A multivalent vaccine for feline infectious rhinotracheitis and its preparation method and application

Abstract

This invention provides a multivalent vaccine for feline infectious rhinoconjunctivitis (FINC), its preparation method, and its application. The multivalent vaccine is prepared using feline calicivirus strains FCV CC475, FCV-CC3, and FCV-CC246, with the gene sequence of FCV CC475 shown in SEQ ID NO.1. This multivalent vaccine is a trivalent inactivated vaccine, which addresses the problem of low vaccine protection due to the rapid mutation rate of feline calicivirus strains. It achieves a protection rate of up to 88.2% against 17 existing FINC strains.

Description

Technical Field

[0001] This invention belongs to the field of animal vaccine preparation, specifically relating to a feline infectious rhinoconjunctivitis multivalent vaccine, its preparation method, and its application. Background Technology

[0002] Infectious feline rhinitis-conjunctivitis (FCV) is a highly contagious disease caused by feline calicivirus (FCV). Its main clinical manifestations include oral ulcers, rhinitis, conjunctivitis, and upper respiratory tract infections. The disease is highly contagious, with infected and carrier cats being the primary sources of infection. Since Fastier first isolated FCV in 1957, researchers have subsequently isolated the virus from other felines such as lions, cheetahs, and tigers.

[0003] Following the first report of fatal viral systemic disease (VSD) associated with FCV in 1998, outbreaks of the disease occurred in countries such as the United States. Infected cats exhibited fever, edema, skin ulcers, jaundice, and a mortality rate of approximately 50%. Due to the high variability of FCV and its persistent shedding in infected individuals, it poses a serious threat to the health of pet cats and other felines.

[0004] Although immunization is widely used to control FCV infection, the presence of multiple hypervariable regions on the FCV capsid protein gives it rich antigenic diversity. Existing vaccines cannot produce complete cross-reactions with newly isolated strains and can only prevent and control the disease to a certain extent, but cannot protect animals from external infection.

[0005] Currently, significant progress has been made in domestic research on the etiology, morphology, and molecular biology of FCV. Research on FCV immunogenicity is helpful for virus screening and the development of novel vaccines, laying the foundation for the prevention and control of the disease. However, since most neutralizing epitopes are located in the hypervariable region of the FCV capsid protein, monovalent FCV vaccines may not completely prevent viral infection.

[0006] Therefore, multivalent vaccines have become a new research direction for FCV vaccines in order to improve cross-neutralization response. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a multivalent vaccine for feline infectious rhinoconjunctivitis, its preparation method, and its application.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention also provides a multivalent vaccine for feline infectious rhinoconjunctivitis, the multivalent vaccine being prepared using feline calicivirus strain FCV CC475, the full-length cDNA sequence of which is shown in SEQ ID NO.1.

[0010] The FCV CC475 strain described in this invention belongs to the Caliciviridae family, Calicivirus genus, and is a non-enveloped, single-stranded positive-sense RNA virus that provides more comprehensive protection against existing FCV strains. Since attenuated and inactivated vaccines from earlier FCV strains no longer provide sufficient protection against recent FCV strains, current calicivirus vaccines must be replaced by vaccines more adapted to the current epidemiological situation and capable of providing greater cross-neutralization against currently identified isolates in feline populations.

[0011] It should be noted that, in addition to being isolated from pathogenic material, the feline calicivirus strain CC475 described in this invention can also be obtained through virus rescue, provided that SEQ ID NO.1 is known. That is, a negative-strand RNA whole virus is rescued from a full-length cDNA clone to simulate the real virus entering the cell for replication and passage, thereby obtaining real virus particles. Then, the viral cDNA can be artificially manipulated in vitro, such as through mutation, deletion, insertion, and substitution. This allows for the study of RNA virus gene expression and regulation mechanisms, virus-host interactions, antiviral and gene therapy, exogenous gene expression, and the development of new vaccines.

[0012] As a preferred technical solution of the present invention, the feline infectious rhinoconjunctivitis multivalent vaccine is a bivalent vaccine, a trivalent vaccine, a quadrivalent vaccine or a pentavalent or higher vaccine, preferably a trivalent vaccine.

[0013] Preferably, the trivalent vaccine includes inactivated or attenuated FCV strains CC475, FCV-CC3, and FCV-CC246.

[0014] Monovalent vaccines are insufficient to provide complete protection against prevalent feline infectious rhinoconjunctivitis virus (FCV). Therefore, this invention collects FCV samples from across the country, isolates multiple FCV strains, prepares multiple strains of FCV rabbit hyperimmune serum, and tests the cross-protective efficacy between different strains. The results show that strains FCV-CC3, FCV-CC246, and FCV-CC475 can achieve a protection rate of nearly 90%. Furthermore, there are no commercially available multivalent FCV vaccines in China or abroad. Consequently, we developed a multivalent FCV vaccine.

[0015] Among them, FCV CC3 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 1, 2022, with accession number CGMCC No. 45218.

[0016] The FCV CC246 strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 1, 2022, with accession number CGMCC No. 45217.

[0017] Preferably, the feline infectious rhinoconjunctivitis multivalent vaccine is an inactivated vaccine.

[0018] In this invention, the multivalent vaccine further includes a vaccine adjuvant, which is an adjuvant acceptable for vaccine preparation. Preferably, the vaccine adjuvant used in this invention is Ge102 adjuvant.

[0019] In a second aspect, the present invention provides a method for preparing a multivalent feline infectious rhinoconjunctivitis vaccine as described in the first aspect, the method comprising:

[0020] Prepare a viral solution containing an inactivated or attenuated FCV CC475 strain, and then mix it with a viral solution containing other inactivated or attenuated feline infectious rhinoconjunctivitis virus strains.

[0021] Adding a vaccine adjuvant yields the aforementioned feline infectious rhinoconjunctivitis multivalent vaccine.

[0022] As a preferred embodiment of the present invention, the virus content in the viral fluid is ≥10. 7.0 TCID 50 / ml; for example, it could be 10 7.00 TCID 50 / ml, 10 7.50 TCID 50 / ml, 10 8.00 TCID 50 / ml, 10 8.50 TCID 50 / ml, 10 9.00 TCID 50 / ml, 10 9.50 TCID 50 / ml, 10 10.00 TCID 50 / ml; preferably ≥10 8.00 TCID 50 / ml, more preferably ≥10 9.00 TCID 50 / ml.

[0023] Preferably, in this invention, the volume ratio of the virus solution is 1:(0.5~2):(0.5~2), for example, it can be 1:1:1, 1:0.5:0.5, 1:1:0.5, 1:0.5:1, 1:1.5:1, 1:1:1.5, 1:2:2, etc.; preferably 1:1:1.

[0024] As a preferred technical solution of the present invention, the preparation method includes:

[0025] Virus solutions containing inactivated FCV CC475 strain, inactivated FCV CC3 strain, and inactivated FCV CC246 strain were prepared separately, with a virus content ≥10 in each solution. 9.5 TCID 50 / ml;

[0026] The viral fluid was then mixed at a volume ratio of 1:(0.5-2):(0.5-2), and a vaccine adjuvant was added to obtain the feline infectious rhinoconjunctivitis multivalent vaccine.

[0027] In this invention, the feline infectious rhinoconjunctivitis multivalent vaccine can be prepared using the following method:

[0028] The specific method is as follows:

[0029] Feline infectious rhinoconjunctivitis virus (FCV) strains FCV CC3, FCV CC246, and FCV CC475 were inoculated into F81 cells at MOIs of 0.01–0.1 and cultured for 24–48 hours. Pathological changes were observed periodically. Cell cultures and viral fluids were harvested after complete cell detachment, with viral concentrations not less than 10-. 9.5 TCID 50 / ml;

[0030] After the virus solution was inactivated at 30°C for 20 hours with BEI at a final concentration of 0.001 mol / L, 1 mol / L sodium thiosulfate (10% of the amount of BEI used) was added to neutralize the BEI. The three inactivated virus solutions were then mixed in a 1:1:1 ratio and then emulsified with Ge102 adjuvant in a 1:1 (V:V) ratio to obtain the corresponding vaccine.

[0031] The preparation method of BEI is as follows: weigh BEA and dissolve it in 0.2 mol / L NaOH. The weight-volume ratio of BEA to 0.2 mol / L NaOH is 2.05 g: 100 ml. Heat the solution in a water bath at 37°C for 1 hour to obtain 0.1 mol / L BEI. This process is accompanied by a change in pH value, which drops from 12.5 to 8.5.

[0032] Thirdly, the present invention provides the use of the feline infectious rhinoconjunctivitis multivalent vaccine as described in the first aspect in the preparation of a medicament for the prevention of feline infectious rhinoconjunctivitis.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] (1) In this invention, three strains with good cross-protection and high antibody titers were screened from 17 strains of feline infectious rhinoconjunctivitis virus (FCV), namely FCV CC3, FCV CC246, and FCV CC475. Among them, the neutralizing antibody titer of FCV CC475 serum against FCV CC10, FCV CC256, FCV CC272, FCV CC313, FCV CC458, and FCV CC463 virus strains (excluding itself) was ≥1:32, indicating that it has a protective effect.

[0035] (2) The homology among the three strains FCV CC3, FCV CC246, and FCV CC475 is low. The sequence homology between FCV CC3 and FCV CC246 is 78.6%, and the homology between FCV CC475 and FCV CC3 and FCV CC246 is 78.1% and 82.9%, respectively. Therefore, their combination can prevent a sufficient number of feline infectious rhinoconjunctivitis virus infections. The combination of these three strains can neutralize 15 different strains of feline infectious rhinoconjunctivitis virus. The trivalent inactivated FCV vaccine prepared using these three strains has a protection rate of up to 88.2% against the existing 17 strains of feline infectious rhinoconjunctivitis virus. In the immune challenge test, after injection of this trivalent FCV vaccine, the test cats can obtain 100% protection and there are no deaths. Detailed Implementation

[0036] The technical solution of the present invention will be further illustrated below through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be determined by the claims.

[0037] In the following embodiments, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the art; unless otherwise specified, all experimental methods and techniques used were conventional methods and techniques in the art.

[0038] Example 1: Screening of virus strains

[0039] This invention utilizes 17 strains of feline calicivirus (FCV) capable of causing feline infectious rhinoconjunctivitis (FIC) isolated and identified from diseased cat samples to prepare rabbit polyantiserum. These strains include FCV CCSN-1, FCV CC3, FCV CC9, FCV CC10, FCV CC85, FCV CC246, FCV CC247, FCV CC256, FCV CC272, FCV CC295, FCV CC313, FCV CC316, FCV CC320, FCV CC456, FCV CC458, FCV CC463, and FCV CC475.

[0040] Rabbit polyantiserum obtained after preparing Feline Infectious Rhinotracheitis, Chimeric Virus Disease, and Panleukopenia Trivalent Inactivated Vaccine was used as a control and cross-protection was performed with existing feline infectious rhinoconjunctivitis virus to determine the neutralizing antibody titer.

[0041] Table 1. Results of strain screening

[0042]

[0043] Table 2 Results of strain screening

[0044]

[0045]

[0046] Table 3. Results of strain screening

[0047]

[0048] Based on Tables 1, 2, and 3, it can be seen that the Miaosanduo antiserum can neutralize 6 different strains of feline infectious rhinoconjunctivitis virus (FIV) (a neutralizing antibody titer of ≥1:32 indicates protection), with a protection rate of 35.3% against these 17 strains of FIV. The FCV CC3, CC246, and FCV CC475 antiserums can neutralize 15 different strains of FIV, with a protection rate of 88.2% against these 17 strains of FIV.

[0049] Furthermore, there is no effective cross-protection between FCV CC3, FCV CC246 and FCV CC475 strains, and the antiserum neutralizing antibody titers of FCV CC3, FCV CC246 and FCV CC475 strains are all not lower than 1:2048, indicating good immunogenicity.

[0050] Therefore, in this embodiment, three strains with good cross-protection and high antibody titers were screened from 17 strains of feline infectious rhinoconjunctivitis virus: FCV CC3, FCV CC246 and FCV CC475.

[0051] Example 2 Homology Comparison

[0052] The genome sequence of FCV CC475 is shown in SEQ ID NO.1.

[0053] FCV CC3 strain belongs to the family Caliciviridae, genus Calicivirus, and is a non-enveloped, single-stranded, positive-sense RNA virus. It was deposited on July 1, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 45218. The viral load is no less than 10^6.9.5 TCID 50 / ml.

[0054] FCV strain CC246 belongs to the family Caliciviridae, genus Calicivirus, and is a non-enveloped, single-stranded, positive-sense RNA virus. It was deposited on July 1, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 45217. The viral load is no less than 10^6. 9.5 TCID 50 / ml.

[0055] Sequence alignment analysis revealed low sequence homology (78.6%) between FCV CC3 and FCV CC246. Similarly, FCV CC475 also showed low homology with both FCV CC3 and FCV CC246, at 78.1% and 82.9%, respectively.

[0056] Example 3: Vaccine Preparation

[0057] Inactivated virus solutions of FCV strains CC3, CC246, and CC475 were prepared separately and then mixed in proportion to prepare a trivalent inactivated FCV vaccine.

[0058] The specific method is as follows:

[0059] Feline infectious rhinoconjunctivitis virus (FCV) strains FCV CC3, FCV CC246, and FCV CC475 were inoculated into F81 cells at an MOI of 0.05 and cultured for at least 24 hours. The lesions were observed periodically. Once the cells had completely detached, the cell culture and viral fluid were harvested, ensuring a viral load of at least 10⁻⁶. 9.5 TCID 50 / ml;

[0060] After the virus solutions were inactivated at 30°C for 20 hours with BEI at a final concentration of 0.001 mol / L, 1 mol / L sodium thiosulfate was added to neutralize the BEI. The three inactivated virus solutions were then mixed in a 1:1:1 ratio and then emulsified with Ge102 adjuvant in a 1:1 (V:V) ratio to obtain the corresponding vaccines.

[0061] The preparation method of BEI is as follows: Weigh BEA and dissolve it in 0.2 mol / L NaOH. The weight-to-volume ratio of BEA to 0.2 mol / L NaOH is 2.05 g: 100 ml. Heat in a water bath at 37°C for 1 hour to obtain 0.1 mol / L BEI.

[0062] Example 4: Evaluation of vaccine efficacy

[0063] The FCV trivalent inactivated vaccine prepared in Example 3 was used to challenge the virus (FCV CC3 / FCV CC246 / FCVCC475) after immunization. The immune protection effect was evaluated after 14 days of observation.

[0064] I. Immunization, challenge, and observation of experimental cats

[0065] 1. Grouping

[0066] Thirty healthy, susceptible test cats aged 2-3 months with no history of immunization (negative FCV and FHV antigen tests in eye and nasal swabs and pharyngeal swabs, neutralizing antibody titers of FCV CC3 / FCV CC246 / FCV CC475 not exceeding 1:4, and weighing 0.8kg-1.3kg) were randomly divided into 6 groups of 5 cats each.

[0067] The groups are: Group 1 - Immune Group (FCV CC3), Group 2 - Immune Group (FCV CC246), Group 3 - Immune Group (FCV CC475), Group 4 - Immune Control Group (FCV CC3), Group 5 - Immune Control Group (FCV CC246), and Group 6 - Immune Control Group (FCV CC475), as shown in Table 4 below.

[0068] Table 4. Grouping of experimental animals and virus strains used for challenge.

[0069]

[0070] 2. Immunity

[0071] The trivalent inactivated FCV vaccine prepared in Example 3 was used to immunize cats in group 1 (FCV CC3), group 2 (FCV CC246), and group 3 (FCV CC475).

[0072] First immunization: The immunized group was subcutaneously injected with FCV trivalent inactivated vaccine, 1.0 ml / animal; the immunized control group was subcutaneously injected with MEM culture medium, 1.0 ml / animal.

[0073] Boosted immunization: The immunization group received a booster immunization 21 days after the initial immunization, administered subcutaneously by injection, 1.0 ml / animal; the immunization control group received a subcutaneous injection of MEM culture medium, 1.0 ml / animal.

[0074] 3. Attacking the poison

[0075] Twenty-one days after booster immunization, all experimental cats were challenged with FCV CC3, FCV CC246, and FCV CC475 viral fluids, which were diluted to 10⁻⁶ in MEM medium. 4.50 TCID 50 / ml;

[0076] The diluted FCV CC3 strain virus was used to challenge Group 1 (FCV CC3) and Group 4 (FCV CC3) immunization control group.

[0077] The diluted FCV CC246 strain was used to challenge the two groups (FCV CC246) and the five groups (FCV CC246) of the immune control group.

[0078] The diluted FCV CC475 strain was used to challenge the three groups of immune groups (FCV CC475) and the six groups of immune control groups (FCV CC475).

[0079] FCV CC3, FCV CC246, and FCV CC475 were administered via nasal drops, 0.25 ml per cat in each group, slowly and alternately instilled into both nostrils. The six experimental cats were isolated and their condition was observed and recorded daily for 14 consecutive days. Nasopharyngeal swabs were collected for PCR testing. The criteria for determining disease in cats were a positive RT-PCR test result and the presence of typical FCV infection symptoms (high fever, oral ulcers, respiratory symptoms).

[0080] II. Post-infection observation and result recording

[0081] 4.1 Clinical symptoms

[0082] Clinical symptoms of all experimental cats were observed at the same time every day for 4 hours before and 1 to 14 days after the challenge. After the observation, the eyes and nose of each cat were wiped clean with sterile gauze for observation the next day.

[0083] Cats in groups 1 (FCV CC3), 2 (FCV CC246), and 3 (FCV CC475) did not show obvious clinical symptoms after challenge with the virus, only a small amount of ocular and nasal discharge and sneezing. Cats in groups 4 (FCV CC3), 5 (FCV CC246), and 6 (FCV CC475) began to show symptoms of FCV infection, such as lethargy, increased ocular and nasal discharge, ulcers, and fever, 2-3 days after challenge.

[0084] 4.2. Body temperature

[0085] After the viral challenge, body temperature was measured at the same time every day.

[0086] The body temperature fluctuations of cats in Group 1 (FCV CC3), Group 2 (FCV CC246), and Group 3 (FCV CC475) were not significant. Cats in Group 4 (FCV CC3), Group 5 (FCV CC246), and Group 6 (FCV CC475) all began to show elevated body temperature on the 3rd day after challenge, and their body temperature gradually returned to normal 10 days after challenge. The results are shown in Tables 5 and 6 below.

[0087] Table 5. Observation of body temperature after viral challenge in the immunized group.

[0088]

[0089] Table 6. Body temperature observation after challenge in the immune control group.

[0090]

[0091] 4.2 PCR detection of eye and nasal swabs

[0092] From day 1 to day 14 after the viral challenge, eye and nasal swabs were collected daily for PCR testing to detect FCV.

[0093] On day 3 after challenge, PCR results from eye and nasal swabs of group 1 (FCV CC3), group 2 (FCV CC246), and group 3 (FCV CC475) were positive, and continuous testing showed that their viral shedding time was less than or equal to 3 days. On days 2-3 after challenge, PCR results from eye and nasal swabs of group 4 (FCV CC3), group 5 (FCV CC246), and group 6 (FCV CC475) were positive, and continuous testing showed that their viral shedding time was greater than 7 days. The experimental cats began to die on day 9 after challenge. The results are shown in Tables 7 and 8.

[0094] Table 7. PCR detection results of the immunized group after challenge.

[0095]

[0096] Note: "+" represents positive, "-" represents negative, and "x" represents the death of the test cat.

[0097] Table 8. PCR detection results of the immune control group after challenge.

[0098]

[0099]

[0100] Note: "+" represents positive, "-" represents negative, and "x" represents the death of the test cat.

[0101] III. Experimental Conclusions

[0102] After the immune challenge test, the experimental cats were 100% protected after being injected with the FCV trivalent vaccine of the present invention, and there were no deaths.

[0103] The incidence rate was 100% in the four immune control groups (FCV CC3), five immune control groups (FCV CC246), and six immune control groups (FCV CC475), the protection rate was 0, and the mortality rates were 20%, 40%, and 40%, respectively.

[0104] The incidence rate was 0 in group 1 (FCV CC3), group 2 (FCV CC246), and group 3 (FCV CC475), the protection rate was 100%, and the mortality rate was 0. The results are shown in Table 9.

[0105] Table 9. Statistical results of morbidity / protection rate / mortality in each group.

[0106]

[0107]

[0108] The above experiments show that the trivalent feline infectious rhinoconjunctivitis vaccine of the present invention has good cross-protection and can provide effective and broader immune protection for cats infected with feline infectious rhinoconjunctivitis virus. It can be used for the protection against feline infectious rhinoconjunctivitis virus and has broad application prospects.

[0109] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A multivalent vaccine for feline infectious rhinoconjunctivitis, characterized in that, The multivalent vaccine was prepared using the feline calicivirus strain FCVCC475, the cDNA sequence of which is shown in SEQ ID NO.

1.

2. The feline infectious rhinoconjunctivitis multivalent vaccine according to claim 1, characterized in that, The feline infectious rhinoconjunctivitis multivalent vaccine is a trivalent vaccine.

3. The feline infectious rhinoconjunctivitis multivalent vaccine according to claim 2, characterized in that, The trivalent vaccine includes inactivated or attenuated FCV strains CC475, FCV-CC3, and FCV-CC246. Among them, FCV CC3 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 1, 2022, with accession number CGMCC No. 45218; The FCV CC246 strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 1, 2022, with accession number CGMCC No. 45217.

4. The feline infectious rhinoconjunctivitis multivalent vaccine according to any one of claims 1 to 3, characterized in that, The feline infectious rhinoconjunctivitis multivalent vaccine is an inactivated vaccine.

5. The feline infectious rhinoconjunctivitis multivalent vaccine according to any one of claims 1 to 4, characterized in that, The multivalent vaccine also includes vaccine adjuvants.

6. A method for preparing a multivalent feline infectious rhinoconjunctivitis vaccine as described in any one of claims 1 to 5, characterized in that, The preparation method includes: Prepare a viral solution containing an inactivated or attenuated FCV CC475 strain, and then mix it with a viral solution containing other inactivated or attenuated viral strains. Adding a vaccine adjuvant yields the aforementioned feline infectious rhinoconjunctivitis multivalent vaccine.

7. The preparation method according to claim 6, characterized in that, The virus content in the virus liquid is ≥ 10 7.0 TCID 50 / ml.

8. The preparation method according to claim 6 or 7, characterized in that, The preparation method includes: Virus solutions containing inactivated FCV CC475 strain, inactivated FCV CC3 strain, and inactivated FCV CC246 strain were prepared separately; the virus content in each virus solution was ≥10. 7.0 TCID 50 / ml; The viral fluid was then mixed at a volume ratio of 1:(0.5-2):(0.5-2), and a vaccine adjuvant was added to obtain the feline infectious rhinoconjunctivitis multivalent vaccine.

9. The use of the feline infectious rhinoconjunctivitis multivalent vaccine as described in any one of claims 1 to 5 in the preparation of a medicament for the prevention of feline infectious rhinoconjunctivitis.

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