WO2007034507A2 - Tetravalent dengue specific domain iii based chimeric recombinant protein - Google Patents

Abstract

A single recombinant Tetravalent Domain III (rTDIII) protein for use in the detection and or diagnosis of any or all of dengue specific Immunoglobin M anti- dengue IgM and Immunoglobin G anti-dengue IgG antibodies is disclosed. Said protein comprises domain III of envelope protein from all four serotypes of dengue virus, namely, Dengue-virus type-1, Dengue-virus type-2, Dengue-virus type-3 and Dengue- virus type-4, linked with each other through penta glycine linkers and codon optimized for expression in an expression vector. The protein of the presentb invention shows a very high degree of sensitivity and specificity to anti-dengue IgM and anti-dengue IgG antibodies.

Description

TETRAVALENT DENGUE SPECIFIC DOIVIAIN HI BASED CHIMERIC RECOMBINANT PROTEIN

Field of invention The present invention relates to tetravalent dengue specific Domain III based chimeric recombinant protein as dengue diagnostic intermediates of high specificity for the detection of both anti-dengue Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies. The present invention also relates to novel kits and reagents for diagnosis of Dengue viral infections. In particular, the present invention relates to novel kits for diagnosis of the four known closely related, antigenically distinct serotypes of Dengue virus. More particularly, the present invention relates to multiepitope recombinant proteins and their use in the diagnosis of dengue and other viral infections. More particularly, the present invention relates to a novel diagnostic reagent and usage thereof for development of IgM and IgG ELISAs for diagnosis of dengue viral infection. In particular, the present invention relates to a single recombinant tetravalent domain III protein which is capable of detecting both anti-dengue IgM and IgG antibodies in human serum specimens, processes for their preparation and uses thereof. Background of the invention

Dengue infection has been one of the most important resurgent mosquito-borne tropical viral diseases in the past two decades, with expanding geographical distribution of both the viruses and the mosquito vectors, increased frequency of epidemics, the development of hyperendemicity and emergence of Dengue Haemorrhagic Fever (DHF) in new areas (Gubler, 1997; Gubler, 1998). The estimated number of 50-100 million infections per year results into 250 000-500 000 cases of DHF and 25 000-50 000 deaths (Gibbons and Vaughan, 2002).

Infants and young children may have an undifferentiated febrile disease, often with a maculopapular rash in the primary infection (WHO, 1997). In older children and adults the more classical Dengue Fever (DF) is seen (Lam, 1995). After an incubation period of 2-7 days, there is sudden onset of fever (39.5-41⁰C), which can last for 5-7 days. This is usually accompanied by severe myalgia, frontal or retro-orbital headache and a flushing of the face. A transient generalized rash occurs in some cases during the first 24 hours of fever. In addition, nausea, vomiting, taste aberrations and pronounced anorexia may occur together with cutaneous hyperesthesia or hyperalgesia (Oh, 1998). In epidemics, DF may be accompanied by bleeding complications, such as epistaxis. gingival bleeding, gastrointestinal bleeding, haematuria and menorrhagia. In the absence of signs of plasma leakage these patients do not meet the criteria for having DHF. Unusually severe bleeding can cause death in some cases (Lam, 1995; WHO, 1997). A clinical definition of DHF was established by WHO based on the presence of high continuous fever, haemorrhagic manifestations (including at least positive tourniquet test), hepatomegaly, thrombocytopenia and haemoconcentration. Haemorrhagic manifestations may vary from a positive tourniquet test to obvious skin patches. The major pathophysiological change that differentiates DHF from DF is the leakage of plasma as manifested by a rising haematocrit value, the presence of serous effusion or hypoproteinaemia. Clinical laboratory findings are important for the presumptive diagnosis of DHF. The 2 important findings are thrombocytopenia (100 000/nim³) and haemoconcentration (haemotocrit increased by >20%) (WHO, 1997).

Dengue Shock Syndrome (DSS) can occur in up to 30% of DHF patients (Oh, 1998). In DSS, the patient's condition suddenly deteriorates after 3-7 days of fever. The patient develops circulatory failure/shock; skin becomes cool, blotchy and congested Avith cold extremities accompanied by dyspnoea, circumoral and peripheral cyanosis and restlessness (Lam, 1995; WHO, 1997; Oh, 1998). DSS is characterized by a rapid and weak pulse, hypotension and narrowing of plasma pressure (<20 mmHg). Diagnosis of dengue infection in endemic areas mainly based on clinical presentation of patients can cause confusions with other viral diseases. Therefore, laboratory-based diagnosis is important for effective treatment which can prevent the onset of irreversible shock and reduce the case fatality rate. Laboratory diagnosis of dengue viral infection can be performed by virus isolation, antigen detection and serology for detection of anti-dengue antibodies and molecular assays for the detection of viral genome (Igarashi et aL, 1995; Gubler, 1997; Ling and Doraisingham, 1998). Virus isolation and Reverse Transcription Polymerase Chain Reaction (RT-PCR) assays are mainly used for the detection of dengue viruses in the blood during viremic/early period of infection (1-5 days of fever)- Virus isolation is important for epidemiological information but few laboratories have access to adult mosquitoes, cell culture facilities and/or a fluorescent microscopy (Tan et aL, 1994; Harris et aL, 1998). Another problem with this method is that it is a time consuming procedure, thus limiting its usefulness for management of patients and the need for specialized training and laboratory conditions (Tan et aL, 1994; Chanyasanha et aL, 1995). Polymerase chain reaction (PCR)-based methods (Morita et al., 1991 ; Chow et al., 1993; Seah et ah, 1995), which offer distinct advantages of accuracy, rapidity, sensitivity and specificity of detection, and typing of dengue virus in clinical specimens, has some disadvantages. The major disadvantage of PCR-based assays is that it is subject to amplicon contamination and it requires technical expertise which may not be available in laboratories that are not research oriented (Vorndam and Kuno, 1997). Economically, however RT-PCR is a rapid, but expensive test (Chanyasanha et al., 1995).

Serological tests which detect dengue specific antibodies are the commonest methods of laboratory confirmation of dengue virus infection in most laboratories (Ling and Doraisingham, 1998) because of the difficulty of virus isolation and PCR-based assays. Anti-dengue (IgM and IgG) antibodies rise significantly in the late phase of infection (after 5-10 days of fever). HaemAgglutination Inhibition (HAI) assay (Clarke and Casals, 1958; Sever, 1962) is considered as the gold standard assay for diagnosis of dengue by the World Health Organization (WHO, 1986). Diagnosis using HAI assay depends upon demonstration of a significant rise in IgG antibodies between acute phase and convalescent phase and therefore, needs paired serum samples (WHO, 1999). The level of HAI antibody in paired serum samples indicates whether it is a primary or secondary dengue infection (Table 1) (WHO, 1997). However, difficulty is encountered with the collection of paired sera (Lam, 1995) and it is time consuming, where at least 2 days are required to complete the assay (Sangkawibha, 1994). This situation prevents the assay from providing clinicians with rapid information about the diagnosis. Results of HAI assay based on a high titre of IgG antibody (>2560), in a single serum sample in the secondary infection provides a probable diagnosis of dengue. Some of the disadvantages of HAI assay have been overcome with the advent of IgM capture ELISA (Burke et al, 1982; Bundo and Igarashi, 1985; Lam et al., 1987; Innis et al., 1989) on a single serum specimen collected between 5-10 days of infection which provides diagnosis of past and/or current dengue viral infection. This is the commonly used method to detect anti-dengue antibodies in patients serum. Immunoglobulin¹ M antibodies are produced transiently during both primary and secondary dengue infections (Lam, 1995). The main advantage in the IgM ELISA is the availability of results within a few hours from an acute serum specimen (Sinniah and Igarashi, 1995). This technique also has the advantage of its sensitivity, potential for automation and the ability to accommodate a large number of samples (Ling and Doraisingham, 1998). Table 1. Interpretation of haemagglutination inhibition antibody response (WHO, 1997)

Most of the currently available commercial dengue diagnostic kits rely on the use of whole dengue virus antigen as dengue diagnostic intermediate, and are consequently associated with false positives due to serologic cross-reactivity with other flaviviruses, high cost of production and biohazard risk (AnandaRao et al., 2005). The dengue viruses are classified within the family Flaviviridae, genus Flavivirus, which consists of more than 60 arboviruses, including other important human pathogens such as yellow fever virus and Japanese encephalitis virus (Lindenbach and Rice, 2001). Because the dengue viruses occur throughout tropical and subtropical areas of the world, their distribution overlaps with other human pathogenic flaviviruses. This can complicate the interpretation of serologic results for dengue infections, since all flaviviruses share antigenic determinants that induce cross-reactive antibodies. (Simmons et al., 1998).

Recombinant proteins can be used in diagnostic assays for dengue to overcome safety issues associated with use of whole virus lysate (Halstead, 1997). The dengue virus has a genome with a single-stranded, positive strand Ribonucleic Acid (RNA) surrounded by an icosahedral nucleocapsid and covered by a lipid envelope. The genome is composed of 3 structural protein genes namely: Core (C), pre-Membrane (prM) and an Envelop (E), and 7 Non Structural (NS) genes (WHO, 1997) (Fig.l). Antibodies to C₅ prM, E, NSl and NS3 have been detected in dengue infected patients (Churdboonchart et αl., 1991; Se-Thoe et αl., 1999; Valdes et αl., 2000; Cardosa et αl., 2002). The E-glycoprotein is the major structural component (Simmons et αl., 2001) and the most immunogenic of the dengue viral proteins, eliciting the long-lasting antibodies (Churdboonchart et αl., 1991; Innis, 1997). From the perspective of developing diagnostic antigens, E protein is important as it carries numerous immunodominant epitopes (Innis et αl., 1989; Trirawatanapong et αl., 1992; Hung et αl,, 1999). Recently, one of the leading manufacturer of dengue diagnostic kits have replaced the 4 whole dengue virus antigens with 4 dengue virus recombinant envelope proteins expressed in a eukaryotic expression system. Although, the diagnostic antigens produced through this approach pose no biohazard risks, they are still associated with higher costs of production and lower specificity. The higher cost is due to the need of expressions and purifications of 4 recombinant E proteins, while the specificity is compromised as these complete E proteins contains immunogenic epitopes which are cross-reactive to other flaviviruses, like YF, JEV and TBE viruses etc. There is thus, currently a need for developing cost-effective, simple and rapid ELISA that combines high sensitivity and specificity. Objects of the invention

It is therefore, an important object of the present invention to provide cost- effective, simple and rapid ELISA that combines high sensitivity with high specificity. It is another object of the present invention to provide a single recombinant diagnostic material, which is simple, cost effective, rapid and capable of detecting both anti-dengue IgM and IgG antibodies in human serum specimens without picking antibodies against other flaviviruses.

It is a further object of the present invention to provide a multi-epitope dengue diagnostic kit capable of detecting both anti-dengue IgM and IgG antibodies in human serum specimens which avoids the drawbacks of the prior art. Summary of the invention

Earlier, the applicants had designed and expressed two novel recombinant multiepitope proteins by assembling key immunodominant, short (7-20 aa residue long), linear and dengue specific epitopes. These epitopes were chosen on the basis of pepscan analysis, phage display and computer predictions. The two proteins were expressed to high levels in Escherichia coli and utilized as dengue diagnostic antigens. One of this antigen contained epitopes from dengue structural and non-structural proteins and it was useful for the detection of anti-dengue IgG response in patient sera. The other antigen was limited to contain linear N terminal immunodominant epitope only from non-structural protein 1 (NSl) of all 4 dengue serotypes. The NSl multiepitope protein was found to be useful for the detection of anti-dengue IgM antibodies in dengue infected patients. Such novel recombinant multiepitope proteins and diagnostic kits based on proteins were described in the applicants' International Patent Publication No.PCT WO 2005/014627 A 1.

The present invention on the other hand, provides a novel single recombinant Tetravalent Domain III (rTDIII) protein, which has the ability to detect both anti- dengue IgM and IgG antibodies with high sensitivity and specificity. This protein contains Domain III of envelope protein from all four serotypes of dengue virus linked with each other through penta glycine linkers. The protein is expressed in E. coli and purified by immobilized-metal affinity chromatography (IMAC). The purified tetravalent protein has been found to have an absolute specificity for the detection of dengue virus infection in an ELISA. This single tetravalent Domain III based protein is capable of being utilized in the detection of anti-dengue IgM and IgG antibodies to all four serotypes of dengue virus. To prove the above fact, before testing this tetravalent protein against patient sera, this protein was tested with polyclonal antibodies raised in mouse against individual dengue viruses (DEN 1-4), which recognized this designer protein equally well. This shows that there is no steric hindrance between adjacent Domain Ills, linked by flexible glycine linker. Individual Domain III fragments of all four dengue serotypes have been expressed as fusion protein with TrpE (Simmons et a!., 1998; Fonseca, 1991) or maltose binding proteins (Simmons et al., 2001) in E. coli. A physical mixture of these four fusion proteins has been found to be useful as a dengue diagnostic intermediate (Simmons et al., 1998). Ludolfs et al. (2002) have been used individual Domain III in immunoblot strips format. Unlike, prior art, the applicants have not employed E. coli proteins as fusion partners. Instead, the applicants have fused these domains with each other by penta glycine flexible linkers. Most surprisingly and unexpectedly, this approach permitted expression of Domain III of envelope protein from all four serotypes of dengue virus as a single protein. Apart from the cost effectivity of single tetravalent antigen, the physical mixture of four individual Domain Ills may contribute to an unanticipated competition between the domains during the binding on to the microwell surface attributed by the physical or chemical characteristics of the four individual Domain Ills. This can be overcome by the usage of single recombinant tetravalent Domain III protein as a capture antigen. This single tetravalent protein as dengue diagnostic intermediate is used to achieve above objects of the present invention.

This novel recombinant protein of the present invention has been validated as a diagnostic reagent in both IgM and IgG ELISAs on a large number of human serum specimens collected from HAI assay confirmed paired (acute and convalescent) serum samples from definite dengue patients (Panel- 1, n=80), confirmed dengue acute serum samples from definite dengue patients (PaneI-2, n=82) by HAI assay / IgM capture ELISA (MRL kit) / virus isolation or PCR based assays and HAI confirmed non dengue acute and/or paired serum samples (Panel-3, n=39). Both IgM and IgG inhouse ELISAs, developed using the single novel recombinant protein showed an excellent agreement with the commercially available IgM capture ELISA (MRL) and HAI respectively for all 3 panels of acute serum specimens and Panel 1 convalescent serum specimens. These findings establish that that this novel dengue specific tetravalent recombinant protein antigen of the present invention is useful as a diagnostic intermediate for dengue viral infection. Brief description of the accompanying drawings In the accompanying drawings:

Fig 1: Structure of dengue virus. A. Schematic representation of dengue virion B. Detailed schematic presentation of dengue virus genome. Dengue virus encodes three structural (C-Capsid, M-Membrane, E-Envelop) and seven non-structural (NSl, NS2a, NS2b, NS3, NS4a, NS4b and NS5) proteins. RNA-Ribonucleic Acid.

Fig. 2: Multiple sequence alignment (ClustalW) of amino acid residues of Domain III of E protein of dengue serotype 1-4. Conserved residues are shown in gray boxes. Protein sequences retrieved from GenBank.

Fig. 3A: DNA Sequence of synthetic rTDIII gene. This sequence has a BamWl restriction site at 5' end and a Hinώλl restriction site at 3' end for cloning in expression vector.

Fig. 3B: Amino Acid sequence of rTDIII in fusion with N-Terminal Hexa- His- Tag and five extra aa at C- terminal from vector backbone. Amino acids from vector backbone are in gray colour. Fig. 3C: Schematic presentation of rTDIII protein in which domain III of envelope protein from all four serotypes of dengue virus are linked by flexible penta glycine linker.

Fig. 4: Expression of rTDIII protein in E. coli. A- Map of prTDIII expression plasmid. In this plasmid, the synthetic rTDIII gene is cloned in-frame with initiator codon and hexa- His Tag provided by pQE30 vector. Restriction sites used for cloning are indicated. Phage T5 promoter directs expression. Other abbreviations are as fellows. TT, transcriptional terminator; Ori, origin of replication; Amp^R, ampicillin resistance gene. The arrows indicate direction of transcription. B- SDS-PAGE analysis of rTDIII protein expression. The panel depicts Coomassie stained polypeptide profiles of E. coli harboring the plasmid p-rTDIII, prepared before (lane 2) and after (lane 3) induction with IPTG. Protein molecular weight markers (M) were run in lane 1. Their sizes (in kDa) are shown at the left of panel. The arrow on the right indicates the position of the rTDIII protein. Abbreviations are as follows, U-uninduced; I-induced. Fig. 5: Purification and characterization of the rTDIII protein. A. SDS-PAGE analysis of Ni-NTA affinity column fractions obtained during purification of rTDIII protein. Protein molecular mass markers (M) were in lane 1. Their sizes in kDa are shown to the left. Samples analyzed in the gel are load (L) in lane 2, Flowthrough (F) in lane 3, and Elution fractions E1-E7 in lanes 4-10. B. Western blot analysis of purified rTDIII protein using penta-His MAb. Prestained markers (P) were in lane 1 and their sizes in kDa are shown to the left. An aliquot of the purified recombinant protein (R) was in lane 2. Negative (a protein without His tag; BSA) was in lane 3. The arrow at the right indicates the position of the ~55 kDa rTDIII protein.

Fig.6: Western blot analysis of rTDIII protein with dengue-infected patient serum. Protein molecular mass markers (P) were in lane 1. Seropositive sample for IgM antibody (M+) was reacted in lane 2. Seronegative sample for IgM antibody (M-) was reacted in lane 3. Seropositive sample for IgG antibody (G+) was reacted in lane 4. Seronegative sample for IgG antibody (G-) was reacted in lane 5. Polyclonal rTDIII antibody (R) was reacted in lane 6. Penta-his antibody (H) was reacted in lane 7. Normal mouse serum (N) was reacted in lane 8; their sizes in kDa are shown to the left. The size of rTDIII protein is -55 kDa.

Fig 7. Evaluation of rTDIII protein as a diagnostic intermediate in indirect ELISAs using three Panels of human serum specimens Detailed description of the present invention

The applicants have designed and expressed a single recombinant tetravalent protein antigen, which contains domain III of envelope protein from all four serotypes of dengue virus, linked with each other through penta glycine linkers. This synthetic gene was expressed in Escherichia coli and the protein was purified using a single affinity chromatographic step. ELISA was done using this novel protein as the capture antigen. The protein was validated as a diagnostic reagent on serum specimens collected from HAI assay confirmed paired (acute and convalescent) serum samples from definite dengue patients (Panel- 1, n=80), confirmed dengue acute serum sample from definite dengue patients (Panel-2, n=82) by HAI assay/ IgM capture ELISA (MRL kit) / virus isolation or PCR based assays and HAI confirmed non dengue acute and/or paired serum samples (Panel-3, n=39). It was possible to purify 30 mg recombinant protein per liter of culture. Both Immunoglobulin M (IgM) and Immunoglobulin G (IgG) ELISAs, developed using this novel recombinant protein showed an excellent agreement with the commercially available IgM capture ELISA (MRL diagnostic) and HAI assay respectively for all 3 panels of acute and convalescent serum specimens from panel 1. Thus, these findings show that this single dengue specific tetravalent recombinant protein antigen is capable of being used as a diagnostic intermediate for detection of dengue viral infection at very low cost with high sensitivity and specificity.

By way of improvement upon the applicants' own PCT International publication referred to above, the present invention provides a novel single recombinant Tetravalent Domain III (rTDIII) protein which has the ability to detect both anti-dengue IgM and IgG antibodies with high sensitivity and specificity. More specifically, the present invention can be differentiated from the applicants' previous invention as shown in the following Table 2:

Table 2. Differences between the diagnostic intermediate of the applicants' PCT Application No. PCT WO 2005/014627 Al and the present invention:

Two multiepitope protein (MEP) New Tetravalent protein contains long contain short (7-20 aa residues) linear (>100 aa residues) and conformational epitopes domains

Two MEP contain epitopes from E, NS 1 New Tetravalent protein contains and NS3 proteins of dengue virus epitopes only from E protein

Two MEP lack epitopes from dengue 3 New Tetravalent contains epitope from serotype all four dengue serotypes rDME-G contains 15 epitopes (8 from E New multiepitope contains one domain and 7 from NS proteins) from E protein of each four serotypes of rDME-M contains four epitopes from dengue virus NSl (one from each 4 dengue serotypes)

Expression and purification of two Expression and purification of a single proteins are required to detect anti- protein is required to detect anti-dengue dengue IgM and IgG antibodies IgM and IgG antibodies

Two proteins do not contain epitopes This protein contains epitopes from only from domain III of E protein of four domain III of all four dengue serotypes dengue serotypes

Materials

The under-mentioned materials were used for the present invention. Escherichia coli host strain DH5a was purchased from Invitrogen Life Technology (Carlsbad, CA.) E.coli. expression strain SGl 3009 (pREP4-kan^r), the expression plasmid pQE30 (amp¹), Ni NTA Super-flow resin and anti-His (penta-His) monoclonal antibody were from QIAGEN (Hilden, Germany). IPTG, anti-mouse IgG- alkaline phosphatase (AP) conjugate and substrate 5-Bromo-4-Chloro-3-Indoryl Phosphate- Nitroblue Tetrazolium (BCIP/NBT) were from Calbiochem, (La Jolla, CA). Anti- human IgM and IgG Alkaline Phosphatase (AP) and anti-human IgM and IgG Horseradish Peroxidase (HRPO) conjugate were from Calbiochem, (San Diego, USA). The HRPO substrate, 3,3\5,5'-Tetramethylbenzidine (TMB) soluble was from Kirkegard Perry laboratories ( U.S.A.). Liquid chromatography column was from Sigma-Aldrich Co. (St. Louis, USA.). Urea GR and Guanidine hydrochloride were from Merck Limited (Mumbai, India). Human serum specimens: Two hundred and one human serum specimens used in this study were from fever patients warded at the North Colombo Teaching Hospital, Ragama, Sri Lanka. Five milliliters of venous blood were drawn from each volunteer patient by a Medical Officer after obtaining informed written consent. Laboratory diagnosis of serum specimens was performed at the Department of Parasitology, Faculty of Medicine, University of Kelaniya, Ragama, Sri Lanka. Depending on the results obtained from the laboratory diagnostic assays, patients were divided into 3 panels as HAI assay confirmed paired (acute and convalescent) serum samples from definite dengue patients (Panel- 1, n=80), confirmed dengue acute serum sample from definite dengue patients (Panel-2, n=82) by HAI assay/ IgM capture ELISA (MRL kit) / virus isolation or PCR based assays and HAI confirmed non dengue acute and/or paired serum samples (Panel-3, n=39).

Panel 1- Haemagglutination inhibition assay confirmed paired serum samples from definite dengue patients (n=80): Eighty patients confirmed as dengue by HAI assay, the gold standard assay for diagnosis of dengue infection were taken under this group. Acute serum specimen was collected from each patient in the early symptomatic phase and a convalescent blood specimen was obtained after 7-14 days of collection of the acute serum specimen. Both acute and convalescent serum specimens were tested by HAI assay (Clarke and Casals, 1958; Saver, 1962). Laboratory confirmation of dengue patient in Panel 1 was defined by positive detection of 4 fold rise in anti-dengue IgG antibody in acute and convalescent serum specimens by HAI assay which confirmed that 10 patients were found to be infected with primary dengue infection while 70 patients were found to be infected with secondary dengue infection. Further, both acute and convalescent serum specimens were also tested by IgM capture ELISA using a commercial kit (MRL diagnostics, U.S.A.). Forty two acute serum specimens and all of convalescent serum specimens showed the presence of IgM antibody which further confirmed that all these patients were infected with dengue virus.

Virus isolation and PCR-based assays were also attempted only for the acute serum specimens for further confirmation of dengue viral infection. Virus isolation from clinical specimens was performed using C6/36 clone of Singh's Ae. albopictiis cells (Singh, 1967; Igarashi, 1978) according to the methods described by Chanyasanha et al. (1995). Infected cell culture fluids were detected by immunofluorescent assay (Gubler et al., 1984). Single step Reverse Transcriptase Polymerase Chain Reaction Agarose Gel Electrophoresis (RT-PCR- AGE)(Chow et al, 1993) was performed. Dengue virus was isolated from 18 serum specimens by virus isolation and dengue viral RNA was detected in 30 serum specimens by RT-PCR-AGE assay. Serotyping of serum specimens was performed by Semi-Nested- PCR- AGE assay (Seah et al., 1995). Two and 31 dengue confirmed patients were found to be infected with dengue 2 and 3 serotypes by Semi-Nested-PCR-AGE assay.

Panel 2: Confirmed dengue acute serum sample from definite dengue patients by haemagglutination inhibition assay (HAI) / IgM capture ELISA (MRL kit) / virus isolation or polymerase chain reaction (PCR) based assays (n=82): Eighty two patients were confirmed to be dengue-infected by any one or more than one laboratory diagnostic assays; HAI assay (depends on only single acute serum specimen), IgM capture ELISA kit (MRL), virus isolation and PCR-based assays were taken under this group. A single serum specimen was collected from each patient at the early symptomatic phase. Laboratory confirmation of dengue in Panel 2 was defined by the positive detection of anti-dengue IgG antibody (in a single acute serum specimen) by HAI assay and/or positive detection of anti-dengue IgM antibody by the same commercial kit and/or positive detection of dengue virus by either viral culture and nucleic acid amplification as described above.

HAI assay confirmed that 13 and 41 out of 82 patients were found to be cases of probable primary and secondary dengue infections, respectively. Sixty serum specimens showed the presence of anti-dengue IgM antibody by IgM capture ELISA performed by the MRL kit. Dengue virus was isolated from 13 serum specimens and dengue viral RNA was detected in 18 serum specimens using RT-PCR-AGE assay. Two and 17 out of 82 dengue confirmed patients were found to be infected with dengue 2 and 3 serotypes, respectively by Semi-Nested-PCR-AGE assay.

Panel 3- Haemagglutination inhibition assay confirmed acute and/or paired serum samples from non dengue patients (n=39):

Thirtynine patients confirmed as non dengue by all laboratory diagnostic assays; HAI assay, IgM capture ELISA kit (MRL), virus isolation and PCR-based assays were taken under this group. Paired serum was collected from 8 patients and only acute serum was from 31 patients. Laboratory confirmation of non dengue patients in Panel 3 required <20 titre of IgG antibody by HAI assay and negative detection of anti-dengue IgM antibody by IgM capture ELISA kit (KOlL) and negative detection of dengue virus by either viral culture and nucleic acid amplification. Thus, according to the present invention there is provided a single recombinant Tetravalent Domain III (rTDIII) protein for use in the detection and or diagnosis of any or all of dengue specific Immunoglobin M anti-dengue IgM and Immunoglobin G anti- dengue IgG antibodies with high sensitivity and specificity, said protein comprising domain III of envelope protein from all four serotypes of dengue virus Dengue-virus type-1, Dengue-virus type-2, Dengue-virus type-3 and Dengue-virus type-4, linked with each other through penta glycine linkers and codon optimized for expression in an E. coli expression vector.

In a preferred feature, said protein-encoding gene has the following nucleotide sequence:

ATGAGAGGATCGCATCACCATCACCATCACGGATCCAGCTATGTGATGTGTΆCCGGCA GCTTCAAACTGGAAAAAGAAGTGGCGGAAACCCAGCATGGCACCGTTCTGGTTCAGGT GAAATACGAAGGCACCGATGCCCCATGTAAAATTCCGTTCAGCACCCAGGATGAAAAA GGCGTTACCCAGAACCGTCTGATTACCGCCΆATCCGATCGTGACCGATAAAAAACCGG TGAACATCGAAACCGAACCGCCGTTTGGCGAAAGCTATATTGTTGTTGGCGCCGGTGA AAAAGCGAAACAGTGGTTCAAAAAAGGCAGCAGCATCGGCAAAATGTTTGΆAGCGACC GCTCGTGGCGCCCGTCGTATGGCGATTCTGGGCGGTGGTGGTGGTATGAGCTATGCCA TGTGCCTGAACACCTTCGTGCTGAΆAAAAGAAGTTAGCGAGACCCAGCACGGTACGAT TCTGATCAAAGTGGAATATAAAGGCGAAGATGCCCCTTGTAAGATCCCGTTTTCCACC GAAGATGGTCΆGGGCAAAGCACATΆACGGTCGCCTGATTACCGCTAΆCCCGGTGGTGA CCAAAΆAAGAAGAACCGGTGAATATTGAAGCGGAACCACCGTTCGGCGAATCCAACAT TGTGATTGGCATCGGCGATAAAGCGCTGAAAATCAACTGGTATCGTAAAGGTAGCTCC ATTGGCAAAATGTTCGAGGCAACGGCACGTGGTGCTCGCCGTATGGCAATCCTGGGTG GCGGCGGTGGCATGAGCTATATGTGTGGCAAATTCAGCGGCAAATTTAGCATCGATAA AGAAATGCGTGAGACCCAACATGGCACCACCGTGGTGAAAGTGAAATATGAAGGCGCT GGCGCCCCTTGTAAAGTGCCGATTGAAATCCGCGATGTGAACAAAGAAAAAGTGGTGG GCCGTATTATTAGCAGCACCCCGCTGGCGGAAAATACCAACAGCGTCACGAACATTGA ACTGGAACGTCCGCTGGATAGCTATATCGTTATTGGCGTTGGCAATGCCCTGACCCTG CATTGGTTTCGTAAAGGCTCCAGCATTGGTAAGATGTTCGAAAGCACCTATCGTGGCG CCAAACGTATGGCCATCCTGGGAGGTGGAGGCGGTATGTCTTATAGCATGTGTACCGG TAAATTCAAAGTGGTGAAAGAAATCGCCGAAACCCAGCACGGAACCATTGTGATTCGC GTCCAGTATGAAGGTGATGGCAGCCCATGTAAAACCCCGTTCGAAATCATGGATCTGG AAAAACGTCATGTTCTGGGTCGTCTGACCACCGTTAATCCGATTGTGACGGAAAAAGA TAGCCCGGTTAACATTGAAGCAGAGCCGCCATTTGGCGATTCCTATATCATCATTGGC GTGGAACCGGGTCAGCTGAAACTGGATTGGTTTAAAAAAGGCTCCTCCATCGGCCAGA TGTTTGAAACCACCATGCGTGGAGCGAAGCGTATGGCAATTCTGAGATCTAAGCTTAA

TTAG Seq I D 1 .

The underlined portion shows the sequences from the vector. In another feature of the present invention, said protein has the following amino acid sequence:

MRGSHHHHHHGSSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSTQDEK GVTQNRLITANPIVTDKKPVNIETEPPFGESYIVVGAGEKAKQWFKKGSSIGKMFEAT ARGARRMAILGGGGGMSYAMCLNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFST EDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDKALKINWYRKGSS IGKMFEATARGARRMAILGGGGGMSYMCGKFSGKFSIDKEMRETQHGTTVVKVKYEGA GAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELERPLDSYIVIGVGNALTL HWFRKGSSIGKMFESTYRGAKRMAILGGGGGMSYSMCTGKFKVVKEIAETQHGTIVIR VQYEGDGSPCKTPFEIMDLEKRHVLGRLTTVNPIVTEKDSPVNIEAEPPFGDSYIIIG VEPGQLKLDWFKKGSSIGQMFETTMRGAKRMAILRSKLN Seq ID 2.

The underlined portion shows the sequences from the vector backbone.

In a preferred embodiment, said nucleotide sequence has a Banttl restriction site at 5' end and a Hiπdlll restriction site at 3' end for cloning in said expression vector.

In another preferred embodiment, said amino acid sequence is in fusion with N- Terminal Hexa- His- Tag and five extra amino acid (aa) residues at the C- terminus from the vector backbone. In another preferred embodiment, said protein has the following schematic structure:

DEN1 Domain III DEN3 Domain III DEN4 Domain III - DEN2 Domain III

In another preferred embodiment, said protein contains more than 100 aa residues and conformational domain. In- another preferred embodiment, the present invention also provides a method for the synthesis of single recombinant Tetravalent Domain III (rTDIII) protein for use in the detection and or diagnosis of any or all of dengue specific Immunoglobin M anti-dengue IgM and Immunoglobin G anti-dengue IgG antibodies, said method comprising (a) synthesizing a gene comprising single recombinant Tetravalent Domain

III (rTDIII) from all four serotypes of dengue virus Dengue-virus type-1, Dengue- virus type-2, Dengue-virus type-3 and Dengue-virus type-4, linked with each other through penta glycine linkers and codon optimizing it for expression in an E. coli expression vector; (b) constructing a recombinant TDIII gene expression vector;

(c) expressing said gene to produce said recombinant Tetravalent Domain III (rTDIII) protein;

(d) purifying said protein.

In another preferred embodiment, said recombinant TDIII gene expression vector is constructed by ligating rTDIII gene into BamUl and HindUl restriction enzyme sites of said expression vector to generate the plasmid prTDIII, inserting said rTDIII gene in frame with ATG codon at the 5' end and the hexa-histidine-tag encoding sequences also at the 5' end) provided by said vector, transforming the ligation mixture so obtained in DH5α E. coli cells, selecting recombinant clones on ampicillin containing LB (Luria-Bertani) plates and subjecting them to direct colony PCR screening, and identifying recombinants harboring the synthetic rTDIII gene using vector specific primers.

In another preferred embodiment, said expression vector is a bacterial expression vector. In another preferred embodiment, said bacterial expression host is a E. coli.

In another preferred embodiment, said gene is synthesised by ligation of oligonucleotides encoding Domain HI from dengue serotypes 1-4.

The present invention also relates to use of a single recombinant Tetravalent Domain III (rTDIII) protein in the detection and or diagnosis of any or all of dengue specific Immunoglobin M (anti-dengue IgM) and Immunoglobin G (anti-dengue IgG) antibodies.

In yet another feature, the present invention relates to a method of detecting or diagnosing any or all of dengue specific Immunoglobin M anti-dengue IgM and Immunoglobin G anti-dengue IgG antibodies in a test sample which comprises subjecting said test sample to ELISA in the presence of a single recombinant Tetravalent Domain III (rTDIII) protein of the present invention. Preferably, said test sample comprises a human serum.

The Domain III of dengue viruses subtypes DEN 1, DEN 2, DEN 3 and DEN 4 are well known. They have the following nucleotide and amino acid sequences respectively:

Amino acid sequences:

Den 1 Domain HI (Dengue 1 Singapore strain S275/90) MSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSTQDEKGVTQNRLITAN PIVTDKKPVNIETEPPFGESYIVVGAGEKAKQWFKKGSSIGKMFEATARGARRMAIL Seq ID 3

Den 2 Domain HI (Dengue 2 Strain PR159)

MSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKTPFEIMDLEKRHVLGRLTTV NPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLDWFKKGSSIGQMFETTMRGAKRM AIL Seq ID 4 Den 3 Domain III (Dengue 3 Strain H87)

MSYAMCLNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITA NPVVTKKEEPVNIEAEPPFGESNIVIGIGDKALKINWYRKGSSIGKMFEATARGARRM AIL Seq ID 5

Den 4 Domain III (Dengue 4 Strain H241) MSYMCGKFSGKFSIDKEMRETQHGTTVVKVKYEGAGAPCKVPIEIRDVNKEKVVGRII SSTPLAENTNSVTNIELERPLDSYIVIGVGNALTLHWFRKGSSIGKMFESTYRGAKRM AIL Seq ID 6

Nucleotide Sequences

Den 1 Domain III (synthetic DNA)

ATGAGCTATGTGATGTGTACCGGCAGCTTCAAACTGGAAAAAGAAGTGGCGGAAACCC AGCATGGCACCGTTCTGGTTCAGGTGAAATACGAAGGCACCGATGCCCCATGTAΆAAT TCCGTTCAGCACCCAGGATGAAAAAGGCGTTACCCAGAACCGTCTGATTACCGCCAAT CCGATCGTGACCGATΆAAAAACCGGTGAACATCGΆAACCGAACCGCCGTTTGGCGAAA GCTATATTGTTGTTGGCGCCGGTGAAAAΆGCGAAACAGTGGTTCAAAAAAGGCAGCAG CATCGGCAAAATGTTTGAAGCGACCGCTCGTGGCGCCCGTCGTATGGCGATTCTG Seq ID 7

Den 2 Domain III (synthetic DNA)

ATGTCTTATAGCATGTGTACCGGTAAATTCAAAGTGGTGAAAGAAATCGCCGAAACCC AGCΆCGGAACCATTGTGATTCGCGTCCAGTATGAAGGTGATGGCAGCCCATGTAAAAC CCCGTTCGAAATCATGGATCTGGAAAAACGTCATGTTCTGGGTCGTCTGACCACCGTT AATCCGATTGTGACGGAAAAAGATAGCCCGGTTAACATTGAAGCAGAGCCGCCATTTG GCGATTCCTATATCATCATTGGCGTGGAACCGGGTCAGCTGAAACTGGATTGGTTTAA AAAAGGCTCCTCCATCGGCCAGATGTTTGAAACCACCATGCGTGGAGCGAAGCGTATG

GCAATTCTG Seq ID 8

Den 3 Domain III (synthetic DNA)

ATGAGCTATGCCATGTGCCTGAACACCTTCGTGCTGAAAAAAGAAGTTAGCGAGACCC AGCACGGTACGATTCTGATCAAAGTGGAΆTATAAAGGCGAΆGATGCCCCTTGTAAGAT CCCGTTTTCCACCGAAGATGGTCAGGGCAAAGCACATAACGGTCGCCTGΆTTACCGCT AACCCGGTGGTGACCAAAAAAGAAGAACCGGTGAATATTGAAGCGGAACCACCGTTCG GCGAATCCAACATTGTGΆTTGGCATCGGCGATAAΆGCGCTGAAAATCAACTGGTATCG TAAAGGTAGCTCCΆTTGGCAAAATGTTCGAGGCAACGGCACGTGGTGCTCGCCGTATG

GCAATCCTG Seq I D 9 Den 4 Domain III (synthetic DNA)

ATGAGCTATATGTGTGGCAAATTCAGCGGCAAATTTAGCATCGATAAAGAAATGCGTG AGACCCAACATGGCACCACCGTGGTGAAAGTGAAATATGAAGGCGCTGGCGCCCCTTG TAAAGTGCCGATTGAAATCCGCGATGTGAACAAAGAAAAAGTGGTGGGCCGTATTATT AGCAGCACCCCGCTGGCGGAAAATACCAACAGCGTCACGAACATTGAACTGGAACGTC CGCTGGATAGCTATATCGTTΆTTGGCGTTGGCAΆTGCCCTGACCCTGCATTGGTTTCG TAAAGGCTCCAGCATTGGTAAGATGTTCGAAAGCACCTATCGTGGCGCCAAACGTATG

GCCATCCTG Seq ID 10 .

The present invention will now be described with reference to the following Examples which are merely illustrative. They are not intended to limit the scope of the invention in any way: Example 1 Synthesis of Tetravalent Domain III (TDIII) gene

A synthetic gene, codon optimized for E. coli expression, was generated by ligation (GeneArt, Germany) of oligonucleotides encoding Domain III from dengue serotypes 1-4. Each of the four Domain III was 115-119 aa residues long and linked by penta glycyl linkers. Example 2 Construction of the recombinant TDIII gene expression vector The resultant 1.47 kb gene, lTDIII, was ligated into BamUl and Hindttl restriction enzyme sites of bacterial expression vector pQE30 to generate the plasmid prTDIII. In this plasmid, the rTDIII gene was inserted in frame with ATG codon (at the 5' end ) and the hexa-histidine-tag encoding sequences (also at the 5' end) provided by the pQE30 vector. Ligation mixture was transformed into DH5α E. coli cells. Recombinant clones were selected on ampicillin containing LB (Luria-Bertani) plates and subjected to direct colony PCR screening, using vector specific primers, to identify recombinants harboring the synthetic rTDIII gene. Recombinants were further verified by restriction analysis of plasmid minipreps. For expression, positive clone plasmid was transformed in E. coli expression strain SG 13009 and selected on ampicillin and kanamycin containing LB agar plates. Example 3 Expression screening of rTDIII gene

Expression screening was done using test tubes cultures. Single SG 13009 colony harboring prTDIII plasmid was inoculated into 3 ml of LB culture medium per tube (containing 100 μg/ml ampicillin + 25 μg/ml kanamycin) and was grown overnight in a shaker at 37⁰C, at 200 rpm. In 3 ml fresh LB medium, 2% (60μl) overnight culture was inoculated and allowed to grow at 37⁰C in a shaker at 200 rpm. When the cultures were in logarithmic growth phase (corresponding to an optical density at 600nm [OD₀₀₀] of -0.6 after 2-2.5h), they were induced with ImM IPTG for 4 hours. After induction, equivalent numbers of cells from different cultures (normalized on the basis of ODeoo values) were lysed in sample buffer and analyzed by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Noninduced controls were analyzed in parallel. All induced clones expressed similar levels of recombinant protein; one clone was selected for further work. Plasmid DNA isolated from this clone and verified by restriction analysis. Example 4 Purification of rTDIII protein

Twenty ml of LB medium containing ampicillin (100 μg/ml) and kanamycin (25 μg/ml) was inoculated with 2 μl glycerol stock of a SG13009 clone (chosen above) harboring the prTDIII plasmid, grown overnight in a shaker at 37⁰C, at 200 rpm, and inoculated into 1 liter LB medium (containing 100 μg/ml amplicillin and 25 μg/ml kanamycin) in a 4-liter Haffkine flask, at 37⁰C for about 2 to 3 hours at 120 rpm. When the ODόoo of the culture reached ~0.6, a small aliquot of noninduced culture was set aside for subsequent SDS-PAGE analysis, and rest of the culture was induced by addition of IPTG to the final concentration of 1 mM. Induction was allowed to proceed for 4 hours before harvesting the cells. Aliquots of induced and noninduced cell cultures were analyzed by SDS-PAGE prior to initiating purification.

The induced culture was centrifuged in a Sorvall GS3 rotor at 6000 rpm for 15 minutes at 4⁰C. About 2.5 g of induced cell pellet (corresponding to 1 liter E. coli culture) was suspended in 25 ml lysis buffer pH 7.8 (6 M guanidine HCl, 100 mM sodium phosphate buffer, 10 mM Tris-HCl, 300 mM NaCl) and sonicated for 5 minutes and kept for stirring for 1 hour at Room Temperature (RT). The Iy sate was clarified by centrifugation (12000 rpm in a Sorvall SS34 rotor at 4⁰C for 30 minutes) and passing the resultant supernatant through a 0.45 μm filter. The filtrate was mixed with 4 ml of Ni-NTA Superflow resin (pre-equilibrated with lysis buffer). This suspension was gently rocked overnight at RT and than packed into a column. After collecting the flowthrough, the column was washed with 100 ml of lysis buffer at the flow rate of 3 ml/min followed by 300 ml wash buffer I pH 6.5 (8 M urea, 100 mM sodium phosphate buffer, 10 mM Tris-Cl) at 3 ml/min and 300 ml wash buffer II pH 6.0 (8 M urea, 10OmM sodium phosphate, 1OmM Tris HCl) at 1 ml/min. The protein was eluted by elution buffer pH 4.5 (8M urea, 10OmM sodium phosphate buffer, 1OmM Tris HCl) at the flow rate of 0.3 ml/min. Fractions of 3 ml were collected and attd-analyzed by SDS- PAGE. Peak fractions were pooled together; concentration was measured by absorbance on 280 nm. In pooled fraction, gentamicin was mixed to final concentration of 20 μg/ml, flash-frozen in liquid nitrogen and stored at -8O⁰C until use. The entire process of washing and elution was controlled and monitored by connecting the column to an AKTA FPLC system (GE Healthcare Bio-Sciences, Uppsala, Sweden). Example 5 Evaluation of rTDIII protein by western blot

The purified rTDIII protein was electrophoresed on 12% denaturing and reducing gel (SDS-PAGE), along with appropriate controls and pre-stained markers and transferred electrophoretically (transfer buffer: 24 mM Tris base, 192 mM glycine, 20% methanol in distilled water) to nitrocellulose membrane. The membrane was blocked with 1% polyvinyl pyrrolidone in Ix phosphate buffered saline, pH 7.2 (PBS) for 2 hours at RT. It was then washed three times with Ix PBS containing 0.1% Tween 20 (Ix PBS plus 0.1% T) and incubated with a commercially available murine Anti- penta-His monoclonal antibody (1 :2 000 dilution) for 90 minutes on flip-flop at RT. The membrane was washed again, as above, and incubated with anti-mouse IgG-AP conjugate (1 :5 000 dilution) for further 45minutes on flip-flop at RT. The blot was washed and developed by incubation with BCIP/NBT substrate solution for 15 minutes on flip-flop at RT. Example 6

Preliminary analysis of rTDIII protein with dengue patient serum by western blot Activity of purified rTDIII protein for detection of anti-dengue IgM and IgG antibodies in human serum samples was also analyzed by a western blot. Strips of nitrocellulose membrane with electrophoretically transferred rTDIII protein as mentioned above were incubated, separately, with dengue seropositive and seronegative serum samples for IgM and IgG antibodies (1:100 dilution in 1% PVP, 5% normal goat serum in PBS), at RT for 1 hour on a flip-flop. The strips were washed as mentioned above and incubated with diluted anti-human IgM or IgG HRPO conjugate (1 : 5 000 in 1% PVP, 5% normal goat serum in PBS) RT for 1 hour on a flip- flop. Then strips were washed as mentioned above and developed by incubating with TMB soluble substrate at RT for 15 minutes. Reaction was stopped by adding strips into water. A control experiment was done with rTDIII polyclonal antibody (raised in mouse) and normal mouse serum samples. Anti-mouse IgG alkaline phosphate (1 : 5 000 in 1% PVP, 5% normal goat serum in PBS) was used as the conjugate for this control experiment. Other steps as mentioned above were followed. After washing strips membrane was developed by incubating with BCIP/NBT substrate at room temperature for 15 min. Reaction was stopped by adding strips into water. Dengue seropositive and seronegative samples for both antibodies were selected based on the results obtained from MRL Kit ELISAs, Panbio cassette and HAI assay. Example 7 Evaluation of rTDIII protein as a diagnostic intermediate by indirect ELISA

In order to evaluate the feasibility of rTDIII protein as a diagnostic reagent to detect anti-dengue IgM and IgG antibodies, the applicants developed ELISA protocols. This protein was used as the capture antigen for both IgM and IgG ELISAs. Human IgM and IgG antibodies bound to rTDIII antigen were revealed using HRPO conjugated anti-human IgM and IgG, respectively. Detailed description of performance of ELISA is given below. A 96-well flat-bottomed ELISA plate (Nunc, Immuno) was coated with 100 μl of diluted rTDIII protein (10 μg/ml in 0.1 M carbonate buffer, pH 9.5) and incubated at 37⁰C for 1 hour. The wells were blocked with 200 μl of 5% skimmed milk in PBS at 37⁰C for 2 hours. After incubation, the wells were washed with washing buffer (0.5% Tween 20, 0.1% CHAPS in PBS) 5 times (1 minutes/each washing step). Hundred microlitres of diluted serum samples (1 : 100 in 5% skimmed milk in PBS) λvere added to each well and incubated at 37⁰C for 15 min. Wells were washed as described above and incubated with 100 μl of anti-human IgM HRPO conjugate (1 :20 000 dilution in 5% skimmed milk in PBS) or anti-human IgG HRPO conjugate (1:40 000 dilution in 5% skimmed milk in PBS) separately at 37⁰C for 30 min. Following washing steps as described above, 100 μl of TMB soluble substrate was added and incubated at 37⁰C for 15 min. The reaction was stopped by adding 100 μl of 1 M H₂SO₄. Optical density value of each sample was measured at 450 nm with 630 nm as the reference wave length. Dengue seropositive or seronegative samples were included in each plate. Dengue seropositive or seronegative samples for both antibodies were selected based on the results obtained from MRL ELISAs, Panbio cassette and HAI assay.

Three panels of human serum specimens collected from Sri Lanka as described under materials were tested by the in-house ELISA developed using the novel single recombinant tetravalent protein. A positive sample in both in-house IgM and IgG ELISAs was defined as having a test absorbance/negative control ratio of >2.0 and a negative sample was defined as having a ratio of <2.0 (Shu et al., 2004; Mohamed et «/., 1995). Activity of novel rTDIII protein in the in-house IgM ELISA for detection of IgM antibody in human serum specimens was compared with IgM ELISA assay performed by MRL diagnostic kits using chi-squre test (Epi-info, version 6.04d software, Centre for Disease Control, U.S.A.). Similarly, activity of the novel rTDIII protein in IgG ELISA for detection of IgG antibody in human serum specimens was compared with HAI assay using the same statistical test. Two variables were analyzed at a 95% confidence interval and p value <0.05 was considered as significant. Example s

Synthesis of tetravalent gene and construction of rTDIII gene expression vector

In order to create multivalent protein that could be useful in early detection of all dengue serotypes infection, focus was maintained on an immunodominant domain of dengue virus, which is very specific to anti dengue antibodies and not reactive with antibodies against other flavi viruses. Domain III of envelope protein from dengue virus was shown to display high antigenecity and was reactive towards sera from patient infected only with dengue virus. A sequence alignment of domain III of all four serotypes, depicted in Fig.2, revealed a high degree of homology among these four domain Ills. The applicants designed a synthetic gene based on these four domains linked by 5 glycine residues. Fig. 3A shows the DNA sequence of synthetic rTDIII gene. Further, Fig. 3B shows amino acid sequence of rTDIII in fusion with N-Terminal Hexa- His- Tag and five extra aa at the C- terminus from vector backbone. Schematic presentation of rTDIII protein comprising in which Domain III of envelope protein from all four serotypes of dengue virus is shown in Fig 3C. A synthetic gene encoding this protein, codon optimized for expression in E.coli, was created and cloned into bacterial expression vector pQE30. The insert was designed to be in frame with the initiator codon and hexa-histidine-tag encoding sequence provided by vector. This expression vector is depicted in Fig. 4A. The synthetic gene, designated rTDIII (recombinant Tetravalent Domain III) was predicted to encode a ~55kDa recombinant protein.

Example 9

Expression of the rTDIII protein In the expression vector, rTDIII gene is under the transcriptional control of the IPTG inducible T5 promoter. This vector was introduced into the E. coli host SG 13009, and the transformants, selected in the presence of ampicillin and kanamycin, were analyzed by expression screening. In this experiment, IPTG induced cells were directly lysed in Laemmli sample buffer (Laemmli et άl. 1970) and analyzed by SDS-PAGE. The IPTG induction strategy resulted in successful expression. Fig. 4B depicts the induction profile of a clone. Induction of the rTDIII gene resulted in the appearance of a ~55 kDa band, consistent with the predicted size of rTDIII protein. When induced cells were lysed by sonication in a native buffer, separated into supernatant and pellet fractions, and analyzed by SDS-PAGE, the protein was found to be present partially in soluble fraction (-10%) with the rest in the insoluble fraction. Example 10 Purification of the r-TDIII protein

As the rTDIII protein was only partially soluble with a major proportion in the insoluble fraction, the applicants proceeded to purify it under denaturing conditions to obtain maximum yields. For purification the applicants opted to take advantage of N- terminal hexa-histidine tag. Ni-NTA affinity purification was done in denaturing condition using AKTA FPLC system. Fractions collected during different steps of purification were analysed by SDS-PAGE. In this experiment, almost all of the induced protein bound to the column, as evident from comparison of the protein profile (Fig.5 A) of initial lysate loaded onto affinity matrix (lane 2) with that of flowthrough material (lane 3). During the experiment, the column was washed with slightly acidic Buffer I (pH 6.5) and Buffer II (pH 6.0) to remove most of the impurities without eluting significant amount of rTDIII protein. Therefore, 300 ml volume wash with buffer II was given. Bound rTDIII protein was eluted with Elution buffer (pH 4.5) result in the emergence of highly purified recombinant protein from column (lane 4-10). An estimated >95% purity was achieved on the basis of SDS PAGE analysis. Starting from 2.5g induced cell pellet (equivalent to 1 liter of E. coli culture), ~ 30mg purified protein (pooled fractions) was obtained. This corresponds to a recovery of >95%, as crude lysate was estimated to contain 31mg of recombinant protein, based on densitometry analysis. A summary of purification is presented in Table 3.

TABLE 3. Summary of rTDIII purification from 1 liter of induced culture

??

a. Protein content was determined by the BioRad method, using BSA as standard. b. Purity was assessed by SDS-PAGE analysis. c. The amount of rTDIII protein in crude lysate (which estimated to be ~ 31mg by densitometry analysis) was designated as 100%. n.a. not applicable

Example 11

Evaluation of rTDIII protein by western blot

The Ni-NTA affinity purified protein was tested in a western blot assay; an aliquot of pooled purified protein was probed with commercially available murine anti- penta-His MAb specific to the engineered His-tag at the amino-terminusal of recombinant protein. In this blot, we also included a negative control (protein lacking the His-tag), we used bovine serum albumin© (BSA). It is evident from the data shown in Fig. 5B that penta-His MAb, which specifically recognizes the hexa-histidine-tagged protein, has recognized the ~55 kDa rTDIII protein purified as described in experiment. Preliminary analysis of rTDIII protein with dengue serum by western blot

Fig. 6 clearly indicates that this novel rTDIII protein is-recognizes«g anti- Dengue IgM and IgG antibodies in dengue infected patients' seraum. Evaluation of rTDIII as a diagnostic intermediate by indirect in-house ELISA

Having demonstrated that this novel recombinant tetravalent protein of the present invention can be used to identify dengue specific antibody in the western blot, utility of this reagent as a diagnostic intermediate in ELISA was tested. Indirect IgM and IgG ELISAs were developed using this protein as the capture antigen. Fig. 7 A₅ B, C, D, E and F shows results of evaluation of rTDIII protein as a diagnostic intermediate in ELISAs on 3 panels of human serum specimens collected from Sri Lanka. Detailed results of validation of the protein in ICGEB IgM and IgG ELISAs together with HAI assay, IgM capture ELISA by MRL kit, virus isolation and PCR-based assays are shown in Table 4A, B and C.

Table 4A. Panel 1-Haemagglutination inhibition assay confirmed paired serum samples from definite dengue patients (n=80)

Table 4C. Panel 3- Haemagglutination inhibition confirmed non dengue acute and/or paired serum samples ( n=39).

Summary of results obtained from different laboratory diagnostic assays are shown in Table 5A. Comparison of ICGEB ELISAs with IgM ELISA-MRL kit and HAI assay for acute serum samples are shown in Table 5B. Comparison of ICGEB IgM ELISA (test method) with MRL IgM capture ELISA (reference method) are shown in Table 6A. Comparison of ICGEB IgG ELISA (test method) with HAI assay (reference method) are shown in Table 6B.

Table 5A. Summary of Ia bora to ry diagnostic assays for acute serum samples

Table 5B. Comparison of ICGEB ELISAs with IgM capture ELISA-MRL kit and HAI assay for acute serum samples

Table 6A. Comparison of ICGEB IgM ELISA (test method) with MRL IgM capture ELISA (reference method)

Table 6B. Comparison of ICGEB IgG ELISA (test method) with HAI assay (reference method)

^a, total number of serum samples tested ^b, capacity to define true positives [number of test method positive sera that also were reference method positive/total number of reference method positive sera X 100%] °, capacity to define true negatives [number of test method negative sera that also were reference method negative/total number of reference method negative sera X 100%] ^d, number of sera positive by both methods + number of sera negative by both methods/ total number of sera tested X 100

Majority of dengue patients in the study population showed definitive, 87% (Panel 1 -70/80), or probable, 50% (Panel 2 - 41/82), secondary dengue infection and dengue serotype 3 infection 30% (48/162). The ICGEB ELISAs showed high sensitivity in detecting dengue viral infection in clinical samples collected from HAI confirmed dengue patients after 5 days of infection. The ICGEB ELISAs could detect 100% (38/38) of HAI confirmed dengue patients in Panel 1, after 5 days of infection indicating the high sensitivity. Further, a greater reactivity to dengue rTDIII protein with convalescent sera, 100% (80/80) by IgM ELISA compared with acute sera 66% (107/162) has been observed in Panels 1 and 2 serum samples including dengue patients. Further, a greater reactivity to dengue rTDIII protein with convalescent sera, 100% (80/80) by IgG ELISA compared with acute sera, 56% (92/162) was also observed in the same two panels of serum samples. This is to be expected since antibody titers to an infecting agent increase over time (Santos et ah, 2004). The overall comparative analysis of the ICGEB ELISAs with IgM ELISA by MRL kit and HAI assay suggest that there is an excellent agreement between ICGEB IgM ELISA with MRL IgM ELISA as well as ICGEB IgG ELISA with HAI assay. Further, ICGEB IgM ELISA picked up 8 additional samples which MRL kit failed to identify. When ICGEB IgM ELISA was compared with the MRL commercial kit, it showed that there was no significant statistical difference in the detection of IgM antibody by both assays (p=0.00). When ICGEB IgG ELISA was compared with the HAI assay there was no significant statistical difference in the detection of IgG antibody by both assays (p=0.00). When sensitivity and specificity of IgM ELISA developed using the novel recombinant tetravalent protein was calculated by comparison with the MRL IgM ELISA kit, it showed 90% sensitivity, 85% specificity and 87% agreement with the commercial kit for acute serum specimens used in 3 panels (n=201). When sensitivity and specificity of IgM ELISA, developed using the novel recombinant tetravalent protein, were calculated by comparison with the MRL IgM capture ELISA kit, it showed 100% sensitivity, 100% specificity and 100% agreement with the commercial kit for convalescent serum specimens used in Panel 1 (n=80) (Table 6A). When sensitivity and specificity of IgG ELISA were calculated by comparison with the gold standard assay (HAI), it showed 90% sensitivity, 91% specificity and 91% agreement for acute serum specimens used in 3 panels (n=201). Similarly, sensitivity and specificity of IgG ELISA developed using the novel recombinant tetravalent protein was calculated by comparison with the HAI assay, it showed 100% sensitivity, 100% specificity and 100% agreement with the HAI assay for convalescent serum specimens used in Panel 1 (n=80) (Table 6B). These results show that specificity of these novel IgG and IgM ELISAs seems to be similar to the available assay. To achieve maximum level of sensitivity, a diagnostic assay that gives the best results depending on the duration of illness should be selected. Virus isolation and PCR-based techniques can be used for serum samples collected at the early phase of infection (1-5 days of fever). As evident from this study, some patients are hospitalized when viremia is at low level or absent. Serological assays are important for diagnosis of dengue infection during this late stage of the illness (more than five days of fever) as illustrated in the present study. Discrepancies between the results of ICGEB IgM and IgG ELISAs with other laboratory diagnostic assays were not visible in the Panel 3 serum collected from non dengue patients as all these specimens were negative by both ICGEB ELISAs.

As mentioned above diagnosis of dengue infection based on clinical symptoms is not reliable and more than half of infected individuals either are asymptomatic or have a mild undifferentiated fever. The clinical presentation and blood counts were similar between patients hospitalized with acute dengue fever and patients with other febrile illness (Buchy et al., 2005). Therefore, there is a great demand for the rapid detection and differentiation of dengue virus infection in the acute phase of illness in order to provide timely clinical treatment, eitiologic investigation and disease control (Shu and Huangy, 2004). Dengue diagnosis in many endemic countries is hindered by the high cost of commercial kits and inaccessibility of reagents. In-house ELISAs require viral antigen, which are produced in limited quantities in cell culture or in suckling mouse brain, and is insufficient for the large-scale serologic screening sometimes necessitated by the epidemic spread of dengue. In this study, a recombinant protein antigens has been introduced, which can be produced without using any infectious agents and it is stable even at room temperature. An ELISA that would enable rapid and definitive diagnosis of dengue viral infection in suspected patients within four hours has been developed in the present invention. No pretreatment of serum samples to remove IgG antibody is required for IgM ELISA developed in the present invention. The recombinant protein identified in this study was found to be a potentially useful diagnostic antigen that is easy to prepare and suitable for mass production as protein was obtained at high levels in E. coli. Further, it is economical to prepare protein using the above-mentioned method. One microlitre of serum sample is sufficient for a single IgM/IgG ELISA developed in this invention. References

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Claims

We claim:

1. A single recombinant Tetravalent Domain III (rTDIII) protein for use in the detection and or diagnosis of any or all of dengue specific Immunoglobin M anti- dengue IgM and Immunoglobin G anti-dengue IgG antibodies with high sensitivity and specificity, said protein comprising domain III of envelope protein from all four serotypes of dengue virus, namely, Dengue-virus type-1, Dengue- virus type-2, Dengue-virus type-3 and Dengue-virus type-4, linked with each other through penta glycine linkers and codon optimized for expression in an expression vector.

2. A single recombinant Tetravalent Domain III (rTDIII) protein as claimed in claim

1 wherein said protein has a nucleotide sequence as shown in Seq ID 1.

3. A single recombinant Tetravalent Domain III (rTDIII) protein as claimed in claim

2 wherein said nucleotide sequence has a BaniHl restriction site at 5' end and Hindlll restriction site at 3' end for cloning in said expression vector.

4. A single recombinant Tetravalent Domain III (rTDIII) protein as claimed in any preceding claim wherein said protein has an amino acid sequence as shown in Seq ID 2.

5. A single recombinant Tetravalent Domain III (rTDIII) protein as claimed in claim 4 wherein said amino acid sequence is in fusion with N-Terminal Hexa- His- Tag and five extra aa at C- terminus from the vector backbone.

6. A single recombinant Tetravalent Domain III (rTDIII) protein as claimed in any preceding claim wherein said Dengue-virus type-1, Dengue-virus type-2, Dengue- virus type-3 and Dengue-virus type-4, linked with each other in any order through penta glycine linkers

7. A single recombinant Tetravalent Domain III (rTDIII) protein as claimed in clam 6 wherein protein has the following preferred schematic structure:

DEN1 Domain III - DEN3 Domain III - DEN4 Domain III - DEN2 Domain III

8. A single recombinant Tetravalent Domain III (rTDIII) protein as claimed in any preceding claim wherein said protein contains more than 100 aa residues and conformational domain.

9. A single recombinant Tetravalent Domain III (rTDIII) protein as claimed in any preceding claim wherein said protein contains epitopes only from E protein.

10. A kit for use in the detection and or diagnosis of any or all of dengue specific Immunoglobin M anti-dengue IgM and Immunoglobin G anti-dengue IgG antibodies with high sensitivity and specificity, said kit characterized by the fact that it includes a single recombinant Tetravalent Domain III (rTDIII) protein as claimed in any one of claims 1 to 9.

1 1. A method for the synthesis of single recombinant Tetravalent Domain III (rTDIII) protein for use in the detection and or diagnosis of any or all of dengue specific Immunoglobin M (anti-dengue IgM) and Immunoglobin G (anti-dengue IgG) antibodies, said method comprising

(a) synthesizing a gene comprising single recombinant Tetravalent Domain III (rTDIII) protein from all four serotypes of dengue virus, namely, Dengue-virus type-1, Dengue-virus type-2, Dengue-virus type-3 and Dengue-virus type-4, linked with each other through penta glycine linkers and codon optimizing it for expression in an expression vector;

(b) constructing a recombinant TDIII gene expression vector; (c) expressing said gene to produce said recombinant Tetravalent Domain III

(rTDIII) protein; (d) purifying said protein.

12. A method as claimed in claim 11 wherein said recombinant TDIII gene expression vector is constructed by ligating rTDIII into Bamϊil and Hindlll restriction enzyme sites of said expression vector to generate the plasmid prTDIII, inserting said rTDIII gene in frame with ATG codon at the 5' end and the hexa-histidine- tag encoding sequences also at the 5' end) provided by said vector, transfoπning the ligation mixture so obtained into DH5α E. coli cells, selecting recombinant clones on ampicillin containing LB (Luria-Bertani) plates and subjecting them to direct colony PCR screening, and identifying recombinants harboring the synthetic rTDIII gene using vector specific primers..

13. A method as claimed in claim U or 12 wherein said expression vector is preferably, a bacteria] expression host.

14. A method as claimed in claim 13 wherein said bacterial expression host is preferably, an E. coli.

15. A method as claimed in claim 1 1 wherein said gene is synthesised by ligation of oligonucleotides encoding Domain III from dengue serotypes 1-4.

16. Use of a single recombinant Tetravalent Domain HI (rTDIII) protein for use in the detection and or diagnosis of any or all of dengue specific Immunoglobin M (anti- dengue IgM) and Immunoglobin G (anti-dengue IgG) antibodies.

17. A method of detecting or diagnosing any or all of dengue specific Immunoglobin M (anti-dengue IgM) and Immunoglobin G (anti-dengue IgG) antibodies in a test sample which comprises subjecting said test sample to ELISA in the presence of a single recombinant Tetravalent Domain III (rTDIII) protein as claimed in any one of claims 1 to 9.

18. A method as claimed in any preceding claim wherein said test sample comprises a human serum.