JPS60248624A - Rotavirus vaccine and manufacture - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to live attenuated human rotavirus (rotavi).
rus) vaccine and a method for producing this vaccine.

The invention further relates to a method for preventing acute diarrhea caused by this human rotavirus.

Rotavirus particles were identified in fecal samples by electron microscopy in 1974 (Bishop et al., “Detection of a new virus by electron microscopy of stool extracts from children with acute gastroenteritis”, Lancela (Lanc)
et) 1974, 149).

Rotavirus is now recognized as an important cause of severe horny diarrhea in children worldwide.

After rotavirus was first identified as a cause of acute diarrhea, efforts were made to culture this virus in various cell lines without success.

Human rotavirus shows its RN in Dull electrophoresis
Based on the mobility of A-danome segments, they are broadly classified into two groups: those with short RNA J(S)-turns and those with long RNA J(LJ-turns).

In 1981, Sato et al. described the successful cultivation of human rotavirus from stool samples using roller cultures of MA-104 cells [1 Human rotavirus in cell culture].
Isolation of rotavirus” Ark.

Bio Mouth, (Arch, Virol, ) 69:
The 155-160'3° sample was pretreated with trypsin and a small amount of trypsin was introduced into the maintenance medium. This technique has been successfully used by others to culture rotavirus from the diarrheal side.

The majority of strains cultured to date are those with the 'lJ no f turn; rsJ/f turn strains have proven difficult to culture, and only two strains of rotavirus with the 'SJ pattern It has been described that the human rotavirus subgroups 1 and 2 were cultured [for example, 1 Kutsuzawa et al., "Isolation of human rotavirus subgroups 1 and 2 in cell culture", J. Clin, Microbiol.

(J, Cl1n, Microbiol, ) 16
: 727-730].

The inventors succeeded in culturing six strains of human rotavirus, which are of great epidemiological importance in Australia and Pazoa New Guinea, and preparing live attenuated viruses that are promising as vaccines.

The strains that caused diarrhea outbreaks in Melpirun, Australia from 1977 to 1979, and in Zoa New Guinea in 1979, were all rSJ pattern strains, but were different under an electron microscope. The inventors, etc.
We succeeded in culturing the i''5Jzf turn virus from two of the six selected samples containing the S'' mother turn virus. was found.

The inventors used a culture method different from the original method of Sato et al.

A 1.0-d stool pie was used for the start of the culture, and a 1.0-d tissue culture medium was used for all subsequent passages. It is surprising that this increased amount of inoculum yielded better results compared to the original method. The increased number of infectious particles in the large inoculum used in this study
This may have increased the chances of successfully culturing strains with 3Jzf turns.

The inventors used strains from whole stools stored at -70°C, PB
Strains from stools stored at -20°C and -70°C in S.
We also succeeded in culturing a Sea Crose cushion pelleted T. tyrus carano strain stored at -70°C. Due to the small number of strains cultured, it is not yet possible to draw conclusions regarding the optimal storage conditions of stool samples for culturing the virus.

The invention also provides a single vaccine that provides immunological protection against acute diarrhea caused by human rotavirus, which vaccine is based on m-Hu/Au5tra.
A live attenuated strain of the virus designated as IIA/10-25-10/77/L, - Hu/Au5tralia/1-9-12/77/S
It consists of a live attenuated strain of the virus called. The invention further provides a vaccine comprising both attenuated strains of said virus, providing enhanced cross-protection.

Since 1973, the inventors have been stockpiling rotavirus strains identified from the stool of children with acute diarrhea. Most of these samples were obtained from Australian children. Additionally, the inventors used samples obtained from a joint epidemiological survey conducted in Indonesia and Zoa New Guinea. Many of these stored strains were
tested by lumi pneuphoresis [e.g., Rodger
etc., determined by electrophoresis of nucleic acids,
"Molecular epidemiology of human rotavirus in Meldern, Australia, 1973-1979", J.

Clin, Microbiol (J, Cl1n, Macrob
iol,) 13:272-278).

Stool samples containing rotavirus particles of known electrophoretic type were selected by the inventors. The aim was to culture the human rotavirus strain that has caused large outbreaks of acute diarrhea in children in Australia and elsewhere.

The sample was whole stool and phosphate buffered saline (PBS).

pi-17,0) as a 20% fecal homogenate, or as a sea-cloth cushion pellet,
Stored at -70°C or -20°C for various periods.

All of the diarrheal sides selected for culture contained a large number of predominantly double-shelled virus particles (more than 24+ or 108 virus particles/d under electron microscopy). Details of the sample are as follows.

1, 8 samples with pneumophoretic Tizo in rR-' (Rod
ger et al., supra). Collected from children in the neonatal unit of the maternity hospital in Merseln in 1977, and PBS
20 mohomononate from 2nd year middle school and stored at -70°C.

2. 5 samples containing “M-electrophoresis type” (Rod
ger et al., see all above). Collected from children in Melbourne in 1977-1978 and
0% homogenate or sea-close cushion pelleted virus stored at -70°C [Reported by Albert et al., 1983, “Dunom RN
“Epidemiology of rotavirus diarrhea in Pazoa New Guinea, I., by electrophoresis in A.,” J. Klin.

Microbiol, (J, Cl1n, Mierobi
ol) OL:162-164).

3. 1 sample containing "rPA-electrophoresis type".

Collected from children affected by an outbreak of rotavirus diarrhea in New Guinea in 1979.
and stored as a 20% homogenate in PBS at -20°C.

4. 1 sample containing rotavirus with rLJ'f turn. Collected from children affected by a rotavirus diarrhea epidemic in Queensland, Australia in 1981 and stored as whole stool at -20°C.

5. One sample containing the predominant electrophoretic type of rotavirus with rLJ A' turn. Collected from a child in Möldern in 1981 and stored as a 20% homogenate in PBS at -70°C.

Culture technique (1) Cultivation techniques generally follow the method of Sato et al.

MA-104 cells [Microbiosocal Associates (
Microbiological As5ociate
s) (MA) Bioprodu
cta), Walkerville, MA, USA], 10
% fetal bovine serum (Fe2. Flora Dalardries, Sitney, Australia) and 12.5 tt&/rL
l of neomycin sulfate and polymyxin B
Dulbecco with added sulfate
modified medium (DMM, Flora Radleys, Sitney, Australia, Cat No 74-013-54
). Confluent cell layers for 3 days in culture tubes were used for virus culture.

Stool samples containing rotavirus were thawed, and whole stool samples were homogenized to form 20 microliters of suspension in PBS. All samples were then vortexed and centrifuged for 10 minutes at 3,0OOX, li+. Bacteria were removed by passing the supernatant through a 0.45 μm membrane filter (Millivore, Bedford, Mass., USA).

The inoculum was treated with 10μ9/rtl trypsin (Sigma,
1) Pretreated with Pushin 1x, Sig 1, St. Louis, MO, USA). Maintenance medium during virus growth (
DMM) contained 1 μg/lte trypsin.

Virus concentration in the inoculum was checked by electron microscopy. Inocula were used only if the concentration of virus particles was 108 particles/d or higher.

1.0 aliquots of trypsin-treated stool were
Two cell cultures were inoculated with MA 104MfU cells grown as confluent cell monolayers in a single layer of Rollerchie. Sato et al. used 0.1 d aliquots. Many attempts by others to culture other rotaviruses using 0.1 aliquots have been unsuccessful. 1
．． The change to 0M aliquots led to successful cultivation of several rotavirus strains. Each sample was inoculated into two tubes. Cells from one tube were used for immunofluorescence staining (immunofluorescence staining).
Viral growth was monitored after each passage by microscopic staining. In this case a rabbit antiserum against simian virus 5A-11.

and goat FITC-conjugated anti-rabbit) IgG (goat immunoglobulin conjugated with fluorescein inthiocyanate, supplied by Riga Co., Pearlingham, Calif., USA). The other culture tube was used for the next passage. At each passage, 10 − undiluted material from the previous passage was used as inoculum. This feature also contrasts with the technique used by Sato et al.

Successive passages were performed until cytopathic effects (CPE) were evident. The cultures were then tested for viruses using an electron microscope (Whitby et al., 1980.
6 Detection of virus particles by electron microscopy using polyacrylamide hydrochloride”, J. Klin, Yanle, J.
(J, Cl1n Patho!,) 33:484-
487].

(ii) F'RhL2 cells (DBS・-FRhL2-
2, ATCC.

Rockbill, Maryland, USA) was grown in minimal essential medium (Iguru), which was supplemented with EarlegBSS.
(BME) medium non-essential amino acids, 10% fetal bovine serum (
p″C8, Flow Laboratories, Sitney, Australia) and 12.5 μs/m each of neomycin sulfate and polymyxin B sulfate were added.

RU=3 virus, previously cultured in MA-104 cells, was inoculated onto 7-day-old confluent monolayers of cells in small plastic flasks. Next, the culture technique described above was performed. Serial passages were carried out at intervals of 2-7 days, and virus growth was monitored by enzyme-linked immunosorbent assay (EIA) and later by immunofluorescence staining.

This method has been described (Rodger et al., supra).

Briefly, stool samples or cell culture fluids were disrupted with sodium dodecyl sulfate and deproteinized using a combination of phenol, chloroform and isoamyl alcohol. [
“Cleavage of structural proteins in the collecting structure of the head of cacteriophage T4”, Nature, (C+
227:68 (1-685'J.) We first identified the electrophoretic tie 1 of all viruses from stool and then the appearance of CPE in cell culture, i.e. due to viruses multiplying in the layers of cells. Cell damage was checked.

Book Formation of the Virus After adaptation to static cell culture, the virus was allowed to form plaques in order to measure the amount of virus (see Urasawa et al., 1981, “Human rotavirus in MA-104 cells”). "Successive Passage of Microbiol, Immutsuru." (M
icrobiol Immunol, ) 25:1025
-10353° Top layer: 0.6 ml of purified agar (Oxoid, UK) and 2 μfj/ml tryptic gel.

A second top layer containing Neutral Red (Q, 067~/ml) was applied 4-5 days after incubation.

Results Six human rotavirus strains from the 16 stool samples studied were successfully adapted to cell culture. Three of these strains were isolated from stool stored at -70°C in PBS, one was isolated from sucrose cushion pelleted virus stored at -70°C, and one was isolated from PBS.
One strain was isolated from stool stored at -20°C in BS, and one strain was isolated from stool stored at -70°C. For these six strains, specific intracytoplasmic fluorescence was observed from the first passage until the appearance of CPE. One other strain showed initial fluorescence for 3 passages, but this disappeared over time.

The remaining 9 samples did not show intracytoplasmic fluorescence from the 1st passage to the 10th passage, and the virus was determined to be unculturable at this stage. CPE appeared between passages 6 and 9 for all culturable strains.

Appearance of CPE occurred 3-5 days after inoculation of MA-104 cell monolayers.
Consists of increased granularity, spherification into clusters, and extreme carrion over the next few days. No differences in CPE were observed between the various strains. C.P.E.
Testing of the cell culture fluid after appearance showed a mixture of unilamellar and double-shelled virus particles.

In this specification, six types of viruses are described in 1979.
Comparison of monkey, bovine and human rotavirus r-thums by lumi aerophoresis and detection of r-thum differences between bovine isolates", J. , pyrrol, (J, Viro
1, ) 30:839-846). This method encrypts the following information.

a, Animal species from which rotavirus was obtained.

b. Geographic origin of the virus.

Strain identification symbol in C0 laboratory.

d, year when the virus was obtained.

e. Electrophoresis, turn, ``S'' or ``L''.

If comparison sera for serotype and subgroup identification are available, this information could be added to the code for each strain.

The codes and laboratory identification symbols for strains cultured according to this invention are shown below.

1, Hu/Au5tralia/ 6-5-7777
/LRV-12, Hu/Au5tralia/11-
20-9777/'L RV-23, Hu/Au5tr
a11a/10-25-1+1777/L RV-34
, Hu/Au5tralia/ 6-6-1/81/L
RV-45, Hu/Au5tralia/ 1-9-
12/77/S f'tV -56, Hu/Papua
New Guinea/RV-1, RV-2 and RV
-3 are identical and correspond to “R-electrophoretic type” and RV-5 is “M-electrophoretic type J (Rodg
er et al., supra), and RV-6 appears to correspond to the "PA-electrophoretic type" (Albert et al., supra).

Human rotavirus RN cultured according to the invention
The A-e turn (using the r-lumi pneumophoresis method of Rodger et al., supra) is shown in FIG. 1 along with the RNA zf turn of the simian virus 5AII routinely cultured in the laboratory. In Figure 1, from left to right: RV-1, RV-6
, shows the turns of RV-5, RV-4 and 5A-11. Simultaneous electrophoresis of the original stool viral RNA and the cell culture adapted viral RNA showed no differences.

Three strains, namely RV-4, RV-5 and RV-6, formed plaques of different sizes (0.25-35 h).

One strain, RV-3, formed plaques consisting of thickening of cells rather than the usual areas of clearing of the cell monolayer. This plaque is related to the ts mutants of animal rotaviruses by Faulkner-Valle et al.
Virol-) 42: 669-677] by 1
It is similar to the one described in 982. Other 2 stocks R
V-1 and RV-2 were unable to adapt to plaque formation. The titers of strains that were able to form plaques varied between 10 and 10 infectious particles/ml.

Figure 2 shows human rotavirus RV-5 (B) and RV
-3 (C) compared to uninfected cell control (N
Compare with. Cleared areas are noticeable in B and thickened areas are noticeable in C.

Human rotavirus RV-3 is known to belong to rotavirus subgroup 2 based on r-luminography of the RNA genome and is known to be serotype 3 based on its response to polyclonal and monoclonal antibodies. It will be done. BY-3 is also particularly
of GaIItroenterology+Roy
al children's Ho5pital mail?
Run.

It is also identified by a monoclonal antibody developed by Australia. This antibody is MabRV-3:3
(previously designated 101AB5-B8). This one was more likely to be used in RV-3 than other rotaviruses tested.
reacts 1,000 times more strongly.

RV-5 is known to belong to rotavirus subgroup 1 by r-lumi electrophoresis of the RNA genome, and is known to be serotype 2 by reaction with polyclonal and monoclonal antibodies.

Part of the unique biochemical structure of RV-5 is now known (Dyall-8mith M, L, and Hol
Mesl, H., “Sequence homology between human and animal rotavirus serotype-specific glycoproteins”, Nueleic Acids Re5.
(19B4), 12:109).

Production of the vaccine The preferred route for the production of the vaccine of this invention follows, with appropriate modifications, the recent WHO standard for poliomyelitis live vaccine (oral) [1 Poliomyelitis vaccine (
Requirements for oral), WHO on biological standards
Expert Committee, 33rd Report (Technical Report Series 68)
7), pp. 107-174, Wao, Shi, Neve, 198
3 years].

Use of the VaccineThe first oral vaccine administration is preferably given before the age of 3 months, especially immediately after birth, and then after 3 months, when maternal antibodies in the child's serum have fallen to low levels. The second vaccination will be administered on the following day. If infection occurs after this time, children appear to be most vulnerable to severe clinical disease.

Maternal vaccination produces high titers of serum antibodies in children at birth, but does not appear to confer protection against rotavirus infection after 3 months of age. The number of rotavirus strains needed in an oral vaccine is not yet clear, but infection with one strain is thought to be sufficient to protect against severe disease caused by other strains. .

However, a preferred vaccine contains a suspension of one or more serotypes of live attenuated rotavirus grown in culture of a human or monkey diploid cell line. Such vaccines will be administered orally once or twice.

Contribution Hu/Au5tralia/10-25-10/ 77
There are 198 live attenuated strains of the virus designated as /L in the American Type Culture Collection (ATCC).
It was donated on February 1, 2005, and the elm≠-4 carving → i Ayuta O Hu/ Au5tralia/i -9-12777/
In 1985, a live attenuated strain of the virus, designated S.
The apology was submitted on February 1, 2017.

[Brief explanation of the drawing]

Figure 1 is a dull electropherogram of RNA of various viruses, where lane 1 is R'V-1, lane 2 is RV-6, lane 3 is RV-5, lane 4 is RV-4, and lane 5 is R'V-1. 5A-11; Figure 2 shows a plaque formed by a virus and is a photograph in place of a drawing showing the form of an organism; the snake is an RV-5 pattern;
, C is RV-3, and -' (A indicates the contrast. Hereinafter, 7jτ White 11M, f, 3 + j (SQ Origin: Author: Ruth Francis, Australia. 7' 31) However, there is a written amendment to the procedure (method) June 7, 1985 Manabu Shiga, Commissioner of the Patent Office 1, Indication of the case 1985 Patent Application No. 22813 No. 2, Name of the invention Four Tavirus vaccine and its manufacturing method 3. Relationship with the amended person's case Name of patent applicant Royal Children's Hospital Research
Foundation fraud 4, agent 6, subject of amendment (1) "Brief explanation of drawings" column in the specification (2) "Applicant's representative" column in the application (3) Power of attorney package = stand = Bone (5) Corporate Certificate 7, Contents of amendment ill
This is a photograph in place of a drawing.'' +2+ +31 +51 As shown in the attached sheet -11 8. List of attached documents (1) Application for correction 1 copy (2) Power of attorney and translation 1 copy each -→Tenbansha-←-Ichiban (4) Corporate certificate and translation 1 copy each (5) Report form 1 copy

Claims (1)

[Scope of Claims] 1. A method for producing a vaccine for providing immunological protection against acute diarrhea caused by human rotavirus, comprising preparing a filtrate from fecal sasansol containing human rotavirus. using a larger amount of the lachrymal fluid than would be the case with known techniques as an inoculum, and using a comparable concentration of inoculum and a comparable amount of a suitable substrate, on which the virus is grown, and the virus obtaining a live attenuated strain of said virus suitable for use as said vaccine by successive passage of said virus. 2. The large amount of F solution is used in known techniques.
The method according to claim 1, wherein the amount of liquid is 5 times or more. 3. Claim 2, in which the same amount of child inoculum used in the first passage is used in the next passage without dilution.
The method described in section. 4. The method according to claim 3, wherein the virus is successively passaged five or more times. 5. The method of claim 4, wherein said substrate comprises cells from a cell line known as MA-104. 6. The method of claim 4, wherein said substrate comprises cells from a cell line known as F Rb L2. 7. A vaccine providing immunological protection against acute diarrhea caused by human rotavirus, - Hu/Au5tralia/10-25-10/77/
A vaccine comprising a live attenuated strain of the virus designated L. 8. A vaccine that provides immunological protection against acute diarrhea caused by human rotavirus, which
“ Hu/Au5tralia/1-9-12/77/S
A vaccine containing a weak strawberry strain of a virus called. 9. A vaccine providing enhanced cross-protection against acute diarrhea caused by human rotavirus, Hu/Au5tralia/10-25-10/7
A vaccine comprising a live attenuated strain of the virus designated 7/L and a live attenuated strain of the virus designated Hu/Au5tralia/1-9-127777S.