AU599895B2 - Polypeptides of the rhinovirus strain HRV2 and the DNA coding therefor - Google Patents

Description

p~ 4r~' 599895 COMMONWEALTH OF AUSTRALIA PATENT ACT 1952 COMPLETE SPECIFICATION (original) FOR OFFICE USE Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority: Related Art: This do-urn~ent cont.ims01 th' ainei-Uynmnts inaide, fli'k ISection 49 and is ccqrrect for 0 jr.44i -j C ;,ameof Applicant: C C C r, t Xtt 9 t ct C Address of Applicant: i9ttctual Inventor(s): 'Address for Service: BOEHRINGER INGELHEIM INTERNATIONAL GmbH D-6507 Ingelheim am Rhein, Federal Republic of Germany Ernst KUCHLER Wolfgang SOMMERGRUBER Peter GRUNDLER MarkusDUCHLER Timothy SKERN Dieter BLAAS Franz-Josef FRAUENDORFER DAVIES COLLISON, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

.:Coplt Specification for the invention entitled: "POILYPEPTIDES OF THE RHINOVIRUS STRAIN HRV2 AND THE DNIA CODING THEREFOR" The following statement is a full description of this invention, iluding the best method of performing it known to us -i

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This invention relates to peptides which correspond wholly or partly to those of the viral proteins of the human rhinovirus strain 2 (HRV2), the deoxyribonucleic acid molecules coding for these peptides and the preparation and use of these substances.

Rhinoviruses are RNA viruses and, according to the conventional classification of viruses, they form a genus within the family of the Picorna viruses (Cooper, Agol, Bachrach, Brown, Ghendon, Gibbs, Gillespie, J.H., Lonberg-Holm, Mandel, Melnick, Mohanty, Povey, Rueckert, Schaffer, F.L.

and Tyrrell, 1978, Intervirology, 10, 165-180; MacNaughton, 1982, Current Top. Microbiol. Immunol.

97, 1 26).

They are widespread, attack the upper respiratory S( tract in humans and cause acute infections which lead to colds, coughs, hoarseness etc. and are Sgenerally referred to as colds (Stott, E.J. and SKillington, 1972, Ann.Rev.Microbiol. 26, 503 524). Infections caused by rhinoviruses are among the most common sicknesses in man. Although 25 the course of these diseases is generally harmless, Ig g tcolds do result in a temporary weakening of the organism. This may give rise to secondary infections S caused by other viruses or bacteria which may, under certain circumstances, result in serious illness. In addition, the economic damage caused S 'by rhinoviruses is considerable. It has been calculated c that; in the USA rhinovirus infections annually cause the loss of more than 200 million working days or school days (Davis, Dulbecco, R., i 35 Eisen, and Ginsberg, 1980, Microbiology, Third Edition, Harper Row, Publ., New York, p.

1114). In addition, in recent years, there has 1 1 1 1 1 been a considerable increase in rhinovirus infections in large conurbations. Whereas most other infectious diseases confer a permanent or longlasting 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 number of strains of rhinovirus.

Hitherto, ever 100 rhinovirus strains have been isolated, showing no or very few immunological cross-reactions with each other (Fox, 1976, American J. Epidemiol. 103, 345 354; Melnick, 1980, Proc. Med. Virol. 26, 214 232).

After the infection has occurred, antibodies against the particular virus strain can be detected, but these confer no protection against other rhinovirus strains. Because of the large number of strains circulating in the population, repeated infections SttI by rhinoviruses are possible.

.t 1 20 One object of this invention was therefore to prepare H agents which give protection from infections caused by rhinoviruses.

Using hyperimmune sera it has been possible to 25 classify 50 of a total of 90 rhinovirus serotypes S. into 16 groups (Conney, Fox, J.P. and Kenny, S, 1982, Infect. Immun. 37, 642 647). Another form of classification is obtained on the basis of bonding to cellular receptors. In fact, despite l 30 the marked heterogeneity in their immunological properties, rhinoviruses behave similarly to one ;another in terms of their bonding to receptors on the surfaces of cells.

By means of competitive experiments it has been established that there are only two different receptors for the 24 rhinovirus strains investigated in HeLa ft

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4-' cells (clone R-19) (Abraham, G. and Colonno, R.J., 1984, J. Virol. 51, 340 344). Since this group consisted of randomly selected strains, it is assumed that these findings could also be extended to other strains of rhinovirus. However, it should be pointed out that these results were obtained with HeLa cells and would not necessarily also apply to the receptors on the natural host cells in the upper respiratory tract in man.

A further object of the invention is to prepare oligopeptides, which correspond wholly or partly to the viral proteins, and which may be used either to stimulate an immune response directed against intact viruses or to bind to and block cellular receptors.

rC V V V V c c C C e t *i V 4,' t C Ci t 4 4 As typical Picorna viruses, rhinoviruses contain a single-stranded RNA which ic surrounded by a 20 capsid consisting of 4 polypeptides known as VP1 (PlD), VP2 (PlB), VP3 (PIC) and VP4 (PlA) (Medappa, McLean, C. and Rueckert, 1971, Virology 44, 259-270; the terms given in brackets are of the new nomenclature corresponding to that proposed 25 by Rueckert, R.R. and Wimmer, 1984, J. Virol.

50, 957 959). A single virus particle contains copies of each of these polypeptides. The relative molecular masses in different rhinoviruses are 34 36000 for VP1, 27 30000 for VP2, 24 28000 for VP3 and 7 8000 for VP4 (MacNaughton, M.R., 1982, loc.cit.). A further characteristic of rhinoviruses is that they are rapidly inactivated at pH values below 5 and are sensitive to high concentrations of salt solution. Moreover, the majority of rhinoviruses show optimum growth at 33 34 0 C (Luria, S.E., Darnell, Jr., Baltimore, D and Campbell, 1978, General Virology, Third Edition, John 4 1 *4 SI t<c

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Wiley Sons, New York, 308 ff.).

The single-stranded RNA with a length of about 7100 nucleotides constitutes the genome of the virus and may simultaneously serve as the matrix for the synthesis of the viral proteins. A large proportion of the nucleotide sequence is first translated into a long polyprotein from which the finished viral proteins are formed by proteolytic cleavage (Butterworth, 1973, Virology 56, 439 453; McLean, C. and Rueckert, 1973, J.Virol. 11, 341 344; McLean, Matthews, T.J.

and Rueckert, 1976, J. Virol. 19, 903 914).

The products of this processing are, in addition to the viral coat proteins VPl, VP2, VP3 and VP4, a number of proteins such as P2C, P3B, P3C and o P3D. P3B (generally known as VPg) is covalently Sbonded to the 5'-terminal nucleotide of the HRV2-RNA.

i Analogously to the polio virus it can be assumed 20 that P3C is a protease which is partly responsible 'c .for the processing of the viral polyprotein (MacNaughton, SM.R., 1982, loc.cit.). Accordingly, P3D is the polymerase for the replication of viral RNA. At present, little is known of the function of the 25 protein P2C.

During the processing of the viral polyprotein, parts of the amino acid sequence are split off and then decomposed (McLean, Matthews, T.J.

J 30 and Reuckert, 1976, loc.cit.).

Regarding the sequence of the rhinovirus strain HRV2, hitherto only the sequence of the 3'-untranslated region, the RNA polymerase (P3D) and the VPg (P3B) l: 35 is known from publications of our working group 1 (Skern, Sommergruber, Blaas, Pieler, Si .I r ini! ii 1- fi wwfisgS C -'6 Ch. and Kuechler, 1984, Virology 136, 125 132; Skern, Sommergruber, Blaas, Pieler, Ch. and Kuechler, 1984, Sixth Int. Congress Virology, Sendai, Abstract, P8-20). A comparison of the nucleotide sequence with that of poliovirus (Type 1) and fooandfo nd-mouth disease virus (subtype A12) shows no significant homologies in the 3'untranslated region. It is striking that the 3'untranslated region in HRV2, with 42 nucleotides, is very much shorter than in other Picorna viruses.

In the region of the RNA polymerase, on the other hand, there is a striking homology in the amino acid sequence between HRV2 and poliovirus (56%) whereas the homology with RNA polymerase of footand-mouth virus is only 27% (Skern, Sommergruber Blaas, Pieler, Ch. and Kuechler, 1984, Virology, loc. cit.). These data indicate a strong similarity between rhinoviruses and enteroviruses.

t Thus, the tyrosine, by means of which bonding to 20 the 5'-terminal nucleotide of the RNA occurs in the poliovirus, is also preserved in the VPg (P3B) ij of HRV2 (Skern, Sommergruber, Blaas, D., Pieler, Ch. and Kuechler, 1984, 6th Int. Congress Virology, Sendai, loc.cit.). The affinity with 25 the enteroviruses is also made clear by a sequence comparison between HRV2, the human rhinovirus strain HRV14 (Stanway, Hughes, Mountford, R.C., Minor, P.D. and Almond, 1984, Nucleic Acids S0 Res. 12, 7859-7877) and polioviruses.

According to one feature of the present invention, there is provided DNA coding for at least one viral protein of rhinovirus strain HRV2.

According to another feature of the invention, 4pt the new DNA as hereinbefore described may be obtained -7 4 1 1 1 1 1 1 As

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C Cf C C ft 7 by isolating the viral RNA of rhinovirus strain HRV2, preparing DNA complementary to the viral RNA, incorporating the viral cDNA/RNA hybrid into a suitable replicable vector transforming said vector into a suitable host organism, culturing the host organism and isolating the desired DNA.

A further feature of the present invention provides a polypeptide having the biological activity of at least one of the viral proteins of the rhinovirus strain HRV2, and which has been coded by DNA as defined above, which corresponds wholly or in part to at least one of the viral proteins of the rhinovirus strain HRV2 or which corresponds to at least two of said viral proteins or portions thereof connected to each other in any desired combination and sequence.

According to a still further feature of the invention, the new polypeptides as hereinbefore described may be obtained by isolating said polypeptide from 20 a host organism transformed with DNA coding for at least one viral protein of rhinovirus strain HRV2.

In view of the biological activity of the novel 25 polypeptides, the present invention also provides pharmaceutical compositions containing, as active ingredient, at least one polypeptide as hereinbefore defined, in association with one or more inert pharmaceutical carriers, diluents or adjuvants.

Moreover, the novel polypeptides of this invention may be used for the therapy or prophylaxis of viral infections or for stimulating the immune system of mammals by administering an effective amount 35 of the polypeptide.

8 I I e I I a *t 4 C* We now describe a preferred procedure for putting our invention into effect.

In order to obtain the viral starting material, HeLa cells were infected with HRV2 in a suitable infection medium and incubated for several hours under optimum growth conditions.

The virus could be obtained both from the cells and from the medium.

Various purification steps yielded a virus preparation, the protein pattern of which is typically as shown in Fig. 1.

The viral RNA was obtained from the virus preparation by, for example, phenol extraction, and purified; it was then transcribed with the aid of reverse transcriptase and oligo dT as primer cDNA. The resulting RNA/cDNA hybrids were extended homopolymerically and incorporated in a suitable plasmid, for example pBR322.

The use of methods for the reverse transcription of RNA, for the integration of RNA-cDNA hybrids into plasmids and for the transformation of bacteria have been sufficiently described in the literature (Kitamura, N. and Wimmer, 1980, Proc. Natl.

Acad. Sci. USA 77, 3196 3200; Nelson, T. and 30 Brutlag, 1979, Methods in Enzymology 68, 41 Zain, Sambrook, Roberts, Keller, Fried, M. and Dunn, 1979, Cell 16, -51 861; Roychoudhury, R. and Wu, 1980, Methods in Enzymology 42 62; Kitamura, Semler, Rothberg, Larsen, Adler, Dorner, A.J., Emini, Hanecak, Lee, van der Werf, Anderson, C.W. and Wimmer, 1981, Nature .4 9 (London) 291, 547 553; van der Werf, Bregegere, Kopecka, Kitamura, Rothberg, P.G. Kourilsky, Wimmer, E. and Girard, 1981, Proc. Natl.

Acad. Sci. USA 78, 5983 5987; Namoto, Omata, Toyoda, Kuge, Hosie, Kataoka, Y., Genba, Nakano, Y. and Imura, 1982, Proc.

Natl. Acad. Sci. USA 79, 5793 5797; Stanway, Cann, Hauptmann, Hughes, Clarke: Mountford, Minor, Schild, G.C.

and Almond, 1983, Nucleic Acids Res. 11, 5629 5643).

After the transfor-ation of a suitable host organism, for example E. coli HB 101, the plasmid DNA was isolated using the "plasmid mini-preparation technique" and the size of the recombinant DNA was determined.

To do this, the recombinant plasmids were cut using the restriction endonuclease PstI, the fragments were separated electrophoretically and compared 20 with known lambda-HindIII marker DNA.

t I t In order to sub-clone the DNA, the DNA fragments obtained by PstI digestion of the recombinant pBR322 clones were purified, incorporated into a suitable 25 vector, for example the plasmid pUC9, and then the recombinant vectors were transformed into a suitable host, for example E. coli JM101. The S" subclones which had been successfully transformed with recombinant vectors were cultivated and their 30 plasmid DNA was isolated and purified.

*i Two primer fragments of different lengths [59 nucleotides (fragment A) and 68 nucleotides (fragment were isolated from two subclones and purified. With 35 the aid of this complementary single stranded DNA a 32 P labelled reverse transcript of HRV2-RNA was prepared.

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In order to detect HRV2 sequences in recombinant DNA, the plasmid DNA was digested with PstI, analysed by electrophoresis and the DNA fragments transferred from the gel onto nitrocellulose filters and fixed.

These DNA-carrying filters were hybridised with the radio.:tively labelled HRV2-cDNA and the filters were then exposed. From the recombinant DNA fragments complementary to the HRV2-RNA obtained in this way, the restriction sites were mapped and sequenced.

Clones were obtained which represent the genome of HRV2.

The sequence of the HRV2 genome is shown in Fig. 4.

It is shown in the form of the DNA as obtained from the sequencing of the recombinant DNA. In order to determine the sequence, 17 clones (shown in Fig. 3) and an additional 25 clones were used. The sequence comprises 7102 nucleotides, not including the 3'-terminal poly-A. It contains an open reading frame which codes for a polyprotein with a length of 2150 amino acids.

20 The reading frame begins with an AUG in position 611 and ends with a UAA stop codon 42 nucleotides before the start of the poly-A.

The 5'-terminal sequence was obtained from the 25 clones nos. 61, 100 and 109, fragment A being used as primer (see Fig. Cleaving with PstI yielded a fragment of constant length. By sequencing it was. established that all three clones contained the sequence TTAAAAC immediately adjacent to oligo-G, which originates from the integration site in the plasmid. From this it was concluded that these clones constitute the 5'-terminus of the HRV2 genome.

This sequence corresponds to the first 7 nucleotides of the three types of polio virus, Coxsackie-virus 35 B1 (Hewlett, M.J. and Florkiewicz,...R.Z., 1980, Proc. Natl. Acad.. Sci, USA 77, 303 307; Stanway, Cann, Hauptmann, Hughes Clarke, 11 11, £11 *4,1 4, t 4 4.

Mountford, Minor, Schild, G.C.

and Almond, 1983, loc. cit.) and HRV-14 (Stanway, Hughes, Mountford, Minor, P.D. and Almond, 1984 loc. cit.). Analysis of the 610 nucleotides between the 5'-terminal and the start of the long open reading frame shows the presence of a number of short reading frames. A comparison of the nucleotide sequence of the 5'-terminal region of HRV2 with that of HRV14 and poliovirus type 1 shows a high degree of homology. Bearing in mind any insertions, homologies of 65% between HRV2 and HRV14 and 55% between HRV2 and poliovirus type 1 were established in this region.

The homology is particularly great in some regions.

In all, 5 different blocks of 16 or more identical nucleotides located one after another are present in the 5'-terminal region of the HRV2 and HRV14 genome.

A comparison of HRV2 and poliovirus type 1 also shows blocks of identical sequence, of which only 2 were found in HRV14. These are a sequence of 16 nucleotides 20 beginning with base 436 and a sequence of 23 nucleotides beginning with base 531 in HRV2.

A comparison of the amino acid sequences showed that the regions of homology are also continued in the region coding for the polyprotein (Fig.

It is particularly noticeable that the homology between HRV2 and HRV14 is in many instances no greater or only a little greater than that between HRV2 and poliovirus type 1. It was surprising to discover that the homology between HRV2 and poliovirus in the region of the VP4 and, to a small extent also in the polymerase, is even greater than that between HRV2 and HRV14. It is all the more remarkable since, according to the classical taxonomy, the rhinoviruses are regarded as a separate genus within the family of the Picorna viruses.

Homologies were also found between HRV14 and poliovirus, :1 ~1

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4 4 4 C Ct t ~4 t '4 IE 4 4 I. SI I S 4 4* 444 4' 45 4 4 444 4 4~ 4C 14 4 t 44 I 5* '12 with the result that it has recently been proposed that rhinoviruses and enteroviruses be grouped together as a genus within the family of the Picorna viruses (Stanway, Hughes, Mountford, Minor, P.D. and Almond, 1984, loc.cit.).

However, in addition there are some genes such as VPI, VP2, VPg and the proteases in which the similarity between HRV2 and HRV14 is considerably greater than that between HRV2 and poliovirus.

It is also remarkable that the smallest homology is to be found in the region of the VP1.

Further support for homologous regions in parts of the sequence obtained as described in the examples was obtained by comparison with a gene fragment obtained from cloned HRV89. HRV89 was grown as described for HRV2. HRV89 was neutralized by a specific antiserum ((ATCC VR-1199 AS/GP) whereas antiserum against HRV2 (ATCC VR-1112 AS/GP) used 20 as control .serum had no effect. The isolation of the viral RNA, characterization, cloning, isolation of clones and sequencing was performed as described for HRV2. Fig. 8 shows the sequence comparison with a partial sequence obtained from the clone 25 34/1 of HRV89, which obviously represents a region corresponding to the genes for P3A and P3B (VPg).

An extensive homology in the amino acid sequence is apparent. Replacements are frequently observed between chemically closely related amino acids such as, for example, arginine versus lysine valine versus isoleucine leucine versus isoleucine etc. As seen from the homology the presumptive cleavage site between P3A and P3B (VPg) is completely conserved. There is, however, a small region (corresponding to the region between nucleotides 5018 and 5029 in HRV2) where the amino acid sequence differs. The significance 7] 7 ~t r. t4 i 0 s4 t i I .t I II 4 t t I 14 4 t .44 .1t I. I 13 of this observation is not yet clear. Nothing is known about the function of P3A. It can not therefore be excluded that other subtypes of HRV2 show a higher degree of homology to HRV89 in this region. For that reason other subtypes of HRV2 and/or rhinoviruses, whose nucleic acids hybridize with HRV2 cDNA as defined by the hybridization conditions given above, and which show a higher degree of homology to HRV89 in this region, are also the subject of this invention.

The viral proteins are obtained from the polyprotein by proteolytic cleavage. In order to determine the cleavage sites, the viral coat proteins were isolated and the N-terminal amino acid sequence was determined (see Fig. In this way, not only were the cleavage sites in the viral coat proteins clearly identified but also the reading frame which had been derived from the. nucleotide sequence was confirmed. It is apparent from a comparison with the amino acid sequence derived from the nucleotide sequence that the VP4/VP2 cleavage takes place between glutamine and serine, the VP2/VP3 cleavage occurs between glutamine and glycine and the VP3/VPl cleavage occurs between glutamine and asparagine.

The present invention makes it possible to produce a DNA which contains the information for the viral RNA of HRV2.

However, the invention relates not only to the 30 genetic sequences coding specifically for the viral proteins but also to the modifications which may be obtained, for example, by mutation, degradation, transposition or addition. Each sequence which is degenerate by comparison with those shown is included. The sequences which hybridise under stringent conditions with the sequences shown or parts thereof, for la<ample under conditions which I~I I

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*4'4LI I a I 4 14 select for more than 85%, preferably for more than homology, and which code for the proteins with the viral activity spectrum, are also included.

The hybridizations are carried out in 6 x SSC/5 x Denhardt's solution/0.1% SDS at 65°C. The degree of stringency is determined in the washing step.

Thus, for selection of DNA sequences with approximately or more homology, suitable conditions are 0.2 x SSC/0.01% SDS/65°C and for selection of DNA sequences *with approximately 90% homology or more, the suitable conditions are 0.1 x SSC/0.01% As shown, this DNA may be incorporated into suitable re-licable i f vehicles such as plasmid vectors both in its entirety and.also in fragments in order either to multiply the DNA or to achieve expression of the proteins themselves ,after the transformation of suitable host organisms.

Suitable hosts, vectors and the conditions for S' these operations are already well known to those skilled in the art. Similarly, a number of studies have been published describing the synthesis of foreign proteins in bacteria by means of genetic engineering (for a survey see Harris, T.J.R. in t "Genetic Engineering", Williamson, Editor, 1983, Vol. 4, Academic Press, London, 127 ff). For this purpose, the foreign DNA is introduced in the vicinity of suitable bacterial control regions (promoters, ribosome binding sites) of plasmids which make it possible to express this information mostly in the form of fusion proteins in high yields and thus obtain the corresponding proteins.

In the field of Picorna viruses there are also already a number of publications describing the expression of viral genes in bacteria (Kdpper, Keller, Kurz, Forss, Schaller, vH., Franzel, Strohmaier, Marquardt, 0., Zaslavsky, V.G. and Hofschneider, 1981, Nature

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0 A: -r '5 (London) 289, 555 559; Kleid, Yansura, Small, Darbenko, Moore, Grubman, McKercher, Morgan, Robertson, B.H. and Bachrach, 1981, Science 214, 1125 1129; Wychowski, van der Werf, Siffert, Crainic, Bruneau, P. and Girard, 1983, EMBO J. 11, 2019 2024; Klump, Marquardt, 0. and Hofschneider, 1984, Proc.Nat.Acad. Sci. USA 81, 3351 3355; Hanecak, Semler, Ariga, Anderson, C.W. and Wimmer, 1984, Cell 37, 1063 1073).

Prokaryotes are particularly preferred for expression, for example E. coli K 12, strain 294 (ATCC No. 31 446) or E. coli Xl 1776 (ATCC No. 31537). Apart from the above mentioned strains it is also possible to use E. coli W 3110 F lambda prototroph, ATCC No. 27325), bacilli such as Bacillus subtilis tX and other enterobacteriaceae, such as Salmonella typhimurium or Serratia marcescens and various 20 pseudomonads.

S In general, plasmid vectors which contain replicon and control sequences originating from species which are compatible with the host cells may be 1 25 used in conjunction with these hosts. The vector usually carries, in addition to a replication site, recognition sequences which make it possible to I I phenotypically select the transformed cells. For example, E. coli is usually transformed with pBR322, a plasmid which originates from the species E. coli S (Bolivar, et al., Gene 2, 95 (1977). pBR322 contains genes coding for ampicillin and tetracycline resistance and thus affords a simple means of identifying Stransformed cells. The pBR322 plasmid or other plasmid must, in addition, contain promoters themselves or-must be. modified so that they contain promoters S -which can be used by the microbial organism for i I~~ '16 the expression of its own proteins. The promoters most frequently used in the preparation of recombinant DNA include the beta-lactamase (penicillinase) and lactose promoter systems (Chang et al., Nature 275, 615 (1978); Itakura et al., Science 198, 1056 (1977); Goeddel et al., Nature 281, 544 (1979)) and tryptophan(trp) promoter systems (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980); EP-A-0,036,776).

Whereas these are the most commonly used promoters, other microbial promoters have also been developed.

The genetic sequence according to the invention may be used, for example, under the control of the leftward promoter of the bacteriophage lambda This promoter is one of the promoters known to be particularly powerful and is also controllable.

Control is made possible by the lambda repressor of which adjacent restriction cutting sites are ,'nt known.

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I 20 A temperature-sensitive allele of this repressor gene may be inserted into a vector which contains a complete viral DNA-sequence. If the temperature Sis increased to 42 0 C, the repressor is inactivated and the promoter is expressed up to its maximum 25 concentration. The total of the mRNA produced under these conditions should be sufficient to obtain a cell which contains, among its new synthetic V ribonucleic acids, approximately 10% originating from the PL promoter. In this way it is possible to establish a clone bank in which a functional viral. DNA sequence is placed in the neighbourhood I of a ribosome binding site at varying distances Sfrom the lambda PL promoter. These clones can then be checked and those with the highest yield 1 35 selected.

The expression and translation of a viral DNA sequence M 1 1 1 1 l l y- 1 r r s l l 1 1 1 1 1 I i i 1 i i a. a a.

a aa a a.i at -'17 may also be effected under the control of other regulating systems which may be regarded as "homologous" to the organism in its untransformed form. Thus, for example, chromosomal DNA from a lactose-dependant E. coli contains a lactose or lac-operon which allows the degradation of lactose by secreting the enzyme beta-galactosidase.

The lac-control elements may be obtained from the bacteriophage lambda-plac5, which is infectious for E. coli. The lac-operon of the phage may be obtained from che same bacterial species by transduction.

Regulating systems which may be used in the process according to the invention may originate from plasmid DNA which is native to the organism. The lac-promoteroperator system may be induced by IPTG.

Other promoter-operator systems or parts thereof may be used with equally good effect: for example, arabinose operator, colicine E 1 -operator, galactose operator, alkaline phosphatase operator, trp operator, xylose-A operator, tac-promoter, etc.

The genes may preferably be expressed in the expression plasmid pER0O3 Rastl-Dworkin et al., Gene 21, 237-248 (1983); also European Patent Application No 83112812.9, deposit DSM 2773, 20th December 1983). These vectors all contain regulating elements which lead to a high expression rate for the cloned 30 genes.

In addition to prokaryotes, eukaryotic microorganisms such as yeast cultures may also be used. Saccharomyces cerevisiae is the most commonly used of the eukaryotic 35 microorganisms, although a number of other species are generally obtainable. For expression in Saccharomyces, for example the plasmid YTp7 (Stinchcomb ac a 'a.

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i B a 1 ra asta a a i li i h.* I i I 1 I I 18 et al., Nature 282, 39 (1979); Kingsman et al., Gene 7, 141 (1979); Tschumper et al., Gene 157 (1980)) and the plasmid YEpl3 (Bwach et al., Gene 8, 121-133 (1979)) are conventionally used.

The plasmid YRp7 contains the TRP1 gene which is a selectable marker in a yeast mutant which is incapable of growing in tryptophan-free medium; for example ATCC No. 44076.

The presence of the TRP1 defect as a characteristic of the yeast host genome constitutes an effective aid to detecting transformation, in which cultivation is carried out without tryptophan. The situation is very similar with the plasmid YEpl3, which contains the yeast gene LEU 2, which can be used to complement a LEU-2-minus mutant. Suitable promoter sequences for yeast vectors contain the 5'-flanking region of the genes of ADH I (Ammerer Methods of Enzymology S101, 192-201 (1983)), 3-phosphoglycerate-kinase' S: 20 (Hitzeman et al., J. Biol. Chem. 255, 2073 (1980), c or other glycolytic enzymes (Kawasaki and Fraenkel, Biochem. Biophys. Res. Comm. 108, 1107-1112 (1982)) such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, and glucokinase.

t By constructing suitable expression plasmids, the i i c termination sequences associated with these genes may also be inserted into the expression vector A at the 3'-end of the sequence which is to be expressed, in order to ensure polyadenylation and termination of the mRNA.

j Other promoters which also have the advantage of transcription controlled by growth conditions are the promoter regions of the genes for alcohol dehydroj /genase-2, isocytochrome C, acid phosphatase, degradation enzymes which are coupled to nitrogen metabolism, 1 1 '19 the above-mentioned glyceraldehyde-3-phosphate dehydrogenase and enzymes which are responsible for the processing of maltose and galactose. Promoters which are regulated by the yeast mating type locus, for example promoters of the genes BAR1, MFal, STE2, STE3 and STES, may be used in temperatureregulated systems by the use of temperature dependant sir mutations. (Rhine, Ph.D. Thesis, University of Oregon, Eugene, Oregon (1979), Herskowitz and Oshima, The Molecular Biology of the Yeast Saccharomyces, Part I, 181-209 (1981), Cold Spring Harbour Laboratory).

These mutations affect the expression of the resting mating type cassettes of yeasts and thus indirectly the mating type dependant promoters. Generally, however, any plasmid vector which contains a yeastcompatible promoter, origin of replication and termination sequences, is suitable.

In addition to microorganisms, cultures of multicellular 20 organisms are also suitable host organisms. In theory, any of these cultures may be used, whether obtained from vertebrate or invertebrate animal cultures. However, the greatest interest has been in vertebrate cells, with the result that the multiplication of vertebrate cells in culture (tissue culture) has become a routine method in recent years (Tissue Culture, Academic Press, Editors Kruse and Patterson, (1973)). Examples of useful host cell lines of this kind include VERO and HeLa cells, hamster ovary (CHO) cells and W138, BHK, COS-7 and MDCK cell lines. Expression vectors for these cells generally contain (when necessary) a replication site, a promoter which is located in front of the gene to be expressed, together with any necessary ribosome binding site, RNA splicing site, polyadenylation site and transcriptional termination sequences.

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When used in mammalian cells, the control functions in the expression vector are often obtained from viral material. For example, the promoters normally used originate from polyoma, adenovirus 2 and particularly frequently from simian virus 40 (SV 40). The early and late promoters of SV 40 are particularly useful since both can be easily obtained from the virus as a fragment which also contains the viral replication site of the SV 40. (Fiers et al., Nature 273, 113 (1978)) It is also possible to use smaller or larger fragments of SV 40, provided that they contain the sequence, approximately 250 bp long, which extends from the HindIII cutting site to the Bgll cutting site in the viral replication site. Furthermore it is also possible and frequently desirable to use promoter or control sequences which are normally linked to the desired genetic sequences, provided that these control sequences are compatible with the host cell systems.

A replication site may either be provided by corresponding vector construction in order to incorporate an exogenic site, for example from SV 40 or other viral sources polyoma, adeno, VSV, etc.) or it may be provided by the chromosomal replication mechanisms of the host cell. if the vector is integrated into the host cell chromosome, the latter measure is usually sufficient.

Transformation of the cells with the vectors can J ~be achieved by a number of methods. For example, *it can be effected using calcium, either by washing the cells in magnesium and adding the DNA to the cells suspended in calcium or by subjecting the cells to a coprecipitate of DNA and calcium phosphate.

Following gene expression, the cells are transferred to media which selects for transformed cells.

"1K

TV

1 1 '21 After transformation of the host, expression of the gene and fermentation or cell cultivation have been carried out under conditions in which the protein according to the invention is expressed, the product may usually be extracted by known chromatographic methods of separation in order to obtain a material which contains the viral protein with or without leader and tailing sequences. The protein according to the invention may be expressed with a leader sequence at the N-terminus (pre-protein) which can be removed by some host cells. If not, the leader polypeptide (if present) must be split off in order to obtain mature protein. Alternatively, the clone may be modified so that the mature protein is produced directly in the microorganism instead of the pre-protein. In this instance, the precursor sequence of the yeast mating pheromone MF-alphao 1. can be used in order to ensure correct "maturation" of the fused protein and precipitation of the products t! 20 into the growth medium or the periplasmic space.

SThe DNA sequence for functional or mature protein can be connected with MF-alpha-1 at the presumed cutting site.

I 25 Apart from using the DNA according to the invention for preparing the relevant proteins in bacteria or eukaryotic cells, it may also be used to prepare E o i the amino acid sequences derived from the nucleotide sequence, synthetically, in their entirety or in parts thereof.

These oligopeptides may then, like the proteins produced by genetic engineering, be used either to stimulate an immune response directed against intact viruses or for binding to and blocking cellular receptors. Studies of the use of oligopeptides for stimulating an immune response directed against i 22 polio viruses have been published (Emini, E.A., Jameson, B.A. and Wimmer, 1983, Nature (London) 699-703; similar investigations have also been carried out in the case of foot and mouth viruses (Bittle, Houghton, Alexander, Shinnick, Sutcliffe, Lerner, Rowlands, D.J. and Brown, 1982, Nature (London), 298, 33; Pfaff, Mussgay, Bohm, Schulz, G.E. and Schaller, 1982, EMBO J. 1, 869 874).

This invention also includes the oligo and polypeptide components of HRV2 proteins which are connected to other oligo or polypeptides as a result of the synthesis or for the purposes of the intended application, e.g. as vaccines.

The term "biological activity" as used herein in conjunction with proteins (polypeptides) indicates Sthat the protein (polypeptide) in question stimulates an immune response in biological tests and/or enters into some reaction with the cellular receptors j for the rhinoviruses.

SFurther features and aspects of our invention will be apparent from the Examples which follow, which are given for illustration only. In the Examples .e reference is made to the accompanying drawings.

The matter depicted in the various Figures is as follows: ^tt, I 1 1 1 1 p. 1 1 1 1 1 m I 1 n 1 I 1 :i i prepared.

U

I

3 Tf" .4 23 Fig. 1 Electrophoretic separation of the coat proteins of HRV2 on a 12.5% polyacrylamide gel in the presence of sodium dodecylsulphate.

VP4 is not visible since it has migrated out of the gel with the front.

Fig. 2 Electrophoresis of HRV2-RNA on a 2% agarose gel in the presence of sodium dodecylsulphate.

The positions of the ribosomal RNA markers are shown on the right.

Fig. 3 Restriction map of the HRV2 genome. 17 overlapping clones which were used for sequencing are shown. Characteristic cutting sites of some restriction enzymes are indicated. The arrows represent restriction fragments which were used as primers for reverse transcription.

Fig. 4 Sequence of the cloned HRV2 genome and the amino acid sequence derived therefrom.

Cleavage sites in the polyprotein are shown by arrows. Solid arrows show cleavage sites determined experimentally, open arrows indicate cleavage sites in the polyprotein predicted on the basis of homology with other Picorna viruses.

Fig. 5 Comparison of the homology in the amino acid sequence (in of individual genes between HRV2, HRV14 and polio virus type 1.

C

CC

C C Ct C C C

C~

C Ci CC C r CC L C C C C LcCC

LC

C.C

C

Fig. 6 N-terminal amino acid sequences of the coat proteins of HRV2.

t-W-'i mm .24 Fig. 7 Identification of the viral capsid protein VP1 as the strongest antigen of HRV2 in rabbits.

Fig. 8 Comparison of the amino acid and nucleotide sequences in the region of the genes for P3A and P3B (VPg) of HRV2 and HRV89. The open arrow indicates the predicted cleavage sites as deduced by comparison with other picornaviruses.

Fig. 9 Complete nucleotide sequence of HRV2.

Fig. 10 Amino acid sequence of the polypeptide HRV2.

t t t t S I: t hr

II

r If *1 g 4 C t t4 t Fig. 11 A:

B:

C:

Fig. 12 A:

B:

C:

nucleotide sequence of the polypeptide VP1 amino acid sequence of the polypeptide VP1 deduced nucleotide sequence of the polypeptide VP1.

nucleotide sequence of the polypeptide VP2 amino acid sequence of the polypeptide VP2 deduced nucleotide sequence of the polypeptide VP2.

nucleotide sequence of the polypeptide VP3 amino acid sequence of the polypeptide VP3 deduced nucleotide sequence of the polypeptide VP3.

nucleotide sequence of the polypeptide VP4 amino acid sequence of the polypeptide VP4 deduced nucleotide sequence of the polypeptide VP4. Fig. 13 A:

B:

C:

Fig, 14 A:

B:

C:

'I

1 25 Example 1 Preparation of HRV2 HeLa cells (strain HeLa-Ohio, 03-147, Flow Laboratories, England) were cultivated in suspension at 37?C.

The suspension medium (Thomas, Conant, R.M.

and Hamparian, 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 5 10 x 104 cells/ml and the volume was 500 ml. At a cell density of 1 x 106 cells/ml the suspension was centrifuged for minutes at 300 g under sterile conditions. The supernatant was suction filtered and the cells were resuspended in 100 ml of infection medium S1 (0 Joklik modification of MEM for suspension culture Swith 2% ahorse serum and 2 mM MgCl 2 By carefully 20 sucking up in a 20 ml pipette several times, the ,2$L cells were homogeneously distributed in the infection Shmedium. This was then made up to 500 ml. The t cell suspension was brought to 34 0 C and infected with HRV2 (twice plaque-purified) with a multiplicity 25 of 0.1 viruses per cell. The HRV2 strain was obtained St, from Dr. D. Tyrrell (Common Cold Centre, Salisbury, England) and may also be obtained from the American s1 Type Culture Collection (ATCC VR-4 82 and ATCC VR-1112).. The strain used was neutralised by antiserum against HRV2 (American Type Culture Collection, Cat. No. ATCC VR-1112 482AS/GP). The control serum used was an antiserum against HRV7 (Cat.No. ATCC 4 V VR-1117 AS/GP), which showed no neutralisation.

t After 40 hours at 34*C the virus was harvested.

SVirus was obtained both from the cells and cell S! fragments and also from the medium. For this purpose, x ifC Enln)admyas eotie rmteAeia J 26 the medium was separated from infected cells and cell fragments by centrifuging for 10 minutes at I 1500 g and then suction filtered. The precipitate was frozen at The cell precipitates from 12 litres of suspension culture were combined, resuspended in 40 ml-of TM buffer (20 mM Tris/HC1, pH 7.5, 2 mM MgCl 2 put on ice for 15 minutes, then broken up in a Dounce homogeniser and the mixture centrifuged for 30 minutes at 6000 g. The precipitate was then washed once again in 10 ml of TM buffer.

The two supernatants were combined and centrifuged for 3 hours at 5000 g in order to pellet the virus.

The virus pellet was then taken up in 10 ml of KTMP buffer (50 mM KC1, 50 mM Tris/HCl, pH mM MgCI 2 2 mM mercaptoethanol, 1 mM puromycin, mM GTP) and after the addition of 150 mcg of 1 DNase I (Sigma, ribonuclease free) incubated on 20 ice for 1 hour.

t- 4 The virus was precipitated out of the infection medium by stirring at 4°C with polyethylene glycol 6000 (PEG 6000; Merck) at a concentration of 7% S 25 and 450 mM NaCI (Korant, Lonberg-Holm, K., Noble, J. and Stasny, 1972, Virology 48, 71 86). After 4 hours in the cold the virus was centrifuged for 30 minutes at 1500 g, the precipitate .was resuspended in 10 ml of KTMP buffer containing l 75 mcg of DNase I, the mixture was incubated on sj^ ,f ,ice for 1 hour and then frozen at -70 0

C.

jI The virus suspensions obtained from the cells and ^l from the medium were combined, incubated at 37°C 35 for 5 minutes, cooled by the addition of 60 ml r of cold TE buffer (10 mM Tris/HCl, pH 7.4, 1 mM I EDTA) and then sonicated for 5 minutes in an ice bath.

1 1 1 1 1 1 1 1 1 1 -'27 It was then centrifuged for 30 minutes at 6000 g.

920 ml of TE buffer containing 7% of PEG 6000 and 450 mM of NaC1 were added to the supernatant which was carefully stirred for 4 hours at 4 0 C and the resulting precipitate was pelleted for 30 minutes at 6000 g. The precipitate was again taken up in 100 ml of TM buffer, the virus was precipitated as above by the addition of PEG 6000 and NaCl and then 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 85000 g. The precipitate was dissolved in 1 ml of TM buffer, incubated for 1 hour at 4 0

C

after the addition of 50 mcg of DNase I and then 1 ml of TE buffer was added. For further purification, the virus suspension was centrifuged on a sucrose gradient (10 30% w/w in TE buffer) for 4 hours Scat 4 0 C and at 85000 g. The fractions containing the virus were determined from the extinction at 260 nm and diluted with TM buffer so that the final concentration of sucrose was 10%. Then they were centrifuged for 8 hours at 85000 g. The virus pellet was taken up in 1 ml of TM buffer and stored at -70°C. To check the purity of the virus preparation, electrophoresis was carried out on a 12.5% polyacrylamide Sgel in the presence of 0.1% sodium dodecylsulphate (Laemmli, 1970, Nature (London) 277, 680-685) and the protein bands were stained with Coomassie 1 Brilliant Blue. A typical image of the protein pattern of a preparation of HRV2 is shown in Fig. 1.

S' Example 2 S f Cloning of cDNA-RNA hybrids RNA extraction from the HRV2 preparation i' The virus was suspended in 1 ml of NTES buffer s i: :li r l' I i s~a~-a~ .(vrC

CC

SC

er cc 4 44 I 'sI: c 4: a 0646 r s -'28 (100 mM NaC1, 10 mM Tris/HCl, pH 9, 1 mM EDTA, 0.1% sodium dodecylsulphate), and extracted with phenol. In order to improve the phase separation, chloroform was added. 20 mcl of 5 M NaCl and 20 mcl of 3 M sodium acetate (pH 5.6) were added to the aqueous phase and the viral RNA was precipitated with twice the volume of ethanol.

In order to remove the VPg covalently bonded to the 5'-end of the viral RNA, the RNA was subsequently digested in NTES buffer with 1 mg/ml of proteinase K (Merck) for 15 minutes at 37 0 C. In order to remove contamination by ribonuclease, the proteinase K strain solution (50 mg/ml) had been previously incubated for 15 minutes at 37 0 C. After the proteinase K digestion had ended, the solution was extracted with phenol/chloroform as described above and the RNA was precipitated by the addition of ethanol.

A small proportion of the RNA was then electrophoretically 20 separated on a 2% agarose gel in 7 -E buffer (10 mM NaAc, 40 mM Tris/acetate, pH 8.2, 2 mM EDTA) with 0.1% sodium dodecylsulphate. After staining with ethidium bromide, the band of intact HRV2-RNA was visible (Fig. A weak diffuse band below it showed a slight degradation of the HRV2-.NA.

Reverse transcription of the HRV2-RNA with oligodT as primer 30 4 mcg of HRV2-RNA were dissolved in 10 mcl of H 2 0, mcl of 10 x RT-buffer (1 x RT buffer 100 mM KC1, 10 mM MgCl 2 50 mM Tris/HCl, pH 5 mcg oligo-dT (12 18) (Pharmacia P-L Biochemicals), 10 mcCi 32 P]-dCTP (3000 Ci/mmol Amersham International, England), 100 U of reverse transcriptase (Anglian Biotechnology Co., Cambridge) and 20 nmol of dATP, dGTP, dTTP, dCTP were added and the whole was incubated i~: 1: i; ;i i Ii: ;I I C /7 ,29 for 2 hours at 42 0 C in a total volume of 50 mcl.

After the addition of 2 mcl of 250 mM EDTA (pH 8) extraction was carried out with phenol/chloroform and the aqueous phase was applied to a Biogel (or Sephadex G-25) column in a Pasteur pipette.

TE buffer was used for elution. The cDNA-RNA hybrid formed was separated from excess [a 32P]dCTP in this way and precipitated after the addition of 1/10 parts by volume of 3 M sodium acetate (pH 5.6) and 2 parts by volume of ethanol.

Homopolymeric extending of the HRV2 RNA-cDNA hybrids ("Tailing") The extending of the HRV2 RNA-cDNA hybrids was carried out using the method of Roychoudhury and Wu (1980, loc.cit.). The HRV2 RNA-cDNA hybrid Swas incubated for 5 minutes at 37 0 C in 50 mcl of 4 TT buffer (200 mM potassium cacodylate, 25 mM Tris/HCl, pH 6.9, 0.5 mM CoCl2, 2 mM dithiothreitol), in the presence of 2 nmol [a P]dCTP (5 Ci/mmol) with S 25 U of terminal transferase (Pharmacia P-L Biochemicals) I After the addition of 2 mcl of 0.25 M EDTA (pH 8) extraction was carried out with phenol/chloroform, the reaction mixture was then chromatographed on a Biogel P30 column as described above and the oligo-dC-carrying RNA-cDNA hybrid was precipitated with ethanol.

S 30 Incorporation of oligo-dC-carrying HRV2 RNA-cDNA hybrid into the plasmid pBR322 ("annealing") and S* transformation of Escherichia coli HB 101 The oligo-dC-carrying RNA-cDNA hybrid was added to 100 mcl of NTE buffer (100 mM NaCl, 10 mM Tris/HCl, SI pH 7.6, 1 mM EDTA) and mixed with 0.3 pmol of pBR322 1 plasmid (which had been cut with PstI and polymerised .plye ised r 4 t ,s r t ta L W I t t 4 6 C t t, t 6 6 0 6* t 1 30 with oligo-dG residues; Bethesda Research Laboratories), heated first to 65 0 C for 5 minutes then to 42 0 C for 2 hours, cooled slowly overnight to ambient temperature and stored at 4°C.

For the cell transformation, the strain HB 101 (DSM 1607) was cultivated in 50 ml of LB medium g of tryptone, 5 g of yeast extract, 10 g of NaCl in 1 litre) (Mandel, M. and Higa, 1970, J.Mol.Biol. 53, 159 162). In order to obtain cells suitable for transformation ("competent cells") the bacteria were pelleted and taken up in 25 ml of TR buffer (150 mM KC1, 50 mM CaCl 2 1 mM Tris/HCl, pH 7, 3 mM MgCl 2 put on ice for 30 minutes, centrifuged again, resuspended once more in 2 ml of TR buffer and put on ice for 1 hour. 100 mcl of the mixture, containing pBR322 with the inserted HRV2 RNA-cDNA hybrid, were added to 200 mcl of the cell suspension, and also 5 mcl of 1 M CaCI 2 were added and the 20 resulting mixture was incubated for 1 hour at 0 C and then for 90 seconds at 42°C. Then 2 ml of LB medium were added and the mixture was incubated for 1 hour at 37 0 C. The cell suspension was applied to LB-agar plates agar in LB medium) which 25 contained 10 mcg/ml of tetracycline (Sigma) and then incubated overnight. Tetracycline-resistant clones were then tested for ampicillin sensitivity on ampicillin agar plates (100 mcg of ampicillin/ml; Sigma).

Characterisation and isolation of the recombinant DNA molecules Clones of tetracycline-resistant, ampicillin-sensitive bacteria were cultivated overnight in 6 ml of LB

I

-31 medium (10 mcg of tetracycline/ml), plasmid DNA was isolated using the "plasmid mini-preparation technique" (Birnboim, H.C. and Doly, 1979, Nucleic Acids Res. 7, 1195 1204) and the size of the recombinant DNA was determined by digestion with the restriction enzyme PstI. The plasmid- DNA was incubated for 2 hours at 37 0 C in 25 mcl of RE buffer (6 mM MgCl 2 10 mM Tris/HC1, pH 6 mM mercaptoethanol) with 50 mM of NaC1 in the presence of 2 U of the restriction enzyme PstI (Bethesda Research Laboratories) and 5 mcg of ribonuclease A. The probes were then electrophoretically separated on a 1.4% agarose gel. The sizes of the insertions could be determined by staining with ethidium bromide and comparison with lambda- HindIII marker DNA. The sizes were between 300 and 2000 base pairs.

In order to isolate larger quantities of the DNA 20 inserts, plasmids were obtained as described above E t from 200 ml cultures of tetracycline-resistant II ampicillin-sensitive bacteria clones and digested with Pstl. The recombinant DNA fragments were separated as described above over a preparative I 25 agarose gel, the bands were cut out, the DNA was 'electroeluted in 0.05 x TBE buffer (1 x TBE buffer 100 mM Tris/borate, pH 8.3, 2 mM EDTA) and precipitated with ethanol.

Subcloning in Escherichia coli strain JM 101 cells L a: with the aid of the pUC9 vector S: The plasmid pUC9 (Vieisa, J. and Messing, J.G., i 35 1982, Gene 19, 259 268) contains a gene for ampicillin t^J resistance, a region for the start of replication which originates from the plasmid pBR322, and part 1 r iS 1 11 1 I. r w t 1 1 1 cC C L C C L t lt I; C C(

CC

C Cl C Vr cC C of the lacZ gene of E. coli. A small DNA fragment which contains a number of restriction sites is located in this lacZ region, with the result that the cloning of DNA at one of these cutting sites interrupts the lacZ gene region. Colonies which contain DNA inserts therefore appear on X-Gal (X- Gal 5-bromo-4-chloro-3-indolyl-$-D-galactoside, Bethesda Research Laboratories) indicator plates as white colonies whilst those without any inserts show up blue (Rdther, 1980, Mol. Gen. Genet.

178, 475 478). In order to subclone DNA inserts in pUC9, recombinant pBR322 clones (about 7 mcg) were digested with PstI and after separation on agarose gel the DNA inserts were separated from the vector DNA. The DNA was recovered from the gel as described above by electroelution and ethanol precipitation. The isolated DNA insert was incubated for 1 hour at 15 0 C in 20 mcl of RE buffer with 0.4 mcg of pUC9 vector (cut with PstI and pretreated with bacterial alkaline phosphatase), in the presence of 1 mM ATP and 3 U of T 4 -ligase (Bethesda Research Laboratories) and stored at 4 0 C (Vieisa, J. and Messing, 1982, loc.cit.).

At the same time E. coli strain JM101 cells (New England Biolabs), competent for transformation, were prepared in the manner described above. 200 mcl of the competent cell suspension was mixed with mcl of the pUC9 ligase reaction mixture and the resulting mixture was incubated for 1 hour at 0 0

C.

After a heat.shock (90 seconds, 42 0 C) the cells were mixed with 10 mcl of 200 mM isopropylthiogalactoside (Sigma), 50 mcl of a solution of 20 mg of X-Gal in 1 ml of dimethylformamide and 1 ml of LB medium and the resulting mixture was incubated for 1 hour at 37°C., 200 mcl of this cell suspension were i I-!1

~B

I

'1 site, polyadenylation site and transcriptional termination sequences.

-~&tr,<jtr I~4J~ 33 then transferred onto ampicillin LB-agar plates (100 mcg/ml) and incubated overnight at 37 0 C in an incubator. Positive transformants were identified as white colonies which were investigated for DNA inserts using the "plasmid mini-preparation technique".

Preparation of pUC9 plasmids with inserts of recombinant

DNA

I

I

The subclones of E. coli JM 101 obtained which hadbeen transformed with pUC9 and contained DNA inserts were cultivated in 200 ml of LB medium (with 100 mcg of ampicillin/ml) and the plasmid DNA was isolated (Birnboim, and Doly, J., 1979, loc. cit.). The plasmid DNA was then dissolved in 100 mcl of TE buffer and the solution was chromatographed over a Sephacryl-1000 column (1 x 20 cm) with TE buffer. The fractions containing the purified plasmid were located by extinction, then combined and lyophilised. The plasmid was taken up in 500 mcl of TE buffer, incubated for 5 minutes at 65 0 C and again extracted with phenol/chloroform and precipitated with ethanol.

25 Recovery of primer fragments from the plasmids C C C C f C C C (c 6k C i it tc *fc C t pHRV2-773 and pHRV2-87 Clones 773 and 87, which contained corresponding DNA inserts, were subcloned in pUC9, cultivated in 200 ml of LB medium (with 100 mcg of ampicillin/ml) and the plasmids were isolated as described above.

The primer fragment (59 nucleotides) from clone 773 was obtained by digestion with the restriction enzymes AhaIII and EcoRI (the primer fragment is 35 designated fragment A in Fig. 3).

For this purpose, 200 mcg of purified plasmid from 2 i pBW'ltWrt^ 34 f Ci C c FC CC C C C C CC C C Oi C CC 0 CF clone 773 were incubated for 15 hours at 37 0 C in 200 mcl of RE buffer in the presence of 50 mM of NaC1 with 30 U of EcoRI (Bethesda Research Laboratories).

After the reaction had ended, precipitation was carried out with ethanol and the cut plasmid was incubated for 15 hours at 37°C in 100 mcl of RE buffer in the presence of 50 mM of NaC1 with 40 U of AhaIII (New England Biolabs). The pattern of digestion with restriction enzymes was checked by electrophoresis on 1.4% agarose gels. The EcoRI/AhaIII fragment was taken up in 100 mcl of OG solution ficoll, 1 mM EDTA, 0.01% orange G) and the DNA was separated on a preparative 15% polyacrylamide.

gel (acrylamide/bisacrylamide 19:1, gel thickness 1.2 mm) in 1 x TBE buffer. The band corresponding to the 59 base-pair EcoRI/AhaIII fragment was cut out after staining with ethidium bromide, the fragment was electroeluted in 0.05 x TBE buffer and the DNA was precipitated with ethanol. The DNA fragment was then incubated for 1 hour at 65 0 C in 50 mcl of 100 mM Tris/HC1, pH 8, with 200 U of bacterial alkaline phosphatase (Bethesda Research Laboratories).

After the reaction, 2.5 mcl of 0.5M EDTA, pH 8, were added, the aqueous phase was extracted twice with phenol/chloroform and the DNA was precipitated by ethanol precipitation. The DNA was dissolved in water and incubated for 30 minutes at 37°C in 50 mcl of K buffer (10 mM MgCl 2 50 mM Tris/HCl, pH 8, 5 mM dithioerythritol) with 20 mcCi -[32p]- ATP (spec. activity 5000 Ci/mmol; Amersham International) and 5 U of polynucleotide kinase (Pharmacia-PL Laboratories). The fragment labelled with 32p was then precipitated and taken up in 30 mcl of dimethylsulphoxide containing 1 mM of EDTA 35 and 0.01% xylene cyanol-bromophenol blue. This solution was incubated for 2 minutes at 90 0 C, cooled in ice, applied to a 15% polyacrylamide gel (acrylamide/bis- 1 1 1 1 1 1 1 1 1 1 1 1 *1: acrylamide 59:1; gel thickness 1.2 mm) in TBE buffer and the two DNA strands were electrophoretically separated from one another (15 hours at 200 Volts).

Using the autoradiogram, the two strands were located, cut out and electroeluted. The strand hybridising with HRV2-RNA was determined by a "dot-blot" experiment.

In this, two nitrocellulose strips measuring 2 x 2 cm (Schleicher Schdll, BA 85, 0.45 mcm) were moistened with H 2 0, washed once with 20 x SSC (1 x SSC 150 mM NaC1, 15 mM sodium citrate, pH 7.4) and dried in the air. About 1 mcg of HRV2-RNA was applied in dots to each strip and then dried and incubated for 2 hours at 80 0 C. The strips were then moistened with 2 x SSC and incubated together for 1 hour at 42 0 C in a plastic film in 1 ml of H buffer (400 mM NaC1, 40 mM PIPES, pH 6.4, 1 mM SEDTA, 80% formamide) with 4 mcg of denatured salmon sperm DNA (Sigma; incubated for 2 minutes at 100°C and cooled to Then the two nitrocellulose strips were separately sealed into plastic films, each with; 0.5 ml of H buffer, 4 mcg of denatured salmon sperm DNA and I aliquot of the isolated strands (20000 cpm) and hybridised overnight at 42°C. After this incubation the filters were washed C 25 twice with 2 x SSC for 10 minutes at 50°C and twice with 0.1 x SSC, 0.1% sodium dodecylsulphate for minutes at 50 0 C and the radioactivity was determined.

The primer fragment from pHRV2-87 was isolated f ~30 in the same way. The fragment is located between two RsaI restriction sites (68 nucleotides) and was obtained by digestion with this restriction enzyme. The RsaI fragment is designated fragment B in Fig. Strand separation and hybridisation l4 35 were carried out as described above.

i '36 Reverse transcription of HRV2-RNA using restriction fragments as primer pmol of the single stranded DNA complementary to HRV2-RNA from the restriction fragments which had been isolated from clones 773 (fragment A) and 87 (fragment B) as described above were precipitated together with 0.25 pmol of HRV2-RNA from an aqueous solution with ethanol. The precipitate was taken up in 20 mcl of H buffer, welded into a capillary, incubated for 10 minutes at 72 0 C, transferred to slowly cooled to 35°C and then put on ice.

The solution was then incubated for 2 hours at 42 0 C in 100 mcl of RT buffer in the presence of 140 U of reverse transcriptase (Anglian Biotechnology Co., Cambridge), 8 U of ribonuclease inhibitor (RNasin, Bethesda Research Laboratories), 0.2 mM 32 of dATP, dCTP, dGTP and dTTP, 30 mcCi [32 P]dc~2 and 5 mM of dithioerythritol. The reverse transcript 20 obtained was worked up as described above.

Detection of HRV2 sequences in recombinant DNA and restriction mapping Plasmid DNA from the recombinants was isolated from 3 ml cultures (Birnboim, H.C. and Doly, J., 1979, loc. cit). The DNA was incubated with the restriction enzyme PstI and the probes were analysed by electrophoresis on 1.4% agarose gels. The gels 30 were stained with ethidium bromide. The DNA was then transferred from the gel to nitrocellulose filters (Southern, 1975, J.Mol. Biol. 98, 503-517) and fixed on the nitrocellulose by incubation for 2 hours at 80 0 C. The filters were pre-incubated in 50% formamide, 1 x Denhardts solution (Denhardt, 1966, Biochem.Biophys. Res. Comm., 23, 641 646), 900 mM NaC1, 50 mM sodium phosphate, pH 7.4, 5 mM r r.-

I

LCe~: LC ~S

I

r z-r a i r; cti s i 4 I Ie t* I

I:

b I P j: j;i Ai 2

U\

-a 37 EDTA with 80 mcg/ml of denatured salmon sperm DNA for 2 hours at 42 0 C in a plastic film. Radioactive HRV2-cDNA was prepared as described above, except 32 that the reaction mixture contained 50 mcCi [a 3 2 P]dCTP.

The cDNA-HRV2-RNA hybrid was denatured at 100 0

C

for 90 seconds. For hybridisation the filters were incubated at 42 0 C for 18 hours with radioactive HRV2-cDNA as described above, then washed twice in 2 x SSC and twice in 0.1% sodium dodecylsulphate for 30 minutes at 50 0 C, dried in the air and exposed at -70 0 C (Kodak XAR-5, 18 to 40 hours with intensifier film). The presence of a radioactive band showed the existence of recombinant DNA which was complementary to the HRV2-RNA.

For mapping, the DNA inserts were digested us .ng restriction enzymes (New England Biolabs and Bethesda SResearch Laboratories), using the incubation conditions Sspecified by the manufacturer. The results of %20 restriction mapping are shown in Fig. 3. The plasmids SpHRV2-1 and pHRV2-24 contained (immediately adjacent "0 i to the oligo-C which can be traced back to the chain lengthening caused by the terminal transferase reaction) a longer sequence of A residues which 25 form part of the 3'-terminal poly-A of the HRV2- RNA. The other plasmids were arranged relative to pHRV2-1 and pHRV2-24 with the aid of characteristic cleavage sites of individual restriction enzymes.

SDNA inserts ismaller than 500 base pairs were not 30 classified by restriction enzyme mapping but were immediately subcloned and sequenced in pUC9 (see Example 3).

Identification of the remaining clones was obtained by colony hybridisation using DNA inserts which 1 had already been mapped by "nick translation" probes i using the method of Grunstein and Hogness (Grunstein, 1 1 1 1 11 1 fHWHWnfvsfiKs ^fr- -38 M. and Hogness, 1975, Proc. Natl. Acad.

Sci USA 72, 3961-3965). 32 P-labelled DNA probes are obtained with the aid of a "nick translation kit" made by Amersham International (England; Amersham Kit No. 5000) in accordance with the manufacturers instructions with [a- 32 P]dCTP (3000 Ci/mnuol).

The labelled DNA was separated using a Biogel column in a Pasteur pipette in TE buffer. Fractions corresponding to the exclusion volume were combined, heated to 100°C for 2 minutes and quickly put into ice water. Hybridisation was effected as described above. 50 ml cultures were prepared (overnight in LB medium with 10 mcg of tetacycline/ml) from colonies showing a positive hybridisation signal and the DNA inserts were isolated from the plasmid DNA with PstI. These inserts were then characterised by digestion with various restriction enzymes and S by sequencing. In this way clones were obtained which represent the genome of HRV2.

SC1... Example 3 DNA sequencing t a f The majority of cDNA clones of HRV2 were sequenced 25 using a modification of the method of Maxam and Gilbert (Maxam, A. and Gilbert, 1980, Methods Enzymol. 65, 499-560). Some of the sequences were also determined using the M13-chain breakage method according to Sanger et al. (Sanger, F., Nicklen, S. and Coulsen, 1977, Proc. Natl.

S. ~Acad. Sci USA 74, 5463 5467).

For sequencing according to Maxam and Gilbert the DNA inserts were subcloned in pUC9 as described above and competent E. coli JM 101 cells were transformed therewith. Positive transformants were isolated as white colonies and cultivated in LB medium (with ,t Bh t i 4 ~I 'wwwia 39

I

l l 100 mcg/ml of ampicillin). 10 20 mcg of DNA were digested in 100 mcl overnight under standard reaction conditions with restriction enzymes which have a cleavage site in the plasmid, e.g. in the polylinker region of pUC9 BamHI, EcoRI, AccI, HindIII). The restricted fragment was then dephosphorylated. For restriction digestion, 5 mcl of 2M Tris/HCl, pH 8, and 100 U of bacterial alkaline phosphatase (Bethesda Research Laboratories) were added and incubated for 3 hours at 65°C. After the addition of EDTA to 20 mM, the mixture was extracted twice with phenol/chloroform and the DNA was precipitated using ethanol. The DNA was then incubated for 30 minutes at 37 0 C in 50 mcl of 50 mM Tris/HCl, pH 8, 10 mM MgCl 2 5 mM dithioery- 32 thritol, with 25 mcCi P]ATP (5000 Ci/mmol, Amersham International) and 4 U T 4 -polynucleotide kinase (Pharmacia-P.L. Biochemicals) and the labelled DNA was precipitated with ethanol. Recombinant 32 DNA labelled with 3 in one strand was obtained with the aid of another restriction enzyme which cleaves in the polylinker region of pUC9.

DNA sequencing according to a modification of the 25 method of Maxam and Gilbert The sequencing reactions were carried out with the following modifications according to Maxam and Gilbert (Maxam, A. and Gilbert, 1980, Methods 30 Enzymol. 65, 499-560): No carrier-DNA was added.

The DNA solution was divided up into aliquots: guanine (G)-specific reaction 7.5 mcl, guanine 35 and adenine (G/A)-specific reaction 10 mcl, cytosine and thymine (C/T)-specific reaction 10 mcl, cytosine (C)-specific reaction 6 mcl.

t t

C

CEL

C r C C CC C C C C t

SCC

C I~t 'Ii~r p..

I

t WW*WWSCS^- 4 4 .40 t S t Ct t A. A A *t A A t t t AG o t S At t A t: GAt C GA a mcl of 96% formic acid were added to the (G/A)-reaction mixture whi:h was then incubated for 4.25 minutes at 19 0 C. In order to stop the reaction 200 mcl of hydrazine-stop solution and 750 mcl of 96% ethanol were added. The (G/A)-reactions were then treated just the same as the other three reactions.

Instead of hydrazine, hydrazinium hydroxide (Merck) was used in the and C-reactions.

The reaction times were 7.5 min.

The piperidine reactions were incubated for minutes at 95 0 C. After lyophilisation the fragments were heated to 95°C for seconds in 3 20 mcl of buffer (80% deionised formamide, 1 x TBE, 0.05% bromophenol blue and 0.05% xylene cyanol) and rapidly cooled to 0°C.

For sequencing, 6% polyacrylamide gels (40 cm x 20 20 cm x 0.4 mm) were used with 8 M urea and 1 x TBE, which had been subjected to electrophoresis for one hour at 50 watts before the application of the probes. Between 1 mcl and 3 mcl of each reaction mixture was applied to the gel. Usually, 25 two. gel charges were carried out at different times.

The first gel passage of a reaction mixture lasted until the bromophenol blue marker reached the end of the gel 'd the second passage lasted until 30 the xylene cyanol marker reached the end of the gel. The gels were then fixed for 20 minutes in 10% acetic acid and 10% methanol (about 2 litres), transferred to 3 MM-filter paper and dried at on a gel drier.. Then the gels were exposed without intensifier film at -70°C using an XAR-Omat film (Kodak) (about 18 to 36 hours).

J~AA~

A

-V

I

41 MI3 Sequencing The recombinant DNA was cut with the restriction enzyme Sau3AI and cloned into the BamHI cutting site of M13 mp9, using a "sequencing pack" (New England Biolabs, catalogue No. 409). The sequencing was carried out using the chain breakage method (Sanger, Nicklen, S. and Coulson, 1977, Proc. Natl. Acad. Sci USA 74, 5463-5467).

Analysis of the sequencing data The sequencing results were analysed with the aid of a Cyber 170 Computer. The programmes used were the Staden programme (Staden, 1980, Nucleic Acids Res. 8, 3673-3694) and the modified programmes of Isono (Isono, 1982, Nucleic Acids Res. 85-89).

Example 4 Proteolytic cleavage sites in the Pl-coat protein region of the polyprotein The viral proteins are obtained by proteolytic 25 cleavage from the polyprotein. In order to identify rI r the cleavage sites the viral coat proteins were isolated and the N-terminal amino acid sequepces Swere determined. For this purpose the proteins from 2 mg of HRV2 were electrophoretically separated a^ 30 on a 12.5% polyacrylamide gel (Laemmli, 1970, C oc l t loc. cit.). The gels were stained with a saturated solution of Coomassie Brillant Blue in 50 mM Tris/HCl, pH 7.4, and the protein bands were cut out. The individual proteins were electroeluted in an ISCO elution apparatus at 50 V for 16 hours and precipitated with trichloroacetic acid. The N-terminal amino acids were determined using an AB-470A protein 1 1 g c C CC C C s 0 1

C

CS c C c C e C c Cs <CC t CC- 42 sequenator (Applied Biosystems, Inc., Foster City, CA, USA). For sequencing, 2 nmol of VP1 and VP2 and 1 nmol of VP3 were used. The derivatised amino acids were analysed by HPLC. The N-terminal sequences of the individual proteins are given in Fig. 6.

Example Identification of the viral capsid protein VP1 as the strongest antigen of HRV2 in rabbits Rabbits were given a subcutaneous injection of mcg of HRV2 in 500 mcl of complete Freund's adjuvant. 21 and 35 days later further immunisations were carried out with 25 mcg of HRV2 in 1.5 ml of incomplete Freund's adjuvant. 50 days after the first immunisation, serum was taken by plasmaphoresis and stored at -20 0 In order to detect the formation 'of antibodies, 2 mcg of virus were applied to a 15% polyacrylamide gel (Laemmli, 1970, loc.

cit.) and the proteins were separated electrophoretically.

The proteins were transferred by electrotransfer from the gel by the "Western Blot" method onto a nitrocellulose filter membrane (Schleicher 25 Schill, BA85, 0.45 mcm) (Burnette, 1981, Analyt. Biochem. 112, 195-203). The filter with the proteins bonded thereon was bathed for 16 hours in 20 ml of PBS (137 mM NaCl, 2.7 mM KC1, 6.6 mM Na 2

HPO

4 1.5 mM KH 2

PO

4 with 3% bovine serum albumin (BSA) and 3% Tween 20. After 2 hours' incubation with antiserum (1:200 dilution in PBS/1% BSA/1% Tween 20) the filter was washed 3 times for minutes in 20 ml of PBS with 1% BSA and 1% Tween (PBSBT) and then incubated for 2 hours in 20 ml of PBSBT with 10 mcCi of 125 I-labelled protein from Staphylococcus aureus (about 1 mCi/mg), washed 3 times for 20 minutes with PBSBT and 3 x 5 min 44CC 7, 7

I

~ii '1 4 '-1 0 C) As is clear from Fig. 7, the antiserum '43 1' in PBS, quickly rinsed twice with H20 and dried overnight between several layers of filter paper.

The radioactivity bound to the filter was determined by autoradiography on Kodak XAR5 X-ray film As is clear from Fig. 7, the antiserum contains, in particular, antibodies against VP1.

Example 6 Comparison of a partial sequence of the genes for the polyneptides P3A and P3B (VPg) of HRV2 with a homolc.gous region from the genome of HRV89 Further support for part of the sequence obtained as described in the previous examples was obtained by comparison with a gene fragment obtained from cloned HRV89. HRV89 was from the American Type Culture Collection (ATCC VR-1199) and was grown as described for HRV2. HRV89 was neutralized by a specific antiserum (ATCC VR-1199 AS/GP) whereas S: antiserum against HRV2 (ATCC VR-1112 AS/GP) used as control serum had no effect. The isolation Sof the viral RNA, characterisation, cloning, isolation of clones and sequencing was performed as described I 'c for HRV2. Fig. 8 shows the sequence comparison with a partial sequence obtained from the clone 34/1 of HRV89, which obviously represents a region corresponding to the genes for P3A and P3B (VPg).

0i

Claims (21)

1. DNA coding for at least one viral protein ofjIrhinovirus strain

2. DNA as claimed in claim 1, which corresponds to the entire viral RNA or parts of the viral RNA of rhinovirus strain HRV2.

3. DNA as claimed in claim 1, which codes for a viral protein comprising at least two of tile viral proteins VPl, VP2, VP3, VP4, P2A, P2B, P2C, P3A and P3C, linked together in any desired combination.

4. DNA as claimed in claim 1, containing a sequence according to any one of Figs. 4, 9 or 11 to 14 or a substantial part thereof or a degenerate variation thereof.

5. DNA as claimed in claim 1, coding for any one of the viral proteins VPl, VP2, VP3, VP4, P2A, P2B, P2C, P3A and P3C.

6. DNA coding for part of a viral protein defined in claim 1, the biological activity of the said part of the viral protein corresponding to at least one of said viral proteins.

7. DNA as claimed in claim 1, whose non-coding sequence 30 hybridizes with DNA as claimed in any one of claims 3 to 6 or with degenerate variations thereof under stringent conditions which make it possible to recognise homology greater than

8. DNA as claimed in any one of the preceding claims, incorporated into a suitable expression vehicle so as to be replicable in microorganisms t. t.r '45 or in mammalian cells.

9. DNA as claimed in claim 8 wherein said expression vehicle is a plasmid. DNA according to claim 1, substantially as described herein.

11. A polypeptide having the biological activity of at least one of the viral proteins of the rhinovirus strain HRV2, and which has been coded by DNA as claimed in any one of claims 1 to 9, which corresponds wholly or in part to at least one of the viral proteins of the rhinovirus strain HRV2 or which corresponds to at least two of said viral proteins or portions thereof connected to each other in any desired combination and sequence.

12. A polypeptide as claimed in claim 11, containing an amino acid sequence according to any one of H Figs. 4 or 10 to 14 or a substantial portion thereof.

13. A polypeptide as claimed in claim 11, containing Sthe amino acid sequence for any one or more of the viral proteins VP1, VP2, VP3, VP4, P2A, P2B, L P2C, P3A and P3C.

14. A polypeptide derived from an amino acid sequence according to claims 11, 12 or 13 or a .,.portion thereof, said polypeptide being effective S, to bind to and/or block the cellular receptors Sfor rhinovirus strain HRV2. A polypeptide according to claim 11 substantially jas described herein.

16. A process for preparing DNA as claimed in I l l 1 I 4 4 T For this purpose, 200 mcg of purified plasmid from iq 1 46 0 IB* SI° *4111 I~ I any one of claims 1 to 10, wherein a) the viral RNA of rhinovirus strain HRV2 is isolated, b) a DNA complementary to the viral RNA is prepared, c) the viral cDNA/RNA hybrid is incorporated in a suitable replicable vector d) a suitable host organism is transformed with the vector prepared in c, and e) the host organism prepared in d is cultured and the desired DNA isolated.

17. A process according to claim 16 substantially as described herein.

18. A host organism transformed with the genetic information coding for a viral protein as defined in any of claims 1 to

19. A transformed host organism as claimed in claim 18 wherein said genetic information is contained in a vehicle which is replicable in the said host. An E. coli bacterium transformed with the genetic information coding for the viral protein as defined in any of claims 1 to

21. A process for preparing the polypeptide as claimed in one of claims 11 to 15, wherein a host organism is transformed with genetic information coding for a viral polypeptide according to any one of claims 11 to 15; said information is expressed ILI AI I I I (1 tO trt, t IC I CtC 47 to produce said viral polypeptide in the host organism; and then said viral polypeptide is isolated.

22. A process according to claim 21 wherein said genetic information is contained in DNA as claimed in any one of claims 1 to 9.

23. A process according to claim 21 substantially as described herein.

24. A pharmaceutical composition containing, as active ingredient, at least one polypeptide as claimed in any one of claims 11 to 15, in association with one or more pharmaceutical carriers, diluents or adjuvants. A method for the therapy or prophylaxis of viral infections or for stimulating the immune system of mammals which comprises administering an effective amount of a polypeptide as claimed in any one of claims 11 to Dated this 28th day of February, 1990 BOEHRINGER INGELHEIM INTERNATIONAL GmbH, By its Patent Attorneys, DAVIES COLLISON. CO i: U