HK1022704B - Subunit respiratory syncytial virus vaccine preparation - Google Patents

Description

Subunit respiratory syncytial virus vaccine formulations

Technical Field

The invention relates to the field of immunology, in particular to a vaccine preparation for resisting respiratory syncytial virus infection.

Reference to related applications

This application is a continuation of U.S. patent application No. 08/679,061 filed 7/12 1996 in aesthetic countries.

Background

Human respiratory syncytial virus is the major cause of lower respiratory tract infections in infants and adolescents (references 1 to 3-in the tables appearing at the end of this disclosure, each reference in the tables is incorporated herein by reference). Worldwide, 65 million infections occur annually (ref 4). In the united states, 100,000 people of a single child in a year are likely to be required to go to a hospital for pneumonia and bronchiolitis caused by RS virus (references 5, 6). The cost of hospitalization and ambulatory treatment offered to RS virus-infected children in the united states is over $340 million each year (reference 7). Severe lower respiratory tract disease caused by RS virus infection occurs mainly in infants between 2 and 6 months (reference 8). Approximately 4,000 infants die each year in the united states from complications arising from severe respiratory disease caused by infection with RS virus and parainfluenza virus type c (PIV-3). The World Health Organization (WHO) and the National Institute of Allergy and Infectious Disease (NIAID) vaccine advisory committee have ranked vaccine development of RS virus second only to HIV.

The structure and composition of RSV has been elucidated and described in detail in the textbook "virology field", Fields, B.N. et al, Raven Press, N.Y. (1996), in particular "respiratory syncytial Virus" Collins, P., McIntosh, K., and Chanock, R.M., chapter 44, page 1313-1351 (reference 9).

The two major protective antigens of RSV are the envelope fusion (F) and attachment (G) glycoproteins (ref 10). The F protein is present as an approximately 68 kilodalton precursor molecule (F)₀) Synthetically produced by proteolytic cleavage into disulfide-linked F₁(about 48 kilodaltons) and F₂(about 20 kilodaltons) (reference 11). The G protein (about 33 kDalton) is heavily 0-glycosylated, resulting in a glycoprotein with an apparent molecular weight of about 90 kDalton (reference 12). Two broad-spectrum RS virus subtypes have been identified as a and B (reference 13). The major antigenic differences among these subtypes are in the G glycoprotein, while the F glycoprotein is highly conserved (ref 7, 14).

In addition to the antibody response produced by the F and G glycoproteins, RSV-produced human cytotoxic T cells have been shown to recognize RSV F protein, matrix protein M, nucleoprotein N, small hydrophobic protein SH, and non-structural protein 1b (reference 15).

A safe and effective RSV vaccine is not yet available and is urgently needed. Approaches to develop vaccines for RS viruses include inactivation of the virus with formalin (ref 16), isolation of cold-adapted and/or temperature-sensitive mutant viruses (ref 17), and purification of the F or G glycoprotein (ref 18, 19, 20). Clinical experimental results have shown that neither live attenuated nor formalin inactivated vaccines are able to properly protect against RS virus infection (references 21 to 23). Problems encountered with attenuated cold-adapted and/or temperature sensitive RS virus mutants administered intranasally include clinical morbidity, genetic instability and over-attenuation (references 24 to 26). Live RS virus vaccines administered subcutaneously are also not amenable (reference 27). Inactivated RS virus vaccines are typically prepared using formaldehyde as the inactivating agent. Murphy et al (ref.28) have reported information on immune responses in infants and children immunized with formalin-inactivated RS-virus. Infants (2 to 6 months) developed high titer antibodies to the F glycoprotein, but responded less to the G protein. Older children (7 to 40 months) developed F and G antibody titers compared to those infected with RS virus in infancy. However, both infants and young children develop lower levels of neutralizing antibodies than individuals of the same age infected with the native RS virus. This unbalanced immune response has high and low neutralizing antibody titers to the F (fusion) and G (attachment) proteins of the major immunogenic RS viruses, in part because important epitopes in the F and G glycoproteins are altered by formalin treatment. In addition, some infants receiving formalin inactivated RS virus vaccine developed more severe lower respiratory tract disease after subsequent exposure to native RS virus than non-immunized individuals (references 22, 23). Therefore, formalin inactivated RS virus vaccines have been identified as unacceptable vaccines for human use.

Evidence of an abnormal immune response was also seen in cotton rats immunized with formalin-inactivated RS virus (reference 29). In addition, evaluation of formalin inactivated vaccines for RS virus in cotton rats showed that the lung organization pathology of immunized animals further developed upon challenge with live virus (reference 30).

The mechanism of enhancement of disease caused by formalin inactivated RS virus vaccine formulations remains to be determined, but is a major obstacle to the development of effective RS virus vaccines. Disease enhancement may be due in part to the effects of formalin on F and G glycoproteins. In addition, specific mechanisms have been proposed by non-RS viruses to potentiate disease, where the immune response to components of contaminating cells or serum present in vaccine formulations may contribute in part to the exacerbation of disease (reference 31). Indeed, mice and cotton rats vaccinated with HEp-2 cell lysates and challenged with RS virus grown on HEp-2 cells developed an increased lung inflammatory response.

In addition, elution of purified RS virus glycoproteins at acidic pH by immunoaffinity chromatography is immunogenic and protective, and induces immune enhancement in cotton rats (references 29, 32).

Clearly, there remains a need for immunogenic formulations, including vaccines, that are not only effective in conferring prophylaxis against RSV-induced disease, but do not produce unwanted side effects, such as immune enhancement. There are both antigens and immunogens for antibody (including monoclonal) production which are required to diagnose RSV infection, and which specifically recognize the RSV protein, and which may be used, for example, to diagnose RS virus-induced disease.

Summary of The Invention

The present invention provides methods for producing vaccine grade cell lines Respiratory Syncytial Virus (RSV), e.g., VERO, MRC5 or WI38 cells, purifying the virus from the fermentor harvest, extracting F, G and M proteins from the purified virus, and co-purifying the F, G and M proteins, but does not involve an affinity step for immunophilins or lentil lectin (lentil lectin) or concanavalin a. In particular, lectin affinity methods, such as those described in WO91/00104 (US 07/773,949, filed by Committee on 28.6.1990, the disclosure of which is incorporated herein by reference), may result in the filtration of the ligand into the product.

In addition, provided herein for the first time is a method for co-isolating and co-purifying the F, G and M proteins of RSV and the immunogenic composition of a co-purified mixture containing RSV proteins.

The co-isolated and co-purified F, G and M RSV proteins are pyrogen-free, non-immunopotentiating, and substantially free of serum and cell contamination. The isolated and purified protein is immunogenic and free of any infectious RSV and other disadvantages.

Thus, in one aspect of the invention, there is provided a mixture of purified fusion (F), attachment (G) and matrix (M) proteins of Respiratory Syncytial Virus (RSV).

Fusion (F) proteins can include multimeric fusion (F) proteins, which can include heterodimers and dimeric and trimeric forms having a molecular weight of about 70 kilodaltons when analyzed under non-reducing conditions.

Attachment (G) proteins, when analyzed under non-reducing conditions, may include oligomeric G proteins, G proteins having a molecular weight of about 95 kilodaltons, and G proteins having a molecular weight of about 55 kilodaltons.

The matrix (M) protein may comprise a protein having a molecular weight of about 28 to 34 kilodaltons when analyzed under non-reducing conditions.

Protein mixtures provided herein include fusion (F) proteins comprising F when analyzed in reducing SDS-PAGE₁A molecular weight of about 48 kilodaltons, and F₂The molecular weight is about 23 kilodaltons, the attachment (G) protein is included to contain a G protein having a molecular weight of about 95 kilodaltons and a G protein having a molecular weight of about 55 kilodaltons, and the matrix (M) protein is included to contain an M protein having a molecular weight of about 31 kilodaltons.

The mixture provided according to this aspect of the invention may comprise F, G and M proteins in the relative proportions:

f about 35 to 70 (by weight)%

G is about 5 to 30 (by weight)%

M about 10 to 40 (by weight)%

When analyzed by SDS-PAGE under reducing conditions and densitometric scanning after silver staining, F with a molecular weight of about 48 kilodaltons was found in this mixture₁And F having a molecular weight of about 23 kilodaltons₂The mixture may be in a ratio of between about 1: 1 and 2: 1. The purity of the mixture of F, G and M proteins may be at least about 75%, preferably at least about

85％。

According to this aspect of the invention, the mixtures provided herein are free of monoclonal antibodies and free of lentil agglutinin and concanavalin a using the isolation method described below.

The RSV proteins provided in the mixtures of proteins provided herein are generally substantially non-denatured under the mild conditions of manufacture and may contain RSV proteins from one or both of RSV a and RSV B subtypes.

In a preferred embodiment of the present invention, there is provided a co-isolated and co-purified mixture of non-denatured Respiratory Syncytial Virus (RSV) proteins, consisting essentially of the fusion (F) protein, attachment (G) protein and matrix (M) protein of RSV, wherein the mixture is free of concanavalin and monoclonal antibodies comprising concanavalin a.

According to another aspect of the invention, there is provided an immunogenic formulation comprising an immunologically effective amount of a mixture provided herein.

The immunogenic compositions provided herein can be formulated as vaccines containing the F, G and M proteins for administration to a host, which can be a primate, specifically a human host, in order to protect the host against RSV-induced disease.

The immunogenic compositions of the invention may be formulated as microparticles, capsules, ISCOMs or liposomes. The immunogenic composition may additionally contain at least one other immunogenic or immunostimulatory substance, which may be at least one adjuvant or at least one immunomodulator, such as a cytokine including ILK.

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 hydroxide, glycolipid analogs, octadecyl esters of amino acids, muramyl dipeptides, polyphosphazenes, lipoproteins, ISCOM matrices, DC-Chol, DDA, and other adjuvants and bacterial toxins, components and derivatives thereof. These substances are described in USSN08/258,228, filed 6/10 of 1994, assigned to attorneys, the disclosure of which is incorporated herein by reference (WO 95/34323). In special cases, an adjuvant that induces a Th1 response is desirable.

The immunogenic compositions provided herein may be formulated to contain at least one additional immunogen, and conveniently it may contain human parainfluenza virus (PIV) proteins from PIV-1, PIV-2 and/or PIV-3, such as PIV F and HN proteins. However, other immunogens such as those from chlamydia, polio, hepatitis b, diphtheria toxin, tetanus toxin, influenza, haemophilus, b.

Other aspects of the invention provide methods of generating an immune response in a host by administering to the host an immunologically effective amount of an immunogenic composition provided herein. Preferably, the immunogenic composition is formulated as a vaccine for administration to a host, including a human, for administration internally to the host and to protect the host against disease caused by RSV. The immune response may be a humoral or a cell-regulated immune response.

In other aspects, the invention provides a method of producing a vaccine against disease caused by Respiratory Syncytial Virus (RSV) infection, the method comprising administering to a test subject an immunogenic composition provided herein, such that the amount and frequency of administration thereof is determined to enable the subject to resist disease caused by RSV; and formulating the immunogenic composition in a form suitable for administration to a host in accordance with the determined amount and frequency of administration. The processing host may be a human.

Other aspects of the invention provide methods for determining the presence of an antibody specifically reactive with the F, G or M protein of Respiratory Syncytial Virus (RSV) in a sample, comprising the steps of:

(a) contacting the sample with the mixture provided herein to produce a complex comprising respiratory syncytial virus protein and any said antibody present in the sample specifically reactive therewith; and

(b) the production of the complex is determined.

In another aspect of the invention, there is provided a method of determining the presence of the F, G or M protein of Respiratory Syncytial Virus (RSV) in a sample, comprising the steps of:

(a) immunizing a subject with an immunogenic composition provided herein so as to generate antibodies specific for F, G and M proteins of RSV;

(b) contacting the sample with an antibody to produce a complex comprising any RSV protein and protein-specific antibodies present in the sample; and

(c) the production of the complex is determined.

Another aspect of the invention provides a diagnostic kit for determining the presence of an antibody specifically reactive with the F, G or M protein of respiratory syncytial virus in a sample, the kit comprising:

(a) mixtures provided herein;

(b) a method of contacting the mixture with a sample to produce a complex comprising respiratory syncytial virus protein and any said antibody present in the sample; and

(c) a method of determining the production of a complex.

In another aspect of the invention, there is provided a method of producing a monoclonal antibody specific for Respiratory Syncytial Virus (RSV), comprising:

(a) administering an immunogenic composition provided herein to at least one mouse so as to produce at least one immunized mouse;

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

(c) fusing B-lymphocytes from at least one immunized mouse with myeloma cells, thereby producing a hybridoma;

(d) cloning hybridomas that produce selected anti-RSV protein antibodies;

(e) culturing colonies that produce the selected anti-RSV protein antibodies; and

(f) anti-RSV protein antibodies were isolated from selected cultures.

In another aspect, the invention provides methods for producing a co-isolated and co-purified mixture of respiratory syncytial virus proteins, comprising growing RSV on cells in culture, isolating the growing virus from the culture, enhancing at least the F, G and M proteins from the isolated virus; and co-isolating and co-purifying the solubilized RSV protein.

Loading of the ion exchange matrix, preferably a calcium phosphate matrix, specifically a hydroxyapatite matrix, with solubilized proteins allows for co-separation and co-purification and optionally co-elution of the F, G and M proteins from the ion exchange matrix. The grown virus can first be washed with urea to remove contamination, but without substantial removal of the F, G and M proteins.

The advantages of the invention include:

-co-isolating and co-purifying F, G and M proteins of RSV;

-immunogenic compositions comprising such proteins;

-a method of isolating such proteins; and

a diagnostic kit for identifying RSV and infected host.

Brief description of the drawings

Figure 1, comprising panels a and b, shows SDS-PAGE analysis of purified RSV a subunit preparations using silver-stained polyacrylamide gels under reducing (panel (a)) and non-reducing (panel (b)) conditions.

Figure 2, comprising panels a, b, c, and d, shows Western blot analysis of purified RSV subunit preparations under reducing conditions.

Figure 3, comprising panels a, b, c and d, shows Western blot analysis of purified RSV subunit preparations under non-reducing conditions.

FIG. 4 shows SDS-PAGE analysis of purified RSV B subunit preparations by silver staining polyacrylamide gels under reducing conditions.

General description of the invention

As discussed above, the present invention provides for the co-purification and co-isolation of F, G and M proteins from RS viruses. The virus is grown on vaccine grade cell lines, such as VERO cells and human diploid cells, such as MRC5 and WI38, and the grown virus is collected. Fermentation was completed in the presence of calf embryo serum (FBS) and trypsin.

The virus harvest is filtered, then concentrated, typically by tangential flow ultrafiltration using a desired molecular weight cut-off membrane, and diafiltered. The virus can be centrifuged to collect the concentrate and the supernatant discarded. The pellet after centrifugation is first washed with a buffer containing urea to remove soluble contaminants without substantially affecting the retention of F, G and M proteins, and then centrifuged. The pellet from centrifugation is then extracted with detergent to enhance the F, G and M proteins from the pellet. Such detergent extracts can be produced by resuspending the pellet in a concentrated volume of the original collection of extraction buffer containing a detergent, such as a nonionic detergent, including TRITON  X-100, the nonionic detergent octadienol (ethylene glycol)₁₀. Other detergents include octyl glucoside and Mega detergents.

After centrifugation to remove insoluble proteins, the F, G and M protein extracts were purified by chromatographic methods. The extract is first applied to an ion exchange chromatography matrix, allowing the F, G and M proteins to bind to the matrix, while the impurities flow through the column. The ion exchange chromatography matrix may be any desired chromatography material, in particular a calcium phosphate matrix, in particular hydroxyapatite, although other materials, such as DEAE and TMAE and others may also be used.

The bound F, G and M proteins are then co-eluted from the column by an appropriate eluent. The resulting co-purified F, G and M proteins may be further processed to increase their purity.

The forms of purified F, G and M proteins used herein may be homo-and hetero-oligomers, including F: g heterodimers and include dimers, trimers and higher forms. The RSV protein formulations prepared according to this procedure proved to be free of any extraneous agents, blood cell adsorbing agents or live viruses.

Use of the formulations provided herein with an agent of the group of drugs^TMGroup of mice immunized intramuscularly were used. As shown in tables 1 and 2 below, strong anti-fusion and neutralization titers were obtained. As shown in tables 3 and 4 below, complete protection against viral infection was obtained in the upper and lower respiratory tract.

In addition, the formulations provided herein are utilized in combination with alum and Iscometrix as adjuvants^TMGroups of mice were immunized intramuscularly with a phosphazene chain polymer and DC-chol. As shown in tables 5 and 6 below, strong neutralizing anti-F antibody titers can be obtained. In addition, complete protection against viral infection was obtained as shown by the absence of virus in lung homogenates (table 7 below).

Use of the formulations provided herein in combination with alum and Iscometrix as adjuvants^TMGroups of immunized monkeys. As shown in tables 8 and 9 below, strong neutralizing titers and anti-F antibody titers were obtained.

The animal immunization data generated herein demonstrate that by using mild detergents to extract the major RSV proteins from the virus, and mild salts to elute the proteins from the ion exchange matrix, a co-purified mixture of F, G and M RSV proteins is obtained that are capable of eliciting an immune response in an experimental animal model, conferring a protective function against RSV challenge.

The invention relates to the use of a mixture of F, G and M proteins from respiratory syncytial virus as a pharmaceutical substance for the active ingredient in vaccines against diseases caused by active multikaryon virus.

In another aspect, the invention provides the use of the F, G and M proteins from respiratory syncytial virus to prepare a vaccine composition for immunizing against disease caused by respiratory syncytial virus infection.

It will be apparent to those skilled in the art that the various embodiments of the present invention have many applications in the fields of vaccines, diagnosis and treatment of respiratory syncytial virus infection, and the generation of immunological reagents. Other non-limiting discussions of this problem are presented further below.

1. Vaccine formulations and uses

Immunogenic compositions suitable for use as vaccines can be prepared from mixtures containing immunogenic F, G and M proteins of RSV disclosed herein. The immunogenic composition elicits an immune response that produces antibodies, including anti-RSV antibodies, including anti-F, anti-G, and anti-M antibodies. Such antibodies may be neutralizing and/or anti-fusing antibodies to the virus.

Immunogenic compositions containing the vaccine may be prepared as injectables, as liquid solutions, suspensions or emulsions. One or more active immunogenic ingredients may be mixed with compatible pharmaceutically acceptable excipients. Such excipients may include water, saline, dextran, glycerol, ethanol, and combinations thereof. The immunogenic compositions and vaccines may further contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance their effectiveness. Immunogenic compositions and vaccines can be administered transdermally by parenteral, subcutaneous, intradermal, or intramuscular injection. Alternatively, the immunogenic compositions formed according to the invention may be formulated and delivered in a manner that elicits an immune response at a mucosal surface. Thus, the immunogenic composition may be administered to a mucosal surface by nasal or oral (intragastric) route. Alternatively, other modes of administration, including suppositories and oral formulations are desirable. For suppositories, binders and carriers may include, for example, polyalkylene glycols or triglycerides. Such suppositories may be formed from mixtures containing the active immunogenic component in the range of about 0.5 to 10%, preferably 1 to 2%. Oral formulations may generally include carriers such as pharmaceutical grades of saccharine, cellulose and magnesium carbonate. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain from about 1 to 95% of the active ingredient, preferably from about 20 to 75%.

The immunogenic formulations and vaccines are administered in a manner compatible with the dosage formulation, in an amount that is therapeutically effective, immunogenic and protective. The amount administered will vary depending on the individual to be treated, including, for example, the ability of the individual's immune system to synthesize antibodies and, if desired, to generate a cell-transmitted immune response. The exact amount of active ingredient to be administered is determined at the discretion of the attendant physician. However, one skilled in the art can readily determine the appropriate dosage range, which may be in the range of micrograms to milligrams of active ingredient per vaccination. Suitable methods of initiating administration and boosting the dose are also variable, but may include the steps of initiating administration followed by boosting. The dosage may also vary depending on the route of administration, and will vary depending on the size of the host.

The concentration of the active ingredient protein in the immunogenic composition of the invention is generally about 1 to 95%. Vaccines containing antigenic material of only one pathogen are monovalent vaccines. Vaccines containing antigenic material of several pathogens are combination vaccines and are also within the scope of the present invention. Such combination vaccines contain, for example, substances from multiple pathogens or from multiple strains of the same pathogen, or from a combination of pathogens. As noted above, the F, G and M proteins of RSV a and RSV B are combined in the present invention into a single multivalent immunogenic composition, which may also contain other immunogens.

If the antigen and the adjuvant are administrated synergistically, the immunogenicity can be obviously improved. Adjuvants enhance the immunogenicity of antigens, but they do not have to be immunogenic themselves. The effect of the adjuvant may be to retain the antigen locally near the site of administration, so as to produce a depot effect, facilitating slow, sustained release of the antigen into the cells of the immune system. Adjuvants may also attract cells of the immune system to antigen storage sites and stimulate such cells to elicit an immune response.

Immunostimulatory agents or adjuvants have been used for many years and are capable of enhancing the immune response of a host to, for example, a vaccine. Intrinsic adjuvants, such as lipopolysaccharides, are often components of killed or attenuated bacteria for use as vaccines. External adjuvants are immunomodulators and can be formulated to enhance the immune response in a host. Therefore, adjuvants have been identified that enhance the immune response to antigens delivered by parenteral administration. Some of these adjuvants are toxic and can cause unwanted side effects, making them unsuitable for use in humans and many animals. Indeed, only aluminium hydroxide and aluminium phosphate (commonly referred to collectively as alum) are commonly used as adjuvants in human and animal husbandry vaccines. The efficacy of alum in enhancing antibody responses to diphtheria and tetanus toxins is well established and HBsAg vaccines have been formulated with alum supplementation. While the use of alum in some applications is well established, its use is limited. For example, for influenza, alum vaccination is ineffective and does not generally elicit a cell-mediated immune response. Antibodies elicited by alum-assisted antigens are predominantly of the IgG1 isotype in mice and are not optimal for protection by some vaccine reagents.

A wide range of external adjuvants can elicit potential immune responses to antigens. These include saponins complexed with membrane protein antigens (immunostimulatory complexes), pluronic polymers containing mineral oil, killed mycobacteria in mineral oil, Freund's incomplete adjuvant, bacterial products such as Muramyl Dipeptide (MDP) and Lipopolysaccharide (LPS), and lipid a, and liposomes.

To efficiently induce both a Humoral Immune Response (HIR) and cell-mediated immunity (CMI), immunogens are often emulsified in adjuvants. Many adjuvants are toxic, induce granulomas, acute and chronic inflammation (Freund's complete adjuvant, FCA), cytolysis (saponin and pluronic multimers) and production of pyrogens, arthritis and anterior uveitis (LPS and MDP). Although FCA is a good adjuvant and widely used in research, it is not allowed for use in human or animal husbandry vaccines because of its toxicity.

2. Immunoassay

The F, G and M proteins of RSV of the invention may be used as immunogens to produce antibodies, as antigens in immunoassays including enzyme-linked immunosorbent assays (ELISAs), RIA and other non-enzyme-linked antibody binding assays or methods known in the art for detecting antibodies. In an ELISA assay, a selected F, G or M protein or mixture of proteins is immobilized to a selected surface, e.g., a surface capable of binding proteins, such as the wells of a polystyrene microtiter plate. After washing and removal of incompletely adsorbed material, a solution of a non-specific protein such as Bovine Serum Albumin (BSA), which is known to be antigenically neutral to the test sample, can be bound to the selected surface. This allows blocking of non-specific adsorption sites on the immobilized surface, thus reducing the background caused by non-specific binding of proteins in the antiserum to the surface.

The fixed surface is then contacted with a sample, such as a clinical or biological substance, which is to be tested in a manner that forms immune complexes (antigen/antibody). This involves diluting the sample with a diluent such as BSA solution, Bovine Gamma Globulin (BGG) and/or Phosphate Buffered Saline (PBS)/Tween. The sample is then allowed to incubate for about 2 to 4 hours at a temperature of about 25 to 37 ℃. After incubation, the surface contacting the sample is washed to remove non-immunocomplexed material. The washing process may involve washing with a solution such as PBS/tween or borate buffered saline. After formation of specific immune complexes between the test sample and the binding protein, followed by washing, the formation, and even the amount, of immune complexes can be determined by contacting the immune complexes with a second antibody specific for the first antibody. If the test sample is of human origin, the second antibody is an antibody specific for a human immunoglobulin, and is typically an IgG. To provide a detection means, the second antibody may have an associated activity, such as an enzymatic activity, for example, developing a color upon incubation with an appropriate chromogenic substrate. Then, quantitative determination was made by measuring the degree of color produced using a spectrophotometer.

Examples

The above disclosure generally illustrates the present invention. References the following specific examples may be more fully understood. These examples are described for illustrative purposes only and are not intended to limit the scope of the present invention. Variations and alternative equivalents are also of interest due to emergency methods that may be proposed or provided for by changes in circumstances. Although specific terms have been employed herein, such terms are intended for use in a descriptive sense and not for purposes of limitation.

The determination of tissue culture infectious doses not explicitly recited in this disclosure has been well reported in the scientific literature₅₀(TCID₅₀Per ml), plaque and neutralizing titers, and are within the purview of those skilled in the art. Protein concentrations were determined by the bicinchoninic acid (BCA) method described in the Pierce handbook (23220, 23225; Pierce chemical company, USA), which is incorporated herein by reference.

CMRL1969 and Iscove Modified Dulbecco's Medium (IMDM) were used for cell culture and virus growth. The cells used in this study were vaccine grade VERO cells from the merienux institute (VERO lot M6). RS virus and RS virus subtype A (Long and A2 strains) used were obtained from the American Type Culture Collection (ATCC), and the most recent subtype A clinical isolate and RSV subtype B clinical isolate were from Baylor's medical college.

Example 1:

this example describes the production of RSV on a mammalian cell line on microcarrier beads in a 150 liter regulated fermentor.

In the presence of 360 g of Cyto60 liters of CMRL1969 medium at pH7.2 in a 150 liter bioreactor containing dex-1 microcarrier beads was added at a concentration of 10⁵Individual cells/ml vaccine grade vero cells, stirred for 2 hours. A further 60 liters of DMRL1969 medium were added to a total volume of 120 liters. Fetal bovine serum was added to a final concentration of 3.5%. Sucrose was added to a final concentration of 3 g/L and L-glutamic acid was added to a final concentration of 0.6 g/L. Dissolved oxygen (40%), pH (7.2), stirring (36rpm), and temperature (37 ℃) were controlled. Cell growth, sucrose, lactate, and glutamate levels were measured. At 4 days, the medium was drained from the fermentor, 100 liters of E199 medium (without fetal calf serum) was added, and stirred for 10 minutes. The fermenter was drained and filled with 120 liters of E199.

An RSV inoculum of RSV subgroup a was added at a multiplicity of infection (m.o.i.) of 0.001, and the culture was then cultured for 3 days, then one third to one half of the medium was drained and replaced with fresh medium. 6 days after infection, stirring was stopped and the beads were allowed to stand still. The fluid of the dry virus culture was filtered through a 20 micron filter paper, then through a 3 micron filter paper, and then further processed.

The clarified virus harvest was concentrated 75 to 150 fold by tangential flow ultrafiltration using 300NMWL membrane and diafiltration against phosphate buffered saline containing 10% glycerol. The virus concentrate was stored frozen at-70 ℃ and then further purified.

Example 2:

this example illustrates the purification of RSV subunits from a viral concentrate of RSV subgroup a.

To an aliquot of the virus concentrate prepared as described in example 1 was added a 50% solution of polystyrene glycol 8000 to give a final concentration of 6%. After stirring at room temperature for 1 hour, the mixture was centrifuged at 15,000RPM for 30 minutes at 4 ℃ in a Sorvall SS-34 centrifuge. The viral pellet was suspended in 1 mM sodium phosphate, pH6.8, 2M urea, 0.15M NaCl, stirred at room temperature for 1 hour, and then centrifuged at 15,000RPM for another 30 minutes. The centrifuge was Sorvall SS-34 at a temperature of 4 ℃. The viral pellet was then suspended in 1 mM sodium sulfate, pH6.8, 50 mM NaCl, 1% Triton X-100 and stirred at room temperature for 30 minutes. The soluble protein supernatant was loaded onto a column of ceramic hydroxyapatite (type II, Bio-Rad laboratories), and the column was washed with 5 volumes of 1 mM sodium phosphate, pH6.8, 50 mM NaCl, 0.02% Triton X-100. The RSV subunit composition from RSV subgroup a containing F, G and M proteins was obtained by elution with 10 column volumes of 1 millimolar sodium phosphate, ph6.8, 400 millimolar NaCl, 0.02% Triton X-100.

Example 3:

this example describes formulations of RSV subunits from RSV subgroup a by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting.

The RSV subunit compositions prepared as described in example 2 were analyzed by SDS-PAGE on a 12.5% polyacrylamide gel. The samples were electrophoresed in the presence or absence of 2-mercaptoethanol (reducing agent). The gels were stained with silver stain and viral proteins were detected (FIG. 1, panels a and b). Immunoblots were prepared for replicating gels, probed with a mouse monoclonal antibody (mAb 5353C75) to the F glycoprotein (FIGS. 2, groups a and 3, group a), a mouse monoclonal antibody (mAb 131-2G) to the G glycoprotein (FIGS. 2, groups b and 3, group b), or a goat antiserum (Virostat #0605) to the RSV M peptide (peptide sequence: LKSKNMLTTVKDLTMKTLNPTHDIIALCEFEN- (SEQ ID NO: 1) (FIGS. 2, groups C and 3, group C), or to whole RSV (FIGS. 2, groups d and 3, group d). This analysis of silver-stained gels of RSV subunit preparations electrophoresed under reducing conditions showed the following distribution of the composition:

g glycoprotein (95 kilodalton form) ═ 10%

F₁Glycoprotein (48 kilodaltons) 30%

M protein (31 kilodaltons) 23%

F₂Glycoprotein (23 kilodalton) ═ 19%

Migration of the F glycoprotein under non-reducing conditions, e.g., a molecular weight of about 70 kilodaltonsDolton heterodimer (F)₀) And higher oligomeric forms (dimers and trimers) (fig. 3, panel a).

Example 4:

this example describes the immunogenicity of RSV subunit formulations in cotton rats.

With the production as described in example 2 and with alum at 1.5 mg/dose or Iscometrix at 5. mu.g/dose^TMGroups of 5 cotton rats were immunized by intramuscular injection (0.1 ml on day 0, 1 microgram or 10 microgram on day 28) of a subunit formulation of RSV formulated (Iscotec, switzerland). Blood samples were obtained at day 41 and tested for anti-fusion and neutralization titers. Mice were challenged intranasally with RSV on day 43 and sacrificed after 4 days. Lavage fluid of lung and nasopharynx is collected and tested for RSV titer. Strong anti-fusion and neutralizing antibody titers were induced as shown in tables 1 and 2 below. In addition, complete protection against viral infection was obtained in the upper and lower respiratory tract, except for one mouse (tables 3 and 4 below).

Example 5:

this example describes the immunogenicity of RSV subunit formulations in mice.

Produced as described in example 2 and treated with 1.5 mg/dose alum, 10. mu.g/dose Iscometrix^TMGroups of 6 BALB/c mice were immunized intramuscularly (0.1 ml) at day 0 and day 28 with 200 micrograms/dose of phosphorus nitrogen chain-containing polymer (PCPP), or various doses of RSV subunit formulations formulated at 200 micrograms/dose DC-chol. The various formulations tested are set forth in tables 5, 6 and 7 below. Blood samples were obtained at 28 days and 42 days and tested for neutralizing antibody titer and anti-F antibody titer. Mice were challenged with RSV on day 44 and sacrificed after 4 days. Lungs were removed and homogenized to determine viral titer. Strong neutralizing titers and anti-F antibody titers were elicited as shown in tables 5 and 6 below. In addition, complete protection against viral infection was obtained as shown by the absence of virus in lung homogenates and nasal washes (table 7 below).

Example 6:

this example describes the immunogenicity of RSV subunits prepared in african green monkeys.

Prepared as described in example 2, with 1.5 mg/dose alum or 50 μ g/dose Iscomatrix^TMThe formulated RSV subunit formulation immunizes (0.5 ml on day 0, 100 μ g on day 21) groups of 4 monkeys injected intramuscularly. Blood samples were taken on days 21, 35 and 49 and tested for neutralizing anti-F antibody titers. Strong neutralizing anti-F antibody titers were obtained as shown in tables 8 and 9 below.

Example 7:

this example further describes the production of RSV on living mammalian cells or microbeads in a 150 liter pilot fermentor.

Vaccine grade Vero cells (Vero cells) were added to 150 liters of Iscove Modified Dulbecco Medium (IMDM) containing 3.5% fetal bovine serum until the final concentration was 2 x 10 in 150 liters of bioreactor containing 450 grams of Cytodex-1 minivector beads (3 grams/liter)⁵One cell/ml (range 1.5 to 3.5 cells/ml). After cell seeding, dissolved oxygen (40% air saturation (range 25 to 40%), pH (7.1. + -. 0.2)), stirring (36. + -. 2rpm), and temperature (37 ℃ C.. + -. 0.5 ℃ C.) were controlled. The process of cell attachment to beads, cell growth (cell number), and glucose and lactate levels in the growth medium was measured daily. The cell concentration is 1.5 to 2.0X 10 days after the start of cell growth⁶Infection of Vero cell cultures occurred per ml of cells. The agitation was stopped, the microcarrier beads allowed to rest for 60 minutes, and a drain line was placed approximately 3 cm above the resting bead volume to drain the media from the bioreactor. 75 liters of IMDM without fetal bovine serum (wash medium) was added and the mixture was stirred at 36RPM for 10 minutes. Agitation was terminated and the microcarrier beads were allowed to stand for 30 minutes. The medium was removed using a drain line and then 75 liters (half volume) was filled with IMDM medium without fetal bovine serum.

For infection, an RSV inoculum of RSV subgroup B was added at a multiplicity of infection (m.o.i.) of 0.001 and the cells were allowed to adsorb virus by stirring at 36RPM for 2 hours in half volume. Then in a bioreactor75 liters of IMDM were added until the final volume was 150 liters. After infection, dissolved oxygen (40% air saturation (range 10 to 40%)), pH (7.25. + -. 0.1), stirring (36. + -. 2rpm) and temperature (37%) were controlled⁰. + -. 0.5 ℃ C.). After infection, the medium was examined daily for cell growth (cell number determination), the course of changes in glucose and lactate levels, RSV F and G antigens and RSV infectivity. At 3 days post infection, agitation was terminated, the small carrier beads were allowed to rest for 60 minutes, and 75 liters (50%) of medium was removed via a drain line and replaced with fresh medium. At 8 days post infection (7 to 9 days), when complete virus-induced cytopathology was observed (i.e., cells were detached from the minicar beads and the medium no longer consumed oxygen), agitation was terminated and the minicar beads were allowed to stand for 60 minutes. The virus-containing culture liquid is removed from the bioreactor and transferred to a storage vessel. 75 liters of IMDM without fetal bovine serum was added to the bioreactor and stirred at 75rpm for 30 minutes. The microcarrier beads were allowed to stand for 30 minutes and the rinse liquid was removed from the bioreactor and combined with the collected material in a storage vessel.

The collected material was concentrated about 20-fold to a final volume of 10 liters using a 500 or 1000 kilodalton (k) ultrafiltration membrane or alternatively tangential flow filtration with a 0.45 micromolar microfiltration membrane (i.e., the membrane retained the virus-containing material). The concentrated material was diafiltered with 10 volumes of phosphate ph 7.2. The diafiltered virus concentrate was stored at-70 ℃ and then further purified.

Example 8:

this example describes the purification of RSV subunits from a viral concentrate of RSV subgroup B.

The virus concentrate prepared as described in example 7 was centrifuged at 15,000RPM for 30 minutes at 4 ℃ in a Sorvall SS-34 centrifuge. The virus pellet was then resuspended in 1 mM sodium phosphate pH6.8, 300 mM NaCl, 2% Triton X-100 and stirred at room temperature for 30 minutes. Insoluble viral cores were removed by centrifugation at 15,000RPM in a Sorvall SS-34 centrifuge at 4 ℃ and the soluble protein supernatant was applied to a ceramic hydroxyapatite column (type I, Bio-Rad laboratories) and washed with 10 column volumes of 1 millimolar sodium phosphate, pH6.8, 10 millimolar NaCl, 0.02% Triton X-100. The RSV subunit compositions containing F, G and M proteins were obtained by elution with 10 column volumes of 1 mM sodium phosphate, pH6.8, 600 mM NaCl, 0.02% Triton X-100. In some cases, further purification of the RSV subunit composition can be achieved by first diluting the eluate from the first ceramic hydroxyapatite column to 400 millimolar NaCl to reduce the NaCl concentration to a lower concentration, and then applying the diluted subunits to the ceramic hydroxyapatite column (type II, Bio-Rad laboratories). From this column was drawn a purified RSV subunit composition from RSV subgroup B.

Example 9:

this example describes the results of an analysis of RSV subunit preparations from RSV subgroup B by SDS polyacrylamide gel electrophoresis.

The RSV subunit formulations prepared as described in example 8 were analyzed by SDS-PAGE using a 15.0% polyacrylamide gel. The sample was electrophoresed in the presence of 2 mercaptoethanol (reducing agent). The gel was stained with silver stain to detect viral proteins (fig. 4). Densitometric analysis of silver-stained gels of RSV subunit formulations under reducing conditions showed the following distribution of protein in the composition:

g glycoprotein (95 kilodalton form) ═ 21%

F₁Glycoprotein (48 kilodaltons) 19%

M protein (31 kilodaltons) 22%

F₂Glycoprotein (23 kilodalton) ═ 20%

Summary of the disclosure

In summary of the present disclosure, the present invention provides a co-isolated and purified mixture of the F, G and M proteins of RSV that is capable of protecting a relevant animal model against infection by RSV. Several modifications are possible within the scope of the invention.

TABLE 1 serum anti-fusion titers in Cotton rats

Group of	Average potency (log2)	Std，Dev(log2)
			Rock placebo	2.0	00
IscomatrixTMPlacebo	2.3	0.5
			Alum containing 1 microgram RSV subunit	8.0	1.0
Alum-containing 10 microgram RSV subunit	7.5	1.0
			Containing IscometrixTM1 microgram RSV subunit	10.4	1.3
Containing IscometrixTM10 micrograms RSV subunit	10.0	1.6

TABLE 2 serum neutralization titers in Cotton rats

Group of	Average potency (log2)	Std.Dev.(log2)
			Placebo for alum	2.0	0.0
IscomatrixTMPlacebo	2.0	0.0
			Alum containing 1 microgram RSV subunit	9.6	1.3
Alum-containing 10 microgram RSV subunit	10.0	1.4
			Containing IscometrixTM1 microgram RSV subunit	10.6	1.1
Containing IscometrixTM10 micrograms RSV subunit	11.2	1.1

TABLE 3 RSV titers of lung washes in Cotton rats

Group of	Mean titer (log)10/g lung)	Std.Dev.(log10/g lung)
			Placebo for alum	3.8	0.4
IscomatrixTMPlacebo	3.7	0.5
			Alum containing 1 microgram RSV subunit	0.4	0.8
Alum-containing 10 microgram RSV subunit	0.0	0.0
			Containing IscometrixTM1 microgram RSV subunit	0.0	0.0
Containing IscometrixTM10 micrograms RSV subunit	0.0	0.0

TABLE 4 nasal wash RSV titers in Cotton rats

Group of	Mean titer (log)10/g lung)	Std.Dev.(log10/g lung)
			Placebo for alum	3.2	0.5
IscomatrixTMPlacebo	3.1	0.3
			Alum containing 1 microgram RSV subunit	0.0	0.0
Alum-containing 10 microgram RSV subunit	0.0	0.0
			Containing IscometrixTM1 microgram RSV subunit	0.0	0.0
Containing IscometrixTM10 micrograms RSV subunit	0.0	0.0

TABLE 5 serum neutralization titers in Balb/c mice

Group of	4 weeks exsanguination		Exsanguination for 6 weeks
						Average potency (log2)	Std.Dev.(log2)	Average potency (log2)	Std.Dev(log2)
Placebo for alum	3.01	0.0	3.0	0.0
					IscomatrixTMPlacebo	3.0	0.0	3.0	0.0
PCPP placebo (200 microgram)	ND	ND	3.0	0.0
					DC-Chol placebo (200 microgram)	ND	ND	3.0	0.0
0.1 microgram RSV subunit without adjuvant	ND	ND	3.0	0.0
					Alum containing 0.1 microgram RSV subunit	ND	ND	10.3	0.9
Alum containing 1 microgram RSV subunit	6.5	0.6	8.7	1.0
					Alum-containing 10 microgram RSV subunit	8.0	1.1	9.5	1.1
Containing IscometrixTM1 microgram RSV subunit	8.2	0.8	13.2	1.0
					Containing IscometrixTM10 microgram RSV subunit	10.4	1.3	13.4	0.6
1 microgram RSV subunit containing PCPP (200 microgram)	ND	ND	15.0	0.6
					0.5 microgram RSV subunit containing DC-Chol (200 microgram)	ND	ND	11.7	1.1

1 minimum detectable potency in the test

ND is not determined

TABLE 6 serum anti-F titers in Balb/c mice

Group of	4 weeks exsanguination		Exsanguination for 6 weeks
						Mean titer (log)3Valence/100)	Std.Dev.(log2Valence/100)	Mean titer (log)2Valence/100)	Std.Dev(log2Valence/100)
Placebo for alum	0.5	1.2	0.0	0.0
					IscomatrixTMPlacebo	1.0	0.0	0.0	0.0
PCPP placebo (200 microgram)	0.0	0.0	0.0	0.0
					DC-Chol placebo (200 microgram)	0.0	0.0	0.0	0.0
0.1 microgram RSV subunit without adjuvant	0.0	0.0	0.0	0.0
					Alum containing 0.1 microgram RSV subunit	7.0	1.0	12.4	0.9
Alum containing 1 microgram RSV subunit	8.7	0.8	11.2	0.8

Alum-containing 10 microgram RSV subunit	9.7	0.8	12.3	1.0
					Containing IscometrixTM1 microgram RSV subunit	8.5	0.6	13.3	0.5
Containing IscometrixTM10 microgram RSV subunit	10.0	0.0	13.0	0.0
					1 microgram RSV subunit containing PCPP (200 microgram)	10.2	0.8	14.0	0.7
0.5 microgram RSV subunit containing DC-Chol (200 microgram)	9.7	1.4	13.0	1.0

TABLE 7 Pneumovirus titers in Balb/c mice

Group of	Mean titer (log)10/g lung)	Std.Dev.(log10/g lung)
			Placebo for alum	4.1	0.2
IscomatrixTMPlacebo	3.5	0.1
			PCPP placebo (200 microgram)	5.2	0.2
DC-Chol placebo (200 microgram)	5.0	0.3
			0.1 microgram RSV subunit without adjuvant	5.3	0.1
Alum containing 0.1 microgram RSV subunit	＜1.71	1.7
			Alum containing 1 microgram RSV subunit	＜1.7	1.7
Alum-containing 10 microgram RSV subunit	＜1.7	1.7
			Containing IscometrixTM1 microgram RSV subunit	＜1.7	1.7
Containing IscometrixTM10 microgram RSV subunit	＜1.7	1.7
			1 microgram RSV subunit containing PCPP (200 microgram)	＜1.7	1.7
0.5 microgram RSV subunit containing DC-Chol (200 microgram)	＜1.7	1.7

1 minimum virus titer detectable in the assay

TABLE 8 serum neutralization titers in African green monkeys

Group of	3 blood serum exsanguination		5 weeks exsanguination		Exsanguination for 7 weeks
								Mean titer (log)2)	Std.Dev(log2)	Mean titer (log)2)	Std.Dev.(log2)	Mean titer (log)2)	Std.Dev.(log2)
Placebo for alum	3.3	0.0	3.3	0.0	3.3	0.0
							IscomatrixTMPlacebo	3.3	0.0	3.3	0.0	3.3	0.0
Alum containing 100 microgram RSV subunit	11.3	1.3	14.6	1.3	11.5	1.4
							Containing IscometrixTM100 micrograms of RSV subunit	10.8	0.7	15.1	0.1	11.9	0.5

TABLE 9 serum anti-F Titers in African Green monkeys

Group of	3 blood serum exsanguination		5 weeks exsanguination		Exsanguination for 7 weeks
								Mean titer (log)2Valence/100)	Std.Dev(log2Valence/100)	Mean titer (log)2Valence/100)	Std.Dev.(log2Valence/100)	Mean titer (log)2Valence/100)	Std.Dev.(log2Valence/100)
Placebo for alum	0.0	0.0	0.0	0.0	0.0	0.0
							IscomatrixTM placebo	0.0	0.0	0.0	0.0	0.0	0.0
Alum containing 100 microgram RSV subunit	6.5	1.9	9.3	1.0	9.0	1.2
							100 microgram RSV subunits containing IscometrixTM	5.5	1.0	9.8	0.5	9.5	1.0

Reference documents

Glezen, w.p., Paredes, a.allison, j.e., Taber, l.h., and frank.a.l. (1981) J. Erichen, 98, 708-.

Chanock, r.m., Parrot, r.h., Connors, m., Collins, p.l., and Murphy, b.r. (1992) pediatrics 90, 137-.

Martin, A.J.Gardiner, P.S., and McQuillin, J. (1978) Lancet ii, 1035-.

Robbins, A., and Freeman, P. (1988) science Americans 259, 126-.

Glezen, W.P., Taber, L.H., Frank, A.L., and Kasel, J.A, (1986) J.Am.Sod.140, 143-.

Katz, s.l. priority for the new vaccine development project, volume one, washington: national academy of academic Press (1985) 397-.

Techniques 20, 151-176 of Wertz, G.W., Sullender, W.M, (1992).

McIntosh, K. and Chanock, R.M. (1990) virology field (Fields, B.M and Knipe, D.M. ed.) 1045-.

9 Collins, P., McIntosh, K., and Chanock, R.M. virology, Fields, B.N., Knipe, D.M. and Howley, P.M., Lippincott-Raven Press, New York (1996) 1313-.

Walsh, E.E., Hall, C.B., Briselli, M., Brandiss, M.W., and Schleinger, J.J, (1987) J.InfectionProgress, 155, 1198-.

Walsh, E.E., Hruska, J. (1983) J. Virol 47, 171-.

Levine, S., Kleiber-France, R., and Paradiso, P.R, (1987) J.Vigenetics, 69, 2521-.

Anderson, l.j.hirerholzer, j.c., Tsou, c., Hendry, r.m., Fernie, b.f., Stone, y, and mcinosh, K. (1985), journal of infectious diseases, 151, 626-.

Johnson, P, R, Olmsted, r.a., Prince, g.a., Murphy, b.r., Alling, d.w., Walsh, e.e., and Collins, P.L, (1987) journal of virology 61(10) 3163-.

Cherrie, A.H., Anderson, K., Wertz, G.W., and Openshaw, P.J.M. (1992) J.Virol 66, 2102-2110.

Kim, h.w., cacchla, j.g., Brandt, c.d., Pyles, g., Chanock, r.m.jensen, k., and parrot, r.h. (1969) journal of epidemiology in usa 89, 422-.

Firedewald, w.t., Forsyth, b.r., Smith, c.b., Gharpure, m.s., and Chanock, r.m. (1989) JAMA 204, 690-charge 694.

Walsh, E.E., Brandsis, M.W., Schlesinger, J.J, (1985) J Virus genetics 66, 409-.

Walsh, E.E., Schleinger, J.J., and Brandrises, M.W, (1984) J.Viral Genet 65, 761-.

Routridge, e.g., Willcocks, m.w., Samsin, a.c.r., Morgan, l., Scott, r., Amderson, j.j., and Toms, g.l. (1988) journal of viral genetics 69, 293-.

Fulgini, V.A., Eller, J.J., Sieber, O.F., Joyner, I.W., Minamitani, M., and Meikejohn, G. (1969) J.Ers.89 (4) 435-448.

Chi, j., magoflfin, r.l., Shearer, l.a., Schieble, j.h., and Lennette, E.H, (1969) american journal of pedigree 89(4)449 463.

Kapikian, a.z., Mitchell, r.h., Chanock, r.m., Shvedoff, r.a., and Stewart, C.E, (1969) journal of pediatrics 89(4) 405-.

Kim, h.w., Arrobio, j.o., Pyles, g., Brandt, c.d.camargo, e., Chanock, r.m., and Parrott, r.h. (1971) pediatrics 48, 745-.

Wright, P.F., Belsche, R.B., Kim, H.W., Van Voris, L.P., and Chanock, R.M. (1982) Immunoantifection 37(1), 397-400.

Wright, p.f., Chinozaki, t. and fleet.w. (1976) journal of pediatrics, 88, 931-.

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

Murphy, b.r., Prince, g.a., Walsh, e.e., Kim, h.w., Parrott, r.h., hem v.g., Rodriguez, w.j., and Chanock, r.m. journal of clinical microbiology (1986), 24(2), 197-.

Connors, m., Collins, p.l., Firestone, c.y., Sotnikov, a.v., Waitze, a.a., Davis, a.r., Hung, p.p., Chanock, r.m., murphy.b. (1992) vaccine, 10, 475-.

Prince, g.a., Jenson, a.b., Hemming, v.g., Murphy, e.r., Walsh, e.e., Horswood, r.l., and Chanock, r.l. (1986) journal of virology 57(3), 721-.

31 Piedra, P.A., Camussi, F. and Ogra, P.L, (1989) J Virus genetics 70, 325- "333.

Walsh, E.E., Hall, C.B., Briselli, M., Brandiss, M.W., and Schleinger, J.J, (1987) J.InfectionsAceol 155(6), 1198-.

Claims (24)

1. Purified fusion (F) protein, attachment (G) protein and matrix (M) protein of Respiratory Syncytial Virus (RSV).

2. The mixture as claimed in claim 1, characterized in that the fusion (F) protein comprises a multimeric fusion (F) protein.

3.A mixture as claimed in claim 2, characterized in that said multimeric fusion (F) protein comprises heterodimers and dimers and trimers having a molecular weight of about 70 kilodaltons, when analyzed by SDS-PAGE under non-reducing conditions.

4. A mixture as claimed in any one of claims 1 to 3, characterized in that said attachment (G) proteins comprise G proteins having a molecular weight of about 95 kilodaltons and G proteins having a molecular weight of about 55 kilodaltons, and oligomeric G proteins, when analyzed by SDS-PAGE under non-reducing conditions.

5. A mixture as claimed in any one of claims 1 to 3, characterised in that said matrix (M) proteins comprise M proteins having a molecular weight of about 28 to 34 kilodaltons, when analysed by SDS-PAGE under non-reducing conditions.

6. A mixture as claimed in any one of claims 1 to 3, characterized in that said fusion (F) protein comprises F having a molecular weight of about 48 kilodaltons, when analyzed by reducing SDS-PAGE₁And F having a molecular weight of about 23 kilodaltons₂The attachment (G) protein includes a G protein having a molecular weight of about 95 kilodaltons and a G protein having a molecular weight of about 55 kilodaltons, and the matrix (M) protein includes an M protein having a molecular weight of about 31 kilodaltons.

7. A mixture as claimed in any one of claims 1 to 3, characterized in that said F, G and M proteins are present in the following ratios:

f35 to 70 (by weight)%

G5 to 30 (by weight)%

M10 to 40% by weight.

8. A mixture as claimed in claim 7, characterized in that F, having a molecular weight of about 48 kilodaltons by scanning the optical density when analysed by SDS-PAGE and silver staining under reducing conditions₁And a molecular weight of about 23Kilodalton F₂In a ratio of 1: 1 to about 2: 1.

9. A mixture as claimed in any one of claims 1 to 3, having a purity of about 75%.

10. A mixture as claimed in any one of claims 1 to 3 which is free of monoclonal antibodies.

11. A mixture as claimed in any one of claims 1 to 3 which is free of lentil lectin and concanavalin a.

12. A mixture as claimed in any one of claims 1 to 3, characterised in that the RSV protein is non-denatured.

13.A mixture as claimed in any one of claims 1 to 3, characterised in that the RSV proteins are from one or both of subtypes RSV a and RSV B.

14. A mixture as claimed in any one of claims 1 to 3 which is co-isolated and co-purified.

15. A mixture of co-isolated and co-purified non-denatured proteins of Respiratory Syncytial Virus (RSV), consisting essentially of the fusion (F) protein, attachment (G) protein and matrix (M) protein of RSV, wherein the mixture is lectin-free and monoclonal antibody-free.

16. An immunogenic composition comprising an immunologically effective amount of a mixture as claimed in any one of claims 1 to 3 and 15.

17. The immunogenic composition according to claim 16, further comprising at least one additional immunogen.

18. An immunogenic composition as claimed in claim 17, characterised in that said at least one further immunogen comprises at least a human parainfluenza virus (PIV) protein selected from the group of PIV-1, PIV-2 and PIV-3.

19. A method for producing a co-isolated and co-purified protein mixture of Respiratory Syncytial Virus (RSV), characterized in that it comprises the steps of:

growing RSV on cells in culture;

isolating the growing virus from the culture medium;

solubilizing at least the fusion (F) protein, the attachment (G) protein and the matrix (M) protein from the isolated virus; and

co-isolating and co-purifying the F, G and M proteins of solubilized RSV.

20. The method as claimed in claim 19, characterized in that said co-separation and co-purification steps are produced as follows:

loading the solubilized protein into the ion exchange matrix; and

the F, G and M proteins are selectively co-eluted from the ion exchange matrix.

21. A method as claimed in claim 20, characterised in that the ion exchange matrix is a hydroxyapatite matrix.

22. A method as claimed in any one of claims 19 to 21 characterised in that prior to the solubilisation step the growing virus is washed with urea to remove contaminants without substantially removing F, G and M proteins.

23. Use of a mixture of a purified fusion (F) protein, attachment (G) protein and matrix (M) protein of Respiratory Syncytial Virus (RSV) for the preparation of a vaccine for combating diseases caused by respiratory syncytial virus infection.

24. Use of a mixture of the fusion (F) protein, the attachment (G) protein and the matrix (M) protein of purified respiratory syncytial virus for the preparation of a vaccine composition for immunizing against a disease caused by respiratory syncytial virus infection.