JPS62201573A - Influenza vaccine for poultry - Google Patents

Abstract

PURPOSE:To obtain the titled vaccine capable of improving persistent immunological activity of poultry against novel antigen-type virus in a short time, by inactivating influenza virus H5N3 strain or an influenza virus having an antigen common with said viral strain. CONSTITUTION:Influenza virus H5N3 strain or an influenza virus strain having an antigen common with said viral strain is inoculated in chorio-allantoic cavity of an incubating egg and proliferated in an incubator at 33 deg.C. Chorio-allantoic liquid is extracted from the egg, added with formalin and left standing. The liquid is centrifuged to remove residues and the obtained H5N3 supernatant liquid is mixed with aluminum hydroxide gel. The objective inactivated vaccine can be produced by adding a phosphoric acid-buffered saline water to the mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] This invention relates to a novel poultry influenza vaccine (inactivated vaccine).

[Background of the Invention] Infection of poultry by influenza virus is extremely severe, resulting in almost 100% death of infected poultry, and is highly contagious. This influenza has long been extremely feared as a poultry vest, and is now designated as a legally infectious disease of poultry.

In foreign countries, outbreaks of poultry influenza, mainly in turkeys, are reported every year, and in particular, the influenza epidemic that began in April 1983 and centered around poultry farms in Pennsylvania, USA, caused great damage to poultry farmers.

[Problems to be solved by the invention] Fortunately, no major outbreaks of poultry diseases caused by influenza viruses have been reported in our country for the past few decades. However, with recent developments in virology, it has become clear that most poultry influenza is transmitted by migratory birds, mainly ducks, so in Japan, where many migratory birds come to visit, it is important to be careful about poultry influenza. It is not allowed to do so. In fact, research by the present inventors has confirmed that migratory birds captured in Japan carry influenza viruses of various antigenic types [Acta
Virol, ) 28: 524, +984], and the serotypes of influenza viruses that infect poultry change over time, so the development of vaccines against new serotype viruses will make a great contribution to the industry. It is expected.

[Means for Solving the Problems] The present inventors isolated a new influenza virus H5N strain, which has a different antigenic type from the conventional classical poultry influenza virus strain, from migratory birds that fly into our country. Successfully [Acta Bioloca (AcLa v
, 1ro1. ) 28: 524.1984] (This H's N 3 strain is located in Ujina Kanda-cho, Minami-ku, Hiroshima City!-590
It is currently being distributed to Naoki Takei, Hiroshima Prefectural Institute of Health's Virus Laboratory. Date of sale: February 21, 1986), and
This Virust 1. An inactivated vaccine was produced by culturing the N3 strain, adding an inactivating agent to the culture solution to inactivate the virus, and then centrifuging to separate the supernatant. It was confirmed that the immune activity of poultry was increased within 2 to 3 days, and that the immune activity lasted for a long time in the body, and it was discovered that such an inactivated vaccine is extremely useful for preventing influenza infection in poultry. Ta.

That is, the present invention provides an influenza vaccine for poultry containing as an active ingredient a centrifuged supernatant of an inactivated influenza virus H8N strain culture.

The method for producing an inactivated influenza vaccine according to the present invention will be described in detail below.

Fresh swan feces isolated from influenza virus Hs, N3 strains were treated with large amounts of antibiotics (penicillin: 8, OOOU/mQ, and streptomycin:
Phosphate buffer [(pH 7,2
) to prepare an emulsion, and this emulsion is inoculated into 10-day-old embryonated chicken eggs. After incubation at 33°C for 3 days, chorioallantoic fluid is collected and the presence or absence of virus isolation is determined by examining the aggregation of corneal hemocytes. When the chorioallantoic fluid having keratin hemagglutinating properties is centrifuged at 3°,000 rpm for about 30 minutes to remove the residue, virus H3N, strain (A/Succulentus/Shimane/499/83 strain) is obtained from the supernatant.

Production of inactivated vaccine The isolated virus Hs N strain is inoculated into the chorioallantoic space of eggs or cultured cells, and heated at 30-40°C (preferably 34-30°C).
9° C.) and inactivated by adding an inactivating agent to the chorioallantoic fluid (a mixture of fetal and chorioallantoic fluid may be used as long as the egg yolk is removed) or the cultured cell growth fluid. The cultured cells used here include, but are not limited to, chicken, swan, quail, pigeon, and turkey cells. Inactivating agents include formalin, carbolic acid,
It is preferable to use marzonin, a mixture of marzonin and carbolic acid, or crystal violet.

When residues or cells of the cape eggs are removed from the inactivated growth solution by centrifugation, the desired inactivated vaccine is obtained as a supernatant (referred to as centrifugation supernatant). Usually, an adjuvant such as aluminum hydroxide gel, aluminum phosphate gel, calcium phosphate gel or alum is added to the inactivated vaccine thus obtained to enhance immune activity. The amount of adjuvant may be any suitable amount, 1 to 9 in 100 volumes of vaccine.
It is enough if it contains 5 volumes. As used herein, the term "inactivated vaccine" refers to both those containing and not including such adjuvants.

The inactivated vaccine of the present invention may contain a preservative to suppress the proliferation of germs. Preferred preservatives include surfactants such as marzonin, tylosin, benzoic acid, formalin, salicylic acid, crystal violet, benzethonium chloride, polymyxin or gramicidin.

The method for producing a poultry influenza vaccine obtained by culturing the influenza virus Hs N 3 strain has been described above, but even if an influenza virus strain having common antigens with the Hs N 3 strain is used, the same inactivated vaccine cannot be produced. can be obtained. Therefore, the present invention provides Hs
The vaccine is not limited to the inactivated vaccine obtained from the culture supernatant of a specific virus called the Ns strain;
It should also be construed to include those obtained from influenza viruses that share antigens with the S strain.

The inactivated vaccine according to the present invention can be used alone or in the form of a combination vaccine mixed with other vaccines for all kinds of poultry such as chickens, turkeys, ducks, and quail. There are no particular restrictions on the administration method, and any of intramuscular injection, subcutaneous injection, or intradermal injection may be used. The dosage is preferably 0.1 to 1 OmQ per poultry.

EXAMPLES The present invention will be explained in more detail by giving examples and test examples below.

[Example covirus Hs N 1 strain approximately 10'EID 5° 9-11
The eggs were inoculated into the chorioallantoic cavity of day-old Misaki eggs, and grown for about 3 days in an ovipositor at 33°C. Take out the chorioallantoic fluid from the eggs, add formalin to this chorioallantoic fluid to a concentration of 0.05%, and add 4
It was left at ℃ for 7 days. This chorioallantoic fluid was centrifuged at 3,000 rpm for about 30 minutes to remove the egg residue.
The supernatant was obtained and the following inactivated vaccine was prepared.

1) 4% inactivated vaccine H s NG supernatant (virus I (S
About 10@~107EI05G/d) 4.0 volume of N5 strain was mixed with 50 volumes of aluminum hydroxide gel, and 46 volumes of sterile phosphate buffered saline was added to the mixture.

2) 7.5% Inactivated Vaccine 7.5 volumes of the above HsN3 supernatant and 50 volumes of aluminum hydroxide gel were mixed, and 42.5 volumes of sterile phosphate buffered saline was added to the mixture.

3) 7.5% Inactivated Vaccine 7.5 volumes of the above HsNs supernatant and 50 volumes of aluminum phosphate gel were mixed, and 42.5 volumes of sterile phosphate buffered saline was added to the mixture.

4) 10% Inactivated Vaccine 10 volumes of the above HaNs supernatant and 50 volumes of aluminum hydroxide gel were mixed, and 40 containers of sterile phosphate buffered saline were added to the mixture.

These inactivated vaccines were intramuscularly inoculated to 8- to 9-week-old chicken chicks, and the immune activity against influenza was determined. Immune activity was determined by measuring Nl antibody titer (hemagglutination inhibition) and Nl antibody titer (neuraminidase inhibition).

(1) Measuring method of Nl antibody titer In a microplate (12 x 8 wells), phosphate buffer solution 0°02
After dispensing 5 ml, antiserum was added to prepare a 2-fold serial dilution series of antiserum. Next, 0.025 mQ of a virus solution prepared with a phosphate buffer solution so as to contain 4 l-(A units) of hemagglutination inhibition was added.

After thoroughly shaking the microplate, it was left at room temperature for 1 hour. Next, add 0.05ml of 0.5% chicken red blood cell suspension.
2 was added, and after shaking, the mixture was allowed to stand at room temperature for 1 hour and evaluated. The value obtained by multiplying the reciprocal of the maximum dilution factor of the antiserum that inhibited hemagglutination by 4 was defined as the HI antibody titer.

(2) Measuring method of Nl antibody titer 0.025 mL of antiserum solution and virus solution prepared in 2-fold serial dilution using 0.15M NaCQ solution were placed in test tubes and sensitized at 37°C for 1 hour. After, O, l
mQ of fetuin was added. After thorough mixing, 37℃
It was placed in a constant temperature chamber. After 18 hours, cool to about 20°C,
O, 1 mQ of periodic acid reagent was added and immediately mixed and then kept at 20°C. After 20 minutes 1. om (2) was added and shaken until the brown color disappeared.Next, 2.5 mQ of thiobarbic acid reagent was added, and after mixing, the mixture was boiled for 15 minutes.

Next, after quenching the mixture in ice water, 4 mf2 of butanol reagent was added and thoroughly mixed. About t,oooppI1
1 for 5 minutes, the supernatant was collected, and the absorbance at a wavelength of 549 nm was measured using a spectrophotometer. The final dilution of the serum at which the ratio of the absorbance of (virus + immune serum) to the absorbance of (virus + normal serum) was suppressed to 50% or less was defined as the Nl antibody titer.

Experimental Results The HI antibody titer and Nl antibody titer were measured for three of the above-mentioned inactivated vaccines, and the results are shown in FIGS. 1 and 2. Figure 1 shows the H I antibody titer (geometric mean of one group) produced when chicken chicks were administered 7.5% (·) and 4% (0) inactivated vaccines prepared using aluminum hydroxide gel. Figure 2 shows the H I antibody titer (○) and Nl antibody titer (・) produced when 7°5% inactivated vaccine produced using aluminum phosphate gel was administered to chicken chicks. also shows the geometric mean value of the first group.

These figures show that the inactivated vaccine of the present invention is effective in preventing influenza in poultry.

[Brief explanation of drawings]

FIG. 1 is a graph showing the F(I antibody titer and its transition) produced when chicken chicks are inoculated with the inactivated vaccine of the present invention. This is a graph showing the T and N antibody titers and their trends when inoculated with an inactivated vaccine. Patent applicant: Shionogi & Co., Ltd. Representative: Patent attorney: Aohaku Ao and one other person Figure 1 Fig. 2

Claims (3)

[Claims] (1) An inactivated virus obtained by inactivating influenza virus H_5N_3 strain or an influenza virus strain having a common antigen with that virus strain. (2) A method for producing an inactivated virus, which comprises inactivating influenza virus H_5N_3 strain or an influenza virus strain having a common antigen with that virus strain. (3) A poultry influenza vaccine containing as an active ingredient an inactivated influenza virus H_5N_3 strain or an inactivated influenza virus strain having a common antigen with that virus strain.