Preparation method and application of novel coronavirus and dengue virus combined vaccine

Abstract

The invention discloses a preparation method and application of a novel coronavirus and dengue virus combined vaccine, and belongs to the technical field of biology. The invention provides an SARS-COV-2 and DENV chimeric VRP vaccine and a DREP vaccine based on an SINV replicon. The vaccine can generate virus protein with immunogenicity in cells, can induce a mouse to generate an antibody aiming at SARS-CoV-2, and shows neutralizing activity on the SARS-CoV-2 virus and DENV virus at the same time. Compared with a mouse inoculated with a DREP vaccine, an immunization method inoculated with a VRP vaccine is simpler and more convenient, and generation of a higher-level antibody can be induced. The chimeric vaccine provided by the invention can protect human beings or mammals from being infected with novel coronavirus and dengue hemorrhagic fever, so that the novel coronavirus and dengue virus are effectively blocked from spreading and prevalence in people.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the prevention and treatment of novel coronavirus and dengue virus, and specifically to a preparation method and application of a combined vaccine of novel coronavirus and dengue virus. Background Art

[0002] The global spread of the novel coronavirus (SARS-CoV-2) and dengue virus (DENV) poses a serious challenge to public health. Dengue is a mosquito-borne viral disease caused by four serotypes of DENV (DENV-1-4). Among them, DENV-1 and DENV-2 are the two most common serotypes, especially DENV-2, which is the main contributor to the global dengue epidemic and poses a major threat to the global health system. The overlapping epidemic areas of the two increase the risk of co-infection. Especially during the COVID-19 pandemic, cases of co-infection have been reported in many places, and the misdiagnosis rate has increased. Therefore, it is crucial to develop vaccines that can control both SARS-CoV-2 and dengue at the same time. Such vaccines may help improve vaccination efficiency, reduce vaccination doses and costs, and manage overlapping outbreaks, thereby helping to enhance global health security.

[0003] Studies have shown that sera from patients with SARS-CoV-2 can neutralize DENV-2 viruses at high dilutions. In addition, injection of anti-S1-RBD IgG antibodies into mice significantly reduced hemorrhagic symptoms after DENV infection and reduced serum NS1 levels. These studies show partial cross-protection between the two RNA viruses, indicating that combined SARS-CoV-2 and DENV vaccines are a feasible and promising option, and these findings provide a theoretical basis for the development of combined vaccines. Significant progress has been made in the development of new crown vaccines. Vaccines targeting the receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 spike (S) protein have been widely vaccinated and can effectively curb the spread of the virus. Other studies have shown that the conserved functional domains of the S protein, the heptad repeat sequences 1 and 2 (HR1 and HR2), can induce broadly neutralizing antibodies and strong immune responses in various animals, and are new and promising immunogenic targets. Vaccines using these proteins almost completely protect against SARS-CoV-2 infection in mouse and non-human primate models. However, the development of an effective dengue vaccine remains challenging. Although several vaccines are currently undergoing clinical trials, safe and effective vaccines for controlling dengue outbreaks are still not available. Dengvaxia (CYD-TDV), the only licensed dengue vaccine, is a live attenuated vaccine that uses the yellow fever virus genome as a backbone, in which the PrM and E protein coding sequences are replaced by those of DENV. However, this vaccine may increase the risk of dengue infection and severe illness and hospitalization in children who have never been infected with dengue after vaccination due to antibody-dependent enhancement (ADE). Therefore, the effectiveness of this vaccine is limited to people who have been previously infected with dengue and live in dengue-endemic areas. Antibodies against the DENV structural protein E, particularly the envelope protein domain 3 (80E) fragment, can prevent viral attachment and invasion. In addition, vaccination with the nonstructural protein NS1 can generate antibodies that reduce viral replication without causing ADE. Therefore, 80E and NS1 are promising candidates for dengue vaccine development.

[0004] In vaccine development, the choice of delivery platform is crucial. Alphavirus-based DNA-initiated self-replicating RNA replicon vaccines (DREPs) show good stability and can be stored for a long time at room temperature without freezing during storage and transportation, so they have significant advantages over traditional vaccines in responding to global pandemics. DREP vaccines based on Sindbis virus (SINV) can activate innate and adaptive immune responses and effectively induce the production of virus neutralizing antibodies. In addition, the viral replicon particle delivery system (VRP) replaces the coding sequence of viral structural proteins with the coding sequence of exogenous target proteins, thereby producing self-amplified RNA and achieving large-scale translation of the corresponding antigen. Replicon RNA lacks viral structural genes and requires co-transfection with auxiliary plasmids for effective packaging, so it does not have replication ability and is suitable for large-scale vaccine production. In addition, the DREP and VRP systems can encode any target antigen and provide a consistent upstream supply chain and downstream process for each vaccine product, thereby accelerating the development and deployment of vaccines.

[0005] Given the significant impact of dengue fever and the new coronavirus on global public health, as well as the key role of vaccines in the prevention and control of infectious diseases, and the fact that a combined vaccine for dengue fever and the new coronavirus has not yet been developed, it is particularly important to develop a combined vaccine that can prevent both diseases at the same time. This combined vaccine can not only improve the efficiency and coverage of vaccination, reduce the number of vaccinations and costs, but also effectively respond to the risks of overlapping epidemics that may occur in the future, providing a more solid guarantee for global public health security. Summary of the invention

[0006] The object of the present invention is to provide an antigen combination containing a novel coronavirus antigen and a dengue virus antigen. The novel coronavirus antigen is selected from the RBD domain or the HR1HR2 domain of the S protein; the dengue virus antigen is selected from the NS1 protein or the 80E protein, and the dengue virus includes DENV-2 and DENV-1.

[0007] In some embodiments, the amino acid sequence of the RBD domain is as shown in SEQ ID NO.3, and the encoding nucleotide sequence is preferably as shown in SEQ ID NO.2; the amino acid sequence of the HR1HR2 domain is as shown in SEQ ID NO.5, and the encoding nucleotide sequence is preferably as shown in SEQ ID NO.4.

[0008] In some embodiments, the amino acid sequence of the DENV-2 NS1 protein (DV2-NS1) is as shown in SEQ ID NO.7, and the encoding nucleotide sequence is preferably as shown in SEQ ID NO.6; the amino acid sequence of the DENV-2 80E protein (DV2-80E) is as shown in SEQ ID NO.9, and the encoding nucleotide sequence is preferably as shown in SEQ ID NO.8.

[0009] In some embodiments, the amino acid sequence of the NS1 protein (DV1-NS1) of DENV-1 is as shown in SEQ ID NO.11, and the encoding nucleotide sequence is preferably as shown in SEQ ID NO.10; the amino acid sequence of the 80E protein (DV1-80E) of DENV-1 is as shown in SEQ ID NO.13, and the encoding nucleotide sequence is preferably as shown in SEQ ID NO.12.

[0010] The present invention also aims to provide a SINV replicon plasmid expressing the above antigen combination and a preparation method thereof.

[0011] The method for preparing the SINV replicon plasmid expressing the above antigen combination comprises the following steps:

[0012] (1) Construction of SINV replicon plasmid: The DNA fragment with the sequence shown in SEQ ID NO.1 was digested with SacⅠ and ApaⅠ and ligated to the pDNA3.1 vector to obtain the SINV replicon plasmid.

[0013] (2) Constructing a SINV replicon plasmid expressing the above antigen combination: inserting nucleotides encoding the above antigen combination into a SINV replicon plasmid to obtain a SINV replicon plasmid expressing the above antigen combination.

[0014] In some embodiments, step (2) specifically includes the following steps: using the RBD synthetic sequence as a template, amplifying it with F1 and R1 primers to obtain PCR1. Using the HR1HR2 synthetic sequence as a template, amplifying it with F2 and R2 primers to obtain PCR2. Using the DV2-NS1 synthetic sequence as a template, amplifying it with F3 and R3 primers to obtain PCR3. Using the DV2-80E synthetic sequence as a template, amplifying it with F4 and R4 primers to obtain PCR4. Using the DV1-NS1 synthetic sequence as a template, amplifying it with F5 and R5 primers to obtain PCR5. Using the DV1-80E synthetic sequence as a template, amplifying it with F6 and R6 primers to obtain PCR6. Using PCR1 and PCR3 as templates, overlapping extension PCR using F1 and R3 primers to obtain PCR7. Using PCR1 and PCR4 as a template, overlapping extension PCR using F1 and R4 primers to obtain PCR8. PCR9 was obtained by overlapping extension PCR using PCR1 and PCR5 as templates and primers F1 and R5. PCR10 was obtained by overlapping extension PCR using PCR1 and PCR6 as templates and primers F1 and R6. PCR11 was obtained by overlapping extension PCR using PCR2 and PCR3 as templates and primers F2 and R3. PCR12 was obtained by overlapping extension PCR using PCR2 and PCR4 as templates and primers F2 and R4. PCR13 was obtained by overlapping extension PCR using PCR2 and PCR5 as templates and primers F2 and R5. PCR14 was obtained by overlapping extension PCR using PCR2 and PCR6 as templates and primers F2 and R6. PCR7, PCR8, PCR9, PCR10, PCR11, PCR12, PCR13 and PCR14 were respectively digested with XbaⅠ and NotⅠ and connected to SINV replicon plasmids to obtain SINV replicon plasmids expressing different antigen combinations.

[0015] The above primer sequences are as follows:

[0016] F1: 5'-TTTGCTCTAGAGCGCGTGCAGCCCACCGA-3';

[0017] R1: 5'-ACCGCTGCTACCGCTGCTGAAGTTCACGCACTT-3'.

[0018] F2: 5'-TTTGCTCTAGAGACCCAGAACGTGCTGTACG-3';

[0019] R2: 5'-accgctgctaccgctgctGTACTTGCCCAGCTCCTG-3'.

[0020] F3: 5'-AGCAGCGGTAGCAGCGGTgatagtggttgcgttgtg-3';

[0021] R3: 5'-TGCTCGCGGCCGCagctgtgaccaaggagtt-3'.

[0022] F4: 5'-AGCAGCGGTAGCAGCGGTatgcgttgcataggaatg-3';

[0023] R4: 5'-TGCTCGCGGCCGCtttcttaaaccagttgagcttca-3'.

[0024] F5: 5'-AGCAGCGGTAGCAGCGGTgattcgggatgtgtaatc-3';

[0025] R5: 5'-CTGCTCGCGGCCGCtgcagagaccattgactt-3'.

[0026] F6: 5'-AGCAGCGGTAGCAGCGGTatgcgatgcgtgggaata-3';

[0027] R6: 5'-CTGCTCGCGGCCGCgcttcctttcttgaacca-3'.

[0028] The present invention also aims to provide a SINV replicon pseudovirus expressing the above antigen combination and a preparation method thereof.

[0029] The method for preparing the SINV replicon pseudovirus expressing the above antigen combination comprises the following steps: transfecting the SINV replicon plasmid expressing the above antigen combination, the SINV replicon helper plasmid, and the plasmid expressing T7 RNA polymerase into cells for cultivation, collecting the cell supernatant, filtering, and obtaining the SINV replicon pseudovirus expressing the above antigen combination.

[0030] In some embodiments, the method for constructing the SINV replicon helper plasmid comprises the following steps: a DNA fragment with a sequence as shown in SEQ ID NO.14 is digested with SacⅠ and ApaⅠ and connected to a pDNA3.1 vector to obtain a SINV replicon helper plasmid.

[0031] In some embodiments, the method for constructing a plasmid expressing T7 RNA polymerase comprises the following steps: using the T7 RNA polymerase coding sequence as a template, amplifying with T7 polymerase-F and T7 polymerase-R primers to obtain a PCR product, and connecting the PCR product to the pDNA3.1 vector via EcoRⅠ and XhoⅠ to obtain a plasmid expressing T7 RNA polymerase.

[0032] The above primer sequences are as follows:

[0033] T7 polymerase-F: 5'-CAGCGAATTCatgaacaccatcaatattgcc-3';

[0034] T7 polymerase-R: 5'-GCTGCTCGAGttaggcaaatgcgaaatcgg-3'.

[0035] A plasmid combination for preparing a SINV replicon pseudovirus expressing the above antigen combination comprises: a SINV replicon plasmid expressing the above antigen combination, a SINV replicon helper plasmid, and a plasmid expressing T7 RNA polymerase.

[0036] The purpose of the present invention is also to provide the use of the above-mentioned antigen combination, the SINV replicon plasmid expressing the above-mentioned antigen combination, the SINV replicon pseudovirus expressing the above-mentioned antigen combination or the above-mentioned plasmid combination in the preparation of a new coronavirus and dengue virus combined vaccine. The vaccine includes a DREP vaccine and a VRP vaccine. RBD-DV2-NS1, RBD-DV1-80E and HR1HR2-DV1-NS1 of the DREP vaccine group, and RBD-DV2-NS1, RBD-DV2-80E and HR1HR2-DV2-NS1 of the VRP vaccine group have a good protective effect against SARS-CoV-2. Except for the HR1HR2-DV-2-80E vaccine in DREP, other vaccines containing DENV-2 antigens have a good protective effect against DENV-2.

[0037] The present invention also aims to provide a novel coronavirus and dengue virus combined vaccine, wherein the combined vaccine comprises the above-mentioned SINV replicon plasmid or the above-mentioned SINV replicon pseudovirus, which can produce the above-mentioned antigen combination in cells.

[0038] Advantages and beneficial effects of the present invention: The present invention provides a chimeric VRP vaccine and DREP vaccine of SARS-COV-2 and DENV based on SINV replicon, which can produce immunogenic viral proteins in cells and can induce mice to produce antibodies against SARS-CoV-2. RBD-DV2-NS1 in the DREP vaccine injection group of the present invention, RBD-DV2-NS1, RBD-DV2-80E and HR1HR2-DV2-NS1 in the VRP vaccine injection group simultaneously show neutralizing activity against both SARS-CoV-2 virus and DENV-2 virus. Whether it is DREP vaccine or VRP vaccine, the antigen combination of RBD-DV2-NS1 has a significant neutralizing effect on the new coronavirus and dengue virus. In addition, compared with mice vaccinated with DREP vaccine, the immunization method of vaccinating with VRP vaccine is simpler and can induce higher levels of antibody production. In general, the chimeric vaccine provided by the present invention can protect humans or mammals from infection with new coronavirus and dengue hemorrhagic fever, thereby effectively blocking the spread and prevalence of new coronavirus and dengue virus in the population. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a diagram of the transformation scheme of the SINV full-length sequence for constructing the SINV replicon plasmid.

[0040] Figure 2 This is a schematic diagram of the combination of different antigens of SARS-CoV-2 and DENV.

[0041] Figure 3 This is a diagram of the transformation scheme for constructing the SINV replicon auxiliary plasmid.

[0042] Figure 4 This is a picture of the antigen expression of DNA replicon vaccine detected by Western blot.

[0043] Figure 5 This is a diagram of DNA replicon vaccine antigen expression detected by immunofluorescence.

[0044] Figure 6 This is a picture of Western blot detection of pseudovirus vaccine antigen expression.

[0045] Figure 7 This is a graph showing the titer of pseudovirus vaccine detected by plaque assay.

[0046] Figure 8 This is a diagram of the replication kinetics of the pseudovirus vaccine.

[0047] Fig. 9 This is a graph of the levels of IgG antibodies in mice after vaccination.

[0048] Fig.10 This is a graph showing the weight changes of mice after vaccination.

[0049] Fig.11 This is a graph of the neutralizing antibody titer of SARS-COV-2 S protein antibodies in the serum of immunized mice.

[0050] Fig.12 This is an immunofluorescence detection chart of the vaccine's effectiveness in neutralizing SARS-CoV-2.

[0051] Fig.13 This is a graph showing the effectiveness of the vaccine in neutralizing SARS-CoV-2 using qPCR detection.

[0052] Fig.14 This is a picture showing the effectiveness of the vaccine in neutralizing DENV-2 using a plaque assay. DETAILED DESCRIPTION

[0053] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0054] Example 1 Construction and verification of DNA and RNA vaccines

[0055] 1. Construction of SINV replicon plasmid

[0056] A DNA fragment of the sequence shown in SEQ ID NO.1 was synthesized. The sequence shown in SEQ ID NO.1 is a modification of the full-length sequence of Sindbis virus (SINV, hrsp strain). The modification scheme is as follows: Figure 1 The synthesized DNA fragment was digested with SacⅠ and ApaⅠ and connected to the commercial vector pDNA3.1 to obtain the SINV replicon plasmid.

[0057] 2. Construction of SINV replicon plasmids with different antigens

[0058] The RBD and HR1HR2 domains of the S protein of SARS-CoV-2, as well as the NS1 and 80E proteins of DENV-1 and DENV-2 were selected for antigen combination, and then plasmid construction was performed. According to the vaccine design plan, the combined sequences of different antigens were inserted into the SINV replicon plasmid. The schematic diagram of the antigen combination design is shown in the figure. Figure 2 shown.

[0059] The nucleotide sequence of RBD is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.3. The nucleotide sequence of HR1HR2 is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.5. The nucleotide sequence of NS1 of DENV-2 is shown in SEQ ID NO.6, and the amino acid sequence is shown in SEQ ID NO.7. The nucleotide sequence of 80E of DENV-2 is shown in SEQ ID NO.8, and the amino acid sequence is shown in SEQ ID NO.9. The nucleotide sequence of NS1 of DENV-1 is shown in SEQ ID NO.10, and the amino acid sequence is shown in SEQ ID NO.11. The nucleotide sequence of 80E of DENV-1 is shown in SEQ ID NO.12, and the amino acid sequence is shown in SEQ ID NO.13.

[0060] Step 1: Using the RBD synthetic sequence as a template, amplify it with primers F1 and R1 to obtain PCR1. Step 2: Using the HR1HR2 synthetic sequence as a template, amplify it with primers F2 and R2 to obtain PCR2. Step 3: Using the DV2-NS1 synthetic sequence as a template, amplify it with primers F3 and R3 to obtain PCR3. Step 4: Using the DV2-80E synthetic sequence as a template, amplify it with primers F4 and R4 to obtain PCR4. Step 5: Using the DV1-NS1 synthetic sequence as a template, amplify it with primers F5 and R5 to obtain PCR5. Step 6: Using the DV1-80E synthetic sequence as a template, amplify it with primers F6 and R6 to obtain PCR6. Step 7: Using PCR1 and PCR3 as templates, overlap extension PCR using primers F1 and R3 to obtain PCR7. Step 8: Using PCR1 and PCR4 as a template, overlap extension PCR using primers F1 and R4 to obtain PCR8. Step 9: Using PCR1 and PCR5 as templates, PCR9 was obtained by overlapping extension PCR using primers F1 and R5. Step 10: Using PCR1 and PCR6 as templates, PCR10 was obtained by overlapping extension PCR using primers F1 and R6. Step 11: Using PCR2 and PCR3 as templates, PCR11 was obtained by overlapping extension PCR using primers F2 and R3. Step 12: Using PCR2 and PCR4 as templates, PCR12 was obtained by overlapping extension PCR using primers F2 and R4. Step 13: Using PCR2 and PCR5 as templates, PCR13 was obtained by overlapping extension PCR using primers F2 and R5. Step 14: Using PCR2 and PCR6 as templates, PCR14 was obtained by overlapping extension PCR using primers F2 and R6. Step 15: PCR7, PCR8, PCR9, PCR10, PCR11, PCR12, PCR13 and PCR14 were respectively connected to the SINV replicon plasmid constructed in the previous step through XbaⅠ and NotⅠ restriction enzyme digestion, that is, eight SINV replicon plasmids carrying different antigens.

[0061] The above primer sequences are as follows:

[0062] F1: 5'-TTTGCTCTAGAGCGCGTGCAGCCCACCGA-3';

[0063] R1: 5'-ACCGCTGCTACCGCTGCTGAAGTTCACGCACTT-3'.

[0064] F2: 5'-TTTGCTCTAGAGACCCAGAACGTGCTGTACG-3';

[0065] R2: 5'-accgctgctaccgctgctGTACTTGCCCAGCTCCTG-3'.

[0066] F3: 5'-AGCAGCGGTAGCAGCGGTgatagtggttgcgttgtg-3';

[0067] R3: 5'-TGCTCGCGGCCGCagctgtgaccaaggagtt-3'.

[0068] F4: 5'-AGCAGCGGTAGCAGCGGTatgcgttgcataggaatg-3';

[0069] R4: 5'-TGCTCGCGGCCGCtttcttaaaccagttgagcttca-3'.

[0070] F5: 5'-AGCAGCGGTAGCAGCGGTgattcgggatgtgtaatc-3';

[0071] R5: 5'-CTGCTCGCGGCCGCtgcagagaccattgactt-3'.

[0072] F6: 5'-AGCAGCGGTAGCAGCGGTatgcgatgcgtgggaata-3';

[0073] R6: 5'-CTGCTCGCGGCCGCgcttcctttcttgaacca-3'.

[0074] 3. Construction of SINV replicon virus vaccine helper plasmid

[0075] A DNA fragment having a sequence as shown in SEQ ID NO.14 was synthesized. The sequence as shown in SEQ ID NO.14 is a modification of the full-length sequence of Sindbis virus (SINV, hrsp strain). The modification scheme is as follows: Figure 3 Then the synthesized DNA fragment was digested with SacⅠ and ApaⅠ and connected to the pDNA3.1 commercial vector to obtain the SINV replicon virus vaccine helper plasmid DHBB.

[0076] 4. In vitro experiments to identify vaccine antigen expression

[0077] (1) BHK-21 cell transfection

[0078] The density is 2×10 6 BHK-21 cells were plated into 12-well plates and transfected with 8 SINV replicon plasmids carrying different antigens as described in step 2 using Lipofectamine 2000 (Invitrogen) according to the instructions.

[0079] (2) WB detection of the expression of various antigen combinations

[0080] The above cell proteins were extracted and immunoblotted with internal reference GAPDH (1:3000; Proteintech, 60004-1-Ig) and Flag tag (1:2000; Cell Signaling Technology, 14793T) to detect the expression of various antigen combinations. Figure 4 As shown, the results show that various antigens can be expressed normally.

[0081] (3) IFA detection of the expression of various antigen combinations

[0082] The cells were fixed with 4% formaldehyde for 30 minutes, washed three times with PBS, and blocked with immunostaining blocking solution. The primary antibody of mouse anti-Flag (Cell Signaling Technology, 14793T) was diluted 1:500 and incubated overnight. The cells were washed three times the next day and stained with DAPI fluorescent dye (1:2000, Sigma, D9542) and goat anti-mouse secondary antibody (1:1000, Invitrogen) for 90 minutes. The cells were washed three times with PBST and sealed. The cells were observed using a Leica confocal microscope (nucleus: blue, antigen: green). Figure 5 As shown, the results show that various antigens can be expressed normally.

[0083] 5. In vitro packaging of SINV replicon pseudovirus

[0084] (1) Construction of a plasmid expressing T7 RNA polymerase

[0085] A plasmid containing full-length T7 RNA polymerase was used as a template. The nucleotide sequence of T7 RNA polymerase is shown in GenBank: MK567984.1, and the amino acid sequence is shown in GenBank: QEQ55972.1. The PCR product was amplified with primers T7 polymerase-F and T7 polymerase-R. The PCR product was then ligated to the pDNA3.1 vector via EcoRI and XhoⅠ to obtain the CMV-T7 polymerase plasmid.

[0086] The above primer sequences are as follows:

[0087] T7 polymerase-F: 5'-CAGCGAATTCatgaacaccatcaatattgcc-3';

[0088] T7 polymerase-R: 5'-GCTGCTCGAGttaggcaaatgcgaaatcgg-3'.

[0089] (2) BHK-21 cell transfection

[0090] BHK-21 cells were passaged into T75 cell culture flasks. When the cell confluence reached 60-80%, Lipofectamine 2000 (Invitrogen) was used to co-transfect the above 8 SINV replicon plasmids with different antigens and the auxiliary plasmid DHBB and CMV-T7 polymerase plasmid according to the instructions.

[0091] (3) Pseudovirus collection and WB detection expression

[0092] 48 hours after transfection, the 8 cell supernatants in the above T75 bottles were collected and filtered with a 0.45 μm filter membrane to obtain 8 bivalent pseudovirus vaccines of new coronavirus and dengue virus based on SINV replicons. To test whether the packaged pseudovirus is effective, the cell supernatant was added with loading buffer, and then the antigen expression was detected by WB. Figure 6 As shown, antigen proteins can be detected in all 8 pseudovirus vaccines.

[0093] (4) Pseudovirus vaccine titer detection

[0094] To further prove the effectiveness of the pseudoviruses, the titers of these eight viruses were tested by plaque assay using Vero E6 cells, as shown in Figure 7 As shown, the infectivity titers of all pseudovirus vaccines ranged from 10 7 PFU / mL.

[0095] (5) Replication dynamics detection

[0096] RNA from BHK-21 cells was collected 2, 4, 8, 12, and 16 hours after infection of BHK-21 cells with the eight pseudovirus vaccines, and the kinetics of RNA transcription from various SINV replicons in infected BHK-21 cells was evaluated by RT-qPCR to detect the replication of the pseudovirus vaccines. Figure 8 As shown, RT-qPCR indicated that RNA transcription in the SINV replicon pseudovirus vaccine construct increased dramatically 4-8 h after infection and tended to a stable trend after 8 h.

[0097] Example 2 Immunization of mice with DREP and VRP vaccines

[0098] 1. Preparation of SARS-CoV-2 and DENV mouse antisera

[0099] This experiment used 7-week-old Balb / c female mice as a model to evaluate the effects of different antigen delivery methods in inducing immune responses. The experimental process is as follows:

[0100] First, the day before each SINV replicon plasmid (DREP vaccine) injection, the mice were injected with the immune adjuvant procaine hydrochloride to enhance the intensity and durability of the immune response. Subsequently, according to the established immunization schedule: on the 1st day (primary immunization), the 7th day and the 14th day, each mouse was injected intramuscularly with 100 μL of SINV replicon plasmid containing different antigens at a concentration of 1 μg / μL each time. 21 days after the completion of the immunization, the mouse serum samples were collected, which will be analyzed as specific antibodies induced by the SINV-based DREP vaccine. At the same time, in order to obtain anti-Pre-immune control antibodies, serum was collected from mice that were normally fed and did not receive any immunization treatment at the same time point.

[0101] On the other hand, the prepared SINV replicon pseudovirus vaccine (VRP) containing the same antigen was administered to mice via tail vein injection with an injection volume of 100 μL and a concentration of 10 7 pfu / mL. Similarly, the mice were vaccinated according to the immunization schedule of primary immunization, boost on day 7, and boost again on day 14. Serum samples were collected from these mice on day 21 after the completion of the immunization to analyze the specific antibody levels induced by the SINV replicon pseudovirus vaccine (VRP).

[0102] 2. ELISA to detect IgG antibody levels

[0103] In order to evaluate the immunogenicity of the chimeric vaccine, this embodiment uses an enzyme-linked immunosorbent assay (ELISA) to quantitatively detect the level of IgG antibodies in mice after immunization. First, the well plate was thoroughly washed three times with PBS (PBST) containing 0.1% Tween-20 to remove any impurities that may affect the experimental results. Subsequently, PBST containing 1% bovine serum albumin (BSA) was added as a blocking solution and incubated at room temperature for 1 hour. After the blocking was completed, the blocking solution was removed, and the mouse serum sample was added, and three duplicate wells were set. After incubation at 37°C for 1 hour, the well plate was washed three times with PBST to remove unbound serum components. Next, the goat anti-mouse IgG horseradish peroxidase (HRP)-labeled antibody was diluted in the blocking solution at a ratio of 1:5000 and added to the well plate and incubated at room temperature for 1 hour. Subsequently, the well plate was thoroughly washed five times with PBST to remove unbound HRP-labeled antibodies. Then, tetramethylbenzidine (TMB) substrate solution was added for color development for 10 minutes. After the color development reaction was completed, 100 μL / well of sulfuric acid (H 2 SO 4 ) solution was used to terminate the reaction, and the absorbance value of each well was immediately measured at a wavelength of 450 nm using a microplate reader within 30 minutes.

[0104] The results are as follows Fig. 9 As shown in the figure, after the initial immunization and subsequent booster vaccination, both SINV-based DREP vaccines and VRP vaccines can induce a certain titer of IgG antibodies in mice. It is worth noting that the IgG antibody titer induced by the VRP vaccine injection group was significantly higher than that of the DREP vaccine injection group, indicating that the VRP vector vaccine may have a superior performance in stimulating the immune response of mice.

[0105] 3. Evaluating vaccine safety in mouse models

[0106] During the entire vaccine immunization experiment, in order to evaluate the safety of the vaccine, the weight of the mice was tested and recorded. Fig.10 As shown, compared with the control group of mice, the body weight of the mice injected with the vaccine in each group did not change significantly, and no adverse signs visible to the naked eye were observed in the mice, indicating that the vaccine constructed by the present invention is safe.

[0107] Example 3 Neutralizing Antibody Titers of DREP and VRP Vaccines

[0108] 1. ELISA to detect SARS-CoV-2 antibody titer

[0109] The neutralizing antibody titer of SARS-COV-2 S protein antibody in the sera of immunized mice was quantified using the SARS-CoV-2 S protein neutralizing antibody ELISA detection kit. Fig.11As shown, an inhibition rate greater than 20% compared with the control group was considered to have neutralizing antibodies. These results show that both vaccine vector delivery methods can stimulate mice to produce antibodies. The six antigen combinations of RBD-DV2-NS1, RBD-DV1-80E, HR1HR2-DV1-NS1 in the DREP vaccine injection group and RBD-DV2-NS1, RBD-DV2-80E, HR1HR2-DV2-NS1 in the VRP vaccine injection group had a high S protein neutralization efficiency, so these six groups were selected for the next step of research.

[0110] 2. IFA testing of the SARS-CoV-2 neutralization effect of the above vaccines

[0111] In order to further verify the neutralizing antibody titer produced by mice, this example conducted a SARS-CoV-2 live virus (Omicron) neutralization test. 100 μL of immunized mouse serum was mixed with 100 μL of cell culture medium (containing 10 4 pfu SARS-CoV-2 Omicron virus) was diluted 1:1, incubated at 37°C for 60 minutes, and the mixture was inoculated into Vero E6 cells. Cultured in a 37°C / 5% CO2 incubator for 1 day, the cells were fixed with 4% formaldehyde for 30 minutes, washed three times with PBS, and blocked with immunostaining blocking solution, and incubated overnight with human anti-SARS-CoV-2N protein primary antibody diluted 1:500. After washing three times the next day, staining with DAPI fluorescent dye (1:2000, Sigma, D9542) and goat anti-human secondary antibody (1:1000, Invitrogen) for 90 minutes, washed three times with PBST, and sealed, and observed with a Leica confocal microscope (cell nucleus: blue, S protein: green). Fig.12 As shown, the results showed that compared with the control group, the RBD-DV2-NS1, RBD-DV1-80E, HR1HR2-DV1-NS1 mouse sera in the DREP vaccine injection group and the RBD-DV2-NS1, RBD-DV2-80E, HR1HR2-DV2-NS1 mouse sera in the VRP vaccine injection group effectively neutralized SARS-CoV-2 virus infection in Vero E6 cells.

[0112] 3. qPCR detection of SARS-CoV-2 neutralization effect in the above vaccines

[0113] In order to further verify the neutralizing antibody titer produced by mice, this example conducted a SARS-CoV-2 live virus (Omicron) neutralization test. 100 μL of immunized mouse serum was mixed with 100 μL of cell culture medium (containing 10 4pfu SARS-CoV-2 Omicron virus) was diluted 1:1, incubated at 37°C for 60 minutes, and the mixture was inoculated into Vero E6 cells. Cultured in a 37°C / 5% CO2 incubator for 1 day. The level of SARS-CoV-2 N protein was detected by qPCR after 1 dpi. Fig.13 As shown, compared with the control group, the RBD-DV2-NS1, RBD-DV1-80E, HR1HR2-DV1-NS1 mouse sera of the DREP vaccine injection group and the RBD-DV2-NS1, RBD-DV2-80E, HR1HR2-DV2-NS1 mouse sera of the VRP vaccine injection group effectively neutralized the SARS-CoV-2 virus infection in Vero E6 cells, and the vaccine of the present invention can be used to prevent and treat new coronavirus infection. It is worth noting that the inhibitory effect of the VRP vaccine on viral replication is greater than that of the DREP vaccine. These findings confirm the high efficiency of the VRP vaccine in expressing SARS-CoV-2 antigens and inducing immune responses.

[0114] The primers used to detect the internal reference GAPDH are as follows:

[0115] Upstream primer: 5′-TGTGTCCGTCGTGGATCTGA-3′;

[0116] Downstream primer: 5′-GCTTCACCACCTTCTTGAT-3′.

[0117] The primers used to detect the SARS-CoV-2 N protein are as follows:

[0118] Upstream primer: 5′-GGGGAACTTCTCCTGCTAGAAT-3′;

[0119] Downstream primer: 5′-CAGACATTTTGCTCTCAAGCTG-3′.

[0120] 4. Plaque assay to detect the DENV-2 neutralization effect of the above vaccines

[0121] In order to further verify the neutralizing antibody titer produced by mice, this example conducted a DENV-2 live virus neutralization test. The immune mouse serum was mixed with 10 μL of the immune mouse serum and 10 μL of DENV-2 virus (10 7 pfu / mL) was diluted 1:1 and incubated at 37°C for 30 minutes. After the incubation, the titers of the vaccines in each group after neutralization of DENV-2 were determined using Vero E6 cells in a 24-well plate. Fig.14 As shown, except for the HR1HR2-DV2-80E vaccine in DREP, other vaccines containing DENV-2 antigens can effectively neutralize DENV-2.

[0122] The above results all show that the RBD-DV2-NS1 of the DREP vaccine injection group of the present invention, and the RBD-DV2-NS1, RBD-DV2-80E, and HR1HR2-DV2-NS1 of the VRP vaccine injection group have synergistic effects on the immune effects of the new coronavirus and dengue virus, and can protect humans or mammals from infection with the new coronavirus and dengue hemorrhagic fever, thereby effectively blocking the spread and prevalence of the new coronavirus and dengue virus in the population. Among them, whether it is DREP vaccine or VRP vaccine, the antigen combination of RBD-DV2-NS1 has a significant neutralizing effect on the new coronavirus and dengue virus.

[0123] The above embodiments are only used to help illustrate the present invention. The implementation methods of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An antigen combination containing a novel coronavirus antigen and a dengue virus antigen, Features: The novel coronavirus antigen is selected from the RBD domain or HR1HR2 domain of the S protein; the dengue virus antigen is selected from the NS1 protein or the 80E protein, and the dengue virus includes DENV-2 and DENV-1.

2. The antigen combination according to claim 1, Features: The amino acid sequence of the RBD domain is shown in SEQ ID NO.3; the amino acid sequence of the HR1HR2 domain is shown in SEQ ID NO.

5.

3. The antigen combination according to claim 1, Features: The amino acid sequence of the NS1 protein of DENV-2 is shown in SEQ ID NO.7; the amino acid sequence of the 80E protein of DENV-2 is shown in SEQ ID NO.9; The amino acid sequence of the NS1 protein of DENV-1 is shown in SEQ ID NO.11; the amino acid sequence of the 80E protein of DENV-1 is shown in SEQ ID NO.

13.

4. A SINV replicon plasmid expressing the antigen combination according to any one of claims 1 to 3.

5. The method for preparing the SINV replicon plasmid according to claim 4, It is characterized in that The following steps are involved: (1) The DNA fragment with the sequence shown in SEQ ID NO.1 was digested with SacⅠ and ApaⅠ and ligated to the pDNA3.1 vector to obtain the SINV replicon plasmid; (2) Inserting the nucleotide encoding the antigen combination into the SINV replicon plasmid to obtain the SINV replicon plasmid expressing the antigen combination.

6. A SINV replicon pseudovirus expressing the antigen combination according to any one of claims 1-3.

7. The method for preparing the SINV replicon pseudovirus according to claim 6, It is characterized in that The method comprises the following steps: transfecting a SINV replicon plasmid expressing the antigen combination, a SINV replicon auxiliary plasmid and a plasmid expressing T7 RNA polymerase into cells for cultivation, collecting cell supernatant, filtering and obtaining a SINV replicon pseudovirus expressing the antigen combination.

8. A plasmid combination for preparing a SINV replicon pseudovirus expressing the antigen combination according to any one of claims 1 to 3, It is characterized in that It comprises: a SINV replicon plasmid expressing the antigen combination, a SINV replicon helper plasmid, and a plasmid expressing T7 RNA polymerase.

9. Use of the antigen combination described in any one of claims 1 to 3, the SINV replicon plasmid described in claim 4, the SINV replicon pseudovirus described in claim 6, or the plasmid combination described in claim 8 in the preparation of a combined vaccine of a new coronavirus and dengue virus.

10. A novel coronavirus and dengue virus combined vaccine, Features: It comprises the SINV replicon plasmid according to claim 4 or the SINV replicon pseudovirus according to claim 6.

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