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Last Updated: March 29, 2024

Claims for Patent: 6,140,059


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Summary for Patent: 6,140,059
Title: Methods for the obtention of human immunodeficiency virsus Type 1 envelope glycoproteins in native and oligomeric form employing recombinant chimeric antigens containing collagenase recognition sites.
Abstract:The present invention is relative to a method for the obtention of native domains of viral membrane proteins, especially of native, that is, oligomeric and glycosylated ectodomains of the surface protein gp160 of the human immunodeficiency virus HIV, the causative agent of AIDS (acquired immune deficiency syndrome), as well as the native protein domains themselves obtained by this method, especially native ectodomains of the env glycoprotein of HIV whose monomers exhibit an electrophoretic mobility of approximately 140 kD as well as their use as vaccine, especially as vaccine against HIV. A nucleotide sequence coding for a recognition sequence for protein-splitting enzymes is inserted at a suitable site into the gene coding for the precursor protein of the protein domain to be obtained. After expression of the gene mutant in eukaryotic cells a digestion with a suitable enzyme is carried out and the protein domain to be obtained is subsequently purified.
Inventor(s): Schawaller; Manfred (D-75210 Keltern-Dietlingen, DE)
Assignee:
Application Number:08/448,619
Patent Claims:1. A method for the production of recombinant truncated HIV envelope glycoprotein gp140 domains that retain their native oligomeric and glycosylated form comprising the following:

(i) providing a nucleotide sequence encoding an amino acid sequence comprising a protease recognition site;

(ii) providing a nucleotide sequence encoding for the HIV envelope glycoprotein precursor gp160, wherein said sequence lacks a functional wildtype gp120/gp41 proteolytic cleavage site;

(iii) inserting the nucleotide sequence of step (i) into the nucleotide sequence of step (ii) at a region corresponding to amino acids 645 to 673 of the transmembrane area of gp41, wherein said numbering scheme is based upon isolate HXB2, thereby creating a mutant HIV envelope gene;

(iv) cloning the mutant HIV envelope gene into a suitable expression vector and expressing said mutant HIV envelope gene in eukaryotic cell line to produce a recombinant mutant HIV envelope glycoprotein gp160 comprising said protease recognition site;

(v) subjecting the expression product of step (iv) to enzymatic digestion with a suitable protease thereby creating HIV gp140 envelope glycoprotein domains that retain their native oligomeric and glycosylated form; and

(vi) isolating and purifying said HIV gp140 from the digestion mixture.

2. The method according to claim 1 wherein said recognition sequence is a recognition sequence for proteases, lipases, or blood coagulating factor Xa.

3. The method according to claim 1 wherein said recognition sequence has the following general form:

in which n is a whole number greater than 1 and X is one of the 20 amino acids determined by the genetic code, said recognition sequence being specific for a collagenase.

4. The method according to claim 1 further comprising supplying a buffer to the enzymatic digestion products of said enzymatic digestion, said buffer comprising borate in a concentration between 1 and 200 mM.

5. The method according to claim 3, wherein said collagenase is a bacterial collagenase.

6. The method according to claim 1 wherein said desired protein domain is an ectodomain of the env-glycoprotein of HIV.

7. The method according to claim 1 comprising the additional step of inserting said recognition sequence into said nucleotide region coding for said HIV env-glycoprotein between amino acids 659 and 660.

8. The method according to claim 4, wherein said concentration is from 30 to 100 mM.

9. The method according to claim 1 wherein a suitable insertion site is identified by the following method:

(i) synthesizing short, overlapping oligopeptide sequences corresponding to the amino acid sequence of wildtype HIV gp160;

(ii) admixing said oligopeptides with HIV gp160-specific antiserum under conditions that facilitate the formation of peptide-antibody complexes;

(iii) identifying those oligopeptide sequences that are antigenic and correspond to surface accessible domains, said surface accessible domains providing suitable insertion sites for the proteolytic cleavage site, within the region corresponding to amino acids 645 to 673 of the transmembrane area of gp41, wherein said numbering scheme is based upon isolate HXB2.

10. The method according to claim 9, wherein said overlapping peptide sequences have a length of between 5 and 30 amino acids.

11. The method according to claim 9, wherein each of said overlapping olipopeptide sequences has a length of 12 to 16 amino acids.

12. The protein domains isolated according to the method of claim 1 comprising: HIV env-glycoproteins in native oligomeric and glycosylated form.

13. The protein domains according to claim 12, wherein said protein domains are ectodomains.

14. The isolated protein domains according to claim 13, wherein said protein domains are in tetrameric form.

15. The isolated protein domains according to claim 13, wherein the monomeric form of said protein domains has an electrophoretic mobility of about 140 kD as measured by 6% SDS-PAGE analysis.

16. The isolated protein domains according to claim 15, said protein domains comprising: domain gp120 of the HIV env-glycoprotein and the aminoterminal portion of domain gp41 of the HIV env-glycoprotein said aminoterminal portion further comprising an aminoterminal amino acid sequence having a length in the range of from 131 to 165 amino acids following said enzymatic digestion.

17. The protein domains according to claim 16, wherein said length of said aminoterminal amino acid sequence is 148 amino acids.

18. A method for diagnosing the presence or absence of an antibody-mediated immune response by a mammal to the presence of HIV env-glycoprotein comprising:

mixing protein domains isolated in accordance with the method of claim 1 with serum from said mammal to form a reaction mixture;

maintaining said reaction mixture under conditions conducive to the formation of antibody-antigen complexes;

analyzing said antibody-antigen complexes to determine the presence or absence in said antibody-antigen complexes of said protein domains.

19. A method for the production of immunogenic, recombinant truncated HIV envelope glycoprotein gp140 domains that retain their native oligomeric and glycosylated form and that are capable of inducing HIV-specific immune responses comprising the following:

(i) providing a nucieotide sequence encoding an amino acid sequence comprising a protease recognition site;

(ii) providing a nucleotide sequence encoding for the HIV envelope glycoprotein precursor gp160, wherein said sequence lacks a functional wildtype gp120/gp41 proteolytic cleavage site;

(iii) inserting the nucleotide sequence of step (i) into the nucleotide sequence of step (ii) at a region corresponding to amino acids 645 to 673 of the transmembrane area of gp41, wherein said numbering scheme is based upon isolate HXB2, thereby creating a mutant HIV envelope gene;

(iv) cloning the mutant HIV envelope gene into a suitable expression vector and expressing said mutant HIV envelope gene in a eukaryotic cell line to produce a recombinant mutant HIV envelope glycoprotein gp160 comprising said protease recognition site;

(v) subjecting the expression product of step (iv) to enzymatic digestion with a suitable protease thereby creating HIV gp140 envelope glycoprotein domains that retain their native oligomeric and glycosylated form;

(vi) isolating and purifying said HIV gp140 from the digestion mixture; and

(vii) mixing said gp140 with a suitable adjuvant wherein said gp140 maintains a spatial and multimeric configuration that is substantially similar to the spatial and multimeric configuration of the wildtype HIV envelope gp160.

20. A method for the detection of HIV-specific antibodies in a sample comprising the following:

(i) providing a nucleotide sequence encoding an amino acid sequence comprising a protease recognition site;

(ii) providing a nucleotide sequence encoding for the HIV envelope glycoprotein precursor gp160, wherein said sequence lacks a functional wildtype gp120/gp41 proteolytic cleavage site;

(iii) inserting the nucleotide sequence of step (i) into the nucleotide sequence of step (ii) at a region corresponding to amino acids 645 to 673 of the transmembrane area of gp41, wherein said numbering scheme is based upon isolate HXB2, thereby creating a mutant HIV envelope gene;

(iv) cloning the mutant HIV envelope gene into a suitable expression vector and expressing said mutant HIV envelope gene in eukaryotic cell line to produce a recombinant mutant HIV envelope glycoprotein gp160 comprising said protease recognition site;

(v) subjecting the expression product of step (iv) to enzymatic digestion with a suitable protease thereby creating HIV gp140 envelope glycoprotein domains that retain their native oligomeric and glycosylated form;

(vi) isolating and purifying said HIV gp140 from the digestion mixture; and

(vii) employing said isolated and purified gp140 as an antigen in a suitable immunoassay to detect HIV-specific antibodies.

21. An expression vector for expressing a glycosylated, oligomeric form of the gp120 env-glycoprotein of HIV comprising:

a recombinant vaccinia virus the genetic material of which comprises the mutant gene according to claim 1.

22. A composition comprising a mixture of recombinant truncated HIV envelope glycoprotein gp140 domains that retain their native oligomeric and glycosylated form comprising the following: at least one HIV envelope glycoprotein fragment, wherein said fragment comprises the gp120 domain, a portion of the amino terminus of gp41, said portion ranging in length from 131 to 165 amino acids, a non-functional wildtype gp120/gp41 proteolytic cleavage site, and a portion of a protease recognition site that remains following proteolytic cleavage, said fragment displaying an approximate molecular weight of 140 kDa as determined by SDS-PAGE and a tertiary and quaternary configuration that is substantially similar to that displayed by the native HIV envelope glycoprotein.

23. An isolated oligomeric and glycosylated ectodomain of the HIV env-glycoprotein, said ectodomain having an N-terminal end and a C-terminal end and having aphysico-chemical configuration substantially similarto the physico-chemical configuration of a native ectodomain of said HIV env-glycoprotein comprising:

a gp120 domain of the HIV env-glycoprotein comprising said N-terminal end of said isolated ectodomain; and

a fragment of a gp41 domain of said HIV env-glycoprotein, said gp41 fragment comprising 148 amino acids and further comprising said C-terminal end of said isolated ectodomain.

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