JPH072650B2 - Rotavirus vaccine - Google Patents

Description

The present invention relates to live attenuated human-rotavirus vaccines and methods of making the vaccines.

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. 1974, 149].

Rotavirus is currently recognized as an important cause of severe acute diarrhea in children worldwide. After the initial identification of rotavirus as the cause of acute diarrhea, efforts were made to cultivate the virus in various cell lines, but unsuccessfully.

Hido-rotavirus is its RNA in gel electrophoresis
It is broadly classified into two groups based on the mobility of genome segments: those with a "short RNA" (S) pattern and those with a "long RNA" (L) pattern.

In 1981, Sato et al. Described the successful cultivation of human-rotavirus from stool samples using roller culture of MA-104 cells ["Human-rotavirus isolation in cell culture" Arc. Bilolo. (Arch.Virol.) 69 : 155-16
0]. The 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 diarrhea stools.

Most strains cultured to date have the "L" pattern. It has been shown that "S" pattern strains are difficult to culture, and that only two strains of rotavirus with the "S" pattern have been cultured [eg, "Kutsuzawa et al., Humans in cell culture". Isolation of Rotavirus Subgroups 1 and 2 ", J. Klin. Microbiol. (J. Clin. Microbiol.) 16 : 727-730].

The inventors have succeeded in culturing six human-rotavirus strains of epidemiologically very important strains in Australia and Papua New Guinea, and preparing live attenuated viruses that are promising as vaccines.

The strains that caused diarrhea in 1977-1979 in Melbourne, Australia and in 1979 in Papua New Guinea were "S" pattern strains, but electron microscopically different. The inventors succeeded in culturing the "S" pattern virus from 2 out of 6 selected samples containing the "S" pattern virus. Others have found that culturing "S" pattern viruses is difficult.

The inventors used a culture method different from the original method such as Sato.
1.0 ml of stool fluid was used for the start of the culture and 1.0 ml of tissue culture fluid for all subsequent passages. It is surprising that this increased amount of inoculum was successful compared to the original method. An increased number of infectious particles in the bulk inoculum used in this study was "S".
It may have increased the chance of successful culture of strains with patterns.

We have found that strains from all stools stored at -70 ° C, strains from stool stored at -20 ° C and -70 ° C in PBS, and -70 ° C.
We have succeeded in culturing strains from sucrose cushion pelleted virus stored at ° C. Due to the small number of strains cultivated, it is not yet possible to draw conclusions about the optimal storage conditions of stool samples for the culture of viruses.

The present invention also provides a vaccine for providing immunological protection against acute diarrhea caused by human-rotavirus, which is internationally deposited under the Budapest Treaty as ATCC VR2104 Hu / Australia / 10- 25-1
Live attenuated strain of virus called 0 / 77L / L, or ATTC VR21
Hu / Austr, which has been internationally deposited under the Budapest Treaty as 05
It comprises a live attenuated strain of the virus called alia / 1-9-12 / 77 / S. The invention further provides a vaccine that provides enhanced cross-protection, comprising both attenuated strains of said virus.

Rotavirus Isolation Since 1973, the inventors have stored a rotavirus strain identified from the stools 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. Many of these stocks were tested by gel electrophoresis of genomic RNA [eg Rodger et al., "Molecules of human-rotavirus in Melbourne, Australia, 1973-1979, determined by electrophoresis of genomic ribonucleic acid." Epidemiology ”, J. Klin. Microbiol (J.Cl
in.Mcrobiol.) 13 : 272-278].

A stool sample containing known electrophoretic type rotavirus particles was selected by the inventors. The aim was to cultivate a human-rotavirus strain that caused a large and explosive epidemic of acute diarrhea in children in Australia and elsewhere.

All samples are phosphate buffered saline solution (PBS, pH 7.0)
They were stored as medium 20% stool homogenate or as sucrose cushion pelleted virus at -70 ° C or -20 ° C for various periods of time.

All of the diarrhea stools selected for culturing were predominantly multi-shelled viral particles (2-4 + or under electron microscopy)
10 ⁸ virus particles / ml or more). The details of the sample are as follows.

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

2. 5 samples containing "M-electrophoresis type" (Rodger et al.,
See above). Collected from children in Melbourne between 1977 and 1978 and stored as 20% homogenate in PBS or as sucrose cushion pelleted virus at -70 ° C [1983 Albert et al., "By electrophoresis of genomic RNA. , Epidemiology of Rotavirus Diarrhea in the Highlands of Papua New Guinea ",
J. Klin. Microbiol. (J.Clin.Microbiol) 17 : 162
-164].

3. One sample containing "PA-electrophoresis type". 20% in PBS, collected from children with a rotavirus diarrhea epidemic in the Highlands of Papua New Guinea in 1979.
Stored at -20 ° C as a homogenate.

4. One sample containing the "L" pattern of rotavirus. 19
Collected from children with a rotavirus diarrhea epidemic in Queensland, Australia in 1981 and stored as whole stools at -20 ° C.

5. One sample containing the predominant electrophoretic type of "L" pattern rotavirus. Collected from children in Melbourne in 1981 and as a 20% homogenate in PBS -70
Stored at ℃.

Culture technique (i) The culture technique generally follows the method of Sato et al. MA-1
04 cells [Microbiological Associates (Microbiolo
gical Associates (MA) Bioproducts (Bioproduct
s), Walkerville, MA, USA] with 10% fetal calf serum (FCS, Flow Laboratories, Sydney, Australia) and 12.25 μg / ml neomycin sulphate and polymyxin B sulphate variants of Dulbecco. Grow in medium (DMM, Flow Laboratories, Sydney, Australia, Cat NO 74-013-54). Cell layers that were confluent for 3 days in culture tubes were used for virus culture.

A stool sample containing rotavirus was thawed and all stool samples were homogenized to form a 20% suspension in PBS. Then all samples are vortexed and 3,000
It was centrifuged at xg for 10 minutes. Cells were removed by passing the supernatant through a 0.45 μm membrane filter (Millipore, Bedford, MA, USA).

Inoculate 10 μg / ml trypsin (sigma, trypsin 1
X, Sigma, St. Louis, MO, USA). 1 μg / ml for maintenance medium (DMM) during virus growth
Of trypsin.

The virus concentration in the inoculum was checked by electron microscopy. The inoculum was used only when the concentration of virus particles was 10 ⁸ particles / ml or more.

A 1.0 ml aliquot of trypsin-treated stool was inoculated into 2.0 ml cell culture of MA104 cells grown as a confluent cell monolayer in roller tubes. For Sato, etc., a 0.1 ml aliquot was used. Many others' attempts to culture other rotaviruses with 0.1 ml aliquots have failed. The change to a 1.0 ml aliquot led to successful cultivation of some rotavirus strains.

Each sample was inoculated in duplicate tubes. Immunofluorescence staining (immunofluorescen) using cells from one tube
The increase in virus was monitored after each passage by staining. In this case rabbit antiserum against simian virus SA-11 and goat FITC-conjugated anti-rabbit IgG (goat immunoglobulin conjugated with fluorescein isothiocyanate, supplied by Tago, Burlingham, CA, USA) I was there. 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.

Sequential passages were performed until the cytopathic effect (CPE) was revealed. The culture was then examined for viruses using an electron microscope [Whitby et al., 1980, "Detection of Virus Particles by Electron Microscopy Using Polyacrylamide Hydrogels", J. Klin. Pasol. (J. Clin Patho
l.) 33 : 484-487].

(Ii) FRh L2 cells (DBS-FRhL ₂ -2, ATCC, rockville,
Maryland, USA) were grown in minimal essential medium (Eagle) with non-essential amino acids in Earles BSS (BME), 10% fetal calf serum (FCS, Flow Laboratories, Sydney, Australia) and 12.5 μg / ml. Neomycin sulphate and polymyxin B sulphate each were added.

RU-3 virus, which was previously cultured in MA-104 cells,
Cells were seeded on day 7 confluent monolayers in small plastic flasks. Next, the culture technique described above was performed. 2 to
Serial passages were performed at 7-day intervals, and viral increase was monitored by enzyme-linked immunosorbent assay (EIA) and later by immunofluorescence staining.

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

Plaque formation of the virus After adaptation to static cell culture, the virus was plaqued to measure the amount of the virus [1981, Urasawa et al., "Human-rota in MA-104 cells. Sequential passage of virus ", Microbiol. Imnor. (Microbiol Im
25 : 1025-1035]. The upper layer consists of 0.6% purified agar (Oxoid, UK) and 2 μg / ml trypsin.
A second top layer containing Neutral Red (0.067 mg / ml) was applied 4-5 days after incubation.

Results Six human-rotavirus strains from the 16 stool samples examined were successfully adapted to cell culture. Three of these strains were isolated from stool stored in PBS at -70 ° C, one strain from sucrose cushion pelleted virus stored at -70 ° C, and one strain in PBS- 2
Isolated from stool stored at 0 ° C, and one strain at -70 ° C
Separated from stool stored at. For these 6 strains,
From the first passage to the appearance of CPE, specific cytoplasmic fluorescence was observed. One other strain showed early fluorescence during the third passage, which eventually disappeared. The remaining 9 samples are from passage 1 to 10
No intracytoplasmic fluorescence was shown until passage, and it was concluded that the virus could not be cultured at this stage. CPE appeared between the 6th and 9th passages for all culturable strains. The appearance of CPE consists of increased granularity, spheroidization into clusters, and extreme sloughing 3-5 days after inoculation of MA-104 cell monolayers. No differences in CPE were observed between different strains. Examination of cell cultures after the appearance of CPE showed a mixture of single-shell and double-shell virus particles.

In this specification, six types of viruses are described in 1979.
The nomenclature first proposed by Rodger and Holmes ["Comparison of monkey, bovine and human-rotavirus genomes by gel electrophoresis and detection of genomic differences between bovine isolates", J. Virol. (J.Virol.) 30 : 839-846]. This method is an encryption of the following information.

The animal species from which the rotavirus was obtained.

b. The historical origin of the virus.

c. Laboratory strain identification code.

d. Age of virus.

e. Electrophoresis pattern, ie "S" or "L".

This information could be added to the code for each strain if comparative sera for serotype and subgroup identification were available.

The code and laboratory identification code of strains cultivated according to the invention are shown below.

RV-1, RV-2 and RV-3 are identical and correspond to "R-electrophoretic type", RV-5 is "M-electrophoretic type" (Rodg
er et al., supra), and RV-6 appears to correspond to the "PA-electrophoretic type" (Albert et al., supra).

Human-rotavirus RNA cultured according to the invention.
The pattern (using the gel electrophoresis method of Rodger et al., Supra) is shown in Figure 1 along with the RNA pattern of monkey-derived virus SA11 routinely cultured in the laboratory. In FIG. 1, the patterns of RV-1, RV-6, RV-5, RV-4 and SA-11 are shown from left to right. No difference was observed in co-electrophoresis of the original fecal viral RNA and the cell culture adapted viral RNA.

The three strains, namely RV-4, RV-5 and RV-6, have different sizes (0.25
mm-3.5 mm) plaques were formed. 1 strain, namely RV-
3 formed plaques consisting of thickening of cells, rather than the normal areas where cell monolayers became clear. This plaque was described by Faulkner-Valle et al. [“Molecular Biology of Rotavirus III. Isolation and Characterization of Bovine-Rotavirus Temperature-Sensitive Mutants”, ts mutants of animal rotavirus, J. Virolo.
(J. Virol.) 42 : 669-677], similar to that described in 1982. The other two strains RV-1 and RV-2 were unable to adapt to plaque formation. The titers of strains that were able to form plaques varied between 10 ^{5 and} 10 ⁶ infectious particles / ml.

In Figure 2, human-rotavirus RV-5 (B) and RV-3
Plaque formation by (C) is compared to uninfected cell control (A). Cleared areas are prominent in B and 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 serotype 3 based on its response to polyclonal and monoclonal antibodies . The RV-3 is also especially the Department of Gastroenterolog
It is also identified by a monoclonal antibody developed by y, Royal children's Hospital Melbourne, Australia. This antibody is designated Mab RV-3: 3 (previously designated 101AB5-B8). It reacts 1,000 times more strongly with RV-3 than with the other rotaviruses tested.

RV-5 is known to belong to rotavirus subgroup 1 by gel electrophoresis of RNA genome, and serotype 2 by reaction with polyclonal and monoclonal antibodies.
Is known to be. Part of the unique biochemical structure of RV-5 is now known [Dyall-Smith ML and Holmes.
IH, "Sequence Homology Between Human and Animal Rotavirus Serotype-Specific Glycoproteins," Nucleic Acids Research (1984), 12 : 109].

Vaccine Production Construction The preferred route for the production of the vaccine of this invention follows the recent WHO standard for live poliomyelitis vaccine (oral) with appropriate modifications ["Paleomyelitis Vaccine (oral)" Requirements, ”WHO Technical Committee on Biological Standards, 33rd Report (Technical Report Series 687), 107
-174, WHO, Geneva, 1983].

Use of Vaccine The first oral dose of the vaccine is preferably given before 3 months of age, particularly preferably immediately after birth, and then 3 months later when the maternal antibody level in the serum of the child falls to low levels. The second vaccination is given. If infection occurs after this, the child appears most vulnerable to severe clinical illness. Vaccination of the mother results in high titers of serum antibodies in the child at birth, but does not appear to confer protection against rotavirus infection after 3 months of age. The number of rotavirus strains required in an oral vaccine is not yet clear, but it appears that infection with one strain is sufficient to protect against severe disease caused by another strain .

However, preferred vaccines contain a suspension of one or more serotypes of 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 efficacy and non-toxicity of the viral vaccine of the present invention will be described with reference to experimental examples.

Experimental The purpose of this experiment is to study the response of human rotavirus to the RV3 strain (seroconversion, clinical signs of virus release, neutralizing antibody titers). This rotavirus strain represents a live attenuated strain of human-rotavirus.

Materials and Methods Viral inoculum: 4 gnotobiotic (Gn) pigs (Gn5,
6,7,8), on the third day after birth, human rotavirus vaccine R
V3 (clone 2-B-3-AGMK, lot C-108V) was orally administered. The titer of RV3 vaccine in the cell culture immunofluorescence assay (CCIF) was 6 × 10 ⁵ FCFU / ml.

All pigs were fed Similac pup diets and maintained in gnotobiotic conditions. On postnatal day 3, pigs were orally inoculated with 1 ml RV3 in 4 ml medium. All four pigs were re-inoculated as the second dose with the same amount of RV3 on day 16 after the first inoculation (0/16 DPE). As the third administration, 5 ml of RV3 was orally administered per pig on day 32 after the first inoculation (0/16/32 DPE). All pigs were observed daily for diarrhea and clinical symptoms.

Sample Collection and Testing (i) Rectal Swabs Collect rectal swabs from each pig 0-10 days (DPE) after inoculation,
And cell culture immunofluorescence measurement (Reference 1: Bohl, EH et al., JC
lin. Microbiol. 19: 105-111) for rotavirus release.

(Ii) Mucosal smear Two pigs were euthanized to 35DPE (3DPE after the third inoculation), and 2nd grade pigs were euthanized to 42DPE (10DPE after the third inoculation), and duodenum, jejunum and ileum. Mucosal smears were prepared from Fixed tissues were tested for the presence of rotavirus antigen by immunofluorescence assay (1) with FITC-conjugated anti-rotavirus serum.

(Iii) Serum samples Blood samples from selected pigs are collected approximately weekly and serum separated and human rotavirus M (G3)
Indirect immunofluorescence assay for cells infected by the strain (II
F) (Reference 1; Reference 2: Kang, SK et al., J. Clin. Microbiol. 27:
2744-2750) for the presence of rotavirus antibodies. Titers were expressed as the reciprocal of the maximum serum dilution that reacted with rotavirus infected cells in this immunofluorescence assay.

(Iv) Serological test Regarding the serum sample obtained as described above, (1) enzyme immunoassay for detecting a specific antiviral serum antibody (EIA Ig) as a group antigen; and (2) same rotavirus (RV3), related serotype 3 animal rotavirus (SA11) and serotypes G1, G2 and G4
Of three standard rotavirus strains, RV4,
Inhibition of fluorescent cell focus measurements to detect rotavirus serum neutralizing antibodies (VN) to RV5 and ST3.

RESULTS: Clinical signs and rotavirus release were analyzed daily after each RV3 vaccination, with no post-vaccination virus release detected by CCIF for 1-10 days and no diarrhea or slight idiopathic diarrhea. It only happened.

Rotavirus antigen by immunofluorescence (Reference 1) was negative in mucosal smears prepared from the duodenum, jejunum, and ileum after euthanizing the animals.

The results of the serological tests are shown in the table below. All 4 pigs showed seroconversion after inoculation with RV3. All pigs produced EIA Ig anti-rotavirus antibodies and neutralizing antibodies against vaccine strain (RV3) and serotype 3 strain (SA11). No neutralizing antibodies were raised against serotype 1, 2 or 4 heterologous viruses.

Rotavirus antibodies (EIA and VN) were not detected or were only present in low titers after the first dose of RV3,
High levels were achieved in 3 of the 4 piglets after the second dose. In these 3 piglets, this level was maintained or decreased slightly after the third dose. The fourth piglet showed the highest titers (EIA and VN) only after the third dose of RV3.

As described above, the vaccine of the present invention shows a high neutralizing antibody titer against syngeneic rotavirus (G3), but after administration, rotavirus is not detected in the visceral mucosa and feces, and does not cause diarrhea, It turns out that it is extremely attenuated.

Deposited Hu / Australia / 10-25-10 / 77 / L, a live attenuated strain of the virus is included in the American Type Culture Collection (ATCC) on 1 February 1985 as ATCC VR2104 under the Budapest Treaty International Deposited.

A live attenuated strain of the virus called Hu / Australia / 1-9-12 / 77 / S was deposited with the ATCC on February 1, 1985 as ATCC VR2105 under the Budapest Treaty.

[Brief description of drawings]

Fig. 1 is a gel electrophoresis diagram of RNA of various viruses.
Lane 1 is for RV-1, Lane 2 is for RV-6, Lane 3 is for RV-5, Lane 4 is for RV-4, and Lane 5 is for SA-11 and is a picture in place of a drawing; FIG. 2 shows a plaque formed by a virus, which is a photograph as a substitute for a drawing showing the morphology of organisms, where B is RV-5 and C
Indicates RV-3, and A indicates control.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ruth Francis Visiyot, Australia, Victoria 3186, Brighton, Kinnere Street 23 (56) Reference US Patent 4341870 (US, A) Journal of Medical Virology, vol. 11, No. 3, PP, 241-250

Claims (3)

[Claims] 1. A vaccine which provides protection against diseases caused by human rotavirus, which is Hu / Australia.
/ 10-25-10 / 77L (ATCC VR2104) and Hu / Australia / 1-9-1
A vaccine comprising at least one live attenuated rotavirus strain selected from the group consisting of 2/77 / S (ATCC VR2105), together with a suitable carrier and / or excipient. 2. The live attenuated rotavirus strain is Hu / Australia.
The vaccine according to claim 1, which is / 10-25-10 / 77 / L (ATCC VR2104). 3. The live attenuated rotavirus strain is Hu / Australia.
The vaccine according to claim 1, which is / 1-9-12 / 77 / S (ATCC VR2105).