IE64964B1 - Production of retroviral envelope proteins in bacteria and use in seroepidemiological survey of human lymphoid malignancies. - Google Patents

Description

Production of Retroviral Envelope Proteins in Bacteria and Use in Seroepidemioloqical Survey of Human Lymphoid Malignancies Two regions of the gene for the HTLV-I envelope were expressed in Escherichia coli by use of the vector pJLA16. One corresponds to the carboxy terminal region of the major envelope protein p46 and the other corresponds to the transmembrane protein p21E. Reactivity of the expressed protein with human sera was tested by Western blot procedure. Each of the sera tested that had been shown to contain anti-HTLV-I or anti-HTLV-II antibodies by ELISA assay recognized the bacterially synthesized 1θ· envelope proteins. There was no reaction detected when control sera were tested. This system is useful for large scale seroepidemiological surveys for this and related human retroviruses.

It has been found, and is the subject of this invention, that where the HTLV envelope protein is isolated into fragments such as the 400 base pair (bp) and 300 bp, these fragments placed in a comnon vector and in bacteria can be utilized in competition testing where the protein here is used as the antigen and mixed with sera of patients to show that said competition analysis will be almost 100% positive for patients of HTLV-I.

Human T-cell leukemia virus subgroup I (HTLV-I) is a retrovirus causatively linked to certain adult lymphoid malignancies, notably adult T-cell leukemia25 lymphoma (ATL). Two other isolates (HTLV-II), including one from a patient with T-cell hairy cell leukemia, are clearly related to HTLV-I but significantly differ in antigen assays and in their genomes. A third subgroup of HTLV (HTLV-III) has recently been described (Popovic, et al, Science, 224:437 , 1984; Gallo et al, ibid., p. 500 ; and Sarngadharan et al, ibid. , p. 506) that is associated with acquired immunodeficiency syndrome (AIDS).

Antibodies that react with HTLV-I proteins have - 2 been found in the sera of ATL patients. These antibodies recognize both the gag core antigens and the envelope proteins of the virus. Viral core proteins were readily purified, sequenced, and extensively used in immunoassays; however, progress with the more important viral envelope proteins was slower. A limiting factor, therefore, in the studies of the immune response to these viruses has been the difficulty in isolating the viral envelope proteins in pure form and in quantity. As an alternative approach, the present invention expresses the virus envelope protein in a bacterial vector. This procedure has the advantage that only a single viral product as defined by the structure of the input DNA is made by the bacteria. HTLV-I was suitable for such an approach since the integrated proviral DNA has been cloned [Seiki e t a 1, Proc. Natl. Acad. Sci. USA, 79:6899 (1982) and Manzari et al, Proc. Natl. Acad. Sci. USA, 80:1574 (1983)] and sequenced [Seiki et al, Proc. Natl. Acad. Sci. USA, 80:3618 (1983)]. The HTLV-I envelope is expressed by placing it into the pJLA16 derivative [Lautenberger et al, Gene Anal. Techniques, 1:63-66 ( 1984)] of plasmid pJL6 [Lautenberger et al, Gene, 23:75 ( 1983)]. This plasmid contains the 13 ami no-terminal codons of the bacteriophage lambda ell gene placed under the transcriptional control of the well-regulated phage lambda p^ promoter. This plasmid is known and has been successfully used to express sequences from myc, myb, and ras oncogenes [Lautenberger et al, Gene, 23:75 (1983) and Lautenberger et al, in Gene Amplification and Analysis, Volume 3, Expression of Cloned Genes in Prokaryotic and Eukaryotic Cells, Papas et al (eds), Elsevier, New York/Amsterdam, pp. 147-174].

Initial attempts to express the entire HTLV-I envelope were unsuccessful, possibly because this protein can interact with the bacterial cell membrane in such a way as to be toxic to the cell. Therefore, individual fragments coding for specific regions of the envelope were inserted into pJLA6 by use of polynucleotide linkers (Fig. 1). Such plasmids were introduced into 12. coli MZ1, a strain that contains a partial lambda prophage bearing the mutant cI857 temperature-sensitive repressor.

At 32°C the repressor is active and p^ promoter on the plasmid is repressed. At 42°C the repressor is inactive and the p^ promoter is induced, allowing high level expression of genes under its transcriptional control.

When lysogens carrying either of the two plasmids containing different portions of the HTLV-I envelope gene (cf. ante) were grown at 32°C and induced by shifting the temperature to 42°C, prominent bands were observed that were not found in uninduced cells or in induced cells containing the pJL6 vector alone (Fig. 2). These proteins were readily observed both in gels of radiolabeled bacterial extracts and gels stained for total protein.

Based on DNA sequence data of the envelope gene fragments utilized in this study, the calculated molecular sizes of the pKS300 and pKS400 proteins are 12.84 Kd and 15.88 Kd, respectively. These sizes include the 1.56 Kd coding sequence contributed by the amino terminal codons of the lambda ell gene. The observed molecular weights of both proteins on SDS-polyacrylamide gels are consistent with those calculated for a 321 base pair (pKS300 insert) and 397 base pair (pKS400 insert) coding sequences or polypeptide sequences.

Statement of Deposit On 24th October 1984 there were deposited in the American Type Culture Collection (ATCC) envelope gene fragments pKS300 and pKS400, under the accession Nos. 39902 and 39903 respectively.

This depository assures permanence of the deposit and availability to the public upon the issuance of a patent related directly to this patent application.

Description of the Drawings Figure 1 is the construction of plasmids pKS300 and pKS400.

Figure 2 is the expression of the HTLV-I envelope gene in E. ooli.

Figure 3 is the recognition of bacterial synthesized HTLV-I envelope protein by antibodies in human serum.

Material Information Disclosure Lautenberger et al, Gene Anal. Techniques, 1:63-66 (1984).

Lautenberger et al, Gene, 23 :75 ( 1983).

Lautenberger et al, Science, 221 :858 ( 1983).

Lautenberger et al, in Gene Amplification and Analysis, Vol. 3, Expression of Cloned Genes in Prokaryotic and Eukaryotic Cells, Papas et al (eds.), New York/Amsterdam: Elsevier, pp. 147-174.

The Invention The HTLV-I env gene codes for a glycoprotein (gp61) of molecular weight 61,000 that is cleaved into the molecular weight 46,000 exterior glycoprotein (gp46) and the molecular weight 21,000 trans membrane protein (gp21E). The precise site of proteolytic cleavage has been determined by locating radiolabeled valine residues with respect to the amino terminal end of gp21. The cleavage of the env gene precursor is adjacent to the residues Arg-Arg that also occur next to the proteolytic cleavage sites in the bovine leukemia virus (BLY) and mouse manmary tumor virus (MtfTV) env precursor. Since the BamHl site that separates the inserted fragments is close to the region coding for proteolytic cleavage site that separates gp46 from p21E, the protein from pKS300 contains sequences corresponding to the carboxy-terminal portion of gp46 and the protein from pKS400 predominantly consists of sequences from p21E. See also preparation in Example 1.

Sera from many patients with HTLV-I associated ATL and certain other lymphoid malignancies contain antibodies to proteins that have been shown to be the product of the viral env gene. In order to see if such antibodies can recognize a bacterially synthesize envelope product, a lysate of induced MZl[pKS400] cells containing this protein was fractionated by SDS-polyacry1amide gel electrophoresis and transferred to nitrocellulose sheets by electrophoretic (Western) blotting. Strips containing the transferred proteins were reacted with diluted human serum and antigen-antibody complexes formed were detected ι o e by incubation of the strips with I-labeled Staphlococcus aureus protein A followed by autoradiography. As shown in Fig. 3, prominent bands corresponding to reaction of antibody to the 15 Kd bacterial envelope produce could readily be observed when the serum used was from patients with HTLV-I associated ATL or from HTLV-I antigen (+) individuals. No such reactions were observed with sera from healthy control individuals. This procedure was used to screen a group of 28 coded sera. Antibodies that recognized the bacterially synthesized HTLV-I envelope protein sequences were found in all sera that had been shown to have anti-HTLV-I antibodies by ELISA assay using disrupted virions as antigen (Table 1). Thus, a method is formulated for serologically testing for the presence in human sera of antibodies directed against HTLV-I or HTLV-II. None of the normal control sera were found to have reacting antibodies. Antibodies from a patient (Mo) with a hairy cell leukemia, whose disease is associated with HTLV-II, strongly reacted to the protein coded for in pKS400 indicating that there is a high degree of relatedness between the p21E region of HTLV-I and HTLV-II.

Since the bacterially synthesized HTLV-I env protein was recognized by antibodies present in sera from AIDS patients, it was also of interest to show that this assay can be utilized to screen for a more distantly related subgroup, namely, HTLV-III (the virus associated with AIDS). Therefore, a number of sera samples of AIDS patients, some of which were also sero-positive for HTLVI, were examined.

TABLE 1 Presence of Antibodies Recognizing Bacterially Synthesized HTLV-I Envelope in Human Sera Status HTLV-I or HTLV-II + /(by ELISA) Number Tested Number Pos i t iv Clinically normal heterosexual + 2 2/2 - 8 0/8 Clinically normal homosexua1 - 5 0/5 AIDS patients + 2 2/2 - 2 0/2 ATL patients + 5 5/5 Mycosis fungoides patient + 1 1/I Hairy cell leukemia patient Mo (HTLV-I + pat i ent) + 1 1/1 Lymphadenopathy syndrome patients 2 0/2 The sera from all positive AIDS which reacted with HTLV-I in ELISA contained antibodies that recognized the bacterial synthesized HTLV-I env protein. None of the sera from AIDS patients that were HTLV-I negative contained antibodies that reacted with the bacterial protein. Since antibodies that react with HTLV-III proteins can be found in the serum of greater than 90% of AIDS patients, this result indicates that there is little or no cross reaction between the carboxy-terminal portion of the envelope proteins of HTLV-I and HTLV-III.

The results presented here demonstrate the importance of using bacterially synthesized proteins to study the properties of antibodies in human serum. Since the structure of the genes for such proteins can be controlled by recombinant DNA techniques, the antigens produced by these methods have a defined structure.

Example 1 Construction of plasmids pKS300 and pKS400. Plasmid pHTLV-I HX-CR was obtained by subcloning the 5.7 kb Hind III-XbaI fragment of lambda CR1 [Manzar i et al, Proc. Natl. Acad. Sci. USA, 79:6899 (1982)] that contained envelope, pX, and LTR sequences. Lambda CR1 contained integrated HTLV-I proviral DNA from mycosis fungoides patient CR. pHTLV-I HX-CR DNA was digested Xhol and BamHI and the 300 bp and 400 bp fragments containing the env sequences were isolated from an agarose gel. The termini of these fragments were converted to blunt ends by the action of Klenow fragment ΙΪ. col i DNA polymerase I and Hind III linkers were attached. Excess linkers were removed by digestion with Hind III and reisolation of the fragments from agarose gels. The pJLA16 [Lautenberger et al, Gene Anal. Techniques, 1:63— 66 (1984)] vector DNA was cleaved with Hindlll and the ends were dephosphorylated by the action of calf intestinal phosphatase. The dephosphorylated vector DNA was ligated to the fragment DNAs and introduced into DC646 cells by transformation using ampicillin selection.

Plasmids containing inserts were identified by hybridization of colonies transferred to nitrocellulose with radiolabelled fragment produced by nick-translation of fragment DNA using [a-33P]dCTP. For protein expression experiments, the plasmids were transferred into a prokaryote host such as by transferring into E. col i (strain MZ1) provided by M. Zuber and D. Court. Recombinant DNA procedures were as described by Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Example 2 Expression of the HTLV-I envelope gene in E. coli. (a) Radiolabeling of bacterial cell proteins. E. coli MZ1 cells were grown at 32°C, induced by shifting the temperature to 41°C, labeled with [35S] -cysteine and lysed. Proteins were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and visualized by autoradiography. (b) Uninduced (U) and induced (I) cell extracts of expression plasmid vectors - Lane 1, pJL6 vector without insert; Lane 2, pJLcII ras; Lane 3, pKS300; Lane 4, pKS400.1; Lane 5, pKS400.2; Lane 6, 400 bp fragment in wrong orientation. Confer Fig. 2.

Example 3 Recognition of bacterial synthesized HTLV-I envelope protein by antibodies in human serum.

MZ1 [pKS400] cells were grown at 32°C, induced at 42eC, and lysed in the presence of 1% SDS-0.1% betamercaptoethano1. Protein in the extracts were resolved by SDS-PAGE and electrophoretically transferred to nitrocellulose paper by the Western blot procedure. After transfer, filters were air dried and soaked in TBS-NDM (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 3% Nonfat Dry Milk). The filters were incubated overnight at room temperature in TBS-NDM plus 1/77 volume human serum as indicated below. Filters were then washed with TBS-NDM for 30 min and then incubated with 10^ cpm [I]—proteίn A (NEN) . The filter was then washed with TBS-NDM and finally with TBS. The filters were air dried and protein bands reacting with antibody were visualized by autoradi5 ography. The sera used were: (1) American ATL patient; (2) T-cell hairy cell leukemia pateint Mo (Ref. 4); (3) Healthy normal; (4) Health normal; (5) Healthy normal; (6) Healthy relative of ATL patient; (7) Healthy normal; (8) Japanese ATL patient; (9) AIDS patients found to be HTLV-II (+) by ELISA (disrupted virus antigen); (10) AIDS patient found to be HTLV-I (+) by ELISA (disrupted virus antigen); (11) Healthy normal; (12) American ATL patient; (13) Mycosis fungoides patient; (14) Healthy normal found to be HTLV-I (+) by ELISA (disrupted virus antigen).

Uninduced and induced extracts pKS400.2 reacted with patients MJ serum (HTLV-I positive by ELISA).

Claims (17)

1. A method of producing retroviral envelope proteins comprising the steps of: (1) Isolating the envelope gene of a retrovirus;

2. (2) Cleaving the envelope gene to provide at least two gene fragments;

3. (3) Attaching polynucleotide linkers to the gene fragments formed in step (2); 4. (4) Inserting the fragments formed in step (3) into vectors; 5. (5) Transferring the vectors formed in step (4) into prokaryote hosts; and (6) Isolating the protein from lysated host cells. 2. A method of claim 1 wherein the envelope gene is cleaved into two fragments; one that codes for glycoprotein and another which codes for transmembrane protein. 3. plasmid. A method of claim wherein the vector of step (4) is a

4. A method of claim 1 wherein the host cell is E. coli.

5. A method of claim 4 wherein the L·. coli bears a temperature-sensitive repressor.

6. A method of claim 5 wherein the E. coli is strain MZ1.

7. A method of claim 1 wherein the vector of step (4) is a pJLA16 plasmid.

8. A method of claim 7 wherein the vector insert is pKS400. - 11

9. A method of claim 7 wherein the vector inserted is pKS300.

10. A composition comprising peptides produced by the method of claim 1 on a solid support or in a carrier. z

11. A substantially pure HTLV-I envelope protein sequence which is a transmembrane protein.

12. A method of detecting antibodies to HTLV-I, comprising Ιθ contacting a composition of claim 10 with sera suspected of containing antibodies to HTLV-I.

13. A method of claim 12, wherein the peptide is attached to a solid support.

14. A method of claim 13, wherein the test used is an ELISA test.

15. A method of claim 12, wherein the peptide is on a carrier. 20

16. A method as claimed in claim 1 substantially as described herein with reference to the Examples and/or the accompanying drawings.

17. A method of detecting antibodies as claimed in claim 12 substantially as described herein with reference to the Examples and/or 25 the accompanying drawings.