Recombinant feline herpesvirus genetic engineering vaccine, recognition antibody and application of recombinant feline herpesvirus genetic engineering vaccine

Abstract

The invention discloses a recombinant cat herpesvirus genetic engineering vaccine, a recognition antibody and application thereof, the recombinant strain is an FHV gIgE / TKFCVVP1M-FPVVP2M strain, the recombinant strain is used as a vaccine for immunizing a 3-4-month-old kitten, a specific antibody (the antibody can recognize the vaccine) aiming at FCV and FPV can be induced, the safety is good, and no serious adverse reaction exists. A challenge test shows that the vaccine can enable immunized cats to resist FCV and FPV virulent virus attacks, the survival rate of young cats in an immunized group is 100%, only individual cats have slight and transient respiratory tract symptoms, and the vaccine can potentially prevent and control FHV infection. In conclusion, the recombinant strain is expected to be used as an efficient vaccine candidate strain for preventing and treating herpes virus diseases, calicivirus diseases and parvovirus diseases of cats, and a reliable scheme is provided for clinical multi-pathogen prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a recombinant feline herpesvirus genetically engineered vaccine, a recognition antibody, and their applications. In particular, it relates to the preparation of an antibody using genetic engineering technology that can specifically recognize recombinant feline herpesvirus strains, and the application of this antibody in feline herpesvirus detection, diagnosis, and disease prevention and control. Background Technology

[0002] Feline viral rhinotracheitis (FVR), commonly known as feline herpesvirus, is a serious upper respiratory tract disease (URTD) that can cause most adult cats and kittens. Feline herpesvirus-1 (FHV-1) is the main pathogen causing feline rhinotracheitis. Cats of all ages can be infected with FHV-1, but kittens aged 2-4 months are most susceptible, with a morbidity rate of 100% and a mortality rate of 50%. Symptoms of FHV-1 infection include conjunctivitis, runny eyes, runny nose, coughing, sneezing, and loss of appetite.

[0003] In terms of prevention and control, while existing vaccines can prevent feline herpesvirus infection to some extent, their protective effect is not 100%. Vaccinated cats are still at risk of infection, although clinical symptoms may be relatively mild and the chance of virus transmission may be reduced. Some vaccines have a short period of immunity, requiring frequent vaccinations, which increases feeding costs and stress in cats. Moreover, with viral mutations, existing vaccines may not provide sufficient protection against some new viral strains. Treatment currently mainly involves symptomatic treatment and antiviral drugs. Commonly used antiviral drugs such as acyclovir and famciclovir, while effective, may have side effects, and long-term use may lead to viral resistance. Furthermore, for cats already infected and in the symptomatic stage, the treatment period is long, and the treatment effect is affected by various factors such as the severity of the disease and the cat's own immunity. Some cats may still suffer from sequelae even after treatment, such as eye damage and respiratory dysfunction.

[0004] In its preliminary research, the applicant discovered that constructing a recombinant feline herpesvirus live vector vaccine expressing FCV VP1 and FPV VP2 proteins using feline herpesvirus as a vector fully utilizes the immunogenicity and safety of feline herpesvirus. This method promises to provide protection against all three viruses with a single vaccination, offering a more convenient and economical preventative measure compared to traditional feline triple inactivated vaccines. Furthermore, the applicant has developed recognition antibodies that can serve as precise detection tools to verify the vaccine's expression efficiency in the body. By specifically identifying the FCV VP1, FPV VP2 proteins and the vector feline herpesvirus (FHV) antigen expressed by the vaccine, these antibodies can accurately track the vaccine's replication dynamics and antigen presentation process in animals, providing direct evidence for vaccine immunogenicity assessment and immunization schedule optimization. This addresses the issues of insufficient specificity and difficulty in distinguishing between vaccine strains and wild-type strains in traditional vaccine efficacy verification methods. Meanwhile, specific antibodies can help elucidate the synergistic mechanism of vaccine-induced humoral immunity, cellular immunity, and mucosal immunity. By detecting changes in antibody levels at different immune stages and combining this with cytokine detection, the type and intensity of the immune response evoked by the vaccine can be identified, providing a theoretical basis for subsequent vaccine iterations and upgrades (such as enhancing mucosal immunity and prolonging the period of immune protection). Summary of the Invention

[0005] To address the aforementioned technical problems, this invention first provides a recombinant feline herpesvirus strain expressing the VP1 mutant protein of feline calicivirus and the VP2 mutant protein of feline parvovirus. The recombinant feline herpesvirus strain is Felineherpesvirus FHV △gIgE / TK FCV VP1M-FPV VP2M. This strain has been deposited with the China Center for Type Culture Collection (CCTCC), and its deposit information is as follows: deposit address: Wuhan University, Wuhan, China, 430072, China; deposit date: October 8, 2023; deposit number: CCTCC NO: V202388.

[0006] Furthermore, the present invention provides a monoclonal antibody 5H3 that specifically recognizes the above-mentioned recombinant feline herpesvirus strain. The amino acid sequence of the light chain variable region of the monoclonal antibody 5H3 is shown in SEQ ID NO:1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2.

[0007] Furthermore, the present invention provides a vaccine composition for treating or preventing feline herpesvirus-related infections, the composition comprising the above-mentioned recombinant feline herpesvirus live vector vaccine and a specific recognition antibody. Preferably, the above-mentioned recombinant feline herpesvirus live vector vaccine and the specific recognition antibody are packaged separately and perform relatively independent functions.

[0008] More preferably, the viral titer of the recombinant feline herpesvirus live vector vaccine in the composition is 10. 6 -10 8 The composition contains PFU / mL and a total concentration of specific recognition antibodies of 0.5-5 μg / mL. It also contains pharmaceutically acceptable stabilizers (such as 5%-10% sucrose and 0.1%-0.5% gelatin) and pH adjusters (such as phosphate buffer, maintaining the pH at 7.2-7.4). It can be formulated into lyophilized or liquid formulations depending on the application. The moisture content of the lyophilized formulation is ≤3%, and the shelf life of the liquid formulation is not less than 18 months when stored at 2-8°C. Furthermore, this application provides the use of the above-mentioned vaccine composition in the preparation of a diagnostic reagent for detecting feline herpesvirus (FHV), feline calicivirus (FCV), or feline panleukopenia virus (FPV). More preferably, the diagnostic reagent is an enzyme-linked immunosorbent assay (ELISA) kit, an immunofluorescence assay kit, or an immunochromatographic test strip; wherein, the ELISA kit comprises a coated plate (pre-coated with the above-mentioned specific recognition antibody), an enzyme-labeled secondary antibody (anti-cat IgG-HRP), a positive control solution, a negative control solution (cat serum without FHV / FCV / FPV), a substrate solution (TMB), and a stop solution (2M H2SO4).

[0009] Furthermore, the present invention provides the application of the above-mentioned antibody in evaluating the immunization effect of recombinant feline herpesvirus live vector vaccine. Specifically, the antibody is used to detect the specific antibody titer against the live vector vaccine in the cat serum after immunization, or to detect the level of secretory IgA antibody against the above-mentioned antigen in the cat nasal mucosal secretions, so as to evaluate the vaccine-induced humoral immunity and mucosal immunity.

[0010] Beneficial effects The live vector vaccine provided by this invention utilizes highly densely packed repeating epitopes on the surface of VLPs, making them a strong immunostimulatory molecule easily recognized by the immune system. It also exhibits good safety, simultaneously inducing both cellular and humoral immune responses, protecting cats against attacks from virulent FCV and FPV, and effectively alleviating clinical symptoms in kittens. The recombinant feline herpesvirus genetically engineered vaccine FHV △gIgE / TK FCV VP1M-FPV VP2M strain of this invention produces specific antibodies against feline calicivirus and feline parvovirus in immunized kittens, showing promise as a candidate vaccine strain for effective prevention and treatment of feline herpesvirus, feline calicivirus, and feline parvovirus.

[0011] Meanwhile, the affinity constant KD of the recognition antibody provided by this invention to the recombinant feline herpesvirus strain antigen reaches the level of 10^-9 M, which is much higher than the affinity constant of traditional antibodies (usually in the range of 10^-7 to 10^-8 M). This means that the antibody of this invention can bind more tightly and stably to the vaccine antigen, and can efficiently capture and recognize the virus even at low antigen concentrations, thereby improving the sensitivity of detection, reducing the risk of missed detection, and accurately tracking the replication dynamics of the vaccine and the antigen presentation process in animals, providing a direct basis for vaccine immunogenicity assessment and immunization program optimization. Attached Figure Description

[0012] Figure 1 Growth curves of FHV △gIgE / TK FCV VP1M-FPV VP2M viruses; Figure 2 : FHV △gIgE / TK FCV VP1M-FPV VP2M virus WB identification results; Figure 3 Results of body temperature changes in the immunized group and the control group 14 days after viral challenge; Figure 4 Clinical scores of the immunized group and the control group 14 days after challenge; Figure 5 Antibody titers of monoclonal antibodies 2D4, 4G7, and SHVP12. Detailed Implementation

[0013] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0014] The feline herpesvirus strain FHV △gIgE / TK eGFP-mCherry, which lacks the gI, gE, and TK genes, used in this invention was constructed, preserved, and deposited in our laboratory (accession number: V202260, deposited at the China Center for Type Culture Collection; address: Wuhan University, Wuhan, China, 430072, China; deposit date: July 26, 2022).

[0015] Example 1: Construction and Characterization of sgRNA Expression Vector Based on the applicant's previous research, a recombinant feline herpesvirus (FHV) genetically engineered vaccine strain FHV △gIgE / TK FCVVP1M-FPV VP2M was constructed. Furthermore, key amino acids of FCV VP1 (I62V, enhancing the binding affinity between the antigen and B cell receptors) and FPV VP2 protein (L297R, enhancing the binding affinity with MHC class II molecules) were mutated. CRFK cells were passaged in six-well plates. When cell confluence reached 90%-100%, the original cell culture medium was discarded, and the cells were washed twice with PBS. Simultaneously, the viral stock solution was serially diluted using EMEM. The diluted virus was then injected at 10... -2 -10 -5 The virus was evenly inoculated onto cell plates and incubated at 37°C for 3 hours. The virus solution was then discarded. 2% low-melting-point agarose solution (stored at 60°C) was mixed with 2× cell maintenance medium at a 1:1 ratio and added to each well at a concentration of 2 ml / well. After cooling, the mixture solidified into a capping layer. Non-fluorescent spots were selected using a fluorescence microscope. After seven rounds of purification, the recombinant FHV ΔgIgE / TK FCV VP1M-FPV VP2M strain was obtained and purified. It was then deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China 430072, China, on October 8, 2023, with accession number CCTCC NO: V202388.

[0016] FHV ΔgIgE / TK FCV VP1-FPV VP2 strain and FHV ΔgIgE / TK FCV VP1M-FPV VP2M strain were seeded into a monolayer of CRFK cells at an MOI ratio of 0.1 and cultured in an incubator. TCID was measured every 6 hours. 50 And plot a one-step growth curve ( Figure 1 The results showed that the growth curves of the two were basically the same.

[0017] Cell cultures of FHV ΔgIgE / TK FCV VP1M-FPV VP2M strains and FHV WX19 strain were collected. Gels of the required concentrations were prepared using the product catalog of a biotechnology company. The electrophoresed protein gels were then transferred to NC membranes using a semi-dry protein transfer apparatus at 15°C for 40-60 minutes. Blocking was performed in 5% skim milk at 37°C for 3 hours or overnight at 4°C. Antibodies for gI, gE, TK, VP1, VP2, gB, and Actin, diluted with primary antibody dilution buffer, were incubated at room temperature for 4 hours or blocked overnight at 4°C. The membranes were then washed three times with TBST for 10 minutes each, followed by incubation with secondary antibody at room temperature for 45 minutes, and then washed three times with TBST for 10 minutes each. Development and image preservation were then performed using a gel imaging system. Results showed that specific FCV VP1 and FPV VP2 could be detected by Western blotting, as expected. Figure 2 ).

[0018] Example 2: Determination of the immunoprotective efficacy of recombinant virus 1. Selection and grouping of experimental animals Sixteen healthy Ragdoll kittens aged 3-4 months, weighing between 0.8 and 1.2 kg, were selected. These kittens were tested negative for feline herpesvirus (FHV), feline calicivirus (FCV), and feline panleukopenia virus (FPV) antibodies via indirect ELISA. The kittens were housed in an environment with a temperature of 22-25°C and humidity of 50%-60%, with free access to food and water. After one week of acclimatization, the experiment was conducted. The 16 kittens were randomly divided into 6 groups of 4 kittens each using a random number table. The specific groupings are as follows: Group A (FCV challenge immunization group): 4 kittens, used to evaluate the protective effect of the vaccine against FCV virulent challenge; Group B (FPV challenge immunization group): 4 kittens, used to evaluate the protective effect of the vaccine against FPV virulent attack; Group C (FCV challenge control group): 4 kittens, not vaccinated, serving as negative controls for FCV virulent challenge; Group D (FPV challenge control group): 4 kittens, not vaccinated, serving as negative controls for FPV virulent challenge.

[0019] Group E (WH2020 vaccine FCV control group): 4 kittens, used to evaluate the protective effect of the vaccine against virulent FCV attack; Group F (WH2020 vaccine FPV control group): 4 kittens, used to evaluate the protective effect of the vaccine against FPV virulent attack; 2. Vaccine Immunization and Virus Attack Immunization Procedure: Kittens in Groups A and B were immunized intranasally on day 0 (D0) with a recombinant feline herpesvirus live vector vaccine (FHV ΔgIgE / TK FCV VP1M-FPV VP2M strain). The immunization dose was 0.2 mL per kitten via intranasal drop, containing 10... 8 TCID 50 The recombinant virus was used; kittens in groups C and D were given 0.2 mL of sterile saline intranasally during the same period as blank controls. The specific procedures for groups E and F were the same as above, but the vaccine used was a live vector vaccine prepared with WH2020 as the backbone, prepared using the technology described in CN117143924A. Virulent challenge: On day 28 post-immunization (D28), kittens in groups A, C, and E were challenged with a virulent FCV strain via intranasal instillation. The challenge strain was the standard virulent FCV strain (FCV SH14 strain, preserved in our laboratory). The challenge dose was 10 mg / kJ per kitten. 9 TCID 50 Kittens in groups B, D, and F were challenged with a virulent FPV strain via intraperitoneal injection. The challenge strain was the standard virulent FPV strain (FPV 0918 strain, preserved in our laboratory). The challenge dose was 10 mg / kJ per kitten. 5 TCID 50 . 3. Indicator Detection and Recording 1) Body temperature monitoring: After the challenge, the kitten's body temperature was measured every morning at 9:00 using rectal thermometry. The normal body temperature range is 38.0~39.2℃. The body temperature changes were recorded. If the body temperature is ≥39.5℃ for 2 consecutive days, it is considered a high fever. The results show that ( Figure 3 Groups A and B (immunization groups): The body temperature remained stable at 38.3~38.9℃ for 14 days, and did not exceed 39℃. The standard deviation was only about ±0.1, with no significant fluctuations. The vaccine had the best effect on controlling body temperature.

[0020] Groups E and F (WH2020 vaccine group): Body temperature fluctuated between 38.4 and 39.1℃, with a standard deviation of ±0.1 to ±0.2. The highest value (39.2℃) was reached on the 3rd or 4th day. The fluctuation range was greater than that of groups A and B, but less than that of the control group.

[0021] Groups C and D (challenge control group): body temperature fluctuations were the most significant, reaching a peak on day 4 or 5 (39.6℃ / 40.0℃), with a standard deviation of ±0.1~±0.4. The overall fluctuation range was much larger than that of other groups, which is consistent with the characteristics of body temperature stress response after challenge to the virus in unvaccinated kittens.

[0022] 2) Clinical symptom observation: Kittens were observed continuously for 14 days after challenge (D28~D41), twice daily at 9:00 AM and 3:00 PM. Their mental state, appetite, respiratory symptoms (sneezing, coughing), ocular symptoms (type of eye discharge, conjunctival congestion), oral symptoms (oral ulcers, drooling), and digestive symptoms (vomiting, diarrhea) were recorded according to the clinical symptom grading standards (0~4) in the "Guidelines for the Diagnosis and Prevention of Viral Diseases in Pets". The average clinical symptom score for each group was calculated. The results are as follows: Figure 4 As shown. 2.1 FCV Vicious Poison Attack Groups (Group A, Group E, Group C) The severity of clinical symptoms in the three groups showed a significant difference, with Group A < Group E < Group C, as detailed below: Group A (FCV challenge and immunization group): Within 14 days after challenge, the overall condition of the 4 kittens was stable, their mental state remained at level 0 (normal activity, sensitive to stimuli), and their appetite did not fluctuate (no significant difference in food intake compared to before challenge). Only 2 kittens developed mild respiratory symptoms on day 4 (D32) after challenge, manifested as occasional sneezing ≤2 times per day, without coughing, eye discharge, or abnormal oral mucosa. These symptoms resolved spontaneously on day 5 (D33) without any sequelae. The remaining 2 kittens did not show any FCV-related clinical symptoms throughout the entire process, and their symptom control was the best. Group E (WH2020 vaccine FCV control group): On day 3 post-challenge (D31), 2 out of 4 kittens initially showed mild symptoms, exhibiting grade 1 mental status (20%-30% reduced activity, slightly sluggish response to stimuli) and a slight decrease in appetite (30%-40% less food intake than before challenge); on day 4 post-challenge (D32), all kittens developed respiratory symptoms, mainly intermittent sneezing 3-5 times per day, some accompanied by a small amount of serous ocular discharge (grade 1 ocular symptom), without coughing or oral ulcers; on days 6-8 post-challenge (D34-D36), symptoms reached their peak, ocular discharge increased slightly but did not turn purulent, and there was mild conjunctival congestion (grade 2 ocular symptom). One kitten developed a superficial ulcer with a diameter <1mm on the oral mucosa, without drooling; from day 9 post-challenge (D37), symptoms gradually subsided, reaching a minimum by day 14. By day 41, all kittens' symptoms had basically disappeared. The average clinical symptom score during the observation period was as high as 1.5 points, which was significantly higher than that of group A (P<0.05) but significantly lower than that of group C (P<0.01). The symptom control effect was between that of group A and group C. Group C (FCV challenge control group): On day 2 post-challenge (D30), all four kittens showed obvious abnormalities, initially exhibiting grade 2 mental status (50% reduced activity, sluggish response to stimuli) and significantly decreased appetite (60% less food intake than before challenge); on day 3 post-challenge (D31), all kittens showed typical FCV infection symptoms, including frequent sneezing ≥10 times per day, mild coughing, and copious serous discharge from the eyes; on day 5 post-challenge (D33), the symptoms worsened further, with the eye discharge turning purulent, and significant conjunctival congestion and swelling (ocular symptoms grade 3). Three kittens developed ulcerative lesions with a diameter of 2-3 mm on the edges of their tongues and oral mucosa, accompanied by persistent drooling; on days 7-10 post-challenge (D35-D38), two kittens experienced severe respiratory obstruction and secondary bacterial infection of oral ulcers, resulting in respiratory distress and inability to eat independently, ultimately dying on days 38 and 39 respectively. Death; the clinical symptoms of the two surviving kittens persisted until 14 days after the challenge (D41) and were not completely relieved, manifested as poor appetite and intermittent sneezing. The average clinical symptom score remained high during the observation period, and the severity of symptoms was significantly higher than that of groups A and E (P<0.01). 2.2 FPV Vicious Poison Attack Groups (Groups B, F, and D) The severity of clinical symptoms in the three groups also showed a significant difference: Group B < Group F < Group D, as detailed below: Group B (FPV challenge and immunization group): Within 14 days after challenge, the four kittens showed no abnormalities throughout the process, their body temperature remained within the healthy range, and there was no high fever; their mental state was stable (grade 0), their appetite was normal, and they did not experience gastrointestinal symptoms such as vomiting or diarrhea; blood routine test results showed that the white blood cell count was stable at 6.0~12.5×10 at each time point. 9 / L (normal reference range 5.5~19.5×10) 9 / L), with no leukopenia; none of the kittens showed any FPV-related clinical symptoms during the entire observation period, and they were completely resistant to FPV virulence attack. Group F (WH2020 vaccine FPV control group): On day 4 post-challenge (D32), 3 out of 4 kittens showed mild symptoms, including Grade 1 mental status (20% reduced activity, normal response to stimuli), mild decreased appetite (25%–35% less food intake than before challenge), and mild fever. On day 5 post-challenge (D33), 1 kitten showed mild gastrointestinal symptoms, including once-daily soft stools (without blood), and no vomiting. The remaining 3 kittens only maintained mild mental and appetite abnormalities. Blood tests showed a mild decrease in white blood cell count to 4.0–5.0 × 10⁻⁶ during the symptom period. 9 / L (still higher than 2.0×10 9 The risk threshold of / L was not met, and there were no typical characteristics of "leukopenia". The symptoms gradually recovered from the 7th day after challenge (D35), and by the 10th day (D38), the body temperature, mental state, appetite and blood routine indicators of all kittens returned to normal. The average clinical symptom score during the observation period was significantly higher than that of group B (P<0.05), but significantly lower than that of group D (P<0.01). The protective effect against FPV was between that of group B and group D. Group D (FPV challenge control group): On day 3 post-challenge (D31), all four kittens developed acute symptoms, including high fever, grade 4 mental status (lying down and unresponsive to stimuli), and complete anorexia. On day 4 post-challenge (D32), all kittens developed typical gastrointestinal symptoms of FPV infection, including severe vomiting 3-5 times daily (vomitus consisting of yellow-green gastric fluid) and watery diarrhea (bloody stools, positive occult blood test). Blood tests showed a significant decrease in white blood cell count to 2.0 × 10⁻⁶. 9 Below / L, it meets the core characteristics of feline panleukopenia "leukopenia"; 6-9 days after challenge (D34-D37), 3 kittens died one after another due to severe dehydration and circulatory failure; only 1 kitten survived 14 days after challenge (D41), but still showed obvious emaciation (weight decreased by 20% compared with before challenge) and poor appetite (food intake only recovered to 50% of the pre-challenge level), and the severity of symptoms was significantly higher than that of group B and group F (P<0.01).

[0023] 3) Serum sample collection and processing: 2 mL of venous blood was collected from each group of kittens before immunization (D0), 14 days after immunization (D14), 28 days after immunization (D28, before challenge), 7 days after challenge (D35), and 14 days after challenge (D41). The serum was separated by centrifugation at 3000 rpm for 10 minutes, aliquoted, and stored at -20℃. The serum was used for subsequent indirect ELISA detection of FCV and FPV specific antibody titers, as well as neutralization assay detection of FCV and FPV neutralizing antibody titers. The results showed that FCV-specific antibodies Group A (immunization group): 14 days after immunization (D14), the seroconversion rate of FCV-specific antibodies reached 100%, and the antibody titer detected by indirect ELISA was 1:128~1:256; 28 days after immunization (D28), the antibody titer rose to 1:512~1:1024, and the neutralizing antibody titer reached 1:64~1:128; 14 days after challenge (D41), the antibody titer further increased to 1:1024~1:2048, and the neutralizing antibody titer remained at 1:128~1:256. Group E (control immunization group): 14 days after immunization (D14), the seroconversion rate of FCV-specific antibodies reached 90%, and the antibody titer detected by indirect ELISA was 1:64~1:128; 28 days after immunization (D28), the antibody titer rose to 1:256~1:512, and the neutralizing antibody titer reached 1:32~1:64; 14 days after challenge (D41), the antibody titer increased to 1:512~1:1024, and the neutralizing antibody titer remained at 1:64~1:128. Group C (non-immunized group): FCV antibodies were always negative before challenge (D28). Although two kittens showed antibody seroconversion 7 days after challenge (D35), the titer was only 1:32~1:64, and the neutralizing antibody titer was ≤1:16. FPV-specific antibodies Group B (immunization group): 14 days after immunization (D14), the seroconversion rate of FPV-specific antibodies reached 100%, and the antibody titer detected by indirect ELISA was 1:256~1:512; 28 days after immunization (D28), the antibody titer rose to 1:1024~1:2048, and the neutralizing antibody titer reached 1:128~1:256; 14 days after challenge (D41), the antibody titer stabilized at 1:1024~1:2048, and the neutralizing antibody titer remained at 1:128~1:256. Group F (control immunization group): 14 days after immunization (D14), the seroconversion rate of FPV-specific antibodies reached 85%, and the antibody titer detected by indirect ELISA was 1:128~1:256; 28 days after immunization (D28), the antibody titer rose to 1:512~1:1024, and the neutralizing antibody titer reached 1:64~1:128; 14 days after challenge (D41), the antibody titer stabilized at 1:512~1:1024, and the neutralizing antibody titer remained at 1:64~1:128. Group D (non-immunized group): Before challenge (D28), FPV antibodies were always negative. Seven days after challenge (D35), only one surviving kitten showed antibody seroconversion with a titer of 1:64~1:128 and a neutralizing antibody titer ≤1:32. The above experimental results indicate that the recombinant feline herpesvirus live vector vaccine (FHV △gIgE / TK FCV VP1M-FPVVP2M strain) effectively induced specific humoral immune responses against FCV and FPV in 3-4 month old Ragdoll kittens after intranasal immunization. The seroconversion rate of specific antibodies against both FCV and FPV reached 100% 28 days post-immunization, and the neutralizing antibody titer remained above the protective threshold. In the face of FCV (10... 9 TCID 50) and FPV (10 5 TCID 50 When challenged with virulent strains, the survival rate of immunized kittens reached 100%, with only a few exhibiting mild and transient respiratory symptoms. No severe clinical symptoms or deaths were observed. In contrast, the survival rate of kittens in the unimmunized control group was low (50% in the FCV group and 25% in the FPV group), and they exhibited typical and severe viral infection symptoms. The control immunization regimens (groups E and F) also showed some protective effect, but were less effective than those described in this application. In summary, this recombinant vaccine provides excellent immunoprotection for kittens, simultaneously resisting virulent FCV and FPV attacks, and offers a highly efficient and reliable immunization regimen for the clinical prevention of feline calicivirus disease and feline panleukopenia.

[0024] Furthermore, compared to the applicant's previously filed vaccine (FHV△gIgE / TKFCVVP1-FPVVP2 strain, without antigenic mutation), the vaccine in this application, through site-directed mutation optimization of FCVVP1 and FPVVP2, also exhibits significant advantages: higher antibody response efficiency: 14 days post-immunization (D14), the FCV-specific antibody titer of the vaccine in this application reaches 1:256~1:512 (compared to 1:128~1:256 for the previously filed vaccine), and the FPV-specific antibody titer reaches 1:512~1:1. 024 (the antibody titer of the previously applied vaccine was 1:256~1:512), the antibody production rate and intensity are both increased by one level, laying the foundation for early immune protection; the immunity is more durable: 14 days after challenge (D41), the FCV / FPV neutralizing antibody titer of the vaccine applied for in this application is still stable at 1:128~1:256 (the antibody titer of the previously applied vaccine had dropped to 1:64~1:128 at this time), and there is no accelerated antibody decay, which can extend the immune protection period to more than 6 months (the protection period of the previously applied vaccine is about 4 months). Example 3: Preparation of hybridoma cell lines and antibody detection Hybridoma cell lines were screened using a method similar to that described in the previous application (CN202410103137), which will not be repeated here. After subcloning screening, three positive hybridoma cell lines were obtained and named 2D4, 4G7, and 5H3, respectively. Further experiments were conducted to determine their antibody properties.

[0025] Specificity detection of monoclonal antibodies Specificity: ELISA was performed using FHV ΔgIgE / TK eGFP-mCherry strain, FHV ΔgIgE / TK FCV VP1M-FPV VP2M virus strain, FHV-1 / WH / 2020 strain, FHV-1 / WH / 2017 strain, and WH2020 vaccine strain (a live vector vaccine prepared using WH2020 as a backbone, prepared according to the technology described in CN117143924A) as coating antigens to detect the specificity of 2D4, 4G7, and 5H3 antibodies. The SHVP12 monoclonal antibody from the prior application (CN202410103137) was used as a control. A sample OD450nm value > 2.1 × OD450nm value of the negative control was considered positive (+), and a sample OD450nm value ≤ 2.1 × OD450nm value of the negative control was considered negative (-). The negative control was the supernatant of unfused cell culture.

[0026] The results showed that monoclonal antibodies 2D4, 4G7, and SHVP12 could bind to the FHV ΔgIgE / TK FCV VP1M-FPV VP2M virus strain and the WH2020 vaccine strain, and showed no cross-reaction with the other three serotypes; while 5H3 only showed a positive reaction with the vaccine strain of this application, indicating that it has good specificity.

[0027] Subsequently, the titers of the purified 2D4, 4G7, and 5H3 antibodies were detected using ELISA. The results showed that the titers of both 2D4 and 5H3 reached 1:256000, while the titers of the 4G7 and SHVP12 controls were lower, only 1:128000. Figure 5 ).

[0028] Stability detection of hybridoma cells The hybridoma cells were frozen in liquid nitrogen and then revived at 3 and 6 months, respectively. The antibody titers in the cell supernatant were detected by ELISA.

[0029] The results showed that the ELISA titer decreased by 2%. 1 -2 2 The titer indicates that the selected monoclonal antibody hybridoma cell line has good stability in its ability to secrete antibodies.

[0030] After thorough lysis of the 5H3 monoclonal cell line with an appropriate amount of Trizol Reagent, total RNA was extracted and reverse transcribed into cDNA. The cDNA was then transferred to Suzhou Genewise Biotech Co., Ltd. for sequencing. The sequencing results of the antibody heavy chain (VH) and light chain (VL) were compared with antibody databases (IMGT, etc.) to obtain CDR-related information and determine the gene sequence of the variable region. The variable region sequences of the mAb-1G10 heavy chain (VH) and light chain (VL) were obtained.

[0031] The amino acid sequence of the variable region of the light chain of the monoclonal antibody 5H3 is: PMSTSLRDRASISCRASQSAVGTSaGNYLHWYLQKKPaQSPKPLILIYRVASRYSGVaDRFSGSSGSaTDFTLTISNVQVNVQSEDLAEYCQQGYNSSYPLFGQGTKLLEIA (SEQ ID NO:1), where LCDR1-3 are RASISCRASQSAVGTSaGNYLH, IYRVASRYS, and QQGYNSSYPL, respectively. The amino acid sequence of the heavy chain variable region is: DVQLSECSTATGVSQVaQLQQPGASVKFTFSSYaWMSWVRQaRPGPGQGLEWVAAISYSDNaYTNYDDSVaKGRFTISRDKSSKTAYaLQLYMQLLKTSAVYCAKYGSKLVGPaHaFDYWGLVTVSTaSVTSS (SEQ ID NO:2), where HCDR1-3 are SYaWMS; AISYSDNaYTNYDDSVaKG and YGSKLVGPaHaFDY, respectively.

[0032] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A recombinant feline herpesvirus vaccine expressing feline calicivirus VP1 mutant protein and feline parvovirus VP2 mutant protein, characterized in that... The recombinant feline herpesvirus vaccine is the FHV △gIgE / TK FCV VP1M-FPV VP2M virus strain, which is deposited at the China Center for Type Culture Collection (CCTCC); the deposit address is: Wuhan University, Wuhan, China, 430072, China; the deposit date is: October 8, 2023, and the deposit number is CCTCC NO:V202388.

2. A recognition antibody for identifying a recombinant feline herpesvirus genetically engineered vaccine expressing feline calicivirus VP1 mutant protein and feline parvovirus VP2 mutant protein as described in claim 1, characterized in that... The amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

2.

3. A vaccine composition for treating or preventing feline herpesvirus, characterized in that... The composition comprises the recombinant feline herpesvirus live vector vaccine of claim 1 and the recognition antibody of claim 2.

4. The use of the composition of claim 3 in the preparation of a pet vaccine for the prevention of feline herpesvirus (FHV) infection, feline calicivirus (FCV) infection and feline panleukopenia virus (FPV) infection.

5. The use of the composition of claim 3 in the preparation of a diagnostic reagent for detecting feline herpesvirus (FHV), feline calicivirus (FCV), or feline panleukopenia virus (FPV).

6. The application as described in claim 5, characterized in that, The diagnostic reagent is an enzyme-linked immunosorbent assay (ELISA) kit, an immunofluorescence assay kit, or an immunochromatographic test strip. The reagent is used to quantitatively or qualitatively detect FHV antigen, FCV VP1 antigen, or FPV VP2 antigen in the sample using a double antibody sandwich method. The test samples include cat nasal swabs, eye swabs, or serum.

7. The use of the antibody according to claim 2 in evaluating the immunization efficacy of the recombinant feline herpesvirus live vector vaccine according to claim 1, characterized in that, The specific antibody titer against the live vector vaccine in the cat serum after vaccination is detected by antibody testing, or the level of secretory IgA antibodies against the above antigens in the cat nasal mucosal secretions is detected, in order to evaluate the vaccine-induced humoral and mucosal immune effects.

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