WO2000078969A1 - Hiv tat peptides and multiple peptide conjugate system - Google Patents

Hiv tat peptides and multiple peptide conjugate system Download PDF

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Publication number
WO2000078969A1
WO2000078969A1 PCT/US2000/010119 US0010119W WO0078969A1 WO 2000078969 A1 WO2000078969 A1 WO 2000078969A1 US 0010119 W US0010119 W US 0010119W WO 0078969 A1 WO0078969 A1 WO 0078969A1
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Prior art keywords
peptide
group
tat
acid
ammo
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PCT/US2000/010119
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French (fr)
Inventor
Subhash Dhawan
Robert A. Boykins
Kenneth M. Yamada
Manju B. Joshi
Chiang Syin
Hira L. Nakhasi
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The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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Priority to AU40823/00A priority Critical patent/AU4082300A/en
Publication of WO2000078969A1 publication Critical patent/WO2000078969A1/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
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the HIV Tat protein a transactivator which regulates gene expression from the HIV long terminal repeat
  • Tat protein of human immunodeficiency virus type-1 (HIV-1 ) is an 86 ammo acid protein released by infected cells and plays a critical role in the progression of HIV disease (Jeang, J Biomed. Sci. 5. 24 (1998); Ensoli et al., J. Virol. 67: 277(1993)). Tra ⁇ sactivation of the HIV-LTR promoter by the Tat protein is essential for both viral gene expression and virus replication.
  • Extracellular Tat released by infected cells during the acute phase of infection enters non-infected cells and disrupts many host immune functions by activating a wide variety of genes regulated by specific viral and endogeneous cellular promoters (Vaishnav and Wong-Staal, Ann
  • Tat mimics many of the effects of HIV infection on monocytes including increased P-9 and cytokine production, and collagen expression in glioblastoma cells (Lafrenie et al., J. Immunol. 159: 4077 (1997);
  • HIV-1 encodes the transactivati ⁇ g protein Tat, which is essential for virus replication and progression of HIV disease.
  • Tat has multiple domains, and consequently the molecular mechanisms by which it acts remain unclear.
  • cellular activation by Tat involves a short core domain, Tat 21 40 , containing only 20 ammo acids including seven cysteine residues highly conserved in most HIV-1 subtypes.
  • Effective induction by Tat 21 0 of both NF- ⁇ B-med ⁇ ated HIV replication and TAR-dependent transactivatio ⁇ of HIV-long terminal repeat indicates that this short sequence is sufficient to promote HIV infection.
  • Tat 21 40 possesses potent angiogemc activity, further underscoring its role in HIV pathogenesis.
  • Figure 2 shows the chemical structure of one embodiment of a core template of the present invention.
  • Figure 3 shows the chemical structure of one embodiment of a base peptide of the present invention.
  • Figure 4 shows the chemical structure of one embodiment of a multiple peptide conjugate of the present invention.
  • Figure 5A and B show the sequences of malarial peptides and HIV-1 peptides used in some embodiments of the multiple peptide conjugates of the present invention.
  • (A) Letters underlined represent Hmb derivatized ammo acids for malaria peptides;
  • B letters underlined show the position of the seven c ⁇ ste ⁇ ne(tButh ⁇ o) residues in the HIV-Tat peptide (HIV-1 -Tat-1 ).
  • Figure 6 illustrates one embodiment of the synthetic process which was used to prepare a multiple peptide conjugate.
  • FIG. 7A illustrates one embodiment of a core template of the present invention.
  • Figure 7B illustrates one embodiment of a base peptide of the present invention.
  • Figure 7C illustrates one embodiment of a multiple peptide conjugate of the present invention.
  • FIGURE 8. Chemical structure of HIV-1 -Tat-MPC comprising functional epitopes of the HIV-1 -Tat protein.
  • FIGURE 9. Effect of HIV-1 -Tat synthetic peptide on HIV replication in monocytes. Monocytes were infected with HIV ⁇ a-L ⁇ n ne presence or absence of recombinant HIV-1 Tat or the group 0 peptide. At day 6, culture supernatants were harvested, and cell-free virus was determined by p24 ELISA. The data are representative of two separate experiments.
  • FIGURE 10 Panel a, Humoral response in mice immunized with various doses (10 ug, 20 ug, and 100 ug) of the HlV-1-Tat-MPC. Antisera were assayed by ELISA on 96-well plates coated with HIV-1-Tat-MPC at a concentration of 1 ug/ml. Titers shown as OD values at 650 nm of 1000-fold diluted serum specimens.
  • Panel b Ant ⁇ -HIV-1 -Tat-MPC titer of sera at various dilutions, where reactivity was determined as described for panel a. ( • ) 10 ug HIV-1 -Tat-MPC;
  • FIGURE 11 Panel a, Binding of ant ⁇ -HIV-1 -Tat-MPC antisera to recombinant (rTat) protein and Tat peptides.
  • rTat recombinant
  • Tat Tat peptides.
  • Ninety-six (96) well plates were coated with 10 ug/ml of rTat protein or Tat peptides as indicated and blocked with
  • FIGURE 12 Lymphoproliferative responses of spleen cells from Balb/c mice vaccinated with HIV 1 Tat- MPC.
  • Splenocytes (2 x 10 ⁇ ) prepared from mice immunized with 20 ug of HIV-1 Tat-MPC were incubated with 10 ug rTat , HIV-1-Tat-MPC, or Tat peptides for 96 h at 37°C, and antigen-induced proliferative responses were determined.
  • the bars represent incorporation, expressed as cpm + SEM from triplicate cultures of pooled spleen cells from two independent experiments.
  • FIGURE 13 Model illustrating a possible mechanism for inhibition of Tat-mduced HIV pathogenesis by anti- HIV-1-Tat MPC antibodies.
  • the present invention relates to the identification of functional domains of the HIV Tat protein which mediate viral activation.
  • functional domains of the HIV Tat protein which mediate viral activation.
  • the present invention uses particular peptide sequences to identify functional domains in Tat.
  • the particular sequences used in the examples below may be substituted with the corresponding sequences derived from consensus sequences for each subtype, corresponding sequences from individual isolates, or corresponding sequences from subtypes other than those specified above. Accordingly, the present invention specifically contemplates the use of the corresponding sequences from such consensus sequences, isolates or other subgroups.
  • purified does not require absolute purity; rather, it is intended as a relative definition.
  • purification of starting material or natural material to at least one order of magnitude preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated as falling within the definition of "purified.”
  • isolated requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally-occurring poly ⁇ ucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Figure 1 lists the consensus sequences of the Tat genes from the B, C, D, F, 0, and U subtypes as well as a consensus sequence derived from the sequences from all of these subtypes.
  • overlapping peptides from various domains of the consensus-B and other HIV-1 subtypes were synthesized. The ability of the peptides to direct HIV replication in monocytes was compared to that of full length recombinant Tat protein as described in Example 1 below.
  • HIV Tat was dissolved at 10 g/ml in treatment buffer (PBS containing 1 mg/ml BSA, 0.1 mM dithiothreitol) and frozen in aliquots at 80°C. Tat preparations were screened and found to be negative for endotoxin contamination.
  • treatment buffer PBS containing 1 mg/ml BSA, 0.1 mM dithiothreitol
  • Monocytes were isolated from the peripheral blood mononuclear cells of donors seronegative for HIV and hepatitis after leukapheresis and purification by countercurrent centrifugal elutnation (Wahl and Smith In Current Protocols in Immunology, J. E. Coligan, A. M. Kruisbeek, and D. H. Margulies, E. M. Shevach, and W. Strober, eds.,
  • Monocytes cultured for 5 days were treated with recombinant full length Tat protein or test peptides.
  • the cells were harvested and the concentration of p24 gag protein (of HIV) in culture super ⁇ atants was determined using a DuPont (Wilmington, DE) p24 ELISA test kit.
  • Data representative of two separate experiments are shown in Table I and expressed as mean + / SEM of triplicate determinations.
  • Tat2l -40 As shown in Table I below, like recombinant full length Tat (rTat), the 20 ammo acid core domain Tat2l -40 containing 7 cysteine residues, all of which are strongly conserved in various subtypes, enhanced HIV replication by greater than four fold.
  • a peptide derived from the basic domain (Tat53 68) induced a lesser increase in viral replication compared to Tat21-40- Likewise, the Tat g 2 ⁇ peptide also greatly enhanced HIV replication.
  • Tat4i-52 a peptide sequence located between the core and the basic domains, and a variety of peptides from other positions in the
  • Tat sequence had no significant effect on HIV replication.
  • the cytopathic effects of the Tat peptides were examined in monocytes as described in Example 2.
  • Example 2 Monocytes were infected with HIV and contacted with recombinant full length Tat or Tat peptides as described above. At five days post oculation, ceils were washed once with PBS, fixed, and Wright-stained. HIV- associated cytopathic effects were determined by examining the formation of multinucleated giant cells.
  • Tat21-40 treatment produced a marked increase in HIV- associated cytopathic effects in monocytes as indicated by formation of multinucleated giant cells; the effects were similar to those induced by rTat protein itself.
  • the effect of Tat53-68 was less than that of Tat21-40- Tat4]-52, the peptide between core and basic domains, and peptides from other Tat domains did not alter HlV-associated cytopathic effects.
  • a major active site for stimulating HIV replication and monocyte dysfunction can be localized to the 20- residue peptide Tat21 -40 and to a lesser extent to Tat53-68 peptide.
  • HIV-Tat potentiates HIV replication involves transactivation of the HIV-1 LTR via its binding to the TAR sequence along with other cellular factors, resulting in increased viral transcription initiation and elongation (Cujec et al, (1997) Mol. Cell. Biol. 17: 1817).
  • CEM lymphoid cells were transfected with wild-type promoter in the presence of various Tat peptides, and the extent of transactivation was determined using CAT assays (Cujec et al., Mol. Cell.
  • Example 3 Transactivation Assays Cells were electroporated as described in (Kashanchi et al, Virology 227: 431 (1997)). CEM cells (12D7) were cultured at a density of 0.5 to 0.8 x10 ⁇ cells/ml with daily media additions. Typically, 5x10 ⁇ cells were electroporated with 5 g of either purified plasmid or Tat protein and 5 g of reporter plasmid. Tat peptides or Tat protein and the reporter HIV LTR-CAT or the TAR mutant HIV TM26 LTR-CAT were mixed with cells and electroporated using a cell porater apparatus (GIBCO/BRL, Gaithersburg, MD). Cell mixtures were electroporated at 800 F, 240V, in RPMI 1640 medium without serum. Following electroporation, cells were plated in 10 ml complete medium, and samples were collected 24 h later for CAT assays.
  • the Tat 21 40 peptide produced a nine-fold induction of HIV-LTR, while full-length rTat produced a 25- foid induction.
  • the actual effectiveness of induction by Tat21-40 might be greater than observed due to the low solubility of this complex hydrophobic peptide in aqueous buffers.
  • buffer, Tat 1 20 , Tat 5368 , Tat 2540 , and Tat 41 52 demonstrated virtually no significant induction of HIV-1 LTR.
  • the presence of Cys22 in core domain Tat21-40 (and 3 adjacent residues) was critical for viral activation, since deletion of these residues substantially reduced the ability of Tat2l -40 to activate HIV infection (Tat25-40 m Table I).
  • Tat and cytokines play a key role in the pathogenesis of HIV infection.
  • Extracellular HIV-Tat causes activation of intracelluiar signal transduction pathways that culminate in the production of various cytokines (Lafrenie et al, J. Immunol. 159: 4077 (1997); Chen et al, J. Biol. Chem. 272: 22385 (1997)). Therefore, because of its ability to induce host factors, Tat is believed to be a key factor for viral enhancement. HIV Tat activates both virai and host cell genes, and the host F transcription factor contributes to immune dysregulation during HIV infection (Conant et al, J Virol.
  • Example 4 Effects of Tat Peptides on F Activity Monocytes (1 x10' /ml) were treated with rTat protein or Tat peptides at 37°C for 15 mm. Nuclear extracts were then prepared and analyzed by gel shift analysis as described in (Dhawan et al, Eur. J. Immunol. 27: 2172 (1997)).
  • Tat53-68- The ability of HIV-Tat to activate F was retained in core peptide Tat21 -40 and to a lesser extent Tat53- 68- Treatment of monocytes with the Tat2l-40 peptide rapidly activated F (within 15 mm. after exposure) by greater than 9-fold as compared to 3 fold induction by Tat53-68- Interestingly, despite inducing NF- B activity, Tat53-
  • Tat is released by HIV-infected cells into the extracellular milieu, and has been implicated as a cofactor in the pathogenesis of Kaposi's sarcoma (Albim et al, Proc. Natl. Acad. Sci. USA 92: 4838 (1995)), an a ⁇ gioprohferative disease frequently seen in HIV-infected individuals. There is increasing evidence that HIV-Tat induces endothelial cell migration, invasion, and angiogemc processes in vivo (Albim et al, Oncoge ⁇ e 12: 289 (1996)).
  • Example 5 In order to test for potential angiogemc activity of the core domain implicated above in viral pathogenesis, the ability of Tat peptides to induce ⁇ eovasculanzation was examined using the chick chonoallantoic membrane (CAM) assay as described in Example 5 below.
  • CAM chick chonoallantoic membrane
  • the chick CAM assay was carried out as described in (Gho and Chae, J. Biol. Chem. 272: 24294 (1997)) to determine the angiogemc activity of rTat and its derived peptides. Briefly, salt-free aqueous solution (5 I) containing
  • Tat Recombinant Tat alone was less effective in inducing an angiogemc response, as reported by others (Albi et a/., Oncogene 12. 289 (1996)). No significant angiogemc response was observed using vehicle alone or the control peptide Tat4i -52 containing sequence between the core and basic domains. Interestingly, Tat53-68 from the Tat basic domain also had substantial activity; as noted above, this peptide had either partial or minimal activities in assays for HIV replication, cytopathic effects, and transactivation of the HIV-LTR promoter. The exact mechanism of neovascula ⁇ zation in vivo is not clear. However, one scenario is that Tat induced cytokines stimulate endothelial cells, degrade basement membrane matrix by local enhancement of MMP 9 secretion, and migrate into adjacent tissue to form new blood vessel networks
  • Detectable levels of Tat have been reported in HIV-infected individuals (Westendorp et al, Nature 375:497 (1995)), suggesting the presence of extracellular HIV-Tat protein in certain phases of HIV infection It has also been shown that high levels of anti-Tat antibodies are directly related to low viral load (Re et al, Clin. Diagn. Lab. Immunol. 3: 230 (1996); Poznansky et a/., Hum. Gene Ther. 9: 487 (1998)) in seropositive non-progressor patients. Therefore, a strategy targeting a required s ⁇ te(s) in Tat may be used to provide a novel therapeutic modality to reduce disease progression in HIV-infected individuals.
  • the above results demonstrate that short core domains of the Tat protein retain activities characteristic of the full length Tat protein.
  • the Tat 21 40 , Tat 536 ⁇ and Tat 920 peptides retain activity.
  • the Tat2i 40 peptide which consists of 7 cysteine residues and only 13 other ammo acids, is a potent inducer of HIV transactivation and replication. This domain is highly conserved in various HIV 1 subtypes, including the newly discovered group 0.
  • the invention provides the active domain Tat 21 40 , Tat 5368 and Tat 920 , singly or in combination, as a novel therapeutic vaccine or a dominant-negative strategy to reduce Tat-mediated progression of disease in individuals with HIV infection.
  • peptide comprising a Tat functional domain refers to fragments of the Tat protein comprising one or more of the Tat 21 40 , Tat 53 B8 or Tat 920 functional domains from any subtype or isolate of HIV and are not limited to functional domains having the exact sequences used in the experiments described above.
  • the terminology "peptide comprising a Tat functional domain” encompasses fragments of the HIV Tat protein comprising the sequence ACTNCYCKKCCFHCQVCFTT (SEQ ID NO: 1), the sequence RQRRRAHQNSQTHQAS (SEQ ID NO: 2), or the sequence PPWHHPGSQPQl (SEQ ID NO: 3).
  • peptide comprising a Tat functional domain also encompasses fragments of the HIV Tat protein comprising the corresponding sequences from the consensus sequence for the B subgroup of HIV, i.e. EPWKHPGSQPKT (SEQ ID NO: 4), ACTNCYCKKCCFHCQVCFTT
  • the terminology "peptide comprising a Tat functional domain” also encompasses fragments of the HIV Tat protein comprising the corresponding sequences from the consensus sequence for the C subgroup of HIV, i.e. EPWNHPGSQPKT (SEQ ID NO: 7), ACTKCYCKKCSYHCLVCFQT (SEQ ID NO: 8), or RQRRSAPQSSEDHQNP (SEQ ID NO: 9).
  • the terminology "peptide comprising a Tat functional domain” also encompasses fragments of the HIV Tat protein comprising the corresponding sequences from the consensus sequence for the D subgroup of HIV, i.e.
  • EPWNHPGSQPKT (SEQ ID NO: 10), PCNKCHCKKCCYHCQVCFIT (SEQ ID NO: 11 ), or RQRRRPPQGGQAHQVP (SEQ ID NO: 12).
  • the terminology "peptide comprising a Tat functional domain” also encompasses fragments of the HIV Tat protein comprising the corresponding sequences from the consensus sequence for the F subgroup of HIV i.e. DPWNHPGSQPTT (SEQ ID NO: 13), PCTKCYCKRCCFHCQWCFTT (SEQ ID NO: 14), or KQRHRTPQSSQIHQDL (SEQ ID NO: 15).
  • peptide comprising a Tat functional domain also encompasses fragments of the HIV Tat protein comprising the corresponding sequences from the consensus sequence for the 0 subgroup of HIV i.e. PPWHHPGSQPQl (SEQ ID NO: 16), PCNNCYCKRCCYHCYVCFVR (ID NO: 17), or KQGRPAAASHPDHKDP (SEQ ID NO: 18).
  • peptide comprising a Tat functional domain also encompasses fragments of the HIV Tat protein comprising the corresponding sequences from the consensus sequence for the U subgroup of HIV i.e. EPWNHPGSQPKT (SEQ ID NO: 19), ACTKCYCKKCCYHCPVCFLN (SEQ ID NO:
  • peptide comprising a Tat functional domain also encompasses fragments of the HIV Tat protein comprising the corresponding sequences from an individual isolate falling within the B, C, D, F, M, 0, and U subgroups or fragments of the HIV Tat protein comprising the corresponding sequences from other subgroups.
  • peptide comprising a Tat functional domain also encompasses peptides that retain at least one activity of the full length Tat protein and which have at least about 60, 70, 80, 90, 95, or 99 % of their ammo acid sequence identical to that of SEQ ID N0:1, SEQ ID NO: 2, or SEQ ID NO: 3 as determined by FASTA or BLAST using default opening and gap penalties and a PAM scoring matrix. Percent sequence identity can be determined by standard methods that are commonly used to compare the similarity in position of the ammo acids of two polypeptides.
  • BLAST or FASTA Two polypeptides are aligned for optimal matching of their respective ammo acids (either along the full length of one or both sequences, or along a predetermined portion of one or both sequences)
  • the programs provide a "default" opening penalty and a "default” gap penalty, and a scoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoff et al., in: Atlas of Protein Sequence and
  • the percent identity can then be calculated as the total number of identical matches/ length of the sequence within the matched span + number of gaps introduced into the longer sequence in order to align the two segments.
  • Peptides that are at least 70 percent identical will typically have one or more ammo acid substitutions, deletions, and/or insertions. Usually, the substitutions will be conservative so as to have little or no effect on the overall net charge, polarity, or hydrophobicity of the peptide but optionally may increase or decrease the activity of the peptide.
  • peptide comprising a Tat functional domain also encompasses fragments of the Tat protein which include one or more am o acids adjacent to the Tat 2 , 40 , Tat 53 68 or Tat 920 functional domains.
  • the fragments may comprise about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, or more than 70 ammo acids adjacent to the functional domains, provided that such additional sequences are consistent with the length of the Tat protein and that the fragments are shorter than the intact Tat protein.
  • peptide comprising a Tat functional domain also includes peptide analogs or peptidomimetics based on the Tat 21 40 , Tat 5368 or Tat 920 functional domains.
  • Peptidomimetics are molecules that have the same effect as a peptide but are not peptides. Peptidomimetics may be resistant to proteases or cheaper to make than peptides.
  • the peptide comprising a Tat functional domain may be administered via any of the routes traditionally used to administer compositions which induce an immune response, including intraperitoneal administration, intramuscular administration, intradermal administration, intravascular administration or nasal administration.
  • the peptide comprising a Tat functional domain may be administered along with an adjuvant to enhance the level of the immune response.
  • adjuvants suitable for use in compositions for inducing an immune response are familiar to those skilled in the art, including aluminums like hydroxide and phosphate.
  • the peptide comprising a Tat functional domain may be administered in a physiologically acceptable earner.
  • a variety of carriers suitable for use in compositions for inducing an immune response are familiar to those skilled in the art. Such carriers are nontoxic to recipients at the dosages and concentrations employed.
  • the carriers may contain saline, buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, ammo acids, carbohydrates including glucose or dextnns or chelatmg agents such as
  • EDTA EDTA, glutathione or other stabilizers and excipients.
  • the peptides comprising a Tat functional domain are administered at a dosage and frequency sufficient to induce a desired level of immune response.
  • the level of immune response is sufficient to protect an umnfected individual from infection or to reduce or eliminate the symptoms of HIV infection in an infected individual.
  • the dosage and frequency of peptide administration will vary depending on the age, weight, and condition of the individual.
  • the dosage sufficient to induce a desired immune response may range from about 1 ⁇ g 100 mg or more, more preferably from about 100 ⁇ g 100mg or more. However, other dosages may also be used.
  • peptides comprising different functional domains of the Tat protein may be administered to the individual.
  • a peptide comprising the Tat 21 40 domain and a peptide comprising the Tat 5368 domain may be administered to the individual.
  • any combination of peptides comprising the Tat 21 40 , Tat 920 , or Tat 5368 may be administered to the individual.
  • the peptides comprising different functional domains of the Tat protein may be administered to the individual simultaneously or at different times.
  • nucleic acids encoding the one or more peptides comprising Tat functional domains may be administered to the individual.
  • the nucleic acids may comprise the nucleic acid sequence from an HIV isolate or subtype consensus sequences or the nucleic acid may be designed to encode the peptide comprising a Tat functional domain using the genetic code.
  • a vector comprising a nucleic acid encoding a peptide comprising a Tat functional domain operably linked to a promoter is administered to the individual.
  • the vector may be any of the expression vectors familiar to those skilled in the art, including viral or retroviral vectors.
  • Viral vectors suitable for use in the present invention include adenovirus, adeno-associated virus, herpes virus, and vaccinia virus.
  • Retroviral vectors suitable for use in the present invention include Moloney murine leukemia virus vectors, Harvey murine sarcoma virus vectors, mouse mammary tumor virus vectors, and Rous sarcoma virus vectors. Other vectors that induce a minimal host response to the vector itself may also be used.
  • the vector may be directly introduced into the individual such that the encoded peptide comprising a Tat functional domain is expressed in the individual.
  • the expression vector may be introduced into cells in vitro. Cells containing the expression vector are thereafter selected and introduced into the individual, where they express the encoded peptide comprising a Tat functional domain.
  • the nucleic acid encoding a peptide comprising the Tat functional domain may be administered as naked DNA. Techniques for administering naked DNA are described in U.S. Patent No. 5,580,859. If desired, the nucleic acid may be administered along with bupivacame.
  • the nucleic acid encoding a peptide comprising a Tat functional domain may be administered at a dosage and frequency sufficient to induce a desired level of immune response in the individual.
  • the level of immune response is sufficient to protect an unmfected individual from infection or to reduce or eliminate the symptoms of HIV infection in an infected individual.
  • the dosage and frequency of peptide administration will vary depending on the age, weight, and condition of the individual.
  • the dosage sufficient to induce a desired immune response may range from about 1 ⁇ g-100mg or more, more preferably from about 100 ⁇ g-200 ⁇ g or more.
  • Another aspect of the invention relates to dominant negative mutants of peptides comprising one or more Tat functional domains.
  • Dominant-negative mutants are mutants which are capable of reducing the level of activity of a wild type Tat protein.
  • Such dominant-negative mutants may be generated by mutagenizmg a nucleic acid encoding a peptide comprising a Tat functional domain using techniques familiar to those skilled in the art, including site directed mutagenesis, chemical mutagenesis, or PCR based methods.
  • such dominant-negative mutations may be generated by synthesizing peptides having the desired sequence in vitro.
  • the dominant negative mutant comprises a substitution of one or more ammo acids in the peptide comprising a Tat functional domain with another ammo acid.
  • the A (Alanine) at position 1 of the peptide may be substituted with an ammo acid selected from the group consisting of cysteine, aspartic acid, glutamic acid, phenylala ⁇ me, giycine, histidme, isoleucine, lysi ⁇ e, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • an ammo acid selected from the group consisting of cysteine, aspartic acid, glutamic acid, phenylala ⁇ me, giycine, histidme, isoleucine, lysi ⁇ e, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • the C (Cysteine) at position 2 of the peptide may be substituted with an amino acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylaia ⁇ me, giycine, histidme, isoleucine, lysi ⁇ e, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • an amino acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylaia ⁇ me, giycine, histidme, isoleucine, lysi ⁇ e, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • the T (threonme) at position 3 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysi ⁇ e, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, valine, tryptophan, and tyrosine.
  • an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysi ⁇ e, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, valine, tryptophan, and tyrosine.
  • the N (asparagine) at position 4 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, prolme, glutamine, argmine, senne, thereomne, valine, tryptophan, and tyrosine.
  • an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, prolme, glutamine, argmine, senne, thereomne, valine, tryptophan, and tyrosine.
  • the C (Cysteine) at position 5 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • the Y (tyrosine) at position 6 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leuc e, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, and tryptophan.
  • an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leuc e, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, and tryptophan.
  • the C (Cysteine) at position 7 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • the K (lysine) at position 8 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • the K (lysine) at position 9 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • the C (Cysteine) at position 10 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • the C (Cysteine) at position 1 1 of the peptide may be substituted with an am o acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, WO 00/78969 PCT/USOO/l 0119
  • the F (phenylalamne) at position 12 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • the H (histidme) at position 13 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • the C (Cysteine) at position 14 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, iysme, leucme, methiomne, asparagine, prol e, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, iysme, leucme, methiomne, asparagine, prol e, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • the Q (glutamine) at position 15 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • the V (valine) at position 16 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, tryptophan, and tyrosine.
  • an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, tryptophan, and tyrosine.
  • the C (Cysteine) at position 17 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • the F (phenylalamne) at position 18 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine.
  • the T (threonme) at position 19 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, valine, tryptophan, and tyrosine.
  • an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, valine, tryptophan, and tyrosine.
  • the T (threonme) at position 20 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, valine, tryptophan, and tyrosine.
  • an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, valine, tryptophan, and tyrosine.
  • the invention contemplates ammo, carboxy and internal truncations in a peptide comprising a Tat functional domain.
  • Examples of ammo terminal truncations in one of the Tat 21 40 sequences are provided in Table II below. However, it will be appreciated that ammo terminal truncations may also be ma ⁇ e in the Tat 53 68 and Tat 92D sequences or in Tat 21 40 sequences from other isolates or subtypes.
  • the truncated peptides can be produced by any of a number of protocols well known to those of skill in the art. Some short examples include, purifying one or more of the peptides taught by the invention, followed by modification of the peptide. Alternatively, the peptides can be produced recombmantly, using molecular biology and biochemical techniques, or synthetically. These peptides may themselves be chemically modified. Table II
  • X-ACTNCYCKKCCFHCQVCFTT-Z (SEQ ID NO: 1 ) "X" may represent an ammo group, a hydrophobic group, including but not limited to carbobenzoxyl, da ⁇ syl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier group including but not limited to hpid-f atty acid conjugates, polyethylene glycol, or carbohydrates.
  • X may represent an ammo group, a hydrophobic group, including but not limited to carbobenzoxyl, da ⁇ syl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier group including but not limited to hpid-f atty acid conjugates, polyethylene glycol, or carbohydrates.
  • Z may represent a carboxyl group; an amido group; a T butyloxycarbo ⁇ yl group; a macromolecular carrier group including but not limited to hpid-f atty acid conjugates, polyethylene glycol, or carbohydrates. Examples of carboxy-termmal truncations in one of the Tat 21 40 sequences are provided in Table III below.
  • carboxy terminal truncations may also be made in the Tat 53 B8 and Tat 920 sequences or in Tat 21 40 sequences from other isolates or subtypes.
  • X-ACTNCYCK-Z (SEQ1DN0:49) X-ACTNCYCKK-Z (SEQ ID NO: 48) X-ACTNCYCKKC-Z (SEQ ID NO: 47) X-ACTNCYCKKCC-Z (SEQ ID NO: 46) X-ACTNCYCKKCCF-Z (SEQ ID NO: 45)
  • X-ACTNCYCKKCCFH-Z (SEQ ID NO: 44) X-ACTNCYCKKCCFHC-Z (SEQ ID NO: 43) X-ACTNCYCKKCCFHCQ-Z (SEQ ID NO: 42) X-ACTNCYCKKCCFHCQV-Z (SEQ ID NO: 41) X-ACTNCYCKKCCFHCQVC-Z (SEQ ID NO: 40)
  • X-ACTNCYCKKCCFHCQVCF-Z (SEQ ID NO: 39)
  • X-ACTNCYCKKCCFHCQVCFT-Z (SEQ ID NO: 38)
  • X-ACTNCYCKKCCFHCQVCFTT-Z (SEQ ID NO: 1)
  • X may represent an amino group, a hydrophobic group, including but not limited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier group includig but not limited to lipid-f atty acid conjugates, polyethylene glycol, or carbohydrates.
  • Z may represent a carboxyl group; an amido group; a T-butyloxycarbonyl group; a macromolecular carrier group including but not limited to iipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
  • the internal truncations include
  • HCQ- and HCQ-16 20 wherein 16-20 means V, VC, VCF...VCFTT (SEQ ID NO: 65) CQV- and CQV-17 20 wherein 17-20 means C, CF, CFT...CFTT- (SEQ ID NO: 66)
  • Another type of dominant-negative mutant peptide combines a functional domain peptide that has been inactivated with another peptide that facilitates its binding to the normal target site.
  • a chimeric dominant negative peptide combines the Tat 21 40 sequence containing an ammo acid substitution that inactivates its function but fails to convert it to an effective dominant negative inhibitor in a peptide chimera with a second peptide sequence such as Tat 5368 in order to enhance binding to a molecular target normally bound by both peptide regions in the full length Tat molecule.
  • the separate peptides may have low or no inhibitory activity, while the composite peptide containing both peptide sequences would have dominant negative activity.
  • a spacer of ammo acids between the two peptides such as repeats of giycine or giycine plus senne, may be added to obtain optimal spacing of functional domains for full activity of the chimera.
  • a second type of chimeric dominant negative peptide uses a functional peptide domain inactivated by ammo acid substitutions containing additional charged ammo acids at the ends of the peptide to strengthen its binding to the target site. For example, 4-8 lysine residues added to the ammo terminus, the carboxy terminus or both termini will extend the length of the peptide by adding more positive charge in order to increase its binding to negatively charged targets such as DNA.
  • a third type of chimeric dominant negative peptide combines an inactivated functional domain with some other type of functional binding peptide sequence besides a known Tat peptide or charged ammo acids.
  • the strategy of each of the above types of chimeric peptides is to use a functional Tat peptide domain that has been inactivated by mutation in combination with some other binding moiety that enhances binding of the chimera in order to prevent the binding or function of native Tat molecules.
  • Dominant-negative mutants produced using the above methods are identified by determining their ability to reduce the activity level of wild type Tat protein.
  • a nucleic acid encoding a prospective dommant- negative mutant may be introduced into a cell containing a gene encoding the wild type full length Tat protein and a
  • Tat-activated reporter gene which encodes a detectable product.
  • a nucleic acid encoding a prospective dominant-negative mutation is operably linked to the hCMV promoter and cotransfected into BALB/c 3T3 cells with a vector encoding wild type full length Tat and a reporter vector in which the CAT gene is operably linked to the HIV LTR.
  • Control cells are transfected with the vector encoding full length Tat and the CAT reporter vector. Those mutants which reduce the levels of CAT expression relative to that in the control cells are dominant-negative mutations
  • Peptides comprising a Tat functional domain containing a dominant-negative mutation may be used in any of the methods for inducing an immune response described above, including methods in which the peptides or nucleic acids encoding the peptides are administered to an individual.
  • peptides comprising a Tat functional domain containing a dominant negative mutation are administered to an individual as described above. The peptides inhibit the activity of the wild type Tat protein in the individual.
  • nucleic acids encoding peptides comprising a Tat functional domain containing a dominant negative mutation may be operably linked to a promoter in any of the vectors described above.
  • the vector does not induce an immunological response to itself.
  • the vector is introduced into the individual or introduced into cells in vitro which are later introduced into the individual as described above.
  • the peptides comprising a Tat functional domain containing a dominant-negative mutation are expressed in the individual and inhibit the activity of wild type Tat protein in the individual.
  • Antibodies to a peptide comprising a Tat functional domain are another embodiment of the present invention.
  • the antibodies may be monoclonal antibodies or polyclonal antibodies.
  • Monoclonal antibody to the peptides comprising a Tat functional domain can be prepared from murine hybndomas according to the classical method of Kohler and Milstem (Nature 256:495 (1975)), or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the peptide over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminoptenn (HAT media).
  • HAT media aminoptenn
  • the successfully fused cells are diluted and a quots of the dilution placed in wells of a microtiter plate where growth of the culture is continued.
  • Antibody producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall [Meth. Enzymol. 70:419 (1980)), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis, L et al. Basic Methods in Molecular Biology Elsevier, New York. Section 21 2. If desired, the antibodies can be humanized to reduce the risk of immune reactions against non-human epitopes on the antibody.
  • Polyclonal antiserum containing antibodies to peptides comprising a Tat functional domain can be prepared by immunizing suitable animals with the peptide, which can be unmodified or modified to enhance immunogemcity.
  • Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunoge ⁇ ic than others and may require the use of carriers and adjuvant.
  • host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable An effective immunization protocol for rabbits can be found in Vaitukaitis, J. et al. (1971 ) J. Clin. Endoc ⁇ nol. Metab.
  • Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined by ELISA or semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony, 0. et al, Chap. 19 in: Handbook of Experimental Immunology
  • Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, D., Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc. For
  • Antibody preparations prepared according to either protocol are useful in quantitative immunoassa ⁇ s which determine concentrations of antigen bearing substances in biological samples; they are also used semi quantitatively or qualitatively to identify the presence of antigen in a biological sample.
  • the antibodies may also be used in therapeutic compositions for reducing the levels of the protein in the body.
  • the peptides comprising a Tat functional domain may be used as reagents to study HIV replication and the HIV life cycle.
  • the peptides may be used to identify proteins which interact with the functional domains in systems such as the yeast two hybrid systems such as the Matchmaker Two Hybrid System 2
  • the peptides comprising a Tat functional domain may also be used in affinity chromatograph ⁇ to identify proteins which interact with the functional domains.
  • the peptides comprising a Tat functional domain are linked to a suitable chromatograph ⁇ matrix.
  • Samples comprising proteins which bind to the functional domain are placed in contact with the matrix under conditions which allow a complex to be formed between the functional domain and the proteins which bind thereto. After a wash to remove non-specifically bound proteins, specifically bound proteins are eluted.
  • the specifically bound proteins can be sequenced and clones encoding them can be identified using techniques familiar to those skilled in the art.
  • the peptides comprising a Tat functional domain may be used to identify agents which disrupt the activity of Tat.
  • a peptide comprising the Tat 2140 functional domain may be bound to the HIV TAR sequences to form a complex.
  • the complex can be contacted with an agent to determine whether the agent disrupts the complex.
  • the cells containing a peptide comprising a functional domain of the Tat protein can be contacted with the agent to determine whether the agent disrupts at least one activity of the peptide, including such activities as activation of the HIV LTR, angiogenesis or NF- ⁇ B activation.
  • the agents tested for disruption of the activity of the peptide comprising the Tat functional domain may include organic or inorganic molecules, biochemical molecules, peptides, or poly ⁇ ucleotides.
  • Another embodiment of the present invention is a method for determining the disease state of an individual infected with HIV.
  • Non progressors have a high titer of antibodies against Tat in their sera, while rapid progressors have low titers of antibodies against Tat in their sera.
  • sera is obtained from an individual.
  • the sera is contacted with a peptide comprising a Tat functional domain, and the amount of antibody in the sera which binds to the peptide is determined to identify the individual as a non progressor or a rapid progressor.
  • the amount of antibody in the sera which binds to a peptide comprising a Tat functional domain may be determined using conventional methods familiar to those skilled in the art, including ELISA analyses or Western blots.
  • a peptide comprising a Tat functional domain is attached to a microtiter well and contacted with serum from an individual.
  • the amount of antibody against the peptide is determined by adding a detectably labeled secondary antibody.
  • the peptide comprising a Tat functional domain is attached to a filter and contacted with the serum from the individual.
  • the amount of antibody against the peptide is determined using a detectably labeled secondary antibody.
  • the present invention relates to multiple peptide conjugates, methods of preparing them, and methods of using them to induce an immune response.
  • t Boc means tert-butoxycarbonyl
  • Maldi-tof means matrix assisted laser desorption time of flight.
  • NMP means N-methylpyrrohdone.
  • TFA means trif luoroacetic acid.
  • HOBt means 1-hydroxybenzotriazole.
  • RP-HPLC reverse phase-high performance liquid chromatography
  • tbu means tert-butyl.
  • Fmoc means 9-fluorenylmethoxycarbonyl.
  • DCM dichloromethane
  • Npys means 3-nitro-2pyridine sulphenyl.
  • Mmt means 4-methoxytrityl
  • TCEP means triscarboxyethylphosphine.
  • DCC means N,N'-dicylohexylcarbodiimide.
  • Trt means trityl
  • 'TIS means triisopropylsilane.
  • Pmc means 2,2,5,7, 8-pentamethylchroman-6-sulfonyl.
  • DMSO dimethylsulfoxide
  • Hmb N-[2-hydroxy-4-methoxybenzyl].
  • HBTU 2-[1-H-Benzotriazole-1-yl]-1,13,3-tetramethyluronium hexafluorophosphate.
  • Oxbu means tert-butyl ester
  • DCU means dicyclohex ⁇ l urea
  • TNBS trinitrobenze ⁇ e sulf onic acid.
  • NHS N-Hydroxysuccimide.
  • EDT means ethanedithiol
  • CMC carboxymethylcysteine
  • Br2Ac2 ⁇ means Bromoacetic anhydride.
  • CI2AC2O Chloroacetic anhydride.
  • BU3P tri-n-butylphosphine.
  • MAP means multiple antigen peptide.
  • MPC multiple peptide conjugate
  • SEC size exclusion chromatography
  • SPPS solid phase peptide synthesis
  • ESI-MS electrospray ionization mass spectroscopy
  • linear peptide refers to a peptide sequence to be attached to the base peptide.
  • base peptide refers to the core peptide to which a peptide has been attached or the core template on which a peptide has been synthesized.
  • vaccines based on inactivated organisms or materials derived from organisms may suffer from a variety of drawbacks. Accordingly, it is desirable to use synthetic compounds to induce an immune response. However, it is generally thought that the conformational B-cell epitopes involved in neutralization of some organisms would be difficult to mimic though the use of a simple synthetic linear molecule (Sesardic, J. Med. Microbiol. 39:241
  • peptides are linked to a core structure to generate a multiple antigen peptide (MAP) (Nardelli et alThe MAP system: A flexible and unambiguous vaccine design of branched peptides in vaccine design: The subumt and adjuvant approach Plenum Press, New York, pp803 819 (1995); D ⁇ jfhout et al. Solid-phase and applications of NfS-acetylmercaptoacetyl) peptides
  • the classical solid phase synthesis methodology employed in the traditional MAP is not without difficulty.
  • the classical solid phase synthesis methodology employed in this approach may not yield a sufficient amount of product for use in a vaccine or may yield a heterogenous product which is not amenable to use in a vaccine.
  • problems may arise as a consequence of microheteroge ⁇ eit ⁇ in the synthetic compounds
  • the present invention relates to multiple peptide conjugates which comprise multiple peptides, such as antigens, epitopes or other peptides, coupled to a core template.
  • peptide refers to compounds comprising two or more ammo acids linked b ⁇ peptide bonds.
  • the peptides may be from about 2 to about 100 ammo acids in length.
  • the peptides ma ⁇ be from about 10 to about 100 ammo acids in length.
  • the core template preferably comprises a branched compound.
  • the core template has at least two branches.
  • the core template may have 3, 4 or more than four branches. In fact, the core template ma ⁇ have an ⁇ number of branches consistent with its intended function.
  • Each branch of the core template contains a first reactive group and a second reactive group, each of which permit peptides to be selectively attached thereto or synthesized thereon.
  • the adjacent branches of the core template are spaced at a sufficient distance from one another to minimize the steric hindrance between peptides being s ⁇ nthesized on or attached to the reactive groups on the branches of the core template.
  • the peptides are s ⁇ nthesized on the core template.
  • the adjacent branches of the core template are separated from one another b ⁇ the distance occupied b ⁇ at least four carbon atoms.
  • the adjacent branches are linked to the ⁇ and ⁇ ammo groups of a lysine residue such that they are separated from one another by four carbons.
  • the core template comprises a peptide.
  • the core template ma ⁇ be s ⁇ nthesized using an ⁇ of the conventional methods for synthesizing peptides, including solid phase s ⁇ nthesis or conventional solution phase chemistr ⁇ .
  • S ⁇ nthesis ma ⁇ include the use of an ⁇ and all protecting strategies, such as the use of t-Boc, Fmoc, Bpoc or other suitable urethane derivatives for blockage of the N-termmai amme prior to coupling or attaching subsequent ammo acids in a continuous chain or arra ⁇ .
  • S ⁇ nthesis ma ⁇ also be performed b ⁇ the methodolog ⁇ of convergent solid phase s ⁇ nthesis using protected fragments utilizing acid labile linkers or resins.
  • Convergent chemical gation of unprotected peptides, fragments or ammo acids ma ⁇ also be used to prepare the core template.
  • the s ⁇ nthesis of the core template ma ⁇ be achieved entirely by direct, indirect or modular means or a combination of these techniques which couple two or more ammo acids together to form conventional peptide bonds or other stable bonds enabling a covalent and stable linkage between two ammo acids.
  • the core template comprises a two branched compound of the following structure:
  • A is absent or an ammo acid
  • B is an ammo acid
  • D is absent or an ammo acid
  • E is absent or an ammo acid
  • G is an am o acid
  • J is an ammo acid
  • M is an ammo acid having a first reactive group and a second reactive group thereon, wherein said first reactive group and said second reactive group are capable of selectively being attached to an ammo acid or peptide,
  • R is an ammo acid having a first reactive group and a second reactive group thereon, wherein said first reactive group and said second reactive group are capable of selectively being attached to an ammo acid or peptide; n is i 10; s is 0 when D is absent or is 1 20 when D is present; and t is 0 when E is absent or is 1 20 when E is present.
  • ammo acids in Structure A i.e. A (if present), B, D (if present), E( ⁇ f present), G, J, M, and R
  • A if present
  • B if present
  • D if present
  • E( ⁇ f present) if present
  • G if present
  • J if present
  • M if present
  • R if present
  • the starting material for synthesizing the core template is an ammo acid linked to a solid support such as a resin.
  • residue A is preferably an ammo acid which can be linked to a solid support such as a resin.
  • residue A of structure A is senne
  • the side chain of the senne used as the starting material for synthesis is t ⁇ tylated to block its reactivity. The use of se ⁇ ne(Trt) allows modification on the solid phase after deprotection of the h ⁇ drox ⁇ l group to produce a highl ⁇ reactive group for chemical ligation.
  • the h ⁇ drox ⁇ l group ma ⁇ be oxidized to an aldeh ⁇ de group or otherwise modified to provide a group suitable for coupling other chemical moieties at this specific site.
  • the C terminal senne modification can be performed in the solid phase state or as a free peptide.
  • the C terminal residue ma ⁇ be an ⁇ residue consistent with its intended use.
  • the senne residue placed at C-termmal end of the core template ma ⁇ be substituted b ⁇ an ⁇ number of ammo or non ammo acids.
  • the C terminal senne ma ⁇ be replaced with L ⁇ s ⁇ ne(Mtt), L ⁇ s(Tfa), L ⁇ s(Dde), C ⁇ s( mt), C ⁇ s(Acm) as well as man ⁇ other suitable ammo acids or compounds capable of side chain modification.
  • Fmoc s ⁇ nthesis ma ⁇ be replaced b ⁇ the conventional t Boc s ⁇ nthesis.
  • the C-termmal ammo acid ma ⁇ be selected from t-Boc ammo acids, such as C ⁇ s(Acm), L ⁇ s(Dde), Lys(Fmoc),
  • peptide synthesis is performed to extend the starting material.
  • the starting material is Ser(Trt) linked to a resin
  • additional ammo acids ma ⁇ be added thereto to s ⁇ nthesize the core template.
  • an initial lysine residue is linked to the Ser(Trt).
  • residue B can be lysine.
  • This initial lysi ⁇ e forms the branch point.
  • n is from 1 10.
  • n is from 1-5.
  • n is 1.
  • residues D and E ma ⁇ be added to extend the chain lengths of the branches.
  • s and t ma ⁇ be from 1-20.
  • s and t are from 10.
  • s and t are from 1 5.
  • residues D and E of structure A are absent (i.e. s and t are 0).
  • Residues G and J of structure A ma ⁇ be a ⁇ ammo acid.
  • G and J are I ⁇ smes.
  • residue B is lysine
  • lysine residues G and J are attached to the ⁇ and ⁇ NH 2 groups of residue B to form a branched structure.
  • residues G, and J are lysmes
  • the ⁇ NH 2 group of these lysmes are blocked with a tert-butox ⁇ carbon ⁇ l (t Boc) group and residues M and R are joined to the ⁇ NH, groups of these lysmes.
  • the ⁇ NH 2 groups of lysmes G and J ma ⁇ be blocked and M and R ma ⁇ be linked to the ⁇ NH 2 of lysmes G and J.
  • residues B, G and J may be derivatives such as Fmoc-Orn(Mtt)-OH, Fmoc
  • Residues B, G, and J ma ⁇ also be other diammo compounds having appropriate steric and chemical properties.
  • the residues B, G, and J bear a reactive side chain.
  • the reactive side chain is spatially separated b ⁇ at least about four carbon atoms from the chiral center of the molecule.
  • residues B, G and J are preferabl ⁇ compatible with standard chemical methodologies, such as solid phase and solution chemistry, used in the preparation of s ⁇ nthetic peptides.
  • residues B, G, and J are preferabl ⁇ suitable for use in an orthogonal or non orthogonal s ⁇ nthesis approach using either Fmoc or t-Boc chemistr ⁇ or using alternate protecting groups for those strategies using both acid and base chemistries.
  • the residues B, G, and J are spatially arranged such that the level of steric hindrance does not exceed that in the compounds in which residues B, G and J are lysmes.
  • Residues M and R have at least two reactive groups thereon.
  • Each of the reactive groups permits a peptide to be specifically s ⁇ nthesized thereon or attached thereto after synthesis.
  • the reactive groups are designed such that a peptide can be synthesized thereon or a fully synthesized peptide can be attached thereto under conditions in which peptides cannot be s ⁇ nthesized on or attached to the remaining reactive group or groups.
  • the reactive groups ma ⁇ be ammo acids modified with appropriate blocking groups such that a single reactive group ma ⁇ be unblocked at a time
  • one of the reactive groups is deprotected upon acid treatment, while the other reactive group is deprotected upon treatment with base.
  • the peptide will be s ⁇ nthesized on or attached to onl ⁇ one reactive group at a time.
  • residues M and R are c ⁇ stemes and the first and second reactive groups comprise the thiol group of the c ⁇ stemes and the ammo groups of the c ⁇ stemes respectively.
  • peptides are s ⁇ nthesized on the ammo groups of the c ⁇ stemes and the cysteine side chain thiol is used to form a stable thioether bond with an incoming haloacet ⁇ l peptide.
  • peptides ma ⁇ be s ⁇ nthesized on the ammo groups of the c ⁇ stemes with a reduced risk of epimenzation, and, if the thiol groups of the c ⁇ stemes are blocked during s ⁇ nthesis of the peptides on the ammo groups of the c ⁇ steines, there is a reduced risk of dime ⁇ zatio ⁇ during s ⁇ nthesis of the peptides on the ammo groups of the c ⁇ steines. In one embodiment, the thiol groups of the c ⁇ steines are blocked with
  • Trt groups while the ammo groups are blocked with Fmoc.
  • M and R may be ammo acids or non-ammo acids other than c ⁇ steme which provide first and second reactive groups which can be selectively attached to an ammo acid or peptide.
  • residues M and R ma ⁇ be ammo acids having reactive side chains, such as lysi ⁇ e, senne, threonme, asparagine, aspartic acid, glutamine, and glutamic acid.
  • M and R are capable of forming a chemical bond with both the preceding and successive ammo acid in the peptide chain.
  • the covalent bonds between the peptide antigens and base peptide need not be the traditional peptide bond (NHCO) formed between the ammo group (NH 2 ) of one ammo acid and the carboxyl (COOH) of another but ma ⁇ be an ⁇ number of other bonds that insures rigidity, and strength for use in this kind of chemical s ⁇ nthesis.
  • Such couplings ma ⁇ include coupling via thioether formation.
  • Other couplings ma ⁇ also include that of the oxime, thiazolidme, h ⁇ drazone, thioester, or other suitable bond formation compatible with all chemistries and groups used in the preparation of the multiple peptide conjugate.
  • the core template has the structure WO 00/78969 PCT/USOO/l 0119
  • Structure B has the chemical structure depicted in Figure 2.
  • the core template ma ⁇ have more than two branches.
  • the core template ma ⁇ have 3, 4, or more than four branches.
  • the core template ma ⁇ have the structure:
  • A is absent or an ammo acid
  • B is an ammo acid
  • D is absent or an ammo acid
  • E is absent or an ammo acid
  • G is an ammo acid
  • J is an ammo acid
  • M is an ammo acid having two reactive groups thereon which can be attached to an ammo acid
  • R is an ammo acid having two reactive groups thereon which can be attached to an ammo acid
  • W is an am o acid having a first reactive group and a second reactive group thereon, wherein said first reactive group and said second reactive group are capable of selectively being attached to an ammo acid or peptide;
  • X is an ammo acid having a first reactive group and a second reactive group thereon, wherein said first reactive group and said second reactive group are capable of selectively being attached to an ammo acid or peptide
  • Y is an ammo acid having a first reactive group and a second reactive group thereon, wherein said first reactive group and said second reactive group are capable of selectively being attached to an ammo acid or peptide
  • Z is an ammo acid having a first reactive group and a second reactive group thereon, wherein said first reactive group and said second reactive group are capable of selectively being attached to an ammo acid or peptide;
  • A, B, D, and E may have an ⁇ of the identities discussed above with respect to Structure A.
  • the values of n, s, and t may be any of those discussed above with respect to Structure A
  • the ammo acids in Structure C i.e. A (if present), B, D (if present) , E( ⁇ f present), G, J, M, R, W, X,
  • Y and Z are linked to one another via peptide bonds.
  • G and J ma ⁇ be an ⁇ ammo acid.
  • G and J are lysmes.
  • M and R may be a ⁇ ammo acids having two reactive groups which can be linked to an ammo acid, such that residues M and R form branch points.
  • M and R are lysmes.
  • M and R may have an ⁇ of the identities discussed above for residues G and J of Structure A.
  • W, X, Y, and Z ma ⁇ have an ⁇ of the identities discussed above for residues M and R of Structure A.
  • W, X, Y, and Z are c ⁇ steines.
  • additional branches may be added b ⁇ adding additional ammo acids having two reactive groups which can be linked to an ammo acid at the penultimate positions of the branches.
  • the penultimate ammo acids are lysmes.
  • the ⁇ or ⁇ ammo groups of the lysmes are then linked to ammo acids having a first reactive group and a second reactive group thereon wherein the first and second reactive groups are capable of being selectively attached to an ammo acid or peptide.
  • the ammo acids having a first reactive group and a second reactive group thereon wherein the first and second reactive groups are capable of being selectively attached to an ammo acid or peptide are c ⁇ stemes.
  • the core template structures provided above are ammo acids linked b ⁇ peptide bonds
  • the core template ma ⁇ be an ⁇ compound, including compounds which are not based on peptide bonds between ammo acids, which allows peptides to be selectively synthesized on or attached directly to a single type of reactive group at a time as discussed in more detail below.
  • peptides are s ⁇ nthesized on the first reactive groups or fully synthesized peptides are attached to the fust reactive groups to generate a base peptide.
  • the peptides are s ⁇ nthesized on the first reactive groups.
  • a first ammo acid residue ma ⁇ be attached to the first reactive groups using an ⁇ of the s ⁇ nthetic methods familiar to those skilled in the art.
  • the peptide chain ma ⁇ then be extended one residue at a time to generate the desired peptide using any of the synthetic methods familiar to those skilled in the art. If the first reactive group is blocked, the blocking group is removed prior to linking the first ammo acid thereto.
  • the desired peptide sequence may be fully s ⁇ nthesized using all conventional methods. Subsequentl ⁇ , the complete peptide is attached to the reactive group on the core template.
  • one or more ammo acids in the peptides being s ⁇ nthesized on or attached to the first reactive groups are modified with agents which reduce steric effects.
  • one or more of the ammo acids added to the peptide chain during s ⁇ thesis or included in a full ⁇ s ⁇ nthesized peptide ma ⁇ be protected using any of the blocking groups familiar to those skilled in the art.
  • one or more ammo acids linked to the carboxy group of asparagine or aspartic acid residues are incorporated as Hmb protected derivatives in the peptides being synthesized on or attached to the first reactive groups.
  • an ⁇ c ⁇ steme in the peptides being s ⁇ nthesized on or attached to the first reactive groups ma ⁇ have their thiol groups modified with a blocking group. In a preferred embodiment, the thiols are blocked with t ⁇ t ⁇ l groups.
  • the s ⁇ nthesis is conducted in a solvent containing DMSO.
  • the DMSO may be present at a concentration from about 1 % to about 20% . In a preferred embodiment, the DMSO is present at a concentration from about 5% to about 12%. In a highly preferred embodiment the DMSO is present at a concentration of about 10%.
  • chaotropic salts such as KSCN, sodium perchlorate, THT (tetrahydrofuran), 1 ,4, dioxane, TFE (trifluoroethanol) or LiCI ma ⁇ be used to increase solvation.
  • the solvent is the N-meth ⁇ lpyroll ⁇ done coupling solvent conventionally used to synthesize peptides.
  • the solvent may be DMF (dimethylformamide). It will be appreciated that any suitable solvent, including aprotic solvents, ma ⁇ be used.
  • the s ⁇ nthesis of peptides on the first reactive groups or the attachment of full ⁇ s ⁇ nthesized peptides to the first reactive groups on the core templates generates the base peptide.
  • the core template has Structure A
  • the base peptide has peptides attached to the first reactive groups of residues M and R.
  • the core template has Structure C
  • the base peptide has peptides attached to the first reactive groups of residues W, X, Y, and Z.
  • the peptides are selectively synthesized on or joined to the ammo groups of the cystemes following deprotection thereof to form the base peptide.
  • the base peptide has the following structure after removal of the blocking groups on the thiols of the cystemes: SH
  • the core template ma ⁇ be cleaved from the solid support following s ⁇ nthesis or attachment of the peptides to the core template.
  • the thiol group of c ⁇ steme is one of the reactive groups on the core template
  • preferabl ⁇ the cysteine thiol groups on the core template are blocked with an appropriate blocking group such that only these groups are deprotected during the resin cleavage of the peptide by acidolysis.
  • the above measures need not be emplo ⁇ ed when the peptides attached to the core template during s ⁇ nthesis of the base peptide do not include c ⁇ steme residues.
  • the thiol blocking group (trit ⁇ l) on the core template ma ⁇ be replaced with a ⁇ blocking group compatible with its intended use.
  • C ⁇ s(Acm), C ⁇ s(tbu), C ⁇ s(Mmt), C ⁇ s(Npys) or other protected residues could be used in this strategy and appropriate chemical methodologies would be used to deprot ⁇ ct the thiol group when desired.
  • the base pept ⁇ de(s) ma ⁇ also be prepared b ⁇ a chemical or solution s ⁇ nthesis process.
  • a free peptide can be generated after an acidolysis or other cleavage procedure is performed on resin bound peptide or protected peptide in solution.
  • An alternate approach may also include the s ⁇ nthesis, whether b ⁇ solid phase or solution chemistr ⁇ , of protected fragment(s) of either the base or core molecule.
  • These components or fragment(s) produced as free peptides or protected fragments can then be used in convergent synthesis by activation and coupling of two ends of the moiecule together followed b ⁇ suitable methods for isolation.
  • the coupling of free peptides to the core molecule or other peptides generally will require chemoselective specificity to ensure reactivity at a specific site or residue.
  • peptide chain elongation ma ⁇ be performed from cystemes on the epsilon position of the I ⁇ sines with the alpha position of the I ⁇ sines in a blocked state.
  • the results would be identical to that obtained when the c ⁇ steines are linked to the alpha positions of the I ⁇ sines, since the spatial construction of the core template would not be changed in an ⁇ wa ⁇ .
  • the ammo groups of the two c ⁇ steines ma ⁇ be protected with different blocking groups such that different peptides ma ⁇ be s ⁇ nthesized thereon or attached thereto.
  • the different peptides are sequentially attached to the first reactive groups following deprotection with the appropriate reagents.
  • peptides are selectively synthesized or attached on the second reactive groups to generate a multiple peptide conjugate.
  • pre-s ⁇ nthesized peptides are attached to the second reactive groups.
  • the peptides s ⁇ nthesized or attached on the second reactive groups ma ⁇ be the same as or different from those s ⁇ nthesized or attached on the first reactive groups.
  • the multiple peptide conjugate has peptides attached to the first and second reactive groups of residues M and R.
  • pre-s ⁇ thesized peptides are attached to the second reactive groups on the base peptide.
  • a haloacet ⁇ l group at the end of a pre-s ⁇ nthesized peptide ma ⁇ be attached to thiol groups on c ⁇ steine residues in the base peptide.
  • the peptides are selectively synthesized or attached on the thiol groups of the cystemes.
  • the peptides s ⁇ nthesized or attached on the thiol groups of the c ⁇ steines ma ⁇ be the same as or different from the peptides s ⁇ nthesized or attached on the ammo groups of the c ⁇ steines.
  • the multiple peptide conjugate has the following structure: L ⁇ s
  • Structure E The chemical structure of one embodiment Structure E is shown in Figure 4
  • the peptides P 3 and P 4 are linked to the c ⁇ steme thiol groups via a haloacet ⁇ l group.
  • other groups ma ⁇ also be used, or the peptides ma ⁇ be attached directly to the thiol groups in the cystemes.
  • reactive groups in the peptides which are to be attached to the base peptide to generate the multiple peptide conjugate are protected with a blocking group which prevents them from being reactive under the conditions in which they are attached to the base peptide.
  • the blocking groups in the peptides which are to be attached to the base peptide to generate the multiple peptide conjugate are different than the blocking groups on the reactive groups in the base peptide such that the reactive groups in the base peptide can be selectively deprotected.
  • C ⁇ s(tbuth ⁇ o) insures site directed coupling b ⁇ the haloacet ⁇ l peptide to the thiols. It will be appreciated that the internal C ⁇ s(tbuth ⁇ o) ma ⁇ be replaced with other SH protection compatible with the chemistr ⁇ emplo ⁇ ed to s ⁇ nthesize the multiple peptide conjugate.
  • one or more of the peptides in the multiple peptide conjugates ma ⁇ also include one or more peptides adjacent to the peptides P1 , P2, P3, and P4.
  • the peptides may comprise one, two, three, four, five or more than five peptides adjacent thereto.
  • the peptides may comprise about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, or more than 70 peptides adjacent to the peptides P1, P2, P3, and P4.
  • the peptides included in the multiple peptide conjugate comprise agents, including antigens or epitopes, which are capable of eliciting an immune response in an individual.
  • the peptides comprise antigens from a pathogenic organism. It will be appreciated that in some embodiments the peptide antigens from pathogenic organisms ma ⁇ have ammo acids or no ⁇ - ammo acids inserted therein which function as linkers between two antigenic peptides which are fused together or which provide an ⁇ desirable attribute to the antigenic peptide.
  • the peptides attached to the core template are from the causative agents of malaria and/or AIDS.
  • the peptide attached to the core template ma ⁇ be from the HIV-
  • the peptide attached to the core template comprises an antigenic epitope from the HIV-1 Tat protein.
  • one or more of the peptides in the multiple peptide conjugate comprises a portion of the Tat protein which is responsible for mediating one or more activities of the Tat protein, such as replication of HIV 1 , transcription from the HIV-1 LTR, induction of c ⁇ tokine production, increased activit ⁇ of the F transcription factor, or increased angiogemc activit ⁇ in assa ⁇ s such as the chick CAM assa ⁇ (Gho and Chae. J. Biol. Chem. 272: 24294 (1997)).
  • the portion of the HIV-1 Tat protein also includes one or more antigenic sites therein.
  • one or more of the peptides in the multiple peptide conjugate constitute SEQ ID N0S:1 21.
  • one or more of the peptides in the multiple peptide conjugates ma ⁇ comprise two, three, four, five, or more than five peptides containing Tat functional domains linked to one another.
  • the two, three, four, five, or more than five peptides are spaced from one another b ⁇ a iinker comprising one or more ammo acids or non-ammo acids.
  • one or more of the peptides in the multiple peptide conjugate comprises a peptide from Plasmodium falcipanum, the causative agent of malaria.
  • the peptide from Plasmodtum falcipanum has at least one antigenic site therein.
  • one or more of the peptides in the multiple peptide conjugate comprises a T3 peptide from Plasmodium falcipanum.
  • the peptide ma ⁇ comprise the sequence NENLDDLDEGIEKSSEELSEEKI (SEQ ID NO: 81 ).
  • one or more of the residues which are linked to the carbox ⁇ l groups of asparagine or aspartic acid residues in the T3 peptide ma ⁇ be Hmb derivatives as indicated b ⁇ underlining in Figure 5A.
  • one or more of the peptides in the multiple peptide conjugate comprises a T1 peptide from Plasmodium falcipanum.
  • the peptide may comprise the sequence LTMSNVKNVQTNFKSLLRNLGVS (SEQ ID NO: 82).
  • one or more of the residues which are linked to the carboxyl groups of asparagine or aspartic acid residues in the T1 peptide may be Hmb derivatives as indicated b ⁇ underlining in Figure 5A.
  • one or more of the peptides in the multiple peptide conjugate comprises an MSP
  • the peptide ma ⁇ comprise the sequence VTHESYQELVKKLEALEDAV (SEQ ID NO: 83).
  • one or more of the residues which are linked to the carbox ⁇ l groups of asparagine or aspartic acid residues in the MSP 1 peptide ma ⁇ be Hmb derivatives as indicated b ⁇ underlining in Figure 5A.
  • one or more of the peptides in the multiple peptide conjugate may comprise the
  • the CSP peptide from Plasmodium falcipanum (Nardin et al., Vaccine 16:590 600 (1998)).
  • the peptide may comprise the sequence NANPNANP (SEQ ID NO: 84).
  • one or more of the residues which are linked to the carboxyl groups of asparagine or aspartic acid residues in the CSP peptide may be Hmb derivatives as indicated by underlining in Figure 5A
  • one or more of the peptides in the multiple peptide conjugates may comprise an immunogemc peptide from an ⁇ isolate or strain of Plasmodium falcipanum.
  • one or more of the peptides in the multiple peptide conjugate comprises a peptide which has one, two, three, four, five or more than five ammo acid substitutions relative to a reference T3, T1, MSP 1 or CSP peptide.
  • the substituted peptides may be produced using conventional techniques of peptide synthesis.
  • one or more of the peptides in the multiple peptide conjugate comprises a fusion between two immunogemc peptides from Plasmodium falcipanum.
  • the two peptides are spaced from one another by a linker comprising one or more ammo acids or non-ammo acids.
  • one or more of the peptides in the multiple peptide conjugate may cormpnse a fusion peptide comprising the CSP and T3 peptides such as a peptide comprising the sequence NANPNANPNENLDDLDEGIEKSSEELSEEKI (SEQ ID NO: 85).
  • one or more of the residues which are linked to the carboxyl groups of asparagine or aspartic acid residues in the fusion peptide may be Hmb derivatives as indicated by underlining in Figure 5A.
  • one or more of the peptides in the multiple peptide conjugate comprises a fusion between more than two immunoge c peptides from Plasmodium falcipanum.
  • one or more residues linked to the carboxy groups of asparagine or aspartic acid residues ma ⁇ be Hmb derivatives.
  • the multiple peptide conjugates may include any peptide from HIV-1 , Plasmodium falcipanum, or any other organism, which is capable of inducing an immune response.
  • the multiple peptide conjugates are not limited to the peptides specifically enumerated above.
  • the multiple peptide conjugates synthesized as described above may be used to induce an immune response in an individual.
  • the multiple peptide conjugates may be administered via any of the routes traditionally used to administer compositions which induce an immune response, including intraperitoneal administration, intramuscular administration, mtradermal administration, intravascuiar administration or nasal administration.
  • the multiple peptide conjugates may be administered along with an adjuvant to enhance the level of the immune response.
  • a variety of adjuvants suitable for use in compositions for inducing an immune response are familiar to those skilled in the art, including aluminums like hydroxide and phosphate.
  • the multiple peptide conjugate may be administered in a physiologically acceptable carrier.
  • a physiologically acceptable carrier suitable for use in compositions for inducing an immune response are familiar to those skilled in the art. Such carriers are nontoxic to recipients at the dosages and concentrations emplo ⁇ ed.
  • the carriers ma ⁇ contain saline, buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, ammo acids, carbohydrates including glucose or dextnns or chelating agents such as EDTA, glutathione or other stabilizers and excipie ⁇ ts.
  • the multiple peptide conjugates are administered at a dosage and frequency sufficient to induce a desired level of immune response.
  • the level of immune response is sufficient to protect an unmfected individual from infection or to reduce or eliminate the s ⁇ mptoms of infection in an infected individual.
  • the dosage and frequenc ⁇ of administration of the multiple peptide conjugates will var ⁇ depending on the age, weight, and condition of the individual.
  • the dosage sufficient to induce a desired immune response ma ⁇ range from about 1 ⁇ g-100 mg or more, more preferabl ⁇ from about 25 ⁇ g-100mg or more.
  • other dosages ma ⁇ also be used.
  • the multiple peptide conjugates ma ⁇ comprise several antigens from a pathogenic organism.
  • two or more different antigenic peptides ma ⁇ be attached to the core template.
  • Figure 6 illustrates one embodiment of the s ⁇ nthetic process which was used to prepare a multiple peptide conjugate
  • solid phase peptide s ⁇ nthesis followed b ⁇ thiol coupling chemistr ⁇ was utilized to synthesize and construct the MPC molecules.
  • a core template that serves as primary anchor for the s ⁇ nthesis of a base peptide and subsequent attachment of additional peptides through specific site directed coupling was s ⁇ nthesized using solid phase methods.
  • the core template may have the structure illustrated in Figure 7A.
  • Ser ⁇ ne(Trt) representing the C-termmal of the MPC was attached to the solid support followed b ⁇ the sequential addition of multiple branched I ⁇ sine residues forming a tetrame ⁇ c core whose epsilon ammo groups remain blocked throughout the s ⁇ nthesis process.
  • Example 6 describes the s ⁇ nthesis of the s ⁇ nthesis core template having the structure shown in Figure 7A.
  • the core template was s ⁇ nthesized on an ABI Model 430 peptide s ⁇ nthesizer using Fmoc chem ⁇ str ⁇ (Mer ⁇ f ⁇ eld, In Peptides: Synthesis, Structure and Applications (Gutte, Ed.), Academic press, San Diego, pp93
  • the second step of assembl ⁇ involves the s ⁇ nthesis of a base peptide consisting of one or more antigens.
  • the base peptide ma ⁇ be generated b ⁇ synthesizing the antigens on the core template.
  • the complete antigenic peptides may be synthesized first and then attached to the core template.
  • the base peptide ma ⁇ have the structure shown in Figure 7B.
  • the base peptide constructs were s ⁇ nthesized b ⁇ synthesizing the antigenic peptides listed in Figure 5A and 5B on the core template as described in Example 7.
  • Example 7 The base peptides consisting of antigens from malaria and HIV-Tat protein were synthesized on the core template following characterization of the core template. These base peptides were subsequently used to prepare four
  • the coupling time for a single coupling was 1 hour. Coupling efficiencies were monitored using the Kaiser or TNBS test after completion of each coupling step to insure greater than 99% completion. Double couple/recouple c ⁇ cles were introduced when necessar ⁇ . C ⁇ cles which failed to achieve at least 99% after a double/recouple c ⁇ cle were capped with 2mmol benzoic anhydride to terminate any unreacted amines. An antigen from Plasmodium falciparum (LSA-1 ) (Connelly et al., Infection and Immunity 65:5082 5087(1997)) previously identified as T3 was used to construct the base peptide T3.
  • LSA-1 Plasmodium falciparum
  • the T3 CSP base peptide was s ⁇ nthesized using T3 base peptide as its template following removal of 1/2 of the T3 peptid ⁇ l resin from the s ⁇ nthesizer reaction flask after deprotection thus allowing the s ⁇ nthesis to be continued with an additional eight residues added per branch of the T3 construct.
  • Both T3 and T3CSP base peptides were subjected to cleavage b ⁇ acidolysis and purification.
  • the HIV-Tat base peptide Tat-2-Tat 3 was obtained b ⁇ direct solid phase s ⁇ nthesis under similar conditions as described at the 0.25 mmol scale followed b ⁇ cleavage and purification.
  • Example 8 Following deprotection of the N terminal ammo group, the base peptides were cleaved with a cocktail of TFA, water, EDT, TIS, phenol, thioanisole (88:4:2:1:1:4) Following cleavage, base peptides were precipitated and washed in cold Tert-but ⁇ l Meth ⁇ l Ether. Prior to further processing, peptides were dissolved in water or 0.1 % TFA or acetic acid followed b ⁇ the addition of 1.0 ml -mercaptoethanol.
  • Example 9 Base peptides were purified b ⁇ RP HPLC using 6.8 x 25 cm V ⁇ dac C4, C8, or C18 columns (The Separation Group, Hespena, CA) with a gradient of 0.1 % TFA/H2O and 0.1 % TFA/acetonitnle. Following HPLC purification, the acetonitnle was removed b ⁇ rotar ⁇ evaporator and the peptides were I ⁇ opholized and stored at -20° or -70°C (haloacet ⁇ l peptides) until needed.
  • Tat-2-Tat-3 (base peptide) 7,303.65 7, 306
  • Peptides comprising malarial antigens were s ⁇ nthesized as described above using standard ABI Fmoc c ⁇ cles with a final c ⁇ cle added for modification of the N terminal ammo group with a haloacet ⁇ l group (Robey and Fields, Anal
  • peptides were cleaved from the resin according to a specific cleavage protocol described (Bo ⁇ kms et al. Cytokine 11 :8-15 (1999)) followed b ⁇ purification b ⁇ RP-HPLC. The peptides were lyophilized and stored at -70°C until needed.
  • the HIV Tat peptides (Boy kins et al, J. Immunol. 163.15-20 (1999)) were synthesized essentially the same way as the malaria peptides, except that all cysteine sulfhydr ⁇ l residues of Tat-1 peptide were protected with the tButhio blocking group (Figure 5B). Chloroacet ⁇ lation of the ammo terminal end of the peptide was chosen specifically for the Tat peptide to minimize reactivity with other highl ⁇ reactive side chains present in both the base and linear peptides during final assembly of the Tat MPC. Cleavage of this molecule produced a stable N chloroacet ⁇ l peptide whose side chain thiol groups remained blocked throughout the purification.
  • the haloacet ⁇ l linear peptide Tat 1 was coupled to the base peptide Tat-2-Tat-3.
  • the sulfh ⁇ dr ⁇ l protection was released following coupling of the haloacet ⁇ l peptide to the previously deprotected c ⁇ steine residues on the base peptide Deprotection was accomplished with an excess of BU3P reagent as used earlier in the assembl ⁇ process.
  • Haloacet ⁇ l peptides were cleaved in a cleavage cocktail consisted of TFA, water, phenol (90:5:5). Thiol scavengers were not used since thiol compounds could react with the haloacet ⁇ l moiet ⁇ as well as could result in the deprotection of the C ⁇ s (tButhio) in the Tat linear peptide.
  • haloacet ⁇ l peptides were purified and characterized as described in Example 9 above.
  • HPLC purified linear or S-protected haloacetyl peptides comprising one or more functional epitopes were then coupled to the cysteine thiol groups on the base peptide to generate an MPC molecule having the structure shown in Figure 7C.
  • Examples 13 and 14 describe the coupling of the malarial or HIV Tat peptides to the base peptide.
  • the base construct and the linear haloacet ⁇ l malarial peptide were coupled as follows. Into a 50 ml teflon flask fitted with a nitrogen tube, the base peptide was dissolved in 0.5M NaHC03 or 0 1 1 Tns/guandine-HCI pH 8.0 buffer as needed. A solution of BU3P in 1 propanol or TCEP was freshl ⁇ prepared. The haloacet ⁇ l peptides were dissolved in 1 2 ml (0-6M) guandinme-HCI and added to the reaction flask under nitrogen. For example, to 10 mg base peptide T3 (1.7 M) 0.6 M reducing agent was added for reduction of the base c ⁇ stei ⁇ l residues.
  • the solution was placed on a stirrer for 1 hour followed b ⁇ the addition of 0.8 mg (0.29 M) haloacet ⁇ l peptide (T1) to the reaction mixture.
  • the coupling reaction was then carried out for 1.5 3 hours at 25°C for a bromoacet ⁇ l peptide and up to 6 hours when using the chloroacetyl derivative.
  • the reaction mixture was monitored with MALDI-MS for the presence of conjugate material, -mercaptoethanol (0.2 ml) was then added to the reaction flask.
  • the conjugate mixture was then desalted by RP-HPLC and further purified by size exclusion chromatography to isolate the desired multiple peptide conjugate.
  • Example 14 The base peptide and linear (sulfhydr ⁇ l protected) HIV-1 Tat haloacetyl peptide were coupled as follows.
  • HIV-Tat peptide (Tat-1 ) peptide contains seven protected cysteine residues.
  • the base peptide was dissolved in 1 Tns/6M guanidine HCl buffer, pH 8.0.
  • a fivefold excess (based on thiol content) of BU3P in 1 -propanol was added to effect reduction of the base cysteine residues.
  • 10 mg (1.4 M) of the base peptide Tat-2-Tat-3 0.25 M BU3P reagent was added.
  • 1.6 mg (0.5 M) chloroacet ⁇ l peptide was dissolved in 6M guanidme-HCI and added to the reaction mixture.
  • the peptide conjugate mixtures were desalted b ⁇ RP-HPLC using a C i 8 column (10 ⁇ , 19 mm x 150 mm) Waters, Milford, MA using a one step gradient of 0.1 % TFA/H2O and 10% aceton ⁇ tr ⁇ le/H2 ⁇ to elute the low molecular weight salts followed b ⁇ 70% acetonitnle to elute the MPC or an ⁇ unconjugated peptides.
  • the conjugated mixtures were then lyophilized and stored at -20°C.
  • the MPC, thus formed was further characterized by mass spectroscopy, size exclusion chromatograph ⁇ , and
  • Example 16 describes the size exclusion chromatograph ⁇ anal ⁇ ses, while Example 17 describes the SDS gel electrophoresis.
  • Size exclusion chromatograph ⁇ was performed using a D ⁇ ol-S5 column (YMC, Wilmington, NC) to isolate the desired MPC peptide from the reaction mixture.
  • the elution buffer consisted of 0.1 M phosphate buffer, 0.2 M NaCI, pH 7.0, containing 0.02 M sodium azide.
  • the column was standardized using a GPC standard molecular weight mixture obtained from Bio Rad (Hercules, CA).
  • Example 17 The MPCs were subjected to SDS-polyacrylamide gel electrophoresis using 10-20% Tricme gels (Novex, San Diego, CA) under reducing conditions. After completion of electrophoresis, gels were stained with Commassie blue to visualize the position of the MPCs.
  • Table C The yields of the various base constructs and the efficiency of conjugation are summarized in Table C and
  • Mass anlaysis of the T1-T3 construct produces an ion signal centered at 11,144 m/z.
  • Analysis of the T1- CSP-T3 produces a similar ion cluster centered at 12,737 m/z.
  • the level of punt ⁇ for each MPC was determined b ⁇ analytical reverse phase HPLC on a Vydac C18, 5 ⁇ column.
  • Molecular weight of the MPCs was determined b ⁇ SDS-PAGE under reducing conditions.
  • the apparent molecular weights of malaria peptide conjugates T1-T3, T1 -MSP-1 , and T1 -CSP-T3 were observed to be 10 kDa, 12 kDa, 13 kDa and that of the HIV-1 -Tat MPC (Tat-1 -Tat-2-Tat-3) to be 12 kDa, respectively.
  • Overall purities of the tetramenc MPC molecules were greater than 80% with the absolute percent dependent upon isolation and removal by size exclusion chromatography (SEC) of the base and linear peptides used as reacta ⁇ ts.
  • SEC size exclusion chromatography
  • assembl ⁇ of a tetramenc core template and coupling to alternate I ⁇ sine residues with the epilson position remaining blocked ma ⁇ provide greater access to the resin bound ammo groups during the ac ⁇ lation and deprotection reactions, thus ensuring a higher degree of coupling efficiency at each cycle especially when coupling residues with bulky side chains.
  • greater than 99% coupling yields for each residue were obtained as determined b ⁇ Kaiser or TNBS assa ⁇ .
  • peptide lengths described above are 31 residues per branch or less on the base peptide, peptide lengths of 50 or more residues per branch ma ⁇ be achieved with similar levels of punt ⁇ and yield using the above-described methodology.
  • the buffer used to solubihze the base and linear peptides should be selected to optimize the conditions for the thiol reaction.
  • the Tat MPC has higher levels of these peptides which ma ⁇ be a factor in suppression of the lomzation of the MPC when anal ⁇ zed b ⁇ MALDI-MS.
  • these low intensity signals observed appear to be consistent with the expected molecular mass indicating the presence of both tetramer and tnmer species.
  • the above strategies for synthesizing MPCs provide several benefits.
  • the above methods facilitate s ⁇ nthesis of a well defined core template moiecule that minimizes steric hindrance b ⁇ coupling to onl ⁇ two branches per s ⁇ nthetic c ⁇ cle. While core molecule spacing is similar to that used in the s ⁇ nthesis of a tetramer, onl ⁇ two residues are assembled per c ⁇ cle at alternate ammo groups on the tetramenc core with the opposite positions remaining protected throughout the synthesis of the base molecule.
  • a senne residue with a t ⁇ tyl group used as side chain protection is incorporated into the core template.
  • the senne residue can be modified on the solid phase for greater functionality without cleavage of the peptide resin link, thereby permitting the attachment of antigens or an adjuvant molecule if desired. Furthermore, as discussed above, synthesis of a base molecule with specific thiol sites for attachment of additional peptides allows site directed coupling of the linear peptides.
  • solubility of the peptide chains ma ⁇ be improved b ⁇ modifying the N-methylp ⁇ roliidone coupling solvent b ⁇ adding 10% DMSO, thereb ⁇ enhancing the ac ⁇ lation and/or deprotection reaction and improving coupling efficiency.
  • addition of Hmb protected ammo acids at specific points in the peptide chain further minimizes aggregation of the growing peptide chains thereb ⁇ minimizing the risk of lower ⁇ ieids in the desired peptide.
  • the homogeneit ⁇ of the product ma ⁇ be enhanced b ⁇ attaching blocking groups to the side chains of one or more ammo acids in the haloacet ⁇ l peptides to be attached to the base peptide.
  • a tButhio side chain blocking group was used on all c ⁇ stem ⁇ l residues in the haloacet ⁇ l peptides. Internal c ⁇ stem ⁇ l residues were selectively deprotected onl ⁇ after formation of the final multiple peptide conjugate.
  • the above methods permit further side chain modification to the i ⁇ sine and c ⁇ steine residues on the core template, thereb ⁇ permitting alternative methods of selectively attaching peptides to the core template or the core peptide.
  • the reactive groups on the core template can be selected so that one reactive group is deprotected upon treatment with base while the other reactive group is deprotected upon acid treatment.
  • an alternate use of the core template functionality is in the replacement of the t-Boc group at the branched lysine position b ⁇ a quasi-orthogonall ⁇ protected I ⁇ sine derivative such as Fmoc-Lys(Dde) or Fmoc L ⁇ s( ⁇ vDde)-OH
  • I ⁇ sine derivative such as Fmoc-Lys(Dde) or Fmoc L ⁇ s( ⁇ vDde)-OH
  • L ⁇ s(Mtt) or L ⁇ s(Tfa) allows selective deprotection with dilute acid solutions.
  • the methodolog ⁇ described herein produces well characterized multiple peptide conjugates useful in the development of subumt vaccines in significant yields for important commercial applications.
  • Tat protein is produced b ⁇ HIV-infected cells and is believed to be involved in the progression of HIV infection, development of Kaposi's sarcoma, immunosuppression, apoptosis of normal cells, and various neurological disorders.
  • Tat protein b ⁇ prophylactic immunization of HIV-infected individuals could reduce viremia and thereby prevent or delay the onset of progression to AIDS.
  • Neutralization of extracellular Tat could also partly restore immune function in AIDS patients.
  • Recent studies have proposed Tat toxoid or oxidized Tat protein as potential candidates for therapeutic AIDS vaccines.
  • the present invention is directed to a novel highl ⁇ immunogemc s ⁇ nthetic multiple peptide conjugate constituting functional domains Tat2140 and Tat53 68 from HIV-1 group M plus Tat9-20 from HIV-1 group 0 of the HIV-Tat protein (HIV-1 Tat-MPC).
  • HIV-1 Tat-MPC HIV-Tat protein
  • Vaccination of mice with HIV-1 -Tat-MPC induces an effective immune response to all three functional domains.
  • a ⁇ t ⁇ -HIV-1 -Tat-MPC antibodies efficiently inhibit Tat-mduced viral activation in monocytes infected with HIV ⁇ a-L as well as with various clinical HIV-1 isolates, and reduce Tat-mediated cytopathicit ⁇ in infected cells b ⁇ greater than 75%.
  • the preferred embodiment of the present invention utilizes three pathogenesis-related functional domains of the HIV Tat protein: the cysteine-nch Tat2l 40 and basic Tat53-68 domains critical for promoting HIV and the Tat 920 domain of HIV-1 group 0.
  • Consensus-B Tat21 40: ACTNCYCKKCCFHCQVCFTT (SEQ ID N0:1)
  • Tat53-68 RQRRRAHQNSQTHQAS (SEQ ID N0:2) Consensus-O:
  • Tat9-20 PPWHHPGSQPQl (SEQ ID N0:3)
  • HIV-1-Tat-MPC The three peptide sequences described above were used to prepare the s ⁇ nthetic construct HIV-1-Tat-MPC, the structure of which is shown in Figure 8.
  • the Tat 920 domain of HIV-1 group 0 enhances HIV infection in monoc ⁇ tes ( Figure 9).
  • Construction of HIV-1 -Tat MPC utilized an Fmoc solid-phase s ⁇ nthesis coupled with conventional solution chemistr ⁇ as described above.
  • the HIV 1 -Tat-MPC was constructed with Tat53 68 + Tatg.20 as a base peptide and Tat2l40 as a linear peptide attached to the tetramenc core template (Figure 8).
  • Final assembl ⁇ of the HIV-1 -Tat-MPC construct involved coupling of purified N-chloroacet ⁇ l (C ⁇ s-tButh ⁇ o)-Tat2l 40 peptide to the c ⁇ steine thiol groups on the core containing the base peptide Tat53.g8+Tatg.20 as described above.
  • the t-Buthio protecting groups were removed and purit ⁇ of HIV-1 Tat-MPC was established b ⁇ reverse-phase HPLC (RP-HPLC) on a 4.6 x 25 cm V ⁇ dac C18 column with a 0-80% gradient of 0.1 % TFA/H2O and 0.1 % TFA/acetonit ⁇ le.
  • HIV-1-Tat MPC eluted as a major peak at 55% acetonitnle.
  • the fractions eluted in the major peak at 9.353 m retention time were pooled, lyophilized, repunfied by HPLC, and stored at -70°C. Homogeneity of this HIV-1-Tat-MPC preparation was further evaluated b ⁇ SDS pol ⁇ acr ⁇ lamide electrophoresis with immunological characterization b ⁇ Western blot analysis.
  • HIV-1 Tat-MPC consisted of a predominantly homogeneous species ( > 90%) with an apparent molecular mass of 12 kDa that showed strong reactivity with rabbit anti Tat antiserum. In contrast, purified recombinant Tat used as a positive control exhibited multiple bands, indicating the presence of polymerized products.
  • HIV-1 -Tat-MPC ability of HIV-1 -Tat-MPC to induce an effective immune response was tested by immunizing three groups of BALB/c mice with various doses of HIV-1 -Tat-MPC b ⁇ intraperitoneal injections. Serum antibod ⁇ responses were monitored b ⁇ ELISA. All three groups of mice immunized with 10-100 ug doses of HIV 1 -Tat-MPC developed antibod ⁇ titers (Fig. 10, panel a). The largest immune response was observed in mice immunized with 100 ug of antigen. Peak antibod ⁇ titer was observed at six weeks (second bleed) for all groups of mice. The antibod ⁇ titers at six weeks after the initial immunization achieved detectable levels even up to 1 :243,000 serum dilutions (Fig. 10, panel b).
  • anti-HIV 1 Tat-MPC antisera The binding of anti-HIV 1 Tat-MPC antisera to rTat and the various s ⁇ nthetic peptides present in the construct was determined b ⁇ ELISA using 96-well plates coated with 10 ug/ml of each antigen. As evident from the data, antisera raised against HIV-1 Tat-MPC showed strong reactivity with rTat as well as with all three Tat peptides.
  • ant ⁇ -HIV-1 Tat-MPC antisera to inhibit Tat-stimulated HIV 1 production was evaluated by infection of monoc ⁇ tes in the presence of endogenous or exogenous HIV-Tat protein. Monoc ⁇ tes were infected with HIV ⁇ a-L in the presence or absence of rTat (20 ng/ml) and ant ⁇ -HIV-1 -Tat-MPC antiserum from each mouse at a final dilution of 1 :100. The medium containing rTat or ant ⁇ -HIV-1-Tat-MPC antiserum was replaced ever ⁇ alternate da ⁇ .
  • HIV ⁇ a-L-asso ⁇ ated c ⁇ topathic effects were observed to be reduced b ⁇ 60-75% in monoc ⁇ tes treated with ant ⁇ -HIV-1 Tat-MPC antisera.
  • the neutralizing anti-HIV- 1 -Tat-MPC antisera were also able to inhibit Tat- mediated induction of HIV replication b ⁇ 15-67% in monoc ⁇ tes infected with a variet ⁇ of clinical isolates (Table Two).
  • Figure 13 depicts a possible mechanism b ⁇ which a ⁇ t ⁇ -HIV-1-Tat-MPC inhibits Tat-mduced HIV replication in monoc ⁇ tes.
  • Extracellular Tat protein produced b ⁇ HIV-infected cells is believed to act on cells both in autocrme and paracnne fashion. Tat activates normal cells for productive HIV infection and then promotes virus replication in HIV- infected cells. It is likely that ant ⁇ -HIV-1-Tat-MPC antibodies target key functional determinants and disrupt the autocrme and paracnne actions induced b ⁇ secreted Tat protein.
  • the three peptides used to generate the HIV-1 -Tat-MPC construct were all immunogemc, and antibodies raised in mice against the HIV-1 -Tat-MPC reacted with recombinant Tat protein from which the peptide sequences had been deduced. Furthermore, Tat-mduced pathogenesis was inhibited b ⁇ antisera against HIV-1 -Tat-MPC, supporting roles for Tat2i 40- Tat53-68/ and Tatg.20 epitopes in viral activation b ⁇ infected cells.
  • this HIV-1 -Tat- MPC containing conserved functional domains from group M of HIV-1 , as well as a new functional sequence from the newl ⁇ discovered HIV-1 strain group 0, serves as an immunogen for various subt ⁇ pes of HIV-1 infection.
  • Vaccination of mice with low doses (10 to 20 ug) of the HIV-1 -Tat-MPC construct elicited antibodies that efficiently inhibited Tat effects; 100 ug-injections did not enhance efficacy and even appeared to significantly decrease effectiveness in supporting HIV-1 viral production. It is practical to optimize the minimal dose of the HIV-1 Tat-MPC to induce maximal immune responses in mice and other animal species.
  • HIV-1 -Tat-MPC is a purel ⁇ s ⁇ nthetic molecule that does not contain a live or inactivated virus, it conve ⁇ s no risk of viral transmission to recipients.
  • Example 18 HIV-1 -Tat rotein The HIV-1 -Tat protein used in these studies was obtained as a gift from Dr. John Brad ⁇ (National
  • HIV-1 Tat was dissolved at 10 ug/ml in 1 ,000X treatment buffer (PBS containing 1 mg/ml BSA, 0.1 mM dithiothreitol) and frozen in ahquots at -80°C. Tat preparations were screened and found to be negative for endotoxm contamination.
  • Tat peptides were s ⁇ nthesized b ⁇ solid-phase s ⁇ nthesis on an Applied Bios ⁇ stems Peptide s ⁇ nthesizer Model 430A (Foster Cit ⁇ , CA) Peptide identities were confirmed b ⁇ ammo acid compositional analysis and plasma desorption mass spectroscopic analysis
  • the HIV-Tat-1 MPC was prepared according to the above-described procedure.
  • mice with HIV-1 -Tat-MPC Immunization of mice with HIV-1 -Tat-MPC.
  • Groups of five BALB/c mice (6 8 weeks old) were inoculated three times at three-week intervals with 10 to 100 ug of the HIV-1 -Tat MPC construct (Strategic BioSolutions, Newark, DE). Constructs were mixed with an equal volume of complete Freund's adjuvant for primary injections and incomplete Freund's adjuvant for secondary and tertiary injections. Sera were prepared from blood taken 15 days after each injection.
  • Enz ⁇ me-lmked immunosorbent assa ⁇ was performed using HIV-1 -Tat-MPC coated on 96 well microtiter plates (Costar, Cambridge, MA) at a concentration of 1 ug/ml (Strategic BioSolutions).
  • Antibod ⁇ titers were expressed as optical densit ⁇ (OD) values measured at 650 nm on 1, 000-fold diluted antiserum specimens.
  • Monocyte isolation and infection with HIV- 1 Monoc ⁇ tes were isolated from peripheral blood mononuclear ceils
  • PBMC PBMC of donors seronegative for HIV and hepatitis b ⁇ leukapheresis and purification b ⁇ countercurrent centrifugal elutnation (Lafrenie et al., J. Immunol., 156:1638 (1996)).
  • monoc ⁇ tes were treated with HIV-1 Tat protein.
  • Pnmar ⁇ monoc ⁇ tes cultured for 5 da ⁇ s were exposed to HIV l R a .
  • Splenoc ⁇ tes (2 X 10 ⁇ cells) were cultured in round-bottom 96 well tissue culture plates (Costar) with 10 ug/ml rTat, various Tat peptides, or medium alone and incubated in a 5% CO2 humidified incubator at 37°C for 96 h and pulsed with 1 uCi ⁇ H th ⁇ midme/well during the last 4 h. Cells were harvested and incorporation was determined b ⁇ liquid scintillation counting. All cultures were performed in triplicate.
  • rTat and HIV- 1- Tat-MPC by Western blot analysis.
  • the lyophilized rTat protein or HIV-1 -Tat-MPC were resuspended in SDS-Laemmli loading buffer [500 mM Tris-HCl, pH 6.8, 10% SDS, 0.01 % bromophe ⁇ ol blue, 20% gl ⁇ cerol], reduced with 1 % mercaptoetha ⁇ ol, heated for 2 mm at 95°C, loaded, and electrophoresed on a 8-16% Tns- gl ⁇ cme gradient poi ⁇ acr ⁇ lamide gel (Novex, San Diego, CA) in SDS running buffer [25 mM Tris-HCl, pH 8.3, 192 mM giycine, 10% SDS].
  • the rHIV-1 -Tat protein and HIV 1 -Tat-MPC were transferred onto 0.45-uM nitrocellulose in a buffer containing 25 mM Tris-HCl [pH 8.3, 192 mM giycine, 20% methanol] and blocked with 50 mM Tris-HCl [pH 7.5, 150 mM NaCI, 0.2% Tween-20 (TBST)] containing 5% nonfat dry milk for at least 1 h.
  • the blots were washed three times with TBST and then incubated for 1 h with rabbit anti Tat antibody followed b ⁇ horseradish peroxidase (HRP)-conjugated protein A (Pierce Chemicals, Rockford, IL; 1 :3,000 dilution in TBST containing 5% nonfat dr ⁇ milk) and developed with SuperSignal chemiluminescence enhancer solution (Pierce).
  • HRP horseradish peroxidase
  • IL horseradish peroxidase
  • the bands were visualized b ⁇ exposure of blots to X-ra ⁇ film.
  • Monocytes (2 x 10 ⁇ cells) cultured in 96-well plates were treated with HIV-Tat (20 ng/ml) in the presence or absence of mouse antisera against HIV-1-Tat-MPC (pooled 20 ug immunizations, 1 :100 dilution) 2 h prior to HIV infection at an MOI of 0.005. After a 2 h exposure, virus was removed, and the culture medium was replaced with fresh medium with or without rTat in the presence or absence of antisera against HIV-1-Tat-MPC (1 :100). On da ⁇ 5, culture medium was replaced with fresh medium with no additions. After 24 h, culture fluid was harvested for p24 determination.
  • Monoc ⁇ tes (2 x 10 ⁇ cells) cultured in 96-well plates were treated with HIV-Tat (20 ng/ml) in the presence or absence of mouse antisera against HIV 1 -Tat-MPC (pooled 20 ug immunizations, 1 :100 dilution) 2 h prior to HIV infection with various HIV-1 isolates (40,000 RT/10 ⁇ cells).
  • virus was removed, and the culture medium was replaced with fresh medium with or without rTat in the presence or absence of antisera against HIV-1 Tat-MPC (1:100).
  • culture medium was replaced with fresh medium with no additions.
  • culture fluid was harvested for p24 determination. The p24 values are expressed as mea ⁇ SEM of three determinations. Values in parentheses represent percent inhibition of HIV production b ⁇ ant ⁇ -HIV-1 -Tat-MPC antisera. ND: not determined.
  • Example 19 Effect of anti-HIV ) ' -Tat-MPC antibody on uninfected or HIV-1 -infected monocyte migration in response to HIV-1 Tat protein.
  • Uninfected or HIV-infected monoc ⁇ tes were suspended at 2 X 10 6 cells/ml.
  • HIV-1 Tat (20 ng/ml) was placed in the lower chamber of a chemotactic chamber in the presence of normal mouse serum or mouse anti-HIV- 1 -Tat-MPC antiserum (1 :100), covered with a pol ⁇ carbonate filter, and 50 ul of the uninfected or HIV-1 -infected monoc ⁇ te suspension was placed in the upper chamber.
  • Part I Subhash Dhawan, Robert A. Bo ⁇ kins and Kenneth M. Yamada
  • Part II Robert A. Bo ⁇ kins, Manju B. Joshi, Chiang S ⁇ in, Subhash Dhawan and Hira L. Nakhasi
  • Part III Subhash Dhawan and Robert A. Bo ⁇ kins.

Abstract

The present invention relates to pathogenic HIV-1-Tat peptides, multiple peptide conjugates, and HIV-1-Tat-multiple peptide conjugate, methods of preparing them, and methods of using them to induce an immune response.

Description

HIV TAT PEPTIDES AND MULTI PLE PEPTIDE CONJUGATE SYSTEM PART 1: PATHOGENIC HIV-1-TAT PEPTIDES
Background of the Invention Infection with HIV, the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is responsible for a large number of deaths annually and represents a significant threat to human health Accordingly, an extensive effort has been mounted to characterize the HIV virus and to identify potential targets for therapeutics.
The HIV Tat protein, a transactivator which regulates gene expression from the HIV long terminal repeat
(LTR) represents one attractive therapeutic target. The Tat protein of human immunodeficiency virus type-1 (HIV-1 ) is an 86 ammo acid protein released by infected cells and plays a critical role in the progression of HIV disease (Jeang, J Biomed. Sci. 5. 24 (1998); Ensoli et al., J. Virol. 67: 277(1993)). Traπsactivation of the HIV-LTR promoter by the Tat protein is essential for both viral gene expression and virus replication. Extracellular Tat released by infected cells during the acute phase of infection enters non-infected cells and disrupts many host immune functions by activating a wide variety of genes regulated by specific viral and endogeneous cellular promoters (Vaishnav and Wong-Staal, Ann
Rev. Biochem. 60: 577 (1991 ); Kumar et a/., J. Immunol. 161 : 776 (1998)) In addition, Tat mimics many of the effects of HIV infection on monocytes including increased P-9 and cytokine production, and collagen expression in glioblastoma cells (Lafrenie et al., J. Immunol. 159: 4077 (1997);
Lafrenie et al., J. Immunol. 156: 1638 (1996); Taylar et al, Proc. Natl. Acad. Set., USA 89:9617 (1992)) These observations correlate with the high levels of cytokines such as IL-1 , IL-6, and TNF found in sera from HIV-infected individuals that lead to an increase in the level of HIV replication, thus suggesting that extracellular Tat promotes the intracellular pathogenic process. However, Tat has multiple domains, and consequently how Tat induces these diverse effects is not clearly understood.
Summary of the Invention
HIV-1 encodes the transactivatiπg protein Tat, which is essential for virus replication and progression of HIV disease. However, Tat has multiple domains, and consequently the molecular mechanisms by which it acts remain unclear. We show that cellular activation by Tat involves a short core domain, Tat21 40, containing only 20 ammo acids including seven cysteine residues highly conserved in most HIV-1 subtypes. Effective induction by Tat21 0 of both NF- κB-medιated HIV replication and TAR-dependent transactivatioπ of HIV-long terminal repeat indicates that this short sequence is sufficient to promote HIV infection. Moreover, Tat21 40 possesses potent angiogemc activity, further underscoring its role in HIV pathogenesis. These data provide the first demonstration that a 20 residue core domain sequence of Tat is sufficient to transactivate, induce HIV replication, and trigger angiogenesis. This short peptide sequence provides a novel therapeutic target for disrupting the functions of Tat and inhibiting progression of HIV disease. Brief Description of the Drawings Figure 1. Ammo acid sequence of HIV-Tat protein from various subtypes, designated consensus B (SEQ ID NO: 74), consensus C (SEQ ID NO: 75), consensus D (SEQ ID N0:76), conseπsus-F (SEQ ID N0:77), coπsensus-0 (SEQ ID N0:81 ); and consensus-U (SEQ ID N0:79). Highly conserved residues are indicated at the bottom (SEQ ID N0:80).
Figure 2 shows the chemical structure of one embodiment of a core template of the present invention. Figure 3 shows the chemical structure of one embodiment of a base peptide of the present invention. Figure 4 shows the chemical structure of one embodiment of a multiple peptide conjugate of the present invention. Figure 5A and B show the sequences of malarial peptides and HIV-1 peptides used in some embodiments of the multiple peptide conjugates of the present invention. (A) Letters underlined represent Hmb derivatized ammo acids for malaria peptides; (B) letters underlined show the position of the seven cγsteιne(tButhιo) residues in the HIV-Tat peptide (HIV-1 -Tat-1 ).
Figure 6 illustrates one embodiment of the synthetic process which was used to prepare a multiple peptide conjugate.
Figure 7A illustrates one embodiment of a core template of the present invention. Figure 7B illustrates one embodiment of a base peptide of the present invention. Figure 7C illustrates one embodiment of a multiple peptide conjugate of the present invention. FIGURE 8. Chemical structure of HIV-1 -Tat-MPC comprising functional epitopes of the HIV-1 -Tat protein. FIGURE 9. Effect of HIV-1 -Tat synthetic peptide on HIV replication in monocytes. Monocytes were infected with HIVβa-L ιn ne presence or absence of recombinant HIV-1 Tat or the group 0 peptide. At day 6, culture supernatants were harvested, and cell-free virus was determined by p24 ELISA. The data are representative of two separate experiments.
FIGURE 10. Panel a, Humoral response in mice immunized with various doses (10 ug, 20 ug, and 100 ug) of the HlV-1-Tat-MPC. Antisera were assayed by ELISA on 96-well plates coated with HIV-1-Tat-MPC at a concentration of 1 ug/ml. Titers shown as OD values at 650 nm of 1000-fold diluted serum specimens. Panel b, Antι-HIV-1 -Tat-MPC titer of sera at various dilutions, where reactivity was determined as described for panel a. ( • ) 10 ug HIV-1 -Tat-MPC;
( o ) 20 ug HIV-1 -Tat-MPC; ( ) 100 ug HIV-1 -Tat-MPC.
FIGURE 11. Panel a, Binding of antι-HIV-1 -Tat-MPC antisera to recombinant (rTat) protein and Tat peptides. Ninety-six (96) well plates were coated with 10 ug/ml of rTat protein or Tat peptides as indicated and blocked with
SuperBlock blocking buffer in PBS (Pierce). Bound antibodies were detected by ELISA. Panel b, Specificity of HIV-1 - Tat-MPC antisera to rTat was evaluated by competition experiments using soluble peptides. The ability of purified peptides to inhibit binding of antι-HIV-1 -Tat-MPC antibodies to rTat protein was evaluated in the presence of the indicated peptide combinations. Each peptide (10 ug/ml) was incubated with 1:100 diluted antisera prior to addition onto rTat-coated plates, and bound antibodies were determined by ELISA Data for triplicate experiments are shown as OD + SEM at 450 nm.
FIGURE 12. Lymphoproliferative responses of spleen cells from Balb/c mice vaccinated with HIV 1 Tat- MPC. Splenocytes (2 x 10^) prepared from mice immunized with 20 ug of HIV-1 Tat-MPC were incubated with 10 ug rTat , HIV-1-Tat-MPC, or Tat peptides for 96 h at 37°C, and antigen-induced proliferative responses were determined.
The bars represent
Figure imgf000005_0001
incorporation, expressed as cpm + SEM from triplicate cultures of pooled spleen cells from two independent experiments. rTat, P< 0.005; HIV-1 Tat-MPC, P < 0.01; Tatg.20, P < 0.001 ; Tat2l-40- P < 0.02; Tat53-68/ P < 0.005; Tat4i .52- P = 0.97 versus the control with medium alone.
FIGURE 13. Model illustrating a possible mechanism for inhibition of Tat-mduced HIV pathogenesis by anti- HIV-1-Tat MPC antibodies.
Detailed Description of the Preferred Embodiment The present invention relates to the identification of functional domains of the HIV Tat protein which mediate viral activation. As will be familiar to those skilled in the art, there are a variety of subtypes of HIV, including the B, C, D, F, M, 0, and U subtypes. The present invention uses particular peptide sequences to identify functional domains in Tat. However, it will be appreciated that the particular sequences used in the examples below may be substituted with the corresponding sequences derived from consensus sequences for each subtype, corresponding sequences from individual isolates, or corresponding sequences from subtypes other than those specified above. Accordingly, the present invention specifically contemplates the use of the corresponding sequences from such consensus sequences, isolates or other subgroups. As used herein, the term "purified" does not require absolute purity; rather, it is intended as a relative definition.
For example, purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated as falling within the definition of "purified."
As used herein, the term "isolated" requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polyπucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
Figure 1 lists the consensus sequences of the Tat genes from the B, C, D, F, 0, and U subtypes as well as a consensus sequence derived from the sequences from all of these subtypes. In order to identify Tat-specific sequences responsible for cellular dysfunction, overlapping peptides from various domains of the consensus-B and other HIV-1 subtypes were synthesized. The ability of the peptides to direct HIV replication in monocytes was compared to that of full length recombinant Tat protein as described in Example 1 below.
Example 1 Evaluation of Effects of Peptides on HIV Replication in Monocytes Tat peptides to be tested for their effects on HIV replication in monocytes were synthesized by solid phase synthesis on an Applied Biosystems Peptide synthesizer Model 430A (Foster City, CA) (Merπfield, J. Am. Chem. Soc.
85: 2149 (1963)). After an initial HPLC purification of the crude cysteme-contammg peptides, they were redissoived in
0.1 M Tris acetate buffer, pH 8.3, and air-oxidized overnight. Peptides were then subjected to desalting and purification by reverse-phase HPLC, lyophilized, and stored at -70°C Peptide identities were confirmed by ammo acid compositional analysis to determine specific molar ratios for each residue and plasma desorption mass spectroscopic analysis.
Recombinant full length Tat protein was obtained through the AIDS Research and Reference Reagent
Program, Division of AIDS, National Institute of Allergies and Infectious Disease, National Institutes of Health, from Dr. Andrew Rice or Dr. John N. Brady (Lafrenie et al, J. Immunol., 156:1638(1996)). HIV Tat was dissolved at 10 g/ml in treatment buffer (PBS containing 1 mg/ml BSA, 0.1 mM dithiothreitol) and frozen in aliquots at 80°C. Tat preparations were screened and found to be negative for endotoxin contamination.
Monocytes were isolated from the peripheral blood mononuclear cells of donors seronegative for HIV and hepatitis after leukapheresis and purification by countercurrent centrifugal elutnation (Wahl and Smith In Current Protocols in Immunology, J. E. Coligan, A. M. Kruisbeek, and D. H. Margulies, E. M. Shevach, and W. Strober, eds.,
2:7.6,1 (1991 )). Primary monocytes cultured for 5 days were exposed to HIV-1 βa-L a monocytotrophic HIV strain
(Advanced Biotechnologies Inc.), at a multiplicity of infection of 0.01 infectious virus particles/target cell (Dhawan et al., J. Immunol. 154: 422 (1995)).
Monocytes cultured for 5 days were treated with recombinant full length Tat protein or test peptides. On day 5, the cells were harvested and the concentration of p24 gag protein (of HIV) in culture superπatants was determined using a DuPont (Wilmington, DE) p24 ELISA test kit. Data representative of two separate experiments are shown in Table I and expressed as mean + / SEM of triplicate determinations.
As shown in Table I below, like recombinant full length Tat (rTat), the 20 ammo acid core domain Tat2l -40 containing 7 cysteine residues, all of which are strongly conserved in various subtypes, enhanced HIV replication by greater than four fold. A peptide derived from the basic domain (Tat53 68) induced a lesser increase in viral replication compared to Tat21-40- Likewise, the Tatg 2α peptide also greatly enhanced HIV replication. In contrast, Tat4i-52, a peptide sequence located between the core and the basic domains, and a variety of peptides from other positions in the
Tat sequence, had no significant effect on HIV replication.
Table I. Effect of various Tat peptides on HIV replication in monocytes
Residue p24 (pg/ml) numbers Tat sequence (Mean +. SEM) Control 471 +. 10
Ti-86 rTat (recombinant full length Tat) 1886 +. 26 ati -20 MEPVDPRLEPWKHPGSQPKT1 421 + 3
Tati O-30 PWKHPGSQPKTACTNCYCKKC2 378 + 4
Tat21 -40 ACTNCYCKKCCFHCQVCFTT3 1958 + 101
Tat31-51 CFHCQVCFTTKGLGISYGRK4 429 + 24 at53.68 RQRRRAHQNSQTHQAS5 969 + 75
Tat25-40 CYCKKCCFHCQVCFTT6 836 + 123
Tat8-19 LDPWNHPGSQPT7 370 + 2
Tat4i-52 KGLGISYGRKKR8 569 + 9
Tat8-19 LEPWNHPGSQPK9 376 + 2
Tat92o PPWHHPGSQPQΓ0 1513 + 12
The cytopathic effects of the Tat peptides were examined in monocytes as described in Example 2.
Example 2 Monocytes were infected with HIV and contacted with recombinant full length Tat or Tat peptides as described above. At five days post oculation, ceils were washed once with PBS, fixed, and Wright-stained. HIV- associated cytopathic effects were determined by examining the formation of multinucleated giant cells.
Consistent with its enhancement of viral replication, Tat21-40 treatment produced a marked increase in HIV- associated cytopathic effects in monocytes as indicated by formation of multinucleated giant cells; the effects were similar to those induced by rTat protein itself. The effect of Tat53-68 was less than that of Tat21-40- Tat4]-52, the peptide between core and basic domains, and peptides from other Tat domains did not alter HlV-associated cytopathic effects. Thus, a major active site for stimulating HIV replication and monocyte dysfunction can be localized to the 20- residue peptide Tat21 -40 and to a lesser extent to Tat53-68 peptide.
1 SEQ ID NO:67
2 SEQ ID NO:68 SEQ ID NO: 1
4 SEQ ID NO:69 3 SEQ ID NO:2 6 SEQ ID NO:70 7 SEQ ID NO:71 8 SEQ ID NO:72 9 SEQ ID NO:73 10 SEQ ID NO:3
-3- One of the mechanisms by which HIV-Tat potentiates HIV replication involves transactivation of the HIV-1 LTR via its binding to the TAR sequence along with other cellular factors, resulting in increased viral transcription initiation and elongation (Cujec et al, (1997) Mol. Cell. Biol. 17: 1817). In order to characterize further the mechanism of Tat transactivation of the HIV LTR, CEM lymphoid cells were transfected with wild-type promoter in the presence of various Tat peptides, and the extent of transactivation was determined using CAT assays (Cujec et al., Mol. Cell.
Biol. 17: 1817 (1997)) as described in Example 3.
Example 3 Transactivation Assays Cells were electroporated as described in (Kashanchi et al, Virology 227: 431 (1997)). CEM cells (12D7) were cultured at a density of 0.5 to 0.8 x10^ cells/ml with daily media additions. Typically, 5x10^ cells were electroporated with 5 g of either purified plasmid or Tat protein and 5 g of reporter plasmid. Tat peptides or Tat protein and the reporter HIV LTR-CAT or the TAR mutant HIV TM26 LTR-CAT were mixed with cells and electroporated using a cell porater apparatus (GIBCO/BRL, Gaithersburg, MD). Cell mixtures were electroporated at 800 F, 240V, in RPMI 1640 medium without serum. Following electroporation, cells were plated in 10 ml complete medium, and samples were collected 24 h later for CAT assays.
The Tat21 40 peptide produced a nine-fold induction of HIV-LTR, while full-length rTat produced a 25- foid induction. The actual effectiveness of induction by Tat21-40 might be greater than observed due to the low solubility of this complex hydrophobic peptide in aqueous buffers. In contrast, buffer, Tat1 20, Tat5368, Tat2540, and Tat41 52 demonstrated virtually no significant induction of HIV-1 LTR. The presence of Cys22 in core domain Tat21-40 (and 3 adjacent residues) was critical for viral activation, since deletion of these residues substantially reduced the ability of Tat2l -40 to activate HIV infection (Tat25-40 m Table I). Since rTat activation of the HIV-LTR promoter is required for productive HIV replication (Yankulov and Bentley, Curr. Biol. 18: R447 (1998)), the demonstration of induction by the Tat21-40 sequence conserved in most HIV-1 subtypes further confirms a functional role of Tat2l-40 m HIV infection. In contrast, transfectioπ of CEM cells with a TAR mutant (HIV TM26 LTR-CAT) construct in the presence of the same peptides failed to induce HIV-LTR activation, confirming that the HIV-LTR activation by Tat peptides was TAR-specific.
Previous studies have demonstrated that the argimne rich basic domain located between residues 49 and 57 constitutes the TAR-bindmg activity (Garcia et al, EMBO J. 8:765 (1989); Weeks et al, Science 249: 1281 (1990); Cordingley et al, Proc. Natl. Acad. Sci., USA 87: 8985 (1990); Feng and Holland, Nature 334:165 (1988)). Mutation or deletion of the basic domain severely diminishes the ability of Tat to transactivate the LTR. The overlapping peptιde(s) from this region tested in the present study were not as active as Tat21-40 peptide. Thus, the Tat21-40 peptide may possess the ability to induce TAR-dependent transcription.
Although the precise mechanism of virus regulation by host factors is not clear, it is generally believed that in addition to other unknown factors, Tat and cytokines play a key role in the pathogenesis of HIV infection. Extracellular HIV-Tat causes activation of intracelluiar signal transduction pathways that culminate in the production of various cytokines (Lafrenie et al, J. Immunol. 159: 4077 (1997); Chen et al, J. Biol. Chem. 272: 22385 (1997)). Therefore, because of its ability to induce host factors, Tat is believed to be a key factor for viral enhancement. HIV Tat activates both virai and host cell genes, and the host F transcription factor contributes to immune dysregulation during HIV infection (Conant et al, J Virol. 70: 1384 (1996); Ott et al, J. Immunol. 160: 2872 (1998)). Since macrophages are a well-known reservoir for HIV in vivo, the ability of Tat peptides to activate the expression of F in these cells was examined. Monocytes were treated with rTat and other peptides, nuclear extracts were prepared, and F activity was examined by gel shift assay using an F consensus oligonucleotide as described in Example 4 below.
Example 4 Effects of Tat Peptides on F Activity Monocytes (1 x10' /ml) were treated with rTat protein or Tat peptides at 37°C for 15 mm. Nuclear extracts were then prepared and analyzed by gel shift analysis as described in (Dhawan et al, Eur. J. Immunol. 27: 2172 (1997)).
The ability of HIV-Tat to activate F was retained in core peptide Tat21 -40 and to a lesser extent Tat53- 68- Treatment of monocytes with the Tat2l-40 peptide rapidly activated F (within 15 mm. after exposure) by greater than 9-fold as compared to 3 fold induction by Tat53-68- Interestingly, despite inducing NF- B activity, Tat53-
68 had little effect on transactivation of HIV-LTR. These observations delineate two distinct mechanisms for viral activation by HIV-Tat: (a) TAR-dependent transactivation of HIV-LTR involving Tat2i40 domain, and (b) TAR- mdependent activation of virus replication involving the host factor NF- B by an tracellular signal transduction pathway. The above results are complementary to those recently reported by Mayne et al, who have demonstrated the involvement of protein kinase A, phospholipase C and protein tyrosine kinase in Tat-mediated induction of NF-kB and cytokine production by monocytes (Mayne et al, Neuroimmunomodulation. 5:184 (1998)).
Tat is released by HIV-infected cells into the extracellular milieu, and has been implicated as a cofactor in the pathogenesis of Kaposi's sarcoma (Albim et al, Proc. Natl. Acad. Sci. USA 92: 4838 (1995)), an aπgioprohferative disease frequently seen in HIV-infected individuals. There is increasing evidence that HIV-Tat induces endothelial cell migration, invasion, and angiogemc processes in vivo (Albim et al, Oncogeπe 12: 289 (1996)). In order to test for potential angiogemc activity of the core domain implicated above in viral pathogenesis, the ability of Tat peptides to induce πeovasculanzation was examined using the chick chonoallantoic membrane (CAM) assay as described in Example 5 below. Example 5
Neovasculaπzation Analysis
The chick CAM assay was carried out as described in (Gho and Chae, J. Biol. Chem. 272: 24294 (1997)) to determine the angiogemc activity of rTat and its derived peptides. Briefly, salt-free aqueous solution (5 I) containing
5.3 pmol of rTat or its derived peptides (Tat2l-40. Tat53-68, or Tat4i-52) was loaded onto a 1/4 piece of 15-mm Thermonox disk (Nunc), and the sample was dried under sterile air; control treatments used water only. The disk loaded with sample was placed on the CAM of a 10 day old chick embryo. After 72 h incubation, negative or positive responses were scored under a microscope. A positive response was characterized as the appearance of a typical radiating network (spokewheel) pattern of new blood vessels around the loaded samples. Assays for each test sample were carried out in 2 sets of eggs, and each set contained 12 to 15 eggs. The results indicate that picomole quantities (5.2 pmol/egg) of Tat2i 0 can induce neovasculaπzation.
Recombinant Tat (rTat) alone was less effective in inducing an angiogemc response, as reported by others (Albi et a/., Oncogene 12. 289 (1996)). No significant angiogemc response was observed using vehicle alone or the control peptide Tat4i -52 containing sequence between the core and basic domains. Interestingly, Tat53-68 from the Tat basic domain also had substantial activity; as noted above, this peptide had either partial or minimal activities in assays for HIV replication, cytopathic effects, and transactivation of the HIV-LTR promoter. The exact mechanism of neovasculaπzation in vivo is not clear. However, one scenario is that Tat induced cytokines stimulate endothelial cells, degrade basement membrane matrix by local enhancement of MMP 9 secretion, and migrate into adjacent tissue to form new blood vessel networks
Detectable levels of Tat have been reported in HIV-infected individuals (Westendorp et al, Nature 375:497 (1995)), suggesting the presence of extracellular HIV-Tat protein in certain phases of HIV infection It has also been shown that high levels of anti-Tat antibodies are directly related to low viral load (Re et al, Clin. Diagn. Lab. Immunol. 3: 230 (1996); Poznansky et a/., Hum. Gene Ther. 9: 487 (1998)) in seropositive non-progressor patients. Therefore, a strategy targeting a required sιte(s) in Tat may be used to provide a novel therapeutic modality to reduce disease progression in HIV-infected individuals. The above results demonstrate that short core domains of the Tat protein retain activities characteristic of the full length Tat protein. In particular, the Tat21 40, Tat536β and Tat920 peptides retain activity. Notably, the Tat2i 40 peptide, which consists of 7 cysteine residues and only 13 other ammo acids, is a potent inducer of HIV transactivation and replication. This domain is highly conserved in various HIV 1 subtypes, including the newly discovered group 0. Some of these results are complementary to those demonstrating the involvement of Tat and the core domain in the process of moπocyte che otaxis in response to Tat (Lafrenie et al, J. Immunol. 157: 974 (1996);
Albi et al, J. Biol. Chem. 273: 15895 (1998)), which may contribute to altered immuπoregulation in HIV-infected individuals. It is important to note that monocytes differentiate into tissue-resident macrophages, which are non- recirculating cells. HIV-infected macrophages could, therefore, continue to infect neighboring normal cells and contribute to the tissue damage typically seen after HIV infection. Accordingly, the invention provides the active domain Tat21 40, Tat5368 and Tat920, singly or in combination, as a novel therapeutic vaccine or a dominant-negative strategy to reduce Tat-mediated progression of disease in individuals with HIV infection.
As used herein the terminology "peptide comprising a Tat functional domain" refers to fragments of the Tat protein comprising one or more of the Tat21 40, Tat53 B8 or Tat920 functional domains from any subtype or isolate of HIV and are not limited to functional domains having the exact sequences used in the experiments described above. In particular, the terminology "peptide comprising a Tat functional domain" encompasses fragments of the HIV Tat protein comprising the sequence ACTNCYCKKCCFHCQVCFTT (SEQ ID NO: 1), the sequence RQRRRAHQNSQTHQAS (SEQ ID NO: 2), or the sequence PPWHHPGSQPQl (SEQ ID NO: 3). The terminology "peptide comprising a Tat functional domain" also encompasses fragments of the HIV Tat protein comprising the corresponding sequences from the consensus sequence for the B subgroup of HIV, i.e. EPWKHPGSQPKT (SEQ ID NO: 4), ACTNCYCKKCCFHCQVCFTT
(SEQ ID NO: 5), or RQRRRAHQNSQTHQAS (SEQ ID NO: 6). The terminology "peptide comprising a Tat functional domain" also encompasses fragments of the HIV Tat protein comprising the corresponding sequences from the consensus sequence for the C subgroup of HIV, i.e. EPWNHPGSQPKT (SEQ ID NO: 7), ACTKCYCKKCSYHCLVCFQT (SEQ ID NO: 8), or RQRRSAPQSSEDHQNP (SEQ ID NO: 9). The terminology "peptide comprising a Tat functional domain" also encompasses fragments of the HIV Tat protein comprising the corresponding sequences from the consensus sequence for the D subgroup of HIV, i.e. EPWNHPGSQPKT (SEQ ID NO: 10), PCNKCHCKKCCYHCQVCFIT (SEQ ID NO: 11 ), or RQRRRPPQGGQAHQVP (SEQ ID NO: 12). The terminology "peptide comprising a Tat functional domain" also encompasses fragments of the HIV Tat protein comprising the corresponding sequences from the consensus sequence for the F subgroup of HIV i.e. DPWNHPGSQPTT (SEQ ID NO: 13), PCTKCYCKRCCFHCQWCFTT (SEQ ID NO: 14), or KQRHRTPQSSQIHQDL (SEQ ID NO: 15). The terminology "peptide comprising a Tat functional domain" also encompasses fragments of the HIV Tat protein comprising the corresponding sequences from the consensus sequence for the 0 subgroup of HIV i.e. PPWHHPGSQPQl (SEQ ID NO: 16), PCNNCYCKRCCYHCYVCFVR (ID NO: 17), or KQGRPAAASHPDHKDP (SEQ ID NO: 18). The terminology "peptide comprising a Tat functional domain" also encompasses fragments of the HIV Tat protein comprising the corresponding sequences from the consensus sequence for the U subgroup of HIV i.e. EPWNHPGSQPKT (SEQ ID NO: 19), ACTKCYCKKCCYHCPVCFLN (SEQ ID NO:
20), or PPPPPSPQNSEDHQNP (SEQ ID NO: 21 ). The terminology "peptide comprising a Tat functional domain" also encompasses fragments of the HIV Tat protein comprising the corresponding sequences from an individual isolate falling within the B, C, D, F, M, 0, and U subgroups or fragments of the HIV Tat protein comprising the corresponding sequences from other subgroups. The terminology "peptide comprising a Tat functional domain" also encompasses peptides that retain at least one activity of the full length Tat protein and which have at least about 60, 70, 80, 90, 95, or 99 % of their ammo acid sequence identical to that of SEQ ID N0:1, SEQ ID NO: 2, or SEQ ID NO: 3 as determined by FASTA or BLAST using default opening and gap penalties and a PAM scoring matrix. Percent sequence identity can be determined by standard methods that are commonly used to compare the similarity in position of the ammo acids of two polypeptides. Using a computer program such as BLAST or FASTA, two polypeptides are aligned for optimal matching of their respective ammo acids (either along the full length of one or both sequences, or along a predetermined portion of one or both sequences) The programs provide a "default" opening penalty and a "default" gap penalty, and a scoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoff et al., in: Atlas of Protein Sequence and
Structure, vol. 5, supp.3 (1978) can be used in conjunction with the computer program. The percent identity can then be calculated as the total number of identical matches/ length of the sequence within the matched span + number of gaps introduced into the longer sequence in order to align the two segments. Peptides that are at least 70 percent identical will typically have one or more ammo acid substitutions, deletions, and/or insertions. Usually, the substitutions will be conservative so as to have little or no effect on the overall net charge, polarity, or hydrophobicity of the peptide but optionally may increase or decrease the activity of the peptide. The terminology "peptide comprising a Tat functional domain" also encompasses fragments of the Tat protein which include one or more am o acids adjacent to the Tat2, 40, Tat53 68 or Tat920 functional domains. For example, the fragments may comprise about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, or more than 70 ammo acids adjacent to the functional domains, provided that such additional sequences are consistent with the length of the Tat protein and that the fragments are shorter than the intact Tat protein.
The terminology "peptide comprising a Tat functional domain" also includes peptide analogs or peptidomimetics based on the Tat21 40, Tat5368 or Tat920 functional domains. Peptidomimetics are molecules that have the same effect as a peptide but are not peptides. Peptidomimetics may be resistant to proteases or cheaper to make than peptides. The peptide comprising a Tat functional domain may be administered via any of the routes traditionally used to administer compositions which induce an immune response, including intraperitoneal administration, intramuscular administration, intradermal administration, intravascular administration or nasal administration.
If desired, the peptide comprising a Tat functional domain may be administered along with an adjuvant to enhance the level of the immune response. A variety of adjuvants suitable for use in compositions for inducing an immune response are familiar to those skilled in the art, including aluminums like hydroxide and phosphate.
The peptide comprising a Tat functional domain may be administered in a physiologically acceptable earner. A variety of carriers suitable for use in compositions for inducing an immune response are familiar to those skilled in the art. Such carriers are nontoxic to recipients at the dosages and concentrations employed. For example, the carriers may contain saline, buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, ammo acids, carbohydrates including glucose or dextnns or chelatmg agents such as
EDTA, glutathione or other stabilizers and excipients.
The peptides comprising a Tat functional domain are administered at a dosage and frequency sufficient to induce a desired level of immune response. Preferably, the level of immune response is sufficient to protect an umnfected individual from infection or to reduce or eliminate the symptoms of HIV infection in an infected individual. It will be appreciated that the dosage and frequency of peptide administration will vary depending on the age, weight, and condition of the individual. For example, the dosage sufficient to induce a desired immune response may range from about 1 μg 100 mg or more, more preferably from about 100μg 100mg or more. However, other dosages may also be used.
In some embodiments, peptides comprising different functional domains of the Tat protein may be administered to the individual. For example, a peptide comprising the Tat21 40 domain and a peptide comprising the Tat5368 domain may be administered to the individual. It will be appreciated that any combination of peptides comprising the Tat21 40, Tat920, or Tat5368 may be administered to the individual. The peptides comprising different functional domains of the Tat protein may be administered to the individual simultaneously or at different times.
Alternatively, rather than administering one or more peptides comprising Tat functional domains to an individual, nucleic acids encoding the one or more peptides comprising Tat functional domains may be administered to the individual. The nucleic acids may comprise the nucleic acid sequence from an HIV isolate or subtype consensus sequences or the nucleic acid may be designed to encode the peptide comprising a Tat functional domain using the genetic code.
In this embodiment, a vector comprising a nucleic acid encoding a peptide comprising a Tat functional domain operably linked to a promoter is administered to the individual. The vector may be any of the expression vectors familiar to those skilled in the art, including viral or retroviral vectors. Viral vectors suitable for use in the present invention include adenovirus, adeno-associated virus, herpes virus, and vaccinia virus. Retroviral vectors suitable for use in the present invention include Moloney murine leukemia virus vectors, Harvey murine sarcoma virus vectors, mouse mammary tumor virus vectors, and Rous sarcoma virus vectors. Other vectors that induce a minimal host response to the vector itself may also be used.
The vector may be directly introduced into the individual such that the encoded peptide comprising a Tat functional domain is expressed in the individual. In another approach, the expression vector may be introduced into cells in vitro. Cells containing the expression vector are thereafter selected and introduced into the individual, where they express the encoded peptide comprising a Tat functional domain. Alternatively, the nucleic acid encoding a peptide comprising the Tat functional domain may be administered as naked DNA. Techniques for administering naked DNA are described in U.S. Patent No. 5,580,859. If desired, the nucleic acid may be administered along with bupivacame.
The nucleic acid encoding a peptide comprising a Tat functional domain may be administered at a dosage and frequency sufficient to induce a desired level of immune response in the individual. Preferably, the level of immune response is sufficient to protect an unmfected individual from infection or to reduce or eliminate the symptoms of HIV infection in an infected individual. It will be appreciated that the dosage and frequency of peptide administration will vary depending on the age, weight, and condition of the individual. For example, the dosage sufficient to induce a desired immune response may range from about 1 μg-100mg or more, more preferably from about 100μg-200μg or more. However, other dosages may also be used. Another aspect of the invention relates to dominant negative mutants of peptides comprising one or more Tat functional domains. Dominant-negative mutants are mutants which are capable of reducing the level of activity of a wild type Tat protein. Such dominant-negative mutants may be generated by mutagenizmg a nucleic acid encoding a peptide comprising a Tat functional domain using techniques familiar to those skilled in the art, including site directed mutagenesis, chemical mutagenesis, or PCR based methods. Alternatively, such dominant-negative mutations may be generated by synthesizing peptides having the desired sequence in vitro. Preferably, the dominant negative mutant comprises a substitution of one or more ammo acids in the peptide comprising a Tat functional domain with another ammo acid. For example, where the peptide comprising the Tat functional domain has the sequence ACTNCYCKKCCFHCQVCFTT (SEQ ID N0:1 ), the A (Alanine) at position 1 of the peptide may be substituted with an ammo acid selected from the group consisting of cysteine, aspartic acid, glutamic acid, phenylalaπme, giycine, histidme, isoleucine, lysiπe, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine. In another embodiment, the C (Cysteine) at position 2 of the peptide may be substituted with an amino acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylaiaπme, giycine, histidme, isoleucine, lysiπe, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine. In yet another embodiment, the T (threonme) at position 3 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysiπe, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, valine, tryptophan, and tyrosine. In another embodiment, the N (asparagine) at position 4 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, prolme, glutamine, argmine, senne, thereomne, valine, tryptophan, and tyrosine. Alternatively, the C (Cysteine) at position 5 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine. In still another embodiment, the Y (tyrosine) at position 6 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leuc e, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, and tryptophan. In a further embodiment, the C (Cysteine) at position 7 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine. In yet another embodiment, the K (lysine) at position 8 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine. In a further embodiment, the K (lysine) at position 9 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine. In yet another embodiment, the C (Cysteine) at position 10 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine. In another embodiment, the C (Cysteine) at position 1 1 of the peptide may be substituted with an am o acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, WO 00/78969 PCT/USOO/l 0119
threonme, valine, tryptophan, and tyrosine. In a further embodiment, the F (phenylalamne) at position 12 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine. In a further embodiment, the H (histidme) at position 13 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine. In still a further embodiment, the C (Cysteine) at position 14 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, iysme, leucme, methiomne, asparagine, prol e, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine. In another embodiment, the Q (glutamine) at position 15 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, argmine, senne, threonme, valine, tryptophan, and tyrosine. In a further embodiment, the V (valine) at position 16 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, tryptophan, and tyrosine. In another embodiment, the C (Cysteine) at position 17 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine. in another embodiment, the F (phenylalamne) at position 18 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine. In yet another embodiment, the T (threonme) at position 19 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, valine, tryptophan, and tyrosine. In yet another embodiment, the T (threonme) at position 20 of the peptide may be substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, valine, tryptophan, and tyrosine.
It will be appreciated that similar single ammo acid substitutions may aiso be made in any of the peptides comprising a Tat functional domain, including the particular Tat21 40, Tat5368, and Tat920 sequences described above.
The invention contemplates ammo, carboxy and internal truncations in a peptide comprising a Tat functional domain. Examples of ammo terminal truncations in one of the Tat21 40 sequences are provided in Table II below. However, it will be appreciated that ammo terminal truncations may also be maαe in the Tat53 68 and Tat92D sequences or in Tat21 40 sequences from other isolates or subtypes.
- I D- The truncated peptides can be produced by any of a number of protocols well known to those of skill in the art. Some short examples include, purifying one or more of the peptides taught by the invention, followed by modification of the peptide. Alternatively, the peptides can be produced recombmantly, using molecular biology and biochemical techniques, or synthetically. These peptides may themselves be chemically modified. Table II
Ammo Terminal Truncations
X FTT-Z (No SEQ ID NO)
X-CFTT-Z (SEQ ID NO: 37)
X-VCFTT-Z (SEQ ID NO: 36) X-QVCFTT-Z (SEQ ID NO: 35)
X-CQVCFTT-Z (SEQ ID NO: 34)
X-HCQVCFTT-Z (SEQ ID NO: 33)
X-FHCQVCFTT Z (SEQ ID NO: 32)
X-CFHCQVCFTT-Z (SEQ ID NO: 31} X-CCFHCQVCFTT-Z (SEQ ID NO: 30)
X-KCCFHCQVCFTT-Z (SEQ ID NO: 29)
X-KKCCFHCQVCFTT-Z (SEQ ID NO: 28)
X-CKKCCFHCQVCFTT-Z (SEQ ID NO: 27)
X-YCKKCCFHCQVCFTT-Z (SEQ ID NO: 26) X-CYCKKCCFHCQVCFTT Z (SEQ ID NO: 25)
X-NCYCKKCCFHCQVCFTT-Z (SEQ ID NO: 24)
X-TNCYCKKCCFHCQVCFTT Z (SEQ ID NO: 23)
X-CTNCYCKKCCFHCQVCFTT-Z (SEQ ID NO: 22)
X-ACTNCYCKKCCFHCQVCFTT-Z (SEQ ID NO: 1 ) "X" may represent an ammo group, a hydrophobic group, including but not limited to carbobenzoxyl, daπsyl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier group including but not limited to hpid-f atty acid conjugates, polyethylene glycol, or carbohydrates.
"Z" may represent a carboxyl group; an amido group; a T butyloxycarboπyl group; a macromolecular carrier group including but not limited to hpid-f atty acid conjugates, polyethylene glycol, or carbohydrates. Examples of carboxy-termmal truncations in one of the Tat21 40 sequences are provided in Table III below.
However, it will be appreciated that carboxy terminal truncations may also be made in the Tat53 B8 and Tat920 sequences or in Tat21 40 sequences from other isolates or subtypes.
Table III Carboxy-termmal Truncations X-ACT-Z(NoSEQIDNO) X-ACTN-Z (SEQ ID NO: 53) X-ACTNC-Z (SEQ ID NO: 52) X-ACTNCY-Z(SEQIDN0:51) X-ACTNCYC-Z (SEQ ID NO: 50)
X-ACTNCYCK-Z(SEQ1DN0:49) X-ACTNCYCKK-Z (SEQ ID NO: 48) X-ACTNCYCKKC-Z (SEQ ID NO: 47) X-ACTNCYCKKCC-Z (SEQ ID NO: 46) X-ACTNCYCKKCCF-Z (SEQ ID NO: 45)
X-ACTNCYCKKCCFH-Z (SEQ ID NO: 44) X-ACTNCYCKKCCFHC-Z (SEQ ID NO: 43) X-ACTNCYCKKCCFHCQ-Z (SEQ ID NO: 42) X-ACTNCYCKKCCFHCQV-Z (SEQ ID NO: 41) X-ACTNCYCKKCCFHCQVC-Z (SEQ ID NO: 40)
X-ACTNCYCKKCCFHCQVCF-Z (SEQ ID NO: 39) X-ACTNCYCKKCCFHCQVCFT-Z (SEQ ID NO: 38) X-ACTNCYCKKCCFHCQVCFTT-Z (SEQ ID NO: 1)
"X" may represent an amino group, a hydrophobic group, including but not limited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier group includig but not limited to lipid-f atty acid conjugates, polyethylene glycol, or carbohydrates.
"Z" may represent a carboxyl group; an amido group; a T-butyloxycarbonyl group; a macromolecular carrier group including but not limited to iipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
Examples of internal truncations in one of the Tat2M0 sequences are provided in below. However, it will be appreciated that internal truncations may also be made in the Tat5368 and Tat920 sequences or in Tat21 40 sequences from other isolates or subtypes.
For example, the internal truncations include
-CTN- and CTN-5-20, wherein 5-20 means C, CY, CYC, ...CYCKKCCFHCQVCFTT- (SEQ ID NO: 54)
-TNC and TNC-6-20, wherein 6-20 means Y, YC, YCK...YCKKCCFHCQVCFTT- (SEQ ID NO: 55) -NCY and NCY-7-20 wherein 7-20 means C, CK, CKK...CKKCCFHCQVCFTT- (SEQ ID NO: 56) -CYC and CYC-8-20 wherein 8-20 means K, KK, KKC...KKCCFHCQVCFTT- (SEQ ID NO: 57) -YCK and YCK-9-20 wherein 9-20 means K, KC, KCC...KCCFHCQVCFTT- (SEQ ID NO: 58) -CKK and CKK-10-20 wherein 10-20 means C, CC, CCF...CCFHCQVCFTT- (SEQ ID NO: 59) -KKC and KKC-11 -20 wherein 11-20 means C, CF, CFH...CFHCQVCFTT-(SEQ ID NO: 60) •KCC and KCC 12-20 wherein 12-20 means F, FH, FHC, ..FHCQVCFTT- (SEQ ID NO: 61) CCF and CCF 13 20 wherein 13-20 means H, HC, HCQ...HCQVCFTT (SEQ ID NO: 62)
CFH- and CFH-14-20 wherein 14-20 means C, CQ, CQV...CQVCFTT (SEQ ID NO: 63)
FHC- and FHC-15 20 wherein 15-20 means Q, QV, QVC...QVCFTT- (SEQ ID NO: 64)
HCQ- and HCQ-16 20 wherein 16-20 means V, VC, VCF...VCFTT (SEQ ID NO: 65) CQV- and CQV-17 20 wherein 17-20 means C, CF, CFT...CFTT- (SEQ ID NO: 66)
-QVC- and QVC- 18-20 wherein 18-20 means F, FT, or FTT
VCF and VCF-19-20 wherein 19-20 means T or TT
CFT and CFTT (SEQ ID NO: 37)
Another type of dominant-negative mutant peptide combines a functional domain peptide that has been inactivated with another peptide that facilitates its binding to the normal target site. One example of such a chimeric dominant negative peptide combines the Tat21 40 sequence containing an ammo acid substitution that inactivates its function but fails to convert it to an effective dominant negative inhibitor in a peptide chimera with a second peptide sequence such as Tat5368 in order to enhance binding to a molecular target normally bound by both peptide regions in the full length Tat molecule. In this case, the separate peptides may have low or no inhibitory activity, while the composite peptide containing both peptide sequences would have dominant negative activity. A spacer of ammo acids between the two peptides, such as repeats of giycine or giycine plus senne, may be added to obtain optimal spacing of functional domains for full activity of the chimera.
A second type of chimeric dominant negative peptide uses a functional peptide domain inactivated by ammo acid substitutions containing additional charged ammo acids at the ends of the peptide to strengthen its binding to the target site. For example, 4-8 lysine residues added to the ammo terminus, the carboxy terminus or both termini will extend the length of the peptide by adding more positive charge in order to increase its binding to negatively charged targets such as DNA.
A third type of chimeric dominant negative peptide combines an inactivated functional domain with some other type of functional binding peptide sequence besides a known Tat peptide or charged ammo acids. The strategy of each of the above types of chimeric peptides is to use a functional Tat peptide domain that has been inactivated by mutation in combination with some other binding moiety that enhances binding of the chimera in order to prevent the binding or function of native Tat molecules.
Dominant-negative mutants produced using the above methods are identified by determining their ability to reduce the activity level of wild type Tat protein. For example, a nucleic acid encoding a prospective dommant- negative mutant may be introduced into a cell containing a gene encoding the wild type full length Tat protein and a
Tat-activated reporter gene which encodes a detectable product. In one method, a nucleic acid encoding a prospective dominant-negative mutation is operably linked to the hCMV promoter and cotransfected into BALB/c 3T3 cells with a vector encoding wild type full length Tat and a reporter vector in which the CAT gene is operably linked to the HIV LTR. Control cells are transfected with the vector encoding full length Tat and the CAT reporter vector. Those mutants which reduce the levels of CAT expression relative to that in the control cells are dominant-negative mutations
Peptides comprising a Tat functional domain containing a dominant-negative mutation may be used in any of the methods for inducing an immune response described above, including methods in which the peptides or nucleic acids encoding the peptides are administered to an individual. In another embodiment, peptides comprising a Tat functional domain containing a dominant negative mutation are administered to an individual as described above. The peptides inhibit the activity of the wild type Tat protein in the individual.
Alternatively, nucleic acids encoding peptides comprising a Tat functional domain containing a dominant negative mutation may be operably linked to a promoter in any of the vectors described above. Preferably, the vector does not induce an immunological response to itself. The vector is introduced into the individual or introduced into cells in vitro which are later introduced into the individual as described above. The peptides comprising a Tat functional domain containing a dominant-negative mutation are expressed in the individual and inhibit the activity of wild type Tat protein in the individual.
Antibodies to a peptide comprising a Tat functional domain are another embodiment of the present invention. The antibodies may be monoclonal antibodies or polyclonal antibodies.
Monoclonal antibody to the peptides comprising a Tat functional domain can be prepared from murine hybndomas according to the classical method of Kohler and Milstem (Nature 256:495 (1975)), or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the peptide over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminoptenn (HAT media). The successfully fused cells are diluted and a quots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall [Meth. Enzymol. 70:419 (1980)), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis, L et al. Basic Methods in Molecular Biology Elsevier, New York. Section 21 2. If desired, the antibodies can be humanized to reduce the risk of immune reactions against non-human epitopes on the antibody.
Polyclonal antiserum containing antibodies to peptides comprising a Tat functional domain can be prepared by immunizing suitable animals with the peptide, which can be unmodified or modified to enhance immunogemcity. Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogeπic than others and may require the use of carriers and adjuvant. Also, host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable An effective immunization protocol for rabbits can be found in Vaitukaitis, J. et al. (1971 ) J. Clin. Endocπnol. Metab.
33:988-991.
Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined by ELISA or semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony, 0. et al, Chap. 19 in: Handbook of Experimental Immunology
D. Wier (ed) Blackwell (1973). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum
(about 12 M). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, D., Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc. For
Microbiol., Washington, D.C. (1980). Antibody preparations prepared according to either protocol are useful in quantitative immunoassaγs which determine concentrations of antigen bearing substances in biological samples; they are also used semi quantitatively or qualitatively to identify the presence of antigen in a biological sample. The antibodies may also be used in therapeutic compositions for reducing the levels of the protein in the body.
In addition to their therapeutic uses, the peptides comprising a Tat functional domain may be used as reagents to study HIV replication and the HIV life cycle. For example, the peptides may be used to identify proteins which interact with the functional domains in systems such as the yeast two hybrid systems such as the Matchmaker Two Hybrid System 2
(Catalog No. K1604 1, Clontech).
The peptides comprising a Tat functional domain may also be used in affinity chromatographγ to identify proteins which interact with the functional domains. In such systems, the peptides comprising a Tat functional domain are linked to a suitable chromatographγ matrix. Samples comprising proteins which bind to the functional domain are placed in contact with the matrix under conditions which allow a complex to be formed between the functional domain and the proteins which bind thereto. After a wash to remove non-specifically bound proteins, specifically bound proteins are eluted. The specifically bound proteins can be sequenced and clones encoding them can be identified using techniques familiar to those skilled in the art. In addition, the peptides comprising a Tat functional domain may be used to identify agents which disrupt the activity of Tat. For example, in the case of the Tat o functional domain, a peptide comprising the Tat2140 functional domain may be bound to the HIV TAR sequences to form a complex. The complex can be contacted with an agent to determine whether the agent disrupts the complex.
Alternatively, the cells containing a peptide comprising a functional domain of the Tat protein can be contacted with the agent to determine whether the agent disrupts at least one activity of the peptide, including such activities as activation of the HIV LTR, angiogenesis or NF-κB activation. The agents tested for disruption of the activity of the peptide comprising the Tat functional domain may include organic or inorganic molecules, biochemical molecules, peptides, or polyπucleotides.
Another embodiment of the present invention is a method for determining the disease state of an individual infected with HIV. Non progressors have a high titer of antibodies against Tat in their sera, while rapid progressors have low titers of antibodies against Tat in their sera. In this embodiment of the present invention, sera is obtained from an individual. The sera is contacted with a peptide comprising a Tat functional domain, and the amount of antibody in the sera which binds to the peptide is determined to identify the individual as a non progressor or a rapid progressor. The amount of antibody in the sera which binds to a peptide comprising a Tat functional domain may be determined using conventional methods familiar to those skilled in the art, including ELISA analyses or Western blots. In an ELISA analysis, a peptide comprising a Tat functional domain is attached to a microtiter well and contacted with serum from an individual.
The amount of antibody against the peptide is determined by adding a detectably labeled secondary antibody.
In a Western analysis, the peptide comprising a Tat functional domain is attached to a filter and contacted with the serum from the individual. The amount of antibody against the peptide is determined using a detectably labeled secondary antibody.
PART II: MULTIPLE PEPTIDE CONJUGATES
Background of the Invention
Traditional vaccines have been prepared using immunogens such as attenuated versions of pathogenic organisms, inactivated pathogenic organisms or preparations containing antigens from the pathogenic organism. For example, the organisms responsible for pertussis, typhoid, and polio have been inactivated and used as vaccines. Cell wall or polysacchande preparations obtained from Haemophilus or Memπgococcus have also been used (Seid et al.,
G/ycocoπjugate J.6:489498 (1989); Frasch, Clin. Microbml. Rev. 2 (Suppl.) 134-138 (1989).
While these approaches have produced highly effective vaccines in some instances, they suffer from a variety of complications. Such complications include the presence of adventitious agents in the vaccine compositions, reversion of the organism used in the vaccine to virulence, variation in the properties of the strains used in the vaccination, and risk of cross-reactivity of antigens in the vaccine with host antigens. In addition, in approaches utilizing inactivated organisms, it may be difficult to completely inactivate the organism.
An alternative strategy to conventional vaccines has been to identify epitopes or immunogens which are responsible for a specific response. Synthetic peptides containing these epitopes or immunogens are then used as vaccines (Mills, Curr. Opinion in Immunol. 2.804-814 (1989)). Such synthetic molecules are devoid of many of the risks associated with vaccines derived from pathogenic organisms. In addition, the synthesis of peptides with pophilic or glycosidic functional groups may improve delivery of the antigenic peptides and aid in targeting the antigenic peptide to antigen presenting cells.
Summary of the Invention The present invention relates to multiple peptide conjugates, methods of preparing them, and methods of using them to induce an immune response.
Definitions
As used herein, "t Boc" means tert-butoxycarbonyl.
As used herein, "Maldi-tof" means matrix assisted laser desorption time of flight. As used herein, "NMP" means N-methylpyrrohdone. As used herein, "TFA" means trif luoroacetic acid.
As used herein, "HOBt" means 1-hydroxybenzotriazole.
As used herein, "RP-HPLC" means reverse phase-high performance liquid chromatography.
As used herein, "tbu" means tert-butyl. As used herein, "Fmoc" means 9-fluorenylmethoxycarbonyl.
As used herein, "DCM" means dichloromethane.
As used herein "Npys" means 3-nitro-2pyridine sulphenyl.
As used herein "Mmt" means 4-methoxytrityl.
As used herein, "TCEP" means triscarboxyethylphosphine. As used herein, "DCC" means N,N'-dicylohexylcarbodiimide.
As used herein, "Trt" means trityl.
As used herein, 'TIS" means triisopropylsilane.
As used herein, "Pmc" means 2,2,5,7, 8-pentamethylchroman-6-sulfonyl.
As used herein, "DMSO" means dimethylsulfoxide. As used herein, "Hmb" means N-[2-hydroxy-4-methoxybenzyl].
As used herein, "HBTU" means 2-[1-H-Benzotriazole-1-yl]-1,13,3-tetramethyluronium hexafluorophosphate.
As used herein, "Otbu" means tert-butyl ester.
As used herein, "DCU" means dicyclohexγl urea.
As used herein, "TNBS" means trinitrobenzeπe sulf onic acid. As used herein, "NHS" means N-Hydroxysuccimide.
As used herein, "EDT" means ethanedithiol.
As used herein, "CMC" , means carboxymethylcysteine.
As used herein, "Br2Ac2θ" means Bromoacetic anhydride.
As used herein, "CI2AC2O" means Chloroacetic anhydride. As used herein, "BU3P" means tri-n-butylphosphine.
As used herein, "MAP" means multiple antigen peptide.
As used herein, "MPC" means multiple peptide conjugate.
As used herein, "SEC" means size exclusion chromatography.
As used herein, "SPPS" means solid phase peptide synthesis. As used herein, "ESI-MS" means electrospray ionization mass spectroscopy.
As used herein, "linear peptide" refers to a peptide sequence to be attached to the base peptide.
As used herein, "base peptide" refers to the core peptide to which a peptide has been attached or the core template on which a peptide has been synthesized. Detailed Description of the Preferred Embodiment As discussed above, vaccines based on inactivated organisms or materials derived from organisms may suffer from a variety of drawbacks. Accordingly, it is desirable to use synthetic compounds to induce an immune response. However, it is generally thought that the conformational B-cell epitopes involved in neutralization of some organisms would be difficult to mimic though the use of a simple synthetic linear molecule (Sesardic, J. Med. Microbiol. 39:241
243 (1993); Arnon and Horwitz, Curr. Opinion in Immunol. 4:449453 (1992)).
One approach to address this issue has been described by Tarn and others. In this approach, peptides are linked to a core structure to generate a multiple antigen peptide (MAP) (Nardelli et alThe MAP system: A flexible and unambiguous vaccine design of branched peptides in vaccine design: The subumt and adjuvant approach Plenum Press, New York, pp803 819 (1995); Dπjfhout et al. Solid-phase and applications of NfS-acetylmercaptoacetyl) peptides
187:349 354 (1990); Nardelli et al, J. Biol. Chem. 148:914 920(1992); Nardin et al., Vaccine 16:590 600 (1998)). However, the classical solid phase synthesis methodology employed in the traditional MAP is not without difficulty. For example, the classical solid phase synthesis methodology employed in this approach may not yield a sufficient amount of product for use in a vaccine or may yield a heterogenous product which is not amenable to use in a vaccine. For example, problems may arise as a consequence of microheterogeπeitγ in the synthetic compounds
(Dπjfhout and Bloemhoff, Int . Peptide Protein Res. 37:27-32 (1991); Grant et al., Methods in Enzymology 289: 395- 419(1997)). Aggregation and steric hindrance of the growing peptide chain during solid phase peptide synthesis is thought to be a major source of the microheterogeπeitγ found in traditional multiple antigen peptides produced by the direct synthesis approach (Nardin et al., Vaccine 16:590-600 (1998); Dπjfhout and Bloemhoff, IntJ Peptide Protein Res. 37 27 32 (1991 )). Many of these problems are thought to be sequence related and may possibly be caused by inter and mtra chain hγdrogen bonding bγ the peptide backbone, forming beta sheets or other secondarγ structures (Kent et al., In: Innovations and perspectives in solid phase synthesis, Epton R, ed., Intercept Ltd., Andover, UK, p. 1 (1992)). This maγ lead to significant steric hindrance, thus reducing the efficiency of the acγlation or deprotection reactions or both (Milton et al., J. Am. Chem. Soc. 112: 6039-6046(1990)). Some groups have shown that peptide aggregation maγ occur as early as the fifth residue depending upon the peptide sequence and side chain blocking groups used (Bedford et al.. Int. J Peptide Protein Res. 40:300 307 (1992)). In order to reduce the effects of hγdrogen bonding, protection of the peptide bonds (i.e. bγ forming a tertiary nitrogen) has been utilized (Johnson et al., J. Chem. Soc. Chem. Co mun. 4:369 372 (1993)). In addition to the factors listed above, the procedures for isolating and purifying the synthetic product resulting from the current methods for preparing multiple peptide conjugates are extremely laborious. Furthermore, in many cases it is impractical to obtain a reasonable amount of the synthetic product for use.
The present invention relates to multiple peptide conjugates which comprise multiple peptides, such as antigens, epitopes or other peptides, coupled to a core template. As used herein the term "peptide" refers to compounds comprising two or more ammo acids linked bγ peptide bonds. In some embodiments, the peptides may be from about 2 to about 100 ammo acids in length. In other embodiments, the peptides maγ be from about 10 to about 100 ammo acids in length. However, it will be appreciated that the peptides maγ have anγ length consistent with their intended use. The core template preferably comprises a branched compound. Preferably, the core template has at least two branches. However, the core template may have 3, 4 or more than four branches. In fact, the core template maγ have anγ number of branches consistent with its intended function.
Each branch of the core template contains a first reactive group and a second reactive group, each of which permit peptides to be selectively attached thereto or synthesized thereon. Preferablγ, the adjacent branches of the core template are spaced at a sufficient distance from one another to minimize the steric hindrance between peptides being sγnthesized on or attached to the reactive groups on the branches of the core template. Preferablγ, the peptides are sγnthesized on the core template. Preferablγ, the adjacent branches of the core template are separated from one another bγ the distance occupied bγ at least four carbon atoms. For example, in some of the embodiments described below, the adjacent branches are linked to the α and ε ammo groups of a lysine residue such that they are separated from one another by four carbons.
In one embodiment, the core template comprises a peptide. In this embodiment, the core template maγ be sγnthesized using anγ of the conventional methods for synthesizing peptides, including solid phase sγnthesis or conventional solution phase chemistrγ. Sγnthesis maγ include the use of anγ and all protecting strategies, such as the use of t-Boc, Fmoc, Bpoc or other suitable urethane derivatives for blockage of the N-termmai amme prior to coupling or attaching subsequent ammo acids in a continuous chain or arraγ.
Sγnthesis maγ also be performed bγ the methodologγ of convergent solid phase sγnthesis using protected fragments utilizing acid labile linkers or resins. Convergent chemical gation of unprotected peptides, fragments or ammo acids maγ also be used to prepare the core template. Thus, the sγnthesis of the core template maγ be achieved entirely by direct, indirect or modular means or a combination of these techniques which couple two or more ammo acids together to form conventional peptide bonds or other stable bonds enabling a covalent and stable linkage between two ammo acids.
In one embodiment of the present invention, the core template comprises a two branched compound of the following structure:
M
(Structure A) wherein:
A is absent or an ammo acid;
B is an ammo acid;
D is absent or an ammo acid;
E is absent or an ammo acid;
G is an am o acid;
J is an ammo acid;
M is an ammo acid having a first reactive group and a second reactive group thereon, wherein said first reactive group and said second reactive group are capable of selectively being attached to an ammo acid or peptide,
R is an ammo acid having a first reactive group and a second reactive group thereon, wherein said first reactive group and said second reactive group are capable of selectively being attached to an ammo acid or peptide; n is i 10; s is 0 when D is absent or is 1 20 when D is present; and t is 0 when E is absent or is 1 20 when E is present.
Preferablγ, the ammo acids in Structure A (i.e. A (if present), B, D (if present), E(ιf present), G, J, M, and R) are linked to one another via peptide bonds.
Two branched compounds maγ be sγnthesized as follows. In a preferred version of the compounds with two branches, the starting material for synthesizing the core template is an ammo acid linked to a solid support such as a resin. Thus, in this embodiment, residue A is preferably an ammo acid which can be linked to a solid support such as a resin. In a preferred embodiment, residue A of structure A is senne Preferably, the side chain of the senne used as the starting material for synthesis is tπtylated to block its reactivity. The use of seπne(Trt) allows modification on the solid phase after deprotection of the hγdroxγl group to produce a highlγ reactive group for chemical ligation. For example, the hγdroxγl group maγ be oxidized to an aldehγde group or otherwise modified to provide a group suitable for coupling other chemical moieties at this specific site. The C terminal senne modification can be performed in the solid phase state or as a free peptide. It will be appreciated that the C terminal residue maγ be anγ residue consistent with its intended use. For example, the senne residue placed at C-termmal end of the core template maγ be substituted bγ anγ number of ammo or non ammo acids. In the case of Fmoc sγnthesis for example, the C terminal senne maγ be replaced with Lγsιne(Mtt), Lγs(Tfa), Lγs(Dde), Cγs( mt), Cγs(Acm) as well as manγ other suitable ammo acids or compounds capable of side chain modification. If desired, Fmoc sγnthesis maγ be replaced bγ the conventional t Boc sγnthesis. In this case, the C-termmal ammo acid maγ be selected from t-Boc ammo acids, such as Cγs(Acm), Lγs(Dde), Lys(Fmoc),
Cys(tbu) or other t Boc ammo acids, with side chain protection corresponding to those listed for Fmoc.
After preparation of the starting material for synthesizing the core template, further peptide synthesis is performed to extend the starting material. For example, if the starting material is Ser(Trt) linked to a resin, additional ammo acids maγ be added thereto to sγnthesize the core template. In one embodiment, an initial lysine residue is linked to the Ser(Trt). Thus, residue B can be lysine. This initial lysiπe forms the branch point. As indicated in Structure A, there maγ be more than one residue between residue A and the branch point (i.e. n is more than 1). In some embodiments, n is from 1 10. In other embodiments, n is from 1-5. In a preferred embodiment, n is 1.
Thereafter, additional residues are added to residue B of structure A. For example, in some embodiments, residues D and E maγ be added to extend the chain lengths of the branches. Thus, s and t maγ be from 1-20. In some embodiments s and t are from 10. In other embodiments, s and t are from 1 5. In one embodiment, residues D and E of structure A are absent (i.e. s and t are 0).
Residues G and J of structure A maγ be aπγ ammo acid. In a preferred embodiment, G and J are Iγsmes. In this embodiment, if residue B is lysine, lysine residues G and J are attached to the α and ε NH2 groups of residue B to form a branched structure. In one version of the embodiment in which residues G, and J are lysmes, the ε NH2 group of these lysmes are blocked with a tert-butoxγcarbonγl (t Boc) group and residues M and R are joined to the α NH, groups of these lysmes. Alternatively, the α NH2 groups of lysmes G and J maγ be blocked and M and R maγ be linked to the ε NH2 of lysmes G and J.
It will be appreciated that the lysmes maγ be substituted with anγ residues consistent with the intended use. For example, rather than being lysiπe, residues B, G and J may be derivatives such as Fmoc-Orn(Mtt)-OH, Fmoc
Lγs(ιvDde)-OH Residues B, G, and J maγ also be other diammo compounds having appropriate steric and chemical properties. Preferablγ, the residues B, G, and J bear a reactive side chain. Preferablγ, the reactive side chain is spatially separated bγ at least about four carbon atoms from the chiral center of the molecule. In addition, residues B, G and J are preferablγ compatible with standard chemical methodologies, such as solid phase and solution chemistry, used in the preparation of sγnthetic peptides. In addition, residues B, G, and J are preferablγ suitable for use in an orthogonal or non orthogonal sγnthesis approach using either Fmoc or t-Boc chemistrγ or using alternate protecting groups for those strategies using both acid and base chemistries. In some embodiments, the residues B, G, and J are spatially arranged such that the level of steric hindrance does not exceed that in the compounds in which residues B, G and J are lysmes.
Residues M and R have at least two reactive groups thereon. Each of the reactive groups permits a peptide to be specifically sγnthesized thereon or attached thereto after synthesis. The reactive groups are designed such that a peptide can be synthesized thereon or a fully synthesized peptide can be attached thereto under conditions in which peptides cannot be sγnthesized on or attached to the remaining reactive group or groups. For example, the reactive groups maγ be ammo acids modified with appropriate blocking groups such that a single reactive group maγ be unblocked at a time In one particular embodiment, one of the reactive groups is deprotected upon acid treatment, while the other reactive group is deprotected upon treatment with base. Thus, the peptide will be sγnthesized on or attached to onlγ one reactive group at a time.
In a preferred embodiment, residues M and R are cγstemes and the first and second reactive groups comprise the thiol group of the cγstemes and the ammo groups of the cγstemes respectively. Preferablγ, peptides are sγnthesized on the ammo groups of the cγstemes and the cysteine side chain thiol is used to form a stable thioether bond with an incoming haloacetγl peptide. In addition, peptides maγ be sγnthesized on the ammo groups of the cγstemes with a reduced risk of epimenzation, and, if the thiol groups of the cγstemes are blocked during sγnthesis of the peptides on the ammo groups of the cγsteines, there is a reduced risk of dimeπzatioπ during sγnthesis of the peptides on the ammo groups of the cγsteines. In one embodiment, the thiol groups of the cγsteines are blocked with
Trt groups, while the ammo groups are blocked with Fmoc.
It will be appreciated that M and R may be ammo acids or non-ammo acids other than cγsteme which provide first and second reactive groups which can be selectively attached to an ammo acid or peptide. For example, residues M and R maγ be ammo acids having reactive side chains, such as lysiπe, senne, threonme, asparagine, aspartic acid, glutamine, and glutamic acid. Preferably, M and R are capable of forming a chemical bond with both the preceding and successive ammo acid in the peptide chain. Thus, the covalent bonds between the peptide antigens and base peptide need not be the traditional peptide bond (NHCO) formed between the ammo group (NH2) of one ammo acid and the carboxyl (COOH) of another but maγ be anγ number of other bonds that insures rigidity, and strength for use in this kind of chemical sγnthesis. Such couplings maγ include coupling via thioether formation. Other couplings maγ also include that of the oxime, thiazolidme, hγdrazone, thioester, or other suitable bond formation compatible with all chemistries and groups used in the preparation of the multiple peptide conjugate. M and R preferablγ have stability and lability characteristics compatible with solid phase or solution syntheses and side chain reactivity capable of protection and deprotection in such manner that the stability and lability of the peptide chains would not be affected. Thus, in one embodiment of the present invention, the core template has the structure WO 00/78969 PCT/USOO/l 0119
Trt
Figure imgf000028_0001
Lγs
/ ^ t-Boc
Ser — Lγs Trt
Trt \ Cγs — Fmoc Lγs ^
\ t-Boc
(Structure B)
Structure B has the chemical structure depicted in Figure 2.
In other embodiments, the core template maγ have more than two branches. For example, the core template maγ have 3, 4, or more than four branches. For example, the core template maγ have the structure:
Figure imgf000028_0002
E, J R /
\ Z
(Structure C) wherein:
A is absent or an ammo acid; B is an ammo acid; D is absent or an ammo acid; E is absent or an ammo acid;
G is an ammo acid; J is an ammo acid;
M is an ammo acid having two reactive groups thereon which can be attached to an ammo acid; R is an ammo acid having two reactive groups thereon which can be attached to an ammo acid; W is an am o acid having a first reactive group and a second reactive group thereon, wherein said first reactive group and said second reactive group are capable of selectively being attached to an ammo acid or peptide;
X is an ammo acid having a first reactive group and a second reactive group thereon, wherein said first reactive group and said second reactive group are capable of selectively being attached to an ammo acid or peptide; Y is an ammo acid having a first reactive group and a second reactive group thereon, wherein said first reactive group and said second reactive group are capable of selectively being attached to an ammo acid or peptide;
Z is an ammo acid having a first reactive group and a second reactive group thereon, wherein said first reactive group and said second reactive group are capable of selectively being attached to an ammo acid or peptide;
s is 0 when D is absent or is 1 -20 when D is present; and t is 0 when E is absent or is 1 -20 when E is present.
A, B, D, and E may have anγ of the identities discussed above with respect to Structure A. The values of n, s, and t may be any of those discussed above with respect to Structure A Preferably, the ammo acids in Structure C (i.e. A (if present), B, D (if present) , E(ιf present), G, J, M, R, W, X,
Y and Z) are linked to one another via peptide bonds.
G and J maγ be anγ ammo acid. In some embodiments, G and J are lysmes.
M and R may be aπγ ammo acids having two reactive groups which can be linked to an ammo acid, such that residues M and R form branch points. In preferred embodiments, M and R are lysmes. However, M and R may have anγ of the identities discussed above for residues G and J of Structure A.
W, X, Y, and Z maγ have anγ of the identities discussed above for residues M and R of Structure A. In a preferred embodiment, W, X, Y, and Z are cγsteines.
It will be appreciated that additional branches maγ be added bγ adding additional ammo acids having two reactive groups which can be linked to an ammo acid at the penultimate positions of the branches. In preferred embodiments, the penultimate ammo acids are lysmes.
As discussed above with respect to Structure A, the α or ε ammo groups of the lysmes are then linked to ammo acids having a first reactive group and a second reactive group thereon wherein the first and second reactive groups are capable of being selectively attached to an ammo acid or peptide. Preferably, the ammo acids having a first reactive group and a second reactive group thereon wherein the first and second reactive groups are capable of being selectively attached to an ammo acid or peptide are cγstemes.
Although the core template structures provided above are ammo acids linked bγ peptide bonds, it will be appreciated that the core template maγ be anγ compound, including compounds which are not based on peptide bonds between ammo acids, which allows peptides to be selectively synthesized on or attached directly to a single type of reactive group at a time as discussed in more detail below. Following preparation of the core template, peptides are sγnthesized on the first reactive groups or fully synthesized peptides are attached to the fust reactive groups to generate a base peptide. Preferably, the peptides are sγnthesized on the first reactive groups. In one embodiment, in which peptides are sγπthesized on the first reactive groups, a first ammo acid residue maγ be attached to the first reactive groups using anγ of the sγnthetic methods familiar to those skilled in the art. The peptide chain maγ then be extended one residue at a time to generate the desired peptide using any of the synthetic methods familiar to those skilled in the art. If the first reactive group is blocked, the blocking group is removed prior to linking the first ammo acid thereto.
Alternatively, rather than synthesizing the desired peptide one residue at a time starting from the reactive groups on the core template, the desired peptide sequence may be fully sγnthesized using all conventional methods. Subsequentlγ, the complete peptide is attached to the reactive group on the core template.
Preferablγ, to reduce steric effects one or more ammo acids in the peptides being sγnthesized on or attached to the first reactive groups are modified with agents which reduce steric effects. Thus, one or more of the ammo acids added to the peptide chain during sγπthesis or included in a fullγ sγnthesized peptide maγ be protected using any of the blocking groups familiar to those skilled in the art. In a preferred embodiment, one or more ammo acids linked to the carboxy group of asparagine or aspartic acid residues are incorporated as Hmb protected derivatives in the peptides being synthesized on or attached to the first reactive groups.
In addition, in some embodiments of the present invention, anγ cγsteme in the peptides being sγnthesized on or attached to the first reactive groups maγ have their thiol groups modified with a blocking group. In a preferred embodiment, the thiols are blocked with tπtγl groups. In another embodiment, in which the peptides are sγnthesized on the first reactive groups, the sγnthesis is conducted in a solvent containing DMSO. The DMSO may be present at a concentration from about 1 % to about 20% . In a preferred embodiment, the DMSO is present at a concentration from about 5% to about 12%. In a highly preferred embodiment the DMSO is present at a concentration of about 10%. Alternatively other chaotropic salts, such as KSCN, sodium perchlorate, THT (tetrahydrofuran), 1 ,4, dioxane, TFE (trifluoroethanol) or LiCI maγ be used to increase solvation.
In one embodiment, the solvent is the N-methγlpyrollιdone coupling solvent conventionally used to synthesize peptides. In another embodiment, the solvent may be DMF (dimethylformamide). It will be appreciated that any suitable solvent, including aprotic solvents, maγ be used.
The sγnthesis of peptides on the first reactive groups or the attachment of fullγ sγnthesized peptides to the first reactive groups on the core templates generates the base peptide. Where the core template has Structure A, the base peptide has peptides attached to the first reactive groups of residues M and R. Where the core template has Structure C, the base peptide has peptides attached to the first reactive groups of residues W, X, Y, and Z.
In the embodiment in which the core template has Structure B, the peptides are selectively synthesized on or joined to the ammo groups of the cystemes following deprotection thereof to form the base peptide. For example, in the embodiment in which the core template has Structure B, the base peptide has the following structure after removal of the blocking groups on the thiols of the cystemes: SH
Cγs — P, /
Lγs x
/ ^ NH2
Figure imgf000031_0001
(Structure D) wherein P, and P2 are peptides which have been linked to the am o groups of the cγsteines. The chemical structure of
Structure D is depicted in Figure 3.
If desired, the core template maγ be cleaved from the solid support following sγnthesis or attachment of the peptides to the core template. In embodiments in which the thiol group of cγsteme is one of the reactive groups on the core template, preferablγ the cysteine thiol groups on the core template are blocked with an appropriate blocking group such that only these groups are deprotected during the resin cleavage of the peptide by acidolysis. Use of an acid labile protecting group on the cysteine residues in the core template and non acid labile group(s) on all cysteine residues in the peptides which are synthesized on or attached to the core template during sγπthesis of the base peptide provides compatibility with the sulfhγdrγl reducing agents used in the final assemblγ of the multiple peptide conjugate.
However, it will be appreciated that the above measures need not be emploγed when the peptides attached to the core template during sγnthesis of the base peptide do not include cγsteme residues. In such instances, the thiol blocking group (tritγl) on the core template maγ be replaced with aπγ blocking group compatible with its intended use.
For example, Cγs(Acm), Cγs(tbu), Cγs(Mmt), Cγs(Npys) or other protected residues could be used in this strategy and appropriate chemical methodologies would be used to deprotεct the thiol group when desired.
The base peptιde(s) maγ also be prepared bγ a chemical or solution sγnthesis process. In either case, a free peptide can be generated after an acidolysis or other cleavage procedure is performed on resin bound peptide or protected peptide in solution. An alternate approach may also include the sγnthesis, whether bγ solid phase or solution chemistrγ, of protected fragment(s) of either the base or core molecule. These components or fragment(s) produced as free peptides or protected fragments can then be used in convergent synthesis by activation and coupling of two ends of the moiecule together followed bγ suitable methods for isolation. Unlike the protected fragments the coupling of free peptides to the core molecule or other peptides generally will require chemoselective specificity to ensure reactivity at a specific site or residue.
In the embodiment where the core template has Structure B, steric effects are lessened by coupling only to the ammo groups of the cystemes on the alpha ammo group of opposite lysine positions and maintaining protection on WO 00/78969 PCT/USOO/l 0119
the epsilon position of each respective Iγsine residue on the tetramer core. Sγnthesis of two identical peptide chains on the ammo groups of the cγsteines occurs using this strategγ. It will be appreciated that the same methodology may be used to attach four peptide chains to an octameric core template such that the epsilon position of the Iγsines is protected while the peptides are coupled to cγsteines on alternate alpha positions on the lysmes in the octameric core template.
Alternatively, peptide chain elongation maγ be performed from cystemes on the epsilon position of the Iγsines with the alpha position of the Iγsines in a blocked state. The results would be identical to that obtained when the cγsteines are linked to the alpha positions of the Iγsines, since the spatial construction of the core template would not be changed in anγ waγ. It will be appreciated that in some embodiments, the ammo groups of the two cγsteines maγ be protected with different blocking groups such that different peptides maγ be sγnthesized thereon or attached thereto. In such embodiments, the different peptides are sequentially attached to the first reactive groups following deprotection with the appropriate reagents.
Following the synthesis or attachment of peptides on the first reactive groups to generate the base peptide, peptides are selectively synthesized or attached on the second reactive groups to generate a multiple peptide conjugate. Preferablγ, pre-sγnthesized peptides are attached to the second reactive groups. The peptides sγnthesized or attached on the second reactive groups maγ be the same as or different from those sγnthesized or attached on the first reactive groups. Thus, in the embodiment in which the core template has Structure A, the multiple peptide conjugate has peptides attached to the first and second reactive groups of residues M and R. In a preferred embodiment, pre-sγπthesized peptides are attached to the second reactive groups on the base peptide. For example, a haloacetγl group at the end of a pre-sγnthesized peptide maγ be attached to thiol groups on cγsteine residues in the base peptide.
For example, in the embodiment in which the base peptide has Structure D, the peptides are selectively synthesized or attached on the thiol groups of the cystemes. The peptides sγnthesized or attached on the thiol groups of the cγsteines maγ be the same as or different from the peptides sγnthesized or attached on the ammo groups of the cγsteines. In the embodiment in which the base peptide has structure D, the multiple peptide conjugate has the following structure: Lγs
Figure imgf000033_0001
"^ H2
/
Figure imgf000033_0002
(Structure E) wherein P„ P2, P3 and P4 are peptides.
The chemical structure of one embodiment Structure E is shown in Figure 4 In this embodiment, the peptides P3 and P4 are linked to the cγsteme thiol groups via a haloacetγl group. However, other groups maγ also be used, or the peptides maγ be attached directly to the thiol groups in the cystemes.
In some embodiments of the present invention, reactive groups in the peptides which are to be attached to the base peptide to generate the multiple peptide conjugate are protected with a blocking group which prevents them from being reactive under the conditions in which they are attached to the base peptide. In other words, the blocking groups in the peptides which are to be attached to the base peptide to generate the multiple peptide conjugate are different than the blocking groups on the reactive groups in the base peptide such that the reactive groups in the base peptide can be selectively deprotected. For example, in the embodiment in which the base peptide has Structure D in which the thiol groups of the cystemes are the reactive groups to which the peptides are to be attached during synthesis of the multiple peptide conjugates, any cγsteines present in the peptides which were attached to the core template during sγnthesis of the base peptide maγ be cγsteine (tbuthio). The use of Cγs(tbuthιo) insures site directed coupling bγ the haloacetγl peptide to the thiols. It will be appreciated that the internal Cγs(tbuthιo) maγ be replaced with other SH protection compatible with the chemistrγ emploγed to sγnthesize the multiple peptide conjugate.
It will be appreciated that one or more of the peptides in the multiple peptide conjugates maγ also include one or more peptides adjacent to the peptides P1 , P2, P3, and P4. For example, the peptides may comprise one, two, three, four, five or more than five peptides adjacent thereto. Alternatively, the peptides may comprise about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, or more than 70 peptides adjacent to the peptides P1, P2, P3, and P4.
In some embodiments of the present invention, the peptides included in the multiple peptide conjugate comprise agents, including antigens or epitopes, which are capable of eliciting an immune response in an individual. In some embodiments of the present invention, the peptides comprise antigens from a pathogenic organism. It will be appreciated that in some embodiments the peptide antigens from pathogenic organisms maγ have ammo acids or noπ- ammo acids inserted therein which function as linkers between two antigenic peptides which are fused together or which provide anγ desirable attribute to the antigenic peptide.
In particular embodiments of the present invention, the peptides attached to the core template are from the causative agents of malaria and/or AIDS. For example, the peptide attached to the core template maγ be from the HIV-
1 Tat protein. Preferablγ, the peptide attached to the core template comprises an antigenic epitope from the HIV-1 Tat protein.
In some embodiments, one or more of the peptides in the multiple peptide conjugate comprises a portion of the Tat protein which is responsible for mediating one or more activities of the Tat protein, such as replication of HIV 1 , transcription from the HIV-1 LTR, induction of cγtokine production, increased activitγ of the F transcription factor, or increased angiogemc activitγ in assaγs such as the chick CAM assaγ (Gho and Chae. J. Biol. Chem. 272: 24294 (1997)). Preferablγ, the portion of the HIV-1 Tat protein also includes one or more antigenic sites therein.
Fragments of the HIV-1 Tat protein which are responsible for mediating one or more activities of the Tat protein are described above. In some embodiments of the present invention, one or more of the peptides in the multiple peptide conjugate constitute SEQ ID N0S:1 21. In other embodiments, one or more of the peptides in the multiple peptide conjugates maγ comprise two, three, four, five, or more than five peptides containing Tat functional domains linked to one another. In some embodiments, the two, three, four, five, or more than five peptides are spaced from one another bγ a iinker comprising one or more ammo acids or non-ammo acids.
In another embodiment, one or more of the peptides in the multiple peptide conjugate comprises a peptide from Plasmodium falcipanum, the causative agent of malaria. Preferablγ, the peptide from Plasmodtum falcipanum has at least one antigenic site therein.
In one embodiment, one or more of the peptides in the multiple peptide conjugate comprises a T3 peptide from Plasmodium falcipanum. For example, the peptide maγ comprise the sequence NENLDDLDEGIEKSSEELSEEKI (SEQ ID NO: 81 ). In some embodiments, one or more of the residues which are linked to the carboxγl groups of asparagine or aspartic acid residues in the T3 peptide maγ be Hmb derivatives as indicated bγ underlining in Figure 5A.
In another embodiment, one or more of the peptides in the multiple peptide conjugate comprises a T1 peptide from Plasmodium falcipanum. For example, the peptide may comprise the sequence LTMSNVKNVQTNFKSLLRNLGVS (SEQ ID NO: 82). In some embodiments, one or more of the residues which are linked to the carboxyl groups of asparagine or aspartic acid residues in the T1 peptide may be Hmb derivatives as indicated bγ underlining in Figure 5A. In still another embodiment, one or more of the peptides in the multiple peptide conjugate comprises an MSP
1 peptide from Plasmodium falcipanum. For example, the peptide maγ comprise the sequence VTHESYQELVKKLEALEDAV (SEQ ID NO: 83). In some embodiments, one or more of the residues which are linked to the carboxγl groups of asparagine or aspartic acid residues in the MSP 1 peptide maγ be Hmb derivatives as indicated bγ underlining in Figure 5A. In yet another embodiment, one or more of the peptides in the multiple peptide conjugate may comprise the
CSP peptide from Plasmodium falcipanum (Nardin et al., Vaccine 16:590 600 (1998)). For example, the peptide may comprise the sequence NANPNANP (SEQ ID NO: 84). In some embodiments, one or more of the residues which are linked to the carboxyl groups of asparagine or aspartic acid residues in the CSP peptide may be Hmb derivatives as indicated by underlining in Figure 5A
As discussed above, one or more of the peptides in the multiple peptide conjugates may comprise an immunogemc peptide from anγ isolate or strain of Plasmodium falcipanum.
In some embodiments of the present invention, one or more of the peptides in the multiple peptide conjugate comprises a peptide which has one, two, three, four, five or more than five ammo acid substitutions relative to a reference T3, T1, MSP 1 or CSP peptide. The substituted peptides may be produced using conventional techniques of peptide synthesis.
In yet another embodiment, one or more of the peptides in the multiple peptide conjugate comprises a fusion between two immunogemc peptides from Plasmodium falcipanum. In some embodiments, the two peptides are spaced from one another by a linker comprising one or more ammo acids or non-ammo acids. For example, one or more of the peptides in the multiple peptide conjugate may cormpnse a fusion peptide comprising the CSP and T3 peptides such as a peptide comprising the sequence NANPNANPNENLDDLDEGIEKSSEELSEEKI (SEQ ID NO: 85). In some embodiments, one or more of the residues which are linked to the carboxyl groups of asparagine or aspartic acid residues in the fusion peptide may be Hmb derivatives as indicated by underlining in Figure 5A.
In yet another embodiment, one or more of the peptides in the multiple peptide conjugate comprises a fusion between more than two immunoge c peptides from Plasmodium falcipanum. In some embodiments, one or more residues linked to the carboxy groups of asparagine or aspartic acid residues maγ be Hmb derivatives.
It will be appreciated that the multiple peptide conjugates may include any peptide from HIV-1 , Plasmodium falcipanum, or any other organism, which is capable of inducing an immune response. Thus, the multiple peptide conjugates are not limited to the peptides specifically enumerated above. In some embodiments, the multiple peptide conjugates synthesized as described above may be used to induce an immune response in an individual. The multiple peptide conjugates may be administered via any of the routes traditionally used to administer compositions which induce an immune response, including intraperitoneal administration, intramuscular administration, mtradermal administration, intravascuiar administration or nasal administration. If desired, the multiple peptide conjugates may be administered along with an adjuvant to enhance the level of the immune response. A variety of adjuvants suitable for use in compositions for inducing an immune response are familiar to those skilled in the art, including aluminums like hydroxide and phosphate.
The multiple peptide conjugate may be administered in a physiologically acceptable carrier. A variety of carriers suitable for use in compositions for inducing an immune response are familiar to those skilled in the art. Such carriers are nontoxic to recipients at the dosages and concentrations emploγed. For example, the carriers maγ contain saline, buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, ammo acids, carbohydrates including glucose or dextnns or chelating agents such as EDTA, glutathione or other stabilizers and excipieπts.
The multiple peptide conjugates are administered at a dosage and frequency sufficient to induce a desired level of immune response. Preferablγ, the level of immune response is sufficient to protect an unmfected individual from infection or to reduce or eliminate the sγmptoms of infection in an infected individual. It will be appreciated that the dosage and frequencγ of administration of the multiple peptide conjugates will varγ depending on the age, weight, and condition of the individual. For example, the dosage sufficient to induce a desired immune response maγ range from about 1 μg-100 mg or more, more preferablγ from about 25 μg-100mg or more. However, other dosages maγ also be used.
In some embodiments, the multiple peptide conjugates maγ comprise several antigens from a pathogenic organism. For example, two or more different antigenic peptides maγ be attached to the core template.
Figure 6 illustrates one embodiment of the sγnthetic process which was used to prepare a multiple peptide conjugate In the embodiment illustrated in Figure 6, solid phase peptide sγnthesis followed bγ thiol coupling chemistrγ was utilized to synthesize and construct the MPC molecules. In the embodiment of Figure 6, a core template that serves as primary anchor for the sγnthesis of a base peptide and subsequent attachment of additional peptides through specific site directed coupling was sγnthesized using solid phase methods. For example, the core template may have the structure illustrated in Figure 7A.
Initially, Serιne(Trt) representing the C-termmal of the MPC was attached to the solid support followed bγ the sequential addition of multiple branched Iγsine residues forming a tetrameπc core whose epsilon ammo groups remain blocked throughout the sγnthesis process. Cγsteme, whose side chain thiol group serves to form a stable thioether in the final assemblγ of the MPC, was then coupled to the dual branched Iγsines forming a completed core template.
Example 6 describes the sγnthesis of the sγnthesis core template having the structure shown in Figure 7A.
Example 6
The core template was sγnthesized on an ABI Model 430 peptide sγnthesizer using Fmoc chemιstrγ(Merπfιeld, In Peptides: Synthesis, Structure and Applications (Gutte, Ed.), Academic press, San Diego, pp93
(1995); Atherton and Sheppard, Solid-phase peptide synthesis, IRL Press, Oxford, (1989)) mediated bγ HBTU/HOBt activation on the Rink Amide [4,2',4'Dιmethoxγphenγl Fmoc-aminomethγl] phenoxγacetamido-norleucγl-MBHA resin,
0.45 mmol/g (Novabiochem, La Jolla, CA) at the 0.25 mmol scale for sγnthesis. Bulk ammo acids derivatives were purchased from Novabiochem and 1.0 mmol of each desired residue was used initially in the coupling reaction. Double couple or recouplmg required two or more millimole per residue. For the core template, ammo acids were incorporated with the following side chain protection: Lys(Fmoc), Lγs(t-Boc), Ser(Trt), and Cγs(Trt). Side chain protection used in all other peptides were: Glu(Otbu), Hιs(Trt), Asp(OtBu), Cγs(Trt), Lγs(t-Boc), Asn(Trt), Arg(P c), Ser(tBu), Cys(tButhιo), Lys(Fmoc), VaKFmoc Hmb), LeufFmoc Hmb), Gln(Trt), Trp(t-Boc).
As illustrated in Figure 6, the second step of assemblγ involves the sγnthesis of a base peptide consisting of one or more antigens. In one embodiment, the base peptide maγ be generated bγ synthesizing the antigens on the core template. However, as discussed above, in other embodiments, the complete antigenic peptides may be synthesized first and then attached to the core template. For example, the base peptide maγ have the structure shown in Figure 7B. The base peptide constructs were sγnthesized bγ synthesizing the antigenic peptides listed in Figure 5A and 5B on the core template as described in Example 7.
Example 7 The base peptides consisting of antigens from malaria and HIV-Tat protein were synthesized on the core template following characterization of the core template. These base peptides were subsequently used to prepare four
MPCs. Hmb derivatives were incorporated at specific residues as indicated bγ underlining in Figure 5A in order to reduce potential aggregation in the growing peptide chains (Johnson et al., J. Chem. Soc. Chem. Commun. 4:369 372(1993), Johnson et al., Tetrahedron Letters 35:463 466 (1994); Packman et al., Peptide Research 7: 125 131(1994); Quibell et al., J. Am. Chem Soc. 117:1 1656 1 1668(1995); Quibell et al., J. Chem.Soc, Perkm Trans. 1 :1227 1234 (1996)). The coupling time for a single coupling was 1 hour. Coupling efficiencies were monitored using the Kaiser or TNBS test after completion of each coupling step to insure greater than 99% completion. Double couple/recouple cγcles were introduced when necessarγ. Cγcles which failed to achieve at least 99% after a double/recouple cγcle were capped with 2mmol benzoic anhydride to terminate any unreacted amines. An antigen from Plasmodium falciparum (LSA-1 ) (Connelly et al., Infection and Immunity 65:5082 5087(1997)) previously identified as T3 was used to construct the base peptide T3. The T3 CSP base peptide was sγnthesized using T3 base peptide as its template following removal of 1/2 of the T3 peptidγl resin from the sγnthesizer reaction flask after deprotection thus allowing the sγnthesis to be continued with an additional eight residues added per branch of the T3 construct. Both T3 and T3CSP base peptides were subjected to cleavage bγ acidolysis and purification. The HIV-Tat base peptide Tat-2-Tat 3 was obtained bγ direct solid phase sγnthesis under similar conditions as described at the 0.25 mmol scale followed bγ cleavage and purification.
Peptides were then deprotected and cleaved from the resin as described in Example 8.
Example 8 Following deprotection of the N terminal ammo group, the base peptides were cleaved with a cocktail of TFA, water, EDT, TIS, phenol, thioanisole (88:4:2:1:1:4) Following cleavage, base peptides were precipitated and washed in cold Tert-butγl Methγl Ether. Prior to further processing, peptides were dissolved in water or 0.1 % TFA or acetic acid followed bγ the addition of 1.0 ml -mercaptoethanol.
The crude peptides were purified bγ RP HPLC as described in Example 9
Example 9 Base peptides were purified bγ RP HPLC using 6.8 x 25 cm Vγdac C4, C8, or C18 columns (The Separation Group, Hespena, CA) with a gradient of 0.1 % TFA/H2O and 0.1 % TFA/acetonitnle. Following HPLC purification, the acetonitnle was removed bγ rotarγ evaporator and the peptides were Iγopholized and stored at -20° or -70°C (haloacetγl peptides) until needed.
Fractions containing the desired peptide as determined bγ mass spectral analysis, were pooled and lyophilized. Identities of the base peptides were established bγ MALDI-MS. Mass spectrometrγ was used to identify and/or confirm the molecular mass of the various peptide species. Mass analysis was performed using a Voyager DE RP MALDI-TOF mass spectrometer (Perseptive Biosystems, San Jose, CA). The nization matrix for the analysis was carried out in 10 mg Sinapimc acid/ CHCA dissolved in 50% acetonιtrιle/0.1 % TFA. All mass analyses were performed in the positive ion mode. The observed molecular ion for each construct was found to be consistent with that obtained from the theoretical sequence (Table A). Table A
Mass spectral analysis of base peptides and multiple peptide conjugates
Mass [MH+]
MPC Theoretical Observed
T3 (base peptide) 5,930.56 5,931
T3-CSP (base peptide) 7,515.15 7, 513
Tat-2-Tat-3 (base peptide) 7,303.65 7, 306
T1-T3 11,139.24 11, 144
T1-CSP-T3 12,723.73 12, 737
T3-MSP 10,615.03 10, 71 1
Tat-1-Tat-2-Tat-3 1 1 ,997.35 12,460
'Theoretical mass reported for the tetrameric MPC molecules.
The amino acid sequence and the relative degree of purity of the base peptides were established bγ Edman sequence analysis (Liu et al., Anal Biochem. 182:383-387 (1989)) on an Applied Biosγstems Model 494 sequencer,
Foster Citγ, CA as well as quantitative amino acid compositional analγses to determine specific molar ratio for each residue. The molar ratio of each amino acid was found to be consistent with that of the theoretical values as indicated in Table B.
Table B
Ammo acid composition of the base peptides
Theoretical Experimental
Ammo Acid T3-CSP Tat-2-Tat-3 T3-CSP Tat-2-Tat-3
Asx 9 1 8.7 0.8
Thr - 1 0.7
Ser 4 4 3.6 3.5
Glx 7 6 6.8 5.7
Pro 2 4 2.0 3.6
Glγ 1 1 0.8 1.2
Ala 2 2 2.0 2.0
Cγs 2 2 1.8 1.8
Val
Met lie 2 1 1.5 0.8
Leu 3 2.7
T r -
Phe -
His - 4 3.7
Lγs 5 3 4.7 2.8
Trp - 1 0.8
Arg - 4 3.8
Theoretical- peptide plus Core molecule **Cysteιne determined as cysteic acid
Linear haloacetγl peptides for attachment to the base peptides were sγnthesized as described in Examples 10 and 11. Example 10
Peptides comprising malarial antigens were sγnthesized as described above using standard ABI Fmoc cγcles with a final cγcle added for modification of the N terminal ammo group with a haloacetγl group (Robey and Fields, Anal
Biochem. 177:373-377 (1989)). Briefly, 1.0 mmol N-bromoacetyl-NHS ester, CI2AC2O or Br2Ac2θ which is prepared by dissolving 1.0 mmol BrCH2C00H in DCM to which 0.5mmol DCC in DCM is added with mixing. The reaction was carried out for 30 minutes at room temperature and the solution is filtered to remove the DCU precipitate formed. The activated ammo acid residue [active ester/anhγdnde] was then added to the reaction vessel containing the resin bound deprotected peptide and allowed to couple for 1 hour at room temperature. Coupling of the anhydride was carried out in DCM. Following sγnthesis, peptides were cleaved from the resin according to a specific cleavage protocol described (Boγkms et al. Cytokine 11 :8-15 (1999)) followed bγ purification bγ RP-HPLC. The peptides were lyophilized and stored at -70°C until needed.
Example 1 1
The HIV Tat peptides (Boy kins et al, J. Immunol. 163.15-20 (1999)) were synthesized essentially the same way as the malaria peptides, except that all cysteine sulfhydrγl residues of Tat-1 peptide were protected with the tButhio blocking group (Figure 5B). Chloroacetγlation of the ammo terminal end of the peptide was chosen specifically for the Tat peptide to minimize reactivity with other highlγ reactive side chains present in both the base and linear peptides during final assembly of the Tat MPC. Cleavage of this molecule produced a stable N chloroacetγl peptide whose side chain thiol groups remained blocked throughout the purification. Subsequentlγ, the haloacetγl linear peptide Tat 1 was coupled to the base peptide Tat-2-Tat-3. The sulfhγdrγl protection was released following coupling of the haloacetγl peptide to the previously deprotected cγsteine residues on the base peptide Deprotection was accomplished with an excess of BU3P reagent as used earlier in the assemblγ process.
The linear haioacetγl peptides were then cleaved as described in Example 12.
Example 12
Haloacetγl peptides were cleaved in a cleavage cocktail consisted of TFA, water, phenol (90:5:5). Thiol scavengers were not used since thiol compounds could react with the haloacetγl moietγ as well as could result in the deprotection of the Cγs (tButhio) in the Tat linear peptide.
The haloacetγl peptides were purified and characterized as described in Example 9 above.
In the final phase of the assemblγ, with identities established bγ mass spectral and ammo acid analγses,
HPLC purified linear or S-protected haloacetyl peptides comprising one or more functional epitopes were then coupled to the cysteine thiol groups on the base peptide to generate an MPC molecule having the structure shown in Figure 7C.
Examples 13 and 14 describe the coupling of the malarial or HIV Tat peptides to the base peptide.
Example 13
The base construct and the linear haloacetγl malarial peptide were coupled as follows. Into a 50 ml teflon flask fitted with a nitrogen tube, the base peptide was dissolved in 0.5M NaHC03 or 0 1 1 Tns/guandine-HCI pH 8.0 buffer as needed. A solution of BU3P in 1 propanol or TCEP was freshlγ prepared. The haloacetγl peptides were dissolved in 1 2 ml (0-6M) guandinme-HCI and added to the reaction flask under nitrogen. For example, to 10 mg base peptide T3 (1.7 M) 0.6 M reducing agent was added for reduction of the base cγsteiπγl residues. The solution was placed on a stirrer for 1 hour followed bγ the addition of 0.8 mg (0.29 M) haloacetγl peptide (T1) to the reaction mixture. The coupling reaction was then carried out for 1.5 3 hours at 25°C for a bromoacetγl peptide and up to 6 hours when using the chloroacetyl derivative. The reaction mixture was monitored with MALDI-MS for the presence of conjugate material, -mercaptoethanol (0.2 ml) was then added to the reaction flask. The conjugate mixture was then desalted by RP-HPLC and further purified by size exclusion chromatography to isolate the desired multiple peptide conjugate.
Example 14 The base peptide and linear (sulfhydrγl protected) HIV-1 Tat haloacetyl peptide were coupled as follows.
HIV-Tat peptide (Tat-1 ) peptide contains seven protected cysteine residues. A similar strategγ, as previously described, was used to couple the N-chloroacetyl modified HIV-Tat peptide to the base peptide Tat-2 Tat-3. The base peptide was dissolved in 1 Tns/6M guanidine HCl buffer, pH 8.0. A fivefold excess (based on thiol content) of BU3P in 1 -propanol was added to effect reduction of the base cysteine residues. Essentially, to 10 mg (1.4 M) of the base peptide Tat-2-Tat-3, 0.25 M BU3P reagent was added. Following reduction of the cysteinyl residues in the base molecule, 1.6 mg (0.5 M) chloroacetγl peptide was dissolved in 6M guanidme-HCI and added to the reaction mixture.
The solution was adjusted to 20% (v/v) with 1 -propanol to increase solubility of the Tat haloacetyl peptide, and allowed to proceed 2-6 hours under nitrogen. Reaction mix was again monitored qualitatively by MALDI-MS bγ observing the presence of a signal consistent with the expected mass of the desired MPC. Following conjugation of the base and haloacetγl peptide, excess BU3P (1.9 mol) was added for final deprotection of the cγsteine residues. The deprotection was carried out for 2 hours at 25°C. The conjugate mixture was then desalted bγ RP-HPLC as described below.
Example 15
The peptide conjugate mixtures were desalted bγ RP-HPLC using a C i 8 column (10 μ, 19 mm x 150 mm) Waters, Milford, MA using a one step gradient of 0.1 % TFA/H2O and 10% acetonιtrιle/H2θ to elute the low molecular weight salts followed bγ 70% acetonitnle to elute the MPC or anγ unconjugated peptides. The conjugated mixtures were then lyophilized and stored at -20°C.
The MPC, thus formed was further characterized by mass spectroscopy, size exclusion chromatographγ, and
SDS-gel electrophoresis. Mass spectroscopγ was performed as described in Example 9 above. Example 16 describes the size exclusion chromatographγ analγses, while Example 17 describes the SDS gel electrophoresis.
Example 16
Size exclusion chromatographγ was performed using a Dιol-S5 column (YMC, Wilmington, NC) to isolate the desired MPC peptide from the reaction mixture. The elution buffer consisted of 0.1 M phosphate buffer, 0.2 M NaCI, pH 7.0, containing 0.02 M sodium azide. The column was standardized using a GPC standard molecular weight mixture obtained from Bio Rad (Hercules, CA). Example 17 The MPCs were subjected to SDS-polyacrylamide gel electrophoresis using 10-20% Tricme gels (Novex, San Diego, CA) under reducing conditions. After completion of electrophoresis, gels were stained with Commassie blue to visualize the position of the MPCs. The yields of the various base constructs and the efficiency of conjugation are summarized in Table C and
Table D, respectively. As shown in Table D, the CMC values indicate that the extent of conjugation was nearly complete. Under ideal conditions, 2 mole of CMC/mole of MPC should be released during hγdrolγsis of the conjugate molecule. Table C Summarγ of yields for the base peptides
Yield (mg) Base peptide Crude_ HPLC Purified
T3 680 258
T3-CSP 845 302
Tat-2-Tat-3 1750 490
* All yields calculated from acid hγdrolγsis of protein content as determined bγ ammo acid analysis.
Table D
Relative Degree of Conjugation
H Haallooaacceettyγll p peeppttiiddee ++ B Baassee ppeeppttiiddee *mole CMC/mole MPC
T1 T3 1.5
MSP-1 T3 1.8
Tat-1 Tat-2-Tat-3 1.4
T1 T3-CSP 1.5
Calculations based on determination of carboxymethγlcγsteine [CMC] after acid hγdrolγsis of conjugated peptide bγ ammo acid analysis.
Mass anlaysis of the T1-T3 construct produces an ion signal centered at 11,144 m/z. Analysis of the T1- CSP-T3 produces a similar ion cluster centered at 12,737 m/z. The level of puntγ for each MPC was determined bγ analytical reverse phase HPLC on a Vydac C18, 5μ column. Molecular weight of the MPCs was determined bγ SDS-PAGE under reducing conditions. The apparent molecular weights of malaria peptide conjugates T1-T3, T1 -MSP-1 , and T1 -CSP-T3 were observed to be 10 kDa, 12 kDa, 13 kDa and that of the HIV-1 -Tat MPC (Tat-1 -Tat-2-Tat-3) to be 12 kDa, respectively. Overall purities of the tetramenc MPC molecules were greater than 80% with the absolute percent dependent upon isolation and removal by size exclusion chromatography (SEC) of the base and linear peptides used as reactaπts. Analysis of the final MPC molecules bγ mass spectroscopγ and SDS-gel electrophoresis indicated the presence of material corresponding to a tnmeric species. Analysis of these data suggested the presence of a tnmer could have been the result of incomplete coupling to the thiol groups on the core molecule. In all the MPCs reported here, the tnmer represented approximately 20% relative to the tetramer. As discussed above, aggregation and steric hindrance have resulted in microheterogeneitγ in multiple peptide conjugates made using other techniques. In order to minimize these possible deleterious effects during chain assemblγ, a small amount of DMSO was added to the NMP solvent and several Hmb ammo acids were incorporated throughout the peptide chain in the syntheses described above. Furthermore, in the methods described above, incorporation of a senne residue as the C-termmal ammo acid with its side chain protected by a tntγl group allows ease of removal and further modification prior to cleavage from the solid support. The side chain of this residue could be useful in the attachment of one or more antigens, adjuvant or other component.
As discussed above, assemblγ of a tetramenc core template and coupling to alternate Iγsine residues with the epilson position remaining blocked maγ provide greater access to the resin bound ammo groups during the acγlation and deprotection reactions, thus ensuring a higher degree of coupling efficiency at each cycle especially when coupling residues with bulky side chains. In the above-described sγntheses of all base peptides, greater than 99% coupling yields for each residue were obtained as determined bγ Kaiser or TNBS assaγ.
It should be noted that while the peptide lengths described above are 31 residues per branch or less on the base peptide, peptide lengths of 50 or more residues per branch maγ be achieved with similar levels of puntγ and yield using the above-described methodology.
The above techniques employ nucleophihc substitution of alkyl hahdes (Lundblad, Techniques in protein modification, CRC Press, pp63 96 (1995); Means and Feeney, Chemical modification of proteins, Holdeπ-Daγ, Inc., pp105-138.( 1971 )). This chemistry has been used extensively in the conjugation of proteins and peptides that result in the formation of stable thioether bonds (Kolodny and Robey, Anal Biochem 187: 136-140 (1990)). A faint lower band consistent with the molecular weight of a tnmeric species (i.e., the base peptide and one linear peptide coupled) was present in the mixture with the desired tetramer molecule in the case of the malaria MPCs. Mass analysis by
MALDI-TOF and ESI-MS also confirmed this finding. In the case of Tat MPC, similar observations were made. However, the average yield of the MPC constructs was greater than 70% tetramer. The presence of additional species may be the result of limited solubility of the reactants or steric effects thereby limiting accessibility to the thiol group and therefore reducing conjugation. In the above methods, both Tris and bicarbonate buffers were utilized in addition to guandine hγdrochloπde in order to carry out the thiol coupling step.
However, the buffer used to solubihze the base and linear peptides should be selected to optimize the conditions for the thiol reaction.
Measurement of the CMC ratio was performed to determine the degree of conjugation (Kolodnγ and Robey, Anal Biochem. 187: 136-140 (1990)). Slightly less than 2 moles CMC/mole of each MPC construct were present. Under ideal conditions 2 moles of CMC/mole of conjugate should be liberated upon acid hydrolysis. Among the many factors that maγ inhibit production of the tetramer molecule are solubility of the peptides and steric hindrance. Some of these factors could contribute to variable yields which, in addition, could be dependent upon the phγsical parameters of the individual species. To facilitate the solubility of the chloroacetγl Tat peptide [Tat-1], increasing amounts of 1 - propanol were added. In the case of the more hγdrophobic peptides, 1 M Trιs/6M guandme-HCI minimized the formation of the tnmeric MPC. This was particularly true for the MPC containing the Tat-3 peptide, where the overall yield was somewhat lower. The lower yield of the Tat MPC may result from the release of a small number of thiols, Cysteιne[tButhιo] in the peptide sequence, following addition of the haloacetγl peptide into the reaction mixture containing the base peptide Tat-2-Tat-3. The release could occur as a result of the initial reduction of the base cγsteinγl residues with BU3P.
Poor solubility of the Tat MPC may be a factor in the SEC analysis to isolate only the MPC and remove the unreacted starting peptides (base and linear peptide). Therefore, the Tat MPC has higher levels of these peptides which maγ be a factor in suppression of the lomzation of the MPC when analγzed bγ MALDI-MS. However, these low intensity signals observed appear to be consistent with the expected molecular mass indicating the presence of both tetramer and tnmer species.
In summary, the above strategies for synthesizing MPCs provide several benefits. The above methods facilitate sγnthesis of a well defined core template moiecule that minimizes steric hindrance bγ coupling to onlγ two branches per sγnthetic cγcle. While core molecule spacing is similar to that used in the sγnthesis of a tetramer, onlγ two residues are assembled per cγcle at alternate ammo groups on the tetramenc core with the opposite positions remaining protected throughout the synthesis of the base molecule. In addition, in some of the embodiments described above, a senne residue with a tπtyl group used as side chain protection is incorporated into the core template. The senne residue can be modified on the solid phase for greater functionality without cleavage of the peptide resin link, thereby permitting the attachment of antigens or an adjuvant molecule if desired. Furthermore, as discussed above, synthesis of a base molecule with specific thiol sites for attachment of additional peptides allows site directed coupling of the linear peptides.
In addition, solubility of the peptide chains maγ be improved bγ modifying the N-methylpγroliidone coupling solvent bγ adding 10% DMSO, therebγ enhancing the acγlation and/or deprotection reaction and improving coupling efficiency. Furthermore, addition of Hmb protected ammo acids at specific points in the peptide chain further minimizes aggregation of the growing peptide chains therebγ minimizing the risk of lower γieids in the desired peptide. In addition, in some instances the homogeneitγ of the product maγ be enhanced bγ attaching blocking groups to the side chains of one or more ammo acids in the haloacetγl peptides to be attached to the base peptide. For example, in the examples above, a tButhio side chain blocking group was used on all cγstemγl residues in the haloacetγl peptides. Internal cγstemγl residues were selectively deprotected onlγ after formation of the final multiple peptide conjugate. The above methods permit further side chain modification to the iγsine and cγsteine residues on the core template, therebγ permitting alternative methods of selectively attaching peptides to the core template or the core peptide. For example, the reactive groups on the core template can be selected so that one reactive group is deprotected upon treatment with base while the other reactive group is deprotected upon acid treatment. For example, an alternate use of the core template functionality is in the replacement of the t-Boc group at the branched lysine position bγ a quasi-orthogonallγ protected Iγsine derivative such as Fmoc-Lys(Dde) or Fmoc Lγs(ιvDde)-OH The use of such derivatives allows selective deprotection with dilute hγdrazine or other similar amines without cleavage from the solid support. The use of Lγs(Mtt) or Lγs(Tfa) allows selective deprotection with dilute acid solutions. Similarly, the use of Cys(Mmt)-OH as a replacement for the current Cγs(Trt) allows the selective deprotection of the thiol with dilute acid, therebγ enabling the sulfhγdrγl to be readily exploited to produce a thioether linkage or other reactions involving the thiol.
Thus, the basic core molecule described above provides increased versatility in the use of multiple strategies for the covalent attachment of additional peptide antigens, adjuvant components, or other desirable functionalities. WO 00/78969 PCT USOO/l 0119
The methodologγ described herein produces well characterized multiple peptide conjugates useful in the development of subumt vaccines in significant yields for important commercial applications.
PART III: HIV-1 TAT MULTIPLE PEPTIDE CONJUGATE
Background of the Invention Infection with HIV is responsible for a large number of deaths annually and represents a significant threat to the public health. Despite continuous efforts bγ a number of laboratories, little progress has been made towards the development of a safe and effective vaccine to control progression of HIV disease. Most strategies have failed due to the huge diversitγ in predominant immunologic epitopes of gp120 and gp41 of this heterogeneous virus; this diversitγ occurs between HIV-infected individuals and sometimes even within a single host. (Berman et al., Nature, 345:622 (1990); Burton and Moore, Nature Med, 4:495 (1998); Stott et al., J. Gen. Virol, 79:423 (1998)).
Neutralization of extracellular Tat, a regulatorγ protein of HIV-1, bγ a therapeutic vaccine approach for the treatment of AIDS has been proposed bγ a number of investigators. (Cafaro et al., Nature Med., 5:643 (1999); Caselli et al., J. Immunol, 162:5631 (1999); Cohen et al., Proc. Natl. Acad. Sci. USA, 96:10842 (1999); Lambert, J. Hum. Virol, 1 :249 (1998); Zagurγ et al., J. Hum. Virol, 1 :282 (1998); Grmgeri et al., J. Hum Virol, 1 :293 (1998); Goldstein, Nature Med, 2:960 (1996)). Tat protein is produced bγ HIV-infected cells and is believed to be involved in the progression of HIV infection, development of Kaposi's sarcoma, immunosuppression, apoptosis of normal cells, and various neurological disorders. (Ensoli et al., J. Virol, 67:277 (1993); Chang et al., J. Biomed. Sci., 2:189 (1995); Chang et al., AIDS, 1 1 :1421 (1997); Chen et al., J. Biol. Chem., 272:22385 (1997)). It has recentlγ been shown that manγ of the effects of HIV infection of monocγtes can be mimicked bγ treatment of these cells with extracellular HIV- Tat protein. (Dhawan et al., J. Immunol, 154:422 (1995); Lafrenie et al., J. Immunol, 156:1638 (1996); Lafrenie et al., J. Immunol, 157:974 (1996); Lafrenie et al., J. Immunol, 159:4077 (1997); Albim et al., J. Biol. Chem., 273:15895 (1998); Weeks, Int. J. Molec. Med, 1 :361 (1998)). Therefore, immunologic obstruction of Tat protein bγ prophylactic immunization of HIV-infected individuals could reduce viremia and thereby prevent or delay the onset of progression to AIDS. Neutralization of extracellular Tat could also partly restore immune function in AIDS patients. Recent studies have proposed Tat toxoid or oxidized Tat protein as potential candidates for therapeutic AIDS vaccines.
(Cafaro et al., Nature Med., 5:643 (1999); Caselli et al., J. Immunol, 162:5631 (1999); Cohen et al., Proc. Natl. Acad. Sci. USA, 96:10842 (1999); Lambert, J. Hum. Virol, 1:249 (1998); Zagury et al., J. Hum. Virol, 1 :282 (1998); Grmgeri et al., J. Hum Virol, 1 :293 (1998); Goldstein, Nature Med, 2:960 (1996); Girard et al., C. R. Acad. Sci. Ill, 322:959 (1999); Le Buanec et al., Biomed. Pharmacother., 52:431 (1999)). However, like other viral proteins, there are mutational changes in Tat protein among various viral isolates. The use of full-length recombinant Tat protein from a single HIV-1 strain as a vaccine maγ, therefore, not be sufficient for effective treatment of individuals infected with different HIV-1 isolates. Moreover, crucial functional epitopes maγ not be immunodommant. A subumt vaccine targeting conserved, functionally critical epitopes could resolve these problems. The mechanisms bγ which HIV-Tat protein promotes viral pathogenesis primarily involve highly conserved cysteine-nch and basic domains in various HIV 1 isolates. (Boγkms et al., J. Immunol, 163:15 (1999); Albim et al., J Hum. Virol, 2:179 (1999); Mitola et al., J Virol, 74:344 (2000)). Neutralization of these domains bγ epitope-specific antibodies could provide selective targeting of Tat to reduce progression of HIV infection. Summary of the Invention
The present invention is directed to a novel highlγ immunogemc sγnthetic multiple peptide conjugate constituting functional domains Tat2140 and Tat53 68 from HIV-1 group M plus Tat9-20 from HIV-1 group 0 of the HIV-Tat protein (HIV-1 Tat-MPC). Vaccination of mice with HIV-1 -Tat-MPC induces an effective immune response to all three functional domains. Aπtι-HIV-1 -Tat-MPC antibodies efficiently inhibit Tat-mduced viral activation in monocytes infected with HIVβa-L as well as with various clinical HIV-1 isolates, and reduce Tat-mediated cytopathicitγ in infected cells bγ greater than 75%. The results indicate that antι-HIV-1 -Tat-MPC antibodies inhibit viral pathogenesis, possibly by blocking functional determinants of Tat and disrupting autocnne and paracnne actions of secreted Tat protein This epitope-specific synthetic Tat construct provides a subumt AIDS vaccine for inducing an effective immunoprophγlaxis response to reduce progression of HIV infection. Description of the Preferred Embodiment
We have constructed a synthetic HIV Tat multiple peptide conjugate constituting the highly conserved cysteine-nch and basic domains bγ which HIV-Tat protein promotes viral pathogenesis. We have demonstrated that antibodies against this immunogen effectively reduce HIV pathogenesis in vitro. Our general approach overcomes many of the problems associated with immuπodommance of non-essential epitopes, viral diversity, and mutational changes in order to design an effective vaccine.
The preferred embodiment of the present invention utilizes three pathogenesis-related functional domains of the HIV Tat protein: the cysteine-nch Tat2l 40 and basic Tat53-68 domains critical for promoting HIV and the Tat920 domain of HIV-1 group 0. Consensus-B: Tat21 40: ACTNCYCKKCCFHCQVCFTT (SEQ ID N0:1)
Tat53-68: RQRRRAHQNSQTHQAS (SEQ ID N0:2) Consensus-O:
Tat9-20: PPWHHPGSQPQl (SEQ ID N0:3)
The three peptide sequences described above were used to prepare the sγnthetic construct HIV-1-Tat-MPC, the structure of which is shown in Figure 8. The Tat920 domain of HIV-1 group 0 enhances HIV infection in monocγtes (Figure 9). Construction of HIV-1 -Tat MPC utilized an Fmoc solid-phase sγnthesis coupled with conventional solution chemistrγ as described above. The HIV 1 -Tat-MPC was constructed with Tat53 68 +Tatg.20 as a base peptide and Tat2l40 as a linear peptide attached to the tetramenc core template (Figure 8). Both the base peptide Tat53 68 +Tatg.20 on the core and Tat2l 40- which was modified as a linear N-chloroacetγl (Cγs-tButhio) derivative, were WO 00/78969 PCT/USOO/l 0119
purified bγ HPLC and subjected to MALDI-MS mass spectroscopic analysis to confirm the molecular mass of the peptide species. Mass spectroscopic profiles of purified peptides bγ MALDI-MS indicated both base peptide Tat53 68 + Tatg 20 and N-chloroacetγl (Cγs tButhιo)-Tat2i 40 peptide to be highlγ purified, and the molecular ions observed for each species were consistent with that of the theoretical masses. Final assemblγ of the HIV-1 -Tat-MPC construct involved coupling of purified N-chloroacetγl (Cγs-tButhιo)-Tat2l 40 peptide to the cγsteine thiol groups on the core containing the base peptide Tat53.g8+Tatg.20 as described above. The t-Buthio protecting groups were removed and puritγ of HIV-1 Tat-MPC was established bγ reverse-phase HPLC (RP-HPLC) on a 4.6 x 25 cm Vγdac C18 column with a 0-80% gradient of 0.1 % TFA/H2O and 0.1 % TFA/acetonitπle. HIV-1-Tat MPC eluted as a major peak at 55% acetonitnle. A minor peak at 7.6 mm, also observed in the reagent blank run, indicated the presence of a trace amount of unrelated product. The fractions eluted in the major peak at 9.353 m retention time were pooled, lyophilized, repunfied by HPLC, and stored at -70°C. Homogeneity of this HIV-1-Tat-MPC preparation was further evaluated bγ SDS polγacrγlamide electrophoresis with immunological characterization bγ Western blot analysis. HIV-1 Tat-MPC consisted of a predominantly homogeneous species ( > 90%) with an apparent molecular mass of 12 kDa that showed strong reactivity with rabbit anti Tat antiserum. In contrast, purified recombinant Tat used as a positive control exhibited multiple bands, indicating the presence of polymerized products.
Next, the ability of HIV-1 -Tat-MPC to induce an effective immune response was tested by immunizing three groups of BALB/c mice with various doses of HIV-1 -Tat-MPC bγ intraperitoneal injections. Serum antibodγ responses were monitored bγ ELISA. All three groups of mice immunized with 10-100 ug doses of HIV 1 -Tat-MPC developed antibodγ titers (Fig. 10, panel a). The largest immune response was observed in mice immunized with 100 ug of antigen. Peak antibodγ titer was observed at six weeks (second bleed) for all groups of mice. The antibodγ titers at six weeks after the initial immunization achieved detectable levels even up to 1 :243,000 serum dilutions (Fig. 10, panel b).
The binding of anti-HIV 1 Tat-MPC antisera to rTat and the various sγnthetic peptides present in the construct was determined bγ ELISA using 96-well plates coated with 10 ug/ml of each antigen. As evident from the data, antisera raised against HIV-1 Tat-MPC showed strong reactivity with rTat as well as with all three Tat peptides.
To evaluate the specificity of anti-HIV 1 -Tat-MPC antisera, the ability of purified peptides to inhibit binding of HIV-1 - Tat-MPC antisera to rTat protein was examined. Preincubation of antι-HIV-1 -Tat-MPC antisera with purified peptides in various combinations substantially inhibited the reactivity of anti HIV 1 Tat-MPC with rTat (Fig. 1 1 , panel b), thus confirming the ability of each epitope on the construct to induce an effective immune response. Cell proliferation in response to Tat or various Tat peptides was evaluated using splenocγtes of mice immunized with HIV-1 -Tat-MPC. Splenocγtes from mice obtained four weeks after the last vaccination were cultured for three days in the presence of rTat, HIV 1 Tat-MPC, or various Tat peptides, and antigen-stimulated cell proliferation was determined by ^H thymidme incorporation. Specific responses were observed in spleen cells from mice immunized with HIV-1 -Tat-MPC (Figure 12). Tat53-β8 and Tatg_20 peptides induced proliferative responses four-fold, and Tat2l - 40 enhanced proliferation two-fold over control Tat4i -52 peptide. Recombinant Tat protein used as the positive control also stimulated proliferation bγ greater than two-fold. Basal levels of proliferation were observed in spleen cells treated with the control peptide or medium oniγ and m cells from unvaccinated control mice.
The ability of antι-HIV-1 Tat-MPC antisera to inhibit Tat-stimulated HIV 1 production was evaluated by infection of monocγtes in the presence of endogenous or exogenous HIV-Tat protein. Monocγtes were infected with HIVβa-L in the presence or absence of rTat (20 ng/ml) and antι-HIV-1 -Tat-MPC antiserum from each mouse at a final dilution of 1 :100. The medium containing rTat or antι-HIV-1-Tat-MPC antiserum was replaced everγ alternate daγ. On daγ five, culture media were replaced with medium without rTat or antisera, and the level of virus production was quantitated 24 h later bγ measuring cell-free p24 antigen bγ ELISA. The data summarized in Table One reveal that 10 ug or 20 ug of the immunogen elicited effective responses in sera of most animals for neutralizing Tat-mduced HIV replication. However, despite the high immune response according to ELISA in mice inoculated with 100 ug of the immunogen, sera from these mice were slightly less effective in neutralization of Tat effects compared to those inoculated with 10 or 20 ug of the antigen (Table One; P< 0.01 vs. 20 ug and P< 0.05 vs. 10 ug).
HIVβa-L-assoαated cγtopathic effects were observed to be reduced bγ 60-75% in monocγtes treated with antι-HIV-1 Tat-MPC antisera. Importantly, the neutralizing anti-HIV- 1 -Tat-MPC antisera were also able to inhibit Tat- mediated induction of HIV replication bγ 15-67% in monocγtes infected with a varietγ of clinical isolates (Table Two). Figure 13 depicts a possible mechanism bγ which aπtι-HIV-1-Tat-MPC inhibits Tat-mduced HIV replication in monocγtes. Extracellular Tat protein produced bγ HIV-infected cells is believed to act on cells both in autocrme and paracnne fashion. Tat activates normal cells for productive HIV infection and then promotes virus replication in HIV- infected cells. It is likely that antι-HIV-1-Tat-MPC antibodies target key functional determinants and disrupt the autocrme and paracnne actions induced bγ secreted Tat protein.
We describe a novel sγnthetic multiple-peptide conjugate sγstem constituting three functional peptide sequences of the HIV-1 Tat protein in a single, homogenous immunogen. All three peptides constituting the construct induced an effective immune response in mice and generated antisera that efficiently inhibited Tat-mduced pathogenesis in monocytes infected with HIVβa-L as we" as various clinical HIV-1 isolates. Importantly, HIV-1-Tat- MPC alone was sufficient to induce an effective immune response without being conjugated to a carrier protein. Thus, this construct represents the first successful model of a sγnthetic immunogen that serves as a basis for designing a multi-epitope subumt vaccine for AIDS.
The three peptides used to generate the HIV-1 -Tat-MPC construct were all immunogemc, and antibodies raised in mice against the HIV-1 -Tat-MPC reacted with recombinant Tat protein from which the peptide sequences had been deduced. Furthermore, Tat-mduced pathogenesis was inhibited bγ antisera against HIV-1 -Tat-MPC, supporting roles for Tat2i 40- Tat53-68/ and Tatg.20 epitopes in viral activation bγ infected cells. Therefore, this HIV-1 -Tat- MPC containing conserved functional domains from group M of HIV-1 , as well as a new functional sequence from the newlγ discovered HIV-1 strain group 0, serves as an immunogen for various subtγpes of HIV-1 infection. Vaccination of mice with low doses (10 to 20 ug) of the HIV-1 -Tat-MPC construct elicited antibodies that efficiently inhibited Tat effects; 100 ug-injections did not enhance efficacy and even appeared to significantly decrease effectiveness in supporting HIV-1 viral production. It is practical to optimize the minimal dose of the HIV-1 Tat-MPC to induce maximal immune responses in mice and other animal species.
Our findings provide a new approach to developing effective and safe subumt vaccines, especially when a conventional vaccine maγ fail to induce an effective immune response due to problems associated with immunodominance, viral diversitγ, and mutational instability. The fact that HIV-1 -Tat-MPC can be readily customized bγ replacing peptides present on the construct with desired peptides from other viral antigens (such as gp120, gp41 or gag regions as needed) provides a versatile tool for generating sγnthetic subumt vaccines. This flexibility is a significant advantage over conventional vaccines, especially for heterogeneous viruses such as HIV. Thus, our observations constitute a major advance in HIV research bγ providing a completely novel strategy to develop a synthetic AIDS vaccine based on a new tγpe of multiple peptide antigen constituting a homogeneous entitγ with multiple epitopes. Because HIV-1 -Tat-MPC is a purelγ sγnthetic molecule that does not contain a live or inactivated virus, it conveγs no risk of viral transmission to recipients. These advances form the basis for immunization of primates and humans.
Example 18 HIV-1 -Tat rotein. The HIV-1 -Tat protein used in these studies was obtained as a gift from Dr. John Bradγ (National
Cancer Institute, National Institutes of Health) or through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergies and Infectious Disease, National Institutes of Health, from Dr. Andrew Rice or Dr. John N. Bradγ (Lafrenie et al., J. Immunol., 156:1638 (1996)). HIV-1 Tat was dissolved at 10 ug/ml in 1 ,000X treatment buffer (PBS containing 1 mg/ml BSA, 0.1 mM dithiothreitol) and frozen in ahquots at -80°C. Tat preparations were screened and found to be negative for endotoxm contamination.
Synthesis of Tat peptides and multiple peptide construct. Tat peptides were sγnthesized bγ solid-phase sγnthesis on an Applied Biosγstems Peptide sγnthesizer Model 430A (Foster Citγ, CA) Peptide identities were confirmed bγ ammo acid compositional analysis and plasma desorption mass spectroscopic analysis The HIV-Tat-1 MPC was prepared according to the above-described procedure.
Immunization of mice with HIV-1 -Tat-MPC. Groups of five BALB/c mice (6 8 weeks old) were inoculated three times at three-week intervals with 10 to 100 ug of the HIV-1 -Tat MPC construct (Strategic BioSolutions, Newark, DE). Constructs were mixed with an equal volume of complete Freund's adjuvant for primary injections and incomplete Freund's adjuvant for secondary and tertiary injections. Sera were prepared from blood taken 15 days after each injection.
Serological analysis. Enzγme-lmked immunosorbent assaγ (ELISA) was performed using HIV-1 -Tat-MPC coated on 96 well microtiter plates (Costar, Cambridge, MA) at a concentration of 1 ug/ml (Strategic BioSolutions). Antibodγ titers were expressed as optical densitγ (OD) values measured at 650 nm on 1, 000-fold diluted antiserum specimens. Monocyte isolation and infection with HIV- 1 Monocγtes were isolated from peripheral blood mononuclear ceils
(PBMC) of donors seronegative for HIV and hepatitis bγ leukapheresis and purification bγ countercurrent centrifugal elutnation (Lafrenie et al., J. Immunol., 156:1638 (1996)). In some experiments, monocγtes were treated with HIV-1 Tat protein. Pnmarγ monocγtes cultured for 5 daγs were exposed to HIV l Ra.|_, a monocγtotrophic HIV strain (Advanced Biotechnologies, Inc., Columbia, MD) at a multiplicity of infection of 0.005 infectious virus particles/target cell, or with various clinical HIV 1 isolates.
Determination of virus production. The levels of cell-free HIV 1 in culture superπatants were determined using DuPont NEN p24 ELISA kits for HIV-1.
Cell proliferation assay. Spleens from vaccinated or non-vaccinated (control) mice were aseptically excised and used to prepare single-cell suspensions in RPMI-1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 2 mM L-glutamme, 5% heat-inactivated FCS, 100 units/ml penicillin, and 100 ug/ml streptomγcin. Cell viability was > 90% as determined by trypan blue exclusion. Splenocγtes (2 X 10^ cells) were cultured in round-bottom 96 well tissue culture plates (Costar) with 10 ug/ml rTat, various Tat peptides, or medium alone and incubated in a 5% CO2 humidified incubator at 37°C for 96 h and pulsed with 1 uCi ^H thγmidme/well during the last 4 h. Cells were harvested and
Figure imgf000053_0001
incorporation was determined bγ liquid scintillation counting. All cultures were performed in triplicate.
Detection of rTat and HIV- 1- Tat-MPC by Western blot analysis. The lyophilized rTat protein or HIV-1 -Tat-MPC were resuspended in SDS-Laemmli loading buffer [500 mM Tris-HCl, pH 6.8, 10% SDS, 0.01 % bromopheπol blue, 20% glγcerol], reduced with 1 % mercaptoethaπol, heated for 2 mm at 95°C, loaded, and electrophoresed on a 8-16% Tns- glγcme gradient poiγacrγlamide gel (Novex, San Diego, CA) in SDS running buffer [25 mM Tris-HCl, pH 8.3, 192 mM giycine, 10% SDS]. After electrophoresis, the rHIV-1 -Tat protein and HIV 1 -Tat-MPC were transferred onto 0.45-uM nitrocellulose in a buffer containing 25 mM Tris-HCl [pH 8.3, 192 mM giycine, 20% methanol] and blocked with 50 mM Tris-HCl [pH 7.5, 150 mM NaCI, 0.2% Tween-20 (TBST)] containing 5% nonfat dry milk for at least 1 h. The blots were washed three times with TBST and then incubated for 1 h with rabbit anti Tat antibody followed bγ horseradish peroxidase (HRP)-conjugated protein A (Pierce Chemicals, Rockford, IL; 1 :3,000 dilution in TBST containing 5% nonfat drγ milk) and developed with SuperSignal chemiluminescence enhancer solution (Pierce). The bands were visualized bγ exposure of blots to X-raγ film.
Table One
Effect of antisera against HIV-1 Tat MPC on Tat-mduced HIV-1 production by monocytes
Mouse Dose of Inhibition of HIV infection
Number Immunogen (% of Control) ean t! SEM
Bleed #1 Bleed #2 Bleed tf 1 Bleed #2
1.1 10 ug 0 2
1.2 10 ug 54 54
1.3 10 ug 68 100 544+ 144 64.2 + 18.1
1.4 10 ug 66 100 (/»< 0.u2) {/»< 0.03)
1.5 10 ug 84 65
2.1 20 ug 84 74
2.2 20 ug 69 81
2.3 20 ug 78 99 46.2 + 19.0 86.0 + 5.6
2.4 20 ug 0 100 lV-0.07) (P< 0.0001
2.5 20 ug 0 76
3.1 100 ug 38 23
3.2 100 ug 35 82
3.3 100 ug 0 33 42.2±13.2 46.2 + 10.0
3.4 100 ug 79 48 V< Q.M) (/'< 0.01 )
3.5 100 ug 59 45
Monocytes (2 x 10^ cells) cultured in 96-well plates were treated with HIV-Tat (20 ng/ml) in the presence or absence of mouse antisera against HIV-1-Tat-MPC (pooled 20 ug immunizations, 1 :100 dilution) 2 h prior to HIV infection at an MOI of 0.005. After a 2 h exposure, virus was removed, and the culture medium was replaced with fresh medium with or without rTat in the presence or absence of antisera against HIV-1-Tat-MPC (1 :100). On daγ 5, culture medium was replaced with fresh medium with no additions. After 24 h, culture fluid was harvested for p24 determination. The data are presented as inhibition of HIV production measured as cell free p24 relative to HIV-infected monocγtes treated with rTat in the absence of antisera P values indicate statistical significance of difference from 0% inhibition by 2-taιl Student's t-test.
Table Two
Effect of antisera against HIV-1 Tat-MPC on Tat-mduced HIV 1 production bγ monocγtes infected with various clinical HIV-1 isolates
HIV-1 Date of Coreceptor p24 (ng/ml) p24 (ng/ml) isolate collection usage -Tat -Tat + Tat +Tat
+ antι- + antι-
MPC MPC antisera antisera
Rapid progressors:
5084 Oct. 86 R5 8.3 + 2.7 4.7 + 1.0 (43) 1 1.8 + 1.8 5.0 0.6 (58)
5108 Oct. 85 ND 1.9+0.7 1.4+0.2 (30) 2.6±0.3 1.9 + 0.5 (26)
5157 Mar. 84 R5 1.8±04 2.0 + 0.6 (-) 2/7 + 0.3 1.6+0.5 (40)
Late progressors:
5048 Mar. 87 ND 3.8 + 0.5 3.0 + 0.4 (20) 6.9 + 0.7 3.2+0.5 (54)
5160 Jul. 84 R5 2.1 ±0.3 1.0+0.1 (53) 3.3 + 0.2 1.8 + 0.4 (47)
5073 Maγ 89 R5 0.7 + 0.1 0.6+0.1 (15) 1.5 + 0.2 Q ±QΛ (51)
Long term nonprogressors:
5096 Mar. 89 ND 10.1 + 1.0 7.2+.0.6 (29) 15.7+0.2 6.7 + 0.4 (57)
5148 Apr. 83 R5 1.3±0.2 1.06±0.3 (16) 1.9+0.4 0.6 + 0.10 (67)
5155 May 95 R5 9.3+.0-5 4.6^0-5 (51) 17.7+0.2 8.1 +0.9 (54)
Monocγtes (2 x 10ϋ cells) cultured in 96-well plates were treated with HIV-Tat (20 ng/ml) in the presence or absence of mouse antisera against HIV 1 -Tat-MPC (pooled 20 ug immunizations, 1 :100 dilution) 2 h prior to HIV infection with various HIV-1 isolates (40,000 RT/10^ cells). After a 2 h exposure, virus was removed, and the culture medium was replaced with fresh medium with or without rTat in the presence or absence of antisera against HIV-1 Tat-MPC (1:100). On daγ 5, culture medium was replaced with fresh medium with no additions. After 24 h, culture fluid was harvested for p24 determination. The p24 values are expressed as meaπ±SEM of three determinations. Values in parentheses represent percent inhibition of HIV production bγ antι-HIV-1 -Tat-MPC antisera. ND: not determined.
Example 19 Effect of anti-HIV ) ' -Tat-MPC antibody on uninfected or HIV-1 -infected monocyte migration in response to HIV-1 Tat protein. Uninfected or HIV-infected monocγtes were suspended at 2 X 106 cells/ml. HIV-1 Tat (20 ng/ml) was placed in the lower chamber of a chemotactic chamber in the presence of normal mouse serum or mouse anti-HIV- 1 -Tat-MPC antiserum (1 :100), covered with a polγcarbonate filter, and 50 ul of the uninfected or HIV-1 -infected monocγte suspension was placed in the upper chamber. The chamber was incubated for 3 h at 37"C and then the filters were removed and stained with Diff-Quick stain. The number of cells attached to the underside of the filter was determined bγ high power microscope field in triplicate for each well. Anti-HIV-Tat-MPC antibodγ substantially inhibited ( > 70%) HIV-1 Tat-induced migration of uninfected and HIV-infected monocytes. Example 20
Effect of anti-HIV- 1 -Tat-MPC on invasion of uninfected or HIV-1 -infected monocytes through basement membrane matrix (Matrigel). Uninfected or HIV-1 -infected monocytes were suspended at 2 X 106 cells/ml. HIV-1 -Tat (20 ng/ml) was placed in the lower chamber in the presence of normal mouse serum (1 :100) or mouse anti-HIV-1-Tat- MPC antiserum (1 :100), covered with a Matrigel-coated polγcarbonate filter, and 50 ul of the uninfected or HIV-1 - infected monocγte suspension was placed in the upper chamber. The chamber was incubated for 12 h at 37°C, and then the filters were removed and stained. The number of cells attached to the underside of the filter was determined bγ high power microscope field in triplicate for each well. Preincubation of HIV-1 -Tat with anti-HIV-1 -Tat-MPC antibodγ inhibited monocγte invasion across the basement membrane matrix bγ greater than 60%. Iπventorship Please note that the inventors of each of the separate parts of this application are as follows:
Part I: Subhash Dhawan, Robert A. Boγkins and Kenneth M. Yamada; Part II: Robert A. Boγkins, Manju B. Joshi, Chiang Sγin, Subhash Dhawan and Hira L. Nakhasi; and Part III: Subhash Dhawan and Robert A. Boγkins.

Claims

WHAT IS CLAIMED IS:
1. An isolated or purified peptide comprising a Tat functional domain, said peptide comprising a portion of the Tat protein selected from the group consisitmg of the portion from ammo acids 21 -40 of the Tat protein, the portion from ammo acids 53-68 of the Tat protein and the portion from ammo acids 9 20 of the Tat protein.
2. The isolated or purified peptide of Claim 1, wherein said isolated or purified peptide has a sequence selected from the group consisting of SEQ ID NOs 1-21.
3. The isolated or purified peptide of Claim 1 , wherein said peptide contains a dominant-negative mutation.
4. An isolated or purified peptide having a sequence selected from the group consisting of SEQ ID NOs. 22 66
5. An isolated or purified peptide having a sequence selected from the group consisting of:
(a) the sequence of SEQ ID NO: 1 wherein the A (Alanine) at position 1 of the peptide has been substituted with an ammo acid selected from the group consisting of cγsteme, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine;
(b) the sequence of SEQ ID NO: 1 wherein the C (Cysteine) at position 2 of the peptide has been substituted with an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, trγptophan, and tγros e;
(c) the sequence of SEQ ID NO: 1 wherein the T (threonme) at position 3 of the peptide has been substituted with an ammo acid selected from the group consisting of alanine, cγsteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, valine, trγptophan, and tγrosme;
(d) the sequence of SEQ ID NO: 1 wherein the N (asparagine) at position 4 of the peptide has been substituted with an am o acid selected from the group consisting of alanine, cγsteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, Iγsine, leucme, methiomne, prolme, glutamine, argmine, senne, thereonme, valine, trγptophan, and tγros e; (e) the sequence of SEQ ID NO: 1 wherein the C (Cγsteine) at position 5 of the peptide has been substituted with an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine; (f) the sequence of SEQ ID NO: 1 wherein the Y (tyrosine) at position 6 of the peptide has been substituted with an ammo acid selected from the group consisting of alanine, cγsteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysiπe, leucme, methio ne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, and tryptophan; (g) the sequence of SEQ ID NO: 1 wherein the C (Cysteine) at position 7 of the peptide has been substituted with an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine;
(h) the sequence of SEQ ID NO: 1 wherein the K (Iγsine) at position 8 of the peptide has been substituted with an ammo acid selected from the group consisting of alanine, cγsteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tγrosme;
(i) the sequence of SEQ ID NO: 1 wherein the K (Iγsiπe) at position 9 of the peptide has been substituted with an ammo acid selected from the group consisting of alanine, cγsteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, trγptophan, and tγrosme;
(j) the sequence of SEQ ID NO: 1 wherein the C (Cγsteine) at position 10 of the peptide has been substituted with an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, Iγsine, leucme, methiomne, asparagine, prol e, glutamine, argmine, senne, threonme, valine, trγptophan, and tγrosme;
(k) the sequence of SEQ ID NO: 1 wherein the C (Cγsteine) at position 1 1 of the peptide has been substituted with an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, Iγsine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, trγptophan, and tyrosine; (I) the sequence of SEQ ID NO: 1 wherein the F (phenylalamne) at position 12 of the peptide has been substituted with an ammo acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, trγptophan, and tγrosme;
(m) the sequence of SEQ ID NO: 1 wherein the H (histidme) at position 13 of the peptide has been substituted with an ammo acid selected from the group consisting of alanine, cγsteine, aspartic acid, glutamic acid, phenylalamne, giycine, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine;
(n) the sequence of SEQ ID NO: 1 wherein the C (Cγsteine) at position 14 of the peptide has been substituted with an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, WO 00/78969 PCT/USOO/l 0119
histidme, isoleucine, Iγsine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, trγptophan, and tyrosine;
(o) the sequence of SEQ ID NO. 1 wherein the Q (glutamine) at position 15 of the peptide has been substituted with an am o acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalamne, glγcine, histidme, isoleucine, Iγsine, leucme, methiomne, asparagine, prolme, argmine, se ne, threonme, valine, trγptophan, and tγrosme;
(p) the sequence of SEQ ID NO: 1 wherein the V (valine) at position 16 of the peptide has been substituted with an ammo acid selected from the group consisting of alanine, cγsteine, aspartic acid, glutamic acid, phenylalamne, glγcine, histidme, isoleucine, Iγsine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, trγptophan, and tyrosine;
(q) the sequence of SEQ ID NO: 1 wherein the C (Cγsteine) at position 17 of the peptide has been substituted with an ammo acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, tryptophan, and tyrosine; (r) the sequence of SEQ ID NO. 1 wherein the F (phenylalamne) at position 18 of the peptide has been substituted with an ammo acid selected from the group consisting of alanine, cγsteine, aspartic acid, glutamic acid, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, threonme, valine, trγptophan, and tγros e;
(s) the sequence of SEQ ID NO: 1 wherein the T (threonme) at position 19 of the peptide has been substituted with an ammo acid selected from the group consisting of alanine, cγsteine, aspartic acid, glutamic acid, phenylala ne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, valine, tryptophan, and tyrosine; and
(t) the sequence of SEQ ID NO. 1 wherein the T (threonme) at position 20 of the peptide has been substituted with an ammo acid selected from the group consisting of alanine, cγsteine, aspartic acid, glutamic acid, phenylalamne, giycine, histidme, isoleucine, lysine, leucme, methiomne, asparagine, prolme, glutamine, argmine, senne, valine, trγptophan, and tyrosine.
6. An isolated or purified peptide that is at least 70 percent identical to a peptide having a sequence selected from the group consisting of SEQ ID NOs: 1 21 wherein percent identity is determined by FASTA or BLAST using default opening and gap penalties and a PAM scordmg matrix
7. A nucleic acid encoding the peptide of any of Claims 1 6.
8. A peptide of anγ of Claims 1 6 or a nucleic acid encoding the peptide of aπγ of Claims 1 6 for use in reducing the activitγ of Tat in an individual infected with HIV or inducing an immune response against the Tat protein
9. A method of reducing the activitγ of Tat in an individual infected with HIV or inducing an immune response in an individual against the Tat protein of HIV comprising the step of administering the peptide of aπγ of Claims 1 6 or a nucleic acid encoding the peptide of any of Claims 1 6 to said individual in an amount sufficient to reduce the activity of Tat or induce an immune response against the Tat protein.
10. An isolated or purified complex comprising the peptide of any of Claims 1 6 and an HIV LTR.
1 1. A compound having the structure:
Figure imgf000060_0001
wherein:
A is absent or an ammo acid; B is an ammo acid;
D is absent or an ammo acid; E is absent or an ammo acid; G is an ammo acid; J is an ammo acid; M is an ammo acid having a first reactive group and a second reactive group thereon, wherein said first reactive group and said second reactive group are capable of selectively being attached to an ammo acid or peptide;
R is an ammo acid having a first reactive group and a second reactive group thereon, wherein said first reactive group and said second reactive group are capable of selectively being attached to an ammo acid or peptide;
s is 0 when D is absent or is 1 20 when D is present; and t is 0 when E is absent or is 1 20 when E is present.
12. The compound of Claim 1 1 , wherein D and E are absent.
13. The compound of Claim 12, wherein A comprises an ammo acid having a functional group thereon which permits attachment to a solid support.
14. The compound of Claim 13, wherein A is senne.
15. The compound of Claim 14, wherein said senne is Ser(Trt).
16. The compound of Claim 15, wherein B is an ammo acid having functional groups which permit two ammo acids to be attached thereto.
17 The compound of Claim 16, wherein B is lysiπe
18. The compound of Claim 17, wherein G and J are lysmes.
19 The compound of Claim 18, wherein the ε ammo groups of G and J are protected with t Boc. WO 00/78969 PCT/USOO/l 0119
20. The compound of Claim 19, further comprising a peptide linked to said first reactive groups of M and R.
21. The compound of Claim 20, wherein said peptide comprises at least one antigenic site.
22. The compound of Claim 21 , further comprising a peptide linked to said second reactive groups of M and R.
23. The compound of Claim 22, wherein the peptide linked to said second reactive groups of M and R comprise at least one antigenic site.
24. The compound of Claim 18, wherein M and R are cγsteines and wherein the ammo and SH groups of said cγsteines are protected with blocking groups which permit ammo acids or peptides to be selectively attached thereto.
25. The compound of Claim 24, wherein the ammo groups of said cystemes are protected with Fmoc.
26. The compound of Claim 25, wherein the SH groups of said cystemes are protected with Trt.
27. The compound of Claim 23, wherein M and R are cystemes, said first reactive groups are the ammo groups of said cystemes, and said second reactive groups are the thiol groups of said cγsteines.
28. The compound of Claim 27, wherein the peptide attached to the ammo group of M is the same as the peptide attached to the ammo group of R.
29. The compound of Claim 27, wherein the peptide attached to the ammo group of M is different than the peptide attached to the ammo group of R.
30. The compound of Claim 27, wherein the peptide attached to the thiol group of M is the same as the peptide attached to the thiol group of R.
31. The compound of Claim 27, wherein the peptide attached to the thiol group of M is different than the peptide attached to the thiol group of R
32. The compound of Claim 21, wherein n is 1 5, s is 1-10, and t is 1-10.
33. The compound of Claim 21 , wherein n is 1 , s is 0, and t is 0.
34. A compound having the structure:
W
Ds G M
• X
B„ Y
E, J z
wherein:
A is absent or an ammo acid; B is an ammo acid; D is absent or an amino acid; E is absent or an amino acid; G is an amino acid; J is an amino acid; M is an amino acid having two reactive groups thereon which can be attached to an amino acid;
R is an amino acid having two reactive groups thereon which can be attached to an amino acid; W is an amino acid having a first reactive group and a second reactive group thereon, wherein said first reactive group and said second reactive group are capable of selectivelγ being attached to an amino acid or peptide; X is an amino acid having a first reactive group and a second reactive group thereon, wherein said first reactive group and said second reactive group are capable of selectivelγ being attached to an amino acid or peptide;
Y is an amino acid having a first reactive group and a second reactive group thereon, wherein said first reactive group and said second reactive group are capable of selectivelγ being attached to an amino acid or peptide;
Z is an amino acid having a first reactive group and a second reactive group thereon, wherein said first reactive group and said second reactive group are capable of selectivelγ being attached to an amino acid or peptide; n is 1 -10; s is 0 when D is absent or is 1-20 when D is present; and t is 0 when E is absent or is 1-20 when E is present.
35. A compound having the structure:
Trt
Cys — Fmoc
Lys / ^ t-Boc
Ser — Lys Trt
Trt \ Cys — Fmoc
Lγs t-Boc
36. A compound having the structure:
SH
Cγs — P,
Lγs ' / ^ NH2
Ser — Lγs SH OH \ Cγs — P2
Lys ^
\
NH2 wherein P, and P2 are peptides.
37. The compound of Claim 36, wherein P, and P2 comprise at least one antigenic site.
38. The compound of Claim 37, wherein P, and P2 are selected from the group consisting of malarial and HIV peptides.
39. The compound of Claim 38, wherein at least one of said malarial peptides comprises a peptide selecting from the group consisting of SEQ ID NOs: 81-85.
40. The compound of Claim 38, wherein at least one of said HIV peptides comprises a peptide selected from the group consisting of SEQ ID NOS.: 1 21.
41. A compound having the structure:
S — P,
Cγs — P,
Lγs x / ^ H2
Ser - - Lγs S — P4
I
I
OH \ Cγs — P2
Lγs ^
\
NH2 wherein P,, P2, P3 and P4 are peptides.
42. The compound of Claim 41 , wherein P,, P2, P3 and P4 comprise at least one antigenic site.
43. The compound of Claim 42, wherein P,, P2, P3 and P4 are selected from the group consisting of malarial and HIV peptides.
44. The compound of Claim 43, wherein at least one of said malarial peptides comprises a peptide selected from the group consisting of SEQ ID NOs: 81-85.
45. The compound of Claim 43, wherein at least one of said HIV peptides comprises a peptide selected from the group consisting of SEQ ID NOs: 1-21.
46. A compound having the structure:
Cys — B
Lys /
Ser — Lys A Cys — B
Lys
wherein A is a blocking group which can be removed without removing B or C, B is a blocking group which can be removed without removing A or C, and C is a blocking group which can be removed without removing A or B.
47. HIV-1 -Tat-MPC having the structure: S— CH2C0NH— Tat2140
Cy Is — Tat53-68+Tat9-20
Lys / ^ NH2
H2N - Ser — Lys S— CH2C0NH— Tat2140
OH \ Cys — Tat53-68+Tat9-20
Lys ^
\ NH2
48. A method of making a multiple peptide conjugate comprising the steps of: obtaining a core template having at least two branches, said at least two branches being separated from one another by at least four carbons, wherein each of said branches have a first reactive group and a second reactive group thereon; sγnthesizing a peptide on said first reactive groups on said at least two branches; and attaching a pre sγπthesized peptide on said second reactive groups on said at least two branches.
49. The method of Claim 48, wherein one or more of the ammo acids used in said synthesizing step are blocked with agents which reduce steric hindrance.
50. The method of Claim 49, wherein said agents which reduce steric hindrance comprise N [2-hydroxγ 4-methoxγbenzγl] groups.
51. The method of Claim 49, further comprising blocking the thiol groups of anγ cγsteme residues used in said synthesizing step or present in said pre-sγnthesized peptide with an agent which prevents said thiol groups from being reactive.
52. The method of Claim 51 , wherein said agent which prevents said thiol groups from being reactive is a tert-butyl group.
53. The method of Claim 52, further comprising removing said agent which prvents said thiol groups from being reactive after said attaching step.
54. The method of Claim 49, further comprising removing said agents which reduce steric hindrance after said attaching step.
55. The method of Claim 48, wherein said synthesizing step is performed in the presence of DMSO.
56. The method of Claim 48, further comprising removing a first blocking group from said first reactive group prior to attaching said ammo acid or peptide to said first reactive group under conditions in which a second blocking group is not removed from said second reactive group.
57. The method of Claim 56, wherein said first reactive group comprises the ammo group of cysteine and said second reactive group comprises the thiol group of cysteine.
58. The method of Claim 57, wherein said first blocking group is removable with base and said second blocking group is removable with acid.
59. The method of Claim 58, wherein said first blocking group comprises Fmoc and said second blocking group comprises Trt
60. The method of Claim 48, wherein said core template is selected from the group consisting of the compound of Claim 1, the compound of Claim 24, and the compound of Claim 25.
61. A compound of anγ of Claims 11-47 for use in inducing an immune response or for use as an intermediate in making a compound for use in inducing an immune response.
62. A method of inducing an immune response in an individual comprising the step of administering a compound of anγ of Claims 11 -47 or a compound made from an intermediate that is a compound of anγ of Claims 1 1
47 to said individual in a sufficient amount to induce an immune response.
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WO2002020555A2 (en) * 2000-09-04 2002-03-14 Bionor Immuno As Hiv peptides from tat, rev and wef conserved regions and their application as e.g.vaccine components
EP1279404A1 (en) * 2001-07-26 2003-01-29 Istituto Superiore di Sanità Use of HIV-1 tat, fragments or derivatives thereof, to target or to activate antigen-presenting cells, to deliver cargo molecules for vaccination or to treat other diseases
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CN1318091C (en) * 2001-07-26 2007-05-30 高等健康研究院 Use of biologically active HIV-1 Tat, fragments or derivatives thereof for preventing or therapeutic vaccination and/or treating other diseases
US7811573B2 (en) 2001-07-26 2010-10-12 Istituto Superiore Di Sanita' Compositions of antigens bound to HIV-1 Tat, fragments or derivatives thereof
EP1578461A2 (en) * 2002-12-18 2005-09-28 University of Maryland Biotechnology Institute Office of Research Admin/Tech. Dev. Vaccines against hiv-1 tat protein to generate neutralizing antibodies
EP1578461A4 (en) * 2002-12-18 2008-09-03 Univ Maryland Biotech Inst Vaccines against hiv-1 tat protein to generate neutralizing antibodies
US7927580B2 (en) 2004-03-16 2011-04-19 Nanirx, Inc. Tat-based immunomodulatory compositions and methods of their discovery and use
US7943140B2 (en) * 2006-08-14 2011-05-17 Thymon, Llc Compositions and methods for the treatment and prophylaxis of multiple strains and subtypes of HIV-1
WO2009139004A3 (en) * 2008-05-14 2010-01-21 Jawaharlal Nehru Centre For Advanced Scientific Research Tat dna sequences, gene constructs, vaccine and processes thereof
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US9439922B2 (en) * 2008-05-14 2016-09-13 Jawaharlal Nehru Centre For Advanced Scientific Research Tat DNA sequences, gene constructs, vaccine and processes thereof
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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