CN112195161A

CN112195161A - Canine parvovirus vaccine strain, vaccine and preparation method thereof

Info

Publication number: CN112195161A
Application number: CN202011126639.6A
Authority: CN
Inventors: 刘艳霞; 刘国英; 舒秀伟; 李凤艳; 王一平; 侯芳芳
Original assignee: Liaoning Yikang Biological Corp ltd
Current assignee: Liaoning Yikang Biological Corp ltd
Priority date: 2020-10-20
Filing date: 2020-10-20
Publication date: 2021-01-08
Anticipated expiration: 2040-10-20
Also published as: CN112195161B

Abstract

The invention discloses a canine parvovirus vaccine strain for treating, preventing, slowing down or controlling canine parvovirus enteritis, wherein the microbial preservation number is CGMCC No. 19398. The vaccine strain has low toxicity and good immunogenicity. The invention also discloses a live vaccine composition taking the canine parvovirus vaccine strain as immunogen. The vaccine composition is safe and effective.

Description

Canine parvovirus vaccine strain, vaccine and preparation method thereof

Technical Field

The invention belongs to the field of prevention veterinarians, relates to a live vaccine for veterinary use, and particularly relates to a canine parvovirus vaccine strain, a vaccine and a preparation method thereof.

Background

With changing environments and dietary patterns of human life, wild animals and companion animals have increasingly intimate contact with humans, thereby increasing the potential risk of human infection with animal-derived diseases. Dogs are one of the important companion animals for humans, and along with humans, it is imperative to strengthen the control of major diseases in dogs.

Canine Parvovirus (CPV) is a causative agent of a virulent infectious disease in dogs, and it causes acute hemorrhagic enteritis and non-suppurative myocarditis, an acute, contact infectious disease that threatens and endangers the canine industry in the world today most seriously. The disease has spread to all dog groups in the world, and CPV is widely spread in both wild and domestic dog groups. With the great increase of the feeding amount of working dogs (such as military dogs, police dogs, guide dogs and the like), experimental dogs and pet dogs in China, the canine parvovirus infection is getting more severe day by day, which brings great economic loss to the breeding dogs and becomes one of the great epidemic diseases harming the breeding dog industry. Therefore, the complete elimination of the disease becomes a difficult problem, so that the prevention and treatment of canine parvoviral enteritis becomes a long-term and difficult task, and the development of safe and effective vaccines is very important.

Disclosure of Invention

The canine parvovirus live vaccine prepared by the invention adopts the domestic F81 cell passage attenuated strain of an epidemic strain, has good toxicity and immunogenicity, high virus content, long immune duration and safe and effective vaccine.

In order to solve the problems in the prior art, the invention provides a vaccine strain for treating, preventing, slowing and/or controlling canine parvovirus enteritis, which is a canine parvovirus vaccine strain, wherein the canine parvovirus vaccine strain is a virus strain with the microbial preservation number of CGMCC No.19398, or a passaged virus strain or a mutant virus strain with unchanged clinical pathogenicity and immunogenicity.

In a second aspect, the present invention provides a vaccine composition for treating, preventing, slowing and/or controlling canine parvoviral enteritis, said vaccine composition comprising a vaccine strain according to the first aspect of the present invention as an immunogen.

In some embodiments, the raw materials of the vaccine composition comprise the immunogen and an adjuvant.

In some embodiments, the vaccine composition is a live vaccine composition.

In some embodiments, the vaccine composition comprises the immunogen and the adjuvant in a dry weight ratio of 1.0 × 10^5.0TCID₅₀: 50-150mg (e.g., 1.0X 10)^5.0TCID₅₀：60mg、 70mg、80mg、90mg、100mg、110mg、120mg、130mg、140mg)。

In some embodiments, the vaccine composition comprises the immunogen and the adjuvant in a dry weight ratio of 1.0 × 10^5.0TCID₅₀：105mg。

In some embodiments, the TCID of the immunogen₅₀Calculated according to the Reed-Muench method based on F81 cell culture.

In some embodiments, the method for culturing the vaccine strain comprises the following steps:

(i) inoculating the vaccine strain into F81 cells for culture, and harvesting crude venom of the vaccine strain;

(ii) and purifying the crude venom of the vaccine strain to obtain purified vaccine strain venom.

In some embodiments, in step (i), the crude venom of the vaccine strain is harvested when CPE reaches 80% (e.g., 85%, 88%, 92%, 95%, 98%) or more.

In some embodiments, in step (i), the crude venom of the vaccine strain is harvested when CPE reaches 90% or more.

In some embodiments, in step (i), the culture solution 10 is incubated according to the virus strain^5.0TCID₅₀: 50-150ml (e.g., 10)^5.0TCID₅₀: 60ml, 70ml, 80ml, 90ml, 100ml, 110ml, 120ml, 130ml, 140ml) in a predetermined ratio.

In some embodiments, in step (i), the inoculation is performed at an MOI of 0.005 to 0.01 (e.g., 0.006, 0.007, 0.008, 0.009).

In some embodiments, in step (i), the culture Medium is RPMI Medium 1640 with 2-8 v/v% (e.g., 3 v/v%, 4 v/v%, 5 v/v%, 6 v/v%, 7 v/v%) newborn bovine serum.

In some embodiments, in step (i), the temperature of the culture is 36.5-37.5 ℃ (e.g., 36.7 ℃, 36.9 ℃, 37.1 ℃, 37.3 ℃).

In some embodiments, in step (i), the pH of the culture is 7.0-7.5 (e.g., 7.1, 7.2, 7.3, 7.4).

In some embodiments, in step (i), the DO value of the culture is 40-50% (e.g., 42%, 44%, 46%, 48%).

In some embodiments, in step (i), when the culture is a spinner flask culture, the culture is performed under conditions of 9-12 rpm (e.g., 10 rpm, 11 rpm).

In some embodiments, in step (i), when the culturing is suspension culturing, the culturing is performed under the condition of 25-35 rpm (e.g., 26 rpm, 28 rpm, 30 rpm, 32 rpm, 34 rpm).

In some embodiments, in step (i), the F81 cells are from a monolayer of F81 cells.

In some embodiments, in step (i), the vaccine strain is inoculated after trypsinizing the monolayer of F81 cells.

In some embodiments, in step (i), the crude venom of the vaccine strain is subjected to a sterility test.

In some embodiments, in step (i), the F81 cells are prepared by: the cultured F81 seed cells were expanded to obtain expanded F81 cells.

In some embodiments, the expansion culture is performed at 36.5-37.5 deg.C (e.g., 36.7 deg.C, 36.9 deg.C, 37.1 deg.C, 37.3 deg.C).

In some embodiments, the expansion culture is performed in a spinner flask at 9-12 rpm (e.g., 10 rpm, 11 rpm).

In some embodiments, the extended culture Medium is RPMI Medium 1640 with 6-10 v/v% (e.g., 7 v/v%, 8 v/v%, 9 v/v%) newborn bovine serum.

In some embodiments, the time for the extended culture is 18-30h (e.g., 20h, 22h, 24h, 26h, 28 h).

In some embodiments, when the culturing is suspension culturing, the expanded cultured F81 cells are trypsinized and then transferred to a bioreactor for continued culturing.

In some embodiments, the temperature of the continued culture in the bioreactor is 36-38 ℃ (e.g., 36.5 ℃, 37 ℃, 37.5 ℃).

In some embodiments, the pH of the continued culture in the bioreactor is 7.0-7.5 (e.g., 7.1, 7.2, 7.3, 7.4).

In some embodiments, the DO value of the further culture in the bioreactor is 40-50% (e.g., 42%, 44%, 46%, 48%).

In some embodiments, the continuing culturing is performed at 25-35 rpm (e.g., 26 rpm, 28 rpm, 30 rpm, 32 rpm, 34 rpm) in the bioreactor.

In some embodiments, in step (ii), the crude venom of the vaccine strain is purified after 1-3 freeze-thawing cycles.

In some embodiments, in step (ii), the method of purification is: filtering with 0.22-0.45 μm filter membrane to remove cells and cell debris to obtain the purified vaccine strain venom.

In some embodiments, in step (ii), the virus content in the purified vaccine strain venom is more than or equal to 10^6.0TCID₅₀/ml。

In some embodiments, the excipient is a lyoprotectant.

In some embodiments, the lyoprotectant is a composition comprising 2-3mg/m (e.g., 2.2mg/mL, 2.4mg/mL, 2.6mg/mL, 2.8mg/mL) L polyvinylpyrrolidone, 8-12mg/mL (e.g., 8.5mg/mL, 9.0mg/mL, 9.5mg/mL, 10.0mg/mL, 10.5mg/mL, 11.0mg/mL, 11.5mg/mL) sorbitol, 3-7mg/mL (e.g., 4.0mg/mL, 5.0mg/mL, 6.0mg/mL) glycine, 30-50mg/mL (e.g., 35mg/mL, 40mg/mL, 45mg/mL) sucrose, 30-60mg/mL (e.g., 35mg/mL, 40mg/mL, 45mg/mL, 50 mg/mL), 55mg/mL) of trehalose in water.

In some embodiments, the method of preparing the lyoprotectant includes the steps of:

preparing a first aqueous solution comprising 4-6mg/mL (e.g., 4.5mg/mL, 5.0mg/mL, 5.5mg/mL) polyvinylpyrrolidone, 16-24mg/mL (e.g., 18.0mg/mL, 20.0mg/mL, 22.0mg/mL) sorbitol;

preparing a second aqueous solution comprising 6-14mg/mL (e.g., 8.0mg/mL, 10.0mg/mL, 12.0mg/mL) glycine, 60-100mg/mL (e.g., 65.0mg/mL, 70.0mg/mL, 75.0mg/mL, 80.0mg/mL, 85.0mg/mL, 90.0mg/mL, 95.0mg/mL) sucrose, 60-120mg/mL (e.g., 65.0mg/mL, 70.0mg/mL, 75.0mg/mL, 80.0mg/mL, 85.0mg/mL, 90.0mg/mL, 95.0mg/mL, 100.0mg/mL, 105.0mg/mL, 110.0mg/mL, 115.0mg/mL) trehalose;

mixing the first aqueous solution and the second aqueous solution in a volume ratio of 1:0.8-1.2 (e.g., 1:0.9, 1.0, 1.1) to obtain the lyoprotectant.

In some embodiments, the first aqueous solution is used after sterilization.

In some embodiments, the first aqueous solution is used after sterilization at 110 ℃ and 120 ℃ (e.g., 112 ℃, 114 ℃, 116 ℃, 118 ℃).

In some embodiments, the second aqueous solution is used after sterilization.

In some embodiments, the second aqueous solution is used after filter sterilization.

In a third aspect, the present invention provides a process for the preparation of a vaccine composition according to the second aspect of the invention, said process comprising the steps of:

preparing the vaccine strain into the vaccine composition.

In some embodiments, the vaccine strain is mixed with the adjuvant to obtain the vaccine composition.

In some embodiments, the vaccine composition is dried to obtain a dried vaccine composition.

In some embodiments, the drying is vacuum freeze drying.

In some embodiments, the freeze vacuum drying comprises the steps of:

(a) freezing the vaccine composition to obtain a frozen vaccine composition;

(b) subjecting the frozen vaccine composition to sublimation drying at a temperature that is increased in stages under vacuum conditions to obtain a preliminary dried vaccine composition;

(c) and (3) carrying out sublimation drying on the preliminarily dried vaccine composition under the vacuum condition at the normal temperature condition to obtain the dried vaccine composition.

In some embodiments, in step (a), the vaccine composition is frozen at-50 ℃ to-40 ℃ (such as, -48 ℃, -46 ℃, -44 ℃, -42 ℃).

In some embodiments, in step (a), the duration of freezing is 1.5-2.5h (e.g., 1.6h, 1.8h, 2.0h, 2.2h, 2.4 h).

In some embodiments, in step (b), the vacuum condition is 8pa to 10pa (e.g., 8.5pa, 9.0pa, 9.5 pa).

In some embodiments, in step (b), the frozen vaccine composition is subjected to a first sublimation at-30 ℃ to-26 ℃ (e.g., -29 ℃, -28 ℃, -27 ℃), a second sublimation at-17 ℃ to-13 ℃ (e.g., -16 ℃, -15 ℃, -14 ℃) and a third sublimation at 6-10 ℃ (e.g., 7 ℃, 8 ℃, 9 ℃).

In some embodiments, in step (b), the first sublimation time is 4-8h (e.g., 5h, 6h, 7 h).

In some embodiments, in step (b), the time required for the temperature to increase from-50 ℃ to-40 ℃ to-30 ℃ to-26 ℃ is 0.5 to 1.5h (e.g., 0.6h, 0.8h, 1.0h, 1.2h, 1.4 h).

In some embodiments, in step (b), the second sublimation time is 4-8h (e.g., 5h, 6h, 7 h).

In some embodiments, in step (b), the time required for the temperature to increase from-30 ℃ to-26 ℃ to-17 ℃ to-13 ℃ is 0.5 to 1.5h (e.g., 0.6h, 0.8h, 1.0h, 1.2h, 1.4 h).

In some embodiments, in step (b), the third sublimation is carried out for a time of 2 to 6 hours (e.g., 3.0 hours, 3.5 hours, 4.0 hours, 4.5 hours, 5.0 hours, 5.5 hours).

In some embodiments, in step (b), the time required for the temperature to increase from-17 ℃ to-13 ℃ to 6-10 ℃ is 0.5 to 1.5h (e.g., 0.6h, 0.8h, 1.0h, 1.2h, 1.4 h).

In some embodiments, in step (c), the vacuum condition is 8pa to 10pa (e.g., 8.5pa, 9.0pa, 9.5 pa).

In some embodiments, in step (c), the temperature of sublimation is from 25 ℃ to 30 ℃ (e.g., 26 ℃, 27 ℃, 28 ℃, 29 ℃).

In some embodiments, in step (c), the sublimation time is 1-3h (e.g., 1.5h, 2.0h, 2.5 h).

In some embodiments, in step (b), the time required for the temperature to rise from 6-10 ℃ to 25-30 ℃ is 0.5-1.5h (e.g., 0.6h, 0.8h, 1.0h, 1.2h, 1.4 h).

In a fourth aspect, the invention provides a vaccine strain according to the first aspect of the invention, a vaccine composition according to the second aspect of the invention, or a method of manufacture according to the third aspect of the invention, for use in the manufacture of a formulation for use alone or in combination with other immunological agents and/or drugs in the treatment, prevention, alleviation and/or management of canine parvoviral enteritis.

In some embodiments, the canine parvoviral enteritis is canine parvoviral enteritis, ferret canine parvoviral enteritis, or feline canine parvoviral enteritis.

In some embodiments, the canine parvoviral enteritis is mink canine parvoviral enteritis, or fox canine parvoviral enteritis.

Drawings

FIG. 1 is a photograph of isolated culture of canine parvovirus to be identified.

FIG. 2 is an electron micrograph of a canine parvovirus isolate to be identified.

FIG. 3 is a schematic diagram of the production process of the canine parvovirus live vaccine (strain P6) of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Defining:

a passaged or mutant strain with no changes in clinical pathogenicity and immunogenicity:

with respect to the strains claimed in the present invention, it is clear that passaged strains without mutations belong to substantially identical strains, with no change in clinical pathogenicity and immunogenicity meaning no substantial change. Due to the detection operation conditions, differences of animal species, age, sex, health condition and the like and predictable systematic errors belong to the passaged virus strain without substantial changes and clinical pathogenicity and immunogenicity changes. In addition, minor mutations are inevitably introduced after the virus is passaged many times, and when the mutations occur in non-coding sequence regions or synonymous mutations of coding regions or mutations that do not affect the pathogenicity and immunogenicity of the virus (for example, there may be a connecting amino acid residue between two domains, or a residue of a minor mutation located in a higher structure of a protein that does not affect the pathogenicity or immunogenicity because it is not in contact with immune cells), these minor mutations remain insubstantial mutations and should be regarded as mutant virus strains without changes in clinical pathogenicity and immunogenicity. For example, the F81 cell line passaged virus strain of the virus strain with the biological preservation number of CGMCC No.19398 of the invention has no substantial change in clinical pathogenicity and immunogenicity, and belongs to the passaged virus strain or mutant virus strain with no change in clinical pathogenicity and immunogenicity.

TCID of the invention₅₀Is calculated according to the Reed-Muench method.

Canine parvovirus virulent CPV-v strain: strain isolated internally by Liaoning Yikang Biometrics Ltd, TCID₅₀Data were obtained by F81 cell assay.

The test dogs of the invention were beagles.

F81 cells were purchased from China institute of veterinary medicine, and were passaged and preserved by Liaoning Yikang biological products GmbH.

RPMI Medium 1640, available from GIBCO, lot No.: 1900496, respectively; newborn bovine serum, purchased from Bovogen Biologicals Pty Ltd, lot No.: 1507D; trypsin, purchased from GIBCO, lot No.: 1902509.

The invention refers to the current 'Chinese animal pharmacopoeia' which is published by the Chinese agriculture publishing company and is divided into three parts of 'Chinese animal pharmacopoeia' 2015 edition.

The scheme of the production process of the canine parvovirus live vaccine (strain P6) is shown in figure 3.

Example 1 acquisition of Primary strains of Canine parvoVirus

(1) Cases of disease

1 case of puppies presenting suspected clinical canine parvovirus disease symptoms are collected in a certain canine farm in Liaoning province, the disease is developed, and the symptoms are as follows: fever, mental depression, mild diarrhea. Judging according to symptoms, the canine parvoviral enteritis is suspected to be suffered.

(2) Virus isolation

Taking intestinal canal tissue of a diseased animal, washing the intestinal canal tissue with sterilized normal saline for 3 times, and mixing the intestinal canal tissue with the normal saline according to the ratio of lg tissue: adding sterilized cold normal saline into intestinal canal tissue according to the proportion of 10ml, putting the intestinal canal tissue into a tissue mixer, fully mashing the intestinal canal tissue at 10000-12000 r/min, repeatedly freezing and thawing at the temperature of below-15 ℃ for 3 times, filtering for 2 times by using 3 layers of sterilized gauze, collecting filtrate, centrifuging for 30 minutes at the temperature of 12000r/min4 ℃, collecting supernatant, adding penicillin with the final concentration of 1000IU/ml and streptomycin with the final concentration of 1000 mu g/ml, and subpackaging to obtain a tissue fluid b.

F81 cell separation was used. Taking F81 cells for digestion culture according to a conventional method, wherein the culture solution is RPMI Medium 1640 containing 8% newborn bovine serum, and synchronously inoculating the tissue fluid b when dispersed cells are to be transferred, wherein the inoculation amount is 1 v/v%, and meanwhile, a healthy cell control without inoculation is arranged. Placing at 37 ℃ with 5% CO₂Culturing and observing in an incubator for 3-5 days, harvesting the culture, and identifying. Visible cytopathic effect appears in the 1 st generation, and the cells have the cell pathological changes of net pulling and shedding (see figure 1), on the basis, the 1 st generation is further expanded, 200ml of cell culture solution b is collected by co-culture, most of the cell culture solution b is added with the sucrose skim milk protective agent and then is frozen and preserved. The virus contained in the cell culture liquid b is referred to as strain b.

Diluting the cell culture solution b with RPMI Medium 1640 cell culture solution containing 8% newborn calf serum 10 times, and collecting 10 times^-1～10^-8Diluting, synchronously inoculating 96-well F81 cell culture plate, inoculating 8-well each diluting, inoculating 0.1ml of normal cell control 8-well, standing at 37 deg.C and containing 5% CO₂The incubator was incubated for 5 days, and the CPE was observed daily. TCID calculation according to Reed-Muench method₅₀. As a result, the virus content was 10^5.2TCID₅₀/0.1ml。

(3) Morphological observation

And (3) staining the strain b in the supernatant of the cell culture solution b in the step (2) by using phosphotungstic acid, then carrying out negative staining, observing by using a transmission electron microscope, and judging from morphological observation by referring to FIG. 2, wherein the strain b is canine parvovirus.

(4) Molecular biological identification

Aiming at the canine parvovirus VP2 gene, primers P1b and P2b with the following sequences are designed, RT-PCR detection is carried out on the strain b in the supernatant of the cell culture solution b, agarose gel electrophoresis is carried out, the size of an electrophoresis band is 390bp, the length of the canine parvovirus VP2 gene is met, and the strain b is further judged to be canine parvovirus. Sequencing the amplified product to obtain the nucleic acid sequence homology of the canine parvovirus strain with the published sequence NCBI of the canine parvovirus strain with the highest 99.3 percent, thereby confirming that the strain b is the canine parvovirus.

P1b：5’-CTCAGCCACCAACTAAAG-3’

P2b：5’-GTAAGCCCAATGCTCTAT-3’

(5) Specificity test

Respectively adopting 0.1ml of canine parvovirus positive serum (the neutralization titer is not less than 1:128), canine distemper virus positive serum (the neutralization titer is not less than 1:128), canine viral hepatitis virus positive serum (the neutralization titer is not less than 1:128), rabies virus positive serum (the neutralization titer is not less than 40IU/ml) and healthy dog negative serum (the negative serum is the serum of 4-month-old healthy dogs which are not immunized with any vaccine) (the serum is given as gift by a virus room of a military veterinary institute), incubating the serum and 0.1ml of cell culture solution b for 1 hour at 37 ℃, synchronously inoculating the serum to F81 cells, wherein the culture solution is RPMI Medium 1640 containing 8% newborn bovine serum, and meanwhile, a normal cell control (F81 cells which are not inoculated with any virus) and a virus control (cell culture solution b is not added with serum and inoculated to F81 cells) at 6 holes respectively, and placing the serum at 37 ℃ and 6 holes containing 5% CO₂The culture in the incubator is observed for 5 days. As a result, cells were subjected to netting, crushing and shedding in the tests except that F81 cells were not cytopathic due to the culture solution b of canine parvovirus-positive serum-incubated cells and the normal cell control, indicating that the isolated virus was canine parvovirus.

Thus, strain b is canine parvovirus.

(6) Regression test

Diluting the cell culture solution b (containing the strain b) in the step (2) by using sterilized normal saline until the virus content is 10^5.0TCID₅₀And (2) inoculating 4 canine parvovirus antibody negative healthy puppies (SN < 1:2) with the inoculation amount of 2.0ml to the canine parvovirus of 2-4 months old in an inoculation mode of: orally, observed time after inoculation was 14 days, and animal symptoms were: on days 5 and 6, the 3 inoculated dogs showed mild enteritis and fever symptoms, the disease course lasted for 5 days, and 4 control dogs which were fed in a consistent feeding mode and were not injected with virus did not suffer from the disease. These symptoms are typical canine parvovirus enteritis, and thus strain b can be judged to be canine parvovirus.

(7) Confirmation of diagnosis

Through the comprehensive judgment of the detection and identification, the strain b is canine parvovirus.

Example 2 domestication of Canine parvoVirus strains and testing of Biochemical and genetic alterations of strains

(1) Subculturing of virus

The cell culture solution b (containing the strain b) obtained in the step (2) of example 1 is continuously subcultured on F81 cells for 220 generations, and the specific steps are as follows:

the culture solution is 8% newborn bovine serum RPMI Medium 1640, and the cell culture solution b is inoculated on the F81 cell monolayer according to the dosage of 1 v/v%, and the culture conditions are as follows: placing at 37 ℃ and 5% CO₂Culturing in an incubator. Culturing in an incubator for 2-3 days, harvesting progeny viruses when CPE reaches more than 90%, continuously carrying out passage for 220 generations, and naming the 220 th generation as a P6 strain.

The 220 th generation virus strain weakened by passage is used as a candidate strain for preparing the canine parvovirus live vaccine.

(2) Virus viability assay

TCID of 180, 200, and 220 generation seed cell culture solutions was measured by the same method as in step (2) of example 1₅₀. As a result: 180. the virus content measured by 200 and 220 generations of virus culture solution is 10 respectively^6.71TCID₅₀/ml、10^6.33TCID₅₀/ml、10^6.41TCID₅₀/ml。

The trend of decreasing virus content, as measured by infectivity, also indicates that the virus is becoming less virulent.

(3) Viral safety test

TCID was calculated by taking 150, 180, 200 and 220-passage virus cell culture solutions and using the same method as in step (2) of example 1₅₀Respectively diluted to 10 with sterilized normal saline^6.0TCID₅₀And/ml, orally inoculating 5 healthy susceptible dogs with CPV neutralizing antibody titer not higher than 1:2 and 2 months old and fasting for 24 hours without water prohibition, wherein 4.0ml of each dog is inoculated, 4 non-inoculated control dogs are arranged, isolated and observed for 14 days, and the clinical performance of each group of dogs is recorded respectively.

As a result: the 150 th generation of the canine 3/5 inoculated with the virus cell culture solution has the symptoms of: mild diarrhea. The dogs inoculated with the 180-220 virus cell culture solution are 5/5 healthy and alive, have no obvious difference with control dogs, have no toxicity to puppies, and show that the virus has no pathogenicity and side effect.

The virus had lost virulence through 180 passages, and thus, the virus was less virulent through 220 passages and could potentially be safely used as a live vaccine.

(4) Immunogenicity and immunoprotection assays

Diluting 180, 220 generation seed cell culture solution with sterilized normal saline to 10^5.0CID₅₀And/ml, respectively inoculating 5 CPV antibody negative healthy dogs susceptible dogs with the age of 6 weeks, 1.0ml for each dog, simultaneously setting 5 control dogs not injected with the virus, collecting blood separated serum 21 days after immunization, and performing neutralizing antibody titer determination by adopting a neutralization test, wherein the method comprises the following steps: the cell culture fluid b (containing the strain b) in the step (2) in example 1 was used as an antigen (containing 200 TCID)₅₀) The serum was diluted 2-fold, and the immunization effect was judged by inoculating F81 cells and observing CPE, and the antibody titer was calculated by the Reed-Muench method.

After blood collection, the immunization group and the control group were each inoculated with 2.0ml (content: 10) of CPV-v strain by oral administration^3.0TCID₅₀/ml), isolated feeding and observed for 14 days, and clinical performance is recorded. As a result, both the 180 th generation and the 220 th generation of CPV can induce the organism to generate better neutralizing antibodies, and the 21-day neutralizing antibodies reach 1:90.5, so that the CPV in cells can be neutralized. The immune dogs are all healthy after being attacked by virulent virus, and the control dogs are all killed after being attacked by virulent virus. See table 1 for details.

TABLE 1 results of immunogenicity assays

Therefore, after the canine parvovirus separated by the invention is passaged for 180 generations, the virulence disappears, and the immunogenicity is good.

(5) And (4) sterile inspection:

and randomly extracting 5 attenuated cell culture solutions (P6 strain) of the canine parvovirus virus seeds frozen in the 220 th generation, and inspecting according to the appendix of the existing Chinese veterinary pharmacopoeia.

As a result: 5 strains of virus cultured in TG medium, GA medium and GP medium were all grown aseptically.

(6) And (3) mycoplasma test:

mycoplasma culture medium was used. Attenuated canine parvovirus seed cell culture fluid (P6 strain) of frozen canine parvovirus of 220 th generation was randomly extracted and examined according to the appendix of the current Chinese veterinary pharmacopoeia.

As a result: agar solid plate cultures are free of "fried egg" mycoplasma colonies.

Positive control (mycoplasma hyorhinis): the solid medium is in the form of a colony of Mycoplasma pannicum. The liquid medium turns yellow in color, pH6.35, and is acidic.

Negative control: the solid medium is free of "fried egg" mycoplasma colonies. The pH of the liquid medium is 7.48.

(7) Exogenous virus assay

The 220 generation virus (P6 strain) is used as a template, PCR amplification is carried out on a canine parvovirus VP2 gene and a canine viral hepatitis virus spike protein gene, RT-PCR amplification is carried out on a canine parainfluenza virus N gene, a rabies virus N gene and a canine distemper virus H gene, and the result is positive only on the canine parvovirus VP2 gene amplification result.

(8) Identification of the specificity of the attenuated virus

The attenuated virus (P6 strain) was diluted to 200TCID with RPMI Medium 1640 containing 8% newborn calf serum instead of 220₅₀0.1ml, and the virus specificity was measured by the same method as in step (5) of example 1, as a result: the cells showed cytopathic effect in all the tests except that the F81 cells were not cytopathic due to the culture solution of the 220 th generation attenuated cells incubated with canine parvovirus positive serum and the normal cell control, indicating that the P6 strain virus is canine parvovirus.

(9) Sequence analysis of attenuated genes

And performing nucleic acid amplification, sequencing and sequence comparison on the attenuated 180 th generation virus and the attenuated 220 th generation virus by using upstream and downstream primers P1b and P2b of the full-length sequence of the canine parvovirus VP2 gene. As a result, the homology was 99.7%.

The 220 th generation attenuated virus strain is used as a candidate strain prepared from the canine distemper live vaccine.

(10) Preservation of microorganisms

The separated and domesticated canine parvovirus P6 strain is submitted to a patent program approved preservation organization for preservation, and the microorganism preservation number is CGMCC No. 19398. The classification is named as: canine parvovirus. The preservation time is as follows: year 2020, 3, 30: the preservation unit is: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, North Chen, Chaoyang, China. The virus strains are referred to as canine parvovirus strain P6, CPV P6 and P6.

Example 3 preparation of Canine parvovirus vaccine

(1) Spinner flask culture of Canine parvoviral enteritis virus strain (P6 strain)

(a) And (3) expanding and culturing cells: expanding the cultured F81 seed cells into a spinner flask for culture, wherein the culture solution is as follows: RPMI Medium 1640 containing 8 v/v% newborn calf serum is cultured for 24 hours at the temperature of 37 ℃ and the rotating speed of 10-11 r/h.

(b) Inoculation: when the cells were transferred to a flask and the monolayer F81 was grown, the culture Medium was discarded, the cells were digested with 240USP U/mg trypsin, and then passaged at a ratio of 1:2, RPMI Medium 1640 containing 8 v/v% newborn calf serum was added, and the parvovirus P6 strain of example 2 was used to produce a seed virus (content: 10)^5.0TCID₅₀And/ml) F81 cells were inoculated simultaneously at an inoculum size of 1 v/v%, and were cultured by rotation at 37 ℃ at a rotation speed of 10-11 rpm.

(c) Harvesting: cytopathic effect was observed day by day, and when CPE reached more than 90%, cell culture fluid was harvested and frozen and thawed 2 times at-20 ℃. Sampling was performed simultaneously for sterility testing and virus content determination. And carrying out subsequent steps after the sterility is ensured.

(d) And (4) sterile inspection: the test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the result is aseptic growth.

(e) Virus content: the virus content is determined by the same method as the step (2) in the embodiment 1, and the virus content is ensured to be more than or equal to 10^6.0TCID₅₀/ml。

(f) And (3) purification: filtering the virus culture solution obtained in the step (c) through a filter membrane with the diameter of 0.22 μm to obtain a cell and debris removed P6 strain canine parvovirus fluid.

(2) Suspension culture of canine parvoviral enteritis virus strain (strain P6)

(a) Preparation of cells

And (3) expanding the recovered and cultured F81 cells into a spinner flask for culture, wherein the culture solution is as follows: 8 v/v% newborn bovine serum RPMI Medium 1640, at the temperature of 37 ℃, the rotating speed is controlled to be 10-11 r/h. After the cells grow to a compact monolayer, digesting and dispersing the cells by 240USP U/mg trypsin until the cell viability is not lower than 95 percent according to the proportion of 2.0-10.0 multiplied by 10⁵Transferring the F81 cells into a bioreactor at the cell/ml inoculation density, wherein the using amount of the carrier is 2-5 g/L, and setting parameters of cell culture in the bioreactor (the temperature is 37 +/-0.5 ℃, the pH value is 7.2 +/-0.1, the DO value is 45 +/-5%, and the rotating speed is 30 r/min). The cells were sampled at 24h intervals for observation and counting.

(b) Inoculation and harvesting

When F81 cells in the bioreactor grow into a compact monolayer, the cell density reaches 4.0 x 10⁶Cells/ml, precipitating and discharging a culture solution, inoculating a parvovirus P6 strain produced virus seed in example 2 according to the MOI of 0.005-0.01, supplementing RPMI Medium 1640 containing 2 v/v% newborn calf serum to perform virus culture, setting parameters of the virus culture in a reactor (the temperature is 37 +/-0.5 ℃, the pH value is 7.2 +/-0.1, the DO value is 45 +/-5%, the rotating speed is 30r/min), culturing for 36-48 h or harvesting when the cytopathic effect reaches more than 90%, freezing and thawing at the temperature of-20 ℃ for 2 times, and sampling for performing sterile inspection and virus content determination. Before the preparation of the vaccine, the virus antigen solution qualified by the inspection is subjected to aseptic centrifugation or filtration to remove the carrier and cell debris.

(c) And (4) sterile inspection: the test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the result is aseptic growth.

(d) Virus content: the virus content is determined by the same method as the step (2) in the embodiment 1, and the virus content is ensured to be more than or equal to 10^6.0TCID₅₀/ml。

(e) And (3) purification: filtering the virus culture solution obtained in the step (b) through a filter membrane with the diameter of 0.22 μm to obtain a cell and debris removed P6 strain canine parvovirus fluid.

(3) Preparation of freeze-drying protective agent

The preparation of the freeze-drying protective agent comprises the following steps: (a) stabilizer macromolecular moiety: dissolving in 100ml water for injection, packaging, and autoclaving at 116 deg.C for 30 min. The components of the composition are as follows: 0.5g polyvinylpyrrolidone (PVP) and 2g sorbitol. (b) Stabilizer small molecule moiety: dissolving with 100ml water for injection, and filtering to remove bacteria. The components of the composition are as follows: 1g of glycine, 8.3g of sucrose and 9.2g of trehalose. (c) And (3) mixing the two solutions according to the volume ratio of 1:1 during seedling preparation to obtain the freeze-drying protective agent.

(4) Preparation of vaccines

The qualified and purified canine parvovirus virus fluid obtained by rotary bottle culture or suspension culture is diluted to the content of 10 by using sterilized normal saline^5.0TCID₅₀/ml) and the sterilized stabilizer are uniformly mixed according to the volume ratio of 1:1, and then the mixture is quantitatively packaged. Each part comprises 1ml of canine parvovirus stock solution and 1ml of freeze-drying protective agent, and the virus content of each part is not less than 10^5.0TCID_50,After subpackaging, quickly freezing and vacuum drying, covering and sealing, and labeling.

The freeze-drying procedure was as follows:

(i) before the products are not packed, the temperature of the shelf of the drying box is reduced to about 0 ℃, after the products are packed, the temperature of the shelf of the drying box is continuously reduced, the temperature of the shelf is set to be-45 ℃, the running time in the process is 1 hour, the temperature is kept for 2 hours, and the products are completely frozen firmly, and the total time is 3 hours.

(ii) And (3) starting cooling the cold trap 1 hour before the product is prefrozen, starting a vacuum pump set to vacuumize when the temperature of the cold trap reaches-40 to-50 ℃, reducing the pressure of a freeze-drying box to 8Pa to 10Pa, and preparing to carry out sublimation drying of the first stage of the product.

(iii) At the beginning of the first stage sublimation, the shelf temperature is-28 deg.C, the sublimation interface does not exceed its disintegration temperature, and the freeze-drying chamber pressure does not exceed 18 pa. The time of the first stage sublimation drying is divided into three stages: the shelf temperature is raised to-28 ℃ at-40 ℃, the process needs to be operated for 1 hour, and the temperature is kept for 6 hours; the temperature of the shelf is increased from minus 28 ℃ to minus 15 ℃, and the process needs to be operated for 1 hour; the sublimation drying time is 6 hours, the temperature is increased to 8 ℃ from minus 15 ℃ of a shelf, and the process needs to be operated for 1 hour; the sublimation drying time was 4 hours, and the first stage drying was completed for a total of 19 hours.

(iv) And (3) analysis and drying stage: and during the second stage of sublimation drying, the temperature of the product reaches about 28 ℃, the process needs to be operated for 1 hour, the temperature is kept for 2 hours, the water content of the product reaches 1-4%, and the freeze-drying process is finished.

(5) Inspection of finished product

Each of the 1 batches of vaccine obtained from the spinner flask culture and the suspension culture was subjected to the following test.

(i) Physical state observation of vaccines

2 the equal sponginess loose piece of trigeminy seedling of batch easily breaks away from with the bottle wall, dissolves rapidly after adding the normal saline of sterilization.

(ii) And (4) sterile inspection:

the test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia.

As a result: 2 batches of triple seedlings cultured in TG medium, GA medium and GP medium all grew aseptically.

(iii) And (3) mycoplasma test:

mycoplasma culture medium was used. The test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia.

As a result: no "fried egg" mycoplasma colony was found in 2 triplicate seedlings cultured on agar solid plates.

Positive control (mycoplasma hyorhinis): the solid culture medium is a mycoplasma colony in a shape of a fried egg; the liquid medium turns yellow in color, pH6.35, and is acidic.

Negative control: the solid culture medium has no mycoplasma colony in the shape of a fried egg; the pH of the liquid medium is 7.48.

(iv) Exogenous virus assay

2 batches of triple-vaccine nucleic acids are respectively used as templates, PCR amplification is carried out on a canine parvovirus VP2 gene and a canine viral hepatitis virus spike protein gene, RT-PCR amplification is carried out on a canine parainfluenza virus N gene, a rabies virus N gene and a canine distemper virus H gene, and the result is positive only on the canine parvovirus VP2 gene amplification result.

Example 4 determination of vaccine safety

The live canine parvovirus vaccines prepared by the method of example 3 through the spinner flask culture of the obtained virus were diluted with sterilized normal saline to 5 injections each, and safety tests were performed by single-dose, single-dose repeat, and single-overdose inoculation of the injections, with 5 clinically healthy dogs of 2 months of age per group.

In one single dose test, the mode of inoculation was subcutaneous injection and the inoculation dose was 1 part per tube.

In a single dose repeat test, the inoculation mode is subcutaneous injection, the inoculation dose is 1 part per tube, and the inoculation is repeated once every 14 days for 2 times.

In one overdose test, the inoculation mode is subcutaneous injection, and the inoculation dose is 10 heads per tube.

5 uninoculated healthy dogs were used as controls and fed under the same conditions.

The state of the dogs within 14 days after each inoculation is observed, and the result shows that the dogs inoculated with the canine parvovirus live vaccines in the above modes are all healthy and alive, have no obvious difference with the health status of the dogs in a healthy control group, have good spirit, have no abnormal behaviors, normal excrement, no abnormal food intake and drinking, normal body temperature and body weight, have good injection local absorption, have no red swelling and no induration, and have no abnormal liver, spleen, kidney, heart and gall bladder.

Therefore, the canine parvovirus live vaccine prepared by the invention is safe for being inoculated to dogs and can be used for clinically preventing canine parvovirus enteritis.

Example 5 vaccine immunopotency and protection test

Taking 7 clinical healthy dogs of 2 months of age, wherein 5 dogs prepared by the method according to the embodiment 3 through spinner bottle culture are subcutaneously injected with 1 part of the canine parvovirus live vaccine 1 part per dog, each part of the vaccine is diluted by 1ml of sterilized normal saline and then used, the immunization is strengthened once by the same dose and way in 21 days after the immunization, the other 2 dogs which are not immunized are used as blank controls and are raised under the same condition, the blood is respectively collected and separated in 21 days after the secondary immunization and 2 control dogs which are not injected with the vaccine, and the neutralizing antibody titer of the serum is respectively determined according to the neutralizing antibody titer determination method in the appendix of the current Chinese veterinary pharmacopoeia, and the steps are as follows: dog prepared as in example 2Parvovirus strain P6 as antigen (containing 200 TCID)₅₀) Each serum was diluted 2-fold, and the immunization effect was judged by inoculating F81 cells and observing CPE, and the antibody titer was calculated by the Reed-Muench method.

Immediately after blood collection, a virulent challenge was performed. The immunity group and the control group are respectively orally taken with 2.0ml of strong toxicity of CPV-v strain (the content is 10)^3.0TCID₅₀/ml), continuously observing for 14 days, and recording the morbidity and mortality of dogs in each immune group and control group. See table 2 for results.

TABLE 2 serum neutralizing antibodies and challenge protection results

The neutralizing antibody titer of the serum of 5 inoculated dogs is averagely 1:128.0, the protection rate is 100 percent, no neutralizing antibody is detected in a control group, and the protection rate is 0.

It will be appreciated by those skilled in the art that the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The embodiments disclosed above are therefore to be considered in all respects as illustrative and not restrictive. All changes which come within the scope of or equivalence to the invention are intended to be embraced therein.

Claims

1. A vaccine strain for treating, preventing, slowing and/or controlling canine parvovirus enteritis, wherein the vaccine strain is a canine parvovirus vaccine strain, and the canine parvovirus vaccine strain is a virus strain with a microorganism preservation number of CGMCC No.19398, or a passaged virus strain or a mutant virus strain with unchanged clinical pathogenicity and immunogenicity.

2. A vaccine composition for treating, preventing, slowing and/or controlling canine parvoviral enteritis, said vaccine composition comprising the vaccine strain of claim 1 as an immunogen.

3. The vaccine composition of claim 2, wherein the raw materials of the vaccine composition comprise the immunogen and an adjuvant;

preferably, the vaccine composition is a live vaccine composition.

4. The vaccine composition of claim 3, wherein the ratio of the amount of said immunogen to the dry weight of said adjuvant in said vaccine composition is 1.0 x 10^5.0TCID₅₀：50-150mg；

Preferably, in the vaccine composition, the ratio of the dosage of the immunogen to the dosage of the adjuvant in dry weight is 1.0 x 10^5.0TCID₅₀：105mg；

Preferably, the TCID of the immunogen₅₀Calculated according to the Reed-Muench method based on F81 cell culture.

5. The vaccine composition according to any one of claims 2 to 4, wherein the method of culturing the vaccine strain comprises the steps of:

(ii) purifying the crude venom of the vaccine strain to obtain purified vaccine strain venom;

preferably, in step (i), when CPE reaches more than 80%, harvesting the crude venom of the vaccine strain;

preferably, in step (i), when CPE reaches above 90%, harvesting the crude venom of the vaccine strain;

preferably, in step (i), the culture broth 10 is incubated according to said strain^5.0TCID₅₀: inoculating with 50-150ml of the seed;

preferably, in step (i), inoculation is carried out in an amount such that the MOI is 0.005 to 0.01;

preferably, in step (i), the culture Medium is RPMI Medium 1640 containing 2-8 v/v% newborn calf serum;

preferably, in step (i), the temperature of the culture is 36.5-37.5 ℃;

preferably, in step (i), the pH of the culture is 7.0-7.5;

preferably, in step (i), the DO value of said culture is 40-50%;

preferably, in step (i), when the culture is a spinner flask culture, the culture is performed under conditions of 9-12 revolutions per hour;

preferably, in step (i), when the culture is a suspension culture, the culture is performed at 25 to 35 rpm;

preferably, in step (i), the F81 cells are from a monolayer of F81 cells;

preferably, in step (i), the monolayer of F81 cells is trypsinized before inoculation with the vaccine strain;

preferably, in step (i), the crude venom of the vaccine strain is subjected to sterility testing;

preferably, in step (i), the F81 cells are prepared by the steps of: carrying out expanded culture on the cultured F81 seed cells to obtain expanded cultured F81 cells;

preferably, the expansion culture is performed at 36.5-37.5 ℃;

preferably, the expansion culture is carried out in a spinner flask at 9-12 rpm;

preferably, the culture Medium for the enlarged culture is RPMI Medium 1640 containing 6-10 v/v% newborn calf serum;

preferably, the time for the expanding culture is 18-30 h;

preferably, when the culture is suspension culture, the expanded cultured F81 cells are trypsinized and then transferred to a bioreactor for continuous culture;

preferably, in the bioreactor, the temperature for the continuous culture is 36-38 ℃;

preferably, in the bioreactor, the pH value of the continuous culture is 7.0-7.5;

preferably, in said bioreactor, said continued culturing has a DO value of 40-50%;

preferably, in the bioreactor, the further culturing is carried out at 25-35 rpm;

preferably, in step (ii), the crude venom of the vaccine strain is purified after being frozen and thawed 1-3 times;

preferably, in step (ii), the purification method is: filtering with a filter membrane with the diameter of 0.22-0.45 μm to remove cells and cell debris to obtain the purified vaccine strain venom;

preferably, in step (ii), the virus content in the purified vaccine strain venom is more than or equal to 10^6.0TCID₅₀/ml。

6. The vaccine composition of claim 3, wherein the adjuvant is a lyoprotectant;

preferably, the freeze-drying protective agent is an aqueous solution containing 2-3mg/mL of polyvinylpyrrolidone, 8-12mg/mL of sorbitol, 3-7mg/mL of glycine, 30-50mg/mL of sucrose and 30-60mg/mL of trehalose;

preferably, the preparation method of the lyoprotectant comprises the following steps:

preparing a first aqueous solution comprising 4-6mg/mL polyvinylpyrrolidone and 16-24mg/mL sorbitol;

preparing a second aqueous solution comprising 6-14mg/mL glycine, 60-100mg/mL sucrose, 60-120mg/mL trehalose;

mixing the first aqueous solution and the second aqueous solution in a volume ratio of 1:0.8-1.2 to obtain the freeze-drying protective agent;

preferably, the first aqueous solution is used after sterilization;

preferably, the first aqueous solution is used after being sterilized at the temperature of 110-120 ℃;

preferably, the second aqueous solution is used after sterilization;

preferably, the second aqueous solution is used after filter sterilization.

7. A process for the preparation of a vaccine composition according to any one of claims 2 to 6, said process comprising the steps of:

preparing the vaccine strain into the vaccine composition.

8. The method according to claim 7, wherein the vaccine composition is obtained by mixing the vaccine strain with the adjuvant;

preferably, the vaccine composition is dried to obtain a dried vaccine composition;

preferably, the drying is vacuum freeze drying;

preferably, the freeze vacuum drying comprises the steps of:

(a) freezing the vaccine composition to obtain a frozen vaccine composition;

(c) sublimating and drying the primarily dried vaccine composition under the vacuum condition at normal temperature to obtain a dried vaccine composition;

preferably, in step (a), the vaccine composition is frozen at-50 ℃ to-40 ℃;

preferably, in step (a), the duration of the freezing is 1.5-2.5 h;

preferably, in step (b), the vacuum condition is 8pa to 10 pa;

preferably, in step (b), the frozen vaccine composition is subjected to a first sublimation at-30 ℃ to-26 ℃, a second sublimation at-17 ℃ to-13 ℃ and a third sublimation at 6-10 ℃;

preferably, in step (b), the time of the first sublimation is 4 to 8 h;

preferably, in step (b), the time required for the temperature to rise from-50 ℃ to-40 ℃ to-30 ℃ to-26 ℃ is 0.5 to 1.5 h;

preferably, in step (b), the time of the second sublimation is 4 to 8 h;

preferably, in step (b), the time required for the temperature to rise from-30 ℃ to-26 ℃ to-17 ℃ to-13 ℃ is 0.5 to 1.5 h;

preferably, in step (b), the time of the third sublimation is 2 to 6 h;

preferably, in step (b), the time required for the temperature to rise from-17 ℃ to-13 ℃ to 6-10 ℃ is 0.5-1.5 h;

preferably, in step (c), the vacuum conditions are 8pa to 10 pa;

preferably, in step (c), the temperature of sublimation is 25-30 ℃;

preferably, in step (c), the sublimation time is 1-3 h;

preferably, in step (b), the time required for the temperature to rise from 6-10 ℃ to 25-30 ℃ is 0.5-1.5 h.

9. Use of the vaccine strain of claim 1, the vaccine composition of any one of claims 2 to 6, or the method of manufacture of claim 7 or 8, for the manufacture of a formulation for use alone or in combination with other immunological agents and/or drugs for the treatment, prevention, alleviation and/or management of canine parvoviral enteritis.

10. The use of claim 9, wherein the canine parvoviral enteritis is canine parvoviral enteritis, ferret canine parvoviral enteritis, or feline canine parvoviral enteritis;

preferably, the canine parvoviral enteritis is mink canine parvoviral enteritis, canine parvoviral enteritis or fox canine parvoviral enteritis.