WO2000058438A2

WO2000058438A2 - Conformationally constrained peptides

Info

Publication number: WO2000058438A2
Application number: PCT/US2000/008232
Authority: WO
Inventors: David Bernstein; Afzal Chowdhury; Alexander Kozhich; Marvin Motsenbocker
Original assignee: David Bernstein; Afzal Chowdhury; Alexander Kozhich; Marvin Motsenbocker
Priority date: 1999-03-29
Filing date: 2000-03-29
Publication date: 2000-10-05
Also published as: AU3774000A; WO2000058438A3

Abstract

Peptides and methods for their synthesis are provided having unique and useful properties for immuno-therapy and prophylaxis of HIV and other infectious diseases. Medical compositions are provided that contain one or more of the peptides. In a particularly advantageous embodiment, a peptide useful for a virus that enters a host cell in a pH independent manner is made from an envelope protein of the virus by replacing an infection enhancing sequence with a sequence that elicits neutralizing antibody. This feature allows the use of new regions of envelope proteins from this class of virus.

Description

Conformationally Constrained Peptides

Field of the Invention

The invention relates to immunology, and particularly to synthetic peptides that mimic T and B cell epitopes from viruses that are useful for vaccines.

Background of the Invention

Despite massive research into the molecular biology of HIN, no vaccine for HIN has yet been proven effective. One reason for the slow progress in this area is that 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).

Of the prominent epitopes involved in forming an antibody response, those from gpl60 envelope protein appear most important. Initial studies of the virus indicated that HIV binds to T-cell receptors via parts of its gpl60 envelope protein and thus, a vaccine should contain this protein. The gplόO 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. Publications in this field generally use "gp36" to mean the transmembrane part of the HIV-2 envelope protein but sometimes differ in the use of "gpl20" to mean the other part. For this reason, the term "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. The term "N3 loop of the HIN-2 envelope protein means the V3 loop of that larger non-transmembrane protein. Analogously, "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.

In the early stages of HIV infection, 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. 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 RΝA into the target cell immediately follows fusion. Because of their role in binding, fusion, and RΝA entry into host target cells, much work on therapy and vaccination for HIV has focused on the gpl20 and gρ41 proteins. It has been thought that identification of one or more epitopes of the envelope protein that will produce neutralizing antibodies or inhibitors of the HIV binding process would lead to a successful vaccine or treatment.

During investigations that matched the specificities of circulating antibodies of infected individuals to HIV envelope protein epitopes, investigators have noted a marked response to peptides of the gp41 transmembrane envelope protein. Despite the fact that most of the epitopes of gp41 are hidden, strong antibody responses to gp41 envelope proteins appear early in infection and persist throughout the course of AIDS.

Prompted by the realization of the role of gp41 epitopes in forming antibodies, attempts have been made to prepare vaccines from gp41 envelope protein. Antibodies found in response to gp41 protein administration, however, did not neutralize infective HIV virus and instead enhanced viral infectivity. Subsequent research has revealed that antibodies directed against the immunodominant cystine loop of the gp41 protein (positions 603-609) in particular are not protective and can enhance viral infectivity in vitro. Gp41, rather than elicit "neutralizing" antibodies (antibodies that protect by neutralizing the effect of HIV infection) elicits "enhancing" antibodies (antibodies that enhance HIV infectivity). Furthermore, other parts of the 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. For example, 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. Unfortunately, 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. No. 5,756,674. Those peptides are short and have sequences that closely resemble the natural gp41 sequence. Unfortunately, the individual peptide sequences proposed correspond with only a tiny fraction of the HIV epitopic repertoire and have been identified in the context of a particular monoclonal antibody. This piecemeal approach to immunization may not provide a suitable long term vaccine, in view of the very rapid and diverse alteration of viral sequences that occurs during HIV infection. A more comprehensive approach is needed for a vaccine that can anticipate a broad range of HIV epitopes.

Both simple primary sequences of gpl20 or gp41 protein as well as more complex three-dimensional epitopes having secondary helix or pleated sheet or tertiary intermolecular structure can elicit neutralizing antibodies against HIV. 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. When 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. In this case, stabilizing the secondary helix structure of the epitope should make the peptide more potent in inducing an immune response against the epitope. Accordingly, 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. Unfortunately, the methods proposed generally are limited to helical secondary structures and 6 amino acid long segments. Epitopes useful for vaccines often have non-helical secondary and tertiary structures made up of differing lengths of peptide sequences. Thus, the problem of stabilizing a three dimensional structure remains and will become increasingly important as more workers in this field attempt to use synthetic peptides as immunogens.

To obtain good cellular immunity 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. More elaborate attempts to engineer a peptide have simply led to fusing a cytotoxic T lymphocyte stimulatory peptide to the antigen such as described by U.S. No. 5,759,769, the contents of which are herein incorporated by reference in their entirety. Publications in the HIV field teach that the gp41 protein generally is a bad immunogen that elicits enhancing antibodies and should be avoided. These publications teach away from using large stretches of gp41, and instead emphasize taking, if anything, a small epitope from gp41, such as the fusion peptide of region 662 through 667. In the latter case, the epitope is used as a small peptide or combined as part of a larger peptide sequence that is not from gp41.

Finally, the general problem of "virus escape" , which is particularly an issue for an RNA virus such as HIV or HCV that replicates and mutates rapidly, has not been solved adequately. 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. Moreover, the inventors believe that a perspective is needed that benefits from the study of many strains having many alternative epitopes, from which a minimum set of antigens can be determined that anticipates a broad range of HIV epitopes. Moveover, although not properly described, if at all noted in the literature, "universal" epitopes that differ from all known sequences and that cross-react broadly across clades may be desirable.

Summary of the Invention

It is an object of an embodiment of the invention to provide a peptide useful for a vaccine, the peptide having a sequence of an envelope protein from a virus that fuses with a host cell in a pH-independent manner, the peptide having the modification wherein an enhancing epitopic sequence of the envelope protein is replaced with a neutralizing sequence from the same virus. In a preferred embodiment 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. In another preferred embodiment 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 teπninal 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 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

SEQ ID No : 1 RARLQAWEKTLEDQARLNCΑLDKNMiC

SEQ ID No : 2 RARLQAWEKTLEDQARLNCE DΕNOiSC

SEQ ID No : 3 RARLQAWEKTLEDQARLNCK DKHςβfC

SEQ ID No : 4 RARLQAWEKTLEDQARLNCQLDKSHΑSC

SEQ ID No : 5 RARLQAWEKTLEDQARLNCELDΈVΆSC SEQ ID No : 6 RARLQAWEKTLEDQARLNCQEMRIG-PMAWYSMC

SEQ ID No : 7 RARLQAWEKTLEDQARLNCQDIYTGPMR RS C

SEQ ID No : 8 RARLQAWEKTLEDQARLNCKS IHIGPGRAFYTC

SEQ ID No : 9 RARLQAWEKTLEDQARLNCQRTHIGPGQALYTC

SEQ ID No : 1 0 RARLQAWEKTLEDQARLNCIM MSGHVFHSRYC

SEQ ID No : 1 1 RARLQAWEKTLEDQARLNC TS ITIGPGQVFYRC

SEQ ID NO : 12 J ARiβAWEKriEDβAR-LNCQEMRIGPMAWYSMCTTTVP NS

SEQ ID NO : 1 3 RARLQAWEKTLEDQARLNCQOI YΥG MRΗRSMCΥT VPVINS

SEQ ID NO . 1 4 RARLQAWEKTLEDQARLNCKSIHIGPGRAFYTC YTS VKWNR

SEQ ID NO 1 5 RARLQAWEKT EDQARLNCQRΥB.IGPGQALYTC YTS VKWNR

SEQ ID NO 1 6 RARLQAWEKTLEDQARLNCIM MSGKVFliS lYC YTS VKWNR

SEQ ID NO 1 1 RARLQAWEKTLEDQARLNCTSITIGPGQVFYRCYTS VKWNR

SEQ ID NO 1 8 RARLQAWEKTLEDQARLNCALDKVIANCΥΥTVPVMS

SEQ ID NO 1 9 RARLQAWEKTLEDQARLNCELΌKWASCTΎΎXPWISIS

SEQ ID NO 20 RARLQAWEKTLEDQARLNCA ΌKVSQNC YTSVKWNR

SEQ ID NO 21 RARLQAWEKTLEDQARLNCQLΌKWASC YTSVKWNR

SEQ ID NO 22 RARLQAWEKTLEDQARLNCE ΌEWASC YTSVKWNR

SEQ ID NO 23 RARLQAWEKTLEDQARLNCELΌKWASC YTSVKWNR

These and other embodiments will be readily appreciated by a skilled artisan from reading the specification.

Detailed Description of the Preferred Embodiments

The inventors have discovered how to engineer advantageous vaccines for viruses that enter cells in a pH independent manner. One embodiment of the invention reduces or eliminates the gp41 enhancing problem while increasing humoral response by designing a peptide construct for HIV vaccination with 3 advantageous features outlined here. 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.

In preferred embodiments 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. In a preferred embodiment 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. Most preferably, 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.

Another embodiment is a peptide useful for vaccinating against viruses that fuse with their host in a pH independent manner. The peptide mimics a part of the viral envelope protein and lacks an enhancing sequence while maintaining desirable B cell and T cell sequences. In a specific embodiment 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. In an embodiment for HCV vaccination a peptide is used having the sequence for the HCV envelope protein wherein a portion is replaced with a neutralizing epitope.

Constructs according to embodiments of the invention have three features that are discussed serially and in more detail below.

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. Most preferably, 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). In preferred embodiments, 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.

In other embodiments of the invention 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.

In a preferred embodiment, 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. Νatl. 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. Furthermore, 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.

The following table lists alternative advantageous sequences relating to a gp41 fusion peptide neutralizing epitope having preferred immunological reactivity and which are contemplated for the constrained loop portion of the inventive constructs.

Clade B: SEQ ID NO: 24 E-L-L-E-L-D-K-W-A-S-L

SEQ ID NO: 25 E-L-L-A-L-D-K-W-A-S-L

Clade D: SEQ ID NO: 26 E-L-L-Q-L-D-K-W-A-S-L

SEQ ID NO: 27 E-L-L-A-L-D-K-W-A-S-L SEQ ID NO: 28 E-L-L-Q-L-D-K-W-T-S-L

SEQ ID NO: 29 E-L-L-A-L-D-K-W-T-S-L

SEQ ID NO: 30 E-L-L-Q-L-D-K-W-K-S-L

SEQ ID NO : 31 E-L-L- A-L-D-K-W-K-S-L

SEQ ID NO: 32 E-L-L-Q-L-D-K-W-A-N-L SEQ ID NO: 33 E-L-L-A-L-D-K-W-A-N-L SEQ ID NO: 34 E-L-L-Q-L-D-K-W-T-N-L SEQ ID NO: 35 E-L-L-A-L-D-K-W-T-N-L SEQ ID NO: 36 E-L-L-Q-L-D-K-W-K-N-L SEQ ID NO: 37 E-L-L-A-L-D-K-W-K-N-L SEQ ID NO: 38 E-L-L-Q-L-D-S-W-A-S-L SEQ ID NO: 39 E-L-L-A-L-D-S-W-A-S-L SEQ ID NO: 40 E-L-L-Q-L-D-S-W-T-S-L SEQ ID NO: 41 E-L-L-A-L-D-S-W-T-S-L SEQ ID NO: 42 E-L-L-Q-L-D-S-W-K-S-L SEQ ID NO: 43 E-L-L-A-L-D-S-W-K-S-L SEQ ID NO: 44 E-L-L-Q-L-D-S-W-A-N-L SEQ ID NO: 45 E-L-L-A-L-D-S-W-A-N-L SEQ ID NO: 46 E-L-L-Q-L-D-S-W-T-N-L SEQ ID NO: 47 E-L-L-A-L-D-S-W-T-N-L SEQ ID NO: 48 E-L-L-Q-L-D-S-W-K-N-L SEQ ID NO: 49 E-L-L-A-L-D-S-W-K-N-L SEQ ID NO: 50 E-L-L-Q-L-G-S-W-A-S-L SEQ ID NO: 51 E-L-L-A-L-G-S-W-A-S-L SEQ ID NO: 52 E-L-L-Q-L-G-S-W-T-S-L SEQ ID NO: 53 E-L-L-A-L-G-S-W-T-S-L SEQ ID NO: 54 E-L-L-Q-L-G-S-W-K-S-L SEQ ID NO: 55 E-L-L-A-L-G-S-W-K-S-L SEQ ID NO: 56 E-L-L-Q-L-G-S-W-A-N-L SEQ ID NO: 57 E-L-L-A-L-G-S-W-A-N-L SEQ ID NO: 58 E-L-L-Q-L-G-S-W-T-N-L SEQ ID NO: 59 E-L-L-A-L-G-S-W-T-N-L SEQ ID NO: 60 E-L-L-Q-L-G-S-W-K-N-L SEQ ID NO: 61 E-L-L-A-L-G-S-W-K-N-L SEQ ID NO: 62 E-L-L-Q-L-G-K-W-A-S-L SEQ ID NO: 63 E-L-L-A-L-G-K-W-A-S-L SEQ ID NO: 64 E-L-L-Q-L-G-K-W-T-S-L SEQ ID NO: 65 E-L-L-A-L-G-K-W-T-S-L SEQ ID NO: 66 E-L-L-Q-L-G-K-W-K-S-L SEQ ID NO: 67 E-L-L-A-L-G-K-W-K-S-L SEQ ID NO: 68 E-L-L-Q-L-G-K-W-A-N-L SEQ ID NO: 69 E-L-L-A-L-G-K-W-A-N-L SEQ ID NO: 70 E-L-L-Q-L-G-K-W-T-N-L SEQ ID NO: 71 E-L-L-A-L-G-K-W-T-N-L SEQ ID NO: 72 E-L-L-Q-L-G-K-W-K-N-L SEQ ID NO: 73 E-L-L-A-L-G-K-W-K-N-L SEQ ID NO: 74 L-L-E-L-D-K-W-A-S-L SEQ ID NO: 75 L-L-E-L-D-K-W-A-S-V SEQ ID NO: 76 L-L-E-L-D-K-W-A-G-L SEQ ID NO: 77 L-L-E-L-D-T-W-A-S-L SEQ ID NO: 78 L-L-E-L-D-T-W-A-S-V SEQ ID NO: 79 L-L-A-L-D-K-W-A-S-L SEQ ID NO: 80 L-L-A-L-D-K-W-A-S-V SEQ ID NO: 81 L-L-A-L-D-T-W-A-S-L SEQ ID NO: 82 L-L-A-L-D-T-W-A-S-V

SEQ ID NO: 83 L-L-Q-L-D-K-W-A-S-L

SEQ ID NO: 84 L-L-Q-L-D-K-W-A-S-V

SEQ ID NO: 85 L-L-Q-L-D-T-W-A-S-L

SEQ ID NO: 86 L-L-Q-L-D-T-W-A-S-V

Group O: SEQ ID NO: 87 E-L-L-E-L-D-E-W-A-S-I

SEQ ID NO: 88 E-L-Q-E-L-D-E-W-A-S-I SEQ ID NO: 89 L-L-E-L-D-E-W-A-S-I

SEQ ID NO: 90 L-L-E-L-D-E-W-A-S-L

HIV-2 SEQ ID NO: 91 L-Q-K-L-N-S-W-D-I-F

SEQ ID NO: 92 L-Q-K-L-N-Q-W-D-I-F SEQ ID NO: 93 L-Q-K-L-N-N-W-D-I-F

SEQ ID NO: 94 L-Q-K-L-N-S-W-D-V-F

SEQ ID NO: 95 L-Q-K-L-N-Q-W-D-V-F

SEQ ID NO: 96 L-Q-K-L-N-N-W-D-V-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.

Without wishing to be bound by any one particular theory of their invention, the inventors theorize that 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. In fact, is was found that when a small 13 mer portion of peptide was excised from a 35 mer V3 loop, stabilized by crosslinking at its ends, and placed near a helical structure, the stabilized construct exhibited very good antibody binding activity compared to the original 35 mer sequence. In another embodiment, three neutralizing epitopes from the above table are used together in vaccine formulations. In this embodiment, it is most preferred to include 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.

B. Representative gp!20 V3 Cross-reactive Sequences

A large number of 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.

The following table lists alternative advantageous sequences relating to a gpl20 V3 loop peptide neutralizing epitope having preferred immunological reactivity and which are contemplated for the constrained loop portion of the inventive constructs.

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

Group O SEQ ID NO: 101 Q-E-I-K-I-G-P-M-A-W-Y-S-M

C. Representative gp!20 CD4 Binding Sequences 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.

Most Preferred Sequences from a V4 gp!20 CD4 Binding Site Region Subtype B: SEQ ID NO: 102 W-Q-E-V-G-K-A-M-Y-A-P-P

SEQ ID NO: 103 W-Q-E-V-G-K-A-M-Y-A-P-P-I-S

SEQ ID NO: 104 W-Q-E-V-G-K-A-M-Y-A-P-P-I-R

SEQ ID NO: 105 W-Q-E-V-G-K-A-M-Y-A-P-P-I-K

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

SEQ ID NO: 108 W-Q-G-V-G-K-A-M-Y-A-P-P-I-E

SEQ ID NO: 109 W-Q-R-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

SEQ ID NO: 111 W-M-R-G-S-G-L-M-Y-A-P-P-I-R SEQ ID NO: 112 W-M-R-G-S-R-L-M-Y-A-P-P-I-R

SEQ ID NO: 113 W-M-K-G-S-G-L-M-Y-A-P-P-I-R

SEQ ID NO: 114 W-M-K-G-S-R-L-M-Y-A-P-P-I-R

SEQ ID NO: 115 W-M-R-G-S-G-I-M-Y-A-P-P-I-R

SEQ ID NO: 116 W-M-R-G-S-R-I-M-Y-A-P-P-I-R SEQ ID NO: 117 W-M-K-G-S-G-I-M-Y-A-P-P-I-R

SEQ ID NO: 118 W-M-K-G-S-R-I-M-Y-A-P-P-I-R

SEQ ID NO: 119 W-M-R-G-S-G-L-M-Y-A-P-P-I-K

SEQ ID NO: 120 W-M-R-G-S-R-L-M-Y-A-P-P-I-K

SEQ ID NO: 121 W-M-K-G-S-G-L-M-Y-A-P-P-I-K SEQ ID NO: 122 W-M-K-G-S-R-L-M-Y-A-P-P-I-K

SEQ ID NO: 123 W-M-R-G-S-G-I-M-Y-A-P-P-I-K

SEQ ID NO: 124 W-M-R-G-S-R-I-M-Y-A-P-P-I-K SEQ ID NO . 125 W-M-K-G-S-G-I-M-Y-A-P-P-I-K

SEQ ID NO 126 W-M-K-G-S-R-I-M-Y-A-P-P-I-K

SEQ ID NO 127 W-M-R-G-S-G-L-M-Y-A-P-P-I-P

SEQ ID NO 128 W-M-R-G-S-R-L-M-Y-A-P-P-I-P

SEQ ID NO 129 W-M-K-G-S-G-L-M-Y-A-P-P-I-P

SEQ ID NO 130 W-M-K-G-S-R-L-M-Y-A-P-P-I-P

SEQ ID NO 131 W-M-R-G-S-G-I-M-Y-A-P-P-I-P

SEQ ID NO 132 W-M-R-G-S-R-I-M-Y-A-P-P-I-P

SEQ ID NO 133 W-M-K-G-S-G-I-M-Y-A-P-P-I-P

SEQ ID NO 134 W-M-K-G-S-R-I-M-Y-A-P-P-I-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

Other Preferred Sequences from a second V4 gp!20 CD4 Binding Site Region Subtype D: SEQ ID NO T-L-Q-C-R-I-K-Q-I-I-N-M SEQ ID NO I-T-L-P-C-R-I-K-Q SEQ ID NO I-T-L-P-C-K-I-K-Q SEQ ID NO I-T-L-Q-C-R-I-K-Q SEQ ID NO I-T-L-Q-C-K-I-K-Q SEQ ID NO I-T-I-P-C-R-I-K-Q SEQ ID NO I-T-I-P-C-K-I-K-Q SEQ ID NO I-T-I-Q-C-R-I-K-Q SEQ ID NO I-T-I-Q-C-K-I-K-Q SEQ ID NO I-R-L-P-C-R-I-K-Q SEQ ID NO I-R-L-P-C-K-I-K-Q SEQ ID NO I-R-L-Q-C-R-I-K-Q SEQ ID NO I-R-L-Q-C-K-I-K-Q SEQ ID NO I-R-I-P-C-R-I-K-Q SEQ ID NO I-R-I-P-C-K-I-K-Q SEQ ID NO I-R-I-Q-C-R-I-K-Q SEQ ID NO I-R-I-Q-C-K-I-K-Q SEQ ID NO I-K-L-P-C-R-I-K-Q SEQ ID NO: 155 I-K-L-P-C-K-I-K-Q

SEQ ID NO: 156 I-K-L-Q-C-R-I-K-Q

SEQ ID NO: 157 I-K-L-Q-C-K-I-K-Q

SEQ ID NO: 158 I-K-I-P-C-R-I-K-Q SEQ ID NO: 159 I-K-I-P-C-K-I-K-Q

SEQ ID NO: 160 I-K-I-Q-C-R-I-K-Q

SEQ ID NO: 161 I-K-I-Q-C-K-I-K-Q Subtype B: SEQ ID NO: 162 T-L-P-C-R-I-K-Q-I-I-N-M

SEQ ID NO: 163 I-T-L-P-C-R-I-K-Q SEQ ID NO: 164 I-I-L-P-C-R-I-K-Q

SEQ ID NO: 165 I-T-L-P-Q-R-I-K-Q

SEQ ID NO: 166 I-I-L-P-Q-R-I-K-Q

SEQ ID NO: 167 I-T-L-P-C-R-K-K-Q

SEQ ID NO: 168 I-I-L-P-C-R-K-K-Q SEQ ID NO: 169 I-T-L-P-Q-R-K-K-Q

SEQ ID NO: 170 I-I-L-P-Q-R-K-K-Q

Group O: SEQ ID NO: 171 Q-A-I-C-K-L-R-Q-V-V-R-S

SEQ ID NO: 172 T-Q-A-I-C-K-L-R-Q

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

Other useful 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.

Most preferred is the combined use of at least nine constrained neutralizing peptide constructs according to the invention, 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. Of course, additional peptides having sequences corresponding to the analogous portions of HIV-2 envelope protein also are desired for inclusion in a vaccine.

In embodiments useful for vaccines effective against other infections and cancer, known epitopic sequences are used by insertion into constrained loop portions in a like manner.

Feature 2: Crosslink the Epitope to Constrain its Structure

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. Of course, in some situations, 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. In some cases, 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. more than half of the amino acids that create the epitope) is formed by amino acids within the bounded portion, or 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. Preferably cross-linking occurs by the formation of a sulphur-sulphur bond via formation of a cystine from oxidation of two cysteines. This type of crosslinking is preferred in cases where the 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.

Other means of cross-linking terminal residues of the epitopic region are contemplated and known to the skilled artisan as, for example, described in WO 98/20036, the content of which is explicitly incorporated by reference in its entirety. For example, 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. In one embodiment the sequence is 6 amino acids long and forms an α-helix within the loop as described in U.S. No. 98/20036. Using these known methods one can, from 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 α-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.

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. carbodiimide) to cyclize the peptide; and (2) 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, and wherein one of the flanking residues is added to the peptide chain carrying a difunctional linker such that one functional group of the linker is coupled to the residue's side chain amide bond-forming groups, followed by coupling of the linker's free functional group to the side chain amide bond-forming group on the other flanking residue to constrain the five to thirteen amino acid long peptide.

Any amino acid that has a side chain containing a group capable of forming an amide bond can be used as a flanking (i.e. "terminal") residue herein. Suitable 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. Of course, the functional groups on either side may be used such as thiol (SH) or hydroxyl (OH) groups.

The other method of cyclizing of a neutralizing epitope is through formation of a thioether bond. This method is known to those skilled in the art as for example described in US Patent 6,031 ,073, the content of which is herewith incorporated by reference in its entirety. For example, 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.

Feature 3: Add Secondary Structure Outside the Constrained Epitope It was further discovered that increasing the amount of secondary structure of a construct outside of a constrained epitope region increases antigenicity of the constrained region. 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. In preferred embodiments, 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. For example, the inventors discovered that 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.

Without wishing to be bound by any one theory of the invention, 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. In alternative embodiments, the alpha helix portion may be 6, 7, 8, 9, 10, 11, 12, 13 or more amino acids long. In other advantageous embodiments alpha helix is added to the carboxyl terminal side of the constrained cross- linked loop.

Of course, other amino acid combinations, for example, that provide beta pleated sheet between different parts of the peptide, or that provide alpha helix extending further away from the amino terminal side of the constrained region are advantageous and are contemplated. The following substitutions of amino acids outside the amino terminal side of the constrained region represent are contemplated that can form and/or increase alpha helix structure.

Position From Amino Acid Possible Amino Acid Substitutions

Constrained Region

21 R QMWANDEGHILKFPST 20 A MRDEHK

19 R MADEHK

18 L QMRANDEGHKI

17 Q MRADEGHLKPS

16 A MDEK

15 W LQMRANDCEGF

14 E KD

13 K QTMWANDEH

12 T QMWRAND

11 L QMWRAEHKFST

10 K EHDAM

9 D NKRHEQWFMILTSCGP

8 Q MRADEHLKS

7 A ME

6 R QMADEHLKFS

In advantageous embodiments, 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. In other advantageous embodiments, 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.

Of course, further extensions of the peptide chain on either side of the constrained cross-linked loop region are possible and form advantageous vaccine reagents in accordance with these principles. 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 teπninal side of the constrained region.

In a preferred embodiment, 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.

The 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 As described in the referenced publications, vaccine constructs preferably contain one or more T helper and/or cytotoxic lymphocyte sequences. In those cases, generally the T cell stimulating sequence is simply added to another sequence by making a fusion peptide.

In a departure from that approach, the inventors realized that certain sequences flanking the immunodominant loop of gp41 can be used as naturally occurring adjuvants to enhance the effect of peptide sequence(s) within the loop that participate in neutralizing epitope formation. In contrast, many publications in the HIN area stress that gp41 peptide enhances viral infectivity and is a very poor, if not dangerous vaccine agent. Nevertheless, the inventors have discovered that they could combine gp41 (and also gp36) adjuvant sequences such as T helper and cytotoxic lymphocyte active sequences with a constrained epitope of neutralizing epitope amino acids to make useful vaccine peptides. Moreover, in most preferred embodiments, 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.

The activities of the 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. In most preferred embodiments, 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. Preferably 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. In further preferred embodiments 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-Ν-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

E-K-T-L-K-D-Q-A-R-L E-R-T-L-K-D-Q-Q-R-R E-R-T-L-K-D-Q-Q-R-L E-R-T-L-K-D-Q-A-R-R E-R-T-L-K-D-Q-A-R-L

Most preferred adjuvant sequences to place at the carboxyl terminus side adjacent to the loop are the following.

C-T-T-TN-P-W-N-A-S-W-S-N

C-T-T-T-V-P-W-N-A-T-W-S-N

C-T-T-T-V-P-W-N-S-S-W-S-N

C-T-T-T-V-P-W-N-S-T-W-S-N C-T-T-T-V-P-W-N-T-S-W-S-N

C-T-T-T-V-P-W-N-T-T-W-S-N

C-T-T-T-V-P-W-N-R-S-W-S-N

C-T-T-T-V-P-W-N-R-T-W-S-N

C-T-T-T-V-K-W-N-A-S-W-S-N C-T-T-T-V-K-W-N-A-T-W-S-N

C-T-T-T-V-K-W-N-S-S-W-S-N

C-T-T-T-V-K-W-N-S-T-W-S-N

C-T-T-T-V-K-W-N-T-S-W-S-N

C-T-T-T-V-K-W-N-T-T-W-S-N C-T-T-T-V-K-W-N-R-S-W-S-N

C-T-T-T-V-K-W-N-R-T-W-S-N

C-T-T-N-V-P-W-N-A-S-W-S-N

C-T-T-N-V-P-W-N-A-T-W-S-N

C-T-T-N-V-P-W-N-S-S-W-S-N C-T-T-N-V-P-W-N-S-T-W-S-N

C-T-T-NN-P-W-N-T-S-W-S-N

C-T-T-N-V-P-W-N-T-T-W-S-N

C-T-T-N-V-P-W-N-R-S-W-S-N

C-T-T-N-V-P-W-N-R-T-W-S-N C-T-T-N-V-K-W-N-A-S-W-S-N

C-T-T-N-V-K-W-N-A-T-W-S-N

C-T-T-N-V-K-W-N-S-S-W-S-N C-T-T-NN-K-W-N-S-T-W-S-N

C-T-T-N-V-K-W-N-T-S-W-S-N

C-T-T-N-V-K-W-N-T-T-W-S-N

C-T-T-N-V-K-W-N-R-S-W-S-N C-T-T-N-V-K-W-N-R-T-W-S-N

C-T-T-A-V-P-W-N-A-S-W-S-N

C-T-T-A-V-P-W-N-A-T-W-S-N

C-T-T-A-V-P-W-N-S-S-W-S-N

C-T-T-A-V-P-W-N-S-T-W-S-N C-T-T-A-V-P-W-N-T-S-W-S-N

C-T-T-A-V-P-W-N-T-T-W-S-N

C-T-T-A-V-P-W-N-R-S-W-S-N

C-T-T-A-V-P-W-N-R-T-W-S-N

C-T-T-A-V-K-W-N-A-S-W-S-N C-T-T-A-V-K-W-N-A-T-W-S-N

C-T-T-A-V-K-W-N-S-S-W-S-N

C-T-T-A-V-K-W-N-S-T-W-S-N

C-T-T-A-V-K-W-N-T-S-W-S-N

C-T-T-A-V-K-W-N-T-T-W-S-N C-T-T-A-V-K-W-N-R-S-W-S-N

C-T-T-A-V-K-W-N-R-T-W-S-N

C-T-T-S-V-P-W-N-A-S-W-S-N

C-T-T-S-V-P-W-N-A-T-W-S-N

C-T-T-S-V-P-W-N-S-S-W-S-N C-T-T-S-V-P-W-N-S-T-W-S-N

C-T-T-S-V-P-W-N-T-S-W-S-N

C-T-T-S-V-P-W-N-T-T-W-S-N

C-T-T-S-V-P-W-N-R-S-W-S-N

C-T-T-S-V-P-W-N-R-T-W-S-N C-T-T-S-V-K-W-N-A-S-W-S-N

C-T-T-S-V-K-W-N-A-T-W-S-N

C-T-T-S-V-K-W-N-S-S-W-S-N C-T-T-S-N-K-W-N-S-T-W-S-N

C-T-T-S-V-K-W-N-T-S-W-S-N

C-T-T-S-V-K-W-N-T-T-W-S-N

C-T-T-S-V-K-W-N-R-S-W-S-N C-T-T-S-V-K-W-N-R-T-W-S-N

C-Y-T-T-V-P-W-N-A-S-W-S-N

C-Y-T-T-V-P-W-N-A-T-W-S-N

C-Y-T-T-V-P-W-N-S-S-W-S-N

C-Y-T-T-V-P-W-N-S-T-W-S-N C-Y-T-T-V-P-W-N-T-S-W-S-N

C-Y-T-T-V-P-W-N-T-T-W-S-N

C-Y-T-T-V-P-W-N-R-S-W-S-N

C-Y-T-T-V-P-W-N-R-T-W-S-N

C-Y-T-T-V-K-W-N-A-S-W-S-N C-Y-T-T-V-K-W-N-A-T-W-S-N

C-Y-T-T-V-K-W-N-S-S-W-S-N

C-Y-T-T-V-K-W-N-S-T-W-S-N

C-Y-T-T-V-K-W-N-T-S-W-S-N

C-Y-T-T-V-K-W-N-T-T-W-S-N C-Y-T-T-V-K-W-N-R-S-W-S-N

C-Y-T-T-V-K-W-N-R-T-W-S-N

C-Y-T-N-V-P-W-N-A-S-W-S-N

C-Y-T-N-V-P-W-N-A-T-W-S-N

C-Y-T-N-V-P-W-N-S-S-W-S-N C-Y-T-N-V-P-W-N-S-T-W-S-N

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C-Y-T-S-V-K-W-N-S-S-W-S-N C-Y-T-S-V-K-W-N-S-T-W-S-N C-Y-T-S-V-K-W-N-T-S-W-S-N C-Y-T-S-V-K-W-N-T-T-W-S-N C-Y-T-S-V-K-W-N-R-S-W-S-N C-Y-T-S-V-K-W-N-R-T-W-S-N

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In most preferred embodiments 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. In preferred embodiments this eight amino acid portion comprises four very hydrophobic residues selected from the group consisting of isoleucine, leucine, valine, tryptophan, tyrosine and phenylalanine. Without wishing to be bound by any one theory of this embodiment of their invention, 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

Anticipate Virus Structures 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.

In selecting multiple sequences for the constrained loop, 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). In the case of the hepatitis C virus, 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. For HIV tests, it was discovered that Subtype D peptides cross-react well with the very rare HIV-1 samples that fell into the second group. Accordingly, 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. Of course, as described above, in selecting 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.

In a preferred embodiment, 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. In a preferred embodiment 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.

The combination of peptide constructs according to this embodiment of the invention can also be used in a confirmatory test for detection of gpl20 response. In this case, 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. Preferably the peptide(s) are immobilized onto a solid phase and are used to detect antibody against gpl20 of HIV.

Optional Improvement 3: Alter Hydrophobic Amino Acids to Hydrophilic Forms

As detailed in co-pending applications 60,091,659 filed July 2, 1998 (attorney docket No. 073294/0160), 60/072,863 filed January 28, 1998 (attorney docket No. 073294/0161), 60/072,981 filed January 29, 1998 (attorney docket No. 073294/0162), 60/098,693 filed August 31, 1998 (attorney docket No. 073294/0172), 60/088,229 filed September 1, 1998 (attorney docket No. 073294/0168), 60/088,229 filed June 5, 1998 (attorney docket No. 073294/0127), 60/100047 filed September 11, 1998 (attorney docket No. 073294/0190), 60/100,047 filed September 16, 1998 (attorney docket No. 073294/0191), 100,422 filed October 19, 1998 (attorney docket No. 073294/0193) and 60/104,681 filed October 19, 1998 (attorney docket No. 073294/0197), which are herein incorporated in their entireties by reference, the inventors discovered that new peptide antigens having altered and useful immunological characteristics could be prepared by changing a hydrophobic amino acid (for example, leucine) to a hydrophilic amino acid (for example, glutamine or argi ine). One demonstrable effect of this change was to remove immunological reactivity of an antigen for Group M strains relative to reactivity for Group O strains. In this case, the alteration made the antigen relatively more specific for strains that are closer in DNA and peptide sequence to the strain from which the antigen originally was derived.

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.

For example, 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. Analogously, and/or at the same time, a leucine on an outside helix can associate (mutually repel water) with a leucine from the constrained portion. These kinds of associations stabilize epitopic structures and are particularly preferred, especially when using the CD4 binding site as a neutralization epitope. It is particularly preferred to place a CD4 binding site region within the constrained region and to place another cooperative CD4 binding site region nearby. The term "cooperative CD4 binding site region" refers to the fact that regions are chosen that normally come together to form at least part of the CD4 receptor binding site.

In a preferred embodiment, 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. By selecting two closely associated CD4 binding site regions, the regions can be made to come together and form a tertiary structure epitopic site to stimulate production of neutralizing antibodies. Most preferably, 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.

Prompted by the discovery that modifications of hydrophobic amino acids to hydrophilic amino acids enhanced antigenicity, intermediate sized peptides were synthesized having additional substitutions of hydrophilic amino acid residues for hydrophobic residues. These peptides have sequences that correspond to (i.e., at least half of the --mino acids correspond in identity with) naturally-occurring sequences. The synthesized peptides showed greater binding with antibody from HIV-1 O Group infected individuals compared to peptides that have sequences that are identical to sequences from naturally occurring peptides.

Preferred Constructs made from Modifying a gp41 or gp36 Peptide Sequence

In a preferred embodiment, 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. In one previous study, 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.

Although such a single mutation provides some relief from the enhancement from the loop epitope, it does not adequately (if at all) reverse the immunosuppressive effect of the region approximately 6 to 21 positions to the amino terminal side of the loop. 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. Such 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. These preferred sequences are particularly useful because they lead to greater predicted alpha helix in the region and help avoid structures that simulate the immunosuppressive structure of the gp41 sequence. The inventors found that increasing alpha helix in this region improved immunoreactivity of epitopes formed at the immunodominant loop region. This immunoreactivity is very desirable in a vaccine agent. Use of the Peptide constructs in Vaccines

As described above, 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. Thus, 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.

Examples of 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.

In a preferred embodiment 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. In addition, 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.

It is understood that 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. For instance, 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. Thus, although preferred amino acid substitutions are shown in the tables, further conservative substitutions are possible and sometimes desirable. By "conservative" 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.

Preferably, 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. Where 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.

In addition, 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. 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. By 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. Generally, 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. As the

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. Further, 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.

Generally, 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

Amino Acids Codons

Alanine Ala A GCA GCC GCG GCU

Cysteine Cys C UGC UGU

Aspartic acid Asp D GAC GAU

Glutamic acid

Glu E GAA GAG

Phenylalanine

Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU

Histidine

His H CAC CAU Isoleucine

He I AUA AUC AUU

Lysine Lys K AAA AAG

Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine

Met M AUG

Asparagine

Asn N AAC AAU

Proline Pro P CCA CCC CCG CCU Glutamine

Gin Q CAA CAG

Arginine Arg R AGA AGG CGA CGC CGG CGU

Serine Ser S AGC AGU UCA UCC UCG UCU

Threonine Thr T ACA ACC ACG ACU

Valine Val V GUA GUC GUG GUU

Tryptophan

Trp W UGG

Tyrosine Tyr Y UAC UAU The conservative changes in amino acid sequence are easily carried out by making such changes, and, in fact, a considerable amount of work in this area has provided algorithms to use in making such changes. For example, 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

(+4.5); valine (+4.2); leucine ( + 3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); Jo methionine ( + 1.9); alanine ( + 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a peptide with similar biological activity, i.e., still obtain a biological functionally equivalent peptide. In making such changes, the substitution of amino acids whose hydropathic indices are within +- 2 is preferred, those which are within +- 1 are particularly preferred, and those within +- 0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a peptide, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Pat. No. 4,554,101, the following 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).

It is understood that an 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. In such changes, the 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. As outlined above, 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. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Site-specific mutagenesis is a technique useful in the preparation of individual 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. Typically, 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.

In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications. As will be appreciated, 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. In general, 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. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement. The preparation of 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. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.

In another embodiment of the invention, 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. In addition to covalent linkages, 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. For example, 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. Also useful are a large number of 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. A variety of such 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. , Milwaukee, Wis.) and include reactive esters of 6-maleimidocaproic acid, 2 bromoacetic acid, 2-iodoacetic acid, 4-(N-maleimido-methyl) cyclohexane-1-carboxylic acid and the like. The carboxyl groups can be activated by combining them with succinimide or l-hydroxy-2-nitro-4-sulfonic acid, sodium salt. A particularly preferred coupling agent is succinimidyl-4-(n-maleimidomethyl) cyclohexane- 1 -carboxylate (SMCC) .

In another aspect of the present invention, 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. Chem. Soc, 105, 6442 (1983); Merrifield, Science, 232, 341-347 (1986); and Barany and Merrifield, The Peptides (Gross and Meienhofer, eds., Academic Press, New York, 1979), 1-284. Alternatively, suitable recombinant DNA technology may be employed for the preparation of the peptides of the present invention, wherein a nucleotide sequence that encodes a peptide of interest is inserted into an expression vector, transformed or transfected into a suitable host cell and cultivated under conditions suitable for expression. These procedures are generally known in the art, as described generally in Sambrook et al. , Molecular Cloning, A Laboratory Manual (2d ed., Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989), and Current Protocols in Molecular Biology (Ausubel et al., eds., John Wiley and Sons, Inc. , New York, 1987), and U.S. Pat. Nos. 4,237,224, 4, 273,875, 4,431,739, 4,363,877 and 4,428,941 , for example.

Thus, 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. For example, 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. By this means, a polyprotein is used that incorporates several antigen epitopes or T cell epitopes into a single polypeptide. As 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. , J. Am. Chem. Soc , 103, 3185 (1981), 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.

For expression of fusion peptides, 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. For example, 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. All of the variations recited hereinabove regarding the molecule of the present invention and the polypeptide that such a molecule includes may be used in the context of the method of provoking an immune response. A preferred preparation of the 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. In particular, 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.

One skilled in the art will appreciate that suitable methods of administering a compound to a patient for the treatment or prophylaxis of HIV infection are available. Although more than one route can be used to administer a particular compound, a particular route can provide a more immediate and more effective reaction than another route. Accordingly, the described methods provided herein are merely exemplary and are in no way limiting. Generally, 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. In therapeutic applications, 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.

Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. In any event, 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. For therapeutic use, 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.

The pharmaceutical 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.

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).

The choice of 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.

The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intramuscular, interperitoneal, rectal, and vaginal administration are merely exemplary and are in no way limiting. Preferably, the pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides 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. Overall, the requirements for effective pharmaceutical carriers for parenteral compositions are well known to those of ordinary skill in the art. See Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250, (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986). 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, or emulsifying agents and other pharmaceutical adjuvants.

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, and 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. The 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. 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. 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.

The molecules and/or peptides of the present invention, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. For aerosol administration, 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. Representative of such agents are the 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. These 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.

Additionally, 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. 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. In some embodiments, it may be desirable to include in the pharmaceutical composition at least one component that primes CTL generally. Lipids have been identified that are capable of priming CTL in vivo against viral antigens, e.g. , tripalmitoyl-S- glycerylcysteinly-seryl-serine (P sub 3 CSS), which can effectively prime virus specific cytotoxic T lymphocytes when covalently attached to an appropriate peptide. See, Deres et al., Nature, 342, 561-564 (1989). Peptides of the present invention can be coupled to P sub 3 CSS, for example and the lipoprotein administered to an individual to specifically prime a cytotoxic T lymphocyte response to HIV.

The 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.

Thus, 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).

It will be appreciated by one of ordinary skill in the art that, in addition to the aforedescribed pharmaceutical compositions, the compounds of the present inventive method may be formulated as 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. In these preparations 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. Thus, 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 variety of methods are available for preparing liposomes, as described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9, 467 (1980), and U.S. Pat. Nos. 4,235,871 , 4,501 ,728, 4,837,028 and 5,019,369. For targeting to the immune cells, 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.

In another aspect 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. Adjuvants such as incomplete Freund"s adjuvant, aluminum phosphate, aluminum hydroxide, or alum or materials well known in the art. And, as mentioned above, cytotoxic T lymphocyte responses can be primed by conjugating peptides of the invention to lipids, such as P sub 3 CSS. 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. Such an amount is defined to be a "immunogenically effective dose" or a "prophylactically effective dose. " In this use, 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.

For therapeutic or immunization purposes, 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. 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). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g. ,

Salmonella typhi vectors and the like, will be apparent to those skilled in the art from the description herein.

The 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. Accordingly, in more detail, ex vivo therapy as used herein 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. The host's WO 00/58438 ^ PCT/USOO/08232

cells also may be exposed to vectors that carry genes encoding the peptides, as described above. Once transfected with the vectors, the cells may be propagated in vitro or returned to the patient. The cells that are propagated in vitro may be returned to the patient after reaching a predetermined cell density. In one method, in vitro CTL responses to HIV are induced by incubating in tissue culture a patient's CTL precursor cells (CTLp) together with a source of antigen-presenting cells (APC) and the appropriate immunogenic peptide. After an appropriate incubation time (typically 1-4 weeks), in which the CTLp are activated and mature and expand into effector CTL, the cells are infused back into the patient, where they will destroy their specific target cell (an HIV infected cell). To optimize the in vitro conditions for the generation of specific cytotoxic T cells, the culture of stimulator cells is typically maintained in an appropriate serum-free medium. Peripheral blood lymphocytes are isolated conveniently following simple venipuncture or leukapheresis of normal donors or patients and used as the responder cell sources of CTLp. In one embodiment, 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. Specifically, the MHC restriction of the CTL of a patient can be determined by a number of methods known in the art. For instance, CTL restriction can be determined by testing against different peptide target cells expressing appropriate or inappropriate human MHC class I. The peptides that test positive in the MHC binding assays and give rise to specific CTL responses are identified as immunogenic peptides.

Methods of reintroducing cellular components are known in the art and include procedures such as those exemplified in U.S. Pat. No. 4,844,893 to Honsik, et al. and U.S. Pat. No. 4,690,915 to Rosenberg. For example, administration of activated CTLs via intravenous infusion is typically appropriate. Certain disadvantages of conventional vaccines are overcome by using what is called "genetic immunization" (Tang, 1992). This technology involves inoculating simple, naked plasmid DNA encoding a pathogen peptide into the cells of the host. The pathogen's antigens are produced intracellularly and, depending on the attached targeting signals, can be directed toward major histocompatibility complex (MHC) class I or II presentation. Risk of infection is essentially eliminated and the DNA can be delivered to cells not normally infected by the pathogen. Compared to conventional vaccines, the production of genetic vaccines is straightforward and DNA is considerably more stable than proteinaceous or live/attenuated vaccines. Genetic immunization (a.k.a. DNA, polynucleotide etc. immunization) with specific genes has shown promise in several model systems of pathogenic disease, and a few natural systems. Use of DNA (or RNA) thus overcomes some of the problems encountered when an animal is presented directly with an antigen. U.S. No. 6,008,200 describes particularly useful information in this context.

Genetic immunization concerns DNA segments, that can be isolated from virtually any non-mammalian pathogen source, that are free from total genomic DNA and that encode the peptides disclosed herein. In addition these DNA segments may be synthesized entirely in vitro using methods that are well-known to those of skill in the art. As used herein, the term "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.

Similarly, 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. In this respect, 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.

The invention can be further understood by reference to the following examples, which illustrates representative embodiments of the invention and are not meant to be limiting in any way.

Example 1

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.

This data indicates that the constrained 13 mer construct having less epitopes retains about as much immuno-reactivity as the entire V3 loop from which it came.

Example 2

The following 27 mer constructs are chemically synthesized. 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.

SEQ ID No : RARLQAWEKTLEDQARLNCALOYΛJANC SEQ ID No : RARLQAWEKTLEDQARLNCEIJOXNASC

SEQ ID No : RARLQAWEKTLEDQARLNCALOKNQVIC

SEQ ID No : RARLQAWEKTLEDQARLNCQLDKWASC

SEQ ID No : RARLQAWEKTLEDQARLNCELDKH&SC

SEQ ID No : RARLQAWEKTLEDQARLNCEKDKHASC

Example 3

The following 33 mer constructs are chemically synthesized. 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. In other contemplated experiments 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.

SEQ ID No : RARLQAWEKTLEDQARLNCQEMRIGFMANYSMC SEQ ID No : RARLQAWEKTLEDQARLNCQOIYTGΕMR^RSMC SEQ ID No : RARLQAWEKTLEDQARLNCKS IHIGPGRAFYTC SEQ ID No : RARLQAWEKTLEDQARLNCQRTEIGΕGQAlbY'TC SEQ ID No : RARLQAWEKT EDQARLNCIM MSGHVFHSHYC

In other experiments, 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.

Example 4

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.

SEQ I D NO RARLQAWEKTLEDQARLNCQEMRIGΕiΛA.VSYSMCΥ'TTVR^NS

SEQ I D NO RARLgArroK-T---EDC-?-- LNCQDIYTGP->--RWRSMCTTTVPWNS

SEQ I D NO RARLQAWEKTLEDQARLNCKS I H I GPGRAF YT C YTS VKWNR

SEQ I D NO RARLQAWEKTLEDQARLNCQRT! H I GPGQAL YT C YTS VKWNR

SEQ I D NO RARLQAWEKTLEDQARLNCIMLMSGEVFESRYC YTSVKWNR

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 and toxicity 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.

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 teπninal 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.

SEQ I D NO RARLQAWEKTLKDQARLNCΑLDKWMiCτ'rTVPVIliS SEQ I D NO RARLQAWEKTLEDQARLNCELOKWASCTΥΥVPVMS SEQ I D NO RARLQAWEKTLKDQARLNCA^'L DK QNC YTSVKWNR SEQ I D NO RARLQAWEKTLEDQARLNCQLOKVIASC YTS VKWNR SEQ I D NO RAR QAWEKTLKDQARLNCE ΩKWASC YTSVKWNR SEQ I D NO RARLQAWEKTLEDQARLNCELΏKWASC YTSVKWNR

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.

SEQ I D NO : RARLQAWEKTLKDQARLNCWQEVGKAMYAPPCTTIVRmS SEQ ID NO: RARLQAWEKTLEDQARLNCWMRGGSGLYAPPCTTTVPVmS

SEQ ID NO: RARLQAWEKTLKDQARLNCTIQCRIKQIINNCYTSVKWNR

SEQ ID NO: RARLQAWEKTLEDQARINCTLPCRIKQIINMCYTSVKWNR

SEQ ID NO: RARLQA WEKTLKDQARLNCQAICKLRQ WR SC YTS VKWNR

SEQ ID NO: RARLQAWEKTLEDQARLNCNYAPCHIRQWRCYTSVKWNR

SEQ ID NO: RARLQAWEKTLKDQARLNCTLQCRIKQIINMCTTTVPWNS

SEQ ID NO: RARLQAWEKTLEDQARLNCTLPCRIKQIINMCTΥΥVRmS

SEQ ID NO: RARLQAWEKTLKDQARLNCQAICKLRQWRSCYTSVKWNR

All publications and patent applications cited in this disclosure are specifically incorporated by reference in their entireties.

It is intended that the specification be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims

We claim:

1. A peptide between 16 and 75 amino acids long comprising a first conformationally constrained portion 5 to 13 amino acids long and a second portion, the second portion comprising a continuous stretch of at least 5 amino acids having predicted secondary structure, wherein the conformationally constrained portion comprises a cross-linked epitope of HIV envelope protein that induces neutralizing antibodies, the cross link formed by a link between two terminal amino acids.

2. The peptide of claim 1, comprising an alpha helical portion between 5 and 20 amino acids long outside the amino terminal end of the conformationally constrained portion..

3. The peptide of claim 1, wherein the second portion is adjacent to a terminal cross- linked amino acid of the conformationally constrained portion.

4. The peptide of claim 1, wherein the conformationally constrained portion comprises at least one 5-13 amino acid long loop formed through a linkage selected from the group consisting of disulfide, thioether and amide and the link is formed directly or through a separate bifunctional linker.

5. The peptide of claim 1 , wherein the HIV neutralizing epitope is selected from the group consisting of a portion of the V3 loop of gpl20, a portion of the V3 loop of the HIV-2 envelope protein, a portion of the fusion peptide of gp41, a portion of the fusion peptide of gp36, and a portion of a CD4 binding site region of the gpl20 HIV envelope protein.

6. The peptide of claim 1, wherein the amino terminal side of the conformationally constrained portion comprises a segment of at least 6 amino acids, the segment having an epitope that stimulates cytotoxic lymphocytes.

7. The peptide of claim 1 comprising at least one portion outside and adjacent to the conformationally constrained portion wherein said at least one portion outside and adjacent to the constrained portion has a sequence at least 50% identical to analogous portions of gp41 protein sequences outside and adjacent to the immunodominant loop of gp41 protein.

8. The peptide of claim 1, wherein the HIV neutralizing epitope has a sequence at least 50% identical to a known epitope sequence selected from the group consisting of HIV-1 Group M Subtype B envelope protein, HIV-1 Group M Subtype D envelope protein, HIV-1 Group M Subtype E envelope protein, HIV-1 Group O envelope protein and HIV-2 envelope protein.

9. The peptide of claim 8, wherein said HIV neutralizing epitope sequence is at least 60% identical to said known epitope sequence.

10. The peptide of claim 8, wherein said HIV neutralizing epitope sequence is at least 80% identical to said known epitope sequence.

11. A pharmaceutical composition useful for prophylactic or therapeutic treatment of a mammal at risk for or infected with HIV, comprising at least one peptide as described in claim 1.

12. The pharmaceutical composition of claim 11, comprising at least two peptides as described in claim 1 wherein the peptides are 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 the

HIV envelope protein.

13. The pharmaceutical composition of claim 12, wherein the neutralizing epitope from the fusion peptide of gp41 has a sequence that corresponds to an HIV-1 Group O strain sequence.

14. The pharmaceutical composition of claim 12, wherein the neutralizing epitope from the fusion peptide of gp41 has a sequence that corresponds to an HIV-1 Group M subtype D strain sequence.

15. The pharmaceutical composition of claim 12, wherein at least one of the peptides is an HIV-1 group M subtype D neutralizing epitope from the fusion peptide of gp41 and at least one of the peptides is an HIV Group O strain sequence.

16. The pharmaceutical composition of claim 12, wherein at least one of the peptides is an HIV-1 group M subtype B neutralizing epitope from the fusion peptide of gp41 and at least one of the peptides is an HIV Group O strain sequence.

17. The pharmaceutical composition of claim 12, wherein at least one of the peptides is an HIV-1 group M subtype E neutralizing epitope from the fusion peptide of gp41 and at least one of the peptides is an HIV Group O strain sequence.

18. The pharmaceutical composition of claim 12, wherein at least one of the peptides is an HIV-2 neutralizing epitope from the fusion peptide of gp36 and at least one of the peptides is an HIV Group O strain neutralizing epitope from the fusion peptide of gp41.

19. The pharmaceutical composition of claim 12, wherein at least one of the peptides is an HIV-2 neutralizing epitope from the fusion peptide of gp36 and at least one of the peptides is an HIV Group M subtype E strain neutralizing epitope from the fusion peptide of gp41.

20. The pharmaceutical composition of claim 12, wherein at least one of the peptides is an HIV-2 neutralizing epitope from the fusion peptide of gp36 and at least one of the peptides is an HIV Group M subtype D strain neutralizing epitope from the fusion peptide of gp41.

21. The pharmaceutical composition of claim 12, wherein at least one of the peptides is an HIV-2 neutralizing epitope from the fusion peptide of gp36 and at least one of the peptides is an HIV Group M subtype B strain neutralizing epitope from the fusion peptide of gp41.

22. The pharmaceutical composition of claim 12, wherein at least one of the peptides is an HIV-1 group M subtype B neutralizing epitope from the V3 loop of gpl20 and at least one of the peptides is an HIV Group M subtype E strain neutralizing epitope from the V3 loop of gρl20.

23. The pharmaceutical composition of claim 12, wherein at least one of the peptides is an HIV-1 group M subtype D neutralizing epitope from the V3 loop of gpl20 and at least one of the peptides is an HIV Group M subtype E strain neutralizing epitope from the V3 loop of gpl20.

24. The pharmaceutical composition of claim 12, wherein at least one of the peptides is an HIV-1 group M subtype B neutralizing epitope from the V3 loop of gpl20 and at least one of the peptides is an HIV Group M subtype D strain neutralizing epitope from the V3 loop of gρl20.

25. The pharmaceutical composition of claim 12, wherein at least one of the peptides is an HIV-1 group M subtype B neutralizing epitope from the V3 loop of gpl20 and at least one of the peptides is an HIV-2 strain neutralizing epitope from the V3 loop of gpl20.

26. The pharmaceutical composition of claim 12, wherein at least one of the peptides is an HIV-1 group M subtype E neutralizing epitope from the V3 loop of gpl20 and at least one of the peptides is an HIV-2 strain neutralizing epitope from the V3 loop of gpl20.

27. The pharmaceutical composition of claim 12, wherein at least one of the peptides is an HIV-1 group M subtype D neutralizing epitope from the V3 loop of gpl20 and at least one of the peptides is an HIV-2 strain neutralizing epitope from the V3 loop of gpl20.

28. The pharmaceutical composition of claim 12, wherein at least one of the peptides is an HIV-1 group M subtype B neutralizing epitope from the CD4 binding site of gpl20 and at least one of the peptides is an HIV Group M subtype E strain neutralizing epitope from the CD4 binding site of gpl20.

29. The pharmaceutical composition of claim 12, wherein at least one of the peptides is an HIV-1 group M subtype D neutralizing epitope from the CD4 binding site of gpl20 and at least one of the peptides is an HIV Group M subtype E strain neutralizing epitope from the CD4 binding site of gpl20.

30. The pharmaceutical composition of claim 12, wherein at least one of the peptides is an HIV-1 group M subtype B neutralizing epitope from the CD4 binding site of gpl20 and at least one of the peptides is an HIV Group M subtype D strain neutralizing epitope from the CD4 binding site of gpl20.

31. The pharmaceutical composition of claim 12, wherein at least one of the peptides is an HIV-1 group M subtype B neutralizing epitope from the CD4 binding site of gpl20 and at least one of the peptides is an HIV-2 strain neutralizing epitope from the CD4 binding site of gpl20.

32. The pharmaceutical composition of claim 12, wherein at least one of the peptides is an HIV-1 group M subtype E neutralizing epitope from the CD4 binding site of gpl20 and at least one of the peptides is an HIV-2 strain neutralizing epitope from the CD4 binding site of gpl20.

33. The pharmaceutical composition of claim 12, wherein at least one of the peptides is an HIV-1 group M subtype D neutralizing epitope from the CD4 binding site of gpl20 and at least one of the peptides is an HIV-2 strain neutralizing epitope from the CD4 binding site of gpl20.

34. The pharmaceutical composition of claim 12, comprising at least two V3 loop peptides wherein each V3 loop peptide contains at least one neutralizing epitope from the V3 loop of gpl20 and further comprising at least two neutralizing peptides wherein each neutralizing peptide contains a portion of the fusion peptide of gp41 or gp36.

35. The pharmaceutical composition of claim 34, comprising a first peptide of the V3 loop of gpl20 that corresponds to a sequence of an HIV-1 group M subtype B, a second peptide of the V3 loop of gpl20 that corresponds to a sequence of an HIV-1 group M subtype E, a third peptide of the neutralizing epitope of gp41 fusion peptide that corresponds to a sequence of an HIV-1 group M subtype D, and a fourth peptide of the neutralizing epitope of gp41 fusion peptide that corresponds to a sequence of HIV-1 Group O.

36. The pharmaceutical composition of claim 12 comprising three peptides having sequences that correspond to sequences of neutralizing epitopes from the V3 loop of gpl20 and comprising three peptides having sequences that correspond to sequences of neutralizing epitopes from the fusion peptide of gp41 or gp36.

37. The peptide of claim 2, wherein the hydrophobic portion comprises a sequence that corresponds to a T-helper stimulating sequence.

38. A method of prophylactic or therapeutic treatment of a mammal for HIV infection, comprising administering a prophylactically or therapeutically effective amount of a peptide as described in claim 1.

39. A method of preparing a peptide as described in claim 1, the method comprising:

(a) synthesizing a linear peptide having a first sequence 5 to 13 amino acids long that corresponds to an HIV neutralizing epitope wherein the neutralizing epitope is terminated at both ends by residues that have unprotected side chain bond-forming groups and having a second sequence that has predicted secondary structure; and

(b) bridging the two terminal residues of the first sequence with a difunctional linker.

40. A method of preparing a peptide as described in claim 1, the method comprising the steps: (a) synthesizing a linear peptide having a first sequence 5 to 13 amino acid residues long that corresponds to an HIV-1 neutralizing epitope wherein both ends of the neutralizing epitope are crosslinked by a bond selected from the group consisting of a disulfide, a thioether, and an amide to form a link directly between the terminal amino acid residues or indirectly through a bifunctional linker, and having a second sequence that has predicted secondary structure; and

(b) bridging the two terminal residues of the first sequence.

41. A peptide between 16 and 75 amino acids long comprising a first conformationally constrained portion between 5 and 13 amino acids long, and a second portion having a sequence and directionality of a segment of a gp41 or gp36 HIV envelope protein between 1 and 26 amino acid residues on the amino terminal side of the immunodominant loop sequence of the envelope protein, wherein the immunodominant loop portion is substituted with an HIV neutralizing epitope selected from the group consisting of the V3 loop of gpl20, the V3 loop of the HIV-2 envelope protein, the fusion peptide of gp41, the fusion peptide of gp36, and a CD4 binding site region of the HIV envelope protein.

42. The peptide of claim 41 having a length of between 25 and 45 amino acid residues.

43. A peptide between 27 and 75 amino acids long comprising a sequence selected from the group consisting of :

SEQ ID No RARLQAWEKTLEDQARLNCALDKWANC SEQ ID No RARLQAWEKTLEDQARLNCELDKWASC SEQ ID No RARLQAWEKTLEDQARLNCALDKWQNC SEQ ID No RARLQAWEKTLEDQARLNCQLDKWASC SEQ ID No RARLQAWEKTLEDQARLNCELDKWASC SEQ ID No RARLQAWEKTLEDQARLNCELDKWASC ;

SEQ ID No: RARLQAWEKTLEDQARLNCQEMRIGPMAWYSMC SEQ ID No: RARLQAWEKTLEDQARLNCQDIYTGPMRWRSMC SEQ ID No: RARLQAWEKTLEDQARLNCKSIHIGPGRAFYTC SEQ ID No: RARLQAWEKTLEDQARLNCQRTHIGPGQALYTC SEQ ID No: RARLQAWEKTLEDQARLNCIMLMSGHVFHSHYC

SEQ ID NO: RARLQAWEKTLEDQARLNCQEMRIGPMAWYSMCTTTVPWNS SEQ ID NO: RARLQAWEKTLEDQARLNCQDIYTGPMRWRSMCTTTVPWNS SEQ ID NO: RARLQAWEKTLEDQARLNCKSIHIGPGRAFYTCYTSVKWNR SEQ ID NO: RARLQAWEKTLEDQARLNCQRTHIGPGQALYTCYTSVKWNR SEQ ID NO: RARLQAWEKTLEDQARLNCIMLMSGHVFHSHYCYTSVKWNR

SEQ ID NO: RARLQAWEKTLEDQARLNCALDKWANCTTTVPWNS SEQ ID NO: RARLQAWEKTLEDQARLNCELDKWASCTTTVPWNS SEQ ID NO: RARLQAWEKTLEDQARLNCALDKWQNCYTSVKWNR SEQ ID NO: RARLQAWEKTLEDQARLNCQLDKWASCYTSVKWNR SEQ ID NO: RARLQAWEKTLEDQARLNCELDKWASCYTSVKWNR SEQ ID NO: RARLQAWEKTLEDQARLNCELDKWASCYTSVKWNR

44. A peptide having a modification of a sequence from an envelope protein of a virus that fuses with a host cell in a pH-independent manner wherein the modification comprises replacement of an enhancing epitopic sequence of the envelope protein with a neutralizing epitopic sequence from the same virus.

45. The peptide of claim 44, wherein the virus is selected from the group consisting of the paramyxo viruses, herpes viruses, corona viruses, human immunodeficiency virus, simian virus 5, hepatitis b virus, hepatitis C virus, hepatitis D virus, hepatitis G virus and respiratory syncytial virus.

46. The peptide of claim 44, wherein the neutralizing sequence is a sequence for a fusion peptide from the virus envelope protein.

47. The peptide of claim 44, wherein the neutralizing sequence is a sequence for a portion of the viral envelope protein that binds to a host cell receptor.

48. The peptide of claim 1 , wherein the HIV neutralizing epitope has a sequence that is identical to a naturally occurring sequence of a protein selected from the group consisting of the V3 loop of gpl20, the V3 loop of the HIV-2 envelope protein, the fusion peptide of gp41, the fusion peptide of gp36, and a CD4 binding site region of the gpl20 HIV envelope protein.

49. The peptide of claim 1, wherein the HIV neutralizing epitope has a sequence that is at least 60% identical to sequence of a protein selected from the group consisting of HIV- 1 Group M Subtype B envelope protein, HIV-1 Group M Subtype D envelope protein, HIV-1 Group M Subtype E envelope protein, HIV-1 Group O envelope protein and HIV-2 envelope protein.

50. The peptide of claim 1, wherein the HIV neutralizing epitope has a sequence that is at least 80% identical to sequence of a protein selected from the group consisting of HIV- 1 Group M Subtype B envelope protein, HIV-1 Group M Subtype D envelope protein, HIV-1 Group M Subtype E envelope protein, HIV-1 Group O envelope protein and HIV-2 envelope protein.

51. A peptide between 16 and 75 amino acids long having at least 50% sequence homology to the immunodominant domain region of an immunodeficiency retroviral envelope protein containing a constrained loop of amino acids and having the native amino acid sequence of said constrained loop replaced with a neutralizing antibody producing amino acid sequence derived from outside the immunodominant domain region.

52. A method of designing a peptide to elicit the production of neutralizing antibodies targeted to a virus that fuses with a host cell in a pH-independent manner, the method comprising the steps:

(a) obtaining a sequence of an envelope protein of the virus and (b) replacing an enhancing epitopic portion of the envelope protein sequence with a neutralizing epitopic portion sequence from the same virus.

53. The peptide of claim 2, further comprising a hydrophobic portion of at least 5 amino acids long at the carboxyl terminal side within the conformationally constrained portion