WO2000078790A2 - Procedes pour inhiber la replication du vih - Google Patents

Procedes pour inhiber la replication du vih Download PDF

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WO2000078790A2
WO2000078790A2 PCT/US2000/016680 US0016680W WO0078790A2 WO 2000078790 A2 WO2000078790 A2 WO 2000078790A2 US 0016680 W US0016680 W US 0016680W WO 0078790 A2 WO0078790 A2 WO 0078790A2
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group
seq
amino acid
lysine
leucine
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PCT/US2000/016680
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WO2000078790A3 (fr
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Kumar Visvanathan
John B. Zabriskie
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The Rockefeller University
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Publication of WO2000078790A3 publication Critical patent/WO2000078790A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This invention relates to methods for protecting against, or reducing the severity of, HIV infections. More particularly it relates to the use of peptides, derived from homologous sequences of the family of staphylococcal and streptococcal pyrogenic toxins to reduce, inhibit or eliminate T-cell proliferation and HIV replication.
  • the peptides useful in the invention are described herein and also in U.S. application Serial No. 08/838,413 filed April 7, 1997, and its related applications, including U.S. Application Serial No. 09/168,303 filed October 7, 1998; International Application PCT/US98/06663, filed April 1, 1998; and U.S. Application Serial No. 09/335,581, filed June 18, 1999. The disclosures of these applications are specifically incorporated herein by reference.
  • peptides may also be useful to induce antibodies which reduce, inhibit or eliminate T-cell proliferation and HIV replication.
  • the invention also relates to methods of using these peptides, or antibodies thereto, prevent, treat, ameliorate or protect against the deleterious effects of HIV infection or replication.
  • NEF is one of the earliest proteins synthesized by the HIV virus after it enters a CD4 cell and a number of studies indicate that it protects infected cells from killing by T cells (Collins, Chen et al. 1998). Though its complete role in HIV infection is not completely understood, its importance in the pathogenesis of infection is clear as seen in the results of Learmont et al. (1992), discussed above, as well as in the results of a study that showed that adult macaques infected with a Simian Immunodeficiency Virus (SIV) vaccine with a deletion in its NEF protein were protected against a pathogenic strain of SIV (Shibata, Maldarelli et al. 1997). In addition it has been suggested that the NEF gene of the cloned SIV virus that had undergone attenuating mutations during propagation in tissue culture were
  • NEF is a myristylated protein of 206 amino acids and acts as a superantigen binding to a particular region of the MHC complex of antigen presenting cells. This brings the antigen presenting cell and the CD4 cell closer together and stimulates the production of Interleukin 2 (IL-2) and Interferon gamma (IFN- ⁇ ) with activation and proliferation of the CD4 cell and subsequent up-regulation of virus production. With the presence of the NEF protein the virus is able to help exhaust the immune defenses of the host and cause AIDS.
  • IL-2 Interleukin 2
  • IFN- ⁇ Interferon gamma
  • NEF protein binds to the MHC to stimulate T cell proliferation and at the same site as SEA and SEE (Torres and Johnson 1994).
  • the sequence of a NEF peptide which has been demonstrated to bind to MHC II is DWQNYTPGPGVRYPLTFGWCYKLVPVEPDKVEEANKGE (SEQ ID NO: 32) which is located at positions 123-160 of the Nef protein. (Torres and Johnson (1994)).
  • Other superantigens have been observed in another retrovirus, Mouse Mammary Tumor Virus (MMTV).
  • MMTV-1 peptide which binds to MHC is DSFNNSSVQDYNLNDSENSTFLLGQGPQPTSSYKPHR LCPSEIE (SEQ ID NO: 33), which is located at positions 76-1 19 of the protein.
  • Baskar and colleagues have shown that SIV induces proliferation of human peripheral blood mononuclear cells (PBMC). They have extended this observation to show that SIV induces proliferation of PBMC from pigtailed macaques. This proliferative response was markedly inhibited by monoclonal antibodies (mAbs) against human class I and II MHC. These antibodies also strongly inhibited viral infectivity. (Baskar, Nagel et al. 1997).
  • T cell stimulation due to Nef is not the only mechanism of polyclonal activation of CD4 T cells for HIV replication.
  • Evidence also points to a superantigen encoded by cytomegalovirus (CMV) that expands V ⁇ l2-bearing T cells, thereby enhancing replication in CMV-infected individuals. Therefore, versatility in polyclonal CD4 T cell expansion via the endogenous superantigen NEF and exogenous superantigens such as that of CMV probably play an important role in HIV pathogenesis.
  • the control of the mitogenic activity of these substances should help reduce the viral load in HIV-infected individuals. (Torres, Tanabe et al., 1996a).
  • Novel peptides developed to have consensus regions of two highly conserved regions of the staphylococcal and streptococcal pyrogenic exotoxins are described in U.S. Application Serial No. 08/838,413, filed April 7, 1997, and its related applications.
  • a polymeric peptide (peptide 6348) was synthesized and used to immunize rabbits to produce high titer antibodies to the peptide.
  • the antibodies also protected against T-cell proliferation of these toxins in an in vitro blastogenesis assay using human mononuclear cell populations, and also completely blocked the lethal effects of staphylococcal toxin SEB and streptococcal pyrogenic toxin SPEA in vivo when the antibodies were mixed with the toxin prior to injection.
  • the present invention relates to the realization that these peptides and antibodies thereto might be useful in other situations in which superantigens are involved.
  • the peptide 6343 is demonstrated to block T-cell proliferation by MMTV superantigen (as measured in an IL2-assay) and to block stimulation of human T-cells by HIV Nef protein.
  • the peptide is also shown to inhibit HIN replication in vitro.
  • the present invention provides methods of using peptides and/or conjugates thereof having amino acid sequences developed to have consensus sequences for two conserved regions of the staphylococcal enterotoxins and streptococcal pyrogenic exotoxins (hereinafter called "region 1 " and "region 2") for reducing, inhibiting, or eliminating proliferation of mononuclear cells in the presence of HIV Nef superantigen or other superantigen and/or for reducing, inhibiting, or eliminating HIV replication. Therefore, these peptides and/or conjugates thereof are expected to be capable of preventing, ameliorating or treating diseases related to HIV infection and/or replication.
  • the peptides and/or conjugates thereof are capable of inducing antibodies that are expected to be capable of reducing, inhibiting or eliminating proliferation of mononuclear cells in the presence HIV Nef superantigen or other superantigen and/or reducing, inhibiting or eliminating HIV replication.
  • Antibodies may be induced by administration of a pharmaceutical composition and/or vaccine containing a composition comprising a peptide and/or conjugate thereof derived from one or both of the two conserved regions described herein, or a structurally and or immunologically related antigen. These antibodies are expected to be capable of preventing, ameliorating or treating diseases related to HIV infection and/or replication.
  • the invention provides methods of preventing and treating symptoms associated with the stimulation of T cells by Nef-1 or other superantigen and associated HIV replication.
  • Such methods include, for example, administering to an individual who is suspected of having an infection with HIV, or is diagnosed as having an infection with HIV, or is at risk for developing an infection with HIV, a compound comprising at least one of the consensus amino acid sequences of this invention in an amount sufficient to inhibit Nef or other superantigen stimulation of T-cells, preferably an amount sufficient to reduce, inhibit or eliminate the deleterious effects of the stimulation of T-cells by Nef or other superantigen, including HIV replication.
  • Such methods also include administering to an individual a compound comprising at least one of the consensus amino acid sequences of this invention in an amount sufficient to elicit the production of antibodies to the amino acid sequences.
  • an individual at risk for developing HIV infection, or an individual with symptoms of HIN infection, or with a diagnosis of HIV infection may be treated by administering to such individual a composition comprising at least one of the peptides useful in this invention and/ or carrier-conjugate thereof.
  • an individual at risk for developing HIV infection may be treated by administering to such individual antibodies which have been generated in a mammal immunized with a composition comprising at least one of the peptides useful in this invention and/or carrier-conjugate thereof.
  • Another object of the invention is to provide a method of using isolated and purified nucleic acids encoding the amino acid sequences of the invention, as well as suitable expression systems, vector components and transformed host cells containing those nucleic acids, to prepare peptides useful in the methods of the invention.
  • Vaccines and pharmaceutical compositions comprising nucleic acids, peptides or carrier conjugates thereof, or antibodies induced by the peptides and carrier-conjugates thereof, and a physiologically acceptable carrier, for use in the methods of this invention, are also considered to be part of the invention.
  • Figure 1 Schematic drawing of unbound HIV-Nef protein and HIV- Nef protein bound to the MHC II complex of a HIV positive T-cell attached to the CD4 molecule of an uninfected T-cell.
  • activation of uninfected T cells occurs by Nef binding to the MHC II complex of the uninfected T-cell.
  • peptide 6343 inhibits T cell proliferation and viral replication by binding to the MHC II molecule, which in turn inhibits Nef binding, leading to decreased T-cell activation and diminished viral replication. HIV cells are not able to infect non-activated cells.
  • Figure 2 Graph showing the results of an MMTV IL-2 assay to determine inhibition of proliferation of peripheral blood mononuclear cells (PBMC) by MMTV by O ⁇ g, 75 ⁇ g, lOO ⁇ g, or 150 ⁇ g of peptide 6343 per well.
  • PBMC peripheral blood mononuclear cells
  • FIG. 3 Bar graph showing the results of blastogenesis assays of human peripheral blood mononuclear cell (PBMC) populations stimulated by 5 ug HIV-1 Nef in the presence or absence of 150 ug monomer peptide 6343.
  • NM normal media;
  • PHA phytohemagglutinin.
  • PBMCs include T-cell, ⁇ -cells and monocytes.
  • Figure 4 Bar graph showing inhibition of HIV replication by peptide 6343 as determined by measuring the level of p24 antigen.
  • the human CD4 HTLV1 transformed T-cell line MT4 was used and a laboratory strain of HIV virus
  • FIG. 5 Graph of the P24 levels showing inhibition of HIV replication by peptide 6343 over a 10 day period in peripheral mononuclear cells obtained from a patient with known HIV infection. The viral load for this patient was 126,000 virus particles per ml.
  • Figure 6 Graph showing P27 levels over a 15 day period in SIV infected Macaque peripheral mononuclear cells with and without the addition of peptide 6343. Triangles show P27 values in the cells infected with SIV with peptide added at day 0 and 3 days. Squares show SIV production in a NEF negative cell line. Diamonds show P27 levels in wild-type virus.
  • the first consensus sequence (“GCG consensus #1 ”) has the amino acid sequence YGG(LIV)TXXXXN, which is rewritten herein as YGGX1TX2X3X4X 5 N (SEQ ID NO: 1), wherein X, is selected from the group consisting of L, I, or V; and X , X 3 , * and X are each independently selected from the group consisting of any amino acid.
  • This pattern is present in the staphylococcal enterotoxins and streptococcal pyrogenic exotoxins, but not in TSST-1.
  • the sequence begins immediately at the COOH -terminal side of the cysteine loop.
  • the second consensus sequence (“GCG consensus #2”) has the amino acid sequence KXX(LIV)XXXX(LIV)DXXXXRXXLXXXXX(LIV)Y, rewritten herein as
  • X 22 and X 23 are each independently selected from the group consisting of any amino acid. This pattern is present in the staphylococcal enterotoxins, streptococcal pyrogenic exotoxins, and TSST-1.
  • One object of the invention is to provide methods of using compositions comprising peptides having amino acid sequences based on these two conserved regions of the staphylococcal enterotoxins and streptococcal pyrogenic toxins. These peptides may be used for eliciting an immunogenic response in mammals, including responses which provide protection against, or reduce the severity, of HIV infection. These peptides may also be useful to protect against, or ameliorate the effects of, autoimmune diseases which are associated with, or are the result of, the presence of HIV.
  • peptides useful in the invention are those derived from either one or both of the following two consensus sequences:
  • X 8 , X ⁇ 3 and X 24 are each independently selected from the group consisting of L, I and V, and X 6 , X 7 , X 9 , X ]0 , Xn, X12, Xi4, X15, Xi6, X ⁇ , Xi8, X19, X 2 0, X 21 , X 22 and X 23 are each independently selected from the group consisting of any amino acid.
  • a preferred consensus sequence from Region 1 has the amino acid sequence X 25 X 26 YGGX ⁇ TX 2 X 3 X 4 X N (SEQ ID NO: 28), wherein Xi is selected from the group consisting of L, I, and V; X 2 , X-t and X 5 are each independently selected from the group consisting of any amino acid; and X 3 , X 2 5 and X 2 6 are each independently selected from the group consisting of any amino acid and of no amino acid; but preferably Xi is selected from the group consisting of I and V; X 2 is selected from the group consisting of L, E, K, P and N; X 3 is selected from the group consisting of H and A and no amino acid; X is selected from the group consisting of D, N, E, Q, and H; X 5 is selected from the group consisting of N, G, S, and R; X 5 is selected from the group consisting of C and Y and no amino acid; and
  • a preferred consensus sequence from region 2 has the amino acid sequence:
  • Table 1 lists the amino acids that are found at each of the variable positions in the sequences, and the number of times they appear at that position: Table 1
  • Xi, X 8 , X ⁇ 3 and X2 may each independently be selected from the group consisting of L, I and V; X 2 , X 3 , X 4 , Xs, X&, X7, X9, Xio, Xn, X12, X14, Xis, Xi6, X ⁇ , Xis X19, X20, X21.
  • X22, X23, X25 and X26 may each independently be any amino acid;
  • X 3 , X 25 and X 26 may also each independently be no amino acid; and
  • X 27 is selected from the group consisting of L and Y.
  • the amino acids present at the positions Xi to X27 in the toxins listed in Table 1 are preferred for those positions, and the amino acids present most often at those positions in the toxins listed in Table 1 are more preferred.
  • H histidine
  • A alanine
  • inosine (I) is used at position X !6 instead of the more frequently found alanine (A).
  • the preferred consensus is larger (consensus #la), and usually includes a C in the first position (X 25 ).
  • the second residue (X 26 ) is most often a M, but this can vary.
  • H is the most highly conserved.
  • the eleventh residue (X 5 ) is most often a G.
  • the preferred consensus (consensus #2a) is much more highly conserved than suggested by the GCG program, especially if one excludes TSST-1 sequences from consideration, as follows:
  • the second position (X 6 ) is more highly conserved than suggested, being almost exclusively a K;
  • the fourth residue (X 8 ) is always a V followed exclusively by a T in the fifth position (X 9 );
  • the sixth position (Xio) is somewhat variable; but the seventh position (Xi i) is always a Q, followed by E (X ⁇ 2 ).
  • the next position is almost always an L (X 13 ), and the second to last position (X 24 ) is almost always an L.
  • Xi is V or I, preferably V;
  • X 2 is L, E, K, P or N, preferably E or L;
  • X4 is D, N, E, Q or H, preferably E.
  • X 7 is N, K, S, E, M, I or Q, preferably N;
  • Xio is V, A, L, F or I, preferably V;
  • Xi is L, Y, I, A, F or C, preferably Y;
  • X 15 is Q, L, K or E, preferably K;
  • Xi 6 is A, T, I or V, preferably I;
  • X 17 is R, H, N or K, preferably K;
  • Xis is Y, F, I, L or Q, preferably Y;
  • X 1 9 is Q, V, I, H, S, T or M, preferably V;
  • X 20 is E, K, N, D, G, S or Q, preferably D;
  • X21 is K, N, D, R or I, preferably N;
  • X22 is Y, K, L, F or H, preferably K;
  • X 23 is N, K, G or Q, preferably K.
  • X27 is L or Y, preferably L.
  • Peptides exemplified herein are CMYGGVTEHEGN (SEQ ID NO: 3), CMYGGVTEHEGNGC* (SEQ ID NO: 5), K-KNNTVQELDYKIRKYLVDNKKLY (SEQ ID NO: 4), CGKKNVTVQELDYKIRKYLVDNKKLYGC* (SEQ ID NO: 6), CMYGGVTEHEGNKKNVTVQELDYKIRKYLVDNKKLY (SEQ ID NO: 7) and CMYGGVTEHEGNKKNVTVQELDYKIRKYLVDNKKLYGC* (SEQ ID NO: 8), wherein an asterisk indicates that the peptide is a randomly cross-linked polymer.
  • the exemplified polymer peptides are at least 6,000 to 8,000 daltons.
  • the average size of the exemplified polymer peptides is about 12,000 to 15,000 daltons.
  • Small peptides and/or contaminants may be removed by dialysis or other methods available in the art.
  • larger aggregates may be removed using, e.g., a 0.25 micron filter, which can also be used to sterilize the peptides.
  • amino acids cysteine and methionine, "CM” are present at the amino terminus of the exemplified region 1 peptides since those amino acids are most often found in that position in nature.
  • amino acids cysteine and glycine, "CG” and “GC” are used at the amino and/or carboxy- termini of some of the exemplified region 2 peptides.
  • the amino acid cysteine “C” is used to facilitate cross-linking through the formation of disulfide bonds.
  • the amino acid glycine, "G” is used as a spacer residue.
  • the preferred peptides useful in the invention are those which exclude full length native toxin molecules.
  • the preferred peptides useful in this invention are not toxic, but toxic peptides maybe useful in this invention, for example, in eliciting antibodies in a non-human system.
  • the most preferred peptides useful in the invention do not contain amino acid sequences in the sequence in which they are found in any particular native toxin molecule.
  • the methods of the present invention encompass using monomers of the peptides derived from either one or both of the two consensus regions described herein. These monomers may comprise one or more sequences derived from either region 1 or region 2 or both, such as consensus sequences #1 and #2, preferably consensus sequences #la and/or #2a, more preferably consensus sequences #lb and/or #2b, most preferably one or more of the exemplified consensus sequence peptides. If the monomer contains more than one consensus sequence, these sequences may be immediately adjacent to each other or separated by a linker. In addition, different orientations of the peptides are within the scope of this invention. Furthermore, the order of the consensus peptides within the full peptide may be variable.
  • the methods of the present invention also encompass using homogeneous or heterogeneous polymers of the peptides disclosed herein (e.g., concatenated, cross-linked and/or fused identical peptide units or concatenated, cross-linked and/or fused diverse peptide units), and mixtures of the peptides, polymers, and/or conjugates thereof.
  • homogeneous or heterogeneous polymers of the peptides disclosed herein e.g., concatenated, cross-linked and/or fused identical peptide units or concatenated, cross-linked and/or fused diverse peptide units
  • Linkers may, for example, be simply peptide bonds, or may comprise amino acids, including amino acids capable of forming disulfide bonds, but may also comprise other molecules such as, for example, polysaccharides or fragments thereof.
  • sequences derived from consensus region 1 and consensus region 2 may be immediately adjacent to each other, linked by peptide bonds, (see, e.g., SEQ ID NO: 7) and/or connected via amino acid linkers capable of forming di-sulfide bonds via cysteine residues (see, e.g., SEQ ID NO: 8).
  • the sequences of region 1 and region 2 are separated by about 27 amino acids.
  • the linkers are additional amino acids, they are most preferably 1 to 27 amino acids in length, although longer linkers may also be used in accordance with this invention.
  • linkers for use with this invention may be chosen so as to contribute their own immunogenic effect which may be either the same, or different, than that elicited by the consensus sequences of the invention.
  • linkers may be bacterial antigens which also elicit the production of antibodies to infectious bacteria.
  • the linker may be a protein or protein fragment of an infectious bacteria, or a bacterial polysaccharide or polysaccharide fragment.
  • a peptide useful in the invention includes any substituted analog or chemical derivative of a peptide derived from one or both of the two consensus regions described herein, most preferably of the exemplified peptides described herein, so long as the peptide is capable of inhibiting binding of Nef superantigen to the MHC complex; inhibiting the stimulation of human mononuclear cells, including T-cells, in the presence of HIV Nef protein; inhibiting HIN replication, and/or infection; or eliciting the production of antibodies which are capable of inhibiting the stimulation of human mononuclear cells, including T-cells, by HIN ⁇ ef protein and, preferably, are capable of inhibiting HIV replication and/or infection.
  • a peptide can be subject to various changes that provide for certain advantages in its use.
  • D amino acids can be substituted for L amino acids to increase in vivo stability of the peptides, while still retaining biological activity.
  • retro-inverso peptides which contain ⁇ H-CO bonds instead of CO- ⁇ H peptide bonds, are much more resistant to proteolysis than L-peptides.
  • they have been shown to mimic natural L-peptides with respect to poly- and monoclonal antibodies (48).
  • peptides having at least one D amino acid on the amino terminal and/or carboxy terminal end of the molecule and which retain biological activity are considered useful in the methods of the invention.
  • retro-inverso peptides which contain one or more of the amino acid sequences of the invention and which retain biological activity are also considered useful in the methods of the invention.
  • the peptides are useful for providing active immunization for the prevention or treatment of disease related to HIV infection or replication, or for preparation of antibodies as a passive immunization therapy.
  • the peptides are designed to induce antibodies for use in therapy to increase resistance to, prevent and/or treat disease related to HIV infection or replication.
  • the peptides may also be useful to protect against, or ameliorate the effects of, autoimmune diseases which are associated with, or are the result of, the presence of HIV.
  • the peptide may be mixed with an adjuvant.
  • the peptide also may be bound to a non-toxic non-host protein carrier to form a conjugate or it may be bound to a saccharide carrier and/or a non-toxic non-host protein carrier to form a conjugate.
  • the molecular weight of the peptide monomers having one consensus sequence of the invention range from about 1000 to 5000 daltons. Such lower molecular weight species are useful themselves to inhibit superantigen induced mononuclear cell, especially T cell, proliferation and/or reduce, inhibit or eliminate the deleterious effects of HIV infection in vivo, either when used alone or in combination with another form of therapy, e.g., nucleoside analogs and/or protease inhibitors.
  • such lower molecular weight species or, more preferably, may be used as haptens conjugated to a larger carrier molecule, such as, for example, a protein.
  • a carrier molecule such as, for example, a protein.
  • the molecular weight of the peptide alone, or when conjugated to a carrier, or in the presence of an adjuvant, is related to its immunogenicity.
  • the peptide may vary in molecular weight in order to enhance its antigenicity or immunogenicity.
  • the molecular weight of the peptide, in polymeric form is greater than about 6000 to 8000 daltons, with an average weight of 12,000 to 15,000 daltons.
  • the total size of the peptide is only limited to its ability to be physiologically tolerated.
  • the invention also relates to the use of isolated and purified nucleic acid molecules which code for the peptides to produce the encoded peptides.
  • the encoded peptides may be monomers, polymers or linked to other peptide sequences (e.g., they may be fusion proteins).
  • Other features of the invention include the use of vectors which comprise the nucleic acid molecules of the invention operably linked to promoters, as well as cell lines, such as prokaryotic (e.g., E. coli) and eukaryotic (e.g., CHO and COS) cells transfected with the nucleic acid molecules of the invention.
  • Vectors and compositions for enabling production of the peptides in vivo, i.e., in the individual to be treated or immunized, are also within the scope of this invention.
  • nucleic acids encoding the peptides of the invention can be introduced into a vector such as a plasmid, cosmid, phage, virus or mini- chromosome and inserted into a host cell or organism by methods well known in the art.
  • the vectors containing these nucleic acids can be utilized in any cell, either eukaryotic or prokaryotic, including mammalian cells (e.g., human (e.g., HeLa), monkey (e.g., COS), rabbit (e.g., rabbit reticulocytes), rat, hamster (e.g., CHO and baby hamster kidney cells) or mouse cells (e.g., L cells), plant cells, yeast cells, insect cells or bacterial cells (e.g., E. coli).
  • mammalian cells e.g., human (e.g., HeLa), monkey (e.g., COS), rabbit (e.g., rabbit reticulocytes), rat, hamster (e.g., CHO and baby hamster kidney cells) or mouse cells (e.g., L cells), plant cells, yeast cells, insect cells or bacterial cells (e.g., E. coli).
  • mammalian cells e.g., human (e.g
  • the vectors which can be utilized to clone and/or express these nucleic acids are the vectors which are capable of replicating and/or expressing the nucleic acids in the host cell in which the nucleic acids are desired to be replicated and or expressed. See, e.g., F. Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley- Interscience (1992) and Sambrook et al. (1989) for examples of appropriate vectors for various types of host cells. Strong promoters compatible with the host into which the gene is inserted may be used. These promoters may be inducible. The host cells containing these nucleic acids can be used to express large amounts of the protein useful in pharmaceuticals, diagnostic reagents, vaccines and therapeutics.
  • the nucleic acids could be used, for example, in the production of peptides for vaccines and therapies for HIV related diseases.
  • vectors expressing high levels of peptide can be used in immunotherapy and immunoprophylaxis, after expression in humans.
  • Such vectors include retroviral vectors and also include direct injection of DNA into muscle cells or other receptive cells, resulting in the efficient expression of the peptide, using the technology described, for example, in Wolff et al., Science 247:1465-1468 (1990), Wolff et al, Human Molecular Genetics l(6):363-369 (1992) and Ulmer et al., Science 259:1745-1749 (1993). See also, for example, WO 96/36366 and WO 98/34640.
  • antibodies which react with peptides of the invention. These antibodies will be useful for passive immunization therapy to increase resistance to HIV infection or prevent
  • the antibodies may also be useful to protect against, or ameliorate the effects of, autoimmune diseases which are associated with, or are the result of, the presence of HIV.
  • the peptides useful in the invention may be prepared by synthetic methods or by recombinant DNA methods, as known in the art and as described in U.S. Application 08/838,413, filed April 7, 1997 and its related applications, which is reproduced hereinbelow in Example 1 for convenience.
  • compositions useful in this invention contain a pharmaceutically and/or therapeutically effective amount of at least one peptide and/or carrier thereof, antibody, or nucleic acid encoding a peptide useful in the methods of the invention, or combination thereof.
  • the effective amount or peptide per unit dose is an amount sufficient to inhibit T-cell proliferation and HIV replication, in particular T-cell proliferation or HIV replication stimulated by HIV
  • the effective amount of peptide per unit dose is an amount sufficient to prevent, treat or protect against deleterious symptoms or diseases related to HIV infection or replication.
  • symptoms and diseases include diarrhea, pneumonia, Karposi's
  • the effective amount of peptide per unit dose depends, among other things, on the species of mammal inoculated, the body weight of the individual and the chosen inoculation regimen, as is well known in the art.
  • inocula for a human or similarly sized mammal typically contain peptide concentrations of 100 to 500 mg/kg body weight of the mammal per inoculation dose.
  • any route of administration may be used that delivers sufficient peptide or antibody to HIV infected or potentially infected tissue.
  • the route of inoculation of the peptide will be subcutaneous, intravenous, or nasal. The dose is administered at least once.
  • the pharmaceutical composition contains an effective, immunogenic, amount of peptide of the invention.
  • the effective amount of peptide per unit dose sufficient to induce an immune response depends, among other things, on the species of mammal inoculated, the body weight of the mammal and the chosen inoculation regimen, as well as the presence or absence of an adjuvant, as is well known in the art.
  • Inocula typically contain peptide concentrations of about 1 micro gram to about 1000 micrograms per inoculation (dose), preferably about 3 micrograms to about 100 micrograms per dose, most preferably about 5 micrograms to 50 micrograms. The use of higher amounts is envisaged.
  • dose micrograms per inoculation
  • Standard procedures to determine dose response relationships known to those skilled in the art may be used to determine optimum doses of peptide to be used either to inhibit mononuclear cell proliferation, HIV replication or infection, or to raise antibodies to inhibit mononuclear cell proliferation, HIV replication or infection, and/or prevent or treat symptoms or diseases related to HIV replication or infection.
  • unit dose refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined quantity of active material (e.g., peptide, antibody or nucleic acid) calculated to produce the desired immunogenic effect in association with the required diluent.
  • active material e.g., peptide, antibody or nucleic acid
  • Inocula are typically prepared as a solution in a physiologically acceptable carrier such as saline, phosphate-buffered saline and the like to form an aqueous pharmaceutical composition.
  • a physiologically acceptable carrier such as saline, phosphate-buffered saline and the like to form an aqueous pharmaceutical composition.
  • the peptides of the invention are generally administered with a physiologically acceptable carrier or vehicle therefor.
  • a physiologically acceptable carrier is one that does not cause an adverse physical reaction upon administration and one in which the peptides are sufficiently soluble and retain their activity to deliver a therapeutically effective amount of the compound.
  • the therapeutically effective amount and method of administration of a peptide of the invention may vary based on the individual patient, the indication being treated and other criteria evident to one of ordinary skill in the art.
  • a therapeutically effective amount of a peptide of the invention is one sufficient to inhibit superantigen stimulation of mononuclear cells, especially T-cells, and is preferably an amount sufficient to decrease or completely inhibit HIV infection or replication without causing significant side effects such as non-specific T cell lysis or organ damage.
  • the route(s) of administration useful in a particular application are apparent to one or ordinary skill in the art.
  • Routes of administration of the peptides include, but are not limited to, parenteral, and direct injection into an affected site.
  • Parenteral routes of administration include but are not limited to intravenous, intramuscular, intraperitoneal, subcutaneous, and nasal.
  • the route of inoculation of the peptides of the invention is typically parenteral and is preferably intramuscular, sub-cutaneous and the like.
  • compositions of the peptides described above suitable for parenteral administration including, but not limited to, pharmaceutically acceptable sterile isotonic solutions.
  • pharmaceutically acceptable sterile isotonic solutions include, but are not limited to, saline and phosphate buffered saline for nasal, intravenous, intramuscular, intraperitoneal, subcutaneous or direct injection into another area.
  • a system for sustained delivery of the peptides of the invention may also be used.
  • a delivery system based on containing a peptide in a polymer matrix of biodegradable microspheres may be used (Jeong, Bae et al., 1997).
  • One such polymer matrix includes the polymer poly(lactide-co-glycolide) (PLG).
  • PLG is biocompatible and can be given intravenously or orally.
  • the encapsulated protein is released by a complex process involving hydration of the particles and drug dissolution. The duration of the release is mainly governed by the type of PLG polymer used and the release of administering modifying excipients (Bartus, Tracy et al., 1998).
  • the dose is administered at least once.
  • at least one booster dose may be administered after the initial injection, preferably at about 4 to 6 weeks after the first dose, in order to increase the antibody level. Subsequent doses may be administered as indicated.
  • antibody titers may be determined. In most instances it will be sufficient to assess the antibody titer in serum or plasma obtained from such an individual. Decisions as to whether to administer booster inoculations or to change the amount of the composition administered to the individual may be at least partially based on the titer.
  • the titer may be based, e.g., on an immunobinding assay which measures the concentration of antibodies in the serum which bind to a specific antigen, e.g., peptide.
  • the ability to neutralize in vitro biological effects of the HIV Nef protein may also be assessed to determine the effectiveness of the treatment.
  • the assessment can also use surrogate markers of HIV replication including P24 antigen and viral load determined by PCR in the patient so treated.
  • Antibodies is used herein to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and portions of an immunoglobulin molecule, including those portions known in the art as Fab, Fab', F(ab') 2 and F(v) as well as chimeric antibody molecules.
  • An antibody useful in the present invention is typically produced by immunizing a mammal with an immunogen or vaccine containing one or more peptides of the invention, or a structurally and/or antigenically related molecule, to induce, in the mammal, antibody molecules having immunospecificity for the immunizing peptide or peptides.
  • the peptide(s) or related molecule(s) may be monomeric, polymeric, conjugated to a carrier, and/or administered in the presence of an adjuvant.
  • the antibody molecules may then be collected from the mammal if they are to be used in immunoassays or for providing passive immunity.
  • the antibody may be polyclonal or monoclonal. Monoclonal antibodies may be produced by methods known in the art. Portions of immunoglobulin molecules may also be produced by methods known in the art.
  • the antibody may be contained in various carriers or media, including blood, plasma, serum (e.g., fractionated or unfractionated serum), hybridoma supematants and the like.
  • the antibody is isolated to the extent desired by well known techniques such as, for example, by using DEAE Sephadex, or affinity chromatography.
  • the antibodies may be purified so as to obtain specific classes or subclasses of antibody such as IgM, IgG, IgA, IgGi, IgG2,
  • IgG 3 IgG 4 and the like.
  • Antibody of the IgG class is preferred for purposes of passive protection.
  • the presence of the antibodies of the present invention can be determined by various assays.
  • Assay techniques include, but are not limited to, immunobinding, immunofluorescence (IF), indirect immunofluorescence, immunoprecipitation, ELISA, agglutination and Western blot techniques.
  • the assays which use the antibodies to detect the presence of peptide in a sample involve contacting the sample with at least one of the antibodies under conditions which will allow the formation of an immunological complex between the antibody and the peptide that may be present in the sample.
  • the formation of an immunological complex if any, indicating the presence of the peptide in the sample, is then detected and measured by suitable means.
  • suitable means include, but are not limited to, radioimmunoassays, (RIA), ELISA, indirect immunofluorescence assay, Western blot and the like.
  • the antibodies may be labeled or unlabeled depending on the type of assay used.
  • Labels which may be coupled to the antibodies include those known in the art and include, but are not limited to, enzymes, radionucleotides, fluorogenic and chromogenic substrates, cofactors, biotin/avidin, colloidal gold and magnetic particles. Modification of the antibodies allows for coupling by any known means to carrier proteins or peptides or to known supports, for example, polystyrene or polyvinyl microliter plates, glass tubes or glass beads and chromatographic supports, such as paper, cellulose and cellulose derivatives, and silica. Similar assays may be conducted using peptides to detect the presence of antibodies in a sample, with appropriate modifications.
  • Assays may be, for example, of direct format (where the labelled first antibody reacts with the antigen), an indirect format (where a labelled second antibody reacts with the first antibody), a competitive format (such as the addition of a labelled antigen), or a sandwich format (where both labelled and unlabelled antibody are utilized), as well as other formats described in the art.
  • the sample is contacted to antibodies of the present invention and a labelled second antibody is used to detect the presence of peptide to which the antibodies are bound.
  • the antibodies of the present invention are useful as therapeutic agents in the prevention and treatment of symptoms or diseases caused by the deleterious effects of HIV infection or replication.
  • the antibodies are generally administered with a physiologically acceptable carrier or vehicle therefor.
  • a physiologically acceptable carrier is one that does not cause an adverse physical reaction upon administration and one in which the antibodies are sufficiently soluble and retain their activity to deliver a therapeutically effective amount of the compound.
  • the therapeutically effective amount and method of administration of the antibodies may vary based on the individual patient, the indication being treated and other criteria evident to one of ordinary skill in the art.
  • a therapeutically effective amount of the antibodies is one sufficient to inhibit superantigen stimulation of mononuclear cells, especially T- cells, and is preferably an amount sufficient to decrease or completely inhibit HIV infection or replication without causing significant side effects such as non-specific T cell lysis or organ damage.
  • the route(s) of administration useful in a particular application are apparent to one or ordinary skill in the art. Routes of administration of the antibodies include, but are not limited to, parenteral, and direct injection into an affected site. Parenteral routes of administration include but are not limited to intravenous, intramuscular, intraperitoneal, subcutaneous, and nasal.
  • compositions of the antibodies described above suitable for parenteral administration including, but not limited to, pharmaceutically acceptable sterile isotonic solutions.
  • pharmaceutically acceptable sterile isotonic solutions include, but are not limited to, saline and phosphate buffered saline for nasal, intravenous, intramuscular, intraperitoneal, subcutaneous or direct injection into another area.
  • Antibodies for use to elicit passive immunity in humans are preferably obtained from other humans previously inoculated with compositions comprising peptides having one or more of the consensus amino acid sequences of the invention. Alternatively, antibodies derived from other species may also be used. Such antibodies used in therapeutics suffer from several drawbacks such as a limited half-life and propensity to elicit an immune response. Several methods have been proposed to overcome these drawbacks. One such method is the "humanizing" of non-human antibodies by cloning the gene segment encoding the antigen binding region of the antibody to the human gene segments encoding the remainder of the antibody. Only the binding region of the antibody is thus recognized as foreign and is much less likely to cause an immune response.
  • the dosage of administered antibodies will vary depending upon such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history and the like.
  • IVIG intravenously
  • IM intramuscularly
  • IVIG intravenous immunoglobulin
  • IVIG can generally be given with a loading dose of 200 mg/kg, with monthly injections of 100 mg/kg.
  • High-dose IVIG may be given at 400-800 mg/kg, especially for antibody-deficient patients. See, e.g., The Merck Manual of Diagnosis and Therapy, 16 th Edition, (Berkow R and Fletcher AJ, Eds.), Merck Research Laboratories, Rahway, NJ (1992).
  • peptides and/or antibodies of the present invention are intended to be provided to the recipient subject in an amount sufficient to prevent, or attenuate the severity, extent or duration of the deleterious effects of HIV infection or replication.
  • the administration of the agents, including peptide and antibody compositions, of the invention may be for either "prophylactic" or "therapeutic" purpose.
  • the agents are provided in advance of any symptom.
  • the prophylactic administration of the agent serves to prevent or ameliorate any subsequent deleterious effects of HIV infection or replication.
  • the agent is provided at (or shortly after) the detection of the virus in the individual and/or the onset of a symptom of infection with HIV.
  • the agent of the present invention may, thus, be provided either prior to the anticipated exposure to HIV (so as to attenuate the anticipated severity, duration or extent of disease symptoms) or after the initiation of the infection.
  • the agent may also be provided to persons at high risk for getting HIV infection.
  • the agents of the invention may be effective against any HIV infected cells, including dendritic cells or antigen presenting cells.
  • therapies based upon vectors, such as viral vectors containing nucleic acid sequences coding for the peptides described herein. These molecules, developed so that they do not provoke a pathological effect, will produce peptides and, thereby, stimulate the immune system to respond to the peptides.
  • the peptides useful in this invention alone or linked to a carrier, as well as antibodies and other necessary reagents and appropriate devices and accessories may be provided in kit form so as to be readily available and easily used.
  • kits may contain a solid support, such as a membrane (e.g., nitrocellulose), a bead, sphere, test tube, rod, and so forth, to which a receptor such as an antibody specific for the target molecule will bind.
  • a solid support such as a membrane (e.g., nitrocellulose), a bead, sphere, test tube, rod, and so forth, to which a receptor such as an antibody specific for the target molecule will bind.
  • kits can also include a second receptor, such as a labelled antibody.
  • Such kits can be used for sandwich assays to detect toxins. Kits for competitive assays are also envisioned.
  • GCG Consensus #1 YGGX,TX 2 X 3 X4X 5 N (SEQ ID NO:l) peptide #1 CMYGGVTEHEGN (SEQ ID NO:3)
  • synthetic peptides #1 and #2 are not native peptides, i.e., their sequences differ from those found in native toxins. Variations of these peptides have also been constructed in order to generate concatenated polymers of the peptides. These polymers were constructed by the addition of glycine and of additional cysteine residues to the amino- and/or carboxyl- termini of the initial 2 peptides, thus facilitating concatenation via disulfide bond formation (Patarroyo, Amador et al, 1988; Lopez Silva et al., 1994; and Rodriguez, Moreno et al., 1990).
  • polymerized molecules were then dialyzed to remove molecules with molecular weights less than 6000-8000 daltons.
  • One polymeric construct is composed of the monomer: CMYGGVTEHEGNGC (SEQ ID NO:5).
  • An additional polymer is composed of the peptide: CGKKNVTVQELDYKIRKYLVDNKKLYGC (SEQ ID NO:6).
  • consensus region #1 precedes consensus region #2 by 27 amino acid residues (e.g. [consensus region 1] x27 [consensus region 2]).
  • CMYGGVTEHEGNKKNVTVQELDYKIRKYLVDNKKLY SEQ ID NO:7.
  • this peptide is representative of the two consensus regions joined together in the proper order (region 1 in the N terminal half, and region 2 in the C-terminal half of the molecule), however they are not separated by an additional 27 residues as they are in the native toxins.
  • concatenated polymers based on the monomer: CMYGGVTEHEGNKKNVTVQELDYKIRKYLVDNKKLYGC (SEQ ID NO:8).
  • Peptides with an (*) are cross-linked polymers composed of the described sequence. It is expected that monomers of these peptides will also be useful in the present invention.
  • MMTV Mouse Mammary Tumor Virus
  • MMTV is a retrovirus which can induce mammary carcinomas in mice late in life by activation of proto-oncogenes after integration in their vicinity.
  • MMTV requires a functional immune system to achieve efficient infection of the mammary gland. This requirement became clear when it was discovered that the MMTV virus has developed strategies to exploit the immune response. Instead of escaping immune detection, it induces a vigorous polyclonal T-B interaction which is required to induce a chronic infection.
  • MMTV peripheral blood mononuclear cells
  • the assay was done using a number of different murine strains of cells:
  • Murine B cells 1. CH 12.1
  • the CH 12.1/VS7 is the MMTV infected cell and KMLS-8 is the appropriate T cell for the MMTV superantigen.
  • KMLS-8 is the appropriate T cell for the MMTV superantigen.
  • the combination of 2 and 3 should produce an IL-2 response.
  • the others are controls.
  • the combination of 2 and 3 with peptide 6343 showed increasing inhibition with increasing dosage of peptide 6343.
  • control wells were the same number of cells without peptide.
  • the negative control were wells with only cord blood added. As seen in Figure 5, there was marked inhibition of HIV replication by the peptide at the higher doses over a period of 10 days. Daily observation of the wells revealed there was no obvious toxicity of the cells by the peptide per se.
  • HIV-1 Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes. Nature 391 (6665): 397- 401.
  • SEB 140 CMYGGVTEHNGN 151 SEQ ID NO 10

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Abstract

L'invention concerne des procédés qui assurent la protection contre les infections par le VIH et les effets secondaires néfastes y associés ou qui en réduisent la gravité. Elle concerne plus particulièrement des procédés d'utilisation de peptides dérivés à partir de séquences homologues de la famille des toxines de staphylocoques et de streptocoques et/ou leurs porteurs/conjugués, ou à partir d'anticorps se liant à ces peptides, et ce afin de réduire, d'inhiber ou d'éliminer la stimulation de blastogenèse de monocytes, y compris les leucocytes T, par la protéine Nef du VIH ou par un autre super-antigène, et afin de réduire, inhiber ou éliminer la réplication du VIH. L'invention concerne aussi des procédés utilisant des acides nucléiques isolés et purifiés codant les peptides de l'invention ou des cellules hôtes transformées contenant ces acides nucléiques pour produire des peptides utiles dans les procédés de l'invention.
PCT/US2000/016680 1999-06-18 2000-06-16 Procedes pour inhiber la replication du vih WO2000078790A2 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998045325A1 (fr) * 1997-04-07 1998-10-15 The Rockefeller University Peptides pouvant reduire efficacement des syntomes du syndrome de choc toxique

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998045325A1 (fr) * 1997-04-07 1998-10-15 The Rockefeller University Peptides pouvant reduire efficacement des syntomes du syndrome de choc toxique

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