WO2005078443A1 - Determination of infection by the immune response to a carbohydrate moiety - Google Patents

Abstract

A method and means for determining an infection with an infective agent in a mammal, by detecting an immune reactivity against a carbohydrate moiety associated with the agent, and a diagnostic kit for carrying out the method are provided.

Description

DETERMINATION OF INFECTION BY THE IMMUNE RESPONSE TO A CARBOHYDRATE MOIETY

FIELD OF THE INNENTION The present invention relates to a method and means for determining an infection with an infective agent in a mammal, by detecting an immune response against a carbohydrate moiety associated with the agent, and a diagnostic kit for carrying out the method. The detection method and kit of the invention can be used to diagnose and monitor infection of a mammal by pathogenic infective agents such as viruses, bacteria, fungi and parasites.

BACKGROUND OF THE INNENTION Diagnosis of Infectious agents Diagnosis of, infectious disease traditionally depends on four basic laboratory techniques: direct visualization of the infectious agent; detection of a "diagnostic" antigen; detection of "diagnostic" host immune response to the infectious agent; and isolation of the infectious agent in culture. While direct visualization is strong evidence of infection, it is very often impractical, due to the need for high concentrations of the agent in the sample, and the cumbersome, time consuming and inexact methods involved. Detection of pathognomonic antigens, while more rapidly and easily done, also requires antibodies of both high affinity and great specificity, high titers of the target antigen in the sample. Isolation and culture of the infectious agent in the laboratory, although conclusive, is an often unattainable goal, being hazardous and requiring complicated equipment and time consuming procedures. Detectϊøη? ?of a diagnostic host immune response can be a rapid, relatively simple and accurate method of diagnosing infectious disease. Histopathological examination of biopsied or excised tissue often reveals patterns of the host inflammatory response that can narrow down diagnostic possibilities. Further, host cell-mediated immune responses can be used for diagnoses: bacterial infection typically provokes, a PMN'leukocytosis, while viral infections produce a lymphocytic pleocytosis; a positive delayed type hypersensitivity skin test for mycobacterial or fungal antigens indicates active or previous infection. A partial list of infective agents commonly diagnosed by host immune response includes viral infections, mycoplasma pneumonia, rikketsial infection, Chlamydia, Lyme Disease, Syphilis,

Leptospirosis, Rheumatic fever, Legionnaire's Disease, Tuleremia, Brucellosis,

Histoplamosis, Coccidiomycosis and Amebiasis. Evaluation of the host immune response is greatly facilitated by accurate and positive detection of specific antibodies produced in response to the invasive agent, which depends on the identification and isolation of the. antigens presented to the host in the course of the infection. This can often be a problem, as has been encountered in the diagnosis of Human Immunodeficiency Virus (HIV). Serology in HIV Serology is one of the means to determine whether an organism is infected with an infective, agent (virus, bacterium, fungi, parasite, etc) by detecting the presence of antibodies to the agents or its components. Despite its shortcomings visa-vis direct detection of the agents, serology is currently still the preferable method for screening blood specimens for the presence of HIN, in view of serology's sensitivity and relative simplicity. Acquired Immunodeficiency Syndrome (AIDS) is an infectious and incurable disease transmitted through sexual contact from HIN infected individuals or by exposure to HIV contaminated blood or blood products. HIV-1 includes the formerly named viruses Human T-cell Lymphofrophic Virus Type HI (HTLV HI), Lymphadenopathy Associated Virus (LAV), and AIDS Associated Retrovirus (ARV). HIV is a. retrovirus related o a group of cytopathic retro viruses, namely lentiviruses, on the basis of . morphologic features, genomic organization, and nucleotide sequence (Gonda et al., Science (1985) 277:177-179; Stephan et al., Science (1986) 231:589-594; Kόrber, B, (ed.) et al., Human Retroviruses and AIDS. A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Published by Theoretical Biology arid Biophysics, Los Alamos National Laboratory, Los Alamos, N, Mex.; Reviewed in, Schochetman, G. and George, J. R., (1994) AIDS Testing. Sprmge?r-Nerlag, New York, Berlin, Heidelberg). HIV is an enveloped virus containing several structural proteins. Of particular relevance, the core of the virus is formed by condensation of cleavage products from a highly processed gag-pol polyprotein precursor (Prl80gag-pol) which is cleaved into apol precursor and a gag precursor (Pr55gag). Subsequently, the core precursor Pr55gag is cleaved into pl7

(myristilated gag protein), p24 (major structural protein), p7 (nucleic acid binding protein), and p9 (proline-rich protein). The envelope contains two structural proteins, gpl20 (envelope glycoprotein) and gp41 (fransmembrane protein) which are cleavage products of the envelope polypr tein precursor, gp 160. Shortly after the discovery of HIV, whole virions or lysates of virus infected cells served as the antigen in serological tests (Gallo et ah, EP 0181374B1, US

4520113). As the knowledge about HIV expanded, defined proteins, polypeptides or synthetic peptides replaced the whole virions as the targets for antibody in modern HIV tests, thus improving sensitivity and specificity. The most common markers of HIV infection are antibodies against viral structural proteins (Dawson, et,al., J. Infect. Dis. (1988) 157:149-155; Montagnier, et al. Virology (1985) 144:283-289; Barin, et al., Science (1985) 228:1094-1096; Schulz, T. F.,.et ^'ai., Lancet (1986) 2:111-112; Sarngadharan, et al., Science (1984) 224:506-508; Allan, et al., Science (1985) 228:1091-1093) and viremia in the form of detectable viral core antigen (antigenemia) (Kessler, et. al., JAMA (1987) 258:1196-1199; Phair, JAMA (1987) 258:pl218; ain, et al., The Lancet (1986) ii: 1233-1236; Kenny, et al., The Lancet (1987) 1 (8532):565-566; Wall, et al., The Lancet (1987) l(8532):p566; Stute, The Lancet (1987) l(8532):p566; Goudsmit, et al., The Lancet (1986) ii: 177-180; vόnSydow, et al, Brit. Med. J. (1988) 296:238- 240; Bowen, et al. Ann. of Int. Med, (1988) 108:46-48) or detectable viral nucleic acid (Mellors, et al., Science (1996) 272: 1167-1170; Saag, et al. Nat. Med. (1996) 2: 625-629; Mulder, et al. J. Clin. Microbiol. (1994) 32:292-300; Zhang, et al., AIDS (1991) 5(6):675-681; Sirnmόnds, et al., J. Virology (1990) 64(2):864-872). For example, in the United States, screening of blood and blood products by tests to detect antibody or antigen is mandated (Federal Food, Drug, and Cosmetic Act, 21 U.S.C. .sctn..sctn30T et seq., Public Health Service Act 42 U.S.C. .sctn..sctn.201 et. seq.). Nucleic, acid testing lias also been implemented in order to attain maximal reduction of the HIV serocόiϊyersion window (www.fda.gov). As a further example, various countries in Europe have begun to evaluate and use tests that detect antibody and antigen simultaneously (Ly, et al. J. Clin. Microbiol. (2000) 38(6): 2459-2461; Gurtler, et ah, ?J. Virol. Methods (1998) 75: 27-38; Weber, et al., J. Clin. Microbiol (1998) 36(8): 2235-2239; Courouce', et al., La Gazette de la Transfusion (1999)

N.degree.l55-Mars-Avril; Van Binsbergen, et al., J. Virol. Methods (1999) 82: 77-

84), in addition to European implementation of nucleic acid testing. An early version of an HIV combo assay is described in Gallarda, et al., 1992, WO93/21346, Assay for Detection 0?fHrV Antigen and Antibody. Within? several weeks after infection with HTV, individuals generally enter a clinical phase characterized by extensive viremia and acute symptoms. During this period, prior to seroconversion, HIV ρ24 core antigen can be detected transiently in serum or plasma specimens (antigenemia) (Devare, et al., (1990) In, Human Immunodeficiency Virus: Innovative Techniques. Monograph in Virology, J. L. Melnick (ed.)_? Basel, Karger, vol 18: 105-121; Kessler, et al. JAMA (1987 258: 1196-1199; Phair, J. P., JAMA (1987) 258: pl218; Allain, et al. The Lancet (1986) ii: 1233-1236; ^'Kenny, ef al., The Lancet (1987) 1(8532): 565-566; Wall, et al., The Lancet (1987) 1(8532): 566; State, R , The Lancet (1987) 1(8532): 566; Goudsmit, et al., The Lancef (1986) ii: 177-180; yonSydow, et al., Brit. Med. J. (1988) 296: 238- 240; Bowen, et al., Ann of Int. Med. (1988) 108: 46-48). However, after seroconversion, the core protein apparently is bound up by antibodies in circulating immune complexes, making core protein detection difficult and requiring immune complex disruption techniques (Schupbach, et al, AIDS (1996) 10:1085-1090; Kageyama, et ah, J. Nirol: Methods (1988) 22: 125-131; Mathiesen, et al., J. Nirol. Methods (1988) 22: 143-148; Steindl, et al, J. Immunol. Methods (1998) 217: 143- 151; Euler, et al.,_. Clin. Exp. Immunol. (1985) 59: 267-275; Gupta, et al., New Eng. J. Med. (1984) 310: 1530-1531; Griffith, et al., J. Clin. Microbiol. (1995) 33: 1348- 1350). After the initial viremic phase and throughout the remainder of the disease, the virus generally, establishes a steady state level (reviewed in Coffin, J. M. Science

Core proteins from isolates of HIV-1 group O, HIV-1 group M, and HIV-2 are antigemcally Siώilar because they share regions of amino acid sequence homology. Human (or mouse) immune polyclonal sera (i.e., immunoglobulin) elicited against the core protein of one group or type will cross react against the core protein of a different group or type (Clavel, et al., Science (1986) 233; 343-346; Guyader, et al., ISTatύre (1987) 326: 662-669; Barin, et al., Lancet (1985) 2: 1387- 1389; Kanki, et al., Science (1986) 232: 238-243; Kanki, et al., Science (1987) 236:

827-831; Clavel,?et al., Nature (1986) 324: 691-695; Hunt, et al., AIDS Res. Human

Retroviruses (1997) 13: 995-1005; Gurtler, et al., J. Virol. Methods (1995) 51: 177-

184; Mauclere, P. AIDS (1997) 11: 445-453). However, in contrast to human (or mouse) immune polyclonal sera, mouse or human monoclonal antibodies raised or elicited against the core protein of one HIV group or type may (Mehta, et al., U.S.

Pat. No. 5,173,399; Butman, et al., U.S. Pat. Nos. 5,210,181; Butman, et al., U.S. Pat.

No. 5,514,541) or may not (Mehta, et al., U.S. Pat. No. 5,173,399; Butman, et al.,

U.S. Pat. No. 5,210,181; Butman,_. et al., U.S. Pat. No. 5,514,541) react against the core protein of a. different HIV group or type. In cases where HIV-1 and HIV-2 core proteins were detected simultaneously (Butman, et al., U.S. Pat. No. 5,210,181; Butman, et al., U.S. Pat. No. 5,514,541), a combination of at least 3 monoclonals were required to achieve quantitative sensitivity against HIV-1 core protein. Typically, monoclonal antibodies display a lower affinity against cross-reactive antigens (epitopes) (Karush, F. (1978) In, Comprehensive Immunology, ed. R. A. Good, S. B. Day, 5: 85-116. New York/London: Plenum; Mariuzza, et al., Rev. Biophys. Biophys. Chem. (1987) 16: 139-159; Tijssen, (1993) hi, Laboratory Techniques in Biochemistry and Molecular Biology. R. H. Burdon and P. H. van ICnippenberg, eds. Vol. 15. Elsevier, Amsterdam). The extensive genetic (and therefore antigenic) variability of HIV has not been predictable, although many scientific papers have sought to supply explanations for the mechanism(s) of variability . (Meyerhans, et al., Cell (1989) 58: 901-910; Wain-Hobson^Cun. Top, Microbiol. Immunol. (1992) 176:181-193; Holland, et al., Curr. Top. Microbiol. Immunol. (1992) 176: 1-20; Gao, F. et al., Nature (1999) 397: 436-441 ; . Sharp, et al., Biol. Bull, (ϊ 999) 196: 338-342; Robertson, et al., Nature (1995) 374: 124-126; Zhu, J. Virol. (1995) 69: 1324-1327). Determination of HIV genetic (and therefore antigenic) variability has relied solely on many empirical observations that, subsequently have led to phylogenetic classification based on variation of HIV nucleic and amino acid sequence (Korber, ibid). Similarly, prediction of many epitopes of HIV jproteins cannot be made because (a) the protein sequences must first be discovered, (b) once discovered, genetic variation provides added complexity and uncertainty to the identification of epitopes and (c) epitope discovery and characterization are required to differentiate cross-reactive from shared epitopes. Carbohydrate antigens Sugar chains are abundantly expressed on the outer surfaces of the vast majority of viral, bacterial, protozoan and fungal pathogens, as well as on the membranes of mammalian cells. Recently, there has been a growing recognition of the importance and diagnostic potential of identifying and classifying characteristic carbohydrate moieties expressed in cells and on cell surfaces (for a recent review, see Wang, Protebmies 2003; 3:2167-75). Diagnostic carbohydrate antigens have been identified as tumor _. markers for detection of cancer [mostly altered products of glycoprotein glyeosylation (Orntoft et al Elecfrophoresis 1999;20:362-71)], such as carbohydrate antigens 15-3, 1-19 and 72-4 (Mohandy et al Postgrad Med Jour. 2003;79:569-74)(see US Patent No. 4,146,603 to Davidson et al, and US Patent Application No..10/486714 to Saarinen et al). Antibodies of the IgA and IgG type against carbohydrate antigenis LAM and PGL-1 are also used in the detection of Leprosy (Mycobacterium- leprάe). C. trichomonas exoglycolipid antigens have been used diagnostically and_. for vaccine development against Chlamydia. Other carbohydrate and glycoprotein antigens recognized by ELISA that have been used for clinical diagnosis of infections are O-antigen of Shigella, C-polysaccharide and Lipooligosacchari.de of Neisseria, and the polysacchari.de capsule of Cryptococcus (Sansanee et al, J? Clin Microb 2003 ;41 :432-34). In the past, the sugar epitopes of HIV were considered "immunologically silent" (Moore .& Sodroski, 1996; Rudd & Dwek, 1997) since they originate from the host synthetic: pathways and were therefore considered non-immunogenic, self antigens. Further, they have been implicated in shielding or masking of otherwise potentially immunogenic peptide epitopes of the glycoprotein polypeptide molecules to which they .are anchored. Hence, carbohydrates such as sugars or saccharides (these terms are , used interchangeably and also include oligosaccharides and polysaccharides) were traditionally not employed for serological monitoring. Indeed, even after the discovery that the broadly HlV-neutralizing human monoclonal antibody 2G12 recognizes such a "silent" carbohydrate epitope on gp 120 of HIN (Roux et al Moϊ nήiunόl 2004, 41:1001-11, Scanlan et al J. Nirol. 2002;76:7306-21, Trkola et al; 1995, 1996; Calarese et al, Science 2003;300:2065-71) and consideration of it's application in vaccine design (Wang et al J. Chem Biol

2004;11:127-134), serological monitoring of HIV using this or other carbohydrate antigens has yet to developed. HIV testing and qnti-HLV Immune response The detection of humoral and cellular immune response to infection is of particular importance in determining prognosis and prescribing treatment. This has been shown to be especially crucial in HIV infection, since it has been shown that measurement of HIV viruses in early detection of HIV infection indicate a dissociation between the strong cellular immune response and the low levels of viremia following infection, leading one researcher to state that "qualitative differences in the primary immune response, and NOT viremia levels, are predictive of the rate of disease ρrogression."(Pantaleo et al PNAS USA 1997;94:254-58). Many methods for detection of HIV antigens and antibodies have been disclosed (see above). Commercially available kits for serodetection of HIV are widely marketed^ even for home use (for example, VIDAS HIV DUO, bioMerieux sa, France, which tests fo HIV-ρ24, and the B-Safe HIV 1-2 kit, USA BioMed Las Vegas NV). . In some studies, anti-HIV antibody has been reported to be more reliable for diagnosis than either HIN culture or HIN antigen detection in patient samples (Steckelberg, J. M. and Cockerill, HI, F. R, Mayo Clin. Proc. 1988, 63:373-380; Schleupner, C. J.,??rin. &,Practice of Infectious Diseases-Update I, 1989 10:3-19). Consequently, anti-HIN antibody detection tests are the most common method of diagnosis of infection. Both EIA and Western blot assays are currently used in the detection of anti-HIV antibody. Unfortunately, a degree of unreliability continues to exist with the use of conventional anti-HIV antibody screening methods, such as by conventional EIAs; Thus, further confirmatory tests, such as a Western Blot (WB) or fixed-cell immunofluorescence assay, have become recommended additional testing procedures. However, a number of studies report the existence of a seemingly silent period of HIN infection during which antibody to the virus is not detectable even after exhaustive testing, anywhere from a few months to as much as two and one-half years before infection is detectable by conventional EIAs and Western blot assays. While not always successful, culturing of peripheral blood lymphocytes to amplify

HIV does provide for detection of the virus when anti-HIV antibody cannot be detected by conventional EIA or WB. However, several recent studies using PCR- based HIV detection methods continue to report the existence of PCR(+)positive, sero(-)negative^; cases in high-risk populations (Brettler, D. B., Somasundaran, M., Forsberg, A. F., Krause, E., et al., Blood, 1992 80:2396-2400; Gupta, P., Kingsley, L, Anderson, R, Ho, M., Enrico, A., Ding, M., et al., AIDS, 1992 6:143-149). The rate of HIV transmission in negatively tested blood, using conventional testing methods, continues to persist at a relatively constant rate (Ward, J. W., Developments in Biological Standardization, 1993 81:41-43). For example, HIV-1 transmission from seemingly "seronegative" blood using EIA conventional methods, continue to be reported. . Donated organs also constitute a source of HIV disease fransmission, with HIV infection being diagnosed in recipients of organs from individuals whom, again, test HIV seronegative by conventional assays (Simonds, R. J., Holmberg, S. D., Hurwitz, R. L, et al., N. Engl. J. Med. 1992;326:726-732). Retrospective studies have reported that early donor education and self- exclusion measures have reduced the rate of disease transmission. However, such exclusion methods . together with antibody testing, while hopefully reducing the probability of at least some false negative results, provides only a partial and imperfect solution to the problem in at least a small subset of reported HIV cases. Some studies have reported the presence of HIN specific T-cells in high risk individuals testing negative with conventional EIA, WB, and PCR based detection techniques (Clerici, M., Berzofsky, J. A., Shearer, G. M., and Tacket, C. O., J. Infect. Dis. 1991;164:178-182; Clerici, M., Levin, J. M., Kessler, H. A., Harris, A., Berzofsky, J.. ., et al, JAMA, 1994:271:42-46). Other reports have identified the existence of B-cells which produce HIV-specific antibodies in vitro that are present in EIA-negatiye, WB-negative, high-risk subjects. While these approaches present possible alternatives, for testing, they are relatively complex and difficult procedures, and are thus impractical for large-scale clinical screening. US Patent o. 6,165,710 to Robinson teaches the detection of antibodies recognizing viral atid. other. pathogen antibodies, in a tissue or blood sample of a subject, hi order to achieve specific and consistent recognition of the target antigens (glycoproteins) by the marker antibodies, Robinson teaches the immobilization of glycoprotein antigens on lectin-coated solid-phase detection substrate. However, no methods for testing for infection by detecting an immune response against a carbohydrate antigen employing native or synthetic carbohydrate moieties unconjugated to their respective glycoprotein backbone were disclosed. There is thus a widely recognized need for, and it would be highly advantageous to have an accurate and reliable method of determining the presence of an infection with an infective agent such as a virus, and specifically HIN, devoid of the above limitations by monitoring the host immune response to carbohydrate moieties of the infectious agent,

SUMMARY OF THE INVENTION According to the present invention there is provided a method of diagnosing a viral infection in a subject, comprising detecting in a biological sample of the subject an immune reactivity to at least one viral-associated carbohydrate antigen, wherein the immune reactivity to the viral-associated carbohydrate antigen is diagnostic of the viral infection, thereby diagnosing the viral infection in the subject. In a preferred embodiment, the viral infection is an HIV infection, and the viral associated carbohydrate antigen is an HIV-associated carbohydrate antigen. According to further features in described preferred embodiments the HIV infection is an HIV-1, HIV-0 or an HIVτ2 infection.; According to further features in described prefened embodiments the viral- associated carbohydrate antigen is a viral-specific carbohydrate antigen. According to further features in described prefened embodiments immune reactivity comprises the presence of anti- viral-associated carbohydrate antigen antibodies. In ..a prefered embodiment the anti- viral-associated carbohydrate antigen antibodies are anti-HIN-associated carbohydrate antigen antibodies. According to further features in described preferred embodiments the detecting is effected by contacting the biological sample with at least one viral- associated carbohydrate antigen under conditions allowing a formation of antigen- antibody complexes; and detecting the formation of the antigen-antibody complexes. According to yet further features in described preferred embodiments the detecting is effected by a method selected from the group consisting of an enzyme linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), an enzyme immunosorbent assay (EIA), Western blotting, a fluorimmunoassay, immune precipitation, FACS and immunohistochemistry. According to still further features in described prefened embodiments the immune reactivity is a cellular, reaction to at least one viral-associated carbohydrate antigen. According to further features in described prefened embodiments the detecting is effected by contacting cells of the biological sample with at least one viral-associated carbohydrate antigen and detecting in the cells an antigen-specific cellular response to the at least one viral-associated antigen. According to yet further features in described prefened embodiments the cellular response is selected from the group consisting of lymphocyte proliferation, immediate and delayed-type hypersensitivity, chemotaxis, extravasation, migration, cytokine secretion, detection of activation cell surface markers and cytotoxicity assay. According to still further features in described prefened embodiments the detecting is preceded by processing the biological sample to substantially dissociate viral-associated carbohydrate antigen-antibody complexes, thereby allowing detection of the dissociated anti- viral-associated carbohydrate antigen antibodies. According to further features in described preferred embodiments the processing is effected by at least one method selected from the group consisting of antigen degradation, competitive displacement and denaturation. The antigen degradation can be chemical, mechanical or enzymatic degradation. According to yet further features in described preferred embodiments the processing further comprises dissociating the viral-associated carbohydrate antigens from the dissociated antibodies. According_. to. still further features in described prefened embodiments the processing further includes separating the viral-associated carbohydrate antigens from the dissociated antibodies. According to further features in described prefened embodiments the at least one viral-associated carbohydrate antigen is a carbohydrate moiety selected from the group consisting of a carbohydrate moiety of a glycoprotein, a carbohydrate moiety of a proteoglycan and a carbohydrate moiety of a glycolipid. According to yet further features in described prefened embodiments the viral infection is selected from the group consisting of a DNA virus infection and an RNA virus infection? Further according to the present invention there is provided a kit for diagnosis, prognosis, Or staging of, a viral infection in a biological sample, the kit comprising at least one viral-associated carbohydrate antigen. Additionally and optionally the kit further comprises an agent capable of detecting an immune reaction to said at least one_. viral-associated carbohydrate antigen. The present invention successfully addresses the shortcomings of the presently known configurations, by providing methods for detecting an infection with an infective _. viral agent by accurately detecting an immune reactivity to a viral- associated carhoϋydrate antigen in a biological sample.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of a method, means and kit for determining an infection with an infective viral agent in a mammal, by detecting an immune reactivity against a carbohydrate moiety associated with the viral agent. Specifically, the method of the present invention can be used for diagnosing, prognosing, staging and/or monitoring HIV infection. Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following- description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. The outer envelope of virus particles is composed of glycoproteins, such as the gpl20 and ?gp41 coat glycoproteins, cleaved from the gp 160 glycoprotein of the HIV virus. Other viral components, containing carbohydrate moieties include glycolipids and proteoglycans. Thus, via such carbohydrate-containing molecules virus particles present a great variety of complex carbohydrate structures to the immune systems of the infected host. Antigenic determinants comprising carbohydrate moieties isolated from these glycosylated macromolecules, have been identified, and are detected by antibodies from infected individuals and monoclonal anti-HIV antibodies. Thus, the immune response to viral-associated carbohydrate antigens can be used as an indicator of, and method monitoring, viral infection. Thus, according to one aspect of the present invention, there is provided a method of diagnosing a viral infection in a subject, comprising detecting in a biological sample of the subject an immune reactivity to at least one viral-associated carbohydrate antigen, where the immune reactivity to the viral-associated carbohydrate antigen is diagnostic of the viral infection. Thus, detecting the immune reactivity to the diagnostic viral-associated carbohydrate antigen is diagnostic and can be prognostic of the viral infection in the subj ect. As nsed herein, the term "biological sample" is defined as a sample of tissue, cells, fluids, exύdates or other material originating from the body of the subject. Biological samples suitable for analysis using the methods of the present invention include tissue samples and biological fluid samples. Fluid samples include, but are not limited to, blood, plasma, serum, tears, urine, lymph, feces, sweat and milk. As used herein, the phrase "viral-associated carbohydrate antigen" is defined as a carbohydrate molecule structurally associated with a virus particle and capable of stimulating an immune response in a mammal. In a preferred embodiment, the viral- associated carbohydrate antigen of the method of the present invention is a viral- specific carbohydrate antigen. As defined herein, the phrase "viral-specific carbohydrate a tigen'' is defined as a viral-associated carbohydrate antigen relating to, or derived from, solely a virus, and not found in non-viral organisms. Viral- associated carbohydrates are synthesized by the infected host cells, and include, but are not limited to, the carbohydrate moieties of glycosylated macromolecules such as viral glycoproteins, glycolipids and proteoglycans. Additionally, virus-associated carbohydrate antigens, can be portions of carbohydrate-containing host molecules incorporated into/the virus particle, which antigens being capable of stimulating an immune response in the infected host. It will be appreciated that carbohydrate moieties bound, covalently and non-covalently, to the virus particle or portions thereof can be. antigenic during the course of an infection with a viral pathogen.

Thus, in yet another embodiment , the viral-associated antigen can be a "viral-bound carbohydrate antigen". As used herein, the term "viral-bound carbohydrate antigen" is defined as a viral-associated carbohydrate antigen being physically associated, covalently or otherwise, with the virus particle. As used herein, the terms "glycan" and "carbohydrate" are interchangeable.

As used herein, the carbohydrates of the present invention include sugars. As used herein, the term "sugars" and "saccharides" are interchangeable. As used herein, the terms "moiety", "sidechain" and "residue" are interchangeable, referring to the portion of a complex molecule belonging to a distinct class of biological molecules different from another portion of the biological molecule. By way of example, it will be understood, that the carbohydrate moiety of a glycoprotein is the carbohydrate chain linked to, the polypeptide portion of the glycoprotein. Examples of such viral-associated carbohydrate antigens are the sugar residues added posttranslationally to glycoproteins, predominantly via N-linkage sites on the polypeptide portion of the glycoprotein. It will be -appreciated, that many classes of viruses are infective in man, and most are antigenic uring the course Of the infection. Infectious virus of both human and non-human vertebrates, , include retro viruses, RNA viruses and DNA viruses.

According to a prefened embodiment the viral infection can be a retroviral infection, an RNA viral infection or a D?NA viral infection. According -to a prefened embodiment of the present invention, the viral infection can be a. human immunodeficiency virus (HIV) infection. HIV infection can be the result of infection with HIV-1, HIV-0 and/or HIV-2 virus. Examples of viruses which, can be detected by the present invention include, but are not limited to: retfόvirμses, RNA viruses and DNA viruses . It will be appreciated that the invention is not limited to the detection of only infection due to the Human Immunodeficiency Virus (HIV). It also encompasses the generalization of the method, of the means and of the kits described below to the detection of infection, and/or the monitoring of infection, due to any sort of infectious virus such as, for example, the viruses responsible for SARS, the various types of hepatitis A, B, C, D or E, the retroviruses responsible for hepatitis C virus in humans (HCV) or monkeys, the cytomegalovirus (CMV), flaviviruses, herpes virus,

Epstein-Barr virus (EBV), herpes simplex, human herpes virus type 6 (HHV-6)), papilloma, poxvirus, picornavirus, adenovirus, rhino virus, human T lymphotropic virus-type 1 and 2 (HTLN-1/-2), human rotavirus, rabies, encephalitis and respiratory viral infections, the dengue viruses, and any other viruses for which detection of an immune response is desirable. Other antigenic viruses suitable for detection and diagnosis with the methods of the present invention include, but are not limited to both simple retroviruses and complex retroviruses. The simple retroviruses include the subgroups of B-type retroviruses, C-type retroviruses and D-type retroviruses. An example of a B-type retrovirus is nipuse mammary tumor virus (MMTN). The C-type retroviruses include subgroups C-type -group A (including Rous sarcoma virus (RSN), avian leukemia virus (ALN), and avian myeloblastosis virus (AMY)) and C-type group B (including murine leukeiriia? virus (MLN), feline leukemia virus (FeLV), murine sarcoma virus (MS V), gibbon ape leukemia virus (GALV), spleen necrosis virus (S?ΝV), reticuloendotheliosis virus (RN) and simian sarcoma virus (SSN)). The D-type retroviruses include Mason-Pfizer monkey virus (MPMN) and simian retrovirus type 1 (SRN-1). The complex retroviruses include the subgroups of lentiviruses, T-cell

including the. genus Enterovirus (poliovirus, Coxsackie virus A and B, enteric cytopatliic human orphan (ECHO) viruses, hepatitis A virus, Simian enteroviruses,

Murine encephalomyelitis (ME) viruses, Poliovirus muris, Bovine enteroviruses,

Porcine enteroviruses, the genus Cardiovirus (Encephalomyocarditis virus (EMC), Mengo virus), the genus Rhino virus (Human rhino viruses including at least 113 subtypes; other rhinoviruses), the genus Apthovirus (Foot and Mouth disease

(FMDN); the family Calciviridae, including Vesicular exanthema of swine virus, San

Miguel sea lion virus, Feline picornavirus and Νorwalk virus; the family

Togaviridae, including the genus Alphavirus (Eastern equine encephalitis virus, Semliki forest virus, Sindbis virus, Chikungunya virus, OTMyong-Νyong virus, Ross river virus, Nene^μelan equine encephalitis virus, Western equine encephalitis virus), the genus Flavirius (Mosquito borne yellow fever virus, Dengue virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Nalley encephalitis virus, West Nile virus, Kunjin virus, Central European tick borne virus, Far Eastern tick borne virus, Kyasanur forest virus, Louping HI virus, Powassan virus, Omsk hemonhagic fever virus), the genus Rubivirus (Rubella virus), the genus Pestivirus (Mucosal disease virus, Hog cholera virus, Border disease virus); the family Bunyaviridae, including the genus Bunyvirus (Bunyamwera and related viruses, California encephalitis group viruses), the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Nalley fever virus), the genus Nairovirus (Crimean-Congo hemonhagic fever virus, Nairobi sheep disease virus), and the genus Uukuvirus (Uukuniemi and related viruses); fhe: family Orthomyxoviridae, including the genus Influenza virus (Influenza virus type A, many human subtypes); Swine influenza virus, and Avian and Equine Influenza viruses; influenza type B (many human subtypes), and influenza type. C (possible separate genus); the family paramyxoviridae, including the genus Paramyxovirus (Parainfluenza virus type 1, Sendai virus, Hemadsorption virus, Parainfluenza viruses types 2 to 5, Newcastle Disease Virus, Mumps virus), the genus Morbillivirus (Measles virus, subacute sclerosing panencephalitis virus, distemper virus, Rinderpest virus), the genus Pneumo virus (respiratory syncytial virus (RSV), ?Bovine respiratory syncytial virus and Pneumonia virus of mice); forest virus, Sindbis virus, Chikungunya virus, OTSfyong-Nyong virus, Ross river virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus), the genus Flavirius (Mosquito borne yellow fever virus, Dengue virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Nalley encephalitis virus, West Nile virus,

Runjin virus,. Central European tick borne virus, Far Eastern tick borne virus,

Kyasanur forest virus, Louping III virus, Powassan virus, Omsk hemonhagic fever virus), the genus Rubivirus (Rubella virus), the genus Pestivirus (Mucosal disease virus, Hog cholera virus, Border disease virus); the family Bunyaviridae, including the genus Bunyvirus (Bunyamwera and related viruses, California encephalitis group viruses), the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Nalley fever virus), the genus Nairovirus (Crimean-Congo hemorrhagic fever virus, Nairobi sheep disease virus), and the genus Uukuvirus (Uukuniemi and related viruses); the family Orthomyxoviridae; iiiclμding the genus Influenza virus (Influenza virus type A, many human subtypes); Swine influenza virus, and Avian and Equine Influenza viruses; influenza type ?B (many human subtypes), and influenza type C (possible separate genus); the family paramyxoviridae, including the genus Paramyxovirus (Parainfluenza virus type 1, Sendai virus, Hemadsorption virus, Parainfluenza viruses types 2 to . 5, Newcastle Disease Virus, Mumps virus), the genus Morbillivirus (Measles virus, subacute sclerosing panencephalitis virus, distemper virus, Rinderpest virus), the genus Pneumovirus (respiratory syncytial virus (RSV), Bovine respiratory syncytial virus and Pneumonia virus of mice); the family Rhabdoviridae, including the! genus? Vesiculovirus (VSV), Chandipura virus, Flanders-Hart Park virus), the genus Lyssavirus (Rabies virus), fish Rhabdoviruses, and two probable Rhabdoviruses (Marburg virus and Ebola virus); the family Arenaviridae, including Lymphocytic choriomeningitis virus (LCM), Tacaribe virus complex, and Lassa virus; the family Coronoayiridae, including Infectious Bronchitis Virus (IBV), Mouse Hepatitis virusi Human enteric corona virus, and Feline infectious peritonitis (Feline coronavirus). Exemplary HIN-specific carbohydrate antigens are the glycan moieties of the gρl20, gp41 and gpl60 HIV glycoproteins, many of which have been shown to be associated with antigenic? determinants of the HIV glycoproteins. The glycan moieties of HI glycoproteins have been analyzed, and, for example, gpl20 contains 33% high mannose glycans, 4%hybrid type, and 63% complex type glycans, of which 90% are fucosylated and 94% sialylated (see Leonard et al., J Biol Chem. 1990; 268:10373-82). ? Further examples include, but are not limited to O-linked oligosaccharides, etc. Identification? of other viral specific and viral-associated carbohydrate antigens can be effected by, for example, the use of glycan arrays. Such glycan anays comprise a wide varieity of candidate carbohydrates, or combinatorial libraries of carbohydrate residues, affixed to a solid matrix, which can be contacted with a putative source (e.g. a blood sample) of carbohydrate binding ligand, such as anti- carbohydrate antibodies. After washing, the bound carbohydrate-ligand complex (e.g. the carbohydrate-anti-carbohydrate antibody complex) can be detected by any suitable means of signal generation, and the identity of the target carbohydrate(s) bound determined: In this regard, it will be appreciated that glycan arrays, recently developed for the identification of carbohydrate-binding molecules and the ligands recognized thereby, can be used to identify viral-associated and viral-specific carbohydrate antigens suitable for use in the method of the present invention, and can themselves be used to identify an immune response to a viral-associated carbohydrate antigen using the methods of the present invention as further described hereinbelow. Glycan arrays, their preparation and their use are disclosed in detail in Galustian et al., Lit Immunol 2004;16:553-66; US Patent Applications 09/783,083, 09/860,488 and 09/860,487 to Dukler et al; Schwartz et al, Glycobiology 2003;13:749-54. Adams et al (Chem Biol 2004;875-881) have demonstrated the use of such glycan arrays for the identification of HIN-viral-associated carbohydrate antigens and epitopes, and Wang et al?(Phys. Genomics 2004;18:245-8) reported the identification of diagnostic SARS-CoN antigenic glycan epitopes with a carbohydrate array. Glycan anays have recently become available from The Consortium for Functional Genomics of the Νffl? (www.functionalgenomics.org), and commercially available from Glycomlnds Ltd. : (Glycominds Ltd., Lod ISRAEL) and KamTek Ine (Gaithersburg, MD). Carbohydrate. antigens suitable for the use with the methods of the present invention should?be viral- associated and viral-specific carbohydrates presented to the immune system of the host, preferably the carbohydrates moieties of viral envelope glycoproteins, viral proteoglycans and viral glycolipids. Preferably, the carbohydrate antigens are antigen? biήdiήjg antibodies from patients infected with the disease in question. By way of example, the viral- associated and viral-specific carbohydrate antigens can be HlV-associated carbohydrate antigens. Preferably, the HIV- associated carbohydrate antigen should be selected from the group of molecules which bind antibodies from HTV patients, including those molecules which are known to bind such antibodies and those which are expected to bind. Preferably such antibodies are. secreted by clones isolated from HIV patients. A non-limiting example of such a clone is designated 2G12 and binds with high affinity to terminal

Manαl-2Man moieties on a cluster of oligomannose-type sugars attached to the gpl20 surface protein of HIV (Scanlan et al, 2002; Sanders et al, 2002; Moore & Sodroski, 1996?; Calarese et al. , 2003). The sugar antigens employed for the method can optionally be derived from virions (e.g., HIV), infected cells, cell cultures, virion fractions or components. Alternatively the sugars can be synthesized or derived from non-HIN related sources and molecules. Thus, mannose-rich oligosaccharides are associated with ovalbumin, soybean agglutiniri, yeast mannan, ribonuclease B and Uromodulin, to name a few (Muchmore et al, 1990; Rudd et al, 1992; Deras et al, 1998; Calarese et al, 2003). For carrying out . the present method, the sugars may be in their free form, or alternatively, attached to their natural carrier molecules, or may be immobilized or bound to any carrier of choice, be it natural or synthetic, soluble, particulate or cellular. As used herein, the term "diagnostic" is defined as having relevance to, and capable of providing information leading to the detection of a viral infection, and to the identification of the infective agent. The term "diagnosing" is thus the use of diagnostic information in order to detect and/or identify an infective agent. The term prognostic is defined: herein as having relevance to the determination of a prognosis (i.e. the probable outcome) of. a viral infection. As used herein, the term "staging" relates to the determination f a stage of the natural history of the viral infection, for example, deterrnining the seroconversion stage of the HIV infection. Such staging of a viral infection, for example, HIV, is crucial to determination of accurate treatment. It will b appreciated, in this regard, that diagnosis of a viral infection may be performed by .the methods of the present invention alone, or in conjunction with other diagnostic methods, .such as clinical examination, additional testing of biological fluids, for example, for viral antigens or viremia. Further, one of ordinary skill in the art will appreciate that in order to provide a conclusive diagnosis of the viral infection, repeated tests and monitoring of the biological samples of subjects can be performed, as is described in detail regarding HIV infections in the Background section hereinabove. As used hefeinj the term "immune reactivity" is defined as a specific response of the host immune system to the presence of a viral-associated antigen. Reflecting the makeup of the irimiune system, immune reactivity can be a humoral or circulating antibody immune reactivity, generally refened to as B-cell response, and/or a cellular immune reactivity, resulting from a T-cell response. In a preferred embodiment of the present invention, the immune reactivity is a B-cell reactivity, comprising the presence in the biological sample, of anti-viral- associated carbohydrate antigen antibodies. In a yet more prefened embodiment, detection of the . anti- viral- associated and viral-specific carbohydrate antigen antibodies is effected by contacting the biological sample with at least one viral- associated caφohydrate antigen under conditions allowing a formation of antigen- antibody complexes; and detecting the formation of antigen-antibody complexes. It will be appreciated that the detection of a B-cell immune response comprising the presence of . anti-viral-associated carbohydrate antigen antibodies is preferably effected in fluid biological samples, most preferably blood, serum, and the like. Methods Tfor the detection of antibody-antigen complexes are well known in the art, including^ but not liriϊited to enzyme linked immunosorbent assay (ELISA), a radioimmurioassay (RIA), an enzyme immunosorbent assay (EIA), Western blotting, immune precipitation, a fluoroimmunoassay, FACS and immunohistochemistry. In a preferred embodiment of the methods of the present invention, the viral-associated carbohydrate antigens are. immobilized to the surface of, for example, a microtiter plate, and the antibody-antigen complexes are detected on an ELISA reader using an enzyme labeled second antibody (for detailed description see Example 1 hereinbelow), . Further examples include the detection of viral-associated carbohydrate antibody-antigen complexes on glycan arrays, or cliips, which are developed and analyzed, as described in Adams et al (Chem Biol 2004;875-881) and Wang et al (Phys. Genoiήics 2004;18:245-8), and allow the analysis of multiple samples, or a multiplicity of antigens for a variety of viral infections. In general, the detection of antibodies to viral-associated carbohydrate antigens can be accomplished by any method intended for the detection of antibodies known in the art. Such methods may include solid and liquid phase immunoassays with enzymatic, fluorescent, luminescent, radioactive or particle labeling. The method can be carried out in test tubes or wells or on strips, sticks and chips (the above list is non-limiting). The choice of method is governed by the location and amenities available to the test operator and by the cost, rapidity, sensitivity and economical considerations. Prior to? detection, processing of the biological sample may be advantageous. In view of the abundance of carbohydrate epitopes in any bio-organism, anti- carbohydrate antigen antibodies can be neutralized by carbohydrate-containing molecules and? therefore may not be directly detectable by the antibody detection methods of the present invention. In this case it would be advantageous to process the sample of ..choice before being subjected to an antibody detection test of the invention. For example, detecting HIN antibodies in a sample may be preceded by processing the. biological sample to substantially dissociate HIN-associated carbohydrate antigen-antibody complexes, thereby allowing detection of dissociated anti-viral-associated carbohydrate antigen antibodies. Such processing can include antigen degradation, competitive displacement of the antigen and denaturation of the antibody-antigen complex. Antigen degradation can be chemical, mechanical or enzymatic. ?Met?hods f chemical, mechanical and enzymatic degradation of carbohydrates are well known in the art. Νon-limiting examples of chemical degradation include periodate . oxidation (see detailed description in the Examples section hereinbelόw) and /3-elimination (treatment with a strong alkali plus a reducing agent). Mechanical degradation can include cavitation (for example, by ultra sound using a Kontes UltraSonic tissue disruptor (Kontes, Nineland, ΝJ) or a Ultrasonic disruptor from Biologies, Ine, Gainsville, NA). Enzymatic degradation includes the use of the enzyme peptide Ν-glycosidase F (PΝGaseF, Ν-Glycanase, EC 3.2.2.18), and other endoglycosidases and glycoamidases, as described in detail hereinbelow. Another useful class of enzymes for carbohydrate release are the endo- beta-N-acetylglueosidases (EC 3.2.1.96) of the Endo H and the Endo F family.

These enzymes show considerable specificity for the types of N-linked structures which they will cleave. The "Endo" enzymes are especially useful for the study of oligomannosyi ("high mannose") and "hybrid" -type oligosaccharides. Competitive displacement of the bound viral-associated carbohydrate antigen can be carried out by incubation of the sample with high concentrations of other carbohydrates having affinity for the antibody. For example, incubation in a 1M solution of methyl of-D-mannopyranoside has been shown to dissociate the liigh affinity complex between an oligo-mannose and Concanavalin A (Deras et al., 1998). Other sugar competition assays are disclosed in Whitehurst et al (J. hnmun. Methods. 1990;131; 15-24). Alternatively or additionally, processing can comprise dissociating the viral- associated carbohydrate antigens firom said dissociated antibodies. In this regard, it will be appreciated by one of ordinary skill in the art, that heating at 80°C for 10 - 30 minutes dissociates antigen-antibody complexes without affecting antibody activity. Thus, in a preferred embodiment, the viral-associated carbohydrate antigen- antibody complex is dissociated with thermal treatment, as described in detail in the Examples section hereinbelow. This treatment can be combined with degradation of the antigen in the pre-existing antigen-antibody complex such as by chemical degradation; such, as periodate, whose activity increases with heating, so that the structure of the released oligo-mannose is immediately destroyed by the chemical degradation upon release. . It will be appreciated by one of skill in the art that immune complexes can be dissociated at extrernes of pH, while preserving antibody activity. Thus, in one embodiment of the present invention, processing to dissociate the antibody-antigen complex is carried out by incubation in acidic or basic solution, prior to detection with carbohydrate antigens. For example, glycine-HCl pH 2.0 buffer can be employed to dissociate the antibody-HIN-associated carbohydrate (glycan) complex. The released carbohydrate antigen can then be degraded by chemical and/or enzymatic treatments as described above, or can be removed by binding to an immobilized lectin such as Concanavalin-A (Sigma Ine, St Louis MO). Further methods for dissociation of the antibody-antigen complex suitable for the present invention include physico-chemical dissociation. For example, molar level concentrations of some salts (e.g. 3M KC1, 3M MgCl₂, 3M KCNS) or high molar concentrations of urea are commonly used to dissociate immune complexes by gentle reversible denaturation. Following dissociation of the antibody-antigen complex, any remaining carbohydrate tnoieties, which are still capable of binding with the antibody in question, can be removed or separated from the sample being tested. Removal and/or separation can be accomplished by various methods known in the art, for example antigen degradation^ dialysis, size exclusion chromatography, lectin binding (for example, Concanavalin A) and the like. It will be appreciated that the abovementioned treatments can be employed individually, or in combination with one another. In one prefened embodiment, chemical or heat denaturation of the antibody-antigen complex is combined with enzymatic or: chemical degradation of the carbohydrate antigen prior to the detection by immobilized carbohydrate antigen. In yet another embodiment of the present invention, the immune reactivity is a T-cell reactivity, comprising a cellular reaction to at least one viral-associated carbohydrate antigen, and detecting the immune reactivity is effected by contacting cells of said biological sample with at least one viral-associated carbohydrate antigen and detecting in the cells an antigen-specific cellular response to said at least one viral-associated antigen. Antigen-specific cellular responses suitable for detection and diagnosis according to the methods of the present invention include, but are not limited to assays of . lymphocyte proliferation, delayed-type hypersensitivity, chemotaxis, extravasation, migration, cytokine secretion, detection of activation cell surface markers ! and cytotoxicity assay. Cellular immune response can also be detected , in other tissues, such as the cells of lymphatic organs (i.e. spleen, lymph nodes). Additional immune responses suitable for detection with the methods of the present invention include, but are not limited to immediate and delayed type hypersensitivity. Methods and assays for the detection of such cellular immune responses (such as T-cell activation) are well known in the art, for example, the lymphocyte proliferation assay commonly measures uptake of radio-labeled nucleotides

(mitogenesis) in immune cells (i.e.peripheral blood lymphocytes) in response to a candidate antigen, as described in detail by Wahren et al (J. Virol 1987 61:2017-23) for the identification of HIV-associated cellular antigens. T lymphocyte activation can be assayed, for example by measuring resultant proliferation (e.g., via XTT or

MTT colorimetric? assay, or [3H]-thymidine incorporation assay), IL-2 secretion (e.g., via ELISA or? CTLL growth stimulation assay), cytotoxicity (e.g., via chromium release assay), or upregulation of activation markers such as, for example, CD25 or

CD69 (e.g., via immunofluorescence assay). Cytotoxicity assays generally measure the ability of cytotoxic T-cells

(CTCs) to lyse target cells, using, for example, a standard microcytotoxicity assay. Briefly, target cells are pelleted and resuspended in a buffer comprising Na51Cr (DuPont NEN, Boston, MA), and incubated for 1-2 hours, washed, resuspended and added to round-bottomed microtiter wells (Becton Dickinson & Company, Franklin Lakes, NJ). Varying numbers of effector cells (such as NK cells) are added at various effector/target (E/T) ratios. Release of cell contents, and the loaded radioactivity due to disruption of the cell membranes can be measured in a gamma counter following precipitation of the cells and cellular debris. Controls include spontaneous 51 Cr- release, and maximum release (achieved by adding a detergent such as 1STP-40 (United States Biochemical) to the target cells). Cytotoxicity is expressed as percent specific 51Cr-release = i00 x [(experimental cpm - spontaneous cpm) / (maximum release cpm - spontaneous cpm)]. Detection of cellular immune response to a viral-associated carbohydrate antigen can be detected in peripheral blood samples of a candidate subjects suspected of, for example, -HIV infection, by isolating a peripheral blood fraction from the samples, and coniparing the lymphocyte proliferation in response to HIV-associated carbohydrate antigen or antigens, using non-antigenic glycans and PMH activation as negative and positive controls, respectively. Detection of a cellular immune response in a biological sample having cells, such as increased lymphocyte proliferation in response to a HIV-associated carbohydrate antigen, such as the oligo-mannose and mannose disaccharides comprising the carbohydrate epitope of gpl20 recognized by mAb 2G12, indicates present or previous active infection of the subject with HIV virus. Means for diagnosis, prognosis, or staging of a viral infection in a biological sample according to the methods of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit containing at least one viral-associated carbohydrate antigen. In a prefened embodiment, the kit can further comprise an agent capable of detecting an immune reaction or reactivity, such as an agent for detection of bound antibodies (for example, enzyme- or fluorophore-liiiked anti-IgG antibody) to said at least one viral-associated carbohydrate antigen. Diagnostic kits are well known in the art, and are commonly used for the detection of pregnancy, glucosuria, and viral, bacterial and other infections (see, for example, the HIV detection kits NED AS HTV DUO, bioMerieux sa, France and the B-Safe HIV 1-2 kit, USA BioMed Las Vegas ?ΝN). The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of diagnostic kits, which notiόe is reflective of approval by the agency of the form of the kits for human or veterinary diagnostics. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Means for performing the methods of the invention may also be prepared, placed in an appropriate container, and labeled for diagnosis, prognosis or staging of an indicated condition, as if further detailed above. Suitable indicia on the label may include detection, diagnosis, prognosis and/or staging of a viral disease, viral infection, AIDS or infection with HIN-1 or HIN-2. One such exemplary test kit includes: 1. A oligosaccharide containing antigen coated microtiter plate(s). Optionally, the plate can include wells, which are coated with an oligosaccharide- devoid antigen, as control. 2. Negative and positive control specimens. 3. Anti-human-immunoglobulin conjugated to a chromogenic enzyme

(second, reporter antibody). Preferable enzymes are: Horseradish-peroxidase (HRP), alkaline phosphatase and β-galacatosidase. 4. Sample and reactant/conjugate diluents. 5. Wash solution. 6. A chromogenic substrate (for enzyme detection) solution. 7. Optional reagent system for dissociating oligosaccharide antigen- antibody immune complexes (see above). 8. Optional chromogenic reaction stop solution. All or parts of the above components are optionally presented in a dry form, to be reconstituted by the operator.

The present invention successfully addresses the shortcomings of the presently known configurations by providing methods and kits for the diagnosis of viral infections by detection of an immune response to at least one viral-associated carbohydrate antigen. Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion. Materials and Experimental Methods Oligo-mannose containing antigens: Native and recombinant HIV, HCN and other viral antigens (HIN gρl20, HIV env, HIV gag, HIV gp41, HIV gpl60; HCV ΝS3, HCN.ΝS4, etc) are obtained commercially (Advanced Biotechnologies Ine, Columbia,, 1VΪD; Researchi Diagnostics, Flanders, NJ). Qvalbumin, soybean agglutinin, yeast mannan, ribonuclease B, and other sources of carbohydrate moiety antigens are obtained from Sigma Chemical Co. (St.

Louis, MO, USA). Antibodies: Anti-carbohydrate epitope antibodies such as the 2G12 monoclonal antibody are obtained from NIH AIDS Research and Reference Reagent Program, Rockville,

MD, USA. Enzymes and chemicals'. Sodium periodate, almond mannosidase, neuroa inidase, additional glycosidases, methyl alpha-?D-mannopyranoside and p-nitrophenyl ct-D-mannoside are obtained from; Sigma Chemical Co. (St. Louis, MO, USA). Microbial, fungal, plant and animal endoglycόsidases (N-Glycanase, O-Glycanase, Ceramide Glycanase, PNGaseF, and the like) and exoglycosidases (Sialidase/Neuraminidase, Beta Galactosidase, Hexosaminidase, Galactosaminidase, Mannosidase, Fucosidase and the like) are obtained from Glyko-Prozyme (San Leandro, CA, USA). Removal of the N- & simple O-linked (including polysialylated) carbohydrates from glycoproteins is also performed using the Enzymatic Deglycosylation Kit (GK80110) (Glyko-Prozyme, San Leandro, CA, USA). Enzymatic reactions are carried out according to manufacturers' directions. Deglycosylation Oligo-mannose containing antigens, such as HIV gρl60, HIV gp41, and HCV glycoproteins are deglycόsylated using one or more chemical or enzymatic processes resulting in removal Of the oligosaccharide moieties. Chemical deglycosylation by periodate oxidation is according to Spiro et al (JBC 1964;239:567). Briefly, periodate oxidation of antigens is carried out in acidic conditions (pH 4.0, 0.025M _. NaIO₄ in 0.1M Na₂COOH buffer) or mild alkaline conditions (pH 7.5, 0.0125M KIO₄in 0.1M Tris) at 25 °C, in the dark, and reaction terminated by 0.1M sodium phosphate buffer with 0.1M ethylene glycol. Optionally the antigen solution is incubated at room temperature for 1 hour with 25mM of NaIO₄ in 50mM. sodium acetate buffer, pH 4.5, in the dark, followed by 1 hr incubation at room temperature with 1% glycine in PBS (Priest et al, 2003). Enzymatic hydrolysis of mannose-containing oligosaccharide moieties from a glycoprotein is performed essentially as previously described (Mao et al,

Elecfrophoresis 2003 24:3273-3278), using commercial mannosidase [alpha mannosidase for terminal (1, 2) mannose, alpha mannosidase IvIANNI for terminal (1-6) mannose] (Glyko Prozyme, San Laurento CA) and manufacturer's recommended, incubation. For complete removal of all Ν- & simple O-linked (including polysialylated) carbohydrates from glycoproteins, the "Enzymatic Deglycosylation Kit" from Glyko (Glyko-Prozyme, San Leandro, CA, USA) is used, according to the manufacturers instructions. Monitoring the enzymatic deglycosylation is performed by the addition of p- nitrophenyl α-D-mannoside to the reaction mixture, the appearance of yellow color indicating the cleavage of the nitrophenyl group by the mannosidase. Degradation or release of the oligpsaccharide from the treated biomolecules is monitored by known carbohydrate detection methods such as periodate oxidation followed by direct staining with Alcian Blue or with silver stain.

EXAMPLE 1 Detection of oligo-mannose binding antibodies in human serum or plasma using ELISA As mentioned hereinabove, a variety of protocols and kits for HIN testing in biological samples from subjects are available, having a range of specificity and sensitivity value,? ?BLISA tests for anti-HIV antibodies have been described in detail, and are presently μsed for institutional and private applications. In order to evaluate the specificity? and sensitivity of. detection of anti-viral-associated carbohydrate antigen antibodies in biological samples, ELISA assay for anti-HIN associated carbohydrate antigen antibodies is calibrated with known antigen and antibody, and then anti-HIN antibody titer in biological fluid samples from HIV positive and control subjects is assayed. Calibration of detection of antibodies to HIV associated carbohydrate antigens: Calibration of the ELISA is performed with a known anti-HIV-associated carbohydrate antigen? antibody, the monoclonal antibody 2G12 (ΝIH AIDS Research and Reference Reagent Program, Rpckville, MD, USA), which recognizes Manc -* 2 Man-linked sugars found on the outer face of HIV gpl20 (Scanlan et al J of Virol 2002; 76:7306-7321), and a variety of sources of the oligo-mannose antigen, including glycosylated gp 120 glycoprotein (Cat No.14-102-050 Advanced Biotechnologies hie, Columbia MD), ovalbumin, soybean agglutinin, yeast mannin, and RNAse B (Sigma Chemicals, St Louis MO), and purified oligo-mannose having αl→ 2 bonds (products 00-025 to 00-033, GlycoTech, Gaithersburg MD). The oligo- mannose containing antigens, and control antigens lacking mannose (recombinant and deglycosylated gpl20) and oligo-mannose having αl→ 6 and αl→ 3 linkage rather than αl→ 2 linkage (GlycoTech, Gaithersburg MD) are affixed to the wells of a 96-well micrόtϊter plate using a bifunctional linker as described by Dukler et al. for preparation of glycan microarrays (US Patent Application No. 09/860,488 to Dukler et al) in a range of concentrations (serial dilutions from 1:10 to 1:1000), washed with TBS containing 0.1% TweenfTBST) (Sigma, St Louis, MO), and blocked with BSA (to prevent non-specific binding). Antibody-carbohydrate antigen binding is performed by addition of 2G12 antibody, in TBS, diluted from 1:10 to 1:1000, into the wells, to a final volume of 100 μl. An identical series of controls are prepared using a control antibody specific for HIV antigen gpl20 aa307-320 (anti-gpl20 M77/3P3, Cat No, 13-105-100 Adv Biotechnologies Ine, Columbia MD), and not cross-reactive with the oligo-mannose epitope, and the microtiter plates are incubated at 25 °C for up to 2 hours. The antibody solutions,, are washed from the wells with TBST, and then the plates are incubated ith a? ?second HRP labeled anti-IgG antibody (Cat No. 115-035-166, Jackson Jmnxύnό ? Research Laboratories fric, West Grove PA), according to manufacturer's recommendations. After removal of the second antibody by washing, plates were developed with a buffer containing a 1:2000 dilution of 30% hydrogen peroxide and .photometric determination and concentration analysis is performed, according to manufacturers instructions, using a microtiter plate ELISA reader (ADI Cat No. MPR-20)?at 450 nm. Concentration dependent color development in the 2G12-glycosylated gp-120 wells indicates sensitivity of the assay to anti-HIV-associated carbohydrate antibody- antigen pairs/ while absence of color development with either the control antibody anti-gpl20 M77/3P3, . or .with the deglycosylated gpl20 and other control antigens indicates specificity of the assay to anti-HIN-associated carbohydrate antibody- antigen pairs. Similar development with other oligo-mannose containing antigens indicates the suitability of their incorporation into the ELISA assay for detection of antibodies to anti-HIV-associated carbohydrate antigens in biological samples. Further calibration is then performed, in order to determine concentrations of reagents within the range of linear response, in the presence of human serum. 5-50 μL of human serum and plasma (HIV negative, Bioreclamation Inc., East Meadow, ΝY) are added in place of TBST buffer along with the 2G12 and M77/3P3 antibody, and the reaction mixtures performed as before. Color development similar to the calibration without serum or plasma indicates the sensitivity and specificity of the assay for detection of an immune response to HIV associated carbohydrates in biological samples?, such as blood and plasma. Detection of oligo-mannose binding antibodies in human serum or plasma:

In order to test the specificity and sensitivity of the ELISA test for immune response to an HIV associated carbohydrate antigen in actual biological samples, samples of

HIN positive serum and plasma, from various stages of infection, and HIV negative control sera are diluted in serial dilution (1:10 - 1:500) with TBS, and assayed as described above (minus the antibodies to HIV-associated carbohydrates) in the

ELISA assay, using a panel of carbohydrate antigens including, but not limited to HlV-specific gpl20 mannose disaccharide and oligo mannose antigens described above. Low numbers of false positives (color detection with HlV-negative samples) indicates specificity of the assay, while low numbers of false negatives (no color detection with. HIV-positive samples) across a range of concentrations indicates sensitivity of the assay.

EXAMPLE 2 Disruption of carbohydrate-antigen antibody complexes for detection ofHW- specific oligo-mannose binding antibodies in human serum or plasma Viruses such as HIV are known to shed their coat glycoprotein components, making detection of exposed epitopes problematic. Such shedding of glycan epitopes can result, in? the formation of antibody-antigen complexes between the HIV carbohydrate (glycan) . antigens arid their respective specific antibodies. Such antibody blocking can interfere with, and even prevent the detection of diagnostic anti HIV carbohydrate (glycan) antibody titers in biological samples of patients being tested. Dissociation of the antibody-antigen complex in the samples before testing for anti- HIV carbohydrate (glycan) antibodies can free such bound antibodies, and make them available to detection using the bound HIV carbohydrate (glycan) antigens assay described above. In order to assess the effect of dissociation of the antibody-antigen complex on the sensitivity of detection of oligo-mannose binding antibodies in human serum or plasma by ELISA, the serum and plasma samples are assayed after treatment to either degrade or release, and subsequently remove the carbohydrate (glycan) antigens before the detection steps. Effect of degradation of the carbohydrate (glycan) antigens on detection of anti- HIV carbohydrate (glycan) antibodies: Samples of HIV positive serum and plasma, from various stages of infection, and HIV negative control sera are reacted with endo-and exoglycosidases as described in Materials and Experimental Methods hereinabove, or deglycosylated with the Enzymatic Deglycosylation Kit (Glyko- Prozyme, San Leandro, CA; USA). Following incubation, the enzymes are deactivated according to manufacturers instructions, the sample diluted in serial dilution (1:10- 1:500) with TBS, and assayed as described above in the ELISA assay, using a panel? of carbohydrate antigens. Control samples are incubated without deglycosylating enzymes. Positive detection of anti- HIV carbohydrate (glycan) antibodies in samples from known HIN positive subjects, which test negative without enzyme treatment, indicates the efficacy of enzyme treatment of the biological samples previous to detection of anti- HIN carbohydrate (glycan) antibodies by the present method. In order tø.test the effectiveness of chemical degradation of HIN carbohydrate (glycan) antigens- in antibody-antigen complexes, the plasma and serum samples are incubated with periodate as described hereinabove. Following periodate treatment, the samples are diluted as described, and assayed for anti-HIN carbohydrate (glycan) antibodies by ELISA, as described. Positive detection of anti-HIN carbohydrate (glycan) antibodies in samples from known HIV positive subjects, which test negative witnout penoαate treatment, mdicates the efficacy of periodate treatment of the biological samples previous to detection of anti- HIV carbohydrate (glycan) antibodies by the present method. Effect of competitive dissociation of the antibody- HIV carbohydrate

(glycan) complex with another carbohydrate on detection of anti- HIV carbohydrate (glycan) antibodies'. Incubation in a 1M solution of methyl α-D- mannopyranoside has been shown to dissociate the high affinity complex between an oligo-riiannose and Concariavalin A (Deras et al., 1998). In order to test the effectiveness of competitive dissociation of HIV carbohydrate (glycan) antigens in antibody-antigen complexes, the plasma and serum samples are incubated with methyl α-D-mannopyranoside. Following dissociation, the mannopyranoside- containing samples are diluted and assayed for anti-HIN carbohydrate (glycan) antibodies with the immobilized HlV-specific oligo-mannose antigen by ELISA, as described. Binding of the antibody to the immobilized antigen will occur in the presence of the soluble mannopyranoside due to the dilution of the methyl α-D- mannopyranoside and the higher affinity of the antibody to a multi-epitope antigen on the surface of the ELISA plate well. Positive detection of anti-HIV carbohydrate (glycan) antibodies in samples from known HIV positive subjects, which test negative without α-D-manriopyranoside treatment, indicates the efficacy of competitive dissociation treatment of the biological samples previous to detection of anti-HIV carbohydrate (glycan) antibodies by the present method. Effect of heat dissociation of the antibody- HIV carbohydrate (glycan) complex on detection of anti- HIV carbohydrate (glycan) antibodies: It will be appreciated, by one of ordinar skill ^'in the art, that heating at 80°C for 10 - 30 minutes dissociates antigen-antibody complexes without affecting antibody activity. This treatment cari be combined with periodate, whose activity increases with heating, so that the structure of the released oligo-mannose is immediately destroyed by the chemical. In order to test the effectiveness of thermal dissociation of HIV carbohydrate (glycan) antigens' in antibodyrantigeri complexes, the plasma and serum samples are incubated for 10,720 and 30 minutes at 80°C, with and without periodate, as described above. Following dissociation, the samples are diluted and assayed for anti- HIN carbohydrate (glycan) antibodies with the immobilized HlV-specific oligo-mannose antigen by ELISA, as described. Positive detection of anti-HIV carbohydrate

(glycan) antibodies in samples from known HIV positive subjects, which test negative without heat and/or heat and periodate treatment, indicates the efficacy of heat treatment for .dissociation of the antibody-antigen complexes in the biological samples previous/ to detection of anti- HIV carbohydrate (glycan) antibodies by the present method. Effect of pH dissociation of the antibody- HD7 carbohydrate (glycan) complex on detection of anti- HIV carbohydrate (glycan) antibodies: Immune complexes can be dissociated at extreme pH, while preserving antibody activity. Thus, incubation with glycine-HCl pH 2.0 buffer can be employed to dissociate the antibody-HIV-specific carbohydrate (glycan) complex. The released oligosacchari.de can then be degraded by chemical and/or enzymatic treatments as described above, or can be removed by binding to an immobilized lectin such as Concanavalin-A (Sigma Ine, St Louis MO). In order to test the effectiveness of pH dissociation of HIN carbohydrate

(glycan) antigens in antibody-antigen complexes, the plasma and serum samples are incubated for 10, 20 and 30 minutes with glycine-HCl pH 2.0 buffer, and then incubated with periodate, as described above, to degrade the HIN carbohydrate (glycan). Following dissociation, the samples are diluted and assayed for anti-HIV carbohydrate (glycan) antibodies with the immobilized HIV-associated oligo- mannose antigen by ELISA, as described. Positive detection of anti-HIV carbohydrate (glycan) antibodies in samples from known HIV positive subjects, which test negative without low pH and/or low pH along with periodate treatment, indicates the efficacy of low pH treatment for dissociation of the antibody-antigen complexes in, the biological samples previous to detection of anti-HIV carbohydrate (glycan) antibodies by the present method. Effect of chemical dissociation of the antibody- HIV carbohydrate (glycan) complex on detection of anti_" HIV carbohydrate (glycan) antibodies: Molar level concentrations; of some salts_. (e.g. 3M KC1, 3M MgCl₂, 3M KCΝS) or high molar concentrations ?of ?urea are commonly used to dissociate immune complexes by gentle reversible denaturation, hi order to test the effectiveness of chemical dissociation of HIV carbohydrate (glycan) antigens in antibody-antigen complexes, the plasma and serum samples are incubated for 10, 20 and 30 minutes with 2M, 4M and 8M urea.

Following dissociation, the samples are diluted and assayed for anti- HIV carbohydrate (glycan) antibodies with the immobilized HlV-specific oligo-mannose antigen by ELISA, as described. Positive detection of anti-HIV carbohydrate (glycan) antigen antibodies in samples from known HIV positive subjects, which test negative without urea treatment, indicates the efficacy of chemical dissociation of the antibody-antigen complexes in the biological samples previous to detection of anti- HIV carbohydrate (glycan) antibodies by the present method. It is appreciated? that certain features of the invention, which are, for clarity, described in the? context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents, patent applications arid sequences identified by an accession number, mentioned in this specification are -herein incorporated in their entirety by reference into the specification, to the? same extent as if each individual publication, patent, patent application or segμence was specifically and individually indicated to be incorporated herein by refererice. In addition, citation or identification of any reference in this application: shall not be construed as ari admission that such reference is available as prior art to the present invention.

Claims

WHAT IS CLAIMED IS:

1. A method of diagnosing a viral infection in a subject, comprising detecting iri a biological sample of the subject an immune reactivity to at least one viral-associated carbohydrate antigen wherein said immune reactivity to said viral- associated carbohydrate antigen is diagnostic of said viral infection, thereby diagnosing said viral infection in the subject.

2. Tbe method of claim 1, wherein said viral-associated carbohydrate antigen is a viral-specific carbohydrate antigen.

3. The method of claim 1, wherein said immune reactivity comprises the presence of anti- viral-associated carbohydrate antigen antibodies.

4. The method of claim 3 , wherein said detecting comprises: (a) contacting said biological sample with at least one viral-associated carbohydrate aritigen under conditions allowing a formation of antigen-antibody complexes; and (b) detecting said formation of said antigen-antibody complexes.

5. The method of claim 3, wherein said detecting is effected by a method selected from the group consisting of an enzyme linked immunosorbent assay (ELISA), a radioirnmunoassay (RIA), an enzyme immunosorbent assay (EIA), a fluoroimriiun assay, Western blotting, immune precipitation, FACS and inimimohistochemistry.

6. The method, of claim 1, wherein said immune reactivity is a cellular reaction to at least one viral-associated carbohydrate antigen.

7. The method of claim 6, wherein said detecting comprises: (a) contacting cells of said biological sample with at least one viral- associated carbohydrate antigen; and (b) detecting in said cells an antigen-specific cellular response to said at least one viral-associated antigen.

8. The method of claim 4, wherein said cellular response is selected from the group consisting of lymphocyte proliferation, delayed-type hypersensitivity, chemotaxis, extravasation, migration, cytokine secretion, detection of activation cell surface markers and cytotoxicity assay.

9. The method of claim 1, wherein said detecting is preceeded by processing said biological sample to substantially dissociate viral-associated carbohydrate antigen-antibody complexes, thereby allowing detection of dissociated anti-viral-associated carbohydrate antigen antibodies.

10. The method of claim 9, wherein said processing is effected by at least one method selected from the group consisting of antigen degradation, competitive displacement and denaturation.

11. The method of claim 10, wherein said antigen degradation is chemical, mechanical or enzymatic degradation.

12. The method of claim 9, wherein said processing further comprises dissociating said viral-associated carbohydrate antigens from said dissociated antibodies.

13. The method of claim 12, further comprising separating said viral- associated carbohydrate antigens from said dissociated antibodies.

14. The method of claim 1, wherein at least one viral-associated carbohydrate antigen is a carbohydrate moiety selected from the group consisting of a carbohydrate moiety of a glycoprotein, a carbohydrate moiety of a proteoglycan and a carbohydrate moiety of a glycolipid.

15. The method of claim 1, wherein said viral infection is selected from the group consisting of a retrovirus infection, an RNA virus infection and a DNA virus infection.

16. A kit for diagnosis, prognosis, or staging of a viral infection in a biological sample, said kit comprising at least one viral-associated carbohydrate antigen.

17. The kit of claim 16, further comprising an agent capable of detecting an immune reaction to said at least one viral-associated carbohydrate antigen.

18. A method of diagnosing an HIV infection in a subject, comprising detecting in a biological sample of the subject an immune reactivity to at least one HIV-associated carbohydrate antigen, wherein said immune reactivity to said HIV- associated carbohydrate antigen is diagnostic of said HIV infection, thereby diagnosing said HIV infection in the subject.

19. The method of claim 18, wherein said viral-associated carbohydrate antigen is a viral-specific carbohydrate antigen.

20. The method of claim 18, wherein said viral infection is selected firom the group consisting of an HIV-1 infection, an HIV-0 infection and an HTV-2 infection.

21. Title method of claim 18, wherein said immune reactivity comprises the presence of anti-HIV-associated carbohydrate antigen antibodies.

22. The method of claim 21, wherein said detecting comprises: (a) contacting said biological sample with at least one HIV-associated carbohydrate antigen under conditions allowing a formation of antigen-antibody complexes; and (b) detecting said formation of said antigen-antibody complexes.

23. The method of claim 22, wherein said detecting is effected by a method selected from the group consisting of an enzyme linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), an enzyme immunosorbent assay (EIA.), a fluoroimmunoassay, Western blotting, immune precipitation, FACS and immunohistocheihistiy.

24. The method of claim 18, wherein said immune reactivity is a cellular reaction to at least one HIV-associated carbohydrate antigen.

25. The method of claim 24, wherein said detecting comprises: (a) contacting cells of said biological sample with at least one HIV- associated carbohydrate antigen; and (b) defecting in said cells an antigen-specific cellular response to said at least one HIV -associated antigen.

26. The method of claim 25, wherein said cellular response is selected from the group consisting of lymphocyte proliferation, delayed-type hypersensitivity, chemotaxis, extravasation, migration, cytokine secretion, detection of activation cell surface markers and cytotoxicity assay.

27., The method of claim 18, wherein said detecting is preceeded. by processing said biological sample to substantially dissociate HIN-associated carbohydrate antigen-antibody complexes, thereby allowing detection of dissociated anti-viral-associated carbohydrate antigen antibodies.

28. he method of claim 27, wherein said processing is effected by at least one method selected from the group consisting of antigen degradation, competitive displacement and denaturation.

29. The -method of claim 28, wherein said antigen degradation is chemical, mechanical, or enzymatic degradation.

30. The method of claim 27, wherein said processing further comprises disociating said HIV-associated carbohydrate antigens from said dissociated antibodies.

31. The method of claim 29, further comprising separating said HIV- associated carbohydrate antigens from said dissociated antibodies.

32. The method of claim 18, wherein at least one HIV-associated carbohydrate antigenis a carbohydrate moiety selected from the group consisting of a carbohydrate moiety of a glycoprotein, a carbohydrate moiety of a proteoglycan and a carbohydrate moiety of a glycolipid.

33. A kit for diagnosis, prognosis, or staging of an HIN infection in a biological sample, said kit comprising at least one HIN-associated carbohydrate antigen.

34. The kit of claim 33, further comprising an agent capable of detecting an immune reaction to said at least one viral-associated carbohydrate antigen.