| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | April 20, 1999 |
| PATENT TITLE |
Recombinant dengue virus envelope protein/maltose-binding protein antigens and subunit vaccine compositions containing said antigens |
| PATENT ABSTRACT |
A recombinant fusion protein (DEN-2 MBP) containing the B domain of the due (DEN 2 envelope protein is disclosed as a candidate subunit immunogen for vaccination against dengue virus infection. A gene fragment encoding amino acid 298 to amino acid 400 of the DEN-2 virus envelope was expressed as a fusion protein with the maltose binding protein (MBP) of Escherichia coli (E. coli). The recombinant fusion protein was purified and analyzed for its antigenicity imunogenicity and ability to protect mice against lethal challenge. This antigen is detected by monoclonal antibody (3H5) which is specific for a neutralizing epitope on the DEN-2 envelope and reacted with homologous polyclonal mouse immune ascitic fluid and DEN-2 immune human sera. A recombinant fusion plasmid bearing the DEN-2 MBP DNA sequence, expressing the fusion product in E. coli is disclosed. The fusion protein when administered to a host elicits a virus neutralizing antibody response which confers partial protection to the recipient animals against challenge infection. Sera from immunized mice revealed no neutralizing antibodies to any of the other DEN serotypes in the plaque reduction neutralization assay (PRNT). |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | June 27, 1996 |
| PATENT REFERENCES CITED |
Mason et al., 1990, J. Gen. Virol. 71:2107-2114. Makino et al., 1991, Am. J. Trop. Med. Hyg. 45:636-643. Megret et al., 1992, Virol. 187:480-491. Stanley et al., 1995, Vaccine 13:947-951. Stephenson, J., 1988, Vaccine 6:471-480. Kurane et al., 1992, Sem. Immunol. 4:121-127. Smucny et al., 1995, Am. J. Trop. Med. Hyg. 53:432-437. Morens et al., 1990, J. Gen. Virol. 71:2909-2914. |
| PATENT CLAIMS |
What is claimed is: 1. A fusion protein comprising a fragment selected exclusively from the B domain of the dengue X envelope protein and the maltose binding protein of E. coli wherein X is a number from 1-4. 2. The fusion protein of claim 1 wherein X is 1. 3. The fusion protein of claim 1 wherein X is 2. 4. The fusion protein of claim 1 wherein X is 3. 5. The fusion protein of claim 1 wherein X is 4. 6. The fusion protein of claim 2, wherein the dengue envelope protein consists of amino acid 293-412. 7. The fusion protein of claim 3, wherein the dengue envelope protein consists of amino acid 298-400. 8. The fusion protein of claim 4, wherein the dengue envelope protein consists of amino acid 297 to 398. 9. The fusion protein of claim 5, wherein the dengue envelope protein consists of amino acid 298 to 400. 10. The fusion protein of claim 7 wherein the maltose binding protein of E. coli contains 366 amino acids. 11. An immunogenic composition capable of inducing a neutralizing antibody response against dengue virus comprising the fusion product of a fragment selected exclusively from the B domain of the dengue X envelope protein and the maltose fusion binding protein of E. coli wherein X is a number from 1-4 in a pharmaceutically effective amount in an injectable solution. 12. The composition of claim 11, wherein X is 2 and the dengue envelope proteins consists of amino acids 298 to 400. 13. A plasmid consisting essentially of a nucleic acid fragment encoding a fusion protein comprising a fragment selected exclusively from the B domain of the dengue X envelope protein, wherein X is a number from 1-4 and the maltose binding protein of E. coli. 14. The plasmid of claim 13 wherein X is 2 and the nucleic acid fragment encodes amino acids 298 to 400 of the dengue envelope protein. |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION 1.Field of the Invention This invention relates to an immunologically active fusion protein, useful in vaccination against dengue (DEN) virus as well as a diagnostic antigen. More particularly, this invention relates to a fusion protein antigen containing 366 amino acids of a maltose binding protein (MBP) from E. coli fused to 103 amino acids of the DEN envelope protein. This fusion protein antigen reacts with mouse monoclonal (3H5) and polyclonal (HIAF) antibody to dengue virus as well as human anti-DEN antibody, making it a useful antigen for diagnostic assays and when administered as a vaccine, can confer protective immunity to a substantial proportion of vaccinated subjects. 2.Description of the Prior Art Prevention of DEN infections would alleviate a major health problem in tropical and subtropical areas of the world. Each of the four DEN serotypes may cause dengue fever and the more severe illnesses, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Infection with any one serotype of DEN appears to confer lifelong immunity to reinfection with the same serotype, but not to the other serotypes. Individuals sequentially infected with a different dengue serotype (secondary infection) appear to be at higher risk for developing more severe disease. Immmune enhancement has been proposed as the mechanism responsible for DHF/DSS. Because of this phenomenon, an effective vaccine must be either serotype specific or be against all four serotypes of dengue virus to insure that vaccination against a single serotype does not enhance infection with a different serotype. Despite more than 50 years of effort, safe and effective vaccines have not been developed. Attempts to produce cell culture-derived live attenuated dengue vaccines by long term passage in primary kidney (PDK) cells were tried unsuccessfully (Ennis et al., J. Inf. Dis., 158:876-880, 1988). A dengue-1 candidate was shown to be genetically unstable (McKee et al., Am. J. Trop. Med. Hyg., 36(2):435-442, 1987). Other dengue 2, 3 and 4 candidates were also not suitable because they were either unstable, caused unmodified dengue fever or produced low levels of neutralizing antibody (Bancroft et al., J. Infect. Dis., 149:1005-1010, 1984, Hoke et al., Am. J. Trop. Med. Hyg., 43(2):219-226, 1990, Innis et al., Am. J. Trop. Med. Hyg., 40(6):676-687, 1989). DEN viruses are composed of a single-stranded RNA molecule of positive polarity that is translated into three structural proteins, the capsid (C) protein, the membrane (M) protein, and the envelope (E) glycoprotein followed by seven nonstructural proteins (Henchal and Putnak, Clin. Microbiol. Rev., 3(4):376-396, 1990). The E protein is the major surface component of the virion membrane, it mediates virus binding to cell receptors and interacts with neutralizing antibody. Neutralizing antibodies are believed to correlate with protection (Henchal and Putnak, Clin. Microbiol. Rev., 3(4):376-396, 1990). Studies have shown that the E protein of DEN viruses contain cross reactive, subgroup specific, and serotype specific antigenic determinants and has been shown to elicit a protective immune response (Gentry et al., Am. J. Trop. Med. Hyg., 31:548-555, 1982, to Mc Cown et al., Am. J. Trop. Med. Hyg., 42(5):491-499, 1990). Immunization of rabbits with a purified native 33 kDa E protein fragment of the 17D Yellow Fever (17DYF) virus produced neutralizing antibodies and protection against intracerebral challenge (Brandriss et al., J. Enf. Dis., 161:1134-1139, 1990). Mice immunized with native purified DEN-2 envelope protein developed neutralizing antibodies and were protected against lethal virus challenge (Feighny et. al., Am. J. Trop. Med. Hyg., 47(4):405-412, 1992). Several laboratories have reported production of recombinant DEN virus structural and nonstructural proteins in expression vectors including baculovirus, vaccinia virus and E. coli (Bray et. al., J. Virol., 63:2853-2856, 1989, Bray et al., J. Virol., 185:505-508, 1991, Lai et al, Vacc., 89:351-356, 1989, Mason et al., Virol., 158:361-372, 1987, Mason et al., J. Gen. Virol., 70:2037-2049, 1989, Men et al., J. Virol., 65:1400-1407, 1991, Putnak et al., Am. J. Trop. Med. Hyg.,45:159-167, 1991, Putnak et al., Virol., 163:93-103, 1988, Zhang et al., J. Virol. 62:3027-3031, 1988). Success with recombinant proteins as vaccines in the murine model has been variable. Some baculovirus recombinants have shown protection against virus challenge, but they were membrane-associated, making them difficult to purify without denaturation and were produced in low yields (Lai et al., Vacc., 89:351-356, 1989, Putnak et al., Virol., 163:93-103, 1988, Zhang et al., J. Virol., 62:3027-3031, 1988). As crude antigens they elicited low levels of neutralizing antibodies in mice, making them unlikely candidate vaccines. Vaccinia virus expressing the E protein and nonstructural protein one (NS1) used as a live recombinant virus failed to elicit a detectable seroresponse to either protein (Zhao et al., J. Virol., 62:4019-4022, 1987). An E. coli produced fusion protein with staphylococcal protein A containing 269 amino acids of the DEN-2 E protein and NS1 protein was recently reported to elicit neutralizing antibodies and protected mice against lethal challenge (Srivastava et al., Vacc., 13:1251-1258, 1995). Staphylococcal protein A however, has a high binding affinity for human immunoglobulin G, eliminating this fusion protein as a candidate vaccine (Kobatake et al., Anal. Biochem., 208(2):300-305, 1993). Recombinants containing the B domain of the E protein as a fusion protein with TrpE were expressed in E. coli as insoluble intracellular inclusion bodies and required detergents for its purification. These purified preparations did not elicit neutralizing antibodies in immunized mice nor did they confer protection against virus challenge (Fonseca et al., Am J. Trop. Med. Hyg., 44(5):500-508, 1991). SUMMARY OF THE INVENTION Accordingly, an object of this invention is recombinant fusion proteins containing a fragment of an envelope protein of a serotype-specific Dengue virus fused to a protein fragment having the amino acid sequence of a maltose binding protein from E. coli. (MBP). Another object of this invention is recombinant subunit vaccines containing the fusion protein of MBP to the fragment of the corresponding region of the serotype-specific DEN envelope protein. An additional object of the invention is the production of virus neutralizing antibodies produced in response to serotype-specific DEN-MBP fusion products for use in passive immunization as well as for other therapeutic purposes. A further object of the invention is a plasmid containing the DNA sequence coding for the serotype-specific DEN-MBP fusion protein. An additional object of this invention is the use of serotype-specific DEN-MBP fusion proteins as diagnostic antigens in immunoassays. These and additional objects of the invention are accomplished by isolating a fragment of the serotype-specific Dengue virus envelope protein (DEN X E Protein) including at least amino acids 298-400. "X" is a number selected from 1, 2, 3, or 4 to denote a specific sero-type of the Dengue virus. The amino acid sequence of the fragment of interest of the DEN E protein for serotype 2 and 4 is the same (298-400), for DEN 1 it is amino acid 293-412, and for DEN 3 it is amino acid 297-398. Using standard techniques, the DEN E protein fragment is inserted into a plasmid containing the nucleotide sequence coding for the amino acids of the Maltose binding protein of E. Coli. The plasmid is inserted into E. coli. and the fusion protein of the Dengue "X" virus fragment and Maltose binding protein (fusion protein) are expressed. The fusion protein can be used as a type-specific diagnostic antigen in any of the accepted diagnostic tests. The fusion protein is maintained and used in the diagnostic solutions usually used to preserve a diagnostic protein such as glycerol etc. Further, the fusion protein of this invention can be injected into a host to produce type-specific virus neutralizing antibodies. These virus neutralizing antibodies can be isolated and reproduced through standard techniques and the virus neutralizing antibodies can be used as type-specific passive immunogens against Dengue fever. In the embodiment of Dengue 2 illustrated in the specific examples, the fusion protein is formed of the fragment containing amino acids 298 to amino acid 400 of DEN 2 E protein fused to 366 amino acids of a maltose binding protein from E. coli. This protein is designated DEN 2 maltose binding protein (DEN-2 MBP). By DEN-2MBP we refer to a DEN virus serotype 2 specific maltose binding protein fusion. BRIEF DESCRIPTION OF THE DRAWINGS A more complete appreciation of the invention will be readily obtained by reference to the following Description of the Preferred Embodiments and the accompanying drawings in which like numerals in different figures represent the same structures or elements. The representations in each of the figures is diagrammatic and no attempt is made to indicate actual scales or precise ratios. Proportional relationships are shown as approximations. FIG. 1 is a schematic diagram of the predicted general features of the amino acid sequence of DEN-2 MBP. DESCRIPTION OF THE PREFERRED EMBODIMENTS One of the problems with Dengue fever is enhancement of susceptibility to death caused by a reinfection of a subject. To avoid antibody-dependent enhancement of DEN virus, a suitable DEN vaccine should induce long-lasting type-specific antibodies. This is accomplished with a subunit vaccine containing proteins representing DEN type-specific protective antigens or combinations of type-specific antigens, but containing few or no DEN group-reactive epitopes. The illustrated embodiment of the invention is a construct containing 366 amino acids of the MBP of E. coli fused to 103 amino acids of the B domain of the DEN 2 E protein. Fonseca et. al described this region as disulfide-bridge dependent, indicating the importance of conformation and Trirawatanapong et. al showed that a 12 amino acid epitope of the DEN envelope protein located in this region was associated with the production of neutralizing antibody (Trirawatanapong et al., GENE, 116:139-150, 1992). Reactivity of the illustrated DEN-2 MBP fusion protein of this invention with hyper immune ascitic fluid, human sera and DEN-2 specific monoclonal antibody by Western blot and ELISA shows proper folding of the DEN-2 MBP fusion protein, as well as confirming the presence of the type-specific neutralizing epitope described by Trirawatanapong. Plaque reduction neutralization assays (PRNT) using sera from mice immunized with the DEN-2 MBP fusion protein revealed no neutralizing antibodies to any of the other serotypes, indicating the achievement of type-specificity required for a successful DEN subunit vaccine. The maltose binding protein used as the carrier protein is involved in the translocation of maltose across the cytoplasmic membrane of E. coli (Duplay et al., J. Biol. Chem., 259(16):10606-10613, 1984). A sequence homology search was performed at the NCBI using the BLAST network service and revealed no sequence similarity with any known eukaryotic proteins. Therefore antibodies produced against MBP would not present a significant risk factor to human health. The enhanced immunogenicity observed with the DEN-2 MBP fusion protein is believed due to its ability to be expressed in a soluble form, resulting in proper folding, as well as providing a protective environment enhancing its stability. Another possible role for the MBP component might be the presence of T-helper cell epitopes aiding B-cells in antibody synthesis. In contrast to the TrpE fusion protein discussed above, this invention is the fusion protein of a maltose binding fusion protein (MBP) and the B domain of the appropriate DEN E protein, expressed as a soluble protein. This fusion protein is easily purified by affinity chromatography. Mice immunized with the E. coli produced DEN-2 MBP fusion protein developed the highest reported neutralizing antibody titers for recombinant DEN subunit imunuogens and conferred significant protection against lethal virus challenge. The recombinant fusion protein antigen DEN-2 MBP containing 103 amino acids of DEN virus E protein is a candidate immunogen for vaccination against DEN virus infections. The DEN-2 MBP antigen is detected by the monoclonal antibody (3H5) which is specific for a neutralizing epitope (Trirawatanapong et al., Gene, 116:139-150, 1992) on the DEN envelope. In addition, the DEN-2 MBP is detected by DEN specific antibodies in HIAFs and human sera when used in immunoassays such as the enzyme linked immunosorbent assay (ELISA), dot immunoassay (DOTIA) and Western blot. A recombinant prokaryotic expression plasmid bearing the DEN-2 MBP DNA sequence expresses the fusion protein in E. coli and introduction of this fusion protein antigen into mice elicits a type-specific virus neutralizing antibody response and confers significant protection to the recipient animals against challenge infection. |
| PATENT EXAMPLES | Available on request |
| PATENT PHOTOCOPY | Available on request |
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