WO2023242155A1

WO2023242155A1 - Compositions and methods for the diagnosis of hiv infection

Info

Publication number: WO2023242155A1
Application number: PCT/EP2023/065719
Authority: WO
Inventors: Ole Siegfrid LAGATIE
Original assignee: Janssen Vaccines & Prevention B.V.
Priority date: 2022-06-14
Filing date: 2023-06-13
Publication date: 2023-12-21

Abstract

The invention provides compositions comprising and diagnostic methods, kits and devices using polypeptide antigens derived from HIV for the detection of HIV infection, in particular in subjects vaccinated with an HIV vaccine.

Description

COMPOSITIONS AND METHODS FOR THE DIAGNOSIS OF HIV INFECTION TECHNICAL FIELD This invention relates to compositions and methods for the detection of human immunodeficiency virus (HIV) infection, especially human immunodeficiency virus-1 (HIV-1) infection. The invention particularly concerns compositions and methods that do not detect vaccine-generated anti-HIV-1 antibodies. BACKGROUND OF THE INVENTION HIV is a lentivirus, part of the retrovirus family. Infection with HIV-1 over time causes acquired immune deficiency syndrome (AIDS) and related disorders. AIDS is a condition in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive. HIV compromises the immune system of infected individuals by targeting and infecting the CD4+ T lymphocytes which play a pivotal role in the recipient’s cellular immune system response . HIV was considered a global pandemic, but the WHO currently describes it as a global epidemic. With 1.7 million new infections and about 690,000 lives lost to AIDS-related illnesses in 2019, it remains a major world health problem [1]. HIV-2 is primarily found in West- Africa and is closely related to the simian immunodeficiency virus. The detection of HIV infection is generally accomplished by either of following methods: i) identification of viral proteins in the blood of infected individuals, ii) detection of viral nucleic acids, or iii) detection of host antibodies produced in response to viral infection. Nucleic acid-based tests (NAT) amplify and detect one or more of several target sequences located in specific HIV genes. These tests are relatively expensive but are very sensitive with detection limits down to 20 copies/mL and have a short detection window of 17 days [2]. The most common screening method for the diagnosis of infection with HIV is the detection of virus-specific antibodies elicited by infected individuals in response to the infection [3-6]. Since the development of the first serological test for detection of the human T cell lymphotropic virus type III (the term ‘HIV’ had not yet been adopted at that time) in 1985, several generations of serological tests have been employed [7]. “First generation” assays used purified viral proteins obtained from infected cells to bind, and identify, such antibodies. However, since diagnostically relevant viral proteins, such as those encoded by the HIV-1 env gene were difficult to obtain in large quantities, "second generation” assays were soon developed that employed recombinantly produced HIV antigens. The addition of IgM detection to the assay procedure through a sandwich ELISA resulted in the third generation HIV test. The IgG/IgM combination reduced the antibody- negative window to approximately 3 weeks post-infection, instead of 4 to 6 weeks in the second 1

generation [8]. In the late 1990s fourth generation tests detecting HIV-1 p24 antigen and antibodies to HIV-1 and HIV-2 were developed [9]. These tests reduced the test-negative window to approximately 2 weeks and are the most widely used for screening nowadays in resource-rich settings. During the past 30 years, numerous HIV vaccine prototypes have been developed but only a few HIV vaccine regimens have been tested in late stage (efficacy) clinical trials [10]. One ongoing late-stage HIV vaccine efficacy trial evaluates a heterologous HIV vaccine regimen based on mosaic Gag, Pol, and Env antigens designed to induce immune responses against a wide variety of global HIV strains, and two adjuvanted HIV Env proteins (“MOSAICO” trial, also referred to as HVTN706, or HPX3002; clinicaltrials.gov identifier: NCT03964415), while several of these vaccine components were tested in various combinations in earlier clinical trials (including HVTN 117, also referred to as HPX2004; and HVTN 118, also referred to as HPX2003). Most of the HIV vaccine candidates are complex products containing multiple viral genes or proteins and were designed to optimize cellular and humoral immune responses. Consequently, vaccine recipients’ sera are often reactive in licensed HIV serodetection assays, generating patterns indistinguishable from HIV-infected individuals [11-14]. Once an HIV vaccine is licensed, it can be expected that vaccine-induced seropositivity (VISP; sometimes referred to as vaccine-induced sero-reactivity or VISR) will cause substantial adoption challenges and implementations barriers. VISP is a major concern for HIV vaccine developers as there are several issues related to the anticipated high prevalence of false positives in vaccinated individuals. These following issues reconfirm the criticality of having a serological test that can distinguish between vaccinated human subjects with an HIV infection and vaccinated individuals who have merely become seroconverted (i.e., have generated anti-HIV antibodies) due to the administration of the vaccine: (a) Prophylactic HIV vaccines might initially be rolled-out in high risk populations only. Since no vaccine is expected to give 100% protection, it will be essential to diagnose break- through infections at the earliest possible times, thereby allowing the patient to get anti- viral therapy. (b) Since HIV infected individuals are excluded from the pool of potential blood donors, it is important to ensure both that infected individuals do not donate blood, and that individuals who have merely become seroconverted due to the administration of the vaccine are not erroneously precluded from serving as blood donors. (c) Since HIV infected individuals face exclusions from health insurance, immigration and travel across countries, employment limitations, and preclusion from military service, it is 2

important to be able to ensure that healthy vaccine recipients are not erroneously scored as HIV-infected individuals. (d) The increased number of false positive results in first-line serological testing will result in a steadily increasing need for confirmatory testing using molecular techniques. These latter tests are far more expensive than serological tests, thereby increasing the overall cost of HIV screening for the healthcare system. Furthermore, it requires specific laboratory infrastructure which is not always present in resource-limited setting. This increased cost and need for centralized lab infrastructure might negatively affect decision-taking at governmental level to start HIV vaccination campaigns. So far, only one test, called HIV-Selectest, was developed with the goal of differentiating between vaccine-generated antibodies and those produced after true HIV infection. This test was based on the detection of antibodies against conserved sequences in Env gp41 and Gag p6 [15-17]. Unfortunately, the development of the test was discontinued. Only one test appeared to be unaffected by the vaccination status: the OraQuick ADVANCE® Rapid HIV 1_2 Antibody Test. Almost all tested vaccinated individuals were found to be negative when using this test [18]. This 3rd generation test is based on the detection of antibodies against the gp41 immunodominant domain [19]. Although being an FDA approved test, its sensitivity to detect HIV-1 antibodies has been reported to be significantly lower than other (4th generation) tests [20,21]. This lower sensitivity might explain why most vaccinated individuals remain negative on this test. Those tests that do not detect anti-HIV-1 antibodies in vaccinated (uninfected) individuals tend to have limited sensitivity for detecting anti-HIV-1 antibodies in infected individuals. Different HIV proteins are expressed at different times during infection and as a consequence lead to different detection windows for antibodies against different proteins [3, 22]. Therefore, there is an important need for the development of a serological test that discriminates VISP from true infection to ensure that the antigens selected are complementary and together enable the detection of an antibody response as early as possible after infection and have the ability to maintain detection of antibody response over all stages of the disease [3, 5, 23]. Additionally, such a test might distinguish between HIV-1 and HIV-2, two variants that share 40-60% homology at the amino acid level [5]. SUMMARY OF THE INVENTION The present invention addresses the above concerns, in particular with respect to VISP, by providing a new composition of HIV polypeptide antigens that fulfills the following criteria: (1) is highly immunogenic in infected individuals; (2) is not recognized by vaccine-generated antibodies; 3

(3) is recognized by antibodies from HIV infected individuals at early times post-infection; (4) maintains detection of antibody response over all stages of the disease and (5) enables detection of antibodies against all clades and subtypes. In particular, the inventors have identified a number of polypeptide antigens derived from HIV which can be used in diagnostic assays to accurately determine the presence of an HIV infection. Indeed, the polypeptide antigens are likely useful in detection of a breakthrough HIV infection in human subjects who have been vaccinated with an HIV vaccine. The polypeptide antigens can effectively distinguish between anti-HIV antibodies generated due to an HIV infection in a human subject and anti-HIV antibodies generated in a human subject as a result of their vaccination with an HIV vaccine (i.e., VISP). Even more surprisingly, the polypeptide antigens provide this distinction even in those subjects who have been vaccinated with complex HIV vaccines incorporating antigens derived from the same viral proteins as the polypeptide antigens described here. Therefore, the invention relates to compositions and methods for the detection of human immunodeficiency virus infection, especially human immunodeficiency virus-1 (HIV-1) infection. The compositions and methods allow accurate detection of HIV-1 infection with striking sensitivity and specificity, thus maximizing the occurrence of true positives and minimizing the occurrence of false negatives. In particular, the compositions and methods are for use in human subjects who have been vaccinated with an HIV vaccine. The invention particularly concerns compositions and methods that do not detect vaccine-generated anti-HIV antibodies (e.g., anti-HIV-1 antibodies). Accordingly, the compositions and methods may be used to detect breakthrough HIV infection in HIV vaccine recipients whose sera may also contain vaccine-generated anti-HIV antibodies. The invention also concerns diagnostics kits and devices using the compositions and methods of the present invention. In a first aspect, the invention provides a composition comprising at least three polypeptide antigens derived from human immunodeficiency virus (HIV), for example HIV-1. The at least three polypeptide antigens may be recognized by an anti-HIV antibody present in a subject infected with HIV. The at least three polypeptide antigens may be selected from: at least one polypeptide antigen derived from the endodomain of HIV gp41, at least one polypeptide antigen derived from HIV Gag p17, at least one polypeptide antigen derived from HIV integrase IN, and at least one polypeptide antigen derived from HIV Nef. The composition may comprise at least one polypeptide antigen derived from the endodomain of HIV gp41; at least one polypeptide antigen derived from HIV Gag p17; and at least one polypeptide antigen derived from HIV integrase IN. A particularly useful composition may comprise at least one polypeptide antigen derived from the endodomain of HIV gp41; at least one polypeptide antigen derived from HIV Gag p17; at least one polypeptide antigen 4

derived from HIV integrase IN; and at least one polypeptide antigen derived from HIV Nef. In some embodiments, the composition does not comprise any further polypeptide antigens. In particular, the composition does not comprise any further polypeptide antigens derived from HIV, especially HIV-1. The polypeptide antigen derived from the endodomain of HIV gp41 may comprise a sequence having at least 90% identity to SEQ ID NO: 1 or fragments thereof; may comprise the sequence of SEQ ID NO: 1 or fragments thereof; or may consist of the sequence SEQ ID NO: 1 or fragments thereof. The polypeptide antigen derived from HIV Gag p17 may comprise a sequence having at least 90% identity to SEQ ID NO: 2 or fragments thereof; may comprise the sequence of SEQ ID NO: 2 or fragments thereof; or may consist of the sequence SEQ ID NO: 2 or fragments thereof. The polypeptide antigen derived from HIV IN may comprise a sequence having at least 90% identity to SEQ ID NO: 3 or fragments thereof; may comprise the sequence of SEQ ID NO: 3 or fragments thereof; or may consist of the sequence SEQ ID NO: 3 or fragments thereof. The polypeptide antigen derived from HIV Nef may comprise a sequence having at least 90% identity to SEQ ID NO: 4, or fragments thereof; may comprise the sequence of SEQ ID NO: 4, or fragments thereof; or may consist of the sequence SEQ ID NO: 4, or fragments thereof. In certain embodiments, the composition of the invention may further comprise an HIV antigen binding molecule capable of binding an HIV antigen. The HIV antigen binding molecule may be an anti-HIV antibody. In a particular example, the anti-HIV antibody may bind HIV p24. The composition of the invention may be used for the in vitro diagnosis of an HIV infection in a human subject, in particular a breakthrough HIV infection. The composition of the invention can be used in in vitro diagnostic assays which display striking sensitivity and specificity (both more than 95%) for the detection of HIV (e.g., HIV-1) infection in an individual. The polypeptide antigens, in certain instances, do not specifically bind to vaccine-generated anti-HIV antibodies. Accordingly, when used in a diagnostic assay of a biological sample from an uninfected human subject who has been vaccinated with an HIV vaccine, the composition of the invention does not generate a detectable signal (i.e., ideally, the signal is not above the background level) in the immunoassay. The composition of the invention can therefore be used in an immunoassay capable of discriminating between anti-HIV antibodies raised in a subject as a consequence of an HIV infection and anti-HIV antibodies raised in a subject in response to administration of an HIV vaccine (i.e., due to VISP in human subjects not infected with HIV). In a second aspect, the invention provides a method for the in vitro diagnosis of an HIV infection, for example an HIV-1 infection, in a subject, for example a human subject. The method may 5

comprise an immunoassay. The immunoassay may detect the presence of anti-HIV antibodies in a biological sample from a subject infected with HIV. Additionally, the immunoassay may detect the presence of HIV antigens in a biological sample from a subject infected with HIV. In a particular example, the immunoassay detects the formation of an immunocomplex between one or more of the at least three polypeptide antigens provided herein and an anti-HIV antibody in the biological sample from a subject. The formation and detection of an immunocomplex may indicate the presence of an HIV infection in the subject. In particular embodiments, vaccine-generated anti- HIV antibodies do not form an immunocomplex with the one or more of the at least three polypeptide antigens provided herein to a detectable level (i.e., ideally, the signal is not above the background level). Therefore, in some embodiments the method does not specifically detect vaccine-generated anti-HIV antibodies (i.e., a signal significantly above background is not generated even in the presence of vaccine-generated anti-HIV antibodies). This minimises the diagnosis of a false positive HIV infection. Accordingly, the methods of the invention are particularly useful for detecting presence of an HIV infection, or lack thereof, in recipients of an HIV vaccine. The method for the in vitro diagnosis of an HIV infection, for example an HIV-1 infection, in a human subject may comprise conducting an immunoassay comprising the steps of: a) contacting a biological sample from the subject with the composition of the invention, the contacting being under conditions sufficient to permit the formation of an immunocomplex; and b) determining whether an immunocomplex is formed. The method for the in vitro diagnosis of an HIV infection, for example an HIV-1 infection, in a human subject may comprise conducting an immunoassay comprising the steps of: a) contacting a biological sample from the subject with at least three polypeptide antigens recognized by an anti-HIV antibody present in a subject infected with HIV, the contacting being under conditions sufficient to permit the formation of an immunocomplex; and b) determining whether an immunocomplex is formed, wherein the at least three polypeptide antigens recognized by an anti-HIV antibody are selected from: at least one polypeptide derived from the endodomain of HIV gp41; at least one polypeptide antigen derived from HIV Gag p17; at least one polypeptide antigen derived from HIV IN; and at least one polypeptide antigen recognized by an anti-HIV antibody is derived from HIV Nef. In some embodiments, the polypeptide antigens recognized by an anti-HIV antibody are at least one polypeptide derived from the endodomain of HIV gp41; at least one polypeptide antigen derived 6

from HIV Gag p17; and at least one polypeptide antigen derived from HIV IN. In a particularly useful embodiment, the polypeptide antigens recognized by an anti-HIV antibody are at least one polypeptide derived from the endodomain of HIV gp41; at least one polypeptide antigen derived from HIV Gag p17; at least one polypeptide antigen derived from HIV IN; and at least one polypeptide antigen derived from HIV Nef. In order to maximise the chance of detecting an HIV infection in a subject within the first diagnostic window (i.e., before anti-HIV antibodies have been generated to a detectable level in the infected subject) or within the second diagnostic window (i.e., during which anti-HIV antibody levels drop during the isotype switch from IgM to IgG), when anti-HIV antibodies may be more difficult to detect, the method of the invention may further comprise detecting an HIV antigen in the biological sample of the subject. This provides a further opportunity to detect HIV infection in a subject, for example if the subject has limited serum concentration of anti-HIV antibodies capable of recognizing the polypeptide antigens of the present invention. The HIV antigen may be p24. Accordingly, the method of the invention may further comprise the steps (simultaneously or sequentially to the method steps above) of: a) contacting the biological sample from the subject with at least one HIV antigen binding molecule capable of binding an HIV antigen from an HIV infected subject, the contacting being under conditions sufficient to permit the formation of a further immunocomplex; and b) determining whether a further immunocomplex is formed. In certain embodiments hereof, the at least one HIV antigen is p24. In a third aspect, the invention provides a kit for the in vitro diagnosis of an HIV infection in a biological sample. The kit comprises at least three polypeptide antigens, which are selected from: at least one polypeptide antigen derived from the endodomain of human immunodeficiency virus (HIV) gp41, at least one polypeptide antigen derived from HIV Gag p17, at least one polypeptide antigen derived from HIV IN, and at least one polypeptide antigen derived from HIV Nef. In some embodiments, the kit comprises at least one polypeptide derived from the endodomain of HIV gp41; at least one polypeptide antigen derived from HIV Gag p17; and at least one polypeptide antigen recognized by an anti-HIV antibody is derived from HIV IN. A particularly useful kit may comprise at least one polypeptide antigen derived from the endodomain of human immunodeficiency virus (HIV) gp41; at least one polypeptide antigen derived from HIV Gag p17; at least one polypeptide antigen derived from HIV IN; and at least one polypeptide antigen derived from HIV Nef. In some embodiments, the kit does not comprise any further polypeptide antigens. 7

In particular, the kit does not comprise any further polypeptide antigens derived from HIV, especially HIV-1. The kit of the invention may further comprise an HIV antigen binding molecule capable of binding an HIV antigen. The HIV antigen binding molecule may be an anti-HIV antibody. In a particular example, the anti-HIV antibody may bind HIV p24. The use may be in human subjects who have been vaccinated with an HIV vaccine. In a fourth aspect, the invention provides the use of the composition or kit of the invention for detecting HIV infection, for example HIV-1, in a biological sample. In particular examples, the HIV infection may be detected by detecting anti-HIV antibodies in the biological sample. As shown in the examples herein, the polypeptide antigens of the compositions of the invention display striking sensitivity and specificity for detecting HIV infection. Indeed, the compositions of the invention comprising one or more of the at least three polypeptide antigens show sensitivity of more than 90%, particularly more than 98%, across a number of clades with signal detectable as soon as or before p24 antigen became undetectable (e.g. signal below cut-off using an ELISA for p24 antigen), as well as showing a specificity of more than 95%, particularly more than 97% irrespective of vaccination status of the subject. Therefore, the compositions, methods and kits of the present invention are particularly useful for accurately determining the presence of an HIV infection in a subject, shortly after infection, and minimising the likelihood of false negatives (i.e., no detection of HIV infection in an infected subject) and false positives (i.e., erroneous detection of HIV infection) even in subjects administered an HIV vaccine. In a further aspect, the invention provides a diagnostic device for use in the method or use, or with the kit, of the invention, wherein the device comprises: a) a first solid phase onto which has been attached one or more anti-HIV antibody binding molecules; b) a second solid phase onto which has been attached: i. at least one polypeptide antigen derived from the endodomain of HIV gp41; ii. at least one polypeptide antigen derived from HIV Gag p17; iii. at least one polypeptide antigen derived from HIV IN; and iv. at least one polypeptide antigen derived from HIV Nef; and c) a chamber configured to contain a liquid phase, wherein the chamber is in liquid communication with the first and second solid phase, and wherein the chamber is configured such that the liquid phase can comprises a biological sample from a subject, 8

wherein the one or more anti-HIV antibody binding molecules are eluted from the first solid phase upon contact with the liquid phase. The compositions, methods, kit and devices of the present invention are for diagnosing HIV infection, for example HIV-1 infection, in a human subject. Of course, the skilled person appreciates that an accurate diagnosis can be either positive (if the human subject has an HIV- infection) or negative (if the human subject does not have an HIV infection). The compositions, methods, kits and devices of the invention may suitably provide either diagnostic outcomes depending on the infection status of the human subject. DETAILED DESCRIPTION OF THE INVENTION The compositions, methods, uses, kits and devices of the present invention may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures. All patents, published patent applications and publications cited herein are incorporated by reference in their entirety. Polypeptide antigens The present invention provides polypeptide antigens for use in the compositions, methods, kits, devices of the invention. The polypeptide antigens are selected on the basis of their preferential recognition by anti-HIV antibodies from subjects infected with HIV compared to vaccine- generated anti-HIV antibodies from subjects uninfected with HIV. Ideally, the polypeptide antigen possesses the following characteristics: (1) is highly immunogenic in infected individuals; (2) is not recognized by vaccine- generated antibodies; (3) is recognized (e.g., in an immunoassay described herein) by antibodies from HIV infected individuals at early times post-infection; (4) maintains detection of antibody response over all stages of the disease; and (5) enables detection of antibodies against all clades and subtypes of HIV. The inventors identified that polypeptide antigens derived from the endodomain of HIV gp41 and from HIV p17, IN, and Nef are particularly useful in determining the presence or absence of HIV infection in a subject irrespective of vaccine status of the subject. In particular, polypeptide antigens derived from these HIV proteins are effectively recognized by anti-HIV antibodies from subjects with an HIV infection, but are not effectively recognized by vaccine-generated anti-HIV antibodies in uninfected subjects. The term “derived from” when in reference to a polypeptide antigen derived from HIV as used herein comprises a polypeptide antigen which has been obtained (e.g., isolated, purified, etc.) from a polypeptide or portion thereof of an HIV virus, and a polypeptide antigen that is genetically 9

(i.e., recombinantly) engineered and/or chemically synthesized to be essentially identical to the native polypeptide antigen or a portion thereof of the source. For example, a polypeptide antigen derived from an HIV polypeptide or a portion thereof will comprise at least a fragment of, and in some instances the entirety of, the sequence of said HIV polypeptide or portion thereof. The term “polypeptide antigen” includes a polypeptide binding partner that specifically binds to an antibody with the corresponding binding site. Accordingly, the polypeptide antigen comprises at least one epitope that is recognized and specifically bound by an anti-HIV antibody, in particular an anti-HIV antibody from a subject infected with HIV, for example HIV-1. Endodomain of HIV gp41 The polypeptide antigen for use in the compositions, methods, kits, and devices of the invention may be derived from the endodomain of HIV gp41. In a particular embodiment, the endodomain of HIV gp41 is a polypeptide antigen covering amino acids 706-856 of the HXB2 Env sequence (i.e., SEQ ID NO: 1), as follows: gp41 endodomain (SEQ ID NO: 1) NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLF SYHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTD RVIEVVQGACRAIRHIPRRIRQGLERILL Polypeptide antigens derived from gp41 are particularly useful in the immunoassays of the invention not least because gp41 displays at least 90% sequence conservation among HIV-1 clades. The polypeptide antigen derived from the endodomain of gp41 may comprise a sequence having at least 70%, 75%, 80%, 85%, or 90% identity to SEQ ID NO: 1 or a fragment thereof. The polypeptide antigen derived from the endodomain of gp41 may comprise a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 1 or a fragment thereof. The polypeptide antigen derived from the endodomain of gp41 preferably corresponds to the natural sequence derived from HIV as this ensures the reliable detection of in anti-HIV antibodies in the immunoassays described herein. Accordingly, the polypeptide antigen derived from the endodomain of gp41 may comprise the sequence of SEQ ID NO: 1 or a fragment thereof. Indeed, the polypeptide antigen derived from the endodomain of gp41 may consist of the sequence of SEQ ID NO: 1 or a fragment thereof. The polypeptide antigen derived from the endodomain of gp41 may comprise or consist of the sequence of SEQ ID NO: 1. Ideally, the polypeptide antigen derived from the endodomain of HIV gp41, or the composition comprising said polypeptide antigen, is not recognized by (i.e., does not specifically bind, 10

generating a detectable signal significantly above background, in an immunoassay, e.g., as described herein, to) anti-HIV antibodies in a biological sample from uninfected subjects vaccinated with an HIV vaccine but is recognized by anti-HIV antibodies in subjects with an HIV infection, for example an HIV-1 infection, irrespective of vaccine status of the infected subject. Accordingly, in particular embodiments, the polypeptide antigen derived from the endodomain of HIV gp41 is recognized by (e.g., generates a signal significantly above background in an immunoassay, e.g., as described herein, in response to) an anti-HIV antibody in a biological sample from less than 15%, 10%, 5%, 4%, 3%, 2% or 1%, preferably from 0%, of uninfected subjects vaccinated with an HIV vaccine. Ideally, the polypeptide antigen derived from the endodomain of HIV gp41 displays equivalent immunoreactivity to biological samples from an uninfected human subject before and after vaccination with an HIV vaccine, and preferably the immunoreactivity corresponds to baseline levels (for example in an immunoassay as described herein). On the other hand, the polypeptide antigen derived from the endodomain of HIV gp41 may be recognized by an anti-HIV antibody in a biological sample from more than 50%, 55%, 60%, particularly more than 65%, of subjects infected with HIV, for example HIV-1. Furthermore, the polypeptide antigen derived from the endodomain of HIV gp41 may have a sensitivity for the detection of HIV infection (i.e., correctly identifying subjects who have an HIV infection) of at least 50%, 55%, 60%, or 65%. In particular instances, the polypeptide antigen derived from the endodomain of HIV gp41 has an identical specificity for determining an absence of HIV infection (i.e., correctly identifying subjects who do not have the infection) in uninfected individuals before and after administration of an HIV vaccine. For example, the specificity in uninfected individuals before and after administration of an HIV vaccine is at least 90%, 95%, 97%, or 98%. Therefore, a polypeptide antigen derived from the endodomain of HIV gp41, in particular comprising or consisting of the sequence of SEQ ID NO: 1, is particularly useful in diagnostic methods, kits and devices for detecting anti-HIV antibodies in subjects infected with HIV, for example HIV-1, while not detecting vaccine-generated anti-HIV antibodies in the sera of uninfected subjects. HIV Gag p17 The polypeptide antigen may be derived from HIV Gag p17. In a particular embodiment, the HIV Gag p17 is the polypeptide antigen Gag-p17 from a group M clade, for example obtained from RPC (AHIV 205). The polypeptide antigen Gag-p17 may have an amino acid sequence (SEQ ID NO: 2), as follows: HIV Gag p17 (SEQ ID NO: 2) 11

GARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPGLLETSEGCRQILG QLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALDKIEEEQNKSKKKAQQAAADTG HSNQVSQNY The polypeptide antigen derived from HIV Gag p17 may comprise a sequence having at least 70%, 75%, 80%, 85%, or 90% identity to SEQ ID NO: 2 or a fragment thereof. The polypeptide antigen derived from HIV Gag p17 may comprise a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 2 or a fragment thereof. The polypeptide antigen derived from HIV Gag p17 preferably corresponds to the natural sequence derived from HIV as this ensures the reliable detection of in anti-HIV antibodies in the immunoassays described herein. Accordingly, the polypeptide antigen derived from HIV Gag p17 may comprise the sequence of SEQ ID NO: 2 or a fragment thereof. Indeed, the polypeptide antigen derived from HIV Gag p17 may consist of the sequence of SEQ ID NO: 2 or a fragment thereof. The polypeptide antigen derived from HIV Gag p17 may comprise or consist of the sequence of SEQ ID NO: 2. Ideally, the polypeptide antigen derived from HIV Gag p17, or the composition comprising said polypeptide antigen, is not recognized by (i.e., does not specifically bind, generating a detectable signal significantly above background, in an immunoassay, e.g., as described herein, to) anti-HIV antibodies in a biological sample from uninfected subjects vaccinated with an HIV vaccine but is recognized by anti-HIV antibodies in subjects with an HIV infection, for example an HIV-1 infection, irrespective of vaccine status of the infected subject. Accordingly, in particular embodiments, the polypeptide antigen derived from HIV Gag p17 is recognized by (e.g., generates a signal significantly above background in an immunoassay, e.g., as described herein, in response to) an anti-HIV antibody in a biological sample from less than 15%, 10%, 5%, 4%, 3%, 2% or 1%, preferably from 0%, of uninfected subjects vaccinated with an HIV vaccine. Ideally, the polypeptide antigen derived from HIV p17 displays equivalent immunoreactivity to biological samples from an uninfected human subject before and after vaccination with an HIV vaccine, and preferably the immunoreactivity corresponds to baseline levels (for example in an immunoassay as described herein). On the other hand, the polypeptide antigen derived from HIV Gag p17 may be recognized by an anti-HIV antibody in a biological sample from more than 40%, 45%, or 48%, of subjects infected with HIV, for example HIV-1. Furthermore, the polypeptide antigen derived from HIV Gag p17 may have a sensitivity for the detection of HIV infection (i.e., correctly identifying subjects who have an HIV infection) of at least 40%, 45%, or 48%. In particular instances, the polypeptide antigen derived from HIV Gag p17 has an identical specificity for determining an absence of HIV infection (i.e., correctly identifying subjects who do not have the infection) in uninfected individuals before and after administration of an HIV vaccine. For example, the specificity in uninfected individuals before and after administration of an HIV vaccine is at least 12

90%, 95%, 97%, 98%, or 99%. Therefore, a polypeptide antigen derived from HIV Gag p17, in particular comprising or consisting of the sequence of SEQ ID NO: 2, is particularly useful in diagnostic methods, kits and devices for detecting anti-HIV antibodies in subjects infected with HIV, for example HIV-1, while not detecting vaccine-generated anti-HIV antibodies in the sera of uninfected subjects. HIV IN The polypeptide antigen may be derived from HIV IN. In a particular embodiment, the HIV IN is the polypeptide antigen p31, for example obtained from Abcam (ab173265). The polypeptide antigen p31 may have an amino acid sequence (SEQ ID NO: 3), as follows: HIV IN (SEQ ID NO: 3) FLDGIDKAQDEHEKYHSNWRAMASDFNLPPVVAKEIVASCDKCQLKGEAMHGQVDCSPGIW QLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTIHTDNGSNFT GATVRAACWWAGIKQEFGIPYNPQSQGVVESMNKELKKIIGQVRDQAEHLKTAVQMAVFIH NFKRKGGIGGYSAGERIVDIIATDIQTKELQKQITKIQNFRVYYRDSRNPLWKGPAKLLWK GEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDED The polypeptide antigen derived from HIV IN may comprise a sequence having at least 70%, 75%, 80%, 85%, or 90% identity to SEQ ID NO: 3 or a fragment thereof. The polypeptide antigen derived from HIV IN may comprise a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 3 or a fragment thereof. The polypeptide antigen derived from HIV IN preferably corresponds to the natural sequence derived from HIV as this ensures the reliable detection of in anti-HIV antibodies in the immunoassays described herein. Accordingly, the polypeptide antigen derived from HIV IN may comprise the sequence of SEQ ID NO: 3 or a fragment thereof. Indeed, the polypeptide antigen derived from HIV IN may consist of the sequence of SEQ ID NO: 3 or a fragment thereof. The polypeptide antigen derived from HIV IN may comprise or consist of the sequence of SEQ ID NO: 3. Ideally, the polypeptide antigen derived from HIV IN, or the composition comprising said polypeptide antigen, is not recognized by (i.e., does not specifically bind, generating a detectable signal significantly above background, in an immunoassay, e.g., as described herein, to) anti-HIV antibodies in a biological sample from uninfected subjects vaccinated with an HIV vaccine but is recognized by anti-HIV antibodies in subjects with an HIV infection, for example an HIV-1 infection, irrespective of vaccine status of the infected subject. Accordingly, in particular embodiments, the polypeptide antigen derived from HIV IN is recognized by (e.g., generates a signal significantly above background in an immunoassay, e.g., as described herein, in response to) an anti-HIV antibody in a biological sample from less than 15%, 10%, 5%, 4%, 3%, 2%, or 1%, 13

preferably from 0%, of uninfected subjects vaccinated with an HIV vaccine. Ideally, the polypeptide antigen derived from HIV IN displays equivalent immunoreactivity to biological samples from an uninfected human subject before and after vaccination with an HIV vaccine, and preferably the immunoreactivity corresponds to baseline levels (for example in an immunoassay as described herein). On the other hand, the polypeptide antigen derived from HIV IN may be recognized by an anti-HIV antibody in a biological sample from more than 85%, 90%, 95% or from 100% of subjects infected with HIV, for example HIV-1. Furthermore, the polypeptide antigen derived from HIV IN may have a sensitivity for the detection of HIV infection (i.e., correctly identifying subjects who have an HIV infection) of at least 80%, 85%, or 90%. In particular instances, the polypeptide antigen derived from HIV IN has an identical specificity for determining an absence of HIV infection (i.e., correctly identifying subjects who do not have the infection) in uninfected individuals before and after administration of an HIV vaccine. For example, the specificity in uninfected individuals before and after administration of an HIV vaccine is at least 90%, 95%, 97%, 98%, or 99%. Therefore, a polypeptide antigen derived from HIV IN, in particular comprising or consisting of the sequence of SEQ ID NO: 3, is particularly useful in diagnostic methods, kits and devices for detecting anti-HIV antibodies in subjects infected with HIV, for example HIV-1, while not detecting vaccine-generated anti-HIV antibodies in the sera of uninfected subjects. HIV Nef The polypeptide antigen may be derived from HIV Nef. In a particular embodiment, the HIV Nef is the polypeptide antigen Nef from HIV-1 clade B, for example obtained from Diatheva (REP0035). The polypeptide antigen Nef may have an amino acid sequence (SEQ ID NO: 4, as follows: HIV Nef (SEQ ID NO: 4) MGGKWSKSSVIGWPTVRERMRRAEPAADRVGAASRDLEKHGAITSSNTAATNAACAWLEAQ EEEEVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQGYFP DWQNYTPGPGVRYPLTFGWCYKLVPVEPDKVEEANKGENTSLLHPVSLHGMDDPEREVLEW RFDSRLAFHHVARELHPEYFKNC The polypeptide antigen derived from HIV Nef may comprise a sequence having at least 70%, 75%, 80%, 85%, or 90% identity to SEQ ID NO: 4, or a fragment thereof. The polypeptide antigen derived from HIV Nef may comprise a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 4, or a fragment thereof. The polypeptide antigen derived from HIV Nef preferably corresponds to the natural sequence derived from HIV as this ensures the 14

reliable detection of in anti-HIV antibodies in the immunoassays described herein. Accordingly, the polypeptide antigen derived from HIV Nef may comprise the sequence of SEQ ID NO: 4, or a fragment thereof. Indeed, the polypeptide antigen derived from HIV Nef may consist of the sequence of SEQ ID NO: 4, or a fragment thereof. The polypeptide antigen derived from HIV Nef may comprise or consist of the sequence of SEQ ID NO: 4. Ideally, the polypeptide antigen derived from HIV Nef, or the composition comprising said polypeptide antigen, is not recognized by (i.e., does not specifically bind, generating a detectable signal significantly above background, in an immunoassay, e.g., as described herein, to) anti-HIV antibodies in a biological sample from uninfected subjects vaccinated with an HIV vaccine but is recognized by anti-HIV antibodies in subjects with an HIV infection, for example an HIV-1 infection, irrespective of vaccine status of the infected subject. Accordingly, in particular embodiments, the polypeptide antigen derived from HIV Nef is recognized by (e.g., generates a signal significantly above background in an immunoassay, e.g., as described herein, in response to) an anti-HIV antibody in a biological sample from less than 15%, 10%, 5%, 4%, 3%, 2%, or 1%, preferably from 0%, of uninfected subjects vaccinated with an HIV vaccine. Ideally, the polypeptide antigen derived from HIV Nef displays equivalent immunoreactivity to biological samples from an uninfected human subject before and after vaccination with an HIV vaccine, and preferably the immunoreactivity corresponds to baseline levels (for example in an immunoassay as described herein). On the other hand, the polypeptide antigen derived from HIV Nef may be recognized by an anti-HIV antibody in a biological sample from more than 50%, e.g. at least 60%, 65%, 70%, 75%, of subjects infected with HIV, for example HIV-1. Furthermore, the polypeptide antigen derived from HIV Nef may have a sensitivity for the detection of HIV infection (i.e., correctly identifying subjects who have an HIV infection) of at least 60%, 65%, 70%, or 75%. In particular instances, the polypeptide antigen derived from HIV Nef has an identical specificity for determining an absence of HIV infection (i.e., correctly identifying subjects who do not have the infection) in uninfected individuals before and after administration of an HIV vaccine. For example, the specificity in uninfected individuals before and after administration of an HIV vaccine is at least 90%, 95%, 97%, or 98%. Therefore, a polypeptide antigen derived from HIV Nef, in particular comprising or consisting of the sequence of SEQ ID NO: 4, is particularly useful in diagnostic methods, kits and devices for detecting anti-HIV antibodies in subjects infected with HIV, for example HIV-1, while not detecting vaccine-generated anti-HIV antibodies in the sera of uninfected subjects. 15

Further embodiments of the polypeptide antigens The polypeptide antigens may be derived from HIV-1 or HIV-2. In particular embodiments, the polypeptide antigens enable the in vitro detection of anti-HIV antibodies in a biological sample from a subject infected with HIV-1 and/or HIV-2. Preferably, the polypeptide antigen is derived from HIV-1. The polypeptide antigen may comprise the entire sequence of the viral polypeptide (e.g., the endodomain of HIV gp41, HIV p17, HIV IN or HIV Nef as described above). The polypeptide antigen should typically be of a length sufficient to allow binding of an anti-HIV antibody (i.e., to comprise an epitope for an anti-HIV antibody) from a subject infected with HIV. Accordingly, the length of the polypeptide antigens may be oriented towards the naturally occurring epitopes of the HIV proteins from which the polypeptide antigens are derived. Therefore, in certain embodiments the polypeptide antigen or fragment thereof may have a minimum length of 50, 60, 70, 80, 90, or 100 amino acids. To maximise the sensitivity and specificity of the immunoassays employing the polypeptide antigens described herein, the polypeptide antigen should contain an epitope for an anti-HIV antibody which specifically binds only the particular HIV protein from which the polypeptide antigen is derived. Accordingly, the polypeptide antigen would ideally not contain an epitope that shows significant reactivity with other anti-HIV antibodies. Nevertheless, the polypeptide antigen may comprise multiple epitopes for its corresponding anti-HIV antibody (i.e., the polypeptide antigen may be a polyhapten). The skilled person can determine the sensitivity and specificity of particular polypeptide antigens in detecting HIV infection using routine methods. For example, the specificity and/or sensitivity can be detected using immunoassays as described in the examples herein. In particular, an indirect ELISA can be used. Alternatively, a double antigen bridging ELISA can be used. The polypeptide antigens may be modified, provided that the capacity for binding to an anti-HIV antibody from an HIV infected subject is preserved. This means that a polypeptide antigen described above may also have N-terminal and/or C-terminal flanking sequences. Accordingly, the polypeptide antigen may also contain sequences that do not occur naturally in the HIV protein from which it was derived. Moreover, it is possible to equip the polypeptide antigen with spacer groups known in the art. The polypeptide antigens can also be modified e.g., by substitution, deletion or insertion of a single amino acid residue. The precondition of such modifications is, however, that the specific binding capacity of the anti-HIV antibodies to be detected is preserved. The polypeptide antigens may comprise naturally or non-naturally occurring amino acid homologs. 16

For the use in immunoassays, for example those used in the methods of in vitro diagnosis, or with the kits and devices of the invention, the polypeptide antigens, for example within a composition, may be provided with a solid phase binding group, for example biotin and haptens like digoxigenin and other label groups familiar to the skilled person. Methods of producing hapten labelled peptides are for example described in WO 96/03423, which is herein incorporated by reference in its entirety. Alternatively or additionally, the polypeptide antigen may be attached to a peptide linker. The linker can be attached at the N or C-terminal domain of the polypeptide antigen. The linker may be further attached to a conjugate. The conjugate may be used for attachment of the polypeptide antigen to a solid phase. Alternatively, the conjugate may be a detectable label used to generate a signal from the polypeptide antigen, for example if the polypeptide antigen is also employed in a detection step (e.g., as the anti-HIV antibody binding molecule) in a bridging ELISA assay or equivalent immunoassay. The skilled person is aware of appropriate linkers and conjugates, and examples of these, along with appropriate detectable labels are provided herein. The precondition for modifying the polypeptide antigen in this way is that it should still be recognized by an anti-HIV antibody from an infected subject. The percent identity between two or more sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions ×100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two or more sequences. The percent identity between two or more amino acid sequences may be determined using the algorithm of E. Meyers and W. Miller (Comput Appl Biosci 4:11-17 (1988)) which has been incorporated into the ALIGN program, using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two or more amino acid sequences may be determined using the Needleman and Wunsch (J Mol Biol 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Polypeptide antigens may be prepared using virtually any known technique for the preparation of polypeptides. For example, the polypeptides may be prepared using conventional step-wise solution or solid phase peptide syntheses, recombinant DNA techniques, proteolysis, or modifications of purified viral proteins/peptides or recombinant proteins. Peptides may be prepared using conventional stepwise solution or solid phase synthesis, or by way of segment condensation. In particular embodiments, the polypeptide antigens are synthetic polypeptide antigens as these offer several potential advantages in particular immunoassays used with the invention for example 17

increasing the sensitivity and specificity of the assay, decreasing its cost, and providing a relatively simple format that would be suitable for testing sizeable number of samples in any laboratory. In certain embodiments, the polypeptide antigens are prepared by recombinant DNA expression, which enables production of large quantities of polypeptides (including longer ones, e.g., longer than 100 amino acids) according to well established procedures in a commercially feasible manner. The invention provides the use of at least one of the polypeptide antigens for detecting HIV infection, for example HIV-1 infection, in a human subject. Preferably, the subject has been vaccinated with an HIV vaccine. The use comprises the detection of anti-HIV antibodies in a biological sample from a human subject infected with HIV, for example HIV-1. The polypeptide antigens described above can be incorporated into compositions of polypeptide antigens according to the invention. Furthermore, the polypeptide antigens can be used in the methods, uses, kits or devices of the invention. Compositions The invention further provides compositions comprising three or more of the polypeptide antigens provided herein, and methods and uses of, and kits and devices comprising these compositions. The inventors have demonstrated that a composition comprising multiple polypeptide antigens (for example three, or preferably four polypeptide antigens recognized, in an immunoassay as described herein, by different anti-HIV antibodies from a subject infected with HIV) achieved remarkable sensitivity for the detection of HIV infection across multiple clades and over all stages of the disease (i.e., from less than 2 weeks to more than 3 years). Without wishing to be bound by theory, the use of a composition comprising two or more of the polypeptide antigens for an in vitro diagnostic assay may reduce the problems associated with the Hook effect in biological samples having high antibody concentrations – such a composition is more likely to contain polypeptide antigens having high affinities as well as polypeptide antigens having low affinities. In addition, the use of such a composition in the diagnostic assays of the invention may allow for reliable detection of anti-HIV antibodies directed towards different clades of HIV. Therefore, the compositions herein are for use in in vitro diagnostic methods for detecting HIV infection, or for kits and devices to be used in such methods. A composition of the invention may comprise any combination of at least three polypeptide antigens, selected from: at least one polypeptide antigen derived from the endodomain of HIV gp41; at least one polypeptide antigen derived from HIV Gag p17; at least one polypeptide antigen derived from HIV IN; and at least one polypeptide antigen derived from HIV Nef. Accordingly, a 18

composition of the invention may comprise at least one polypeptide antigen derived from the endodomain of HIV gp41, at least one polypeptide antigen derived from HIV Gag p17 and at least one polypeptide antigen derived from HIV IN. A preferred composition comprises at least one polypeptide antigen derived from the endodomain of HIV gp41; at least one polypeptide antigen derived from HIV Gag p17; at least one polypeptide antigen derived from HIV IN; and at least one polypeptide antigen derived from HIV Nef. In particular embodiments, the HIV is HIV-1. In some embodiments, the composition does not comprise any further polypeptide antigens. In particular, the composition does not comprise any further polypeptide antigens derived from HIV, especially HIV-1. Alternatively, in some embodiments, the composition may comprise no more than 3, 2, or 1 further polypeptide antigens derived from HIV, especially HIV-1. In certain embodiments, the composition comprises three further polypeptide antigens derived from HIV, especially HIV-1; preferably the composition comprises two further polypeptide antigens derived from HIV, especially HIV-1; more preferably the composition comprises one further polypeptide antigen derived from HIV, especially HIV-1. In another most preferred embodiment, the composition comprises no further polypeptide antigens derived from HIV, especially HIV-1. In certain preferred embodiments, the composition does not comprise full length gp41 polypeptide antigen. In certain preferred embodiments, the composition does not comprise polypeptide antigen comprising gp41 ectodomain. In certain preferred embodiments, the composition does not comprise gp120 or gp160 polypeptide antigen. In preferred embodiments, the composition does not comprise any of gp120, gp160, and full length gp41 polypeptide antigen. The composition may be useful for the detection of anti-HIV antibodies in a biological sample from a human subject infected with HIV, for example HIV-1, while not detecting anti-HIV antibodies in the sera of uninfected human subjects generated by an HIV vaccine (i.e., minimising the background signal in a diagnostic assay (e.g., an immunoassay as described herein) detecting anti-HIV antibodies for determining presence of an HIV infection, wherein the background signal may be caused by vaccine-generated anti-HIV antibodies). The polypeptide antigens for incorporation into the compositions of the invention can be any of the polypeptide antigens described herein, including fragments thereof. Accordingly, a particular composition of the invention comprises at least one polypeptide antigen comprising the sequence of SEQ ID NO: 1; at least one polypeptide antigen comprising the sequence of SEQ ID NO: 2; at least one polypeptide antigen comprising the sequence of SEQ ID NO: 3; and at least one polypeptide antigen comprising the sequence of SEQ ID NO: 4. The composition of the invention may consist essentially of at least one polypeptide antigen derived from the endodomain of HIV gp41 (e.g., comprising or consisting of SEQ ID NO: 1); at least one polypeptide antigen derived 19

from HIV Gag p17 (e.g., comprising or consisting of SEQ ID NO: 2); at least one polypeptide antigen derived from HIV IN (e.g., comprising or consisting of SEQ ID NO: 3); and at least one polypeptide antigen derived from HIV Nef (e.g. comprising or consisting of SEQ ID NO: 4). Like the polypeptide antigens provided herein, the compositions of the invention are particularly useful in in vitro diagnostic assays, for example an immunoassay, for detecting HIV infection, for example HIV-1 infection, in a human subject. The human subject may have been vaccinated with an HIV vaccine. Accordingly, the one or more polypeptide antigens within the composition may be modified, as described herein, such that they can be attached to a solid phase of an immunoassay device or equivalent assay surface. For certain applications, having all of the one or more polypeptide antigens within the same composition simplifies the preparation of the immunoassay (i.e., the polypeptide antigens can be attached to the same solid phase simultaneously ready for application of the biological sample from a subject). Alternatively, the one or more polypeptide antigens may be modified for conjugation of a detectable label, allowing their detection in an immunoassay. In order to allow additional sensitivity at the very early stages of disease progression (i.e., 0-2 weeks) the composition may further comprise at least one HIV antigen binding protein capable of binding an HIV antigen in a biological sample from a subject with an HIV infection. The at least one HIV antigen binding protein may be an antibody. The antibody may be an antibody binding to HIV p24. For completeness, an exemplary HIV p24, derived from HIV-1, comprises the sequence (SEQ ID NO: 5), as follows: HIV p24 (SEQ ID NO: 5) HQALSPRTLNAWVKVIEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEEAAEWDRMHPV QAGPIPPGQIREPRGSDIAGTTSTLQEQITWMTSNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPF RDYVDRFFRVLRAEQATQEVKNWMTETLLVQNANPDCRTILKALGSGATLEEMMTAC Therefore, in a particular embodiment, the composition may comprise at least one polypeptide antigen derived from the endodomain of HIV gp41 (e.g., comprising SEQ ID NO: 1); at least one polypeptide antigen derived from HIV Gag p17 (e.g., comprising SEQ ID NO: 2); at least one polypeptide antigen derived from HIV IN (e.g., comprising SEQ ID NO: 3); at least one polypeptide antigen derived from HIV Nef (e.g., comprising SEQ ID NO:4); and at least one HIV antigen binding protein (e.g., an antibody specifically binding HIV p24). Similar to the polypeptide antigens, the at least one HIV antigen binding protein may be modified, as described herein, such 20

that it can be attached to a solid phase of an immunoassay device or equivalent assay surface or conjugated to a detectable signal. The invention provides the use of one or more compositions of the invention for detecting HIV infection, for example HIV-1 infection, in a human subject. The human subject may have been vaccinated with an HIV vaccine. The use comprises the detection of anti-HIV antibodies in a biological sample from a human subject infected with HIV, for example HIV-1. The compositions of the invention are particularly useful for the detection (e.g., in an immunoassay as described herein) of anti-HIV antibodies in a biological sample from a human subject infected with HIV, for example HIV-1, while not detecting (e.g., generating a signal significantly above background in an immunoassay as described herein) anti-HIV antibodies in the sera of uninfected human subjects generated by administration of an HIV vaccine. Therefore, in some embodiments, the compositions of the invention, for example when employed in an in vitro diagnostic method (e.g., as described herein), do not generate a signal significantly above background in response to vaccine-generated anti-HIV antibodies. The compositions may be highly immunogenic in infected individuals. Ideally, the composition comprises polypeptide antigens not recognized by (e.g., not generating a signal significantly above background in an immunoassay, e.g., as described herein, in response to) vaccine-generated anti- HIV antibodies. Furthermore, preferably compositions may: (i) be recognized by anti-HIV antibodies from HIV infected human subjects at early times post-infection; (ii) be capable of detecting anti-HIV antibodies in HIV infected human subjects over all stages of the disease; and/or (iii) enable the detection of anti-HIV antibodies against all HIV clades and subtypes. The composition may comprise at least one polypeptide antigen that alone detects HIV infection in a human subject (i.e., by the detection of anti-HIV antibodies) with a % sensitivity of at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%. Preferably, the composition of the invention may comprise at least three polypeptide antigens (e.g., four polypeptide antigens), wherein the combined % sensitivity of the polypeptide antigens to detect HIV infection in a human subject is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. Ideally, the combined % sensitivity of the one or more polypeptide antigens within the composition of the invention to detect HIV infection in a human subject is maintained across one or more clades of HIV, for example HIV-1 clades B, A, complex, AG, C, F, AE, G, BF, D, BC, AB and/or B. In 21

particular embodiments, the combined % sensitivity of the polypeptide antigens within the composition of the invention to detect HIV infection is at least 85% at or before p24 antigen becomes undetectable (e.g. signal below cut-off using an ELISA for p24 antigen), or within two weeks post initial diagnosis, i.e. in early stages of infection. The combined % sensitivity of the polypeptide antigens within the composition of the invention to detect HIV infection may be at least 95% within 9-12 weeks post initial diagnosis. The combined % sensitivity of the polypeptide antigens within the composition of the invention to detect HIV infection may be at least 99% at least 12 weeks post initial diagnosis. Thus the sensitivity increases over time post infection. It is often difficult to determine the time since first infection, not least because infection may be undetectable in its very early stages, and so in certain instances, time since first diagnosis may act as a suitable surrogate for time since first infection. The % specificity of the at least three, preferably four, polypeptide antigens within the composition of the invention or the combined % specificity of the polypeptide antigens within the composition of the invention to determine the absence of HIV infection is at least 95%, at least 96%, at least 97%, at least 98%, at least 98%, at least 99%, or 100%. Ideally, the % specificity is determined after the subject has been vaccinated with an HIV vaccine. In particular embodiments, the % specificity after vaccination is within less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5% or identical to the % specificity before vaccination. Accordingly, in a particularly preferred embodiment, the three or more, preferably four, polypeptide antigens within the composition of the invention do not detect (e.g., in an immunoassay described herein) anti-HIV-1 antibodies from an uninfected subject after the uninfected subject has been vaccinated with an HIV vaccine. The skilled person can determine the sensitivity and specificity of particular compositions in detecting HIV infection using routine methods. For example, the specificity and/or sensitivity can be detected using immunoassays as described in the examples herein. In particular, an indirect ELISA can be used. Alternatively, a double antigen bridging ELISA can be used. Preferably the HIV is HIV-1. Diagnostic methods The invention provides methods for the in vitro diagnosis of an HIV infection, for example HIV-1 infection, in a human subject. The methods of the invention are particularly useful for the detection of anti-HIV antibodies in a biological sample from an infected subject while not detecting anti-HIV antibodies generated by vaccination with an HIV vaccine in an uninfected subject (i.e., minimising the background signal caused by vaccine-generated anti-HIV antibodies in an HIV-uninfected subject). Accordingly, the methods are equally applicable for detecting HIV infection (for example, 22

HIV-1 infection) in vaccinated human subjects (i.e., having breakthrough HIV infection) and unvaccinated human subjects. Ideally, the method of the invention does not specifically detect vaccine-generated anti-HIV antibodies (e.g., the method does not generate a signal significantly above background in response to vaccine-generated anti-HIV antibodies in an immunoassay e.g., as described herein). The diagnostic methods of the invention are highly sensitive and specific for in vitro detection of HIV (e.g., HIV-1) infection. The skilled person would understand that any diagnostic method is not 100% accurate and that all diagnostic methods are subject to a certain amount of error (e.g., a low percentage of false positives). Nevertheless, as demonstrated herein, the diagnostic methods of the invention are significantly improved and are capable of detecting HIV (e.g., HIV-1) infection in a subject with remarkable sensitivity and specificity (i.e., upwards of 95%). Indeed, the methods herein are so advanced that they can be used to detect HIV infection even in individuals having vaccine-generated anti-HIV-1 antibodies. Moreover, the methods described herein only give a very low percentage of VISP, which is a big improvement over several previously described methods for HIV diagnosis. As will become clear, the methods of the invention can be performed on any number of appropriate assay platforms. In a particularly preferred form, the method comprises conducting an immunoassay (i.e., an assay measuring the presence or concentration of a molecule in solution through the use of an antibody or antigen). Depending on the content of the biological sample being assayed, the immunoassay will comprise formation and detection of an immunocomplex. Herein, the term “immunocomplex” comprises the complex formed between an antigen and an antibody. Herein, the term “biological sample” comprises fluids typically used for diagnostic assays (e.g., sera, blood, urine, saliva, pancreatic juice, cerebrospinal fluid, semen, etc.). However, the term also includes any fluidic biological sample (e.g., tissue or biopsy extracts, extracts of feces, sputum, etc.). Most preferably, the biological sample being assayed will be serum or plasma. In certain embodiments, the biological sample may be saliva. Detecting the presence of anti-HIV antibodies The methods of the invention may employ three or more (e.g., three, four, or more), preferably four, of the polypeptide antigens described herein. The selected polypeptide antigens, alone or in combination, or compositions thereof, can be used to differentiate between vaccine-generated antibodies and infection-generated antibodies (including those generated by breakthrough HIV infection in vaccinated subjects). Therefore, the methods can be used to detect HIV infection in a population of individuals irrespective of the vaccination status of the individuals, to assess the 23

efficacy of vaccine clinical trials, or to monitor potential infections during those trials, or once an HIV vaccine is approved and used in the population. The method may comprise conducting an immunoassay comprising the steps of: a) contacting a biological sample from the subject with at least three polypeptide antigens, the contacting being under conditions sufficient to permit the formation of an immunocomplex (i.e., in the event that the subject is infected with HIV and thus the biological sample contains anti-HIV antibodies); and b) determining whether an immunocomplex is formed. The at least three polypeptide antigens are recognized by an anti-HIV antibody present in a biological sample of a subject infected with HIV, for example HIV-1, irrespective of vaccine status of the subject. Ideally, the at least three polypeptide antigens are not recognized by (i.e., do not specifically bind to) vaccine-generated anti-HIV antibodies present in a biological sample of a subject who has been vaccinated with an HIV vaccine. Accordingly, ideally, the polypeptide antigens do not generate a signal in response to vaccine-generated anti-HIV antibodies significantly above background in an immunoassay, for example an immunoassay as described herein. The at least three polypeptide antigens may comprise at least one polypeptide antigen derived from the endodomain of HIV gp41. The at least three polypeptide antigens may comprise at least one polypeptide antigen derived from HIV Gag p17. The at least three polypeptide antigens may comprise at least one polypeptide antigen derived from HIV IN. The at least three polypeptide antigens may comprise at least one polypeptide antigen derived from HIV Nef. In preferred embodiments, the polypeptide antigens comprise at least one polypeptide antigen derived from the endodomain of HIV gp41, at least one polypeptide antigen derived from HIV Gag p17, at least one polypeptide antigen derived from HIV IN, and at least one polypeptide antigen derived from HIV Nef. In some embodiments, the immunocomplex is formed between one or more of the at least three, preferably four, polypeptide antigens and an anti-HIV antibody in the biological sample. In some embodiments the immunoassay detects the presence of an anti-HIV antibody in the biological sample from the subject. The inventors have demonstrated that a diagnostic assay using multiple polypeptide antigens in a single assay achieved remarkable sensitivity for the detection of HIV infection across multiple clades and over all stages of the disease (i.e., from early stages, e.g. before p24 antigen becomes undetectable, to more than 3 years post infection), while also minimising the occurrence of false positives in uninfected subjects, even in uninfected subjects vaccinated with an HIV vaccine. 24

Therefore, the method of the invention may employ any combination of three or more, preferably four, of the polypeptide antigens described herein, or a composition described herein comprising any combination of three or more, preferably four, of the polypeptide antigens. For example, the immunoassay may use at least one polypeptide antigen derived from the endodomain of HIV gp41, at least one polypeptide antigen derived from HIV Gag p17 and at least one polypeptide antigen derived from HIV IN. In a preferred embodiment, the immunoassay may use at least one polypeptide antigen derived from the endodomain of HIV gp41, at least one polypeptide antigen derived from HIV Gag p17, at least one polypeptide antigen derived from HIV IN, and at least one polypeptide antigen derived from HIV Nef. In an exemplary embodiment, the immunoassay may use a composition comprising polypeptide antigens derived from four viral components of HIV, for example HIV-1. Therefore, the immunoassay may comprise the steps of: a) contacting a biological sample from the subject with four polypeptide antigens recognized by an anti-HIV antibody present in a subject infected with HIV, the contacting being under conditions sufficient to permit the formation of an immunocomplex (i.e., in the event that the subject is infected with HIV and thus the biological sample from the subject contains anti-HIV antibodies); and b) determining whether an immunocomplex is formed, wherein the four polypeptide antigens recognized by an anti-HIV antibody comprise at least one polypeptide antigen derived from the endodomain of HIV gp41 (e.g., comprising SEQ ID NO: 1); at least one polypeptide antigen derived from HIV Gag p17 (e.g., comprising SEQ ID NO: 2); at least one polypeptide antigen derived from HIV IN (e.g., comprising SEQ ID NO: 3); and at least one polypeptide antigen derived from HIV Nef (e.g., comprising SEQ ID NO: 4). In some embodiments, the immunoassay does not comprise contacting the biological sample from the subject with any further polypeptide antigens derived from HIV, especially HIV-1. In some embodiments, the immunoassay does not comprise contacting the biological sample from the subject with any further polypeptide antigens derived from HIV, especially HIV-1. If appropriate, the immunoassay may comprise contacting the biological sample from the subject with no more than 3, 2, or 1 further polypeptide antigens derived from HIV, for example HIV-1, and recognized by an anti-HIV antibody present in a biological sample of a subject infected with HIV, the contacting being under conditions sufficient to permit the formation of an immunocomplex. In certain embodiments, the immunoassay may comprise contacting the biological sample from the subject with three further polypeptide antigens derived from HIV, for example HIV-1; preferably the immunoassay may comprise contacting the biological sample from 25

the subject with two further polypeptide antigens derived from HIV, for example HIV-1; more preferably the immunoassay may comprise contacting the biological sample from the subject with one further polypeptide antigen derived from HIV, for example HIV-1. In another most preferred embodiment, the immunoassay may comprise contacting the biological sample from the subject with no further polypeptide antigens derived from HIV, for example HIV-1. In certain preferred embodiments, the immunoassay does not comprise contacting the biological sample from the subject with any of full length gp41 polypeptide antigen, polypeptide antigen comprising gp41 ectodomain, gp120 polypeptide antigen, or gp160 polypeptide antigen. In some embodiments, the immunoassay comprises contacting the biological sample from the subject with polypeptide antigens derived from HIV, especially HIV-1, wherein the polypeptide antigens consist essentially of at least one polypeptide antigen derived from the endodomain of HIV gp41 (e.g., comprising or consisting of SEQ ID NO: 1); at least one polypeptide antigen derived from HIV Gag p17 (e.g., comprising or consisting of SEQ ID NO: 2); at least one polypeptide antigen derived from HIV IN (e.g., comprising or consisting of SEQ ID NO:3); and at least one polypeptide antigen derived from HIV Nef (e.g., comprising or consisting of SEQ ID NO: 4). The immunoassay may comprise contacting the biological sample from the subject with one or more (e.g., no more than 4, 3, 2, or 1) polypeptide antigens derived from HIV-2 and recognized by an anti-HIV antibody present in a biological sample of a subject infected with HIV, the contacting being under conditions sufficient to permit the formation of an immunocomplex. Such an immunoassay may allow the detection of both HIV-1 and HIV-2 infection within the same immunoassay format, thus increasing ease and efficacy of diagnosis irrespective of HIV infection subtype. The detection of HIV infection may be achieved via the detection of the presence of an anti-HIV antibody in the biological sample of a human subject. Accordingly, the immunocomplex is formed between one or more of the at least three, preferably four, polypeptide antigens and an anti-HIV antibody in the biological sample. Based on the selection of polypeptide antigens described herein, the immunocomplex is more likely to form between the one or more of the at least three, preferably four, polypeptide antigens and an anti-HIV antibody generated by an HIV infection in the subject, rather than an anti-HIV antibody generated by vaccination of the subject with an HIV vaccine. As outlined above, the polypeptide antigens are recognized by anti-HIV antibodies present in an infected subject, but ideally are not recognized by (i.e., do not specifically bind to and thus does 26

not generate a signal significantly above background in an immunoassay, e.g., as described herein, in response to) anti-HIV antibodies in an uninfected vaccinated subject. The immunoassay, therefore, may comprise the steps of: a) contacting the biological sample with the at least three, preferably four, polypeptide antigens, the contacting being under conditions sufficient to permit the anti-HIV antibody, if present in the biological sample, to bind to one or more of the at least three, preferably four, polypeptide antigens and form a polypeptide antigen-anti-HIV antibody complex; b) contacting the formed polypeptide antigen-anti-HIV antibody complex with an anti-HIV antibody binding molecule, the contacting being under conditions sufficient to permit the anti-HIV antibody binding molecule to bind to the anti- HIV antibody of the formed polypeptide antigen-anti-HIV antibody complex and form an extended complex; and c) determining the presence or concentration of the anti-HIV antibody in the biological sample by determining the presence or concentration of the formed extended complex. The anti-HIV antibody binding molecule employed in step b) may be any molecule capable of binding to an anti-HIV antibody and generating a detectable signal, therefore determining the presence or concentration of the formed extended complex. For example, the anti-HIV antibody binding molecule may be an anti-human IgG antibody or an antigen binding fragment thereof; an anti-human IgM antibody or an antigen binding fragment thereof; protein A, protein G, protein A/G, and/or a polypeptide comprising an antigen recognized by the anti-HIV antibody. Preferably the anti-HIV antibody binding molecule is a polypeptide comprising an antigen recognized by the anti-HIV antibody because such a molecule could detect the presence of an anti-HIV antibody irrespective of the isotype of the antibody (i.e., the polypeptide comprising an antigen recognized by the anti-HIV antibody will bind a free paratope of the anti-HIV antibody, while the other paratope of the anti-HIV antibody is bound to the polypeptide antigen according to step a)). The polypeptide comprising an antigen recognized by the anti-HIV antibody may be of the same type as the polypeptide antigen employed in step a) of the immunoassay, further comprising a modification to allow the polypeptide antigen employed in step b) to generate a detectable signal. A detectable signal may be generated by a label conjugated to the anti-HIV antibody binding molecule. For example, the conjugated label may be an enzyme; a fluorescent label; a radioisotope; a chemiluminescent label; and/or a chromophore label. Accordingly, the immunoassay may be an enzyme-linked immunosorbent assay (ELISA), an immunofluorescence assay (IFA), a 27

radioimmunoassay (RIA), a chemiluminescent microparticle immunoassay (CMIA), or a radioimmunoprecipitation assay (RIPIA). The skilled person would appreciate the applicability and utility of these different formats. The immunoassay may be a sandwich immunoassay, for example a bridging ELISA. Alternatively, the immunoassay may be an indirect ELISA. Of course, to the extent that an immunoassay used in the method permits, the anti-HIV antibody binding molecule may be modified such that it can be attached to a solid phase of the immunoassay or immunoassay device. Depending on the immunoassay employed, the anti-HIV antibodies detected in the methods, kits, or devices of the invention, may be the low affinity antibody IgM and/or the high affinity antibody IgG, IgA and/or IgE. In a particular embodiment, the method, kit or device detects IgM and IgG isotype anti-HIV antibodies generated by HIV infection in a human subject. The skilled person would appreciate that the embodiments above apply equally to the use of compositions of the invention comprising polypeptide antigens provided herein in the diagnostic methods disclosed herein. In particular embodiments, the HIV from which the polypeptide antigen is derived is HIV-1. The HIV-1 infection being diagnosed may be HIV-1 or HIV-2, but is preferably HIV-1. Detecting the presence of HIV antigens In order to allow additional sensitivity at the very early stages of disease progression, the method of the invention may further detect the presence of at least one HIV antigen in a biological sample from a human subject with an HIV infection. The immunoassay may detect the presence of an HIV antigen in the biological sample simultaneously or sequentially to detecting the presence of an anti-HIV antibody in the biological sample. Therefore, the immunoassay may further comprise the steps of: a) contacting the biological sample from the subject with at least one HIV antigen binding molecule capable of binding an HIV antigen from an HIV infected subject, the contacting being under conditions sufficient to permit the formation of a further immunocomplex (i.e., in the event that the subject is infected with HIV and thus the biological sample contains HIV antigens); and b) determining whether a further immunocomplex is formed. The further immunocomplex is formed between one or more of the at least one HIV antigen binding molecules and an HIV antigen in the biological sample. The further immunocomplex is 28

so-called because the immunoassay used in the method of the invention is configured to (simultaneously or sequentially) detect at least one first immunocomplex formed between at least one polypeptide antigen and an anti-HIV antibody in the biological sample from a subject infected with HIV. The immunoassay may comprise the steps of: a) contacting the biological sample with at least one HIV antigen binding molecule, the contacting being under conditions sufficient to permit the HIV antigen binding molecule to bind to the HIV antigen, if present in the biological sample, and form an HIV antigen binding molecule-HIV antigen complex; b) contacting the formed HIV antigen binding molecule-HIV antigen complex with a further HIV antigen binding molecule, the contacting being under conditions sufficient to permit the further HIV antigen binding molecule to bind to the HIV antigen within the HIV antigen binding molecule-HIV antigen complex and form a further extended complex; and c) determining the presence or concentration of the HIV antigen in the biological sample by determining the presence or concentration of the formed further extended complex. The HIV antigen binding molecule may be an anti-HIV antigen antibody or an antigen binding fragment thereof. In particular, the HIV antigen may be p24, and the anti-HIV antigen antibody or an antigen binding fragment thereof may specifically bind to HIV p24. Preferably, the anti- HIV antigen antibody or an antigen binding fragment thereof does not bind to the precursor of p24 (i.e., p55) – in other words, the anti-HIV antibody or an antigen binding fragment thereof binds specifically to the mature form of p24. Alternatively, the HIV antigen may be p1, p2, p6 or p7. Accordingly, the anti-HIV antigen antibody or an antigen binding fragment thereof specifically binds to p1, p2, p6 or p7. As noted above, the HIV antigen binding molecule, for example an antibody, may be modified such that it can be attached to a solid phase or equivalent surface of the immunoassay or immunoassay device. The further HIV antigen binding molecule employed in step b) may be a molecule capable of binding to an HIV antigen and generating a detectable signal, therefore determining the presence or concentration of the formed further extended complex of HIV antigen binding molecule-HIV antigen. Therefore, the further HIV antigen binding molecule may be a further anti-HIV antigen antibody or an antigen binding fragment thereof. The anti-HIV antigen antibody or an antigen 29

binding fragment thereof used in step b) may have a different binding epitope on the HIV antigen compared to the epitope bound by an anti-HIV antigen antibody used in step a). This allows both anti-HIV antigen antibodies to bind the HIV antigen concurrently, thus permitting use of the components in a sandwich ELISA, for example. The detectable signal may be generated by a label conjugated to the anti-HIV antigen binding molecule. For example, the conjugated label may be an enzyme; a fluorescent label; a radioisotope; a chemiluminescent label; and/or a chromophore label. Accordingly, the immunoassay may be an enzyme-linked immunosorbent assay (ELISA), an immunofluorescence assay (IFA), a radioimmunoassay (RIA), a chemiluminescent microparticle immunoassay (CMIA), or a radioimmunoprecipitation assay (RIPIA). Of course, to the extent that the immunoassay used in the method permits, the HIV antigen binding molecule or further HIV antigen binding molecule may be modified such that it can be attached to a solid phase or equivalent surface of the immunoassay or immunoassay device. As well as using the polypeptide antigens and/or compositions described herein, the methods of the invention may employ additional polypeptide antigens or compositions thereof for targeting further anti-HIV antibodies and/or HIV antigens in a biological sample from a subject infected with HIV, for example HIV-1. Immunocomplexes and extended complexes The present invention also provides immunocomplexes and extended complexes generated in the methods of the invention, and the kits and devices employing the methods of the invention. In particular, the present invention provides an immunocomplex comprising a polypeptide antigen provided herein bound to an anti-HIV antibody from a subject infected with HIV, for example HIV-1. Additionally, the present invention provides an extended complex comprising the immunocomplex of the invention bound to an anti-HIV antibody binding molecule, for example a complex of a polypeptide antigen provided herein, an anti-HIV antibody from a subject infected with HIV, for example HIV-1, and an anti-HIV antibody binding molecule. Immunoassay formats Any of a wide variety of assay formats may be used in accordance with the methods of the invention and with the kits and devices of the invention. Such formats may be heterogeneous or homogeneous, sequential or simultaneous, competitive or noncompetitive. U.S. Patent Nos. 5,563,036; 5,627,080; 5,633,141; 5,679,525; 5,691,147; 5,698,411; 5,747,352; 5,811,526; 5,851,778; and 5,976,822 illustrate several different assay 30

formats and applications, each of which is herein incorporated by reference in its entirety. Such assays can be formatted to be quantitative, to measure the concentration or amount of an anti-HIV antibody (or HIV antigen), or they may be formatted to be qualitative, to measure the presence or absence of an anti-HIV antibody (or HIV antigen). Additional descriptions of immunoassays that may be adapted for use in accordance with the methods, kits and devices, of the invention are available in the scientific literature and known to the skilled person. The methods of the invention can be performed as a wet or a dry test. In the wet tests, all test reagents are present in liquid phase. But all usual dry test formats suitable for the detection of antigens or antibodies can be used too. The dry tests, for example those disclosed in European Patent Appln. No.186,799 combine all test components on one single carrier. The methods of the invention may be performed using immunoassays having both solid and liquid phases. Heterogeneous immunoassays Heterogeneous immunoassay techniques typically involve the use of a solid phase material to which the reaction product becomes bound, but may be adapted to involve the binding of non- immobilized antigens and/or antibodies (i.e., a solution-phase immunoassay). The reaction product is separated from excess sample, assay reagents, and other substances by removing the solid phase from the reaction mixture (e.g., by washing). One type of solid phase immunoassay that may be used in accordance with the methods or kits of the invention is a sandwich immunoassay. In the sandwich assay, the more analyte present in the sample, the greater the amount of label present on the solid phase. This type of assay format is generally preferred, especially for the visualization of low analyte concentrations, because the appearance of label on the solid phase is more readily detected. Accordingly, in a preferred immunoassay for use in the methods or with the kits of the invention, at least one, preferably at least three, preferably four, polypeptide antigen provided herein, for example within a composition of the invention, that is recognized by an anti-HIV antibody in a biological sample from a human subject infected with HIV, is bound to a solid support (i.e., immobilized) and incubated in contact with the biological sample being tested for the presence of an anti-HIV antibody. A blocking agent may be added to reduce non-specific binding. As will be appreciated, the polypeptide antigen may be incubated with the biological sample in an unbound state and then subsequently bound to the solid support (i.e., immobilizable). The solid supports are then preferably extensively treated (e.g., by washing, etc.) to substantially remove non-HIV antibodies and/or anti-HIV antibodies (e.g., vaccine-generated antibodies) that may be present but that failed to bind to the bound polypeptide antigen. In consequence of such treatment, an 31

immunocomplex forms between the polypeptide antigen and anti-HIV antibody. A detectably labelled anti-HIV antibody binding molecule (capable of binding to the anti-HIV antibody in the immunocomplex) is then preferably added and the support is incubated under conditions sufficient to permit the second antibody to bind to any anti-HIV antibody that may be present. As noted above, the anti-HIV antibody binding molecule may be an anti-human IgG antibody or an antigen binding fragment thereof; an anti-human IgM antibody or an antigen binding fragment thereof; protein A; protein G; protein A/G; and/or a polypeptide comprising an antigen recognized by the anti-HIV antibody. The solid support may then be extensively treated (e.g., by washing, etc.) to substantially remove any unbound labelled anti-HIV antibody binding molecule. If an anti-HIV antibody generated from an HIV infection is present in the biological sample, then the anti-HIV antibody binding molecule, anti-HIV antibody and polypeptide antigen will form an extended complex. Any signal from the detectably labelled anti-HIV antibody binding molecule can be detected, for example using an enzyme; a fluorescent label; a radioisotope; a chemiluminescent label; and/or a chromophore label. If the anti-HIV antibody binding molecule is an anti-human immunoglobulin antibody (i.e., an anti-human IgG or anti-human IgM antibody), the extended complex is an anti-human immunoglobulin antibody/anti-HIV antibody/immobilized polypeptide antigen sandwich. The anti-human immunoglobulin antibody may be a natural immunoglobulin isolated from nonhuman species (e.g., anti-human IgG murine antibody, anti-human IgG goat antibody, anti-human IgM goat antibody, etc.), or it can be produced recombinantly or synthetically. It may be an intact immunoglobulin, or an immunoglobulin fragment (e.g., Fab etc.). In a preferred embodiment, the anti-human immunoglobulin antibody is an anti-human IgG or, more preferably, an anti-human IgM antibody, the latter of which is generated earlier during HIV infection. Alternatively, the anti-human immunoglobulin antibody is both an anti-human IgG and an anti-human IgM antibody. If the anti-HIV antibody binding molecule is a polypeptide comprising an antigen recognized by the anti-HIV antibody, which is preferred, the extended complex is a polypeptide comprising an antigen recognized by the anti-HIV antibody/anti-HIV antibody/polypeptide antigen sandwich, or double antigen bridge (see Fig.3). In this double antigen bridge, the antigens may be identical, except for the presence of a detectable label on the antigen employed to act as the anti-HIV antibody binding molecule. In such assays, the detection of a detectable label bound to the solid support is indicative of the presence of an anti-HIV antibody in the biological sample generated by HIV infection in the human subject. In the double antigen bridge format, one binding site of the anti-HIV antibody is required for binding to the polypeptide antigen attached to the solid support, while another binding site of the anti-HIV antibody is required for binding to the labelled polypeptide comprising an antigen recognized by the anti-HIV antibody. 32

Therefore, anti-HIV antibodies in the IgM format are particularly well detected in such an assay given the 10 antigen binding paratopes on IgM antibodies. Sandwich assay formats and other appropriate assays for use in and with the present invention are for instance described by Schuurs et al.; U.S. Patent Nos. 3,791,932 and 4,016,043, Pankratz, et al., U.S. Patent No. 5,876,935; European Patent Appln. No. 386,713; and UK Patent Application 2,313,666, each of which is herein incorporated by reference in its entirety. Such immunoassays might also be appropriate for the embodiments of the invention which detect the presence of an HIV antigen simultaneously or sequentially to detecting the presence of an anti- HIV antibody in a biological sample from a subject. In such instances, the HIV antigen binding molecule may be bound to the solid support. Upon addition of the biological sample, if a corresponding HIV antigen is present, it will bind to the HIV antigen binding molecule on the solid support forming an HIV antigen binding molecule-HIV antigen complex. A detectably labelled further HIV antigen binding molecule can then bind to the HIV antigen in the HIV antigen binding molecule-HIV antigen complex forming an extended complex (i.e., a further HIV antigen binding molecule/HIV antigen/HIV antigen binding molecule sandwich). The further HIV antigen binding molecule can be labelled as described herein and thus can be used to detect any HIV antigen in the biological sample. The additional detection of an HIV antigen besides detecting anti-HIV antibodies raised against another polypeptide antigen may allow detection of the presence of an HIV infection in a subject at an earlier stage of infection, than with an assay detecting anti-HIV antibodies only, thereby closing the first diagnostic window slightly. Homogenous immunoassays To eliminate the bound-free separation (i.e., washing) step and reduce the time and equipment needed for a chemical binding assay, a homogeneous assay format may alternatively be employed. In such assays, one component of the binding pair may still be immobilized; however, the presence of the second component of the binding pair is detected without a bound-free separation. Examples of homogeneous optical methods are the EMIT method of Syva, Inc. (Sunnyvale, CA), which operates through detection of fluorescence quenching; the laser nephelometry latex particle agglutination method of Behringwerke (Marburg, Germany), which operates by detecting changes in light scatter; the LPIA latex particle agglutination method of Mitsubishi Chemical Industries (Tokyo, Japan); the TDX fluorescence depolarization method of Abbott Laboratories (Abbott Park, IL); and the fluorescence energy transfer method of Cis Bio International (Paris, France). In preferred embodiments, the immunoassay may be an immunochromatographic assay (e.g., a lateral flow assay (LFA)) in which all components required for the assay steps are within a single reaction 33

environment (e.g., a diagnostic device) and do not require intervention beyond addition of the biological sample. Any such assays may be adapted for use in accordance with the methods or with the kits or devices of the invention and may employ any one or more of the polypeptide antigens or compositions of the invention. Competitive immunoassays The binding assay of the present invention may be configured as a competitive assay. In a competitive assay, the more anti-HIV antibody present in the test sample, the lower the amount of label present on the solid phase. The competitive assay can be conducted by providing a defined amount of a labelled anti-HIV antibody, capable of binding a polypeptide antigen attached to a solid support, and determining whether the biological sample being tested contains anti-HIV antibody that would compete with the labelled antibody for binding to the immobilized polypeptide antigen. In such a competitive assay, the amount of captured labelled antibody is inversely proportional to the amount of analyte (i.e., anti-HIV antibody from an infected human subject) present in the test sample. Alternative competitive immunoassays are for instance described in U.S. Patent Nos. 4,401,764; 4,746,631; 4,661,444; 4,185,084; and 4,243,749; European Patent Appln. No. 177,191; and GB Patent No.2,084,317, all of which are incorporated herein by reference in their entirety and may be applicable for use in accordance with the methods or with the kits or devices of the invention and may employ any one or more of the polypeptide antigens or compositions of the invention. Immunochromatographic assay formats Immunochromatographic assay formats may be used to detect anti-HIV antibodies in the biological samples of human subjects having HIV infection, in accordance with the methods of the invention and with the kits and devices of the invention. The immunochromatographic assay may be a lateral flow assay (LFA) format. In one immunochromatographic assay format, two contacting, but spatially distinct, solid phases are employed. The first solid phase may contain at least one non-immobilized, labelled, anti-HIV antibody binding molecule and the second solid phase may contain at least one immobilized, unlabelled, polypeptide antigen provided herein, for example within a composition of the invention, that is recognized by anti-HIV antibodies from a human subject infected with HIV, for example HIV-1. Upon introduction of a biological sample (e.g., serum, plasma, or whole blood test sample) the at least one non-immobilized, labelled, anti-HIV antibody binding molecule will enter the liquid phase and can permeate to the second solid phase. If an anti-HIV antibody generated to an HIV infection is present in the biological sample, the anti-HIV antibody will bind to the at least one 34

immobilized polypeptide antigen in the second solid phase. The immobilized anti-HIV antibody will then be bound by the anti-HIV antibody binding molecule in the liquid phase. Therefore, an extended complex of polypeptide antigen/ anti-HIV antibody/anti-HIV antibody binding protein will be formed in the second solid phase and can be detected by detecting the label on the anti-HIV antibody binding molecule. In such an instance, the second solid phase can further contain at least one immobilized, unlabelled HIV antigen binding molecule and the first solid phase can further contain at least one non-immobilized, labelled, further HIV antigen binding molecule. If the biological sample contains an HIV antigen, the HIV antigen will bind to the immobilized HIV antigen binding molecule in the second solid phase, and the further HIV antigen binding molecule in the first solid phase will enter the liquid phase and travel to the second solid phase to bind to the immobilized HIV antigen forming an HIV antigen binding protein/HIV antigen/further HIV antigen binding protein extended complex. The extended complex can be detected by detecting the label on the further HIV antigen binding molecule. In another immunochromatographic assay format, two contacting, but spatially distinct, solid phases are employed. The first solid phase may contain at least one non-immobilized, labelled, polypeptide antigen provided herein, for example within a composition of the invention, that is recognized by anti-HIV antibodies from a human subject infected with HIV, for example HIV-1, and the second solid may contain at least one immobilized, unlabelled, anti-HIV antibody binding molecule. Upon introduction of a biological sample (e.g., serum, plasma, or whole blood test sample) the at least one non-immobilized, labelled, polypeptide antigen will enter the liquid phase and can permeate to the second solid phase. If an anti-HIV antibody generated to an HIV infection is present in the biological sample, the at least one polypeptide antigen will bind to the anti-HIV antibody as it permeates to the second solid phase. The anti-HIV antibody will then be bound by the immobilized anti-HIV antibody binding molecule in the second solid phase. Therefore, an extended complex of anti-HIV antibody binding molecule/ anti-HIV antibody/polypeptide antigen will be formed in the second solid phase and can be detected by detecting the label on the polypeptide antigen. In such an instance, the second solid phase can further contain at least one immobilized, unlabelled HIV antigen binding molecule and the first solid phase can further contain at least one non-immobilized, labelled, further HIV antigen binding molecule. If the biological sample contains an HIV antigen, the HIV antigen will bind to the immobilized HIV antigen binding molecule in the second solid phase, and the further HIV antigen binding molecule in the first solid phase will enter the liquid phase and travel to the second solid phase to bind to the immobilized HIV antigen forming an HIV antigen binding protein/HIV antigen/further HIV antigen binding 35

protein extended complex. The extended complex can be detected by detecting the label on the further HIV antigen binding molecule. The skilled person would appreciate that such formats achieve useful results irrespective of which component of the assay is attached to the solid phase and which component having a detectable label is eluted into the liquid phase. As noted above, the polypeptide antigens can be modified so as to allow their attachment to one or more solid phase of an immunoassay format. The skilled person would understand that it would be possible to attach the modified one or more polypeptide antigens to the one or more solid phases individually. If two or more polypeptide antigens (recognized by different anti-HIV antibodies from a subject infected with HIV) are to be attached to the same solid phase, the attachment can occur sequentially. In preferred embodiments, three or more, for example four, of the polypeptide antigens provided herein (recognized by different anti-HIV antibodies from a subject infected with HIV) are attached to the one or more solid phases, for example the same solid phase, simultaneously. Therefore, the compositions of the present invention, comprising three or more, for example four, of the polypeptide antigens (recognized by different anti-HIV antibodies from a subject infected with HIV) are particularly useful for quick and easy preparation of one or more solid phases of an immunoassay format or device. Further features of the immunoassay formats In all such assay formats, at least one component of the assay reagents will preferably be labelled or otherwise detectable by the evolution or quenching of light. Such component may be the anti-HIV antibody binding molecule and may be for example, an anti-human immunoglobulin antibody, protein A, protein G, protein A/G, or a polypeptide comprising an antigen recognized by the anti-HIV antibody, depending on the immunoassay format employed. The skilled person is aware of suitable assay formats appropriate for use in the methods and devices of the invention (e.g., ELISA, IFA, RIA, CMIA, or RIPIA). Radioisotopic-binding assay formats (e.g., a radioimmunoassay, etc.) employ a radioisotope as such label; the signal is detectable by the evolution of light in the presence of a fluorescent or fluorogenic moiety (see e.g. Lucas et al., U.S. Patent No.5,698,411 and Landrum et al., U.S. Patent No.5,976,822, each of which is herein incorporated by reference in its entirety). Enzymatic-binding assay formats (e.g., an ELISA, etc.) employ an enzyme as a label; the signal is detectable by the evolution of color or light in the presence of a chromogenic or fluorogenic moiety. Other labels, such as paramagnetic labels, materials used as colored particles, latex particles, colloidal metals such as selenium and gold, and dye particles (see e.g. U.S. Patent Nos.4,313,734; 4,373,932, and 36

5,501,985, each of which is herein incorporated by reference in its entirety) may also be employed. The use of enzymes (especially alkaline phosphatase, p-galactosidase, horse radish peroxidase, or urease) as the detectable label (e.g., an ELISA) is preferred. The presence of enzymatic labels may be detected through the use of chromogenic substrates (including those that evolve or adsorb fluorescent, W, visible light, etc.) in response to catalysis by the enzyme label. Chemical labels also may be employed (e.g., colloidal gold, latex bead labels, etc.). Detection of label can be accomplished using multiple detectors, multipass filters, gratings, or spectrally distinct fluors (see e.g., U.S. Patent No. 5,759,781, which is herein incorporated by reference in its entirety), etc. It is particularly preferred to employ peroxidase as an enzyme label, especially in concert with the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB), OPD, or ABTS. In the case of labelling of the antibodies with peroxidase as enzyme, it is possible to use the periodate technique (Nakane, P.K. et al. (1974) J Histochem Cytochem.22: 1084-90, which is herein incorporated by reference in its entirety) or a method reported in which the partners are linked with a heterobifunctional reagent (Ishikawa, E. et al. (1983) J Immunoassay.49(3):209-327, which is herein incorporated by reference in its entirety). Any of a wide variety of solid supports may be employed in the immunoassays of the methods, kits and devices of the present invention. Suitable materials for the solid support are synthetics such as polystyrene, polyvinyl chloride, polyamide, or other synthetic polymers, natural polymers such as cellulose, as well as derivatized natural polymers such as cellulose acetate or nitrocellulose, and glass, especially glass fibers. The support can take the form of spheres, rods, tubes, and microassay or microtiter plates. Sheet-like structures such as paper strips, small plates, and membranes are likewise suitable. The surface of the carriers can be permeable and impermeable for aqueous solutions. Herein, the terms solid support and solid phase are used interchangeably. The immunoassay may be an indirect ELISA, in which at least one polypeptide antigen is attached to a solid phase of the immunoassay (see Fig. 2). The binding of an anti-HIV antibody to the at least one polypeptide antigen is detected by an anti-human immunoglobulin antibody (e.g., an anti- human IgG antibody or anti-human IgM antibody) which is conjugated to a detectable label, preferably an enzyme for ELISA. In a preferred embodiment of the present invention, the immunoassay is a double antigen bridging assay ELISA (see Fig.3), in which at least three, preferably four, polypeptide antigens (preferably a polypeptide antigen derived from the endodomain of HIV gp41, a polypeptide antigen derived from HIV p17, a polypeptide antigen derived from HIV IN, and a polypeptide antigen derived from HIV Nef), are attached to a solid phase of the immunoassay. The binding of an anti-HIV antibody 37

to any one of the at least one polypeptide antigens is detected by corresponding labelled polypeptides comprising antigens recognized by the anti-HIV antibody. The labelled polypeptides may be identical to the polypeptide antigens attached to the solid phase, but additionally comprising a detectable label, preferably an enzyme for ELISA. Positive and negative controls may be included in or alongside the immunoassays described herein to ensure reliable results. Positive controls can include a control for each analyte (i.e., anti-HIV antibody and/or HIV antigen) which is tested separately, or a combined positive control wherein the presence of all of the analytes to be detected in the assay are determined. Negative samples (i.e., serum or plasma from an individual not infected with HIV) can be included as a negative control. The skilled person would understand that the concentrations of the components of the immunoassay attached to the solid phase can be optimized to ensure maximal detection of anti-HIV antibody and/or HIV antigen. Diagnostic kits The polypeptide antigens and compositions of the invention are ideally suited for the preparation of a diagnostic kit. The components of such a kit may be used in the methods and uses of the invention or with the diagnostic device of the invention. Accordingly, the invention provides a kit for the in vitro diagnosis of an HIV infection, for example HIV-1, in a biological sample. Such a kit may comprise a carrier means being compartmentalized to receive in close confinement; one or more containers means (e.g., vials, tubes and the like). Each of the containers means may comprise one of the separate elements to be used in the method. Alternatively each of the container means may comprise multiple elements (e.g., a composition comprising a combination of the polypeptide antigens provided herein). The kit may comprise at least three polypeptide antigens, wherein the at least three polypeptide antigens are selected from: at least one polypeptide antigen derived from the endodomain of HIV gp41; at least one polypeptide antigen derived from HIV Gag p17; at least one polypeptide antigen derived from HIV IN; and at least one polypeptide antigen derived from HIV Nef. As outlined above, particularly preferred compositions of the invention comprise a combination of four polypeptide antigens provided herein. Accordingly, a preferred kit may comprise at least one polypeptide antigen derived from the endodomain of HIV gp41 (e.g., comprising or consisting of SEQ ID NO: 1); at least one polypeptide antigen derived from HIV Gag p17 (e.g., comprising or 38

consisting of SEQ ID NO: 2); at least one polypeptide antigen derived from HIV IN (e.g., comprising or consisting of SEQ ID NO: 3); and at least one polypeptide antigen derived from HIV Nef (e.g., comprising or consisting of SEQ ID NO: 4). In some embodiments, the kit does not comprise any further polypeptide antigens. In particular, the kit does not comprise any further polypeptide antigens derived from HIV, especially HIV-1. Alternatively, the kit may comprise no more than 3, 2, or 1 further polypeptide antigens derived from HIV. In certain embodiments, the kit comprises three further polypeptide antigens derived from HIV, especially HIV-1; preferably the kit comprises two further polypeptide antigens derived from HIV, especially HIV-1; more preferably the kit comprises one further polypeptide antigen derived from HIV, especially HIV-1. In another most preferred embodiment, the kit comprises no further polypeptide antigens derived from HIV, especially HIV-1. In certain embodiments, the kit does not comprise any of full length gp41 polypeptide antigen, antigen comprising gp41 ectodomain, gp120 polypeptide antigen, or gp160 polypeptide antigen. The three or more, preferably four, polypeptide antigens may be provided in individual containers or as a composition in a single container. The kit may comprise further containers comprising other components required for the immunoassays used in the methods of the invention. For example, the kit may comprise one or more anti-HIV antibody binding molecules. As outlined above, the anti-HIV antibody binding molecule may be an anti-human IgG antibody; an anti-human IgM antibody; protein A; protein G; protein A/G; and/or a polypeptide comprising an antigen recognized by the anti-HIV antibody. The anti-HIV antibody binding molecule may be conjugated to a label selected from the group consisting of: an enzyme; a fluorescent label; a radioisotope; a chemiluminescent label; and/or a chromophore label. Furthermore, the kit may comprise a reaction environment for the production of an immunocomplex between the one or more polypeptide antigens and an anti-HIV antibody if present. The kit may further comprise an HIV antigen binding molecule capable of binding an HIV antigen. The HIV antigen binding molecule may be an anti-HIV antigen antibody or an antigen binding fragment thereof. In a preferred embodiment, the anti-HIV antigen antibody binds HIV p24. The kit may further comprise one or more further HIV antigen binding molecules for the detection of an immunocomplex formed between the HIV antigen binding molecule and the HIV antigen. The further HIV antigen binding molecule may be conjugated to a label selected from the group consisting of: an enzyme; a fluorescent label; a radioisotope; a chemiluminescent label; and/or a chromophore label. Accordingly, the kit may further comprise a reaction environment for the 39

production of an immunocomplex between the HIV antigen binding molecule and an HIV antigen if present. The reaction environment for the production of an immunocomplex between the one or more of the polypeptide antigens and an anti-HIV antibody, if present, and an immunocomplex between the HIV antigen binding molecule and an HIV antigen, if present, may be the same or different reaction environment. The kit may comprise a biological fluid to be used as a reference sample, for example a biological fluid having no anti-HIV antibodies or HIV antigens recognised by the other components of the kit. Accordingly, the kit may provide an appropriate positive and/or negative control for use in the methods of the invention. In addition, the kit may also contain one or more containers, each of which comprises a (different) predetermined amount of an anti-HIV antibody. These latter containers can be used to prepare a standard curve into which can be interpolated the results obtained from the biological sample containing the unknown amount of anti-HIV antibodies. Furthermore, the kit may comprise appropriate buffers and blocking reagents in order to perform the methods of the invention. The kit may also comprise appropriate instructions for using the kit to perform the methods of the invention. Accordingly, the invention provides the use of a kit of the invention for detecting HIV infection, for example HIV-1 infection, in a human subject. Preferably, the human subject has been vaccinated with an HIV vaccine. The use comprises the detection of anti-HIV antibodies in a biological sample from a human subject infected with HIV, for example HIV-1. Diagnostic device The invention also provides a diagnostic device for use with the methods, uses, or kits, of the invention. The device may use one or more of the polypeptide antigens provided herein and/or a composition of the invention for the in vitro diagnosis of an HIV infection in a human subject. In particular, the device detects anti-HIV antibodies in a biological sample from a human subject infected with HIV, for example HIV-1. The diagnostic device may comprise one or more solid phases and a chamber configured to contain a liquid phase. The solid and liquid phases are in fluid communication upon introduction of a biological sample. In a particular embodiment, the diagnostic device comprises two contacting, but spatially distinct, solid phases. The first solid phase may contain at least one non-immobilized, 40

labelled, anti-HIV antibody binding molecule and the second solid phase may contain at least three, preferably four, immobilized, unlabelled, polypeptide antigens provided herein, for example within a composition of the invention, that is recognized by anti-HIV antibodies from a human subject infected with HIV, for example HIV-1. The device may further comprise a multilayer filter system allowing fluid communication between a sample region and the one or more solid phases of the device once a biological sample is applied directly to the filter system of the sample region. In such a device, the liquid phase generated by the application of the biological sample will permeate through the filter system travelling through the first solid phase to the second solid phase. In permeating the first solid phase, the liquid phase elutes the at least one non-immobilized, labelled, anti-HIV antibody binding molecule which can become bound to any migrating anti-HIV antibodies generated by an HIV infection, if present in the biological sample. The anti-HIV antibody continues to migrate to the second solid phase where it becomes immobilized by its recognition of the at least one of the polypeptide antigens. Accordingly, an extended complex is formed between the polypeptide antigen immobilized in the second solid phase, the anti-HIV antibody from an infected subject, and the anti-HIV antibody binding molecule. As the anti-HIV antibody binding molecule is labelled, the formation of the extended complex at the second solid phase can be detected. The device may further comprise an HIV antigen binding molecule immobilized at the second solid phase, thus allowing the detection of a HIV antigen upon the binding of a further HIV antigen binding molecule conjugated to an appropriate label. The further HIV antigen binding molecule may be non-immobilized at the first solid phase and may elute upon introduction of the biological sample, allowing migration of the further HIV antigen binding molecule to the immunocomplex of HIV antigen and HIV antigen binding molecule immobilized at the second solid phase. Accordingly, in a preferred embodiment, the diagnostic device comprises: a) a first solid phase onto which has been attached one or more anti-HIV antibody binding molecules; b) a second solid phase onto which has been attached at least three polypeptide antigens, selected from: i. at least one polypeptide antigen derived from the endodomain of human immunodeficiency virus (HIV) gp41; and/or ii. at least one polypeptide antigen derived from HIV Gag p17; and/or iii. at least one polypeptide antigen derived from HIV integrase IN; and/or 41

iv. at least one polypeptide antigen derived from HIV Nef; and c) a chamber configured to contain a liquid phase, wherein the chamber is in liquid communication with the first and second solid phase, and wherein the chamber is configured such that the liquid phase can comprise a biological sample from a subject, wherein the one or more anti-HIV antibody binding molecules are eluted from the first solid phase upon contact with the liquid phase. In some embodiments, the one or more anti-HIV antibody binding molecules and/or the one or more HIV antigen binding molecules are conjugated to a label. The label may be selected from the group consisting of: an enzyme, a fluorescent label, a radioisotope, a chemiluminescent label. and/or a chromophore label. In a preferred instance, the second solid phase has attached at least one polypeptide antigen derived from the endodomain of HIV gp41 (e.g., comprising SEQ ID NO: 1); at least one polypeptide antigen derived from HIV Gag p17 (e.g., comprising SEQ ID NO: 2); at least one polypeptide antigen derived from HIV IN (e.g., comprising SEQ ID NO: 3); and at least one polypeptide antigen derived from HIV Nef (e.g., comprising SEQ ID NO: 4). Furthermore, the second solid composition may also have attached at least one HIV antigen binding protein (e.g., an antibody specifically binding HIV p24). In certain embodiments, no more than 3, 2, or 1 further polypeptide antigens derived from HIV, especially HIV-1, are attached onto the second solid phase. In certain embodiments, three further polypeptide antigens derived from HIV are attached onto the second solid phase; preferably two further polypeptide antigens derived from HIV are attached onto the second solid phase; more preferably one further polypeptide antigen derived from HIV is attached onto the second solid phase. In another most preferred embodiment, no further polypeptide antigens derived from HIV, especially HIV-1, are attached onto the second solid phase. Advantages of the invention The compositions, methods, kits, uses and devices of the invention are particularly useful for the detection of anti-HIV antibodies in a biological sample from a human subject infected with HIV, for example HIV-1. Strikingly, the compositions, methods, kits, uses and devices of the invention are capable of detecting anti-HIV antibodies in a biological sample from a human subject infected with HIV, for example HIV-1, while not detecting (i.e., not generating a signal significantly above background in an immunoassay, e.g., as described herein, in response to) anti-HIV antibodies in the sera of uninfected human subjects generated by an HIV vaccine. This diagnostic utility is due to the fact that the one or more polypeptide antigens are not recognized by (i.e., do not generate a 42

signal significantly above background in an immunoassay, e.g., as described herein, in response to) anti-HIV antibodies from uninfected human subjects vaccinated with an HIV vaccine, but are recognized by anti-HIV antibodies in human subjects infected with HIV, for example HIV-1, irrespective of vaccine status of the infected human subject. Accordingly, in particular instances the compositions, methods, kits, uses and devices of the invention may be for minimising the background signal in a diagnostic assay detecting anti-HIV antibodies for determining presence of an HIV infection, wherein the background signal may be caused by vaccine-generated anti-HIV antibodies. Therefore, the compositions, methods, kits, uses and devices of the invention may be for the detection of an HIV infection in a human subject. Additionally, the compositions, methods, kits, uses and devices of the invention may be for the detection of a breakthrough HIV infection in a human subject vaccinated with an HIV vaccine (i.e., the sensitivity and specificity of the immunoassay are not compromised by interference from vaccine-generated anti-HIV antibodies). On the other hand, by having limited reactivity with vaccine-generated anti-HIV antibodies, the compositions, methods, kits, uses and devices of the invention are particularly useful for minimising the rate of false positives in an in vitro diagnostic assay of a biological sample from a human subject not infected with HIV. The reduction in the rate of false positives is observed when using the compositions, methods, kits, uses and devices of the invention even if the subject has been vaccinated with an HIV vaccine. Minimising the occurrence of false positives in in vitro HIV diagnostic assays is of critical importance given the stigma that remains associated with a positive diagnosis of HIV. Accordingly, the compositions, methods, kits, uses and devices of the invention can be used for differentiating between vaccine-generated anti-HIV antibodies and anti-HIV antibodies generated in human subjects infected with HIV (e.g., anti-HIV antibodies generated by a breakthrough HIV infection in human subjects vaccinated with an HIV vaccine). Therefore, the compositions, methods, kits, uses and devices of the invention can be used for distinguishing between human subjects infected with HIV, for example HIV-1, and uninfected human subjects who have merely become seroconverted (i.e., have generated anti-HIV antibodies) due to the administration of an HIV vaccine (i.e., VISP). In other words, in some embodiments, the compositions, methods, kits, uses and devices of the invention can distinguish between anti-HIV antibodies generated due to an HIV infection in a human subject and anti-HIV antibodies generated in an uninfected human subject as a result of their vaccination with an HIV vaccine. Such assays will be critically important to assess the efficacy of vaccine clinical trials by accurately monitoring infections, while not 43

erroneously diagnosing uninfected participants of the trials as positive for HIV infection, the latter which has significant negative socio-economic implications for the individual. Therefore, given the surprising and advantageous properties of the polypeptide antigens provided herein and the compositions of the present invention, the methods of the invention may be performed on biological samples from human subjects who have not been or who have been vaccinated with an HIV vaccine, i.e., the methods are suitable for samples from any human subjects, irrespective of HIV vaccination status. In certain embodiments, the methods of the invention are performed on biological samples from human subjects who have been vaccinated with an HIV vaccine. Accordingly, the methods of the invention may be performed on biological samples containing at least one vaccine-generated anti-HIV antibody. Ideally, the methods of the invention do not detect (i.e., do not generate a signal significantly above background in an immunoassay, e.g., as described herein, in response to) vaccine-generated anti-HIV antibodies in the biological samples of human subjects who have been vaccinated with an HIV vaccine. Similarly, preferably, the compositions, kits and devices of the invention are used in diagnostic assays of biological samples from human subjects who have been vaccinated with a HIV vaccine. The human subjects may or may not be infected with HIV, for example HIV-1. In certain embodiments, the methods of the invention detect (e.g., via an immunoassay as described herein) an HIV infection in a human subject with a % sensitivity of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, preferably at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or ideally 100%. Ideally, the % sensitivity is maintained across one or more clades of HIV, for example HIV-1 clades B, A, complex, AG, C, F, AE, G, BF, D, BC, AB and/or B. The % sensitivity may be at least 85% before or at the moment p24 antigen becomes undetectable (e.g. signal below cut-off using an ELISA for p24 antigen), or within two weeks post initial diagnosis, i.e. in early stages of infection. The % sensitivity may be at least 95% within 9- 12 weeks post initial diagnosis. The % sensitivity may be at least 99% at least 12 weeks post initial diagnosis, and the sensitivity thus increases over time post infection. As noted above, time since first diagnosis may act as a suitable surrogate for time since first infection. The % specificity of the methods of the invention (e.g., as determined via an immunoassay as described herein) to determine the absence of HIV infection may be at least 95%, at least 96%, at least 97%, preferably at least 98%, at least 98%, at least 99%, or 100%. Typically, the % specificity (i.e., the true negative rate) is determined on a healthy uninfected control population. The impact of vaccination-induced seropositivity on % specificity may be determined by a comparison of the % specificity observed before and after a population of subjects has been vaccinated with an HIV vaccine. In particular 44

embodiments, the % specificity of the methods of the invention after vaccination is within less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5% of or identical to the % specificity before vaccination. Accordingly, the methods of the invention preferably do not detect anti-HIV-1 antibodies from an uninfected subject after the uninfected subject has been vaccinated with an HIV vaccine. The HIV infection can be HIV-1 and/or HIV-2, but is preferably HIV-1. The compositions, methods, kits, uses and devices of the invention may be for distinguishing between HIV-1 and HIV-2 infection in a human subject. The HIV vaccine can be any HIV vaccine generating anti-HIV antibodies. The HIV vaccine can be a complex vaccine comprising a number of viral antigens. As demonstrated herein, the vaccine may even comprise fragments of the polypeptide antigens described herein, for example p17 or IN. Ideally, the HIV vaccine does not comprise the endodomain of gp41 or a fragment thereof. Preferably, the HIV vaccine does not comprise Nef or a fragment thereof. In certain instances, the HIV vaccine is a vaccine that encodes and/or comprises HIV-1 antigens Gag, Pol, Env and/or gp140. In particular instances, the HIV vaccine comprises the Ad26.Mos4.HIV (encoding HIV mosaic Gag, Pol, and Env antigens having sequences set forth in SEQ ID NOs: 6, 7, 8, and 9) and aluminium phosphate-adjuvanted isolated recombinant Clade C gp140 (SEQ ID NO: 10) and Mosaic gp140 (SEQ ID NO: 11) vaccine, as described in WO2017102929A1, WO2020064621A1 and US2019231866A1, each of which is herein incorporated by reference in its entirety. Mos1.Env (SEQ ID NO: 6): MRVTGIRKNYQHLWRWGTMLLGILMICSAAGKLWVTVYYGVPVWKEATTTLFCASDAKAYD TEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLT PLCVTLNCTDDVRNVTNNATNTNSSWGEPMEKGEIKNCSFNITTSIRNKVQKQYALFYKLD VVPIDNDSNNTNYRLISCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNDKKFNGTGPC TNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSENFTNNAKTIMVQLNVSVEINCTRPN NNTRKSIHIGPGRAFYTAGDIIGDIRQAHCNISRANWNNTLRQIVEKLGKQFGNNKTIVFN HSSGGDPEIVMHSFNCGGEFFYCNSTKLFNSTWTWNNSTWNNTKRSNDTEEHITLPCRIKQ IINMWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGNDTSGTEIFRPGGGDMRDNWRSEL YKYKVVKIEPLGVAPTKAKRRVVQSEKSAVGIGAVFLGFLGAAGSTMGAASMTLTVQARLL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICT TTVPWNASWSNKSLDKIWNNMTWMEWEREINNYTSLIYTLIEESQNQQEKNEQELLELDKW ASLWNWFDISNWLW Mos2SEnv (SEQ ID NO: 7): MRVRGMLRNWQQWWIWSSLGFWMLMIYSVMGNLWVTVYYGVPVWKDAKTTLFCASDAKAYE KEVHNVWATHACVPTDPNPQEIVLGNVTENFNMWKNDMVDQMHEDIISLWDASLEPCVKLT PLCVTLNCRNVRNVSSNGTYNIIHNETYKEMKNCSFNATTVVEDRKQKVHALFYRLDIVPL DENNSSEKSSENSSEYYRLINCNTSAITQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNG 45

TGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNETVNITC TRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNLSRDGWNKTLQGVKKKLAEHFPNKTI KFAPHSGGDLEITTHTFNCRGEFFYCNTSNLFNESNIERNDSIITLPCRIKQIINMWQEVG RAIYAPPIAGNITCRSNITGLLLTRDGGSNNGVPNDTETFRPGGGDMRNNWRSELYKYKVV EVKPLGVAPTEAKRRVVEREKRAVGIGAVFLGILGAAGSTMGAASITLTVQARQLLSGIVQ QQSNLLRAIEAQQHMLQLTVWGIKQLQTRVLAIERYLQDQQLLGLWGCSGKLICTTAVPWN TSWSNKSQTDIWDNMTWMQWDKEIGNYTGEIYRLLEESQNQQEKNEKDLLALDSWNNLWNW FSISKWLWYIKIFIMIVGGLIGLRIIFAVLSIVNRVRQGY Mos1.Gag-Pol (SEQ ID NO: 8): MGARASVLSGGELDRWEKIRLRPGGKKKYRLKHIVWASRELERFAVNPGLLETSEGCRQIL GQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALEKIEEEQNKSKKKAQQAAADT GNSSQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQD LNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQ EQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFYKTLR AEQASQDVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVLAEA MSQVTNSATIMMQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCT ERQANFLGKIWPSNKGRPGNFLQNRPEPTAPPEESFRFGEETTTPSQKQEPIDKEMYPLAS LKSLFGNDPSSQMAPISPIETVPVKLKPGMDGPRVKQWPLTEEKIKALTAICEEMEKEGKI TKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTV LAVGDAYFSVPLDEGFRKYTAFTIPSTNNETPGIRYQYNVLPQGWKGSPAIFQCSMTRILE PFRAKNPEIVIYQYMAALYVGSDLEIGQHRAKIEELREHLLKWGFTTPDKKHQKEPPFLWM GYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWASQIYPGIKVRQLCKLLRGAKALTD IVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGHDQWTYQIYQEPFKNLKTG KYAKMRTAHTNDVKQLTEAVQKIAMESIVIWGKTPKFRLPIQKETWETWWTDYWQATWIPE WEFVNTPPLVKLWYQLEKDPIAGVETFYVAGAANRETKLGKAGYVTDRGRQKIVSLTETTN QKTALQAIYLALQDSGSEVNIVTASQYALGIIQAQPDKSESELVNQIIEQLIKKERVYLSW VPAHKGIGGNEQVDKLVSSGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEI VASCDQCQLKGEAMHGQVDCSPGIWQLACTHLEGKIILVAVHVASGYIEAEVIPAETGQET AYFILKLAGRWPVKVIHTANGSNFTSAAVKAACWWAGIQQEFGIPYNPQSQGVVASMNKEL KKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQIIK IQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKVKIIKDYGKQMAGADC VAGRQDED Mos2.Gag-Pol (SEQ ID NO: 9): MGARASILRGGKLDKWEKIRLRPGGKKHYMLKHLVWASRELERFALNPGLLETSEGCKQII KQLQPALQTGTEELRSLFNTVATLYCVHAEIEVRDTKEALDKIEEEQNKSQQKTQQAKEAD GKVSQNYPIVQNLQGQMVHQPISPRTLNAWVKVIEEKAFSPEVIPMFTALSEGATPQDLNT MLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPVAPGQMREPRGSDIAGTTSNLQEQI AWMTSNPPIPVGDIYKRWIILGLNKIVRMYSPTSILDIKQGPKEPFRDYVDRFFKTLRAEQ ATQDVKNWMTDTLLVQNANPDCKTILRALGPGATLEEMMTACQGVGGPSHKARVLAEAMSQ TNSTILMQRSNFKGSKRIVKCFNCGKEGHIARNCRAPRKKGCWKCGKEGHQMKDCTERQAN FLGKIWPSHKGRPGNFLQSRPEPTAPPAESFRFEETTPAPKQEPKDREPLTSLRSLFGSDP LSQMAPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPY NTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLAVGDAYFS VPLDEDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRKQNPDI VIYQYMAALYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKW TVQPIVLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVKQLCKLLRGTKALTEVVPLTEEAE LELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMRGAH TNDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPPL 46

VKLWYQLEKEPIVGAETFYVAGAANRETKLGKAGYVTDRGRQKVVSLTDTTNQKTALQAIH LALQDSGLEVNIVTASQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAHKGIGG NEQVDKLVSRGIRKVLFLDGIDKAQEEHEKYHSNWRAMASEFNLPPIVAKEIVASCDKCQL KGEAIHGQVDCSPGIWQLACTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAG RWPVKTIHTANGSNFTSATVKAACWWAGIKQEFGIPYNPQSQGVVASINKELKKIIGQVRD QAEHLKTAVQMAVFIHNFKRKGGIGEYSAGERIVDIIASDIQTKELQKQITKIQNFRVYYR DSRDPLWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDED Clade C gp140 protein (SEQ ID NO: 10): AENLWVGNMWVTVYYGVPVWTDAKTTLFCASDTKAYDREVHNVWATHACVPTDPNPQEIVL ENVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNATFKNNVTNDMNKE IRNCSFNTTTEIRDKKQQGYALFYRPDIVLLKENRNNSNNSEYILINCNASTITQACPKVN FDPIPIHYCAPAGYAILKCNNKTFSGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEKEI IIRSENLTDNVKTIIVHLNKSVEIVCTRPNNNTRKSMRIGPGQTFYATGDIIGDIRQAYCN ISGSKWNETLKRVKEKLQENYNNNKTIKFAPSSGGDLEITTHSFNCRGEFFYCNTTRLFNN NATEDETITLPCRIKQIINMWQGVGRAMYAPPIAGNITCKSNITGLLLVRDGGEDNKTEEI FRPGGGNMKDNWRSELYKYKVIELKPLGIAPTGAKERVVEREERAVGIGAVFLGFLGAAGS TMGAASLTLTVQARQLLSSIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQTRVLAIERYLKD QQLLGIWGCSGKLICTTNVPWNSSWSNKSQTDIWNNMTWMEWDREISNYTDTIYRLLEDSQ TQQEKNEKDLLALDSWKNLWSWFDISNWLWYIKSRIEGRGSGGYIPEAPRDGQAYVRKDGE WVLLSTFL Mosaic gp140 protein (SEQ ID NO: 11): AGKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVTE NFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTDDVRNVTNNATNTNSSWGEP MEKGEIKNCSFNITTSIRNKVQKQYALFYKLDVVPIDNDSNNTNYRLISCNTSVITQACPK VSFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEE EVVIRSENFTNNAKTIMVQLNVSVEINCTRPNNNTRKSIHIGPGRAFYTAGDIIGDIRQAH CNISRANWNNTLRQIVEKLGKQFGNNKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTKLF NSTWTWNNSTWNNTKRSNDTEEHITLPCRIKQIINMWQEVGKAMYAPPIRGQIRCSSNITG LLLTRDGGNDTSGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKERVVQREERA VGIGAVFLGFLGAAGSTMGAASMTLTVQARLLLSGIVQQQNNLLRAIEAQQHLLQLTVWGI KQLQARVLAVERYLKDQQLLGIWGCSGKLICTTTVPWNASWSNKSLDKIWNNMTWMEWERE INNYTSLIYTLIEESQNQQEKNEQELLELDKWASLWNWFDISNWLWYIKSRIEGRGSGGYI PEAPRDGQAYVRKDGEWVLLSTFL General The term “comprising” encompasses “including” as well as “consisting” e.g., a composition “comprising” X may consist exclusively of X or may include something additional e.g., X + Y. Accordingly, embodiments described in terms of the phrase “comprising” (or its equivalents) also provide as embodiments those independently described in terms of “consisting of”. The term “about” in relation to a numerical value x means, for example, x±10%. 47

The word “substantially” does not exclude “completely” e.g., a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention. Where the invention provides a method involving multiple sequential steps, the steps are carried out in the indicated order, i.e., in numerical or alphabetical order. However, the skilled person will understand that the order of steps may be altered while still achieving useful results. BRIEF DESCRIPTION OF DRAWINGS Fig. 1 shows the detection of anti-HIV antibodies using individual polypeptide antigens in HIV infected subjects and subjects before and 12 weeks after vaccination with an HIV vaccine. The polypeptide antigen is derived from: (A) gp41 endodomain; (B) IN; (C) p17 and (D) Nef. Fig. 2 shows a schematic representation of an exemplary indirect immunoassay for use in the methods, kits and devices and with the compositions of the invention. Fig.3 shows a schematic representation of the theoretical sensitivity of the different combinations of individual indirect ELISAs based on gp41 endodomain, IN, p17, and Nef. Fig.4 shows a schematic representation of an exemplary double antigen bridging immunoassay for use in the methods, kits and devices and with the compositions of the invention. Fig.5 shows the performance of a combo double-antigen bridging ELISA with the combination of four antigens (IN, Nef, gp41 endodomain, and p17) (indicated in Figure as ‘DHIVAx’) in seroconversion panels, including p24 antigen data and 2 reference tests (Enzygnost Anti HIV1/2 Plus, and Vironostika HIV Ag/Ab); for details see example 4). Fig.6 shows the assessment of a combo double-antigen bridging ELISA in longitudinal follow- up samples collected from individuals with known HIV infection. MODES FOR CARRYING OUT THE INVENTION Example 1 Generation of recombinantly produced antigens His₆-Gp41e, MBP-Gp41e, His₆-Nef, His₆-p17 and His6-p31, and their respective biotinylated versions a) Generation of His6-Gp41e 48

The sequence coding for gp41 endodomain (SEQ ID NO:1) was synthesized, including a His₆-tag and a cleavage site for TEV protease between the tag and the protein of interest to allow the subsequent elimination of the tag. The synthesized gene coding for His₆-gp41e antigen was cloned into IPTG inducible prokaryotic expression vector pT7 using NdeI and XhoI restriction sites, generating pT7-His₆-HIVgp41e. An E. coli BL21 (DE3) pLysS strain was transformed with the pT7-His₆-HIVgp41e expression vector. Next, the transformed bacterial strain was amplified in LB medium in presence of ampicillin and chloramphenicol, at 37°C overnight. The bacterial pellet was then recovered by centrifugation and resuspended in LB/glycerol 50% before to be aliquoted and stored at -70°C. These aliquots correspond to the working cell bank (WCB). WCB was grown in 1 liter of LB medium in presence of ampicillin and chloramphenicol, at 37°C and cells were induced with IPTG 1 mM at 37°C for 4h. Bacteria were harvested by centrifugation and bacterial pellets were stored at -70°C. The pellets were resuspended in phosphate buffer with 0.5% Triton X-100 and cell lysis was performed by sonication. The obtained lysate was centrifuged to obtain the soluble fraction and further purified on a Ni++ Sepharose column. After column loading and two intermediate washing steps, the elution of His6- Gp41e was performed with an imidazole gradient from 20 mM to 500 mM.0.1% of Triton X-100 was present all along the purification process in wash and elution buffers to maintain the solubility of His₆-gp41e. After SDS-PAGE analysis, selected elution fractions were pooled and dialyzed against phosphate buffer 20mM pH7.4, NaCl 500mM, glycerol 10% and Triton X-100 to eliminate imidazole and were stored frozen at -20°C. The total amount of His₆-gp41e recovered after purification was estimated around 1.1mg by BCA in a total volume of ~17ml (67.32 µg/ml). SDS- PAGE analysis was performed to evaluate the purity of affinity purified His₆-gp41e. Purity of His6-gp41e was evaluated by densitometry to 79%. b) Generation of MBP-Gp41e The sequence coding for gp41 endodomain (SEQ ID NO:1) was synthesized, including an MBP- tag and a cleavage site for TEV protease between the tag and the protein of interest to allow the subsequent elimination of the tag. The synthesized gene coding for MBP-gp41e antigen was cloned into IPTG inducible prokaryotic expression vector pET17b using NdeI and XhoI restriction sites, generating pET17b-MBP- HIVgp41e. An E. coli BL21 (DE3) strain was transformed with the pET17b-MBP-HIVgp41e expression vector. Next, the transformed bacterial strain was amplified in LB medium in presence of ampicillin, at 37°C overnight. The bacterial pellet was then recovered by centrifugation and resuspended in LB/glycerol 50% before to be aliquoted and stored at -70°C. These aliquots correspond to the working cell bank (WCB). 49

WCB was grown in 1 liter of LB medium in presence of ampicillin and chloramphenicol, at 37°C and cells were induced with IPTG 1 mM at 25°C overnight. Bacteria were harvested by centrifugation and bacterial pellets were stored at -70°C. The pellets were resuspended in phosphate buffer with 200 mM NaCl, and cell lysis was performed by sonication. The obtained lysate was centrifuged to obtain the soluble fraction and further purified on a Dextrin sepharose column. After column loading and two intermediate washing steps (20 mM Tris pH 7.4, 200 mM NaCl, 1mM EDTA), the elution of MBP-Gp41e was performed with 10 mM maltose. An additional 40 mM maltose step was performed to ensure no protein of interest remained on the column. After purification, selected elution fractions were pooled and dialyzed against phosphate buffer 50mM pH7.4, NaCl 200mM to eliminate maltose and were stored frozen at -20°C. The total protein concentration was estimated with UV (A₂₈₀) measurement to 0.93 mg/ml (6.5 ml). SDS- PAGE analysis was performed to evaluate the purity of affinity purified MBP-gp41e. Purity of MBP-gp41e was evaluated by densitometry to 93.9%. c) Generation of His6-p17 The sequence coding for p17 (SEQ ID NO:2) was synthesized, including a His₆-tag and a cleavage site for enterokinase between the tag and the protein of interest to allow the subsequent elimination of the tag. The synthesized gene coding for His₆-p17 antigen was cloned into IPTG inducible prokaryotic expression vector pET17b using NdeI and XhoI restriction sites, generating pET17b-His₆-p17. An E. coli BL21 (DE3) strain was transformed with the pET17b-His₆-p17 expression vector. Next, the transformed bacterial strain was amplified in LB medium in presence of ampicillin, at 37°C overnight. The bacterial pellet was then recovered by centrifugation and resuspended in LB/glycerol 50% before to be aliquoted and stored at -70°C. These aliquots correspond to the working cell bank (WCB). WCB was grown in 1 liter of LB medium in presence of ampicillin, at 37°C and cells were induced with IPTG 1 mM at 37°C for 4h. Bacteria were harvested by centrifugation and bacterial pellets were stored at -70°C. The pellets were resuspended in phosphate buffer with 700 mM trehalose and cell lysis was performed by sonication. The obtained lysate was centrifuged to obtain the soluble fraction and further purified on a Ni++ Sepharose column. After column loading and two intermediate washing steps (50 mM phosphate pH 8.2, 10 mM imidazole, trehalose 700 mM), the elution of His6-p17 was performed with an imidazole gradient from 40 mM to 250 mM. After SDS-PAGE analysis, selected elution fractions were pooled and dialyzed against phosphate buffer 50mM pH8.2 and trehalose 700 mM to eliminate imidazole and were stored frozen at -20°C. The total protein concentration was estimated with UV (A₂₈₀) measurement to 0.69 mg/ml (32 ml). SDS-PAGE analysis was performed to evaluate the purity of affinity purified His₆-p17. Purity of His₆-p17 was evaluated by densitometry to 76.5%. 50

d) Generation of His6-p31 The sequence coding for p31 (SEQ ID NO:3) was synthesized, including a His₆-tag and a cleavage site for enterokinase between the tag and the protein of interest to allow the subsequent elimination of the tag. The synthesized gene coding for His₆-p31 antigen was cloned into IPTG inducible prokaryotic expression vector pET17b using NdeI and XhoI restriction sites, generating pET17b-His₆-p31. An E. coli BL21 (DE3) strain was transformed with the pET17b-His₆-p31 expression vector. Next, the transformed bacterial strain was amplified in LB medium in presence of ampicillin, at 37°C overnight. The bacterial pellet was then recovered by centrifugation and resuspended in LB/glycerol 50% before to be aliquoted and stored at -70°C. These aliquots correspond to the working cell bank (WCB). WCB was grown in 1 liter of LB medium in presence of ampicillin, at 37°C and cells were induced with IPTG 1 mM at 37°C for 4h. Bacteria were harvested by centrifugation and bacterial pellets were stored at -70°C. The pellets were resuspended in phosphate buffer with 8M urea and 1mM DDT and cell lysis was performed by sonication. The obtained lysate was centrifuged to obtain the soluble fraction and further purified on a Ni++ Sepharose column. After column loading and two intermediate washing steps (50 mM phosphate pH 8, 10 mM/25 mM imidazole, Urea 8M, 1mM DDT), the elution of His6-p31 was performed with an imidazole gradient from 25 mM to 250 mM. After SDS-PAGE analysis, selected elution fractions were pooled and dialyzed against phosphate buffer 50mM pH8, Urea 8M, 1mM DDT to eliminate imidazole and were stored frozen at -20°C. The total protein concentration was estimated with UV (A₂₈₀) measurement to 1.46 mg/ml (68 ml). For renaturation of His₆-p31, protein was diluted to 0.1 mg/mL in 50 mM phosphate pH 8, Urea 8M, 1mM DDT and dialyzed against phosphate buffer 50mM pH7.2, 0.5M arginine, 0.05% sarkosyl, 2mM DTT, 0.1 mM NaCl and was stored frozen at -20°C. The total protein concentration was estimated with UV (A₂₈₀) measurement to 0.151 mg/ml (68 ml). e) Generation of His6-Nef The sequence coding for Nef (SEQ ID NO:4) was synthesized, including a His₆-tag and a cleavage site for enterokinase between the tag and the protein of interest to allow the subsequent elimination of the tag. The synthesized gene coding for His6-Nef antigen was cloned into IPTG inducible prokaryotic expression vector pET17b using NdeI and XhoI restriction sites, generating pET17b-His₆-Nef. An E. coli BL21 (DE3) strain was transformed with the pET17b-His₆-Nef expression vector. Next, the transformed bacterial strain was amplified in LB medium in presence of ampicillin, at 37°C overnight. The bacterial pellet was then recovered by centrifugation and resuspended in 51

LB/glycerol 50% before to be aliquoted and stored at -70°C. These aliquots correspond to the working cell bank (WCB). WCB was grown in 1 liter of LB medium in presence of ampicillin, at 37°C and cells were induced with IPTG 1 mM at 25°C overnight. Bacteria were harvested by centrifugation and bacterial pellets were stored at -70°C. The pellets were resuspended in phosphate buffer, and cell lysis was performed by sonication. The obtained lysate was centrifuged to obtain the soluble fraction and further purified on a Ni++ Sepharose column. After column loading and two intermediate washing steps (50 mM phosphate pH 7.4, 10 mM imidazole, 200mM NaCl), the elution of His₆-p31 was performed with an imidazole gradient from 40 mM to 250 mM. After SDS-PAGE analysis, selected elution fractions were pooled in 2 different pools and dialyzed against Phosphate buffer 50mM pH7.4 and NaCl 250 mM to eliminate imidazole and were stored frozen at -20°C. The total protein concentration was estimated with UV (A₂₈₀) measurement to 0.61 mg/ml (68 ml) and 0.88 mg/mL (35.5 mL) for pool 1 and pool 2, respectively. SDS-PAGE analysis was performed to evaluate the purity of affinity purified His₆-Nef. Purity of His₆-Nef was evaluated by densitometry to 78.1% and 90.3%, for pool 1 and pool 2, respectively. f) Generation of biotinylated proteins The water soluble EZ-Link Sulfo-NHS-LC-Biotin (Pierce) was used to biotinylate the protein. The biotin reagent allows biotinylation on primary amine (N-terminus and Lysine lateral chain) with a mid-length spacer (22.4 Å). In order to reach the amount of biotinylated protein required, the following amounts of proteins were used: - MBP-gp41e (0,93 mg/ml) ^ 2,5ml ^ 2,32 mg - His₆-p17 (0,69 mg/ml) ^ 4 ml ^ 2,76 mg - His₆-Nef (0,88 mg/ml) ^ 3,5 ml ^ 3,08 mg Biotin reagents were dissolved in pure water at 10 mM and a 25 times molar excess were added to all protein samples. After 2 hours incubation at 4°C, protein samples were dialyzed 2 times against their conservation buffer to remove free biotin: - His₆-p17: Phosphate 50 mM pH 8,2; Trehalose 700 mM - MBP-gp41e and His₆-Nef: Phosphate 50 mM pH 7,4; NaCl 200 mM Sample concentrations at the end of the process were estimated by with UV (A₂₈₀) measurement.

In order to validate biotinylation and to quantify the number of biotins per protein the “HABA- avidin assay” was used. For the 3 biotinylated proteins, the following results were obtained: 52

Example 2 Identification of polypeptide antigens In order to identify polypeptide antigens that (1) are highly immunogenic in infected individuals; (2) are not recognized by vaccine-generated antibodies; (3) are recognized by antibodies from HIV infected individuals at early times post-infection; (4) maintain detection of antibody response over all stages of the disease and (5) enable detection of antibodies against all clades and subtypes, a set of viral proteins of HIV-1 were evaluated individually for immunoreactivity in a set of serum/plasma samples from 10 healthy uninfected individuals, 10 HIV-1 infected individuals and 10 individuals that participated in HIV vaccine trials (HVTN 117 [also referred to as HPX2004; HIV vaccination with 3 Ad26 vectors together encoding HIV antigenic mosaic polypeptides derived from Gag, Pol, and Env (SEQ ID NOs: 6, 8, and 9) or with 4 Ad26 vectors together encoding HIV antigenic polypeptides derived from Gag, Pol, and Env (SEQ ID NOs: 6, 7, 8, and 9), in both cases combined with vaccination with isolated recombinant HIV clade C gp140 protein (SEQ ID NO: 10)]; and HVTN 118 [also referred to as HPX2003; HIV vaccination with 4 Ad26 vectors together encoding HIV antigenic mosaic polypeptides derived from Gag, Pol, and Env (SEQ ID NOs: 6, 7, 8, and 9) combined with either isolated recombinant HIV clade C gp140 protein (SEQ ID NO: 10) or with bivalent isolated recombinant HIV gp140 proteins, i.e., a mixture of clade C gp140 protein (SEQ ID NO: 10) and mosaic gp140 protein (SEQ ID NO: 11)]), collected 12 weeks post-last vaccination. The proteins included in the immunoreactivity assay were HIV-1 Gag- p55, Gag-p17, Gag-p15, RT, IN, PR, Nef and gp41 endodomain (Table 1). The latter was produced as a recombinant protein covering amino acids 706-856 of the HXB2 Env sequence (SEQ ID NO:1), prepared as described in example 1. Table 1. Proteins used for evaluation of immune response in healthy uninfected individuals, HIV-1 infected individuals and individuals that participated in HIV vaccine trial.

53

IgG antibody levels against the different antigens were assessed using indirect enzyme-linked immunosorbent assay (ELISA, see an exemplary schematic in Fig. 2). These ELISAs were performed as follows. Flat bottomed polystyrene plates (Maxisorp Immuno Plate, Nunc, Denmark) were coated with the individual proteins, diluted to the concentration indicated in Table 1 in PBS (except for gp41 endodomain, which was diluted in 0.1M carbonate buffer) and incubated overnight at 4°C. Next, after washing with PBS-T (PBS with 0.05% Tween-20) the plate was blocked with block buffer (Blocker Casein in PBS, Thermo Fisher Scientific, Breda, the Netherlands) for 1 hour at room temperature. Serum samples, diluted 100-fold in sample diluent (block buffer + 5% Triton X-100), were added and incubated for 1 hour at room temperature. Subsequently, plates were washed 5 times with PBS-T. Then, the secondary antibody solution was added to each well. The solution contained an affinity purified Donkey anti-human IgG (H+L) peroxidase conjugate (Jackson Immuno Research Europe Ltd., Newmarket, UK) diluted 1:10,000 in block buffer. The reaction mixture was incubated at room temperature for 30 minutes. At the end of the incubation period, the plates were rinsed 5 times with washing buffer and treated with 100 µL 1-Step™ Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific, Breda, the Netherlands). After 10 minutes of incubation the colorimetric reaction was stopped with 100 µL 1N HCl. The plate was then read by a microplate reader at a wavelength (λ) of 450 nm. For each antigen, a cutoff for positivity was defined as the absorbance that resulted in all healthy control samples to be negative (i.e., 100% specificity). Table 1 shows the outcome of this evaluation. IN, p17, PR, Nef and the Gp41 endodomain were of particular interest because these proteins were found to have no immunoreactivity in vaccinated individuals but were immunoreactive in at least half of the HIV-1 infected individuals. The gp41 endodomain, IN, p17 and Nef polypeptide antigens were taken forward for further evaluation. 54

Example 3 Performance evaluation of the individual antigens using indirect ELISA The individual ELISAs for the selected antigens (IN, p17, Nef and gp41 endodomain) were further optimized in order to lower non-specific binding of antibodies from uninfected individuals, hence lowering the cutoff for positivity. For each polypeptide antigen, the respective supplier and coating concentration is listed in Table 2 (the ones that were not obtained from a commercial supplier (indicated by “N.A.” in the table), were prepared as described in example 1). The other parameters of the ELISA’s were as described above for the initial evaluation. These optimized assays were used to determine the immunoreactivity in a set of serum/plasma samples from 600 treatment naïve HIV-1 infected individuals and 109 healthy uninfected individuals that participated in HIV vaccine trials (HVTN 117 and HVTN 118), with samples collected pre-vaccination and 12 weeks post-last vaccination. This analysis indicated that the individual polypeptide antigens had a sensitivity to detect HIV infection of 93.5%, 68.7%, 55.2% and 76.2%, for IN, gp41 endodomain, Gag p17 and Nef, respectively (Fig. 1, Table 2). The specificity in healthy uninfected individuals both before and after vaccination was > 98.0% for all polypeptide antigens, except for Nef for which a specificity > 96.0% was observed. Of the 109 healthy uninfected individuals, only 7 (6.4%) were found to be positive for reactivity to any of the polypeptide antigens before vaccination and this remained similar after vaccination, where 8 (7.3%) were found to be positive for any of the polypeptides, indicating that no vaccine-induced seroreactivity is present. Although no individual polypeptide antigen provided adequate sensitivity on its own, combinations of multiple antigens resulted in an increased sensitivity (Fig.3). The combined sensitivity of IN, gp41 endodomain, Gag p17, and Nef, was determined to be 98.5%. Importantly, these polypeptide antigens were similarly recognized by samples from seropositive individuals from different geographical origins and infected with different clades of HIV-1 (Table 3). Grouping of the samples based on the time since first diagnosis indicated that most of the samples that were found to be negative on all three polypeptide antigens were from recently diagnosed individuals (< 12 weeks since first diagnosis, Table 4). Sensitivity in the group of samples > 12 weeks since first diagnosis was 100.0%. Within the group of IgG negative individuals, 33.3 % (i.e., 3 out of 9) were found to be positive for p24, indicating that these samples were collected at or before seroconversion and therefore remained negative on the IgG assays. The overall sensitivity, including p24 antigen detection was found to be 99.0%. 55

Table 2. Proteins used for performance evaluation of the individual polypeptide antigens.

Table 3. Combined sensitivity for the four markers (gp41 endodomain, Gag-p17, IN and Nef), per clade.

56

Table 4. Combined sensitivity for the four markers (gp41 endodomain, Gag-p17, IN, and Nef), grouped based on time since diagnosis.

Strikingly, the composition comprising polypeptide antigens derived from the endodomain of gp41, p17, IN, and Nef, showed remarkable 98.5% sensitivity in an immunoassay to the majority of clades of HIV-1 and through all stages of the infection. In addition, the polypeptide antigens of the composition are not reactive to vaccine-generated anti-HIV antibodies in healthy vaccinated subjects. Therefore, this composition is particularly useful for detecting HIV infection in vaccinated subjects without interference from vaccine-generated anti-HIV antibodies (i.e., the composition can accurately detect breakthrough infections). Additionally, in light of the limited reactivity to vaccine-generated anti-HIV antibodies (i.e., not more than background), the composition will be useful in diagnostic assays, for example those used in parallel with a vaccination program, because it will minimize the number of false positives detected in vaccinated individuals. This will maximize engagement with the vaccine trials and vaccine use after a vaccine is approved and employed in a population, without concerns over incorrect diagnoses of HIV infection. Therefore, the composition provides a way of accurately and reliable distinguishing between anti-HIV antibodies generated by an HIV infection and anti-HIV antibodies generated in response to an HIV vaccine (i.e., VISP). These observations are particularly surprising for p17 and IN because both proteins are encoded by the vaccine used in the human subjects studied in this example and so these proteins were expected to be immunogenic. 57

Example 4 Performance evaluation of a composition of polypeptide antigens using double-antigen bridging ELISA In order to study the performance of a composition of the preferred polypeptide antigens, the four selected antigens (IN, Nef, gp41 endodomain, and p17) were studied in a single assay, a combo double-antigen bridging ELISA (see an exemplary schematic in Fig. 4). This also offers the additional advantage of being isotype independent, so also detecting the IgM response which typically precedes the IgG response as determined by the indirect ELISA described above. These ELISAs were performed as follows. Flat bottomed polystyrene plates (Maxisorp Immuno Plate, Nunc, Denmark) were coated with a mixture of the proteins (proteins prepared as described in example 1, or obtained from commercial supplier as indicated in Table 5), diluted to the concentration indicated in Table 5 in 0.1M carbonate buffer and incubated overnight at 4°C. Next, after washing with PBS-T (PBS with 0.05% Tween-20) the plate was blocked with block buffer (Blocker Casein in PBS, Thermo Fisher Scientific, Breda, the Netherlands) for 1 hour at room temperature.100 µL of serum samples, diluted 5-fold in sample diluent (block buffer + 0.5% Triton X-100), were added and incubated for 30 minutes at room temperature. Subsequently, 50 µL of the biotinylated detection proteins, diluted to the concentration indicated in Table 5 in block buffer, were added and plates were further incubated for 30 minutes at room temperature. Then, plates were washed 3 times with PBS-T and the streptavidin-HRP solution was added to each well. The solution contained a peroxidase-conjugated streptavidin (Jackson Immuno Research Europe Ltd., Newmarket, UK) diluted 1:20,000 in block buffer. The reaction mixture was incubated at room temperature for 30 minutes. At the end of the incubation period, the plates were rinsed 3 times with washing buffer and treated with 100 µL 1-Step™ Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific, Breda, the Netherlands). After 10 minutes of incubation the colorimetric reaction was stopped with 100 µL 1N HCl. The plate was then read by a microplate reader at a wavelength (λ) of 450 nm. A cutoff for positivity was defined as the absorbance that resulted in > 98.0% of all healthy control samples to be negative (i.e., >98.0% specificity). Table 5. Proteins used for performance evaluation of the combo double-antigen bridging ELISA.

58

Using this single combined assay, a specificity of 98.1% and sensitivity of 98.5% was observed and no vaccine-induced seroreactivity was apparent (specificity of 97.1% 12 weeks post- vaccination). The combo double-antigen bridging ELISA similarly detected antibodies in samples from seropositive individuals from different geographical origins and infected with different clades of HIV-1 (Table 6). Grouping of the samples based on the time since first diagnosis indicated that most of the samples that were found to be negative in the combo double-antigen bridging ELISA were from recently diagnosed individuals (< 12 weeks since first diagnosis, Table 7). Sensitivity in the group of samples > 12 weeks since first diagnosis was 99.7%. Within the group of IgG negative individuals, 81.8% (i.e. 9 out of 11) were found to be positive for p24, indicating that these samples were collected at or before seroconversion and therefore remained negative on the IgG assays. The overall sensitivity, including p24 antigen detection was found to be 99.7%. 59

Table 6. Sensitivity of the combo double-antigen bridging ELISA, per clade.

Table 7. Sensitivity of the combo double-antigen bridging ELISA, grouped based on time since diagnosis.

To determine how soon post-infection HIV-specific antibodies are detected with the double- antigen bridging ELISA, several well-characterized seroconversion panels were obtained from LGC SeraCare. As shown in Fig. 5, the assay reacted positively as soon as or before the p24 antigen became undetectable for the 0600-0271, PRB-926, PRB-968, and PRB-952 panels. For the PRB-969 and PRB-930 panels, the p24 antigen remained detectable at the last day of sampling, while the ELISA remained negative. For the PRB-965 panel, both p24 antigen and 60

our ELISA remained negative across all timepoints, while the Enzygnost Anti HIV1/2 Plus and Vironostika HIV Ag/Ab assays were positive on Days 12, 14, and 21, indicating that in some individuals there might be a delay in detection of infection with the double-antigen bridging ELISA, especially when p24 antigen remains negative. Assessment of the response dynamics in the double-antigen bridging ELISA was performed using a longitudinal sample series from 18 patients with documented seroconversion (Fig.6). In most patients, the signal increased strongly over time and all samples, except 1, that were still negative in the ELISA were found to be positive for p24 antigen. The data in this example demonstrate that the composition comprising the polypeptide antigens of gp41 endodomain (e.g., SEQ ID NO: 1), Gag p17 (e.g., SEQ ID NO: 2), HIV IN (e.g., SEQ ID NO: 3), and HIV Nef (SEQ ID NO: 4) can be used in a single combined immunoassay for the specific and sensitive detection of HIV-1 infection. Additionally, this composition failed to generate a signal significantly above background in vaccinated subjects not infected with HIV-1 (i.e., minimized the occurrence of false positives). The assay is sensitive across clades and across timepoints after infection, and sensitivity at early timepoints can be further enhanced by adding p24 antigen detection (i.e. as in 4th generation HIV assays). Example 5 Performance evaluation of a composition of polypeptide antigens using the double-antigen bridging format in the Abbott ARCHITECT^® instrument Three selected antigens (IN, MBP-Gp41e, and Gag p17) were implemented on a chemiluminescent microparticle immunoassay (CMIA) using the Abbott ARCHITECT® instrument in a one-step double-antigen bridging format. Based on the individual indirect ELISA data a combined sensitivity of 96.3% and a specificity of 96.3% was calculated (see example 3). The antigens (MBP-Gp41e (see example 1), IN (AHIV-108, available from RPC), and Gag p17 (AHIV-205, available from RPC)) were coated on the beads (Dynabeads MyOne Tosylactivated) as follows. For each antigen, beads were coated at a concentration of 19.6 µg protein / mg beads by adding 98 µg protein, diluted in a total volume of 150 µL Coat Buffer (100 mM borate, pH 9.5) to 5 mg beads and 100 µL Coupling Buffer (100 mM borate + 3 M Ammonium Sulfate, pH 9.5). This mixture was incubated for 12-18 hours at 37°C on a roller bank. The beads were then blocked with 10 mM PBS + 2% Bovine Gamma Globulin (BGG), 61

pH 7.4 for 12-18 hours at 37°C on a roller bank. Next, the beads were washed 3 times for 2 hours at room temperature with 10 mM PBS + 0.05% bovine serum albumin (BSA), pH 7.4 and stored in MP storage buffer (13.6% sucrose in 50 mM Tris + 0.1 M NaCl, pH 8.0) at 2- 8°C. The three sets of beads were mixed by diluting each of them in MP assay buffer (13.6% sucrose in 50 mM Tris, 0.1 M NaCl, 1% Casein ATC, pH 8.0) to a final concentration of 0.083 mg beads/mL, for all three antigens. The biotinylated antigens (Gp41-1-biotin (SEQ ID NO: 12, with a C-terminal biotin tag, Pepperprint), IN-biotin (AHIB-b-108, available from RPC), biotinylated His₆-P17 (see example 1) were mixed by diluting each of them in sample diluent (10 mM PBS, 3% Casein ATC, 1% ProClin-300, 1% Blockmaster CE500, 1.5% Triton X-100) to a final concentration of 0.33 µg/mL, 0.33 µg/mL and 1 µg/mL, respectively. Gp41-1 (SEQ ID NO: 12): LIAARIVELLGHSSLKGLRRGWEALKYLWNLLQYWGQELKNSAISL The beads and biotinylated antigen mixture described above were used in an assay for detection of HIV-1 antibody in a test sample, as follows. Each sample was diluted in a vial by adding 50 µL of the serum sample to 150 µL to the sample diluent (10 mM PBS, 3% Casein ATC, 1% ProClin-300, 1% Blockmaster CE500, 1.5% Triton X-100). Then, these samples were placed in the Abbott ARCHITECT® instrument where the test protocol was as follows. In the first step, 50 µL of the beads mixture described above and 100 µL of the diluted sample was added to 50 µL of biotinylated antigen mixture. These vials were incubated for 22.5 minutes. Following incubation, the beads were washed 3 times with Abbott ARCHITECT®wash buffer. In the second step, 50 µL of streptavidin conjugated to acridinium (1 µg/mL in conjugate buffer (10 mM PBS, 3% Casein ATC, 1% ProClin-300, 1% Blockmaster CE500, 1.5% Triton X-100)) was added to the beads and incubated for 7.5 minutes and beads were wash another 3 times with Abbott ARCHITECT® wash buffer. Subsequently activation and luminescence readout was performed by the Abbott ARCHITECT® instrument. Assessment of the performance of this prototype assay showed a sensitivity of 95.3% which further increased to 97.2% when p24 antigen detection was included to the dataset. Specificity was found to be 96.3% in a healthy control population pre-vaccination and 97.2% in the same population post- vaccination. Of interest, when using the currently available 4th generation ARCHITECT® HIV 62 Ag/Ab Combo assay, this same vaccinated population was misclassified as HIV infected in 83.5% of the cases. Therefore, the composition comprising the polypeptide antigens of gp41 endodomain (e.g., SEQ ID NO: 1), Gag p17 (e.g., SEQ ID NO: 2) and HIV IN (e.g., SEQ ID NO: 3) can be incorporated into an established assay platform and provides impressive sensitivity and specificity. Critically, the particular composition achieved 97% specificity even post-vaccination, something which the current 4th generation combination assays fail to achieve on the same platform. On the basis of the observations in examples 3 and 4, without wishing to be bound by theory, the inclusion of a polypeptide antigen derived from Nef (e.g. SEQ ID NO: 4) as an additional polypeptide antigen would be expected to further improve the sensitivity of the immunoassay in this format. Example 6 Performance evaluation of a composition of polypeptide antigens using the double-antigen bridging ELISA in a lateral flow assay (LFA) format Using a similar double-antigen bridging format, the four selected antigens (IN, gp41 endodomain, Gag p17, and Nef) were implemented on a lateral flow assay (LFA). In a small set of HIV infected individuals, healthy controls and HIV vaccinated individuals, excellent discrimination of HIV positivity was achieved. Therefore, the composition comprising the polypeptide antigens of gp41 endodomain (e.g., SEQ ID NO: 1), Gag p17 (e.g., SEQ ID NO: 2), HIV IN (e.g., SEQ ID NO: 3), and HIV Nef (e.g., SEQ ID NO: 4) can be incorporated into the established lateral flow assay format while retaining its excellent diagnostic capacity. Lateral flow assay formats are well known for their ease of use and cost-effectiveness. It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention. 63

REFERENCES 1. UNAIDS Data 2020. Geneva, Switzerland: UNAIDS, 2020. 2. Fiebig EW, Wright DJ, Rawal BD, Garrett PE, Schumacher RT, Peddada L, et al. Aids. 2003;17(13):1871-9. 3. Dopel SH, Schubert U, Grunow R, Pas P, Ronspeck W, Pauli G, et al. Eur J Clin Chem Clin Biochem.1991;29(5):331-7. 4. Manocha M, Chitralekha KT, Thakar M, Shashikiran D, Paranjape RS, Rao DN. Immunology letters.2003;85(3):275-8. 5. Alcaro MC, Peroni E, Rovero P, Papini AM. Curr Protein Pept Sci.2003;4(4):285-90. 6. Beristain CN, Rojkin LF, Lorenzo LE. J Clin Lab Anal.1995;9(6):347-50. 7. Alexander TS. Clinical and vaccine immunology : CVI.2016;23(4):249-53. Epub 2016/03/05. doi: 10.1128/CVI.00053-16. 8. Chappel RJ, Wilson KM, Dax EM. Future microbiology.2009;4(8):963-82. 9. Ly TD, Martin L, Daghfal D, Sandridge A, West D, Bristow R, et al. Journal of clinical microbiology.2001;39(9):3122-8. 10. Ng'uni T, Chasara C, Ndhlovu ZM. Front Immunol. 2020;11:590780. 11. Ackers ML, Parekh B, Evans TG, Berman P, Phillips S, Allen M, et al. J Infect Dis. 2003;187(6):879-86. 12. Pitisuttithum P, Nitayaphan S, Thongcharoen P, Khamboonruang C, Kim J, de Souza M, et al. J Infect Dis.2003;188(2):219-27. 13. Chuenchitra T, Wasi C, Louisirirojchanakul S, Nitayaphan S, Sutthent R, Cox JH, et al. AIDS research and human retroviruses.2003;19(4):293-305. 14. Schwartz DH, Mazumdar A, Winston S, Harkonen S. Clinical and diagnostic laboratory immunology.1995;2(3):268-71. 15. Khurana S, Needham J, Park S, Mathieson B, Busch MP, Nemo G, et al. Journal Acquir Immune Defic Syndr.2006;43(3):304-12. 16. Khurana S, Needham J, Mathieson B, Rodriguez-Chavez IR, Catanzaro AT, Bailer RT, et al. J Virol.2006;80(5):2092-9. 17. Khurana S, Norris PJ, Busch MP, Haynes BF, Park S, Sasono P, et al. J. Clin. Microbiol. 2010;48(1):281-5. 18. Oganezova K, Fontana-Martinez EJ, Gothing JA, Pandit A, Kwara E, Yanosick K, et al. Open Forum Infect Dis.2021;8(1):ofaa606. 19. O'Connell RJ, Merritt TM, Malia JA, VanCott TC, Dolan MJ, Zahwa H, et al. 2003;41(5):2153-5. 20. Pavie J, Rachline A, Loze B, Niedbalski L, Delaugerre C, Laforgerie E, et al. PLoS One. 2010;5(7):e11581. 21. Chavez PR, Bradley HM, Wesolowski LG, Violette LR, Katz DA, Niemann LA, et al. J Clin Virol.2020;124:104282. 22. Gallerano D, Cabauatan CR, Sibanda EN, Valenta R. Int Arch Allergy Immunol. 2015;167(4):223-41. 23. Dopel SH, Porstmann T, Henklein P, von Baehr R. Journal of virological methods. 1989;25(2):167-77. 64

Claims

CLAIMS 1) A composition comprising at least one polypeptide antigen derived from the endodomain of human immunodeficiency virus (HIV) gp41; at least one polypeptide antigen derived from HIV Gag p17; at least one polypeptide antigen derived from HIV integrase (IN); and at least one polypeptide antigen derived from HIV Nef. 2) The composition of claim 1, wherein the composition comprises no more than 3, 2, or 1 further polypeptide antigens derived from HIV. 3) The composition of claim 1, wherein the composition comprises not more than one further polypeptide antigen derived from HIV. 4) The composition of claim 1, wherein the composition comprises no further polypeptide antigens derived from HIV. 5) The composition of any one of claims 1-4, wherein the composition is a) for detecting an HIV infection in a human subject; and/or b) for distinguishing between anti-HIV antibodies generated due to an HIV infection in a human subject and anti-HIV antibodies generated in a human subject as a result of their vaccination with an HIV vaccine, optionally wherein the HIV infection is a breakthrough HIV infection in a subject vaccinated with an HIV vaccine. 6) A method for the in vitro diagnosis of an HIV infection in a human subject comprising conducting an immunoassay comprising the steps of: a) contacting a biological sample from the subject with at least four polypeptide antigens recognized by an anti-HIV antibody present in a subject infected with HIV, the contacting being under conditions sufficient to permit the formation of an immunocomplex; and b) determining whether an immunocomplex is formed, wherein the at least four polypeptide antigens recognized by an anti-HIV antibody are at least one polypeptide antigen derived from the endodomain of human immunodeficiency virus (HIV) gp41; at least one polypeptide antigen derived from HIV Gag p17; at least one polypeptide antigen derived from HIV integrase (IN); and at least one polypeptide antigen derived from HIV Nef. 7) The method of claim 6, wherein the at least four polypeptide antigens comprise no more than 3, 2, or 1 further polypeptide antigens derived from HIV. 65

8) The method of claim 6, wherein the at least four polypeptide antigens comprise not more than one further polypeptide antigen derived from HIV. 9) The method of claim 6, wherein the at least four polypeptide antigens comprise no further polypeptide antigens derived from HIV. 10) The method of any one of claims 6-9, wherein the immunoassay comprises the steps of: a) contacting the biological sample with the at least four polypeptide antigens, the contacting being under conditions sufficient to permit an anti-HIV antibody, if present in the biological sample, to bind to one or more of the at least four polypeptide antigens and form a polypeptide antigen-anti-HIV antibody complex; b) contacting the formed polypeptide antigen-anti-HIV antibody complex with an anti-HIV antibody binding molecule, the contacting being under conditions sufficient to permit the anti-HIV antibody binding molecule to bind to the anti-HIV antibody of the formed polypeptide antigen-anti-HIV antibody complex and form an extended complex; and c) determining the presence or concentration of the anti-HIV antibody in the biological sample by determining the presence or concentration of the formed extended complex. 11) The method of claim 10, wherein the immunoassay further detects the presence of an HIV antigen in the biological sample simultaneously or sequentially to detecting the presence of an anti-HIV antibody in the biological sample, optionally wherein the immunoassay further comprises the steps of: a) contacting the biological sample with at least one HIV antigen binding molecule capable of binding an HIV antigen from an HIV infected subject, the contacting being under conditions sufficient to permit the formation of a further immunocomplex; and b) determining whether a further immunocomplex is formed, further optionally wherein the HIV antigen binding molecule is an anti-HIV antigen antibody or fragment thereof, for example wherein the HIV antigen is HIV p24. 12) The method of any one of claims 6-11, wherein the method does not specifically detect vaccine-generated anti-HIV antibodies. 66

13) The method of any one of claims 6-12, wherein the immunoassay is an enzyme-linked immunosorbent assay (ELISA), an immunofluorescence assay (IFA), a radioimmunoassay (RIA), a chemiluminescent microparticle immunoassay (CMIA), a radioimmunoprecipitation assay (RIPIA), or an immunochromatographic assay, optionally wherein the immunoassay is a sandwich immunoassay, for example a bridging ELISA. 14) A kit for the in vitro diagnosis of an HIV infection in human subject, the kit comprising at least one polypeptide antigen derived from the endodomain of human immunodeficiency virus (HIV) gp41; at least one polypeptide antigen derived from HIV Gag p17; at least one polypeptide antigen derived from HIV integrase (IN); and at least one polypeptide antigen derived from HIV Nef. 15) The kit of claim 14, wherein the kit comprises no more than 3, 2, or 1 further polypeptide antigens derived from HIV. 16) The kit of claim 14, wherein the kit comprises not more than one further polypeptide antigen derived from HIV. 17) The kit of claim 14, wherein the kit comprises no further polypeptide antigens derived from HIV. 18) The kit of any one of claims 14-17, further comprising: a) a reaction environment for the production of an immunocomplex between the polypeptide antigen and an anti-HIV antibody if present; and/or b) one or more anti-HIV antibody binding molecules for the detection of the formation of the immunocomplex. 19) The composition of any one of claims 1-5 or the kit of any one of claims 14-18, further comprising an HIV antigen binding molecule capable of binding an HIV antigen, optionally wherein the HIV antigen binding molecule is an anti-HIV antigen antibody or fragment thereof, further optionally wherein the anti-HIV antigen antibody binds HIV p24. 20) The kit of claim 19, comprising: a) a reaction environment for the production of an immunocomplex between the HIV antigen binding molecule and an HIV antigen if present; and/or b) one or more further HIV antigen binding molecules for the detection of the formation of the immunocomplex. 67

21) Use of the composition of any one of claims 1–5 or 19, or the kit of any one of claims 14-20, for detecting HIV infection in a human subject. 22) The composition, method, kit, or use of any one of the preceding claims, wherein the HIV is HIV-1. 23) The composition, method, kit, or use of any one of the preceding claims, wherein: a) the polypeptide antigen derived from the endodomain of gp41 comprises a sequence having at least 90% identity to SEQ ID NO: 1, optionally wherein the polypeptide antigen derived from the endodomain of gp41 comprises the sequence of SEQ ID NO: 1; b) the polypeptide antigen derived from Gag p17 comprises a sequence having at least 90% identity to SEQ ID NO: 2, optionally wherein the polypeptide antigen derived from Gag p17 comprises the sequence of SEQ ID NO: 2; c) the polypeptide antigen derived from integrase (IN) comprises a sequence having at least 90% identity to SEQ ID NO: 3, optionally wherein the polypeptide antigen derived from IN comprises the sequence of SEQ ID NO: 3; and d) the polypeptide antigen derived from Nef comprises a sequence having at least 90% identity to SEQ ID NO: 4, optionally wherein the polypeptide antigen derived from Nef comprises the sequence of SEQ ID NO: 4. 24) The method, kit, or use of any one of claims 6-23, wherein (a) the polypeptide antigen does not detect anti-HIV-1 antibodies from an uninfected subject after the uninfected subject has been vaccinated with an HIV vaccine; (b) the method, use, or kit is for the in vitro diagnosis of a breakthrough HIV infection in a subject vaccinated with an HIV vaccine, and/or (c) the method, use, or kit is for distinguishing between (i) a subject infected with HIV and (ii) a subject vaccinated with an HIV vaccine and not infected with HIV. optionally wherein the HIV vaccine is an HIV-1 vaccine, for example a vaccine that expresses or comprises HIV-1 antigens Gag, Pol, Env and/or gp140. 25) A diagnostic device for use in the method or use, or with the kit of any one of claims 6-24, wherein the device comprises: 68

a) a first solid phase onto which has been attached one or more anti-HIV antibody binding molecules; b) a second solid phase onto which has been attached: i) at least one polypeptide antigen derived from the endodomain of human immunodeficiency virus (HIV) gp41; ii) at least one polypeptide antigen derived from HIV Gag p17; iii) at least one polypeptide antigen derived from HIV integrase (IN); and iv) at least one polypeptide antigen derived from HIV Nef; and c) a chamber configured to contain a liquid phase, wherein the chamber is in liquid communication with the first and second solid phase, and wherein the chamber is configured such that the liquid phase can further comprise a biological sample from a subject, wherein the one or more anti-HIV antibody binding molecules are eluted from the first solid phase upon contact with the liquid phase, optionally wherein further attached onto the first solid phase is at least one HIV antigen binding molecule, for example an anti-HIV antigen antibody or fragment thereof, optionally an antibody to HIV p24, further optionally wherein further attached onto the second solid phase is one or more further HIV antigen binding molecules, wherein the one or more further HIV antigen binding molecules are eluted from the second solid phase upon contact with the liquid phase. 26) The diagnostic device of claim 25, wherein no more than 3, 2, or 1 further polypeptide antigens derived from HIV are attached onto the second solid phase, preferably not more than one further polypeptide antigen derived from HIV is attached onto the second solid phase. 27) The diagnostic device of claim 25, wherein no further polypeptide antigens derived from HIV are attached onto the second solid phase. 69