Method for testing efficacy of porcine pseudorabies GE gene deletion inactivated vaccine
By constructing the pig pseudorabies virus HNQYY2012△gE strain and preparing it into an inactivated vaccine, using guinea pigs and pigs for immunization and efficacy testing, the problem of existing vaccines not being able to provide complete protection and difficulty in screening of pigs is solved, and cost reduction and efficacy evaluation are simplified.
Patent Information
- Application Number
- CN202311545655.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-20
- Publication Date
- 2025-05-20
AI Technical Summary
Existing pseudorabies vaccines for pigs cannot provide full protection, and screening of pseudorabies negative pigs is difficult.
By constructing the porcine pseudorabies virus HNQYY2012△gE strain and preparing it into an inactivated vaccine, guinea pigs and pigs were used as alternative animals for immunization and efficacy tests, and the correlation between neutralizing antibody levels and challenge protection was studied.
It effectively reduces the cost of inactivated vaccine development of pseudorabies gE gene deletion, solves the difficulty of screening pseudorabies negative pigs, and provides a simpler and more effective vaccine efficacy evaluation method.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of vaccine research and development, and particularly relates to a method for detecting the potency of a porcine pseudorabies gE gene-deleted inactivated vaccine. Background Art
[0002] Porcine pseudorabies is an acute infectious disease of pigs caused by porcine pseudorabies virus. The disease shows an explosive epidemic in pigs, which can cause abortion and stillbirth of pregnant sows, infertility of boars, massive death of newborn piglets, dyspnea and growth stagnation of commercial pigs, etc. It is one of the major infectious diseases endangering the global pig industry. Porcine pseudorabies virus is widely distributed throughout the world. Pseudorabies occurs naturally in pigs, cattle, sheep, dogs and cats. In addition, a variety of wild animals, minks, ferrets and carnivores are also susceptible. Among experimental animals, rabbits are the most sensitive, and mice, rats, guinea pigs, etc. can also be infected;
[0003] Over the years, porcine pseudorabies has caused huge economic losses to the global pig industry. In the early stage in China, attenuated vaccines were used to control this disease. However, after 2011, porcine pseudorabies reappeared in China, and new epidemic strains emerged, causing huge economic losses to the aquaculture industry in China. Research shows that compared with classical strains, the epidemic strains of porcine pseudorabies have mutated, and traditional vaccines cannot provide complete protection. Our laboratory isolated the epidemic strain HNQYY2012, and then constructed the porcine pseudorabies virus HNQYY2012△gE strain by genetic engineering means, and prepared it into an inactivated vaccine;
[0004] After the porcine pseudorabies inactivated vaccine immunizes the body, it can stimulate the body to produce high levels of neutralizing antibodies, and neutralizing antibodies are the recognized gold standard for evaluating the effect of porcine pseudorabies inactivated vaccines and can be used as an important means for vaccine effect evaluation. In addition, using alternative animals for potency testing reduces the requirements for sites and the difficulty of work, and at the same time can objectively evaluate the effectiveness of the vaccine, providing a simpler and more effective way for vaccine evaluation;
[0005] The object of the present invention is to use the porcine pseudorabies inactivated vaccine (HNQYY2012△gE strain) to simultaneously immunize alternative animals guinea pigs and target animals pigs, and conduct research on the correlation between the immune antibody level and challenge protection of the two animals, so as to provide a basis for the correlation between the antibody level and challenge protection of the target animal and the alternative animal in the potency evaluation method;
[0006] The method provided by the present invention can effectively reduce the cost of developing porcine pseudorabies gE gene-deleted inactivated vaccines, and can solve the problem that it is very difficult to screen porcine pseudorabies virus-negative pigs at present. Summary of the Invention
[0007] To solve the problems raised in the above background art, the present invention provides a method for testing the efficacy of a porcine pseudorabies GE gene-deleted inactivated vaccine, which has the characteristic of simple screening of porcine pseudorabies virus-negative pigs.
[0008] To achieve the above object, the present invention provides the following technical solution: A method for testing the efficacy of a porcine pseudorabies GE gene-deleted inactivated vaccine, comprising the following steps:
[0009] Inoculate the porcine pseudorabies virus HNQYY2012ΔgE strain seed virus (the virus content per 1 ml should be ≥108.5 TCID50) into suspended ST cells according to MOI = 0.05.
[0010] After inoculation, samples are taken every 12 hours, the cell lesions are counted and observed, and the TCID50 titer is measured at the same time.
[0011] 42 hours after inoculation, samples are taken for counting and observing cell lesions. When the cell lesions reach about 80%, the virus solution is harvested.
[0012] The virus solution is carried out according to the appendix of the current "Chinese Veterinary Pharmacopoeia" and should be sterile.
[0013] The virus content of the porcine pseudorabies virus HNQYY2012ΔgE strain should be ≥108.5 TCID50 / ml.
[0014] Autoclave the Montanide ISA 206VG commercial adjuvant and cool it to room temperature for standby.
[0015] The inactivated virus solution of the porcine pseudorabies virus HNQYY2012ΔgE strain (the virus content before inactivation is 108.5 TCID50 / ml) that has passed the inspection is stored at 2-8°C for standby.
[0016] Add the oil phase to the emulsification tank in a ratio of 1 part, and then slowly add the water phase in a ratio of 1 part (mass ratio). Under the condition of 30-32°C, emulsify for 5 minutes at 500 r / min, stir and homogenize sufficiently, and import it into the filling tank after emulsification.
[0017] After passing the inspection, two animals, guinea pigs and pigs, are used for efficacy testing.
[0018] Compared with the prior art, the beneficial effects of the present invention are:
[0019] 1. In this invention, by using the inactivated porcine pseudorabies vaccine (HNQYY2012△gE strain), guinea pigs as substitute animals and pigs as target animals are simultaneously immunized to study the correlation between the immune antibody levels and challenge protection of the two animals, providing a basis for the correlation between the antibody levels and challenge protection of the target animal and the substitute animal in the potency evaluation method, effectively reducing the cost of developing the inactivated porcine pseudorabies gE gene-deleted vaccine, and solving the problem that it is very difficult to screen negative pigs for porcine pseudorabies virus at present. Specific Embodiments
[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
[0021] Example 1
[0022] The present invention provides the following technical solutions:
[0023] Materials
[0024] Virus strain: Porcine pseudorabies virus HNQYY2012 strain, preserved by Henan Agricultural University;
[0025] Vaccine: 3 batches of inactivated porcine pseudorabies vaccines (HNQYY2012△gE strain) were prepared by Henan Agricultural University, with batch numbers HN01, HN02, and HN03 respectively; Specification: 100 ml / bottle; Store at 2 - 8°C;
[0026] Test animals: Guinea pigs weighing 350 g - 400 g were purchased from Wuhan Wanqian Jiaxing Biotechnology Co., Ltd.;
[0027] Biosafety matters: The experimental materials and experimental instruments after the experiment were all treated by high-pressure sterilization.
[0028] Methods
[0029] Determination of the challenge dose for guinea pigs: 50 guinea pigs were randomly divided into 10 groups, with 5 guinea pigs in each group. The first group, the second group, and the third group were immunized with the inactivated vaccine against porcine pseudorabies of batch HN01 (HNQYY2012 △gE strain), and the immunization dose was 0.1 ml; the fourth group, the fifth group, and the sixth group were immunized with the inactivated vaccine against porcine pseudorabies of batch HN01 (HNQYY2012 △gE strain), and the immunization dose was 0.2 ml; the seventh group, the eighth group, and the ninth group were immunized with the inactivated vaccine against porcine pseudorabies of batch HN01 (HNQYY2012 △gE strain), and the immunization dose was 0.4 ml; the tenth group was the control group immunized with "water + adjuvant". Fourteen days after the first immunization, the immunized groups were boosted with the same method. Fourteen days after the boost, together with the 5 guinea pigs in the control group, they were challenged with virulent virus. The challenge virus strain was porcine pseudorabies virus HNQYY2012 strain. The challenge doses for the first group to the third group were 25 LD50, 50 LD50, and 75 LD50; the challenge doses for the fourth group to the sixth group were 25 LD50, 50 LD50, and 75 LD50; the challenge doses for the seventh group to the ninth group were 25 LD50, 50 LD50, and 75 LD50, and the challenge dose for the tenth group was 25 LD50. Observe the clinical symptoms of each group of guinea pigs and calculate the challenge protection rate of each group of guinea pigs;
[0030] Immune efficacy test for guinea pigs: 50 guinea pigs were randomly divided into 10 groups, with 5 guinea pigs in each group. The first group, the second group, and the third group were immunized with the inactivated vaccine against porcine pseudorabies of batch HN01 (HNQYY2012 △gE strain), and the immunization doses were 0.1 ml, 0.2 ml, and 0.4 ml respectively; the fourth group, the fifth group, and the sixth group were immunized with the inactivated vaccine against porcine pseudorabies of batch HN02 (HNQYY2012 △gE strain), and the immunization doses were 0.1 ml, 0.2 ml, and 0.4 ml respectively; the seventh group, the eighth group, and the ninth group were immunized with the inactivated vaccine against porcine pseudorabies of batch HN03 (HNQYY2012 △gE strain), and the immunization doses were 0.1 ml, 0.2 ml, and 0.4 ml respectively. Fourteen days after the first immunization, the immunized groups were boosted with the same method and dose. Fourteen days after the boost, the 0.1 ml immunized groups together with the 5 guinea pigs in the control group were challenged with virulent virus. The challenge virus strain was porcine pseudorabies virus HNQYY2012 strain, and the dose was 50 LD50. Observe the clinical symptoms and health status of each group of guinea pigs and calculate the challenge protection rate of each group of guinea pigs.
[0031] Results
[0032] After immunization with the 0.1 ml dose group and challenge with different doses, the protection rates were all lower than 100%; when challenged with a dose of 75LD50, neither the 0.2 ml nor the 0.4 ml immunization dose group could provide protection; after challenge of the 0.2 ml and 0.4 ml immunization dose groups, when challenged with the challenge doses of 25LD50 and 50LD50 respectively, the protection rates could reach 100%. In order to more effectively evaluate the effectiveness of the vaccine, a higher challenge dose of 50LD50 was selected as the optimal challenge dose. Based on the above test results, in the potency test of guinea pigs, an immunization dose of 0.2 ml and a challenge dose of 50LD50 were used. The detailed results are shown in Table 1;
[0033] Three batches of inactivated porcine pseudorabies vaccines (HNQYY2012 △gE strain) were used to immunize guinea pigs. The results are shown in Table 2. When the three batches of inactivated vaccines were used to immunize guinea pigs with 0.1 ml, the average serum neutralization titers after immunization were 1:97.7, 1:103.7 and 1:105.9 respectively. When the three batches of vaccines were used to immunize guinea pigs with 0.2 and 0.4 ml, the average serum neutralizing antibodies after the second immunization were not less than 1:128;
[0034] The relationship between different neutralizing antibody titers (in guinea pigs) and the challenge protection rate in the 0.1 ml immunization dose group was studied. The results are shown in Table 3. For guinea pigs (9) with a neutralizing antibody < 1:128, the protection rate after challenge was 44%. For guinea pigs (6) with a neutralizing antibody ≥ 1:128, all were 100% protected after challenge, proving that when the neutralizing antibody ≥ 1:128, it can provide complete protection, and there is an obvious correlation between the neutralizing antibody level and the challenge protection rate.
[0035] Conclusion
[0036] The three batches of inactivated porcine pseudorabies vaccines (HNQYY2012 △gE strain) prepared in the laboratory can all provide good protection for guinea pigs. The immunization dose is 0.2 ml, the neutralizing antibody can reach above 1:128, and the challenge protection rate of guinea pigs can reach 100%. In this experiment, by immunizing the surrogate animal guinea pigs, the correlation between the neutralizing antibody level and the challenge protection was studied, providing a basis for the correlation between the antibody level and the challenge protection of the potency evaluation method in the target animal and the surrogate animal.
[0037] Example 2
[0038] The present invention provides the following technical solutions:
[0039] Materials
[0040] Virus strain: The challenge virus strain is porcine pseudorabies virus HNQYY2012 strain, preserved by Henan Agricultural University;
[0041] Vaccine: 3 batches of inactivated porcine pseudorabies vaccine (HNQYY2012 △gE strain) were prepared by Henan Agricultural University; the batch numbers are HN01, HN02, and HN03 respectively; specification: 100 ml / bottle; stored at 2 - 8°C;
[0042] Experimental animals: Piglets aged 28 - 35 days (screened according to Appendix 2 of the protocol) were purchased from the healthy pig herd of Henan Xinda Animal Husbandry Co., Ltd. and raised under isolation conditions;
[0043] Biosafety matters: After the experiment, the experimental materials and experimental instruments were all autoclaved.
[0044] Methods
[0045] Immunopotency test on guinea pigs: 20 guinea pigs weighing 350 g - 400 g were taken and immunized with 0.2 ml per dose of batches HN01, HN02, and HN03 of inactivated porcine pseudorabies vaccine (HNQYY2012 △gE strain), 5 guinea pigs for each batch. The second immunization was carried out 14 days after the first immunization, and blood was collected and challenged 14 days after the second immunization. The challenge dose was 50 LD50. Another 5 guinea pigs were immunized with 0.2 ml of "water + adjuvant" as the control group;
[0046] Immunopotency test on piglets aged 28 - 35 days: 35 piglets aged 28 - 35 days were randomly divided into 7 groups, 5 piglets in each group. The first to third groups were immunized with 1 ml per dose of batches HN01, HN02, and HN03 of inactivated porcine pseudorabies vaccine (HNQYY2012 △gE strain), 5 piglets for each batch. The fourth to sixth groups were immunized with 2 ml per dose of batches HN01, HN02, and HN03 of inactivated porcine pseudorabies vaccine (HNQYY2012 △gE strain), 5 piglets for each batch. Another 5 piglets were immunized with 2 ml of "water + adjuvant" as the control group. The second immunization was carried out 14 days after the first immunization in the immunized groups according to the same method, and 14 days after the second immunization, 5 piglets in the control group were also included for nasal drip challenge with a dose of 1 ml (containing 106.0 TCID50). The clinical symptoms and health status of each group of piglets were observed, and the protection rate after challenge was calculated.
[0047] Results
[0048] Results of serum neutralizing antibody determination after immunizing guinea pigs: Guinea pigs were immunized with 3 batches of inactivated porcine pseudorabies vaccine (HNQYY2012 △gE strain), and the results are shown in Table 4. The results showed that after immunizing guinea pigs with 0.2 ml per guinea pig for each of the 3 batches of inactivated vaccines, the average value of serum neutralizing antibody in each batch was not less than 1:128, and all were 100% protected after challenge;
[0049] Results of the challenge protection test for piglets immunized at 28 - 35 days old: Three batches of inactivated porcine pseudorabies vaccines (HNQYY2012 △gE strain) were used to immunize piglets. The results are shown in Tables 5 and 6. The results showed that after immunizing piglets with 2 ml per head for each of the three batches of inactivated vaccines, the protection rate was 100%;
[0050] Parallel relationship between the serum neutralizing antibody level of guinea pigs immunized with inactivated porcine pseudorabies vaccine (HNQYY2012 △gE strain) and the challenge protection of piglets: After immunizing guinea pigs with 0.2 ml per guinea pig for each of the three batches of inactivated vaccines, the average value of serum neutralizing antibody for each batch was not less than 1:128, and the protection rate was 100% after challenge; for the immunized groups with a neutralizing antibody titer lower than 128, the protection rate was 60%;
[0051] After immunizing piglets with 2 ml per piglet for each of the three batches of inactivated vaccines, the protection rate was 100%; for the immunized groups with 1 ml, the protection rate was between 60% and 80%;
[0052] According to the above tests, as shown in Table 7, when immunizing guinea pigs with 0.2 ml per guinea pig and piglets with 2 ml per piglet, the potency tests were all qualified and there was a good correlation, proving that guinea pigs can be used as substitute animals for the potency evaluation of inactivated porcine pseudorabies vaccine (HNQYY2012 △gE strain). Therefore, both guinea pigs and pigs can be used as animals for the potency evaluation of inactivated porcine pseudorabies vaccine (HNQYY2012 △gE strain), and guinea pigs can be used as substitute animals for vaccine potency evaluation.
[0053] Conclusion
[0054] After immunizing guinea pigs with 0.2 ml per guinea pig for each of the three batches of inactivated porcine pseudorabies vaccines (HNQYY2012 △gE strain), the average value of serum neutralizing antibody for each batch was not less than 1:128, and the protection rate was 100% after challenge. The tests proved that the three batches of inactivated vaccines were all qualified when using guinea pigs for immunization;
[0055] After immunizing piglets with 2 ml per piglet for each of the three batches of inactivated porcine pseudorabies vaccines (HNQYY2012 △gE strain) prepared in the laboratory, the challenge protection rate could reach 100%. Therefore, it was proved that the inactivated porcine pseudorabies vaccine (HNQYY2012 △gE strain) was qualified in the potency test on piglets;
[0056] Based on the above test results, for the inactivated porcine pseudorabies vaccine (HNQYY2012 △gE strain), when immunizing guinea pigs with 0.2 ml per guinea pig and piglets with 2 ml per piglet, the potency tests were all qualified and there was a good correlation between the neutralizing antibodies, proving that guinea pigs can be used as substitute animals for the potency evaluation of inactivated porcine pseudorabies vaccine (HNQYY2012 △gE strain). Therefore, both guinea pigs and pigs can be used as animals for the potency evaluation of inactivated porcine pseudorabies vaccine (HNQYY2012 △gE strain), and guinea pigs can be used as substitute animals for vaccine potency evaluation.
[0057] In summary
[0058] Testing with guinea pigs: Use 10 healthy guinea pigs weighing 350 - 400 g. Each guinea pig is subcutaneously injected with 0.2 ml of the vaccine. After 14 days, a second immunization is carried out in the same manner. 14 days after the second immunization, together with 5 control guinea pigs raised under the same conditions, blood is collected respectively, serum is separated, and neutralizing antibodies are measured. The neutralizing antibodies of the immunized group of guinea pigs should not be lower than 1:128, and the neutralizing antibodies of the control group of guinea pigs should all not be higher than 1:2;
[0059] Testing with pigs: Use 5 healthy susceptible pigs (Note 2) at 28 - 35 days old. Each pig is intramuscularly injected with 2 ml of the vaccine in the neck. After 14 days, a second immunization is carried out in the same manner. 14 days after the second immunization, together with 5 control pigs raised under the same conditions, each is intranasally challenged with 1 ml of PRV HNQYY2012 strain (containing 106.0 TCID50). Observe continuously for 10 days. All pigs in the control group should develop the disease and at least 2 pigs should die, and at least 4 pigs in the immunized group should be protected;
[0060] The inactivated porcine pseudorabies vaccine (HNQYY2012△gE strain) can use both guinea pigs and pigs as animals for potency evaluation. Guinea pigs can be used as alternative animals for vaccine potency evaluation.
[0061] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.
[0062]
[0063] Table 1: Determination of different challenge doses on guinea pigs;
[0064]
[0065] Table 2: Neutralizing antibody levels in guinea pigs immunized with the inactivated porcine pseudorabies vaccine (HNQYY2012△gE strain);
[0066]
[0067] Table 3: Relationship between different neutralizing antibody titers (guinea pigs) and the protection rate against challenge with porcine pseudorabies virus HNQYY2012 strain;
[0068]
[0069] Table 4: Efficacy results after immunizing guinea pigs with inactivated porcine pseudorabies vaccine (HNQYY2012 △gE strain);
[0070]
[0071] Table 5: Results of challenge protection in piglets after immunizing with inactivated porcine pseudorabies vaccine (HNQYY2012 △gE strain);
[0072]
[0073] Table 6: Clinical symptoms after challenging piglets;
[0074]
[0075] Table 7: Comparison of the parallel relationship between the serum neutralizing antibody levels in immunized guinea pigs and the challenge protection in immunized piglets.
Claims
1. A method for testing the efficacy of a porcine pseudorabies GE gene-deficient inactivated vaccine, comprising the following steps, characterized in that: The porcine pseudorabies virus HNQYY2012△gE strain (the virus content per 1 ml should be ≥108.5TCID50) was inoculated into the suspended ST cells at an MOI of 0.05; After infection, samples were taken every 12 hours to count and observe the cytopathic effects, and the TCID50 titer was determined. 42 hours after infection, samples were taken for counting and observation of cytopathic effect. When the cytopathic effect reached about 80%, the virus solution was harvested. The virus solution should be sterile and grow in accordance with the current appendix of the Chinese Veterinary Pharmacopoeia; The virus content of porcine pseudorabies virus HNQYY2012△gE strain should be ≥108.5TCID50 / ml; The commercial adjuvant Montanide ISA 206VG was sterilized by high pressure and then cooled to room temperature for use; The qualified inactivated virus solution of pseudorabies virus HNQYY2012△gE strain (the virus content before inactivation is 108.5TCID50 / ml) is placed at 2-8°C for standby use; Add 1 part of the oil phase into the emulsification tank, and then slowly add 1 part of the water phase (mass ratio), emulsify at 500r / min for 5 minutes at 30-32°C, stir and emulsify thoroughly, and introduce into the sub-packaging tank after emulsification; After passing the test, guinea pigs and pigs were used for efficacy testing.