PCR identification and sequencing identification, and then extract the plasmid. The construction process of the recombinant plasmid is shown in Figure 10 . The primer sequences used are shown in Table 3.

[0174] Table 3: Primers for constructing chimpanzee adenovirus ZIKV mutant vaccines with MutB and MutC antigens

[0175]

[0176] The plasmids pAdC7-M / E-MutB and pAdC7-M / E-MutC were digested into linear form with the PacI (NEB, R0547S) restriction endonuclease, and then the endonuclease was inactivated by heating in a constant temperature bath at 65°C for 20 minutes. The plasmids were transfected into HEK293 cells using the Fugene-6 transfection reagent (Promega, E2691), and the cells were cultured in an incubator at 37°C for at least 7 days. Thereafter, the cells were examined daily under a microscope for the appearance of plaques. When the plaque cells detached, all the cells and the supernatant were collected, which was the first-generation recombinant adenovirus. The culture could be successively expanded at a ratio of 1:10 until 40 plates of cells were cultured. All the cells were collected, and the cells were lysed by freezing and thawing 3 times to release the virus. Then, it was purified by cesium chloride density gradient centrifugation and desalted and purified by polyacrylamide gel (Bio-Gel P-6DG Media, BIO-RAD, 1500738). The OD260 of the sample was detected using NANODROP, and the concentration of the sample was the OD260 value multiplied by 1.1x 10 12 , and the unit is vp (viral particle) / ml. It was aliquoted and stored at -80°C.

[0177] Example 8 Evaluation of the Humoral Immune Response Induced by Recombinant Adenovirus Vaccines AdC7-M / E-MutB and AdC7-M / E-MutC in BALB / c Mice

[0178] Twenty-four BALB / c mice were randomly divided into 4 groups and immunized with 3 recombinant adenovirus vaccines by intramuscular injection, namely AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC. The immunization dose of the adenovirus vaccine was 1.6x10 11 vp, and 1 group of mice was immunized with PBS as a negative control. Four weeks later, blood was collected to isolate serum, and the neutralizing antibody titer in the serum was detected using a micro-neutralization assay.

[0179] The results are as Figure 11 shown. The wild-type AdC7-M / E-WT and the mutant adenovirus vaccines AdC7-M / E-MutB and AdC7-M / E-MutC could all induce the production of neutralizing antibodies in mice. The average value of Log(MN 50 ) was between 2.0 and 2.5, and there was no significant difference in the neutralizing antibody titers between the two groups of AdC7-M / E-MutB and AdC7-M / E-MutC and the AdC7-M / E-WT group, indicating that the mutation did not significantly reduce the immunogenicity of the antigen and could still activate the production of neutralizing antibodies well. Among them, Sham in the figure refers to the group immunized with PBS, M / E-WT refers to the group immunized with the AdC7-M / E-WT vaccine, M / E-MutB refers to the group immunized with the AdC7-M / E-MutB vaccine, and M / E-MutC refers to the group immunized with the AdC7-M / E-MutC vaccine.

[0180] Example 9. Evaluation of Humoral Immune Responses Induced by AdC7-M / E-MutB and AdC7-M / E-MutC Vaccines in Ifnar1 - / - Mice

[0181] Since ZIKV infection in BALB / c mice does not cause mouse death or obvious disease symptoms, to better verify the effectiveness of the vaccine, we selected immunodeficient Ifnar1 - / - Mice as a ZIKV infection model (Lazear, Helen M et al. (2016), vol.19, 5:720 - 30.), and evaluated the humoral immune levels of Ifnar1 - / - Mice induced by AdC7-M / E-MutB and AdC7-M / E-MutC vaccines

[0182] The Ifnar1 - / - Mice were randomly divided into 4 groups and were respectively injected with AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC adenovirus vaccines by intramuscular injection. The immunization dose of the adenovirus vaccine was 1.6x10 11 vp. One group of mice was immunized with PBS as a negative control. After 28 days, blood was collected to isolate serum, and the micro-neutralization assay was used to detect the titer levels of neutralizing ZIKV antibodies in the serum of Ifnar1 - / - Mice. The results are as Figure 12 shown

[0183] As Figure 12 can be seen, both AdC7-M / E-MutB and AdC7-M / E-MutC vaccines can induce a high level of neutralizing antibody differences in mice. Among them, Sham in the figure refers to the group immunized with PBS, M / E-WT refers to the group immunized with AdC7-M / E-WT vaccine, M / E-MutB refers to the group immunized with AdC7-M / E-MutB vaccine, and M / E-MutC refers to the group immunized with AdC7-M / E-MutC vaccine

[0184] Example 10. Challenge Protection Experiment in Ifnar1 - / - Mice after Immunization

[0185] The Ifnar1 - / - Mice immunized in Example 9 were challenged with ZIKV on the 30th day after immunization by intraperitoneal injection of 5x10 6 PFU ZIKV (SMGC-1 strain). Then, the status of the mice was observed daily and the body weight changes were monitored. The experimental results are shown in Figure 13 .

[0186] From Figure 13It can be seen that the body weight of the sham group gradually decreased starting from the 4th day after virus challenge ( Figure B in Figure 13 ), and all the mice in the sham group died on the 6th and 7th days ( Figure A in Figure 13 ). However, for the mice immunized with AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC, there was no weight loss after virus challenge ( Figure B in Figure 13 ), and none of the mice in the three groups died ( Figure A in Figure 13 ). This indicates that the AdC7-M / E-MutB and AdC7-M / E-MutC vaccines can provide complete protection during mouse virus challenge, just like the wild-type AdC7-M / E-WT vaccine. Among them, Sham in the figure refers to the group immunized with PBS, M / E-WT refers to the group immunized with the AdC7-M / E-WT vaccine, M / E-MutB refers to the group immunized with the AdC7-M / E-MutB vaccine, and M / E-MutC refers to the group immunized with the AdC7-M / E-MutC vaccine.

[0187] To detect whether the AdC7-M / E-MutB and AdC7-M / E-MutC vaccines can protect mice against viremia caused by virus infection, the blood of mice on the 3rd and 6th days after virus challenge was collected in this experiment, the serum was separated, and then RNA was extracted using the MagaBio Plus Viral RNA Kit (Bioer Technology, BSC58S1B). Then, the FastKing One-Step RT-qPCR Kit (Tiangen Biochemical Technology, FP314) was used to quantify the viral RNA. The probe and primer sequences used for quantification are shown in Table 4, and the quantification results are as shown in Figure 14 .

[0188] Table 4: Probe and primer sequences used for RT-PCR quantification of ZIKV-SMGC-1 nucleic acid

[0189]

[0190] By Figure 14It can be seen that relatively high viral loads could be detected in the mice of the Sham group on the 3rd and 6th days after infection, while no virus could be detected in the sera of the mice immunized with the AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC adenovirus vaccines on the 3rd and 6th days after infection, indicating that the AdC7-M / E-MutB and AdC7-M / E-MutC vaccines could protect mice against viremia as well as the wild-type AdC7-M / E-WT vaccine. Among them, Sham in the figure refers to the group immunized with PBS, M / E-WT refers to the group immunized with the AdC7-M / E-WT vaccine, M / E-MutB refers to the group immunized with the AdC7-M / E-MutB vaccine, and M / E-MutC refers to the group immunized with the AdC7-M / E-MutC vaccine.

[0191] Example 11 Detection of the sterilizing immunity effect of the ZIKV vaccine

[0192] The above experiments demonstrated that immunization of mice with the AdC7-M / E-MutB and AdC7-M / E-MutC vaccines could provide protection against viremia and death caused by ZIKV infection. Further experiments were designed to detect whether the AdC7-M / E-MutB and AdC7-M / E-MutC vaccines could provide sterilizing immunity, and the specific steps were as follows.

[0193] The Ifnar1 - / - mice were randomly divided into 4 groups and immunized with 3 recombinant adenovirus vaccines, AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC, by intramuscular injection. The immunization dose of the adenovirus vaccine was 1.6x10 11 vp. One group of mice was immunized with PBS as a negative control. Blood was collected on the 28th day after immunization to separate serum, and 5x10 4 FFU of ZIKV (SMGC-1 strain) was injected intraperitoneally on the 30th day after immunization. Blood was collected again on the 6th day after challenging with ZIKV, and then on the same day, part of the liver, spleen, testis, brain, and spinal cord were dissected. The dissected tissues and organs were added to PBS solution, then ground using a grinder (Tiangen Biochemical Technology, OSE-Y30), centrifuged to take the supernatant, and RNA was extracted using the MagaBio Plus viral RNA kit, and then RT-PCR was performed on ZIKV RNA. The results are as Figure 15 shown.

[0194] From Figure 15It can be seen that a certain amount of virus was detected in 5 organs of the mice in the Sham group, while no virus was detected in all tissues and organs of the three groups of mice immunized with the AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC adenovirus vaccines. Among them, Sham in the figure refers to the group immunized with PBS, M / E-WT refers to the group immunized with the AdC7-M / E-WT vaccine, M / E-MutB refers to the group immunized with the AdC7-M / E-MutB vaccine, and M / E-MutC refers to the group immunized with the AdC7-M / E-MutC vaccine.

[0195] The neutralizing antibody titers in the sera on the 28th day after immunization and the 7th day after virus challenge were detected using a micro-neutralization assay, and the results are as Figure 16 shown.

[0196] As Figure 16 can be seen, no neutralizing antibody was detected in the Sham group before virus challenge, and a relatively high titer of neutralizing antibody could be detected in each mouse in the Sham group on the 7th day after virus challenge; there was no significant difference in the serum neutralizing antibody titers of the two groups immunized with the AdC7-M / E-WT and AdC7-M / E-MutB vaccines before and after virus challenge, maintaining at almost the same level, which indicates that both the AdC7-M / E-WT vaccine and the AdC7-M / E-MutB and AdC7-M / E-MutC vaccines can provide complete immunity against virus challenge. The AdC7-M / E-MutB vaccine has a slightly better effect than the AdC7-M / E-MutC vaccine and is a complete clearance immunity. Among them, Sham in the figure refers to the group immunized with PBS, M / E-WT refers to the group immunized with the AdC7-M / E-WT vaccine, M / E-MutB refers to the group immunized with the AdC7-M / E-MutB vaccine, and M / E-MutC refers to the group immunized with the AdC7-M / E-MutC vaccine; Figure 16 The results of the Sham group, M / E-WT group, M / E-MutB group, and M / E-MutC group are shown from left to right in

[0197] Example 12 Detection of the cross-reactivity of sera from ZIKV vaccine-immunized BALB / c mice against DENV

[0198] A major reason for ADE against DENV caused by ZIKV infection is that the FL epitopes of the E proteins of ZIKV and DENV are relatively conserved, resulting in some antibodies induced by ZIKV having cross-reactivity with DENV (Stettler, K., et al. Science (2016): science.aaf8505.). Therefore, we further detected the cross-reactivity of sera from BALB / c mice immunized with the AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC adenovirus vaccines against 4 serotypes of DENV by ELISA, and the results are asFigure 17 as shown

[0199] It can be seen from Figure 17 that the sera of BALB / c mice in the immune AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC vaccine groups all had strong binding ability to the ZIKV E protein. However, their binding abilities to the DENV E proteins of 4 serotypes were different: the sera of mice in the M / E-MutB group and the M / E-MutC group had very low binding ability to the DENV E proteins of 4 serotypes, while the sera of mice in the M / E-WT group still maintained a relatively high binding ability to the DENV E proteins of 4 serotypes. This shows that the mutant disrupted the FL epitope of the E protein, reducing the amount of antibodies induced by this epitope, thus showing that the sera of mice immunized with the AdC7-M / E-MutB and AdC7-M / E-MutC adenovirus vaccines had weakened binding ability to the DENV E protein.

[0200] Among them, Figure 17 Sham refers to the group immunized with PBS, M / E-WT refers to the group immunized with the AdC7-M / E-WT vaccine, M / E-MutB refers to the group immunized with the AdC7-M / E-MutB vaccine, and M / E-MutC refers to the group immunized with the AdC7-M / E-MutC vaccine; Figure 17 In figure A, it is the result of the binding ability of mouse sera to the Zika virus (ZIKV) E protein, in figure B, it is the result of the binding ability of mouse sera to the dengue virus type 1 (DENV1) E protein, in figure C, it is the result of the binding ability of mouse sera to the dengue virus type 2 (DENV2) E protein, in figure D, it is the result of the binding ability of mouse sera to the dengue virus type 3 (DENV3) E protein, and in figure E, it is the result of the binding ability of mouse sera to the dengue virus type 4 (DENV4) E protein.

[0201] Example 13 In vitro experiment to detect the antibody-dependent enhancement (ADE) of DENV by sera from ZIKV-immunized BALB / c mice

[0202] The experimental results of Example 12 demonstrated that the AdC7-M / E-MutB and AdC7-M / E-MutC vaccines reduced the induction of cross-antibodies against DENV. We further designed experiments to prove whether the AdC7-M / E-MutB and AdC7-M / E-MutC vaccines could reduce the ADE of DENV, and the specific steps are as follows.

[0203] The sera from the BALB / c mice immunized in Example 8 were serially diluted, incubated with DENV1, DENV2, DENV3, and DENV4 respectively, then K562 cells were added. After culturing for 4 days, they were stained with FITC-labeled Z6 antibody, and then the proportion of positive cells was detected by flow cytometry. The results are asFigure 18 as shown

[0204] In the absence of antibody mediation, DENV virus cannot infect K562 cells. From Figure 18 it can be seen that since the serum of the Sham group does not contain antibodies that can bind to DENV, the infection rate of the detected samples is at the background level; the mouse serum of the M / E-WT group can mediate the infection of K562 cells by DENV viruses of 4 serotypes at a certain concentration; while the infection rates of the two groups of samples of M / E-MutB and M / E-MutC are significantly lower than that of the M / E-WT group, showing a weakening or even elimination of ADE against DENV, indicating that the AdC7-M / E-MutB and AdC7-M / E-MutC vaccines achieved a good effect of reducing ADE.

[0205] Among them, Sham in the figure refers to the group immunized with PBS, M / E-WT refers to the group immunized with the AdC7-M / E-WT vaccine, M / E-MutB refers to the group immunized with the AdC7-M / E-MutB vaccine, and M / E-MutC refers to the group immunized with the AdC7-M / E-MutC vaccine.

[0206] Figure 18 Figure A in shows the detection of the enhanced infection of K562 cells by mouse serum against ZIKV, Figure B shows the detection of the enhanced infection of K562 cells by mouse serum against DENV1, Figure C shows the detection of the enhanced infection of K562 cells by mouse serum against DENV2, Figure D shows the detection of the enhanced infection of K562 cells by mouse serum against DENV3, and Figure E shows the detection of the enhanced infection of K562 cells by mouse serum against DENV4.

[0207] The above series of in vitro experiments have all proved that the AdC7-M / E-MutB and AdC7-M / E-MutC adenovirus vaccines can reduce or even eliminate the ADE against DENV after immunization.

[0208] Example 14 In vivo experiment to detect the ADE of ZIKV vaccine-immunized BALB / c mouse serum against DENV

[0209] To further prove whether the AdC7-M / E-MutB and AdC7-M / E-MutC vaccines can reduce the ADE against DENV under physiological conditions, we used a model based on Ifnα / βr - / - Ifnγr - / - mice to verify the ADE effect against DENV.

[0210] First, 80 BALB / c mice were randomly divided into 4 groups of 20 mice each, and were immunized with AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC adenovirus vaccines and PBS, respectively. The immunization dose of the adenovirus vaccine was 1.6 x 10 11 vp per mouse. After 4 weeks, blood was collected, serum was separated, and heated at 56 °C for 30 minutes. The sera of the 20 mice in each group were mixed together for subsequent passive immunization of Ifnα / βr - / - Ifnγr - / - mice. The Ifnα / βr - / - Ifnγr - / - mice were randomly divided into 4 groups and were injected intraperitoneally with AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC adenovirus vaccines or sera from BALB / c mice immunized with PBS (the sera were diluted with PBS at a ratio of 1:10, and 200 μl of the diluted sera was injected per mouse). 24 hours later, DENV2 virus was injected subcutaneously at 5000 FFU per mouse, and each mouse was weighed and recorded before injection. Subsequently, the status, survival, and body weight of the mice were observed daily, and the results are as Figure 19 shown.

[0211] As Figure 19 can be seen, compared with the mice in the Sham group, the overall death time of the mice in the M / E-WT group was earlier, indicating that the sera of the mice immunized with the AdC7-M / E-WT vaccine enhanced the pathogenesis of DENV, while the death time and trend of the mice in the M / E-MutB and M / E-MutC groups were consistent with those in the Sham group ( Figure 19 Figure A in). This indicates that the mutation of the ZIKV E protein weakened the production of antibodies that can cause the ADE effect on DENV, making the performance of the mice in the M / E-MutB and M / E-MutC groups similar to that in the Sham group. The trend of body weight change also showed that the body weight of the mice in the M / E-WT group decreased faster, while the trend of body weight change of the mice in the M / E-MutB and M / E-MutC groups was consistent with that in the Sham group ( Figure 19 Figure B in). These in vivo experimental results well demonstrated that the AdC7-M / E-MutB and AdC7-M / E-MutC vaccines could significantly reduce the ADE of DENV after immunization with the wild-type vaccine.

[0212] Among them, Figure 19 Sham refers to the PBS-immunized group, M / E-WT refers to the AdC7-M / E-WT vaccine-immunized group, M / E-MutB refers to the AdC7-M / E-MutB vaccine-immunized group, and M / E-MutC refers to the AdC7-M / E-MutC vaccine-immunized group.

[0213] Analysis of the antibody repertoire induced by the 15ZIKV vaccine in BALB / c mice

[0214] Since the ZIKV vaccine with FL substitution induced protective immunity while reducing the ADE response to DENV, we further analyzed the B cell repertoire of ZIKV E in mice to explain how the mutant vaccine affects the antibody response.

[0215] BALB / c mice were randomly divided into 3 groups and immunized intramuscularly with AdC7-M / E-WT adenovirus vaccine (WT group), AdC7-M / E-MutB adenovirus vaccine (MutB group), and AdC7-M / E-MutC adenovirus vaccine (MutC group), respectively, at a dose of 1.6 x 10 11 vp. On the 20th day after immunization, the lymph nodes were dissected and placed in 1640 medium containing 1% FBS. The lymph nodes of all mice in each vaccine group were pooled together, ground using the rough surface of a glass slide, and then filtered through a 0.45 μm filter. The lymphocytes were centrifuged at 400 g for 15 minutes at 4°C, the supernatant was discarded, and the cells were resuspended in 1 ml of FACS buffer. FACS buffer is PBS solution supplemented with 0.5% FBS. After resuspension, the cells were transferred to a 1.5 ml EP tube and centrifuged at 400 g for 10 minutes at 4°C, and the supernatant was discarded. The cells were resuspended in 200 μl of FACS buffer, and a mixture of 4 μg of biotin-labeled ZIKV E monomeric protein and dimeric protein was added, and the cells were incubated in the dark at 4°C for 30 min. Then 1 ml of FACS buffer was added, mixed well, and the cells were centrifuged to precipitate. The cells were washed once with 1 ml of FACS buffer, and then stained with antibodies. The antibodies were diluted with FACS buffer, and each 200 μl of antibody solution contained: FITC-GL7, 2 μl (BD, 553666); PE-CD138, 4 μl (BD, 553714); PE / CY7-CD38, 4 μl (BioLegend, 102718); APC-CD93, 4 μl (BioLegend, 136510); BV421-B220, 16 μl (BioLegend, 103240); BV510–IgD, 2 μl (BD, 563110); BV711, 4 μl (BD, 563262). 200 μl of antibody solution was added to each sample, and the samples were incubated in the dark at 4°C for 30 min, and then washed twice with FACS buffer. 2 ml of FCAS buffer was added to resuspend the cells, filtered through a 0.45 μm filter, transferred to a flow tube, and the sorting condition was GL-7 + B220 hi CD38 lo IgD - CD93- CD138 -+ (as shown in Figure 20 ), the GC B cells reactive to ZIKV E were screened out and then library construction was performed before sequencing.

[0216] To obtain the paired B cell receptor (BCR) sequences of single cells, we used single cell sequencing technology. The Chromium Single Cell V(D)J Enrichment Kit, Mouse B Cell, 96rxns (10x genomics, PN-1000072) kit was used for library construction before sequencing, and then high-throughput sequencing was performed to analyze the full-length sequences of the V(D)J segments of the heavy and light chains of each cell. The results are as shown in Figure 21 and Table 5.

[0217] As can be seen from Figure 21 and Table 5, the WT group samples obtained 451 heavy chain variable region sequences and 661 light chain variable region sequences, and 334 pairs could be paired; the MutB group samples obtained 310 heavy chain variable region sequences and 379 light chain variable region sequences, and 234 pairs could be paired; the MutC group samples obtained 664 heavy chain variable region sequences and 776 light chain variable region sequences, and 515 pairs could be paired. Among them, Figure 21 in

[0218] and Table 5, WT refers to the group immunized with the AdC7-M / E-WT vaccine, MutB refers to the group immunized with the AdC7-M / E-MutB vaccine, and MutC refers to the group immunized with the AdC7-M / E-MutC vaccine.

[0219]

[0220] Analysis of the antibody spectra induced by WT, MutB and MutC in mice found that the AdC7-M / E-WT vaccine had a preference for activating the variable (V) region genes of mice. Approximately 60% of the heavy chains used IGHV9-2-1, IGHV1-22 and IGHV7-3, and approximately 60% of the light chains used IGKV10-96, IGKV14-111 and IGKV6-23 ( Figure 21 ). However, for the AdC7-M / E-MutB and AdC7-M / E-MutC vaccines with the FL replaced, the BCR spectra showed more diverse and dispersed in the heavy chain variable region (HV) and light chain variable region (LV) ( Figure 21 ). In the WT group, several of the most significantly activated variable region genes were significantly reduced or even absent in the antibody spectra of the MutB and MutC groups of vaccines with the FL replaced.Figure 21 )。

[0221] Then we analyzed the paired HV and LV results in the antibody repertoire, and the results are shown respectively as Figure 22 , Figure 23 and Figure 24-1 and Figure 24-2 shown.

[0222] As Figure 22 can be seen, AdC7-M / E-WT vaccine-activated GC B cell clones in mice have an obvious preference for HV:LV, among which the frequencies of IGHV9-2-1:IGKV10-96 (29.9%), IGHV1-22:IGKV14-111 (14.4%) and IGKV1-22:IGKV6-23 (7.5%) are relatively high, and the sum of these three is about 50%.

[0223] As Figure 23 and Figure 24-1 and 24-2 can be seen, AdC7-M / E-MutB and AdC7-M / E-MutC vaccines activate more types of HV:LV in mouse GCB cell clones, and the frequency distribution is also more dispersed. The highest IGHV9-2-1:IGKV10-96 was detected in the WT group and did not appear in the MutB and MutC groups.

[0224] The results of antibody repertoire analysis show that after replacing the FL region of AdC7-M / E-WT vaccine, the immunodominant epitopes of B cell responses are transferred.

[0225] Example 16 Identification of the main FLE antibody types with ADE response to DENV infection

[0226] Among the antibodies causing ADE response to DENV, a large part targets the FLE of the E protein, and the antibodies induced by ZIKV infection can also cause ADE response to DENV. Therefore, we identified the binding characteristics of the above isolated monoclonal antibodies to detect which ones bind to FLE and whether they can induce ADE response to DENV.

[0227] According to the classification based on the similarity of GC B cell clones, some representative monoclonal antibody genes were synthesized (Genewiz, Suzhou), covering 63.38%, 46.57%, and 43.88% of the total of the three groups of AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC, respectively (Table 6). Subsequently, the HV and LV genes were cloned into murine IgG2A and Igκ expression vectors. Monoclonal antibodies derived from the AdC7-M / E-WT vaccine group (denoted as M / E-WT in Table 6) were named ZWT.1-10, and monoclonal antibodies derived from the AdC7-M / E-MutB vaccine group (denoted as M / E-MutB in Table 6) and AdC7-M / E-MutC vaccine group (denoted as M / E-MutC in Table 6) were named ZMutB.1-8 and ZMutC.1-13, respectively (Table 6).

[0228] Table 6 Murine monoclonal antibodies expressed

[0229]

[0230]

[0231]

[0232] : Considering 7 gene components: HV, HD, and HJ genes of the heavy chain and the length of CDRH3, and LV, LJ, and CDRL3 lengths of the light chain, when 4 or more elements of the clone are the same, it is considered to belong to the same gene cluster.

[0233] b : ELISA was used to detect the binding of each monoclonal antibody to ZIKV E (monomer or dimer). When the OD of the sample 450 is more than 5 times higher than the negative control value, it is considered to bind; otherwise, it is considered not to bind.

[0234] We co-transfected 293T cells with plasmids expressing the heavy and light chains of the monoclonal antibody. After 3 days, the supernatant was collected, and the binding ability of the antibody in the supernatant to ZIKV-E protein was detected by ELISA.

[0235] The experimental method of ELISA is as follows: Dilute the protein to 3 μg / ml with ELISA coating buffer (sodium carbonate-sodium bicarbonate buffer, pH 9.6), add 100 μl to each well of a 96-well ELISA plate, and place it at 4 °C overnight. The next day, pour out the coating buffer, block the ELISA plate with 5% skim milk prepared with PBS, and place it at room temperature for 1 hour. Pour out the blocking solution, add 100 μl of the culture supernatant expressing monoclonal antibody to each well of the ELISA plate, incubate at room temperature for 2 hours, and wash 3 times with PBST. Then add the secondary antibody Goat Anti-Mouse HRP (ab6789) diluted 1:2000 with the blocking solution, incubate at room temperature for 1.5 hours, and wash 4 times with PBST. Add 50 μl of TMB chromogenic solution for color development, add 50 μl of 2M hydrochloric acid to terminate the reaction after 30 minutes, and detect the OD450 reading on an enzyme-linked immunosorbent assay (ELISA) reader.

[0236] The ELISA detection results are as Figure 25 shown. Most of the monoclonal antibodies can bind to the monomeric or dimeric form of the ZIKV sE protein; as can be seen from Table 6, the positive rates of binding reach 90% (9 / 10) in the ME-WT group, 87.5% (7 / 8) in the ME-MutB group, and 76.9% (10 / 13) in the ME-MutC group.

[0237] Twenty-six positive monoclonal antibodies that bind to the ZIKV sE protein were further evaluated. From Figure 25 , we found that all the monoclonal antibodies derived from the ME-WT group did not react with the ZIKV sE-MutC protein, but had cross-reactivity with the sE proteins of DENV serotypes 1 to 4, indicating that the monoclonal antibodies derived from the ME-WT group are monoclonal antibodies that bind to FLE; while the monoclonal antibodies derived from the ME-MutB and ME-MutC groups are mainly ZIKV-specific antibodies, and they hardly bind to DENV sE.

[0238] We expressed and purified the representative FLE monoclonal antibodies derived from the ME-WT group to further evaluate the ADE effect of these antibodies on DENV in K562 cells. The results are as Figure 26 shown. From Figure 26 , it can be seen that all the antibodies detected from the ME-WT group have a certain degree of enhanced infection effect on DENV of 4 serotypes. Among them, Figure 26 , ZWT.1, 4, 6, 8, 9, 10 are monoclonal antibodies from the M / E-WT group respectively; DENV1, DENV 2, DENV 3, DENV 4 are DENV viruses of serotypes 1-4 respectively; from A to D are the results of antibodies ZWT.1, 4, 6, 8, 9, 10 promoting the enhanced infection of K562 cells by DENV of 4 serotypes.

[0239] The FLE monoclonal antibodies (mAbs) derived from the ME-WT group mainly consist of 4 types of HV:LV gene combinations. We tried to search for previously reported flavivirus FLE murine mAbs in the literature and databases, and then compared them with the antibody loci and sequences we isolated. The following 4 mAbs were found:

[0240] 6B6C-1, which was isolated after infection with Tick-borne encephalitis virus (TBEV) (Crill et al., (2004) Journal of Virology, 78.24:13975-13986.);

[0241] Both 4G2 and 2A10G6 were isolated after DENV infection (Bennett et al. (2015), BMC Biotechnology, 15.1:71-71.; Deng, Yongqiang, et al. (2011), PLOS ONE 6.1);

[0242] E53 was isolated after WNV infection (Oliphant et al. (2006), Journal of Virology 80.24:12149-12159.).

[0243] After analysis, it was found that the FLE mAbs we isolated from the ME-WT group had many similarities in loci and sequences with these 4 reported mAbs, such as Figure 27 、 Figure 28-1 and Figure 28-2 as shown. As can be seen from Figure 27 , 6B6C-1 and 4G2 use the same HV:LV gene pairs as ZWT.1-3 and ZWT.4-5 respectively, and the sequence similarity is relatively high; 2A10G6 and E53 use the same HV genes as ZWT.6 and ZWT.8 respectively.

[0244] Although 2A10G6 and ZWT.6 use different LV genes, the LV sequences of the two antibodies are somewhat similar. As can be seen from Figure 28-2 , the CDRL3 and FR4 of 2A10G6 and ZWT.6 are exactly the same.

[0245] From the above analysis, it was found that the FLE mAbs cloned from the lymph node GC B cells of mice immunized with the AdC7-M / E-WT vaccine use loci that are close to or even the same as those of the previously reported murine FLE mAbs, and are relatively similar in sequence, indicating that there is a preference for the loci used to induce antibodies in mice that bind to the FL epitope, and the characteristics of the generated FLE antibodies are also relatively similar.

[0246] Figure 28-1Among them, A is the sequence alignment analysis of the heavy chains of antibodies ZWT.1, ZWT.2, ZWT.3, 6B6C-1 with the mouse locus, B is the sequence alignment of the light chains of antibodies ZWT.1, ZWT.2, ZWT.3, 6B6C-1 with the mouse locus, and C is the sequence alignment of the heavy chains of antibodies ZWT.4, ZWT.5, ZWT.6, 4G2, 2A10G6 with the mouse locus; Figure 28-2 Among them, D is the sequence alignment of the light chains of antibodies ZWT.4, ZWT.5, 4G2 with the mouse locus, and E is the sequence alignment of the light chains of antibodies ZWT.6, 2A10G6 with the mouse locus.

[0247] Example 17 Detection of the Affinity of Wild-Type and Mutant ZIKV E Proteins for ZIKV Antibodies by SPR Experiment

[0248] Since the AdC7-M / E-MutB and AdC7-M / E-MutC vaccines can provide complete protection for mice while avoiding ADE to DENV, to explain the potential molecular mechanism, we expressed and purified the soluble sE-MutC protein as a representative and compared it with sE-WT.

[0249] BIOCORE8000 is based on the principle of Surface Plasmon Resonance (SPR), which can detect the interaction between molecules, reflect the dynamic changes during the molecular binding process in real time, and obtain the kinetic parameters of the interaction.

[0250] Use BIOCORE8000 to detect the affinity of ZIKV sE-WT protein and ZIKV sE-MutC protein for FLE antibody and non-FLE neutralizing antibodies. Using the amino coupling method, fix ZIKV sE-WT protein and ZIKV sE-MutC protein to the CM5 chip respectively, and then serially dilute 4 kinds of antibodies as the mobile phase. The test antibodies flow through the immobilized ZIKV sE protein in turn to obtain different binding corresponding signals. The collected data is calculated by fitting, and the results are as Figure 29 and Figure 30 shown.

[0251] Figure 29 Among them, from A to D are the binding results of ZIKV sE-WT protein and 2A10G6 antibody (sE-WT-2A10G6), the binding results of ZIKV sE-MutC protein and 2A10G6 antibody (sE-MutC-2A10G6), the binding results of ZIKV sE-WT protein and Z6 antibody (sE-WT-Z6), and the binding results of ZIKV sE-MutC protein and Z6 antibody (sE-MutC-Z6). Among them, by Figure 29As can be seen from Figure A in [reference], the affinity of the 2A10G6 antibody for the ZIKV sE-WT protein is 9.13 nM; from Figure 29 As can be seen from Figure C in [reference], the affinity of the Z6 antibody for the ZIKV sE-WT protein is 7.14 nM; from Figure 29 As can be seen from Figure B in [reference], the ZIKV sE-MutC protein does not bind to the 2A10G6 antibody at all; from Figure 29 As can be seen from Figure D in [reference], the ZIKV sE-MutC protein does not bind to the Z6 antibody at all; this result is also consistent with the theoretical analysis.

[0252] Figure 30 In [reference], from A to D are the binding results of the ZIKV sE-WT protein and the Z3L1 antibody (sE-WT-Z3L1), the binding results of the ZIKV sE-MutC protein and the Z3L1 antibody (sE-MutC-Z3L1), the binding results of the ZIKV sE-WT protein and the Z23 antibody (sE-WT-Z23), and the binding results of the ZIKV sE-MutC protein and the Z23 antibody (sE-MutC-Z23). Among them, from Figure 30 Figures A and C in [reference], the affinities of the Z3L1 and Z23 antibodies for the ZIKV E-WT protein are 9.48 μM and 0.625 μM respectively; from Figure 30 Figures B and D in [reference], the affinities of the Z3L1 and Z23 antibodies for the ZIKV sE-MutC protein are 8.01 μM and 0.701 μM respectively. The affinities of the mutant protein ZIKV sE-MutC for Z23 and Z3L1 are almost unchanged compared with the wild-type ZIKV sE-WT protein; this indicates that the designed mutant can maintain the overall conformation of the protein and does not cause changes in other epitopes outside the mutant sites.

[0253] Example 18 Structural Analysis of the Complex of Mutant ZIKV sE-MutC Protein and Z3L1 Antibody

[0254] To further explain the mechanism of action of the mutant vaccine, we purified the complex protein of ZIKV sE-MutC and the single-chain variable fragment (scFv) of Z3L1, then performed crystal screening, and finally obtained the atomic structure of the complex with a resolution of as shown in Figure 31 The data collection and refinement parameters of the complex are shown in Table 7.

[0255] Table 7 Data Collection and Refinement Parameters of the ZIKV sE MutC-Z3L1 Complex

[0256]

[0257]

[0258] From Figure 31 It can be seen that although five point mutations were introduced into the FL sequence of the ZIKV E protein, sE-MutC still binds to the scFV of Z3L1 in the form of a dimer in the pre-fusion state. A detailed analysis of the dimer contact interface revealed that the mutated FL amino acid residues established new interactions between two adjacent E protein protomers, generating four and one hydrogen bonds at N98 and W108 respectively, which is beneficial to the stability of the E protein dimer, as shown in Table 8.

[0259] From Figure 32 It can be seen that the folding form of sE-MutC is very similar to that of the wild-type protein, showing normal secondary, tertiary and quaternary epitope structures.

[0260] To analyze the conformation of the neutralizing epitope, we superimposed the complex structure of ZIKV sE-MutC and the scFv of Z3L1 (Z3L1 / ZIKV sE MutC) with the complex structure of Z3L1 and ZIKV sE-WT protein (Z3L1 / ZIKV sE WT, PDB:5GZN), and the results are as Figure 33 shown. From Figure 33 it can be seen that the binding modes of the ZIKV wild-type protein sE-WT and the mutant protein sE-MutC to the Z3L1 antibody are the same. The binding sites are mainly the D0, E0 and F0 strands in DI and the 150 loop, as well as the kl hairpin in DII (Wang, et al. (2016) Science translational medicine 8.369:369ra179.).

[0261] The FL region of sE-MutC was superimposed with sE-WT for comparative analysis, and the results are shown in Figure 34 . From Figure 34 it can be seen that the conformations of the FL epitopes of the two are very similar, showing differences only in the side chains of the mutation sites.

[0262] To analyze the possibility of the mutant antigen sE-MutC inducing FLE antibodies, we superimposed the structures of the FLE antibodies with known structures (Z6 antibody, 2A10G6 antibody and E53 antibody) binding to the flavivirus E protein with the DII structure of sE-MutC for analysis, and the results are as Figure 35 shown.

[0263] Figure 35Among them, the complex structures of the Z6 antibody and ZIKV sE protein, the 2A10G6 antibody and ZIKV sE protein (PDB: 5JHL), and the E53 antibody and WNV sE protein (PDB: 3I50) were used from A to C respectively. From Figure 35 It can be seen that after the mimic monoclonal antibody binds to sE-MutC, the mutations of G106, L107, and F108 will significantly hinder the binding of the antigen to the antibody; the long side chain protruding from G106L will cause a conflict with the binding of these FLE antibodies; the L107E mutation results in the formation of a charged side chain, which may disrupt the local hydrophobic interaction in the interaction; in addition, the F108W mutation will form a steric hindrance to affect the interaction between the FL epitope of sE-MutC and the 2A10G6 antibody. Therefore, on the FL epitope of the ZIKV E protein, the mutations of these 3 key amino acids, G106, L107, and F108, play a synergistic role and eliminate the induction of FLE antibodies.

[0264] Table 8 Amino acid analysis of the interaction between two protomers in the ZIKV E dimer

[0265]

[0266]

[0267] Example 19 Construction of DENV vaccines with MutA, MutB, and MutC mutations

[0268] In the design of DENV vaccines, how to avoid ADE remains an unresolved issue (REF). The FL sequences of ZIKV and DENV are very conservative. Through the above systematic experiments, it has been proved that the AdC7-M / E-MutB and AdC7-M / E-MutC adenovirus vaccines can provide complete protection to mice while avoiding ADE to DENV. Therefore, DENV vaccines with MutA, MutB, and MutC mutations were constructed to verify whether the vaccines have similar effects.

[0269] First, the signal peptide gene from JEV (Japanese encephalitis virus) (SEQ ID NO.17) and the M / E gene expressing DENV2 New Guinea C virus strain (GenBank: KM204118.1) (SEQ ID NO.18) were constructed into the pshuttle vector to obtain the plasmid pshuttle-DV2-M / E-WT expressing wild-type DENV2 M / E. The signal peptide gene from JEV (SEQ ID NO.17) and the gene expressing the prM / E protein of DENV2 New Guinea C virus strain (SEQ ID NO.19) were constructed into the pshuttle vector to obtain the plasmid pshuttle-DV2-prM / E-WT expressing wild-type DENV2 prM / E.

[0270] All mutants were constructed based on the M / E-WT antigen. Using the pshuttle-DV2-M / E-WT plasmid as a template, plasmids of mutants MutA (D98N, N103T, G106F, L107E, and F108W), MutB (D98N, N103T, G106F, L107K, and F108W), and MutC (D98N, N103T, G106L, L107E, and F108W) were constructed. The primer sequences used in the construction process are shown in Table 9.

[0271] Table 9 Primers used for constructing pshuttle-DV2-M / E-WT, pshuttle-DV2-prM / E-WT, and pshuttle-DV2-M / E-MutA / B / C

[0272]

[0273] The plasmids pshuttle-DV2-M / E-WT and pshuttle-DV2-prM / E-WT expressing wild-type proteins and three mutant plasmids were transfected into 293T cells respectively. After 48 hours, the supernatant was removed, the cells were washed once with PBS, then digested with trypsin into single cells, centrifuged and resuspended with DMEM medium, and washed once with DMEM medium. Then, the Fixation and Permeabilization solution from BD was added and placed on ice for 20 minutes. Then, the cells were collected by centrifugation at 800g for 10 minutes and washed twice with 1×Perm / Wash buffer from BD. Each sample was divided into 5 parts, and the antibodies Z6, 2A10G6, and mAb11 that bind to the FL epitope and the antibodies mAb513 and D448 that bind to the non-FL epitope were added respectively, and placed in the refrigerator at 4°C for 1 hour. The cells were collected by centrifugation and washed twice with 1×Perm / Wash buffer. Then, the Goat Anti-Human FITC (Proteintech, 00003-12) antibody was added and placed in the refrigerator at 4°C for 1 hour. The cells were collected by centrifugation and washed twice with 1×Perm / Wash buffer. The cells were resuspended with PBS (200 μl per well), and the positive ratio of the samples was detected using a flow cytometer. The experimental results are as Figure 36 shown.

[0274] As Figure 36 can be seen, the antibodies Z6 and 2A10G6 that bind to the FL epitope can recognize the M / E-WT and prM / E-WT proteins of DENV2. However, after introducing three mutant combinations into the FL epitope of the E protein of DENV2, the binding ability of the Z6 and 2A10G6 antibodies to the M / E-MutA or M / E-MutB or M / E-MutC proteins of DENV2 was significantly weakened or even non-binding. For the mAb513 and D448 antibodies that bind to the non-FL epitope and have relatively high neutralizing activity, they can still bind to the M / E-MutA or M / E-MutB or M / E-MutC proteins of DENV2 with the MutA, MutB, and MutC mutations introduced, indicating that the mutations in the FL epitope of the DENV2 E protein did not have a significant impact on other epitopes. The above results show that these three mutant combinations can be applied in DENV vaccines, and the vaccine obtained from the DENV2 E protein antigen based on the mutant combination at the G106, L107, and F108 sites can reduce the ADE effect caused by subsequent DENV virus infection after vaccination.

[0275] Example 20 Detection of the effect of mutating the W101 position of ZIKV E into other 19 amino acids on antigen activity

[0276] In the structures of Z6 / ZIKV sE and 2A10G6 / ZIKV sE, the amino acid of the E protein that interacts most with antibodies is W101. There are also literature reports that the binding of most FLE antibodies to the E protein depends on W101 (Dejnirattisai, W. et al. (2015). Nat Immunol 16, 170 - 177.). Therefore, we tried to mutate W101 into the other 19 amino acids and then use flow cytometry to detect whether the epitopes of the cell-expressed antigens changed, in order to screen out the most suitable mutations.

[0277] The signal peptide gene from JEV (SEQ ID NO.17) and the M / E gene of wild-type ZIKV (SEQ ID NO.31) were constructed into the pCAGGS vector (Addgene) to obtain the plasmid pCAGGS-M / E-WT that can express the M / E protein of wild-type ZIKV. Using this plasmid as a template, the tryptophan at position 101 of the E protein was mutated into the other 19 amino acids. Taking the mutation of tryptophan into alanine as an example, using the plasmid pCAGGS-M / E-WT as a template, PCR was performed with W101-WT-F and W101-1A-R as primers to obtain the product W101-1A-1, and the primer sequences are shown in Table 10. Using the plasmid pCAGGS-M / E-WT as a template, PCR was performed with W101-WT-R and W101-1A-F as primers to obtain the product W101-1A-2. Then, W101-1A-1 and W101-1A-2 were mixed in a molar ratio of 1:1 as a template, and PCR was performed with W101-WT-F and W101-WT-R as primers to obtain the PCR product W101-1A. The pCAGGS vector was digested with XhoI (Thermo, FD0694) and EcoRI (Thermo, FD0274) to obtain a linear plasmid with sticky ends. The digested linear plasmid was mixed with W101-1A in a molar ratio of 1:5, and recombination was performed using the In-Fusion kit (Takara, 639648). The recombinant product was transformed into DH5α competent cells, spread on an ampicillin-resistant plate and cultured at 37°C. Then, clones were picked for PCR identification and sequencing identification.

[0278] Table 10 Primers for mutating tryptophan at position 101 of the E protein of ZIKV

[0279]

[0280]

[0281] After extracting the plasmids of 19 mutants of W101, the wild-type plasmid and the 19 mutant plasmids were transfected into 293T cells respectively. After 48 hours, the supernatant was removed, and the cells were washed once with PBS. Then the cells were digested into single cells with trypsin, centrifuged, resuspended with DMEM medium, and washed once with DMEM medium. Then, Fixation and Permeabilization solution from BD was added, and the cells were placed on ice for 20 minutes. Then, the cells were collected by centrifugation at 800g for 10 minutes and washed twice with 1×Perm / Wash buffer from BD. Each sample was divided into 5 parts, and Z6 and 2A10G6 antibodies were added respectively, and the samples were placed in a refrigerator at 4°C for 1 hour. The cells were collected by centrifugation and washed twice with 1×Perm / Wash buffer. Then, Goat Anti-Human FITC (Proteintech, 00003-12) antibody was added, and the samples were placed in a refrigerator at 4°C for 1 hour. The cells were collected by centrifugation and washed twice with 1×Perm / Wash buffer. The cells were resuspended with PBS (200 μl per well), and the positive ratio of the samples was detected by flow cytometry. The experimental results are as Figure 37 shown.

[0282] As Figure 37 can be seen, the wild-type M / E antigen of ZIKV can bind to Z6 and 2A10G6 antibodies, but almost none of the 19 mutants can bind to Z6 and 2A10G6 antibodies, indicating that the tryptophan at position 101 of the ZIKV E protein is crucial for activating antibodies targeting FL. The vaccine prepared by mutating the W101 site will reduce or avoid the production of antibodies induced by the FL epitope, thus avoiding the ADE effect on DENV after vaccination.

[0283] Example 21 Detection of the binding ability of ZIKV E protein mutants at positions G106, L107, and F108 to FLE antibodies

[0284] Based on the above pCAGGS-ZIKV-M / E expression plasmid, the following single-site and double-site mutations were made. The method for constructing mutant plasmids was referred to Example 20, and the primers used in the process of constructing mutant plasmids are shown in Table 11.

[0285] Table 11 Primers used for mutating positions G106, L107, and F108 of the E protein of ZIKV

[0286]

[0287]

[0288]

[0289] The wild-type plasmid pCAGGS-ZIKV-M / E-WT and the successfully constructed mutant plasmids were transfected into 293T cells respectively. After 48 hours, the cells were collected, digested into single cells, fixed and permeabilized, and incubated with the ADE antibodies Z6 and 2A10G6 that bind to the FL epitope. The Goat Anti-Human(mouse)FITC secondary antibody was incubated, and finally the positive ratio of the samples was detected by flow cytometry. If the positive ratio was less than 10% of the wild-type positive rate, it was considered non-binding; if it was between 10% and 50%, it was considered weak binding. The results are shown in Table 12.

[0290] As can be seen from Table 12, the above mutations can basically prevent the binding of the representative ADE antibodies (Z6 antibody and 2A10G6 antibody) of the FL epitope, indicating that the vaccines prepared by single-site or cooperative mutations at the G106, L107 and F108 sites represented by these mutations will be able to reduce or avoid the generation of ADE antibodies induced by the FL epitope, thus avoiding the ADE effect on DENV after vaccination.

[0291] Table 12 Binding results of ZIKV E protein mutants with Z6 antibody and 2A10G6 antibody

[0292]

[0293]

[0294] Note: -- indicates non-binding.

[0295] Example 22 Detection of the binding ability of mutations at the G106, L107 and F108 sites of DENV E protein to FLE antibody

[0296] Based on the above pCAGGS-DENV2-M / E expression plasmid, the following single-site and double-site mutations were made. The construction method of the mutant plasmids was referred to Example 19, and the primers used in the process of constructing the mutant plasmids are shown in Table 13.

[0297] Table 13 Primers used for mutation of the G106, L107 and F108 sites of the E protein of DENV

[0298]

[0299]

[0300]

[0301]

[0302] The wild-type plasmids pCAGGS-ZIKV-M / E-WT, pCAGGS-DENV2-M / E-WT and the mutants constructed in the above table were transfected into 293T cells respectively. After 48 hours, the cells were collected, digested into single cells, fixed and permeabilized, and incubated with the ADE antibodies Z6 and 2A10G6 that bind to the FL epitope. The Goat Anti-Human (mouse) FITC secondary antibody was incubated, and finally the positive ratio of the samples was detected by flow cytometry. If the positive ratio is less than 10% of the wild-type positive rate, it is considered non-binding; if it is 10%-50%, it is considered weak binding. The results are shown in Table 14.

[0303] As can be seen from Table 14, the above mutations basically blocked the binding of the representative ADE antibodies to the FL epitope, indicating that the vaccines prepared by single-site or cooperative mutations of G106, L107 and F108 represented by these mutations will reduce or avoid the generation of ADE antibodies induced by the FL epitope, thus avoiding the ADE effect on DENV after vaccination.

[0304] Table 14 Binding results of DENV E protein mutants with Z6 antibody and 2A10G6 antibody

[0305]

[0306]

[0307] Note: -- indicates non-binding.

[0308] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention. SEQUENCE LISTING <110> Institute of Microbiology, Chinese Academy of Sciences <120> A Zika / Dengue vaccine and its application <130> 1087-190438F-1-D2 <160> 191 <170> PatentIn version 3.5 <210> 1 <211> 504 <212> PRT <213> zika virus <400> 1 Ile Arg Cys Ile Gly Val Ser Asn Arg Asp Phe Val Glu Gly Met Ser 1 5 10 15 Gly Gly Thr Trp Val Asp Val Val Leu Glu His Gly Gly Cys Val Thr 20 25 30 Val Met Ala Gln Asp Lys Pro Thr Val Asp Ile Glu Leu Val Thr Thr 35 40 45 Thr Val Ser Asn Met Ala Glu Val Arg Ser Tyr Cys Tyr Glu Ala Ser 50 55 60 Ile Ser Asp Met Ala Ser Asp Ser Arg Cys Pro Thr Gln Gly Glu Ala 65 70 75 80 Tyr Leu Asp Lys Gln Ser Asp Thr Gln Tyr Val Cys Lys Arg Thr Leu 85 90 95 Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser 100 105 110 Leu Val Thr Cys Ala Lys Phe Ala Cys Ser Lys Lys Met Thr Gly Lys 115 120 125 Ser Ile Gln Pro Glu Asn Leu Glu Tyr Arg Ile Met Leu Ser Val His 130 135 140 Gly Ser Gln His Ser Gly Met Ile Val Asn Asp Thr Gly His Glu Thr 145 150 155 160 Asp Glu Asn Arg Ala Lys Val Glu Ile Thr Pro Asn Ser Pro Arg Ala 165 170 175 Glu Ala Thr Leu Gly Gly Phe Gly Ser Leu Gly Leu Asp Cys Glu Pro 180 185 190 Arg Thr Gly Leu Asp Phe Ser Asp Leu Tyr Tyr Leu Thr Met Asn Asn 195 200 205 Lys His Trp Leu Val His Lys Glu Trp Phe His Asp Ile Pro Leu Pro 210 215 220 Trp His Ala Gly Ala Asp Thr Gly Thr Pro His Trp Asn Asn Lys Glu 225 230 235 240 Ala Leu Val Glu Phe Lys Asp Ala His Ala Lys Arg Gln Thr Val Val 245 250 255 Val Leu Gly Ser Gln Glu Gly Ala Val His Thr Ala Leu Ala Gly Ala 260 265 270 Leu Glu Ala Glu Met Asp Gly Ala Lys Gly Arg Leu Ser Ser Gly His 275 280 285 Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Arg Leu Lys Gly Val Ser 290 295 300 Tyr Ser Leu Cys Thr Ala Ala Phe Thr Phe Thr Lys Ile Pro Ala Glu 305 310 315 320 Thr Leu His Gly Thr Val Thr Val Glu Val Gln Tyr Ala Gly Thr Asp 325 330 335 Gly Pro Cys Lys Val Pro Ala Gln Met Ala Val Asp Met Gln Thr Leu 340 345 350 Thr Pro Val Gly Arg Leu Ile Thr Ala Asn Pro Val Ile Thr Glu Ser 355 360 365 Thr Glu Asn Ser Lys Met Met Leu Glu Leu Asp Pro Pro Phe Gly Asp 370 375 380 Ser Tyr Ile Val Ile Gly Val Gly Glu Lys Lys Ile Thr His His Trp 385 390 395 400 His Arg Ser Gly Ser Thr Ile Gly Lys Ala Phe Glu Ala Thr Val Arg 405 410 415 Gly Ala Lys Arg Met Ala Val Leu Gly Asp Thr Ala Trp Asp Phe Gly 420 425 430 Ser Val Gly Gly Ala Leu Asn Ser Leu Gly Lys Gly Ile His Gln Ile 435 440 445 Phe Gly Ala Ala Phe Lys Ser Leu Phe Gly Gly Met Ser Trp Phe Ser 450 455 460 Gln Ile Leu Ile Gly Thr Leu Leu Val Trp Leu Gly Leu Asn Thr Lys 465 470 475 480 Asn Gly Ser Ile Ser Leu Met Cys Leu Ala Leu Gly Gly Val Leu Ile 485 490 495 Phe Leu Ser Thr Ala Val Ser Ala 500 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 ttttggcaaa gaattcgccg 20 <210> 3 <211> 41 <212> DNA <213> Artificial Sequence <400> 3 gatctgctag ctcgagtcaa gcgctcacag ctgtggacag a 41 <210> 4 <211> 55 <212> DNA <213> Artificial Sequence <400> 4 gtgtaagagg accctggtga acaggggctg gggaacaggc tgcttcgaat gggga 55 <210> 5 <211> 54 <212> DNA <213> Artificial Sequence <400> 5 gtgaacaggg gctggggaac aggctgcttc gaatggggaa agggctccct ggtg 54 <210> 6 <211> 55 <212> DNA <213> Artificial Sequence <400> 6 gtgtaagagg accctggtga acaggggctg gggaacaggc tgcttcaagt gggga 55 <210> 7 <211> 54 <212> DNA <213> Artificial Sequence <400> 7 gtgaacaggg gctggggaac aggctgcttc aagtggggaa agggctccct ggtg 54 <210> 8 <211> 55 <212> DNA <213> Artificial Sequence <400> 8 gtgtaagagg accctggtga acaggggctg gggaacaggc tgcctggaat gggga 55 <210> 9 <211> 54 <212> DNA <213> Artificial Sequence <400> 9 gtgaacaggg gctggggaac aggctgcctg gaatggggaa agggctccct ggtg 54 <210> 10 <211> 37 <212> DNA <213> Artificial Sequence <400> 10 aaacgggccc tctagagcca ccatgggcaa gaggagc 37 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <400> 11 tttaacttaa gcttggtacc tcaagcgctc acagctgtgg 40 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <400> 12 gtataactat aacggtccta aggtagcgaa 30 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <400> 13 tcattacctc tttctccgca cccgacatag 30 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <400> 14 ccacacctct gccggcacac 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <400> 15 ttggctggcc tatcaggttg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <400> 16 cacctcggtt tgagcactct 20 <210> 17 <211> 24 <212> PRT <213> Japanese encephalitis virus <400> 17 Met Gly Lys Arg Ser Ala Gly Ser Ile Met Trp Leu Ala Ser Leu Ala 1 5 10 15 Val Val Ile Ala Cys Ala Gly Ala 20 <210> 18 <211> 570 <212> PRT <213> Dengue virus <400> 18 Ser Val Ala Leu Val Pro His Val Gly Met Gly Leu Glu Thr Arg Thr 1 5 10 15 Glu Thr Trp Met Ser Ser Glu Gly Ala Trp Lys His Ala Gln Arg Ile 20 25 30 Glu Thr Trp Ile Leu Arg His Pro Gly Phe Thr Ile Met Ala Ala Ile 35 40 45 Leu Ala Tyr Thr Ile Gly Thr Thr His Phe Gln Arg Ala Leu Ile Phe 50 55 60 Ile Leu Leu Thr Ala Val Ala Pro Ser Met Thr Met Arg Cys Ile Gly 65 70 75 80 Ile Ser Asn Arg Asp Phe Val Glu Gly Val Ser Gly Gly Ser Trp Val 85 90 95 Asp Ile Val Leu Glu His Gly Ser Cys Val Thr Thr Met Ala Lys Asn 100 105 110 Lys Pro Thr Leu Asp Phe Glu Leu Ile Lys Thr Glu Ala Lys Gln Pro 115 120 125 Ala Thr Leu Arg Lys Tyr Cys Ile Glu Ala Lys Leu Thr Asn Thr Thr 130 135 140 Thr Asp Ser Arg Cys Pro Thr Gln Gly Glu Pro Ser Leu Asn Glu Glu 145 150 155 160 Gln Asp Lys Arg Phe Val Cys Lys His Ser Met Val Asp Arg Gly Trp 165 170 175 Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Gly Ile Val Thr Cys Ala 180 185 190 Met Phe Thr Cys Lys Lys Asn Met Lys Gly Lys Val Val Gln Pro Glu 195 200 205 Asn Leu Glu Tyr Thr Ile Val Ile Thr Pro His Ser Gly Glu Glu His 210 215 220 Ala Val Gly Asn Asp Thr Gly Lys His Gly Lys Glu Ile Lys Ile Thr 225 230 235 240 Pro Gln Ser Ser Ile Thr Glu Ala Glu Leu Thr Gly Tyr Gly Thr Val 245 250 255 Thr Met Glu Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val 260 265 270 Leu Leu Gln Met Glu Asn Lys Ala Trp Leu Val His Arg Gln Trp Phe 275 280 285 Leu Asp Leu Pro Leu Pro Trp Leu Pro Gly Ala Asp Thr Gln Gly Ser 290 295 300 Asn Trp Ile Gln Lys Glu Thr Leu Val Thr Phe Lys Asn Pro His Ala 305 310 315 320 Lys Lys Gln Asp Val Val Val Leu Gly Ser Gln Glu Gly Ala Met His 325 330 335 Thr Ala Leu Thr Gly Ala Thr Glu Ile Gln Met Ser Ser Gly Asn Leu 340 345 350 Leu Phe Thr Gly His Leu Lys Cys Arg Leu Arg Met Asp Lys Leu Gln 355 360 365 Leu Lys Gly Met Ser Tyr Ser Met Cys Thr Gly Lys Phe Lys Val Val 370 375 380 Lys Glu Ile Ala Glu Thr Gln His Gly Thr Ile Val Ile Arg Val Gln 385 390 395 400 Tyr Glu Gly Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu Ile Met Asp 405 410 415 Leu Glu Lys Arg His Val Leu Gly Arg Leu Ile Thr Val Asn Pro Ile 420 425 430 Val Thr Glu Lys Asp Ser Pro Val Asn Ile Glu Ala Glu Pro Pro Phe 435 440 445 Gly Asp Ser Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln Leu Lys Leu 450 455 460 Asn Trp Phe Lys Lys Gly Ser Ser Ile Gly Gln Met Ile Glu Thr Thr 465 470 475 480 Met Arg Gly Ala Lys Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp 485 490 495 Phe Gly Ser Leu Gly Gly Val Phe Thr Ser Ile Gly Lys Ala Leu His 500 505 510 Gln Val Phe Gly Ala Ile Tyr Gly Ala Ala Phe Ser Gly Val Ser Trp 515 520 525 Thr Met Lys Ile Leu Ile Gly Val Ile Ile Thr Trp Ile Gly Met Asn 530 535 540 Ser Arg Ser Thr Ser Leu Ser Val Ser Leu Val Leu Val Gly Val Val 545 550 555 560 Thr Leu Tyr Leu Gly Val Met Val Gln Ala 565 570 <210> 19 <211> 661 <212> PRT <213> Dengue virus <400> 19 Phe His Leu Thr Thr Arg Asn Gly Glu Pro His Met Ile Val Ser Arg 1 5 10 15 Gln Glu Lys Gly Lys Ser Leu Leu Phe Lys Thr Glu Asp Gly Val Asn 20 25 30 Met Cys Thr Leu Met Ala Met Asp Leu Gly Glu Leu Cys Glu Asp Thr 35 40 45 Ile Thr Tyr Lys Cys Pro Phe Leu Arg Gln Asn Glu Pro Glu Asp Ile 50 55 60 Asp Cys Trp Cys Asn Ser Thr Ser Thr Trp Val Thr Tyr Gly Thr Cys 65 70 75 80 Thr Thr Thr Gly Glu His Arg Arg Glu Lys Arg Ser Val Ala Leu Val 85 90 95 Pro His Val Gly Met Gly Leu Glu Thr Arg Thr Glu Thr Trp Met Ser 100 105 110 Ser Glu Gly Ala Trp Lys His Ala Gln Arg Ile Glu Thr Trp Ile Leu 115 120 125 Arg His Pro Gly Phe Thr Ile Met Ala Ala Ile Leu Ala Tyr Thr Ile 130 135 140 Gly Thr Thr His Phe Gln Arg Ala Leu Ile Phe Ile Leu Leu Thr Ala 145 150 155 160 Val Ala Pro Ser Met Thr Met Arg Cys Ile Gly Ile Ser Asn Arg Asp 165 170 175 Phe Val Glu Gly Val Ser Gly Gly Ser Trp Val Asp Ile Val Leu Glu 180 185 190 His Gly Ser Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr Leu Asp 195 200 205 Phe Glu Leu Ile Lys Thr Glu Ala Lys Gln Pro Ala Thr Leu Arg Lys 210 215 220 Tyr Cys Ile Glu Ala Lys Leu Thr Asn Thr Thr Thr Asp Ser Arg Cys 225 230 235 240 Pro Thr Gln Gly Glu Pro Ser Leu Asn Glu Glu Gln Asp Lys Arg Phe 245 250 255 Val Cys Lys His Ser Met Val Asp Arg Gly Trp Gly Asn Gly Cys Gly 260 265 270 Leu Phe Gly Lys Gly Gly Ile Val Thr Cys Ala Met Phe Thr Cys Lys 275 280 285 Lys Asn Met Lys Gly Lys Val Val Gln Pro Glu Asn Leu Glu Tyr Thr 290 295 300 Ile Val Ile Thr Pro His Ser Gly Glu Glu His Ala Val Gly Asn Asp 305 310 315 320 Thr Gly Lys His Gly Lys Glu Ile Lys Ile Thr Pro Gln Ser Ser Ile 325 330 335 Thr Glu Ala Glu Leu Thr Gly Tyr Gly Thr Val Thr Met Glu Cys Ser 340 345 350 Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val Leu Leu Gln Met Glu 355 360 365 Asn Lys Ala Trp Leu Val His Arg Gln Trp Phe Leu Asp Leu Pro Leu 370 375 380 Pro Trp Leu Pro Gly Ala Asp Thr Gln Gly Ser Asn Trp Ile Gln Lys 385 390 395 400 Glu Thr Leu Val Thr Phe Lys Asn Pro His Ala Lys Lys Gln Asp Val 405 410 415 Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu Thr Gly 420 425 430 Ala Thr Glu Ile Gln Met Ser Ser Gly Asn Leu Leu Phe Thr Gly His 435 440 445 Leu Lys Cys Arg Leu Arg Met Asp Lys Leu Gln Leu Lys Gly Met Ser 450 455 460 Tyr Ser Met Cys Thr Gly Lys Phe Lys Val Val Lys Glu Ile Ala Glu 465 470 475 480 Thr Gln His Gly Thr Ile Val Ile Arg Val Gln Tyr Glu Gly Asp Gly 485 490 495 Ser Pro Cys Lys Ile Pro Phe Glu Ile Met Asp Leu Glu Lys Arg His 500 505 510 Val Leu Gly Arg Leu Ile Thr Val Asn Pro Ile Val Thr Glu Lys Asp 515 520 525 Ser Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Asp Ser Tyr Ile 530 535 540 Ile Ile Gly Val Glu Pro Gly Gln Leu Lys Leu Asn Trp Phe Lys Lys 545 550 555 560 Gly Ser Ser Ile Gly Gln Met Ile Glu Thr Thr Met Arg Gly Ala Lys 565 570 575 Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Leu Gly 580 585 590 Gly Val Phe Thr Ser Ile Gly Lys Ala Leu His Gln Val Phe Gly Ala 595 600 605 Ile Tyr Gly Ala Ala Phe Ser Gly Val Ser Trp Thr Met Lys Ile Leu 610 615 620 Ile Gly Val Ile Ile Thr Trp Ile Gly Met Asn Ser Arg Ser Thr Ser 625 630 635 640 Leu Ser Val Ser Leu Val Leu Val Gly Val Val Thr Leu Tyr Leu Gly 645 650 655 Val Met Val Gln Ala 660 <210> 20 <211> 31 <212> DNA <213> Artificial Sequence <400> 20 aaacgggccc tctagagcca ccatgctcaa c 31 <210> 21 <211> 42 <212> DNA <213> Artificial Sequence <400> 21 tttaacttaa gcttggtacc ttaagcttgc accatgactc cc 42 <210> 22 <211> 73 <212> DNA <213> Artificial Sequence <400> 22 aaacgggccc tctagagcca ccatgctcaa cattttaaac agaaggagga gaaccgccgg 60 aatgatcatc atg 73 <210> 23 <211> 71 <212> DNA <213> Artificial Sequence <400> 23 ggagaaccgc cggaatgatc atcatgctga tccccaccgt gatggccagc gtggctctgg 60 tgccccatgt c 71 <210> 24 <211> 73 <212> DNA <213> Artificial Sequence <400> 24 aaacgggccc tctagagcca ccatgctcaa cattttaaac agaaggagga gaaccgccgg 60 aatgatcatc atg 73 <210> 25 <211> 59 <212> DNA <213> Artificial Sequence <400> 25 gtgaacagag gctggggcac aggatgcttc gaatggggaa agggaggcat cgtgacttg 59 <210> 26 <211> 59 <212> DNA <213> Artificial Sequence <400> 26 tccccattcg aagcatcctg tgccccagcc tctgttcacc atggagtgct tgcacacga 59 <210> 27 <211> 59 <212> DNA <213> Artificial Sequence <400> 27 gtgaacagag gctggggcac aggatgcttc aagtggggaa agggaggcat cgtgacttg 59 <210> 28 <211> 59 <212> DNA <213> Artificial Sequence <400> 28 tccccacttg aagcatcctg tgccccagcc tctgttcacc atggagtgct tgcacacga 59 <210> 29 <211> 59 <212> DNA <213> Artificial Sequence <400> 29 gtgaacagag gatggggcac aggatgcctg gaatggggca agggctcttt aatcacttg 59 <210> 30 <211> 59 <212> DNA <213> Artificial Sequence <400> 30 gccccattcc aggcatcctg tgccccatcc tctgttcacg aaggtcctac gacacacga 59 <210> 31 <211> 579 <212> PRT <213> zika virus <400> 31 Ala Val Thr Leu Pro Ser His Ser Thr Arg Lys Leu Gln Thr Arg Ser 1 5 10 15 Gln Thr Trp Leu Glu Ser Arg Glu Tyr Thr Lys His Leu Ile Arg Val 20 25 30 Glu Asn Trp Ile Phe Arg Asn Pro Gly Phe Ala Leu Ala Ala Ala Ala 35 40 45 Ile Ala Trp Leu Leu Gly Ser Ser Thr Ser Gln Lys Val Ile Tyr Leu 50 55 60 Val Met Ile Leu Leu Ile Ala Pro Ala Tyr Ser Ile Arg Cys Ile Gly 65 70 75 80 Val Ser Asn Arg Asp Phe Val Glu Gly Met Ser Gly Gly Thr Trp Val 85 90 95 Asp Val Val Leu Glu His Gly Gly Cys Val Thr Val Met Ala Gln Asp 100 105 110 Lys Pro Thr Val Asp Ile Glu Leu Val Thr Thr Thr Val Ser Asn Met 115 120 125 Ala Glu Val Arg Ser Tyr Cys Tyr Glu Ala Ser Ile Ser Asp Met Ala 130 135 140 Ser Asp Ser Arg Cys Pro Thr Gln Gly Glu Ala Tyr Leu Asp Lys Gln 145 150 155 160 Ser Asp Thr Gln Tyr Val Cys Lys Arg Thr Leu Val Asp Arg Gly Trp 165 170 175 Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser Leu Val Thr Cys Ala 180 185 190 Lys Phe Ala Cys Ser Lys Lys Met Thr Gly Lys Ser Ile Gln Pro Glu 195 200 205 Asn Leu Glu Tyr Arg Ile Met Leu Ser Val His Gly Ser Gln His Ser 210 215 220 Gly Met Ile Val Asn Asp Thr Gly His Glu Thr Asp Glu Asn Arg Ala 225 230 235 240 Lys Val Glu Ile Thr Pro Asn Ser Pro Arg Ala Glu Ala Thr Leu Gly 245 250 255 Gly Phe Gly Ser Leu Gly Leu Asp Cys Glu Pro Arg Thr Gly Leu Asp 260 265 270 Phe Ser Asp Leu Tyr Tyr Leu Thr Met Asn Asn Lys His Trp Leu Val 275 280 285 His Lys Glu Trp Phe His Asp Ile Pro Leu Pro Trp His Ala Gly Ala 290 295 300 Asp Thr Gly Thr Pro His Trp Asn Asn Lys Glu Ala Leu Val Glu Phe 305 310 315 320 Lys Asp Ala His Ala Lys Arg Gln Thr Val Val Val Leu Gly Ser Gln 325 330 335 Glu Gly Ala Val His Thr Ala Leu Ala Gly Ala Leu Glu Ala Glu Met 340 345 350 Asp Gly Ala Lys Gly Arg Leu Ser Ser Gly His Leu Lys Cys Arg Leu 355 360 365 Lys Met Asp Lys Leu Arg Leu Lys Gly Val Ser Tyr Ser Leu Cys Thr 370 375 380 Ala Ala Phe Thr Phe Thr Lys Ile Pro Ala Glu Thr Leu His Gly Thr 385 390 395 400 Val Thr Val Glu Val Gln Tyr Ala Gly Thr Asp Gly Pro Cys Lys Val 405 410 415 Pro Ala Gln Met Ala Val Asp Met Gln Thr Leu Thr Pro Val Gly Arg 420 425 430 Leu Ile Thr Ala Asn Pro Val Ile Thr Glu Ser Thr Glu Asn Ser Lys 435 440 445 Met Met Leu Glu Leu Asp Pro Pro Phe Gly Asp Ser Tyr Ile Val Ile 450 455 460 Gly Val Gly Glu Lys Lys Ile Thr His His Trp His Arg Ser Gly Ser 465 470 475 480 Thr Ile Gly Lys Ala Phe Glu Ala Thr Val Arg Gly Ala Lys Arg Met 485 490 495 Ala Val Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val Gly Gly Ala 500 505 510 Leu Asn Ser Leu Gly Lys Gly Ile His Gln Ile Phe Gly Ala Ala Phe 515 520 525 Lys Ser Leu Phe Gly Gly Met Ser Trp Phe Ser Gln Ile Leu Ile Gly 530 535 540 Thr Leu Leu Val Trp Leu Gly Leu Asn Thr Lys Asn Gly Ser Ile Ser 545 550 555 560 Leu Met Cys Leu Ala Leu Gly Gly Val Leu Ile Phe Leu Ser Thr Ala 565 570 575 Val Ser Ala <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <400> 32 ttttggcaaa gaattcgccg 20 <210> 33 <211> 41 <212> DNA <213> Artificial Sequence <400> 33 gatctgctag ctcgagtcaa gcgctcacag ctgtggacag a 41 <210> 34 <211> 36 <212> DNA <213> Artificial Sequence <400> 34 cgcagccatt tccggcgccc ctgtccacca gggtcc 36 <210> 35 <211> 38 <212> DNA <213> Artificial Sequence <400> 35 ggtggacagg ggcgccggaa atggctgcgg cctgtttg 38 <210> 36 <211> 36 <212> DNA <213> Artificial Sequence <400> 36 cgcagccatt tccccggccc ctgtccacca gggtcc 36 <210> 37 <211> 38 <212> DNA <213> Artificial Sequence <400> 37 ggtggacagg ggccggggaa atggctgcgg cctgtttg 38 <210> 38 <211> 36 <212> DNA <213> Artificial Sequence <400> 38 cgcagccatt tccgttgccc ctgtccacca gggtcc 36 <210> 39 <211> 38 <212> DNA <213> Artificial Sequence <400> 39 ggtggacagg ggcaacggaa atggctgcgg cctgtttg 38 <210> 40 <211> 36 <212> DNA <213> Artificial Sequence <400> 40 cgcagccatt tccgtcgccc ctgtccacca gggtcc 36 <210> 41 <211> 38 <212> DNA <213> Artificial Sequence <400> 41 ggtggacagg ggcgacggaa atggctgcgg cctgtttg 38 <210> 42 <211> 36 <212> DNA <213> Artificial Sequence <400> 42 cgcagccatt tccgcagccc ctgtccacca gggtcc 36 <210> 43 <211> 38 <212> DNA <213> Artificial Sequence <400> 43 ggtggacagg ggctgcggaa atggctgcgg cctgtttg 38 <210> 44 <211> 36 <212> DNA <213> Artificial Sequence <400> 44 cgcagccatt tccctggccc ctgtccacca gggtcc 36 <210> 45 <211> 38 <212> DNA <213> Artificial Sequence <400> 45 ggtggacagg ggccagggaa atggctgcgg cctgtttg 38 <210> 46 <211> 36 <212> DNA <213> Artificial Sequence <400> 46 cgcagccatt tccctcgccc ctgtccacca gggtcc 36 <210> 47 <211> 38 <212> DNA <213> Artificial Sequence <400> 47 ggtggacagg ggcgagggaa atggctgcgg cctgtttg 38 <210> 48 <211> 36 <212> DNA <213> Artificial Sequence <400> 48 cgcagccatt tccgccgccc ctgtccacca gggtcc 36 <210> 49 <211> 38 <212> DNA <213> Artificial Sequence <400> 49 ggtggacagg ggcggcggaa atggctgcgg cctgtttg 38 <210> 50 <211> 36 <212> DNA <213> Artificial Sequence <400> 50 cgcagccatt tccgtggccc ctgtccacca gggtcc 36 <210> 51 <211> 38 <212> DNA <213> Artificial Sequence <400> 51 ggtggacagg ggccacggaa atggctgcgg cctgtttg 38 <210> 52 <211> 36 <212> DNA <213> Artificial Sequence <400> 52 cgcagccatt tccgatgccc ctgtccacca gggtcc 36 <210> 53 <211> 38 <212> DNA <213> Artificial Sequence <400> 53 ggtggacagg ggcatcggaa atggctgcgg cctgtttg 38 <210> 54 <211> 36 <212> DNA <213> Artificial Sequence <400> 54 cgcagccatt tcccaggccc ctgtccacca gggtcc 36 <210> 55 <211> 38 <212> DNA <213> Artificial Sequence <400> 55 ggtggacagg ggcctgggaa atggctgcgg cctgtttg 38 <210> 56 <211> 36 <212> DNA <213> Artificial Sequence <400> 56 cgcagccatt tcccttgccc ctgtccacca gggtcc 36 <210> 57 <211> 38 <212> DNA <213> Artificial Sequence <400> 57 ggtggacagg ggcaagggaa atggctgcgg cctgtttg 38 <210> 58 <211> 36 <212> DNA <213> Artificial Sequence <400> 58 cgcagccatt tcccatgccc ctgtccacca gggtcc 36 <210> 59 <211> 38 <212> DNA <213> Artificial Sequence <400> 59 ggtggacagg ggcatgggaa atggctgcgg cctgtttg 38 <210> 60 <211> 36 <212> DNA <213> Artificial Sequence <400> 60 cgcagccatt tccgaagccc ctgtccacca gggtcc 36 <210> 61 <211> 38 <212> DNA <213> Artificial Sequence <400> 61 ggtggacagg ggcttcggaa atggctgcgg cctgtttg 38 <210> 62 <211> 36 <212> DNA <213> Artificial Sequence <400> 62 cgcagccatt tccggggccc ctgtccacca gggtcc 36 <210> 63 <211> 38 <212> DNA <213> Artificial Sequence <400> 63 ggtggacagg ggccccggaa atggctgcgg cctgtttg 38 <210> 64 <211> 36 <212> DNA <213> Artificial Sequence <400> 64 cgcagccatt tccgctgccc ctgtccacca gggtcc 36 <210> 65 <211> 38 <212> DNA <213> Artificial Sequence <400> 65 ggtggacagg ggcagcggaa atggctgcgg cctgtttg 38 <210> 66 <211> 36 <212> DNA <213> Artificial Sequence <400> 66 cgcagccatt tccggtgccc ctgtccacca gggtcc 36 <210> 67 <211> 38 <212> DNA <213> Artificial Sequence <400> 67 ggtggacagg ggcaccggaa atggctgcgg cctgtttg 38 <210> 68 <211> 36 <212> DNA <213> Artificial Sequence <400> 68 cgcagccatt tccgtagccc ctgtccacca gggtcc 36 <210> 69 <211> 38 <212> DNA <213> Artificial Sequence <400> 69 ggtggacagg ggctacggaa atggctgcgg cctgtttg 38 <210> 70 <211> 36 <212> DNA <213> Artificial Sequence <400> 70 cgcagccatt tcccacgccc ctgtccacca gggtcc 36 <210> 71 <211> 38 <212> DNA <213> Artificial Sequence <400> 71 ggtggacagg ggcgtgggaa atggctgcgg cctgtttg 38 <210> 72 <211> 32 <212> DNA <213> Artificial sequence(Artificial sequence) <400> 72 ctgctgtttg gaaagggctc cctggtgacc tg 32 <210> 73 <211> 32 <212> DNA <213> Artificial Sequence <400> 73 aaacagcagg cagccatttc cccagcccct gt 32 <210> 74 <211> 32 <212> DNA <213> Artificial Sequence <400> 74 ttcctgtttg gaaagggctc cctggtgacc tg 32 <210> 75 <211> 32 <212> DNA <213> Artificial Sequence <400> 75 aaacaggaag cagccatttc cccagcccct gt 32 <210> 76 <211> 32 <212> DNA <213> Artificial Sequence <400> 76 tggctgtttg gaaagggctc cctggtgacc tg 32 <210> 77 <211> 32 <212> DNA <213> Artificial Sequence <400> 77 aaacagccag cagccatttc cccagcccct gt 32 <210> 78 <211> 32 <212> DNA <213> Artificial Sequence <400> 78 tacctgtttg gaaagggctc cctggtgacc tg 32 <210> 79 <211> 32 <212> DNA <213> Artificial Sequence <400> 79 aaacaggtag cagccatttc cccagcccct gt 32 <210> 80 <211> 32 <212> DNA <213> Artificial Sequence <400> 80 atcctgtttg gaaagggctc cctggtgacc tg 32 <210> 81 <211> 32 <212> DNA <213> Artificial Sequence <400> 81 aaacaggatg cagccatttc cccagcccct gt 32 <210> 82 <211> 32 <212> DNA <213> Artificial Sequence <400> 82 ggcgagtttg gaaagggctc cctggtgacc tg 32 <210> 83 <211> 32 <212> DNA <213> Artificial Sequence <400> 83 aaactcgccg cagccatttc cccagcccct gt 32 <210> 84 <211> 32 <212> DNA <213> Artificial Sequence <400> 84 ggcaagtttg gaaagggctc cctggtgacc tg 32 <210> 85 <211> 32 <212> DNA <213> Artificial Sequence <400> 85 aaacttgccg cagccatttc cccagcccct gt 32 <210> 86 <211> 32 <212> DNA <213> Artificial Sequence <400> 86 ggccggtttg gaaagggctc cctggtgacc tg 32 <210> 87 <211> 32 <212> DNA <213> Artificial Sequence <400> 87 aaaccggccg cagccatttc cccagcccct gt 32 <210> 88 <211> 32 <212> DNA <213> Artificial Sequence <400> 88 ggcgactttg gaaagggctc cctggtgacc tg 32 <210> 89 <211> 32 <212> DNA <213> Artificial Sequence <400> 89 aaagtcgccg cagccatttc cccagcccct gt 32 <210> 90 <211> 32 <212> DNA <213> Artificial Sequence <400> 90 ggcacctttg gaaagggctc cctggtgacc tg 32 <210> 91 <211> 32 <212> DNA <213> Artificial Sequence <400> 91 aaaggtgccg cagccatttc cccagcccct gt 32 <210> 92 <211> 32 <212> DNA <213> Artificial Sequence <400> 92 ggcctgtggg gaaagggctc cctggtgacc tg 32 <210> 93 <211> 32 <212> DNA <213> Artificial Sequence <400> 93 ccacaggccg cagccatttc cccagcccct gt 32 <210> 94 <211> 32 <212> DNA <213> Artificial Sequence <400> 94 ggcctgcacg gaaagggctc cctggtgacc tg 32 <210> 95 <211> 32 <212> DNA <213> Artificial Sequence <400> 95 gtgcaggccg cagccatttc cccagcccct gt 32 <210> 96 <211> 32 <212> DNA <213> Artificial Sequence <400> 96 ggcctgtacg gaaagggctc cctggtgacc tg 32 <210> 97 <211> 32 <212> DNA <213> Artificial Sequence <400> 97 gtacaggccg cagccatttc cccagcccct gt 32 <210> 98 <211> 32 <212> DNA <213> Artificial Sequence <400> 98 ggcctgcccg gaaagggctc cctggtgacc tg 32 <210> 99 <211> 32 <212> DNA <213> Artificial Sequence <400> 99 gggcaggccg cagccatttc cccagcccct gt 32 <210> 100 <211> 32 <212> DNA <213> Artificial Sequence <400> 100 ggcctggccg gaaagggctc cctggtgacc tg 32 <210> 101 <211> 32 <212> DNA <213> Artificial Sequence <400> 101 ggccaggccg cagccatttc cccagcccct gt 32 <210> 102 <211> 32 <212> DNA <213> Artificial Sequence <400> 102 ctggagtttg gaaagggctc cctggtgacc tg 32 <210> 103 <211> 32 <212> DNA <213> Artificial Sequence <400> 103 aaactccagg cagccatttc cccagcccct gt 32 <210> 104 <211> 32 <212> DNA <213> Artificial Sequence <400> 104 ctgaagtttg gaaagggctc cctggtgacc tg 32 <210> 105 <211> 32 <212> DNA <213> Artificial Sequence <400> 105 aaacttcagg cagccatttc cccagcccct gt 32 <210> 106 <211> 32 <212> DNA <213> Artificial Sequence <400> 106 ttcgagtttg gaaagggctc cctggtgacc tg 32 <210> 107 <211> 32 <212> DNA <213> Artificial Sequence <400> 107 aaactcgaag cagccatttc cccagcccct gt 32 <210> 108 <211> 32 <212> DNA <213> Artificial Sequence <400> 108 ttcaagtttg gaaagggctc cctggtgacc tg 32 <210> 109 <211> 32 <212> DNA <213> Artificial sequence(Artificial sequence) <400> 109 aaacttgaag cagccatttc cccagcccct gt 32 <210> 110 <211> 32 <212> DNA <213> Artificial Sequence <400> 110 ttccggtttg gaaagggctc cctggtgacc tg 32 <210> 111 <211> 32 <212> DNA <213> Artificial Sequence <400> 111 aaaccggaag cagccatttc cccagcccct gt 32 <210> 112 <211> 32 <212> DNA <213> Artificial Sequence <400> 112 ttcctgtggg gaaagggctc cctggtgacc tg 32 <210> 113 <211> 32 <212> DNA <213> Artificial Sequence <400> 113 ccacaggaag cagccatttc cccagcccct gt 32 <210> 114 <211> 32 <212> DNA <213> Artificial Sequence <400> 114 ctgctgtggg gaaagggctc cctggtgacc tg 32 <210> 115 <211> 32 <212> DNA <213> Artificial Sequence <400> 115 ccacagcagg cagccatttc cccagcccct gt 32 <210> 116 <211> 32 <212> DNA <213> Artificial Sequence <400> 116 ttcctgcacg gaaagggctc cctggtgacc tg 32 <210> 117 <211> 32 <212> DNA <213> Artificial Sequence <400> 117 gtgcaggaag cagccatttc cccagcccct gt 32 <210> 118 <211> 32 <212> DNA <213> Artificial Sequence <400> 118 tacctgtggg gaaagggctc cctggtgacc tg 32 <210> 119 <211> 32 <212> DNA <213> Artificial Sequence <400> 119 ccacaggtag cagccatttc cccagcccct gt 32 <210> 120 <211> 32 <212> DNA <213> Artificial Sequence <400> 120 tggctgtacg gaaagggctc cctggtgacc tg 32 <210> 121 <211> 32 <212> DNA <213> Artificial Sequence <400> 121 gtacagccag cagccatttc cccagcccct gt 32 <210> 122 <211> 32 <212> DNA <213> Artificial Sequence <400> 122 ggcgagtggg gaaagggctc cctggtgacc tg 32 <210> 123 <211> 32 <212> DNA <213> Artificial Sequence <400> 123 ccactcgccg cagccatttc cccagcccct gt 32 <210> 124 <211> 32 <212> DNA <213> Artificial Sequence <400> 124 ggcaagtggg gaaagggctc cctggtgacc tg 32 <210> 125 <211> 32 <212> DNA <213> Artificial Sequence <400> 125 ccacttgccg cagccatttc cccagcccct gt 32 <210> 126 <211> 32 <212> DNA <213> Artificial Sequence <400> 126 ggccggtggg gaaagggctc cctggtgacc tg 32 <210> 127 <211> 32 <212> DNA <213> Artificial Sequence <400> 127 ccaccggccg cagccatttc cccagcccct gt 32 <210> 128 <211> 32 <212> DNA <213> Artificial Sequence <400> 128 ggcgactggg gaaagggctc cctggtgacc tg 32 <210> 129 <211> 32 <212> DNA <213> Artificial Sequence <400> 129 ccagtcgccg cagccatttc cccagcccct gt 32 <210> 130 <211> 32 <212> DNA <213> Artificial Sequence <400> 130 ggcaagtacg gaaagggctc cctggtgacc tg 32 <210> 131 <211> 32 <212> DNA <213> Artificial Sequence <400> 131 gtacttgccg cagccatttc cccagcccct gt 32 <210> 132 <211> 32 <212> DNA <213> Artificial Sequence <400> 132 ctgttattcg gaaagggagg catcgtgact tg 32 <210> 133 <211> 32 <212> DNA <213> Artificial Sequence <400> 133 gaataacagg catccattgc cccagcctct gt 32 <210> 134 <211> 32 <212> DNA <213> Artificial Sequence <400> 134 ttcttattcg gaaagggagg catcgtgact tg 32 <210> 135 <211> 32 <212> DNA <213> Artificial Sequence <400> 135 gaataagaag catccattgc cccagcctct gt 32 <210> 136 <211> 32 <212> DNA <213> Artificial Sequence <400> 136 tggttattcg gaaagggagg catcgtgact tg 32 <210> 137 <211> 32 <212> DNA <213> Artificial Sequence <400> 137 gaataaccag catccattgc cccagcctct gt 32 <210> 138 <211> 32 <212> DNA <213> Artificial Sequence <400> 138 tacttattcg gaaagggagg catcgtgact tg 32 <210> 139 <211> 32 <212> DNA <213> Artificial Sequence <400> 139 gaataagtag catccattgc cccagcctct gt 32 <210> 140 <211> 32 <212> DNA <213> Artificial Sequence <400> 140 atcttattcg gaaagggagg catcgtgact tg 32 <210> 141 <211> 32 <212> DNA <213> Artificial Sequence <400> 141 gaataagatg catccattgc cccagcctct gt 32 <210> 142 <211> 32 <212> DNA <213> Artificial Sequence <400> 142 ggtgagttcg gaaagggagg catcgtgact tg 32 <210> 143 <211> 32 <212> DNA <213> Artificial Sequence <400> 143 gaactcaccg catccattgc cccagcctct gt 32 <210> 144 <211> 32 <212> DNA <213> Artificial Sequence <400> 144 ggtaagttcg gaaagggagg catcgtgact tg 32 <210> 145 <211> 32 <212> DNA <213> Artificial Sequence <400> 145 gaacttaccg catccattgc cccagcctct gt 32 <210> 146 <211> 32 <212> DNA <213> Artificial Sequence <400> 146 ggtcggttcg gaaagggagg catcgtgact tg 32 <210> 147 <211> 32 <212> DNA <213> Artificial Sequence <400> 147 gaaccgaccg catccattgc cccagcctct gt 32 <210> 148 <211> 32 <212> DNA <213> Artificial Sequence <400> 148 ggtgacttcg gaaagggagg catcgtgact tg 32 <210> 149 <211> 32 <212> DNA <213> Artificial Sequence <400> 149 gaagtcaccg catccattgc cccagcctct gt 32 <210> 150 <211> 32 <212> DNA <213> Artificial Sequence <400> 150 ggtaccttcg gaaagggagg catcgtgact tg 32 <210> 151 <211> 32 <212> DNA <213> Artificial Sequence <400> 151 gaaggtaccg catccattgc cccagcctct gt 32 <210> 152 <211> 32 <212> DNA <213> Artificial Sequence <400> 152 ggtttatggg gaaagggagg catcgtgact tg 32 <210> 153 <211> 32 <212> DNA <213> Artificial Sequence <400> 153 ccataaaccg catccattgc cccagcctct gt 32 <210> 154 <211> 32 <212> DNA <213> Artificial Sequence <400> 154 ggtttacacg gaaagggagg catcgtgact tg 32 <210> 155 <211> 32 <212> DNA <213> Artificial Sequence <400> 155 gtgtaaaccg catccattgc cccagcctct gt 32 <210> 156 <211> 32 <212> DNA <213> Artificial Sequence <400> 156 ggtttatacg gaaagggagg catcgtgact tg 32 <210> 157 <211> 32 <212> DNA <213> Artificial Sequence <400> 157 gtataaaccg catccattgc cccagcctct gt 32 <210> 158 <211> 32 <212> DNA <213> Artificial Sequence <400> 158 ggtttacccg gaaagggagg catcgtgact tg 32 <210> 159 <211> 32 <212> DNA <213> Artificial Sequence <400> 159 gggtaaaccg catccattgc cccagcctct gt 32 <210> 160 <211> 32 <212> DNA <213> Artificial Sequence <400> 160 ggtttagccg gaaagggagg catcgtgact tg 32 <210> 161 <211> 32 <212> DNA <213> Artificial Sequence <400> 161 ggctaaaccg catccattgc cccagcctct gt 32 <210> 162 <211> 32 <212> DNA <213> Artificial Sequence <400> 162 ctggagttcg gaaagggagg catcgtgact tg 32 <210> 163 <211> 32 <212> DNA <213> Artificial Sequence <400> 163 gaactccagg catccattgc cccagcctct gt 32 <210> 164 <211> 32 <212> DNA <213> Artificial Sequence <400> 164 ctgaagttcg gaaagggagg catcgtgact tg 32 <210> 165 <211> 32 <212> DNA <213> Artificial sequence <400> 165 gaacttcagg catccattgc cccagcctct gt 32 <210> 166 <211> 32 <212> DNA <213> Artificial Sequence <400> 166 ttcgagttcg gaaagggagg catcgtgact tg 32 <210> 167 <211> 32 <212> DNA <213> Artificial Sequence <400> 167 gaactcgaag catccattgc cccagcctct gt 32 <210> 168 <211> 32 <212> DNA <213> Artificial Sequence <400> 168 ttcaagttcg gaaagggagg catcgtgact tg 32 <210> 169 <211> 32 <212> DNA <213> Artificial sequence <400> 169 gaacttgaag catccattgc cccagcctct gt 32 <210> 170 <211> 32 <212> DNA <213> Artificial Sequence <400> 170 ttccggttcg gaaagggagg catcgtgact tg 32 <210> 171 <211> 32 <212> DNA <213> Artificial Sequence <400> 171 gaaccggaag catccattgc cccagcctct gt 32 <210> 172 <211> 32 <212> DNA <213> Artificial Sequence <400> 172 ttcttatggg gaaagggagg catcgtgact tg 32 <210> 173 <211> 32 <212> DNA <213> Artificial Sequence <400> 173 ccataagaag catccattgc cccagcctct gt 32 <210> 174 <211> 32 <212> DNA <213> Artificial Sequence <400> 174 ctgttatggg gaaagggagg catcgtgact tg 32 <210> 175 <211> 32 <212> DNA <213> Artificial Sequence <400> 175 ccataacagg catccattgc cccagcctct gt 32 <210> 176 <211> 32 <212> DNA <213> Artificial Sequence <400> 176 ttcttacacg gaaagggagg catcgtgact tg 32 <210> 177 <211> 32 <212> DNA <213> Artificial Sequence <400> 177 gtgtaagaag catccattgc cccagcctct gt 32 <210> 178 <211> 32 <212> DNA <213> Artificial Sequence <400> 178 tacttatggg gaaagggagg catcgtgact tg 32 <210> 179 <211> 32 <212> DNA <213> Artificial Sequence <400> 179 ccataagtag catccattgc cccagcctct gt 32 <210> 180 <211> 32 <212> DNA <213> Artificial Sequence <400> 180 tggttatacg gaaagggagg catcgtgact tg 32 <210> 181 <211> 32 <212> DNA <213> Artificial Sequence <400> 181 gtataaccag catccattgc cccagcctct gt 32 <210> 182 <211> 32 <212> DNA <213> Artificial Sequence <400> 182 ggtgagtggg gaaagggagg catcgtgact tg 32 <210> 183 <211> 32 <212> DNA <213> Artificial Sequence <400> 183 ccactcaccg catccattgc cccagcctct gt 32 <210> 184 <211> 32 <212> DNA <213> Artificial Sequence <400> 184 ggtaagtggg gaaagggagg catcgtgact tg 32 <210> 185 <211> 32 <212> DNA <213> Artificial Sequence <400> 185 ccacttaccg catccattgc cccagcctct gt 32 <210> 186 <211> 32 <212> DNA <213> Artificial Sequence <400> 186 ggtcggtggg gaaagggagg catcgtgact tg 32 <210> 187 <211> 32 <212> DNA <213> Artificial Sequence <400> 187 ccaccgaccg catccattgc cccagcctct gt 32 <210> 188 <211> 32 <212> DNA <213> Artificial Sequence <400> 188 ggtgactggg gaaagggagg catcgtgact tg 32 <210> 189 <211> 32 <212> DNA <213> Artificial Sequence <400> 189 ccagtcaccg catccattgc cccagcctct gt 32 <210> 190 <211> 32 <212> DNA <213> Artificial Sequence <400> 190 ggtaagtacg gaaagggagg catcgtgact tg 32 <210> 191 <211> 32 <212> DNA <213> Artificial Sequence <400> 191 gtacttaccg catccattgc cccagcctct gt 32

Claims

1. An antigen, wherein the immunogenic portion of the antigen consists of the full-length E protein sequence of Zika virus or Dengue virus and the full-length M protein or full-length prM protein sequence of Zika virus or Dengue virus, wherein, the FL fusion region of the E protein has a mutation, and the mutations in the FL fusion region of the E protein are selected from the following mutations: ； wherein: when the antigen comprises the full-length E protein of Zika virus, the antigen further comprises the full-length M protein or full-length prM protein sequence of Zika virus; when the antigen comprises the full-length E protein of Dengue virus, the antigen further comprises the full-length M protein or full-length prM protein sequence of Dengue virus.

2. A polynucleotide encoding the antigen according to claim 1.

3. An expression cassette, recombinant vector, transgenic cell line, recombinant bacterium or virus particle comprising the polynucleotide according to claim 2.

4. The expression cassette, recombinant vector, transgenic cell line, recombinant bacterium or virus particle according to claim 3, characterized in that the virus particle is an adenovirus particle or a lentivirus particle.

5. An mRNA encoding the antigen according to claim 1.

6. A vaccine, which comprises the antigen according to claim 1, the polynucleotide according to claim 2, the expression cassette, recombinant vector, transgenic cell line, recombinant bacterium or virus particle according to claim 3 or 4, or the mRNA according to claim 5 as an active ingredient.

7. The vaccine according to claim 6, characterized in that the vaccine comprises one or more of inactivated vaccine, attenuated vaccine, DNA vaccine, mRNA vaccine, virus vector vaccine, subunit vaccine or virus particle; and / or, the vaccine further comprises a pharmaceutically or veterinarily acceptable vehicle.

8. The vaccine according to claim 7, characterized in that the vaccine further comprises a pharmaceutically or veterinarily acceptable diluent, adjuvant or excipient.

9. The vaccine according to claim 7 or 8, characterized in that the vaccine is an adenovirus vaccine.

10. A recombinant adenovirus vaccine, characterized in that it comprises chimpanzee adenovirus type 7, and the chimpanzee adenovirus type 7 is packaged with a recombinant vector; the recombinant vector comprises: a backbone vector and a nucleic acid sequence containing a target gene linked to the backbone vector; the backbone vector is a replication-defective chimpanzee adenovirus type 7 vector; the target gene is: the polynucleotide according to claim 2 or the mRNA according to claim 5.

11. Use of the antigen according to claim 1, the polynucleotide according to claim 2, the expression cassette, recombinant vector, transgenic cell line, recombinant bacterium or virus particle according to claim 3 or 4, or the mRNA according to claim 5 in the preparation of a vaccine for preventing Zika virus or Dengue virus infection.

Images