WO1998041619A1 - Poliovirus attenues - Google Patents

Poliovirus attenues Download PDF

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WO1998041619A1
WO1998041619A1 PCT/GB1998/000798 GB9800798W WO9841619A1 WO 1998041619 A1 WO1998041619 A1 WO 1998041619A1 GB 9800798 W GB9800798 W GB 9800798W WO 9841619 A1 WO9841619 A1 WO 9841619A1
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positions
poliovirus
type
sabin
attenuated
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PCT/GB1998/000798
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Philip David Minor
Andrew Joseph Macadam
Jeffrey William Almond
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Btg International Limited
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Priority to CA002283638A priority Critical patent/CA2283638A1/fr
Priority to JP54025498A priority patent/JP2001519655A/ja
Priority to EP98909658A priority patent/EP0968278A1/fr
Publication of WO1998041619A1 publication Critical patent/WO1998041619A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A61K39/13—Poliovirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32611—Poliovirus
    • C12N2770/32634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32611—Poliovirus
    • C12N2770/32661—Methods of inactivation or attenuation
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to attenuated polioviruses, to their preparation and to vaccines containing them. More specifically, the invention relates to polioviruses which are attenuated by the introduction of defined mutations into their genomes.
  • Vaccine strains derived from each of the three poliovirus serotypes known as Sabin types 1, 2 and 3, were prepared by passage of wild-type viruses in cell cultures and whole animals until attenuated strains were obtained. These attenuated viruses are substantially less able to cause poliomyelitis in humans than the original wild-type strains. They are administered orally and replicate in the gut to induce a protective immune response.
  • the Sabin vaccine strains were developed by essentially empirical procedures. The genetic basis of their attenuation is not completely understood. Over the past several years, however, scientists have employed a number of molecular biological techniques in an attempt to elucidate the mechanism by which the neurovirulence of these vaccine strains is reduced. Most of the work has concentrated on serotypes 1 and 3. For both of these the complete nucleotide sequences of the vaccine strains have been compared with those of their neuro virulent progenitors.
  • the vaccine strain differs from its progenitor at 47 positions in the 7441 base genome (Nomoto et al, Proc. Natl. Acad. Sci. USA 79, 5793-5797, 1982). All of these are simple point mutations and 21 of them give rise to amino acid changes in virus- coded proteins. Although several mutations are thought to contribute to the attenuation phenotype of the vaccine strain, direct evidence has been presented that the mutation of A to G at position 480 in the 5' non-coding region of the genome has a marked attenuating effect on the virus (Nomoto et al. , UCLA Symp. Mol. Cell. Biol., New Series, 54 (Eds M.A. Brinton and R.R. Rueckert), 437- 452, New York: Alan R. Liss Inc., 1987).
  • the construction of defined recombinants between the type 3 Sabin vaccine strain and its progenitor strain has allowed the identification of the mutations which contribute to the attenuation phenotype.
  • One of these is at position 2034 and causes a serine to phenylalanine change in virus protein VP3.
  • the other mutation of interest is C (progenitor) to U (vaccine strain) at position 472 in the 5' non-coding region of the genome.
  • This 472U mutation has been observed to revert to the progenitor (wild type) 472 C rapidly upon replication of the virus in the human gut (Evans et al., Nature 314, 548-550, 1985). This reversion is associated with an increase in neuro virulence.
  • C at position 472 has also been shown to be essential for growth of a mouse/human polio recombinant virus in the mouse brain (La Monica et al, J. Virol. 57, 515-525, 1986). More recently it has been observed that A changes to G at position 481 in poliovirus type 2, again upon replication of the virus in the gut of vaccinees (Macadam et al, Virology 181, 451- 458, 1991).
  • stem (a) 471-473/538-536) and stem (b) (477-483/534-528).
  • stem (a) 471-473/538-536)
  • stem (b) 477-483/534-528
  • a type 3 poliovirus with the base pair 472-537 reversed i.e. 472 G and 537 C
  • this attenuated virus had a slightly lower LD 50 value than the corresponding poliovirus which only had the mutation C to G at position 472 but which retained the wild-type G at position 537.
  • Attenuated polioviruses in which a base pair of stem (a) or stem (b) of domain V is reversed are disclosed in EP-A-0383433.
  • subsequent experiments showed that the type 3 poliovirus in which the 472-537 base pair is reversed is not as attenuated as the type 3 Sabin vaccine strain.
  • the solution of the present invention comprises providing an attenuated poliovirus which does not have a U-G base pair or other base pair mismatch in stem (a) or (b) of domain V of the 5' non-coding region of the poliovirus genome.
  • the poliovirus may be a type 1, type 2 or type 3 poliovirus. A type 2 or type 3 poliovirus is preferred.
  • Attenuated type 3 poliovirus in which the 5' non-coding region of the genome of the poliovirus contains a U-A base pair at position 472-537 or 480-531, or both.
  • “Attenuated” means attenuated with respect to the wild type poliovirus which is the progenitor of the relevant Sabin vaccine strains (each strain has its own progenitor). Overall, the virus must be sufficiently attenuated to be safe in use.
  • WHO monkey neurovirulence test World Health Organization (WHO): Requirements for poliomyelitis vaccine (oral), WHO Tech. Rep. Ser. 687, 107-175, 1983.
  • FIGURE 1 shows the predicted R A secondary structure of domain V (nucleotides 471-538) of the type 3 Sabin strain (adapted from Skinner et al, 1989). Arrows show attenuating nucleotide changes found in the three Sabin vaccine strains (attenuating bases are in bold, followed by the relevant serotype in parentheses). Due to slight differences in lengths of the 5' non-coding regions, nucleotide 480 in Sabin 1 is at position 483 in this Figure and nucleotide 481 in Sabin 2 is at position 484. The base-paired stem regions are designated (a), (b), (c) and (d).
  • FIGURE 2 shows the sequence of stems (a) and (b) of domain V of the Sabin vaccine strains of each type of poliovirus.
  • Domain V of a type 3 poliovirus extends from positions 471 to 538.
  • Domain V of a type 2 or a type 1 poliovirus extends from positions 468 to 535.
  • FIGURE 3 shows the sequence of stems (c) and (d) of domain V of the Sabin vaccine strains of each type of poliovirus.
  • FIGURE 4 to 6 show the results of genetic stability testing of the Sabin 3 strain with a point mutation to C at 472 (S3/472C), the Sabin vaccine strain (S3) and mutants SI 5 and S16 of the present invention, the three last-mentioned before passage and after passages 1-5 (pl-p5) in cells.
  • the passaged virus was assayed and a parameter based on plaque-forming units (y axis) plotted against assay temperature (x axis).
  • the graphs show loss of temperature sensitivity, used as a temperature attenuation phenotype, with increasing passage.
  • Attenuated polioviruses of the invention have been modified so that stems (a) and (b) of domain V do not contain a U-G base pair or other base pair mismatch such as the U-U mismatch in the type 1 Sabin vaccine strain.
  • stems (c) and (d) also do not contain a U-G base pair or other base pair mismatch.
  • An alternative base pair is provided in place of the pair mismatch, chosen to ensure sufficient attenuation of the resulting poliovirus. This is as described above or, more preferably, substantially the same level of attenuation as the Sabin vaccine strain of the same poliovirus type.
  • a U-A base pair is present instead of a U-G base pair or a base pair mismatch.
  • a U-A base pair is preferably present at positions 472-537 and/or 480-531 of domain V of a type 3 poliovirus, and at position 527-478 of a type 2 poliovirus, replacing U-G base pairs (refer to Figure 2).
  • mutations are present as necessary to modify the thermodynamic stability of domain V so that the above-mentioned desired level of attenuation is achieved.
  • these mutations are in any one or more of stems (a), (b), (c) and (d) of domain V. They may include, for example, a base pair reversal and/or the replacement of a C-G base pair which is present in a neurovirulent wild-type poliovirus by a U-A or A-U base pair.
  • a U-A or A-U base pair may also be present in domain V of poliovirus type 3 at positions 478-533 and/or positions 481-530 and/or positions 482-529.
  • Preferred type 3 polioviruses of the invention contain the following U-A base pairs:
  • type 1 and 2 polioviruses can be correspondingly derived from the sequence of stems (a) and (b) of the wild-type neurovirulent type 1 and 2 polioviruses.
  • All strains are preferably Sabin.
  • the present invention provides a process for the preparation of an attenuated poliovirus of the invention, which process comprises: (i) introducing the or each desired mutation by site-directed mutagenesis into a sub-cloned region, which includes the or each position it is wished to mutate, of a DNA copy of a poliovirus genome; (ii) reintroducing the thus modified region into a complete copy DNA from which the region was derived; and
  • a mutation can thus be introduced into a strain of a poliovirus, normally a Sabin strain, by site-directed mutagenesis of a copy DNA corresponding to the genomic RNA of a poliovirus. This may be achieved by sub-cloning an appropriate region from an infectious DNA copy of a poliovirus genome into the single strand DNA of a bacteriophage such as Ml 3.
  • the modified sub-cloned copy DNAs are reintroduced into the complete copy DNA from which they were derived.
  • Live virus is recovered from the mutated full length copy DNA by production of a positive sense RNA typically using a T7 promoter to direct transcription in vitro (Van der Werf et al, Proc. Natl. Acad. Sci. USA 83, 2330-2334, 1986).
  • the recovered RNA may be applied to tissue cultures using standard techniques (Koch, Curr. Top. Microbiol. Immunol. 61: 89-138, 1973). After 2-3 days of incubation, virus can be recovered from the supernatant of the tissue culture. The level of neurovirulence and thus of attenuation of the modified virus may then be compared with that of the unmodified virus using a standard LD50 test in mice or the above-mentioned WHO-approved vaccine safety test in monkeys.
  • Attenuation of domain V has also been shown to correlate approximately with temperature sensitivity in BGM cells (Macadam et al., Virology 181, 451-458, 1991) or in L20B cells (as described for CM-1 cells in Macadam et al., Virology 189, 415- 422, 1992).
  • the temperature sensitivity of modified virus can thus be determined as a preliminary screen to determine the level of attenuation expected. This can be expressed as the temperature (T) at which the number of plaque forming units (pfti) is reduced by a power of 10 (1.0 log 10 ) from the number obtained at 35°C in the same cells.
  • the attenuated polioviruses can be used as live vaccines. They may therefore be formulated as pharmaceutical compositions further comprising a pharmaceutically acceptable carrier or diluent. Any carrier or diluent conventionally used in live vaccine preparations may be employed.
  • the attenuated polioviruses can be stabilised in 1M aqueous MgCl 2 and administered as a mixture of the three serotypes.
  • the attenuated polioviruses can therefore be used to prevent poliomyelitis in a human patient.
  • they may be administered orally, as a nasal spray, or parenterally, for example by subcutaneous or intramuscular injection.
  • a dose corresponding to the amount administered for a conventional Sabin vaccine strain, such as from 10 4 to 10 6 TCID 50 , may be administered.
  • HEp2C, BGM and Vero cells were grown in monolayers as described in
  • L20B cells mouse L-cells expressing the human poliovirus receptor were grown as described in Pipkin et al, J. Virol.
  • MRC-5 cells were grown in monolayers in the same growth medium as BGM cells.
  • Virus passage MRC-5, Vero or L20B cell monolayers in 25cm 2 flasks were inoculated with viruses at multiplicities of infection (moi) of 0.1 or 10.
  • Infected cell sheets were incubated in Minimum Essential Medium (MEM) containing antibiotics but no serum at 33°C or 37°C until complete cytopathic effect (cpe) was apparent.
  • Flasks were frozen at -70°C and thawed, three times, and cell debris removed by centrifugation. Supematants were used for further passage, as above, or for biological and molecular analysis.
  • MEM Minimum Essential Medium
  • BGM cells were used for temperature-sensitivity (ts) assays as described in Macadam et al., 1991.
  • L20B cells were used for ts assays as described for CM-1 cells in Macadam et al., Virology 189, 415-422, 1992. Briefly, viruses were assayed by plaque-formation at different temperatures. These were controlled by incubation of inoculated plates in sealed plastic boxes submerged in water baths whose temperatures fluctuated by ⁇ 0.01°C. All viruses were assayed at least twice and control viruses with known phenotypes were always included for validation.
  • Viruses with a Leon Lansing genomic background were assayed in BGM cells; Sabin 3, Leon and mutants of these viruses were assayed in L20B cells.
  • the range of temperatures over which variation in domain V stability influences viral growth depends on the cell substrate used (Macadam et al, 1991 and 1992 cited above) and is lower in L20B cells than BGM cells.
  • the major ts determinant is at VP3 91 (Minor et al, J. Gen. Virol. 70, 1117-1123, 1989). This influences ts at temperatures higher than domain V in L20B cells, but approximately the same temperatures as domain V in BGM cells. Consequently, ts assays in L20B cells can be used to distinguish viruses with different mutations in domain V irrespective of the residue at VP3 91.
  • Mutants Leon U-A and Leon U-G were constructed by partial Sstl/Satl fragment exchanges between the relevant Leon/Lansing mutant and T7/Leon full-length clones. Mutants in Table 3 were constructed in two steps. Mlul-Sstl fragments from relevant Leon/Lansing mutants were ligated into Sabin 3 clones digested to completion with Mlul (279) and Sstl (751 and 1900). Since there are two Sstl sites in the Sabin 3 genome, resultant clones lacked the Sstl-Sstl (751-1900) region. Full-length infectious clones were generated by digestion with Sstl/Smal (2768) and insertion of a partial SstllSmal (751-2768) fragment of the Sabin 3 clone.
  • Viruses were recovered by transfection of HEp2C monolayers with >2 ⁇ g T7 transcripts (Van der Werf et al, cited above), followed by incubation at 34°C for 24 to 48h, by which time complete cpe was apparent. Sequences of nucleotides 450-550 of all mutants were confirmed following RNA extraction and reverse transcription- polymerase chain reaction (RT-PCR).
  • Neurovirulence Viruses were assayed by the above-cited standard WHO approved test for vaccine safety (WHO, 1983) except that fewer animals were used per virus. It is expected that the results obtained using the required number of animals would not be materially different.
  • Mutant LI with 472-537 U-A, confers a ts phenotype intermediate between those of Leon-Lansing, with C-G at 472-537, and Mutant L2 with U-G at 472-537, all being in a Leon-Lansing genomic background.
  • a further mutation in the L3L2 472- 537 U-A background of C-G to U-A or A-U at 478-533, 481-530 or 482-529 resulted in a large reduction of the T value, i.e. increased temperature-sensitivity, (compare mutant LI with mutants L5, L8 and L10). This was considered likely to result in over-attenuation.
  • mutants LI 4, LI 5 and LI 6 were introduced into a Sabin 3 genomic background and viruses were assayed for temperature-sensitivity in L20B cells.
  • Ts phenotypes of these strains (mutants SI 4, SI 5 and SI 6) were similar to that of Sabin 3, although strain S14 was slightly more ts than Sabin 3 (Table 3).
  • Mutants SI 5 and S16 were also tested for neurovirulence in monkeys and results are shown in Table 3 along with two sets of results for the cloned Sabin 3 strain ("T7/Sabin 3”), one set from a concurrent test and another derived from all neurovirulence tests of this virus carried out to date.
  • strain SI 5 The attenuation phenotype of strain SI 5 hardly differed from that of T7/Sabin 3. No animals were paralysed by any of the viruses.
  • the mean lesion score of strain S 15 was almost identical to the score for T7/Sabin 3 in the concurrent test, although higher than the overall score.
  • Strain SI 6 appeared slightly more attenuated than T7/Sabin 3. On the preliminary basis of these results both strains SI 5 and strain SI 6 would have passed the above-cited WHO monkey neurovirulence test. Strain SI 4, being more ts than Sabin 3, would also have done so.
  • the current type 3 vaccine strain of poliovirus reverts rapidly in the human gut and is associated with a low level of paralytic poliomyelitis in vaccine recipients or their contacts. It has not yet been possible to compare the stabilities of these strains with that of Sabin 3 during growth in the human alimentary tract, so a cell culture model was sought. Conditions which favoured rapid reversion at nucleotide 472 were presumed to mimic selection pressures in the human gut. Reversion of nucleotide 472 from U to C on passage of the Sabin 3 strain in cell culture is temperature- and cell- dependent and it was found that growth at 37°C in L20B cells strongly selects for this reversion.
  • T7/Sabin 3 and strains SI 5 and S16 were passaged five times in L20B cells at 37°C using a multiplicity of infection (moi) of 10. All passage stocks were assayed for temperature sensitivity in L20B cells and fifth passage stocks were tested for neurovirulence.
  • viral RNA was extracted and sequenced through domain V after RT-PCR. Sequencing and MAPREC (Mutant Analysis by Polymerase Chain Reaction and Restriction Enzyme Cleavage) assays (Lu et al, PCR Methods Appl.
  • passage 3 virus was only slightly more temperature-sensitive than the passage 5 or the fully reverted virus (S3/472C).
  • the ts phenotypes of strains S15 and S16 were much more stable (Figs 5 and 6).
  • a gradual loss of temperature sensitivity did occur on passage of both these strains, which must have been due to mutation outside domain V.
  • the fifth passage stock of Sabin 3 was significantly more virulent than the Sabin 3 vaccine strain, as shown by numbers of clinically affected animals and histological lesion scores, whereas the fifth passage stocks of strains SI 5 and S16 were as attenuated as the original stocks and the Sabin 3 vaccine strain (Table 3).

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Abstract

L'invention se rapporte à un poliovirus atténué approprié à la vaccination contre la poliomyélite. Ledit virus ne présente pas de paire de bases U-G ou d'autres mauvais appariements de paire de bases dans la souche (a) ou (b) du domaine V de la région non codante 5' du génome du poliovirus. Le poliovirus atténué peut être un poliovirus de type 3 dont la région non codante 5' contient les paires de bases U-A suivantes: (a) U-A aux emplacements 472-537, U-A aux emplacements 478-533 et U-A aux emplacements 480-531; (b) U-A aux emplacements 472-537, U-A aux emplacements 480-531 et U-A aux emplacements 481-530; ou (c) U-A aux emplacements 472-537, U-A aux emplacements 480-531 et A-U aux emplacements 482-529.
PCT/GB1998/000798 1997-03-19 1998-03-18 Poliovirus attenues WO1998041619A1 (fr)

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CA002283638A CA2283638A1 (fr) 1997-03-19 1998-03-18 Poliovirus attenues
JP54025498A JP2001519655A (ja) 1997-03-19 1998-03-18 弱毒化ポリオウイルス
EP98909658A EP0968278A1 (fr) 1997-03-19 1998-03-18 Poliovirus attenues

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GBGB9705669.1A GB9705669D0 (en) 1997-03-19 1997-03-19 Attenuated polioviruses

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008017870A1 (fr) * 2006-08-10 2008-02-14 National Biological Standards Board Poliovirus atténués
WO2012090000A1 (fr) 2010-12-29 2012-07-05 Health Protection Agency Vaccin inactivé contre la poliomyélite
EP2139515B1 (fr) 2007-03-30 2018-06-13 The Research Foundation of the State University of New York Virus attenues utiles pour des vaccins

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EP0325768A2 (fr) * 1988-01-11 1989-08-02 American Cyanamid Company Virus à ARN non retournant
EP0383434A1 (fr) * 1989-01-18 1990-08-22 Btg International Limited Virus atténués
EP0508783A1 (fr) * 1991-04-10 1992-10-14 British Technology Group Ltd Poliovirus atténués et vaccines

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
EP0325768A2 (fr) * 1988-01-11 1989-08-02 American Cyanamid Company Virus à ARN non retournant
EP0383434A1 (fr) * 1989-01-18 1990-08-22 Btg International Limited Virus atténués
EP0508783A1 (fr) * 1991-04-10 1992-10-14 British Technology Group Ltd Poliovirus atténués et vaccines

Non-Patent Citations (2)

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Title
HALLER A A ET AL: "ATTENUATION STEM-LOOP LESIONS IN THE 5' NONCODING REGION OF POLIOVIRUS RNA: NEURONAL CELL-SPECIFIC TRANSLATION DEFECTS", JOURNAL OF VIROLOGY, vol. 70, no. 3, March 1996 (1996-03-01), pages 1467 - 1474, XP002037413 *
MACADAM A J ET AL: "CORRELATION OF RNA SECONDARY STRUCTURE AND ATTENUATION OF SABIN VACCINE STRAINS OF POLIOVIRUS IN TISSUE CULTURE", VIROLOGY, vol. 189, no. 2, August 1992 (1992-08-01), pages 415 - 422, XP002037412 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008017870A1 (fr) * 2006-08-10 2008-02-14 National Biological Standards Board Poliovirus atténués
US20100158945A1 (en) * 2006-08-10 2010-06-24 Macadam Andrew Attenuated polio viruses
AU2007283199B2 (en) * 2006-08-10 2012-11-15 Secretary of State for Health and Social Care Attenuated polio viruses
US8557252B2 (en) 2006-08-10 2013-10-15 The Secretary Of State For Health Genetically stable attenuated polioviruses comprising multiple mutations in domain V of the 5′ noncoding region
US9408903B2 (en) 2006-08-10 2016-08-09 The Secretary Of State For Health Vaccination method employing genetically stable attenuated polioviruses comprising multiple mutations in domain V of the 5′ noncoding region
EP2139515B1 (fr) 2007-03-30 2018-06-13 The Research Foundation of the State University of New York Virus attenues utiles pour des vaccins
EP2139515B2 (fr) † 2007-03-30 2023-12-20 The Research Foundation of the State University of New York Virus attenues utiles pour des vaccins
WO2012090000A1 (fr) 2010-12-29 2012-07-05 Health Protection Agency Vaccin inactivé contre la poliomyélite
US9402892B2 (en) 2010-12-29 2016-08-02 The Secretary Of State For Health Inactivated poliovaccine

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EP0968278A1 (fr) 2000-01-05
JP2001519655A (ja) 2001-10-23
GB9805810D0 (en) 1998-05-13
CA2283638A1 (fr) 1998-09-24

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