DE1617288B - Process for the preparation of a Gnppe virus vaccine - Google Patents

Description

Table I.

treatment Anti-HA titer (log ₁₀ units / ml) 300 Primary 30,000 'for different Vaccine doses * ') 30,000 virus HA units 3000 HA units Secondary HA units 3.21 HA units n. U. 300 3000 n. u. Intact 3.00 3.54 3.63 HA units HA units 4.29 SW Sub-unit 3.06 3.30 3.63 4.47 4.47 3.99 Intact 1.89 3.30 3.27 4.20 4.38 3.78 BEL Sub-unit 3.00 3.21 4.02 4.02 4.11 · 4.59 Intact 2.10 3.48 3.42 2.79 3.84 3.96 AS Sub-unit 3.24 4.41 4.53 3.27 4.11

*) The numbers are the geometric mean values for groups of six rabbits on the 21st, 25th, 36th (primary reaction) and 42nd and 49th (secondary reaction) day.
nu = not investigated.

These results show that the vaccine prepared according to the protocol of this example is antigenic.

The antisera obtained by immunizing rabbits with influenza virus subunit vaccines such as were prepared above differed in activity and neutralization potency not from antisera made by immunizing rabbits with intact influenza viruses had been. Similarly, the antibodies produced in the rabbits by each of the vaccine types showed The same cross-reactions had been induced.

Example 2

Manufacture of bivalent flu virus subunit vaccines by deoxycholate treatment and demonstration of their immunogenicity in mice.

The procedures described below were separately applied to the following strains of influenza virus: A ² / SA / 64 (A2 / 64), a typical A ² strain isolated in Australia in 1964; B / Vic / 65 (B / 65), a type B strain isolated in Australia in 1965. Both strains are used in the manufacture of commercial influenza virus vaccines.

The viruses were grown in the allantoic cavity of 10 day old embryonic ones. The virus was separated from the cooled allantoic fluid by passing it through Sharples supercentrifuges, resuspended in a phosphate buffer and inactivated with formaldehyde. The virus concentrates contained 4,470 chap cell agglutination units (CCA units) and 12,100 CCA units ( ⁴ × ⁷⁰ or 10 ^{5 '26} HA units).

A volume of 5% sodium deoxycholate solution corresponding to a quarter of the volume of the virus concentrate was added to each. The mixture was allowed to stand at 37 ° C for 30 minutes and then dialyzed at pH 8.2 against a potassium dihydrogen phosphate buffer containing formaldehyde until the sodium deoxycholate concentration of the vaccines reached an undetectable level. Appropriate volumes of each subunit vaccine were then mixed and diluted with phosphate buffer and physiological saline so that each ml of the final mixture contained the equivalent of 300 CCA units of the original vaccine with intact virus of each type.

A similar mixture of preparations with intact Virus was produced, also in this case 300 C.C.A. units of each type per ml contained.

Table II

Virus- Bivalent Anti-HA titer (log ₁₀ < Sub-unit Intact of the reciprocal) *) Sub-unit Intact staxnm vaccine Primary 2.13 2.52 Secondary 3.41 3.16 dilution 1.84 2.27 3.15 3.11 Α2 / 64 Undiluted 1.86 2.04 2.82 2.91 1: 2 1.59 1.85 2.72 2.85 1: 4 (5/9) 0 **) 1.73 2.50 2.70 1: 8 (6/10) **) 1.44 (9/10) 2.32 2.46 1:16 2.29 2.35 3.38 3.02 1:32 2.04 2.15 3.19 2.91 Β / 65 Undiluted 2.13 2.03 3.10 2.76 1: 2 1.89 (9/10) 1.84 2.97 2.61 1: 4 1.42 (7/10) 1.67 2.54 2.37 1: 8 1.57 (8/10) 1.60 (7/10) 2.64 2.33 1:16 1:32

*) The numbers are the geometric mean values for groups of 10 mice from blood samples taken on day 20 (primary reaction) and taken on the 32nd day (secondary reaction).

0 Where not all mice in a group had detectable antibody at a 1:20 dilution of the sera is the number of the reacting animals given as a counter.

**) Insufficient animal reaction to determine a valid geometric mean titer.

Twofold dilutions of each of these final Vaccines over a range Vöii undiluted to 1.32 fold were made for inoculation into mice. Groups of 10 mice were given 0.25 ml of a injected each dilution of each vaccine, after which A blood sample was taken 20 days later. Each mouse received a second dose on day 21, and another blood sample was taken on the 32nd day.

The antihemagglutin titers of the sera of each mouse would be as given in Table ΙΓ:

The results show that the vaccine prepared according to the protocol in this example is antigenic.

From the above it can be seen that the present invention does not have a-V-record ^ '- does-do-that-which in the older method, occurred, does not have, and at the same time - ^! the distribution of an influenza virus vaccine that is immune and, at the same time, minimally rectal.

Claims (1)

1 2 Animal is antigenic and has lost its specific toxicity Claim: has. However, the use of ether brings in large-scale process dangers with it, and it Process for the production of an antigenic, only therefore there is a need for another process, still little reactogenic influenza virus subunits - 5 in which the undesirable, with the use vaccine, characterized by ether-linked dangers are avoided that you can get a suspension of flu viruses. a pH of 7.5 to 8.2 and a temperature of The present invention was accordingly the need 20 to 37 ° C with a soluble salt of the deoxy would be based on such an improved process for cholic acid added until the virus suspension is a production of an influenza virus subunit vaccine, Concentration of 0.5 to 2% deoxycholate, which is antigenic but non-toxic, is available too holds, lets stand for 1 to 60 minutes and then sets. by dialysis the concentration of deoxychol- So according to the present invention, a ver acid salt to less than 0.01%. drive to the production of an antigenic, very little 15 reactogenic influenza virus subunits proposed which is characterized in that one a suspension of influenza viruses at a pH of 7.5 to 8.2 and a temperature of 20 to 37 ° C a soluble salt of deoxycholic acid added until So that a vaccine in the field of preventive 20 the virus suspension has a concentration of 0.5 to The medicine is of maximum value, it is necessary that 2% contain deoxycholate, let stand for 1 to 60 minutes He is highly immunogenic and only in one and then by dialysis the concentration wrestling measure is reactogenic. of the deoxycholic acid salt to less than 0.01% If the virus vaccine is inactivated, the immune system is lowered. nogeneity, of course, the most important quality; 25 is preferably used as the salt of deoxycholic acid in it is a function of the antigenic mass produced in a method according to the invention sodium deoxycholate Dose of vaccine is present. used. In the case of parenteral administration, the invention is illustrated by the following examples People explained by a number of factors in one go
Vaccine reactions are evoked. Some 30 the same are: (a) the cell system that is used for cultivating the virus has been used; for example can cause allergenic reactions in people who have been exposed to the production of influenza virus subunit vaccines are sensitive to eggs, evoked by vaccines - through deoxycholate treatment and demonstration of their immunogenicity in rabbits, bred in embryonic eggs,
killed viruses have been produced; (b) Fumed Sub- The procedures described below were performed punch in the vaccine; for example, viable isolates to the following strains of influenza viruses - influenza viruses elicit pyrogenic responses; (c) toxic turned: Swine (SW), strain 15 from Shope; BEL is a substance associated with microorganisms, fast-growing type A strain; and Asian AS from whom the vaccine is made, such as B. 40 (A ² / AA / 23/57), a typical A ² strain of the strain the specific toxic substance associated with intact Study Center, Commission on Influenza, School of Influenza Virus Particles. Public Health, Ann Arbor, USA. The reactogenicity of a vaccine can be modified The viruses were in the allantoic cavity for 11 days or be eliminated by, for example, the ... old embryonic eggs bred and partially through Vaccine for removal of non-specific protein 45 adsorption-elution purified on chicken erythrocytes cleans or by centrifuging pyrogen-free dilution and subsequently differentiated. The finished using fluids or by taking the dose of virus preparations (which were in no way pure) by checking the administered antigenic mass were in 0.15 sodium chloride containing limited. 0.01 m Tris buffer and a pH of 7.5 in one In the case of influenza virus vaccines, the egg protein content of approximately ΙΟ ⁵ x ⁶⁰ hemagglutins in concentration can be suspended by centrifugation to an acceptable level (HA) per ml. and those with the viable viruses Any suspension of virus particles (2.0 ml) Associated pyrogenicity can be inactivated with a 5% solution of sodium deoxyeinem Means such as B. formaldehyde, are eliminated. cholate (0.5 ml) and the mixture 5 minu-Es However, the specific toxicity still remains, which was left to stand at 35 ° C for 55th. The mixture the intact, although inactivated, virus particles were then dialyzed against water in order to combine the concentrations is which the effectiveness of the vaccine tration of sodium deoxycholate to less than limited in adults and reducing its use by 0.01%. Contraindicated to children. The vaccines were then approved for injection into restrictions are due to the fact that 60 rabbits are diluted accordingly,
a flu virus vaccine whose antigen levels were given to groups of six rabbits with 300, is reduced to a value that in most cases 3,000 or 30,000 HA units of an intact SW, is of acceptable reactogenicity, not a useful BEL or AS virus and corresponding dilution immunogenicity indicates. vaccinated against the treated vaccines. Every rabbit It is known that the treatment of myxoviruses, 65 chen, was vaccinated 35 days after administration of the first including the influenza viruses, the dose with a second dose with ether.
Splits virus particles into subunits. The antihemagglutinin titre of the sera from the showed that the material obtained in humans and rabbits were as shown in Table I.