METHODS AND COMPOSITIONS FOR DENGUE VIRUS SEROTYPE 4 EPITOPES

RELATED APPLICATIONS

This application claims the benefit, under 35 U.S.C. § 119(e), of U.S. Provisional Application Serial No. 62/510,133, filed May 23, 2017, and U.S. Provisional Application Serial No. 62/561,942, filed September 22, 2017, the entire contents of each of which are incorporated by reference herein.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos. All 07731, All 06695 and All 09761 awarded by the National Institutes of Health. The United States government has certain rights in the invention.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE

LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. § 1.821, entitled 5470-816WO_ST25.txt, 139,037 bytes in size, generated on May 23, 2018 and filed via EFS- Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated herein by reference into the specification for its disclosures

BACKGROUND OF THE INVENTION

Dengue is a mosquito-borne flavivirus that is spreading at an unprecedented rate and has developed into a major health and economic burden in over 50 countries. Even though infected individuals develop potent and long-lasting serotype-specific neutralizing antibodies (Abs), the epitopes engaged by human neutralizing Abs have not been identified.

The four serotypes of dengue virus are the causative agents of dengue fever and dengue hemorrhagic fever. People exposed to primary DENV infections develop long-term neutralizing antibody responses principally only to the infecting serotype. An effective vaccine against dengue needs to elicit long lasting protective antibody responses to all four serotypes simultaneously. We and others have defined antigenic sites on the envelope (E) protein of viruses of dengue serotypes 1 , 2 and 3 targeted by human neutralizing antibodies. The mechanisms of serotype 4 neutralization by human antibodies are poorly understood. Here, we report on the properties of human antibodies that neutralize dengue serotype 4. People exposed to serotype 4 infections or a live attenuated serotype 4 vaccine developed strongly neutralizing antibodies that bound to similar sites on the viral E protein. These studies provide a foundation for developing and evaluating DENV4 vaccines.

SUMMARY OF THE INVENTION

The present invention provides a chimeric dengue virus E glycoprotein comprising the amino acid sequence:

The present invention further provides a chimeric dengue virus E glycoprotein comprising the amino acid sequence:

Also provided herein is a chimeric dengue virus E glycoprotein comprising the amino acid sequence:

In addition, the present invention provides a chimeric dengue virus E glycoprotein comprising the amino acid sequence:

Further provided herein is a chimeric dengue virus E glycoprotein comprising the amino acid sequence:

Additionally provided herein is a chimeric dengue virus E glycoprotein comprising the amino acid sequence:

Further provided herein is a chimeric dengue virus E glycoprotein comprising the amino acid sequence:

The present invention also provides a chimeric dengue virus E glycoprotein comprising the amino acid sequence:

A chimeric dengue virus E glycoprotein is also provided herein, comprising the amino acid sequence:

A chimeric dengue virus E glycoprotein is also provided herein, comprising the amino acid sequence:

A chimeric dengue virus E glycoprotein is also provided herein, comprising the amino acid sequence:

A chimeric dengue virus E glycoprotein is also provided herein, comprising the amino acid sequence:

The present invention also provides a nucleic acid molecule encoding the chimeric dengue virus E glycoprotein of this invention, a vector comprising the nucleic acid molecule of this invention, a flavivirus particle comprising the chimeric dengue virus E glycoprotein of this invention and/or the nucleic acid molecule of this invention, a virus like particle (VLP) comprising the chimeric dengue virus E glycoprotein of this invention, and a composition comprising the chimeric dengue virus E glycoprotein of this invention and/or the nucleic acid molecule of this invention, the vector of this invention, the flavivirus particle of this invention and/or the VLP of this invention, in a pharmaceutically acceptable carrier.

Further provided herein is a method of producing an immune response to a dengue virus in a subject, the method comprising administering to the subject an effective amount of the chimeric dengue virus E glycoprotein of this invention, the nucleic acid molecule of this invention, the vector of this invention, the flavivirus particle of this invention, the VLP of this invention, the composition of this invention, and any combination thereof.

Also provided herein is a method of treating a dengue virus infection in a subject in need thereof, the method comprising administering to the subject an effective amount of the chimeric dengue virus E glycoprotein of this invention, the nucleic acid molecule of this invention, the vector of this invention, the flavivirus particle of this invention, the VLP of this invention, the composition of this invention, and any combination thereof.

In a further embodiment, the present invention provides a method of preventing a dengue virus infection in a subject, the method comprising administering to the subject an effective amount of the chimeric dengue virus E glycoprotein of this invention, the nucleic acid molecule of this invention, the vector of this invention, the flavivirus particle of this invention, the VLP of this invention, the composition of this invention, and any combination thereof.

Additionally provided herein is a method of protecting a subject from the effects of dengue virus infection, the method comprising administering to the subject an effective amount of the chimeric dengue virus E glycoprotein of this invention, the nucleic acid molecule of this invention, the vector of this invention, the flavivirus particle of this invention, the VLP of this invention, the composition of this invention, and any combination thereof.

A method is also provided herein of detecting a neutralizing antibody to a dengue virus, the method comprising administering to the subject an effective amount of the chimeric dengue virus E glycoprotein of this invention, the nucleic acid molecule of this invention, the vector of this invention, the flavivirus particle of this invention, the VLP of this invention, the composition of this invention, and any combination thereof.

In addition, the present invention provides a method of identifying a neutralizing antibody to a dengue virus, comprising: (a) contacting an antibody with the E glycoprotein of this invention; and (b) determining if the antibody binds to the E glycoprotein, wherein binding by the antibody to the E glycoprotein identifies the antibody as a neutralizing antibody to a dengue virus.

Furthermore the present invention provides a method of identifying an immunogenic composition that induces a neutralizing antibody to a dengue virus in a subject, the method comprising: (a) contacting a biological sample from a subject that has been administered the immunogenic composition with the E glycoprotein of this invention; (b) determining if the biological sample comprises an antibody that binds the E glycoprotein; and (c) identifying the immunogenic composition as inducing a neutralizing antibody to a dengue virus in the subject if the biological sample comprises an antibody that binds to the E glycoprotein.

In another embodiment, the present invention provides a method of identifying an immunogenic composition that induces a neutralizing antibody to a dengue virus in a subject, the method comprising: (a) administering an immunogenic composition comprising a dengue virus antigen to a subject in an amount effective to induce antibodies against the dengue virus antigen; (b) contacting a biological sample from the subject with the E glycoprotein of any of claims 1-9; (c) determining if the biological sample comprises an antibody that binds the E glycoprotein; and (d) identifying the immunogenic composition as inducing a neutralizing antibody to a dengue virus in the subject if the biological sample comprises an antibody that binds to the E glycoprotein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1D. DENV4 is neutralized by type-specific antibodies in human primary immune and vaccine sera. A primary DENV4-immune sample 112 (Figure 1 A) and (Figure IB) and a DENV4 NIH monovalent vaccine sample 256.01.36 (Figure 1C) and (Figure ID) were depleted of antibodies binding to DENV4 antigen (Figure 1A) and (Figure 1C) or DENV2 antigen (Figure IB) and (Figure ID). Control depletions were performed using bovine serum albumin as an antigen. Results presented here for antibody depletions are representative of data obtained with three primary DENV4 immune and four DENV4 monovalent vaccine sera (Table 2).

Figures 2A-2B. Properties of DENV4 neutralizing human monoclonal antibodies D4- 126 and D4-131. (Figure 2A) Neutralization of DENV1, 2, 3 and 4. (Figure 2B) Binding to DENV4 whole virions, recombinant E protein ectodomain (rE) and recombinant E protein domain III (rEDIII). Both antibodies bound well to whole DENV4 virions and strongly neutralized DENV4. The MAbs bound poorly to rE or EDIII.

Figures 3A-3B. Neutralization of different DENV4 strains by hMAbs D4-126 and D4-131. U937+DC-SIGN flow based neutralization assays were conducted to compare the ability of D4-126 (Figure 3 A) and D4-131 (Figure 3B) hMAbs to neutralize different laboratory adapted and clinical strains of DENV4. The MAbs neutralized all variants except for the Cambodia 2010 genotype I strain, which was not neutralized by D4-126 and weakly neutralized by D4- 131.

Figures 4A-4B. HMAbs D4-126 and D4-131 bind to epitopes near the hinge region between E protein domains I and II. (Figure 4A) Binding of hMAbs D4- 126, D4-131 to DENV4, DENV3 and rDENV4/3 viruses. (Figure 4B) DENV4 and rDENV4/3

neutralization by hMAbs D4- 126, D4- 131. Both hMAbs failed to bind and neutralize the rDENV4/3 virus.

Figures 5A-5B. DENV4 immune sera block the binding of hMAbs D4-126 and D4- 131 to DENV4. Blockade of binding assays were performed with DENV immune sera and MAbs D4-126 (Figure 5 A), D4-131 (Figure 5B). Blockade assays were performed using 2 primary DENV4 immune sera (002 and 112), a primary DENV2 immune serum (08/90) and a primary DENV3 immune serum (118). The epitope specific response in the plasma was calculated by determining the titer of the plasma that leads to a 50% reduction in MAb binding to DENV4. The primary DENV4 immune sera efficiently blocked the binding of hMAbs D4-126 and D4-131 to DENV4. Primary DENV2 and DENV3 immune sera failed to block binding.

Figures 6A-B. The DENV4 E protein domain I/II hinge region is a target of long- lived plasma cell derived serum neutralizing antibodies in people exposed to DENV4 infections or a vaccine. A U937+DC-SIGN flow based neutralization assay was performed with primary DENV4 infection sera (Figure 6A) or DENV4 monovalent vaccine sera

(Figure 6B) and WT DENV4 and rDENV4/3 viruses. Neut ₅₀ titers were calculated and plotted. Samples that did not block 50% of infection at the highest concentration were assigned a value of 5.

Figure. 7. Alignment of E glycoprotein sequences of dengue virus serotypes and other fiaviviruses.

Figure 8. Alignment of E glycoprotein sequences of dengue virus serotypes and chimeras thereof. DENV4 residues were implanted into the DENV2 E glycoprotein to generate the chimeras.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part on the unexpected discovery that amino acid residues that define an epitope specific for a particular DENV serotype can be transferred into the backbone amino acid sequence of a different DENV serotype to create chimeric molecules. Thus, in one embodiment, the present invention provides a chimeric dengue virus E glycoprotein comprising the amino acid sequence:

The present invention further provides a chimeric dengue virus E glycoprotein comprising the amino acid sequence:

Also provided herein is a chimeric dengue virus E glycoprotein comprising the amino acid sequence:

In addition, the present invention provides a chimeric dengue virus E glycoprotein comprising the amino acid sequence:

Further provided herein is a chimeric dengue virus E glycoprotein comprising the amino acid sequence:

Additionally provided herein is a chimeric dengue virus E glycoprotein comprising the amino acid sequence:

In some embodiments, the chimeric dengue virus E glycoprotein comprising the amino acid sequence identified herein as DENV 2/4 M12 (SEQ ID NO:8), DENV 2/4 M14 (SEQ ID NO:9), and/or DENV 2/4 M complete (SEQ ID NO:10) can comprise additional substitutions (e.g., from the DENV 4 amino acid sequence) into the DENV 2 sequence at residues K64, E71 and/or K247. In some embodiments, the substitutions can be K64S, E71 A and/or K247R. Additional amino acid sequences of this invention are included herein and in the attached SEQUENCE LISTING.

Further provided herein is a chimeric dengue virus E glycoprotein comprising the amino acid sequence:

The present invention also provides a chimeric dengue virus E glycoprotein comprising the amino acid sequence:

A chimeric dengue virus E glycoprotein is also provided herein, comprising the amino acid sequence:

Additional embodiments of the invention include an amino acid sequence:

Some embodiments of the invention can include any portion, fragment, domain, N- terminal, C-terminal of SEQ ID NO: 14. For example, some embodiments of the invention include a fragment of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100,, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3,000 contiguous amino acids, including any values not explicitly recited herein. These fragments can be at either end and/or at any internal location in the amino acid sequence (e.g., residues 1-50 and/or residues 75-100).

Some embodiments of the invention can include any portion, fragment, domain, N- terminal, C-terminal of SEQ ID NO: 15. For example, some embodiments of the invention include a fragment of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200,

1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3,000 contiguous amino acids, including any values not explicitly recited herein. These fragments can be at either end and/or at any internal location in the amino acid sequence (e.g., residues 1-50 and/or residues 75-100).

Additional embodiments of the invention include a chimeric dengue virus E glycoprotein comprising the amino acid sequence:

Additional embodiments of the invention include a chimeric dengue virus E l co rotein com risin the amino acid se uence:

Additional embodiments of the invention include a chimeric dengue virus E glycoprotein comprising the amino acid sequence:

Additional embodiments of the invention include a chimeric dengue virus E glycoprotein comprising the amino acid sequence:

Figure 8 is an alignment of the amino acid sequences of DENV2, DENV4 and the chimeras of this invention, showing the specific residues that are substituted in each amino acid sequence of the respective chimeras. It is also contemplated that embodiments of this invention can include any fragment of the amino acid sequence of the chimeric E glycoprotein sequences provided herein. For example, some embodiments of the invention can include a fragment of any 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300 or 350 contiguous amino acids, including any values not explicitly recited herein. These fragments can be at either end and/or at any internal location in the amino acid sequence (e.g., residues 1-50 and/or residues 75-100).

The present invention also provides a nucleic acid molecule encoding the chimeric dengue virus E glycoprotein of this invention, a vector comprising the nucleic acid molecule of this invention, a flavivirus particle comprising the chimeric dengue virus E glycoprotein of this invention and/or the nucleic acid molecule of this invention, a virus like particle (VLP) comprising the chimeric dengue virus E glycoprotein of this invention, and a composition comprising the chimeric dengue virus E glycoprotein of this invention and/or the nucleic acid molecule of this invention, the vector of this invention, the flavivirus particle of this invention and/or the VLP of this invention, in a pharmaceutically acceptable carrier.

Further provided herein is a method of producing an immune response to a dengue virus in a subject, the method comprising administering to the subject an effective amount of the chimeric dengue virus E glycoprotein of this invention, the nucleic acid molecule of this invention, the vector of this invention, the flavivirus particle of this invention, the VLP of this invention, the composition of this invention, and any combination thereof.

Also provided herein is a method of treating a dengue virus infection in a subject in need thereof, the method comprising administering to the subject an effective amount of the chimeric dengue virus E glycoprotein of this invention, the nucleic acid molecule of this invention, the vector of this invention, the flavivirus particle of this invention, the VLP of this invention, the composition of this invention, and any combination thereof.

In a further embodiment, the present invention provides a method of preventing a dengue virus infection in a subject, the method comprising administering to the subject an effective amount of the chimeric dengue virus E glycoprotein of this invention, the nucleic acid molecule of this invention, the vector of this invention, the flavivirus particle of this invention, the VLP of this invention, the composition of this invention, and any combination thereof.

Additionally provided herein is a method of protecting a subject from the effects of dengue virus infection, the method comprising administering to the subject an effective amount of the chimeric dengue virus E glycoprotein of this invention, the nucleic acid molecule of this invention, the vector of this invention, the flavivirus particle of this invention, the VLP of this invention, the composition of this invention, and any combination thereof.

A method is also provided herein of detecting a neutralizing antibody to a dengue virus, the method comprising administering to the subject an effective amount of the chimeric dengue virus E glycoprotein of this invention, the nucleic acid molecule of this invention, the vector of this invention, the flavivirus particle of this invention, the VLP of this invention, the composition of this invention, and any combination thereof.

In addition, the present invention provides a method of identifying a neutralizing antibody to a dengue virus, comprising: (a) contacting an antibody with the E glycoprotein of this invention; and (b) determining if the antibody binds to the E glycoprotein, wherein binding by the antibody to the E glycoprotein identifies the antibody as a neutralizing antibody to a dengue virus.

Furthermore the present invention provides a method of identifying an immunogenic composition that induces a neutralizing antibody to a dengue virus in a subject, the method comprising: (a) contacting a biological sample from a subject that has been administered the immunogenic composition with the E glycoprotein of this invention; (b) determining if the biological sample comprises an antibody that binds the E glycoprotein; and (c) identifying the immunogenic composition as inducing a neutralizing antibody to a dengue virus in the subject if the biological sample comprises an antibody that binds to the E glycoprotein.

In another embodiment, the present invention provides a method of identifying an immunogenic composition that induces a neutralizing antibody to a dengue virus in a subject, the method comprising: (a) administering an immunogenic composition comprising a dengue virus antigen to a subject in an amount effective to induce antibodies against the dengue virus antigen; (b) contacting a biological sample from the subject with the E glycoprotein of the invention; (c) determining if the biological sample comprises an antibody that binds the E glycoprotein; and (d) identifying the immunogenic composition as inducing a neutralizing antibody to a dengue virus in the subject if the biological sample comprises an antibody that binds to the E glycoprotein.

In some embodiments, the flavivirus E glycoprotein backbone can be from any flavivirus, including but not limited to, yellow fever virus (YFV), Japanese encephalitis virus (JEV) or West Nile virus (WNV). The present invention also provides a flavivirus particle or virus like particle (VLP) comprising the chimeric dengue virus E glycoprotein or chimeric flavivirus E glycoprotein of this invention.

In addition, the present invention provides an isolated nucleic acid encoding the chimeric dengue virus E glycoprotein or the chimeric flavivirus E glycoprotein of this invention, as well as an isolated nucleic acid encoding the isolated dengue virus epitope of this invention, an isolated nucleic acid encoding the polypeptide of this invention, an isolated nucleic acid encoding the flavivirus particle, VLP or viral coat of the chimeric flavivirus of this invention.

Further provided herein is a composition comprising the isolated dengue virus epitope this invention, the polypeptide of this invention, the chimeric VLP of this invention, the chimeric dengue virus E glycoprotein or chimeric flavivirus E glycoprotein of this invention, the flavivirus particle or VLP of this invention, the nucleic acid of this invention and any combination thereof, in a pharmaceutically acceptable carrier.

The term "dengue virus E protein domain I and domain II hinge region" and similar terms would be understood in the art to include the three-dimensional interface between domain I and II in the dengue virus E glycoprotein and, optionally, the adjacent amino acid residues. In addition, those skilled in the art will appreciate that certain amino acid residues in the hinge region may facilitate proper folding and presentation of the epitope, even if they do not form part of the epitope per se. In representative embodiments, the dengue virus E protein domain I and domain II hinge region comprises, consists essentially of, or consists of amino acid positions 47-59, 124-133, 199-222 and/or 206-228 of the E protein of dengue virus serotype 3 (DENV3; e.g., GenBank ^® Database Accession No. JQ411814) or the corresponding positions of the E protein of other dengue viruses {e.g., dengue virus serotype 1 (DENV1 ; e.g., GenBank ^® Database Accession No. U88535), serotype 2 (DENV2; e.g.,

GenBank ^® Database Accession No. NC 001474) or serotype 4 (DENV4); full E glycoprotein sequences are shown in Figures 7 and 8 and corresponding amino acid numbers are provided in Table 7).

The term "at least a portion of a dengue virus E protein domain III" and similar terms refer to those portions of E protein domain III that form part of the epitope as well as those amino acid residues that facilitate proper folding and presentation of the epitope, even if they do not form part of the epitope per se. In representative embodiments, the dengue virus E protein domain III comprises, consists essentially of, or consists of amino acid positions 305- 308, 323-325, 359-362 and/or 389-390 of the E protein of dengue virus serotype 3 or the corresponding positions of the E protein of other dengue viruses (e.g., dengue virus serotypes 1 (DENV1), 2 (DENV2) or DENV4; full E glycoprotein sequences are shown in Figure 7 and corresponding amino acid numbers are provided in Table 7). Likewise, in some embodiments, amino acid positions of the E protein of dengue serotype 2 (DENV2) were replaced with amino acid residues of dengue serotype 4 (DENV4) (Figure 8).

In some embodiments, production of the chimera of this invention can be carried out by introducing some (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) or all of the amino acid

substitutions identified in Table 7. Not every amino acid identified in Table 7 is required to be substituted to produce a chimeric protein of this invention. For example, in some embodiments further substitutions and/or omission of substitutions of about 1 , 2, 3, 4 or 5 amino acids at either end of the contiguous amino acid sequences identified in Table 7 as the respective epitope regions can be included in production of a chimera of this invention. The number of substitutions necessary to produce the desired conformational epitope can be readily determined by one of ordinary skill in the art according to the teachings herein and according to protocols well known in the art.

In some embodiments, the present invention provides a chimeric flavivirus E glycoprotein in which amino acid substitutions are made to introduce a dengue virus epitope into a flavivirus E glycoprotein from a flavivirus that is not a dengue virus. Nonlimiting examples of flaviviruses that can be used include yellow fever virus (YFV) (e.g., GenBank® Database Accession No. JX503529) Japanese encephalitis virus (JEV) (e.g., GenBank ^® Database Accession No. U14163), West Nile virus (WNV) (e.g., GenBank® Database Accession No. DQ211652) and any other flavivirus now known or later identified. Thus, the present invention provides, for example a chimeric flavivirus E glycoprotein comprising a DENV1, DENV2, DENV3, or DENV4 domain I and domain II hinge region in a YFV, JEV or WNV E glycoprotein backbone. Also provided is a chimeric dengue virus E glycoprotein comprising a DENV1, DENV2, DENV3 or DENV4 domain I and domain II hinge region as well as a domain III region in a YFV, JEV or WNV E glycoprotein backbone.

In other embodiments, "at least a portion of a dengue virus E protein domain III" (and similar terms) comprises, consists essentially of, or consists of at least about 6, 8, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids, optionally contiguous amino acids, and/or less than about 12, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids, optionally contiguous amino acids, including any combination of the foregoing as long as the lower limit is less than the upper limit. In representative embodiments, the peptide spacer comprises, consists of, or consists essentially of about 1 , 2, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 1 1 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, 20 or less, 25 or less, 30 or less, 35 or less, 40 or less, 45 or less, 50 or less, 55 or less, 60 or less, 70 or less, 80 or less, 90 or less or 100 or less amino acids. In some embodiments, the peptide spacer comprises, consists of, or consists essentially of about 1 to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90 or 100 amino acids. In some embodiments, the peptide spacer comprises, consists of, or consists essentially of about 3 to about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90 or 100 amino acids. In some embodiments, the peptide spacer comprises, consists of, or consists essentially of about 4 to about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90 or 100 amino acids. In some embodiments, the peptide spacer comprises, consists of, or consists essentially of about 5 to about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90 or 100 amino acids. In some embodiments, the peptide spacer comprises, consists of, or consists essentially of about 10 to about 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90 or 100 amino acids. In some embodiments, the peptide spacer comprises, consists of, or consists essentially of about 15 to about 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90 or 100 amino acids. In some embodiments, the peptide spacer comprises, consists of, or consists essentially of about 20 to about 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90 or 100 amino acids.

In some embodiments, the spacer brings the E protein domain Vll hinge region and the domain III region involved in the quaternary epitope about 10 or less, 15 or less, 20 or less, 25 or less, 30 or less, 35 or less, 40 or less, 45 or less, 50 or less, 60 or less or 70 or less angstroms apart. In some embodiments, the spacer brings the E protein domain Vll hinge region and the domain III region involved in the quaternary epitope about 10 to 20, 25, 30, 35, 40, 45, 50, 60 or 70 angstroms apart. In some embodiments, the spacer brings the E protein domain I/II hinge region and the domain III region involved in the quaternary epitope about 15 to 25, 30, 35, 40, 45, 50, 60 or 70 angstroms apart. In some embodiments, the spacer brings the E protein domain I/II hinge region and the domain III region involved in the quaternary epitope about 20 to 30, 35, 40, 45, 50, 60 or 70 angstroms apart. In some embodiments, the spacer brings the E protein domain I/II hinge region and the domain III region involved in the quaternary epitope about 25 to 35, 40, 45, 50, 60 or 70 angstroms apart. In embodiments, the spacer brings the E protein domain I/II hinge region and the domain III region involved in the quaternary epitope about 30 to 40, 45, 50, 60 or 70 angstroms apart. In some embodiments, the spacer brings the E protein domain I/II hinge region and the domain III region involved in the quaternary epitope about 35 to 45, 50, 60 or 70 angstroms apart.

The peptide spacer can be derived in whole or in part from a native E protein, or can be partially or wholly synthetic.

In some embodiments, the peptide spacer forms a secondary structure, e.g., a beta- sheet, beta-barrel and/or an alpha helical structure. In some embodiments, the peptide spacer comprises one or more disulfide bonds (e.g. , cystine residues).

It is known in the art that many attempts to produce dengue virus vaccines result in the production of non-neutralizing antibodies, which may increase the likelihood of pathology upon subsequent exposure to natural infection or vaccine. Another approach to provide an engineered epitope is to deliver all or a portion of the dengue virus E protein incorporated into another flavivirus particle or VLP. In representative embodiments, the heterologous flavivirus can be West Nile virus or Yellow Fever virus. Portions of the E protein can be grafted into the E protein of the heterologous flavivirus backbone, e.g., to reduce the generation of non-neutralizing dengue virus antibodies to non-neutralizing epitopes present in the dengue virus E protein and/or other dengue virus structural proteins.

Thus, a chimeric flavivirus or chimeric flavivirus VLP can present the quaternary dengue virus epitope in proper conformation while reducing the generation of non- neutralizing antibodies to other portions of the dengue virus E protein and/or other structural proteins that are not presented in the chimeric flavivirus or flavivirus VLP.

Thus, as another aspect, the invention provides a chimeric flavivirus particle or chimeric flavivirus VLP comprising a chimeric flavivirus E protein, the chimeric flavivirus E protein comprising a dengue virus E protein domain I and domain II hinge region and at least a portion of the dengue virus E protein domain III. In embodiments of the invention, the dengue virus E protein region(s) are substituted for the corresponding region(s) of the heterologous flavivirus E protein. In embodiments, amino acid sequences from the dengue virus prM protein and/or the dengue virus C protein are not incorporated into the chimeric flavivirus or chimeric flavivirus VLP.

In some embodiments of the invention the individual and conformational epitopes of the flavivirus E glycoprotein or dengue virus E glycoprotein can be presented on a synthetic backbone or support structure so that the epitopes within the synthetic backbone or support structure mimic the conformation and arrangement of the epitopes within the structure of the E glycoprotein, virus particle or VLP. In still further embodiments of the invention, the present invention provides peptide mimitopes (see, Meloen et al. (2000) J. Mol. Recognit. 13, 352-359) that mimic the individual and conformational epitopes of the E glycoproteins of the invention. Mimitopes may be identified using any technique known in the art, such as by surface stimulation, random peptide libraries or phage display libraries, using an antibody or antibodies to the individual and conformational epitopes of the E glycoproteins of the invention.

The invention further provides a nucleic acid molecule (e.g., isolated nucleic acid molecule) encoding a dengue virus epitope or a polypeptide of the invention.

The invention further provides a nucleic acid molecule {e.g., an isolated nucleic acid molecule) encoding a chimeric flavivirus VLP or a viral coat of a chimeric flavivirus particle of the invention.

Also provided are vectors encoding the nucleic acid molecules of the invention.

Also provided are cells comprising the vectors, nucleic acid molecules, dengue virus epitopes, polypeptides, chimeric flavivirus VLPs or chimeric flavivirus particles of the invention.

The invention also provides immunogenic compositions comprising the cells, vectors, nucleic acid molecules, dengue virus epitopes, polypeptides, chimeric flavivirus VLPs or chimeric flavivirus particles of the invention. In some embodiments, the immunogenic composition is monovalent. In some embodiments, the immunogenic composition is multivalent (e.g., tetravalent) for dengue virus serotypes DEN1, DEN2, DEN 3 and/or DEN4.

The invention encompasses methods of producing an immune response to a dengue virus in a subject, the method comprising administering to the subject an effective amount of a dengue virus epitope, a polypeptide, a chimeric flavivirus VLP or chimeric flavivirus particle, nucleic acid, vector, cell or immunogenic composition of the invention.

Further, the present invention can advantageously be practiced to induce an immune response against one, two, three or all four of DEN1, DEN2, DEN3 and DEN4. It is well- known in the art that effective and safe multivalent dengue vaccines have been a challenge to design because of the problem of interference among serotypes. For example, the immune response may be predominantly directed against only some of the target serotypes. Multiple vaccinations are then required to try to achieve a response against all serotypes; however, in the case of dengue virus, this approach can be dangerous because repeated administrations to a subject with pre-existing antibodies can lead to dengue hemorrhagic fever.

A still further aspect of the invention is a method of treating a dengue virus infection, comprising administering to the subject an effective amount of a dengue virus epitope, a polypeptide, a chimeric flavivirus VLP or chimeric flavivirus particle, nucleic acid molecule, vector, cell, or immunogenic composition of the invention.

A still further aspect of the invention is a method of preventing a dengue virus infection, comprising administering to the subject an effective amount of a dengue virus epitope, a polypeptide, a chimeric flavivirus VLP or chimeric flavivirus particle, nucleic acid molecule, vector, cell, or immunogenic composition of the invention.

A still further aspect of the invention is a method of protecting a subject from the effects of dengue virus infection, comprising administering to the subject an effective amount of a dengue virus epitope, a polypeptide, a chimeric flavivirus VLP or chimeric flavivirus particle, nucleic acid, vector, cell, or immunogenic composition of the invention.

The invention can also be practiced to identify antibodies that bind (e.g., specifically bind) to the quaternary dengue virus epitope, e.g., to identify neutralizing antibodies to a dengue virus. For example, the invention can be employed as a diagnostic to qualitatively determine if a vaccine candidate is inducing neutralizing antibodies. In general, due to the abundance of non-neutralizing antibodies induced by many candidate dengue virus vaccines, antibody titers alone without further characterization of the antibody specificity provides incomplete information.

In representative embodiments, the invention provides a method of detecting a neutralizing antibody to a dengue virus, the method comprising the step of determining whether an antibody binds to a dengue virus epitope, a polypeptide, or a chimeric VLP or chimeric flavivirus of the invention, wherein binding by the antibody to the dengue virus epitope, the polypeptide, the chimeric VLP or the chimeric flavivirus indicates that the antibody is a neutralizing antibody to a dengue virus.

In further representative embodiments, the invention provides a method of identifying a neutralizing antibody to a dengue virus, the method comprising: (a) contacting an antibody to a dengue virus epitope, a polypeptide, or a chimeric VLP or chimeric flavivirus of the invention; and (b) determining if the antibody binds to the dengue virus epitope, the polypeptide, the chimeric VLP or the chimeric flavivirus; wherein binding by the antibody to the dengue virus epitope, the polypeptide, the chimeric VLP or the chimeric flavivirus indicates that the antibody is a neutralizing antibody to a dengue virus.

The invention also provides a method of identifying a neutralizing antibody to a dengue virus, the method comprising: (a) contacting an antibody to a dengue virus epitope, a polypeptide, or a chimeric VLP or chimeric flavivirus of the invention; (b) determining if the antibody binds to the dengue virus epitope, the polypeptide, the chimeric VLP or the chimeric flavivirus; and (c) identifying the antibody as a neutralizing antibody to a dengue virus if the antibody binds to the dengue virus epitope, the polypeptide, the chimeric VLP or the chimeric flavivirus.

Still further, the invention provides a method of identifying an immunogenic composition that induces a neutralizing antibody to a dengue virus in a subject, the method comprising the step of determining whether a biological sample obtained from a subject that has been administered the immunogenic composition comprises an antibody that binds to a dengue virus epitope, a polypeptide, or a chimeric VLP or chimeric flavivirus of the invention, wherein if the biological sample comprises an antibody that binds to the dengue virus epitope, the polypeptide, the chimeric VLP or the chimeric flavivirus, it indicates that the immunogenic composition induces a neutralizing antibody to a dengue virus in the subject.

The invention also provides a method of identifying an immunogenic composition that induces a neutralizing antibody to a dengue virus in a subject, the method comprising: (a) contacting a biological sample from a subject that has been administered the

immunogenic composition with a dengue virus epitope, a polypeptide, or a chimeric VLP or chimeric flavivirus of the invention; and (b) determining if the biological sample comprises an antibody that binds to the dengue virus epitope, the polypeptide, the chimeric VLP or the chimeric flavivirus; wherein if the biological sample comprises an antibody that binds to the dengue virus epitope, the polypeptide, the chimeric VLP or the chimeric flavivirus, it indicates that the immunogenic composition induces a neutralizing antibody to a dengue virus in the subject.

In yet another embodiment, the invention provides a method of identifying an immunogenic composition that induces a neutralizing antibody to a dengue virus in a subject, the method comprising: (a) contacting a biological sample from a subject that has been administered the immunogenic composition with a dengue virus epitope, a polypeptide, or a chimeric VLP or chimeric flavivirus of the invention; (b) determining if the biological sample comprises an antibody that binds to the dengue virus epitope, the polypeptide, the chimeric VLP or the chimeric flavivirus; and (c) identifying the immunogenic composition as inducing a neutralizing antibody to a dengue virus in the subject if the biological sample comprises an antibody that binds to the dengue virus epitope, the polypeptide, the chimeric VLP or the chimeric flavivirus.

In other representative embodiments, the invention provides a method of identifying an immunogenic composition that induces a neutralizing antibody to a dengue virus in a subject, the method comprising: (a) administering an immunogenic composition comprising a dengue virus antigen to a subject in an amount effective to induce antibodies against the dengue virus antigen; (b) contacting a biological sample from the subject with a dengue virus epitope, a polypeptide, or a chimeric VLP or chimeric flavivirus of the invention; (c) determining if the biological sample comprises an antibody that binds to the dengue virus epitope, the polypeptide, the chimeric VLP or chimeric flavivirus; and (d) identifying the immunogenic composition as inducing a neutralizing antibody to a dengue virus in the subject if the biological sample comprises an antibody that binds to the dengue virus epitope, the polypeptide, the chimeric VLP or the chimeric flavivirus.

There are four serotypes of dengue virus (DEN1 , DEN2, DEN3 and DEN4). Within each serotype there are a number of different strains or genotypes. The dengue virus antigens and epitopes of the invention can be derived from any dengue virus, including all serotypes, strains and genotypes, now known or later identified.

In embodiments of the invention, the dengue virus is UNC1017 strain (DEN1), West Pacific 74 strain (DEN1), S16803 strain (DEN2), UNC2005 strain (DEN2), UNC3001 strain (DEN3), UNC3043 (DEN3 strain 059.AP-2 from Philippines, 1984), UNC3009 strain (DEN3, D2863, Sri Lanka 1989), UNC3066 (DEN3, strain 1342 from Puerto Rico 1977), CH53489 strain (DEN3), UNC4019 strain (DEN4), or TVP-360 (DEN4).

In embodiments of the invention, an "immunogenically active fragment" of a dengue virus polypeptide (e.g., the E protein, or the EDI, ED II or EDIII domain) comprises, consists essentially of or consists of at least about 6, 8, 10, 12, 15, 20, 30, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450 or more amino acids, optionally contiguous amino acids, and/or less than about 495, 475, 450, 425, 400, 350, 300, 250, 200, 150, 100, 75 or 50 amino acids, optionally contiguous amino acids, including any combination of the foregoing as long as the lower limit is less than the upper limit, and the "immunogenically active fragment" induces an immune response (e.g., IgG and/or IgA that react with the native antigen), optionally a protective immune response, against dengue virus in a host and induces the production of antibodies that specifically bind to the quaternary dengue virus epitope newly identified by the inventors.

The term "epitope" as used herein means a specific combination of amino acid residues in an amino acid sequence that, when present in the proper conformation, provides a reactive site for an antibody (e.g., B cell epitope) or T cell receptor (e.g., T cell epitope).

Portions of a given polypeptide that include a B-cell epitope can be identified using any number of epitope mapping techniques that are known in the art. (See, e.g. , Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed., 1996, Humana Press, Totowa, N.J.). For example, linear epitopes can be determined by, e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871 ; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81 :3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715.

Similarly, conformational epitopes can be readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance. Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using, e.g., the Omiga version 1.0 software program available from the Oxford Molecular Group. This computer program employs the Hopp/Woods method (Hopp et al., Proc. Natl. Acad. Sci USA (1981) 78:3824-3828) for determining antigenicity profiles and the Kyte-Doolittle technique (Kyte et al., J. Mol. Biol. (1982) 157:105-132) for hydropathy plots.

Generally, T-cell epitopes that are involved in stimulating the cellular arm of a subject's immune system are short peptides of about 8-25 amino acids. A common way to identify T-cell epitopes is to use overlapping synthetic peptides and analyze pools of these peptides, or the individual ones, that are recognized by T cells from animals that are immune to the antigen of interest, using, for example, an enzyme-linked immunospot assay

(ELISPOT). These overlapping peptides can also be used in other assays such as the stimulation of cytokine release or secretion, or evaluated by constructing major

histocompatibility (MHC) tetramers containing the peptide. Such immunogenically active fragments can also be identified based on their ability to stimulate lymphocyte proliferation in response to stimulation by various fragments from the antigen of interest.

The present invention can be practiced for prophylactic, therapeutic and/or diagnostic purposes. In addition, the invention can be practiced to produce antibodies for any purpose, such as diagnostic or research purposes, or for passive immunization by transfer to another subject.

The present invention further provides a kit comprising one or more compositions of this invention. It would be well understood by one of ordinary skill in the art that the kit of this invention can comprise one or more containers and/or receptacles to hold the reagents (e.g., antibodies, antigens, nucleic acids) of the kit, along with appropriate buffers and/or diluents and/or other solutions and directions for using the kit, as would be well known in the art. Such kits can further comprise adjuvants and/or other immunostimulatory or immunomodulating agents, as are well known in the art.

The compositions and kits of the present invention can also include other medicinal agents, pharmaceutical agents, carriers, diluents, immunostimulatory cytokines, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art.

Administration to a subject can be by any route known in the art. As non-limiting examples, the route of administration can be by inhalation (e.g., oral and/or nasal inhalation), oral, buccal (e.g., sublingual), rectal, vaginal, topical (including administration to the airways), intraocular, transdermal, by parenteral (e.g., intramuscular [e.g., administration to skeletal muscle], intravenous, intra-arterial, intraperitoneal and the like), subcutaneous (including administration into the footpad), intradermal, intrapleural, intracerebral, and/or intrathecal routes.

The epitopes, polypeptides, VLPs and viral vectors of the invention can be delivered per se or by delivering a nucleic acid (e.g. , DNA) that encodes the same.

Immunomodulatory compounds, such as immunomodulatory chemokines and cytokines (preferably, CTL inductive cytokines) can be administered concurrently to a subject.

Cytokines may be administered by any method known in the art. Exogenous cytokines may be administered to the subject, or alternatively, a nucleic acid encoding a cytokine may be delivered to the subject using a suitable vector, and the cytokine produced in vivo. In particular embodiments, a viral adjuvant expresses the cytokine.

In embodiments of the invention, multiple dosages (e.g., two, three or more) of a composition of the invention can be administered without detectable pathogenicity (e.g., Dengue Shock Syndrorne/Dengue Hemorrhagic Fever).

In embodiments of the invention, the multivalent vaccines of the invention do not result in immune interference, e.g., a balanced immune response is induced against all antigens presented. In embodiments of the invention, the balanced response results in protective immunity against DEN1, DEN2, DEN3 and DEN4.

In embodiments of the invention, the multivalent vaccine can be administered to a subject that has anti-dengue maternal antibodies present.

It should be appreciated that the invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

As used herein, "a," "an" or "the" can mean one or more than one. For example, "a" cell can mean a single cell or a multiplicity of cells.

Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of

combinations when interpreted in the alternative ("or").

The term "about," as used herein when referring to a measurable value such as an amount of dose (e.g., an amount of a fatty acid) and the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

As used herein, the transitional phrase "consisting essentially of means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. See, In re Her z, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus, the term "consisting essentially of when used in a claim of this invention is not intended to be interpreted to be equivalent to "comprising."

As used herein, the terms "nucleic acid" or "nucleic acid molecule" encompass both RNA and DNA, including cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA and chimeras of RNA and DNA. The nucleic acid molecule may be double-stranded or single-stranded. The nucleic acid molecule may be synthesized using nucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acid molecules that have altered base-pairing abilities or increased resistance to nucleases.

As used herein, the term "polypeptide" encompasses both peptides and proteins (including fusion proteins), unless indicated otherwise.

A "fusion protein" is a polypeptide produced when two heterologous nucleotide sequences or fragments thereof coding for two (or more) different polypeptides not found fused together in nature are fused together in the correct translational reading frame. A fusion protein can also comprise two or more heterologous amino acid sequences connected or linked by a spacer or linker amino acid sequence.

A "recombinant" nucleic acid, polynucleotide or nucleotide sequence is one produced by genetic engineering techniques.

A "recombinant" polypeptide is produced from a recombinant nucleic acid, polypeptide or nucleotide sequence.

As used herein, an "isolated" polynucleotide (e.g., an "isolated nucleic acid" or an "isolated nucleotide sequence") means a polynucleotide at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polynucleotide. Optionally, but not necessarily, the "isolated"

polynucleotide is present at a greater concentration (i.e., is enriched) as compared with the starting material (e.g., at least about a two-fold, three- fold, four- fold, ten-fold, twenty- fold, fifty-fold, one-hundred-fold, five-hundred-fold, one thousand-fold, ten thousand-fold or greater concentration). In representative embodiments, the isolated polynucleotide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more pure.

An "isolated" polypeptide means a polypeptide that is at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide. Optionally, but not necessarily, the

"isolated" polypeptide is present at a greater concentration (i.e., is enriched) as compared with the starting material (e.g., at least about a two-fold, three-fold, four-fold, ten-fold, twenty-fold, fifty-fold, one-hundred-fold, five-hundred-fold, one thousand-fold, ten thousandfold or greater concentration). In representative embodiments, the isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more pure.

Furthermore, an "isolated" cell is a cell that has been partially or completely separated from other components with which it is normally associated in nature. For example, an isolated cell can be a cell in culture medium and/or a cell in a pharmaceutically acceptable carrier.

The terms "immunogen" and "antigen" are used interchangeably herein and mean any compound (including polypeptides) to which a cellular and/or humoral immune response can be directed. In particular embodiments, an immunogen or antigen can induce a protective immune response against the effects of dengue virus infection. "Effective amount" as used herein refers to an amount of a vector, nucleic acid, epitope, polypeptide, cell, composition or formulation of the invention that is sufficient to produce a desired effect, which can be a therapeutic and/or beneficial effect. The effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular agent administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art. As appropriate, an "effective amount" in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation.

The term "immunogenic amount" or "effective immunizing dose," as used herein, unless otherwise indicated, means an amount or dose sufficient to induce an immune response (which can optionally be a protective response) in the treated subject that is greater than the inherent immunity of non-immunized subjects. An immunogenic amount or effective immunizing dose in any particular context can be routinely determined using methods known in the art.

The terms "vaccine," "vaccination" and "immunization" are well-understood in the art, and are used interchangeably herein. For example, the terms vaccine, vaccination or immunization can be understood to be a process or composition that increases a subject's immune reaction to an immunogen (e.g., by providing an active immune response), and therefore its ability to resist, overcome and/or recover from infection (i.e., a protective immune response).

By the term "treat," "treating" or "treatment of (and grammatical variations thereof) it is meant that the severity of the subject's condition is reduced, at least partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or disorder. In representative embodiments, the term "treat,", "treating" or "treatment of (and grammatical variations thereof) refer to a reduction in the severity of viremia and/or a delay in the progression of viremia, with or without other signs of clinical disease.

A "treatment effective" amount as used herein is an amount that is sufficient to treat (as defined herein) the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

The term "prevent," "preventing" or "prevention of (and grammatical variations thereof) refer to prevention and/or delay of the onset and/or progression of a disease, disorder and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset and/or progression of the disease, disorder and/or clinical symptom(s) relative to what would occur in the absence of the methods of the invention. In representative embodiments, the term "prevent," "preventing" or "prevention of (and grammatical variations thereof) refer to prevention and/or delay of the onset and/or progression of viremia in the subject, with or without other signs of clinical disease. The prevention can be complete, e.g., the total absence of the disease, disorder and/or clinical symptom(s). The prevention can also be partial, such that the occurrence of the disease, disorder and/or clinical symptom(s) in the subject and/or the severity of onset and/or the progression is less than what would occur in the absence of the present invention.

A "prevention effective" amount as used herein is an amount that is sufficient to prevent (as defined herein) the disease, disorder and/or clinical symptom in the subject.

Those skilled in the art will appreciate that the level of prevention need not be complete, as long as some benefit is provided to the subject.

The efficacy of treating and/or preventing dengue virus infection by the methods of the present invention can be determined by detecting a clinical improvement as indicated by a change in the subject's symptoms and/or clinical parameters (e.g., viremia), as would be well known to one of skill in the art.

Unless indicated otherwise, the terms "protect," "protecting," "protection" and

"protective" (and grammatical variations thereof) encompass both methods of preventing and treating dengue virus infection in a subject, whether against one or multiple strains, genotypes or serotypes of dengue virus.

The terms "protective" immune response or "protective" immunity as used herein indicates that the immune response confers some benefit to the subject in that it prevents or reduces the incidence and/or severity and/or duration of disease or any other manifestation of infection. For example, in representative embodiments, a protective immune response or protective immunity results in reduced viremia, whether or not accompanied by clinical disease. Alternatively, a protective immune response or protective immunity may be useful in the therapeutic treatment of existing disease.

An "active immune response" or "active immunity" is characterized by "participation of host tissues and cells after an encounter with the immunogen. It involves differentiation and proliferation of immunocompetent cells in lymphoreticular tissues, which lead to synthesis of antibody or the development of cell-mediated reactivity, or both." Herbert B.

Herscowitz, Immunophysiology: Cell Function and Cellular Interactions in Antibody

Formation, in IMMUNOLOGY: BASIC PROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively stated, an active immune response is mounted by the host after exposure to immunogens by infection or by vaccination. Active immunity can be contrasted with passive immunity, which is acquired through the "transfer of preformed substances (antibody, transfer factor, thymic graft, interleukin-2) from an actively immunized host to a non-immune host." Id.

A "subject" of the invention includes any animal susceptible to dengue virus infection. Such a subject is generally a mammalian subject {e.g., a laboratory animal such as a rat, mouse, guinea pig, rabbit, primates, etc.), a farm or commercial animal {e.g., a cow, horse, goat, donkey, sheep, etc.), or a domestic animal {e.g., cat, dog, ferret, etc.). In particular embodiments, the subject is a primate subject, a non-human primate subject {e.g., a chimpanzee, baboon, monkey, gorilla, etc.) or a human. Subjects of the invention can be a subject known or believed to be at risk of infection by dengue virus. Alternatively, a subject according to the invention can also include a subject not previously known or suspected to be infected by dengue virus or in need of treatment for dengue virus infection.

Subjects may be treated for any purpose, such as for eliciting a protective immune response or for eliciting the production of antibodies in that subject, which antibodies can be collected and used for other purposes such as research or diagnostic purposes or for administering to other subjects to produce passive immunity therein, etc.

Subjects include males and/or females of any age, including neonates, juvenile, mature and geriatric subjects. With respect to human subjects, in representative embodiments, the subject can be an infant {e.g., less than about 12 months, 10 months, 9 months, 8 months, 7 months, 6 months, or younger), a toddler {e.g., at least about 12, 18 or 24 months and/or less than about 36, 30 or 24 months), or a child {e.g., at least about 1 , 2, 3, 4 or 5 years of age and/or less than about 14, 12, 10, 8, 7, 6, 5, or 4 years of age). In embodiments of the invention, the subject is a human subject that is from about 0 to 3, 4, 5, 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 3 to 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 6 to 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 9 to 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 12 to 18, 24, 36, 48 or 60 months of age, from about 18 to 24, 30, 36, 48 or 60 months of age, or from about 24 to 30, 36, 48 or 60 months of age.

In embodiments of the invention, the subject has maternal antibodies to dengue virus.

A "subject in need" of the methods of the invention (e.g., a subject in need thereof) can be a subject known to be, or suspected of being, infected with, or at risk of being infected with, dengue virus. Pharmaceutical formulations (e.g., immunogenic formulation) comprising the dengue virus epitopes, polypeptides, chimeric flavivirus VLPs or chimeric flavivirus particles, nucleic acids, vectors, cells or compositions of the invention and a pharmaceutically acceptable carrier are also provided, and can be formulated for administration in a

pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (latest edition). In the manufacture of a pharmaceutical composition according to embodiments of the present invention, the composition of the invention is typically admixed with, inter alia, a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant a carrier that is compatible with other ingredients in the pharmaceutical composition and that is not harmful or deleterious to the subject. The carrier may be a solid or a liquid, or both, and is preferably formulated with the composition of the invention as a unit-dose formulation, for example, a tablet, which may contain from about 0.01 or 0.5% to about 95% or 99% by weight of the composition. The pharmaceutical compositions are prepared by any of the well-known techniques of pharmacy including, but not limited to, admixing the components, optionally including one or more accessory ingredients. In certain embodiments, the pharmaceutically acceptable carrier is sterile and would be deemed suitable for administration into human subjects according to regulatory guidelines for pharmaceutical compositions comprising the carrier.

Furthermore, a "pharmaceutically acceptable" component such as a salt, carrier, excipient or diluent of a composition according to the present invention is a component that (i) is compatible with the other ingredients of the composition in that it can be combined with the compositions of the present invention without rendering the composition unsuitable for its intended purpose, and (ii) is suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are "undue" when their risk outweighs the benefit provided by the composition. Non-limiting examples of pharmaceutically acceptable components include any of the standard

pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsion, microemulsions and various types of wetting agents.

In some embodiments, the compositions of the invention can further comprise one or more than one adjuvant. The adjuvants of the present invention can be in the form of an amino acid sequence, and/or in the form or a nucleic acid encoding an adjuvant. When in the form of a nucleic acid, the adjuvant can be a component of a nucleic acid encoding the polypeptide(s) or fragment(s) or epitope(s) and/or a separate component of the composition comprising the nucleic acid encoding the polypeptide(s) or fragment(s) or epitope(s) of the invention. According to the present invention, the adjuvant can also be an amino acid sequence that is a peptide, a protein fragment or a whole protein that functions as an adjuvant, and/or the adjuvant can be a nucleic acid encoding a peptide, protein fragment or whole protein that functions as an adjuvant. As used herein, "adjuvant" describes a substance, which can be any immunomodulating substance capable of being combined with a composition of the invention to enhance, improve or otherwise modulate an immune response in a subject.

In further embodiments, the adjuvant can be, but is not limited to, an

immunostimulatory cytokine (including, but not limited to, GM/CSF, interleukin-2, interleukin-12, interferon-gamma, interleukin-4, tumor necrosis factor-alpha, interleukin-1, hematopoietic factor flt3L, CD40L, B7.1 co-stimulatory molecules and B7.2 co-stimulatory molecules), SYNTEX adjuvant formulation 1 (SAF-1) composed of 5 percent (wt/vol) squalene (DASF, Parsippany, N.J.), 2.5 percent Pluronic, L121 polymer (Aldrich Chemical, Milwaukee), and 0.2 percent polysorbate (Tween 80, Sigma) in phosphate-buffered saline. Suitable adjuvants also include an aluminum salt such as aluminum hydroxide gel (alum), aluminum phosphate, or algannmulin, but may also be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatized polysaccharides, or polyphosphazenes.

Other adjuvants are well known in the art and include without limitation MF 59, LT- K63, LT-R72 (Pal et al., Vaccine 24(6):766-75 (2005)), QS-21, Freund's adjuvant (complete and incomplete), aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr- MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor- MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(l '-2'-dipalmitoyl-sn - glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE) and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trealose dimycolate and cell wall skeleton (MPL+TDM+CWS) in 2% squalene/Tween 80 emulsion.

Additional adjuvants can include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl. lipid A (3D-MPL) together with an aluminum salt. An enhanced adjuvant system involves the combination of a monophosphoryl lipid A and a saponin derivative, particularly the combination of QS21 and 3D-MPL as disclosed in PCT publication number WO 94/00153, or a less reacto genie composition where the QS21 is quenched with cholesterol as disclosed in PCT publication number WO

96/33739. A particularly potent adjuvant formulation involving QS21 3D-MPL & tocopherol in an oil in water emulsion is described in PCT publication number WO 95/17210. In addition, the nucleic acid compositions of the invention can include an adjuvant by comprising a nucleotide sequence encoding the antigen and a nucleotide sequence that provides an adjuvant function, such as CpG sequences. Such CpG sequences, or motifs, are well known in the art.

An adjuvant for use with the present invention, such as, for example, an

immunostimulatory cytokine, can be administered before, concurrent with, and/or within a few hours, several hours, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10 days before and/or after the administration of a composition of the invention to a subject.

Furthermore, any combination of adjuvants, such as immunostimulatory cytokines, can be co-administered to the subject before, after and/or concurrent with the administration of an immunogenic composition of the invention. For example, combinations of

immunostimulatory cytokines, can consist of two or more immunostimulatory cytokines, such as GM/CSF, interleukin-2, interleukin-12, interferon-gamma, interleukin-4, tumor necrosis factor-alpha, interleukin-1, hematopoietic factor flt3L, CD40L, B7.1 co-stimulatory molecules and B7.2 co-stimulatory molecules. The effectiveness of an adjuvant or combination of adjuvants can be determined by measuring the immune response produced in response to administration of a composition of this invention to a subject with and without the adjuvant or combination of adjuvants, using standard procedures, as described herein and as known in the art.

In embodiments of the invention, the adjuvant comprises an alphavirus adjuvant as described, for example in U.S. 7,862,829, which is hereby incorporated by reference in its entirety.

Boosting dosages can further be administered over a time course of days, weeks, months or years. In chronic infection, initial high doses followed by boosting doses may be advantageous.

The pharmaceutical formulations of the invention can optionally comprise other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, diluents, salts, tonicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

For injection, the carrier will typically be a liquid. For other methods of

administration, the carrier may be either solid or liquid. For inhalation administration, the carrier will be respirable, and is typically in a solid or liquid particulate form. The compositions of the invention can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (9 ^th Ed. 1995). In the manufacture of a pharmaceutical composition according to the invention, the VLPs are typically admixed with, inter alia, an acceptable carrier. The carrier can be a solid or a liquid, or both, and is optionally formulated with the compound as a unit-dose formulation, for example, a tablet. A variety of

pharmaceutically acceptable aqueous carriers can be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, pyrogen-free water, pyrogen-free phosphate-buffered saline solution, bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, N. J.), and the like. These compositions can be sterilized by conventional techniques. The formulations of the invention can be prepared by any of the well-known techniques of pharmacy.

The pharmaceutical formulations can be packaged for use as is, or lyophilized, the lyophilized preparation generally being combined with a sterile aqueous solution prior to administration. The compositions can further be packaged in unit/dose or multi-dose containers, for example, in sealed ampoules and vials.

The pharmaceutical formulations can be formulated for administration by any method known in the art according to conventional techniques of pharmacy. For example, the compositions can be formulated to be administered intranasally, by inhalation (e.g., oral inhalation), orally, buccally (e.g., sublingually), rectally, vaginally, topically, intrathecally, intraocularly, transdermally, by parenteral administration (e.g., intramuscular [e.g., skeletal muscle], intravenous, subcutaneous, intradermal, intrapleural, intracerebral and intra-arterial, intrathecal), or topically (e.g., to both skin and mucosal surfaces, including airway surfaces).

For intranasal or inhalation administration, the pharmaceutical formulation can be formulated as an aerosol (this term including both liquid and dry powder aerosols). For example, the pharmaceutical formulation can be provided in a finely divided form along with a surfactant and propellant. Typical percentages of the composition are 0.01-20% by weight, preferably 1-10% by weight. The surfactant is generally nontoxic and soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1-20% by weight of the composition, preferably 0.25-5% by weight. The balance of the composition is ordinarily propellant. A carrier can also be included, if desired, as with lecithin for intranasal delivery. Aerosols of liquid particles can be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Patent No.

4,501,729, which is hereby incorporated by reference in its entirety. Aerosols of solid particles can likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art. Intranasal administration can also be by droplet administration to a nasal surface.

Injectable formulations can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, one can administer the pharmaceutical formulations in a local rather than systemic manner, for example, in a depot or sustained-release formulation.

Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. For example, an injectable, stable, sterile formulation of the invention in a unit dosage form in a sealed container can be provided. The formulation can be provided in the form of a lyophilizate, which can be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject. The unit dosage form can be from about 1 μg to about 10 grams of the formulation. When the formulation is substantially water-insoluble, a sufficient amount of emulsifying agent, which is pharmaceutically acceptable, can be included in sufficient quantity to emulsify the formulation in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.

Pharmaceutical formulations suitable for oral administration can be presented in discrete units, such as capsules, cachets, lozenges, or tables, as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water- in-oil emulsion. Oral delivery can be performed by complexing a compound(s) of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers include plastic capsules or tablets, as known in the art. Such formulations are prepared by any suitable method of pharmacy, which includes the step of bringing into association the protein(s) and a suitable carrier (which may contain one or more accessory ingredients as noted above). In general, the pharmaceutical formulations are prepared by uniformly and intimately admixing the compound(s) with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet can be prepared by compressing or molding a powder or granules, optionally with one or more accessory ingredients. Compressed tablets are prepared by compressing, in a suitable machine, the formulation in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Molded tablets are made by molding, in a suitable machine, the powdered protein moistened with an inert liquid binder.

Pharmaceutical formulations suitable for buccal (sub-lingual) administration include lozenges comprising the compound(s) in a flavored base, usually sucrose and acacia or tragacanth; and pastilles in an inert base such as gelatin and glycerin or sucrose and acacia.

Pharmaceutical formulations suitable for parenteral administration can comprise sterile aqueous and non-aqueous injection solutions, which preparations are preferably isotonic with the blood of the intended recipient. These preparations can contain antioxidants, buffers, bacteriostats and solutes, which render the composition isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions, solutions and emulsions can include suspending agents and thickening agents. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water,

alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

Pharmaceutical formulations suitable for rectal administration are optionally presented as unit dose suppositories. These can be prepared by admixing the active agent with one or more conventional solid carriers, such as for example, cocoa butter and then shaping the resulting mixture.

Pharmaceutical formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers that can be used include, but are not limited to, petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof. In some embodiments, for example, topical delivery can be performed by mixing a pharmaceutical formulation of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.

Pharmaceutical formulations suitable for transdermal administration can be in the form of discrete patches adapted to remain in intimate contact with the epidermis of the subject for a prolonged period of time. Formulations suitable for transdermal administration can also be delivered by iontophoresis {see, for example, Pharmaceutical Research 3:318 (1986)) and typically take the form of a buffered aqueous solution of the compound(s).

Suitable formulations can comprise citrate or bis\tris buffer (pH 6) or ethanol/water and can contain from 0.1 to 0.2M active ingredient.

In embodiments of the invention, the dosage of a virus particle of this invention can be in a range of about 10 ⁴ to about 10 ⁷ plaque forming units (PFUs). In embodiments of this invention, the dosage of a VLP of this invention can be in a range of about 500 micrograms to about 5 milligrams. In embodiments of this invention, the dosage of a protein of this invention can be in a range of about 10° to about 10 ⁴ micrograms +/- adjuvant.

Further, the composition can be formulated as a liposomal formulation. The lipid layer employed can be of any conventional composition and can either contain cholesterol or can be cholesterol-free. The liposomes that are produced can be reduced in size, for example, through the use of standard sonication and homogenization techniques.

The liposomal formulations can be lyophilized to produce a lyophilizate which can be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.

The immunogenic formulations of the invention can optionally be sterile, and can further be provided in a closed pathogen-impermeable container.

EXAMPLES

EXAMPLE 1

Dengue virus is an important human pathogen responsible for considerable morbidity and mortality worldwide. Four DENV serotypes circulate worldwide and vaccines must protect against each serotype. Current vaccines under development or on the market are tetravalent live virus attenuated vaccines containing the E and the prM glycoproteins of each serotype DEN VI -4. However in humans, many vaccines elicit unbalanced responses, especially concerning as current vaccines elicit weak immune responses to either DENV 2 or DENV 4. Consequently, a new strategy is needed to enhance the immune response simultaneously to both DENV 2 and DENV 4 in a balanced formulation. The DENV E glycoprotein forms a dimer and is divided into three discrete domains designated EDI, ED II and EDIII. The major DENV4 neutralizing sites are encoded by human monoclonal antibodies hml26 and hml 31. The goal of these studies was to use reverse genetics to build recombinant chimeric viruses that encode the major neutralizing epitopes of DENV2 and DENV4 in the same recombinant virus. This new chimeric virus is designed to enhance the response to DENV 4 neutralizing epitopes presented in the context of the DENV2 E glycoprotein, while maintaining functional neutralizing epitopes that elicit robust immune responses against DENV 2.

Using serotype specific antibodies to guide immunogen design, we have made chimeric dengue viruses that contain neutralizing epitopes from both of the 2 different serotypes in a single virus. To accomplish this, we present the structure of the DENV E glycoprotein, which forms a dimer that assembles into 30 larger rafts composed of three dimers of E protein. Consequently, a total of 180 E proteins cover the surface of each dengue virus particle. Importantly, DENV2 neutralizing monoclonals 2D22 and 3F9 have been shown to specifically neutralize DENV 2 and their quaternary epitopes have been determined to lie mainly on domains EDI and ED III respectively of the E protein. DENV 4 has been shown to be specifically neutralized by monoclonals hmAB126 and hmAB131 which primarily bind across dimers on ED II. We designed recombinant DENV2/4 chimeras by insertion of residues that reconstitute DENV4 neutralizing antibody sites 126/131 in DENV2 ( M12) encodes residues of hmAB 126 epitope and M14 encodes residues for hniAB126 and hmAB 131). The combined epitopes of hmAB126 and hmAB131 can be transplanted from DENV 4 to DENV 2 by changing those variable amino acids of DENV 2 into residues encoded by DENV 4 for that specific area of the E protein. We have designed DENV 2/4 viruses with transplants of increasing size; DENV 2/4 M12 (SEQ ID NO:8), DENV 2/4 M14 (SEQ ID NO:9) and DENV 2/4 M Complete (SEQ ID NO:10) (Table 5), simply by moving residues from DENV4 into DENV2. The smallest, DENV M12, converts only a small area of DENV 2 into DENV 4, encoding the hm126 antibody epitope. This virus can be used to measure DENV 4 specific antibodies to this single epitope in polyclonal sera. DENV Ml 4 reconstructs both DENV4 hml26 and hml31 targeted epitopes into DENV 2. The largest, DENV 2/4 M complete (SEQ ID NO:10), converts nearly all of domain 2 into DENV 4. This final design incorporates new features into epitope exchange by taking into

consideration the interactions between the E proteins both within each dimers and then between dimers encoded in the larger rafts. The impact is potentially profound as this approach captures the larger antigenic site while stabilizing interactions within and between dimers of the larger raft, enhancing stability and recombinant virus viability. Since EDI and ED III occur in an alternating pattern on the virus surface and are adjacent to other EDI and EDIII domains, this forms a large surface area that is solely DENV 2. In contrast, ED II mostly boarders other EDII encoding monomers and thus forms a large contiguous surface area of DENV4 residues made of 6 ED lis that run down the center of the larger raft. Since contiguous areas and borders present critical epitopes of DENV4 and DENV2, this approach has the potential to increase the stability of the chimeric virus. This virus may be used in a vaccine to simultaneously immunize people against both DENV 2 and DENV 4 because it contains immune dominant epitopes from both viruses. The DENV 2/4 M Complete (SEQ ID NO: 10), encoding the largest DENV4 epitope transplant, has been electroporated into Vero cells and produced virus that is recognizable by dengue monoclonal antibodies. In addition, DENV 1/4 viruses (Table 4) and DENV 3/4 viruses (Table 6) with transplants of increasing size were prepared in an analogous manner.

In summary, we have designed DENV 2/4 viruses with small to large DENV4 epitope transplants. The viruses with small transplants can be used to precisely map antibody responses to each epitope and combined antigenic site in polyclonal sera. The DENV 2/4 M Complete (SEQ ID NO: 10) virus with the largest transplant is a good vaccine candidate, because it carries the immune dominate epitopes in a format most similar to that seen in the native virus and, thus, may be best able to present these epitopes to the immune system of the largest variety of people. In addition, these viruses may have increased stability, which could make them useful vaccine candidates.

EXAMPLE 2

The four serotypes of dengue virus are the causative agents of dengue fever and dengue hemorrhagic fever. People exposed to primary DENV infections develop long-term neutralizing antibody responses principally only to the infecting serotype. An effective vaccine against dengue needs to elicit long lasting protective antibody responses to all four serotypes simultaneously. We and others have defined antigenic sites on the envelope (E) protein of viruses of dengue serotypes 1, 2 and 3 targeted by human neutralizing antibodies. The mechanisms of serotype 4 neutralization by human antibodies are poorly understood. Here, we report on the properties of human antibodies that neutralize dengue serotype 4. People exposed to serotype 4 infections or a live attenuated serotype 4 vaccine developed strongly neutralizing antibodies that bound to similar sites on the viral E protein. These studies provide a foundation for developing and evaluating DENV4 vaccines.

DENV4 is neutralized by type-specific antibodies in human immune serum

To study the properties of serum polyclonal DENV4 neutralizing human antibodies, we assembled a panel of blood samples from people exposed to DENV4 infections or people who had received a monovalent live-attenuated DENV4 vaccine (Table 1). Individuals exposed to DENV have specific antibodies in circulation as well as DENV-specific memory B cells (MBCs). Some of the dengue specific antibodies in circulation bind only to viruses of the serotype of infection (type-specific), while others cross-react with two or more serotypes. Using serum samples from people exposed to DENV4 natural infections or a monovalent live-attenuated DENV4 vaccine, we performed antibody depletion studies to determine the relative contributions of serotype cross-reactive and type-specific antibodies to DENV4 neutralization. Polystyrene beads coated with the homotypic (DENV4) or heterotypic (DENV2) DENV serotypes were incubated with the immune serum samples to deplete specific populations of antibodies. Depletion of DENV4 or DENV2 binding antibodies was confirmed by ELISA before using the samples in DENV4 neutralization assays. Depleting the DENV4 immune serum samples with the homotypic DENV4 antigen led to the removal of nearly all the DENV-specific (serotype cross-reactive and DENV4 type-specific antibodies) antibodies in the sample. As anticipated, depleting with DENV4 antigen led to a large drop in DENV4 neutralizing antibodies in both DENV4 infection and vaccine sera (Figures 1A-1D and Table 2). Depleting DENV4 immune sera with heterotypic DENV2 antigen led to the removal of serotype cross reactive but not DENV4 type-specific antibodies. There was minimal to no loss in neutralization of DENV4 in sera depleted with DENV2 antigen (Figures 1A-1D and Table 2). These results demonstrate that type-specific antibodies were mainly responsible for DENV4 neutralization in primary infection and monovalent DENV4 vaccination serum samples.

Isolation of DENV4 neutralizing human monoclonal antibodies

To further characterize the B cell response to DENV4, we transformed B cells from two DENV4 immune individuals (subjects 002 and 112) and isolated human monoclonal antibodies (hMAbs), as previously described. The transformed B cell culture supernatants were screened for binding to DENV4. Based on the number of positive wells and the number of transformed B cells tested (determined by average colony counts in transformed wells), the frequency of DENV-specific B cells in circulation was estimated to be 0.19 and 0.2% of transformable B cells for subjects 002 and 112, respectively. Previously, it was reported that there maybe a long-term set point frequency of 0.1-0.2% DENV specific B cells in the circulating memory B cell pool following DENV infection, and the frequencies of 0.19 and 0.2% in the two subjects studied here are consistent with these previous reports.

We also determined the DENV serotype specificity of all the positive B cell culture supernatants from subject 112. Of the 34 DENV antigen-reactive supernatants, antibodies in 32% bound only to DENV4 (type-specific) and in 68%) bound to two or more serotypes (cross-reactive). From the EBV-transformed B cell lines secreting DENV antigen-reactive antibodies, we isolated 8 human B cell hybridoma cell lines, as previously described. Two lines, designated D4-126 or D4-131, secreted DENV4 type- specific and strongly neutralizing MAbs with neut ₅₀ values of 0.54 μg/mL or 0.43 μg/mL, respectively (Figure 2A). To characterize the binding properties of D4-126 and D4-131 MAbs further, we performed binding assays with whole DENV4 virions, rE or rEDIII proteins of DENV4 and increasing concentrations of hMAbs D4-126 or D4-131. Both hMAbs bound to whole DENV4 virus particles similarly (Figure 2B). HMAb D4-126 did not bind to rE protein, whereas D4-131 exhibited low levels of binding to rE protein at high concentrations (>10 μg/mL) (Figure 2B). The hMAbs did not bind to rEDIII protein (Figure 2B). These studies revealed that neutralizing hMAbs D4-126 and D4-131 bound best to intact DENV4 virions.

HMAb neutralization of different DENV4 strains

To determine if hMAbs D4-126 and D4-131 neutralized diverse strains of DENV4, we used a panel of recombinant isogenic DENV4 viruses expressing the E protein from different DENV4 genotypes and laboratory strains (Table 3). The hMAbs equally neutralized all variants tested except for a Cambodia 2010 genotype 1 (GI) strain, which was not neutralized by D4-126 and was neutralized weakly by D4-131 (Figure 3 A and Figure 3B).

Mapping the epitopes of DENV4 neutralizing hMAbs

Human DENV1, 2 and 3 type-specific neutralizing antibodies often bind to quaternary structure epitopes centered on the EDI/II hinge and/or the ED III region. Recently, we demonstrated that it is possible to recover recombinant chimeric DENVs displaying E protein domains or epitopes from viruses of two different serotypes. We used a recombinant DENV4 with a mutated EDI/II hinge region (rDENV4/3) to map the binding sites of hMAbs D4-126 and D4- 131. We did not detect any binding or neutralizing activity for the hMAbs D4- 126 or D4-131 with the rDENV4/3 virus, indicating that the DENV4 EDI/II hinge residues are part of the epitope recognized by these MAbs (Figure 4 A and Figure 4B).

As an alternate approach to mapping the epitopes of D4-126 and D4- 131, both hMAbs were screened by shotgun mutagenesis against a comprehensive mutation library in which nearly every residue within prM and E was individually mutated to alanine, as described previously. Residues were identified as critical to binding of the DENV4 hMAb if they were required for the binding of DENV4 MAb, but did not other conformation- dependent MAbs. Six amino acids (K51, V53, K124, L135, K200, K234) in the EDI/II hinge and ED II regions were critical for binding of D4-126. Four amino acids (K51, K124, K200, K202) within the EDI/II hinge and ED II region were critical for binding of D4-131. These data validated our observations that the EDI/II hinge region residues are critical for binding of D4-126 and D4-131, and also indicated that the epitopes differ slightly between the two hMAbs.

DENV4 neutralizing hMAbs define epitopes targeted by serum antibodies in DENV4 immune individuals

HMAbs D4-126 and D4-131 were isolated from circulating memory B cells. Serum antibodies are thought generally to derive from secretion of long-lived plasma cells (LLPCs) residing in the bone marrow. To determine if DENV4 polyclonal serum neutralizing antibodies in immune sera secreted by LLPCs also targeted the D4-131 and D4-126 epitopes, we performed competition-binding assays with DE V4 immune serum samples and labeled DENV4-specific MAbs. As depicted in Figure 5A and Figure 5B, DENV4 immune sera effectively blocked the binding of each of the MAbs, whereas DENV2 or DENV3 immune sera had marginal effects on MAb binding. The magnitude of the DENV4 immune responses (based on their neutralization titers to DENV4) in serum samples correlated with the ability to block 50% of the binding of D4-126 or D4-131. These findings indicated that there are type- specific antibodies in DENV4 polyclonal antibodies in immune donor serum samples that bind to sites similar to the epitopes of hMAbs D4-126 and D4-131.

To further assess the epitope specificity of functionally neutralizing antibodies in DENV4 sera collected following infection or experimental vaccination, we performed neutralization assays with the rDENV4/3 virus, which had lost the epitopes recognized by hMAbs 126 and 131. Neutralization assays using the rDENV4/3 virus were performed with sera from six subjects with prior natural DENV4 infection and sera from 4 subjects previously immunized with a DENV4 live attenuated vaccine (Figure 6 A and Figure 6B). Sera from both types of immune donors neutralized the rDENV4/3 strain poorly compared to WT DENV4 (Figure 6B). The mean percent loss in neutralization with rDENV4/3 in both naturally infected immune and vaccine sera were significant (p=0.005 and p=0.002 respectively) as determined by one sample t-tests. Of the six DENV4 post-infection sera tested, three subjects (002, 102, 07/333) showed > 60% loss in neutralization against rDENV4/3, while the remaining infection sera displayed a more modest to no reduction in neutralization (Figure 6A). All of the vaccine sera tested neutralized the rDENV4/3 strain poorly compared to WT DE V4 (Figure 6B). These results suggest that the EDI/II hinge region is a major target of type-specific DENV4 neutralizing antibodies. Sustained humoral immunity depends on LLPCs to maintain protective levels of antibody and on memory B cells (MBCs), which comprise a subset of cells poised to expand and adapt in response to subsequent exposure to the infecting pathogen. In this study we characterized the properties of MBC- and LLPC-derived human antibodies that neutralize DENV4. Although people exposed to DENV4 infections developed serotype cross-reactive and type-specific antibodies, our results established that the type-specific antibodies were the principal determinants of neutralization of DENV4. Using MAbs isolated from the MBCs of people exposed to DENV4, we identified epitopes centered around the EDI/II hinge that were best displayed on intact virions as major targets of DENV4 neutralizing antibodies. In people exposed to DENV4 infections or a live attenuated vaccine candidate, both MBC- and LLPC- derived neutralizing antibodies recognized complex epitopes centered around the EDI/II hinge of DENV4.

Substantial progress has been made in understanding the epitopes targeted by human antibodies that neutralize DENV serotypes 1, 2, and 3, whereas serotype 4 is relatively understudied. The two DENV4 neutralizing MAbs reported in this study were sensitive to changes in or near the EDI/II hinge region. The hinge region plays a critical role in the conformational change that E protein undergoes at low pH to fuse to the endosomal membrane allowing viral uncoating and the release of viral RNA into the cellular cytoplasm. Because these epitopes are located in this region, we hypothesize that these DENV4 hMAbs act by preventing conformational changes in E protein required for fusion and a productive viral infection.

Some DENV4 epitopes targeted by neutralizing mouse MAbs vary between strains of DENV4. We evaluated if hMAb D4-126 and D4-131 effectively neutralized different strains of DENV4. All strains studied here were neutralized well, except for one GI strain

(Cambodia 2010) that was resistant or partially resistant to hMAb D4- 126 and D4- 131 respectively. There are 16 amino acid differences between the E proteins of SL1992 (Gil) neutralization sensitive and the Cambodian 2010 (GI) neutralization resistant strains. Three of the mutations in EDII (122 L->S; 203 T->K; 233 H->Y) overlapped with the region identified by shotgun mutagenesis as the binding sites of D4-126 and D4-131. We propose that natural variation between DENV4 strains leads to poor or altered binding of D4-126 and D4-131 and neutralization escape. Moreover, recent studies demonstrate that some DENV strains flex and "breathe" more than other strains, which can also lead to better exposure of partially hidden epitopes. Mutations outside the main footprints of D4-126 and D4-131 may also indirectly alter epitope exposure and contribute to strain specific differences in neutralization sensitivity.

The LLPC-derived polyclonal serum antibodies likely provide the first line of defense against re-infection in vivo. Our studies using blockade of antibody binding demonstrated that the DENV4 polyclonal immune sera contained antibodies that specifically blocked the binding of MAbs D4-126 and D4-131 to their epitopes. Additionally, a recombinant DENV4 strain missing the D4-126 and D4-131 epitopes was less sensitive to neutralization by DENV4 infection and vaccine sera compared to WT DENV4. These results establish that the region/epitopes defined using MAbs are important targets of the LLPC-derived polyclonal serum antibody response. In some individuals, a fraction or most of the serum DENV4 neutralizing antibody response was unaffected by EDI/II hinge mutations, indicating other regions and epitopes likely are involved in DENV4 neutralization. A chimpanzee DENV4 type-specific strongly neutralizing MAb 5H2 was directed to the EDI region. Cockburn et al. demonstrated that at least a portion of antibodies in DENV4 convalescent patient sera bound to epitopes on DI that overlapped with that of MAb 5H2.

In summary, we propose that the EDI/II hinge region is a target of DENV4 neutralizing human antibodies in both the MBC and LLPC compartments. The EDI/II hinge region is also a target of human type-specific antibodies that neutralize DEN VI and DENV3.

Cells. Aedes albopictus C6/36 cells were maintained in MEM (Gibco) medium at 32°C. Vero cells (American Type Culture Collection; CCL-81) were maintained in .

Dulbecco's modified Eagle's (DMEM-F12) medium at 37°C. A human monocyte lymphoma cell line U937 ectopically expressing dendritic cell-specific intercellular adhesion molecule- 3 -grabbing non-integrin DC-SIGN (U937+DC-SIGN)(36, 37) was maintained in RPMI-1640 (Gibco) medium at 37°C supplemented with 50 mM beta mercaptoethanol. All growth and maintenance media used were supplemented with 5% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, 0.1 mM non-essential amino acids (Gibco) and 2 mM glutamine. All cells were incubated in the presence of 5% C0 ₂ . The 5% FBS was reduced to 2% to make infection medium for each cell line.

Viruses, rE and rEDIII. The DEN VI (American genotype; strain West Pac74), DENV2 (Asian genotype; strain S-16803), DENV3 (Asian genotype; strain CH-53489),

DENV4 (American genotype; strain TVP-376) viruses (provided by Robert Putnak, Walter Reed Army Institute of Research, Silver Spring, MD) were used for both binding enzyme- linked immunosorbent assays (ELISAs) and neutralization assays. All viruses used in the neutralization assays were grown in C6/36 Aedes albopictus mosquito cells at 32°C, as previously described (38). DENV4 virus was purified as previously described. DENV2 (New Guinea C) purified live virus was purchased from Microbix Biosystems, Inc. (Mississauga, Ontario, Canada). Recombinant envelope (rE) proteins (80% of E protein) from each of the four serotypes were produced within our laboratory or purchased from Hawaii Biotech, Inc. Recombinant ED III proteins were obtained as described previously.

DENV4 immune sera. Convalescent DENV4 immune serum samples were obtained from ongoing studies in dengue endemic countries or from travelers visiting dengue endemic countries. DENV4 immune sera were also obtained from people who received a live attenuated monovalent DENV4 vaccine under development by the US National Institutes of Health. The protocol for obtaining dengue immune blood samples was approved by the

Institutional Review Board of the University of North Carolina at Chapel Hill (protocol 08- 0895).

Depletion of DENV4-specific antibodies from human immune sera collected from subjects with prior DENV4 infection or vaccination. Purified DENV was absorbed onto 4.5- μm Polybead polystyrene microspheres (Polysciences, Inc.) at a bead (μL) to ligand μg) ratio of 5:2. Polystyrene beads were washed three times with 0.1 M borate buffer (pH 8.5) and incubated with the relevant purified DENV (DENV4 for homotypic depletions and DENV2 for heterotypic depletions) overnight at room temperature (RT). Control beads were incubated overnight with an equivalent amount of bovine serum albumin (BSA). The control and virus-adsorbed beads were blocked with BSA (10 mg/mL) in borate buffer for 30 min at RT three times and washed four times with PBS. DENV4 immune sera from naturally infected individuals or NIH vaccine candidate recipients were depleted of virus-specific antibodies by incubating the samples with virus-adsorbed beads for lh at 37°C with end- over-end mixing. Samples were subjected to at least three sequential rounds of depletions before confirming successful removal of the respective antibodies by ELIS A. The ability of the depleted samples to neutralize viruses of all of the four serotypes then was tested after the confirmation ELISA.

Generation of DENV4-specific MAbs. Previously cryopreserved peripheral blood mononuclear cells (PBMCs) were thawed rapidly in a 37°C water bath and washed prior to transformation with Epstein-Barr virus (EBV) and incubated with CpG and additional supplements, as described previously. Cultures were incubated at 37°C with 5% C0 ₂ for 10 days prior to screening for DENV4-reactive cell lines with ELISA. The minimal frequency of DENV4-reactive B cells was estimated on the basis of the number of wells with DENV4- reactive supernatants as compared to the total number of lymphoblastoid cell line colonies in the transformation plates, as follows: [number of wells _^ with DENV4-reactive supernatants]/[number of LCL colonies in the plate]. Cells from wells with supernatants reacting in the DENV4 capture ELISA were subjected to cytofusion with HMMA2.5 non- secreting myeloma cells, as previously described. Following cytofusion, hybridomas were selected for growth in HAT medium containing ouabin. Wells containing hybridomas producing DENV4-reactive antibodies were cloned biologically by 3 rounds of limiting dilution plating. Once clonal, the cell lines were used to produce MAb immunoglobulin G (IgG) in cell supernatants, using serum-free medium, followed by protein G column purification.

Virus, rE and rEDIII ELISA. Equivalent quantities of DENV (as previously titrated by ELISA) virus was captured by anti-E mouse mAb 4G2, or rE proteins were directly coated (rE - 100 ng/well; rEDIII - 200 ng/well) on ELISA plates overnight at 4°C. Plates were blocked with 3% (vol/vol) normal goat serum (Gibco - Thermo Fisher, USA), in Tris- buffered saline (TBS) containing 0.05% (vol/vol) Tween 20 (blocking buffer). Primary antibodies were diluted serially to generate a range of concentrations. Alkaline-phosphotase conjugated secondary antibodies were used to detect binding of primary antibodies with p- nitrophenyl phosphate substrate, and reaction color changes were quantified by

spectrophotometry.

Blockade of binding assays. Blockade of binding assays were performed as described previously. Briefly, DENV4 was captured using mouse anti-E MAb 4G2, and blocked as described above. Serial dilutions of DENV serum were added to the DENV4-coated plates and incubated at 37°C for lh. The plates were washed and alkaline-phosphatase conjugated DENV4 hMAbs were added and incubated at 37°C for lh. P-nitrophenyl phosphate substrate was added and reaction color changes were quantified by spectrophotometry. Percentage of blockade of binding was calculated as follows: (100-[OD of sample/OD of negative control]* 100).

Flow-based U937+DC-SIGN neutralization assay. Neutralization potential of the DENV4 immune sera and hMAbs were measured using a flow cytometry-based

neutralization assay with U937+DC-SIGN cells as previously described. Briefly, virus and antibody mixtures or serum were incubated for 1 h at 37°C, prior to the addition of

U937+DC-SIGN cells. After 2 h of incubation, cells were washed twice with infection media. Cells were then fixed and permeabilized 24h after infection, probed with 2H2 (anti- prM MAb) conjugated to Alexa-Fluor 488 and infected cells quantified using a Guava flow cytometer (EMD Millipore). Stained cells were analyzed to calculate 50% neutralization titers.

Construction of the rDENV viruses. rDENV4/3 viruses were generated as described previously. Briefly, the DENV4 genome was split into 4 separate plasmids (A, B, C, D), allowing production of genomic cDNA. Plasmids were digested, and genome fragments were ligated together into a full-length-cDNA genome from which RNA transcripts were derived. These transcripts were electroporated into cells, and cell culture supernatant containing viable virus was harvested. Virus was passaged two times on C6/36 cell monolayer cultures and stored at -80°C. To generate rDENVs, the nucleotide sequence of the envelope glycoprotein was changed to alter the amino acid residues. rDENV4/3 contains EDI/II hinge residues (25 amino acids) from DENV3.

Generation of DENV4 strains displaying diverse E glycoproteins. In order to examine genetic diversity within a serotype, a panel of near-isogenic rDENV4 viruses was generated by replacing the E gene of WT genotype II infectious clone virus (Sri Lanka 1992; Accession: KJ 160504.1 ) with that of E glycoprotein genes representing diverse strains within DENV4. Subgenomic A plasmids were synthesized encoding E protein genes only (all others proteins remained Sri Lanka 1992) representing genotype I (GI; Cambodia 2010; Accession: KF543272.1), genotype II (Gil; Puerto Rico 1999; Accession: FJ882599.1), or a sylvatic E sequence (Malaysia 1973; Accession: JF262780). Recombinant subgenomic A plasmids were synthesized, and viral assembly and rescue were performed as described above for generation of rDENV4 viruses.

Shotgun mutagenesis epitope mapping. Shotgun mutagenesis epitope mapping was performed as described previously. Briefly, a DENV4 prM-E protein expression construct was subjected to high-throughput alanine-scanning mutagenesis to generate a comprehensive mutation library (each residue mutated to alanine, and alanine residues mutated to serine). Mutant proteins (97% coverage) were generated and arrayed into 384-well plates. Mutants were transfected into HEK-293T cells and allowed to express for 22h. Cells were fixed in 4% (vol/vol) paraformaldehyde (Electron Microscopy Sciences) and permeabilized with 0.1% (wt/vol) saponin (Sigma) in PBS containing calcium and magnesium. Cells were stained with purified anti-DENV4 hMAbs (D4- 126, D4- 131 ) diluted in 10% normal goat serum

(NGS; Sigma) and 0.1% saponin, pH 9.0. Antibody binding was detected using Alexa Fluor 488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) in 10% NGS (Sigma) and 0.1 % saponin. Cells were washed three times with PBS supplemented with 0.1%) saponin, 1 mM MgCl ₂ , and CaCl ₂ followed by two washes in PBS. The mean cellular fluorescence was detected using a high-throughput flow cytometer (HTFC; Intellicyt).

Mutations were identified as critical to the hMAb epitope if they did not bind the test hMAb but did bind other conformation-dependent MAbs. This counter screen strategy facilitated the exclusion of E mutants that were locally misfolded or had expression defects.

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

All publications, patent applications, patents, nucleotide sequences, amino acid sequences, GenBank accession numbers and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.