WO1999038887A1 - Peptides de vih-1 divergents - Google Patents

Peptides de vih-1 divergents Download PDF

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Publication number
WO1999038887A1
WO1999038887A1 PCT/US1999/001726 US9901726W WO9938887A1 WO 1999038887 A1 WO1999038887 A1 WO 1999038887A1 US 9901726 W US9901726 W US 9901726W WO 9938887 A1 WO9938887 A1 WO 9938887A1
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Prior art keywords
hiv
peptide
sequence
amino acids
group
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PCT/US1999/001726
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English (en)
Inventor
Mohammed A. Chowdhury
David Bernstein
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Universal Healthwatch, Inc.
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Application filed by Universal Healthwatch, Inc. filed Critical Universal Healthwatch, Inc.
Priority to AU23453/99A priority Critical patent/AU2345399A/en
Priority to AU45463/99A priority patent/AU4546399A/en
Priority to PCT/US1999/012446 priority patent/WO1999062945A2/fr
Publication of WO1999038887A1 publication Critical patent/WO1999038887A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to diagnostic reagents, test kits, vaccines and therapeutics for the detection, prevention and treatment of acquired immune deficiency syndrome (AIDS) caused by human immunodeficiency virus infection. More specifically, the invention relates to peptide sequences that diverge from known sequences and that cross-react with a wide range of HIV strains.
  • AIDS acquired immune deficiency syndrome
  • HIV Human Immunodeficiency Virus
  • HTLV-HI Human Immunodeficiency Virus
  • AIDS Acquired Immunodeficiency Syndrome
  • Various HIV components have been analyzed for their ability to react with sera obtained from HIV infected patients. Serological methods such as latex agglutination, ELISA, IFA have been developed for diagnosis and screening of HIV infection from blood. The first diagnostic and screening tests utilizing whole viral ly sates resulted in many nonspecific reactions, requiring all positive test results to be further confirmed by other methods such as Western Blot analysis.
  • the HIN envelope protein particularly the gp41 immunodominant region, has long been recognized as playing a key antigenic role for detection of HIN infection (Gnann et al. J. Infect Dis, 1987; 156:261-267 and J. Virol 1987; 61:2639-2641).
  • the immunodominant region contains a heptapeptide loop as a major epitope (Wang et al. Proc. ⁇ atl. Acad. Sci. USA, 1986, 83:6159-6163, Dopel et al. J. Virol Methods 1990, 28:189-98, Bugge et al. J. Virol, 1990, 64:4123-9. Oldstone et al. J.
  • Virol 65:1727-34 (1991) reported that the conformation of the heptapeptide loop CSGKLIC at amino acid positions 603-609 of classical HIV-1 (i.e. HIV strains known at that time) has an intramolecular disulfide bond essential for loop formation.
  • Aleanzi et al. J Mol. Recog. 1996 9:631-638 studied a series of sequences that partially overlap in the region vicinal to the immunodominant epitope, and concluded that 15 mer peptides were more reactive than a 23mer peptide. The authors attributed the cause of this phenomenon to unfavorable conformation of the larger peptide.
  • synthetic peptides mimic mainly sequential epitopes, conformational preferences in fluid or solid phase (i.e.
  • the gp 36 protein is analogous to the gp41 Env fragment of HIV-1 and shares some amino acid sequence similarity. Synthetic peptides corresponding to reported sequences of HIV-2 gp36 membrane containing this heptapeptide loop were reactive with all strains of HIV-2 but failed to react with strains of HIV-1.
  • Broliden employed a 20 amino acid peptide (sequence 594- 613) derived from an HIV-1 gp41 sequence, and a second peptide derived from an HIV-2 gp36 sequence as the capture antigen (antibody binder).
  • MVP5180 displays Env proteins that reacted "faintly” or moderately with sera from German, Ivoirian, and Malawian patients that were infected with HIV-1.
  • the Env gene of MVP-5180 showed 53% similarity to an HIV- 1 consensus sequence and 49% similarity to an HIV-2 consensus sequence.
  • This peptide was added to existing HIV-1 antigens in a test for the purpose of detecting group O strains that might be missed in the ELISA method and was successful in identifying antibody to one of the group O strains that was nonreactive in the absence of this peptide.
  • Virol Methods 67:85-91 reported on an ELISA using a 25mer synthetic peptide derived from the gp41 of MVP5180 strain and having the amino acid sequence ALETLIQNQQRLNLWGCKGKLICYT.
  • the ELISA employing this peptide failed to detect 10/111 confirmed HIV-1 (non Group O) positive samples but detected 42/42 group O positive sera. All of the undetected HIV-1 non group O samples were reactive by a third generation ELISA using gp41 and gpl20 recombinant proteins of HIV-1 group M. (Enzygnost HIV- 1/2, Behringwerke, Marburg, Germany. Twenty five anti-HIV negative samples were included as controls. The authors reported that there is no (single group O) consensus sequence antigen available to be added to the current screening assays.
  • HIV-1 surface envelope (env) glycoprotein coding sequences vary with unusually high ratio of mutations, indicative of strong selection for viral surface change, and numerous small in-frame nucleotide deletions and insertions.
  • the highly variable and continuously evolving nature of HIV-1 within individuals contributes to the high level of genetic diversity observed between viral strains identified worldwide.
  • All serological assays require an appropriate choice of an antigen that is both sensitive in reactivity with antibodies from all kinds of HIV infections and is so specific that it does not react with sera from other non HIV infections or with any compounds that might interfere with an accurate test result such as autoimmune phenomena, hemolyzed sera, icteric sera, etc.
  • the difficulties associated with selecting an appropriate HIV antigen(s) that does not cross react with antibodies of other disease states are compounded when one recognizes that the tertiary structure of the antigen can undergo conformational changes when the peptides are free, bound to solid phase, or conjugated to other materials resulting in loss or enhancement of antibody binding to specific epitopes that may become hidden or exposed as a result of folding.
  • Klasse et al. Mol. Immunol. (1991) 28:613-22 reported that amino acid substitutions as well as deletions in the sequence 589-596 (AVERYLKD) can abrogate the antigenicity of the immunodominant domain.
  • the authors state that epitopes may be exposed on the surface of a native protein, or exposed only after cleavage or unfolding of the protein. Thus a decrease in antigenicity by a deletion or substitution of a residue does not demonstrate that this residue is part of the antigenic structure.
  • the authors also state that antigenic structures of larger peptides are conformationally dependent.
  • HIV-1 and HIV-2 we have two distinct types of HIV believed to have arisen through mutations of SIV and resulted in significantly divergent strains, in which specific peptide sequences of gp41 and gp36 are different and result in unique host immune responses to HIV-1 and HIV-2 infection.
  • a mutant strain of HIV-2 arise as divergent from classical HIV-2 as the group ODs are from classical HIV-1
  • using an extended peptide containing the heptapeptide loop and sequences similar to the divergent HIV-2 strain could confer broader reactivity in immunoassays to all strains of HIV-2 and may also be useful as a vaccine to produce some protective antibodies against strains of HIV-2.
  • immunoassay tests for HIV infection must contain an antigen that recognizes antibodies from individuals that have been exposed to classical "M” subtype of HIV-1, an antigen that recognizes the newer “O” outlier subtype of HIV-1 and an antigen that recognizes type HIV-2.
  • HIV Env protein diversity affects not only diagnostic testing for exposure to HIV but also hinders development of therapies and prophylatics to prevent HIV infection.
  • One basic problem in this context is that the strategy of administering one or more antigens to stimulate an immune response, is easily sidestepped by the virus mutating new forms of Env protein.
  • no reliable vaccine or prophylatic peptide based on an HIV Env protein sequence has been developed.
  • the difficulty in obtaining an effective vaccine and prophylatic may stem from the s.ame problem that affects diagnostic testing for HIV.
  • individual antigens derived from consensus sequences from an examination of sequenced HIV Env protein genes, fail to work as expected and new strains are continually evolving in nature.
  • the antigen diversity problem is even seen within an individual patient during progression of HIV infection. At the early stages of infection, many antibodies appear to be directed to epitopes of the Env gp 41 protein. As the disease progresses, more antibodies appear that are directed against other HIV proteins.
  • Embodiments of invention reduce or eliminate the problems of (i) insufficient reactivity of peptide antigen with antibody made against HIV, (ii) insufficient stimulation of an immune treatment to protect against infection and to cure infection, (iii) HIV mutation and variation, which allows HIV to escape detection and treatment, (iv) insufficient reactivity, thus allowing detection of HIV infection at an earlier stage, and (v) awkward production requirements necessitated by using a lysate or a combination of two or more peptides to obtain suitable reactivity to a wide variety of HIV strains.
  • the advantage of a using a defined peptide sequence for antibody detection and for vaccine allows for simpler production than required for lysate or recombinant antigen production.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (i) a mutant peptide that reacts with blood samples from individuals infected with HIV-1 group O virus and from individuals infected with HIV-1 group M virus, said mutant peptide having a sequence selected from the sequences depicted in Figure 1; and (ii) a medically acceptable powder or liquid carrier, wherein said pharmaceutical composition stimulates the formation of antibodies against HIV-1 virus upon administration of said pharmaceutical composition to a patient.
  • the mutant peptide is complexed with another peptide portion to form a fusion protein.
  • the pharmaceutical composition includes an additional molecule that separately stimulates cytotoxic T lymphocytes.
  • such additional molecule is combined with a peptide antigen depicted in Figure 1 to form a fusion peptide.
  • the invention reduces or eliminates the problems of the prior art by providing a paradigm and method to obtain peptides that have very wide immunological specificity.
  • the inventors in studying alternative peptide sequences to cover the range of HIV strains, realized that, contrary to common wisdom, they could solve the specificity problem if they chose a peptide sequence to differ from the accepted consensus sequences. More particularly, they realized that they could use a broader specificity peptide if they chose a sequence that differs from the accepted sequence by at least 25 % .
  • a preferred embodiment of this idea is a 36 amino acid sequence given in SEQ ID NO: 1.
  • the peptide must be comprised of at least 26 amino acids and not more than 100 amino acids, preferably 26 amino acids to 36 amino acids.
  • the particular amino acid sequence of the peptide will influence it's tertiary structure and the resultant conformation will effect the presentation of epitopes to specific antibodies. Therefore amino acid substitutions are chosen that correspond most closely with sequence derived from HIV-1 group O strains than classical group M HIV-1 strains.
  • the invention provides a peptide based on amino acid sequences published in database of Los Alamos National Laboratory derived from a segment of immunodominant gp41 region of a HIV-0 strain containing a heptapeptide loop and amino acid sequences extending from the loop.
  • the peptide comprises at least 26 amino acids and contains, as a minimum, a core sequence as shown in Figure 1.
  • A Alanine
  • R Arginine
  • N Asparagine
  • D Aspartic acid
  • C Cysteine
  • Q Glutamine
  • E Glutamic acid
  • G Glycine
  • H Histidine
  • W Tryptophan
  • Y Tyrosine
  • V Valine.
  • amino acid substitutions in the peptide can effect the presentation of these epitopes for antibody recognition. What is evident is that a peptide derived from sequences of group O strains and containing at least 25 % amino acid substitutions from the classical M strains of HIV-1 becomes reactive to sera derived from group M and group O HIV-1 infected patients.
  • a peptide is chosen, according to a preferred embodiment of the invention, by selecting a sequence which is more closely related to sequences of the HIV-1 O strains than to the most likely sequence of M strains. Such "divergence" can be defined numerically from the % of amino acid substitutions compared to the most likely sequence of HIV-1 reported in the Los Alamos National Laboratory Data Base, Human Retroviruses and AIDS 1996, Los Alamos, New Mexico.
  • the % diversity of a given sequence is defined as (the number of amino acid substitutions from the M sequence appearing in the selected peptide) % by (the total number of amino acids in the selected peptide).
  • the selected peptide has 25% diversity.
  • the selected peptide "differs" from the M sequence by 25% .
  • the immunodominant region from aa 594-609 is comprised of sixteen amino acids of which substitutions can occur at positions 597, 599, 605, 606, or 608 or up to 31 % of the most likely group M sequences.
  • substitutions can occur at positions 597, 599, 605, 606, or 608 or up to 31 % of the most likely group M sequences.
  • up to 10 substitutions can occur in the 25 amino acid residues or up to 40% of the most likely group M sequences.
  • the immunodominant domain toward the Carboxy terminal end representing > 25 % increase in diversity.
  • up to 4 substitutions can occur in the 8 amino acid residues or up to 50% of the most likely group M sequences are substituted.
  • the tertiary structure of the molecule is transformed and the immunodominant domain is expressed in a more universal form comprised of one or more epitopes that are capable of binding antibodies arising from the immune response from all types of HIV-1 infection.
  • these peptides are useful as vaccines for protection against HIV-1 infection or in the case of the HIV-2 peptide, as a vaccine for protection against HIV-2 infection.
  • the peptides of the instant invention for creating a universal HIV-1 target analyte are selected to have a sequence from any of the sequences presented in Figure 2.
  • the peptide should contain the immunodominant region from positions 594-609 (shown in bold), and the length of the peptide should be at least 26 amino acids.
  • the criteria for selection of the extended sequences is that the extended sequences should be equal to or greater in diversity than the immunodominant region compared to the classical M subtypes as shown by Los Alamos National Laboratory Data Base Human Retroviruses and AIDS (1996).
  • the immunodominant region extends from positions 594-609 and consists of 16 amino acids of which 4-10 can be substituted.
  • the C terminal end adjacent to the immunodominant region from 569-593 contains 25 amino acids of which up to 7-13 can be substituted.
  • additional amino acids are added to the termini of a peptide of the present invention to provide for ease of linking peptides one to another, for coupling to a carrier, support or a larger peptide, for reasons discussed herein, or for modifying the physical or chemical properties of the peptide, and the like.
  • Suitable amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, and the like, can be introduced at the C- or N-terminus of the peptide.
  • the peptide of the present invention can differ from the natural sequence by being modified by terminal-NH sub 2 acylation, e.g., acetylation, or thioglycolic acid amidation, terminal-carboxy amidation, e.g., ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule, thereby providing a linker function.
  • terminal-NH sub 2 acylation e.g., acetylation, or thioglycolic acid amidation
  • terminal-carboxy amidation e.g., ammonia, methylamine, etc.
  • these modifications may provide sites for linking to a support or other molecule, thereby providing a linker function.
  • the peptides of the present invention or analogs or homologs thereof may be further modified beyond the sequence considerations given above, as necessary to provide certain other desired attributes, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially the biological activity of the unmodified peptide.
  • the peptides can be modified by extending, decreasing or substituting amino acids in the peptide sequence by, for ex.ample, the addition or deletion of suitable amino acids on either the amino terminal or carboxy terminal end, or both, of peptides derived from the sequences disclosed herein.
  • suitable amino acids are shown in Figures 1 and 2
  • further conservative substitutions are possible and sometimes desirable.
  • substitutions is meant replacing an amino acid residue with another that is biologically and/or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another.
  • the substitutions include combinations such as Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • the portion of the sequence that is intended to mimic substantially a peptide from HIV in the body will not differ by more than about 20% from a sequence given in Figure 2, except where additional amino acids may be added at either terminus for the purpose of modifying the physical or chemical properties of the peptide for, for example, ease of linking or coupling, and the like.
  • additional amino acids may be added at either terminus for the purpose of modifying the physical or chemical properties of the peptide for, for example, ease of linking or coupling, and the like.
  • sequences are highly variable, it may be desirable to vary one or more particular amino acids to mimic more effectively differing epitopes of different HIV strains.
  • the contributions made by the side chains of the residues can be probed via a systematic replacement of individual residues with a suitable amino acid, such as Gly or Ala.
  • a suitable amino acid such as Gly or Ala.
  • Systematic methods for determining which residues of a linear amino acid sequence of a peptide are required for binding to a specific MHC protein, (or other component of the immune system) are known. See, for instance, Allen et al., Nature, 327, 713-717; Sette et al., Nature, 328, 395-399; Takahashi et al., J. Exp. Med., 170, 2023-2035 (1989); and Maryanski et al., Cell, 60, 63-72 (1990).
  • Peptides that tolerate multiple amino acid substitutions generally incorporate small, relatively neutral molecules, e.g., Ala, Gly, Pro, or similar residues.
  • the number and types of residues that can be substituted, added or subtracted will depend on the spacing necessary between the essential epitopic points and certain conformational and functional attributes that are sought.
  • types of residues it is intended, e.g., to distinguish between hydrophobic and hydrophilic residues, among other attributes. If desired, increased binding affinity of peptide analogs to can also be achieved by such alterations.
  • any spacer substitutions, additions or deletions between epitopic and/or conformationally important residues will employ amino acids or moieties chosen to avoid stearic and charge interference that might disrupt intramolecular binding of the peptides and intermolecular binding of peptides to other molecules.
  • D-amino acid-containing peptides Peptides that tolerate multiple substitutions while retaining the desired immunological activity also may be synthesized as D-amino acid-containing peptides.
  • Such peptides may be synthesized as "inverso" or “retro-inverso” forms, that is, by replacing L-amino acids of a sequence with D-amino acids, or by reversing the sequence of the amino acids and replacing one or more L-amino acids with D-amino acids.
  • the D-peptides are substantially more resistant to peptidases, and therefore are more stable in serum and tissues compared to their L-peptide counterparts, the stability of D-peptides under physiological conditions may more than compensate for a difference in affinity compared to the corresponding L-peptide.
  • L-amino acid-containing peptides L-amino acid-containing peptides
  • 15 with or without substitutions can be capped with a D-amino acid to inhibit exopeptidase destruction of the antigenic peptide.
  • modifications including conservative modifications, are best carried out by changing a DNA sequence that codes for a recombinant form of the peptide.
  • the following is a discussion based upon changing the .amino acids of a protein to create an equivalent, or even an improved, second-generation molecule.
  • the amino acid changes may be achieved by changing the codons of the DNA sequence, according to the following codon table:
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art as cited in U.S. No. 5,703,057 (citing Kyte and Doolittle, 1982, incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant peptide which in turn defines the interaction of the peptide with other molecules, for example, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+ 1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4-5).
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 +- 1); glutamate (+3.0 +- 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2) glycine (0); threonine (-0.4); proline (-0.5 +- 1); alanine (-0.5); histidine (-0.5) cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8) tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent peptide.
  • substitution of amino acids whose hydrophilicity values are within +- 2 is preferred, those which are within +- 1 are particularly preferred, and those within +- 0.5 are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional
  • the technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
  • the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications.
  • the technique typically employs a phage vector which exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art.
  • Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.
  • site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide.
  • An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically.
  • This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • E. coli polymerase I Klenow fragment DNA polymerizing enzymes
  • a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation.
  • This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and
  • 19 clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
  • sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained.
  • recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • a peptide may be modified to enhance substantially its ability to induce cytotoxic T lymphocyte stimulating ("CTL") activity, such that the modified peptide analog has CTL activity greater than a peptide of the wild-type sequence.
  • CTL cytotoxic T lymphocyte stimulating
  • the peptides of the invention can be combined via linkage to form polymers (multimers), or can be formulated in a composition without linkage, as an admixture. Where the same peptide is linked to itself, thereby forming a homopolymer, a plurality of repeating epitopic units are presented. When the peptides differ, heteropolymers with repeating units are provided, forming a cocktail of, for example, epitopes specific to HIV-1 as well as HIV-2 types, different epitopes to the same protein or gene region within a type, different epitopes to different proteins or gene regions within a type, different HIV restriction specificities, and/or a peptide that contains T helper epitopes.
  • noncovalent linkages capable of forming intermolecular and intrastructural bonds are included.
  • Linkages for homo- or hetero-polymers or for coupling to carriers can be provided in a variety of ways.
  • cysteine residues can be added at both the amino- and carboxy-termini, where the peptides are covalently bonded via controlled oxidation of the cysteine residues.
  • hetero-bifunctional agents that generate a disulfide link at one functional group end and a peptide link at the other, including N-succidimidyl-3-(2-pyridyl-dithio) propionate (SPDP).
  • SPDP N-succidimidyl-3-(2-pyridyl-dithio) propionate
  • the carboxyl groups can be activated by combining them with succinimide or l-hydroxy-2-nitro-4-sulfonic acid, sodium salt.
  • a particularly preferred coupling agent is succinimidyl-4-(n-maleimidomethyl) cyclohexane-1-carboxylate (SMCC).
  • the peptides of the invention can be combined or coupled with other suitable peptides that present HIV T-helper cell epitopes, i.e., epitopes that stimulate T cells that cooperate in the induction of cytotoxic T cells to HIV.
  • the T-helper cells can be either the T-helper 1 or T-helper 2 phenotype, for example.
  • the peptides of the invention can be prepared using any suitable means. Because of their relatively short size (generally, less than 100 amino acids, preferably less than 50 and more preferably less than 40), the peptides can be synthesized in solution or on a solid support in accordance with conventional peptide synthesis techniques. Various automatic synthesizers are commercially available (for example, from Applied Biosystems) and can be used in accordance with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis (2d. ed., Pierce Chemical Co., 1984); Tam et al., J. Am. Chem.
  • suitable recombinant DNA technology may be employed for the preparation of the peptides of the present invention, wherein a nucleotide sequence that encodes a peptide of interest is inserted into an expression vector, transformed or transfected into a suitable host cell and cultivated under conditions suitable for
  • recombinant DNA-derived proteins or peptides which comprise one or more peptide sequences of the invention, can be used to prepare the HIV cytotoxic T cell epitopes identified herein or identified using the methods disclosed herein.
  • a recombinant peptide of the present invention is prepared in which the amino acid sequence is altered so as to present more effectively epitopes of peptide regions described herein to stimulate a cytotoxic T lymphocyte response.
  • a polypeptide is used that incorporates several T cell epitopes into a single polypeptide.
  • coding sequence for peptides of the length contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al., J. Am. Chem. Soc, 103, 3185 (1981), modification can be made simply by substituting the appropriate base(s) for those encoding the native peptide sequence.
  • the coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the desired fusion protein. A number of such vectors and suitable host systems are now available.
  • the coding sequence will be provided with operably linked start and stop codons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in a suitable cellular host.
  • promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the desired coding sequence.
  • the resulting expression vectors are transformed into suitable bacterial hosts.
  • Yeast or mammalian cell hosts may also be used, employing suitable vectors and control sequences.
  • Another aspect of the present invention is directed to a method of provoking an immune response to an HIV Env gp 41 epitope, comprising contacting a suitable
  • cytotoxic T lymphocyte with an immune response provoking effective amount of peptide having a stimulatory sequence selected from Figure 2.
  • All of the variations recited hereinabove regarding the molecule of the present invention and the polypeptide that such a molecule includes may be used in the context of the method of provoking an immune response.
  • a preferred preparation of the HIV gp 41 epitope, in whatever form, or, for that matter, of the in vitro stimulated CTL's intended to be reintroduced to a host, is as a pharmaceutical composition.
  • a pharmaceutical composition of the present invention is comprised of a molecule that includes a polypeptide having substantial homology with an epitope from one of the peptide sequences shown in Figure 1 or 2, or the peptide itself, and a pharmaceutically acceptable carrier.
  • Suitable methods of administering a compound to a patient for the treatment or prophylaxis of HIV infection are available. Although more than one route can be used to administer a particular compound, a particular route can provide a more immediate and more effective reaction than another route. Accordingly, the described methods provided herein are merely exemplary and are in no way limiting.
  • a peptide of the present invention as described above will be administered in a pharmaceutical composition to an individual already infected with HIV or at high risk of HIV infection. Those in the incubation phase or the acute phase of infection can be treated with the immunogenic peptides separately or in conjunction with other treatments, as appropriate.
  • compositions are administered to a patient in an amount sufficient to elicit an effective cytotoxic T lymphocyte response to HIV and to cure or at least partially arrest its symptoms and/or complications.
  • Amount adequate to accomplish this is defined as a “therapeutically or prophylactically effective dose” which is also an “immune response provoking amount.” Amounts effective for a therapeutic or prophylactic use will depend on, e.g., the stage and severity of the disease the age, weight, and general state of health of the patient, and the judgment of the prescribing physician. The size of the dose will also be determined by the peptide composition, method of administration, timing and frequency of
  • Suitable doses and dosage regimens can be determined by conventional range-finding techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages that are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached.
  • the present inventive method typically will involve the administration of about 0.1 mg to about 50 mg of one or more of the compounds described above per kg body weight of the individual. For a 70 kg patient, dosages of from about 10 mg to about 100 mg of peptide would be more commonly used, followed by booster dosages from about 0.01 mg to about 1 mg of peptide over weeks to months, depending on a patient's CTL response.
  • peptides and compositions of the present invention may generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, in view of the minimization of extraneous substances and the relative nontoxic nature of the peptides, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions.
  • compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • the pharmaceutical formulations should provide a quantity of cytotoxic T-lymphocyte stimulatory peptides of the invention sufficient to effectively treat the patient.
  • administration should begin at the first sign of HIV infection or shortly after diagnosis in cases of acute infection, and continue until at least symptoms are substantially abated and for a period thereafter. In well established and chronic cases, loading doses followed by maintenance or booster doses may be required.
  • compositions for therapeutic treatment are intended for parenteral, topical, oral or local administration and generally comprise a pharmaceutically acceptable carrier and an amount of the active ingredient sufficient to reverse or prevent the bad effects of HIV infection.
  • the carrier may be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the compound, and by the route of administration.
  • Examples of pharmaceutically acceptable acid addition salts for use in the present inventive pharmaceutical composition include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, gly colic, gluconic, succinic, p-toluenesulphonic acids, and arylsulphonic, for example.
  • mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids
  • organic acids such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, gly colic, gluconic, succinic, p-toluenesulphonic acids, and arylsulphonic, for example.
  • pharmaceutically acceptable excipients described herein for example, vehicles, adjuvants, carriers or diluents, are well-known to those who are skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one that is chemically inert to the active compounds and one that has no detrimental side effects or toxicity under the conditions of use.
  • excipient will be determined in part by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention.
  • compositions for parenteral administration that comprise a solution of the cytotoxic T-lymphocyte stimulatory peptides dissolved or suspended in an acceptable carrier suitable for parenteral administration, including aqueous and non-aqueous, isotonic sterile injection solutions.
  • Such solutions can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the compound may be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene gly col or polyethylene glycol, dimethylsulf oxide, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, such as poly (ethylenegly col) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, mefhylcellulose, hydroxypropylmethylcellulose, or carboxymethyl
  • Oils useful in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils useful in such formulations include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
  • Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts
  • suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, cationic
  • alkyl- beta -aminopropionates and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.
  • the parenteral formulations typically will contain from about 0.5 to about 25% by weight of the active ingredient in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene gly col.
  • HLB hydrophile-lipophile balance
  • parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
  • sterile liquid excipient for example, water
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Topical formulations including those that are useful for transdermal drug release, are well-known to those of skill in the art and are suitable in the context of the present invention for application to skin.
  • Formulations suitable for oral administration equire extra considerations considering the peptidyl nature of the epitopes and the likely breakdown thereof if such compounds are administered orally without protecting them from the digestive secretions of the gastrointestinal tract.
  • Such a formulation can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions.
  • Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically
  • Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch.
  • Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients.
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
  • a flavor usually sucrose and acacia or tragacanth
  • pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
  • the molecules and/or peptides of the present invention can be made into aerosol formulations to be administered via inhalation.
  • the cytotoxic T-lymphocyte stimulatory peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are 0.01 %-20% by weight, preferably 1 %-10% .
  • the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
  • esters or partial esters of fatty acids containing from 6 to 22 carbon atoms such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • the surfactant may constitute 0.1 %-20% by weight of the composition, preferably 0.25-5% .
  • the balance of the composition is ordinarily propellant.
  • a carrier can also be included as desired, e.g., lecithin for intranasal delivery.
  • aerosol formulations can be placed into acceptable pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for
  • non-pressured preparations such as in a nebulizer or an atomizer.
  • Such spray formulations may be used to spray mucosa.
  • the compounds and polymers useful in the present inventive methods may be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases.
  • bases such as emulsifying bases or water-soluble bases.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.
  • Lipids have been identified that are capable of priming CTL in vivo against viral antigens, e.g., tripalmitoyl-S- glycerylcysteinly-seryl-serine (P sub 3 CSS), which can effectively prime virus specific cytotoxic T lymphocytes when covalently attached to an appropriate peptide. See, Deres et al., Nature, 342, 561-564 (1989).
  • Peptides of the present invention can be coupled to P sub 3 CSS, for example and the lipopeptide administered to an individual to specifically prime a cytotoxic T lymphocyte response to HIV.
  • the concentration of cytotoxic T-lymphocyte stimulatory peptides of the present invention in the pharmaceutical formulations can vary widely, i.e., from less than about 1 % , usually at or at least about 10% to as much as 20 to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 100 mg of peptide.
  • Actual methods for preparing parenterally administrable compounds will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science (17th ed., Mack Publishing Company, Easton, Pa., 1985).
  • the compounds of the present inventive method may be formulated as inclusion complexes, such as cyclodextrin inclusion
  • Liposomes serve to target the peptides to a particular tissue, such as lymphoid tissue or HIV-infected cells. Liposomes can also be used to increase the half-life of the peptide composition. Liposomes useful in the present invention include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
  • the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, e.g., a receptor, prevalent among lymphoid cells, such as monoclonal antibodies which bind to the antigen, or with other therapeutic or immunogenic compositions.
  • liposomes filled with a desired peptide of the invention can be directed to the site of infection, where the liposomes then deliver the selected therapeutic/immunogenic peptide compositions.
  • Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, for example, liposome size and stability of the liposomes in the blood stream.
  • a ligand to be incorporated into the liposome can include, for example, antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells.
  • a liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose that varies according to the mode of administration, the peptide being delivered, the stage of disease being treated, etc.
  • the present invention is directed to vaccines that contain as an active ingredient an immunogenically effective amount of a cytotoxic T-lymphocyte stimulating gp 41 peptide having a sequence selected from Figure 1 or 2.
  • the peptide(s) may be introduced into a patient linked to its own carrier or as a homopolymer or heteropolymer of active peptide units.
  • Such a polymer has the advantage of increased immunological reaction and, where different peptides are used to make up the polymer,
  • cytotoxic T cells that react with different antigenic determinants of HIV.
  • Useful carriers include, e.g., keyhole limpet hemocyanin, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly(D-lysine:D-glutamic acid), and the like.
  • the vaccines can also contain a physiologically tolerable (acceptable) diluent such as water, phosphate buffered saline, or saline, and further typically include an adjuvant.
  • Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum or materials well known in the art.
  • cytotoxic T lymphocyte responses can be primed by conjugating peptides of the invention to lipids, such as P sub 3 CSS.
  • the immune system of the host Upon immunization with a peptide composition as described herein, via injection, aerosol, oral, transdermal or other route, the immune system of the host responds to the vaccine by producing large amounts of cytotoxic T-lymphocytes specific for HIV antigen, and the host becomes at least partially immune to HIV infection, or resistant to developing chronic HIV infection.
  • Vaccine compositions containing the peptides of the invention are administered to a patient susceptible to or otherwise at risk of HIV infection to enhance the patient's own immune response capabilities.
  • a patient susceptible to or otherwise at risk of HIV infection is defined to be a "immunogenically effective dose” or a “prophylactically effective dose.”
  • the precise amounts again depend on the patient's state of health and weight, the mode of administration, the nature of the formulation, etc., but generally range from about 1.0 mg to about 500 mg per 70 kilogram patient, more commonly from about 50 mg to about 200 mg per 70 kg of body weight.
  • the peptides of the invention can also be expressed by attenuated viral hosts, such as vaccinia.
  • vaccinia This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode an HIV peptide of the invention.
  • the recombinant vaccinia virus Upon introduction into an HIV-infected host or into a non-infected host, the recombinant vaccinia virus expresses the HIV peptide and thereby elicits a host cytotoxic T lymphocyte response to HIV.
  • Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector
  • BCG Bacille Calmette Guerin
  • Salmonella typhi vectors and the like will be apparent to those skilled in the art from the description herein.
  • compositions and methods of the claimed invention may be employed for ex vivo therapy, wherein, as described briefly above, a portion of a patient's lymphocytes are removed, challenged with a stimulating dose of a peptide of the present invention, and the resultant stimulated CTL's are returned to the patient.
  • ex vivo therapy concerns the therapeutic or immunogenic manipulations that are performed outside the body on lymphocytes or other target cells that have been removed from a patient. Such cells are then cultured in vitro with high doses of the subject peptides, providing a stimulatory concentration of peptide in the cell medium far in excess of levels that could be accomplished or tolerated by the patient.
  • the cells are returned to the host, thereby treating the HIV infection.
  • the host's cells may also be exposed to vectors that carry genes encoding the peptides, as described above. Once transfected with the vectors, the cells may be propagated in vitro or returned to the patient. The cells that are propagated in vitro may be returned to the patient after reaching a predetermined cell density.
  • in vitro CTL responses to HIV are induced by incubating in tissue culture a patient's CTL precursor cells (CTLp) together with a source of antigen-presenting cells (APC) and the appropriate immunogenic peptide. After an appropriate incubation time (typically 1-4 weeks), in which the CTLp are activated and mature and expand into effector CTL, the cells are infused back into the patient, where they will destroy their specific target cell (an HIV infected cell). To optimize the in vitro conditions for the generation of specific cytotoxic T cells, the culture of stimulator cells is typically maintained in an appropriate serum-free medium. Peripheral blood lymphocytes are isolated conveniently following simple venipuncture or leukapheresis of normal donors or patients and used as the responder cell sources of CTLp. In one
  • the appropriate APC's are incubated with about 10-100 mu M of peptide in serum-free media for four hours under appropriate culture conditions.
  • the peptide-loaded APC are then incubated with the responder cell populations in vitro for 5 to 10 days under optimized culture conditions.
  • Positive CTL activation can be determined by assaying the cultures for the presence of CTLs that kill radiolabeled target cells, both specific peptide-pulsed targets as well as target cells expressing endogenously processed form of HIV antigen as further discussed below.
  • the MHC restriction of the CTL of a patient can be determined by a number of methods known in the art. For instance, CTL restriction can be determined by testing against different peptide target cells expressing appropriate or inappropriate human MHC class I. The peptides that test positive in the MHC binding assays and give rise to specific CTL responses are identified as immunogenic peptides. Methods of reintroducing cellular components are known in the art and include procedures such as those exemplified in U.S. Pat. No. 4,844,893 to Honsik, et al. and U.S. Pat. No. 4,690,915 to Rosenberg. For example, administration of activated CTLs via intravenous infusion is typically appropriate.
  • DNA segment refers to a DNA molecule that has been isolated free of total genomic DNA of a particular host species. Therefore, a DNA segment encoding an HIV gp 41 peptide having a desired sequence refers to a DNA segment that contains these peptide coding sequences yet is isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment has been cloned. Included within the term "DNA segment”, are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.
  • a DNA segment contemplated here refers to a DNA segment which may include in addition to peptide encoding sequences, certain other elements such as, regulatory sequences, isolated substantially away from other naturally occurring genes or protein-encoding sequences.
  • the term "gene” is used for simplicity to refer to a functional protein-, polypeptide- or peptide-encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, cDNA sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides.
  • isolated substantially away from other coding sequences means that the gene of interest, in this case, a gene encoding HIV epitopes forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
  • test antigens were dissolved in a low ionic strength buffer and applied to a nitrocellulose membrane (pore size 0.6 microns) as a 0.5 microliter spot and allowed to air dry.
  • the membranes were used in Quix TM HIV test devices (Universal HealthWatch Inc, Columbia, MD) replacing classical HIV-1 antigen coated membranes.
  • Known panels of HIV-1 positive and negative sera were tested using the following procedure for HIV testing: The following reagents were added in sequential order to the test device: two drops (approximately 40ul/drop) of blocking buffer containing detergent/BSA , one drop of test sample, three drops of blocking buffer, two drops of Wash reagent, two drops of Protein A colloidal gold reagent, two drops of Wash reagent. After addition of all the reagents, the membrane was visually examined for any red to purple colored spot indicating the binding of antibody to the immobilized antigens on the membrane.
  • a 36 mer peptide was custom synthesized to specifications and purified (Peninsula Laboratories) and had the following sequence (SEQ ID NO: 1): RARLQALETLIQNQQRLNLWGCKGKLICYTSVKWNT
  • test sample serum, plasma, or whole
  • Umversal PeptideR36T detected all 30 samples while ANT70 peptide reacted with only 22 of them.
  • R-36-T gp41 peptide from recombinant: Control ⁇ -
  • a dilution panel was prepared by making a 1:10 dilution of a known HTV-l positive sample with normal
  • HIV-O positive C-10 4+ +

Abstract

L'invention concerne un paradigme permettant d'obtenir des antigènes peptidiques améliorés pour analyses diagnostiques et thérapies du VIH, et une méthode utilisant ce paradigme, afin de créer des antigènes peptidiques différant des peptides connus par leurs séquences d'acides aminés. Cette méthode détermine une séquence en utilisant la séquence de la région consensus immunodominant de la gp 41 pour décider d'une séquence divergente convenable pour un peptide long de 25 à 100 acides aminés, en particulier long de 30 à 40 acides aminés. Les séquences peptidiques obtenues diffèrent d'au moins 25 % des séquences consensus et réagissent, plus généralement, avec des souches du VIH. Le paradigme découvert et son mode d'utilisation constituent un moyen de réduire le problème de la diversité de souches qui a freiné le développement de thérapies et de diagnostics d'infections à VIH.
PCT/US1999/001726 1998-01-28 1999-01-28 Peptides de vih-1 divergents WO1999038887A1 (fr)

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AU23453/99A AU2345399A (en) 1998-01-28 1999-01-28 Divergent hiv-1 peptides
AU45463/99A AU4546399A (en) 1998-06-05 1999-06-04 Universal peptides
PCT/US1999/012446 WO1999062945A2 (fr) 1998-06-05 1999-06-04 Peptides universels

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WO2010022740A2 (fr) * 2008-08-28 2010-03-04 Aarhus Universitet Polypeptides d’enveloppe du vih-1 pour un vaccin contre le vih

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EP0591914A2 (fr) * 1992-10-06 1994-04-13 BEHRINGWERKE Aktiengesellschaft Rétrovirus du groupe HIV et son application
DE4405810A1 (de) * 1994-02-23 1995-08-24 Behringwerke Ag Von einem Retrovirus aus der HIV-Gruppe abgeleitete Peptide und deren Verwendung
WO1996012809A2 (fr) * 1994-10-20 1996-05-02 Institut Pasteur Sequences nucleotidiques d'antigenes retroviraux vih-1 groupe (ou sous-groupe) o
WO1996027013A1 (fr) * 1995-02-27 1996-09-06 Institut National De La Sante Et De La Recherche Medicale-Inserm Vih-1 de groupe o, fragments desdits virus, ainsi que leurs applications

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Publication number Priority date Publication date Assignee Title
EP0591914A2 (fr) * 1992-10-06 1994-04-13 BEHRINGWERKE Aktiengesellschaft Rétrovirus du groupe HIV et son application
DE4405810A1 (de) * 1994-02-23 1995-08-24 Behringwerke Ag Von einem Retrovirus aus der HIV-Gruppe abgeleitete Peptide und deren Verwendung
WO1996012809A2 (fr) * 1994-10-20 1996-05-02 Institut Pasteur Sequences nucleotidiques d'antigenes retroviraux vih-1 groupe (ou sous-groupe) o
WO1996027013A1 (fr) * 1995-02-27 1996-09-06 Institut National De La Sante Et De La Recherche Medicale-Inserm Vih-1 de groupe o, fragments desdits virus, ainsi que leurs applications

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010022740A2 (fr) * 2008-08-28 2010-03-04 Aarhus Universitet Polypeptides d’enveloppe du vih-1 pour un vaccin contre le vih
WO2010022740A3 (fr) * 2008-08-28 2010-05-14 Aarhus Universitet Polypeptides d’enveloppe du vih-1 pour un vaccin contre le vih

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