WO1995031999A1 - Compositions of transactivating proteins of human immunodeficiency virus - Google Patents

Compositions of transactivating proteins of human immunodeficiency virus Download PDF

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Publication number
WO1995031999A1
WO1995031999A1 PCT/US1995/006077 US9506077W WO9531999A1 WO 1995031999 A1 WO1995031999 A1 WO 1995031999A1 US 9506077 W US9506077 W US 9506077W WO 9531999 A1 WO9531999 A1 WO 9531999A1
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Prior art keywords
seq
immunogen
tat
protein
arg
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PCT/US1995/006077
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French (fr)
Inventor
Gideon Goldstein
Michael D. Culler
Ponniah Shenbagamurthi
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Immunogiology Research Institute, Inc.
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Application filed by Immunogiology Research Institute, Inc. filed Critical Immunogiology Research Institute, Inc.
Priority to EP95921262A priority Critical patent/EP0767678A1/en
Priority to AU26382/95A priority patent/AU2638295A/en
Publication of WO1995031999A1 publication Critical patent/WO1995031999A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates generally to methods for immunizing humans or other mammals against infection by viruses that produce proteins that are released extracellularly and are taken up by other noninfected cells, preparing them for infection by the virus.
  • HIV human immunodeficiency virus
  • HTLV human T cell lymphocytotropic virus
  • SIV simian immunodeficiency virus
  • FV feline immunodeficiency virus
  • the TAT protein also activates other host cellular genes which contribute to cellular activation and sustenance of the virus. For example, there is evidence that the TAT protein activates host cell genes, such as bcl-2, c-myc, IL-6, TGF- ⁇ , and TNF [G. Zauli et al, Cancer Res. , 51:4481-4485 (1993); J. Laurence et al, Proc. Natl. Acad. Sci.. USA. 88:7635-7639 (1991); G. Scala et al, J. EXP. Med.. 179:961-971 (1994); G. Zauli et al, Blood. 0:3036-3043 (1992); and L.
  • host cell genes such as bcl-2, c-myc, IL-6, TGF- ⁇ , and TNF
  • TAT of HIV is also released extracellularly by infected cells and taken up by other non-infected cells [see, e.g., A. D. Frankel et al, Cell. 55: 1189-1193 (1988); G. Barillari et al, J. Immunol.. 149:3727-3734 (1992); B. Ensoli et al, Nature, 345:84-86 (1990)].
  • TAT protein has been shown to selectively depress antigen induced T cell proliferation in vitro [R. P. Viscidi et al. Science. 246:1606-1608 (1989)]. This latter immunological abnormality has been demonstrated in T cells from asymptomatic HIV infected subjects early in the disease.
  • Uptake of TAT by cells is very strong, and appears to be mediated by a short basic sequence of the protein.
  • U. S. Patent No. 5,158,877 discloses synthetic DNA coding for the TAT protein of HIV-1, and provides its cDNA sequence.
  • U. S. Patent No. 5,238,882 refers to a transformed yeast cell capable of expressing TAT for use in screening for agents which inhibit the function of the protein.
  • U. S. Patent No. 5,110,802 refers to an antiviral agent capable of attacking the first splice acceptor site of tat gene.
  • International Patent Application No. WO92/07871, published May 14, 1992 refers to oligopeptide inhibitors of HIV replication, which operate by direct competition to prevent activation by the TAT protein, while International Patent Application No.
  • O91/10453, published July 25, 1991 refers to inhibition of TAT function by DNA sequences. See, also International Patent Application No. O91/09958, published July 11, 1991. International Patent Application No. WO87/02989, published May 21, 1987, provides an E. coli expression vector for producing TAT protein. International Patent Application No. 092/14755, published September 3, 1992, refers to in vitro blocking of TAT uptake by a selected integrin cell surface receptor by fragments of TAT, fragments of integrin or antibodies to integrin. Also described in this publication is the immunization of rabbits with a TAT peptide and Complete Freund's adjuvant to generate antibodies to TAT for experimental work.
  • the invention provides an immunogen capable of eliciting a humoral and/or antibody response in a mammal to which the immunogen is administered, the immune response directed against a native extracellular transactivating protein from a selected chronic virus, such as HIV.
  • the immunogen comprises an HIV TAT protein sequence or peptide fragment thereof including the sequence involved in cellular uptake of the TAT.
  • this latter sequence will be referred to as the "cellular uptake region" [in HIV TAT, this region spans about AA46 to about AA63 of SEQ ID NO: 2] as defined by S. Fawell et al, cited above.
  • the immunogen itself will be referred to as the TAT immunogen.
  • the immunogen comprises analogous sequences of similar extracellular proteins for other viruses, e.g., SIV, HTLV, and the like.
  • this immunogenic protein or peptide sequence has the characteristic of inactivated biological function vis-a- vis the intact TAT protein.
  • the invention provides a DNA sequence encoding the TAT immunogen described above for use as a 'naked DNA* composition capable of eliciting an immune response in an animal, preferably a mammal, to which it is administered.
  • the invention provides a DNA molecule containing regulatory sequences which control the replication and expression of the inserted DNA sequence encoding the HIV TAT immunogen.
  • regulatory sequences may direct the expression of the TAT immunogen in cell cultures for recombinant production and manipulation of the DNA.
  • a vector may also be administered as an immunogenic composition, such as a vaccine, for expression of the TAT immunogen in vivo in a host mammal and the elicitation of an immune response thereto.
  • the invention provides immunogenic compositions, such as vaccine compositions, useful for producing an immune response to, and for immunizing a patient against infection with, a virus characterized by a transactivating protein or analogous extracellular protein.
  • immunogenic compositions such as vaccine compositions, useful for producing an immune response to, and for immunizing a patient against infection with, a virus characterized by a transactivating protein or analogous extracellular protein.
  • One embodiment of such an immunogenic composition comprises the protein or peptide immunogen described herein in a suitable pharmaceutical carrier.
  • Another embodiment of the immunogenic composition comprises a DNA sequence capable of expressing the immunogen in vivo upon direct administration of the DNA into a host animal.
  • the DNA sequence encoding the immunogen is present in a vector with associated regulatory sequences.
  • Still a further aspect of this invention is a method for producing an immune response, such as a protective immune response, in a patient against infection with a virus characterized by a transactivating or analogous extracellular protein.
  • the method comprises administering to said patient an HIV TAT immunogen or tat nucleic acid sequence as described above in an amount sufficient to evoke production of high titer antibodies capable of specifically binding the native HIV TAT protein secreted by said virus.
  • the antibodies would bind to the cellular uptake region of the TAT protein.
  • the invention contemplates the production of analogous methods and compositions designed similarly for other proteins which function in a manner similar to that of the TAT protein of HIV, including proteins of other viruses.
  • this method and compositions are anticipated to be useful in the prophylaxis of various chronic viruses including, HIV-1, HTLV-I, HTLV-II, HIV-2, SIV, and FIV.
  • Fig. 1 illustrates the cDNA sequence [SEQ ID NO: 1] and amino acid sequence [SEQ ID NO: 2] of the HIV-1 TAT protein.
  • Fig. 2 is a graph illustrating the purification of the I 125 labelled SIV-Tyr 0 TAT ⁇ sequence using reverse phase HPLC. The purified label will be used to detect the presence of antibodies to TAT following immunization. See, Example 2 below.
  • the present invention provides novel immunogens, immunogenic compositions, vaccine compositions, and methods of eliciting an immune response in mammals, particularly humans, against viruses that produce proteins that are released extracellularly, which proteins are in turn taken up by other uninfected, or latently infected, cells, thereby rendering the uninfected cells susceptible to viral infection.
  • One such viral protein capable of facilitating infectivity from one cell to another uninfected cell is the transactivating TAT protein of HIV (and in the monkey virus, SIV) ; other such proteins exist in other viruses.
  • TAT protein of HIV and in the monkey virus, SIV
  • the following description relates to the TAT proteins of HIV and SIV as specific examples of extracellular proteins which perform the above-described role of perpetuating viral infection.
  • analogous extracellular proteins necessary for the development and spread of the viral infection in HIV, SIV, and other viruses may be manipulated similarly to provide other immunogenic compositions and methods encompassed by this invention.
  • the inventors have determined that the novel method of this invention and the related compositions herein described permit the elicitation of an immune response in a mammal directed against these extracellular or transactivating proteins. Because infection is perpetuated either by activation of latently infected cells or by activation of host genes of uninfected cells which render the cells susceptible to viral infection, the mechanism underlying this invention is significant.
  • An important feature of the invention and an advantage over other therapeutic or vaccinal compositions useful for the treatment of viral infections, such as HIV, is that this invention elicits and directs an immunized host's immune response against the extracellular TAT protein.
  • Active immunization against a selected virus, e.g., HIV-1 may be accomplished by eliciting high titer antibodies that will complex with the native secreted TAT protein produced by the virus and prevent its uptake by cells.
  • the immune response produced by the methods and compositions of this invention interdicts the TAT protein extracellularly before it can be taken up by another cell.
  • This immunization has a particularly desirable advantage in contrast to other treatments and prophylactic methods employed against such viruses. Because the immunization is not directed against the virus itself, interdiction of the TAT protein extracellularly does not create a selective pressure on the parent virus itself, which would encourage the development of mutant virus variants producing a TAT protein that is not recognized by the induced antibodies.
  • any viral strain which is not producing the same TAT protein, or an immunologically cross-reactive protein will be unaffected by the use of the immunogenic compositions of this invention.
  • treatment with another immunogenic composition reactive with that TAT protein and prepared according to this invention is contemplated.
  • blocking the incorporation of TAT protein by the patient's cells may also reduce the level of viremia.
  • A. Protein/Peptide Immunogens of the Invention involves the development of a selected TAT immunogen.
  • immunogen is meant any molecule which elicits an immune response, either cellular or humoral, in an animal exposed to that molecule in vivo .
  • An immunogen of this invention is desirably an HIV TAT protein or peptide fragment thereof, which comprises cellular uptake region of the TAT protein or fragments thereof. The cellular uptake region is that region which mediates the binding of TAT protein to the cell to be infected.
  • An immunogen of this invention is desirably an HIV TAT protein or peptide fragment thereof, which comprises cellular uptake region of the TAT protein or fragments thereof.
  • the cellular uptake region is that region which mediates the binding of TAT protein to the cell to be infected.
  • the cellular uptake region is identified as spanning amino acids 46 to 63 of that sequence SYGRKKRRZRRRAPZGSQ of SEQ ID NO: 2.
  • the cellular uptake region includes a smaller fragment, such as amino acids 46 through 59 thereof.
  • the cellular uptake region as defined herein can include all or a portion of the sequence identified above, as well as variants thereof.
  • the TAT protein or peptide fragment useful as the immunogen is biologically inactive; i.e., it does not share the biological function of the intact TAT protein.
  • the immunogen may be made inactive by deletion of amino acids at the amino terminus or carboxy terminus, or the deletion or replacement of native Cysteine residues to interfere with naturally-occurring disulfide bonds in the protein.
  • One embodiment of a TAT immunogen may consist of a TAT protein characterized by deletion of at least 3 or more amino acids at the amino terminus.
  • TAT protein immunogen examples include a peptide fragment from a selected region of TAT protein, which peptide fragment alone is biologically inactive, and may be optionally coupled to a carrier.
  • a desired size for such an immunogenic peptide may be between about 12 to about 22 amino acids. However, other sizes may be desired, depending on the peptide construct, i.e., for a multiple antigenic peptide (also referred to as an octameric lysine core peptide) as described in detail below, the peptide may desirably be about 20 amino acids in length.
  • a selected region of the TAT immunogen of this invention may be a conserved region or an immunogenic region of TAT protein.
  • this peptide sequence includes the cellular uptake region.
  • the selected TAT immunogen may include, among other sequences, the following peptide sequences and variants:
  • SYGRKKRRQRRRPP [SEQ ID NO 11] SYGRKKRRQRRRAH.
  • SEQ ID NO 12 Larger peptides incorporating the above-identified sequences of amino acids of TAT protein of HIV or smaller fragments and fragments thereof may also be employed to form the TAT immunogen of this invention.
  • smaller fragments of the cellular uptake region such as QRRRAP [SEQ ID NO: 16] and its variants or GRKKRRQ [SEQ ID NO: 17] and its variants may also be " employed alone or repeated in one peptide in multiple antigenic peptides.
  • the virus is other than HIV-1, e.g., a non-human virus, such as SIV
  • a non-human virus such as SIV
  • the selected TAT protein region may include, among other sequences, the cellular uptake region occurring at about amino acids 79 through about amino acids 100 of the SIV TAT protein, as well as the following peptide sequences and variants: RRRTPKKTKANTSSASY [SEQ ID NO:13] which spans the SIV TAT sequence from amino acids 80 through 95; and
  • YEQQRRRTPKKTKANTSSAS [SEQ ID NO:14], which spans SIV TAT 76 _ 95 .
  • Other variants in size and amino acid composition may also be employed in this invention, such as the peptide motifs PKKTK [SEQ ID NO: 18], PKKAK [SEQ ID NO: 19] and QRRRTP [SEQ ID NO: 20] alone or repeated in a single peptide, desirably in multiple antigenic peptides as disclosed below.
  • the immunogen of the invention is in the form of a multiple antigenic peptide construct comprising a plurality of peptides from a TAT protein.
  • a construct may be designed employing the multiple antigenic peptide system described by Tarn, Proc. Natl. Acad. Sci. USA. 85:5409-5413 (1988). This system makes use of a core matrix of lysine residues onto which multiple copies of an immunogenic peptide are synthesized [D. Posnett et al, J. Biol. Chem. , 263 . (4) : 1719-1725 (1988); J. Tarn, "Chemically DefinedSynthetic Immunogens and Vaccines by the Multiple Antigen Peptide Approach", Vaccine Research and
  • the invention also encompasses immunogens prepared from other amino acid sequences of viral transactivating or analogous extracellular proteins.
  • selected TAT peptides may be developed from allelic or engineered variations of viral genes or gene fragments encoding the particular TAT protein.
  • the immunogens employed in this invention may also have amino acid sequences which are analogs or derivatives of TAT or related extracellular protein sequences of a selected virus.
  • the immunogens of this invention may also be prepared to reflect variations in the native DNA and amino acid sequences of selected TAT proteins.
  • immunogens may typically include TAT protein analogs, and the DNA encoding them, that differ by only 1 to about 4 codon changes.
  • analogs include polypeptides with minor amino acid variations from the natural amino acid sequence of TAT proteins, in particular, conservative amino acid replacements.
  • the TAT protein/peptide immunogens of this invention are prepared by chemical synthesis techniques, such as described by Merrifield, J. Amer. Che . Soc.. .85:2149-2154 (1963), and including the multiple antigenic techniques described above.
  • TAT protein fragments can be prepared synthetically or can be derived from viral RNA by known techniques, or from available cDNA-containing plasmids.
  • TAT immunogen in various microorganisms and cells, including, for example, E ⁇ . coli. Bacillus. Streptomyces. Saccharo yces. mammalian, yeast and insect cells, and suitable vectors therefor are known and available from private and public laboratories and depositories and from commercial vendors.
  • the most preferred host is a mammalian cell such as Chinese Hamster ovary cells (CHO) or COS-1 cells. These hosts may be used in connection with poxvirus vectors, such as vaccinia or swinepox.
  • poxvirus vectors such as vaccinia or swinepox.
  • the selection of other suitable host cells and methods for transformation, culture, amplification, screening and product production and purification can be performed by one of skill in the art by reference to known techniques. See, e.g., Gething and Sambrook, Nature, 293:620-625 (1981) .
  • Another preferred system includes the baculovirus expression system and vectors. Bacterial expression may also be desired.
  • the TAT immunogens When produced by conventional recombinant means, the TAT immunogens may be isolated either from the cellular contents by conventional lysis techniques or from cell medium by conventional methods, such as chromatography. See, e.g., Sambrook et al, Molecular Cloning. A Laboratory Manual. , 2d Edit. , Cold Spring Harbor Laboratory, New York (1989) .
  • the resulting TAT protein or peptide immunogens, or multiple antigenic peptides may be screened for efficacy as a immunogen by in vivo assays, employing immunization of an animal, e.g., a simian, with the TAT protein or fragment, and evaluation of titers of antibody to the native TAT protein of the selected virus.
  • immunization of an animal e.g., a simian
  • Examples 3 and 4 disclose suitable assay systems for such evaluation.
  • Still another aspect of this invention involves nucleic acid sequences which encodes the TAT protein immunogens described above.
  • the nucleic acid sequences such as that described as SEQ ID NO: 1 or allelic variants thereof, or DNA sequences containing certain preference codons for the species of the indicated patient preferably in the form of DNA, may be used as so- called 'naked DNA' to express the protein/peptide immunogen in vivo in a patient. See, e.g., J. Cohen, Science. 259:1691-1692 (March 19, 1993); E. Fynan et al, Proc. Natl. Acad. Sci.. USA. £0:11478-11482 (Dec. 1993) ;and J. A.
  • the DNA sequences encoding the selected TAT immunogen of this invention may also be employed in a plasmid for either direct injection into a host animal or in a vector under suitable regulatory sequences for expression of the protein in vitro .
  • the latter form of expression is conventional (see, Sambrook et al, cited above and the references above to production of the protein) .
  • the former type of plasmid for in vivo expression of the immunogen may be designed as described in the references above or, e.g., as described in International Patent Application PCT WO94/01139, published January 20, 1994.
  • the DNA encoding the TAT protein or desired fragment thereof is inserted into a nucleic acid cassette.
  • This cassette is engineered to contain, in addition to the tat sequence to be expressed, other optional flanking sequences which enable its insertion into a vector.
  • This cassette is then inserted into an appropriate DNA vector downstream of a promoter, an mRNA leader sequence, an initiation site and other regulatory sequences capable of directing the replication and expression of that sequence in vivo .
  • This vector permits infection of patient's cells and expression of the TAT immunogen in vivo.
  • the present invention provides immunogenic compositions containing a TAT protein or peptide immunogen of the invention or the 'naked DNA' of the invention. These immunogenic compositions are capable of eliciting in an immunized host mammal, e.g., a human, an immune response capable of interdicting extracellular TAT protein.
  • such immunogenic compositions contain at least one such protein/peptide immunogen directed to a selected native TAT protein according to the invention or a fragment thereof, together with a carrier suitable for administration as a composition for prophylactic treatment of virus infections.
  • the compositions may contain more than one immunogen, the amino acid sequences of the immunogens differing to encompass allelic variants of the TAT protein.
  • a multiple antigenic peptide of the invention may contain repeats of the same peptide, e.g., for HIV, RRRAP [SEQ ID NO: 21].
  • the composition may preferably contain a number of MAPs, each bearing copies of different TAT peptides as described herein.
  • novel TAT immunogens may be designed by resort to this disclosure.
  • the HIV strain of a patient not responsive to a composition of this invention may be isolated and the TAT protein sequence determined by conventional methods, e.g., polymerase chain reaction. If the sequence is indeed different from that sequence which was employed in the first composition, another TAT immunogen may be obtained and administered with the first immunogen in a single immunogenic composition.
  • an immunogenic or vaccine composition of this invention may contain several different TAT immunogens of this invention, each directed to a different TAT protein produced as described herein.
  • the TAT immunogen(s) of the invention or fragments thereof can be employed in a vaccine composition containing a carrier, such as saline, and a selected adjuvant, such as aqueous suspensions of aluminum and magnesium hydroxides, liposomes and others.
  • a carrier such as saline
  • a selected adjuvant such as aqueous suspensions of aluminum and magnesium hydroxides, liposomes and others.
  • Such compositions may optimally contain other conventionalvaccine components.
  • suitable carriers and adjuvants are known to the art and the selection thereof is expected to be routine.
  • the preparation of a pharmaceutically acceptable vaccine composition, having appropriate pH isotonicity, stability and other conventional characteristics is within the skill of the art.
  • These immunogenic or vaccine compositions according to the present invention can be administered by an appropriate route, e.g., by the oral, intranasal, subcutaneous, intravenous, intraperitoneal or intramuscular routes. The presently preferred route of administration is intramuscular.
  • each vaccine dose is selected with regard to consideration of the patient's age, weight, sex, general physical condition and the like.
  • the amount required to induce an immune response, preferably a protective response, in the patient without significant adverse side effects may vary depending upon the immunogen employed and the optional presence of an adjuvant.
  • each dose will comprise between about 50 ⁇ g to about 1 mg of TAT protein/peptide immunogen per L of a sterile solution.
  • a more preferred dosage may be about 200 ⁇ g of TAT immunogen of this invention.
  • Other dosage ranges may also be contemplated by one of skill in the art.
  • Initial doses may be optionally followed by repeated boosts, where desirable.
  • immunogenic compositions of this invention may be designed for direct administration of •naked DNA' encoding one or more TAT immunogens of this invention.
  • nucleic acid sequences encoding one or more desired TAT immunogens of this invention may be directly delivered into the patient, alone or as part of a plasmid.
  • Suitable vehicles for direct DNA administration include, without limitation, saline, or sucrose, prota ine, polybrene, polylysine, polycations, proteins, CaP0 4 or spermidine. See e.g, PCT application WO94/01139 and the references cited above.
  • the amounts of components in the 'naked DNA 1 composition and the mode of administration e.g., injection or intranasal, may be selected and adjusted by one of skill in the art.
  • the immunogenic compositions of this invention are designed to prevent infection by the selected virus of an uninfected mammal, e.g. , human. Such immunogenic compositions thus have utility as vaccines.
  • compositions of this invention may also be employed therapeutically to treat already infected patients.
  • the mechanism of the present invention as described above may be useful in impeding the course of viral infection and producing desirable clinical results. More specifically, the compositions of this invention are capable of reducing viremia in patients already infected with the virus by blocking further uptake of the TAT protein by uninfected cells.
  • the compositions of the present invention are anticipated to assist in the reduction of viremia and prevention of clinical deterioration.
  • the formulations and modes of administration are substantially identical to those described specifically above and may be administered concurrently or simultaneously with other conventional therapeutics for the specific viral infection. For therapeutic use, repeated dosages may be desirable.
  • the TAT immunogens of this invention may have a number of other utilities.
  • the TAT peptide or protein immunogens of this invention may be employed in conventional assays, e.g.. Western blots, ELISA, and other protein binding assays, for use in identifying antibodies which are capable of detecting HIV strains with different extracellular TAT proteins.
  • the DNA sequences encoding the TAT immunogens or complementary strands thereto may be used in nucleic acid assays, such as Northern and Southern blots, and other nucleic acid hybridization assays for the same purpose.
  • the nucleic acid sequences encoding the TAT immunogens of the invention may be employed as polymerase chain reaction sequences to identify HIV strains on the basis of the sequence of the tat gene.
  • proteins are in the development of antibodies which are directed to the cellular uptake regions.
  • Polyclonal antibodies may be produced by conventional methods, i.e., by injecting a laboratory animals with sufficient quantities of the TAT protein or fragments thereof and collecting the antibodies from the animal's tissue.
  • a “monoclonal antibody” refers to homogenous populations of immunoglobulins which are capable of specifically binding to the TAT protein of this invention. It is understood that the TAT protein may have one or more antigenic determinants. The antibodies of the invention may be directed against one or more of these determinants.
  • Hybridomas capable of secreting these monoclonal antibodies are produced by conventional techniques by employing as antigen, the TAT protein described herein.
  • the hybridoma process involves generating a B- lymphocyte which produces a desired polyclonal antibody.
  • Techniques for obtaining the appropriate lymphocytes from mammals injected with the target antigen are well known.
  • the peripheral blood lymphocytes (PBLs) are used if cells of human origin are desired. If non-human sources are desired, spleen cells or lymph nodes from other mammalian sources are used.
  • a host animal e.g. a rabbit, is injected with repeated doses of the purified antigen, and the mammal is permitted to generate the desired polyclonal antibody producing cells.
  • Immortalizing cell lines are usually transformed mammalian cells, particularly cells of rodent, bovine and human origin. Most frequently, rat or mouse myeloma cells are employed. Techniques for fusion are also well known in the art and generally involve mixing the cells with a fusing agents, e.g. polyethylene glycol.
  • Immortalized hybridoma cell lines are selected by standard procedures, such as HAT selection. From among these hybridomas, those secreting the desired monoclonal antibody are selected by assaying the culture medium by standard immunoassays, such as Western blotting, ELISA, or RIA. Antibodies are recovered from the medium using standard purification techniques. See, generally, Kohler et al. Nature, 256:495 (1975) for antibody production techniques. Alternatively, non-fusion techniques for generating an immortal antibody-producing hybridoma cell line is possible, e.g. virally induced transformation.
  • Recombinant techniques such as described by Huse et al. Science, 246:1275-1281 (1988), or any other modifications thereof known to the art may also be employed to produce recombinant antibodies and chimeric antibodies, where desired.
  • These antibodies are likewise useful in diagnostic methods for detecting and identifying different HIV strains based on the differences in their extracellular tat genes. These antibodies may perform such detection in conventional assays known to one of skill in the art.
  • TAT protein and peptide immungens are conventional and may include use of the TAT protein and peptide immatoriums as molecular weight markers.
  • Side chain protecting groups were as follows: benzyl ether (Bzl) for the hydroxy1 group of threonine and serine; benzyl ester for the carboxyl group of aspartic and glutamic acids; tosyl (Tos) for the guanidine of arginine; benzyloxymethyl (Bom) for the imidazole of histidine; 2- chlorobenzyloxycarbonyl (Cl-Z) for the e-amine of lysine; 2-bromobenzyloxycarbonyl (Br-Z) for the phenolic hydroxy1 of tyrosine. Asparagine, glutamine, and arginine were coupled as their 1-hydroxybenzotriazole esters.
  • the peptide was cleaved from the resin support (1.48 g) by stirring in liquid HF (50 L) , p-cresol (1.2 mL) , p-thiocresol (1.2 L) , and dimethyl sulfide (1.2 mL) for 1 hour at 0°C. After the removal of excess HF under reduced pressure, the resin-peptide mixture was extracted with anhydrous diethyl ether (3 x 200 mL) . The ether extracts were discarded. The cleaved peptide was then extracted with 30% aqueous acetic acid (3 X 70 mL) . After removal of solvents under reduced pressure, the residue obtained was dissolved in water and freeze-dried.
  • liquid HF 50 L
  • p-cresol 1.2 mL
  • p-thiocresol 1.2 L
  • dimethyl sulfide 1.2 mL
  • the crude peptide was dissolved in 30% acetic acid and passed through an Amberlite IRA-68 (acetate form) ion exchange column (60 g, 1.6 meq/mL, 2.73 cm i.d. X 18 cm length) in 30% aqueous HOAc at a flow rate of 60 mL/h.
  • the appropriate fractions from two such experiments were combined and freeze-dried (1.59 g) .
  • the crude peptide was dissolved in 0.1% TFA/H 2 0 and purified by preparative RP-HPLC using a Vydac 218TP1022 column (22 X 250 mm) .
  • the mobile phases employed were as shown below:
  • Amino acid analysis (AAA) : Ala 2.16 (2), Arg 3.23 (3), Asx 1.23 (1),
  • This peptide was synthesized as described above, using the appropriate protected amino acid derivatives.
  • the characteristics of the peptide are as follows.
  • the protected peptide-resin was treated with liquid hydrogen fluoride, in the presence of p-cresol, p- thiocresol and dimethylsulfide as scavengers, at 0°C for 1 hour with constant stirring. Excess HF was removed by vacuum and the residue treated with ether to remove scavenger products. The peptide was extracted with 50% acetic acid (3 X 50 mL) , the solvents evaporated in vacuo, and the product freeze-dried.
  • the crude peptide was initially purified on an Amberlite IRA-68 ion-exchange column; further purification was accomplished by RP-HPLC on a preparative C 18 column.
  • the solvents used were: water containing 0.1% TFA (Buffer A) and CH 3 CN - H 2 0 (4:1) containing 0.1% TFA (Buffer B) .
  • a linear gradient of 0-20% B over 100 minutes was used.
  • the appropriate fractions containing the peptide were pooled, the solvents evaporated in vacuo , and the product freeze-dried (644 mg) .
  • the purified peptide gave satisfactory amino acid analysis.
  • AAA Ala 18.42 (16) , Arg 23.74 (24), ASX 10.41 (8) , Gly 1.47 (1), Lys 25.91 (31) , Pro 8.14 (8), Ser 23.68 (24), Thr 23.99 (24) .
  • This peptide was synthesized as described above, using the appropriate protected amino acid derivatives. The purified peptide gave satisfactory amino acid analysis.
  • the following multiple antigenic peptides can be produced using the techniques described above.
  • the following MAPs can be produced using methods analogous to those described above.
  • the animals are fed a commercial primate diet, optionally supplemented with fresh fruit. Water is provided ad libitum. Animals are randomized into two treatment groups (peptide or placebo) .
  • the animals Prior to challenge infection with SIV, the animals are immunized intramuscularly (by thigh injection) on day 0, day 10 and monthly as necessary with either 200 micrograms of test peptide mixed with alum as an adjuvant or a saline-adjuvant placebo, until suitable antibody titers are attained.
  • Serum for antibody testing is collected prior to initial immunization, on day 7 and on day 17 post immunization and then 7 days after each monthly injection until suitable antibody titers are attained.
  • SIV antibody in serum is determined by both radioimmunoassay and ELISA and is confirmed by Western Blot analysis.
  • the B670 SIV strain is a well-characterized viral isolate originally isolated at Tulane Regional Primate Research Center (TRPRC) .
  • the clinical, immunological and virological status of the animals are evaluated. For example, a lymph node biopsy is performed at 2 and 4 weeks after challenge, and a sample of blood is collected for the determination of antibody titers at week 4 after challenge and then monthly until death or completion of study. For all measurements of antibody. the mean titer level, expressed on a log 10 scale is computed at each time point.
  • Results of this protocol are anticipated to reveal significant protection of the animals by use of the peptide immunogens of this invention.
  • EXAMPLE 4 EVALUATION OF ANTIBODY TITERS OF IMMUNIZED MONKEYS WITH TAT IMMUNOGEN FOR SIV
  • Serum from monkeys immunized with the SIV TAT i0.95 multiple antigenic peptide are tested for the presence of anti-SIV TAT binding activity. This activity is assessed by two independent methods.
  • the antibodies present in the samples are precipitated, along with any bound [ 15 I-TYRo]-SIV TAT i( . 95 , by subsequent incubation with goat anti-monkey serum followed by centrifugation. The supernatant is removed and the remaining pellet is counted in a gamma counter to determine the amount of [ 125 I-TYRo]-SIV TAT ia . 95 bound by the monkey serum dilutions.
  • titer is defined as the dilution of monkey serum required to bind 50% of the added [ 125 I-TYRo]-SIV TAT iM5 tracer.
  • the monkey serum is titrated by enzyme- linked immunoassay (ELISA) on polystyrene microtiter plates that have previously been coated with both the SIV TAT i0 _ 95 multiple antigenic peptide to determine the total binding activity, and with ovalbumin to determine amount of the binding that is not specific for SIV TAT i0 .
  • ELISA enzyme- linked immunoassay
  • the monkey serum is serially diluted using 0.01M phosphate buffered saline containing 0.01% Tween 20 and 0.5% BSA (PBS/Tween 20) .
  • AGT CAG ACT CAT CAA GTT TCT CTA TCA 210 Arg Pro Pro Gin Gly Ser Gin Thr His Gin Val Ser Leu Ser 60 65 70

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Abstract

The inventions provides compositions and a novel method of immunization directed against released transactivating proteins of certain target viruses that are taken up by other cells, including particularly HIV and other integrating and chronically infecting viruses. The method employs TAT immunogens, which are capable of eliciting high titer antibody to the native TAT protein, especially the regions involved in cellular uptake.

Description

COM POSITIO NS O F TRAN SACTI VATI NG PROTEI NS O F HUM IMMUNODEFICIENCY VIRUS
Field of the Invention
The present invention relates generally to methods for immunizing humans or other mammals against infection by viruses that produce proteins that are released extracellularly and are taken up by other noninfected cells, preparing them for infection by the virus.
Background of the Invention
Certain viruses possess genes which are involved in the expression and secretion of viral proteins which function to transfer susceptibility to infection by the virus to other non-infected host cells. One primary example of such a viral gene is the transactivator gene (tat) of the human immunodeficiency virus (HIV) , which is essential for viral replication. Similar transactivating genes are found in other viruses such as human T cell lymphocytotropic virus (HTLV) I and II, simian immunodeficiency virus (SIV) , and feline immunodeficiency virus (FIV) . The tat gene of HIV is essential for viral replication; the protein it encodes (TAT) activates transcription of HIV through the HIV long terminal repeat (LTR) . The TAT protein also activates other host cellular genes which contribute to cellular activation and sustenance of the virus. For example, there is evidence that the TAT protein activates host cell genes, such as bcl-2, c-myc, IL-6, TGF-β, and TNF [G. Zauli et al, Cancer Res. , 51:4481-4485 (1993); J. Laurence et al, Proc. Natl. Acad. Sci.. USA. 88:7635-7639 (1991); G. Scala et al, J. EXP. Med.. 179:961-971 (1994); G. Zauli et al, Blood. 0:3036-3043 (1992); and L. Buonaguro et al, J. Virol.. .66:7159-7167 (1992)]. TAT of HIV is also released extracellularly by infected cells and taken up by other non-infected cells [see, e.g., A. D. Frankel et al, Cell. 55: 1189-1193 (1988); G. Barillari et al, J. Immunol.. 149:3727-3734 (1992); B. Ensoli et al, Nature, 345:84-86 (1990)].
Picomolar to nanomolar extracellular concentrations can be demonstrated to affect the functions of certain cells. Furthermore, TAT protein has been shown to selectively depress antigen induced T cell proliferation in vitro [R. P. Viscidi et al. Science. 246:1606-1608 (1989)]. This latter immunological abnormality has been demonstrated in T cells from asymptomatic HIV infected subjects early in the disease.
Uptake of TAT by cells is very strong, and appears to be mediated by a short basic sequence of the protein. S. Fawell et al, Proc. Natl. Acad. Sci.. USA. 91:664-668 (1994) identified this sequence as Tat 37.72 (CFITKALGISYGRKKRRQRRRPPQGSGTHQVSLSKQ) [SEQ ID NO: 15] and utilized it in fusion proteins to enable the uptake of other proteins into the cells and, more specifically, into the nucleus. If taken up by cells latently infected with HIV, TAT protein would likely activate HIV production. If taken up by uninfected cells, TAT would likely activate host genes that would render the cell more receptive to fresh HIV infection, thus enabling the expansion of HIV infection in the host.
The tat gene and its protein have been sequenced and examined for involvement in proposed treatments of HIV. U. S. Patent No. 5,158,877 discloses synthetic DNA coding for the TAT protein of HIV-1, and provides its cDNA sequence. U. S. Patent No. 5,238,882 refers to a transformed yeast cell capable of expressing TAT for use in screening for agents which inhibit the function of the protein. U. S. Patent No. 5,110,802 refers to an antiviral agent capable of attacking the first splice acceptor site of tat gene. International Patent Application No. WO92/07871, published May 14, 1992 refers to oligopeptide inhibitors of HIV replication, which operate by direct competition to prevent activation by the TAT protein, while International Patent Application No. O91/10453, published July 25, 1991, refers to inhibition of TAT function by DNA sequences. See, also International Patent Application No. O91/09958, published July 11, 1991. International Patent Application No. WO87/02989, published May 21, 1987, provides an E. coli expression vector for producing TAT protein. International Patent Application No. 092/14755, published September 3, 1992, refers to in vitro blocking of TAT uptake by a selected integrin cell surface receptor by fragments of TAT, fragments of integrin or antibodies to integrin. Also described in this publication is the immunization of rabbits with a TAT peptide and Complete Freund's adjuvant to generate antibodies to TAT for experimental work. Despite the growing knowledge about the expression and proposed use of TAT in HIV therapies, there remains a need in the art for the development of a successful therapeutic or immunization regimen useful in humans (and where appropriate, other mammals) that utilizes active immunization with TAT protein or peptides in the treatment or prophylaxis of HIV, as well as for similar regimens utilizing other analogous essential extracellular proteins of other viruses, or their immunogenic peptides.
Summary of the Invention
In one aspect, the invention provides an immunogen capable of eliciting a humoral and/or antibody response in a mammal to which the immunogen is administered, the immune response directed against a native extracellular transactivating protein from a selected chronic virus, such as HIV. In one embodiment, the immunogen comprises an HIV TAT protein sequence or peptide fragment thereof including the sequence involved in cellular uptake of the TAT. Hereafter this latter sequence will be referred to as the "cellular uptake region" [in HIV TAT, this region spans about AA46 to about AA63 of SEQ ID NO: 2] as defined by S. Fawell et al, cited above. The immunogen itself will be referred to as the TAT immunogen. In other embodiments, the immunogen comprises analogous sequences of similar extracellular proteins for other viruses, e.g., SIV, HTLV, and the like. Preferably, this immunogenic protein or peptide sequence has the characteristic of inactivated biological function vis-a- vis the intact TAT protein.
In another aspect, the invention provides a DNA sequence encoding the TAT immunogen described above for use as a 'naked DNA* composition capable of eliciting an immune response in an animal, preferably a mammal, to which it is administered.
In still a further aspect, the invention provides a DNA molecule containing regulatory sequences which control the replication and expression of the inserted DNA sequence encoding the HIV TAT immunogen. Such regulatory sequences may direct the expression of the TAT immunogen in cell cultures for recombinant production and manipulation of the DNA. Alternatively, such a vector may also be administered as an immunogenic composition, such as a vaccine, for expression of the TAT immunogen in vivo in a host mammal and the elicitation of an immune response thereto.
In still a further aspect, the invention provides immunogenic compositions, such as vaccine compositions, useful for producing an immune response to, and for immunizing a patient against infection with, a virus characterized by a transactivating protein or analogous extracellular protein. One embodiment of such an immunogenic composition comprises the protein or peptide immunogen described herein in a suitable pharmaceutical carrier. Another embodiment of the immunogenic composition comprises a DNA sequence capable of expressing the immunogen in vivo upon direct administration of the DNA into a host animal. In still another embodiment of such an immunogenic composition, the DNA sequence encoding the immunogen is present in a vector with associated regulatory sequences.
Still a further aspect of this invention is a method for producing an immune response, such as a protective immune response, in a patient against infection with a virus characterized by a transactivating or analogous extracellular protein. The method comprises administering to said patient an HIV TAT immunogen or tat nucleic acid sequence as described above in an amount sufficient to evoke production of high titer antibodies capable of specifically binding the native HIV TAT protein secreted by said virus. Preferably the antibodies would bind to the cellular uptake region of the TAT protein.
In yet a further aspect, the invention contemplates the production of analogous methods and compositions designed similarly for other proteins which function in a manner similar to that of the TAT protein of HIV, including proteins of other viruses. Thus this method and compositions are anticipated to be useful in the prophylaxis of various chronic viruses including, HIV-1, HTLV-I, HTLV-II, HIV-2, SIV, and FIV.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof. Brief Description of the Drawings
Fig. 1 illustrates the cDNA sequence [SEQ ID NO: 1] and amino acid sequence [SEQ ID NO: 2] of the HIV-1 TAT protein.
Fig. 2 is a graph illustrating the purification of the I125 labelled SIV-Tyr0 TAT^^ sequence using reverse phase HPLC. The purified label will be used to detect the presence of antibodies to TAT following immunization. See, Example 2 below.
Detailed Description of the Invention
The present invention provides novel immunogens, immunogenic compositions, vaccine compositions, and methods of eliciting an immune response in mammals, particularly humans, against viruses that produce proteins that are released extracellularly, which proteins are in turn taken up by other uninfected, or latently infected, cells, thereby rendering the uninfected cells susceptible to viral infection. One such viral protein capable of facilitating infectivity from one cell to another uninfected cell is the transactivating TAT protein of HIV (and in the monkey virus, SIV) ; other such proteins exist in other viruses. For ease of discussion, the following description relates to the TAT proteins of HIV and SIV as specific examples of extracellular proteins which perform the above-described role of perpetuating viral infection. However, it should be understood by one of skill in the art that analogous extracellular proteins necessary for the development and spread of the viral infection in HIV, SIV, and other viruses may be manipulated similarly to provide other immunogenic compositions and methods encompassed by this invention.
While not wishing to be bound by theory, the inventors have determined that the novel method of this invention and the related compositions herein described permit the elicitation of an immune response in a mammal directed against these extracellular or transactivating proteins. Because infection is perpetuated either by activation of latently infected cells or by activation of host genes of uninfected cells which render the cells susceptible to viral infection, the mechanism underlying this invention is significant.
An important feature of the invention and an advantage over other therapeutic or vaccinal compositions useful for the treatment of viral infections, such as HIV, is that this invention elicits and directs an immunized host's immune response against the extracellular TAT protein. Active immunization against a selected virus, e.g., HIV-1, may be accomplished by eliciting high titer antibodies that will complex with the native secreted TAT protein produced by the virus and prevent its uptake by cells. That is, once TAT protein has been expressed by the virus in an infected cell and is separated from the virus itself and is secreted from the infected cell in which it was produced, the immune response produced by the methods and compositions of this invention interdicts the TAT protein extracellularly before it can be taken up by another cell. This immunization has a particularly desirable advantage in contrast to other treatments and prophylactic methods employed against such viruses. Because the immunization is not directed against the virus itself, interdiction of the TAT protein extracellularly does not create a selective pressure on the parent virus itself, which would encourage the development of mutant virus variants producing a TAT protein that is not recognized by the induced antibodies. However, any viral strain which is not producing the same TAT protein, or an immunologically cross-reactive protein, will be unaffected by the use of the immunogenic compositions of this invention. In this case treatment with another immunogenic composition reactive with that TAT protein and prepared according to this invention is contemplated. Additionally, it is anticipated that blocking the incorporation of TAT protein by the patient's cells may also reduce the level of viremia.
A. Protein/Peptide Immunogens of the Invention One embodiment of this invention involves the development of a selected TAT immunogen. By "immunogen" is meant any molecule which elicits an immune response, either cellular or humoral, in an animal exposed to that molecule in vivo . An immunogen of this invention is desirably an HIV TAT protein or peptide fragment thereof, which comprises cellular uptake region of the TAT protein or fragments thereof. The cellular uptake region is that region which mediates the binding of TAT protein to the cell to be infected. One variant of the HIV TAT protein is illustrated in SEQ ID NO: 2 and in Fig. 1; the cellular uptake region is identified as spanning amino acids 46 to 63 of that sequence SYGRKKRRZRRRAPZGSQ of SEQ ID NO: 2. Preferably, the cellular uptake region includes a smaller fragment, such as amino acids 46 through 59 thereof. However it is known that other variants of HIV exist which differ from that sequence at one or more amino acids. Thus, the cellular uptake region as defined herein can include all or a portion of the sequence identified above, as well as variants thereof.
Preferably, the TAT protein or peptide fragment useful as the immunogen is biologically inactive; i.e., it does not share the biological function of the intact TAT protein. The immunogen may be made inactive by deletion of amino acids at the amino terminus or carboxy terminus, or the deletion or replacement of native Cysteine residues to interfere with naturally-occurring disulfide bonds in the protein. One embodiment of a TAT immunogen may consist of a TAT protein characterized by deletion of at least 3 or more amino acids at the amino terminus.
Additional embodiments of a TAT protein immunogen include a peptide fragment from a selected region of TAT protein, which peptide fragment alone is biologically inactive, and may be optionally coupled to a carrier. A desired size for such an immunogenic peptide may be between about 12 to about 22 amino acids. However, other sizes may be desired, depending on the peptide construct, i.e., for a multiple antigenic peptide (also referred to as an octameric lysine core peptide) as described in detail below, the peptide may desirably be about 20 amino acids in length.
A selected region of the TAT immunogen of this invention may be a conserved region or an immunogenic region of TAT protein. Preferably, this peptide sequence includes the cellular uptake region. For example, where the virus selected for use in this invention is HIV-1, the selected TAT immunogen may include, among other sequences, the following peptide sequences and variants:
SYGRKKRRQRRRAPQGSQ [SEQ ID NO 3]
SYGRKKRRQRRRAP [SEQ ID NO 4]
SYGKKKRRQRRRAP [SEQ ID NO 5]
SYGRKKRKQRRRAP [SEQ ID NO 6] SYGRKKRRPRRRAP [SEQ ID NO 7]
SYGRKKRRQRQRAP [SEQ ID NO 8]
SYGRKKRRQRRGAP [SEQ ID NO 9]
SYGRKKRRQRRRTP [SEQ ID NO 10]
SYGRKKRRQRRRPP [SEQ ID NO 11] SYGRKKRRQRRRAH. [SEQ ID NO 12] Larger peptides incorporating the above-identified sequences of amino acids of TAT protein of HIV or smaller fragments and fragments thereof may also be employed to form the TAT immunogen of this invention. For example, smaller fragments of the cellular uptake region, such as QRRRAP [SEQ ID NO: 16] and its variants or GRKKRRQ [SEQ ID NO: 17] and its variants may also be"employed alone or repeated in one peptide in multiple antigenic peptides. Similarly, where the virus is other than HIV-1, e.g., a non-human virus, such as SIV, one of skill in the art is expected to be able to select analogous regions of the transactivating or analogous extracellular protein of the virus for use as an immunogen in accordance with this invention. For example, where the virus selected for use in this invention is SIV, the selected TAT protein region may include, among other sequences, the cellular uptake region occurring at about amino acids 79 through about amino acids 100 of the SIV TAT protein, as well as the following peptide sequences and variants: RRRTPKKTKANTSSASY [SEQ ID NO:13] which spans the SIV TAT sequence from amino acids 80 through 95; and
YEQQRRRTPKKTKANTSSAS [SEQ ID NO:14], which spans SIV TAT76_95. Other variants in size and amino acid composition may also be employed in this invention, such as the peptide motifs PKKTK [SEQ ID NO: 18], PKKAK [SEQ ID NO: 19] and QRRRTP [SEQ ID NO: 20] alone or repeated in a single peptide, desirably in multiple antigenic peptides as disclosed below.
In a preferred embodiment, the immunogen of the invention is in the form of a multiple antigenic peptide construct comprising a plurality of peptides from a TAT protein. For example, such a construct may be designed employing the multiple antigenic peptide system described by Tarn, Proc. Natl. Acad. Sci. USA. 85:5409-5413 (1988). This system makes use of a core matrix of lysine residues onto which multiple copies of an immunogenic peptide are synthesized [D. Posnett et al, J. Biol. Chem. , 263.(4) :1719-1725 (1988); J. Tarn, "Chemically DefinedSynthetic Immunogens and Vaccines by the Multiple Antigen Peptide Approach", Vaccine Research and
Developments. Vol. 1, ed. W. Koff and H. Six, pp. 51-87 (Marcel Deblau, Inc., New York 1992)]. These constructs are designed for use in the present invention, as described in detail in Example 1 below. Other conventional protein/peptide immunogenic constructs may be designed by resort to known techniques.
In addition to the amino acid sequences of the specifically-recited HIV-1 TAT proteins described herein, the invention also encompasses immunogens prepared from other amino acid sequences of viral transactivating or analogous extracellular proteins. For example, selected TAT peptides may be developed from allelic or engineered variations of viral genes or gene fragments encoding the particular TAT protein. The immunogens employed in this invention may also have amino acid sequences which are analogs or derivatives of TAT or related extracellular protein sequences of a selected virus.
The immunogens of this invention may also be prepared to reflect variations in the native DNA and amino acid sequences of selected TAT proteins. For example, such immunogens may typically include TAT protein analogs, and the DNA encoding them, that differ by only 1 to about 4 codon changes. Other examples of analogs include polypeptides with minor amino acid variations from the natural amino acid sequence of TAT proteins, in particular, conservative amino acid replacements.
Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into four families: (1) acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, histidine; (3) non-polar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamatic acid, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a significant effect on peptide structure or activity. Preferably, the TAT protein/peptide immunogens of this invention are prepared by chemical synthesis techniques, such as described by Merrifield, J. Amer. Che . Soc.. .85:2149-2154 (1963), and including the multiple antigenic techniques described above. Alternatively, they may be prepared by known recombinant DNA techniques by cloning and expressing within a host microorganism or cell a DNA fragment carrying a coding sequence for the selected TAT immunogen. Coding sequences for the TAT protein fragments can be prepared synthetically or can be derived from viral RNA by known techniques, or from available cDNA-containing plasmids.
Systems for cloning and expressing the TAT immunogen in various microorganisms and cells, including, for example, E^. coli. Bacillus. Streptomyces. Saccharo yces. mammalian, yeast and insect cells, and suitable vectors therefor are known and available from private and public laboratories and depositories and from commercial vendors. Currently, the most preferred host is a mammalian cell such as Chinese Hamster ovary cells (CHO) or COS-1 cells. These hosts may be used in connection with poxvirus vectors, such as vaccinia or swinepox. The selection of other suitable host cells and methods for transformation, culture, amplification, screening and product production and purification can be performed by one of skill in the art by reference to known techniques. See, e.g., Gething and Sambrook, Nature, 293:620-625 (1981) .
Another preferred system includes the baculovirus expression system and vectors. Bacterial expression may also be desired.
When produced by conventional recombinant means, the TAT immunogens may be isolated either from the cellular contents by conventional lysis techniques or from cell medium by conventional methods, such as chromatography. See, e.g., Sambrook et al, Molecular Cloning. A Laboratory Manual. , 2d Edit. , Cold Spring Harbor Laboratory, New York (1989) .
The resulting TAT protein or peptide immunogens, or multiple antigenic peptides may be screened for efficacy as a immunogen by in vivo assays, employing immunization of an animal, e.g., a simian, with the TAT protein or fragment, and evaluation of titers of antibody to the native TAT protein of the selected virus. Examples 3 and 4 disclose suitable assay systems for such evaluation.
B. 'Naked DNA' Compositions of the Invention
Still another aspect of this invention involves nucleic acid sequences which encodes the TAT protein immunogens described above. The nucleic acid sequences, such as that described as SEQ ID NO: 1 or allelic variants thereof, or DNA sequences containing certain preference codons for the species of the indicated patient preferably in the form of DNA, may be used as so- called 'naked DNA' to express the protein/peptide immunogen in vivo in a patient. See, e.g., J. Cohen, Science. 259:1691-1692 (March 19, 1993); E. Fynan et al, Proc. Natl. Acad. Sci.. USA. £0:11478-11482 (Dec. 1993) ;and J. A. Wolff et al, Biotechniques. 11:474-485 (1991) , all incorporated by reference herein. The DNA sequences encoding the selected TAT immunogen of this invention may also be employed in a plasmid for either direct injection into a host animal or in a vector under suitable regulatory sequences for expression of the protein in vitro . The latter form of expression is conventional (see, Sambrook et al, cited above and the references above to production of the protein) . The former type of plasmid for in vivo expression of the immunogen may be designed as described in the references above or, e.g., as described in International Patent Application PCT WO94/01139, published January 20, 1994. Briefly, the DNA encoding the TAT protein or desired fragment thereof is inserted into a nucleic acid cassette. This cassette is engineered to contain, in addition to the tat sequence to be expressed, other optional flanking sequences which enable its insertion into a vector. This cassette is then inserted into an appropriate DNA vector downstream of a promoter, an mRNA leader sequence, an initiation site and other regulatory sequences capable of directing the replication and expression of that sequence in vivo . This vector permits infection of patient's cells and expression of the TAT immunogen in vivo.
C. Immunogenic Compositions of the Invention Thus, the present invention provides immunogenic compositions containing a TAT protein or peptide immunogen of the invention or the 'naked DNA' of the invention. These immunogenic compositions are capable of eliciting in an immunized host mammal, e.g., a human, an immune response capable of interdicting extracellular TAT protein.
In one embodiment, such immunogenic compositions contain at least one such protein/peptide immunogen directed to a selected native TAT protein according to the invention or a fragment thereof, together with a carrier suitable for administration as a composition for prophylactic treatment of virus infections. Alternatively, the compositions may contain more than one immunogen, the amino acid sequences of the immunogens differing to encompass allelic variants of the TAT protein. For example, a multiple antigenic peptide of the invention may contain repeats of the same peptide, e.g., for HIV, RRRAP [SEQ ID NO: 21]. The composition may preferably contain a number of MAPs, each bearing copies of different TAT peptides as described herein.
Further, where the occurrence of more than one strain of a particular virus is suspected, which strain produces a different TAT protein, novel TAT immunogens may be designed by resort to this disclosure. For example, the HIV strain of a patient not responsive to a composition of this invention may be isolated and the TAT protein sequence determined by conventional methods, e.g., polymerase chain reaction. If the sequence is indeed different from that sequence which was employed in the first composition, another TAT immunogen may be obtained and administered with the first immunogen in a single immunogenic composition. Thus, an immunogenic or vaccine composition of this invention may contain several different TAT immunogens of this invention, each directed to a different TAT protein produced as described herein. The TAT immunogen(s) of the invention or fragments thereof can be employed in a vaccine composition containing a carrier, such as saline, and a selected adjuvant, such as aqueous suspensions of aluminum and magnesium hydroxides, liposomes and others. Such compositions may optimally contain other conventionalvaccine components. Other suitable carriers and adjuvants are known to the art and the selection thereof is expected to be routine. The preparation of a pharmaceutically acceptable vaccine composition, having appropriate pH isotonicity, stability and other conventional characteristics is within the skill of the art. These immunogenic or vaccine compositions according to the present invention can be administered by an appropriate route, e.g., by the oral, intranasal, subcutaneous, intravenous, intraperitoneal or intramuscular routes. The presently preferred route of administration is intramuscular.
The amount of the TAT immunogen of the invention present in each vaccine dose is selected with regard to consideration of the patient's age, weight, sex, general physical condition and the like. The amount required to induce an immune response, preferably a protective response, in the patient without significant adverse side effects may vary depending upon the immunogen employed and the optional presence of an adjuvant. Generally, it is expected that each dose will comprise between about 50 μg to about 1 mg of TAT protein/peptide immunogen per L of a sterile solution. A more preferred dosage may be about 200 μg of TAT immunogen of this invention. Other dosage ranges may also be contemplated by one of skill in the art. Initial doses may be optionally followed by repeated boosts, where desirable.
Alternatively, immunogenic compositions of this invention may be designed for direct administration of •naked DNA' encoding one or more TAT immunogens of this invention. As discussed above with reference to the recited art on the administration of DNA vaccines, it is anticipated that nucleic acid sequences encoding one or more desired TAT immunogens of this invention may be directly delivered into the patient, alone or as part of a plasmid. Suitable vehicles for direct DNA administration include, without limitation, saline, or sucrose, prota ine, polybrene, polylysine, polycations, proteins, CaP04 or spermidine. See e.g, PCT application WO94/01139 and the references cited above. As with the protein immunogenic compositions, the amounts of components in the 'naked DNA1 composition and the mode of administration, e.g., injection or intranasal, may be selected and adjusted by one of skill in the art.
Thus, the immunogenic compositions of this invention are designed to prevent infection by the selected virus of an uninfected mammal, e.g. , human. Such immunogenic compositions thus have utility as vaccines.
D. Therapeutic Uses of the Invention
It is also further anticipated that the compositions of this invention may also be employed therapeutically to treat already infected patients. The mechanism of the present invention as described above may be useful in impeding the course of viral infection and producing desirable clinical results. More specifically, the compositions of this invention are capable of reducing viremia in patients already infected with the virus by blocking further uptake of the TAT protein by uninfected cells. In conjunction with other therapeutic regimens for HIV infected patients, for example, the compositions of the present invention are anticipated to assist in the reduction of viremia and prevention of clinical deterioration. For such therapeutic uses, the formulations and modes of administration are substantially identical to those described specifically above and may be administered concurrently or simultaneously with other conventional therapeutics for the specific viral infection. For therapeutic use, repeated dosages may be desirable.
E. Other Utilities of the TAT Immunogens of this Invention
As with other peptides, proteins, and nucleic acid sequences in general, the TAT immunogens of this invention may have a number of other utilities. For example, the TAT peptide or protein immunogens of this invention may be employed in conventional assays, e.g.. Western blots, ELISA, and other protein binding assays, for use in identifying antibodies which are capable of detecting HIV strains with different extracellular TAT proteins.
Similarly, the DNA sequences encoding the TAT immunogens or complementary strands thereto may be used in nucleic acid assays, such as Northern and Southern blots, and other nucleic acid hybridization assays for the same purpose. The nucleic acid sequences encoding the TAT immunogens of the invention may be employed as polymerase chain reaction sequences to identify HIV strains on the basis of the sequence of the tat gene.
Other uses for the proteins are in the development of antibodies which are directed to the cellular uptake regions.
Polyclonal antibodies may be produced by conventional methods, i.e., by injecting a laboratory animals with sufficient quantities of the TAT protein or fragments thereof and collecting the antibodies from the animal's tissue.
A "monoclonal antibody" refers to homogenous populations of immunoglobulins which are capable of specifically binding to the TAT protein of this invention. It is understood that the TAT protein may have one or more antigenic determinants. The antibodies of the invention may be directed against one or more of these determinants.
Hybridomas capable of secreting these monoclonal antibodies are produced by conventional techniques by employing as antigen, the TAT protein described herein. Generally, the hybridoma process involves generating a B- lymphocyte which produces a desired polyclonal antibody. Techniques for obtaining the appropriate lymphocytes from mammals injected with the target antigen are well known. Generally, the peripheral blood lymphocytes (PBLs) are used if cells of human origin are desired. If non-human sources are desired, spleen cells or lymph nodes from other mammalian sources are used. A host animal, e.g. a rabbit, is injected with repeated doses of the purified antigen, and the mammal is permitted to generate the desired polyclonal antibody producing cells.
Thereafter the B-lymphocytes are harvested for fusion with the immortalizing cell line. Immortalizing cell lines are usually transformed mammalian cells, particularly cells of rodent, bovine and human origin. Most frequently, rat or mouse myeloma cells are employed. Techniques for fusion are also well known in the art and generally involve mixing the cells with a fusing agents, e.g. polyethylene glycol.
Immortalized hybridoma cell lines are selected by standard procedures, such as HAT selection. From among these hybridomas, those secreting the desired monoclonal antibody are selected by assaying the culture medium by standard immunoassays, such as Western blotting, ELISA, or RIA. Antibodies are recovered from the medium using standard purification techniques. See, generally, Kohler et al. Nature, 256:495 (1975) for antibody production techniques. Alternatively, non-fusion techniques for generating an immortal antibody-producing hybridoma cell line is possible, e.g. virally induced transformation.
Recombinant techniques, such as described by Huse et al. Science, 246:1275-1281 (1988), or any other modifications thereof known to the art may also be employed to produce recombinant antibodies and chimeric antibodies, where desired.
These antibodies are likewise useful in diagnostic methods for detecting and identifying different HIV strains based on the differences in their extracellular tat genes. These antibodies may perform such detection in conventional assays known to one of skill in the art.
Other utilities are conventional and may include use of the TAT protein and peptide immungens as molecular weight markers.
The following examples illustrate preferred methods for preparing TAT immunogens of the invention and utilizing these immunogens to induce immune response to the virus in an immunized host. These examples are illustrative only and do not limit the scope of the invention.
EXAMPLE 1 - SYNTHESIS OF PEPTIDES OF THE INVENTION - SIV TAT AND HIV-1 TAT SEQUENCES The peptides of the SIV TAT and HIV-1 TAT were synthesized on an Applied Biosystems Model 430A peptide synthesizer by the solid-phase method developed by Merrifield, cited above. Peptide assembly was carried out starting with the p-methylbenzhydrylamine resin andthe Boc-protected amino acids using a symmetrical anhydride activation procedure. All the amino acids were double coupled to ensure complete coupling. Side chain protecting groups were as follows: benzyl ether (Bzl) for the hydroxy1 group of threonine and serine; benzyl ester for the carboxyl group of aspartic and glutamic acids; tosyl (Tos) for the guanidine of arginine; benzyloxymethyl (Bom) for the imidazole of histidine; 2- chlorobenzyloxycarbonyl (Cl-Z) for the e-amine of lysine; 2-bromobenzyloxycarbonyl (Br-Z) for the phenolic hydroxy1 of tyrosine. Asparagine, glutamine, and arginine were coupled as their 1-hydroxybenzotriazole esters.
A. Synthesis of Tyr-Arg-Arg-Arg-Thr-Pro-Lγs-Lγs- Thr-Lys-Ala-Asn-Thr-Ser-Ser-Ala-Ser-NH2: rTyrP-SIV ΓAΓB0951-
Figure imgf000023_0001
The protected peptide, Boc-Tyr(Br-Z)-Arg(Tos)- Arg(Tos) -Arg(Tos) -Thr(Bzl) -Pro-Lys(Cl-Z)-Lys(Cl-Z) - Thr(Bzl) -Lys(Cl-Z) -Ala-Asn-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)- Ala-Ser(Bzl)-(Me-BHA)-resin was synthesized by the symmetrical anhydride coupling technique, utilizing standard coupling protocols (Std 1 cycle, version 1.40) on the ABI 430A peptide synthesizer. [Note that the initial Tyr is referred to as Tyr0, since the first SIV tat amino acid is Arg (SIV TAT protein amino acid 80) . The Tyr is added to permit ligand attachment.] The synthesis was initiated with p-methylbenzhydrylamine resin (0.6 mmole, 780 mg, 0.77 meq/g) . The N-terminal Boc-group was removed by the end-NH2 program (version 1.40). The resin was then washed with DMF (5 X 10 mL) and CH2C12 (5 X 10 mL) , and dried in a vacuum oven at 30°C (2.96 g) .
The peptide was cleaved from the resin support (1.48 g) by stirring in liquid HF (50 L) , p-cresol (1.2 mL) , p-thiocresol (1.2 L) , and dimethyl sulfide (1.2 mL) for 1 hour at 0°C. After the removal of excess HF under reduced pressure, the resin-peptide mixture was extracted with anhydrous diethyl ether (3 x 200 mL) . The ether extracts were discarded. The cleaved peptide was then extracted with 30% aqueous acetic acid (3 X 70 mL) . After removal of solvents under reduced pressure, the residue obtained was dissolved in water and freeze-dried. The crude peptide was dissolved in 30% acetic acid and passed through an Amberlite IRA-68 (acetate form) ion exchange column (60 g, 1.6 meq/mL, 2.73 cm i.d. X 18 cm length) in 30% aqueous HOAc at a flow rate of 60 mL/h. The appropriate fractions from two such experiments were combined and freeze-dried (1.59 g) .
The crude peptide was dissolved in 0.1% TFA/H20 and purified by preparative RP-HPLC using a Vydac 218TP1022 column (22 X 250 mm) . The mobile phases employed were as shown below:
A = 0.1% TFA/H20
B = 0.1% TFA/CHjCN - H20, 4:1, v/v A linear gradient of 0% B to 8% B over 80 minutes at a flow rate of 8 mL/minute was used. The fractions were analyzed by HPLC and those containing pure peptide were combined and the organic solvents were removed under reduced pressure. The residue was dissolved in water, converted into the acetate form by passing through an ion-exchange column, and freeze-dried (327 mg) .
Thin layer chromatography (TLC) was performed on Merck F-254 plates (5 X 10 cm) in the following solvent systems (v/v) : Rf(l) = 0.65 (1-BuOH:Pyr:HOAc:H20,
5:4:8:2)
Rf(2) = 0.25 (1-BuOH:Pyr:HOAc:H20, 5:4:4:2)
Amino acid analysis (AAA) : Ala 2.16 (2), Arg 3.23 (3), Asx 1.23 (1),
Lys 3.21 (3), Pro 1.07 (1), Ser 2.27 (3), Thr 2.77 (3), Tyr 1.09 (1). During the hydrolysis of the peptide, Ser undergoes decomposition resulting in low recovery. Liquid Chromatography - Mass Spectrometry (LC-
MS) :
[MH+] at m/z = 651.9 a.m.u., charge 3+; 977.1 a.m.u., charge 2+. Observed mol. wt. 1952 (Theoretical mol. wt. 1951.2).
B. Synthesis of Ser-Tyr-Gly-Arg-Lvs-Lvs-Arg-Arg- Gln-Arg-Arg-Arg-Ala-Pro-Gln-Glv-Ser-Gln-NH,: THIV-l TAT^
Figure imgf000025_0001
This peptide was synthesized as described above, using the appropriate protected amino acid derivatives. The characteristics of the peptide are as follows.
TLC: Rf(l) - 0.24 (1-BuOH:Pyr:HOAc:H20, 5:4:8:2, v/v) Rf(2) - 0.09 (1-BuOH:Pyr:HOAc:H20,
5:4:4:2, v/v)
AAA: Ala 1.71 (1), Arg 5.85 (6), Glx 2.97 (3), Gly 1.87 (2), Lys 2.03 (2), Pro 1.37 (1), Ser 1.30 (2), Tyr 0.93 (l) . LC-MS: [MH+] at m/z = 1108.2 a.m.u., charge
2+. Observed mol. wt. 2214.4 (Theoretical mol. wt. 2214.5) .
EXAMPLE 2 - SYNTHESIS OF MULTIPLE ANTIGENIC PEPTIDES OF THE SIV TAT AND HIV-1 TAT SEQUENCES
Traditionally, small peptides have been conjugated to larger carrier proteins or self-polymerized to render them immunogenic. Both of these techniques suffer from significant limitations that reduce their utility for the production of highly specific antibodies. These obstacles were overcome by using the multiple antigen peptide system of D. N. Posnett et al, J. Biol. Chem.. 263:1719-1725 (1988) . The peptide was synthesized directly onto a branching lysine core with 8 copies of the peptide sequence linked to the core by the carboxy terminal amino acid. The octameric multiple antigenic peptide was highly immunogenic in mice and rabbits, allowing production of monoclonal and polyclonal antibodies of high titer. The resultant antibodies were capable of recognizing the specific peptide sequence within a larger synthetic peptide fragment or natural SIV TAT protein.
A. Synthesis of Multiple Antigenic Peptide: f (Boc)-Lys(Boc) 1ι-Lvs-,-Lys-Glv-0CH?-PAM Resin The synthesis of a multiple antigenic peptide was accomplished manually by a stepwise solid-phase procedure on Boc-Gly-OCH2-PAM resin. The synthesis was initiated with 1.5 mmoles (1.97 g, 0.76 mmol/g) of the resin. The synthesis of the first and every subsequent level of the carrier core was achieved using a 3 molar excess of Boc-Lys(Boc)-OH. All the couplings were done by the DCC-HOBt method following the protocol given below: Step Reagent Time (minutes) 1. CH2C12 wash 3 X 1 min .
2. 40% TFA - 10% anisole in CH2C12 1 X 5 min .
3. 40% TFA - 10% anisole in CH2C12 1 X 25 min .
4. CH2C12 wash 3 X 1 min .
5 . 10% NMM in CH2C12 2 X 5 min . 6 . CH2C12 wash 3 X 1 min .
7 . DMF wash 2 X 1 min .
8. Boc-Lys(Boc)-OH (4.5 mmol) and 1 X 3 min . HOBt (1.5 mmol) in DMF
9. DIC (4.5 mmol) added to the 1 X 180 min . above and shaken 10. Recouple, if necessary by repeating steps 4-9
11. DMF wash 3 X 1 min. After three successive couplings of Boc- Lys(Boc)-OH, the resin was washed with DMF (3 X 50 mL) and CH2C12 (3 X 50 mL) and dried in a vacuum oven at 30°C (3.71 g). B. Synthesis of [Arg-Arg-Arg-Thr-Pro-Lys-Lys-Thr- Lvs-Ala-Asn-Thr-Ser-Ser-Ala-Ser1 -Lys^-Lys?-Lvs-Glv-0H: f (SIV TAT^n<) Multiple Antigenic Peptidel fSEO ID NO: 231
The protected precursor of (SIV TATS0_9S) multiple antigenic peptide, [Boc-Arg(Tos)-Arg(Tos)- Arg(Tos)-Thr(Bzl)-Pro-Lys(Cl-Z)-Lys(Cl-Z)-Thr(Bzl)- Lys(Cl-Z)-Ala-Asn-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Ala- Ser(Bzl) ]8-Lys4-Lys2-Lys-Gly-OCH2-PAM resin was synthesized by growth from both the a and e amino groups using the symmetrical anhydride coupling technique, utilizing the standard coupling protocols (std 1 cycle, software version 1.40) on the ABI 430A peptide synthesizer. All the amino acids were double coupled to ensure complete coupling. The synthesis was initiated with [Boc- Lys(Boc) ]4-Lys2-Lys-Gly-OCH2-PAM resin (0.22 mmol, 94 mg) prepared as described in A. above. The N-terminal Boc group was removed by the end-NH2 program (version 1.40). The resin was then washed with DMF (5 X 10 mL) and CH2C12 (5 X 10 mL) , and dried in a vacuum oven at 30°C (1.9 g) . The protected peptide-resin was treated with liquid hydrogen fluoride, in the presence of p-cresol, p- thiocresol and dimethylsulfide as scavengers, at 0°C for 1 hour with constant stirring. Excess HF was removed by vacuum and the residue treated with ether to remove scavenger products. The peptide was extracted with 50% acetic acid (3 X 50 mL) , the solvents evaporated in vacuo, and the product freeze-dried.
The crude peptide was initially purified on an Amberlite IRA-68 ion-exchange column; further purification was accomplished by RP-HPLC on a preparative C18 column. The solvents used were: water containing 0.1% TFA (Buffer A) and CH3CN - H20 (4:1) containing 0.1% TFA (Buffer B) . A linear gradient of 0-20% B over 100 minutes was used. The appropriate fractions containing the peptide were pooled, the solvents evaporated in vacuo , and the product freeze-dried (644 mg) . The purified peptide gave satisfactory amino acid analysis.
AAA: Ala 18.42 (16) , Arg 23.74 (24), ASX 10.41 (8) , Gly 1.47 (1), Lys 25.91 (31) , Pro 8.14 (8), Ser 23.68 (24), Thr 23.99 (24) . C. Synthesis of rSer-Tyr-Glv-Arg-Lys-Lys-Arg-Arg- Gln-Arg-Arg-Arg-Ala-Pro-Gln-Glv-Ser-Glnlo-Lvs^Lys-i-Lvs- Glv-OH: r (HIV-1 TAT^ ^ Multiple Antigenic Peptidel TSEO ID NO: 24]
This peptide was synthesized as described above, using the appropriate protected amino acid derivatives. The purified peptide gave satisfactory amino acid analysis.
AAA: Ala 9.74 (8) , Arg 46.5 (48) , Glu 26.55 (24) , Lys 23.21 (23) , Pro 8.56 (8) , Ser 10.95 (16) , Tyr 7.52 (8) . During the hydrolysis of the peptide, Ser undergoes decomposition resulting in low recoveries. D. Other Multiple Antigenic Peptides
Making use of repeating fragments of the SIV TAT protein, the following multiple antigenic peptides can be produced using the techniques described above.
(Gln-Arg-Arg-Arg-Thr-Pro-Gln-Arg-Arg-Arg-Thr- Pro-Gln-Arg-Arg-Arg-Thr-Pro-Gln)g-MAP [SEQ ID NO: 25].
(Pro-Lys-Lys-Thr-Lys-Thr-His-Pro-Lys-Lys-Thr- Lys-Thr-His-Pro-Lys-Lys-Thr-Lys-Thr-His)g-MAP
[SEQ ID NO: 26] . (Pro-Lys-Lys-Ala-Lys-Thr-His-Pro-Lys-Lys-Ala- Lys-Thr-His-Pro-Lys-Lys-Ala-Lys-Thr-His)g-MAP [SEQ ID NO: 27] .
Similarly, making use of repeating fragments of the HIV TAT protein, the following MAPs can be produced using methods analogous to those described above.
(Gln-Arg-Arg-Arg-Ala-Pro-Gln-Arg-Arg-Arg-Ala- Pro-Gln-Arg-Arg-Arg-Ala-Pro-Gln)g-MAP [SEQ ID NO: 28]. (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Gly-Arg-Lys-
Lys-Arg-Arg-Gln-Gly-Arg-Lys-Lys-Arg-Arg-Gln)8- MAP [SEQ ID NO: 29] .
EXAMPLE 3: IMMUNIZATION OF ANIMALS WITH ANTI-TAT MULTIPLE ANTIGENIC PEPTIDES
The following experiment is performed to evaluate whether immunization with a multiple antigenic peptide of this invention in alum adjuvant induces a high titer antibody response and whether the presence of such antibodies will prevent or ameliorate acute viremia and subsequent disease progression in rhesus monkeys inoculated with infectious SIV.
Young adult, male, colony born rhesus monkeys (Maraca mulatta) older than 2 years of age, but weighing less than 10 kg (a species commonly used in experimental models of SIV infection) are employed. The animals are fed a commercial primate diet, optionally supplemented with fresh fruit. Water is provided ad libitum. Animals are randomized into two treatment groups (peptide or placebo) .
Prior to challenge infection with SIV, the animals are immunized intramuscularly (by thigh injection) on day 0, day 10 and monthly as necessary with either 200 micrograms of test peptide mixed with alum as an adjuvant or a saline-adjuvant placebo, until suitable antibody titers are attained.
TABLE 1 Peptide
Group No. Immunogen Route Dose Vol.
1 10 placebo1 i. . 0 μg 400 μL 2 10 test peptide i.m. 200μg2 400 μL
1 Saline adjuvant only (unimmunized control)
2 Dissolved in saline and mixed 1:1 in an alum suspension
Serum for antibody testing is collected prior to initial immunization, on day 7 and on day 17 post immunization and then 7 days after each monthly injection until suitable antibody titers are attained. SIV antibody in serum is determined by both radioimmunoassay and ELISA and is confirmed by Western Blot analysis.
After suitable antibody titers are attained in the peptide immunized animals, all animals are challenged intravenously with 50 AIDJ0SIV delta B670 virus by way of the saphenous vein using a 23 gauge butterfly needle. Needles are flushed with PBS before and after virus to assure accurate delivery of material. The B670 SIV strain is a well-characterized viral isolate originally isolated at Tulane Regional Primate Research Center (TRPRC) .
At selected times after infection the clinical, immunological and virological status of the animals are evaluated. For example, a lymph node biopsy is performed at 2 and 4 weeks after challenge, and a sample of blood is collected for the determination of antibody titers at week 4 after challenge and then monthly until death or completion of study. For all measurements of antibody. the mean titer level, expressed on a log10 scale is computed at each time point.
Results of this protocol are anticipated to reveal significant protection of the animals by use of the peptide immunogens of this invention.
EXAMPLE 4: EVALUATION OF ANTIBODY TITERS OF IMMUNIZED MONKEYS WITH TAT IMMUNOGEN FOR SIV
Serum from monkeys immunized with the SIV TATi0.95 multiple antigenic peptide are tested for the presence of anti-SIV TAT binding activity. This activity is assessed by two independent methods.
First [Tyr0]-SIV TAT8r95 is radiolabeled with 125I by the pulse labeling method of Culler et al, Proc. Soc. EXP. Biol. Med.. 173:264-268 (1985) and purified by partition chromatography [QUSO, Degussa Corporation, Dublin, Ohio] and subsequent high pressure liquid chromatography. The HPLC elution profile of [125I-TYRo]- SIV TATi0_9S is illustrated in Fig. 1. Serial dilutions of the monkey serum are incubated with the [125I-TYRo]-SlV TATi0_95 for 18-24 hours at room temperature. The antibodies present in the samples are precipitated, along with any bound [15I-TYRo]-SIV TATi(.95 , by subsequent incubation with goat anti-monkey serum followed by centrifugation. The supernatant is removed and the remaining pellet is counted in a gamma counter to determine the amount of [125I-TYRo]-SIV TATia.95 bound by the monkey serum dilutions. For comparison, titer is defined as the dilution of monkey serum required to bind 50% of the added [125I-TYRo]-SIV TATiM5 tracer.
Second, the monkey serum is titrated by enzyme- linked immunoassay (ELISA) on polystyrene microtiter plates that have previously been coated with both the SIV TATi0_95 multiple antigenic peptide to determine the total binding activity, and with ovalbumin to determine amount of the binding that is not specific for SIV TATi0.9S . Briefly, the monkey serum is serially diluted using 0.01M phosphate buffered saline containing 0.01% Tween 20 and 0.5% BSA (PBS/Tween 20) . 100 μl of each serial dilution of monkey serum is added in triplicate to the wells of a 96-well microtiter plate coated with either SIV TATi0_9S multiple antigenic peptide or ovalbumin. The plates are incubated for 2 hours at 37°C with rocking. Following incubation, the plates are washed 5 times with the PBS/Tween 20 buffer. Goat anti-monkey IgG, labeled with horse-radish peroxidase (HRPO) , is added to each well at the appropriate dilution in PBS/Tween 20 buffer. Following an incubation at 37°C for 1 hour, the plates are again washed 5 times. The specific HRPO substrate, Ultrablue (Transgenic Sciences, Inc., Milford, MA), is added and incubated in the dark for 30 minutes at room temperature. The reaction is terminated by adding 2.5N HCl to each well and the optical density of each well is measured at 450 nm. For comparison, the titer is defined as the concentration of monkey serum which results in an optical density reading of 1.0. The titers from each monkey are followed over time to determine empirically when the plateau antibody response against the SIV TATi0_9S multiple antigenic peptide is attained. Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Immunobiology Research, Institute Inc.
(ii) TITLE OF INVENTION: Vaccine Interdiction of
Extracellular Transactivating Proteins of Human Immunodeficiency Virus and Other Chronically Infecting Viruses Employing Similar Intercellular Transactivating Strategies
(iii) NUMBER OF SEQUENCES: 38
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Howson and Howson
(B) STREET: Spring House Corporate Cntr, PO Box 457
(C) CITY: Spring House
(D) STATE: Pennsylvania
(E) COUNTRY: USA
(F) ZIP: 19477
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/247,991
(B) FILING DATE: 23-MAY-1994
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Bak, Mary E.
(B) REGISTRATION NUMBER: 31,215
(C) REFERENCE/DOCKET NUMBER: IRI44PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 215-540-9200
(B) TELEFAX: 215-540-5818 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 261 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..258
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
ATG GAG CCA GTA GAT CCT AGA CTA GAG CCC TGG AAG CAT CCA 42 Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro 1 5 10
GGA AGT CAG CCT AAA ACT GCT TGT ACC AAT TGC TAT TGT AAA 84 Gly Ser Gin Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys 15 20 25
AAG TGT TGC TTT CAT TGC CAA GTT TGT TTC ATA ACA AAA GCC 126 Lys Cys Cys Phe His Cys Gin Val Cys Phe lie Thr Lys Ala 30 35 40
TTA GGC ATC TCC TAT GGC AGG AAG AAG CGG AGA CAG CGA CGA 168 Leu Gly lie Ser Tyr Gly Arg Lys Lys Arg Arg Gin Arg Arg 45 50 55
AGA CCT CCT CAA GGC AGT CAG ACT CAT CAA GTT TCT CTA TCA 210 Arg Pro Pro Gin Gly Ser Gin Thr His Gin Val Ser Leu Ser 60 65 70
AAG CAA CCC ACC TCC CAA TCC CGA GGG GAC CCG ACA GGC CCG 252 Lys Gin Pro Thr Ser Gin Ser Arg Gly Asp Pro Thr Gly Pro
75 80
AAG GAA TAG 261
Lys Glu 85
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 86 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
( i) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly 1 5 10 15
Ser Gin Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys
20 25 30
Cys Phe His Cys Gin Val Cys Phe lie Thr Lys Ala Leu Gly lie
35 40 45
Ser Tyr Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg Pro Pro Gin
50 55 60
Gly Ser Gin Thr His Gin Val Ser Leu Ser Lys Gin Pro Thr Ser
65 70 75
Gin Ser Arg Gly Asp Pro Thr Gly Pro Lys Glu
80 85
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ser Tyr Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg Ala Pro 1 5 10
Gin Gly Ser Gin 15
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Ser Tyr Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg Ala Pro 1 5 10
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Ser Tyr Gly Lys Lys Lys Arg Arg Gin Arg Arg Arg Ala Pro 1 5 10
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Ser Tyr Gly Arg Lys Lys Arg Lys Gin Arg Arg Arg Ala Pro 1 5 10
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Ser Tyr Gly Arg Lys Lys Arg Arg Pro Arg Arg Arg Ala Pro 1 5 10 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: "
Ser Tyr Gly Arg Lys Lys Arg Arg Gin Arg Gin Arg Ala Pro 1 5 10
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Ser Tyr Gly Arg Lys Lys Arg Arg Gin Arg Arg Gly Ala Pro 1 5 10
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Ser Tyr Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg Thr Pro 1 5 10
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Ser Tyr Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg Pro Pro 1 5 10
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Ser Tyr Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg Ala His 1 5 10
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Arg Arg Arg Thr Pro Lys Lys Thr Lys Ala Asn Thr Ser Ser 1 5 10
Ala Ser Tyr 15
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Tyr Glu Gin Gin Arg Arg Arg Thr Pro Lys Lys Thr Lys Ala 1 5 10
Asn Thr Ser Ser Ala Ser 15 20
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Cys Phe lie Thr Lys Ala Leu Gly lie Ser Tyr Gly Arg Lys 1 5 10
Lys Arg Arg Gin Arg Arg Arg Pro Pro Gin Gly Ser Gly Thr 15 20 25
His Gin Val Ser Leu Ser Lys Gin 30 35
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Gin Arg Arg Arg Ala Pro 1 5
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Gly Arg Lys Lys Arg Arg Gin 1 5
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Pro Lys Lys Thr Lys 1 5
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Pro Lys Lys Ala Lys 1 5
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Gin Arg Arg Arg Thr Pro 1 5 (2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Arg Arg Arg Ala Pro 1 5
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
( i) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Tyr Arg Arg Arg Thr Pro Lys Lys Thr Lys Ala Asn Thr Ser 1 5 10
Ser Ala Ser 15
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Arg Arg Arg Thr Pro Lys Lys Thr Lys Ala Asn Thr Ser Ser 1 5 10
Ala Ser Lys Lys Lys Lys Lys Lys Lys Gly 15 20 (2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Ser Tyr Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg Ala Pro 1 5 10
Gin Gly Ser Gin Lys Lys Lys Lys Lys Lys Lys Gly 15 20 25
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Gin Arg Arg Arg Thr Pro Gin Arg Arg Arg Thr Pro Gin Arg 1 5 10
Arg Arg Thr Pro Gin 15
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Pro Lys Lys Thr Lys Thr His Pro Lys Lys Thr Lys Thr His 1 5 10
Pro Lys Lys Thr Lys Thr His 15 20 (2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Pro Lys Lys Ala Lys Thr His Pro Lys Lys Ala Lys Thr His 1 5 10
Pro Lys Lys Ala Lys Thr His 15 20
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Gin Arg Arg Arg Ala Pro Gin Arg Arg Arg Ala Pro Gin Arg 1 5 10
Arg Arg Ala Pro Gin 15
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Tyr Gly Arg Lys Lys Arg Arg Gin Gly Arg Lys Lys Arg Arg 1 5 10
Gin Gly Arg Lys Lys Arg Arg Gin 15 20 (2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Arg Arg Arg Ala Pro 1 5
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
Arg Gin Arg Ala Pro 1 5
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Arg Arg Gly Ala Pro 1 5
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Arg Arg Arg Thr Pro 1 5
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
Arg Arg Arg Pro Pro 1 5
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
Arg Arg Arg Ala His 1 5
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Arg Lys Lys Arg Arg 1 5 (2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37
Lys Lys Lys Arg Arg 1 5
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
Arg Lys Lys Arg Lys 1 5

Claims

WHAT IS CLAIMED IS:
1. An isolated immunogen comprising an amino acid sequence comprising the cellular uptake region or a fragment thereof of a native transactivating (TAT) protein from a selected virus, said immunogen capable of eliciting antibody against said native protein.
2. The immunogen according to claim 1 wherein said protein is a TAT protein of human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) .
3. The immunogen according to claim 2 wherein said sequence comprises a TAT protein having amino acid deletions at the amino terminus.
4. The immunogen according to claim 2 wherein said sequence comprises a TAT protein sequence having amino acid deletions or substitutions at one or more cysteine residues which normally participate in disulfide bonding.
5. The immunogen according to claim 3 wherein said deletion comprises about 3 to about 10 amino acids at the amino terminus.
6. The immunogen according to claim 1 wherein said cellular uptake region is an HIV TAT sequence selected from the group consisting of
SYGRKKRRQRRRAPQGSQ SEQ ID NO: 3,
SYGRKKRRQRRRAP SEQ ID NO: 4,
SYGKKKRRQRRRAP SEQ ID NO: 5,
SYGRKKRRQRRRAP SEQ ID NO: 6,
SYGRKKRRPRRRAP SEQ ID NO: 7,
SYGRKKRRQRQRAP SEQ ID NO: 8,
SYGRKKRRQRRGAP SEQ ID NO: 9, SYGRKKRRQRRRTP SEQ ID NO: 10,
SYGRKKRRQRRRPP SEQ ID NO: 11,
SYGRKKRRQRRRAH, and SEQ ID NO: 12, fragments and allelic variants thereof.
7. The immunogen according to claim 6 wherein said fragment is selected from the group consisting of RRRAP, RQRAP, RRGAP, RRRTP, RRRPP, RRRAH, RKKRR, KKKRR, and RKKRK (SEQ ID NOS: 30- 38) .
8. The immunogen according to claim 1 wherein said cellular uptake region is an SIV TAT sequence selected from the group consisting of RRRTPKKTKANTSSASY (SEQ ID NO: 13), YEQQRRRTPKKTKANTSSAS (SEQ ID NO: 14), and fragments and allelic variants thereof.
9. The immunogen according to claim 8 wherein said fragment is selected from the group consisting of PKKAK (SEQ ID NO: 19), PKKTK (SEQ ID NO: 18) and RRRTP (SEQ ID NO: 33).
10. The immunogen according to claim 1 comprising a synthetic peptide from a selected region of said protein coupled to a carrier.
11. The immunogen according to claim 10 comprising a construct comprising a plurality of synthetic peptides from said protein in the form of a multiple antigenic peptide.
12. The immunogen according to claim 1 wherein said sequence does not share the biological activity of the native protein.
13. A composition useful for inducing an immune response in a patient protective against infection with a virus characterized by a transactivating protein, the composition comprising an immunogen capable of eliciting antibody against a native transactivating protein from said virus, said immunogen comprising an amino acid sequence comprising the cellular uptake region of said protein or a fragment thereof in a selected pharmaceutical carrier.
14. The composition according to claim 13 wherein said carrier comprises an adjuvant selected from the group consisting of alum, liposomes, and magnesium hydroxide.
15. The composition according to claim 13 comprising an immunogen of any of claims 2-12.
16. The composition according to claim 13 comprising at least two different immunogens.
17. An isolated nucleic acid sequence encoding the immunogen of any of claims 1 to 12.
18. The sequence according to claim 17, said sequence suitable for direct administration into a mammal and in vivo expression of said immunogen.
19. A nucleic acid molecule comprising the sequence of claim 17 in association with nucleic acid sequences capable of regulating the replication and expression of said TAT sequence in vivo in said mammal or in vitro in a host cell culture.
20. An antibody capable of identifying a virus strain having a selected extracellular TAT protein, wherein said antibody is capable of binding to an immunogen of any of claims 1-12.
21. A method of producing an antibody capable of identifying a virus strain having an extracellular TAT protein, said method comprising immunizing an animal with an immunogen of any of claims 1-12 and isolating said antibody from the tissue of said animal.
22. The use of an immunogen of any of claim 1 to 12 or a nucleic acid sequence of claim 17 in preparing a medicament for immunizing a patient against infection with a virus characterized by a transactivating protein.
23. The use of an immunogen of any of claim 1 to 12 or a nucleic acid sequence of claim 17 in preparing a medicament for reducing viremia in a mammal infected with a virus characterized by a transactivating protein.
PCT/US1995/006077 1994-05-23 1995-05-16 Compositions of transactivating proteins of human immunodeficiency virus WO1995031999A1 (en)

Priority Applications (2)

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EP95921262A EP0767678A1 (en) 1994-05-23 1995-05-16 Compositions of transactivating proteins of human immunodeficiency virus
AU26382/95A AU2638295A (en) 1994-05-23 1995-05-16 Compositions of transactivating proteins of human immunodeficiency virus

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US08/247,991 1994-05-23

Publications (1)

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US5877282A (en) * 1996-09-20 1999-03-02 Bristol-Myers Squibb Company Peptide inhibitors of nuclear protein translocation having nuclear localization sequences and methods of use thereof
US6525179B1 (en) 1997-07-11 2003-02-25 Thymon L.L.C. Methods and compositions for impairing multiplication of HIV-1
US5891994A (en) * 1997-07-11 1999-04-06 Thymon L.L.C. Methods and compositions for impairing multiplication of HIV-1
US7008622B2 (en) 1997-07-11 2006-03-07 Thymon, L.L.C. Methods and compositions for impairing multiplication of HIV-1
EP0994724A4 (en) * 1997-07-11 2004-12-08 Thymon Llc Methods and compositions for impairing multiplication of hiv-1
EP0994724A1 (en) * 1997-07-11 2000-04-26 Thymon L.L.C. Methods and compositions for impairing multiplication of hiv-1
US6193981B1 (en) 1997-07-11 2001-02-27 Thymon L.L.C. Methods and compositions for impairing multiplication of HIV-1
WO1999027958A3 (en) * 1997-12-01 1999-07-22 Ist Superiore Sanita Hiv-1 tat, or derivatives thereof for prophylactic and therapeutic vaccination
US8197820B2 (en) 1997-12-01 2012-06-12 Istituto Superiore di Sanità HIV-1 Tat, or derivatives thereof for prophylactic and therapeutic vaccination
AU762893B2 (en) * 1997-12-01 2003-07-10 Istituto Superiore Di Sanita HIV-1 Tat, or derivatives thereof for prophylactic and therapeutic vaccination
EP1698347A2 (en) * 1997-12-01 2006-09-06 Istituto Superiore di Sanità hiv-1 tat, or derivatives thereof for prophylactic and therapeutic vaccination
WO1999027958A2 (en) * 1997-12-01 1999-06-10 Istituto Superiore Di Sanita' Hiv-1 tat, or derivatives thereof for prophylactic and therapeutic vaccination
US7744896B1 (en) 1997-12-01 2010-06-29 Istituto Superiore Di Sanita' HIV-1 Tat compositions
EP1698347A3 (en) * 1997-12-01 2006-12-20 Istituto Superiore di Sanità hiv-1 tat, or derivatives thereof for prophylactic and therapeutic vaccination
US7022326B1 (en) 1997-12-26 2006-04-04 Biovacs, Inc. Carboxymethylated retroviral regulatory proteins and interferon-α
EP1975173A1 (en) 1997-12-26 2008-10-01 Neovacs Anti-Retroviral Immunogens, Preparation and Use
US7022482B2 (en) 1998-07-15 2006-04-04 Neovacs Use of immunogenic immunosuppressive and/or angiogenic proteins which have been rendered inactive, process for their preparation and pharmaceutical or vaccinal uses
US7504479B2 (en) 1998-07-15 2009-03-17 Neovacs Use of immunogenic immunosuppressive and/or angiogenic proteins which have been rendered inactive, process for their preparation and pharmaceutical or vaccinal uses
US8323928B2 (en) 1999-08-12 2012-12-04 Pin Pharma, Inc. Vaccines and immunotherapeutics derived from the human immunodeficiency virus (HIV) transactivator of transcription protein for the treatment and prevention of HIV disease
US6399067B1 (en) 2000-04-28 2002-06-04 Thymon L.L.C. Methods and compositions for impairing multiplication of HIV-1
US6524582B2 (en) 2000-04-28 2003-02-25 Thymon L.L.C. Methods and compositions for impairing multiplication of HIV-1
US7927580B2 (en) 2004-03-16 2011-04-19 Nanirx, Inc. Tat-based immunomodulatory compositions and methods of their discovery and use
US7563437B2 (en) 2005-02-15 2009-07-21 Thymon, Llc Methods and compositions for impairing multiplication of HIV-1
US9206239B2 (en) 2009-03-23 2015-12-08 Pin Pharma, Inc. Treatment of cancers with immunostimulatory HIV Tat derivative polypeptides
US9663556B2 (en) 2013-10-04 2017-05-30 Pin Pharma, Inc. Treatment of cancers with immunostimulatory HIV tat derivative polypeptides
US10159707B2 (en) 2013-10-04 2018-12-25 Pin Pharma, Inc. Treatment of cancers with immunostimulatory HIV Tat derivative polypeptides

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