O-type foot-and-mouth disease virus strain containing vp1 t193a mutation site, construction method thereof and application in vaccine antigen escape research

Abstract

This invention belongs to the field of veterinary biological products, and relates to an O-type foot-and-mouth disease virus strain containing the VP1 T193A mutation site, its construction method, and its application in vaccine antigen escape research. The present invention provides an O-type foot-and-mouth disease virus mutant strain, which mutates threonine at position 193 of the VP1 protein of the wild-type O-type foot-and-mouth disease virus strain to alanine. The amino acid sequence of the mutated VP1 protein is shown in SEQ ID NO:2. The mutant strain rVP1-T193A constructed in this invention has highly similar lesion time, viral titer, and growth curve to rWT, but the neutralizing antibody titer to immune serum is reduced by 4.26 times, corresponding to an r value of 0.23, which is less than 0.3, potentially causing immune escape. This mutation significantly reduces the virus's sensitivity to neutralizing antibodies and can be used to study viral immune escape mechanisms and optimize vaccine design.

Description

Foot-and-mouth disease virus strain O containing the VP1 T193A mutation site, its construction method, and its application in vaccine antigen escape research. Technical Field

[0001] This invention belongs to the field of veterinary biological products, specifically relating to type O foot-and-mouth disease virus strains containing the VP1 T193A mutation site, their construction methods, and their application in vaccine antigen escape research. Background Technology

[0002] Foot-and-mouth disease (FMD) is a highly contagious and deadly disease caused by the foot-and-mouth disease virus (FMDV), infecting major livestock such as pigs, cattle, and sheep, as well as wild cloven-hoofed animals. The disease spreads rapidly, is highly infectious, and has an extremely high morbidity rate. The World Organisation for Animal Health (OIE) lists it as a reportable animal disease. Outbreaks and epidemics of FMD severely damage livestock productivity and the quality of livestock products, impact international trade in livestock and their products, and cause enormous economic losses to livestock farming in affected areas. Therefore, effective prevention and control of FMD is of significant strategic importance for the sustainable and healthy development of global livestock farming.

[0003] Currently, the most important means of controlling and preventing FMD is immunization with inactivated vaccines. Inactivated vaccines play a very important role in the prevention and eradication of FMD. However, there are many FMD serotypes, and due to the lack of proofreading function of RNA-dependent RNA polymerase (RDRP), the virus genome is highly variable. Furthermore, immune pressure can also promote the development of FMDV in the direction of vaccine escape. In recent years, mutant strains have been able to evade vaccine immunization.

[0004] The VP1 protein of foot-and-mouth disease virus (FMDV) is a major antigenic target, and its GH loop (residues 141-160) and C-terminal linear epitope (residues 200-213) are the core recognition regions for neutralizing antibodies. Studies have shown that amino acid mutations in VP1 (such as mutations at key sites in the GH loop) may affect antibody binding ability by altering epitope conformation or charge distribution, leading to immune escape. Summary of the Invention

[0005] This invention describes a mutant strain of type O foot-and-mouth disease virus (FMDV) constructed using reverse genetics technology. The mutant strain has a threonine (T) mutation at position 193 of the VP1 protein to an alanine (A). This mutant strain reveals that the virus has a certain immune evasion ability and significantly reduces the virus's sensitivity to neutralizing antibodies.

[0006] The present invention specifically adopts the following technical solution:

[0007] This invention provides a mutant strain of type O foot-and-mouth disease virus, which is obtained by mutating threonine to alanine at position 193 of the VP1 protein of a wild-type O foot-and-mouth disease virus strain. The wild-type O foot-and-mouth disease virus strain is FMDV / O / 17002, with the accession number CCTCC NO:V202060. The amino acid sequence of the mutated VP1 protein is shown in SEQ ID NO:2. The nucleotide sequence encoding the mutated VP1 protein is shown in SEQ ID NO:4.

[0008] This invention also provides a method for constructing the above-mentioned type O foot-and-mouth disease virus mutant strain, which involves introducing the T193A mutation using reverse genetics technology. This invention introduces the VP1 T193A mutation into the infectious clonal backbone of the FMDV strain FMDV / O / 17002 to construct the mutant strain rVP1-T193A. Specific steps include:

[0009] Step 1: Primers were designed to introduce the mutation site into a half-length plasmid containing the VP1 gene of the wild-type O foot-and-mouth disease virus strain, resulting in a mutant half-length plasmid. The wild-type O foot-and-mouth disease virus strain is FMDV / O / 17002, and the amino acid sequence of the VP1 protein of this wild-type O foot-and-mouth disease virus strain is shown in SEQ ID NO:1. The half-length plasmid was obtained by ligating the L-VP4-VP3-VP2-VP1 nucleotide sequence of the wild-type O foot-and-mouth disease virus strain to the vector pcDNA3.1. The mutation involved changing the base ACG encoding the 193rd amino acid of the VP1 protein to GCG.

[0010] Step 2: The mutant half-length plasmid obtained in Step 1 and the full-length plasmid containing the full-length gene of the wild-type O foot-and-mouth disease virus strain are ligated after double enzyme digestion to obtain the mutant full-length plasmid. The full-length plasmid is obtained by ligating the nucleotides of the full-length gene of the wild-type O foot-and-mouth disease virus strain to the vector pcDNA3.1. The double enzyme digestion is performed using SbfⅠ and PacⅠ restriction endonucleases.

[0011] Step 3: Transfect the full-length mutant plasmid obtained in Step 2 into BHK-21 cells to rescue the virus and obtain the recombinant virus rVP1-T193A, which is the O-type foot-and-mouth disease virus mutant strain.

[0012] The present invention also provides the application of the mutant strain in evaluating the protective efficacy of type O foot-and-mouth disease virus vaccine and / or in the study of antigen escape of type O foot-and-mouth disease virus vaccine.

[0013] In addition, the present invention provides the application of the mutant strain in the preparation of a broad-spectrum vaccine against foot-and-mouth disease virus.

[0014] The present invention also provides an inactivated vaccine comprising the mutant strain, which induces a neutralizing antibody titer that is more than 4 times lower than that of the wild type.

[0015] The beneficial effects of this invention are as follows:

[0016] 1. The mutant strain constructed in this invention has reduced sensitivity to neutralizing antibodies: Through the micro-neutralization test (VNT), it was found that the neutralizing titer (NT50) of rVP1-T193A against polyclonal antibodies was 4.26 times lower than that of wild type (rWT) (p<0.001), and the antigen ratio (r1) was 0.23, indicating that it significantly escapes the immune response induced by existing vaccines.

[0017] 2. Structural mechanism of the mutant strain constructed in this invention: Molecular dynamics simulations show that the T193A mutation disrupts the hydrogen bond network between VP1 and VP3 Q96, resulting in a conformational change of the C-terminal epitope and hindering antibody binding (Figure 2).

[0018] 3. The present invention demonstrates through an immunized pig challenge model that rVP1-T193A can overcome rWT vaccine immunization, with a protection efficiency of 20% (1 / 5).

[0019] 4. Application of the mutant strain constructed in this invention in vaccine antigen escape research: This mutant strain can be used to assess vaccine protective efficacy gaps and guide the design of broad-spectrum vaccines.

[0020] 5. Application of the mutant strain constructed in this invention in vaccine optimization: By introducing the T193A mutation in reverse, conserved epitopes can be screened or multivalent vaccines can be designed to improve cross-protection capabilities. Attached Figure Description

[0021] Figure 1 is a schematic diagram of the FMDV VP1 mutation site of the present invention.

[0022] Figure 2 shows a schematic diagram of hydrogen bonds before and after the VP1 193 mutation.

[0023] Figure 3 shows the electrophoresis diagram of the enzyme digestion products of the recombinant plasmid. M is the DNA marker of DL 15000, 1 is the enzyme digestion band of the original full-length plasmid pSK-HB2017P1, and 2 and 3 are the enzyme digestion bands of mutant plasmids 1 and 2 of pSK-HB2017P1-VP1-T193A.

[0024] Figure 4 shows the cell morphology of BHK-21 cells 24 hours after transfection with the original full-length plasmid pOHB2017 and the recombinant plasmid pOHB2017-VP1-T193A.

[0025] Figure 5 shows the indirect immunofluorescence results of the rWT and rVP1-T193A mutant strains.

[0026] Figure 6 shows the plaque phenotypes of the rWT and rVP1-T193A mutant strains.

[0027] Figure 7 shows the multi-step growth curves of the rWT and rVP1-T193A mutant strains.

[0028] Figure 8 shows the neutralizing antibody titers of rWT and rVP1-T193A mutant strains and rWT vaccine-immunized serum.

[0029] Figure 9 shows hoof lesions after immunization with rWT inactivated vaccine and challenged with rWT and rVP1-T193A.

[0030] Preserved biological material: FMDV / O / 17002;

[0031] Date of preservation: September 17, 2020;

[0032] Name of depositary institution: China Center for Type Culture Collection;

[0033] Accession number: CCTCC NO:V202060;

[0034] Address of the depositary institution: Wuhan University, Wuhan, China;

[0035] Classification and naming: Foot-and-mouth disease virus strain O / 17002 (FMDV / O / 17002 strain). Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0037] CutSmart, SbfⅠ, and PacⅠ mentioned in the examples were all purchased from New England Biotechnology (Beijing) Co., Ltd. Three-day-old Kunming suckling mice were purchased from the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.

[0038] Example 1

[0039] 1. Construction of type O foot-and-mouth disease virus strain rVP1-T193A containing the VP1 T193A mutation site

[0040] The FMDV VP1 mutation sites are shown in Figures 1 and 2. The amino acid sequences of the original VP1 and the mutant VP1 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0041] 1.1 Construction of recombinant plasmids

[0042] Using the half-length plasmid pSK-HB2017P1 (pSK-HB207P1 is obtained by ligating the nucleotide sequence of L-VP4-VP3-VP2-VP1 from FMDV / O / 17002 (accession number: CCTCC NO: V202060) to the vector pcDNA3.1) as a template, the VP1 gene fragment containing the T193A mutation was amplified by PCR. The PCR amplification system was as follows: 10 μL of 2×M5-Mutase Mix (Beijing Polymer Biotechnology Co., Ltd.), 0.4 μL of forward mutation primer (10 μM), 0.4 μL of reverse mutation primer (10 μM), 1-10 ng of half-length plasmid pSK-HB2017P1, and ddH2O to a final volume of 20 μL. The PCR program was as follows: 95℃ for 2 min, (94℃ for 25 s, 60℃ for 25 s, 68℃ for 3 min, 25 cycles), 68℃ for 5 min. Primer sequences used: Forward mutation primer: 5'-CCCTATTG G CTGTCCACCCGAGTGAGGC-3' (containing ACT→GCT mutation); reverse mutation primer: 5'-GTGGACAG C CAATAGGGGCCGAGGACAGT-3'.

[0043] Take 8 μL of the above PCR product, add 1 μL of 10×M5-Remase Buffer and 1 μL of M5 Remase enzyme (Beijing Polymer Biotechnology Co., Ltd.), incubate at 37℃ for 1 h to remove the original plasmid. Transform the PCR product after removing the original plasmid into E. coli DH5α, plate, pick single colonies, extract plasmid, and obtain the mutant plasmid pSK-HB2017P1-VP1-T193A.

[0044] The recombinant mutant plasmid pSK-HB2017P1-VP1-T193A and the full-length plasmid pOHB2017 (pSK-HB2017 was obtained by ligating the full-length nucleotide sequence of FMDV / O / 17002 to the vector pcDNA3.1) were double-digested using SbfⅠ and PacⅠ. The double-digestion system was: 5 μL CutSmart, 1 μg plasmid, 1 μL SbfⅠ, 1 μL PacⅠ, and ddH2O to a final volume of 50 μL. The reaction conditions were 37℃ for 20 min. After double digestion, the digestion products were subjected to agarose gel electrophoresis. The gel was excised, and the 6000 bp target band of pSK-HB2017P1-VP1-T193A and the 8000 bp target band of the full-length plasmid pOHB2017 were recovered and ligated to obtain the recombinant plasmid pOHB2017-VP1-T193A.

[0045] The recombinant plasmid pOHB2017-VP1-T193A was identified by double digestion with SbfⅠ and PacⅠ, and the correctly identified plasmids were sent to Qingke Biotechnology Co., Ltd. for sequencing identification.

[0046] The results showed that the restriction enzyme digestion bands of the recombinant plasmid pOHB2017-VP1-T193A were consistent with those of the original full-length plasmid pSK-HB2017P1, both yielding two bands of 8000bp and 6000bp, which were consistent with the expected size (see Figure 3). Sequencing results of the recombinant plasmid pOHB2017-VP1-T193A also indicated that the VP1 T193A mutation was successfully introduced into the target band of 8000bp in the full-length plasmid pOHB2017.

[0047] The nucleotide sequences of the original VP1 and the mutant VP1 are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0048] 1.2 Rescue of Recombinant Viruses

[0049] Standard cultured BHK-21 monolayers were transferred to T25 cell flasks. Once the cell density reached 70%-80%, the full-length plasmid pOHB2017 and the recombinant plasmid pOHB2017-VP1-T193A were transfected into BHK-21 cells using the transfection reagent jetPRIME (purchased from Polyplus Transfection®) (refer to the instruction manual for specific procedures). Four hours after transfection, the transfection medium was replaced with DMEM containing 2% FBS, and the cells were continuously cultured at 37°C with 5% CO2, observing for cytopathic effects. Seventy-two hours after transfection, the cells were harvested, subjected to two freeze-thaw cycles, continuously passaged in BHK-21 cells, and stored at -80°C for future use.

[0050] The results showed that both the original full-length plasmid pOHB2017 and the recombinant plasmid pOHB2017-VP1-T193A exhibited typical cytopathic effects (CPE) 24 hours after transfection into BHK-21 cells. Cells in the transfection control (MOCK) showed a fibrous distribution and were nearly confluent, while the diseased cells became larger, rounder, and grape-like in shape, with a large number dying (see Figure 4). The genetically engineered viruses rescued from BHK-21 cells after transfection with the original full-length plasmid pOHB2017 and the recombinant plasmid pOHB2017-VP1-T193A were named rWT and rVP1-T193A, respectively.

[0051] 1.3 PCR identification of recombinant virus

[0052] Viral supernatants from the fourth generation of rWT and rVP1-T193A were collected and viral RNA was extracted using the Omega Viral RNA Kit (Omega Bio-Tek). RT-PCR amplification was performed using primers VP1-3253-F: 5'-CGCTCGGCAACAGACCAC-3'; VP1-3907-R: 5'-GTTCAAGGACTGTTTCACAGGTG-3' (RT-PCR kit purchased from Takara, RR055A). The PCR amplification products of VP1 were obtained and sent to Qingke Biotechnology Co., Ltd. for sequencing identification to verify the correctness of the recombinant virus. The RT-PCR reaction system was as follows: PrimeScript 1 Step EnzymeMix 2 μL, 2×1 Step Buffer 25 μL, VP1-3253-F (20 μM) 1 μL, VP1-3907-R (20 μM) 1 μL, template RNA 1 μg, and RNase-free dH2O to a final volume of 50 μL.

[0053] The RT-PCR reaction program was as follows: 50℃ for 30 min; 94℃ for 2 min; 94℃ for 30 sec, 60℃ for 30 sec, 72℃ for 90 sec, for 30 cycles.

[0054] Sequencing results showed that the rVPT193A recombinant virus contained the VP1 T193A mutation, indicating that the present invention successfully constructed a recombinant FMDV containing the VP1 T193A substitution. The sequencing sequences of VP1 of rWT and rVPT193A are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. It should be noted that the sequencing sequences are partial sequences of the PCR amplification products of the above-mentioned VP1.

[0055] 1.4 Indirect immunofluorescence identification of recombinant virus

[0056] When BHK-21 monolayer cells in six-well plates reached a confluence density of 70%-80%, they were inoculated with parental virus rWT and recombinant virus rVPT193A, respectively. The expression of specific proteins in the virus-inoculated cells was detected using indirect immunofluorescence. The specific steps were as follows:

[0057] S1: After inoculating cells with the virus for 12 hours, discard the culture medium, wash three times with PBS, add pre-cooled anhydrous ethanol, and fix at -20℃ for 30 minutes.

[0058] S2: Wash 3 times with PBS, add 5% BSA and block at room temperature for 1 hour;

[0059] S3: Wash 3 times with PBS, add 1:500 diluted polyclonal antibody against FDMV nonstructural protein 3D (FMDV-3D polyclonal antibody, which is obtained by expressing and purifying FMDV 3D protein from E. coli, and collecting serum after immunizing 8-week-old New Zealand rabbits twice, which is the rabbit anti-polyclonal antibody against FMDV 3D), and incubate at 37°C for 1 hour;

[0060] S4: Wash 3 times with PBS, add FITC-labeled goat anti-rabbit IgG secondary antibody diluted 1:1000, and incubate at 37°C for 1 hour;

[0061] S5: Wash 3 times with PBS, add DAPI diluted 1:1000, incubate at room temperature for 10 min, wash 3 times with PBS to remove excess DAPI, and take pictures under a fluorescence microscope.

[0062] The results showed that BHK-21 cells inoculated with parental virus rWT and recombinant virus rVP-T193A specifically bound to the FMDV-3D polyclonal antibody and showed green fluorescence, while control cells showed no fluorescence (see Figure 5). This indicates that the present invention successfully constructed recombinant FMDV, and the mutation of VP1 T193A did not affect the rescue of infectious FMDV.

[0063] 2. Plaque phenotype and multi-step growth curve of recombinant virus

[0064] The fourth-generation parental virus rWT and recombinant virus rVP1-T193A were serially diluted 10-fold. Then, the different dilutions of virus were seeded into 6-well plates of BHK-21 or PK-15 cells that had formed a confluent monolayer, 200 μL / well. The cells were incubated at 37°C in a 5% CO2 incubator for 1 h, with shaking every 10 min to prevent the cells from drying out. After incubation, the virus solution was discarded, and 2 mL of astragalus gum mixture (MEM and 1.2% astragalus gum mixed 1:1, with 1% FBS added) was added. The cells were incubated at 37°C for 48 h. After incubation, the culture medium was discarded, and the cells were gently washed 3 times with PBS. Pre-cooled anhydrous ethanol was added, and the cells were fixed at -20°C for 30 min. The anhydrous ethanol was discarded, and the cells were gently washed 3 times with PBS. The cells were stained overnight with 1% crystal violet, and washed 5 times with PBS. The viral plaques were observed, and the viral plaque-forming units (PFU / mL) were calculated.

[0065] Fourth-generation parental virus rWT and recombinant virus rVP1-T193A were used to infect 6-well monolayers of BHK-21 or PK-15 cells with an MOI of 0.1. After 1 hour of adsorption, the virus solution was removed, and the cells were washed three times with PBS. DMEM was added, and the cells were incubated at 37°C in a 5% CO2 incubator. The supernatant was collected at 4, 8, 12, 16, 20, and 24 hours after inoculation, and the viral titer (TCID) was measured in 96-well monolayers of BHK-21 (or PK-15) cells. 50), and plotted the multi-step growth curve of the virus.

[0066] The results showed that the plaque phenotype and multi-step growth curves of the parental virus rWT and the recombinant virus rVP1-T193A were similar (see Figures 6 and 7). This indicates that the VP1 T193A mutation did not significantly affect the replication ability of the recombinant FMDV.

[0067] 3. Neutralizing antibody titer detection

[0068] Neutralizing antibody titers against the 4th generation parental rWT virus and recombinant virus rVP1-T193A were determined using laboratory-preserved porcine serum immunized with rWT vaccine (rWT was cultured in suspension BHK-21 cells, inactivated and emulsified after 146 seconds of assay, immunized with 8-week-old healthy pigs, and immune serum was obtained 28 days post-immunization). The specific steps are as follows:

[0069] (1) Inactivate the immunized pig serum at 56℃ for 30 min, then perform a 2-fold serial dilution to dilute the virus to 2000 TCID. 50 / mL, mix the diluted virus with serum of different dilutions in equal volumes, incubate at 37°C for 1h, then add 100μL of BHK-21 cells to each well of a 96-well plate and incubate at 7°C and 5% CO2 for 72h.

[0070] (2) After 72 hours, observe and record the lesion status of cells in each well, and use the Reed-Muench method to calculate the neutralizing antibody titers of the parental virus rWT and recombinant virus rVP1-T193A.

[0071] The results showed that the neutralizing antibody titer of the parental virus rWT to serum was 1:23442.3, and the neutralizing antibody titer of the recombinant virus rVP1-T193A to serum was 1:5495.4, with a corresponding r1 value of 0.23, which is less than 0.3. This indicates that the VP1 T19A mutation leads to a mismatch between FMDV and the vaccine, which may result in immune evasion (see Figure 8).

[0072] 4. Animal immune challenge protection experiment

[0073] Nine 8-week-old pigs (purchased from a pig farm in Kanglu County, Linxia Hui Autonomous Prefecture, Gansu Province) that were negative for common pathogen antibodies were selected and immunized with a vaccine containing inactivated and emulsified parental rWT virus. Forty-two days post-immunization, the pigs were tested for type O FMDV-specific antibodies. After confirming a positive antibody result, the immunized pigs were challenged with parental rWT virus and recombinant rVP1-T193A virus (8000 LD50 / head). Clinical symptoms were observed and scored, and the incidence of disease was recorded.

[0074] The results showed that the vaccine provided 100% protection against infection with the parental virus rWT (4 / 4), while the protection against the recombinant virus rVP1-T193A was only 20% (1 / 5). This indicates that the VP1 T193A mutation allows FMDV to evade vaccine immunization (see Figure 9).

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A mutant strain of type O foot-and-mouth disease virus, characterized in that, The method involves mutating threonine at position 193 of the VP1 protein of the wild-type O foot-and-mouth disease virus strain to alanine; the amino acid sequence of the VP1 protein of the wild-type O foot-and-mouth disease virus strain is shown in SEQ ID NO:1; the wild-type O foot-and-mouth disease virus strain is FMDV / O / 17002 strain, and the preservation number of the wild-type O foot-and-mouth disease virus strain is CCTCC NO:V202060.

2. The O-type foot-and-mouth disease virus mutant strain according to claim 1, characterized in that, The nucleotide sequence encoding the mutated VP1 protein is shown in SEQ ID NO:

4.

3. The method for constructing a mutant strain of type O foot-and-mouth disease virus as described in claim 1, characterized in that, Mutation sites are introduced using reverse genetics techniques.

4. The construction method according to claim 3, characterized in that, Includes the following steps: Step 1: Design primers to introduce the mutation site into a half-length plasmid containing the VP1 gene of wild-type O foot-and-mouth disease virus strain to obtain the mutant half-length plasmid. Step 2: The mutant half-length plasmid obtained in Step 1 and the full-length plasmid containing the full-length gene of wild type O foot-and-mouth disease virus strain are ligated after double enzyme digestion to obtain the mutant full-length plasmid; Step 3: The full-length mutant plasmid obtained in Step 2 is transfected into BHK-21 cells to rescue the virus and obtain the type O foot-and-mouth disease virus mutant strain.

5. The construction method according to claim 4, characterized in that, In step 1, the wild type O foot-and-mouth disease virus strain is FMDV / O / 17002 strain, and the preservation number of this wild type O foot-and-mouth disease virus strain is CCTCC NO:V202060.

6. The construction method according to claim 4, characterized in that, In step 1, the mutation is to change the base ACG encoding the 193rd amino acid of the VP1 protein to GCG.

7. The use of the mutant strain according to any one of claims 1-2 in the preparation of foot-and-mouth disease virus vaccine.

8. An inactivated vaccine comprising the mutant strain according to any one of claims 1-2.

Images