JPH0470288B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a highly attenuated mumps-Rubini strain obtained from the urine of young acute mumps patients and which is compatible with human tissue. This invention relates to a live virus-containing vaccine against mumps.

Mumps, Parotitis epidemica, is an extremely widespread contagious disease that often has few symptoms, usually causes swelling of the parotid glands, and is common in childhood. The process progresses mostly slowly.

After puberty, many complications occur. For example, orchitis in men or oophoritis in women. This can sometimes result in atrophy to the point of infertility. Other complications of this viral infection affect the central nervous system, resulting in encephalitis, encephalomyelitis, neuritis, and brain membrane inflammation.

The greatest incidence of disease is seen in school children. The contact infection index is the highest at this age. After an incubation period of 18-21 days, acute fever symptoms begin. Infectivity begins two days before glandular swelling and continues until the swelling subsides. Recovery of disease or inoculation with attenuated mumps virus confers lifelong immunity.

In view of the possible complications of the mumps-disease, especially in adults, it is imperative to use optimally tolerated mumps-vaccines that produce high antibody titers. Vaccinations should be given as low as 1 to 1 if possible.
Must be performed at the age of 3.

Although mumps is well known to be a rather harmless disease,
Opponents of vaccination only emphasize the accidents that may occur due to vaccination. As a result, the number of people who receive mumps vaccinations continues to decline, and the risk of developing mumps infection is as high as ever. There is. Therefore, it is extremely important to use mumps vaccines with satisfactory results in terms of safety and efficacy, so that they are fully attenuated and safe for human use. A genetically homogeneous mumps virus population adapted to propagation in polyploid cells, which contains no foreign virus and no remnants of animal protein. , the safest and at the same time the most effective.

Swiss Patent No. 475,355 describes a method for culturing viruses that produce mumps-vaccines. The method described there is characterized in that the virulent mumps virus is passed several times into chicken embryo tissue culture until it becomes correspondingly attenuated.

This method exhibits the following fundamental drawbacks. Vaccines produced accordingly are compatible with chicken embryo tissue-culture and still contain remnants of this culture.

Foreign proteins from the inoculum can induce unexpected hypersensitivity reactions. After all, vaccines produced in chicken embryo tissue-culture contain antibiotics, such as neomycin, to avoid microbial contamination, which is not considered desirable.

U.S. Pat. No. 3,961,046 describes the first passage inoculation with chicken eggs and the
Attenuation of mumps virus by 1st generation human amniotic cells-turf by 18 passages followed by 3 consecutive passages has been described. Mumps-vaccines produced according to this method have the following important drawbacks: 1. Possible contamination with foreign viruses; 2. Mumps-vaccines produced according to this method are genetically heterologous. 3. Contamination with foreign proteins that cause allergic side reactions in hypersensitive humans; 4. Unknown safety and immunogenicity in humans.

French Patent No. 2455628
147227 (corresponding to Publication No. 147227), the virus containing the mumps vaccine was added to the culture solution of chicken egg-chorioallantoic membrane, human diploid cells, or chicken embryo-fibroblast cells. A method is described for production from the Urabe strain by attenuation of the virus by serial passages of 18 generations and propagation of one or all of the above cellular materials. Urabe vaccines produced according to this method sometimes cause mumps-meningitis. Therefore, it can no longer be used in Canada.

The object of the present invention is to produce a mumps vaccine. It does not exhibit the disadvantages described, is compatible with growth in human diploid cells, is optimally compatible, does not stimulate local or systemic hypersensitivity reactions, and does not cause neurological side effects when mixed inoculation. It does not produce any pharmacologically active ingredients other than mumps virus.

The present invention resides in a new attenuated mumps virus strain called "Rubini". This strain is highly attenuated, compatible with growth in human diploid cells and does not cause neurological side effects (encephalomyelopathy, meningitis, meningitis).

The present inventors have now cultured mumps virus derived from patients with acute mumps inflammation in diploid human tissue and by repeatedly passing it through this tissue and injected into fertilized chicken eggs. The pathogenicity is attenuated and adapted by passage through the amniotic and allantoic membranes, followed by further passage through diploid human tissue culture, thereby increasing the potential for use against mumps. It was discovered that it is possible to produce raw materials for vaccines.

In order to obtain an effective vaccine, it is advantageous to select passages with particularly high titers for further processing of the virus, respectively, when passed through various tissue cultures.

Also, according to this principle method, the mumps virus strain Rubini obtained from the urine of an 8-year-old boy with acute mumps inflammation was passed into diploid human cell culture. Attenuated pathogenicity and adaptation. From the raw materials thus obtained, a Rubini type seed strain suitable for vaccine production is selected.

The obtained and selected mumps virus strain exhibits a very good growth rate in human tissues, a rapid formation of particularly high antibody titers in humans as well as its problem-free and good compatibility. Excellent in that respect.

The resulting live human diploid cell (HDC) vaccine against mumps is free of animal xenoproteins and antibiotics.

Therefore, the subject of the present invention is diploid human tissue.
A live vaccine against mumps, characterized in that it contains a live, productive mumps-virus whose pathogenicity is attenuated by passing it through culture. . This vaccine is a pathogenic attenuated mumps-virus of the strain Rubini (80MUP4, deposited at the Institut Pasteur, CNCM, Paris, January 14, 1986,
Deposit number-503).

The production method for this live vaccine involves culturing the virus obtained from the urine of acute mumps patients in diploid human tissue and repeatedly passing it through such tissue and into hatched chicken egg cells. and further attenuated pathogenicity by further passage through diploid human tissue, thereby making it compatible with human tissue, isolated, and subsequently cultured (propagated) and grown. It is characterized by being processed into vaccines.

In addition, this method uses an attenuated, virulence-reduced and adapted to human tissue culture strain Rubini mumps virus (80MUP4, Institut Pasteur, CNCM, Paris). Deposited on January 14, 1986, deposit number -503) is cultured and processed into a live vaccine.

The production of HDC-mumps vaccine is described in detail.

A. Isolation and repurposing of virulent mumps virus.

Isolation of virus from urine of patients with mumps is performed by post-treatment in an ultracentrifuge (4).
time, 25000U/min). The resulting mumps-virus-containing fraction (determined by a hemagglutination test) was then collected in diploid human cell tissue cultures (e.g. Wi-38 type cultures) and in hatched chicken eggs. Cultivate in cells. The isolated mumps-virus is then purified and concentrated by centrifugation (4 hours, 25000 U/min).

B Pathogenic attenuation, attenuation and adaptation.

For pathogenic attenuation and adaptation, the mumps virus is further passed into fertilized chicken eggs and again into human diploid cells: 1. amnion of hatched chicken egg cells - or allantoic membrane or alternately at 30-35°C (e.g.
(at 32°C) several times.

2 Further passage at 30°C into diploid human cell tissue of type MRC-5.

3 Pass fresh cells of the same type (MRC-5) at 35°C in tissue culture.

C. Manufacture of vaccines The seed virus obtained after this sequential passage is transferred to human diploid cells, e.g. MRC-5.
- Proliferates in cells.

A virus suspension is obtained and worked up into a finished vaccine for use.

The resulting virus suspension is clarified, for example by centrifugation or filtration, and the virus content is quantified, for example against VERO-cells;
The raw material is stabilized by the addition of sugars, such as glucose, lactose and/or sucrose, and finally freeze-dried.

The number of times the virus is passed through diploid human tissues and through fertilized chicken eggs can be varied. Similarly, the temperature can vary between approximately 30-38°C during incubation and culture.

Two main objectives always keep in mind the attenuation (attenuation of pathogenicity) and the culturing of viruses: (a) Attenuation of the disease-causing virulence of the virus while preserving its antigenic nature, i.e. its ability to reduce the virulence of antibodies; induce formation.

(b) Production of pathogenicity-attenuated virus cultures.

It does not contain foreign proteins - in the present case no chicken protein - and very little antibiotics.

Example 1 HDC - Manufacture of mumps vaccine A Manufacture of seed virus Mumps virus was isolated from the urine of patients with mumps - obtained at the end of virusemia. centrifuge (4 hours,
25000U/min) and concentrate. The mumps virus-containing fraction was determined by a hemagglutination test and prepared using a protein-containing medium [e.g. Gibco + 0.4% human serum albumin (HSA)].
Basel medium-agar (BME)] with a certain amount of alcohol, and a certain amount is taken for actual measurement.

The aliquots are further processed as follows: Fresh confluent human diploid cell turf (e.g. from Wi-38) is inoculated with the mumps suspension and grown on a nutrient medium [e.g. BME+10]. Incubate at 37 °C after addition of % fetal bovine serum (FBS).
A few days later, a cell fungus lawn is obtained. The resulting harvest is passed through human diploid cells one to four additional times.

The SPF-egg cells are then subjected to three passes. In this case, different inoculation and harvesting methods are used: (a) inoculation into and harvesting from the allantoic cavity; (b) inoculation into and harvesting from the amniotic cavity; (c) inoculation into the amniotic cavity. Inoculated and harvested from the allantoic or amniotic cavity. Eggs are incubated at 32-35°C. After passage of these three egg cells, the viruses obtained in the eight inoculation/harvest variants are combined.

Ten further oocyte passages are performed with this combination: inoculation, amniotic cavity and harvest, allantoic fluid.
Maintain temperature at 32°C. The mumps antigen titer is determined by a hemagglutination test, respectively.

Then human diploid cells (MRC-5) at 30°C
Make four rapid passes at (approximately 7 per pass)
Day). Nine further passages (10-20 days per passage) in MRC-5 cells at 35°C yield an attenuated and pathogenic reduced seed virus.

B. Testing of the seed virus The seed virus obtained by attenuation was tested for mumps-identification (VERO-on neutralization test) and virus concentration (VERO-cell neutralization test).
- Titrate into cells: test for 5.6log ₁₀ ID ₅₀ /ml). The seed virus was identified as mumps virus, and titrations show that production in MRC-5- cells is possible by "Rubini"-mumps virus.

In addition, the seed virus is considered microbiologically pure by the following tests: Tested in liquid thioglycollate medium at -30-32°C: free of microorganisms. Tested in liquid soybean medium at -20-25°C: mold free. - Tested in liquid and solid media: free of mycoplasma. -Tested in guinea pigs (in vivo): Free of tuberculosis bacteria. - liquid (according to Santon) and solid (Lo wenstein)
Tested (in vitro) in culture medium by Jensen:
Contains no tuberculosis bacteria. - Tested on chicken leukocytes: free of retrovirus (Retrovinen). - Test for heterologous viruses in monkeys (crab eaters), guinea pigs, mice, baby mice (younger than 24 hours) and fertilized chicken eggs (in vivo): no heterologous viruses. −Primary Monkey Kidney and
Heterologous virus testing (in vitro) with HDC (MRC-5) and Langu 18: Contains no xenovirus.

C HDC - Manufacture of mumps vaccine Seed virus is injected into human diploid cells (e.g.
MRC-5 cells from a cell bank tested according to WHO-requirements) are grown in culture bottles suitable for mass production (eg roller bottles).
After 7-10 days of incubation at 35° C. and microscopic evaluation (detection of cytopathogenic effects), virus suspensions are harvested daily for about 7 days. Large chunks of the virus obtained were kept until after the test results were retained.
Store at 190 °C (liquid nitrogen gas phase).

A large mass of the appropriate virus is rethawed and clarified through a filter with a pore size of approximately 5 nm.

After dilution with a solution of lactose or lactose and glucose containing a stabilizer consisting of human serum albumin, the resulting "final mass" is
Fill 0.5 ml into 3 ml small bottles and freeze-dry under reduced pressure.

D. Clinical Trials Conventionally conducted trials show that vaccination with the above-mentioned vaccines is optimally tolerated. There is no fever or any signs of whereabouts. The organ in question, such as the parotid gland or the gland, does not show swelling, inflammation or any painful reaction. All but one case exhibits benign seroconversion. The only cases that do not have mumps-specific antibodies upon reconstitution will react negatively to the previously commercially available mumps vaccine.

HDC obtained in other clinical trials in the form of a mumps-measles-rubella-live virus-human diploid cell-vaccine (HDCV) mixed with mumps vaccine. Tested against mumps, measles and rubella in a double-blind clinical trial.

In total, 120 children aged 15-20 months were studied. 60 children were given the novel vaccine while a control group of 60 children received the known mumps-measles-rubella vaccine N-M-R.
(R) (Merck, Schap & Daume). Six to eight weeks after vaccination, an exceptionally high seroconversion rate (95-100%) is evident in the two groups. Multiple X ² -tests do not result in statistically significant differences in the immune efficacy of the two vaccines. (P>0.05). No reports of any side effects due to HDCV were obtained from any of the medical cases involved in the clinical trial.

However, it was observed that all components of the HDC-vaccine have a high degree of pathogenicity attenuation and the vaccine does not contain chicken protein, animal protein extracts, or antibiotics. All theoretical and practical incompatibility of corresponding hypersensitivity are therefore eliminated. No side effects were observed in all cases.

Example 2 Production of HDC-Mumps Vaccine (A) Isolation of mumps virus as described in Example 1. (B) Growth of virus in diploid human cell tissue. (C) Amniotic and allantoic cells of chicken egg cells incubated in diploid human cell tissue for 10 passes at 30-35°C, alternately. Pathogenicity was attenuated by 6 passes at 35-38°C in the refrigerator and finally 6 rapid and 10 passes at 30-35°C in the diploid human cell tissue MRC-5. (D) Propagation (production) of the seed virus obtained by A/B/C in human diploid cells. (E) Virus harvest, media isolation, purification, concentration, testing, stabilization and lyophilization as described in Example 1.

Claims (1)

[Scope of Claims] 1 Mumps-Rubini strain virus obtained from the urine of young patients with acute mumps inflammation, highly attenuated and compatible with human tissue [Pasteur] Institute, CNCM, Paris, 19861
A live vaccine against mumps containing [deposited on May 14, deposit number -503].