Disclosed are compositions and methods for the prevention and treatment of viral infections. The identification of distinct classes of peptides for use in both anti-viral vaccines and therapeutic formulations is reported. Peptide formulations are disclosed which enhance the systemic distribution, activity, and longevity of anti-viral cytotoxic T cells, and/or which protect human cells from HIV infection.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention addresses one or more of the foregoing or other disadvantages in the prior art by providing, firstly, compositions and methods for immunization against viral diseases, and secondly, therapeutic compositions and methods for use in inhibiting HIV infection. The improved immunization compositions, or vaccines, of the invention comprise synthetic peptide combinations which enhance the systemic distribution, level of activity, and longevity of virus-specific cytotoxic T cells (CTLs). The therapeutic formulations of the invention comprise synthetic peptides which function to protect human cells from HIV infection. The present invention therefore widely encompasses anti-viral, and particularly anti-HIV, compositions which have both preventative and therapeutic applications.

In important embodiments, the present invention concerns compositions and methods for enhancing the CTL response of an animal to a given immunogen, and particularly to a CTL-inducing peptide. A CTL-inducing peptide is a peptide, bearing a CTL-inducing epitope, which is capable of stimulating the formation, or increasing the activity, of specific cytotoxic T cells following its administration to an animal. The term "enhancing the CTL response", is used herein to encompass improvements in all aspects of the CTL response, such as for example, improving the systemic distribution, level of activity, and longevity of the response to CTL-inducing peptides.

The method of the present invention for enhancing a CTL response to an immunogen, such as a peptide, involves the addition of a composition including a separate and distinct class of peptides to the immunization mixture that possess T helper cell-inducing activity. To achieve enhancement of the CTL response in this manner one would therefore administer to the animal, in addition to the immunogen itself, an immunologically effective amount of a peptide bearing a T helper cell epitope.

In a related embodiment, the present invention provides a method of identifying a candidate substance capable of enhancing a CTL response. To identify such an enhancing substance, such as a peptide, one would administer to an animal both the candidate substance and an immunogen capable of inducing a CTL response. The CTLs would then be recovered from the animal and their activity, including systemic distribution and longevity, would be determined. A candidate substance capable of enhancing a CTL response would be identified as a substance that increased the CTL response, using any of the above parameters, over that observed in the presence of the immunogen alone.

The immunization methods of the present invention are generally applicable to enhancing CTL responses towards immunogens from any source, including those from a wide variety of infectious agents such as viruses or parasites. However, in preferred embodiments, the invention is exemplified by enhancing the CTL response against components of the HIV viral family. CTL responses may be generated against epitopes located within the products of any viral gene, such as, for example, the gag, pol, nef and env genes, with the products of the env genes being preferred targets.

Any one of a wide variety of peptide sequences may be employed as a CTL-inducing epitope in accordance with the present invention. These include the peptides listed in Table 1 (see Original Patent), which is, of course, an exemplary and not exhaustive list. In preferred embodiments, it is contemplated that peptides from the env (envelope) gene product, and more preferably, those derived from the V3 loop of HIV gp120, will be employed as CTL-inducing peptides. An exemplary list of V3 loop peptides from a variety of HIV-1 isolates is provided in Table 2 ((see Original Patent, SEQ ID NO:11 through SEQ ID NO:27, respectively, and SEQ ID NO:1). A specific example of a V3 loop-derived peptide found to be successful in generating CTL responses is the peptide R15K, having the sequence RIQRGPGRAFVTIGK (seq id no:1).

Various methods are available to identify T helper cell-inducing epitopes suitable for use in accordance herewith. For example, the amphipathicity of a peptide sequence is known to effect its ability to function as a T helper cell inducer. In preferred embodiments, it is contemplated that one would wish to employ a T helper cell-inducing epitope having an amphipathicity value of from about plus 10 to about plus 20. Other quantifiable characteristics include, for example, having an alpha helix turn of 10015 degrees, or a 3.sub.10 helix turn with 12015 degrees. In embodiments relating specifically to HIV, T helper cell-inducing peptides including sequences derived from an HIV gp120 sequence which have an amphipathicity value within the above range will be preferred.

Again in relation to HIV, the use of peptides containing the sequence CRIKQIINMWQGVGKAMYA (C19A, seq id no:2) is preferred as this peptide was found to be particularly effective at enhancing CTL responses. However, peptides including other T helper cell-inducing epitopes may be employed. A full discussion of T helper cell-inducing epitopes is given in U.S. Pat. No. 5,128,319, incorporated herein by reference.

Related embodiments of this invention concern compositions for effecting improved CTL responses. Such compositions will generally include at least two peptides, the first of which will contain the CTL-inducing epitope against which the immune response is specifically desired, and second of which will comprise a T helper cell-inducing epitope. Naturally, a combination of such a composition in which the peptides are dispersed in a pharmacologically acceptable vehicle would be an ideal formulation for use as a vaccine.

The present invention further relates to the identification of synthetic peptides which protect human cells from HIV-1 infection. Such inhibitory peptides may be employed to inhibit HIV infection of cells, for use, for example, in assay protocols and as therapeutic agents for use in the treatment of AIDS.

As used herein, the term "HIV infection-inhibiting sequence" refers to a peptide sequence which prevents entry of the HIV virus into its target cell. As such, an inhibitory peptide may be characterized as including a peptide sequence that is involved in the infection process, or that functions to contact the target cell. Infection-inhibiting peptides particularly include peptides that comprise a sequence wherein antibodies against that sequence are capable of inhibiting HIV cellular infection.

The present invention discloses that synthetic peptides with sequences derived from the HIV-1 env gene product, gp120, have the capacity to inhibit HIV cellular infection. In particular, the inventors have identified HIV infection-inhibiting sequences within the V3 loop and at the N-terminal regions of gp120. It is also contemplated that HIV infection-inhibiting sequences may prove to be located within the CD4 binding region.

In preferred embodiments, the inventors contemplate the use of HIV infection-inhibiting peptides with sequences derived from the gp120 V3 loop. As detailed herein, peptides found to be of use in this regard include, for example, those peptides of Table 11 (see Original Patent), and particularly, peptides D23 (SEQ ID NO:11), D24 (SEQ ID NO:12), D25 (SEQ ID NO:13), D26 (SEQ ID NO:14), D30 (SEQ ID NO:18), D35 (SEQ ID NO:23), D38 (SEQ ID NO:25), D39 (SEQ ID NO:26), D40 (SEQ ID NO:27) and D44 (R15K, SEQ ID NO:1) the sequences of which are shown in Table 11A (see Original Patent). These peptides have sequences derived from the V3 loops of a variety of strains such as mn, rf, wmj-3, sc, z6, eli, mn (y-1) and mn (y-p). Preferred V3-derived infection-inhibiting peptides are contemplated to include those listed in Table 11A, and those such as R15K, having the sequence RIQRGPGRAFVTIGK (seq id no:1), and also N24G, having the sequence NNTRKSIRIQRGPGRAFVTIGKIG (seq id no:3).

An important aspect of the present invention is the discovery that peptides with sequences that correspond to sequences from a variety of different HIV isolates have the ability to inhibit HIV infection of cells. Further to those shown in Table 11A, these peptides include, for example, H13N (HIGPGRAFYTTKN, seq id no:7), a V3-loop peptide from HIV-1mn strain, and T13Q (TKGPGRVIYATGQ, seq id no:6), a V3-loop peptide from HIV-1rf strain. However, in regard to Table 11, it is important to note that this data was compiled from assays specifically directed to inhibiting infection by one HIV strain, namely HIV-1 IIIB. Therefore peptides such as D27 (SEQ ID NO:15), D28 (SEQ ID NO:16), D29 (SEQ ID NO:17), D31 (SEQ ID NO:19), D32 (SEQ ID NO:20), D33 (SEQ ID NO:21), D34 (SEQ ID NO:22) and D37 (SEQ ID NO:24), listed Table 11B, which may not show activity in one specific inhibitory assay may still have utility in preventing the infection of target cells by a variety of other HIV strains, for example against HIV strains ny-5, rf, cdc4, z3, sf2, ma1, z321 and jy1.

The inventors have also identified peptides from the gp120 N-terminus, such as E13V, having the sequence EQLWVTVYYGVPV (seq id no:4), as infection-inhibiting peptides. This is an important finding as this area of gp120 is known to be relatively conserved between different HIV strains.

It should also be noted that smaller peptides which include portions of these sequences, such as, for example, R8K, having the sequence RAFVTIGK (seq id no:5), have also been identified as having infection-inhibiting sequences. As such, smaller peptides and peptide fragments are considered to be useful in accordance herewith and to be within the scope of this invention.

The infection-inhibiting peptides of the present invention may include natural, or engineered, sequence variations and yet still function to prevent HIV entry into cells. As such, the biological functional equivalents of the peptides in Tables 1, 2 and 11, and peptides such as R15K (SEQ ID NO:1), N24G (SEQ ID NO:3), E13V (SEQ ID NO:4), H13N (SEQ ID NO:7) and T13Q (SEQ ID NO:6), are also considered to fall within the scope of the present invention.

Such biological functional peptide equivalents may include changes based upon hydropathic index of amino acids. It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant protein or peptide, which in turn defines its interaction with other molecules, such as for example, receptors, antibodies, and the like. It is generally known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still result in a peptide with similar biological activity (Kyte & Doolittle, 1982).

The hydropathic indices of the amino acids are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). Biological functional equivalents are considered to be those peptides which include the substitution of amino acids whose hydropathic indices are within 2, and more preferably, within 1, and even more preferably, within 0.5.

Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly where the biological functional equivalent protein or peptide thereby created is intended for use in immunological embodiments, as is the case for the present invention. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. 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 (+3.01); glutamate (+3.01); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.51); threonine (-0.4); 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). Similarly, biological functional equivalents are considered to be those peptides which include the substitution of amino acids whose hydrophilicity values are within 2, and more preferably, within 1, and even more preferably, within 0.5.

As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. 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.

In addition to the specific sequences described above, and their biological functional equivalents, other sequences may be employed as HIV infection-inhibiting peptides. For example, as mentioned above, it is contemplated that any sequence where anti-peptide antibodies against that sequence have been shown to inhibit viral entry into cells will be a suitable HIV infection-inhibiting peptide for use in accordance herewith. The advantages of the present invention include the demonstration that such peptides can function effectively at low concentrations, such as from between about 1g/ml to about 1 ng/ml.

In any event, in light of the present disclosure, candidate sequences with the potential to function as HIV infection-inhibiting peptides can now be identified and tested. Such candidate peptides will likely include sequences located within the gp120 V3 loop or N-terminal region, or even within the CD4 binding region of gp120, and preferably, will be sequences against which antibodies have been raised and have been shown to inhibit or reduce HIV cellular infection. Candidate sequences may be synthesized as peptides and tested for the ability to inhibit HIV cellular infection.

As discussed above, in the compositions of the present invention, peptide(s) containing CTL-inducing epitope(s) may be combined with peptide(s) including T helper cell-inducing epitope(s) to create immunization, or vaccine, formulations. In regard to peptide cocktails for immunization, the use of peptides with sequences which correspond to the more conserved areas of HIV gp120 is generally preferred. Additionally, and specifically relating to HIV, the CTL-inducing peptide(s) may be combined with HIV infection-inhibiting sequence(s) as therapeutic formulations.

However, and importantly, compositions comprising all three of the distinct classes of peptide identified herein are also contemplated by the present invention. Such a single formulation would be capable of preventing infection of human cells by HIV-1 and of inducing HIV-1 specific CTL responses. Since all of these formulations are completely synthetic and defined, these reagents will be both economical and safe.

Naturally, any one of the peptides may have more than one immunological activity. This phenomenon is exemplified by the peptide R15K which has CTL-inducing and infection-inhibiting properties. In the most preferred embodiments, it is contemplated that peptides which do not induce the significant production of antibodies that bind to native HIV will be employed. In addition to being used directly as monomers, the peptides of the present invention may also be formulated as polymers or lipid-tailed peptides.

In further embodiments, the present invention concerns methods for assaying a composition for its ability to induce a cytotoxic T cell (CTL) response in an animal. Related to this, are methods for assaying a composition for its ability to enhance a CTL response induced by a distinct component. These screening methods include generally immunizing an animal, such as a human subject or an experimental animal such as a mouse, rat, rabbit, guinea pig, goat, rhesus monkey, or chimpanzee, thereafter collecting cells from lymph nodes or other lymphoid tissue from the animal, and then testing the tissue for the presence of CTLs that are primed to kill or lyse cells producing a component of an infectious agent, such as a viral envelope, core protein, one of the functional proteins (e.g., reverse transcriptase) or the like.

Thus, the method of the invention includes generally three steps, with the first step involving immunizing an animal with the composition to be tested. While it is believed that any accepted mode and route of immunization can be employed and nevertheless achieve some advantages in accordance herewith, the inventors have found particular advantages to be associated with intradermal immunization. Intradermal immunization is believed to be preferred because it serves to activate more efficiently the cell mediated arm of the immune system. Moreover, although where desired one may choose multiple injections of the immunizing substance, and even multiple sites of injection, the inventors have found that a particular advantage of the invention is that a single injection of the candidate will usually be sufficient to achieve detectable CTL activation in a nearby lymph node. Furthermore, although where desired one may employ immunization modifiers such as T-helper cell peptide sequences, lipid structures that induce micelle formation, peptide polymerization methods, or the like in association with the candidate composition, the inventors have found that the sensitivity of the assay is such that the use of such modifiers will generally not be required in order to achieve CTL priming.

Once immunization has been effected, it will then be necessary to recover cytotoxic T cells from lymphoid tissue of the immunized animal. The preferred lymphoid tissue will be lymph node tissue, and most preferably tissue from draining lymph nodes proximal to the site of injection. As used herein, the word "proximal node" is intended to refer to the node or nodes that are located proximal to the site of injection, e.g. the popliteal node of the mouse following foot pad injection. It is believed that the use of proximal or draining nodal tissue to identify CTL activation is one reason for the rapidity of the more preferred aspects of the invention. Such nodes are physically located in the proximity of the immunization site, or in the area draining the site of immunization, and also included are those draining nodes that are physically at a greater distance from the immunization site.

The final step of the assay in its most general sense involves determining whether said cytotoxic T cells have been activated by the composition. Although it is not generally required, it will typically be preferred to actually measure the level of activation, through, e.g., radioactive chromium-release assays, or other radioisotope assays, or single cell assays; also single cell cytotoxic assays using vital stains and/or cell-sorters could be employed.

Where measurement of activation of cytotoxic T cells is desired, a preferred method involves contacting a killing effective amount of said cytotoxic T cells with MHC-matched target cells that exhibit the candidate epitope on their cell surfaces; maintaining said contact for a time period sufficient for said cytotoxic T cells to lyse said target cells; and determining the degree of T cell-mediated lysis of said target cells. However, any method capable of detecting a specific CTL response may be employed, including but not limited to chromium-release assays, single-cell assays or even determination of cell-cell conjugates.

An advantage of the present invention is the speed with which one is able to determine the ability of the candidate substance to activate CTLs. Prior techniques have generally required a wait of a few weeks (Kast et al., 1991; Aichele et al., 1990). The present technique, though, typically requires only about 7 to 10 days following immunization. The reason for the reduced time necessary to achieve a CTL response with the assay of the present invention is believed to be the result of the route of immunization and the use of draining or proximal lymph node cells. A further advantage is the ability to test peptide candidates without the use of an associated modifier, such as carrier molecules, lipid tails, or T helper epitopes, to enhance its CTL activity.

Typically, the composition to be tested for CTL priming capability will comprise one or more peptides, or peptide multimers, believed to have or suspected of having useful activities. Through the application of the techniques of the present invention to such peptides, one will thus be enabled to determine whether such peptides do in fact have CTL priming activity. If so, then the peptide will be a candidate for inclusion in a CTL priming vaccine. It will be appreciated by those of skill in the art that while candidates for CTL priming activity will generally comprise peptides (or proteins), the use of the assay of the present invention in the context of non-peptidyl compounds is certainly not excluded.

It is proposed that the method of the present invention will find a broad range of application, particularly in the identification of components for use in the preparation of vaccines for the treatment and/or prevention of viral diseases such as AIDS, influenza, feline leukemia, bovine leukemia, Herpes virus infections, and even in the case of non-viral infectious diseases such as parasitic and bacterial infections. Therefore, in the case of embodiments directed to the identification of epitopes for promoting a specific anti-HIV CTL response, the invention will generally be concerned with the identification of peptides having the ability to direct a CTL response to HIV-infected cells.

In the context of vaccine development, the method will include first identifying a CTL-reactive composition in accordance with the foregoing method, and admixing the composition with one or more pharmaceutically acceptable diluents or additives, such as water, salts, emulsifiers and/or adjuvants. Of course, the amount of the composition added to the vaccine will vary depending on its ability to induce a specific CTL response, it solubility, and other biological responses. The selection of an appropriate amount of the identified CTL-priming composition will therefore be well within the skill of the art in light of the present disclosure.

In other embodiments, the invention involves a method of preparing cytotoxic T cells. In its most general sense, this method includes immunizing, preferably intradermally, an animal with a composition having the ability to induce a cytotoxic T cell response to a preselected epitope of a targeted protein. The epitope or epitopes employed may or may not be specific for CTL priming, and thus may or may not substantially induce antibodies that will cross react with the targeted protein. However, in the practice of this aspect of the invention, one will typically desire to recover cytotoxic T cells from lymph nodes of the animal for further use. Numerous potential uses of specifically primed CTLs are envisioned. For example, in the case of human therapy, it is contemplated that specifically primed CTLs may be cultured and administered to humans for the treatment of viral infections or patients with cancer. In this case, the CTLs are prepared by immunizing the species in vivo and isolating the immune cells to expand in vitro in the presence of appropriate peptide, cytokines and presenting cells.

Typically, for the induction of an HIV-directed CTL response, the invention will involve the use of peptides which comprise from 7 to about 30 amino acid residues, and have a sequence that corresponds to a domain of an HIV protein such as the gp16O envelope and core proteins, reverse transcriptase, tat, rev or other gene products expressed by the virus, which peptide includes within its structure a conserved region.

For the preparation of vaccines, peptide multimers are generally preferred in order to include multiple CTL epitopes within a single complex. Two specific classes of peptide multimers are disclosed. In one class, the amino-terminal residue of a peptide is peptide-bonded to a spacer peptide that contains an amino-terminal lysyl residue and one to about five amino acid residues such as glycyl residues to form a composite polypeptide. Those added residues of the spacer peptide do not interfere with the immunizing capacity of the multimer, nor with its capacity to form surfactant-like micelles in aqueous compositions. The alpha- and epsilon-amino groups of the amino-terminal lysyl residue are amidified with a C.sub.12-C.sub.18. fatty acid such as palmitic acid to form the reaction product that is used. The di-amide so formed forms surfactant-like micellular multimers in an aqueous composition.

A second class of multimer is a polymer having a peptide as a repeating unit. Here, each peptide is synthesized to contain a cysteine (Cys) residue at each of its amino- and carboxy-termini. The resulting di-cysteine-terminated (di-Cys) peptide is then oxidized to polymerize the di-Cys peptide monomers into a polymer or cyclic peptide multimers in which the peptide repeating units are linked by cystine (oxidized cysteine) residues.

A peptide multimer of either class can contain one or a plurality of different peptide sequences. A peptide of a multimer is an "active" peptide in that when used in a composition discussed below, the multimer can induce cell mediated immunity such as production of cytotoxic T cells. A multimer can also include an inactive peptide, for example to assist in dispersing the multimer in the aqueous medium. The lysyl-containing peptide spacer discussed before can be viewed as such an inactive peptide.

The peptide multimer is utilized in an aqueous composition (inoculum). That composition contains water having a before-described multimer dispersed therein. The composition, when used to immunize an immunocompetent host animal such as a mouse, has the capacity of inducing cell mediated immunity such as cytotoxic T cell activation to the native HIV protein corresponding in sequence to that of an active peptide of the multimer, but does not substantially induce production of antibodies that immunoreact with that corresponding native HIV protein. The composition thus contains an immunizing effective amount of a before-discussed multimeric peptide.
 

Claim 1 of 14 Claims

1. A method for directly inhibiting HIV entry into a cell comprising the step of contacting said cell with an effective amount of a composition comprising a peptide of 8 to 24 residues comprising the sequence RAFVTIGK (SEQ ID NO:5), wherein said cell is in a human subject.

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