Methods and kits for determining the presence and / or amount of human IGG3 antibodies specific for flavivirus antigens in a sample - Patents.com

Abstract

The present invention relates to a method for determining the presence and / or amount of human IgG3 antibodies, preferably specific for a viral antigen, in a sample. The present invention also relates to the use of said method in the quality control of viral vaccines and in the diagnosis of viral infections. Furthermore, a kit for use in said method is described.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This international PCT application claims priority to and the benefit of European Application No. 21216535.1, filed December 21, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to a method for determining the presence and / or amount of human IgG3 antibodies, preferably specific for a viral antigen, in a sample. The present invention also relates to the use of said method in the quality control of viral vaccines and in the diagnosis of viral infections. Furthermore, a kit for use in said method is described. [Background technology]

[0003] The antibody response to viral infections in humans is diverse and has a wide range of clinical significance. Pre-existing reactive antibodies, or antibodies formed early during infection, can bind to viral particles, forming immune complexes that can neutralize the virus or mediate its clearance. On the other hand, immune complexes can also promote inflammation and exacerbate disease symptoms. How antibodies in immune complexes regulate infection depends in part on their Fc domain structure. The Fc structure, in turn, dictates their interaction with Fcγ receptors (FcγR), which are expressed by various cells that are activated during infection.

[0004] Antibody isotypes IgG, IgA and IgM are the major determinants of Fc structure and therefore activity. Early B cell responses are characterized by the production of IgM antibodies. This is followed by the production of class-switched IgA and IgG antibodies, with IgA playing a central role in mucosal immunity, while IgG is the predominant isotype involved in systemic antiviral immunity. IgG function is regulated by interactions between immune complexes and effector immune cells expressing FcγR, the receptor for IgG. The residual amount of FcγR engaged by immune complexes determines the degree of inflammatory effector cell response. Activating low affinity FcγR (FcγRIIa and FcγRIIIa) mediates inflammatory signaling via immunoreceptor tyrosine-dependent activation motifs (ITAMs), whereas in health, ITAM signaling is counterbalanced by immunoreceptor tyrosine-dependent inhibitory motif (ITIM) signaling via inhibitory FcγRIIb.

[0005] The strength of the interaction between immune complexes and various FcγRs is determined by the structural diversity within IgG subclasses (IgG1, IgG2, IgG3, and IgG4) and post-translational modifications of their Fc domains. Importantly, individuals produce distinct structural repertoires of IgG Fc domains, with some producing highly activating / pro-inflammatory repertoires enriched for IgG1, IgG3, etc., and / or with reduced core fucosylation of the IgG1 Fc domain. Others produce IgG repertoires characterized by higher levels of IgG2 and / or sialylated Fc with reduced activating / inflammatory FcγR signaling potential (Chakaborty et al., Nature Immunol. 22 (2021), 67-73).

[0006] Nascimento et al., J. Virol. Methods 257(2018), 62-68, describe anti-dengue NS1-specific IgG and IgG3 as potential biomarkers of long-term and recent (within 6 months) dengue virus infection, respectively. IgG3 was also used as a marker of recent infection with HIV (Viana et al., Epidemiology & Infection 146(2018), 1293-1300. Rodriguez-Barraquer et al., Science 363(6427), 607-610, describe the measurement of IgG3 responses against the Zika virus NS1 protein.

[0007] Trend et al., Front. Immunol. 9 (2018), Article 1590:1-13, review that higher serum IgG3 levels may predict the development of multiple sclerosis in individuals with clinically isolated syndrome.

[0008] Chakaborty et al., supra, described that IgG3 and IgG1 with F0N0 glycoform modifications are elevated in a greater proportion of severe COVID-19 patients.

[0009] There is a need to provide improved assays for determining IgG3 subtype antibodies, particularly assays for determining IgG3, and in particular human IgG3 specific for viral antigens, that have improved sensitivity and / or specificity over prior art assays. Summary of the Invention

[0010] In a first aspect, the present invention relates to a method for determining the presence and / or amount of human IgG3 antibodies specific for a flavivirus antigen in a sample, comprising the steps of: Step 1: contacting a volume of sample with protein A bound to beads to allow binding of human IgG1, human IgG2, human IgG4, human IgA, human IgE and human IgM antibodies to the protein A bound to the beads; Step 2: separating the human IgG1, human IgG2, human IgG4, human IgA, human IgE and human IgM antibodies that are bound to the bead-bound Protein A from the remainder of the sample, thereby producing a human IgG3 antibody-enriched supernatant; Step 3: Removing the human IgG3 antibody enriched supernatant from human IgG1, human IgG2, human IgG4, human IgA, human IgE and human IgM antibodies that are bound to the bead-bound Protein A; Step 4: contacting the viral antigens with human IgG3 antibody enriched supernatant to allow binding of human IgG3 antibodies to the flavivirus antigens; Step 5: contacting the human IgG3 antibody bound to the viral antigen with a primary IgG-specific antibody or a primary IgG3-specific antibody to allow binding of the primary IgG-specific antibody or the primary IgG3-specific antibody to the human IgG3 antibody bound to the flavivirus antigen; Step 6: contacting the bound primary IgG3-specific antibody or the bound primary IgG-specific antibody of step 4 with a labeled secondary antibody specific for the primary IgG3-specific antibody or the primary IgG-specific antibody to allow binding of the labeled secondary antibody to the primary antibody; and Step 7: detecting a signal from the labeled secondary antibody bound to the primary antibody in step 5, wherein the signal indicates the presence and / or amount of the labeled secondary antibody, which indicates the presence and / or amount of human IgG3 antibody specific for a Flavivirus antigen in the sample; The method includes the steps of:

[0011] In a second aspect, the present invention provides the use of in vitro pretreatment of a sample containing human IgG3 and one or more of human IgG1, IgG2, IgG4, IgA and IgM antibodies specific for a Flavivirus antigen with immobilized or immobilizable Protein A to improve the sensitivity of a method for determining the presence and / or amount of human IgG3 antibodies in the sample.

[0012] In a third aspect, the present invention provides the use of the method according to the invention for the in vitro diagnosis of a flavivirus infection, preferably a dengue virus infection, within the past 6 months.

[0013] In a fourth aspect, the present invention provides the use of the method according to the invention for determining the human IgG3 response to a flavivirus vaccine.

[0014] In a fifth aspect, the present invention relates to a kit for determining the presence and / or amount of human IgG3 antibodies in a sample, comprising: (a) Protein A bound to magnetic beads; (b) a primary IgG-specific antibody or a primary IgG3-specific antibody; and (c) A labeled secondary antibody specific for the primary IgG-specific antibody or the primary IgG3-specific antibody. The kit includes:

[0015] In a sixth aspect, the present invention relates to a kit for determining the presence and / or amount of human IgG3 antibodies in a sample, comprising: (a) Protein A bound to magnetic beads; and (b) Labeled primary IgG-specific antibody or labeled primary IgG3-specific antibody The kit includes:

[0016] Prior art methods for determining IgG3 subtype antibodies, such as ELISA or immunofluorescence assays, are based on the use of IgG3-specific primary antibodies. The percentage of IgG3 in biological samples is relatively low, typically less than 5%. In contrast, the percentages of IgG1 and IgG2 are 60% and 32%, respectively; see Vidarsson et al., Front.Immunol.5(2014),Article 520:1-17. The inventors found that the sensitivity / specificity of prior art assays is adversely affected by the coexistence of large amounts of IgG1 and IgG2. The inventors found that a prior step of removing human IgM, human IgA, human IgG1, human IgG2 and human IgG4 by using Protein A can significantly improve the sensitivity of the detection of human IgG3 in a sample. [Brief description of the drawings]

[0017] [Figure 1] Determination of IgG3 amount in different patient samples by the method according to the invention (denoted Protein A) and by a method that did not include a Protein A removal step before the actual determination of IgG3 (denoted Pre-removal). DENV1 VLP (top left panel), DENV2 VLP (top right panel), DENV3 VLP (bottom left panel), and DENV4 VLP (bottom right panel) were used as antigens, respectively. Putative Dengue samples used in Figures 1-6: PLA-102, PLA-116, PLA-108, and PLA-117. Putative Zika samples used in Figures 1-6: PARS_64, PARS_55, PARS_71, PARS_70, and PARS_97. [Diagram 2]Determination of IgG3 amount in different patient samples by the method according to the invention (denoted Protein A) and by a method that did not include a Protein A removal step before the actual determination of IgG3 (denoted Pre-removal). DENV1 NS1 protein (top left panel), DENV2 NS1 protein (top right panel), DENV3 NS1 protein (bottom left panel), and DENV4 NS1 protein (bottom right panel) were used as antigens, respectively. Putative Dengue samples used in Figures 1-6: PLA-102, PLA-116, PLA-108, and PLA-117. Putative Zika samples used in Figures 1-6: PARS_64, PARS_55, PARS_71, PARS_70, and PARS_97. [Diagram 3] Determination of IgG3 amount in different patient samples by the method according to the invention (denoted Protein A) and by a method that did not include a Protein A removal step before the actual determination of IgG3 (denoted Pre-removal). DENV1 VLP (top left panel), DENV2 VLP (top right panel), DENV3 VLP (bottom left panel), and DENV4 VLP (bottom right panel) were used as antigens, respectively. Putative Dengue samples used in Figures 1-6: PLA-102, PLA-116, PLA-108, and PLA-117. Putative Zika samples used in Figures 1-6: PARS_64, PARS_55, PARS_71, PARS_70, and PARS_97. [Figure 4] Determination of IgG3 amount in different patient samples by the method according to the invention (denoted Protein A) and by a method that did not include a Protein A removal step before the actual determination of IgG3 (denoted Pre-removal). DENV1 NS1 protein (top left panel), DENV2 NS1 protein (top right panel), DENV3 NS1 protein (bottom left panel), and DENV4 NS1 protein (bottom right panel) were used as antigens, respectively. Putative Dengue samples used in Figures 1-6: PLA-102, PLA-116, PLA-108, and PLA-117. Putative Zika samples used in Figures 1-6: PARS_64, PARS_55, PARS_71, PARS_70, and PARS_97. [Diagram 5]Determination of IgG3 amounts in different patient samples by the method according to the invention (denoted Protein A) and by a method that did not include a Protein A removal step before the actual determination of IgG3 (denoted Pre-removal). Zika VLPs were used as antigen. Putative Dengue samples used in Figures 1-6: PLA-102, PLA-116, PLA-108, and PLA-117. Putative Zika samples used in Figures 1-6: PARS_64, PARS_55, PARS_71, PARS_70, and PARS_97. [Figure 6] Determination of IgG3 amounts in different patient samples by the method according to the invention (denoted Protein A) and by a method that did not include a Protein A removal step before the actual determination of IgG3 (denoted Pre-removal). Zika NS1 protein was used as antigen. Putative Dengue samples used in Figures 1-6: PLA-102, PLA-116, PLA-108, and PLA-117. Putative Zika samples used in Figures 1-6: PARS_64, PARS_55, PARS_71, PARS_70, and PARS_97. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] In a first aspect, the present invention relates to a method for determining the presence and / or amount of human IgG3 antibodies specific for a flavivirus antigen in a sample, comprising the steps of: Step 1: contacting a volume of sample with protein A bound to beads to allow binding of human IgG1, human IgG2, human IgG4, human IgA, human IgE and human IgM antibodies to the protein A bound to the beads; Step 2: separating the human IgG1, human IgG2, human IgG4, human IgA, human IgE and human IgM antibodies that are bound to the bead-bound Protein A from the remainder of the sample, thereby producing a human IgG3 antibody-enriched supernatant; Step 3: Removing the human IgG3 antibody enriched supernatant from human IgG1, human IgG2, human IgG4, human IgA, human IgE and human IgM antibodies that are bound to the bead-bound Protein A; Step 4: contacting the viral antigens with human IgG3 antibody enriched supernatant to allow binding of human IgG3 antibodies to the flavivirus antigens; Step 5: contacting the human IgG3 antibody bound to the viral antigen with a primary IgG-specific antibody or a primary IgG3-specific antibody to allow binding of the primary IgG-specific antibody or the primary IgG3-specific antibody to the human IgG3 antibody bound to the flavivirus antigen; Step 6: contacting the bound primary IgG3-specific antibody or the bound primary IgG-specific antibody of step 4 with a labeled secondary antibody specific for the primary IgG3-specific antibody or the primary IgG-specific antibody to allow binding of the labeled secondary antibody to the primary antibody; and Step 7: detecting a signal from the labeled secondary antibody bound to the primary antibody in step 5, wherein the signal indicates the presence and / or amount of the labeled secondary antibody, which indicates the presence and / or amount of human IgG3 antibody specific for a Flavivirus antigen in the sample; The method includes the steps of:

[0019] Step 1 of the method of the present invention involves contacting a volume of sample with Protein A bound to beads to allow binding of human IgG1, human IgG2, human IgG4, human IgA, human IgE and human IgM antibodies to the Protein A bound to the beads.

[0020] "Protein A" as used herein includes Protein A itself. Protein A is a 49 kDa surface protein found originally in the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene and its regulation is controlled by DNA topology, cell osmolarity, and a two-component system called ArlS-ArlR. It has found use in biochemical research due to its ability to bind immunoglobulins. As used herein, the term "Protein A" also includes sequence variants of Protein A that differ in having at least one amino acid, preferably no more than 10 amino acids, more preferably no more than 5 amino acids, and most preferably no more than 1 amino acid addition, substitution, and / or deletion. The term also includes conjugates of Protein A with peptides or proteins.

[0021] Protein A bound to beads can be commercially available. Alternatively, it can be prepared by binding Protein A to beads. Beads are commercially available. The material of the beads can be Sepharose or Sephadex. Binding can be done by using activated moieties on the surface of the beads. Alternatively, magnetic beads can be used. Protein A bound to magnetic beads is commercially available (AmMag™ Protein A Magnetic Beads; Genscript).

[0022] "Sample" as used herein includes blood samples. Preferably, the sample is serum from a human individual. The individual may be a patient with a virus infection or a healthy individual immunized with a virus vaccine. The virus may be a DNA or RNA virus. The virus may be either double-stranded or single-stranded.

[0023] More preferably, the DNA virus is a herpes virus, a pox virus, an adenovirus, a papilloma virus and a polyoma virus.

[0024] More preferably, the RNA virus is a flavivirus, a reovirus, a retrovirus, an orthomyxovirus, a deltavirus, a rhabdovirus, a filovirus or a paramyxovirus.Even more preferably, the RNA virus is a dengue virus, a yellow fever virus, a Japanese encephalitis virus, a tick-borne encephalitis virus, a West Nile virus, a Zika virus, an HIV virus, an influenza virus, a rotavirus, an RSV, a coronavirus, a measles, a mumps or a rabies.Most preferred RNA viruses are a dengue virus, a yellow fever virus, a West Nile virus, a Zika virus and a coronavirus.In particular, the RNA virus is a dengue virus or a Zika virus.When the RNA virus is a dengue virus, the virus can be selected from DENV1, DENV2, DENV3 and / or DENV4.

[0025] The vaccine used for vaccination of an individual may preferably be a dengue vaccine, such as a quadrivalent vaccine, such as TAK-003 (Takeda).

[0026] The contacting step can be carried out in a plate format or a tube format. Alternatively, it can be carried out in a column format. Preferably, a plate with 24 or 96 wells is used. In the first step, the plate is incubated with the virus antigen to allow the binding of human antibodies other than human IgG3 to the protein A-bound beads.

[0027] Conditions for binding of human IgG1, human IgG2, human IgG4, human IgA, human IgE and human IgM to bead-bound Protein A are known to those skilled in the art. Strongly acidic or strongly basic conditions must be avoided to prevent denaturation of Protein A and / or human antibodies. Preferably, loading of Protein A is performed at a pH in the range of about pH 8.0 to about pH 10.0. More preferably, loading is performed in 1 M potassium phosphate at pH 9.0.

[0028] Step 2 of the method of the present invention involves separating the human IgG1, human IgG2, human IgG4, human IgA, human IgE and human IgM antibodies that are bound to the bead-bound Protein A from the remainder of the sample, thereby producing a human IgG3 antibody-enriched supernatant.

[0029] Separating the antibody-bound protein A bound to the beads from the remainder of the sample can be done by centrifugation. If the beads are magnetic beads, separation can also be done by magnetic force. This can be accomplished, for example, by using a magnetic stand.

[0030] Step 3 of the method of the present invention involves removing the human IgG3 antibody enriched supernatant from the human IgG1, human IgG2, human IgG4, human IgA, human IgE and human IgM antibodies that are bound to the bead-bound Protein A.

[0031] The remainder of the sample, depleted of human IgG1, human IgG2, human IgG4, human IgA, human IgE and human IgM, can then be removed from the beads, which can be done by aspiration or decantation.

[0032] Step 4 of the method of the present invention involves contacting the flavivirus antigen with human IgG3 antibody enriched supernatant to allow binding of human IgG3 antibodies to the viral antigen.

[0033] "Viral antigen" as used herein refers to any substance that can be bound by Ab. An antigen can induce an immune response in a subject. An antigen can have one or more epitopes. An antigen can be a protein, a polypeptide, a carbohydrate, a polynucleotide, a lipid, or a combination thereof. An antigen can be a truncated form of a protein, a protein tagged with an affinity tag such as His or STREP tag, or a single domain of a protein. As used herein, an antigen can refer to, for example, DENV1 VLP, DENV2 VLP, DENV3 VLP, DENV4 VLP, ZIKV VLP; ZIKV NS1, DENV1 NS1, DENV2 NS1, DENV3 NS1, DENV4 NS1, DENV1 E, DENV2 E, DENV3 E, and / or DENV4 E. Thus, the term flavivirus antigen refers to an antigen derived from a flavivirus.

[0034] The term "virus like particle (VLP)" or "virus like particle(s) (VLPs)" refers to molecules that closely resemble viruses but do not contain viral genetic material and are therefore non-infectious. VLPs can be prepared recombinantly by expression of viral structural proteins that can then self-assemble into VLPs. Examples of VLPs are ZIKV VLPs and DENV VLPs.

[0035] The contacting step can be carried out in a plate format. Preferably, 48-well or 96-well plates can be used. In a first step, the plate is incubated with a viral antigen to allow binding of the viral antigen to the surface of the plate. When testing different viral antigens, for example from different flaviviruses such as dengue or zika, or from different dengue virus serotypes, the antigens can be in different wells (single-plex format) or in the same well (multiplex format). Methods for performing single-plex and multiplex assays are known to those skilled in the art.

[0036] The contacting step may be followed by a blocking step known to those skilled in the art. Conditions for incubation and blocking must be such that the viral antigens are not significantly denatured. Any of these steps may be followed by a washing step.

[0037] Step 5 of the method of the present invention involves contacting the human IgG3 antibody bound to the flavivirus antigen with a first IgG-specific antibody or a first IgG3-specific antibody to allow binding of the first IgG-specific antibody or the first IgG3-specific antibody to the human IgG3 antibody bound to the flavivirus antigen.

[0038] The primary IgG-specific antibody is an anti-IgG antibody that has the ability to specifically bind to more than one IgG subtype. Preferably, the primary IgG-specific antibody is a pan-IgG antibody that specifically binds to each of the IgG subtypes, i.e., IgG1, IgG2, IgG3 and IgG4. Pan-IgG antibodies are commercially available.

[0039] Alternatively, a primary IgG3 specific antibody may be used. Such antibodies are likewise commercially available or can be produced by methods known in the art. Preferably, the primary IgG specific antibody and the primary IgG3 specific antibody are of non-human origin. More preferably, goat, mouse, rat, hamster antibodies may be used.

[0040] In a separate embodiment, the primary IgG-specific antibody or the primary IgG3-specific antibody is linked to a detection label. Thus, in this embodiment, steps 5 to 7 defined above are: Step 5': contacting the human IgG3 antibody bound to the viral antigen with a labeled primary IgG-specific antibody or a labeled primary IgG3-specific antibody to allow binding of the labeled primary IgG-specific antibody or the labeled primary IgG3-specific antibody to the human IgG3 antibody bound to the flavivirus antigen; and Step 6': detecting a signal from the labeled primary IgG-specific antibody or the labeled primary IgG3-specific antibody bound to the viral antigen in step 5', the signal indicating the presence and / or amount of the labeled primary IgG-specific antibody or the labeled primary IgG3-specific antibody, and the presence and / or amount of the labeled primary antibody indicating the presence and / or amount of human IgG3 antibody specific for the viral antigen in the sample. Replace with.

[0041] Step 6 of the method of the invention comprises contacting the bound primary IgG3-specific antibody or the bound primary IgG-specific antibody of step 4 with a labeled secondary antibody that is specific for the primary IgG3-specific antibody or the primary IgG-specific antibody to allow binding of the labeled secondary antibody to the primary antibody.

[0042] If the primary IgG-specific antibody or the primary IgG3-specific antibody is not labeled, a secondary antibody that is specific for the primary IgG-specific antibody or the primary IgG3-specific antibody is labeled.

[0043] The term "label" as used herein refers to any compound or moiety, including one or more suitable chemicals or enzymes, that directly or indirectly generate a detectable compound or signal in a chemical, physical or enzymatic reaction. Labeling can be accomplished by methods well known in the art (see, for example, Lottspeich, F., and Zorbas H., Springer Spektrum 2012, Bioanalytik).

[0044] As used herein, the term "labeled antibody" refers to an Ab linked to a detection label. The linkage can be a covalent linkage, for example, resulting from the formation of an amide bond between the antibody and the detection label. The type of linkage depends on the functional groups available on the Ab and on the label. In a preferred embodiment, the antibody is linked to the label through the heavy chain constant region of the antibody.

[0045] The label may be selected from chemiluminescent, fluorescent or enzymatic labels. The enzyme may be horseradish peroxidase, alkaline oxidase, glucose oxidase, or alkaline phosphatase. The substrate for alkaline phosphatase may be adamantyl 1 or 2-dioxetane aryl phosphate (AMPPD), and the substrate for horseradish peroxidase may be luminol, or a derivative thereof, as the substrate.

[0046] In a preferred embodiment, the label may be a fluorescent label selected from the group consisting of xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin, cyanine, coumarin and any derivatives thereof.

[0047] In a further preferred embodiment, the label may be a chemiluminescent label selected from acridinium and ruthenium esters.

[0048] Step 7 of the method of the present invention comprises detecting a signal from the labeled secondary antibody bound to the primary antibody in step 5, the signal indicating the presence and / or amount of the labeled secondary antibody, which in turn indicates the presence and / or amount of human IgG3 antibody specific for the Flavivirus antigen in the sample.

[0049] The signal from the labeled secondary antibody is detected using a detection system. Any system suitable for determining a value indicative of the presence and / or amount of labeled secondary antibody bound to the bound primary IgG-specific or primary IgG3-specific antibody may be used. The detection system includes one or more light sources. Detection systems are commercially available and are well known to those skilled in the art.

[0050] In a further aspect, the present invention provides the use of in vitro pretreatment of a sample containing human IgG3 and one or more of human IgG1, human IgG2, human IgG4, human IgA and human IgM antibodies with immobilized or immobilizable Protein A to improve the sensitivity of a method for determining the presence and / or amount of human IgG3 antibodies specific for a flavivirus antigen in the sample. The pretreatment step may be performed as described above for steps 1 to 3. Protein A may be immobilized on the solid support by covalent or non-covalent attachment. Preferably, Protein A may be bound to beads as outlined above. The sample may be a blood sample from a human subject. Preferably, the sample may contain human serum. The sample may further contain water, buffer salts, and / or protein stabilizing agents.

[0051] In a further aspect, the invention provides the use of immobilized or immobilizable Protein A for the enrichment of human IgG3 in a biological sample containing human IgG3 and one or more of human IgG1, human IgG2, human IgG4, human IgA and human IgM antibodies.

[0052] In a further aspect, the present invention provides the use of the method according to the present invention for determining the human IgG3 response to a viral vaccine. The vaccine used for vaccination of an individual may preferably be a dengue or Zika vaccine, such as a quadrivalent dengue vaccine, such as TAK-003 (Takeda). In a preferred embodiment, the human IgG3 response to at least one, preferably each, of the different dengue serotypes is determined. The dengue serotypes include DENV1, DENV2, DENV3, and DENV4.

[0053] In a further aspect, the present invention provides the use of the method according to the invention for the in vitro diagnosis of a flavivirus infection, preferably a dengue virus or a Zika virus infection, within the past 6 months in a human subject. In a preferred embodiment, the human IgG3 response to at least one, preferably each, of the different dengue serotypes is determined. The dengue serotypes include DENV1, DENV2, DENV3 and DENV4.

[0054] Dengue and Zika virus infections mostly cause asymptomatic symptoms, so it can be difficult to accurately assess disease events without laboratory confirmation. Direct confirmation of infection using tools such as PCR and detection of NS1 antigen has a limited time window (within the first few days after symptoms begin) for maximum sensitivity. In contrast, anti-dengue NS1-specific IgG3 is detected very early after symptoms begin and has a wider detection window (4-6 months).

[0055] In a separate preferred embodiment, the human subject is infected with at least two different flaviviruses. Preferably, the human subject is infected with Dengue virus and Zika virus. The infection can be acute or convalescent. For example, the subject is first infected with DENV and then several months later with ZIKV. The in vitro diagnostic method of the present application is capable of diagnosing at least two different flavivirus infections, for example, DENV infection and ZIKV infection. As a result, the in vitro diagnostic method of the present application is capable of determining whether a subject is infected with one or more flaviviruses and which flaviviruses the subject is infected with. This can be very useful in practice, since multiple infections with different flaviviruses are common due to co-circulation of flaviviruses in some regions.

[0056] Alternatively, the human subject is infected with at least two different dengue virus serotypes. For example, the subject may be infected with DENV serotype 1 and DENV serotype 2. The infection may be acute or convalescent. For example, the subject is first infected with DENV serotype 1 and then several months later with DENV serotype 2. The in vitro diagnostic method of the present application is capable of diagnosing at least two different dengue virus serotype infections, for example, DENV serotype 1 and DENV serotype 2 infections. As a result, the in vitro diagnostic method of the present application is capable of determining whether the subject is infected with one or more DENV serotypes and which DENV serotype the subject is infected with.

[0057] In a further aspect, the present invention provides a kit for determining the presence and / or amount of human IgG3 antibodies in a sample.

[0058] In one embodiment, a kit for determining the presence and / or amount of human IgG3 antibodies in a sample comprises: (a) Protein A bound to magnetic beads; (b) a primary IgG-specific antibody or a primary IgG3-specific antibody; and (c) A labeled secondary antibody specific for the primary IgG-specific antibody or the primary IgG3-specific antibody. Includes.

[0059] In a further embodiment, the kit for determining the presence and / or amount of human IgG3 antibodies in a sample comprises: (a) Protein A bound to magnetic beads; (b) Labeled primary IgG-specific antibody or labeled primary IgG3-specific antibody Includes.

[0060] Specific embodiments regarding beads, IgG specific antibodies and labels are as defined above. The kit may include further components. It may also include instructions for the user.

[0061] Item of the present invention 1. A method for determining the presence and / or amount of human IgG3 antibodies specific for a viral antigen in a sample, comprising: Step 1: contacting a volume of the sample with Protein A bound to beads to allow binding of human IgG1, human IgG2, human IgG4, human IgA, human IgE and human IgM antibodies to the Protein A bound to the beads; Step 2: separating the human IgG1, the human IgG2, the human IgG4, the human IgA, the human IgE and the human IgM antibodies that are bound to the bead-bound Protein A from the remainder of the sample, thereby producing a human IgG3 antibody-enriched supernatant; Step 3: Removing the human IgG3 antibody enriched supernatant from the human IgG1, the human IgG2, the human IgG4, the human IgA, the human IgE and the human IgM antibodies that are bound to the Protein A bound to the beads; Step 4: contacting a viral antigen with the human IgG3 antibody enriched supernatant to allow binding of the human IgG3 antibody to the viral antigen; Step 5: contacting the human IgG3 antibody bound to the viral antigen with a first IgG-specific antibody or a first IgG3-specific antibody to allow binding of the first IgG-specific antibody or the first IgG3-specific antibody to the human IgG3 antibody bound to the viral antigen; Step 6: contacting the bound primary IgG3-specific antibody or the bound primary IgG-specific antibody of step 4 with a labeled secondary antibody specific for the primary IgG3-specific antibody or the primary IgG-specific antibody to allow binding of the labeled secondary antibody to the primary antibody; and Step 7: detecting a signal from the labeled secondary antibody bound to the primary antibody in step 5, wherein the signal indicates the presence and / or amount of the labeled secondary antibody, and the presence and / or amount of the labeled secondary antibody indicates the presence and / or amount of human IgG3 antibody specific for a viral antigen in the sample. The method comprising the steps of:

[0062] 2. The method according to item 1, wherein the IgG3 antibody is specific for a flavivirus antigen, more preferably a dengue virus antigen.

[0063] 3. The method of item 1 or 2, wherein the antigen is selected from the group consisting of virus-like particle (VLP), nonstructural protein 1, envelope protein, precursor membrane protein, membrane protein, capsid protein, nonstructural protein 2A, nonstructural protein 2B, nonstructural protein 3, nonstructural protein 4A, nonstructural protein 4B and nonstructural protein 5, and any derivatives thereof, preferably wherein the antigen is a VLP.

[0064] 4. The method according to any one of items 1 to 3, wherein step 1 is carried out in a microplate format, preferably a 96-well microplate.

[0065] 5. The method according to any one of items 1 to 4, wherein the Protein A is bound to magnetic beads and the separating step 2 is carried out by applying a magnetic force.

[0066] 6. The method according to any one of items 1 to 5, wherein said Protein A is bound to beads and said separating step 2 is carried out by gravity, preferably by applying a centrifugation step.

[0067] 7. The method according to any one of items 1 to 6, wherein the human IgG3 antibody is concentrated in the IgG3 supernatant at least 2-fold, preferably at least 5-fold, more preferably at least 10-fold compared to its content in the starting sample.

[0068] 8. The method according to any one of items 1 to 7, wherein the label of the secondary antibody in step 6 comprises a chemiluminescent, fluorescent or enzymatic label.

[0069] 9. The method according to claim 8, wherein the enzyme is horseradish peroxidase, alkaline oxidase, or glucose oxidase.

[0070] 10. The method according to item 8, wherein the label is a fluorescent label selected from the group consisting of xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin, cyanine, coumarin and any derivatives thereof.

[0071] 11. The method according to any one of items 1 to 10, wherein steps 4 to 6 are carried out in the format of a sandwich assay.

[0072] 12. The method according to any one of items 1 to 11, wherein steps 4 to 7 are carried out in a microplate format, preferably in a 96-well plate format.

[0073] 13. Use of the method according to any one of items 1 to 12 for determining the response of said human IgG3 to a viral vaccine.

[0074] 14. Use of the method according to any one of items 1 to 12 for the in vitro diagnosis of a viral infection, preferably a dengue virus infection, within the past 6 months.

[0075] 15. A kit for determining the presence and / or amount of human IgG3 antibodies in a sample, comprising: (a) Protein A bound to beads; (b) a primary IgG-specific antibody or a primary IgG3-specific antibody; and (c) a labeled secondary antibody specific for the primary IgG-specific antibody or the primary IgG3-specific antibody; The kit comprising:

[0076] 16. The kit according to item 15, wherein the Protein A is bound to magnetic beads.

[0077] 17. The kit according to item 15 or 16, wherein in step 5, the label of the secondary antibody comprises a chemiluminescent, fluorescent or enzymatic label.

[0078] 18. The kit according to item 17, wherein the enzyme is horseradish peroxidase, alkaline oxidase, or glucose oxidase.

[0079] 19. The kit according to item 17, wherein the label is a fluorescent label selected from the group consisting of xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin, cyanine, coumarin and any derivative thereof. EXAMPLES

[0080] Materials and Methods An orbital plate shaker Heidolph set was used. Protein A beads - AmMag™ Protein A magnetic beads were obtained commercially (Genscript; Catalog No. L00695). Magnetic plates were obtained from Life Technology (Cat. No. 032513). Benchtop centrifuge was obtained from Thermo Scientific (Tag #305395; S / N 42204931). Microplates were obtained from Costar (Ref. No. 39155). Dynamag magnetic stands for 2mL and 50mL tubes were obtained from Invitrogen (Cat. Nos. 12321D and 12302D, respectively).

[0081] sample The samples were commercially obtained presumptive samples: Dengue samples: PLA-102, PLA-116, PLA-108, PLA-117; and Zika samples: PARS_64, PARS_55, PARS_71, PARS_70, PARS_97. Samples were purchased from ABO Pharmaceuticals.

[0082] Preparation of magnetic beads Before use, the magnetic beads were resuspended and the resulting slurry was transferred to a 50 mL conical tube. The magnetic beads were washed twice using binding / washing buffer DPBS (1x) (Gibco). The magnetic beads were then washed twice with 0.1 N NaOH. The above washing with binding / washing buffer was then repeated twice.

[0083] Removal of non-human IgG3 human antibodies from samples The bead suspension, pretreated as described above, was resuspended. 120 μl of suspension was placed in each of the wells of a 96-well plate according to the plate layout provided. The plate was placed on a magnetic plate for at least 30 seconds and then the supernatant was removed by flicking. 70 μl of sample was added to the wells of the microplate according to the plate layout provided and mixed with a pipette. The plate was incubated at 37°C overnight (16-20 hours) with mixing on a plate shaker (600 rpm). The plate was spun down at 1000 rpm for 1 minute at 4°C. The suspension was transferred to a 0.5 mL tube and the beads were separated from the sample using a magnetic stand. The supernatant was carefully removed from the suspension in the plate. The supernatant was then further analyzed for the presence of IgG3 as described below.

[0084] Detection of anti-dengue NS1-specific IgG3 antibodies and anti-Zika NS1-specific IgG3 antibodies by ELISA Half-area 96-well plates (Coming, USA) were coated overnight at 4°C with either ZIKV NS1 (for ZIKV NS1 IgG3 assay) at 2.27ug / mL in equimolar ratios for all DENV serotypes (for DENV NS1 IgG3 assay; Native Antigen, UK). Plates were then blocked for 15 minutes at room temperature (RT). Serum samples and assay controls were diluted 1:50 in blocking / dilution buffer, added to wells in duplicate, and incubated for 1 hour at room temperature. Serum from recent ZIKV infection, collected 20-30 days after symptom onset, was used as a ZIKV positive control. Pooled serum from recent dengue infection, collected 20-30 days after symptom onset, was used as a DENV positive control. Pooled serum from healthy individuals naïve to both ZIKV and DENV was used as a negative control for both ZIKV and DENV assays. After washing five times, horseradish peroxidase (HRP)-conjugated mouse monoclonal antibody anti-human IgG3 (Invitrogen, USA) was added to the wells and incubated at room temperature for 1 h. After another washing cycle, TMB (KPL, USA) was added to the wells and incubated at room temperature for 30 min. The reaction was stopped with 1N HCl (Sigma, USA) and the optical density was determined at a wavelength of 450 nm (OD450) using a SpectraMax Plus PC380 microplate spectrophotometer and SoftMax Pro software version 6.4 (Molecular Devices, USA). The OD450 of the sample and control wells was subtracted from the average OD450 of the conjugate blank wells before analysis.

[0085] For DENV IgG3 analysis, the DENV ratio was determined by dividing the OD450 of the sample by the mean OD450nm of the negative controls. For ZIKV IgG3 analysis, the ZIKV ratio was determined by dividing the OD450 of the sample by the mean OD450 of the recent DENV infection.

[0086] The cutoff values ​​for DENV IgG3 and ZIKV IgG3 ratios were 1.5 and 0.586, respectively. Samples were defined as eligible if their mean ratios were above the cutoff values ​​(antigen-specific IgG3 positive samples) and the coefficient of variation of the replicates [(standard deviation of replicates / mean of replicates) × 100] was less than 20%.

[0087] Detection of anti-Dengue VLP-specific IgG3 antibodies and anti-Zika VLP-specific IgG3 antibodies by ELISA The same procedure outlined above was followed, changing the antigens to Dengue and Zika VLPs, respectively, in place of the NS1 antigen.

[0088] result Figure 1 shows the results obtained using the method according to the invention (labeled Protein A) compared to a method that did not include a Protein A removal step prior to the actual determination of IgG3 (labeled Pre-Removal). It can be seen that for each of the four different DENV VLPs as antigens, the method according to the invention provided significantly improved sensitivity compared to the method without a prior Protein A removal step.

[0089] Figure 2 shows the results obtained using the method according to the invention (denoted Protein A) compared to a method that did not include a Protein A removal step prior to the actual determination of IgG3 (denoted Pre-removal). It can be seen that for each of the four different DENV NS1 proteins as antigens, the method according to the invention provided significantly improved sensitivity compared to the method without a prior Protein A removal step.

[0090] Figure 3 shows the results obtained using the method according to the invention (labeled Protein A) compared to a method that did not include a Protein A removal step before the actual determination of IgG3 (labeled Pre-removal). Figure 3 differs from Figure 1 in that the same four different DENV VLPs were used as antigens, but different patient samples were tested. Again, the method according to the invention provided significantly improved sensitivity.

[0091] Figure 4 shows the results obtained using the method according to the invention (labeled Protein A) compared to a method that did not include a Protein A removal step prior to the actual determination of IgG3 (labeled Pre-removal). Figure 4 differs from Figure 1 in that the same four different DENV NS1 proteins were used as antigens, but different patient samples were tested. Similarly, the method according to the invention resulted in significantly improved sensitivity.

[0092] Figure 5 shows the application of the method of the invention to an IgG3 antibody specific for Zika virus VLPs. It may be demonstrated that the method of the invention also provides improved sensitivity for such antibodies.

[0093] Figure 6 shows that the method of the invention can be successfully applied to IgG3 antibodies specific for the Zika NS1 protein. The method of the invention provided improved sensitivity compared to methods lacking prior removal of non-IgG3 antibodies from the sample.

Claims

1. 1. A method for determining the presence and / or amount of human IgG3 antibodies specific for a flavivirus antigen in a sample, comprising: Step 1: contacting a quantity of the sample with protein A bound to beads to allow binding of human IgG1, human IgG2, human IgG4, human IgA, human IgE, and human IgM antibodies to the protein A bound to the beads; Step 2: separating the human IgG1, the human IgG2, the human IgG4, the human IgA, the human IgE, and the human IgM antibodies that are bound to the bead-bound Protein A from the remainder of the sample, thereby producing a human IgG3 antibody-enriched supernatant; Step 3: removing the human IgG3 antibody-enriched supernatant from the human IgG1, the human IgG2, the human IgG4, the human IgA, the human IgE, and the human IgM antibodies that are bound to the bead-bound Protein A; Step 4: contacting a viral antigen with the human IgG3 antibody-enriched supernatant to allow binding of the human IgG3 antibody to the flavivirus antigen; Step 5: contacting the human IgG3 antibody bound to the viral antigen with a primary IgG-specific antibody or a primary IgG3-specific antibody to allow binding of the primary IgG-specific antibody or the primary IgG3-specific antibody to the human IgG3 antibody bound to the flavivirus antigen; Step 6: contacting the bound primary IgG3-specific antibody or the bound primary IgG-specific antibody of step 4 with a labeled secondary antibody specific for the primary IgG3-specific antibody or the primary IgG-specific antibody to allow binding of the labeled secondary antibody to the primary antibody; and Step 7: Detecting a signal from the labeled secondary antibody bound to the primary antibody in step 5, wherein the signal indicates the presence and / or amount of the labeled secondary antibody, and the presence and / or amount of the labeled secondary antibody indicates the presence and / or amount of human IgG3 antibody specific for the flavivirus antigen in the sample. or Steps 5 to 7 Step 5': contacting the human IgG3 antibody bound to the viral antigen with a labeled primary IgG-specific antibody or a labeled primary IgG3-specific antibody to allow binding of the labeled primary IgG-specific antibody or the labeled primary IgG3-specific antibody to the human IgG3 antibody bound to the flavivirus antigen; and Step 6': detecting a signal from the labeled primary IgG-specific antibody or the labeled primary IgG3-specific antibody bound to the flavivirus antigen in step 5', wherein the signal indicates the presence and / or amount of the labeled primary IgG-specific antibody or the labeled primary IgG3-specific antibody, and the presence and / or amount of the labeled primary antibody indicates the presence and / or amount of human IgG3 antibody specific for the flavivirus antigen in the sample. was replaced with The method.

2. 10. The method of claim 1, wherein the IgG3 antibody is specific for a Dengue virus antigen or a Zika virus antigen.

3. The antigen is Virus-like particles (VLPs), nonstructural protein 1, envelope protein, precursor membrane protein, membrane protein, capsid protein, nonstructural protein 2A, nonstructural protein 2B, nonstructural protein 3, nonstructural protein 4A, nonstructural protein 4B, and nonstructural protein 5, and any derivatives thereof. The method according to any one of claims 1 to 2, wherein the compound is selected from the group consisting of:

4. The method according to any one of claims 1 to 2, wherein step 1 is carried out in a plate or tube format.

5. the Protein A is bound to magnetic beads; the separating step 2 is performed by applying a magnetic force; The method according to any one of claims 1 to 2.

6. the Protein A is bound to beads; The separating step 2 is performed by gravity. The method according to any one of claims 1 to 2.

7. The method according to any one of claims 1 to 2, wherein the human IgG3 antibodies are enriched in the IgG3 supernatant by at least two-fold compared to their content in the starting sample.

8. 2. The method of claim 1, wherein the label of the secondary antibody in step 6 comprises a chemiluminescent, fluorescent, or enzyme label.

9. The method of claim 1 , wherein the label of the primary antibody in step 5′ comprises a chemiluminescent, fluorescent, or enzyme label.

10. 10. The method of claim 8 or 9, wherein the enzyme is horseradish peroxidase, alkaline oxidase, or glucose oxidase.

11. The label is Xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin, cyanine, coumarin and any derivatives thereof 11. The method of claim 10, wherein the fluorescent label is selected from the group consisting of:

12. 10. The method of any one of claims 1, 2, 8 or 9, wherein steps 4 to 6 are carried out in the format of a sandwich assay.

13. 10. The method of any one of claims 1, 2, 8 or 9, wherein steps 4-7 are performed in a microplate format.

14. 10. The method of claim 1, wherein steps 4, 5' and 6' are performed in a microplate format.

15. 1. A method for improving the sensitivity of a method for determining the presence and / or amount of human IgG3 antibodies specific for a flavivirus antigen in a sample, comprising: performing a step of pre-treatment of the sample containing the human IgG3 and one or more of human IgG1, human IgG2, human IgG4, human IgA and human IgM antibodies specific for a flavivirus antigen with immobilized or immobilizable protein A. The method comprising:

16. 16. The method of claim 15, wherein the Protein A is bound to beads.

17. 17. The method of claim 15 or 16, wherein the sample is a blood sample from a human subject.

18. 15. A method for determining the human IgG3 response to a flavivirus vaccine, comprising performing the method of any one of claims 1, 2, 8, 9 or 14.

19. 19. The method of claim 18, wherein the human IgG3 response to at least one dengue serotype selected from DENV1, DENV2, DENV3 and DENV4 is determined.

20. 16. A method for the in vitro diagnosis of a flavivirus infection within the past six months in a human subject, comprising carrying out the method of any one of claims 1, 2, 8, 9 or 14.

21. 21. The method of claim 20, wherein the flavivirus infection is an infection with at least one dengue serotype selected from DENV1, DENV2, DENV3 and DENV4, or a Zika virus infection.

22. 21. The method of claim 20, wherein the human subject is infected with at least two different flaviviruses.

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