Title:  Methods and compositions for impairing multiplication of HIV-1

United States Patent:  6,193,981

Assignee:  Thymon L.L.C. (Short Hills, NJ)

Filed:  July 10, 1998

A composition which elicits antibodies to greater than 95%, and even greater than 99%, of the known variants of HIV-1 Tat protein contains at least one peptide or polypeptide of the formula of Epitope I (based on amino acids 2-10 of HIV-1 Tat consensus sequence) and optionally one or more of a peptide or polypeptide of Epitope II (based on amino acids 41 to 51 of that sequence), of Epitope III (based on amino acids 52-62 of that sequence), or of Epitope IV (based on amino acids 62 through 72 of that sequence with a C-terminal Pro). Vaccinal and pharmaceutical compositions can contain one or more such peptides associated with carrier proteins, in multiple antigenic peptides or as part of recombinant proteins. Various combinations of the Epitope I through IV peptides can provide other compositions useful in eliciting anti-Tat antibodies which cross-react with multiple strains and variants of HIV-1 Tat protein. Vaccinal and pharmaceutical compositions can contain the antibodies induced by the peptide compositions for use in passive therapy. Diagnostic compositions and uses are described for assessing the immune status of vaccinated patients.

SUMMARY OF THE INVENTION

In one aspect, the invention provides as a novel composition comprising a peptide or polypeptide, which comprises an amino acid sequence selected from the formula referred to as Epitope I: R1-Val-Asp-Pro-Y-Leu-Glu-Pro-R2 [SEQ ID NO: 36], wherein Y is variously Arg, Lys, Ser or Asn. The N-terminal R1 may represent hydrogen (i.e., the hydrogen on the unmodified N terminal amino acid), or a lower alkyl, or a lower alkanoyl. R1 may also include a sequence of between 1 to about 5 amino acids, optionally substituted with a lower alkyl or lower alkanoyl. In one embodiment, R1 is -X-Pro-, wherein X is Glu or Asp. Preferably, R1 represents 2 amino acids. The C-terminal R2 can also represent the hydroxyl group on the C terminal amino acid or an amide. To enhance titer R2 is preferably a sequence of between 1 to about 14 additional amino acids amidated at the carboxyl terminus. In a preferred embodiment, R2 is -Trp-Lys-His-Pro-Gly-Ser- amide [SEQ ID NO: 10]. The peptides or polypeptides of these compositions are produced synthetically or recombinantly. This composition may take the form of one or more of the above-described peptides expressed as a synthetic peptide coupled to a carrier, or expressed as a multiple antigenic peptide, or the selected peptides may be expressed within a recombinantly produced protein. This composition is designed to induce antibodies reactive with greater than 95% of the known variants of the HIV-1 Tat protein.

In another aspect, the above-described composition further contains one or more additional peptide or polypeptide(s) which represent other amino acid sequences which correspond to amino acid residues 2 or 4 to 10 of an HIV-1 Tat protein. These optional amino acid sequences are described in detail below. These sequences are preferably from an HIV-1 strain with a Tat protein variant at that location.

In another aspect, the invention provides a novel composition comprising a peptide or polypeptide of the formula referred to as Epitope II: R3-Lys-X-Leu-Gly-Ile-Ser-Tyr-Gly Arg-Lys-Lys-R4 [SEQ ID NO: 37]. According to this formula, X is Gly or Ala. The N terminal R3 may represent hydrogen (i.e., the hydrogen on the unmodified N terminal amino acid), or may be a lower alkyl, or a lower alkanoyl. R3 may also include a sequence of between 1 to about 5 amino acids, optionally substituted with a lower alkyl or lower alkanoyl. The C terminal R4 may be the free hydroxyl of the C terminal amino acid, or an amide, or a sequence of one or up to about 5 additional amino acids, optionally substituted with an amide. The peptides or polypeptides of these compositions are produced synthetically or recombinantly, provided that the recombinant Epitope II peptide is situated at the C terminus of the recombinant protein. This composition may take the form of one or more of the above-described peptides expressed as a synthetic peptide coupled to a carrier, or expressed as a multiple antigenic peptide. This composition is designed to induce antibodies reactive with greater than about 95% of the known variants of HIV-1 Tat protein.

In yet a further aspect, this invention provides a composition comprising a peptide or polypeptide of the formula referred to as Epitope III: R5-Arg-Arg-X-Z-A-Y-Ser-R6 [SEQ ID NO: 38], wherein X is selected from the group consisting of Ala, Pro, Ser and Gln; wherein Y is selected from the group consisting of Asp, Asn, Gly and Ser; wherein Z is selected from the group consisting of Pro and His; and wherein A is selected from the group consisting of Gln and Pro. The N terminal R5 is hydrogen, a lower alkyl, a lower alkanoyl, or a sequence of between 1 to about 3 amino acids, optionally substituted with a lower alkyl or lower alkanoyl. In a preferred embodiment R5 is -Gln-Arg-, optionally modified as above. The C terminal R6 is either a free hydroxyl or an amide. A preferred embodiment of such a composition contains at least three Epitope III peptides, i.e., -Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp-Ser- (amino acids 54-62 of SEQ ID NO: 1), -Gln-Arg-Arg-Arg-Ala-His-Gln-Asp-Ser- (amino acids 2-10 of SEQ ID NO: 65), and -Gln-Arg-Arg-Arg-Ala-Pro-Pro-Asp-Ser- (amino acids 264-272 of SEQ ID NO: 3), optionally modified as above. Other peptides or polypeptides representative of amino acids 56-62 of Tat, but having different sequences from that of the above formula may also be included in the composition. The peptides or polypeptides of these compositions are produced synthetically or recombinantly. This composition may take the form of one or more of the above-described peptides expressed as a synthetic peptide coupled to a carrier, or expressed as a multiple antigenic peptide, or the selected peptides may be expressed within a recombinantly produced protein. This composition is designed to induce antibodies reactive with greater than about 75% of all known variants of HIV-1 Tat protein.

In still a further aspect, this invention provides a composition comprising a peptide or polypeptide of the formula referred to as Epitope IV: R7-Ser-Gln-X-His-Gln-Y-Ser-Leu-Ser-Lys-Gln-Pro-R8 [SEQ ID NO: 39], wherein X is selected from the group consisting of Asn and Thr; and wherein Y is selected from the group consisting of Ala and Val. The N terminal R7 may be hydrogen, a lower alkyl, a lower alkanoyl, or a sequence of between 1 to about 3 amino acids, optionally substituted with a lower alkyl or lower alkanoyl. The C terminal R8 may be a free hydroxyl, an amide, or a sequence of one or up to about 3 additional amino acids, optionally substituted with an amide. A preferred Epitope IV peptide is -Ser-Gln-Thr-His-Gln-Ala-Ser-Leu-Ser-Lys-Gln-Pro- [SEQ ID NO: 40]. The peptides or polypeptides of these compositions are produced synthetically or recombinantly. This composition may take the form of one or more of the above-described peptides expressed as a synthetic peptide coupled to a carrier, or expressed as a multiple antigenic peptide, or the selected peptides may be expressed within a recombinantly produced protein. This composition is designed to induce antibodies reactive with greater than 64% of all known variants of HIV-1 Tat protein.

In still another aspect, this invention provides composition described above that contains peptides or polypeptides which comprise one or more Epitope I peptides in combination with one or more Epitope II peptides, and/or one or more Epitope III peptides, and/or one or more Epitope IV peptides. Such compositions can combine appropriate Epitope peptides, so as to provide for a composition than induces antibodies reactive with greater than about 99% of all known HIV-1 Tat proteins.

In yet a further aspect, the invention provides a synthetic gene which encodes sequentially a peptide or polypeptide that contains at least one Epitope I amino acid sequence defined above, optionally with a carboxy terminal Epitope II peptide, or contains at least two Epitope I amino acid sequences. The synthetic gene may contain each amino acid sequence separated by a spacer sequence, or may express each peptide/polypeptide in an open reading frame with a carrier protein. The synthetic gene may be separated from the carrier protein by a spacer if the spacer is fused to an Epitope I sequence, leaving an Epitope II sequence at the carboxy terminus of the recombinant protein. Further embodiments include multiple Epitope I peptides fused together and to the carrier protein.

In yet a further aspect, the invention provides a synthetic molecule, e.g., a vector, comprising the above-described synthetic gene, operatively linked to regulatory nucleic acid sequences, which direct and control expression of the product of the synthetic gene in a host cell.

In another aspect, the invention provides a recombinant virus which contains the above described synthetic gene or synthetic molecule, which virus is capable of expressing multiple copies of the product of the gene or molecule in a host cell. The virus is non-pathogenic to humans.

In yet another aspect, the invention provides a commensal bacterium which contains the above described synthetic gene or synthetic molecule, which bacterium is capable of expressing multiple copies of the product of the gene or molecule and inducing antibodies in a mammalian host.

In still a further aspect, the invention provides an isolated antibody composition which is directed against a peptide or polypeptide of the compositions described above. Antibodies may also be obtained against multiple components of the compositions described above. This antibody is produced by immunizing a mammal with a peptide/polypeptide composition of the invention, a synthetic gene or synthetic molecule of the invention; a recombinant virus or commensal bacterium of the invention; and isolating and purifying antibody from said immunized mammal. Alternatively, the antibody may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, or mixtures thereof.

Thus, another aspect of the invention is a pharmaceutical composition useful for inducing antibodies that react with greater than 95%, and preferably greater than 99%, of the known HIV-1 Tat proteins. These induced antibodies can impair the multiplication of HIV-1. The pharmaceutical composition comprises at least one of the recombinant or synthetic peptide/polypeptide compositions described above; the synthetic gene/molecule described above; the recombinant virus described herein; or the commensal bacterium described herein, in a pharmaceutically acceptable carrier.

Still a further aspect of the invention is a pharmaceutical composition useful for impairing the multiplication of HIV-1, this composition containing an above described antibody composition.

In yet a further aspect of the invention, a method for reducing the viral levels of HIV-1 involves exposing a human to antibody-inducing pharmaceutical compositions described above, actively inducing antibodies that react with most HIV-1 Tat proteins, and impairing the multiplication of the virus in vivo. This method is appropriate for an HIV-1 infected subject with a competent immune system, or an uninfected or recently infected subject. The method induces antibodies which react with HIV-1 Tat proteins, which antibodies reduce viral multiplication during any initial acute infection with HIV-1 and minimize chronic viremia which leads to AIDS.

In still another aspect, the invention provides a method for reducing the viral levels of HIV-1 by administering to a human, who is incapable of mounting an effective or rapid immune response to infection with HIV-1, a pharmaceutical composition containing the antibody compositions described above. The method can involve chronically administering the composition.

Yet other aspects of the invention include methods for producing the compositions described above, as well as host cells transfected with such compositions.

Still another aspect of this invention is a kit useful for the measurement and detection of titers and specificities of antibodies induced by vaccination with the compositions described above. The kit of the invention includes peptides of Epitopes I through IV, and coated solid supports, a labelled reagent for detecting the binding of antibodies to these peptides, and miscellaneous substrates and apparatus for evoking or detecting the signals provided by the labels, as well as conventional apparatus for taking blood samples, appropriate vials and other diagnostic assay components.

In yet a further aspect, the invention provides a method for detecting the titers and reactivity patterns of antibodies in subjects vaccinated with the compositions of this invention. The method includes the steps of incubating dilutions of the subject's biological fluid, e.g. serum, with plates or beads on which are bound one or more peptides of the Epitopes I through IV, washing away unbound biological materials, and measuring any antibody binding to the peptides with labeled reagent, e.g., an anti-human immunoglobulin to which is associated an enzyme. Depending on the type of label employed, the signal produced by the label may be evoked by further adding a substrate which reacts with the enzyme, e.g., producing a color change. Other conventional labels may also be incorporated into this assay design.

Claim 1 of 55 Claims

What is claimed is:

1. A composition comprising at least two variants of a peptide or polypeptide of the formula R1-Val-Asp-Pro-Y-Leu-Glu-Pro-R2 SEQ ID NO: 36,

wherein each variant has a different amino acid residue at Y,

wherein Y is selected from the group consisting of Arg, Lys, Ser and Asn;

wherein R1 is selected from the group consisting of hydrogen, a lower alkyl, a lower alkanoyl, a sequence of between 1 to about 5 additional amino acids, and a sequence of 1 to about 5 amino acids with at least one of said additional amino acids being substituted with a lower alkyl or lower alkanoyl;

wherein R2 is selected from the group consisting of a free hydroxyl, an amide, a sequence of one to about 14 additional amino acids, and a sequence of 1 to about 14 amino acids with at least one of said additional amino acids being substituted with an amide.

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