GB2532488B

GB2532488B - Use of AMP1 or AMP8 to diagnose HIV infection

Info

Publication number: GB2532488B
Application number: GB1420695.7A
Authority: GB
Inventors: Pretorius Ashley; Nur Gabere Musa; Belmondo Tincho Marius; Edward Williams Monray
Original assignee: University of the Western Cape
Current assignee: University of the Western Cape
Priority date: 2014-11-21
Filing date: 2014-11-21
Publication date: 2019-07-31
Anticipated expiration: 2034-11-21
Also published as: GB201420695D0; ZA201703730B; AU2015348948A1; WO2016079716A1; GB2532488A

Description

USE OF AMP1 or AMP8 TO DIAGNOSE HIV INFECTION

BACKGROUND OF THE INVENTION

This invention relates to one or more isolated or synthetic HIV p24 N-terminal binding peptides for diagnosis of HIV infection in a subject by detecting binding of the peptide to HIV p24 in a sample from the subject. The invention further relates to a test device and a kit comprising the one or more peptides of the invention for detection of HIV in a sample from a subject and a method of use of the one or more peptides, the test device and/or the kit.

Acquired Immunodeficiency Syndrome (AIDS) is a disease that attacks the human immune system and is caused by the Human Immunodeficiency Virus (HIV). HIV can be transmitted to a healthy individual through body fluids or via mucosal surfaces. Despite the effort of scientific research on HIV therapeutic regiments and to reduce the rate of HIV infection and transmission, AIDS still remains the major cause of death around the world, particularly in Sub-Saharan Africa.

Though much advancement has been made in HIV research to date, neither a cure, nor an HIV vaccine had been found and the disease can only be managed by using High Active Antiretroviral Therapy (HAART), which can only slow the course of the disease and reduce both deaths and new infections. However, the early detection and treatment of HIV can reduce the transmission of the virus.

The internal structural protein of HIV-1, p24, which appears in the serum of infected individuals within 2 weeks after infection, is one of the HIV antigens used in the development of diagnostic kits, including HIV-1 p24 ELISA assays. However, these diagnostics systems are not very sensitive because of competitive binding between the p24 antigen and the p24 antibody in the serum of HIV-infected individuals. Consequently, these kits require the heating of the tested sample (or booster step) to separate the complex formed between p24 and the p24 antibody so as to enable the detection of p24.

There is therefore a need to improve the current HIV p24 diagnostic test, in particular to enable the use of such a kit in a point-of-care setting.

SUMMARY OF THE INVENTION

We disclose one or more isolated or synthetic HIV p24 N-terminal binding peptides for detection of HIV infection in a subject.

The HIV p24 N-terminal binding peptides may be AMP1 or AMP8, or a modification or variant thereof.

The subject may be infected with HIV-1 or HIV-2. Preferably, the subject is infected with HIV-2.

The peptide may be conjugated to an indicator molecule. For example, the indicator molecule may be a molecular biological, microfluidic or nano technology indicator a such as a gold nanoparticle, a silver nanoparticle, a cadmium nanoparticle, or other nanoparticles known to those skilled in the art. In particular, the indicator may be a chemical, radiochemical, florescent, colourmetric or chromatographic indicator. Preferably, the indicator is a chromatographic indicator, such as a colloidal gold indicator.

Preferably, the peptide may be a variant peptide of AMP1, comprising a mutation at position F62W.

Alternatively preferably, the peptide may be a variant peptide of AMP 8, comprising a mutation at position F12H.

The peptide may be a variant peptide of AMP1, comprising conserved amino acid residues Gln10; Trp13; Lys18; Arg20; Cys22; Pro23; Thr36; Arg41; Met56; Asn58; Asn61; Glu64; Met65; Gly67; Gln68; and Cys69, wherein the variant amino acid residues have equivalent hydrophobicity to the parent amino acid residues.

The peptide may be a variant peptide of AMP8, comprising conserved amino acid residues Ala6; Ile7; Cys13; Pro14; Tyr17; and Lys33, wherein the variant amino acid residues have equivalent hydrophobicity to the parent amino acid residues.

We disclose a test device for detecting HIV in a sample from a subject, the test device comprising one or more isolated or synthetic HIV p24 binding peptides according to the invention.

The test device may further comprise a capture antibody or antibody fragment thereof capable of binding to HIV p24 or the HIV p24 binding peptide of the invention. The capture antibody or antibody fragment thereof may be conjugated to an indicator molecule.

The antibody conjugate or the capture antibody may be a monoclonal antibody or a polyclonal antibody.

The indicator may be a molecular biological, microfluidic or nano technology indicator. In particular, the indicator may be a chemical, radiochemical, florescent, chromatographic or colourmetric indicator. Preferably, the indicator is a chromatographic indicator, such as a colloidal gold indicator.

The test device may be used to detect HIV-1 or HIV-2 infection in the subject. Preferably the test device is used to detect HIV-2 infection.

The test device may be a lateral flow assay device, an ELISA plate, a flow cytometry device or a bioanalyser device. For example, the test device is a strip-test device or an 8-well ELISA plate. Preferably, the test device is a point-of-care test device.

The sample may be a blood or serum sample.

We disclose a kit for detecting HIV in a sample from a patient, the kit comprising: i) one or more isolated or synthetic HIV p24 binding peptides according to the invention; and ii) optionally instructions for use.

The kit may further comprise a capture antibody or antibody fragment thereof capable of binding to p24 or an HIV p24 peptide of the invention. The capture antibody or antibody fragment thereof may be conjugated to an indicator molecule.

The kit may be used to detect HIV-1 or HIV-2 infection in the subject. Preferably the kit is used to detect HIV-2 infection.

The sample may be a blood or serum sample.

We disclose a method for detecting HIV in a sample from a patient, the method comprising the steps of: i) providing one or more isolated or synthetic HIV p24 binding peptides, a test device or a kit according to the invention; ii) providing a sample from the subject, which if the subject has HIV, comprises p24; iii) reacting the isolated or synthetic HIV p24 binding peptide of i) with the sample of the subject; and iv) detecting whether or not there is binding of the HIV p24 binding peptide with HIV p24 protein in the sample.

The method may be used to detect HIV-1 or HIV-2 infection in the subject. Preferably, the method is used to detect HIV-2 infection.

The sample may be a blood or serum sample.

The method may be a point-of-care method.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows an illustrative demonstration of how the results are interpreted. A negative result is indicated by the absence of a dot in the window. A positive result is indicated by the presence of a grey dot in the window.

DETAILED DESCRIPTION OF THE INVENTION

According to an aspect of the invention there is provided the use of an isolated or synthetic HIV p24 N-terminal binding peptide selected from AMP1 or AMP8, or variant thereof, in the diagnosis of HIV infection in a subject.

In a preferred embodiment of the invention said HIV p24 N-terminal binding peptide is a variant peptide of AMP1, comprising conserved amino acid residues Gln10; Trp13; Lys18; Arg20; Cys22; Pro23; Thr36; Arg41; Met56; Asn58; Asn61; Glu64; Met65; Gly67; Gln68; and Cys69, and wherein comprising a mutation at position F62W.

In a further preferred embodiment of the invention said HIV p24 N-terminal binding peptide is a variant peptide of AMP8, comprising conserved amino acid residues Ala6; Ile7; Cys13; Pro14; Tyr17; and Lys33 and comprising a mutation at position F12H.

In a preferred embodiment of the invention said isolated or synthetic HIV p24 N-terminal binding peptide is for detection of HIV-1 or HIV-2 infection in a subject.

In a preferred embodiment of the invention said HIV p24 N-terminal binding peptide is conjugated to a chemical, radiochemical, fluorescent, colourmetric or chromatographic indicator.

In a preferred embodiment of the invention said HIV p24 N-terminal binding peptide is conjugated to a colloidal gold indicator molecule.

According to an aspect of the invention there is provided a test device selected from the group consisting of lateral flow assay device, an ELISA plate, a flow cytometry device or a bioanalyser device or point-of-care test device for detecting HIV in a sample from a subject, the test device comprising one or more isolated or synthetic AMP1 or AMP8 HIV p24 binding peptides.

In a preferred embodiment of the invention said capture HIV p24 binding peptide is conjugated to an indicator molecule.

In a preferred embodiment of the invention said indicator molecule is a chemical, radiochemical, florescent, chromatographic or colourmetric indicator.

In a preferred embodiment of the invention said AMP1 or AMP8 HIV p24 N-terminal binding peptide is conjugated to a colloidal gold indicator molecule.

In a preferred embodiment of the invention said test device is for detection of HIV-1 or HIV-2 infection in the subject.

In a preferred embodiment of the invention said test device is for detection of HIV-2 infection in the subject.

According to an aspect of the invention there is provided a kit for detecting HIV in a sample from a patient, the kit comprising: (i) an isolated or synthetic AMP1 or AMP8 HIV p24 binding peptide conjugated to a chemical, radiochemical, fluorescent, chromatographic or colourmetric indicator; and (ii) instructions for use.

In a preferred embodiment of the invention said AMP1 or AMP8 HIV p24 binding peptide is conjugated to a colloidal gold indicator.

In a preferred embodiment of the invention said kit is for detection of HIV-1 or HIV-2 infection in the subject.

In a preferred embodiment of the invention said kit is for detection of HIV-2 infection in the subject.

According to as aspect of the invention there is provided a method for detecting HIV in a sample from a patient, the method comprising the steps of: (i) providing an isolated or synthetic AMP1 or AMP8 HIV p24 binding peptide, a test device according to any one of claims 7 to 12, or a kit according to any one of claims 13 to 16; (ii) providing a sample from the subject, which if the subject has HIV, comprises p24; (iii) contacting the isolated or synthetic AMP 1 or AMP8 HIV p24 binding peptide of i) with the sample of the subject; and (iv) detecting whether or not there is binding of the AMP1 or AMP8 HIV p24 binding peptide with HIV p24 protein in the sample.

In a preferred embodiment of the invention said method is for detection of HIV-1 or HIV-2 infection in the subject.

In a preferred embodiment of the invention said method is for detection of HIV-2 infection in the subject.

In a preferred embodiment of the invention said method is a point-of-care method.

According to an aspect of the invention there is provided an isolated or synthetic HIV p24 N-terminal binding peptide, wherein the HIV p24 N-terminal binding peptide is a variant peptide of AMP1, comprising conserved amino acid residues Gln10; Trp13; Lys18; Arg20; Cys22; Pro23; Thr36; Arg41; Met56; Asn58; Asn61; Glu64; Met65; Gly67; Gln68; and Cys69, and comprising a mutation at position F62W.

In a preferred embodiment of the invention said HIV p24 N-terminal binding peptide is a variant peptide of AMP8, comprising conserved amino acid residues Ala6; IIe7; Cys13; Pro14; Tyr17; and Lys33, and comprising a mutation at position F12H.

As used herein, the phrase “Point of Care Testing (POCT)” means medical diagnostic testing performed outside the clinical laboratory in close proximity to where the patient is receiving care. POCT is typically performed by non-laboratory personnel and the results are used for clinical decision making.

There are currently two general approaches for the diagnosis of HIV. These are either by monitoring the viral load within the plasma which indicates viral replication and virus particles in the infected individuals, or by looking for the antibodies which are produced to resist the viral infection. While the diagnostic system is able to detect these molecules in the blood of an infected individual, the main goal is to detect with confirmation these molecules at the onset of infection. As a consequence, early medications can be prescribed to prevent the replication of the virus and consequently slow the progression of the virus.

The diagnostic approach to detect viral replication or virus particles include the Ultrasensitive p24 assay, the ExaVir™ RT viral load and Real Time Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR). The Ultrasensitive p24 assay makes use of a specific antibody which binds to the p24 viral core protein in a serological sample via a EIA technique to diagnose an individual but also requires a window period of a few days, an average of 7 days, and the result of the test is received within an hour. However, the results of the test are insensitive in comparison with the nucleic acid based assay (Butto et al., 2010).

The ExaVir™ RT viral load and RT-qPCR techniques amplify the HIV RNA extracted from the patients’ blood. The method operates by converting the HIV RNA into DNA via an enzyme reverse transcriptase which makes it easier for quantification of the amount of viral particles present (Wang et al., 2010).

Whist these techniques are important for the diagnosis of HIV, they require a window period after infection has occurred, to be able to detect either the antibodies or the viral particles within a serological sample. Most of these assays such as the ExaVir™ RT viral load, RT-qPCR, the ElAs and western blots are expensive and require specialized trained staff, regular and expensive maintenance of equipment, are laborious, and suitable for centralized laboratories, but not for district clinics in resource-limited settings (Wang et al., 2010). Inexpensive techniques such as the Ultrasensitive p24 assay and rapid tests though insensitive fulfil the point-of-care purpose for a proper global diagnostics system. However, a more sensitive and specific technique needs to be put in place so as to reduce the window period, a common drawback of all the above-mentioned assays.

The applicant has identified a group of novel peptides which bind specifically to the HIV p24 protein in an HIV-infected subject. The applicant has further developed a test device and a diagnostic kit for detection of HIV comprising one or more of the group of peptides. In particular, the test device or kit is a point-of-care device or kit.

These novel peptides are members of a family of peptides called antimicrobial peptides (or AMPs). AMPs are components of the first line of defence for prokaryotes and eukaryotes and have a wide range of activities against gram-negative and grampositive bacteria, fungi, cancer cells, protozoa as well as viruses (Andreu and Rivas, 1998).

The novel p24 protein binding peptides were initially identified by the applicant from AMP databases: APD, CAMP, Bactibase and UniprotKB using an in silico mathematical algorithm with the use of biophysical modelling, virtual genome screening, prediction of peptides three-dimensional structures and structural docking assessment between the putative binding peptides and a number of HIV proteins, p17, p24, gp41 and gp120 protein. A number of putative binding peptides were identified that bind with high specificity and sensitivity to the N-terminal portion of the HIV p24 protein. These were then tested by in vitro testing in a lateral flow device using sandwich ELISA to validate functionality of the peptides. Two of the putative binding peptides, AMP1 and AMP8, were found to be of high affinity to the HIV p24 antigen. AMP1 is a 79 amino acid peptide (CLRYKKPECQ SDWQCPGKKR CCPDTCGIKC LDPVDTPNPT RRKPGKCPVT YGQCLMLNPP NFCEMDGQCK RDLKCCMGM) (SEQ ID NO:1) having a 43 amino acid binding interface with the HIV p24 protein. AMP8 is a 34 amino acid peptide (CLKSGAICHP VFCPRRYKQI GTCGLPGTKC CKKP) (SEQ ID NO:2) with a 20 amino acid binding interface with the HIV p24 protein.

The applicant then performed further in silico analysis of the two peptides in order to identify amino acids considered to be “hotspots” which cannot be mutated in order for the peptides to maintain the binding interface with the HIV p24 protein.

For AMP1,63% (27/43) of the amino acids within the binding interface between AMP1 and p24 can tolerate mutation as these amino acids contribute least to binding free energy of the interaction within that interface. The amino acids required to remain conserved in AM P1 are as follows: Gin 10; Trp13; Lys18; Arg20; Cys22; Pro23; Thr36; Arg41; Met56; Asn58; Asn61; Glu64; Met65; Gly67; Gln68; and Cys69.

For AMP 8, 70% (14/20) of the amino acids within the binding interface between AMP 8 and p24 are able to tolerate mutation. The amino acids required to remain conserved in AMP8 are as follows: Ala6; IIe7; Cys13; Pro14; Tyr17; and Lys33.

However, there is a proviso that the amino acid substitution in the variant sequence is required to be conservative in nature compared to the parent amino acids in order to maintain the functioning and binding of that AMP. A conservative substitution is a substitution of one amino acid for another with similar characteristics. Conservative substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic or acidic groups by another member of the same group can be deemed a conservative substitution.

Preferably, the conservative substitution comprises the use of amino acids with similar hydrophobicity and longer side chains (to bridge gap between AMP and p24 protein) or similar hydrophobicity and increased positive charged amino acid (to increase overall net charge on the AMP to increase binding activity).

Furthermore, the applicants have identified two variants where site-directed mutagenesis is predicted to increase the binding affinity of the anti-HIV p24 peptide. The particular variants identified are AMP1 F62W and AMP8 F12H. A particular advantage of the use of the novel peptides identified for detection of the p24 protein is that the window period after the first testing of anti-p24 antibodies which is usually required, will not be required, since the viral p24 protein is expressed within the human body at the earliest stage of HIV replication. Very early detection is therefore possible, allowing any necessary treatment to commence earlier. A further advantage of the peptides identified is that use of these novel p24 N-terminal binding peptides also reduces the possibility of a false positive result encountered in the traditional anti-HIV p24 diagnostic assay which occurs due to a competitive binding between the p24 antigen and the p24 antibody in the serum of HIV-infected individuals. Competitive binding in the p24 test occurs due to the test antibody in some instances competing for the C-terminus of the p24 protein. The C-terminus of p24 shows antibody promiscuity and thus may be coated prior to an infected person taking the test with antibodies produced in response to infection. Based on the stage of infection, the window period can be missed or the test may provide false results. However, since the anti-HIV p24 peptides identified target the N-terminus of the p24 protein, this antibody promiscuity is circumvented.

Moreover, since the traditional methods require either heating of the sample or a booster step to mitigate the problem set out above, the traditional assays do not lend themselves to use in a point-of-care setting.

The applicant has further developed a test device and a diagnostic assay for use with the novel p24 binding peptides. The peptides were conjugated to colloidal gold nanoparticles, although alternative types of nanoparticles that could be used by a person skilled in this field may be silver particles, cadmium particles, graphene, carbon nanotubes and the like. The labelled peptides were then coated onto a standard lateral flow device for detection of the presence of p24 viral protein in human serum and blood samples. However, any peptide binding assay known to those skilled in the art may be used, including EIA (enzyme immunoassays) and ELISA (enzyme linked immunosorbent assays), and the test device may include a lateral flow assay device, an ELISA plate, a flow cytometry device ora bioanalyser device. In particular, the assay may be a point-of-care test (POCT) for the detection of HIV p24 viral protein found in HIV infected individuals at an early stage of HIV infection.

In summary, an assay comprising the novel p24 N-terminal binding peptides identified by the applicant would provide a means for detection of HIV infection at an early stage, with more sensitivity and specificity than the traditional tests. Furthermore, the assay, in particular a POCT assay version, may be used at clinics in a rural setting.

The invention will be described by way of the following examples which are not to be construed as limiting in any way the scope of the invention. EXAMPLE 1: In silico analysis 1.1. Methods and materials 1.1.1. Models construction and identification of novel anti-HIV AMPs

Firstly, the modelling was constructed by performing a data mining to identify all possible experimentally validated anti-HIV AMPs from repository databases (APD, CAMP, DAMPD, Bactibase and UniprotKB). Thereafter, literature mining was performed to verify that all the retrieved anti-HIV AMPs were experimentally validated or predicted. Duplicate experimentally validated anti-HIV AMPs were then removed from the list.

The final list of the experimentally validated anti-HIV AMPs was classified according to super-family: Amphibian, Microorganism, Human defensin, Fish and Crab, Insect, Vertebrate and Plant super-families.

Each super-family was then divided into two portions: three-quarters of each superfamily was used for the training set and one-quarter was used as the test of the model built for that super-family.

The Hidden Markov Models (HMMER) algorithm (Brahmachary et al., 2004; Fjell et ai., 2007) was used to construct seven super-families models using the training set. The training sets were used to construct the profiles and the testing sets were used to query the profiles. Statistical performances were then calculated (sensitivity, specificity, accuracy and Mathew Correlation Coefficient) for each model. A negative data set was also used to confirm the strength of the models.

More than 1059 genome sequences were queried by the respective super-families profiles with the list of all genome sequences searched was retrieved from the Ensembl database (http://www.ensembl.org/index.html) and the Uniprot database (http://www.uniprot.org/).

The matches of the query profiles against the genome sequences are shown with scores (bits) and E-values. The E-value, which is calculated from the bits score, shows the number of false positives that is expected to be seen at or above this bit score. Therefore an E-value of 0.01 and 0.05 indicates that there is only a 1% and 5% chance respectively that the hit is false or has come up by chance. Hence, a low E-value is considered appropriate with the lowest E-value appearing at the top of the result list. The cut-off E-value was set to be 0.01.

The “Query db” was to identity peptides which had the same signatures/motifs and properties as the profiles of the various super-families. The identified peptides were considered as putatively having activity against HIV. The final list of putative anti-HIV AMPs contained 30 peptide sequences and is shown in Table 1.

The applicant then selected the 10 top ranked putative anti-HIV AMPs based on the lowest E-value scores for further analysis. These are listed in Table 2. 1.1.2. In silico validation of putative anti-HIV AMPs for HIV diagnostic testing

To confirm the role of the putative HIV binding peptides identified in the genome scan, the sequence of each of the ten peptides was docked against HIV proteins useful for diagnostic methods.

The following parameters of the putative anti-HIV AMPs and the HIV proteins were calculated: the charge, Boman index, the instability index and many more using the prediction interface of the Bactibase and Antimicrobial Peptides Database with the amino acid sequences of the peptides and the proteins as input (http://bactibase.pfba-lab-tun.org/physicochem and http://aps.unmc.edu/AP/design/design_improve.php) (Hammami et al., 2007; Wang and Wang, 2009; Hammami et al., 2010).

First, the prediction of the 10 putative anti-HIV AMPs and the HIV proteins 3-D structures were performed using l-TASSER (Iterative Threading ASSembly Refinement) server which is an example of a de novo method of structure prediction (Schwede et al., 2008). This method was chosen because the 10 putative anti-HIV AMP sequences were considered novel and thus had no homologous structures available for comparative modelling. The l-TASSER server is a free on-line tool which computationally predicts the 3-D structure of protein or peptide from its amino acid sequence. The server can be found at the URL (http://zhanglab.ccmb.med.umich.edu/l-TASSER/) and it is held at the University of Michigan, USA. The server implements various mathematical algorithms to predict 3-D structure of proteins and peptides.

Table 2: List of 10 top ranked putative anti-HIV AMPs according to the E-values.

The 3-D structures of the anti-HIV AMPs and HIV proteins were predicted by uploading each sequence onto the l-TASSER website. The output from the I-TASSER server includes: a full-length secondary and tertiary structure prediction as well as functional annotations on ligand-binding sites, Enzyme Commission numbers, Gene Ontology terms and most importantly provides an estimate of accuracy scoring of the predicted peptides and the proteins 3-D structures based on the C-score, TM-score and RMSD (Roy et al., 2010). The visualisations of the 3-D structures were done using the PyMOL 1.3. Software as PDB files.

Secondly, the structural docking between the putative peptides and the HIV proteins (p 17, p24, gp41 and gp120) were performed. Docking is a method used to predict the most favourable orientation of protein-protein or protein-ligand complexes (Lengauer, and Rarey, 1996). The docking of the 10 putative anti-HIV AMPs to the HIV proteins gp120, gp41, p24 and p17 were done using PatchDock Beta 1.3 version. PatchDock

is a free online web-server that allows for protein-protein and protein-small ligand molecule docking and is available at http://bioinfo3d.cs.tau.ac.il/PatchDock/.

Docking was done by uploading the respective PDB files of the HIV proteins and the putative anti-HIV AMPs onto the PatchDock server website, after which the user enters an email address. The cluster RMSD was set to 4.0 A and the complex type was selected as “protein-small ligand”. The docking results are sent via an email notification, containing the web link to the docking results. The result provides the highest scoring complexes between the HIV protein and the anti-HIV AMP as a PDB output file (Schneidman-Duhovny et al., 2005). Besides the geometric scoring system given in the result section of PatchDock, additional information includes the Atom Contact Energy (ACE), the area covered between the two molecules, the transformation coordinates during the molecular interaction and the PDB file of the complex formed as a ball and stick structure (Schneidman-Duhovny et al., 2005). Interaction analysis of the complex formation between the HIV protein and the putative anti-HIV AMP was done using PyMOL 1.3. Software and the distance between the interacting residues calculated.

The results for l-TASSER showed that the C-score of 3 HIV proteins (p24, p17 and gp120) were 1.41, 1.70 and 2.00 respectively, whilst the C-score of gp41 was -0.17. Additionally, the TM-score of p24, p17, gp120 and gp41 were 0.91, 0.95, 0.99 and 0.69 respectively. All proteins had a TM-score above 0.5. The Root Means Square Deviation (RMSD) of p24, p17, gp120 and gp41 were 2.7, 1.4, 1.7 and 3.6 respectively. The RMSD of p17 and gp120 were less than 2k. Although the RMSD is not less that 1 A, the topologies of the predicted 3-D structures are a consequence of them having a TM-score above 0.5, since there is a strong correlation between the RMSD and the TM-score of a predicted 3-D protein structure (Roy et al., 2010).

The prediction of the putative anti-HIV AMP 3-D structures gave C-score values which ranged from -1.83 to 0.95. All the peptides had C-score values which were higher than -1.5, except for AMP1, which had a C-score of-1.83. The TM-score of the 10 putative anti-HIV AMPs ranged from 0.49 to 0.84 and were all above 0.5 except again for AMP1 which had a TM-score of 0.49. Whilst AMPs 8 and 9, the positive and the negative controls had RMSD scores which were less that 1 A, AMP AMPs 2, 3, 4, 5, 6, 7 and 10 predicted 3-D structures have RMSD scores higher than the positive and negative controls, ranging between 2.0 to 2.2k. The RMSD value of AMP 1 was above 4A and was reported to be 7.3k.

The anti-HIV AMPs represented different secondary structures. AMP 1 represented an extended or loop structure whilst AMPs 2, 3, 4, 5, 6, 7 and 10, the positive and negative each had α-helical conformation. AMP 9 represented a very short a-helical structure with some coil structure. Only AMP 8 had an anti-parallel β-sheeted secondary structure arrangement, mix with a loop structure.

As part of the results output delivered by the l-TASSER server, the 3-D structural models of each AMP predicted was confirmed by superimposing the secondary structure of the predicted AMP to the closest similar protein 3-D structure found in the Protein Data Bank. The PDB ID of the known 3-D structures which were used for the superimposition was attached to the l-TASSER result page. Besides AMP1 which had a TM-score less than 0.5, all the superimposed 3-D structures had TM-scores which was greater than 0.5, meaning that the structures had structural similarity with the templates which were used for their prediction. Also, all the 3-D structures except AMP1, had RMSDs less than 2A, thus the superimposition of the 3-D structures and the templates are closely related and there is little atomic deviation between the predicted AMPs and their templates (results not shown).

The results from the PatchDock docking study indicated that all the putative anti-HIV AMPs as well as positive and the negative control, binds to the four HIV proteins gp120, gp41, p24 and p17. The geometric scores of the binding affinities of the putative anti-HIV AMPs and HIV proteins ranged from 14926 to 6710 (data not shown). It was observed that the 10 putative anti-HIV AMPs have higher binding affinity scores than the positive and negative controls. Most importantly, the 10 putative anti-HIV AMPs geometric scores are higher than the positive control kn2-7 that has been demonstrated to possess potent anti-HIV activity (Chen et al., 2012). AMP1 showed a very strong binding affinity geometric score to all the HIV proteins. This confirms the probability of this peptide to be a putative anti-HIV peptide as it also showed the lowest E-value prediction score and fulfils all the physicochemical property requirements of a good AMP. However, AMP9 has the lowest binding affinity for all the HIV proteins.

The in silico docking predictions of the peptides showed that all ten of the peptides putatively bind to the N-terminal domain of p24. In particular, AMP molecules 1,2, 3, 5, 6, and 8 had the highest binding geometric score to p24 at its N-terminal. No sequence similarity was shown between the putative anti-HIV AMP sequences with other known peptide sequences, thus these AMPs have not yet been implicated as anti-HIV peptides and they were therefore regarded as novel putative AMPs.

The specific interaction of the 10 putative anti-HIV peptides to the p24 proteins was shown to be promising. This is as a result of all the putative anti-HIV AMPs binding to the N-terminal rather than the C-terminal of p24 HIV protein. Binding of Fab13B5 (used for the p24 assay for HIV detection) at the p24 C-terminal has shown to be of a low affinity due to the small surface interaction between the two molecules. However, the N-terminal of p24 offers a larger surface interaction with other molecules (Monaco-Malbet et al., 2000). Additionally, in 50% of the cases testing for presence of HIV with the p24 assay system, the C-terminal of p24 is already occupied by the antibodies produced in response to the HIV infection. Thus, the binding of the Fab13B5 to the already occupied C-terminal is prevented and can indicate a false negative test. Binding of the novel putative AMPs to the unoccupied N-terminal with high specificity, should result in 100% accuracy of the p24 assay even if the C-terminal is already occupied. EXPERIMENT 2: Molecular validation of the in silico studies 2.1. Methods and Materials 2.1.1. AMPs used for detection of p24 in a diagnostic tool

The putative anti-HIV AMPs identified in the in silico phase of the discovery (i.e. AMP1 to AMP10) were molecularly validated as follows. The AMPs were used in a sandwich type ELISA assay for the detection of HIV in human blood or serum. In particular, a lateral flow device was used in this method.

Strips were assembled in accordance with standard methods for lateral flow testing with a sandwich type ELISA assay. The capture anti-HIV AMPs were blotted onto nitrocellulose membranes of the lateral flow test in triplicate at a concentration of 0.ΙΟ.5 mg/ml.

Next, matching indicator anti-HIV AMPs were conjugated to gold nanoparticles and blotted onto the conjugate pads of the lateral flow strips containing the membrane blots. The same anti-HIV AMPs were used as the capture and indicator AMPs in the sandwich ELISA. The strips were then placed into lateral flow test cassettes and run by adding 10 pi of human blood or serum to the sample well (top well) and 120 μΙ of malaria running buffer to the buffer well (bottom well). The results were read after 15 minutes but before 30 minutes had elapsed. Positive and negative control blood and serum standards for HIV-1 and HIV-2 were also included in the experiment procedure. 2.2. Results 2.2.1. AMPs used for detection of p24 in a diagnostic tool

Figure 1 describes how the results for detection of HIV p24 protein are interpreted from the cassettes, either for the negative or positive HIV samples. The criteria for evaluating the Dot-Blot intensity of the test were as follows. Where there is a significant difference between the intensities of the dots between negative and positive, this indicates a difference in dose response between the negative and positive samples and can be viewed as a confirmation result irrespective of whether a faint dot appears or not when a negative sample is used. Signal intensities were visually interpreted using a G1-G10 Gold Colour Chart, with the higher the G number, the greater the signal intensity.

The results of the tests were assessed, including for the negative and positive control AMPs. Negative and positive controls (HIV-1 and 2) and the results of the dot intensities are reported in Table 3.

Table 3: Results from the evaluation of the putative HIV p24 binding peptides. Significant results are provided in greyed out boxes.

At the end of the molecular validation, two putative novel anti-HIV AMPs (molecules 1 (AMP1) and 6 (AMP8) were demonstrated to bind the HIV p24 protein. The positive control AMP (Kn2-7) also showed a strong binding to the HIV p24 protein, thus this molecule could also be included as a third peptide to diagnose HIV infection in the blood or serum of a subject. In particular, any one or more of the three putative anti-HIV AMPs may be used for detection of HIV-2 in a subject. 2.2.2. Variations and modifications to the peptides for use in the detection of p24

After the in silico validation of the AMPs putatively identified for use in a diagnostic tool for early detection of HIV through a docking study, the molecular validation of two AMPs as diagnostic molecules to bind to HIV protein p24 was confirmed to react with human HIV positive serum. In order to increase the sensitivity and the specificity of the binding affinity of the two AMPs, in silico mutation by substitution was performed on the parental AMPs and novel variants of AMPs for HIV diagnostic were generated. To obtain these variants, the following steps were achieved: a) Identification of the “Hotspot” residues “Hotspot” residues were identified from the structural complex between two AMPs (AMP1 and AMP8) and HIV protein p24. Before the commencement of site-directed mutagenesis, essential amino acids were identified. “Hotspot” residues are residues, which are likely to be sensitive to mutation. The knowledge-based FADE and Contacts (KFC) server (http://kfc.mitchell-lab.org/upload.php) (Darnell et al., 2008) was used to identify hotspot residues. This tool improves the ability to predict “hotspots” or residues, which are responsible for most of the interfaces binding free energy. KFC is based on two predictive models, namely (i) basis of shape specificity features and (ii) biochemical contacts (Darnell et al., 2008). It is stated that KFC displays better

predictive accuracy than computational alanine scanning (Robetta-Ala) (Darnell et al., 2008). b) In silico site-directed mutagenesis

Hotspot residues were identified, and based on those results certain amino acids were subjected to site-directed mutagenesis in silico to attempt to increase binding affinity for the N-terminal of the HIV p24 protein. All mutations introduced were conservative, thus ensuring the integrity and functioning of AMPs. Only the two putative anti-HIV p24 AMPs (AMP1 and AMP8) validated by in vivo molecular testing were subjected to in silico site directed mutagenesis analysis.

Table 4 shows the possible Hotspots at the interface of interaction between AMP1 and p24 and AMP8 and p24. Only the amino acids that are not Hotspots were subjected to mutations.

Table 4: All amino acids within the interface of interaction between p24 and two selected AMPs (AMP1 and AMP8). Amino acids that are considered as “hotspot” residues are provided in grey boxes.

In silico binding validation has confirmed that the variant AMPs bind to the correct pocket. Any mutations introduced outside of the hotspot regions are required to be conservative compared to the parent amino acids to maintain the functioning and binding of that AMP, i.e. with similar hydrophobicity and longer side chains (to bridge gap between AMP and p24 protein) or similar hydrophobicity and increased positive

charged amino acids (to increase overall net charge on AMP to increase binding activity).

In particular, two variant peptides were identified by in silico site-directed mutagenesis that demonstrated higher p24 affinity binding than the parent sequences. The results are set out in Table 5.

Table 5: Table displaying the amino acid position for selected mutation for each AMP.

Following in silico mutation of the parental AMP1 and AMP8 to the variant sequences, these mutated putative anti-HIV AMPs were subjected to l-TASSER to predict their 3-D structures and then to PatchDock for an in silico docking study as previously described.

The results of these in silico studies suggest that the binding affinity of these AMPs has increased significantly (Table 6). These mutated AMPs identified may therefore be synthesised and used in an HIV diagnostic tool for detection of HIV with greater affinity that with the parental molecules.

Table 6: Binding affinities; position of AMPs 1 and 8 on HIV protein p24 and percentage difference

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Claims

CLAIMS: 1 Use of an isolated or synthetic HIV p24 N-terminal binding peptide selected from AMP1 or AMP8, or variant thereof, in the diagnosis of HIV infection in a subject.
2. The use of the isolated or synthetic HIV p24 N-terminal binding peptide according to claim 1, wherein the HIV p24 N-terminal binding peptide is a variant peptide of AMP1, comprising conserved amino acid residues Gln10; Trp13; Lys18; Arg20; Cys22; Pro23; Thr36; Arg41; Met56; Asn58; Asn61; Glu64; Met65; Gly67; Gln68; and Cys69, and wherein comprising a mutation at position F62W.
3. The use of the isolated or synthetic HIV p24 N-terminal binding peptide according to claim 1, wherein the HIV p24 N-terminal binding peptide is a variant peptide of AMP8, comprising conserved amino acid residues Ala6; Ile7; Cys13; Pro14; Tyr17; and Lys33, and comprising a mutation at position F12H.
4. The use of the isolated or synthetic HIV p24 N-terminal binding peptide according to claims 1 to 3 for detection of HIV-1 or HIV-2 infection in a subject.
5. The use of the isolated or synthetic HIV p24 N-terminal binding peptide according to claims 1 to 4, wherein the HIV p24 N-terminal binding peptide is conjugated to a chemical, radiochemical, florescent, colourmetric or chromatographic indicator.
6. The use of the isolated or synthetic HIV p24 N-terminal binding peptide according to claims 1 to 5, wherein the HIV p24 N-terminal binding peptide is conjugated to a colloidal gold indicator molecule.
7. A test device selected from the group consisting of lateral flow assay device, an ELISA plate, a flow cytometry device or a bioanalyser device or point-of-care test device for detecting HIV in a sample from a subject, the test device comprising one or more isolated or synthetic AMP1 orAMP8 HIV p24 binding peptides.
8. The test device according to claim 7, wherein the HIV p24 binding peptide is conjugated to an indicator molecule.
9. The test device according to claim 8, wherein the indicator molecule is a chemical, radiochemical, florescent, chromatographic or colourmetric indicator.
10. The test device according to any one of claims 7 to 9, wherein the AMP1 or AMP8 HIV p24 N-terminal binding peptide is conjugated to a colloidal gold indicator molecule.
11. The test device according to any one of claims 7 to 10, wherein the test device is for detection of HIV-1 or HIV-2 infection in the subject.
12. The test device according to any one of claims 7 to 10, wherein the test device is for detection of HIV-2 infection in the subject.
13. A kit for detecting HIV in a sample from a patient, the kit comprising: (i) an isolated or synthetic AMP1 or AMP8 HIV p24 binding peptide conjugated to a chemical, radiochemical, fluorescent, chromatographic or colourmetric indicator; and (ii) instructions for use.
14. The kit according to either claim 13, wherein the AMP1 or AMP8 HIV p24 binding peptide is conjugated to a colloidal gold indicator.
15. The kit according to claim 13 or 14, which is for detection of HIV-1 or HIV-2 infection in the subject.
16. The kit according to claim 15, which is for detection of HIV-2 infection in the subject.
17. A method for detecting HIV in a sample from a patient, the method comprising the steps of: (i) providing an isolated or synthetic AMP1 or AMP8 HIV p24 binding peptide, a test device according to any one of claims 7 to 12, or a kit according to any one of claims 13 to 16; (ii) providing a sample from the subject, which if the subject has HIV, comprises p24; (iii) contacting the isolated or synthetic AMP1 or AMP8 HIV p24 binding peptide of i) with the sample of the subject; and (iv) detecting whether or not there is binding of the AMP1 or AMP8 HIV p24 binding peptide with HIV p24 protein in the sample.
18. The method according to claim 17 which is for detection of HIV-1 or HIV-2 infection in the subject.
19. The method according to claim 18 which is for detection of HIV-2 infection in the subject.
20. The method according to any one of claims 17 to 19 which is a point-of-care method.
21. An isolated or synthetic HIV p24 N-terminal binding peptide, wherein the HIV p24 N-terminal binding peptide is a variant peptide of AMP1, comprising conserved amino acid residues Gln10; Trp13; Lys18; Arg20; Cys22; Pro23; Thr36; Arg41; Met56; Asn58; Asn61; Glu64; Met65; Gly67; Gln68; and Cys69, and comprising a mutation at position F62W.
22. An isolated or synthetic HIV p24 N-terminal binding peptide, wherein the HIV p24 N-terminal binding peptide is a variant peptide of AMP8, comprising conserved amino acid residues Ala6; Ile7; Cys13; Pro14; Tyr17; and Lys33, and comprising a mutation at position F12H.