CA2436126A1 - Subunit respiratory syncytial virus vaccine preparation - Google Patents

Abstract

The fusion (F) protein, attachment (G) pro-tein and matrix (M) protein of respiratory syncy-tial virus (RSV) are isolated and purified from res-piratory syncytial virus by mild detergent extrac-tion of the proteins from concentrated virus, load-ing the protein onto a hydroxyapatide or other ion-exchange matrix column and eluting the protein using mild salt treatment. The F, G and M pro-teins, formulated as immunogenic compositions, are safe and highly immunogenic and protect rele-vant animal models against disease caused by res-piratory syncytial virus infection.

Description

TITLE OF INVENTION
SUBUNIT RESPIRATORY SXNCXTIAL VIRUS VACCINE PREPARATION
FIELD OF INVENTION
The present invention is related to the field of immunology and is particularly concerned with vaccine preparations against respiratory syncytial virus to infection.
REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part of copending United States Patent Application No.
081679,060 filed July 12, 1996.
z5 BACKGROUND OF THE INVENTION
Human respiratory syncytial virus is the main cause of lower respiratory tract infections among infants and young children (refs. 1 to 3 - a list of references appears at the end of the disclosure and each of the ao references in the list is incorporated herein by reference thereto). Globally, 55 million infections occur every year resulting in 160,000 deaths (ref. 4).
In the USA alone 100,000 children may require hospitalization for pneumonia and bronchiolitis caused 2s by RS virus in a single year (refs. 5, 6). Providing inpatient and ambulatory care for children with RS virus infections costs in excess of $340 million annually in the USA (ref. 7). Severe lower respiratory tract disease due to RS virus infection predominantly occurs so in infants two to six months of age (ref. 8).
Approximately 4,000 infants in the USA die each year from complications arising from severe respiratory tract disease caused by infection with RS virus and Parainfluenza type 3 virus (PIV-3). The t~TOrld Health 3s Organization (WHO) and the National Institute of Allergy and Infectious Disease (NIAID) vaccine advisory WO 98102457 PCTICA9'1100497

2 committees have ranked RS virus second only to HIV for vaccine development.
The structure and composition of RSV has been elucidated and is described in detail in the textbook s "Fields Virology", Fields, B.N. et al. Raven Press, N.Y. (1996), in particular, Chapter 44, pp 1313-1351 "Respiratory Syncytial Virus" by Collins, P., McIntosh, K., and Chanock, R.M. (ref. 9).
The two major protective antigens of RSV are the is envelope fusion (F) and attachment (G) glycoproteins (ref. 10). The F protein is synthesized as an about 68 kDa precursor molecule (Fo) which is proteolytically cleaved into disulfide-linked F1 (about 98 kDa) and Fz (about 20 kDa) polypeptide fragments (ref. 1I). The G
1s protein (about 33 kDa) is heavily O-glycosylated giving rise to a glycoprotein of apparent molecular weight of about 90 kDa ( ref . 12 ) . Two broad subtypes of RS virus have been defined A and B (ref. 13). The major antigenic differences between these subtypes are found 2o in the G glycaprotein while the F glycoprotein is more conserved (refs. 7, 14).
In addition to the antibody response generated by the F and G glycoproteins, human cytotoxic T cells produced by RSV infection have been shown to recognize 2s the RSV F protein, matrix protein M, nucleoprotein N, small hydrophobic protein SH, and the nonstructural protein 1b (ref. I5).
A safe and effective RSV vaccine is not available and is urgently needed. Approaches to the development so of RS virus vaccines have included i.naetivation of the virus with formalin (ref. 16), isolation of cold-adapted and/or temperature-sensitive mutant viruses (ref. 17) and purified F or G glycoproteins refs. 18, 19, 20).
Clinical trial results have showy. that both live 3s attenuated and formalin-inactivated vaccines failed to adequately protect vaccines against RS virus infection

3 (refs. 21 to 23). Problems encountered with attenuated cold-adapted and/or temperature-sensitive RS virus mutants administered intranasally included clinical morbidity, genetic instability and overat tenuation s (refs. 24 to 26). A live RS virus vaccine administered subcutaneously also was not efficacious (ref. 27).
Inactivated RS viral vaccines have typically been prepared using formaldehyde as the inactivating agent.
Murphy et al. (ref. 28} have reported data on the immune so response in infants and children immunized with formalin-inactivated RS virus. Infants (2 to 6 months of age) developed a high titre of antibodies to the F
glycoprotein but had a poor response to the G protein.
Older individuals (7 to 40 months of aqe) developed i5 titres of F and G antibodies comparable to those in children who were infected with RS virus. However, both infants and children developed a lower level of neutralizing antibodies than did individuals of comparable age with natural RS virus infections. The 2o unbalanced immune response, with high titres of antibodies to the main immunogenic RS virus proteins F
(fusion) and G (attachment) proteins but. a low neutralizing antibody titre, may be in part due to alterations of important epitopes in the F and G
2s glycoproteins by the formalin treatment. Furthermore, some infants who received the formalin-inactivated RS
virus vaccine developed a more serious lower respiratory tract disease following subsequent exposure to natural RS virus than did non-immunized individuals (refs. 22, 30 23). The formalin-inactivated RS virus vaccines, therefore, have been deemed unacceptable for human use.
Evidence of an aberrant immune response also was seen in cotton rats immunized with formalin-inactivated RS virus (ref_ 29). Furthermore, evaluation of RS
3s virus formalin-inactivated vaccine in cotton rats also WO 98102457 fCTlCA97100497

4 showed that upon live virus challenge, immunizs=d animals developed enhanced pulmonary histopathology (ref. 30).
The mechanism of disease potentiation caused by formalin-inactivated RS virus vaccine preparations s remains to be defined but is a major obstacle in the development of an effective RS virus vaccine. The potentiation may be partly due to the action of formalin on the F and G glycoproteins. Additionally, a non--RS
virus specific mechanism of disease potentiation has to been suggested, in which an immunological response to contaminating cellular or serum components present in the vaccine preparation could contribute, in part, to the exacerbated disease (ref. 31). Indeed, mice and cotton rats vaccinated with a lysate of HEp-2 cells and is challenged with RS virus grown on HEp-2 cells developed a heightened pulmonary inflammatory response.
Furthermore, RS virus glycoproteins purified by immunoaffinity chromatography using elution at acid pH
were immunogenic and protective but also induced 2o immunopotentiation in cotton rats (refs. 29, 32).
There clearly remains a need for immunogenic preparations, including vaccines, which are not only effective in conferring protection against disease caused by RSV but also do not produce unwanted side-2s effects, such as immunopotentiation. There is also a need for antigens for diagnosing RSV infection and immunogens for the generation of antibodies (.including monoclonal antibodies) that specifically recognize RSV
proteins for use, for example, in diagnosis of disease 3o caused by RS virus.
SUMMARY OF THE INVENTION
The present invention provides the production of respiratory syncytial virus ;RSV) on a vaccine quality cell line, for example, VERO, MRC5 or WI38 cells, 3s purification of the virus from fermentor harvests, extraction of the F, G and M proteins from the purified virus and copurification of the F, G and M proteins without involving immunoaffinity or lentil lectin or concanavalin A affinity steps. In particular, the lectin affinity procedure, described, for example, in WO
s 91/00109 (US 07/773,999 filed June 28, 1990) assigned to the assignee hereof and the disclosure of which. is incorporated herein by reference), could lead to leaching of the ligand into the product.
In addition, there is provided herein, for the io first time, a procedure for the coisolation and copurification of the F, G and M proteins of RSV and also immunogenic compositions comprising copurified mixtures of the RSV proteins.
The coisolated and copurified F, G and M RSV
is proteins are non-pyrogenic, non-immunopotentiating, and substantially free of serum and cellular contaminants.
The isolated and purified proteins are immunogenic, free of any infectious RSV and other adventitious agents.
Accordingly, in one aspect of the present zo invention, there is provided a mixture of purified fusion (F) protein, attachment (G) protein and matrix (M) protein of respiratory syncytial virus (RSV).
The fusion. (F) protein may comprise multimeric fusion (F) proteins, which may include, when analyzed zs under non-reducing conditions, heterodimers of molecular weight approximately 70 kDa and dimeric and trimeric forms .
The attachment (G) protein may comprise, when analyzed under non-reducing conditions, oligomeric G
3o protein, G protein of molecular weight approximately 95 kDa and G protein of molecular weight approximately 55 kDa.
The matrix (M) protein may comprise, when analyzed under non-reducing conditions, protein of molecular 3s weight approximately 28 to 34 kDa.

5? PCTIC.~97I00497

6 The protein mixture provided herein, when analyzed by reduced SDS-PAGE analysis, may comprise the fusion (F) protein comprising F1 of molecular weight approximately 48 kDa and F2 of about 23 kDa, the s attachment (G) protein comprising a G protein of molecular weight approximately 95 kDa and a G protein of molecular weight approximately 55 kDa, and the matrix (M) protein comprising an M protein of approximately 31 kDa.
io The mixture provided in accordance with Iris aspect of the invention may comprise the F, G and M proteins in the relative proportions of:
F about 35 to about 70 wt%
G about 5 to about 30 wt%
is M about 10 to about 40 wt°~
When analyzed by SDS-PAGE under reducing conditions and densitometric scanning following silver staining, the ratio of F1 of molecular weight approximately 48 kDa to F2 of molecular weight approximately 23 kDa in this 2o mixture may be approximately between 1:l and 2:1. The mixture of F, G and M proteins may have a purity of at least about 750, preferably at least about 850.
The mixture provided herein in accordance with this aspect of the invention, having regard to the method of 25 isolation employed herein as described below, is devoid of monoclonal antibodies and devoid of. lentil lectin and concanavalin A.
The RSV proteins provided in the mixture of proteins provided herein generally are substantially so non-denatured by the mild conditions of preparation and may comprise RSV proteins from one or both of subtypes RSV A and RSV B.
In accordance with a preferred embodiment of the invention, there is provided a coisolated and copurified 35 mixture of non-denatured proteins of respiratory syncytial virus (RSV), consisting essentially of the WO 9810245'7 PC'~/CA97/U0497

7 fusion (F) protein, attachment (6) protein and matrix (M) protein of RSV, wherein the mixture is free from lentil-lectins including concanavalin A and from monoclonal antibodies.
s In accordance with another aspect of the present invention, there is provided an immunogenic preparation comprising an immunoeffective amount of the mixtures provided herein.
The immunogenic compositions provided herein may be io formulated as a vaccine containing the F, G and M
proteins for in vivo administration to a host, which may be a primate, specifically a human host, to confer protection against disease caused by RSV.
The immunogenic compositions of the invention may zs be formulated as microparticles, capsules, ISCOMs or liposomes. The immunogenic compositions may further comprise at least one other immunogenic or immunostimulating material, which may be at least one adjuvant or at least one immunomodulator, such as 2o cytokines including ILK.
The at least one adjuvant may be selected from the group consisting of aluminum phosphate, aluminum hydroxide, QS21, Quil A or derivatives or components thereof, calcium phosphate, calcium hydroxide, zinc 2s hydroxide, a glycolipid analog, an octodecyl ester of: an amino acid, a muramyl dipeptide, polyphosphazene, a lipoprotein, ISCOM matrix, DC-Chol, DDA, and other adjuvants and bacterial toxins, Components and derivatives thereof as, for example, described in USSN
ao 08/258,228 filed ,June 10, 1994, assigned to the assignee hereof and the disclosure of which is incorporated herein by reference thereto (WO 95/34323). Under particular circumstances, adjuvants that induce a Th1 response are desirable.
35 The immunogenic compositions pro~aided herein may be formulated to comprise at least one additional

8 immunogen, which conveniently may comprise a human parainfluenza virus (PIV) protein from PIV-1, PIV-2 and/or PIV-3, such as the PIV F and HN proteins.
However, other immunogens, such as from Ch.~amydia, s polio, hepatitis B, diphtheria toxoid, tetanus toxoid, influenza, haemophilus, B. pertussis, pneumococci, mycobacteria, hepatitis A and Moraxella also may be incorporated into tine compositions, as polyvalent (combination) vaccines.
1o An additional aspect of the present invention provides a method of generating an immune response in a host by administering thereto an immunoeffective amount of the immunogenic composition provided herein.
Preferably, the immunogenic composition is formulated as 15 a vaccine for in viVO administration to the host and the administration to the host., including humans, confers protection against disease caused by RSV. The immune response may be humoral or a cell-mediated immune response.
2o The present invention provides, in an additional aspect thereof, a method of producing a vaccine for protection against disease caused by respiratory syncytial virus (RSV) infection, comprising administering the immunogenic composition provided 2s herein to a test host to determine the amount of and frequency of administration thereof to confer protection against disease caused by a RSV; and formulating the immunogenic composition in a form suitable for administration. to a treated host in accordance with the so determined amount and frequency of administration. The treated host may be a human.
A further aspect of the invention provides a method of determining the presence in a sample of antibodies specifically reactive with an F, U or M protein of 35 respiratory syncytial virus (RSV), comprising the steps of

9 (a) contacting the sample with the mixture as provided herein to produce complexes comprising a respiratory syncytial virus protein and any said antibodies present in s the sample specifically reactive therewith;
and (b) determining producai.on of the complexes.
In a further aspect of the invention, there is provided a method of determining the presence in a Zo sample of a F, G or M protein of respiratory syncytial virus (RSV) comprising the steps of:
(a) immunizing a subject with the immunogenic composition as provided herein, to produce antibodies specific for the F, G and M
i5 proteins of RSV;
(b) contacting the sample with. the antibodies to produce complexes comprising any RSV protein present in the sample and the protein specific antibodies; and 20 (c) determining production of the complexes.
A further aspect of the invention provides a diagnostic kit for determining the presence of antibodies in a sample specifically reactive with a F~ G
or M protein of respiratory syncytial virus, comprising:
25 (a) a mixture as provided herein;
(b) means for contacting the mixture with the sample to produce complexes comprising a respiratory syncytial virus protein and any said antibodies present in the sample; and 30 (c) means for determining production of the complexes.
In an additional aspect of the invention, there is provided a method of producing monoclonal antibodies specific for F, G or M proteins of respiratory syncytial 35 virus (RSV), comprising:

WO 98102457 PC'fIGA97100497 (a) administrating an immunogenic composition as provided herein to at least one mouse to produce at least one immunized mouser (b) removing B-lymphocytes from the at least one s immunized mouse;
(c) fusing the B-lymphocytes from the at least one immunized mouse with myeloma cells, thereby producing hybridomas;
(d) cloning the hybridomas which produce a to selected anti-RSV protein antibody;
(e) culturing the selected anti-RSV protein antibody-producing clones; and (f) isolating anti-RSV protein antibodies from the selected cultures.
is The present invention, in a further aspect, provides a method of producing a coisolated and copurified mixture of proteins of respiratory syncytial virus, which comprises growing RSV on cells in a culture medium, separating the grown virus from the culture zo medium, solubilizing at least the F, G and M proteins from the separated virus: and coisolating and copurifying the solubilized RSV proteins.
The coisolation and copurification may be effected by loading the solubilized proteins onto an ion-exchange 2s matrix, preferably a calcium phosphate matrix, specifically a hydroxyapatite matrix, and selectively coeluting the F, G ar._d M proteins from the ion-exchange matrix. The grown virus may first be washed with urea to remove contaminants without substantially removing F, 3o G and M proteins.
Advantages of the present invention include:
- coisolated and copurified mixtures of F, G and M proteins of RSV;
- immunogenic compositions containing such 35 proteins;
- procedures for isolating such protein; and diagnostic kits for identification of RSV and hosts infected thereby.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1, containing panels a and b, shows SDS-PAGE
analysis of a purified RSV A subunit preparation using acrylamide gels stained with silver, under both reduced (panel (a)) and non-reduced (panel (b)) conditions;
Figure 2, containing panels a, b, c and d, shows Western blot analysis of a purified RSV subunit io preparation under reduced conditions;
Figure 3, containing panels a, b, c and d, shows Western blct analysis of a purified RSV subunit preparation under non-reduced conditions: and Figure 4 shows SDS-PAGE analysis of a purified RSV
B subunit preparation using acrylamide gels stained with silver under reduced conditions.
GENERAL DESCRIPTION OF INVENTION
As discussed above, the present invention provides the F, G and M proteins of RSV coisolated and copurified 2~ from RS virus. The virus is grown on a vaccine quality cell line, such as VERO cells and human diploid cells, such as MRCS and WI38, and the grown virus is harvested.
The fermentation may be effected in the presence of fetal bovine serum (FBS) and trypsin.
The viral harvest is filtered and then concentrated, typically using tangential flow ultrafiltration with a membrane of desired molecular weight cut-off, and diafiltered. The virus harvest concentrate may be centrifuged and the supernatant ao discarded. The pellet following centrifugation may first be washed with a buffer containing urea to remove soluble contaminants while leaving the F, G and M
proteins substantially unaffected, and then recentrifuged. The pellet from the centrifugation then 3s is detergent extracted to solubilize the F, G and M
proteins from the pellet. Such detergent extraction may be effected by resuspending the pellet to the original harvest concentrate volume in an extraction buffer containing a detergent, such as a non-ionic detergent, including TRITON~ X-100, a non-ionic detergent which is s octadienyl phenol (ethylene glycol}lo. Other detergents include octylglucoside and Mega detergents.
Following centrifugation to remove non-soluble proteins, the F, G and M protein extract is purified by chromatographic procedures. The exi~ract may first be io applied to an ion exchange chromatography matrix to permit binding of the F, G and M proteins to the matrix while impurities are permitted to flow through the column. The ion-exchange chromatography matrix may be any desired chromatography material, particularly a is calcium phosphate matrix, specifically hydroxyapatite, although other materials, such as DEAF and TMAE and others, may be used.
The bound F, G and M proteins then are coeluted from the column by a suitable eluant. The resulting zo copurified F, G and M proteins may be further processed to increase the purity thereof.
The purified F, G and M proteins employed herein may be in the form of homo and hetero oligomers including F:G heterodimers and including dimers, 2s tetramers and higher species. The RSV protein preparations prepared following this procedure demonstrated no evidence of any adventitious agent, hemadsorbing agent or live virus.
Groups of cotton rats were immunized so intramuscularly with the preparations provided herein in combination with alum or IscomatrixTM as adjuvant.
Strong anti-fusion and neutralization titres were obtained, as shown in Tables 1 and 2 below. Complete protection against virus infection was obtained in the 3s upper and lower respiratory tracts, as shown in Tables 3 and 4 below.

WO 98!02457 PCT/CA97/0049?

In addition, groups of mice were immunized intramuscularly with the preparation provided herein in combination with alum, IscomatrixT'~, polyphosphazene and DC-chol as adjuvant. Strong neutralizing and anti-F
s antibody titres were obtained, as shown in Tables 5 and 6 below. In addition, complete protection against virus infection was obtained, as shown by the absence of virus in lung homogenates (Table 7 below).
Groups of monkeys also were immunized with the 1o preparations provided herein in combination with alum or IscomatrixT"' as adjuvant. Strong neutralizing titres and anti-F antibody titres were obtained, as shown in Tables 8 and 9 below.
The animal immunization data generated herein demonstrate that, by employing mild detergent extraction of the major RSV proteins from virus and mild salt elution of the proteins from the ion-exchange matrix, there are obtained copurified mixtures of the F, G and M
RSV proteins which are capable of eliciting an immune zo response in experimental animals models that confers protection against RSV challenge.
The invention extends to the mixture of F, G and M
proteins from respiratory syncytial virus for use as a pharmaceutical substance as an active ingredient in a 2s vaccine against disease caused by infection with respiratory syncytial virus.
In a further aspect, the invention provides the use of F, G and M proteins from respiratory syncytial virus for the preparation of a vaccinal composition for s~ immunization against disease caused by infection with respiratory syncytial virus.
It is clearly apparent to one skilled in the art, that the various embodiments of the present invention have many applications in the fields of vaccination, as diagnosis and treatment of respiratory syncytial virus infections, and the generation of immunological agents.

WO 98102457 PC~1CA97I00497 A further non-limiting discussion of such issue is further presented below.
L. Vaccine Preparation and Use Immunogenic compositions, suitable to be used as s vaccines, may be prepared from mixtures comprising immunogenic F, G and M proteins of RSV as disclosed herein. The immunogenic composition elicits an immune response which produces antibodies, including anti-RSV
antibodies including anti-F, anti-G and anti-M
1o antibodies. Such antibodies may be viral neutralizing and/or anti-fusion antibodies.
Immunogenic compositions including vaccines may be prepared as injectables, as liquid solutions, suspensions or emulsions. The active immunogenic is ingredient or ingredients may be mixed with pharmaceutically acceptable excipients which are compatible therewith. Such excipients may include water, saline, dextrose, glycerol, ethanol, and combinations thereof. The immunogeni.c compositions and 2o vaccines may further contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness thereof.
Immunogenic compositions and vaccines may be administered parenterally, by injection subcutaneous, 25 intradermal or intramuscularly injection. Alternatively, the immunogenic compositions formed according to the present invention, may be formulated and delivered in a manner to evoke an immune response at mucosal surfaces.
Thus, the immunogenic composition may be administered 3o to mucosal surfaces by, for example, the nasal or oral (intragastric) routes. Alternatively, other modes of administration including suppositories and oral formulations may be desirable. For suppositories, binders and carriers may include, for example, 35 polyalkalene glycols or triglycerides. Such suppositories may be formed from mixtures containing the active immunogenic ingredients) in the range of about 0.5 to about 100, preferably about 1 to 2~. Oral formulations may include normally employed carriers such as, pharmaceutical grades of saccharine, cellulose and 5 magnesium carbonate. These compositions can take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 1 to 950 of the active ingredient(s), preferably about 20 to about 75~.
to The immunogenic preparations and vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, immunagenic and protective.
The quantity to be administered depends on the subject 15 to be treated, including, for example, the capacity of the individual's immune system 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 2o practitioner. However, suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of micrograms to milligrams of the active ingredients) per vaccination. Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent booster administrations. The dosage may also depend on the route of administration and will vary according to the size of the host.
The concentration of the active ingredient protein 3o in an immunogenic composition according to the invention is in general about 1 to 950. A vaccine which contains antigenic material of only one pathogen is a monovalent vaccine. Vaccines which contain antigenic material, of several pathogens are combined vaccines and also belong as to the present invention. Such combined vaccines contain, for example, material from various pathogens or from various strains of the same pathogen, or from combinations of various pathogens. In the present invention, as noted above, F, G and ~I proteins of RSV A
and RSV B are combined in a single multivalent s immunogenic composition which also may contain other immunogens.
Immunogenicity can be significantly improved if the antigens are co-administered with adjuvants. Adjuvants enhance the immunogenicity of an antigen but are not to necessarily immunogenic themselves. Adjuvants may act by retaining the antigen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of antigen to cells of the immune system. Adjuvants can also attract cells of the 15 immune system to an antigen depot and stimulate such cells to elicit immune responses.
Immunostimulatory agents or adjuvants have been used for many years to improve the host immune responses to, for example, vaccines. Intrinsic adjuvants, such as 20 lipopolysaccharides, normally are the components of the killed or attenuated bacteria used as vaccines.
Extrinsic adjuvants are immunomodulators which are formulated to enhance the host immune responses. Thus, adjuvants have been identified that enhance the immune 25 response to antigens delivered parenterally. Some of these adjuvants are toxic, however, and can cause undesirable side-effects, making them unsuitable for use in humans and many animals. Indeed, only aluminum hydroxide and aluminum phosphate {collectively commonly so referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum in increasing antibody responses to diphtheria and tetanus toxoids is well established and a HBsAg vaccine has been adjuvanted with alum. While the usefulness of alum is s5 well established for some applications, it has limitations. For example, alum is ineffective for influenza vaccination and usually does not elicit a cell mediated immune response. The antibodies elicited by alum-adjuvanted antigens are mainly of the IgG1 isotype in the mouse, which may not be optimal for protection by s some vaccinal agents.
A wide range of extrinsic adjuvants can provoke potent immune responses to antigens. These include saponins complexed to membrane protein antigens (immune stimulating complexes), pluronic polymers with mineral 2o oil, killed mycobacteria in mineral oil, Freund's incomplete adjuvant, bacterial products, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A, and liposomes.
To efficiently induce humoral immune responses i5 (HIR) and cell-mediated immunity (CMI), immunogens are often emulsified in adjuvants. Many adjuvants are toxic, inducing granulomas, acute and chronic inflammations (Freund's complete adjuvant, FCA), cytolysis (saponins and Pluronic polymers) and za pyrogenicity, arthritis and anterior uveitis (ZPS and MDP). Although FCA is an excellent adjuvant and widely used in research, it is not licensed for use in human or veterinary vaccines because of its toxicity.
2 . Inonnunoas says 2s The F, G and M proteins of RSV of the present invention are useful as immunogens for the generation of antibodies thereto, as antigens in immunoassays including enzyme-linked immunosorbent assays (ELISA), RIAs and other non-enzyme :Linked antibody binding assays 30 or procedures known in the art for the detection of antibodies. In ELISA assays. the selected F, G or M
protein or a mixture of proteins is immobilized onto a selected surface, for example, a surface capable of binding proteins such as the wells of a polystyrene 3s microtiter plate. After washing to remove incompletely adsorbed material, a nonspecific protein, such as a solution of bovine serum albumin (BSA) that is known to be antigenically neutral with regard. to the test sample may be bound to the selected surface. This allows for blocking of nonspecific adsorption sites on the s immobilizing surface and thus reduces the background caused by nonspecific binding of proteins in the antisera onto the surface.
The immobilizing surface is they contacted with a sample, such as clinical or biological materials, to be 1o tested in a manner conducive to immune complex (antigen/antibody) formation. This may include diluting the sample with diluents, such as solutions of BSA, bovine gamma globulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sample is then allowed to ~5 incubate for from about 2 to 4 hours, at temperatures, such as of the order of about 25~ to 37~C. Following incubation, the sample-contacted surface is washed to remove non-immunocomplexed material. The washing procedure may include washing with a solution, such as 2o PBS/Tween or a borate buffer. Following formation of specific immunocomplexes between the test sample and the bound protein, and subsequent washing, the occurrence, and even amount, of immunocomplex formation may be determined by subjecting the immunocomplex to a second 25 antibody having specificity for the first antibody. If the test sample is of human origin, the second antibody is an antibody having specificity for human immunoglobulins and in general IgG. To provide detecting means, the second antibody may have an ao associated activity such as an enzymatic activity that will generate, for example, a color development upon incubating with an appropriate chromogenic substrate.
Quantification may then be achieved by measuring the degree of color generation using, for example, a 3s spectrophotometer.

WO 98/02457 PC'f/CA97100497 EXA~3P?aES
The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific s Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although Zo specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.
Methods of determining tissue culture infectious dose5o (TCIDSO/mL), plaque and neutralization titres, not ~.s explicitly described in this disclosure are amply reported in the scientific literature and well within the scope of those skilled in the art. Protein concentrations were determined by the bicinchoninic acid (BCA) method as described in the Pierce Manual {23220, 20 23225; Pierce Chemical company, U.S.A.), incorporated herein by reference.
CMRL 1969 and Iscove's Modified Dulbecco"s Medium {IMDM) culture media were used for cell culture and virus growth. The cells used in this study are vaccine 2s quality African green monkey kidney cells (VERO lot M6) obtained from Institut Merieux. The RS viruses used were the RS virus subtype A (Long and A2 strains) obtained from the American Type culture Collection (ATCC), a recent subtype A clinical isolate and RSV
so subtype B clinical isolate from Baylor College of Medicine.
Example l:
This Example illustrates the production of RSV on a mammalian cell line on microcarrier beads in a 150 L
3s controlled fermenter.

WO 98!02457 PC'fJCA97JOd1497 Vaccine quality African green monkey kidney cells (VERO) at a concentration of 105 cellslmL were added to 60 L of CMRL 1969 medium, pH 7.2 in a 150 L bioreactor containing 360 g of Cytodex-1 microcarrier beads and s stirred for 2 hours. An additional 60 L of CMRL 1969 was added to give a total vclume of 120 L. Fetal bovine serum was added to achieve a final concentration of 3.50. Glucose was added to a final concentration of 3 g/L and L-glutamine was added to a final concentration so of 0.6 g/L. Cissolved oxygen (40~), pH {7.2), agitation (36 rpm), and temperature {37~C) were controlled. Cell growth, glucose, lactate, and glutamine levels were monitored. At day 9, the culture medium was drained from the fermenter and 100 L of E199 media (no fetal i5 bovine serum) was added and stirred for 10 minutes. The fermentor was drained and filled again with 120 L of E199.
An RSV inoculum of RSV subtype A was added at a multiplicity of infection (M.O.S.) of 0.001 and the 2o culture was then maintained for 3 days before one-third to one-half of the medium was drained and replaced with fresh medium. On day 6 post-infection, the stirring was stopped and the beads allowed to settle. The viral culture fluid was drained and filtered through a 20 ~m 2s filter followed by a 3 ~m filter prior to further processing.
The clarified viral harvest was concentrated 75- to 150-fold using tangential flow ultrafiltration with 300 NMWL membranes and diafiltered with phosphate buffered so saline containing loo glycerol. The viral concentrate was stored frozen at -70°~C prior to further purification.
Example 2:
This Example illustrates the process of purifying 35 RSV subunit from a viral concentrate of RSV subtype A.

A solution of 50°s polyethylene glycol-8000 was added to an aliquot of virus concentrate prepared as described in Example 1 to give a final concentration of 6~. After stirring at room temperature for one hour, s the mixture was centrifuged at 15,000 RPM for 30 min in a Sorvall SS-34 rotor, at 4'C. The viral pellet was suspended in 1 mM sodium phosphate, pH 6.8, 2 M urea, 0.15 M NaCl, stirred for 1 hour at room temperature, and then recentrifuged at 15,000 RPM for 30 min. in a Zo Sorvall SS-34 rotor at 4~C_ The viral pellet was then suspended in 1 mM sodium phosphate, pH 6.8, 50 mM NaCl, 1~ Triton X-1.00 and stirred for 30 minutes at room temperature. The insoluble virus core was removed by centrifugation at 15,000 RPM for 30 min. in a Sorval SS-i5 34 rotor at 4'C. The soluble protein supernatant was applied to a column of ceramic hydroxyapatite (type II, Bio-Rad Laboratories) and the column was then washed with five column volumes of 1 mM sodium phosphate, pH
6.8, 50 mM NaCl, 0.02 Triton X-100. The RSV subunit zo composition from RSV subtype A, containing the F, G and M proteins, was obtained by eluting the column with 10 column volumes of 1 mM sodium phosphate, pH 6.8, 400 mM
NaCl, 0.020 Triton X-100.
Example 3:
25 This Example illustrates the analysis of RSV
subunit preparation obtained from RSV subtype A by SDS
polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting.
The RSV subunit composition prepared as described 3o in Example 2 was analyzed by SDS-PAGE using 1.2.5 acrylamide gels. Samples were electrophoresed in the presence or absence of 2-mercaptoethanol (reducing agent) . Gels were stained with silver stain to detect the viral proteins (Figure 1, panels a and b).
ss Immunoblots of replicate gels were prepared and probed with a mouse monoclonal antibody (mAb 5353C75) to F

dV0 98!02457 PCT/CA97/UU497 glycoprotein (Figures 2, panel a and 3, panel a), or a mouse monoclonal antibody (mAb 131-2G), to G
glycoprotein (Figures 2, panel b and 3, panel b} or guinea pig anti-serum (gp178) against an RSV M peptide s (peptide sequence: LKSKNMLTTVKDLTMKTLNPTHDIIALCEFEN -SEQ ID No:1) (Figures 2, panel c and 3, panel c), or goat antiserum (Virostat #0605) against whole RSV
(Figures 2, panel d and 3, panel d). Densitometric analysis of the silver-stained gel of the RSV subunit to preparation electrophored under reducing conditions indicated a compositional distribution as follows:
G glycoprotein (95 kDa form} - 10$
F1 glycoprotein (48 kDa) - 300 M protein (31 kDa) - 23%
is F2 glycoprotein (23 kDa) - 190 The F glycoprotein migrates under non-reducing conditions as a heterodimer of approximately 70 kDa (Fo) as well as higher oligomeric forms (dimers and trimers) (Figure 3, panel a).
2o Example 4:
This Example illustrates the immunogenicity of the RSV subunit preparation in cotton rats.
Groups of five cotton rats were immunized intramuscularly (0.1 mL on days 0 and 28 with ~. )Zg or 10 2s pig the RSV subunit preparation, produced as described in Example 2 and formulated with either 1.5 mg/dose alum or ~.g/dose IscomatrixTM (Iscotec, Sweden). Blood samples were obtained on day 41 and assayed for anti-fusion titres and neutralization titres. The rats were so challenged intranasally on day 43 with RSV and sacrificed four days later. Lavages of the lungs and naso pharynx were collected and assayed for RSV titres.
Strong anti-fusion and neutralizing antibody titres were induced as shown in Tables 1 and 2 below. In 3s addition, complete protection against virus infection was obtained with the exception of one rat, in both the WO 98102457 PC'~°/CA9710049'~
2~
upper arid lower respiratory tracts (Tables 3 and 4 below).
Example 5:
This Example illustrates the immunagenicity of the s RSV subunit preparation in mice.
Groups of six BALB/c mice were immunized intramuscularly (0.1 mL) on days 0 and 28 with various doses of the RSV subunit preparation, produced as described in Example 2 and formulated with either 1.5 1o mg/dose alum, 10 ~.g/dose IscomatrixTM, 200 ~,g/dose polyphosphazene (PCPP) or 200 ~g/dose DC-chol. The various preparations tested are set Earth in Tables 5, 6 and 7 below. Blood samples were obtained on days 28 and 42 and assayed for neutralizing antibody titres and i5 anti-F antibody titres. The mice were challenged on day 44 with RSV and sacrificed four days later. Lungs were removed and homogenized to determine virus titres.
Strong neutralization titres and anti-F antibody titres ware elicited as shown in Tables 5 and 6 below. In 2o addition, complete protection against virus infection was obtained as shown by the absence of virus in lung homogenates and nasal washes (Table l below).
Example 6;
This Example illustrates the immunogenicity of RSV
2s subunit preparation in African green monkeys.
Groups of four monkeys were immunized intramuscularly (0.5 mL on days 0 and 21 with 100 ~,g of the RSV subunit preparation, produced as described in Example 2 and formulated with either 2.5 mg/dose alum or 30 50 ~.g/dose IscomatrixTM. Blood samp~.es were obtained on days 21, 35 and 49 and assayed for neutralizing and anti-F antibody titres. Strong neutralizing and anti-F
antibody titres were obtained as shown in Tables 8 and 9 below.

Example 7:
This Example further illustrates the production of RSV or a mammalin cell live or microbeads in a 150L
controlled fermenter.
s Vaccine quality African green monkey kidney cells (Vero cells) were added to 150L of: Iscove's Modified Dulbecco°s Medium (IMDM) containing 3.5% fetal bovine serum, pH 7.2, to a final concentration of 2 x 105 cells/mL (range 1.5 to 3.5 cells/mL), in a 150 L
Zo bioreactor containing 450 g of Cytodex-1 microcarrier beads (3 g/L). Following cell inoculation, dissolved oxygen (40 percent air saturation (range 25 to 400), pH
(7.1 ~ 0.2)), agitation (36 ~ 2 rpm), and temperature (37° ~ 0.5°C) were controlled. Initial cell attachment 15 to beads, cell growth (cell number determination), and growth medium levels of glucose and lactate were monitored on a daily basis. Infection of the Vero cell culture occurred three to four days following initiation of cell growth, when the concentration of cells was in 2o the range 1.5 to 2.0 x 106 cells/mL. Agitation was stopped and the microcarrier beads were allowed to settle for 60 minutes and the culture medium was drained from the bioreactor using a drain line placed approximately 3 cm above the settled bead voi.ume.
z5 Seventy-five L of IMDM without fetal bovine serum (wash medium) was added and the mixture stirred at 36 rprr~ for minutes. The agitation was stopped and the microcarrier beads allowed to settle for 30 minutes. The wash medium was removed using the drain line and then 3a the bioreactor was filled to 75 L (half volume) with IMDM without fetal bovine serum.
For infection, an RSV inoculum of RSV subtype F3 was added at a multiplicity of infection (M.O.I.) of 0.001 and virus adsorption to cells at half volume was carried ss out for 2 hours with stirring at 36 rpm. Seventy-five L
of IMDM was then added to the bioreactor to a final W~ 98/02457 PCf'/CA97l00497 volume of 150 L. Following infection, dissolved oxygen (40 percent air saturation (range 10 -- 400)), pH (7.25 ~
0.1) , agitation (36 -!- 2 rpm) and temperature (3'7° ~
0.5°C) were controlled. Following infection, cell growth s (cell number determination) medium, glucose and lactate levels, RSV F and G antigens and RSV infectivity were monitored on a daily basis. On day 3 following infection, agitation was stopped, the microcarrier beads were allowed to settle for 60 minutes, and 75 L (500) of 1o the medium was removed via the drain line and replaced with fresh medium. Eight days (range seven to nine days) following infection, when complete virus-induced cytopathic effect was observed (i.e. cells were detached from the microcarrier beads, and oxygen was no longer 15 being consumed by the culture), the agitator was stopped and the microcarrier beads were allowed to settle for 60 minutes. The virus containing culture fluid was removed from the bioreactor and transferred to a holding vessel.
Seventy-five L of IMDM without fetal bovine serum was 2o added to the bioreactor and agitated at 75 rpm for 30 minutes. The microcarrier beads were allowed to settle for 30 minutes, the rinse fluid was removed from the bioreactor and combined with the harvested material in the holding vessel.
2s The harvested material was concentrated approximately 20-fold by tangential flow filtration (i.e. virus-containing material was retained by the membrane) using a 500 or 1000 kilodalton (K) ultrafiltration membrane or alternatively a 0.45 ~.M
3o microfiltration membrane to a final volume of 10L. The concentrated material was diafiltered with 10 volumes of phosphate-buffered saline, pH 7.2. The diafiltered viral concentrate was stared frozen at -70°C prior to further purification.

1~V~ 98/02457 PCT'/CA97100497 Example 8:
This Example illustrates the process of purifying RSV subunit from a viral concentrate of RSV subtype B.
A virus concentrate, prepared. as described in s Example 7, was centrifuged at 15,000 rpm for 30 min in a Sorvall SS-39 rotor at 4°C. The viral pellet was then suspended in 1 mM sodium phosphate, pH 6.8, 300 mM NaCl, 2o Triton X-100 and stirred for 30 minutes at room temperature. The insoluble virus core was removed by Zo centrifugation at 15,000 RPM for 30 min in a Sorval SS-34 rotor at 4°C. The soluble protein supernatant was applied to a column of ceramic hydroxyapatite (type I, Bio-Rad Laboratories) and the column was then washed with ten column volumns of 1 mM sodium phosphate, pH
Zs 6.8, 10 mM NaCl, 0.020 Triton X-100. The RSV subunit composition, containing the F, G and M protein, was obtained by eluting the column with 10 column volumes of 1 mM sodium. phosphate, pH 6.8, 600 mM NaCl, 0.02% Triton X-100. In some instances, the RSV subunit composition 2o was further purified by first diluting the eluate from the first ceramic hydroxyapatite column to 7_ower the NaCl concentration to 900 mM NaCl and then applying the diluted subunit onto a column ef ceramic hydroxyapatite (type II, Bio-Rad Laboratories). The flowthrough from zs this column is the purified RSV subunit composition from RSV subtype B.
Example 9:
This Example illustrates the analysis of RSV
subunit preparation obtained from RSV subtype~B by SDS
3o polyacryamide gel electrophoresis (SDS-PAGE).
The RSV subunit composition prepared as described in Example 8 was analyzed by SDS-PAGE using a 15.0%
acrylamide gel. The sample was electrophoresed in the presence of 2-mercaptoethanol (reducing agent). The gel as was stained with silver stain to detect the viral proteins (Figure 4). Densitometric analysis of the WO 98/02457 fC7~/CA97IOU497 silver--stained gel of the RSV subunit preparation under reducing conditions indicated a compositional distribution of the proteins as follows:
G glycoprotein {95 kDa form) - 210 s F1 glycoprotein (48 kDa) - 19~
M protein {31 kDa) - 220 F2 glycoprotein (23 kDa) - 20o SUMMARY OF DISCLOSURE
In summary of this disclosure, the present Zo invention provides a coisolated and purified mixture of F, G and M proteins of RSV which is able to protect against RSV in relevant animal models of infection.
Modifications are possible within the scope of this invention.

Table 1 - Serum Ant i-Fusion Titres in Cotton Rats ~Crroup :Mean titre Std. Dev llo~i} (loRa}

Alum lacebo 2.0 0 0 Iscomatrix''" facebo 2.3 O.S

RSV Subunit 1 with Alum 8.0 1.4 ~ 7.5 1.C~
RSV Subunit IO with Alum RSV Subunit 1 with Iscomatrix~'"10.4 1.3 RSV Subunit 10 ~t with Iscomatrix~"10.0 I.6 Table 2 - Serum lYeutralization T'strrs in Cotton Rats Grou ~ Mean titre _Std, Dev.
o ~ to z Alum lacebo 2.0 0.0 Iscomatrix'"' lacebo 2.0 0.0 RSV Subunit 1 with Alum 9.6 1.3 RSV Subuttit 10 with Alum 10.0 1.4 RSV Subunit I with Iscomatrix~I0.6 1. 1 RSV Subunit 10 ~cg with 1 1.2 ~ 1.!
Iscotnatrixn'' ~

Table 3 ~ Putmoas Wash RSV Titres en Cottoa Rats Group Mean titre Std. Dev.
Alum lacebo 3.8 0.4 ~

Iscomatrix lacebo 3,7 0.5 _ -RSV Subunit 1 with Alum 0.4 0.8 ~

RSV Subutvi 10 with Alum 0.0 0.0 R5V Subutut 1 with Iscomatrix~0.0 0.0 RSV Subutut 10 ua with Iscomattix4.0 I 0.0 Tgble d . Ngs't Wish R.SV Titr~t ie Cettnn Rats Group Mean titre Std Dev.

~!o ,d Jun (Io td lun Alum lacebo 3.2 0.5 Iscomatrix tatcebo 3.1 0.3 RSV Subuttit ! with Alum 0.0 0.0 RSV Suburut 10 with Alum 0.0 0.0 RSV Subunit ! with Iscomatrix'~0.0 0.0 RSV Subuttit t0 a with 0.0 0.0 iscomatrix~

Table 5 ~ Serum Neutralization Titrrs in Balb/c i~Iice 4 Week ~~ 6 Bleed Week Bleed Group Mean Std. Meaa Std.
titre Dev. titre Dev.
(lo to z) to z) (lo z) z Alum lacebo 3.0 0.0 3.0 0.0 Iscomatrix 3.0 0.0 3.0 0.0 ' lacebo PCPP lacebo ND ND 3.0 0.0 200 ND ND 3.0 0.0 DC-Chol lacebo RSV Subunitwith no ad'uvantND ND 3.0 0.0 0.1 RSV Subunitwith Alum ND ND 10.3 0.9 0.1 RSV Subunitwith Alum 6.5 O.b 8.7 I.0 RSV Subunitwith Alum 8.0 1.1 9.5 1.1 RSV SubunitwithIscomatrix~8.2 0.8 _ 1.0 1 t3.2 RSV Subuaitwith Iscomatrix10.4 1.3 13.4 0.6

10 RSV Subuaitwith PCPP 200 ND ND 15.0 0.6 RSV Subunitwith DC-Chol ND STD 11.7 1.1 0.5 200 minimal detectable titre in assay ND = not determined Table 6 - Serumt1-F _ Ac~ Titres in BalbJc Mice 4 Week 6 Week Bleed Bleed Group Mean Std. Mean Std.
titre Dev. titre Dev.
to ~titrel100to ititrcf100(to ~tiuel100to nitrd?00 Ah,tm lacebo 0.5 I.2 0.0 0.0 Iscomatrix 1.0 0.0 0.0 0.0 lacebo PCPP lacebo 0.0 0.0 0.0 0.0 DC-Chol 200 0.0 0.0 0.0 0.0 lacebo RSV Subunitwith no ad' 0.0 0.0 0Ø 0.0 0.1 vent RSV Subunitwith Alum 7.0 1.0 i 2.4 0.9 0.1 RSV Subunitwith Alum 8.7 0.8 1 I.2 0.8 RSV Subunitwith Alum 9.7 0.8 12.3 1.0 RSV Subunitwith Iscomatrix'~8.5 0.6 13.3 0.5 RSV Subunitwith Iscomatrix10.0 0.0 13.0 0.0 10 with PCPP 200 10.2 0.8 14.0 0.7 RSV Subunit LRSV Subunitw:; with DC-Chol9.7 ~ 1.4 13.0 1,0 0.5 {200 ug) ' ~ ~

VVO 98/02457 PCT'/CA97/00497 Table 7 - Lung Virus 'Titres in l3alb/c Mice Group Mean titre Std. Dev.

_ (lo ,o/~ lun ) to ,o/ lun Alum lacebo 4.I 0.2 Iscomatrix 3.5 0.I
lacebo PCPP lacebo 5.2 0.2 DC-Chol lacebo200 S.0 0.3 RSV Subunit with no ad'uvant5.3 0.1 0. I

RSV Subunit with Alum <1.7 1.7 0.1 RSV Subunit with Alum <1.7 1.7 RSV Subunit with Alum <1.7 1.7 RSV Subunit with Iscomatrix~<1.7 1.7 RSV Subunit with Iscomatrix~<1.7 1.7 RSV Subunit with PCPP 200 <1.7 1.7 RSV Subunit g with DC-Chol <1.7 1.7 0.5 u {200 ~,g) minimal detectable virus titre in assay W~ 98102457 PCT"lCA97100497 y o o ~ 6n D ~ ~ ~ - o a v ...
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WO 98!02457 PC7.'/CA97100497 -~ n r,r, ro r.nm. r.~ n i. Gle2en, W.P., Pareses, A. Allison., J.E., Taber, L.H.
and Frank, A.L. (1981). J. Pediatr. 98, 708-715.
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Lance! ii, 1035-1038.
4. Robbins, A., and Freeman, P. (1988) Sci. Am. 259, 126-i33.
5. Glezen, W.P., Taber, L.H., Frank, A.L, and Kasel, J.A. (1986) Am. J. Dis. Child. 140, 143-146.
6. Katz, S.L. New vaccine development establishing priorities. Vol. 1. Washington National Academic Press. (1985) pp. 397-909.
7. Wertz, G.W., Sullender, W.M. (1992) Biotech. 20, 151-176.
8. McIntosh, K. and Chanock, R.M. (1990) in Fields Virology (Fields, B.M., and Knipe, D.M. eds.) pp.
1045-1075, Raven Press, Ltd., New York.
9. Collins, P., McIntosh, K., and Chanock, R.M. in "Fields Virology" ed. by Fields, B.N., Kni.pe, D.M., and Howley, P.M., Lippincott-Raven Press, New York, (1995) pp. 1313-1351.
10. Walsh, E.E., Hall, C.B., Briselli,. M., Brandiss, M.W.
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12. Levine, S., Kleiber-France, R., and Paradiso, P.R.
(1987) J. Gen. Virol. 69, 2521-2524.

13. Anderson, L.J. Hierholzer, J.C., Tsou, C., Hendry, R.M., Fernie, B.F., Stone, Y. and Mclntosh, K.
(1985), J. Infect. Dis. 151, 626-a33.

14. Johnson, P.R., Olmsted, R.A., Prince, G.A., Murphy, B.R., Alling. D.W., Walsh, E.E. and Collins, P.L.
(1987) J. Virol. 61 (10), 3163-316.

15. Cherrie, A.H., Anderson, K., Wertz, G.W., and Openshaw, P.J.M. (1992) J. Virology 65, 2102-2110.

WO 98102457 PC'~'/CA97100497

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17. Firedewald, W.T., Forsyth, B.R., Smith, C.B., Gharpure, M.S., and Chanock, R.M. (1968) JAMA 204, 690-694.

18. Walsh, E.E., Brandriss, M.W., Schlesinger, J.J.
(1985) J. Gen. Virol. 66, 409-415.

19. Walsh, E.E., Schlesinger, J.J. and Brandriss, M.W.
(1984) J. Gen. Virol. 65, 761-766.

20. Routledge, E.G., Willcocks, M.M., Samson, A.C.R., Morgan, L., Scott, R., Anderson, J.J., and Toms, G.L.
(1988) J. Gen. Virology 69, 293-303.

21. Fulginiti, V.A., Elier, J.J., Sieber, O.F., Joyner, I.W., Minamitani, M. and Meiklejohn, G. (1969) Am J.
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22. Chin, J., Magoffin, R.L., Shearer, L.A., Schieble, J.
H. and Lennette, E.H. (1969) Am. J. Epidemiol. 89 (4}, 449-463.

23. Kapikian, A.Z., Mitchell, R.H., Chanock, R.M., Shvedoff, R.A. and Stewart, C.E. (1969) Am. J.
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24. Kim, H.W., Arrobio, J.O., Pyles,, G., Brandt, C.D.
Camargo, E., Chanock, R.M. and Parrott, R.H. (1971) Pediatrics 48, 745-755.

25. Wright, P.F., Belshe, R.B., Kim, H.W., Van Voris, L.P. and Chanock, r.M. (1982) Infect. Immun. 3? (1), 397-400.

26. Wright, P.F., Chinozaki, T. and 1~leet, W. (1976) J.
Pediatr. 88, 931-936.

27. Belshe, R.B., Van Voris, P. and Mufson, M.A. (1982) J. Infect. Dis. 145, 311-319.

28. Murphy, B.R., Prince, G.A., Walsh, E.E., Kim, H.W., Parrott, R.H., Hemming V.G., Rodriguez, W.J., and Chanock, R.M.. J. Clin. Microbiol. (1986), 24(2), 197-202.

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WO 98102457 ~C'~YCA97100497

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(1986b} J. Vircl. 57 (3}, 721-728.

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Claims (36)

What we claim is:

1. A mixture of purified fusion (F) protein, attachment (G) protein and matrix (M) protein of respiratory syncytial virus (RSV).

2. The mixture of claim 1 wherein said fusion (F) protein comprises multimeric fusion (F) proteins.

3. The mixture of claim 2 wherein, when analyzed under non-reducing conditions, said multimeric fusion (F) protein includes heterodimers of molecular weight approximately 70 kDa and dimeric and trimeric forms.

4. The mixture of claim 1 wherein, when analyzed under non-reducing conditions, said attachment (G) protein comprises G protein of molecular weight approximately 95 kDa arid G protein of molecular weight approximately 55 kDa and oligomeric G protein.

5. The mixture of claim 1 wherein, when analyzed by SDS-PAGE under non-reducing conditions, said matrix (M) protein comprises M protein of molecular weight approximately 28 to 34 kDa.

6. The mixture of claim 1 wherein, when analyzed by reduced SDS-PAGE analysis, said fusion (F) protein comprises F1 of molecular weight approximately 48 kDa and F2 of molecular weight approximately 23 kDa, said attachment (G) protein comprises a G protein of molecular weight approximately 95 kDa and a G protein of molecular weight approximately 55 kDa, and said matrix (M) protein comprises an M protein of approximately 31 kDa.

7. The mixture of claim 2 wherein said F, G and M
proteins are present in the relative; proportions of:
F from about 35 to about 70 wt%
G from about 5 to about 30 wt%
M from about 10 to about 40 wt%

8. The mixture of claim 7 wherein, when analyzed by SDS-PAGE under reducing conditions and silver stained, the ratio of F1 of molecular weight approximately 48 kDa to F2 of molecular weight approximately 23 kDa is between 1:1 to about 2:1 by scanning densitometry.

9. The mixture of claim 7 which is at least about 75% pure.

10. The mixture of claim 1 which is devoid of monoclonal antibodies.

11. The mixture of claim 1 which is devoid of lentil lectin and concanavalin A.

12. The mixture of claim 1 wherein said RSV proteins are non-denatured.

13. The mixture of claim 1 wherein said RSV proteins are from one or both of subtypes RSV A and RSV B.

14. A coisolated and copurified mixture of non-denatured proteins of respiratory syncytical virus (RSV), consisting essentially of the fusion (F) protein, attachment (G) protein and matrix (M) protein of RSV, wherein the mixture is free from lectins and is free from monoclonal antibodies.

15. An immunogenic composition comprising an immunoeffective amount of the mixture of claim 1.

16. The immunogenic composition of claim 15 formulated as a vaccine for in vivo administration to a host to confer protection against RSV.

17. The immunogenic composition of claim 15 further comprising at least one adjuvant or at least one immunomodulator.

18. The immunogenic composition of claim 17 wherein the at least one adjuvant is selected from the group consisting of aluminum phosphate, aluminum hydroxide, QS21, Quil A or derivatives or components thereof, calcium phosphate, calcium hydroxide, zinc hydroxide, a glycolipid analog, an octodecyl ester of an amino acid, a muramyl dipeptide, a lipoprotein, polyphosphazene, ISCOM matrix, DC-chol, DDA and bacterial toxins or derivatives thereof.

19. The immunogenic composition of claim 16 wherein the host is a primate.

20. The immunogenic composition of claim 19 wherein the primate is a human.

21. The immunogenic composition of claim 15 further comprising at least one additional immunogen.

22. The immunogenic composition of claim 21 wherein said at least one additional immunogen comprises at least one human parainfluenza virus (PIV) protein selected from the group consisting of PIV-1, PIV-2 and PIV-3.

23. A method of generating an immune response in a host, comprising administering thereto an immunoeffective amount of the immunogenic composition of claim 15.

24. The method of claim 23 wherein said immunogenic composition is formulated as a vaccine for in vivo administration to the host and said administration to the host confers protection against respiratory syncytial virus.

25. A method for producing a vaccine for protection against respiratory syncytial virus (RSV), comprising:
administering the immunogenic composition of claim 15 to a test host to determine the amount of and frequency of administration thereof to confer protection against disease caused by RSV; and formulating the immunogenic composition in a form suitable for administration to a treated host in accordance with said determined amount and frequency of administration.

26. The method of claim 25 wherein the treated host is a human.

27. A method of producing monoclonal antibodies specific for fusion (F) protein, attachment (G) protein and matrix (M) protein of respiratory syncytial virus (RSV), comprising:

(a) administering an immunogenic composition of claim 15 to at least one mouse to produce at least one immunized mouse;

(b) removing B-lymphocytes from the at least one immunized mouse;

(c) fusing the B-lymphocytes from the at least one immunized mouse with myeloma cells, thereby producing hybridomas;
(d) cloning the hybridomas which produce a selected anti-RSV protein antibody;
(e) culturing the selected anti-RSV protein antibody-producing clones; and (f) isolating anti-RSV protein antibodies from the selected cultures.

28. A method of producing a coisolated and copurified mixture of proteins of respiratory syncytial virus (RSV), which comprises:

growing RSV on cells in a culture medium;
separating the grown virus from the; culture medium;
solubilizing at least the fusion (F) protein, attachment (G) protein and the matrix (M) protein from the separated virus; and coisolating and copurifying the solubilized RSV
proteins.

29. The method of claim 28 wherein said coisolation and copurification are effected by:

loading the solubilized proteins onto an ion-exchange matrix; and selectively coeluting the F, G and M proteins from the ion-exchange matrix.

30. The method of claim 29 wherein said ion-exchange matrix is a hydroxyapatite matrix.

31. The method of claim 28 wherein said grown virus is washed with urea to remove contaminants without substantial removal of F, G and M proteins prior to solubilization step.

32. A method of determining the presence in a sample of antibodies specifically reactive with a fusion (F) protein, attachment (G} protein or matrix (M) protein of respiratory syncytial virus (RSV), comprising the steps of:

(a) contacting the sample with the mixture of claim 1 to produce complexes comprising a respiratory syncytial virus protein and any said antibodies present in the sample specifically reactive therewith; and (b) determining production of the complexes.

33. A method of determining the presence in a sample of an F, G or M protein of respiratory syncytial virus, comprising the steps of:

(a) immunizing a subject with the immunogenic composition of claim 15 to produce antibodies specific for F, G and M proteins of RSV;
(b) contacting the sample with the antibodies to produce complexes comprising any RSV protein present in the sample and said protein specific antibodies% and (c) determining production of complexes.

34. A diagnostic kit for determining the presence of antibodies in a sample specifically reactive with a fusion (F) protein, attachment (G) protein or a matrix (M) protein of respiratory syncytial virus comprising:
(a) a mixture of claim 1;
(b) means for contacting the immunogenic composition with the sample to produce complexes comprising a respiratory syncytial virus protein a.nd any said antibodies present in the sample; and (c) means for determining production of the complexes.

35. A mixture of purified fusion (F) protein, attachment (G) protein and matrix (M) protein of respiratory syncytial virus (RSV) for use as a pharmaceutical substance in a vaccine against disease caused by infection with respiratory syncytial virus.

36. The use of a mixture of purified fusion (F) protein, attachment (G) protein and matrix (M) protein of respiratory syncytial virus far the preparation of a vaccinal composition for immunization against disease caused by infection with respiratory syncytial virus.