Zika-dengue vaccine and its uses

Abstract

The present invention provides a Zika / dengue vaccine and its use. The present invention introduces a mutation into the FL fusion region of the E protein of Zika virus or dengue virus, so that antigens with this mutation cannot bind to antibodies that cause ADE. A vaccine obtained from the antigen provided by the present invention can avoid the production of antibodies induced by FL epitopes after immunization, thereby reducing or eliminating the ADE phenomenon.

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, specifically to Zika-Dengue vaccines and uses thereof. [Background technology]

[0002] Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus in the family Flaviviridae. The ZIKV outbreak that occurred in the United States in 2015–2016 has spread to 84 countries worldwide, including China. However, to date, no vaccine or drug is available. Although the global incidence of ZIKV infection is currently declining, ZIKV remains a threat to people living in endemic areas, making the development of a vaccine for ZIKV an urgent task.

[0003] The dengue virus (DENV) is a mosquito-borne virus of the genus Flavivirus in the family Flaviviridae, and there are four serotypes.

[0004] The ZIKV and DENV viruses have similar structures: both have an icosahedral spherical structure and an envelope containing envelope (E) protein on the surface of the envelope. The internal viral genome is a single-stranded positive-stranded RNA approximately 11 kb in length, containing only one open reading frame. The genome is translated into a polyprotein, which can then be cleaved into three structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).

[0005] The approximately 53-kD E protein, a major surface protein of ZIKV and DENV, mediates viral entry into cells and membrane fusion, making it an important target for activating neutralizing antibodies. The E protein is also an important target protein for vaccine design. The E protein contains 504 amino acids and exists as a dimer, with each monomer containing three domains: DI, DII, and DIII. The DII domain (amino acids 98–109) contains a highly conserved fusion loop (FL). The sequences of the FL domains of ZIKV and DENV viruses are completely identical: D98-R99-G100-W101-G102-N103-G104-C105-G106-L107-F108-G109. The FL domain plays a key role in the membrane fusion process of viral entry, and immune cells produce large amounts of antibodies against the FL during viral infection.

[0006] The approximately 26 kD prM protein assists in the correct folding of the E protein, and the 3' transmembrane region of prM / E acts as an endoplasmic reticulum retention signal, supporting the formation of heterodimers between prM and E. One of the main functions 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, which masks the E protein's fusion peptide. In this case, steric hindrance makes it difficult for prM to contact and be cleaved by furin. Subsequently, a translocation reaction is induced in the acidic environment of the Golgi apparatus, exposing the furin cleavage site, allowing prM protein to be cleaved into M protein by furin. The cleaved pr polypeptide does not immediately dissociate from the virus particle; instead, it is released after exposure to the neutral pH of the cellular environment, resulting in the formation of mature virus particles.

[0007] The genetic organization and antigenic characteristics of ZIKV are similar to those of four serotypes of dengue virus. ZIKV is highly similar to DENV (Development of Novel Coronary Syndrome (DENV)), sharing approximately 56% amino acid similarity. Therefore, antibodies induced by ZIKV infection may exhibit stronger cross-reactivity against DENV, which should also be considered in vaccine safety. Many studies have shown that pre-existing antibodies after ZIKV infection can cross-react with DENV and enhance subsequent DENV infection (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document 4; Non-Patent Document 5; Non-Patent Document 6). 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 present at sub-neutralizing concentrations (Non-Patent Document 7; Non-Patent Document 8). Despite the lack of epidemiological studies, pre-existing ZIKV antibodies from humans, monkeys, and mice have all been shown to enhance DENV infection in cell experiments (Non-Patent Document 2; Non-Patent Document 4; Non-Patent Document 5; Non-Patent Document 6). Furthermore, in monkey and mouse models, ZIKV infection, vaccination, or maternal antibodies to fetuses has been shown to exacerbate the symptoms of DENV infection (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 4; Non-Patent Document 5). Therefore, after acquiring immunity with a ZIKV vaccine, there may be an ADE phenomenon against future DENV infection. This should be taken into consideration when designing a ZIKV vaccine.

[0008] Antibodies that induce ADE responses are primarily induced by the FL fusion region of the virus (Non-Patent Document 7; Non-Patent Document 8). In flavivirus infections, these antibodies account for the majority of all induced antibodies. Because their epitopes are well conserved, these antibodies generally exhibit cross-reactivity between different serotypes. Many of them have low neutralizing activity and are prone to induce ADE responses, but most antibodies with high neutralizing activity bind to other epitopes of the E protein. A series of ZIKV-neutralizing monoclonal antibodies targeting domain I (Domain I, DI), domain II (Domain II, DII), and domain III (Domain III, DIII) of the E protein or quaternary structure epitopes have been identified (Non-Patent Document 9; Non-Patent Document 5; Non-Patent Document 10; Non-Patent Document 11; Non-Patent Document 12). Therefore, an ideal ZIKV vaccine design strategy would be to shift the immune hotspot epitopes from the FL region to other neutralizing epitopes.

[0009] Antibodies bind to virus particles and then bind to Fcγ receptor proteins on the surface of bone marrow cells, leading to their internalization and subsequent viral infection. Because DENV exists in four serotypes, reinfection with a different serotype from the initial infection is highly likely to result in ADE. Therefore, humans exhibit more severe symptoms after DENV infection (Non-Patent Document 13). The only currently available DENV vaccine, Dengvaxia®, has limitations due to the ACE phenomenon. Dengvaxia® can only be used in DENV-seropositive individuals; in seronegative individuals, vaccination can actually worsen dengue fever infection (Non-Patent Document 14; Non-Patent Document 15). Therefore, avoiding ADE is also a challenge in developing a DENV vaccine.

[0010] The information disclosed in this background section is intended only to enhance understanding of the general background of the present invention and should not be construed as an admission or any form of suggestion that it constitutes prior art already known to those skilled in the art. [Prior art documents]

Non-licensed literature

[0011] [Non-licensed document 1] Fowler et al., 2018 [Non-licensed document 2] George et al., 2017 [Non-licensed document 3] Li et al., 2017

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[0012] The objective of the present invention is to provide a Zika / dengue vaccine that avoids ADE reactions and its use. The present invention obtains information on the epitopes of the major antibodies that cause ADE through crystal structure analysis and other structural and functional analyses. The antigens provided in the present invention are antigens in which mutations have been introduced into the FL fusion region of the E protein of Zika virus or dengue virus, and neither antigen with this mutation can bind to antibodies that cause ADE (FLE antibodies). Vaccines obtained from the antigens provided by the present invention can avoid the production of antibodies induced by FL epitopes after immunization, thereby reducing or eliminating ADE. [Means for solving the problem]

[0013] To achieve the object of the present invention, an embodiment of the present invention provides an antigen comprising a FL fusion loop of an envelope protein of an E protein of a Zika virus or a Dengue virus, The FL fusion region of the E protein is (1) A combination of one or two of the mutations at D98 and N103 and the mutations at G106, L107, and F108; (2) one or a combination of a mutation at G106, a mutation at L107, and a mutation at F108; (3) a single-site mutation at W101; The present invention provides an antigen having one of the mutations.

[0014] (1) The combination of one or two of the mutations at D98 and N103 and the mutations at G106, L107, and F108 is Mutations at five sites: D98, N103, G106, L107, and F108; Mutations at four sites: D98, G106, L107, and F108; This is one of the four mutations at N103, G106, L107, and F108.

[0015] (2) One or a combination of a mutation at G106, a mutation at L107, and a mutation at F108 is a single-site mutation selected from a mutation at position G106, a mutation at position L107, and a mutation at position F108; Mutations at two sites selected from a mutation at G106, a mutation at L107, and a mutation at F108; This is one of the three mutations at G106, L107, or F108.

[0016] The D98 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 mutation refers to the substitution of asparagine (N) at position 103 of the E protein with any amino acid other than asparagine. The G106 mutation refers to the substitution of glycine (G) at position 106 of the E protein with any amino acid other than glycine. The L107 mutation refers to the substitution of leucine (L) at position 107 of the E protein with any amino acid other than leucine. The F108 mutation refers to the substitution of phenylalanine (F) at position 108 of the E protein with any amino acid other than phenylalanine. The W101 mutation refers to the substitution of tryptophan (W) at position 101 of the E protein with any amino acid other than tryptophan.

[0017] The D98, W101, N103, G106, L107, or F108 site is located in the FL fusion region of the E protein. The FL (fusion region) sequence is highly conserved among Flaviviruses, and the FL sequences of ZIKV and DENV are completely identical: 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 their positions in the E protein sequences of Zika virus and Dengue virus, specifically, the 98, 101, 103, 106, 107, and 108 sites shown in SEQ ID NO. 1 of the E protein of Zika virus (ZIKV FSS13025 strain, for example, GenBank: JN860885.1).

[0018] In one possible embodiment, the mutations in the FL fusion region of the E protein of the antigen consist of mutations at five sites: D98, N103, G106, L107, and F108. Selected, Or, the mutation in the FL fusion region of the E protein is selected from the group consisting of mutations at three sites: G106, L107, and F108; Or, the mutation in the FL fusion region of the E protein is selected from mutations at two sites, G106 and L107; Alternatively, the mutation in the FL fusion region of the E protein is selected from mutations at two sites, G106 and F108; Alternatively, the mutation in the FL fusion region of the E protein is selected from mutations at two sites, L107 and F108; Or, the mutation in the FL fusion region of the E protein is selected from a single site mutation at G106; Or, the mutation in the FL fusion region of the E protein is selected from a single site mutation at L107; Or, the mutation in the FL fusion region of the E protein is selected from a single-site mutation in F108; Alternatively, the mutation in the FL fusion region of the E protein is selected from single-site mutations in W101.

[0019] In one possible embodiment, the mutation in the FL fusion region of the E protein of the antigen is selected from the following mutations or combinations of mutations:

[0020] [Table 1]

[0021] [Table 2]

[0022] In the table, the D98N mutation refers to a substitution of aspartic acid (D) at position 98 of the E protein with asparagine (N).

[0023] The N103T mutation refers to the substitution of asparagine (N) at position 103 of the E protein with threonine (T).

[0024] The G106F mutation refers to a substitution of glycine (G) at position 106 of the E protein with phenylalanine (F), and the G106L mutation refers to a substitution of glycine (G) at position 106 of the E protein with leucine (L).

[0025] The L107E mutation refers to a substitution of leucine (L) at position 107 of the E protein with glutamic acid (E), and the L107K mutation refers to a substitution of leucine (L) at position 107 of the E protein with lysine (K).

[0026] The F108W mutation refers to the substitution of phenylalanine (F) at position 108 of the E protein with tryptophan (W).

[0027] Amino acid mutations can be inferred in this manner, and the types of amino acids represented by a single alphabetic letter are generally understood by those skilled in the art.

[0028] In one possible embodiment, when the antigen comprises the FL fusion region of the E protein of Zika virus, the antigen further comprises the full length or a portion of the sequence of the M protein of Zika virus, and optionally, the antigen further comprises the full length sequence of the M protein of Zika virus.

[0029] When the antigen has the FL fusion region of the E protein of a dengue virus, the antigen further comprises the full-length or part of the sequence of the M protein of a dengue virus, and optionally, the antigen further comprises the full-length sequence of the M protein of a dengue virus.

[0030] M protein is formed after prM structural protein is cleaved by furin. The full length or a portion of the M protein sequence refers to 0.5% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100% of the M protein sequence, and the sequence may be a sequence selected consecutively from the M protein, or a combination of fragments individually selected from the M protein sequence.

[0031] In one possible embodiment, when the antigen comprises the FL fusion region of the E protein of Zika virus, the antigen further comprises the full-length or a portion of the sequence of the prM protein of Zika virus, and optionally, the antigen further comprises the full-length sequence of the prM protein of Zika virus.

[0032] When the antigen has the FL fusion region of the E protein of a dengue virus, the antigen further comprises the full-length or part of the sequence of the prM protein of a dengue virus, and optionally, the antigen further comprises the full-length sequence of the prM protein of a dengue virus.

[0033] The prM protein is a structural protein of Zika or Dengue virus of approximately 26 kD, and is used to assist in the correct folding of the E protein. The full-length or partial sequence of the prM protein refers to 0.5% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100% of the sequence of the prM protein, and the sequence may be a sequence selected consecutively from the prM protein, or a combination of fragments individually selected from the prM protein sequence.

[0034] In one possible embodiment, when the antigen comprises the FL fusion region of the E protein of Zika virus, the antigen comprises the full length or a portion of the sequence of the E protein of Zika virus, and optionally, the antigen comprises the full length of the E protein sequence of Zika virus.

[0035] When the antigen has the FL fusion region of the E protein of dengue virus, the antigen is The antigen may have the full length or a portion of the sequence of the E protein of a dengue virus, and optionally the antigen comprises the full length sequence of the E protein of a dengue virus.

[0036] The full length or a portion of the E protein sequence refers to 0.5% to 100%, 50 to 100%, 60 to 100%, 70 to 100%, 80 to 100%, or 90 to 100% of the E protein sequence, and the sequence may be a sequence selected consecutively from the E protein, or a combination of fragments individually selected from the E protein sequence.

[0037] The sequences of the E protein, prM protein, and M protein of the Zika virus can be obtained from the complete sequences of each Zika virus strain disclosed in NCBI and prior art reports, and the sequences of the E protein, prM protein, and M protein of the Dengue virus can be obtained from the complete sequences of each of the four serotypes of Dengue virus strains disclosed in NCBI and prior art reports.

[0038] In one possible embodiment, the Zika virus is any Zika virus strain, for example, ZIKV FSS13025 strain (GenBank: JN860885.1), ZIKK Including the SMGC-1 strain.

[0039] In one possible embodiment, the dengue virus includes each dengue virus strain of the four serotypes of 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, China, GenBank: AF289029).

[0040]

[0013] An embodiment of the present invention also provides an antigen binding epitope of an FL fusion region of an E protein of a Zika virus, wherein the FL fusion region of the E protein of a Zika virus comprises the amino acid sequence D98-R99-G100-W101-G102-N103-G104-C105-G106-L107-F108-G109; (1) A combination of one or two of the mutations at D98 and N103 and the mutations at G106, L107, and F108; (2) one or a combination of a mutation at G106, a mutation at L107, and a mutation at F108; (3) a single-site mutation at W101; It has one of the following mutations:

[0041] Embodiments of the present invention also provide Zika virus antigens containing the antigen-binding epitopes.

[0042] In one possible embodiment, the Zika virus antigen further comprises: a full-length or partial sequence of the E protein of Zika virus; a full-length or partial M protein sequence of Zika virus; a full-length or partial sequence of the prM protein of Zika virus; The sequence includes one or more of the following:

[0043]

[0013] An embodiment of the present invention also provides an antigen-binding epitope of an FL fusion region of an E protein of a dengue virus, wherein the FL fusion region of the E protein of a dengue virus comprises the amino acid sequence D98-R99-G100-W101-G102-N103-G104-C105-G106-L107-F108-G109; (1) one or two of the mutations at D98 and N103, and G106; combination with the three site mutations of L107 and F108; (2) one or a combination of a mutation at G106, a mutation at L107, and a mutation at F108; (3) a single-site mutation at W101; It has one of the following mutations:

[0044] Embodiments of the present invention also provide dengue virus antigens comprising the antigen-binding epitopes.

[0045] In one possible embodiment, the dengue virus antigen further comprises: the full length or a part of the sequence of the E protein of the dengue virus, the full length or a part of the sequence of the M protein of dengue virus, the full length or a part of the sequence of the prM protein of the dengue virus, The sequence includes one or more of the following:

[0046] Embodiments of the present invention also provide antibodies that bind to the antigens, Zika virus antigens, and Dengue virus antigens.

[0047] Embodiments of the present invention also provide polynucleotides encoding the antigens, antigen-binding epitopes, Zika virus antigens, and Dengue virus antigens.

[0048] Embodiments of the present invention also provide an expression cassette, recombinant vector, transgenic cell line, recombinant bacterium, adenovirus, lentivirus or viral particle comprising the polynucleotide.

[0049] Embodiments of the present invention also provide mRNAs encoding the antigens, antigen-binding epitopes, Zika virus antigens, and Dengue virus antigens.

[0050] An embodiment of the present invention also provides a vaccine comprising, as an active ingredient, the antigen, the Zika virus antigen, the Dengue virus antigen, the polynucleotide, the expression cassette, the recombinant vector, the transgenic cell line, the recombinant bacterium, the adenovirus, the lentivirus or viral particle, or the mRNA.

[0051] In one possible embodiment, the vaccine comprises one or more of an inactivated vaccine, an attenuated vaccine, a DNA vaccine, an mRNA vaccine, an adenovirus vaccine, other viral vector vaccines, subunit vaccines, and viral particles.

[0052] In one possible embodiment, the vaccine is an adenovirus vaccine.

[0053] In one possible embodiment, the vaccine further comprises a pharmaceutically or veterinarily acceptable vehicle, diluent, adjuvant or excipient.

[0054] Embodiments of the present invention also provide use of the antigen, the antigen-binding epitope, the antibody, the polynucleotide, the expression cassette, the recombinant vector, the transgenic cell line, the recombinant bacterium, the adenovirus, the lentivirus or the viral particle, or the mRNA in the preparation of a vaccine for preventing and / or treating infection with a virus belonging to the Flavivirus genus.

[0055] The present invention also provides a method for detecting infection by a virus belonging to the Flavivirus genus. The present invention provides use of the antigen, the antigen-binding epitope, the antibody, the polynucleotide, the expression cassette, the recombinant vector, the transgenic cell line, the recombinant bacterium, the adenovirus, the lentivirus or the viral particle, or the mRNA in the preparation of a detection reagent or kit for detecting a target gene. [Effects of the Invention]

[0056] The present invention obtains information on the epitopes of major antibodies that cause ADE through crystal structure analysis and other structural and functional analyses. In the present embodiment, the antigens provided in the FL fusion region of the E protein of Zika or Dengue virus contain one of the following mutations: i. a D98 mutation, a N103 mutation, and a combination of three mutations at G106, L107, and F108; ii. a G106 mutation, a L107 mutation, and a F108 mutation, or a combination thereof; or iii. a W101 single mutation. Antigens with these mutations cannot bind to ADE-causing antibodies (FLE antibodies). Vaccines derived from the antigens provided by the present invention can avoid the production of FL epitope-induced antibodies after immunization, thereby reducing or eliminating ADE.

[0057] (2) The antigens provided in the examples of the present invention do not bind to antibodies that cause ADE (FLE antibodies), but do not affect the antibody binding ability of other epitopes. Using several Zika virus adenovirus vaccines derived from the antigens as examples, the present invention demonstrates that the resulting recombinant adenovirus vaccines do not reduce the immunogenicity of the antigens and can still effectively stimulate the production of neutralizing antibodies. The vaccines also provide complete protection in mouse challenge tests, effectively protect mice from viremia and tissue and organ infection, and, after immunization, can reduce and even eliminate the ADE phenomenon against four serotypes of DENV virus.

[0058] The present invention further demonstrated that the E protein after mutation maintained a dimeric form, with only the FL-mutated amino acid side chains undergoing changes. Analysis of mouse antibody responses induced by recombinant adenovirus vaccines by single-cell sequencing of germinal center (GC) B cells demonstrated that recombinant adenovirus vaccines with FL mutations significantly reduced antibodies induced by the FL epitope compared with vaccines with wild-type FL regions, demonstrating the transfer of the dominant epitope of the antigen. This demonstrates the mechanism of action that vaccines derived from the antigens provided by the present invention can eliminate the ADE phenomenon.

[0059] (3) The present invention further demonstrates that, using expression plasmids of several dengue virus adenoviruses derived from the antigens, none of the resulting antigens can bind to antibodies that cause ADE (FLE antibodies). Therefore, the antigens of the present invention can avoid the production of antibodies induced by FL epitopes after immunization with a dengue virus vaccine, thereby reducing or eliminating the ADE phenomenon caused by DENV virus infection after immunization.

[0060] (4) Vaccines obtained from the antigens provided by the present invention can include various forms, such as nucleic acid vaccines, mRNA vaccines, adenovirus vector vaccines, other virus vector vaccines, virus-like particles, attenuated or inactivated vaccines based on the antigen sequences, and chimeric vaccines with other backbones, and can be used to produce Zika vaccines and tetravalent dengue vaccines that eliminate the ADE phenomenon. [Brief explanation of the drawings]

[0061] One or more embodiments are illustrated by way of example in the accompanying drawings, corresponding figures, and these illustrative descriptions are not to be construed as limiting the embodiments. As used herein, the term "exemplary" means "serving as an example, example, or illustration." Any embodiment described as "exemplary" herein should not be construed as preferred or better than other embodiments. [Figure 1] FIG. 1 shows the results of a neutralization assay of mice immunized with wild-type recombinant adenovirus vaccines AdC7-M / E-WT and AdC7-prM / E-WT. [Figure 2A] FIG. 2A shows experimental results showing that sera from mice immunized with wild-type recombinant adenovirus vaccines AdC7-M / E-WT and AdC7-prM / E-WT enhance DENV1-infected cells. [Figure 2B] FIG. 2B shows experimental results showing that sera from mice immunized with wild-type recombinant adenovirus vaccines AdC7-M / E-WT and AdC7-prM / E-WT enhance DENV2-infected cells. [Figure 2C] FIG. 2C shows experimental results showing that sera from mice immunized with wild-type recombinant adenovirus vaccines AdC7-M / E-WT and AdC7-prM / E-WT enhance DENV3-infected cells. [Figure 2D]FIG. 2D shows experimental results showing that sera from mice immunized with wild-type recombinant adenovirus vaccines AdC7-M / E-WT and AdC7-prM / E-WT enhance DENV4-infected cells. [Figure 3] FIG. 3 shows that the Z6 antibody has some degree of ADE against cells infected with all four serotypes of DENV. [Figure 4] Figure 4 shows the structure of the complex between the Fab fragment of the Z6 antibody and the ZIKV E protein. [Figure 5A] Figure 5A shows the structure of the complex between the Z6 antibody and the ZIKV E protein. [Figure 5B] Figure 5B shows the structure of the complex between the 2A10G6 antibody and the ZIKV E protein. [Figure 5C] FIG. 5C shows the structure of the complex between E53 antibody and WNV E protein. [Figure 5D] Figure 5D is a superimposition of the three antibodies and ZIKV E protein structures in Figures 5A-5C. [Figure 6] Figure 6 shows amino acid site analysis of the binding between ZIKV E protein and Z6 antibody, the binding between ZIKV E protein and 2A10G6 antibody, and the binding between WNV E protein and E53 antibody. [Figure 7] FIG. 7 is a diagram showing a phylogenetic tree of viruses of the genus Flavivirus. [Figure 8] Figure 8 shows the mutation sites and sequences of the ZIKV M / E MutA / B / C mutant. [Figure 9] FIG. 9 shows the results of detection of M / E-MutA / B / C antigen activity using a flow cytometer. [Figure 10] FIG. 10 shows a flow chart for constructing the pAdC7-M / E-MutB / MutC recombinant plasmid. [Figure 11] FIG. 11 shows neutralizing antibody titers in the serum of BALB / c mice immunized with ZIKV recombinant adenovirus vaccines. [Figure 12]Figure 12 shows neutralizing antibody titers in the serum of Ifnar1− / − mice immunized with ZIKV recombinant adenovirus vaccines. [Figure 13A] Figure 13A shows the mortality results after challenge of Ifnar1− / − mice immunized with ZIKV recombinant adenovirus vaccines. [Figure 13B] Figure 13B shows the results of body weight changes after a challenge test in Ifnar1− / − mice immunized with a ZIKV recombinant adenovirus vaccine. [Figure 14] Figure 14 shows the experimental results of protection of Ifnar1− / − mice from viremia caused by ZIKV infection by ZIKV recombinant adenovirus vaccine. [Figure 15] Figure 15 shows the experimental results of ZIKV recombinant adenovirus vaccine protection of Ifnar1− / − mice from tissue and organ infection after ZIKV challenge. [Figure 16] Figure 16 shows the changes in serum neutralizing antibody titers before and after ZIKV challenge in Ifnar1− / − mice immunized with a ZIKV recombinant adenovirus vaccine. [Figure 17A] Figure 17A shows the results of a cross-reactivity experiment between serum from BALB / c mice immunized with a ZIKV recombinant adenovirus vaccine and ZIKV. [Figure 17B] Figure 17B shows the results of a cross-reactivity experiment of serum from BALB / c mice immunized with a ZIKV recombinant adenovirus vaccine against DENV1. [Figure 17C] Figure 17C shows the results of a cross-reactivity experiment of serum from BALB / c mice immunized with a ZIKV recombinant adenovirus vaccine against DENV2. [Figure 17D] Figure 17D shows the results of a cross-reactivity experiment of serum from BALB / c mice immunized with a ZIKV recombinant adenovirus vaccine against DENV3. [Figure 17E]Figure 17E shows the results of a cross-reactivity experiment of serum from BALB / c mice immunized with a ZIKV recombinant adenovirus vaccine against DENV4. [Figure 18A] Figure 18A shows the results of an ADE experiment using serum from BALB / c mice immunized with a ZIKV recombinant adenovirus vaccine against ZIKV-infected cells. [Figure 18B] Figure 18B shows the results of an ADE experiment using serum from BALB / c mice immunized with a ZIKV recombinant adenovirus vaccine against DENV1-infected cells. [Figure 18C] Figure 18C shows the results of an ADE experiment using serum from BALB / c mice immunized with a ZIKV recombinant adenovirus vaccine against DENV2-infected cells. [Figure 18D] Figure 18D shows the results of an ADE experiment using serum from BALB / c mice immunized with a ZIKV recombinant adenovirus vaccine against DENV3-infected cells. [Figure 18E] Figure 18E shows the results of an ADE experiment using serum from BALB / c mice immunized with a ZIKV recombinant adenovirus vaccine against DENV4-infected cells. [Figure 19A] Figure 19A shows the results of an in vivo experiment detecting the effect of ZIKV recombinant adenovirus vaccine on enhancing DENV type 2 infection in Ifnα / βr− / −Ifnγr− / − mice. [Figure 19B] Figure 19B shows the results of an in vivo experiment detecting the effect of ZIKV recombinant adenovirus vaccine on enhancing DENV type 2 infection in Ifnα / βr− / −Ifnγr− / − mice. [Figure 20] Figure 20 shows the conditions for selecting GC B cells that bind to ZIKV E protein from lymph node cells of BALB / c mice immunized with a ZIKV recombinant adenovirus vaccine by flow cytometry. [Figure 21]Figure 21 shows the results of an analysis of the antibody repertoire of GC B cells that bind to ZIKV E protein in BALB / c mice immunized with a ZIKV recombinant adenovirus vaccine. [Figure 22] FIG. 22 shows the results of paired HV and LV on the antibody repertoire elicited by BALB / c mice immunized with AdC7-M / E-WT vaccine. [Figure 23] FIG. 23 shows the results of paired HV and LV on the antibody repertoire elicited by BALB / c mice immunized with AdC7-M / E-MutB vaccine. [Figure 24-1] FIG. 24-1 shows the results of paired HV and LV in the antibody repertoire induced by BALB / c mice immunized with AdC7-M / E-MutC vaccine. [Figure 24-2] FIG. 24-2 shows the results of paired HV and LV in the antibody repertoire induced by BALB / c mice immunized with AdC7-M / E-MutC vaccine. [Figure 25] Figure 25 shows the identification of the binding ability of antibodies induced by BALB / c mice immunized with ZIKV recombinant adenovirus vaccines to different ZIKV E proteins and DENV E proteins. [Figure 26A] Figure 26A shows the experimental results of detecting the ADE-promoting effect of antibodies induced by mice immunized with a representative AdC7-M / E-WT vaccine on DENV1-infected cells. [Figure 26B] Figure 26B shows the experimental results of detecting the ADE-promoting effect of antibodies induced by mice immunized with a representative AdC7-M / E-WT vaccine on DENV2-infected cells. [Figure 26C] Figure 26C shows the experimental results of detecting the ADE-promoting effect of antibodies induced by mice immunized with a representative AdC7-M / E-WT vaccine on DENV3-infected cells. [Figure 26D]Figure 26D shows the experimental results of detecting the ADE-promoting effect of antibodies induced by mice immunized with a representative AdC7-M / E-WT vaccine on DENV4-infected cells. [Figure 27] FIG. 27 shows the alignment of the V loci used in the monoclonal antibodies induced by mice immunized with the AdC7-M / E-WT vaccine with reported FLE monoclonal antibodies. [Figure 28A] FIG. 28A shows the sequence alignment of monoclonal antibodies induced by mice immunized with AdC7-M / E-WT vaccine with reported FLE monoclonal antibodies. [Figure 28B] FIG. 28B shows the sequence alignment of monoclonal antibodies induced by mice immunized with AdC7-M / E-WT vaccine with reported FLE monoclonal antibodies. [Figure 28C] FIG. 28C shows a sequence alignment of monoclonal antibodies induced by mice immunized with AdC7-M / E-WT vaccine with reported FLE monoclonal antibodies. [Figure 28D] FIG. 28D shows the sequence alignment of monoclonal antibodies induced by mice immunized with AdC7-M / E-WT vaccine with reported FLE monoclonal antibodies. [Figure 28E] FIG. 28E shows the sequence alignment of monoclonal antibodies induced by mice immunized with AdC7-M / E-WT vaccine with reported FLE monoclonal antibodies. [Figure 29A] Figure 29A shows the results of detecting the affinity of ZIKV sE-WT protein for FLE antibodies by BIAcore. [Figure 29B] Figure 29B shows the results of BIAcore detection of the affinity of ZIKV sE-MutC protein for FLE antibodies. [Figure 29C]Figure 29C shows the results of detecting the affinity of ZIKV sE-WT protein for FLE antibodies by BIAcore. [Figure 29D] Figure 29D shows the results of BIAcore detection of the affinity of ZIKV sE-MutC protein for FLE antibodies. [Figure 30A] Figure 30A shows the results of detecting the affinity of ZIKV sE-WT protein for non-FLE neutralizing antibodies by BIAcore. [Figure 30B] Figure 30B shows the results of detecting the affinity of ZIKV sE-MutC protein for non-FLE neutralizing antibodies by BIAcore. [Figure 30C] Figure 30C shows the results of detecting the affinity of ZIKV sE-WT protein for non-FLE neutralizing antibodies by BIAcore. [Figure 30D] Figure 30D shows the results of BIAcore detection of the affinity of ZIKV sE-MutC protein for non-FLE neutralizing antibodies. [Figure 31] Figure 31 shows the protein structure of the complex between ZIKV sE-MutC and Z3L1 single-chain variable region fragment (scFv). [Figure 32] FIG. 32 shows the dimeric structure of the ZIKV sE-MutC protein. [Figure 33] Figure 33 shows a comparative analysis of the Z3L1 / ZIKV sE MutC complex structure and the Z3L1 / ZIKV sE WT (PDB:5GZN) complex structure after superposition. [Figure 34] Figure 34 shows a comparative analysis of the FL region of ZIKV sE-MutC after superimposition with the FL region of ZIKV sE-WT (PDB: 5JHM). [Figure 35A] Figure 35A shows a comparative structural analysis of ZIKV sE MutC FL superimposed on the complex of flavivirus FLE antibody and E protein, using Z6 antibody and ZIKV sE protein. [Figure 35B]Figure 35B shows a comparative structural analysis of ZIKV sE MutC FL superimposed onto the complex of flavivirus FLE antibody and E protein, using the 2A10G6 antibody and ZIKV sE protein (PDB: 5JHL). [Figure 35C] Figure 35C shows a comparative structural analysis of ZIKV sE MutC FL superimposed onto the complex of flavivirus FLE antibody and E protein, using E53 antibody and WNV sE protein (PDB: 3I50). [Figure 36] FIG. 36 shows the results of measuring the antigenic activity of DENV2 wild-type and mutant E proteins by flow cytometry. [Figure 37] Figure 37 shows the results of flow cytometry detection of antigen activity when tryptophan at position 101 of the ZIKV E protein is mutated to one of 19 other amino acids. DETAILED DESCRIPTION OF THE INVENTION

[0062] In order to clarify the objectives, technical solutions and advantages of the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It is clear that the described embodiments are only some of the embodiments of the present invention, and not all of the embodiments. All other embodiments that can be obtained by those skilled in the art based on the embodiments of the present invention without creative effort also fall within the scope of the protection claims of the present invention. Unless otherwise specified in the present specification and claims, the term "comprises" or variations such as "contains" or "has" should be understood to include the described elements or components but not to exclude other elements or components.

[0063] In the following detailed description, numerous specific details are described to better explain the present invention. Those skilled in the art should understand that the present invention can be similarly practiced without certain details. In some examples, materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail in order to emphasize the gist of the present invention.

[0064] Example 1: Detection of humoral immune responses in mice induced by a recombinant chimpanzee adenovirus vaccine composed of ZIKV wild-type M / E and prM / E antigens

[0065] The M / E antigen of the ZIKV FSS13025 virus strain (GenBank: JN860885.1) was constructed into the vector chimpanzee adenovirus type 7, and the adenovirus was packaged, cultured, and purified to obtain the recombinant adenovirus vaccine AdC7-M / E-WT (for the construction of the recombinant adenovirus vaccine AdC7-M / E-WT, see Non-Patent Document 16). Experiments have demonstrated that this vaccine has a good protective effect in mice. Adenovirus vaccines constructed with ZIKV prM / E antigens have also been reported to have a protective effect. Therefore, the prM / E antigen of the ZIKV-SMGC-1 virus strain was constructed into the vector chimpanzee adenovirus type 7, packaged, and used to produce the recombinant adenovirus AdC7-prM / E-WT (recombinant adenovirus AdC7-prM / E-WT). For the construction of recombinant adenovirus AdC7-prM / E-WT, see Non-Patent Document 17) was obtained and used as a control in subsequent experiments.

[0066] The humoral immune responses of mice induced by the recombinant adenovirus vaccines AdC7-M / E-WT and AdC7-prM / E-WT were evaluated.

[0067] Fifteen BALB / c mice were randomly divided into three groups, each containing 1.6 x 10 11 Mice were immunized with vp (virus particles) AdC7-M / E-WT adenovirus vaccine, AdC7-prM / E-WT adenovirus vaccine, or PBS. Four weeks later, blood was collected, centrifuged, and serum was isolated. The serum was inactivated by heating at 56°C for 30 minutes, and neutralizing antibody titers in the serum were measured using a microneutralization test.

[0068] The microneutralization test was performed as follows. VERO cells were seeded into 96-well plates one day prior to the test. The following day, serum was serially diluted in DMEM medium (Invitrogen, C11995500BT) containing 1% FBS (Gibco, 10270-106) using a 96-well plate. Virus was also diluted in DMEM medium containing 1% FBS. The serum and virus were mixed uniformly, and 100 FFU ZIKV-SMGC-1 was added to each well and incubated at 37°C for 2 hours. The culture supernatant was removed from the VERO cell plate, and the serum-virus mixture was added. After 2 hours of incubation, DMEM containing 10% FBS was added. The cells were cultured in a 37°C incubator for 4 days. After 4 days, the cell culture plate was removed, the supernatant was discarded, washed once with PBS, and fixed with 150 μL of methanol. The plate was then placed in a -20°C refrigerator for 15–20 minutes and then washed twice with PBS. After blocking with 2% nonfat milk (blocking solution) prepared in PBS and incubation at room temperature for 30 minutes, the primary antibody was added. The primary antibody was a Z6 antibody, which binds to the ZIKV E protein. This primary antibody was diluted to a working concentration of 5 μg / mL in the blocking solution and incubated for 2 hours at room temperature. After washing three times with PBST, the secondary antibody was added. The secondary antibody was an HRP-conjugated goat anti-human antibody (Proteintech, SA00001-17). This secondary antibody was diluted 1:1500 in the blocking solution and incubated for 2 hours at room temperature, followed by four washes with PBST. 50 μl of TMB color development solution (Beyotime, P0209) was added, and the mixture was incubated at room temperature for approximately 20 minutes. The color change was observed, and the reaction was stopped by adding 50 μl of 2 M hydrochloric acid. The OD450 absorbance was measured using a microplate reader. Using GraphPad Prism software, nonlinear fitting calculations were performed on the data, and the serum dilution factor required to neutralize 50% of the cell infection was calculated to obtain the neutralization titer (MN 50 The lowest serum dilution that could not neutralize 50% of the cell infection was determined to be the MN of the sample. 50 was defined as half the lowest dilution factor.

[0069] The results of the neutralization test are shown in Figure 1. The group immunized with PBS was the negative control group, the Sham group (shown as Sham in the figure), and no neutralizing antibodies were detected in the serum of this group. Mice in the AdC7-M / E-WT group (shown as M / E in the figure) and the AdC7-prM / E-WT group (shown as prM / E in the figure) both had Log MN neutralizing antibodies. 50 The mean values of β-glucan (β) and β-glucan (β) were between 2 and 2.5, and statistical analysis showed no significant difference between the two groups. This indicates that both the recombinant adenovirus vaccines AdC7-M / E-WT and AdC7-prM / E-WT induced mice to produce higher levels of neutralizing antibodies.

[0070] Example 2: Detection of DENV ADE in serum from mice immunized with wild-type recombinant adenovirus vaccines AdC7-M / E-WT and AdC7-prM / E-WT in in vitro experiments

[0071] Because the fusion loop (FL) sequence of the flavivirus envelope (E) protein is highly conserved, ZIKV infection or ZIKV E protein immunophenotyping induces antibodies that cross-react with DENV, resulting in antibody-dependent enhancement (ADE) against DENV (Non-Patent Document 18). Therefore, we examined whether sera from mice immunized with the recombinant adenovirus vaccines AdC7-M / E-WT and AdC7-prM / E-WT constructed as described above could produce ADE against DENV.

[0072] Serum (collected from mice immunized with the recombinant adenovirus vaccine of Example 1) was serially diluted in RPMI-1640 medium (Invitrogen, C11875500BT) containing 1% FBS, and the diluted samples were added to a 96-well plate at 10 μl / well, followed by the corresponding DENV (DENV2, GenBank: KM204118.1; DENV3, GenBank: KF824903; DENV4, GenBank: AF289029; DENV1 is a virus strain isolated from an infected patient sample at Shenzhen Third People's Hospital), and placed in a cell incubator at 37°C for 1 hour.

[0073] Cultured K562 cells expressing FcγRIIA receptors on the cell surface were centrifuged at 800 g for 5 minutes, resuspended in RPMI-1640 medium containing 1% FBS, and counted. The cell density was further adjusted to 3x10 6 The resulting solution was adjusted to cells / ml. 10 μl / well of the resulting solution was added to the virus and serum mixture and incubated for 2 hours in a cell incubator at 37°C. 100 μl / well of RPMI-1640 medium containing 2% FBS was added, and the cells were cultured for 4 days in a cell incubator at 37°C. After 4 days, the cells were transferred to a 96-well plate, centrifuged at 800 g for 5 minutes, and the supernatant was removed. The cells were washed once with PBS and centrifuged to recover the cells. 100 μl / well of Fixation and Permeabilization solution (BD, 554722) was added to the 96-well plate and placed in a refrigerator at 4°C for 20 minutes. The cells were then centrifuged at 800 g for 5 minutes to recover the cells and washed twice with 1x Perm / Wash buffer (BD, 554723). 50 μl / well of FITC-labeled Z6 antibody (Z6-FITC) was added and placed in a refrigerator at 4°C for 1 hour. The cells were collected by centrifugation and washed twice with 1x Perm / Wash buffer. The cells were resuspended in PBS (200 μl / well), and the percentage of virus-infected positive cells was detected using a flow cytometer.

[0074] The results are shown in Figure 2A–D. K562 cells expressing the FcγRIIA receptor on their cell surface were used as a cell infection model in the experiments. Sera from mice immunized with the recombinant adenovirus vaccine AdC7-M / E-WT group (indicated by M / E in the figure) and the AdC7-prM / E-WT group (indicated by prM / E in the figure) exhibited an enhancing effect on K562 cells infected with four DENV serotypes, whereas ADE did not occur with sera from the control and sham groups (indicated by Sham in the figure). Sera from mice immunized with the recombinant adenovirus vaccine AdC7-M / E-WT and AdC7-prM / E-WT were cross-reactive with ZIKV and DENV, suggesting that some of the antibodies in the sera bind to DENV and induce ADE responses.

[0075] Example 3: Detection of ADE of monoclonal antibodies against DENV1-4 in vitro experiments

[0076] The Z6 antibody is a monoclonal antibody capable of binding to the ZIKV E protein, which was obtained by sequencing B cells isolated from the blood of a patient infected with ZIKV, followed by recombination, expression, and purification. Previous studies have demonstrated that the antibody primarily binds to the FL epitope of the ZIKV E protein (Non-Patent Document 19). The FL sequences of ZIKV and DENV are highly conserved, and the Z6 antibody has lower neutralizing activity (Non-Patent Document 20). Therefore, it was speculated that the Z6 antibody is likely to cause ADE against DENV, whereas the recombinant chimpanzee adenovirus vaccines AdC7-M / E-WT and AdC7-prM / E-WT, constructed with ZIKV wild-type M / E and prM / E antigens, are likely to activate the production of antibodies targeting the FL epitope, resulting in ADE.

[0077] We examined whether the Z6 antibody induces ADE, and the results are shown in Figure 3. The results in Figure 3 indicate that the Z6 antibody exhibits some degree of ADE against all four serotypes of DENV. Z6 antibodies with lower neutralizing activity cross-reacted with DENV. We speculated that the Z6 antibody likely induces ADE by binding to the FL epitope of the DENV E protein, and further investigated this possibility.

[0078] Example 4 Structural analysis of the complex between ZIKV E protein and Z6 antibody

[0079] Reported structures of antibody-antigen complexes of the FL epitope in the Flavivirus genus include the structure of the complex between the 2A10G6 antibody and the ZIKV soluble E protein (soluble E, sE) (Non-Patent Document 20) and the structure of the complex between the E53 antibody and the WNV E protein (Non-Patent Document 21). 2A10G6 is a broad-spectrum neutralizing antibody against flaviviruses that can neutralize DENV1-4, WNV, YFV, and ZIKV, and binds to the FL and bc loops of the ZIKV E protein (Non-Patent Document 20).

[0080] To further analyze the antibody binding mechanism of the ZIKV FL epitope, we combined protein crystallization, X-ray diffraction, and data analysis to obtain a 3 Å resolution structure of the complex between the Fab fragment of the Z6 antibody and the ZIKV E protein. The results are shown in Figure 4, and the data collection and optimization parameters for the complex structure are listed in Table 1.

[0081] As shown in Figure 4, the Z6 antibody, like the 2A10G6 antibody, bound to the tip of the DII domain of the E protein at a nearly perpendicular angle and interacted with the FL and bc loops of the E protein. However, as shown in Figures 5A-D, the difference between the Z6 and 2A10G6 antibodies is that both the heavy and light chains of Z6 interact with the ZIKV E protein, whereas 2A10G6 primarily binds its heavy chain to the ZIKV E protein. Analysis of the binding sites, as shown in Figure 6, revealed that the Z6 antibody interacted more with W101, G106, L107, and F108 of the ZIKV E protein. These four amino acid sites also play an important role in the binding of the 2A10G6 antibody to the ZIKV E protein. The interaction of E53 with the E protein was independent of W101, but it made more contact with G106 and L107.

[0082] Therefore, it is predicted that modification of the immunogen at these four amino acid sites may avoid the generation of antibodies induced by the FL epitope, thereby reducing or eliminating ADE against DENV.

[0083] It has been reported in the literature that ZIKV vaccines based on M / E antigens have excellent protective effects (Non-Patent Document 22). Therefore, the M / E antigen was selected for subsequent mutation design.

[0084] Furthermore, it has been reported in the literature that ZIKV vaccines based on prM / E antigens also have excellent protective effects (Non-Patent Document 23), and previous data also demonstrate that mutation designs based on M / E antigens have similar effects to prM / E antigens. Therefore, the experimental results of mutation designs based on prM / E antigens are not described in the examples of the present invention. The mutation designs for M / E antigens in the examples of the present invention can also be applied to prM / E antigens.

[0085] [Table 3]

[0086] Example 5 Construction of a chimeric virus antigen protein using FLs from evolutionarily distant Flaviviruses

[0087] By comparing the amino acid sequences of the FL epitope of the E protein of Flaviviruses, we found that the FL sequence of most viruses is conserved, but that the FL sequences of some viruses that are evolutionarily distant from ZIKV are distinct from that of ZIKV. To disrupt the ZIKV FL epitope without affecting normal protein folding or the expression of other neutralizing epitopes, we constructed a chimeric viral antigen protein using the FL amino acid sequence of a Flavivirus that is evolutionarily distant from ZIKV (the Flavivirus phylogenetic tree is shown in Figure 7). We speculated that this would maintain the overall conformation of the antigen and reduce the impact on epitopes other than the mutation site.

[0088] Using the FL sequences of the E proteins of these viruses as references, mutants of the ZIKV M / E antigen were designed, where M and E are full-length, and the mutation sites and sequences of the FL fusion region are shown in Figure 8. Here, MutA is derived from AEFV (Aedes flavivirus, GenBank: KC181923.1), and the mutation sites are D98N, N103T, G106F, L107E, and F108W. MutB is derived from CFAV (Cell fusing agent virus, GenBank: NC_001564.2), and the mutation sites are D98N, N103T, G106F, L107K, and F108W. MutC is derived from NAKV (Nakiwogo virus, GenBank: NC_030400.1), and the mutation sites are D98N, N103T, G106L, L107E, and F108W.

[0089] The primers used to construct the pCAGGS-M / E-MutA / B / C plasmid are listed in Table 2. For example, in the construction of MutA, the pCAGGS-M / E-WT plasmid was used as a template, and WT-F and mutA-R were used as primers to generate the mutA-1 product by PCR. The pCAGGS-M / E-WT plasmid was used as a template, and WT-R and mutA-F were used as primers to generate the mutA-2 product by PCR. Furthermore, a 1:1 molar mixture of mutA-1 and mutA-2 was used as a template, and WT-F and WT-R were used as primers to generate the mutA product by PCR. The pCAGGS plasmid was digested with XhoI and EcoRI to obtain a linearized plasmid with both cohesive ends. The enzyme-cleaved linearized plasmid and the PCR product mutA were mixed at a molar ratio of 1:5, and recombination was performed using the In-Fusion kit. The recombination product was transformed into DH5α competent cells, plated on ampicillin-resistant plates, and cultured at 37°C. The cells were selected, cloned, PCR identified, and sequenced. The plasmid (pCAGGS-M / E-MutA) was then extracted and used in subsequent experiments.

[0090] [Table 4]

[0091] Example 6 Detection of M / E-MutA / B / C antigen activity

[0092] Wild-type plasmid pCAGGS-M / E-WT and mutant plasmids pCAGGS-M / E-MutA, pCAGGS-M / E-MutB, and pCAGGS-M / E-Mu tC was transfected into 293T cells. After 48 hours, the cells were harvested, digested, and single-celled. They were then fixed and permeabilized, incubated with ZIKVE-binding antibodies, and then incubated with a goat anti-human FITC secondary antibody. Finally, the positive rate of the samples was detected using a flow cytometer. The results are shown in Figure 9.

[0093] Z3L1, Z20, and Z23 are all ZIKV-specific antibodies with high neutralizing activity, and bound to the DI, DII, and DIII regions of the ZIKV E protein, respectively (Non-Patent Document 19).

[0094] As can be seen in Figure 9, antibodies (Z6 and 2A10G6) that bind to the FL epitope of the ZIKV E protein were able to bind to cells expressing wild-type M / E-WT antigen but not to cells expressing the three mutant antigens. In contrast, antibodies Z3L1, Z23, and Z20, which have high neutralizing activity and bind to non-FL epitopes, bound to both cells expressing wild-type M / E-WT antigen and cells expressing the three mutant antigens. As can be seen, the FL epitopes of the M / E-MutA, M / E-MutB, and M / E-MutC antigens were disrupted, preventing binding by the corresponding antibodies. However, the epitopes bound by other potent ZIKV neutralizing antibodies remained unchanged, allowing the corresponding antibodies to still bind.

[0095] That is, the M / E-MutA, M / E-MutB, and M / E-MutC antigens induced little or no production of antibodies that bind to DENV FL, thereby reducing ADE against DENV, and at the same time, the M / E-MutA, M / E-MutB, and M / E-MutC antigens did not affect other antibody epitopes.

[0096] Example 7 Construction of recombinant chimpanzee adenovirus vaccines AdC7-M / E-MutB and AdC7-M / E-MutC using ZIKV M / E-MutB and M / E-MutC antigens

[0097] As can be seen from Figure 9 , the cell-positive intensity of MutB and MutC was slightly higher than that of MutA, so subsequent experiments were mainly performed using MutB and MutC.

[0098] First, the M / E-MutB and M / E-MutC antigens were cloned into the pshuttle vector. Using the pCAGGS-M / E-MutB or pCAGGS-M / E-MutC plasmid as template and the to_pshuttle-F and to_pshuttle-R primers, PCR products to_pshuttle-mutB and to_pshuttle-mutC were obtained. The pshuttle plasmid was digested with XbaI (Thermo, FD0684) and KpnI (Thermo, FD0524) to obtain linearized plasmids with both cohesive ends. The digested linearized plasmids were mixed with the PCR products to_pshuttle-mutB or to_pshuttle-mutC at a molar ratio of 1:5, respectively, and recombined using the In-Fusion kit. The recombinant products were transformed into DH5α competent cells, plated on canna-resistant plates, and cultured at 37°C. The cells were then selected and cloned, PCR identified, and sequenced, and the plasmids were extracted. A cassette expressing M / E-MutB and M / E-MutC on the pshuttle plasmid was then constructed in the AdC7 vector. The construction procedure was described in Non-Patent Document 16.

[0099] The PCR products to_AdC7-MutB and to_AdC7-MutC were obtained by PCR using the plasmid pshuttle-M / E-MutB or MutC as a template and to_AdC7-F and to_AdC7-R as primers. The AdC7 plasmid was digested with restriction enzymes PI-SceI (NEB, R0696S) and I-CeuI (NEB, R0699S) to obtain a linearized plasmid with both cohesive ends. The digested linearized plasmid and the PCR product to_AdC7-MutB or to_AdC7-MutC were mixed at a molar ratio of 1:5 and recombined using the In-Fusion kit. The recombinant product was transformed into stbl2 competent cells, plated on ampicillin-resistant plates, and cultured at 30°C. The cells were selected and cloned, followed by PCR identification and sequencing. The plasmid was then extracted. The construction flow of the recombinant plasmid is shown in Figure 10. The primer sequences used are listed in Table 3.

[0100] [Table 5]

[0101] The pAdC7-M / E-MutB and pAdC7-M / E-MutC plasmids were linearized with the restriction enzyme PacI (NEB, R0547S) and then inactivated by heating at 65°C for 20 minutes in a constant temperature bath. The plasmids were transfected into HEK293 cells using Fugene-6 transfection reagent (Promega, E2691). After culturing in a 37°C incubator for at least 7 days, the cells were examined daily for the appearance of plaques under a microscope. When plaques shed, all cells and supernatant were collected to obtain first-generation recombinant adenovirus. The cells were then expanded at a ratio of 1:10 until they reached 40 plates of cells. Afterwards, all cells were collected and lysed by freezing and thawing three times to release the virus, which was then purified by cesium chloride density gradient centrifugation, desalted on polyacrylamide gel (Bio-Gel P-6 DG Media, BIO-RAD, 1500738), and purified. The OD260 of the sample was detected by NANODROP. When the concentration of the sample reached an OD260 value of 1.1x10, the OD260 value was 1.1x10. 12 The unit is vp (viral particle) / ml, and the resultant was aliquoted and stored at -80°C.

[0102] Example 8 Evaluation of humoral immune responses in BALB / c mice induced by recombinant adenovirus vaccines AdC7-M / E-MutB and AdC7-M / E-MutC

[0103] Twenty-four BALB / c mice were randomly divided into four groups and immunized intramuscularly with three recombinant adenovirus vaccines, AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC, respectively. The adenovirus vaccine immunization dose was 1.6 x 10 11 One group of mice was immunized with PBS as a negative control. After 4 weeks, blood was collected and serum was separated, and the neutralizing antibody titer in the serum was detected using a microneutralization test.

[0104] The results are shown in Figure 11. Both wild-type AdC7-M / E-WT and mutant adenovirus vaccines AdC7-M / E-MutB and AdC7-M / E-MutC were able to induce mice to produce neutralizing antibodies, with Log(MN 50 The mean value of the neutralizing antibody titer was between 2.0 and 2.5, and there was no significant difference in the neutralizing antibody titer between the AdC7-M / E-MutB and AdC7-M / E-MutC groups and the AdC7-M / E-WT group, which indicates that the mutations did not significantly reduce the immunogenicity of the antigen and could still effectively stimulate the production of neutralizing antibodies. In the figure, 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.

[0105] Example 9 Ifnar1 induced by AdC7-M / E-MutB and AdC7-M / E-MutC vaccines - / - Assessment of humoral immune responses in mice

[0106] Since ZIKV infection of BALB / c mice did not result in death or obvious symptoms, we used immunodeficient mice, Ifnar1, to better verify the efficacy of the vaccine. - / - Mice were selected as a ZIKV infection model (Lazear, Helen M et al. (2016), vol. 19, 5: 720-30.), and the Ifnar1 expression induced by AdC7-M / E-MutB and AdC7-M / E-MutC vaccines was investigated. - / - The humoral immunity levels of the mice were evaluated.

[0107] Ifnar1 - / - The mice were randomly divided into four groups and intramuscularly injected with AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC adenovirus vaccines, respectively. The adenovirus vaccine immunization dose was 1.6 × 10 11 One group of mice was immunized with PBS as a negative control. After 28 days, blood was collected and serum was isolated. The serum was tested for Ifnar1 activity using a microneutralization test. - / - The titer levels of neutralizing ZIKV antibodies in mouse serum were detected, and the results are shown in Figure 12.

[0108] As can be seen from Figure 12, both the AdC7-M / E-MutB and AdC7-M / E-MutC vaccines were able to induce high levels of neutralizing antibodies in mice, where 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.

[0109] Example 10 Ifnar1 - / - Challenge protection test after mouse immunization

[0110] Ifnar1 immunized in Example 9 - / - Mice were challenged with ZIKV on day 30 post-immunization, with 5x10 intraperitoneally 6After injection of PFU ZIKV (SMGC-1 strain), the condition of the mice was observed daily and changes in body weight were monitored. The experimental results are shown in Figures 13A and 13B.

[0111] As can be seen from Figures 13A and 13B, the sham group gradually lost weight starting on day 4 after challenge (Figure 13B), and all mice in the sham group died on day 6 or 7 (Figure 13A). In contrast, mice in the three groups immunized with AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC did not lose weight after challenge (Figure 13B), and none of the mice in these three groups died (Figure 13A). This indicates that the AdC7-M / E-MutB and AdC7-M / E-MutC vaccines, like the wild-type AdC7-M / E-WT vaccine, provided complete protection in mice upon challenge. Here, 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.

[0112] To determine whether AdC7-M / E-MutB and AdC7-M / E-MutC vaccines could protect mice from viremia induced after viral infection, For this experiment, blood was collected from mice on days 3 and 6 after challenge, serum was separated, and RNA was extracted using the MagaBio Plus viral RNA kit (Bioer Technology, BSC58S1B). The viral RNA was then quantitatively measured using the FastKing one-step reverse transcription-fluorescence quantification kit (TIANGEN BIOTECH, FP314). The probe and primer sequences used for quantification are listed in Table 4, and the quantitative results are shown in Figure 14.

[0113] [Table 6]

[0114] As can be seen from Figure 14, mice in the sham group had high virus loads on both days 3 and 6 post-infection, while mice immunized with AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC adenovirus vaccines had no virus detected in their sera on days 3 and 6 post-infection, indicating that the AdC7-M / E-MutB and AdC7-M / E-MutC vaccines, like the wild-type AdC7-M / E-WT vaccine, could effectively protect mice from viremia. In the figure, "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.

[0115] Example 11: Detection of the sterilizing immunity effect of ZIKV vaccine

[0116] The above experiments demonstrated that mice immunized with AdC7-M / E-MutB and AdC7-M / E-MutC vaccines could be protected from viremia and death caused by ZIKV infection. Further experiments were designed to determine whether AdC7-M / E-MutB and AdC7-M / E-MutC vaccines could provide sterilizing immunity. The specific steps were as follows:

[0117] Ifnar1 - / - Mice were randomly divided into four groups and immunized with three recombinant adenovirus vaccines, AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC, by intramuscular injection. The adenovirus vaccine immunization dose was 1.6x10 11 One group of mice was immunized with PBS as a negative control. Blood was collected on day 28 after immunization, serum was separated, and 5x10 ZIKV (SMGC-1 strain) was intraperitoneally administered on day 30 after immunization. 4On day 6, blood was collected again after FFU injection and challenge with ZIKV. The liver, spleen, testes, brain, and spinal cord were dissected and removed. The dissected tissues were added to PBS solution, then crushed in a grinder (TIANGEN BIOTECH, OSE-Y30), centrifuged, and the supernatant was removed. RNA was extracted using the MagaBio Plus viral RNA kit. ZIKV RNA was then subjected to RT-PCR detection. The results are shown in Figure 15.

[0118] As can be seen from Figure 15, a certain amount of virus was detected in all five organs of mice in the sham group, but AdC7-M / E-WT, AdC7-M / E-MutB, and Ad In the three groups of mice immunized with the C7-M / E-MutC adenovirus vaccine, virus was not detected in any tissues or organs. In the figure, 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.

[0119] A microneutralization test was used to detect neutralizing antibody titers in the serum on day 28 after immunization and day 7 after challenge, and the results are shown in FIG.

[0120] As can be seen from Figure 16, no neutralizing antibodies were detected in the sham group before challenge, and high titers of neutralizing antibodies were detected in all mice in the sham group 7 days after challenge. There was no significant difference in the serum neutralizing antibody titers before and after challenge between the two groups immunized with AdC7-M / E-WT and AdC7-M / E-MutB vaccines, and they remained at approximately the same levels. This indicates that the AdC7-M / E-WT vaccine, AdC7-M / E-MutB, and AdC7-M / E-MutC vaccines were all able to provide complete immunity to challenge, and that the AdC7-M / E-MutB vaccine was slightly more effective than the AdC7-M / E-MutC vaccine, providing complete sterilizing immunity. Here, Sham in the figure refers to the group immunized with PBS, M / E-WT refers to the immunized AdC7-M / E-WT vaccine group, M / E-MutB refers to the immunized AdC7-M / E-MutB vaccine group, and M / E-MutC refers to the immunized AdC7-M / E-MutC vaccine group. In Figure 16, the results are from left to right for the Sham group, M / E-WT group, M / E-MutB group, and M / E-MutC group.

[0121] Example 12 Detection of cross-reactivity of serum from BALB / c mice immunized with ZIKV vaccine to DENV

[0122] One of the main reasons why ZIKV infection leads to DENV ADE is that the FL epitope of the E protein of ZIKV and DENV is more conserved, and some antibodies elicited by ZIKV cross-react with DENV (Non-Patent Document 18). Therefore, further ELISA experiments detected cross-reactivity of sera from BALB / c mice immunized with AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC adenovirus vaccines against four serotypes of DENV. The results are shown in Figures 17A-E.

[0123] As shown in Figures 17A-E, sera from BALB / c mice immunized with AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC vaccines all strongly bound to the ZIKV E protein. However, their binding abilities to the four serotypes of DENV E protein differed. While sera from the M / E-MutB and M / E-MutC groups showed low binding to the four serotypes of DENV E protein, sera from the M / E-WT group maintained high levels of binding to the four serotypes of DENV E protein. As can be seen, the mutations disrupted the FL epitope in the E protein, reducing the amount of antibodies induced by this epitope, resulting in reduced binding abilities of sera from mice immunized with AdC7-M / E-MutB and AdC7-M / E-MutC adenovirus vaccines to DENV E protein.

[0124] In Figures 17A-E, 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 17A shows the binding ability of mouse sera to the Zika virus (ZIKV) E protein, Figure 17B shows the binding ability of mouse sera to the E protein of dengue virus type 1 (DENV1), and Figure 17C shows the binding ability of mouse sera to the E protein of dengue virus type 1 (DENV1). Figure 17A shows the results of the binding ability of serum to the E protein of dengue virus type 2 (DENV2), Figure 17B shows the results of the binding ability of mouse serum to the E protein of dengue virus type 3 (DENV3), and Figure 17C shows the results of the binding ability of mouse serum to the E protein of dengue virus type 4 (DENV4).

[0125] Example 13 Detection of ADE against DENV in serum from BALB / c mice immunized with ZIKV vaccine in an in vitro experiment

[0126] The experimental results of Example 12 demonstrated that AdC7-M / E-MutB and AdC7-M / E-MutC vaccines reduced the induction of cross-antibody against DENV. Further experiments were designed to determine whether AdC7-M / E-MutB and AdC7-M / E-MutC vaccines could reduce ADE against DENV. The specific steps were as follows:

[0127] Serum from BALB / c mice immunized in Example 8 was serially diluted and mixed with DENV1, DENV2, DENV3, and DENV4, respectively, and incubated. K562 cells were then added and cultured for 4 days. The cells were then stained with FITC-labeled Z6 antibody and the percentage of positive cells was then detected using a flow cytometer. The results are shown in Figures 18A-E.

[0128] Without antibody mediation, DENV virus was unable to infect K562 cells. As can be seen from Figures 18A-E, the serum from the Sham group did not contain antibodies capable of binding to DENV, and thus the infection rate of the detected samples was at background levels. At a certain concentration, serum from mice in the M / E-WT group was able to mediate the infection of K562 cells with four serotypes of DENV virus. In contrast, the infection rates of samples from both the M / E-MutB and M / E-MutC groups were significantly lower than those of the M / E-WT group, indicating a reduction or elimination of DENV ADE. This indicates that the AdC7-M / E-MutB and AdC7-M / E-MutC vaccines achieved a good effect in reducing ADE.

[0129] Here, 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.

[0130] 18A-E, FIG. 18A shows mouse serum detection of enhanced infection of K562 cells by ZIKV, FIG. 18B shows mouse serum detection of enhanced infection of K562 cells by DENV1, FIG. 18C shows mouse serum detection of enhanced infection of K562 cells by DENV2, FIG. 18D shows mouse serum detection of enhanced infection of K562 cells by DENV3, and FIG. 18E shows mouse serum detection of enhanced infection of K562 cells by DENV4.

[0131] All of the above series of in vitro experiments demonstrated that the AdC7-M / E-MutB and AdC7-M / E-MutC adenovirus vaccines could reduce or eliminate ADE against DENV.

[0132] Example 14 Detection of ADE against DENV in serum from BALB / c mice immunized with ZIKV vaccine in an in vivo experiment

[0133] To further demonstrate whether AdC7-M / E-MutB and AdC7-M / E-MutC vaccines can reduce ADE against DENV under physiological conditions, Ifnα / βr - / - Ifnγr - / - Using a mouse-based model, DENV The ADE phenomenon was examined.

[0134] First, 80 BALB / c mice were randomly divided into 4 groups, each with 20 mice. They were immunized with AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC adenovirus vaccines and PBS, respectively. The adenovirus vaccine immunization dose was 1.6 × 10 per mouse. 11 After 4 weeks, blood was collected and serum was separated. The serum was heated at 56°C for 30 minutes. The serum from 20 mice in each group was mixed and used for subsequent passive immunization with Ifnα / βr. - / - Ifnγr - / - Used in mice. Ifnα / βr - / - Ifnγr - / -Mice were randomly divided into four groups and intraperitoneally injected with serum from BALB / c mice immunized with AdC7-M / E-WT, AdC7-M / E-MutB, or AdC7-M / E-MutC adenovirus vaccines or PBS (serum was diluted 1:10 with PBS, and 200 μl of diluted serum was injected into each mouse). Twenty-four hours later, each mouse was subcutaneously injected with 5000 FFU of DENV type 2 virus. Each mouse's weight was recorded before injection. Thereafter, the mice were monitored daily for condition and survival, and their weights were measured. The results are shown in Figures 19A and 19B.

[0135] As can be seen from Figures 19A and 19B, the overall time to death for mice in the M / E-WT group was earlier than that for mice in the sham group. This indicates that serum from mice immunized with the AdC7-M / E-WT vaccine enhances the pathogenesis of DENV. In contrast, the time to death and trends for mice in the M / E-MutB and M / E-MutC groups were consistent with those for the sham group (Figure 19A). This indicates that the mutation in the ZIKV E protein reduces the production of antibodies that cause ADE events against DENV, making the appearance of mice in the M / E-MutB and M / E-MutC groups similar to that of the sham group. Furthermore, while mice in the M / E-WT group lost weight more quickly, the trends in weight change for mice in the M / E-MutB and M / E-MutC groups were consistent with those for the sham group (Figure 19B). The results of these in vivo experiments clearly demonstrate that the AdC7-M / E-MutB and AdC7-M / E-MutC vaccines can significantly reduce ADE against DENV after immunization with the wild-type vaccine.

[0136] Here, in Figures 19A and 19B, 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.

[0137] Example 15 Analysis of antibody repertoire produced by BALB / c mice induced by ZIKV vaccine

[0138] Because the FL mutant ZIKV vaccine induced protective immunity while reducing ADE responses to DENV, we further analyzed ZIKV E B cell lineages in mice to explain how the mutant vaccine affected antibody responses.

[0139] BALB / c mice were randomly divided into three 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). Each mouse received 1.6 × 10 11 The mice were immunized with 1640 medium containing 1% FBS. On the 20th day after immunization, the lymph nodes were dissected and removed. The lymph nodes from all mice in each vaccine group were mixed, crushed on the rough surface of a 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 resuspended in 1 ml of FACS buffer (PBS solution supplemented with 0.5% FBS). After resuspension, the lymphocytes were transferred to a 1.5 ml EP tube and spun at 400 g for 4°C. The cells were centrifuged at RT for 10 minutes and the supernatant discarded. The cells were resuspended in 200 μl of FACS buffer, and 4 μg of a mixture of biotin-labeled ZIKV E monomer and dimer proteins was added. The mixture was incubated at 4°C in the dark for 30 minutes. 1 ml of FACS buffer was then added, mixed thoroughly, and centrifuged to pellet the cells. After washing once with 1 ml of FACS buffer, the cells were stained with antibodies diluted in FACS buffer (200 μl each): 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), and BV711 (4 μl, BD, 563262). Each sample was incubated at 4°C for 30 minutes in the dark and then washed twice with FACS buffer. Add 2 ml of FCAS buffer, suspend the cells, filter them through a 0.45 μm filter, transfer them to a flow tube, and incubate them in GL-7. + B220 hi CD38 lo IgD - CD93 - CD138 -+ GC B cells reactive with ZIKV E were selected under the selection conditions (shown in Figure 20), and a database was constructed before sequencing.

[0140] To obtain the paired sequences of the B cell receptor (BCR) of single cells, we used single cell sequencing technology. A pre-sequencing database was constructed using the V(D)J Enrichment Kit, Mouse B Cell, 96 rxns (10xgenomics, PN-1000072), followed by high-throughput sequencing to analyze the full-length sequences of the V(D)J fragments of the heavy and light chains of each cell line. The results are shown in Figure 21 and Table 5.

[0141] As can be seen from Figure 21 and Table 5, 451 heavy chain variable region sequences and 661 light chain variable region sequences were obtained in the WT group sample, resulting in 334 possible pairings; 310 heavy chain variable region sequences and 379 light chain variable region sequences were obtained in the MutB group sample, resulting in 234 possible pairings; and 664 heavy chain variable region sequences and 776 light chain variable region sequences were obtained in the MutC group sample, resulting in 515 possible pairings. In Figure 21 and Table 5, WT refers to the group immunized with the AdC7-M / E-WT vaccine, the group immunized with the MutB-immunized AdC7-M / E-MutB vaccine, and MutC refers to the group immunized with the AdC7-M / E-MutC vaccine.

[0142] [Table 7]

[0143] Analysis of the repertoires generated by mice induced with WT, MutB, and MutC revealed that the variable region (V) genes of mice stimulated with AdC7-M / E-WT vaccine showed a preference, with approximately 60% of heavy chains using IGHV9-2-1, IGHV1-22, and IGHV7-3, and approximately 60% of light chains using IGKV10-96, IGKV14-111, and IGKV6-23 (Figure 21). In contrast, the FL mutant AdC7-M / E-MutB and AdC7-M / E-MutC vaccines showed a preference for V. The BCR profiles were more diverse and dispersed in both the heavy chain variable region (HV) and light chain variable region (LV) (Figure 21). Some of the most significantly activated variable region genes in the WT group were reduced or not present at all in the antibody repertoires of the FL mutant vaccine, MutB, and MutC groups (Figure 21).

[0144] The paired HV and LV results in the antibody repertoire were then analyzed, and the results are shown in Figures 22, 23, and 24-1 and 24-2, respectively.

[0145] As can be seen from Figure 22, GC B cell clones from mice activated with AdC7-M / E-WT vaccine showed a significant HV:LV preference, with the highest frequencies of IGHV9-2-1:IGKV10-96 (29.9%), IGHV1-22:IGKV14-111 (14.4%), and IGKV1-22:IGKV6-23 (7.5%), totaling approximately 50%.

[0146] As can be seen in Figures 23 and 24-1 and 24-2, the GC B cell clones from mice stimulated with AdC7-M / E-MutB and AdC7-M / E-MutC vaccines used a greater number of HV:LV species and their frequency distribution was more divergent. The highest IGHV9-2-1:IGKV10-96 was detected in the WT group, but not in the MutB or MutC groups.

[0147] The results of antibody repertoire analysis indicate that the immunodominant epitopes of B cell responses were transferred after substitution of the FL region of the AdC7-M / E-WT vaccine.

[0148] Example 16 Identification of the major FLE antibody types responsible for ADE responses to DENV infection

[0149] Although most of the antibodies that induced ADE responses to DENV targeted the FLE of the E protein, antibodies induced by ZIKV infection also induced ADE responses to DENV. Therefore, we identified the binding characteristics of the isolated monoclonal antibodies to determine which of them bound to FLE and whether they induced ADE responses to DENV.

[0150] Based on the similarity classification of GC B cell clones, several representative monoclonal antibody genes were synthesized (by Jin Weizhi, Suzhou) covering 63.38%, 46.57%, and 43.88% of the total number of the three groups: AdC7-M / E-WT, AdC7-M / E-MutB, and AdC7-M / E-MutC, respectively (Table 6). The HV and LV genes were then cloned into mouse IgG2A and Igκ expression vectors, respectively. The 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 the monoclonal antibodies derived from the AdC7-M / E-MutB vaccine group (denoted as M / E-MutB in Table 6) and the 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).

[0151] [Table 8]

[0152] [Table 9]

[0153] The heavy and light chain plasmids expressing monoclonal antibodies were transfected into 293T cells, and the supernatant was collected after 3 days. ELISA experiments were performed to confirm that the antibodies in the supernatant were ZIKV-E. The ability to bind to proteins was detected.

[0154] The ELISA experiment method was as follows: The protein was diluted to 3 μg / ml with ELISA coating solution (sodium carbonate-sodium bicarbonate buffer, pH 9.6), and 100 μl was added to each well of a 96-well ELISA plate and left overnight at 4°C. On the day of the assay, the coating solution was discarded, and the ELISA plate was blocked with 5% nonfat milk prepared in PBS and left at room temperature for 1 hour. The blocking solution was discarded, and 100 μl of culture supernatant expressing the monoclonal antibody was added to each well of the ELISA plate. The plate was then incubated at room temperature for 2 hours and washed three times with PBST. Goat Anti-Mouse HRP (ab6789) secondary antibody, diluted 1:2000 in blocking solution, was then added, and the plate was incubated at room temperature for 1.5 hours and washed four times with PBST. 50 μl of TMB color development solution was added, and after 30 minutes, 50 μl of 2M HCl was added to stop the reaction. OD450 values were measured using a microplate reader.

[0155] The ELISA detection results are shown in Figure 25. Most of the monoclonal antibodies can bind to either the monomeric or dimeric forms of the ZIKV sE protein. As can be seen in Table 6, the binding positive rates were 90% (9 / 10) for the ME-WT group, 87.5% (7 / 8) for the ME-MutB group, and 76.9% (10 / 13) for the ME-MutC group, respectively.

[0156] We further evaluated the 26 positive monoclonal antibodies that bound to the ZIKV sE protein. Figure 25 shows that none of the monoclonal antibodies from the ME-WT group reacted with the ZIKV sE-MutC protein but cross-reacted with the sE proteins of DENV serotypes 1 to 4. This indicates that the monoclonal antibodies from the ME-WT group were FLE-binding monoclonal antibodies, while the monoclonal antibodies from the ME-MutB and ME-MutC groups were primarily ZIKV-specific antibodies, with most not binding to DENV sE.

[0157] Representative FLE monoclonal antibodies from the ME-WT group were purified and their activity against DENV in K562 cells was further evaluated. The results are shown in Figures 26A-D. As can be seen from Figures 26A-D, all ME-WT antibodies detected enhanced infection to some extent against all four serotypes of DENV. In Figures 26A-D, ZWT.1, 4, 6, 8, 9, and 10 are monoclonal antibodies from the M / E-WT group, respectively. DENV1, DENV2, DENV3, and DENV4 are serotypes 1-4 of DENV viruses, respectively. Figures 26A-D show, respectively, that ZWT.1, 4, 6, 8, 9, and 10 antibodies enhanced infection of K562 cells with the four serotypes of DENV.

[0158] The FLE monoclonal antibodies derived from the ME-WT group were mainly four types of HV:LV gene panel. We attempted to search for previously reported flavivirus FLE mouse-derived monoclonal antibodies in the literature and databases, and further compared the isolated antibody gene loci and sequences, and discovered the following four monoclonal antibodies.

[0159] 6B6C-1 was isolated after infection with tick-borne encephalitis virus (TBEV) (Non-Patent Document 24), and 4G2 and 2A10G6 were both isolated after infection with DENV (Non-Patent Document 25; Non-Patent Document 26). E53 was isolated after WNV infection (Non-patent Document 27).

[0160] As shown in Figure 27 and Figures 28A-E, analysis revealed that the FLE monoclonal antibodies isolated from the ME-WT group shared many similarities in gene loci and sequences with the four reported monoclonal antibodies. As can be seen from Figure 27, 6B6C-1 and 4G2 share the same HV:LV gene pair as ZWT.1-3 and ZWT.4-5, respectively, showing high sequence similarity. 2A10G6 and E53 share the same HV gene as ZWT.6 and ZWT.8, respectively.

[0161] Although 2A10G6 and ZWT.6 used different LV genes, the LV sequences of the two antibodies were somewhat similar, and as can be seen in Figure 28E, the CDRL3 and FR4 of 2A10G6 and ZWT.6 were completely identical.

[0162] As can be seen from the above analysis, the FLE monoclonal antibodies cloned from lymph node GC B cells of mice immunized with the AdC7-M / E-WT vaccine use almost the same gene locus as reported mouse FLE monoclonal antibodies and have similar sequences. This indicates that the gene locus used to generate antibodies binding to FL epitopes in the induced mice is preferred, and the properties of the generated FLE antibodies are similar.

[0163] Figure 28A is an alignment analysis of the antibody heavy chains of ZWT.1, ZWT.2, ZWT.3, and 6B6C-1 with the mouse locus sequences; Figure 28B is an alignment of the antibody light chains of ZWT.1, ZWT.2, ZWT.3, and 6B6C-1 with the mouse locus sequences; Figure 28C is an alignment of the antibody heavy chains of ZWT.4, ZWT.5, ZWT.6, 4G2, and 2A10G6 with the mouse locus sequences; Figure 28D is an alignment of the antibody light chains of ZWT.4, ZWT.5, and 4G2 with the mouse locus sequences; and Figure 28E is an alignment of the antibody light chains of ZWT.6 and 2A10G6 with the mouse locus sequences.

[0164] Example 17 Detection of the affinity of wild-type and mutant ZIKV E proteins for ZIKV antibodies by SPR experiments

[0165] Because the AdC7-M / E-MutB and AdC7-M / E-MutC vaccines were able to provide complete protection to mice while avoiding ADE against DENV, we expressed and purified a representative soluble sE-MutC protein and compared it with sE-WT to explain the potential molecular mechanism.

[0166] Based on the principle of surface plasmon resonance (SPR), BIOCORE8000 can detect interactions between molecules, reflect the dynamic changes in the molecular binding process in real time, and obtain the kinetic parameters of the interaction.

[0167] The affinity of ZIKV sE-WT and ZIKV sE-MutC proteins to FLE and non-FLE neutralizing antibodies was detected using a BIOCORE8000. ZIKV sE-WT and ZIKV sE-MutC proteins were immobilized on a CM5 chip using the amino coupling method. Four antibodies were then serially diluted and used as the mobile phase. The test antibodies were sequentially run over the immobilized ZIKV sE protein, yielding signals corresponding to different binding events. The collected data was subjected to fitting calculations, and the results are shown in Figures 29A-D and 30A-D.

[0168] 29A to 29D show, in order, the binding results between the ZIKV sE-WT protein and the 2A10G6 antibody (sE-WT-2A10G6), the binding results between the ZIKV sE-MutC protein and the 2A10G6 antibody (sE-MutC-2A10G6), and the ZIKV The binding results for the sE-WT protein and the Z6 antibody (sE-WT-Z6) and the ZIKV sE-MutC protein and the Z6 antibody (sE-MutC-Z6) are shown. Figure 29A shows that the 2A10G6 antibody binds to the ZIKV sE-WT protein with an affinity of 9.13 nM. Figure 29C shows that the Z6 antibody binds to the ZIKV sE-WT protein with an affinity of 7.14 nM. Figure 29B shows that the ZIKV sE-MutC protein does not bind to the 2A10G6 antibody at all. Figure 29D shows that the ZIKV sE-MutC protein does not bind to the Z6 antibody at all. These results are consistent with theoretical analysis.

[0169] Figures 30A to 30D show, respectively, the binding results between the ZIKV sE-WT protein and the Z3L1 antibody (sE-WT-Z3L1), the binding results between the ZIKV sE-MutC protein and the Z3L1 antibody (sE-MutC-Z3L1), the binding results between the ZIKV sE-WT protein and the Z23 antibody (sE-WT-Z23), and the binding results between the ZIKV sE-MutC protein and the Z23 antibody (sE-MutC-Z23). As can be seen from Figures 30A and 30C, the binding affinities of the Z3L1 and Z23 antibodies to the ZIKV E-WT protein are 9.48 μM and 0.625 μM, respectively. As can be seen from Figures 30B and 30D, the binding affinities of the Z3L1 and Z23 antibodies to the ZIKV sE-MutC protein are 8.01 μM and 0.701 μM, respectively. The binding affinities of the mutant protein ZIKV sE-MutC to Z23 and Z3L1 were almost unchanged compared to the wild-type ZIKV sE-WT protein, indicating that the designed mutants were able to maintain the overall conformation of the protein and that there were no changes in other epitopes other than the mutation sites.

[0170] Example 18 Structural analysis of the complex between mutant ZIKV sE-MutC protein and Z3L1 antibody

[0171] To further elucidate the mechanism of action of the mutant vaccine, we purified the complex protein between ZIKV sE-MutC and the Z3L1 single-chain variable fragment (scFv). After crystallographic screening, we obtained the atomic structure of the complex at 3 Å resolution through X-ray diffraction and structural analysis. The structure is shown in Figure 31, and the data collection and optimization parameters for the complex are listed in Table 7.

[0172] [Table 10]

[0173] As shown in Figure 31, despite the introduction of five point mutations into the FL sequence of the ZIKV E protein, sE-MutC bound to the Z3L1 scFV in a dimeric form, which is the prefusion state. As shown in Table 8, detailed analysis of the dimer contact interface revealed that the mutated FL amino acid residues reconstructed the interaction between two adjacent E protein promoters, generating four and one hydrogen bonds at positions N98 and W108, respectively, which favored the stabilization of the E protein dimer.

[0174] As can be seen from Figure 32, the folding of sE-MutC was very similar to the wild-type protein, displaying normal secondary, tertiary and quaternary epitope structures.

[0175] To analyze the conformation of the neutralizing epitope, the complex structure of ZIKV sE-MutC and Z3L1 scFv (Z3L1 / ZIKV sE MutC) was compared with the complex structure of Z3L1 and ZIKV sE-WT protein (Z3L1 / ZIKV sE WT). , PDB:5GZN), and the results are shown in Figure 33. As can be seen from Figure 33, the ZIKV wild-type protein sE-WT and the mutant protein sE-MutC have the same binding profile with the Z3L1 antibody, with the binding sites being mainly the DO, EO, and FO chains in DI and the 150 loop, as well as the k1 hairpin in DI (Non-Patent Document 19).

[0176] The FL region of sE-MutC was superimposed with that of sE-WT for comparative analysis, and the results are shown in Figure 34. As can be seen from Figure 34, the conformations of the FL epitopes of both are very similar, with differences only in the side chains of the mutation sites.

[0177] To analyze the possibility that the mutant antigen sE-MutC induces FLE antibodies, the structures of FLE antibodies with known structures (Z6, 2A10G6, and E53) binding to flavivirus E protein were analyzed by superimposing them on the DII structure of sE-MutC. The results are shown in Figure 35A-C.

[0178] Figures 35A-C show 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), respectively. As can be seen from Figures 35A-C, when the monoclonal antibody bound to sE-MutC, the G106, L107, and F108 mutations significantly inhibited antigen-antibody binding. The protruding long side chain of G106L could inhibit the binding of these FLE antibodies. After the L107E mutation, a charged side chain was formed, which may disrupt local hydrophobic interactions. Furthermore, the F108W mutation created a steric hindrance, affecting the interaction of the FL epitope of sE-MutC with the 2A10G6 antibody. Thus, mutation of three key amino acids, G106, L107, and F108, in the FL epitope of ZIKV E protein acted synergistically to abolish the induction of FLE antibodies.

[0179] [Table 11]

[0180] Example 19 Construction of MutA, MutB, and MutC mutant DENV vaccines

[0181] How to avoid ADE in the design of DENV vaccines remains an open question (REF), as the FL sequences of ZIKV and DENV are highly conserved. The above systematic study demonstrated that AdC7-M / E-MutB and AdC7-M / E-MutC adenovirus vaccines can avoid ADE against DENV while providing complete protection to mice. Therefore, MutA, MutB, and MutC antigens are highly conserved. By constructing a mutated DENV vaccine, we verified whether the vaccine would have the same effect.

[0182] First, the signal peptide gene (SEQ ID NO. 17) derived from Japanese encephalitis virus (JEV) and the M / E gene (SEQ ID NO. 18) expressing the DENV2 New Guinea C virus strain (GenBank: KM204118.1) were constructed into the pshuttle vector, resulting in the plasmid pshuttle-DV2-M / E-WT expressing wild-type DENV2 M / E. The signal peptide gene (SEQ ID NO. 17) derived from JEV and the gene expressing the prM / E protein of the DENV2 New Guinea C virus strain (SEQ ID NO. 19) were constructed into the pshuttle vector, resulting in the plasmid pshuttle-DV2-prM / E-WT expressing wild-type DENV2 prM / E.

[0183] All mutants were constructed based on the M / E-WT antigen. MutA (D98N, N103T, G106F, L107E, and F108W), MutB (D98N, N103T, G106F, L107K, and F108W), and MutC (D98N, N103T, G106L, L107E, and F108W) mutant plasmids were constructed using the pshuttle-DV2-M / E-WT plasmid as a template. The primer sequences used during construction are shown in Table 9.

[0184] [Table 12]

[0185] The wild-type protein-expressing plasmids pshuttle-DV2-M / E-WT and pshuttle-DV2-prM / E-WT, as well as the three mutant plasmids, were transfected into 293T cells. 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 in DMEM medium, and washed once more with DMEM medium. Next, the cells were transfected with BD Fixation and Permeabilization solution was added and the cells were placed on ice for 20 minutes. Cells were then collected by centrifugation at 800 g for 10 minutes and washed twice with BD's 1x Perm / Wash buffer. Each sample was divided into five portions, and the Z6, 2A10G6, and mAb11 antibodies, which bind to FL epitopes, and the mAb513 and D448 antibodies, which bind to non-FL epitopes, were added, respectively, and the cells were placed in a refrigerator at 4°C for 1 hour. The cells were collected by centrifugation and washed twice with 1x Perm / Wash buffer. Goat Anti-Human FITC (Proteintech, 00003-12) antibody was then added and the cells were placed in a refrigerator at 4°C for 1 hour. The cells were collected by centrifugation and washed twice with 1x Perm / Wash buffer. The cells were resuspended in PBS (200 μl / well) and the positive rates of the samples were detected using a flow cytometer. The experimental results are shown in Figure 36.

[0186] As shown in Figure 36, antibodies Z6 and 2A10G6, which bind to the FL epitope, can recognize the DENV2 M / E-WT and prM / E-WT proteins. However, when the three mutation combinations were introduced into the FL epitope of the DENV2 E protein, the binding ability of the Z6 and 2A10G6 antibodies to the DENV2 M / E-MutA, M / E-MutB, or M / E-MutC proteins was significantly reduced or even eliminated. The mAb513 and D448 antibodies, which bind to non-FL epitopes and have higher neutralizing activity, were still able to bind to the DENV2 M / E-MutA, M / E-MutB, or M / E-MutC proteins containing the MutA, MutB, and MutC mutations. This indicates that the mutations in the FL epitope of the DENV2 E protein did not significantly affect other epitopes. These results suggest that this combination of three mutations can be used in DENV vaccines. A vaccine derived from a DENV2 E protein antigen with a combination of mutations at positions G106, L107, and F108 can reduce the ADE phenomenon caused by subsequent DENV virus infection after immunization with the vaccine.

[0187] Example 20: Detection of the effect on antigen activity after mutating ZIKV E W101 to 19 other amino acids

[0188] In the structures of Z6 / ZIKV sE and 2A10G6 / ZIKV sE, the amino acid with the greatest interaction between the E protein and antibodies is W101. It has also been reported that the binding of most FLE antibodies to the E protein is dependent on W101 (Non-Patent Document 28). Therefore, we mutated W101 to 19 other amino acids and then used flow cytometry to detect whether the epitope on which the cells express the antigen changed, thereby screening for the most optimal mutations.

[0189] The JEV signal peptide gene (SEQ ID NO. 17) and wild-type ZIKV M / E gene (SEQ ID NO. 31) were inserted into the pCAGGS vector (Addgene) to obtain the plasmid pCAGGS-M / E-WT, which expresses the wild-type ZIKV M / E protein. Using this plasmid as a template, tryptophan at position 101 of the E protein was mutated to one of 19 other amino acids. For example, tryptophan to alanine mutation was performed by PCR using the pCAGGS-M / E-WT plasmid as a template and primers W101-WT-F and W101-1A-R to obtain product W101-1A-1. The primer sequences are listed in Table 10. Using the pCAGGS-M / E-WT plasmid as a template and primers W101-WT-R and W101-1A-F, product W101-1A-2 was obtained by PCR. Furthermore, a mixture of W101-1A-1 and W101-1A-2 in a molar ratio of 1:1 was used as a template, and PCR was performed using W101-WT-F and W101-WT-R as primers to obtain the PCR product W101-1A. The CAGGS vector was digested with restriction enzymes XhoI (Thermo, FD0694) and EcoRI (Thermo, FD0274) to obtain a linearized plasmid with both cohesive ends. The digested linearized plasmid and W101-1A were mixed at a molar ratio of 1:5 and recombined using the In-Fusion kit (Takara, 639648). The recombinant product was transformed into DH5α competent cells, plated on ampicillin-resistant plates, and cultured at 37°C. The cells were selected, cloned, and subjected to PCR identification and sequencing.

[0190] [Table 13]

[0191] [Table 14]

[0192] After extracting the 19 W101 mutant plasmids, the wild-type and 19 mutant plasmids were transfected into 293T cells. After 48 hours, the supernatant was removed, and the cells were washed once with PBS. Subsequently, the cells were digested into single cells with trypsin, centrifuged, resuspended in DMEM medium, and washed once more with DMEM medium. BD Fixation and Permeabilization solution was added and the cells were placed on ice for 20 minutes. Cells were then harvested by centrifugation at 800g for 10 minutes and washed twice with BD 1x Perm / Wash buffer. Each sample was divided into five, and Z6 and 2A10G6 antibodies were added, respectively, and the cells were placed in a refrigerator at 4°C for 1 hour. The cells were then harvested by centrifugation and washed twice with 1x Perm / Wash buffer. Goat Anti-Human FITC (Proteintech, 00003-12) antibody was added and the cells were placed in a refrigerator at 4°C for 1 hour. The cells were collected by centrifugation and washed twice with 1x Perm / Wash buffer. The cells were resuspended in PBS (200 μl / well), and the positive rate of the samples was detected using a flow cytometer. The experimental results are shown in Figure 37.

[0193] As can be seen from Figure 37, the ZIKV wild-type M / E antigen can bind to the Z6 and 2A10G6 antibodies, but none of the 19 mutants can bind to the Z6 and 2A10G6 antibodies at all. This indicates that tryptophan at position 101 of the ZIKV E protein is important for the activation of antibodies targeting FL, and vaccines prepared by mutating the W101 site reduce the production of antibodies induced by the FL epitope. Alternatively, it is possible to avoid causing ADE phenomenon against DENV after immunization with a vaccine.

[0194] Example 21: Detection of the binding ability of mutations at G106, L107, and F108 sites of ZIKV E protein to FLE antibodies

[0195] Based on the pCAGGS-ZIKV-M / E expression plasmid, the following single-site and double-site point mutations were made. The construction method of the mutant plasmids is described in Example 20. The primers used in constructing the mutant plasmids are listed in Table 11.

[0196] [Table 15]

[0197] [Table 16]

[0198] [Table 17]

[0199] The wild-type plasmid pCAGGS-ZIKV-M / E-WT and the constructed mutant plasmids were transfected into 293T cells. After 48 hours, the cells were harvested, digested into single cells, fixed, permeabilized, and incubated with ADE antibodies Z6 and 2A10G6, which bind to the FL epitope. They were then incubated with a goat anti-human (mouse) FITC secondary antibody. Finally, the positive rate of the samples was detected using a flow cytometer. A positive rate of less than 10% of the wild-type positive rate was considered non-binding, and a positive rate of 10% to 50% was considered weak binding. The results are shown in Table 12.

[0200] As can be seen from Table 12, the above mutations can essentially inhibit the binding of representative ADE antibodies (Z6 antibody and 2A10G6 antibody) to the FL epitope. As can be seen from this, vaccines made with single or synergistic mutations at the G106, L107, and F108 sites represented by these mutations can reduce or avoid ADE antibodies induced by the FL epitope, and avoid the ADE phenomenon against DENV after immunization with the vaccine.

[0201] [Table 18]

[0202] Example 22: Detection of the binding ability of mutations at G106, L107, and F108 sites of DENV E protein to FLE antibodies

[0203] Based on the above pCAGGS-DENV2-M / E expression plasmid, the following single-site and double-site point mutations were made. The construction method of the mutant plasmids is shown in Example 19. The primers used in the process of constructing the mutant plasmids are listed in Table 13.

[0204] [Table 19]

[0205] [Table 20]

[0206] [Table 21]

[0207] The wild-type plasmids pCAGGS-ZIKV-M / E-WT and pCAGGS-DENV2-M / E-WT, as well as the mutant constructs listed above, were transfected into 293T cells. After 48 hours, the cells were harvested, digested into single cells, fixed, permeabilized, and incubated with the ADE antibodies Z6 and 2A10G6, which bind to the FL epitope. They were then incubated with a goat anti-human (mouse) FITC secondary antibody. Finally, the positive rates of the samples were detected using a flow cytometer. A positive rate of less than 10% of the wild-type positive rate was considered non-binding, and a positive rate of 10% to 50% was considered weak binding. The results are shown in Table 14.

[0208] As can be seen from Table 14, the above mutations can essentially inhibit the binding of representative ADE antibodies to the FL epitope. As can be seen from this, vaccines made with single or synergistic mutations of G106, L107, and F108, represented by these mutations, can reduce or avoid ADE antibodies induced by the FL epitope, and avoid the ADE phenomenon against DENV after immunization with the vaccine.

[0209] [Table 22]

[0210] Finally, it should be noted that the above embodiments are used to explain the technical solutions of the present invention, and are not limiting. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or make equivalent substitutions for some of the technical features thereof, and such modifications and substitutions will not deviate from the spirit and scope of the technical solutions of the embodiments of the present invention. [Industrial Applicability]

[0211] Embodiments of the present invention relate to Zika-dengue vaccines and uses thereof.

[0212] The present invention provides epitope information of the major antibodies that cause the ADE phenomenon based on crystal structure analysis and other structural and functional analyses. Mutations are introduced into the FL fusion region of the E protein of dengue virus or flu virus, and antigens with these mutations cannot bind to antibodies that cause ADE (FLE antibodies). Vaccines obtained from the antigens provided by the present invention can avoid the production of antibodies induced by FL epitopes after immunization, thereby reducing or eliminating the ADE phenomenon.

Claims

1. An antigen having an E protein of Zika virus, The amino acid sequence of the Zika virus E protein is the amino acid sequence shown in SEQ ID NO. 1, The Zika virus E protein is (1) D98N / N103T / G106F / L107K / F108W, (2) D98N / N103T / G106L / L107E / F108W, An antigen having one of the mutations.

2. The antigen of claim 1, further comprising the full-length sequence of the M protein of Zika virus.

3. The antigen of claim 1, further comprising the full-length sequence of the prM protein of Zika virus.

4. A polynucleotide encoding the antigen according to any one of claims 1 to 3.

5. An expression cassette comprising the polynucleotide of claim 4.

6. A recombinant vector comprising the polynucleotide of claim 4.

7. A transgenic cell line comprising the polynucleotide of claim 4.

8. A recombinant bacterium containing the polynucleotide of claim 4.

9. A recombinant adenovirus comprising the polynucleotide of claim 4.

10. A recombinant lentivirus comprising the polynucleotide of claim 4.

11. A recombinant viral particle comprising the polynucleotide of claim 4.

12. An mRNA encoding the antigen according to any one of claims 1 to 3.

13. A vaccine comprising, as an active ingredient, an antigen according to any one of claims 1 to 3, a polynucleotide according to claim 4, an expression cassette according to claim 5, a recombinant vector according to claim 6, a transgenic cell line according to claim 7, a recombinant bacterium according to claim 8, a recombinant adenovirus according to claim 9, a recombinant lentivirus according to claim 10, a recombinant viral particle according to claim 11, or an mRNA according to claim 12.

14. The vaccine comprises one or more of an inactivated vaccine, an attenuated vaccine, a DNA vaccine, an mRNA vaccine, an adenovirus vaccine, another viral vector vaccine, a subunit vaccine, and a viral particle; and / or 14. The vaccine of claim 13, further comprising a pharmaceutically or veterinarily acceptable vehicle, diluent, adjuvant or excipient.

15. 15. The vaccine of claim 14, wherein the vaccine comprises an adenovirus vaccine.

16. the recombinant vector comprises packaged chimpanzee adenovirus type 7; The recombinant vector comprises a backbone and a nucleic acid sequence comprising a target gene inserted into the backbone; the backbone is a replication-deficient chimpanzee adenovirus type 7 vector; A recombinant adenovirus vaccine, characterized in that the target gene is the polynucleotide of claim 4 or the mRNA of claim 12.

17. Use of the antigen according to any one of claims 1 to 3, the polynucleotide according to claim 4, the expression cassette according to claim 5, the recombinant vector according to claim 6, the transgenic cell line according to claim 7, the recombinant bacterium according to claim 8, the recombinant adenovirus according to claim 9, the recombinant lentivirus according to claim 10, the recombinant viral particle according to claim 11, or the mRNA according to claim 12 in the preparation of a vaccine for preventing and / or treating Zika virus infection, or in the preparation of a detection reagent or kit for detecting Zika virus infection.

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