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WO1997038138A1 - Virus respiratoire syncytial attenue - Google Patents

Virus respiratoire syncytial attenue Download PDF

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Publication number
WO1997038138A1
WO1997038138A1 PCT/US1997/005588 US9705588W WO9738138A1 WO 1997038138 A1 WO1997038138 A1 WO 1997038138A1 US 9705588 W US9705588 W US 9705588W WO 9738138 A1 WO9738138 A1 WO 9738138A1
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WIPO (PCT)
Prior art keywords
amino acid
codon encoding
rsv
virus
respiratory syncytial
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PCT/US1997/005588
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English (en)
Inventor
Hunein F. Maassab
M. Louise Herlocher
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The Regents Of The University Of Michigan
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Application filed by The Regents Of The University Of Michigan filed Critical The Regents Of The University Of Michigan
Priority to EP97920111A priority Critical patent/EP0917592A4/fr
Priority to AU24385/97A priority patent/AU2438597A/en
Priority to US08/882,358 priority patent/US6077514A/en
Publication of WO1997038138A1 publication Critical patent/WO1997038138A1/fr
Priority to CA002292448A priority patent/CA2292448A1/fr
Priority to EP98914473A priority patent/EP0975363A4/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • 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
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18534—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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18561—Methods of inactivation or attenuation
    • C12N2760/18564—Methods of inactivation or attenuation by serial passage

Definitions

  • the present invention relates generally to attenuated respiratory syncytial viruses and, more particularly, to live attenuated respiratory syncytial virus vaccines and methods of protecting against disease caused by infection with respiratory syncytial virus
  • Respiratory syncytial virus a member of the paramyxovi ⁇ dae family, is the leading cause of viral pneumonia and bronchitis in infants and young children worldwide, and is a major cause of fatal respiratory tract disease
  • Serious disease is most prevalent in infants 6 weeks to 6 months of age and in children with certain underlying illnesses (e g , immunodeficiencies, congenital heart disease and bronchopulmonary dysplasia)
  • underlying illnesses e g , immunodeficiencies, congenital heart disease and bronchopulmonary dysplasia
  • Virtually all children are infected by two years of age
  • Most infections are symptomatic and are generally confined to mild upper respiratory tract disease
  • a decrease in severity of disease is associated with two or more prior infections and, in some studies, with high levels of serum antibody, suggesting that protective immunity to RSV disease will accumulate following repeated infections (Lamprecht, C L et al , J Inf Dis 134 21 1-217 (1976), Henderson, F W et al , N Eng
  • RSV was cold-adapted to 25-26°C in several laboratories in the mid-1960's, but was found to be under-attenuated in vaccine trials (Kim, H.W. et al., Pediatrics
  • Live attenuated vaccines offer several advantages over inactivated vaccines. These include the possible use of a single dose and administration by the natural route of infection i.e., intranasally. In addition, live attenuated vaccines stimulate a wide range of immune responses, including local and serum antibody responses and cellular immunity. Furthermore, these vaccines are cost-effective and can be rapidly produced and updated in the event of antigenic changes.
  • Attenuated RSV strains which exhibit the cold-adapted [ca) and/or temperature sensitive (ts) phenotype are provided Samples of viruses as embodiments of the present invention have been deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20582, under the terms of the Budapest Treaty, and have been accorded the following ATCC designation numbers
  • the deposit of the viruses will be maintained in the ATCC depository, which is a public depository, for a period of 30 years, or 5 years after the most recent request, or for the effective life of a patent, whichever is longer, and will be replaced if the deposit becomes depleted or nonviable during that period Samples of the deposited strains will become available to the public and all restrictions imposed on access to the deposits will be removed upon grant of a patent on this application
  • the present invention also provides methods for immunizing a subject against disease caused by infection by RSV comprising administering to the subject an immunoeffective amount of an attenuated RSV and in particular, cold-adapted and/or temperature sensitive RSV Methods of making and using such attenuated RSV in a pharmaceutical composition e g , a vaccine, are also provided
  • Figure 1A is a graph showing the total anti-F IgG response of mice immunized with an immunogenic composition of an aspect of the present invention
  • Figure 1 B is a graph showing the anti-F IgG, response of mice immunized with an immunogenic composition of an aspect of the present invention
  • Figure 1C is a graph showing the anti-F lgG 2a response of mice immunized with an immunogenic composition of an aspect of the present invention
  • Figure 2A is a graph showing the anti-RSV-F antibody titers (after 4 weeks) of mice immunized with an immunogenic composition of an aspect of the present invention
  • Figure 2B is a graph showing the anti-RSV-F antibody titers (after 8 weeks) of mice immunized with an immunogenic composition of an aspect of the present invention
  • Figure 3 is a graph showing RSV specific neutralizing antibody titers (after 4 and 8 weeks) of mice immunized with an immunogenic composition of an aspect of the present invention
  • Figure 4 is a graph showing cytotoxic T cell (CTL) activity of mice immunized with an immunogenic composition of an aspect of the present invention
  • Figure 5 is a graph showing CTL activity of mice immunized with immunogenic compositions of aspects of the present invention.
  • CTL cytotoxic T cell
  • Attenuated RSV including cold-adapted and/or temperature sensitive RSV are provided and in particular, eleven subgroup A RSV lines, have been deposited with the ATCC and are described in detail herein.
  • cold- adapted means a virus that has been attenuated by propagation at lower than optimal growth temperatures and the term "temperature sensitive” means that replication of the virus is impeded as temperature is elevated.
  • the deposited lines of the present invention have been successfully attenuated using three different approaches: adaption to suboptimal temperature by direct and stepwise passage; high passages at 35°C; and, adaption to a heterologous host (i.e., host-range restricted).
  • the attenuated RSV of the present invention are genetically-stable, immunogenic and protective, and avirulent, and are thus particularly useful in the formulation of live, attenuated RSV vaccines which are capable of eliciting a protective immune response without causing unacceptable symptoms of severe respiratory disease.
  • the immune response which is achieved in the subject by the method of an embodiment of the present invention preferably includes the production of virus specific neutralizing antibodies and the virus specific cytotoxic T-cell responses. The invention is therefore particularly effective to provide protection against respiratory tract diseases caused by RSV.
  • Methods of attenuating RSV as well as methods of making and using attenuated RSV vaccines are also provided by the present invention, including the preparation of pharmaceutical compositions.
  • Nucleic acid molecules encoding the attenuated RSV are also within the scope of the present invention. These nucleic acids may be DNA molecules, cDNA molecules or RNA molecules e.g., antisense RNA.
  • the present invention further includes nucleic acid molecules which differ from that of the nucleic acid molecules which encode the RSV of the present invention, but which produce the same cold- adapted and temperature sensitive phenotypic effect. These altered, phenotypically equivalent nucleic acid molecules are referred to as "equivalent nucleic acids.”
  • the present invention also encompasses nucleic acid molecules characterized by changes in non-coding regions that do not alter the phenotype of the polypeptide produced therefrom, when compared to the nucleic acid molecules of the RSV described herein
  • nucleic acid molecules comprising noncoding sequences of the RSV of the present invention
  • These non- coding regions are to include 5' noncoding regions, 3' noncoding regions, intergenic sequences, and other noncoding regions of the viral genome These include, but are not limited to, transcriptional, translational and other regulatory regions
  • nucleic acid molecules also may be DNA molecules, cDNA molecules or RNA molecules
  • Nucleic acid molecules which hybridize under stringent conditions to the nucleic acid molecules described herein are also within the scope of the present invention as will be appreciated by those skilled in the art, multiple factors are considered in determining the stringency of hybridization including species of nucleic acid, length of nucleic acid probe, T m (melting temperature), temperature of hybridization and washes, salt concentration in the hybridization and wash buffers, aqueous or formamide hybridization buffer, and length of time for hybridization and for washes
  • An example of stringent conditions are DNA-DNA hybridization with a probe greater than 200 nucleotides in 5 x SSC,
  • polypeptides isolated from the RSV described herein or from cells infected with these same virus are also encompassed by the present invention
  • the polypeptides (or fragments thereof) may be of varying length, and preferably will be capable of exhibiting immunological activity.
  • RSV polypeptides can be isolated in substantially pure form from RSV or cultures of cells infected with RSV.
  • RSV polypeptides can be isolated from a recombinant system or are vector-engineered to produce these polypeptides.
  • RSV polypeptides can be chemically synthesized by methods well known to those of skill in the art. All derived RSV strains are also encompassed by the present invention, including, without limitation, those attenuated by cold adaptation (including both direct and stepwise passage), high in vitro passage, host-range restriction and chemical or genetic modification e.g., site-directed mutagenesis.
  • subgroup B virus can be produced by biologically cloning wild-type subgroup B virus in an acceptable cell substrate using methods known in the art. The subgroup B virus may then be attenuated as described herein.
  • compositions comprising any of the RSV described herein or polypeptides, either alone or in combination, and a pharmaceutically acceptable carrier, are also provided by the present invention.
  • pharmaceutically acceptable carrier encompasses any of the standard pharmaceutical carriers, such as physiologically balanced culture medium, phosphate buffered saline solution, water, and emulsions, such as an oil/water emulsion, various types of wetting agents and protein stabilizers. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • Formulations include those suitable for oral, nasal, topical (including transdermal, buccal and sublingual), parenteral (including subcutaneous) and pulmonary administration.
  • the pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any method known in the art.
  • the pharmaceutical composition is intended for use as a vaccine.
  • a virus may be mixed with cryoprotective additives or stabilizers such as proteins (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate), saline, or other protective agents.
  • proteins e.g., albumin, gelatin
  • sugars e.g., sucrose, lactose, sorbitol
  • amino acids e.g., sodium glutamate
  • saline saline
  • Lyophilized virus will typically be maintained at about 4°C and when ready for use, reconstituted in a stabilizing solution, with or without adjuvant.
  • the virus may be inactivated and may be mixed with an adjuvant, saline and a detergent such as phosphate Tween buffer.
  • a detergent such as phosphate Tween buffer.
  • Immunogenicity can be significantly improved if the virus is co-administered with an immunostimulatory agent or adjuvant.
  • Adjuvants enhance immunogenicity but are not necessarily immunogenic themselves.
  • Immunostimulatory agents or adjuvants have been used for many years to improve the host immune responses to, for example, vaccines.
  • Suitable adjuvants are well known to those skilled in the art and include, without limitation, aluminum phosphate, aluminum hydroxide, QS21 , Quil A, derivatives and components thereof, calcium phosphate, calcium hydroxide, zinc hydroxide, a glycolipid analog, an octodecyl ester of an amino acid, a muramyl dipeptide, polyphosphazene, a lipoprotein, ISCOM matrix, DC-Choi, DDA, and other adjuvants and bacterial toxins, components and derivatives thereof.
  • Pharmaceutical compositions comprising any of the attenuated RSV of the present invention are useful to immunize a subject against disease caused by RSV infection.
  • this invention further provides methods of immunizing a subject against disease caused by RSV infection, comprising administering to the subject an immunoeffective amount of a pharmaceutical composition of the invention.
  • This subject may be an animal, for example a mammal, such as a primate or preferably a human.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, immunogenic and protective.
  • the quantity to be administered depends on the subject to be treated, including, for example, the capacity of the immune system of the individual to synthesize antibodies, and, if needed, to produce a cell-mediated immune response. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and may be monitored on a patient-by-patient basis.
  • suitable dosage ranges are readily determinable by one skilled in the art and generally range from about 10 2 to about 10 9 plaque forming units (PFU) or more of virus per patient, more commonly, from about 10 4 to about 10 5 PFU of virus per patient
  • PFU plaque forming units
  • the dosage may also depend, without limitation, on the route of administration, the patient's state of health and weight and the nature of the formulation
  • administration of the vaccines of the present invention will be by procedures well established in the pharmaceutical arts, such as mtranasally, parenterally, intravenously, orally, or topically applied to any mucosal surface such as intranasal, oral, eye or rectal surface
  • more than one route of administration may be employed either simultaneously or sequentially (e g , boosting)
  • live, attenuated viral vaccines are administered mtranasally, orally, parenterally or applied to any mucosal surface (nasal, oral, eye, rectal)
  • the attenuated RSV of the present invention can be combined with viruses of other subgroups or strains to achieve protection against multiple strains of RSV Typically the viruses will be in an admixture and administered simultaneously, but may also be administered separately Due to the phenomenon of cross-protection among certain strains of RSV, immunization with one strain may protect against several different strains of the same or different subgroup
  • the vaccine compositions of the present invention can be administered simultaneously, separately or sequentially with other vaccines such as parainfluenza virus vaccine, as desc ⁇ bed in Clements, et al , J Clm Microbiol 29 1175-1185 (1991 )
  • a multivalent preparation may be employed comprising for example, the attenuated RSV of the present invention (including subgroups A and B), parainfluenza virus type 1 , 2 and 3 and influenza virus types A and B.
  • single or multiple administrations of the vaccine compositions of the present invention may be carried out.
  • multiple administration may be required to elicit sufficient levels of immunity.
  • Administration should begin within the first month of life, and continue at intervals throughout childhood, such as at two months, six months, one year and two years, as necessary to maintain sufficient levels of protection against wild-type RSV disease.
  • adults who are particularly susceptible to repeated or serious RSV infection such as, for example, health care workers, day care workers, elderly and individuals with compromised cardiopulmonary function, may require multiple immunizations to establish and/or maintain protective immune responses.
  • Levels of induced immunity can be monitored by measuring amounts of neutralizing secretory and serum antibodies, and dosages adjusted or vaccinations repeated as necessary to maintain desired levels of protection.
  • viruses of the present invention may be used in diagnostic applications.
  • a method of determining the presence of antibodies specifically reactive with an RSV of the present invention comprises the steps of contacting a sample with the RSV to produce complexes comprising the virus and any antibodies present in the sample specifically reactive therewith, and determining production of the complexes.
  • a similar method of determining the presence of RSV is provided wherein the sample is contacted with an antibody specifically reactive with an RSV to produce complexes comprising the antibody and the virus present in the sample that is specifically reactive with the antibody, and determining production of the complexes.
  • the virus of the present invention are characterized by a level of attenuation such that they do not produce RSV disease in a host immunized therewith, evoke a protective immune response and do not lead to immunopotentiation or exacerbated disease. They lack transmissibility, are genetically stable and exhibit cold-adapted and temperature sensitive markers. They are immunogenically protective and induce protective levels of humoral and cell mediated immunity. In particular, a balanced anti-RSV F lgG1/lgG2a response is seen in hosts immunized with attenuated viruses of the present invention. They can be administered by the natural route i.e., intranasally.
  • the RSV of the present invention may be tested in in vitro and in vivo models to demonstrate these characteristics
  • a variety of animal models have been described and are summarized in Meignier et al , eds , Animal Models of Respiratory Syncytial Virus Infection, Me ⁇ eux Foundation Publication (1991 )
  • a cotton rat model of RSV infection is described in U S 4,800,078 and Prince et al , Virus Res 3 193-206 (1985), and is believed to be predictive of attenuation and efficacy in humans
  • a primate model of RSV infection using a chimpanzee is also useful in examining attenuation and protection and is desc ⁇ bed in detail in Richardson et al , J Med Virol 3 91-100 (1978) and Wright et al , Infect Immun 37 397-400 (1982) SPECIFIC EXAMPLES
  • Example 1 describes the production and characterization of the virus of the present invention including the passage status and procedures for developing the strains
  • Example 2 describes temperature sensitivity studies, wherein the deposited strains were found to have the ts phenotype (see Tables 2 and 3)
  • the results are shown in Figures 1A-3 and Tables 4-7 and show that the intranasal immunization with the attenuated RSV produces a substantial anti-F antibody response
  • a balanced anti-RSV F lgG1/lgG2a response demonstrating the induction of both Th-1 and Th-2 type responses was achieved
  • the generation of lgG2A antibodies in the murine model is indicative of a Th1-type immune response
  • the level of virus-neutralizing antibodies was also determined, by plaque reduction assays
  • a study was performed to evaluate the effect of boosting by a route of administration that differs from the initial inoculation route of administration In particular, mice were inoculated intrana
  • RSV lines Specific Example 4A. sets forth the comparison of the sequences for wild type RSV (referred to herein as WRSV) and two attenuated lines, Ca19V and 19H (see Table 10).
  • Specific Example 4B. illustrates the comparison of the sequences for the line 19 progenitor strain (referred to herein as wt 19) and the same two lines, lines Ca19V and 19H (see Table 11).
  • the F genes of the two attenuated lines both differ from the WRSV as well as the wt 19, but have 66 nucleotides and 11 amino acids in common. With respect to the amino acid differences between the attenuated lines and the wt 19, none of the amino acid differences are shared by the two attenuated lines.
  • Specific Example 5 describes the plaque purification of the attenuated RSV lines of the present invention. Table 12 illustrates the temperature sensitivity of the plaque purified lines.
  • Example 6 describes RSV therapeutic protocols for administering the pharmaceutical compositions of the present invention to humans.
  • MRC5 cells were purchased in tubes from Bio Whittaker Laboratories. Media was removed. 1.2 ml EMEM + 5% FBS was added to each tube. 0.3 ml virus was added to each of 4 tubes. Tubes were incubated at 35°C and observed for development of cytopathic effect (CPE). Tubes were frozen at -70°C. Virus was harvested and passed to fresh cells. 2. Ca19S
  • Virus was diluted 1 :5 in 1 x 199 + 5% FBS. 1 ml of virus was added to each of 2 flasks (1 flask is used as a control) Virus was adsorbed on a rocker at room temperature for 2 hours. 4 ml of 1 x 100 +5% FBS was added to each flask. Flasks were incubated at the appropriate temperature until 80% CPE was observed. Flasks were frozen at -70°C. Virus was harvested and passed to fresh cells. Virus was passed 16 times at 25°C; 6 times at 20°C; and, 2 times at 33°C. 4. Ca48V
  • Line 19H is ts when assayed in both MRC cells and under agarose in Vero cells.
  • Line Ca19S has a 5 log reduction in growth at 39°C versus that at 33°C.
  • Line CRIa is also ts in MRC cells. WRSV grows as well at 39°C as it does at 33°C.
  • Lines Ca19S and Ca48V are both ts when assayed in Vero cells using the second antibody technique and under agarose.
  • Line Ca19S has a 5 log reduction in growth at 39°C versus that at 33°C and line Ca48V has a 3 log reduction in growth at the non-permissive temperature.
  • the following tables further set forth the results of the temperature sensitivity study.
  • TCID S0 in MRC5 Cells Virus to be titrated was diluted 10 "1 in EMEM + 5% FBS. Confluent MRC5 tubes were used, for each dilution and for each temperature (total of 96 tubes for 3 temperatures). 1 ml of a viral dilution was added to each tube. Tubes were incubated at 33°C, 37°C or 38°C, and 39°C. Tubes were read daily to day 14 for CPE. TCID 50 was calculated using the method of Reed and Muench.
  • HEP2 Cells or VERO cells
  • HEP2 Cells or VERO cells
  • Virus was diluted in 1 x 199 + 5% FBS 10 ⁇ 1 to 10 "7 .
  • Cells were inoculated in triplicate, 0.5 ml/well and allowed to adsorb at 35°C for 2 hours. Inoculum was then removed.
  • the cells were overlaid with 2 ml of the 1 :1 mixture of 2 x EMEM with 6% FBS and the 4% Methyl Cellulose (4 gm Methyl Cellulose and 100 ml Type I Deionized Water, autoclaved to sterilize; final concentration 2% Methyl Cellulose) and incubated at 35°C with 5% C0 2 for 7 days. Overlay media was then discarded. The cells were fixed with cold 80% methanol at -70°C for 1 hour. The methanol was then removed and the plates were frozen at -70°C. Plates were allowed to thaw at room temperature.
  • Anti-F antibody titer was determined as follows: immunoaffinity purified RSV-F antigen was coated on wells of Nunc-immuno Maxi Sorp flat bottom microtiter plates, by incubating antigen overnight at room temperature in 0.05M Carbonate - Bicarbonate buffer, pH 9.6. Wells were blocked for non-specific binding by adding 0.1% BSA in PBS for 30 min. at room temperature, followed by two washes in a washing buffer of 0.1% BSA in PBS + 0.1 % Tween 20. Mice sera was diluted in two or four-fold serial dilutions, and added to wells.
  • the total IgG assay used F(ab') 2 . goa anti-mouse IgG (H+L specific)-HRP from Jackson Immuno Research Laboratory Inc., Baltimore.
  • the IgG 1 assay used sheep anti-mouse lgG1-HRP from Serotec.
  • the IgG 2a and IgA assays used goat anti-mouse IgG 2a-HRP and goat-anti-mouse IgA- HRP respectively, from Caltag Laboratories, San Francisco.
  • Line 19H elicited levels of anti-F IgG antibodies that were equivalent to those induced by the A2 mouse adapted virus (see Figures 1A, 1B and 1C). Sera from animals that received two doses of line 19H had a balanced anti-RSV F IgJlgG ⁇ response. As outlined in the table below, the sera of animals that were immunized with two doses of 19H had RSV-specific neutralizing antibodies that were comparable to those obtained following inoculation with live mouse adapted RSV. Thus, line 19H was immunogenic in the mouse model.
  • Viruses used were not plaque purified. b Reported as reciprocal of last dilution which reduced viral plaques by 60%. c Reported as reciprocal of last dilution which had an OD of 0.1 or greater after adjustment for normal serum.
  • Pathogen-free BALB/c mice (approximately 8 weeks old) were immunized intranasally with either 2 X 10 4 TCID 50 of the lines or their respective progenitor viruses (designated wt 19 and WRSV), 2.0 X 10 4 pfu of mouse-adapted virus (designated live virus), or medium +5% FBS +5% glycerol (designated placebo). Animals were bled 4 weeks after the primary inoculation and boosted at 4 weeks with an equivalent dose of the vaccine formulation. Serum samples were also taken 4 weeks after the booster dose.
  • mice were anesthetized IP with 200 ⁇ ketoset diluted 1 :10 in PBS and inoculated IN with 50 ⁇ undiluted virus, WRSV (VERO 35°, Titer 2.00 x 10 6 Pfu's/ml in VERO cells), CRIa, CaBCV, 19H and Ca48V.
  • Mice were boosted with 100 ⁇ l virus via footpad injection. Approximately three weeks later, mice were boosted with 200 ⁇ half virus and half complete Freund's adjuvant intramuscularly. Approximately ten days later, mice were bled for serum. Neutralization titers were done in VERO cells in the presence of complement. The following table sets forth the results of the study. Table 8
  • CTL Spleens from two BALB/c mice from each group that were immunized with either live mouse adapted A2 virus, line 19H or placebo, (see A. above, Immunogenicity of RSV Lines (Study 1 )), were removed three weeks after the booster dose.
  • Single cell suspensions were prepared and incubated at 2.5 x 10 7 cells in RPM1 1640 plus 10% FBS.
  • Gamma-irradiated (3,000 rads) syngeneic spleen cells were infected with RSV at an MOI of 1 for 2 h. The cells were washed twice to remove free virus and 2.5 x 10 7 spleen cells in a final volume of 10 ml of complete medium. CTL activity was tested 5-6 days following re-stimulation.
  • Cytoxicity assay On the date of the assay, effector cells were washed twice with fresh medium and viable cell counts were determined by the Trypan blue dye exclusion method. BC cells (2 x 10 6 cells), a BALB/c fibroblast cell line, as well as BCH4 cells (2 x 10 6 cells), a BALB/c fibroblast T cell line persistently infected with RSV, were pulsed with 200 ⁇ Ci of Sodium 5 chromate (Dupont) for 90 min. The targets were washed three times with medium to remove free 51 chromium. Viable cell counts of the target cells were determined and target cell suspensions were prepared at 2 x 10 4 cells/mL.
  • Washed responder T-cells (in 100 ⁇ ) were incubated with 2 x 10 3 target cells (in 100 ⁇ ) at various Effector:Target cell ratios in triplicate in 96-wel! V-bottomed tissue-culture plates for 4 h at 37 °C with 6% C0 2 .
  • Spontaneous and total release of 51 chromium were determined by incubating target cells with either medium or 2.5% Triton-X100 in the absence of responder lymphocytes. Six replicates of each were prepared. After 4 h plates were centrifuged at 200 x g for 2 min and 100 ⁇ l supernatant was removed from each well to determine the amount of 51 chromium released.
  • CTL Generation of CTL.
  • Spleens from two BALB/c mice from each group that were immunized with either the mutants or their respective progenitor viruses, live mouse adapted virus or medium (placebo) were removed three weeks after the booster dose.
  • Single cell suspensions were prepared and incubated at 2.5 X 10 7 cells in RPMI 1640 plus 10% FBS.
  • Gamma-irradiated (3,000 rads) syngeneic spleen cells were infected with RSV at an MOI of 1 for 2h. The cells were washed twice to remove free virus and 2.5 X 10 7 spleen cells in a final volume of 10 mL of complete medium.
  • CTL activity was tested 5-6 days following re-stimulation. Cytotoxicity assay.
  • effector cells were washed twice with fresh medium and were resuspended in 2 mL of complete medium.
  • BC cells (2 X 10 6 cells), a BALB/c fibroblast cell line, as well as BCH4 cells (2 X 10 6 cells), a BALB/c fibroblast T cell line persistently infected with RSV, were pulsed with 200 ⁇ Ci of Sodium 51 chromate (Dupont) for 90 min.
  • the targets were washed three times with medium to remove free 51 chromium. Viable cell counts of the target cells were determined and target cell suspensions were pared at 2 X 10 4 cells/mL.
  • Washed responder T-cells at various dilutions were incubated with 2 X 10 3 target cells (in 100 ⁇ l) in triplicate in 96-well V bottomed tissue-culture plates for 4 h at 37°C with 6% C0 2 .
  • Spontaneous and total release of 51 chromium were determined by incubating target cells with either medium of 2.5% Triton -X100 in the absence of responder lymphocytes.
  • Six replicates of each were prepared. After 4 h plates were centrifuged at 200 X g for 2 min. and 100 ⁇ l of supernatant was removed from each well to determine the amount of 5 , chromium related.
  • chromium release was calculated as (Experimental Release-Spontaneous Release)/ (Total release - Spontaneous release) X 100.
  • the spontaneous release of 51 chromium in the absence of effector cells was found to be between 10-15% in these studies.
  • the lysis in cultures is directly proportional to the number of effector cells present in the culture, which in turn is proportional to the number of CTL precursors activated in vivo by that particular immunogen. The results of this cytotoxicity study are shown in Figure 5.
  • lysis of BC (open symbols) and BCH4 cells (filled symbols) by CTL generated from BALB/c mice immunized with either placebo, 19HL 3PI, 19H 3PI, 19H MD, wt 19, CRIa MD or WRSV, or live mouse adapted virus is shown.
  • RSV infected lysed BCH4 cells
  • mice that were immunized with either the plaque purified viruses, progenitor viruses or medium alone were challenged with 10 6 pfu of RSV A2 immediately after the 8 week bleed. Lungs were harvested four days after virus challenge and virus titers in lung homogenates were determined by the plaque assay. As shown in the table below, mice immunized with the viruses of the present invention were protected against live virus challenge. The protective ability was comparable to that observed with mice that were inoculated with live mouse adapted virus.
  • the F gene of the wild type (WRSV), line Ca19V and line 19H were sequenced using polymerase chain reaction (PCR)
  • the F gene is composed of 1899 nucleotides, 13 of which are non- coding at the 3' end
  • Both viruses were grown in Vero cells to isolate the RNA for sequencing
  • Comparison of the F genes of line Ca19V and line 19H revealed 73 nucleotide and 15 ammo acid differences
  • Comparison of the F genes of line 19H and WRSV revealed 72 nucleotide and 13 ammo acid differences
  • the F genes of the two attenuated line 19 viruses have 66 nucleotides and 11 ammo acids in common but differ from that of WRSV (ammo acid positions 66, 76, 79, 97,
  • the Gamier Osguthorpe Robson (GOR) predicted F protein structures of the two line 19 viruses are nearly identical, however, the GOR F protein structure of WRSV differs at ammo acid 97, 1 19, 191 , 357 and 522 from both attenuated viruses and differs at ammo acid 294 only from line Ca19V
  • Am o acid 97 (threonme in both line 19's and methionme in WRSV) predicts a turn in the attenuated line 19 viruses not present in WRSV.
  • Amino acid 119 phenylalanine in both line 19's and leucine in WRSV predicts an additional turn in the attenuated line 19 viruses not present in WRSV.
  • Amino acid 191 (lysine in the attenuated lines 19 viruses and arginine in WRSV) predicts the formation of an alpha helix in the attenuated line 19 viruses while WRSV continues a beta sheet fold and then turns.
  • Amino acid 357 predicts the formation of an alpha helix in WRSV not present in either attenuated line
  • amino acid 19 virus and amino acid 522 predicts a turn in WRSV not present in either attenuated line 19 viruses.
  • Amino acid 294 predicts the formation of a beta sheet in WRSV and 19H not present in Ca19V. It is interesting that, although there are 6 amino acid differences between the two attenuated line 19 viruses, the 2 attenuated viruses have the same predicted protein fold whereas the WRSV fold is quite different. Thus, amino acids 97, 119, 191 , 357 and 522 are good candidates for attenuating lesions in the F protein.
  • Amino acid 235 is basic arginine to uncharged glycine; amino acid 294 is negatively charged glutamic acid to positively charged lysine; amino acid 368 is negatively charged aspartic acid to hydrophobic valine; and amino acid 398 is uncharged serine to hydrophobic leucine.
  • nucleotide 1249 (which does not code for an amino acid change) is C in wt 19 and A in both Ca19V and 19H.
  • Line 19HL 3PI was passed 72 times in MRC5 cells, then plaque purified three times in Vero cells.
  • Line 19H 3PI was passed 70 times in MRC5 cells, then plaque purified three times in Vero cells and passed 2 times in MRC5 cells.
  • 19H MD was passed 92 times in MRC5 cells, then purified by limiting dilution three times.
  • Line CRIa MD was passed 28 times at 25°C and 1 time at 33°C then purified by limiting dilution five times.
  • the following table sets forth the titers of the strains at 33°C and 39°C, illustrating temperature sensitivity.
  • the attenuated virus of the present invention is administered to human subjects according to well established human RSV protocols, for example, those described in Wright et al., Infect. Immun. 37:397-400 (1982); Kim et al., Pediatrics 52:56-63 (1973) and Wright et al., J. Pediatr. 88:931-936 (1976). Briefly, adults or children are inoculated intranasally via droplet with 10 2 to 10 9 PFU, preferably 10 4 to 10 5 PFU, of attenuated virus per ml in a volume of 0.5 ml. Antibody response is evaluated by complement fixation, plaque neutralization, and/or enzyme-linked immunosorbent assay. Individuals are monitored for signs and symptoms of upper respiratory illness. Subsequent immunizations are administered periodically to the individuals as necessary to maintain sufficient levels of protective immunity.

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Abstract

On décrit des virus respiratoires syncytiaux (RSV) atténués et notamment de tels virus sensibles à la température. On peut utiliser les virus de la présente invention dans des compositions pharmaceutiques telles que des vaccins. On décrit également des procédés de préparation et d'utilisation de telles compositions pharmaceutiques.
PCT/US1997/005588 1996-04-04 1997-04-03 Virus respiratoire syncytial attenue WO1997038138A1 (fr)

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EP97920111A EP0917592A4 (fr) 1996-04-04 1997-04-03 Virus respiratoire syncytial attenue
AU24385/97A AU2438597A (en) 1996-04-04 1997-04-03 Attenuated respiratory syncytial virus
US08/882,358 US6077514A (en) 1996-04-04 1997-06-25 Attenuated respiratory syncytial virus
CA002292448A CA2292448A1 (fr) 1997-04-03 1998-04-02 Virus syncytial respiratoire attenue
EP98914473A EP0975363A4 (fr) 1997-04-03 1998-04-02 Virus syncytial respiratoire attenue

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Cited By (4)

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WO1999015672A1 (fr) * 1997-09-19 1999-04-01 American Cyanamid Company Virus syncytiaux respiratoires attenues
WO2000012684A3 (fr) * 1998-09-01 2000-07-27 Univ St Louis Virus parainfluenza humain de type 2 (hpiv-2) thermosensible et adapte au froid; vaccins a base d'un tel virus
EP0975363A4 (fr) * 1997-04-03 2002-04-17 Univ Michigan Virus syncytial respiratoire attenue
WO2010053883A1 (fr) * 2008-11-05 2010-05-14 Merck Sharp & Dohme Corp. Virus syncytial respiratoire vivant atténué

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J.E.CROWE ET AL.: "Satisfactorily Attenuated and Protective Mutants Derived from a Partially Attenuated Cold-Passaged Respiratory Syncytial Virus Mutant by Introduction of Additional Attenuating Mutations During Chemical Mutagenesis", VACCINE, vol. 12, no. 8, June 1994 (1994-06-01), pages 691 - 699, XP002910145 *
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0975363A4 (fr) * 1997-04-03 2002-04-17 Univ Michigan Virus syncytial respiratoire attenue
WO1999015672A1 (fr) * 1997-09-19 1999-04-01 American Cyanamid Company Virus syncytiaux respiratoires attenues
WO2000012684A3 (fr) * 1998-09-01 2000-07-27 Univ St Louis Virus parainfluenza humain de type 2 (hpiv-2) thermosensible et adapte au froid; vaccins a base d'un tel virus
US6946136B2 (en) 1998-09-01 2005-09-20 St. Louis University Temperature-sensitive and cold-adapted human parainfluenza virus type 2 (HPIV-2) and vaccines based on such virus
WO2010053883A1 (fr) * 2008-11-05 2010-05-14 Merck Sharp & Dohme Corp. Virus syncytial respiratoire vivant atténué
JP2011250798A (ja) * 2008-11-05 2011-12-15 Merck Sharp & Dohme Corp 生弱毒化呼吸器合胞体ウイルス
JP2012507302A (ja) * 2008-11-05 2012-03-29 メルク・シャープ・エンド・ドーム・コーポレイション 生弱毒化呼吸器合胞体ウイルス
US9011876B2 (en) 2008-11-05 2015-04-21 Merck Sharp & Dohme Corp. Live, attenuated respiratory syncytial virus

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