WO2014064707A1

WO2014064707A1 - Pichia pastoris -expressed dengue virus like particles

Info

Publication number: WO2014064707A1
Application number: PCT/IN2012/000707
Authority: WO
Inventors: Shailendra MANI; Lav TRIPATHI; Karthik DHATCHINAMOORTHY; Poornima Tyagi; Sathyamangalam Swaminathan; Navin Khanna; Rahendra RAUT
Original assignee: International Centre For Genetic Engineering And Biotechnology
Priority date: 2012-10-26
Filing date: 2012-10-26
Publication date: 2014-05-01

Abstract

The present invention provides virus like particles comprising recombinant envelop (E) protein of Dengue virus. It also provides gene constructs capable of expressing a polypeptide that elicit type specific immune response in a host against dengue virus. A method for producing recombinant envelop (E) protein is also disclosed.

Description

PICHIA PASTORIS -EXPRESSED DENGUE VIRUS LIKE PARTICLES

FIELD OF THE INVENTION

The present invention relates to highly immunogenic virus like particles (VLPs) comprising recombinant envelop (E) protein of the Dengue virus (DENV). More particularly, the invention relates to the development of recombinant DENV envelope E based Dengue vaccine candidate and a method for producing it using Pichia pastoris expression.

BACKGROUND OF THE INVENTION

Dengue viruses (DENV), members of flavi virus genus of the Flaviviridae family, impose one of the largest social and economic burdens of any mosquito-borne viral pathogens. There are four serologically and genetically related viruses (DENV-1, DENV-2, DENV -3, and DENV-4), each of which can produce dengue disease. DENV infection may be asymptomatic or result in a range of manifestations, from mild dengue fever (DF) to more severe and potentially fatal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Over 2.5 billion people, representing a staggering 40% of world population, in more than 100 countries, are at risk from DENV infection, with at least 50-100 million infections each year.

Several candidate vaccines are now in the pipeline, including live attenuated, inactivated, recombinant, DNA, and viral-vector vaccines, some of which are at advanced stages of clinical development [Swaminathan, Khanna, N. (2009) Dengue; Recent advances in biology and current status of translational research, Current Mol. Med. 9: 152-173]. Despite several years of effort, safe and effective vaccines have not been developed. Attempts to develop live attenuated dengue vaccines or genetically manipulated recombinant flaviviruses have all been unsuccessful because they were either being unstable, or produced low levels of neutralizing antibodies or had the potential to undergo genetic recombination [Seligman, S.J., Gould, E.A. (2004). Live flavivirus vaccines: reasons for caution. Lancet, 363: 2073-2075].

Numerous alternative approaches for the development of recombinant DNA- and protein-based subunit vaccines either using naked plasmid DNA or vectors encoding DENV antigens or proteins expression systems have been made. For a majority of protein based recombinant subunit vaccine developments, the envelope E protein being the major structural protein present on the surface of mature Dengue virions has been the focus. The E protein binds to host cell surface receptor and contains type specific and dominant neutralization determinants [Lindenbach BD, Thiel HJ, Rice CM. (2007). Flavivindae: The viruses and their replication. In Fields of Virology, 5th edition (Eds.-in chief nipe, D.M., Howley, P. M.), pp. 1101-1 152; Wolters Kluwer and Lippincott Williams & Wilkins, Philadelphia.] making it highly efficacious. The E protein contains multiple conformational neutralizing epitopes and can more importantly elicit virus-neutralizing antibodies.

There are several reports of production of recombinant DENV proteins in expression vectors including baculovirus, vaccinia virus and E. colt. However, success with recombinant proteins as vaccines has been variable with problems of purification, or low yield or failure to elicit neutralizing antibodies. Recombinant E proteins of DENVs have been expressed using different expressions systems in the past but most have been associated with issues of low expression levels and improper conformation. Hawaii Biotech has produced a DENV E protein based vaccine candidate using Drosophila Schneider-2 (S2) cell expression system [Clements, D.E., Coller, B.A., Lieberman, M.M., Ogata, S., Wang, G., Harada, K.E., Putnak, J.R., Ivy, J.M., McDonell, M., Bignami, G.S., Peters, I.D., Leung, J., Weeks-Levy, C, Nakano, Ε.Τ.,Μ Humphreys, T. (2010). Development of a recombinant tetravalent dengue virus vaccine: Immunogenicity and efficacy studies in mice and monkeys. Vaccine 28: 2705- 2715]. While the insect cell-expressed recombinant E protein is in its native conformation, it does not form VLPs. The E protein expressed in insect cells using a baculovirus vector tends to form large unorganized aggregates [Kelly, E. P., Greene, J; J. King, A. D., Innis, B. L. (2000). Purified dengue 2 virus envelope glycoprotein aggregates produced by baculovirus are immunogenic in mice. Vaccine 18: 2549- 2559]. Therefore, the design of an effective dengue recombinant antigen with an easy and inexpensive expression system leading to the development of a safe, efficacious, and cost effective dengue vaccine is of utmost importance and urgently needed.

OBJECTS OF THE INVENTION

It is an object of the invention to produce recombinant virus like particles comprising surface (envelop) protein of the Dengue virus. Another object of the invention is to provide an efficient method for the expression of recombinant polynucleotide encoding envelope (E) genes of Dengue virus.

Another object of the invention is to provide an expression system for the expression of recombinant E protein in higher level and with proper confirmation.

Still another object of the invention is to provide a safe, efficacious and inexpensive dengue vaccine.

SUMMARY OF THE INVENTION

The present invention provides highly immunogenic virus like particles (VLPs) comprising recombinant envelop (herein after E) protein of Dengue virus (DENV).

In an embodiment the envelop protein E comprises 393-395 amino acid residues of the ecto-domain of Dengue virus serotypes selected from DENV-1, DENV- 2, DENV-3 or DENV- 4. The ecto-domain has amino acid sequence selected from the sequences having SEQ IDs 1, 2, 3 and 4.

In another embodiment the virus like particles have particle size between 20nm-

80nm.

Another embodiment of the invention provides a polynucleotide sequence encoding a recombinant envelop protein E comprising a pre-membrane (prM) carboxy terminal sequence; a sequence encoding ecto-domain of envelop protein E; and a sequence encoding 6x histidine tag.

Yet another embodiment of the invention provides a recombinant envelop protein E encoded by a polynucleotide sequence comprising a pre-membrane carboxy terminal sequence; a sequence encoding ecto-domain of envelop protein E; and a sequence encoding 6x histidine tag.

In another embodiment said ecto-domain nucleotide sequence is linked to said histidine tag nucleotide sequence through a nucleotide sequence encoding penta- glycine linker.

In another embodiment the ecto-domain nucleotide sequence is selected from sequences having SEQ IDs 5, 6, 7, and 8.

In another embodiment the pre-membrane carboxy terminal sequence is selected from sequences having SEQ IDs 9, 10, I I and 12.

Yet another embodiment of the invention provides a method for producing virus like particles comprising recombinant envelop protein E, said method comprising: transforming host cells with an expression vector comprising a recombinant polynucleotide, wherein the polynucleotide comprises a nucleotide sequence encoding 36 carboxy terminal amino acid residues of pre-membrane domain, followed by a sequence encoding ecto-domain of the Envelop protein and 6x histidine tag; and culturing the transformed host cell under conditions whereby envelop (E) proteins are expressed and assembled into VLPs.

In another embodiment the host cell is Pichia pastoris.

In preferred embodiment the transformed cultures are grown in an environment of methanol at a temperature of 30°C.

Still another embodiment of the invention provides a recombinant envelop protein E having 393-395 amino acid residues.

In another embodiment the recombinant envelop proteins assemble into virus like particles (VLPs).

Yet another embodiment of the invention provides an expression vector comprising a polynucleotide sequence for the expression of recombinant envelop protein E. The vector used is pPICZ A and is a shuttle vector.

In another embodiment the polynucleotide sequence is integrated at the EcoRI and Notl sites of the vector. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Vector construct for expression of recombinant DENV E proteins in P. pastoris. DENV. E gene is cloned in pPICZ A vector (Invitrogen) at EcoRI and Notl sites. The box at N terminus denotes 3' prM sequences and the box at C terminus denotes the 6x histidine tag for purification.

Figure 2: Western blot analyses of DENV E protein expression in P. pastoris.

Total lysates from un-induced (lanes 'UI') and methanol-induced (lanes 'Induced') cultures of P.pastoris clones were subjected to SDS-PAGE, electro-transferred to membranes, and probed using DENV E-specific mAb as a primary antibody and anti IgG HRPO conjugate as a secondary antibody; pre-stained markers were run in the first lane of each panel. Their sizes (in kDa) are indicated to the left of the panels.

Figure 3: Purification profile of DENV- 1 (A), DENV-2 (B), DENV-3 (C) and

DENV-4 (D) E proteins from total cell lysate by affinity-chromatography. The solid curve indicates UV absorbance at 280nm. The dashed curve indicates the imidazole step gradient. Purified peak fractions were analysed by SDS-PAGE gel (inset, iane 2) and confirmed by Western blot analysis (inset, second lane 3). The dashed line in the insets demarcates the SDS-PAGE and Western blot lanes.

Figure 4: The recombinant E protein preparations were negatively stained with uranyl acetate and observed by electron microscopy, DENV E proteins were found to assemble into VLPs, with particle size varying from 20nm to 80nm.

Figure 5: Evaluation of the immunogenic ity of DENV-3 VLPs in Balb/c mice. Anti-DENV-3 antibodies in sera from DENV-3 E VLP-immunized mice collected after the 1st (curve 'b') and second (curve 'c') boosts were analyzed using DENV-3 VLPs coated on the microtiter wells. Captured anti-DENV-3 antibodies were revealed using anti-mouse IgG-HRPO conjugate and TMB substrate. The ELISA reactivity of sera from PBS-immunized control mice is represented by curve 'a'. The y-axis represents ELISA reactivity (absorbance at 450 nm) and x-axis represents sera dilution in thousands.

Figure 6: Evaluation of the immunogenicity of DENV-2 VLPs in Balb/c mice. Anti-DENV-2 antisera from DENV-2 E VLP-immunized mice, collected after the 1st (curve 'b') and second (curve V) boosts were analyzed by ELISA using DENV-2 VLPs coated on the microtiter wells. Captured anti-DENV-2 antibodies were revealed using anti-mouse IgG-HRPO conjugate (1 : 10,000 dilution) and TMB substrate. The ELISA reactivity of sera from PBS-immunized control mice is represented by curve 'a'. The y-axis represents ELISA reactivity (absorbance at 450 nm) and x-axis represents sera dilution in thousands (lk-64k).

Figure 7: Evaluation of the immunogenicity of DENV E VLPs through ELISA: (A) anti-DENV-1 E response (curve a), (B) anti-DENV-4 E response (curve b). The ELISA reactivity of Mock sera (PBS-immunized mice) is represented by curve "c" in both graphs. The y-axis represents ELISA reactivity (absorbance at 450 nm) and x-axis represents sera dilution in thousands.

Figure 8: Determination of serum virus-neutralizing antibody titres in Balb/C mice immunized with recombinant DENV-2 E VLPs. Serial dilutions of immune sera were separately pre-incubated with each of the four DENV serotypes, followed by determination of inhibition of virus infectivity using a FACS-based assay. The serum dilution causing 50% inhibition is expressed as FNT50 value on the y-axis.

Figure 9: DENV-2 enhancing antibody titers in antisera raised using recombinant DENV-2 and DENV-3 E VLPs. DENV-2 was pre-incubated with serial dilutions of anti-DENV-2 E antiserum (curve 'b'), anti-DENV-3 E (curve 'c') antiserum or PBS-immunized antiserum (curve 'a') and used to infect 562 cells. A control DENV-2 infection (spot'd') in which antibody pre-incubation was omitted (No Ab) was done in parallel. FACS analysis was done 24 hours later to determine the percentage of infected cells.

Figure 10: DENV-2 enhancing antibody titers in antisera raised using recombinant DENV-3 E VLPs and recombinant DENV-3 EDIII. DENV-2 was pre- incubated with serial dilutions of anti-DENV-3 E antiserum (curve 'c'), anti-DENV-3 EDIII antiserum (curve 'b') or PBS-immunized antiserum (curve 'a') and used to infect K562 cells. A control DENV-2 infection (spot d) in which antibody pre-incubation was omitted (No Ab) was done in parallel. FACS analysis was done 24 hours later to determine the percentage of infected cells.

Figure 1 1 : AG 129 mice that were immunized with PBS (diamonds) and recombinant DENV-2 E VLPs (circles) were challenged with a lethal dose of virulent DENV-2, followed by monitoring of survival rates over a period of 18 days post- challenge. The upper-most curve (squares) shows the corrected survival curve (taking into account only the 6 mice that displayed anti-DENV-2 antibodies in ELISA).

Figure 12: Nucleotide sequence of the synthetic DENV-1 E gene, codon- optimized for expression in P.pastoris. The prM carboxy terminal sequence, the penta- glycine linker and 6x His tag are highlighted in grey, green and yellow, respectively. The start and stop codons are underlined. Restriction sites for cloning are shown in italics.

Figure 13: Nucleotide sequence of the synthetic DENV-2 E gene, codon- optimized for expression in P.pastoris. The prM carboxy terminal sequence, the penta- glycine linker and 6x His tag are highlighted in grey, green and yellow, respectively. The start and stop codons are underlined. Restriction sites for cloning are shown in italics.

Figure 14: Nucleotide sequence of the synthetic DENV-3 E gene, codon- optimized for expression in P.pastoris. The prM carboxy terminal sequence, the penta- glycine linker and 6x His tag are highlighted in grey, green and yellow, respectively. The start and stop codons are underlined. Restriction sites for cloning are shown in italics.

Figure 15: Nucleotide sequence of the synthetic DENV-4 E gene, codon- optimized for expression in P.pastoris. The prM carboxy terminal sequence, the penta- glycine linker and 6x His tag are highlighted in grey, green and yellow, respectively. The start and stop codons are underlined. Restriction sites for cloning are shown in italics.

Figure 16: Amino acid sequences of recombinant DENV-1 E (A), DENV-2 E (B), DENV-3 E (C) and DENV-4 E (D) proteins predicted to be encoded by the P.^astora-optimized DENV-1 E, DENV-2 E, DENV-3 E and DENV-4 E synthetic genes, respectively, shown in the preceding pages. The C-terminal prM sequences preceding the E sequences are highlighted in grey. The penta-glycine linker and 6x-His tag engineered into the C-terminus are highlighted in green and yellow, respectively. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides highly immunogenic virus like particles (VLPs) comprising recombinant envelop (E) protein of Dengue virus (DENV). The recombinant protein comprises ecto-domain of the envelop protein E of Dengue virus of serotypes 1, 2, 3 and 4. The envelope E protein being the major structural protein present on the surface of mature Dengue virions.

In an embodiment the invention provides gene constructs capable of expressing a polypeptide that can elicit type specific immune response in a host against dengue virus. The gene constructs comprise sequences encoding carboxy terminal amino acid residues for pre-membrane (prM) sequence linked to the encoding sequence corresponding to ecto-domain of the dengue envelope E proteins and a 6x histidine tag encoding sequence. The ecto-domain of said envelope E protein is linked to said histidine tag (SEQ ID 26) through a penta-glycine linker (SEQ ID 25). The nucleotide sequences encoding penta-rglycine linker are given in sequence listing under SEQ IDs 17, 18, 19 and 20.The nucleotide sequences encoding histidine tag are given in sequence listing under SEQ IDs 21, 22, 23, and 24.

The polynucleotide encoding the ecto-domain of the envelop/surface protein of either serotype of the Dengue virus is selected from DENV-1, DENV-2, DENV-3 or DENV- 4 has sequence of SEQ IDs 5, 6, 7 and 8, respectively. The corresponding amino acid residues have sequences with SEQ IDs 1, 2, 3 and 4.

The polypeptide expressed by the gene constructs of present invention possesses

36 carboxy terminal amino acids of pre-membrane (prM), which is the signal sequence for the envelop protein. This sequence helps in the translocation of protein from cytosol to ER lumen. The signal sequence gets cleaved at trans golgi network during process. The signal sequences used in the gene constructs are different and are specific for the ectodomain sequence taken from serotype of the Dengue virus.

The nucleotide sequences and amino acid sequences of the prM carboxy terminal sequence used for different type of DENV serotypes are given below:

DENV -1

Nucleotide: sequence (SEQ ID 9)

ATGGTCTTTACTGTTATTGCCTTGTTCCTTGCTCATGCCATTGGTACTTCA ATCACTCAAAAGGGAATCATCTTTATrrTGCTTATGTTGGTTACTCCAAG TATGGCT

Amino acid sequence (SEQ ID 13)

MVFTVIALFLAHAIGTSITQKGIIFILL LVTPSMA

DENV -2

Nucleotide sequence (SEQ ID 10)

ATGGTTT TTACAATCATGGCTGCCATT CTTGCTTACACCATTGGTACTA CACATTTTCAAAGAGCCTTGATCTTCATTTTGCTTACTGCAGTTGCTCCA TCTATGACA

Amino acid sequence (SEQ ID 14)

MVFTIMAAILAYTIGTTHFQRALIFILLTAVAPSMT

DENV -3

Nucleotide sequence (SEQ ID 11)

ATGGTCTTTACGATCTTAGCCTTATTTCTTGCCCACTATATCGGTACATC ATTAACTC AAAAGGTAG TC AT ΑΤΊΓΓ ATTCTTTTG ATGCTTGTC ACTCC A TCTATGACA

Amino acid sequence (SEQ ID 15)

MVFTILALFLAHYIGTSLTQKVVIFILLMLVTPSMT DENV -4 ;

Nucleotide sequence (SEQ ID 12)

ATGGTTTTTGCCTTGCTTGCAGGTTTCATGGCCTACATGATTGGTCAAACA GGAATC CAGAGAACCG TCTTTTTCGT TCTTATGATGTTGGTTGCTCCATC ATATGGT Amino acid sequence (SEQ ID 16)

MVFALLAGFMAYMIGQTGIQRTVFFVLMMLVAPSYG The invention provides encoding nucleotide sequences that express recombinant

DENV E proteins of dengue virus-subtype 1, or dengue virus-subtype 2 or dengue virus-subtype 3 or dengue virus-subtype 4 or a combination of all. The sequences of the four synthetic DENV E genes are given in Figures 12-15. The amino acid sequences encoded by synthetic DENV E genes are given in Figure 16.

Another aspect of the invention provides vector constructs containing the

DENV E genes. The gene constructs are codon optimized for expression in P.pastoris and cloned into pPICZ A at EcoRl and Notl site. The plasmid vectors are electroporated into P.pastoris and analyzed for expression. Clones of P.pastoris harbouring the E genes of each subtype are identified. Plasmid pPICZ A is a shuttle vector.

The recombinant envelope E protein . specific for a dengue virus subtype expressed by the gene constructs form yet another aspect of the invention. P.pastoris clones harbouring the E gene of each subtype are cultured and analyzed for expression of the DENV E proteins by immunoassay using monoclonal antibody (mAb) specific for each of the E protein for DENV-1, DENV-2, DENV-3 and DENV-4. The expressed DENV E proteins are purified by affinity chromatography and fractions analyzed by Western blot method. The amino acid sequences of the proteins encoded by these .genes are given in Figure 16.

In some embodiment of the invention, the purified DENV recombinant E proteins further assembles into highly immunogenic virus like particles (VLPs). The formation of VLP without the co-expression of prM is unexpected. These VLPs are highly immunogenic and have the potential to elicit DENV neutralizing antibodies and confer protection against lethal DENV challenge in AG 129 model. The VLPs consist of 393-395 amino acid residues of ecto-domain of Dengue virus serotypes viz DENV-1, DENV-2, DENV-3 or DENV- 4.

A VLP-based tetravalent dengue vaccine can also be developed by a person skilled in the art from the teachings of the present invention.

Another aspect of the invention provides methods of producing virus neutralizing antibodies against specific subtype of dengue virus comprising immunization of a host with the DENV E VLP of the invention. Immunization can comprise the use of one or more adjuvants well known in the art. immune sera of mice immunized with recombinant proteins could neutralize the infectivity of DENV in Plaque Reduction Neutralization Tests (PRNT). The type-specific antibodies produced against the recombinant E proteins of this invention can also be used for the passive immunization as well as for other therapeutic use. The use of recombinant E proteins of the present invention as diagnostic antigens in immunoassays forms another aspect of this invention.

The recombinant E proteins of the invention may also be used directly in vaccine formulations by a person skilled in the art. Compositions containing DENV rE proteins of the invention in conventional forms well known in the art are also encompassed in this invention. The vaccines of the invention contain an immunogenically effective amount of the recombinant E protein as an active ingredient. The vaccine may be introduced into a subject optionally with adjuvant.

The present invention is described with reference to the figures and examples, which are explained by way of illustration only and should not be construed to limit the scope of the present invention.

Example 1: Cloning of DENV E proteins

The E genes of DENVs, codon-optimized for P.pastoris expression, were synthesized by Genscript and Biobasic Inc. The genes contain sequences encoding 36 carboxy terminal amino acid (aa) residues of prM followed by the first 395 aa residues of E (393 aa in the case of DENV-3 E), representing the ectodomain, and a 6x histidine tag. A penta-glycine linker was inserted between the ectodomain and the 6x histidine tag. The sequences of the four synthetic DENV E genes are presented in Figures 12-15. The amino acid sequences of the proteins encoded by these genes are given in Figure 16. The DENV E genes were cloned into pPICZ A (shuttle vector for P.pastoris) at EcoRl and Notl sites (Figure 1). The plasmids were electroporated into P.pastoris (KM71H cells) and analyzed for gene integration by colony PCR and for expression of the recombinant DENV E proteins using DENV E-protein specific monoclonal antibodies (mAbs). Based on this four P.pastoris clones, each harbouring the E gene of one DENV serotype have been identified.

Example 2: Expression of DENV E proteins Shake-flask cultures of the four P.pastoris clones were set up to reach log phase. Expression of DENV E protein was carried out at 30°C, 250 rpm by adding 1% methanol every 12 hrs. The cultures were induced for 72 hours and 1 ml of each induced culture was collected every 24 hrs. Expression of DENV E (~48kDa) proteins was analysed in induced biomass and confirmed by Western blot analysis using an in- house mAb specific for the carboxy-terminal part of the E protein for DENV-1, -2 and - 3 and a commercial mAb for DENV-4 (Figure 2).

Example 3: Purification

Induced pellets were suspended in lysis buffer (Tris/NaCl buffer pH8.5) and lysed in a Dynomill (WAB). The resultant lysate was clarified and the DENV E proteins were purified by affinity Ni-NTA chromatography under denaturing condition using an imidazole step gradient (from 0 to 500 mM) (Figure 3). Fractions were analyzed by SDS-PAGE and peak fractions were pooled and dialysed against PBS and stored at -80°C. SDS-PAGE profiles and Western blot analyses of the purified recombinant DENV-E proteins are also shown in the insets.

Example 4: Electron microscopic study

Electron microscopic study showed that DENV E proteins of all four serotypes assemble into VLPs with particles sizes varying from 20 nm-80 nm (Figure 4). This is an interesting discovery given the notion that co-expression of prM is also required to observe VLP formation [(Wang, P. G., Kudelko, M, Lo, J, Siu, S. Y. L., Kwok, K. T. H., Sachse, M., Nicholls, J. M., Bruzzone, R., Altmeyer, R. M., Nal, B. (2009). Efficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prM and E proteins. PLoS One 4: e8325; Kuwahara, M.m Konishi, E. (2010). Evaluation of extracellular subviral particles of dengue virus type 2 and Japanese encephalitis virus produced by Spodoptera fugiperda cells for use as vaccine and diagnostic antigens. Clin. Vac. Immunol. 17: 1560-1566)]. Example 5: Immunological evaluation

In order to evaluate the immunogenicity of these DENV E VLPs produced in

P.pastoris, these were used to immunize Balb/c mice on days 0, 30 and 60. The antigens were formulated in alum. Immune sera were collected 1 week after the 1st and

2nd booster immunizations and assessed by ELISA. Figure 5 shows the results of ELISA performed with sera from DENV-3 E VLP-immunized mice. In this ELISA, the microtiter wells were coated with DENV-3 VLPs to capture polyclonal DENV-3 antibodies from the immune sera and revealed with anti-mouse IgG-HRPO in conjunction with TMB substrate. This showed that the DENV VLPs are highly immunogenic. A comparison of the ELISA titers of sera obtained from the first and second booster immunizations show that there is a clear boosting effect. This was evident with DENV-2 VLP immunization as well, as shown in Figure 6. The immunization schedule in this experiment was slightly different in that the first and second booster immunizations were spaced 2 months apart. Taken together, the data in Figures 5 and 6 suggest that while a second boost clearly augments ELISA titers, the time gap between the booster immunization is not very critical. A similar observation was made with the remaining two DENV-1 and DENV-4 proteins as well (Figure 7).

To assess if the antibodies induced by the recombinant DENV-E proteins can effectively block DENV infection, sera from Balb/C mice immunized with the different antigens (on days 0, 30 and 90) were analyzed in a FACS-based virus neutralization assay, A typical experiment using sera from DENV-2 E immunized mice is shown in Figure 8. In this experiment, Vero cells were infected with the WHO reference strains of DENV-1, DENV-2, DENV-3 and DENV-4, separately, before and after preincubation with serial dilutions of the anti-DENV-2 E antiserum. The number of infected cells in each case was determined by FACS analysis. These data were used to calculate the neutralization titers in terms of the antiserum dilution required to inhibit DENV infectivity by 50% in the FACS assay. The FACS based Neutralization titers required for 50% inhibition is designated as the FNT50 titer. The data in Figure 8 show that recombinant DENV-2 E protein-immunized Balb/C mice mount potent virus- neutralizing antibodies against DENV-2 (FNT50=1328), with relatively very low titers against DENV-4. There was no neutralizing activity against the remaining two DENV serotypes. The data suggest that the P.pastoris-expressed recombinant DENV E proteins have the potential to elicit homotypic neutralizing antibodies. This, in turn, suggests that a mixture of the 4 recombinant DENV E proteins could elicit a tetravalent immune response targeting each one of the four DENV serotypes.

In the next set of experiments, we analyzed the immune sera for its potential to mediate antibody-dependent enhancement (ADE) of DENV infection. For this, DENV-

2 was pre-incubated, separately, with serial dilutions of murine antiserum raised against recombinant DENV-2 E (homologous) or DENV-3 E (heterologous) and used to infect K562 cells which contain Fc receptors (FcR). The magnitude of infection was monitored by FACS analysis. The data are presented in Figure 9. K562 cells are not very receptive to DENV infection. Baseline infectivity of K562 cells by DENV-2 was determined either in the absence of any serum (spot 'd') or in the presence of sera from PBS (mock)-immunized animals (curve 'a'). When DENV-2 was pre-incubated with heterologous antiserum (anti-DENV-3 E), an enhancement effect was noticed at a dilution of 1:10⁴. On the other hand, pre-incubation with homologous anti-DENV-2 E antiserum, produced a similar effect at a dilution of 1:10⁵. This suggested that the ADE capacity of homologous antiserum is an order of magnitude lower than that of heterologous antiserum. Interestingly, even this potential for ADE was considerably minimized when DENV-2 was pre-incubated with antiserum raised against P.pastoris- expressed recombinant DENV-3 EDIII antigen.

Exam pie 6 : Protective efficacy in AG 129 challenge model

To test the protective efficacy of the recombinant DENV E antigens produced using P.pastoris, the DENV-susceptible AG129 mice were used (Johnson, A. J., Roehrig, J. T. (1999). New mouse model for dengue virus vaccine testing. J. Virol. 73: 783-786). These mice were immunized with recombinant DENV-2 E antigen formulated in alum on days 0, 30 and 90 and challenged on day 102 with a virulent strain of DENV-2 (1.4xl0⁸ PFU/mouse) obtained by alternate passaging of DENV-2 strain NGC between C6/36 mosquito cells and AG 129 mice as described previously (Shresta, S., Sharar, K. L., Prigozhin, D. M., Beatty, P. R., Harris, E. (2006) Murine model for dengue virus-induced lethal disease with increased vascular permeability. J. Virol. 80: 10208-10217). The data are shown in Figure 11. The data showed that all PBS (mock)-immunized mice succumbed to DENV-2 challenge by day 5. In contrast, recombinant DENV-2 E immunized mice showed -50% survival. Analysis of the sera from AG 129 mice (drawn one week after the final immunization) by ELISA showed that 3 of the 9 immunized mice failed to produce any antibodies against DENV-2 E. Excluding these three non-responders resulted in a protective efficacy of ~70%. The data indicate that P.pastoris expressed recombinant DENV-2 E protein can elicit antibodies that can confer protection against lethal virus challenge in this model.

Claims

We Claim:

1. A virus like particle comprising recombinant envelop E protein of Dengue virus (DENV).

2. The virus like particle as claimed in claim 1, wherein said envelop protein E comprises 393-395 amino acid residues of the ecto-domain of Dengue virus serotypes selected from DENV-1, DENV-2, DENV-3 or DENV- 4.

3. The virus like particle as claimed in claims 1 and 2, wherein said ecto-domain has amino acid sequence selected from the sequences having SEQ IDs 1, 2, 3 and 4.

4. The virus like particle as claimed in claims 1-3, having particle size ranging from 20nm-80nm.

5. A polynucleotide sequence encoding a recombinant envelop protein E comprising: a pre-membrane carboxy terminal sequence;

a sequence encoding ecto-domain of erivelop protein E; and

a sequence encoding 6x histidine tag.

6. The polynucleotide sequence as claimed in claim 5, wherein said ecto-domain sequence is linked to said histidine tag sequence through a sequence encoding penta-glycine linker.

7. The polynucleotide sequence as claimed in claims 5-6, wherein said ecto- domain sequence is selected from sequences having SEQ IDs 5, 6, 7, and 8.

8. The polynucleotide sequence as claimed in claim 5, wherein said pre-membrane carboxy terminal sequence is selected from sequences having SEQ IDs 9, 10, 11 and 12.

9. A recombinant envelop protein E encoded by a polynucleotide sequence comprising: a pre-membrane carboxy terminal sequence; a sequence encoding ecto-domain of envelop protein E;

and a sequence encoding 6x histidine tag.

10. The recombinant protein E as claimed in claim 9, wherein in said polynucleotide sequence the ecto-domain sequence is linked to said histidine tag sequence through a sequence encoding penta-glycine linker.

11. The recombinant protein E as claimed in claim 9, wherein in said polynucleotide sequence the ecto-domain sequence is selected from sequences- having SEQ IDs 5, 6, 7, and 8.

12. The recombinant protein E as claimed in claim 9, wherein in said polynucleotide sequence the pre-membrane carboxy terminal sequence is selected from sequences having SEQ IDs 9, 10, 1 1 and 12.

13. The recombinant protein E as claimed in claims 9-12, wherein said protein comprises 393-395 amino acid residues.

14. The recombinant protein as claimed in claim 13, wherein said proteins assemble into virus like particles (VLPs).

15. A method for producing virus like particles comprising recombinant envelop protein E , said method comprising: a) transforming host cells with an expression vector comprising a recombinant polynucleotide, wherein the polynucleotide comprises a nucleotide sequence encoding 36 carboxy terminal amino acid residues of pre-membrane domain, followed by a sequence encoding ecto-domain of the Envelop protein and 6x histidine tag.

b) culturing the transformed host cell under conditions whereby envelop (E) proteins are expressed and assembled into VLPs.

16. The method as claimed in claim 15, wherein said ecto-domain is selected from the envelop protein of the Dengue virus of either serotypes 1, 2, 3, and 4 having nucleotide sequences of SEQ IDs 5, 6, 7, and 8.

17. The method as claimed in claims 15-16, wherein the polynucleotide of ecto- domain encodes 393-395 amino acid residues.

18. The method as claimed in claim 15, wherein said host cell is Pichia pastoris.

19. The method as claimed in claim 15, wherein in step (b) the transformed cultures are grown in an environment of methanol at a temperature of 30°C.

20. An expression vector comprising a polynucleotide sequence for the expression of recombinant envelop protein E claimed in claims 5-8, wherein said vector is a shuttle vector pPICZ A.

21. An expression vector as claimed in claim 20 wherein said polynucleotide sequence is integrated at the EcoRl and Notl sites of the vector.