Recombinant bacillus calmette-guerin expressing foot-and-mouth disease virus o, a type multi-epitope fusion peptide and application thereof

Abstract

This invention discloses a recombinant BCG vaccine expressing a multi-epitope fusion peptide of foot-and-mouth disease virus (FMD) types O and A and its application, belonging to the fields of biotechnology and veterinary vaccines. The amino acid sequence of the multi-epitope fusion peptide is shown in SEQ ID NO:1, containing dominant immune T-cell and B-cell antigenic epitopes from multiple circulating FMD virus types O and A, and fused with a mycobacterial signal peptide at the N-terminus. This invention successfully constructed the recombinant strain rBCG-MIPGA by codon optimization of the fusion gene, cloning it into the pMV306 vector, and electroporating it into BCG. This recombinant BCG can simultaneously stimulate high-titer specific antibodies and significant T-cell immune responses against FMD virus types O and A, exhibiting durable and broad-spectrum protective potential, and has significant application value in the preparation of safe, efficient, and broad-spectrum FMD vaccines.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and veterinary vaccines, specifically relating to a recombinant BCG vaccine expressing a multi-epitope fusion protein of foot-and-mouth disease virus, its construction method, a pharmaceutical composition containing the recombinant BCG, and its application in the prevention of foot-and-mouth disease. Background Technology

[0002] Foot-and-mouth disease (FMD) is a highly contagious disease caused by the foot-and-mouth disease virus (FMDV), primarily affecting cloven-hoofed animals such as cattle, sheep, and pigs, causing significant economic losses to the global livestock industry. Currently, the main means of controlling this disease is through inactivated vaccination. However, traditional inactivated vaccines have the following inherent drawbacks: (1) The production process requires the use of live viruses, posing biosafety risks and demanding extremely high standards for production facilities; (2) They primarily induce humoral immunity, with weaker cellular immune responses and incomplete immune protection; (3) The period of immune protection is relatively short, requiring multiple immunizations; (4) Most are monovalent or bivalent vaccines, offering limited cross-protection against viruses of different serotypes or different topotypes within the same serotype.

[0003] BCG is a long-established, well-proven safe attenuated live vaccine used not only for tuberculosis prevention but also widely considered a highly effective immune adjuvant. Its powerful adjuvant effect stems from its ability to strongly activate the host's innate and cellular immunity, particularly inducing Th1-type immune responses and cytokines (such as IFN-γ). Therefore, using BCG as a vector to express exogenous antigens and develop novel recombinant live vaccines has become a hot topic in infectious disease vaccine research.

[0004] While there are reports of using BCG to express a single exogenous antigen in the existing technology, how to design a multivalent foot-and-mouth disease vaccine that can simultaneously cover multiple serotypes and effectively stimulate a comprehensive immune response (including highly efficient humoral immunity and strong cellular immunity) remains a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a recombinant BCG that can simultaneously induce a broad-spectrum, efficient, and long-lasting humoral and cellular immune response against foot-and-mouth disease virus types O and A.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a foot-and-mouth disease virus multi-epitope fusion protein, the multi-epitope fusion protein comprising dominant immune T cell and B cell antigenic epitopes from type O and type A foot-and-mouth disease viruses, the amino acid sequence of which is shown in SEQ ID NO:1.

[0008] Secondly, the present invention provides a gene encoding the foot-and-mouth disease virus multi-epitope fusion protein, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0009] Thirdly, the present invention provides a recombinant expression vector containing the foot-and-mouth disease virus multi-epitope fusion protein.

[0010] Fourthly, the present invention provides a host cell comprising the foot-and-mouth disease virus multi-epitope fusion protein.

[0011] Fifthly, the present invention provides a recombinant BCG that expresses the foot-and-mouth disease virus multi-epitope fusion protein.

[0012] Sixthly, the present invention provides a method for preparing the recombinant BCG, comprising the following steps:

[0013] 1) The gene encoding the multi-epitope fusion protein of foot-and-mouth disease virus, as shown in SEQ ID NO:2;

[0014] 2) The gene encoding the foot-and-mouth disease virus multi-epitope fusion protein described in step 1) is cloned into an expression vector to construct a recombinant expression vector containing the foot-and-mouth disease virus multi-epitope fusion protein.

[0015] 3) The recombinant expression vector obtained in step 2) is introduced into BCG competent cells to obtain a recombinant BCG strain expressing the foot-and-mouth disease virus multi-epitope fusion protein;

[0016] 4) Cultivate and identify the recombinant BCG strain.

[0017] The expression vector is the pMV306 vector.

[0018] In a seventh aspect, the present invention provides a pharmaceutical composition comprising the recombinant BCG and a pharmaceutically acceptable adjuvant and / or carrier.

[0019] Eighthly, the present invention provides the use of the recombinant BCG or the pharmaceutical composition described herein in the preparation of a foot-and-mouth disease vaccine, the vaccine being capable of simultaneously inducing specific humoral and cellular immune responses against foot-and-mouth disease virus types O and A.

[0020] The beneficial effects of this invention are:

[0021] BCG serves as both a live bacterial vector and a natural immune adjuvant, and is widely used due to its reliable immunogenicity, high safety, low toxicity, and strong thermal stability. This invention leverages these advantages of BCG to screen for immunodominant epitopes of prevalent FMDV strains, construct multi-epitope fusion genes using genetic engineering, and build recombinant strains using BCG as a vector. By stably expressing the protective antigen of FMDV within BCG, specific humoral and cellular immune responses against foot-and-mouth disease virus are simultaneously induced in a single vaccine system, while retaining the protective effect of BCG against Mycobacterium bovis infection, thus achieving a "one-shot, two-protection" immunogenicity. This strategy can significantly reduce animal immune stress, lower breeding costs, and provide a safe, efficient, and sustainable new approach for the prevention and control of major animal diseases such as foot-and-mouth disease and bovine tuberculosis.

[0022] This invention constructs a multi-epitope fusion antigen by tandemly connecting key T-cell and B-cell epitopes from multiple prevalent FMDV strains, enabling the vaccine to possess broad-spectrum protective potential against various FMDV strains. Using BCG as a live vector, its natural adjuvant effect strongly activates cellular immunity, inducing high levels of IFN-γ; simultaneously, the outer membrane-localized antigen expression facilitates B-cell recognition, generating high-titer neutralizing antibodies. Experiments demonstrate that the vaccine induced by this invention provides a comprehensive and durable immune response.

[0023] The vaccine prepared by this invention does not contain complete FMDV, poses no risk of viral shedding, does not require high-level biosafety facilities in the production process, has relatively low cost, is suitable for large-scale production, and has significant industrialization advantages. Attached Figure Description

[0024] Figure 1 Electrophoresis diagram of PCR identification results of recombinant plasmid pMV306-MIPGA. M represents the molecular weight standard of protein, and 1-4 are BCG bacterial cultures after electroporation.

[0025] Figure 2 Western blot results of rBCG-MIPGA. 1 is the empty vector strain rBCG-pMV306, and 2 is the recombinant strain rBCG-MIPGA.

[0026] Figure 3 : Immunofluorescence identification results of outer membrane localization of rBCG-MIPGA.

[0027] Figure 4 Western blot results of the reactivity of rBCG-MIPGA to foot-and-mouth disease positive serum. 1 is recombinant strain rBCG-MIPGA, and 2 is empty vector strain rBCG-pMV306.

[0028] Figure 5 Dynamic changes in the titers of type O (A) and type A (B) FMDV antibodies in mice immunized with rBCG-MIPGA.

[0029] Figure 6 Results of spleen T lymphocyte proliferation activity detection in mice immunized with rBCG-MIPG.

[0030] Figure 7 Results of INF-γ cytokine level detection in mice immunized with rBCG-MIPG. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions or conditions described in reference books such as *Molecular Cloning: A Laboratory Guide*, or according to the methods recommended in the manufacturer's operating manual.

[0032] Key material descriptions:

[0033] pMV306 vector: pMV306hsp (Plasmid #26155) is a mycobacterial integrative plasmid used for the expression of recombinant antigen genes. Purchased from Addgene and stored in our laboratory;

[0034] Wild-type BCG: The wild-type BCG is BCG Danish 1331 (GenBank: CP039850.1), which is a WHO reference strain and was passaged and preserved in our laboratory.

[0035] Foot-and-mouth disease commercial vaccine (O / A bivalent inactivated vaccine): Foot-and-mouth disease type O and type A bivalent inactivated vaccine (veterinary drug production license number 250047543), used as a control for the commercial vaccine. Purchased from Zhongpu Biopharmaceutical Co., Ltd.;

[0036] Foot-and-mouth disease liquid phase blocking ELISA kits: The improved foot-and-mouth disease virus type O antibody liquid phase blocking ELISA detection kit (01.0002A) and the improved foot-and-mouth disease virus type A antibody liquid phase blocking ELISA detection kit (01.0003A) are used for foot-and-mouth disease antibody detection and were purchased from the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.

[0037] Example 1: Screening and gene optimization of foot-and-mouth disease virus antigenic epitopes

[0038] Based on the gene sequences of VP1 of type O foot-and-mouth disease virus strains (O / May-98, O / PanAsia, O / Ind-2001, O / GZSD / CHA) and VP1 / VP4 of type A foot-and-mouth disease virus strain (A / Sea-97) from the NCBI database, bioinformatics software (such as DNASTAR, ANTHEPROT, etc.) was used for analysis. Taking into account antigenicity, hydrophilicity, plasticity, surface accessibility and secondary structure characteristics, immune dominant T cell epitopes (Table 1) and B cell epitopes (Table 2) were determined.

[0039] Table 1: Prediction results of T-cell antigenic epitopes

[0040]

[0041] Table 2: Prediction Results of B-cell Antigenic Epitopes

[0042]

[0043] To cover a wide range of viral variants, enhance immunogenicity, particularly T-cell responses, and simultaneously address both cellular and humoral immunity, we employed a redundant design with multiple epitopes, multiple strains, and partial duplication. This involved tandemly connecting dominant antigenic epitopes from different strains to construct antigen-peptide complexes. Multiple dominant epitopes were tandemly linked using flexible spacers GGGS and GGSSGG as shown in Table 3 to avoid the formation of new epitopes, constructing multi-epitope fusion fragments for foot-and-mouth disease types O and A. Furthermore, a mycobacterial signal peptide sequence was introduced at the N-terminus of the fusion fragment to enable antigen secretion or surface localization within BCG, and an HA tag was added at the C-terminus. The tandem fragment was ultimately named MIPGA, and its sequence is shown in SEQ ID NO:1.

[0044] Table 3. Schematic diagram of antigenic epitope connections.

[0045]

[0046] The amino acid sequence of the fusion antigen epitope MIPGA (SEQ ID NO:1):

[0047] MSFVVTIPEALAAVATDLAGIGSTIGTAANAAAAVPTTTVLAAAADEVSAAMAALFSGGGGSETQVQRRHHTDVSFILDRFVGGGSLTRLALPYTGGGSLTNVRGDLQVLAQKAARPLPGGSSGGVHPSEARHKQKIVAPVKQSLGGGSETQVQRRQHTDVSFILD RFVGGGSLTRLALPYTGGGSVTNVRGDLQVLAQKAARTLPGGSSGGIHPEQARHKQKIVAPVKQLLGGGSETQVQRRQHTDVSFILDRFVGGGSLTRLALPYTGGGSV TNVRGDLQVLAQKAARTLPGGSSSGGIHPSEARHKQKIVAPVKQLLGGGSETQVQRRQHTDVAFILDRFVGGGSETALDNNTNGGGSANNVRGDLHVLAKNAERTLPGG SSGGIQPNTARHKQKIVAPAKQLLGGGSETQAQRRHTDVGFIMDRFVGGGSSIINNYYMQQYQNSMDGGGSTRRGDLGSLAARLATQLPASGGGSYKQKIIAPAKQLL YPYDVPDYA*

[0048] Note: The bolded part is the flexible peptide; the underlined part is the PE signal peptide and HA tag.

[0049] To improve its expression efficiency in mycobacterial hosts, the codons of the fusion gene were optimized. The optimized MIPGA gene nucleotide sequence is shown in SEQ ID NO:2 and was sent to the company for synthesis.

[0050] MIPGA gene nucleotide sequence (SEQ ID NO:2):

[0051]

[0052] Example 2: Construction of Mycobacterial Expression Vector

[0053] The MIPGA gene nucleotide sequence (SEQ ID NO:2) was synthesized and cloned into the pUC57 vector, resulting in a recombinant plasmid named pUC57-MIPGA. Using this plasmid as a template, the amplified MIPGA gene fragment was ligated into the pMV306 vector to obtain the mycobacterial expression plasmid pMV306-MIPGA. The specific procedures are as follows:

[0054] Using the mycobacterial expression vector pMV306 containing a strong promoter (hsp60 promoter) as a template, the vector was amplified at 60℃ for 5 min using primers pMV306-F: 5'-TATCCATCAAGCTTATCGATGTCGACG-3' and pMV306-R: 5'-AGAGAGTCCTCCTGTCGACG-3' to obtain the linearized pMV306 vector with a size of 4369bp. Using pUC57-MIPGA as a template, primers MIPGA-pMV306-F (5'-CCAATTCGTCGACAGGAGGACTCTCTATGTCATTTGTGGTCACGATCCCG-3') and MIPGA-pMV306-R (5'-CGATAAGCTTGATGGATACTAGGCGTAGTCCGGCAC-3') were used for amplification at 60℃ for 2 min, yielding a 1544 bp gene containing a homologous arm of the pMV306 fragment. The PCR amplification products were collected, and the MIPGA and pMV306 vectors were ligated using homologous recombinase. Using primers MIPGA-pMV306-F and MIPGA-pMV306-R, amplification was performed at 60℃ for 2 min. Positive clones were identified by bacterial culture PCR, and the results were as expected. The sequencing results were completely consistent with the original sequence, indicating successful construction of the recombinant plasmid pMV306-MIPGA.

[0055] Example 3: Preparation and Identification of Recombinant BCG Strains

[0056] 1. Preparation of BCG competent cells

[0057] Activated fresh BCG single colonies were picked from 7H11 plates and inoculated into 5 mL of 7H9 medium. The culture was incubated statically at 37°C for 14–21 days until the OD600 reached 0.8–1.0. The culture was then transferred 1:100 to 100 mL of fresh 7H9 medium and incubated statically at 37°C for approximately 14 days until the OD600 reached 0.6. The cells were collected by centrifugation at 4000 rpm for 10 min at room temperature. The cells were resuspended in 50 mL of 10% sterile glycerol and centrifuged again at 4000 rpm for 10 min. The cells were washed 1–2 times with 10% sterile glycerol, and then resuspended again by adding 5 mL of 10% sterile glycerol. The suspension was then aliquoted into 200 μL portions in 1.5 mL centrifuge tubes and stored at -80°C.

[0058] 2. Electroporation of recombinant plasmid into BCG competent cells and PCR identification of bacterial culture.

[0059] The correctly identified pMV306-MIPGA recombinant plasmid was electroporated into BCG competent cells under conditions of 2.5 kV, 1000 Ω, and 25 μF. After 14 days of culture, the plasmid was amplified for 2 min using MIPGA-pMV306-F and MIPGA-pMV306-R at an annealing temperature of 60℃. The rBCG-pMV306-MIPGA plasmid was then identified by colony PCR, and its size was 1544 bp. Figure 1 This indicates that the recombinant plasmid pMV306-MIPGA was successfully electrotransferred into BCG competent cells.

[0060] 3. Western blot detection of recombinant BCG expression

[0061] Recombinant strain rBCG-MIPGA and empty vector strain rBCG-pMV306 were inoculated into 10 mL of 7H9 liquid medium and cultured at 37℃ and 110 rpm in a shaker until the logarithmic growth phase (OD600 0.8-1.0). 10 mL of bacterial suspension was collected, centrifuged at 10000 rpm for 5 min at 4℃, washed three times with 1×PBS, and finally resuspended in 1 mL of 1×PBS. The bacteria were then sonicated to disrupt their structure. 40 μL of the disrupted bacterial suspension was added to 10 μL of 5×SDS loading buffer, incubated in a boiling water bath for 10 min, and centrifuged at 12000 rpm for 10 min. The supernatant was used for SDS-PAGE and Western blot analysis to detect protein expression. The primary antibody was HA-tagged antibody, and the secondary antibody was goat anti-mouse antibody (10000-fold dilution). rBCG-MIPGA showed a clear positive band at 55 kDa. Figure 2 This indicates that MIPGA was successfully expressed in BCG.

[0062] 4. Immunofluorescence identification of the outer membrane localization of rBCG-MIPGA

[0063] Non-permeable antibody fluorescent staining for mycobacteria is suitable for the identification of bacterial outer membrane proteins. After centrifugation of rBCG-MIPGA and empty vector strain rBCG-pMV306, the cultures were resuspended in Anti-HA-tag mAb (primary antibody) and incubated overnight. After washing off the antibody, the cultures were incubated with FITC-labeled goat anti-mouse IgG (secondary antibody) for 1 hour. After washing off the antibody, 50 μL of the bacterial culture was transferred to a 96-well cell plate for observation using a confocal high-content cell imaging system. The results showed that fluorescent signals were observed only in rBCG-MIPGA. Figure 3 This indicates that MIPGA is expressed on the surface of the bacterial outer membrane.

[0064] 5. Western blot detection of the reactivity of rBCG-MIPGA

[0065] Protein expression was detected by Western blot. Bovine foot-and-mouth disease positive serum was used as the primary antibody (5000-fold dilution), and goat anti-bovine IgG antibody was used as the secondary antibody (10000-fold dilution). The recombinant strain rBCG-MIPGA showed a clear positive band at 55 kDa. Figure 4 This indicates that rBCG-MIPGA has specific reactivity to foot-and-mouth disease positive serum.

[0066] Example 4: Vaccine Immunopotency Testing

[0067] 1. Experimental Grouping

[0068] Forty 6-week-old female Balb / C mice were randomly divided into four groups of 10 each. Each group was immunized subcutaneously with PBS, wild-type BCG, a commercial foot-and-mouth disease vaccine (O / A bivalent inactivated vaccine), or rBCG-MIPGA, respectively, at a dose of 10. 6 CFU / animal, PBS as blank control. Immunize once every two weeks, for a total of 3 times.

[0069] 2. Dynamic detection of O-type and A-type foot-and-mouth disease specific antibodies

[0070] Mouse serum was collected every 14 days after immunization, and antibody levels at each stage were detected using a foot-and-mouth disease liquid-phase blocking ELISA kit. Results are as follows: Figure 5 As shown, commercial vaccines can rapidly induce the production of specific antibodies. Type O and Type A antibodies rise rapidly at 28 days and 14 days, respectively, and then tend to stabilize. In contrast, the titers of type O and Type A antibodies in the rBCG-MIPGA recombinant vaccine continue to rise after immunization and continue to rise until the end of the experiment. This indicates that rBCG-MIPGA can continuously stimulate the body to produce antibodies and is expected to achieve more durable immune protection.

[0071] 3. Evaluation of cellular immunity levels

[0072] 3.1 Mouse spleen T lymphocyte proliferation experiment

[0073] At weeks 4 and 8 post-immunization, five mice from each experimental group were euthanized, and spleen T lymphocytes were isolated. Lymphocyte proliferation was then performed using 0.5 mg / mL PHA as a stimulant. Results are as follows: Figure 6 As shown, compared with the PBS group, splenic T lymphocytes in the commercial foot-and-mouth disease vaccine group, BCG group, and rBCG-MIPGA group all showed significant proliferation (p<0.005), and the T lymphocyte proliferation activity in the rBCG-MIPGA group was comparable to that in the commercial vaccine group. This indicates that the rBCG-MIPGA recombinant vaccine can increase the proliferation activity of splenic T lymphocytes after immunization.

[0074] 3.2 Cytokine Detection

[0075] At weeks 4 and 8, five mice in each experimental group were euthanized, and fresh anticoagulated blood was collected and stimulated with PPD for 20 hours before the in vitro release of IFN-γ was measured. Results are as follows: Figure 7 As shown, the INF-γ levels in the BCG group and the rBCG-MIPGA group were significantly higher than those in the PBS group (p<0.005); both wild-type BCG and recombinant BCG inoculation could induce an increase in IFN-γ, and the IFN-γ level in the recombinant BCG group was higher than that in the wild-type BCG group at week 4 (p<0.01), indicating that the recombinant strain with BCG as a live vector can more effectively induce the host's cellular immune response after animal immunization.

[0076] 3.3 Vaccine Safety

[0077] During the experiment, the clinical manifestations of the mice were monitored. The mice were in good mental condition, with normal appetite and water intake, and their body temperature fluctuated between 37.08±0.67℃. The weight of the mice in each immunization group showed a steady growth trend, increasing from an initial 15g to 22g, with no significant difference in growth compared to the blank control group. Anatomical observation showed that no visible abnormalities were observed in the major organs such as the lungs, kidneys, and spleen of the experimental group mice. Eight weeks after immunization, the lungs of the mice were fixed, sections were prepared, and microscopic observation revealed that, except for one mouse in the foot-and-mouth disease commercial vaccine group with slightly thickened alveolar walls, the alveolar structures of the mice in the PBS, BCG, and rBCG-MIPGA groups were intact, with normal alveolar wall thickness, no abnormal inflammatory infiltration, and normal lung tissue structure.

Claims

1. A multi-epitope fusion protein of foot-and-mouth disease virus (FMDV), characterized in that: The multi-epitope fusion protein contains dominant immune T-cell and B-cell antigenic epitopes derived from type O and type A foot-and-mouth disease virus, and its amino acid sequence is shown in SEQ ID NO:

1.

2. The gene encoding the foot-and-mouth disease virus multi-epitope fusion protein of claim 1, the nucleotide sequence of which is shown in SEQ ID NO:

2.

3. A recombinant expression vector comprising the foot-and-mouth disease virus multi-epitope fusion protein of claim 1.

4. A host cell containing the foot-and-mouth disease virus multi-epitope fusion protein of claim 1.

5. A recombinant BCG vaccine, characterized in that: The recombinant BCG vaccine expresses the foot-and-mouth disease virus multi-epitope fusion protein of claim 1.

6. A method for preparing the recombinant BCG vaccine of claim 5, characterized in that, Includes the following steps: 1) The gene encoding the multi-epitope fusion protein of foot-and-mouth disease virus, as shown in SEQ ID NO:2; 2) The gene encoding the foot-and-mouth disease virus multi-epitope fusion protein described in step 1) is cloned into an expression vector to construct a recombinant expression vector containing the foot-and-mouth disease virus multi-epitope fusion protein. 3) The recombinant expression vector obtained in step 2) is introduced into BCG competent cells to obtain a recombinant BCG strain expressing the foot-and-mouth disease virus multi-epitope fusion protein of claim 1. 4) Cultivate and identify the recombinant BCG strain.

7. The preparation method according to claim 6, characterized in that: The expression vector is the pMV306 vector.

8. A pharmaceutical composition, characterized in that, It includes the recombinant BCG vaccine as described in claim 5, as well as a pharmaceutically acceptable adjuvant and / or carrier.

9. Use of the recombinant BCG vaccine of claim 5 or the pharmaceutical composition of claim 8 in the preparation of a foot-and-mouth disease vaccine.

10. The use according to claim 9, characterized in that, The vaccine can simultaneously induce specific humoral and cellular immune responses against foot-and-mouth disease virus types O and A.

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