Methods for identifying flavivirus infections, and related peptides, kits, and compositions

Abstract

A method for identifying a flavivirus infection selected from Zika virus (ZIKV), Dengue virus (DENV), and combinations thereof in a subject is provided, comprising determining whether a sample from the subject reacts with a peptide associated with a specific relative binding affinity. Also claimed are kits, isolated peptides, immune system stimulating compositions comprising the specific peptides, and methods for distinguishing ZIKV infection from DENV infection.

Description

[Technical field]

[0001] The present disclosure relates generally to methods for identifying Flavivirus infection, as well as related peptides, kits, and compositions. [Background technology]

[0002] Flaviviridae is a family of positive-sense, single-stranded, enveloped RNA viruses. Flaviviruses have been reported to cause widespread morbidity and mortality worldwide. Some flaviviruses are transmitted by mosquitoes, and these include yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV).

[0003] Dengue fever, caused by DENV, is found in tropical and subtropical climates around the world. In recent decades, the global incidence of dengue fever has increased dramatically, and now about half of the world's population is at risk. There are four DENV serotypes: DENV1, DENV2, DENV3, and DENV4. Because the four serotypes are distinct, a person can be infected with DENV up to four times in their lifetime.

[0004] ZIKV was first identified in Uganda in 1947 and isolated from infected monkeys. ZIKV has re-emerged as an important flavivirus causing multiple Zika fever (ZIKF) epidemics worldwide. ZIKV outbreaks in French Polynesia and Brazil in 2013 and 2015 resulted in unexpected severe neurological and congenital complications.

[0005] Current diagnostics of flaviviruses such as DENV and ZIKV, which rely heavily on molecular methods, have several limitations because patients exhibit short viremic phases with low levels of viremia that may escape detection even in symptomatic patients. Serological testing as an alternative diagnostic approach is hampered by antibody cross-reactivity between flaviviruses, e.g., DENV and ZIKV, which share high amino acid identity (55%) and are structurally homologous. Furthermore, both viruses are transmitted by the same mosquito vector and are therefore often found in overlapping geographical areas.

[0006] Some antigens can probably distinguish between ZIKV and DENV infections, as well as other flavivirus infections. Several differential epitopes have been predicted by computational studies, but validation in patient samples remains a challenge.

[0007] Thus, there is a need to provide alternative methods for identifying Flavivirus infections, as well as related peptides, kits, and compositions. Summary of the Invention

[0008] In one aspect, a method for identifying a flavivirus infection selected from Zika virus (ZIKV), Dengue virus (DENV), and combinations thereof in a subject, comprising: The subject's sample has a relative binding capacity BC relative 0.05 or ≧0.1,

[0009]

number

[0010] (i) When peptide P contains a ZIKV-derived peptide,

[0011]

number

[0012] or (ii) When peptide P contains a DENV-derived peptide,

[0013]

number

[0014] A method is provided in which

[0015] In one embodiment, peptide P comprises an epitope in the prM protein, E glycoprotein, or NS1 protein of ZIKV or DENV.

[0016] In one embodiment, the epitope is located in a solvent exposed region of the prM protein, the E glycoprotein, or the NS1 protein.

[0017] In one embodiment, the peptide P is 5 to 25 amino acids in length.

[0018] In one embodiment, peptide P shares sequence similarity with the corresponding peptide C of 50% or less.

[0019] In one embodiment, the peptide P is SEQ ID NO:7 (LDEGVEPDDVDCWCNTT); SEQ ID NO:36 (SVEGELNAILEENGVQ); SEQ ID NO:38 (GKSYFVRAAKTNNSFVVD); SEQ ID NO:39 (GDTLKECPLKHRAWNSFL); SEQ ID NO:46 (KVHVEETCGTRGPSLRST); SEQ ID NO:49 (ECTMPPLSFRAK); SEQ ID NO:3 (SFPTTLGMNKCYIQIMDL); SEQ ID NO:26 (GALEAEMDGAKGRLSSGH); SEQ ID NO:32 (FKSLFGGMSWFSQILIGT); SEQ ID NO:9 (YGTCHHKKGEARRSR); SEQ ID NO:17 (PENLEYRIMLSVHGSQHS); SEQ ID NO:43 (REGYRTQMKGPWHSEELE); SEQ ID NO:45 (IRFEECPGTKVHVEETCG); a peptide sharing at least 75% sequence identity with these; or a peptide that differs from these by 1, 2, 3, or 4 amino acids; or Some of these; and / or one or more ZIKV-derived peptides selected from the group consisting of: SEQ ID NO:58 (HITEVEPEDIDCWCNLT); SEQ ID NO:87 (QIANELNYILWENNIK); SEQ ID NO:89(GKAKIVTAETQNSSFIID); SEQ ID NO:90(GPNTPECPSASRAWNVWE); SEQ ID NO:97 (TVVITENCGTRGPSLRTT); SEQ ID NO:100(SCTLPPLRYMGE); SEQ ID NO:54 (LFKTASGINMCTLIAMDL); SEQ ID NO:77 (GATEIQNSGGTSIFAGH); SEQ ID NO:83(YTALFSGVSWVMKIGIGV); SEQ ID NO:60 (TSTWVTYGTCNQAG); SEQ ID NO:68(YENLKYTVIITVHTGDQH); SEQ ID NO:94 (RPGYHTQTAGPWHLGKLE); SEQ ID NO: 96 (LDFNYCEGTTVVITENCG); or a peptide sharing at least 75% sequence identity with these; or a peptide that differs from these by 1, 2, 3, or 4 amino acids; or Some of these. One or more DENV-derived peptides selected from the group consisting of Includes.

[0020] In one embodiment, the method comprises the steps of: SEQ ID NO:7 (LDEGVEPDDVDCWCNTT); SEQ ID NO:36 (SVEGELNAILEENGVQ); SEQ ID NO:38 (GKSYFVRAAKTNNSFVVD); SEQ ID NO:39 (GDTLKECPLKHRAWNSFL); SEQ ID NO:46 (KVHVEETCGTRGPSLRST); SEQ ID NO:49 (ECTMPPLSFRAK); SEQ ID NO:58 (HITEVEPEDIDCWCNLT); SEQ ID NO:87 (QIANELNYILWENNIK); SEQ ID NO:89(GKAKIVTAETQNSSFIID); SEQ ID NO:90(GPNTPECPSASRAWNVWE); SEQ ID NO:97 (TVVITENCGTRGPSLRTT); SEQ ID NO:100(SCTLPPLRYMGE); a peptide sharing at least 75% sequence identity with these; or a peptide that differs from these by 1, 2, 3, or 4 amino acids; or Some of these; and / or determining whether the subject reacts with one or more peptides selected from the group consisting of: If the sample reacts with one or more of the peptides or portions thereof, the subject is indicated for treatment of a Flavivirus infection.

[0021] In one embodiment, the method is a method for identifying an acute phase flavivirus infection, the method comprising: SEQ ID NO:7 (LDEGVEPDDVDCWCNTT); SEQ ID NO:36 (SVEGELNAILEENGVQ); SEQ ID NO:38 (GKSYFVRAAKTNNSFVVD); SEQ ID NO:58 (HITEVEPEDIDCWCNLT); SEQ ID NO:87 (QIANELNYILWENNIK); SEQ ID NO:89(GKAKIVTAETQNSSFIID); determining whether the sample reacts with one or more peptides selected from the group consisting of If the sample reacts with one or more peptides or portions thereof, the subject is indicated for treatment of acute flavivirus infection.

[0022] In one embodiment, the method comprises the steps of: SEQ ID NO:3 (SFPTTLGMNKCYIQIMDL); SEQ ID NO:26 (GALEAEMDGAKGRLSSGH); SEQ ID NO:32 (FKSLFGGMSWFSQILIGT); a peptide sharing at least 75% sequence identity with these; or a peptide that differs from these by 1, 2, 3, or 4 amino acids; or Some of these; determining whether the peptide reacts with one or more peptides selected from the group consisting of: If the sample reacts with one or more peptides or portions thereof, the subject is eligible for treatment of a ZIKV infection.

[0023] In one embodiment, the method is a method for identifying acute phase ZIKV infection, the method comprising: SEQ ID NO:32 (FKSLFGGMSWFSQILIGT); a peptide that shares at least 75% sequence identity with it; or a peptide which differs therefrom by 1, 2, 3, or 4 amino acids; or Some of it; determining whether the antibody reacts with If the sample reacts with the peptide or a portion thereof, the subject is indicated for treatment of acute ZIKV infection.

[0024] In one embodiment, the method comprises the steps of: SEQ ID NO:60 (TSTWVTYGTCNQAG); SEQ ID NO:68(YENLKYTVIITVHTGDQH); SEQ ID NO:94 (RPGYHTQTAGPWHLGKLE); SEQ ID NO: 96 (LDFNYCEGTTVVITENCG); and / or a peptide sharing at least 75% sequence identity with these; or a peptide that differs from these by 1, 2, 3, or 4 amino acids; or Some of these; determining whether the sample reacts with one or more peptides selected from the group consisting of If the sample reacts with one or more peptides or portions thereof, the subject is indicated for treatment of DENV infection.

[0025] In one embodiment, determining whether a sample reacts with peptide P comprises performing an immunoassay to assess whether an antigen-binding protein capable of binding to the peptide is present in the sample.

[0026] In one embodiment, the method further includes administering a ZIKV and / or DENV treatment regimen to the subject if the subject is eligible for treatment of a ZIKV and / or DENV infection.

[0027] In one aspect, a kit for identifying a flavivirus infection selected from Zika virus, Dengue virus, and combinations thereof in a subject is provided, comprising a relative binding affinity BC relative ≦0.05 or ≧0.1,

[0028]

number

[0029] When peptide P comprises a ZIKV-derived peptide,

[0030]

number

[0031] and When peptide P contains a DENV-derived peptide,

[0032]

number

[0033] A kit is provided,

[0034] In one embodiment of the kit, the peptide P is SEQ ID NO:7 (LDEGVEPDDVDCWCNTT); SEQ ID NO:36 (SVEGELNAILEENGVQ); SEQ ID NO:38 (GKSYFVRAAKTNNSFVVD); SEQ ID NO:39 (GDTLKECPLKHRAWNSFL); SEQ ID NO:46 (KVHVEETCGTRGPSLRST); SEQ ID NO:49 (ECTMPPLSFRAK); SEQ ID NO:3 (SFPTTLGMNKCYIQIMDL); SEQ ID NO:26 (GALEAEMDGAKGRLSSGH); SEQ ID NO:32 (FKSLFGGMSWFSQILIGT); SEQ ID NO:9 (YGTCHHKKGEARRSR); SEQ ID NO:17 (PENLEYRIMLSVHGSQHS); SEQ ID NO:43 (REGYRTQMKGPWHSEELE); SEQ ID NO:45 (IRFEECPGTKVHVEETCG); a peptide sharing at least 75% sequence identity with these; or a peptide that differs from these by 1, 2, 3, or 4 amino acids; or Some of these; and / or one or more ZIKV-derived peptides selected from the group consisting of: SEQ ID NO:58 (HITEVEPEDIDCWCNLT); SEQ ID NO:87 (QIANELNYILWENNIK); SEQ ID NO:89(GKAKIVTAETQNSSFIID); SEQ ID NO:90(GPNTPECPSASRAWNVWE); SEQ ID NO:97 (TVVITENCGTRGPSLRTT); SEQ ID NO:100(SCTLPPLRYMGE); SEQ ID NO:54 (LFKTASGINMCTLIAMDL); SEQ ID NO:77 (GATEIQNSGGTSIFAGH); SEQ ID NO:83(YTALFSGVSWVMKIGIGV); SEQ ID NO:60 (TSTWVTYGTCNQAG); SEQ ID NO:68(YENLKYTVIITVHTGDQH); SEQ ID NO:94 (RPGYHTQTAGPWHLGKLE); SEQ ID NO: 96 (LDFNYCEGTTVVITENCG); or a peptide sharing at least 75% sequence identity with these; or a peptide that differs from these by 1, 2, 3, or 4 amino acids; or Some of these One or more DENV-derived peptides selected from the group consisting of Includes.

[0035] In one embodiment, the kit comprises: Plates coated with anti-ZIKV and / or anti-DENV capture agents; and A detection agent for detecting the presence of captured anti-ZIKV and / or anti-DENV; and The capture agent and the detection agent include ZIKV and / or DENV antigens and / or anti-immunoglobulins.

[0036] In one embodiment, the subject comprises an Asian subject.

[0037] In one aspect, SEQ ID NO:7 (LDEGVEPDDVDCWCNTT); SEQ ID NO:36 (SVEGELNAILEENGVQ); SEQ ID NO:38 (GKSYFVRAAKTNNSFVVD); SEQ ID NO:39 (GDTLKECPLKHRAWNSFL); SEQ ID NO:46 (KVHVEETCGTRGPSLRST); SEQ ID NO:49 (ECTMPPLSFRAK); SEQ ID NO:3 (SFPTTLGMNKCYIQIMDL); SEQ ID NO:26 (GALEAEMDGAKGRLSSGH); SEQ ID NO:32 (FKSLFGGMSWFSQILIGT); SEQ ID NO:9 (YGTCHHKKGEARRSR); SEQ ID NO:17 (PENLEYRIMLSVHGSQHS); SEQ ID NO:43 (REGYRTQMKGPWHSEELE); SEQ ID NO:45 (IRFEECPGTKVHVEETCG); SEQ ID NO:58 (HITEVEPEDIDCWCNLT); SEQ ID NO:87 (QIANELNYILWENNIK); SEQ ID NO:89(GKAKIVTAETQNSSFIID); SEQ ID NO:90(GPNTPECPSASRAWNVWE); SEQ ID NO:97 (TVVITENCGTRGPSLRTT); SEQ ID NO:100(SCTLPPLRYMGE); SEQ ID NO:54 (LFKTASGINMCTLIAMDL); SEQ ID NO:77 (GATEIQNSGGTSIFAGH); SEQ ID NO:83(YTALFSGVSWVMKIGIGV); SEQ ID NO:60 (TSTWVTYGTCNQAG); SEQ ID NO:68(YENLKYTVIITVHTGDQH); SEQ ID NO:94 (RPGYHTQTAGPWHLGKLE); SEQ ID NO: 96 (LDFNYCEGTTVVITENCG); or a peptide sharing at least 75% sequence identity with these; or a peptide that differs from these by 1, 2, 3, or 4 amino acids; or Some of these An isolated peptide is provided that is selected from the group consisting of:

[0038] In one aspect, SEQ ID NO:7 (LDEGVEPDDVDCWCNTT); SEQ ID NO:36 (SVEGELNAILEENGVQ); SEQ ID NO:38 (GKSYFVRAAKTNNSFVVD); SEQ ID NO:39 (GDTLKECPLKHRAWNSFL); SEQ ID NO:46 (KVHVEETCGTRGPSLRST); SEQ ID NO:49 (ECTMPPLSFRAK); SEQ ID NO:3 (SFPTTLGMNKCYIQIMDL); SEQ ID NO:26 (GALEAEMDGAKGRLSSGH); SEQ ID NO:32 (FKSLFGGMSWFSQILIGT); SEQ ID NO:9 (YGTCHHKKGEARRSR); SEQ ID NO:17 (PENLEYRIMLSVHGSQHS); SEQ ID NO:43 (REGYRTQMKGPWHSEELE); SEQ ID NO:45 (IRFEECPGTKVHVEETCG); SEQ ID NO:58 (HITEVEPEDIDCWCNLT); SEQ ID NO:87 (QIANELNYILWENNIK); SEQ ID NO:89(GKAKIVTAETQNSSFIID); SEQ ID NO:90(GPNTPECPSASRAWNVWE); SEQ ID NO:97 (TVVITENCGTRGPSLRTT); SEQ ID NO:100(SCTLPPLRYMGE); SEQ ID NO:54 (LFKTASGINMCTLIAMDL); SEQ ID NO:77 (GATEIQNSGGTSIFAGH); SEQ ID NO:83(YTALFSGVSWVMKIGIGV); SEQ ID NO:60 (TSTWVTYGTCNQAG); SEQ ID NO:68(YENLKYTVIITVHTGDQH); SEQ ID NO:94 (RPGYHTQTAGPWHLGKLE); SEQ ID NO: 96 (LDFNYCEGTTVVITENCG); or a peptide sharing at least 75% sequence identity with these; or a peptide that differs from these by 1, 2, 3, or 4 amino acids; or Some of these An immune system stimulating composition is provided comprising a peptide selected from the group consisting of:

[0039] In one embodiment, there is provided a method for distinguishing ZIKV infection from DENV infection in a subject, comprising: determining whether the subject's sample reacts with a peptide of SEQ ID NO: 32 (FKSLFGGMSWFSQILIGT), a peptide sharing at least 75% sequence identity therewith; or a peptide differing therefrom by 1, 2, 3, or 4 amino acids; or a portion thereof, and if the sample reacts with the peptide or a portion thereof, the subject is indicated for treatment of ZIKV infection; and / or A method is provided for determining whether a sample from a subject reacts with a peptide of SEQ ID NO: 9 (YGTCHHKKGEARRSR); a peptide sharing at least 75% sequence identity therewith; or a peptide that differs therefrom by 1, 2, 3, or 4 amino acids; or a portion thereof, and if the sample reacts with the peptide or portion thereof, the subject is a candidate for treatment of DENV infection.

[0040] definition The term "identifying" as used herein in reference to an infection should be interpreted broadly to include determining the presence, absence, amount, or level of disease burden of an infection. The infection may be an acute, early convalescent phase, late convalescent phase, early recovery phase, late convalescent phase, or full convalescent phase infection.

[0041] The term "peptide" as used herein broadly refers to any chain of amino acid residues connected via peptide bonds. Peptides can be naturally occurring or synthetic (e.g., produced by chemical synthesis or recombinant DNA technology). The term "peptide" does not imply a particular size. In some instances, a peptide may not include an entire virus or full-length antigen.

[0042] The term "derived" as used herein in relation to a peptide is intended to indicate that the amino acid sequence of the peptide originates from, but is not necessarily obtained directly from, a specified source. For example, a "DENV-derived peptide" refers to a peptide whose amino acid sequence is similar or identical to a portion or fragment of naturally occurring DENV, or similar or identical to a portion or fragment of a consensus sequence of multiple naturally occurring DENV strains. A "DENV-derived peptide" may be directly isolated from naturally occurring DENV, such as by enzymatic cleavage of DENV, or more typically, may be synthesized using the amino acid sequence of naturally occurring DENV or the consensus sequence of multiple naturally occurring DENV strains as a prototype / reference. Synthesis may be performed according to standard procedures in the art, such as recombinant production techniques, genetic engineering techniques, or chemical synthesis. A "DENV-derived peptide" may include artificial and non-natural amino acids (e.g., D-amino acids, amino acid analogs, etc.). In some embodiments, a "DENV-derived peptide" may further include one or more conservative substitutions to an amino acid sequence originating from a naturally occurring DENV strain(s). The term "ZIKV-derived peptide" should be interpreted accordingly.

[0043] The term "isolated" as used herein in reference to a peptide refers to a peptide that has been removed from its natural environment (e.g., a cell, tissue, culture medium, body fluid, etc.) or that has been otherwise purified to any degree (e.g., isolated from a synthetic medium). Thus, an "isolated" peptide may be naturally produced or synthetic. A peptide may be "isolated" by separation from some or all of the materials with which it is associated in nature or as a result of synthetic production. Thus, an "isolated" peptide is typically at least partially purified.

[0044] The term "antigen binding protein" as used herein broadly refers to any peptide-based molecule that recognizes and binds to a target, e.g., a virus, such as a flavivirus, e.g., Zika virus (ZIKV) or Dengue virus (DENV). Examples of antigen binding proteins include antibodies, including antibodies of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), and antigen-binding fragments thereof. In some examples, the antigen binding protein comprises an anti-ZIKV IgG. In some examples, the antigen binding protein comprises an anti-DENV IgG.

[0045] The term "reacts with" as used herein broadly means that an antigen binding protein (such as an antibody or fragment thereof) binds to an antigen / epitope at a level higher than that of a non-specific antigen binding protein (such as an antibody or fragment thereof from a healthy sample, and / or a non-flavivirus-infected sample, and / or a non-ZIKV-infected sample, and / or a non-DENV-infected sample, etc.) to the same antigen / epitope. For example, a sample "reacts with" a peptide if it contains an antigen binding protein that exhibits higher binding activity for the peptide compared to any antigen binding protein from a control sample (e.g., a non-flavivirus-infected control sample). The term "reacts" should be interpreted accordingly.

[0046] The term "response" as used herein may be understood to be a measure of the binding activity between an antigen-binding protein and a peptide. The binding activity between an antigen-binding protein and a peptide can be measured by immunoassay.

[0047] The term "recognizes more strongly," when used herein to compare the response of an antigen binding protein to two or more peptides derived from different flaviviruses (e.g., a ZIKV-derived peptide and a DENV-derived peptide), encompasses the meaning that the binding activity between the antigen binding protein and one peptide (e.g., a ZIKV-derived peptide) is higher than the binding activity (if any) between the antigen binding protein and another peptide (e.g., a DENV-derived peptide). In some examples, an antigen binding protein recognizes a ZIKV-derived peptide more strongly than a DENV-derived peptide when the binding activity between the antigen binding protein and the ZIKV-derived peptide is higher than the binding activity between the antigen binding protein and the DENV-derived peptide, as measured by immunoassay.

[0048] The term "immune response" as used herein encompasses both cellular and humoral immune responses. In some embodiments, the immune response is sufficient to provide immune protection against a flavivirus, such as ZIKV or DENV.

[0049] The term "stimulate" as used herein in reference to an immune response broadly means an increase, amplification, or boost of an immune response. The term "stimulate" encompasses the initial stimulation of a new immune response or the enhancement of an existing immune response.

[0050] The terms "treatment," "treat," and "therapy," and their equivalents, as used herein, refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow down (attenuate) medical conditions, including, but not limited to, diseases (such as flavivirus infections), symptoms, and disorders. A medical condition also includes the body's response to a disease or disorder, such as inflammation. Those in need of such treatment include those who already have a medical condition, as well as those who are prone to a medical condition or those in whom a medical condition is desired to be prevented.

[0051] The term "subject" as used herein includes patients and non-patients. The term "patient" refers to an individual who is suffering from or likely to suffer from a medical condition, such as a flavivirus infection, while "non-patient" refers to an individual who is not suffering from and likely not suffering from a medical condition. "Non-patient" includes healthy individuals, non-diseased individuals, and / or individuals who do not have a medical condition. The term "subject" includes humans and animals. Animals include mice, etc. "Rodent" refers to any mammal of the murine family, such as mice, rats, etc.

[0052] The term "micro" as used herein should be interpreted broadly to include dimensions from about 1 micrometer to about 1000 micrometers.

[0053] The term "nano" as used herein should be interpreted broadly to include dimensions less than about 1000 nm.

[0054] The term "particle" as used herein broadly means a discrete entity or a discrete body. Particles as described herein may include organic particles, inorganic particles, or biological particles. Particles as used herein may also be macroparticles formed by the aggregation of multiple subparticles or fragments of smaller objects. Particles of the present disclosure may be spherical, substantially spherical, or non-spherical, such as irregularly shaped particles or ellipsoidal particles. The term "size", when used to refer to a particle, broadly means the largest dimension of the particle. For example, if the particle is substantially spherical, the term "size" may refer to the diameter of the particle, or if the particle is substantially non-spherical, the term "size" may refer to the largest length of the particle.

[0055] The terms "coupled" or "connected," as used herein, unless otherwise specified, are intended to encompass both direct connection and connection through one or more intermediate means.

[0056] The term "associated" as used herein when referring to two elements refers to a broad relationship between the two elements. The relationship includes, but is not limited to, a physical, chemical, or biological relationship. For example, if element A is associated with element B, elements A and B may be directly or indirectly bound to each other, element A may contain element B, or vice versa.

[0057] The term "adjacent" as used herein when referring to two elements refers to the close proximity of one element to another, which may be, but is not limited to, elements that are in contact with each other, or may further include elements that are separated by one or more additional elements disposed therebetween.

[0058] The term "and / or", e.g., "X and / or Y", should be understood to mean either "X and Y" or "X or Y" and should be considered to provide explicit support for both meanings or either meaning.

[0059] Further, in the description herein, the term "substantially", whenever used, is understood to include but is not limited to "entirely" or "completely", etc. Furthermore, terms such as "comprising", "comprise", etc., whenever used, are intended to be open-ended descriptive language in that they broadly include the elements / components listed after such term, in addition to other components not expressly listed. For example, when "comprising" is used, a reference to "a" feature is also intended to be a reference to "at least one" of that feature. Terms such as "consisting", "consist", etc., may be considered subsets of terms such as "comprising", in appropriate context. Thus, in embodiments disclosed herein using terms such as "comprising", it will be understood that these embodiments provide teachings of corresponding embodiments using terms such as "consisting". Additionally, terms such as "about," "approximately," and the like, whenever used, typically refer to a reasonable variation, such as ±5% variation of the disclosed value, or a 4% variation of the disclosed value, or a 3% variation of the disclosed value, or a 2% variation of the disclosed value, or a 1% variation of the disclosed value.

[0060] Further, in the description herein, certain values ​​may be disclosed in ranges. The values ​​indicating the end points of the range are intended to indicate the preferred range. Whenever a range is described, it is intended that the range covers and teaches all possible subranges in addition to the individual values ​​within the range. That is, the end points of the range should not be interpreted as invariant limits. For example, a description of a range of 1% to 5% is intended to specifically disclose subranges such as 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3%, etc., in addition to the values ​​within the range, such as 1%, 2%, 3%, 4%, and 5%, respectively. The above specific disclosure intent is applicable to ranges of any depth / width.

[0061] Furthermore, when describing some embodiments, the present disclosure may disclose a method and / or process as a particular sequence of steps. However, it will be understood that unless specifically required, the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may be possible. The particular order of steps disclosed herein should not be construed as unduly limiting. Unless specifically required, the method and / or process disclosed herein should not be limited to steps performed in the order described. The sequence of steps may be varied and still be within the scope of the present disclosure.

[0062] Furthermore, while the present disclosure provides embodiments having one or more of the features / characteristics discussed herein, it will be understood that one or more of these features / characteristics may be denied in other alternative embodiments, and the present disclosure provides support for such denials and these related alternative embodiments. [Brief description of the drawings]

[0063] [Figure 1-1] Antibody profiles of healthy controls and ZIKV patients from the 2016 Singapore cohort. Total (a) IgM and (b) IgG antibody titers were determined in 45 healthy donors by virion-based ELISA using purified ZIKV, DENV, or CHIKV virions and a plasma dilution of 1:2000. Data are presented as mean values, with dotted lines indicating the respective mean + SD values. Samples with OD values ​​below the mean + SD for both IgM and IgG and across all three viruses are highlighted in black (n = 22) and combined together to form pooled healthy controls for subsequent experiments. Samples with OD values ​​above the cutoff are indicated by open symbols (n = 23). [Figure 1-2]Antibody profiles of healthy controls and ZIKV patients from the 2016 Singapore cohort. (c) Total anti-ZIKV IgM and IgG were determined in ZIKV patients at acute (n=58), early convalescent (n=43), late convalescent (n=45), early convalescent (n=41), late convalescent (n=38), and complete convalescent (n=32) time points in a virion-based ELISA using purified ZIKV virions at plasma dilutions of 1:200, 1:1000, 1:2000, 1:4000, and 1:8000. Pooled plasma from healthy donors served as a negative control. Data are presented as mean ± SEM. [Figure 1-3] Antibody profiles of healthy controls and ZIKV patients from the Singapore cohort in 2016. (d-e) Total anti-DENV (d)IgM and (e)IgG antibody titers in plasma samples from ZIKV patients at the time of first collection were determined by virion-based ELISA using purified DENV virions in a similar manner as in (c). Data are presented as mean ± SEM. All ELISA readings were performed in duplicate. (f) Number and percentage of patients who were positive or negative for anti-DENV IgM and IgG at the time of first plasma specimen collection (1:200 and 1:2000 dilutions, respectively). [Figure 2-1] Time course of antibody profiles of ZIKV patients in the Singapore cohort in 2016. (a-c) Total anti-ZIKV (a) IgM and (b) IgG antibody titers were determined in patient plasma samples by virion-based ELISA using purified ZIKV virions at dilutions of 1:200 and 1:2000, respectively. Pooled plasma from healthy donors served as negative control. Data are presented as mean ± SEM, with dotted lines indicating the mean of pooled healthy controls. (c) Number and percentage of patients positive or negative for anti-ZIKV IgM and IgG at each time point. [Figure 2-2]Time course of antibody profile of ZIKV patients in the Singapore cohort in 2016. (a-c) Total anti-ZIKV (a) IgM and (b) IgG titers were determined in patient plasma samples at 1:200 and 1:2000 dilutions, respectively, by virion-based ELISA using purified ZIKV virions. Pooled plasma of healthy donors served as negative control. Data are presented as mean ± SEM, with the dotted line indicating the mean of the pooled healthy controls. (c) Number and percentage of patients positive or negative for anti-ZIKV IgM and IgG at each time point. (d) IgG isotype titers were determined in patient plasma samples at 1:200 dilution in a ZIKV virion-based ELISA. Data are presented as mean ± SEM, with the dotted line indicating the mean of the pooled healthy controls. All ELISA readings were performed in duplicate or triplicate. (e–f) In vitro neutralization capacity of pooled ZIKV patients and pooled healthy controls was tested at 1:1000 plasma dilution via flow cytometry. (e) Plasma samples were pooled according to the level of anti-ZIKV IgG antibody titers for each time point: acute phase [low (n=37), high (n=21)], early convalescent phase [low (n=29), high (n=14)], and late convalescent phase [low (n=28), high (n=17)] [low titer patient group is indicated by square symbols, while high titer group is indicated by triangle symbols, as shown in (b)]. (f) Plasma samples collected during the recovery phase were pooled together for each time point [early recovery (n=41), late recovery (n=38), complete recovery (n=32)]. Results are expressed as percentage of control infection. Data are presented as mean ± SD and are representative of two independent experiments. Statistical analysis between virus only and pooled healthy or patient samples was performed using two-tailed Mann-Whitney U test with Bonferroni correction for multiple testing (*P<0.05). [Diagram 3]Neutralizing capacity of ZIKV patient antibodies against ZIKV and DENV. In vitro neutralizing capacity of pooled ZIKV patients against (a) ZIKV and (b) DENV was tested at plasma dilutions of 1:500, 1:1000, and 1:2000 via flow cytometry. Plasma samples were pooled according to the level of anti-ZIKV IgG antibody titers at each time point: acute phase [low (n=37), high (n=21)], early convalescent phase [low (n=29), high (n=14)], and late convalescent phase [low (n=28), high (n=17)] (shown in Figure 2). Plasma samples collected during the convalescent phase were pooled together at each time point [early convalescent phase (n=41), late convalescent phase (n=38), complete convalescent phase (n=32)]. Pooled healthy plasma was used as a negative control. Results are expressed as a percentage of control infection. Data are presented as mean ± SD and are representative of two independent experiments. [Figure 4]Preliminary mapping of common flavivirus, ZIKV-specific, and DENV-specific linear B cell pool epitopes within the ZIKV and DENV proteomes using ZIKV and DENV patient samples. (a) Polyprotein of ZIKV H / PF / 2013 (UniProtKB accession: A0A024B7W1) was consistent with the results of pooled peptide-based ELISA experiments. Duplicate 1:2000 diluted ZIKV patient plasma samples (n=30) and DENV patient serum samples (n=20) were subjected to peptide-based ELISA using pooled peptides covering the membrane precursor (prM; pools 1–4), envelope (E; pools 5–17), and nonstructural 1 (NS1; pools 18–25) proteins of the ZIKV and DENV proteomes. Each pool consisted of five peptides of 18-mer length, containing overlapping sequences of 10 amino acids. Patient IgG responses were normalized to the mean of pooled healthy controls. Patient responses to ZIKV and DENV pooled peptide pairs were compared and the average binding capacity is presented in a heatmap. A value of 0 on the scale means that the patient exhibits equivalent binding responses to ZIKV and DENV pooled peptide pairs, while values ​​greater than 0 indicate that the patient preferentially binds to ZIKV pooled peptides. Values ​​less than 0 indicate that the patient preferentially binds to DENV pooled peptides. (b) Recognition rates of ZIKV and DENV patients to peptide pools were calculated. (c) Schematic diagram showing potential common flavivirus, ZIKV-specific, and DENV-specific pools across prM, E, and NS1 based on the heatmap analysis above. [Diagram 5]Mapping of common flavivirus, ZIKV-specific, and DENV-specific linear B cell epitopes using ZIKV and DENV patient samples. (a) ZIKV H / PF / 2013 polyprotein (UniProtKB accession: A0A024B7W1). Late convalescent ZIKV patient plasma samples (n=30-44) and DENV patient serum samples (n=20) were tested at 1:2000 dilution in a peptide-based ELISA in duplicate with pooled peptides covering the membrane precursor (prM; pools 1-10), envelope (E; pools 11-32), and nonstructural 1 (NS1; pools 33-51) proteins of the ZIKV and DENV proteomes. Patient IgG responses were normalized to the mean of pooled healthy controls. Patient responses to ZIKV and DENV peptide pairs are compared and mean binding capacities are presented in a heatmap. A value of 0 on the scale means that the patient shows equivalent binding responses to the ZIKV and DENV peptide pairs, while values ​​greater than 0 indicate that the patient preferentially binds to the ZIKV peptide. Values ​​less than 0 indicate that the patient preferentially binds to the DENV peptide. (b) Schematic showing common flavivirus, ZIKV-specific, and DENV-specific peptides across prM, E, and NS1 based on the heatmap analysis above. (c-e) Genomic organization of ZIKV prM, E, and NS1. Regions of amino acids corresponding to identified linear B-cell epitopes in (c) prM, (d) E, and (e) NS1 are shown. Boxed numbers indicate the number of peptides and the position of the amino acid in the respective proteomes. [Figure 6-1]Peptide binding capacity of ZIKV and DENV patients for potential common flavivirus, ZIKV-specific, and DENV-specific linear B-cell epitopes. Late convalescent-phase ZIKV patient plasma samples (n = 30–44) and DENV patient serum samples (n = 20) were tested in duplicate at 1:2000 dilution in peptide-based ELISA, and pooled plasma from healthy donors served as a negative control. IgG binding capacity of patients positive for each ZIKV and DENV peptide pair was calculated as [(ZIKV peptide response-DENV peptide response) / DENV peptide response], and mean ± SEM values ​​for potential common flavivirus, ZIKV-specific, and DENV-specific linear B-cell epitopes are presented in (a). Distribution of binding capacity of individual ZIKV and DENV patients is shown in (b) for common flavivirus peptides, (c) for ZIKV-specific peptides, and (d) for DENV-specific peptides. Data are presented as mean ± SEM. Statistical analysis was performed using a two-tailed Mann-Whitney U test with Bonferroni correction for multiple testing (**P<0.01). [Figure 6-2]Peptide binding capacity of ZIKV and DENV patients for potential common flavivirus, ZIKV-specific, and DENV-specific linear B-cell epitopes. Late convalescent-phase ZIKV patient plasma samples (n = 30–44) and DENV patient serum samples (n = 20) were tested in duplicate at 1:2000 dilution in peptide-based ELISA, and pooled plasma from healthy donors served as a negative control. IgG binding capacity of patients positive for each ZIKV and DENV peptide pair was calculated as [(ZIKV peptide response-DENV peptide response) / DENV peptide response], and mean ± SEM values ​​for potential common flavivirus, ZIKV-specific, and DENV-specific linear B-cell epitopes are presented in (a). Distribution of binding capacity of individual ZIKV and DENV patients is shown in (b) for common flavivirus peptides, (c) for ZIKV-specific peptides, and (d) for DENV-specific peptides. Data are presented as mean ± SEM. Statistical analysis was performed using a two-tailed Mann-Whitney U test with Bonferroni correction for multiple testing (**P<0.01). [Figure 6-3]Peptide binding capacity of ZIKV and DENV patients for potential common flavivirus, ZIKV-specific, and DENV-specific linear B-cell epitopes. Late convalescent-phase ZIKV patient plasma samples (n = 30–44) and DENV patient serum samples (n = 20) were tested in duplicate at 1:2000 dilution in peptide-based ELISA, and pooled plasma from healthy donors served as a negative control. IgG binding capacity of patients positive for each ZIKV and DENV peptide pair was calculated as [(ZIKV peptide response-DENV peptide response) / DENV peptide response], and mean ± SEM values ​​for potential common flavivirus, ZIKV-specific, and DENV-specific linear B-cell epitopes are presented in (a). Distribution of binding capacity of individual ZIKV and DENV patients is shown in (b) for common flavivirus peptides, (c) for ZIKV-specific peptides, and (d) for DENV-specific peptides. Data are presented as mean ± SEM. Statistical analysis was performed using a two-tailed Mann-Whitney U test with Bonferroni correction for multiple testing (**P<0.01). [Figure 7]Characterization of ZIKV patient antibody profile kinetics against common flavivirus and ZIKV-specific linear B cell epitopes and localization of potential epitopes within the ZIKV and DENV proteomes. (a-b) Acute, late convalescent, and full recovery ZIKV patient plasma samples (n=27) were tested for IgG in duplicate at 1:2000 dilution using ZIKV and DENV peptides in a peptide-based ELISA. Pooled plasma of healthy donors was used as a negative control, and patient data were normalized to the mean of pooled healthy controls. (a) The percentage of ZIKV patients positively binding to ZIKV and DENV peptides and (b) the binding capacity of ZIKV patients positively binding to peptides were calculated and presented in heatmaps. (c–e) Schematic diagram showing the localization of common flavivirus, ZIKV-specific, and DENV-specific epitopes in (c) the prM proteins of ZIKV and DENV (PDB: 3C6E), (d) the E glycoproteins of ZIKV (PDB: 5JHM) and DENV (PDB: 1UZG), (e) the stem transmembrane (TM) domains of the E glycoproteins of ZIKV (PDB: 5IZ7) and DENV (PDB: 3J2P), and (f) the NS1 proteins of ZIKV (PDB: 5K6K) and DENV (PDB: 4O6B). [Figure 8-1] Preliminary diagnostic validation of identified linear B-cell epitopes using patient cohorts. Convalescent plasma samples from ZIKV patients (n=10) and serum samples from DENV patients (n=10) from Singapore, as well as DENV (n=5), bacterial (n=5), and unknown (n=8) patients from Thailand, were tested in the peptide-based ELISA at 1:2000 dilution in duplicate. Pooled healthy plasma was used as a negative control. (a) Sensitivity and specificity were determined for individual peptides. [Figure 8-2]Preliminary diagnostic validation of the identified linear B-cell epitopes using a patient cohort. Convalescent plasma samples (n=10) from ZIKV patients and serum samples (n=10) from DENV patients from Singapore, as well as DENV (n=5), bacterial (n=5), and unknown (n=8) patients from Thailand, were tested in a peptide-based ELISA at 1:2000 dilution in duplicate. Pooled healthy plasma was used as a negative control. (b) Sensitivity and specificity of peptide mixes of selected epitopes were determined. (c) Principal component analysis (PCA) of pooled healthy and patient anti-IgG peptide responses (OD values) was plotted on a graph to show the percentage of variance. (d-e) Peptide binding capacity of patients positively binding to peptides was calculated and statistically analyzed by using Kruskal-Wallis test with Bonferroni correction for multiple testing. Post-hoc testing was performed using Dunn's multiple comparison test to determine (d) discriminatory peptides and (e) peptide binding capacity distribution of patients. Data are presented as mean ± SD. (*P<0.05, **P<0.01). [Figure 8-3] Preliminary diagnostic validation of identified linear B-cell epitopes using patient cohorts. Convalescent plasma samples (n=10) from ZIKV patients and serum samples (n=10) from DENV patients from Singapore, as well as DENV (n=5), bacterial (n=5), and unknown (n=8) patients from Thailand, were tested in the peptide-based ELISA at 1:2000 dilution in duplicate. Pooled healthy plasma was used as negative control. (d-e) Peptide binding capacity of patients positively binding to peptides was calculated and statistically analyzed by using Kruskal-Wallis test with Bonferroni correction for multiple testing. Post-hoc tests were performed using Dunn's multiple comparison test to determine (d) discriminatory peptides and (e) peptide binding capacity distribution of patients. Data are presented as mean ± SD. (*P<0.05, **P<0.01). [Figure 9-1]Correlation analysis of antibody and peptide responses. (a) Plasma samples (n = 65) of ZIKV patients were pooled according to the level of anti-ZIKV IgG antibody titer (shown in Fig. 2b). The mean neutralization capacity of pooled ZIKV patients (shown in Fig. 2e-f) and the mean anti-ZIKV IgG levels of ZIKV patients were calculated for all time points (from acute to full recovery) and correlation analysis was performed. (b-d) ZIKV patient plasma samples (n = 30-44) and DENV patient serum samples (n = 20) at the late convalescent time point were tested for anti-ZIKV or anti-DENV IgG at 1:2000 dilution, respectively, using purified ZIKV or DENV virions in a virion-based ELISA. Additionally, plasma / serum samples were tested in duplicate at 1:2000 dilution for peptide-specific IgG using ZIKV and DENV peptides in a peptide-based ELISA. Patient responses to ZIKV and DENV peptide pairs were compared, and peptide binding capacities were calculated. [Figure 9-2] Correlation analysis of antibody and peptide responses. (b) Antibody responses of both ZIKV and DENV patients to common flavivirus peptides. Correlation analysis was performed using Spearman rank correlation. Spearman's rho (ρ) and P-values ​​(P) are presented. [Figure 9-3] Correlation analysis of antibody and peptide responses. (c) Correlation of antibody responses of ZIKV patients to ZIKV-specific peptides, and (d) antibody responses of DENV patients to DENV-specific peptides. Correlation analysis was performed using Spearman rank correlation. Spearman's rho (ρ) and P-values ​​(P) are presented. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] Exemplary, non-limiting embodiments of methods for identifying a Flavivirus infection are disclosed below.

[0065] In various embodiments, a method of identifying a Flavivirus infection is provided that includes determining whether a sample from a subject reacts with a peptide.

[0066] In various embodiments, the peptides are associated with or can confer a relative binding affinity of about 0.10 or less, about 0.09 or less, about 0.08 or less, about 0.07 or less, about 0.06 or less, about 0.05 or less, about 0.04 or less, about 0.03 or less, about 0.02 or less, about 0.01 or less, less than about or about 0.10, less than about 0.09 or about 0.09, less than about or about 0.08, less than about or about 0.07, less than about or about 0.06, less than about or about 0.05, less than about or about 0.04, less than about or about 0.03, less than about or about 0.02, or less than or about 0.01.

[0067] In various embodiments, the peptides are associated with or can confer a relative binding affinity of about 0.50 or greater, about 0.45 or greater, about 0.40 or greater, about 0.35 or greater, about 0.30 or greater, about 0.25 or greater, about 0.20 or greater, about 0.15 or greater, about 0.10 or greater, about 0.05 or less, greater than or equal to about 0.50, greater than or equal to about 0.45, greater than or equal to about 0.40, greater than or equal to about 0.35, greater than or equal to about 0.30, greater than or equal to about 0.25, greater than or equal to about 0.20, greater than or equal to about 0.15, greater than or equal to about 0.10, or about or equal to about 0.05.

[0068] In various embodiments, the relative binding affinity is a function of one or more of the following: the binding affinity of a Zika virus (ZIKV)-induced antigen binding protein, the binding affinity of a Dengue virus (DENV)-induced antigen binding protein, the response of a ZIKV-induced antigen binding protein to a peptide, the response of a ZIKV-induced antigen binding protein to a corresponding peptide, the response of a DENV-induced antigen binding protein to a peptide, and the response of a DENV-induced antigen binding protein to a corresponding peptide. In various embodiments, the relative binding affinity is the difference between the binding affinity of a ZIKV-induced antigen binding protein and the binding affinity of a DENV-induced antigen binding protein. In various embodiments, the relative binding affinity can be calculated by subtracting the binding affinity of a DENV-induced antigen binding protein from the binding affinity of a ZIKV-induced antigen binding protein. In various embodiments, the binding affinity of a DENV-induced antigen binding protein is the binding affinity of a DENV-induced antigen binding protein for ZIKV compared to DENV. In some embodiments, the binding ability of the DENV-induced antigen binding protein is calculated by [(response of the DENV-induced antigen binding protein to the corresponding peptide-response of the DENV-induced antigen binding protein to the peptide) / response of the DENV-induced antigen binding protein to the peptide]. In some embodiments, the binding ability of the DENV-induced antigen binding protein is calculated by [(response of the DENV-induced antigen binding protein to the peptide-response of the DENV-induced antigen binding protein to the corresponding peptide) / response of the DENV-induced antigen binding protein to the corresponding peptide]. In various embodiments, the binding ability of the ZIKV-induced antigen binding protein is the binding ability of the ZIKV-induced antigen binding protein for ZIKV compared to DENV. In some embodiments, the binding ability of the ZIKV-induced antigen binding protein is calculated by [(response of the ZIKV-induced antigen binding protein to the corresponding peptide-response of the ZIKV-induced antigen binding protein to the peptide) / response of the ZIKV-induced antigen binding protein to the peptide].In some embodiments, the binding ability of the ZIKV-induced antigen binding protein is calculated by [(response of the ZIKV-induced antigen binding protein to a peptide - response of the ZIKV-induced antigen binding protein to the corresponding peptide) / response of the ZIKV-induced antigen binding protein to the corresponding peptide].

[0069] In various embodiments, the peptide comprises a ZIKV-derived peptide or a DENV-derived peptide. In various embodiments, when a peptide comprises a ZIKV-derived peptide, the corresponding peptide comprises a DENV-derived peptide having a sequence homologous to the peptide / ZIKV-derived peptide. In various embodiments, when a peptide comprises a DENV-derived peptide, the corresponding peptide comprises a ZIKV-derived peptide having a sequence homologous to the peptide / DENV-derived peptide.

[0070] In some examples, when a peptide comprises a ZIKV-derived peptide, a corresponding peptide comprising a DENV-derived peptide having a sequence homologous to the peptide / ZIKV-derived peptide can be identified by aligning the ZIKV sequence and the DENV sequence. In some examples, the homologous regions of ZIKV and DENV are at approximately the same amino acid position. Thus, in some examples, a corresponding peptide comprising a DENV-derived peptide having a sequence homologous to the peptide / ZIKV-derived peptide can be identified by aligning the ZIKV sequence and the DENV sequence, determining the region on ZIKV to which the ZIKV-derived peptide is mapped, and identifying the corresponding amino acid residue in the same region or at the same amino acid position in DENV. For example, when a peptide / ZIKV-derived peptide has a sequence corresponding to amino acids 56 to 72 of the prM protein of ZIKV (the first amino acid of the prM protein is counted as 1), the corresponding peptide may have a sequence corresponding to approximately the same position of the prM protein of DENV, i.e., amino acids 56 to 72 of the prM protein of DENV. Corresponding peptides, including ZIKV-derived peptides with sequences homologous to DENV-derived peptides, can also be identified in a similar manner. Homologous sequences of ZIKV and DENV may or may not share high sequence similarity or a common evolutionary origin. In various embodiments, in the case of a flavivirus with multiple strains, such as DENV, the amino acid sequence may be obtained from a consensus sequence of the flavivirus, such as DENV. As will be understood, a consensus sequence represents an "average" sequence where each position represents the most frequently occurring amino acid when multiple sequences (e.g., sequences of different strains of flavivirus) are compared / aligned.

[0071] In various embodiments, the flavivirus infection comprises Zika virus (ZIKV) and / or Dengue virus (DENV).

[0072] In various embodiments, a method for identifying a flavivirus infection in a subject selected from Zika virus (ZIKV), Dengue virus (DENV), and combinations thereof, comprising:relative ≦0.05 or ≧0.1,

[0073]

number

[0074] When peptide P comprises a ZIKV-derived peptide,

[0075]

number

[0076] and When peptide P contains a DENV-derived peptide,

[0077]

number

[0078] A method is provided in which

[0079] Without being bound by theory, it is believed that ZIKV and / or DENV antigen binding proteins are produced in the subject's body in response to ZIKV and / or DENV infection and are therefore present in samples from subjects with various stages of disease. In some embodiments, ZIKV and / or DENV-induced antigen binding proteins include antibodies, optionally serum antibodies, produced in the subject in response to ZIKV and / or DENV infection. Examples of ZIKV-induced antigen binding proteins include anti-ZIKV IgM (e.g., human anti-ZIKV IgM), anti-ZIKV IgG (e.g., human anti-ZIKV IgG1, IgG2, IgG3, and IgG4), and anti-ZIKV IgA (e.g., human anti-ZIKV IgA). Examples of DENV-derived antigen binding proteins include anti-DENV IgM (e.g., human anti-DENV IgM), anti-DENV IgG (e.g., human anti-DENV IgG1, IgG2, IgG3, and IgG4), and anti-DENV IgA (e.g., human anti-DENV IgA).

[0080] ZIKV and / or DENV-induced antigen binding proteins may be present or detectable in a subject during various phases of ZIKV and / or DENV infection, including the acute phase, early convalescent phase, late convalescent phase, early recovery phase, late recovery phase, and full recovery phase, although the levels of ZIKV and / or DENV-induced antigen binding proteins may vary during different phases. For example, anti-ZIKV IgM may become detectable during the acute phase of ZIKV infection, peak during the early convalescent phase, and decline during the convalescent phase. For example, anti-ZIKV IgG may peak during the early convalescent phase, remain at high levels during the late convalescent phase, and remain detectable one year after infection. Variability may also occur within IgG isotypes. For example, anti-ZIKV IgG1 may peak during the early convalescent phase, while anti-ZIKV IgG3 may peak during the late convalescent phase. Thus, for example, detection of ZIKV and / or DENV-induced antigen binding proteins using the peptides disclosed herein can identify ZIKV and DENV infection in a subject, and can also identify infection at or prior to infection.

[0081] In various embodiments, the ZIKV and / or DENV-induced antigen binding protein is protective against ZIKV and / or DENV, in some instances, the ZIKV-induced antigen binding protein can substantially neutralize ZIKV in a neutralization assay.

[0082] In various embodiments, the binding affinity of the ZIKV-induced antigen binding protein is about -0.50 to about 0.50, about -0.40 to about 0.40, about -0.30 to about 0.40, about -0.25 to about 0.35, about -0.21 to about 0.34, about -0.20 to about -0.01, about -0.10 to about -0.01, about 0.01 to about 0.35, about 0.01 to about 0.30, about 0.01 to about 0.20, or about 0.01 to about 0.10.

[0083] In various embodiments, the binding affinity of the DENV-derived antigen binding protein is about -0.60 to about 0.30, about -0.50 to about 0.20, about -0.455 to about 0.173, about -0.50 to about -0.01, about -0.40 to about -0.01, about -0.30 to about -0.01, about -0.20 to about -0.01, about -0.10 to about -0.01, about 0.01 to about 0.30, about 0.01 to about 0.20, or about 0.01 to about 0.10.

[0084] In various embodiments, a ZIKV-derived antigen binding protein or a DENV-derived antigen binding protein with a binding affinity greater than zero indicates that the ZIKV- or DENV-derived antigen binding protein recognizes the ZIKV / ZIKV-derived peptide more strongly than the DENV / DENV-derived peptide and / or has a higher binding activity for the ZIKV / ZIKV-derived peptide than the DENV / DENV-derived peptide. For example, the ZIKV- or DENV-derived antigen binding protein can provide a stronger intensity / magnitude signal / readout, e.g., a stronger intensity chemiluminescent signal / readout, for the ZIKV / ZIKV-derived peptide compared to the DENV / DENV-derived peptide in an immunoassay measuring binding activity for the peptide(s).

[0085] In various embodiments, a ZIKV-derived antigen binding protein or a DENV-derived antigen binding protein with a binding affinity of less than 0 indicates that the ZIKV- or DENV-derived antigen binding protein recognizes DENV / DENV-derived peptides more strongly than ZIKV / ZIKV-derived peptides and / or has a higher binding activity for DENV / DENV-derived peptides than ZIKV / ZIKV-derived peptides. For example, a ZIKV- or DENV-derived antigen binding protein can provide a stronger intensity / magnitude signal / readout, e.g., a stronger intensity chemiluminescent signal / readout, for a DENV / DENV-derived peptide compared to a ZIKV / ZIKV-derived peptide in an immunoassay measuring binding activity for a peptide(s).

[0086] In various embodiments, the peptide can react with or be recognized by ZIKV and / or DENV-induced antigen binding proteins (e.g., in a subject's sample) at various stages of infection, optionally with similar strength / intensity at various stages. In some embodiments, the peptide can react with or be recognized by ZIKV and / or DENV-induced antigen binding proteins only at a particular stage(s) of infection (optionally with similar strength / intensity), but not at other stage(s). In some embodiments, the peptide can react with or be recognized by ZIKV and / or DENV-induced antigen binding proteins at the acute stage of infection. In some embodiments, the peptides can react with or be recognized by ZIKV and / or DENV-induced antigen binding proteins (optionally with similar strength / intensity) at or after the late convalescent phase of infection. In some embodiments, the peptides can react with or be recognized by ZIKV and / or DENV-induced antigen binding proteins (optionally with similar strength / intensity) at or after the acute and late convalescent phases of infection. In some embodiments, the peptides can react with or be recognized by ZIKV and / or DENV-induced antigen binding proteins (optionally with similar strength / intensity) at or after the acute to full convalescent phases of infection. In some embodiments, the peptides can react with or be recognized by ZIKV and / or DENV-induced antigen binding proteins (optionally with similar strength / intensity) at or after the acute to full convalescent phases of infection or all phases of infection.

[0087] In various embodiments, the peptide comprises an epitope of ZIKV and / or DENV. In various embodiments, the peptide comprises an immunodominant epitope of ZIKV and / or DENV.

[0088] In various embodiments, the peptide comprises an antigen / epitope in the prM protein, E glycoprotein, or NS1 protein of ZIKV or DENV. In some embodiments, the peptide comprises an epitope of a ZIKV structural protein selected from the prM protein and the E glycoprotein. In some embodiments, the peptide comprises an epitope of a ZIKV nonstructural protein selected from the NS1 protein. In some embodiments, the peptide comprises a linear antigen / epitope in the prM protein, E glycoprotein, or NS1 protein of ZIKV or DENV.

[0089] In some embodiments, the antigen / epitope comprises an exposed residue on the prM protein, the E glycoprotein, or the NS1 protein. In some embodiments, the antigen / epitope is located in a solvent-exposed region of the prM protein, the E glycoprotein, or the NS1 protein. Thus, the antigen / epitope may have high solvent accessibility. The antigen / epitope may be located in an accessible / exposed region of ZIKV or DENV.

[0090] In some embodiments, the antigen / epitope comprises a non-exposed, buried, or semi-buried residue on the prM protein, E glycoprotein, or NS1 protein. In some embodiments, the antigen / epitope is located in a solvent-non-accessible region of the prM protein, E glycoprotein, or NS1 protein. Thus, in some embodiments, the antigen / epitope may have low solvent accessibility. The antigen / epitope may be located in a non-accessible / buried region of ZIKV or DENV. In one example, the antigen / epitope is present in the relatively non-accessible stem region of the E glycoprotein. In one example, an antigen / epitope in the non-contiguous stem region of E glycoprotein comprises SEQ ID NO:32 (FKSLFGGMSWFSQILIGT) or SEQ ID NO:83 (YTALFSGVSWVMKIGIGV), or a peptide sharing at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with SEQ ID NO:32 or 83, or a portion thereof, or a peptide that differs from SEQ ID NO:32 or 83 by about 1, about 2, about 3, about 4, about 5, about 6, or more amino acids, or a portion thereof, or a portion of SEQ ID NO:32 or 83.

[0091] A variety of structural data and software programs are available to assess the accessibility of epitopes or sequences on proteins. For example, the accessibility of epitopes on E glycoprotein or NS1 protein can be assessed based on structural data available in Protein Data Bank. For example, the accessibility of epitopes on prM protein can be predicted using T-TASSER queries in conjunction with visualization using UCSF Chimera software. As used herein, the phrase "solvent-exposed region" refers to solvent accessibility, which is defined as the degree of burial or exposure of amino acid residues in a three-dimensional protein structure. In some examples, solvent-exposed regions can be visualized using software known in the art, such as, but not limited to, PyMOL (Schroendinger).

[0092] In various embodiments, the length / size of the peptide is about 30 or less, about 29 or less, about 28 or less, about 27 or less, about 26 or less, about 25 or less, about 24 or less, about 23 or less, about 22 or less, about 21 or less, about 20 or less, about 19 or less, about 18 or less, about 17 or less, about 16 or less, about 15 or less, about 14 or less, about 13 or less, about 12 or less, about 11 or less, about 10 or less, about 9 or less, about 8 or less, about 7 or less, about 6 or less, or about 5 or less amino acids in length. In various embodiments, the length / size of the peptide is about 5 to about 30, about 10 to about 25, or about 15 to about 20 amino acids in length. In one embodiment, the peptide is about 5 to about 25 amino acids in length. In some embodiments, the length / size of the peptide is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or about 21 amino acids in length. In various embodiments, the peptide comprises an oligopeptide. In various embodiments, the peptide comprises a short oligopeptide. In various embodiments, the peptide comprises a linear peptide.

[0093] In some embodiments, the sequence similarity / identity between the peptide and the corresponding peptide is about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0094] In some embodiments, the peptide shares high sequence similarity / identity with the corresponding peptide. In various embodiments, the sequence similarity / identity between the peptide and the corresponding peptide is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%. In various embodiments, the sequence similarity / identity between the peptide and the corresponding peptide flavivirus is about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, or about 90% to about 100%. In some embodiments, high sequence similarity / identity between a peptide and a corresponding peptide may contribute to or be associated with the respective abilities to be recognized and / or bound by both the ZIKV-induced antigen binding protein and the DENV-induced antigen binding protein. In some embodiments, the ZIKV-induced antigen binding protein and the DENV-induced antigen binding protein can recognize and / or bind the peptide and / or the corresponding peptide with substantially equal strength / intensity.

[0095] In some embodiments, the peptide and / or corresponding peptide may be recognized and / or bound by both the ZIKV-induced antigen binding protein and the DENV-induced antigen binding protein. In some embodiments, the peptide and / or corresponding peptide may react with both the ZIKV-induced antigen binding protein and the DENV-induced antigen binding protein. In some embodiments, the ZIKV-induced antigen binding protein and the DENV-induced antigen binding protein may recognize and / or bind to and / or react with the peptide and / or corresponding peptide with substantially equal strength / intensity.

[0096] Thus, advantageously, embodiments of the peptides may function as markers, e.g., serological markers, to distinguish flavivirus infections from other infections, or to distinguish either ZIKV or DENV infections, or to distinguish ZIKV or DENV infections from other flavivirus infections (e.g., Yellow Fever Virus (YFV), Japanese Encephalitis Virus, West Nile Encephalitis Virus, St. Louis Encephalitis Virus, Tick-Borne Encephalitis Virus, Kyasanur Forest Disease Virus, and Alkuruma Hemorrhagic Fever Virus, etc.).

[0097] In some embodiments, the peptide shares low sequence similarity / identity with the corresponding peptide. In various embodiments, the sequence similarity / identity between the peptide and the corresponding peptide is about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, or about 75% or less. In various embodiments, the sequence similarity / identity between the peptide and the corresponding peptide is about 5% to about 75%, about 5% to about 50%, about 15% to about 75%, or about 15% to about 50%. In some embodiments, the low sequence similarity / identity between the peptide and the corresponding peptide may contribute to or be associated with the respective abilities to be differentially recognized and / or bound by the ZIKV-induced antigen binding protein and the DENV-induced antigen binding protein. Therefore, the cross-reactivity between ZIKV-induced and DENV-induced antigen-binding proteins against the same or corresponding peptides may be reduced, and the discrimination performance in detecting flaviviruses, such as ZIKV and DENV, may be improved accordingly.

[0098] In some embodiments, the peptides share no more than about 50% sequence similarity with the corresponding peptide.

[0099] In some embodiments, the peptide and / or corresponding peptide may be specifically recognized and / or specifically bound by a ZIKV-induced antigen binding protein, but not specifically recognized and / or specifically bound by a DENV-induced antigen binding protein. In some embodiments, the peptide and / or corresponding peptide may react with a ZIKV-induced antigen binding protein, but not with a DENV-induced antigen binding protein. Thus, advantageously, embodiments of the peptide can function as a marker, e.g., a serological marker, for identifying ZIKV infection and / or for distinguishing ZIKV infection from DENV infection or other flavivirus infection.

[0100] In some embodiments, the peptide and / or corresponding peptide may be specifically recognized and / or specifically bound by a DENV-induced antigen binding protein, but not by a ZIKV-induced antigen binding protein. In some embodiments, the peptide and / or corresponding peptide may react with a DENV-induced antigen binding protein, but not with a ZIKV-induced antigen binding protein. Thus, advantageously, embodiments of the peptide can function as a marker, e.g., a serological marker, for identifying DENV infection and / or distinguishing DENV infection from ZIKV infection or other flavivirus infection.

[0101] In various embodiments, the peptide is selected from a peptide of Table 8, a peptide or portion thereof sharing at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with the peptide, a peptide or portion thereof that differs from the peptide by about 1, about 2, about 3, about 4, about 5, about 6, or more amino acids, or a portion of the peptide.

[0102] In various embodiments, the peptide comprises a sequence corresponding to the amino acid sequence at the following positions in the prM protein of ZIKV and / or DENV (the first amino acid of the prM protein is counted as 1): about 3 to about 26, about 6 to about 23, about 12 to about 35, about 15 to about 32, about 21 to about 44, about 24 to about 41, about 30 to about 53, about 33 to about 50, about 39 to about 62, about 42 to about 59, about 48 to about 71, about 51 to about 68, about 53 to about 75, about 56 to about 72, about 66 to about 89, about 69 to about 86, about 75 to about 95, about 78 to about 92, about 97 to about 121, or about 100 to about 118. In various embodiments, the peptide includes a peptide or portion thereof that shares at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with the amino acid sequence, or that differs from the amino acid sequence by about 1, about 2, about 3, about 4, about 5, about 6 or more amino acids.

[0103] In various embodiments, the peptide is selected from the group consisting of: SEQ ID NO:1 (RGSAYYMYLDRNDAGEAI), SEQ ID NO:52 (RDGEPRMIVGKNERGKSL), SEQ ID NO:2 (DRNDAGEAISFPTTLGMN), SEQ ID NO:53 (GKNERGKSLLFKTASGIN), SEQ ID NO:3 SFPTTLGMNKCYIQIMDL), SEQ ID NO:54 (LFKTASGINMCTLIAMDL), SEQ ID NO:4 (KCYIQIMDLGHMCDATMS), SEQ ID NO:55 (MCTLIAMDLGEMCDDTVT), SEQ ID NO:5 (GHMCDATMSYECPMLDEG), SEQ ID NO:56 (GEMCDDTVTYKCPHITE), SEQ ID NO:6 (YECPMLDEGVEPDDVDCW), SEQ ID NO:57 (YKCPHITEVEPEDIDCW), SEQ ID NO:7 (LDEGVEPDDVDCWCNTT), SEQ ID NO:58 (HITEVEPEDIDCWCNLT), SEQ ID NO:8 (CNTTSTWVVYGTCHHKKG), SEQ ID NO:59 (CNL TSTWVTYGTCNQAG), SEQ ID NO:9 (YGTCHHKKGEARRSR), SEQ ID NO:60 (TSTWVTYGTCNQAG), SEQ ID NO:10 (HSTRKLQTRSQTWLESREY), SEQ ID NO:61 (VGMGLDTRTQTWMSAEGAW), a peptide or portion thereof sharing at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with the above sequences, a peptide or portion thereof that differs from the above sequences by about 1, about 2, about 3, about 4, about 5, about 6 or more amino acids, or a portion of the above sequences.

[0104] In various embodiments, the peptide comprises a sequence corresponding to the amino acid sequence at the following positions in the E glycoprotein of ZIKV and / or DENV (the first amino acid of the E glycoprotein is counted as 1): about 34 to about 57, about 37 to about 54, about 58 to about 75, about 61 to about 72, about 70 to about 93, about 73 to about 90, about 88 to about 111, about 91 to about 108, about 110 to about 133, about 113 to about 130, about 120 to about 143, about 123 to about 140, about 128 to about 152, about 131 to about 149, about 146 to about 169, about 149 to about 166, about 154 to about 177, about 157 to about 174, about 163 ~ about 186, about 166 to about 183, about 188 to about 206, about 191 to about 203, about 196 to about 219, about 199 to about 216, about 214 to about 237, about 217 to about 234, about 232 to about 248, about 235 to about 245, about 241 to about 264, about 244 to about 261, about 268 to about 291, about 27 1 to about 288, about 303 to about 322, about 306 to about 319, about 322 to about 345, about 325 to about 342, about 340 to about 358, about 343 to about 355, about 358 to about 381, about 361 to about 378, about 399 to about 422, about 402 to about 419, about 450 to about 473, or about 453 to about 470. In various embodiments, the peptide comprises a peptide or a portion thereof sharing at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with the amino acid sequence, or a peptide or a portion thereof that differs from the amino acid sequence by about 1, about 2, about 3, about 4, about 5, about 6, or more amino acids, or a portion of the amino acid sequence.

[0105] In various embodiments, the peptide is selected from the group consisting of SEQ ID NO:11 (DKPTVDIELVTTTVSNMA), SEQ ID NO:62 (NKPTLDIELQKTEATQLA), SEQ ID NO:12 (YEASISDMASDS), SEQ ID NO:63 (IEGKITNITTDS), SEQ ID NO:13 (RCPTQGEAYLDKQSDTQY), SEQ ID NO:64 (RCPTQGEAVLPEEQDQNY), SEQ ID NO:14 (VCKRTLVDRGWGNGCGLF), SEQ ID NO:65 (VCKHTYVDRGWGNGCGLF), SEQ ID NO:15 (LVTCAKFA CSKKMTGKSI), SEQ ID NO:66 (LVTCAKFQCLEPIEGKVV), SEQ ID NO:16 (KKMTGKSIQPENLEYRIM), SEQ ID NO:67 (EPIEGKVVQYENLKYTVI), SEQ ID NO:17 (PENLEYRIMLSVHGSQHS), SEQ ID NO:68 (YENLKYTVIITVHTGDQH), SEQ ID NO:18 (SGMIVNDTGHETDENRAK), SEQ ID NO:69 (GDQHQVGNETQGVTAEIT), SEQ ID NO:19 (GHETDENRAKVEITPNSP), SEQ ID NO:70 (GNETQGVTAEITPQA STT), SEQ ID NO: 20 (KVEITPNSPRAEATLGGF), SEQ ID NO: 71 (TAEITPQASTTEAILPEY), SEQ ID NO: 21 (EPRTGLDFSDLYY), SEQ ID NO: 72 (SPRTGLDFNEMIL), SEQ ID NO: 22 (SDLYYLTMNNKHWLVHKE), SEQ ID NO: 73 (NEMILLTMKNKAWMVHRQ), SEQ ID NO: 23 (WFHDIPLPWHAGADTGTP), SEQ ID NO: 74 (WFFDLPLPWTSGATTETP), SEQ ID NO: 24 (HWNNKEALVEF), SEQ ID NO: 75 (TWNRKELLVTF) , SEQ ID NO: 25 (EFKDAHAKRQTVVVLGSQ), SEQ ID NO: 76 (TFKNAHAKKQEVVVLGSQ), SEQ ID NO: 26 (GALEAEMDGAKGRLSSGH), SEQ ID NO: 77 (GATEIQNSGGTSIFAGH), SEQ ID NO: 27 (SLCTAAFTFTKIPA), SEQ ID NO: 78 (AMCTNTFVLKKEVS), SEQ ID NO: 28 (TVTVEVQYAGTDGPCKVP), SEQ ID NO: 79 (TILIKVEYKGEDAPCKIP), SEQ ID NO: 29 (AQMAVDMQTLTPV), SEQ ID NO: 80 (FSTEDGQGKAHN),SEQ ID NO:30 (ANPVITESTENSKMMLEL), SEQ ID NO:81 (ANPVVTKKEEPVNIEA), SEQ ID NO:31 (RSGSTIGKAFEATVRGAK), SEQ ID NO:82 (KKGSSIGKMFEATARGAR), SEQ ID NO:32 (FKSLFGGMSWFSQILIGT), SEQ ID NO:83 (YTALFSGVSWVMKIGIGV), a peptide or a portion thereof sharing at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with the above sequence, a peptide or a portion thereof differing from the above sequence by about 1, about 2, about 3, about 4, about 5, about 6, or more amino acids, or a portion of the above sequence.

[0106] In various embodiments, the peptide comprises a sequence corresponding to the amino acid sequence at the following positions in the NS1 protein of ZIKV and / or DENV (the first amino acid of the NS1 protein is counted as 1): about 1 to about 21, about 1 to about 18, about 16 to about 39, about 19 to about 36, about 52 to about 75, about 55 to about 72, about 67 to about 88, about 70 to about 85, about 88 to about 115, about 91 to about 112, about 116 to about 139, about 119 to about 136, about 134 to about 157, about 137 to about 154, about 152 to about 179, about 155 to about 176, about 236 to about 259, about 239 to about 256, about 245 to about 268, about 248 to about 265, about 254 to about 277, about 257 to about 274, about 267 to about 284, about 270 to about 281, about 272 to about 295, about 275 to about 292, about 281 to about 304, about 284 to about 301, about 290 to about 313, about 293 to about 310, about 299 to about 322, about 302 to about 319, about 312 to about 329, about 315 to about 326, about 317 to about 340, about 320 to about 337, about 335 to about 356, or about 338 to about 353. In various embodiments, the peptide includes a peptide or portion thereof that shares at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with the amino acid sequence, or that differs from the amino acid sequence by about 1, about 2, about 3, about 4, about 5, about 6, or more amino acids.

[0107] In various embodiments, the peptide is selected from the group consisting of SEQ ID NO:33 (DVGCSVDFSKKETRCGTG), SEQ ID NO:84 (DMGCVINWKGKELKCGSG), SEQ ID NO:34 (VFVYNDVEAWRDRYKYHP), SEQ ID NO:85 (IFVTNEVHTWTEQYKFQA), SEQ ID NO:35 (CGISSVSRMENIMWRSVE), SEQ ID NO:86 (CGIRSTTRMENLLWKQIA), SEQ ID NO:36 (SVEGELNAILEENGVQ), SEQ ID NO:87 (QIANELNYILWENNIK), SEQ ID NO:37 (SEQ ID NO:39), SEQ ID NO:40 (SEQ ID NO:41), SEQ ID NO:42 (SEQ ID NO:43), SEQ ID NO:44 (SEQ ID NO:45), SEQ ID NO:46 (SEQ ID NO:47), SEQ ID NO:48 (SEQ ID NO:49), SEQ ID NO:50 (SEQ ID NO:51), SEQ ID NO:52 (SEQ ID NO:53), SEQ ID NO:53 (SEQ ID NO:54), SEQ ID NO:55 (SEQ ID NO:56), SEQ ID NO:57 (SEQ ID NO:58), SEQ ID NO:59 (SEQ ID NO:59), SEQ ID NO:60 (SEQ ID NO:61), SEQ ID NO:61 (SEQ ID NO:62), SEQ ID NO:63 (SEQ ID NO:64), SEQ ID NO:65 (SEQ ID NO:65), SEQ ID NO:66 (SEQ ID NO:67), SEQ ID NO:68 (SEQ ID NO:69), SEQ ID NO:69 (SEQ ID NO:61), SEQ ID NO:64 (SEQ ID NO:65), SEQ ID NO:65 (SEQ ID NO:66), SEQ ID NO:67 ( 7 (GSVKNPMWRGPQRLPVPVNELP), SEQ ID NO: 88 (GDIIGVLEQGKRTLTPQPMELK), SEQ ID NO: 38 (GKSYFVRAAKTNNSFVVD), SEQ ID NO: 89 (GKAKIVTAETQNSSFIID), SEQ ID NO: 39 (GDTLKECPLKHRAWNSFL), SEQ ID NO: 90 (GPNTPECPSASRAWNVWE), SEQ ID NO: 40 (VEDHGFGVFHTSVWLKVREDYS), SEQ ID NO: 91 (VEDYGFGVFTTNIWLKLREVYT), SEQ ID NO: 41 (SDLIIPKSLAGPLS HHNT), SEQ ID NO: 92 (SDMIIPKSLAGPISQHNH), SEQ ID NO: 42 (AGPLSHHNTREGYRTQMK), SEQ ID NO: 93 (AGPISQHNHRPGYHTQTA), SEQ ID NO: 43 (REGYRTQMKGPWHSEELE), SEQ ID NO: 94 (RPGYHTQTAGPWHLGKLE), SEQ ID NO: 44 (SEELEIRFEECP), SEQ ID NO: 95 (LGKLELDFNYCE), SEQ ID NO: 45 (IRFEECPGTKVHVEETCG), SEQ ID NO: 96 (LDFNYCEGTTVVITENCG), SEQ ID NO: 46 (KVHV EETCGTRGPSLRST), SEQ ID NO: 97 (TVVITENCGTRGPSLRTT), SEQ ID NO: 47 (TRGPSLRSTTASGRVIEE), SEQ ID NO: 98 (TRGPSLRTTTVSGKLIHE), SEQ ID NO: 48 (TASGRVIEEWCCRECTMP), SEQ ID NO: 99 (TVSGKLIHEWCCRSCTLP), SEQ ID NO: 49 (ECTMPPLSFRAK), SEQ ID NO: 100 (SCTLPPLRYMGE), SEQ ID NO: 50 (PLSFRAKDGCWYGMEIRP), SEQ ID NO: 101 (PLRYMGEDGCWYGMEIRP),SEQ ID NO:51 (RKEPESNLVRSMVTAG), SEQ ID NO:102 (ISEKEENMVKSLVSAG), a peptide or a portion thereof that shares at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with the above sequence, a peptide or a portion thereof that differs from the above sequence by about 1, about 2, about 3, about 4, about 5, about 6, or more amino acids, or a portion of the above sequence.

[0108] In various embodiments, the peptide is one or more ZIKV-derived peptides selected from the group consisting of: SEQ ID NO:7 (LDEGVEPDDVDCWCNTT); SEQ ID NO:36 (SVEGELNAILEENGVQ); SEQ ID NO:38 (GKSYFVRAAKTNNSFVVD); SEQ ID NO:39 (GDTLKECPLKHRAWNSFL); SEQ ID NO:46 (KVHVEETCGTRGPSLRST); SEQ ID NO:49 (ECTMPPLSFRAK); SEQ ID NO:3 (SFPTTLGMNKCYIQIMDL); SEQ ID NO: SEQ ID NO:26 (GALEAEMDGAKGRLSSGH); SEQ ID NO:32 (FKSLFGGMSWFSQILIGT); SEQ ID NO:9 (YGTCHHKKGEARRSR); SEQ ID NO:17 (PENLEYRIMLSVHGSQHS); SEQ ID NO:43 (REGYRTQMKGPWHSEELE); SEQ ID NO:45 (IRFEECPGTKVHVEETCG); a peptide sharing at least 75% sequence identity therewith; or a peptide differing therefrom by 1, 2, 3, or 4 amino acids; or a portion thereof. and / or one or more DENV-derived peptides selected from the group consisting of: SEQ ID NO:58 (HITEVEPEDIDCWCNLT); SEQ ID NO:87 (QIANELNYILWENNIK); SEQ ID NO:89 (GKAKIVTAETQNSSFIID); SEQ ID NO:90 (GPNTPECPSASRAWNVWE); SEQ ID NO:97 (TVVITENCGTRGPSLRTT); SEQ ID NO:100 (SCTLPPLRYMGE); SEQ ID NO:54 (LFKTASGINMCTLIAMDL); SEQ ID NO:77 (G ATEIQNSGGTSIFAGH); SEQ ID NO:83 (YTALFSGVSWVMKIGIGV); SEQ ID NO:60 (TSTWVTYGTCNQAG); SEQ ID NO:68 (YENLKYTVIITVHTGDQH); SEQ ID NO:94 (RPGYHTQTAGPWHLGKLE); SEQ ID NO:96 (LDFNYCEGTTVVITENCG); or a peptide sharing at least 75% sequence identity therewith; or a peptide which differs therefrom by 1, 2, 3 or 4 amino acids; or a portion thereof.

[0109] Method embodiments can identify flavivirus infection, one or both of ZIKV and DENV infection, ZIVK infection or DENV infection.

[0110] In various embodiments, the method comprises the step of: providing a sample comprising the sequence of SEQ ID NO:7 (LDEGVEPDDVDCWCNTT); SEQ ID NO:36 (SVEGELNAILEENGVQ); SEQ ID NO:38 (GKSYFVRAAKTNNSFVVD); SEQ ID NO:39 (GDTLKECPLKHRAWNSFL); SEQ ID NO:46 (KVHVEETCGTRGPSLRST); SEQ ID NO:49 (ECTMPPLSFRAK); SEQ ID NO:58 (HITEVEPEDIDCWCNLT); SEQ ID NO:87 (QIANELNYILWENNIK); SEQ ID NO:89 (GKAKIVTAETQNSSFIID); SEQ ID NO:90 (GPNTPECPSASRAWNVWE ); SEQ ID NO: 97 (TVVITENCGTRGPSLRTT); SEQ ID NO: 100 (SCTLPPLRYMGE); a peptide sharing at least 75% sequence identity therewith; or a peptide differing therefrom by 1, 2, 3, or 4 amino acids; or a portion thereof; and / or if the sample reacts with the one or more peptides or portions thereof, the subject is indicated for treatment of a flavivirus infection, or for treatment of one or both of a ZIKV and DENV infection.

[0111] In various embodiments, the method is for identifying an acute phase flavivirus infection, the method comprising determining whether a sample reacts with one or more peptides selected from the group consisting of SEQ ID NO:7 (LDEGVEPDDVDCWCNTT); SEQ ID NO:36 (SVEGELNAILEENGVQ); SEQ ID NO:38 (GKSYFVRAAKTNNSFVVD); SEQ ID NO:58 (HITEVEPEDIDCWCNLT); SEQ ID NO:87 (QIANELNYILWENNIK); SEQ ID NO:89 (GKAKIVTAETQNSSFIID), and if the sample reacts with the one or more peptides or portions thereof, the subject is indicated for treatment of an acute phase flavivirus infection, or for treatment of one or both of ZIKV and DENV infection.

[0112] In various embodiments, the method includes determining whether the sample reacts with one or more peptides (e.g., ZIKV-specific peptides) selected from the group consisting of SEQ ID NO:3 (SFPTTLGMNKCYIQIMDL); SEQ ID NO:26 (GALEAEMDGAKGRLSSGH); SEQ ID NO:32 (FKSLFGGMSWFSQILIGT); a peptide sharing at least 75% sequence identity therewith; or a peptide differing therefrom by 1, 2, 3, or 4 amino acids; or a portion thereof, and if the sample reacts with the one or more peptides or a portion thereof, the subject is indicated for treatment of a ZIKV infection.

[0113] In various embodiments, a method is a method for identifying an acute phase ZIKV infection, the method comprising determining whether a sample reacts with SEQ ID NO: 32 (FKSLFGGMSWFSQILIGT); a peptide sharing at least 75% sequence identity therewith; or a peptide differing therefrom by 1, 2, 3, or 4 amino acids; or a portion thereof, and if the sample reacts with the peptide or portion thereof, the subject is indicated for treatment of an acute phase ZIKV infection.

[0114] In various embodiments, the method includes determining whether the sample reacts with one or more peptides (e.g., DENV-specific peptides) selected from the group consisting of SEQ ID NO: 60 (TSTWVTYGTCNQAG); SEQ ID NO: 68 (YENLKYTVIITVHTGDQH); SEQ ID NO: 94 (RPGYHTQTAGPWHLGKLE); SEQ ID NO: 96 (LDFNYCEGTTVVITENCG); and / or peptides sharing at least 75% sequence identity therewith; or peptides differing therefrom by 1, 2, 3, or 4 amino acids; or portions thereof, and if the sample reacts with the one or more peptides or portions thereof, the subject is indicated for treatment of DENV infection.

[0115] Method embodiments may include the use of multiple or a plurality of peptides described herein. In some embodiments, at least about two, at least about three, at least about four, or at least about five different peptides may be used to detect ZIKV and / or DENV-induced antigen binding proteins in a sample. In some embodiments, the plurality of peptides includes at least one peptide from one or more of the following groups: common flavivirus peptides, ZIKV-specific peptides, and DENV-specific peptides.

[0116] In various embodiments, determining whether a subject's sample reacts with a peptide includes performing an immunoassay to assess whether an antigen binding protein (e.g., an antibody) capable of recognizing or binding the peptide is present in the sample. Examples of immunoassays include radioimmunoassays (RIA), chemiluminescent and fluorescent immunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunosorbent spot (ELISPOT), Luminex-based bead arrays, protein microarray assays, immunochromatographic (ICG)-based assays, and rapid test formats such as immunochromatographic strip tests. The assays may be competitive and non-competitive sandwich assays.

[0117] In some embodiments, the immunoassay comprises an ELISA, such as, but not limited to, a direct ELISA, an indirect ELISA, a competitive ELISA, a sandwich ELISA, etc. In some embodiments, the immunoassay comprises a sandwich ELISA. The suitability of the specific ELISA to be used will be within the skill of one of ordinary skill in the art. However, as an example of the present invention, in some embodiments, the assay is in the form of a non-competitive sandwich assay in which the ZIKV and / or DENV antibodies or fragments thereof to be detected and / or quantified bind to antibodies of ZIKV and / or DENV antibodies or fragments thereof and / or ZIKV and / or DENV antigens. In some examples, the ZIKV and / or DENV antigens may be labeled, for example with biotin, and bound to a solid phase, such as, for example, a bead, well or other inclusion surface, chip, or strip, to capture any ZIKV and / or DENV antibodies or fragments thereof, and antibodies of ZIKV and / or DENV antibodies are used to detect any captured ZIKV and / or DENV antibodies. The detection antibody may be labeled, for example, with a dye, a radioisotope, or a reactive or catalytically active moiety. In one example, the detection antibody is labeled with horseradish peroxidase (HRP) and a tetramethylbenzidine (TMB) substrate may be added for visualization. It is understood that other suitable labels and substrates may also be used. In some examples, a streptavidin-coated plate (e.g., a microplate, a microtiter plate, etc.) is used to capture biotin-labeled ZIKV and / or DENV antigens, which are then used to capture any ZIKV and / or DENV antibodies or fragments thereof, and a labeled anti-immunoglobulin is used for detection. The anti-immunoglobulin may be anti-IgG, anti-IgM, or anti-IgA. In some examples, the anti-immunoglobulin includes anti-human IgM, anti-human IgG, anti-human IgG1, anti-human IgG2, anti-human IgG3, and / or anti-human IgG4.

[0118] In various embodiments, the sample comprises a biological sample. In various embodiments, the biological sample comprises a fluid or liquid biological sample. The fluid or liquid biological sample may be blood, serum, plasma, sputum, lavage fluid, cerebrospinal fluid, urine, semen, sweat, tears, saliva, etc. In some embodiments, the fluid or liquid biological sample comprises whole blood, serum, plasma, or processed fractions thereof. In some embodiments, the fluid biological sample comprises serum or plasma. In some embodiments, the fluid biological sample comprises an antigen binding protein, such as an antibody.

[0119] In various embodiments, samples include samples collected from a subject during the acute phase (about 2 days to about 7 days after onset of illness (pio)), early recovery phase (about 10 days to about 14 days pio), late recovery phase (about 1 month pio), early recovery phase (about 3 months pio), late recovery phase (about 5 months to about 6 months pio), or full recovery phase (about 1 year pio). In some embodiments, samples are collected from a subject when the subject exhibits symptoms associated with ZIKV infection, such as fever, arthritis / joint pain, skin rash, conjunctivitis, joint pain, headache, Guillain-Barré syndrome (GBS), and congenital central nervous system (CNS) abnormalities. In some embodiments, samples are collected from a subject when the subject is asymptomatic or has become asymptomatic for ZIKV infection. In some embodiments, samples are collected from subjects when they exhibit symptoms associated with DENV infection, such as fever (typically high fever), headache, muscle, bone, and joint pain, nausea, vomiting, pain behind the eyes, swollen glands, rash, severe abdominal pain, persistent vomiting, bleeding from the gums or nose, blood in the urine, stool, or vomit, bleeding under the skin, difficulty or rapid breathing, cold skin (shock), fatigue, and irritability or restlessness. In some embodiments, samples are collected from subjects when they are asymptomatic or have become asymptomatic for DENV infection.

[0120] In various embodiments, the method includes a diagnostic or prognostic method. For example, the reaction of a subject sample with a peptide can be indicative of ZIKV and / or DENV infection in the subject. For example, a decreased level of reaction of a subject sample compared to a previous sample (e.g., a sample collected at an earlier time point from the same subject) can be indicative of a prognosis of ZIKV and / or DENV infection in the subject.

[0121] In various embodiments, the method has high sensitivity and / or specificity, ie, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sensitivity.

[0122] In various embodiments, the method has a specificity of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%.

[0123] In various embodiments, the methods include in vitro or ex vivo methods.

[0124] In some embodiments, the method can distinguish a flavivirus infection from other infections, such as bacterial infections, other viral infections, or other non-flavivirus infections. In some embodiments, the method can distinguish a flavivirus infection from other infections that share similar symptom(s). For example, the method can distinguish a flavivirus infection from influenza, chikungunya infection, malaria, typhoid fever, etc. In some embodiments, the method can distinguish a ZIKV infection and / or a DENV infection from other flavivirus infections. In some embodiments, the method can distinguish a ZIKV infection from a DENV infection and / or a DENV infection from a ZIKV infection.

[0125] In some embodiments, there is provided a method for distinguishing a flavivirus infection in a subject from other infections, the method comprising determining whether a sample of the subject reacts with a peptide capable of giving a relative binding ability BC relative ≦0.05. In some embodiments, there is provided a method for distinguishing a ZIKV and / or DENV infection in a subject from other flavivirus infections, the method comprising determining whether a sample of the subject reacts with a peptide capable of giving a relative binding ability BC relative ≦0.05. In some embodiments, there is provided a method for distinguishing a ZIKV infection from a DENV infection and / or a DENV infection from a ZIKV infection in a subject, the method comprising determining whether a sample of the subject reacts with a peptide capable of giving a relative binding ability BC relative ≧0.1.

[0126] In some embodiments, a method of distinguishing ZIKV infection from DENV infection in a subject is provided, comprising determining whether a sample from the subject reacts with a peptide of SEQ ID NO: 32 (FKSLFGGMSWFSQILIGT); a peptide sharing at least 75% sequence identity therewith; or a peptide differing therefrom by 1, 2, 3, or 4 amino acids; or a portion thereof, and if the sample reacts with the peptide or a portion thereof, the subject is indicated for treatment of a ZIKV infection; and / or determining whether a sample from the subject reacts with a peptide of SEQ ID NO: 9 (YGTCHHKKGEARRSR); a peptide sharing at least 75% sequence identity therewith; or a peptide differing therefrom by 1, 2, 3, or 4 amino acids; or a portion thereof, and if the sample reacts with the peptide or a portion thereof, the subject is indicated for treatment of a DENV infection.

[0127] In some embodiments, the method further includes administering to the subject a ZIKV and / or DENV treatment regimen if the subject is eligible for treatment of a flavivirus infection, a ZIKV infection, and / or a DENV infection.

[0128] In some embodiments, the method further includes administering a ZIKV and / or DENV treatment regimen to the subject if the subject is eligible for treatment of a ZIKV and / or DENV infection. Thus, in some embodiments, a method of treating a flavivirus infection, a ZIKV infection, and / or a DENV infection is provided, the method comprising determining whether a sample from the subject reacts with a peptide, and if the sample reacts with the peptide, administering a ZIKV and / or DENV treatment regimen to the subject.

[0129] In some embodiments, a method is provided for identifying a common flavivirus antigen / epitope, a ZIKV-specific antigen / epitope, and / or a DENV-specific antigen / epitope, comprising contacting a test peptide and its corresponding peptide with a ZIKV-derived antigen binding protein and a DENV-derived antigen binding protein under conditions suitable for binding, and determining a relative binding affinity, wherein if the relative binding affinity is ≦0.05, the test peptide is identified as a common flavivirus antigen / epitope, and if the relative binding affinity is ≧0.1, the test peptide is identified as a ZIKV-specific antigen / epitope or a DENV-specific antigen / epitope.

[0130] In some embodiments, the method further comprises generating a library of short linear peptides, optionally short overlapping linear peptides, from a flavivirus, such as ZIKV and / or DENV, for use as test peptides and / or corresponding peptides. In some embodiments, the method comprises an ELISA method.

[0131] In various embodiments, a kit is provided for identifying a flavivirus infection in a subject selected from Zika virus, Dengue virus, and combinations thereof, the kit comprising one or more peptides disclosed herein. In some embodiments, a kit for identifying a flavivirus infection in a subject selected from Zika virus, Dengue virus, and combinations thereof, the kit comprising one or more peptides disclosed herein, the kit comprising one or more peptides comprising one or more peptides disclosed herein. In some embodiments, a kit for identifying a flavivirus infection in a subject selected from Zika virus, Dengue virus, and combinations thereof, the kit comprising one or more peptides ... relative ≦0.05 or ≧0.1,

[0132]

number

[0133] When peptide P comprises a ZIKV-derived peptide,

[0134]

number

[0135] and When peptide P contains a DENV-derived peptide,

[0136]

number

[0137] A kit is provided,

[0138] In various embodiments of the kit, the peptides are: SEQ ID NO:7 (LDEGVEPDDVDCWCNTT); SEQ ID NO:36 (SVEGELNAILEENGVQ); SEQ ID NO:38 (GKSYFVRAAKTNNSFVVD); SEQ ID NO:39 (GDTLKECPLKHRAWNSFL); SEQ ID NO:46 (KVHVEETCGTRGPSLRST); SEQ ID NO:49 (ECTMPPLSFRAK); SEQ ID NO:3 (SFPTTLGMNKCYIQIMDL); SEQ ID NO:26 (GALEAEMDGAKGRLSSGH); One or more ZIKV-derived peptides selected from the group consisting of sequence number 32 (FKSLFGGMSWFSQILIGT); SEQ ID NO:9 (YGTCHHKKGEARRSR); SEQ ID NO:17 (PENLEYRIMLSVHGSQHS); SEQ ID NO:43 (REGYRTQMKGPWHSEELE); SEQ ID NO:45 (IRFEECPGTKVHVEETCG); a peptide sharing at least 75% sequence identity therewith; or a peptide differing therefrom by 1, 2, 3, or 4 amino acids; or and / or a portion of SEQ ID NO:58 (HITEVEPEDIDCWCNLT); SEQ ID NO:87 (QIANELNYILWENNIK); SEQ ID NO:89 (GKAKIVTAETQNSSFIID); SEQ ID NO:90 (GPNTPECPSASRAWNVWE); SEQ ID NO:97 (TVVITENCGTRGPSLRTT); SEQ ID NO:100 (SCTLPPLRYMGE); SEQ ID NO:54 (LFKTASGINMCTLIAMDL); SEQ ID NO:77 (GATEIQNSGGTSIFAGH); SEQ ID NO:83 (YT One or more DENV-derived peptides selected from the group consisting of: ALFSGVSWVMKIGIGV; SEQ ID NO:60 (TSTWVTYGTCNQAG); SEQ ID NO:68 (YENLKYTVIITVHTGDQH); SEQ ID NO:94 (RPGYHTQTAGPWHLGKLE); SEQ ID NO:96 (LDFNYCEGTTVVITENCG); or a peptide sharing at least 75% sequence identity therewith; or a peptide differing therefrom by 1, 2, 3, or 4 amino acids; or a portion thereof.

[0139] The kit may also include one or more of a plate coated with an anti-ZIKV and / or anti-DENV capture agent and a detection agent for detecting the presence of the captured anti-ZIKV and / or anti-DENV. The capture and detection agents may be independently selected from purified ZIKV and / or DENV, ZIKV and / or DENV antigens, anti-ZIKV and / or anti-DENV antibodies, or anti-immunoglobulins. The anti-immunoglobulins may be one or more of anti-IgA, anti-IgM, anti-IgG, anti-IgG1, anti-IgG2, anti-IgG3, and anti-IgG4, anti-human IgA, anti-human IgM, anti-human IgG, anti-human IgG1, anti-human IgG2, anti-human IgG3, and anti-human IgG4. In some embodiments, the capture agent includes a ZIKV and / or DENV antigen, and the detection agent includes a labeled anti-immunoglobulin. In some embodiments, the capture agent comprises an anti-immunoglobulin and the detection agent comprises a labeled ZIKV and / or DENV antigen. In some embodiments, the capture agent and detection agent comprise a ZIKV and / or DENV antigen and / or an anti-immunoglobulin. In some embodiments, the capture agent and detection agent are selected from the group consisting of (i) a ZIKV and / or DENV antigen and one or more of a labeled anti-immunoglobulin, and (ii) an anti-immunoglobulin and a labeled ZIKV and / or DENV antigen.

[0140] In some embodiments, the kit further comprises one or more of a plate coated with an anti-ZIKV and / or anti-DENV capture agent and a detection agent for detecting the presence of captured anti-ZIKV and / or anti-DENV, wherein the capture agent and the detection agent comprise a ZIKV and / or DENV antigen and / or an anti-immunoglobulin.

[0141] The kit may be a diagnostic kit or a prognostic kit.

[0142] In various embodiments, the subject comprises a mammal. In various embodiments, the subject comprises a human subject. In various embodiments, the subject comprises an Asian subject. In some embodiments, the subject comprises a Southeast Asian subject. In various embodiments, the subject (whether a Southeast Asian subject or not) is infected with a flavivirus in Asia, or optionally in Southeast Asia. Southeast Asian countries include Indonesia, Malaysia, Singapore, Philippines, Timor-Leste, Brunei, Cambodia, Laos, Myanmar (Burma), Thailand, and Vietnam.

[0143] The ZIKV may be an African or Asian strain, have an African or Asian genotype, or be of an African or Asian lineage. In some embodiments, the ZIKV comprises an Asian strain, optionally a Southeast Asian strain. In some embodiments, the ZIKV comprises an Asian genotype, optionally a Southeast Asian genotype. In some embodiments, the ZIKV comprises an Asian lineage, optionally a Southeast Asian lineage ZIKV. Non-limiting examples of ZIKV / Asia lineage ZIKV with Asian ZIKV strain / Asia genotype include H / PF / 2013 (KJ776791), SPH2015, PRVABC59, R103451, P6-740, and FSS13025. Non-limiting examples of ZIKV / Africa lineage ZIKV with African ZIKV strain / African genotype include MR-766 and DakAr41524.

[0144] DENV may be serotype 1 (DENV-1), 2 (DENV-2), 3 (DENV-3), or 4 (DENV-4), or a consensus sequence of one or more serotypes. DENV-1 may be genotypes I, II, III (sylvatic), IV, V, and VI. DENV-2 may be Asian-I, Asian-II, Asian / American, American, Cosmopolitan, and sylvatic. DENV-3 may be genotypes I, II, III, IV, and V. DENV-4 may be genotypes I, IIA, IIB, III, and sylvatic. In some embodiments, DENV comprises DENV of strains / genotypes / lineages found and / or originating from and / or predominant in an Asian country, optionally a Southeast Asian country. Non-limiting examples of such DENV include strains KR296743, KF973487, EU081181, KF041254, JF808120, JF808121, KJ189293, KC762692, KC425219, KJ830751, KF973479, and AY099336.

[0145] In various embodiments, peptides, optionally isolated peptides, are provided, including peptides disclosed herein. In various embodiments, SEQ ID NO: 7 (LDEGVEPDDVDCWCNTT); SEQ ID NO: 36 (SVEGELNAILEENGVQ); SEQ ID NO: 38 (GKSYFVRAAKTNNSFVVD); SEQ ID NO: 39 (GDTLKECPLKHRAWNSFL); SEQ ID NO: 46 (KVHVEETCGTRGPSLRST); SEQ ID NO: 49 (ECTMPPLSFRAK); SEQ ID NO: 3 (SFPTTLGMNKCYIQIMDL); SEQ ID NO: 26 (GALEAEMDGAKGRLSSGH); SEQ ID NO: 32 (FKSLFGGMSWFSQILIGT); SEQ ID NO: 9 (YGTCHHKKGEARRSR); SEQ ID NO: 17 (PENLEYRIMLSVHGSQHS); SEQ ID NO: 43 (REGYRTQMKGPWHSEELE); SEQ ID NO: 45 (IRFEECPGTKVHVEETCG); SEQ ID NO: 58 (HITEVEPEDIDCWCNLT); SEQ ID NO: 87 (QIANELNYILWENNIK) ;SEQ ID NO:89 (GKAKIVTAETQNSSFIID);SEQ ID NO:90 (GPNTPECPSASRAWNVWE);SEQ ID NO:97 (TVVITENCGTRGPSLRTT);SEQ ID NO:100 (SCTLPPLRYMGE);SEQ ID NO:54 (LFKTASGINMCTLIAMDL);SEQ ID NO:77 (GATEIQNSGGTSIFAGH);SEQ ID NO:83 (YTALFSGVSWVMKIGIGV);SEQ ID NO:60 (TSTWVTYGTCNQAG);SEQ ID NO:68 (YENLKYTVIITVHTGDQH);SEQ ID NO:94 (RPGYHTQTAGPWHLGKLE);SEQ ID NO:96 (LDFNYCEGTTVVITENCG), optionally an isolated peptide; or a peptide sharing at least 75% sequence identity therewith; or a peptide differing therefrom by 1, 2, 3 or 4 amino acids; or a portion thereof.

[0146] In various embodiments, compositions comprising the peptides disclosed herein, optionally immune system modulating compositions, and optionally immune system stimulating / activating compositions, and optionally vaccine compositions are provided. The compositions, when administered to a subject, can stimulate, e.g., increase, amplify, or boost, an immune response to flavivirus and / or ZIKV and / or DENV infection in the subject. In various embodiments, the compositions can increase the immune response to flavivirus and / or ZIKV and / or DENV infection in the subject by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold after administration to the subject. In some examples, the immune response of the subject can be determined by measuring the amount of ZIKV and / or DENV antigen binding protein in a sample from the subject. The composition may further include an adjuvant. Examples of suitable adjuvants include aluminum hydroxide and aluminum phosphate, which can enhance the body's immune response when administered.

[0147] In some embodiments, SEQ ID NO:7 (LDEGVEPDDVDCWCNTT); SEQ ID NO:36 (SVEGELNAILEENGVQ); SEQ ID NO:38 (GKSYFVRAAKTNNSFVVD); SEQ ID NO:39 (GDTLKECPLKHRAWNSFL); SEQ ID NO:46 (KVHVEETCGTRGPSLRST); SEQ ID NO:49 (ECTMPPLSFRAK); SEQ ID NO:3 (SFPTTLGMNKCYIQIMDL); SEQ ID NO:26 (GALEAEMDGAKGRLSSGH); SEQ ID NO:32 (FKSLFGGMSWFSQILIGT); SEQ ID NO:9 (YGTCHHKKGEARRSR); SEQ ID NO:17 (PENLEYRIMLSVHGSQHS); SEQ ID NO:43 (REGYRTQMKGPWHSEELE); SEQ ID NO:45 (IRFEECPGTKVHVEETCG); SEQ ID NO:58 (HITEVEPEDIDCWCNLT); SEQ ID NO:87 (QIANELNYILWENNIK); SEQ ID NO:89 (GKAKIVTAET QNSSFIID); SEQ ID NO:90 (GPNTPECPSASRAWNVWE); SEQ ID NO:97 (TVVITENCGTRGPSLRTT); SEQ ID NO:100 (SCTLPPLRYMGE); SEQ ID NO:54 (LFKTASGINMCTLIAMDL); SEQ ID NO:77 (GATEIQNSGGTSIFAGH); SEQ ID NO:83 (YTALFSGVSWVMKIGIGV); SEQ ID NO:60 (TSTWVTYGTCNQAG); SEQ ID NO:68 (YENLKYTVIITVHTGDQH); SEQ ID NO:94 (RPGYHTQTAGPWHLGKLE); SEQ ID NO:96 (LDFNYCEGTTVVITENCG); or a peptide sharing at least 75% sequence identity therewith; or a peptide differing therefrom by 1, 2, 3 or 4 amino acids; or a composition comprising a portion thereof, optionally an immunomodulatory composition, and optionally an immune system stimulating / activating composition, and optionally a vaccine composition, is provided.

[0148] In various embodiments, a method of inducing an immune response in a subject is provided, comprising administering to the subject a composition, optionally an immunomodulatory composition, and optionally an immune system stimulating / activating composition, and optionally a vaccine composition.

[0149] In various embodiments, provided herein are methods, kits, peptides, or compositions. EXAMPLES

[0150] Exemplary embodiments of the present disclosure will be better understood and readily apparent to those skilled in the art from the following discussion and, where applicable, in conjunction with the drawings. It should be understood that other modifications relating to structural, electrical, and optical changes may be made without departing from the scope of the present invention. The exemplary embodiments are not necessarily mutually exclusive, as some may be combined with one or more embodiments to form new exemplary embodiments.

[0151] ZIKV patients mount a robust and protective humoral response First, we used virion-based ELISA to screen 45 healthy donors for the presence of IgM and IgG against ZIKV, DENV, and Chikungunya virus (CHIKV), the three major arboviruses co-circulating in Singapore and several parts of Asia. Twenty-two donors (Fig. 1a-b) who had antibody levels below the assigned cutoffs (mean + SD) for all three viruses were used as a healthy control pool and set as the baseline standard.

[0152] Anti-ZIKV IgM and IgG levels were assessed longitudinally in ZIKV patients from the 2016 Singapore outbreak using virion-based ELISA. The majority of patients displayed robust ZIKV-specific humoral responses (Fig. 2a–c, Fig. 1c). Anti-ZIKV IgM was detectable as early as the acute phase (days 2–7 after onset of illness (pio)), peaked during the early convalescent phase (days 10–14 pio), and then declined during the convalescent phase (3 months–1 year pio) (Fig. 2a and c, Fig. 1c). ZIKV-specific IgG antibody titers peaked during the early convalescent phase, remained at high levels during the late convalescent phase, and remained detectable 1 year after infection (Fig. 2b–c, Fig. 1c). These patients were also screened for the presence of DENV-specific antibodies, and 80% of patients were anti-DENV IgM negative in samples taken during the acute phase (Fig. 1d and 1f). However, 75% of patients were found to have anti-DENV IgG (Fig. 1e–f), suggesting that ZIKV IgG, but not IgM, cross-reacts with DENV.

[0153] We then examined the IgG isotypes produced by ZIKV patients, and found that the highest titers of anti-ZIKV IgG1 and IgG3 subtypes occurred during the early convalescent phase for IgG3 and the late convalescent phase for IgG1 (Fig. 2d). To examine whether the antibodies produced in these patients were protective against ZIKV, we performed neutralization assays via flow cytometry (Fig. 2e-f, Fig. 3a). Efficient neutralization (71%-93%) was observed during the early and late convalescent phases (Fig. 2e), whereas only weak neutralization (37%-47%) was observed during the late convalescent and full convalescent phases (Fig. 2f). The neutralization capacity of ZIKV patients correlated with the levels of anti-ZIKV IgG (Fig. 1c and Fig. 9a). Plasma from these patients only minimally neutralized DENV (Fig. 3b), indicating ZIKV specificity.

[0154] Identification of specific B cell linear epitopes recognized by antibodies from ZIKV and DENV patients First, we performed preliminary mapping of specific ZIKV and DENV epitopes in peptide-based ELISAs against the most antigenic flavivirus antigens, prM, E, and NS1, using pooled linear ZIKV and consensus DENV peptides. We used plasma / serum samples from ZIKV and DENV patients collected in the late convalescent phase because IgG levels were highest at this time point (Fig. 1c). Results specifically showed that within the ZIKV and DENV proteomes, two common flavivirus pools (pools 1 and 21), six potential ZIKV-specific pools (pools 6, 10, 11, 16, 17, and 24), and one potential DENV-specific pool (pool 19) were identified (Table 1, Fig. 4).

[0155] [Table 1-1]

[0156] [Table 1-2]

[0157] Then, novel peptides selectively designed based on exposed residues and computational predictions were resynthesized for further experiments (Table 2).

[0158] [Table 2-1]

[0159] [Table 2-2]

[0160] [Table 2-3]

[0161] Interestingly, the results showed differences between the pooled and individual peptides (Table 3, Figure 5).

[0162] [Table 3-1]

[0163] [Table 3-2]

[0164] [Table 3-3]

[0165] Although these differences may be due to the interference of pool peptides, a single peptide could more strongly enhance the specific binding. Nevertheless, six potential common flavivirus peptides with relative differences in binding ability between ZIKV and DENV patients were identified (peptides 7, 36, 38, 39, 46, and 49) (Table 3, Figure 5, Figure 6). These peptides were also selected based on the close similarity between ZIKV and DENV peptide sequences (Table 2). Furthermore, three potential ZIKV-specific peptides (peptides 3, 26, and 32) and four potential DENV-specific peptides (peptides 9, 17, 43, and 45) with differences in binding ability greater than 0.1 were identified (Table 3, Figure 5, Figure 6).

[0166] Time course of epitope recognition by ZIKV patients To characterize the time course of epitope recognition by ZIKV patients, we screened common flavivirus peptides and ZIKV-specific peptides using plasma from ZIKV patients in the acute, late convalescent, and full recovery phases. For common flavivirus hits, more than 60% of ZIKV patients were able to recognize six peptide pairs at the late convalescent phase and beyond (Fig. 7a). However, only peptides 7, 36, and 38 were recognized by ZIKV patients during the acute phase (Fig. 7a). In terms of binding capacity, there was comparable binding between ZIKV and DENV peptide pairs over time for peptides 7, 36, 38, and 49 (Fig. 7b).

[0167] For ZIKV-specific epitopes, more than 60% of ZIKV patient samples were able to recognize peptides 3 and 26 (Fig. 7a) and had positive peptide binding capacity at the late convalescent phase (Fig. 7b). On the other hand, peptide 32, in addition to showing strong recognition by patient samples (Fig. 7a), also showed high binding capacity at various time points from the acute phase to the full convalescent phase (Fig. 7b). The localization of all potential epitopes within the viral proteins is shown in Fig. 7c-f.

[0168] Epitope evaluation using patient cohorts To evaluate the diagnostic performance of the identified epitopes, we screened the 13 peptides using patient serum samples from a cohort of patients who had DENV, bacterial, or unknown infections, and analyzed in parallel the results of a randomized selection of ZIKV and DENV patients in Singapore (Table 4).

[0169] [Table 4-1]

[0170] [Table 4-2]

[0171] Interestingly, the results showed a wide range of specificity and sensitivity for each peptide (Table 5, Figure 8a).

[0172] [Table 5-1]

[0173] [Table 5-2]

[0174] The 26 ZIKV-specific peptides (amino acid residues 271-288) in domain I / II (EDI / II) of the E protein had the best sensitivity and specificity profiles (80% and 85.7%, respectively) (Table 5, Fig. 8a). However, eight peptides (common flavivirus peptides 36, 38, 46, 49; ZIKV-specific peptides 3, 26, 32; and DENV-specific peptide 9) showed sensitivity and specificity greater than 50% (Table 5, Fig. 8a) and were selected for further evaluation. These peptides were used to "diagnose" patients (Table 6) and collectively determine the performance of peptide combinations based on epitope groupings (Table 5, Fig. 8b).

[0175] [Table 6-1]

[0176] [Table 6-2]

[0177] [Table 6-3]

[0178] The ZIKV-specific peptide mix showed a robust specificity of 96.4%, while the common flavivirus and DENV-specific groups showed intermediate measurements (Table 5, Fig. 8b). Furthermore, when the patients' anti-peptide IgG responses were plotted by principal component analysis (PCA), it was observed that patients with different diagnoses and cohorts formed separate clusters, with ZIKV patients standing out compared to healthy controls (Fig. 8c). To identify peptides with discriminatory power, the binding capacity of positive peptides was calculated. Virus-specific ZIKV and DENV epitopes were significantly different (Fig. 8d). Peptide 32 (amino acid residues 453–470 on the E protein) was the best-performing ZIKV-specific epitope and was able to distinguish ZIKV patients in Singapore from bacterial and unknown infections in Thailand (Fig. 8d–e). DENV-specific peptide 9 (amino acid residues 78–92 of prM) was used to distinguish DENV patients in Singapore from bacterial infection patients in Thailand (Fig. 8e). Overall, we were able to identify optimal differential epitopes for distinguishing between DENV and ZIKV patients.

[0179] conclusion ZIKV patients were shown to produce high levels of ZIKV-specific IgG antibodies. Specifically, IgG1 and IgG3 were the subclasses induced after ZIKV infection, very similar to DENV-infected patients. Although patients in this cohort had detectable DENV IgG levels, due to high levels of cross-reactivity between flaviviruses, neutralization of DENV was significantly less efficient compared to ZIKV, indicating that the antibodies were ZIKV specific (Fig. 2e-f, Fig. 3). This observation is supported by another study in which the profile of ZIKV neutralizing antibodies in patients from Nicaragua, Sri Lanka, and Thailand was not influenced by past DENV infection. Nevertheless, it is inevitable to consider the possible involvement of enhanced viral infection. Moreover, in our study, no ZIKV patients showed severe symptoms suggesting the occurrence of antibody-dependent enhancement (ADE), and similar observations have also been reported from Brazil.

[0180] Peptides identified from B cell epitope mapping have been reported in flavivirus E, prM NS1, and NS3 antigens from antibodies in patients and animal models. Identification of antigenic epitopes and characterization of cross-reactive epitopes are crucial for vaccine and immunodiagnostic development. Although various reports have shown the specificity of NS1 antigen for distinguishing ZIKV from DENV, the majority of common flavivirus peptides identified in this disclosure are in the NS1 protein, likely due to the conservation of regions of NS1 among flaviviruses. For example, common flavivirus peptides 36 (amino acid residues 70-85), 38 (amino acid residues 119-136), and 49 (amino acid residues 315-326) have been identified as ZIKV specific in other patient cohorts in South America. However, it remains unclear whether these peptides can be used to detect all flaviviruses, such as yellow fever virus (YFV) and Japanese encephalitis virus (JEV).

[0181] The identified differential ZIKV and DENV epitopes were located across prM, E, and NS1. Notably, DENV-specific peptide 17 (amino acid residues 131–149) and ZIKV-specific peptide 26 are found in the EDI and EDII of the E glycoprotein, which share 35% and 51% amino acid identity between ZIKV and DENV, respectively, whereas ZIKV-specific peptide 32 is found in the transmembrane domain of the anchor region (Fig. 7e). Interestingly, peptide 32 (amino acid residues 453–470) maps to a region overlapping with a DENV-2 epitope (amino acid residues 451–468) described in immune sera from DENV-2-infected patients. Computational analysis of ZIKV-specific peptide 32 and the DENV-2 equivalent epitope showed that they remain moderately accessible to the viral particle. As they share low sequence identity (43.75%), this epitope is structurally distinct and therefore may be recognized differently by ZIKV and DENV-specific antibodies. It will also be useful to evaluate the use of the identified peptides, especially peptides 26 and 32, as targets for ZIKV vaccines. Interestingly, despite the sequence similarity of these ZIKV and DENV peptide pairs (Table 2), we were able to distinguish between ZIKV and DENV patients. Furthermore, since ZIKV patients at different disease stages have different peptide recognition, in our setting we were able to identify ZIKV infection at any time point, independent of the patient's level of ZIKV-specific antibodies (Fig. 9b-C). However, given that the identified epitopes were screened and validated using samples from adult patients, it will be important to evaluate how these epitope profiles perform in other patient cohorts, especially in ZIKV-infected pregnant women in Brazil.

[0182] The identified putative epitopes were preliminarily evaluated in 38 patient samples. Interestingly, the Singapore and Thai DENV patients did not cluster together in the PCA (Fig. 8c). The majority of the Singapore DENV patients selected for validation had moderate to severe plasma leakage, a clinical feature of severe manifestations of DENV infection, while the Thai DENV patients showed mild symptoms (unpublished data). Thus, the latter "negative" in our assay may be due to differences in epitope recognition in different DENV pathologies, as well as differences in the strains of viruses circulating in Singapore and Thailand. Nevertheless, further improvements are needed to identify serotype-specific DENV epitopes.

[0183] Furthermore, comparison of these results with computationally predicted diagnostic peptide regions revealed differences. First, the majority of computationally predicted peptide regions were not ZIKV specific. For example, NS1 peptide 36 was predicted to be differential but was actually common flavivirus. Furthermore, peptide 26 of the E glycoprotein, predicted to recognize both ZIKV and DENV, was shown in this disclosure to be ZIKV specific. Despite the differences in the various approaches, computational prediction remains a useful tool.

[0184] Overall, this disclosure provides important valuable information about human antibody responses to ZIKV and insights into epitope cross-reactivity. Notably, several novel differential ZIKV and DENV epitopes with promising diagnostic utility were identified in the prM and E proteins. These results provide useful insights for the development of diagnostics or vaccines.

[0185] method Ethics statement Written informed consent was obtained from participants in accordance with the tenets of the Declaration of Helsinki. Study protocols for the Singapore ZIKV (2016–2018) and DENV (2010–2012) patient cohorts were approved by the SingHealth Centralised Institutional Review Board (CIRB Ref2016 / 2219) and the National Healthcare Group (NHG) Domain Specific Review Board (DSRB-E-2009 / 432), respectively. Samples from Singapore healthy donors (2010–2015) and Thai patients (2011–2013) were collected in accordance with the research guidelines approved by NUS-IRB 09-256 and NUS-IRB 10-445, respectively; MUTM 2011-008-01, OXTREC 42-10, and TCAB-01-11.

[0186] Research subjects and sample collection ZIKV patients in Singapore Specimen collection from subjects during the 2016 ZIKV outbreak has been previously described. 30 Briefly, 65 patients with positive RT-PCR for ZIKV and negative RT-PCR for DENV in whole blood or urine were enrolled. Whole blood samples were collected in EDTA-coated vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ, USA) after peripheral venipuncture and centrifuged at 12000 rpm for 10 min. Plasma was collected and stored at −80°C after heat inactivation at 56°C for 30 min. Samples were obtained across six time points: (1) acute phase [2–7 days after illness onset (pio)], (2) early convalescent phase (10–14 days pio), (3) late convalescent phase (1 month pio), (4) early convalescent phase (3 months pio), (5) late convalescent phase (5–6 months pio), and (6) complete convalescent phase (1 year pio).

[0187] DENV patients in Singapore This study used serum samples from 20 DENV patients collected before the ZIKV outbreak (2010-2012). Patients were positive for DENV PCR and / or NS1 on admission and included a combination of 1 patient with unknown serotype, 6 patients with DENV-1, 7 patients with DENV-2, 3 patients with DENV-3, and 3 patients with DENV-4. Serum samples used were obtained in the late convalescent phase (days 21-37 p.o.).

[0188] Thai patient Archived serum samples from an undifferentiated fever study conducted at the Shoklo Malaria Research Unit (SMRU) were used. Five DENV patients were confirmed by gold-standard paired serology tests, and all but one were DENV PCR positive. Five bacterial infection patients were diagnosed with leptospirosis, scrub typhus, murine typhoid, or S. pneumoniae infection, or a combination of the above, and all were DENV PCR and DENV NS1, IgM, and IgG RDT negative. Eight patients with unknown diagnoses were negative for the above pathogens by serology, blood culture, and PCR. Convalescent serum samples used were collected on days 14–20 p.i.

[0189] virus The ZIKV Polynesian isolate (H / PF / 2013) was obtained from the European Virus Archive (EVA, Marseille, France). DENV-3 was used as the reference serotype because of its prevalence in Southeast Asia and was kindly provided by the National Public Health Laboratory (NPHL), Singapore. CHIKV SGP011 was isolated from a patient in Singapore. Virus was propagated in VeroE6 cells (ATCC, Manassas, VA, USA) and purified via ultracentrifugation before titering by standard plaque assay on VeroE6 cells.

[0190] Virion-based ELISA As mentioned above 18、24~27Antibody titers were determined by virion-based ELISA. Briefly, purified viruses were immobilized overnight in 96-well maxisorp microtiter plates (Thermo Fisher Scientific, Waltham, MA, USA). Wells were blocked with 0.05% PBST [0.05% Tween-20 in PBS (Sigma-Aldrich, Saint Louis, MO, USA)] containing 5% skim milk (Nacalai Tesque, Kyoto, Japan) for 1.5 h at 37°C. Heat-inactivated patient and pooled healthy control plasma samples at 1:200 to 1:8000 dilutions prepared in PBST containing 2.5% milk were incubated for 1 h at 37°C. Detection was performed with HRP-conjugated goat anti-human IgM or IgG (H+L) (Thermo Fisher Scientific) or mouse anti-human IgG1, IgG2, IgG3, and IgG4 (Thermo Fischer Scientific) antibodies. Reactions were developed using TMB (3,3,5,5-tetramethylbenzidine) substrate (Sigma-Aldrich), terminated with stop reagent (Sigma-Aldrich), and absorbance was measured at 450 nm in a microplate autoreader (Tecan, Mannedorf, Zurich, Switzerland). ELISA readings were performed in triplicate or duplicate.

[0191] Serum Neutralization The neutralizing capacity of ZIKV patient-derived antibodies was determined via flow cytometry. Briefly, pooled patient and healthy plasma samples diluted 1:500, 1:1000, and 1:2000 were incubated with ZIKV or DENV-3 at an MOI of 10 for 2 h at 37 °C with gentle agitation (350 rpm). The virus-antibody suspension was then added in duplicate to HEK 293T cells (ATCC) at 37 °C. After 2 h, the medium was removed and Dulbecco's modified Eagle's medium (DMEM; GE Healthcare Life Sciences) containing 10% fetal bovine serum (FBS; GE Healthcare Life Sciences, Pittsburgh, PA, USA) was added. After 48 h, cells were harvested and neutralized with a ZIKV NS3 protein-specific rabbit polyclonal antibody or the DENV human monoclonal antibody 1B25 as previously described. 54 Cells were stained and counterstained with fluorophore-tagged goat anti-rabbit or anti-human IgG (H+L) (Thermo Fisher Scientific), respectively. Cells were acquired using a MacsQuant Analyser 10 (Miltenyi-Biotec, Bergisch Gladbach, Germany). Assays were performed in duplicate in two independent experiments. Flow cytometry results were analyzed using FlowJo (version 10.4.1, Tree Star Inc, Ashland, OR, USA). Data from neutralization assays of patients and pooled healthy controls were normalized to the respective untreated infections and calculated as a percentage of virus-only control infections.

[0192] Epitope determination Linear Peptide Libraries The sequences used to design biotinylated linear peptides of prM, E, and NS1 proteins were derived from the consensus sequences of ZIKV Polynesian isolates (KJ776791) and DENV-3 strains (KR296743, KF973487, EU081181, KF041254, JF808120, JF808121, KJ189293, KC762692, KC425219, KJ830751, KF973479, and AY099336). Peptides were generated as ZIKV and DENV peptide pairs of corresponding sequences. A library of peptides consisting of 18-mer overlapping sequences (Mimotopes, Mulgrave, Victoria, Australia) was used for preliminary epitope screening. Five peptides were combined to form one pooled peptide set. Patient screening and validation were performed using higher purity peptides (≥90%, EMC microcollections GmbH, Tuebingen, Germany) with lengths of 11–22 mer (Table 2). Peptides were dissolved in DMSO (Sigma-Aldrich) to a stock concentration of 3.75 μg / μL.

[0193] Peptide-based ELISA As mentioned above 24、25、27Epitope determination was performed via peptide-based ELISA. Briefly, streptavidin-coated plates (Thermo Fisher Scientific) were blocked overnight at 4°C with 0.1% PBST (0.1% Tween-20 in PBS) containing 1% sodium caseinate (Sigma-Aldrich) and 1% bovine serum albumin (BSA; Sigma-Aldrich), followed by the addition of biotinylated peptides (1:1000 dilution in 0.1% PBST) followed by the addition of heat-inactivated pooled healthy control and patient plasma / serum samples (1:2000 dilution in 0.1% PBST). Antibodies bound to the peptides were detected with HRP-conjugated goat anti-human IgG (H+L) antibody (Thermo Fisher Scientific) prepared in 0.1% blocking buffer. After color development using TMB substrate and stop reagent (Sigma-Aldrich), absorbance at 450 nm was measured (Tecan). All incubation steps were performed for 1 h at room temperature on a rotating shaker and ELISA readings were performed in duplicate.

[0194] 4. Data Analysis The OD values ​​obtained in the peptide-based ELISA experiments for ZIKV and DENV were first normalized to the average OD value of pooled healthy donors. Patient samples were considered positive if the normalized response was greater than 1.01. The normalized values ​​were then used to calculate peptide binding capacity as [(ZIKV peptide response-DENV peptide response) / DENV peptide response]. A binding capacity with a positive value means that the sample binds preferentially to the ZIKV peptide, while a negative value means that it binds preferentially to the corresponding DENV peptide. The difference in the average peptide binding capacity of peptide pairs (i.e., ZIKV and DENV peptides with complementary sequences) of ZIKV and DENV patients was calculated. Peptides with a relative difference of 0.1 or more are considered to be differential ZIKV and DENV epitopes of interest, whereas peptides with a difference of 0.05 or less and sharing amino acid similarity between the peptide pairs (Table 2) are considered to be common flavivirus epitopes.

[0195] Data visualization and statistical analysis Heatmaps were generated using Multi Experiment Viewer (version 4.8, Microarray Software Suite TM4, Boston, MA, USA). For structural localization, simulations of ZIKV prM were performed using Phyre (version 2, Structural Bioinformatics Group, London, UK). The structures of DENV-3 prM, ZIKV E glycoprotein, DENV-3 E glycoprotein, stem transmembrane domain of ZIKV E glycoprotein, stem transmembrane domain of DENV-3 E glycoprotein, ZIKV NS1, and DENV-3 NS1 were modeled based on PDB 3C6E, 5JHM, 1UZG, 5IZ7, 3J2P, 5K6K, and 4O6B, respectively. All structures were visualized using PyMol (Schroedinger, Cambridge, MA, USA). Principal component analysis (PCA) was performed using the OD values ​​of anti-peptide IgG responses for each patient using the prcomp function in R (version 3.3.1; R Foundation for Statistical Computing, Vienna, Austria).

[0196] Statistical analysis was performed using GraphPad Prism (version 7.03, San Diego, CA, USA). Any statistical significance was derived using a two-tailed Mann-Whitney U test with Bonferroni correction for multiple testing or a Kruskal-Wallis test with Bonferroni correction for multiple testing, and post-hoc testing with Dunn's multiple comparison test. Correlation analysis was performed using Spearman's rank correlation. P values ​​less than 0.05 were considered significant.

[0197] Those skilled in the art will appreciate that other variations and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the present disclosure as broadly described. For example, as described herein, features of different illustrative embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, subsumed, etc. among the different illustrative embodiments. The present embodiments are therefore considered in all respects to be illustrative and not restrictive.

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patients.J Infect Dis 2018;218:814-824. 31 Vaughan K,Greenbaum J,Blythe M,Peters B,Sette A.Meta-analysis of all immune epitope data in the Flavivirus genus:inventory of current immune epitope data status in the context of virus immunity and immunopathology.Viral Immunol 2010;23:259-284. 32 Low JGH,Ooi E-E,Tolfvenstam T et al.Early dengue infection and outcome study(EDEN)- study design and preliminary findings.Ann Acad Med Singapore 2006;35:783-789. 33 Koraka P,Suharti C,Setiati TE et al.Kinetics of dengue virus-specific serum immunoglobulin classes and subclasses correlate with clinical outcome of infection.J Clin Microbiol 2001;39:4332-4338. 34 Montoya M,Collins M,Dejnirattisai W et al.Longitudinal analysis of antibody cross-neutralization following Zika virus and dengue virus infection in Asia and the Americas.J Infect Dis 2018;218:536-545. 35 Martin-Acebes MA,Saiz J-C,Jimenez de Oya N.Antibody-dependent enhancement and Zika:real threat or phantom menace? 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Biology of Zika virus infection in human skin cells. J Virol 2015;89:8880 - 8896. 54 Ang LW, Kam YW, Lin C et al. Seroprevalence of antibodies against chikungunya virus in Singapore resident adult population. PLoS Negl Trop Dis 2017;11:1 - 13. 55 Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 2015;10:845.

[0199]

Table 7 - 1

[0200]

Table 7 - 2

[0201]

Table 8 - 1

[0202]

Table 8 - 2

[0203]

Table 8 - 3

[0204] Use In this disclosure, we longitudinally characterized antibody and neutralizing responses from ZIKV patients in Singapore. We then identified common and differential linear B cell epitopes recognized by antibodies from ZIKV and DENV patients in Singapore. Importantly, we further evaluated the potential value of these identified epitopes in a diagnostic context using sera from patients in Thailand who had previously been diagnosed with DENV, bacteria (as well as those with unknown infections).

[0205] This disclosure will facilitate the development of serological test-based differential diagnostics between flaviviruses, particularly ZIKV and DENV, allowing accurate diagnosis to improve patient management, and its application can be further extended to seroprevalence and vaccine strategy studies.

[0206] Method embodiments advantageously provide a suitable serodiagnostic tool that can accurately identify flavivirus infections, such as ZIKV and / or DENV, or differentiate between two flavivirus infections, e.g., ZIKV and DENV.

Claims

1. 1. A method for identifying a flavivirus infection selected from Zika virus (ZIKV), Dengue virus (DENV), and combinations thereof in a subject, comprising: The subject sample is subjected to a relative binding affinity BC ｒｅｌａｔｉｖｅ ≦0.05 or ≧0.1, [0010] (i) When the peptide P comprises a ZIKV-derived peptide, [0025] or (ii) When the peptide P comprises a DENV-derived peptide, [0030] 1. A method according to claim 1, wherein the binding affinity of the ZIKV-induced antigen-binding protein is about -0.50 to about 0.50; the binding affinity of the DENV-derived antigen-binding protein is between about -0.60 and about 0.30; and A ZIKV-derived antigen-binding protein or a DENV-derived antigen-binding protein having a binding affinity of greater than 0 indicates that the ZIKV or DENV-derived antigen-binding protein recognizes ZIKV / ZIKV-derived peptides more strongly than DENV / DENV-derived peptides and / or has higher binding activity to ZIKV / ZIKV-derived peptides than DENV / DENV-derived peptides; The peptide P is A method comprising the sequence shown in sequence number 7 (LDEGVEPDDVDCWCNTT).

2. The method of claim 1, wherein if the sample reacts with the peptide, the subject is eligible for treatment of a flavivirus infection.

3. 1. A method for identifying acute-phase flavivirus infection, comprising: The method of claim 1 or 2, wherein if the sample reacts with the peptide, the subject is indicated for treatment of an acute flavivirus infection.

4. The sample is SEQ ID NO:3 (SFPTTLGMNKCYIQIMDL); SEQ ID NO:26 (GALEAEMDGAKGRLSSGH); or SEQ ID NO:32 (FKSLFGGMSWFSQILIGT); and further determining whether the sample reacts with one or more peptides selected from the group consisting of: The method of any one of claims 1 to 3, wherein if the sample reacts with the one or more peptides, the subject is eligible for treatment of ZIKV infection.

5. A method for identifying acute-phase ZIKV infection, comprising: SEQ ID NO:32 (FKSLFGGMSWFSQILIGT); and further determining whether the antibody reacts with The method of any one of claims 1 to 4, wherein if the sample reacts with the peptide, the subject is indicated for treatment of acute ZIKV infection.

6. The sample is SEQ ID NO:60 (TSTWVTYGTCNQAG); SEQ ID NO:68 (YENLKYTVIITVHTGDQH); SEQ ID NO:94 (RPGYHTQTAGPWHLGKLE); and / or SEQ ID NO:96 (LDFNYCEGTTVVITENCG); determining whether the peptide reacts with one or more peptides selected from the group consisting of The method of any one of claims 1 to 5, wherein if the sample reacts with the one or more peptides, the subject is indicated for treatment of DENV infection.

7. 7. The method of any one of claims 1 to 6, wherein determining whether the sample reacts with peptide P comprises performing an immunoassay to assess whether an antigen binding protein capable of binding to the peptide is present in the sample.

8. A peptide P comprising the sequence shown in SEQ ID NO: 7 (LDEGVEPDDVDCWCNTT) for use in a method for treating ZIKV and / or DENV infection in a subject, the method comprising the steps of: (i) identifying a flavivirus infection selected from ZIKV, DENV and combinations thereof in a subject using the method of any one of claims 1 to 7; and (ii) a ZIKV and / or DENV treatment regimen administered to the subject if ZIKV and / or DENV infection is indicated; Peptide P.

9. A kit for identifying a flavivirus infection selected from Zika virus, Dengue virus, and combinations thereof in a subject, comprising: ｒｅｌａｔｉｖｅ ≦0.05 or ≧0.1, [0045] When the peptide P comprises a ZIKV-derived peptide, [0050] and When the peptide P comprises a DENV-derived peptide, [006] A kit comprising: The binding affinity of the ZIKV-induced antigen-binding protein is about -0.50 to about 0.50; the binding affinity of the DENV-derived antigen-binding protein is between about -0.60 and about 0.30; and A ZIKV-derived antigen-binding protein or a DENV-derived antigen-binding protein having a binding affinity of greater than 0 indicates that the ZIKV or DENV-derived antigen-binding protein recognizes ZIKV / ZIKV-derived peptides more strongly than DENV / DENV-derived peptides and / or has higher binding activity to ZIKV / ZIKV-derived peptides than DENV / DENV-derived peptides; The peptide P is A kit comprising the sequence shown in sequence number 7 (LDEGVEPDDVDCWCNTT).

10. A plate coated with an anti-ZIKV and / or anti-DENV capture agent, and Detection agents for detecting the presence of captured anti-ZIKV and / or anti-DENV and The kit of claim 9 , wherein the capture agent and the detection agent comprise a ZIKV and / or DENV antigen and / or an anti-immunoglobulin.

11. The method of any one of claims 1 to 7, wherein the subject comprises an Asian subject.

12. The kit of claim 9 or 10, wherein the subject comprises an Asian subject.

13. Relative binding capacity BC ｒｅｌａｔｉｖｅ ≦0.05 or ≧0.1, [0070] An isolated peptide P, which is (iii) When the peptide P comprises a ZIKV-derived peptide, [0080] or (iv) When peptide P comprises a DENV-derived peptide, [0090] and The binding affinity of the ZIKV-induced antigen-binding protein is about -0.50 to about 0.50; the binding affinity of the DENV-derived antigen-binding protein is between about -0.60 and about 0.30; and The peptide P is Isolated peptide P, which is a peptide having the sequence shown in SEQ ID NO: 7 (LDEGVEPDDVDCWCNTT).

14. Relative binding capacity BC ｒｅｌａｔｉｖｅ A composition comprising peptide P, wherein the P is ≦0.05 or ≧0.1, [0010] and (v) When the peptide P comprises a ZIKV-derived peptide, ##EQU00011## or (vi) When peptide P comprises a DENV-derived peptide, ##EQU00012## and The binding affinity of the ZIKV-induced antigen-binding protein is about -0.50 to about 0.50; the binding affinity of the DENV-derived antigen-binding protein is between about -0.60 and about 0.30; and The peptide P is A composition which is a peptide having the sequence shown in SEQ ID NO: 7 (LDEGVEPDDVDCWCNTT).

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