CA2250883A1 - Attenuated respiratory syncytial virus - Google Patents

Abstract

Attenuated respiratory syncytial viruses (RSV) and in particular temperature sensitive RSV are provided. The viruses of the present invention may be used in pharmaceutical compositions such as vaccines. Methods of making and using such pharmaceutical compositions are also provided.

Description

CA 022~0883 1998-10-02 W O 97/38138 PCT~US97/05588 ATTENUATED RESPIRATORY SYNCYTIAL VIRUS
FIELD OF THE INVENTION
The present invention relates generally to attenuated respiratory syncytial viruses and, more particularly, to live attenuated respiratory syncytial virus vaccines 5 and methods of protecting against disease caused by infection with respiratory syncytial virus.
BACKGROUND OF THE INVENTION
Respiratory syncytiai virus (RSV), a member of the paramyxoviridae family, is the leading cause of viral pneumonia and bronchitis in infants and young children 10 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). Virtually all children are infected by two years of age.
Most infections are symptomatic and are generally confined to mild upper respiratory 15 tract disease. A decrease in severity of disease is associated with two or more prior infections andl 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. 134:211-217 (1976); Henderson, F.W. et al., N.
Eng. J. Med. 300:530-534 (1979); Glezen, W.P. et al., J. Ped. 98:706-715 (1981);20 Glezen, W.P. et al., Am. J. Dis. Child. 140:543-546 (1986); Kasel, J.A. et al., Vir.
Immunol. 1:199-205 (1987/88); Hall, C.B. et al., J. Inf. Dis. 163:693-698 (1991)).
Two major subgroups of RSV have been identified, A and B, as well as antigenic variants within each subgroup (Anderson, L.J. et al., J. Inf. Dis. 151 :626-633 (1985); Mufson, M.A. et al., J. Gen. Virol. 66:2111-2124 (1985)). Multiple 25 variants of each subgroup have been found to co-circulate in epidemics which occur annually during late fall, winter, and spring months (Anderson, L.J. et al., J. Inf. Dis.
163:687-692 (1991)). There is evidence that children infected with one of the two major RSV subgroups may be protected against reinfection by the homologous subgroup (Mufson, M.A. et al., J. Clin. Microbiol. 26:1595-1597 (1987)). This, along 30 with evidence that protective immunity will accumulate following repeated infections, suggests that it is feasible to develop an RSV vaccination regiment for infants and young children which would provide sufficient immunity to protect against disease and death.
A previous attempt to vaccinate young children against RSV employed a 35 parenterally administered formalin-inactivated RSV vaccine. Unfortunately, administration of this vaccine in several field trials was shown to be associated with CA 022',0883 1998-10-02 W O97/38138 PCT~US97/05588 the development of a significantly exacerbated illness following subsequent natural infection with RSV (Kapikian, A.Z. et al., Am. J. Epidemiol. 89:405-421 (1968); Kim, H.W. et al., Am. J. Epidemiol. 89:422~34 (1g69); Fulginiti, V.A. et al., Am. J.
Epidemiol. 89:435-448 (1969); Chin, J. et al., Am. J. Epidemiol. 89:44g463 (1969)).
Following the lack of success with the formalin-inactivated RSV vaccine, emphasis was placed on the development of live attenuated vaccines. For example,cold adaptation, a process by which virus is adapted to grow at temperatures colder than those at which it normally optimally grows, has been used to develop temperature sensitive, attenuated RSV mutants for consideration as vaccines (Ma~ssab, H.F. et al., Vaccine 3:355-369 (1985)). Unlike chemical mutagenesis inwhich the genetic lesions are usually single, this method generally results in the accumulation of multiple genetic lesions. These multiple lesions would help to confer phenotypic stability by reducing the probability that reversion of any one lesion will result in reversion to virulence. Stepwise cold adaptation, wherein the virus is passaged multiple times at progressively lower temperatures, has been used to successfully develop several influenza vaccine candidates currently in clinical trials (M~ss~h, H.F. etal., Viral VaccinesWiley-Liss, Inc. (1990); Obrosova-Serova, N.P.
et al., Vaccine 8:57-60 (1990); Edwards, K.M. et al., J Inf. Dis. 163:740-745 (1991)).
These mutants, which bear attenuating mutations in at least four different genes, appear to be attenuated, immunogenic, and phenotypically stable.
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 48:745-755 (1971); Maass~h, H.F. et al., Vaccine 3:355-369 (1985)). However, it is of note that administration of these live RSV vaccine candidates was never associated with disease enhancement following natural infection.
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 andcellular immunity. Furthermore, these vaccines are cost-effective and can be rapidly produced and updated in the event of antigenic changes.
It would thus be desirable to provide avirulent (attenuated), immunoprotective and genetically-stable live attenuated RSV strains. It would further be desirable to provide a vaccine comprising such attenuated strains. It would further be desirable CA 022~0883 1998-10-02 to provide methods of making and using said RSV vaccine to protect against disease caused by infection with RSV.
SUMMARY OF THE INVENTION
Attenuated RSV strains which exhibit the cold-adapted (ca) and/or 5 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:

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.' ~ 5 ~ I -CA 022~0883 1998-10-02 W O 97138138 PCT~US97/05588 -6- _ 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 5 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 10 temperature sensitive RSV. Methods of making and using such attenuated RSV in a pharmaceutical composition e.g., a vaccine, are also provided.
Additional objects, advantages, and features of the present invention will become apparent from the following description, drawings and appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
The various advantages of the present invention will become apparent to one skilled in the art by reading the following specification and subjoined claims and by referencing the following drawings in which:
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 1B 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 IgG2a 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; and CA 022~0883 1998-10-02 Figure 5 is a graph showing CTL activity of mice immunized with immunogenic compositions of aspects of the present invention.
DETAILED DESCRIPTION OF TH~ INVENTION
Attenuated RSV including cold-adapted and/ortemperature sensitive RSV are 5 provided and in particular, eleven subgroup A RSV lines, have been deposited with the ATCC and are described in detail herein. As used herein, the term "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). Four of the deposited lines have also been plaque purified (19HL 3PI, 19H 3PI, 19H MD and CRla MD). The attenuated 15 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 withoutcausing unacceptable symptoms of severe respiratory disease. The immune response which is achieved in the subject by the method of an embodiment of the 20 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 25 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 30 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 35 changes in non-coding regions that do not alter the phenotype of the polypeptide CA 022~0883 1998-10-02 W O97/38138 PCTrU$97105588 produced therefrom, when compared to the nucleic acid molecules of the RSV
described herein.
Also encompassed by the present invention are the nucleic acid molecules comprising noncoding sequences of the RSV of the present invention. These non-5 coding regions are to include 5' noncoding regions, 3' noncoding regions, intergenicsequences, and other noncoding regions of the viral genome. These include, but are not limited to, transcriptional, translational and other regulatory regions. These 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 presentinvention. As will be appreciated by those skilled in the art, multiple factors are considered in determining the stringency of h~,bricli~lion including species of nucleic acid, length of nucleic acid probe, Tm (melting temperature), temperature of hybridization and washes, salt concentration in the hybridization and wash buffers, aqueous or ro""al"ide 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, at 65~C in aqueous solution or 42~C
in formamide, followed by washing with 0.1 x SSC, at 65~C in aqueous solution.
(Other experimental conditions for controlling stringency are described in Maniatis, T. et al., Molecu/ar Cloning: a Laboratory Manual, Cold Springs Harbor Laboratory, Cold Springs, N.Y. (1982) at pages 387-389 and Sambrook, J. et al., Molecular Cloning: a Laboratory Manual, Second Edition, Volume 2, Cold Springs Harbor Laboratory, Cold Springs, N.Y., at pages 8.46-8.47 (1989)).
The nucleic acid molecules of the present invention may be operatively-linked to a promoter of RNA transcription, as well as other regulatory sequences. As used herein, the term "operatively-linked" means positioned in such a manner that thepromoter and other regulatory sequences will direct the transcription off of thenucleic acid molecule. An example of a promoter is the T7 promoter. Vectors which contain both a promoter and a cloning site to which an inserted piece of nucleic acid is operatively-linked to the promoter, are well known in the art. Preferably, these vectors are capable of transcribing nucleic acid in vitro and in vivo.
Purified polypeptides isolated from the RSV described herein or from cells infected with these same virus, are also encompassed by the present invention. The CA 022~0883 1998-10-02 W O97/38138 PCTrUS97/05588 g polypeptides (or fragments thereof) may be of varying length, and preferably will be capable of exhibiting immunological activity.
Methods for producing polypeptides of the present invention are also within the scope of the present invention. In one embodiment, RSV polypeptides can be 5 isolated in substantially pure form from RSV or cultures of cells infected with RSV.
In an alternative embodiment, RSV polypeptides can be isolated from a recombinant system or are vector-engineered to produce these polypeptides. In yet another embodiment, 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.
Although the deposited RSV of the present invention are subgroup A virus, 15 it will be appreciated by those skilled in the art that 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.
Pharmaceutical compositions comprising any of the RSV described herein or 20 polypeptides, either alone or in combination, and a pharmaceutically acceptable carrier, are also provided by the present invention. As used herein, the phrase "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 25 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 30 compositions may conveniently be presented in unit dosage form and may be prepared by any method known in the art.
~ In one embodiment of the present invention, the pharmaceutical composition is intended for use as a vaccine. In such embodiment, a virus may be mixed with cryoprotective additives orstabilizers such as proteins (e.g., albumin, gelatin), sugars 35 (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate), saline, or CA 022~0883 1998-10-02 WO97/38138 PCT~US97/05588 other protective agents. This mixture may then be desiccated or Iyophilized for transport and storage, and reconstituted prior to administration. Lyophilized virus will typically be maintained at about 4~C and when ready for use, reconstituted in a stabilizing solution, with or without adjuvant. In yet another embodiment of thepresent invention, the virus may be inactivated and may be mixed with an adjuvant, saline and a detergent such as phosphate Tween buffer. For further methods of vaccine preparation, see Duffy, J.l., Vaccine Preparation Techniques, Noyes DataCorporation, (1980).
Immunogenicity can be significantly improved if the virus is co-administered 10 with an immunostimulatory agent or adjuvant. Adjuvants enhance immunogenicitybut 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, 15 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-Chol, DDA, and otheradjuvants 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 RSVinfection. Thus, this invention further provides methods of immunizing a subjectagainst disease caused by RSV infection, comprising administering to the subjectan immunoeffective amount of a pharmaceutical composition of the invention. This25 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 30 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-patientbasis. However, suitable dosage ranges are readily determinable by one skilled in 35 the art and generally range from about 102 to about 109 plaque forming units (PFU) CA 022~0883 l998-l0-02 or more of virus per patient, more commonly, from about 104 to about 105 PFU of virus per patient. 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 .
It will be appreciated that administration of the vaccines of the present invention will be by procedures well established in the pharmaceutical arts, such as intranasally, parenterally, intravenously, orally, or topicaliy applied to any mucosal surface such as intranasal, oral, eye or rectal surface. Moreover, as described in more detail in Specific Example 3E., more than one route of ad",;~ ,l,d~ion may be employed either simultaneously or sequentially (e.g., boosting). In a preferred embodiment of the present invention, live, attenuated viral vaccines are administered intranasally, orally, parenterally or applied to any mucosal surface (nasal, oral, eye, rectal). Inactivated whole virus vaccine is preferably adr"i"i;,lered parenterally or to any mucosal surface.
Upon inoculation with an attenuated RSV pharmaceutical composition as described herein, the immune system of the host responds to the vaccine by producing antibodies, both secretory and serum, specific for RSV proteins. As a result of the vaccination, the host becomes at least partially or completely immune to RSV infection, or resistant to developing moderate or severe RSV infection, particularly of the lower respiratory tract.
It will be appreciated that 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 thephenomenon of cross-protection among certain strains of RSV, immunization with one strain may protect against several different strains of the same or different subgroup.
In some instances it may be desirable to combine the attenuated RSV
vaccines of the present invention with vaccines which induce protective responses to other agents, particularly other childhood viruses. For example, the vaccine compositions of the present invention can be administered simultaneously, separately or sequentially with other vaccines such as parainfluenza virus vaccine, as described in Clements, et al., J. Clin. Microbiol. 29:1 175-1 185 (1991 ). Moreover, a multivalent preparation may be employed comprising for example, the attenuated CA 022~0883 l998-l0-02 W O97/38138 PCT~US97/05588 RSV of the present invention (including subgroups A and B), parainfluenza virus type 1, 2 and 3 and influenza virus types A and B.
It will also be appreciated that single or multiple administrations of the vaccine compositions of the present invention may be carried out. In neonates and infants, 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. Similarly, adults who are particularly susceptible to repeated or serious 10 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 ofinduced immunity can be monitored by measuring amounts of neutralizing secretoryand serum antibodies, and dosages adjusted or vaccinations repeated as necessary15 to maintain desired levels of protection.
Those skilled in the art will further appreciate that the viruses of the presentinvention may be used in diagnostic applications. For example, a method of determining the presence of antibodies specifically reactive with an RSV of the present invention is provided. Such a method comprises the steps of contacting asample with the RSV to produce complexes co~n~rising 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 samplethat 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 IgG1/lgG2a response is seen in hosts immunizedwith 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 CA 022~0883 1998-10-02 WO 97/38138 PCT~US97/05588 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, Merieux Foundation Publication (1991). A cotton rat model of RSV infection is described in U.S. 4,800,078 and 5 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 described in detail in Richardson et al., J. Med. Virol. 3:91-100 (1978) and Wright et al., Infect.
Immun. 37:397400 (1982).
SPECIFIC EXAMPLES
The following Specific Examples illustrate practice of the invention. These examples are for illustrative purposes only and are not intended in any way to limit the scope of the claimed invention.
Specific Example 1 describes the production and characterization of the virus of the present invention including the passage status and procedures for developing the strains.
Specific Example 2 describes temperature sensitivity studies, wherein the deposited strains were found to have the ts phenotype (see Tables 2 and 3).
Specific Example 3 describes immunogenicity studies wherein mice were immunized by administering an RSV strain of the present invention. As described in Specific Examples 3A.-3D., mice were immunized and sera examined four weeks after boosting for anti-F, total IgG antibodies, IgG1 and IgG2a antibodies. 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. In particular, a balanced anti-RSV F IgG1/lgG2a response demonstratingthe induction of both Th-1 and Th-2 type responses was achieved. The generation of IgG2A 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.
In Specific Example 3E., a study was performed to evaluate the effect of boosting by a route of admiui~l~ation that differs from the initial inoculation route of ad"linistlation. In particular, mice were inoculated intranasally and boosted in the footpad and intramuscularly. Neutralization titer data is set forth in Table 8.
As described in Specific Examples 3F. and 3G., the generation of RSV-specific cytotoxic T cells (CTL) following immunization was determined and the CA 022~0883 l998-l0-02 W O97/38138 PCTrUS97/05588 results shown in Figures 4 and 5. Immunizing animals with the attenuated RSV of the present invention induced significant levels of CTL activity. In addition, as shown in Table 9, mice immunized with the plaque purified viruses of the present invention were protected against live virus challenge.
Specific Example 4 describes sequence analysis of the F gene of several 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.
Specific Example 6 describes RSV therapeutic protocols for a~ll"inis~ering the pharmaceutical compositions of the present invention to humans.

A. The following sets forth the deposited RSV strains and their titers.
1. 19H
in EMEM 5% FBS 5% Glycerol 1.00 x 108 TClDso in MRC Tubes on Day 14 1.85 x 105 Pfu's/ml in VERO cells ATCC Designation No. VR-2513 2. Ca 1 9S
in EMEM 5% FBS 5% Glycerol 7.00 X 104 Pfu's/ml in VERO cells ATCC Designation No. VR-2512 CA 022~0883 1998-10-02 W O 97t38138 PCTrUS97/05588 3. Ca19V
in 199 5% FBS 5% Glycerol 1.00 x 107 Pfu's/ml in VERO cells ATCC Designation No. VR-2515 4. Ca48V
in 199 5% FBS 5% Glycerol 9.00 X 104 Pfu's/ml in VERO cells ATCC Designation No. VR-2514 5. CaBCV
in 199 5% FBS 5% Glycerol 1.30 x 104 Pfu's/ml in VERO cells ATCC Designation No. VR-2516 6. CRla in 100 5% FBS 5% Glycerol 6.00 x 103 Pfu's/ml in VERO cells ATCC Designation No. VR-2511 7. CaBCL
in 199 5% FBS 5% Glycerol 1.70 x 104 in VERO cells ATCC Designation No. VR-2517 8. 19HL 3PI
in 5% Glycerol 3.2 x 105 TClDsO/ml in MRC5 cells ATCC Designation No.

9. 19H 3PI
in 5% Glycerol 4 x 105 TClDsJml in MRC5 cells ATCC Designation No.

CA 022~0883 1998-10-02 W O97/38138 PCT~US97105588 10. 19H MD
in 5% Glycerol 3.2 x 105 TClD50/ml in MRC5 cells ATCC Designation No.

11. CRla MD
in 5% Glycerol 4 x 105 TClD5Jml in MRC5 cells ATCC Designation No.

B. The following depicts the passage status of exemplary derivative RSV strains 10 of the present invention.
1. Line 19 MRC5 72-35~
2. Line 19 MRC5 24-25~ st 1-33~
3. Line 19 MRC5 10-25~ VERO 16-25~ 6-20~ 3-33~
4. Line 48 MRC 14-25~ st 1-33~ VERO 10-25~ 1-33~
5. CRSV-BC5 CL17 MRC 30-25~
6. Ia-CRSV-5 CL15 MRC 27-25~
7. CRSV-BC13 MRC 19-25~ 1-33~
8. Line 19 MRC5 P-70-35~, VERO P-3-35~, MRL5, P-2-35~ clone 2,3PI
9. Line 19 MRC5, P-92-35~, clone 5-1, purified by minimal limited dilution 20 (MD) 10. Ia-CRSV-5, MRC5, P-38-25~, MRC5, P-1-33~, purified by MD
11. Line 19 HP clone 4-1, MRC5, P-103-33~, purified by MD

C. The following are the procedures for developing the deposited RSV strains of the present invention (see Specific Example 5 for plaque purification).
1. 19H
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 30 harvested and passed to fresh cells.

CA 022~0883 1998-10-02 WO97/38138 PCT~S97/05588 2. Ca 19S
The same steps as in 1. above were performed, except that tubes were passed 10 times at 35~C; 10 times at 30~C; 15 times at 25~C, and 2 times at 33~C.
3. Ca19V
The first 10 passages in MRC5 cells were performed as described in 1. above except that tubes were incubated at 25~C. Media was removed from 3 confluent 25cm2 VERO flasks. 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
The first 14 passages in MRC5 cells were performed as described in 2. above. Passage in VERO cells was performed as in 3. above. Virus was passed 10 times in VERO cells at 25~C and 1 time at 33~C.
5. CaBCV, CRla and CaBCL
Same as 2. above except that 199 + 5% FBS was used.

To screen the cold-adapted and high passage virus for the presence of temperature sensitive (ts) variants, viruses were tested at 39~C,37~C and 33~C by one of two methods of titration: plaque immunoassay or TClDso.
Results 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 CRla 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.

CA 022~0883 l998-l0-02 The following tables further set forth the results of the temperature sensitivity study.
Table 2 TClDso in MRC Cells on Day 14 Virus Lines 33~C 37~C 38~C 39~C
19H 1.00 x 108 3.16 x 105 1gH 4.68 x 106 2.15 x 105 4.68 x 104 Ca19S 3.16 x 107 3.16 x 103 3.16x 102 CRla 4.68x 103 4.68 x 102 3.16x 10' WRSV 3.16 x 104 3.16 x 104 1.00 x 104 Table 3 Pfu's in Vero Cells on Day 7 Virus Lines33~C 37~C 38~C 39~C
Ca19V 3.16 x 10'~ 5.85 x 109 2.54 x 105 pinpoint plaques Ca48V 2.50 x 107 8.00 x 106 6.00 x 104 pinpoint plaques Materials and Methods TClDso in MRC5 Cells. Virus to be titrated was diluted 10-' 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 20 tube. Tubes were incubated at 33~C, 37~C or 38~C, and 39~C. Tubes were read daily to day 14 for CPE. TClDso was calculated using the method of Reed and Muench.
Plaque Immunoassay - Pfu's in HEP2 Cells or Vero Cells. HEP2 Cells (or VERO cells) were grown in a 12 well microtiter plate until semi-confluent and 25 media was removed. Virus was diluted in 1 x 199 + 5% FBS 10-' to 10-7. Cells were inoculated in l,i,u'.c~te, 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 CA 022~0883 1998-10-02 100 ml Type I Deionized Water, autoclaved to sterilize; final concentration 2% Methyl Cellulose) and incubated at 35~C with 5% CO2 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 5 allowed to thaw at room temperature. 1 ml of 5% Blotto media (25 gm Milk and 500 ml PBS) was added to each well and the wells were incubated at 35~C for 30 minutes. 5% Blotto media was then removed and 1 ml of 5% Blotto media with 1/100 dilution of anti-RSV antibody was added. Incubation at 35~C for 30 minutestook place. After incubation, 5% Blotto media with antibody was removed and cells 10 were washed with 5% Blotto media. 1 ml of 5% Blotto media with 1/100 dilution of conjugate antibody was then added and incubation took place at 35~C for 30 minutes. 5% Blotto media with conjugate antibody was removed and cells were washed with PBS. 1 ml of 1:1 mix of peroxide solution substrate (4 chloro-1 napthol) + H2O2 was added and incubation took place at room temperature for 1-5 15 minutes. During this period color development was watched carefully. Cells were then washed with PBS. Plaques were counted and Pfu's recorded.

A. Imm~JnG~nicity of RSV Lines ~Study 1) A study was performed to determine the immunogenicity of line 1 9H.
20 Pathogen-free BALB/c mice (approximately 8 weeks old) were immunized intranasally with either 1.6 X10fi TClDso of 19H, 2.5 X 105 pfu of A2 mouse adapted virus (designated live virus), or 5% glycerol (designated placebo). Animals werebled 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 25 weeks after the booster dose. 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 30 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. After 1 hour incubation at room temperature, plates were washed five times with washing buffer, and horseradish peroxidase labeled conjugate was added at the appropriate optimal dilution in washing buffer. The total IgG assay used F(ab')2, goat 35 anti-mouse IgG (H+L specific)-HRP from Jackson Immuno Research Laboratory Inc., CA 022~0883 1998-10-02 W O97/38138 PCT~US97/05588 Baltimore. The IgG 1 assay used sheep anti-mouse IgG1-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. Following 1 hour incubation at room temperature, the plates were washed five times with washing 5 buffer, and a substrate hydrogen peroxide in the presence of tetramethylbenzidine was added. The color reaction was stopped by adding 2M sulfuric acid. The color was read in a Multiscan Titertek plate reader at an optical density (OD) of 450nm.
The titre was taken as the reciprocal of the last dilution at which the OD was approximately double. This OD must be greater than the negative control of the 10 assay at the starting dilution. The pre-immune serum of each animal was used as the negative control.
Line 1 9H elicited levels of anti-F IgG antibodies that were equivalent to thoseinduced 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 lg,/lgG2a 15 response. As outlined in the table below, the sera of animals that were immunized with two doses of 1 9H had RSV-specific neutralizing antibodies that were comparable to those obtained following inoculation with live mouse adapted RSV.
Thus, line 1gH was immunogenic in the mouse model.
Table 4 20 Serum a"lil~GJy response of BALB/c mice immunized with ts mutant Formulation Neutralization titera~b (lOg2 + s.d,) 4 Week Bleed 8 Week Bleed 19H 8.2 + 0.9 9.5 + 1.2 Live RSV (mouse- 8.5 + 0.8 10.6 + 0.7 adapted A2 virus) Placebo <3.3 + 0 0 <3 3 + 0 0 aNeutralization titer determined by complement-enhanced 60% plaque-reduction assay bEach value represents the reciprocal mean titer of at least 6 animals B. Immunogenicity of RSV Lines (Study 2) A second study was performed to determine the immunogenicity of the following RSV lines: WRSV (Vero 35~1~ titer 2.00 x 106 Pfu's/ml in Vero cells), CRla, CaBCV, 19H, Ca19V and Ca48V. Balb/c/AnNTacfBR three week old male mice CA 022~0883 l998-l0-02 W O97/38138 PCT~US97/05588 from Taconic, 272 Hoover Avenue, Germantown, NY 12526, were used. Mice were anesthetized IP with 200 ~/1 ketoset diluted 1:10 and inoculated IN with 50 ~l undiluted virus, 6 mice/group. Mice were boosted with 50 /11 virus diluted 1:2 in 199 media. Approximately one week later, mice were bled for serum. Neutralization 5 titers were done in VERO cells.
All of the lines tested for neutralization antibodies had titers of at least 1:20.
Line 19H had a titer of 1:80, however, WRSV had a titer of 1:320. The following tables further set forth the results of the immunogenicity study.
Table 5 Neutralization Titers (60% Reduction) without Com;~le.,~entt Antiserum Titers to Virus 19H 1:80 CRla 1 :40 Ca19V 1:20 CaBCV 1 :40 Ca48V 1 :40 WRSV >1 :320 Normal Serum c1:20 tSerum periodate and heat treated (56~
for 1 hour); serum diluted in presence of 2.5% FBS.
Table 6 ELISA Titers*
Antiserum to Titers' Virus 19H 1 :1280 CRla 1 :320 Ca19V 1 :1280 CaBCV 1:2560 Ca48V neg W RSV 1:640 *Antigen - Whole RSV Virus 'Titers corrected for normal serum CA 022~0883 l998-l0-02 W O 97/38138 PCT~US97/05588 C. Immunogenicity Of RSV Lines (Study 3) A third study was performed to determine the immunogenicity of the following RSV lines: Ca19S, 19H, Ca19V, Ca48V and WRSV 223227 and Line 19 MRC5 28 25~ (not deposited). Mice were infected with wild type and the RSV lines with twice 5 boosted procedures, similar to the procedure described above. As shown in the table below, serum neuL~c~ lion titers, carried out in the presence of complement, were reported as the reciprocal of the last dilution which reduced the number of viral plaques by 60%. Also shown in the table below, ELISA titers were reported as thereciprocal of the last dilution which had an OD reading of 0.1 or greater after 10 correction for normal serum. All the lines tested for neutralization antibodies had titers of at least 320 and ELISA titers ranging from 160 to 40,960 after the second boost. Although line Ca19S produced the highest neul,dli~alion titer, all lines were similar in neutralizing ability and reached peak titer after the first boost. Line Ca19V
produced the highest ELISA IgG titer. All RSV attenuated lines were immunogenic.
Table 7 Neutrali~ali~-~ and ELISA Titers of Mice Immunized with Vaccine Lines Virusesa Titers Of Neutralizationb ELISA IgGC
Inoculum Weeksd Weeks Weeks Weeks Weeks Weeks WRSV 5.3 80 640 320 160 2560 80 Line 19 5.7 80 640 1280 160 640 1280 25~

Ca19S 7.5 80 640 640 320 2560 1280 19H 7.3 80 320 320 80 2560 320 Ca19V 6.3 320 640 640 320 5120 40960 Ca48V 7.4 80 320 320 160 5120 10240 aViruses used were not plaque purified.
bReported as reciprocal of last dilution which reduced viral plaques by 60%.
CReported as reciprocal of last dilution which had an OD of 0.1 or greater after30 adjustment for normal serum.
dWeeks after Initial Vaccination.

CA 022C.0883 1998-10-02 D. Immunogenicity Of RSV Lines (Study 4~
A fourth study was performed to determine the immunogenicity of the following plaque purified RSV lines: 19HL 3PI, 19H 3PI, 19H MD and CRla MD.
Pathogen-free BALB/c mice (approximately 8 weeks old) were immunized . intranasally with either 2 X 104 TCID50 of the lines or their respective progenitor viruses (designated wt 19 and WRSV), 2.0 X 104 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 10 4 weeks after the booster dose. All plaque purified lines elicited anti-F IgGantibodies at 4 and 8 weeks (see Figures 2A and 2B, wherein each value represents the mean titer of antisera from 6 animals). At the 8 week time point, the sera of animals that had received 2 doses of the various plaque purified lines had anti-RSV
F IgG antibody titres that were comparable to that observed in the sera of mice that 15 were immunized with the mouse-adapted virus (designated live virus). As shown in Figure 3 (values represent the mean titer of antisera from 6 animals), the sera of animals that were immunized with two doses of the various plaque purified lines had high levels of RSV-specific neutralizing antibodies. Thus, all RSV attenuated lines tested were immunogenic in the mouse model.
20 E. Boostin~ By Alt~r,-dti-Je Route of Adminisl,dlion A study was performed to determine the effect of boosting by a route of administration that differs from the initial inoculation route of administration. Taconic Balblc/AnNTacfBR 3 week old male mice were anesthetized IP with 200 ~I ketoset diluted 1:10 in PBS and inoculated IN with 50~1 undiluted virus, WRSV (VERO 35~,25 Titer 2.00 x 1 o6 Pfu's/ml in VERO cells), CRla, CaBCV, 19H and Ca48V. Mice were boosted with 100,ul virus via footpad injection. Approximately three weeks later, mice were boosted with 200 ~I 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. The30 following table sets forth the results of the study.

CA 022~0883 1998-10-02 W O 97/38138 PCTrUS97/05588 Table 8 Neutralization Data in Presence of Complement (60% Reduction) Antiserum to: Titer with Complement+
Normal Serum CaBCV 1: 160 Ca48V ~1 :640 CRla >1 :640 WRSV >1 :640 10 +Titer reported as last dilution with 60% reduction over positive controls with complement average positive control = 100.33 colonies/well.
F. Cytotoxicity Study Generation of 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 15 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 107 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 2 h. The cells were washed twiceto remove free virus and 2.5 x 107 spleen cells in a final volume of 10 ml of complete 20 medium. CTL activity was tested 5-6 days following re-stimulation.
Cytoxicify 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 106 cells), a BALB/c fibroblast cell line, as well as BCH4 cells (2 x 106 cells), a BALB/c fibroblast T cell line persistently infected with 25 RSV, were pulsed with 200 IJCi of Sodium S1chromate (Dupont) for 90 min. The targets were washed three times with medium to remove free 5'chromium. Viable cell counts of the target cells were determined and target cell suspensions wereprepared at 2 x 104 cells/mL. Washed responder T-cells (in 100 ~I) were incubated with 2 x 103 target cells (in 100 IJI) at various Effector:Target cell ratios in triplicate 30 in 96-well V-bottomed tissue-culture plates for 4 h at 37~C with 6% CO2.
Spontaneous and total release of 5'chromium were determined by incubating targetcells with either medium or 2.5% Triton-X100 in the absence of responder Iymphocytes. Six replicates of each were prepared. After 4 h plates were centrifuged at 200 x 9 for 2 min and 100 ~I supernatant was removed from each well 35 to determine the amount of 51chromium released. Percentage specific 51chromium release was calculated as (Experimental Release - Spontaneous Release) (Total CA 022~0883 1998-10-02 Release - Spontaneous Release) x 100. The Spontaneous Release of S'chromium in the absence of effector cells was found to be between 10-15% in these studies.
Figure 4 shows the results of the study. In Figure 4, Iysis of BC (filled symbols) and BCH4 (empty symbols) by CTL generated from BALBlc mice immunized with placebo (Triangle), live RSV (Square) or line 19H (Circle) is shown.
Mice immunized with live RSV (empty square) or 19H (empty circle) Iysed BCH4 cells (RSV infected) significantly at all effector to target cell ratios when compared to the Iysis of BC (un-infected) cells. There was no significant levels of Iysis by effector cells from the placebo indicating that line 19H is c~p~bt~ of inducing significant levels of CTL activity.
G. Protection Study Gc~erRtiG~ 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 107 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 107 spleen cells in a final volume of 10 mL of complete medium. CTL activity was tested 5-6 days following re-stimulation.
Cytotoxicity assay. On the day of the assay, effector cells were washed twice with fresh medium and were resuspended in 2 mL of complete medium. BC
cells (2 X 106 cells), a BALB/c fibroblast cell line, as well as BCH4 cells (2 X 106 cells), a BALB/c fibroblast T cell line persistently infected with RSV, were pulsed with 200~Ci of Sodium 51chromate (Dupont) for 90 min. The targets were washed three times with medium to remove free S'chromium. Viable cell counts of the target cells were determined and target cell suspensions were pared at 2 X 104 cells/mL.
Washed responder T-cells at various dilutions (in 100 ~I) were incubated with 2 X
103 target cells (in 100 ~I) in triplicate in 96-well V bottomed tissue-culture plates for 4 h at 37DC with 6% CO2. Spontaneous and total release of S'chromium were determined by incubating target cells with either medium of 2.5% Triton -X100 in the absence of responder Iymphocytes. Six replicates of each were prepared. After 4 h plates were centrifuged at 200 X g for 2 min. and 100~1 of supernatant was removed from each well to determine the amount of S'chromium related. Percentagespecific S'chromium release was calculated as (Experimental Release-Spontaneous Release)/ (Total release - Spontaneous release) X 100. The spontaneous release CA 022~0883 1998-10-02 WO 97/38138 PCT~US97/05588 of S'chromium in the absence of effector cells was found to be between 10-15% inthese studies. The Iysis in cultures is directly proportional to the number of effector cells present in the culture, which in turn is proportional to the number of CTLprecursors activated in vivo by that particular immunogen.
The results of this cytotoxicity study are shown in Figure 5. In Figure 5, Iysisof 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, CRla MD or WRSV, or live mouse adapted virus is shown. Mice immunized with live mouse adapted RSV, 19HL 3PI, 19H 3PI, 19H MD, CRla MD, wt 19 and WRSV, 10 Iysed BCH4 cells (RSV infected) at all effector cell dilutions when compared to the Iysis of BC (non-infected) cells. There were no significant level of Iysis by effector cells from the placebo indicating that all the tested viruses are capable of inducing significant levels of CTL activity.
To evaluate the ability of the plaque purified deposited viruses to protect mice15 against live virus challenge, mice that were immunized with eitherthe plaque purified viruses, progenitor viruses or medium alone (see D. above, Immunogenicity of RSVLines (Study 4)), were challenged with 106 pfu of RSV A2 immediately after the 8week 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 20 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.

CA 022~0883 l998-l0-02 W O97/38138 PCTrUS97/05588 Table 9 Protective Ability Of The Plaque Purified Viruses And Progenitor Viruses Virus Mean Virus Lung Titre % Animals Protected (log pfu/~ + s.d.) Placebo 5.2 + .06 o 19HL 3PI ~ 1.7 + 0 100 19H 3PI ~ 1.7 + 0 100 19H MD ~ 1.7 + 0 100 wt 19 ~ 1.7 + 0 100 CRla MD s 1.7 + 0 100 WRSV 1.9 + 2.2 50 Live virus s 1.7 + 0 100 Represents the mean value of 6 animals.

15 A. Sequence Comparison -WRSV
In identifying the molecular basis for the ts phenotype, 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 20 sequencing. Comparison of the F genes of line Ca19V and line 19H revealed 73 nucleotide and 15 amino acid differences. Comparison ofthe F genes of line 19H
and WRSV revealed 72 nucleotide and 13 amino acid differences. There are 11 nucleotide changes and 6 amino acid changes between the F genes of the two line 19 attenuated viruses, Ca19V and 19H. Only base changes (no insertions or 25 deletions) were found. The F genes of the two attenuated line 19 viruses have 66 nucleotides and 11 amino acids in common but differ from that of WRSV (amino acid positions 66, 76, 79, 97, 119, 129, 191, 357, 384, 522 and 530).
The Garnier Osguthorpe Robson (GOR) predicted F protein structures of the two line 19 viruses are nearly identical; however, the GOR F protein structure of 30 WRSV differs at amino acid 97,119,191,357 and 522 from both attenuated viruses and differs at amino acid 294 only from line Ca19V. Amino acid 97 (threonine in both line 19's and methionine in WRSV) predicts a turn in the attenuated line 19 CA 022~0883 l998-l0-02 viruses not present in WRSV. Amino acid 119 (phenylalanine in both line 19's andleucine in WRSV) predicts an additional turn in the attenuated line 19 viruses not present in WRSV. Amino acid 191 (Iysine in the aUenuated 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 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 10 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.
Table 10 Sequence Comparison of the Genes Coding for the F Proteins of WRSV, Ca1 9V and 1 9H
Nucleo- WRSV C 19V 19H Ancnidn~# WRSV Ca19V 19H

T C C

131 G G A 40 Val Val lle 209 A G G 66 Lys Glu Glu 240 T C C 76 Val Ala Ala 250 G A A 79 Met lle lle 303 T C C 97 Met Thr Thr CA 02250883 l998-l0-02 Nucleo- wRsV C 19V 19H AAcjdn# WRSV Ca19V 19H

368 C T T 119 Leu Phe Phe 398 T A A 129 Leu lle lle 585 G A A 191 _~ Lvs Lvs 716 A G C 235 Arg Gly Arg 893 G A G 294 Glu Lys Glu 906 C C A 298 Ala Ala Glu 1083 A C C 357 LYS Thr Thr 1116 A T A 368 Asp Val Asp W O 97/38138 PCTrUS97/05588 Nucleo-WRSV C 19V 19HAcid # WRSV Ca19V 19H

1163 G A A 384 Val lle lle 1206 C T C 398 Ser Leu Ser 1577 A G G 522 Thr Ala Ala 1603 A G G 530 lle Met Met 30 Differences between Ca19V and 19H are italicized and bolded. Amino acids which impact on the predicted protein folds are underlined.

CA 022~0883 l998-l0-02 W O97/38138 PCT~US97/05588 - 31 - _ B. Sequence Comparison - wt 19 The nucleotide sequence of the F genes of line 19 wild type (wt 19), Ca19V
and 1 gH, were compared by f-mol sequencing. The following table lists the nucleotide and amino acid changes in the F genes between lines Ca19V, 19H and 5 wt 19.
Table 11 Sequence Differences Behveen The F Genes Of wt 19, Ca19V and 19H

Nucleo- wt 1919H Ca19V Acid # wt 19 19H Ca19V

131 G A G 40 val iso val 716 C C G 235 arg arg gly 893 G G A 294 glu glu Iys 906 C A C 298 ala glu ala 15 1116 A A T 368 asp asp val 1206 C C T 398 ser ser leu 1655 C c T
Differences between 19H and wt 19 are bolded.
Differences between Ca19V and wt 19 are italicized.

Between wt 19 and 19H, there were 4 nucleotide differences, 2 of which 25 coded for amino acid differences. Amino acid 40, valine in wt 19 and isoleucine in 19H, is a conserved change since both are hydrophobic and neither is charged. The difference at amino acid 298 is not conserved. Alanine, in wt 19, is hydrophobic and not charged while glutamic acid in 19H, is not hydrophobic and is negatively charged. Chou Fasman analysis predicts that the glutamic acid of 19H extends an 30 alpha helix thus postponing the formation of a beta sheet predicted by the alanine at 298 of the wt 19 F protein~

CA 022~0883 1998-10-02 W O97/38138 PCTrUS97/05588 Between wt 19 and Ca19V, there are 9 nucleotide differences and 4 amino acid differences, none of which is a conserved change. Amino acid 235 is basic arginine to uncharged glycine; amino acid 294 is negatively charged glutamic acid to positively charged Iysine; amino acid 368 is negatively charged aspartic acid to 5 hydrophobic valine; and amino acid 398 is uncharged serine to hydrophobic leucine.
The structure of the two F proteins, as predicted by Chou Fasman, differs only at amino acid 234; Ca19V extends an alpha helix, thus losing a turn predicted for the wt 19 F protein.
None of the amino acid differences is shared by the two line 19 attenuated 10 viruses, but nucleotide 1249 (which does not code for an amino acid change) is C
in wt 19 and A in both Ca19V and 19H.

As indicated above, several of the deposited strains were plaque purified. In particular, Line 19HL 3PI was passed 72 times in MRC5 cells, then plaque purified 15 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. Line19H MD was passed 92 times in MRC5 cells, then purified by limiting dilution three times. Line CRla MD was passed 28 times at 25~C and 1 time at 33~C then purifiedby limiting dilution five times. The following table sets forth the titers of the strains 20 at 33~C and 39~C, illustrating temperature sensitivity.
Table 12 TClDso In MRC5 Cells Virus 33~C 39~C
19HL 3PI 1.0 x 106 1.0 x 105 19H 3PI 3.2 x 105 2.0 x 103 19H MD 3.2 x 105 1.0 x 103 CRla MD >1.0 x 106 2.0 x 103 19H 3.16 x 106 WRSV 2232272.0 x 105 The attenuated virus of the present invention is administered to human subjects according to well established human RSV protoGols, for example, those W O 97/38138 PCTrUS97/OSSB8 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCTRule 13bis) A. The i ~ rqlion~ made below relale ~o the ~ie~uu~ referred to in the description onpages 4 and 5 line B. IDENTIFICATION OF DEPOSIT 15VMRc2a7(;~v 5 C;L Further deposits are identified on an addilional sheel [~
Name of depositary ins~ilu~ion Anterican Type Cu1tutre Co11ecticn Address of depositary institution ~including postal code and country) 12301 Park1awn Drive Rockville, MarylcLnd 20852 United States of America Date of deposit Accession Number 20 SepteTnlber 1995 (20.09.95) VR-2511 (see additional sheet) C. ADDITIONAL INDICATIONS Qeave blank if not ~Ir~ - P) This r ' jS continued on an addhional sheet In respect of the desi~At;~t of the EPO, samples of the deposited micro-or~;An; ~rn.Ci wi11 be made available until the publ ;c~t;~n of the T~t;C~l of the grant of the European patent or until the date on which the appl;cAti~n is refused or with~ir.~ n or is deemed to be withdrawn, as provided in Rule 28(~) of the I~,plt~rPnt;n~ Pe~t~ t;ons under the EPC only by the issue of a samPle toan e~pert nrlm;n~teci by requester (Rllle ~8(4) EPC).

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE ~if the i . ' - . a ore notfor all designated S~ates) E~PO

E. SEPAiRATE FURNISHING OF INDICATIONS Qeave blank If not trplicn~
Thein~lir~inn~listedbelowwillbesubmittedtothe~ ollalBureaulater(specifythegeneralnatureoftheind~catio~e.g.~rAccession Number oJDeposit ~

For receiving Of fice use oniy For ll~t. .. '1~ Bureau use only ~3 This sheet v.as~~~eived with the~ ' ~rrlirq~ion O This sheet was received by the International Bureau on Authorized officer Authorized of ficer Forrn PCT/RO/134 (July 1992) CA 022~0883 l998-l0-02 W O97/38138 PCT~US97/05588 -33 -described in Wright et al., tnfect. 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 102 to 109 PFU, preferably 104 to 105 PFU, of attenuated virus per ml in a volume of 0.5 ml. Antibody response is 5 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.
The foregoing discussion discloses and describes merely exemplary 10 embodiments of the present invention. One skilled in the art will readily recognize from such discussion, and from the accompanying claims, that various changes, modifications and variations can be made therein without departing from the spirit and scope of the invention as defined in the following claims.
All patents and other references cited herein are expressly incorporated by reference.

CA 022~0883 1998-10-02 W O97/38138 PCT~US97/05588 Continuation Of Indications Relating To A Deposited Microoryanism (PCT Rule 13bis) For EPO

American Type Culture Collection /~ccession Identification Of Deposit Number Date of Deposit RSV, Line 19 VR-2512 20 September 1995 MRC5-15-25~ st-33~ (20.09.95) RSV, Line 19 VR-2513 20 September 1995 MRC5-60-35~ (20.09.95) RSV, Line 48 VR-2514 20 September 1995 MRC5-14-25~ st (20.09.95) MRC1-33~ VERO
10-25~ VERO 1-33~
RSV, Line 19 VR-2515 20 September 1995 MRC5-10-25~ (20.09.95) VERO16-25~ VERO
6-20~ VERO 3-33~
RSV, CRSV-BC5 VR-2516 20 September 1995 CL-17 MRC30 (20.09.95) RSV, CRSV-BC13 VR-2517 20 September 1995 MRC19-25~ MRC1-33~ (20.09.95) RSV, line 19 2 April 1997 MRC5 72-35~C, Vero 3- (02.04.97) 35~C, Large Clone 6-35~C (3PI) Plaque Purified RSV, line 19 MRC 70-35~C, 2 April 1997 Vero 3-35~C, MRC5 2-35~C (02.04.97) Clone 2-35~C (3PI) Plaque purified RSV, line 19 MRC5 92-35~C 2 April 1997 Clone 5-1 Purified by (02.04.97) minimal dilution RSV, la-CRSV-5 MRC-38- 2 April 1997 25~C, MRC1-33~C Purified (02.04.97) by minimal dilution INDICATIONS RELATIN(~ TO A DEPOSITED MICROORGANISM
(PCT Rule 1 3bis) A. The i ' onC made below relate to the ~ uu~y~n~"~ referred to in the description onpageS 4 and 5 line B. IDEI~TIFICATION OF DEPOSIT RSVMRl~a7CRSV 5 CL Funher deposits are identified on an additional sheet Narne of depositarv institution American Type Culture Collection Address of depositary institution ~Including postal code and counrry) 12301 Parklawn Drive Rockville, Maryland 20852 United States of America Date of deposit Accession Number 20 September l99S (2U.09.9S) VR-2511 (see additional sheet) C. ADDITIONAL INDICATIONS (/eave blank if not a' F' ' l~) This i.,fu. is continued on an additional sheet O
In respect of the ~s;~ n of Finland, samples of the deposited micro-or~n;~n~ will be made available until the application has been laid oPen to public inspection (by the NAt;nnAl Board of Patents and Re~istration), or has been finally ~t~ ed upon by the National Board of Patents and ~e~istrati~n without having been laid open to public inspection, the filrn;~h;n~ of a sample shall only be effected to an expert in the art.

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (ifthe jn~irn~in~S are notfor all designated States) Finland E. SEPARATE FURNISHING OF INDICATIONS ~leave blank iSnot applicable) Thei ' -listedbelowwillbesubminedtothelnternationalBureaulater(specis!?thegeneralnattlreofthemdlcationseg~ "Accession Number of Deposit") For receiving Of~Ice use only For 1.. ?~ n~1 Bureau use only ~s sheet was received ~h~ Ihe -' application O This sheet was received by the International Bureau on:
O f~O~
Authorized officer Aulhorized officer Forrn PCT/RO/134 (July 1992~

CA 022~0883 1998-10-02 W O97/38138 37 PCT~US97/05588 Continuation Of Indications Relating To A Deposited Microorganism (PCT Rule 13bis) For Finland American Type Culture Collection Accession Ide.,lificalion Of DePosit Number Date of DePosit RSV, Line 19 VR-2512 20 September 1995 MRC5-15-25~ st-33~ (20.09.95) RSV, Line 19 VR-2513 20 September 1995 MRC5-60-35~ (20.09.95) RSV, Line 48 VR-2514 20 September 1995 MRC5-14-25~ st (20.09.95) MRC1-33~ VERO
10-25~ VERO 1-33~
RSV, Line 19 VR-2515 20 September 1995 MRC5-10-25~ (20.09.95) VERO16-25~ VERO
6-20~ VERO 3-33~
RSV, CRSV-BC5 VR-2516 20 September 1995 CL-17 MRC30 (20.09.95) RSV, CRSV-BC13 VR-2517 20 September 1995 MRC19-25~ MRC1-33~ (20.09.95) RSV, line 19 2 April 1997 MRC5 72-35~C, Vero 3- (02.04.97) 35~C, Large Clone 6-35~C (3PI) Plaque Purified RSV, line 19 MRC 70-35~C, 2 April 1997 Vero 3-35~C, MRC5 2-35~C (02.04.97) Clone 2-35~C (3PI) Plaque purified RSV, line 19 MRC5 92-35~C 2 April 1997 Clone 5-1 Purified by (02.04.97) minimal dilution RSV, la-CRSV-5 MRC-38- 2 April 1997 25~C, MRC1-33~C Purified (02.04.97) by minimal dilution W O 97/38138 PCTrUS97/05588 INDICATIO~S RELATING TO A DEPOSITED MICROORGA~ISM
(PCTRule 13bis) A. The in-lir~irmc made below relate to the ~ luolL referred tû in thc d~
onpageS 4 and 5 ,line B. IDENTIFICATION OF DEPOSIT 15v~127cRsv 5 CL Further deposits are identified on an additional sheet Narne of depositary institution American Type Culture Collection Address of depositary institution (including posral code and country) 12301 Parklawrt Drive Rockville, M~ryland 20852 United States of America Date of deposit Accession Number 20 September 1995 (20.09.95)VR-2511 (see additional sheet) C. ADDITIONAL INDICATIONS (/eave blank yno~ arp' ' ' ) This ~ is continued on an additional sheet O
In respect of the designation of Singapore, the ~ni~h;np of a sample of a microorganism shall only be made available to an expert.

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if thc indicarions ore notfor all ~lr~h~ated Srates) S~ ~ .e E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not arpli( ~lo) Thei ~ listedbelowwillbesubminedtothelnt~ ' 'Bureaulater(specifvthegeneralnarureoftheind~canonseg~"Accesslon Number of Dcposit") For receiving Of fice use only For International Bureau use onlv ~3 This sheet was received with the -, I a~ This sheet was received by the International Bureau on:

Authorized officer Authorized officer Form PCT/RO/134 (Julv 1992) . , ~ .

CA 022~0883 1998-10-02 Continuation Of IndicaliGns Relating To A neposited Micro~ryanism (PCT Rule 13bis) For Singapore American Type Culture Collection Accession Identification Of Deposit Number Date of DePosit RSV, Line 19 VR-2512 20 September 1995 MRC5-15-25~ st-33~ (20.09.95) RSV, Line 19 VR-2513 20 September 1995 MRC5-60-35~ (20.09.95) RSV, Line 48 VR-2514 20 September 1995 MRC5-14-25~ st (20.09.95) MRC1-33~ VERO
10-25~ VERO 1-33~
RSV, Line 19 VR-2515 20 September 1995 MRC5-10-25~ (20.09.95) VERO16-25~ VERO
6-20~ VERO 3-33~
RSV, CRSV-BC5 VR-2516 20 September 1995 CL-17 MRC30 (20.09.95) RSV, CRSV-BC13 VR-2517 20 September 1995 MRC19-25~ MRC1-33~ (20.09.95) RSV, line 19 2 April 1997 MRC5 72-35~C, Vero 3- (02.04.97) 35~C, Large Clone 6-35~C (3PI) Plaque Purified RSV, line 19 MRC 70-35~C, 2 April 1997 Vero 3-35~C, MRC5 2-35~C (02.04.97) Clone 2-35~C (3PI) Plaque purified RSV, line 19 MRC5 92-35~C 2 April 1997 Clone 5-1 Purified by (02.04.97) minimal dilution RSV, la-CRSV-5 MRC-38- 2 April 1997 25~C, MRC1-33~C Purified (02.04.97) by minimal dilution CA 022~0883 l998-l0-02 w 097t38138 PCTrUS97/05588 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCTRule 13bis) A. The jn~i~s~ nc made beiow relate to the ~ Uol~ referred to in the description onpageS 4 and S .line B. IDENTIFICATION OF DEPOSIT ~ MRl2a7cRsv 5 CL Further deposits are identified on an additional sheet Name of depositary institution American Iype Culture Collection Address of depositarv institution (incl~ding postal code and coun~
12301 Parklawn Drive ~ockville, Maryland 20852 United States of ~ erica Date of deposit Accession Number 20 September 1995 (20.09.95) VR-~511 (see additional sheet) C. ADDITIONAL INDICATIONS (leave blank if not nrpli- ~.'r) This ;~ru~ aliu.. is cûntinued on an additional sheet O
In respect of the des;~nAtion of Sweden, samples of the deposited micro-org~n;~m~ will be made available until the applio~t;~n has been laid open to public inspection (by the ~wedish Patent Office), or has been finally ~ec;~d upon by the Swedish Patent Office without having been laid oPen to public inspection, the filrn;.~h;ng of a sample shall only be effected to an expert in the art.

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the in~lienrions are not Jor all Jesi~ States) Sweden E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not arrlicnhle) The ' listedbelowwillbesubminedtothellit~ lBureaulaterfspeclfythegeneralnarureof~heindicahonseg~"Accession humber oJl~eposit'7 For receivin~ Of fice use only For International Bureau use onl~
This sheet uas received uith the ~ pplics~ n O This sheet was received by the International Bureau on:

Authorized officer Authorized officer Forrn PCT/RO/134 (luly 199~) CA 022~0883 1998-10-02 W 097/38138 41 PCT~US97/05588 Continuation Of Indicalio"s Relating To A Deposited Microor~anism (PCT Rule 13bis) For Sweden American Type Culture Collection Accession "tificalion Of Deposit Number Date of Deposit RSV, Line 19 VR-2512 20 September 1995 MRC5-15-25~ st-33~ (20.09.95) RSV, Line 19 VR-2513 20 September 1995 MRC5-60-35~ (20.09.95) RSV, Line 48 VR-2514 20 September 1995 MRC5-14-25~ st (20.09.95) MRC1-33~ VERO
10-25~ VERO 1-33~
RSV, Line 19 VR-2515 20 September 1995 MRC5-10-25~ (20.09.95) VERO16-25~ VERO
6-20~ VERO 3-33~
RSV, CRSV-BC5 VR-2516 20 September 1995 CL-17 MRC30 (20.09.95) RSV, CRSV-BC13 VR-2517 20 September 1995 MRC19-25~ MRC1-33~ (20.09.95) RSV, line 19 2 April 1997 MRC5 72-35~C, Vero 3- (02.04.97) 35~C, Large Clone 6-35~C (3PI) Plaque Purified RSV, line 19 MRC 70-35~C, 2 April 1997 Vero 3-35~C, MRC5 2-35~C (02.04.97) Clone 2-35~C (3PI) Plaque purified RSV, line 19 MRC5 92-35~C 2 April 1997 Clone 5-1 Purified by (02.04.97) minimal dilution RSV, la-CRSV-5 MRC-38- 2 April 1997 25~C, MRC1-33~C Purified (02.04.97) by minimal dilution WO 97/38138 42 PCTrUS97/05588 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 1 3bis) A. The i ' ~q~ ns made below relate to the mie,oo,L referred to in the dc~ io..
on page,s 4 and 5 . Iine B. IDENTIFICATION OF DEPOSIT 15~V la27CR~\~ 5 CL Further deposits are idenlified on an additional sheet Name of depositary institution Am~rican Type Culture Collection Address of depositary institution (incl~ldmg postal code and coun~ry 12301 Parklawn Drive Rockville, Maryland 20852 United States of America Date of deposit Accession Number 20 September 1995 (20.09.95) VR-2511 (see additional sheet) C. ADDITIONAL INDICATIONS fleave blank if not ~l rl; /~1~) This ' ~ , ' is continued on an additional sheet O
In respect of the ~e.C;~n~t;on of the United ~;np,~nm, a sample should be made available only to an expert.

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if Ihe ~ irn~ions are notfor all ~i.~5 e /States) United ~;n~r~

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicableJ
The;...1; -uo..clistedbelowwillbesubminedtotheInternation81Bureaulater (specifythegeneralr~ctureoftheindicahon5e.g., "Accession Number of Deposit") For receivin~ Of fice use only For In~rrs~i -' Bureau use only This shee~ was received with the international spplics~ion O This sheet was received by the Intemational Bureau on:

Authorized officer Authorized officer Fomn PCT/R01134 (3uly 199~) CA 022~0883 1998-10-02 W O97/38138 43 PCTrUS97/05588 Continuation Of Inclicalions Relating To A Deposited Microoryanism (PCT Rule 13bis) For United Ki"ydGri, American Type Culture Collection Accession Ide~,liricalion Of Deposit Number Date of DePosit RSV, Line 19 VR-2512 20 September 1995 MRC5-15-25~ st-33~ (20.09.95) RSV, Line 19 VR-2513 20 September 1995 MRC5-60-35~ (20.09.95) RSV, Line 48 VR-2514 20 September 1995 MRC5-14-25~ st (20.09.95) MRC1-33~ VERO
10-25~ VERO 1-33~
RSV, Line 19 VR-2515 20 September 1995 MRC5-10-25~ (20.09.95) VERO16-25~ VERO
6-20~ VERO 3-33~
RSV, CRSV-BC5 VR-2516 20 September 1995 CL-17 MRC30 (20.09.95) RSV, CRSV-BC13 VR-2517 20 September 1995 MRC19-25~ MRC1-33~ (20.09.95) RSV, line 19 2 April 1997 MRC5 72-35~C, Vero 3- (02.04.97) 35~C, Large Clone 6-35~C (3PI) Plaque Purified RSV, line 19 MRC 70-35~C, 2 April 1997 Vero 3-35~C, MRC5 2-35~C (02.04.97) Clone 2-35~C (3PI) Plaque purified RSV, line 19 MRC5 92-35~C 2 April 1997 Clone 5-1 Purified by (02.04.97) minimal dilution RSV, la-CRSV-5 MRC-38- 2 April 1997 25~C, MRC1-33~C Purified (02.04.97) by minimal dilution

Claims (12)

WE CLAIM:

1. A respiratory syncytial virus selected from the group consisting of the viruses having ATCC Designation Nos. VR-2511, VR-2512, VR-2513, VR-2514, VR-2515, VR-2516, VR-2517 and 19HL 3PI, 19H 3PI, 19H MD and CRIa MD, and derivative viruses thereof.

2. The attenuated virus of Claim 1 for use as an active pharmaceutical substance.

3. The use of the attenuated virus of Claim 1 for the preparation of a medicament for the treatment or prevention of disease caused by infection by respiratory syncytial virus.

4 A method of producing a vaccine against disease caused by infection by respiratory syncytial virus, comprising:
a) administering the virus of Claim 1 to a test host to determine an amount and a frequency of administration thereof to elicit a protective immune response in said host, and b) formulating said virus in a form suitable for administration to a treatable host in accordance with said determined amount and frequency of administration.

5. A method of determining the presence of antibodies specifically reactive with a respiratory syncytial virus in a sample comprising the steps of:a) contacting the sample with the virus of Claim 1 to produce complexes comprising the virus and antibodies present in the sample specifically reactive therewith; and b) determining production of the complexes,

6. A method of determining the presence of a respiratory syncytial virus in a sample comprising the steps of:
a) contacting the sample with an antibody specifically reactive with a respiratory syncytial virus of Claim 1 to produce complexes comprising the antibody and the virus present in the sample specifically reactive therewith; and b) determining production of the complexes.

7. A nucleic acid molecule encoding the respiratory syncytial virus of Claim 1 and equivalent nucleic acid molecules thereof.

8. A vaccine composition comprising an attenuated respiratory syncytial virus and a pharmaceutically acceptable carrier.

9. The vaccine composition of Claim 8, wherein the attenuated respiratory syncytial virus is selected from the group consisting of the viruses of Claim 1 and derivative viruses thereof.

10. A method of immunizing a host against disease caused by infection by respiratory syncytial virus which comprises administering to the host an immunoeffective amount of the vaccine of Claim 8 or the nucleic acid molecule ofClaim 7.

11. An attenuated respiratory syncytial virus characterized by having a codon encoding an amino acid in the F protein chosen from the group consisting of codon encoding isoleucine at amino acid 40, a codon encoding glutamic acid at amino acid 66, a codon encoding alanine at amino acid 76, a codon encoding isoleucine at amino acid 79, a codon encoding threonine at amino acid 97, a codon encoding phenylalanine at amino acid 119, a codon encoding isoleucine at amino acid 129, a codon encoding lysine at amino acid 191, a codon encoding glycine atamino acid 235, a codon encoding arginine at amino acid 235, a codon encoding lysine at amino acid 294, a codon encoding glutamic acid at amino acid 294, a codon encoding glutamic acid at amino acid 298, a codon encoding alanine at amino acid 298, a codon encoding threonine at amino acid 357, a codon encoding valine at amino acid 368, a codon encoding aspartic acid at amino acid 368, a codon encoding isoleucine at amino acid 384, a codon encoding leucine at amino acid 398, a codon encoding shrine at amino acid 398, a codon encoding alanine at amino acid 522 and a codon encoding methionine at amino acid 530.

12. An attenuated respiratory syncytial virus characterized by having a codon encoding an amino acid in the F protein chosen from the group consisting of a codon encoding isoleucine at amino acid 40, a codon encoding glycine at amino acid 235, a codon encoding arginine at amino acid 235, a codon encoding lysine at amino acid 294, a codon encoding glutamic acid at amino acid 294, a codon encoding glutamic acid at amino acid 298, a codon encoding alanine at amino acid298, a codon encoding valine at amino acid 368, a codon encoding aspartic acid at amino acid 368, a codon encoding leucine at amino acid 398 and a codon encoding serine at amino acid 398.