WO1998018819A1

WO1998018819A1 - Purification of respiratory syncytial virus antigens

Info

Publication number: WO1998018819A1
Application number: PCT/EP1997/006016
Authority: WO
Inventors: Alex Bollen; Dirk Gheysen; Sophie Houard; Jean-Paul Prieels; Moncef Mohamed Slaoui; Omar Van Opstal; Christophe Jacques Robert Andre
Original assignee: Smithkline Beecham Biologicals S.A.
Priority date: 1996-10-29
Filing date: 1997-10-27
Publication date: 1998-05-07
Also published as: AU6908598A; AR011270A1

Abstract

The present invention relates to purification of respiratory syncytial virus (RSV) proteins and for use in vaccines.

Description

Purification of Respiratory Syncytial Virus Antigens

Background

The present invention relates to purification of respiratory syncytial virus (RSV) proteins and for use in vaccines that are able to clear the lungs from a subsequent viral infection without inducing significant pulmonary pathology. In the late 1960's infants immunized with a RSV formalin inactivated vaccine formulated on alum (herein referred to as Fi RSV Alum) and subsequently exposed to a natural RSV infection showed a high increase in hospitalization due to an enhanced respiratory and pulmonary pathology. Post-mortem histological examination of the lungs of the two Fi RSV Alum vaccinated infants who died following natural RSV infection showed the presence of an exacerbated inflammatory process.

U.S. patent 5,194,595 (Upjohn) describes chimeric glycoproteins containing immunogemc segments of the F and G glycoproteins of RSV and suggests that such proteins can be expressed from a variety of systems including bacterial, yeast, mammalian (e.g., CHO cells) and insect cells (using for example a baculovirus). Wathen et al (J. Gen. Virol. 1989, 70, 2625-2635) describes a particular RSV F-G chimeric glycoprotein expressed using a baculovirus vector consisting of amino acids 1-489 of the F protein linked to amino acids 97-279 of the G protein. Although early studies by Wathen et al (J. Infectious Diseases, 1991, 163, 477-482) suggested that such baculovirus-produced RSV F-G antigens induced minimal pulmonary pathology on challenge in cotton rats, later workers obtained contrary results. For example, Connors et al (Vaccine, 1992, 10, 475-484) reported that a baculovirus-produced RSV F-G antigen caused significant enhanced pulmonary pathology in cotton rats when subsequently infected with RSV.

It is thus an object of the present invention to purifiy respiratory syncytial virus (RSV) proteins, and for their use in vaccines which are able to clear the lungs from a subsequent viral infection without inducing significant pulmonary pathology. Summary of the Invention

In one aspect, the present invention is an improved method to purifiy RSV

F-G chimeric proteins which comprises contacting an impure solution containing said protein with a filter to remove viruses (viral clearance); passing the filtered solution through a cation exchange, a metal chelate and anion exchange resin

(chromatography) and recovering said protein; followed by inactivation of said filtered solution; and then filter sterilization to produce a protein of suitable purity for (human) clinical use.

In another embodiment, the present invention is an improved method to purifiy RSV F-G chimeric proteins, wherein the viral clearance step occurs after one or more of the following steps: cation exchange, metal chelate and/or anion exchange chromatography.

In yet another related aspect, the present invetion comprises RSV F-G chimeric proteins obtained by the methods of the invention and a method to prepare a pharmaceutical composition comprising purification of RSV F-G chimeric proteins obtained by the methods of the invention and admixing said proteins with an adjuvant.

Brief Description of the Figures Figure 1 is the study on the induction of enhanced pulmonary pathology upon vaccination of RSV F-G. Figure 2 is an outline of the Cotton rat experiment.

Detailed Description The present invention comprises an improved method to purify proteins that aggregate or form viral-like particles. In particular, the present invention relates to RSV F-G chimeric proteins where it was discovered that such molecules readily aggregate in a purified state. Hence, the present invention relates to a method to purify RSV F-G chimeric proteins, suitable for clinical use, by conducting a viral clearance step very early in the purification scheme, rather than as the last step, or one of the last steps, as is typically done. The early viral clearance step can also be useful for purification of other antigens that aggregate. For example, many viral surface proteins (e.g., Hepatitis B surface antigen) tend to form particles. The chimeric proteins of the instant invention comprise at least one immunogenic fragment from both human respiratory syncytial virus glycoproteins F and G. Preferably, the chimeric protein contains 80% (contiguous sequence) or more of the F and G extracellular domains, while lacking their respective cytoplasmic domains. More preferably, the G protein is lacking the signal sequence region. Still more preferably, the respective F and G protein transmembrane regions are also missing. A preferred example is a RSV F-G chimeric protein comprising amino acids 1-526 of the F protein fused to amino acids 69-298 of the G protein.

The purification scheme of the instant invention involves the following steps. Starting with an impure solution (e.g., cell culture supernatant) of RSV F-G, a viral clearance step is conducted, followed by (in any order) a cation exchange, metal chelate, and anion exchange chromatography. As a regulatory guideline, the solution containing RSV F-G is then treated (e.g., by altering the pH, temperature, etc.) to inactivate any potential bacterial or viral contaminants. Lastly, the solution is filter sterilized. As noted in the preceding paragraph, the viral clearance step is a requisite for ensuring safety of materials for clinical use. Products for viral clearance are know in the art such as hollow fiber membranes (e.g., Planova 15 & Planova 35 (ASHAI), Viresolve (Millipore) and DV50 (PALL)). Planova 15 is a preferred membrane because it removes small non-enveloped viruses. It has the advantage over Planova 35 in that it flters smaller particle sizes. However, unlike Planova 35, this membrane (PLANOVA 15) cannot be used at the end of the purification scheme as the chimeric F-G protein aggregates are the same size as small viruses that the membrane is intended to remove. Preferably, Planova 15 is loaded less than 1 mg protem per cm Optionally, clarifying filters or prefilters are used prior to the viral clearance step to remove contaminants that could otherwise plug the membrane and thus reduce the membrane's effectiveness and service life.

Preferably the viral clearance step is the first, or amongst the first few (e.g., 1-4) steps in the purification process. Alternatively, if the viral clearance step is not the first significant step (e.g., occurs after the cation exchange, anion exchange and/or metal chelate chromatography), then it may be desirable to add detergent to the impure solution just prior to the viral clearance step, particularly if the impure solution containing RSV F-G has ever been frozen. Detergents are well known in the art (e.g., Triton-X, Tween, etc.). A preferred detergent is Tween 20.

Ion exchange resins are well known in the art. A preferable resin for cation exchange chromatography is SP Sepaharose (Pharmacia). A preferable resin for anion exchange chromatography is Q Sepaharose (Pharmacia).

For metal chelate affinity chromatography (MCAC) (also known as immobilized metal ion adsorption chromatography (IMAC)), the gel (resin) is first charged with metal ions such as Cu2+ or Zn2+ to form a chelate. Proteins and other biomolecules will bind to the gel depending on the presence of surface histidine groups which have an affinity for the chelated metal ions. The binding strength is mainly affected by the nature of the metal ion and the pH of the buffers. Since the metal ions are strongly bound to the matrix, the adsorbed proteins are eluted by competitive elution, lowering the pH, or the use of strong chelating agents.

Optionally, the solution containing the chimeric RSV F-G protein can undergo an adsorption/desorption/gel filtration step prior to the inactivation step. The step can be performed with one resin (e.g., CM EMD TSK (Merck) or via a combination of resins (e.g., Ether Toyopearl (ToyoHaas) followed by a gel filtration (sizing) step with, for example, Sephacryl S resin (such as S-200 HR) or G25 (Pharmacia).

In addition to the purification of RSV F-G chimeric proteins, it has now been discovered that a RSV F-G protein (comprising amino acids 1-526 of the F protein fused to amino acids 69-298 of the G protein) expressed from CHO cells, when solely adjuvanted with an aluminum salt, does not induce any enhanced pulmonary pathology in the cotton rat model. This is unlike the baculovirus- produced RSV F-G protein (antigen) which when solely adjuvanted with an aluminum salt does indeed result in significant pulmonary pathology as found by Connors et al (Vaccine, 1992, 10, 475-484).

Advantageously such CHO-expressed protein is purified under mild conditions to ensure that the recombinant protein retains its native structure as much as possible. When used as a vaccine such antigen should mimic the natural virus so to minimize the induction of pulmonary pathology following subsequent exposure to RSV. Such mild conditions involve the use suitable buffers having a pH in the range of approximately 4-10.5, preferably 5-10.

It has further been shown in the present invention that the use of the adjuvant, 3-De-O-acylated monophosphoryl lipid A (3D-MPL) known from GB-A- 2220211 (Ribi), prevents pulmonary pathology of RSV vaccines.

Although, WO 94/27636 (American Cyanamid) describes RSV vaccines comprising, as an adjuvant, QS21, MPL, 3D-MPL or a mixture thereof, there is no suggestion that the use of 3D-MPL as a component of a RSV vaccine would prevent such vaccine from inducing any pulmonary pathology, especially in naive recipients (i.e., seronegative individuals) upon their first infection with RSV.

Accordingly, the present invention relates to the use of 3D-MPL in the manufacture of a vaccine comprising a RSV antigen for preventing an infection associated with RSV without inducing pulmonary pathology during subsequent RSV infection. (In a related aspect the present invention provides a method of preventing an infection associated with RSV without inducing pulmonary pathology during subsequent RSV infection which comprises adrriinistering to an individual a vaccine comprising an immunoprotective amount of a RSV antigen and 3D-MPL.) Such vaccines can be used to prevent first infection or subsequent re-infection with RSV. They do not suffer from the disadvantage of inducing any lower respiratory tract morbidity associated with RSV infection following vaccination. Such RSV vaccines may be administered to seropositive individuals to boost their immune response, for example to pregnant women and especially to ύrrimunocomprornized individuals such as the elderly.

Advantageously such 3D-MPL adjuvanted RSV vaccines are administered to seronegative individuals, such as infants, to prevent any pulmonary pathology associated with RSV infection following vaccination.

Suitable antigens include an inactivated RSV virus, such as a formalin inactivated RSV virus, or antigens derived from the RSV virus such as the RSV F or G protein or immunogemc fragments thereof as disclosed for example in U.S. Patent 5,149,650, or a chimeric polypeptide comprising at least one immunogenic fragment from both RSV F and G proteins, advantageously an RSV F-G chimeric protein as disclosed, for example, in U.S. Patent 5,194,595, preferably expressed from CHO cells. The CHO-expressed polypeptide is preferably a RSV F-G chimeric protein comprising amino acids 1-526 of the F protein fused to the amino acids 69-298 of the G protein.

The vaccine used in the present invention will contain an immunoprotective quantity of RSV antigen and may be prepared by conventional techniques.

Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, USA 1978 and in Vaccine Design: The Subunit and Adjuvant Approach, edited by Powell and Newman, Plenum Press, New York, 1995. Encapsulation within liposomes is described, for example, by Fullerton, US Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example by Likhite, US Patent 4,372,945 and by Armor et al, US Patent 4,474,757. The amount of protein in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees. Such amount will vary depending upon which specific immunogen is employed and how it is presented. Generally, it is expected that each dose will comprise 1-1000 μg of protein, preferably 2-100 μg, most preferably 5-50 μg. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of appropriate immune responses in subjects. Following an initial vaccination, subjects may receive one or several booster immunizations adequately spaced.

Suitably the 3D-MPL will be present in the range 10 μg per dose to 100 μg, preferably 25-75 μg, such as 50 μg per dose. Advantageously small particles of 3D- MPL may be utilized as disclosed in WO 9421292.

Suitably the vaccine used in the present invention may comprise a carrier such as an aluminum salt, e.g. aluminum hydroxide (Al(OH)₃), aluminum phosphate or alurninum phosphate sulfate (alum), or a non-toxic oil in water emulsion or a mixture thereof.

If an aluminum salt (preferably aluminum hydroxide) is used as a carrier it is generally present in the range of 50 to 1000 μg, preferably 100 to 500 μg per dose.

Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g. squalene and an emulsifier such as Tween 80, in an aqueous carrier such as phosphate buffered saline.

If desired the vaccine used in the present invention may comprise an additional adjuvant, preferably a saponin adjuvant such as QS21 as described for example in WO 9517210, optionally in the presence of a sterol, such as cholesterol as described for example in PCT/EP96/01464.

In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only, and are not to be construed as limiting the scope of the invention in any manner.

EXAMPLES

I. CLONING AND EXPRESSION OF RSV F-G PROTEIN. 1. Description of RSV F-G

A. BACULOVIRUS-F-G The F-G fusion protein described by Wathen et al. J. Gen. Virol. , 1989, Vol.

70, 2625-2635 and disclosed in US Patent 5,194,595 is different from the F-G-CHO fusion protein described below with respect to the fusion junction and the length of the chimeric protein. F-G-Baculovirus comprises amino acids 1-489 from F followed by arnino acids 97-279 from G and contains a total of 672 amino acids.

B. CHO-F-G

The plasmid pEE14-FG containing a chimeric construct comprising of a fusion between arnino acid sequences of F (1-526) and G (69-298) was received from a collaboration with A. BOLLEN (ULB/CRI, Belgium). This F-G fusion protein contains a total of 755 amino acids. It starts at the N-terminal signal sequence of F and lacks the C-terminal transmembrane domain (526-574) -anchor domain- of F glycoprotein. Then, followed the extracellular region of G glycoprotein, without the ammo-terminal region that contains the Signal/ Anchor domain of G, a typical class ϋ glycoprotein.

The pEE14-FG expression plasmid was generated by the insertion of the F- G coding sequence from pNIV2857 (A. Bollen, ULB/CRI, Belgium) as an Asp7181 (blunt) 5' - Hindiπ (blunt) 3' restriction fragment (2188 bp) into the Smal site of pEE14 (Celltech). A Kozak sequence in lieu of the F-G start ATG was generated into the pNIV 2857 construction as follows:

pEE14— ccc gtacc ATG GAG x CAG TAG aagct ggg — pEE14

(Smal) Metl Gln(298)Stop

Asp718I(klenow) Hindm(klenow)

5 'F(l -526) x G(69-298)3 '

The F sequence in pEE14-FG is from SS2 RSV strain, and was kindly made available by Dr. PRINGLE as a cDNA construct in a Vaccinia vector (Baybutt and Pringle, J.Gen. Virol., 1987, 2789-2796). The G sequence is from A2 RSV strain and was generated from a recombinant G Vaccinia virus obtained from Dr. G. WERTZ (Alabama, USA). 2. CHO Kl transfection and stable F-G protein expression.

CHO Kl cells derived from MCB O24M (Celltech) were transfected with 20 ug of pEE14-FG plasmid DNA twice CsCl purified using the Ca-phosphate - glycerol transfection procedure. Cell clones were selected according to the procedure of the GS (glutamine synthetase ) expression system (Crocett et al BioTechn., 1990, Vol8, 662) and amplified in the presence of 25 micro molar mettoonine sulphoximine (MSX) in G MEM medium containing no glutamine and supplemented with 10% dialyzed FBS (Foetal Bovine Serum). Following transfection, 39 MSX resistant clones were screened in 24-well plates and their supematants were tested for secretion of the FG fusion protein. All transfectants proved to be positive for F antigen expression using a specific 'Sandwich' ELISA assay (i.e. rabbit polyclonal anti FG serum / Antigen / mAB19). Monoclonal antibody 19 recognizes a conformational FI - epitope and is neutralizing.

The 3 best FG-producer clones (n° 7, 13 and 37) were single-cell subcloned in a limiting dilution assay using 0.07 cells per well in a 96- well plates. A total of 59 positive subclones were obtained and the 16 best FG-producers were further characterized by western blot and ELISA. Again, the 8 best FG-subclones were further amplified and their FG expression was evaluated in presence and absence of sodium butyrate (2mM) or DMSO (1 or 2%). Six subclones were amplified and cell vials were made and stored at -80°C and liquid N2. Finally, the 3 best FG- subclones were selected. These are CHOK1 FG ° 7.18, ° 13.1 , and ° 37.2.

Westernblot analyses (non-reducing conditions) with monoclonal mAB19 indicated a major band of FG at about 135 kDa. The purified FG protein from recombinant Baculovirus FG infected cells (UPJOHN) appeared as major broad bands at +/-100kDa and other bands at +/-70kDa under similar blot conditions. The addition of sodium butyrate in CHO-FG cell culture medium increased the expression level of FG 3 to 12 fold depending on the subclone and cell culture growth conditions. In particular, subclone CHO-FG 13.1 expressed 8-10 fold more FG protein in the presence of butyrate (WB/ELISA). Expression level determination was performed by ELISA (mAB19 or MoAb AK13) using purified FG baculo protein as standard, as well as by western blot analysis using serial dilution.

Depending of the ELISA assay and cell culture conditions, the expression level of CHO-FG 13.1 is 5-12 ug of FG/ml after treatment with butyrate. Under accumulation conditions and medium replacements (3 to 5 days ) yields of 16 to 28 ug of secreted FG protein /ml were obtained.

CHOK1 FG 13.1 cell line was subsequently adapted to grow in suspension and serum-free (S/SF) conditions using a proprietary growth medium. The elimination of sodium butyrate may be desirable as butyrate is suspected to affect glycosylation patterns of glycoproteins. Cell line CHO-FG 13.1 S/SF grown in a medium without butyrate expressed higher yields than the parental adherent cell line grown in medium with butyrate. In addition, FG protein produced in CHO appears to be more stable than the baculovirus produced FG. Long term expression evaluation and preliminary genetic characterization showed that CHO-FG 13.1 and the S/SF adapted 13.1 cell line were stable, contained intact FG expression cassettes giving rise to one single mRNA band of about 3000 nucleotides long (Southern and Northern analyses). The CHO-FG clone 13.1 S/SF was further used for production of FG antigen.

II. PRELIMINARY PURIFICATION OF RSV F-G.

Two recombinant RSV surface glycoproteins have been included in an immunological evaluation. Both antigens are fusion proteins between F and G but differ in primary amino acid structure, in expression system (Baculovirus versus rec. CHO cells) and in the purification procedure.

A. BACULOVIRUS-FG

FG has been expressed in Sf9 cells with recombinant baculovirus as described by Wathen et al. J. Gen. Virol. (1989), 70, 2625-2635. In our immunological studies, such antigen prepared by the procedure as described by Wells et al. in Protein Expression and Purification (1994), 5, 391-401 was used. Briefly, cell free culture supernatant containing secreted FG protein is made pH 8 to precipitate contaminants and the antigen is further chromatographed on a S- Sepharose cation-exchanger, a copper chelating Sepharose and a Vydac C4 reverse phase column using acetonitrile/TFA as the eluant. After a refolding step out of 6M GuHCl by dialysis, a final polishing step is performed on a S-Sepharose column in the presence of Tween 80. The purified antigen is heavily glycosylated (83% O- linked; 17% N-linked) and migrates on SDS-PAGE as a broad doublet around 100- 130 kDa with additional antigen related bands of lower MM. The expression level at 20L scale is relatively low, ranging from 0.08 to 0.85 mg FG/L culture and the antigen is purified to 95% homogeneity with an overall yield of 21 % . This material was used in cotton rat experiment described below.

The purification process includes very harsh treatments such as reversed phase chromatography (organic solvent in strongly acidic conditions). In order to restore a more native conformation, a refolding step by dialysis out of GuHCl in the presence of detergent (Tween 80) is included into the scheme. This treatment leads, according to the authors, to native antigen as it is recognized in-vitro by a panel of 14 monoclonal antibodies directed against linear and conformational epitopes on the F and G sequences of the molecule

B. CHO-FG

Example 1 (serum)

The frozen cell culture supernatant is thawed in the cold room (4°C-7°C). Protease inhibitors (for example, 1/1000 Aprotinin, 0.5 mg/litre leupeptin, 0.5 mM Pefabloc) and 25 mM Na Caprylate are added. In presence of Na Caprylate, BSA, the major contaminant, could be separated from FG protein on Blue Hyper D chromatography.

Chimeric F-G from CHO cells exists as a doublet, with apparent molecular weight of 150 kD and 130 kD on SDS-PAGE. Step I : Blue Hyper D chromatography The thawed cell culture supernatant is loaded onto a column packed with

Biosepra Blue Hyper D resin. The Blue Hyper D resin is equilibrated with 20 mM TRIS-Cl pH 7.5, 25 mM Na Caprylate (buffer A). After loading, the column is first washed with buffer A and than with 20 mM TRIS-Cl pH 7.5 (buffer B) until the absorbance at 280 nm returns to baseline.

FG antigen is eluted with 20 mM TRIS-Cl pH 7.5, 3M NaCl, 1 % Tween 80. FG positive fractions are pooled and dialyzed at 4 °C against buffer B.

Step II : Anion Exchange Chromatography

The dialyzed eluate from step I is loaded onto a column packed with Pharmacia Q-Sepharose HP or fast flow resin. The Q Sepharose HP or f . fl. resin is equilibrated with 20 mM TRIS pH 7.5, 1 % Tween 80 (buffer C). After loading, the column is washed with buffer C until the absorbance at 280 nm returns to baseline.

The column is eluted successively with buffer C containing 150 mM NaCl, than 250 mM NaCl and finally 3M NaCl. FG antigen is eluted with buffer C containing 250 mM NaCl. The eluate is dialyzed at 4°C against 20 mM MES pH 6.5.

Step III : Cation Exchange Chromatography

The dialyzed eluate from step II is loaded onto a column packed with Pharmacia SP Sepharose HP or fast flow resin. The SP Sepharose HP or fast flow resin is equilibrated with 20 mM MES pH 6.5, 1 % thesit (buffer D). After loading, the column is washed with buffer D until the absorbance at 280 nm returns to baseline. The column is eluted successively with buffer D containing 150 mM NaCl, than 300 mM NaCl and finally 1 M NaCl. FG is eluted with buffer D containing 300 mM NaCl. The eluate could be, if necessary, concentrated on a Filtron OMEGA 30 or 50 kDa membrane. Step JTV : Gel Filtration Chromatography

Eluate from step III, concentrated or not, is loaded onto a Pharmacia Superdex 200 16/60 column equilibrated and eluted in PBS, 1 % Tween 80. 1-3 mg of total protein (Lowry) are injected per run. Example 2, FG/1 (serum free)

FG antigen is purified as described in example 1 , except that the thawed supernatant is directly loaded onto Q Sepharose fast flow resin. In this case, no Na caprylate is added. This led to the purification of FG/1 used in cotton rat experiment described below.

Example 3, FG/3 (serum free)

The thawed supernatant is directly applied onto Pharmacia SP Sepharose fast flow cation exchanger at pH 6.5, followed by a Pharmacia Q Sepharose fast flow anion exchanger at pH 9.5. FG containing fractions from this step are dialyzed against PBS.

Step I : Cation Exchange Chromatography

The thawed cell culture supernatant is loaded onto a column packed with Pharmacia SP Sepharose fast flow resin equilibrated in 20 mM Na acetate pH 6.5 (buffer E).

After loading, the column is washed with buffer E until the absorbance at 280 nm returns to baseline.

The column is eluted with 50 mM Ethanolamine pH 9.5, 0.51 M NaCl. FG positive fractions are pooled, diluted 1.5-2 times with 50 mM Ethanolamine pH 9.5 till the conductivity reaches 17 m S/cm2.

Step II : Anion Exchange Chromatography

Diluted sample from step I is loaded onto a column packed with Pharmacia Q Sepharose fast flow resin equilibrated in 50 mM Ethanolamine pH 9.5, 0.17 M NaCl (buffer F).

After loading, the column is washed with buffer F, until the absorbance at 280 nm returns to baseline.

The column is eluted with 50 mM Ethanolamine pH 9.5, 0.4 M NaCl. FG positive fractions are pooled and dialyzed at 4°C against PBS. Dialyzed sample is filtered through 0.22 μm. This led to the purification of FG/3 used in the cotton rat experiment described below.

III. ADMINISTRATION OF FG BAC AND FG CHO TO COTTON RATS. The animal test protocol was as follows:

Groups of 24 cotton rats were administered twice, at Day 0 and at Day 21, IM, with 200 μl of 25 ng of FG antigen from Baculovirus (FG bac) infected cells, or from CHO-FG cells (FG/1 and FG/3, see purification schemes described above), formulated with 100 μg of Al(OH)3. Two groups were controls. A first control group was administered intra-nasaly with 10E6 p.f.u. of live RSV/Long strain on DO and D21 as well. This was the control for absence of enhanced pathology. The second control group was administered twice, same timing, with Fi RSV (Formalin inactivated RSV). This group was used as control for enhanced pathology. Animals were challenged under anaesthesia by the intra-nasal route on day 49 using 106pfu of live RSV/Long strain. Lung tissues from the challenged animals were taken for virus titration in those organs. Pieces of lungs were also cryo preserved for histopathological analysis and the enhanced pathology, that is the alveolar (intra- and interstitial) inflammation, was scored.

Data of two experiments, one with FG bac and the second with FG CHO, e.g. FG/1 and FG/3, are presented on Figure 1.

In both experiments, a non sterilizing immunological response was induced by prototype RSV FG vaccines as monitored by appearance of anti-FG Abs in vaccinated rats, which allowed some replication of the challenge virus in the lungs as demonstrated by titration of the RSV in the lungs. As expected administration of live RSV did not induce immunopathology after challenge and the scoring of alveolar inflammation varied from 0.33 to 1. As predicted by the cotton rat model, the administration of FiRSV followed by challenge induced alveolar inflammation with score of 10.25 in the first experiment and 8.58 in the second. The FG bac antigen formulated with Al(OH)3 gave an alveolar score of

6.75, compared to 10.25 of the FiRSV. The two FG-CHO adsorbed on Al(OH)3 gave an alveolar score of 0.5 and 1 for FG/1 and FG/3, respectively. These scores were similar to the live RSV negative control.

This demonstrated that FG CHO Al(OH)3 antigen is superior to FGbac Al(OH)3 in that its administration to naive cotton rats did not induce any enhanced pathology, scored as alveolar inflammation, after challenge with a high dose of virulent RSV.

Evaluation of Enhanced Pathology in the Cotton Rat Model; Alveolar Scoring.

FG bac: FG produced in recombinant baculovirus infected cells and purified by Upjohn, lot 109-A03. FG/1 FG produced in CHO cells and purified through Q (pH7.5), SP Sepharose (pH6.5) and Superdex 200, Tween 1%. FG/3 has been produced in CHO cells and purified through SP (pH9.5) and Q Sepharose (pH9.5).

IV, PURIFICATION OF CLINICAL GRADE RSV F-G Example 1 (serum free)

Culture from serum-free adapted CHO cell line 13.1 (33°C) is centrifuged and the supernatant is filtered through 0.2 μm cartridge. The supernatant is frozen at -20°C before purification. 20 litres of cell free solution are purified as follows. 1050 ml of SP Sepharose resin (Pharmacia) are equilibrated with 50 mM borate buffer (pH 7.6) containing 0.1 M NaCl. The FG protein is eluted with 50 mM borate buffer (pH 7.6), containing 0.5 M NaCl.

The pool containing the antigen is loaded onto 200 ml of Zn-chelate column (Pharmacia). The flow-through fraction contains the antigen. The flow-through fraction of the Zn-chelate column is diluted height times in Tris 50 mM pH 7.4 buffer. This diluted solution is loaded onto 700 ml of Q Sepharose FF column (Pharmacia). The antigen is eluted in 20 mM piperazine, 0.25 M NaCl, pH 5.0.

To the Q eluate fraction containing the F-G antigen, ammonium sulphate is added so to reach 1.4 M. 400 ml of Ether Toyopearl resin (TosoHaas) is equilibrated with 10 mM phosphate buffer, pH 6.8, containing 2.5 M ammonium sulphate. The Q eluate containing 1.4 M ammonium sulphate is loaded onto the Ether Toyopearl column. The antigen elutes in a phosphate 10 mM pH 6.8 buffer.

The antigen solution containing trace amounts of ammonium sulphate is loaded onto 1000 ml of Sephacryl S-200 HR resin (Pharmacia), equilibrated in 10 mM ethanolamine, pH 10, containing 150mM NaCl.

The pH of the alkaline antigen solution is adjusted to 10, if necessary. This solution is incubated 16 hours at 25°C.

The alkaline antigen solution is loaded onto 1500 ml of Sephacryl S-200 HR resin (Pharmacia), equilibrated in 10 mM PBS Mod adjusted to pH 6.8 (NaCl 137 mM, KC1 2.7 mM, Na2HPO4 8.1 mM, KH2PO4 1.47 mM).

This antigen solution is filtered on a 0.2 μm Millipak 20 membrane. The filtrate is then passed through a specific virus removal hollow fibber module, operating in deadend mode (PLANOVA 35, ASAHI). The filtrate is finally sterile filtered through a 0.2 μm filter (Millipak 20).

121 mg of FG antigen are purified by this method.

Comments:

Material is suitable for clinical use, but viral clearance step is the second to the last step.

Example 2

Culture from serum-free adapted CHO cell line 13.1(33°C) is clarified by tangential microfiltration device. The permeate is filtered through 0.2 μm cartridge. 4 litres of cell free solution are purified as follows. 200 ml of SP Sepharose resin (Pharmacia) are equilibrated with 50 mM borate buffer (pH 7.6) containing 0.1 M NaCl. The F-G protein is eluted with 50 mM borate buffer (pH 7.6), containing 0.5 M NaCl. The pool containing the antigen is loaded onto 40 ml of Zn-chelate column (Pharmacia). The flow-through fraction contains the antigen.

The flow-through fraction of the Zn-chelate column is diluted eight times in Tris 50 mM pH 7.4 buffer. This diluted solution is loaded onto 140 ml of Q Sepharose FF column (Pharmacia). The antigen is eluted in 10 mM piperazine 10 mM, pH 5.0, containing 0.25 M NaCl.

The eluate of the Q Sepharose is diluted 3 times in phosphate 10 mM pH 7.0. This antigen solution is loaded onto CM EMD TSK resin (Merck), equilibrated in phosphate 10 mM pH 7, NaCl 50 mM. The antigen fraction is recovered in phosphate 10 mM, pH 7, containing 400 mM NaCl.

The CM EMD TSK pool is adjusted to pH 10 by addition of concentrated NaOH solution. After 16 hour at 25°C, this alkaline solution is neutralised to pH 6.8 by 1 M NaK phosphate buffer.

This antigen solution is filtered on a 0.2 μm Millipak 20 membrane. The filtrate is then passed through a specific virus removal hollow fibber module, operating in deadend mode (PLANOVA 35, ASAHI).

The filtrate is finally sterile filtered through a 0.2 μm filter (Millipak 20).

Comments: Example 2 provides an improved purification method over Example 1 : the cell supernatant is not frozen before purification, the Ether Toyopearl resin and the two Sephacryl resins(S-HR200) are replaced by a CM EMD TSK resin.

Example 3

Culture from serum-free adapted CHO cell line 13.1(33°C) is clarified by tangential microfiltration device. The permeate is filtered through 0.2 μm cartridge. 4 litres of cell free solution are to be purified as follows. The cell culture will be filtered through a PLANOVA 15 membrane (ASAHI). The purification of the filtrate, free of adventitious viruses, is to be as follows:

200 ml of SP Sepharose resin (Pharmacia) will be equilibrated with 50 mM borate buffer (pH 7.6) containing 0.1 M NaCl. The FG protein will be eluted with 50 mM borate buffer (pH 7.6), containing 0.5 M NaCl.

The pool containing the antigen will be loaded onto 40 ml of Zn-chelate column (Pharmacia). The flow-through fraction contains the antigen.

The flow-through fraction of the Zn-chelate column will be diluted eight times in Tris 50 mM pH 7.4 buffer. This diluted solution will be loaded onto 140 ml of Q Sepharose FF column (Pharmacia). The antigen will be eluted in 10 mM piperazine 10 mM, pH 5.0, containing 0.25 M NaCl.

The eluate of the Q Sepharose will be diluted 3 times in phosphate 10 mM pH 7.0. This antigen solution will be loaded onto CM EMD TSK resin (Merck), equilibrated in phosphate 10 mM pH 7, NaCl 50 mM. The antigen fraction will be recovered in phosphate 10 mM, pH 7, containing 400 mM NaCl.

The CM EMD TSK pool will be adjusted to pH 10 by addition of concentrated NaOH solution. After 16 hour at 25°C, this alkaline solution will be neutralised to pH 6.8 by 1 M Na/K phosphate buffer.

The filtrate will be finally sterile filtered through a 0.2 μm filter (Millipak 20). Comments:

Example 3 illustrates an improved process in view of the viral clearance efficiency. Using a PLANOVA 15 on the cell supernatant, where FG is less aggregated, improves the efficiency of the viral clearance.

Example 4

Culture from serum-free adapted CHO cell line 13.1(33°C) is clarified by tangential microfiltration device. The permeate is filtered through 0.2 μm cartridge. 4 litres of cell free solution will be purified as follows. buffer (pH 7.6) containing 0.1 M NaCl. The F-G protein will be eluted with 50 mM borate buffer (pH 7.6), containing 0.5 M NaCl.

Optionally Tween 20 (0.1 to 1% V/V) will be added to the flow-through fraction and incubated 1 hour at 4°C. This solution will be filtered through PLANOVA 15 membrane.

The filtrate of the PLANOVA 15 will be diluted eight times in Tris 50 mM pH 7.4 buffer. This diluted solution will be loaded onto 140 ml of Q Sepharose FF column (Pharmacia). The antigen will be eluted in 10 mM piperazine 10 mM, pH 5.0, containing 0.25 M NaCl.

The CM EMD TSK pool will be adjusted to pH 10 by addition of concentrated NaOH solution. After 16 hour at 25°C, this alkaline solution will be neutralised by 1 M Na/K phosphate buffer. The neutralised antigen solution will be sterile filtered through a 0.2 μm filter (Millipak 20).

Comments:

In this example, the PLANOVA 15 is placed after the second chromatographic step. It is an alternative way of improving the viral clearance efficiency. To dissociate F-G aggregates, detergent (Tween 20 as 0.1 to 1 % V/V) may be added to the antigen solution. This detergent will then be eliminated on the two following chromatographic steps.

20 - Example 5

Culture from serum-free adapted CHO cell line 13.1(33 °C) is clarified by tangential microfiltration device. The permeate is filtered through 0.2 μm cartridge. 4 litres of cell free solution are purified as follows. 200 ml of SP Sepharose resin (Pharmacia) will be equilibrated with 50 mM borate buffer (pH 7.6) containing 0.1 M NaCl. The F-G protein will be eluted with 50 mM borate buffer (pH 7.6), containing 0.5 M NaCl.

The pool containing the antigen will be loaded onto 40 ml of Zn-chelate column (Pharmacia). The flow-through fraction contains the antigen. Optionally Tween 20 (0.1 to 1% V/V) will be added to the flow-through fraction and incubated 1 hour at 4°C. This solution will then be filtered through PLANOVA 15 membrane.

The filtrate of the PLANOVA 15 will be diluted eight times in Tris 50 mM pH 7.4 buffer containing 1 % V/V Tween 20. This diluted solution will be loaded onto 140 ml of Q Sepharose FF column (Pharmacia). The antigen will be eluted in 10 mM piperazine 10 mM, pH 5.0, containing 0.25 M NaCl and 1 % V/V Tween 20.

The eluate of the Q Sepharose will be diluted 3 times in phosphate 10 mM pH 7.0. This antigen solution is loaded onto CM EMD TSK resin (Merck), equilibrated in phosphate 10 mM pH 7, NaCl 50 mM. The antigen fraction will be recovered in phosphate 10 mM, pH 7, containing 400 mM NaCl.

The neutralised antigen solution will be sterile filtered through a 0.2 μm filter (Millipak 20). Comments:

In this case, the Q Sepharose step following the PLANOVA 15 is performed in presence of Tween 20.

V. Evaluation of Protection Against the Enhanced Pulmonary Inflammatory Pathology The cotton rat model

The cotton rat model (see Example III) was chosen for evaluation of protection against the enhanced pulmonary irvflammatory pathology occurring in the presence of an inadequate immune response. The pathology observed upon RSV challenge of animals vaccinated with Fi RSV Al(OH)₃ closely resembles what was observed in the infants described previously.

An initial experiment was carried out in cotton rats to determine the experimental conditions which would allow to measure the induction of the typical enhanced inflammatory pulmonary pathology resulting from the vaccination by the candidate vaccine. To allow such observation, a dose of immunogen was determined which induces a sub optimal neutralizing immune response to allow the challenge virus to replicate into the lung tissues. The presence of intra- and interstitial alveolar infiltration, known to be related to enhanced inflammation of the lung (as was observed in the lungs of the two deceased infants in the 1960s) is then typically observed by histological examination, (Figure 1, group 1). All other histological parameters (peribronchiolar infiltration, perivascular infiltration, PMN infiltration of bronchiolar epithelium, eosinophilic infiltration around bronchioles and eosinophilic infiltration of lymph nodes), which are scored in parallel to the two alveolar parameters are considered as normal indicators of an inflammatory process which occurs upon natural infection.

In contrast, when Fi RSV is formulated on 3D-MPL/Al(OH)₃ (hereinafter referred to as SB AS4) a significant reduction of the enhanced pulmonary pathology was observed as compared to Fi RSV Al(OH)₃ demonstrating that the addition of 3D- MPL is able to limit the occurrence of enhanced pulmonary pathology. This significant observation suggests that the Fi RSV Al(OH)₃ vaccine administered in the 1960s to seronegative children most probably induced an inadequate cell mediated immune response rather than what has long been hypothesized as an inadequate humoral response.

The observation that Fi RSV formulated on SB AS4 significantly reduces the post infection enhanced disease as compared to Fi RSV Al(OH) ₃ suggests that the use of 3D-MPL as an adjuvant can provide a safe formulation for the development of an anti-RSV vaccine, especially in infants.

This observation lead us to evaluate the use of 3D-MPL for a chimeric FG recombinant antigen produced in CHO mammalian cells, for example as described in US Patent 5194 595. The FG SB AS4 vaccine showed very promising results since, under experimental conditions similar to those described for the Fi RSV Al(OH)₃ it was observed that clearance of the virus from the lungs by the cell mediated immune response induced upon vaccination with a suboptimal dose of vaccine, (that is a vaccine dose which allows viral replication in the lungs), no alveolar nor peribronchiolar inflammation are induced (Figure 1). These results are comparable to what is observed in naturally immune animals. The RSV FG vaccine comprising 3D- MPL is therefore expected to be safe and protective against RSV primary infection.

The cotton rat experiment was carried out according to the protocol as detailed below. Figure 1 illustrates the obtained results.

The upper part of the figure shows that the sub optimal immunogenicity experimental conditions were respected. Indeed, a non sterilizing immune response was induced as monitored by appearance of anti-FG Abs in vaccinated cotton rats allowing some viral replication in the lung following challenge of the immunized animals.

The lower part of the figure shows the respective scoring of the alveolar (intra- and interstitial) and "other" inflammation induced by Al(OH)₃ and SB AS4 formulated Fi RSV or FG. As discussed above, the candidate vaccine FG SB AS4 shows no enhanced pathology and the reduced alveolar inflammation observed for Fi RSV SB AS4 as compared to Fi RSV Al(OH)₃ illustrates the drastic positive effect of SB AS4 on the enhanced pulmonary disease. The level of "other" inflammation reached by SB AS4 formulations is of the same order of magnitude as that seen for live RSV which is considered as the control for absence of enhanced pathology since it does not induce enhanced pathology upon subsequent exposure to RSV.

The protocol for the cotton rat experiment

- Cotton rats (24/group) were vaccinated IM with 200 μl of the Ag formulation on days 0 and day 21.

- Ag formulations are as follows:

A control group immunized with the live RSV/Long strain given by the intranasal route was included as a control for absence of enhanced pathology. The untreated animals served as a control for normal disease read-outs.

The animals were challenged under anesthesia by the intranasal route on day 49 using 10⁶pfu of live RSV/Long strain.

Lung tissues from the challenged animals were taken for virus titration in those organs. Pieces of lungs were also cryo preserved for histopathological analysis and evaluation of enhanced pathology.

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

Claims

What is claimed is:

1. A method to improve purification of RSV F-G chimeric protein comprising: a) contacting an impure solution containing said protein with a filter which removes viruses; b) passing the filtered solution through cation exchange, metal chelate and anion exchange chromatography and recovering said protein; c) inactivation of said filtered solution; and d) filter sterilization.

2. The method of claim 1 wherein an adsorption and gel filtration step occur after step (b) and before step (d).

3. The method of claim 2 wherein the adsorption and gel filtration step occur on CM EMD TSK resin.

4. The method of claim 2 wherein the adsorption and gel filtration step occur on an Ether Toyopearl resin followed by a Sephacryl S or (Pharmacia) G25 resin.

5. The method of claim 1 wherein step (b) is carried out in the following order: cation exchange chromatography, metal chelate chromatography, and anion exchange chromatography.

6. The method of claim 1 wherein said filter in step (a) is a hollow fiber filter.

7. The method of claim 1 wherein the cation exchange chromatography is carried out with SP Sepharose resin.

8. The method of claim 1 wherein the anion exchange chromatography is carried out Q Sepharose resin.

9. The method of claim 1 wherein the metal chelate chromatography is carried out with zinc-chelate resin.

10. A method to improve purification of RSV F-G chimeric protein comprising: a) contacting an impure solution containing said protein with one of the following: cation exchange, metal chelate or anion exchange chromatography and recovering said protein; b) passing a solution containing said protein through a filter which removes viruses; c) passing the filtered solution through the two other chromatography steps not used in (a); d) inactivation of said filtered solution; and e) filter sterilization.

11. The method of claim 10 wherein detergent is added after step (a) and before step (b).

12. The method of claim 11 wherein the detergent is Tween 20.

13. The method of claim 12 wherein the detergent is added to an amount of 0.1 to 1 % (v/v).

14. The method of claim 10 wherein an adsorption and gel filtration step occur after step (c) and before step (d).

15. The method of claim 14 wherein the adsorption and gel filtration step occur on CM EMD TSK resin.

16. The method of claim 14 wherein the adsorption and gel filtration step occur on an Ether Toyopearl resin followed by a Sephacryl S resin.

17. The method of claim 10 wherein step (a) is cation exchange chromatography.

18. The method of claim 10 wherein step (c) is carried out in the following order: metal chelate chromatography, and anion exchange chromatography.

19. The method of claim 10 wherein said filter in step (b) is a hollow fiber filter.

20. The method of claim 10 wherein the cation exchange chromatography is carried out with SP Sepharose resin.

21. The method of claim 10 wherein the ion exchange chromatography is carried out with Q Sepharose resin.

22. The method of claim 10 wherein the metal chelate chromatography is carried out with zinc-chelate resin.

23. The method of claim 1 or 10 wherein the RSV F-G chimeric protein comprises amino acids 1-526 of the F protein fused to amino acids 69-298 of the G protein.

24. The method of claim 1 or 10 wherein the RSV F-G chimeric protein comprises amino acids 1-489 of the F protein fused to amino acids 79-279 of the G protein.

25. A RSV F-G chimeric protein obtained by a method as claimed in any one of claims 1 - 24.

26. A method to prepare a pharmaceutical composition comprising purification of a RSV F-G chimeric protein as claimed in any one of claims 1 - 24 and admixing said protein with an adjuvant.

27. A RSV F-G chimeric protein comprising amino acids 1-526 of the F protein fused to amino acids 69-298 of the G protein.