WO1999062945A2

WO1999062945A2 - Peptide antigens for detection of hiv, hcv and other microbial infections

Info

Publication number: WO1999062945A2
Application number: PCT/US1999/012446
Authority: WO
Inventors: Mohammed Afzal Chowdhury; David Bernstein; Marvin A. Motsenbocker
Original assignee: Peptide Solutions, Inc.
Priority date: 1998-06-05
Filing date: 1999-06-04
Publication date: 1999-12-09
Also published as: WO1999062945A3; WO1999062945A9

Abstract

Improved peptide antigens for diagnostic testing and therapy of disease are provided which differ from naturally occurring peptide sequences. These peptides immunologically cross-react with a wide variety of mutated forms of antigens and are particularly useful for testing and treatment of retroviral disease such as HIV infection, where rapid mutation of the disease vector is a concern. The peptides are from about 25 to about 100 amino acid residues long and, in addition to advantageous reactivity, also may possess one or more other advantageous characteristics such as improved water solubility or improved immunological reactivity as compared to naturally occurring strains such as ANT70 and MVP5180. Advantageous alterations include substitution of one or more hydrophobic residues with hydrophilic residues, removal of a basic amino acid positive charge to decrease non-specific binding and increase in peptide secondary structure by adding one or more amino acids to form or extend an alpha helix within the peptide. The claimed invention is exemplified by substitution of leucine by glutamine and by arginine in HIV envelope protein peptides. Peptides that have been modified according to principles of the claimed invention differ greatly from the naturally occurring forms and are not identified as belonging to any particular viral Group or strain.

Description

UNIVERSAL PEPTIDES

Field of the Invention

The invention relates in part, to diagnostic tests and antigens used in such tests.

Embodiments of the invention relate to peptide sequences and sequence modifications of such peptides that cross react immunologically with a wide range of antigens such as from HIV-1 Group O virus strains.

Background of the Invention

The retro virus known as Human Immunodeficiency Virus (HIV, also HTLV-ffl), causes Acquired Immunodeficiency Syndrome (AIDS) in humans (Gallo, et al, Science 224:500 (1984); Sarngadharan, et al , Science, 224:506 (1984), Popovic, et al , Science 224:497 (1984). Infection by this virus results in the appearance of antibodies in the blood that react against various molecular parts of the virus, particularly the envelope proteins gp41 and gpl20. The antibody-based binding reaction between antibodies from an infected patient and a viral antigen(s) is used in various methods for detection of HIV infection, such as latex agglutination and ELISA. Of the retro viruses, HIV has received the most attention recently because of the widespread damage caused by this virus.

The first tests developed to detect HIV infection contained whole viral lysates for reaction with antibodies from a blood sample. Occasionally, however, these tests yield false results due to nonspecific binding reactions with one or more antibody binding sites, i.e. , "epitopes" of the various proteins found in a lysate. Consequently, all positive results from these tests must be confirmed by further testing with another method such as Western Blot assay.

Improved versions of tests to detect HIV infection now are available that use purified viral lysate components, recombinant peptides, and even chemically synthesized peptides, that lack one or more non-specific binding regions. These improvements were prompted by the discovery that certain portions of the HIV envelope proteins are highly immunogenic. Proteins and peptides that substantially possess a highly immunogenic region and, even more preferably, lack other non-immunogenic regions, often perform better in these assays.

For these tests, improved peptides have been studied that share sequence identity with the HIV envelope protein gp41 immunodominant region but that lack other portions of HIV envelope protein. In this context, the gp41 immunodominant region is known to be important due to its key role in presenting epitopes during HIV infection (Gnann et al , J. Infect. Dis., 156:261-267 1987 and /. Virol, 61:2639-2641 1987). Peptides that share sequence similarity with this region should be cross-reactive with HIV. The immunodominant region of gp41 comprises a major heptapeptide loop epitope that has been well studied (Wang et al, Proc. Natl Acad. Sci. USA, 83:6159-6163 1986, Dopel et al , J. Virol. Meth. , 28:189-98 1990, Bugge et al, J. Virol , 64:4123-9 1990). Oldstone et al, J. Virol. 65:1727-34 (1991) reported that this loop epitope (CSGKLIC) in classic "M" type HIV-1 strains (i.e. , HIV strains known in 1991) occupies amino acid positions 603-609. An immunodominant region of another envelope protein, gpl20 also exists and is useful for antigen-based tests.

Following the discoveries of these immunodominant regions, many research groups have focused their investigations on small peptides of 25 or fewer amino acids, which contain one or more epitopes from the gp41 and gpl20 proteins. Such peptides are thought to react more favorably with anti-HTV antibodies than do larger peptides because, among other things, non-specific binding reactions occur more often with epitopes present in the larger peptide. This may be the reason why, despite massive efforts to understand HIV, no significant large chemically synthesized peptide has been reported that detects accurately HIV infection from all samples. Another reason is that aqueous solutions comprised of smaller peptides are more reactive than equivalent protein concentrations comprised of larger length peptides. Aleanzi et al, J. Mol. Recog. , 9:631-638 (1996), for example, studied peptide sequences that overlap partially in the region vicinal to the immunodominant epitope, and concluded that shorter, 15 mer peptides, were more reactive than a 23mer peptide. Aleanzi concluded that adding a residue to a known immunodominant sequence does not reliably improve antibody recognition. Accordingly, only small peptides have been studied intensively for their use in HIV detection and therapy.

Despite these efforts with peptides that simulate immunodominant region(s) of the HIV retro virus, no peptide has been found that reliably reacts with antibodies from all patients infected with HIV-1. Thus, until now there has not existed a screening test for HIV infection that uses a single peptide antigen and that reliably detects a wide range of HIV-1 infections. Likewise, no therapeutic peptide has been found that stimulates effectively the immune system to ward off HIV infections, or to cure an HIV infection.

One explanation for these failures in the search for an immunologically active peptide seems to be that the virus mutates rapidly. An RNA virion such as an HIV virion, has a very short generation time and forms many copies of genetic material within a cell by RNA replication. Furthermore, most RNA polymerases (replicases) lack proof-reading activities and have an inherent error frequency of about one in 10,000. Consequently, the virus yield of a single cell generally is a population of genomes, each with one or more (usually subtle or silent) changes from an average sequence. VIROLOGY 164 (Bernard N. Fields ed., Lippincott-Raven Publishers, 1996). In one instance of HIV-1 infection, the polypeptide sequence of a virus was isolated twice from an individual over a 3 month interval and was shown to have had radically changed its amino acid sequence in an important (normally) conserved region of envelope protein that is known to be highly immunogenic. Eberle et al. , J. Vir. Meth. 67: 85,88 (1997). This kind of rapid and drastic change in the immunogenic portion of the envelope protein is a major detection and therapy problem. The HIV antigen diversity problem is seen at different levels of viral classification.

At the highest level, genetic diversity studies indicate that two types of HIV exist, HIV-1 and HIV-2. Antibodies made against one type do not cross-react well, if at all, against proteins of the other and DNA sequences of strains from one type differ as a group from that of the other type. At a lower viral classification level, HIV-1 has been subdivided into several types (from A to I) with a more distinct Group O. Group O exhibits 55-70% homology with the other HIV-1 Groups, and is regarded by some researchers as a new group. Accordingly, and to reflect the close relation of types A-I, which were first found, these types are grouped together as Group M (for major).

Accurate detection of infection by Group O strains is a major challenge in HIV testing. Accordingly, the discovery of a new or improved antigen sequence useful for Group O detection would advance this art. An antigen sequence capable of substantially differentiating infections from group M and Group O likewise would be useful. A publication in this area by Eberle et. al, (Id.) describes Group O strains that have two strongly basic amino acids (lysine and arginine) within the immunologically important gp41 immunodominant loop region. These authors reported test results from using a 25 amino acid peptide of strain MVP5180-91 as an antigen but failed to obtain broad reactivity with HIV M infected blood specimens. At a still lower classification level, closely related strains are grouped together.

However, even these strains have epitopes that differ. This diversity problem was reported by Eberle et al , (Id.) who compared gp41 envelope protein sequences from 27 different HIV-1 Group O samples. Eberle 's data reveal that an immunologically important 25 amino acid peptide segment of the gp41 protein that begins with the tripeptide "ALE," varies from strain to strain by an average of 12 percent with respect to a consensus sequence. The term "consensus sequence" as used here means a sequence containing the most likely amino acid at each position, based on a comparison of all known sequences.

Much of the amino acid sequence diversity found within Group O as shown by Eberle, arises from conservative amino acid substitutions. A conservative amino acid substitution in this context is replacement of one amino acid with a similar one such as arginine with lysine, or leucine, isoleucine and valine with each other. After accounting for these conservative changes however, the average amino acid variation from each O Group strain to a consensus sequence is about 5% . A direct comparison of the 27 Group O sequences shown by Eberle reveals that the important gp41 immunodominant region sequence of an individual "strain" differs from that of another strain by about 5%, or 10% if conservative amino acid substitutions are taken into account. Of course, in some cases, the difference in amino acid sequence is less than this average. For example, the 25 amino acid long portion of variant CM.4974-95 differs from variant MVP5180 by a single conservative substitution of arginine for lysine. These results are consistent with that of other studies which show that a skilled artisan can expect to isolate a new and different genome sequence which merits a new name simply by sequencing new genetic material from an HIV infected patient. In other words, a given peptide sequence obtained from an infected individual will not be found in another individual. Moreover, as seen above, merely changing one amino acid within a 25 amino acid sequence the immunodominant region of gp41 creates a uniquely different sequence that may represent an entirely new strain of HIV, having altered immunological properties.

This high level of genetic diversity seen in HIV infected patients adversely affects the ability of antibodies to recognize the virus. Because of this mis-recognition, a diagnostic test that uses only one polypeptide antigen may fail to immunologically react with one or more groups or strains. The mis-recognition problem may be affected by the stage of infection by the virus. For example, double-antigen assays are more sensitive in the early phase of seroconversion but have a lower cross-reactivity with antibodies directed against variant HIVs. Consequently, non-M strains of HIV-1 may be missed during early seroconversion. Most worrisome, infections with Group O strains may be missed even at later stages of infection.

The genetic diversity problem extends to the use of peptides for therapeutics because it is difficult for the immune system to mount a proper defense against an infectious organism that can change its antigenic character so readily. Thus, any information leading to a more appropriate antigen would improve not only diagnostic technology but therapy as well. One solution to the diversity problem in the diagnostic area has been to include multiple polypeptide antigens in each test. For example, peptide antigens that react with HIV-1, HIV-1 Group O and HIV-2, when present as a mixture, can react with most sera of patients infected with HIV. Drawbacks of this strategy include, among other things, a higher degree of non-specific binding reactions due to a greater variety of proteins used and a limitation in the amount of total antigen that can be added to a test that typically has a small solid phase surface or reaction volume. If a single peptide having broad reactivity could be used in place of two or more peptides, significant improvements to assay quality thus can be achieved. Reasons for this include, inter alia, a greater amount of peptide can be dissolved and used in an assay, and less unnecessary peptide sequence (which causes non-specific binding) is exposed to the sample.

An analogous effort to combine antigens is conceivable for therapy of HIV as well. Immunogens comprising more than one polypeptide sequence and which represent alternative forms of epitopes can be envisioned. Unfortunately, no such vaccine has been tested successfully. The great mutation rate of retro viruses such as HIV, thus hampers the development of both diagnostic tests and therapies for viral infection. Workers in this field have attempted to organize study of the mutation problem by sequencing the genomes of multiple strains of HIV and arriving at "consensus" sequences of the viral envelope proteins. Consensus sequences for the retro virus HIV are presented in HUMAN RETRO VIRUSES AND AIDS 1996, Theoretical Biology and Biophysics, Los Alamos National Laboratory (1996). This reference is herein incorporated by reference in its entirety.

These consensus sequences have been useful in comparing strains for the study of HIV diversity. However, the notion that using a consensus sequence to achieve a more broadly immunoreactive peptide has not worked and even may be misleading. Although workers in this field believe in the use of consensus sequences, despite much experimentation to date no single peptide has been found that will react substantially with sera from patients infected with a range of HIV-1 strains, regardless of sequence identity between such test peptide and a consensus sequence.

The need to detect a wide range of samples is particularly important for the O Group strains. However, attempts to detect such strains have focused on peptides that comprise naturally occurring sequences. The unpredictability of such antigens to detect broadly different strains is, for example, explained by Hunt et al , in AIDS Res. Human Retro. 13: 995-1005 (1997). Hunt produced data obtained with peptide antigens corresponding to immunodominant sequences of specific new strains of Group O HIV-1. Hunt found that synthetic peptides best detected strains from which they were derived. In the search for peptides that can react with multiple HIV strains, much work has focused on peptides of 25 amino acid residues or less because, as shown by Aleanzi et al. , longer peptides may have unfavorable conformation (J. Mole. Recog. 9: 631-8 (1996)). In a related report, Eberle et al. have reported using a 25 amino acid long antigen from the strain MVP5180 gp41 immunodominant region to test 111 anti-HIV-1 Group M specimens and found that ten of these specimens were not reactive (J. Vir. Meth. 67: 85 (1997)). In testing Group O samples, this group found 2 of 42 samples only reacted weakly. This indicates that even using a peptide with a sequence from a member of the O Group provides insufficient reactivity with other members of the group. A reason for these failures in using peptides for diagnosis (and therapy) of HIV infection is that the peptides used do not cross-react sufficiently with mutated strains of HIV- 1. This failure, and failures in using consensus sequences to guide deriving suitable antigens is a central problem blocking the development of new strategies to detect and treat HIV infection. Eberle et al. commented on this failure, stating that "there is no consensus sequence antigen available to be added to current screening assays" (Id. p.89).

Thus, efforts over the years to alter existing native sequences to make new immunogenic sequences which maintain epitopes of the natural protein have failed. In fact, a basic understanding, or dogma in the art, is that making an amino acid substitution in a peptide sequence will, if anything generally destroy the epitopic character of that sequence. The best example of this dogma, as it pertains to peptides used in HIV tests, is presented by Horal et al. , J. Vir. 65: 2718-23 (1991). Horal found an important epitopic site in the immunodominant region of the HIV-1 gp41 envelope protein, which comprises the sequence GKLICT, and that reacts with sera of patients infected with HIV-1. Horal systematically replaced the leucine (L) residue of this epitopic sequence with every other naturally occurring amino acid and found that in every case, the new sequence performed more poorly as a diagnostic test antigen for detection of antibody in blood compared with the native leucine sequence. Replacement of this leucine with glutamine, for example, decreased the immunoreactivity of the epitope. Others have tried making these kinds of radical changes to peptides and found similar degradation in immunoreactivity. Accordingly, efforts to make improved antigens have centered around finding new naturally occurring sequences, and classifying these sequences into immunoreactive groups based on similarity with consensus sequences that represent all known sequences for a given protein. The dominance of this paradigm, however, actually may have delayed the development of improved antigens for infectious disease testing and therapy. Thus, despite recent advances in HIV treatment the genetic mutation problem remains and inhibits both HIV diagnosis and therapy. In fact, as new drugs emerge into the marketplace, the central mutation problem has emerged as a significant obstacle to full recovery of HIV infected patients. Accordingly, the lack of understanding of how to devise polypeptides that broadly represent HIV strain epitopes has emerged as a central problem with the new therapies. A fundamental understanding is needed of how antigens could be improved to better simulate a wide range of peptide sequences.

Summary of the Invention

It is an object of embodiments of the invention to provide peptides having improved biological or immunological reactivity for in vivo use transgenically, in vivo use as therapeutic agents, and for in vitro use as diagnostic agents. It is another object of embodiments of the invention to provide methods for improving stability of peptides and thus allow the peptides to react more advantageously with other molecules via natural in vivo binding reactions or immune system reactions such as in antibody antigen binding reactions. It is another object of embodiments of the invention to reduce or eliminate false test results by providing one or more peptide sequences that react broadly with sera from patients infected with an infectious disease agent such as HCV or HIV-1 Group O. It is another object of embodiments of the invention to provide peptide sequences that improve HIV-1 test sensitivity over existing tests using previously known peptides. It is another object of embodiments of the invention to provide peptide sequences that are relatively specific for Group O infection such that the peptides can differentiate between group M and Group O infections. It is yet another object of embodiments of the invention to provide peptide vaccines against infectious agents. It is another object of embodiments of the invention to provide a method for improving peptides used as antigens in diagnostic testing and in therapy and to provide peptides useful for other diagnostic tests such as hepatitis C, HTLV-I and HTLV-II virus. Further objects of the invention readily will be apparent from the disclosure herein.

One embodiment of the invention is a peptide useful for detecting HIV-1 Group O infection, having a length of between 16 and 100 amino acid residues, and comprising a core sequence SEQ ID NO: 1, wherein XI is selected from the group consisting of N, Q, G, S, T, and A; X2 is R or K; X3 is selected from the group consisting of N, Q, G, S, T, H, A, L, I, V, P and M; and X4 is selected from the group consisting of N, Q, G, S, T, H, A, L, I, V, P and M, and wherein at least one of the two X4 residues is selected from the group consisting of N, Q, G, S, T, H, A, P and M.

Another embodiment of the invention is a peptide useful for detecting HIV-1 Group O infection, having a length between 26 and 100 amino acid residues, and comprising a core sequence SEQ ID NO: 2, wherein X is a helix of at least 5 amino acids, XI is selected from the group consisting of N, Q, G, S, T, D, N, H and A, X2 is R, K, P or E, X3 is selected from the group consisting of I, L, and V, X4 is selected from the group consisting of T, S and A, and X5 is at least one amino acid long.

Another embodiment of the invention is a peptide useful for detecting HIV-1 Group O infection, having a length between 36 and 100 amino acid residues, and comprising a core sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 and derivatives thereof that contain one or more conservative amino acid substitutions

Another embodiment of the invention is a peptide between 26 and 100 amino acids long that substantially reacts with Group O HIV-1 test specimens and that comprises a 17 amino acid long immunodominant region, the region having a sequence shown by SEQ ID NO: 8. Another embodiment of the invention is a peptide useful for detecting HIV-1 Group

O infection, having a length of between 16 and 100 amino acid residues, and comprising a core sequence SEQ ID NO: 1, wherein XI is selected from the group consisting of N, Q, G, S, T, and A; X2 is selected from the group consisting of R, K, T and N; X3 is selected from the group consisting of N, Q, G, S, T, H, A, L, I, V, P and M; and X4 is selected from the group consisting of N, Q, G, S, T, H, A, L, I, V, P and M, and wherein at least one of the two X4 residues is selected from the group consisting of N, Q, G, S, T, H, A, P and M.

Another embodiment of the invention is a peptide useful for detecting HIV-1 Group O infection, having a length of between 17 and 100 amino acid residues, and comprising a core sequence SEQ ID NO: 8, wherein X2 is selected from the group consisting of R, K, T and N; X5 is selected from the group consisting of N, Q, G, S, T, H, A, L, I, V, P and M; and wherein at least one of the two X5 residues is selected from the group consisting of N, Q, G, S, T, H, A, P and M.

Yet another embodiment of the invention is a peptide useful for detecting HIV-1 Group O infection, having a length of between 17 and 100 amino acid residues, and comprising a core sequence SEQ ID NO: 17, wherein X4 is selected from the group consisting of R, K, N, E and A; X5 is selected from the group consisting of G, N, E and D; X6 is R or K; X7 is selected from the group consisting of N, Q, L, I, V and P; X8 is selected from the group consisting of N, Q, L, I and V; X12 is selected from the group consisting of S, T and A; X13 is selected from the group consisting of I, L, V and A; X14 is selected from the group consisting of K, R and E; X15 is W or T; X16 is N or H; and X17 is R or K.

Yet another embodiment of the invention is a peptide useful for detecting HIV-1 infection, comprising a sequence of at least 35 amino acids from a gp41 immunodominant region, wherein the peptide sequence comprises at least 5 amino acids in an alpha helix structure on the amino terminal side of the immunodominant region, and wherein the alpha helix structure is determined by Chou-Fasman Conformational parameters from a peptide analysis computer program.

Yet a further embodiment of the invention is a reagent for immunological detection of anti-HIV antibody in a blood sample, comprising a dried antigen that, upon rewetting with water or a clinical sample, substantially reacts with antibodies from patients exposed to HIV- 1 group M virus and with antibodies from patients exposed to HIV-1 Group O virus, wherein the antigen is between 16-50 amino acids long and possesses a sequence described herein.

Yet another embodiment of the invention is a method of detecting HIV-1 Group O infection, comprising incubating a blood sample or blood derivative with a peptide described herein, followed by determination of binding between antibody in the blood sample or blood derivative and the peptide.

Another embodiment of the invention is a kit for determining infection with HIV-1 Group O, comprising, an instruction booklet and a device for detecting the presence of anti- HIV- 1 Group O antibody in a blood sample or blood derivative, wherein the device comprises a peptide described herein.

Another embodiment of the invention is a method of improving reactivity of an antigenic peptide for a diagnostic test of an infectious agent, comprising replacing a hydrophobic amino acid in a known immunoactive portion of the peptide with a hydrophilic amino acid. Yet another embodiment of the invention is an improved diagnostic test peptide antigen for the detection of an infectious agent, wherein the peptide has a sequence that corresponds to a naturally occurring sequence and wherein at least one hydrophobic amino acid residue from the naturally occurring sequence is replaced by a hydrophilic amino acid residue

Yet another embodiment of the invention is an improved diagnostic test peptide antigen for the detection of an infectious agent, wherein the peptide has a sequence that corresponds to a naturally occurring sequence and wherein two hydrophobic amino acid residues from the naturally occurring sequence are replaced by hydrophilic amino acid residues.

Yet another embodiment of the invention is an improved diagnostic test peptide antigen for the detection of an infectious agent, wherein the peptide has a sequence that corresponds to a naturally occurring sequence and wherein three hydrophobic amino acid residues from the naturally occurring sequence are replaced by hydrophilic amino acid residues.

Yet another embodiment of the invention is an improved diagnostic test peptide antigen for the detection of an infectious agent, wherein the peptide has a sequence that corresponds to a naturally occurring sequence and wherein four hydrophobic amino acid residues from the naturally occurring sequence are replaced by hydrophilic amino acid residues.

A further embodiment of the invention is a peptide between 26 and 100 amino acids long having a central portion of at least 16 amino acids that corresponds in sequence identity to an immunodominant region of an antigen protein, and at least one amino acid at each end of the central portion, wherein the peptide is chemically synthesized and at least one hydrophobic amino acid of the immunodominant region has been replaced with a hydrophilic amino acid.

Another embodiment of the invention is an improved immunogenic therapeutic peptide for the treatment or prevention of infection by an infectious agent, the improvement comprising replacing at least a L, I, or V of a naturally occurring sequence of the peptide with an amino acid selected from the group consisting of A, S, T, G and N. Another embodiment of the invention is a peptide antigen that comprises an amino acid sequence selected from the sequences listed in Figure 2.

Brief Description of the Figures

Figure 1 shows allowable amino acid substitutions in an antigenic sequence of gp41 in accordance with one embodiment of the claimed invention.

Figure 2 shows representative advantageous sequences for HIV-1 peptide antigens, (SEQ ID NOs: 1-26) and for hepatitis C peptide antigens (SEQ ID NOs: 28-30, 32-229) in accordance with the claimed invention. SEQ ID Nos. 7 and 27 are naturally occurring sequences of proteins that correspond to HIV-1 and hepatitis C respectively.

Figure 3 shows data obtained from testing HIV-1 infected blood samples with known antigen sequences and with sequences according to embodiments of the claimed invention, as described in Example 1.

Figure 4 shows data obtained from testing HIV negative, HIV-1 positive, HIV-1 O group positive, and HIV-2 positive samples with an antigen having a native sequence from HIV-1 (MVP5180) and with an antigen (SEQ ID NO: 5) that differs from this native sequence by three amino acid substitutions.

Figure 5 is a representative set of peptide Chou Fasman conformational parameters for a peptide obtained by software in accordance with one embodiment of the claimed invention.

Detailed Description of the Preferred Embodiments

The inventors studied natural sequence variation of gp41 envelope peptides obtained from HIV-1 strains as a model system and made several discoveries relating to improving peptides for testing and therapy of disease. Generally, the inventors discovered that increasing the structural stability of epitope region(s) in intermediate sized peptides yielded peptides that react more advantageously with antibodies to allow more advantageous diagnostic assays. More, specifically, the structural stability can be increased by at least four different strategies. One strategy is to replace a hydrophobic amino acid with a less hydrophilic amino acid. A second is to increase the amount of secondary structure such as alpha helix in the peptide. A third strategy is to remove a positive charge from the peptide. This third strategy is particularly helpful to limit undesirable binding of the peptide to a negatively charged surface, which destabilizes the structure. A fourth strategy is to constrain the structure of an epitopic sequence by forming a covalent crosslink (such as a cystine bridge) within the peptide. Embodiments of each method and representative peptides produced from the method are presented separately below.

Embodiments of the claimed invention provide peptide antigens for diagnostic testing and therapy of disease having sequences that differ from naturally occurring peptide sequences. These peptides immunologically cross-react with a wide variety of mutated forms of antigens and are particularly useful for testing and treatment of retroviral disease such as HIV or HCV infection, where rapid mutation of the disease vector is a concern. The peptides are from about 16 (e.g. 16) to about 100 (e.g. 100) amino acid residues long and preferably from 25 to 50 amino acids long. In addition to their advantageous reactivities, the antigens also may possess one or more other advantageous characteristics such as improved water solubility or improved immunological reactivity compared to antigens having epitopic sequences from naturally occurring strains such as (in the case of HIV-1) ANT70 and MVP5180. Preferred peptides that have been modified according to principles of the claimed invention differ greatly from the naturally occurring forms and are not identified as belonging to any particular viral Group or strain.

These advantageous features are useful as well for peptides made and/or used in vivo. For example, many new transgenic therapies transgenically express a protein that binds specifically to (one or more) binding partners such as other proteins or peptide hormones. By expressing a protein having suitable binding characteristics, the desired binding reactions are favored. In many cases it is desired that an expressed protein (such as a therapeutic transgenic protein) having a structure that participates in the binding reaction actually leave the cell where it is synthesized and to enter the general circulation. In this case, it is very helpful to express not the entire protein, but only a small peptide having the desired binding characteristic and the smaller size. The small size allows the peptide to more easily leave the cell. The methods of the invention are particularly useful for these applications because they alleviate problems that occur when a shorter intermediate sized peptide is prepared from a larger protein. That is, methods of the invention can stabilize a peptide structure, allowing the peptide to remain more soluble and even to bind more advantageously to its intended target. In embodiments of the invention, intermediate peptides obtained from a larger binding protein are improved by alterations as described below. In this context, the inventive methods can enhance the biological and/or immunological reactivity of a peptide by improving its solubility and/or by increasing the association (binding constant) between the peptide and an intended binding partner. The inventors have obtained data that verifies the use of certain embodiments for improving binding between certain peptides and antibodies made against various strains of HIV. However, binding between peptides and other molecules can be improved as well by practice of the methods.

The methods of the invention, outlined below in four categories for convenience, are applicable to practically any peptide type that is intermediate in size. That is, embodiments provide improved and novel intermediate sized peptides in virtually all categories. Examples of such peptides include, for example, portions (or complete sequences) that correspond to the following peptides. Lymphokines and Interferons: IL-1, IL-2, IL-3, IL- 4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IFN- alpha , IFN- beta , IFN- gamma. Cluster Differentiation Antigens and MHC Antigens: CD2, CD3, CD4, CD5, CD8, CDl la, CDl lb, CDl lc, CD16, CD18, CD21, CD28, CD32, CD34, CD35, CD40, CD44, CD45, CD54, CD56, K2, Kl, P beta , O alpha , M alpha , M beta 2, M beta 1, LMPl, TAP2, LMP7, TAPl, O beta , IA beta , IA alpha , IE beta , IE beta 2, IE alpha , CYP21 , C4B, CYP21P, C4A, Bf, C2, HSP, G7a/b, TNF- alpha , TNF-62 , D, L, Qa, Tla, COL11A2, DP beta 2, DP alpha 2, DP beta 1, DP alpha 1, DN alpha , DM alpha , DM beta , LMP2, TAPl, LMP7, DO beta , DQ beta 2, DQ beta 2, DQ beta 3, DQ beta 1, DQ alpha 1, DR beta , DR alpha , HSP-70, HLA-B, HLA-C, HLA-X, HLA-E, HLA-J, HLA-A, HLA-H, HLA-G, HLA-F. Hormones and Growth Factors: nerve growth factor, somatotropin, somatomedins, parathormone, FSH, LH, EGF, TSH, THS-releasing factor, HGH, GRHR, PDGF, IGF-I, IGF-II, TGF- beta , GM-CSF, M-CSF, G-CSF, erythropoetin. Tumor Markers and Tumor Suppressors: beta -HCG, 4-N- acetylgalactosaminyltransferase, GM2, GD2, GD3, MAGE-1, MAGE-2, MAGE-3, MUC- 1, MUC-2, MUC-3, MUC-4, MUC-18, ICAM-1, C-CAM, V-CAM, ELAM, NM23, EGFR, E-cadherin, N-CAM, CEA, DCC, PSA, Her2-neu, UTAA, melanoma antigen p75, K19, HKer 8, pMel 17, tyrosinase related proteins 1 and 2, p97, ρ53, RB, APC, DCC, NF-1, NF-2, WT-1 , MEN-I, MEN-II, BRCA1 , VHL, FCC and MCC. Products of oncogenes: ras, myc, neu, raf, erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl. Complement Cascade Proteins and Receptors: Clq, Clr, Cls, C4, C2, Factor D, Factor B, properdin, C3, C5, C6, C7, C8, C9, Cllnh, Factor H, C4b-binding protein, DAF, membrane cofactor protein, anaphylatoxin inactivator S protein, HRF, MIRL, CR1, CR2, CR3, CR4, C3a/C4a receptor, C5a receptor. Other viral Antigens: HIV (gag, pol, gpl20, vif, tat, rev, nef, vpr, vpu, vpx) , HSV (ribonucleotide reductase, alpha -TIF, ICP4, ICP8, ICP35, LAT-related proteins, gB, gC, gD, gE, gH, gl, gJ), influenza (hemagluttinin, neuraminidase, PB1, PB2, PA, NP, Ml, M2, NS1, NS2), papillomaviruses (El, E2, E3, E4, E5a, E5b, E6, E7, E8, LI, L2) adenovirus (E1A, E1B, E2, E3, E4, E5, LI, L2, L3, L4, L5), Epstein-Barr Virus (EBNA), Hepatitis B Virus (gp27, gp36, gp42, p22, pol, etc.). Although for brevity, details of the invention provided herein describe particular uses for the therapy and diagnosis of infectious disease agents, the invention pertains to all intermediate sized peptides having biological activity, such as those listed above. In preferred embodiments, a desired epitope (or binding site) of the protein or peptide is selected and then altered and/or combined with another sequence(s) to produce a new peptide according to four strategies outlined below.

(1) Change a Hydrophobic Amino Acid to a Hydrophilic Amino Acid in the Intermediate Sized Peptide

During studies with peptides and blood samples that contain alternate Group O strains of HIV-1, it was 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 arginine). It was further discovered that such a modification could be made outside the immunodominant region, outside the cysteine loop but within the immunodominant region, or even within the cysteine loop itself. 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 protein sequence to the strain from which the antigen originally was derived.

Another consequence of the hydrophobic to hydrophilic amino acid residue shift can be that the new peptide may have greater solubility in water. An increase in water solubility can lead directly to improved diagnostic assay or 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.

A peptide antigen according to the invention is greater than 16 amino acid residues long but smaller than 100 amino acid residues 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 peptide 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 an intact 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 an alteration according to the claimed embodiment.

Prompted by this discovery, 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 amino acids correspond in identity with) naturally-occurring sequences. The synthesized peptides showed greater specificity for HIV-1 O Group specimens compared to peptides that have sequences that are identical to sequences from naturally occurring proteins. That is, the peptide sequences of the claimed invention exhibit different immunological characteristics than the corresponding sequences of naturally occurring proteins. The different characteristics can include a loss of one or more immunological properties, exemplified by the loss of HIV-2 reactivity for peptides obtained from a naturally-occurring HIV-1 envelope protein sequence.

Although not wishing to be bound by any one theory of their invention, the inventors theorize that altering a hydrophobic amino acid such as leucine, valine and isoleucine etc. to a hydrophilic amino acid such as glutamine, asparagine, serine, threonine etc., particularly in an immunodominant (or biologically active) region of a protein, helps prevent structural instability when present in an intermediate sized (16-100 residue-long) peptide that lacks complex protein (i.e. tertiary structure). The inventors theorize that hydrophobic amino acid residues in a large protein come together to form an interior oily pocket that excludes water and stabilize the structure of the complete large protein. However, when a peptide antigen less than about 100 amino acids (e.g. less than 100 amino acids), particularly less than about 75 amino acids (e.g. less than 75 amino acids) and more particularly less than about 50 amino acids (e.g. less than 50 amino acids) is prepared to mimic antigenically this same protein, individual hydrophobic residues no longer can avoid water by optimally coming together and instead randomly are exposed to water and increase disorder of the peptide in water. The disorder contributes to less stable and unrecognizable epitopic structures which react less well or react less specifically with antibodies directed against the native undenatured protein, which is more ordered. The increased disorder is alleviated by decreasing the hydrophobic character of the hydrophobic residue, preferably by substituting the amino acid with a more hydrophilic residue. Of the proposed amino acid changes, it is particularly advantageous to alter leucine or isoleucine to glutamine because of the similarity in sizes of these amino acids, although other related changes are desirable and contemplated as described elsewhere.

Some embodiments of the invention pertain specifically to epitopes useful for diagnosis and therapy of infectious disease agents. However, many biological activities arise from binding reactions between a protein or peptide and another agent, such as a cell surface receptor, or specific binding protein inside a cell. The invention is useful for making intermediate sized peptides having improved binding activity for these biological effects because the improved stability of the intermediate sized peptides provides greater opportunity for binding between the peptide and the in-vivo binding partner, such as a cell surface receptor or intracellular receptor. One embodiment in this vein, is to improve the binding reactivity of a peptide having a sequence obtained from a larger protein, wherein the larger protein acts as a binding partner in vivo. The binding partner may be for example, a membrane protein that has a portion that binds to a blood factor.

This embodiment of the invention is particularly useful to modify leucine zipper regions of proteins when preparing a peptide portion (less than the whole protein sequence) that lacks at least some of the secondary or tertiary (folding) structure of the protein. In particular, the existence of a "hydrophobic zipper" binding mechanism with leucines playing a major role, is believed to be important in protein folding dynamics, as described in Proc. Nat'l Acad. Sci. USA 90: 1953, (1993) and Science 263:536, 1994). This embodiment of the invention is particularly applicable for obtaining intermediate sized peptides from proteins of this protein class described in these two publications.

The embodiment of replacing one or more hydrophobic amino acids with one or more hydrophilic amino acids particularly relates to intermediate sized peptides from 16 amino acids to 100 amino acids in length, and more particularly to peptides between 25 to 50 amino acids, 36 to 50 amino acids and 41 to 50 amino acids. The improved effect is seen particularly with intermediate sized peptides because, at very small sizes of less than about 16 (e.g., 16), and particularly less than 10 amino acids, the epitope recognized by an antibody more closely resembles the primary structure of the short segment, namely, the individual amino acid residues themselves. That is, antibody reactivity (if any) to such a short peptide arises primarily from chemical characteristics of the amino acid residues themselves. In contrast, secondary structures such as alpha helix and beta pleated sheet, and tertiary structure, such as that resulting from ionic bonding, hydrogen bonding and hydrophobic effect "bonding" (actually, association driven by an increase in entropy) between residues of the same chain have little role in these small peptides. Most studies have evaluated short peptides of less than about 20 amino acids, partly because it has been difficult to chemically synthesize intermediate sized peptides greater than this size. Another reason is that publications in this field emphasize, as briefly reviewed above, that small peptides less than about 25 amino acids long (e.g. less than 25 amino acids long) perform better as antigens than do larger peptides.

In one embodiment of the claimed invention, peptides between about 25 to 100 amino acid residues long, and particularly 25-50 amino acids long advantageously are used. These intermediate sized antigens are larger than short pieces studied by Horal, Aleanzi and others, and have more advantageous secondary structure in water solution. In this case, altering a hydrophobic amino acid to a hydrophilic amino acid provides an advantage to the peptide. Thus, peptide antigens of most interest for diagnostics and therapy generally have more advantageous secondary and tertiary structures which are more sensitive to disruption by a hydrophobic residue, yet the hydrophobic residue(s) present in these peptides need a large protein for proper orientation.

The claimed invention is exemplified by, for example, altering a leucine to a glutamine but works well with shifts of other hydrophobic amino acids such as I, V, M, F and W to hydrophilic amino acids, and even to hydrophilic charged amino acids. In making these substitutions, it is particularly advantageous to replace a hydrophobic amino acid residue with a hydrophilic (preferably uncharged) amino acid residue having a similar overall size. Most advantageous in this aspect is to replace a leucine, which has a three carbon long residue with a methyl group attached, with a glutamine, which also has a three carbon long residue with an additional amine group attached. In another embodiment, a peptide between 25 and 50 amino acids long is used for diagnostic tests that has only one hydrophobic residue within an 8 residue long portion. Altering this hydrophobic residue to a hydrophilic residue improves reactivity (sensitivity and/or selectivity). In yet another embodiment, 2 hydrophobic residues within an 8 amino acid long portion exist and at least one of these is altered to a hydrophilic amino acid to provide the benefit. Altering 2 or more residues within a short region can provide great improvement to solubility and the ability to incorporate the peptide, alone or with other peptide(s) in a diagnostic test reagent or therapeutic agent.

In another embodiment, an isoleucine, leucine, valine, or methionine is replaced with glutamine. In another embodiment, any of these hydrophobic amino acids is replaced with asparagine. In another embodiment, any of these hydrophobic amino acids is replaced with threonine, serine, alanine or glycine. In another embodiment, any of these hydrophobic amino acids is replaced with histidine or proline. In another embodiment, any of these hydrophobic amino acids is replaced with aspartic acid, glutamic acid, arginine or lysine.

Other hydrophobic to hydrophilic amino acid changes are possible in accordance with the claimed invention. For example, in one embodiment, a phenyl alanine can be converted to a glutamine. In another embodiment, a phenyl alanine can be converted to any of the other hydrophilic amino acids. For preferred embodiments, methods are contemplated in which:

(1) in intermediate peptide sequence is reviewed to determine the presence of a leucine, isoleucine, valine, methionine, or other hydrophobic amino acid; (2) at least one such hydrophobic amino acid in the sequence is changed to a less hydrophobic, or preferably, hydrophilic amino acid as described herein; and (3) an intermediate peptide is synthesized having the new sequence. In a preferred embodiment, at least one leucine or isoleucine is changed to an arginine and/or arginine.

In considering any specific alteration, a computer modeling software program, such as "Peptide Companion" advantageously is used and a specific alteration is chosen, using the program, to maintain the predicted pre-existing secondary or tertiary structure of the protein.

(2) Increase Secondary Structure of the Intermediate Sized Peptide

It was further discovered that increasing the amount of secondary structure in an antigen improved test results, when the antigen was used in a test for detection of viral infection from blood samples. Secondary structure in this context refers to polypeptide helix or pleated sheet that forms 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. In further study of the immunodominant region of gp41 antigen from HIV-1, the inventors discovered that the alpha helix on the amino terminal side of this region is important to stabilize the antigen structure. The degree of stabilization has a great influence on performance of a peptide used in diagnosis or therapy. For example, the inventors learned that the 25 amino acid peptide of sequence ALETLIQNQQRLNLWGCKGKLICYT fails to detect some HIV-1 infected samples in an immunoassay. However, a longer peptide having an extra 5 amino acids that form a more extended alpha helix at the amino terminus: RARLQALETLIQNQQRL-NLWGCKGKLICYTSVKWNT, successfully detected all HIV-1 samples tested. The extra 5 amino acids, "RARLQ" provide a more stable peptide by virtue of extending the alpha helix at the amino terminal side of the immunodominant region.

Other amino acid combinations can provide helix at the amino terminal side of the immundominant region and are contemplated. In the case of the 36 amino acid long peptide exemplified above, the following substitutions for the first 8 positions are possible and can form alpha helix structure:

Position Number Amino Acid Possible Amino Acid Substitutions

R QMWANDEGHILKFPST

2 A MRDEHK

3 R MADEHK

4 L QMWRANDEGHIKFPST

5 Q MWRANDEGHILKFPST

6 A MDEK

7 L QMWRANDCEGHKFPST

8 E (no substitution)

QMWRANDEGHLKFPS Peptides that comprise one or more of these combinations are contemplated in the invention. Of course, further extensions of the peptide chain on either side of the immunodominant region can form advantageous antigens in accordance with these principles. Most advantageous amino acids may be, for example, determined by predictions 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.

In the case of the HIV-1 gp41 immunodominant region exemplified here, it was discovered that an antigen works better if it includes at least about 5 amino acids (e.g. five) to the amino terminal side of the immunodominant region. In alternative embodiments, this portion may be 6, 7, 8, 9, 10, 11, 12 or more amino acids long. In advantageous embodiments this added portion, (or at least a part that is adjacent to the immunodominant region) is in the form of a helix as described above. In preferred embodiments, methods are contemplated for preparing improved intermediate sized peptides for diagnostics and therapy of disease, comprising: (1) obtaining the sequence of an intermediate-sized peptide, or portion of a larger protein; (2) computing secondary structure information for at least a portion of the sequence from step (1); (3) determining one or more specific amino acid substitutions to the portion studied, that allow greater predicted secondary structure compared to the sequence from step (1); and (4) synthesizing an intermediate sized peptide having the sequence determined from step (3). In a preferred embodiment alpha helix information is obtained from step (2) and step (3) is carried out by substituting various amino acids until at least one is found that gives a sequence with greater predicted alpha helix structure.

(3) Remove a Positive Charge from the Intermediate Sized Peptide

It was further discovered that a peptide antigen could be improved to provide greater specificity if a basic amino acid such as arginine or lysine in the antigen is replaced with another non-basic hydrophilic amino acid. Such substitution most advantageously is made for an arginine or lysine that is in an alpha helix but not in an immunodominant region. When making a substitution within a helix, the new amino acid should be chosen to maintain the helix structure.

One embodiment of the invention, accordingly, is a method to improve an intermediate sized peptide reagent used in a diagnostic assay, comprising the step of replacing an arginine or lysine of the peptide with another amino acid that lacks a positive charge in the buffer or aqueous solution used. In a preferred method, the arginine or lysine is at the amino terminus of the peptide and the peptide has two positive charges at the amino terminus, by virtue of the amino terminus amino group and the side group of the lysine or arginine. In this embodiment, one of the positive charges is removed when the basic lysine or arginine is replaced with the non-basic amino acid. In another embodiment, the entire peptide sequence is reviewed to determine whether two or more positively charged amino acids are present within a 8 amino acid long portion. If found, at least one of the two positive charges is removed by replacing basic amino acid(s) with a non-basic amino acid such as glutamine. In yet another embodiment of this method, a basic amino acid within an intermediate sized peptide but outside of a known epitopic sequence of the peptide is altered to a non-basic amino acid. In another embodiment arginine is altered to a glutamine.

The inventors have used the method to improve a peptide for HIV-1 diagnosis by converting an arginine of the peptide into a non-basic acid. However, lysine and even histidine (which forms a positive charge in many physiological pH solutions) profitably may be altered by the method.

Although not wishing to be bound by any particular theory of the invention, the inventors theorize that a basic amino acid residue (having a positive charge at physiological pH) forms undesirable ionic bonds with negative charges of other molecules or with negative surfaces. These bonds can result in non-specific binding, thus causing a false positive assay result, or improper non-specific binding in the case of a therapeutic application. The inventors further point out that a positive charge (from a basic amino acid) within an intact protein, when positioned near a negative charge, would not readily form a strong ionic bond with another molecule or with a negatively charged surface. However, when an intermediate sized peptide is prepared from this portion of the entire protein sequence, but lacking the positioned negative charge, the basic amino acid is more free to react outside the molecule that contains it, leading to high background and/or taise positives when the peptide is used as an immobilized binding partner in an assay. Thus, removing the positive charge will improve assay performance. Of course, the method works best for a basic amino acid(s) outside the desired known epitopic sequence because in most cases background ionic binding is to be alleviated while maintaining epitopic reactivity of the peptide. In this context, the inventors further note that the method works best for removing positive charge(s) in particular. Removing a negative charge from the peptide is less helpful, probably because most surfaces of interest for diagnostics tests are negatively charged.

In one advantageous embodiment, false positive assay results were alleviated by replacing an arginine residue at the position 12 residues to the amino terminal side of the gp41 immunodominant loop region, with the non-basic amino acid glutamine. Data was obtained showing that replacement of this arginine with glutamine provides enhanced antigen selectivity and somewhat less antigen sensitivity, associated with removal of false positive results.

(4) Constrain an Epitopic Sequence by Covalent Crosslinking

The inventors further learned to stabilize the structure of an epitope within an intermediate sized peptide by constraining the structure within a covalent crosslink. This technique is preferred because the constrained epitope portion of the peptide can bind an antibody or other component of the immune system with greater affinity. A constrained "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 an 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 an 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-diaminopropanoicacid (2,3-diaminopropionicacid), 2,4-diaminobutanoicacid (2,4-diaminobutyricacid), 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. Further Improvements by Varying the Sequence Outside the Immunodominant Region of the Peptide for Broader Reactivity

Yet another discovery leading to the claimed invention is that a native peptide antigen sequence can be varied outside of an immunodominant epitopic region in order to react immunologically with a more diverse range of antibodies. This is particularly important where the disease causing organism is an RNA virus and rapidly mutates new antigen structures. In this embodiment of the claimed invention, sequence variation is added to a peptide containing a gp41 immunodominant region according to a formula shown by Figure 1, to form a peptide that reacts with a wider variety of HIV-1 infected blood specimens. Figure 1 shows allowable changes to the core immunoreactive part of a peptide (positions 594-609) according to this embodiment. These embodiments are described in co-pending utility application entitled "Multiple Readout Immunoassay with Improved Resistance to Interferences," filed April 30, 1998 (Attorney docket No. 073294/0173), "Universal HIV-1 Peptide Antigens," filed January 28, 1998 (attorney docket No. 073294/0161) and co-pending U.S. application "Universal HIV-1 Peptide Antigens," filed March 2, 1998 (attorney docket No. 073294/0164), all of which are herein incorporated in their entireties by reference.

This discovery originated with studies of natural sequences of gp41 envelope protein from the HIV-1 virus. This protein has a core immunodominant region between positions 594-609. Many peptides have been made and studied that span positions 567 to 617 from this protein. According to the paradigm summarized in Figure 1 , advantageous peptides for HIV diagnostic tests and therapy in accordance with the claimed invention differ from previously studied peptides because they have sequences that more closely relate to sequences of the HIV-1 O strains than to the most likely (i.e. consensus) sequence of M strains. To make this comparison, an M consensus sequence and an O consensus sequence published in the Los Alamos National Laboratory Data Base, are used as described in the co-pending provisional application "Universal HIV-1 Peptide Antigens" (U.S. No. 60/072,863).

Advantageously, an HIV peptide antigen according to this embodiment of the claimed invention is from about 25 (e.g., 25) to about 100 (e.g., 100) amino acids long. More advantageously, the HIV peptide antigen is from about 36 (e.g., 36) to about 50 (e.g., 50) amino acids long. Even more preferably, this antigen includes at least about 5 amino acids, from about 21 amino acids to about 29 amino acids (e.g., 21 to 29 amino acids) from the cysteine at the N-terminal side of the heptapeptide loop. This segment on the N-terminal side of the loop preferably forms an alpha helix. Furthermore, the Hopp acrophilicity scale peptide profile should be about at least in 75% agreement (e.g., 75% or more) with the profile of the classical HIV-1 group M strain B sequence, as determined by peptide analysis with "Peptide Companion Version 1.24 for Windows" software from Peptides International, Inc. Louisville, Kentucky 40299 U.S.A. In this context, the skilled artisan will appreciate that "strain B" also means the same as "Group B" or "clade B. " The sequence denoted as strain B of group M has been published by the Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (HUMAN RETRO VIRUSES AND AIDS 1996).

A universal peptide sequence according to this embodiment preferably has a Janin accessibility scale peptide profile that is in about at least 80% agreement (eg. , 80% or more) with the sequence profile of the classical HIV-1 M strain B Group, as determined by this software. Also preferred is a sequence having a Hopp and Woods hydrophilicity scale peptide profile that is at least in 75% agreement with the profile of the classical HIV-1 M strain B Group. Furthermore, the Kyte and Doolittle hydropathy scale profile of the peptide should be in at least 80% agreement with the profile of the classical HIV-1 M strain B Group (all determined by the Peptide Companion software.) When selecting a peptide sequence in accordance with the algorithm shown in Table 1 , and in accordance with the method detailed herein, it is advantageous to use these computer derived profiles to help determine which alterations of which amino acid(s) will work best in the sequence.

Peptide Antigens that Cross-react with Envelope Protein from HIV-1

Antigens that cross-react with the immunodominant region of the gp41 envelope protein of HIV are contemplated as embodiments of the claimed invention as exemplified above. Combinations of substitutions are particularly advantageous. Specific examples of these antigens are peptides that comprise (i.e. contain in whole or in part) peptide sequences shown as SEQ ID Nos. 1 through 20 in Figure 2. The inventors realized that they could mix one or more peptide antigens according to the invention with recombinant antigen at a higher concentration if the peptide is made more hydrophilic by amino acid substitution as described herein.

Of course, other antigens can be devised and used for diagnostics and/or therapy of

HIV infection by following the selection methods described above. In this context, antigens that have at least one substitution of a hydrophilic amino acid (such as glutamine or arginine) for an aliphatic amino acid (such as leucine, isoleucine or valine) from a naturally occurring sequence are particularly advantageous.

An amino acid substitution outside of a known epitope does not remove the epitope but may modulate the immunoreactivity of the epitope with respect to the native (unmodified) form. When used in diagnostics for binding to antibodies in infected samples for example, the substitution may increase or decrease binding between the peptide and with antibodies in one or more variant samples. Experimental data have been obtained, as exemplified by Figure 3 and Figure 4 that indicate such alteration can alter or even remove immunoreactivity from the native sequence. Figure 3 shows that small changes in a peptide sequence can improve reactivity for specific infected samples. Figure 4 shows that two alterations of leucine to glutamine and alteration of arginine to glutamine in a 36 mer portion of a natural HIV-1 sequence (MVP5180) removed reactivity of this sequence to an entire group of non HIV-1 samples, in this case, HIV-2 infected samples. In this context, substitution of a valine for an isoleucine within the cysteine loop region of the HIV-1 gp 41 protein, and substitution of a basic amino acid such as arginine or lysine at the eighth position to the carboxy terminal side of the cystine loop is particularly advantageous and can lead to a peptide having altered immunological characteristics. Sequences of representative altered peptides having these and related changes are shown in Figure 2, and described more generally by the language of the claims. Of course, other alterations are contemplated in accordance with the discoveries detailed herein.

In making a change to the peptide within the immunodominant region of HIV-1 gp 41 antigen, it is most advantageous to maintain a "t-h-s-s-s-s-s" helix turn profile, as modeled by the peptide analysis software program, "Peptide Companion Version 1.24 for Windows" from Peptides International, Inc. Louisville, Kentucky 40299 U.S.A. and as exemplified in Figure 5 for a peptide having the sequence SEQ ID NO: 15. More specifically, this program indicates that the immunodominant loop of a suitable antigenic sequence forms a helix with a kink at the first cysteine at the amino terminal side. The helix turn profile and Chou Fasman conformational parameters shown in Figure 5 are useful for predicting an advantageous peptide that contains a cystine loop sequence found in many naturally existing HIV strains. In advantageous embodiments of peptides useful for HIV-1 diagnosis and therapy, the following sequences are particularly useful to form the cystine loop region of HIV-1 having the advantageous peptide turn profile: SEQ ID NO: 21: CAGKQVC; SEQ ID NO: 22: CAGRLVC; SEQ ID NO: 23: CADRQVC; SEQ ID NO: 24: CANRQVC; SEQ ID NO: 25: CAGRQVC; and SEQ ID NO: 26: CAGKLVC. A peptide that comprises a sequence chosen from this list advantageously further comprises one or more amino acids at both ends of the cystine loop region.

Peptide Antigens that Cross-React with Protein from Hepatitis C Virus

A variety of peptide sequences from Hepatitis C Virus ("HCV") are known, as for example, described by U.S. Nos. 5,698,390, 5,712,087, 5,350,671, 5,191,064, 5,866,139, 5,709,995, 5,856,437 and by EP Nos. 970305 Bl, 318216 Bl and 450931 Bl, the disclosures of which are incorporated by reference in their entireties. The sequences shown in these patents can be modified and improved by preferred embodiments of the present disclosure. Furthermore, such modifications often lead to new peptides having new properties and which are not identifiable as belonging to any particular virus species or strain.

In a particularly preferred embodiment, an epitope sequence of HCV that is within an intermediate sized peptide is cross-linked as described in section 4 above. The cross- linking stabilizes the epitopic structure, increasing its reactivity with antibodies and with other components of the immune system. It is further preferred to modify one or more hydrophobic residues within the epitope by substituting a hydrophilic form of an amino acid for a hydrophobic form, and to add (or increase) secondary structure outside the chosen epitopic region. SEQ ID NOs: 43 through 229 depict representative examples of sequences according to the invention that specify intermediate length peptides useful as antigens for detecting HCV infection. SEQ ID NOs: 35 through 42 show sequences from the HCV core region residues 5 through 21, and particularly residues 5 through 15, wherein the desired epitopic region is constrained by placement within a cystine loop. These sequences also show a helix region outside of the loop, which is preferred to further improve reactivity of the selected epitopic sequence. SEQ ID NOs: 108-115 show related sequences wherein one or more hydrophobic amino acid has been replaced with a hydrophilic amino acid.

When used as an antigen for immunodiagnostics testing of HCV infection, it is particularly preferred that the peptide having two or more basic amino acids within an 8 amino acid long segment, and particularly having 3 or 4 basic amino acids within a 6 amino acid long segment, as shown in this group, be used together with a glycosaminoglycan such as heparin or chondroitin sulfate. That is, to prevent non-specific binding of the antigen to a negatively charged surface used in the assay, it is preferred to add a glycosaminoglycan to the assay kit, solid phase, wash solution or the like. Co- pending application U.S. No. 08/912,580 entitled DIAGNOSTIC TEST DEVICE WITH IMPROVED FLUID MOVEMENT AND RESISTANCE TO INTERFERENCE, which is herein incorporated by reference in its entirety, describes further details of how to use one or more glycosaminoglycans to improve diagnostic tests that utilize antigens with a local concentration of basic amino acid residues. The inventors specifically contemplate methods and materials that improve HCV tests by alleviating false positive (high background) test results via including glycosaminoglycan in water solutions of antigen peptides such as those described here.

SEQ ID NOs: 43 through 50 show sequences from the HCV core region residues 44 through 55, and particularly residues 44 through 53, wherein the desired epitopic region is constrained by placement within a cystine loop. These sequences also show a helix region outside of the loop, which is preferred to further improve reactivity of the selected epitopic sequence. SEQ ID NOs: 116 through 123 show related sequences wherein one or more hydrophobic amino acid has been replaced with a hydrophilic amino acid.

SEQ ID NOs: 51 and 52 show sequences from the HCV core region residues 61 through 74 wherein a terminal arginine has been replaced with a non-basic amino acid in accordance with an embodiment of the invention. SEQ ID NOs: 124 and 125 show related sequences wherein one or more hydrophobic amino acid has been replaced with a hydrophilic amino acid.

SEQ ID NOs: 53 through 60 show sequences from the HCV core region residues 62 through 71, wherein the desired epitopic region is constrained by placement within a cystine loop. These sequences also show a helix region outside of the loop, which is preferred to further improve reactivity of the selected epitopic sequence. SEQ ID NOs: 126 through 133 show related sequences wherein one or more hydrophobic amino acid has been replaced with a hydrophilic amino acid.

SEQ ID NOs: 61 through 70 show sequences from the HCV non-structural 4 region (NS4) residues 1933 through 1947, particularly residues 1937 through 1945, wherein, as shown in SEQ ID NOs: 63 through 70, the desired epitopic region preferably is constrained by placement within a cystine loop. These sequences also show a helix region outside of the loop, which is preferred to further improve reactivity of the selected epitopic sequence. SEQ ID NOs: 134 through 143 show related sequences wherein one or more hydrophobic amino acid has been replaced with a hydrophilic amino acid.

SEQ ID NOs: 71 through 99 show sequences from the HCV El region residues 208 through 226, and particularly residues 211 through 222, wherein the desired epitopic region is constrained by placement within a cystine loop. In the examples shown here, 18 amino acid long regions are terminated by cysteines. Preferably, the cysteines are oxidized to form intra-chain cystines in order to constrain and stabilize the epitope structure. SEQ ID NOs: 144 through 172 show related sequences wherein one or more hydrophobic amino acid has been replaced with a hydrophilic amino acid.

SEQ ID NOs: 100 through 107 show sequences from the HCV El region residues 208 through 226, and particularly residues 211 through 222, wherein the desired epitopic region preferably is constrained by terminal cystines. Preferably, the cysteines are oxidized to form intra-chain cystines in order to constrain and stabilize the epitope structure. These sequences also show a helix region outside of the loop, which is preferred to further improve reactivity of the selected epitopic sequence. SEQ ID NOs: 173 through 180 show related sequences wherein one or more hydrophobic amino acid has been replaced with a hydrophilic amino acid. SEQ ID NOs: 181 through 184 show representative sequences useful for detecting antibodies against the HCV core region. SEQ ID NOs: 185 through 194 show related sequences for the NS3 protein. SEQ ID NOs: 195 through 209 show related sequences for the NS4 protein. SEQ ID NOs: 210 through 229 show related sequences for the NS5 protein.

Of course, other sequences can be determined according to methods of the invention and are contemplated. In further embodiments, combinations of two or more, particularly three or more, more particularly 4 or more peptides are used together. The embodiment of altering a peptide to make it more hydrophilic as described above is particularly helpful to obtain multiple peptides of the same epitopic site but of alternative viral strains. It is preferred that peptides used in combination be made more hydrophilic so that they could be used at higher concentration.

Peptide Antigens that Correspond to Other Disease Organisms The antigens contemplated for HIV testing, in accordance with the discoveries enumerated above, differ from naturally occurring proteins and peptides, and provide improved diagnostic assay and therapeutic results compared to the use of sequences obtained from naturally occurring molecules. Further embodiments according to the invention provide improved tests and vaccines for intermediate size antigens that correspond to other disease agents such as HTLV-I and HTLV-II virus as well.

Disease agents such as those exemplified herein as well as others contain antigens with hydrophobic amino acid residues of leucine, isoleucine, valine, methionine and phenyl alanine. These residues can contribute to structural instability when a peptide antigen mimic from about 25 to about 100 amino acids long, advantageously from about 36 to about 50 amino acids and more advantageously between about 41 to about 50 amino acids long is prepared (de novo or by removal) from a larger protein sequence. Altering such a hydrophobic residue to a more hydrophilic form such as glutamine, serine, threonine, asparagine, proline or even to a less hydrophobic form such as alanine provides improved diagnostic tests of greater sensitivity and improved therapeutic agents of greater potency. In some cases, the hydrophobic amino acid can be replaced with a charged amino acid such as arginine for leucine. Generally, however, the hydrophobic amino acid most advantageously is replaced with a hydrophilic uncharged amino acid having a similar size to the original hydrophobic amino acid. Replacement of an individual L amino acid with another L amino acid is emphasized for convenience, however, alteration of the amino acid, or replacement with a D amino acid or other compound also is contemplated, as reviewed below under "Further Modifications to the Antigen. "

Multiple changes of hydrophobic amino acids to less hydrophobic, or to hydrophilic amino acids are advantageous, particularly when more than one hydrophobic amino acid is present in an 8 amino acid long section such as a leucine finger domain. The inventors have obtained data showing that such multiple changes to a natural sequence of an intermediate sized peptide yield improved performance when the peptide is used for diagnostic tests of HIV-1. The skilled artisan will readily appreciate that alterations can be made to a wide range of antigens of intermediate size.

The other strategies for improving intermediate sized antigens, by for example, removing a positive charge and increasing the amount of alpha helix are useful to prepare antigens corresponding to other disease-causing organisms and are contemplated. These antigens generally are more stable than the corresponding natural sequence antigens and can be used advantageously in improved immunoassays and immunotherapies.

Embodiments of the claimed invention advantageously allow an increase in the amount of antigen used in an immunodiagnostic assay (or therapy) by making the antigen more hydrophilic. This increase in antigen used for specific binding reaction(s) can lead directly to more advantageous sensitivity as well as more advantageous reactivity with a broader range of HIV-1 Group O specimens when applied to HIV infection testing. Analogous improvements in the use of other peptide antigens for other disease organisms such as HTLV-I and HTLV-II virus are possible.

Test Methods that Use Peptide Antigens

Peptides of the invention can be used in diagnostic tests that employ antigen-antibody binding for detection of a disease agent. It is preferred to use a very easy, rapid (three minutes) dot-blot assay method as described in co-pending application U.S. App. Ser. No. 09/069,935 "Multiple Readout Immunoassay with Improved Resistance to Interferences" (Attorney Docket No. 073294/0173 filed April 30, 1998, incorporated herein in its entirety by reference.) However, the inventive antigens also can be used in diagnostic methods that require these very long incubation time periods and multiple steps. The test device has a housing comprised of a water impermeable material in which other test components such as an absorbent pad with a reagent layer, filter and a reagent used to obtain a test result are held. The housing has an opening to admit a fluid sample. The housing comes apart during use so that the user can remove the filter to expose the reagent layer for application of a reagent and/or wash fluid. A sleeve that holds the filter is removably attached to the housing such that contact of the filter is favored over contact of the sleeve with the surface of the reagent layer. The sleeve is attached to the housing by a bayonet mount. After a sample is applied, and an optional wash solution added, the sleeve is removed and further optional reagent solution and a wash solution are added directly to the reagent layer. In some applications a sample is added to the device and further processing is carried out at a separate location or after storage of the device for a few hours. In these situations, the sleeve remains attached to the housing to prevent or delay the release of moisture from the device until the later processing steps are carried out. The housing also may contain a cover to protect the opening and further guard against the release of moisture.

Multiple housings can be incorporated into a multi-test unit to allow high volume testing. The latter embodiment is acceptable for infectious disease testing of blood samples at blood banks. Especially acceptable in this context is a 32 well multiple-test device having overall dimensions of 3.5 inches by 6.75 inches, a 48 well multiple-test device having overall dimensions of 5.125 inches by 6.75 inches and a ninety six well multiple-test device having overall dimensions of 6.75 inches by 9.875 inches. Each of these multiple-test devices has a well-size (for admission of a sample) of 0.75 inches. The 32 well device is particularly advantageous and is desirably configured as a single array of 4 eight member rows. In one embodiment 4 (or 8) test devices that correspond in size to a column (or row) of a microtiter plate are used in applications where intermediate numbers of samples are processed.

The housing and other parts of the test device are constructed from well-known materials in accordance with well-known methods of the prior art. Material suitable for the invention should not interfere with the production of a detectable signal and should have a reasonable inherent strength, or strength can be provided by means of a supplemental support, such as, for example, by forming a nitrocellulose layer onto an absorbent pad, by means of a suspension of nitrocellulose. The test device positions parts with a positioning "sleeve" to allow even fluid flow between the parts without interference by the sleeve itself, and the parts are arranged to minimize transverse flow. The device uses friction-held parts and water swellable parts to allow fluid to more evenly flow through junctions between the parts and a dispersing layer downstream of the filter to help disperse fluid more evenly to the reagent layer, where the reagent layer is integrated with absorbent material to form a single unit. The physical assembly of components from known materials within the housing generally will be understood to a skilled artisan but for clarity, further details are provided in the above- referenced applications in the form of definitions of some terms used in the claims.

An antigen for an HIV test is immobilized onto the reagent layer portion of the absorbent pad by absorption, via spotting a water solution of the antigen. The optimum amount of antigen to use is determined by methods accepted in the art. The inventors used approximately 100 ng of antigen per test for the HIV-1 embodiments.

Acceptable antigens for use as a device for hepatitis C testing include, for example, peptides of modified "HCV regions" known as core, NS3, NS4 and NS5, as discussed by Feucht et al. in J. Clin. Microbiol. 33:620-624 (1995), having one or more amino acid substitutions as described for HIV test antigens in the present specification. Representative examples taken from an immunoreactive region of the NS4 protein are shown in Figure 2 as SEQ ID NO: 27-30. The three intermediate size peptides of SEQ ID NO: 28-30 are derived from the natural sequence shown in SEQ ID NO: 27. Representative examples taken from the core protein of hepatitis C virus are shown as SEQ ID NO: 31-34. The three intermediate size peptides of SEQ ID NO: 32-34 are derived from the natural sequence shown in SEQ ID NO: 31. The claimed invention of replacing one or more hydrophobic residue(s) with hydrophilic residue(s), as exemplified by SEQ ID NO: 28-30 and SEQ ID NO: 32-34 is particularly advantageous for hepatitis C testing because a mixture of several antigens typically are used together in order to detect a suitably wide range of hepatitis C infections. By altering a peptide to make it more soluble, the claimed invention allows a larger amount and/or variety of peptide to be employed as binding agent for testing hepatitis C.

Antigens useful for testing of exposure to other pathogens such as those responsible for lyme disease, toxoplasmosis, and other microorganisms such as rubella, mycoplasma, cytomegalo virus, herpes, HTLVI, HTLVII, Hepatitis B, and chlamydia are known and also can be modified according to the principles enumerated herein. Chemically synthesized peptides and recombinant proteins can be immobilized within devices as claimed by routine methods, such as spotting a water solution of the antigen onto a nitrocellulose membrane or membrane layer. Antigens in accordance with the invention can be used for therapy (prevention and/or treatment) of infection according to well known methods in the art, such as those described in the above-cited co-pending applications.

Preparation of the Peptides The peptides of the invention can be prepared using any suitable means. Because of their relatively short size (generally, less than 100 amino acids, advantageously less than 75, more advantageously less than 50 and conveniently less than 40), the peptides can be synthesized in solution or on a solid support in accordance with conventional peptide synthesis techniques. Various automatic synthesizers are commercially available (for example, from Applied Biosystems) and can be used in accordance with known protocols. See, for example, Stewart and Young, SOLID PHASE PEPTIDE SYNTHESIS (2d. ed. , Pierce Chemical Co., 1984); 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 claimed 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 erf. , 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 proteins or peptides, which comprise one or more peptide sequences of the invention, can be used to prepare the HIV cytotoxic T cell epitopes identified herein or identified using the methods disclosed herein. For example, a recombinant peptide of the claimed 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 polypeptide is used that incorporates several T cell epitopes into a single polypeptide.

As the coding sequence for peptides of the length contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et. al. , J. Am. Chem. Soc , 103, 3185 (1981), modification can be made simply by substituting the appropriate base(s) for those encoding the native peptide sequence. The coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the desired fusion protein. A number of such vectors and suitable host systems are now available. For expression of the fusion proteins, 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.

Further Modifications to the Peptides

In another embodiment at least one additional amino acid is added to at least one terminus of a peptide of the claimed invention. Such added amino acid(s) facilitates linking the peptide to another peptide, coupling to a carrier, or coupling to a support. The added amino acid(s) also can be chosen to alter the physical, chemical or biological properties of the peptide, such as, for example adding another epitope for T-cell stimulation. 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 another embodiment, a peptide of the invention can differ from the natural sequence by being modified by terminal-NH sub 2 acylation, e.g. , acetylation, or thioglycolic acid amidation, terminal-carboxyl 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 claimed 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 carboxyl terminal end, or both, of peptides derived from the sequences disclosed herein.

Thus, although preferred amino acid substitutions for HIV-1 testing are described by, for example, SEQ ID Nos. 1-6, further conservative substitutions are possible and sometimes desirable for HIV-1 testing. 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. Other amino acid substitutions are provided as groups within individual claims. Preferably, the portion of the peptide sequence that is intended to mimic an antigen of HIV will not differ by more than about 30% from any of the sequences provided herein, 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 protein, (or other component of the immune system) are known. See, for instance, Allen et. al , Nature, 327, 713-717; Sette et. al , Nature, 328, 395-399; Takahashi et. al , J. Exp. Med. , 170, 2023-2035 (1989); and Maryanski et. al , Cell, 60, 63-72 (1990).

A considerable amount of work in this area has provided algorithms to use in making conservative changes to individual amino acids without altering a peptide 's biological activity. 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 protein is generally understood in the art as cited in U.S. No. 5,703,057 (citing Kyte and Doolittle, 1982, incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant peptide which in turn defines the interaction of the peptide with other molecules, for example, receptors, DNA, antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

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 hydrophihcity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophihcity of a protein, as governed by the hydrophihcity 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 hydrophihcity 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 hydrophihcity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent peptide. In such changes, the substitution of amino acids whose hydrophihcity 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 substituents, for example, their hydrophobicity, hydrophihcity, 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.

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 to can also 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-peptides are substantially more resistant to peptidases, and therefore are more stable in serum and tissues compared to their L-peptide counterparts, the stability of D-peptides under physiological conditions may more than compensate for a difference in affinity compared to the corresponding L-peptide. 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.

An advantageous embodiment is to prepare the peptide by chemical synthesis. In another embodiment, the peptide is made recombinantly. In the latter case, modifications, including conservative modifications, are best carried out by changing a DNA sequence that codes for the peptide. The following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. The amino acid changes may be achieved by changing the codons of the DNA sequence, according to the following codon table:

Amino Acids Svmbol 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

Biologically functional universal peptides can be prepared 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.

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 M13 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage. Site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. 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 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.

The following examples are provided to illustrate an embodiment of the invention and are not intended to limit the specification or scope of the claims in any way. Example 1

In this Example, peptides were prepared from sequences shown in Figure 2 and were tested as antigens for HIV-1 testing of blood samples. The test method is described in co- pending U.S. patent application "Multiple Readout Immunoassay for Improved Resistance to Interferences. " Five HIV Group O blood specimens obtained from Africa, two HIV Group O blood specimens obtained from the United States and one non-Group O blood specimen were tested. Each blood specimen was tested at no dilution, 10 times dilution, 100 times dilution and 1000 times dilution with peptides according to embodiments of the claimed invention and with two previously known peptides, as shown in Figure 3.

Data were obtained as exemplified by the data presented in Figure 3 and Figure 4, indicating that peptides of the claimed invention provide more sensitive HIV tests, and that peptides according to the invention provide HIV tests of more specific reactivity for HIV-1 Group O compared to a control test.

Example 2

In this Example, a peptide is prepared having a sequence shown by SEQ ID NO: 9.

The peptide is incorporated into a diagnostic test according to the procedure of Example 1. Ten HIV-1 group O infected samples are tested according to the procedure of Example 1, and all test results are positive, indicating the presence of antibodies to HIV-1 Group O virus antigen.

Example 3

In this Example, a peptide is prepared having a sequence shown by SEQ ID NO: 10.

The peptide is incorporated into a diagnostic test according to the procedure of Example 1.

Ten HIV-1 group O infected samples are tested according to the procedure of Example 1, and all test results are positive, indicating the presence of antibodies to HIV-1 Group O virus antigen. Example 4

In this Example, a peptide is prepared having a sequence shown by SEQ ID NO: 11. The peptide is incorporated into a diagnostic test according to the procedure used in Example

1. Ten HIV-1 group O infected samples are tested according to the procedure used in

Example 1, and all test results are positive, indicating the presence of antibodies to HIV-1

Group O virus antigen.

Example 5

In this Example, a peptide is prepared having a sequence shown by SEQ ID NO: 12.

The peptide is incorporated into a diagnostic test according to the procedure used in Example

1. Ten HIV-1 group O infected samples are tested according to the procedure used in Example 1, and all test results are positive, indicating the presence of antibodies to HIV-1

Group O virus antigen.

Example 6

In this Example, a peptide is prepared having a sequence shown by SEQ ID NO: 13.

The peptide is incorporated into a diagnostic test according to the procedure used in Example 1. Ten HIV-1 group O infected samples are tested according to the procedure used in Example 1, and all test results are positive, indicating the presence of antibodies to HIV-1 Group O virus antigen.

Example 7

In this Example, a peptide is prepared having a sequence shown by SEQ ID NO: 14.

Example 8

In this Example, a peptide is prepared having a sequence shown by SEQ ID NO: 15.

Group O virus antigen.

Example 9

In this Example, a peptide is prepared having a sequence shown by SEQ ID NO: 16.

Example 10

In this Example, a peptide is prepared having a sequence shown by SEQ ID NO: 18.

The peptide is incorporated into a diagnostic test according to the procedure used in Example 1. Ten HIV-1 group O infected samples are tested according to the procedure used in

Group O virus antigen. Example 11

In this Example, a peptide is prepared having a sequence shown by SEQ ID NO: 19.

Group O virus antigen.

Example 12

In this Example, a peptide is prepared having a sequence shown by SEQ ID NO: 20.

Example 1, and all test results are positive, indicating the presence of antibodies to HIV-1 Group O virus antigen.

Example 13

Peptides having a substitution for an isoleucine within the cystine loop and for a threonine at eight positions to the carboxyl terminal side of the cystine loop are useful for

HIV diagnostic tests. Substitutions at these two positions by valine and arginine, respectively, yield useful peptides that have altered immunological activity compared with the unsubstituted peptide, as determined by procedures outlined in Example 1.

Example 14

In this Example, a peptide is prepared having a sequence shown by SEQ ID NO: 30. The peptide is incorporated into a diagnostic test according to the procedure used in Example 1. A hepatitis C infected sample is tested according to the procedure used in Example 1 , and the test result is positive, indicating the presence of antibodies to hepatitis C virus antigen. All publications and filed applications referenced herein are specifically incorporated by reference in their entireties.

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein.

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 useful for detecting HIV-1 Group O infection, having a length of between 16 and 100 amino acid residues, and comprising a core sequence SEQ ID NO: 1, wherein XI is selected from the group consisting of N, Q, G, S, T, and A; X2 is R or K; X3 is selected from the group consisting of N, Q, G, S, T, H, A, L, I, V, P and M; and X4 is selected from the group consisting of N, Q, G, S, T, H, A, L, I, V, P and M, and wherein at least one of the two X4 residues is selected from the group consisting of N, Q, G, S, T, H, A, P and M.

2. A peptide as described in claim 1, wherein both X4 residues are selected from the group consisting of N, Q, G, S, T, H, P and A.

3. A peptide as described in claim 1, wherein at least one of the X4 residues is Q.

4. A peptide as described in claim 1, wherein the X3 residues are selected from the group consisting of I, L , V, H and A.

5. A peptide as described in claim 4, wherein the X3 residues are selected from the group consisting of I, L and V.

6. A peptide as described in claim 5, wherein the X3 residues are L.

7. A peptide as described in claim 1, wherein at least one of the X3 residues is Q.

8. A peptide as described in claim 1, further comprising at least 8 additional amino acid residues at the amino terminal end of SEQ ID NO: 1, wherein the eighth additional residue is selected from the group consisting of N, Q, G, S, T, H, and A.

9. A peptide as described in claim 6, wherein the eighth additional residue is Q.

10. A peptide as described in claim 1, comprising at least 12 additional amino acids at the amino terminal end of SEQ ID NO: 1, wherein the twelfth additional residue is arginine or glutamine.

11. A peptide as described in claim 10, wherein the twelfth additional residue is glutamine.

12. A peptide as described in claim 1, further comprising a Y residue added to the carboxyl side of the C residue at the carboxyl terminus of SEQ ID NO: 1.

13. A peptide as described in claim 1, wherein the core 16 residue sequence of SEQ ID NO: 1 comprises a maximum of five hydrophobic residues selected from the group consisting of I, L, V, M and W.

14. A peptide as described in claim 13, wherein the core 16 residue sequence of SEQ ID NO: 1 comprises a maximum of four hydrophobic residues selected from the group consisting of l, L, V, M and W.

15. A peptide as described in claim 13, wherein the core 16 residue sequence of SEQ ID NO: 1 comprises a maximum of three hydrophobic residues selected from the group consisting of l, L, V, M and W.

16. A peptide useful for detecting HIV-1 Group O infection, having a length between 26 and 100 amino acid residues, and comprising a core sequence SEQ ID NO: 2, wherein X is a helix of at least 5 amino acids, XI is selected from the group consisting of N, Q, G, S, T, D, N, H and A, X2 is R, K, P or E, X3 is selected from the group consisting of I, L, and V, X4 is selected from the group consisting of T, S and A, and X5 is at least one amino acid long.

17. A peptide as described in claim 16, wherein X5 comprises at least one amino acid beginning with an amino acid selected from the group consisting of R, K and T.

18. A peptide useful for detecting HIV-1 Group O infection, having a length between 36 and 100 amino acid residues, and comprising a core sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 and derivatives thereof that contain one or more conservative amino acid substitutions.

19. A peptide as described in claim 18, wherein said conservative amino acid substitutions consist of l, L, V, and M for each other, and S, T and A for each other.

20. A peptide as described in claim 18, wherein at least one amino acid selected from the group consisting of L, I and V is replaced with an amino acid selected from the group consisting of N, Q, G, S, T, H, and A.

21. A peptide useful for detecting HIV-1 Group O infection, having a length of between 17 and 100 amino acid residues, and comprising a core sequence SEQ ID NO: 17, wherein X4 is selected from the group consisting of R, K, N, E and D; X5 is selected from the group consisting of G, N, E and D; X6 is R or K; X7 is selected from the group consisting of N, Q, L, I, V and P; X8 is selected from the group consisting of N, Q, L, I and V; X12 is selected from the group consisting of S, T and A; X13 is selected from the group consisting of l, L, V and A; X14 is selected from the group consisting of K, R and E; X15 is W or T; X16 is N or H; and X17 is R or K.

22. A peptide between 26 and 100 amino acids long that substantially reacts with Group O HIV-1 test specimens and that comprises a 16 amino acid long immunodominant region, said region having a sequence shown by SEQ ID NO: 8.

23. The peptide of claim 22, further comprising an arginine or glutamine residue that is linked to the amino terminal side of said immunodominant region by an 11 amino acid residue sequence.

24. The peptide of claim 22, further comprising a serine or threonine residue that is linked to the carboxyl terminal side of said immunodominant region by a 7 amino acid residue sequence.

25. The peptide of claim 22, having a sequence selected from the sequences depicted in Figure 1.

26. The peptide of claim 25, wherein said selected sequence differs from the M type sequence shown in Figure 1 by at least 25 % .

27. The peptide of claim 26, wherein said selected sequence differs from said M type sequence by at least 50% .

28. The peptide of claim 22, wherein at least 4 amino acids within the immunodominant region differ from the M type sequence.

29. The peptide of claim 22, further comprising a lysine or arginine within the immunodominant region and separated from the amino terminus of said region by 3 amino acids, and a hydrophilic amino acid within the immunodominant region and separated from the amino terminus of said immunodominant region by 7 amino acids.

30. The peptide of claim 29, further comprising a carboxyl terminal serine or threonine residue separated from said immunodominant region by 7 amino acids.

31. The peptide of claim 30, comprising between 30 and 40 amino acids, wherein said selected sequence differs from the M type sequence by between 24% and 35%.

32. A peptide useful for detecting HIV-1 Group O infection, having a length of between 16 and 100 amino acid residues, and comprising a core sequence SEQ ID NO: 1, wherein XI is selected from the group consisting of N, Q, G, S, T, and A; X2 is selected from the group consisting of R, K, T and N; X3 is selected from the group consisting of N, Q, G, S, T, H, A, L, I, V, P and M; and X4 is selected from the group consisting of N, Q, G, S, T, H, A, L, I, V, P and M, and wherein at least one of the two X4 residues is selected from the group consisting of N, Q, G, S, T, H, A, P and M.

33. A peptide useful for detecting HIV-1 Group O infection, having a length of between 16 and 100 amino acid residues, and comprising a core sequence SEQ ID NO: 8, wherein X2 is selected from the group consisting of R, K, T and N; X5 is selected from the group consisting of N, Q, G, S, T, H, A, L, I, V, P and M; and wherein at least one of the two X5 residues is selected from the group consisting of N, Q, G, S, T, H, A, P and M.

34. A peptide useful for detecting HIV-1 infection, comprising a sequence of at least 35 amino acids from a gp41 immunodominant region, wherein said peptide sequence comprises at least 5 amino acids in an alpha helix structure on the amino terminal side of said immunodominant region, and wherein said alpha helix structure is determined by Chou- Fasman Conformational parameters from a peptide analysis computer program.

35. A peptide as described in claim 34, wherein said peptide structure comprises at least 6 amino acids in said alpha helix structure.

36. A peptide as described in claim 34, wherein said peptide structure comprises at least 8 amino acids in said alpha helix structure.

37. A peptide as described in claim 36, wherein said peptide comprises at least 12 amino acids in said alpha helix structure and further comprises an arginine or lysine amino acid residue at the twelfth position on the amino terminal side of the immunodominant region.

38. A peptide as described in claim 37 having the sequence shown in SEQ ID NO: 7.

39. A reagent for immunological detection of anti-HIV antibody in a blood sample, comprising a dried antigen that, upon rewetting with water or a clinical sample, substantially reacts with antibodies from patients exposed to HIV-1 group M virus and with antibodies from patients exposed to HIV-1 Group O virus, wherein said antigen is between 16-50 amino acids long and possesses a sequence described by any of claims 1-38.

40. A method of detecting HIV-1 Group O infection, comprising incubating a blood sample or blood derivative with a peptide described any of claims 1-38, followed by determination of binding between antibody in the blood sample or blood derivative and the peptide.

41. A kit for determining infection with HIV-1 Group O, comprising, an instruction booklet and a device for detecting the presence of anti-HIV- 1 Group O antibody in a blood sample or blood derivative, wherein the device comprises a peptide described by any of claims 1-38.

46. A diagnostic test peptide antigen for the detection of an infectious agent, wherein the peptide has a sequence that corresponds to a naturally occurring sequence and wherein at least one hydrophobic amino acid residue from the naturally occurring sequence is replaced by a hydrophilic amino acid residue.

47. An antigen as described in claim 46, wherein said hydrophobic amino acid residue is selected from the group consisting of leucine, isoleucine and valine.

48. An antigen as described in claim 46, wherein said hydrophilic amino acid residue is selected from the group consisting of glutamine, asparagine, serine, threonine and alanine.

49. An antigen as described in claim 48, wherein said hydrophilic amino acid residue is glutamine and said hydrophobic amino acid is leucine.

50. A peptide between 26 and 100 amino acids long having a central portion of at least 16 amino acids that corresponds in sequence identity to an immunodominant region of an antigen protein, and at least one amino acid at each end of said central portion, wherein said peptide is chemically synthesized and at least one hydrophobic amino acid of the immunodominant region has been replaced with a hydrophilic amino acid.

51. A peptide as described by claim 50, wherein said antigen protein is the gp36 or gp41 protein of HIV.

52. A modified peptide for the treatment or prevention of infection by an infectious agent, the modification comprising replacing at least a L, I, or V in a naturally occurring sequence of the peptide with an amino acid selected from the group consisting of A, S, T, G and N.

53. A modified peptide as described in claim 52, wherein at least one leucine amino acid is replaced with a glutamine amino acid.

54. A peptide useful for detecting hepatitis C infection, having a length between 24 and 100 amino acid residues, and comprising a core sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34 and derivatives thereof that contain one or more conservative amino acid substitutions.

55. A peptide useful for detecting hepatitis C infection, having a length between 24 and 100 amino acid residues, and comprising at least one sequence selected from the group consisting of SEQ ID NOs: 27, 31, and 32 though 229, and derivatives thereof that contain one or more conservative amino acid substitutions.

56. A peptide as described in claim 55, wherein at least one hydrophobic residue is modified to a less hydrophobic residue.

57. A method of enhancing the biological or immunological reactivity of an intermediate sized peptide, comprising replacing a hydrophobic amino acid in the peptide with a hydrophilic amino acid.

58. A method as described in claim 57, wherein the hydrophobic amino acid is selected from the group consisting of leucine, isoleucine, and valine.

59. A method as described in claim 57, wherein the hydrophilic amino acid is glutamine or arginine.

60. A method as described in claim 57, wherein at least two hydrophobic amino acids within an eight amino acid long region of the peptide are replaced.

61. A method of enhancing the biological or immunological reactivity of an intermediate sized peptide that comprises the steps:

(a) selecting a first region within the peptide that is associated with the biological or immunological reactivity;

(b) selecting a second region of at least 5 amino acids outside the first region selected in step (a);

(c) calculating the tendency of the second region to form alpha helix with one or more amino acid alterations within the region and determine a new sequence within the second region that provides a greater predicted alpha helix; and

(d) synthesizing a peptide comprising the new sequence determined from step (c).

62. A method as described in claim 61, wherein the peptide comprises an antigen of an infectious disease agent selected from the group consisting of HIV, HTLV-I, HTLV-II, syphilis organism and hepatitis C virus.

63. A method as described in claim 61, wherein the region selected in step (b) is contiguous to the first region.

569 570 571 572 573 574 575 576

Most likely M H L L Q L T V w Substitution H/Q R L/P T/S

579 580 581 582 583 584 585 586

Most likely M K Q L Q A R V L Substitution K/R R L L/Q/I

587 588 589 590 591 592 593 594

Most likely M A V E R Y L K D Substitution L T L F L/I/M Q N

597 598 599 600 601 602 603 604

Most likely M L L G I w G C S Substitution L/R N S/N L K N

607 608 609 610 61 1 612 613 614

Most likely M L I C T T T V P Substitution L/I I/V Y s K

617 618 619

Most likely M S S w Substitution T/R K S/T

SEQ ID NO: 1 X1-X1-X1-X2-X3-X1-X4-W-G-C-X2-G-X2-X4-X3-C

SEQ ID NO: 2 X-X1-X1-X1-X2-X3-X1-X1-W-G-C-X2-G-X2-X1-X3-C-Y-X4-X4-X3-X2-W-X1-X5

SEQ ID NO: 3 Q-A-R-L-Q-A-L-E-T-L-I-Q-N-Q-Q-R-L-N-L-W-G-C-K-G-K-Q-I-C-Y-T-S-V-K-W-N-T

SEQ ID NO: 4 R-A-R-L-Q-A-L-E-T-L-I-Q-N-Q-Q-R-L-N-Q-W-G-C-K-G-K-Q-I-C-Y-T-S-V-K-W-N-T

SEQ ID NO: 5 Q-A-R-L-Q-A-L-E-T-L-I-Q-N-Q-Q-R-L-N-Q-W-G-C-K-G-K-Q-I-C-Y-T-S-V-K-W-N-T

SEQ ID NO: 6 Q-A-R-L-Q-A-L-E-T-L-I-Q-N-Q-Q-R-L-N-L-W-G-C-K-G-R-L-I-C-Y-T-S-L-K-W-N-T

SEQ ID NO: 7 R-A-R-L-Q-A-L-E-T-L-I-Q-N-Q-Q-R-L-N-L-W-G-C-K-G-K-L-I-C-Y-T-S-V-K-W-N-T

SEQ ID NO: 8 N-Q-Q-X2-L-N-X5-W-G-C-K-G-K-X5-I-C-Y

SEQ ID NO: 9 Q-A-Q-L-Q-A-L-E-T-L-I-Q-N-Q-Q-R-L-N-Q-W-G-C-K-G-K-Q-I-C-Y-T-S-V-K-T-N-K

SEQ ID NO: 10 Q-A-Q-L-Q-A-L-E-T-L-L-Q-N-Q-Q-R-L-N-Q-W-G-C-K-G-K-Q-I-C-Y-T-S-V-K-T-N-K

SEQ ID NO: 11 Q-A-Q-L-Q-A-L-E-T-L-I-Q-N-Q-Q-R-L-N-Q-W-G-C-K-G-K-Q-V-C-Y-T-S-V-K-T-N-R

SEQ ID NO: 12 Q-A-Q-L-Q-A-L-E-T-L-L-Q-N-Q-Q-R-L-N-Q-W-G-C-K-G-K-Q-V-C-Y-T-S-V-K-T-N-R

SEQ ID NO: 13 R-A-R-L-Q-A-L-E-T-L-I-Q-N-Q-Q-R-L-N-L-W-G-C-K-G-K-L-V-C-Y-T-S-V-K-W-N-R

SEQ ID NO: 14 Q.A-Q-L-Q-A-L-E-T-L-I-Q-N-Q-Q-R-L-N-Q-W-G-C-K-G-K-Q-V-C-Y-T-S-V-K-W-N-R

SEQ ID NO: 15 R-A-R-L-Q-A-L-E-T-L-I-Q-N-Q-Q-R-L-N-I-W-G-C-K-G-K-L-V-C-Y-T-S-V-K-W-N-R SEQ ID NO: 16 R-A-R-L-Q-A-L-E-T-L-I-Q-N-Q-Q-R-I-N-L-W-G-C-K-G-K-L-V-C-Y-T-S-V-K-W-N-R

SEQ ID NO: 17 W-G-C-X4-X5-X6-X7-X8-C-Y-T-X12-X13-X14-X15-X16-X17

SEQ ID NO: 18 W-G-C-K-G-K-Q-V-C-Y-T-S-V-K-W-N-R

SEQ ID NO: 19 Q-N-Q-Q-R-L-N-Q-W-G-C-K-G-K-Q-V-C-Y-T-S-V-K-W-N-R

SEQ ID NO: 20 Q-A-Q-L-Q-A-L-E-T-L-I-Q-N-Q-Q-R-L-N-Q-W-G-C-K-G-K-Q-V-C-Y-T-S-V-K-W-N-R

SEQ ID NO: 21 C-A-G-K-Q-V-C

SEQ ID NO: 22 C-A-G-R-L-V-C

SEQ ID NO: 23 C-A-D-R-Q-V-C

SEQ ID NO: 24 C-A-N-R-Q-V-C

SEQ ID NO: 25 C-A-G-R-Q-V-C

SEQ ID NO: 26 C-A-G-K-L-V-C.

SEQ ID NO: 27 A-F-A-S-R-G-N-H-V-S-P-T-H-Y-V-P-E-S-D-A-A-A-R-V-T-A-I-L

SEQ ID NO: 28 A-F-A-S-R-G-N-H-V-S-P-T-H-Y-V-P-E-S-D-A-A-A-R-V-T-A-Q-Q

SEQ ID NO: 29 A-F-A-S-R-G-N-H-V-S-P-T-H-Y-V-P-E-S-D-A-A-A-R-V-T-A-I-I

SEQ ID NO: 30 A-F-A-S-R-G-N-H-V-S-P-T-H-Y-V-P-E-S-D-A-A-A-R-Q-T-A-I-L

SEQ ID NO: 31 S-T-N-G-K-P-Q-R-K-T-K-R-N-T-N-R-R-P-Q-D-V-K-F-P-G-G-G-Q-I-V-G SEQ ID NO: 32 S-T-N-G-K-P-Q-R-K-T-K-Q-N-T-N-R-R-P-Q-D-V-K-F-P-G-G-G-Q-I-V-G

SEQ ID NO: 33 S-T-N-G-K-P-Q-R-K-T-K-R-N-T-N-R-R-P-Q-D-V-K-F-P-G-G-G-Q-Q-V-G

SEQ ID NO: 34 S-T-N-G-K-P-Q-R-K-T-K-R-N-T-N-R-R-P-Q-D-V-K-F-P-G-G-G-Q-L-Q-G

SEQ ID NO: 35 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-P-K-P-Q-R-K-T-K-R-N-C

SEQ ID NO: 36 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-C-P-K-P-Q-R-K-T-K-R-N-C

SEQ ID NO: 37 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-G-K-P-Q-R-K-T-K-R-N-C

SEQ ID NO: 38

Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-C-G-K-P-Q-R-K-T-K-R-N-C

SEQ ID NO: 39 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-P-K-P-Q-R-K-T-K-R-N-C

SEQ ID NO: 40 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-G-C-P-K-P-Q-R-K-T-K-R-N-C

SEQ ID NO: 41

R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-G-K-P-Q-R-K-T-K-R-N-C

SEQ ID NO: 42

R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-G-K-P-Q-R-K-T-K-R-N-C

SEQ ID NO: 43 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-Q-V-R-A-T-R-K-T-S-C

SEQ ID NO: 44 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-G-V-R-A-T-R-K-T-W-C

SEQ ID NO: 45 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-C-Q-V-R-A-T-R-K-T-S-C

SEQ ID NO: 46

Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-C-G-V-R-A-T-R-K-T-W-C

SEQ ID NO: 47 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-L-Q-V-R-A-T-R-K-T-S-C SEQ ID NO: 48 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-G-C-L-Q-V-R-A-T-R-K-T-S-C

SEQ ID NO: 49 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-L-Q-V-R-A-T-R-K-T-S-C

SEQ ID NO: 50 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-G-C-L-Q-V-R-A-T-R-K-T-S-C

SEQ ID NO: 51 O-R-O-P-I-P-K-A-R-O-P-E-G-R-S-G

SEQ ID NO: 52 O-R-O-P-I-P-K-A-R-R-P-E-G-R-T-A

SEQ ID NO: 53 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-R-Q-P-I-P-K-A-R-Q-C

SEQ ID NO: 54 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-R-Q-P-I-P-K-A-R-R-C

SEQ ID NO: 55 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-C-R-Q-P-I-P-K-A-R-Q-C

SEQ ID NO: 56 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-C-R-Q-P-I-P-K-A-R-R-C

SEQ ID NO: 57 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-R-Q-P-I-P-K-A-R-Q-C

SEQ ID NO: 58 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-G-C-R-Q-P-I-P-K-A-R-R-C

SEQ ID NO: 59 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-R-Q-P-I-P-K-A-R-Q-C

SEQ ID NO: 60 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-G-C-R-Q-P-I-P-K-A-R-R-C

SEQ ID NO: 61 G-T-V-E-S-D-A-A-A-R-V-T-A-I-L-S

SEQ ID NO: 62 H-Y-V-P-E-S-D-A-A-A-R-V-T-O-I-L

SEQ ID NO: 63

Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-O-E-S-D-A-A-A-R-V-T-A-C SEQ ID NO: 64 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-O-E-S-D-A-A-A-R-V-T-A-C

SEQ ID NO: 65 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-C-P-E-S-D-A-A-A-R-V-T-Q-C

SEQ ID NO: 66 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-C-P-E-S-D-A-A-A-R-V-T-Q-C

SEQ ID NO: 67 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-O-E-S-D-A-A-A-R-V-T-A-C

SEQ ID NO: 68 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-G-C-O-E-S-D-A-A-A-R-V-T-A-C

SEQ ID NO: 69 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-P-E-S-D-A-A-A-R-V-T-Q-C

SEQ ID NO: 70

R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-G-C-P-E-S-D-A-A-A-R-V-T-Q-C

SEQ ID NO: 71 C-P-N-S-S-I-V-Y-E-A-A-D-A-I-L-H-T-P-G-C

SEQ ID NO: 72 C-S-N-S-S-I-V-Y-E-A-A-D-M-I-M-H-T-P-G-C

SEQ ID NO: 73 C-S-N-N-S-I-T-W-Q-L-T-N-A-V-L-H-L-P-G-C

SEQ ID NO: 74 C-S-N-S-S-I-T-W-Q-L-T-N-A-V-L-H-L-P-G-C

SEQ ID NO: 75

C-S-N-N-S-I-T-W-Q-L-T-D-A-V-L-H-L-P-G-C

SEQ ID NO: 76 C-S-N-N-S-I-T-W-Q-L-T-R-A-V-L-H-L-P-G-C

SEQ ID NO: 77 C-S-N-S-S-I-V-W-Q-L-E-Q-A-V-L-H-T-P-G-C

SEQ ID NO: 78 C-S-N-D-S-I-T-W-Q-L-Q-A-A-V-L-H-V-P-G-C

SEQ ID NO: 79

C-S-N-S-S-I-V-Y-E-A-D-D-V-I-L-H-T-P-G-C SEQ ID NO: 80 C-S-N-S-S-I-V-Y-E-A-A-D-M-I-M-H-T-P-G-C SEQ ID NO: 81

C-S-N-S-S-V-V-Y-E-T-A-D-M-I-M-H-T-P-G-C

SEQ ID NO: 82 C-S-N-S-S-I-V-Y-E-A-V-D-V-I-L-H-T-P-G-C

SEQ ID NO: 83 C-S-N-L-S-I-V-Y-E-T-T-D-M-I-M-H-T-P-G-C

SEQ ID NO: 84 C-S-N-S-S-I-V-F-E-A-A-D-I-I-M-H-T-P-G-C

SEQ ID NO: 85 C-S-N-S-S-I-V-Y-E-A-V-D-V-I-M-H-T-P-G-C

SEQ ID NO: 86

C-S-N-S-S-I-V-Y-E-T-A-D-M-I-M-H-T-P-G-C

SEQ ID NO: 87 C-P-N-S-S-I-V-Y-E-A-A-D-A-I-L-H-T-P-G-C

SEQ ID NO: 88 C-P-N-S-S-I-V-Y-E-A-A-D-A-I-L-H-S-P-G-C

SEQ ID NO: 89 C-P-N-S-S-I-V-Y-E-A-A-D-A-I-L-H-A-P-G-C

SEQ ID NO: 90 C-P-N-S-S-I-V-Y-E-T-A-D-T-I-L-H-S-P-G-C

SEQ ID NO: 91

C-P-N-S-S-I-V-Y-E-A-D-H-H-I-L-H-L-P-G-C

SEQ ID NO: 92 C-P-N-S-S-I-V-Y-E-A-E-H-Q-I-L-H-L-P-G-C

SEQ ID NO: 93 C-P-N-S-S-I-M-Y-E-A-E-H-H-I-L-H-L-P-G-C

SEQ ID NO: 94 C-P-N-S-S-I-V-Y-E-T-D-Y-H-I-L-H-L-C-P-G

SEQ ID NO: 95 C-P-N-T-S-I-V-Y-E-T-E-H-H-I-M-H-L-P-G-C SEQ ID NO: 96 C-P-N-S-S-I-V-Y-E-A-D-N-L-I-L-H-A-P-G-C

SEQ ID NO: 97

C-P-N-S-S-I-V-Y-E-A-D-S-L-I-L-H-A-P-G-C

SEQ ID NO: 98 C-P-N-S-S-I-V-Y-E-A-D-D-L-I-L-H-A-P-G-C

SEQ ID NO: 99 C-P-N-S-S-I-V-L-E-A-D-A-M-I-L-H-L-P-G-C

SEQ ID NO: 100 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-V-Y-E-A-A-D-A-I-L-H-T-C

SEQ ID NO: 101 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-T-W-Q-T-D-H-M-V-M-H-L-C SEQ ID NO: 102

Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-C-V-Y-E-A-A-D-A-I-L-H-T-C

SEQ ID NO: 103 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-C-T-W-Q-T-D-H-M-V-M-H-L-C

SEQ ID NO: 104 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-V-Y-E-A-A-D-A-I-L-H-T-C

SEQ ID NO: 105 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-G-C-V-Y-E-A-A-D-A-I-L-H-T-C

SEQ ID NO: 106 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-T-W-Q-T-D-H-M-V-M-H-L-C

SEQ ID NO: 107

R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-G-C-T-W-Q-T-D-H-M-V-M-H-L-C

SEQ ID NO: 108 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-P-K-P-Q-R-K-T-K-R-N-C

SEQ ID NO: 109 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-C-P-K-P-Q-R-K-T-K-R-N-C

SEQ ID NO: 110 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-G-K-P-Q-R-K-T-K-R-N-C

SEQ ID NO: 111 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-C-G-K-P-Q-R-K-T-K-R-N-C SEQ ID NO: 112 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-A-Q-G-C-P-K-P-Q-R-K-T-K-R-N-C SEQ ID NO: 113

R-A-R-L-Q-A-W-E-K-T-Q-E-D-Q-A-R-L-G-C-P-K-P-Q-R-K-T-K-R-N-C

SEQ ID NO: 114 R-A-R-L-Q-A-S-E-K-T-L-E-D-Q-A-R-L-N-C-G-K-P-Q-R-K-T-K-R-N-C

SEQ ID NO: 115 R-A-R-L-Q-A-S-E-K-T-L-E-D-Q-A-R-L-N-C-G-K-P-Q-R-K-T-K-R-N-C

SEQ ID NO: 116 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-Q-G-I-W-G-C-Q-N-R-A-T-R-K-T-S-C

SEQ ID NO: 117 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-G-Q-R-A-T-R-K-T-W-C

SEQ ID NO: 118

Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-C-Q-S-R-A-T-R-K-T-S-C

SEQ ID NO: 119 Q-A-R-L-L-A-W-E-K-T-N-K-D-Q-Q-L-L-G-C-G-Q-R-A-T-R-K-T-W-C

SEQ ID NO: 120 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-L-Q-T-R-A-T-R-K-T-S-C

SEQ ID NO: 121 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-G-C-L-Q-Q-R-A-T-R-K-T-S-C

SEQ ID NO: 122 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-L-Q-S-R-A-T-R-K-T-S-C

SEQ ID NO: 123

R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-G-C-L-Q-Q-R-A-T-R-K-T-S-C

SEQ ID NO: 124 R-R-O-P-N-P-K-A-R-O-P-E-G-R-S-G

SEQ ID NO: 125 R-R-O-P-O-P-K-A-R-R-P-E-G-R-T-A

SEQ ID NO: 126 Q-A-R-L-L-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-R-Q-P-Q-P-K-A-R-Q-C

SEQ ID NO: 127 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-R-Q-P-Q-P-K-A-R-R-C SEQ ID NO: 128 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-C-R-Q-P-N-P-K-A-R-Q-C

SEQ ID NO: 129 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-C-R-Q-P-Q-P-K-A-R-R-C

SEQ ID NO: 130 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-R-Q-P-H-P-K-A-R-Q-C

SEQ ID NO: 131 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-G-C-R-Q-P-R-P-K-A-R-R-C

SEQ ID NO: 132 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-R-Q-P-N-P-K-A-R-Q-C

SEQ ID NO: 133 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-G-C-R-Q-P-R-P-K-A-R-R-C SEQ ID NO: 134

G-T-V-E-S-D-A-A-A-R-O-T-A-I-L-S

SEQ ID NO: 135 H-Y-V-P-E-S-D-A-A-A-R-N-T-O-I-L-O-R-S

SEQ ID NO: 136 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-O-E-S-D-A-A-A-R-Q-T-A-C

SEQ ID NO: 137 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-O-E-S-D-A-A-A-R-T-T-A-C

SEQ ID NO: 138 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-C-P-E-S-D-A-A-A-R-Q-T-Q-C

SEQ ID NO: 139

Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-C-P-E-S-D-A-A-A-R-T-T-Q-C

SEQ ID NO: 140 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-O-E-S-D-A-A-A-R-Q-T-A-C

SEQ ID NO: 141 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-G-C-O-E-S-D-A-A-A-R-S-T-A-C

SEQ ID NO: 142 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-P-E-S-D-A-A-A-R-A-T-Q-C

SEQ ID NO: 143 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-G-C-P-E-S-D-A-A-A-R-Q-T-Q-C SEQ ID NO: 144 C-P-N-S-S-I-N-Y-E-A-A-D-A-I-Q-H-T-P-G-C SEQ ID NO: 145

C-S-N-S-S-I-Q-Y-E-A-A-D-Q-I-M-H-T-P-G-C

SEQ ID NO: 146 C-S-N-N-S-I-T-Q-Q-L-T-N-A-V-Q-H-L-P-G-C

SEQ ID NO: 147 C-S-N-S-S-I-T-N-Q-L-T-N-A-V-T-H-L-P-G-C

SEQ ID NO: 148 C-S-N-N-S-I-T-Q-Q-L-T-D-A-V-Q-H-L-P-G-C

SEQ ID NO: 149 C-S-N-N-S-I-T-T-Q-L-T-R-A-V-Q-H-L-P-G-C

SEQ ID NO: 150

C-S-N-S-S-I-V-Q-Q-L-E-Q-A-V-Q-H-T-P-G-C

SEQ ID NO: 151 C-S-N-D-S-I-T-H-Q-L-Q-A-A-V-R-H-V-P-G-C

SEQ ID NO: 152 C-S-N-S-S-I-V-H-E-A-D-D-V-I-R-H-T-P-G-C

SEQ ID NO: 153 C-S-N-S-S-I-Q-Y-E-A-A-D-M-I-Q-H-T-P-G-C

SEQ ID NO: 154 C-S-N-S-S-V-V-Q-E-T-A-D-M-I-Q-H-T-P-G- C

SEQ ID NO: 155

C-S-N-S-S-I-V-Y-E-A-V-D-V-I-Q-H-T-P-G-C

SEQ ID NO: 156

C-S-N-L-S-I-Q-Y-E-T-T-D-Q-I-M-H-T-P-G-C

SEQ ID NO: 157 C-S-N-S-S-I-Q-F-E-A-A-D-I-I-Q-H-T-P-G-C

SEQ ID NO: 158 C-S-N-S-S-I-V-Q-E-A-V-D-V-I-Q-H-T-P-G-C

SEQ ID NO: 159 C-S-N-S-S-I-V-Q-E-T-A-D-Q-I-M-H-T-P-G-C SEQ ID NO: 160 C-P-N-S-S-I-Q-Y-E-A-A-D-A-I-Q-H-T-P-G-C

SEQ ID NO: 161

C-P-N-S-S-I-V-Y-E-A-A-D-A-I-Q-H-S-P-G-C

SEQ ID NO: 162

C-P-N-S-S-I-V-Y-E-A-A-D-A-I-Q-H-A-P-G-C

SEQ ID NO: 163 C-P-N-S-S-I-V-Y-E-T-A-D-T-I-Q-H-S-P-G-C

SEQ ID NO: 164 C-P-N-S-S-I-V-Y-E-A-D-H-H-I-Q-H-L-P-G-C

SEQ ID NO: 165 C-P-N-S-S-I-V-Y-E-A-E-H-Q-I-Q-H-L-P-G-C

SEQ ID NO: 166

C-P-N-S-S-I-M-Y-E-A-E-H-H-I-Q-H-L-P-G-C

SEQ ID NO: 167

C-P-N-S-S-I-V-Y-E-T-D-Y-H-I-Q-H-L-C-P-G

SEQ ID NO: 168 C-P-N-T-S-I-V-Y-E-T-E-H-H-I-Q-H-L-P-G-C

SEQ ID NO: 169 C-P-N-S-S-I-V-Y-E-A-D-N-L-I-Q-H-A-P-G-C

SEQ ID NO: 170 C-P-N-S-S-I-V-Y-E-A-D-S-L-I-Q-H-A-P-G-C

SEQ ID NO: 171

C-P-N-S-S-I-V-Y-E-A-D-D-L-I-Q-H-A-P-G-C

SEQ ID NO: 172 CPNSSIVLEADAMIQHLPGC

SEQ ID NO: 173 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-V-Y-E-A-A-D-A-I-Q-H-T-C

SEQ ID NO: 174 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-T-W-Q-T-D-H-Q-V-M-H-L-C

SEQ ID NO: 175 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-C-V-Q-E-A-A-D-A-I-Q-H-T-C SEQ ID NO: 176 Q-A-R-L-L-A-W-E-K-T-L-K-D-Q-Q-L-L-G-C-T-W-Q-T-D-H-M-Q-M-H-L-C

SEQ ID NO: 177 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-A-Q-G-C-V-Y-E-A-A-D-A-Q-L-H-T-C

SEQ ID NO: 178 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-G-C-V-Y-E-A-A-D-A-I-Q-H-T-C

SEQ ID NO: 179 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-T-W-Q-T-D-H-Q-V-M-H-L-C

SEQ ID NO: 180 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-G-C-T-Q-Q-T-D-H-M-Q-M-H-L-C

SEQ ID NO: 181 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-K-R-N-T-N-R-R-P-Q-D-V-K-F-P-G-C SEQ ID NO: 182

R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-V-K-F-P-G-G-G-Q-I-V-G-G-V-Y-L-C

SEQ ID NO: 183 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-R-S-Q-P-R-G-R-R-Q-P-I-P-K-A-R-C

SEQ ID NO: 184 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-R-S-Q-PR-G-R-R-Q-P-I-P-K-D-R-C

SEQ ID NO: 185 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-L-N-P-S-V-A-A-T-L-G-F-G-A-Y-M-C

SEQ ID NO: 186 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-A-A-T-L-G-F-G-A-Y-M-S-K-A-H-G-C

SEQ ID NO: 187

R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-L-T-H-I-D-A-H-F-L-S-Q-T-K-Q-A-C

SEQ ID NO: 188 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-L-T-H-I-D-A-H-F-L-S-Q-T-K-Q-S-C

SEQ ID NO: 189 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-L-T-H-I-D-A-H-F-L-S-Q-T-K-Q-G-C

SEQ ID NO: 190 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-D-A-H-F-L-S-Q-T-K-Q-A-G-D-N-F-C

SEQ ID NO: 191 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-D-A-H-F-L-S-Q-T-K-Q-A-G-E-N-L-C SEQ ID NO: 192 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-D-A-H-F-L-S-Q-T-K-Q-A-G-E-N-F-C

SEQ ID NO: 193 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-D-A-H-F-L-S-Q-T-K-Q-S-G-D-N-F-C

SEQ ID NO: 194 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-D-A-H-F-L-S-Q-T-K-Q-G-G-D-N-F-C

SEQ ID NO: 195 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-K-P-A-I-I-P-D-R-E-V-L-Y-R-E-F-C

SEQ ID NO: 196 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-K-P-A-V-V-P-D-R-E-V-L-Y-Q-E-F-C

SEQ ID NO: 197 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-K-P-A-V-I-P-D-R-E-V-L-Y-R-E-F-C SEQ ID NO: 198

R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-R-P-A-V-I-P-D-R-E-V-L-Y-R-E-F-C

SEQ ID NO: 199 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-R-P-A-I-V-P-D-R-E-L-L-Y-R-E-F-C

SEQ ID NO: 200 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-R-V-V-V-A-P-D-K-E-I-L-Y-E-A-F-C

SEQ ID NO: 201 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-R-V-A-I-A-P-D-K-E-V-L-Y-E-A-F-C

SEQ ID NO: 202 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-R-V-V-I-V-P-D-K-E-V-L-Y-E-E-F-C

SEQ ID NO: 203

R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-K-P-A-L-V-P-D-K-E-V-L-Y-Q-Q-Y-C

SEQ ID NO: 204 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-L-V-P-D-K-E-V-L-Y-Q-Q-Y-D-E-M-C

SEQ ID NO: 205 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-A-F-A-S-R-G-N-H-V-S-P-T-H-Y-V-C

SEQ ID NO: 206 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-A-F-A-S-R-G-N-H-V-A-P-T-H-Y-V-C

SEQ ID NO: 207 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-R-G-N-H-V-S-P-T-H-Y-V-P-E-S-D-C SEQ ID NO: 208 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-R-G-N-H-V-A-P-T-H-Y-V-V-E-S-D-C

SEQ ID NO: 209 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-R-G-N-H-V-A-P-T-H-Y-V-P-E-S-D-C

SEQ ID NO: 210 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-V-G-S-Q-L-P-C-E-P-E-P-D-T-A-V-C

SEQ ID NO: 211 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-V-G-S-Q-L-P-C-E-P-E-P-D-V-A-V-C SEQ ID NO: 212

R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-V-G-S-Q-L-P-C-E-P-E-P-D-T-E-V-C

SEQ ID NO: 213

R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-V-G-S-Q-L-P-C-D-P-E-P-D-V-A-V-C

SEQ ID NO: 214 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-R-K-S-K-K-F-P-A-A-M-P-I-W-A-R-C

SEQ ID NO: 215 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-R-K-R-K-K-F-A-A-A-L-P-V-W-A-R-C

SEQ ID NO: 216 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-R-K-S-K-K-F-P-A-A-M-P-I-WA-R-C

SEQ ID NO: 217

R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-K-F-P-A-A-M-P-I-W-A-R-P-D-Y-N-C

SEQ ID NO: 218

RA-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-K-F-P-A-A-M-P-I-W-A-R-P-D-Y-N-C

SEQ ID NO: 219 RA-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-R-F-A-Q-A-L-P-V-W-A-R-P-D-Y-N-C

SEQ ID NO: 220 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-K-F-P-P-A-L-P-P-W-A-R-P-D-Y-N-C

SEQ ID NO: 221 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-L-I-E-T-W-K-R-P-G-Y-E-P-P-T-V-C

SEQ ID NO: 222

R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-L-I-E-S-W-K-D-P-D-Y-V-P-P-V-V-C

SEQ ID NO: 223 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A

SEQ ID NO: 224

R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-L-L-E-P-W-K-R-P-G-Y-E-P-P-T-V-C

SEQ ID NO: 225 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-K-G-S-D-V-E-S-Y-S-S-M-P-P-L-E-C

SEQ ID NO: 226 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-A-G-S-D-V-E-S-Y-S-S-M-P-P-L-E-C

SEQ ID NO: 227 R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-P-D-S-D-A-E-S-Y-S-S-M-P-P-L-E-C

SEQ ID NO: 228

R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-A-A-S-E-A-G-S-L-S-S-M-P-P-L-E-C

SEQ ID NO: 229

R-A-R-L-Q-A-W-E-K-T-L-E-D-Q-A-R-L-N-C-K-G-S-D-A-E-S-Y-S-S-M-P-P-L-E-C