VACCINE

Abstract

The invention provides a recombinant polypeptide comprising the EDIII domain of each of Dengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4 linked to the N-terminal of HBsAg.

Claims

1. A recombinant polypeptide comprising the EDIII domain of each of Dengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4 linked to the N-terminal of HBsAg.

2. A nucleic acid sequence encoding a recombinant protein comprising the EDIII domain of Dengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4 linked to the N-terminal of HBsAg.

3. A host cell transformed or transfected with a nucleic acid of claim 2, wherein the host cell expresses HBsAg.

4. A bio-nanoparticle comprising the recombinant polypeptide of claim 1.

5. A method of producing a bio-nanoparticle comprising the recombinant polypeptide of claim 1, comprising culturing the host cell of claim 4 under appropriate conditions and recovering the expressed recombinant protein or bio-nanoparticle.

6. A vaccine comprising the recombinant polypeptide of claim 1.

7. A vaccine comprising the bio-nanoparticle of claim 4.

8. A method of treating or preventing Dengue virus, comprising administering to a subject the recombinant polypeptide of claim 1, the bio-nanoparticle, of claim 4 or the vaccine of claim 6 or claim 7.

9. The recombinant polypeptide of claim 1, the bio-nanoparticleof claim 4 or the vaccine of claim 6 or claim 7 for use in treating or preventing Dengue virus.

Description

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0032] FIG. 1: (A) Design of DSV.sup.4: EDIII-T consisting of four EDIIIs corresponding to the four DENVs linked through hexaglycyl linker was genetically fused with HBsAg (S) to encode for EDIII-T-HBsAg, which was cloned in vector carrying four expression cassettes of HBsAg. The recombinant plasmid was linearized with Bg1 II and electroporated into P. pastoris cells to obtain a clone co-expressing EDIII-T-HBsAg and HBsAg.

[0033] (B) Methanol induction: Uninduced (U) and induced (I) biomass of selected clone was prepared by methanol induction and analysed for expression by silver staining and western blotting. Silver stained gel shows expression of both EDIII-T-HBsAg (˜72 kDa) and HBsAg (˜25 kDa) in induced sample. Western blot with EDIII specific mAb detects EDIII-T-HBsAg, and with HBsAg specific mAb detects both EDIII-T-HBsAg and HBsAg.

[0034] (C) Purification of DSV.sup.4: Three preps of purified DSV.sup.4 from induced biomass.

[0035] FIG. 2: (A) Gel filtration chromatography of DSV.sup.4: DSV.sup.4 eluted in void volume when subjected to gel filtration chromatography. Analysis of protein in void volume by silver staining indicates presence of both EDIII-T-HBsAg and HBsAg.

[0036] (B) CsCl ultracentrifugation: Co-migration of EDIII-T-HBsAg and HBsAg on ultracentrifugation on CsCl column.

[0037] (C) Electron microscopic view of DSV.sup.4 VLPs: 25-35 nm sized DSV.sup.4 VLPs as observed on negative staining under an electron microscope.

[0038] FIG. 3: (A) Source of EDIII sequence: Genotype of each DENV serotype from which corresponding EDIII sequence was derived.

[0039] (B) ELISA reactivity of DSV.sup.4 antiserum: Reactivity of pooled DSV.sup.4 antiserum against HBsAg, EDIII-1, EDIII-2, EDIII-3 and EDIII-4. Purple curve represents reactivity of un-immunized serum against DSO.

[0040] (C) DSV.sup.4 generates balanced neutralizing titres: FACS-based neutralization titres of DSV.sup.4 Antiserum against specified genotype(s) of each serotype.

[0041] (D) Genotype neutralization breadth with pooled sera FIG. 4: (a) the amino acid sequence of the envelope domain III of DENV-1 (SEQ ID NO:1) and the encoding nucleic acid (SEQ ID NO:5);

[0042] (b) the amino acid sequence of the envelope domain III of DENV-2 (SEQ ID NO:2) and the encoding nucleic acid (SEQ ID NO:6);

[0043] (c) the amino acid sequence of the envelope domain III of DENV-3 (SEQ ID NO:3) and the encoding nucleic acid (SEQ ID NO:7);

[0044] (d) the amino acid sequence of the envelope domain III of DENV-4 (SEQ ID NO:4) and the encoding nucleic acid (SEQ ID NO:8);

[0045] (e) the amino acid sequence of the N-terminal of HBsAg (SEQ ID NO:9) and the encoding nucleic acid (SEQ ID NO:10);

[0046] (f) the amino acid sequence of a recombinant polypeptide of the invention comprising EDIII's from DENV-1, 2, 3 and 4 linked to the N-terminal of HBsAg (SEQ ID NO:11) and the encoding nucleic acid (SEQ ID NO:12), wherein the italicized nucleic acid and amino acid sequences are hexa-glycine linkers and the underlined nucleic acid and amino acid sequences are resulting from translation of KpnI restriction site. The EDIII' s are sequentially order from N-terminus to C-terminus DNV-1, DNV-3, DNV-4 and DNV-2.

DETAILED DESCRIPTION OF THE INVENTION

[0047] The present invention provides a dengue subunit vaccine against all the four serotypes-DENV-1, DENV-2, DENV-3, and DENV-4 serotypes of Dengue virus. The subunit vaccine comprises a recombinant protein comprising tetravalent EDIII-T and HBsAg. The present invention also relates to a subunit vaccine comprising VLP-based quadrivalent vaccine candidate for the prevention of dengue disease against all the four serotypes of DENV.

[0048] In one aspect the invention provides a nucleic acid sequence encoding a recombinant protein comprising the EDIII domain of Dengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4 linked in frame to the N-terminal of HBsAg. The nucleotide sequences encoding each of the EDIII domains of Dengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4 can be linked with the N-terminus of HBsAg in any sequential order.

[0049] Preferably, the nucleic acid sequence encodes EDIII domains of each of Dengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4, which have the amino acid sequences of SEQ ID NO's: 1, 2, 3 and 4 respectively. Preferably, the nucleic acid sequence encoding each of the EDIII domains of Dengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4 is SEQ ID NO's: 5, 6, 7 and 8 respectively.

[0050] Preferably, the nucleic acid sequence encodes a HBsAg having the amino acid sequence of SEQ ID NO:9. Preferably, the nucleic acid sequence encoding HBsAg is SEQ ID NO:10.

[0051] In one embodiment the nucleic acid sequence comprises each of the nucleotide sequences SEQ ID NO's: 5, 6, 7 and 8 linked in frame with the N-terminus of SEQ ID NO: 10. The nucleotide sequences SEQ ID NO's: 5, 6, 7 and 8 can be linked with the N-terminus of SEQ ID NO: 10 in any sequential order.

[0052] Preferably, the nucleic acid sequence encodes a linker that links each of the EDIII domains of Dengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4. Preferably, the nucleic acid encodes a flexible linker, most preferably a hexa-glycine linker. Preferably, the nucleic acid sequence encodes a linker that links the EDIII domains of Dengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4 to the N-terminal of HBsAg. Preferably, the nucleic acid encodes a flexible linker, most preferably a hexa-glycine linker.

[0053] Preferably, the nucleic acid sequence encodes a recombinant polypeptide having the amino acid sequence of SEQ ID NO: 11. Preferably, the nucleic acid sequence encoding the recombinant polypeptide is the nucleic acid sequence of SEQ ID NO:12.

[0054] In one embodiment the nucleic acid sequence is codon optimized for expression in yeast, preferably for expression in P. pastoris. In one embodiment the nucleic acid is an expression vector.

[0055] In one aspect the invention relates to a host cell transformed or transfected with a nucleic acid of the invention, wherein the host cell expresses HBsAg. In one embodiment the host cell is transformed or transfected with a nucleic acid sequence that encodes HBsAg. Preferably, the host cell is transformed or transfected with 1, 2, 3, 4 or more nucleic acid sequences expressing HBsAg. In one embodiment the host cell is a yeast. Most preferably, the host cell is P. pastoris.

[0056] In one aspect the invention provides a recombinant polypeptide comprising the EDIII domain of each of Dengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4 linked to the N-terminal of HBsAg. The amino acid sequences encoding each of the EDIII domains of Dengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4 can be linked with the N-terminus of HBsAg in any sequential order. Preferably, EDIII domains of Dengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4 are fused sequentially, N-terminal to C-terminal, in the sequence DENV-1, DENV-3, DENV-4 and DENV-2

[0057] Preferably, the amino acid sequence of the EDIII domains of each of Dengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4 is SEQ ID NO's: 1, 2, 3 and 4 respectively. Preferably, the N-terminal of HBsAg has the polypeptide sequence of SEQ ID NO:9. Preferably, the recombinant polypeptide comprises each of the amino acid sequences SEQ ID NO's: 1, 2, 3 and 4 linked to the N-terminus of SEQ ID NO:9. The amino acid sequences SEQ ID NO's: 1, 2, 3 and 4 can be linked with the N-terminus of SEQ ID NO: 9 in any sequential order. Preferably, the recombinant polypeptide comprises the amino acid sequences SEQ ID NO's:1, 2, 3, and 4 sequentially, N-terminal to C-terminal, in the sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:2.

[0058] Preferably, the EDIII domains of each of Dengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4 are linked by a linker, preferably a flexible linker, most preferably a hexa-glycine linker. Preferably, the EDIII domains are linked to the N-terminal of HBsAg by a linker, preferably a flexible linker, most preferably a hexa-glycine linker.

[0059] In one aspect the recombinant polypeptide has the amino acid sequence of SEQ ID NO: 11.

[0060] The transformed or transfected host cells of the invention synthesises both HBsAg and the recombinant polypeptide of the invention. The inventors have shown that these two polypeptides spontaneously co-assemble into bio-nanoparticle. In one aspect the invention includes bio-nanoparticle comprising HBsAg and the recombinant polypeptide of the invention of the invention.

[0061] In one aspect the invention provides a method of preparing a recombinant protein or bio-nanoparticle comprising culturing the host cell of the invention under appropriate conditions and recovering the expressed recombinant protein or bio-nanoparticle.

[0062] In one aspect, the invention relates to a vaccine comprising the recombinant polypeptide or the bio-nanoparticles of the invention. Preferably, the vaccine comprises the recombinant polypeptide or the bio-nanoparticle of the invention in a pharmaceutically acceptable carrier or suitable diluent.

[0063] In one aspect, the invention provides a method of treating or preventing Dengue virus, comprising administering to a subject a recombinant protein, a bio-nanoparticle or a vaccine of the invention. In one embodiment the Dengue virus is serotype DENV-1, DENV-2, DENV-3, or DENV-4.

[0064] The tetravalent EDIII-based molecule, EDIII-T, was developed, which was designed to contain all the four EDIIIs linked together through flexible glycyl linkers as depicted in FIG. 1A. EDIII-T was expressed in Pichia pastoris, purified and was found to be immunogenic in mice. The construction of the EDIII-T molecule is provided in the Indian Patent No. 261749.

[0065] The ability of surface antigen of Hepatitis B virus (HBsAg) to serve as a platform for the presentation and display of foreign epitopes is illustrated well by the success of malarial vaccine candidate RTS,S in the Patent Application No. WO 93/10152.

[0066] The present invention explores the possibilities whether HBsAg could serve to increase the immunogenicity of EDIII-T. Therefore, EDIII-T was cloned in fusion with HBsAg and in a background of four expression cassettes of HBsAg in P. pastoris vector as depicted in FIG. 1A. This design of EDIII-T and HBsAg is termed as “DSV.sup.4” and is similar to that of RTS,S in the Patent application no. WO9310152 which displays malarial epitope on HBsAg VLPs.

[0067] The recombinant plasmid was electroporated in P. pastoris and the colonies were screened for the co-expression of EDIII-T-HBsAg and HBsAg proteins by methanol induction of clones. One of the positive clones co-expressing the two proteins as shown in FIG. 1B, was selected for further study. Induced biomass was lysed and the proteins associated with the membrane were extracted and subjected to diafiltration through 300 kDa membrane. This step was designed to allow enrichment of large sized protein considering that the two co-expressed proteins assemble into DSV.sup.4 VLPs. The retentate was purified through phenyl 600M resin with high purity as depicted in FIG. 1C.

[0068] Ability of the co-expressed proteins to assemble into VLPs was evaluated through gel filtration as shown in FIG. 2A, CsCl Ultra-centrifugation as shown in FIG. 2B and electron microscopy as shown in FIG. 2C. It was observed that both the protein components of DSV.sup.4 eluted together in the void volume during gel filtration as shown in FIG. 2A and co-migrated during CsCl ultra-centrifugation as shown in FIG. 2B. On visualization under electron microscope, they were observed to assemble into 25-35 nm VLPs as shown in FIG. 2C.

[0069] The conformational integrity of EDIII of all four DENVs in DSV.sup.4 VLPs was evaluated through recognition of critical EDIII epitopes by well characterized mAbs in sandwich ELISA format.

[0070] Capability of these VLPs to mount a strong immune response against the four DENV serotypes was evaluated by immunization in BALB/c mice as shown in FIG. 3. Purified DSV.sup.4 VLPs were immunized (20 μg/500 μg Al as alhydrogel/100 μl in PBS) in a group of six BALB/c mice intraperitoneally on days 0, 30 and 90. Terminal bleed was taken on day 100 and analysed for response against DSV.sup.4 by ELISA. Sera from positive responders were pooled and characterized for the presence of antibodies against all its five components namely EDIII-1, EDIII-2, EDIII-3, EDIII-4 and HBsAg (FIG. 3B). It was observed that a strong immune response was generated against all of them. It was essential to determine whether anti-dengue response was capable of neutralizing the four DENVs. Therefore, the pooled serum was evaluated for its neutralization capacity through FACS-based assay and it was observed that DSV.sup.4-antiserum was indeed capable of neutralizing all four DENVs (FIG. 3C). FIG. 3A illustrates the design of DSV.sup.4 and the strain from which corresponding EDIII aa sequences were acquired. FIG. 3C illustrates the neutralization titre of DSV.sup.4-antiserum against the four DENVs (of specified strain) and strains of two additional genotypes of DENV-3. It is evident that DENV-2, -3 and -4 genotypes used in neutralization assay varied from the genotypes from which EDIII sequence was acquired, and it did not adversely affect the neutralization capability of DSV.sup.4 antiserum, indicating the high strength of the generated immune response. Moreover, the overall response against the various genotypes also appeared to be balanced. highlighting the candidacy of DSV.sup.4 as a potential dengue vaccine as depicted in FIG. 3D. DSV4 appeared to be efficacious to comparable extent with various adjuvants evaluated (FIG. 3D)

[0071] The present invention is described with reference to the following examples, which are included merely to illustrate and demonstrate the invention. These specific examples should not be construed to limit the scope of the invention in any way.

EXAMPLE 1

Construction of Recombinant VLP-Based Dengue Quadrivalent Vaccine Candidate

[0072] EDIII-T was cloned in fusion with HBsAg and in a background of four expression cassettes of HBsAg in P. pastoris vector as depicted in FIG. 1A. This design of EDIII-T and HBsAg is termed as “DSV.sup.4” The recombinant plasmid was electroporated in P. pastoris and the colonies were screened for the co-expression of EDIII-T-HBsAg and HBsAg proteins by methanol induction of clones.

EXAMPLE 2

Characterization of Dengue Quadrivalent Vaccine Candidate-DSV.SUP.4

[0073] One of the positive clones co-expressing the two proteins as shown in FIG. 1B, was selected for further study. Induced biomass was lysed and the proteins associated with the membrane were extracted and subjected to diafiltration through 300 kDa membrane. This step was designed to allow enrichment of large sized protein considering that the two co-expressed proteins assemble into DSV.sup.4 VLPs. The retentate was purified through phenyl 600M resin with high purity as depicted in FIG. 1C.

EXAMPLE 3

Identification and Characterization of VLPs

[0074] Ability of the co-expressed proteins to assemble into VLPs was evaluated through gel filtration as shown in FIG. 2A, CsCl Ultra-centrifugation as shown in FIG. 2B and electron microscopy as shown in FIG. 2C. It was observed that both the protein components of DSV.sup.4 eluted together in the void volume during gel filtration as shown in FIG. 2A and co-migrated during CsCl ultra-centrifugation as shown in FIG. 2B. On visualization under electron microscope, they were observed to assemble into 25-35 nm VLPs as shown in FIG. 2C.

EXAMPLE 4

Evaluation of Conformational Integrity of All Four DENVs in DSV.SUP.4 .VLPs by mAbs

[0075] The conformational integrity of EDIII of all four DENVs in DSV.sup.4 VLPs was evaluated through recognition of critical EDIII epitopes by well characterized mAbs in sandwich ELISA format. Dengue specific mAbs were coated in microtiter wells and DSV.sup.4 VLPs were added. Bound VLPs were revealed through peroxidase labelled anti-HBsAg Hepnostika. Most of these mAbs were against A-strand and lateral ridge region of EDIII, which are believed to be essential in generating a strong neutralizing immune response. ELISA reactivity of DSV.sup.4 by 21 dengue mAbs (EDIII and non-EDIII specific mAbs) is illustrated in Table 1 and the results indicate that EDIII epitopes of all four DENVs are intact in DSV.sup.4 VLPs.

EXAMPLE 5

Immunization of Mice by Purified DSV.SUP.4 .VLPs

[0076] Capability of these VLPs to mount a strong immune response against the four DENV serotypes was evaluated by immunization in BALB/c mice as shown in FIG. 3. Purified DSV.sup.4 VLPs were immunized (20 μg/500 μg Al as alhydrogel/100 μl in PBS) in a group of six BALB/c mice intraperitoneally on days 0, 30 and 90. Terminal bleed was taken on day 100 and analysed for response against DSV.sup.4 by ELISA. Sera from positive responders were pooled and characterized for the presence of antibodies against all its five components namely EDIII-1, EDIII-2, EDIII-3, EDIII-4 and HBsAg as depicted in FIG. 3B and further Genotype neutralization breadth of DSV.sup.4 antisera generated with various adjuvants was determined as depicted in FIG. 3D

Immune Response

[0077] It was observed that a strong immune response was generated against all of the five components namely EDIII-1, EDIII-2, EDIII-3, EDIII-4 and HBsAg. It was essential to determine whether anti-dengue response was capable of neutralizing the four DENVs. Therefore, the pooled serum was evaluated for its neutralization capacity through FACS-based assay and it was observed that DSV.sup.4-antiserum was indeed capable of neutralizing all four DENVs as shown in FIG. 3C which illustrates the neutralization titre of DSV.sup.4-antiserum against the four WHO reference strains DENVs and strains of two additional genotypes of DENV-3. It is evident that DENV-2, -3 and -4 genotypes used in neutralization assay varied from the genotypes from which EDIII sequence was acquired, and it did not adversely affect the neutralization capability of DSV.sup.4 antiserum, indicating the high strength of the generated immune response. Moreover, the overall response against the various serotypes also appeared to be balanced, highlighting the candidacy of DSV.sup.4 as a potential dengue vaccine. Table 2 below illustrates FNT post depletion on EDIII-3-MBP eliciting serotype specific neutralizing Abs.

[0078] The invention also includes the following specific aspects:

[0079] Aspect 1. A recombinant VLP-based dengue quadrivalent vaccine candidate comprising a tetravalent EDIII-T molecule and the surface antigen of Hepatitis B virus (HBsAg).

[0080] Aspect 2. A recombinant VLP-based dengue quadrivalent vaccine candidate designated DSV.sup.4.

[0081] Aspect 3. The recombinant VLP-based dengue quadrivalent vaccine candidate, DSV.sup.4, wherein the tetravalent EDIII molecule comprises EDIII of DENV-1, DENV-2, DENV-3, and DENV-4.

[0082] Aspect 4. The recombinant VLP based dengue quadrivalent vaccine candidate as recited in aspect 2 of the invention, wherein DSV.sup.4 generates DENV serotype specific neutralizing antibodies against DENV-1, DENV-2, DENV-3 and DENV-4 .

[0083] Aspect 5. A process for the production of recombinant VLP-based dengue quadrivalent vaccine candidate as claimed in claim 1 comprises the steps of:

[0084] i) cloning of EDIII-T in fusion with HBsAg in a recombinant construct carrying four expression cassettes of HBsAg;

[0085] ii) electroporation of recombinant plasmid into Pichia pastoris cells to obtain a clone co-expressing EDIII-T-HBsAg and HBsAg;

[0086] iii) screening for the co-expression of EDIII-T-HBsAg and HBsAg proteins;

[0087] iv) analyzing the expression of EDIII-T-HBsAg and HBsAg proteins by silver staining and western blotting;

[0088] v) lysis of the induced biomass;

[0089] vi) extraction of the proteins associated with the membrane and subjected to diafiltration;

[0090] vii) purification of DSV.sup.4

[0091] Aspect 6. The process as recited in aspect 5, wherein the screening in step (iii) is done by methanol induction of clones.

[0092] Aspect 7. A dengue subunit vaccine comprising a recombinant VLP-based dengue quadrivalent vaccine candidate as recited in Aspect 1.

[0093] Aspect 8. The dengue subunit vaccine as recited in Aspect 7, wherein said vaccine is active against DENV-1, DENV-2, DENV-3, and DENV-4 serotypes of Dengue virus.

[0094] Aspect 9. The dengue subunit vaccine as recited in aspect 8 wherein said vaccine can be administered intraperitoneally or intramuscularly.

[0095] Aspect 10. A recombinant VLP-based dengue quadrivalent vaccine candidate for use as a dengue subunit vaccine candidate comprising of tetravalent EDIII-T molecule and the surface antigen of Hepatitis B virus (HBsAg).