WO2001029177A2 - Hiv-1 subtype d peptides - Google Patents

Abstract

Improved peptide antigens for HIV-1 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. The peptides are from about 16 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. Advantageous alterations include substitution of one or more hydrophilic residues with hydrophilic residues and increase in peptide secondary structure by adding or substituting one or more amino acids to form or extend and alpha helix within the peptide. Peptides that have been modified according to principles of the claimed invention differ from the naturally occurring forms and are not identified as belonging to any particular viral Group or subtype.

Description

HIV-1 SUBTYPE D PEPTIDES

Field of the Invention

The claimed invention relates to diagnostic tests and antigens used in such tests. More specifically, embodiments of the invention relates to peptide sequences and sequence modifications of peptides from gp 41 envelope protein that cross react immunologically with antibodies from HIN-1 subtype D.

Background of the Invention

The retro virus known as Human Immunodeficiency Virus (HIV, also HTLV-III), 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 J. 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. Sc 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-HIV 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, little information exists concerning how to make peptides that react well with HIV- 1 subtype D infected blood samples. Thus, until now attempts to detect or treat infections of HIV-1 subtype D have centered on the use of peptides and proteins having sequence(s) that correspond to known sequences obtained from naturally occurring samples, particularly relating to other subtypes within the M group. However, intermediate sized peptides between from about 24 to about 100 amino acids long obtained from these natural sequences generally are not optimized for detecting subtype D. One problem with peptide antigens is that these peptides comprise naturally occurring sequences that literally have been taken out of context from a larger protein where hydrophobic amino acids are employed to form tertiary protein structure within the intact protein. Removing or synthesizing such peptides and using them out of context often does not work well. For example, a peptide may not have enough stability or solubility to adequately react with an antibody. Furthermore, a natural sequence may be very specific to a particular subtype. Hunt et al, for example, described such a problem 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. These data indicate that even using a peptide with a sequence from a member of the O Group provides insufficient reactivity with other members of this group.

Thus, efforts to extirpate portions of native proteins to make peptides often fail to maintain epitopes of the natural protein. Most such efforts concern unmodified sequences from an intact naturally occurring protein. In fact, a basic understanding, or dogma in the art, is that making an amino acid substitution in a natural peptide sequence will, if anything generally destroy the epitopic character of that sequence. An 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 and solubility of the peptide antigen. 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. Furthermore, although negative data reporting poor solubility of peptides that mimic portions of an intact protein is seldom reported, poor solubility of such peptides is a problem that arises from using sequences that are optimized for placement inside a large protein. Accordingly, the problem of detecting a particular sub-type of HIV such as sub-type D remains an obstacle to the development of HIV tests.

Summary of the Invention

It is an object of embodiments of the invention to provide peptide sequences that yield improved reactivity for detecting and treating HIV-1 subtype D infection compared to detection and treatment using previously known peptides of natural sequences. It is another object of embodiments of the invention to provide peptides having improved solubility for the detection and treatment of HIV-1 subtype D infection compared to detection and treatment using previously known peptides of natural sequences. It is another object of embodiments of the invention to provide methods for improving peptides used as antigens in diagnostic testing and in therapy. It is yet another object of embodiments of the invention to provide diagnostic test devices for detecting HIV infection. It is yet another object of embodiments of the invention to provide methods of detecting HIV-1 infection. Further objects of embodiments of the invention readily will be apparent from the disclosure herein.

Brief Description of the Figures

Figure 1 shows representative amino acid sequences according to embodiments of the claimed invention. Figure 2 is a representative set of amino acid substitutions that advantageously produce secondary structure in HIV-1 gp 41 peptides according to embodiments of the claimed invention.

Figure 3 shows representative data obtained for detecting infection with HIV-1 Subtype D using a peptide according to embodiments of the invention, and comparison with the use of other more broadly reactive peptides.

Figure 4 shows data concerning the analytical sensitivity and specificity of SEQ ID 10 peptide as a single peptide as well as in combination with SEQ ID 23 in detecting anti- HIV antibodies in ELISA.

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 improved peptides for testing and therapy of HIV-1. These discoveries are discussed separately, together with related embodiments of the claimed invention.

An embodiment of the claimed invention provides peptide antigens for diagnostic testing and therapy of disease having sequences that differ from naturally occurring peptide sequences. These sequence differences from naturally occurring sequences provide greater stability and reactivity of peptides that are not in their customary large protein environment. Such peptides advantageously are from about 24 to about 100 amino acid residues long and more particularly, from about 24 to about 45 amino acid residues long. The peptides in accordance with one embodiment of the claimed invention are more reactive than peptides having a known natural sequence. Peptides in accordance with yet another embodiment of the claimed invention have improved water solubility compared with peptides that have a natural sequence.

The methods of altering naturally occurring sequences and peptides created thereby provide improved diagnostic tests and improved therapeutic agents. Advantageous alterations in this context include the substitution of one or more hydrophobic residues with one or more less hydrophobic residues and alteration of the amino acid sequence to increase peptide secondary structure, particularly on the amino terminal side of the cystine loop. The claimed invention is exemplified, inter alia, by substitution of tyrosine with threonine, substitution of isoleucine with serine and substitution of leucine with glutamine. Peptides that have been modified according to principles of the claimed invention differ from the naturally occurring forms and are not identified as belonging to any particular viral Group or subtype.

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 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 less hydrophobic amino acid (for example, alanine) or 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 consequence of the hydrophobic to less hydrophobic, or hydrophilic amino acid residue shift is 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 embodiments of the invention is greater than 16 amino acid residues long but smaller than 100 amino acid residues long and particularly from 24 to 45 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 embodiments of 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 embodiments of 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 exhibit different immunological characteristics than the corresponding sequences of naturally occurring proteins. The different characteristics can, for example, include a loss of one or more immunological properties, or an increase in reactivity.

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, tryptophan and isoleucine etc. to a hydrophilic (or less hydrophobic) amino acid such as glutamine, asparagine, serine, threonine, alanine etc., particularly in the immunodominant region, helps prevent structural instability when the amino acid is 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), more particularly less than about 50 amino acids (e.g. less than 50 amino acids) and especially less than about 45 amino acids (e.g. less than 45 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, in contrast, 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.

The embodiment of replacing one or more hydrophobic amino acids with one or more less hydrophobic, 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 24 to 50 amino acids, and 36 to 45 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 reviewed briefly 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 16 to about 100 amino acid residues long, and particularly 24-45 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 less hydrophobic amino acid or 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 tyrosine to a threonine amino acid but works well with a shift of another hydrophobic amino acid such as I, V, M, F and W to a less hydrophobic or to a hydrophilic amino acid. In making a substitution, 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 24 and 45 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 less hydrophobic 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 less hydrophobic or 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. Some representative sequences having one or more alterations from a known peptide of HIV-1 subtype D envelope gp41 protein are shown in Figure 1. A consensus "subtype D" sequence has been published by the Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (HUMAN RETROVIRUSES AND AIDS 1996) and is used for making sequence comparisons because some naturally occurring strains of HIV contain protein having this sequence within the respective part of the gp41 antigen.

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

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 can have 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.

It was further discovered that one or more amino acids to the amino terminal side of the cystine loop region could be altered to increase the amount of alpha helix of the peptide. The inventors extended the predicted length of alpha helix to 13 amino acids this way to make an advantageous peptide for detecting HIV-1 exposed blood samples. Some alternative sequences useful for making or extending alpha helix structure in this context are depicted in Figure 2. The amino acid combinations shown in Figure 2 are useful as a guide to increase secondary structure of an HIV peptide antigen for detection or treatment of HIV infection. For example, a consensus sequence of QARILAV for HIV-1 subtype D can be altered to become QARLLAW (SEQ ID No. 22) to increase the length of alpha helix in a peptide antigen for detecting and treating HIV-1 infection.

In other embodiments, less than 13 amino acids may form a continuous predicted secondary structure in the peptide antigen. Preferably at least 5 amino acids, more preferably at least 7, yet more preferably at least 8, and yet more preferably at least 9 contiguous amino acids are predicted to form the structure. By way of example, the peptide of SEQ ID No. 10 was synthesized and found to have greater reactivity with samples of HIV-1 subtype D infected blood compared to reactivity obtained with more native antigen sequences having less alpha helix structure, as shown in Example 1.

Other combinations of amino acids in accordance with the selection shown in Figure 2 are advantageous and are contemplated as embodiments. Particularly advantageous are peptides formed by substituting at least one of isoleucine, valine, arginine and tyrosine, which are often in the natural protein as shown by positions 4, 7, 9 and 10 of Figure 2. At least one of these amino acids advantageously is altered to an acceptable amino acid as shown in Figure 2, to provide a peptide having a greater predicted secondary structure. "Predicted" in the sense used herein, means that analysis of the sequence by the peptide analysis software program, "Peptide Companion Version 1.24 for Windows" from Peptides International, Inc. Louisville, Kentucky 40299 U.S.A. indicates the peptide should have secondary structure. The Chou- Fasman Conformational parameters are used in determining which amino acids can be changed within a helix (the preferred type of secondary structure) 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 also 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.

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).

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 HTV-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 1. The inventors found that they could mix one or more peptide antigens according to embodiments of the invention with recombinant antigen at a higher concentration if the peptide is made more hydrophilic or less hydrophobic 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 as described can improve diagnosis and/or therapy of HIV infection as was found experimentally in Experiment 1. Figure 4 shows data that indicates a peptide according to embodiments of the invention can detect an HIV-1 subtype D blood sample better than two other HIV-1 peptides of the same length. Sequences of representative altered peptides having these and related changes are shown in Figure 1 and described by the language of the claims.

The hydrophobic amino acid residues in a peptide can contribute to structural instability when a peptide antigen mimic from about 16 to about 100 amino acids long, advantageously from about 24 to about 50 amino acids and more advantageously between about 36 to about 45 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 skilled artisan will readily appreciate that alterations can be made to a wide range of antigens of intermediate size.

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. Methods and representative examples for engineering immunoreactive peptides of greater solubility and stability are provided in co-pending applications 60/098,693 filed August 31, 1998 (attorney docket No. 073294/0127), 60/091,659 filed July 2, 1998 (attorney docket No. 073294/0160), 60/072,863 filed January 28, 1998 (attorney docket No. 073294/0161), 60/072,981 filed January 29, 1998 (attorney docket No. 073294/0162), 60/098,705 filed September 1, 1998 (attorney docket No. 073294/0168), 60/088,229 filed June 5, 1998 (attorney docket No. 073294/0172), 60/100,047 filed September 11, 1998 (attorney docket No. 073294/0190), 60/100,422 filed September 15, 1998 (attorney docket No. 073294/0191), 60/104,686 filed October 16, 1998 (attorney docket No. 073294/0197) and 60/104,685 filed October 16, 1998 (attorney docket No. 073294/0198), which are herein incorporated in their entireties by reference. Such antigens generally are more stable than the corresponding natural sequence antigens and can be used advantageously in improved immunoassays and immunotherapies. Particularly preferred sequences useful for peptides that react with anti-HIV-1 Subgroup D antibodies are described in U.S. No. 60/104,681 filed 10/16/98, the contents of which are explicitly incorporated by reference in their entirety.

Embodiments of the claimed invention advantageously allow an increase in the amount of antigen used in an HIV diagnostic assay (or therapy) by making the antigen less hydrophobic. The increase in antigen that can be 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 specimens when applied to HIV infection testing.

Test Methods that Use The Antigens

Antigens of the instant 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 embodiments of 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.

Preparation of the Antigens

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 45), 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 ed., Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989), and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel et. al, eds., John Wiley and Sons, Inc., New York, 1987), and U.S. Pat. Nos. 4,237,224, 4, 273,875, 4,431,739, 4,363,877 and 4,428,941, for example. Thus, recombinant DNA-derived proteins or peptides, which comprise one or more peptide sequences of the invention, can be used to prepare the HIV cross-reacting antigens contemplated 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, along with epitope(s) of a D subtype gp41 peptide.

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 Antigen

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 advantageous amino acid substitutions for HIV testing are described by, for example, SEQ ID Nos. 1-20 and in U.S. No. 60/104,681, 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 hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 +- 1) glutamate (+3.0 +- 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0) threonine (-0.4); proline (-0.5 +- 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0) methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent peptide. In such changes, the substitution of amino acids whose hydrophilicity values are within +- 2 is preferred, those which are within +- 1 are particularly preferred, and those within +- 0.5 are even more particularly preferred. As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

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 Symbol 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. Such phages 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 mutagemc agents, such as hydroxylamine, to obtain sequence variants.

The Use of Peptides in Test Devices The peptides are particularly useful in dot blot immunoassays. An advantageous device configuration in this context comprises an absorbent pad, reagent layer and a two-part filter within a single housing as described in co-pending application No. 08/933,943 filed on September 19, 1997. Acceptable materials for the device housing include water impermeable plastics such as polystyrene, polypropylene, polyvinyl chloride and the like. Acceptable materials for the filtering portion of the filter, the dispersing portion of the filter, the reagent layer and the absorbent pad are glass fiber, ethyl cellulose, nitrocellulose and ethyl cellulose respectively. When nitrocellulose and ethyl coeeulose respectively. When nitrocellulose is used for the reagent layer, however, the material of the filter should be chosen for its ability to premix the test sample and any test reagent that may be present in the filter. Two or more materials can be physically combined to make up a filter, reagent layer or absorbent pad. Two materials are acceptable for the filter, an upper filtering material and a lower dispersal material. Most acceptable is a dispersal layer that passes fluid at a slower flow rate compared to passage of fluid through an upper filtering material. The applicants have found that the use of a dispersal layer is surprisingly superior over the use of a single part filter. Although applicants wish not to be bound by one particular theory of how their dispersal layer works in the claimed invention, the layer may remover irregularities in the flow that arise from irregular collection of particles in the upper regions of the filter. A second advantage of using a two-part filter is that the dispersal layer, by virtue of its slow fluid flow rate, can prolong one or more reaction times for substances that react before entering the reaction layer, thereby increasing sensitivity. Advantageously, the lower portion of the filter, i.e. the "dispersant layer," extends beyond the sleeve that holds the filter such that when the filter is attached to the container, the dispersant layer portion exerts a greater pressure onto the reagent layer than does the sleeve "Greater pressure" in this context means that the mechanical force per unit area exerted by the filter onto the reagent layer exceeds the mechanical force per unit area exerted by the sleeve onto the reagent layer. Preferably, the filter exerts at least twice as much mechanical pressure onto the reagent layer compared to the sleeve. More preferably, the sleeve does not contact the reagent layer. By relying principally on the filter itself to contact the reagent layer, the sleeve does not deform the reagent layer. This allows a more even and reproducible transfer of fluid from the filter to the reagent layer. Further advantageous features of a test device are found in co-pending application No. 08/933,943 filed on September 19, 1997.

The following example is 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

For this Example, peptides were prepared having the sequences SEQ ID No. 8 and SEQ ID No. 10, (sequences related to Subtype D in accordance with one embodiment of the invention) and sequences 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 and sequence 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. These peptides were used in tests of serum and plasma samples. All of the "D" typed samples reacted well with peptides of SEQ ID Nos. 8 and 10 and much better than reaction with the other two peptides. The test method described in the above-cited co-pending U.S. patent application "Multiple Readout Immunoassay for Improved Resistance to Interferences" was used. Each blood specimen was tested at no dilution, 10 times dilution, 100 times dilution and 1000 times dilution with the peptides.

Evidence was obtained showing that peptides according to the invention provide HIV tests of more specific reactivity for HIV-1 subtype D compared to previously known tests.

All references to publications and filed applications 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 subtype D infection, having a length of between 26 and 100 amino acid residues, and comprising a sequence X1-X2-X3-X4-X5-X6-X6-X6-X6- X6-X6-X6-X6-X6-X6-X6-X6-G-C-S-G-X7-H-I-C-T (SEQ ID No. 1), wherein XI is selected from the group consisting of L, Q, M, R, A, N, D, E, G, H, K, F, P and S; X2 is selected from the group consisting of Q, M, R, A, D, E, G, H, L, K, P and S; X3 is selected from the group consisting of A, M, D, E and K; X4 is selected from the group consisting of W, L, Q, M, R, A, N, D, C, E, G, H, K, F, P, S and T; X5 is selected from the group consisting of E, K and D; X6 is an amino acid and X7 is selected from the group consisting of R and K.

2. A peptide as described in claim 1, wherein the total number of the aliphatic hydrophobic amino acids I, L and V is less than 8.

3. A peptide useful for detecting HIV-1 subtype D infection, having a length of between 24 and 100 amino acid residues, and comprising a sequence X1-X2-X3-X4-X5-X6-X6-X6-X6- X6-X6-X6-X6-X6-X6-G-C-S-G-X7-H-I-C-T (SEQ ID No. 2), wherein XI is selected from the group consisting of A, M, D, E, and K; X2 is selected from the group consisting of W, L, Q, M, R, A, N, D, C, E, G, H, K, F, P, S and T; X3 is selected from the group consisting of E, K and D; X4 is selected from the group consisting of K, T, Q, M, W, R, A, N, D, E, and H; X5 is selected from the group consisting of T, Q, M, W, R, A, N and D; X6 is an amino acid and X7 is selected from the group consisting of R and K.

4. A peptide as described in claim 3, wherein the total number of the aliphatic hydrophobic amino acids I, L and V is less than 6.

5. A method of improving a peptide of HIV-1 gp 41 useful for detecting HIV-1 subtype D infection, comprising replacing a tyrosine with a more hydrophilic amino acid, wherein the peptide comprises the cystine loop region of gp 41 protein and the tyrosine is twelve positions to the amino terminal side of the cystine loop region.

6. A method as described in claim 5, wherein the more hydrophilic amino acid is threonine.

7. A method of improving a peptide of HIV-1 gp 41 useful for detecting HIV-1 subtype D infection, comprising replacing an isoleucine with a more hydrophilic amino acid, wherein the peptide comprises the cystine loop region of gp 41 protein and the isoleucine is three positions to the amino terminal side of the cystine loop region.

8. A method as described in claim 7, wherein the more hydrophilic amino acid is serine.

9. A method of improving a peptide of HIV-1 gp 41 useful for detecting HIV-1 subtype D infection, comprising replacing a leucine with a more hydrophilic amino acid, wherein the peptide comprises the cystine loop region of gp 41 and the leucine is on the amino terminal side of the cystine loop region.

10. A method as described in claim 9, wherein the more hydrophilic amino acid is glutamine.

11. A peptide useful for detecting HIV-1 subtype D infection, having a length of between 26 and 100 amino acid residues, and comprising a sequence selected from the group of sequences consisting of SEQ ID No's 1 through 130,580.

12. A reagent for immunological detection of anti-HIV subtype D 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 subtype D virus, wherein said antigen is between 16-50 amino acids long and possesses a sequence described by any of claims 1-11.

13. A method of detecting HIV-1 subtype D infection, comprising incubating a blood sample or blood derivative with a peptide described any of claims 1-12, followed by determination of binding between antibody in the blood sample or blood derivative and the peptide.

14. A method of detecting HIV-1 subtype D infection from a blood sample with a whole blood assay device, the device comprising a blood filter positioned on top of a reagent layer that contains at least one peptide described by any of claims 1-12, wherein the method comprises applying one or more drops of whole blood to the blood filter, removing the filter, and determining the presence of one or more visual reactions in response to the presence of anti-HIV antibody in the blood sample.

15. A device for detecting HIV-1 subtype D infection from a blood sample, comprising a blood filter that has at least one fibrous pad, a reagent layer having immobilized at least one peptide as described by any of claims 1-12 and an absorbent pad, wherein a fibrous pad of the blood filter contacts the reagent layer without deforming the reagent layer, and the reagent layer contacts the absorbent pad.

16. A kit for determining infection with HIV-1 subtype D, comprising, an instruction booklet and a device for detecting the presence of anti-HIV- 1 subtype D antibody in a blood sample or blood derivative, wherein the device comprises a peptide described by any of claims 1-12.