Methods for detection of flavivirus antibodies
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-11353454-B2
Publication Date
2022-06-07
Expiration Date
2040-03-26
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Abstract
Isolated peptides that include one or more antigenic sites of Zika virus (ZIKV) and methods of their use and production are disclosed. The peptides can be used, for example, to detect exposure of a subject to a flavivirus infection, such as a ZIKV infection.
Core Innovation
The invention provides isolated peptides comprising antigenic sites of the Zika virus (ZIKV) polyprotein, which can be used to detect exposure of a subject to flavivirus infections, including ZIKV infection. These peptides can be less than 100 amino acids in length and may be linked to a solid support, fused to a heterologous protein, or conjugated to a heterologous carrier for use in diagnostic and detection assays. Methods are also disclosed for detecting anti-flavivirus antibodies in biological samples by contacting the sample with the ZIKV peptides and detecting the formation of immune complexes, indicating the presence of anti-flavivirus antibodies.
The problem addressed by the invention is the difficulty in accurately diagnosing ZIKV infection due to pre-existing cross-reactive antibodies against other flaviviruses that circulate in the same geographic areas. Conventional serologic tests have limitations in sensitivity and specificity, complicated by antibody cross-reactivity and the time-consuming nature of confirmatory assays like plaque reduction neutralization tests (PRNT). There is a need for identifying new antigenic targets within the ZIKV polyprotein that are recognized by antibodies at early stages post-exposure to improve diagnostic accuracy and speed for flavivirus infections, including ZIKV.
The disclosed peptides encompass antigenic sites recognized by IgM, IgG, and IgA antibodies in serum and urine following acute ZIKV infection, with differential antibody binding patterns and immune focusing over time. Structural analyses show that the identified antigenic sites are predominantly surface-exposed on ZIKV structural and non-structural proteins. These findings enable development of serodiagnostic assays using peptides derived from specific non-structural proteins such as NS1, NS2B, NS3, NS4B, and NS5 that exhibit high sensitivity and specificity in detecting ZIKV infection versus other flaviviruses. The invention also contemplates immunogenic compositions and vaccination methods based on these peptides to induce protective immune responses.
Claims Coverage
The patent contains multiple independent claims covering isolated peptides, solid supports linked to peptides, and methods for detecting anti-flavivirus antibodies using specific peptides.
Isolated peptides comprising defined ZIKV antigenic sequences linked or conjugated to carriers
The isolated peptides consist or consist essentially of amino acid sequences selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 or 10. The peptides can be linked to a solid support, fused to a heterologous protein, or conjugated to a heterologous carrier.
Linking peptides to solid supports via specific linkers
The peptides can be linked to solid supports by linkers such as biotin, streptavidin, maleimide, polyethylene glycol (PEG), peptide linkers, or combinations thereof. The solid supports include beads, membranes, reaction trays, multi-well plates, or test tubes.
Fusion of peptides to heterologous proteins or conjugation to heterologous carriers
The peptides can be fused to heterologous proteins comprising tags or linkers, or conjugated to heterologous carriers which include proteins from bacteria, viruses, keyhole limpet hemocyanin (KLH), ovalbumin (OVA), or bovine serum albumin (BSA).
Solid supports linked to specific peptides for immunoassay use
Solid supports linked to one or more peptides consisting or consisting essentially of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 or 10, especially peptides SEQ ID NOs: 2, 5, 7 or 8, intended for use in detection assays.
Methods for detecting anti-flavivirus antibodies via specific peptide binding
Methods involve contacting a biological sample with one or more peptides consisting or consisting essentially of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 or 10 under conditions sufficient to form an immune complex between peptides and antibodies in the sample, and detecting the immune complex to indicate presence of anti-flavivirus antibodies.
Use of peptides to identify subjects with flavivirus infection
The detection methods can be applied to human or non-human samples containing antibodies from subjects at risk or suspected of flavivirus infection, including ZIKV.
The claims cover isolated antigenic ZIKV peptides conjugated to carriers or fused to heterologous proteins, solid supports linked to these peptides, and the use of such peptides and conjugates in methods to detect anti-flavivirus antibodies in biological samples. The focus is on specific sequences identified by SEQ ID NOs and their application in diagnostics and immunoassays.
Stated Advantages
Identification of new ZIKV antigenic sites that improve sensitivity and specificity of diagnostic tests by discriminating ZIKV infection from other flavivirus infections.
Improved accuracy and speed of serologic diagnosis for flaviviruses including ZIKV compared to conventional methods.
Use of peptides for early detection of antibodies post ZIKV exposure, enabling detection across different stages of infection and in different biological fluids such as serum and urine.
Capability to distinguish natural flavivirus infection from vaccination based on antibody binding to non-structural proteins absent in vaccines.
Potential for development of immunogenic compositions and vaccines based on identified peptides inducing protective immune responses.
Documented Applications
Detection of exposure and diagnosis of flavivirus infections, including ZIKV, by detecting anti-flavivirus antibodies in biological samples such as blood, serum, plasma, urine, saliva, tears, feces, semen, mucous, tissue extracts, and spinal fluid.
Use of the isolated peptides conjugated or linked to solid supports in immunoassays including ELISA, surface plasmon resonance, lateral flow assays and point-of-care assays for serodiagnostics.
Differential diagnosis to distinguish between naturally infected subjects and vaccinated subjects by detecting antibodies against flavivirus non-structural proteins.
Evaluation of immune responses induced by vaccines through antibody binding to specific ZIKV peptides to monitor vaccine efficacy.
Development of immunogenic compositions and methods of administering peptides or encoding nucleic acids to induce immune responses against ZIKV for prophylactic or therapeutic purposes.
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