Anti-dengue vaccine and antibodies

Abstract

To provide an antibody that enables neutralization of more than one serotype of Dengue virus.SOLUTION: The present invention provides an antibody that binds to a dengue virus envelope dimer epitope (EDE), the antibody comprising a heavy chain and a light chain each including a specific sequence having no, one, or two amino acid substitutions, insertions, or deletions, wherein the heavy chain comprises a CDR including a specific sequence and the light chain comprises a CDR including a specific sequence, and a pharmaceutical composition comprising the antibody.SELECTED DRAWING: Figure 32

Description

[Technical Field]

[0001] This invention relates to the field of treatment and prevention of dengue fever virus infection, as well as related compounds and methods. [Overview of the project]

[0002] Dengue fever infects nearly 400 million people every year. 1 Symptoms range from mild illness (dengue fever) to severe cases such as dengue hemorrhagic fever in 25% of infections. The virulence factors are four serologically related viruses from the Flavivirus genus. 2 These are referred to as dengue virus serotypes 1-4 (DENV1-4). Infection with one serotype provides lifelong protection against that serotype, but not against other serotypes. There is epidemiological evidence that severe illness is more likely to occur during secondary infection than during the first or primary DENV infection. 3、4 The increased risk of disease during secondary infections and the need for protection against four different serotypes set high standards for vaccines urgently needed to prevent the estimated 400 million infections that occur annually. 1、5、6 .

[0003] Most DENV vaccines in development, which aim to produce virus-neutralizing antibodies and DENV envelopes, have focused their efforts on this. 8、9, this envelope protein is involved in receptor binding and subsequent receptor-mediated endocytosis. In the acidic environment of endosomes, the envelope protein catalyzes a membrane fusion reaction between the viral envelope and the endosomal membrane, thereby releasing the viral genomic RNA into the cytoplasm. The envelope protein is about 500 amino acids in length, with a large N-terminal ectodomain and two transmembrane (TM) helices at the C-terminus. Its overall structure is conserved among all flaviviruses, with about 65% amino acid sequence identity between the most distant viruses within the dengue fever group, all of which exhibit two conserved N-linked glycosylation sites at positions N67 and N153. The 400 amino-terminal residues of the ectodomain of the envelope protein (referred to as "sE" for "soluble E") fold into a three-domain structure rich in β-sheets, which is characteristic of class II viral fusion proteins, and the mature virion 8、9 head-to-tail homodimer that coats the surface of 5~7 is formed.

[0004] The crystal structures of the envelope glycoprotein ectodomains from dengue virus serotypes 2, 3, and 4 are available in the PDB (Protein Data Bank) database under accession numbers 1OAN, 1OK8 for dengue virus serotype 2, 1UZG for dengue virus serotype 3, and 3UAJ for dengue virus serotype 4.

[0005] However, antibody recognition of DENV particles is complicated by many significantly different compositions and conformations of the viral capsid presented at different stages of the viral life cycle 11、12 . "Immature" viral particles have a full complement of precursor membrane protein (prM) in a 1:1 association with the envelope and virion, adopting a characteristic spiky appearance, and each spike is composed of a trimer of prM / E proteins 11~15 .

[0006] Following furin-mediated prM cleavage, “mature” virus particles adopt a smooth appearance with nearly 90 packed dimers of envelope proteins arranged around 2, 3, and 5 times the symmetrical axes. The expansion of these mature particles into an “uneven” morphology upon exposure to temperatures above 34°C, where the envelope protein dimers rearrange relative to each other, has also been described in recent years. 16、17 After early internalization into endosomes, the acidic environment triggers a major conformational change in the envelope protein, which exposes the fusion loop and then irreversibly trimers to induce membrane fusion. 18、19 .

[0007] An important and further level of complexity is that prM cleavage is often incomplete, which leads to varying degrees of cleavage and the resulting viral population. 15、20 The study showed that viruses containing high levels of prM are not infectious, while viruses with low levels of prM remain infectious, and furthermore, non-infectious particles with high levels of prM can be subjected to antibody-dependent enhancement leading to infection. 21、22 .

[0008] It is not yet clear whether epitopes are the most effective targets for human neutralizing antibodies, for example, de Alwis 27 This suggests that epitopes require viral assembly for formation, while Rey 81 This suggests that the envelope dimer itself is the target.

[0009] Antibody-dependent enhancement (ADE) of DENV infection is one of the mechanisms that presupposes an increase in disease severity during secondary infections. 23 Antibodies formed during primary infection optimize the virus but do not completely neutralize it, and it has been suggested that they promote Fc receptor-mediated uptake into myeloid cells, which leads to a higher viral load in secondary infection. ADE can be observed at almost all secondary neutralization concentrations of antibodies, and its perceived risk poses a challenge to vaccine strategies in DENV.

[0010] The leading dengue vaccine candidates currently being tested in clinical trials consist of a tetravalent formulation of either live attenuated dengue virus or dengue / yellow disease chimeric virus. 24、25 It has been confirmed that creating a balanced quadrivalent immune response free from unacceptable reactiongenicity is difficult. The most advanced vaccine candidate is currently in Phase II clinical trials. 4 While the vaccine showed much lower efficacy than expected in the initial study and did not protect against DENV-2, in a Phase III trial, the vaccine reduced the incidence of the disease by 56% (http: / / sanofipasteur.com / en / articles / theworld-s-first-large-scale-dengue-vaccine-efficacy-study-successfully-achieved-its-primary-clinical-endpoint.aspx), nearly half of the disease-exposed population in Capeding et al (2014) Lancet Published online July 11, 2014 http: / / dx.doi.org / 10.1016 / S0140-6736(14)61060-6). This discrepancy creates an urgent need to understand the human antibody response in natural dengue infection and post-vaccination, specifically identifying the epitopes recognized by the most potent cross-reactive antibodies in humans and understanding their correlation with protection from the disease. Therefore, it is extremely important to provide a dengue vaccine that contains an epitope recognized by the most potent cross-reactive antibodies generated in humans.

[0011] Recent evidence shows that the serological antibody response specific to dengue virus (DENV) in humans consists of a large proportion of cross-reactive, poorly neutralizing antibodies and only a small number of serotype-specific, potent inhibitory antibodies. 27 This means that intact virus particles are required to bind to quaternary structural epitopes of complexes, which are only expressed when envelope proteins are assembled on virus particles, in order to stimulate an effective immune response.

[0012] In contrast to this recent evidence, the inventors of the present invention have surprisingly identified and characterized human antibodies obtained by isolating rearranged heavy and light chain genes from classified single plasmablasts of patients infected with dengue virus, which were found to be potently neutralizing and cross-reactive antibodies. Furthermore, they found that the epitopes to which these antibodies bind are limited to envelope protein dimers and do not require the assembly of the complete virus. Therefore, subunit vaccines containing stabilized soluble protein E dimers are good candidates in a successful dengue vaccine that avoids antibodies that would be induced against poorly immunogenic regions on the surface of infectious virions that are normally inaccessible.

[0013] The present invention, as described below, provides compounds, compositions, methods, uses, and vaccines relating to newly identified antibodies and antigens.

[0014] The inventors characterized 145 human monoclonal antibodies from patients suffering from dengue fever infection. Acute human antibody reactions were found to focus on two major epitopes, one of which is well-described in the fusion loop, and a second, novel epitope found in intact virions or envelope protein dimers, which are contained within domains I, II, and III. Antibodies reacting with this epitope, the envelope dimer epitope, or EDE were found to be able to completely neutralize viruses produced in both intact human cells and primary human cells in the low picomolecular range. This novel epitope has various implications for the treatment and prevention of diseases caused by the dengue virus.

[0015] The present invention will be described below with respect to various embodiments of different aspects of the invention. For clarity, it will be understood that certain characteristics of the invention described in the context of individual embodiments may also be provided in combination with one or more embodiments or in a single embodiment. Conversely, for brevity, various characteristics of the invention described in the context of a single embodiment may also be provided individually or in any suitable subcombination. All combinations of these embodiments are specifically encompassed by the invention and disclosed herein as if each and all combinations were individually and expressly disclosed. In addition, all subcombinations are also specifically encompassed by the invention and disclosed herein as if each and all such subcombinations were individually and expressly disclosed herein.

[0016] Therefore, in a first aspect of the present invention, a compound is provided that neutralizes dengue virus of one or more serotypes.

[0017] Preferably, the compound neutralizes dengue viruses of two serotypes, more preferably three, and most preferably four serotypes; that is, all serotypes of dengue viruses neutralize two or more serotypes from a list including, for example, DENV-1, DENV-2, DENV-3, and DENV-4.

[0018] A compound means any compound capable of neutralizing one or more serotypes of dengue virus. The compound may be, for example, a small molecule, a polypeptide, or a protein (these terms are used synonymously herein), and may include glycoproteins, nucleic acids, carbohydrates, fats, atoms, such as metals. In preferred embodiments, the compound is a polypeptide, preferably an antibody or its antigen-binding moiety. This antigen-binding moiety may be an Fv fragment, a Fab-like fragment (e.g., a Fab fragment, a Fab' fragment, an F(ab)2 fragment, an Fv fragment, or an scFv fragment), or a domain antibody. This antigen-binding moiety may derive from a linear amino acid sequence present in an intact antibody, or may contain a series of discontinuous amino acids, or may contain a series of discontinuous amino acids optionally scattered with other amino acids, for example, certain amino acids required for contact with an epitope, but may not contain amino acids required for the framework of a native antibody, which may, for example, be replaced in some cases by heterologous skeletal proteins. Antibodies according to the present invention are obtained by a method that includes a step of immunizing mammals such as humans, monkeys, rabbits, or mice, as will be known to those skilled in the art, and / or by an in vitro method that includes, for example, a phage display selection step.

[0019] The term "antibody" includes substantially intact antibody molecules, as well as chimeric antibodies, humanized antibodies (antibodies in which at least one amino acid is mutated relative to a non-human antibody, e.g., naturally occurring non-human antibodies or antibodies assembled from non-human antibody sequences), single-chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers or heterodimers of antibody heavy and / or light chains, and their antigen-binding moieties and derivatives.

[0020] If the compound is a protein, for example, if an antibody or a fragment thereof is administered to a human subject and this antibody is not a human antibody or fragment thereof, it may be humanized to reduce its immunogenicity in humans. Methods for producing humanized antibodies or fragments thereof are well known in the art (Vinckle et al., 2009).

[0021] Furthermore, the bioavailability of the antibody or fragment thereof according to the present invention can be improved by conjugating the neutralizing antibody or fragment thereof to an inert carrier such as albumin (Coppieters et al., 2006) or immunoglobulin (Harmsen et al., 2005).

[0022] The term antibody also includes all classes of antibodies, including IgG, IgA, IgM, IdD, and IgE. The term antibody also includes any defined antibody and variants, fusions, and derivatives of its antigen-binding moiety.

[0023] This compound can be alternatively found in, for example, MILLWARD STEVEN W ET AL: “Design of cyclic peptides that bind protein surfaces with antibody-like affinity”, ACS CHEMICAL BIOLOGY, vol.2, no.9, 1 January 2007 (2007-01-01), pages 625-634, XP002616292, AMERICAN CHEMICAL SOCIETY, WASHINGTON, DC, US ISSN:1554-8929, DOI:10.1021 / CB7001126, HEINIS CHRISTIAN ET AL: “Phage-encoded combinatorial chemical libraries based on bicyclic peptides” NATURE CHEMICAL BIOLOGY, vol.5, no.7, July 2009 (2009-07), pages As described in 502-507, XP007913181, the peptides may be cyclic peptides, such as polycyclic peptides, or bicyclic peptides. See also, for example, WO2009098450. Bicyclic peptides with the required binding properties may be selected, for example, by phage presentation methods.

[0024] Neutralization means reducing the virus's ability to infect cells that have not been infected before.

[0025] Those skilled in the art will be well aware of appropriate techniques for observing the ability of a compound to neutralize a virus. One such method is detailed in Example 3, in which one or more serotypes of dengue virus can infect a population of potential host cells, the compound is mixed with the virus under assay conditions, and the mixture is then incubated in the potential host cells. The number of infected cells is assayed to obtain a criterion for the neutralizing ability of the compound, i.e., the compound's ability to prevent infection. In certain cases, the potential of a compound, such as an antibody or its antigen-binding moiety, can be determined using a focus reduction neutralization test (FRNT) when the reduction in the number of infected foci is compared to a control (no compound). 22 In short, the compound is mixed with the virus and incubated at 37°C for 1 hour. The mixture is then transferred to Vero cells (a kidney epithelial cell line from African green monkeys) and incubated for 3 days. The focus formation assay can be performed using an anti-E mAb (4G2), followed by HRP-coupled rabbit anti-mouse IgG. This reaction can be visualized by adding a DAB substrate. The percentage of focus reduction is calculated for each compound. The 50% FRNT value can be determined from a graph of reduction rate versus compound concentration using a probit program from the SPSS package. Typically, this assay can be performed in the absence of the test compound, for example, with confluent cells, e.g., about 100 foci in a 96-well plate well containing just confluent cells.

[0026] For example, other such examples, such as the Focus Reduction Neutralization Test (FRNT), Plaque Reduction Neutralization Test (PRNT), see WHO document http: / / whqlibdoc.who.int / hq / 2007 / who_ivb_07.07_eng.pdf, and in vivo techniques in mice and monkeys using flow cytometry, will be well known to those skilled in the art. For example, see Figure 30 for an example of FRNT and flow cytometry methods.

[0027] In one embodiment, the compound neutralizes the virus by at least 80%, preferably 90%, more preferably 95%, and most preferably 100%. In a further preferred embodiment, the compound neutralizes all serotypes of dengue virus by at least 80%, preferably 90%, more preferably 95%, and most preferably 98%, 99%, or 100%. The virus may be produced by insect cells or in human cancer cell lines (typically considered to produce high pr-M content viruses, as will be discussed further below), or in human primary cells, such as primary human dendritic cells, or in cell lines overexpressing furin (considered to produce low pr-M content viruses).

[0028] Neutralizing to a specific level includes neutralizing a compound to a specific level relative to a given concentration. It will be understood that the appropriate concentration of a given compound may vary depending on the actual compound. For example, the concentrations of a given compound used in the above assay could be 100 mM, 10 mM, 1 mM, 100 μM, 10 μM, 1 μM, 100 nM, 10 nM, or 1 nM; or 0.01 ug / ml, 0.02 ug / ml, 0.04 ug / ml, 0.05 ug / ml, 0.06 ug / ml, 0.075 ug / ml, 0.1 ug / ml, 0.25 ug / ml, 0.5 ug / ml, 0.75 ug / ml, 1 ug / ml, 1.25 ug / ml, 1.5 ug / ml, 1.75 ug / ml, 2 ug / ml, 2. The concentration may be 25 ug / ml, 2.5 ug / ml, 2.75 ug / ml, 3 ug / ml, 3.25 ug / ml, 3.5 ug / ml, 3.75 ug / ml, 4 ug / ml, 4.25 ug / ml, 4.5 ug / ml, 4.75 ug / ml, 5 ug / ml, 5.25 ug / ml, 5.5 ug / ml, 5.75 ug / ml, 6 ug / ml, 6.5 ug / ml, 7 ug / ml, 7.5 ug / ml, 8 ug / ml, 8.5 ug / ml, 9 ug / ml, 9.5 ug / ml, or less than 10 ug / ml, or less than 0.01 ug / ml. Typically, the concentration of a compound, such as an antibody, may be less than 1 ug / ml.

[0029] For example, a compound (e.g., an antibody) can neutralize 80% of one or more serotypes of virus at a compound concentration of 0.1 ug / ml, and can neutralize at least 98%, for example 100%, of one or more serotypes of virus at a compound concentration of 1 ug / ml. Preferably, the compound (e.g., an antibody) neutralizes 80% of one or more serotypes of virus at a concentration of 0.05 μg / ml, or neutralizes at least 98%, for example 100%, of one or more serotypes of virus at a concentration of 0.5 μg / ml.

[0030] It will also be understood that the observed level of neutralization for a given concentration of a compound may vary depending on the number of viral particles in the assay. For example, for a given concentration of the compound, if the number of viral particles in the assay is doubled, the level of neutralization may be expected to decrease (for a given host cell population). The number of viral particles in the assay would typically be such that, in the absence of the test compound, for example, in a 96-well microtiter plate well, it provides approximately 100 foci using, for example, confluent cells.

[0031] For example, in one embodiment, the compound neutralizes one or more serotypes of virus to at least 80% or 100% of a level at a concentration of 1 μg / ml or 0.05 ug / ml or less, when the viral concentration is sufficient to provide approximately 100 foci using, for example, confluent cells, in a 96-well microtiter plate well, in the absence of the test compound.

[0032] The number of cells that can be infected by the virus in an assay can also affect the apparent level of neutralization. For example, a small number of cells may show a greater infection rate per cell than a larger cell population. Therefore, the ratio of compound, virus, and host cell number can also be important. The cells used in the assay can also be important. The assay may be performed in a microtiter plate, for example, a 96-well microtiter plate. The cells may be confluent in the container in the microtiter plate, for example, a 96-well microtiter plate, e.g., just confluent.

[0033] Preferably, the compound can neutralize viruses produced in both insect cells, e.g., C6 / 36 insect cells, or human tumor cell lines (typically capable of producing high pr-M content viruses) and human cells, e.g., primary human cells, e.g., primary human dendritic cells, or cells overexpressing furin (considered to produce low pr-M content viruses). The production of viral particles, subviral particles, or virus-like particles in different cell types will be known to those skilled in the art. For example,

[0034] The ability of the compound to neutralize viruses can be tested in the examples described above. In one embodiment, the compound can neutralize viruses produced in primary human cells, such as primary human dendritic cells, or insect cells. In another embodiment, the compound can neutralize viruses produced in primary human cells and insect cells to the same level. To the same level means that, for a given concentration of the compound and / or a given concentration of the virus and / or a given number of host cells, the level of neutralization reaction attributable to the compound is not significantly different for viruses produced in both insect cells and primary human cells, or the level of neutralization reaction attributable to the compound exceeds a certain threshold for viruses from both insect cells and primary human cells, for example, greater than 80%, 90%, 95%, or 98%. For example, for a given concentration of virus particles and a given number of potential host cells, the 50% FRNT is the same (not significantly different) for viruses produced in insect cells and primary human cells, for example, 0.05 μg / ml or less, or 0.5 μg / ml or less, or 1 μg / ml or less, or 5 μg / ml or less. In preferred embodiments, the compound can neutralize dengue virus produced in primary human cells and insect cells, preferably two serotypes, preferably three serotypes, more preferably four serotypes, or all serotypes, or more than one serotype. In most preferred embodiments, the compound can completely (i.e., 100%) neutralize dengue virus of all serotypes produced in both insect cells and primary human cells. For example, as discussed above, the compound can 100% neutralize viruses produced in both primary human cells and insect cells at virus concentrations sufficient to obtain about 100 foci at a compound concentration of 0.05 ug / ml. To be produced in both primary human cells and insect cells includes viruses produced independently in primary human cells (for example), viruses produced independently in insect cells using the same procedure as a particular population of viral particles produced using both primary human cells and insect cells.

[0035] The cross-reactive, highly neutralizing compounds identified in this invention have been found to bind to specific epitopes that can be found in both intact viruses and dimers of the envelope protein, independently of virus formation. Therefore, the compounds of this invention can be defined in relation to their ability to bind to these specific epitopes.

[0036] Therefore, further embodiments of the present invention provide compounds that bind to the dengue virus envelope dimer epitope (EDE). EDE includes any EDE as defined herein.

[0037] A compound that binds to the envelope dimer epitope (EDE) means any compound that can bind to the EDE of one or more serotypes of dengue virus. This compound may be a small molecule, polypeptide, nucleic acid, carbohydrate, fat, atom, or, for example, a metal. In preferred embodiments, the compound is a polypeptide, preferably an antibody or its antigen-binding moiety. The preference for this compound is as described above.

[0038] There are four serotypes of dengue virus. Therefore, it will be understood that this compound may bind to the EDE of one serotype of dengue virus. In preferred embodiments, this compound may bind to the EDE of more than one serotype of dengue virus, and may be considered to bind to two, three, or four serotypes of dengue virus, i.e., all serotypes of dengue virus.

[0039] "Binding" means any form of non-covalent bond between the compound of the present invention and an epitope or molecule or macromolecule or compound, and means any significant degree of binding to EDE as evaluated by methods commonly used in the art. In preferred embodiments, the compound selectively binds to EDE. Selective binding to EDE means that the compound does not bind to or significantly binds to any dengue virus or envelope protein other than EDE. It also means that the compound does not bind to or significantly binds to any other compound, molecule or macromolecule, and it will be well within the skill of those skilled in the art to present an EDE that determines whether the compound binds to EDE. For example, an ELISA-type assay, as known to those skilled in the art, may be used. A non-limiting example of a method for determining whether the compound binds to EDE is one or more intact viruses of dengue fever virus, preferably all serotypes, and / or envelope dimers of one or more dengue fever viruses, preferably all serotypes, and / or EDE according to any of the definitions described herein, e.g., stabilized envelope dimers, or EDE containing residues from envelope proteins held within a heterologous skeleton. The pseudo-uninfected supernatant is separately captured on a solid support, e.g., a MAXISORP immunoassay plate (NUNC) coated with anti-E Ab(4G2). The capture wells are then incubated with the compound, e.g., an antibody or its antigen-binding moiety, e.g., a human monoclonal antibody, e.g., 1 ug / ml human mAb, followed by incubation with a reporter-conjugated secondary antibody (to bind to the compound), e.g., ALP-conjugated anti-human IgG. This reaction is visualized by adding a suitable substrate, e.g., a PNPP substrate, and stopped with NaOH. For ALP / PNPP, absorbance is measured at 405 nm.

[0040] The term "compound that binds to EDE" includes any compound that binds to a well containing the virus or EDE, such as a stabilized soluble protein E dimer, to any degree exceeding the background level of binding to the well containing the uninfected supernatant. Preferably, the level of binding obtained to the virus or EDE, such as a stabilized soluble protein E dimer, is twice, preferably four times, preferably six times, and more preferably ten times, the background level of binding to the uninfected supernatant well. To determine whether this compound binds to the virus or envelope protein at a site other than EDE, the compound's ability to bind to a denatured, monomeric, or recombinant envelope protein may be evaluated. If this compound binds to a denatured, monomeric, or recombinant envelope protein to a significant level, it is considered to bind to the virus or envelope protein at a site other than EDE. To determine whether this compound selectively binds to EDE other than any other molecule, macromolecule, or compound, the ability of the compound to bind to EDE can be compared with the ability of the compound to bind to a molecule, macromolecule, or compound using the method described above. A compound selectively binds to EDE if it binds to EDE over a significantly broader range than it would to another molecule, macromolecule, or compound, such as a denatured or monomeric envelope protein, for example, if the compound has at least 2, 4, 6, 8, or 10 times greater affinity to EDE than it would to another molecule, macromolecule, or compound, such as a denatured or monomeric or recombinant envelope protein.

[0041] EDEs are epitopes that are thought to form on intact virus particles that spread across the envelope protein dimer, or on free dimers of the envelope protein that spread across two polypeptides, such as free dimers of a soluble envelope protein. The envelope protein sequences are detailed in Figure 29, as well as in SEQ ID NOs. 29, 31, 33, and 35.

[0042] In preferred embodiments, the compounds of the present invention bind to intact viruses or free envelope dimers (i.e., having twice the molecular weight of the envelope polypeptide monomer), or to EDE in any other structural manner as described above and further discussed below, and do not bind to monomeric envelope proteins or denatured envelope proteins. In one embodiment, if the compound binds to monomeric envelope proteins or denatured envelope proteins, it is not considered a useful compound and is not a compound of the present invention. Therefore, one non-limiting method for determining whether a compound is a compound of this embodiment of the present invention is, for example, by assaying the compound, for example, an antibody or its antigen-binding moiety, for its ability to bind to denatured envelope proteins in a Western blot and / or recombinant (monomeric) envelope proteins in an ELISA, for example, and to intact virus particles and / or envelope protein dimers (e.g., dimers of soluble envelope proteins) in an ELISA, for example. Preferred compounds of the present invention are considered to bind to intact viruses or undenatured dimers, but not to denatured or monomeric envelope proteins (or to a significantly lesser extent). The degree of bonding can be evaluated as described above.

[0043] Compounds that bind to the fusion loop but not to EDE are not considered compounds of the present invention. The fusion loop is a restrictive set of residues inside and outside 101W that defines the above-described or classical fusion loop epitope (FL). In the fusion loop, residues 101-WGNG-104 create a distorted α-helical rotation that causes the W101 side chain to protrude toward domain III opposite the dimer interface. If a compound binds to an envelope monomer or denatured envelope protein (determined, for example, as described above), it is possible that this antibody could instead bind to a different portion of the envelope polypeptide (which can be identified by binding to an envelope polypeptide that is mutated in the fusion loop region), but it can still be considered to bind to the fusion loop.

[0044] In another embodiment, the compound that binds to the fusion loop is one that is not affected (or not significantly affected) by mutations in any one or more of the following residues in the envelope protein, particularly DENV-1:E49, Q77, I161, T200, W391, or F392.

[0045] In some embodiments, the compounds of the present invention do not bind to denatured EDE or denatured envelope proteins.

[0046] In one embodiment, the EDE is considered to extend to the dengue virus envelope polypeptide dimer, for example, to the polypeptide of a soluble envelope polypeptide dimer. In a particular embodiment, the EDE comprises the envelope polypeptide dimer of domains I, II, and III. It will be understood that the EDE contains a quaternary structure-dependent epitope at the dimer interface of one or more serotypes of dengue virus envelope proteins.

[0047] It will be understood that envelope proteins from different dengue serotypes can dimerize, forming hybrid dimers. Therefore, in one embodiment, the EDE to which the compound binds is made from envelope monomers derived from different dengue serotypes, and thus the EDE may contain homodimers or heterodimers.

[0048] It will also be understood that EDE can be presented on a compound as part of a virion, subviral particle, or virus-like particle, just as dimers of envelope proteins are found on intact virions or virus-like particles. When EDE is presented as part of a virion, subviral particle, or virus-like particle, the compounds of the present invention bind to the intact virion, subviral particle, or virus-like particle but not to the monomer or denatured envelope protein.

[0049] Alternatively, EDE may be presented in a compound not as part of a virion; for example, EDE formed from a dimer of two envelope proteins may be presented in a compound as a free dimer. Therefore, in one embodiment, the compound of the present invention is a compound that binds to EDE when EDE is a free dimer of an envelope or soluble envelope (sE) protein. In another embodiment, the compound of the present invention is a compound that binds to EDE when EDE is a stabilized dimer of an envelope or sE protein.

[0050] Free dimers may be presented as part of a composition containing components that stabilize protein dimerization. For example, certain buffer components considered to promote protein association may be used. Alternatively, envelope proteins may be presented at high concentrations that promote dimerization (see Example 7).

[0051] In addition to external agents that stabilize the envelope dimer, the envelope protein can be manipulated to have increased stability in the dimeric configuration. For example, the dimer is -The two envelopes or sE monomers can be covalently stabilized by at least one, optionally 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more disulfide interchain bonds, and / or -The two sE monomers can be covalently stabilized by at least one, optionally 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more sulfhydryl reactive crosslinking agents, and / or -Can be covalently stabilized by linking two envelopes or sE monomers via a modified sugar, and / or -The dimer interface or the domain 1(D1) / domain 3(D3) linker of each monomer can be non-covalently stabilized by substituting at least one envelope or at least one amino acid residue in the amino acid sequence of the sE monomer with at least one bulky side-chain amino acid.

[0052] The dengue virus envelope glycoprotein E external domain (sE; soluble envelope polypeptide / glycoprotein) refers to the amino acid fragments 1-395 of the envelope glycoprotein E of dengue virus serotypes 1, 2, and 4, and the amino acid fragments 1-393 of the envelope glycoprotein E of dengue virus serotype 3.

[0053] In a preferred embodiment, the compound binds to EDE, which is a stabilizing dimer of sE, and the recombinant dengue virus envelope glycoprotein E external domain (recombinant sE) monomer is selected from the group consisting of DENV-1 sE of SEQ ID NO: 132, DENV-2 sE of SEQ ID NO: 133, DENV-3 sE of SEQ ID NO: 134, DENV-4 sE of SEQ ID NO: 135, and its mutant sE having at least one mutation (substitution) selected from H27F, H27W, L107C, F108C, H244F, H244W, S255C, A259C, T / S262C, T / A265C, L278F, L292F, L294N, A313C (S313C in DEN3), and T315C. These mutations are considered to contribute to increased stability in the dimer configuration, as described below.

[0054] Optionally, the mutant sE further has at least one mutation (substitution) selected from Q227N, E174N, and D329N, preferably three mutations of Q227N, E174N, and D329N. These mutations cover unsuitable immunogenic regions, and in a subject, the stabilized recombinant sE dimer of the present invention is considered capable of preferentially inducing neutralizing antibodies targeting all four dengue virus serotypes.

[0055] The aforementioned mutagenesis of the sE dimer, which induces mutations, introduces mutations that do not impede its immunogenicity but provide higher dimer affinity, including cysteine ​​mutations at dimer contact to provide stabilization by crosslinking, and / or introduces novel glycosylation that enables chemical crosslinking between adjacent sugars in the dimer by clicking chemistry and / or by substituting at least one amino acid residue in the amino acid sequence of at least one sE monomer with at least one bulky side-chain amino acid to enable cavity formation at the dimer interface or in the domain 1(D1) / domain 3(D3) linker of each monomer.

[0056] Unless otherwise specified, these amino acid residue positions are numbered according to the sE amino acid sequence alignment shown in Figure 15.

[0057] The nucleic acid sequences encoding DENV-1 sE (sequence number 132), DENV-2 sE (sequence number 133), DENV-3 sE (sequence number 134), and DENV-4 sE (sequence number 135) are shown as sequence numbers 136, 137, 138, and 139, respectively.

[0058] As used herein, the term “recombinant” refers to the use of a genetic engineering method (cloning, amplification) for producing the dengue virus envelope glycoprotein E external domain, antibody, or antibody fragment thereof according to the present invention.

[0059] The dimer may be a homodimer of two identical recombinant sE as defined above, or a heterodimer of two different recombinant sE as defined above, and this dimer is preferably a homodimer.

[0060] For example, it could be a heterodimer of DENV-1 sE and DENV-2 sE as defined above. It could also be a heterodimer of DENV-1 sE and a mutant sE of DENV-1 sE as defined above.

[0061] In one embodiment, the compound is bonded to EDE, which is a stabilized dimer of sE, and the envelope or recombinant sE stabilized dimer is covalently stabilized by at least one, two, or three disulfide interchain bonds between two sE monomers.

[0062] Advantageously, the stabilized dimer may consist of a single cysteine ​​mutant sE positioned by a twice-across molecular axis of the dimer, resulting in a single interchain disulfide bond, or multiple (e.g., double) cysteine ​​mutant sEs capable of forming multiple (e.g., two) disulfide bonds away from the twice-across molecular axis. These disulfide bonds can be synthesized under acidic conditions, for example, using a DMSO solution (O. Khakshoor et al., 2009) or an oxidizing agent such as CdCl2 or CuSO4. Thus, the stabilized dimer may consist of monomers in which one amino acid residue of each monomer is substituted with cysteine ​​by a (nearly) twice-across molecular axis of the dimer. The stabilized dimer may also consist of monomers in which two amino acid residues of each monomer are substituted with cysteine ​​away from the twice-across molecular axis of the dimer. The stabilized dimer can also consist of monomers in which three amino acid residues in each monomer, located twice as far from the molecular axis as the dimer, are substituted with cysteine.

[0063] Such a configuration may restrict access to the FLE region, and for this purpose, it may be desirable to have more than one interchain disulfide bond to reduce the ability of molecules to produce anti-FLE reactions, as will be further discussed in Example 17.

[0064] In another preferred embodiment, the compound is bound to EDE, which is a stabilized dimer of sE, and the envelope or recombinant sE stabilized dimer is a homodimer of mutant sE having the above-defined mutations A259C or S255C, respectively, with residues 259C or 255C linked together by disulfide interchain bonds.

[0065] In another preferred embodiment, when the EDE contains a stabilized dimer of recombinant sE, the stabilized recombinant sE dimer is a heterodimer of mutant sE having the above-defined mutation A259C and mutant sE having the above-defined mutation S255C, wherein residues 259C and 255C are joined together by a disulfide interchain bond.

[0066] In another preferred embodiment, when the EDE contains a stabilized dimer of recombinant sE, the stabilized recombinant sE dimer is a homodimer of mutant sE having mutations F108C and T315C as defined above, respectively, or a homodimer of mutant sE having mutations L107C and A313C as defined above, respectively, wherein residues 108C and 315C or residues 107C and 313C are linked together by disulfide interchain bonds.

[0067] In one embodiment, the compound is bound to EDE, which is a stabilizing dimer of sE, and the envelope or recombinant sE stabilizing dimer is a heterodimer of mutant sE having the above-defined mutations F108C and A313C and mutant sE having the above-defined mutations L107C and T315C, with residues 108C and 313C bound to residues 315C and 107C, respectively, by disulfide interchain bonds between the two sE monomers.

[0068] In another preferred embodiment, the EDE comprises a stabilized recombinant sE dimer, which is selected from the group consisting of homodimers of mutant sE having mutations A259C, F108C, and T315C, respectively; homodimers of mutant sE having mutations S255C, F108C, and T315C, respectively; homodimers of mutant sE having mutations A259C, L107C, and A313C, respectively; and homodimers of mutant sE having mutations A255C, L107C, and A313C, respectively, wherein residues 259C, 255C, 108C, 315C, 107C, and 313C are linked to residues 259C, 255C, 315C, 108C, 313C, and 107C, respectively, by disulfide interchain linkages.

[0069] In another preferred embodiment, the compound is bound to EDE, which comprises a stabilized dimer of recombinant sE, the stabilized recombinant sE dimer being a heterodimer of mutant sE having the above-defined mutations A259C, F108C, and T315C and mutant sE having the above-defined mutations S255C, F108C, and T315C, wherein residues 259C, 108C, and 315C are bound to residues 255C, 315C, and 108C, respectively, by disulfide interchain bonds.

[0070] In another preferred embodiment, the EDE comprises a stabilized dimer of recombinant sE, the stabilized recombinant sE dimer being a heterodimer of mutant sE having the above-defined mutations S255C, L107C, and A313C and mutant sE having the above-defined mutations A259C, L107C, and A313C, wherein residues 255C, 107C, and 313C are linked to residues 259C, 313C, and 107C, respectively, by disulfide interchain bonds.

[0071] It will be understood that, similar to stabilization by disulfide bonds, stabilization can also be achieved by sulfhydryl-reactive crosslinking agents. Therefore, in one embodiment, EDE comprises a stabilized dimer of recombinant sE, the stabilized recombinant sE dimer of the present invention is covalently stabilized between sE monomers by at least one, two, or three sulfhydryl-reactive crosslinking agents (also called thiol-reactive crosslinking agents).

[0072] Chemical crosslinking of proteins is well known in the art (see Hemaprabha, 2012 for an overview).

[0073] Naturally, the sE dimer has two distinct sides: one exposed to the extracellular culture medium to which the antibody binds, and the other exposed to the viral membrane.

[0074] Advantageously, the stabilized recombinant sE dimer contains candidate amino acid residues present on the sE surface exposed to the viral membrane and is therefore not the epitope portion. One of each candidate amino acid residue in each monomer is mutated (substituted) with cysteine ​​to produce a free sulfhydryl group of appropriate length, which is the target of a sulfhydryl-reactive crosslinking agent.

[0075] Thr / Ser262 and Thr / Ala265 are candidate residues. In the context of dimerization, the distances between them are 12 and 22 Å, respectively. Furthermore, these residues (Thr / Ser262, Thr / Ala265) are not completely conserved. Therefore, they may be resistant to point mutations.

[0076] In a preferred embodiment, the compound is bound to EDE, which comprises a stabilized dimer of recombinant sE, the stabilized recombinant sE dimer being a homodimer of mutant sE having the above-defined mutations T / S262C or T / A265C, respectively, with residues 262C or 265C bound together by a sulfhydryl-reactive crosslinking agent.

[0077] In another preferred embodiment in which the EDE includes a stabilized dimer of recombinant sE, the stabilized recombinant sE dimer is a heterodimer of mutant sE having the above-defined mutation T / S262C and mutant sE having the above-defined mutation T / A265C, wherein residues 262C and 265C are linked together by a sulfhydryl-reactive crosslinking agent.

[0078] The regions of recombinant sE that are not considered part of the epitope and can be crosslinked are region A, consisting of sE residues 1-9; region B, consisting of sE residues 25-30; region C, consisting of sE residues 238-282; region D, consisting of sE residues 96-111; and region E, consisting of sE residues 311-318. Any one of the residues in these five regions (A-E) of the monomer is less than 25-30 Å from other residues of the other monomer in the recombinant sE dimer, and therefore these residues can be crosslinked.

[0079] Advantageously, one or more of the candidate amino acid residues in these five regions of each monomer are mutated (substituted) with cysteine, producing a free sulfhydryl group that is the target of a sulfhydryl-reactive crosslinking agent of the appropriate length as defined above.

[0080] In another embodiment, the compound is bound to EDE, which comprises a stabilized dimer of recombinant sE, the stabilized recombinant sE dimer being a homodimer or heterodimer of mutant sE, wherein at least one of the amino acid residues 1-9, 25-30, 238-282, 96-111, or 311-318 of sE is mutated (substituted) with cysteine, and the mutated cysteine ​​residues are bound together by a sulfhydryl-reactive crosslinking agent.

[0081] The sulfhydryl reactive crosslinking agent is preferably a homobifunctional reagent containing identical or non-identical reactive groups, enabling the construction of intermolecular crosslinks between two monomers. Homobifunctional crosslinking agents have identical reactive groups at either end of the spacer arm, and generally they can be used in a one-step reaction procedure. The sulfhydryl reactive crosslinking agent of the present invention may be maleimide, haloacetyl (preferably bromo- or iodo-acetyl), pyridyl disulfide, vinyl sulfone, alkyl halide or aziridine compounds, acryloyl derivatives, arylating agents, or thiol-disulfide exchange reagents (Hermanson, 2010, Hemaprabha, 2012), such as bis(methanethiosulfonate) (Haberz et al., 2006).

[0082] Examples of maleimide homobifunctional sulfhydryl reactive crosslinking agents according to the present invention, having spacers of different lengths, include BMOE (1,2-bis-maleimideethane), BMB (1,4-bis-maleimidebutane), BMH (1,6-bis-maleimidehexane), TMEA (tris-(2-maleimideethyl)amine), BM(PEG)2 (1,8-bismaleimidediethylene glycol), BM(PEG)3 (1,11-bismaleimidetriethylene glycol), BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane), DTME (dithio-bis-maleimideethane), and preferably BMH, BM(PEG)2, and BM(PEG)3. [ka]

[0083] The specific reaction of the sulfhydryl group with the maleimide group is carried out under mild buffering and pH conditions to minimize the degree of structural shift due to the crosslinking reaction. Preferably, the pH of the reaction mixture is 6.5 to 7.5, which leads to the formation of a stable, non-reversible thiol-ether bond (this bond cannot be cleaved by a reducing agent).

[0084] In addition to stabilization via disulfide bonds and sulfhydryl-reactive crosslinking agents, it will be understood that stabilization can also be achieved by the linking of two monomers by modified sugars. To achieve this objective, glycosylation sites are inserted into them and reacted with modified sugars to link them by click chemistry.

[0085] According to this embodiment, the compound is bound to EDE, and EDE comprises a stabilized dimer of recombinant sE, the stabilized recombinant sE dimer being a homodimer or heterodimer of mutant sE. -One sE monomer has at least one mutation that introduces a glycosylation site, and the mutated amino acid residue is glycosylated with a modified sugar supporting an X functional group. -The other sE monomer has at least one mutation that introduces a glycosylation site, and the mutated amino acid residue is glycosylated with a modified sugar supporting a Y functional group. Both mutated residues are linked together via a modified sugar, specifically by Crick chemistry, which involves reacting the X functional group of the sugar of the first sE monomer with the Y functional group of the sugar of the other sE monomer.

[0086] An X functional group refers to a chemical group carried by a sugar that reacts with a Y functional group and forms a covalent bond by click chemistry, and the Y functional group is preferably an azide functional group.

[0087] A Y functional group refers to a chemical group carried by a sugar that reacts with an X functional group and forms a covalent bond by click chemistry, and the X functional group is preferably a terminal alkyne functional group.

[0088] Modified sugars can be synthesized and introduced into sE monomers, as described by Laughlin et al., 2007, and can be bonded together, as described by Speer et al., 2003.

[0089] In addition to the covalent bonding methods described above for stabilizing the dimer, non-covalent bonding means may also be used. Therefore, in another embodiment where EDE includes a stabilized dimer of recombinant sE, the dimer is non-covalently stabilized by substituting at least one amino acid in the amino acid sequences of one or two monomers, preferably two monomers, with a bulky side-chain amino acid, thereby filling the dimer cavity at the dimer interface. According to this embodiment, the cavity characteristic of the quaternary structure of the recombinant sE dimer is identified and filled by hydrophobic substitutions manipulated in the monomers.

[0090] According to this embodiment, the stabilized recombinant sE dimer is non-covalently stabilized at the dimer interface or in the domain 1(D1) / domain 3(D3) linker of each monomer, within a cavity-forming region, by substituting at least one amino acid residue in the amino acid sequence of at least one sE monomer with at least one bulky side-chain amino acid. Such substitution can increase the hydrophobic interaction between the two sE monomers.

[0091] In a preferred embodiment in which the EDE includes a stabilized dimer of recombinant sE, the stabilized recombinant sE dimer is a homodimer or heterodimer of two recombinant sEs defined above, preferably a homodimer, where one or two of the recombinant sEs have at least one mutation (substitution) selected from the group consisting of H27F, H27W, H244F, H244W, and L278F. The mutations H27F, H27W, H244F, H244W, and L278F stabilize the cavity around F279 in the recombinant sE dimer, strengthen the dimer interface, and allow it to mimic the three-dimensional structure of F279 in the virion.

[0092] Other means of non-covalently stabilizing the dimer include, for example, non-covalent stabilization by substituting one or two, preferably two, amino acids in the amino acid sequence of each monomer with at least one bulky side-chain amino acid in the domain 1(D1) / domain 3(D3) linker of each monomer.

[0093] In a preferred embodiment, the compound is bound to EDE, which comprises a stabilized dimer of recombinant sE, the stabilized recombinant sE dimer being a homodimer or heterodimer of the two recombinant sEs defined above, preferably a homodimer, and one or two of the recombinant sEs having at least one mutation (substitution) selected from the group consisting of L292F and L294N. These mutations L292F, L294N are considered to enable the stabilization of the D1-D3 linkers in the sE dimer stereostructure.

[0094] In a preferred embodiment in which the EDE is stabilized in the dimeric configuration by manipulation, the manipulation described above does not result in a change in the overall 3D structure of the dimer, or substantially alters the overall 3D structure, and the residues in the natural dimer spatially correspond to those in the manipulated dimer. Where the natural dimer spatially corresponds to the manipulated dimer, this means that when the 3D model of the manipulated dimer (or a portion thereof, e.g., particularly important residues in characterizing the VDR, e.g., those shown in and / or further discussed below) is superimposed on the 3D model of the natural dimer, the coordination characterizing the spatial positions of the skeletal atoms in the natural dimer differs from the coordination characterizing similar skeletal atoms in the manipulated dimer by less than about 10 angstroms. Skeletal atoms are either skeletal atoms of amino acids that form the peptide skeleton, or the 3D folding pattern does not include side chain atoms, i.e., the positions of some or all of the side chain atoms do not change similarly significantly. The 3D structure is key to determining the immunogenicity of VDE, and therefore, in a preferred embodiment, the operation does not result in a dimer with reduced immunogenicity. In one embodiment, the operation does not result in a dimer with a different 3D stereostructure. Preferably, the operation results in a dimer with increased immunogenicity. Such an approach is referenced. 84 It is used in [the following context]. Therefore, in one embodiment, the compound binds to the manipulated EDE as described above.

[0095] 3D models of the natural dimer can be formed, for example, using information on the crystalline structure of the envelope glycoprotein outer domain from dengue virus serotypes, e.g., serotypes 2, 3, and 4, which are available in the Protein Databank under the aforementioned acceptance numbers 1OAN, 1OK8, 1UZG, and 3UAJ.

[0096] Whether a particular mutation or modification alters or substantially alters the 3D structure can be assessed by different techniques, including observing whether antibodies described herein, known to bind to VDE, can still bind to the manipulated version of VDE.

[0097] Those skilled in the art may, for example, use a computer program that helps identify potential stabilizing modifications.

[0098] The effect of manipulation on the immunogenicity of EDE can be evaluated by comparing antibody responses in subjects when manipulated and unmanipulated EDE are administered, or by comparing binding to known anti-EDE antibodies.

[0099] Alternatively, the modified envelope protein can be expressed in the dengue virus, and its ability to neutralize the virus can be evaluated.

[0100] To present a stabilized EDE, a non-EDE heterologous protein having a similar three-dimensional structure to each EDE (referred to as a backbone protein) can be modified to contain appropriate residues that allow the modified protein to hold the EDE. Therefore, in one embodiment, the compound binds to EDE, which is presented as part of an epitope backbone protein. The epitope backbone protein is a chimeric protein containing an epitope sequence that is fused to a heterologous "receptor" backbone protein. The design of the epitope backbone is performed computerically, for example, in a manner that preserves the native structure and three-dimensional structure of the epitope when the epitope is fused to the heterologous backbone protein. The use of such backbone proteins is known in the art, and such methods and techniques are described in WO 2011 / 050168 and reference 54、82、83 It is described therein, and those skilled in the art can apply the methods described therein to the present invention.

[0101] Therefore, in one embodiment, the EDE comprises a portion of an epitope backbone protein, which comprises a heterologous backbone protein covalently bound to the envelope dimer epitope. The backbone protein is useful in constructing the EDE of the present invention by holding the contact residues of the EDE in an appropriate spatial orientation to facilitate interactions between such residues and the compound, for example, between contact residues of the compound, when the compound is a protein, optionally an antibody or its antigen-binding moiety. The contact residues are any amino acids present in a molecule that directly or indirectly interact with an amino acid of another molecule (e.g., directly or indirectly form an ionic bond through a salt bridge). In at least DENV-1, the envelope protein residues considered potentially important in the compound binding to the EDE are detailed in Table 2, and the backbone protein may represent the entire dimer or only the selected residues above. 3D models of the natural dimer or a portion thereof may be formed, for example, using information on the crystalline structure of the envelope glycoprotein outer domain from dengue virus serotypes, e.g., serotypes 2, 3, and 4, which are available in the Protein Databank under the aforementioned acceptance numbers 1OAN, 1OK8, 1UZG, and 3UAJ.

[0102] Mutation analysis revealed specific residues of DENV1 and DENV2 that are important for binding to the antibodies identified in the present invention. These residues are DENV1: E49, K64, Q77, W101, V122, N134, N153, T155, I161, A162, P169, T200, K202, E203, L308, K310, Q323, W391, F392, DENV2: Q77, W101, N153, T155, K310.

[0103] All of these residues are considered important for binding and for Q77, W101, N153, T155, and K310.

[0104] Therefore, in one embodiment, the compound binds to EDE, which is part of a backbone protein, and this backbone protein holds at least one residue corresponding to one or more of E49, K64, Q77, W101, V122, N134, N153, T155, I161, A162, P169, T200, K202, E203, L308, K310, Q323, W391, F392 of the dengue virus envelope protein, or residues equivalent to those in the dengue virus envelope protein, specifically DENV-1 and DENV-2. Certain residues are considered more important, and therefore, further embodiments of EDE include a backbone protein that holds at least one or more residues corresponding to Q77, W101, N153, T155, K310 of the dengue virus envelope protein, or residues equivalent to those in the dengue virus envelope protein, specifically DENV-1 and DENV-2.

[0105] The envelope protein residues considered important for epitope contact are shown in Figure 31, for example, The B7 antibody is thought to contact DENV2 EDE at residues N67, T68, T69, T70, E71, S72, R73, L82, V97, D98, R99, W101, G102, N103, G104, I113, G152, N153, D154, T155, G156, K246, K247, Q248, and D249. The A11 antibody is considered to contact DENV2 EDE at residues N67, T68, T69, T70, E71, S72, R73, C74, E84, V97, D98, R99, G102, N103, G104, C105, V114, N153, D154, T155, G156, H158, K246, K247, Q248, D249, and V250. The C10 antibody is considered to contact DENV2 EDE at residues R2, H27, G28, E44, L45, I46, K47, N67, T68, T69, T70, E71, S72, R73, C74, Q77, S81, L82, N83, E84, V97, R99, W101, G102, N103, G104, C105, G106, L113, T115, K246, K247, Q248, Q271, V309, K310, R323, Q325, and D362. The C10 antibody is considered to contact DENV4 EDE at residues R2, H27, G28, G29, E44, L45, T46, N67, T69, T70, A71, T72, R73, C74, Q77, V97, R99, W101, G102, N103, G104, C105, G106, V113, R247, Q248, D249, D271, M278, D309, K310, V324, K323, K325, T361, and N362. The C8 antibody is thought to contact DENV2 EDE at residues N67, T68, T69, T70, E71, S72, R73, C74, Q77, N83, E84, V97, D98, R99, W101, G102, N103, G104, C105, G106, L113, E148, H158, K246, K247, Q248, D249, I308, K310, E311, R323, D362, and G374.

[0106] Therefore, the residues of the envelope proteins that are considered important in binding to the compound, specifically DENV2 and DENV4, A71, C105, C74, D154, D249, D271, D309, D362, D98, E148, E311, E44, E71, E84, G102, G104G106, G152, G156, G28, G29, G374, H158, H27, I113, I308, I46, K246, K247, K310, K323, K325K47, L113, Is it L45, L82, M278, N103, N153, N362, N67, N83, Q248, Q271, Q325, Q77, R2, R247, R323, R73, R99, S72, S81, T115, T155, T361, T46, T68, T69, T70, T72, V113, V114, V250, V309, V324, V97, W101? Alternatively, it is an equivalent residue in the dengue virus envelope protein.

[0107] Backbone proteins are, Dengue virus envelope proteins E49, K64, Q77, W101, V122, N134, N153, T155, I161, A162, P169, T200, K202, E203, L308, K310, Q323, W391, F392, A71, C105, C74, D154, D249, D271, D309, D362, D98, E148, E311, E44, E71, E84, G102, G104, G106, G152, G156, G28, G29, G374, H158, H27, I113, I308, I46, K246, K247, K323, K This may represent one or more residues selected from both of the following sets of residues: 325K47, L113, L45, L82, M278, N103, N362, N67, N83, Q248, Q271, Q325, R2, R247, R323, R73, R99, S72, S81, T115, T361, T46, T68, T69, T70, T72, V113, V114, V250, V309, V324, V97, or their equivalent residues, or at least one of these, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or all of them.

[0108] In addition, the skeletal protein may represent any one or more, or all, of the set of residues that are considered to increase the stability of the dimer configuration, as mentioned above: H27F, H27W, L107C, F108C, H244F, H244W, S255C, A259C, T / S262C, T / A265C, L278F, L292F, L294N, A313C, and T315C.

[0109] The skeletal protein may retain a dimer or a fragment of a dimer, may contain any of the above modifications considered essential for immunogenicity, and / or may result in increased dimer stability, such as increased disulfide bonds.

[0110] Furthermore, this skeleton may be such that an improved EDE is presented. In one embodiment, the compound thus binds to the improved EDE. For example, as described below and in Examples 2 and 5, patients with dengue fever infection tend to have either antibodies directed toward VDE, which is considered a useful antibody, or antibodies directed toward a fusion loop (anti-FL antibody) that is not considered useful. Therefore, the skeleton may be manipulated to present only EDE and to eliminate the possibility of the antibody or its antigen-binding moiety being produced on the compound, e.g., FL. Thus, in a preferred embodiment, the EDE can optionally be incorporated into the backbone protein to produce antibodies on EDE rather than FL.

[0111] Independent of the backbone protein, the envelope protein can be manipulated to produce an improved EDE. As mentioned above, an EDE that cannot be recognized by an anti-FL antibody and that cannot produce such an antibody is considered an improved EDE. This can be achieved by one or more mutations, deletions, or insertions in the envelope protein, or by generating a hybrid protein in which a specific epitope is fused to the backbone protein without the use of any antigen capable of producing an anti-FL antibody.

[0112] In one embodiment, the envelope protein is manipulated by modifying the inner surface of the dimer (which protrudes into the inside of the virus) by adding an N or O-linked glycan sequence, thereby making it less immunogenic.

[0113] The broad mutagenicity that allows for the creation of new surfaces on dimers may be useful in further reducing the generation of non-ED suboptimal reactions through residue mutations and / or the addition of glycans.

[0114] For example, the L278F mutation is thought to reshape the kl loop and mimic a virion-like three-dimensional structure.

[0115] Modeling and optimizing core EDE epitopes can also be useful for generating optimal sequences to induce desired EDE reactions and for providing binding and neutralizing antibodies.

[0116] It will be understood that EDE may be a native envelope protein held within a backbone that influences the increased stability of the dimer. EDE may also be manipulated to increase the stability of the dimer independently of any backbone. In one embodiment, these two may be combined such that EDE includes a dimer in which the envelope protein held within a heterologous backbone protein is manipulated to have improved stability in the dimer configuration. Alternatively, the envelope protein may be manipulated so that only the relevant portion of the protein exists, which may be held within the heterologous backbone protein.

[0117] The dimer structure may be stabilized, for example, by creating a long linker, such as a glycine-serine-rich liner, between two envelope monomers, which are represented as a single polypeptide chain containing two envelope polypeptide domains. Alternatively, the dimer structure may be stabilized by any antibody (e.g.) that binds to a surface facing the interior of the dimer or to a tag associated with the dimer.

[0118] Any reference to envelope protein, sE, sE dimer, or envelope protein dimer also includes, within that scope, a skeletal protein or structure, and includes specific residues that are retained in a particular three-dimensional structure in order to produce an EDE and exhibit a suitable EDE.

[0119] The envelope nucleotide sequence may be manipulated so that the envelope protein has one or more mutations, insertions, or deletions. The nucleotide sequence may have at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% homology to the native sequence of a particular envelope protein (or portion thereof).

[0120] In further embodiments, the envelope protein may be engineered to have at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% homology to an envelope protein (or portion thereof, e.g., one or more portions of at least 8, 9, or 10 consecutive amino acids) from another serotype of dengue virus. In preferred embodiments, the envelope protein may be engineered to have at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% homology to two different envelope proteins (or portions thereof, e.g., one or more of at least 8, 9, or 10 consecutive amino acids) from all serotypes of dengue virus, more preferably four different envelope proteins (or portions thereof, e.g., one or more of at least 8, 9, or 10 consecutive amino acids), and most preferably all envelope proteins (or portions thereof, e.g., one or more of at least 8, 9, or 10 consecutive amino acids).

[0121] As described above, envelope proteins can actually be manipulated to have very low homology to the native envelope protein, while maintaining the integrity and conformation of the EDE, or modifying the EDE in a way that improves it, for example, in a way that prevents the growth of anti-FL antibodies. Therefore, the level of sequence homology does not necessarily represent 3D structural homology or functional homology. For example, a particular sequence encoding a structure containing the EDE may actually have a very low level of homology to the native envelope protein, but may still be considered a useful compound of the present invention. For example, a protein may have 10%, 20%, 30%, 40%, 50%, or 60% homology to the native envelope protein, and the nucleotide sequence encoding this structure may have low sequence identity corresponding to the native envelope sequence.

[0122] In preferred embodiments, the envelope protein or structure containing EDE is at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% homologous to the dengue virus envelope protein (or portions thereof, e.g., one or more portions of at least 8, 9, or 10 consecutive amino acids), or two different envelope proteins (or portions thereof, e.g., one or more portions of at least 8, 9, or 10 consecutive amino acids), more preferably four different envelope proteins, most preferably all envelope proteins. In contrast, a protein or structure having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% homology, or containing EDE, has at least 10%, 20%, 30%, 40%, 50%, or 60% homology to the native envelope protein of one or more serotypes of dengue virus, and this protein contains one or more, or optionally all, of the following residues of the dengue virus envelope protein: E49, K64, Q77, W101, V122, N134, N153, T155, I161, A162, P169, T200, K202, E203, L308, K310, Q323, W391, F392, or equivalent residues of the dengue virus envelope protein.

[0123] Some of these residues are considered more important than others, and therefore, in further embodiments of EDE, the envelope protein or structure comprising EDE includes one or more, or optionally all, of the residues Q77, W101, N153, T155, K310, or equivalent residues of the dengue virus envelope protein.

[0124] Residues E49, K64, Q77, W101, V122, N134, N153, T155, I161, A162, P169, T200, K202, E203, L308, K310, Q323, W391, F392 of the envelope protein, or equivalent residues of the dengue virus protein, are considered necessary for the binding of compounds to EDE. Therefore, in one embodiment, the envelope protein or structure containing EDE includes one or more of these residues, or all of them.

[0125] Anti-FL antibodies appear to require only residue W101 from among the mutated residues in alanine scanning analysis in most cases (Example 2), and are not affected by mutations in any of the other residues. On the other hand, anti-EDE antibodies require a much larger epitope and, like anti-FL antibodies, require the presence of residue W101, but are affected by mutations in many other residues. Therefore, in one embodiment, EDE is defined as an epitope in which residue W101 and at least one of the following residues at positions E49, K64, Q77, W101, V122, N134, N153, T155, I161, A162, P169, T200, K202, E203, L308, K310, Q323, W391, F392, or equivalent residues are required for binding to the compound in the envelope dimer epitope.

[0126] In one particular embodiment, the envelope dimer epitope contains residue K310 of domain III.

[0127] In one embodiment, EDE is glycosylated, for example, at each envelope, e.g., sE, at position 67 (Asn67 glycan) and / or position 153 (Asn153 glycan) of the monomer, preferably at least at position 67 (Asn67 glycan) of each monomer.

[0128] According to one embodiment, the compound of the present invention comes into contact with either the N67 glycan chain of an envelope protein dimer or the N153 glycan chain of an envelope protein dimer. It will be understood that the compound can come into contact with both the N67 and N153 glycan chains of an envelope protein dimer.

[0129] In certain cases, this compound is an antibody in which CDR H2 interacts with the N67 glycan chain of the envelope protein.

[0130] In one embodiment, the compound contains one or more serotypes of dengue virus, preferably all serotypes of dengue virus envelope proteins, for example, A71, C105, C74, D154, D249, D271, D309, D362, D98, E148, E311, E44, E71, E84, G102, G104, G106, G152, G156, G28, G29, G374, H158, H27, I113, I3 in DENV-2 or DENV-4. One or more of the following contacts will make contact with EDE: 08, I46, K246, K247, K310, K323, K325, K47, L113, L45, L82, M278, N103, N153, N362, N67, N83, Q248, Q271, Q325, Q77, R2, R247, R323, R73, R99, S72, S81, T115, T155, T361, T46, T68, T69, T70, T72, V113, V114, V250, V309, V324, V97, W101.

[0131] In one embodiment, the envelope dimer epitope includes a region located in the center of a depression formed by the b strand on the domain II side and the “150 loop” on the domain I side (see, for example, Figure 29) (opposite the dimer interface), where the 150 loop extends to residues 148-159, connecting the b strands E0 and F0 of domain I and supporting the N153 glycan, which covers the fusion loop of the partner subunit in the dimer. The 150 loop is considered to include Denv-1 QHQVGNETTEHG of SEQ ID NO: 148, Denv 2 EHAVGNDTGKHG of SEQ ID NO: 1149, Denv 3 QHQVGNETQG of SEQ ID NO: 150, and Denv 4 THAVGNDIPNHG of SEQ ID NO: 151.

[0132] In some cases, the envelope dimer epitope comprises the envelope protein of domain II and optionally further comprises any one or more of the following characteristics of domain II: the b chain (residues 67-74), the fusion loop and residues immediately upstream (residues 97-106), and the ij loop (residues 246-249), as well as any one or more of residues 243-251 and 307-314.

[0133] In one embodiment, the EDE comprises a dengue virus glycoprotein E external domain (sE) consisting of five polypeptide segments: residues 67-74, 97-106, 148-159, 243-251, and 307-314.

[0134] Therefore, in one embodiment, the present invention also provides an isolated neutralizing antibody or antigen-binding fragment thereof against a compound, for example, the stabilized recombinant sE dimer defined above, the antibody or fragment thereof, which binds to the dengue virus glycoprotein E external domain (sE) of five polypeptide segments consisting of residues 67-74, 97-106, 148-159, 243-251, and 307-314.

[0135] Characterization of the binding of the antibody fragment according to the present invention to polypeptide segments or amino acid residues can be performed, for example, by crystallization tests as described in the following examples.

[0136] Preferably, in addition to binding to EDE, the compound can neutralize the virus. In preferred embodiments, the compound can neutralize all serotypes of dengue virus, preferably at least 90% or at least 98%, for example 100%, and preferably at least 90% or at least 98%, for example 100%, of all serotypes of dengue virus produced in both insect cells and human cells. Preferences for neutralization reactions and neutralization assay techniques are as described above.

[0137] In one embodiment, the EDE comprises a dimer of the full-length envelope protein. In another embodiment, the EDE comprises a dimer of the envelope outer domain (sE). In a further embodiment, the envelope protein comprises (as discussed above, approximately) 400 amino-terminal residues of the outer domain of the envelope protein. See, for example, Figure 28. The preference for the stability of the full-length envelope protein dimer described above also applies to the cleaved outer domain of the envelope protein. Thus, the dimer of the outer domain of the envelope protein may be stabilized through manipulation, or by being incorporated into a backbone protein, or may comprise a hybrid dimer.

[0138] In further embodiments, the compounds of the present invention will not bind to dengue virus or virion or subviral particles or virus-like particles when incubated at an acidic pH. The acidic pH causes the envelope protein to irreversibly introduce a trimer configuration. The inventors have found that the compounds of the present invention do not bind to virus particles at low pH (see Example 4). Therefore, in one embodiment, the compound, for example, an antibody or its antigen-binding moiety, will not bind to dengue virus or virion or subviral particles or virus-like particles when incubated at an acidic pH. Acidic pH means any pH lower than 7, preferably pH 5.5.

[0139] Therefore, those skilled in the art can easily determine whether a particular compound is a compound of the present invention according to this embodiment of the present invention by simply identifying whether the compound cannot bind to one or more of the following: a) virions or subviral particles or virus-like particles produced in cells lacking furin activity, b) virions or subviral particles or virus-like particles having a high proportion of prM protein, and / or c) virions or subviral particles or virus-like particles incubated under acidic conditions.

[0140] The methods for assaying the binding ability of the compound to virions, subviral particles, or virus-like particles described above are provided above with respect to the ability of the compound to bind to EDE and are detailed in Example 4, and generally involve simply using an ELISA on a specific virion or virus-like particle to assay whether the compound can bind. The compound is considered useful if it binds to or significantly binds to natural EDE or virions or virus-like particles, and if it does not bind to virions or subviral particles or virus-like particles that are a) produced in cells lacking furin activity, b) have a high percentage of prM protein, and / or c) are incubated under acidic conditions.

[0141] The present invention further includes certain compounds. For example, in one embodiment, the compound is an antibody containing the sequences of heavy chain SEQ ID NO: 1 and light chain SEQ ID NO: 37, heavy chain SEQ ID NO: 2 and light chain SEQ ID NO: 38, heavy chain SEQ ID NO: 3 and light chain SEQ ID NO: 39, or heavy chain SEQ ID NO: 4 and light chain SEQ ID NO: 40. The present invention also includes cleavage and mutation of these antibodies, so that the compound may be understood to be its antigen-binding portion. Antibodies having sequence homology of at least 90% or at least 95% homology to the above sequences are included in the present invention. The light and heavy chains, which are specific sequences of the antibody, are given in SEQ ID NOs: 1-4, 37-141, 141-147, for example, in Figure 29.

[0142] In further embodiments, the compound is an antibody comprising heavy chain SEQ ID NO: 1 and any of light chain SEQ ID NOs: 37, 38, 39, or 40; heavy chain SEQ ID NO: 2 and any of light chain SEQ ID NOs: 37, 38, 39, or 40; heavy chain SEQ ID NO: 3 and any of light chain SEQ ID NOs: 37, 38, 39, or 40; or heavy chain SEQ ID NO: 4 and any of light chain SEQ ID NOs: 37, 38, 39, or 40.

[0143] Specific heavy and light chain residues are considered important for binding to EDE. Therefore, in one embodiment, if the sequence homology is at least 90% with respect to the above sequence, the following residues, if present, are: Sequence ID 1 - T52, E54, D56, S57, A58, K65, G66, T69, E82, N84, S85, Y100, N102, F103, Y104, Y105, Y106, Sequence ID 2 - G554, N55, N57, K59, Q62, Q65, G66, R94, R98, F99, Y100, Y101, D102, S103, T104, Y106, Y107, P108, D109, S110, D117, V118, Sequence ID 3 - V2, S28, N31, D54, S56, T57, R58, K65, G66, R94, R98, F99, Y100, Y101, D102, S103, T104, Y106, Y107, P108, D109, S110, D117, V118, Sequence ID 4 - V2, T28, S31, D54, S56, S57, T58, G66, F68, M69, R94, R98, Y99, Y100, Y101, D102, S103, T104, Y106, Y107, P108, D109, N110, D117, V118, Sequence ID 37 - S30, T31, F32, Y49, D50, S52, R54, R66, R91, Y92, N93, W94, Sequence ID 38 - S26, S27, G30, G31, F32, N33, Y34, D52, T54, S55, R56, S62, S95, R96, G97, Sequence ID 39 - Y51, R56, P57, S58, G59, S96, R97, Sequence ID 40 corresponds to Y51, R56, P57, S58, and K97.

[0144] Antibodies consist of a light chain and a heavy chain, each containing three variable regions. The most variable portion of each of these regions is the complementarity-determining region, which is considered essential for antigen binding and recognition. Therefore, in one embodiment, the compound comprises one or more of the following amino acid sequences, which have one or two amino acid substitutions, insertions, or deletions, and which do not have the following amino acid substitutions, insertions, or deletions: Sequence ID 5, Sequence ID 8, Sequence ID 11, or Sequence ID 14 and / or Sequence ID 6, Sequence ID 9, Sequence ID 12, or Sequence ID 15 and / or Sequence ID 7, Sequence ID 10, Sequence ID 13, or Sequence ID 16 and / or Sequence ID 17, Sequence ID 20, Sequence ID 23, or Sequence ID 26 and / or Sequence ID 18, Sequence ID 21, Sequence ID 24, or Sequence ID 27 and / or Sequence ID 19, or Sequence ID 22, or Sequence ID 25, or Sequence ID 28.

[0145] Certain compounds may contain the following sequences, which have one or two amino acid substitutions, insertions, or deletions, or which do not have any amino acid substitutions, insertions, or deletions. Heavy chain: Sequence IDs 5, 6, and 7 or Sequence IDs 8, 9, and 10 or Sequence IDs 11, 12, and 13 or Sequence IDs 14, 15, and 16 Furthermore / or Light chain: Sequence IDs 17, 18, and 19 or Sequence IDs 20, 21, and 22 or Sequence IDs 23, 24, and 25 or Sequence IDs 26, 27, and 28.

[0146] In preferred embodiments, certain compounds may include the following sequences having one or two amino acid substitutions, insertions, or deletions, or having no amino acid substitutions, insertions, or deletions. Heavy chain SEQ ID NOs. 5, 6, and 7, and light chain SEQ ID NOs. 17, 18, and 19 or Heavy chain SEQ ID NOs. 8, 9, and 10, and light chain SEQ ID NOs. 20, 21, and 22 or Heavy chain SEQ ID NOs. 11, 12, and 13, and light chain SEQ ID NOs. 23, 24, and 25 or Heavy chain SEQ ID NOs. 14, 15, and 16, and light chain SEQ ID NOs. 26, 27, and 28 or Heavy chain sequence numbers 11, 12, and 13, and optionally light chain sequence number 25, and optionally amino acid sequence numbers 23 and 24. or Heavy chain SEQ ID NOs. 14, 15, and 16, and optionally light chain SEQ ID NOs. 28, and optionally amino acid sequence SEQ ID NOs. 26 and 27 or Heavy chain sequence number 3 and optionally light chain sequence number 25, preferably sequence number 140, in the light chain variable region. or A heavy chain variable region of sequence number 4, and optionally a light chain variable region of sequence number 28, preferably sequence number 141.

[0147] In further embodiments, certain residues in the above sequence are considered important for antigen binding. Therefore, in this embodiment, if present, the following residues are: In sequence number 6, residue 3 is T, residue 5 is E, residue 7 is D, residue 8 is S, residue 9 is A, residue 16 is K, and residue 17 is G. In sequence number 7, residue 2 is Y, residue 4 is N, residue 5 is F, residue 6 is Y, residue 7 is Y, and residue 8 is Y. In sequence number 9, residue 5 is G, residue 6 is N, residue 10 is K, residue 13 is Q, residue 16 is Q, and residue 17 is D. In sequence number 10, residue 5 is D, residue 6 is Y, residue 8 is D, residue 10 is W, residue 11 is F, residue 12 is P, and residue 14 is L. The residue 1 in sequence number 11 is N, In sequence number 12, residue 5 is D, residue 7 is S, residue 8 is T, residue 9 is R, residue 16 is K, and residue 17 is G. In sequence number 13, residue 4 is R, residue 5 is F, residue 6 is Y, residue 7 is Y, residue 8 is D, residue 9 is S, residue 10 is T, residue 12 is Y, residue 13 is Y, residue 14 is P, residue 15 is D, and residue 16 is S. The residue 1 of sequence number 14 is S, In sequence number 15, residue 5 is D, residue 7 is S, residue 8 is S, residue 9 is T, and residue 17 is G or H. In sequence number 16, residue 4 is R, residue 5 is Y, residue 6 is Y, residue 7 is Y, residue 8 is D, residue 9 is S, residue 10 is T, residue 12 is Y, residue 13 is Y, residue 14 is P, residue 15 is D, and residue 16 is N. In sequence number 17, residue 7 is S, residue 8 is T, and residue 9 is F. In sequence number 18, residue 1 is D, residue 3 is S, and residue 5 is R. In sequence number 19, residue 3 is R, residue 4 is Y, and residue 5 is N. In sequence number 20, residue 4 is S, residue 5 is S, residue 8 is G, residue 9 is G, residue 10 is F, residue 11 is N, and residue 12 is Y. In sequence number 21, residue 1 is D, residue 3 is T, residue 4 is S, and residue 5 is R. In sequence number 22, residue 5 is S, residue 6 is R, and residue 7 is G. In sequence number 24, residue 5 is R, residue 6 is P, and residue 7 is S. Residue 6 of sequence number 25 is S, and residue 7 is R. In sequence number 27, residue 5 is R, residue 6 is P, and residue 7 is S.

[0148] As described above regarding the presentation of antigenic EDEs in the protein backbone, the compound, for example, a protein, or for example, an antibody, may also be part of a larger structure, for example, held within the protein backbone. The preference for this backbone is as previously stated. For example, in one embodiment, the antibody or its antigen-binding portion is located within a larger polypeptide.

[0149] In preferred embodiments, the compounds that bind to EDE as described in any of the embodiments above also neutralize the dengue virus by at least 80%, preferably 90%, more preferably 95% or 98%, and most preferably 100%. In even more preferred embodiments, the compounds neutralize all serotypes of the dengue virus by at least 80%, preferably 90%, more preferably 95% or 98%, and most preferably 100%. The preferences for the neutralization reaction, including the concentrations of the compound, the virus or subvirus or virus-like particles, and the host cells, are as described above in the first aspect of the present invention.

[0150] In a first aspect of the present invention, it is preferable that a compound capable of binding to EDE can neutralize viruses produced in both insect cells, e.g., C6 / 36 insect cells, and human cells, e.g., primary human cells, e.g., dendritic cells. Preferably, the compound neutralizes dengue virus produced in both insect cells, e.g., C6 / 36 insect cells, and human cells, e.g., primary human cells, e.g., dendritic cells, to the same level, as discussed above. The virus-neutralizing ability of the compound can be tested in the examples as described above. In the most preferred embodiment, the compound can completely (i.e., 100%) neutralize all serotypes of dengue virus produced in both insect cells and human cells.

[0151] In preferred embodiments, the compound is an antibody or its antigen-binding moiety. The antigen-binding moiety may be an Fv moiety, a Fab-like fragment (e.g., a Fab fragment, a Fab' fragment, or an F(ab)2 fragment), or a domain antibody.

[0152] In one embodiment, the antibody or its antigen-binding portion is a monoclonal antibody or derived from a monoclonal antibody. In another embodiment, the antibody or its antigen-binding portion is a polyclonal antibody or derived from a polyclonal antibody. In a further embodiment, the compound is a) A mixture of monoclonal antibodies or their antigen-binding moieties, b) A mixture of polyclonal antibodies or their antigen-binding moieties, c) A composition comprising a mixture of monoclonal and polyclonal antibodies or their antigen-binding moieties, for example, a mixture of antibodies or their antigen-binding moieties in which the ratio of monoclonal antibody to polyclonal antibody or its antigen-binding moiety is 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:6, 1:8, or 1:10.

[0153] It will be understood that this compound may be a recombinant protein, such as a recombinant antibody or its antigen-binding portion. This compound may also be synthesized. This compound may be a combination of recombinant and synthetically produced compounds.

[0154] The present invention also includes means for producing such compounds, such as proteins, such as antibodies or their antigen-binding portions.

[0155] Therefore, the compound can be produced by recombinant means, for example, the compound, for example, a polypeptide, for example, an antibody or its antigen-binding portion. a) Human cell lines, optionally CHO cells, or b) Mammals, optionally humans, or c) Microorganisms, or d) It will also be understood that it can be produced, isolated, or purified from a variety of organisms, including insect cell lines.

[0156] To isolate or purify means that the drug has been removed from its natural environment and is no longer reflected in the degree to which it is purified.

[0157] Therefore, the present invention includes the isolation or purification of the compounds of the present invention from various organisms, including human cell lines, optionally CHO cells, or mammals, optionally humans, or microorganisms or insect cell lines.

[0158] If the compound is a polypeptide, for example, an antibody or its antigen-binding portion, or is included in, for example, a protein backbone, the compound may be encoded by a nucleic acid. Nucleic acid includes both DNA and RNA in single- or double-stranded and all their various forms. Therefore, the present invention includes nucleic acids encoding any of the proteinaceous compounds of the present invention. Specifically, Sequence IDs 41-48 are included in the present invention. This includes any sequence, including Sequence IDs 41-48, that encompasses any of the possibilities mentioned above, for example, a nucleic acid sequence containing a portion encoding any of the following, which may result in silent mutations, such as those derived from or containing mutations: Sequence ID 1, or a sequence having at least 90% homology to Sequence ID 1, Sequence ID 2, or a sequence having at least 90% homology to Sequence ID 2, Sequence ID 3, or a sequence having at least 90% homology to Sequence ID 3, Sequence ID 4, or a sequence having at least 90% homology to Sequence ID 4, Sequence ID 37, or a sequence having at least 90% homology to Sequence ID 37, Sequence ID 38, or a sequence having at least 90% homology to Sequence ID 38, Sequence ID 39, or a sequence having at least 90% homology to Sequence ID 39, Sequence ID 40, or a sequence having at least 90% homology to Sequence ID 40, A sequence having at least 90% homology to SEQ ID NO: 1, with residues T52, E54, D56, S57, A58, K65, G66, T69, E82, N84, S85, Y100, N102, F103, Y104, Y105, Y106. A sequence having at least 90% homology to SEQ ID NO: 2, with residues G554, N55, N57, K59, Q62, Q65, G66, R94, R98, F99, Y100, Y101, D102, S103, T104, Y106, Y107, P108, D109, S110, D117, V118. A sequence having at least 90% homology to Sequence ID No. 3, with residues V2, S28, N31, D54, S56, T57, R58, K65, G66, R94, R98, F99, Y100, Y101, D102, S103, T104, Y106, Y107, P108, D109, S110, D117, V118. A sequence having at least 90% homology to SEQ ID NO: 4, with residues V2, T28, S31, D54, S56, S57, T58, G66, F68, M69, R94, R98, Y99, Y100, Y101, D102, S103, T104, Y106, Y107, P108, D109, N110, D117, V118. A sequence having at least 90% homology to sequence number 37, with residues S30, T31, F32, Y49, D50, S52, R54, R66, R91, Y92, N93, W94. A sequence having at least 90% homology to sequence number 38, with residues S26, S27, G30, G31, F32, N33, Y34, D52, T54, S55, R56, S62, S95, R96, G97. A sequence having at least 90% homology to sequence number 39, with residues Y51, R56, P57, S58, G59, S96, and R97. A sequence having at least 90% homology to sequence number 40, with residues Y51, R56, P57, S58, and K97. Sequences resulting in polypeptides having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID No. 5, Sequence ID 6, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID 6, where residue 3 is T, residue 5 is E, residue 7 is D, residue 8 is S, residue 9 is A, residue 16 is K, and residue 17 is G. Sequence ID 7, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID 7, where residue 2 is Y, residue 4 is N, residue 5 is F, residue 6 is Y, residue 7 is Y, and residue 8 is Y. Sequences resulting in polypeptides having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID No. 8, Sequence ID 9, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID 9, where residue 5 is G, residue 6 is N, residue 10 is K, residue 13 is Q, residue 16 is Q, and residue 17 is D. Sequence ID 10, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID 10, where residue 5 is D, residue 6 is Y, residue 8 is D, residue 10 is W, residue 11 is F, residue 12 is P, and residue 14 is L. Sequence ID 11, or optionally, a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID 11, where residue 1 is N. Sequence ID 12, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID 12, where residue 5 is D, residue 7 is S, residue 8 is T, residue 9 is R, residue 16 is K, and residue 17 is G. Sequence ID 13, or optionally a sequence having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID 13, where residue 4 is R, residue 5 is F, residue 6 is Y, residue 7 is Y, residue 8 is D, residue 9 is S, residue 10 is T, residue 12 is Y, residue 13 is Y, residue 14 is P, residue 15 is D, and residue 16 is S. Sequence ID 14, or optionally, a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID 14, where residue 1 is S. Sequence ID 15, or optionally, a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID 15, where residue 5 is D, residue 7 is S, residue 8 is S, residue 9 is T, and residue 17 is G or H. Sequence ID 16, or optionally, a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID 16, where residue 4 is R, residue 5 is Y, residue 6 is Y, residue 7 is Y, residue 8 is D, residue 9 is S, residue 10 is T, residue 12 is Y, residue 13 is Y, residue 14 is P, residue 15 is D, and residue 16 is N. Sequence ID 17, or optionally, a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID 17, where residue 7 is S, residue 8 is T, and residue 9 is F. Sequence ID 18, or optionally, a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID 18, where residue 1 is D, residue 3 is S, and residue 5 is R. Sequence ID 19, or optionally, a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID 19, where residue 3 is R, residue 4 is Y, and residue 5 is N. Sequence ID 20, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID 20, where residue 4 is S, residue 5 is S, residue 8 is G, residue 9 is G, residue 10 is F, residue 11 is N, and residue 12 is Y. Sequence ID 21, or optionally, a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID 21, where residue 1 is D, residue 3 is T, residue 4 is S, and residue 5 is R. Sequence ID 22, or optionally, a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID 22, where residue 5 is S, residue 6 is R, and residue 7 is G. Sequences resulting in polypeptides having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID No. 23, Sequence ID 24, or optionally, a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID 24, where residue 5 is R, residue 6 is P, and residue 7 is S. Sequence ID 25, or optionally, a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID 25, where residue 6 is S and residue 7 is R. Sequences resulting in polypeptides having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID No. 26, Sequence ID 27, or optionally, a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to Sequence ID 27, where residue 5 is R, residue 6 is P, and residue 7 is S. Furthermore Sequences resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 28.

[0159] Nucleic acids may or may not contain introns. Nucleic acids may also be modified to allow for subsequent purification of the translated polypeptide; for example, the open reading frame of the target polypeptide may be modified to incorporate tags, such as a myc tag or a his tag, to allow for subsequent purification.

[0160] Nucleic acids may also be modified, for example, with codons optimized to be better translated by the organism to be translated, without affecting the final polypeptide sequence.

[0161] The nucleic acids of this disclosure can be produced or modified using many methods well known to those skilled in the art, such as classical mutagenesis, chemical treatment, restriction digestion, ligation, and PCR.

[0162] The nucleic acids of the present invention may be incorporated into a vector. Therefore, the present invention may also include a vector containing nucleic acids. A vector means a vehicle for cloning amplification of nucleic acids or for insertion into a target organism, and for example, the vector may be a plasmid or a nucleic acid used to target a target organism, for example, the genome of the target organism, with the nucleic acids of the present invention. The vector may further include a nucleotide sequence required for the expression of a polypeptide encoded by the nucleic acids of the present invention, for example, a promoter sequence or a stop sequence which may be operably ligated to the nucleic acids of the present invention, and may include a reporter gene, for example, an antibiotic resistance cassette. The vector may be single-stranded or double-stranded, linear or circular. In one embodiment, the vector is a plasmid.

[0163] In addition to providing compounds that can bind to the EDE described above, further embodiments of the present invention also provide EDE compounds as defined below. The present invention also provides nucleic acids or vectors encoding the EDE compounds of the present invention, in addition to host cells comprising nucleic acids or vectors. For example, the preferences for the nucleic acids and vectors described above may also be relevant to these embodiments of the present invention, as will be apparent to those skilled in the art. Therefore, the present invention provides EDE compounds as defined below, nucleic acids encoding such EDE compounds, or vectors comprising such nucleic acids, or host cells comprising such nucleic acids or vectors.

[0164] The EDE compound is intended to provide the epitope described above as an envelope-dependent epitope. The EDE compound may specifically bind to one or more EDE-specific antibodies of the present invention, e.g., preferred neutralizing antibodies, as discussed above or illustrated in the examples. The EDE compound is typically a polypeptide or comprises a polypeptide. In one embodiment, the EDE compound is a dimer of an envelope protein, or the outer domain of an envelope protein, or the 400th amino-terminal residue of the outer domain of an envelope protein. As used herein, “400 amino-terminal residues” includes about 400 amino-terminal residues, e.g., 350–450 residues, 320–470 residues, or 330–480 residues (or a combination thereof), e.g., 380–420 residues, e.g., 390–410 residues, e.g., 395 or 393 e.g., 320–470 residues, e.g., 330–480 residues, e.g., 380–420 residues, e.g., 390–410 residues, e.g., 395 or 393 residues, e.g., 395 or 393 residues, e.g., 390–420 residues, e.g., 390–410 The envelope protein may be any of the envelope proteins from DENV-1, DENV-2, DENV-3, and DENV-4 (SEQ ID NOs. 29, 31, 33, or 35), or any protein having at least 90% homology to the sequence of SEQ ID NOs. 29, 31, 33, or 35. This dimer may be a homodimer or a heterodimer. In preferred embodiments, this dimer is not incorporated into intact virus particles, subvirus particles, or virus-like particles, but rather is a free dimer having, for example, twice the molecular weight of the monomeric envelope polypeptide. It will be understood that, for example, any form of the EDE or EDE compounds described herein, such as an engineered envelope protein, may potentially be presented as part of a virus, virus-like particle, or subvirus particle, for example, as part of a protein backbone.

[0165] In another embodiment, the EDE compound comprises a dimer of an envelope protein, or an envelope outer domain, or about 400 amino-terminal residues of the outer domain of an envelope protein, which are manipulated to have increased stability in a dimeric configuration, for example, to have increased levels of covalent and / or non-covalent bonds between the dimers.

[0166] In preferred embodiments, the EDE compound is a stabilized recombinant dengue virus envelope glycoprotein E external domain (recombinant sE) dimer according to the above-described aspects of the present invention, for example, a stabilized recombinant dengue virus envelope glycoprotein E external domain (recombinant sE) dimer, and this dimer is - Covalently stabilized by at least one disulfide interchain bond between two sE monomers, and / or - Covalently stabilized by at least one sulfhydryl-reactive crosslinking agent between two sE monomers, and / or - Covalently stabilized by linking two sE monomers via a modified sugar, and / or - Non-covalent stabilization occurs at the dimer interface or in the domain 1(D1) / domain 3(D3) linker of each monomer by substituting at least one amino acid residue in the amino acid sequence of at least one sE monomer with at least one bulky side-chain amino acid.

[0167] The dengue virus envelope glycoprotein E external domain (sE) refers to the amino acid fragments 1-395 of the envelope glycoprotein E of dengue virus serotypes 1, 2, and 4, and the amino acid fragments 1-393 of the envelope glycoprotein E of dengue virus serotype 3.

[0168] Therefore, the EDE compound mentioned above is a stabilized dimer. a) A monomer selected from the group consisting of DENV-1 sE of SEQ ID NO: 132, DENV-2 sE of SEQ ID NO: 133, DENV-3 sE of SEQ ID NO: 134, DENV-4 sE of SEQ ID NO: 135, and mutant sE having at least one mutation (substitution) selected from H27F, H27W, L107C, F108C, H244F, H244W, S255C, A259C, T / S262C, T / A265C, L278F, L292F, L294N, A313C, and T315C, and optionally, the mutant sE further has at least one mutation (substitution) selected from Q227N, E174N, and D329N, preferably three mutations Q227N, E174N, and D329N, b) The dimer may be a homodimer of two identical recombinant sEs defined above, or a heterodimer of two different recombinant sEs defined above, and this dimer is preferably a homodimer, for example, a heterodimer of DENV-1 sE and DENV-2 sE as defined above. It may also be a heterodimer of DENV-1 sE and a mutant sE of DENV-1 sE as defined above. c) Dimers that are glycosylated at the 67th position (Asn67 glycan) and / or the 153rd position (Asn153 glycan) of each sE monomer, preferably at least at the 67th position (Asn67 glycan) of each monomer. d) A dimer, covalently stabilized by at least one, two, or three disulfide interchain bonds between two sE monomers. e) A homodimer of mutant sE having the above-defined mutations A259C or S255C, respectively, in which these residues 259C or 255C are linked together via disulfide interchain bonds, f) A heterodimer of mutant sE having the above-defined mutation A259C and mutant sE having the above-defined mutation S255C, wherein residues 259C and 255C are linked together via disulfide interchain bonds, g) A homodimer of mutant sE having the mutations F108C and T315C as defined above, respectively, or a homodimer of mutant sE having the mutations L107C and A313C as defined above, wherein residues 108C and 315C or residues 107C and 313C are linked together via disulfide interchain bonds, h) A heterodimer of mutant sE having the mutations F108C and A313C defined above, and mutant sE having the mutations L107C and T315C defined above, wherein residues 108C and 313C are bound to residues 315C and 107C, respectively, via disulfide interchain bonds between the two sE monomers. i) A dimer selected from the group consisting of homodimers of mutant sE having mutations A259C, F108C, and T315C respectively, homodimers of mutant sE having mutations S255C, F108C, and T315C respectively, homodimers of mutant sE having mutations A259C, L107C, and A313C respectively, and homodimers of mutant sE having mutations A255C, L107C, and A313C respectively, wherein residues 259C, 255C, 108C, 315C, 107C, and 313C are each linked to residues 259C, 255C, 315C, 108C, 313C, and 107C via disulfide interchain bonds, j) A heterodimer of mutant sE having the above-defined mutations A259C, F108C, and T315C and mutant sE having the above-defined mutations S255C, F108C, and T315C, wherein residues 259C, 108C, and 315C are linked to residues 255C, 315C, and 108C, respectively, via disulfide interchain bonds. k) A heterodimer of mutant sE having the mutations S255C, L107C, and A313C defined above, and mutant sE having the mutations A259C, L107C, and A313C defined above, wherein residues 255C, 107C, and 313C are linked to residues 259C, 313C, and 107C, respectively, via disulfide interchain bonds. l) Dimers covalently stabilized between sE monomers by at least one, two, or three sulfhydryl reactive crosslinking agents (also called thiol reactive crosslinking agents), m) A homodimer of mutant sE having the mutations T / S262C or T / A265C as defined above, wherein residues 262C or 265C are linked together by a sulfhydryl-reactive crosslinking agent, n) A heterodimer of mutant sE having the above-defined mutation T / S262C and mutant sE having the above-defined mutation T / A265C, wherein residues 262C and 265C are linked together by a sulfhydryl-reactive crosslinking agent, o) A homodimer or heterodimer of mutant sE, wherein at least one of the amino acid residues 1-9, 25-30, 238-282, 96-111, 311-318 of sE is mutated (substituted) to cysteine, and the mutated cysteine ​​residues are linked together by a sulfhydryl-reactive crosslinking agent, forming a dimer. p) A dimer that is covalently stabilized by bonding to two monomers via a modified sugar. q) A homodimer or heterodimer of mutant sE, - A dimer in which one sE monomer has at least one mutation introducing a glycosylation site, and the mutated amino acid residue is glycosylated with a modified sugar supporting an X functional group, and the other sE monomer has at least one mutation introducing a glycosylation site, and the mutated amino acid residue is glycosylated with a modified sugar supporting a Y functional group, and both mutated residues are linked together via modified sugars, specifically by click chemistry, which reacts the X functional group of the sugar of the first sE monomer with the Y functional group of the sugar of the other sE monomer. The X functional group means a chemical group supported by a sugar that can react with a Y functional group and form a covalent bond by click chemistry, and the Y functional group is preferably an azide functional group. The Y functional group means a chemical group supported by a sugar that can react with an X functional group and form a covalent bond by click chemistry, and the X functional group is preferably a terminal alkyne functional group. r) By filling the dimer cavity at the dimer interface, a dimer is stabilized non-covalently by substituting at least one amino acid in the amino acid sequence of one or two monomers, preferably two monomers, with a bulky side-chain amino acid. s) Non-covalently stabilized at the dimer interface or in the domain 1(D1) / domain 3(D3) linker of each monomer, within the region forming the cavity, by substituting at least one amino acid residue in the amino acid sequence of at least one sE monomer with at least one bulky side-chain amino acid. Such substitution can increase the hydrophobic interaction between the two sE monomers, dimer, t) A homodimer or heterodimer of two recombinant sEs defined above, preferably a homodimer, wherein one or two of the recombinant sEs have at least one mutation (substitution) selected from the group consisting of H27F, H27W, H244F, H244W, and L278F, u) In the domain 1(D1) / domain 3(D3) linker of each monomer, a dimer is non-covalently stabilized by substituting one or two, preferably two, amino acids in the amino acid sequence of the monomer with at least one bulky side-chain amino acid. v) A homodimer or heterodimer, preferably a homodimer, of two recombinant sEs defined above, where one or two of the recombinant sEs have at least one mutation (substitution) selected from the group consisting of L292F and L294N, and one or more of these dimers may be any one or more of these.

[0169] In yet another embodiment, the EDE compound presents an improved epitope beyond the naturally occurring envelope dimer in the virus, virus-like particle, or subviral particle. Improved epitope means that it surpasses any improved epitope naturally presented on intact virus particles. Improved means that it can elicit a more beneficial immune response than naturally intact dengue virus particles. For example, an EDE compound having increased stability in the dimer configuration through the modifications described in a) to v) above is considered an improved epitope. EDE may be improved in other ways, for example, the EDE compound may be an EDE in which the FL is manipulated so that it cannot be recognized by itself by a compound, such as a polypeptide, such as an antibody or its antigenic moiety, or an EDE inserted into the backbone, for example, the EDE may be manipulated so that the FL cannot be recognized by an antibody in isolation from the direct adjacency of the fusion loop, i.e., the induction loop cannot be recognized in a context independent of quaternary tissue.

[0170] In another embodiment, the EDE compound is incorporated into a heterogeneous protein backbone that preserves the dimeric configuration by increasing the level of covalent and / or noncovalent bonding between the dimers, as described above. Furthermore, the VDE compound may include a heterogeneous protein backbone that can be presented only on a portion of the (approximately) 400 amino-terminal residues of the envelope protein dimer, or envelope outer domain, or the outer domain of the envelope protein, and this portion is either a continuous portion of the (approximately) 400 amino-terminal residues of the envelope protein dimer, or envelope outer domain, or the outer domain of the envelope protein, or this portion includes a selection of discontinuous residues of the (approximately) 400 amino-terminal residues of the envelope protein dimer, or envelope outer domain, or the outer domain of the envelope protein, as described above.

[0171] For example, in one embodiment, the EDE compound is located at positions E49, K64, Q77, W101, V122, N134, N153, T155, I161, A162, P169, T200, K202, E203, L308, K310, Q323, W391, F392, A71, C105, C74, D154, D249, D271, D309, D362, D98, E148, E311, E44, E71, E84, G102, G104 G106, G152, G156, G28, G29, G374, H158, H27, I113, I308, I46, K246, K247, K310, K323, K325 K47, L113, L45, L82, M278, N103, N153, N362, N67, N83, Q248, Q271, Q325, Q77, R2, R247, R 323, R73, R99, S72, S81, T115, T155, T361, T46, T68, T69, T70, T72, V113, V114, V250, V309 The EDE compound comprises one or more of V324, V97, W101, or equivalent residues of the dengue virus envelope polypeptide that are in substantially the same spatial arrangement as the residues adopted at the (approximately) 400 amino-terminal residues of the envelope protein's natural dimer, or the envelope's outer domain, or the outer domain of the envelope protein. These residues may be present in the naturally occurring envelope protein, or, in alternative embodiments, they are retained within the backbone protein in appropriate arrangements. In preferred embodiments, the EDE compound comprises the aforementioned W101 and at least one other residue. In more preferred embodiments, the EDE compound comprises all of the above residues. In one embodiment, the EDE compound comprises the N153 glycan. In alternative embodiments, the EDE does not contain the N153 glycan.

[0172] In a more preferred embodiment, the EDE compound comprises residues observed both in terms of amino acids and spatial position across more than one serotype of dengue virus, preferably residues observed both in terms of amino acids and spatial position across all serotypes of dengue virus, i.e., four serotypes of dengue virus.

[0173] The EDE compound may contain a dimer of the envelope protein, or the outer domain of the envelope protein, or approximately 400 amino-terminal residues of the outer domain of the envelope protein, which have increased stability in the dimeric configuration and are manipulated to be retained within the protein backbone as described above.

[0174] The inventors have found that a specific region of the envelope dimer is important for contact with the compound of the present invention, such as an antibody or its antigen-binding moiety. Therefore, in some embodiments, the VDE compound comprises a dimer of the envelope protein, or the envelope outer domain, or a specific antigenic moiety at the 400-amino terminal residue of the outer domain of the envelope protein.

[0175] The EDE compound may be a dimer of an envelope protein, or an envelope outer domain, or a specific fragment containing (approximately) 400 amino-terminal residues of the envelope protein outer domain, which includes a region considered necessary for antigenicity. This fragment may also be manipulated to maintain a specific three-dimensional structure, or retained within a protein backbone, or manipulated and retained within a protein backbone.

[0176] For example, in one embodiment, the EDE compound includes a region located in the center of a depression formed by the b strand on the domain II side and the “150 loop” on the domain I side (opposite the dimer interface), where the 150 loop extends to residues 148-159, connecting the b strands E0 and F0 of domain I and supporting the N153 glycan, which covers the fusion loop of the partner subunit in the dimer. In one embodiment, this region includes three polypeptide segments of domain II of the reference subunit, which are defined as subunits contributing to the FL to the epitope. These three segments are the b strand (residues 67-74 supporting the N67 glycan), the fusion loop and residues immediately upstream (residues 97-106), and the ij loop (residues 246-249).

[0177] In another embodiment, in addition to the region described above (the region containing the three polypeptide segments of domain II of the reference subunit), the EDE compound further comprises 150 loops and the N153 glycan chain of the second subunit.

[0178] Further embodiments of the EDE compound include the region described above (the region containing the three polypeptide segments of domain II of the reference subunit) at a specific residue K310, as well as the 150 loop and the A strand of domain III of the second subunit. The inventors have found that the 150 loop of the second subunit interferes with the binding of the subunits of the useful compounds defined herein to EDE. Therefore, in one embodiment, the 150 loop may be in a native configuration found in the native dimer of the envelope protein, or in the envelope outer domain, or at the 400 amino-terminal residue of the outer domain of the envelope protein, or in another embodiment, the 150 loop may be in an irregular configuration that the 150 loop adopts when binding to one of the compounds of the present invention.

[0179] N67 glycan is considered particularly important for dengue infection of dendritic cells. Therefore, as described herein, EDE compounds containing this residue in the correct epitope environment are considered preferred embodiments.

[0180] In preferred embodiments, once administered to a subject, preferably a human, the EDE compound produces antibodies that preferably can bind to all four serotypes of dengue virus, optionally neutralize all four serotypes of dengue virus, preferably neutralize 100% of all four serotypes of dengue virus, optionally neutralize viruses produced in both human and insect cells, preferably neutralize 100% of all four serotypes of dengue virus produced in both human and insect cells.

[0181] The VDE compound may be an anti-idiotype antibody (or a fragment thereof, or a molecule sharing binding specificity as discussed above) developed against one or more of the high-affinity / neutralizing antibodies provided herein, as is known to those skilled in the art, for example as shown in the examples.

[0182] The present invention also provides a method for synthesizing EDE, which is a stabilized recombinant sE dimer of the present invention, and this method is a) The step of bringing one or more cysteine ​​mutants sE as defined above into contact under oxidative conditions, and / or b) The step of contacting two sE monomers with at least one, two, or three sulfhydryl reactive crosslinking agents as defined above, and / or c) The step of contacting two sE monomers having glycosylation sites as defined above by click chemistry, and / or d) comprising at least one of the steps of bringing at least one amino acid residue in the amino acid sequence of at least one sE monomer into contact with two sE monomers that substitute for the bulky side-chain amino acids defined above.

[0183] The present invention also provides stabilized recombinant sE dimers obtained by the methods defined above.

[0184] To ensure proper formation of stabilized recombinant sE dimers according to the present invention, the affinity for the antibodies described below can be measured by ELISA (for covalently and non-covalently stabilized dimers) or by surface plasmon resonance (for covalently stabilized dimers).

[0185] The present invention also includes a host cell comprising either the nucleic acid or vector of the present invention, for example, a nucleic acid or vector comprising an EDE compound or a portion of a nucleic acid encoding a compound of the present invention. For example, the present invention includes any host cell known to be useful for the expression of heterologous proteins, such as C6 / 36 insect cells, human dendritic cells, CHO cells, or microorganisms, such as Pichia pastris cells, comprising a vector, such as a plasmid. The host cell may also optionally include the nucleic acid of the present invention, which is incorporated into the genome of the host cell by the use of a viral vector to target the nucleic acid of the present invention to a genome, such as an adenovirus, adeno-associated virus, cytomegalovirus, herpesvirus, poliovirus, retrovirus, Sindbisvirus, vaccinia virus, or any other DNA or RNA viral vector.

[0186] The present invention further includes a non-human transgenic animal comprising at least one cell transformed by the nucleic acid or vector of the present invention, or by a host cell of the present invention, for example, by the nucleic acid or vector comprising a portion of the nucleic acid encoding the VDE compound or the compound of the present invention.

[0187] A process for producing the compounds of the present invention, preferably polypeptides, preferably antibodies or their antigen-binding moieties, or the EDE compounds of the present invention is provided herein. This process consists of the following steps: ii) Culture of the host cells of the present invention in an appropriate culture medium, ii) Recovery of the compound, preferably the produced antibody or its antigen-binding portion, or the EDE compound, wherein the recovery is from either the culture medium or the cultured cells.

[0188] For the purification or isolation of polypeptides, for example, if the compound is a polypeptide, or if the EDE compound is a polypeptide, those skilled in the art will understand that the nucleotide coding sequence can be easily manipulated to include nucleotides useful for purification, such as an affinity tag for an epitope tag. Therefore, in one embodiment, a process for producing the compound of the present invention or an EDE compound includes a culture of host cells comprising a nucleotide sequence encoding the compound or an EDE compound, further comprising nucleotides encoding a portion useful for the purification of the compound or an EDE compound, or a vector comprising a nucleotide sequence encoding the compound or an EDE compound, and further comprising nucleotides encoding a portion useful for the purification of the compound or an EDE compound.

[0189] It will be understood that, if the compound is a polypeptide, such as an antibody or its antigen-binding portion, and is similarly produced by recombinant means, polypeptide production may be triggered by administration of EDE, optionally an EDE compound as defined above, to a subject. Following administration of EDE (optionally an EDE compound), a native host response may produce antibodies that can be recovered from the subject's blood. Preferably, the EDE is not presented as part of an intact virus, virus-like particle, or subviral particle. Preferably, the EDE is an envelope polypeptide dimer as discussed above, or other EDE compounds as discussed above or below.

[0190] For example, the present invention provides a method for producing the compound of the present invention, the compound being the antibody of the present invention, and this method is a) A step of contacting a mammal with the stabilized recombinant sE dimer of the present invention or the immunogenic composition of the present invention, b) A step of detecting the presence of antibodies targeting the sE in one or more serum samples derived from the mammal, c) The step of collecting pancreatic cells from a mammal, d) A step of fusing pancreatic cells with myeloma cells in order to produce hybridoma cells, e) A step of identifying hybridoma cells capable of producing antibodies, f) A step of culturing hybridoma cells capable of producing antibodies, g) optionally includes the step of isolating the antibody.

[0191] The present invention also provides antibodies obtained by any of the methods defined above.

[0192] The present invention also provides hybridoma cells obtained by the method defined above.

[0193] The present invention also provides the use of the stabilized recombinant sE dimer of the present invention for the preparation of hybridoma cells capable of producing neutralizing antibodies targeting the dimer defined above.

[0194] In preferred embodiments, the present EDE or EDE compound is one that has already been determined to be capable of producing highly cross-reactive and potently neutralizing antibodies. The antibodies identified in Examples (Examples 1-6) were produced on intact viruses in natural infection with the dengue virus. More specific and improved antibodies are considered to be possible by administration of a specific EDE antigen, which may be the EDE compound of the present invention. For example, in natural infection, some patients did not produce anti-EDE antibodies, but instead produced anti-FL antibodies that are considered less useful, less cross-reactive, and less neutralizing. Administration of an EDE antigen is considered to have a higher probability of producing useful anti-EDE antibodies. As described above, in some embodiments, the present EDE or EDE compound is engineered to have increased stability in a dimer configuration, which is considered to increase the chances of producing anti-VDE antibodies in the subject. In addition, in some embodiments, the EDE or EDE compound is manipulated, for example, by mutating itself within the envelope protein or by using a skeletal protein to present an improved epitope, thereby concealing the fusion loop to reduce the likelihood of anti-FL antibody production. Administration of EDE or an EDE compound common to all serotypes of dengue virus is likely to produce highly cross-reactive and potently neutralizing antibodies. These antibodies can be recovered from subjects and used for further analysis or in the treatment of dengue fever or in dengue fever clinical trials.

[0195] Therefore, one embodiment provides a process for producing a compound according to the present invention, wherein the compound is a polypeptide, or an antibody or its antigen-binding portion, and the process is as follows: a. Administration of an envelope dimer epitope or EDE compound as defined in any of the embodiments described above to the subject. b. Including the recovery and isolation of the antibody or its antigen-binding portion from the blood of the subject.

[0196] It will be understood that the above method of producing a compound, for example, an antibody or its antigen-binding moiety, according to the present invention, which involves administering EDE or an EDE compound to a subject, can also be used as part of a method for selecting a suitable antigen for a vaccine. Current vaccines utilize attenuated versions of all four serotypes of dengue fever and are not particularly effective. Such vaccines may also induce the production of useless anti-FL antibodies. A preferred vaccine may contain a single antigen capable of eliciting an immune response against all serotypes of dengue fever virus, and this immune response is considered to be capable of neutralizing all serotypes of dengue fever virus, i.e., all four serotypes of dengue fever virus.

[0197] The inventors of this invention have for the first time identified highly cross-reactive and potently neutralizing antibodies, as well as specific epitopes (EDEs) to which they bind. Therefore, the use of these epitopes in vaccines is likely to be more preferable than current vaccine strategies.

[0198] Therefore, the present invention provides a method for selecting a suitable antigen for a vaccine against dengue virus, the method comprising characterizing one or more antibodies produced in a subject responsive to the antigen, and optionally, the antigen has been found to bind to a panel of antibodies known to bind to an envelope dimeric epitope as defined in any of the embodiments described above.

[0199] The identification of highly cross-reactive and potently neutralizing antibodies in subjects administered with dengue antigens indicates antigens that are likely to be useful for vaccines. In one embodiment, this antigen is not presented as part of an intact virus. In a preferred embodiment, this antigen is an EDE compound as defined in any of the above embodiments, optionally as part of a scaffold protein, preferably a dimer of envelope protein, preferably a stabilized dimer. In a preferred embodiment, this antigen is one that has already been determined to be capable of binding to a highly cross-reactive and potently neutralizing antibody capable of binding to EDE, for example as identified in the Examples, such as the antibody of the present invention.

[0200] By administering such an antigen known to be capable of binding to highly useful antibodies, the antibodies generated in response to the antigen in the subject can be characterized. Such antigens result in the production of such useful antibodies within the subject, and thus are likely to be suitable candidate antigens for use in vaccine compositions. Characterizing includes the meaning of determining whether these antibodies are considered to bind to the fusion loop, for example by determining the ability of the antibodies to bind to linear or denatured or recombinant envelope proteins, for example the ability to bind to envelope proteins in Western blot or ELISA, and the ability of the antibodies to bind to the dimer of the envelope protein, or the EDE or EDE compound described above in the preceding embodiments. The ability of the antibodies to bind to all four serotypes of dengue virus can also be evaluated, such that the ability of the antibodies to neutralize all four dengue viruses can be assessed. Methods for determining the neutralizing ability of antibodies have been described above. The ability of the antibodies to neutralize dengue virus produced in both human cells and insect cells can also be determined as described above and in the Examples.

[0201] In one embodiment, an antigen is not considered useful as a vaccine if it predominantly produces anti-FL antibodies. For example, an antigen is considered useful when the ratio of antibodies raised against FL to antibodies raised against EDE is no more than 1:2, 1:4, 1:5, 1:10, 1:50, 1:100, 1:500, or 1:1000. The relative amounts of anti-FL antibodies and anti-EDE antibodies can be determined by methods known to those skilled in the art, for example, by using techniques based on ELISA. An antigen is considered useful if it produces antibodies capable of binding to the EDE of more than one serotype of dengue virus, preferably all four dengue virus serotypes. An antigen is considered useful if it produces antibodies capable of neutralizing more than one serotype of dengue virus, preferably capable of neutralizing all four dengue virus serotypes, preferably 100%. Furthermore, an antigen is considered useful if it produces antibodies capable of neutralizing dengue virus produced in both human cells and insect cells, preferably to the same level (as discussed above), preferably neutralizing at least 95% or at least 98%, for example 100% of the virus. The antigen is, a) does not produce, or does not significantly produce anti-FL antibodies, and b) binds to all four serotypes of dengue virus to a fairly significant extent, and c) most considered useful when it 100% neutralizes all four serotypes of dengue virus produced in both human cells and insect cells to a fairly significant extent.

[0202] Furthermore, in another embodiment, this antigen is considered suitable for use in vaccination if the produced antibodies are capable of binding to the EDE as defined in the above embodiments.

[0203] In a further embodiment, the antigen administered to a subject may comprise an additional agent to help prevent antibodies from being generated against the fusion loop.

[0204] Antibodies produced by a subject exposed to an antigen may be obtained from a single, classified plasma cell of the subject.

[0205] The identification of antibodies that are new to or highly cross-reactive and potently neutralize the dengue virus will be understood to provide a unique opportunity to treat or prevent this viral disease. In addition, since there has been no reliable method to treat infections that occur during testing until the present invention, it will be possible to conduct clinical trials involving live dengue virus. Therefore, further aspects of the present invention provide a method for treating or preventing dengue virus infection in a subject.

[0206] This method comprises the administration of one or more compounds according to the present invention, preferably polypeptides, preferably antibodies or fragments thereof. The present invention also provides one or more compounds according to the present invention, preferably polypeptides, preferably antibodies or fragments thereof, for use in the prevention or treatment of dengue fever virus infection. The present invention also provides the use of the compounds of the present invention in the manufacture of agents for the treatment or prevention of dengue fever infection.

[0207] With regard to administration, it will be understood that the compounds of the present invention may be part of a composition, for example, a pharmaceutical composition. This composition may further include one or more therapeutic agents that are considered useful in themselves for treating an infection, for example, an additional antiviral agent, or one or more agents that are considered useful in treating symptoms of dengue fever infection.

[0208] The term "treat" includes administering any of the compounds of the present invention, such as the compounds of the present invention, EDE compounds, vaccine compositions, antibodies, stabilized recombinant sE dimers, or immunogenic compositions of the present invention, to a patient having dengue virus infection or symptoms of dengue virus infection, for the purpose of curing, resolving, alleviating, reducing, altering, relieving, improving, enhancing, or influencing dengue virus infection and / or symptoms of dengue infection. Treating any one or more of the symptoms of dengue infection includes alleviating. Treatment also includes preventing new cells from infecting. Whether a patient is being treated well will be obvious to those skilled in the art. For example, the viral load may decrease.

[0209] The term "prevent" means that the progression of dengue virus infection is slowed and / or eliminated, or that the onset of dengue virus infection is delayed or eliminated.

[0210] Dengue virus infection and the symptoms of dengue fever are presented, for example, in WHO Fact Sheet 117. As described therein, dengue fever is a severe influenza-like illness that affects infants, toddlers, and adults, but is rarely fatal. Dengue fever should be suspected when high fever (40°C / 104°F) is accompanied by two or more of the following symptoms: severe headache, pain behind the eyes, muscle and joint pain, nausea, vomiting, glandular swelling, or rash. Symptoms usually last for 2 to 7 days, following an incubation period of 4 to 10 days after being bitten by an infected mosquito. Severe forms of dengue fever are potentially fatal complications, including plasma leakage, fluid retention, respiratory distress, severe bleeding, and organ failure. Dangerous signs occur 3 to 7 days after the onset of initial symptoms and include severe abdominal pain, persistent vomiting, rapid breathing, bleeding gums, malaise, restlessness, and hematemesis, along with a simultaneous decrease in body temperature (below 38°C / 100°F). Death can occur 24 to 48 hours after reaching critical condition, and appropriate medical treatment is necessary to avoid complications and the risk of death.

[0211] Administration of the compound or a composition containing the compound is an amount, such as a therapeutically effective dose, that results in the inhibition of cell infection when the compound is used prophylactically, or the inhibition of further cell infection when used therapeutically, and / or the reduction of signs and / or symptoms of the disease.

[0212] The therapeutically effective dose is the amount that results in a subjective reduction of other symptoms or an objectively identifiable improvement as noted by a clinician or qualified observer.

[0213] Preventing dengue fever infection means reducing the level of infection to any significant degree. In one embodiment, the compounds of the present invention prevent infection by 30%, 50%, 70%, 80%, 90%, 95%, preferably 100%, from one serotype of dengue virus. In a preferred embodiment, the compounds of the present invention prevent infection by 30%, 50%, 70%, 80%, 90%, 95%, preferably 100%, from two serotypes of dengue virus, three serotypes of dengue virus, or all four serotypes of dengue virus. In the most preferred embodiment, the compounds of the present invention completely prevent infection by all four serotypes of dengue virus. This may be evaluated by techniques known to those skilled in the art, for example, by measuring the viral load.

[0214] The present invention provides the use of EDE, preferably a stabilized recombinant sE dimer, or an immunogenic composition according to the present invention, for conferring immunity to animals (non-human), preferably mammals, such as monkeys, rabbits, mice, or camelids (e.g., llamas).

[0215] Further embodiments provide one or more compounds, preferably polypeptides, preferably antibodies or fragments thereof, according to the present invention for use in a live dengue vaccine test, for example, with the intention of terminating infection.

[0216] Preferably, the compounds of the present invention can neutralize at least 95% or at least 98%, for example 100%, all four serotypes of dengue virus produced in both insect and human cells. Pre-administration of the compounds before exposure to the virus is considered to prevent viral infection.

[0217] For example, a compound according to the present invention, such as an antibody or fragment thereof, that can neutralize all four serotypes of dengue virus described above, may be administered before exposure to the virus, for example, as a preventative measure in the vicinity of travelers or in the home of people who are likely to have been bitten by mosquitoes, in the vicinity of or in the home of a large outbreak or an outbreak or an outbreak of one or more infected people. Alternatively, the compound may be administered when the patient first develops a fever or when the symptoms become severe.

[0218] All preferences for this compound are as described above in the embodiments of the present invention.

[0219] It will be understood that the compounds of the present invention, for example, antibodies or their antigen-binding moieties, may be administered together with further therapeutic agents, such as one or more T-cell vaccines or other antiviral agents. These may be administered as part of the same composition as the compounds of the present invention, or separately. For example, T-cell vaccines have been proposed for protection against influenza. 85 .

[0220] The compounds of the present invention may be administered once, twice, or several times. Administration may be carried out over a period of one day, two days, one week, two weeks, one month, six months, one year or longer. For post-infection treatment, a shorter period, for example, up to one month, may be appropriate. For prevention, a longer period, for example, six months or more out of one year, may be appropriate.

[0221] The present compounds, such as antibodies or antigen-binding portions thereof, for use in preventing or treating dengue virus infection may be selected using the methods of the present invention. Accordingly, the present invention provides a method of selecting a suitable antibody or fragment thereof for use in preventing or treating dengue virus, which method comprises characterizing an antibody or fragment thereof produced in a subject in response to an antigen comprising an envelope dimer epitope as defined in any of the above-described embodiments.

[0222] The EDE compound according to any of the above-described embodiments is highly likely to be capable of generating a suitable antibody after administration of the EDE to a subject. Therefore, antibodies generated in such subjects are highly likely to be useful in the treatment or prevention of dengue virus infection.

[0223] In a preferred embodiment, the EDE is an EDE compound of the present invention, such as a dimer of an envelope protein, preferably a stabilized dimer, optionally as part of a scaffold protein. In a preferred embodiment, the antigen / EDE compound is a highly cross-reactive and potently neutralizing antibody capable of binding to EDE, for example, as is already well known to be capable of binding to the antibodies of the present invention. In a preferred embodiment, this antigen is considered to be improved over the native envelope dimer, for example, by comprising residues in the specific arrangement required to generate cross-reactive and potentially neutralizing anti-EDE antibodies, but not comprising residues that generate anti-FL antibodies or residues of a specific conformation.

[0224] In another embodiment, the subject is administered, for example, a compound or agent that blocks the formation of anti-FL antibodies, similarly to the administration of EDE, optionally an EDE compound. For example, a stabilized sE dimer may be useful.

[0225] Characterization means determining whether these antibodies are considered to bind to the fusion loop by determining, for example, the ability of the antibody to bind to linear, denatured, or recombinant envelope proteins, such as the ability to bind to envelope proteins in Western blotting or ELISA, and the ability of the antibody to bind to envelope protein dimers, or, in the embodiments described above, to bind to the EDE or EDE compounds. The ability of antibodies to bind to all four serotypes of dengue virus can also be evaluated so as to the ability of antibodies to neutralize all four serotypes of dengue virus. Methods for determining the neutralizing ability of antibodies are described in detail above and in the examples. The ability of antibodies to neutralize dengue virus produced in both human and insect cells can also be evaluated.

[0226] In one embodiment, the antibody is not considered useful if it binds to FL. The antibody is considered useful if it can bind to one or more serotypes of dengue virus, preferably four serotypes, or if it can bind to one or more serotypes of EDE as described in any of the embodiments above. The antibody is considered useful if it can neutralize one or more serotypes of dengue virus, optionally two serotypes, or optionally three serotypes, preferably four serotypes, preferably at least 95% or at least 98%, for example 100%. Furthermore, the antibody is considered useful if it can neutralize dengue virus produced in both human cells, optionally dendritic cells, and insect cells, optionally C6 / 36 cells, preferably to the same level, preferably at least 95% or at least 98%, for example 100% of the virus. This antibody, a) If anti-FL antibodies are not produced, or are not produced in significant quantities, and b) When binding to all four serotypes of dengue virus is significant, and c) It is considered most useful when it neutralizes all four serotypes of dengue virus produced in both human and insect cells to a fairly significant degree, reaching 100%.

[0227] The inventors have found that patients suffering from dengue fever infection produce either useful anti-EDE antibodies or useless anti-FL antibodies. Therefore, a further method for identifying antibodies that may be useful for treating or preventing dengue fever infection is simply to identify those antibodies that cannot bind to their denatured or linear envelope proteins. Any antibodies that cannot bind to these envelope proteins are likely to be useful compounds of the present invention.

[0228] Patients should also understand that they may be treated with nucleic acids, vectors, or host cells expressing polypeptides, preferably antibodies or their antigen-binding moieties. For example, nucleic acids containing polypeptides may be inserted into a suitable delivery system, such as a viral vector, e.g., adenovirus, adeno-associated virus, cytomegalovirus, herpesvirus, poliovirus, retrovirus, Sindbisvirus, vacciniavirus, or any other DNA or RNA viral vector, so that the compounds of the present invention are endogenously expressed within the patient being treated.

[0229] The present invention also provides a method for stratifying patients according to their estimated need to receive therapeutic or prophylactic treatment with one or more of the compounds of the present invention. Accordingly, the Specified provides a method for identifying patients suffering from dengue virus infection who are likely to require treatment with the compound or composition, or an increasing dose of the compound or composition, according to any one of the embodiments described above, the method comprising determining the levels of anti-enveloped dimer epitope antibodies and anti-fusion loop antibodies in the subject, wherein the enveloped dimer epitope is as described in any of the embodiments described above.

[0230] As identified by the inventors, patients infected with dengue fever primarily produce either anti-EDE antibodies or anti-FL antibodies. Anti-FL antibodies are not considered useful, while anti-EDE antibodies are. If a subject has anti-EDE antibodies but may still require some further treatment with the compounds of the present invention, subjects primarily with anti-FL antibodies are likely to require higher doses because they do not have a congenitally useful antibody. Therefore, patients with only anti-FL antibodies are considered likely to require treatment with the compounds of the present invention. Patients who already produce anti-EDE antibodies may not require treatment. In addition, patients who do not produce anti-EDE antibodies but only anti-FL antibodies are likely to require higher doses of treatment than patients with anti-EDE antibodies. Furthermore, patients may produce only low levels of anti-EDE antibodies and therefore may require higher doses of the compound.

[0231] A higher dose means that this patient requires 2, 3, 4, 5, 10, 20, or 50 times the dose of the compound of the present invention compared to a patient producing anti-EDE antibodies.

[0232] "Producing low levels of anti-EDE antibodies" means that this patient has lower-than-average levels of anti-EDE antibodies compared to other patients who produce anti-EDE antibodies.

[0233] For example, means for determining whether these antibodies bind to EDE, such as determining whether an antibody binds to intact dengue virus or EDE but does not denature or bind to the linear envelope protein, are as described above and in the examples. The ability of an antibody to bind to a protein can be evaluated when the envelope protein is manipulated to have the stability of an increased dimer, or when the envelope protein or its residues are presented as part of the backbone.

[0234] The levels of anti-FL antibodies and anti-EDE antibodies within a subject can also be used to assess the subject's need for dengue virus vaccination. Therefore, in further embodiments, a method is provided for assessing a patient's need for dengue virus vaccination, the method comprising identifying levels of anti-enveloped dimer epitope antibodies and anti-fusion loop antibodies, the enveloped dimer epitope being as described in any of the embodiments described above. Similar to the criteria for patients requiring treatment with the compound of the present invention, or higher doses thereof, if a patient is determined to have anti-enveloped dimer epitope antibodies, vaccination is likely not required.

[0235] Furthermore, if the patient is determined to have anti-enveloped dimer epitope antibodies, a boost dose may be administered to this patient.

[0236] In another embodiment, if the patient does not have anti-enveloped dimer epitope antibodies, total vaccination is required.

[0237] The present invention also provides the use of the stabilized recombinant sE dimer (used as an antigen) defined above for preparing prophylactic or therapeutic immunogenic (or vaccine) compositions for the purpose of preventing and / or treating dengue virus infection in susceptible mammalian subjects, such as humans.

[0238] Significantly, the inventors have identified, for the first time, a specific epitope recognized by a highly cross-reactive and potently neutralizing antibody that was previously unknown, as described above. This epitope is considered to provide a particularly effective antigen in dengue virus vaccination. A method for selecting a suitable antigen for use in dengue virus vaccination is described in the embodiments described above. Accordingly, the present invention provides compositions presenting an envelope dimer epitope of dengue virus, optionally an EDE compound, for use in preparing prophylactic or therapeutic immunogenic (or vaccine) compositions for the purpose of preventing and / or treating dengue virus infection in susceptible mammalian subjects, e.g., humans, the envelope dimer epitope and the EDE compound as defined in any of the embodiments described above and identified according to the methods described above, for example, EDE or the EDE compound may be identified, for example, by characterizing the antibody produced after administration of a potential vaccine candidate EDE / EDE compound to a subject, in the embodiments described above which a method for selecting a suitable antigen for use in a vaccine is presented. This may be sufficient within the authority of those skilled in the art. Alternatively, EDE or the EDE compound may be as presented in the embodiments described above, for example, in one embodiment, EDE or the EDE compound is a dimer of an envelope protein, or the outer domain of an envelope protein, or the (approximately) 400 amino-terminal residues of the outer domain of an envelope protein. The envelope protein may be any of the envelope proteins from DENV-1, DENV-2, DENV-3 and DENV-4, (SEQ ID NOs. 29, 31, 33, or 35), or any protein having at least 90% homology to the sequence of SEQ ID NOs. 29, 31, 33, or 35. This dimer may be a homodimer or a heterodimer. In a preferred embodiment, this dimer is not incorporated into intact virus particles, subvirus particles, or virus-like particles, but rather is a free dimer.For example, it will be understood that any form of EDE or EDE compound described herein, such as an engineered envelope protein, may be presented as part of a virus, virus-like particle, or subviral particle, as part of a protein backbone. In preferred embodiments, the EDE compound is a stabilized recombinant sE dimer, as described in the embodiments described above.

[0239] In yet another embodiment, EDE or EDE compounds for use in vaccine compositions present improved epitopes that surpass naturally occurring envelope dimers. For example, the EDE / EDE compound is engineered so that the FL cannot be recognized by itself by a compound, such as a polypeptide, such as an antibody, or has an insertion into its backbone, for example, the EDE / EDE compound is engineered so that the FL cannot be recognized by an antibody in isolation from the direct adjacency of the fusion loop, i.e., the induction loop cannot be recognized in a context independent of quaternary tissue.

[0240] In another embodiment, EDE or an EDE compound for use in a vaccine composition comprises an envelope protein dimer, or an envelope outer domain, or about 400 amino-terminal residues of the envelope protein outer domain, which are manipulated to have increased stability in the dimeric configuration, for example, to have increased levels of covalent and / or non-covalent bonding between the dimers, or are incorporated into a heterogeneous protein backbone that preserves the dimeric configuration by increasing the levels of covalent and / or non-covalent bonding between the dimers, for example, as described above. Furthermore, the EDE / EDE compound may include a heterologous protein backbone that can be presented only on a portion of the (approximately) 400 amino-terminal residues of the envelope protein dimer, envelope outer domain, or outer domain of the envelope protein, and this portion may be a continuous portion of the (approximately) 400 amino-terminal residues of the envelope protein dimer, envelope outer domain, or outer domain of the envelope protein, or this portion may include a selection of discontinuous residues of the (approximately) 400 amino-terminal residues of the envelope protein dimer, envelope outer domain, or outer domain of the envelope protein as described above.

[0241] For example, in one embodiment, the EDE / EDE compound is located at positions E49, K64, Q77, W101, V122, N134, N153, T155, I161, A162, P169, T200, K202, E203, L308, K310, Q323, W391, F392, A71, C105, C74, D1 54, D249, D271, D309, D362, D98, E148, E311, E44, E71, E84, G102, G104G106, G15 2, G156, G28, G29, G374, H158, H27, I113, I308, I46, K246, K247, K310, K323, K325 One or more of the following residues are included: K47, L113, L45, L82, M278, N103, N153, N362, N67, N83, Q248, Q271, Q325, Q77, R2, R247, R323, R73, R99, S72, S81, T115, T155, T361, T46, T68, T69, T70, T72, V113, V114, V250, V309, V324, V97, W101, or the equivalent residues of the dengue virus envelope polypeptide in substantially similar spatial arrangement to adopt the native dimer of the envelope protein, or the envelope outer domain, or (approximately) 400 amino-terminal residues of the outer domain of the envelope protein. These residues may be present in naturally occurring envelope proteins, or, in alternative embodiments, they may be retained within the backbone protein in appropriate configuration. In preferred embodiments, the EDE / EDE compound comprises the aforementioned position W101 and at least one other residue. In more preferred embodiments, the EDE / EDE compound comprises all of the above residues. In one embodiment, the EDE / EDE compound comprises the N153 glycan. In an alternative embodiment, the EDE / EDE compound does not contain the N153 glycan.

[0242] In a more preferred embodiment, the EDE / EDE compound for use in the vaccine composition comprises residues observed both in terms of amino acids and spatial position across one or more serotypes of dengue virus, preferably residues observed both in terms of amino acids and spatial position across all serotypes of dengue virus, i.e., four serotypes of dengue virus.

[0243] The EDE / EDE compound may contain a dimer of the envelope protein, or the outer domain of the envelope protein, or approximately 400 amino-terminal residues of the outer domain of the envelope protein, which have increased stability in the dimeric configuration and are manipulated to be retained within the protein backbone as described above.

[0244] The inventors have found that a specific region of the envelope dimer is important for contact with the compound of the present invention, such as an antibody or its antigen-binding moiety. Therefore, in some embodiments, the EDE / EDE compound comprises a dimer of the envelope protein, or the envelope outer domain, or a specific antigenic moiety of approximately 400 amino-terminal residues of the outer domain of the envelope protein. The EDE / EDE compound for use in the vaccine composition may be a dimer of an envelope protein, or an envelope outer domain, or a specific fragment containing specific residues of the (approximately) 400 amino-terminal residues of the envelope protein outer domain, including a region considered necessary for antigenicity. This fragment may also be manipulated to maintain a specific three-dimensional structure, or retained within a protein backbone, or manipulated and retained within a protein backbone.

[0245] For example, in one embodiment, the EDE / EDE compound for use in a vaccine composition includes a region located in the center of a depression formed by the b strand on the domain II side and the “150 loop” on the domain I side (opposite the dimer interface), where the 150 loop extends to residues 148-159, connecting the b strands E0 and F0 of domain I and supporting the N153 glycan, which covers the fusion loop of the partner subunit in the dimer. In one embodiment, this region includes three polypeptide segments of domain II of the reference subunit, which are defined as subunits contributing to the FL to the epitope. These three segments are the b strand (residues 67-74 supporting the N67 glycan), the fusion loop and residues immediately upstream (residues 97-106), and the ij loop (residues 246-249).

[0246] In another embodiment, in addition to the region described above (the region containing the three polypeptide segments of domain II of the reference subunit), the EDE / EDE compound further comprises 150 loops and the N153 glycan chain of the second subunit.

[0247] Further embodiments of the EDE / EDE compounds include the aforementioned region (the region containing the three polypeptide segments of domain II of the reference subunit) at a specific residue K310, as well as the 150 loop and the A strand of domain III of the second subunit. The inventors have found that the 150 loop of the second subunit interferes with the binding of the subunits of the useful compounds defined herein to VDE. Therefore, in one embodiment, the 150 loop may be in a native configuration found in the native dimer of the envelope protein, or in the envelope outer domain, or at approximately 400 amino-terminal residues of the outer domain of the envelope protein, or in another embodiment, the 150 loop may be in an irregular configuration adopted when the 150 loop binds to one of the compounds of the present invention.

[0248] N67 glycan is considered particularly important for dengue infection of dendritic cells. Therefore, as described herein, EDE / EDE compounds containing this residue in the correct epitope environment are considered preferred embodiments.

[0249] In preferred embodiments, once administered to a subject, preferably a human, the EDE / EDE compound produces antibodies that preferably can bind to all four serotypes of dengue virus, optionally neutralize all four serotypes of dengue virus, preferably neutralize 100% of all four serotypes of dengue virus, optionally neutralize viruses produced in both human and insect cells, preferably neutralize 100% of all four serotypes of dengue virus produced in both human and insect cells.

[0250] The immunogenic composition containing EDE, which includes the stabilized recombinant sE dimer described above, is used in the subject. - Recognizes the envelope dimeric epitope (EDE) independently (does not show binding to recombinant E protein monomers in ELISA). - There is cross-reactivity, - Particularly suitable for inducing neutralizing antibodies that neutralize dengue fever virus from four serotypes (DENV1-4).

[0251] The present invention also provides a dengue virus immunogenic composition comprising an effective amount of the stabilized recombinant sE dimer (used as an antigen) as defined above.

[0252] The composition may include the EDE / EDE compound itself, or it may include means for expressing the EDE / EDE compound within a subject to be vaccinated. For example, the present invention includes a nucleic acid encoding an envelope dimer epitope or an EDE compound for use in vaccination against dengue virus infection, the envelope dimer epitope or EDE compound being as described in any of the embodiments described above. Furthermore, the nucleic acid may be part of a vector. Preferences for vectors and vector components are as described above.

[0253] For example, it is known in the art that vaccination can be carried out using nucleic acids encoding a specific antigen, for example, via direct immunization by plasmid DNA. Such nucleic acids can be delivered via liposomes and immunostimulatory constructs. Alternatively, attenuated viral hosts or vectors or bacterial vectors may be used, for example, adenoviruses, adeno-associated viruses, cytomegaloviruses, herpesviruses, polioviruses, retroviruses, Sindbisviruses, vaccinia viruses, or any other DNA or RNA viral vectors.

[0254] When the composition for use in vaccination against dengue virus infection is nucleic acid, the nucleic acid can be delivered to the patient in the form of a viral vector, such as an adenovirus, adeno-associated virus, cytomegalovirus, herpesvirus, poliovirus, retrovirus, Sindbisvirus, vacciniavirus, or any other DNA or RNA viral vector.

[0255] For use in vaccination against dengue fever virus infection, a) Envelope dimer epitope or EDE compound, b) Nucleic acids encoding EDE or EDE compounds, c) A vector containing nucleic acids, A composition comprising one or more of the above is also part of the present invention if the envelope dimer epitope or EDE compound is as described in any of the embodiments described above.

[0256] In one embodiment, a) Envelope dimer epitope or EDE compound, b) Nucleic acids encoding EDE or EDE compounds, c) A vector containing nucleic acids, This refers to or codes for dengue virus of any more than one, any two, any three, or any four serotypes.

[0257] In a preferred embodiment, a) Envelope dimer epitope or EDE compound, b) Nucleic acids encoding EDE or EDE compounds, c) A vector containing nucleic acids, This is a single epitope capable of producing antibodies that neutralize all four serotypes of dengue virus, preferably antibodies that can neutralize all four serotypes of dengue virus 100%, or results in the production of a single epitope.

[0258] The use of the composition of the present invention in vaccination against dengue virus is intended to reduce or prevent infection by dengue virus.

[0259] Reducing or preventing dengue fever infection means reducing the level of infection to any degree. In one embodiment, the compounds of the present invention reduce infection by 30%, 50%, 70%, 80%, 90%, 95%, preferably 100%, from one serotype of dengue virus. In a preferred embodiment, the compounds of the present invention reduce infection by 30%, 50%, 70%, 80%, 90%, 95%, preferably 100%, from two serotypes of dengue virus, three serotypes of dengue virus, or all four serotypes of dengue virus. In the most preferred embodiment, the compounds of the present invention completely prevent infection by all four serotypes of dengue virus.

[0260] The present invention, which induces neutralizing antibodies against dengue virus infection, includes EDE or EDE compounds, such as stabilized recombinant sE dimers, which are administered to mammalian subjects, preferably humans, in amounts sufficient to prevent or attenuate the severity of dengue virus infection over the course of the infection period.

[0261] The therapeutically effective dose varies depending on other factors, including the subject being treated, the subject's age and general condition, the capacity of the subject's immune response to synthesize antibodies, the desired degree of protection, the severity of the condition being treated, the specific VDE compound, e.g., a selected specific stabilized recombinant sE dimer, and the mode of administration. An appropriate effective dose can be readily determined by those skilled in the art. The therapeutically effective dose will likely fall within a relatively broad range, which can be determined through periodic testing.

[0262] More specifically, the EDE compound, for example, the stabilized recombinant sE dimer of the present invention, is administered in a therapeutically effective dose containing 1 to 1000 μg of dimer, preferably 1 to 50 μg.

[0263] The optimal dose for a particular vaccine can be determined by standard studies that include measuring the antibody titer of the anti-sE dimer in the target population.

[0264] The immunogenic composition of the present invention may be administered with or without an adjuvant. The adjuvant may be added directly to the immunogenic composition or administered separately at the same time as or immediately after administration of the vaccine. Examples of such adjuvants include, but are not limited to, detoxified mutants of bacterial toxins such as aluminum salts (aluminum hydroxide), oil-in-water emulsion formulations with or without specific stimulants such as muramil peptide, saponin adjuvants, cytokines, cholera toxin, pertussis toxin, or Escherichia coli thermolucious toxin.

[0265] The immunogenic composition of the present invention may be administered together with other immunogens or immunomodulators, such as immunoglobulins, cytokines, lymphokines, and chemokines.

[0266] Vaccination programs often include boost strategies. After initial vaccination, subjects may receive one or two booster injections at appropriate intervals determined by those skilled in the art. In one embodiment, vaccination may include a prime, followed by one or two boosts. Antigens, compositions, nucleic acids, or vectors that result in the expression of an antigen are included in the present invention for use in boost strategies for vaccination against dengue virus infection, and optionally, this antigen, compound, nucleic acid, vector, or composition is for (prime) pre-administration or (boost) post-administration of dengue virus, optionally attenuated dengue virus, and / or dengue virus-like particles, and this dengue virus or dengue virus-like particle may be a group of dengue viruses of one or more serotypes, and may include or present EDE, e.g., the non-natural EDE or EDE compounds described above. As a further example, heterologous flaviviruses such as kimmeribax associated with yellow fever may be used, for example, by one or more of dimers, DNA, vaccinia, and adenoviruses, and as will be apparent to those skilled in the art, different orders and timings of administration of different antigens and / or nucleic acids encoding the antigens may be possible, and the present invention is not limited to any particular combination or order of administration.

[0267] The present invention also includes a vaccination strategy for providing protection against the dengue virus, which, for example, a) A single dose of an envelope dimer epitope or EDE compound capable of producing antibodies against all four serotypes as described in any of the embodiments described above, or a vaccine composition according to the embodiments described above, or a nucleic acid for use in vaccination, or a vector for use in vaccination, optionally followed by administration of attenuated dengue virus, or b) Administration of two envelope dimer epitopes or EDE compounds from two serotypes as described in any of the embodiments described above, followed by administration of envelope dimer epitopes or EDE compounds from two other serotypes, optionally followed by administration of attenuated dengue virus, or c) Administration of a weakened dengue virus, followed by administration of an envelope dimer epitope capable of producing antibodies against all four serotypes, as described in any of the embodiments described above, or d) The administration of a weakened dengue virus, followed by the administration of two envelope dimer epitopes or EDE compounds from two serotypes as described in any of the embodiments described above, followed by the administration of two other envelope dimer epitopes or EDE compounds from two other serotypes.

[0268] It is also conceivable that patients who have been vaccinated according to the present invention may still require subsequent treatment with compounds or compositions according to the present invention for use in treating or preventing dengue fever infection.

[0269] Therefore, the compounds of the present invention are intended for use in treating or preventing dengue fever infection in patients who have previously received dengue fever vaccination or in patients who have not previously received dengue fever vaccination.

[0270] Since this vaccination is considered to provide protection against more than one serotype of dengue virus, preferably protection against all four serotypes, vaccination is still considered useful if the patient has already contracted dengue fever; however, the vaccine is preferably administered before the onset of symptoms of dengue fever or before the patient is known to have contracted dengue fever.

[0271] Therefore, this vaccine is intended for patients who have never been infected with dengue fever, and for patients who are not currently infected with dengue fever and are not infected at the time of vaccination. Alternatively, this vaccine is intended for patients who have been infected with one or more serotypes of dengue fever in the past, but are not infected at the time of vaccination, or for patients who have been infected with one or more serotypes of dengue fever in the past, are not currently infected with one or more serotypes of dengue fever, and are not infected at the time of vaccination.

[0272] This vaccine is also intended for use in patients who have previously been treated with the compounds of the present invention but are not currently being treated with them, and it is also intended for use in patients who have previously been treated with the compounds of the present invention and are currently being treated with them. This vaccine is also intended for use in patients who are being treated with the compounds of the present invention for the first time.

[0273] The present invention also provides EDE compounds, such as stabilized recombinant sE dimers or immunogenic compositions, as defined above, for pharmaceutical use, preferably for the prevention and / or treatment of dengue virus infection.

[0274] The present invention also provides EDE compounds, such as stabilized recombinant sE dimers or immunogenic compositions, as defined above, for the manufacture of a drug, preferably a vaccine for the prevention or treatment of dengue virus infection in a subject.

[0275] The present invention also provides a method for preventing and / or treating dengue virus infection, the method comprising administering to a subject in need of prevention and / or treatment an EDE compound, such as a stabilized recombinant sE dimer or immunogenic composition, as defined above, in an amount effective in inhibiting dengue virus infection of susceptible cells, thereby preventing or treating the infection.

[0276] The present invention also provides diagnostic agents comprising or comprising the EDE compounds of the present invention, such as stabilized recombinant sE dimers, or compounds of the present invention, such as antibodies or fragments thereof according to the present invention.

[0277] In one embodiment of the diagnostic agent, the compound, for example, an antibody or fragment thereof according to the present invention, is directly or indirectly bound to a detectable marker, either covalently or noncovalently.

[0278] A detectable marker may be directly and covalently bound to the compound, for example, an antibody or a fragment thereof, to one of its ends (N or C terminus) or to one of its amino acid side chains. The detectable marker may also be directly and covalently bound to the antibody or fragment via a connecting arm (i.e., a crosslinking reagent) to one of its ends or to one of its amino acid side chains. Methods for binding target compounds to peptides or antibodies are known in the art.

[0279] Advantageously, the detectable marker is - Enzymes such as horseradish peroxidase, alkaline phosphatase, glucose-6-phosphatase, or beta-galactosidase, - Fluorescent dye molecules such as green fluorescent protein (GFP), blue fluorescent dyes excited by wavelengths in the ultraviolet (UV) part of the spectrum (e.g., AMCA (7-amino-4-methylcoumarin-3-acetic acid), Alexa Fluor 350), green fluorescent dyes excited by blue light (e.g., FITC, Cy2, Alexa Fluor 488), red fluorescent dyes excited by green light (e.g., rhodamine, Texas Red, Cy3, Alexa Fluor dyes 546, 564, and 594), or dyes excited by far-infrared light visualized by an electron detector (CCD camera, photomultiplier tube) (e.g., Cy5), - Heavy metal chelates such as Europium, Lanthanum, or Yttrium, - [ 18 F]Fluorodeoxyglucose, 11 C-, 125 I-, 131 I-, 3 H-, 14 C-, 35 S, or 99 It is selected from the group consisting of radioactive isotopes such as Tc-labeled compounds.

[0280] The present invention also provides the use of EDE compounds, such as stabilized recombinant sE dimers, antibodies or fragments thereof, or diagnostic agents according to the present invention, for diagnosing or observing dengue virus infection in a subject.

[0281] The present invention also provides an in vitro method for diagnosing dengue virus infection in a subject, the method being: a) A step of in vitro contacting a suitable biological sample from the subject with an antibody or a fragment thereof, or a diagnostic agent containing or consisting of an antibody or a fragment thereof according to the present invention, b) The step of determining the presence or absence of dengue virus envelope glycoprotein E in a biological sample, The presence of dengue virus envelope glycoprotein E indicates that the subject has been infected with the dengue virus.

[0282] Step b) may be performed by determining the presence or absence of an antibody-antigen complex (i.e., an antibody directed towards the dengue virus envelope glycoprotein E-dengue virus envelope glycoprotein E complex).

[0283] The present invention also provides an in vitro method for determining the presence of dengue virus envelope glycoprotein E in a suitable biological sample from a subject, and this method is a) The step of in vitro contacting the appropriate biological sample from the subject with an antibody or a fragment thereof, or a diagnostic agent containing or consisting of an antibody or a fragment thereof according to the present invention, b) The step of determining the presence or absence of dengue virus envelope glycoprotein E in the biological sample.

[0284] The present invention also provides an in vitro method for diagnosing dengue virus infection in a subject, the method being: a) A step of contacting a suitable biological sample from the subject in vitro with a stabilized recombinant sE dimer according to the present invention, b) A step of determining the presence or absence of an antibody targeting a dimer in a biological sample, The presence of antibodies indicates that the subject has been infected with the dengue virus.

[0285] The present invention also provides an in vitro method for determining the presence of an antibody targeting dengue virus envelope glycoprotein E in a suitable biological sample from a subject, the method being: a) The step of bringing the appropriate biological sample from the subject in vitro with a stabilized recombinant sE dimer according to the present invention, b) The step of determining the presence or absence of an antibody targeting the dimer in the biological sample.

[0286] The present invention also provides an in vitro method for observing the progression or recurrence of dengue virus infection in a subject, and this method is a) A step of in vitro contacting a suitable biological sample from the subject with an antibody or a fragment thereof, or a diagnostic agent containing or consisting of an antibody or a fragment thereof according to the present invention, b) A step of determining the amount of dengue virus envelope glycoprotein E in a biological sample, c) The step of comparing the amount determined in step (b) with the amount of dengue virus envelope glycoprotein E previously obtained in the subject, A significant increase in the amount of dengue virus envelope glycoprotein E constitutes a marker of progression of dengue virus infection, while a significant decrease in dengue virus envelope glycoprotein E constitutes a marker of regression of dengue virus infection.

[0287] As used herein, the terms “significant increase” and “significant decrease” refer to a higher or lower amount of dengue virus envelope glycoprotein E in a suitable biological sample, respectively, compared to the amount of dengue virus envelope glycoprotein E in the suitable biological sample from which the amount has already been determined and used as a reference amount.

[0288] Step b) may be performed by determining the presence or absence of an antibody-antigen complex (i.e., an antibody directed towards the dengue virus envelope glycoprotein E-dengue virus envelope glycoprotein E complex).

[0289] The present invention also provides an in vitro method for predicting a favorable prognosis of exacerbation of dengue virus infection in a subject, and this method is a) A step of contacting a suitable biological sample from the subject in vitro with a stabilized recombinant sE dimer according to the present invention, b) A step of determining the amount of antibody that neutralizes the dimer in the biological sample, c) The step of comparing the amount determined in step (b) with the amount of antibody to the target that was previously obtained as a dimer in the target, A significant increase in the amount of antibody neutralizing the dimer in question constitutes a marker of a favorable prognosis for exacerbation of dengue virus infection.

[0290] The present invention also provides an in vitro method for observing the success of a dengue virus vaccination protocol for dengue virus infection in subjects who have received a dengue virus prophylactic injection, and this method is a) A step of contacting a suitable biological sample from the subject in vitro with a stabilized recombinant sE dimer according to the present invention, b) A step of determining the amount of antibody that neutralizes the dimer in the biological sample, c) The step of comparing the amount determined in step (b) with the amount of antibody to the target that was previously obtained as a dimer in the target, A significant increase in the amount of antibody neutralizing the dimer constitutes a marker of success for the vaccination protocol.

[0291] The appropriate biological sample may be blood, serum, urine, or liver biopsy, preferably blood.

[0292] Immunological methods for detecting and determining the amount of proteins or antibodies are known in the art. For example, EIA, ELISA, RIA, or immunofluorescence assays can be used.

[0293] The present invention also provides isolated polynucleotides encoding the mutant sE defined above, or polypeptides of SEQ ID NOs: 28, 142, 140, 143, 144, 145, 146, or 147.

[0294] Polynucleotides according to the present invention can be obtained by known methods of recombinant DNA technology and / or chemical DNA synthesis.

[0295] The present invention also provides kits of several components. One embodiment provides a kit for diagnosing or observing dengue virus infection in a subject, comprising a stabilized recombinant sE dimer, or an antibody or fragment thereof according to the present invention, and a suitable diagnostic reagent.

[0296] Appropriate diagnostic reagents are necessary for performing assays to diagnose or observe dengue virus infection in subjects. Appropriate diagnostic reagents may include solvents, buffers, dyes, and anticoagulants.

[0297] This kit may also include a microtiter plate.

[0298] In one embodiment, the component kit includes means for identifying patients who require treatment with the compounds of the present invention or who require higher doses of the compounds of the present invention according to the embodiments described above. The kit may also provide methods for determining the presence or absence of anti-EDE antibodies and anti-FL antibodies, for example, the kit may include a microtiter plate, optionally coated with a linear or denatured envelope protein, coated with an EDE epitope according to any of the embodiments described above, and / or reagents for performing an ELISA test, optionally a colorimetric test on a rod. Preferably, the kit includes means for simply determining the presence or absence of antibodies on a solid support. The kit may also further include compounds or compositions of the present invention for use in treating or preventing dengue fever infection.

[0299] A kit of further components, including means for identifying patients requiring vaccination, is also provided. Patients are deemed to require vaccination based on the presence or absence of anti-EDE antibodies and anti-FL antibodies, as well as their levels. The kit may therefore provide a method for identifying the presence or absence of anti-EDE antibodies and anti-FL antibodies, for example, the kit may include a microtiter plate, optionally coated with a linear or denatured envelope protein, coated with an EDE epitope according to any of the embodiments described above, and / or reagents for performing an ELISA test, optionally a colorimetric test on a rod. Preferably, the kit includes means for simply identifying the presence or absence of antibodies on a solid support. The kit may also further include compositions for use in vaccination, as described in the embodiments described above.

[0300] Further kits encompass means for treating or preventing dengue fever infection and include one or more compounds of the present invention that bind to EDE, or compositions comprising compounds of the present invention that bind to EDE, and optionally include further therapeutic agents, such as further antiviral agents.

[0301] Any compound or composition or antigen or antibody referred to herein may be part of a composition. This composition may contain stabilizers such as PEG. Polypeptide components may be covalently modified or conjugated, for example, as known in the art, such as PEGylation.

[0302] Therefore, for example, any compound or antibody, or any polypeptide or antigen, or nucleic acid or vector encoding such antigen or antibody, for use in the treatment or prevention of dengue fever infection, may be conjugated to one or more further components, for example, to a reporter portion, or to one or more further therapeutic agents.

[0303] One such further therapeutic agent is one that prevents Fc receptor binding. It is well known that dengue virus causes antibody-dependent enhancement, which is thought to be due to the production of certain antibodies that bind to the virus but cannot neutralize it. This leads to the internalization of the antigen via the Fc receptor, resulting in an increased response upon reinfection. It is thought that agents that can block Fc receptor binding can prevent antibody-dependent enhancement. Examples of such agents include the compounds of the present invention for use in treating or preventing dengue infection, and are considered useful when administered together with (or separately from) antigens for use in vaccination. It may also be useful, as is well known to those skilled in the art, to modify or select antibody molecules that reduce interaction with the Fc receptor.

[0304] Any administration of any agent described herein is typically administered as part of a pharmaceutical composition together with pharmaceutically acceptable excipients, diluents, adjuvants, or carriers. Therefore, any mention of compounds, polypeptides, antibodies, their antigen-binding moieties, compositions, nucleic acids, vectors, antigens, host cells, and any mention of further therapeutic agents applies equally to pharmaceutically acceptable compositions comprising compounds, compositions, nucleic acids, vectors, antigens, host cells, and / or further therapeutic agents (e.g., formulations).

[0305] This compound, polypeptide, antibody, its antigen-binding moiety, composition, and nucleic acid may be part of a nanoparticle.

[0306] The routes of administration will be well known to those skilled in the art. For example, the agents of the present invention (compounds, polypeptides, antibodies, their antigen-binding moieties, compositions, nucleic acids, vectors, antigens, host cells, and further therapeutic agents) may be administered orally, orally, or sublingually, in the form of tablets, capsules, ovules, elixirs, solutions, or suspensions, which may contain fragrances or colorants, for immediate, delayed, or controlled-release application. The compounds of the present invention may also be administered by intravenous injection. Compound polypeptides, antibodies, their antigen-binding moieties, compositions, nucleic acids, vectors, antigens, host cells, and further therapeutic agents according to the present invention may be administered orally to mammalian subjects, preferably humans. They may also be administered to such subjects by injection, such as intravenous, intraperitoneal, intramuscular, intradermal, or subcutaneous injection.

[0307] Drugs may be administered orally or via any parenteral route in the form of a pharmaceutical agent containing the active ingredient, optionally in the form of a non-toxic organic or inorganic, acid or group, or added salt, in a pharmaceutically acceptable dosage form. Depending on the disease being treated, the patient, and the route of administration, drugs may be administered in different dosages.

[0308] Preferably, the formulation is a unit dose containing a drug or active ingredient in the form of a daily dose or unit, a daily subdose or an appropriate fragment thereof, a once-weekly dose, a once-monthly dose, or a once-six-months dose.

[0309] In human therapeutics, drugs (compounds, polypeptides, antibodies, their antigen-binding moieties, compositions, nucleic acids, vectors, antigens, host cells, and further therapeutic agents) can be administered alone, but generally, they will be administered mixed with appropriate pharmaceutical excipients, diluents, or carriers, selected in accordance with the intended route of administration and standard pharmaceutical practice.

[0310] The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium hydrogen phosphate, and glycine; disintegrants such as starch (preferably corn, potato, or tapioca starch), sodium starch glycolate, and croscarmellose sodium; certain complex silicates; granular binders such as polyvinylpyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropylcellulose (HPC), sacrose, gelatin, and acacia. Furthermore, lubricants such as magnesium stearate, stearic acid, glyceryl behenate, and talc may be included. The capsules or tablets may also be enterically coated to enhance gastric stability.

[0311] Similar types of solid compositions may also be used as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, lactose, or high molecular weight polyethylene glycol. For aqueous suspensions and / or elixirs, the compounds of the present invention may be mixed with various sweeteners or flavorings, colorants or pigments, emulsifiers and / or suspending agents, and diluents such as water, ethanol, propylene glycol, and glycerin, as well as combinations thereof.

[0312] Drugs (compounds, polypeptides, antibodies, their antigen-binding moieties, compositions, nucleic acids, vectors, antigens, host cells, further therapeutic agents, vaccines) can also be administered parenterally, for example, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly, or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of sterile aqueous solutions, which may contain other substances, such as salts or glucose in sufficient quantities to make the solution isotonic with blood. This aqueous solution must be appropriately buffered (preferably to pH 3 to 9) if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily achieved by standard pharmaceutical techniques known to those skilled in the art.

[0313] Preparations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the preparation isotonic with the blood of the recipient, as well as aqueous and non-aqueous sterile injection solutions which may contain suspending agents and thickeners. Preparations may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials, and may be stored in a freeze-dried state requiring only the addition of a sterile liquid carrier, such as distilled water for injection, immediately before use. Immediate injections and suspensions may be prepared from the previously described types of sterile powders, granules, and tablets.

[0314] For oral and parenteral administration to human subjects, the daily dose level of a drug (compound, polypeptide, antibody, its antigen-binding portion, composition, nucleic acid, vector, antigen, host cell, further therapeutic agent, vaccine) is typically 1 to 5000 mg per adult and will be administered as a single dose or in divided doses.

[0315] Therefore, for example, tablets or capsules containing the compounds, polypeptides, antibodies, their antigen-binding moieties, compositions, nucleic acids, vectors, antigens, host cells, further therapeutic agents, and vaccines of the present invention may be administered, as needed, in single, double, or more doses, in amounts ranging from 1 mg to 1000 mg (i.e., approximately 60 to 120 mg / m³) of the active compound at once. 2 ) may contain. In any case, the physician will determine the actual dosage that would be most appropriate for any individual subject, and the dosage will vary depending on the age, weight, and response of the particular subject. The above dosages are illustrative of average cases. Naturally, there will be individual cases where a range of higher or lower dosages is advantageous, and such cases are within the scope of the present invention.

[0316] Drugs (compounds, polypeptides, antibodies, their antigen-binding moieties, compositions, nucleic acids, vectors, antigens, host cells, further therapeutic agents, vaccines) may also be administered intranasally or by inhalation using a suitable high-pressure gas, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, 1,1,1,2-tetrafluoroethane (HFA 134A3) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 134A3). The compounds are conveniently delivered in the form of dry powder inhalers or aerosol spray offerings from pressurized vessels, pumps, sprays, or atomizers using hydrofluoroalkanes such as 227EA3), carbon dioxide, or other suitable gases. In the case of pressurized aerosols, the dose unit may be determined by equipping a valve for delivering the measured amount. Pressurized vessels, pumps, sprays, or atomizers may contain solutions or suspensions of compounds, polypeptides, antibodies, their antigen-binding moieties, compositions, nucleic acids, vectors, antigens, host cells, further therapeutic agents, vaccines, etc., using a mixture of ethanol and a high-pressure gas as a solvent, which may further contain a lubricant, such as sorbitan trioleate. Capsules and cartridges (e.g., made of gelatin) for use in inhalers or injectors may be formulated to contain a powder mixture of the compounds of the present invention and a suitable powder base such as lactose or starch.

[0317] Aerosol or dry powder formulations are preferably prepared such that each measured dose or "puff" contains at least 1 mg of the drug (compound, polypeptide, antibody, its antigen-binding portion, composition, nucleic acid, vector, antigen, host cell, further therapeutic agent, vaccine) for delivery to the subject. It will be understood that the total daily dose using aerosols will vary from subject to subject and may be administered as a single dose or, more generally, as divided doses throughout the day.

[0318] Alternatively, the drugs (compounds, polypeptides, antibodies, their antigen-binding moieties, compositions, nucleic acids, vectors, antigens, host cells, further therapeutic agents, vaccines) may be administered in the form of suppositories or pessaries, or they may be applied topically in the form of lotions, solutions, creams, ointments, or powders. The compounds, polypeptides, antibodies, their antigen-binding moieties, compositions, nucleic acids, vectors, antigens, host cells, further therapeutic agents, and vaccines of the present invention may also be administered percutaneously, for example, by the use of skin patches. They may also be administered via the ocular route, specifically to treat eye diseases.

[0319] For use in the eyes, pharmaceuticals (compounds, polypeptides, antibodies, their antigen-binding portions, compositions, nucleic acids, vectors, antigens, host cells, further therapeutic agents, vaccines) can be formulated as an ultrafine suspension in isotonic, pH-adjusted sterile saline, or preferably as a solution in isotonic, pH-adjusted sterile saline, and optionally combined with preservatives such as benzylalkonium chloride. Alternatively, they may be formulated in ointments such as petrolatum.

[0320] For topical application to the skin, drugs (compounds, polypeptides, antibodies, their antigen-binding moieties, compositions, nucleic acids, vectors, antigens, host cells, further therapeutic agents, vaccines) can be formulated as suitable ointments containing active compounds, polypeptides, antibodies, their antigen-binding moieties, compositions, nucleic acids, vectors, antigens, host cells, further therapeutic agents, vaccines suspended or dissolved in a mixture of, for example, one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compounds, emulsifying wax, and water. Alternatively, they can be formulated as suitable lotions or creams suspended or dissolved in a mixture of, for example, one or more of the following: mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0321] Formulations suitable for topical administration in the mouth include lozenges containing the active ingredient in aromatic bases, typically sucrose and acacia or tragacanth, inert bases such as gelatin and glycerin, or scented tablets containing the active ingredient in sucrose and acacia, as well as mouthwashes containing the active ingredient in a suitable liquid carrier.

[0322] In general, oral or topical administration of drugs (compounds, polypeptides, antibodies, their antigen-binding moieties, compositions, nucleic acids, vectors, antigens, host cells, further therapeutic agents, vaccines) in humans is the most convenient and preferred route. In situations where the recipient suffers from dysphagia or reduced drug absorption after oral administration, the drug may be administered parenterally, for example, sublingually or buccally.

[0323] For use in livestock, the drugs (compounds, polypeptides, antibodies, their antigen-binding portions, compositions, nucleic acids, vectors, antigens, host cells, further therapeutic agents, vaccines) are formulations that are appropriately accepted according to the standards of normal veterinary practice, and the veterinarian will determine the administration regimen and route of administration that would be most appropriate for the particular animal.

[0324] For convenience, this preparation is a pharmaceutical preparation. This preparation may also be a veterinary preparation.

[0325] It should be understood that the term "administration" is not limited to a single dose. The term "administration" encompasses, but is not limited to, single doses, multiple doses over a long period, variable doses over a long period, variable means of administration over a long period, and administration in combination with one or more additional therapeutic agents. Administration may be by any means known in the art, and includes, but is not limited to, oral, intravenous, topical, sublingual, or suppository administration directly to the tumor.

[0326] Any listing or discussion of previously published documents in this specification should not necessarily be interpreted as confirmation that such documents are part of the latest technology and common general knowledge.

[0327] Any preferences and choices in a given aspect, feature, or parameter of the present invention should be considered to be disclosed in conjunction with any and all preferences and choices in all other aspects, features, and parameters of the present invention unless otherwise specifically indicated in the context. For example, various definitions of VDE are relevant to all aspects of the present invention, and for example, an epitope containing VDE for use in vaccination against dengue virus infection may comprise any one or more epitope backbone proteins, the backbone proteins comprising heterologous backbone proteins covalently bound to a virion-dependent epitope; envelope proteins of at least Q77, W101, N153, T155, K310; or optionally, any one or more envelope proteins of domain II further comprising any one or more of the following characteristics of domain II: b chain (residues 67-74), the fusion loop and residues immediately upstream (residues 97-106), and ij loop (residues 246-249). 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[0329] [Figure 1] Characterization of human anti-DENV monoclonal antibodies: Serotype specificity and reaction to DENV envelope protein by Western blotting (WB) of 145 DENV mAbs. (b) Serotype specificity of WB-positive and EDE (WB-negative) antibodies. (c) Schematic diagram of epitope mapping using a panel of mutant VLPs. Mutation locations are shown relative to the domain structure of the dengue envelope protein, with red, yellow, and blue representing domains I, II, and III, respectively. Represents the fusion loop around W101 and shows the location of mutations that disrupt the N153 glycosylation motif. [Figure 2] In neutralization assays, EDE antibodies exhibit potent and highly cross-reactivity. Neutralization assays were performed on Vero cells for nine representative mAbs against all four DENV serotypes produced in C6 / 36 insect cells (three FL, EDE1, and EDE2 cells, respectively). Data were from three independent experiments. [Figure 3]EDE-specific antibodies exhibit excellent neutralizing activity. Neutralization assays using Vero as target cells were performed on DENV2 cells produced from C6 / 36 insect cells C6 / 36-DENV(a) or dendritic cells DC-DENV(b). Red, blue, and green bars represent FRNT (reduction rate (%)) at mAbs of 0.05, 0.5, and 5 ug / ml (final concentration), respectively. EDE mAbs used in the attached paper are marked below the histogram. Antibodies are classified into FL and EDE, while antibodies that are positive for WB and do not map to VLP are referred to as non-FL. Based on the results of VLP mapping, five subgroups of EDE were identified and referred to as EDE15. (c) Titer curves for binding measured by capture ELISA, and neutralization reactions of virus-producing DC and C6 / 36 cells with two representative antibodies from FL and EDE1 and 2 groups. This data comes from two independent experiments, each representing results from nine anti-FL antibodies, nine anti-EDE1 antibodies, and seven anti-EDE2 antibodies, respectively. [Figure 4] Antibody binding to viral particles at different maturation states. (a) Anti-prM and anti-E ELISAs were used to calculate the prM:E ratio in various viral particles and compared to viruses from LoVo cells, defined as having 100% prM content. (b) Binding of representative mAbs to C6 / 36, DC, 293T, 293T transfected with fluin, DENV2 produced from LoVo cells, or acid-treated DENV2 was measured by capture ELISA. Two FL, VDE1, and VDE2 mAbs are shown, along with a VDE4 mAb that is sensitive to acid-treated viruses. This data is from two independent experiments and represents the results from eight anti-FL, ten anti-VDE1, and eight anti-VDE2 antibodies. [Figure 5]FL vs. EDE mAbs from individual patients. (a) Distribution of FL and EDE responses between patients is shown in FL-blue and EDE-red. The number in the center indicates the number of Abs from each patient, and one copy of three replicated antibodies (1 EDE1 and 2 EDE2) from patient 752 with identical amino acid sequences was excluded from this and all other analyses. (b) ADE, U937 cells were infected with DENV2 grown in either C6 / 36 cells or DCs in the presence of titers of anti-E mAbs that respond to FL or EDE. These results are expressed as median peak enhancement (times) from two independent experiments. The purple, blue, green, and red symbols indicate the 752-2C8, 753(3)C10, 747(4)A11, and 747(4)B7 mAbs, respectively, used for NT50 and NT90 titers between FL and EDE mAbs in attached paper (c)FL, which are compared for C6 / 36 and DC viruses. [Figure 6] Schematic diagram of epitope mapping using a panel of mutant VLPs. The locations of mutations are shown relative to the domain structure of the dengue fever envelope protein, with red, yellow, and blue representing domains I, II, and III, respectively. The fusion loop around W101 is shown, indicating the location of the mutation that disrupts the N153 glycosylation motif. [Figure 7] Germ cell analysis of EDE1 and EDE2 anti-EDE antibodies. [Figure 8]Recombinant E dimers bind to both EDE1 and EDE2 antibodies. a) SEC / MALS analysis of recombinant sE protein from four dengue serotypes, either as a complex with or uncomplexed with BNA (broadly neutralizing antibody) Fab fragments. MALS showed that the SEC chromatogram of sE protein (green curve) corresponds only to the monomer fraction, and no dimers were detected. The two peaks observed for sE serotypes 1, 3, and 4 correspond to monomers. The most likely explanation is that the affinity for the dimer is not sufficiently high, disrupting the dimer-monomer equilibrium in the gel filtration column, resulting in dimer dissociation. Complexation with Fab fragments clearly stabilizes the dimer morphology, which allows for the elution of sE dimers complexed with two Fab fragments (red curve). We noted that only the slow-eluting sE monomer fraction was converted into a complex, while the other peaks remained unchanged (this was most evident in DENV-3 sE, but it also applies to other serotypes). For DENV-2 sE, there is a single sE peak with a tail toward small molecular weight, which disappears upon complex formation. The explanation for this behavior is, as with our previous information regarding alphavirus fusion protein El28, that the exposed fusion loop of the fraction containing the dimer-forming sE competent tends to interact with the support, thus causing the elution fraction to be so slow. b) Real-time SPR profiles corresponding to the interaction of sE with tethered Fab fragments of EDE2 all, EDE2 B7, EDE1 C8, and EDE1 C10. Binding of Fab 5H2, which is specific to DENV-4, is shown as a positive control, considering that its well-characterized epitope29 is not at the dimer interface and does not require dimerization for binding. Fab fragments were immobilized at similar densities on a Proteon XPR36 chip. A 2 μM solution of DENV sE protein from four serotypes (as shown) was injected simultaneously across all Fabs (see the section on Online Extended Methods). The SPR signal is presented in reaction units (RUs) as a function of time in seconds.Note that the binding levels are generally consistent with the SEC / MALS plots of the corresponding antibodies in panel (a). [Figure 9] crystallographic statistics [Figure 10] Crystal structure of the unlinked DENV-2 FGA02 sE dimer. a) Comparison with available structures of sE. Of the three available structures of sE in its pre-active form (PDB codes 10AN, 10KE, 1TG8), the one with PDB code 10AN showed a smaller standard deviation in the unlinked FGA02 sE structure. b) Phylogenetic tree positioning the two genotypes of DENV-2 indicated by the structure. [Figure 11]DENV2 sE conjugated with four types of EDE anti-EDE antibodies. a) Complex with EDE2 A11 Fab fragment. The sE dimer is oriented below the vertical double molecular axis and the side facing the viral membrane. The sE promoter is shown as a surface colored by domains (I, II, and III are shown in red, yellow, and blue, respectively), and the fusion loop is shown in purple (in b, labeled for a certain promoter). Foreground and background sE subunits are distinguished by vivid and faint colors. Two N-linked glycan chains (not included on the surface) are shown as ball-shaped rods colored by atom type (carbon in white, oxygen in red, nitrogen in blue) and labeled (N67 and N153). The A11 Fab is shown as ribbon representations with the heavy and light chains in green and gray, respectively. b) Unlinked DENV2 FGA02 sE dimer seen below the double axis (labeled "2" in the center). The green and gray empty ellipses (labeled VH and VL) roughly indicate the heavy and light chain contact sites, respectively, where VH is closer due to the 2x molecular axis. Polypeptide portions and loops relevant to the epitope description are labeled c). The lower part of the diagram with the white arrow in (b) highlights the fusion loop "indentation" covered between two vertices, with a b strand on one subunit and 150 loops on the other. d-f) are the same diagrams as in c, showing complexes with the anti-EDE antibodies EDE2 B7 (d), EDE1 C8 (e), and EDE1 C10 (f) (only the variable domains are shown). The white stars in (e) and (f) mark the 150-loop region, which is irregular in these complexes. Note that in the B7 and A11 complexes, the light chain is too far from domain III to interact with it compared to the C8 and C10 complexes. [Figure 12]Overall complex and imprint of anti-EDE antibody and sE dimer. Each row corresponds to a different sE / BNA (broad-spectrum neutralizing antibody) complex (except the first one showing an unbound sE dimer), and each column shows the same orientation as indicated. In the first two columns, the sE dimer is shown as a ribbon, and the BNA variable domain is shown as a colored surface as in Figure 11. In the side view (left column), the viral membrane may be at the bottom, while the bottom view (center column) corresponds to the sE dimer seen from the viral membrane, and these antibodies are visible across the entire sE ribbon. The top view (right column) shows the sE surface presented to the immune system on a viral particle, with the antibody (green) imprint indicated by a white depth-cue fog. For clarity, the white contour lines define the extent of the green imprint on the blue surface of domain III. For reference, in the upper left panel, the glycan chains of the foreground and background subunits are indicated in red and black, respectively. Fusion loops may be observed in other rows in contact with anti-EDE antibodies, where the loop is indicated in the upper center panel. In the left panel of rows c-e, a red star marks the position of loop 150, which is irregular in complex with the EDE1 anti-EDE antibody. This loop carries N153 glycan, recognized by the EDE2 anti-EDE antibody, as seen in the left panel of row b (glycan shown as a rod with carbon atoms colored red). In contrast, all anti-EDE antibodies appear to be in contact with N67 glycan, with C8 showing the most contact (left panel of row c, N67 glycan as a rod with carbon atoms (yellow)). The blue star in row c indicates an irregular loop in domain III. Note that EDE2 C10 (rows d and e) inserts deeper into the sE dimer than the other anti-EDE antibodies. [Figure 13] Embedding surface area in various BNA composites [Figure 14]The electrostatic potential of the DENV-2 sE complex, epitope, and paratope. A double-page presentation of the complex with negative and positive potential is shown, colored according to the bar below. Because certain regions are irregular in the complex, the electrostatic potential on the surface of the sE dimer was calculated using a DENV-2 sE dimer model constructed as described in the Online Methods section. Contacting and corresponding regions are indicated by the same colored ellipses. [Figure 15]Residues related to BNA / antigen interaction. a) Amino acid sequence alignment of sE from four DENV serotypes, each with residues on a black or light blue background to highlight identity and similarity across the entire serotype. Secondary structural elements are shown and labeled below the alignment, with tertiary structures color-coded as shown in Figure 11. DENV2 sE residues contacted by various anti-EDE antibodies are shown above the alignment, indicating the BNA regions contacted by coding provided at key points. All symbols correspond to contacts in the reference subunit (defined as the fusion loop contributing to the epitope), and empty symbols correspond to contacts across the entire dimer interface. Colored boxes on the sequence highlight five different regions of the sE that make up the epitope. Figure 24 provides a histogram with the number of atomic contacts per sE residue in the complex with each BNA. Since the EDE2 B7 and A11 contacts are very similar, only the B7 contact is shown here. The question marks on the 150 loop indicate that these residues are likely to contact the EDE1 anti-EDE antibody, but are not visible in the structure due to the irregularity of this loop. Alignments of four anti-EDE antibodies were crystallized and numbered according to the Kabat definition, with FRW and CDR regions on gray and white backgrounds, respectively. The CDR corresponding to IMGT conversion 31 is marked with a blue line on the sequence. Somatic mutations are in red, and the corresponding residues in germline are written below in a smaller font. Residues resulting from the VDJ (or VJ) recombination process are in green. Contacted sE regions (corresponding to the colored boxes in Figure 15a) are shown above each sequence and coded to be supported in key places. Secondary structure elements of the EDE2 C8 Ig β-barrel are shown above each sequence for reference. A histogram of the number of contacts per BNA residue in each crystallized complex is provided in Figure 26. [Figure 16]Comparison of interactions between various BNAs and DENV-2 sE. a) Structures of all scFv (red, unbound, 1.7A resolution) of unbound EDE2 superimposed on all variable domains of Fab in complex with DENV-2 sE (yellow, 3.8A resolution) to show that the same three-dimensional structure is maintained in the sE / Fab fragment complex. b) Three-dimensional diagrams showing the variable domains of B7 (green) and A11 (yellow) superimposed together with 150 loops extracted from the corresponding Fab / DENV-2 sE complex structure. Note that the main chain of the 150 loops adopts different three-dimensional structures in the two complexes, mainly because the hydroxyl group of the Y99 side chain at CDR H3 of B7 is hydrogen-bonded at sE T155. All have phenylalanine at this position and therefore lack a hydroxyl group. In complex with A11, the sE protein exhibits the same three-dimensional structure as unbound sE (not shown). c) Histogram of atomic contacts between B7 (above the sE sequence) and A11 (below this sequence). d) Similar to c), but showing the contact patterns created by BNA C10 (above this sequence) and C8 (below) in the sE protein primary structure. e) Similar to c), but comparing C10 (EDE1) and B7 (EDE2) according to the E protein sequence. [Figure 17] H3 loop in broad-spectrum neutralizing antibody PG9 for EDE anti-EDE and anti-HIV. Fab or scFv fragments are similarly oriented, with light chains in gray, heavy chains in green, and H3 loops highlighted in red. For comparison, a Fab fragment of potent anti-HIV-1 BNA PG951, which recognizes glycan chains over a wide range and has a very long H3 loop (30 aa calculated by IMGT), is shown in the same manner. [Figure 18]BNA-CDR interaction at the sE dimer interface. a) The right panel shows the sE dimer as a ribbon, and the left panel shows the epitope magnified with key features indicated. Rows b-d show the sE surface in a translucent display where the ribbon is visible. Glycan residues are shown as rods (these were not included in the surface calculation). The associated CDR loop of the anti-EDE antibody is shown as a ribbon with side chains as rods on top of the sE protein, colored as in Figure 11. The orientation of the left panel in rows b-d corresponds to the left panel in row a, and the right panel follows the arrows in Figure 11b. Hydrogen bonds are shown as dotted lines. [Figure 19] Interaction with glycan chains. a) The EDE1 C8 / DENV-2 sE complex is shown as a ribbon, with selected side chains as rods, and the sE surface is shown in a translucent display to highlight the interaction with the N67 glycan. A few hydrogen bonds are shown as dotted lines. The 150 loop is irregular (indicated by density interruptions). In both (a) and (b), the right panel is a diagram along the glycan chain, pointed to by the arrows in the left panel. b) To highlight that EDE1 and EDE2 anti-EDE antibodies bind in a similar manner, the EDE2 B7 BNA / DENV-2 sE complex is shown in (a) with B7 in the same orientation as C8. EDE2 B7 inserts its long CDR H3 into the recess of the fusion loop, while its side contacts the two glycan chains, as seen in the left panel. The H3 α-helion packs against the N153 glycan. Numerous hydrogen bonds between the anti-EDE antibody and sE are also shown, as enumerated in Figure 21c). Key points for sugar connectivity and nomenclature used in the text and panels (a) and (b). d) Contact between antibody and sugar residue (similar to in Figure 24), as encoded according to key points. e) Dengue variants lacking N153 glycan are more readily recognized by EDE1 anti-EDE antibody, while they exhibit resistance to neutralization by EDE2 anti-EDE antibody. Mean and sem values ​​were estimated from three independent experiments. [Figure 20]Experimental electron density for glycan chains. Figure 11 shows ribbons of a) all EDE2 Fab and b) EDE2 C8 EDE1 Fab complexed with DENV2 sE, color-coded as shown. Simulated annealing omit maps, outlined with 1 sigma (blue-green) or 0.6 sigma (gold), clearly show densities for N153 (in a) and N67 glycans (in a and b) (red and yellow arrows, respectively). To create an unbiased map, all glycan atoms were removed from the structure, all B factors were initialized to 20A2, and the structure was refined using simulated annealing with torsional dynamics. Note that antibody footprints spread across the entire dimer interface between the two glycans (also shown in Figure 11). c) Enlarged views of the complexes for DENV-2 / B7 (left panel) and DENV-2 / C8 (right panel), as seen below the red (left panel) and yellow (right panel) arrows in panels a) and b), respectively. The heavy and light chains are shown as the surface, and the glycans (along with the corresponding asparagine) are labeled with mean factor B for each residue. The glycans are also colored with a lamp from blue (cold) to red (hot). Note that in (c), electron density is shown for DENV-2 / B7 instead of DENV-2 / A11 as in (a) to show the omit map for the glycans in the three complexes. [Figure 21] Polar interaction between antibodies and antigens. [Figure 22]C10 BNA imprints in sE dimers of dengue serotypes 2 and 4. a) Surface representation of DENV-2 sE as seen from outside the virion, exposed main chain atoms orange (upper panel), or main chain atoms + strictly conserved side chains (orange), and highly conserved side chains (yellow) (lower panel). Epitopes of EDE1 BNA C10 (black outline) and EDE2 BNA B7 (green outline) are shown. b) Surface of sE dimers of DENV-2 (upper panel) and DENV-4 (lower), extracted from complexes with C10, color-coded by domain as shown in Figure 11. C10 footprints in each case are shown. Note the asymmetry in three-dimensional structure. The large "hole" on the right side of the dimer (in both panels) is due to irregular ij loops, and the small hole on the left (lower panel, DENV-4 sE / C10) is due to irregular kl loops. [Figure 23] Comparison of the interaction of C10 with DENV-2 and DENV-4 sE. a) The structures of sE dimers of DENV-2 (red) and DENV-4 (green) complexed with C10 are superimposed on the C10 portion. The axis of the sE dimer is drawn in the center, which is colored as appropriate. For clarity, only the superimposed C10 scFv is shown for each complex. When the antibodies are superimposed, the sE dimers coincide only locally, resulting in slightly different orientations of the dimer axis, as depicted. The sE dimers rotate approximately 6 degrees relative to each other around the axis drawn in blue (indicated by the curved gray / red arrows), and have significantly different orientations when the superimposition is performed on the scFv on the left (binding to epitope A) rather than on the scFv on the right (epitope B), highlighting the asymmetry of C10 binding to the sE dimer. b) C10 contact is shown in the alignments of DENV-2 (top) and DENV-4 sE (bottom), indicating a very similar contact pattern in the two complexes. The background of the sequences corresponds to the background of Figure 15 in the main text and shows conservation in the four serotypes. [Figure 24]The detected asymmetric BNA binding to the sE dimer. This figure provides histograms of the number of contacts per sE residue in the structure of all independent complexes analyzed here in panels (a) to (e): a) EDE1 C8 / DENV-2 sE, b) EDE1 Cl0 / DENV-2 sE, c) EDE2 A11 / DENV-2 sE, d) EDE2 B7 / DENV-2 sE, e) EDE1 Cl0 / DENV-4 sE. Each panel is divided into two parts, part (I) showing the immunocomplexes seen under the double axis of the sE dimer on the left (for clarity, the contact domain of the Fab fragment has been removed) and on the right with the antibody completely removed, to indicate the epitope. This part also defines epitopes A and B used in II. Part (II) shows histograms of contacts corresponding to epitopes A and B in the dimer, with histogram bars color-coded as shown in key places, to map the antibody regions involved in contact (in parentheses, the symbols used in Figure 15a are used to mark the corresponding contacts). Note that although the contact patterns remain the same, the number of contacts is not identical for the two epitopes of the sE dimer. The DENV-2 sE / EDE2 C10 crystal has two complexes in an asymmetric unit (i.e., two sE dimers, each with two C10 scFv), and therefore, in total, there is a diagram of four independent epitopes labeled A-D, as shown in (b). [Figure 25]The EDE1 C10 residue Y100 (CDR H3) is likely to interact with F279 in mature dengue virions. a) One of the 90E / M heterotetramers containing a mature DENV-2 particle is extracted from a 3.5Å low-temperature EM reconstitution and shown in a side view (drawn as a white bar labeled "2", perpendicular to the 2x axis) with two E subunits in gray and yellow, and two M subunits in red and salmon. Two black arrows indicate connections between the E external domains (corresponding to sE) having α-helical membrane interaction regions (horizontal "stem" α-helical and vertical TM α-helical). The N-terminal portion of M appears to interact below the E dimer (pink arrow). b) Lower part of the 2x axis with the region enlarged in (c) enclosed. c) This figure is slightly tilted for clarity with respect to the figure in (b) which shows the structures of both (DENV-2 and -4 sE) of the C10 complex superimposed on virion E. These markings correspond to the colors of the corresponding structures (DENV-2 sE / C10 green / mustard yellow, DENV-4 sE / C10 blue / beige), as well as to virion E as shown in panels (a) and (b). The M protein is shown as a salmon-colored surface (marked in white). The M protein lies beneath the E dimer and, throughout the dimer interface, highlights the kl hairpin (contained between arrows and marked) and the base of the ij hairpin, inducing a different three-dimensional structure of the kl hairpin, so that F279 (marked) points away from the hydrophobic core (dark gray bar) of the E protomer, whereas in the sE protein structure (including unbound sE, not shown), it is part of the hydrophobic core (green and blue bars, marked). The side chain of Y100 (labeled) in C10's CDR H3 has an alternative conformation because it does not find a partner in sE (Y100 is also illustrated in Figure 18d, left panel). The CDR H3 loop is flexible enough for Y100 to perform stacking interactions with F279 (conserved across the serotype, see Figure 15a) in the conformation observed in virions. [Figure 26]Histogram of contacts at antibody residues. This figure is reflected in Figure 24, where contacts are shown on the antibody side. (a) C8, b) C10 (from complex with DENV-2 sE), c) all, d) B7, e) C10 (from complex with DENV-4 sE). In each panel, Part I shows the BNA variable domains extracted from the corresponding complexes, colored red for somatic mutations, green for conjugation residues resulting from combinations, and gray (VH dark gray, VL light gray). Side chains involved in contact are represented and indicated by spheres and bars. Part II shows a histogram of the number of atomic contacts per residue, color-coded according to the key points to indicate the regions of contacting sE (in parentheses, symbols used in Figure 15b are used to mark the corresponding contacts). Sequence numbering and background correspond to Kabat transformations (as shown in Figure 15b). CDRs corresponding to IMGT transformations are shown on the sequence with orange dotted lines. Somatic mutations are shown in red, and residues resulting from VDJ (or VI) combinations are shown in green. [Figure 27] Comparison with the binding properties of potent anti-EDE antibodies that target other viruses. [Figure 28] Sequences of dengue fever envelope proteins from serotypes 1-4 [Figure 29] Sequences of EDE1 and EDE2 type antibodies identified in Example 1. [Figure 30] Method for conducting a neutralization test. [Figure 31] Contact residues in envelope proteins derived from crystal structure. [Figure 32] Covalently bridged DV2 E dimer [Figure 33] rE DENV2 WT vs MT A259C coupling of EDE1 [Figure 34] rE DENV2 WT vs MT A259C binding of EDE2 [Figure 35] FLE coupling of rE DENV2 WT to MT A259C [Figure 36] Non-FLE coupling of rE DENV2 WT to MT A259C [Figure 37]rE DENV2 WT vs MT L107C, EDE1 coupling to A313C [Figure 38] rE DENV2 WT vs MT L107C, EDE2 coupling to A313C [Figure 39] FLE coupling of rE DENV2 WT to MT L107C, A313C [Figure 40] Non-FLE coupling of rE DENV2 WT to MT L107C, A313C [Figure 41] Antibody titers in C6 / 36 DENV2: Group 1: Monomer / monomer E WT prime and boost E WT Group 2: Dimer / dimer E A259C mutant prime and boost Group 3: VLP / VLP prM / E virus-like particles (VLP) prime and boost Group 4: Dimer / VLP E A259C mutant prime, followed by VLP boost Group 5: VLP / dimer VLP prime, followed by E A259C mutant boost Group 6: Fake, unvaccinated [Figure 42] Cross-reactivity: Binding to live virus (pooled serum) Group 1 Monomer / monomer E WT prime and boost E WT Group 2 Dimer / dimer E A259C mutant prime and boost Group 3 VLP / VLP prM / E virus-like particles (VLP) prime and boost Group 4 Dimer / VLP E A259C mutant prime, followed by boost with VLP Group 5 VLP / dimer VLP prime, followed by boost with E A259C mutant Group 6 Counterfeit Unvaccinated [Figure 43]Mouse serum: C6 / 36 DENV2 neutralization reaction Group 1: Prime and boost using monomer / monomer E WT Group 2: Prime and boost using dimer / dimer E A259C mutant Group 3: Prime and boost using VLP / VLP prM / E virus-like particles (VLPs) Group 4: Prime using dimer / VLP E A259C mutant, followed by a boost using VLPs Group 5: Prime using VLP / dimer VLP, followed by a boost using E A259C mutant Group 6: Placebo, unvaccinated [Figure 44] Mouse serum: DC DENV2 neutralization reaction Group 1 Monomer / monomer E WT prime and boost E WT Group 2 Monomer / monomer E A259C mutant (dimer / dimer) prime and boost Group 3 VLP / VLP prM / E virus-like particles (VLP) prime and boost Group 4 Dimer / VLP E A259C mutant prime, followed by VLP boost Group 5 VLP / dimer VLP prime, followed by E A259C mutant boost Group 6 Placebo Unvaccinated [Figure 45] ADE: Pooled mouse serum: U937 Group 1 Monomer / monomer E WT Prime and boost E WT Group 2 Monomer / monomer E A259C mutant (dimer / dimer) Prime and boost Group 3 VLP / VLP prM / E virus-like particles (VLP) Prime and boost Group 4 Dimer / VLP E A259C mutant prime, followed by boost with VLP Group 5 VLP / dimer VLP prime, followed by boost with E A259C mutant Group 6 Placebo Non-vaccinated example Example 1 - Human DENV antibodies form two distinct groups based on their ability to bind to the dengue envelope protein in Western blotting.

[0330] Using samples from seven patients (Table 1), 145 human monoclonal antibodies that react to the DENV envelope protein were produced. 32、33Plasma blast (CD3 - CD20 lo / -、 CD19 + CD27 hi CD38 hi The cells were classified from peripheral blood, and Elispot showed that 50-90% of these cells secreted anti-DENV antibodies, which is consistent with the frequency reported elsewhere. 34、35 Of these antibodies, 84% reacted to all four DENV serotypes, 13% reacted to two or three serotypes, and only 3% reacted to a single serotype (Figure 1a). [Table 1] Table 1. Overview of DENV-infected patients enrolled in this study

[0331] Initial antibody screening was performed by ELISA using captured whole virions rather than recombinant proteins or fixed cells to ensure a completely representative panel of antibodies was obtained. Only 57% of the antibodies reacted against the DENV envelope by Western blotting (Figure 1a), and the WB-negative mAbs also failed to react against recombinant E by ELISA. This grouped the antibodies into two broad groups: those that were WB-reactive and those that recognized only epitopes present in intact virions, and from here on, these antibodies are referred to as reactivity to virion-dependent epitopes or envelope dimeric epitopes (VDE or EDE). The majority of the WB-positive antibodies were fully cross-reactive among the four viral serotypes, while for the EDEEDE mAbs, 41 out of 62 were fully cross-reactive, and furthermore, 17 out of 62 reacted against DENV-1, 2, and 3 (Figure 1b). Neutralization assays of three antibodies from each group against viruses produced in C6 / 36 insect cells are shown in Figure 2. The fusion loop and EDEEDE antibodies broadly neutralized all four viral serotypes. EDE2 747(4)A11 and 747(4)B7 showed lower activity against Den4, which is related to the deletion of N-linked glycan at position N153 in Den4 strain H241. Methods related to this embodiment and other embodiments

[0332] Samples. Blood samples were collected from hospitalized patients in accordance with written informed consent. The study protocol was approved in the UK by the Scientific Ethics Committee of the Hospital for Tropical Diseases, the Oxford Tropical Research Ethics Committee, and the Riverside Ethics Committee. Laboratory confirmation of dengue infection was determined by RT-PCR detection of DENV nucleic acid (including the infectious serotype), NS1 antigen lateral flow test, or seroconversion in IgM ELISA. Disease severity was classified according to the 1997 World Health Organization criteria. Of the patients enrolled in this study, two were classified as mild dengue fever (DF), and five were classified as severe dengue fever with plasma leakage and bleeding of dengue hemorrhagic fever (DHF) (Table 1). Secondary infection was defined based on a ratio of dengue-specific IgM to IgG less than 1.8. 7 Blood samples for B cell sorting were collected during hospitalization when the hematopoietic plasmablast population was evident. PBMCs were isolated from whole blood by Ficoll-Hypaque density gradient centrifugation and resuspended in 10% FCS / RPMI for direct use.

[0333] Cells and antibodies. C6 / 36 cell lines derived from the mosquito Aedes arbopictus were cultured in Leibovitz L-15 at 28°C. Vero, U937, and 293T cells transfected with 293T or furin were grown in MEM, RPMI 1640, and DMEM at 37°C, respectively. All media were supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units / ml penicillin, 100 μg / ml streptomycin, and 2 mM L-glutamine. Furin-deficient LoVo cell lines were purchased from ATCC and maintained in F-12 as recommended. Monocyte-derived dendritic cells (DCs) were prepared as described above. 10 .

[0334] Antibodies against human CD3, CD19, CD20, CD27, and CD38 (BD Pharmingen), anti-human IgG-ALP (Sigma), and anti-human or mouse IgG-HRP (DAKO) were used in these experiments. Anti-DENV envelope, 4G2, and anti-DENV prM, 1H10, mouse monoclonal Ab were donated by Dr. C. Puttikhunt and Dr. W. Kasinrerk (Puttikhunt, 2003). Anti-DENV NS3, E1D8 was donated by Dr. Eva Harris.

[0335] Viral resources. Dengue virus serotypes 1 (Hawaii), 2 (16681), 3 (H87), and 4 (H241) were grown in C6 / 36 cells. Furthermore, DENV2 was propagated in DC, LoVo, 293T, and 293T transfected with furin, and the cell-free supernatant was collected and stored at -80°C. Viral titer was determined by foci formation in Vero cells and expressed as foci-forming units (FFU) per ml. 26

[0336] Production of DENV-specific human monoclonal mAbs. DENV-specific human mAbs were produced from activated B cells / plasmablasts. 32、33 In short, PBMCs were stained with anti-CD3, CD19, CD20, CD27, and CD38. Then, activated antibody-secreting cells (ASCs) were stained with CD19 + CD3 - CD20 低 / - CD27 高 CD38 高The cells were gated. A single ASC was separated into each well of a 96-well PCR plate containing an RNase inhibitor (Promega). The plates were briefly centrifuged and frozen on dry ice before storage at -80°C. RT-PCR and nested PCR were then performed to amplify the gamma, lambda, and kappa genes using a cocktail of IgG-specific primers. The heavy and light chain PCR products were then digested with appropriate restriction endonucleases and cloned into IgG1, Igκ, or Igλ expression vectors, a gift from Dr. Hedda Wardemann. To express antibodies, the heavy and light chain plasmids were co-transfected into 293T cells by polyethyleneimine, and the antibody supernatant was harvested for further characterization.

[0337] ELISPOT assay. Elispot plates (Millipore) were coated with either anti-human Ig (Invitrogen) or UV-inactivated DENV1-4. The plates were washed with RPMI and blocked with 1% BSA / RPMI for 1 hour. Selected ASCs were added to wells coated with anti-Ig and DENV with 500 cells each and incubated overnight in 5% CO2 at 37°C. The plates were washed and incubated with biotinylated anti-human IgG and IgM (Sigma) at room temperature for 2 hours, followed by incubation with streptavidin-ALP (Sigma). The reaction products were developed and the spots were counted using an AID Elispot plate reader.

[0338] Detection of DENV specificity and serotype cross-reactivity by ELISA. Non-infected supernatants of DENV1-4 and placebos were separately captured on MAXISORP immunoassay plates (NUNC) coated with anti-E Abs(4G2). The DENV capture wells were then incubated with 1 ug / ml human mAb, followed by incubation with ALP-conjugated anti-human IgG. This reaction was visualized by adding PNPP substrate and stopped with NaOH. Absorbance was measured at 405 nm.

[0339] Recombinant soluble DENV envelope protein ELISA. Plates were directly coated with 150 ng of recombinant soluble E, and bovine serum albumin (BSA) was used as a negative control antigen. The protein-coated wells were then incubated with 1 ug / ml human monoclonal Abs, followed by incubation with ALP-coupled anti-human IgG. PNPP substrate was added last, and the reaction was measured at 405 nM.

[0340] Western blot analysis. For Western blot analysis, DENV supernatant from C6 / 36 cells was prepared under non-heating and non-reducing conditions, electrophoresed on a 12% SDS polyacrylamide gel, and electrophoresed on a nitrocellulose membrane (Amersham). The membrane was then blocked with 5% skim milk, probed with DENV-specific human mAbs, subsequently probed with HRP-coupled anti-human IgG Abs, and grown on a membrane-enhanced chemiluminescent substrate (Amersham). Example 2 - Mutation analysis shows that the EDE antibody and the WB-reactive antibody bind to different epitopes.

[0341] To gain greater insight into epitopes recognized by mAbs, 65 virus-like particles (VLPs) containing alanine substitutions at solvent-exposed residues predicted to be present on the virion surface were constructed. These were derived from the 3D structures of mature virus particles. 4、7、8 These mutant VLPs were screened against 145 monoclonal antibodies by ELISA. 22、36 Mutations resulting in a reduction of over 80% in antibody binding were considered significant. Using this panel, 112 of the 145 mAbs were assigned to epitopes in the envelope protein. 33 antibodies, all of which reacted to E by WB, remained unmapped using the mutant VLP panel. The epitope mapping results are shown in Figure 1c, and in more detail in Figure 6. These epitopes can be broadly clustered into two groups:

[0342] Group 1: Fusion loop, a restrictive set of residues inside and outside 101W that defines the above-mentioned or classical fusion loop epitope (FL). 46 out of 83 antibodies that bind to the envelope in WB have been shown to be sensitive to mutations at position 101W and are key residues for binding of many anti-DENV mAbs. 37、38 Of the 46 FL-specific mAbs, 40 were sensitive only to the W101 mutation, while the other epitopes contained combinations of residues W101, G106, and L107. The crystal structure of FL mAb E53, which binds to the envelope protein from West Nile virus, shows contact with residues 104-110 in the bc loop, but not with 101W, and shows contact with residues 74-79. 39 Similar to the 1C19 mAb, only two of the FL-specific mAbs were sensitive to changes in the bc-loop loop where binding was lost when amino acids 76-79 were changed to alanine. 40 .

[0343] Group 2: EDE antibodies; these can be subdivided into five distinct subgroups based on their reactivity patterns to VLP mutants (Figures 1c and 6). While most EDE antibodies were sensitive to the alteration at fusion loop residue 101, they should not be confused with the classical "fusion loop" specific antibodies described above, as they have a more extensive epitope and possess further determinants in domains I, II, and III. Most EDE antibodies can be divided between two distinct subgroups, EDE1 and EDE2, differentiated by sensitivity to alterations at residues 153 and 155 in EDE2, which would interfere with the N-linked glycosylation site. Twelve antibodies constitute three further subgroups with different epitopes and functions; EDE3 mAbs were similar to EDE1 mAbs but were also sensitive to alterations at 107L and 295K. EDE4 was insensitive to the alteration at 101W and best responded to acidified viral particles (Figure 4b). Finally, EDE5 mAb constitutes a group of 5 mAbs, which bind only to the fusion loop 101W, but this epitope is replicated only in intact virions, while EDE3, 4, and 5 are all antibodies that incompletely neutralize the epitope (Figures 3a and 3b).

[0344] VLP mutagenesis experiments suggest that EDE is a composite quaternary epitope containing more than one envelope protomer. method

[0345] Antibody epitope mapping using virus-like particle (VLP) mutants. The full-length prM / E of DENV1 was cloned into the expression vector pHLsec to create a VLP (constructed by Dr. Aleksandra Flanagan). 55 VLP mutations were generated by PCR-based site-directed mutagenesis. 62Mutagenic PCR was performed using Pfx DNA polymerase (Invitrogen) to substituted selected amino acid residues in the E protein with alanine; if alanine was already present, the mutation was made to glycine. After DpnI (NEB) treatment, the PCR products were transformed into E. coli. All mutations were confirmed by sequencing. The plasmids were transfected into the 293T cell line by polyethyleneimine, and the culture supernatant was harvested for epitope mapping.

[0346] To identify epitope-specific mAbs, WT and mutant VLPs were captured with mouse anti-prM(1H10). Then, DENV-specific anti-E mAb was added at 1–5 ug / ml, followed by anti-human IgG-ALP. Finally, a PNPP substrate was added, the reaction was stopped with NaOH, and absorbance was measured at 405 nm. The relative recognition index was calculated as [absorbance of mutant VLP / absorbance of WT VLP] (recognized by the test mAb) / [absorbance of mutant VLP / absorbance of WT VLP] (recognized by a series of four mixed mAbs). Example 3 - Unlike the EDE antibody, the WB-reactive antibody cannot completely neutralize viruses produced in human dendritic cells.

[0347] During DENV infection, the host is present with two forms of the virus: initial exposure is due to the virus produced in insect cells, while the virus produced in human cells drives the subsequent infection cycle and exhibits the enormous viral load generated during infection. To observe these two distinct viral forms, we compared DENV-2 viruses produced in C6 / 36 insect cells (C6 / 36-DENV) or monocyte-derived dendritic cells (DC-DENV), which are thought to be the sites of viral replication in the infected human host after infection following a mosquito bite and subsequent injection of the virus into the skin. 20 .

[0348] Of the 83 WB-positive mAbs, 46 were mapped to FL, and 37 were identified as unmapped binding sites. Surprisingly, none of the 83 WB-positive antibodies could completely neutralize DC-DENV, even at high concentrations; only one neutralized over 80% at 5 ug / ml (Figures 3a and 3b). On the other hand, most of the EDE1 and EDE2 antibodies could neutralize DC-DENV over 80%, with many reaching 100% neutralization. The complete binding and neutralization curves for representative mAbs (Figure 3c) show that anti-FL mAbs reduced binding to DC-DENV (blue X marks) by ELISA and could not completely neutralize DC virus infection (blue circles), while the neutralization reaction and binding curves for EDE mAbs were more closely inverse to those for C6 / 36 and DC-DENV. method

[0349] Neutralization and enhancement assays. The potential for mAb neutralization was determined using a focus reduction neutralization assay (FRNT) when the number of infected lesions was reduced compared to the control (no antibody). 22 In short, serially diluted Ab was mixed with the virus and incubated at 37°C for 1 hour. The mixture was then transferred to Vero cells and incubated for 3 days. Focus formation assays were then performed using anti-E mAb (4G2), followed by HRP-coupled rabbit anti-mouse IgG. This reaction was visualized by the addition of DAB substrate. The percentage of focus reduction was calculated for each antibody dilution. The 50% FRNT value was determined from a graph of reduction rate versus Ab concentration using a probit program from the SPSS package. Example 4 - Anti-EDE antibodies cannot bind to the virus when they have a high proportion of prM or when the envelope protein adopts a trimer structure.

[0350] To illustrate these different viral morphologies, we compared antibodies that bind to six DENV-2 preparations. To assess the degree of prM cleavage, we measured the prM:E ratio by ELISA and normalized this to DENV produced in LoVo cells, which lack furin activity and produce almost completely incomplete, mature viral particles with complete prM components. 17 (Figure 4a). The virus preparations were: 1) C6 / 36-DENV with a relatively high prM content of 56%, 2) DC-DENV with a prM content of 13%, and 3) viruses produced in furin-deficient LoVo cells (LoVo-DENV) with a prM content close to 100%. 22 4) Virus produced in 293T cells overexpressing furin (furin-293T-DENV) with a 5% prM content, 5) Virus produced in natural 293T cells (293T-DENV) with a 60% prM content, and 6) Virus incubated at pH 5.5 (acid DENV) that irreversibly adopts the E trimer three-dimensional structure. 42 .

[0351] EDE1 and 2 mAbs were unable to bind to acidic DENV, presumably because trimerization interfered with the steric epitope, or presumably because the complete complement of prM supported the prM / E spike, which could again interfere with the mature envelope dimer epitope or sterically hinder access to EDE (Figure 4b). Anti-FL mAbs showed reduced binding to viruses with low prM content, shifting the binding curves for DC-DENV and 293T-Fulin-DENV 1.5–2 log-to-right for DENV-producing C6 / 36. Furthermore, binding to LoVo-DENV was even more efficient than for C6 / 36DENV, highlighting the importance of prM in fusion loop antibody exposure and effective binding. 39、43 Four EDE4 antibodies isolated from three different individuals bound most effectively to the acid-treated virus, but they showed negligible neutralization reactions (Figure 4b). method

[0352] DENV-binding ELISA. To determine the binding affinity of abs to DENV generated from different cell types, DENV2 produced from sham, C6 / 36, DC, 293T, 293T or LoVo cells transfected with fluyn, and acid-treated C6 / 36 DENV2 were captured on 4G2-coated plates and subsequently incubated with serial dilutions of DENV-specific human monoclonal abs, followed by incubation with ALP-coupled anti-human IgG. This reaction was induced by the addition of a PNPP substrate and stopped with NaOH. Absorbance was measured at 405 nm. Antigen loading of different viral morphologies and inter-experimental variability in OD readout were normalized by a control ELISA using a well-described humanized version of 3H5 mAb specific to DENV2 domain III. Example 5 - Antibodies in a specific patient indicate immune dominance.

[0353] The anti-DENV mAbs described herein are complex ensembles with overlap specificity when EDE overlaps with the more restricted epitopes of FL antibodies. When comparing these antibody groups (FL vs. EDE) within individual patients, we found asymmetric repertoires showing preference for either the FL epitope or the EDE epitope (Figure 5a). This immunodominance of recognition within an individual was surprising. When epitopes overlap, the antibody with the strongest binding affinity may be able to competitively remove other antibodies and achieve affinity maturation, thus dominating the response that results in a stochastic selection between FL and EDE. However, the response to EDE or FL is polyclonal (recombination of different VDJs) within the individual, which is not a very likely explanation. Example 6 - Anti-EDE antibody results in antibody-dependent enhancement of reduced levels of infection.

[0354] The ability of antibodies to enhance DENV infection at Fc receptors expressing U937 cells was tested. All antibodies tested induced ADE, but the enhancement was approximately 4–8 times lower in the EDE group compared to the FL group. The mean peak enhancement between the FL and EDE groups was 3745:545 for C6 / 36-DENV and 2070:480 for DC-DENV (Figure 5b). method

[0355] For the ADE assay, serially diluted Ab was pre-incubated with the virus at 37°C for 1 hour, transferred to U937 cells (a human monocyte cell line carrying the Fc receptor), and incubated for 4 days. The supernatant was harvested and titrated onto Vero cells using a focus formation assay. Viral titer was expressed as focus formation units (FFU) per ml, and the enhancement factor was calculated by comparing it with the viral titer in the absence of the antibody. Example 7 - Anti-EDE antibody binds to recombinant sE dimer.

[0356] For structural studies, from the four most potent anti-EDE antibodies identified, designated A11, B7, C8, and C10, were selected: 747(4)A11 and 747 B7(EDE2) and 752-2 C8 and 753(3)C10(EDE1). Both EDE2 anti-EDE antibodies were isolated from the same patient (who had a secondary infection with DENV-2) and are the same IgG clones of somatic mutations derived from the IGHV3-74 and IGLV2-23 germline cells. The heavy chain has a very long (26 amino acid) complementarity-determining region 3 (CDR H3). EDE1 anti-EDE antibodies were isolated from different patients, with the corresponding germline derived from the VH and VL genes IGHV3-64 and IGKV3-11 (EDE1 C8, this patient had primary infection of an undetermined serotype) and IGHV1-3* and IGLV2-14 (EDE1 C10, from a patient with secondary DENV-1 infection). Genetic analysis of these antibodies is summarized in Figure 7.

[0357] Anti-EDE antibodies against recombinant sE protein (the 400 amino-terminal residue of the outer domain of the envelope protein, referred to as "sE" for "soluble E"), the antigen-binding moiety (Fab), and the side-chain variable domain (scFv) were produced in Drosophila S2 cells. 44、45 Since the anti-EDE antibody did not react with recombinant sE protein in a standard ELISA assay, we tested the interaction of the antibody fragment with purified recombinant DENV sE in solution at high concentrations to favor dimerization. Size exclusion chromatography (SEC) combined with multi-angle light scattering (MALS) experiments showed that the dimer / monomer equilibrium of recombinant DENV-1, -2, -3, and -4 sE shifted to dimerization by antibody fragments that eluted as a complex corresponding to the sE dimer with two antibody fragments in most cases, despite dissociation effects that induced size exclusion during the separation of various species, as shown in Figure 8a. This was further confirmed by surface plasmon resonance (SPR) analysis (Figure 8b). Example 8 - Crystal Structure

[0358] To identify the determinants of cross-reactivity, a total of seven crystal structures were determined, including DENV-2 sE dimers complexed with four selected anti-EDE antibody fragments, and DENV-4 sE complexed with EDE1 C10. Since the DENV-2 sE dimer used belonged to a different strain than those whose structure was already available, unbound sE dimers were crystallized to detect possible conformational changes induced by the antibody. Furthermore, the structure of unbound A11 scFv was determined because it was unclear whether the long CDR H3 of unbound A11 scFv maintained the same conformation in the absence of the antigen. The crystallization procedure is described in Example 15, and crystallographic statistics are listed in Figure 9. DENV-2 sE strain FGA02, genotype III

[0359] Asian / American genotype III with serotype 2 11The majority of the structural studies were performed using recombinant sE from the DENV-2 field strain FGA02 (isolated in French Guiana in 2002), which belongs to the same group. FGA02 sE was found to be scattered over 394 residues (3%) of the outer domain, along with pre-crystallized DENV-2 sE. 5、7 In comparison, it shows a difference of 13 amino acids. As expected, the 3 Å resolution structure of FGA02 sE falls within the range of three-dimensional structures observed in various structures deposited in the PDB (acceptance numbers 1OAN, 1OKE, 1TG8), showing only small differences from the already available structure of its pre-fusion form DENV-2 sE (Figure 10). By demonstrating that the structure of unbound FGA02 sE is not due to a specific amino acid sequence of the E protein of this strain, it was useful in evaluating the region where antibody binding induces disease, specifically the 150 loops (see below). Example 9 - Envelope dimer epitope Anti-EDE antibodies bind at the sE dimer interface.

[0360] The crystal structure of the FGA02 sE immune complex shows that four anti-EDE antibodies bind in a similar manner to both subunits of the dimer (see also ED in Figure 12, which provides the imprint of each anti-EDE antibody in the sE dimer) (Figure 11). The heavy chain binds closer to the double axis (i.e., the center of the dimer), while the light chain is positioned peripherally. The epitope is low pH 46 In immature DENV particles exposed to the saturates, the prM protein imprint significantly overlaps the E dimer. These overlaps are centered in a depression formed by the b strand on the domain II side and the “150 loop” on the domain I side (opposite the dimer interface, Figure 11c). The 150 loop extends to residues 148159, connecting to the β strands E0 and F0 of domain I and carrying the N153 glycan on the fusion loop of the partner subunit in the dimer. The heavy chain extends the distance between the two glycan chains, N67 and N153, across the dimer interface (Figures 11c-f). The total embedded surface per epitope is 1050 Å. 2 ~1400Å 2The range extends to the surface complementation coefficient. 13 The values ​​were 0.67–0.74, which are typical values ​​for antibody / antigen complexes (Figure 13). The surface electrostatic potentials of the epitope and paratope were lightly charged and had a relatively complementary charge distribution (Figure 14). Conserved residues create epitopes.

[0361] Anti-EDE antibodies essentially cluster at highly conserved residues across four serotypes (Figure 15), which explains their cross-reactivity. The 26-residue CDR H3 at B7 and A11 accounts for the majority of EDE2 anti-EDE antibody contact in both sE subunits that form the epitope. The H3 loop creates a protrusion in the paratope, adopting a convex shape complementary to the concave surface of the antigen (Figure 11d). The H3 protrusion is pre-formed in the antibody, as shown by the 1.7 Å degraded structure of unbound EDE2 A11 scFv, indicating no entropy consumption for binding (Figures 9, 16, and 17). In the reference subunit, defined as contributing to the fusion loop to the epitope, both EDE1 and EDE2 anti-EDE antibodies target the same serotype invariant residues clustered in the three main polypeptide segments of domain II (circled in Figure 15a): the b strand (residues 67-74 carrying the N67 glycan), the fusion loop and residues immediately upstream (aa97-106), and the ij loop (aa246-249). While both the light and heavy chains of the EDE1 anti-EDE antibody interact with the reference subunit via all three CDRs, the EDE2 anti-EDE antibody essentially interacts with the heavy chain and contacts only a few light chains from CDR L3 (Figure 18). In the opposite subunit across the interface, the targeted sE segment is distinct from the two EDE groups. EDE2 anti-EDE antibodies interact with the 150 loop and N153 glycan chain (see below), while EDE1 anti-EDE antibodies induce 150 loop damage upon binding. This is due to cross-reactivity with mouse DENV antibodies. 47、48In the region of the structurally characterized "A strand" epitope, the light chain in the EDE1 anti-EDE antibody can be brought closer to the sE residue and interact with domain III. These domain III contact surfaces are centered at the conserved sE residue K310, and at the crucial stabilizing sE dimer contact surface, the side chain forms a lid covering the indole ring at W101 of the fusion loop (Figure 18). Although the light chains originate from different VL genes (Figure 7), both EDE1 C10 and C8 use CDR L1 and L2 residues to contact domain III (Figures 15 and 18). In domain I, EDE1 C10 inserts its relatively long CDR H3 (21aa, Figure 7) to interact with a conserved residue under 150 loops (see the area circled in Figure 15a or Figure 18d, left panel, scattered over the N-terminal 50 amino acids of the E protein), whereas the shorter H3 loop of EDE1 C8 cannot reach this region. Example 10 - Antibody recognition of glycan chains

[0362] Anti-EDE antibodies create extensive contact surfaces with the glycan chain at both E positions N67 and N153 (Figures 19 and 20). All four anti-EDE antibodies interact with the N67 glycan via the CDR H2 contact surface, and therefore with the N67 glycan. 49 It has been shown to specifically interact with dendritic cells, which would likely prevent binding to the DC-SIGN receptor. The DENV-2 sE / EDE1 C8 complex exhibits a top-level N67 glycan structure with a distal mannose residue in contact with framework region 3 of the heavy chain (FRW H3, Figures 15b and 18c). Except for EDE1 C10 (which is very close to its germline, Figure 7), many FRW H3 residues are altered (Figure 15b), suggesting affinity maturation that recognizes sugars. If further glycan residues are visible in the structure, other anti-EDE antibodies may also exhibit the same FRW H3 residue interactions.

[0363] Although the N150 loop and N153 glycan are irregular in the EDE1 complex, the limited space between the antibody and domain I (Figure 19a, left panel) suggests that this glycopeptide segment engages in a variable local conformation in each complex to ensure that there is no average degraded electron density in the crystal, even though it is in contact with the antibody (as indicated by the question mark above the 150 loop in Figure 15A). If the 150 loop remains in place, the CDR H3 loop of the EDE1 anti-EDE antibody may collide with the N153 glycan, e.g., the first GlcNAc residue in DENV-2 sE / EDE1 C10 (sugar 1 in Figure 19).

[0364] Electron density is evident in the core 6 sugar residues of the N153 glycan of sE in the crystal of the complex with the EDE2 anti-EDE antibody (shown in Figure 20, including the omitted map). The CDR H3 of the EDE2 anti-EDE antibody constructs a two-turn α-helical (referred to as the H3 helix, Figure 11d) using one of its C-terminal carbonyl groups, which is covered by a hydrogen bond provided by the N2 atom of the first N153 GlcNAc residue (Figure 21). The H3 helix laterally protrudes the aromatic side chains Y99 (F99 in EDE2 A11) and Y100, enclosing sugar residues 1, 3, and 4 of the N153 glycan. The most distal residues of the glycan and mannose 4, 5, and 6 are in contact with the light chain via residues from the CDR L2, including several hydrogen bonds (Figures 19 and 21).

[0365] The different types of interactions that EDE1 and EDE2 anti-EDE antibodies produce at the 150 loop and N153 glycan are reflected in the contrasting effects of the glycan's absence on their neutralization efficiency. For example, the DENV-4 isolate with isoleucine at position 155 (i.e., a native glycosylated mutant lacking the 153-NDT-155 glycosylation motif) is more susceptible to neutralization by the EDE1 anti-EDE antibody due to the absence of CDR H3 collisions by the glycan chain. In contrast, this mutant is more resistant to neutralization by the EDE2 anti-EDE antibody (Figure 19e), highlighting the importance of the observed specific recognition of the N153 glycan. Example 11 - Main chain structure of fusion loop as a binding determinant

[0366] In the fusion loop, residues 101-WGNG-104 create a distorted α-helical rotation, with the W101 side chain protruding toward domain III opposite the dimer interface. In the complex with the EDE2 anti-EDE antibody, the helical rotation of the fusion loop is H3 helical-underside, such that the carbonyl groups face each other at the C-terminus of the two helices. Furthermore, S100C of CDR H3 covers the helical rotation by forming a main-chain and side-chain hydrogen bond with the carbonyl group of G102 in the complex loop. In the complex with the EDE1 anti-EDE antibody, the fusion loop is located to the right and below the VH / VL interface, with side chains of several aromatic residues in both the heavy and light chains packing it. Specifically, the VL backchain provides a hydrogen bond to the backchain carbonyl group G104 in close proximity at EDE1 C8, where the backchain amide proton donor belongs to N93 from CDR L3, while at EDE1 C10, it belongs to N31 from CDR L1. Residue D50 in CDR L2 of both C10 and C8 forms a salt bridge at K310 (Figures 19a and 18d), which is part of an extensive network of polar interactions in this region (listed in Figure 21).

[0367] The three-dimensional structure of the glycine-rich fusion loop in the E dimer essentially exposes the backchain, while the side chains are almost embedded. Along with the backchain of the ij loop, the backchain atoms form a large surface patch augmented by one end of the b strand, resulting in an invariant exposed surface recognized by the anti-EDE antibody. Along with the exposed backchain on the E dimer surface (Figure 22a, lower panel), the invariant side chains in this region result in an epitope in the core region that is essentially an invariant serotype with non-conserved residues around it. The reason for this transformation is likely related to the interaction with prM during particle maturation. 46 The smallest conserved region is the surface of domain III within the EDE1 epitope. Example 12 - Structure of DENV-4 sE formed in a complex with EDE1 C10

[0368] To gain a detailed understanding of how anti-EDE antibodies can effectively recognize multiple viral serotypes, we returned to DENV-4 because it differs most significantly from other dengue serotypes in its amino acid sequence (Figure 15a) and is strongly neutralized by EDE anti-EDE antibodies (Figure 19e). DENV-4 sE, complexed with C10 scFv at 2.7 Å resolution, exhibited the same general pattern observed in complex with DENV-2 sE (Figures 22b and 23), which has an irregular 150-loop structure. As expected, the anti-EDE antibody exhibited the same interaction between the main chain and the conserved side chain of the epitope. In the more variable lateral region of the EDE1 epitope in domain III (Figure 22a), the contact site includes the side chain of residue 309, which is aspartic acid in DENV-4 but valine in DENV-2 (Figure 15a). In the latter complex, there is Van der Waals packing between the CDR L2 side chain V309 and T52, whereas in the former complex, there is a polar interaction in which D309 accepts a hydrogen bond from the T52 side chain (Figures 23b and 21). Other contacts with domain III are also maintained, specifically at position 362, which involves a hydrogen bond to the main chain carbonyl (Figure 21).

[0369] EDE1 C10 induces 150 loop irregularities in DENV-2 sE, but in DENV-4 sE, this loop appears to exhibit inherently higher mobility, as suggested by its crystal structure when complexed with the Fab fragment of an unrelated chimpanzee antibody called 5H2 (see reference). 17 ). In fact, the 5H2 epitope is also present in domain I, but on the side of the sE dimer and does not overlap with the anti-EDE antibody epitopes described herein, although the 150 loops were irregular in their structure. Furthermore, the structure of the DENV-4 sE / EDE1 C10 complex highlighted a degree of asymmetry in the contact of the anti-EDE antibody by the two epitopes of the dimer (Figures 23, 13, and 21). This asymmetry was also detectable in the complex with DENV-2 sE, as shown in Figure 24. Probabilistically, binding of the first antibody fragment is likely to induce a modification of the asymmetric conformation of the sE dimer, and when the second antibody fragment binds, it is likely to affect the second site to adapt to the available conformation of the second epitope. Taken together, these observations strongly suggest that the binding determinant of the EDE1 anti-EDE antibody resides in a conserved core of the epitope in a region shared by the EDE2 anti-EDE antibody, and that contact at either end adapts to specific side chains present in each serotype that do not impede binding. This observed flexibility of the E dimer is consistent with reports of respiration of the E dimer's conformation in virions, which exposes normally hidden epitopes in antibody interactions. Example 13 - Further putative EDE1 C10 / E dimer interactions in mature virions

[0370] Precise structural inspection is performed using EDE1 The tip of C10's CDR H3 reaches the "bottom" of the sE dimer (circled in Figure 18d, left panel; see also Figures 15a and 12d-e), indicating that in the context of intact virions, the region is reinforced by the lower protein M (Figure 25). Low-temperature EM structure of intact mature DENV-2 particles at 3.5 Å resolution (see reference). 9This indicates that the interaction with M is exposed at the dimer interface instead of being embedded in the hydrophobic core of domain II (Figure 25c), resulting in the conserved residue F279 at the bottom of the kl hairpin of E (Figure 15a). The superposition by the DENV-2 sE / C10 structure indicates that, in the context of virions, the exposed F279 side chain can interact with Y100 in the H3 loop. Y100 appears to interact differently with DENV-2 sE compared to DENV-4 (Figure 25C; also compare panels b and e in ED Figure 26), suggesting that it does not find its exact partner. Therefore, the EDE1 C10 binding site on the E dimer in mature virions is not likely to be fully replicated by the recombinant sE dimer. This observation likely explains the clear difference between the weak binding of EDE1 C10 to the sE dimer (Figure 8), as well as the strong binding and neutralization reactions of viruses from four dengue serotypes (NT50 in the low nM range; see attached manuscript). Importantly, the three-dimensional structure of F279 in mature virions is similar to that observed in sE bound to the hydrophobic ligand. 4 This suggests that it is possible to induce the precise three-dimensional structure of this region of recombinant sE dimer as an immunogen. Example 14

[0371] We provide a snapshot of anti-EDE antibodies interacting with a major novel epitope targeted by human monoclonal antibodies induced in patients infected with dengue fever. These antibodies appear to converge towards the same specificity via entirely different evolutionary pathways, either requiring a heavy chain with a very long CDR H3 that performs most of the interaction, as in the case of EDE2, or obtaining a finely tuned combination of light and heavy chains with backchain contact, or, in the case of the EDE1 anti-EDE antibody analyzed here, a fusion loop and backchain contact to domain III. The EDE1 and EDE2 anti-EDE antibodies comprise nearly one-third of the antibodies isolated from dengue patients in the accompanying study and constitute the majority of those that recognize a structure-specific quaternary epitope on the virion surface. Their general characteristics from alanine scanning experiments (attached manuscript) strongly suggest that they all target the same quaternary epitope described herein.

[0372] Importantly, the binding determinant of EDE anti-EDE antibodies is localized to the E dimer overall, and differs from that of different flaviviruses, such as West Nile virus. 51 Based on research in [location], DENV particles 50 As recently suggested for the quaternary epitopes above, the higher-order configuration of the dimers on the virion surface does not differ. Recent low-temperature EM analysis of DENV-2 particles suggests that the herringbone pattern may be disrupted at physiological temperatures in humans by dimers that reorient each other, relax symmetry, and / or exhibit different surface patterns. 52、53 Therefore, the epitopes described herein are approachable to the E-dimer independently of expansion or particle and may be preferred targets for next-generation vaccines. As a natural consequence, our results are for respiratory syncytial virus (RSV) 54 As recently proposed, this method stabilizes dimer contact in such a way that only the E dimer is presented to the immune system, thus avoiding the induction of antibodies against poorly immunogenic regions on the surface of infectious virions that are normally inaccessible, making it suitable for designing potent immunogens.

[0373] The primary binding determinants of the EDE anti-EDE antibody appear to be the three-dimensional structure of the fusion loop and its neighboring main chain in the context of an intact E dimer. This is in striking contrast to other major classes of antibodies isolated from humans in the attached manuscript, which recognize the fusion loop sequence in a context independent of quaternary tissues. The latter antibodies exhibit cross-reactivity but poor neutralization and potentially potent enhancement of infection. 56 A noteworthy feature of the epitopes described herein is the number of exposed backbone atoms, which account for approximately 30% of the total surface area embedded in the complex in the case of EDE1 and 20% for EDE2 anti-EDE antibodies (this lower proportion for EDE2 is generally corrected for 40% of the invariant glycan composition) (Figure 13), while general backbone atoms contribute 5% to 15% of most immunocomplexes analyzed. For example, three-dimensional structure 57 In this regard, certain very potent neutralizing antibodies against respiratory syncytial virus (RSV), such as D25 antibodies against RSV, which bind to "antigen site 0" that is stabilized and presented only to the pre-fusion form of the RSV fusion protein, as is the case with EDE anti-EDE antibodies, also recognize a high percentage (approximately 30%) of the main chain atoms in the antigen (Figure 27). Hemagglutinin (HA) 58 BNA CH65, or H1, neutralizes a wide range of H1 influenza virus isolates by binding to its receptor-binding pocket. 59 A similar pattern is found in which CR8020, a potent group 2 reactive anti-influenza human BNA with neutralizing activity against H3, H7, and H10 isolates, binds to the base of the stem. CR8020 also recognizes the main chain structure of the fusion peptide in the context of the quaternary structure, as well as the EDE anti-EDE antibodies described herein. Finally, two of the very broad anti-HIV-1 anti-EDE antibodies, B12 (see reference), are found. 60 ) and VRC01 (see reference) 61These antibodies recognize the CD4 binding site on the envelope (ENV) protein and identify 36% and 33% of the main chain atoms in the epitope (Figure 27), suggesting that recognition of the main chain structure is an important aspect shared by many (but not all) anti-EDE antibodies. These broad-spectrum anti-HIV-1 neutralizing antibodies exhibit effective binding. 29 This requires the precise quaternary structure of the ENV trimer, undergoes a long affinity maturation process, and exhibits more than 20% differences from the germline. In contrast, EDE anti-EDE antibodies against dengue fever exhibit a maximum difference of 9% from the germline (Figure 27), indicating that they occur relatively easily in individuals when appropriate immunogens are used for vaccination.

[0374] In conclusion, we described highly conserved binding sites in highly cross-reactive antibodies against the dengue virus. The poor efficacy of recent attenuated live polyvalent dengue vaccines creates an urgent need to better understand the protective response in humans and design effective vaccines for the next generation. Serotype-specific immunity is often a goal of dengue vaccines, requiring their quadrivalent formulations. Our results suggest that subunit vaccines containing stabilized E dimers should be evaluated, that a single optimized universal immunogen may be possible, and that the induction of anti-EDE antibodies should be considered a feasible goal in successful dengue vaccines. Example 15 - Further Methods

[0375] Recombinant sE proteins from DENV serotypes 1-4, as well as Fab and scFv BNA fragments, are used according to the above protocol. 44、45、29The complex was produced in Drosophila melanogaster Schneider 2 using [method / tool ​​name]. Binding of the BNA fragment to the sE protein was monitored by SEC / MALS and SPR (Figure 8). Crystals of the sE / BNA complex were obtained by isolating the complex from the mixture using SEC, or, in the case of EDE1 C10, by mixing sE:antibody at a two-stage burning ratio of 1:2. Crystallization tests were performed using a robotic environment facility. Diffraction data were recovered using the synchrotron sources SOLEIL and ESRF, and the structures were determined by molecular substitution using the search models enumerated in Figure 9, which also provide relevant crystallographic statistics. Neutralization tests in the DENV-4 mutant were performed using the same procedure outlined in the accompanying documentation. Produced in recombinant sE protein

[0376] Recombinant DENV-1 FGA / 89 sE (1-395), DENV-2 FGA02 sE (1-395), and DENV-3 PAH881 sE (1-393), with several modifications, were essentially produced in the aforementioned Drosophila S2 cells for DENV-4 sE (Den4_Burma / 63632 / 1976). 29In short, sE expression was activated by a metallothionein promoter and induced by 5 μM CdCl2 in insect XPRESS medium (Lonza). This construct had a Drosophila BiP signaling sequence fused at the N-terminal end of the prM-sE construct for effective translocation to the ER of transfected S2 cells. prM acts as a chaperone, presenting its N-terminus to sE (which has its N-terminus attached to the prM) and to sE generated by signalase cleavage in the ER, in the case of DENV polyprotein precursors, etc., with prM (in a membrane-fixed state) covering the fusion loop of sE. The prM / sE complex is transported across the acidic fraction when prM is cleaved by furin into pr (the N-terminal half that binds to sE) and M (the membrane-fixed C-terminal half). Upon reaching the external environment, sE, and pr dissociated products, the sE component is purified by affinity chromatography from the cell supernatant. DENV-3 and -4 sE constructs had a C-terminal fusion via twin-strep-tag (IBA, http: / / www.iba-lifesciences.com / twin-strep-tag.html), while DENV-1 and 2 sE had a 6xHis C-terminal tag. The purified cell supernatant was concentrated 20-fold using a Vivaflow tangential filtration cassette (Sartorius, cutoff 10 kDa), and adjusted to 0.5 M NaCl after buffer exchange to remove divalent ions, depending on the construct, before purification using either StrepTactin affinity purification or HisTrap-HP chromatography on an AKTA FPLC system. His-tagged proteins (DENV-1 and -2 sE) were desalted after elution from the HisTrap column and further purified by ion exchange chromatography on MonoQ. The final purification SEC step was performed for all constructs using a Superdex 200 10 / 300 GL column equilibrated in 50 mM Tris pH 8 and 500 mM NaCl. Production of Fab and ScFv

[0377] BNA fragments were used in Drosophila S2 cells, Fab 62and scFv 63 The construct was cloned into a plasmid for expression. The construct contains a twin strep tag fused at the C-terminus (heavy chain only in the case of Fab) for affinity purification. The purification protocol for the strep-tagged sE protein involved the same steps as described above and used the same buffer. Formation and isolation of immune complexes

[0378] The purified DENV sE protein was mixed with Fab or ScFv (in approximately 2x molar excess) in a standard buffer (500 mM NaCl, Tris 50 mM pH 8.0 buffer). This volume was reduced to 0.2 ml by centrifugation at a Vivaspin 10 kDa cutoff, and after incubation at 4°C for 30 minutes, the complex was separated from the excess Fab or scFv by SEC unless a clear peak was obtained in the complex (see Figure 8, similar to BNA C10). In this case, a mixture with an antigen:antibody molar ratio of 1:2 (i.e., with excess antibody) was used directly for crystallization. In all cases, the buffer was replaced with 150 mM NaCl, 15 mM Tris, pH 8 for crystallization testing. The protein concentrations used for crystallization, determined by measuring the optical density at 280 nm and using the decay coefficient estimated from the amino acid sequence, are listed in Figure 9. MALS analysis

[0379] 150 μg of purified DENV-1, -2, -3, and -4 sE were mixed with 300 μg of A11, B7, C8, and C10 Fab fragments, and the total volume was adjusted to 100 μl. (DENV sE or Fab) was also run separately as a control at the same concentration. Samples were incubated at room temperature for 15 minutes, and analyzed by MALS when eluted from a SDX200 10 / 300 GL gel filtration column at a flow rate of 0.4 ml / min. This elution was subsequently subjected to refractive index measurement and MALS detection with the DAWN Heleos-Optilab T-rEX setup (Wyatt Technology). Surface plasmon resonance

[0380] Real-time SPR measurement of the binding of sE dimers to captured Fab fragments of anti-EDE antibodies was performed using a ProteOn XPR36 instrument (BioRad).

[0381] The Fab fragment of the DENV-4 specific neutralizing antibody 5H2 was used as a control. A biotinylated anti-human CH1-specific antibody (Life Technologies) was immobilized on a Neutravidin ProteOn NLC sensor chip and used to capture similar densities (400~500 RU) of different Fab fragments. This anti-CH1 antibody recognizes all IgG subclasses (1, 2, 3, and 4) regardless of the light chain subclass (kappa / lambda). This anti-CH1 antibody was also found to cross-react with the chimpanzee antibody 5H2, albeit with lower affinity. The Fab fragment of an anti-HCV E2 antibody was used as a negative control. After Fab capture, the chip was rotated 90°, and sE of the 4 DENV serotypes was injected at a concentration of 2 μM. A blank injection using running buffer (50 mM Tris pH 8, 500 mM NaCl, 0.01% Tween 20) was used for double referencing. The SPR signal was normalized to the amount of captured Fab. A control injection of the ectodomain of rubella virus E1 glycoprotein at similar concentrations across all Fabs showed no apparent binding (data not shown). Neutralization assay using DENV-4 glycosylation mutants

[0382] The neutralization potential of anti-EDE antibodies was determined using a focus reduction neutralization test (FRNT), when the reduction in the number of infected foci was compared to the control (no antibody). 22DENV-4 strains H241 (possessing Ile at position 155), 1-0093, and 1-0554 (both possessing Thr at position 155, and therefore restoring glycosylation at Asn153) were grown in C6 / 36 cells. Viral titers were determined by foci formation in Vero cells. 64 In short, serially diluted anti-EDE antibodies were mixed with the virus and incubated at 37°C for 1 hour. The mixture was then transferred to Vero cells and incubated for 3 days. A focus formation assay was then performed using mouse monoclonal 4G2 antibody (which cross-reacts with E protein from all flaviviruses), followed by rabbit anti-mouse IgG conjugated with horseradish peroxidase. This reaction was visualized by the addition of a diaminobenzidine substrate. The percentage of focus reduction was calculated for each antibody dilution. 50% FRNT was determined from a graph of reduction rate versus Ab concentration using "Probit" (http: / / www.statisticalassociates.com / probitregression.htm) with the statistical package SPSS. Crystallization and 3D structure determination

[0383] Crystallization tests were performed with 400 nl drops. Droplets were formed by mixing equivolumes of protein and reservoir solution using a Mosquito robot in the form of a 96 Greiner plate and monitored with a Rock-Imager. Crystallization was optimized in 400 nl drops using robotic Matrix Maker and Mosquito settings, or manually in 24-well plates using 2-3 μl suspended drops (Figure 9). Crystallization and cryo-cooling conditions for diffraction data acquisition are listed in Figure 9.

[0384] X-ray diffraction data were collected at the SOLEIL Synchrotron (St Aubin, France) on beamlines PROXIMA-1 and PROXIMA-2, and at the European Synchrotron Radiation Facility (Grenoble, France) on ID23-2 and ID29 (Figure 9). The diffraction data were processed using the XDS package 65 and, in combination with other programs in the CCP4 package suite 67 were measured with SCALA or AIMLESS 66 . The structure was determined by molecular replacement with PHASER 68 and / or AMoRe 69 using the search models listed in Figure 9.

[0385] Subsequently, alternating cycles of careful model building with COOT 70 and crystallographic refinement with the program BUSTER / TNT 71 were performed to yield the final model. Refinement was restrained to respect non-crystallographic symmetry, and target restraints (comprising portions of the high-resolution structure complex) and TLS refinement were used depending on the crystal resolution 72 (see Figure 9). The final omit map was calculated using Phenix.Refine 73 . Analysis and annotation of the atomic model

[0386] Each complex was analyzed using the CCP4 package suite of programs 67 . For intermolecular interactions, the maximum cutoff distance used for interactions was 4.75 Å. The contacts of each residue of the Fab / ScFv or DENV sE protein were then counted and plotted as proportional bars above the corresponding residues.

[0387] Antibody sequences were mapped to CDR / FWR regions according to Kabat 30 and IMGT numbering using Abysis (www.bioinf.org.uk / software) and IMGT (www.imgt.org)31 Each was analyzed accordingly. Analysis of estimated germline and somatic cell maturation events was performed using the IMGT website (www.imgt.org).

[0388] Multiple sequence alignments and phylogenetic trees are available in ClustalW (ClustalW and ClustalX version 2 (see reference)). 74 Using the EBI server 75 The above calculation was performed. This phylogenetic tree was calculated using the amino acid sequences of the sE proteins used in this study: DENV-1 FGA / 89(1-395), DENV-2 FGA02, DENV-3 PAH881(1-393), and DENV-4(DEN_Burma / 63632 / 1976). To map the DENV-2 genotype, 76 The database was used to extract the amino acid sequences of the sE external domains and extended to include DENV2 FGA02 sE and DENV-2 10AN sE. For simplicity of labeling, the subroots were folded down to the level of individual genotypes for DENV-2. This phylogenetic tree was then rooted with the DENV-4 sE sequence and the MEGA5 software package. 77 We instructed them to use [this method] for measurement.

[0389] For Figures 22a and 14, and for analytical purposes, a model of a DENV-2 sE dimer without gaps in the sequence was used. This model was constructed using complete protomer A of the DENV-2 sE / B7 complex.

[0390] The diagram shows Program ESPript 78 and APBS 79 and PDB2PQR tool 80 The system was prepared using PyMOL Molecular Graphics System version 1.5.0.4 Schrodinger, LLC (pymol.sourceforge.net), which includes [specific components / features].

[0391] Finally, current vaccine strategies employ tetravalent formulations to produce balanced, type-specific responses against all four serotypes. The description here of such potent and cross-reactive antibodies suggests a method for subunit vaccines, including heterogeneous prime-boost strategies, that replicate the responses seen in naturally occurring sequential infections, targeting the desired epitope and potentially other types. Example 16: Sequence Information Scalar Sequence ID 1 Complete sequence of antibody C8 heavy chain EVQLVESGGGLVQPGGSLRLSCSASGFTFSTYSMHWVRQAPGKGLEYVSAITGEGDSAFYADSVKGRFTISRDNSKNTLYFEMNSLRPEDTAVYYCVGGYSNFYYYYTMDVWGQGTTVTV Sequence ID 2 Complete sequence of antibody C10 heavy chain EVQLVESGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNGNTKYSQKFQDRVTITRDTSASTAYMELSSLRSEDTAIYYCARDKVDDYGDYWFPTLWYFDYWGQGTLVTV Sequence ID 3 Complete sequence of antibody A11. EVQLVESGGGLVRPGGSLRLSCAASGFSYSNHWMHWVRQAPGKGLVWVSRINSDGSTRNYADFVKGRFTISRDNAENTLYLEMNSLTADDTAVYYCVRDGVRFYYDSTGYYPDSFFKYGMDVWGQGTTVTV Sequence ID 4 Complete sequence of antibody B7 heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSSHWMHWVRQAPGKGLVWVSRTNSDGSSTSYADSVKGRFMISRDNSKNTVYLHMNGLRAEDTAVYFCARDGVRYYYDSTGYYPDNFFQYGLDVWGQGTT Sequence ID 5 C8 CDR H1 TYSMH Sequence ID 6 C8 CDR H2 AITGEGDSAFYADSVKG Sequence ID 7 C8 CDR H3 GYSNFYYY Sequence ID 8 C10 CDR H1 SYAMH Sequence ID 9 C10 CDR H2 WINAGNGNTKYSQKFQD Sequence ID 10 C10 CDR H3 DKVDDYGDYWFPTLW Sequence ID 11 A11 CDR H1 NHWMH Sequence ID 12 A11 CDR H2 RINSDGSTRNYADFVKG Sequence ID 13 A11 CDR H3 DGVRFYYDSTGYYPDSFFKY Sequence ID 14 B7 CDR H1 SHWMH Sequence ID 15 B7 CDR H2 RTNSDGSSTSYADSVKG Sequence ID 16 B7 CDR H3 DGVRYYYDSTGYYPDNFFQY Sequence ID 17 C8-CDR L1 RASQSISTFLA Sequence ID 18 C8 CDR L2 DASTRAT Sequence ID 19 C8 CDR L3 QQRYNWPPYT Sequence ID 20 C10 CDR L1 TGTSSDVGGFNYVS Sequence ID 21 C10 CDR L2 DVTSRPS Sequence ID 22 SSHTSRGTWVF Sequence ID 23 A11 CDR L1 TGTSSNADTYNLVS Sequence ID 24 A11 CDR L2 EGTKRPS Sequence ID 25 A11 CDR L3 CSYATSRTLVF Sequence ID 26 B7 CDR L1 TGISSDVETYNLVS Sequence ID 27 B7 CDR L2 EASKRPS Sequence ID 28 B7 CDR L3 CSYAGGKSLV Sequence ID 29 Full-length envelope protein sequence DENV1 >DENV1st Stock Hawaii MRCVGIGNRDFVEGLSGGTWVDVVLEHGSCVTTMAKDKPTLDIELLKTEVTNPAVLRKLCIEAKISNTTTDSRCPTQGEATLVEEEQDANFVCRRTFVDRGWGNGCGLFGKGSLITCAKFKCVT KLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTIATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNEMVLLTMKEKSWLVHKQWFLDLPLPWTSGASTPQETWNREDLLVTFKTAHAKK QEVVVLGSQEGAMHTALTGATEIQTSGTTKIFAGHLKCRLKMDKLTLKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSTQDEKGVTQNGRLITANPIVTDKEKPVNIEAEP PFGESYIVVGAGEKALKLSWFKKGSSIGKMLEATARGARRMAILGDTAWDFGSIGGVFTSVGKLVHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGLNSRSTSLSMTCIAVGMVTLYLGVMVQA Sequence ID 30 Full-length envelope nucleotide sequence DENV1 Sequence ID 31 Full-length envelope protein sequence DENV2 >DENV2 shares 16681 MRCIGMSNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFRCKK NMEGKVVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKK QDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEP PFGDSYIIIGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVIITWIGMNSRSTSLSVTLVLVGIVTLYLGVMVQA Sequence ID 32 Full-length envelope nucleotide sequence DENV2 Sequence ID 33 Full-length envelope protein sequence DENV3 >DENV3 shares H87 MRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRKLCIEGKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLESIEGKVVQHENLKYTVIITVHTGDQHQVGNETQGVTAEITSQASTAEAILPGYGTLG LECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRRELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGGTSIFAGHLKCRLKMDKLELKGMSYAMCLNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKE EPVNIEAEPPFGESNIVIGIGDKALKINWYRKGSSIGKMFEATARGARRMAILGDTAWDFGSVGGVLNSLGKMVHQIFGSAYTALFSGVSWIMKIGIGVLTWIGLNSKNTSMSFSCIAIGIITLYLGVVVQA Sequence ID 34 Full-length envelope nucleotide sequence DENV3 Sequence ID 35 Full-length envelope protein sequence DENV4 >DENV4 shares 241 MRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELIKTTAKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTCAKFSCSG KITGNLVQIENLEYTVVVTVHNGDTHAVGNDIPNHGVTATITPRSPSVEVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWAAGADTSEVHWNYKERMVTFKVPHAKR QDVIVLGSQEGAMHSALTGATEVDSGDGNHMFAGHLKCKVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGAGAPCKVPIEIRDVNKEKVVGRIISSTPFAEYTNSVTNIELEP PFGDSYIVIGVGDSALTLHWFRKGSSIGKMLESTYRGVKRMAILGETAWDFGSVGGLFTSLGKAVHQVFGSVYTTMFGGVSWMVRILIGFLVLWIGTNSRNTSMAMTCIAVGGITLFLGFTVHA Sequence ID 36 - Full-length envelope nucleotide sequence DENV4 Sequence ID 37 C8 light chain - See the table below. Sequence ID 38 Complete sequence of antibody C10 light chain - see the table below. Sequence ID 39 Complete sequence of antibody A11 light chain - see the table below. Sequence ID 40 B7 Light Chain See the table below. Sequence IDs 37-131: Antibody light and heavy chain sequences from the table below. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] [Table 2-15] [Table 2-16] [Table 2-17] Sequence ID 132 Envelope outer domain protein sequence DENV1 FHLTTRGGEPHMIVSKQERGKSLLFKTSAGVNMCTLIAMDLGELCEDTMTYKCPRITEAEPDDVDCWCNATDTWVTYGTCSQTGEHRRDKRSVALAPHVGLGLETRTETWMSSEGAWKQIQKVETWALRHPGFTVIALFL AHAIGTSITQKGIIFILLMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPAVLRKLCIEAKISNTTTDSRCPTQGEATLVEEQDANFVCRRTVVDRGWGNGCGLFGKGSLL TCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTIATITPQAPTSEIQLTDYGTLTLDCSPRTGLDFNEVVLLTMKEKSWLVHKQWFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLG SQEGAMHTALTGATEIQTSGTTTIFAGHLKCRLKMDKLTLKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSTQDEKGVTQNGRLITANPIVTDKEKPINIETEPPFGESYIIVGAGEKALKLSWFKKG Sequence ID 133 Envelope outer domain protein sequence DENV2 MRCIGISNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPT LDFELIKTEAKQPATLRKYCIEAKLTNTTTESRCPTQGEPSLNEEQDKRFICKHSMVDRGWGNGCGLFGKGGIVTCAKFTCKKNMEGKIVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSTTEAELTGYGTVTMECSPRTGLDFNEMVLLQMEDKAWLVHRQWFLDLP LPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKIVKEIAETQHGTIVIRVQYEGDGSPCKIPFEITDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIVGVEPGQLKLNWFKRG Sequence ID 134 Envelope outer domain protein sequence DENV3 FHLTSRDGEPRMIVGKNERGKSLLFKTASGINMCTLIAMDLGEMCDDTVTYKCPHITEVEPEDIDCWCNLTSTWVTYGTCNQAGEHRRDKRSVALAPHVGMGLDTRTQTWMSAEGAWRQVEKVETWALRHPGFTILALF LAHYIGTSLTQKVVIFILLMLVTPSMTMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRKLCIEGKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSL VTCAKFQCLESIEGKVVQHENLKYTVIITVHTGDQHQVGNETQGVTAEITSQASTAEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTKTPTWNRKELLVTFKNAHAKKQEVVVLGS QEGAMHTALTGATEIQTSGGTSIFAGHLKCRLKMDKLKLKGMSYAMCLNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTED...

Claims

1. An antibody that binds to the dengue virus E dimer epitope (EDE), The antibody comprises a heavy chain containing an amino acid sequence that has no amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 3, or has one or two amino acid substitutions, insertions, or deletions, and a light chain containing an amino acid sequence that has no amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 140, or has one or two amino acid substitutions, insertions, or deletions. Herein, the antibody comprises a heavy chain containing a CDR including the sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, and a light chain containing a CDR including the sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO:

25.

2. The antibody according to claim 1, wherein the antibody comprises a heavy chain containing the sequence of SEQ ID NO: 3 and a light chain containing the sequence of SEQ ID NO:

140.

3. The antibody according to claim 1, wherein the antibody comprises a heavy chain containing the sequence of SEQ ID NO: 3 and a light chain containing the sequence of SEQ ID NO:

39.

4. The antibody according to any one of claims 1 to 3, wherein the antibody is conjugated to a further drug.

5. The antibody according to claim 4, wherein the further agent is a therapeutic agent or a stabilizer.

6. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 5, further comprising a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition comprises one or more further therapeutic agents.

8. The pharmaceutical composition according to claim 7, wherein the therapeutic agent is an agent that prevents Fc receptor binding.

9. A composition comprising an antibody according to any one of claims 1 to 5, or a pharmaceutical composition according to any one of claims 6 to 8, for use in a method of treating and / or preventing a disease.

10. A composition comprising an antibody according to any one of claims 1 to 5, or a pharmaceutical composition according to any one of claims 6 to 8, for use in a method for preventing and / or treating dengue fever infection.

11. A nucleic acid encoding the antibody according to any one of claims 1 to 5.

12. A vector comprising the nucleic acid described in claim 11.

13. A host cell comprising the nucleic acid according to claim 11 and / or the vector according to claim 12.

14. The host cell according to claim 13, wherein the host cell is a C6 / C36 cell, a human dendritic cell, a CHO cell, or a Pichia pastris cell.

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