WO2000058438A2 - Peptides a contrainte de conformation - Google Patents

Peptides a contrainte de conformation Download PDF

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Publication number
WO2000058438A2
WO2000058438A2 PCT/US2000/008232 US0008232W WO0058438A2 WO 2000058438 A2 WO2000058438 A2 WO 2000058438A2 US 0008232 W US0008232 W US 0008232W WO 0058438 A2 WO0058438 A2 WO 0058438A2
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hiv
peptide
sequence
group
peptides
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PCT/US2000/008232
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WO2000058438A3 (fr
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David Bernstein
Afzal Chowdhury
Alexander Kozhich
Marvin Motsenbocker
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David Bernstein
Afzal Chowdhury
Alexander Kozhich
Marvin Motsenbocker
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Priority to AU37740/00A priority Critical patent/AU3774000A/en
Publication of WO2000058438A2 publication Critical patent/WO2000058438A2/fr
Publication of WO2000058438A3 publication Critical patent/WO2000058438A3/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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • 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 invention relates to immunology, and particularly to synthetic peptides that mimic T and B cell epitopes from viruses that are useful for vaccines.
  • HIV-1 has several mechanisms that allow it to escape the body's natural defense(s). For example, exposed portions of the HIV protein are highly glycosylated, masking its epitopes, and the virus replicates rapidly, quickly altering its sequences in response to selection pressure. Accordingly, extreme strain divergence of HIN greatly complicates its biology due to the fact that HIV epitopes are continuously changing and escapes components of the host immune system. The population creates new binding site epitopes to infect target cells (i.e., T-cells).
  • the gpl ⁇ O envelope protein contains two parts, a gpl20 (120,000 Da) glycosylated part and a gp41 (41,000 Da) transmembrane part that is minimally exposed, but participates in binding, fusion and entry of viral material into host cells.
  • the analogous envelope protein precursor of HIV-2 has a similar size and has two parts, a gpl20 glycosylated part and a gp36 transmembrane part.
  • gp36 transmembrane part of the HIV-2 envelope protein
  • gpl20 HIV envelope protein is used herein to mean the larger non-transmembrane part of the envelope protein from either HIV-1 or HIN-2.
  • N3 loop of the HIN-2 envelope protein means the V3 loop of that larger non-transmembrane protein.
  • a CD4 binding site region of the HIV envelope protein refers to a section of that larger non- transmembrane protein, from either HIV-1 or HIV-2 that participates in binding the CD4 receptor.
  • HIV attaches to the surface of a target cell such as a T cell, and sometimes to a small number of M type (ie. macrophages, monocytes) lymphocyctes, via binding of gpl20 protein to particular host cell receptors.
  • M type ie. macrophages, monocytes
  • This binding causes a conformational change in the gpl20 protein.
  • the conformational change involves movement of the protein away from the surface of the virus, rearrangement of "fusion peptide domains" within the gp41 protein and their tight formation into a coiled-coil structure, mediating fusion of the HIV and the target cell membranes. Transfer of HIV R ⁇ A into the target cell immediately follows fusion.
  • gp41 polypeptide sequence may suppress T cell stimulation and a portion of -gp41 to the amino terminal side of the immunodominant region (positions 587 to 591) may be cross-reactive with alpha interferon protein, and interfere with the apha interferon anti-viral activity by virtue of this cross-reactivity.
  • the undesirable epitope problem has been addressed by mutating the gp41 loop region into a form that does not elicit enhancing antibodies.
  • application PCT/US97/11667 describes an HIV virus mutant wherein a tryptophan at residue 596 of the gp41 protein has been altered to a tyrosine, and in which the undesirable enhancement effect allegedly was removed.
  • a protein or peptide having a single amino acid change as described in that patent application may not be suitable for a vaccine, partly because a virus population can escape the effect of a single mutation by rapid mutation of multiple epitopes simultaneously.
  • Another strategy proposed to side step the enhancing antibody problem has been to determine alternative gp41 epitopes that do not stimulate enhancing antibodies and that can be used alone as immunogens.
  • One candidate in this regard has been the epitope centered at positions 662 to 667, within the "fusion peptide" region of gp41.
  • the fusion peptide region participates in the viral fusion to the host target cell through which the transfer of the viral genome occurs.
  • the 662-667 epitope was revealed indirectly from studies of a single monoclonal antibody found to have neutralizing activity.
  • the 662-667 epitope sequence previously had not been perceived as particularly important but short peptides that simulate this region were proposed as vaccines after discovery of their reactivity with the neutralizing monoclonal antibody, as described in U.S.
  • a synthetic peptide may have, for example a short hexapeptide region within it that corresponds to a short alpha helix epitope which normally is stabilized (maintains helix shape) within a large peptide.
  • this same epitope is placed within a synthetic peptide of less than 100 amino acids, particularly less than 75 amino acids, and especially less than 50 amino acids in length, it flops around and presents a less consistent epitope for binding with other molecules.
  • stabilizing the secondary helix structure of the epitope should make the peptide more potent in inducing an immune response against the epitope.
  • a separate challenge in making a vaccine based on small peptides is creating higher order structure such as helix, pleated sheet, or other interactions from short sequences obtained from a larger gp41 or gpl20 peptide.
  • One attempt to create a higher order structure from a small peptide segment is described by Braisted et al. in WO 98/20036. This document discusses the formation of helical six-amino acid long structures within peptides by cross-linking the amino acids terminal to the six amino acid long regions.
  • a vaccine also should contain adjuvants that stimulate one or more cell types involved in immunity such as cytotoxic T cell and helper T cell lymphocytes. That is, a vaccine should contain epitopes that stimulate T cells which cooperate in the induction of cytotoxic T cells to HIV.
  • the T-helper cells can be either the T-helper-1 phenotype to stimulate humoral response or T-helper-2 phenotype to stimulate cellular immunity, for example.
  • Most vaccine formulation attempts have centered on first finding a suitable antigen and then, almost as an afterthought, adding an adjuvant, and or other molecule that separately stimulates cytotoxic T lymphocytes.
  • virus escape is when a virus, that is capable of stimulating a host response sufficient to eliminate the virus, escapes that response by rapid mutation into form(s) that are not recognized by the host.
  • a successful vaccination strategy should, in the inventors' opinion, begin with an analysis of the range of epitopes that a population can assume and then to determine a set of sequences for a given epitope that should anticipate the entire range.
  • the virus is selected from the group consisting of the paramyxo viruses, herpes viruses, corona viruses, human immunodeficiency virus, simian virus 5, and respiratory syncytial virus.
  • the neutralizing sequence is a fusion peptide from the same envelope protein.
  • Another object of an embodiment of the invention is to provide a synthetic or recombinant peptide between 16 and 75 amino acids long comprising a conformationally constrained portion between 5 and 13 amino acids long and a second portion having secondary structure.
  • the conformationally constrained portion comprises a cross-linked neutralizing epitope of HIV envelope protein formed by cross links between two te ⁇ ninal amino acids, and the second portion comprises a continuous stretch of at least 5 amino acids that are predicted to have alpha helix structure.
  • Yet another embodiment of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising three or more peptides selected from the group consisting of a neutralizing epitope from the V3 loop of gpl20, a neutralizing epitope from the fusion peptide of gp41 or gp36, a neutralizing epitope from a CD4 binding site region of gpl20.
  • Yet another embodiment of the invention is a pharmaceutical composition useful for prophylactically or therapeutically treating an animal at risk for or infected with HIV, comprising at least one of the peptides described herein.
  • Yet another embodiment of the invention is a method of preparing a peptide useful for an HIV vaccine, the method comprising: a. synthesizing a linear peptide comprising a sequence of an HIV neutralizing epitope that is between 5 and 13 amino acid residues in length, the neutralizing epitope being bound by two terminal cysteine residues, the linear peptide further comprising a second portion having secondary structure; and b. oxidizing the terminal cysteine residues within the same peptide to cyclize the neutralizing epitope of the peptide.
  • Yet another embodiment of the invention is a synthetic or recombinant peptide between 26 and 75 amino acids long comprising a conformationally constrained portion between 5 and 13 amino acids long, and further comprising a second portion having sequence and directionality of a gp41 or gp36 HIV envelope protein between 1 and 26 amino acid residues to the amino terminal side of the immunodominant loop sequence, wherein the immunodominant loop portion is substituted with an HIV neutralizing epitope selected from the group consisting of the V3 loop of gpl20, the fusion peptide of gp41, the fusion peptide of gp36, and a CD4 binding site portion of gpl20.
  • Yet another embodiment of the invention is a synthetic or recombinant peptide between 27 and 50 amino acids long comprising a sequence selected from the group consisting of
  • a construct according to this embodiment of the invention has (1) a neutralizing epitope sequence, which may be from gp41and/or gp36 or gpl20, but lacks the gp41 and/or gp36 immunodominant loop enhancing sequence, (2) a conformational constraint on the neutralizing epitope which maintains immunologic reactivity of the epitope, and (3) an alpha helix positioned next to or near the neutralizing epitope to increase further its antigenic effect.
  • a neutralizing epitope sequence which may be from gp41and/or gp36 or gpl20, but lacks the gp41 and/or gp36 immunodominant loop enhancing sequence
  • a conformational constraint on the neutralizing epitope which maintains immunologic reactivity of the epitope
  • an alpha helix positioned next to or near the neutralizing epitope to increase further its antigenic effect.
  • the potencies of the constructs as vaccines are further improved three ways.
  • One, at least one T helper and/or cytotoxic lymphocyte stimulatory sequence is incorporated into the construct, preferably in a manner that mimics its sequence and placement in the gp41 protein.
  • Two, neutralizing epitopes that correspond to both the group O and group M subtype D strains, and preferably, additional neutralizing epitopes corresponding to the Group M subtype B strain, and Group M subtype E strain are used to anticipate the myriad epitopic structures that a viral population may assume.
  • Three, at least 2 epitopes from the fusion peptide of gp41, the V3 loop of gpl20 and the CD4 binding site of gpl20, are used simultaneously in a vaccine.
  • neutralizing epitopes substitute for the enhancing immunodominant loop region of a gp41 protein sequence, and the amino terminal side of the loop region is modified both to (1) increase secondary structure and (2) alleviate the immunoinhibitory activity of the sequence to the amino terminal side of the immunodominant loop.
  • multiple peptides selected from the group consisting of neutralizing epitope sequences corresponding to an HIV-1 group O, HIV-1 group M subtype D, HIV-1 group M subtype B, and group M subtype E are used together in a vaccine formulation. In regions of the world where HIV-2 poses a problem, neutralizing epitopes of HIV-2 corresponding to HIV-1 can be added to the vaccine.
  • a vaccination peptide for HIN comprises the immunodominant region of gp41 from HIN wherein the immunodominant cystine loop is replaced with a neutralizing epitope selected from the group consisting of the CD4 binding site, fusion peptide, and the V3 loop of gpl20.
  • a peptide is used having the sequence for the HCV envelope protein wherein a portion is replaced with a neutralizing epitope.
  • Feature 1 Include a Neutralizing Epitope Sequence and Avoid a Major gp41 Enhancing Sequence
  • Peptide constructs according to the invention lack an enhancing sequence from gp41 protein and contain a neutralizing sequence of an epitope from gp41, gp36 or gpl20 protein.
  • Preferred constructs contain at least one epitope that is at least 5 amino acids long.
  • the epitope is selected from the group consisting of the V3 loop of HIV envelope protein (this is envelope protein gpl20, when from HIV-1), the fusion peptide of gp41, the fusion peptide of gp36 and a CD4 binding site from gpl20.
  • Other regions of HIV peptide are known to stimulate neutralizing antibody production and also can be used.
  • Many neutralizing epitopes now are known. More neutralizing epitopes may be found by researchers in the field of HIV molecular biology and are suitable for peptide constructs of the invention.
  • Peptide constructs according to the invention do not include the immunodominant cysteine loop epitope of gp41 protein of HIN-1 or of gp36 of HIV-2, as these epitopes generally can generate enhancing antibodies (antibodies that enhance HIV infection).
  • a vaccine peptide corresponds to (i.e. has at least 60% sequence identity with, preferably at least 70% sequence identity with and preferably at least 80% sequence identity with) the gp41 (or gp36) immunodominant region that begins at the amino terminal side of the immunodominant loop and extends up to 26 amino acid residues away from the immunodominat loop.
  • the immunodominant loop itself is replaced with a neutralizing epitope sequence.
  • peptide constructs and vaccines are provided for other viruses that utilize pH independent fusion, such as the paramyxoviruses, herpesviruses, and coronaviruses.
  • This group of viruses is described, for example, in Annu. Rev. Physiol. 52: 675-97 (1990).
  • the term "pH independent manner" in the context of a class of viruses means viruses that enter a cell, not by the lysozomal pathway via a mechanism that requires low pH, but rather by direct fusion with the plasma lemma in a pH independent process.
  • peptide constructs as described using HIV neutralizing epitopes are prepared in a like manner for hepatitis C vaccines.
  • Hepatitis C neutralizing epitopes are known, and epitopes are described for example in Farci et al 1996 Proc. ⁇ atl. Acad. Sci, Volume 93, pp. 15394-15399.
  • a peptide that contains an enhancing epitope would facilitate viral infection if administered in a vaccine.
  • Superior vaccine constructs on the other. hand, can be made for these other viruses according to the present invention by swapping out an enhancing epitope sequence with a neutralizing epitope sequence.
  • the sequence when using such a sequence in an intermediate-sized peptide (preferably between 26 and 50 amino acids long), the sequence optionally is (1) constrained by cross- linking as taught herein, and (2) further improved by adding alpha helix adjacent to it or separated by a small sequence, as taught herein.
  • Neutralizing epitope sequences are chosen from gp41 (or gp36) fusion peptide (at or near sequence positions 662-667) cross-reacting sequences, gpl20 V3 cross-reacting sequences, and gpl20 CD4 binding sequences.
  • the neutralizing epitope sequence is between 5 and 13 amino acid residues long. Representative sequences contemplated for each of these three categories are summarized below.
  • A. Representative Fusion Peptide Sequences Upon studying the expected antigenicity of sequences in gp41 with a computer program (Peptide Companion, Peptide International Inc. , Louisville, KY, USA), it was discovered that the fusion peptide region of gp41 to the carboxyl terminal side of the immunodominant loop is unusually antigenic. Furthermore, peptides having sequences from this region produce neutralizing antibodies, as described for example by U.S. No. 5,756,674, the content of which is herein incorporated in its entirety by reference. Accordingly, alterations were made to this sequence that can stimulate neutralizing antibodies associated with different strains of HIV, in particular the HIV-1 Group M subtype B, D, and E strains and the Group O strain. It was further realized that a more complete repertoire of alternative HIV epitopes is anticipated by a vaccine having a combination of peptides (or epitopes within a single peptide) of fusion peptide sequences that stimulate production of neutralizing antibodies.
  • Clade B SEQ ID NO: 24 E-L-L-E-L-D-K-W-A-S-L
  • Clade D SEQ ID NO: 26 E-L-L-Q-L-D-K-W-A-S-L
  • HIV-2 SEQ ID NO: 91 L-Q-K-L-N-S-W-D-I-F
  • each sequence listed here can elicit the production of neutralizing antibodies and is useful for constructs according to the invention. Moreover, portions of these listed sequences as small as four amino acids which contain at least the fourth through seventh amino acids of each listed sequence are useful as epitopes. Still further, constraining the structure of an epitope will improve its use for generating neutralizing antibodies, and that formation of such constrained epitopic structures, particularly of the longer, (preferably 11 amino acid long) sequences is preferred in constructs according to embodiments of the invention.
  • a peptide sequence of amino acids such as a portion of the V3 loop presents its epitope(s) poorly because the primary sequence flops around into many possible different conformations, only a tiny proportion of which are immunologically active. This is particularly true when taking a smaller piece of a larger V3 loop sequence. When such a piece becomes constrained by crosslinking its ends, for example by forming a cystine bridge at the ends, a desirable epitopic structure is favored.
  • peptide constructs having a constrained neutralizing epitope selected from the group consisting of epitopes of HIV Group M subtype B, HIV group M subtype D, HIV group M subtype E, and HIV Group O strain sequences.
  • a vaccine composition that contains at least two neutralizing sequences from this group can anticipate an usually wide variety of epitopes that a virus population may generate during infection and thus alleviate the virus escape problem.
  • Preferred embodiments contain LXXW, wherein L and W are leucine and tryptophan respectively, and X represents an amino acid.
  • V3 sequences are known, as summarized in the Los Alamos Data Base, and as referenced in the co-pending patent applications and publications cited herein. All of these V3 sequences have neutralizing epitopes that are useful for the invention. It is particularly preferred to include at least three different peptide constructs that contain constrained gpl20 V3 loop sequences selected from the group consisting of sequences from HIV Group M subtype B, HIV Group M subtype D, HIV Group M subtype E, HIV Group O, and HIV-2 strain sequences, in order to anticipate a wider variety of epitopes that a population of virus may generate and alleviate the virus escape problem.
  • HIV-1 Group M subtype B SEQ ID NO: 97 R-S-I-R-I-G-R-A-F-Y-A-T-G SEQ ID NO: 98 K-S-I-H-I-G-P-G-R-A-F-Y-T
  • Group M subtype D SEQ ID NO: 99 Q-S-T-H-I-G-P-G-Q-A-L-Y-T
  • Group M subtype E SEQ ID NO: 100 T-S-I-T-I-G-P-G-Q-V-F-Y-R
  • the gpl20 protein of HIV-1 (and the analogous protein of HIV-2) contain multiple sites along its primary amino acid sequence that participate in binding CD4 and which advantageously can be used to elicit neutralizing antibodies. Two such regions are particularly useful and, in preferred embodiments, a sequence between 5 and 13 amino acids long is chosen and used after constraining. Sequences that contain neutralizing epitopes are known to the skilled artisan. A representative sample of preferred sequences for each of three HIV-1 strains is given here.
  • Subtype D SEQ ID NO: 106 W-Q-G-A-G-Q-A-M-Y-A-P-P SEQ ID NO: 107 W-Q-E-V-G-K-A-M-Y-A-P-P-I-E
  • Group O SEQ ID NO: 110 W-M-R-G-G-S-G-L-Y-A-P-P
  • HIV-2 SEQ ID NO 135 W-H-K-V-G-R-N-V-Y-L-P-P-R-E
  • HIV-2 SEQ ID NO 136 W-H-K-I-G-R-N-V-Y-L-P-P-R-E
  • HIV-2 SEQ ID NO: 173 N-Y-A-P-C-H-I-K-Q SEQ ID NO: 174 N-Y-A-P-C-H-I-R-Q
  • CD4 binding site region sequences also are known, as summarized in the Los Alamos Data Base and as referenced in the publications referenced herein. It is particularly preferred to include peptide constructs in a vaccine that contain constrained sequences selected from the group of V3 sequences of the subtype B, subtype D, subtype E, and Group O strain sequences, to anticipate a wider variety of epitopes that a population of virus may generate. Of course, two or more constrained neutralizing sequences can be fused into a single peptide.
  • constrained neutralizing peptide constructs selected from the group of neutralizing epitopes corresponding to subtype E, subtype B and Group O sequences of the V3 loop of gpl20 respectively, neutralizing epitopes corresponding to subtype D, subtype B and Group O sequences of the fusion peptide of gp41, and neutralizing epitopes corresponding to subtype E, subtype B and Group O sequences of the CD4 binding site region of the gpl20 HIV envelope protein respectively.
  • additional peptides having sequences corresponding to the analogous portions of HIV-2 envelope protein also are desired for inclusion in a vaccine.
  • known epitopic sequences are used by insertion into constrained loop portions in a like manner.
  • At least one portion of a peptide construct according to this embodiment comprises a neutralizing epitope as described above in a conformationally constrained form.
  • a "portion” in this context means a segment of at least 5 amino acids, preferably at least 6, more preferably at least 8 and in some cases 13 amino acids long or more.
  • the term “conformationally constrained” used herein means that the conformational movement of the portion (and thus the structure of the epitope) is restrained by cross-linking between two terminal amino acids, one at each end of the portion that comprises the epitope.
  • the peptide structure that is recognized by the immune system after administration of the construct in a vaccine may be larger than the portion that is bound by cross-linked terminal amino acids.
  • the constrained portion may form a larger epitope site with another section of the peptide construct as a tertiary structure (complex between different regions of the peptide construct) although in preferred embodiments the constrained portion, which optionally includes the terminal amino acids, itself forms the epitope.
  • the epitope may be smaller than the portion between the terminal amino acids and, in some cases a helix is formed within the constrained portion. In most embodiments a complete helix does not form, and in some cases no helix structure would form. In every case, however, the epitope primarily (i.e.
  • a tertiary structure is formed wherein the bounded portion forms a stable complex (non-covalently formed) with a peptide region outside the constrained portion and a sequence from the constrained portion by a spacer region.
  • the spacer region if used, preferably is between 3 and 10 amino acids and more preferably between 5 and 6 amino acids long.
  • the terminal amino acids of a neutralizing epitopic region are cross-linked by forming at least one covalent bond between them.
  • cross-linking occurs by the formation of a sulphur-sulphur bond via formation of a cystine from oxidation of two cysteines.
  • cystine bridge itself forms part of a desired epitope.
  • Formation of a cystine cross-link from two cysteines is readily carried out by known procedures that cause two thiol groups on the same peptide to oxidize and form a dicysteine (cystine) in the presence of oxygen.
  • a cystine bridge is particularly preferred for use with some V3 loop epitopes, as illustrated in the examples.
  • a side chain amide bond-forming group may be placed at the N-terminus of a neutralizing epitope sequence and another amino acid with a side chain amide bond-forming group is placed at the C-terminus of the peptide.
  • the side chain amide bond-forming groups of the N- terminal and C-terminal residues are joined to form a cyclized structure which constrains the epitopic sequence.
  • the sequence is 6 amino acids long and forms an ⁇ -helix within the loop as described in U.S. No. 98/20036.
  • a larger peptide (less than 75 amino acids, particularly less than 50 amino acids) lock any sequence of, for example, six amino acid residues within a larger peptide into, for example, a helix by importing two residues with side chain amide bond-forming groups into the N-terminal amino acid position and the C-terminal position amino acid position flanking the sequence of six amino acid residues.
  • the side chain amide bond- forming groups of the N-terminal and C-terminal flanking residues are made to form a cyclic structure which mimics the conformation of the ⁇ -helix. Regions 5 amino acids long and regions greater than 6 amino acids long, of course, also can be used as exemplified in this specification and often will form particular helix structures.
  • constrained peptides of this embodiment there are at least two general methods for constructing constrained peptides of this embodiment: (1) synthesis of a linear peptide comprising a pair of residues that flank an amino acid sequence that is five to thirteen residues in length, wherein the two flanking residues are independently selected from amino acid residues having side chain amide bond-forming groups, followed by bridging the side chain amide bond-forming groups of the flanking residues with a linker or peptide coupling reagent (i.e.
  • flanking amino acid residues include amino acids with side chains carrying a free carboxy group, such as aminopropanedioic acid, aspartate, glutamate, 2-aminohexanedioic acid, and 2- aminoheptanedioic acid, and amino acids with side chains carrying a free amino group, such as 2,3-diaminopropanoic acid (2,3-diaminopropionic acid), 2,4-diaminobutanoic acid (2,4-diaminobutyric acid), 2,5-diaminopentanoic acid, lysine and ornithine.
  • the functional groups on either side may be used such as thiol (SH) or hydroxyl (OH) groups.
  • a C-terminal residue may be a cysteine-like amino acid which has a thiol group, while N-terminal residue may have a halo-alkyl group in its side chain, and cyclization occurs by addition of a basic reagent.
  • Secondary structure in this context refers to polypeptide helix or pleated sheet (that may form from disparate regions of the peptide) primarily by multiple hydrogen bonding between peptide bond hydrogen and oxygen. Most advantageous is alpha helix structure that forms within a stretch of the peptide outside but near (spaced by 4-10, preferably 5-6 amino acids) to the cross-linked region.
  • the secondary structure preferably a helix, begins a short distance away from a cystine loop portion as for example described in co-pending patent applications U.S. Nos.
  • 60/111,995 and 60/088,229 which are herein incorporated in their entireties by reference.
  • the inventors experimentally discovered that placing or increasing alpha helix on the amino terminal side of a cystine loop in particular stabilizes the antigen structure, as described in those patent applications.
  • the degree of stabilization has a great influence on the ability of the epitope to react specifically with components of the immune system.
  • adding a five amino acid long helix portion "A-W-E-K-T" to the amino terminal end of an 18 mer peptide provided greater antigenicity for an HIV-1 cross-reacting epitope, as described in the co-pending applications.
  • the inventors theorize that adding this 5 amino acid long helix, which is not a known epitope of HIV, increases structural stability of the constrained epitope, allowing the epitope to more optimally react with component(s) of the immune system such as IgG.
  • the increased stability allows the peptide to react more favorably and improve immunological characteristics of the antigen.
  • This theory is supported by the experimental observation that when this same peptide helix was lengthened by an additional 5 amino acids (to 10 amino acids at the amino terminal portion), the antigen thus formed from the gp41 immunodominant loop, showed yet even better sensitivity for binding antibodies.
  • the alpha helix portion may be 6, 7, 8, 9, 10, 11, 12, 13 or more amino acids long.
  • alpha helix is added to the carboxyl terminal side of the constrained cross- linked loop.
  • amino acids outside the amino terminal side of the constrained region represent are contemplated that can form and/or increase alpha helix structure.
  • positions 9, 10 and 11 from the constrained region as shown in this table contain the amino acids D, K and L respectively. This combination is preferred to facilitate the T cell response based on the activity of cytotoxic T lymphocytes.
  • positions 6, 7 and 8 do not contain L, Q and Q respectively, as avoiding these amino acids at these positions helps prevent immunosuppressive effects that can occur when sequences from peptides of gp41 protein are used as vaccines.
  • Advantageous amino acids in this regard may be determined by prediction from a peptide analysis software program, "Peptide Companion Version 1.24 for Windows" from Peptides International, Inc. Louisville, Kentucky 40299 U.S.A.
  • the Chou- Fasman Conformational parameters are used in determining which amino acids can be changed within the helix in a manner to preserve the helix, with corresponding advantageous antigenicity of the peptide.
  • Most preferred in this context is the addition of the sequence Q- K-I-G at positions 23 - 26 away, respectively, from the amino te ⁇ ninal side of the constrained region.
  • a sequence when selecting a sequence to form a helix on the amino terminal side of the constrained loop, a sequence is chosen that has more alpha helix in this region compared to M clade strains of HIV. Examples of such sequences are shown in U.S. Nos. 60/088,229, 60/098,705, 60/098,693 and 60/100,047, which are herein incorporated by reference in their entireties.
  • Advantageous alpha helices in accordance with this embodiment of the invention comprise at least 50%, and more advantageously at least 65% sequence similarity to a sequence described in one or more of the referenced applications.
  • inventive peptides as described above acquire greater potency as vaccines by the following optional improvements, that may be used separately or in combination.
  • Optional Improvement 1 Integrate at Least one T helper and/or Cytotoxic Lymphocyte Sequence within the Construct
  • vaccine constructs preferably contain one or more T helper and/or cytotoxic lymphocyte sequences.
  • the T cell stimulating sequence is simply added to another sequence by making a fusion peptide.
  • a construct combines one or more naturally occurring adjuvants from gp41 with a neutralizing epitope of HIV-1 envelope protein. It was discovered that certain changes can be made in a portion of gp41, particularly between 6 and 21 residues away from the amino terminal side of the immunodominant loop, that eliminate the immunosuppressing function of the sequence, while preserving the immunostimulating function. Many of these changes are options that additionally, provide increasing predicted secondary structure and are listed in the preceding table. Furthermore, the immunodominant loop sequence, which stimulates the production of antibodies that enhance HIV infection, can be replaced with epitope sequences that stimulate the production of neutralizing antibodies. Most preferred in this context are sequences from the fusion peptide portion of gp41, the V3 portion of gpl20 and parts of the CD4 binding site of gpl20 as described above.
  • cytotoxic T lymphocyte (CTL) active region of gp41 centered at positions 590-594 and of the T helper stimulatory region centered at positions 610 to 617 are important features of adjuvants and are preferred.
  • the position of these adjuvants within the peptide constructs of the invention are the same as or similar to (ie. within three amino acid positions of) their relative positions from the immunodominant loop of gp41.
  • these adjuvant sequences maintain their same relative position with respect to a constrained epitope sequence as they have with respect to the naturally occurring immunodominant cystine loop sequence.
  • these constructs contain extensive alpha helix structure on the N terminal side, which, the inventors found from experimental results, stabilizes the protein and improves antigenicity of the amino acid sequence within the loop.
  • the adjuvants according to the invention preferably are used together with contiguous alpha helix but can be used with alpha helix that begins up to 12 positions away from the constrained neutralizing epitope sequence.
  • Preferred adjuvant sequences for stimulating a T helper cell response are T-T-A-N— P-W- ⁇ -A-S-W and variants such as this sequence with one or more conservative amino acid changes. Other substitutions are known and can be determined from publications.
  • Other preferred sequences are E-K-T-L-K-D-Q-Q-R-R E-K-T-L-K-D-Q-Q-R-L E-K-T-L-K-D-Q-A-R-R
  • an L-K-D adjuvant sequence is separated from the amino terminal side of the constrained loop portion by an eight amino acid portion that is not hydrophilic.
  • this eight amino acid portion comprises four very hydrophobic residues selected from the group consisting of isoleucine, leucine, valine, tryptophan, tyrosine and phenylalanine.
  • the inventors theorize that placing this eight amino acid long very hydrophobic segment between the constrained epitope and the adjuvant sequence improves the activity of the adjuvant by forcing the adjuvant sequence to reversibly come close to the very active antigenic epitope within the loop.
  • the hydrophobic portion curls and tries to form a micelle while avoiding water, and this allows the adjuvant sequence to meet the stimulating epitope and form a tertiary structure.
  • Optional Improvement 2 include Universal Epitopes from at Least Two Clades to
  • Epitopic sequences preferably are selected from alternative clades to make peptide constructs so that, when used together, the constructs elicit broadly neutralizing antibodies to HIV.
  • the problem addressed in using epitopes from multiple clades is the ability of the virus to escape the neutralizing ability of the immune system by mutation.
  • the first step is to determine a suitable neutralizing epitope of a peptide from the target virus.
  • a first peptide then is chosen that comprises at least a portion that corresponds to the chosen epitope. It is most advantageous to pick a peptide that cross reacts broadly with many types of strains, such as taught, in the case of HIV, in the co-pending applications cited above.
  • Particularly advantageous in this context for HIN are Group O reactive peptides, such as those that comprise one or more of the alternative sequences listed within the sequence listing of a co- pending application entitled “Universal Peptides" filed on on September 11, 1998 (Attorney docket No. 073294/0190).
  • an immunodominant region of the NS3 peptide may be advantageously chosen, or another well known immunodominant region of a hepatitis C protein. Although not sufficiently described elsewhere, an even more advantageous immunodominant region would be from the gp70 envelope protein region that has a high rate of mutation.
  • the first peptide then is challenged with a diverse set of strains in binding assays to determine one or more strains that react less well with the peptide.
  • the selected strain(s) constitute a second group.
  • a second peptide that corresponds to the same epitope but which cross-reacts with the second group better than the first peptide then is chosen based on known characteristics of the group and optionally on experimental results.
  • one embodiment of the claimed invention with respect to vaccines for HIV infection is a combination of Group O neutralizing epitope peptide with Subtype D neutralizing epitope peptide.
  • Specific examples of Subtype D peptide sequences are found in co-pending application entitled "HIV-1 Subtype D Peptides" filed October 16, 1998 (Attorney docket No. 073294/0193).
  • the invention, as practiced with HIV testing and therapy is particularly advantageous with Group O and Subtype D peptides as described in the two above referenced applications.
  • a neutralizing epitope particularly from the gp41 immunodominant region, it is necessary to avoid the immunodominant loop sequence, and the immunosuppressive region to the amino terminal side of this sequence.
  • gp41 neutralizing epitopes of Group O and of Group M subtype D sequences are further combined, either in the same peptides or in different peptides, with neutralizing epitopes of another Group M subtype, preferably subtype B.
  • the inventors realized that vaccine compositions which comprise epitopes from all three clades are more efficacious than vaccines having epitopes from only two clades.
  • gpl20 neutralizing epitopes of group M subtype B and subtype E sequences are further combined either in the same peptide or in different peptides with neutralizing epitopes of another HV-1 strain preferably group O.
  • peptide constructs according to this embodiment of the invention can also be used in a confirmatory test for detection of gpl20 response.
  • constructs that contain epitope sequences from alternative strains such as Group O and Group M Subtype E are used together, as fused peptide, or separately, as separate peptide, in a diagnostic test.
  • the peptide(s) are immobilized onto a solid phase and are used to detect antibody against gpl20 of HIV.
  • a preferred change from a hydrophobic to hydrophilic amino acid is alteration of L that is 6 amino acids from the amino terminal end of the constrained portion, (and 6 positions away from the gp41 immunodominant loop) to R. Another change is for this amino acid to become Q. Such alteration, particularly from L to R, helps prevent immunosuppression properties of the sequence.
  • Another consequence of the hydrophobic to hydrophilic amino acid residue shift can be greater solubility of the peptide in water. An increase in water solubility can lead directly to improved vaccine performance by allowing a greater amount of peptide to be used. This attribute also facilitates the use of more than one peptide together in the same solution without causing a precipitate at higher concentrations of one or more of the peptides. Furthermore, such changes alleviate non-specific aggregation or complexing of peptides with each other in vaccine formulations, allowing a greater amount of peptide to be administered.
  • a peptide antigen according to the invention is greater than 16 amino acid residues long but smaller than 100 amino acid residues long, preferably less than 75 amino acids long and more preferably between 26 and 50 amino acids long. This size range is termed "intermediate size. " The upper size limit reflects the fact that an intermediate size peptide according to the invention is shorter than most proteins, which have tertiary structure due to folding of the protein sequence. In a protein, the polypeptide chain folds upon itself (forms tertiary structure) to, among other things, allow mutual association of hydrophobic residues in order to maximize entropy of a water solution that contains the polypeptide.
  • Intermediate sized peptides in accordance with the invention on the other hand, generally are smaller, generally fold less and have less tertiary structure than a whole protein but have secondary structure.
  • Their minimum size limit of 16 amino acids reflects the fact that peptides smaller than 16 residues long generally have little structure outside the primary structure of amino acid sequence and are less improved by making a substitution according to the claimed embodiment.
  • a preferred minimum size of 26 reflects the fact that vaccine constructs according to the invention work better if a constrained neutralizing epitope is combined with a helical structure of at least 5 amino acids long. Furthermore, in preferred embodiments the helical structure begins approximately 5 amino acids away (for example, 5 amino acids away) from the constrained epitope. Without wishing to be bound by any particular theory of their invention, the inventors theorize that placing a non-helical portion of between 3 and 10 amino acids, and preferably 5 to 6, and most preferably 5 amino acids between the constrained epitope and the helix outside the epitope allows amino acid side chains of the helix to interact with the epitope.
  • the non-helical spacer region sterically permits the helix to move around and contact the epitope, and even to participate in a tertiary structure epitope formed from the helix and the constrained portion. This latter event is particularly facilitated when the outer helix and the constrained portion have opposite charges that can approach each other (for example within 5 angstroms distance), or both have hydrophobic residues that can come together.
  • a positive charge of an arginine in the constrained neutralizing sequence can combine with a helix sequence outside the cross-linked portion via an aspartate carboxyl group on the outside helix to form a salt bridge.
  • a leucine on an outside helix can associate (mutually repel water) with a leucine from the constrained portion.
  • two CD4 binding sequences are chosen that have some affinity for each other and that associate to form an epitope for the CD4 receptor binding site.
  • the regions can be made to come together and form a tertiary structure epitopic site to stimulate production of neutralizing antibodies.
  • the spacer between the two CD4 binding site regions is the same length, and has the same sequence as the spacer normally found in the envelope protein from which the CD4 binding site regions came. This embodiment of the invention provides tertiary structure epitopes within an intermediate sized peptide.
  • a construct of the invention comprises a section of the gp41 peptide from the carboxyl end cysteine of the immunodominant loop to 26 positions to the amino side, of the immunodominant loop, with two functional substitutions.
  • One functional substitution is replacement of the enhancing epitope sequence from the immunodominant loop with a neutralizing epitope as described herein.
  • the second substitution is replacement of at least one amino acid within the amino terminal portion from between 6 to 21 positions on the amino side of the loop with another amino acid(s) that alleviates the immunosuppressive activity of the region while maintairiing some T cell stimulatory activity of the region.
  • This embodiment provides vaccine formulations that contain useful T cell stimulatory sequence portions obtained from the gp41 (or gp36) peptide.
  • Previous vaccine formulations generally used the entire unmodified sequence.
  • complement activated enhancement of HIV infection based on an epitope near the immunodominant loop of gp41 (or gp36) was alleviated by mutation of a tryptophan at position 596, as described by PCT/US97/11667.
  • the present invention in contrast, completely removes the enhancement activity from the loop portion by replacing this portion with a neutralizing epitope and furthermore removes the immunosuppressive effect of the amino terminal portion by making multiple substitutions.
  • substitutions particularly are made at positions 19 to 21 to the amino terminal side of the loop and 6 to 8 positions to the amino terminal side of the loop.
  • Most preferred is a change in the amino acid at the 21 st position from the amino terminal end of the constrained portion from a Q to an R.
  • Preferred sequences that contain such modifications are provided in the referenced co-pending applications.
  • Most preferred amino acids for the 19 th position are M, A, D, and E.
  • Most preferred amino acids for the 20 th position are M, R, D, E, H, and K.
  • Most preferred amino acids for the 21 st position are R, M, W, N, D, E, H, I, L, K, F, and P.
  • constructs according to the invention are useful for vaccines generally and are particularly useful for vaccines against viruses that fuse with host membranes by a pH-independent fashion. Some epitopes of envelope proteins from such viruses are dangerous for vaccine formulations because they stimulate the production of enhancing antibodies that actually help the virus infect cells.
  • the invention is particularly directed for use in vaccines against those viruses.
  • the present disclosure is directed to peptide constructs for HIV as a model system but the principles explained herein are applicable to other viral vaccines as well.
  • sequences for peptide constructs given herein 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.
  • Classical methods of constructing peptide vaccines are well known to those skilled in the Art and are well described in the textbook VACCINES, Stanley A. Plotkin and Edward A. Mortimer, Jr Ed, 1994 Edition, WB Saunders Company, Phil., Pa.
  • 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 example, 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 the tables, 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 neutralizing epitope or an adjuvant (T cell stimulating) sequence will not differ by more than about 20% from a known sequence, 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.
  • regions of the peptide 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 peptide, (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 also can 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.
  • 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.
  • D-containing 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-containing peptides under physiological conditions may more than compensate for a difference in affinity compared to the corresponding L-peptide.
  • L-amino acid-containing peptides 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 peptide 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: TABLE 1
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a peptide is generally understood in the art as cited in U.S. No. 5,703,057 (citing Kyte and Doolittie, 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 Doolittie, 1982), these are: isoleucine
  • 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 groups, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Site-specific mutagenesis is a technique useful in the preparation of individual proteins, or biologically functional equivalent proteins, through specific mutagenesis of the underlying DNA. 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.
  • DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment
  • This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
  • appropriate cells such as E. coli cells
  • 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 WO 00/58438 4Q PCT/USOO/08232
  • 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 CTL stimulating activity, such that the modified peptide analog has CTL activity greater than a peptide of the wild-type sequence.
  • 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 peptide or gene region within a type, different epitopes to different peptides 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).
  • This reagent creates a disulfide linkage between itself and a cysteine residue in one peptide and an amide linkage through the amino on a lysine or other free amino group in the other.
  • disulfide/amide forming agents are known. See, for example, Immun. Rev., 62, 185 (1982).
  • Other bifunctional coupling agents form a thioether rather than a disulfide linkage. Many of these thioether forming agents are commercially available (from, for example, Aldrich Chemical Company, Inc.
  • 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.
  • 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 75 and more preferably less than 50), the peptides can be synthesized in solution or on a solid support in accordance with conventional protein 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 Protein Synthesis (2d. ed. , Pierce Chemical Co. , 1984); Tarn et al. , J. Am.
  • recombinant DNA-derived peptides or proteins which comprise at least one constrained neutralizing epitope with added secondary structure such as alpha helix can be used to prepare a construct 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 polyprotein is used that incorporates several antigen epitopes or T cell epitopes into a single polypeptide.
  • the coding sequence for proteins of the length contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al.
  • modification can be made simply by substituting the appropriate base(s) for those encoding the native protein 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 peptide. A number of such vectors and suitable host systems now are 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 a neutralizing epitope, comprising contacting a suitable cytotoxic T lymphocyte with an immune response provoking effective amount of peptide having a neutralizing epitope.
  • a preferred preparation of the 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 may comprise a molecule that includes a polypeptide having substantial homology with an epitope from one of the peptide sequences described herein or the protein itself, and a pharmaceutically acceptable carrier.
  • 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 B cell and/or T cell response to HIV and to cure or at least partially arrest its symptoms and/or complications.
  • An 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 administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound(s) and the desired physiological effect. It will be appreciated by one of skill in the art that various conditions or disease states may require prolonged treatment involving multiple administrations. 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 immune response. It must be kept in mind that the 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 B cell and/or T cell 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, glycolic, 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, glycolic, gluconic, succinic, p-toluenesulphonic acids, and arylsulphonic, for example.
  • the 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.
  • Such carriers can include immuno- stimulating complexes (i.e. cholesterol, saponin, phospholipid peptide complexes), aluminum hydroxide (alum), heat shock proteins, linkage to synthetic microspheres (polyamino-microspheres).
  • 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 stimulatory peptides dissolved or suspended in an acceptable carrier suitable for parenteral administration, including aqueous and non-aqueous, isotonic sterile injection solutions.
  • 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 glycol or polyethylene gly col, dimethylsulfoxide, glycerol ketals, such as 2,2-dimethyl-l ,3-dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 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, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose,
  • 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, 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 glycol.
  • 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 acceptable surfactant, suspending agent, or emulsifying agent.
  • diluents such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent.
  • 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 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.
  • Mixed esters, such as mixed or natural glycerides may be employed.
  • 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.
  • pressurized propellants such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • 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.
  • tripalmitoyl-S- glycerylcysteinly-seryl-serine P sub 3 CSS
  • Peptides of the present invention can be coupled to P sub 3 CSS, for example and the lipoprotein administered to an individual to specifically prime a cytotoxic T lymphocyte response to HIV.
  • concentration of peptide constructs 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 complexes, or liposomes.
  • inclusion complexes such as cyclodextrin inclusion complexes, or liposomes.
  • Liposomes serve to target the proteins 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.
  • 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 WO 00/58438 ⁇ PCT/USOO/08232
  • 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 invention is directed to vaccines that contain as an active ingredient an immunogenically effective amount of a cytotoxic T-lymphocyte stimulating peptide having a sequence as described herein.
  • Other immunomodulators may be added such as interleukin-1 , beta (IL-1 beta) peptide and interleukin 12 (IL-12) peptide.
  • Peptides may be for example, complexed to cholera toxin B subunit to stimulate mucosal immunity.
  • 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, the additional ability to induce antibodies and/or cytotoxic T cells that react with different antigenic determinants of HIV.
  • Useful carriers are well known in the art, and 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.
  • a physiologically tolerable (acceptable) diluent such as water, phosphate buffered saline, or saline
  • 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.
  • 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 is BCG (bacille Calmette Guerin). BCG vectors are described in Stover et al. , Nature, 351, 456-460 (1991).
  • BCG vectors are described in Stover et al. , Nature, 351, 456-460 (1991).
  • 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. Following treatment to stimulate the CTLs, the cells are returned to the host, thereby treating the HIV infection.
  • 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).
  • CTLp CTL precursor cells
  • APC antigen-presenting 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.
  • 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.
  • 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 a 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 peptide-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.
  • a 35 mer peptide was made having the sequence SEQ ID No: RARLQAWEKTLEDQARLNCKSIHIGPGRAFYTC, which contains a constrained 13 mer fragment of the HIV-1 V3 loop sequence SEQ ID No: CTRPNNNTRKSIHIGPGRAFYTTGEIIGDIRQAHC.
  • the unconstrained 35 mer peptide (having a greater number of epitopes) and the construct having a constrained 13 mer portion (with fewer epitopes) were tested for cross reactivity with blood samples from HIV infected patients.
  • the methods of the assay are described in the co-pending applications and immobilized 400ng portions of each peptide were tested. The results are shown below. As explained in the co-pending applications, results were scored with an increasing number meamng a greater positive result. Partial increases are noted with + and + + for increased reactivity respectively.
  • Each construct comprises (1) a predicted alpha helix portion and (2) a neutralizing fusion peptide of gp41 that has a constrained conformation by virtue of formation of a cystine loop between terminal cysteines.
  • Mice are immunized according to procedures described in Brown et al., Arch. Virol. 140: 6335-654 (1995). After immunization, enzyme-linked immunosorbent assays and immunoblotting assays are performed as described in Brown et al. Results will indicate that each construct induces formation of neutralizing antibody that binds to the fusion domain of gp41 protein.
  • Each construct comprises an 18 amino acid long chain with a predicted alpha helix portion that is immunologically non-reactive and serves to stabilize and to make the peptide loop at the C- terminus more accessible to components of the immune system.
  • the amino acids in this fragment are substituted with amino acids that are predicted, by software, to maintain a helical structure.
  • the 19 th and the 33 rd amino acids are added to induce a formation of a cyclic loop so that the flanking epitope would be properly exposed & available to the binding site.
  • a 13 amino acid long gpl20 V3 loop epitope, that represents variants of HIV-1 and HIV-2, is flanked by a disulfide Bridge.
  • mice are immunized according to procedures described in Brown et al., Arch. Virol. 140: 6335-654 (1995). After immunization, enzyme-linked immunosorbent assays and immunoblotting assays, and T-cell proliferation assays are performed as described in Brown et al. Results will indicate that each construct induces formation of neutralizing antibody that inhibits binding of HIV to target T cells in a viral neutralization assay.
  • the constructs are used in combination in diagnostic tests. Mixtures of two of each construct in turn is immobilized at a total peptide amount of 500ng in the HIV assay device described in the co-pending applications. Blood samples that contain antibodies against HIV-1 gpl20 protein are tested and yield positive test results.
  • the following 41 mer constructs are chemically synthesized. These constructs are similar to the above constructs but additionally contain carboxyl terminal portions having helper T cell epitopes.
  • mice are immunized according to procedures described in Brown et al., Arch. Virol.
  • Example 5 The following 35 mer constructs are chemically synthesized. Each construct comprises (1) a predicted alpha helix portion, (2) a neutralizing fusion peptide of gp41 that has a constrained conformation by virtue of formation of a cystine loop between te ⁇ ninal cysteines, and (3) a helper T-cell epitope on the carboxyl terminal side of the constrained epitope.
  • Mice are immunized according to procedures described in Brown et al., Arch. Virol. 140: 6335-654 (1995). After immunization, enzyme-linked immunosorbent assays and immunoblotting assays, and T-cell proliferation assays are performed as described in Brown et al. Results will indicate that each construct induces formation of neutralizing antibody that inhibits binding of HIV to target T cells in a viral neutralization assay. In addition T cell proliferation is expressed compared to un-immunized mice.
  • Example 6 The following 40 mer constructs are chemically synthesized. Each construct comprises (1) a predicted alpha helix portion, (2) a neutralizing CD4 binding site sequence of gpl20 that has a constrained conformation by virtue of formation of a cystine loop between terminal cysteines, and (3) a helper T-cell epitope on the carboxyl terminal side of the constrained epitope.
  • Mice are immunized according to procedures described in Brown et al., Arch. Virol. 140: 6335-654 (1995). After immunization, enzyme-linked immunosorbent assays and immunoblotting assays, and T-cell proliferation assays are performed as described in Brown et al. Results will indicate that each construct induces formation of neutralizing antibody, that binds to gp41 protein, and that each construct elicits a proliferative T-cell response in BALB/c and CBA mice.

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Abstract

L'invention concerne des peptides ainsi que leurs méthodes de synthèse, ces peptides possédant des propriétés remarquables et utiles d'immunothérapie et de prophylaxie du VIH et d'autres maladies infectieuses. L'invention concerne également des compositions médicamenteuses contenant un ou plusieurs de ces peptides. Dans un mode de réalisation particulièrement avantageux, on produit un peptide utile dans le cas d'un virus pénétrant dans une cellule hôte de manière indépendante du pH à partir d'une protéine d'enveloppe du virus en remplaçant une séquence favorisant l'infection par une séquence déclenchant un anticorps neutralisant. Cette caractéristique permet d'utiliser de nouvelles régions de protéines d'enveloppe à partir de cette catégorie de virus.
PCT/US2000/008232 1999-03-29 2000-03-29 Peptides a contrainte de conformation WO2000058438A2 (fr)

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Cited By (4)

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EP1791556A2 (fr) * 2004-08-27 2007-06-06 The Henry M. Jackson Foundation Proteines d'enveloppe du vih-1 modifiees
JP2007533645A (ja) * 2003-10-23 2007-11-22 エヌエムケー・リサーチ・エルエルシー Ｈ因子結合部位に基づく免疫原性組成物およびワクチンの開発方法
WO2010022740A2 (fr) * 2008-08-28 2010-03-04 Aarhus Universitet Polypeptides d’enveloppe du vih-1 pour un vaccin contre le vih
US8044185B2 (en) 2005-09-06 2011-10-25 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Conformationally stabilized HIV envelope immunogens and triggering HIV-I envelope to reveal cryptic V3-loop epitopes

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007533645A (ja) * 2003-10-23 2007-11-22 エヌエムケー・リサーチ・エルエルシー Ｈ因子結合部位に基づく免疫原性組成物およびワクチンの開発方法
EP1791556A2 (fr) * 2004-08-27 2007-06-06 The Henry M. Jackson Foundation Proteines d'enveloppe du vih-1 modifiees
JP2008511331A (ja) * 2004-08-27 2008-04-17 ザ ヘンリー エム ジャクソン ファウンデーション 修飾されたｈｉｖ−１エンベロープタンパク質
EP1791556A4 (fr) * 2004-08-27 2010-07-21 Jackson H M Found Military Med Proteines d'enveloppe du vih-1 modifiees
US8017126B2 (en) 2004-08-27 2011-09-13 Henry M. Jackson Foundation for the Advanvement of Military Medicine Inc. Modified HIV-1 envelope proteins
AU2005280004B2 (en) * 2004-08-27 2012-02-23 Institute Of Tropical Medicine Modified HIV-1 envelope proteins
US8044185B2 (en) 2005-09-06 2011-10-25 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Conformationally stabilized HIV envelope immunogens and triggering HIV-I envelope to reveal cryptic V3-loop epitopes
US8268323B2 (en) 2005-09-06 2012-09-18 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Conformationally stabilized HIV envelope immunogens
US8715686B2 (en) 2005-09-06 2014-05-06 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Conformationally stabilized HIV envelope immunogens
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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