JPS5939831A - Inactivated vaccine of influenzal virus for pig - Google Patents

Abstract

PURPOSE:A vaccine for pigs, obtained by adding macrogol to an influenzal virus multiplied with in allantoic cavities of grown hen's eggs, purifying and concentrating the resultant allantoic fluid, and inactivating the resultant concentrated allantoic fluid. CONSTITUTION:An inactivated vaccine of influenzal virus obtained by inoculating Hong Kong A type influenzal viruses. A/Swine/Wadayama/5/68 (H3N2) and A/Swine/Kyoto/3/79 (H1N1; H2W1N1) respectively into separately allantoic cavities of grown hen's eggs of 9-11 days in age, incubating the eggs at 37 deg.C for 2-3 days to collect an allantoic fluid, adding ''Macrogol #6,000'' in an amount of 10-18W/V% thereto, stirring the resultant mixture for 2-18hr, centrifuging the stirred mixture at 7,000r.p.m. for 20min, resuspending the resultant centrifuged precipitate with a phosphoric acid buffer common salt solution to an amount of (1/5)-(1/10), adding formalin in an amount of 0.05V/V% to the resultant suspension, sensitizing the resultant mixture at 22 deg.C for 18hr to inactivate the virus, mixing both strains, and adding ammonium phosphate gel in an amount of 10V/V% to the mixed strains.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an inactivated swine influenza virus vaccine aimed at preventing respiratory diseases in swine caused by swine influenza virus infection.

Influenza virus infection in pigs in Japan is 19
Hong Kong type A (f+)l/L) was known from around 1968, but pig type A (14,Nti H5Vt) was known from around 1977.
'r) mosquitoes invaded the country, and outbreaks of respiratory illnesses caused by this virus continued throughout the country. We have also clarified the important role of this virus in swine respiratory disease through several years of research on respiratory disease at a certain group pig farm.

In other words, infection with this virus often overlaps with secondary bacterial infection and causes mild or severe respiratory disease, resulting in decreased appetite, loss of energy, exclusion from fattening, and death in severe cases, making it a serious problem in the pig farming industry. The damage caused by this virus infection is not insignificant. Furthermore, as the pig farming industry becomes larger in size, the damage caused by pig farming tends to become more severe, so there has been a strong desire to establish a preventive method using vaccines, but to date there has been no development of an effective vaccine. Not talked about.

On the other hand, various methods have been applied to purify and concentrate influenza viruses, but there are no examples yet of using macrogol. We have succeeded in developing an inactivated vaccine by adding macrogol to a relatively high concentration of 10 to 18% to facilitate the efficient purification and concentration of influenza viruses. We provide an inactivated influenza virus vaccine for swine that contains as an active ingredient an inactivated virus purified and concentrated by adding macrogol 10-18'j;r to influenza viruses isolated from infected pigs. state

First, A Hong Kong type A/5w1ne/Wadayam
a 15 /68 (J,?N2-) and A/5
w-1,ne/Kyot○ /3 /79 CHtN
Add 10~18iX of +6,000 macrogol to the collected allantois-vaginal fluid and leave it for 2~18 hours, centrifuge at 7,000 rpm for 20 minutes, and soak the sediment in phosphate buffered saline. After adding 0.05% formalin and sensitizing at 22°C for 18 hours to inactivate the virus, rainforest was mixed, and aluminum phosphate gel was further added for up to 10 minutes. 1, an inactivated influenza virus vaccine can be obtained, and this vaccine has the following properties.

(1) Inject 0.51 intraperitoneally into 3 appropriate age mice to 7F
(No weight loss was observed during observation.)

In addition, the serum collected from lΦHiguchi inhibited hemagglutination by more than 8 times (
(Hereinafter, this will be abbreviated as "ToJ").

(2) H-antibody negative 2-4. 211 IQ for a 1 month old pig
When given two subcutaneous or intramuscular injections 2 to 3 weeks apart, pigs do not show any abnormalities, and 2 to 8 weeks after the second vaccination, 4
It produces 0 to 64 Iθ times more 1] engineering antibodies.

(3) When these immunized pigs are challenged with live virus, for example, by intranasal inoculation, virus recovery from the nasal cavity is significantly lower than in untreated control pigs, and it is known that they are protected against infection. .

The above-mentioned invention of an inactivated influenza virus vaccine for swine is effective and safe against respiratory diseases in pigs caused by infection with this virus, so it can be said that this invention has great value. .

Examples are shown below.

〔Example〕

Influenza virus A/5Wlne/Wada;
yama15/68 and A/5w1ne/Kyoto/8
/79 was separately inoculated into the allantoic cavity of a 10-day-old chicken egg, and the allantoic fluid collected after the egg was heated to 87°C for 2 days.
After sensitization by stirring at 4°C for 2 hours, add 0.00 lv of macrogol, resuspend in 80 layers of allantois vaginal fluid at 4°C and 7,000 revolutions per minute, and remove formalin to 0. .05q7
The virus was inactivated by adding the virus at a ratio of 10 to 100 ml and sensitizing it at 22°C for 18 hours.

Equal amounts of the above two strains of purified, concentrated, and inactivated viruses were mixed, and 10% aluminum phosphate gel was added to obtain an inactivated vaccine.

Inject this inactivated vaccine subcutaneously or intramuscularly into pigs at 21 to 2
If you give two injections ~3 weeks apart, immunity will last for more than 6 months,
If the vaccine is stored in a cool and dark place at 2 to 5 degrees Celsius, its shelf life is 1.
It was 2 months.

Additionally, the following results were obtained regarding the safety and efficacy of this vaccine.

(1) Regarding safety and duration of immunity, we used 10 pigs of about 2 months old who were negative for H-antibodies and administered the inactivated vaccine subcutaneously to 4 pigs and intramuscularly to another 4 pigs, as shown in Table 1. The injections were performed twice at 2-week intervals, and the remaining two pigs were kept together as untreated controls to observe the reaction and clinical symptoms at the time of inoculation, and no abnormalities were observed in either the injected pigs or the control pigs.

In addition, two weeks after vaccination, a portion of blood was collected from the first month to the seventh month, and the respective II antibody titers were measured, and the results are shown in Table 1. It was found that immunity lasted for at least 6 months. .

(2) To evaluate efficacy, 12 antibody-negative pigs of approximately 2 months of age were tested. As shown in Table 2, the inactivated vaccine was administered subcutaneously to 4 pigs, and 2 times intramuscularly to another 41 pigs at 2-week intervals. Three weeks after the first injection, each immunogen was divided into two groups: A Hong type and A swine type virus%16.
The virus was recovered from the nasal swab for 10 days after intranasal challenge with 3 mu of 50/'mu solution from E. The results are shown in Table 2. It became clear that it also has effectiveness.

Table 1 Note 2: The upper row shows the antibody titer for A pig type, and the lower row shows the antibody titer for A Hong Kong type.

Claims (1)

[Claims] A vaccine for pigs obtained by adding 10 to 18 g of macrogol to influenza virus grown in the allantoic cavity of embryonated chicken eggs, purifying and concentrating it, and inactivating it with formalin.