Dengue and / or zika virus genetically engineered vaccine and preparation method and application thereof

Abstract

This invention provides a dengue / Zika virus genetically engineered vaccine and its application. The dengue / Zika virus vaccine comprises an open reading frame encoding envelope protein domain III (EDIII) and the non-structural protein NS1, and displays EDIII monomers in the delivery vector shell. Immunization with this vaccine primarily induces a type-specific antibody response, reducing the production of cross-antibodies and thus effectively avoiding or eliminating the risk of antibody-dependent enhancement of infection (ADE). This vaccine can be used to prevent dengue virus and Zika virus infection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and relates to vaccines, specifically to a dengue and / or Zika virus genetically engineered vaccine, its preparation method, and its application. Background Technology

[0002] Dengue virus (DENV) and Zika virus (ZIKV) both belong to the genus Flavivirus of the family Flaviviridae and are transmitted by mosquitoes such as Aedes aegypti and Aedes albopictus. There are four serotypes of dengue virus (named dengue 1, 2, 3, and 4, i.e., DENV-1, 2, 3, and 4) that can infect humans. Primary infection usually presents with mild symptoms, but reinfection with different dengue virus serotypes can lead to severe dengue fever (dengue hemorrhagic fever, dengue shock syndrome). In recent years, the global dengue epidemic has become increasingly serious. The World Health Organization (WHO) estimates that approximately 390 million people are infected globally each year, with over 6.5 million reported cases in 2023 and over 13 million in 2024. Zika virus is closely related to dengue virus, possessing only one serotype but containing two genotypes: the African lineage and the Asian lineage. Zika virus infection in adults can cause Guillain-Barré syndrome (GBS), while infection in pregnant women can cause microcephaly in newborns. From 2013 to 2016, the Zika virus caused a large-scale outbreak in the Pacific Islands, the Americas, and Southeast Asia, infecting millions of people and affecting 89 countries and regions worldwide. Subsequently, the Zika epidemic gradually subsided, but sporadic outbreaks still occur.

[0003] Similar to Zika virus, dengue virus particles are approximately icosahedral in shape and have an envelope containing envelope proteins (E). The viral genome is a single-stranded positive-sense RNA, approximately 11 kb in length, encoding a large polyprotein that, after translation, can be cleaved into three structural proteins (C, prM / M, E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). The E protein contains 504 amino acids and exists as a dimer in mature particles. The E monomer consists of three domains: EDI, EDII, and EDIII. The EDII head contains a highly conserved fusion loop (FL), which is sequence-identical between dengue and Zika viruses and primarily mediates the fusion of the viral envelope with the endosome membrane. EDIII, however, varies considerably among dengue serotypes and Zika viruses and primarily mediates the binding of viral particles to cell surface receptors. The pre-membrane protein prM acts as a molecular chaperone, assisting the E protein in folding to the correct conformation and forming a heterodimer with E. prM also maintains the stability of the E protein. In the early stages of viral assembly, E and prM form a dimer in the endoplasmic reticulum, covering the surface of immature viral particles. Upon transport to the Golgi apparatus, the low pH environment causes prM-E rearrangement, exposing furin cleavage sites. prM is then cleaved by furin into pr and M proteins. Therefore, in immature viral particles, the pr peptide is located at the tip of E, concealing the fusion peptide of E. After viral particle maturation, the pr peptide does not immediately dissociate to protect the fusion peptide in its pre-fusion conformation under acidic conditions. During release, the viral particle is exposed to a neutral pH environment, at which point the pr peptide dissociates, and the viral particle fully matures. It is generally believed that only partially or fully mature viral particles have the ability to infect host cells in the absence of antibodies.

[0004] Currently, there are no specific drugs against dengue virus or Zika virus globally. Only two quadrivalent dengue vaccines are approved for use: Dengvaxia and TAK-003. The Dengvaxia vaccine uses the yellow fever virus vaccine strain YF-17D as its backbone, incorporating the pre-membrane protein prM and envelope protein E of dengue virus types 1 to 4. Clinical trials and real-world data show that this vaccine exhibits uneven immunization, poor protection against DENV-2, and a risk of exacerbating infection in previously uninfected individuals; therefore, its use is limited to individuals who are positive for dengue antibodies. The TAK-003 vaccine uses attenuated dengue virus type 2 as its backbone, incorporating the prM / M and E antigens of dengue virus types 1, 3, and 4. Clinical trial results show that the overall protection rate after 3 years of vaccination is only 62%, exhibiting uneven immunization and a tendency to promote DENV-3 infection and severe illness in dengue antibody-negative individuals. Furthermore, both vaccines are chimeric attenuated live vaccines, posing safety risks in immunocompromised individuals; they also require 2 or 3 doses, making widespread use inconvenient. It is evident that the existing dengue vaccine provides insufficient protection and carries the risk of inducing antibody-dependent dengue virus enhancement (ADE).

[0005] Dengue / Zika virus vaccines under investigation include subunit vaccines, nanoparticle vaccines, recombinant viral vector vaccines, and mRNA vaccines. These vaccines primarily use prM / M and E as the main antigens, aiming to induce the body to produce a neutralizing antibody response against E. However, these vaccines also produce non-neutralizing or weakly neutralizing antibodies targeting prM and E. Since certain regions of prM and E are highly conserved between dengue and Zika viruses, these antibodies often exhibit cross-reactivity. Furthermore, their neutralizing activity is often weak, failing to block viral invasion and instead promoting viral infection through FcγR receptors on the target cell surface after forming virus-antibody complexes, thus leading to adverse drug reactions (ADE). Antibodies known to pose an ADE risk primarily target the pr peptide and the EDII fusion loop. To mitigate the production of ADE antibodies, researchers have attempted various techniques: 1) avoiding the use of pr; 2) mutating the fusion loop region; 3) introducing mutations into the E protein to mask the ADE antibody epitope; etc. However, these strategies are still insufficient to completely prevent the production of ADE antibodies. Antibodies against other conserved epitopes within the M protein, EDI, and EDII may still induce ADE. Some researchers have also attempted to use EDIII as an antigen to induce type-specific neutralizing antibody responses, but EDIII has weak immunogenicity, and EDIII-based vaccines often fail to provide complete protection.

[0006] Researchers have also explored the feasibility of using non-structural proteins such as NS1 and NS3 as vaccine antigens. NS1 is a key pathogenic factor for dengue and Zika viruses. During the viral life cycle, NS1 exists in three forms: monomer, dimer, and hexamer. Dimeric NS1 is mainly located in the endoplasmic reticulum and cell membrane, and can assist in the formation of viral replication complexes in the endoplasmic reticulum. Hexamer NS1 is secreted extracellularly, and can regulate immune responses and promote endothelial cell permeability. NS1emia is closely related to the severity of dengue fever. Antibody responses against NS1 can exert protective effects by blocking NS1 toxicity and mediating ADCC. NS3 has protease and helicase activities, playing a key role in viral replication and transcription. NS3 contains T-cell dominant epitopes, and vaccines using NS3 as an antigen have also shown some protective effects. However, using only NS1 and NS3 as antigens, or combining natural EDIII and NS1 antigens, is insufficient to achieve complete protection. Summary of the Invention

[0007] Based on this, the purpose of this invention is to provide a dengue and / or Zika virus vaccine without ADE risk and its application. By screening the expression forms and display methods of dengue and / or Zika virus EDIII, this invention obtains an antigen combination that can significantly enhance EDIII immunogenicity and a corresponding vaccine, namely, an open reading frame encoding the EDIII dimer is integrated into the genome of the delivery vector, while the EDIII protein is displayed in the shell of the delivery vector.

[0008] The vaccine provided by this invention also integrates an open reading frame encoding the NS1 antigen into the genome of the delivery vector. Immunization with this vaccine generates a protective immune response against dengue and Zika viruses, but does not induce the antidepressant effect (ADE). This vaccine is suitable for multiple routes of administration, including intramuscular injection, nasal spray, and oral administration.

[0009] In a first aspect, the present invention provides an antigen combination selected from at least one combination of groups 1)-3):

[0010] 1) a antigen: The EDIII domain of dengue virus type 1 fused to the C' terminus with a dimerized tag, the amino acid sequence of which is shown in SEQ ID NO: 10, or its encoding DNA sequence as shown in SEQ ID NO: 1.

[0011] b antigen: The NS1 protein of dengue virus type 1 with a signal peptide fused to its N' terminus, the amino acid sequence of which is shown in SEQ ID NO: 11, or its encoding DNA sequence as shown in SEQ ID NO: 2, and

[0012] c antigen: EDIII monomer of dengue virus type 1 fused with N'-terminal pairing linker peptide, said EDIII monomer as shown in SEQ ID NO: 7;

[0013] 2) a antigen: The EDIII domain of dengue virus type 2 fused to the C' terminus with a dimerized tag, the amino acid sequence of which is shown in SEQ ID NO: 12, or its coding sequence as shown in SEQ ID NO: 3.

[0014] b antigen: The NS1 protein of dengue virus type 2 with a signal peptide fused to its N' terminus, the amino acid sequence of which is shown in SEQ ID NO: 13, or its coding sequence as shown in SEQ ID NO: 4, and

[0015] c antigen: EDIII monomer of dengue virus type 2 fused with N'-terminal pairing linker peptide, said EDIII monomer as shown in SEQ ID NO: 8;

[0016] 3) a antigen: The EDIII domain of Zika virus with a dimerized tag fused to its C' terminus, the amino acid sequence of which is shown in SEQ ID NO: 14, or its coding sequence as shown in SEQ ID NO: 5.

[0017] b antigen: The Zika virus NS1 protein with a signal peptide fused to its N' terminus, the amino acid sequence of which is shown in SEQ ID NO: 15, or its coding sequence as shown in SEQ ID NO: 6, and

[0018] c antigen: Zika virus EDIII monomer with a pairing linker peptide fused to its N' terminus, said EDIII monomer as shown in SEQ ID NO: 9;

[0019] The pairing linker peptide is used to link the EDIII monomer to a carrier of either the a antigen or the b antigen.

[0020] In some embodiments, the pairing linker peptide is a DogCatcher, SpyCatcher, or SnoopCatcher that can pair with a DogTag, SpyTag, or Snooptag.

[0021] In some embodiments, the coding sequences of the a antigen and the b antigen are encoded by the same open reading frame and are linked by a sequence encoding a self-cleaving linker peptide, or are encoded by two separate open reading frames.

[0022] In some embodiments, the self-cleaving linker peptide may be a 2A linker peptide.

[0023] In some embodiments, the antigen combination includes antigen combinations corresponding to dengue virus type 1, such as group 1) above, and / or antigen combinations corresponding to dengue virus type 2, such as group 2) above.

[0024] In some embodiments, the antigen combination also includes an antigen combination against Zika, as described in group 3) above.

[0025] In a second aspect, the present invention provides a nucleic acid molecule for encoding combinations of antigens as described in any of the first aspects.

[0026] In some embodiments, the nucleic acid molecule is DNA encoding an antigen, or it may be mRNA or circular RNA encoding an antigen.

[0027] Thirdly, the present invention provides a vector comprising an expression vector and a display vector, wherein the expression vector carries a nucleic acid molecule encoding the a antigen and / or the b antigen; and the display vector displays one or a combination of amino acid sequences represented by the c antigen on its surface; the expression vector and the display vector are the same vector or two different vectors.

[0028] In some implementations, the expression vector is a recombinant viral vector, an RNA vector, or a DNA vector, with the recombinant viral vector preferably being a replication-defective adenovirus vector.

[0029] In some implementations, the replication-defective adenovirus vector is a replication-defective type 5 adenovirus vector (Ad5), and the open reading frame of the nucleic acid molecule is inserted into the E1 or E3 region of the Ad5 vector.

[0030] In some implementations, the RNA vector is a circular RNA or mRNA.

[0031] In some implementations, the expression vector and the display vector are the same vector, more preferably an adenovirus vector.

[0032] Fourthly, the present invention provides the use of the antigen combination described in the first aspect, or the nucleic acid molecule described in the second aspect, or the vector described in the third aspect in the preparation of dengue virus and / or Zika virus vaccines.

[0033] Fifthly, the present invention provides a vaccine, characterized in that its active ingredient comprises the antigen combination described in one aspect, or the nucleic acid molecule described in the second aspect, or the carrier described in the third aspect.

[0034] In some implementations, pharmaceutically acceptable diluents, excipients, adjuvants, or drug carriers for delivery into cells are also included.

[0035] Sixthly, the present invention provides a method for preparing the vaccine described in the fifth aspect, comprising the following steps:

[0036] S1.DogTag was used as a surface linker peptide for the display vector. Its encoding DNA sequence was inserted into the pGK5 plasmid. Through homologous recombination in E. coli, this DNA sequence was inserted into the HVR1, 2, and 5 regions of the pAd5ΔE1ΔE3 genome plasmid, resulting in pAd5ΔE1ΔE3-Hm1, pAd5ΔE1ΔE3-Hm2, and pAd5ΔE1ΔE3-Hm5 plasmids.

[0037] S2. In the E1 / E3 region of adenovirus, open reading frames encoding antigens a and b are integrated into the shuttle plasmids pGA1 or pGK53, respectively, and homologous recombination is performed with plasmids pAd5ΔE1ΔE3-Hm1, pAd5ΔE1ΔE3-Hm2, and pAd5ΔE1ΔE3-Hm5 to obtain the pAd5(EfcN) plasmid carrying the EDIII dimer and NS1 open reading frame;

[0038] S3. The obtained pAd5(EfcN) plasmid was linearized and transfected into HEK293 cells to rescue and produce Ad(EfcN) recombinant virus;

[0039] S4. Link antigen c expressed in vitro by Escherichia coli or mammalian cells to the surface of the obtained Ad(EfcN) recombinant virus.

[0040] In a seventh aspect, the present invention provides the use of the vaccine described in the fifth aspect in the preparation of a medicament for the prevention of dengue virus and / or Zika virus infection.

[0041] In some implementations, step S2 includes:

[0042] S2.1 The DNA coding sequences of the EDIII dimer of dengue virus type 1, dengue virus type 2, and Zika virus are respectively constructed into the E1 region shuttle plasmid to obtain shuttle plasmids carrying the open reading frames of the EDIII dimer.

[0043] S2.2 After the shuttle plasmid is linearized by double enzyme digestion, it is homologously recombinated with the linearized pAd5ΔE1ΔE3-Hm5 plasmid to obtain pAd5(Efc) carrying the open reading frame of the viral EDIII dimer.

[0044] S2.3 The DNA coding sequences of NS1 of dengue virus type 1, dengue virus type 2, and Zika virus are respectively constructed into the E3 region shuttle plasmid pGK53 to obtain pGK53-NS1 shuttle plasmids carrying the NS1 open reading frame.

[0045] After double digestion and linearization of the S2.43 pGK53-NS1 shuttle plasmid, the linearized pAd5(Efc) was subjected to homologous recombination to obtain the pAd5(EfcN) plasmid carrying the EDIII dimer and NS1 open reading frame.

[0046] In some embodiments, the coupling ratio of the antigen c to the surface of the recombinant virus Ad (EfcN) is 20%-90%, preferably 30%-50%, and most preferably 40%.

[0047] In some embodiments, in step S4, the purified Ad(EfcN) recombinant virus is mixed with the EDIII monomer in the following ratio, calculated according to the number of Hexon protein monomers and the number of EDIII monomers: 1:0.25-3; preferably: 1:0.25-1.

[0048] Eighthly, this application provides a method for preventing or treating dengue virus and / or Zika virus infection, the method comprising administering an effective amount of the vaccine to an individual.

[0049] In some implementations, the administration method includes at least one of intramuscular injection, nasal drop, and oral administration.

[0050] In some embodiments, the administered vaccines include a group of vaccines corresponding to dengue virus type 1 (with antigen combinations as described in 1 above) and / or a group of vaccines corresponding to dengue virus type 2 (with antigen combinations as described in 2 above). Preferably, when the two vaccines are used in combination, the dosage of each is 1:1.

[0051] In some implementations, the administration of the vaccine also includes a vaccine against Zika, with the antigen combination as described in group 3) above.

[0052] The present invention has the following beneficial effects:

[0053] (1) The antigen combination provided by this invention can induce strong neutralizing and protective antibody responses. After EDIII dimerization, its ability to induce neutralizing antibodies is significantly enhanced compared to the traditional monomeric form. After displaying EDIII on the vector surface, the neutralizing antibody response is further enhanced. The antibody levels induced by the vaccine expressing EDIII and NS1 and displaying EDIII are at least 100 times higher than those of the EDIII subunit vaccine and nearly 10 times higher than those of the vaccine expressing only EDIII and NS1. The vaccine carrying the antigen combination can completely protect the recipient from dengue virus and Zika virus infection.

[0054] (2) The vaccine provided by this invention effectively avoids the generation of ADE antibodies. The antigen combination designed in this invention does not contain a fusion loop or prM component, and will not generate cross-non-neutralizing antibodies against these two immunodominant epitopes. The antigen combination does not contain the EDI and EDII regions, and will not generate antibodies against these two regions, further reducing the risk of ADE. Antibodies generated against the NS1 antigen have a certain protective effect, and since the NS1 antigen does not appear on the surface of virus particles, NS1 antibodies will not induce the risk of ADE. Since EDIII has low similarity among various dengue virus serotypes and Zika virus, the antibodies it induces are mainly type-specific antibodies, and the risk of ADE is low.

[0055] This invention demonstrates that the Zika virus vaccine using this antigen combination primarily induces Zika virus-specific antibodies, with no adverse drug reaction (ADE) against dengue virus types 1-4; the dengue virus type 1 vaccine using this antigen combination primarily induces dengue virus type 1-specific antibodies, with no ADE against dengue virus types 2-4 and Zika virus; the dengue virus type 2 vaccine using this antigen combination primarily induces dengue virus type 2-specific antibodies, with no ADE against dengue virus types 1, 3, 4 and Zika virus. The type-specific antibody titers induced by these vaccines are tens to 100 times higher than the cross-antibody titers.

[0056] (3) This invention creatively provides nasal and oral administration methods for mosquito-borne flavivirus vaccines. Existing dengue and other flavivirus vaccines are administered via intramuscular injection, which relies on professional medical personnel, is costly, and has relatively significant side effects, hindering widespread adoption. This invention is the first to demonstrate that dengue and Zika virus vaccines, administered via nasal spray or orally, can induce a sufficiently effective protective immune response, making it highly valuable for widespread application. Areas where dengue, Zika, and other viruses are prevalent are typically economically underdeveloped regions; therefore, the aforementioned vaccines are of great significance to public health in these areas. Attached Figure Description

[0057] Figure 1 This is a schematic diagram of the gene structure of the EDIII dimer and NS1 antigen combination.

[0058] Figure 2 This is a flowchart illustrating the construction and production process of an adenovirus vector vaccine carrying the antigen combination and displaying the EDIII antigen.

[0059] Figure 3 The results are SDS-PAGE gel electrophoresis and Western blot analysis of Ad(EN)-E virus particles carrying Zika virus antigen combinations after lysis.

[0060] Figure 4The results are obtained by transmission electron microscopy observation of Ad(EN)-E virus particles carrying Zika virus antigen combinations after labeling with EDIII antibody and colloidal gold secondary antibody.

[0061] Figure 5 This refers to the immune response induced in C57BL / 6 mice after intramuscular injection of vaccines such as Ad(E)-E carrying Zika virus antigens. Here, A represents the titer of Zika virus EDIII-specific IgG antibodies, B represents the titer of Zika virus neutralizing antibodies, and C represents the intensity of the Zika virus-specific T-cell immune response.

[0062] Figure 6 This study describes the results of immunizing C57BL / 6 mother mice with a vaccine such as Ad(E)-E carrying Zika virus antigen via intramuscular injection, followed by challenging the offspring of these immunized mother mice with Zika virus. Figure A shows the weight growth curve of the offspring after challenge, Figure B shows the neurological symptoms of the offspring on day 15 after challenge, and Figure C shows the weight of the brain tissue of the offspring after dissection on day 15 after challenge.

[0063] Figure 7 This diagram shows the expression of Zika virus EDIII and NS1 antigens in HEK293 cells and their culture supernatant after infection with Ad(EfcN) carrying the Zika virus antigen combination. A represents the results of EDIII monomer and dimer assays, and B represents the results of NS1 protein assays.

[0064] Figure 8 This refers to the antibody response induced in C57BL / 6 mice after intramuscular injection of vaccines such as Ad(EfcN)-E carrying Zika virus antigen combinations. Here, A represents the Zika virus EDIII-specific IgG antibody titer, B represents the Zika virus NS1-specific IgG antibody titer, and C represents the Zika virus neutralizing antibody titer.

[0065] Figure 9 This study describes the results of immunizing C57BL / 6 mother mice with a vaccine such as Ad(EfcN)-E carrying the Zika virus antigen combination via intramuscular injection, followed by challenging the offspring of these immunized mother mice with Zika virus. In the figures, A represents the weight growth curve of the offspring after challenge, B represents the neurological symptoms of the offspring on day 15 post-challenge, and C represents the Zika virus genome copy number in the brain tissue of the offspring on day 15 post-challenge.

[0066] Figure 10 IFNaR is administered via intramuscular injection of an Ad(EfcN)-E or Ad(EfcN) vaccine carrying a combination of dengue virus type 1 antigens. - / - The antibody response induced in C57BL / 6 mice. A represents the titer of dengue virus type 1 EDIII-specific IgG antibody, B represents the titer of dengue virus type 1 NS1-specific IgG antibody, and C represents the titer of dengue virus type 1 neutralizing antibody.

[0067] Figure 11 IFNaR is administered via intramuscular injection of an Ad(EfcN)-E or Ad(EfcN) vaccine carrying a combination of dengue virus type 1 antigens. - / - The results of challenging C57BL / 6 mice with dengue virus type 1. A represents the weight change curve of the mice after challenge, and B represents the viral load in the peripheral blood of the mice on days 1, 4, and 7 after challenge.

[0068] Figure 12 IFNaR is administered via intramuscular injection of an Ad(EfcN)-E or Ad(EfcN) vaccine carrying a combination of dengue virus type 2 antigens. - / - The antibody response induced in C57BL / 6 mice. A represents the titer of dengue virus type 2 EDIII-specific IgG antibody, B represents the titer of dengue virus type 2 NS1-specific IgG antibody, and C represents the titer of dengue virus type 2 neutralizing antibody.

[0069] Figure 13 IFNaR is administered via intramuscular injection of an Ad(EfcN)-E or Ad(EfcN) vaccine carrying a combination of dengue virus type 2 antigens. - / - The results of challenging C57BL / 6 mice with dengue virus type 2 were shown. Figure A shows the weight change curve of the mice after challenge, and Figure B shows the viral load in the peripheral blood of the mice on days 1, 4, and 7 after challenge.

[0070] Figure 14 The cross-reactivity of antibodies induced in C57BL / 6 mice after intramuscular injection of vaccines such as Ad(EfcN)-E carrying Zika virus antigen combinations with dengue virus serotypes and Zika virus is determined.

[0071] Figure 15 The cross-reactivity of antibodies induced in C57BL / 6 mice after intramuscular injection of an Ad(EfcN)-E vaccine carrying a combination of dengue virus type 1 or 2 antigens with dengue virus against various serotypes and Zika virus was determined.

[0072] Figure 16 This presents the ADE (Aspect-dependent enhancement) effect of Ad(EfcN)-E Zika virus vaccine immune sera against various dengue virus serotypes and Zika virus in cell lines. Specifically, A represents the ADE effect of each immune sera against Zika virus at different dilutions; B represents the ADE effect of each immune sera against dengue virus type 1; C represents the ADE effect of each immune sera against dengue virus type 2; D represents the ADE effect of each immune sera against dengue virus type 3; and E represents the ADE effect of each immune sera against dengue virus type 4.

[0073] Figure 17 It is the Ad(EfcN)-E Zika virus vaccine immune serum in IFNaR- / - The ADE effect of the C57BL / 6 mouse model on dengue virus type 2 was detected. A represents the survival curve of mice after challenge, and B represents the viral load in mouse plasma on days 1, 4, and 7 after challenge.

[0074] Figure 18 This presents the ADE (Aspect-dependent enhancement) effect of Ad(EfcN)-E dengue type 1 vaccine immune sera against various dengue virus serotypes and Zika virus in cell lines. Specifically, A represents the ADE effect of each immune sera against Zika virus at different dilutions; B represents the ADE effect of each immune sera against dengue virus type 1; C represents the ADE effect of each immune sera against dengue virus type 2; D represents the ADE effect of each immune sera against dengue virus type 3; and E represents the ADE effect of each immune sera against dengue virus type 4.

[0075] Figure 19 It is the Ad(EfcN)-E dengue type 1 vaccine immune serum in IFNaR - / - The ADE effect of the C57BL / 6 mouse model on dengue virus type 2 was detected. A represents the survival curve of mice after challenge, and B represents the viral load in mouse plasma on days 1, 4, and 7 after challenge.

[0076] Figure 20 This presents the ADE (Aspect-dependent enhancement) effect of Ad(EfcN)-E dengue type 2 vaccine immune sera against various dengue virus serotypes and Zika virus in cell lines. Specifically, A represents the ADE effect of each immune sera against Zika virus at different dilutions, B represents the ADE effect of each immune sera against dengue virus type 1, C represents the ADE effect of each immune sera against dengue virus type 2, D represents the ADE effect of each immune sera against dengue virus type 3, and E represents the ADE effect of each immune sera against dengue virus type 4.

[0077] Figure 21 It is the Ad(EfcN)-E dengue type 1 and 2 vaccine immune serum in IFNaR - / - The results of ADE detection of Zika virus in the C57BL / 6 mouse model. A represents the survival curve of mice after challenge, and B represents the viral load in mouse plasma on days 1, 4, and 7 after challenge.

[0078] Figure 22 The titers of anti-dengue virus type 2 binding antibodies induced in C57BL / 6 mice after oral administration of the Ad(EfcN)-E dengue type 2 vaccine are given. A represents the serum antibody titer of mice 2 weeks after one vaccination, B represents the serum antibody titer of mice 2 weeks after two vaccinations, and C represents the serum antibody titer of mice 2 weeks after three vaccinations.

[0079] Figure 23The titers of anti-dengue virus type 2 binding antibodies induced in C57BL / 6 mice after intranasal administration of the Ad(EfcN)-E dengue type 2 vaccine are given. A represents the serum antibody titer of mice 2 weeks after a single vaccination, and B represents the serum antibody titer of mice 3 weeks after a single vaccination.

[0080] Figure 24 It is a circular RNA vaccine encoding EDIII, EDIII-Fc, and EDIII-Fd that immunizes IFNaR. - / - Neutralizing antibody response and protective effect induced in C57BL / 6 mice. In this study, A represents the serum neutralizing antibody titer of mice three weeks after two vaccinations; B represents the survival curve of mice after challenge; and C represents the viral load in the brain of mice five days after challenge.

[0081] Figure 25 This diagram illustrates the insertion regions of Hexon HVR1, 2, 5, and 7 as linker peptides, located after amino acids 136 (HVR1), 188 (HVR2), 268 (HVR5), and 422 (HVR7) of the Hexon protein, respectively. It shows the lesions in virus-infected cells and the expression of the reporter gene Tomato under a fluorescence microscope. Detailed Implementation

[0082] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0083] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual* (2013), edited by Green and Sambrook, or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0084] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0085] Display ratio: In display technologies (such as phage display, yeast display, and viral surface display), the number or percentage of fusion proteins or tags that can be correctly folded and function on the surface of vector particles (such as viruses or cells). In this invention, the display ratio is also the coupling ratio, which is the ratio of EDIII monomers to Hexon on the surface of recombinant adenovirus with inserted Dogtag, that is, the proportion of EDIII monomers successfully coupled to Hexon on the surface of the adenovirus.

[0086] This invention provides a dengue / Zika virus genetically engineered vaccine and its application. The invention also provides a specific method for using the vaccine. The vaccine can be used to prevent dengue virus and Zika virus infection.

[0087] The dengue / Zika virus vaccine of this invention comprises an open reading frame encoding envelope protein domain III (EDIII) and the non-structural protein NS1, and EDIII is displayed in the delivery vector shell. Immunization with this vaccine primarily induces a type-specific antibody response, reducing the production of cross-antibodies and thus effectively avoiding or eliminating the risk of antibody-dependent enhancement of infection (ADE).

[0088] This invention provides an antigen combination and its method of use. The antigen combination includes: a protein encoding the open reading frames of SEQ ID NO:1, SEQ ID NO:2 and the protein corresponding to SEQ ID NO:7, or an open reading frame of SEQ ID NO:3, SEQ ID NO:4 and the protein corresponding to SEQ ID NO:8, or an open reading frame of SEQ ID NO:5, SEQ ID NO:6 and the protein corresponding to SEQ ID NO:9.

[0089] Specifically, SEQ ID NO:1 encodes the EDIII domain of dengue virus type 1 with a dimerization tag fused to its C' terminus; SEQ ID NO:2 encodes the NS1 protein of dengue virus type 1 with a signal peptide fused to its N' terminus; SEQ ID NO:3 encodes the EDIII domain of dengue virus type 2 with a dimerization tag fused to its C' terminus; SEQ ID NO:4 encodes the NS1 protein of dengue virus type 2 with a signal peptide fused to its N' terminus; SEQ ID NO:5 encodes the EDIII domain of Zika virus with a dimerization tag fused to its C' terminus; SEQ ID NO:6 encodes the NS1 protein of Zika virus with a signal peptide fused to its N' terminus; SEQ ID NO:7 is the EDIII of dengue virus type 1 with a linker peptide fused to its N' terminus; SEQ ID NO:8 is the EDIII of dengue virus type 2 with a linker peptide fused to its N' terminus; and SEQ ID NO:9 is the EDIII of Zika virus with a linker peptide fused to its N' terminus.

[0090] In one possible implementation of the above antigen combination, SEQ ID NO:1 and SEQ ID NO:2 can be encoded by the same open reading frame and linked together by a sequence encoding a self-splicing linker peptide, or they can be encoded by two separate open reading frames; SEQ ID NO:3 and SEQ ID NO:4 can be encoded by the same open reading frame and linked together by a sequence encoding a self-splicing linker peptide, or they can be encoded by two separate open reading frames; SEQ ID NO:5 and SEQ ID NO:6 can be encoded by the same open reading frame and linked together by a sequence encoding a self-splicing linker peptide, or they can be encoded by two separate open reading frames. In some embodiments, the self-splicing linker peptide can be a 2A linker peptide.

[0091] In one possible implementation of the above antigen combination, the proteins corresponding to SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, after being fused with paired linker peptides, can be produced in prokaryotic or eukaryotic cells and purified by affinity chromatography. In some embodiments, the prokaryotic cells can be *Escherichia coli*, and the eukaryotic cells can be HEK293 cells and their derivative cell lines.

[0092] The present invention also provides an expression vector carrying the above-mentioned nucleic acid sequence. The expression vector may be a recombinant viral vector, an RNA vector, or a DNA vector. In some embodiments, the recombinant viral vector may be a replication-defective adenovirus vector. Optionally, the replication-defective adenovirus vector may be a replication-defective adenovirus type 5 vector (Ad5), and the open reading frame containing the above-mentioned nucleic acid sequence may be inserted into the E1 or E3 region of the Ad5 vector. In some embodiments, the RNA vector may be circular RNA or mRNA, carrying the above-mentioned nucleic acid sequence and the desired translation element, wherein the translation element may be an internal ribosome entry site (IRES). In some embodiments, the DNA vector may be a plasmid carrying an open reading frame containing the above-mentioned nucleic acid sequence.

[0093] This invention also provides a display vector for surface display of the proteins corresponding to SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. The display vector can be protein particles, nucleic acid particles, lipid particles, or recombinant viral particles.

[0094] In some embodiments, the recombinant viral particle may be a replication-deficient adenovirus particle. Optionally, the replication-deficient adenovirus particle may be a replication-deficient Ad5 particle. Optionally, the above-mentioned protein may be linked to the hypervariable region (HVR) of the Ad5 vector capsid protein Hexon via a pairing linker peptide.

[0095] The pairing linker peptide is used to link the EDIII monomer to the a antigen or the b antigen or their expression vector.

[0096] Optionally, the paired linker peptide can be DogTag / DogCatcher, SpyTag / SpyCatcher, SnoopTag / SnoopCatcher, or other covalently linked chemical structures. Optionally, the HVR can be HVR1, HVR2, or HVR5.

[0097] The expression vector and display vector described above can be the same vector. In some embodiments, the vector is a replication-defective Ad5 vector. Optionally, the open reading frame containing SEQ ID NO:1 is inserted into the E1 region of the Ad5 vector, the open reading frame containing SEQ ID NO:2 is inserted into the E3 region of the Ad5 vector, and the protein corresponding to SEQ ID NO:7 is ligated to the HVR 5 of the Ad5 Hexon protein. Optionally, the open reading frame containing SEQ ID NO:3 can also be inserted into the E1 region of the Ad5 vector, the open reading frame containing SEQ ID NO:4 can also be inserted into the E3 region of the Ad5 vector, and the protein corresponding to SEQ ID NO:8 can be ligated to the HVR 5 of the Ad5 Hexon protein. Optionally, the open reading frame containing SEQ ID NO:5 can also be inserted into the E1 region of the Ad5 vector, the open reading frame containing SEQ ID NO:6 can also be inserted into the E3 region of the Ad5 vector, and the protein corresponding to SEQ ID NO:9 can be ligated to the HVR 5 of the Ad5 Hexon protein.

[0098] When the surface displays the proteins corresponding to SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, the ratio of the proteins to the adenovirus vector Hexon monomer can be from 0% to 100%.

[0099] The present invention also provides a vaccine comprising the above-mentioned antigen combination, the above-mentioned dengue virus type 1 antigen, the above-mentioned dengue virus type 2 antigen, the above-mentioned Zika virus antigen, the above-mentioned polynucleotide, the above-mentioned open reading frame, adenovirus vector, or the above-mentioned RNA as active ingredients.

[0100] In one possible implementation of the aforementioned vaccine, the vaccine includes one or more of the following: DNA vaccine, RNA vaccine, adenovirus vector vaccine, other viral vector vaccines, subunit vaccine, virus-like particle vaccine, and nanoparticle vaccine.

[0101] In one possible implementation of the aforementioned vaccine, the vaccine further includes a pharmaceutically or veterinarily acceptable medium, diluent, adjuvant, or excipient, or delivery vector.

[0102] The present invention also provides the use of the above-mentioned antigen combination, the above-mentioned dengue virus type 1 antigen, the above-mentioned dengue virus type 2 antigen, the above-mentioned Zika virus antigen, the above-mentioned polynucleotide, the above-mentioned open reading frame, adenovirus vector, or the above-mentioned RNA in the preparation of a vaccine for the prevention of dengue or Zika virus infection.

[0103] The present invention also provides specific methods for using the above-mentioned vaccine. In some embodiments, the vaccine can be administered by intramuscular injection, nasal drops, or oral administration.

[0104] The HEK293, 293T, and 293F cells used in the following experiments were purchased from the ATCC cell bank. Adenovirus-associated plasmids pGA1, pGK53, pAd5ΔE1ΔE3, and pcDNA3.4 were stored in our laboratory. E. coli competent cells DH5α, BJ5183, and XL1-Blue were all prepared in our laboratory.

[0105] The present invention will be further described in detail below with reference to specific embodiments, but it is not intended to limit the scope of protection of the present invention.

[0106] Example 1: Design of dengue / Zika virus antigen combination.

[0107] Based on the E-coding region sequences of the envelope protein of dengue virus type 1 (Strain: Hawaii; GeneBank No: EU848545.1), dengue virus type 2 (Strain: 16681; GeneBank No: U87411.1), and Zika virus (Strain: GZ02; GeneBank No: KX056898.1), the EDIII region of each strain was extracted (dengue virus type 1, amino acids 297-393; dengue virus type 2, amino acids 297-394; Zika virus, amino acids 298-409). Figure 1 As shown, a signal peptide (tPA, the tissue plasminogen activator signal peptide in this embodiment) is fused to the N' terminus of EDIII to form the EDIII monomer; simultaneously, a dimerization tag (the constant region of human immunoglobulin G, IgG Fc in this embodiment) is fused to the C' terminus of EDIII to form the EDIII dimer. Based on mammalian codon usage preferences, the coding sequences for the above antigens are designed as follows:

[0108] The coding sequence of dengue virus type 1 EDIII dimer (5'-3', the same below) (SEQ ID NO:1):

[0109]

[0110] Amino acid sequence, SEQ ID NO:10: MDAMKRGLCCVLLLCGAVFVSAMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSTQDEKGVTQNGRLITANPIVTDKEKPVNIEAEPPFGESYIVVGAGEKALKLSWFKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0111] Dengue virus type 2 EDIII dimer coding sequence (SEQ ID NO:3):

[0112]

[0113] Amino acid sequence, SEQ ID NO:11: MDAMKRGLCCVLLLCGAVFVSAMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0114] Zika virus EDIII dimer coding sequence (SEQ ID NO:5):

[0115]

[0116] Amino acid sequence, SEQ ID NO:12:MDAMKRGLCCVLLLCGAVFVSALRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0117] Simultaneously, based on the NS1 coding region sequences of the non-structural protein of dengue virus type 1, type 2, and Zika virus, the signal peptide tPA was fused to its N' terminus. According to mammalian codon usage preferences, the NS1 antigen coding sequences were designed as follows:

[0118] The coding sequence of dengue virus type 1 NS1 (SEQ ID NO:2):

[0119]

[0120] Amino acid sequence, SEQ ID NO: 13: MDAMKRGLCCVLLLCGAVFVSADSGCVINWKGRELKCGSGIFVTNEVHTWTEQYKFQADSPKRLSAAIGKAWEEGVCGIRSATRLENIMWKQISNELNHILLENDMKFTVVVGDVSGILTQGRKMIGPQPMEHKYSWKSWGKAKIIGADVQNTTFIIDGPNTPECPDDQRAWNIWEVEDYGFGIFTTNIWLKLRDSYTQVCDPRLMSAAIKDSKAVHADMGYWIESEKNETWKLARASFIEVKTCVWPKSHTLWSNGVLESEMIIPKIYGGPISQHNYRPGYSTQTAGPWHLGKLELDFDLCEGTTVVVDEHCGNRGPSLRTTTVTGKIIHEWCCRSCTLPPLRFKGEDGCWYGMEIRPVKDKEENLVKSLVSA

[0121] Dengue virus type 2 NS1 coding sequence (SEQ ID NO: 4):

[0122]

[0123] Amino acid sequence, SEQ ID NO:14: MDAMKRGLCCVLLLCGAVFVSASGCVVSWKNKELKCGSGIFITDNVHTWTEQYKFQPESPSKLASAIQKAHEEGICGIRSVTRLENLMWKQITPELNHILSENEVKLTIMTGDIKGIMQAGKRSLQPQPTELKYSWKTWGKAKMLSTESHNQTFLIDGPETAECPNTNRAWNSLEVEDYGFGVFTTNIWLKLREKQDVFCDSKLMSAAIKDNRAVHADMGYWIESALNDTWKIEKASFIEVKSCHWPKSHTLWSNGVLESEMIIPKNFAGPVSQHNYRPGYHTQTAGPWHLGKLEMDFDFCEGTTVVVTEDCGNRGPSLRTTTASGKLITEWCCRSCTLPPLRYRGEDGCWYGMEIRPLKEKEENLVNSLVTAGHG。

[0124] Zika virus NS1 coding sequence (SEQ ID NO:6):

[0125]

[0126] Amino acid sequence, SEQ ID NO:15:MDAMKRGLCCVLLLCGAVFVSAVGCSVDFSKKETRCGTGVFVYNDVEAWRDRYKYHPDSPRRLAAAVKQAWEDGICGISSVSRMENIMWRSVEGELNAILEENGVQLTVVVGSVKNPMWRGPQRLPVPVNELPHGWKAWGKSYFVRAAKTNNSFVVDGDTLKECPLKHRAWNSFLVEDHGFGVFH TSVWLKVREDYSLECDPAVIGTAVKGKEAVHSDLGYWIESEKNDTWRLKRAHLIEMKTCEWPKSHTLWTDGIEESDLIIPKSLAGPLSHHNTREGYRTQMKGPWHSEELEIRFEECPGTKVHVEETCGTRGPSLRSTTASGRVIEEWCCRECTMPPLSFRAKDGCWYGMEIRPRKEPESNLVRSMVTAGS.

[0127] Simultaneously, a pairing linker peptide (DogCatcher in this embodiment) is fused to the N' terminus of the aforementioned secretory EDIII, which is an EDIII monomer antigen (DogCatcher-EDIII) that can be displayed (DogCatcher is linked to the EDIII monomer via GGSGGSGGSGGS) to the surface of the display vector. Its amino acid sequence (N' to C' terminus) is as follows:

[0128] DogCatcher (SEQ ID NO:16):

[0129] MKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTGEDGKLTFTNLSDGKYRL IENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQ

[0130] Dengue virus type 1 EDIII monomeric amino acid sequence (SEQ ID NO:7):

[0131] MSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSTQDEKGVTQNGRLITANPIVTDKEKPVNIEAEPPFGESYIVVGAGEKALKLSWFKGS

[0132] Dengue virus type 2 EDIII monomeric amino acid sequence (SEQ ID NO:8):

[0133] MSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKKGS

[0134] Zika virus EDIII monomer amino acid sequence (SEQ ID NO:9):

[0135] LRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKGS.

[0136] The C-terminus of the proteins displayed in EDIII above is marked with a His tag for purification: HHHHHHHH.

[0137] Example 2: Construction of recombinant adenovirus expression vector and display vector.

[0138] Sequence alignment analysis of Hexon proteins from different adenovirus types identified Hexon HVR1, 2, and 5 as insertion regions for the linker peptides, located after amino acids 136 (HVR1), 188 (HVR2), and 268 (HVR5) of the Hexon protein, respectively. In this embodiment, DogTag was selected as the linker peptide on the display vector surface. The DogTag-encoding DNA sequence was artificially synthesized and inserted into the pGK5 plasmid using a seamless cloning method. This DNA sequence was then inserted into the HVR1, 2, and 5 regions of the pAd5ΔE1ΔE3 genome plasmid via homologous recombination in *E. coli*. The open reading frames encoding the antigen were then integrated into the E1 / E3 regions of the adenovirus via shuttle plasmids pGA1 or pGK53, respectively. After linearization, the obtained recombinant pAd5 genome plasmid was transfected into HEK293 cells to rescue and produce recombinant virus. A simplified flowchart is as follows: Figure 2 As shown.

[0139] The specific steps are as follows:

[0140] 1) Construct pAd5ΔE1ΔE3-Hm.

[0141] The codon-optimized DogTag coding sequence was synthesized and constructed into the shuttle plasmid pGK5-Hexon carrying the Hexon HVR region using a seamless cloning method, yielding pGK5-Hexon-HVR-1R, pGK5-Hexon-HVR-2R, and pGK5-Hexon-HVR-5R. This plasmid has homologous recombination arms flanking the Hexon region at the multiple cloning site. The recombinant products were transformed into DH5α competent bacteria, and the correct clones were identified by enzyme digestion. After amplification and small-scale extraction of the correct clones, they were linearized using SgrAI and Bstz17I enzymes. The pAd5ΔE1ΔE3 genomic plasmid was linearized using AsiSI enzyme. The two were then mixed and co-transformed into BJ5183 competent cells. All transformation products were plated on ampicillin-resistant LB agar plates and incubated upside down at 37°C for 8–10 h. After the clones emerge, positive clones are identified by colony PCR and then cultured for 8-12 hours. Plasmids are manually extracted from BJ5183 cells and dissolved in an appropriate volume (usually 200 μL) of ultrapure water. Clones containing large plasmids (~35 kb) are identified by agarose gel electrophoresis and transformed into XL1-Blue competent cells. All transformation products are plated onto ampicillin-resistant LB plates and incubated upside down for 8-10 hours. Single clones are picked, plasmids are extracted manually, and the correct recombinant plasmids are identified by double digestion with XbaI and HindIII to obtain the full-length recombinant adenovirus plasmids pAd5ΔE1ΔE3-Hm1, pAd5ΔE1ΔE3-Hm2, and pAd5ΔE1ΔE3-Hm5.

[0142] DogTag coding sequence: GGAGTGGAGGTTCTGATATACCTGCTACATATGAATTCACTGATGGAAAGCATTATATCACAAATGAACCAATTCCTCCAAAGGGAGGTGGAGGTTCT (SEQ ID NO: 17).

[0143] 2) Construct pAd5(EN) and pAd5(EfcN).

[0144] In this embodiment, the effect of EDIII was demonstrated on Hexon HVR5. The EDIII dimer coding sequences of dengue virus type 1, dengue virus type 2, and Zika virus were constructed into the E1 region shuttle plasmid pGA1 using a seamless cloning method, resulting in pGA1 shuttle plasmids carrying the open reading frames of the EDIII dimers of the respective strains. The shuttle plasmids were linearized by double digestion with BstZ17I and SgrA1, and then homologously recombinated with pAd5ΔE1ΔE3-Hm5 (which was linearized by ClaI unless otherwise specified in subsequent vaccine development), yielding pAd5(Efc) carrying the open reading frames of the EDIII dimers of the respective viruses. Then, the NS1 coding sequences of dengue virus type 1, dengue virus type 2, and Zika virus were constructed into the E3 region shuttle plasmid pGK53 using a seamless cloning method, resulting in pGK53-NS1 shuttle plasmids carrying the open reading frames of the NS1 of the respective strains. The shuttle plasmid was linearized by double digestion with BstZ17I and SgrA1, and then homologously recombined with pAd5(Efc) linearized by SrfI to obtain pAd5(EfcN) carrying the viral EDIII dimer and NS1 open reading frame.

[0145] To fully illustrate the technical advantages of the present invention, pAd5(E) carrying the Zika virus EDIII monomeric open reading frame was constructed using the above method as a comparison for subsequent embodiments.

[0146] 3) Rescue and produce Ad(E) and Ad(EfcN).

[0147] pAd5(EN) and pAd5(EfcN) plasmids were linearized by PacI digestion and then recovered by ethanol precipitation. HEK293 cells were pre-seeded in 6-well plates and transfected with plasmids when the cells reached 60-70% confluency. Transfection dose: 4 μg plasmid per well; transfection reagent: Lipo2000. After transfection, cells were incubated at 37°C (5% CO2) for 6 hours, then the maintenance medium (2% FBS DMEM) was replaced, and the cells were observed every 2-3 days. If the medium turned yellow, fresh medium was added. After 7-10 days, cells were collected and frozen at -80°C, designated as P0 generation virus.

[0148] The P0 generation virus was blindly passaged in HEK293 cells for 2-3 passages. Cytopathic effects were observed after passage; the presence of obvious cytopathic effects indicated successful virus rescue. After the viral titer increased, the cells were transferred to a large container for expanded culture. Cells were collected at an appropriate time point, i.e., when the viral load was maximized. The collected cell mixture was subjected to three freeze-thaw cycles (liquid nitrogen / 37°C), cooled to 4°C, and centrifuged at 2000×g for 5 min. The supernatant viral solution was collected.

[0149] Then, the virus particles were purified using CsCl density gradient centrifugation. Using sterile Beckman centrifuge tubes, 3 mL of pre-chilled (4°C) low-density CsCl solution (1.2 g / mL) was added to each tube, followed by 3 mL of high-density CsCl solution (1.4 g / mL), and the separation was observed. Using a Pasteur pipette, 6-7 mL of virus solution was gently added to the centrifuge tube along the upper layer, until the final liquid level was 2 mm from the tube opening. The centrifuge tubes containing the liquid were gently placed into a centrifuge tube sleeve and centrifuged at 28,000 rpm for 4 hours at 4°C. After centrifugation, the white band containing the virus solution was aspirated using a Pasteur pipette. The solution was desalted, aliquoted, and stored at -70°C for later use.

[0150] Using the above methods, dengue virus type 1 vaccine Ad(EfcN), dengue virus type 2 vaccine Ad(EfcN), Zika virus vaccine Ad(EfcN), and Zika virus vaccine Ad(EfcN) carrying EDIII monomer were produced and purified, as well as Zika virus vaccine Ad(E) carrying EDIII monomer as a control and empty vector Ad empty without antigen gene as a control.

[0151] The number of viral particles in the above-mentioned vaccine was determined by measuring DNA content using a spectrophotometer. 95 μL of purified adenovirus was added to 5 μL of 10% SDS solution, mixed well, and incubated at 56°C for 5 min. A blank control group was set up. OD260 and OD280 were measured three times, and the average value was taken. The OD260 / OD280 ratio was calculated to assess viral purity. The formula for calculating viral titer is:

[0152] Titer (vp / mL) = OD260 × dilution factor × 10 12 ×36 / Length (Length is the length of the viral genome, in Kb).

[0153] Example 3: Expression, purification and surface display of EDIII monomers and construction of a vaccine.

[0154] This embodiment aims to utilize the HEK293F cell transient transfection system to efficiently express the Zika virus EDIII protein monomer fused with the DogCatcher linker peptide (e.g., Figure 1 As shown in the figure, a surface-displaying nanovaccine was constructed by covalently coupling the nanovaccine with adenovirus particles that have integrated DogTags with Hexon proteins.

[0155] 1) Expression and purification of EDIII protein.

[0156] The encoding DNA sequence of Zika virus DogCatcher-EDIII-HisTag was artificially synthesized and constructed into the pcDNA3.4 expression vector. After extraction of the expression plasmid, it was transfected into HEK293F cells. 18 hours after transfection, 293F fed medium was added, and the cells were cultured for another 72 hours before the cell supernatant was collected. The supernatant was collected after centrifugation at 5000×g, 4℃ for 30 min, filtered through a 0.22 μm filter, and then purified using a nickel column affinity assay according to standard methods. The target protein was concentrated using ultrafiltration and quantified using the BCA method.

[0157] Using the above method, the Dogcatcher-EDIII fusion proteins (virus EDIII monomers fused with DogCatcher linker peptides) of dengue virus type 1, dengue virus type 2 and Zika virus were prepared respectively.

[0158] 2) Surface display of vaccine construction.

[0159] The coupling effect of DogCatcher-EDIII with Ad5(E) recombinant virus under different coupling ratios was explored. Purified Ad5(E) viral particles (Ad(EfcN)) were mixed with EDIII monomers at the following ratios: 1:0, 1:0.25, 1:0.5, 1:1, and 1:3. These ratios were calculated based on the number of Hexon protein monomers and DogCatcher-EDIII monomers, with the amount of Ad(E) used being 1 × 10⁻⁶. 10 vp. After incubating overnight at 4°C, take a small sample, denature it, and then examine the protein coupling effect using SDS-PAGE gel electrophoresis. For example... Figure 3As shown in Figure A, the size of the Hexon protein increases after conjugation, and the number of Hexon-EDIII conjugates increases with the increase of the EDIII ratio. The ratio of Hexon to Hexon-EDIII was calculated using grayscale analysis to obtain the corresponding conjugation ratio. Furthermore, using anti-EDIII antibody (7B3, Niu, Xuefeng et al. “Convalescent patient-derived monoclonal antibodies targeting different epitopes of E protein confer protection against Zika virus in a neonatal mouse model.”Emerging microbes & infections vol. 8,1 (2019): 749-759. doi:10.1080 / 22221751.2019.1614885) and anti-Hexon antibody (EPR28237-57, Abcam), Western blot experiments confirmed that EDIII successfully conjugated to the adenovirus surface. Figure 3 (As shown in B).

[0160] Using the above method, Zika virus vaccines Ad(E)-E with different conjugation ratios and Ad-E with a conjugation rate of over 90% were prepared.

[0161] 3) Visual observation of the vaccine surface.

[0162] To determine whether the morphology of adenovirus particles was affected after surface antigen display, we incubated them with anti-EDIII monoclonal antibody 7B3 and Ad(E)-E, followed by incubation with colloidal gold-labeled anti-human IgG secondary antibody (JAC-109-205-088, Jackson ImmunoResearch). Transmission electron microscopy revealed that surface antigen display did not affect the typical structure of the adenovirus particles. The proportion of antibody-labeled particles surrounding the virus increased with increasing antigen conjugation ratio. Figure 4 ).

[0163] Example 4: Determination of the optimal surface display ratio.

[0164] This study aims to evaluate the immunogenicity of Ad(E)-E Zika virus vaccines with different antigen display ratios in C57BL / 6 mice, and to compare the immunoprotective effect of adenovirus vector vaccines without display ratios on Zika virus in a "maternal immunization + pup challenge" model, thereby screening for the optimal display ratio that can induce a strong protective immune response.

[0165] 1) Immunogenicity assessment of Ad(E)-E Zika virus vaccines with different display ratios.

[0166] Female C57BL / 6 mice aged 6-8 weeks were immunized with Ad(E)-E vaccines and control vaccines at different display ratios. Groups: Ad(E)-E 0% (i.e., no display, antigen expressed only by open reading frames), Ad(E)-E 40%, Ad(E)-E 90%, Ad-E 90% (i.e., only displayed, without open reading frames), EDIII protein (with aluminum adjuvant), Ad empty (empty adenovirus vector control). Immunization method: intramuscular injection. Immunization dose: all adenovirus-containing groups, adenovirus dose 1×10⁻⁶. 10 VP / mouse, protein control group 1μg / mouse (containing 50μg aluminum adjuvant). A booster immunization was administered four weeks after the initial immunization, with the same immunization protocol and dosage. Two weeks after the final immunization, mice were euthanized, serum was collected, and spleens were isolated. Serum collection method: Non-anticoagulated whole blood was allowed to stand for 3 hours, centrifuged at 5000 rpm for 30 minutes, the supernatant was collected and inactivated in a 56℃ water bath for 30 minutes, and the serum was aliquoted and stored at -80℃. Brief method for splenic lymphocyte isolation: After euthanizing the mice, the spleen was isolated. The spleen was placed in 4 mL of lymphocyte separation medium, passed through a 200-mesh filter, and gently ground with a 5 mL syringe until completely ground. The cell suspension was then transferred to a 15 mL centrifuge tube, and 3 mL of 1640 medium was slowly added to the upper layer. Centrifuged at 800g at room temperature for 30 minutes, with an acceleration / deceleration setting of 3. After centrifugation, the middle layer of cells was aspirated, resuspended in 4 mL of 1640 medium, and washed by centrifugation at 300g at room temperature for 10 minutes. Finally, based on the lymphocyte count, the cells were resuspended in R10 medium for use in the next ELISA plate experiment. We detected the binding antibody levels using an enzyme-linked immunosorbent assay (ELISA). After incubating the ELISA plate with purified Zika virus EDIII, the plate was blocked, washed, and serially diluted immune serum was added. The plate was then incubated with horseradish peroxidase-labeled anti-mouse IgG secondary antibody, developed with TMB substrate, and the reaction was terminated with H2SO4 solution. The OD450 values ​​of each well were read, and the binding antibody titer in the immune serum was calculated. The results are as follows: Figure 5 As shown, the binding antibody level induced by Ad(E)-E with a display ratio of 40% was 100 times higher than that of the EDIII protein vaccine and 10 times higher than that of other Ad(E)-E vaccines with display ratios. Figure 5 A).

[0167] We detected neutralizing antibody titers using a flow cytometry-based neutralization assay. The method is briefly described below: Vero cells were loaded at 2 × 10⁶ cells per well. 4Cells were seeded at a density of [number] cells per well in 96-well plates and cultured overnight. Mouse serum was serially diluted 3-fold in DMEM (starting at 1:50) and incubated with ZIKV (200 FFU per well) at 37°C for 1 hour. Mouse serum immunized with the empty vector vaccine served as a negative control. The mixture was added to the cells and incubated at 37°C for 2 hours, then replaced with fresh DMEM containing 2% FBS. After three days, cells were fixed and permeabilized with Cytofix / Cytoperm buffer (BD). Cells were then labeled with mAb 8D10, stained with Alexa Fluor 647-conjugated goat anti-human IgG antibody, and analyzed on an Accuri C6 flow cytometer (BD). Neutralizing antibody titers were calculated as the dilution at which 50% of infected positive cells were reduced relative to the negative control. Results are as follows: Figure 5 As shown in B, consistent with the trend of binding antibodies, the neutralizing antibody titer induced by Ad(E)-E with a proportion of 40% was significantly higher than that of other groups.

[0168] We detected vaccine-induced cellular immune responses using an enzyme-linked immunospot (ELISpot) assay. The method is briefly described below: 96-well PVDF membrane plates were pre-coated with IFN-γ-coated antibody and incubated overnight at 4°C, followed by blocking with R10. Then, isolated spleen lymphocytes were added to the plates at a rate of 300,000 cells / well, along with stimulation with a ZIKV EDIII peptide library (final peptide concentration 1 μg / mL). After 24 hours, the cell suspension was discarded, the cells were washed, and then incubated overnight at 4°C with IFN-γ detection antibody. The IFN-γ detection antibody was then further bound to streptomycin-conjugated alkaline phosphatase (BD PharMingen). Finally, BCIP / NBT substrate was used for color development, and after drying, the plates were read using an ELISpot reader to count the number of spots per well. Results are shown below. Figure 5 As shown in C, adenovirus expressing 40% or displaying EDIII antigen on its surface can induce a cellular immune response, which is significantly stronger than that of EDIII protein vaccines.

[0169] Therefore, displaying 40% Ad(E)-E can induce a stronger antibody and cellular immune response.

[0170] 2) Evaluation of the protective efficacy of Ad(E)-E Zika virus vaccines with different display ratios.

[0171] To evaluate the protective effect of Ad(E)-E vaccines with different display ratios against Zika virus challenge, this embodiment used a "maternal immunization + pup challenge" model. Six- to eight-week-old female C57BL / 6 mice were immunized with Ad(E)-E vaccine and a control vaccine. The route of immunization, dosage, and groups were as described above. Two weeks after two immunizations, immunized mothers were mated with unimmunized males, and the offspring were challenged with Zika virus (GZ02 strain, GeneBank No. KX056898.1) (subcutaneous injection, 1×10⁻⁶). 4 PFU / mouse was used to monitor the growth and neurological symptoms of the pups. The pups were euthanized on day 15 post-infection to assess brain development. Results were as follows: Figure 6 As shown in Figure A, immunization of maternal mice with the EDIII protein vaccine offered almost no protection to their offspring; immunization of maternal mice with different forms of adenovirus vector vaccines showed some protection, but there appeared to be no significant difference in offspring weight growth indicators. Figure 6 As shown in Figure B, immunization of maternal mice with both Ad(E)-E (40%) and Ad(E)-E (90%) vaccines significantly reduced neurological symptoms in pups after challenge, with the Ad(E)-E (40%) vaccine showing the most significant effect. Correspondingly, as... Figure 6 As shown in C, immunization of mother mice with EDIII protein vaccine has almost no protective effect on the development of brain tissue in offspring after challenge with the virus, while immunization with different forms of adenovirus vector vaccines all showed some protection. Among them, offspring born to mother mice immunized with Ad(E)-E (40%) vaccine showed better brain tissue growth after challenge with the virus than those of other immunization groups.

[0172] comprehensive Figure 5 , Figure 6 The results show that the Ad(E)-E vaccine with a surface display ratio of 40% exhibits good immunogenicity and immune protection. Therefore, unless otherwise specified, the display ratio used in the following examples is 40%.

[0173] Example 5: Construction of Ad(EfcN)-E Zika virus vaccine.

[0174] To further enhance the immunogenicity and protective efficacy of the vaccine, as described in Example 2, the sequence encoding the EDIII monomer was replaced with the sequence encoding the EDIVII dimer, i.e., SEQ ID NO:1; then, an open reading frame encoding the NS1 antigen was inserted into the E3 region of the adenovirus vector, i.e., SEQ ID NO:4. Zika virus vaccines such as Ad(Efc) and Ad(EfcN) were rescued, produced, purified, and titrated using the method described in Example 2. To verify the expression characteristics of the vaccine antigen gene, HEK293 cells were infected with the above vaccine at a multiplicity of infection of 5 × 10⁻⁶. 2vp / cell. Forty-eight hours post-infection, cells and culture supernatants were collected separately and Western blotted with monoclonal antibodies against Zika virus EDIII (7B3) and NS1 (749-A4) (Wessel, Alex W et al. “Antibodies targeting epitopeson the cell-surface form of NS1 protect against Zika virus infection during pregnancy.” Nature communications vol. 11, 1 5278. 19 Oct. 2020, doi:10.1038 / s41467-020-19096-y). Figure 7 As shown in Figure A, unlike the EDIII monomer expressed in Ad(E)-infected cells, Ad(Efc) and Ad(EfcN) can express EDIII dimers after cell infection. Both the EDIII monomers and dimers expressed by the vaccine can be effectively secreted into the culture supernatant. Meanwhile, as... Figure 7 As shown in Figure B, Ad(EfcN) infected cells can also express NS1 dimers, and NS1 dimers can be effectively secreted into the culture supernatant.

[0175] Furthermore, using the method described in Example 3, a Zika virus vaccine Ad(Efc)-E that simultaneously expresses EDIII dimer and displays EDIII monomer, and a Zika virus vaccine Ad(EfcN)-E that simultaneously expresses EDIII dimer and NS1 and displays EDIIII monomer were constructed.

[0176] Example 6: Evaluation of the immunogenicity and protective effect of the Ad(EfcN)-E Zika virus vaccine.

[0177] The immunogenicity of the Ad(EfcN)-E Zika virus vaccine was evaluated in a C57BL / 6 mouse model using a method similar to that described in Example 4. Groups: Ad empty, Ad(E)-E, Ad(Efc)-E, Ad(EfcN), Ad(EfcN)-E, Ad(EfcN)-E l / h. Immunization route: intramuscular injection. Immune dose: 1 × 10⁻⁶ for all groups except Ad(EfcN)-E l / h. 10 vp / animal; Ad(EfcN)-E l / h group, initial immunization 2×10 9 vp / animal, booster immunization 1×10 10 vp / mouse. A booster immunization was administered four weeks after the initial immunization, using the same regimen and dosage as above. Two weeks after the final immunization, the mice were euthanized, and serum was collected.

[0178] The levels of EDIII-specific and NS1-specific binding antibodies were detected using an ELISA assay. Figure 8 As shown in Figure A, compared to Ad(EfcN) without antigen display, the vaccines displaying the antigen induced significantly stronger EDIII binding antibodies, with an average titer approximately 10-fold higher. There were no significant differences in l / h between Ad(E)-E and Ad(Efc)-E, or between Ad(EfcN)-E and Ad(EfcN)-E. Figure 8 As shown in Figure B, only the Ad(EfcN), Ad(EfcN)-E, and Ad(EfcN)-E l / h vaccines induced NS1-specific antibody responses, and there were no significant differences among them. Figure 8 As shown in Figure C, the neutralizing antibody responses induced by Ad(E)-E and Ad(EfcN) were slightly weaker than those in other groups, and some mice did not produce significant neutralizing antibodies. In contrast, Ad(Efc)-E, Ad(EfcN)-E, and Ad(EfcN)-E l / h induced significant neutralizing antibody responses in all mice. These results indicate that both dimerization and surface display of EDIII significantly enhance its ability to induce antibody responses.

[0179] The protective efficacy of each vaccine was evaluated using a "maternal immunization + pup challenge" model. Six- to eight-week-old female C57BL / 6 mice were immunized with the aforementioned vaccines and a control vaccine. The route of immunization, dosage, and groups were as described above. Two weeks after two immunizations, immunized mothers were mated with unimmunized males, and the offspring were challenged with Zika virus (GZ02 strain, GeneBank No. KX056898.1) via subcutaneous injection (1×10⁻⁶). 4 PFU / mouse), monitor the growth status and neurological symptoms of the pups, and euthanize them on day 15 after challenge to detect viral load in the pups' brains. Figure 9 As shown in Figure A, immunization of maternal mice with the Ad(E)-E vaccine only showed a certain protective effect on pups, with their weight gain being slightly better than the control vaccine group; immunization of maternal mice with Ad(Efc)-E, Ad(EfcN), Ad(EfcN)-E, and Ad(EfcN)-E l / h showed better protection, with no significant difference in pup weight gain compared to the healthy group. Figure 9 As shown in Figure B, immunization of mother mice with Ad(Efc)-E, Ad(EfcN), Ad(EfcN)-E, and Ad(EfcN)-E l / h significantly reduced neurological symptoms in pups after challenge, with the Ad(EfcN)-E1 / h vaccine showing the best results, in which all pups exhibited no obvious neurological symptoms; immunization of mice with the Ad(E)-E vaccine only slightly reduced neurological symptoms. Figure 9As shown in Figure C, immunization of maternal mice with all vaccines significantly reduced viral load in the brain tissue of pups after challenge. No viral genome was detected in the brains of pups in the Ad(EfcN)-E l / h vaccine group, and in most pups in the Ad(EfcN)-E vaccine group, no viral genome was detected. Therefore, the Ad(EfcN)-E vaccine has a strong preventive effect against Zika virus infection.

[0180] Example 7: Evaluation of the immunogenicity and protective efficacy of the Ad(EfcN)-E dengue virus type 1 vaccine.

[0181] Using a method similar to that described in Example 4, in IFNaR - / - Immunogenicity of Ad(EfcN)-E dengue virus type 1 vaccine was evaluated using a C57BL / 6 mouse model. Groups: Ad empty, Ad(EfcN), Ad(EfcN) Mix, Ad(EfcN)-E, Ad(EfcN)-E Mix; where Ad(EfcN) Mix refers to a mixture of dengue virus type 1 and dengue virus type 2 vaccines without surface display, and Ad(EfcN)-E Mix refers to a mixture of dengue virus type 1 and dengue virus type 2 vaccines with surface-displayed EDIII antigen. Immunization route: intramuscular injection. Immune dose: 1×10⁻⁶ 10 vp / vaccine / mouse. A booster immunization is given four weeks after the initial immunization, with the same immunization schedule and dosage. Blood is collected from the orbital region of the mice two weeks after the final immunization.

[0182] The levels of EDIII-specific and NS1-specific binding antibodies were detected using an ELISA assay. Figure 10 As shown in Figure A, compared to Ad(EfcN) without antigen display, Ad(EfcN)-E with antigen display induced significantly stronger EDIII-binding antibodies, with an average titer approximately 6-fold higher. There were no significant differences between Ad(EfcN) and Ad(EfcN) Mix, or between Ad(EfcN)-E and Ad(EfcN)-E Mix, indicating that the bivalent vaccine combination did not affect vaccine immunogenicity. Figure 10 As shown in B, all vaccines can induce NS1-specific antibody responses, with little difference between them. Figure 10 As shown in C, the neutralizing antibody titers induced by Ad(EfcN)-E and Ad(EfcN)-E Mix were significantly stronger than those induced by Ad(EfcN) or Ad(EfcN) Mix, indicating that the EDIII surface exhibits a significant ability to enhance the antibody response induced by dengue virus type 1 vaccine.

[0183] Three weeks after the last immunization, patients were challenged with dengue virus type 1 (Hawaii strain, GeneBank No. EU848545.1) (subcutaneous injection, 2 × 10⁻⁶). 6PFU / mouse), monitor mouse weight, and collect blood samples from the orbital sinus on days 1, 4, and 7 after challenge to detect serum viral load. Figure 11 As shown in Figure A, dengue virus type 1 exhibits weak pathogenicity in this mouse model; mice in the control vaccine group showed only a 5% decrease in body weight after challenge. Except for the Ad(EfcN) Mix vaccine group, mice in the other vaccine groups did not show any decrease in body weight. However, as... Figure 11 As shown in Figure B, all vaccines significantly reduced serum viral load in mice after immunization. On day 7 post-challenge, the virus was still detectable in the control group mice, while the serum viral load in the vaccine group mice decreased to below the detection limit, indicating that the vaccine immunization had a good protective effect.

[0184] Example 8: Evaluation of the immunogenicity and protective efficacy of the Ad(EfcN)-E dengue virus type 2 vaccine.

[0185] Using a method similar to that described in Example 7, in IFNaR - / - Immunogenicity of Ad(EfcN)-E dengue virus type 2 vaccine was evaluated using a C57BL / 6 mouse model. Groups: Ad empty, Ad(EfcN), Ad(EfcN) Mix, Ad(EfcN)-E, Ad(EfcN)-E Mix; where Ad(EfcN) Mix refers to a mixture of dengue virus type 1 and dengue virus type 2 vaccines without surface display, and Ad(EfcN)-E Mix refers to a mixture of dengue virus type 1 and dengue virus type 2 vaccines with surface-displayed EDIII antigen. Immunization route: intramuscular injection. Immune dose: 1×10⁻⁶ 10 vp / vaccine / mouse. A booster immunization is given four weeks after the initial immunization, with the same immunization schedule and dosage. Blood is collected from the orbital region of the mice two weeks after the final immunization.

[0186] The levels of EDIII-specific and NS1-specific binding antibodies were detected using an ELISA assay. Figure 12 As shown in Figure A, compared to Ad(EfcN) without antigen display, Ad(EfcN)-E with antigen display induced significantly stronger EDIII-binding antibodies, with an average titer approximately 3 times higher. There were no significant differences between Ad(EfcN) and Ad(EfcN) Mix, or between Ad(EfcN)-E and Ad(EfcN)-E Mix, indicating that the bivalent vaccine combination did not affect vaccine immunogenicity. Figure 12 As shown in B, all vaccines can induce NS1-specific antibody responses, with little difference between them. Figure 12 As shown in C, the neutralizing antibody titers induced by Ad(EfcN)-E and Ad(EfcN)-E Mix were significantly stronger than those induced by Ad(EfcN) or Ad(EfcN) Mix, indicating that the EDIII surface exhibits a significant ability to enhance the antibody response induced by dengue virus type 2 vaccine.

[0187] Three weeks after the last immunization, patients were challenged with dengue virus type 2 (mp6-GZ-2022 strain, GeneBank No. PQ008452.1) (subcutaneous injection, 2 × 10⁻⁶). 6 PFU / mouse), monitor mouse weight, and collect blood samples from the orbital sinus on days 1, 4, and 7 after challenge to detect serum viral load. Figure 13 As shown in Figure A, dengue virus type 2 is highly pathogenic in this mouse model; most mice in the control vaccine group experienced a rapid weight loss of over 20% after challenge. Mice in all vaccine groups, however, did not show any weight loss. Figure 13 As shown in Figure B, all vaccines significantly reduced viral load in mouse serum after immunization. However, on days 1 and 4 post-challenge, some mice in the Ad(EfcN) and Ad(EfcN)Mix vaccine groups still had detectable viral load, while mice in the Ad(EfcN)-E and Ad(EfcN)-E Mix vaccine groups showed no detectable viral load, indicating that both the Ad(EfcN)-E and Ad(EfcN)-E Mix vaccines had strong protective effects.

[0188] Based on the results of Examples 6, 7, and 8, the Ad(EfcN)-E vaccine design has a good immune protection effect against dengue virus and Zika virus.

[0189] Example 9: Evaluation of cross-reactivity of Ad(EfcN)-E Zika virus vaccine immune serum.

[0190] Given that cross-antibodies against envelope proteins are a significant cause of adverse drug reaction (ADE), this embodiment detected the cross-antibody titers against dengue virus induced after Ad(EfcN)-E Zika virus vaccine immunization and compared them with antibody titers against Zika virus. ELISA plates were coated with dengue virus type 1, 2, 3, and 4 E proteins and Zika virus E protein, respectively. After blocking and washing, titer-diluted immune serum or control serum was added. The immune serum was serum collected after two immunizations of mice with Ad(EfcN), Ad(EfcN)-E, and Ad(EfcN)-E l / h vaccines; the control serum was serum collected after two immunizations of mice with Adempty. After incubation with horseradish peroxidase-labeled human anti-mouse IgG secondary antibody, color development was performed using TMB substrate, and the reaction was terminated with H2SO4. The OD450 value was measured, and the IgG antibody titer was calculated. Figure 14As shown, no IgG was detected in the control serum. Ad(EfcN), Ad(EfcN)-E, and Ad(EfcN)-E l / h immune sera showed high levels of Zika virus-specific antibodies, but low cross-reactivity with dengue serotypes 1-4 (E protein). Cross-antibody titers were generally nearly 100-fold lower than specific antibody titers. Therefore, the Ad(EfcN)-E Zika virus vaccine primarily induces Zika virus-specific antibodies.

[0191] Example 10: Evaluation of cross-reactivity of Ad(EfcN)-E dengue virus vaccine immune serum.

[0192] This embodiment detected the cross-antibody titer against Zika virus induced after immunization with the Ad(EfcN)-E dengue virus vaccine and compared it with the antibody titer against dengue virus. As described in Example 9, the E proteins of dengue virus types 1, 2, 3, and 4, and the E protein of Zika virus were used as antigens, and the IgG antibody titer in immune serum or control serum was detected by ELISA. The immune serum was serum collected after mice were immunized twice with Ad(EfcN)-E dengue type 1 vaccine, Ad(EfcN)-E dengue type 2 vaccine, or a mixture of both in equal proportions. The control serum was serum collected after mice were immunized twice with an Ad empty vaccine. Figure 15 As shown, no IgG was detected in the control serum. The Ad(EfcN)-E dengue 1 vaccine-immunized serum showed high levels of dengue virus type 1 specific antibodies, but low cross-reactivity with dengue types 2-4 and Zika virus E proteins. The Ad(EfcN)-E dengue 2 vaccine-immunized serum showed high levels of dengue virus type 2 specific antibodies, but low cross-reactivity with dengue types 1, 3, 4 and Zika virus E proteins. Cross-antibody titers were generally tens to hundreds of times lower than specific antibody titers. Therefore, the Ad(EfcN)-E dengue virus vaccine primarily induces dengue virus-specific antibodies.

[0193] Example 11: Evaluation of ADE effect in cell model of Ad(EfcN)-E Zika virus vaccine immune serum.

[0194] This embodiment investigated the effect of Ad(EfcN)-E Zika virus vaccine-immunized serum on promoting dengue virus infection at the cellular level, i.e., the ADE effect in a cell model. The sera used included Ad(EfcN)-immunized serum and Ad(EfcN)-E-immunized serum, with Ad empty serum as a negative control and serum from recovered Zika virus-infected mice (ZIKV sera) as a positive control. After serial dilution, the sera were incubated with Zika virus and dengue virus types 1-4, respectively, to infect K562 cells. K562 cells are generally not infected by mosquito flaviviruses in the absence of antibodies, but they express Fcγ receptors on their surface and can therefore be infected in the presence of ADE antibodies. Three to four days post-infection, cells were collected, ruptured, and labeled with the broad-spectrum monoclonal antibody ZK8-4. The cells were then stained with a fluorescently labeled secondary antibody, and the percentage of positive cells was detected by flow cytometry, representing the percentage of infected cells. Results are as follows: Figure 16 As shown, within a certain dilution range, Ad(EfcN) immune serum, Ad(EfcN)-E immune serum, and Zika virus infection convalescent serum can promote Zika virus infection (…). Figure 16 (See Figure A). Ad(EfcN) immune serum and Ad(EfcN)-E immune serum did not significantly promote dengue virus infection of types 1-4, but Zika virus convalescent serum could significantly enhance the ability of dengue virus to infect K562 cells. Figure 16 (B, C, D, E). This result indicates that the content of ADE antibodies in Ad(EfcN)-E immune serum is low, significantly lower than that in Zika virus infection convalescent serum.

[0195] Example 12: Evaluation of ADE effect in mouse model of Zika virus vaccine-immunized serum using Ad(EfcN)-E.

[0196] This embodiment further investigated the ADE effect (anti-deduction effect) of Ad(EfcN)-E Zika virus vaccine immune serum in promoting dengue virus type 2 infection in a mouse model. The sera used included Ad(EfcN) immune serum and Ad(EfcN)-E immune serum, with Ad empty immune serum as a negative control and Zika virus convalescent serum (ZIKV sera) as a positive control. A healthy control group was also included. Each serum was diluted 10-fold with physiological saline and injected intraperitoneally into IFNaR- / -C57BL / 6 mice at a dose of 200 μL / mouse. Twenty-four hours later, the mice were injected intraperitoneally with dengue virus type 2 infection at a dose of 2 × 10⁻⁶. 6 FFU / mouse. After challenge, mouse survival was monitored, and blood was collected from the orbital sinus on days 1, 4, and 7 to detect serum viral load. Figure 17As shown in Figure A, 40% of mice transferred with control serum survived after challenge; 40% of mice transferred with Ad(EfcN)-E immune serum also survived, while 60% of mice transferred with Ad(EfcN) immune serum survived; in contrast, mice transferred with convalescent serum from Zika virus infection rapidly developed the disease and all died. Figure 17 As shown in Figure B, on days 1 and 4 post-challenge, mice transferred with Zika virus convalescent serum had significantly higher viral loads than other mice, indicating a clear ADE effect. Mice transferred with Ad(EfcN) immune serum and Ad(EfcN)-E immune serum had viral loads comparable to those transferred with negative serum, showing no ADE effect. Therefore, Zika virus immunization with Ad(EfcN) and Ad(EfcN)-E vaccines does not lead to an ADE effect against dengue virus.

[0197] Based on the results of Examples 9, 11, and 12, the Ad(EfcN)-E Zika virus vaccine provided by this invention has no risk of inducing dengue virus ADE.

[0198] Example 13: Evaluation of ADE effect in cell model of Ad(EfcN)-E dengue virus type 1 vaccine immune serum.

[0199] This embodiment investigated the effect of Ad(EfcN)-E dengue virus type 1 vaccine-immunized serum on promoting Zika virus and other dengue virus types infection at the cellular level, i.e., the ADE effect in a cell model. The serum used included Ad(EfcN)-E immune serum, with Ad empty immune serum as a negative control and serum from mice recovered from dengue virus type 1 infection (DENV-1sera) as a positive control. The serum was serially diluted and incubated with Zika virus and dengue virus types 1-4, respectively, to infect K562 cells. Three to four days post-infection, cells were collected, ruptured, and labeled with the broad-spectrum monoclonal antibody ZK8-4. The cells were then stained with a fluorescently labeled secondary antibody, and the percentage of positive cells was detected by flow cytometry, representing the percentage of infected cells. Results are as follows: Figure 18 As shown, within a certain dilution range, Ad(EfcN)-E immune serum and dengue virus type 1 infection convalescent serum can promote dengue virus type 1 infection (…). Figure 18 (B). Ad(EfcN)-E immune serum did not significantly promote the infection of Zika virus and dengue virus types 2-4, but convalescent serum from dengue virus type 1 infection could significantly enhance the infection ability of Zika virus and dengue virus types 2-4 on K562 cells. Figure 18 (A, C, D, E). This result is consistent with... Figure 16 The results shown are similar, indicating that the ADE antibody content in Ad(EfcN)-E immune serum is low, significantly lower than that in convalescent serum from dengue virus type 1 infection.

[0200] Example 14: Evaluation of ADE effect in mouse model of Ad(EfcN)-E dengue virus type 1 vaccine immune serum.

[0201] This embodiment further investigated the adverse drug reaction (ADE) effect of Ad(EfcN)-E dengue virus type 1 vaccine-immunized serum in promoting dengue virus type 2 infection in a mouse model, i.e., the in vivo ADE effect. The sera used included Ad(EfcN) immune serum and Ad(EfcN)-E immune serum, with Ad empty immune serum as a negative control and convalescent serum from dengue virus type 1 infection (DENV-1sera) as a positive control. A healthy control group was also included. Experimental grouping and methods were as described in Example 12. Figure 19 As shown in Figure A, 55% of mice transferred with control serum survived after challenge; 55% of mice transferred with Ad(EfcN) immune serum and Ad(EfcN)-E immune serum also survived; in contrast, mice transferred with convalescent serum from dengue virus type 1 infection rapidly developed the disease and all died. Figure 19 As shown in Figure B, on days 1 and 4 post-challenge, mice transferred with convalescent serum from dengue virus type 1 infection had significantly higher viral loads than other mice, indicating a clear ADE effect. Mice transferred with Ad(EfcN) immune serum and Ad(EfcN)-E immune serum had viral loads comparable to those transferred with negative serum, showing no ADE effect. Therefore, immunization with dengue virus type 1 vaccines such as Ad(EfcN) and Ad(EfcN)-E does not lead to an ADE effect against dengue virus type 2.

[0202] Example 15: Evaluation of ADE effect in cell model of Ad(EfcN)-E dengue virus type 2 vaccine immune serum.

[0203] This embodiment investigated the effect of Ad(EfcN)-E dengue virus type 2 vaccine-immunized serum on promoting Zika virus and other dengue virus types infection at the cellular level, i.e., the ADE effect in a cellular model. The serum used included Ad(EfcN)-E immune serum, with Ad empty serum as a negative control and serum from recovered dengue virus type 2 mice (DENV-2 sera) as a positive control. After serial dilution, the serum was incubated with Zika virus and dengue virus types 1-4, respectively, to infect K562 cells. Three to four days post-infection, cells were collected, ruptured, and labeled with the broad-spectrum monoclonal antibody ZK8-4. The cells were then stained with a fluorescently labeled secondary antibody, and the percentage of positive cells was detected by flow cytometry, representing the percentage of infected cells. Results are as follows: Figure 20 As shown, within a certain dilution range, Ad(EfcN)-E immune serum and dengue virus type 2 infection convalescent serum can promote dengue virus type 2 infection (…). Figure 20(C). Ad(EfcN)-E immune serum did not promote the infection of Zika virus and dengue virus types 1, 3, and 4, but convalescent serum from dengue virus type 2 infection significantly enhanced the ability of Zika virus and dengue virus types 1, 3, and 4 to infect K562 cells. Figure 20 (A, B, D, E). This result is consistent with... Figure 16 , Figure 18 The results shown are similar, indicating that the content of ADE antibodies in Ad(EfcN)-E immune serum is low, significantly lower than that in convalescent serum from dengue virus type 2 infection.

[0204] Example 16: Evaluation of ADE effect in mouse model of Ad(EfcN)-E dengue virus vaccine immune serum.

[0205] This embodiment further investigated the ADE effect (anti-deduction effect) of Ad(EfcN)-E dengue virus type 1 and 2 vaccine-immunized serum in promoting Zika virus infection in a mouse model. The serum used included Ad(EfcN)-E Mix immune serum, with Ad empty immune serum as a negative control, and convalescent serum from dengue virus type 1 and 2 infections (DENV-1 sera, DENV-2 sera) as positive controls. A healthy control group was also included. Experimental grouping and methods were as described in Example 12. After serum transfer, mice were challenged with Zika virus via intraperitoneal injection at a dose of 1 × 10⁵ FFU / mouse. Figure 21 As shown in Figure A, all mice transferred to control serum died on day 9 after challenge; all mice transferred to Ad(EfcN)-E Mix immune serum also died on day 9; in contrast, all mice transferred to convalescent serum from dengue virus type 1 infection died on day 9, while all mice transferred to convalescent serum from dengue virus type 2 infection died on day 7. Figure 21 As shown in Figure B, on day 4 post-challenge, mice transferred with convalescent serum from dengue virus type 1 or 2 infection had significantly higher viral loads than other mice, indicating a clear ADE effect. Mice transferred with Ad(EfcN)-E Mix immune serum had viral loads comparable to those transferred with negative serum, showing no ADE effect. Therefore, Ad(EfcN)-E dengue virus vaccine immunization does not lead to an ADE effect against Zika virus.

[0206] Example 17: Evaluation of antibody response induced by oral immunization with Ad(EfcN)-E dengue virus type 2 vaccine.

[0207] Oral vaccination offers several advantages over intramuscular injection: convenient administration, low cost, and fewer toxic side effects. This example evaluates the immunization efficacy of oral administration of Ad(EfcN)-E and Ad(EfcN). Six-week-old female C57BL / 6 mice were divided into four groups and received the following vaccination regimens: Group 1 received three oral doses of Ad(EfcN) at three-week intervals; Group 2 received three oral doses of Ad(EfcN)-E at three-week intervals; Group 3 received two intramuscular injections of Ad(EfcN)-E at three-week intervals; and Group 4 received one intramuscular injection of Ad(EfcN)-E followed by two oral doses at three-week intervals. The vaccine was dengue virus type 2 vaccine, with an immunization dose of 1×10⁻⁶. 10 vp / animal / dose. Two weeks after each immunization, blood was collected from the orbital rim and serum was separated. The titer of anti-dengue virus EDIII IgG antibodies in the serum was detected by ELISA. Results are as follows: Figure 22 As shown, a single oral dose of Ad(EfcN)-E or Ad(EfcN) vaccine induces only a weak IgG antibody response, with a slightly higher titer in the Ad(EfcN) vaccine group. A single intramuscular injection of Ad(EfcN)-E induces a stronger IgG antibody response. After two oral doses of Ad(EfcN)-E or Ad(EfcN) vaccine, the IgG antibody titer increases significantly, but remains lower than that induced by two intramuscular injections or an intramuscular injection plus oral administration regimen. Notably, three oral doses of Ad(EfcN)-E or Ad(EfcN) vaccine induce IgG antibody titers comparable to those induced by two intramuscular injections of Ad(EfcN)-E vaccine, while the intramuscular injection plus oral administration regimen induces the highest IgG antibody titer. These results indicate that oral Ad(EfcN)-E or Ad(EfcN) vaccine can be used as a booster shot to an intramuscular vaccine. We have found that the vaccine described in this invention has the potential to revolutionize the application of dengue virus vaccines and similar treatments.

[0208] Example 18: Evaluation of antibody response induced by nasal drop immunization with Ad(EfcN)-E dengue virus type 2 vaccine.

[0209] Nasal drops or sprays are another simple, convenient, and inexpensive method of vaccination. This example evaluates the immunogenicity of Ad(EfcN)-E administered via nasal drops. Six-week-old female C57BL / 6 mice were divided into three groups and received the following vaccination regimens: Group 1 received a single intramuscular injection of Ad(EfcN)-E; Group 2 received a single nasal drop of Ad(EfcN)-E; and Group 3 received a single nasal drop of Ad(EfcN)-E combined with CpG1018 adjuvant (Cat.#5132506560). The vaccine was a dengue virus type 2 vaccine prepared according to the preparation method described in Examples 2-3 of this invention, with an immunization dose of 1×10⁻⁶. 10 vp / animal. Blood was collected from the orbital rim of the animal 2 and 3 weeks post-immunization, and serum was separated. The serum IgG antibody titer against dengue virus EDIII was detected by ELISA. Results are as follows: Figure 23 As shown, two weeks post-immunization, intramuscular Ad(EfcN)-E vaccine induced a strong IgG antibody response; nasal drop vaccination also produced a significant antibody response, but the titer was lower than that of intramuscular vaccination; CpG adjuvant enhanced the immunogenicity of the nasal drop vaccine. Three weeks post-immunization, the IgG antibody level in the nasal drop group increased, approaching the antibody level in the intramuscular injection group.

[0210] Therefore, the Ad(EfcN)-E vaccine can also induce a specific antibody response via nasal drop immunization.

[0211] Example 19: Evaluation of the immunogenicity and protective effect of the circular RNA dengue virus type 2 vaccine.

[0212] This embodiment further tested the immunogenicity and protective effect of the dengue virus EDIII antigen encoded by circular RNA. Three EDIII antigens were designed: EDIII monomer; EDIII dimer (SEQ ID NO:3), i.e., EDIII-Fc; and EDIII trimer (EDIII monomer C-terminus fused with a trimerizing tag), i.e., EDIII-Fd. The coding sequences of the above antigens and the type I intron circularized sequence of thymidylate synthase (td) derived from T4 phage (Chen, Robert et al. “Engineering circular RNA for enhanced protein production.” Nature biotechnology vol. 41,2 (2023): 262-272. doi:10.1038 / s41587-022-01393-0) were integrated into the circular RNA expression plasmid backbone pGEM-T-Easy (purchased from addgene). The plasmid was linearized, and linear precursor RNA was obtained by in vitro transcription. 2+In the presence of [specific conditions], linear precursor RNA spontaneously circularizes to form circular RNA. After purification by HPLC, the circular RNA was encapsulated with lipid nanoparticles (LNPs) (the preparation of circRNA-LNPs follows existing techniques, e.g., see: Liu, Xinglong et al. “A single-dose circular RNA vaccine prevents Zika virus infection without enhancing dengue severity in mice.” Nature communications vol. 15, 18932. 16 Oct. 2024, doi:10.1038 / s41467-024-53242-0). IFNaR- / - C57BL / 6 mice were immunized with the above vaccine formulation twice, three weeks apart. Three weeks after the last immunization, the serum neutralizing antibody titer was measured. LNPs served as the control group without RNA encapsulation. Figure 24 As shown in Figure A, the EDIII-Fc circular RNA vaccine induced the strongest neutralizing antibody response, followed by EDIII-Fd, while the EDIII monomeric vaccine response was weaker. When immunized mice were challenged with dengue virus type 2, all mice in the EDIII-Fc and EDIII-Fd circular RNA vaccine groups survived completely, while only a portion of the mice in the EDIII monomeric circular RNA vaccine group survived. Figure 24 Mice were dissected on day 5 after challenge with the virus to detect viral load in the brain. All three vaccines significantly reduced viral infection, with the EDIII-Fc circular RNA vaccine showing the best protective effect. Figure 24 (C). This result indicates that the EDIII-Fc antigen can also be prepared as a circular RNA vaccine.

[0213] The EDIII monomer is fused to a trimerized tag at its C-terminus, T4-folden, which is derived from the C-terminal domain of phage T4 fibrin.

[0214] The amino acid sequence is: GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:18)

[0215] The nucleic acid sequence is: GGCTACATCCCTGAGGCCCCCAGGGATGGCCAGGCCTATGTGAGGAAGGATGGCGAGTGGGTGCTGCTGTCCACCTTCCTG (SEQ ID NO:19).

[0216] Example 20: Connecting peptide insertion site.

[0217] In our study, we found that the appropriate insertion sites for the linker peptides on the Hexon protein are HVR1, HVR2, and HVR5.

[0218] Sequence alignment analysis of Hexon proteins from different adenovirus types identified Hexon HVR1, 2, 5, and 7 as insertion regions for the linker peptides, located after amino acids 136 (HVR1), 188 (HVR2), 268 (HVR5), and 422 (HVR7) of the Hexon protein, respectively. In this embodiment, DogTag was selected as the linker peptide on the surface of the display vector. The DogTag-encoding DNA sequence was artificially synthesized and inserted into the pGK5 plasmid using a seamless cloning method. This DNA sequence was then inserted into the HVR1, 2, 5, and 7 regions of the pAd5ΔE1ΔE3 genome plasmid via homologous recombination in *E. coli*. Subsequently, the open reading frames encoding the reporter genes Gluc and Tomato were integrated into the E1 region of the adenovirus via the shuttle plasmid pGA1. The resulting recombinant pAd5 genome plasmid was linearized and transfected into HEK293 cells to rescue the recombinant virus. After the P0 generation virus was collected, it was passaged in HEK293 cells. After 24 hours, observation of cell pathology and the expression of the reporter gene Tomato under a fluorescence microscope determined that the virus with DogTag inserted at HVR1, 2, and 5 was successfully rescued, while the virus with DogTag inserted at HVR7 would affect viral assembly and lead to rescue failure. Figure 25 The insertion sites for the linker peptides on the Hexon protein are HVR1, HVR2, and HVR5.

[0219] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. An antigen combination, characterized in that, It is selected from at least one combination of the following groups 1)-3): 1) a antigen: The EDIII domain of dengue virus type 1 fused to the C' terminus with a dimerized tag, the amino acid sequence of which is shown in SEQ ID NO: 10, or its encoding DNA sequence as shown in SEQ ID NO:

1. b antigen: The NS1 protein of dengue virus type 1 with a signal peptide fused to its N' terminus, the amino acid sequence of which is shown in SEQ ID NO: 11, or its encoding DNA sequence as shown in SEQ ID NO: 2, and c antigen: EDIII monomer of dengue virus type 1 fused with N'-terminal pairing linker peptide, said EDIII monomer as shown in SEQ ID NO: 7; 2) a antigen: The EDIII domain of dengue virus type 2 fused to the C' terminus with a dimerized tag, the amino acid sequence of which is shown in SEQ ID NO: 12, or its encoding DNA sequence as shown in SEQ ID NO:

3. b antigen: The NS1 protein of dengue virus type 2 with a signal peptide fused to its N' terminus, the amino acid sequence of which is shown in SEQ ID NO: 13, or its encoding DNA sequence as shown in SEQ ID NO: 4, and c antigen: EDIII monomer of dengue virus type 2 fused with N'-terminal pairing linker peptide, said EDIII monomer as shown in SEQ ID NO: 8; 3) a antigen: The EDIII domain of Zika virus with a dimerized tag fused to its C' terminus, the amino acid sequence of which is shown in SEQ ID NO: 14, or its coding sequence as shown in SEQ ID NO:

5. b antigen: The Zika virus NS1 protein with a signal peptide fused to its N' terminus, its amino acid sequence as shown in SEQ ID NO: 15, or its encoding DNA sequence as shown in SEQ ID NO: 6, and c antigen: Zika virus EDIII monomer with a pairing linker peptide fused to its N' terminus, said EDIII monomer as shown in SEQ ID NO: 9; The pairing linker peptide is used to link the EDIII monomer to a carrier of either the a antigen or the b antigen.

2. The antigen combination according to claim 1, characterized in that The a antigen and b antigen are encoded by two separate open reading frames; or the coding sequences of the a antigen and b antigen are encoded by the same open reading frame, and the two are linked by a sequence encoding a self-splicing linker peptide, preferably the self-splicing linker peptide is a 2A linker peptide.

3. The antigen combination according to claim 1, characterized in that The pairing linker peptide is a DogCatcher, SpyCatcher, or SnoopCatcher, which can pair and link with the corresponding DogTag, SpyTag, or Snooptag.

4. A nucleic acid molecule for encoding an antigen combination according to any one of claims 1-3, wherein the nucleic acid molecule is DNA encoding an antigen, mRNA encoding an antigen, or circular RNA encoding an antigen.

5. A vector comprising an expression vector and a display vector, characterized in that, The expression vector carries the nucleic acid molecule encoding the a antigen and / or b antigen as described in claim 1; and the display vector displays the amino acid sequence shown by the c antigen on its surface; the expression vector and the display vector are the same vector or two different vectors.

6. The carrier according to claim 5, characterized in that, The expression vector is a recombinant viral vector, an RNA vector, or a DNA vector, with the preferred recombinant viral vector being a replication-defective adenovirus vector.

7. The carrier according to claim 6, characterized in that, The replication-defective adenovirus vector is a replication-defective type 5 adenovirus vector (Ad5), and the open reading frame of the nucleic acid molecule is inserted into the E1 or E3 region of the Ad5 vector.

8. The carrier according to claim 6, characterized in that, The RNA vector is a circular RNA or mRNA.

9. The carrier according to claim 5, characterized in that, The expression vector and the display vector are the same vector, preferably an adenovirus vector.

10. The use of the antigen combination according to any one of claims 1-3, or the nucleic acid molecule according to claim 4, or the vector according to any one of claims 5-9 in the preparation of dengue virus and / or Zika virus vaccines.

11. A vaccine characterized by, Its active ingredients include the antigen combination according to any one of claims 1-3, or the nucleic acid molecule according to claim 4, or the carrier according to any one of claims 5-9.

12. The vaccine according to claim 11, characterized in that, It also includes pharmaceutically acceptable diluents, excipients, adjuvants, or delivery carriers.

13. The method for preparing the vaccine according to claim 11, characterized in that, The preparation method includes the following steps: S1. Insert the coding DNA sequence of DogTag into the pGK5 plasmid carrying the Hexon HVR region. Through homologous recombination in E. coli, insert the coding DNA sequence into the HVR1, HVR2, or HVR5 region of the pAd5ΔE1ΔE3 genome plasmid to obtain the pAd5ΔE1ΔE3-Hm1, pAd5ΔE1ΔE3-Hm2, or pAd5ΔE1ΔE3-Hm5 plasmids. S2. In the E1 and E3 regions of the adenovirus, the open reading frames encoding antigen a and antigen b are integrated into the shuttle plasmids pGA1 or pGK53, respectively, and homologous recombination is performed with the plasmids pAd5ΔE1ΔE3-Hm1, pAd5ΔE1ΔE3-Hm2, or pAd5ΔE1ΔE3-Hm5 to obtain the pAd5(EfcN) plasmid carrying the EDIII dimer and NS1 open reading frame; S3. The obtained pAd5(EfcN) plasmid was linearized and transfected into HEK293 cells to rescue and produce Ad(EfcN) recombinant virus; S4. The antigen c expressed in vitro by Escherichia coli or mammalian cells is linked to the surface of the recombinant virus Ad(EfcN) obtained in S3.

14. The preparation method according to claim 13, characterized in that, Step S2 includes: S2.1 The DNA coding sequences of the EDIII dimer of dengue virus type 1, dengue virus type 2, and Zika virus are respectively constructed into the E1 region shuttle plasmid to obtain shuttle plasmids carrying the open reading frames of the EDIII dimer. S2.2 After the shuttle plasmid is linearized by double enzyme digestion, it is homologously recombinated with the linearized pAd5ΔE1ΔE3-Hm5 plasmid to obtain pAd5(Efc) carrying the open reading frame of the viral EDIII dimer. S2.3 The DNA coding sequences of NS1 of dengue virus type 1, dengue virus type 2, and Zika virus are respectively constructed into the E3 region shuttle plasmid pGK53 to obtain pGK53-NS1 shuttle plasmids carrying the NS1 open reading frame. After double digestion and linearization of the S2.4 pGK53-NS1 shuttle plasmid, the linearized pAd5(Efc) was subjected to homologous recombination, and after purification, Ad5(EfcN) viral particles carrying the EDIII dimer and NS1 open reading frame were obtained.

15. The preparation method according to claim 13 or 14, characterized in that, The conjugation ratio of antigen c to the surface of the recombinant virus Ad (EfcN) is 20%-90%.

16. The preparation method according to claim 15, characterized in that, In step S4, the Ad(EfcN) recombinant virus and the EDIII monomer are mixed in the following ratio according to the number of Hexon protein monomers and DogCatcher-EDIII monomers: 1:0.25-3; preferably: 1:0.25-1.

17. The use of the vaccine according to any one of claims 11-12 in the preparation of a medicament for the prevention or treatment of dengue virus and / or Zika virus infection.

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