A porcine Gtavirus and porcine encephalomyocarditis virus dual genetic engineering subunit vaccine, and a preparation method and application thereof

Abstract

The application discloses a porcine Gertvirus and porcine encephalitis virus B double gene engineering subunit vaccine and a preparation method and application thereof. The core components of the vaccine are Gertvirus P6E recombinant protein and encephalitis virus B DFN fusion protein, and a recombinant nanoparticle loaded with the antigens. The obtained recombinant protein or recombinant nanoparticle is compounded with an adjuvant to prepare the GETV / JEV double gene engineering vaccine, and the GETV / JEV double gene engineering vaccine can induce strong humoral and cellular immune responses of a host after two immunizations, and effectively protects the body from GETV and JEV infection. The technical scheme of the application provides important reference and technical support for developing the GETV / JEV double vaccine with high safety, high immunogenicity and suitable for large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a bivalent genetically engineered subunit vaccine of porcine gettavirus and porcine encephalitis virus, its preparation method and application. Background Technology

[0002] Getah virus (GETV) and Japanese encephalitis virus (JEV) are both mosquito-borne zoonotic viruses that pose a significant threat to my country's pig industry. GETV infection can cause abortion and stillbirth in sows, as well as diarrhea and death in piglets, while JEV causes abortion and stillbirth in sows and orchitis in boars. The similar epidemiological characteristics and clinical symptoms of the two diseases severely hinder accurate diagnosis and control.

[0003] Currently, there is no commercially available vaccine for GETV, while JEV control mainly relies on inactivated vaccines (P3 strain) and attenuated vaccines (SA14-14-2 strain). However, both JEV vaccines have inherent drawbacks, such as insufficient cross-immunity. Global warming is exacerbating the expansion of mosquito activity ranges, driving the continued spread of GETV and JEV epidemic areas. Furthermore, the broadening of host ranges and genotypic mutations are new epidemic trends, increasing not only the risk of infection in pig herds but also the cross-regional transmission of the virus, posing a serious threat to livestock production safety. Therefore, the development of safe and effective new vaccines is urgently needed.

[0004] Subunit vaccines have attracted widespread attention due to their high safety, controllable purity, and precise display of key antigenic epitopes. To enhance the immunogenicity of subunit vaccines, the rapidly developing artificially designed protein nanoparticle platforms have provided significant breakthroughs for multivalent antigen display in recent years. Among them, mi3 nanoparticles are computationally designed dodecahedral protein nanostructures composed of 60 self-assembled subunits, exhibiting advantages such as high stability, engineerability, and multi-site antigen presentation. By covalently linking antigens to the surface of mi3 nanoparticles, antigen density and immunogenicity can be significantly increased, thereby enhancing the host's immune response.

[0005] In summary, given the lack of a commercially available vaccine for Getta virus (GETV) and the insufficient cross-immunity of existing vaccines against Japanese encephalitis virus (JEV), coupled with the frequent occurrence of co-infections of the two viruses and their similar clinical symptoms leading to difficulties in prevention and control, this invention aims to provide a novel genetically engineered subunit bivalent vaccine based on artificially designed protein nanoparticles (mi3 nanoparticles). By displaying antigens at high density and multiple valences on the surface of stable nanoparticles, immunogenicity is significantly enhanced, thereby achieving safe and efficient "one-shot multi-protection." This is the technical problem that this invention urgently needs to solve. Summary of the Invention

[0006] To overcome the shortcomings and drawbacks of existing technologies, the present invention aims to provide a bivalent genetically engineered subunit vaccine against porcine Gettavirus and Japanese encephalitis virus, its preparation method, and its application. This vaccine uses recombinant Gettavirus P6E protein and Japanese encephalitis virus DFN fusion protein expressed by a mammalian cell expression system as antigens. These antigens are covalently bound to nanoparticles to form recombinant nanoparticles displaying the P6E recombinant protein and the DFN fusion protein. A bivalent genetically engineered subunit vaccine against porcine Gettavirus and Japanese encephalitis virus is prepared using a mixture of the two antigens and an adjuvant, enabling simultaneous prevention of both porcine Gettavirus disease and porcine Japanese encephalitis virus disease.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] In a first aspect, the present invention claims protection for an immunogenic composition, which is the composition described in (a1) or (a2) below:

[0009] (a1) A composition comprising porcine Gettavirus P6E recombinant protein and porcine Japanese encephalitis virus DFN fusion protein;

[0010] (a2) A composition comprising recombinant nanoparticles loaded with porcine Getta virus P6E recombinant protein and recombinant nanoparticles loaded with porcine Japanese encephalitis virus DFN fusion protein;

[0011] The porcine Gettavirus P6E recombinant protein is formed by linking the GETV p62 protein and E1 protein (after removing the transmembrane domain) with a 4×GGGGS Linker sequence.

[0012] The porcine encephalitis virus DFN fusion protein is formed by adding five B cell epitopes of D1 and D2 domains to the N-terminus of the third domain of the encephalitis virus E protein, and then linking it to the 172-352 region of the NS1 protein through a 4×GGGGS Linker sequence.

[0013] The recombinant nanoparticles loaded with porcine Geyta virus P6E recombinant protein are formed by covalently linking porcine Geyta virus P6E recombinant protein to nanoparticles via the SpyTag003 / SpyCatcher003 system.

[0014] The recombinant nanoparticles loaded with porcine encephalitis virus DFN fusion protein are formed by covalently linking the DFN fusion protein to the nanoparticles via the SpyTag003 / SpyCatcher003 system.

[0015] Furthermore, the porcine Geyta virus P6E recombinant protein is a protein with the amino acid sequence shown in SEQ ID NO: 2 or a fusion protein with the same function obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO: 2.

[0016] The porcine encephalitis virus DFN fusion protein is a protein with the amino acid sequence shown in SEQ ID NO: 4 or a fusion protein with the same function obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO: 4.

[0017] The nanoparticles are SpyCatcher003-mi3 nanoparticles, SpyCatcher003-ferritin nanoparticles, or any nanoparticles; the SpyCatcher003-mi3 nanoparticles are proteins with amino acid sequences as shown in SEQ ID NO: 6, or fusion proteins with the same function obtained by attaching protein tags to the N-terminus and / or C-terminus of proteins as shown in SEQ ID NO: 6.

[0018] Secondly, the present invention claims protection for a nucleic acid molecule that is a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO: 1 encoding the porcine Geyta virus P6E recombinant protein in the aforementioned immunogenic composition.

[0019] (b2) A nucleic acid molecule encoding the porcine Japanese encephalitis virus DFN fusion protein in the immunogenic composition of claim 1, as shown in SEQ ID NO: 3.

[0020] Thirdly, the present invention claims protection for a nucleic acid molecule whose nucleotide sequence, as shown in SEQ ID NO: 3, encodes the DFN fusion protein of the aforementioned immunogenic composition of porcine encephalitis virus.

[0021] Fourthly, the present invention seeks protection for expression cassettes, recombinant vectors, or host cells containing the aforementioned nucleic acid molecules.

[0022] Fifthly, the present invention claims protection for the use of the above-described immunogenic composition in the preparation of a bivalent vaccine against porcine Geytavirus and porcine Japanese encephalitis virus.

[0023] Sixthly, the present invention claims protection for a combined vaccine of porcine Gettavirus and porcine Japanese encephalitis virus, the vaccine comprising the aforementioned immunogenic composition and adjuvant.

[0024] Furthermore, in the vaccine, the concentrations of the porcine Geetavirus P6E recombinant protein and the porcine Japanese encephalitis virus DFN fusion protein are each independently 50–200 μg / mL; the concentrations of the recombinant nanoparticles loaded with the porcine Geetavirus P6E recombinant protein and the recombinant nanoparticles loaded with the porcine Japanese encephalitis virus DFN fusion protein are each independently 100–200 μg / mL.

[0025] Furthermore, in the vaccine, the concentration of porcine Gettavirus P6E recombinant protein is 100 μg / mL, and the concentration of porcine Japanese encephalitis virus DFN fusion protein is 100 μg / mL; or, the concentration of recombinant nanoparticles loaded with porcine Gettavirus P6E recombinant protein is 145 μg / mL, and the concentration of recombinant nanoparticles loaded with porcine Japanese encephalitis virus DFN fusion protein is 182 μg / mL.

[0026] Furthermore, the volume ratio of the adjuvant to the immunogenic composition is 1:1 to 1:4; the adjuvant is SMMMIT™ adjuvant, ISA adjuvant, or aluminum salt adjuvant.

[0027] Seventhly, the present invention claims protection for a method for preparing the above-mentioned porcine gettavirus and porcine Japanese encephalitis virus bivalent vaccine, comprising the following steps:

[0028] (1) The porcine gettavirus P6E recombinant protein and the porcine Japanese encephalitis virus DFN fusion protein were expressed and purified in a eukaryotic expression system, respectively;

[0029] (2) Express and purify the SpyCatcher003-mi3 nanoparticles or SpyCatcher003-ferritin nanoparticles in a prokaryotic expression system;

[0030] (3) Under in vitro conditions, the antigen protein purified in step (1) was covalently coupled with the SpyCatcher003-mi3 nanoparticles or SpyCatcher003-ferritin nanoparticles purified in step (2) through the SpyTag003 / SpyCatcher003 system to obtain recombinant nanoparticles loaded with antigens.

[0031] (4) The antigen protein obtained in step (1) and / or the recombinant nanoparticles loaded with antigen obtained in step (3) are mixed with adjuvants in proportion to prepare a vaccine.

[0032] In a specific embodiment of the present invention, the preparation method of the porcine gettavirus P6E recombinant protein and the porcine Japanese encephalitis virus DFN fusion protein includes the following steps:

[0033] Step 1: Take gene fragments with nucleotide sequences as described in SEQ ID NO:1 and SEQ ID NO:3 respectively, ligate them with the expression vector, transform them into competent cells, extract the recombinant plasmid, transfect suspension cells, culture at 37°C for 4 days, and collect the supernatant.

[0034] Step 2: Bind the collected supernatant to strep agarose beads at 4°C for 4-6 hours. Collect the agarose beads by passing the supernatant through a column, remove impurities with Bμffer W, and elute the target protein with 1x BXT. Concentrate the eluted target protein through a Millipore ultrafiltration tube, replace it with binding buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0), and store at -80°C for later use.

[0035] Preferably, the expression vector in step 1 is pcDNA3.4(+) vector, the competent cells are DH5α competent cells, and the suspension cells are CHO cells.

[0036] In a specific embodiment of the present invention, the preparation method of SpyCatcher003-mi3 nanoparticles includes the following steps:

[0037] Step 1: Take the gene fragment with the nucleotide sequence shown in SEQ ID NO:5, ligate it with the expression vector, transform it into DH5α competent cells, extract the plasmid and then transform it into competent cells of the expression strain.

[0038] Step 2: Select single colonies for induction of expression. After expression, collect the bacterial cells, sonicate to disrupt, centrifuge to remove the precipitate, and purify the supernatant by attaching it to a nickel column. Collect the purified protein after a elution process. Replace the eluted target protein with binding buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0) using a Millipore ultrafiltration tube and store at 4°C.

[0039] Preferably, the expression vector in step 1 is the pCold II vector, and the expression strain used is BL21(DE3) with the addition of the TF16 molecular chaperone.

[0040] Eighthly, the present invention claims protection for the use of the described porcine Geeta virus and porcine Japanese encephalitis virus bivalent vaccine in the preparation of a medicament for the prevention of porcine Geeta virus and / or porcine Japanese encephalitis virus infection.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] 1. The recombinant protein prepared in this invention uses a highly safe and stable mammalian cell expression system to express the GETV P6E and JEV DFN fusion proteins. The expressed protein has a high yield, simple purification steps, high purity, is easy to mass-produce, and has controllable product quality. Verification has shown that the expressed protein has good immunogenicity. Compared with currently available attenuated and inactivated vaccines, this invention has significant advantages in safety and production performance. The nanoparticle backbone is expressed solublely using a prokaryotic expression system, resulting in a high-purity, high-yield nanoparticle backbone that maintains the nanoparticle structure. The antigen portion and the nanoparticle backbone are covalently linked via the SpyTag003 / SpyCatcher003 system, exhibiting high linkage efficiency, uniform antigen display, mild reaction conditions, and stable complex structure.

[0043] 2. The antigenic portion of this bivalent genetically engineered subunit vaccine comprises two proteins. One is a recombinant Gettavirus P6E protein expressed in eukaryotic cells. This protein has a structure similar to the membrane proteins on the viral surface and contains immunogenic p62 and E1 proteins, exhibiting high affinity for antibodies and ensuring a natural spatial structure and modification. The other protein expressed in eukaryotic cells is the JEV DFN protein. This protein links the third domain (D3) of the JEV E protein to the 172-352 region of the NS1 protein via a linker and adds five additional B-cell epitopes belonging to the D1 and D2 domains. The E protein is the main antigenic protein of JEV, while the NS1 protein plays a crucial role in the replication and immune evasion of Japanese encephalitis virus, and is closely related to the virus's infectivity and pathogenicity. The NS1 protein not only stimulates the body to produce specific antibodies but also induces cellular immunity, providing broad-spectrum protection against JEV infection in pigs. The eukaryotically expressed protein has glycosylation modifications similar to viral proteins, further enhancing the antigenicity of the fusion protein.

[0044] 3. The recombinant protein-derived nanoparticles of this invention employ a strategy of separately expressing the antigen moiety and the nanoparticle backbone, and covalently conjugating them in vitro using the SpyTag003 / SpyCatcher003 system. This method can optimize the expression conditions of the two types of proteins separately, ensuring that the antigen folds correctly and the nanoparticle backbone maintains good self-assembly performance, avoiding conformational abnormalities or particle structure damage caused by fusion expression. After conjugation, the antigen can be uniformly and stably displayed on the particle surface, exhibiting a conformation close to the native epitope, thereby improving immunogenicity. Simultaneously, the covalent bonds formed in this system are stable and reliable, the reaction conditions are mild, and the operation is simple, making it suitable for efficient, controllable, and large-scale vaccine preparation processes.

[0045] 4. Currently, there are no bivalent vaccines for GETV and JEV on the market. This invention prepares GETV-P6E protein and JEV-DFN fusion protein separately using a mammalian cell expression system, and efficiently loads them onto the surface of a nanoparticle backbone to construct recombinant nanoparticles with multi-copy antigen presentation characteristics. After the recombinant protein and recombinant nanoparticles are combined with an adjuvant, the bivalent genetically engineered subunit vaccine obtained by this invention can significantly enhance antigen immunogenicity and induce a strong and durable humoral and cellular immune response. Compared with traditional monovalent or subunit vaccines, this invention has significant advantages in antigen display density, immune response strength, and dual protection capabilities, and can effectively reduce the infection risk of GETV and JEV in swine herds, showing significant potential for industrial application. Attached Figure Description

[0046] Figure 1 SDS-PAGE analysis of porcine gettavirus P6E recombinant protein and porcine Japanese encephalitis virus DFN fusion protein and their nanoparticles.

[0047] Figure 2 Immunogenicity assessment of the GETV / JEV bivalent subunit vaccine in mice. In the figures, a represents the detection of IgG antibodies against GETV; b represents the detection of neutralizing antibodies against GETV; c represents the detection of spleen cell proliferation response against GETV; d represents the detection of IgG antibodies against JEV; e represents the detection of neutralizing antibodies against JEV; and f represents the detection of spleen cell proliferation response against JEV.

[0048] Figure 3 Body temperature detection in pigs immunized with the GETV / JEV bivalent subunit vaccine.

[0049] Figure 4 Antibody detection was performed on pigs immunized with the GETV / JEV bivalent subunit vaccine. Specifically, a) GETV IgG antibody assay was performed after vaccine immunization; b) GETV neutralizing antibody assay was performed after vaccine immunization; c) JEV IgG antibody assay was performed after vaccine immunization; and d) JEV neutralizing antibody assay was performed after vaccine immunization. Detailed Implementation

[0050] The following examples further illustrate the content of the present invention, but should not be construed as limiting the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from its spirit and essence are within the scope of the invention. Reagents or instruments used without specifying a manufacturer are considered to be conventional products that can be purchased on the market.

[0051] Example 1: Preparation of GETV P6E recombinant protein

[0052] The gene fragment with the nucleotide sequence shown in SEQ ID NO:1 was synthesized into the pcDNA3.4(+) vector (between the XbaI and EcoRV restriction enzyme sites), transformed into DH5α competent cells, and positive bacteria that were correctly identified by bacterial PCR and sequencing were selected to extract the plasmid, and the recombinant plasmid pcDNA3.4-GETV-P6E was obtained.

[0053] CHO suspension cells were cultured in culture flasks at 37°C, 8% CO2, and a shaker speed of 130 rpm / min. The target cell count for the cultured CHO suspension cells was 2.5 × 10⁻⁶. 6 At a cell / mL concentration, the constructed positive recombinant plasmid was transfected into CHO cells. The transfection system consisted of 100 μg plasmid, 1 mL Opti-MEM, and 300 μL PEI. After mixing the plasmid and transfection reagent, the mixture was incubated at room temperature for 15 min, then slowly added dropwise to the cell culture flask, shaken well, and incubated at 37°C in a CO2 incubator. 24 h after transfection, 3.5 mL / 0.1 L of CHO cell culture enhancement medium was added, and the cells were incubated at 30°C in an 8% CO2 incubator for 3 to 4 days. After incubation, the cells and culture medium were collected, centrifuged at 6000 rpm / min for 10 min at 4°C, and the cell culture supernatant was collected. The recombinant protein was purified using IBA strep II affinity chromatography.

[0054] The specific steps for purifying the recombinant P6E protein using the IBA strep II tag affinity chromatography method (IBA Strep-Tactin®XT 4flow®) described above are as follows:

[0055] ① Add 5 volumes of PBS to the affinity chromatography column to replace its storage buffer.

[0056] ② After centrifuging the collected cell culture supernatant at 4℃ and 8000rpm for 20 min, add the supernatant to the affinity chromatography column and incubate overnight on a rotating shaker.

[0057] ③ Release the supernatant from the culture medium after binding and wash the column with 10 times the volume of washing buffer. The washing buffer formula is: 100mM Tris, 150mM NaCl, 1mM EDTA.

[0058] ④ Add 2-4 mL of biotin-containing eluent for elution. The eluent formulation is: 100 mM Tris, 150 mM NaCl, 1 mM EDTA, 50 mM biotin. Collect the eluent and concentrate it to replace the buffer.

[0059] ⑤ After concentrating the harvested purified protein sample using a Millipore ultrafiltration tube, 15 mL of binding buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0) was added to replace the storage solution. This process was repeated twice, and the final concentration was brought to 200-300 μL. The purified protein concentration was determined using the Bradford method, and then aliquoted and stored at -80°C for later use.

[0060] After the above expression and purification steps, purified recombinant GETV P6E protein was obtained. SDS-PAGE analysis showed a purity of over 90%. The SDS-PAGE molecular weight (e.g., [details omitted]) was [details omitted] Figure 1 It is approximately 100 kDa, which is in line with the expected size.

[0061] Example 2: Preparation of JEV-DFN fusion protein

[0062] The gene fragment with the same nucleotide sequence as shown in SEQ ID NO:3 was directly synthesized into the pcDNA3.4(+) vector (between the XbaI and EcoRV restriction enzyme sites) by Shanghai Sangon Biotech Sequencing Co., Ltd., and then transformed into DH5α competent cells. Positive bacteria that were correctly identified by bacterial culture PCR and sequencing were selected and the plasmid was extracted to obtain the recombinant plasmid pcDNA3.4-JEV-DFN.

[0063] The expression and protein purification steps of the recombinant plasmid pcDNA3.4-JEV-DFN were the same as in Example 1, and the purified JEV-DFN protein was finally obtained and stored at -80℃ for later use.

[0064] After the above expression and purification steps, purified recombinant JEV-DFN protein was obtained. SDS-PAGE analysis showed a purity of over 90%. The SDS-PAGE molecular weight (e.g., [details omitted]) was [details omitted] Figure 1 It is approximately 58 kDa, which is in line with the expected size.

[0065] Example 3: Preparation of SpyCatcher003-mi3 nanoparticles

[0066] (1) The gene fragment with the same nucleotide sequence as shown in SEQ ID NO:5 was synthesized by Shanghai Sangon Biotech Sequencing Co., Ltd. and directly ligated between the NdeI and XbaI restriction endonuclease sites of the pCold II vector and transformed into DH5α competent cells. 0.5 μg of plasmid was transformed into BL21(DE3) competent cells with TF16 molecular chaperone. Single clones were picked and seeded into LB liquid medium containing ampicillin and chloramphenicol resistance for overnight activation. The next day, the cells were seeded into LB liquid medium containing ampicillin and chloramphenicol resistance at a volume ratio of 1:100, and arabinose was added to a final concentration of 2 g / L. The cells were cultured at 37°C and 200 rpm with shaking until OD. 600 The concentration was increased to 0.6, and then the culture system was cooled to 16°C. IPTG was added to a final concentration of 0.1 mM to induce expression, and the expression was continued at 16°C for 12–16 h to ensure that the target protein was fully soluble and expressed.

[0067] (2) After induction, the bacterial cells were collected by centrifugation at 4°C and 8000 rpm for 30 min, and resuspended in ice-cold lysis buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0), with an appropriate amount of protease inhibitor added. The bacterial cells were lysed by sonication, and the lysate was centrifuged at 4°C and 10000 rpm for 30 min. The supernatant was collected as the crude soluble protein extract.

[0068] The supernatant was filtered through a 0.45 μm filter and then loaded into pre-equilibrated Ni. 2+ -NTA affinity chromatography column, using His tag for affinity purification. After removing non-specifically bound proteins with wash buffer containing 80 mM imidazole, the target protein is obtained by stepwise elution with elution buffer containing 250–300 mM imidazole. The eluted product is further filtered through Millipore ultrafiltration tubes to change the buffer to binding buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0). The concentrated protein is stored at 4°C for later use.

[0069] After the above expression and purification steps, purified SpyCatcher003-mi3 nanoparticles were obtained. SDS-PAGE analysis showed a purity of over 90%. The SDS-PAGE molecular weight (e.g., [missing information]) was [missing information]). Figure 1 It is approximately 45 kDa, which is in line with the expected size.

[0070] Example 4: In vitro coupling and assembly of nanoparticles

[0071] The GETV-P6E recombinant protein purified in Example 1, the JEV-DFN fusion protein purified in Example 2, and the SpyCatcher003-mi3 nanoparticles purified in Example 3 were replaced in the same reaction buffer system (50 mM Tris-HCl, 150 mM NaCl, pH 8.0). Two antigen proteins were added separately at a molar ratio of mi3 nanoparticles to antigen protein binding sites of 1:1, and in vitro coupling reactions were carried out with the mi3 nanoparticles. Incubation was performed at 4°C for 6-8 h to allow the antigen protein to form stable covalent isopeptide bonds with the mi3 surface through the SpyTag003 / SpyCatcher003 system. After the reaction, a small amount of precipitate was removed by centrifugation at 8000 rpm. The supernatant was loaded onto a equilibrated gel filtration column (Superose 6 Increase) for separation. High molecular weight peaks were collected and combined to obtain the fully loaded GETV-P6E-mi3 recombinant nanoparticles and JEV-DFN-mi3 recombinant nanoparticles. The obtained nanoparticles can be further ultrafiltered to a buffer solution of 50 mM Tris-HCl, 150 mM NaCl, and pH 8.0. The assembly integrity and antigen loading are verified by SDS-PAGE and transmission electron microscopy, thus obtaining nanoparticles with uniform structure and stable antigen presentation.

[0072] The molecular weights of GETV-P6E-mi3 recombinant nanoparticles and JEV-DFN-mi3 recombinant nanoparticles purified by in vitro coupling and molecular sieve gel filtration were 145 kDa and 100 kDa, respectively, according to SDS-PAGE analysis. The protein size was as expected, and the purity was greater than 90%. Transmission electron microscopy analysis showed that both GETV-P6E-mi3 and JEV-DFN-mi3 formed nanoparticles with obvious protrusions on the surface and a diameter of about 35 nm.

[0073] Example 5: Preparation of GETV / JEV bivalent genetically engineered subunit vaccine

[0074] The recombinant GETV P6E protein prepared in Example 1 and the recombinant JEV-DFN protein prepared in Example 2 were diluted to 250 μg / mL, respectively. The two recombinant nanoparticles prepared in Example 4 were diluted to 363 and 455 μg / mL, respectively. 20 mL of each diluted recombinant protein was taken and 10 mL of SMMIT was added. TM Adjuvant: Take 20 mL of each type of recombinant nanoparticle and add 10 mL of SMMMIT. TMThe adjuvant, after thorough mixing, constitutes the GETV / JEV bivalent genetically engineered subunit vaccine, wherein the concentrations of both the GETV-P6E recombinant protein and the JEV-DFN fusion protein are 100 μg / mL; and the concentrations of the GETV-P6E-mi3 nanoparticles and JEV-DFN-mi3 nanoparticles are 145 μg / mL and 182 μg / mL, respectively. Sterility testing is performed according to the current Chinese Veterinary Pharmacopoeia, and the qualified vaccine is stored at 2–8°C for later use.

[0075] Example 6: Immunogenicity assessment of the GETV / JEV bivalent genetically engineered subunit vaccine in mice.

[0076] Three-week-old ICR mice were randomly divided into 5 groups: 10 mice in the JEV commercial vaccine group and 20 mice in each of the other groups. The mice were immunized by intramuscular injection and booster immunization was performed on day 14 after the first immunization. The specific immunization grouping is shown in Table 1 below.

[0077] Table 1. Mouse Immunization Groups

[0078]

[0079] (1) Blood samples were collected on days 7, 14, 21, 28 and 35 after the second immunization to collect serum for antibody titer testing. JEV antibody levels were detected using a self-developed JEV antibody detection ELISA kit (coated with recombinant DFN protein, concentration 1ug / mL) and GETV antibody levels were detected using a self-developed GETV indirect ELISA antibody detection kit (coated with recombinant P6E protein, concentration 0.5ug / mL). Neutralization antibody titers of GETV and JEV were determined by neutralization assay.

[0080] (2) On the 14th day after the second immunization, the spleens of 3 mice from each group were isolated for spleen cell proliferation experiments. The specific steps are as follows:

[0081] Spleen cells were aseptically isolated using a 70-mesh cell sieve, treated twice with erythrocyte lysis buffer, and then diluted to 2.5 × 10⁶ cells in RPMI 1640 medium. 6Cells were seeded at a density of 100 μL / mL in 96-well plates. 100 μL of RPMI 1640 medium was added to each well as a blank control. Each sample consisted of three replicates. Subsequently, 10 μL of recombinant protein (10 μg / mL) was added to each well (GETV stimulated with P6E recombinant protein, JEV with DFN protein) or RPMI 1640 medium (control). Cells were incubated at 37 °C for 36 h. Then, 10 μL of CCK8 reagent was added to each well, and the cells were incubated at 37 °C for 4 h. The OD450 values ​​of different wells were measured using a multi-mode microplate reader. The spleen cell stimulation index (SI) was calculated as follows: SI index = (OD value of recombinant protein-stimulated wells - OD value of blank control wells) / (OD value of RPMI 1640 medium-stimulated wells - OD value of blank control wells).

[0082] (3) On day 14 after the second immunization, 5 mice from each of groups 2, 3, 4 and 5 were randomly selected for GETV challenge. Each of the 20 mice in the challenge group was subcutaneously inoculated with 100 μL of a solution containing 10 6 GETV-HN strain virus with TCID50. Blood was collected daily for 3 days after challenge to monitor viremia, and mice were necropsy on day 3 after challenge to detect viral load in the spleen.

[0083] Simultaneously, 5 mice from each of groups 1, 2, 3, 4, and 5 were selected for JEV challenge experiments. In this challenge group, 25 mice were intracranially inoculated with 50 μL of JEV containing 10... 5 TCID 50 The JEV-202401 strain of virus was tested for viremia by collecting blood serum on days 7, 10 and 14 after challenge.

[0084] The results showed that the level of specific antibodies against GETV increased rapidly after the second immunization, reaching its highest level 14 days after the second immunization. Subsequently, the antibody level remained high. The neutralizing titer also reached its highest level 14 days after the second immunization, with an average neutralizing titer of 1:560, significantly higher than that of the control group. Figure 2 (a and b in the original text) The antibody levels of the recombinant protein group were slightly lower than those of the nanoparticle group, and the antibody levels of the recombinant protein group were significantly lower than those of the nanoparticle group in the early stages. Specific antibody levels against JEV reached their highest level 14 days after the second immunization, and then remained at a high level. The neutralizing titer peaked 14 days after the second immunization, with an average neutralizing titer of 1:560, higher than that of the commercial live vaccine. The antibody levels of the recombinant protein group were lower than those of the nanoparticle and commercial live vaccine groups (…). Figure 2 (d and e in the text). These results indicate that mice immunized with the bivalent genetically engineered subunit vaccine produced a significant humoral immune response with high antibody titers.

[0085] Lymphocyte proliferation assay results showed that after stimulation with GETV recombinant protein, the bivalent nanoparticle vaccine group exhibited significant spleen cell proliferation, with stimulation indices all greater than 2, significantly different from the control group. The stimulation index of the recombinant protein group was greater than 1.5. After stimulation with JEV recombinant protein, both the commercial vaccine group and the bivalent vaccine group showed significant spleen cell proliferation responses, with stimulation indices greater than 1.5. The commercial vaccine group showed slightly lower rates than the bivalent nanoparticle vaccine group. Figure 2 (c and f in the text). These results indicate that immunization of mice with the bivalent genetically engineered subunit vaccine can trigger a significant cellular immune response.

[0086] Results of GETV and JEV challenge studies showed that no GETV viremia was detected in the bivalent genetically engineered subunit vaccine group within 1-3 days post-challenge, while significant viremia was detected in the control group. Furthermore, no infectious GETV was detected in the spleen of the bivalent genetically engineered subunit vaccine group on day 3 post-challenge, while the control group showed a high viral load in the spleen. No JEV viremia was detected in the bivalent genetically engineered subunit vaccine group within 7, 10, and 14 days post-challenge, and the commercial live vaccine group also showed no viremia, while the control group showed a high viral load. Post-mortem analysis of brain tissue from mice surviving 14 days post-challenge revealed significantly lower viral loads in the bivalent vaccine and commercial live vaccine groups compared to the control group. These results indicate that the GETV / JEV bivalent genetically engineered subunit vaccine can protect mice from GETV and JEV challenges.

[0087] Example 7: Safety Trial of GETV / JEV Genetically Engineered Subunit Vaccine

[0088] Two GETV / JEV bivalent genetically engineered vaccines and a commercially available attenuated live vaccine (Wuhan Keqian Biotechnology, SA14-14-2 strain live vaccine) were administered to 3-week-old antibody-negative piglets. Five piglets were in the bivalent recombinant protein group, five in the bivalent nanoparticle vaccine group, and five in the commercial vaccine group. Each piglet received an intramuscular injection of 1 mL of the corresponding vaccine in the neck. A control group of five piglets was administered PBS. The piglets' body temperature and weight were monitored daily for 7 consecutive days after injection. The piglets were also observed for adverse reactions such as swelling and nodules at the injection site. Their health status was also observed daily for 28 consecutive days.

[0089] The results showed that both groups of vaccines were safe and effective. The injection site absorbed the vaccines well, with no adverse reactions such as swelling or nodules. The pigs' body temperature remained between 38.5℃ and 39.6℃, and there were no abnormal symptoms such as fever. Figure 3 During the 14-day observation period, the pigs were in good spirits, had a normal appetite, and showed no abnormal symptoms.

[0090] Example 8: Evaluation of the protective efficacy of GETV / JEV bivalent genetically engineered subunit vaccine in pigs.

[0091] In Example 7, serum samples were collected from each group of immunized pigs at 7, 14, 21, 28, and 35 days post-immunization, and antibody levels were detected according to the method described in Example 4. Piglets immunized in Example 5 were used for GETV and JEV challenge experiments, with each piglet receiving one dose (each dose containing 5 x 10^6 viruses). 7 TCID 50 The GETV-HN strain P1 generation virus was used to challenge pigs. Clinical symptoms and body temperature changes were observed daily for 14 days post-challenge, and serum viremia was monitored on days 1-3 post-challenge. Simultaneously, pigs were challenged with JEV, with each piglet inoculated with 10 dV / v. 5 For the JEV-202401 strain of virus with TCID50, clinical symptoms and body temperature changes were observed daily for 14 days after challenge, and viremia was tested on days 7, 10 and 14 after challenge.

[0092] Post-immunization antibody test results Figure 4 The bivalent nanoparticle vaccine rapidly increased specific antibodies against GETV after immunization, stabilizing around day 14 post-secondary immunization, and then remaining at a high level. The average neutralizing antibody level reached 1:480 on day 14 post-secondary immunization. The control group had extremely low antibody levels, with a neutralizing titer less than 1:10. The antibody levels in the bivalent recombinant protein group were significantly lower than those in the nanoparticle group in the early stages. On day 14, the IgG antibody levels were not significantly different from those in the nanoparticle group, but the neutralizing antibody levels were lower in the bivalent nanoparticle group. Specific IgG antibodies against JEV from the bivalent nanoparticle vaccine reached their highest level 14 days post-secondary immunization, with an average neutralizing titer of 1:480. The antibody titer in the live vaccine group was lower than that in the bivalent genetically engineered subunit vaccine group, especially the nanoparticle group, while the control group had extremely low titers.

[0093] After challenge with GETV, none of the pigs in the bivalent genetically engineered vaccine group showed obvious clinical symptoms; their body temperature and mental state were normal, and no viremia was detected (Table 2). The control group exhibited obvious symptoms such as diarrhea and weight loss, and significant viremia was detected in their serum. After challenge with JEV, none of the pigs in the bivalent genetically engineered vaccine group showed obvious clinical symptoms, and no clinical symptoms of JEV infection were observed in the commercial live vaccine group. The control group exhibited obvious symptoms, including depression, unsteady gait, and weight loss. Viremia monitoring of the challenged pigs showed no viremia in the immunized group, while the control group showed high levels of viremia. These results indicate that the GETV / JEV bivalent genetically engineered subunit vaccine effectively resists GETV and JEV challenges in pigs and provides good immunoprotection.

[0094] Table 2. Results of viremia detection after challenge.

[0095]

[0096] Note: "+" represents positive, "-" represents negative, and " / " represents not involved.

Claims

1. An immunogenic composition, characterized in that, The immunogenic composition is the composition described in (a1) or (a2) below: (a1) A composition comprising porcine Gettavirus P6E recombinant protein and porcine Japanese encephalitis virus DFN fusion protein; (a2) A composition comprising recombinant nanoparticles loaded with porcine Getta virus P6E recombinant protein and recombinant nanoparticles loaded with porcine Japanese encephalitis virus DFN fusion protein; The porcine Gettavirus P6E recombinant protein is formed by linking the transmembrane domain-removed GETV p62 protein and E1 protein with a 4×GGGGSLinker sequence. The porcine encephalitis virus DFN fusion protein is formed by adding five B cell epitopes of D1 and D2 domains to the N-terminus of the third domain of the encephalitis virus E protein, and then linking it to the 172-352 region of the NS1 protein through a 4×GGGGS Linker sequence. The recombinant nanoparticles loaded with porcine Geyta virus P6E recombinant protein are formed by covalently linking porcine Geyta virus P6E recombinant protein to nanoparticles via the SpyTag003 / SpyCatcher003 system. The recombinant nanoparticles loaded with porcine encephalitis virus DFN fusion protein are formed by covalently linking the DFN fusion protein to the nanoparticles via the SpyTag003 / SpyCatcher003 system. The porcine Geyta virus P6E recombinant protein is a protein with the amino acid sequence shown in SEQ ID NO: 2 or a fusion protein with the same function obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO:

2. The porcine encephalitis virus DFN fusion protein is a protein with the amino acid sequence shown in SEQ ID NO: 4 or a fusion protein with the same function obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO:

4. The nanoparticles are SpyCatcher003-mi3 nanoparticles or SpyCatcher003-ferritin nanoparticles; the SpyCatcher003-mi3 nanoparticles are proteins with amino acid sequences as shown in SEQ ID NO: 6 or fusion proteins with the same function obtained by attaching protein tags to the N-terminus and / or C-terminus of proteins as shown in SEQ ID NO:

6.

2. An expression cassette, recombinant vector, or host cell containing nucleic acid molecules, characterized in that, The nucleic acid molecules described herein include nucleic acid molecules as shown in SEQ ID NO: 1 and nucleic acid molecules as shown in SEQ ID NO:

3.

3. The use of the immunogenic composition according to claim 1 in the preparation of a bivalent vaccine against porcine Getta virus and porcine Japanese encephalitis virus.

4. A bivalent vaccine against porcine Gettavirus and porcine Japanese encephalitis virus, characterized in that, The vaccine comprises the immunogenic composition and adjuvant as described in claim 1.

5. The porcine gettavirus and porcine Japanese encephalitis virus bivalent vaccine according to claim 4, characterized in that, The concentrations of recombinant porcine Geetavirus P6E protein and porcine Japanese encephalitis virus DFN fusion protein in the vaccine were each independently 50–200 μg / mL; the concentrations of recombinant nanoparticles loaded with porcine Geetavirus P6E protein and recombinant nanoparticles loaded with porcine Japanese encephalitis virus DFN fusion protein were each independently 100–200 μg / mL.

6. The porcine gettavirus and porcine Japanese encephalitis virus bivalent vaccine according to claim 4, characterized in that, The volume ratio of the adjuvant to the immunogenic composition is 1:1 to 1:4; the adjuvant is SMMMIT. TM Adjuvants, ISA adjuvants, or aluminum salt adjuvants.

7. The method for preparing the porcine gettavirus and porcine Japanese encephalitis virus bivalent vaccine according to claim 4, characterized in that, Includes the following steps: (1) The porcine gettavirus P6E recombinant protein and the porcine Japanese encephalitis virus DFN fusion protein were expressed and purified in a eukaryotic expression system, respectively; (2) Express and purify the SpyCatcher003-mi3 nanoparticles or SpyCatcher003-ferritin nanoparticles in a prokaryotic expression system; (3) Under in vitro conditions, the antigen protein purified in step (1) was covalently coupled with the SpyCatcher003-mi3 nanoparticles or SpyCatcher003-ferritin nanoparticles purified in step (2) through the SpyTag003 / SpyCatcher003 system to obtain recombinant nanoparticles loaded with antigens. (4) The antigen protein obtained in step (1) or the recombinant nanoparticles loaded with antigen obtained in step (3) are mixed with adjuvants in proportion to prepare a vaccine.

8. The use of the porcine Geytavirus and porcine Japanese encephalitis virus bivalent vaccine as described in claim 4 in the preparation of a drug for preventing porcine Geytavirus and porcine Japanese encephalitis virus infection.

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