WO2016130786A2 - Flaviviridae proteins and virions and methods of use thereof - Google Patents

Abstract

Embodiments described herein provide polypeptides, such as polypeptides from dengue comprising one or more mutations that can be used, for example, as pharmaceutical compositions and, for example, to induce an immune response in a subject. Embodiments provided herein also provide pharmaceutical compositions that can be used as vaccines against dengue or other flaviviruses.

Description

FLAVIVIRIDAE PROTEINS AND VIRIONS AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62/116,524, filed February 15, 2015, which is hereby incorporated by reference in its entirety.

GOVERNMENT RIGHTS

[0002] Certain embodiments described herein were made with government support under

NIH contract HHSN272200900055C awarded by the National Institutes of Health. Accordingly, the government has certain rights in embodiments.

BACKGROUND

[0003] Live attenuated vaccines (LAVs), which reproduce natural immunity, have been used for the development of vaccines against many diseases, including some viruses belonging to the same genus as dengue (examples of commercially available flavivirus live-attenuated vaccines include yellow fever and Japanese encephalitis vaccines). The advantages of live- attenuated virus vaccines are their capacity to induce both humoral and cellular immune responses. In addition, the immune response induced by a whole virion vaccine against the different components of the virus (structural and non- structural proteins) reproduce those induced by natural infection. Thus, attenuated vaccines are often a preferred method of inducing immunity against infectious agents, such as dengue.

[0004] Often attenuated vaccines are made by passing live un-attenuated vaccines under various conditions to produce an attenuated virus that can be used to induce an immune response in a subject without the subject developing, or developing less severe, symptoms of the infection. The identification of attenuated viruses can be time consuming and laborious. Accordingly, there is a need for the identification of mutant dengue viruses that can be used as an attenuated live-virus to induce an immune response in a subject. The embodiments disclosed herein provided for these needs and others. SUMMARY

[0005] Embodiments described herein provide polyprotein, including isolated polyproteins, comprising a DENV1, DENV2, DENV3, or DENV4 polypeptide comprising at least one mutation in a E-protein that decreases infectivity, retains or increases budding and retains or increases native antibody reactivity of a DENV1, DENV2, DENV3, or DENV4 virus.

[0006] Embodiments described herein also provide DENV3 E-proteins comprising at least one mutation at position G406, D415, G421, 1452, M453, K454, G456, or 1464 of SEQ ID of the DENV3 E-Protein (SEQ ID NO: 1). In some embodiments, DENV3 E-protein comprising a mutation selected from the group consisting of G406R, D415G, G421A, I452N, M453T, K454R, G456S, and I464T of the DENV3 E-Protein (SEQ ID NO: 1), or any combinations thereof, are provided. In some embodiments, DENV3 E-proteins comprising a mutation selected from the group consisting of E233G, R186H, H207L, A265V, A203D, G406R, D415G, G421A, I452N, I452S , H345P, I452V, H435R, E366V, I452N, F446L, P330L, M453T, K454R, K454E, P354L, I478T, K454T, V423A, G456S, N364S, I464T, and I464L of the DENV3 E-Protein (SEQ ID NO: 1), or any combinations thereof, are provided. In some embodiments, DENV3 E- proteins comprising a mutation selected from the group consisting of: E233G, R186H, H197L, A265V, A203D, V206E, E366V, P330L, P354L, and N364S, or any combinations thereof, are provided

[0007] In some embodiments, DENV4 E-proteins comprising at least one mutation at a position selected from the group consisting of: H261, M278, S298, G408, G423, 1455, L458, 1466, R188, A267, M454, R456, L458, V6, G14, V53, R73, D98, 1132, N134, F193, H209, Q211, D225, Q256, E269, R350, P356, E370, F373, S396, 1398, G399, F402 of the DENV4 E Protein (SEQ ID NO: 2) are provided.

[0008] In some embodiments, DENV1 E-proteins comprising a mutation that corresponds to a mutation at position of G406, D415, G421, 1452, M453, K454, G456, R186, A265, G406, D415, 1452, M453, K454, G456, N364, H259, 1276, or S296 of DENV 3 are provided.

[0009] In some embodiments, DENV1 E-proteins comprising a mutation that corresponds to a mutation at position of H261, M278, S298, G408, G423, 1455, L458, 1466, R188, A267, M454, R456, L458, V6, G14, V53, R73, D98, 1132, N134, F193, H209, Q211, D225, Q256, E269, R350, P356, E370, F373, S396, 1398, G399, F402, or V439 of DENV 4 are provided.

[0010] In some embodiments, DENV2 E-proteins comprising a mutation that corresponds to a mutation at position of G406, D415, G421, 1452, M453, K454, G456, R186, A265, G406, D415, 1452, M453, K454, G456, N364, H259, 1276, or S296 of DENV 3 are provided.

[0011] In some embodiments, DENV2 E-proteins comprising a mutation that corresponds to a mutation at position of H261, M278, S298, G408, G423, 1455, L458, 1466, R188, A267, M454, R456, L458, V6, G14, V53, R73, D98, 1132, N134, F193, H209, Q211, D225, Q256, E269, R350, P356, E370, F373, S396, 1398, G399, F402, or V439 of DENV 4 are provided.

[0012] In some embodiments, virions comprises the proteins described herein are provided. In some embodiments, pharmaceutical compositions comprising the proteins described herein are provided. In some embodiments, pharmaceutical compositions comprising the virions described herein are provided.

[0013] In some embodiments, methods of eliciting an immune response in a subject are provided. In some embodiments, the methods comprise administering to a subject a pharmaceutical composition comprising the proteins and/or the virions described herein.

[0014] In some embodiments, methods of preventing or ameliorating flavivirus infections are provided. In some embodiments, the methods comprise administering to a subject pharmaceutical compositions comprising proteins or virions as described herein.

[0015] In some embodiments, vaccines are provided. In some embodiments, the vaccines comprise a flavivirus comprising a mutation that corresponds to a mutation in a DENV1, DENV2, DENV3, or DENV4 E-protein that decreases infectivity, increases budding and optionally retains native antibody reactivity. In some embodiments, recombinant dengue viruses with reduced infectivity as compared to a wild-type virus are provided. In some embodiments, the virus comprises a peptide described herein. [0016] In some embodiments, isolated polyproteins comprising a DENV1, DENV2,

DENV3, or DENV4 polypeptide comprising one or more mutations at a position described herein are provided.

[0017] In some embodiments, isolated polyproteins comprising a DENV1, DENV2,

DENV3, or DENV4 polypeptide comprising a mutations at one or more positions described herein are provided.

[0018] In some embodiments, pharmaceutical compositions comprising a DENV virion comprising a mutation at one or more positions as described herein are provided.

[0019] In some embodiments, pharmaceutical compositions comprising a DENV virion comprising one or more mutations in a polyprotein as described herein are provided.

[0020] In some embodiments, methods of eliciting an immune response comprising administering to a subject a virus particle (virion) and/or a polypeptide comprising a mutations at one or more positions described herein or one or more mutations described herein are provided.

[0021] In some embodiments, methods of preventing or ameliorating a dengue infection comprising administering to a subject a pharmaceutical composition comprising a dengue virus comprising a mutation at one or more positions in a polyprotein as described herein or comprising one or more mutations in a polyprotein as described herein are provided.

[0022] In some embodiments, vaccines comprising a dengue virus comprising a mutation at one or more positions in a polyprotein as described herein or comprising one or more mutations in a polyprotein as described herein are provided.

[0023] In some embodiments, dengue virus with reduced infectivity as compared to a wild-type virus, wherein the virus comprises one or more mutations as described herein or a mutation at one or more of positions described herein are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1. Identification of critical stem and TM residues required for DENV infectivity. Each clone in the DENV-3 mutation library was tested for (A) DENV E-protein expression levels by detecting cellular expressed E-protein with a cocktail of diverse E-protein MAbs, (B) viral particle budding levels by capturing and then detecting DENV virions released by producer cells, and (C) infectivity levels by detecting luciferase expression levels in target cells infected with DENV reporter virus particles (RVPs) made with each mutated prM/E protein. RVPs are antigenically identical to live virus and have been used in numerous studies of DENV function (Austin et al., Structural basis of differential neutralization of DENV-1 genotypes by an antibody that recognizes a cryptic epitope, 2012, PLoS Pathog, 8 :el 002930; de Wispelaere and Yang, Mutagenesis of the DI/DIII linker in dengue virus envelope protein impairs viral particle assembly, 2012, J Virol, 86:7072-7083; Puschnik et al., Correlation between dengue-specific neutralizing antibodies and serum avidity in primary and secondary dengue virus 3 natural infections in humans, 2013, PLoS Negl Trop Dis, 7:e2274; Shrestha et al., Complex phenotypes in mosquitoes and mice associated with neutralization escape of a Dengue virus type 1 monoclonal antibody, 2012, Virology, 427: 127-134; Sukupolvi-Petty et al., Structure and function analysis of therapeutic monoclonal antibodies against dengue virus type 2, 2010, J Virol, 84:9227-9239; Zompi et al., Dominant cross-reactive B cell response during secondary acute dengue virus infection in humans, 2012, PLoS Negl Trop Dis, 6:el568). RVPs carry a reporter gene in place of structural genes so are capable of only a single round of infection (Ansarah-Sobrinho et al., Temperature-dependent production of pseudoinfectious dengue reporter virus particles by complementation, 2008, Virology, 381 :67-74; Mattia et al., Dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes, 2011, PLoS One, 6:e27252). (D) Clones with wild-type levels (>75%) of expression and budding, but low (<25%) infectivity were selected for confirmation in repeat assays. These screens identified eight residues as critical for infectivity. Shown are the values and ranges (max-min) for E-protein expression levels (n=3), viral budding (n=2), and viral infectivity (n=2), all as a percentage of wild-type activities.

[0025] Figure 2. Mutation of G406 perturbs adjacent structures in the Env dimer.

(A) Budding values for E-protein stem mutations critical for infectivity, G406R, D415G, and G421A, obtained from different capture and detection protocols. G406R demonstrates a 2-3 fold increase with 1A1D-2 detection, and low activities with anti-prM capture or anti fusion loop (F loop) detection (arrows). Budding assays used RVPs with mouse cocktail capture (mus)/human cocktail 1 (huml) detection, human cocktail 2 (hum2) capture/mouse 1A1D-2 detection, mouse anti-prM capture/human 1A1D-2 detection, and mouse cocktail capture/anti-fusion loop detection. [0026] Figure 3. Model for the effect of G406R on the structure of the E dimer on the mature virion. Perturbation of E-Hl structure by G406R disrupts the tethering interaction with Dili, shielding the fusion loop and prM (where present) from MAb interactions, while increasing the exposure of the 1 A1D-2 binding site on DHL

[0027] Figure 4. A model for the functions of critical stem and TM residues in E- protein triggering and membrane fusion. In the mature virion, the E-protein dimer is held close to the virus surface by interactions between E-protein ectodomains DI and Dili with stem helices E-Hl and E-H2, which are tethered to the membrane by TM helices E-Tl and E-T2. Critical residues are shown in red, highlighted in yellow at stages where they are important for function. (A) The E-H2/E-H3 inter-helical interaction is stabilized in a pre-trigger conformation by critical residue D415 contacting H435 and by critical residue G421 stabilizing the E-H2/E-H3 turn. (B) In the low pH environment of the host endosome, protonation of H435 disrupts its interaction with D415, resulting in E-Hl and E-H2 extending outward and pushing DI and Dili from the virion surface to disrupt the dimer. (C) E monomers form a fusion trimer whose fusion loops (shown in cyan) insert into the endosome membrane. The position and helix breaking properties of critical residue G406 enable the proximal portion of stem helix E-Hl (residues 394- 401) to unwind and zipper along the monomer interfaces at DI, which pulls the endosome and viral membranes together. (D) In the late stages of fusion, critical residue G421 at the distal end of E-H2 packs against the trimer. TM critical residues 1452, M453, K454, G456 and G464 facilitate oligomerization of TM regions in E-protein (E-Tl, E-T2) and M protein (M-Tl, M-T2), forming an extensively cross-linked membrane anchor that promotes the final stages of membrane fusion. Critical residue K454 is positioned to mediate the disruption of the viral lipid bilayer as the final step in membrane fusion. (E) After fusion, the E-protein fusion loops, E- protein TM helices, and M protein TM helices lie adjacent in the same membrane.

DETAILED DESCRIPTION

[0028] The embodiments disclosed herein can also be understood from the following detailed description when read in connection with the accompanying drawings. For the purpose of illustrating the embodiments, it is understood that the illustrated embodiments are representative only and are not intended to be limiting. [0029] This description is not limited to the particular processes, compositions, or methodologies described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and it is not intended to limit the scope of the embodiments described herein. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. However, in case of conflict, the patent specification, including definitions, will prevail.

[0030] It must also be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise.

[0031] As used in this document, terms "comprise," "have," and "include" and their conjugates, as used herein, mean "including but not limited to." While various compositions, methods, and devices are described in terms of "comprising" various components or steps (interpreted as meaning "including, but not limited to"), the compositions, methods, and devices can also "consist essentially of or "consist of the various components and steps, and such terminology should be interpreted as defining essentially closed-member groups.

[0032] The terms "mutation" means any detectable change in genetic material, e.g. DNA,

RNA, cDNA, or any process, mechanism, or result of such a change. Mutations include substitution of one or more nucleotides. The mutations can result in a change at the protein level as well. The mutation can refer to a residue change at the protein level.

[0033] The term "recombinant" refers to molecules or viruses that are formed by laboratory methods of genetic recombination. For example, a recombinant dengue virus is one that is formed in the laboratory and is not naturally occurring.

[0034] A "nucleic acid molecule" refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. [0035] The embodiments provide flaviviruses, such as but not limited to, Dengue, Zika,

Yellow Fever, Japanese encephalitis, and west nile, having one or more mutations, such as those described herein, that result in decreased or no infectivity while allowing for the virus to be expressed and bud, thereby allowing for a virion to induce an immune response in the subject and, in some embodiments, provide a protective or therapeutic immune response. In some embodiments, the immune response is a humoral response. In some embodiments, the immune response is a cellular immune response. In some embodiments, the immune response induced by the mutant virus is both a humoral and a cellular immune response. In some embodiments, the mutation leaves the antigenic reactivity of the strain the same (i.e. reactivity with MAbs that are sensitive to its conformation). In some embodiments, the mutation decreases or inhibits infectivity, does not effect, or improves, antigenicity, increases budding and/or increases expression.

[0036] The viruses (including chimeras) described herein (e.g., those that include one or more of the mutations described herein) can be made using standard methods in the art. For example, an RNA molecule corresponding to the genome of a virus can be introduced into primary cells, chick embryos, or diploid cell lines, from which (or the supernatants of which) progeny virus can then be purified. Another method that can be used to produce the viruses employs heteroploid cells, such as Vero cells (Yasumura et al., Nihon Rinsho 21, 1201-1215, 1963). In this method, a nucleic acid molecule (e.g., an RNA molecule) corresponding to the genome of a virus is introduced into the heteroploid cells, virus is harvested from the medium in which the cells have been cultured, harvested virus is treated with a nuclease (e.g., an endonuclease that degrades both DNA and RNA, such as Benzonase.TM.; U.S. Pat. No. 5, 173,418), the nuclease-treated virus is concentrated (e.g., by use of ultrafiltration using a filter having a molecular weight cut-off of, e.g., 500 kDa), and the concentrated virus is formulated for the purposes of vaccination. One example of this method is provided in U.S. Patent Application Ser. No. 10/342,681, which is incorporated herein by reference.

[0037] A virus or protein that is described herein as having a mutation can also be referred to as a recombinant virus or protein. That is, the mutation is made due to human intervention. [0038] The viruses described herein, including those that comprise one more of the mutations described herein can be administered as primary prophylactic agents in adults or children at risk of infection, or can be used as secondary agents for treating infected patients. For example, the viruses can be used as a vaccine or to induce an immune response in adults or children at risk of infection from a flavivirus (e.g. Dengue, Zika, Yellow Fever, Japanese encephalitis, and west nile), or can be used as secondary agents for treating flavivirus-infected patients. Examples of patients who can be treated using the flavivirus-related pharmaceutical compositions, including, but not limited to, vaccines, and methods embodied herein include, but are not limited to, (i) subjects in areas in which flavivirus is endemic, such as Asia, Latin America, and the Caribbean, (ii) foreign travelers, (iii) military personnel, and (iv) patients in areas of a flavivirus epidemic. Moreover, inhabitants of regions into which the disease has been observed to be expanding (e.g., Argentina, Chile, Australia, parts of Africa, southern Europe, the Middle East, and the United States), or regions in which it may be observed to expand in the future (e.g., regions infested with Aedes aegypti or mosquito strains), can be treated according to the embodiments described herein. These are exemplary only and not intended to limit the uses or regions where the compositions described herein can be used as a therapeutic or prophylactic agent.

[0039] Formulation of the viruses or virions can be carried out using methods that are standard in the art. Numerous pharmaceutically acceptable solutions for use in vaccine or pharmaceutical preparation are well known and can readily be adapted for use with the present embodiments by those of skill in this art. (See, e.g., Remington's Pharmaceutical Sciences (18.sup.th edition), ed. A. Gennaro, 1990, Mack Publishing Co., Easton, Pa.) For example, the viruses can be formulated in Minimum Essential Medium Earle's Salt (MEME) containing 7.5% lactose and 2.5% human serum albumin or MEME containing 10% sorbitol. However, the viruses can simply be diluted in a physiologically acceptable solution, such as sterile saline or sterile buffered saline. In another non-limiting example, the viruses can be administered and formulated, for example, in the same manner as the yellow fever 17D vaccine, e.g., as a clarified suspension of infected chicken embryo tissue, or a fluid harvested from cell cultures infected with the virus.

[0040] The vaccines (viruses) or other pharmaceutical compositions described herein can be administered using methods that are well known in the art, and appropriate amounts administered can be readily be determined by those of skill in the art. For example, the viruses can be formulated as sterile aqueous solutions containing between 10² and 10⁷ infectious units (e.g., plaque-forming units or tissue culture infectious doses) in a dose volume of 0.1 to 1.0 ml, to be administered by, for example, intramuscular, subcutaneous, or intradermal routes. In addition, because flaviviruses may be capable of infecting the human host via the mucosal routes, such as the oral route (Gresikova et al., "Tick-borne Encephalitis," In The Arboviruses, Ecology and Epidemiology, Monath (ed.), CRC Press, Boca Raton, Fla., 1988, Volume IV, 177- 203), the viruses can be administered by mucosal routes as well. Further, the vaccines can be administered in a single dose or, optionally, administration can involve the use of a priming dose followed by a booster dose that is administered, e.g., 2-6 months later, as determined to be appropriate by those of skill in the art.

[0041] Optionally, adjuvants that are known to those skilled in the art can be used in the administration of the viruses. Adjuvants that can be used to enhance the immunogenicity of the viruses include, for example, liposomal formulations, synthetic adjuvants, such as (e.g., QS21), muramyl dipeptide, monophosphoryl lipid A, or polyphosphazine. Although these adjuvants are typically used to enhance immune responses to inactivated vaccines, they can also be used with live vaccines. In the case of a virus delivered via a mucosal route, for example, orally, mucosal adjuvants such as the heat-labile toxin of E. coli (LT) or mutant derivations of LT can be used as adjuvants. In addition, genes encoding cytokines that have adjuvant activities can be inserted into the viruses. Thus, genes encoding cytokines, such as GM-CSF, IL-2, IL-12, IL-13, IL-5, or IL-12 can be inserted together with foreign antigen genes to produce a vaccine that results in enhanced immune responses, or to modulate immunity directed more specifically towards cellular, humoral, or mucosal responses.

[0042] Mutations in DENV3 and/or DENV4 E-protein are described herein. These positions are made in reference to the sequences as shown in the following table: Strain SEQ ID Sequence of E-protein

NO.

DENV3 SEQ ID RCVGYG RDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQK

NO: 1 TEATQLATLRKLCIEGKIT ITTDSRCPTQGEAILPEEQDQNYVCKH

TYVDRG GNGCGLFGKGSLVTCAKFQCLESIEGKWQHENLKYTVI I TVHTGDQHQVGNETQGVTAEI PQASTVEAILPEYGTLGLECSPRTG LDFNEMILLTMKNKAW VHRQWFFDLPLPWTSGATTETPTWNRKELL VTFK AHAKKQEVVVLGSQEGAiHTALTGATEIQ SGGTSIFAGHLK CRLKMDKLSLKGMSYAMCLNTFVLK EVSETQHGTILIKVSYKGSDA PCKIPFSTEDGQGKAHNGRLITANPyV KEEPVNIEAEPPFGES I VIGIGDKALKIN YKKGSSIGKMFΞ ARGARRM ILGDTAWDFGSV GGVLNSLGKMVHQIFGSAYTALFSGVSWIMKIGIGVLLTWIGLNSK TS SFSCIAIG11TLYLGAVVQA

DENV4 SEQ ID RCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELTK

NO: 2 TTAKEVALLRTYCIEASISNI TATRCPTQGEPYLKEEQDQQYICRR

DWDRG GNGCGLFGKGGVVTCAKFSCSGKITGNLVQIENLEYTVW TVHNGDTHAVGNDTSNHGVTAMI PRSPS EVKLPDYGELTLDCEPR SGIDF EMIL KMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNYKE RMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGH LKCKVR EKLRIKG SYT CSGKFSIDKEMAETQHGTTWKVKYEGA GAPCKVPIEIRDVNKEKWGRI ISSTPLAENTNSVTNIELEPPFGDS YIVIGVGSSALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAW^'DFG SVGGLFTSLGKAVKOVFGSVYTTMFGGVSWMIRILIGFLVLWIGTNS RNTS AMTCIAVGGITLFLGFTVQA

[0043] As described herein flavivirus, virions, or polypeptides are provided comprising one or more of the mutations described herein. Also provided are dengue polypeptides comprising one or more of the mutations described herein. The mutations described herein can also be applied to other viruses besides Dengue viruses. The envelope protein is highly conserved across flavivirus. Therefore, a mutation in one protein that provides for a beneficial therapeutic can be applied to a different virus because of the conservation. The residues in the other viruses can be identified by using routine comparison tools such as, but not limited to, BLASTP, Clustal, T-coffee, MUSCLE, or Kalign, using, for example, default settings. The wild-type sequence of DENV3 and DENV4 envelope proteins are known. For example, U.S. Patent No. 8,691,961 describes the wild-type DENV3 E-protein (Envelope) sequences, which is hereby incorporated by reference in its entirety. DENV3 Envelope is also referred to as SEQ ID NO: 1 and DENV4 Envelope is referred to as SEQ ID NO: 2. [0044] In some embodiments, the DENV3 vims comprises a mutation in one or more of the following residues of DENV3 E-protein.

In some embodiments, the DENV3 vims comprises a mutation in the E-protein as shown in the following table:

In some embodiments, the DENV3 vims comprises a mutation at one or more positions as shown in the following table:

In some embodiments, the DENV3 virus comprises one or more mutations as shown in the following table:

[0045] The positions and mutations described herein can be used alone or in combination with one another. As described in the Examples, the mutations can be used to decrease or inhibit infectivity while maintaining, having a sufficient amount of, or increasing expressing, budding, and antigenicity. In some embodiments, the antigenicity of the protein and/or virus is the same or substantially similar to the wild-type virus and/or protein. In some embodiments, the antigenicity is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the antigenicity of the wild-type protein or virus. The antigenicity can be measured different manners. In some embodiments, the antigenicity is determined by measuring the immune response that is elicited when the protein and/or virus is introduced into an organism, such as a mouse, sheep, goat, human, or other mammal. In some embodiments, the antigenicity is measured by comparing the binding of an antibody that is known to recognize the protein and/or virus and determining whether the binding is the same, increased or decreased as compared to the wild-type protein or virus. In some embodiments, the mutation(s) does not substantially affect or arrogate the binding of one antibody that is known to bind to the wild-type protein or virus. In some embodiments, the mutation(s) does not substantially affect or arrogate the binding of two different antibodies that are known to bind to the wild-type protein or virus. In some embodiments, the mutation(s) does not substantially affect or arrogate the binding of three different antibodies that are known to bind to the wild-type protein or virus. In some embodiments, the mutation(s) does not substantially affect or arrogate the binding of four different antibodies that are known to bind to the wild-type protein or virus. The antibodies can bind to the same or different epitopes. In some embodiments, antibody(ies) bind to the mutant with at least 50%, 60%, 70%, 80%, 90%, 95, or 99% affinity as compared to the wild-type. In some embodiments, the antibody(ies) bind with the same affinity to the mutant as compared to the wild-type. The binding can be done under various conditions. Measuring affinities of antibodies to target molecules is routine in the art. Accordingly, in some embodiments, the virus retains antigenicity as compared to the wild-type virus. In some embodiments, the virus with reduced infectivity is able to be recognized by, or at least, 1, 2, 3, 4, 5, 6, 7, or 8 antibodies that are also able to recognize (bind to) the wild-type virus. Determining whether an antibody can bind to or recognize a certain virus strain or mutant virus can be performed by any known method as the method is not critical.

[0046] Also provided herein are DENV3 peptides or polypeptides comprising a mutation at one or more of the positions described herein. In some embodiments, DENV3 peptides or polypeptides comprising a mutation as described herein.

[0047] The mutations in DENV3 can also be extrapolated to other serotypes of dengue virus. Accordingly, in some embodiments, a DENV4 virus comprising a corresponding mutation at a corresponding position is provided. In some embodiments, a DENV1 or DENV2 virus comprising a corresponding mutation in DENV3 at a corresponding position is provided. The corresponding position can be determined using methods known to one of skill in the art, such as, but not limited to, the methods described herein. In some embodiments, the mutation in DENV4 is at a position or is the mutation as shown in the following table:

[0048] In some embodiments, a DENV4 virus is provided with a corresponding mutation to the DENV3 virus, which can be at a residue or with the specific mutation shown in the following table.

[0049] In some embodiments, a DENV4 virus is provided with a mutation as shown herein. These mutations can be used to generate non-infectious DENV4 particles. These particles can, however, retain budding and antigenicity as described herein.

[0050] Also provided herein are DENV4 peptides or polypeptides comprising a mutation at one or more of the positions described herein. In some embodiments, DENV4 peptides or polypeptides comprising a mutation as described herein.

[0051] Also provided herein are DENV1 and/or DENV2 peptides or polypeptides comprising a mutation at one or more of the positions described herein that correspond to the mutation in DENV3 and/or DENV4 as described herein. Methods of determining a position that corresponds to the positions described herein are known in the art and any method can be used.

[0052] In addition to the pharmaceutical compositions described herein, pharmaceutical compositions comprising a DENV1, DENV2, DENV3 virus and/or a DENV4 virus are provided. That is, a composition comprising dengue viruses of different serotypes are provided to induce a broader immune response, which can be both humoral and/or cellular based. In some embodiments, a pharmaceutical composition comprising one or more DENV1, DENV2, DENV3 and/or DENV4 peptides or polypeptides comprising one or more of the mutations described herein are provided. Pharmaceutical compositions are also provided that comprise any protein or flavivirus described herein.

[0053] Also provided are vaccines or therapeutic compositions comprising a DENV1,

DENV2, DENV3 and/or DENV4 virus comprising one or more of the mutations described herein.

[0054] In some embodiments, methods of inducing an immune response are provided. In some embodiments, the method comprises administering one more DENV1, DENV2, DENV3 or DENV4 viruses described herein. In some embodiments, the DENV1, DENV2, DEN3 or DENV4 virus comprises one or more mutations as described herein. In some embodiments, the method comprises inducing an immune response with a DENV1, DENV2, DENV3, or DENV4 virus, peptide, or polypeptide comprising one or more mutations at one or more of the positions as described herein, including, but not limited to those described herein and above.

[0055] In some embodiments, dengue virus with reduced infectivity as compared to a wild-type virus are provided, wherein the virus comprises one or more mutations as described herein or a mutation at one or more of positions described herein. In some embodiments, the virus with reduced infectivity is still able to bud out of a cell. In some embodiments, the virus retains at least 70, 80, 90, or 95% budding efficiency as compared to the wild-type virus. Measuring budding efficiency can be determined using any known method, including those described herein. In some embodiments, the virus retains at least 70, 80, 90, or 100 % expression as compared to the wild-type virus. In some embodiments, the mutant virus expresses more than the wild-type virus. [0056] Accordingly, in some embodiments, a polyprotein is provided, comprising a

DENV1, DENV2, DENV3, or DENV4 polypeptide comprising at least one mutation in E- protein that decreases infectivity, increases budding and retains native antibody reactivity of a DENV1, DENV2, DENV3, or DENV4 virus. In some embodiments, the polyprotein is isolated from its viral environments, such as being purified from the mutant (or recombinant) form. In some embodiments, the polypeptide comprises a mutation in the E-protein in DI domain, a DII domain, a Dili domain, a E-Hl domain, a E-H2 domain, or a E-Tl domain, or any combinations thereof. These domains can also be mapped to other flaviviruses, such as those described herein, and, therefore, other proteins can be made with mutations in these domains that reduce infectivity, increase or maintain budding, and/or retain antigenicity as compared to the wild-type. In some embodiments, the domain is a DENV3 E-protein domain. . In some embodiments, the domain is a DENV4 E-protein domain. In some embodiments, the domain is a DENV 1 or DENV2 E-protein domain. The domains and their boundaries are known. For example, for DENV3, domain DI consists of E residues 1-52,133-193, and 281-296, domain DII residues consist of 53-132 and 194-280, and domain Dili residues consist of 297-398. For DENV1, DENV2 and DENV4, domain DI consists of E residues 1-52 and 133-195, and 283-298, domain DII residues consists of 53-132,196-282, and domain Dili residues consists of 299-400.

[0057] The mutations and positional mutations described herein can be present alone or combined with one another. The residue can be substituted with any naturally human amino acid residue. For example, the mutation can be to an Ala, Arg, Asn, Asp, Cys, Gin, Glu, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val. In some embodiments, the residue is substituted with a non-naturally occurring amino acid residue. In some embodiments, the mutation is as indicated herein at the particular positions. Additionally, although a specific mutation may be shown, an equivalent substitution may also be made. For example, if a charged residue is present as the mutation it can be substituted with another charged amino acid residue. For example, for G406R, this could also be G406K, G406D, or G406E. In some embodiments, the positive or negative charge is retained. Thus, if the mutant is a positively charged residue then the substitution can be positive and the same if the mutant is negatively charged. Similar substitutions can be made based upon whether the mutant is a polar residue or a hydrophobic residue. The following table can be used as a guide for substitutions that can be made based upon the non-limiting and exemplary mutations and substitutions described herein. Amino Acid Residue Type Residues

Charged Arginine - Arg - R;

Lysine - Lys - K;

Aspartic acid - Asp - D; or

Glutamic acid - Glu - E

Polar Glutamine - Gin - Q

Asparagine - Asn - N

Histidine - His - H

Serine - Ser - S

Threonine - Thr - T

Tyrosine - Tyr - Y

Cysteine - Cys - C

Methionine - Met - M

Tryptophan - Trp - W

Hydrophobic Alanine - Ala - A

Isoleucine - He - 1

Leucine - Leu - L

Phenylalanine - Phe - F

Valine - Val - V

Proline - Pro - P

Glycine - Gly - G

Tryptophan - Trp - W

[0058] In some embodiments, the DENV3 polypeptide comprises at least one mutation at position G406, D415, G421, 1452, M453, K454, G456, H259, R186, A265, N364, 1276, S296, or 1464 of SEQ ID of the DENV3 E-Protein (SEQ ID NO: 1). In some embodiments, the polypeptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations at the positions referenced here.

[0059] In some embodiments, the DENV3 polypeptide comprises a mutation selected from the group consisting of G406R, D415G, G421A, I452N, M453T, K454R, G456S, and I464T of the DENV3 E-Protein (SEQ ID NO: 1), or any combinations thereof. In some embodiments, the DENV3 polypeptide comprises a mutation selected from the group selected consisting of: R186L, E233G, R186H, H207L, A265T, A265V, M258L, A203D, V206E, G406R, D415G, G421A, I452N, I452S , H345P, I452V, H435R, E366V, I452N, F446L, P330L, M453T, K454R, K454E, P354L, I478T, K454T, V423A, G456S, N364S, I464T, I464L, H259R, I276T, and S296G of the DENV3 E-Protein (SEQ ID NO: 1), or any combinations thereof. In some embodiments, the DENV3 polypeptide comprises a mutation selected from the group consisting of: 1) R186L, 2) R186L and E233G, 3) E233G, 4) R186H and H207L, 5) H207L, 6) A265T, 7) A265V and M258L, 8) M258L, 9) A265T and A203D, 10) A265T and V206E, 11) G406R, 12) D415G, 13) G421A, 14) I452N, 15) I452S and H345P, 16) I452V and H435R, 17) I452N and E366V, 18) I452N, F446L and P330L, 19) M453T, 20) K454R, 21) K454E, 22) K454R and P354L, 23) K454E and I478T, 24) K454T and V423A, 25) G456S and N364S, 26) N364S, 27) I464T, 28) I464L, 29) H259R, 30) I276T, 31) S296G of the DENV3 E-Protein (SEQ ID NO: 1), or any combination thereof. In some embodiments, the polypeptide comprises, or comprises at least, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the mutations described herein.

[0060] In some embodiments, the DENV4 polypeptide comprises at least one mutation at a position selected from the group consisting of: H261, M278, S298, G408, G423, 1455, L458, 1466, R188, A267, M454, R456, L458, V6, G14, V53, R73, D98, 1132, N134, F193, H209, Q211, D225, Q256, E269, R350, P356, E370, F373, S396, 1398, G399, F402 of the DENV4 E Protein (SEQ ID NO: 2). In some embodiments, the polypeptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations at the positions referenced here. In some embodiments, the DENV4 polypeptide comprises a mutation selected from the group consisting of H261A, M278A, S298A, G408A, G423A, 1455 A, L458A, I466A, R188A, A267A, M454A, R456A, V6A, G14A, V53A, R73A, D98A, I132A, N134A, F193A, H209A, Q211A, D225A, Q256A, E269A, R350A, P356A, E370A, F373A, S396A, I398A, G399A, F402A, and V439A of the DENV4 E Protein (SEQ ID NO: 2), or any combinations thereof. In some embodiments, the polypeptide comprises, or comprises at least, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the mutations described herein.

[0061] In some embodiments, the DENV1 and/or DENV2 polypeptide comprises a mutation that corresponds to a mutation at position of G406, D415, G421, 1452, M453, K454, G456, H259, R186, A265, G406, D415, G421, 1452, M453, K454, G456, N364, H259, 1276, or S296 of DENV 3. In some embodiments, the DENV1 and/or DENV2 polypeptide comprises a mutation that corresponds to a mutation at position of H261, M278, S298, G408, G423, 1455, L458, 1466, R188, A267, M454, R456, L458, V6, G14, V53, R73, D98, 1132, N134, F 193, H209, Q21 1, D225, Q256, E269, R350, P356, E370, F373, S396, 1398, G399, F402, or V439 of DENV 4. As described herein, the mutations that correspond to the DENV3 and/or DENV4 positions can be determined by routine methods.

[0062] As described herein, virions comprising any of the polypeptides described herein are provided for. The methods for making such virions are well known in the art and any method can be used. Such methods are also described herein and below. In some embodiments, the virion comprises a polyprotein as described herein. Although some embodiments refers to DENV3 or DENV4 peptides, the mutations in these proteins can also be applied to other flaviviruses. Accordingly, in some embodiments, a flavivirus with a mutation in its E-protein that corresponds to one or mutations as described herein is provided. Mapping a mutation from the DENV3 and/or DENV4 peptide to another flavivirus E-protein can be accomplished according to known methods, such as the comparison methods described herein and known to one of skill in the art. In some embodiments, the virion is a flavivirus that is dengue virus, zika virus, Yellow Fever virus, Japanese encephalitis, or a west nile virus. In some embodiments, the virion is DENV1, DENV2, DENV3, or DENV4.

[0063] Pharmaceutical compositions comprising any protein and/or virion (virus) described herein is also provided. The pharmaceutical compositions are described herein, but they should be suitable for administration to an animal subject (e.g. human) for its intended result, such as eliciting a therapeutic or prophylactic immune response.

[0064] Accordingly, and as described elsewhere herein, methods of eliciting an immune response are provided, the method comprising administering to a subject a pharmaceutical composition comprising a polyprotein and/or virion as described herein is provided. The immune response can be any type of immune response such as, but not limited to, those described herein. In some embodiments, methods of preventing or ameliorating a dengue infection are provided, the method comprising administering to a subject a pharmaceutical composition comprising a polyprotein or virion described herein. In some embodiments, methods of preventing or ameliorating a flavivirus infection are provided, the method comprising administering to a subject a pharmaceutical composition comprising a virion or protein described herein. In some embodiments, the flavivirus is a dengue virus, zika virus, Yellow Fever virus, Japanese encephalitis virus, or a west nile virus. In some embodiments, the virion is a DENV3 virion, a DENV4 virion, a DENV1 virion, or a DENV2 virion. As described herein, the mutations described herein can be mapped onto the corresponding E-proteins of the other flaviviruses. Therefore, such methods are provided for by making such mutations in the proteins and eliciting an immune response as descried herein for the different flaviviruses.

[0065] In some embodiments, vaccines or pharmaceutical compositions are provided comprising a flavivirus comprising a mutation that corresponds to a mutation in a DENV1, DENV2, DENV3, or DENV4 polypeptide that decreases infectivity, increases budding and optionally retains native antibody reactivity. In some embodiments, the mutation corresponds to a, or is present in the, DENV3 envelope polypeptide. In some embodiments, the mutation corresponds to a, or is present in the, DENV4 envelope polypeptide. Examples of such mutations are provided herein. In some embodiments, the mutation is in a DENV1 envelope polypeptide. In some embodiments, the mutation is in a DENV2 envelope polypeptide. In some embodiments, the mutation is in a zika, Yellow Fever, Japanese encephalitis, or a west nile envelope protein. In some embodiments, the mutation is in a domain that corresponds to a mutation of DENV3 polypeptide domain, wherein the domain selected from the group consisting of DI domain, a DII domain, a Dili domain, a E-Hl domain, a E-H2 domain, or a E-Tl domain, or any combinations thereof. In some embodiments, the flavivirus comprises a mutation that corresponds to a DENV3 mutation selected from the group consisting of: G406, D415, G421, 1452, M453, K454, G456, H259, or 1464 of DENV3. In some embodiments, the flavivirus comprises a mutation that corresponds to a DENV3 mutation selected from the group consisting of: G406R, D415G, G421A, I452N, M453T, K454R, G456S, and I464T/L. In some embodiments, the flavivirus comprises a mutation that corresponds to a DENV3 mutation selected from the group consisting of: R186, A265, G406, D415, G421, 1452, M453, K454, G456, N364, H259, 1276, and S296. In some embodiments, the flavivirus comprises a mutation that corresponds to a DENV3 mutation selected from the group consisting of: R186L, E233G, R186H, H207L, A265T, A265V, M258L, A203D, V206E, G406R, D415G, G421A, I452N, I452S , H345P, I452V, H435R, E366V, I452N, F446L, P330L, M453T, K454R, K454E, P354L, I478T, K454T, V423A, G456S, N364S, I464T, I464L, H259R, I276T, and S296G, or any combinations thereof. In some embodiments, the flavivirus comprises a mutation that corresponds to a DENV3 mutation selected from the group consisting of: 1) R186L, 2) R186L and E233G, 3) E233G, 4) R186H and H207L, 5) H207L, 6) A265T, 7) A265V and M258L, 8) M258L, 9) A265T and A203D, 10) A265T and V206E, 1 1) G406R, 12) D415G, 13) G421A, 14) I452N, 15) I452S and H345P, 16) I452V and H435R, 17) I452N and E366V, 18) I452N, F446L and P330L, 19) M453T, 20) K454R, 21) K454E, 22) K454R and P354L, 23) K454E and I478T, 24) K454T and V423A, 25) G456S and N364S, 26) N364S, 27) I464T, 28) I464L, 29) H259R, 30) I276T, 31) S296G or any combination thereof. In some embodiments, the flavivirus comprises a mutation that corresponds to a DENV4 mutation selected from the group consisting of: H261, M278, S298, G408, G423, 1455, L458, 1466, R188, A267, M454, R456, L458, V6, G14, V53, R73, D98, 1132, N134, F 193, H209, Q21 1, D225, Q256, E269, R350, P356, E370, F373, S396, 1398, G399, F402, and V439. In some embodiments, the flavivirus comprises a mutation that corresponds to a DENV4 mutation selected from the group consisting of: H261A, M278A, S298A, G408A, G423A, 1455 A, L458A, I466A, R188A, A267A, M454A, R456A, V6A, G14A, V53A, R73A, D98A, I132A, N134A, F 193A, H209A, Q21 1A, D225A, Q256A, E269A, R350A, P356A, E370A, F373A, S396A, I398A, G399A, F402A, and V439A, or any combinations thereof.

[0066] Also provided, as described herein, are dengue viruses with reduced infectivity as compared to a wild-type virus, wherein the virus comprises a polyprotein as described herein. In some embodiments, the virus retains, retains at least, or retains about 70%, 80%, 85%, 90%, 95%), or 99% expression as compared to the wild-type virus. Expression can be measured by the number of virions produced under similar conditions for the mutant and wild-type virions.

[0067] In some embodiments, flaviviruses are provided comprising a mutation in the envelope polypeptide that corresponds to a mutation at position of G406, D415, G421, 1452, M453, K454, G456, H259, R186, A265, G406, D415, G421, 1452, M453, K454, G456, N364, H259, 1276, or S296 of DENV 3. In some embodiments, flaviviruses are provided comprising a mutation in the envelope polypeptide that corresponds to a mutation at position of H261, M278, S298, G408, G423, 1455, L458, 1466, R188, A267, M454, R456, L458, V6, G14, V53, R73, D98, 1132, N134, F193, H209, Q21 1, D225, Q256, E269, R350, P356, E370, F373, S396, 1398, G399, F402 and V439 of DENV 4. These mutations, as described herein, can be alone or combined in any combination. [0068] The present disclosure also provides for flaviviruses comprising a mutation that inhibits flavivirus entry into a cell, wherein the mutation in the flavivirus corresponds to a mutation in the E-Tl domain. In some embodiments, the mutation corresponds to a mutation at position M453, 1464, 1452 and G456 of DENV3.

[0069] In some embodiments, a flavivirus is provided comprising a mutation that corresponds to a mutation at position G406 of DENV3. In some embodiments, the mutation partially triggers an E-protein conformational change. The virus can be Dengue Fever (e.g. DENVl, DENV2, DENV3, or DENV4), Yellow Fever, Japanese encephalitis, Zika, or West Nile.

[0070] This description is not limited to the particular processes, compositions, or methodologies described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and it is not intended to limit the scope of the embodiments described herein. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. However, in case of conflict, the patent specification, including definitions, will prevail.

[0071] The following examples are not to be limiting and are only some of the embodiments encompassed by the presently disclosed subject matter.

Examples

[0072] Example 1.

[0073] Flavivirus entry, like that of other enveloped viruses, proceeds through a membrane-fusion step, catalyzed by a viral fusion protein. Flavivirus genomes, including that of dengue virus (DENV), encode a single polyprotein that is processed into three structural proteins, capsid, pre-membrane (prM), and envelope (E), and seven non- structural proteins that are required for viral replication. The capsid protein and the viral RNA genome form a nucleocapsid that buds at the ER in association with 180 copies each of prM and E, as well as host-derived lipids, to form the immature virion (Welsch et al., Composition and three- dimensional architecture of the dengue virus replication and assembly sites, 2009, Cell Host Microbe, 5:365-375). This non-infectious virion passes through the cell's secretory pathway, maturing in the Golgi where most of the prM proteins are cleaved by the host protease furin into pr peptides that are released from the virion and M proteins that remain in the viral membrane (Stadler et al., Proteolytic activation of tick-borne encephalitis virus by furin, 1997, J Virol, 71 :8475-8481). The mature virions bud from the cell via exocytosis and are then available to infect new cells.

[0074] DENV infection of target cells is initiated by binding to one or more receptors on the cell surface, followed by clathrin-mediated endocytosis and transport to endosomes, the site of viral-host membrane fusion (van der Schaar et al., Dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells, 2008, PLoS Pathog, 4:el000244). Membrane fusion is mediated by the viral envelope protein E, consisting of three distinct ectodomains (DI, DII, and Dili) connected by a stem region to a transmembrane (TM) anchor. In the acidic environment of the endosome, protonation of E-protein histidine residues is thought to mediate conformational changes which trigger the dissociation of the E dimer into monomers and the subsequent rearrangements among DI, DII, and Dili (Fritz et al., Identification of specific histidines as pH sensors in flavivirus membrane fusion, 2008, J Cell Biol, 183 :353-361 ; Sanchez- San Martin et al., Dealing with low pH: entry and exit of alphaviruses and flaviviruses, 2009, Trends Microbiol, 17:514-521). These conformational changes enable formation of the fusogenic E trimer and insertion of E-protein into the endosomal membrane by means of a fusion loop structure (Kaufmann et al., Capturing a flavivirus pre-fusion intermediate, 2009, PLoS Pathog, 5:el000672; Stiasny et al., Probing the flavivirus membrane fusion mechanism by using monoclonal antibodies, 2007, J Virol, 81 : 11526-11531).

[0075] The final stages of membrane fusion require essential contributions from the stem and TM regions, which comprise the C-terminus of the E-protein (residues 392 to 493 in DENV- 3). A recent cryo-EM study shows that the stem consists of three amphipathic a-helices (E-Hl, E-H2, and E-H3) lying close to the viral membrane surface and tethered to it by two TM a- helices (E-Tl and E-T2) in an antiparallel coiled-coil hairpin conformation (Zhang et al., Cryo- EM structure of the mature dengue virus at 3.5-A resolution, 2013, Nat Struct Mol Biol, 20: 105- 110). The E-protein stem and TM regions are necessary for viral fusion, but details of their exact contributions remain largely unknown. Following insertion of the fusion loops into the endosome membrane, the stem connects the viral membrane to the fusion trimer. Fusion between the viral and endosome membranes is driven by the stem "zippering" up the trimer, initiated by a portion of stem helix E-Hl interacting with a groove formed by the DII domains of neighboring E- proteins (Bressanelli et al., Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation, 2004, Embo J, 23 :728-738; Klein et al., Structure of a dengue virus envelope protein late-stage fusion intermediate, 2013, J Virol, 87:2287-2293). This zippering pulls the viral and endosome membranes together, facilitating their fusion and the subsequent release of the DENV genome into the cytoplasm. Complete membrane fusion depends on the presence of both E-Tl and E-T2 that form a TM hairpin, suggesting a functional role for the TM region in fusion (Fritz et al., The unique transmembrane hairpin of flavivirus fusion protein E is essential for membrane fusion, 2011, J Virol, 85:4377-4385). The importance of the TM region to the general structure of E-protein has also been implicated by mutations that severely diminished budding and infectivity of Yellow Fever Virus (Op De Beeck et al., The transmembrane domains of the prM and E-proteins of yellow fever virus are endoplasmic reticulum localization signals, 2004, J Virol, 78: 12591-12602).

[0076] While the available structures of DENV E-protein provide static images of the stem and TM regions in the mature virion and fusogenic trimer (Klein et al., 2013; Zhang et al., 2003; Zhang et al., 2013; Zhang et al., 2004), the key functional residues of stem and TM that mediate the dynamic fusion process remain to be determined. Indeed, the mechanism by which the stem region triggers and dissociates from a robust network of contacts with the E ectodomain is unknown, the exact role of E TMs in the fusion process has only been speculative to date, no previous evidence has suggested a role for mature M protein during entry, and little is known about how the E TMs might contribute to the physical fusion between the viral and host membranes. Here we identify critical residues that help explain how stem and TM regions of DENV E-protein mediate structural changes and interactions that lead to membrane fusion, including evidence that suggests the mature M protein contributes directly to this process. Using a comprehensive library of functional point mutations covering the E-protein stem and TM region, we identified key residues that are required for infectivity but that did not otherwise disrupt E-protein expression, folding, virion assembly, or budding. Our results suggest that key stem residues (e.g. G406, D415, G421, and H435) play an important, previously undescribed role both in maintaining E-protein Domains I and III in a constrained conformation to prevent premature exposure of the fusion loops, and in the triggering process that frees these E ectodomains from the viral membrane to form the fusogenic trimer. The interactions of residues within the TM domains (e.g. M453, K454, and 1464) suggest a novel role for an eight-helix oligomeric E/M protein membrane anchor in promoting the final stages of membrane fusion. Our results integrate the findings from structural studies of DENV E-protein into a cohesive functional model that describes how the stem and TM anchor regions of a class II viral fusion protein mediate entry.

[0077] Identification of stem and anchor residues critical for DENV infectivity

[0078] To identify E-protein critical residues required for DENV infectivity, we created a

'shotgun mutagenesis' mutation library (Paes et al., Atomic-level mapping of antibody epitopes on a GPCR, 2009, J Am Chem Soc, 131 :6952-6954) of DENV-3 prM/E variants, with at least one mutation at each of 90 residues (391-480) of the E-protein stem and TM region. The library contains an average of 2.3 substitutions per residue, representing multiple substitutions at each amino acid position. The entire mutation library was transfected into human HEK-293T cells in a 384-well array format (one clone per well) and evaluated in parallel for Env expression, virus budding, and infectivity. Env expression was measured by immunofluorescent antibody staining and detection by flow cytometry (Figure 1 A). Viral budding was measured by ELISA capture of released virus particles (Figure IB). Finally, infection of target cells was measured by luciferase expression by replication-incompetent dengue reporter viruses (Mattia et al., Dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes, 2011, PLoS One, 6:e27252) (Figure 1C).

[0079] The results of these assays were compared, and clones were selected that demonstrated no signs of structural impairment (i.e. wild-type levels of Env expression, reactivity with multiple MAbs, and assembly and release of intact DENV virions), yet were deficient for infection (<25% infectivity). This identified a total of eight positions in the E stem and TM regions as critical for DENV infectivity (Figure ID). To further confirm that these mutant proteins were not misfolded, each was tested for individual reactivity with eight diverse DENV E-protein-specific MAbs that recognize seven distinct epitopes on the protein. Each mutant Env protein demonstrated reactivity (>50% wild type) with all eight MAbs (Table 1). Infectivity MAb Serotype Flavivirus

Domain Mutation Expression % Budding %

% Reactivity % Conserv % Conserv

E-H1 G406R 148 (2) 217 (212) 0 (0) 8/8 100 91

E-H2 D415G 129 (7) 150 (30) 5 (3) 8/8 75 55

E-H2 G421A 98 (4) 79 (1 1) 7 (6) 8/8 100 85

E-T1 I452N 105 (17) 242 (37) 0 (0) 8/8 25 52

E-T1 M453T 150 (24) 124 (45) 2 (1) 8/8 75 6

E-T1 K454R 107 (9) 170 (36) 6 (1) 8/8 75 52

E-T1 G456S 128 (6) 189 (94) 1 (1) 8/8 25 3

E-T1 I464T 102 (5) 76 (27) 0 (0) 8/8 75 9

Table 1. E-protein stem and TM residues critical for DENV infectivity. Activities for E- protein expression (n=3), viral budding (n=2), and viral infectivity (n=3) are shown as a percentage of wild-type, with ranges (max-min) in parentheses. MAb reactivity indicates the number of MAbs that reacted with the given mutation (>50% wild type) among the eight different E-protein MAbs tested. Conservation among all four DENV serotypes is shown as Serotype %. Flavivirus % shows the extent of conservation among 33 flaviviruses most closely related to DENV (NCBI); residue 415 is a negatively charged residue (Asp or Glu) in 87% of flaviviruses.

[0080] Each of these mutants was also tested in additional virus capture assays using pr- or E-protein-specific MAbs, confirming that the pr to E-protein ratio for each mutant is not significantly different (relative to wild type), and thus that lack of prM cleavage (which significantly increases this ratio) cannot explain the lack of infectivity of these mutants (Table 2). Several mutations demonstrated increased viral budding that was not explained by expression levels, consistent with previous studies demonstrating that the stem/anchor region of flaviviruses contains ER retention signals that can modulate the efficiency of budding (de Wispelaere and Yang, 2012; Hsieh et al., 2008; Hsieh et al., 2010; Purdy and Chang, 2005).

Capture: Mouse Human2 Mouse prM Mouse prM Mouse

Domain Mutation Detect: Humanl m-1A1 D2 h-1A1 D2 Human2 F-loop

E-H1 G406R 217 (212) 377 (46) 6 (4) 7 (1) 13 (1)

E-H2 D415G 150 (30) 219 (25) 141 (39) 150 (85) 163 (1 14)

E-H2 G421A 79 (1 1) 66 (1 1) 39 (18) 35 (15) 54 (4)

E-T1 I452N 242 (37) 314 (24) 483 (141) 643 (94) 167 (130)

E-T1 M453T 124 (45) 84 (37) 252 (60) 242 (70) 96 (14)

E-T1 K454R 170 (36) 371 (45) 273 (90) 347 (69) 208 (285)

E-T1 G456S 189 (94) 70 (6) 256 (19) 282 (1 19) 109 (27)

E-T1 I464T 76 (27) 141 (5) 121 (6) 98 (50) 106 (67)

Table 2. Budding activities for E-protein stem/anchor residues critical for DENV infectivity. Mutation prM avg 1A1 D-2 D004 D173

G406R 105 50 13 153

D415G 95 77 128 1 17

G421 A 109 78 54 92

S440A 80 89 70 1 18

I452N 98 99 167 1 14

M453T 1 12 136 96 158

M453V 82 123 81 82

K454R 88 98 208 83

G456S 96 92 109 1 19

I464T 62 96 106 100

Table 3. IF activities of anti-Env MAbs. Interactions of Mabs with critical clones, expressed as a % of WT. 1A1D-2 recognizes Dili, D004 recognizes the fusion loop, and D173 recognizes DII.

[0081] Five critical residues identified (1452, M453, K454, G456, and 1464) are located in E-Tl, the proximal helix of the TM anchor, and three (G406, D415, and G421) are located in the helical stem regions. The ribbon structures illustrating this can be found in Figure 2 in U. S. Provisional Application No. 62/1 16,524, filed February 15, 2015, which is hereby incorporated by reference in its entirety. Several of these residues, including G406, D415, G421, and K454, are highly conserved among DENV serotypes and other flaviviruses (Table 1), consistent with crucial functional roles. Based on the selection criteria used, these residues represent critical amino acids that are individually required for E-protein functionality during entry but that do not disrupt its expression, folding, assembly, or budding, thus localizing the functional importance of these residues to late stages of infectivity.

[0082] Anchoring interactions between E-Tl and M protein are vital for E-protein function

[0083] A cryo-EM structure is available for the entire E-protein, including stem and TM anchor regions, in its dimer conformation on the mature virion (Zhang et al., Cryo-EM structure of the mature dengue virus at 3.5-A resolution, 2013, Nat Struct Mol Biol, 20: 105-1 10), but critical stem and TM residues that mediate the functionality of these structures have not previously been determined. To understand the contribution of the identified critical residues to E-protein functionality, their positions and individual interatomic contact points (within 4.2 A) were evaluated on the DENV mature virion E-protein structure (PDB ID 3J27) (Table 4), revealing interactions with critical residues that are necessary for stem and TM functionality.

F446 stem 3.6

V449 stem 2.4

S450 E-T1 2.0

W451 E-T1 2.5

K454 I452 E-T1 2.7

L456 E-T1 2.6

I457 E-T1 2.3

G458 E-T1 2.0

L487 E-T2 3.1

G488 E-T2 3.6

V491 E-T2 3.8

I452 E-T1 1.9

M453 E-T1 2.4

K454 E-T1 2.6

L/G456 I455 E-T1 2.7

I457 E-T1 3.0

G458 E-T1 2.6

V459 E-T1 2.5

I460 E-T1 2.0

I53 (M) M-T1 3.8

I460 E-T1 2.0

1461 E-T1 2.4

T462 E-T1 3.1

W463 E-T1 3.2

1464 G465 E-T1 2.4

M466 E-T1 2.3

N467 E-T1 2.4

S468 E-T1 2.9

Table 4. Shortest interatomic contacts for infectivity-critical residues. Shown are E-protein stem and TM critical residue contacts (distance in Angstroms), obtained from a DENV-2 cryo- EM structure of the virion that includes proteins E and M in their mature conformation (PDB ID: 3J27) (Zhang et al., 2013). Interatomic contacts of critical residues were determined using Chimera software. Distances less than 4.2 Angstroms were considered contacts, although a structure with improved resolution could change some of these contact points. Numbering follows that of the DENV-3 dimer crystal structure (PDB ID: 1UZG) except for the numbering of M protein amino acids which starts at mature M protein residue 1 (i.e. after the furin cleavage site). Seven of the eight residues critical for infectivity are identical between the available DENV-2 structure and the DENV-3 mutation library we used for analysis, the exception being G456 (L456 in DENV-2).

[0084] All five critical residues that were identified in the TM region are located on E-

Tl, one of two TM helices that anchor E-protein in the virus membrane These residues can be visualized in the ribbon structures illustrated in Figure 3 in U.S. Provisional Application No. 62/116,524, filed February 15, 2015, which is hereby incorporated by reference in its entirety. Interestingly, four of these critical residues (1452, M453, K454, and G456) cluster at the proximal end of E-Tl, while 1464 lies at the distal end, thus forming functionally critical anchors on both ends of the TM helix. K454 makes cross-helix contacts with residues L487, G488, and V491 at the top of E-T2, while M453 and 1464 collectively make contacts with M protein residues F42, 153, V70, A71, and P72 on both TM helices of M protein, cumulatively forming a heavily cross-bonded complex of four TM helices per E-protein monomer.

[0085] In total, M453 and 1464 contact 5 of the 9 residues within M that are contacted by

E-Tl, suggesting that the E-Tl interaction with M is vital for late stage infectivity, and that M453 and 1464 are essential for this functionality. 1452 and G456 contact only other E-Tl residues, suggesting they are important for stabilizing the local helical structure and potentially mediating its changes during infectivity. Substitution of any these five TM anchor critical residues does not disrupt Env expression, folding, viral assembly, or viral budding, but does reduce infectivity, consistent with these residues being critical in later stages of E-protein function. The locations and interactions of residues at the top and bottom of the E and M TM helices, and their ability to disrupt late stages of E-protein functionality if substituted, suggest that these residues are involved late in viral entry and possibly directly involved in the membrane fusion reaction. Although the role of TM in E-protein function has been speculated previously, direct evidence for its involvement and the role of specific residues in its function have never previously been demonstrated. Similarly, the role of prM/M in E-protein folding and viral assembly has been well documented (Lin et al., 2011), but there has been no prior evidence suggesting the potential involvement of M protein in viral entry.

[0086] Critical atomic contacts for stem region tethering, triggering, and rearrangement

[0087] Three critical residues are located in two of the three helices that make up stem in the mature virion, G406, D415, and G421. The ribbon structures illustrating this can be found in Figure 4, A in U.S. Provisional Application No. 62/116,524, filed February 15, 2015, which is hereby incorporated by reference in its entirety. D415 makes cross-helix contacts with K432, H435, and Q436 on E-H3. The ribbon structures illustrating this can be found in Figure 4, B in U.S. Provisional Application No. 62/116,524, filed February 15, 2015, which is hereby incorporated by reference in its entirety. The relative positions of D415 and H435 suggest that a D415-H435 salt bridge can make a major contribution to the connection between E-H2 and E-H3 to influence stem region rearrangement. Consistent with these residues determining the tethering of E-H2 to E-H3, D415 and H435 comprise 5 of the 8 amino acid connections between the two helices.

[0088] The other two stem critical residues are both glycine residues, known for their helix-breaking properties due to torsional flexibility. G421, at the distal end of E-H2, contacts adjacent residues W418, D419, and L423 to form the E-H2/E-H3 turn. The ribbon structures illustrating this can be found in Figure 4, C in U.S. Provisional Application No. 62/116,524, filed February 15, 2015, which is hereby incorporated by reference in its entirety. G421 also makes contact with residue D22 on Domain I (DI) of E-protein, one of a number of network interactions between stem and DI and Dili which stabilize the E ectodomain's pre-trigger conformation. The highly conserved critical residue G406 (present in 91% of related flaviviruses) lies towards the distal end of helix EH-1, and, in the mature virion, makes contact with every adjacent residue on EH-1 from residue 402 to 410, consistent with G406 playing an important role in maintaining E- Hl in its prefusion metastable state and then mediating its rearrangement after triggering. The ribbon structures illustrating this can be found in Figure 4, D in U.S. Provisional Application No. 62/116,524, filed February 15, 2015, which is hereby incorporated by reference in its entirety. Both G406 and G421 are located at critical hinge positions where structural changes in orientation between the helices are expected to take place upon triggering, so may play important roles in mediating the conformational changes that occur in late stages of Env function.

[0089] Mutation of stem residue G406 partially triggers the E-protein dimer

[0090] To verify that the critical mutations identified here did not result in grossly misfolded proteins, we repeated the DENV virus budding assays using diverse capture and detection antibodies. For seven of the eight critical mutations identified in this study, the different antibodies (or antibody cocktails) used to capture or detect virions demonstrated equivalent results (Table 2), suggesting that each of these seven stem mutants is correctly folded and antigenically equivalent to wild-type. However, substitution of critical residue G406 with arginine resulted in strikingly divergent responses, depending on the antibody combination used. In cases where G406R-RVPs were captured or detected with antibodies (mouse or human) against Domain II (DII), high levels of RVPs were detected (217-377% of wild-type). In contrast, when G406R-RVPs were captured or detected with antibodies against prM or the fusion loop, few or no RVPs were detected (6-13% of wild-type) (Figure 2). These results suggest that the prM and fusion loop epitopes are not accessible in G406R-RVPs due to an altered conformation of E-protein on the virion. The relatively high reactivity with 1 AlD-2 detection (377%) of wild-type, when captured using an anti-DII huMAb cocktail) also suggests an increased availability of the 1 AlD-2 binding site on Domain III (Dili), consistent with exposure of 1A1D-2 epitopes that are normally occluded on the mature virion (Lok et al., Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins, 2008, Nat Struct Mol Biol, 15:312-317). Taken together, these data suggest that G406R induces an altered conformation of E-Hl, the fusion loop, DI, and Dili due to an altered interaction of E-Hl with Dili (Figure 3).

[0091] A model for stem function during triggering and fusion

[0092] The location and inter-atomic contacts of each of the eight identified critical residues on stem and TM, complemented by previous structural and mutagenesis studies, allow us to form a hypothesis-based functional model of how this region of DENV E-protein may contribute to infectivity. In this model, on the mature virion, the inter-atomic contacts of the stem critical residues maintain the three stem helices in a pre-fusion metastable state, held close to the virus surface. In particular, the E-H2/E-H3 inter-helix interaction appears to be stabilized in a pre-trigger conformation by critical residue D415 contacting the highly conserved H435 and by G421 stabilizing the E-H2/E-H3 turn (Figure 4, A). This conformation enables E-Hl and E-H2 interactions with E ectodomain DI and Dili, contacts that maintain E in a constrained conformation close to the virus membrane until it is freed by triggering at low pH. Our data suggest that critical residue D415 on helix E-H2 may be a component of a major 'switch' that governs the tethering of E-H2 to E-H3.

[0093] Upon virus internalization to the endosome, low pH-induced protonation of H435 is likely to break its interaction with D415, initiating the disconnection of stem E-H2 and E-H3, and resulting in displacement of E-H2 from the virus surface (Figure 4, B). Concurrently, conformational changes in E-protein and dissociation of the E-protein dimer, driven by protonation of additional histidine residues (see below), would disrupt interactions that tether E ectodomains to stem helices E-Hl and E-H2, resulting in the complete release of E ectodomain from stem and translocation away from the virus surface. This model is consistent with a West Nile Virus pre-fusion intermediate structure which suggests that stem plays an early role in pH- induced transformations, with E-Hl, E-H2, and E-H3 extending outward from the virion surface prior to formation of the fusogenic trimer (Kaufmann et al., Capturing a flavivirus pre-fusion intermediate, 2009, PLoS Pathog, 5:el000672).

[0094] After dissociation of the E dimer, the resulting monomers form a fusogenic E trimer which inserts in the endosome membrane via the fusion loops (Figure 4, C). Residues 394 to 401 in the proximal region of stem helix E-Hl then unfold in an extended conformation and pack along the interstices of E monomers to pull the viral and host membranes together (Klein et al., Structure of a dengue virus envelope protein late-stage fusion intermediate, 2013, J Virol, 87:2287-2293). G406, by virtue of its location, the helix-breaking propensity of glycine residues, and its conservation (91% among 33 different flaviviruses, the highest level of conservation among the eight critical residues identified), is positioned to facilitate the unwinding of the proximal region of E-Hl to enable this zippering, thus linking the zippered residues (394-401) and the unzippered portion of stem (residues 402-419) to the TM anchor. Consistent with the importance of G406 are the results of a previous study where a G406P mutation greatly reduced assembly of virus-like particles (Lin et al., 2011). Our data suggest that in G406R-RVPs, the relationship of Dili to stem mimics that of the post-trigger state, with Dili no longer moored by interactions with stem and displaced from its normal position relative to the virion surface. This results in a conformational strain sufficient to partially trigger Env, exposing Dili and causing the fusion loop to be buried in a non-accessible position, but not so severe as to diminish budding of virions. As the G406R partially-triggered virion exposes epitopes for potently neutralizing but normally occluded MAbs, such as 1A1D-2, its use as an immunogen to elicit novel antibodies may be of particular interest. A crystal structure of G406R may also provide valuable structural information about the intermediate and post-fusion conformations of stem.

[0095] Critical residue G421, at the junction between E-H2 and E-H3, may participate late in the fusion process by binding the fusion trimer and zippering to promote the final stages of membrane fusion (Figure 4, D). Supporting this model, peptides corresponding to stem residues 417-438 bind strongly to a soluble fusion trimer (Schmidt et al., Peptide inhibitors of dengue-virus entry target a late-stage fusion intermediate, 2010, PLoS Pathog, 6:el000851), suggesting that the distal stem region, which includes E-H3, interacts with the trimer. Our results thus identify critical residues and inter-atomic connections that may explain how stem both helps maintain E-protein in a constrained conformation to prevent premature triggering and subsequently triggers the conformational changes that unfold the helical stem region to facilitate the formation of the fusogenic trimer.

[0096] Histidine latches mediate pH-induced stem triggering

[0097] Triggering reactions that involve the DENV stem region have not been previously investigated in detail, but the dissociation of E stem-ectodomain contacts may be particularly important since a network of contacts between stem E-Hl and ectodomain DI are maintained in both the immature and mature virus but are lost to enable formation of the fusogenic trimer (Kostyuchenko et al., Immature and mature Dengue serotype 1 virus structures provide insight into the maturation process, 2013, J Virol). Our results and others' implicate at least three of E- protein's thirteen histidine residues (H280, H315, and H435) in mediating the low pH-sensing function that triggers the dissociation of stem-ectodomain interactions. These three are the only E-protein histidine residues which directly contact the stem region (<4.2A) in the mature conformation, and each has characteristics consistent with the ability to functionally "latch" stem to the E ectodomains, analogous to that proposed for M protein-E ectodomain interactions that mediate triggering of contacts between M and DII (Zhang et al., Cryo-EM structure of the mature dengue virus at 3.5-A resolution, 2013, Nat Struct Mol Biol, 20: 105-110).

[0098] H280, H315 and H435 are highly conserved (94%, 97%, and 88%, respectively) among 33 related Flaviviruses. H280 on ectodomain DI contacts stem helix E-H2 via residue T416, which contacts adjacent critical residue D415. Protonation of H280 would be predicted to eliminate the hydrogen bonds connecting these residues. H315 on ectodomain Dili contacts stem residue K398 (Q400 in DENV-2) on helix E-Hl, but these contacts are lost after triggering, in part by protonation of H315 and repulsion from K398, with new interactions formed between stem and DII (Klein et al., Structure of a dengue virus envelope protein late-stage fusion intermediate, 2013, J Virol, 87:2287-2293), suggesting a mechanism for the triggered release of stem-DIII interactions in the endosome during infection. H315 has also been implicated in our own structure-function study of the E-protein ectodomain (Christian, An Atomic-level Mechanism of Dengue Virus Envelope Infectivity, 2013) and as one of the critical pH sensors for initiating membrane fusion in TBEV (Fritz et al., Identification of specific histidines as pH sensors in flavivirus membrane fusion, 2008, J Cell Biol, 183 :353-361). As discussed above, residue H435 interacts with critical residue D415 in the mature E cryo-EM structure, potentially forming a critical switch that maintains tethering of E-H2 to E-H3. These histidines, and possibly others, may act in concert to promote the low pH-triggered dissociation of viral Envelope proteins (Nelson et al., Protonation of individual histidine residues is not required for the pH- dependent entry of west nile virus: evaluation of the "histidine switch" hypothesis, 2009, J Virol, 83 : 12631-12635).

[0099] Oligomerization of TM during membrane fusion

[00100] Critical residues in the E-protein TM region are likely crucial for the final stages of membrane fusion and our data suggest that oligomerization of the E and M protein TM regions, facilitated by residues 1452, M453, K454, G456, and G464, may contribute to this part of Envelope functionality (Figure 4, D). In the available cryo-EM structure of the mature virion, critical residue K454 participates in cross-helix interactions between E-Tl and E-T2 to stabilize the E TM anchor. Critical residues M453 and 1464 mediate interactions between the TM regions of E-protein and M protein through their contacts with M-Tl and M-T2. Cumulatively, these interactions result in a heavily cross-linked oligomer of eight TM helices (ET-1, E-T2, M-Tl, and M-T2), each on two connected monomers.

[00101] The importance of an oligomerized TM region is consistent with complete fusion between virus and host endosome membranes requiring the presence not only of both E-protein TM helices, E-Tl and E-T2, but also the interactions between them (Fritz et al., The unique transmembrane hairpin of flavivirus fusion protein E is essential for membrane fusion, 201 1, J Virol, 85:4377-4385); substitution of either one of the DENV E TM domains with a different TM domain from the closely related Japanese Encephalitis Virus did not support fusion. Our data is also consistent with the changes that occur between the E-protein and M protein TM domains transitioning from the immature to mature DENV structures. In the immature virus, the E TM helices are not in close proximity to the M TM helices. However, during maturation, the E- protein and M protein TMs rearrange and associate, suggesting a functional interaction relevant to fusion (Kostyuchenko et al., Immature and mature Dengue serotype 1 virus structures provide insight into the maturation process, 2013, J Virol; Zhang et al., Cryo-EM structure of the mature dengue vims at 3.5-A resolution, 2013, Nat Struct Mol Biol, 20: 105-110; Zhang et al., Conformational changes of the flavivirus E glycoprotein, 2004, Structure, 12: 1607-1618). Our data supports this conclusion, demonstrating that the interactions between E-Tl and M protein at both ends of the TM helices are vital for late-stage E-protein functionality.

[00102] Critical TM anchor residues may be contributing to the final step of fusion when the two lipid membranes are in close physical proximity. Little is known about how the TM region of DENV E-protein contributes to the physical fusion between the viral and host membranes. However, studies of haemagglutinin in influenza virus have suggested an important role for the TM region in class I virus Envelope fusion (Chang et al., Membrane interaction and structure of the transmembrane domain of influenza hemagglutinin and its fusion peptide complex, 2008, BMC Biol, 6:2; Victor et al., Structural determinants for the membrane insertion of the transmembrane peptide of hemagglutinin from influenza virus, 2012

[00103] J Chem Inf Model, 52:3001-3012). Specifically, a highly conserved lysine residue at the proximal end of the influenza hemagglutinin TM region is thought to mediate lipid mixing during membrane fusion (Ge and Freed, Two conserved residues are important for inducing highly ordered membrane domains by the transmembrane domain of influenza hemagglutinin, 2011, Biophys J, 100:90-97), and E-protein K454 is in a comparable position to play a similar role for DENV. In this model, critical residue K454 would mediate the disruption of the viral lipid bilayer as the ultimate step in membrane fusion by interacting with negatively charged lipid phosphate head groups to promote the intermixing of the viral lipid bilayer with the target cell lipid bilayer (whose own disruption is mediated by the fusion peptide). Consistent with this model, positively charged K454 does not appear to interact with any negative charges within the protein structure and is not accessible to soluble ions (within the resolution of the structure available), suggesting that its epsilon-amino group would be able to interact with negatively charged phospholipid head groups at the membrane interface. Interestingly, in the post-fusion trimer conformation, many of the critical TM residues would end up positioned adjacent to the fusion loop (Figure 4, E), also consistent with these TM residues mediating the final stages of membrane fusion.

[00104] The mechanistic model described herein incorporates all DENV stem, TM, and M protein structural information to date into a cohesive functional model that describes how these regions of E-protein contribute to infectivity. Critical residues were identified using stringent criteria: their mutation did not significantly disrupt expression, folding, viral assembly, or budding; many of our critical residues form interconnections around Env structures known to be important during infectivity; and the importance and functionality of many of these residues is confirmed by individual mutagenesis and structural studies of DENV or related flaviviruses by other investigators. Other important residues likely also participate in stem and TM functionality, but could not be definitively distinguished here based on the criteria used to select critical mutants that are not misfolded. Our studies do not experimentally distinguish between different steps of late-stage Env functionality, such as receptor binding, pH-dependence, triggering, zippering, and fusion, but infer the dynamic role of specific residues from previous studies that have functionally characterized these structures. This work independently validates and incorporates the residues into a cohesive model to explain the residues' inter-dependent functionality. Related flaviviruses, including West Nile Virus, Japanese Encephalitis Virus, and Tick-Borne Encephalitis Virus, share the same overall structural features as DENV E-protein, suggesting that the functional model derived here for DENV can be used to apply to other similar flavivirus and class II viral fusion proteins. The identification of functionally critical residues throughout stem and TM also identifies candidate structures for antibody and/or drug-mediated inhibition of the virus.

[00105] Construction of DENV-3 Chimera Plasmid

[00106] To generate a DENV construct capable of high-level CprM/E expression and viral production, we created a chimera construct encoding residues 1-100 of the capsid protein from DENV-2 (SI 6803) and codon-optimized residues 101-760 from DENV-3 (CH53489). These residues encode the ER anchor for capsid (residues 101-114) and prM/E (residues 115-760). The wild type DENV-2 Capsid gene fragment was cloned by PCR, and the codon-optimized prM/E genes from DENV-3 were cloned in-frame in pTRex-pBR322, a modified version of expression plasmid pTRex-DEST-30 (Invitrogen).

[00107] Construction of DENV-3 prM E Mutation Library.

[00108] A shotgun mutagenesis mutation library of prM/E was created as previously described (Paes et al., Atomic-level mapping of antibody epitopes on a GPCR, 2009, J Am Chem Soc, 131 :6952-6954)(Christian et al., 2013). Briefly, the parental plasmid expressing the DENV- 3 CH53489 CprM/E polyprotein was used as a template to make a library of random mutations across prM/E (residues 115-760, excluding capsid), created using PCR-based mutagenesis (Diversify PCR Random Mutagenesis Kit, Clontech). Each mutant clone was sequence-verified. A complete mutation library was assembled by selection of at least two mutant clones per residue, preferably representing a conserved and non-conserved residue at each position. 64% of DENV-3 prM/E variants contained single mutations and the remaining clones contained mutations at two or more positions.

[00109] Immunofluorescence Assay

[00110] Mutation libraries and controls were expressed in human HEK-293T cells.

Twenty-two hours post-transfection, cells were washed and fixed in 4% PFA, permeabilized with 0.1% saponin, incubated with a cocktail of diverse MAbs recognizing the DENV Envelope protein (4.8A, D11C, 4.2C-13.7F, 9.4F-8.10E) (Costin et al., Mechanistic study of broadly neutralizing human monoclonal antibodies against dengue virus that target the fusion loop, 2013, J Virol, 87:52-66), followed by AlexaFluor 488-conjugated secondary antibody (Jackson, West Grove, PA). Microplates were measured using the Intellicyt HTFC screening system. Antibody reactivities against each mutant prM/E protein clone were calculated relative to wild-type protein reactivity by subtracting the signal from mock-transfected controls and normalizing to the signal from wild-type prM/E-transfected controls.

[00111] Critical clones were also tested individually against eight different E-protein- specific MAbs (D11C and 4.2C-13.7F (Costin et al., Mechanistic study of broadly neutralizing human monoclonal antibodies against dengue virus that target the fusion loop, 2013, J Virol, 87:52-66); and 1C17, 2D7, 5J7, 1B 13, 1D7, and 1M12 (de Alwis et al., Identification of human neutralizing antibodies that bind to complex epitopes on dengue virions, 2012, Proc Natl Acad Sci U S A, 109:7439-7444; Smith et al., Persistence of circulating memory B cell clones with potential for dengue virus disease enhancement for decades following infection, 2012, J Virol, 86:2665-2675)), representing seven different epitopes, to confirm that the expressed mutant E- proteins are not misfolded.

[00112] DENV Reporter Virus Particle (RVP) Production

[00113] The BHK-DRRZ cell line, which stably propagates a DENV replicon, was produced by transduction of BFD 21 cells with DENV reporter virus particles (RVPs) that encapsidate a replicon encoding both Renilla luciferase and the S Ble gene (conferring resistance to zeocin). The DENV replicon was described previously (Ansarah-Sobrinho et al., Temperature- dependent production of pseudoinfectious dengue reporter virus particles by complementation, 2008, Virology, 381 :67-74), and DENV RVPs have been extensively characterized for antigenic equivalence to live virus (Ansarah-Sobrinho et al., Temperature-dependent production of pseudoinfectious dengue reporter virus particles by complementation, 2008, Virology, 381 :67- 74; Mattia et al., Dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes, 2011, PLoS One, 6:e27252). Following infection, selection of infected cells was conferred by supplementing complete DMEM with 300 μg/ml zeocin. For RVP production, BFD -DRRZ cells were added to mutation array microtiter plates in DMEM complete medium with FEPES added to 25 mM, pH 8.0. Plates were incubated at 37°C at 5% C0₂ to allow for transfection and RVP production initiation. RVP supernatant from each well was then transferred to a 384-well white flat-bottom microtiter plate and stored at -80°C for at least 4 hours to eliminate any viable BFDC-DRRZ cells that were present, before use for RVP Budding ELISA or RVP Infectivity Assays.

[00114] DENV RVP Budding ELISA

[00115] A 384-well white flat-bottom microtiter plate was coated with a cocktail of mouse anti-Env monoclonal antibodies or mouse prM monoclonal antibody 2H2 (ATCC #FIB-114) (5 μg/ml) in 0.1M NaHC0_3; pH 8.6, and incubated overnight at 4°C. The plate was blocked with 3% BSA in PBS for 2 hours at room temperature. A 384-well RVP production plate containing frozen RVPs was thawed and 6-10 μΐ supernatant was transferred to the blocked ELISA plate and incubated overnight at 4°C to allow capture of RVPs. A human monoclonal antibody cocktail (D163, D168, D381, and D384; final 1 μg/ml), in blocking buffer was allowed to incubate on the plate for 1 hour at room temperature, followed by addition of rabbit anti-human HRP-conjugated secondary antibody (1 :5000) in blocking buffer for 1 hour at room temperature. Reactivity was detected using SuperSignal West Femto Chemiluminescent Substrate (Pierce). Serial dilutions of both capture and detection antibodies were used to optimize detection conditions (Z' >0.3). All luminescence values were background- subtracted and normalized to the average luciferase signal generated from wild-type RVPs. [00116] The RVP budding assay antibody combinations used were as follows: mouse cocktail capture, human cocktail #1 detection: mouse monoclonal antibody cocktail (4G2, 1A1D-2, D015, D022; final 1 μg/ml); human monoclonal antibody cocktail (D003, D004, D173, D178; final 1 μg/ml). mouse prM capture, human 1A1D-2 detection: mouse monoclonal prM antibody 2H2 (ATCC #HB-114); 5 μg/ml); humanized 1A1D-2 unpurified supernatant was diluted 1 : 100 in blocking buffer, mouse prM capture, human cocktail #2: mouse monoclonal prM antibody 2H2 (5 μg/ml); human monoclonal antibody cocktail (D163, D168, D381, D384). human cocktail #1 capture, mouse 1A1D-2 detection: human monoclonal antibody cocktail (D003, D004, D173, D178; final 2.5 μg/ml); mouse 1A1D-2 in ascites fluid diluted 1 :8000 in blocking buffer. mouse cocktail detection/anti-fusion loop detection mouse monoclonal antibody cocktail (4G2), 1A1D-2, D015, D022; final 1 μg/ml); human anti-fusion loop MAb D004.

[00117] DENV-3 RVP Infectivity Assay. RVPs produced from the mutation array were used for infectivity assays, as described previously (Mattia et al., Dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes, 2011, PLoS One, 6:e27252), except that a Renilla luciferase reporter replaced GFP. 20 μΐ of frozen RVPs were used to infect BHK DC-SIGN cells at a density of 10,000 cells per well in DMEM with HEPES added to 10 mM, pH 8.0 in a 384-well microplate format. The plate was then incubated at 37°C at 5% C0₂. 72 hours post-infection, cells were washed with PBS and lysed for 20 minutes at room temperature (IX Lysis Buffer, Promega). Luciferase substrate was diluted 1 : 100 in assay buffer (Promega) and luminescence was detected on a Wallac or Envision luminometer. All luminescence values were background- subtracted and normalized to the average signal from wild-type RVP infection.

[00118] Example 2. This example provides a summary of mutations of DENV3 and

DENV4. The mutations were analyzed for expression, budding, infectivity, and antigenic reactivity as described in Example 1.

[00119] DENV3 Critical Residues - Infectivity MAb Serotype Flavivirus

Domain Mutation Expression % Budding %

% Reactivity % Conserv % Conserv

Dl R186L 111 (47) 262 (48) 1 (1) 8/8 100 39

186L , E233G 35 7 2

E233G 43 2 29

R186H, H207L 72 27 0

H207L 52 10 31 100 82

DM A265T 91 (3) 319 (15) 2 (2) 8/8

A265V, M258L 43 3 1

M258L 107 95 32

A265T , A203D 42 36 2

A265T, V206E 5 2 0

V206E 10 0 0

E-H1 G406R 148 (2) 217 (212) 0 (0) 8/8 100 91

E-H2 D415G 129 (7) 150 (30) 5 (3) 8/8 75 57

E-H2 G421A 98 (4) 79 (11) 7 (6) 8/8 100 86

E-T1 I452N 105 (17) 242 (37) 0 (0) 8/8 25 49

I452S , H345P 88 55 0

I452V, H435R 107 9 0

I452N , E366V 33 1 1

I452N, F446L, P330L 11 2 0

E-T1 M453T 150 (24) 124 (45) 2 (1) 8/8 75 11

E-T1 K454R 107 (9) 170 (36) 6 (1) 8/8 75 54

K454E 94 9 0

K454R, P354L 52 2 0

K454E, I478T 81 8 0

K454T, V423A 101 7 -1

E-T1 G456S, N364S 128 (6) 189 (94) 1 (1) 8/8 25 6

N364S 117 219 82

E-T1 I464T 102 (5) 76 (27) 0 (0) 8/8 75 14

I464L 94 70 102

Table 5. E-protein ectodomain, stem, and TM residues critical for DENV infectivity. R186L and A265T are ectodomain mutations (Christian et al. 2013) of interest. The remainder are stem/TM critical residues. Activities for E-protein expression (n=3), viral budding (n=2), and viral infectivity (n=3) are shown as a percentage of wild-type, with ranges (max-min) in parentheses. MAb reactivity indicates the number of MAbs that reacted with the given mutation (>50% wild type) among the eight different E-protein MAbs tested. Conservation among all four DENV serotypes is shown as Serotype %. Flavivirus % shows the extent of conservation among 33 flaviviruses most closely related to DENV (NCBI); residue 415 is a negatively charged residue (Asp or Glu) in 87% of flaviviruses. Capture: Mouse Human2 Mouse prM Mouse prM Mouse

Domain Mutation Detect: Humanl m-1A1 D2 h-1A1 D2 Human2 F-loop

E-H1 G406R 217 (212) 377 (46) 6 (4) 7 (1) 13 (1)

E-H2 D415G 150 (30) 219 (25) 141 (39) 150 (85) 163 (1 14)

E-H2 G421A 79 (1 1) 66 (1 1) 39 (18) 35 (15) 54 (4)

E-T1 I452N 242 (37) 314 (24) 483 (141) 643 (94) 167 (130)

E-T1 M453T 124 (45) 84 (37) 252 (60) 242 (70) 96 (14)

E-T1 K454R 170 (36) 371 (45) 273 (90) 347 (69) 208 (285)

E-T1 G456S 189 (94) 70 (6) 256 (19) 282 (1 19) 109 (27)

E-T1 I464T 76 (27) 141 (5) 121 (6) 98 (50) 106 (67)

Table 6. Budding activities for E-protein stem/anchor residues critical for DENV infectivity.

The low reactivities seen when prM or fusion loop mAbs are used suggest a non-native conformation.

[00120] From a DENV4 Ala-scan library data, there are about 140 critical residues where mutation to alanine reduces infectivity, but gives normal budding (budding >75% of WT, infectivity <25% of WT, average env >50%). There is a small overlap with DENV3 critical residues (Christian, et al) and one, G456/L458, is in a site of interest. This suggests that these mutations bud but are non-infective for both DENV3 and DENV4 and can be used to engineer and generate a DENV vaccine.

DENV3 DENV4 Mutation Env avg (n=10) budding (n=2) infectivity (n=3)

H259R H261A 91 88 0

I276T M278A 104 133 9

S296G S298A 98 97 1

G406R G408A 119 103 12

G421A G423A 108 101 0

M453T I455A 98 83 11

G456S L458A 93 78 24

I464T I466A 99 72 2

Table 7. Overlap between DENV3 and 4 infectivity critical residues (budding >75% of WT, infectivity <25% of WT). Data for DENV4 alanine scan library, showing equivalent mutation in

DENV3.

DENV3 DENV4 Env # Env avg budding infectivity

186L R188 105 166 73

A265T A267 93 97 56

I452N M454 98 81 62

K454R R456 97 52 0

G456S L458 93 78 24

Table 8. Effect of Ala mutations at the equivalent mutation sites in DENV4. [00121] DENV4 alanine scanning data identifies a number of Ala mutations with high budding, low infectivity. The residue number refers to the E Protein amino acid sequence (SEQ ID NO: 2).

DENV4 Env avg (n=10) budding (n=2) infectivity (n=3)

V6 102 141 8

G14 113 146 0

V53 118 158 6

R73 113 180 5

D98 67 190 1

1132 124 148 11

N134 100 208 16

F193 95 227 2

H209 96 204 19

Q211 101 170 1

D225 106 147 17

Q256 82 164 1

E269 97 142 12

R350 70 167 0

P356 61 171 3

E370 85 147 7

F373 77 182 0

S396 98 144 0

1398 106 260 0

G399 109 233 1

F402 118 240 0

V439 99 144 1

Table 9. DENV4 mutations resulting in high budding (>140% of WT) and low infectivity <20% of WT).

[00122] The results described herein demonstrate that the mutant proteins can lead to attenuated viruses or antigenic proteins that can be used to elicit an effective therapeutic response, including an immune response, for the treatment and/or prevention of flavivirus infections.

[00123] Various references and patents are disclosed herein, each of which are hereby incorporated by reference for the purpose that they are cited. [00124] From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications can be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting.

Claims

What is claimed is:

1. An isolated polyprotein comprising a DENVl, DENV2, DENV3, or DENV4 polypeptide comprising at least one mutation in a E-protein that decreases infectivity, retains or increases budding and retains or increases native antibody reactivity of a DENVl, DENV2, DENV3, or DENV4 virus.

2. The isolated polyprotein of claim 1, wherein the E-protein is a DENV3 E-protein.

3. The isolated polyprotein of claim 1, wherein the E-protein is a DENV4 E-protein.

4. The isolated polyprotein of claim 1, wherein the E-protein is a DENVl E-protein.

5. The isolated polyprotein of claim 1, wherein the E-protein is a DENV2 E-protein.

6. The isolated polyprotein of claim 1, wherein the E-protein comprises a mutation in a DI domain, a DII domain, a Dili domain, a E-Hl domain, a E-H2 domain, or a E-Tl domain, or any combinations thereof.

7. The polyprotein of claim 6, wherein the domain is a DENV3 E-protein domain.

8. The polyprotein of claim 6, wherein the domain is a DENV4 E-protein domain.

9. The polyprotein of claim 6, wherein the domain is a DENV 1 or DENV2 E-protein domain.

10. A DENV3 E-protein comprising at least one mutation at position G421, M453, G406, D415, , 1452, K454, G456, or 1464 of SEQ ID of the DENV3 E-Protein (SEQ ID NO: 1).

11. The DENV3 E-protein of claim 10, further comprising a mutation at position R186, H259, A265, 1276, S296.

12. The DENV3 E-protein of claim 10, further comprising a mutation at position N364.

13. A DENV3 E-protein comprising a mutation selected from the group consisting of G421A, M453T, G406R, D415G, I452N, K454R, G456S, and I464T of the DENV3 E-Protein (SEQ ID NO: 1), or any combinations thereof.

14. A DENV3 E-protein comprising a mutation selected from the group consisting of G421A, M453T, E233G, R186H, H207L, A265V, A203D, G406R, D415G, I452N, I452S , H345P, I452V, H435R, E366V, I452N, F446L, P330L, K454R, K454E, P354L, I478T, K454T, V423A, G456S, N364S, I464T, and I464L of the DENV3 E-Protein (SEQ ID NO: 1), or any combinations thereof.

15. A DENV3 E-protein comprising a mutation selected from the group consisting of: E233G, R186H, H197L, A265V, A203D, V206E, E366V, P330L, P354L, and N364S, or any combinations thereof.

16. The DENV3 E-protein of claim 14, further comprising a mutation selected from the group consisting of: R186L A265T, M258LH259R, I276T, and S296G.

17. The DENV3 E-protein of claim 15, further comprising a mutation selected from the group consisting of: R186L A265T, M258LH259R, I276T, and S296G.

17. A DENV4 E-protein comprising at least one mutation at a position selected from the group consisting of: H261, M278, S298, G408, G423, 1455, L458, 1466, R188, A267, M454, R456, L458, V6, G14, V53, R73, D98, 1132, N134, F193, H209, Q211, D225, Q256, E269, R350, P356, E370, F373, S396, 1398, G399, F402 of the DENV4 E Protein (SEQ ID NO: 2).

18. The DENV4 E-protein of claim 17, wherein the protein comprises a mutation selected from the group consisting of H261A, M278A, S298A, G408A, G423A, I455A, L458A, I466A, R188A, A267A, M454A, R456A, V6A, G14A, V53A, R73A, D98A, I132A, N134A, F193A, H209A, Q211A, D225A, Q256A, E269A, R350A, P356A, E370A, F373A, S396A, I398A, G399A, F402A, and V439A of the DENV4 E Protein (SEQ ID NO: 2), or any combinations thereof.

19. A DENV1 E-protein comprising a mutation that corresponds to a mutation at position of G421, M453, G406, D415, 1452, K454, G456, R186, A265, G406, D415, 1452, M453, K454, G456, N364, H259, 1276, or S296 of DENV 3.

20. A DENV1 E-protein comprising a mutation that corresponds to a mutation at position of H261, M278, S298, G408, G423, 1455, L458, 1466, R188, A267, M454, R456, L458, V6, G14, V53, R73, D98, 1132, N134, F193, H209, Q211, D225, Q256, E269, R350, P356, E370, F373, S396, 1398, G399, F402, or V439 of DENV 4.

21. A DENV2 E-protein comprising a mutation that corresponds to a mutation at position of G421, M453, G406, D415, 1452, M453, K454, G456, R186, A265, G406, D415, 1452, K454, G456, N364, H259, 1276, or S296 of DENV 3.

22. A DENV2 E-protein comprising a mutation that corresponds to a mutation at position of H261, M278, S298, G408, G423, 1455, L458, 1466, R188, A267, M454, R456, L458, V6, G14, V53, R73, D98, 1132, N134, F193, H209, Q211, D225, Q256, E269, R350, P356, E370, F373, S396, 1398, G399, F402, or V439 of DENV 4.

23. A virion comprising the polyprotein or the protein according to any one of claim 1-22.

24. The virion of claim 23, wherein the virion is a flavivirus.

25. The virion of claim 23, wherein the flavivirus is dengue virus, zika virus, Yellow Fever virus, Japanese encephalitis, or a west nile virus.

26. The virion of claim 25, wherein the dengue virus is DENV1, DENV2, DENV3, or DENV4.

27. A pharmaceutical composition comprising a polyprotein or protein according to any one of claims 1-22.

28. A pharmaceutical composition comprising a virion according to any one of claims 23-26.

29. A method of eliciting an immune response comprising administering to a subject a pharmaceutical composition comprising a polyprotein or protein according to any one of claims 1-22.

30. A method of eliciting an immune response comprising administering to a pharmaceutical composition comprising a virion according to any one of claims 23-26.

31. A method of preventing or ameliorating a flavivirus infection comprising administering to a subject a pharmaceutical composition comprising a polyprotein or protein according to any one of claims 1-22.

32. A method of preventing or ameliorating a flavivirus infection comprising administering to a subject a pharmaceutical composition comprising a virion according to any one of claims 23-26.

33. The method of claim 32, wherein the virion is a dengue virion, zika virion, Yellow Fever virion, Japanese encephalitis virion, or a west nile virion.

34. The method of claim 33, wherein the virion is a DENV3 virion, a DENV4 virion, a DEN VI virion, or a DENV2 virion.

35. A vaccine comprising a flavivirus comprising a mutation that corresponds to a mutation in a DENV1, DENV2, DENV3, or DENV4 E-protein that decreases infectivity, increases budding and optionally retains native antibody reactivity.

36. The vaccine of claim 35, wherein the mutation is in a DENV3 E-protein.

37. The vaccine of claim 35, wherein the mutation is in a DENV4 E-protein.

38. The vaccine of claim 35, wherein the mutation is in a DENV1 E-protein.

39. The vaccine of claim 35, wherein the mutation is in a DENV2 E-protein.

40. The vaccine of claim 35, wherein the mutation is in a zika, Yellow Fever, Japanese encephalitis, or a west nile E-protein.

41. The vaccine of claim 35, wherein the mutation is in a domain that corresponds to a mutation of DENV3 E-protein domain, wherein the domain selected from the group consisting of DI domain, a DII domain, a Dili domain, a E-Hl domain, a E-H2 domain, or a E-Tl domain, or any combinations thereof.

42. The vaccine of claim 35, wherein the flavivirus comprises a mutation that corresponds to a DENV3 E-protein mutation selected from the group consisting of: G421, M453, G406, D415, 1452, K454, G456, H259, or 1464 of DENV3.

43. The vaccine of claim 35, wherein the flavivirus comprises a mutation that corresponds to a DENV3 E-protein mutation selected from the group consisting of: G421 A, M453T, G406R, D415G, I452N, K454R, G456S, and I464T/L.

44. The vaccine of claim 35, wherein the flavivirus comprises a mutation that corresponds to a DENV3 E-protein mutation selected from the group consisting of: R186, A265, G406, D415, G421, 1452, M453, K454, G456, N364, H259, 1276, and S296.

45. The vaccine of claim 35, wherein the flavivirus comprises a mutation that corresponds to a DENV3 E-protein mutation selected from the group consisting of: G421A, M453T, R186L, E233G, R186H, H207L, A265T, A265V, M258L, A203D, G406R, D415G, I452N, I452S , H345P, I452V, H435R, E366V, I452N, F446L, P330L, K454R, K454E, P354L, I478T, K454T, V423A, G456S, N364S, I464T, I464L, H259R, I276T, and S296G, or any combinations thereof.

46. The vaccine of claim 35, wherein the flavivirus comprises a mutation that corresponds to a DENV3 E-protein mutation selected from the group consisting of: 1) G421 A, 2) M453T, 3) E233G, 4) R186H and H207L, 5) H207L, 6) A265T, 7) A265V and M258L, 8) M258L, 9) A265T and A203D, 11) G406R, 12) D415G, 13) R186L, 14) I452N, 15) I452S and H345P, 16) I452V and H435R, 19) R186L and E233G, 20) K454R, 21) K454E, 22) K454R and P354L, 23) K454E and I478T, 24) K454T and V423A, 25) G456S and N364S, 26) N364S, 27) I464T, 28) I464L, 29) H259R, 30) I276T, 31) S296G or any combination thereof.

47. The vaccine of claim 35, wherein the flavivirus comprises a mutation that corresponds to a DENV4 E-protein mutation selected from the group consisting of: H261, M278, S298, G408, G423, 1455, L458, 1466, R188, A267, M454, R456, L458, V6, G14, V53, R73, D98, 1132, N134, F193, H209, Q211, D225, Q256, E269, R350, P356, E370, F373, S396, 1398, G399, F402, and V439.

48. The vaccine of claim 35, wherein the flavivirus comprises a mutation that corresponds to a DENV4 E-protein mutation selected from the group consisting of: H261 A, M278A, S298A, G408A, G423A, 1455 A, L458A, I466A, R188A, A267A, M454A, R456A, V6A, G14A, V53A, R73A, D98A, I132A, N134A, F193A, H209A, Q211A, D225A, Q256A, E269A, R350A, P356A, E370A, F373A, S396A, I398A, G399A, F402A, and V439A, or any combinations thereof.

49. A recombinant dengue virus with reduced infectivity as compared to a wild-type virus, wherein the virus comprises a polyprotein or protein according to any one of claims 1-22.

50. The recombinant virus of claim 49, wherein the virus retains at least 70% expression as compared to the wild-type virus.

51. The recombinant virus of claim 49, wherein the virus retains antigenicity as compared to the wild-type virus.

52. The recombinant virus of claim 49, wherein the virus binds to at least 4 different antibodies that bind to at least 4 different epitopes or domains of the wild-type virus.

53. A recombinant flavivirus comprising a mutation in the E-protein that corresponds to a mutation at position of G421, M453, G406, D415, 1452, K454, G456, G406, D415, 1452, M453, K454, G456, or N364, of DENV 3.

54. The recombinant flavivirus of claim 54, further comprising a mutation in the E-protein that corresponds to a mutation at position of H259, R186, A265, 1276, or S296.

55. A recombinant flavivirus comprising a mutation in a E-protein that corresponds to a mutation at position of H261, M278, S298, G408, G423, 1455, L458, 1466, R188, A267, M454, R456, L458, V6, G14, V53, R73, D98, 1132, N134, F193, H209, Q211, D225, Q256, E269, R350, P356, E370, F373, S396, 1398, G399, F402 and V439 of DENV 4.

56. A recombinant flavivirus comprising a mutation in a E-protein that inhibits flavivirus entry into a cell, wherein the mutation in the flavivirus E-protein corresponds to a mutation in the E-Tl domain.

57. The recombinant flavivirus of claim 56, wherein the mutation corresponds to a mutation at position M453, 1464, 1452 and G456 of DENV3 E-protein.

58. A recombinant flavivirus comprising a mutation that corresponds to a mutation at position G406 of DENV3 E-protein.

59. The recombinant flavivirus of claims 58, wherein the virus is a Dengue Fever, Yellow Fever, Japanese encephalitis, Zika, or West Nile.

60. A pharmaceutical composition comprising the recombinant flavivirus of claims 58 or 59.