MX2008006703A - Methods and proteins for the prophylactic and/or therapeutic treatment of the four serotypes of dengue virus and other flaviviruses - Google Patents
Methods and proteins for the prophylactic and/or therapeutic treatment of the four serotypes of dengue virus and other flavivirusesInfo
- Publication number
- MX2008006703A MX2008006703A MXMX/A/2008/006703A MX2008006703A MX2008006703A MX 2008006703 A MX2008006703 A MX 2008006703A MX 2008006703 A MX2008006703 A MX 2008006703A MX 2008006703 A MX2008006703 A MX 2008006703A
- Authority
- MX
- Mexico
- Prior art keywords
- protein
- dengue
- sequence
- virus
- flaviviruses
- Prior art date
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Abstract
The invention relates to the pharmaceutical industry and describes a conserved area of the surface of the E protein, which can be used in the development of broad spectrum molecules which can be used in the prevention and/or treatment of infections caused by dengue virus 1-4 and other flaviviruses. The invention also relates to chimeric proteins which are intended for use as vaccines and for the prophylactic and/or therapeutic treatment of the four serotypes of dengue virus and other flaviviruses.
Description
METHODS AND PROTEINS FOR THE PROPHYLACTIC AND / OR THERAPEUTIC TREATMENT OF THE FOUR SERIES OF THE DENGUE VIRUS AND
OTHER FLAVIVIRUS
FIELD OF THE INVENTION
The present invention is related to the pharmaceutical industry, a conserved surface area of the protein E is described, employable in the development of broad spectrum molecules useful in the prevention and / or treatment of dengue virus infections 1-4 and other flaviviruses The present invention describes methods and proteins useful for the prophylactic and / or therapeutic treatment of the four serotypes of the dengue virus and alternatively, of other flaviviruses.
BACKGROUND OF THE INVENTION
The Dengue virus (DV) complex belongs to the Flaviviridae family and is composed of four viruses or serotypes (DV1 -DV4) related genetically and antigenically. DV is transmitted to man by mosquito, mainly Aedes aegypti. The infection causes clinical manifestations ranging from asymptomatic and benign as an undifferentiated febrile disease to more severe manifestations such as hemorrhagic fever (DHF) and the potentially fatal shock syndrome.
Dengue (DSS). The most severe clinical manifestations are usually associated with sequential infections with two different serotypes of the virus (Halstead.SB Neutralization and antibody-dependent enhancement of dengue viruses, Adv. Virus Res. 60: 421-67., 421-467, 2003. Hammon WMc, New haemorragic fever in children in the Philippines and Thailand, Trans Assoc Physicians 1960; 73: 140-155). Several epidemiological studies have been carried out, evidencing as a risk factor the sequential infection by two different viral serotypes (Kourí GP, Guzmán MG, Bravo JR, Why dengue hemorrhagic fever in Cuba, 2. An integral analysis, Trans Roy Soc Trop Med Hyg 1987; 72: 821-823). This phenomenon has been explained by the theory of antibody-mediated potentiation (ADE), which is based on an increase in viral infectivity due to an increase in the entrance of the virus-antibody complex to the cell, assisted by the cell's Fc receptors. diana (monocytes) (? a / stea SB, Pathogenesis of dengue: challenges to molecular biology, Science 1988; 239: 476-481). The envelope glycoprotein (protein E) is the major structural protein in the envelope of the virus. The three-dimensional structure of a fragment of the E protein ectodomain of the DEN2 and DEN3 viruses has been recently resolved by x-ray diffraction (Modis, Y., Ogata, S., Clements, D. &Harrison, SC A ligand -binding pocket in the dengue virus envelope glycoprotein Proc. Nati. Acad. Sci. US A 100, 6986-6991, 2003. Modis, Y., Ogata, S., Clements, D., and Harríson, SC Variable Surface
Epitope s in the Crystal Structure of Dengue Virus Type 3 Envelope Glycoprotein. J. Virol. 79, 1223-1231, 2005), showing great structural similarities with the crystal structure of the E protein of tick-borne encephalitis virus (Rey, FA, Heinz, FX, Mandl, C, Kunz, C. &Harrison, SC The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution, Nature 375, 291-298, 1995). The high structural similarity is in agreement with the similarity between the sequences of the E protein, the conservation of 6 disulfide bridges and the coincidence of the localization of residues identified as functional in different flaviviruses such as antigenic determinants, attenuation mutants and escape mutants. . Protein E is composed of three structural domains: domain I, N-terminal in sequence and central in the 3D structure, domain II or dimerization, which contains the highly conserved fusion peptide in flavivirus and the lll domain, folding type IgG and involved in the interaction with cellular receptors. Protein E is a multifunctional glycoprotein, which plays a key role in several stages of virus replication. This protein is the fundamental target of neutralizing antibodies against the virus, mediates the interaction with cellular receptors and is the fusion protein that mediates the fusion of the viral membrane and the host's plasma membrane (Heinz, FX, and SL Allison. 2003. Flavivirus structure and membrane fusion, Adv. Virus Res. 59: 63-97, Modis, Y., S. Ogata, D. Clements, and SC Harríson, 2004. Structure of the dengue virus envelope protein after
fusion membrane Nature 427: 313-319. King 2004. Chen, Y., T. Maguire, R. E. Hileman, J. R. Fromm, J. D. Esko, R. J. Linhardt, and R. M. Marks. 1997. Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate. Nat. Med. 3: 866-871. Navarro-Sánchez, E., R. Altmeyer, A. Amara, O. Schwartz, F. Fieschi, J. L. Virelizier, F. Arenzana-Seisdedos, and P. Despres. 2003. Dendrític-cell-specifíc ICAM3-grabbing non-integrin is essential for the production of human dendritic cells by mosquito-cell-derived dengue viruses. EMBO Rep. 4: 1-6. Tassaneetrithep, B., TH Burgess, A. Granelli-Piperno, C. Trumpfheller, J. Finke, W. Sun, MA Eller, K. Pattanapanyasat, S. Sarasombath, DL Birx, RM Steinman, S. Schlesinger, and MA Marovich . 2003. DC-SIGN (CD209) mediates dengue virus infection of human dendrític cells. J. Exp. Med. 197: 823-829). This protein is anchored to the virus membrane and its functions are linked to important conformational changes of both tertiary and quaternary structure. When the virus is still intracellular, this protein is found in the form of heterodimers associated with the preM protein (Allison, S. L, Stadler K., Cw Mandl, C. Kunz, and FX Heinz, 1995. Synthesis and secretion of recombinant tick -borne encephalitis virus protein E in soluble and particulate form, J. Virol 69: 5816-5820. Rice, CM 1996. Flaviviridae: the viruses and their replication, pp. 931-959.In BN Fields, DN Kni e, PM Howley, RM Chanock, JL Melnick, TP Monath, B. Roizman, and SE Straus (ed.), Virology, 3rd ed. Lippincott-Raven, Philadelphia, Pa.). At this viral stage, it is called immature virions, these virions are
defective for fusion of membranes and it has been shown that their in vitro infectivity is much lower than mature extracellular virions (Guirakhoo, F., Heinz, FX, Mandl, CW, Holzmann, H. &Kunz, C. Fusion activity of flaviviruses: comparison of mature and immature (prM containing) tick-borne encephalitis virions J. Gen. Virol. 72, 1323-1329, 1991. Guirakhoo, F., Bolín, RA &Roehrig, JT The Murray Valley encephalitis virus prM protein confers acid resistance to virus particles and alters the expression of epitopes within the R2 domain of E glycoprotein, Virology 191, 921-931, 1992). It is postulated that the function of heterodimers is to inhibit the binding of protein E to the membrane during the passage of immature virions by intracellular compartments that, due to their acid pH, could activate the membrane fusion process. In addition, the preM protein could function as a chaperone in the folding and assembly of the E protein (Lorenz, I. C, Allison, S. L, Heinz, FX &Helenius, A. Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum, J. Virol. 76, 5480-5491, 2002). At the time of secretion of the virions outside the host cells, the preM protein is processed proteolytically by host proteases (furins), the E protein forms homodimers, and the envelope is reorganized giving rise to the mature virions (Stadler, K , Allison, S. L, Schalich, J. &Heinz, FX Proteolytic activation of tick-borne encephalitis virus by furín J. Virol 71, 8475-8481, 1997. Elshuber, S., Allison, SL, Heinz , FX &Mandl, CW Cleavage of protein prM is necessary for infection of BHK-21 cells by tick-borne encephalitis virus J. Gen.
Virol. 84, 183-191, 2003). The structure of mature virions at a resolution of 9.5 A was determined by electronic cryomicroscopy (Zhang W, Chipman PR, Corver J, Johnson PR, Zhang Y, Mukhopadhyay S, Baker TS, Strauss JH, Rossmann MG, Kuhn RJ. of protein protein domains by cryo-electron microscopy of dengue virus, Nat Struct Biol. 2003, 10: 907-12, Kuhn, RJ et al., Structure of dengue virus: implications for flavivirus organization, maturation, and fusion., 717-725, 2002) and immature virions at 12.5 A (Zhang, Y. et al Structures of immature flavivirus particles, EMBO J. 22, 2604-2613, 2003). These virions have icosahedral symmetry T = 3. In mature virions the dimers of the E protein are located parallel to the virus membrane, covering almost the entire surface. These mature virions are completely infective and in this conformation interact with cellular receptors and with antibodies. Once they interact with their receptors, the virions are internalized through a process of receptor-mediated endocytosis. Once located in endosomes, at a slightly acidic pH the protein E changes its conformation and induces the membrane fusion process (Allison, SL et al., Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH, J. Virol 69, 695-700, 1995). In this process, the protein passes from dimers to trimers. The postfusogenic trimeric structure of protein E has recently been elucidated (Modis, Y., Ogata, S., Clements, D. &; Harríson, S.C. Structure of the dengue virus envelope protein after membrane fusion. Nature 427, 313-319 (2004). Bressanelli, S. et al. Structure of a
flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation. EMBO J. 23, 728-738 (2004), showing that the formation of trimers is accompanied by important changes of tertiary structure, the monomers are associated in parallel with the end of domain II, or fusion peptide region interacting with the membranes. The joint analysis of the resolved crystal structures and the structures of the virions indicate that during the different stages of the viral replication cycle, the virions undergo reorganizations in which the tertiary and quaternary structure of the E protein and the virion as a whole change deeply. The E protein is the fundamental target of neutralizing antibodies generated during viral infection. During the infection with a serotype, long-lasting neutralizing antibodies are generated against infection with the homologous serotype. During the first months after infection, these antibodies are also neutralizing against heterologous serotypes, however this capacity is lost after 9 months of infection (Halstead SB Neutralization and antibody-dependent enhancement of dengue viruses Adv Virus Res. 2003; 60: 421-67, Sabin, AB 1952. Research on Dengue World War II, Am. J. Trop.Med. Hyg.1: 30-50.) At the molecular level, the amino acid variations in the surface of the E protein between the different serotypes of the dengue virus, cause that the antibodies generated against a serotype usually have less affinity for the rest of the serotypes. A sufficiently low affinity of the antibodies can result in the loss of the capacity of
neutralization, but these can still bind to the surface of the virions in sufficient quantity to facilitate the binding of the virus to cells carrying FC receptors (Halstead, SB, and EJ O'Rourke, 1977. Dengue viruses and mononuclear phagocytes. Infection enhancement by non-neutralizing antibody J. Exp. Med. 146: 201-217, Littaua, R., I. Kurane, and FA Ennis, 1990. Human IgG Fc receptor II mediates antibody-dependent enhancement of dengue virus infection. J. Immunol., 144: 3183-3186). Additionally, during a secondary infection the increase in titer of the population of antibodies of low affinity surpasses that of the new antibodies with high affinity for the new DV serotype, due to the fact that memory B cells and pre-existing plasma cells are activated more rapidly than virgin B cells. The pattern of antibody response during convalescence in secondary infections is greatly influenced by the DV serotype of the primary infection, a phenomenon referred to as "original antigenic sin" (Halstead, SB, Rojanasuphot, S., and Sangkawibha, N. 1983. Original antigenic without in dengue, Am. J. Trop. Med. Hyg. 32: 154-156). On the other hand, high titre neutralizing monoclonal antibodies can cause immuno amplification in vitro at sufficiently low dilutions (Brandt, WE, JM McCown, MK Gentry, and PK Russell, 1982. Infection enhancement of dengue type 2 virus in the U-937 human monocyte cell line by antibodies to flavivirus cross-reactive determinants, Infecí Immun 36: 1036-1041, Halstead, SB, CN
Venkateshan, M. K. Gentry, and L. K. Larsen. 1984. Heterogeneity of infection enhancement of dengue 2 strains by monoclonal antibodies. J. Immunol. 132: 1529-1532. Morens, D. M., S. B. Halstead, and N. J. Marchette. 1987. Profiles of antibody-dependent enhancement of dengue virus type 2 infection. Microb. Pathog 3: 231-237). The antigenic structure of flavivirus E protein has been studied intensively using panels of murine monoclonal antibodies and a set of biochemical and biological analyzes, which include competence tests, recognition sensitivity to procedures such as disulfide bridge reduction and SDS treatment, recognition of fragments of proteolytic digestions and synthetic peptides, virological tests of neutralization and inhibition of haemagglutination, obtaining escape mutants, serological tests of Mabs, etc. (Heinz T. Roehrig, JT, Bolin, RA and Kelly, RG Monoclonal Antibody Mapping of the Envelope Glycoprotein of the Dengue 2 Virus, Jamaica, VIROLOGY 246, 317-328, 1998. Heinz, FX, and Roehrig, J. T (1990) Flaviviruses, In "Immunochemistry of Viruses, II, The Basis for Serodiagnosis and Vaccines" (MHV Van Regenmortel and AR Neurath, Eds.), Pp. 289-305, Elsevier, Amsterdam, Mandl, CW, Guirakhoo, FG , Holzmann, H., Heinz, FX, and Kunz, C. (1989) .Antigenic structure of the flavivirus envelope protein E at the molecular level, using tick-borne encephalitis virus as a model J. Virol. 63, 564- 571. IL Serafin and JG Aaskov Identification of epitopes on the envelope (E) protein of dengue 2 and dengue 3 viruses using monoclonal
antibodies. Arch Virol (2001) 146: 2469-2479. Three antigenic domains A, B and C have been defined which correspond to the three structural domains II, III and I respectively. Antibodies that recognize a particular epitope show very similar functional characteristics. The epitopes of domain A (equivalent to structural domain II) are destroyed by the reduction of disulfide bridges and Mabs that recognize these epitopes are inhibitors of haemagglutination, neutralize viral infection and inhibit membrane fusion mediated by the virus. In particular, the epitope A1, defined in the dengue virus is recognized by antibodies of group type specificity, that is, they show high cross-reactivity between flaviviruses. Mabs 4G2 (anti-DV2) and 6B6C (anti-JEV) recognize this epitope. The recognition of the epitope decreases in several orders in the case of immature virions and does not increase with treatment of the mature virions at acidic pH (Guirakhoo, F., RA Bolin, and JT Roehrig, 1992. The Murray Valley encephalitis virus prM protein confers acid resistance to virus particles and alters the expression of epitopes within the R2 domain of E glycoprotein, Virology 191: 921-931).
Development of vaccines At present, there are no specific treatments available for DV and its severe manifestations. Mosquito control is expensive and mostly inefficient. Clinical treatment based on the proper management of fluids to correct hypovolemia has allowed the reduction of
mortality by DHF, however treatments are still problematic in many underdeveloped countries. An estimated 30,000 deaths per year per DHF and the rate of morbidity and expenses associated with the disease is comparable with other diseases of first priority in public health (Shepard DS, Suaya JA, Halstead SB, Nathan MB, Gubler DJ, Mahoney RT, Wang DN, Meltzer Ml.Cost-effectiveness of a pediatric dengue vaccine, Vaccine, 2004, 22 (9-10): 1275-80). Several vaccine candidates are currently in different stages of development (Barrett, AD 2001. Current status of flavivirus vaccines, Ann. NY Acad. Sci. 951: 262-271, Chang GJ, Kuno G, Purdy DE, Davis BS. advancement in flavivirus vaccine development, Expert Rev Vaccines, 2004 3 (2): 199-220). The strategies addressed include live attenuated vaccines, chimeric viruses, plasmid DNA and subunit vaccines. Attenuated strains of the four serotypes have been developed with the standard propagation methodologies in primary kidney cells of dogs and monkeys (Bhamarapravati, N., and Sutee, Y. 2000. Live attenuated tetravalent dengue vaccine., Vaccine 18: 44-47 Eckels, KH, et al 2003. Modification of dengue virus strains by passage in primary dog kidney cells: preparation of candidate vaccines and immunization of monkeys, Am. J. Trop. Med. Hyg. 69: 12-16. BL, and Eckels, KH 2003. Progress in development of a live-attenuated, tetravalent dengue virus vaccine by the United States Army Medical Research and Materiel Command, Am. J. Trop. Med. Hyg. 69: 1-4). The advancement of this strategy has been affected by the
absence of animal models and in vitro attenuation markers in humans. In this sense, cDNA clones have been obtained from the four serotypes to which mutations and attenuating variations have been introduced, which in theory decrease the probability of reversion to virulence phenotypes (Blaney, JE, Jr., Manipon, GG, Murphy , BR, and Whitehead, SS 2003. Temperature sensitive mutations in the genes encoding the NS1, NS2A, NS3, and NS5 nonstructural proteins of dengue virus type 4 restrict replication in the brains of mice Arch. Virol. 148: 999-1006. Durbin, AP, et al 2001. Attenuation and immunogenicity in humans of a Uve dengue virus type-4 vaccine candidate with a nucleotide deletion in its 3'-untranslated region, Am. J. Trop. Med. Hyg. 65: 405 -413 Patent: Zeng L, Markoff L, WO0014245, 1999) Another strategy has been the creation of chimeric flavivirus variants for the four serotypes, each consisting of the preM and E structural genes of a DV serotype and the genes of the Core and non-structural proteins of the irus of yellow fever or of an attenuated strain of DV or another virus (Guirkhoo F, Arroyo J, Pugachev KV et al. Construction, safety, and immunogenicity in non-human primates of a chimeric yellow fever-dengue virus tetravalent vaccine. J Virol 2001; 75: 7290-304. Huang CY, Butrapet S, Fierro DJ et al. Chimeric dengue type 2 (vaccine strain PDK-53) / dengue type 1 virus as a potential candidate dengue type 1 virus vaccine. J Virol 2000; 74: 3020-28. Markoff L, Pang X, Houng HS, et al. Derivation and characterization of a dengue 1 host-range restricted mutant
rivs that is attenuated and highly immunogenic in monkeys. J Virol 2002; 76: 3318-28. Patent: Stockmair and schwan Hauesser, WO9813500, 1998. Patent: Clark and Elbing: W09837911, 1998. Patent: Lai CJ, US6184024, 1994.) In general, with regard to strategies based on live attenuated vaccines, there are multiple unknowns about the possible benefits of these by the phenomena of virulence reversion, viral interference and intergenomic recombinations (Seligman SJ, Gould EA 2004 Live flavivirus vaccines: reasons for caution, Lancet, 363 (9426): 2073-5). Plasmid DNA vaccines expressing DV proteins are in early stages of development, as are those based on recombinant proteins (Chang, GJ, Davis, BS, Hunt, AR, Holmes, DA, and Kuno, G. 2001. Flavivirus DNA vaccines: current status and potential, Ann, NY Acad Sci 951: 272-285 Simmons, M., Murphy, GS, Kochel, T, Raviprakash, K., and Hayes, CG 2001. Characterization of antibody responses to combinations of a dengue-2 DNA and dengue-2 recombinant subunit vaccine Am. J. Trop. Med. Hyg. 65: 420-426 Patent: Hawaii Biotech Group, Inc. WO9906068 1998. Feighny, R., Borrous, J. and Putnak R. Dengue type-2 virus envelope protein made using recombinant baculovirus protects mice against virus challenge, Am. J. Trop. Med. Hyg. 1994. 50 (3) .322-328; Deubel, V, Staropoli, i, Megret, F., et al., Affinity-purified dengue-2 envelope virus glycoprotein induces neutralising antibodies and protective immunity in mice, Vaccine, 1997. 15, 1946-1954)
Several candidates based on the above strategies have shown protection in animal models, and some have shown safety and immunogenicity in early stages of clinical trials. The main obstacle in the development of vaccines lies in the need to achieve protection against the four serotypes. It is considered that in humans, infection with a serotype of dengue virus induces lifelong immunity against the same serotype. Immunization against one serotype only achieves protection against the rest of the serotypes (heterotypic immunity) for a short period of time between 2-9 months (Sabin, AB 952. Research on dengue during World War II, Am. J. Trop. Med. Hyg. 1: 30-50). In addition, an inefficient protection against a serotype could sensitize the organism and put it at risk of severe manifestations of the disease associated with a heterologous immune response of a pathological nature in case of subsequent infection with that serotype (Rothman AL 2004 Dengue: defíning protective versus pathologic Immunity J. Clin Invest. 113: 946-951). However, the development of effective tetravalent formulations of the live attenuated vaccines and the available recombinant proteins has been a difficult task, requiring the use of complicated multidose immunization regimes.
Antibodies: Passive immunization An alternative strategy to the use of vaccines for the prevention of dengue is passive immunization with neutralizing antibodies. With
This purpose has been described obtaining humanized chimpanzee antibodies such as anti-dengue neutralizing 5H2 (Men, R., T. Yamashiro, AP Goncalvez, C. Wernly, DJ Schofield, SU Emerson, RH Purcell, and CJ Lai. 2004. Identification of chimpanzee Fab fragments by repertoire cloning and production of a full-length humanized immunoglobulin G1 antibody that is highly efficient for neutralization of dengue type 4 virus, J. Virol. 78: 4665-4674) and crossneutralizing against the four serotypes such as 1A5 (Goncalvez, AP, R. Men, C. Wernly, RH Purcell, and CJ Lai. 2004. Chimpanzee Fab fragments and a humanized immunoglobulin G1 antibody that cross-neutralize dengue type 1 and type 2 viruses. Virol. 78: 12910-12918). The use of passive immunization could be useful both for prophylactic and therapeutic purposes taking into account that it has been shown that the level of viremia is an important factor in the severity of the disease (Wang, W. K. D. Y. Chao, C. L Kao, H. C. Wu, Y. C. Liu, C. M. Li, S. C. Lin, J. H. Huang, and C. C. King. 2003. High levéis of plasma dengue viral load during defervescence in patients with dengue haemorrhagic fever: implications for pathogenesis. Virology 305: 330-338. Vaughn, D W., Green, S., Kalayanarooj, S., Innis, B.L., Nimmannitya, S., Suntayakorn, S., Endy, T.P., Raengsakulrach, B., Rothman, A.L., Ennis, F.A. and Nisalak, A. Dengue Titer Viremia, Antibody Response Pattern, and Virus Serotype Correlate with Disease Severity. J. Infect. Dis. 2000; 181: 2-9). However, the application of antibodies can also have its difficulties. According to the hypothesis
of antibody-mediated infection increase (ADE), at the time that the antibody concentration drops to subneutralizing levels, the virus-antibody immunocomplexes can amplify the entry of virus into cells carrying FC receptors and increase virus replication in these cells. Therefore it would be necessary to maintain high levels of antibody to avoid putting the patient at risk of suffering severe manifestations of the disease. One possibility is to obtain modified antibody chains in the FC to decrease the interaction with its receptors. A particularly attractive strategy is the mutation of FC residues that specifically affect the interaction with FC? R-1, FC? R-ll and FC? RIII, but not the interaction with FCRn involved in the recycling of the antibodies and therefore determinant of the average life time in vivo. Another alternative is the identification of neutralizing antibodies incapable of causing ADE. An antibody with this characteristic has been described (Patent: Bavarian Nordic Res. Inst. W09915692, 1998), which neutralizes DV2 without causing ADE in an in vitro model. However, similar antibodies against other serotypes have not been described. There are also no data on the characterization of this antibody in in vivo models. An additional difficulty is that the available animal models do not reproduce the characteristics of the infection in humans. Also in relation to the use of antibodies, a strategy based on obtaining bispecific antibody complexes has been proposed.
anti-dengue and anti-receptor 1 of the complement of erythrocytes. These complexes called heteropolymers cause the virus to bind to erythrocytes and in this way greatly increase the clearance of the virus from the bloodstream, being redirected to the tissues (Hahn CS, French OG, Foley P, Martin EN, Taylor RP, 2001. Bispecific monoclonal antibodies mediated binding of dengue virus to erythrocytes in a monkey model of passive viremia, J Immunol., 2001, 166: 1057-65.).
DETAILED DESCRIPTION OF THE INVENTION
In the invention, an area or epitope is defined on the surface of the E protein (of the envelope) highly conserved in flaviviruses and the application of this epitope as a target for obtaining efficient molecules for the prophylactic and therapeutic treatment against the four serotypes of the Dengue virus and other flaviviruses. In its application for the design of vaccines the invention demonstrates that, generating an antibody response focused in this region of protein E, a neutralizing and protective effect of similar magnitude is achieved against the four dengue serotypes. In this way, the response against the rest of the protein, of greater variability and potentially neutralizing antibodies of a specific serotype nature (or specific subcomplex) and potentially immuno-amplifying against the rest of the serotypes, is eliminated. As the antigenic properties of the area described are topographic, the invention includes the design of mutations and
stabilizing connections that guarantee the correct folding and secretion of the subdomain of protein E that includes the mentioned epitope. In its application for the development of passive immunization agents, for both prophylactic and therapeutic purposes, the invention defines recombinant molecules capable of simultaneously joining two, three or multiple symmetric copies of this epitope on the surface of the mature flavivirus virions, and they have neutralizing and protective properties superior to natural antibodies and their FAb fragments, due to a combined effect of greater avidity, and interference with the structural changes to which virions are subjected in the early stages of the viral replication cycle. In a first object of the invention the design of recombinant proteins that reproduce the antigenic and structural properties of the mentioned epitope of protein E is described. One of the recombinant proteins described is recognized by a mouse monoclonal antibody capable of neutralizing the four serotypes of the dengue virus, and that also recognizes other flaviviruses. Immunization with this chimeric recombinant protein manages to induce a neutralizing and protective antibody response against the four serotypes of dengue virus and other flaviviruses. The invention describes a way to design the chimeric recombinant protein, which allows the correct folding of the E protein domain containing a neutralizing epitope common to flaviviruses. This epitope is topographic in nature, and its antigenic properties are dependent on the 3D structure. The resulting molecules of this invention are applicable to
the pharmaceutical industry in obtaining vaccine preparations against the dengue virus and other flaviviruses as well as diagnostic means containing these proteins. In the second object of the invention, the design of other recombinant proteins with a potent neutralizing character against the four serotypes of dengue virus and other flaviviruses is described. The amino acid sequence of these proteins contains a binding domain, a spacer segment and a domain called the multimerization domain. The binding domain possesses the ability to bind to an epitope of the envelope protein highly conserved in all flaviviruses and contained in the proteins of the first object of the invention described above. In a variant, the binding domains consist of fragments of single chain antibodies capable of recognizing the conserved epitope. The spacer segments are sequences of 3-20 residues, rich in preferably hydrophilic, polar, small side chain residues, which give the segment great mobility. These segments must not interfere with the independent folding of the binding and multimerization domains and must also be resistant to hydrolysis by the serum proteases. The multimerization domains of the present invention are proteins or domains thereof which, in their native state, are associated, preferably forming dimers or trimers, although quaternary structures with a higher degree of association are not discarded. These domains are selected from human or extracellular human proteins,
to avoid the generation of autoantibodies. An essential property of the multimerization domains considered in this invention is that they do not possess the property of interacting with the Fc receptors involved in the immunoamplification process of the infection of the dengue virus mediated by antibodies. The quaternary structure of the multimerization domains may be dependent on covalent and / or non-covalent interactions. In one of the variants, the multimerization domain is based on the Fc fragment of human antibodies, including the region of the hinge that mediates interchain disulfide bridges that stabilize the dimeric structure. These Fc fragments are devoid of glycosidation, either by chemical or enzymatic deglicosidation, or by their expression in a non-glycosidant organism, such as E. coli bacteria. The non-glycosidated Fc domains can also be obtained in cells of higher organisms when they contain mutations in their sequence that alter the NXT / S pattern. The non-glycosidated Fc domains lose the ability to bind to the Fc? R-1 receptors on lll, capable of mediating immuno-amplification in vitro; however, the interaction with the FcRn receptor is not affected, a favorable property to achieve high half-lives in vivo. In another variant, the multimerization domain is a helical fragment of human matrilin that forms trimers. The connection of the binding domain to the multimerization domain by flexible spacers allows the simultaneous binding of the chimeric protein to multiple adjacent E protein monomers in the structure
icosahedral of the mature virions of flaviviruses. Thus, for a sequence variant [binding domain] - [spacer] - [multimerization domain], which results in a dimeric protein, simultaneous binding of two E protein monomers can be achieved. If a trimeric protein is produced, it would be possible to achieve join three monomers. The neutralization titre of chimeric proteins described in the second object of the present invention is superior to that of Fabs and even whole antibodies. These recombinant proteins bind the virions with greater avidity, and the simultaneous binding of several monomers interferes with the quaternary structure changes required in the membrane fusion process. The molecules resulting from this invention are applicable to the pharmaceutical industry in obtaining prophylactic and / or therapeutic agents against dengue virus and other flaviviruses as well as diagnostic means containing these proteins.
Design of chimeric protein with vaccine objectives. The currently accepted conception is that an effective vaccine against dengue should be able to generate a neutralizing antibody response against the four serotypes. However, glycoprotein E of the viral envelope is variable among serotypes. The sequence variability causes the overall response against the protein to be neutralizing against the homologous serotype, but not against the serotypes
heterologous, at the same time increasing the possibility of immunopotentiating antibodies to the infection. The present invention describes a method for the design of vaccines by subunits against dengue, which generate a uniformly neutralizing and protective response against the four serotypes. The design is based firstly on the identification of areas or epitopes on the surface of the protein, whose conservation is total or very high among serotypes and which are also exposed on the surface of mature virions. By means of a conservation analysis of the protein residues, a cluster of exposed conserved residues was identified (Figures 1 and 2, Table 1). The total surface area of the cluster is 417 Á2, contributed by 25 residues. This area is comparable with the typical values of the interaction surface between antibodies and proteins. The epitope is topographic in nature, including distant residues in the primary structure of the E protein, but close in the three-dimensional structure. Secondly, the invention describes the design of chimeric recombinant proteins containing the conserved epitope, maximizing the ratio between conserved / variable residues presented to the immune system and achieving the stabilization of the three-dimensional structure of the epitope in a manner similar to that found in the context of the complete protein E. Two possible topologies are described: B-L-C and C-L-B,
where B is the Leu237-Val252 segment and C is the Lys64-Thr120 segment of dengue 2 glycoprotein E or the homologous segments of the other serotypes or of another flavivirus, or similar sequences with more than 80% residue identity with respect any of the above. Segments homologous to B and C in flavivirus sequences are defined by the use of sequence pair alignment programs or multiple sequences such as BLAST, FASTA and CLUSTAL (Altschul, SF, Gish, W., Miller, W. , Myers, EW &Lipman, DJ 1990, Basic local alignment search tool, J. Mol. Biol. 215: 403-410, Pearson WR, Lipman DJ, Improved tools for biological! Sequence compar. Proc Nati Acad Sci US A. 1988; 85: 2444-8, Higgins D., Thompson J., Gibson T. Thompson JD, Higgins DG, Gibson TJ 1994; CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Res. 22: 4673-4680). These sequence alignments also make it possible to define homologous residues in other flavivirus sequences (also referred to as equivalent or corresponding) to the high conservation residues identified in Table 1 of Example 1 for the particular case of the dengue 2 virus sequence. both described topologies, L are spacer sequences typically between 1 and 10 residues whose role is to connect the segments B and C in a stabilizing manner with respect to the folding of the chimeric protein, so that the 3D structure of the epitope is similar to the
structure adopted in the context of complete protein E. In the two topological variants of the chimeric protein, the conserved epitope is included in its entirety and the rest of the most variable protein is excluded. The chimeric protein represents a subdomain of structural domain II of the envelope glycoprotein. This subdomain located at the end of domain II is structurally composed of two antiparallel beta sheets packed against each other. The largest is composed of three strands (segment C) and the smaller one is a beta fork (segment B). The subdomain contains two disulfide bridges and is connected to the rest of the glycoprotein E by four points, consistent with the topographic nature of the conserved epitope. However, the contact surface between the subdomain and the rest of the protein is 184 Á2, which represents only 12% of the total accessible surface of the subdomain, consistent with the feasibility of obtaining a correct folding of the subdomain through stabilizing connections described in the two previous topological variants. The invention includes the possibility of increasing the thermodynamic stability of the chimeric protein by mutations in residues not accessible on the surface of the virion and therefore not involved in the interaction with antibodies. A central and novel idea of the present invention is that it is possible to develop a subunit vaccine based on a single protein chain, which is effective against the four dengue serotypes. Current strategies based on recombinant candidates presuppose the use
of at least four recombinant E proteins, one for each serotype combined in a vaccine formulation (Patent: Hawaii Biotech Group, Inc., WO9906068 1998). Potential candidates for fragments of the E protein have also been evaluated, but until now the works have focused on the III domain, expressed in the form of fusion proteins with carrier proteins (Patent: Center for Genetic Engineering and Biotechnology, WO / 2003 / 008571 Simmons M, Murphy GS, Hayes CG Short report: Antibody responses of immunized with tetravalent dengue recombinant protein subunit vaccine Am J Trop Med Hyg. 2001; 65: 159-61 Hermida L, Rodríguez R, Lazo L, Silva R, Zulueta A, Chinea G, López C, Guzman MG, Guillen G. A dengue-2 Envelope fragment inserted within the structure of the P64k meningococcal protein carrier enables a functional immune response against the virus in mice J Virol Methods 2004 Jan; 115 (1): 41-9). The lll domain is able to generate neutralizing but specific response to each serotype, so a vaccine preparation should include the sequences of the four serotypes. The PMEC1 chimeric protein of example 1 of the present invention corresponds to a topology B-L-C, with sequences of fragments B and C of dengue virus 2 and the two-residue linker sequence Gly-Gly. A gene encoding the chimeric protein PMEC1 is described. Plasmid pET-sPMEC1-His6 encodes the PMEC1 protein fused at the N-terminus to the leader peptide pe / B, and at the C-terminus to a coding sequence for 6 histidines (Sequence No. 12).
The chimeric protein PMEC1 was obtained in a soluble form in the periplasm of the E. coli bacterium. An easily scalable purification process was developed by metal chelate chromatography (IMAC) that allowed obtaining the pure protein for subsequent studies. The purified protein was analyzed by mass spectrometry; the mass / z signal obtained corresponds to the theoretical value calculated from the sequence of PMEC1 and assuming the formation of two disulfide bridges. A strong recognition of the PMEC1 protein was demonstrated by the hyperimmune ascites obtained against the four serotypes of the dengue virus and the 4G2 mAb. This recognition is dependent on the correct formation of disulfide bridges, which suggests that the PMEC1 protein is in a conformation similar to that adopted by the corresponding region of the native protein E. When immunizing with the recombinant PMEC1 chimeric protein, neutralizing and protective response was obtained in mice, and high titers against the four serotypes of dengue virus. In parallel, the IHA test was carried out, obtaining positive titers against the four serotypes. In the in vitro neutralization assay, titers of 1: 1280 were achieved against the four serotypes of the virus. Finally, a protection test was carried out in mice, achieving protection with PMEC1 in 80 to 90% of the animals, against the four serotypes.
Modeling of the Mab 4G2-protein E complex Example 8 shows the modeling result of the structure of the Fab 4G2 complex with protein E. This antibody recognizes and neutralizes the four serotypes of dengue virus and other flaviviruses. The model was obtained by molecular coupling using the CLUSPRO method (http://structure.bu.edu/Projects/PPDocking/cluspro.html). The crystallographic structure of Fab 4G2 (file PDB 1 uyw) and PDB loan and 1oam files corresponding to the dimeric structure of dengue E protein 2 were used. Table 8 shows the values of characteristic parameters of the interface between protein E and the Fab; The values calculated for the modeled complex are similar to the protein-antibody complexes whose crystallographic structure has been determined experimentally (Table 9). The obtained model indicates that the Mab 4G2 epitope includes the region of high conservation in flavivirus identified in this invention. Table 1 shows the set of residues that make up the predicted structural epitope (E residues in contact with the antibody) and those that make up the high conservation area. 71% of the residues that make up the structural epitope recognized by the antibody according to the model obtained belong to the high conservation area. Subsequently, a model of the Fab 4G2-protein E complex was obtained in the context of the mature virion, coupling the complex structure previously predicted in the structure of the dengue 2 virus determined by
electronic cryomicroscopy. In this way, a model was obtained in which all the epitopes of the Mab 4G2 (180) present in the virion are occupied by Fab. The interatomic distance of the C-terminal residues of the heavy chain of the Fabs bound to the 90 dimers of protein E in the virion, is 100 A. This same distance measured for Fabs associated with the monomers of the asymmetric unit that are not associated as dimers is 120 Á in one case and 80 A in another. These distances are not compatible stereochemically with the sequence and structural characteristics of the IgG molecules, suggesting that the 4G2 antibody binds to the virus in a monovalent manner. This prediction is supported by the results shown in Example 12, where it is shown that Fab 4G2 and Mab 4G2 have similar neutralizing titers. This contrasts with data obtained for other antiviral antibodies in which the divalent binding causes an increase in the neutralization capacity of up to 2-3 orders (Drew PD, Moss MT, Pasieka TJ, Grose C, Harris WJ, Porter AJ. varicella-zoster virus antibody fragments to gH neutralize virus while monomeric fragments do not J Gen Virol., 2001; 82: 1959-63 Lantto J, Fletcher JM, Ohlin M. A divalent antibody format is required for neutralization of human cytomegalovirus via antigenic domain 2 on glycoprotein B. J Gen Virol., 2002; 83: 2001-5).
This characteristic of the 4G2 antibody could be common to several anti-flavivirus antibodies, as is the case of the chimpanzee antibody 1A5 against the A domain of the E protein (Goncalvez AP, Men R, Wernly C, Purcell RH, Lai CJ, Chimpanzee Fab fragments and a derived humanized immunoglobulin G1 antibody that transforms into the cross-neutralize dengue type 1 and type 2 viruses J Virol 2004; 78: 12910-8). In general, the balance of the neutralizing power between the antibody and its Fab will depend on the epitope recognized by the antibody, the identity of the antibody and the stereochemical details of the complex. Thus, Mab 4E11, which recognizes an epitope of domain B, is 50 times more neutralizing than its corresponding Fab (Thullier, P., P. Lafaye, F. Megret, V. Deubel, A. Jouan, and JC Mazie, 1999. A recombinant Fab neutralizes dengue virus in vitro, J. Biotechnol 69: 183-190).
Design of multivalent neutralizing molecules. The present invention describes the design and obtaining of molecules capable of simultaneously joining two or three copies of the conserved epitope on the surface of the virion. In total, the virion exhibits 180 copies of the conserved epitope described in the present invention, which can be grouped into 90 pairs of epitopes corresponding to protein E dimers or in 60 trios of epitopes corresponding to the copies of the three protein E monomers that make up the asymmetric unit of the virion. These molecules are able to bind di- or trivalently and present
an affinity for the virion and superior neutralizing power in several orders to the neutralizing antibodies that recognize the conserved epitope defined in this invention. These molecules neutralize the four serotypes of the dengue virus and other flaviviruses, so they are useful for the prophylactic and / or therapeutic treatment of dengue and alternatively of other flaviviruses. The sequence of the di- or trivalent molecules (proteins) of the present invention are described with the following formula: [S] - [L] - [D] or [S] - [L] - [T] where [S] is the sequence of a single chain antibody fragment (scFv) that recognizes the conserved epitope described in this invention, [L] is a spacer sequence typically between 3 and 20 amino acids, [D] is the sequence of a protein or fragment of this able to dimerize, and [T] is the sequence of a protein or fragment of this able to trimerize. The sequences of [D] and [T], are proteins or domains of proteins that do not interact with Fc receptors capable of mediating the phenomenon of immunopotentiation of viral infection. In this way the possibility of provoking the increase of viral infection in cells carrying Fc receptors at subneutralizing concentrations of di / trivalent molecules is avoided. Therefore, the molecules described are superior to the antibodies in their inability to cause ADE. In addition, these molecules have a larger size than the fragments of scFv antibodies and therefore a longer half-life in vivo.
The sequences [D] and [T] correspond to proteins and / extracellular human protein fragments, preferably serum, avoiding the possibility of the induction of an autoantibody response generated against intracellular proteins or of other species. In general, the domains [D] / [T] can be replaced by multimerization domains with a higher degree of oligomerization, provided that spacer sequences compatible with the multivalent binding are chosen. With the multimerization (including dimerization and trimerization) designed it is possible to increase the avidity of the fragments and their intrinsic neutralization capacity since the multipuntal binding to the virus stabilizes the structure of the mature virion, interfering with the changes of quaternary structure associated with the process of membrane fusion. In addition, the increase in the average life time in vivo is achieved by increasing the molecular size. These recombinant proteins, which include Fv fragments of the antibody, can become therapeutic and / or immunoprophylactic agents, effective for the control of foci of epidemics. The present invention describes a gene that codes for a chimeric protein called TB4G2. The plasmid pET-TB4G2-LH codes for the TB4G2 protein fused at the N-terminus to the leader peptide pe / B, and at the C-terminus to a coding sequence for 6 histidines (Sequence No. 16).
The chimeric protein TB4G2 contains the following elements in the amino-to-carboxy-terminal direction: (a) the variable region of the light chain of monoclonal antibody 4G2 (Sequence No. 25), (b) a flexible spacer sequence (Sequence No. 26) ), (c) the heavy chain variable region of monoclonal antibody 4G2 (Sequence No. 27), (d) a 15-residue flexible spacer sequence (Sequence No. 28), (e) a fragment of human matrilin which it gives the molecule the ability to trimerize in solution (Sequence No. 51). The chimeric protein TB4G2 corresponds to the topological variant [S] - [L] - [T], where [S] is a scFv fragment of the 4G2 antibody, [L] is a spacer of 15 residues composed of GLY residues and SER , and [T] is a trimerization domain of human matrilin that forms a helical coiled-coil trimeric structure, where the alpha helices are aligned in parallel (Dames SA, Kammerer RA, Wiltscheck R, Engel J, Alexandrescu AT. NMR structure of a parallel homotrimeric coiled coil Nat Struct Biol. 1998; 5: 687-91). This segment of matrilin covalently trimerizes by forming disulfide bridges between cysteines located at the N-terminal end of the helix. The pelB leader peptide allows the periplasmic localization of the TB4G2 protein and therefore its folding in vivo, with the correct formation of the disulfide bridges of the binding and trimerization domains. According to the Fv 4G2-virion complex models, the distances separating the C-terminal ends of the variable region of the
heavy chain of the Fv fragments attached to the three monomers of the asymmetric unit are 36, 58 and 70 A. These three atoms are circumscribed in a sphere of radius 35 Á, so the spacer segment [L] has to be able to adopt conformations compatible with this distance. Theoretically a segment of 15 residues in extended conformation has dimensions of 52.5 Á. However, such conformation is not necessarily the most stable, and in general the structural properties of the peptides are determined by their sequence. The peptides rich in GLY and SER are intrinsically flexible, capable of adopting multiple conformations in solution. Prediction of conformations with PRELUDE (Rooman MJ, Kocher JP, Wodak SJ Prediction of protein backbone conformation based on seven structure assignments, Influence of local interactions.J Mol Biol. 1991; 221: 961-79) performed in example 9, indicates that the most favorable conformations for the sequence of [L] (sequence No 28) have a distance between the amino and carboxy-terminal ends of approximately 35 A. Therefore, the design of the chimeric protein TB4G2 is structurally compatible with the simultaneous binding of the three monomers of E the asymmetric unit. The chimeric protein TB4G2 was obtained in a soluble form in the periplasm of the E. coli bacterium. An easily scalable purification process was developed by metal chelate chromatography (IMAC) that allowed obtaining the pure proteins. The purified protein was analyzed
in SDS-PAGE electrophoresis. The protein treated under reducing conditions migrates to a band corresponding to the mass of the monomer of TB4G2, and to a band corresponding to a trimer under non-reducing conditions. Finally, the neutralization test against the four serotypes of the dengue virus in BHK-21 cells was carried out, in order to compare the neutralizing capacity of TB4G2 with respect to the original antibody 4G2 and its proteolytic fragments Fab and (Fab ') 2. The TB4G2 protein showed similar neutralization titers against the four serotypes and higher in 2-3 orders to the antibody and its fragments. The present invention describes a gene (Sequence No. 17) that codes for a chimeric protein called MA4G2. The chimeric protein MA4G2 (Sequence No. 56) contains the following elements in the amino-to-carboxy-terminal direction: (a) the variable region of the light chain of monoclonal antibody 4G2 (Sequence No. 25), (b) a spacer sequence flexible (Sequence No. 26), (c) the heavy chain variable region of monoclonal antibody 4G2 (Sequence No. 27), (d) a flexible 3-residue spacer sequence (Gly-Gly-Gly), (e) the hinge, CH2 and CH3 domains of the human IgG1 immunoglobulin (Sequence No. 52). In the CH2 domain of human IgG1, the protein has been mutated ASN297- > GLN. The chimeric protein MA4G2 corresponds to the topological variant [S] - [L] - [D], defined in the present invention, where [S] is a
scFv fragment of a 4G2 antibody chain, [L] is a 3-residue spacer, of sequence GLY-GLY-GLY, and [D] the hinge, CH2 and CH3 domains of human IgG1 immunoglobulin. The hinge region mediates the formation of interchain disulfide bridges between two identical proteins, which stabilize a dimeric structure. The mutation ASN297- > GLN in the CH2 domain of human IgG1 prevents its glycosylation in eukaryotic cells and its ability to bind to Fo? R l-III receptors, which mediate immuno-amplification in vitro. Thus, unlike the original 4G2 antibody, the designed protein presents no risk of causing ADE at subneutralizing concentrations. However, it retains the ability to interact with the FcRn receptor, a favorable property to achieve long half-lives in vivo, similar to natural antibodies. Plasmid pET-MA4G2-LH (Sequence No. 20) codes for the MA4G2 protein fused at the N-terminal end to the leader peptide pe B (Sequence No. 24) and at the C-terminal end to a tail of 6 Histidines. The pelB leader peptide allows the periplasmic localization of the MA4G2 protein, where the correct formation of intrachain disulfide bridges (the binding domains, CH2 and CH3) and between the hinge (interchain) regions occurs. The histidine tail allows purification by metal chelate chromatography. The 3D model of the complex formed between MA4G2 and the protein E dimer (example 9), as well as the results of the neutralization assays (example 12) indicate that the MA4G2 chimeric protein is
stereochemically compatible with the simultaneous binding of the associated monomers in dimers in the structure of the mature virions, achieving a considerable potentiation in the biological activity. A central aspect of the present invention is that molecules capable of contacting the high conservation area of the E protein, interfere with the function of this protein, constituting potential candidates as broad-spectrum antiviral agents against flaviviruses. As shown in Example 12, the fragments of the 4G2 antibody, including the scFv, possess a neutralizing activity similar to the complete antibody, indicating that the bivalence in the antiviral activity is not necessary and this depends on the interference in the function of the E protein upon binding to the antiviral activity. area of high conservation and that this activity is broad spectrum against flavivirus. Therefore attractive methods for the identification of molecules with these characteristics, are those that allow the identification of protein molecules, peptides and small molecules that join the high conservation area. Such is the case, of methods based on blocking the binding of antibodies that recognize the high conservation area, such as Mab 4G2 and its Fab, fab2, scFv fragments or the chimeric proteins described in the present invention, which they are based on the antibody binding site. These assays can be immunoenzymatic assays, radioimmunoassays, fluorescent probe assays that allow the quantification of the binding of the molecules to the E protein, virions or chimeric proteins described in the present invention that contain the high area.
conservation. These assays can be useful for the identification of potential molecules with broad spectrum antiviral activity against flaviviruses, through in vitro screening, of libraries of chemical compounds, including those generated by combinatorial chemistry methods. Likewise, the identification of candidate molecules can be done by virtual screening methods, assisted by computers. These methods are based on computational procedures of molecular coupling, with which complexes of molecules linked to proteins can be modeled and the strength or energy of the union quantified by means of scoring functions, calculated from the coordinates of the complex. Examples of these computational molecular coupling procedures are the GOLD programs (Jones, G. et al., 1997. Development and validation of a genetic algorithm for flexible docking, J. Mol. Biol. 267, 727-748), DOCK (Kuntz. , ID, et al., 1982 A geometric approach to macromolecule-ligand interactions, J. Mol, Biol. 161, 269-288) and FLEXX (Olender, R. and Rosen feld, R., 2001. A fast algorithm for searching for molecules containing a pharmacophore in very large combinatorial virtual libraries, J. Chem. Inf. Comput. Sci. 41, 731-738). By means of these methods, virtual libraries of molecules such as the base of ZINC compounds can be screened (Irwing, JJ and Scoichet, BK, 2005. Zinc - A free Datbase of commercially available compounds for virtual screening J. Chem. Inf. Model 45, 177-182) and determine those that are
anticipated as potential binding partners of the selected active site in the receptor protein. In the case corresponding to the present invention, the binding site consists of the area of high conservation of the previously defined protein E. The atomic coordinates of the crystallographic structures of the E protein can be used, available in the PDB database or modeled by means of computational procedures such as the homology modeling method.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Representation in color scheme of the conservation of surface residues of flavivirus E protein. Dark blue means residues of greater degree of conservation in flavivirus sequences, red are residues of greater variability. In the ellipsis region of high conservation of the end of domain II of the protein is indicated. The conservation values were calculated using the CONSURF program, considering the multiple sequence alignment of all the flaviviruses available in the SWISSPROT database. These values were plotted on the surface of the protein using the PyMol program. Figure 2. Representation in color scheme of the conservation of surface residues of the E protein of dengue virus. Dark blue means residues of greater degree of conservation in the sequences of dengue virus isolates, Red are residues of greater
variability. In the ellipsis region of high conservation of the end of domain II of the protein is indicated. The conservation values were calculated using the CONSURF program, considering the multiple sequence alignment of the four serotypes of the dengue virus, available in the SWISSPROT database. These values were plotted on the surface of the protein using the PyMol program. Figure 3. Model of the three-dimensional structure of the PMEC1 chimeric protein. B is the segment Leu237-Val252 and C is the segment
Lys64-Thr120 dengue glycoprotein E 2. L is the spacer segment of two residues. The 3D model of the protein was obtained with the program package WHATIF and graphed with PyMol. Figure 4. Plasmid pET-sPMEC1. Figure 5: Plasmid pET-scFv 4G2 LH. Figure 6A: Plasmid pET-TB4G2 LH. Figure 6B: Plasmid pET-MA4G2 LH. Figures 7A-7C. Physical-chemical characterization of the chimeric protein PMEC1-His6. A: SDS-PAGE electrophoresis of the purified protein by affinity chromatography for metal chelates (lane 1) and reduced chemistry and carbamidomethylated (lane2). B: Analysis of the protein by reverse phase chromatography RP-C4, the arrow indicates the majority peak. C: Mass spectrum of the majority peak collected in reverse phase chromatography Figure 8. Schematic representation of the results of 13 molecular coupling computational simulations (program
CLUSPRO) between the Fv fragment of Mab 4G2 and the E protein of dengue 2. The columns show in a color scheme the structural characteristics of the first 30 best solutions (clusters) obtained in each simulation. Each solution is represented with three properties. The first sample in which domain of protein E is located the epitope corresponding to the solution (yellow, red and blue for domain I, II and III respectively), two colors means interface in two domains and the color violet interaction with three domains. The letters L and T mean that the epitope involves the spacer segment (between domain I and III) or the fusion peptide of protein E respectively. The second property represented with the colors white, gray or black indicates whether the epitope is located on the surface of the E protein towards the interior of the virion, laterally or externally, being the latter only relevant, assuming that the binding of the Fab does not depend of major changes in the structure of the virion. The third property corresponds to the paratope of the antibody, green if it involves the CDR (relevant solution) or does not involve the CDR (incorrect solution). The solutions compatible with experimental data are those represented with the colors yellow-black-green and are indicated with arrows. The first two rows, located on the columns of the solutions indicate the definition of ligand and receptor used in each simulation, including the identifier of the pdb file of the structure of the E protein analyzed. The molecular coupling program used (Dot or Zdock) is shown below each column.
Figure 9. Complex model between mature dengue virion 2 and 180 chains of Fab4G2. The model was obtained by coupling the Fab4G2-protein E complex in the structure of the mature virion obtained by electron cryomicroscopy (1THD). The figure shows the distances between the residues of the C-terminal end of the Fab fragments associated with three monomers of the asymmetric unit of the virion. Figure 10. Model of the complex formed by the chimeric protein MA4G2 and the dimer of the protein E. The figure was obtained with the PyMol program. Figure 11. Prediction of stability of conformers of sequence peptides (GGGS) 3GGG as a function of the distance between the N and C-terminal ends. The predictions were made with the PRELUDE program.
EXAMPLES OF REALIZATION
EXAMPLE 1 Design of the PMEC chimeric protein
In order to identify conserved regions on the surface of protein E, a conservation analysis was carried out with the CONSURF method (ConSurf: identification of functional regions in proteins by surface-mapping of phylogenetic information) Glaser, F., Pupko, T. , Peace, i,
Bell, R.E., Bechor-Shental, D., Matiz, E. and Ben-Tal, N .; 2003; Bioinformatics
79: 163-164). At the end of domain II, a portion of the highly conserved surface is observed both among the four serotypes of the dengue virus and among all the flaviviruses (figures 1 and 2). The high conservation area defines a topographic epitope, formed by nearby residues in the three-dimensional structure but far in the sequence of the protein E. The area is comprised of a structural subdomain located at the end of domain II, which is made up of two Linear segments of protein E, Leu237-Val252 (segment B) and Lys64-Thr120 (segment C). Table 1 shows the list of residues of the subdomain that are located on the outer surface of the virion and therefore accessible to the interaction with antibodies. The residues with high conservation define the area or epitope identified in this invention. The inspection of the structure of domain II of the protein E, indicates that the subdomain presents structural characteristics similar to the structurally independent domains. The area of contact with the rest of the protein is 184 Á2, which represents only 12% of the accessible area of the subdomain. In addition, this portion of the structure is defined as a domain in the CATH database (domain CATH 1 svb03, http://www.biochem.ucl.ac.uk/bsm/cath/cath.html).
TABLE 1
Definition of relevant residues in the present invention, located at the end of domain II of protein E and exposed on the surface of the virion.
No. E No. ACC AA CONS epitope
HIS 244 8 40.8 -1.074 / -0.792 LYS 246 10 43 -0.539 / -0.792 X LYS 247 1 1 41.2 -0.667 / -0.334 X GLN 248 12 4 -0.702 / -0.792 X ASP 249 13 19.5 0.366 / -0.298 X VAL 251 15 13.8 0.726 / 0.835 X VAL 252 16 1 1 .7 -0.724 / -0.792 X
LYS 64 19 26.5 0.426 / 0.271 X
LEU 65 20 9.3 -0.335 / -0.383 X
THR 66 21 11 .7 0.210 / -0.152 X
ASN * 67 22 30.2 0.417 / -0.792 X
THR 68 23 22.3 0.952 / 1.062 X
THR 69 24 14.6 -0.745 / -0.792 X
THR 70 25 23.7 -0.781 / -0.792 X
GLU 71 26 13.3 2.146 / 2.661 X
SER 72 27 12.4 -0.431 / -0.492 X
ARG 73 28 21.5 -0.284 / -0.792 X
CYS 74 29 12.7 -1.074 / -0.792 X
LEU 82 37 6.2 -0.811 / -0.792 X
ASN 83 38 39.4 4.302 / 2.327 X
GLU 84 39 1 1.2 -0.677 / -0.792 X
GLU 85 40 17 -0.861 / -0.792 ASP 87 42 1 1 .7 -0.486 / -0.792 ARG 89 44 30.8 2.051 / 0.179 PHE 90 45 6.4 2.777 / 4.283 X
VAL 97 52 1.7 -0.766 / -0.792 X
ARG 99 54 10.5 -0.898 / -0.792 X
GLY 100 55 1.3 -0.796 / -0.792 X
TRP 101 56 15.4 -1.074 / -0.792 X
GLY 102 57 14.8 -1.074 / -0.792 X
ASN 103 58 21.7 -1.074 / -0.792 X
GLY 104 59 18 -0.775 / -0.792 X
CYS 105 60 1.8 -1.074 / -0.792 X
GLY 106 61 16.3 -1.074 / -0.792 X
MET 1 18 73 13.6 -0.877 / -0.349 X
AA: amino acid, No. E DEN2: number of the residue in the sequence of dengue 2 protein E, No. PMCE1: number of the residue in the sequence of the chimeric protein PMEC1, ACC: area accessible to the solvent calculated with WHATIF (Vriend G. WHAT IF: a molecular modeling and drug design program, J Mol Graph, 1990; 8: 52-6, 29). An atomic model of the protein E dimer obtained by the independent coupling of the 3D structure of the structural domains I, II and III (pdb loan file) in the structure of the mature virion (pdb 1THD file), CONS: Score of conservation calculated with CONSURF taking into account an alignment of flavivirus sequences and the four dengue serotypes respectively. Negative values indicate greater conservation and in bold letters the corresponding values are indicated for residues defined as high conservation, epitope: residues in contact with Fab 4G2 according to the 3D model obtained by molecular coupling in example 8. We consider the residues that have at least one atom whose van der waals sphere is separated less than 3 A from the van der waals sphere of an atom of the Fab, * ASN22 glycosidated in DEN2 virus To obtain the subdomain folded independently first, it is necessary to connect the two segments in a single polypeptide chain. Two possible connections or topologies are possible: B-L-C and C-L-B where L is a spacer segment. The spacer segment must be stereochemically compatible with the three-dimensional structure
of the subdomain and in the ideal case provide a stabilizing effect on the thermodynamic stability of the chimeric protein. The distance between the alpha carbons of the Val252 and Lys64 residuals is 6.6 A, so the topology B-L-C can be obtained with spacers of one or more residues. An analysis of the structures of possible connector turns in the PDB database, which are compatible with the structure of the anchor segments (DGINS command in the DGLOOP menu of the WHATIF program package), indicates that connections of two residues are more common than connections of a waste. In the case of the topology C-L-B, the distance between the alpha carbons of the residues Thr120 and Leu237 is 11.1 Á, consistent with connections of 3-4 residues or more. The chimeric protein PMEC1 (sequence 14) of the present invention corresponds to a topology B-L-C, with sequences of fragments B and C of the dengue virus 2 and the two-residue linker sequence Gly-Gly. As sequences B and C not only the sequences corresponding to the DEN2 virus but also the homologous sequences of other flaviviruses can be chosen, including but not limited to DEN1, DEN3, DEN4, Japanese Encephalitis virus, Garrapata-transmitted Encephalitis virus, West Nile, Murray Valley virus, St. Louis Encephalitis virus, LANGAT virus, Yellow fever virus, Powassan virus, (sequences 29-42)
Furthermore, the chimeric proteins designed according to the method described above can be mutated in one or multiple residues in order to increase the thermodynamic stability of the protein or efficiency in the correct folding. Residues that are not accessible to the interaction with antibodies on the surface of the mature virions described in Table 1 can be mutated. The residues susceptible to be mutated are internal residues in the structure and / or on the lateral and internal surface of the structure. 3D / 4D of protein E in the mature virion. Mutated proteins can be obtained using experimental combinatorial methods such as filamentous phage libraries. They can also be designed using theoretical methods such as FOLDX, POPMUSIC, Rosseta. The sequences 43-50 correspond to analogs of the PMEC1 chimeric protein mutated in multiple positions. Three-dimensional models of these proteins show good compaction and quality of the models. Mutations of the exposed face of the protein are also possible, especially in positions that are not strictly conserved between dengue and / or flavivirus viruses, provided that the mutations do not affect the interaction with the neutralizing and protective antibodies directed against the conserved subdomain of the protein E.
EXAMPLE 2 Construction of plasmid PET-SPMEC1
To obtain a recombinant gene coding for the PMEC1 protein (Sequence No. 1), we started with the envelope E protein of the DEN2 virus (Sequence No. 2) of strain 1409, genotype Jamaica, present in plasmid p30 -VD2 (Deubel V., Kinney RM, Trent DW; "Nucleotide sequence and deduced amino acid sequence of the structural proteins of dengue type 2 virus, Jamaica genotype", Virology 155 (2) .365-377, 1986). This gene codes for the protein shown in Sequence No. 3. By the method of Agarwal et al. (Agarwal KL, Büchi H, Caruthers MH, Gupta N, Khorana HG, Kumas A, Ohtsuka E, Rajbhandary UL, Van de Sande JH, Sgaramella V, Weber H, Yamada T, Total synthesis of the Gene for an alanine transfer ribonucleic acid from yeast, 1970, Nature 227, 27-34) and starting from oligonucleotides synthesized in solid phase by the phosphoramidite method (Beaucage SL, Caruthers MH, Deoxynucleoside phosphoramidites- A new class of key intermediates for deoxypolynucleotide synthesis., Tetrahedron Letters, 1981, 22, 1859) a double-stranded DNA molecule (Sequence No. 4) coding for the PMEC1 protein was obtained, and consists of the following elements: 1) A recognition site for the restriction enzyme Neo I, where the initiation codon encoding the amino acid methionine (M) is found followed by a codon coding for the amino acid alanine (A) (Sequence No. 5); 2) A
fragment corresponding to the sequence comprised from position 709 to position 756 of the gene for E protein of Dengue virus 2 strain Jamaica 1409 (Sequence No. 6), which codes for the peptide sequence shown in Sequence No. 7, included in positions 237 to 252 of Sequence No. 3, 3) A coding binding segment for 2 glycines in succession (Sequence No. 8), 4) A fragment corresponding to the sequence comprised from position 190 to position 360 of Sequence No. 2, which codes for the peptide sequence shown in Sequence No. 9 (comprised between positions 64-120 of Sequence No. 3), where a silent mutation has been introduced that eliminates the Neo I restriction site present at position 284-289 of said sequence (Sequence No. 10) and 5) A recognition site for the restriction enzyme Xho I, where two codons encoding the amino acids leucine and acid are found lutámico, respectively (Sequence No. 11). This synthetic molecule was digested with the restriction enzymes Neo I and Xho I (Promega Benelux b.v., Holland) under the conditions specified by the manufacturer, and ligated with T4 DNA ligase (Promega Benelux, b.v., Netherlands), under the conditions specified by the manufacturer, to plasmid pET22b (Novagen, Inc.) previously digested in the same manner. The obtained reaction was transformed into Escherichia coli strain XL-1 Blue (Bullock WO, Fernandez JM, Short JM, XL-1Blue: A high efficieney plasmid transforming recA Escherichia coli K12 strain with beta-galactosidase selection, Biotechniques 1987; 5: 376-8) according to Sambrook et al. (Sambrook J,
Fritsch EF, Maniatis T. Molecular cloning: A laboratory manual. New York, USA: Cold Spring Harbor Laboratory Press; 1989) and the plasmid ios present in the colonies obtained in selective medium were investigated by restriction analysis. One of the resulting recombinant plasmids was named pET-sPMEC1 (Figure 4) and its DNA sequence was verified by automatic sequencing (Sequence No. 12) Plasmid pET-sPMEC1 codes for the fused PMEC1 protein, by the N-terminus, to the leader peptide of the pelB gene, and by the C-terminal to a coding sequence for 6 histidines (Sequence No. 13). This allows, on the one hand, that said protein be processed by removal of the leader peptide and secreted to the periplasm of E. coli, whose oxidizing conditions allow the correct folding and formation of disulfide bonds of PMEC1, and on the other hand allows an easy purification of said protein using metal chelate affinity chromatography (IMAC) (Suikowski, E. (1985) Purification of proteins by IMAC, Trends Biotechnol.3, 1-7). The final sequence of the protein, called PMEC1-His6, after its processing and secretion to the periplasm, is shown in Sequence No. 14.
EXAMPLE 3 Expression and purification of PMEC1-HIS6
Plasmid pET-sPMEC1 was transformed (Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A laboratory manual, New York, USA: Cold Spring Harbor Laboratory Press, 1989) into E. coli strain BL21 (DE3) (Studier , FW and BA Moffatt. "Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes." J. Mol. Biol. 189.1 (1986): 113-30) and a culture was inoculated from an isolated colony. of 50 mL of Luria-Bertani medium supplemented with ampicillin at 50 μg / mL (LBA) which was grown 12 hours at 30 ° C with a shaking of 350 rpm From this culture 1 L of LBA medium was inoculated with an optical density at 620 nm (OD620) of 0.05, which was grown for 8 h at 28 ° C until late exponential phase and then induced by the addition of isopropyl thiogalactoside (IPTG), following growth in the same conditions for 5 more hours. The induced culture thus obtained was centrifuged at 5000 x g for 30 min. at 4 ° C and from the resulting biomass the periplasmic fraction was extracted by the method of Ausubel et al. (Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K (1989) in Current Protocols in Molecular Biology, John Wiley &Sons, New York). The periplasmic fraction was dialyzed in 50 mM phosphate buffer pH 7/20 mM imidazole using a membrane with a cut-off weight of 1000 Da and the PMEC1-His6 protein was obtained therefrom by affinity chromatography
metal chelates (Suikowski, E. 1985, Purification of proteins by IMAC, Trends Biotechnol., 3, 1-7) using Ni-NTA agarose (Qiagen Benelux B.V., The Netherlands) according to the manufacturer's instructions.
EXAMPLE 4 Physical-chemical characterization of the chimeric protein PMEC1-HIS6
The purified PMEC1-His6 preparation by metal chelate chromatography shows a majority band on SDS-PAGE (FIG. 7A) that migrates with an apparent mass corresponding to the mass expected for the protein (approximately 9500 Da), the high degree of purity achieved in the preparation. In this same figure it is seen that the band corresponding to reduced PMEC1-His6 and carbamidomethylated (lane 2, figure 7A) shows a slightly lower electrophoretic migration than under non-reducing conditions (Lane 1, figure 7A). This behavior indicates that the protein is folded with the cysteines involved in intrachain disulfide bridges. An aliquot of 80 μg of PMEC1-His6 was analyzed on a C44.6x250 mm reverse phase column (J.T. Baker, USA). The chromatographic run was performed at 37oC using a high pressure chromatographic system equipped with two pumps and a controller. For the elution of the protein, a gradient of 10 to 60% (v / v) of acetonitrile in 0.1% (v / v) of trifluoroacetic acid was applied at a flow of 0.8 mL / min and the detection was made to
226 nm. In the chromatogram a single peak was obtained confirming the high degree of homogeneity of the preparation (Figure 7B). The majority peak of the RP-HPLC analysis was analyzed by mass spectrometry in order to obtain the molecular mass of the protein with greater accuracy and verify the disulfide bridge formation. The mass spectra were acquired in a hybrid mass spectrometer with octagonal geometry QT0F-2 ™ (Micromass, UK) equipped with Z-spray electronebulization ionization source. The mass spectral processing software used was the MassLynx version 3.5 (Micromass, UK). The mass spectrum of the majority species of the preparation of PMEC1-His6 has a molecular mass of 9219.51 Da (Figure 7C), this value is differentiated in 0.05 Da of the expected average mass according to the sequence of the gene. This analysis confirms that the cysteine residues of the molecule are involved in the formation of disulfide bridges. At the same time, this analysis rules out the presence of other unwanted post-translational modifications, such as degradation at the ends or modification of susceptible amino acids.
EXAMPLE 5 Antigenic Characterization of PMEC1
The purified fraction of PMEC1 was characterized by both dotblott recognition with different polyclonal and monoclonal sera
murine samples obtained by immunization with the corresponding viral preparations, as well as by human sera positive for Dengue (Table 2 and 3). A recombinant protein consisting of the lll domain of the envelope protein of the DEN-2 virus of the Jamaican genotype, fused to a sequence of six His (Dllle2), was included as control in the assays. Unlike the PMEC1 protein, Dllle2 comprises a region of greater variability of the envelope protein. The lll domain expressed recombinantly in E. coli is strongly recognized by anti-DEN hyperimmune ascites fluid (LAH) presenting a marked specificity for the homologous serotype and significantly loses its reactivity by reducing the only disulfide bridge present in that domain. This specific reactivity to the homologous serotype has also been found in the case of human antibodies. Also as a control, the recognition was included in the assays with the 3H5 mAb which, unlike the 4G2 mAb, has a specific serotype recognition by an epitope present in the lll domain of DEN-2. Like the 4G2 mAb, the recognition of 3H5 to DEN-2 is affected by the treatment of the preparation with a reducing agent (Roehrig JT, Volpe KE, Squires J, Hunt AR, Davis BS, Chang GJ.) Contribution of disulfide bridging to epitope expression of the dengue type 2 virus envelope glycoprotein J Virol., 2004; 78: 2648-52).
TABLE 2
Reactivity of the PMEC1 protein against monoclonal antibodies and
polyclonal in Dotblott.
cs ** Specified PMEC PMCE1- il \ e2 Dllle2_
DEN-1 +++ THERE
DEN-2 +++ +++ THERE
DEN-3 +++ THERE
DEN-4 +++ THERE
TBE ++ THERE
YFV ++ THERE
SLV +++ AHÍ
GF +++ cM 4G2
DEN-2 +++ CM 3H5
* 10 Dg of the purified proteins PMCE1 and DIII were applied
e2. RC: previously reduced and carboxymethylated protein. The intensity of the
obtained signal was evaluated from + to +++.
** Hyperimmune ascites liquids (LAHI) were used 1: 100 while monoclonal antibodies 4G2 and 3H5 were used at a concentration of 10 μg / ml. *** TBE: Encephalitis virus transmitted by Garrapata, YFV: Yellow Fever Virus; SLV: Encefalitis virus of San Luis. GF: cross-reactive to flavivirus group. The hyperimmune ascites obtained against the four serotypes of the dengue virus and the mAb 4G2 recognized the protein in a similar magnitude. In the case of the rest of the flaviviruses tested, the greatest recognition corresponded to the St Louis encephalitis virus, with a signal similar to that obtained for dengue 1-4. The anti-TBE and anti-YF LAIH also recognized the protein although to a lesser extent. The recognition is dependent on the formation of disulfide bridges, indicating that the protein is correctly folded, in a conformation similar to that adopted in the context of the native structure of protein E. The PMEC1 protein was further characterized through Dot-blotting using human sera of different qualities. Sera from individuals with primary infection by DEN-1, DEN-2, DEN-3 and DEN-4 were used, sera from individuals with secondary infection by DEN-2 and DEN-3 were also evaluated. Serum mixtures from three infected individuals were used in the same epidemic, with clinical symptoms and similar serology results.
The IgM Acs reactivity was determined in all sera.
viral antigens and PMEC1.
TABLE 3 Reactivity of the PMt protein: C1 against anticorbodies of the human origin in Dot-blott.
IP * IS ** - AcM AcM DEN DEN DEN DEN DEN-DEN-3 4G2 3H5 -1 -2 -3 -4 2 P +? MEC1 * ++ ++ ++ +++ +++ +++ PMCE1- RC Dllle2 ++ ++ ++ - +++ Dllle2- RC Ag DEN +++ +++ +++ +++ +++ +++ +++ ++ Ctrl Neg - - - * Mixed sera of convalescent individuals of primary infection (PI) by DEN-1, DEN-2, DEN-3 and DEN-4.
** Mixtures of sera from individuals with secondary infection (IS)
by DEN-2 and DEN-3.
*** 10 Dg of the purified PMCE1 proteins were applied and
Dllle2. RC: previously reduced and carboxymethylated protein. The intensity of
the signal obtained was evaluated from + to +++.
Ag DEN: Mixture of viral antigens of the four serotypes
obtained from supernatants of infected Vero cells.
Ctrl Neg: Control preparation obtained from supernatants of Vero cells without infecting. Mixtures of human sera were used at a 1/400 dilution while monoclonal antibodies 4G2 and 3H5 were used at a concentration of 10 μg / ml. Human sera from infections with the different serotypes of the virus recognized the protein in a similar magnitude. The most intense signals were obtained for sera belonging to individuals with secondary infection that correspond to the sera of higher titer in ELISA assays for anti-viral recognition.
EXAMPLE 6 Characterization of the antibody response generated by PMEC1
80 Balb / c mice were immunized with 20 μg intraperitoneally of the purified preparation of PMEC1, using Freund's adjuvant. A part of the animals (10 mice) were bled after the fourth dose and the presence of anti-DEN antibodies was determined by ELISA. High titers were obtained against the four serotypes of the dengue virus (Table 4). In parallel, the IHA test was carried out, obtaining positive titers against the four serotypes (Table 5). Finally, the in vitro neutralization test was carried out, achieving titers of 1/1280 equally against the four serotypes of the virus (Table 6).
TABLE 4 Anti-DEN antibody titers of sera obtained after immunization with PMEC1
* The titles were determined by dilution to the final point. Each serum was evaluated in parallel with a viral antigen preparation obtained in infant mouse brain (Clarke, DM, Casáis, J. Techniques for hemagglutination and hemagglutination-inhibition with arthropode-bome viruses, American Journal of Tropical Medicine and Hygiene 1958. 7: 561-573.) And with an uninfected brain control preparation processed in the same way.
TABLE 5 Titers by IHA of the sera of the animals immunized with the PMEC1 protein.
* The IHA titers were defined as the highest dilution capable of inhibiting the hemagglutination of goose erythrocytes against 8 viral hemagglutinating units.
TABLE 6 Viral neutralization assay with sera from animals immunized with PMEC1.
* Neutralizing titers were defined as the highest serum dilution where a 50% reduction in the number of viral plaques was obtained in BHK-21 cells.
EXAMPLE 7 Protection test
For the evaluation of the protection conferred to the mice against challenge with lethal DEN by immunization with PMEC1, mice that were not bled were used for the assays described above for characterization of the antibody response. Animals immunized with
PMEC1 were divided into four groups (15 animals per group) that were challenged each with a different viral serotype and a control group of 10 animals that did not undergo the challenge. Each of the animals received a dose of 100 LD50 of lethal DEN by intracranial inoculation and were observed for 21 days to obtain the lethality percentages. Groups of 15 mice immunized with the four viral preparations (DEN-1) were used as positive controls., DEN-2, DEN-3 and DEN-4). All the mice of these groups survived while the mice of the control group (-) for each serotype, fell ill in the 7-11 days after the challenge, obtaining a 100% mortality. Finally, the groups immunized with the PMEC1 protein presented between 80% and 90% protection, obtaining in all cases significant differences with respect to the control group (Table 7).
TABLE 7 Percentages of survival in mice immunized with the protein variants tested against challenge with lethal DEN virus.
* Calculated: (# of surviving mice) / (# of total mice). Survivor data is taken 21 after the challenge.
EXAMPLE 8 Prediction of the structure of the 4G2-protein E complex
In order to model the structure of the antigen-antibody complex, a molecular coupling study of the crystallographic structure of the Fab fragment of the 4G2 antibody (1uyw) and two crystallographic structures of the E protein of the envelope of the dengue virus 2 (files of the PDB l oan and 1oam). The CLUSPRO method was used (http://nrc.bu.edu/cluster/ S.R. Comeau, D.W. Gatchell, S.Vajda, C.J.
Camacho ClusPro: an automated docking and discription method for the prediction of protein complexes. (2004) Bioinformatics, 20, 45-50), including two different generation programs for possible complex structures: DOT and ZDOCK (Mandell JG, Roberts VA, Pique ME, Kotlovyi V, Mitchell JC, Nelson E, Tsigelny I, Ten Eyck LF. (2001) Protein docking using electrostatics and geometric fit continuum Protein Eng 14: 105-13, Chen R, Li L, Weng Z (2003) ZDOCK: An Initial-stage Protein-Docking Algorithm, Proteins 52: 80- 87). 13 simulations of molecular coupling were carried out, varying the following parameters: the coupling program (DOT or ZDOCK), the definition of ligand / receptor (Fv fragment of the antibody or protein E), the crystallographic structure of the E protein (loan or 1oam) , the quaternary structure of protein E (monomer or dimer), filtered from solutions involving the binding site of the Fv or the N-terminal segment 1-120 of protein E (Attract option in DOT). Figures 7A-7C present schematically the results of the simulations. Possible solutions were considered complexes in which the epitope was located in domain II of protein E, accessible to the interaction with the antibodies in the virion structure and the paratope consisted of the hypervariable region of the antibody. The location of epitope A1 (epitope recognized by Mab 4G2) in domain II is supported by experimental data and it has also been determined that antibodies related to this epitope recognize the proteolytic fragment constituted by amino acids 1-120 of protein E
(Roehríg, J. T., Bolín, R.A. and Kelly, R.G. Monoclonal Antibody Mapping of the Envelope Glycoprotein of the Dengue Virus 2, Jamaica, 1998, Virology 246: 317-328). Six possible solutions were obtained, structurally very similar. Table 8 shows the values of characteristic parameters of the interface between the E protein and the Fv, the values calculated for the modeled complex are similar to the protein-antibody complexes whose crystallographic structure have been determined experimentally (Table 9). The contact surface of the E protein with the antibody involves 4 segments of the sequence which agrees with the topographic nature of the epitope dependent on the correct folding of the protein, the recognition being susceptible to the reduction of the disulfide bridges. The structural epitope defined by the three-dimensional model contains the region of high conservation in the flaviviruses, which is consistent with the broad cross-reactivity of this antibody and with the recognition of the chimeric protein PMEC1 in example 5. The model also suggests that the The neutralization mechanism of the antibody is mediated by steric impediments of the binding of protein E to membranes and / or interference with the trimerization associated with the fusion process. In addition, the epitope recognized by the antibody coincides with the zone of interaction between the E protein and the preM protein inferred from the electron density corresponding to preM observed in the electronic cryomicroscopy studies of the immature virions. The evolutionary pressure
for the conservation of the intermolecular surface they can explain the high conservation of this epitope of the protein E. On the other hand the generation of escape mutants in this surface is less probable since such mutations would need to be compensated with simultaneous stabilizing mutations in the surface of the preM protein In fact, escape mutants obtained against the antibody are located in the hinge region between domain II and I, and the mutant viruses have a high degree of attenuation and defects in the ability to fuse membranes (Aaskov). This constitutes a favorable property of the PMEC chimeric proteins of the present invention as recombinant vaccinia candidates against flaviviruses. Next, we modeled the interaction between Fab4G2 and protein E in the context of mature virions. With this objective we combine the structure of the complex obtained previously in the structure of the mature virion obtained by electronic cryomicroscopy. To obtain the model we used: 1) PDB file 1THD corresponding to the structure of the virion obtained by electronic cryomicroscopy, 2) the coordinates of the complex between Fab4G2 and the monomer of protein E previously obtained by molecular coupling and 3) applied the icosahedral symmetry operations of the 1THD file to the last one. In this way, a model was obtained in which all the epitopes of Mab 4G2 (180) present in the virion are occupied by the Fab (figure 9).
TABLE 8 Properties of the model of the Fv4G2-protein complex model
* the parameters of the protein-protein interface was calculated using the server http://www.biochem.ucl.ac.uk/bsm/PP/server/ The analysis of the distance between the C-terminal ends of the Fabs indicates that the bivalent binding of the antibody is not possible without major modifications in the structure of the virions. This observation agrees with the results obtained in example 12, which show that the neutralizing titers of equimolar amounts of Fab and Mab are
Similar. This contrasts with the typical increases (2-3 orders) in avidity of the antibodies involved in bivalent unions.
TABLE 9 Characteristic properties in protein-antibody complexes *
* Jones, S. and Thornton, J.M. (nineteen ninety six). Principles of Protein-Protein Interactions Derived from Structural Studies PNAS. Vol. 93 p.13-20. http://www.biochem.ucl.ac.uk/bsm/PP/server/
EXAMPLE 9 Design of the chimeric proteins MA4G2 (bivalent) and TB4G2 (trivalent)
Next, we proceeded to design chimeric proteins based on the binding site of the 4G2 antibody, which allow simultaneous binding to two or more monomers of the E protein in the mature virion. The model of the Fabs bound to the virion obtained in example 8 shows that the distances between the residues of the c-terminal end of the heavy chain of the Fabs associated with the monomers of E in the asymmetric unit are 80, 100 and 120 Á , very distant to allow the bivalent binding of the antibody (figure 9). The same distances measured between molecules of different asymmetric units are even greater. In contrast, the distances between the C-terminal ends of the heavy chain of the Fv fragments are 36, 58 and 70 Á. These three atoms are circumscribed in a sphere of radium A which is an indicator that trivalent binding is possible with the fusion to trimerization domains using spacing peptides of medium size (10-15 residues). The distances between the c-terminal ends of the Fv bound to dimers of E in the virion structure is 36 A, whereby bivalent binding is possible by fusing the Fv fragments to dimerization domains with small spacers (5). -10 waste).
Design of miniantibody type molecule (bivalent binding) As an example of bivalent binding, the MA4G2 chimeric protein was designed whose sequence contains, in order N to C-terminal, the following elements: 1- scFV: single chain Fv fragment of Mab 4G2, with sequence VL-spacer-VH (Sequences No. 25, 26 and 27), VL is the variable region of the light chain of Mab 4G2 and VH is the variable region of the heavy chain of this antibody. 2- GGG: spacer of three Gly 3- Hinge-CH2-CH3: corresponds to the sequence of human IgG1, mutated in the glycosidation site N297 >; Q (Sequence No. 52) The MA4G2 protein is a dimer when expressed in prokaryotes or eukaryotes, because the hinge domain of the antibody allows dimerization by formation of interchain disulfide bridges and results in a human Fc. The hinge region also provides sufficient spacing and flexibility that allows the incorporation of a spacer sequence of only 3 residues (GGG) between the scFv domain and the Fc. Figure 10 shows a 3D model of the MA4G2-protein E dimer complex, consistent with bivalent binding. The presence of the mutation in the glycosidation site allows obtaining non-glycosidated Fc in cells of higher organisms. The non-glycosidated Fc domains lose the ability to bind to FcγRI-III receptors, capable of mediating immuno-amplification in vitro (Lund, J.,
Takahashi, N., Pound, J. D., Goodall, M., and Jefferis, R. 1996, J. Immunol. 157, 4963-4969. Lund, J., Takahashi, N., Pound, J. D., Goodall, M., Nakagawa, H., and Jefferis, R. 1995, FASEB. J. 9, 115-119). In this way, unlike the original antibody, the designed protein presents no risk of causing ADE at subneutralizing concentrations. However, the protein retains the ability to interact with the FcRn receptor, a favorable property to achieve a high half-life in vivo, similar to natural antibodies.
Trivalent Chimeric Protein TB4G2 As an example of trivalent binding the chimeric protein TB4G2 of scFv-spacer-T sequence was designed where: 1- scFv is the single chain Fv fragment of Mab 4G2, with sequence vL-spacer-vH (Sequences No. 25 , 26 and 27), vL is the variable region of the light chain of Mab4G2 and vH is the variable region of the heavy chain 2- spacer is a segment of sequence (GGGS) 3GGG (sequence 28) 3- T is a domain of helical trimerization of the human matrilin protein (sequence 51). The trilization domain of matrilin consists of an alpha helix that trimerizes into a parallel trimeric "coiled-coil" structure, also forming six disulfide bridges in which two end cysteines participate.
N-terminal. The helical trimeric structure is highly stable dG = 7 kcal / mol at 50 ° C (Wiltscheck R, Kammerer RA, Dames SA, Schulthess T, Blommers MJ, Engel J, Alexandrescu AT, Heteronuclear NMR assignments and secondary structure of the coiled coil trimerization Protein Sci. 1997; 6: 1734-45) from cartilage matrix protein in oxidized and reduced forms. Disulfide bridges guarantee trimerization even at very low concentrations, an advantage over trimerization based only on non-covalent interactions. The spacer segment composed of Gly and Ser, is highly flexible. Sequences of similar composition have been used on multiple occasions as spacer sequences in protein engineering. Although a sequence of 10 residues can promote a spatial distance of 35 A, compatible with the trivalent binding to the virion, this would only be achieved for a fully extended conformation of the segment. In solution the spacer segment can adopt a large number of possible conformations, which are in thermodynamic equilibrium, so that the adoption of a fully extended conformation would mean a loss of considerable entropic character. To explore the structural properties of the spacer segment, a prediction of structure of the peptide sequence (GGGS) 3GGG of 15 residues was made, using the PRELUDE program. This method takes into account statistical potentials of local interactions for what has been used for the prediction of peptide structure. In the figure
11 shows the relationship between the energy values and the distance between the N and C-terminal ends for the most probable segment conformations. The most favorable energetic conformations correspond to dimensions of 35 A, with the most extended conformations (more than 40 Á) being very unfavorable. Therefore, the sequence of spacer chosen is structurally adequate for the design of trivalent binding, according to the computational simulation.
EXAMPLE 10 Obtaining coding plasmids for a single chain antibody fragment (scFv 4G2), a trivalent molecule (TB4G2), and a single chain miniantibody (MA4G2) with the variable regions of the 4G2 antibody
To obtain a single chain antibody fragment, a multimeric protein, and a single chain miniantibody (MA4G2) with the variable regions of the monoclonal antibody 4G2, were synthesized, by the method of Agarwal et al. (Agarwal KL, Büchi H, Caruthers MH, Gupta N, Khorana HG, Kumas A, Ohtsuka E, Rajbhandary UL, van de Sande JH, Sgaramella V, Weber H, Yamada T, Total synthesis of the Gene for an alanine transfer ribonucleic acid from yeast, (1970), Nature 227, 27-34) and starting from oligonucleotides synthesized in solid phase by the method of
phosphoramidites (Beaucage SL, Caruthers MH, Deoxynucleoside phosphoramidites- A new class of key intermediates for deoxypolynucleotide synthesis., Tetrahedron Letters, (1981), 22, 1859) double-stranded DNA molecules (Sequence No. 15, Sequence No. 16 and Sequence No. 17), each of which was digested with the restriction enzymes Neo I and Xho I (Promega Benelux bv, The Netherlands) under the conditions specified by the manufacturer. Each digested molecule was subsequently ligated using T4 DNA ligase (Promega Benelux, b.v., The Netherlands), under the conditions specified by the manufacturer, to plasmid pET22b (Novagen, Inc.) previously digested in the same manner. The obtained reactions were transformed into Escherichia coli strain XL-1 Blue (Bullock WO, Fernandez JM, Short JM, XL-1 Blue: A high efficieney plasmid transforming recA Escherichia coli K12 strain with beta-galactosidase selection, Biotechniques 1987; : 376-8) according to Sambrook et al. (Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A laboratory manual, New York, USA: Cold Spring Harbor Laboratory Press, 1989) and the plasmids present in the colonies obtained in selective medium were investigated by restriction analysis. The sequence of several recombinant plasmids resulting from each transformation was verified by automatic sequencing, and for each reaction a representative molecule was chosen whose sequence corresponded to that expected. These plasmids were designated pET-scFv 4G2 LH (Figure 5, Sequence No. 18) for the expression of the single chain antibody fragment, pET-TB4G2 LH (Figure 6A, Sequence No. 19) for the expression of the molecule
multimer, and pET-MA4G2 LH (Figure 6B, Sequence No. 20) for the expression of the single chain miniantibody that carries the variable regions of 4G2. These plasmids allow the expression in Escherichia coli, inducible by isopropyl-thio-galactoside (IPTG) and under the T7 promoter, of the proteins encoded by the synthetic bands described above (Sequence No. 15, Sequence No. 16 and Sequence No. 17 ) and that in their respective immature or unprocessed forms (Sequence No. 21, Sequence No. 22 and Sequence No. 23) contain the following elements in amino-to-carboxy-terminal direction: For the immature protein scFv 4G2 LH, a) The signal peptide pelB (Sequence No. 24), b) The amino acids M (Methionine) and A (Alanine), introduced due to the cloning strategy, c) the variable region of the light chain of the monoclonal antibody 4G2 (Sequence No. 25), d) a flexible linker sequence (Sequence No. 26), e) the variable region of the heavy chain of the monoclonal antibody 4G2 (Sequence No. 27), f) the amino acids L (Leucine) and E (Glutamic acid), introduced due to the cloning strategy ion, and g) a C-terminal segment of 6 histidines; for the immature protein TB4G2 LH: a) The signal peptide pelB (Sequence No. 24), b) The amino acids M (Methionine) and A (Alanine), introduced due to the cloning strategy, c) the variable region of the chain light of monoclonal antibody 4G2 (Sequence No. 25), d) a flexible linker sequence (Sequence No. 26), e) the variable region of the heavy chain of monoclonal antibody 4G2 (Sequence No. 27), f ) a link sequence
(linker) flexible (Sequence No. 28), g) a fragment of human matrilin that gives the molecule the ability to trimerize in solution (Sequence No. 51), h) amino acids L (Leucine) and E (Acid) glutamic), introduced due to the cloning strategy, and e) a C-terminal segment of 6 histidines; and for the immature protein MA4G2 LH: a) The signal peptide pelB (Sequence No. 24), b) the amino acids M (Methionine) and A (Alanine), introduced due to the cloning strategy, c) the variable region of the light chain of monoclonal antibody 4G2 (Sequence No. 25), d) a flexible linker sequence (Sequence No. 26), e) the heavy chain variable region of monoclonal antibody 4G2 (Sequence No. 27), f) a flexible linker sequence formed by 3 glycines (G) in succession, g) a constant region fragment of the human IgG1 immunoglobulin gene consisting of the hinge region, the CH2 domain and the CH3 domain , where the amino acid C (Cysteine) of the hinge region has been mutated to S (Serine) and the potential glycosylation site of the CH2 domain has been eliminated by mutation of an N (Asparagine) to Q (Glutamine) (Sequence No. 52), h) the amino acids L (Leucine) and E (glutamic acid), introduced due to the cloning strategy, and e) a C-terminal segment of 6 histidines. These elements allow, on the one hand, that said proteins, known respectively scFv 4G2 and TB4G2, be processed by removal of the leader peptide and secreted to the periplasm of E. coli, whose oxidizing conditions allow the correct folding and formation of its disulfide bonds, and On the other hand they allow its easy purification using
affinity chromatography for metal chelates (IMAC) (Suikowski, E. (1985) Purification of proteins by IMAC, Trends Biotechnol.3, 1-7). The final sequence of both scFv 4G2 and TB4G2 and MA4G2 after processing and secretion to the periplasm is shown in Sequence No. 53, Sequence No. 54 and Sequence No. 55.
EXAMPLE 11 Expression and purification of scFv 4G2, TB4G2 and MA4G2
For the purification of scFv 4G2, TB4G2 and MA4G2 from pET-scFv4G2 LH, pET-TB4G2 LH and pET-MA4G2, respectively, the same process was followed, which is described below. The corresponding plasmid was transformed (Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A laboratory manual, New York, USA: Cold Spring Harbor Laboratory Press, 1989) into E. coli strain BL21 (DE3) (Studier, FW and BA Moffatt. "Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes." J. Mol.Biol.189.1 (1986): 113-30) and a culture of 50 was inoculated from an isolated colony. mL of Luria-Bertani medium supplemented with ampicillin at 50 μg / mL (LBA) that was grown 12 hours at 30 ° C with a shaking of 350 rpm From this culture 1 L of LBA medium was inoculated with an optical density at 620 nm (OD620) of 0.05, which was grown for 8 h at 28 ° C until late exponential phase and then induced
by the addition of isopropylthiogalactoside (IPTG), followed by growth under the same conditions for a further 5 hours. The induced culture thus obtained was centrifuged at 5000 x g for 30 min. at 4 ° C, and from the resulting biomass the periplasmic fraction was extracted by the method of Ausubel et al. (Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K (1989) in Current Protocols in Molecular Biology, John Wiley &Sons, New York). The periplasmic fraction was dialyzed in 50 mM phosphate buffer pH 7/20 mM imidazole using a membrane with a cut-off weight of 6000 Da, and the TB4G2 protein was purified therefrom by affinity chromatography for metal chelates (Suikowski, E. ( 1985) Purification of proteins by IMAC, Trends Biotechnol 3, 1-7) using Ni-NTA agarose (Qiagen Benelux BV, The Netherlands) according to the manufacturer's instructions.
EXAMPLE 12 Neutralization of the viral infection of the MAG4G2 and TB4G2 proteins
In order to characterize the in vitro biological activity of the MA4G2 and TB4G2 chimeric proteins, and to make their comparison with the Mab 4G2 and its Fab, Fab2 and scFv4G2 fragments, a neutralization test was carried out by reducing the number of plates in BHK cells. -21 (Table 10). The Fab and Fab2 fragments of Mab 4G2 were obtained by digestion with papain and pepsin respectively of the complete antibody and
purified by protein A affinity chromatography. The isoforms were isolated by ion exchange chromatography. The neutralizing titers were defined as the highest dilution of the molecule where a 50% reduction in the number of viral plaques was obtained. The initial concentration of the different molecules tested was adjusted to an equimolar concentration.
TABLE 10 Viral neutralization assay of MA4G2, TB4G2, Mab4G2 and its Fab, Fab2 and scFv4G2 fragments
* Neutralizing titers were defined as the highest dilution of the molecule where a 50% reduction in the number of viral plaques was obtained in BHK-21 cells.
Claims (38)
1. - A topographic and highly conserved area, characterized by being exposed in the mature virion, and representing a common epitope to flaviviruses, which can be used in the development of broad-spectrum molecules useful in the prevention and / or treatment of dengue virus infections 1-4 and other flaviviruses.
2. A highly conserved topographic area according to claim 1, wherein said area is an epitope of flavivirus envelope E protein and is defined by the following residues of the dengue 2 E protein or the corresponding residues in other flaviviruses: ASN67, THR69, THR70, SER72, ARG73, CYS74, LEU82, GLU84, GLU85, ASP87, VAL97, ARG99, GLY100, TRP101, GLY102, ASN103, GLY104, CYS105, GLY106, MET118, HIS244, LYS246, LYS247, GLN248, VAL252.
3. The topographic area according to claims 1 and 2, characterized by being exposed on the surface of protein E of the following flaviviruses: West Nile virus, St Louis encephalitis virus, dengue, dengue2, dengue3, dengue4, Japanese encephalitis virus, yellow fever virus, kunjin virus, Kyasanur jungle disease virus, Encephalitis virus Transmitted by Garrapata, Murray Valley virus, LANGAT virus, Louping disease virus, Powassan virus.
4. - Molecules according to claim 1, useful in the prevention and / or treatment of infections by dengue virus 1-4 and other flaviviruses, based on the topographic area described in claims 1-3, characterized by being capable of inducing a neutralizing antibody response and cross-reactivity to the four serotypes of dengue virus and other flaviviruses in individuals immunized therewith.
5. Molecules according to claim 4, which are recombinant or synthetic proteins or chimeric peptides identified in the sequence listing as SEQ ID 14, SEQ ID 29 to 50.
6. Protein molecules according to claim 4, wherein Primary structure corresponds to the ABLC sequence where A is the sequence of a peptide between 0 and 30 amino acids, B is the sequence of the Leu237-Val252 fragment of the E protein of the dengue virus 2 or the sequence homologous to it of the dengue viruses 1, Dengue 3 or Dengue 4 or any flavivirus, L is a sequence between 3 and 10 amino acids whose function is a stabilizing spacer and C is the sequence of the Lys64-Thr120 fragment of the E protein of dengue virus 2 or the homologous sequence of the dengue 1, dengue 3 or dengue 4 virus or any flavivirus.
7 '.- Protein molecules according to claim 4, whose primary structure corresponds to the sequence ACLB where A is the sequence of a peptide between 0 and 30 amino acids, B is the sequence of the Leu237-Val252 fragment of the virus E protein dengue 2 or the homologous sequence of dengue 1, dengue 3 or dengue 4 viruses or any flavivirus, L is a sequence between 3 and 10 amino acids whose function is a stabilizing linker and C is the sequence of the Lys64-Thr120 fragment of the E protein of the dengue virus 2 or the homologous sequence of the dengue 1, dengue 3 or dengue 4 viruses or any flavivirus.
8. A protein according to claims 6 and 7 wherein A is a signal peptide of bacterial secretion.
9. A protein according to claims 6 and 7 wherein A is a signal peptide of yeast or mammalian cells.
10. A fusion protein, synthetic or recombinant, characterized by being formed by the molecules described in claims 4-9, and the N- or C-terminal fusion of one or more peptide or protein segments that are capable of increasing its protective and / or therapeutic effect, or facilitate their purification and / or detection.
11. A fusion protein, synthetic or recombinant according to claim 10, wherein the N- or C-terminal fusion is one or more peptide or protein segments containing T-cell epitopes.
12. A fusion protein, synthetic or recombinant, according to claim 10, wherein the N- or C-terminal fusion is a tail of histidines.
13. A nucleic acid encoding a protein corresponding to claims 4 to 12.
14. A prokaryotic or eukaryotic host cell containing the nucleic acid of claim 13.
15. - A pharmaceutical composition characterized in that it contains one or more proteins according to claims 4 to 12, capable of inducing in the recipient organism an immune response of neutralizing and protective antibodies with cross-reactivity against dengue viruses 1-4.
16. A pharmaceutical composition characterized in that it contains one or more proteins of claims 4 to 12, capable of inducing in the recipient organism an immune response of neutralizing and protective antibodies with cross-reactivity against other flaviviruses.
17. A pharmaceutical composition characterized by being able to induce in the recipient organism an immune response of neutralizing and protective antibodies with cross-reactivity against dengue 1 -4 and other flaviviruses, at the base of which are live vectors or naked DNA, containing genes that encode for the proteins described in claims 4 to 12.
18. Protein molecules and peptides, synthetic or recombinant, according to claims 5 to 12, useful as diagnostic reagents for the detection of anti-flavivirus antibodies.
19. Molecules according to claim 1, useful in the prevention and / or treatment of infections by dengue virus 1-4 and other flaviviruses, based on the topographic area described in claims 1-3, characterized by being capable of prevent or attenuate viral infection due to its interaction with this topographic area.
20. - Molecules according to claim 19, wherein this molecule is a human antibody or produced in another species.
21. An antibody according to claim 20, wherein this molecule has a cross-reactivity between different flaviviruses and is neutralizing the viral infection.
22. Molecules of use in the prevention and / or treatment of infections by dengue virus 1-4 and other flaviviruses, according to claims 19 to 21, wherein this molecule is a recombinant or proteolytic fragment of the antibody.
23. A molecule according to claim 22, wherein said molecule is a recombinant fragment of the single chain Fv type antibody (scFv).
24. A molecule according to claim 23, characterized by being linked with or without spacers to a region of protein origin that allows its assembly as a multivalent recognition molecule of mature virions.
25. A molecule according to claim 24, wherein the protein region bound to this molecule contains a spacer and the hinge, CH2 and CH3 regions of a human immunoglobulin, as described in SEQ ID: 55 and 56.
26.- A The molecule according to claim 24, wherein the associated protein region contains a spacer and a trimerization domain, as described in SED ID: 54.
27. - A nucleic acid coding for a protein according to claims 18 to 26.
28.- A prokaryotic or eukaryotic host cell containing a nucleic acid according to claim 27.
29.- A pharmaceutical composition characterized in that it contains one or more proteins of those described in claims 18 to 26, capable of preventing or attenuating viral infection with dengue viruses 1-4.
30. A pharmaceutical composition characterized in that it contains one or more proteins of those described in claims 18 to 26, capable of preventing or attenuating viral infection with other flaviviruses.
31.- Protein molecules and peptides, synthetic or recombinant, according to claims 18 to 26, useful as diagnostic reagents for the detection of flaviviruses.
32. A molecule useful as a broad spectrum therapeutic candidate against flavivirus which is identified by a method that comprises the contact of said molecule with the conserved area or epitope of the protein E, according to claims 1-3, where the The binding of said molecule indicates a broad-spectrum therapeutic candidate.
33. A molecule according to claim 32, wherein said molecule is selected from the following class of compounds: proteins, peptides, peptidomimetics and small molecules.
34.- A method according to claim 32, wherein said molecule is included in a library of compounds. .
35. - A method according to claim 34, wherein said library of compounds is generated by combinatorial methods.
36. A method according to claim 32, wherein said binding is determined by an in vitro assay.
37. A method according to claim 36, wherein said test consists of blocking the binding of molecules according to claims 19-24, to the conserved area according to claims 1-3.
38. A method of claim 36, wherein said assay consists of blocking the binding of molecules according to claims 19-24, the protein molecules according to claims 4-7. 39.- A method according to claim 32, wherein said binding is determined by an in vivo test. 40.- A method of claim 32, which consists of a computer-assisted method comprising: 1) the corresponding atomic coordinates to the residues that make up the high conservation area of the protein E according to claims 1-3, and said coordinates are available in protein structure databases or modeled by computational methods or determined by experimental methods 2) the atomic coordinates of molecules, which have been determined experimentally or modeled by computational methods 3) a computational molecular coupling procedure that allows determining if said molecule is anticipated to contact the high conservation area.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CU2005-0229 | 2005-11-22 |
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| MX2008006703A true MX2008006703A (en) | 2008-09-02 |
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