IE61992B1 - Receptor of the small rhinovirus receptor group - Google Patents

Abstract

A receptor from the small receptor group of human rhinoviruses, its purification and use.

Description

RECEPTOR OF THE SMALL RHINOVIRUS: RECEPTOR GROUP This invention relates to the receptor of the small receptor group of human rhinoviruses, the purification and use thereof.

Human rhinoviruses constitute a large genus within the family of Picorna viruses and contain over 90 different serotypes (6,11). These RNA viruses affect the respiratory tract of humans and cause acute infections which may lead to colds, coughs, hoarseness, etc., and are generally known as colds (15). Infections caused by rhinoviruses are among the most common diseases in man. Although the course of the diseases is generally harmless, colds do nevertheless result in general weakening of the organism. This may then give rise to secondary infections caused by other pathogens.

The large group of human rhinoviruses can be subdivided into two sub-groups if the competition for binding sites on the cell surface in human cell culture cells (generally HeLa cells) is used as the criterion for classification. This original classification of a few representatives of the rhinoviruses (10) has been extended to 88 representatives as a result of a wide range of experiments (4,1). The result of these experiments was to indicate that in spite of the large number, there are surprisingly only two different receptors on the cell surface to which representatives of one or other group of rhinoviruses can bind. Up till new, 78 serotypes of the large rhinovirus receptor group and 8 of the small rhinovirus receptor group have been classified (RVRG). 2 other representatives did not behave clearly so that they could-not be definitively classified (1).

Abraham, G. and Colonno, R.J., (1984), J. Virol. 51 340-345) established, by competitive binding assays, I.) that there are probably two families of receptors for receiving human rhinoviruses, the majority of the rhinoviruses binding to one receptor. Rhinoviruses of one receptor group might competitively inhibit binding of the receptor. However, these competitive experiments do not allow any conclusions to be drawn as to the nature of the receptors.

In recent years, a considerable increase in rhinovirus IQ infections has been discovered in densely populated areas. Whereas the majority of other infectious diseases result in a long-lasting or permanent'immunity from the pathogen in question, infections caused by rhinoviruses may recur again and again. The reason for the absence of any lasting immunity is the large variety of strains of rhinovirus which show little or no immunological inter-reaction with one another (6,11). After infection has occurred, antibodies against the strain of virus in question can be detected but these do not confer any protection against other rhinovirus strains. In view of the large number of strains circulating in the population, repeated infections by rhinoviruses are possible.

Therefore, the presence of only two receptors offers promising possibilities for the successful combating of rhinoviral infections.

Since receptors are generally highly specific, there is a’possibility of achieving controlled influence on the receptors by means of suitable substances, for example by blocking the receptors. If substances which block the receptor are used, the penetration of receptorspecific viruses into the cell can be prevented. The same substances which can prevent infection in this way can also be used for the treatment of a manifest rhinovirus infection. The production of such substances is made substantially easier and in some cases made possible for the first time, if the receptor in question is characterised.

One aim of this invention was therefore to isolate and purify the receptor for the small RVRG.

The only information on rhinoviral receptors available hitherto has concerned the receptor of the large RVRG.

The purification and characterisation of the receptor of the large RVRG was effected using a monoclonal'antibody obtained by immunising mice with HeLa cells. This receptor is glycosylated and has a molecular weight of about 440 kD in its native state; denaturing with sodium laurylsulphate results in a dissociation into subunits of 90 kD, leading one to conclude that the functional receptor is present as a pentamer (17) . Hitherto, the receptor for the small RVRG has neither been characterised nor purified. The only data on this receptor indicate its protein structure and also show that these or similar proteins are also present on cells in a number of other species. This distinguishes the receptor of the small RVRG essentially from the receptor of the large group, which has only been found in human cells and, in a few cases, in monkey cells. Influencing of this receptor, for example blocking the receptor with substances which prevent penetration of the virus into the cell, would appear to be suitable as a possible prevention or even treatment for an existing infection.

The aim of this invention was therefore to provide the prerequisites for preparing substances which give protection against infections by rhinoviruses of the small receptor group.

This is achieved in the present invention by isolating the receptor from cell membrane, for example HeLa cell membranes. These cells were cultivated in suspension by methods known per se. the cells were broken up, the nuclei removed and the membranes purified. The receptors found in the membranes were then solubilised. To achieve optimum solubilisation of active receptors from purified HeLa cells, various detergents were tested at different concentrations. The critical factor in choosing a specific detergent was its ability to solubilise as much membrane material as possible with the highest possible virus binding activity. 1% l-O-noctyl-/3-D-glucopyranoside proved to be the most suitable (Fig. 0).

The insoluble constituents were removed and the receptors in solution were further purified. In order to be able to monitor the virus binding activity, a sensitive filter binding test was developed which makes it possible for 35S-labelled virus to bind to receptors which had been immobilised on nitrocellulose paper.

The viruses required for the test were cultivated and purified in a manner known per se (13) .

The receptors according to the invention were purified by chromatographic methods.

Since it is known that the majority of membrane proteins are glycosylated, the receptor was purified on a Lens Gulinaris lectin column. This lectin has specificity for α-D-glucose and α-D-mannose units (16). Bound material was eluted with 1M α-D-methylglucoside in phosphate-buffered NaCl solution with 1% octylglucoside.

Aliquots were applied in duplicate to nitrocellulose and incubated both with native and with heated virus.

Autoradiography of the fractions showed strong binding to the native virus in the case of the material which had been eluted, compared with the fractions from the material which had run through. The heated virus showed weak binding to the run-through material. This indicates nonspecific interactions which are caused by the high proportion of hydrophobic proteins; heated rhinovirus has greater hydrophobicity (9). Since it had been established that, on being stored for a fairly long time at 4’C, purified virus gradually changes into particles which have the same antigenicity as heated virus (C-determinants), these contaminations were separated off by immunoprecipitation with C-determinantspecific monoclonal antibodies, for example mAK 2G2, immediately before the binding test was carried out. These monoclonal antibodies are obtained in a manner known per se by immunising mice or rabbits with Cdeterminants and subsequently cloning according to Kohler and Milstein (18).

In addition to the L. culinaris lectin column, concanavalin A, ricin and heparin-Sepharose columns were also used to purify the receptor. The run-through and eluted material were tested as above. Con.A Sepharose columns were eluted with 1 M a-D-methyl-mannoside, approximately 20% of the binding activity being recoverable; elution of the ricin column with 1 M galactose resulted in approximately 100% recoverable binding activity. By contrast with the receptor for the Coxsackie B virus group (7) heparin-Sepharose did not fetard the binding activity.

The eluate from the L. culinaris column was separated on a Superose column by FPLC (Pharmacia) (gel permeation chromatography). By comparison with marker proteins, the molecular weight of the active receptor was determined as 450 kD. At the same time, a substantial proportion of contaminating proteins could be removed (Figure 1).

The receptor of the small rhinovirus receptor group migrates in a polyacrylamide gel in the presence of SDS with an apparent molecular weight of 120 kD. In some cases a scarcely visible band with a slightly lower molecular weight can also be seen which may possibly be a modification of the main quantity of the receptor protein (Fig. 5, column 1). The molecular weights of both forms of the receptor are considerably higher than that which was found for the large rhinovirus receptor (90 kD) .

Since the receptor proteins of both groups have a molecular weight of about 450 kD in their native form, it is not improbable that their construction from subunits is similar. The actual molecular weight may, however, deviate from that which is determined by gel permeation chromatography, as a result of the small difference in the retention volumes of proteins in this high molecular weight range. The Picorna virus structure shows a deep bifurcation (the Canyon) which runs around the five-fold axes of the icosahedral symmetry and possibly contains the receptor binding site (19). It is assumed that the receptor of the large rhinovirus receptor group and the receptor for the Coxsackie B virus group which bind the viruses via the five-fold axes (20). The question whether the receptor of the small group is a pentamer remains unanswered since the molecular weight of its subunits is very high, compared with the receptor of the large group.

By sucrose gradient centrifugation, it was possible to determine the sedimentation constant of the receptor.

For this purpose, L. culinaris purified receptor was applied to a sucrose gradient and centrifuged.

The activity peak was found to be at the position of the gradient which corresponds to the sedimentation constant of 28.4 S (Figure 2).

Preliminary tests had shown that the receptor could no longer be eluted from an anion exchange chromatography column. Since the receptor is insensitive to neuraminidase, -sialic acid was removed from the glycoprotein in order to reduce the ionic interaction with the column material. The sample was then applied to a mono Q column (Pharmacia) and the receptor was eluted with an NaCl gradient. The binding activity could be detected as a broad peak at about 250 mM NaCl (Figure 3).

It is also possible to purify the receptors according to the invention by a combination of chromatographic purification steps on different chromatography materials.

The chemical properties and structural requirements of the receptor according to the invention for viral binding were determined with the aid of enzymes and chemical reagents (Table 1).

Trypsin treatment destroys the binding activity entirely. This agrees with known results from enzymatic treatment of cell surfaces (14) and indicates the protein nature of the receptor.

..Treatment of the solubilised receptor with neuraminidase resulted, in reproducible manner, in a slight increase in the binding activity. This treatment may possibly lead to better accessibility of the region on the receptor molecule which is the target of the virus interaction. As a result, sialic acid is not necessary for the virus binding.

Dithiothreitol (DTT) destroys the binding activity, leading one to conclude that disulphide bridges are involved in maintaining the correct folding of the protein. The surprisingly high molecular weight, determined by gel permeation chromatography and gradient centrifugation, indicates an oligomeric structure for the receptor molecule. The sensitivity to DTT might lead one to conclude that intermolecular disulphide bridges are necessary for the association of the hypothetical sub-units.

Treatment with iodacetamide reduces the binding activity only slightly and indicates that free sulphydryl groups are not necessary for efficient binding.

In the presence of ethylenediaminotetraacetic acid (EDTA) during incubation of the nitrocellulose filters 35 with S-labelled virus, no binding could be detected.

This agrees with earlier investigations which showed the need for the presence of divalent cations for interaction of the rhinoviruses with the cell surface (12).

Competitive binding assays between pairs of serotypes had been used in order to classify the human rhinoviruses into the two receptor classes (10,1). In the present invention, therefore, HRV2 and HRV49 were used as representatives of the small receptor group and HRV89 as representative of the large receptor group in competitive experiments to discover the specificity of the receptors according to the invention. The nitrocellulose filters with immobilised receptors were incubated in the presence of an approximately 20-fold excess of either HRV2 or HRV89 with labelled HRV2. As shown in Table 2, the binding was massively suppressed in the presence of non-labelled HRV2 but unaffected by HRV89. In order to check these results, the tests were repeated with labelled HRV49, using HRV2 and HRV89 as competitors. Once again, it is obvious that HRV2 reduces binding on a massive scale but HRV89 has no effect.

Although HRV2 and HRV89 bind to different receptors, their capsid proteins are surprisingly similar (5).

A detailed comparison of structure between HRV2 and HRV14, based on the X-ray structure analysis of HRV14, was recently set up (2). Both HRV14 and HRV89 bind to the receptor of the large RVRG. It can therefore be expected that a cluster of preserved amino acids will be found at the hypothetical receptor binding site. Up till now, however, it has not yet been possible to discover a simple pattern of conservative amino acids within the Canyon region.

The present invention makes it possible for the first time to produce receptors for the small RVRG.

Using the receptors according to the invention it is possible for the first time to carry out controlled investigations on the virus/receptor interactions.

Of particular importance is the locating of the regions on the receptors which are finally responsible for the viral activity. Once these areas are known, it should be possible to produce substances which are directed specifically against these areas, and thereby possibly block the receptors for a variety of different rhinoviruses.

T-he present invention relates to receptors which can be prepared by the process described and which bind representatives of the small RVRG.

The present invention also relates to the receptors which can be produced from the natural receptors by methods known to those skilled in the art. By way of example, there may be mentioned the sub-units of the natural receptor, which are obtained by treating with reducing agents, and which can be purified for example by electrophoretic methods. These sub-units may be used, for example, to produce polyclonal and/or monoclonal antibodies which can be used preparatively, diagnostically and/or therapeutically in a similar manner to the corresponding antibodies against the natural receptors. The receptor sub-units may also be used in a similar manner to the natural receptors.

The present invention also relates to the modifications of the natural receptors and/or the sub-units thereof which can be obtained by controlled enzymatic treatment. As has been shown in this invention, treatment with trypsin destroys the binding activity of the receptors according to the invention, whilst neuraminidase caused a slight increase in activity. Therefore it is conceivable, and anyone skilled in the art can check this in a non-inventive manner, that specific enzymes and/or chemical reagents result in receptors which either have an improved activity and/or are easier to apply and use and/or have better stability compared with the natural receptors. These modifications may, for example, result in parts of the protein chain being severed or cut out and/or the protein chains being cut up, in all or some of the sub-units of the natural receptors.

JjO addition to these modifications it is also possible to convert the natural receptors either wholly or partially into the sub-units, for example by controlled reduction. These large or small sub-units may also be linked together, for example by controlled oxidation, to form large or small units which are rearranged compared with the natural receptors.

Suitable reducing agents for cleaving disulphide bridges include, for example, thiol compounds such as thiophenol, 4-nitrothiophenol, 1,4-butanedithiol and particularly 1,4-dithiothreitol. The reduction is advantageously carried out in an aqueous/alkaline medium, for example in the dilute aqueous solution of an alkali metal hydroxide, e.g. sodium hydroxide, alkali metal carbonate, e.g. sodium carbonate or an organic base, more particularly a tri-lower alkylamine, e.g. triethylamine, at ambient temperature.

Suitable oxidising agents for the re-linking of disulphide bonds in the reduced polypeptides include, for example, oxygen from the air, which is passed through an aqueous solution of the polypeptide to which a catalytic amount of a transition metal salt, e.g. iron(III)sulphate, iron(III)-chloride or copper(II)-sulphate, may have been added; iodine, including iodine in the form of the potassium iodide adduct KI^, which is preferably used in alcoholic, e.g. methanolic, or aqueous-alcoholic, e.g. aqueous-methanolic solution; potassium hexacyanoferrate(III) in aqueous solution; 1,2-diiodoethane or dimethyl or diethyl azodicarboxylate, which are reacted in water or in a mixture consisting of water and a water-miscible alcohol, e.g. methanol. Oxidation is more particularly carried out at ambient temperature.

The removal of the reagents, particularly the salts _and the oxidants and reducing agents and their secondary products, from the desired compound is carried out by methods known per se, for example by molecular weight filtration, e.g. on Sephadex or Biogel.

All the modifications may be used in the same way as the natural receptors according to the invention.

The products obtainable in this way, such as the antibodies, like the modifications, fall within the scope of the present invention.

The receptor according to the invention is soluble, so that it is easy to handle and use.

However, it is also possible to bind the receptors to a solid carrier and to use them in this form for diagnostic and preparative purposes. Suitable carriers include all the usual solid carriers such as polystyrene, glass, dextrans and also biological membranes and lipid vesicles.

It is also possible to bind conventional labels to the receptors and to use them in this form for diagnostic purposes. It is also possible to use the receptors for the therapeutic treatment of viral infections. If the receptors according to the invention are bound to a carrier, they may be used both diagnostically and also preparatively to bind the viral protein, for example by means of so-called affinity chromatography. Diagnostically, a viral protein can be detected in the usual way by a receptor bound to a carrier, e.g. by means of antibodies or labelled antibodies. The labelling used may be, for example, radioactive labelling, an enzyme or a fluorescent group.

When the receptors according to the invention are used therapeutically, they may be ejected in suitably Jhighly refined form, so that they can then inhibit competitively against the natural receptor. Preferably, soluble receptors will be used for this purpose.

Such solutions may also be used for diagnosis and differential diagnosis.

An exceptionally important application is the use of the receptors according to the invention for producing polyclonal and/or monoclonal antibodies which act specifically against the receptors located in the cell membranes. Antibodies of this kind may first of all be used diagnostically to show up and determine the receptors on cells or biological cell material. Furthermore, they may be used therapeutically to block the receptors in the cell membranes. Consequently, they open up totally new methods and possibilities.

Legend relating to the Figures Figure 0 Solubilisation of the receptors with various detergents (A: nat. virus, B: denat. virus).

Figure 1 Gel permeation chromatography of the solubilised receptor on a Superose 6 HR 10/30 column. p Membranes equivalent to 10 cells were solubilised in OG (15 mM sodium phosphate pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2 (PBS) with 1% octyl glucoside), applied to a 1 ml L. culinaris column and the adsorbed material was eluted with 1 Μ aD-methylglucoside in OG. The eluate was concentrated in a Centricon test tube to 0.5 ml and applied to the Superose column.

The column was developed with 0.2 ml/min OG and 0.5 ml fractions were collected.

The binding activity of the individual fractions, the positions of the marker proteins (determined in a separate experiment) and the extinction at 280 nm are shown.

Figure 2 Sucrose gradient centrifugation of the solubilised receptor. Material which had been eluted from an L. culinaris column was concentrated (see the legend to Figure 1) and separated on a 10 - 40% sucrose gradient in OG. The binding activity of the fractions (0.4 ml) , the position of the marker proteins catalase (Cat) and aldolase (Aid) and the extinction at 280 nm are shown.

Figure 3 Mono Q anion exchange chromatography.

Fractions 14 to 16 of the gel permeation chromatography (Figure 1) were treated with neuraminidase and the material was Figure 4 Figure 5 separated by FPLC on mono Q. The binding activity of the individual fractions (0.5 ml), the extinction at 280 nm and the path of the gradient are shown.

Detection of the receptor on Western blots. Virus binding activity of the Mono P anion exchange column was applied to a Superose 6 HR 10/30 column. Fractions of 1.2 ml were collected. The proteins were monitored as Α2θθ, the positions of protein markers (thyroglobulin, 670k; apoferritin 440k; βamylase, 200k) are also shown (a). The proteins in the fractions from the Superose column were concentrated and applied to three 6% SDS-polyacrylamide gels. One gel was stained with Coomassie blue (b), the proteins of the second and third gels were applied to nitrocellulose membrane (c, d). The blot was incubated with 35S-labelled HRV2 in the absence (c) or in the presence of an excess of unmarked HRV2s (d); the positions of the marker proteins (2-macroglobulin, 180k; βgalactosidase, 116k; fructose-e-phosphatekinase, 84k) and of the receptor band are indicated by arrows.

Autoradiography of Western blots which were obtained from the combined fractions with virus binding activity from the Superose column (see Fig. 4). The blots were incubated with 35S-labelled HRV2. Before being applied to the gel the samples were incubated with SDS at ambient temperature, trace 1; the sample was boiled in SDS, trace 2, the sample was incubated with 10 mM dithiothreitol, trace 3; a blot identical to trace l was incubated with 35S-labelled HRV2 in the presence of 10 mM EDTA, trace 4, or was incubated with 35Slabelled HRV2 which had been heated to 56°C for 10 minutes, trace 5.

Mater ials l-0-n-Octyl-6-glucopyranoside, Tween 40 and 3-(3cholamidopropyl)-dimethylammonio-l-propane sulphonate (CHAPS) were obtained from Sigma, N-tetradecyl-N,N-dimethyl-ammonio-3-propane sulphonate (Zwittergent 3 - 14) was obtained from Serva. The other detergents came from Merck, trypsin from Miles 35 and S-methionine (1350 Ci/mmol) from Amersham.

Example 1: Preparation of the viruses HRV2, HRV49 and HRV89 were cultivated essentially as described in HeLa cell suspension and then purified (13). The cultivation, isolation and purification of HRV2 will be described here by way of example.

HeLa cells (strain HeLa Ohio, 03-147, Flow Laboratories, England) were grown in suspension at 37°C. The suspension medium (Thomas, D.C., Conant, R.M. and Hamparian, V.U., 1970, Proc. Soc. Exp. Biol. Med. 133, 62 - 65; Stott, E.J. and Heath, G.F., 1970, J. Gen. Virol, 6, 15 - 24) consisted of a Joklik modification of MEM for suspension (Gibco 072-1300) and 7% horse serum (Seromed 0135). The inoculation density was 4 - 10 x 10 cells/ml and the volume was 500 ml.

The suspension was centrifuged at a cell density -of 1 x 10° cells/ml under sterile conditions at 300 g for 10 minutes. The supernatant was removed by suction filtering and the cells were resuspended in 100 ml of infection medium (Joklik modification of MEM for suspension culture with 2% horse serum and 2 mM MgC^)· By carefully sucking up several times in a 20 ml pipette, the cells were homogeneously distributed in the infection medium. The medium was then made - 18 up to 500 ml. The cell suspension was brought to 34°C and infected with HRV2 (twice plaque-purified) at a multiplicity of 0.1 viruses per cell. The HRV2 strain was obtained from the American Type Culture Collection, (ATCC VR-482 and VR-1112). The strain used was neutralised with antiserum against HRV2 (American Type Culture Collection, Cat.No. ATCC VR-1112 -AS/GP). The control serum used was an antiserum against HRV7 (Cat.No. ATCC VR-1117 AS/GP) which showed no neutralisation. After 60 hours at 34°C the virus was harvested. Virus was obtained both from the cells and cell fragments and also from the medium.

For this purpose, the medium was separated from infected cells and cell fragments by centrifuging for 10 minutes at 1500 g and then suction filtering. The precipitate was frozen at -70°C. ........

The cell precipitates from 12 litres of suspension culture were combined, resuspended in 40 ml of TM buffer (20 mM Tris/HCl, pH 7.5, 2 mM MgC^) , put on ice for 15 minutes, then broken up in a Dounce homogeniser and the mixture was centrifuged for 30 minutes at 6000 g. The precipitate was then washed once again in 10 ml of TM buffer. The two supernatents were combined and centrifuged for 3 hours at 110,000 g in order to pellet the virus. The virus pellet was then taken up in 10 ml of KTMP buffer (50 mM KC1, mM Tris/HCl, pH 7.5, 5 mM MgCl2» 2 mM mercaptoethanol, JL mM puromycin, 0.5 mM GTP) and after the addition of 150 meg of DNase I (Sigma, ribonuclease-free) it was incubated for 1 hour on ice.

The virus was precipitated from the infection medium with stirring at 4°C with polyethylene glycol 6000 (PEG 6000; Merck) at a concentration of 7% and 450 mM NaCI (Korant, B.D., Lonberg-Holm, K., Noble, J. and - 19 Stasny, J.T., 1972, Virology £8, 71 - 86). After 4 hours in the cold, the virus was centrifuged off for 30 minutes at 1500 g, the precipitate was resuspended in 10 ml of KTMP buffer containing 75 meg of DNase I, the mixture was incubated for 1 hour on ice and then frozen at -70°C.

The virus suspensions obtained from the cells and from the medium were combined, incubated for 5 min. at 37°C, cooled by the addition of 60 ml of cold TE buffer (10 mM Tris/HCl, pH 7.4, 1 mM EDTA) and then sonicated for 5 min in an ice bath.

The suspension was then centrifuged for 30 minutes at 6000 g. 920 ml of TE buffer containing 7% PEG 6000 and 450 mM NaCl were added to the supernatant, this was stirred carefully for 4 hours at 4°C and the precipitate formed was pelleted for 30 minutes at 6000 g. The precipitate was once again taken up in 100 ml of TM buffer, the virus was precipitated as above by the addition of PEG 6000 and NaCl and pelleted. The precipitate was resuspended in 40 ml of TM buffer, the suspension was centrifuged for 30 minutes at 6000 g and the virus was pelleted for 3 hours at 110,000 g. The precipitate was dissolved in 1 ml TM buffer, incubated for 1 hour at 4°C after the addition of 50 meg of DNase I and then 1 ml of TE buffer was added. For further purification, the virus suspension was centrifuged on sucrose gradients -{10 - 30% w/w in TE buffer) for 4 hours at 4°C and at 110,000 g. From the extinction at 260 nm, the fractions containing the virus were discovered and diluted with TM buffer so that the final sucrose concentration was 10%. Then centrifuging was carried out for 8 hours at 85,000 g.

The virus pellet was taken up in 1 ml TM buffer and stored at -70°C. To check the purity of the virus preparation, electrophoresis was carried out on a 12.5% polyacrylamide gel in the presence of 0.1% sodium dodecylsulphate (Laemmli, U.K., 1970 Nature (London) 277, 680-685) and the protein bands were stained with Coomassie Brilliant Blue.

Example 2: Preparation of 33S-methionine-labelled human rhinovirus serotype 2 (HRV2) HeLa cell mono layers in 165 cm Petri dishes were infected with an MOI (Multiplicity of Infection) of 40 with HRV2 for 1 hour at 34°C in methioninefree MEM medium (Gibco) with 2% dialysed foetal calf serum (Flow). The cells were washed twice with PBS and incubation was continued at 34°C in fresh medium.

After 3 hours, 1 mCi S-methionine (1350 Ci/mmol, Amersham) was added to each mono layer and incubation was continued for a total of 24 hours. The medium of infected cells and cell fragments was separated by 10 minutes' centrifuging at 1500 g and suction filtered. The precipitate was frozen at -70°C in 5 ml of 10 mM Tris, 10 mM EDTA, pH 7.5 (Tris/EDTA) and thawed again. The supernatant and frozen/thawed precipitate were combined and centrifuged for 30 min. at 45,000 x g. The supernatant from this centrifug -at ion was centrifuged for 2 hours at 140,000 x g.

The virus pellet was resuspended in 300 mcl Tris/EDTA and the virus was purified over a 10-30% sucrose gradient as above. The individual fractions of the gradient were analysed by SDS electrophoresis in 12% polyacrylamide gels. The pure virus fractions were combined and stored in the presence of 1% BSA (bovine serum albumin) at 4°C for at most 4 weeks.

In order to remove virus with an altered capsid structure from the preparations from Examples 1 and 2, 20 mcl of immunoadsorbant, containing monoclonal antibodies against the C-determinant, for example mAK 2G2, were incubated with the purified virus for 30 min. and pelleted before the viral probes were removed.

•Preparation of the immunoadsorbant Staphylococcus aureus (BRL) cells were suspended as a 10% w/w suspension in water. The cells were washed twice with PBS, 1/5 vol rabbit-anti-mouse IgG serum (Behring) was added; the suspension was incubated for 1 hour at ambient temperature. The cells were washed twice with PBS and incubated again 15 for 1 hour with 1/5 vol mouse ascites fluid, which contained monoclonal antibodies against the C-determinants (e.g. mAK 2G2). After washing twice, it was pelleted, the pellet was resuspended in PBS (10% w/w) and the preparations of the radioactive viruses were added.

Example 3: Solubilisation of the receptor from HeLa cells HeLa cells (strain HeLa-Ohio, 03-147, Flow Laboratories, England) were cultivated in suspension at 37°C.

The suspension medium (Thomas, D.C., Conant, R.M. and Hamparian, V.U., 1970, Proc. Soc. Exp. Biol. 41ed. 133, 62 - 65; Stott, E.J. and Heath, G.F., 1970, J. Gen. Virol. 6, 15 - 24) consisted of a Joklik modification of MEM for suspension (Gibco 072-1300) and 7% horse serum (Seromed 0135). The inoculation 4 density was 5 - 10 x 10 cells/ml and the volume was 500 ml. The suspension was centrifuged at a cell density of 1 x 10θ cells/ml under sterile conditions at 300 g for 10 min. The supernatant was removed t* by suction filtering and the cells were washed twice g with phosphate-buffered saline solution (PBS). 10 cells were suspended in 20 ml of isotonic buffer (10 mM HEPES-KOH, pH 7.9, 140 mM KCl, 1.5 mM MgCl2, 0.5 mM EDTA, 0.2 mM phenylmethylsulphonylfluoride) and broken up in the cold with 200 pulses of a 50 ml Dounce homogeniser. Cell nuclei were removed by centrifuging for 3 minutes at 1000 x g. The membranes were then further purified by the two-phase method p (3). Membranes corresponding to 2x10 cells per ml were taken up in PBS and stored in liquid nitrogen. p To solubilise them, 2 x 10 cell equivalents were suspended in 1 ml of 1% octylglucoside in PBS (OG) and any insoluble material was removed by centrifuging at 80,000 x g for 1 hour. The supernatant was used for column chromatography.

Example 4: Filter binding test Fractions from column chromatography to be tested for activity were applied to a nitrocellulose membrane (BA85, Schleicher and SchOll) in a dot-blot apparatus (Bio-Rad). The probes were left to seep in at ambient temperature. Then liquid residues were suction filtered under a gentle water jet vacuum and non-specific protein binding sites were saturated with 2% bovine serum albumin (BSA) in PBS at 4°C overnight. The filters were then incubated with 10 cpm S-methionine labelled HRV2 in 1% Tween 40, 0.5% sodium deoxycholate and 10 mM (3-(3-cholamidopropyl)-dimethylammonio1-propane sulphonate) in PBS for 1 hour. The membranes were washed twice with 2% BSA in PBS, then dried and the round areas corresponding to the probes were stamped out; the radioactivity was measured in a liquid scintillation counter.

As a specificity control, HRV2 was heated to 56°C for 10 minutes before the incubation of the nitrocellulose filters (8). After this treatment, no binding to any of the probes could be detected (Fig. IB) . From this it was concluded that the binding of native HRV2 to the immobilised material can actually be ascribed to a specific interaction of the virus with the receptor.

Example 5: Affinity chromatography on Lens culinaris lectin columns p cell equivalents were solubilised as described and applied to an L. culinaris column (1 ml) equilibrated with OG. The column was washed with 5 ml of OG and bound material was eluted with 2 ml of 1 M a-D-methylglucoside in OG. The binding test showed that almost 100% of the binding activity could be recovered, whereas about 90% of the total protein had been removed.

Example 6: Gel permeation chromatography The eluate from the L. culinaris column was concentrated down to 0.5 ml with a Centricon tube (exclusion 30 kD) and separated on a Superose 6 HR 10/30 column (equilibrated with OG-buffer) by FPLC (Pharmacia). By comparison with marker proteins the molecular weight of the active receptor could be determined as 450 kD. At the same time a large proportion of contaminating proteins could be removed (Figure 1).

Example 7: Sucrose gradient centrifugation L. culinaris purified receptor (as above) was applied to a sucrose gradient (10 - 40% in OG) and centrifuged for 8 hours at 38 krpm at 4°C. The activity peak •was found at the position on the gradient corresponding to the sedimentation constant of 28.4 S. The positions of the marker proteins were determined in another gradient (Figure 2). As a result of the presence of detergents, the markers sedimented at calculated sedimentation coefficients of 15.0 S and 21.9 S (7.3 S and 11.3 S in the absence of detergents).

Example 8: Anion exchange chromatography It had been found in preliminary tests that the receptor could no longer be eluted by mono Q HR 5/5 columns (Pharmacia) . Therefore, receptor which, had been subjected to preliminary purification using L. culinaris and gel permeation was treated with 1 U neuraminidase/mg of protein for 60 min at 37°C in order to remove the strongly acidic neuramino acid groups. The sample was then diluted with 10 mM sodium phosphate buffer, pH 7, 1% octylglucoside, to twice the quantity and applied to a mono Q column. The column was developed .with a gradient of 0 to 1 M NaCl in the same buffer.

The binding activity could be detected as a broad peak at about 250 mM NaCl (Figure 3) .

Example 9 Isolation and purification of the receptor Plasma membranes from 2 x 109 HeLa cells were dissolved in 5 ml of PBS, containing 1% w/v l-0-n-octyl-/3-Dglucopyranoside and 0.01% w/v each of L-a-p-tosyl-Llysine chloromethylketone (TLCK), L-l-tosylamide-2phenylethylchloromethylketone (TPCK) and phenylmethylsulphonylfluoride (PMSF) (all obtained from SIGMA), at ambient temperature within 10 minutes. Any insoluble material was centrifuged off for 30 minutes at'30 krpm in a Beckmann 65 Fixed Angle Rotor. The supernatant was applied to a 25 ml L. culinaris lectin column which had been equilibrated with OG (PBS, 1% w/v octylglucoside), and the bound material was eluted with 5 ml of OG containing 1 M α-methylglucoside. The eluate was 50% saturated with an equal volume of saturated ammonium sulphate solution. The material precipitated was dissolved in 2 ml of buffer A (10 mM TRIS-HCl (pH 7.5), mM EDTA, 1% w/v octyl-glucoside), and injected into a Mono P anion exchange column connected to an FPLC-system (Pharmacia).

The proteins were separated by means of a gradient from 0 to 100% buffer B (like buffer A but containing 1.5 M NaCl). The fractions which contained virus binding activity were combined, concentrated to 0.5 ml using a Centricon test tube and separated on a Superose 6 column which had been equilibrated with OG containing 5 mM EDTA. The protein concentration was monitored as an extinction at 280 nm (Fig. 4a).

The fractions from this column were concentrated to μΐ, applied to 0.1% w/v SDS and aliquots were separated on three 6% polyacrylamide gels which contained 5 mM EDTA (21). The proteins separated in the gel were then either stained with Coomassie blue (Fig. 4b) or transferred electrophoretically to a nitrocellulose membrane (22) which had been incubated with 4 x 105 cpm of 35S-labelled HRV2 (see above) . The nitrocellulose was dried and autoradiographed (Fig. 4c). As the control for specific binding, a nitrocellulose replica produced in identical manner was incubated in the presence of a 20-fold excess of unmarked HRV2s (Fig. 4d). It was established that fractions 6 and 7 from the Superose column contained material capable of binding HRV2 when it was transferred to nitrocellulose. The autoradiography shows some bands with an apparent molecular weight greater than 300 kD in addition to a position which corresponds to about 120 kD, compared with marker proteins which were developed on the same gel (Fig. 4c). Only this 120 kD band disappears in the control which contains excess unlabelled virus (Fig. 4d) and thus exhibits a specific interaction of this protein with HRV2. The polyacrylamide gel which contains identical samples and was stained with Coomassie blue exhibited a very weak band at a position corresponding to the radioactive band on the Western blot. This band is found exclusively in the samples from fractions 6 and 7 which exhibited virus binding activity (Fig. 4b).

Example 10 Binding tests The re-creation of an active receptor depends on mild conditions; boiling in SDS, for example, irreversibly destroys the activity (cf. Fig. 5, columns 1 and 2). If the receptor preparation was incubated with 10 mM dithiothreitol before being applied to the gel, no binding could be observed (Fig. 5, column 3). Since the specific interactions of the rhinoviruses with their receptors depend on the presence of divalent cations (12), the blot of a sample which was identical to that applied to trace 1 (Fig. 5) was incubated with virus in the presence of EDTA. No binding was observed under these conditions (Fig. 5, column 4).

As a further test, incubation of the nitrocellulose membrane was carried out with HRV2 heated to 56 °C. This treatment results in structural changes in the viral capsid which makes it impossible for the virus to bind to the HeLa cell surface (23). As can be seen in Fig. 5, column 5, no binding occurs under these conditions.

Table I Sensitivity of the small rhinovirus group receptor Pretreatment of the solubilised small receptor group Binding assay (% Bind.-act.) NO pretreatment 100 10 meg trypsin 6 50 mU neuraminidase 170 10 mM dithiothreitol 15 10 mM iodacetamide 80 10 mM sodium periodate 70 10 mM EDTAa 5 All pre-incubations were carried out at 37°C for 30 minutes.

Note: (a) No pretreatment; instead, incubation was carried out with labelled HRV2 in the presence of 10 mM EDTA.

Table 2 Competition between various rhinovirus serotypes for the small receptor group.

Competition of radioactively labelled HRV2 with: nothing HRV2 HRV89 Binding assay (% Bind.-act.) 100 Competition of radioactively labelled HRV49 with: nothing 100 HRV2 15 HRV89 90 The filters were incubated with a 20-fold excess of non-labelled virus as described.

Bibliography 1. Abraham, G. , and Colonno, R.J. (1984). Many rhinovirus serotypes share the same cellular receptor. J. Virol. 51, 340 - 345. 2". Blaas, D. , Kuechler, E., Vriend, G., Arnold, E., Griffith, J.P., Luo, Μ., Rossmann, M.G. (1987). Comparison of three-dimensional structure of two human rhinoviruses (HRV2 and HRV14). Proteins (in press). 3. Brunette, D.M., and Till, J.E. (1971). A rapid method for the isolation of L-cell surface membranes using an aqueous two-phase polymer system. J. Membr. Biol. 5., 215 - 224. 4. Colonno, R.J., Callahan, P.L., and Long, W.J. (1986), Isolation of a monoclonal antibody that blocks attachment of the major group of human rhinoviruses. J. Virol. 57, 7 - 12.

. Duechler, Μ., Skern, T., Sommergruber, W., Neubauer, Ch., Gruendler, P., Fogy, I., Blaas, D. and Kuechler, E. (1987). Evolutionary relationships within the human rhinovirus genus; comparison of serotypes 89, 2 and 14. Proc. Natl. Acad. Sci. U.S.A. (in press). 6. Fox, J.P. (1976). Is a rhinovirus vaccine possbible? American J.Epidemiol. 103, 345 - 354. 7. Krah, D.L., and Crowell, R.L. (1985). Properties of the desoxycholate-solubilized HeLa cell plasma membrane receptor for binding group B coxsackieviruses. J. Virol. 53.: 867 - 870. 8. Lonberg-Holm, K., and Yin, F.H. (1973). Antigenic determinants of infective and inactivated human rhinovirus type 2. J. Virol., 12 , 114 - 123. 9. Lonberg-Holm, K., and Whiteley, N.M. (1976). Physical and metabolic requirements for early interaction of poliovirus and human rhinovirus with HeLa cells. J. Virol. 19, 857 - 870.

. Lonberg-Holm, K., Crowell. R.L., and Philipson, L. (1976). Unrelated animal viruses share receptors. Nature 259, 679 - 681. 11. Melnick, J.L. (1980). Taxonomy of Viruses. Proc. Med. Virol. 26., 214 - 232. 12. Noble-Harvey, J., and Lonberg-Holm, K. (1974). Sequential steps in attachment of human rhinovirus type 2 to HeLa cells. J. Gen. Virol. 25., 83 - 91. 13. Skern, T., Soramergruber, W., Blaas, D., Pieler, Ch., and Kuechler, E. (1984). The sequence of the polymerase gene of human rhinovirus type 2.

Virology 136., 125 - 132. 14. Stott, E.J., and Heath, G.F. (1970). Factors affecting the growth of rhinovirus 2 in suspension cultures of L132 cells. J. gen. Virol. 6., 15 - 24.

T5. Stott, E.J., Killington, R.A. (1972) Rhinoviruses. Ann. Rev. Microbiol. 26., 503 - 525. 16. Young, N.M., Leon, M.A., Takahashi, T., Howard, I.K., and Sage, H.J. (1971). Studies on a phytohemagglutinin from the lentil. III. Reaction of Lens culinaris hemagglutinin with polysaccharides, glycoproteins, and lymphocytes. J. Biol. Chem. 271, 1596 - 1601. 17. Tomassini, J.E. and Colonno, R.J. (1986). Isolation of a receptor protein involved in attachment of human rhinoviruses. J. Virol. 58 , 290 - 295. 18. Kohler, G. and Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of - predefined specificity. Nature (London) 256, 495 497. 19. Rossraann, M.G., Arnold, E., Erickson, J.W., Frankenberger, E.A., Griffith, J.P., Hecht, H-J., Johnson, J.E., Kamer, G., Luo, Μ., Mosser, A.M., Rueckert, R.R., Sherry, B.A, & Vriend, G. (1985). Structure of a common cold virus, human rhinovirus 14 (HRV14), Nature (London) 317, 145-154.

. Mapoles, J.E., Krah, D.L. S> Crowell, R.L. (1985). Purification of a HeLa cell receptor protein for group B coxsackieviruses. Journal of Virology 55, 560-566. 21. Laemmli, U.K. (1979). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 277. 680-685. 22. Burnette, W.N. (1981). Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate - polyacrylamide gels to unmodified -nitrocellulose and radiographic detection with -antibody and radioiodinated protein A. Analytical Biochemistry 112, 195-203. 23. Lonberg-Holm, K. & Yin, F.H. (1973). Antigenic determinants of infective and inactivated human rhinovirus type 2. Journal of Virology 9, 29-40.

Claims (21)

1. A receptor in substantially pure form, characterised in that - it has binding activity for rhinoviruses of the small receptor group, - the molecular weight, determined by gel permeation chromatography, is about 450 kD, - the sedimentation constant, determined by sucrose 10 ... gradient centrifugation m the presence of detergents, corresponds to about 28.4 S, - it is bound by Lens culinaris lectin, - it is not bound by heparin-Sepharose, - it binds irreversibly to an anion exchanger, - its binding activity is insensitive to neuraminidase, - it consists of sub-units associated by intermolecular disulphide bridges, - it shows no binding to rhinoviruses in the presence of EDTA, - its binding activity to rhinoviruses is only slightly influenced by iodacetamide.

2. A receptor sub-unit, characterised in that it can be produced by complete reduction of a receptor as claimed in claim 1.

3. A receptor, characterised in that it consists of at least two of the receptor sub-units according to claim 2 and is not the natural receptor.

4. A receptor capable of being produced-by controlled reduction of the receptor as claimed in claim 1.

5. A receptor capable of being prepared from a receptor as claimed in claim 1 by controlled treatment - 34 with enzymes and/or chemicals which have not already been specified in any of the preceding claims.

6. A receptor capable of being produced by controlled oxidation of at least two of the receptor sub-units as claimed in claim 2.

7. A process for preparing receptors with binding activity for rhinoviruses of the small receptor group according to claim 1, characterised in that a. membranes are isolated from cells, preferably HeLa cells, and purified, b. the receptors in the membranes are solubilised with detergents, preferably Triton-X100, CHAPS, Zwittergent, octyl glucoside or DOC, but particularly octyl glucoside, the insoluble constituents are removed and the receptors are purified by chromatography, preferably on a concanavalin-A, ricin-Sepharose or Lens culinaris lectin column.

8. A process as claimed in claim 7, characterised in that the receptors are chromatographed on an anion exchange column, preferably a Mono Q column, after treatment with neuraminidase.

9. A process as claimed in claim 7 or 8, characterised in that a second chromatographic purification is carried out on a Superose column after the first chromatographic purification.

10. A process for preparing the receptor sub-units as claimed in claim 2, characterised in that the receptor as claimed in claim 1 is reduced in controlled manner.

11. A process for preparing the receptor as claimed in claim 3, characterised in that at least two of the receptor sub-units as claimed in claim 2 are oxidised in controlled manner.

12. Use of the receptors according to one of claims 1 to 6 5 - for the qualitative and/or quantitative detection of rhinoviruses, in which the receptors may preferably be bound to a solid carrier; - for the analytical characterisation or purification of rhinoviruses; - for the preparation of polyclonal and/or monoclonal antibodies; - for the production of pharmaceutical preparations.

13. Agent for therapeutic treatment, characterised in 20 . that it contains m addition to pharmaceutically inert excipients and/or carriers an effective amount of a receptor according to one of claims 1 to 6.

14. Hybrid cell line, characterised in that it secretes 25 monoclonal antibodies against one of the receptors according to one of claims 1 to 6.

15. Monoclonal antibodies, characterised in that they specifically neutralise the receptors according to one of claims 1 to 6 or specifically bind to one of said receptors.

16. Polyclonal antibodies, characterised in that they neutralise the receptors according to one of claims 1 to 6 or bind to one of said receptors.

17. Use of the polyclonal and/or monoclonal antibodies according to claim 15 or 16 for the qualitative and/or quantitative determination of one of the receptors according to one of claims l to 6 or for purifying one 5 of the receptors according to one of claims 1 to 6.

18. Test kit for determining rhinoviruses, characterised in that it contains receptors according to one of claims 1 to 6.

19. Process for preparing monoclonal antibodies according to claim 15, characterised in that host animals are immunised with one of the receptors according to one of claims 1 to 6, B-lymphocytes of these host animals are fused with myeloma cells, the 15 hybrid cell lines secreting the monoclonal antibodies are sub-cloned and cultivated in vitro or in vivo.

20. A receptor according to claim 1 substantially as described 2Q herein by way of Example.

21. A process according to claim 7 substantially as described herein by way of Example.