JP2574645B2 - Method for producing rotavirus vaccine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE FIELD The inventions is a live attenuated human - a process for the production of rotavirus (rotavirus) vaccine and the vaccine.

[0002]

BACKGROUND OF THE INVENTION Rotavirus particles were identified in stool samples by electron microscopy in 1974 [Bishop
Et al., "Detection of new viruses by electron microscopy of stool extracts from children with acute gastroenteritis", Lancet (La
ncet) 1974, 149].

[0003] Rotavirus is now recognized as an important cause of severe acute diarrhea in children worldwide. After the rotavirus was first identified as the cause of acute diarrhea, efforts to culture the virus in various cell lines have been unsuccessful. Human-rotavirus is
There are two broad groups based on the mobility of their RNA genome segments in gel electrophoresis: "short RN
"A" (S) pattern and "long RNA"
(L) It is classified as having a pattern.

In 1981, Sato et al. Described the successful cultivation of human rotavirus from stool samples using roller culture of MA-104 cells ["Separation of human rotavirus in cell culture" Arc. . Biroro.
(Arch. Virol.) 69 : 155-160]. Samples were 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 diarrheal stool.

[0005] Most strains cultured to date have an "L" pattern. The "S" pattern strain proved to be difficult to culture and it was described that only two strains of rotavirus having the "S" pattern were cultured [eg, "Kutzawa et al. -Isolation of rotavirus subgroups 1 and 2 ",
J. J. Clin. Microbiol. 1
6 : 727-730].

[0006]

DESCRIPTION OF THE INVENTION The inventors have successfully cultured six epidemiologically important human-rotavirus strains in Australia and Papua New Guinea and prepared live attenuated viruses which are promising vaccines.

The strains that transmitted diarrhea between 1977 and 1979 in Melbourne, Australia and in 1979 in Papua New Guinea were all "S" pattern strains but were electron microscopically different. We have successfully cultured the "S" pattern virus from two of the six selected samples containing the "S" pattern virus. Others have found it difficult to culture the "S" pattern virus.

The inventors used a culture method different from the original method of Sato et al. Use 1.0 ml stool filtrate for the start of culture and 1.0 ml for all subsequent passages
Was used. It is surprising that this increased amount of inoculum yielded better results than the original method. The increased number of infectious particles in the large inoculum used in this study may have increased the chances of successfully culturing strains with an "S" pattern.

We have found strains from whole stool stored at -70 ° C, strains from stool stored at -20 ° C and -70 ° C in PBS, and sucrose cushions stored at -70 ° C. The strain from the pelleted virus was successfully cultured. Due to the small number of strains cultured, no conclusions can be drawn yet on the optimal storage conditions of stool samples for virus culture.

[0010] The present invention also provides a vaccine for providing immunological protection against acute diarrhea caused by human-rotavirus, the vaccine comprising ATCC V
Deposited internationally under the Budapest Treaty as R2104
A live attenuated strain of the virus designated Hu / Australia / 10-25-10 / 77 / L, or Hu / Australi deposited internationally under the Budapest Treaty as ATCC VR2105.
a / 1-9-12 / 77 / S comprising a live attenuated strain of the virus. The present invention further provides a vaccine that provides enhanced cross protection, comprising both attenuated strains of the virus.

Rotavirus Isolation Since 1973, the inventors have stocked rotavirus strains identified from the stool of children with acute diarrhea. Most of these samples were obtained from Australian children. In addition, the inventors used samples obtained from a joint epidemiological study conducted in Indonesia and Papua New Guinea.

[0012] Many of these stored strains are
A was tested by gel electrophoresis [eg, Rodger et al.
"197 determined by electrophoresis of genomic ribonucleic acid.
Molecular Epidemiology of Human-Rotavirus in Melbourne, Australia, 3-1979, "J. Clin.
Microbiol (J. Clin. Mcrobiol.) 13 : 272-2
78].

A stool sample containing rotavirus particles of a known electrophoretic type was selected by the inventors. The purpose of this was to culture human-rotavirus strains that caused a large outbreak of acute diarrhea in children in Australia and elsewhere.

The sample was prepared as a 20% stool homogenate in phosphate buffered saline (PBS, pH 7.0)
Alternatively, it was stored as sucrose cushion pelleted virus at -70 ° C or -20 ° C for various periods of time. All diarrheal selected for culture contained primarily number of viral particles of the double shell (2-4 + or 10 ⁸ virus particles / ml or more under electron microscope). The details of the sample are as follows.

1. 8 samples with "R-electrophoresis type" (Rodger et al., See above). Collected from infants in a neonatal room at a maternity hospital in Melbourne in 1977, and
Stored as a 20% homogenate in S at -70 ° C.

2. 5 containing "M-electrophoresis type"
Sample (Rodger et al., See above). 1977-1978
Collected from children in Melbourne and stored at −70 ° C. as 20% homogenate in PBS or as sucrose cushion pelleted virus [reported by Albert et al., 1983, “Genomic RNA”.
Epidemiology of rotavirus diarrhea in the highlands of Papua New Guinea by electrophoresis ", J. Clin. Microbiol. 17 : 162-16.
4].

3. One sample containing "PA-electrophoresis type". Collected from children affected by rotavirus diarrhea epidemics in the Highlands of Papua New Guinea in 1979 and at −20 ° C. as a 20% homogenate in PBS.
Stored in 4. One sample containing "L" pattern rotavirus.
Collected from a child with a rotavirus diarrhea epidemic in Queensland, Australia in 1981, and stored at -20 ° C as whole stool. 5. One sample containing the dominant electrophoretic type of rotavirus in the "L" pattern. Collected from children in Melbourne in 1981 and stored at -70 ° C as a 20% homogenate in PBS.

Culture technique (i) The culture technique generally follows the method of Sato et al. MA-
104 cells [Microbological Associates (Microb
iological Associates) (MA) Bioprodus
cts), Walkerville, MA, USA] from Dulbecco supplemented with 10% fetal calf serum (FCS, Flow Laboratories, Sydney, Australia) and 12.5 μg / ml of neomycin sulfate and polymyxin B sulfate. Deformed media (DMM,
Flow Laboratories, Sydney, Australia, C
at NO 74-013-54). Confluent cell layers for 3 days in culture tubes were used for virus culture.

Thawing a stool sample containing rotavirus,
All stool samples were then homogenized to form a 20% suspension in PBS. All samples were then vortexed and centrifuged at 3,000 xg for 10 minutes.
Cells were removed by passing the supernatant through a 0.45 μm membrane filter (Millipore, Bedford, Mass., USA).

The inoculum was pretreated with 10 μg / ml trypsin (Sigma, Trypsin 1 ×, Sigma, St. Louis, MO, USA). The maintenance medium (DMM) during virus growth contained 1 μg / ml trypsin. The virus concentration in the inoculum was checked by electron microscope. The inoculum was used only when the concentration of virus particles was greater than 10 ⁸ / ml.

A 1.0 ml aliquot of stool treated with trypsin was inoculated into a 2.0 ml cell culture of MA-104 cells grown as a confluent cell monolayer in roller tubes. Sato et al. Used 0.1 ml aliquots. Many others' attempts to culture other rotaviruses using 0.1 ml aliquots have failed. Changing to an aliquot of 1.0 ml led to successful culture of several rotavirus strains.

Each sample was inoculated into two tubes.
Immunofluorescence staining using cells from one tube (immu
No increase in virus was monitored after each passage by nofluorescent staining. In this case, Simian virus SA-11
Rabbit antiserum against goat and FITC-conjugated anti-
Rabbit IgG (goat immunoglobulin conjugated with fluorescein isothiocyanate, supplied by Tago, Burlingham, CA, USA) was used.

The other culture tube was used for the next passage. At each passage, 1.0 ml of undiluted material from the previous passage was used as inoculum. This feature is also in contrast to the technique used by Sato et al. Passaging was performed sequentially until the cytopathic effect (CPE) became apparent. The culture was then tested for the virus using an electron microscope [Whitby et al., 1980, "Detection of virus particles by electron microscopy using polyacrylamide hydrogel", J. Am. Klin Pasol. (J. Clin. Patho
l.) 33 : 484-487].

(Ii) FRhL2 cells (DBS-FRhL
₂ -2, ATCC, Rockville, Maryland, USA)
Was grown in minimal essential medium (Eagle),
noles amino acids in arles BSS (BME), 10
% Fetal calf serum (FCS, Flow Laboratories, Sydney, Australia) and 12.5 μg / ml neomycin sulfate and polymyxin B sulfate were added.

RU-3 virus, previously cultured in MA-104 cells, was inoculated into day 7 confluent monolayers of cells in small plastic flasks. Next, the culture technique described above was performed. Successive passages at 2-7 day intervals,
Virus increase was monitored by enzyme immunoassay (EIA), and later by immunofluorescence staining.

Gel Electrophoresis of the RNA Genome of the Virus This method has been described (Rodger et al., Supra). Briefly, stool samples or cell cultures were disrupted with sodium dodecyl sulfate and deproteinized using a combination of phenol, chloroform and isoamyl alcohol. Electrophoresis of the deproteinized RNA was performed in a 10% acrylamide slab gel using a discontinuous buffer system as described by Laemmli, 1970 ["Cleavage of structural proteins during assembly of bacteriophage T4 head". Nature, (London) 227 : 680-.
685]. First, the electrophoretic type of all viruses from stool was identified, and the appearance of CPE in cell culture,
That is, cell damage due to virus growing in the cell layer was checked.

After adaptation of the virus to plaque-forming static cell culture, the virus was plaque-formed in order to determine the amount of the virus [Urasawa et al., 1981, "MA-104 cells". Sequential passage of human-rotavirus ", Microbiol. Imnor. (Mi
crobiol Immunol.) 25 : 1025-1035]. Top layer is 0.6% purified agar (Oxoid, UK) and 2 μg
/ Ml of trypsin. Neutral Red (0.
(067 mg / 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 strain was isolated from sucrose cushion pelleted virus stored at -70 ° C, and one strain was
It was isolated from stool stored at −20 ° C. in BS, and one strain was isolated from stool stored at −70 ° C.

From these six strains, the first passage to CPE
Until the appearance of, specific intracytoplasmic fluorescence was observed. One other strain showed initial fluorescence for three passages, which disappeared over time. The remaining nine samples did not show cytoplasmic fluorescence from passage 1 to passage 10, at which point the virus was determined to be uncultivable. For all culturable strains, CPE appeared between passages 6 and 9.

The appearance of CPE was attributed to increased granularity, spheroidization into clusters, and extreme sloughing 3-5 days after inoculation of the MA-104 cell monolayer.
Consists of No CPE differences were observed between the various strains. Testing of the cell culture after the appearance of CPE,
A mixture of single and double shell virus particles was shown.

In this specification, six types of viruses
Was first proposed by Rodger and Holmes in 1979.
Nomenclature [monkey, bovine and human by gel electrophoresis
-Comparison of rotavirus genomes and between bovine isolates
Detection of genomic differences ", J. Virol.3
0: 839-846]. This one
The law is an encrypted version of the following information:

A. Animal species from which rotavirus was obtained. b. Geographic origin of the virus. c. Laboratory identification symbol in the laboratory. d. Age when the virus was obtained. e. Electrophoresis pattern, "S" or "L". If comparative sera are available for serotype and subgroup identification, this information could be added to the code of each strain. The code and laboratory identification of the strain cultured according to the invention are shown below.

[0033]

[Table 1]

RV-1, RV-2 and RV-3 are identical and correspond to the "R-electrophoresis type";
5 corresponds to the “M-electrophoresis type” (Rodger et al., Supra) and RV-6 corresponds to the “PA-electrophoresis type” (Albe
rt etc. above). RNA pattern of human-rotavirus cultured according to the invention (Rodg
The above-mentioned gel electrophoresis method is shown in FIG. 1 together with the RNA pattern of the monkey-derived virus SA11 routinely cultured in a laboratory. In FIG. 1, RV-1, RV-6, RV-5, RV-4 and SA from left to right.
11 shows a pattern of -11. RN of the original fecal virus
Co-electrophoresis of A with the RNA of the virus adapted to the cell culture showed no differences.

The three strains, RV-4, RV-5 and R
V-6 formed plaques of different sizes (0.25 mm to 3.5 mm). One strain, RV-3, formed a plaque consisting of thickened cells, rather than the normal areas where the cell monolayer was clarified. This plaque was described by Faulkner-Valle et al. ["Molecular biology of rotavirus III. Isolation and characterization of temperature-sensitive bovine-rotavirus mutants" for ts mutants of animal rotavirus. Biroro. (J. Viro
l.) 42 : 669-677] and similar to those described in 1982. The other two strains RV-1 and RV-
2 failed to adapt to plaque formation. The strain that was able to form plaques had a titer of 10 ⁵ to 10 ⁶
It varied between infectious particles / ml.

FIG. 2 shows that the human rotavirus RV-5
Plaque formation by (B) and RV-3 (C) is compared to uninfected cell control (A). The transparent areas are prominent in B and the thickened areas are prominent in C. Human-rotavirus RV-3 is known to belong to rotavirus subgroup 2 based on gel electrophoresis of the RNA genome, and is known to be serotype 3 based on its response to polyclonal and monoclonal antibodies. .

RV-3 is also, in particular, the Department of Gast
roenterology, Royal children's Hospital Also identified by a monoclonal antibody developed by Melbourne, Australia. This antibody is a Mab RV-
3: 3 (previously referred to as 101AB5-B8). It has a higher R than other rotaviruses tested.
Reacts 1,000 times more strongly with V-3.

RV-5 is known to belong to rotavirus subgroup 1 by gel electrophoresis of RNA genome,
And it is known that it is serotype 2 by the reaction with the polyclonal antibody and the monoclonal antibody. Now, RV
-5 unique biochemical structures are known [Dyal
l-Smith ML and Holmes IH, "Sequence homology between human and animal rotavirus serotype-specific glycoproteins", Nucleic Acids Research.
search) (1984), 12 : 109].

Vaccine Preparation The preferred route for the preparation of the vaccine of the invention follows the recent WHO standards for live poliomyelitis vaccine (oral), with appropriate modifications ["Pomeculomyelitis vaccine (oral) Requirements for biological standards)
O Technical Committee, 33rd Report (Technical Report Series 6)
87), pp. 107-174, WHO, Geneva, 19
1983].

Use of the Vaccine The first oral administration of the vaccine is preferably carried out before 3 months of age, particularly preferably immediately after birth, and after 3 months the maternal antibodies in the serum of the child have fallen to low levels. When the second vaccination is given. If infection occurs later, children appear most vulnerable to serious clinical illness. Vaccination of mothers results in high titers of serum antibodies in children at birth, but does not appear to provide protection against rotavirus infection after 3 months of age. The number of rotavirus strains required in oral vaccines is not yet clear, but infection by one strain may be sufficient to protect against serious illness caused by other strains .

However, preferred vaccines contain a suspension of one or more serotypes of a live attenuated rotavirus grown in culture of a human or monkey diploid cell line. Such a vaccine would be administered orally once or twice. Next, the effectiveness and non-toxicity of the vaccine of the present invention will be described by experimental examples.

The experimental purposes of this experiment, human - response to RV3 strain of rotavirus (seroconversion, virus release, clinical symptoms, neutralizing antibody titer) is to study. This rotavirus strain represents a live attenuated strain of human-rotavirus.

Materials and Methods Viral inoculum : Four gnotobiotic (Gn) pigs (Gn5, 6, 7, 8) were given 3 days after birth to the human-rotavirus vaccine RV3 (clone 2-13-3-A).
GMK, lot C-108V) was administered orally. The titer of the RV3 vaccine in cell culture immunofluorescence (CCIF) was 6 × 10 ⁵ FCFU / ml.

All pigs were fed a standard diet for Similac pups and maintained under gnotobiotic conditions. On the third day after birth, pigs were orally inoculated with 1 ml of RV3 in 4 ml of medium. All four pigs were vaccinated a second time with the same amount of RV3 16 days after the first inoculation (0 / 16D
PE). As a third dose, 5 ml of RV3 was orally administered per pig on day 32 after the first inoculation (0/16/3).
2DPE). All pigs were observed daily for diarrhea and clinical symptoms.

Sample collection and testing : (i) Rectal swabs were collected from each pig from 0 to 10 days after inoculation of the rectal swab (DPE) and cell culture immunofluorescence measurements (Literature 1: Bohl, E .;
H. et al., J. Clin. Microbiol. 19: 105-111). (Ii) Mucosal smear Two pigs were euthanized to 35 DPE (3 DPE after the third inoculation), two more pigs were euthanized to 42 DPE (10 DPE after the third inoculation), and duodenum, jejunum and ileum A mucosal smear was prepared from. Fix the tissue
The presence of rotavirus antigen was tested by immunofluorescence measurement using ITC-conjugated anti-rotavirus serum (1).

(Iii) Serum samples Blood samples were collected approximately weekly from selected pigs and serum was separated. (Iv) Serological test Serum samples obtained as described above were subjected to (1) a specific antiviral serum antibody (EIA) as a group antigen.
Enzyme immunoassay to detect Ig); and (2) the same rotavirus (RV3), related serotype 3 animal rotavirus (SA11) and serotypes G1, G2.
And inhibition of fluorescent cell focus measurements to detect rotavirus serum neutralizing antibodies (VN) against three standard rotavirus strains, representative of G4 and R4, RV5 and ST3, respectively.

Results Clinical symptoms and rotavirus release were analyzed daily after each RV3 vaccination.
No diarrhea occurred, or only slight idiopathic diarrhea occurred during the day. Immunofluorescence on mucosal smears prepared from duodenum, jejunum and ileum after euthanizing animals (Reference 1)
Was negative for Rotavirus antigen.

The results of the serological tests are shown in the table below. All four pigs showed seroconversion after inoculation with RV3. All pigs produced EIA Ig anti-rotavirus antibodies, as well as neutralizing antibodies against vaccine strain (RV3) and serotype 3 strain (SA11). No neutralizing antibodies were raised against heterologous viruses of serotype 1, 2 or 4. Rotavirus antibody (EIA and VN)
Was not detected or was only present at low titers after the first dose of RV3, but high levels were achieved in three of the four piglets after the second dose. In these three piglets, this level was maintained or decreased slightly after the third dose. The fourth piglet has the highest titer (EIA and V) only after the third dose of RV3.
N).

[0049]

[Table 2]

As described above, the vaccine of the present invention shows a high neutralizing antibody titer against the syngeneic rotavirus (G3), but after administration, no rotavirus is detected in the visceral mucosa and stool, and diarrhea is reduced. It does not wake up, indicating that it is extremely attenuated.

A live attenuated strain of the virus, designated Hu / Australia / 10-25-10 / 77 / L, was deposited with the American Type Culture Collection (ATCC) on February 1, 1985, based on the Budapest Treaty. Deposited accession number ATCC
VR2104 was provided. Hu ／ Australia ／ 1-9
A live attenuated strain of the virus designated -12 / 77 / S is AT
It was deposited internationally with the CC on February 1, 1985 under the Budapest Treaty and given the accession number ATCC VR2105.

[Brief description of the drawings]

FIG. 1 is a gel electrophoresis diagram of RNAs of various viruses, wherein lane 1 is RV-1, lane 2 is RV-6, lane 3 is RV-5, lane 4 is RV-4, and lane 5 Is the pattern for SA-11;

FIG. 2 shows a plaque formed by a virus.
V-5, C indicate RV-3, and A indicates the control, respectively.

 ────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ruth Francis Bishop, Victoria 3186, Brighton, Kinnan Street, Australia 23 (56) References MED. VIOL. , VOL. 11, NO. 3 (1983), PP. 241-250 J.P. BIOL. STAND. , VOL. 5, NO. 1 (1977), PP. 19−30

Claims (6)

(57) [Claims] 1. A method for the preparation of a vaccine which confers immunological protection against a disease caused by human rotavirus, comprising preparing a filtrate from a stool sample containing human rotavirus, equivalent to 2 ml. The virus is grown on the substrate using an amount of the filtrate containing filtered material from 0.1 g or more of the sample as an inoculum for an appropriate amount of the substrate, and the virus is serially inoculated. how to vitro passages, you select the live attenuated strain of the appropriate the virus for use as a vaccine, comprising the - in in. 2. The method of claim 1, wherein said amount of filtrate contains 0.2 g or more of filtered material from said sample. 3. The method according to claim 1, wherein an inoculum of a similar amount to that used in the first passage is used without dilution for the next passage.
Or the method of 2. 4. The method of claim 1, wherein said substrate comprises cells from cell line MA-104. 5. The method according to claim 1, wherein said substrate comprises cells from the cell line FRhL2. 6. The method according to claim 1, wherein the virus is sequentially passaged 5 times or more.