This application claims priority to US Provisional Application No. 61/447,582 filed February 28, 2011.

Field of the Invention

The present invention relates to improved flavivirus vaccines and the design and making of such vaccines that enhance immunogenicity of the vaccine. The present invention relates to improved dengue vaccines and the design and making of such vaccines that enhance immunogenicity of the vaccine. In particular the vaccines and immunogenic compositions of the present invention relate to mixed head bivalent flagellin-dengue antigen fusion proteins which can be combined to produce tetravalent vaccines.

Background of the Invention

Infection from flaviviruses, such as Dengue, West Nile virus, tick borne encephalitis, yellow fever virus and several other viruses that cause encephalitis can cause serious illness, and in some case death. Dengue virus infection can generally result in flu-like illness that lasts for several weeks. In certain instances, infection from Dengue virus can result in Dengue hemorrhagic fever, which is characterized by acute vascular leakage, hemorrhagic phemomena (e.g., bleeding, bruising) and a high mortality rate. The treatment for Dengue viral infection includes rest, hydration and electrolyte replacement. Currently, there are no compositions that prevent infection caused by Dengue virus. Thus, there is a need to develop new, improved and effective methods for treatment for preventing and managing disease associated with flavivirus infection.

Summary of the Invention

The present invention relates to immunogenic compositions that include flavivirus proteins, such as Dengue viral antigens. The present invention relates to immunogenic fusion proteins comprising constructs that comprise monovalent, bivalent, trivalent and/or tetravalent immunogenic compositions that can be combined to produce a multivalent vaccine against flavivirus.

The present invention relates to immunogenic fusion proteins comprising constructs that comprise monovalent, bivalent, trivalent and/or tetravalent immunogenic compositions that can be combined to produce a multivalent vaccine against dengue virus.

The present invention relates to immunogenic fusion proteins comprising constructs that comprise two mixed head bivalent immunogenic compositions that can be combined to produce a tetravalent vaccine against dengue virus. The present invention relates to immunogenic fusion proteins comprising constructs that comprise two monovalent and one bivalent immunogenic composition that can be combined to produce a tetravalent vaccine against dengue virus.

The present invention relates to immunogenic fusion proteins comprising flagellin and two distinct dengue virus antigens. The present invention relates to immunogenic compositions where a dengue antigen is linked to the C- terminus of the flagellin and another dengue antigen replaces the D3 domain of the flagellin.

The present invention relates to immunogenic compositions where the dengue antigens in the compositions are chosen from dengue serotype 1, 2, 3, or 4.

The present invention relates to immunogenic compositions comprising a fusion protein wherein a DENV-2 antigen is linked to the C terminus of flagellin and a DENV-4 antigen replaces the D3 domain of flagellin.

The present invention relates to immunogenic compositions comprising a fusion protein wherein a DENV-1 antigen is linked to the C terminus of flagellin and a DENV-3 antigen replaces the D3 domain of flagellin. The present invention relates to immunogenic compositions comprising fusion proteins of dengue antigens and flagellin where at least one of the fusion proteins comprises a DENV-2 antigen linked to the C terminus of the flagellin and a DENV-4 antigen replaces the D3 domain of flagellin and at least one of the fusion proteins comprises a DENV-1 antigen linked to the C terminus of the flagellin and a DENV-3 antigen replaces the D3 domain of flagellin.

The present invention relates to immunogenic fusion proteins comprising STF2R3.D3EIII-C.D1EIII.

The present invention relates to immunogenic fusion proteins comprising STF2R3.D4EIII-C.2EIII.

The present invention relates to immunogenic fusion proteins comprising STF2R3.D3EIII-C.D1EIII and STF2R3.D4EIII-C.D2EIII.

Description of the Figures

Figure 1. Graphic representation of Dengue-1 West Pac 74 virus neutralization titers after two immunization of DENV-3 Elll fused to the R3 and DENV-1 Ell I fused to C-terminal position of flagellin.

Figure 2. Graphic representation of Dengue-3 CH53489 virus neutralization titers after two immunization of DENV-3 Elll fused to the R3 and DENV-1 Elll fused to C-terminal position of flagellin.

Figure 3. Graphic representation of Dengue-2 S-16803 virus neutralization titers after three immunization of DENV-4 Elll fused to the R3 and DENV-2 Elll fused to C-terminal position of flagellin.

Figure 4. Graphic representation of Dengue-4 TVP 360 virus neutralization titers after three immunization of DENV-4 Elll fused to the R3 and DENV-2 Elll fused to C-terminal position of flagellin.

Figure 5. Graphical representation of summary of GMTs against DENV-4 component in DENV-4 in either the R3 or C term and DENV-2 in either the C term position or R3 position + DENV-3 in either the R3 or C term and DENV-1 in either the C term or R3 position.

Figure 6. - A schematic representation of three different vaccine formats (C-term, R3 and R3.2x). DO, Dl, D2 and D3 are the four domains of flagellin and the genetically fused antigen is encircled. The primary TLR5 binding site is located in Dl. Figure 7. Neutralizing antibody responses elicited by tetravalent dengue vaccine composed of two bivalent components.

Figure 8. Neutralizing antibody responses elicited by tetravalent dengue vaccine composed of two monovalent and one bivalent component.

Detailed Description of the Invention

The present invention relates to improved flavivirus immunogenic compositions and vaccines and the design and making of such vaccines that may enhance the immunogenicity of the vaccine. The present invention also relates to improved Dengue immunogenic compositions and vaccines and the design and making of such vaccines that may enhance immunogenicity of the vaccine.

The flaviviruses are RNA viruses comprising a single stranded RNA having a length, among the various species, of about 10 kilobases. The nucleotide sequences of the genomes of several flaviviruses are known and are summarized in US Patent No. 5,494,671 which is incorporated by reference. The Dengue disease is caused by four mosquito-borne, serologically related flaviviruses know as DENV-1 (also referred herein as DENV-1, DENV-2, DENV-3 or DENV-4. The compositions, fusion proteins, and polypeptides of the invention include antigens sequences disclosed in WO 2009/128949 (PCT/US2009/002427), Izquierdo et al., 2008 - 814669, Mota et al., 2005, Khanam et al., 2006, Pattnaik et al., 2007, Tripathi et al., 2008; A. Zulueta, et al., Virus Research 121 (2006) 65-73, Deubel, et al. Virology 155; 365-377 (1986); Gruenberg, et al., J. Gen. vir. 69: 1391-1398 (1988); Osatomi, et al. Virus Genes 2: 99-108 (1988); Mackow, et al. Virology 159: 217-228 (1987), Zhao et al., Virology 155: 77-88 (1986) herein incorporated by reference. The nucleotide sequences of the West Nile Virus genome can be found in Lanciotti, et al. Science 286: 2331-2333 (1999) and of the yellow fever virus in Rice et al. Science 229: 726-733 (1985) also incorporated by reference. Constructs of the present invention may comprise heterologous nucleic acid molecule(s) expressed in vivo in the animal WNV antigen, immunogen or epitope such as WNV E; WNV prM and E; WNV M and E; WNV prM, WNV M and E, WNV polyprotein prM-E, WNV polyprotein M-E, or WNV polyprotein prM-M-E. The genome of the dengue virus comprises a single stranded positive RNA which serves as the messenger RNA for translation of one long open reading frame which is processed into a number of structural and non-structural proteins including the capsid protein (C-protein), premembrane protein (prM protein) and the envelope protein (E protein), domains l-lll. The C terminus of the C protein includes a hydrophobic domain that functions as a signal sequence for translocation of the prM protein into the lumen of the endoplasmic reticulum. The E protein is a membrane protein and likely interacts with viral receptors. Dengue virus enters into host cells by binding the envelope glycoprotein (E) to a receptor and causes human disease. Domain III of dengue virus E glycoprotein (Elll) is immunogenic and capable of inducing neutralizing antibodies. These proteins, portions thereof and mutants thereof may be used alone or in combination in the immunogenic compositions of the present invention. In particular the vaccines and immunogenic compositions of the present invention relate to mixed head bivalent flagellin-dengue antigen fusion proteins which can be combined together with other bivalent antigen fusion proteins and/or monovalent antigen fusion proteins to produce multivalent vaccines including tetravalent vaccines. The mixed head bivalent compositions may be mixed with other mixed head bivalent compositions as well as monovalent compositions. In particular, the improved dengue vaccines of the present invention relate to R3.2x format vaccines (Figure 6) wherein one antigen is linked to the C terminus of flagellin and a different antigen replaces domain 3 of flagellin. Such embodiments are mixed head or heterologous constructs. An embodiment of the present invention is a fusion protein in which a DENV-3 antigen is fused to the R3 position of flagellin (replaced the D3 domain of flagellin and DENV-1 antigen is fused to the C terminus of flagellin. Another embodiment of the present invention is a fusion protein in which a DENV-4 antigen is fused to the R3 position of flagellin (replaced the D3 domain of flagellin) and DENV-2 antigen is fused to the C terminus of flagellin. An embodiment of the present invention is a fusion protein in which a DENV-1 antigen is fused to the R3 position of flagellin (replaced the D3 domain of flagellin) and DENV-3 antigen is fused to the C terminus of flagellin. Another embodiment of the present invention is a fusion protein in which a DENV-2 antigen is fused to the R3 position of flagellin (replaced the D3 domain of flagellin) and DENV-4 antigen is fused to the C terminus of flagellin. An embodiment of the present invention is a fusion protein in which a DENV-3 Elll antigen is fused to the R3 position of flagellin (replaced the D3 domain of flagellin and a DENV-1 Elll antigen is fused to the C terminus of flagellin. Another embodiment of the present invention is a fusion protein in which a DENV-4 Elll antigen is fused to the R3 position of flagellin (replaced the D3 domain of flagellin) and DENV-2 Elll antigen is fused to the C terminus of flagellin. An embodiment of the present invention is a fusion protein in which a DENV-1 Elll antigen is fused to the R3 position of flagellin (replaced the D3 domain of flagellin) and DENV-3 Elll antigen is fused to the C terminus of flagellin. Another embodiment of the present invention is a fusion protein in which a DENV- 2 Elll antigen is fused to the R3 position of flagellin (replaced the D3 domain of flagellin) and DENV-4 Elll antigen is fused to the C terminus of flagellin. The present invention also contemplates homologous double head constructs (R3.2x homologous) in which the constructs carry two copies of the same antigen. In some embodiments the dengue antigen is Elll domain. Titers to all four DENV serotypes are elicited by co-delivery of two mixed head vaccines which comprise fusion proteins where one fusion protein contains two of the four dengue antigens and the other fusion protein contains the other 2 dengue antigens. Titers to all four DENV serotypes are elicited by co-delivery of one mixed head fusion protein which contains two of the four dengue antigens two different monovalent constructs that each contains one of the other two dengue antigens. The Elll antigen in the R3 position may be more immunogenic than that in the C-terminus of flagellin. The present invention relates to constructs that include dengue antigens derived from the various dengue proteins including the capsid protein (C- protein), premembrane protein (prM protein) and the envelope protein (E protein), portions thereof and mutants thereof either alone or in combination. In one embodiment of the compositions of the present invention the El and Elll domain are part of a fusion protein in combination with flagellin as shown in SEQ ID nos. 53-63..

The compositions that include the fusion proteins can be administered alone or as admixtures with conventional excipients, for example, pharmaceutically, or physiologically, acceptable organic, or inorganic carrier substances suitable for enteral or parenteral application which do not deleteriously react with the composition. Suitable pharmaceutically acceptable carriers indue water, salt solutions (such as Ringer's solution), alcohols, oils, gelatins and carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, and polyvinyl pyrolidine. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring and/or aromatic substances and the like which do not deleteriously react with the compositions administered to the human. Preferred diluents for diluting the vaccines of the present invention include but are not limited to 150mM NaCI with histidine and trehalose. The compositions, fusion proteins and proteins of the invention can be administered to a subject on a support that presents the compositions, proteins and fusion proteins of the invention to the immune system of the subject to generate an immune response in the subject. The presentation of the compositions, proteins and fusion proteins of the invention would preferably include exposure of antigenic portions of the fusion protein to generate antibodies. The support is biocompatible. "Biocompatible" as used herein, means that the support does not generate an immune response in the subject (e.g., the production of antibodies).

The dosage and frequency (single or multiple doses) administered to a subject can vary depending upon a variety of factors, including, for example, prior exposure to an infection consequent to exposure to the antigen: health, body weight, body mass index, and diet of the subject or health-related problems. Other therapeutic regimens or agents can be used in conjunction with the methods and compositions, proteins or polypeptides of the present invention.

The composition can be administered to the human in a single dose or in multiple doses, such as at least two doses. When multiple doses are administered to the subject, a second or third dose can be administered days (e.g., 1, 2, 3, 4, 5, 6, 7), weeks (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), months (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) or years (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) after the initial dose. For example, a second dose of the composition can be administered about 7 days, about 14 days or about 28 days following administration of a first dose of the composition that includes the fusion protein.

The dose of the fusion protein may be administered to the human within a range of doses including from about Ο.ΐμ^ to about 500ug, lug to about 100pg, lug to about 50ug, from about l ig to about 30ug, from about lpg to about 25ug, from about lμg to about 20ug, from about lpg to about 15ug, from about lug to about 10μg, from about 2ug to about 50ug, 2ug to about 30pg, from about 2pg to about 20ug, from about 2ug to about 10pg, from about 2ug to about 8ug, from about 3ug to about 50ug, 3pg to about 30ug, from about 3μg to about 20pg, from about 3ug to about lOug, from about 3μg to about 8pg, from about 3pg to about 5pg, from about 4μg to about 50μg, 4ug to about 30ug, from about 4ug to about 20ug, from about 4ug to about lOug, from about 4pg to about 8ug, from about 5μg to about 50μg, 5^ to about 30μg, from about 5ug to about 20ug, from about 5^ to about lOug, from about 5μ§ to about 9ug, and from about 5μg to about 8μζ. The immunogenic compositions for use according to the present invention may be delivered as a standard 0.5 ml injectable dose and contain from about 0.1|ig to about 50vg of antigen. In a preferred embodiment of the immunogenic compositions for use according to the present invention is a standard 0.5 ml injectable dose and contains from about 3 ig to about 20μ£ of antigen. The vaccine volume may be between 0.25 and 1.0 ml, suitably between 0.5 ml and 1.0 ml, in particular a standard 0.5ml. A vaccine dose according to the present invention may be provided in a smaller volume than conventional dosing. Low volume doses according to the present invention are suitably below 0.5ml, typically below 0.3ml and usually not less than 0.1 ml.

With respect to compositions in which the flagellin and antigen are delivered via particles the dosage will depend on the type of particle used and the ratio of flagellin to antigen present in the composition. With respect to compositions in which flagellin and the antigen are present in ratios other than 1:1 the dosage will depend on the ratio of components. With respect to antigens other than influenza antigens the dosage will depend on the antigen used and the ratio of flagellin to the antigen. One of skill in the art may readily be able to determine the optimal dosing of the flagellin compositions based on the carrier, antigen and ratio of flagellin to antigen.

Within this disclosure, any indication that a feature is optional is intended provide adequate support (e.g., under 35 U.S.C. 112 or Art. 83 and 84 of EPC) for claims that include closed or exclusive or negative language with reference to the optional feature. Exclusive language specifically excludes the particular recited feature from including any additional subject matter. For example, if it is indicated that A can be drug X, such language is intended to provide support for a claim that explicitly specifies that A consists of X alone, or that A does not include any other drugs besides X. "Negative" language explicitly excludes the optional feature itself from the scope of the claims. For example, if it is indicated that element A can include X, such language is intended to provide support for a claim that explicitly specifies that A does not include X. Non-limiting examples of exclusive or negative terms include "only," "solely," "consisting of," "consisting essentially of," "alone," "without", "in the absence of (e.g., other items of the same type, structure and/or function)" "excluding," "not including", "not", "cannot," or any combination and/or variation of such language.

Similarly, referents such as "a," "an," "said," or "the," are intended to support both single and/or plural occurrences unless the context indicates otherwise. For example "a dog" is intended to include support for one dog, no more than one dog, at least one dog, a plurality of dogs, etc. Non-limiting examples of qualifying terms that indicate singularity include "a single", "one," "alone", "only one," "not more than one", etc. Non-limiting examples of qualifying terms that indicate (potential or actual) plurality include "at least one," "one or more," "more than one," "two or more," "a multiplicity," "a plurality," "any combination of," "any permutation of," "any one or more of," etc. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. Where ranges are given herein, the endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that the various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

A description of example embodiments of the invention follows.

Examples

Example 1

Testing of Bivalent Mixed Constructs Two constructs STF2R3.D3EIII-C.D1EIII and STF2R3.D4EIII-C.D2EIII were made by recombinant techniques and expressed. STF2R3.D3EIII-C.D1EIII is a bivalent mixed construct in which a flageilin molecule is fused with 2 different dengue antigens, DENV-3EIII and DENV-1EIII. The DENV-1 antigen is linked to the C-terminus of the flageilin and the DENV-3 antigen replaces the D3 domain of flageilin. STF2R3.D4EIII-C.D2EIII is a bivalent mixed construct in which a flageilin molecule is fused with 2 different dengue antigens, DENV4EIII and DENV-2EIII. The DENV-2 antigen is linked to the C-terminus of the flageilin and the DENV-4 antigen replaces the D3 domain of flageilin. The fusion proteins resulting from the constructs were used to immunize BALB/c mice (N=10) subcutaneously three times: day 0, day 14 and day 28. Blood was collected at day 26, 35 and at 1, 3 and 6 months. The mice were divided into 4 group: 1) PBS which is a negative control; 2) 0.6 pg - where the mice were administered 0.6 pg of the vaccine; 3) 3.0 pg - where the mice were administered 3.0 pg of the vaccine; and 4) 15 pg - where the mice were administered 15 pg of the vaccine. The results of these tests can be seen in figures 1-4 and the tables below. Table 1 Bivalent Dengue Vaccines: DENV-3 Elll fused to the R3 and DENV-1 Elll fused to the C term Position; Summary of GMTs with the DENV 1 & 3 Mixed Bivalent Construct

The mixed DENV-3 and DENV-1 bivalent construct demonstrate titer ratios that are nearly balanced.

Table 2 Bivalent Dengue Vaccines: DENV-4 Elll fused to the R3 and DENV-2 Elll fused to the C term Position; Summary of GMTs with the DENV 2 & 4 Mixed Bivalent Construct

The mixed DENV-4 and DENV-2 bivalent construct demonstrate titer ratios that favor DENV-2 Elll.

The bivalent R3.D4EIII.C-D2EIII construct elicited DENV-4 Elll and DENV-2 Elll virus neutralizing antibodies. The bivalent R3.D3EIII.C-D1EIII construct elicited DENV-3 Elll and DENV-1 Elll virus neutralizing antibodies.

Example 2

Co-Delivery of Bivalent Mixed Constructs

Constructs with DENV-4 in either the R3 or C term and DENV-2 in either the C term position or R3 position + DENV-3 in either the R3 or C term and DENV-1 in either the C term or R3 position were made and expressed. The R3 DENV-4/ C-term DENV-2 bivalent construct (R3 D4b+C.D2) or R3 DENV-2/ C-term DENV-4 bivalent construct (R3 D2 + D4b) was delivered alone or in combination at 10 ug (blue symbols) or in combination with the bivalent construct of DENV-3 in either R3 or C-term position and DENV-1 either in C- term or R3 position. 2.5 pg each (red symbols) or 10 \g each (green symbols) were delivered. Comparing the responses of 10 ig of DENV-4 delivered alone (blue) or in combination (green) there is a slight reduction in the titer of the DENV- 4 component (Figure 5).

Example 3

Tetravalent vaccine comprising two bivalent components Groups of 10 BALB/c mice were immunized s.c. on days 0, 21, and 42 with TDV2, TDV3, and TDV4 (2.5 ug R3.D3DIII-C.D1EIII + 10 pg R3.D4EIII-C.D2EIII) listed in Table 2. Animals were bled on days 56. Serum PRNT50 titers were expressed as GMTs (number above each group) ± 95%Cls. GMTs titers of F147 group are 35, 23, 92, and 26 for DENV-1, -2, -3, and -4, respectively. *, p < 0.05 in 2-way ANOVA/Bonferroni tests. Neutralizing antibody responses elicited by the vaccines are shown in Figure 7 which demonstrates that a tetravalent dengue vaccine (TDV4) consisting of 2 bivalent candidates elicited good neutralizing antibody titers to 4 serotypes of DENV that are comparable to the titers elicited by other TDVs.

Table 3.

Vaccine components Total Dose

TDV2 R3.2xD1EIII, R3.2xD3EIII, R3.2xD3EIII, R3.2xD4(H241)EIII 15.5

TDV3 R3.2xD1EIII, R3.2xD3EIII, R3.2xD3EIII, R3.2xD4(341750)EIII 16

TDV4 R3.D3DIII-C.D1 EIII + R3.D4(341750)EIII-C.D2EIII 12.5 Example 4

Tetravalent vaccine comprising one bivalent and two monovalent components

Groups of 10 BALB/c mice were immunized s.c. on days 0, 21, and 42 with a TDVl (2.5 ug of R3.D1EIII- C.D2EIII + 2.5 pg R3.2xD3EIII + 10 pg R3.2xD4EIII). Animals were bled on days 35 and 56. Serum PRNT ₅₀ titers are expressed as GMTs (number above each group) ± 95%Cls. GMTs titers of F147 group are 35, 23, 92, and 26 for DENV-1, -2, -3, and -4, respectively. Results in Figure 1 demonstrated that a tetravalent dengue vaccine (TDVl) consisting of a bivalent DENV-l/DENV-2 (R3.D1EII-C.D2EIII) plus 2 monovalent components (R3.2xD3EIII & R3.2xD4EIII) elicits good neutralizing antibody responses to 4 serotypes of DENV.