Anti-dengue vaccines 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] The present invention relates to the field of treatment and prevention of dengue virus infections and related compounds and methods. Summary of the Invention

[0002] Dengue fever infects nearly 400 million people each year 1 Dengue fever has a clinical manifestation in 25% of infections, ranging from mild illness (dengue fever) to severe cases such as dengue hemorrhagic fever. The causative agents are four serologically related viruses from the Flavivirus genus: 2 These are designated dengue virus serotypes 1-4 (DENV1-4). Infection with one serotype confers lifelong protection against that serotype but not against other serotypes. There is epidemiological evidence that severe disease is more likely to occur during secondary infections than during primary or initial DENV infections. 3、4 The rise in disease during secondary infections and the need for protection against four different serotypes sets a high bar for a vaccine that is urgently needed to prevent the estimated 400 million infections that occur each year. 1、5、6 .

[0003] This effort has been the primary focus for most DENV vaccines in development, with the aim of generating virus-neutralizing antibodies and the DENV envelope. 8、9This envelope protein is involved in receptor binding and subsequent receptor-mediated endocytosis. In the acidic environment of the endosome, the envelope protein catalyzes the membrane fusion reaction between the viral envelope and the endosomal membrane, thereby releasing the viral genomic RNA into the cytoplasm. The envelope protein is approximately 500 amino acids long and has a large N-terminal ectodomain and two transmembrane (TM) helices at the C-terminus. Its overall structure is conserved among all flaviviruses, with approximately 65% ​​amino acid sequence identity between the most distant viruses within the dengue 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 (termed "sE" for "soluble E") fold into a β-sheet-rich three-domain structure characteristic of class II viral fusion proteins, resulting in the formation of the mature virion. 8、9 Head-to-tail homodimers coating the surface of 5~7 Form.

[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 the many strikingly different compositions and conformations of the viral capsid exhibited at different stages of the viral life cycle. 11、12 "Immature" virus particles have a full complement of precursor membrane proteins (prM) in a 1:1 association of the envelope and virion, and adopt a characteristic spiky appearance, with each spiky segment composed of a trimer of prM / E proteins. 11~15 .

[0006] After 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-fold axes of symmetry; expansion of these mature particles to a "rough" morphology upon exposure to temperatures above 34°C, in which the envelope protein dimers rearrange relative to each other, has also been described recently. 16、17 After internalization into early endosomes, the acidic environment triggers a major conformational change in the envelope protein, which exposes the fusion loop and then irreversibly trimerizes to trigger membrane fusion. 18、19 .

[0007] An important additional level of complexity is that prM cleavage is often incomplete, resulting in a population of viruses with varying degrees of cleavage. 15、20 Viruses containing high levels of prM were not infectious, whereas viruses with low levels of prM remained infectious, further demonstrating that non-infectious particles with high prM could be driven to infection by antibody-dependent enhancement. 21、22 .

[0008] It is not yet clear which epitopes are the targets of most human neutralizing antibodies; see, for example, de Alwis et al. 27 suggested that the epitope requires virus assembly for formation, whereas Rey 81 suggest that the envelope dimer itself is the target.

[0009] Antibody-dependent enhancement of DENV infection (ADE) is one of the mechanisms underlying increased disease severity during secondary infection. 23 It has been proposed that antibodies formed during primary infection optimize but do not completely neutralize the virus, promoting Fc receptor-mediated uptake into myeloid cells, which drives higher viral loads during secondary infections. ADE can occur at almost any secondary neutralizing antibody concentration, and this perceived risk poses a challenge to vaccine strategies against DENV.

[0010] The leading dengue vaccine candidates currently being tested in clinical trials consist of a tetravalent formulation of live attenuated dengue or dengue / yellows chimeric viruses. 24、25 Creating a balanced tetravalent immunity without unacceptable reactogenicity has proven challenging. The most advanced vaccine candidates have recently completed Phase II clinical trials. 4 While the vaccine demonstrated much lower efficacy than expected and did not protect against DENV-2, in a Phase III trial, the vaccine reduced disease incidence by 56% (http: / / sanofipasteur.com / en / articles / theworld-s-first-large-scale-dengue-vaccine-efficacy-study-successfully-achieved-its-primary-clinical-endpoint.aspx), and in Capeding et al. (2014) Lancet Published online July 11, 2014 http: / / dx.doi.org / 10.1016 / S0140-6736(14)61060-6, it nearly halved disease in the exposed population. This discrepancy raises an urgent need to understand human antibody responses in natural dengue infection and after vaccination, specifically to identify epitopes recognized by the most potent cross-reactive antibodies generated in humans and understand correlations with protection from disease. It is therefore extremely important to provide a dengue vaccine that contains epitopes recognized by the most potent cross-reactive antibodies generated in humans.

[0011] Recent evidence indicates that the dengue virus (DENV)-specific serum Ab response in humans consists largely of cross-reactive, poorly neutralizing Abs and only a few serotype-specific, potent inhibitory Abs. 27 These bind to complex quaternary structural epitopes that are only expressed when the envelope proteins are assembled on the viral particle, meaning that intact viral particles are required to stimulate an effective immune response.

[0012] In contrast to this recent evidence, the present inventors have surprisingly identified and characterized human antibodies obtained by isolating rearranged heavy and light chain genes from single sorted plasmablasts of patients infected with dengue virus, which were found to be potently neutralizing and cross-reactive. Furthermore, they found that the epitopes bound by these antibodies were restricted to the envelope protein dimer and did not require assembly of the entire virus. Therefore, a subunit vaccine containing stabilized soluble protein E dimers is a good candidate for a successful dengue vaccine, avoiding the elicitation of antibodies against poorly immunogenic regions that are normally inaccessible on the surface of infectious virions.

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

[0014] We characterized 145 human monoclonal antibodies from patients suffering from dengue infection. The acute human antibody response was found to be focused on two major epitopes: one well-described in the fusion loop, and a second, novel epitope found in intact virions or dimers of the envelope protein, encompassing domains I, II, and III. Antibodies reactive with this epitope, the envelope dimer epitope, or EDE, were found to be able to completely neutralize virus produced in both intact and primary human cells in the low picomolar range. This novel epitope has several implications for the treatment and prevention of disease caused by dengue virus.

[0015] The present invention will be described below with reference to various embodiments of different aspects of the invention. For clarity, it will be understood that certain features of the invention that are described in the context of a separate embodiment may also be provided in combination in one or more embodiments or in a single embodiment. Conversely, for brevity, various features of the invention that are 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 embraced by the present invention and disclosed herein, just as if each and every combination were individually and explicitly disclosed herein. In addition, all subcombinations are also specifically embraced by the present invention and disclosed herein, just as if each and every such subcombination were individually and explicitly disclosed herein.

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

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

[0018] By compound is meant any compound capable of neutralizing more than one serotype of dengue virus. The compound may be, for example, a small molecule, polypeptide, or protein (these terms are used interchangeably herein), including glycoproteins, nucleic acids, carbohydrates, fats, and atoms, such as metals. In a preferred embodiment, the compound is a polypeptide, preferably an antibody, or an antigen-binding portion thereof. The antigen-binding portion may be an Fv fragment, a Fab-like fragment (e.g., a Fab fragment, a Fab' fragment, a F(ab)2 fragment, an Fv fragment, or an scFv fragment), or a domain antibody. The antigen-binding portion may be derived from the linear amino acid sequence present in an intact antibody, or may comprise a non-contiguous stretch of amino acids, optionally interspersed with other amino acids, including, for example, certain amino acids required for contacting an epitope, but not, for example, amino acids required for the framework of a natural antibody, which may in some cases be replaced, for example, by a heterologous scaffold protein. Antibodies according to the present invention can be obtained by methods known to those skilled in the art, including immunizing a mammal, such as a human, monkey, rabbit, or mouse, and / or by in vitro methods, including, for example, a phage display selection step.

[0019] By antibody, we include the meaning of substantially intact antibody molecules, as well as chimeric antibodies, humanized antibodies (in which at least one amino acid is mutated relative to a non-human antibody, e.g., a naturally occurring non-human antibody 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 antigen-binding portions and derivatives thereof.

[0020] When the compound is a protein, for example, an antibody or a fragment thereof is administered to a human subject, and if the antibody is not a human antibody or a fragment thereof, it can be humanized to reduce 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 antibodies or fragments thereof according to the invention can be improved by conjugating the neutralizing antibodies or fragments thereof to inert carriers such as albumin (Coppieters et al., 2006) or immunoglobulins (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 variants, fusions, and derivatives of any defined antibody and antigen-binding portion thereof.

[0023] Alternatively, the compound can be prepared by the method described 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 The peptide may be a cyclic peptide, for example, a multicyclic peptide, for example, a bicyclic peptide, as described in WO2009098450, 502-507, XP007913181. See also, for example, WO2009098450. Bicyclic peptides with the required binding properties can be selected, for example, by phage display.

[0024] Neutralizing means reducing the ability of the virus to infect previously uninfected cells.

[0025] Those skilled in the art will be familiar with suitable techniques for observing the virus neutralizing ability of a compound. One example of such a method is detailed in Example 3, in which one or more serotypes of dengue virus are allowed to infect a latent host cell population, the compound under assay is mixed with the virus, and the mixture is then incubated with the latent host cells. The neutralizing ability of the compound, i.e., the number of infected cells, is assayed to obtain a measure of the compound's ability to prevent infection. In certain examples, the neutralizing potential of a compound, e.g., an antibody or antigen-binding portion thereof, can be determined using a focus reduction neutralization test (FRNT), where the reduction in the number of infected foci is compared to a control (no compound). 22 Briefly, 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. Focus formation assays are performed using an anti-E mAb (4G2) followed by an HRP-conjugated rabbit anti-mouse IgG. The reaction can be visualized by adding 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 the probit program from the SPSS package. Typically, this assay can be performed in the absence of test compound, for example, so that approximately 100 foci are present in a 96-well plate well containing confluent cells, e.g., just confluent cells.

[0026] Other such examples will be known to those skilled in the art, such as the Focus Reduction Neutralization Test (FRNT), the Plaque Reduction Neutralization Test (PRNT, see WHO document http: / / whqlibdoc.who.int / hq / 2007 / who_ivb_07.07_eng.pdf, and in vivo techniques such as FRNT, mice and monkeys using flow cytometry. See, e.g., Figure 30 for examples 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 can be produced by insect cells or in human cancer cell lines (typically considered to produce high pr-M content viruses, as discussed further below), or alternatively, 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 to a specific level for a compound at a given concentration.It is understood that the appropriate concentration of a given compound may vary depending on the actual compound.For example, the concentration of a given compound used in the above assay may be 100mM, 10mM, 1mM, 100μM, 10μM, 1μM, 100nM, 10nM, or 1nM; or 0.01ug / ml, 0.02ug / ml, 0.04ug / ml, 0.05ug / ml, 0.06ug / ml, 0.075ug / ml, 0.1ug / ml, 0.25ug / ml, 0.5ug / ml, 0.75ug / ml, 1ug / ml, 1.25ug / ml, 1.5ug / ml, 1.75ug / ml, 2ug / ml, 2. The concentration may be 25 μg / ml, 2.5 μg / ml, 2.75 μg / ml, 3 μg / ml, 3.25 μg / ml, 3.5 μg / ml, 3.75 μg / ml, 4 μg / ml, 4.25 μg / ml, 4.5 μg / ml, 4.75 μg / ml, 5 μg / ml, 5.25 μg / ml, 5.5 μg / ml, 5.75 μg / ml, 6 μg / ml, 6.5 μg / ml, 7 μg / ml, 7.5 μg / ml, 8 μg / ml, 8.5 μg / ml, 9 μg / ml, 9.5 μg / ml, or 10 μg / ml or less, or less than 0.01 μg / ml. Typically, the concentration of a compound, for example, an antibody, may be, for example, less than 1 μg / ml.

[0029] For example, a compound (e.g., an antibody) may neutralize 80% of one or more serotypes of virus at a compound concentration of 0.1 μg / ml, or may neutralize at least 98%, e.g., 100%, of one or more serotypes of virus at a compound concentration of 1 μg / 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%, e.g., 100%, of one or more serotypes of virus at a concentration of 0.5 μg / ml.

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

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

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

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

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

[0035] The cross-reactive, highly neutralizing compounds identified in this invention have been found to bind to a specific epitope that can be found on both intact virus and dimers of envelope proteins, independent of virus formation. Therefore, the compounds of the invention can be defined in terms of their ability to bind to this specific epitope.

[0036] Therefore, in a further aspect of the present invention there is provided a compound that binds to a dengue virus envelope dimeric epitope (EDE), which includes the meaning of any EDE as defined herein.

[0037] A compound that binds to an envelope dimeric epitope (EDE) refers to any compound that can bind to the EDE of one or more serotypes of dengue virus. The compound can be a small molecule, a polypeptide, a nucleic acid, a carbohydrate, a fat, or an atom, such as a metal. In a preferred embodiment, the compound is a polypeptide, preferably an antibody or an antigen-binding portion thereof. The preferences for the compound are as described above.

[0038] There are four serotypes of dengue virus.Therefore, it is understood that the compound can bind to the EDE of one serotype of dengue virus.In a preferred embodiment, the compound binds to the EDE of more than one serotype of dengue virus, and binds to the above-mentioned two serotypes of dengue virus, or three serotypes of dengue virus, or four serotypes of dengue virus, that is, it will be considered to be all serotypes of dengue virus.

[0039] "Binding" includes any form of non-covalent binding between the compounds of the present invention and an epitope, molecule, macromolecule, or compound, and includes any significant degree of binding to the EDE as assessed by methods routine in the art. In a preferred embodiment, the compound selectively binds to the EDE. Selectively binding to the EDE means that the compound does not bind to or significantly bind to dengue virus or envelope proteins other than the EDE. It also means that the compound does not bind to or significantly bind to another compound, molecule, or macromolecule other than the one in question, and it would be well within the skill of one of ordinary skill in the art to present the EDE to determine whether the compound binds to the EDE. For example, an ELISA-type assay, known to those skilled in the art, may be used. In one non-limiting example of a method for determining whether the compound binds to an EDE, intact virus of one or more dengue viruses, preferably of all serotypes, and / or envelope dimers of one or more dengue viruses, preferably of all serotypes, and / or EDEs according to any of the definitions described herein, such as stabilized envelope dimers or EDEs containing residues from an envelope protein held within a heterologous scaffold, and mock non-infected supernatants are separately captured on a solid support, such as a MAXISORP immunoplate (NUNC) coated with anti-E Ab (4G2). The capture wells are then incubated with the compound, e.g., an antibody or antigen-binding portion thereof, e.g., a human monoclonal antibody, e.g., a human mAb, at 1 μg / ml, followed by incubation with a reporter-conjugated secondary antibody (which binds to the compound), e.g., ALP-conjugated anti-human IgG. The reaction is visualized by the addition of an appropriate substrate, e.g., PNPP substrate, and stopped with NaOH. For ALP / PNPP, absorbance is measured at 405 nm.

[0040] A compound that binds to the EDE includes any compound that binds to wells containing a virus or EDE, such as a stabilized soluble protein E dimer, to any degree above the background level of binding to wells containing uninfected supernatant. Preferably, the level of binding obtained to the virus or EDE, such as a stabilized soluble protein E dimer, is 2-fold, preferably 4-fold, preferably 6-fold, and more preferably 10-fold greater than the background level of binding to uninfected supernatant wells. To determine whether the compound binds to the virus or envelope protein at a site other than the EDE, the compound's ability to bind to a denatured, monomeric, or recombinant envelope protein can be assessed. If the 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 the EDE. To determine whether the compound selectively binds to an EDE other than any other molecule, macromolecule, or compound, the compound's ability to bind to the EDE can be compared to the compound's ability to bind to a molecule, macromolecule, or compound using the methods described above. A compound selectively binds to an EDE if it binds to an EDE to a significantly greater extent than it binds to another molecule or macromolecule or compound, e.g., denatured or monomeric envelope protein, e.g., if the compound binds to an EDE with at least 2-fold, 4-fold, 6-fold, 8-fold, or 10-fold greater affinity than it binds to another molecule, macromolecule, or compound, e.g., denatured or monomeric or recombinant envelope protein.

[0041] The EDE is an epitope that is considered to be formed on an intact virus particle spanning a dimer of envelope protein, or on a free dimer of envelope protein spanning two polypeptides, such as a free dimer of a soluble envelope protein. The envelope protein sequences are detailed in Figure 29 and in SEQ ID NOs: 29, 31, 33, and 35.

[0042] In a preferred embodiment, a compound of the present invention binds to EDE either in intact virus or free envelope dimers (i.e., having twice the molecular weight of the envelope polypeptide monomer), or in other structures as discussed above and further below, but does not bind to monomeric or denatured envelope proteins. In one embodiment, if a compound binds to monomeric or denatured envelope proteins, it is not considered a useful compound and is not a compound of the present invention. Thus, one non-limiting way to identify whether a compound is a compound of this embodiment of the present invention is by, for example, assaying the compound, e.g., an antibody or antigen-binding portion thereof, for its ability to bind to modified envelope proteins, e.g., in Western blots, and / or recombinant (monomeric) envelope proteins, e.g., in ELISAs, as well as to intact virus particles and / or dimers of envelope proteins (e.g., dimers of soluble envelope proteins), e.g., in ELISAs. Preferred compounds of the present invention are considered to bind to intact virus or non-denatured dimers, but not (or to a significantly lesser extent) to denatured or monomeric envelope proteins. The degree of binding can be assessed as described above.

[0043] Compounds that bind to the fusion loop but not to the EDE are not considered to be compounds of the present invention. The fusion loop is a restricted set of residues inside and outside 101W that defines the above-mentioned or classical fusion loop epitope (FL). In the fusion loop, residues 101-WGNG-104 make a distorted alpha-helical turn that protrudes the W101 side chain toward domain III, opposite the dimer interface. If a compound binds to an envelope monomer or denatured envelope protein (e.g., as determined above), it can be considered to bind to the fusion loop, although it is possible that the antibody may alternatively bind to a different part of the envelope polypeptide (which can be confirmed by binding to an envelope polypeptide mutated in the fusion loop region).

[0044] In another embodiment, compounds that bind to the fusion loop are those that are 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 invention do not bind to denatured EDE or denatured envelope proteins.

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

[0047] It will be appreciated that envelope proteins from different dengue serotypes can dimerize to form hybrid dimers. Thus, in one embodiment, the EDE to which the present compounds bind is made of envelope monomers from different dengue serotypes, and thus the EDE can comprise a homodimer or a heterodimer.

[0048] It will also be understood that the EDE can be presented in a compound as part of a virion, subviral particle, or virus-like particle, such that a dimer of envelope protein is found in an intact virion or virus-like particle. When the EDE is presented as part of a virion, subviral particle, or virus-like particle, the compound of the invention will bind to the intact virion, subviral particle, or virus-like particle, but will not bind to the monomeric or denatured envelope protein.

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

[0050] The free dimer can be presented as part of a composition containing components that stabilize protein dimerization. For example, certain buffer components that are thought to promote protein association can be used. Alternatively, the envelope protein can be presented at a high concentration that promotes dimer formation (see Example 7).

[0051] In addition to external agents that stabilize envelope dimers, envelope proteins can be engineered to have increased stability in the dimeric configuration. For example, dimers can be engineered to have increased stability in the dimeric configuration. - may be covalently stabilized by at least one, and optionally 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more disulfide interchain bonds between two envelope or sE monomers; and / or -covalently stabilized by at least one, and optionally 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more sulfhydryl-reactive crosslinkers between two sE monomers; and / or -covalently stabilized by linking two envelope or sE monomers via modified sugars; and / or -It can be non-covalently stabilized by replacing at least one amino acid residue in the amino acid sequence of at least one envelope or sE monomer with at least one bulky side chain amino acid at the dimer interface or in the domain 1 (D1) / domain 3 (D3) linker of each monomer.

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

[0053] In a preferred embodiment, the compound binds to an EDE, which is a stabilized dimer of sE, and the recombinant dengue virus envelope glycoprotein E ectodomain (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 mutant sEs thereof having at least one mutation (substitution) selected from among H27F, H27W, L107C, F108C, H244F, H244W, S255C, A259C, T / S262C, T / A265C, L278F, L292F, L294N, A313C (S313C in DENV3), and T315C. These mutations are considered to contribute to increased stability in the dimeric configuration, as described below.

[0054] Optionally, the mutant sE further comprises at least one mutation (substitution) selected from among Q227N, E174N, and D329N, preferably the three mutations Q227N, E174N, and D329N, which are considered to cover non-immunogenic regions and enable the stabilized recombinant sE dimer of the present invention to preferentially elicit neutralizing antibodies directed against all four dengue virus serotypes in a subject.

[0055] The above-described mutagenesis of the sE dimer involves introducing mutations that provide higher dimer affinity without interfering with its immunogenicity, including cysteine ​​mutations at dimer contacts to provide stabilization by cross-linking, and / or introducing new glycosylation that allows chemical cross-linking between adjacent sugars in the dimer by click chemistry and / or by replacing 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 allow the formation of a cavity at the dimer interface or in the domain 1 (D1) / domain 3 (D3) linker of each monomer.

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

[0057] The nucleic acid sequences encoding the DENV-1 sE of SEQ ID NO: 132, the DENV-2 sE of SEQ ID NO: 133, the DENV-3 sE of SEQ ID NO: 134, and the DENV-4 sE of SEQ ID NO: 135 are set forth as SEQ ID NOs: 136, 137, 138, and 139, respectively.

[0058] As used herein, the term "recombinant" refers to the use of genetic engineering methods (cloning, amplification) to produce the dengue virus envelope glycoprotein E ectodomain, antibody, or antibody fragment thereof of 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, with the dimer preferably being a homodimer.

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

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

[0062] Advantageously, the stabilized dimer contains a single cysteine ​​mutant sE positioned along the dimer's two-fold molecular axis, resulting in a single interchain disulfide bond, or multiple (e.g., double) cysteine ​​mutants sE that can create multiple (e.g., two) disulfide bonds away from the two-fold molecular axis. The disulfide bonds can be synthesized under acidic conditions, for example, using DMSO solution (O. Khakshoor et al., 2009) or using an oxidizing agent such as CdCl or CuSO. Thus, the stabilized dimer can consist of monomers in which one amino acid residue of each monomer is substituted with cysteine ​​along the (approximately) two-fold molecular axis of the dimer. The stabilized dimer can also consist of monomers in which two amino acid residues of each monomer away from the dimer's two-fold molecular axis are substituted with cysteine. The stabilized dimer may also consist of monomers in which three amino acid residues of each monomer away from the two-fold molecular axis of the dimer are substituted with cysteines.

[0063] It may be desirable to have more than one interchain disulfide bond, as such an arrangement may limit access to the FLE region and therefore reduce the ability of the molecule to generate an anti-FLE response, as further discussed in Example 17.

[0064] In another preferred embodiment, the compound binds to EDE, and the EDE is a stabilized dimer of sE, and the stabilized dimer of envelope or recombinant sE is a homodimer of mutant sE having the mutation A259C or S255C, respectively, as defined above, wherein residues 259C or 255C are linked together by a disulfide interchain bond.

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

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

[0067] In one embodiment, the compound binds to an EDE, which is a stabilized dimer of sE, and the stabilized dimer of envelope or recombinant sE is a heterodimer of a mutant sE having the mutations F108C and A313C as defined above and a mutant sE having the mutations L107C and T315C as defined above, wherein residues 108C and 313C are linked 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 dimer of recombinant sE, wherein the stabilized recombinant sE dimer is selected from the group consisting of a homodimer of mutant sE having the mutations A259C, F108C, and T315C, respectively, a homodimer of mutant sE having the mutations S255C, F108C, and T315C, respectively, a homodimer of mutant sE having the mutations A259C, L107C, and A313C, respectively, and a homodimer of mutant sE having the mutations A255C, L107C, and A313C, respectively, as defined above, wherein residues 259C, 255C, 108C, 315C, 107C, and 313C are linked to residues 259C, 255C, 315C, 108C, 313C, and 107C, respectively, by disulfide interchain bonds.

[0069] In another preferred embodiment, the compound binds to EDE, and the EDE comprises a stabilized dimer of recombinant sE, wherein the stabilized recombinant sE dimer is a heterodimer of a mutant sE having the mutations A259C, F108C, and T315C as defined above and a mutant sE having the mutations S255C, F108C, and T315C as defined above, wherein residues 259C, 108C, and 315C are linked 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, wherein the stabilized recombinant sE dimer is a heterodimer of a mutant sE having the mutations S255C, L107C, and A313C as defined above and a mutant sE having the mutations A259C, L107C, and A313C as defined above, wherein residues 255C, 107C, and 313C are linked to residues 259C, 313C, and 107C, respectively, by disulfide interchain bonds.

[0071] It will be appreciated that, as well as stabilization by disulfide bonds, stabilization can also be achieved by sulfhydryl-reactive crosslinkers. Thus, in one embodiment, EDE comprises a stabilized dimer of recombinant sE, and the stabilized recombinant sE dimer of the present invention is covalently stabilized by at least one, two, or three sulfhydryl-reactive crosslinkers (also called thiol-reactive crosslinkers) between the sE monomers.

[0072] Chemical cross-linking of proteins is known in the art (for review see Hemaprabha, 2012).

[0073] Of course, the sE dimer has two distinct faces, one exposed to the extracellular medium where antibodies bind, and the other exposed to the viral membrane.

[0074] Advantageously, the stabilized recombinant sE dimer contains candidate amino acid residues that are present on the face of sE exposed to the viral membrane and therefore not part of the epitope. One of each candidate amino acid residue in each monomer is mutated (substituted) to a cysteine ​​to generate a free sulfhydryl group that is the target of a sulfhydryl-reactive crosslinker of appropriate length.

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

[0076] In a preferred embodiment, the compound binds to EDE, wherein the EDE comprises a stabilized dimer of recombinant sE, wherein the stabilized recombinant sE dimer is a homodimer of mutant sE having the mutations T / S262C or T / A265C, respectively, as defined above, wherein residues 262C or 265C are linked together by a sulfhydryl-reactive crosslinker.

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

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

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

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

[0081] Sulfhydryl-reactive crosslinkers are preferably homobifunctional reagents containing identical or non-identical reactive groups, allowing for the construction of intermolecular crosslinks between two monomers. Homobifunctional crosslinkers have identical reactive groups at either end of a spacer arm, and generally, they can be used in a one-step reaction procedure. Sulfhydryl-reactive crosslinkers of the present invention can be maleimides, haloacetyls (preferably bromo- or iodoacetyls), pyridyl disulfides, vinyl sulfones, alkyl halides, 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 crosslinkers according to the present invention with spacers of different lengths include BMOE (1,2-bis-maleimidoethane), BMB (1,4-bis-maleimidobutane), BMH (1,6-bis-maleimidohexane), TMEA (tris-(2-maleimidoethyl)amine), BM(PEG)2 (1,8-bismaleimidodiethylene glycol), BM(PEG)3 (1,11-bismaleimidotriethylene glycol), BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane), DTME (dithio-bis-maleimidoethane), and preferably BMH, BM(PEG)2, and BM(PEG)3. [ka]

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

[0084] It will be appreciated that in addition to stabilization via disulfide bonds and sulfhydryl-reactive crosslinkers, stabilization can be obtained by linking two monomers with modified sugars. To this end, glycosylation sites are inserted therein and reacted with modified sugars to link them by click chemistry.

[0085] In accordance with this embodiment, the compound binds to an EDE, wherein the EDE comprises a stabilized dimer of recombinant sE, wherein the stabilized recombinant sE dimer is a homodimer or heterodimer of a 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 bearing an X functional group; the other sE monomer has at least one mutation introducing a glycosylation site, wherein the mutated amino acid residue is glycosylated with a modified sugar carrying a Y functional group; Both mutated residues are linked together via modified sugars, specifically by click chemistry, by 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] By X functionality is meant a chemical group carried by the sugar that can react with the Y functionality to form a covalent bond by click chemistry, said Y functionality being preferably an azide functionality.

[0087] By Y functionality is meant a chemical group carried by the sugar that can react with the X functionality to form a covalent bond by click chemistry, said X functionality being preferably a terminal alkyne functionality.

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

[0089] In addition to the covalent bonding methods described above for stabilizing dimers, non-covalent bonding means may also be used. Thus, in another embodiment in which the EDE comprises a stabilized dimer of recombinant sE, the dimer is non-covalently stabilized 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, thereby filling the cavity of the dimer at the dimer interface. In accordance with this embodiment, the cavity unique to the quaternary conformation of the recombinant sE dimer is identified and filled by engineered hydrophobic substitutions in the monomers.

[0090] According to this embodiment, the stabilized recombinant sE dimer is non-covalently stabilized 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 within the cavity-forming region at the dimer interface or in the domain 1 (D1) / domain 3 (D3) linker of each monomer. Such substitution can increase hydrophobic interactions between the two sE monomers.

[0091] In a preferred embodiment, the EDE comprises a stabilized dimer of recombinant sE. The stabilized recombinant sE dimer is a homodimer or heterodimer, preferably a homodimer, of two recombinant sEs as defined above, wherein one or two of the recombinant sEs has 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 mimic the conformation of F279 in virions.

[0092] Other means of non-covalently stabilizing dimers include, for example, non-covalent stabilization by substituting one or two, preferably two, amino acids in the amino acid sequences of the monomers 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 binds to an EDE, which comprises a stabilized dimer of recombinant sE, the stabilized recombinant sE dimer being a homodimer or heterodimer, preferably a homodimer, of two recombinant sEs as defined above, one or two of which have at least one mutation (substitution) selected from the group consisting of L292F and L294N. These mutations, L292F and L294N, are considered to enable stabilization of the D1-D3 linkers in the sE dimeric conformation.

[0094] In a preferred embodiment in which the EDE is stabilized in a dimeric configuration by engineering, such engineering does not result in a change in the overall 3D structure of the dimer or does not substantially alter the overall 3D structure, and the residues in the native dimer spatially correspond to those in the engineered dimer. When the native dimer spatially corresponds to the engineered dimer, this means that when a 3D model of the engineered dimer (or a portion thereof, e.g., reflecting residues of particular importance in characterizing the VDR, e.g., residues shown in Table 2 and / or further discussed below) is superimposed on the 3D model of the native dimer, the coordinates characterizing the spatial positions of backbone atoms in the native dimer differ by less than about 10 angstroms from the coordinates characterizing the analogous backbone atoms in the engineered dimer. The backbone atoms may be those of the amino acids forming the peptide backbone, or the 3D folding pattern may not include side chain atoms, i.e., the positions of some or all of the side chain atoms may not be significantly changed as well. The 3D structure is key to whether VDE is immunogenic, and therefore, in a preferred embodiment, the engineering does not result in a dimer with reduced immunogenicity. In one embodiment, the engineering does not result in a dimer with a different 3D conformation. Preferably, the engineering results in a dimer with increased immunogenicity. Such an approach is described in Ref. 84 Thus, in one embodiment, the compounds bind to engineered EDEs as described above.

[0095] A 3D model of the native dimer can be generated using information on the crystal structure of the envelope glycoprotein ectodomain from dengue virus serotypes, e.g., serotypes 2, 3, and 4, available in the Protein Data Bank under the aforementioned accession 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 an antibody described herein that is known to bind to VDE can still bind to an engineered version of VDE.

[0097] Those skilled in the art can use, for example, computer programs to help identify potential stabilizing modifications.

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

[0099] Alternatively, modified envelope proteins can be expressed in dengue virus and the ability of compounds to neutralize the virus assessed.

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

[0101] Thus, in one embodiment, the EDE comprises a portion of an epitope scaffold protein, the scaffold protein comprising a heterologous scaffold protein covalently linked to an envelope dimer epitope. A scaffold protein is useful for generating the EDEs of the present invention in that it holds the contact residues of the EDE in the appropriate spatial orientation to facilitate interactions between such residues and the compound, e.g., between the contact residues of the compound, when the compound is a protein, optionally an antibody or antigen-binding portion thereof. A contact residue is any amino acid present in a molecule that directly or indirectly interacts with an amino acid of another molecule (e.g., forms an ionic bond directly or indirectly through a salt bridge). Residues of envelope proteins considered potentially important in compounds binding to the EDE, at least for DENV-1, are detailed in Table 2, and the scaffold protein may represent the entire dimer or only selected residues above. A 3D model of the native dimer or a portion thereof can be generated using information on the crystal structure of the envelope glycoprotein ectodomain from dengue virus serotypes, e.g., serotypes 2, 3, and 4, available in the Protein Data Bank under the aforementioned accession numbers 1OAN, 1OK8, 1UZG, and 3UAJ.

[0102] Mutational analysis revealed specific residues in DENV1 and DENV2 that are important for binding to the antibodies identified in this 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, as well as Q77, W101, N153, T155, and K310.

[0104] Thus, in one embodiment, the compound binds to an EDE, where the EDE is part of a scaffold protein that retains at least residues 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 equivalent residues in the dengue virus envelope proteins, specifically DENV-1 and DENV-2. Certain residues are considered more important, and thus further embodiments of the EDE include scaffold proteins that retain at least one or more residues corresponding to Q77, W101, N153, T155, K310 of the dengue virus envelope protein, or equivalent residues in the dengue virus envelope proteins, specifically DENV-1 and DENV-2.

[0105] Residues of the envelope protein that are considered important for contacting the epitope are shown in Figure 31, e.g., The B7 antibody appears to contact the 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 believed to contact the 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 believed to contact the 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 believed to contact the 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 believed to contact the 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 protein that are considered to be important in binding to compounds, specifically DENV2 and DENV4, are: 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, 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 or the equivalent residues of the dengue virus envelope protein.

[0107] The skeletal protein is 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, and K or may represent one or more residues selected from both a series 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, V309V324, V97, or equivalent residues, or may represent at least one or more 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 scaffold protein may exhibit any one or more or all of the following set of residues that are considered to increase the stability of the dimeric configuration: H27F, H27W, L107C, F108C, H244F, H244W, S255C, A259C, T / S262C, T / A265C, L278F, L292F, L294N, A313C, and T315C, as described above.

[0109] The scaffold protein may retain a dimer, or a fragment of a dimer, may contain any of the modifications described above that are considered essential for immunogenicity, and / or may result in increased dimer stability, e.g., increased disulfide bonds.

[0110] Furthermore, the scaffold can be one that exhibits an improved EDE. 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 tend to have either antibodies directed toward the VDE, which are considered useful antibodies, or antibodies directed toward the fusion loop (anti-FL antibodies), which are not considered useful. Therefore, the scaffold can be engineered to display only the EDE, and in a manner that eliminates the possibility of antibodies or antigen-binding portions thereof being generated in the compound, e.g., FL. Thus, in a preferred embodiment, the EDE can be optionally incorporated into the scaffold protein, thereby generating antibodies to the EDE rather than the FL.

[0111] Independently of the scaffold protein, the envelope protein can be engineered to produce an improved EDE. As mentioned above, an EDE that cannot be recognized by anti-FL antibodies and cannot generate such antibodies is considered to be 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 scaffold protein without using any antigen that can generate anti-FL antibodies.

[0112] In one embodiment, the envelope protein is engineered by adding N- or O-linked glycan sequences to modify the inner surface of the dimer (which protrudes into the inside of the virus) and make it less immunogenic.

[0113] Extensive mutagenesis to attach new surfaces to the dimer may be useful to further reduce the generation of non-ED suboptimal reactions by mutating residues and / or adding glycans.

[0114] As an example, the L278F mutation is thought to reshape the kl loop and mimic a virion-like conformation.

[0115] Modeling and optimization of core EDE epitopes can also be useful to generate optimal sequences for eliciting the desired EDE response and to provide binding and neutralizing antibodies.

[0116] It will be appreciated that the EDE may be a native envelope protein retained within a scaffold, which affects increased dimer stability. The EDE may also be engineered to increase dimer stability independent of any scaffold. The two may be combined, in one embodiment, to include dimers in which the envelope protein retained within a heterologous scaffold protein is engineered to have improved stability in the dimer configuration. Alternatively, the envelope protein may be engineered so that only the relevant portion of the protein is present, which may be retained in the heterologous scaffold protein.

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

[0118] Any reference to an envelope protein, sE, sE dimer, or envelope protein dimer also includes within its scope the scaffold protein or structure, including specific residues that are held in a particular conformation to make an EDE and represent a suitable EDE.

[0119] The envelope nucleotide sequence may be engineered so that the envelope protein has any one or more mutations, insertions, or deletions. The nucleotide sequence may be such that it has 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(s) thereof, e.g., one or more portions of at least 8, 9, or 10 consecutive amino acids) from another dengue virus serotype. 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 portion(s), e.g., one or more portions of at least 8, 9, or 10 consecutive amino acids) from all serotypes of dengue virus, more preferably four different envelope proteins (or portion(s), e.g., one or more portions of at least 8, 9, or 10 consecutive amino acids), and most preferably all envelope proteins (or portion(s), e.g., one or more portions of at least 8, 9, or 10 consecutive amino acids).

[0121] As described above, envelope proteins can actually be engineered to have very low homology to native envelope proteins, while maintaining the integrity and conformation of the EDE, or the EDE can be modified in such a way that it is not possible to raise anti-FL antibodies, for example. Therefore, the level of sequence homology does not necessarily indicate 3D structural homology or functional homology. For example, a particular sequence encoding a structure containing an EDE may actually have a very low level of homology to native envelope proteins, but 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 native envelope proteins, and the nucleotide sequence encoding this structure may have low sequence identity relative to the native envelope sequence.

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

[0123] Some of these residues are considered more important than others, and therefore in further embodiments of the EDE, the envelope protein or structure comprising the EDE includes one or more, or optionally all, of Q77, W101, N153, T155, K310, or the 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 a dengue virus protein, are considered to be required for binding of the compound to the EDE. Thus, in one embodiment, an envelope protein or structure comprising an EDE comprises one or more, or all, of these residues.

[0125] Anti-FL antibodies appear in most cases to require only residue W101 among the mutated residues in alanine scanning analysis (Example 2) and are unaffected by mutations of any of the other residues, whereas anti-EDE antibodies require a much larger epitope and require the presence of residue W101, as do anti-FL antibodies, but are affected by mutations at many other residues. Thus, in one embodiment, EDE is defined as an epitope in which residue W101 and at least one of positions E49, K64, Q77, W101, V122, N134, N153, T155, I161, A162, P169, T200, K202, E203, L308, K310, Q323, W391, F392, or equivalent residues in the envelope dimer epitope are required for binding of the compound.

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

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

[0128] According to one embodiment, the compound of the present invention contacts the N67 glycan chain of the envelope protein dimer or the N153 glycan chain of the envelope protein dimer. It is understood that the compound can contact both the N67 and N153 glycan chains of the envelope protein dimer.

[0129] In a particular example, the compound is an antibody whose CDR H2 interacts with the N67 glycan chain of an envelope protein.

[0130] In one embodiment, the compound is directed against one or more serotypes of dengue virus, preferably all serotypes of dengue virus envelope proteins, if present, such as 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, I374 in DENV-2 or DENV-4. Contact with EDE at any one or more of 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 comprises a region centered in the cleft formed by the b-strand on the domain II side and the "150 loop" on the domain I side (opposite the dimer interface) (see, e.g., Figure 29), where the 150 loop spans residues 148-159, connects b-strands E0 and F0 of domain I, and carries 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 in the 150 loop of SEQ ID NO: 148, Denv 2 EHAVGNDTGKHG in the 150 loop of SEQ ID NO: 1149, Denv 3 QHQVGNETQG in the 150 loop of SEQ ID NO: 150, and Denv 4 THAVGNDIPNHG in the 150 loop of SEQ ID NO: 151.

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

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

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

[0135] Characterization of the binding of the antibody fragment thereof according to the invention to the polypeptide segment or amino acid residues can be carried out, for example, by crystallization studies as described in the examples below.

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

[0137] In one embodiment, the EDE comprises a dimer of a full-length envelope protein. In another embodiment, the EDE comprises a dimer of an envelope ectodomain (sE). In a further embodiment, the envelope protein comprises the 400 amino-terminal residues (about, as discussed above) of the ectodomain of the envelope protein. See, e.g., Figure 28. The above preference for stability of a dimer of a full-length envelope protein also applies to a truncated ectodomain of an envelope protein. Thus, a dimer of an ectodomain of an envelope protein may be stabilized through engineering, or by incorporation into a scaffold protein, or may comprise a hybrid dimer.

[0138] In a further embodiment, the compounds of the present invention will not bind to dengue viruses or virions or subviral particles or virus-like particles incubated at acidic pH. Acidic pH causes envelope proteins to irreversibly adopt a trimeric configuration. The inventors have found that compounds of the present invention do not bind to virus particles at low pH (see Example 4). Thus, in one embodiment, a compound, e.g., an antibody or antigen-binding portion thereof, does not bind to dengue viruses or virions or subviral particles or virus-like particles incubated at acidic pH. By acidic pH is meant any pH below 7, preferably pH 5.5.

[0139] Thus, one of skill in the art can readily identify whether a particular compound is a compound of the invention according to this embodiment of the invention by simply determining whether the compound is unable to bind to one or more of: a) virions or subviral or virus-like particles made in cells lacking furin activity, b) virions or subviral or virus-like particles having a high proportion of prM protein, and / or c) virions or subviral or virus-like particles incubated under acidic conditions.

[0140] Methods for assaying the ability of the compounds to bind to the virions, subviral particles, or virus-like particles described above are provided above and detailed in Example 4 with respect to assaying the ability of the compounds to bind to EDE, and generally simply involve an ELISA against the particular virion or virus-like particle to assay whether the compound is able to bind. Compounds are considered useful if they bind or significantly bind to native EDE or virions or virus-like particles, as well as if they do not bind to virions or subviral particles or virus-like particles that: a) are made in cells lacking furin activity, b) have a high proportion of prM protein, and / or c) are incubated under acidic conditions.

[0141] The present invention further includes specific compounds. For example, in one embodiment, the compounds are antibodies comprising 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 truncations and mutations of these antibodies, and therefore, the compounds are understood to be antigen-binding portions thereof. Antibodies having at least 90% or at least 95% sequence identity to the above sequences are included in the present invention. Specific sequences of the light and heavy chains of the antibodies are given in SEQ ID NOs: 1-4, 37-141, 141-147, e.g., in Figure 29.

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

[0143] Certain residues of particular heavy and light chains are considered important for binding to EDE. Thus, in one embodiment, where sequence homology is at least 90% to the above sequences, the following residues, if present: SEQ ID NO: 1 - T52, E54, D56, S57, A58, K65, G66, T69, E82, N84, S85, Y100, N102, F103, Y104, Y105, Y106, SEQ ID NO: 2 - G554, N55, N57, K59, Q62, Q65, G66, R94, R98, F99, Y100, Y101, D102, S103, T104, Y106, Y107, P108, D109, S110, D117, V118, SEQ ID NO: 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, SEQ ID NO: 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, SEQ ID NO: 37 - S30, T31, F32, Y49, D50, S52, R54, R66, R91, Y92, N93, W94, SEQ ID NO: 38 - S26, S27, G30, G31, F32, N33, Y34, D52, T54, S55, R56, S62, S95, R96, G97, SEQ ID NO: 39 - Y51, R56, P57, S58, G59, S96, R97, SEQ ID NO: 40 - Y51, R56, P57, S58, K97.

[0144] Antibodies consist of a light chain and a heavy chain, and within each light and heavy chain there are three variable regions. The most variable parts of each of these regions are the complementarity determining regions, which are considered to be the most essential for antigen binding and recognition. Thus, in one embodiment, the compound comprises one or more of the following amino acid sequences with none, one or two amino acid substitutions, insertions, or deletions: SEQ ID NO: 5 or SEQ ID NO: 8 or SEQ ID NO: 11 or SEQ ID NO: 14 and / or SEQ ID NO: 6 or SEQ ID NO: 9 or SEQ ID NO: 12 or SEQ ID NO: 15 and / or SEQ ID NO: 7 or SEQ ID NO: 10 or SEQ ID NO: 13 or SEQ ID NO: 16 and / or SEQ ID NO: 17 or SEQ ID NO: 20 or SEQ ID NO: 23 or SEQ ID NO: 26 and / or SEQ ID NO: 18 or SEQ ID NO: 21 or SEQ ID NO: 24 or SEQ ID NO: 27 and / or SEQ ID NO: 19 or SEQ ID NO: 22 or SEQ ID NO: 25 or SEQ ID NO: 28.

[0145] Particular compounds may include the following sequences with no amino acid substitutions, insertions, or deletions, one or two amino acid substitutions, insertions, or deletions. Heavy chain: SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 or SEQ ID NO: 8 and SEQ ID NO: 9 and SEQ ID NO: 10 or SEQ ID NO: 11 and SEQ ID NO: 12 and SEQ ID NO: 13 or SEQ ID NO: 14 and SEQ ID NO: 15 and SEQ ID NO: 16 and / or Light chain: SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19 or SEQ ID NO: 20 and SEQ ID NO: 21 and SEQ ID NO: 22 or SEQ ID NO: 23 and SEQ ID NO: 24 and SEQ ID NO: 25 or SEQ ID NO:26 and SEQ ID NO:27 and SEQ ID NO:28.

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

[0147] In a further embodiment, certain residues in the above sequence are considered to be important for antigen binding. Thus, in this embodiment, the following residues, if present: Residue 3 of SEQ ID NO:6 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; Residue 2 of SEQ ID NO:7 is Y, residue 4 is N, residue 5 is F, residue 6 is Y, residue 7 is Y, and residue 8 is Y; Residue 5 of SEQ ID NO:9 is G, residue 6 is N, residue 10 is K, residue 13 is Q, residue 16 is Q, and residue 17 is D; Residue 5 of SEQ ID NO: 10 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; Residue 1 of SEQ ID NO:11 is N; Residue 5 of SEQ ID NO:12 is D, residue 7 is S, residue 8 is T, residue 9 is R, residue 16 is K, and residue 17 is G; Residue 4 of SEQ ID NO: 13 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; Residue 1 of SEQ ID NO:14 is S; Residue 5 of SEQ ID NO: 15 is D, residue 7 is S, residue 8 is S, residue 9 is T, and residue 17 is G or H; Residue 4 of SEQ ID NO: 16 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; Residue 7 of SEQ ID NO: 17 is S, residue 8 is T, and residue 9 is F; Residue 1 of SEQ ID NO:18 is D, residue 3 is S, and residue 5 is R; Residue 3 of SEQ ID NO:19 is R, residue 4 is Y, and residue 5 is N; Residue 4 of SEQ ID NO:20 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; Residue 1 of SEQ ID NO:21 is D, residue 3 is T, residue 4 is S, and residue 5 is R; Residue 5 of SEQ ID NO:22 is S, residue 6 is R, and residue 7 is G; Residue 5 of SEQ ID NO:24 is R, residue 6 is P, and residue 7 is S; Residue 6 of SEQ ID NO:25 is S and residue 7 is R; Residue 5 of SEQ ID NO:27 is R, residue 6 is P, and residue 7 is S.

[0148] As described above with respect to the presentation of antigenic EDEs in a protein scaffold, the compound, e.g., a protein, e.g., an antibody, may also be part of a larger structure, e.g., held within a protein scaffold. Preferences for this scaffold are as described above. For example, in one embodiment, an antibody, or antigen-binding portion thereof, is present within a larger polypeptide.

[0149] In a preferred embodiment, a compound that binds to an EDE according to any of the above embodiments also neutralizes dengue virus, preferably by at least 80%, preferably 90%, more preferably 95% or 98%, 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% or 98%, and most preferably 100%. Preferences for neutralization, including the concentration of compound, virus or subvirus or virus-like particle, and host cell, are as described above for the first aspect of the invention.

[0150] Regarding the first aspect of the present invention, it is preferred that the compound capable of binding to EDE can neutralize viruses produced in both insect cells, such as C6 / 36 insect cells, and human cells, such as primary human cells, such as dendritic cells. Preferably, the compound neutralizes dengue viruses produced in both insect cells, such as C6 / 36 insect cells, and human cells, such as primary human cells, such as dendritic cells, to the same level, as discussed above. The ability of the compound to neutralize viruses 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 viruses produced in both insect cells and human cells.

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

[0152] In one embodiment, the antibody or antigen-binding portion thereof is or is derived from a monoclonal antibody. In another embodiment, the antibody or antigen-binding portion thereof is or is derived from a polyclonal antibody. In a further embodiment, the compound is a) a mixture of monoclonal antibodies or antigen-binding portions thereof; b) a mixture of polyclonal antibodies or antigen-binding portions thereof, or c) A composition comprising a mixture of monoclonal and polyclonal antibodies or antigen-binding portions thereof, for example, a mixture of antibodies or antigen-binding portions thereof, wherein the ratio of monoclonal antibodies to polyclonal antibodies or antigen-binding portions thereof 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 the compound may be a recombinant protein, such as a recombinant antibody or antigen-binding portion thereof. The compound may also be synthetically produced. The compound may be a combination of recombinant and synthetically produced.

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

[0155] Thus, the compounds may be produced by recombinant means, e.g., the compounds, e.g., polypeptides, e.g., antibodies or antigen-binding portions thereof, a) a human cell line, optionally CHO cells, or b) a mammal, optionally a human, or c) a microorganism, or It will also be appreciated that the polypeptides may be produced and isolated or purified from a variety of organisms, including d) insect cell lines.

[0156] By isolated or purified, it is meant that the agent is removed from its natural environment and does not reflect the extent to which the agent has been purified.

[0157] Thus, the present invention includes the isolation or purification of the compounds of the present invention from a variety of organisms, including human cell lines, optionally CHO cells, or mammalian, optionally human, or microbial, or insect cell lines.

[0158] When the compound is a polypeptide, e.g., an antibody or antigen-binding portion thereof, or is, for example, contained in a protein scaffold, the compound may be encoded by a nucleic acid. Nucleic acid includes both DNA and RNA, single- or double-stranded, and in all their various forms. Thus, the present invention includes nucleic acids encoding any of the proteinaceous compounds of the present invention. Specifically, SEQ ID NOS: 41-48 are included in the present invention. Included are any sequences comprising SEQ ID NOS: 41-48 derived from or containing mutations that may result in silent mutations, such as sequences encompassing any of the possibilities mentioned above, e.g., nucleic acid sequences containing portions encoding any of the following: SEQ ID NO: 1 or a sequence having at least 90% homology to SEQ ID NO: 1; SEQ ID NO: 2 or a sequence having at least 90% homology to SEQ ID NO: 2; SEQ ID NO: 3 or a sequence having at least 90% homology to SEQ ID NO: 3; SEQ ID NO: 4 or a sequence having at least 90% homology to SEQ ID NO: 4; SEQ ID NO: 37, or a sequence having at least 90% homology to SEQ ID NO: 37; SEQ ID NO: 38, or a sequence having at least 90% homology to SEQ ID NO: 38; SEQ ID NO: 39, or a sequence having at least 90% homology to SEQ ID NO: 39; SEQ ID NO: 40, or a sequence having at least 90% homology to SEQ ID NO: 40; a sequence having at least 90% homology to SEQ ID NO: 1, wherein the residues are 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, wherein the residues are G554, N55, N57, K59, Q62, Q65, G66, R94, R98, F99, Y100, Y101, D102, S103, T104, Y106, Y107, P108, D109, S110, D117, V118; a sequence with at least 90% homology to SEQ ID NO: 3, wherein the residues are 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 with at least 90% homology to SEQ ID NO: 4, wherein the residues are 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 with at least 90% homology to SEQ ID NO: 37, wherein the residues are S30, T31, F32, Y49, D50, S52, R54, R66, R91, Y92, N93, W94; A sequence with at least 90% homology to SEQ ID NO: 38, wherein the residues are S26, S27, G30, G31, F32, N33, Y34, D52, T54, S55, R56, S62, S95, R96, G97; a sequence having at least 90% homology to SEQ ID NO: 39, wherein the residues are Y51, R56, P57, S58, G59, S96, R97; a sequence having at least 90% homology to SEQ ID NO: 40, wherein the residues are Y51, R56, P57, S58, K97; SEQ ID NO: 5 or a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions or deletions compared to SEQ ID NO: 5; SEQ ID NO: 6, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 6, wherein 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; SEQ ID NO: 7, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 7, wherein 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; SEQ ID NO: 8 or a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions or deletions compared to SEQ ID NO: 8; SEQ ID NO: 9, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 9, wherein 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; SEQ ID NO: 10, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 10, wherein 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; SEQ ID NO: 11, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 11, wherein residue 1 is N; SEQ ID NO: 12, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 12, wherein 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; SEQ ID NO: 13, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 13, wherein 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; SEQ ID NO: 14, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 14, wherein residue 1 is S; SEQ ID NO: 15, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 15, wherein residue 5 is D, residue 7 is S, residue 8 is S, residue 9 is T, and residue 17 is G or H; SEQ ID NO: 16, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 16, wherein 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; SEQ ID NO: 17, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 17, wherein residue 7 is S, residue 8 is T, and residue 9 is F; SEQ ID NO: 18, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 18, wherein residue 1 is D, residue 3 is S, and residue 5 is R; SEQ ID NO: 19, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 19, wherein residue 3 is R, residue 4 is Y, and residue 5 is N; SEQ ID NO: 20, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 20, wherein 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; SEQ ID NO: 21, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 21, wherein residue 1 is D, residue 3 is T, residue 4 is S, and residue 5 is R; SEQ ID NO: 22, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 22, wherein residue 5 is S, residue 6 is R, and residue 7 is G; SEQ ID NO: 23 or a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions or deletions compared to SEQ ID NO: 23; SEQ ID NO: 24, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 24, wherein residue 5 is R, residue 6 is P, and residue 7 is S; SEQ ID NO: 25, or a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 25, optionally wherein residue 6 is S and residue 7 is R; SEQ ID NO: 26 or a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions or deletions compared to SEQ ID NO: 26; SEQ ID NO: 27, or optionally a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 27, wherein residue 5 is R, residue 6 is P, and residue 7 is S; and SEQ ID NO:28, or a sequence resulting in a polypeptide having one or two amino acid substitutions, insertions, or deletions compared to SEQ ID NO:28.

[0159] The nucleic acid may or may not contain introns. The nucleic acid may also be modified to allow for subsequent purification of the translated polypeptide, for example, the open reading frame of the polypeptide of interest may be modified to incorporate a tag, such as a myc tag or a his tag, to allow for subsequent purification.

[0160] The nucleic acids may also be modified, for example, to optimize codons for better translation by the organism in which they are to be translated, without affecting the final polypeptide sequence.

[0161] The nucleic acids of the present disclosure can be produced or modified using a number of methods well known to those of skill 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 a nucleic acid. A vector refers to a vehicle for cloning or amplifying a nucleic acid or for insertion into a target organism. For example, a vector may be a plasmid or a nucleic acid used to target a nucleic acid of the present invention into a target organism, for example, into the genome of the target organism. A vector may further contain a nucleotide sequence required for expression of a polypeptide encoded by a nucleic acid of the present invention. For example, a promoter sequence or a termination sequence may be operably linked to the nucleic acid of the present invention, or may contain a reporter gene, such as an antibiotic resistance cassette. A 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 capable of binding to the above-mentioned EDEs, a further aspect of the present invention also provides EDE compounds as defined below. The present invention also provides nucleic acids or vectors encoding the EDE compounds of the present invention, as well as host cells comprising the nucleic acids or vectors. For example, the preferences for the nucleic acids and vectors described above may also be relevant to this aspect of the present invention, as will be apparent to those skilled in the art. Thus, the present invention provides EDE compounds as defined below, nucleic acids encoding such EDE compounds, or vectors comprising the nucleic acids, or host cells comprising the nucleic acids or vectors.

[0164] EDE compounds are intended to provide epitopes described above as envelope-dependent epitopes. EDE compounds may specifically bind to antibodies specific for one or more EDEs of the invention, such as preferred neutralizing antibodies, as discussed above or illustrated in the Examples. EDE compounds are typically polypeptides or include polypeptides. In one embodiment, the EDE compound is an envelope protein dimer, or an envelope ectodomain, or the 400 amino-terminal residues of an envelope protein ectodomain. As used herein, "400 amino-terminal residues" includes approximately 400 amino-terminal residues, e.g., 350-450 residues, 320-470 residues, or 330-480 residues (or combinations thereof), e.g., 380-420 residues, e.g., 390-410 residues, e.g., 395 or 393 residues, as described above and as would be apparent to one skilled in the art. The envelope protein may be any of the envelope proteins from DENV-1, DENV-2, DENV-3, and DENV-4 (SEQ ID NO: 29, 31, 33, or 35), or proteins having at least 90% homology to the sequence of SEQ ID NO: 29, 31, 33, or 35. The dimer may be a homodimer or a heterodimer. In preferred embodiments, the dimer is not incorporated into an intact virus, subviral, or virus-like particle, but rather is a free dimer having, for example, twice the molecular weight of the monomeric envelope polypeptide. It will be understood that any form of the EDE or EDE compound described herein, e.g., an engineered envelope protein, may potentially be presented as part of a virus, virus-like particle, or subviral particle, e.g., as part of a protein scaffold.

[0165] In another embodiment, the EDE compound comprises a dimer of an envelope protein, or an envelope ectodomain, or the (approximately) 400 amino-terminal residues of an envelope protein ectodomain, that has been engineered to have increased stability in the dimeric configuration, e.g., engineered to have an increased level of covalent and / or non-covalent bonding between the dimers.

[0166] In a preferred embodiment, the EDE compound is a stabilized recombinant dengue virus envelope glycoprotein E ectodomain (recombinant sE) dimer in accordance with the above-described aspects of the invention, such as a stabilized recombinant dengue virus envelope glycoprotein E ectodomain (recombinant sE) dimer, which 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 crosslinker between two sE monomers; and / or -covalently stabilized by linking two sE monomers via a modified sugar group; and / or - non-covalently stabilized by replacing 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 at the dimer interface or in the domain 1 (D1) / domain 3 (D3) linker of each monomer.

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

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

[0169] In yet another embodiment, the EDE compound presents an improved epitope beyond that of naturally occurring envelope dimers within a virus, virus-like particle, or subviral particle. An improved epitope includes an epitope that exceeds any epitope naturally presented on intact virus particles. Improved includes the ability to elicit a more beneficial immune response than native, intact dengue virus particles. For example, an EDE compound with increased stability in a dimeric configuration, such as through the modifications described in a) through v) above, is considered to have an improved epitope. The EDE may also be improved by other methods, such as an EDE engineered to have an FL that cannot be recognized by a compound, such as a polypeptide, for example, an antibody or antigenic portion thereof, or an EDE inserted into a scaffold, such that the FL cannot be recognized by an antibody in isolation from the immediate vicinity of the fusion loop, i.e., the fusion loop cannot be recognized in a context independent of quaternary organization.

[0170] In another embodiment, the EDE compound is incorporated into a heterologous protein scaffold that preserves the dimer configuration by increasing the level of covalent and / or non-covalent bonds between dimers, e.g., as described above. Furthermore, the EDE compound may comprise a heterologous protein scaffold that can display only a portion of the (approximately) 400 amino-terminal residues of an envelope protein dimer, or an envelope ectodomain, or an envelope protein ectodomain, where this portion is a contiguous portion of the (approximately) 400 amino-terminal residues of an envelope protein dimer, or an envelope ectodomain, or an envelope protein ectodomain, or this portion includes selected non-contiguous residues of the (approximately) 400 amino-terminal residues of an envelope protein dimer, or an envelope ectodomain, or an envelope protein ectodomain, as described above.

[0171] For example, in one embodiment, the EDE compound may comprise a nucleotide 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 may comprise one or more of V324, V97, W101, or the equivalent residues of a dengue virus envelope polypeptide in a substantially similar spatial arrangement to the residues adopted by the naturally occurring dimer of the envelope protein, or the envelope ectodomain, or the (approximately) 400 amino-terminal residues of the ectodomain of the envelope protein. These residues may be present in the naturally occurring envelope protein, or in alternative embodiments, they are retained in the scaffold protein in the appropriate arrangement. In a preferred embodiment, the EDE compound comprises the above-mentioned position W101 and at least one other residue. In a further preferred embodiment, the EDE compound comprises all of the above residues. In one embodiment, the EDE compound comprises an N153 glycan. In an alternative embodiment, the EDE does not comprise an N153 glycan.

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

[0173] The EDE compound may comprise a dimer of an envelope protein, or an envelope ectodomain, or the (approximately) 400 amino-terminal residues of an envelope protein ectodomain, which has increased stability in the dimeric configuration and is engineered to be retained within the protein scaffold described above.

[0174] The inventors have found that specific regions of the envelope dimer are important for contact with compounds of the invention, such as antibodies or antigen-binding portions thereof. Thus, in some embodiments, the VDE compound comprises a dimer of the envelope protein, or the envelope ectodomain, or a specific antigenic portion of the 400 amino-terminal residues of the envelope protein ectodomain.

[0175] The EDE compound may be a dimer of the envelope protein, or the envelope ectodomain, or a specific fragment containing specific residues of the (approximately) 400 amino-terminal residues of the ectodomain of the envelope protein, including the region deemed necessary for antigenicity. The fragment may also be engineered to maintain a specific conformation, or may be retained within the protein scaffold, or may be both engineered and retained within the protein scaffold.

[0176] For example, in one embodiment, an EDE compound comprises a region centered in the cleft formed by the b-strand on the domain II side and the "150 loop" on the domain I side (opposite the dimer interface). The 150 loop spans residues 148-159, connects b-strands E0 and F0 of domain I, and carries 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, defined as the subunit that contributes FL to the epitope. These three segments are the b-strand (residues 67-74, bearing the N67 glycan), the immediately upstream fusion loop and residues (residues 97-106), and the ij loop (residues 246-249).

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

[0178] Further embodiments of EDE compounds include the aforementioned region (the region comprising the three polypeptide segments of domain II of the reference subunit) at the 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 when a subunit of a useful compound defined herein binds to an EDE, it disrupts the 150 loop of the second subunit. Thus, in one embodiment, the 150 loop may be in its native configuration found in a natural dimer of an envelope protein, or in the envelope ectodomain, or in the 400 amino-terminal residues of the ectodomain of an envelope protein, or in another embodiment, the 150 loop may be in a promiscuous configuration that the 150 loop adopts upon binding to one of the compounds of the present invention.

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

[0180] In a preferred embodiment, the EDE compound, once administered to a subject, preferably a human, generates antibodies such that these antibodies are preferably capable of binding to all four serotypes of dengue virus, optionally neutralizing all four serotypes of dengue virus, preferably neutralizing 100% of all four serotypes of dengue virus, optionally neutralizing virus made in both human and insect cells, preferably neutralizing 100% of all four serotypes of dengue virus made in both human and insect cells.

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

[0182] The present invention also provides a method for the synthesis of a stabilized recombinant sE dimer, EDE, of the present invention, comprising: a) contacting under oxidizing conditions single or multiple cysteine ​​mutants sE as defined above, and / or b) contacting the two sE monomers with at least one, two, or three sulfhydryl-reactive crosslinkers as defined above; and / or c) contacting two sE monomers carrying a glycosylation site as defined above by click chemistry, and / or and d) contacting two sE monomers with each other, wherein at least one amino acid residue in the amino acid sequence of at least one sE monomer is replaced with a bulky side chain amino acid as defined above.

[0183] The present invention also provides a stabilized recombinant sE dimer obtained by the method defined above.

[0184] To ensure proper formation of stabilized recombinant sE dimers according to the present invention, 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 host cells containing either a nucleic acid or a vector of the present invention, e.g., 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 expressing heterologous proteins, including vectors, e.g., plasmids, such as C6 / 36 insect cells, human dendritic cells, CHO cells, or microorganisms, e.g., Pichia pastoris cells. Host cells may also optionally contain a nucleic acid of the present invention integrated into the genome of the host cell by use of a viral vector to target the nucleic acid of the present invention to the genome, e.g., adenovirus, adeno-associated virus, cytomegalovirus, herpesvirus, poliovirus, retrovirus, Sindbis virus, vaccinia virus, or any other DNA or RNA viral vector.

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

[0187] Provided herein is a process for producing a compound of the invention, preferably a polypeptide, preferably an antibody or antigen-binding portion thereof, or an EDE compound of the invention, comprising the following steps: ii) culturing the host cells of the invention in a suitable medium; ii) recovering the compound, preferably the produced antibody or antigen-binding portion thereof, or the EDE compound, wherein the recovery is from either the culture medium or the cultured cells.

[0188] For polypeptide purification or isolation, for example, when the compound is a polypeptide or the EDE compound is a polypeptide, one skilled in the art will understand that the nucleotide coding sequence can be readily manipulated to include nucleotides useful for purification, for example, an affinity tag such as an epitope tag. Thus, in one embodiment, a process for producing a compound or EDE compound of the invention comprises culturing a host cell that includes a nucleotide sequence encoding the compound or EDE compound, further includes nucleotides encoding a portion useful for purifying the compound or EDE compound, or a vector that includes a nucleotide sequence encoding the compound or EDE compound, and further includes nucleotides encoding a portion useful for purifying the compound or EDE compound.

[0189] It will be understood that when the compound is a polypeptide, such as an antibody or an antigen-binding portion thereof, and is produced by recombinant means, polypeptide production can be induced by administering an EDE as defined in any of the above embodiments, optionally an EDE compound as defined above, to a subject. After administering the EDE (optionally an EDE compound), the natural host response can 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 invention provides a method for producing a compound of the invention, wherein the compound is an antibody of the invention, the method comprising: a) contacting a mammal with a stabilized recombinant sE dimer of the invention or an immunogenic composition of the invention; b) detecting the presence of antibodies directed against said sE in one or more serum samples from said mammal; c) harvesting pancreatic cells from the mammal; d) fusing the pancreatic cells with myeloma cells to produce hybridoma cells; e) identifying hybridoma cells capable of producing antibodies; f) culturing hybridoma cells capable of producing antibodies; g) optionally isolating the antibody.

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

[0192] The present invention also provides a hybridoma cell obtainable by the method defined above.

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

[0194] In preferred embodiments, the EDE or EDE compound has been determined to be capable of generating highly cross-reactive and potently neutralizing antibodies. The antibodies identified in the Examples (Examples 1-6) were generated against intact dengue virus during natural infection. It is believed that more specific and improved antibodies can be generated by administering a specific EDE antigen, which may be an EDE compound of the present invention. For example, during natural infection, some patients did not generate anti-EDE antibodies, but instead produced anti-FL antibodies, which are considered less useful and have less cross-reactivity and are less neutralizing. Administration of an EDE antigen is considered more likely to generate useful anti-EDE antibodies. As described above, in some embodiments, the EDE or EDE compound is engineered to have increased stability in the dimeric configuration, which is believed to increase the chances of anti-VDE antibodies being generated in the subject. In addition, in some embodiments, the EDE or EDE compound is engineered, for example, by mutations within the envelope protein itself or by using a scaffolding protein to present an improved epitope, for example, by hiding the fusion loop so that anti-FL antibodies are less likely to be generated. Administration of an EDE or EDE compound common to all serotypes of dengue virus is likely to produce highly cross-reactive and potently neutralizing antibodies. These antibodies can be collected from the subject and used for further analysis or in dengue treatment or dengue clinical trials.

[0195] Thus, one embodiment provides a process for the production of a compound according to the invention, wherein the compound is a polypeptide, or an antibody or antigen-binding portion thereof, the process comprising the steps of: a. administering to a subject an envelope dimer epitope or EDE compound as defined in any of the preceding embodiments; b. Recovering and isolating the antibody or antigen-binding portion thereof from the blood of the subject.

[0196] It will be understood that the above-described methods of producing compounds, e.g., antibodies or antigen-binding portions thereof, of the present invention, which include administering an EDE or EDE compound to a subject, can also be used as part of a method for selecting appropriate antigens for a vaccine. Current vaccines utilize attenuated versions of all four serotypes of dengue virus and are not particularly effective. Such vaccines may also induce the production of non-useful anti-FL antibodies. A preferred vaccine may contain a single antigen capable of eliciting an immune response against all serotypes of dengue virus, which immune response is capable of neutralizing all serotypes of dengue virus, i.e., considered to be all four serotypes of dengue virus.

[0197] The present inventors are the first to identify highly cross-reactive, potently neutralizing antibodies and the specific epitopes (EDEs) to which they bind, and therefore, the use of these epitopes in vaccines is likely to be preferable to 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 generated in a subject that respond to the antigen, optionally wherein the antigen has previously been found to bind to a panel of antibodies known to bind to an envelope dimer epitope as defined in any of the preceding embodiments.

[0199] The identification of highly cross-reactive, potently neutralizing antibodies in subjects administered a dengue antigen indicates an antigen that is likely to be useful in a vaccine. In one embodiment, the antigen is not presented as part of an intact virus. In a preferred embodiment, the antigen is a dimer, preferably a stabilized dimer, of an EDE compound, preferably an envelope protein, as defined in any of the above embodiments, optionally as part of a scaffold protein. In a preferred embodiment, the antigen is a highly cross-reactive, potently neutralizing antibody capable of binding to an EDE, such as those identified in the Examples, e.g., those previously determined to be capable of binding to an antibody of the present invention.

[0200] By administering such an antigen known to be capable of binding highly useful antibodies, the antibodies produced in a subject in response to the antigen can be characterized. Such antigens are likely to result in the production of such useful antibodies in the subject and, therefore, are suitable candidate antigens for use in vaccine compositions. Characterization includes determining whether these antibodies are considered to bind to the fusion loop, for example, by determining the antibody's ability to bind to linear, denatured, or recombinant envelope protein, e.g., by determining the antibody's ability to bind to the envelope protein in Western blots or ELISA, and to a dimer of the envelope protein, or to the EDE or EDE compound described above in the preceding embodiments. The ability of an antibody to bind to all four serotypes of dengue virus can also be evaluated, as can the ability of an antibody to neutralize all four dengue virus types. Methods for determining the neutralizing ability of an antibody have been described above. The ability of antibodies to neutralize dengue virus produced in both human 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 primarily elicits anti-FL antibodies. For example, an antigen is considered useful if the ratio of antibodies elicited against FL to antibodies elicited against EDE is 1:2, 1:4, 1:5, 1:10, 1:50, 1:100, 1:500, 1:1000, or less. The relative amounts of anti-FL and anti-EDE antibodies can be determined by methods known to those skilled in the art, such as ELISA-based techniques. An antigen is considered useful if it elicits antibodies capable of binding to the EDE of more than one serotype of dengue virus, preferably all four dengue virus types. An antigen is considered useful if it elicits antibodies capable of neutralizing more than one serotype of dengue virus, preferably all four dengue virus types, preferably 100% neutralization. An antigen is also considered useful if it produces antibodies capable of neutralizing dengue virus made in both human 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. a) do not produce or do not significantly produce anti-FL antibodies; and b) binds to a highly significant extent to all four serotypes of dengue virus, and c) It is considered most useful if it neutralizes 100% of all four serotypes of dengue virus made in both human and insect cells to a highly significant extent.

[0202] Furthermore, in another embodiment, if the antibodies generated are able to bind to the EDE defined in the above embodiments, the antigen is considered suitable for use in vaccination.

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

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

[0205] It will be appreciated that the identification of first or highly cross-reactive and potently neutralizing antibodies to dengue virus provides a unique opportunity to be able to treat or prevent this viral disease. In addition, it will be possible to conduct clinical trials involving live dengue virus, since until the present invention, there was no way to reliably treat infections that occurred during the trials. Accordingly, a further aspect of the present invention provides a method of treating or preventing dengue virus infection in a subject.

[0206] The method comprises administering one or more compounds, preferably polypeptides, preferably antibodies or fragments thereof, according to the invention. The invention also provides one or more compounds, preferably polypeptides, preferably antibodies or fragments thereof, according to the invention for use in the prevention or treatment of dengue virus infection. The invention also provides use of a compound of the invention in the manufacture of a medicament for the treatment or prevention of dengue infection.

[0207] For administration, it will be appreciated that the compounds of the invention may be part of a composition, e.g., a pharmaceutical composition, which may further comprise one or more therapeutic agents deemed useful in themselves for treating the infection, e.g., an additional antiviral agent, or one or more agents deemed useful for treating the symptoms of, e.g., a dengue infection.

[0208] The term "treating" includes administering a compound of the present invention, e.g., a compound of the present invention, an EDE compound, a vaccine composition, an antibody, a stabilized recombinant sE dimer, or any of the immunogenic compositions of the present invention, to a patient having dengue virus infection or symptoms of dengue virus infection with the intent to cure, cure, alleviate, relieve, alter, relieve, improve, ameliorate, or affect dengue virus infection and / or symptoms of dengue virus infection. Alleviating means treating any one or more of the symptoms of dengue virus infection. Treating also means preventing new cells from becoming infected. It will be apparent to one of skill in the art whether a patient has been successfully treated. For example, the viral load may be reduced.

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

[0210] Dengue virus infection and symptoms are presented, for example, in WHO Fact Sheet 117. As described there, dengue fever is a severe, flu-like illness that affects infants, young children, and adults but rarely causes death. Dengue fever should be suspected when a 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, swollen glands, or a rash. Symptoms usually last 2–7 days after an incubation period of 4–10 days after the bite of an infected mosquito. Severe dengue fever is a potentially fatal complication due to plasma leakage, fluid retention, difficulty breathing, severe bleeding, and organ failure. Danger signs occur 3–7 days after the initial symptoms and include severe abdominal pain, continuous vomiting, shortness of breath, bleeding gums, fatigue, restlessness, and vomiting blood, along with a drop in body temperature (below 38°C / 100°F). Death can occur after 24 to 48 hours in critical condition, and appropriate medical treatment is required to avoid complications and risk of death.

[0211] Administration of the compound or a composition comprising the compound is an amount, e.g., a therapeutically effective amount, that results in inhibition of infection of cells when the compound is used prophylactically, or inhibition of further infection of cells when used therapeutically, and / or reduces the signs and / or symptoms of the disease.

[0212] A therapeutically effective amount is that amount which produces a subjective alleviation of other symptom(s) or an objectively discernible improvement as noted by a clinician or qualified observer.

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

[0214] The present invention provides the use of an EDE, preferably a stabilized recombinant sE dimer, or an immunogenic composition according to the invention, to immunize an animal (non-human), preferably a mammal, such as a monkey, rabbit, mouse, or camelid (e.g., a llama).

[0215] A further embodiment provides one or more compounds, preferably polypeptides, preferably antibodies or fragments thereof, according to the invention for use in live dengue vaccine trials, e.g., with the aim of terminating infection.

[0216] Preferably, the compounds of the invention are capable of neutralizing at least 95%, or at least 98%, e.g., 100%, all four serotypes of dengue virus produced in both insect and human cells. It is believed that pre-administration of the compound prior to exposure to the virus will prevent viral infection.

[0217] For example, a compound according to the invention, e.g., an antibody or fragment thereof, capable of neutralizing all four serotypes of dengue virus mentioned above may be administered prior to exposure to the virus, e.g., as a prophylactic, either for travelers or in the vicinity or residence of people likely to be in close contact with an outbreak or one or more infected people, e.g., mosquito bites. Alternatively or additionally, the compound may be administered when the patient first develops a fever or when symptoms become severe.

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

[0219] It will be understood that the compounds of the present invention, e.g., antibodies or antigen-binding portions thereof, may be administered with additional therapeutic agents, e.g., 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 may be administered 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 over a period of one day, two days, one week, two weeks, one month, six months, one year, or more. For treatment after infection, shorter periods, such as up to one month, may be appropriate. For prophylaxis, longer periods, such as six months or more out of a year, may be appropriate.

[0221] The compounds, e.g., antibodies or antigen-binding portions thereof, for use in the prevention or treatment of dengue infection may be selected using the methods of the invention. Thus, the present invention provides a method for selecting a suitable antibody or fragment thereof for use in the prevention or treatment of dengue virus, the method comprising 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 embodiments.

[0222] The EDE compounds according to any of the above embodiments are likely to be capable of generating suitable antibodies following administration of the EDE to a subject, and the antibodies generated in such a subject are therefore likely to be useful in treating or preventing dengue infection.

[0223] In a preferred embodiment, the EDE is an EDE compound of the present invention, e.g., a dimer, preferably a stabilized dimer, of an envelope protein, 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, e.g., one already known to be capable of binding to an antibody of the present invention. In a preferred embodiment, the antigen is considered to be improved over the native envelope dimer, for example, by containing residues in a specific arrangement required to generate a cross-reactive and potentially neutralizing anti-EDE antibody, but not containing residues or residues in a specific conformation that generate an anti-FL antibody.

[0224] In another embodiment, in addition to administering the EDE, optionally the EDE compound, the subject is also administered a compound or agent that blocks the formation of anti-FL antibodies, for example. For example, stabilized sE dimers can be useful.

[0225] Characterization includes determining whether these antibodies are considered to bind to the fusion loop, for example, by determining the antibody's ability to bind to linear, denatured, or recombinant envelope protein, e.g., by determining the antibody's ability to bind to the envelope protein in a Western blot or ELISA, and to a dimer of the envelope protein, or to the EDE or EDE compound described above in the preceding embodiments. The ability of the antibody to bind to all four serotypes of dengue virus can also be assessed, as can the ability of the antibody to neutralize all four dengue virus. Methods for determining the neutralizing ability of an antibody are described above and in detail in the Examples. The ability of the antibody to neutralize dengue virus produced in both human cells and insect cells can also be determined.

[0226] In one embodiment, an antibody is not considered useful if it binds to FL. An antibody is considered useful if it can bind to more than one serotype of dengue virus, preferably four serotypes of dengue virus, or more than one serotype of EDE as described in any of the previous embodiments. An antibody is considered useful if it can neutralize more than one serotype of dengue virus, optionally two serotypes of dengue virus, optionally three serotypes of dengue virus, preferably four serotypes of dengue virus, preferably at least 95% or at least 98%, for example 100%. An antibody is also considered useful if it can neutralize dengue virus made in both human cells, optionally dendritic cells, and insect cells, optionally C6 / 36 cells, preferably to the same level, preferably neutralizing the virus by at least 95% or at least 98%, for example 100%. An antibody is considered useful if it can neutralize more than one serotype of dengue virus, optionally two serotypes of dengue virus, optionally three serotypes of dengue virus, preferably four serotypes of dengue virus, preferably at least 95% or at least 98%, for example 100%. a) do not produce or do not significantly produce anti-FL antibodies; and b) binds to a highly significant extent to all four serotypes of dengue virus, and c) It is considered most useful if it neutralizes 100% of all four serotypes of dengue virus made in both human and insect cells to a highly significant extent.

[0227] Because the inventors have found that patients suffering from dengue infection produce either useful anti-EDE antibodies or ineffective anti-FL antibodies, a further method for identifying antibodies that may be useful for treating or preventing dengue infection is to simply identify those antibodies that are unable to bind to the denatured or linear form of the envelope protein. Any antibodies that are unable to bind to these envelope proteins are likely to be useful compounds of the present invention.

[0228] It should be understood that a patient may also be treated with a nucleic acid, vector, or host cell expressing a polypeptide, preferably an antibody or antigen-binding portion thereof. For example, a nucleic acid comprising a polypeptide may be inserted into an appropriate delivery system, e.g., a viral vector, e.g., an adenovirus, adeno-associated virus, cytomegalovirus, herpes virus, poliovirus, retrovirus, Sindbis virus, vaccinia virus, or any other DNA or RNA viral vector, such that the compound of the invention is expressed endogenously within the treated patient.

[0229] The present invention also provides methods for stratifying patients according to their likely need for therapeutic or prophylactic treatment with one or more compounds of the present invention. Thus, provided herein is a method for identifying patients suffering from dengue virus infection who are likely to require treatment with the compound or composition, or escalating doses of the compound or composition, according to any one of the preceding embodiments, the method comprising determining the levels of anti-envelope dimer epitope antibodies and anti-fusion loop antibodies in the subject, wherein the envelope dimer epitope is as described in any of the preceding embodiments.

[0230] As identified by the present inventors, patients suffering from dengue fever infection mainly produce anti-EDE or anti-FL antibodies. Anti-FL antibodies are not considered useful, while anti-EDE antibodies are considered useful. If a subject has anti-EDE antibodies but may still require some additional treatment with the compound of the present invention, a subject with mainly anti-FL antibodies is likely to require a higher dose because they do not have inherently useful antibodies. Therefore, patients with only anti-FL antibodies are considered to be patients who are likely to require treatment with the compound 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 and only produce anti-FL antibodies are likely to require a higher dose of treatment than patients with anti-EDE antibodies. Also, patients may only produce low levels of anti-EDE antibodies and therefore may require a higher dose of the compound.

[0231] By higher dose, it is meant that the patient will require 2, 3, 4, 5, 10, 20, 50 times the dose of the compound of the invention than would be required by a patient who develops anti-EDE antibodies.

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

[0233] For example, determining whether an antibody binds to intact dengue virus or the EDE but not to a denatured or linear envelope protein, means for identifying whether these antibodies bind to the EDE are described above and in the Examples. The ability of an antibody to bind to an envelope protein can be assessed when the envelope protein has been engineered to have increased dimer stability, or when the envelope protein or residues thereof are presented as part of a scaffold.

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

[0235] Additionally, if the patient is determined to have anti-envelope dimer epitope antibodies, the patient may be given a booster dose.

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

[0237] The present invention also provides the use of a stabilized recombinant sE dimer (used as an antigen) as defined above for the preparation of a prophylactic or therapeutic immunogenic (or vaccine) composition for the prevention and / or treatment of dengue virus infection in a susceptible mammalian subject, e.g., a human.

[0238] Significantly, the inventors have for the first time identified a specific epitope recognized by a previously unknown, highly cross-reactive, and potently neutralizing antibody, as described above. This epitope is believed to provide a particularly effective antigen for vaccination against dengue virus. Methods for selecting a suitable antigen for use in vaccination against dengue virus have been described in the preceding embodiments. Accordingly, the present invention provides a composition presenting a dengue virus envelope dimer epitope, and optionally an EDE compound, for use in preparing a prophylactic or therapeutic immunogenic (or vaccine) composition for preventing and / or treating dengue virus infection in a susceptible mammalian subject, e.g., a human, where the envelope dimer epitope and EDE compound are as defined in any of the preceding embodiments and identified according to the preceding methods. For example, the EDE or EDE compound may be identified in the preceding embodiment presenting a method for selecting a suitable antigen for use in a vaccine, e.g., by characterizing antibodies generated after administration of a potential vaccine candidate EDE / EDE compound to a subject. This may be well within the purview of one skilled in the art. Alternatively, the EDE or EDE compound may be as set forth in the preceding embodiments, e.g., in one embodiment, the EDE or EDE compound is a dimer of an envelope protein, or an envelope ectodomain, or the (approximately) 400 amino-terminal residues of the ectodomain 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 NO: 29, 31, 33, or 35), or proteins having at least 90% homology to the sequence of SEQ ID NO: 29, 31, 33, or 35. The dimer may be a homodimer or a heterodimer. In a preferred embodiment, the dimer is not incorporated into an intact virus particle, subviral particle, or virus-like particle, but rather is a free dimer.It will be understood that any form of EDE or EDE compound described herein, e.g., engineered envelope protein, may be presented as part of a virus, virus-like particle, or subviral particle, e.g., as part of a protein scaffold. In a preferred embodiment, the EDE compound is a stabilized recombinant sE dimer, as described in the preceding embodiment.

[0239] In yet another embodiment, the EDE or EDE compound for use in the vaccine composition presents an improved epitope over the naturally occurring envelope dimer. For example, the EDE / EDE compound is engineered or inserted into a backbone so that the FL cannot be recognized by a compound, such as a polypeptide, such as an antibody or an antigenic portion thereof, by itself; for example, the EDE / EDE compound is engineered so that the FL cannot be recognized by an antibody in isolation from the immediate vicinity of the fusion loop, i.e., the induction loop cannot be recognized in a context independent of the quaternary organization.

[0240] In another embodiment, the EDE or EDE compound for use in the vaccine composition comprises a dimer of an envelope protein, or an envelope ectodomain, or the (approximately) 400 amino-terminal residues of an envelope protein ectodomain, that has been engineered to have increased stability in the dimeric configuration, as described above, e.g., engineered to have an increased level of covalent and / or non-covalent bonds between dimers, or is incorporated into a heterologous protein scaffold that preserves the dimeric configuration, e.g., by increasing the level of covalent and / or non-covalent bonds between dimers, as described above. Furthermore, the EDE / EDE compound may comprise a heterologous protein scaffold that may be present on only a portion of the (approximately) 400 amino-terminal residues of an envelope protein dimer, or an envelope ectodomain, or an envelope protein ectodomain, wherein this portion is a contiguous portion of the (approximately) 400 amino-terminal residues of an envelope protein dimer, or an envelope ectodomain, or an envelope protein ectodomain, or this portion comprises carefully selected non-contiguous residues of the (approximately) 400 amino-terminal residues of the above-mentioned envelope protein dimer, or an envelope ectodomain, or an envelope protein ectodomain.

[0241] For example, in one embodiment, the EDE / EDE compound is selected from the group consisting of: 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 or one or more of 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 a dengue virus envelope polypeptide in a substantially similar spatial arrangement so that the residues adopt the native dimer of the envelope protein, or the envelope ectodomain, or the (approximately) 400 amino-terminal residues of the ectodomain of the envelope protein. These residues may be present in naturally occurring envelope proteins, or in alternative embodiments, they are retained in the scaffold protein in the appropriate configuration. In preferred embodiments, the EDE / EDE compound comprises position W101 and at least one other residue as described above. In even more preferred embodiments, the EDE / EDE compound comprises all of the above residues. In one embodiment, the EDE / EDE compound comprises an N153 glycan. In an alternative embodiment, the EDE / EDE compound does not comprise an N153 glycan.

[0242] In a further preferred embodiment, the EDE / EDE compounds for use in the vaccine compositions comprise residues that are observed in both amino acid and spatial position across more than one serotype of dengue virus, preferably residues that are observed in both amino acid and spatial position across all serotypes of dengue virus, i.e., across all four serotypes of dengue virus.

[0243] The EDE / EDE compound may comprise a dimer of an envelope protein, or an envelope ectodomain, or the (approximately) 400 amino-terminal residues of an envelope protein ectodomain, which has increased stability in the dimeric configuration and is engineered to be retained within the protein scaffold described above.

[0244] The inventors have found that specific regions of the envelope dimer are important for contact with the compounds of the present invention, such as antibodies or antigen-binding portions thereof. Thus, in some embodiments, the EDE / EDE compound comprises a dimer of the envelope protein, or the envelope ectodomain, or a specific antigenic portion of the (approximately) 400 amino-terminal residues of the ectodomain of the envelope protein. The EDE / EDE compound for use in the vaccine composition may be a dimer of the envelope protein, or the envelope ectodomain, or a specific fragment containing specific residues of the (approximately) 400 amino-terminal residues of the envelope protein ectodomain, including the region deemed necessary for antigenicity. This fragment may also be engineered to maintain a specific conformation, or may be retained within the protein scaffold, or may be both engineered and retained within the protein scaffold.

[0245] For example, in one embodiment, an EDE / EDE compound for use in a vaccine composition comprises a region centered in the cleft formed by the b-strand on the domain II side and the "150 loop" on the domain I side (opposite the dimer interface). The 150 loop spans residues 148-159, connects b-strands E0 and F0 of domain I, and carries 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, defined as the subunit that contributes FL to the epitope. These three segments are the b-strand (residues 67-74, bearing the N67 glycan), the immediately upstream fusion loop and residues (residues 97-106), and the ij loop (residues 246-249).

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

[0247] Further embodiments of EDE / EDE compounds include the aforementioned region (the region comprising the three polypeptide segments of domain II of the reference subunit) at the 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 when a subunit of a useful compound defined herein binds to VDE, the 150 loop of the second subunit is disrupted. Thus, in one embodiment, the 150 loop may be in its natural configuration found in the natural dimer of an envelope protein, or in the envelope ectodomain, or in the (approximately) 400 amino-terminal residues of the ectodomain of an envelope protein, or in another embodiment, the 150 loop may be in a promiscuous configuration that the 150 loop adopts upon binding to one of the compounds of the present invention.

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

[0249] In a preferred embodiment, the EDE / EDE compounds, once administered to a subject, preferably a human, generate antibodies such that these antibodies are preferably capable of binding to all four serotypes of dengue virus, optionally neutralizing all four serotypes of dengue virus, preferably neutralizing 100% of all four serotypes of dengue virus, optionally neutralizing virus made in both human and insect cells, preferably neutralizing 100% of all four serotypes of dengue virus made in both human and insect cells.

[0250] The immunogenic composition comprising the EDE comprising the stabilized recombinant sE dimer described above can be used to treat a subject, - recognizes the envelope dimer epitope (EDE) alone (does not show binding to the recombinant E protein monomer in ELISA tests), - cross-reactive, and - Particularly suitable for eliciting neutralizing antibodies that neutralize dengue viruses from four serotypes (DENV1-4).

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

[0252] The composition may comprise the EDE / EDE compound itself, or it may comprise a means for expressing the EDE / EDE compound in a subject to be vaccinated. For example, the invention comprises a nucleic acid encoding an envelope dimeric epitope or EDE compound for use in vaccination against dengue virus infection, wherein the envelope dimeric epitope or EDE compound is as described in any of the preceding embodiments. Furthermore, the nucleic acid may be part of a vector. Preferences for the vector 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 specific antigens, for example, via direct immunization with plasmid DNA. Such nucleic acids can be delivered via liposomes and immunostimulatory constructs. Alternatively, attenuated viral hosts or vectors or bacterial vectors can be used, for example, adenovirus, adeno-associated virus, cytomegalovirus, herpes virus, poliovirus, retrovirus, Sindbis virus, vaccinia virus, or any other DNA or RNA viral vector.

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

[0255] For use in vaccination against dengue virus infection a) envelope dimer epitopes or EDE compounds; b) a nucleic acid encoding an EDE or EDE compound; c) a vector comprising the nucleic acid; Compositions comprising any one or more of are also part of the present invention, when the envelope dimer epitope or EDE compound is as described in any of the preceding embodiments.

[0256] In one embodiment, a) envelope dimer epitopes or EDE compounds; b) a nucleic acid encoding an EDE or EDE compound; c) a vector comprising the nucleic acid; are or encode more than one, optionally two, optionally three, optionally four serotypes of dengue virus.

[0257] In a preferred embodiment, a) envelope dimer epitopes or EDE compounds; b) a nucleic acid encoding an EDE or EDE compound; c) a vector comprising the nucleic acid; is a single epitope or results in the production of a single epitope that can generate antibodies that neutralize all four serotypes of dengue virus, preferably capable of 100% neutralization of all four serotypes of dengue virus.

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

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

[0260] An EDE or EDE compound of the present invention, e.g., a stabilized recombinant sE dimer, that induces neutralizing antibodies against dengue virus infection is administered to a mammalian subject, preferably a human, in an amount sufficient to prevent or attenuate the severity of infection by dengue virus over the course of the infection.

[0261] The therapeutically effective amount will vary depending on, among other factors, the subject being treated, the age and general condition of the subject being treated, the capacity of the subject's immune response to synthesize antibodies, the degree of protection desired, the severity of the condition being treated, the particular VDE compound, e.g., the particular stabilized recombinant sE dimer selected, and the mode of administration. An appropriate effective amount can be easily determined by one skilled in the art. The therapeutically effective amount will fall in a relatively broad range that can be determined through routine testing.

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

[0263] The optimal amount for a particular vaccine can be ascertained by standard studies involving measuring anti-sE dimer antibody titers in subjects.

[0264] The immunogenic compositions of the present invention may be administered with or without an adjuvant. The adjuvant may be added directly to the immunogenic composition or may be administered separately, simultaneously with or immediately after administration of the vaccine. Such adjuvants include, but are not limited to, aluminum salts (aluminum hydroxide), oil-in-water emulsion formulations with or without specific stimulants such as muramyl peptides, saponin adjuvants, cytokines, and detoxified mutants of bacterial toxins such as cholera toxin, pertussis toxin, or E. coli heat-labile toxin.

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

[0266] Vaccination programs often include a boost strategy. After the initial vaccination, subjects may receive one or two booster injections at appropriate intervals, as determined by those skilled in the art. In one embodiment, vaccination can 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 a boost strategy for vaccination against dengue virus infection, and optionally, the antigens, compounds, nucleic acids, vectors, or compositions are for administration before or after the (prime) or (boost) administration of dengue virus, optionally an attenuated dengue virus, and / or dengue virus-like particles, which may be a group of one or more serotypes of dengue virus and may contain or display an EDE, such as a non-natural EDE or EDE compound as described above. As a further example, heterologous flaviviruses such as Chimerivax associated with yellow fever may be used, e.g., by one or more of dimers, DNA, vaccinia, adenovirus, and as will be apparent to one skilled in the art, different sequences and timing of administration of different antigens and / or nucleic acids encoding antigens may be possible, and the present invention is not limited to any particular combination or sequence of administration.

[0267] The present invention also includes vaccination strategies for providing protection against dengue virus, such as by administering a) a single administration of an envelope dimeric epitope or EDE compound capable of raising antibodies against all four serotypes, or a vaccine composition according to any of the preceding embodiments, or a nucleic acid for use in vaccination, or a vector for use in vaccination, optionally followed by administration of an attenuated dengue virus, or b) administration of two envelope dimeric epitopes or EDE compounds from two serotypes, followed by administration of envelope dimeric epitopes or EDE compounds from two other serotypes, optionally followed by administration of an attenuated dengue virus, according to any of the preceding embodiments; or c) administration of an attenuated dengue virus followed by administration of an envelope dimeric epitope capable of raising antibodies against all four serotypes according to any of the previous embodiments; or d) administration of an attenuated dengue virus, followed by administration of two envelope dimeric epitopes or EDE compounds from two serotypes as described in any of the preceding embodiments, followed by administration of envelope dimeric epitopes or EDE compounds from two other serotypes.

[0268] It is also envisaged that a patient who has been vaccinated in accordance with the present invention may still require subsequent treatment with a compound or composition in accordance with the present invention for use in treating or preventing dengue infection.

[0269] The compounds of the invention are therefore for use in treating or preventing dengue infection in patients who have previously received a dengue vaccination or in patients who have not previously received a dengue vaccination.

[0270] Because this vaccination is considered to provide protection against more than one serotype of dengue virus, preferably against all four serotypes of dengue virus, the vaccination is still considered useful if the patient already has a dengue infection, but the vaccine is preferably administered before symptoms of dengue infection or before the patient is known to have a dengue infection.

[0271] Thus, the vaccination is for use in patients who have not previously been infected with dengue fever, and in patients who have not currently been infected with dengue fever and are uninfected at the time of administration of the vaccine, or the vaccination is for use in patients who have previously been infected with one or more serotypes of dengue fever infection but are uninfected at the time of administration of the vaccine, or the vaccination is for use in patients who have previously been infected with one or more serotypes of dengue fever infection but are not currently infected with one or more serotypes of dengue fever and are uninfected at the time of administration of the vaccine.

[0272] This vaccination is also for use in patients who have been previously treated with a compound of the invention but are not currently being treated with a compound of the invention, and it is also for use in patients who have been previously treated with a compound of the invention and are currently being treated with a compound of the invention. This vaccination is also for use in patients who are being treated for the first time with a compound of the invention.

[0273] The present invention also provides an EDE compound, such as a stabilized recombinant sE dimer or an immunogenic composition, as defined above, for use in medicine, preferably for the prevention and / or treatment of dengue virus infection.

[0274] The present invention also provides an EDE compound, such as a stabilized recombinant sE dimer or an immunogenic composition, as defined above, for the manufacture of a medicament, preferably a prophylactic or therapeutic vaccine against dengue virus infection in a subject.

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

[0276] The present invention also provides diagnostic agents comprising or consisting of an EDE compound of the present invention, such as a stabilized recombinant sE dimer, or a compound of the present invention, such as an antibody according to the present invention or a fragment thereof.

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

[0278] The detectable marker may be directly and covalently bound to the compound, e.g., an antibody or fragment thereof, either at one of the termini (N- or C-terminus) of the antibody or fragment thereof, or to a side chain of one of the amino acids of the antibody or fragment thereof. The detectable marker may also be directly and covalently bound to the antibody or fragment thereof through a connecting arm (i.e., a cross-linking reagent) either at one of the termini of the antibody or fragment thereof, or to a side chain of one of the amino acids of the antibody or fragment thereof. Methods for binding a compound of interest to a peptide or antibody are known in the art.

[0279] Advantageously, the detectable marker is enzymes such as horseradish peroxidase, alkaline phosphatase, glucose-6-phosphatase, or beta-galactosidase, - fluorophores 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 (e.g. Cy5) that are visualized with electronic detectors (CCD cameras, photomultiplier tubes), - 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 and radioisotopes such as Tc-labeled compounds.

[0280] The present invention also provides the use of an EDE compound, such as a stabilized recombinant sE dimer, an antibody or fragment thereof, or a diagnostic agent according to the present invention, for diagnosing or monitoring 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 comprising: a) contacting in vitro a suitable biological sample from the subject with an antibody or fragment thereof, or a diagnostic agent comprising or consisting of an antibody or fragment thereof according to the invention; b) determining the presence or absence of dengue virus envelope glycoprotein E in the biological sample; The presence of dengue virus envelope glycoprotein E indicates that the subject has a dengue virus infection.

[0282] Step b) may be carried out by determining the presence or absence of antibody-antigen complexes (ie antibodies directed against dengue virus envelope glycoprotein E-dengue virus envelope glycoprotein E complexes).

[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, the method comprising: a) contacting in vitro said suitable biological sample from said subject with an antibody or fragment thereof, or a diagnostic agent comprising or consisting of an antibody or fragment thereof according to the invention; b) 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 comprising: a) contacting in vitro a suitable biological sample from the subject with a stabilized recombinant sE dimer according to the present invention; b) determining the presence or absence of antibodies directed against the dimer in the biological sample; The presence of antibodies indicates that the subject has a dengue virus infection.

[0285] The present invention also provides an in vitro method for determining the presence of antibodies directed against dengue virus envelope glycoprotein E in a suitable biological sample from a subject, the method comprising: a) contacting said suitable biological sample from said subject in vitro with a stabilized recombinant sE dimer according to the present invention; b) determining the presence or absence of antibodies directed against the dimer in the biological sample.

[0286] The present invention also provides an in vitro method for monitoring the progression or regression of a dengue virus infection in a subject, the method comprising: a) contacting in vitro a suitable biological sample from the subject with a diagnostic agent comprising or consisting of an antibody or fragment thereof, an antibody or fragment thereof according to the invention; b) determining the amount of dengue virus envelope glycoprotein E in the biological sample; c) comparing the amount determined in step (b) with an 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, and 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, respectively, of dengue virus envelope glycoprotein E in a suitable biological sample compared to the amount of dengue virus envelope glycoprotein E in a suitable biological sample from said subject, previously determined and used as a reference amount.

[0288] Step b) may be carried out by determining the presence or absence of antibody-antigen complexes (ie antibodies directed against dengue virus envelope glycoprotein E-dengue virus envelope glycoprotein E complexes).

[0289] The present invention also provides an in vitro method for predicting a favorable prognosis of exacerbation of dengue virus infection in a subject, the method comprising: a) contacting in vitro a suitable biological sample from the subject with a stabilized recombinant sE dimer according to the present invention; b) determining the amount of neutralizing antibodies directed against the dimer in the biological sample; c) comparing the amount determined in step (b) with an amount of dimer-directed antibody previously obtained in the subject; A significant increase in the amount of neutralizing antibodies directed against the dimer constitutes a good prognostic marker for the progression of the dengue virus infection.

[0290] The present invention also provides an in vitro method for monitoring the success of a vaccination protocol against dengue virus infection in a subject vaccinated against dengue virus, the method comprising: a) contacting in vitro a suitable biological sample from the subject with a stabilized recombinant sE dimer according to the present invention; b) determining the amount of neutralizing antibodies directed against the dimer in the biological sample; c) comparing the amount determined in step (b) with an amount of dimer-directed antibody previously obtained in the subject; A significant increase in the amount of neutralizing antibodies directed against the dimer constitutes a marker of the success of the vaccination protocol.

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

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

[0293] The present invention also provides an isolated polynucleotide encoding a mutant sE as defined above, or a polypeptide of SEQ ID NO: 28, 142, 140, 143, 144, 145, 146, or 147.

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

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

[0296] Suitable diagnostic reagents are necessary to perform an assay to diagnose or monitor dengue virus infection in a subject. Suitable diagnostic reagents may be solvents, buffers, dyes, anticoagulants.

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

[0298] In one embodiment, the kit of parts includes a means for identifying patients in need of treatment with a compound of the invention or in need of a higher dose of a compound of the invention according to the preceding embodiments. The kit may also provide a method for identifying the presence or absence of anti-EDE and anti-FL antibodies; for example, the kit may include a microtiter plate, optionally coated with linear or denatured envelope protein, coated with an EDE epitope according to any of the preceding embodiments, and / or reagents for performing an ELISA test, optionally a colorimetric test on a stick. Preferably, the kit includes a means for simply identifying the presence or absence of antibodies, preferably on a solid support. The kit may also further include a compound or composition of the invention for use in treating or preventing dengue infection.

[0299] A kit of additional parts is also provided, including a means for identifying patients in need of vaccination. Patients are considered to be in need of vaccination based on the presence or absence and levels of anti-EDE and anti-FL antibodies. Thus, the kit may provide a method for identifying the presence or absence of anti-EDE and anti-FL antibodies; for example, the kit may include a microtiter plate, optionally coated with linear or denatured envelope protein, coated with an EDE epitope according to any of the aforementioned embodiments, and / or reagents for performing an ELISA test, optionally a colorimetric test on a stick. Preferably, the kit includes a means for simply identifying the presence or absence of antibodies on a solid support. The kit may also further include a composition for use in vaccination, as described in the aforementioned embodiments.

[0300] Further kits include means for treating or preventing dengue infection and include one or more compounds of the invention that bind to an EDE, or a composition comprising a compound of the invention that binds to an EDE, and optionally an additional therapeutic agent, such as an additional antiviral agent.

[0301] It will be understood that any compound or composition or antigen or antibody mentioned herein may be part of a composition. The composition may include a stabilizing agent such as PEG. It will be understood that the polypeptide component may be covalently modified or conjugated, e.g., PEGylated, for example, as known in the art.

[0302] Thus, for example, any compound or antibody, or any polypeptide or antigen, or nucleic acid or vector encoding the antigen or antibody for use in treating or preventing dengue infection, may be conjugated to one or more further components, for example, conjugated to a reporter moiety or to one or more further therapeutic agents.

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

[0304] Any agent described herein is typically administered as part of a pharmaceutical composition together with a pharmaceutically acceptable excipient, diluent, adjuvant, or carrier. Thus, any reference to a compound, polypeptide, antibody, antigen-binding portion thereof, composition, nucleic acid, vector, antigen, host cell, and any reference to an additional therapeutic agent applies equally to a pharmaceutically acceptable composition comprising the compound, composition, nucleic acid, vector, antigen, host cell, and / or additional therapeutic agent (e.g., formulation).

[0305] The compound, polypeptide, antibody, antigen-binding portion thereof, composition, nucleic acid can be part of a nanoparticle.

[0306] Routes of administration will be well known to those skilled in the art. For example, the agents of the present invention (compounds, polypeptides, antibodies, antigen-binding portions thereof, compositions, nucleic acids, vectors, antigens, host cells, additional therapeutic agents) can be administered orally, bucally, or sublingually in the form of tablets, capsules, ovules, elixirs, solutions, or suspensions, which may contain flavorings or coloring agents, for immediate, delayed, or controlled-release applications. The compounds of the present invention may also be administered by intravenous injection. The compounds of the present invention (polypeptides, antibodies, antigen-binding portions thereof, compositions, nucleic acids, vectors, antigens, host cells, additional therapeutic agents) can be administered orally to mammalian subjects, preferably humans. They can also be administered to the subject by injection, such as intravenous, intraperitoneal, intramuscular, intradermal, or subcutaneous injection.

[0307] The drug may be administered orally or by any parenteral route in the form of a pharmaceutical agent containing an active ingredient, optionally in the form of a non-toxic organic or inorganic acid or base addition salt, in a pharmaceutically acceptable dosage form. Depending on the disease or patient to be treated and the route of administration, the drug may be administered in different dosages.

[0308] Preferably, the formulations are unit dosages containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, a weekly dose, a monthly dose, or a semi-annual dose of the agent or active ingredient.

[0309] In human therapy, an agent (compound, polypeptide, antibody, antigen-binding portion thereof, composition, nucleic acid, vector, antigen, host cell, additional therapeutic agent) can be administered alone, but will generally be administered in admixture with a suitable pharmaceutical excipient diluent, or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

[0310] 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; and granule binders such as certain complex silicates, polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin, and acacia. Additionally, lubricants such as magnesium stearate, stearic acid, glyceryl behenate, and talc may be included. Capsules or tablets may also be enteric coated to enhance gastric stability.

[0311] Solid compositions of a similar type can also be used as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, milk sugar, or high molecular weight polyethylene glycols. For aqueous suspensions and / or elixirs, the compounds of the present invention may be mixed with various sweeteners or flavoring agents, coloring agents or pigments, emulsifying and / or suspending agents, and diluents such as water, ethanol, propylene glycol, and glycerin, as well as combinations thereof.

[0312] Agents (compounds, polypeptides, antibodies, antigen-binding portions thereof, compositions, nucleic acids, vectors, antigens, host cells, additional therapeutic agents, vaccines) can also be administered parenterally, for example, intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly, or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solution should be suitably buffered (preferably to a pH of 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques known to those skilled in the art.

[0313] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile injectable solutions which may contain suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules or vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example, water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.

[0314] For oral and parenteral administration to human subjects, daily dosage levels of the agents (compounds, polypeptides, antibodies, antigen-binding portions thereof, compositions, nucleic acids, vectors, antigens, host cells, further therapeutic agents, vaccines) will typically be in the range of 1 to 5000 mg per adult human, administered in single or divided doses.

[0315] Thus, for example, tablets or capsules containing the compounds, polypeptides, antibodies, antigen-binding portions thereof, compositions, nucleic acids, vectors, antigens, host cells, additional therapeutic agents, vaccines of the invention may contain from 1 mg to 1000 mg of active compound (i.e., about 60-120 mg / m), for single or two or more administrations at a time, as appropriate. 2 ) may be included. In any event, a physician will determine the actual dosage that will be most suitable for any individual subject, and dosage will vary with the age, weight, and response of the particular subject. The above dosages are exemplary of the average case. There will, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

[0316] Agents (compounds, polypeptides, antibodies, antigen-binding portions thereof, compositions, nucleic acids, vectors, antigens, host cells, further therapeutic agents, vaccines) can also be administered intranasally or by inhalation using suitable propellants such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, 1,1,1,2-tetrafluoroethane (HFA 134A3) or 1,1,1,2,3,3,3-heptafluoropropane (HFA The compounds are conveniently delivered in the form of a dry powder inhaler or aerosol spray dispenser from a pressurized container, pump, sprayer, or nebulizer using a hydrofluoroalkane such as 227EA3), carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container, pump, sprayer, or nebulizer can contain a solution or suspension of the compound, polypeptide, antibody, antigen-binding portion thereof, composition, nucleic acid, vector, antigen, host cell, additional therapeutic agent, or vaccine, using, for example, a mixture of ethanol as a solvent and a propellant, which may further contain a lubricant, such as sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator can be formulated to contain a powder mix of a compound of the invention and a suitable powder base, such as lactose or starch.

[0317] Aerosol or dry powder formulations are preferably prepared so that each metered dose or "puff" contains at least 1 mg of agent (compound, polypeptide, antibody, antigen-binding portion thereof, composition, nucleic acid, vector, antigen, host cell, additional therapeutic agent, vaccine) for delivery to a subject. It will be understood that the total daily dosage using an aerosol will vary from subject to subject and may be administered in a single dose, or more commonly, in divided doses throughout the day.

[0318] Alternatively, the agents (compounds, polypeptides, antibodies, antigen-binding portions thereof, compositions, nucleic acids, vectors, antigens, host cells, additional therapeutic agents, vaccines) can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment, or dusting powder. The compounds, polypeptides, antibodies, antigen-binding portions thereof, compositions, nucleic acids, vectors, antigens, host cells, additional therapeutic agents, and vaccines of the present invention may also be administered transdermally, for example, by the use of a skin patch. They may also be administered via the ocular route, specifically to treat eye diseases.

[0319] For ophthalmic use, agents (compounds, polypeptides, antibodies, antigen-binding portions thereof, compositions, nucleic acids, vectors, antigens, host cells, additional therapeutic agents, vaccines) can be formulated as ultrafinely divided suspensions in isotonic, pH-adjusted, sterile saline, or preferably as solutions in isotonic, pH-adjusted, sterile saline, optionally combined with a preservative such as benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.

[0320] For topical application to the skin, agents (compounds, polypeptides, antibodies, antigen-binding portions thereof, compositions, nucleic acids, vectors, antigens, host cells, additional therapeutic agents, vaccines) can be formulated into a suitable ointment containing the active compound, polypeptide, antibody, antigen-binding portion thereof, composition, nucleic acid, vector, antigen, host cell, additional therapeutic agent, vaccine suspended or dissolved in a mixture of one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax, and water. Alternatively, they can be formulated into a suitable lotion or cream suspended or dissolved in a mixture of one or more of the following: mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0321] Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0322] Generally, oral or topical administration of an agent (compound, polypeptide, antibody, antigen-binding portion thereof, composition, nucleic acid, vector, antigen, host cell, further therapeutic agent, vaccine) 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 also be administered parenterally, for example, sublingually or buccally.

[0323] For use in veterinary animals, the agent (compound, polypeptide, antibody, antigen-binding portion thereof, composition, nucleic acid, vector, antigen, host cell, further therapeutic agent, vaccine) will be in a formulation that is appropriately accepted in accordance with normal veterinary practice, and the veterinarian will determine the dosage regimen and route of administration that will be most appropriate for a particular animal.

[0324] Conveniently, the formulation is a pharmaceutical formulation. The formulation may be a veterinary formulation.

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

[0326] The listing or discussion of a published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0327] Preferences and alternatives in a given aspect, feature, or parameter of the invention should be considered as disclosed in combination with any and all preferences and alternatives in all other aspects, features, and parameters of the invention, unless the context indicates otherwise. For example, various definitions of VDE are relevant to all aspects of the invention, e.g., an epitope comprising VDE for use in vaccination against dengue virus infection may include any one or more of epitope scaffold proteins, where the scaffold protein may include a heterologous scaffold protein covalently linked to a virion-dependent epitope; an envelope protein of at least Q77, W101, N153, T155, K310; or an envelope protein of domain II, optionally further including any one or more of the following characteristics of domain II: the b strand (residues 67-74), the immediately upstream fusion loop and residues (residues 97-106), and the ij loop (residues 246-249). 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[0329] [Figure 1] Characterization of human anti-DENV monoclonal antibodies. (b) Serotype specificity and reactivity of 145 DENV mAbs to DENV envelope proteins by Western blot (WB). (c) Schematic of epitope mapping using a panel of mutant VLPs. The positions of mutations are indicated relative to the domain structure of the dengue envelope protein, with red, yellow, and blue representing domains I, II, and III, respectively. The fusion loop around W101 is depicted, and the position of the mutation that disrupts the N153 glycosylation motif is indicated. [Figure 2] In neutralization assays, EDE antibodies are potent and highly cross-reactive. Neutralization assays were performed on Vero cells with nine representative mAbs against all four DENV serotypes produced in C6 / 36 insect cells (three FL, EDE1, and EDE2 serotypes each). Data were from three independent experiments. [Figure 3]EDE-specific antibodies have excellent neutralizing activity. Neutralization assays using Vero as target cells were performed on DENV2 produced from C6 / 36 insect cells (C6 / 36-DENV) (a) or dendritic cells (DC-DENV) (b). The red, blue, and green bars represent the FRNT (percent reduction) at 0.05, 0.5, and 5 μg / ml (final concentration) of mAbs, respectively. The EDE mAbs used in the accompanying paper are marked below the histogram. Antibodies are classified as FL and EDE, while WB-positive antibodies that do not map on VLPs are referred to as non-FL. Based on the VLP mapping results, five subgroups of EDEs were identified and designated EDE15. (c) Titer curves for binding measured by capture ELISA, and neutralization responses of DC and C6 / 36 produced virus with FL and two representative antibodies from EDE1 and EDE2 groups. The data are from two independent experiments and are representative of the results from nine anti-FL and anti-EDE1 antibodies, and seven anti-EDE2 antibodies, respectively. [Figure 4] Antibody binding to virus particles at different maturation states. (a) Anti-prM and anti-E ELISAs were used to calculate the prM:E ratio in various virus particles compared to virus from LoVo cells, defined as 100% prM content. (b) Binding of representative mAbs to DENV2 produced from C6 / 36, DC, 293T, furin-transfected 293T, LoVo cells, or acid-treated DENV2 was measured by capture ELISA. Two FL, VDE1, and VDE2 mAbs are shown, along with the VDE4 mAb, which is sensitive to acid-treated virus. The data are from two independent experiments and are representative of 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 reactivity among patients is shown, with FL—blue and EDE—red. The numbers in the middle indicate the number of Abs from each patient; one copy of three replicate 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 on either C6 / 36 cells or DCs in the presence of titers of anti-E mAbs reactive to FL or EDE. Results are expressed as median peak enhancement (fold) from two independent experiments. Purple, blue, green, and red symbols represent the 752-2C8, 753(3)C10, 747(4)A11, and 747(4)B7 mAbs used in the accompanying paper (c) NT50 and NT90 titers between FL and EDE mAbs, respectively, which are compared for C6 / 36 and DC viruses. [Figure 6] Schematic of epitope mapping using a panel of mutant VLPs. The locations of mutations are shown relative to the domain structure of the dengue envelope protein, with red, yellow, and blue representing domains I, II, and III, respectively. The fusion loop around W101 is depicted, and the location of the mutation that disrupts the N153 glycosylation motif is indicated. [Figure 7] Germline 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 proteins from four dengue serotypes, uncomplexed or in complex with BNA (broadly neutralizing antibody) Fab fragments. MALS showed that the SEC chromatogram of sE proteins (green curve) corresponds only to the monomer fraction; no dimers were detected. The two peaks observed for sE serotypes 1, 3, and 4 correspond to the monomer. The most likely explanation is that the affinity of the dimer is not high enough, and the dimer-monomer equilibrium is disturbed in the gel filtration column, resulting in dimer dissociation. Complexation with Fab fragments apparently stabilizes the dimeric form, which allows the elution of the sE dimer complexed with two Fab fragments (red curve). We noted that only the late-eluting sE monomer fraction was converted to complex, while the other peaks remained unchanged (this was most evident with DENV-3 sE, but is also true for other serotypes). For DENV-2 sE, there is a single sE peak with a tail toward a smaller molecular weight that disappears upon complex formation. An explanation for this behavior is that, similar to our previous information on the alphavirus fusion protein El, the exposed fusion loops in the fraction containing sE competent to form dimers tend to interact with the support, resulting in such a slower elution fraction. 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, specific for DENV-4, is shown as a positive control, given that its well-characterized epitope is not at the dimer interface and does not require dimer formation for binding. Fab fragments were immobilized at similar densities on a Proteon XPR36 chip. A 2 μM solution of DENV sE protein from the four serotypes (as indicated) was injected simultaneously across all Fabs (see Online Extended Methods). SPR signals are presented in response units (RU) as a function of time in seconds (s).Note that the level of binding is generally consistent with the SEC / MALS plot of the corresponding antibody 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-validated 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 represented by the structures. [Figure 11]DENV2 sE complexed with four EDE anti-EDE antibodies. a) Complex with the EDE2 A11 Fab fragment. The sE dimer is oriented with its perpendicular 2-fold molecular axis down and its side facing the viral membrane. The sE promoter is shown as a surface colored by domain (I, II, and III, respectively, red, yellow, and blue) and the fusion loop as purple (labeled for certain promoters in b). Foreground and background sE subunits are distinguished by bright and dark colors. Two N-linked glycan chains (not included on the surface) are shown as ball-and-sticks colored by atom type (carbon white, oxygen red, nitrogen blue) and labeled (N67 and N153). The A11 Fab is shown as a ribbon representation with the heavy and light chains colored green and gray, respectively. b) Unlinked DENV2 FGA02 sE dimer seen down the 2-fold axis (labeled "2" in the center). The green and gray empty ellipses (labeled VH and VL), respectively, roughly indicate the contact sites of the heavy and light chains, with VH closer to the 2-fold molecular axis. The polypeptide portions and loops relevant to epitope interpretation are labeled c). The diagram below the open arrow shown in (b) highlights the fusion loop "cavity" covered between the two vertices, the b-strand on one subunit, and the 150 loop on the other. d-f) The same diagram as in c, showing complexes with the anti-EDE antibodies EDE2 B7 (d), EDE1 C8 (e), and EDE1 C10 (f) (only variable domains are shown). The open star in (e) and (f) marks the region of the 150 loop that is disordered in these complexes. Note that in the B7 and A11 complexes, the light chain is too far away 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 (broadly neutralizing antibody) complex (except the first, which shows the unlinked sE dimer), and each column shows the same orientation, as indicated. The first two columns show the sE dimer as a ribbon and the BNA variable domain as a surface colored as in Figure 11. In the side view (left column), the viral membrane can be seen at the bottom, while the bottom view (center column) corresponds to the sE dimer seen from the viral membrane, with the antibodies visible across the entire sE ribbon. The top view (right column) shows the sE surface presented to the immune system on the virus particle, with the antibody (green) imprint shown in white depth-cue fog. For clarity, a white outline defines the extent of the green imprint on the blue surface of domain III. For reference, in the top left panel, the glycan chains of the foreground and background subunits are labeled red and black, respectively. The fusion loop can be seen in other rows in contact with the anti-EDE antibody, as indicated in the upper center panel. In the left panels of rows c-e, a red star marks the position of the 150 loop, which is disordered in complex with the EDE1 anti-EDE antibody. This loop carries the N153 glycan recognized by the EDE2 anti-EDE antibody, as seen in the left panel of row b (glycan shown as a stick with red carbons). In contrast, all anti-EDE antibodies are seen to contact the N67 glycan with C8 (left panel of row c, N67 glycan as a stick with yellow carbon atoms), which shows the most contacts. The blue star in row c indicates the disordered 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] Buried surface area in various BNA complexes [Figure 14]Electrostatic potential of the DENV-2 sE complex, epitope, and paratope. A double-sided representation of the complex with negative and positive potential is shown and colored according to the underlying bar. Because certain regions are disordered in the complex, the electrostatic potential at the surface of the sE dimer was calculated using the DENV-2 sE dimer model generated as described in the Online Methods section. Corresponding contact regions are indicated by ovals of the same color. [Figure 15]Residues involved in BNA / antigen interactions. a) Amino acid sequence alignment of sE from four DENV serotypes, with residues in black or light blue backgrounds highlighting identity and similarity across serotypes, respectively. Secondary structure elements are indicated and labeled below the alignment, with tertiary structure indicated by color coding as in Figure 11. DENV2 sE residues contacted by various anti-EDE antibodies are shown above the alignment, indicating the contacted BNA regions by the codes provided where appropriate. Full symbols correspond to contacts in the reference subunit (defined as contributing the fusion loop to the epitope), and empty symbols correspond to contacts across the dimer interface. Colored boxes above the sequence highlight the five distinct regions of sE that make up the epitope. Figure 24 provides a histogram with the number of atomic contacts per sE residue in complex with each BNA. Because the EDE2 B7 and A11 contacts are very similar, only the B7 contact is shown here. Question marks on the 150 loop indicate that these residues likely contact the EDE1 anti-EDE antibody but are not visible in the structure due to the disordered nature of this loop. An alignment of four anti-EDE antibodies was crystallized and numbered according to the Kabat definition, with the FRW and CDR regions in gray and white backgrounds, respectively. CDRs corresponding to IMGT changes are marked with blue lines on the sequences. Somatic mutations are in red, with the corresponding germline residues written in smaller font below. Residues resulting from the VDJ (or VJ) recombination process are in green. The contacted sE moieties (corresponding to the colored boxes in Figure 15a) are indicated above each sequence and are coded as supported where appropriate. Secondary structure elements of the EDE2 C8 Ig β-barrel are indicated 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 the interactions of various BNAs with DENV-2 sE. a) Structure of all untethered EDE2 scFvs (red, unbound, 1.7A resolution) superimposed on all Fab variable domains in complex with DENV-2 sE (yellow, 3.8A resolution) to show that the same conformation is maintained in the sE / Fab fragment complex. b) Stereoview showing the variable domains of B7 (green) and A11 (yellow) superimposed with the 150 loop extracted from the corresponding Fab / DENV-2 sE complex structure. Note that the main chain of the 150 loop adopts different conformations in the two complexes, primarily due to the hydrogen bonding of the hydroxyl group of the Y99 side chain in CDR H3 of B7 with sE T155. All have a phenylalanine at this position, thus missing a hydroxyl group. In complex with A11, the sE protein exhibits the same conformation as untethered sE (not shown). c) Histogram of atomic contacts of B7 (above the sE sequence) and A11 (below this sequence). d) As in c), but showing the pattern of contacts made by BNAs C10 (above this sequence) and C8 (below) in the sE protein primary structure. e) As in c), but comparing C10 (EDE1) and B7 (EDE2) according to the E protein sequence. [Figure 17] The H3 loops of the EDE anti-EDE antibody and the broadly neutralizing anti-HIV antibody PG9. The Fab or scFv fragments are similarly oriented, with the light chain in gray, the heavy chain in green, and the H3 loop highlighted in red. For comparison, the Fab fragment of the potent anti-HIV-1 BNA PG951, which recognizes a wide range of glycans and has a very long H3 loop (30 aa as calculated by IMGT), is shown in the same manner. [Figure 18]BNA CDR interactions at the sE dimer interface. a) The right panel shows the sE dimer as a ribbon, and in the left panel, the epitope is expanded and key features are labeled. Rows b-d show the sE surface in a semi-transparent view, showing the ribbons. Glycan residues are shown as sticks (they were not included in the surface calculations). The relevant CDR loops of the anti-EDE antibody are shown as ribbons with their side chains as sticks on top of the sE protein, colored as in Figure 11. The orientation of the left panels 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 ribbons, with selected side chains as sticks and the sE surface in semi-transparent view, highlighting the interaction with the N67 glycan. A few hydrogen bonds are shown as dotted lines. The 150 loop is irregular (marked by density interruptions). In both (a) and (b), the right panel shows the arrowhead in the left panel along the glycan chain. b) The EDE2 B7 BNA / DENV-2 sE complex, shown with B7 in the same orientation as C8 in (a) to emphasize that EDE1 and EDE2 anti-EDE antibodies bind in a similar manner. EDE2 B7 inserts its long CDR H3 into the cavity of the fusion loop, while its side contacts two glycan chains, as seen in the left panel. The H3 α-helix packs against the N153 glycan. Also shown are the numerous hydrogen bonds between anti-EDE antibodies and sE, listed in Figure 21c). Key to the sugar connectivity and nomenclature used in the text and in panels (a) and (b). d) Contacts between the antibody and sugar residues (as in Figure 24), coded according to the key. e) Dengue mutants lacking the N153 glycan are more easily recognized by EDE1 anti-EDE antibodies, whereas they are more resistant to neutralization by EDE2 anti-EDE antibodies. Mean and sem values ​​were estimated from three independent experiments. [Figure 20]Experimental electron density for glycan chains. Ribbon representation of a) EDE2 all Fab and b) EDE2 C8 EDE1 Fab complexed with DENV2 sE, colored as in Figure 11. Simulated annealing omit maps, contoured at 1 sigma (turquoise) or 0.6 sigma (gold), show clear density for the N153 (in a) and N67 glycans (in a and b) (red and yellow arrows, respectively). To generate unbiased maps, all glycan atoms were removed from the structure, all B factors were initialized to 20A2, and the structure was re-refinemented using simulated annealing with torsional dynamics. Note that the antibody footprint spreads the two glycans across the dimer interface (also shown in Figure 11). c) Zoomed-in views of the complexes seen under the red (left panel) and yellow (right panel) arrows in panels a) and b) for DENV-2 / B7 (left panel) and DENV-2 / C8 (right panel), respectively. Heavy and light chains are shown as surfaces, and glycans (along with the corresponding asparagine) are labeled with the average B-factor for each residue. Glycans are also ramp-colored 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 omit maps for the glycans in the three complexes. [Figure 21] Polar interactions between antibodies and antigens. [Figure 22]C10 BNA imprints on sE dimers of dengue serotypes 2 and 4. a) Representation of the surface of DENV-2 sE as seen from outside the virion, with exposed main-chain atoms orange (top panel), or main-chain atoms plus strictly conserved side chains (orange), and highly conserved side chains (yellow) (bottom panel). The epitopes of the EDE1 BNA C10 (black outline) and EDE2 BNA B7 (green outline) are shown. b) Surfaces of DENV-2 (top panel) and DENV-4 (bottom) sE dimers extracted from complex with C10, color-coded by domain as in Figure 11. The C10 footprint in each case is shown. Note the asymmetry in the conformation. The large "hole" on the right-hand side of the dimer (in both panels) is due to the irregular ij loop, while the smaller hole on the left (bottom panel, DENV-4 sE / C10) is due to the irregular kl loop. [Figure 23] Comparison of C10 interactions with DENV-2 and DENV-4 sE. a) The structures of the DENV-2 (red) and DENV-4 (green) sE dimers complexed with C10 were superimposed onto the C10 moiety. The axis of the sE dimer is drawn at the center, appropriately colored. For clarity, only the C10 scFv on which the superposition was performed is shown for each complex. When the antibodies were superimposed, the sE dimers only locally coincided, 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 gray / red curved arrow), resulting in a significantly different orientation when the superposition is performed with the scFv on the left (binding to epitope A) than with the scFv on the right (binding to epitope B), highlighting the asymmetry of C10 binding to the sE dimer. b) C10 contacts are displayed in the alignment of DENV-2 (top) and DENV-4 sE (bottom), showing a very similar pattern of contacts in the two complexes. The sequence background corresponds to that of Figure 15 in the main text, demonstrating conservation among the four serotypes. [Figure 24]Detected asymmetry of BNA binding to sE dimers. This figure provides a histogram of the number of contacts per sE residue in the structures of all independent complexes analyzed here in panels (a)-(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, and e) EDE1 Cl0 / DENV-4 sE. Each panel is divided into two parts. Part (I) shows the immune complex seen below the 2-fold axis of the sE dimer on the left (with the contact domain of the Fab fragment removed for clarity) and on the right with the antibody completely removed to show the epitope. This part also defines epitopes A and B used in II. Part (II) shows a histogram of contacts corresponding to the A and B epitopes in the dimer, with histogram bars color-coded as indicated, to map the antibody regions involved in contacts (in brackets are the symbols used in Figure 15a to mark the corresponding contacts). Note that although the contact pattern remains 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 the asymmetric unit (i.e., two sE dimers, each with two C10 scFvs), so there are a total of four independent epitope diagrams, labeled A through D, as shown in (b). [Figure 25]EDE1 C10 residue Y100 (CDR H3) likely interacts with F279 in mature dengue virions. a) One of the 90 E / M heterotetramers comprising the mature DENV-2 particle, extracted from a 3.5 Å cryo-EM reconstruction, is displayed in a side view (drawn as a white bar labeled "2," 2-fold axis vertical) with two E subunits in gray and yellow and two M subunits in red and salmon. Two black arrows indicate the connection between the E ectodomain (corresponding to sE) with its α-helical membrane-interacting region (the horizontal "stem" α-helix and the vertical TM α-helix). The N-terminal portion of M appears to interact underneath the E dimer (pink arrow). b) View of the lower 2-fold axis, with the region enlarged in (c) boxed. c) This view is slightly tilted for clarity with respect to the view in (b) with the structures of both C10 complexes (DENV-2 and -4 sE) superimposed on virion E. These labels correspond to the color of the corresponding structure (DENV-2 sE / C10 green / mustard, DENV-4 sE / C10 blue / beige, and virion E as in panels (a) and (b)). The M protein is shown as a salmon-colored surface (labeled white). The M protein underlies the E dimer and across the dimer interface highlights the base of the kl hairpin (contained between the arrows and labeled), inducing a different conformation of the kl hairpin; thus, F279 (labeled) points away from the hydrophobic core of the E protomer (dark gray bar), whereas in the sE protein structure (containing unlinked sE, not shown), it is part of the hydrophobic core (green and blue bars, labeled). The side chain of Y100 (labeled) in CDR H3 of C10 has an alternative conformation because it does not find its partner in sE (Y100 is also illustrated in Figure 18d, left panel). The CDR H3 loop is flexible enough that Y100 can make stacking interactions with F279 (conserved across serotypes, see Figure 15a) in the conformation observed in virions. [Figure 26]Histogram of antibody residue contacts. This figure mirrors Figure 24, this time showing contacts 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 complex, colored in gray (VH dark gray, VL light gray), with somatic mutations in red and interface residues resulting from the combination in green. Side chains involved in contacts are represented and labeled with balls and sticks. Part II shows a histogram of the number of atomic contacts per residue, color-coded accordingly to indicate the contacting regions of sE (in brackets are the symbols used in Figure 15b to mark the corresponding contacts). The sequence numbering and background correspond to the Kabat transformation (as in Figure 15b). CDRs corresponding to the IMGT transformation are indicated on the sequence by orange dotted lines. Somatic mutations are in red, residues resulting from VDJ (or VI) combinations are in green. [Figure 27] Comparison with the binding properties of potent anti-EDE antibodies targeting other viruses. [Figure 28] Sequences of dengue envelope proteins from serotypes 1 to 4 [Figure 29] Sequences of EDE1 and EDE2 type antibodies identified in Example 1. [Figure 30] How to perform a neutralization test. [Figure 31] Contact residues in envelope proteins from crystal structures. [Figure 32] Covalently cross-linked DV2 E dimers [Figure 33] Binding of EDE1 to rE DENV2 WT versus MT A259C [Figure 34] Binding of EDE2 to rE DENV2 WT versus MT A259C [Figure 35] Binding of FLE to rE DENV2 WT vs. MT A259C [Figure 36] rE Binding of non-FLE to DENV2 WT vs. MT A259C [Figure 37]rE EDE1 binding to DENV2 WT vs. MT L107C, A313C [Figure 38] rE EDE2 binding to DENV2 WT vs. MT L107C, A313C [Figure 39] rE Binding of FLE to DENV2 WT vs. MT L107C, A313C [Figure 40] rE Binding of non-FLE to DENV2 WT vs. MT L107C, A313C [Figure 41] Antibody titers in C6 / 36 DENV2 Group 1 Monomer / monomer E Prime and boost with WT E WT Group 2 Dimer / dimer E Prime and boost with A259C mutant Group 3 VLP / VLP Prime and boost with prM / E virus-like particles (VLPs) Group 4 Dimer / VLP E Prime with A259C mutant followed by boost with VLPs Group 5 VLP / dimer Prime with VLP followed by boost with E A259C mutant Group 6 Sham Non-vaccinated [Figure 42] Cross-reactivity: Binding to live virus (pooled serum) Group 1 Monomer / monomer E Prime and boost with WT E WT Group 2 Dimer / dimer E Prime and boost with A259C mutant Group 3 VLP / VLP Prime and boost with prM / E virus-like particles (VLPs) Group 4 Dimer / VLP E Prime with A259C mutant followed by boost with VLPs Group 5 VLP / dimer Prime with VLP followed by boost with E A259C mutant Group 6 Sham Non-vaccinated [Figure 43]Neutralization of mouse sera: C6 / 36 DENV2 Group 1 Monomer / monomer E Prime and boost with WT Group 2 Dimer / dimer E Prime and boost with A259C mutant Group 3 VLP / VLP Prime and boost with prM / E virus-like particles (VLPs) Group 4 Dimer / VLP E Prime with A259C mutant followed by boost with VLPs Group 5 VLP / dimer Prime with VLP followed by boost with E A259C mutant Group 6 Sham Non-vaccinated [Figure 44] Mouse serum:DC DENV2 neutralization Group 1 Monomer / monomer E Prime and boost with WT E WT Group 2 Monomer / monomer E Prime and boost with A259C mutant (dimer / dimer) Group 3 VLP / VLP Prime and boost with prM / E virus-like particles (VLPs) Group 4 Dimer / VLP E Prime with A259C mutant followed by boost with VLPs Group 5 VLP / dimer Prime with VLP followed by boost with E A259C mutant Group 6 Sham Non-vaccinated [Figure 45] ADE: Pooled mouse sera: U937 Group 1 Monomer / monomer E Prime and boost with WT E WT Group 2 Monomer / monomer E Prime and boost with A259C mutant (dimer / dimer) Group 3 VLP / VLP Prime and boost with prM / E virus-like particles (VLPs) Group 4 Dimer / VLP E Prime with A259C mutant followed by boost with VLPs Group 5 VLP / dimer Prime with VLP followed by boost with E A259C mutant Group 6 Sham Non-vaccinated Examples Example 1 - Human DENV antibodies form two distinct groups based on their ability to bind to dengue envelope proteins in Western blots.

[0330] Samples from seven patients (Table 1) were used to generate 145 human monoclonal antibodies reactive to DENV envelope proteins. 32、33Plasmablasts (CD3 - , CD20 lo / -、 CD19 + , CD27 hi , CD38 hi ) were sorted 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 (Fig. 1a). [Table 1] Table 1. Summary 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 fully representative panel of antibodies. Only 57% of the antibodies reacted to DENV envelope by Western blot (Fig. 1a), and WB-negative mAbs also failed to react to recombinant E by ELISA. This grouped the antibodies into two broad groups: WB-reactive and those that only recognized epitopes present on intact virions. From here on, we refer to these antibodies as reactive to virion-dependent epitopes or envelope dimer epitopes (VDE or EDE). The majority of WB-positive antibodies were fully cross-reactive among the four virus serotypes, while 41 of 62 EDEDE mAbs were fully cross-reactive, and a further 17 of 62 reacted to DENV-1, -2, and -3 (Fig. 1b). Neutralization assays of three antibodies from each group on viruses produced in C6 / 36 insect cells are shown in Fig. 2. Fusion loop and EDEDE antibodies were broadly neutralizing against all four virus serotypes. EDE2 747(4)A11 and 747(4)B7 had lower activity against Den4, which is related to the loss of the N-linked glycan at position N153 in Den4 strain H241. Methods Related to This and Other Embodiments

[0332] Samples. Blood samples were collected from hospitalized patients following written informed consent. The study protocol was approved by the UK Tropical Diseases Hospital Scientific Ethics Committee, 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 (infecting serotype was also identified), NS1 antigen lateral flow test, or seroconversion in IgM ELISA tests. Disease severity was classified according to the 1997 World Health Organization criteria. Of the patients enrolled in this study, two patients were classified as having mild dengue fever (DF), and five patients were classified as having severe symptoms with plasma leakage and hemorrhage of dengue hemorrhagic fever (DHF) (Table 1). Secondary infection was defined based on a dengue-specific IgM to IgG ratio of less than 1.8. 7 Blood samples for B cell sorting were collected during hospitalization when blood plasmablast populations were 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. The C6 / 36 cell line, derived from the mosquito Aedes albopictus, was cultured in Leibovitz L-15 at 28°C. Vero, U937, and 293T or furin-transfected 293T cells were grown in MEM, RPMI 1640, and DMEM, respectively, at 37°C. 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. The furin-deficient LoVo cell line was 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 Abs were gifts from Dr. C. Puttikhunt and Dr. W. Kasinrerk (Puttikhunt, 2003). Anti-DENV NS3, E1D8, was a gift from Dr. Eva Harris.

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

[0336] Generation of DENV-specific human monoclonal Abs. DENV-specific human mAbs were generated from activated B cells / plasmablasts. 32、33 Briefly, PBMCs were stained with anti-CD3, CD19, CD20, CD27, and CD38. Activated antibody-secreting cells (ASCs) were then isolated by immunoblotting using CD19 + , CD3 - , CD20 低 / - , CD27 高 , CD38 高The ASCs were gated as . Single ASCs were sorted into each well of a 96-well PCR plate containing 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 the appropriate restriction endonucleases and cloned into IgG1, Igκ, or Igλ expression vectors, a gift from Dr. Hedda Wardemann. To express the antibodies, the heavy and light chain plasmids were cotransfected into 293T cells using the polyethylenimine method, and antibody supernatants were harvested for further characterization.

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

[0338] Detection of DENV-specificity and serotype cross-reactivity by ELISA. DENV1-4 and mock-uninfected supernatants were separately captured on MAXISORP immunoplates (NUNC) coated with anti-E Abs (4G2). DENV-captured wells were then incubated with 1 μg / ml human mAb, followed by ALP-conjugated anti-human IgG. The reaction was visualized by the addition of 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 DENV envelope protein, and bovine serum albumin (BSA) was used as a negative control antigen. Protein-coated wells were then incubated with 1 μg / ml of human monoclonal Abs, followed by ALP-conjugated 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 supernatants from C6 / 36 cells were prepared under nonheated and nonreducing conditions, run on 12% SDS polyacrylamide gels, and electrophoresed onto nitrocellulose membranes (Amersham). The membranes were then blocked with 5% skim milk and probed with DENV-specific human mAbs, followed by HRP-conjugated anti-human IgG Abs. The membranes were developed with an enhanced chemiluminescence substrate (Amersham). Example 2 - Mutational analysis shows that EDE and WB-reactive antibodies bind to different epitopes.

[0341] To gain more insight into the epitopes recognized by the mAbs, we generated 65 virus-like particles (VLPs) containing alanine substitutions at solvent-exposed residues predicted to be on the virion surface. These were derived from the 3D structure of mature virus particles. 4、7、8 These mutant VLPs were screened against 145 monoclonal antibodies by ELISA. 22、36 Mutations that resulted in a greater than 80% reduction in antibody binding were considered significant. Using this panel, 112 of 145 mAbs were assigned to epitopes on the envelope protein. Thirty-three antibodies, all of which reacted to E by WB, remained unmapped using the mutant VLP panel. The results of epitope mapping 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 restricted set of residues inside and outside 101W that define the aforementioned or classical fusion loop epitope (FL). 46 of 83 antibodies that bind to the envelope in WB are sensitive to mutations at position 101W, previously shown to be a key residue for binding of many anti-DENV mAbs. 37、38 Of the FL-specific mAbs, 40 of 46 were sensitive to the W101 mutation alone, while 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 contacts with residues 104-110, but not with 101W, and with residues 74-79 in the bc loop. 39 Similar to the 1C19 mAb, only two of the FL-specific mAbs were sensitive to changes in the bc-loop, resulting in loss of binding 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 patterns of reactivity to VLP mutants (Figures 1c and 6). The majority of EDE antibodies were sensitive to changes in fusion loop residue 101 but should not be confused with the classic "fusion loop"-specific antibodies mentioned above, in which the epitope is much broader and contains additional determinants in domains I, II, and III. The majority of EDE antibodies can be divided between two distinct subgroups, EDE1 and EDE2, differentiated by their sensitivity to changes at residues 153 and 155 in EDE2, which would disrupt an N-linked glycosylation site. Twelve antibodies comprised three further subgroups with distinct epitopes and functions; EDE3 mAb was similar to EDE1 mAb but was also sensitive to changes at 107L and 295K. EDE4 was insensitive to changes at 101W and reacted best to acidified virus particles (Figure 4b). Finally, EDE5 mAb constitutes a group of 5 mAbs that bind exclusively to fusion loop 101W, an epitope that is only repeated in intact virions; EDE3, 4, and 5 are all poorly neutralizing antibodies (Figure 3a and b).

[0344] VLP mutagenesis experiments suggest that the EDE is a composite quaternary epitope involving 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 generate VLPs (constructed by Dr. Aleksandra Flanagan). 55 VLP mutants were generated by PCR-based site-directed mutagenesis. 62Mutagenic PCR was performed using Pfx DNA polymerase (Invitrogen) to replace selected amino acid residues in the E protein with alanine; if already alanine, the mutation was made to glycine. After treatment with DpnI (NEB), the PCR product was transformed into E. coli. All mutations were confirmed by sequencing. The plasmid was transfected into 293T cell line by the polyethylenimine method, and the culture supernatant was harvested for epitope mapping.

[0346] To identify epitope-specific Abs, WT and mutant VLPs were captured with mouse anti-prM (1H10). Then, DENV-specific anti-E Abs were added at 1–5 μg / ml, followed by anti-human IgG-ALP. Finally, 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 the 4-component mAb series). Example 3 - WB-reactive antibodies, unlike EDE antibodies, are unable to completely neutralize virus produced in human dendritic cells.

[0347] During DENV infection, the host is presented with two forms of virus: initial exposure is with virus produced in insect cells, while virus produced in human cells drives subsequent infection cycles and represents the vast amount of virus produced during infection. To examine these two distinct virus forms, we compared DENV-2 virus produced in C6 / 36 insect cells (C6 / 36-DENV) or monocyte-derived dendritic cells (DC-DENV), which are infected after injection of virus into the skin following a mosquito bite and are thought to be the site of virus replication in infected human hosts. 20 .

[0348] Of the 83 WB-positive mAbs, 46 were mapped to FL, and 37 identified binding sites that had yet to be mapped. Surprisingly, all 83 WB-positive antibodies were unable to completely neutralize DC-DENV, even at high concentrations; only one neutralized DC-DENV by more than 80% at 5 μg / ml (Figures 3a and 3b). Meanwhile, most EDE1 and 2 antibodies were able to neutralize DC-DENV by more than 80%, with many reaching 100% neutralization. The full binding and neutralization curves for representative mAbs (Figure 3c) show that anti-FL mAbs reduced binding to DC-DENV (blue crosses) by ELISA and failed to completely neutralize DC viral infection (blue circles), whereas the neutralization and binding curves for EDE mAbs were more closely aligned with those of C6 / 36 and DC-DENV. method

[0349] Neutralization and enhancement assays. The neutralizing potential of mAbs was determined using a focus reduction neutralization test (FRNT) where the reduction in the number of infected foci was compared to the control (no antibody). 22 Briefly, serially diluted Abs were mixed with 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 an anti-E mAb (4G2) followed by an HRP-conjugated rabbit anti-mouse IgG. The 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 the reduction rate versus Ab concentration using the Probit program from the SPSS package. Example 4 - Anti-EDE antibodies are unable to bind to viruses at high proportions of prM or when the envelope protein adopts a trimeric conformation.

[0350] To represent these different viral forms, antibody binding to six DENV-2 preparations was compared. To assess the degree of prM cleavage, the prM:E ratio was measured by ELISA and normalized to DENV produced in LoVo cells, which lack furin activity and produce almost entirely incomplete mature virus particles with intact prM. 17 (Fig. 4a). The virus preparations were: 1) C6 / 36-DENV, which has a relatively high prM content of 56%, 2) DC-DENV, which has a prM content of 13%, and 3) virus produced in furin-deficient LoVo cells (LoVo-DENV), which has a prM content approaching 100%. 22 , 4) virus produced in 293T cells overexpressing furin with 5% prM content (furin-293T-DENV), 5) virus produced in native 293T cells with 60% prM (293T-DENV), and 6) virus incubated at pH 5.5 (acidic DENV) that irreversibly adopts the E trimer conformation. 42 .

[0351] EDE1 and 2 mAbs were unable to bind acidic DENV, presumably because trimerization disrupts conformational epitopes, or to LoVo-DENV, presumably because the full complement of prM supports the prM / E spike, which may again disrupt mature envelope dimer epitopes or sterically hinder access to the EDE (Fig. 4b). Anti-FL mAbs showed reduced binding to viruses with low prM content, shifting the binding curves for DC-DENV and 293T-furin-DENV by 1.5 to 2 logs to the right of C6 / 36 produced DENV. Furthermore, binding to LoVo-DENV was even more efficient than that to C6 / 36 DENV, highlighting the importance of prM in the exposure and effective binding of fusion loop antibodies. 39、43 Four EDE4 antibodies isolated from three separate individuals bound most effectively to acid-treated virus, but these showed negligible neutralization (Fig. 4b). method

[0352] DENV-binding ELISA. To determine the binding affinity of Abs to DENV produced from different cell types, DENV2 produced from mock, C6 / 36, DC, 293T, furin-transfected 293T, or LoVo cells, and acid-treated C6 / 36 DENV2 were captured on 4G2-coated plates and then incubated with serial dilutions of DENV-specific human monoclonal Abs, followed by ALP-conjugated anti-human IgG. The reaction was triggered by the addition of PNPP substrate and stopped with NaOH. Absorbance was measured at 405 nm. Day-to-day variations in OD readings due to antigen loading of different viral forms and between experiments were normalized by a control ELISA using a humanized version of the well-described 3H5 mAb, which is specific for domain III of DENV2. Example 5 - Antibodies in specific patients show immunodominance

[0353] The anti-DENV mAbs described herein are complex ensembles with overlapping specificities, where 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 a preference for either FL or EDE epitopes (Fig. 5a). This immunodominance of recognition within individuals was surprising. When epitopes overlap, it is possible that the most avid antibodies can compete out other antibodies and undergo affinity maturation, thus dominating the response, leading to stochastic selection between FL and EDE. However, responses to EDE or FL are polyclonal within individuals (recombination of different VDJs), making this an unlikely explanation. Example 6 - Anti-EDE antibodies result in reduced levels of antibody-dependent enhancement of infection

[0354] We tested the ability of antibodies to enhance DENV infection in Fc receptor expressing U937 cells. All antibodies tested induced ADE, but it was approximately 4-8-fold lower in the EDE group compared to the FL group. The mean peak enhancement in the FL vs. 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 Abs were preincubated with virus for 1 h at 37 °C, transferred to U937 cells (a human monocytic cell line bearing Fc receptors), and incubated for 4 days. Supernatants were harvested and titrated onto Vero cells by focus-forming assay. Viral titers were expressed as focus-forming units (FFU) per ml, and fold enhancement was calculated by comparing the viral titer with that in the absence of antibody. Example 7 - Anti-EDE antibodies bind to recombinant sE dimers

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

[0357] Recombinant sE protein (400 amino-terminal residues of the ectodomain of the envelope protein, referred to as "sE" for "soluble E") and antigen-binding portion (Fab) and side chain variable domain (scFv) anti-EDE antibodies were produced in Drosophila S2 cells. 44、45 Because anti-EDE antibodies did not react with recombinant sE protein in standard ELISA assays, we tested the interaction of the antibody fragments with purified recombinant DENV sE in solution at high concentrations to favor dimer formation. Size-exclusion chromatography (SEC) coupled with multi-angle light scattering (MALS) experiments showed that the dimer / monomer equilibrium of recombinant DENV-1, -2, -3, and -4 sE was mostly shifted toward dimers, with the antibody fragments eluting as complexes corresponding to the sE dimer with two antibody fragments, despite the size-exclusion-induced dissociation effect during the separation of the 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 confirm the cross-reactive determinants, a total of seven crystal structures were determined, including a DENV-2 sE dimer complexed with fragments of four selected anti-EDE antibodies and a DENV-4 sE dimer complexed with EDE1 C10. Because the DENV-2 sE dimer used belongs to a different strain than those for which structures are already available, an untethered sE dimer was crystallized to detect possible conformational changes induced by the antibody. Furthermore, because it was unclear whether the long CDR H3 of the untethered A11 scFv maintained the same conformation in the absence of antigen, the structure of the untethered A11 scFv was determined. The crystallization procedure is described in Example 15, and the crystallographic statistics are listed in Figure 9. DENV-2 sE strain FGA02, genotype III

[0359] Serotype 2 Asian / American genotype III 11The majority of structural studies were performed using recombinant sE from DENV-2 field strain FGA02 (isolated in French Guiana in 2002), belonging to the DENV-2 sE family. FGA02 sE was scattered across 394 residues (3%) of the ectodomain, similar to previously crystallized DENV-2 sE. 5、7 It shows 13 amino acid differences compared to the 3 Å resolution structure of FGA02 sE. As expected, the 3 Å resolution structure of FGA02 sE shows only minor differences with the previously available structure of DENV-2 sE in its prefusion form, falling within the range of conformations observed in various structures deposited in the PDB (accession numbers 1OAN, 1OKE, and 1TG8) (Figure 10). By demonstrating that the structure of uncoupled FGA02 sE is not due to a specific amino acid sequence in the E protein of this strain, it was useful in assessing the region where antibody binding induces disease, specifically the 150 loop (see below). Example 9 - Envelope dimer epitopes Anti-EDE antibodies bind at the sE dimer interface

[0360] The crystal structure of the FGA02 sE immune complex shows that the four anti-EDE antibodies bind in a similar manner (Figure 11), interacting with both subunits of the dimer (see also ED in Figure 12, which provides the imprint of each anti-EDE antibody on the sE dimer). The heavy chains bind closer to the 2-fold axis (i.e., the center of the dimer), while the light chains are peripherally positioned. The epitopes are located at low pH. 46 In immature DENV particles exposed to UVB radiation, the imprints of the prM protein on the E dimer largely overlap. They are centered in a cleft 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 spans residues 148-159, connects to the β-strands E0 and F0 of domain I, and carries the N153 glycan located on the fusion loop of the partner subunit in the dimer. The heavy chain extends the distance between the two glycan strands, N67 and N153, across the dimer interface (Figures 11c-f). The total buried surface per epitope is 1050 Å. 2 ~1400Å 2The surface complement coefficients range from 13 is 0.67-0.74, which are typical values ​​for antibody / antigen complexes (Figure 13). The surface electrostatic potentials of the epitope and paratope are lightly charged and have relatively complementary charge distributions (Figure 14). Conserved residues make up the epitope

[0361] Anti-EDE antibodies essentially contact a cluster of highly conserved residues across the four serotypes (Figure 15), explaining their cross-reactivity. The 26-residue CDR H3 of B7 and A11 accounts for the majority of EDE2 anti-EDE antibody contacts in both sE subunits, forming 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 preformed in the antibody, as shown by the 1.7 Å resolution structure of the untethered EDE2 A11 scFv, indicating no entropic cost for binding (Figures 9, 16, and 17). In the reference subunit, defined as contributing the fusion loop to the epitope, both the EDE1 and EDE2 anti-EDE antibodies target the same serotype-invariant residues clustered in three major polypeptide segments of domain II (boxed in Figure 15a): the b-strand (residues 67-74 bearing the N67 glycan), the immediately upstream fusion loop and residues (aa 97-106), and the ij-loop (aa 246-249). While both the light and heavy chains of the EDE1 anti-EDE antibody interact with the reference subunit through all three CDRs, the EDE2 anti-EDE antibody interacts primarily with the heavy chain and contacts only a small portion of the light chain from CDR L3 (Figure 18). In the opposite subunit, across the interface, the targeted sE segment differs from the two EDE groups. The EDE2 anti-EDE antibody interacts with the 150 loop and the N153 glycan chain (see below), whereas the EDE1 anti-EDE antibody induces a disturbance of the 150 loop upon binding, which is consistent with the murine DENV cross-reactive antibody 47、48It is possible that the light chain in the EDE1 anti-EDE antibody is brought closer to sE and interacts with domain III in the region of the previously structurally characterized "A-strand" epitope for sE. These domain III interfaces are centered around the conserved sE residue K310, whose side chain creates a lid over the indole ring of fusion loop W101 in a critical stabilizing sE dimer interface (Figure 18). Although the light chains are derived 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 (21 aa, Fig. 7) to interact with conserved residues under the 150-loop (scattered over the N-terminal 50 amino acids of the E protein, see circled areas in Fig. 15a or Fig. 18d, left panel), whereas the shorter H3 loop of EDE1 C8 cannot reach this region. Example 10 - Antibody recognition of glycan chains

[0362] The anti-EDE antibodies make extensive contacts with the glycan chain at both positions N67 and N153 of E (Figures 19 and 20). All four anti-EDE antibodies interact with the N67 glycan through the CDR H2 interface, thus enhancing the N67 glycan. 49 This would prevent binding to the DC-SIGN receptor on dendritic cells, which was shown to specifically interact with the sE / EDE1 C8 complex. The DENV-2 sE / EDE1 C8 complex shows the highest N67 glycan structure with a distal mannose residue that contacts framework region 3 of the heavy chain (FRW H3, Figures 15b and 18c). With the exception of EDE1 C10 (which is very close to its germline, Figure 7), many FRW H3 residues are altered (Figure 15b), suggesting affinity maturation of the saccharide recognition. If additional glycan residues were visible in the structure, it is possible that the same FRW H3 residue interactions could be seen in other anti-EDE antibodies.

[0363] Although the N150 loop and N153 glycan are disordered in the EDE1 complex, the limited space between the antibody and domain I (Figure 19a, left panel) suggests that this glycopeptide segment contacts the antibody (as indicated by the question mark above the 150 loop in Figure 15A) but adopts a variable local conformation in each complex to avoid, on average, resolved electron density in the crystal. If the 150 loop remained in place, the CDR H3 loop of the EDE1 anti-EDE antibody could clash 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 for the core six sugar residues of the N153 glycan of sE in a crystal of the complex with the EDE2 anti-EDE antibody (shown in Figure 20, including an abbreviated map). The CDR H3 of the EDE2 anti-EDE antibody creates a two-turn α-helix (designated the H3 helix, Figure 11d) with one of the carbonyl groups at its C-terminus end shielded by a hydrogen bond provided by the N2 atom of the first N153 GlcNAc residue (Figure 21). The aromatic side chains of Y99 (F99 in EDE2 A11) and Y100 project laterally to encase sugar residues 1, 3, and 4 of the N153 glycan. The most distal residues of the glycan, mannose 4, 5, and 6, make contact with the light chain through residues from CDR L2, involving several hydrogen bonds (Figures 19 and 21).

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

[0366] In the fusion loop, residues 101-WGNG-104 create a distorted α-helical turn that projects the W101 side chain toward domain III, across from the dimer interface. In complex with the EDE2 anti-EDE antibody, the helical turn of the fusion loop is under the H3 helix, with the carbonyl groups at the C-terminal ends of the two helices pointing toward each other. Furthermore, S100C of CDR H3 shields the helical turn by making a main-chain and side-chain hydrogen bond to the carbonyl group of G102 in the complex loop. In complex with the EDE1 anti-EDE antibody, the fusion loop is located just below and to the right of the VH / VL interface, with the side chains of several aromatic residues from both the heavy and light chains packing against it. Specifically, the VL backbone is in close proximity by providing a hydrogen bond to the backbone carbonyl group of G104; at EDE1 C8, the backbone 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 at both C10 and C8 forms a salt bridge with K310 (Figures 19a and 18d), which is part of an extensive network of polar interactions in this region (listed in Figure 21).

[0367] The conformation of the glycine-rich fusion loop in the E dimer leaves the main chain essentially exposed, while the side chains are mostly buried. Together with the main chain of the ij loop, the main chain atoms form a large surface patch augmented by one end of the b strand, which results in an invariant exposed surface recognized by anti-EDE antibodies. The invariant side chains in this region, together with the exposed main chain at the E dimer surface (Figure 22a, bottom panel), result in a core region epitope that is essentially invariant serotype with surrounding non-conserved residues. The reason for this conversion is likely related to the interaction with prM during particle maturation. 46 The smallest conserved region is on the surface of domain III within the EDE1 epitope. Example 12 - Structure of DENV-4 sE complexed 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 is the most divergent in amino acid sequence from other dengue serotypes (Fig. 15a) and is potently neutralized by anti-EDE antibodies (Fig. 19e). The 2.7 Å resolution crystal structure of DENV-4 sE complexed with C10 scFv confirmed the general pattern observed in the complex with DENV-2 sE (Figs. 22b and 23), which has an irregular 150 loop. As expected, the anti-EDE antibody exhibits the same interactions between the main chain and the conserved side chains of the epitope. In the more variable lateral region of the EDE1 epitope in domain III (Fig. 22a), a contact site involves the side chain of residue 309, which is an aspartic acid in DENV-4 but a valine in DENV-2 (Fig. 15a). In the latter complex, there is Van der Waals packing of T52 with the side chain V309 of CDR L2, whereas in the former complex there is a polar interaction in which D309 accepts a hydrogen bond from the T52 side chain (Fig. 23b and Fig. 21). Other contacts with domain III are also maintained, specifically that at position 362, including a hydrogen bond to the main-chain carbonyl (Fig. 21).

[0369] EDE1 C10 induces disorder of the 150 loop in DENV-2 sE, whereas in the case of DENV-4 sE, this loop appears to exhibit an intrinsic higher mobility, as suggested by its crystal structure complexed with the Fab fragment of an unrelated chimpanzee antibody designated 5H2 (see 17 ). In fact, the 5H2 epitope, also located in domain I, flanks the sE dimer and does not overlap with the anti-EDE antibody epitopes described herein, although the 150 loop is disordered in its structure. Furthermore, the structure of the DENV-4 sE / EDE1 C10 complex highlighted a non-negligible degree of asymmetry in the contact of the anti-EDE antibody with 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 induces an asymmetric conformational adjustment of the sE dimer, likely affecting the second site to accommodate the available conformation of the second epitope when the second antibody fragment binds. Taken together, these observations strongly suggest that the binding determinants of EDE1 anti-EDE antibodies lie in a conserved core of the epitope, in a region shared with EDE2 anti-EDE antibodies, with contacts at either end accommodating specific side chains present in each serotype that do not interfere with binding. This observed flexibility of the E dimer is consistent with reports of conformational breathing of the E dimer in virions, exposing normally hidden epitopes upon antibody interaction. Example 13 - Putative additional EDE1 C10 / E dimer interactions in mature virions

[0370] A detailed structural examination is performed on EDE1 The tip of CDR H3 of C10 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 the intact virion, the region is reinforced by the underlying protein M (Figure 25). The 3.5 Å resolution cryo-EM structure of an intact mature particle of DENV-2 (see 9) shows that the interaction with M results in the conserved residue F279 at the bottom of the E kI hairpin (Fig. 15a) being exposed at the dimer interface (Fig. 25c) instead of being buried in the hydrophobic core of domain II. Superposition with the DENV-2 sE / C10 structure indicates that in the virion context, the exposed F279 side chain may interact with Y100 in the H3 loop. Y100 appears to interact differently with DENV-2 sE compared to DENV-4 (Fig. 25c; ED; also compare panels b and e in Fig. 26), suggesting that it does not find its exact partner. Therefore, it appears that the EDE1 C10 binding site on the E dimer in mature virions is not perfectly replicated by the recombinant sE dimer. This observation likely explains the clear discrepancy between the weak binding of EDE1 C10 to sE dimers (Fig. 8) and the strong binding and neutralization of viruses from four dengue serotypes (NT50 in the low nM range, see accompanying manuscript). Importantly, the conformation of F279 in mature virions is similar to that observed for sE bound to hydrophobic ligands. 4 This suggests that it may be possible to induce the correct conformation of this region of the recombinant sE dimer as an immunogen. Example 14

[0371] We provide a snapshot of anti-EDE antibodies interacting with the major novel epitopes targeted by human monoclonal antibodies elicited in dengue-infected patients. These antibodies appear to converge toward the same specificity through distinct evolutionary paths, either requiring heavy chains with very long CDR H3s that perform most of the interactions, as in the case of EDE2, or obtaining finely tuned combinations of light and heavy chains with main chain contacts to the fusion loop and domain III in the case of the EDE1 anti-EDE antibody analyzed here. 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 conformationally specific quaternary epitopes on the virion surface. Their common characteristics from alanine scanning experiments (accompanying manuscript) strongly suggest that they all target the same quaternary epitope described herein.

[0372] Importantly, the binding determinants of EDE anti-EDE antibodies are entirely localized to the E dimer and are not specific to a different flavivirus, West Nile virus. 51 Based on studies in DENV particles 50 It does not differ depending on the higher-order arrangement of dimers on the virion surface, as has been recently suggested for quaternary epitopes. Recent cryo-EM analysis of DENV-2 particles suggests that the herringbone pattern may be disturbed at physiological temperatures in humans, with dimers reorienting with each other, relaxing their symmetric arrangement, and / or displaying different surface patterns. 52、53 Thus, the epitopes described herein are accessible to E dimers, either expansion-independent or particle-independent, and may be preferred targets for next-generation vaccines. Corollary, our results suggest that the epitopes described herein may be accessible to E dimers, either expansion-independent or particle-independent, and may be preferred targets for next-generation vaccines. 54 As recently proposed for , stabilizing dimer contacts in such a way that only E dimers are presented to the immune system and therefore suitable for designing potent immunogens by avoiding eliciting antibodies against poorly immunogenic regions that are not normally accessible on the surface of infectious virions.

[0373] The primary binding determinant of EDE anti-EDE antibodies appears to be the conformation of the fusion loop and its adjacent main chain in the context of the intact E dimer. This is in stark contrast to the other major class of antibodies isolated from humans in the accompanying manuscript, which recognize the fusion loop sequence in a quaternary tissue-independent context. The latter antibodies are cross-reactive but poorly neutralizing, with potentially strong infection enhancement. 56 A notable feature of the epitopes described herein is the number of exposed backbone atoms, which in the case of EDE1 account for approximately 30% of the total surface area buried in the complex, and 20% for the EDE2 anti-EDE antibody (this lower proportion of EDE2 is generally compensated for by the 40% invariant glycan composition) (Figure 13), while the general backbone atoms contribute 5%-15% for most of the analyzed immune complexes. 57 In this regard, certain highly potent neutralizing antibodies, such as the D25 antibody against respiratory syncytial virus (RSV), which bind to "antigenic site 0" that is only present on the stabilized, pre-fusion form of the RSV fusion protein, as do EDE anti-EDE antibodies, also recognize a high proportion (approximately 30%) of the main chain atoms in the antigen (Figure 27). 58 BNA CH65, or H1, which neutralizes a broad range of H1 influenza virus isolates by binding to the receptor binding pocket of 59 A similar pattern is found with CR8020, a potent group 2 reactive anti-influenza human BNA with neutralizing activity against H3, H7, and H10 isolates by binding to the base of the stem. CR8020 also recognizes the main chain conformation of the fusion peptide in the pre-fusion trimeric conformation, without the context of the quaternary structure, similar to the EDE anti-EDE antibodies described herein. Finally, B12 (see references), two of the very broad anti-HIV-1 anti-EDE antibodies, 60 ) and VRC01 (see 61) recognize the CD4 binding site in the envelope (ENV) protein and exhibit 36% and 33% of the main-chain atoms in the epitope (Figure 27), suggesting that recognition of the main-chain conformation is an important aspect shared by many (but not all) anti-HIV-1 antibodies. These broadly neutralizing anti-HIV-1 antibodies exhibit effective binding. 29 EDE also requires the correct quaternary structure of the ENV trimer, undergoes a long affinity maturation process, and shows more than 20% divergence from the germline, whereas EDE anti-EDE antibodies to dengue fever show a maximum 9% divergence from the germline (Figure 27), indicating that they can be generated relatively easily in individuals when appropriate immunogens are used for vaccination.

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

[0375] Recombinant sE proteins from DENV serotypes 1 to 4, as well as Fab and scFv BNA fragments, were synthesized using the above-described protocols. 44、45、29The sE / BNA complex was produced in Drosophila melanogaster Schneider 2 using a chromatographic assay. 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 by SEC or, in the case of EDE1 C10, by mixing the two in a 1:2 sE:antibody ratio. Crystallization studies were performed using a robotic facility. Diffraction data were collected at the synchrotron sources SOLEIL and ESRF, and the structure was determined by molecular replacement using the search model listed in Figure 9, which also provides relevant crystallographic statistics. Neutralization studies with DENV-4 mutants were performed using the same procedures outlined in the accompanying document. 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) were produced in Drosophila S2 cells essentially as described above for DENV-4 sE (Den4_Burma / 63632 / 1976), with some modifications. 29Briefly, sE expression was driven by a metallothionein promoter and induced by 5 μM CdCl in insect XPRESS medium (Lonza). This construct had a Drosophila BiP signal sequence fused to the N-terminal end of the prM-sE construct for efficient translocation to the ER of transfected S2 cells. prM, in the case of the DENV polyprotein precursor, presents the N-terminus of sE with the prM N-terminus and sE generated by signalase cleavage in the ER. prM (membrane-anchored) acts as a chaperone by covering the fusion loop of sE. The prM / sE complex is transported across an acidic fraction when prM is cleaved by furin into pr (the N-terminal half that binds to sE) and M (the membrane-anchored C-terminal half). Upon reaching the external environment, sE, and pr dissociation products, the sE components are purified by affinity chromatography from the cell supernatant fluid. DENV-3 and -4 sE constructs had C-terminal fusions with 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. Clarified cell supernatants were concentrated 20-fold using a Vivaflow tangential filtration cassette (Sartorius, 10 kDa cutoff) and, depending on the construct, adjusted to 0.5 M NaCl after buffer exchange to remove divalent ions before purification on an AKTA FPLC system using either StrepTactin affinity purification or HisTrap-HP chromatography. His-tagged proteins (DENV-1 and -2 sE) were desalted after elution from the HisTrap column and further purified by ion-exchange chromatography on a MonoQ column. A final purification SEC step using a Superdex 200 10 / 300 GL column equilibrated in 50 mM Tris pH 8, 500 mM NaCl was performed for all constructs. Production of Fab and ScFv

[0377] The BNA fragments were purified by Fab synthesis in Drosophila S2 cells. 62and scFv 63 The construct contained a twin strep tag fused to the C-terminus (in the case of the Fab, of the heavy chain only) for affinity purification. The purification protocol included the same steps and used the same buffers as described above for the strep-tagged sE protein. Immune complex formation and isolation

[0378] Purified DENV sE protein was mixed with Fab or ScFv (at approximately a two-fold molar excess) in standard buffer (500 mM NaCl, Tris 50 mM pH 8.0 buffer). The volume was brought to 0.2 ml by centrifugation in a Vivaspin 10 kDa cutoff, and after 30 min of incubation at 4 °C, the complex was separated from the excess Fab or scFv by SEC, unless no clear peak was obtained in the complex (similar to BNA C10; see Figure 8). In this case, a 1:2 antigen:antibody molar ratio mixture (i.e., with excess antibody) was used directly for crystallization. In all cases, the buffer was exchanged into 150 mM NaCl, 15 mM Tris, pH 8 for crystallization trials. Protein concentrations used for crystallization, determined by measuring the optical density at 280 nm and using the extinction 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 was 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 at the same concentration as a control. Samples were incubated for 15 min at room temperature and analyzed by MALS upon elution from an SDX200 10 / 300 GL gel filtration column run at a flow rate of 0.4 ml / min, followed by refractometry and MALS detection on a DAWN Heleos-Optilab T-rEX setup (Wyatt Technology). Surface plasmon resonance

[0380] Real-time SPR measurements of the binding of sE dimers to the capture Fab fragment of the anti-EDE antibody were 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 the anti-HCV E2 antibody was used as a negative control. After Fab capture, the chip was rotated 90°, and sE from four 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. Control injections 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 neutralizing potential of anti-EDE antibodies was determined using a focus reduction neutralization test (FRNT) where the reduction in the number of infected foci was compared to the control (no antibody). 22DENV-4 strains H241 (which has an Ile at position 155), 1-0093, and 1-0554 (both of which have a Thr at position 155, thus restoring glycosylation at Asn153) were grown in C6 / 36 cells. Viral titers were determined by focus formation in Vero cells. 64 Briefly, serially diluted anti-EDE antibodies were mixed with 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 mouse monoclonal 4G2 antibody (which cross-reacts with E proteins from all flaviviruses) followed by rabbit anti-mouse IgG conjugated with horseradish peroxidase. The reaction was visualized by the addition of diaminobenzidine substrate. The percentage of focus reduction was calculated for each antibody dilution. The 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 experiments were performed in 400 nl drop fixtures. Droplets were formed by mixing equal volumes of protein and reservoir solution in a 96-well Greiner plate format using a Mosquito robot and monitored by a Rock-Imager. Crystals were optimized manually in 400 nl drop fixtures with the robotized Matrix Maker and Mosquito setup, or in 24-well plates using 2-3 μl hanging drops (Figure 9). Crystallization and cryo-cooling conditions for diffraction data collection 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 beamlines ID23-2 and ID29 (Figure 9). Diffraction data were analyzed using the XDS package. 65 Processing was performed using the CCP4 package software. 67 Combine with other programs from SCALA or AIMLESS 66 The structure was measured using PHASER ® with the search models listed in Figure 9. 68 and / or AMoRe 69 was determined by molecular replacement with

[0385] Next, COOT 70 Careful model construction with the program BUSTER / TNT 71 Alternating cycles of crystallographic refinement with TLS refinement resulted in the final model. The refinement was constrained to respect non-crystallographic symmetries and, depending on the resolution of the crystal, targeted restraints (with parts of the complex in the high-resolution structure) and TLS refinement. 72 (See Figure 9.) The final abbreviated map was generated using Phenix.Refine. 73 was calculated using Analysis and explanation of atomic models

[0386] Each complex is programmed using the CCP4 package software 67 The contacts of each residue of the Fab / ScFv or DENV sE protein were then counted and plotted as proportional bars over the corresponding residue.

[0387] Ab sequences are 30 and IMGT 31 To map the CDR / FWR regions according to the conversion, Abysis (www.bioinf.org.uk / software) and IMGT (www.imgt.org) were used.31 Analysis of putative germline and somatic maturation events was performed on the IMGT website (www.imgt.org).

[0388] Multiple sequence alignments and phylogenetic trees were generated using ClustalW (ClustalW and ClustalX version 2 (see 74 ) to the EBI server 75 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). 76 The database from [link to original text] was used to extract the amino acid sequences of the sE ectodomain and extended to include DENV2 FGA02 sE and DENV-2 10AN sE. For ease of presentation, sub-roots were collapsed to the level of individual genotypes for DENV-2. This phylogenetic tree was then rooted at the DENV-4 sE sequence and analyzed using the MEGA5 software package. 77 It was decided to measure using

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

[0390] The figure shows Program ESPript 78 and APBS 79 and PDB2PQR tool 80 The data were prepared using the PyMOL Molecular Graphics System, version 1.5.0.4, Schrodinger, LLC (pymol.sourceforge.net), including:

[0391] Finally, current vaccine strategies employ tetravalent formulations with the goal of generating a balanced type of specific response against all four serotypes. The description here of such potent and cross-reactive antibodies points the way to subunit vaccines containing desired epitopes and, optionally, heterologous prime-boost strategies that recapitulate responses seen in natural sequential infections. Example 16: Sequence information Sequence number SEQ ID NO: 1 Complete sequence of antibody C8 heavy chain EVQLVESGGGLVQPGGSLRLSCSASGFTFSTYSMHWVRQAPGKGLEYVSAITGEGDSAFYADSVKGRFTISRDNSKNTLYFEMNSLRPEDTAVYYCVGGYSNFYYYYTMDVWGQGTTVTVSEQ ID NO: 2 Complete sequence of antibody C10 heavy chain EVQLVESGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNGNTKYSQKFQDRVTITRDTSASTAYMELSSLRSEDTAIYYCARDKVDDYGDYWFPTLWYFDYWGQGTLVTV SEQ ID NO: 3 Complete sequence of antibody A11 EVQLVESGGGLVRPGGSLRLSCAASGFSYSNHWMHWVRQAPGKGLVWVSRINSDGSTRNYADFVKGRFTISRDNAENTLYLEMNSLTADDTAVYYCVRDGVRFYYDSTGYYPDSFFKYGMDVWGQGTTVTVSEQ ID NO: 4 Complete sequence of antibody B7 heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSSHWMHWVRQAPGKGLVWVSRTNSDGSSTSYADSVKGRFMISRDNSKNTVYLHMNGLRAEDTAVYFCARDGVRYYYDSTGYYPDNFFQYGLDVWGQGTT SEQ ID NO: 5 C8 CDR H1 TYSMH SEQ ID NO:6 C8 CDR H2 AITGEGDSAFYADSVKG SEQ ID NO:7 C8 CDR H3 GYSNFYYY SEQ ID NO:8 C10 CDR H1 SYAMH SEQ ID NO:9 C10 CDR H2 WINAGNGNTKYSQKFQD SEQ ID NO: 10 C10 CDR H3 DKVDDYGDYWFPTLW SEQ ID NO: 11 A11 CDR H1 NHWMH SEQ ID NO: 12 A11 CDR H2 RINSDGSTRNYADFVKG SEQ ID NO: 13 A11 CDR H3 DGVRFYYDSTGYYPDSFFKY SEQ ID NO: 14 B7 CDR H1 SHWMH SEQ ID NO: 15 B7 CDR H2 RTNSDGSSTSYADSVKG SEQ ID NO: 16 B7 CDR H3 DGVRYYYDSTGYYPDNFFQY SEQ ID NO: 17 C8-CDR L1 RASQSISTFLA SEQ ID NO: 18 C8 CDR L2 DASTRAT SEQ ID NO: 19 C8 CDR L3 QQRYNWPPYT SEQ ID NO: 20 C10 CDR L1 TGTSSDVGGFNYVS SEQ ID NO: 21 C10 CDR L2 DVTSRPS SEQ ID NO: 22 SSHTSRGTWVF SEQ ID NO: 23 A11 CDR L1 TGTSSNADTYNLVS SEQ ID NO: 24 A11 CDR L2 EGTKRPS SEQ ID NO: 25 A11 CDR L3 CSYATSRTLVF SEQ ID NO: 26 B7 CDR L1 TGISSDVETYNLVS SEQ ID NO: 27 B7 CDR L2 EASKRPS SEQ ID NO: 28 B7 CDR L3 CSYAGGKSLV SEQ ID NO: 29 Full-length envelope protein sequence DENV1 >DENV1 strain Hawaii MRCVGIGNRDFVEGLSGGTWVDVVLEHGSCVTTMAKDKPTLDIELLKTEVTNPAVLRKLCIEAKISNTTTDSRCPTQGEATLVEEEQDANFVCRRTFVDRGWGNGCGLFGKGSLITCAKFKCVT KLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTIATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNEMVLLTMKEKSWLVHKQWFLDLPLPWTSGASTPQETWNREDLLVTFKTAHAKK QEVVVLGSQEGAMHTALTGATEIQTSGTTKIFAGHLKCRLKMDKLTLKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSTQDEKGVTQNGRLITANPIVTDKEKPVNIEAEP PFGESYIVVGAGEKALKLSWFKKGSSIGKMLEATARGARRMAILGDTAWDFGSIGGVFTSVGKLVHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGLNSRSTSLSMTCIAVGMVTLYLGVMVQA SEQ ID NO: 30 Full-length envelope nucleotide sequence DENV1 SEQ ID NO: 31 Full-length envelope protein sequence DENV2 >DENV2 strain 16681 MRCIGMSNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFRCKK NMEGKVVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKK QDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEP PFGDSYIIIGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVIITWIGMNSRSTSLSVTLVLVGIVTLYLGVMVQA SEQ ID NO: 32 Full-length envelope nucleotide sequence DENV2 SEQ ID NO: 33 Full-length envelope protein sequence DENV3 >DENV3 strain H87 MRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRKLCIEGKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLESIEGKVVQHENLKYTVIITVHTGDQHQVGNETQGVTAEITSQASTAEAILPGYGTLG LECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRRELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGGTSIFAGHLKCRLKMDKLELKGMSYAMCLNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKE EPVNIEAEPPFGESNIVIGIGDKALKINWYRKGSSIGKMFEATARGARRMAILGDTAWDFGSVGGVLNSLGKMVHQIFGSAYTALFSGVSWIMKIGIGVLTWIGLNSKNTSMSFSCIAIGIITLYLGVVVQA SEQ ID NO: 34 Full-length envelope nucleotide sequence DENV3 SEQ ID NO: 35 Full-length envelope protein sequence DENV4 >DENV4 strain 241 MRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELIKTTAKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTCAKFSCSG KITGNLVQIENLEYTVVVTVHNGDTHAVGNDIPNHGVTATITPRSPSVEVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWAAGADTSEVHWNYKERMVTFKVPHAKR QDVIVLGSQEGAMHSALTGATEVDSGDGNHMFAGHLKCKVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGAGAPCKVPIEIRDVNKEKVVGRIISSTPFAEYTNSVTNIELEP PFGDSYIVIGVGDSALTLHWFRKGSSIGKMLESTYRGVKRMAILGETAWDFGSVGGLFTSLGKAVHQVFGSVYTTMFGGVSWMVRILIGFLVLWIGTNSRNTSMAMTCIAVGGITLFLGFTVHA SEQ ID NO: 36 - Full length envelope nucleotide sequence DENV4 SEQ ID NO: 37 C8 light chain - see table below SEQ ID NO: 38 Full sequence of antibody C10 light chain - see table below SEQ ID NO: 39 Full sequence of antibody A11 light chain - see table below SEQ ID NO: 40 B7 light chain See table below SEQ ID NOs: 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] SEQ ID NO: 132 DENV1 envelope ectodomain protein sequence FHLTTRGGEPHMIVSKQERGKSLLFKTSAGVNMCTLIAMDLGELCEDTMTYKCPRITEAEPDDVDCWCNATDTWVTYGTCSQTGEHRRDKRSVALAPHVGLGLETRTETWMSSEGAWKQIQKVETWALRHPGFTVIALFL AHAIGTSITQKGIIFILLMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPAVLRKLCIEAKISNTTTDSRCPTQGEATLVEEQDANFVCRRTVVDRGWGNGCGLFGKGSLL TCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTIATITPQAPTSEIQLTDYGTLTLDCSPRTGLDFNEVVLLTMKEKSWLVHKQWFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLG SQEGAMHTALTGATEIQTSGTTTIFAGHLKCRLKMDKLTLKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSTQDEKGVTQNGRLITANPIVTDKEKPINIETEPPFGESYIIVGAGEKALKLSWFKKG SEQ ID NO: 133 DENV2 envelope ectodomain protein sequence MRCIGISNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPT LDFELIKTEAKQPATLRKYCIEAKLTNTTTESRCPTQGEPSLNEEQDKRFICKHSMVDRGWGNGCGLFGKGGIVTCAKFTCKKNMEGKIVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSTTEAELTGYGTVTMECSPRTGLDFNEMVLLQMEDKAWLVHRQWFLDLP LPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKIVKEIAETQHGTIVIRVQYEGDGSPCKIPFEITDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIVGVEPGQLKLNWFKRG SEQ ID NO: 134 Envelope ectodomain protein sequence DENV3 FHLTSRDGEPRMIVGKNERGKSLLFKTASGINMCTLIAMDLGEMCDDTVTYKCPHITEVEPEDIDCWCNLTSTWVTYGTCNQAGEHRRDKRSVALAPHVGMGLDTRTQTWMSAEGAWRQVEKVETWALRHPGFTILALF LAHYIGTSLTQKVVIFILLMLVTPSMTMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRKLCIEGKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSL VTCAKFQCLESIEGKVVQHENLKYTVIITVHTGDQHQVGNETQGVTAEITSQASTAEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTKTPTWNRKELLVTFKNAHAKKQEVVVLGS QEGAMHTALTGATEIQTSGGTSIFAGHLKCRLKMDKLKLKGMSYAMCLNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDKALKINWYRKG SEQ ID NO: 135 Envelope ectodomain protein sequence DENV4 FSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLGEMCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQSGERRREKRSVALTPHSGMGLETRAETWMSSEGAWKHAQRVESWILRNPGFALLAGFM AYMIGQTGIQRTVFFVLMMLVAPSYGMRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELTKTTAKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVV TCAKFSCSGKITGNLVQIENLEYTVVVTVHNGDTHAVGNDTSNHGVTAMITPRSPSVEVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNYKERMVTFKVPHAKRQDVTVLG SQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGAGAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELEPPFGDSYIVIGVGNSALTLHWFRKG SEQ ID NO: 136 Envelope ectodomain nucleotide sequence DENV1 ttccatttga ccacacgagg gggagagcca cacatgatag ttagtaagca ggaaagagga aagtcactct tgttcaagac ctctgcaggt gtcaatatgt gcactctcat tgcgatggat ttgggagt tatgtgagga cacaatgact tacaaatgcc cccggatcac tgaggcggaa ccagatgacg ttgactgctg gtgcaatgcc acagacacat gggtgaccta tgggacgtgt tctcaaccg gtgaaccg acgacaaa cgttccgtgg cactggcccc acacgtggga cttggtctag aacaagac cgaaacatgg atgtccctg aaggcgcctg gaacaata caaaaagtgg agacttgggc tttgagacac ccaggattca cggtgatagc tcttttta gcacatgcca taggaacatc catcactcag aagggatca tttcattct gctgatgctg gtaacaccat caatggccat gcgatgcgtg ggaataggca acagactt cgttgaagga ctgtcaggag caacgtgggt ggacgtggta ttggagcatg gaagctgcgt caccaccatg gcaaaaata aaccaacatt ggacattgaa ctcttgaaga cggaggtcac gaaccctgcc gtcttgcgca aattgtgcat tgaagctaaaatcaaca ccaccaccga ttcagatgt ccaacacag gagaggctac actggtggaa gaacagacg cgaactttgt gtgtcgacga acggttgtgg acagaggctg gggcaatggc tgcggactat tggaaagg aagcctactg acgtgtgcta agttcaagtg tgtgacaaaa ctggaaggaa agatagttca attgaaaac ttaaatatt cagtgatagt cactgtccac acagggacc acaggggt gggaaacgag actacagaac atggaacaat tgcaaccata acacctcaag ctcctacgtc ggaaatacag ttgacagact acggaaccct tacactggac tgctcaccca gaacagggct ggactttaat gaggtggtgc tattgacaat gaaagaaaaa tcatggcttg tccacaaaca atggtttcta gacttaccac tgccttggac ttcgggggct tcaacatccc aagagacttg gaacagacaa gatttgctgg tcacattcaa gacagctcat gcaaagaagc aggaagtagt cgtactggga tcacaggaag gagcaatgca cactgcgttg accggggcga cagaaatcca gacgtcagga<000"2027>acgacaacaa tctttgcagg acacctgaaa tgcagattaa aaatggataa actgacttta aaagggatgt catatgtgat gtgcacaggc tcatttaagc tagagaagga agtggctgag acccagcatg gaactgtcct agtgcaggtt aaatacgaag gaacagatgc gccatgcaag atcccctttt cgacccaaga tgagaaagga gtgacccaga atgggagatt gataacagcc aatcccatag ttactgacaa agaaaaacca atcaacattg agacagaacc accttttggt gagagctaca tcatagtagg ggcaggtgaa aaagctttga aactaagctg gttcaagaaa<000"2033> gga SEQ ID NO: 137 Envelope outer domain nucleotide sequence DENV2 ttccattaa cacacgtaa cggagaacca cacatgatcg tcagtagaca agagaaaggg aaagtcttc tgtttaaac...

Claims

[Claim 1] The invention described in this application.

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